Journa

C A L I F O R N I A

D E N TA L

February 2016
Intraoral Lesions
Oropharyngeal Cancer
Oral Cancer Chemoprevention

A S S O C I AT I O N

ORAL CANCER:
NOVEL CONCEPTS
FOR THE ORAL
HEALTH CARE
PRACTITIONER

Diana V. Messadi,
DDS, MMSc, DMSc

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it at The Dentists Insurance Company, TDIC. Take our Risk
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Feb. 2016

C D A J O U R N A L , V O L 4 4 , N 2

D E PA R T M E N T S

73 The Associate Editor/Stepping Outside the Operatory

75 Impressions
129 RM Matters/Getting the All-Clear Signal: Medical
Clearance Forms and Follow-Up

135 Regulatory Compliance/Infection Control Q-and-A

75

139 Periscope
142 Tech Trends

F E AT U R E S

82 Oral Cancer: Novel Concepts for the Oral Health Care Practitioner
An introduction to the issue.
Diana V. Messadi, DDS, MMSc, DMSc

85 Managing Intraoral Lesions in Oral Cancer Patients in a General Dental Practice: An Overview
Patients with active cancer or a history of cancer may present with multiple side effects that dental practitioners
can manage or prevent. The authors discuss some of these concerns and provide management strategies.
Reuben Han-Kyu Kim, DDS, PhD; Paul Yang, BS, MS; and Eric C. Sung, DDS

93 Current Trends in the Incidence and Presentation of Oropharyngeal Cancer

This article reviews the current knowledge about oropharyngeal cancers for their epidemiology, pathogenesis,
clinical behavior, treatment and prevention.
Fariba S. Younai, DDS

101 Oral Cancer Chemoprevention: Current Status and Future Direction

The aim of this study is to review the current status of cancer chemoprevention and its effectiveness in
treatment of oral premalignant lesions and prevention of their progression to oral cancer.
Diana V. Messadi, DDS, MMSc, DMSc, and Kazumichi Sato, DDS, PhD

112 Targeting Cancer Stem Cells in Oral Cancer

This article reviews the current knowledge of the cancer stem cells (CSCs) hypothesis in oral cancer and the
traits displayed by CSCs.
Qilin Xu, MD, PhD, and Anh D. Le, DDS, PhD

121 A Chemopreventive Nanodiamond Platform for Oral Cancer Treatment

In this paper, the authors propose a chemopreventive nanodiamond platform for the delivery of celecoxib to
oral cancer lesions.
Albert Yen, BS; Kangyi Zhang, PhD; Giulia Daneshgaran, BS; Ho-Joong Kim, PhD; and Dean Ho, PhD

F E B R U A R Y 2 0 1 6 71

C D A J O U R N A L , V O L 4 4 , N 2

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Volume 44, Number 2

February 2016

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Journal of the California Dental Association (ISSN 10432256) is published monthly by the
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Periodicals postage paid at Sacramento, Calif. Postmaster: Send address changes to Journal
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72F E B R U A R Y 2 01 6

Assoc.
Editor
Editor

C D A J O U R N A L , V O L 4 4 , N 2

Stepping Outside the Operatory

Ruchi K. Sahota, DDS, CDE

hen was the last time

someone asked you,
How can I make
your life easier?
It was incredibly
reassuring to hear this from a member of our
CDA Executive Committee. She asked me
what CDA could do to ensure that parents
of young children, like me, felt supported
and able to serve in leadership positions at
our association. Long emails in paragraph
form ensued. We discussed challenges,
including difficulty attending face-toface meetings while nursing a baby. We
re-examined processes that were already
in place. We reviewed other options.
At the same time, she brought
a few other young mothers into the
conversation. We swapped stories. We
strengthened connections. We confirmed
that one thing that always makes a
person feel better we confirmed that
we were not alone. There were other
young mother CDA volunteers out there.
In that moment, I realized organized
dentistrys sincere intentions to help. Here
was another tangible member benefit. This
isnt anything new. Time and time again,
organized dentistry has helped me be a
better leader and a better person. It
continues to renew its special place in my
heart. CDA has become like family. When
organized dentistry connected me to other
young mother leaders, I was filled with
emotions. I felt relief there were others
out there trying to balance work, family,
volunteerism, friends and all things life. I
felt pride there were others out there
trying to set an example of strength, poise
and diligence for their little ones. And I
felt support there were other dentists
from around the state willing to lend me
their time, experience and encouragement.
After almost 15 years of volunteering,
I can value how much it has given back to
my life. So it is natural to wonder: Why are
there not more young parents involved?
Of course, it may be simpler to stay
within the confines of the office walls and

Time and time again, organized

dentistry has helped me be a better
leader and a better person.

9-to-5 schedule. At times, involvement

in organized dentistry takes us away from
our normal day-to-day lives. Arthur A.
Dugoni, DDS, MSD, dean emeritus of
the University of the Pacific, Arthur A.
Dugoni School of Dentistry, recently
spoke at CDA Presents in San Francisco.
He encouraged attendees to step out of
their comfort zone, saying, You have to
live beyond the nine-by-nine operatory
and your high-speed handpiece.
The volunteer life cycle starts off
as we apply for positions in our dental
society or maybe a CDA council. We
may aim to represent our personal
demographic, voice our opinions
and share our experiences. Slowly
leadership in organized dentistry
enhances our ability to see. Within a
short period of time, volunteering starts
giving back. Motivation seeps into
the consciousness. It makes us want
to do better in our own offices. We
are inspired to find ways to give back
to our communities at home. And we
become more productive in our overall
lives. What started as a mission to serve
transforms itself into a generous gift.
Many may not apply for volunteer
positions because of the lack of monetary
compensation. Napoleon Hill said, The
[wo]man who does more than [s]he is paid
for will soon be paid for more than [s]he
does. Volunteer leadership gives us the
opportunity to be a part of something
bigger than ourselves. Volunteering
involves us in the movement to bring
positive change to the oral health of the
general public isnt that a part of the
Hippocratic Oath we take?

Dr. Dugoni alluded to the many

benefits of getting involved in his speech.
But he repeatedly went back to the same
piece of advice. Dr. Dugoni reminded
us to smile. He reminded us to laugh as
much as possible. More often than not,
we smile and laugh when we are amongst
friends and family when we are with
those who wish us joy and happiness.
We belong to a magnificent profession
with some pretty amazing colleagues
many of whom are kind, virtuous and
ultimately inspirational. We will never
know them unless we step outside
the comfort zone and get involved. We
will have more of a chance to follow Dr.
Dugonis advice if we leave our nine-bynine operatories. We will have more of
a chance to laugh and smile when we
connect with other dentists in organized
dentistry. They will restore our sense
of purpose in our profession. They will
connect with us, share their experiences
and inevitably remind us that we are not
alone. There are others out there going
through many of the same challenges.
And there are others out there who we
will meet through organized dentistry who
will offer to make your life easier.
Ruchi K. Sahota, DDS, CDE, practices
family dentistry in Fremont, Calif., and serves
as faculty at the University of the Pacific,
Arthur A. Dugoni School of Dentistry. She
is also a certified dental editor, a consumer
advisor for the American Dental Association,
past president of the Southern Alameda County
Dental Society and a fellow of the American
College of Dentists, International College of
Dentists and the Pierre Fauchard Academy.
F E B R U A R Y 2 0 1 6 73

You are the protector of the smile. You enable people to

laugh without shame, eat their favorite foods and experience
the dignity of aging with grace. Thats why this association
tirelessly advocates for the profession and stands up for those
in need of care. Because the world is a better place when
people are smiling, and thats thanks to you.

Renew today.

cda.org/member

Impressions

C D A J O U R N A L , V O L 4 4 , N 2

A Cry for Help

David W. Chambers, EdM, MBA, PhD

The nub:
1. Social fatigue is damaging
our willingness to help others.
2. Help is more likely to come
from one who shares your
problems than from experts.
3. No one cares how much you
know until he or she knows how
much you care.

David W. Chambers, EdM, MBA, PhD, is professor

of dental education at the University of the Pacic, Arthur
A. Dugoni School of Dentistry, San Francisco, and editor
of the Journal of the American College of Dentists.

I am one of those very peculiar men who regularly asks for

directions when driving. I believe in the African saying, If you
want to go fast, go alone. If you want to go far, go together.
But my system is failing. I find it harder these days to
get help. As a country, we prefer to sell solutions instead of
answer questions. It seems it is becoming unfashionable in
America to help others.
Part of the reason was obvious when I signed onto
my computer this morning. Several minutes of
protecting myself against unwanted spam put me
in such a negative mood I had to pump myself up
to be civil to those who were
asking for my help.
Of course, I do need
help from time to time
with my computer. Very
occasionally, I will go to the IT department at the school
where I teach. They are knowledgeable when my number
eventually comes up in the cue. I am much more inclined to chat
informally with one of my colleagues. There is a person in HR
who is a wizard with Word. A new faculty member in the basic
sciences is a master of graphics. I prefer the users to the experts
for several reasons. First, they are almost always able to help
immediately, e.g., when I need the help. Second, they know the
context of the problem I am trying to solve and help me with
the problem I should be working on instead of the one I asked
about. Third, they make certain I understand the answer rather
than taking my computer and changing something so it no longer
misbehaves in exactly the same way. I believe the same is true for
dentists who recognize that they face ethical issues: they would
prefer to get help from a colleague rather than from an academic
expert. And for the same reasons.
In an article in the February 2015 issue of the Academy of
Management Journal, I read about helping behavior in a software
engineering firm that uses teams to develop projects. Among
colleagues who were expected to help each other, 26 percent of
the email requests for assistance and 28 percent of the phone
calls for help were ignored. OK, I have a better record than that.
However, the computer has become an all-purpose labor saving
device. Just ignore inconvenient requests.
Calls for help are more successful when begun by establishing
or reaffirming existing social relationships. Another way to boost
success is to explain why it matters to the person from whom
help is sought. Acknowledging the status of the helper is useful.
Finally, only a tiny fraction of requests for help that are made in
person are refused.
F E B R U A R Y 2 0 1 6 75

F E B . 2 0 16

IMPRESSIONS
C D A J O U R N A L , V O L 4 4 , N 2

Image: Oregon State University

New Patent on Synthetic Biochemical Compound

for Gum Disease Treatment

Bioactive Glass May Be the

Future of Fillings
Bioactive glass can be used in tooth
fillings to help reduce the ability of
bacteria to attack composite tooth
fillings and perhaps even provide some
of the minerals needed to replace
those lost to tooth decay, according
to new research from engineers
at Oregon State University.
Bioactive glass, which is a type of
crushed glass that is able to interact with
the body, has been used in some types
of bone healing for decades, said Jamie
Kruzic, a professor and expert in advanced
structural and biomaterials in the OSU
College of Engineering, in a news release.
This type of glass is only beginning
to see use in dentistry, and our research
shows it may be very promising for tooth
fillings, he said. The bacteria in the
mouth that help cause cavities dont seem
to like this type of glass and are less likely
to colonize on fillings that incorporate
it. This could have a significant
impact on the future of dentistry.
Bioactive glass is made with
compounds such as silicon oxide, calcium
oxide and phosphorus oxide, and looks
like powdered glass. It is referred to as
bioactive because the body notices it
is there and can react to it, as opposed to
other biomedical products that are inert.
Bioactive glass is very hard and stiff and
can replace some of the inert glass fillers
that are currently mixed with polymers to
make modern composite tooth fillings.
76F E B R U A R Y 2 01 6

University of Louisville researchers recently received a patent on a synthetic

biochemical compound and its variants, moving science closer to a treatment
for gum disease. This discovery could lead to the formulation of a mouth rinse
or tooth varnish to prevent the pathogen, P. gingivalis, from colonizing in the
mouth and establishing itself orally.
The researchers developed and tested 40 dierent molecular compounds,
and the three most potent compounds are being developed further. This patent
builds on previous work of researchers who developed a series of peptides,
the building blocks of protein in a cell, that prevent interaction between P.
gingivalis and S. gordonii.
When P. gingivalis enters the oral environment, it initially interacts with the
bacterium S. gordonii an otherwise benign organism in order to lay the
ground work to propagate and ultimately gain a foothold below the gum line,
leading to periodontal disease, said Donald Demuth, PhD, associate dean for
research and enterprise at the UofL School of Dentistry.
Demuth found that administering the peptide in an animal model
prevented P. gingivalis-related bone loss and prevented the spread of the
bacterium in the mouth. Creating peptides is expensive, Demuth said, but
synthetic compounds that mimic the active peptides are easier to formulate
and less costly to produce on a large scale.
Bacterial biolm in the presence
of the inhibitory compounds.

Bacterial biolm in the absence of

the inhibitory compounds. (Images
courtesy of University of Louisville.)

New tooth decay often begins at the

interface of a filling and the tooth, and is
called secondary tooth decay. The tooth
is literally being eroded and demineralized
at that interface, Kruzic said.
Bioactive glass may help prolong
the life of fillings because, according
to researchers, the new study showed
that the depth of bacterial penetration
into the interface with bioactive glasscontaining fillings was significantly

smaller than for composites lacking the

glass. Fillings made with bioactive glass
should slow secondary tooth decay and
provide minerals that could help replace
those being lost. The combination of
these two forces should result in a tooth
filling that works just as well, but lasts
longer, according to the authors.
For more, see the study in the
journal Dental Materials, January
2016, vol. 32, issue 1, pp. 73-81.

C D A J O U R N A L , V O L 4 4 , N 2

How Teeth Find Their Way to the Right Spot in the Jaw
Led by scientists at the University of
California, San Francisco, researchers
recently showed in mice that molar
progenitor cells migrate to their final
locations during development, rather
than forming the teeth in place, as
researchers had previously thought.
According to a news release from the
university, this is the first time researchers
have captured on video how teeth find
their way to the right spot in the jaw.

The authors report that by

combining lineage tracing, genetic cell
ablation and confocal live imaging,
they were able to identify a migratory
population of Fgf8-expressing epithelial
cells in the embryonic mandible.
These findings are published in the
journal Developmental Cell and suggest
that the progenitor cells that produce
other organs could also exhibit asyet unrecognized wanderlust, and

Osteoporosis and Marginal Bone Loss in

Osseointegrated Implants
Researchers from the University of Seville recently conducted a study to
evaluate the possibility of a correlation between osteoporosis, as measured
by the mandibular cortical index (MCI), and marginal bone loss (MBL) and to
assess how various systemic diseases, periodontitis and placement of implants
in regenerated bone are correlated with MBL and MCI.
MBL and MCI were assessed from panoramic radiographs and the
retrospective study, which examined 212 implants inserted in 67 patients, found
that when the total sample implant was evaluated, a signicant association
was found between the presence of osteoporosis and MCI and between the
presence of diabetes mellitus and MCI. According to the authors, signicant
associations were also found between MBL and placement of implants in
regenerated sites and between MBL and a previous history of periodontitis.
When the sample is evaluated only in selected implants (one per patient),
signicant dierences appear to relate only to the MBL with the placement of
implants in regenerated bone sites, the authors wrote.
Osteoporosis (as evaluated by MCI) does not pose a risk for the
development of greater MBL, the study concluded, noting
that parameters adversely aecting the development
of increased MBL are a previous history of periodontitis
and especially the placement of implants at sites of bone
regeneration.
For more, see the study in the Journal of
Periodontology, January 2016, vol. 87, no. 1, pp. 14-20.

could help explain how teeth and

other organs ended up in such diverse
configurations in different species
over the course of evolution. The
insights may even have implications
for understanding how cancerous cells
migrate to invade other tissues.
Its a crucial part of development,
said Ophir Klein, MD, PhD, chair of
the divisions of craniofacial anomalies
and orthodontics at UCSF, and senior
author of the new study. For example,
you need to get the eyes in the right
part of the face. The limb positioning
needs to be perfectly balanced. For each
particular species, the teeth need to be at
the right place in the jaw for the animal
to be able to eat or to defend itself. But
we knew little to nothing about how
they get there, at least in mammals.
For more, see the study published
in the journal Developmental Cell,
vol. 35, issue 6, pp. 713-724.
CORREC TION .
In the January 2016 issue of the Journal, the conict
of interest disclosure for Dr. Peter M. Milgrom was
inadvertently omitted. His disclosure should have
read Dr. Milgrom is a principal in ADP Silver Dental
Arrest LLC, which licenses permission to market
Advantage Arrest to Elevate Oral Care LLC. The
online version of the article has been corrected. We
apologize for the error.
F E B R U A R Y 2 0 1 6 77

F E B . 2 0 16

IMPRESSIONS
C D A J O U R N A L , V O L 4 4 , N 2

Reconstructing the Jaw With Free-Flap Fibula Graft

Sound of Bristles Impacts

Tooth Brushing Satisfaction
How effectively we clean our
teeth and how satisfied we are with
the brushing job depends a lot on
the sound of the bristles scrubbing
against the enamel, according to new
research from scientists in Japan.
In trials with volunteer teeth cleaners,
the researchers used a tiny microphone
in a modified toothbrush to sample
the sound being made in the mouth
during brushing and to modulate it
and then feed that sound back to the
volunteer. The study, which is published
in the International Journal of Arts and
Technology, explains how modulating the
brush sound affects brushing efficacy and
satisfaction. The authors found that if
they manipulated the pitch, or frequency
and loudness, of the brushing sound, they
could alter the volunteers perception of
comfort and accomplishment. They also
found that if they gradually increased the
frequency as teeth cleaning progressed,
the volunteers felt like the process was
more comfortable and that their teeth
were cleaner at the end of the process.
Tooth brushing provides a negative
reward for users as they brush their teeth
to avoid developing caries, the research
team explains. Subsequently, users do
not consider the impact of omitting
the action until suffering from caries or
other dental diseases, the team adds.
80F E B R U A R Y 2 01 6

In an article published in the Journal of Oral Implantology, researchers

describe a step-by-step process to completely reconstruct the jaw using a bone
graft and an implant-supported denture. Their case report is one of only a few
studies that focus on the nal, prosthetic phase of the process and a technique
known as a free-ap bula graft.
Free-ap reconstruction is the standard technique used after part or all of a
patients jaw has been removed. Surgeons prefer to use bone from the patients
bula to rebuild the jaw.
The case report focuses on a 24-year-old woman who had undergone
surgery to remove a tumor in her jaw three years earlier with a bular graft to
rebuild her jawbone, but the graft was not successful. A second surgery was
performed using the free bular vascular ap of her other leg, and four implants
and a denture were inserted.
Over the next two years, the patient reported great comfort and ability to
function with the prosthesis. She could wear the denture easily and eat a mostly
normal diet, except for nuts and meats. Her speech was clear and her facial
proportions and symmetry were closer to normal.
For this patient, the plastic attachments had to be replaced once due to
routine wear and tear. She had no problems in the leg that had been used as
the donor site. The patient felt good about her rehabilitation, which made her
condent in her appearance and daily activities.
The authors concluded that their method is safe and reliable, stating that it
resulted in a functional and esthetically acceptable jaw that greatly enhanced the
patients quality of life.
For details, see the study published in the Journal of Oral Implantology,
vol. 41, no. 6, pp. 740-745.

Study results show that it is possible

to motivate users by interactively
manipulating the frequency of brushing
sounds, so that the task becomes more
satisfying. Importantly, the system can
tell, through a built-in force sensor,
whether a person is brushing too hard,
which can damage the gum line, and
give them aural feedback to encourage
them to clean their teeth more gently.

The prototype system requires the

teeth cleaner to wear headphones, which
is impractical in real life. However, there
are bone conduction speaker systems
that might be incorporated into the
smart toothbrush so that the amplified
feedback loop is created in the mouth.
For more, see the study in the
International Journal of Arts and
Technology, vol. 8, no. 4, pp. 307-324.

C D A J O U R N A L , V O L 4 4 , N 2

Adults With Diabetes Lose Twice the Number of Teeth

Adults with diabetes lose
approximately twice the number
of teeth as those without diabetes,
according to a recent study from the
U.S. Centers for Disease Control
and Prevention. Published in the
CDCs Preventing Chronic Disease,
the study aimed to assess the trends
in tooth loss among adults with and

without diabetes mellitus in the

United States as well as racial/ethnic
disparities in tooth loss patterns.
Research shows a bidirectional
relationship between diabetes and
periodontal disease. Periodontal disease
is considered the sixth complication of
diabetes and has been identified as a
risk factor for poor metabolic control in

Multiplying Teeth
Working with colleagues from the Tokyo Medical and Dental University,
researchers have found a way to, literally, multiply teeth. In mice, the researchers
were able to extract teeth germs the groups of cells formed early in life that later
develop into teeth split them into two and then implant the teeth into the mices
jaws where they developed into two fully functional teeth, according to a new
study in Scientic Reports, an online journal of the publishers of Nature.
To manipulate the teeth development process, the researchers removed
teeth germs from mice and grew them in culture. At an appropriate point in
the development process, which according to the authors turned out to be
14.5 days, they nearly sliced the germs in two with nylon thread, leaving
just a small portion attached, and continued to culture them. The hope was
that signaling centers, which control the wave of molecules that regulate the
development of the tooth, would arise in each part and this, in fact, was the
case. The ligated germs developed naturally into two teeth, which the team
transplanted into holes drilled into the jaws of the mice.
Though they were only half the size of normal teeth, the split teeth erupted
into the oral cavity and restored physiological tooth functions, including
mastication, periodontal ligament function and responsiveness to noxious
stimuli. Signicantly, the researchers also report that they were able to
manipulate the teeth using orthodontic methods, equivalent to braces, and the
bone properly remodeled to accommodate the movement of the teeth.
For details on this research, see the
Dec. 17, 2015, edition of Scientic Reports.
Photo of a tooth germ with a nylon noose (left)
and the noose tightened (right).

people with diabetes, the

authors explained in their report.
The study used data from
nine waves of the National Health
and Nutrition Examination Survey
(NHANES) from 1971 through 2012
and included an analytical sample of
37,609 dentate (i.e., with at least one
permanent tooth) adults aged 25 years
or older. Because of insufficient sample
size, the study excluded those with
complete tooth loss and other racial/
ethnic groups (Asians, Native Americans
and Hispanics whose country of origin
was not Mexico). The researchers found
that the estimated number of teeth lost
among non-Hispanic blacks with diabetes
increased more with age than that among
non-Hispanic whites with diabetes or
Mexican Americans with diabetes.
The researchers also evaluated trends
in tooth loss by age, birth cohorts and
survey periods and, according to the
report, found that non-Hispanic blacks
with diabetes lost the largest number
of teeth, and they had the greatest
increase in tooth loss as they aged.
The importance of necessary dental
care and tooth retention needs to be
further promoted among patients and
clinical providers, the authors wrote.
For more details from this
research, see the study published in
the CDCs Preventing Chronic Disease,
Dec. 3, 2015, vol. 12:150309.
F E B R U A R Y 2 0 1 6 81

introduction
C D A J O U R N A L , V O L 4 4 , N 2

Oral Cancer: Novel

Concepts for the Oral
Health Care Practitioner
Diana V. Messadi, DDS, MMSc, DMSc

GUEST EDITOR
Diana V. Messadi,
DDS, MMSc, DMSc,
is a professor and chair
of the section of oral
medicine and orofacial
pain and the associate
dean for education and
faculty development at the
University of California,
Los Angeles, School of
Dentistry.
Conict of Interest
Disclosure: None reported.

ral cancer is a significant

global health concern,
responsible for more
than 600,000 new cases
per year worldwide and
approximately 42,000 annual incident
cases in the U.S. Most cases are diagnosed
at a late stage, resulting in poor fiveyear survival rates between 30 and 60
percent.1,2 Hence, the need is high for
early detection and development of new
management strategies to reduce the
mortality and morbidity of late-stage
diagnosis.3 The disease burden of oral
cancer is significant; patients require
intensive multimodality treatments and
prolonged rehabilitation with longterm support to achieve an adequate
recovery. General dental practitioners
frequently encounter cancer survivors
for their routine dental care and these
patients are likely to present with

oral side effects that are associated

with multiple cancer therapies.4
This issue includes insightful
articles related to oral cancer emerging
technologies in early intervention,
potential therapies and dental
management of cancer survivors. The
first article by Reuben Kim, DDS, PhD,
Paul Yang, BS, MS, and Eric Sung, DDS,
describes the most common oral effects of
associated cancer therapies and discusses
how general dental practitioners can
manage and treat these conditions.
The increasing incidence of human
papillomavirus-positive head and neck
cancers highlights the need to better
understand the role of HPV in the
development of these cancers. Fariba
Younai, DDS, focuses on the role of HPV
in head and neck cancer development
especially among HIV-seropositive
individuals. More specifically, she
F E B R U A R Y 2 0 1 6 83

introduction
C D A J O U R N A L , V O L 4 4 , N 2

describes the epidemiologic trends for

HPV-related oropharyngeal squamous
cell carcinoma and provides an update
on the HPV life cycle, oncogenic
properties and prognostic role in
carcinogenesis. Furthermore, she
emphasizes the role of dental health
care providers in prevention, early
detection and expert referral for this
category of head and neck cancers.
Cancer chemoprevention is defined
as the use of a systemic agent to halt
the carcinogenesis process. This has
been an attractive topic in head
and neck squamous cell carcinoma
(HNSCC) for the past three decades.

Progress toward identifying an effective

chemopreventive agent to reduce the
incidence of oral cancer has been limited
by poor efficacy and intolerable toxicity
profiles. Kazumichi Sato, DDS, PhD,
and I review the current status of cancer
chemoprevention and its effectiveness
in treatment of oral premalignant
lesions (OPL) and prevention of their
progression to oral cancer. Unfortunately,
despite the significant efforts over
the past decades and the substantial
gain in knowledge of the biology of
oral premalignant lesions, no tangible
indications for chemoprevention
have emerged for this disease.

Cancer stem cells (CSCs) may be

involved in oral cancer progression,
metastasis, treatment resistance, and
recurrence. In their article, Anh Le,
DDS, and Qilin Xu, MD, PhD, discuss
the biological properties of CSCs and
their implication in oral carcinogenesis,
which could lead to the development of
novel antitumor drugs that specifically
target oral cancer stem cells.
Nanodiamonds are promising
biomedical agents that have markedly
enhanced theefficacy and safety of
drug delivery and imaging. They
combine several properties that
include uniquely faceted surfaces,
biocompatibility, and scalable
manufacturing parameters, making
them applicable toward oncology and
dentistry. Dean Ho, PhD, et al. explore
the potential use of nanodiamondchemotherapeutic agents to increase
intratumoral retention while markedly
reducing dose-limiting toxicity in an
experimental cancer mouse model.
These advancements have opened the
doors to developing nanodiamond-based
therapies for oral health indications.
I would like to extend my deepest
appreciation to all the authors for sharing
their expertise and knowledge on the
topic of oral cancer. I hope that you, the
readers, find this issue educational and
useful in your dental practice in regard
to the current roles of HPV, stem cells,
nanodiamonds and chemoprevention in
oral cancer detection and management.
RESOURCES

1. American Cancer Society. Cancer Facts and Figures 2014.

Atlanta: American Cancer Society, 2014.
2. Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C,
Rebelo M, et al. Cancer incidence and mortality worldwide:
Sources, methods and major patterns in GLOBOCAN 2012.
Int J Cancer 2015;136:E359-8.
3. Messadi DV. Diagnostic aids for detection of oral
precancerous conditions. Int J Oral Sci 2013; 5:59-65.
4. Jawad H, Hodson NA, Nixon PJ. A review of dental
treatment of head and neck cancer patients, before, during and
after radiotherapy: Part 1. Br Dent J 2015; 218, 65-68.

84F E B R U A R Y 2 01 6

intraoral lesions
C D A J O U R N A L , V O L 4 4 , N 2

Managing Intraoral Lesions

in Oral Cancer Patients in
a General Dental Practice:
An Overview
Reuben Han-Kyu Kim, DDS, PhD; Paul Yang, BS, MS; and Eric C. Sung, DDS

A B S T R A C T As medical technology advances in the area of cancer therapeutics,

dental practitioners will encounter patients with active cancer or a history of cancer.
Typically, these patients may have had or are undergoing therapies such as surgery,
radiation, chemotherapy or a combination of therapies. These patients may present
with multiple side effects that dental practitioners can manage or prevent. We
discuss some of these concerns and provide management strategies.

AUTHORS
Reuben Han-Kyu Kim,
DDS, PhD, is an associate
professor, vice chair in the
section of restorative dentistry
and the course chair for
direct restorations for
predoctoral students at the
University of California, Los
Angeles, School of Dentistry.
He is actively engaged in
research related to oral
diseases including wound
healing and oral cancers.
Conict of Interest
Disclosure: None reported.
Paul Yang, BS, MS, is a
rst-year student in the DDS/
PhD combined program at
the University of California,
Los Angeles, School of
Dentistry. Mr. Yang earned
his bachelors and masters
degrees in biology from
UCLA. He is interested in

pursuing oral maxillofacial

and facial surgery training
and oral medicine after his
DDS/PhD training.
Conict of Interest
Disclosure: None reported.
Eric C. Sung, DDS, is a
professor of clinical
dentistry, chair in the
section of special needs
patients and the program
director of the general
practice residency program
at the University of
California, Los Angeles,
School of Dentistry. His
background is in training
and providing
comprehensive treatment
for medically, physically
and psychologically
complex individuals.
Conict of Interest
Disclosure: None reported.

recent study showed

that nearly 14.5 million
Americans had experiences
with cancers as of 2014,
and almost 1.7 million
new cancer cases were expected to
be diagnosed in 2015.1 Among them,
2 percent is attributed to oral and
oropharyngeal cancers, ranking them
as the sixth most commonly occurring
cancer in the U.S. with 63 percent and
51 percent of overall five- and 10-year
survival rates, respectively. A similar
trend can also be seen worldwide,2
suggesting that oral and oropharyngeal
cancers in the oral cavity impose
significant health issues not only in the
U.S., but also throughout the world.
Approximately 45,780 new
diagnoses of oral and oropharyngeal
cancers alone were expected in 2015

in the U.S.1 While some of these patients

may seek a large cancer center where
their dental needs can be addressed in a
hospital-based setting before, during or
after cancer therapy, significant numbers
of these patients are being referred to
local general dental practitioners for
their dental care.3 With progressive
increase in life expectancy due to the
advancement in medical technology,
these cancer patients seeking general
dentists to address their dental needs
will only escalate. Therefore, as a general
dental practitioner it is important to
know about, and to be better prepared
for, any disease or pathology that may
specifically develop in the oral cavity
in patients who are undergoing or who
have undergone therapy for their cancer.
Depending on the treatment
modality, the side effects commonly
F E B R U A R Y 2 0 1 6 85

intraoral lesions
C D A J O U R N A L , V O L 4 4 , N 2

found in the oral cavity are diverse,

ranging from xerostomia, oral mucositis,
opportunistic infection and trismus
to osteoradionecrosis (ORN). These
side effects may be overlooked because
some of them are asymptomatic in
nature, but can be severe such that
normal functioning in daily life may
be significantly compromised. Detailed
descriptions of screening and examining
oral cancers in patients can be found
on the Foundation for Oral-Facial
Rehabilitations website (ffofr.org) and
in other reviews.4 We primarily focus
on the etiology, clinical presentation,
treatment and management of these
oral lesions in patients who are
undergoing or underwent therapy for
their cancers. The use of adjuvant
cancer therapeutic agents such as
antiresorptive (e.g., bisphosphonates
and denosumab) or anti-angiogenic
(e.g., sunitinib or bevacizumab) drugs is
increasingly common to treat metastatic
cancers. These patients are at risk of
developing oral-specific lesions called
medication-related osteonecrosis of
the jaw (MRONJ). We also discuss
the management of MRONJ lesions.

cancers. However, this approach is often

limited due to compromised functions and
esthetics.5 Surgical removal of a cancer
mass in the oral cavity often creates large
structural defects, and the outcomes
may be disfiguring. In addition, intraoral
surgical removal may result in significantly
altered oral functions for speech and
mastication. Therefore, patients may opt
out of this therapeutic modality because
of these functional and esthetic concerns.

Radiation therapy has the advantage

of inducing DNA damage in highly
proliferating cancer cells by ionizing
radiation via generating reactive
oxygen species (ROS).6 Because cancer
cells constantly replicate DNA for
their continual proliferation, DNA
damage by ionizing radiation through
radiation therapy leads to cell death.
Radiation is delivered to the tumor
sites by fractionating the doses with
different radiation paths in multiple
visits. Typically, an average of 2 gray
(Gy) per fraction is delivered over a
course of six to seven weeks, resulting
in a total dose of 60-72 Gy.

Although these treatment modalities

are specifically formulated to reduce
the cancer burden by inducing cancer
cell death, normal cells responsible
for maintaining body homeostasis by
continually proliferating, differentiating
and replenishing tissue structures and
functions are also affected. As such, there
are multiple complications associated
with cancer therapy, such as nausea,
vomiting, hair loss, myelosuppression and
stomatotoxicity. Among them, several side
effects are observed in the oral-specific
manner and compromise the quality
of patients lives. These complications
include oral mucositis, xerostomia,
ORN, trismus and secondary infection.

Cancer Therapy Options

Chemotherapy

Oral Mucositis

To better manage oral-specific

side effects that are induced during
or after cancer therapy, it is helpful
to understand the nature of each
therapeutic modality. These treatment
modalities include surgical therapy,
radiation therapy, chemotherapy
or a combination of therapies.

Chemotherapy is usually treated on

an outpatient basis, but hospitalization
may be required if serious sequelae
develop. The modality of chemotherapy
is largely dependent on the cytotoxicity
of the drug and the patients body defense
condition. Combinations of different drugs
(e.g., alkylating agents, antimetabolites,
antitumor antibiotics, antineoplastics
and monoclonal antibody such as
cetuximab) are preferred to avoid the
development of single-agent resistance
in cancer cells. In addition, combination
chemotherapy as well as radiation
therapy can lower the drug doses and
result in better remission and cure rate.7

General description: Oral mucositis,

the breakdown of oral epithelial tissues
leading to painful ulcerative lesions, is
one of the most common side effects in
patients undergoing radiotherapy and/or
chemotherapy. The degree of mucositis
severity varies depending on fields, doses
and fractionation of radiation. Ulcerative
mucositis lesions are more severe in
patients receiving adjunctive or concurrent
chemotherapy. The etiology of oral
mucositis is primarily due to the generation
of reactive oxygen species (ROS) by
radiation and/or chemotherapy, which
cause direct DNA damage to actively
proliferating stem cells that are responsible

Surgical Therapy
Surgical therapy for cancer is a
treatment choice, as it allows for the
physical removal of the entire tumor
mass. Following surgical removal, patients
may undergo adjuvant radiation or
chemotherapy for complete eradication of
86F E B R U A R Y 2 01 6

Radiation Therapy

FIGURE 1. Mucositis covered by a pseudomembranous

layer with areas of erythema and ulceration.

Oral Complications Associated

With Cancer Therapy

C D A J O U R N A L , V O L 4 4 , N 2

Xerostomia
FIGURE 2A . Xerostomia causing gross decay

FIGURE 2B . Gross decay leading to fracture at the

(arrows).

gumline (arrow).

FIGURES 2 . Rampant caries secondary to xerostomia.

for replenishing the tissues, leading to

oral mucosal damages.8,9 Ironically, this
is also the basic principle behind the
use of radiation and/or chemotherapy
to target cancer cells. The severity of
mucosal reaction is more evident in the
less keratinized oral mucosa, such as under
the tongue. The ulceration escalates in
patients with chronic alcoholism, liver
cirrhosis and insulin-dependent diabetes.
Clinical manifestation: Mucositis
initially presents as an erythematous lesion
as early as seven to 10 days after the initial
treatment dose. These initial erythematous
mucositis will soon develop into ulcerative
mucositis that is typically covered by
pseudomembranes (FIGURE 1 , arrows).
These lesions are usually confined to the
tissues associated with the initial tumor
site. Ulcerative mucositis lasts throughout
the treatment period, but the lesions
are usually self-limiting after two to four
weeks following the completion of therapy
during which they are re-epithelialized and
covered by normal-appearing oral mucosa.
Caution should be taken when
managing the irradiated oral mucosal
tissues as they can be easily perforated
by trauma, resulting in secondary
ulceration that could take months to
heal. The practitioner should carefully
examine the localized ulcerative mucositis
in oral mucosal tissues particularly
around the metallic crown that is in
the path of the radiation beam due
to backscatter effects of radiation.
Management: As these lesions are
often self-limiting, the primary goal of
managing patients with oral mucositis
should be focused on alleviating pain.

Topical anesthetics in the form of sprays,

ointments, gels or rinses, such as lidocaine,
benzocaine, dyclonine or capsaicin, can
be used. The practitioner should examine
loss of oral function, weight loss and
secondary infection.10 It should be noted
that patients with severe mucositis may
require hospitalization. Patients should
be instructed to avoid hot, spicy or acidic
foods or beverages. Any sharp or hard
food intake should be curtailed, as they
can be traumatic to the oral mucosal
tissues. If oral mucositis is generalized
throughout the oral cavity, analgesics
can be administered systemically, which
may require hospitalization. Emphasizing
good oral hygiene practices to patients
is important to reduce the chances
of developing infection secondary
to mucositis. Fungal and bacterial
infections are common with these lesions
and antifungal and/or antibacterial
medications may be prescribed as needed.
Some of these patients may have
already undergone preventive therapeutic
treatment, such as cryotherapy, palifermin
or amifostine, so practitioners should
be aware of these methods. Palifermin,
a truncated human keratinocyte
growth factor (KGF) recombinant
protein, is FDA approved and currently
available to use in the clinic; however,
recent clinical trials demonstrated
the modest effects of palifermin.11,12
Amifostine, a radioprotectant, can
be administered intravenously or
subcutaneously before therapy to reduce
the severity of oral mucositis, but it
may induce several side effects such as
headaches, nausea or hypotension.

General description: Xerostomia is

another commonly occurring side effect
in cancer patients undergoing radiation
therapy or concomitant chemotherapy.
Xerostomia occurs because of partial or
complete damages, which may be either
recoverable or irreversible to the salivary
glands (e.g., parotid, submandibular
and sublingual glands) especially when
these glands are in the radiation path.
Histologically, early changes at the tissue
level include interstitial fibrosis, progressive
loss of the fine vasculature and vacuolization
of serous acinar cells. Of note, serous acinar
cells seem to be more readily affected by
radiation when compared to the mucous
cells, presumably because of the relatively
rapid turnover rate and profuse vasculature
of serous cells. As such, saliva is more acidic
and viscous with less buffering capacity.
During the late stages of radiation therapy,
glands become progressively fibrotic,
leading to almost complete loss of acinar
elements and the striated duct system.
Ultimately, no saliva may be present.
Because such environmental alterations
make the oral cavity more susceptible to
rampant caries, acute and chronic fungal
infections and compromised tolerance
to prosthesis such as dentures, early
detection and management of xerostomia
in these patients are critical to alleviate
discomforts and possible permanent
structural damage in the oral cavity.
Clinical manifestation: Practitioners
should actively look for signs and symptoms
related to salivary hypofunction including
fissures at the lip commissures, difficulties
in swallowing or chewing as well as with
speech. Salivary reduction up to 80 percent
of its original flow13 and xerostomia can
be specifically noted in cancer patients
two weeks after initial radiation therapy
or at a cumulative dose of 20 Gy. The
diminished salivary flows bring changes
to the oral flora, increasing the chances
F E B R U A R Y 2 0 1 6 87

intraoral lesions
C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 3A . Custom trays for maxillary and

mandibular dentition.

FIGURE 3B . Custom trays on the maxillary and

mandibular dentition.

FIGURES 3 . Stannous uoride gel application with custom tray.

of bacterial and/or fungal infection.

These changes predispose the patient to
radiation caries, which is typically located
at the incisal edge and cervical third of
the teeth. Rampant caries progress rapidly
and extensively such that teeth become
nonrestorable (FIGURE 2A ) or fractured
at the gingival margin (FIGURE 2B );
therefore, early detection and immediate
restorations are highly recommended.
Management: Early symptoms of
xerostomia include thick or ropey saliva in
the oral cavity. Carboxymethylcellulose-,
mucin-, water- or glycerin-based saliva
substitutes may be used, although the
effectiveness of these agents is somewhat
questionable. In mild cases, a simple
increase in the frequency of water intake
is helpful. If salivary glands are spared from
complete eradication by radiation therapy,
salivary stimulants such as pilocarpine or
cevimeline may be used. Xylitol-based
chewing gum that has a nonfermentable
sugar alcohol may also be used to increase
salivary flow. The early use of stannous
fluoride gel applied with custom carriers
(FIGURE 3A ) and five-minute daily
applications is highly recommended to
reduce caries risk (FIGURE 3B ). If significant
numbers of rampant caries are noted,
dental treatment should be performed
without any delay. Due to the high caries
risk, fluoride-releasing glass ionomers
and/or amalgam restorations are more
predictable when compared to composite
restorations. Patients should be counseled
about eliminating sucrose from their diet
and reducing the frequency of meals.
88F E B R U A R Y 2 01 6

Osteoradionecrosis (ORN)
General description: Osteoradionecrosis
(ORN), as the name implies, means bone
death because of radiation. The incidence
of ORN ranges from 8 percent to 35 percent,
largely depending on observational periods
that range from months to years.14,15 Most
of ORN (about 75 percent) occurs within
the first three years of radiation therapy
treatment.16 ORN is more prevalent in the
mandible than the maxilla, owning to the
poor vascularization and increased density
that allows for absorbing more radiation
in the mandible. The cause of ORN is
still unclear although there are several
hypotheses, such as bacterial infection,
hypoxia and fibroatrophy.17-19 Risk factors
include location of primary tumor, cancer
staging, dose of radiation (> 60 Gy), poor
oral hygiene, alcohol and tobacco use and
invasive dental procedures such as tooth
extraction.20 It is noteworthy that, once
radiation therapy is delivered, cancer patients
have the risk of developing ORN that is
lifelong and does not decrease over time.
Therefore, thorough examination at each
visit for periodic examination is essential.
Clinical manifestation: ORN is clinically
defined as an area of exposed bone that
persists for more than three months
(FIGURE 4 ). However, radiographic
findings of irregular radio-opacity that is
indicative of sequestrum formation without
breached overlaying mucosal closure is
also common (FIGURE 5 ). Ulcerative
or necrotic soft tissues can also be seen
frequently around the exposed area.
Long-term exposure without proper oral

FIGURE 4 . Osteoradionecrosis lesion with exposed

bone in the lower right mandibular arch. Note plaque
accumulation around the exposed bone.

care may lead to the accumulation of

plaque that covers the exposed bone.
Management: In one study, tooth
extractions were found to be responsible
for 50 percent of all ORN cases.14
Therefore, invasive dental procedures
should be reserved. Periodontal procedures
such as deep scaling and flap surgery
are also contraindicated particularly in
heavily irradiated patients. Instead, more
conservative treatment approaches, such as
endodontic therapy with or without coronal
restorations, are preferable (FIGURE 6 ).
When bone exposure is evident, a patients
chief complaint is typically pain associated
with bacterial infection secondary to
exposed bone. Prescribing antibiotics may
help resolve the pain. Regular checkups and
dental prophylaxis every four months are
highly recommended to maintain optimal
conditions in the oral cavity along with
giving the clinician ample opportunity to
catch dental disease at the early stages.
Hyperbaric oxygen (HBO) therapy that
provides high contents of oxygen has
been used to manage ORN conditions
but without drastic improvement.21 If
possible, it is highly encouraged to remove
any sources of dental diseases including
advanced caries, periapical infection and
pathologic periodontal bone loss before
a patient undergoes radiation therapy.
New alternatives to HBO treatments
have been introduced, such as the use of
pentoxifylline and/or tocopherol,22,23 and
the use of these medications may hold
promising results in reducing the risk of
and managing patients with ORN.

C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 5A . Sclerotic changes around No. 31 area

(arrows) after radiation therapy.

FIGURE 5B . After two years, sequestrum was

pushed out spontaneously.

FIGURE 6 . Endodontically treated Nos. 18 and 19

that are domed with amalgam restoration.

FIGURES 5 . Radiographic ndings of irregular radio-opacity.

FIGURE 7. Trismus. Note the limited mouth opening.

FIGURE 8 . Candida albicans.

Trismus

worse and are often irreversible, early

identification of trismus and immediate
initiation of an exercise program using
devices such as Therabite are critical to
improving the condition significantly.27,28

General description: Trismus refers to

limited mouth opening caused by any
etiological reason related to sustained
contraction of one or more mastication
muscles.24 The most common etiological
factors involve radiation-induced fibrosis
and postsurgical scar formation.25 Trismus
occurs most commonly when radiation
is combined with a surgical procedure
(e.g., radical maxillectomy) that affects
the temporal mandibular joint (TMJ)
and the muscles of mastication.
Clinical manifestation: Clinically,
mouth opening less than 35 mm is
considered to be trismus, although the
degree of limited mouth opening may
be subjective.26 In severe cases, the
maximum opening may be reduced
to less than 10 mm (F I G U R E 7 ). The
severity of trismus depends on age
and concomitant chemotherapy.
Management: The treatment consists
of exercise and the use of dynamic
bite openers. Because fibrosis and
scar formation become progressively

Oral Candidiasis
General description: Oral candidiasis is
fungal lesions predominantly mediated by
the yeast Candida albicans. C. albicans occurs
naturally in the body including the oral
cavity. In a normal setting, it does not give
rise to any lesions, but candidiasis occurs
when there is a drastic change within the
oral environment (e.g., immunosuppression)
that favors its reproduction. As such, it is
often called an opportunistic infection.
Brown, et al. reported that irradiation
caused up to a hundredfold increases in
fungal populations.29 This increase in
fungal populations has significant clinical
implications and is often overlooked. During
the administration of radiation therapy,
acute candidiasis is likely to occur due to
altered immunity and xerostomia secondary
to hyposalivary functions in the oral cavity.30

Upon completion of radiation, progressive

fibrosis of the salivary glands will continue,
which increases the risk of xerostomia.
As this continues, the increase in the
fungal population will continue. Much
of this will be determined by the amount
of xerostomia the patient experiences.
Clinical manifestation: Candidiasis is one
of the most frequent lesions experienced
by patients undergoing cancer therapy. It
presents as pseudomembranous (thrush),
erythematous and angular cheilitis (FIGURE
8 ). Severe oral infections can result in
discomfort and morbidity during and
after radiation therapy.31 Postradiationtherapy chronic candidiasis is known to
occur in up to 27 percent of patients.32
These patients may complain of a burning
sensation in the oral mucosa and cracked
lips along the commissure; however,
these symptoms are often overlooked.
Management: Nystatin is a drug of
choice, which can be dispensed in a
number of configurations, such as lozenges,
powder, creams or oral suspension. Oral
suppositories may be the best route of
drug delivery to treat acute oral fungal
infections. Many nystatin lozenges
contain large of amounts of sucrose and
should be used with caution in edentulous
patients. In this population, vaginal/
rectal suppositories can be considered
because they have no sucrose. However,
taste or difficulty in use may affect
compliance. Systemic administration of
ketoconazole or fluorconozole is favored
by many clinicians 33 and is preferred
for potentially noncompliant patients.
F E B R U A R Y 2 0 1 6 89

intraoral lesions
C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 9. MRONJ

Prolonged use of antifungal medications

is discouraged because of the risk of
developing fungal resistance to these drugs.

Altered Taste Buds

Alterations in taste acuity are first
noticed as early as the second week of
radiation therapy (approximately 30 Gy
of radiation). Perception of bitter and acid
flavors is more susceptible to impairment
than salt and sweet. The architecture
of the taste buds is almost completely
eliminated at 50 Gy. However, taste
generally returns to normal two to four
weeks after the completion of therapy
as long as salivary flow is reasonable. In
the case of severe xerostomia following
radiation therapy, the number of buds
are significantly decreased and their
morphology is altered. The perception
of taste may be permanently altered.

Oral Complications Associated With

Adjuvant Chemotherapy
Cancers in advanced stages typically
metastasize to other parts of the body.
In particular, some cancers, including
breast, prostate, lung, thyroid and kidney,
are more prone to metastasize to other
areas including bone. These lesions
may also lead to high calcium levels in
the blood stream called hypercalcemia.
Medications commonly prescribed for
the management of metastasis include
90F E B R U A R Y 2 01 6

lesions induced by longterm bisphosphonate use.

(A) Note radiolucency
around the aected area
(arrows), which is an
indicative of nonviable
bone. (B) A typical
MRONJ lesion with
plaque formation induced
by the long-term use of
bisphosphonates on the
lower left mandibular arch.
(C) Bony sequelae that fell
out spontaneously from
the upper right maxillary
arch (A, arrowhead)
that the patient brought
to the clinic.

antiresorptive (e.g., bisphosphonates or

denosumab) or anti-angiogenic (e.g.,
sunitinib or bevacizumab) drugs. The use
of these medications is associated with
MRONJ that specifically occurs in the oral
cavity. Therefore, general practitioners
should be aware of these MRONJ
lesions when managing patients who are
receiving such adjuvant chemotherapy.

MRONJ
General description: The first formal
report on osteonecrosis of the jaw (ONJ)
by bisphosphonates was published in
2003,34 but the etiology is still unknown.
Multiple hypotheses have been suggested,
including suppression of bone remodeling,
inflammation, inhibition of angiogenesis
and soft tissue toxicity.35 The terminology
of bisphosphonate-related osteonecrosis
of the jaw, or simply BRONJ, was recently
updated to MRONJ in order to be more
inclusive of medications other than
bisphosphonates, such as denosumab or
bevacizumab.36 MRONJ is clinically defined
as patients with a history of treatment with
antiresportive or anti-angiogenic agents,
exposed bone for more than eight weeks
and no history of radiation therapy to
the head and neck regions.36 A detailed
classification of MRONJ can be found
elsewhere.36 Individuals with advancedstage cancers that invade bone may take
these medications, usually intravenously or

subcutaneously, to prevent the cancer from

spreading to bone. As such, practitioners
should keep in mind that osteoporosis
patients also take these drugs orally, and
that although the incidence of MRONJ
occurring by this route is relatively less, these
patients may still develop MRONJ lesions
when they have been on these medications
for more than four years.37 Practitioners
should also be aware of the risk factors
associated with MRONJ, such as high (e.g.,
IV or subcutaneous administration) and
long duration (e.g., > four years) of doses,
pre-existing inflammatory dental diseases
(e.g., periodontal disease or periapical
lesions), dentoalveolar surgery (e.g., tooth
extraction), age and corticosteroids,37-43 all
of which may exacerbate ONJ lesions.
Clinical manifestation: A typical
clinical presentation is very similar to
that of ORN. Long-term exposure of
bone is almost inevitably accompanied
by plaque accumulation (FIGURE 9B ).
Practitioners should consider this for
patients who have pain with unidentifiable
origin as it may indicate stage 0 MRONJ.
Abnormal findings (e.g., sclerosis) from
radiography and computed tomography
(CT) should also be noted, but interpreted
with caution, as it may be suggestive of
MRONJ.44,45 Bone exposure is likely to
be seen in previously extracted areas but
can also occur spontaneously in thin oral
mucosal areas such as tori. Spontaneous
bone exposure may be associated with
chronic inflammation (e.g., periodontal
or periapical diseases) and previously
traumatized areas. Radiographically,
nonviable bone can be predicted based on
radiolucent periphery around the affected
area (FIGURE 9A ). A periodontal probe
instrument can be used to detect bony
surface through mucosal fistulas, which is
indicative of MRONJ at the stage 1, 2 or 3.
Management: Similar to ORN, invasive
dental procedures should be refrained
from, but conservative approaches are

C D A J O U R N A L , V O L 4 4 , N 2

TABLE

Summary of Oral-Specific Side Effects and Management in Cancer Patients

Types of side
eects

Types of
cancer therapy

Common
sites in the
oral cavity

Patients
chief
complaints

Clinical
manifestation

Goals of
management

Management

Oral mucositis

Radio/chemo

All

Pain

Erythematous
Ulcerative

To alleviate pain

Self-limiting after cancer therapy

Topical anesthetics (lidocaine,
benzocaine, dyclonine, capsaicin)
Avoid irritating food

Xerostomia

Radio/chemo

All

Thick or
ropey saliva

Rampant caries,
decalcication

To increase salivary ow
or substitutes
To prevent secondary
pathological events
(e.g., rampant caries,
infections)

Saliva substitutes
(carboxymethylcellulose, mucin,
water, glycin)
Salivary stimulants (pilocarpine,
cevimeline)

Osteoradionecrosis

Radio

Mandible

Nonhealing
and/or pain at the
exposed bony sites
Unpleasant mouth
smell

Exposed
bone, plaque
accumulation

To prevent further bone

exposure
To refer to specialist for
surgical intervention for
stage 3 patients

Analgesics
Antibiotics
Antimicrobial rinse (0.12%
chlorhexidine)
Avoid invasive dental procedures
HBO chamber
Pentoxifylline/tocopherol

Trismus

Surgical/radio/
chemo

Mastication
muscles

Cannot open
mouth

Limited mouth
opening

To identify limited mouth

opening early

Mouth opening exercise

(Therabite)

Oral candidiasis

Radio/chemo

All

Burning sensation

White patches
that can rub o

To eradicate candidiasis
lesions

Nystatin (lozenges, powder,

creams, oral suspension)

Altered taste

Radio/chemo

All

Cannot taste

None

None

Self-limiting after cancer therapy

MRONJ

Chemo
(for advanced
cancer)

Maxilla/
mandible

Nonhealing
and/or pain at the
exposed bony sites
Unpleasant mouth
smell

Exposed
bone, plaque
accumulation

To prevent further bone

exposure
To refer to specialist for
surgical intervention for
stage 3 patients

Analgesics
Antimicrobial rinse (0.12%
chlorhexidine)
Good oral hygiene
Avoid invasive dental procedures

recommended. It is important to know

whether cancer patients are taking the
aforementioned medications, as dental
treatment options are significantly limited
due to increased risk of MRONJ after
invasive dental treatment. Once identified,
patients with MRONJ should be managed
according to the MRONJ staging. As a
general practitioner, the primary goals of
managing these patients are to maintain
good oral hygiene in a nonsurgical manner
in patients with stage 2 or less and to
monitor progression of lesions such that,
when the lesions meet the stage 3 criteria,
the patients can be referred to oral surgeons
for possible surgical interventions. For
patients who are taking these mediations

without signs/symptoms of MRONJ,

routine oral hygiene including scaling and
root planning should be continued. For
stage 0 patients with the chief complaint
of pain with unidentifiable origins, the use
of medication to control pain is helpful. In
stage 1 or 2 patients with exposed bone, the
use of oral antimicrobial rinses (e.g., 0.12%
chlorhexidine) is recommended. Although
infection as a primary etiological factor
in causing MRONJ is still controversial,
the use of antibiotics is also recommended
in order to reduce bacterial colonization
particularly at the area with exposed
bone. It is not uncommon to observe a
tooth with class 3 mobility. In such cases,
extraction should be avoided; instead,

the occlusal plane can be reduced as

needed to eliminate occlusal contacts and
contact-associated pain until the tooth
falls out spontaneously. In certain instance,
patients may present with bony sequelae
that naturally sequester out (FIGURE 9C ),
and such a sign is usually accompanied by
reepithelialization at the healing site. These
sites should be continuously monitored.

Conclusion
Once established, the relationship
between dentists and patients can last for
many years. As life expectancy increases
and advancement of medical technology
continues to grow, these relations may
potentially be lifelong. During that time,
F E B R U A R Y 2 0 1 6 91

intraoral lesions
C D A J O U R N A L , V O L 4 4 , N 2

dentists are likely to encounter patients

who are undergoing or who have a history
of cancer therapy. As general dental
practitioners, knowing different cancer
therapeutic modalities (e.g., surgical,
radiation, chemo, or combination
therapies) is essential for assessing
and managing these cancer patients.
Many side effects from cancer therapy,
including oral mucositis, xerostomia,
ORN, trismus and secondary infection,
are inevitable but manageable, and to a
certain degree, treatable (TABLE ). This
holds true for MRONJ lesions in cancer
patients undergoing adjuvant therapy
with bisphosphonates and denosumab. As
such, it is of paramount importance for the
general dental practitioner to know how
cancer therapy can affect oral health and
to manage these patients accordingly in
order to provide the full spectrum of dental
services. Because managing cancer patients
successfully in the general dental practice
is a team effort, it is equally important for
general practitioners to communicate not
only with the patients but also with medical
practitioners to determine the optimal
management plan for each patient.
ACKNOWLEDGMENT

This study was supported by grants R01DE023874 and

R01DE023348 from the National Institute of Dental and
Craniofacial Research/National Institutes of Health.
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THE CORRESPONDING AUTHORS,

Reuben Han-Kyu Kim, DDS, PhD,

and Eric C. Sung, DDS, can be reached at
rkim@dentistry.ucla.edu and esung@dentistry.ucla.edu.

oropharyngeal cancer
C D A J O U R N A L , V O L 4 4 , N 2

Current Trends in the

Incidence and Presentation
of Oropharyngeal Cancer
Fariba S. Younai, DDS

A B S T R A C T The prevalence and behavior of oropharyngeal cancers have

dramatically changed over the past 30 years. It is now clear that human
papillomavirus (HPV) plays a major role in the incidence of head and neck cancers
in the general population and among patients with HIV infection. This article
reviews the current knowledge about oropharyngeal cancers for their epidemiology,
pathogenesis, clinical behavior, treatment and prevention. This review further
examines the subset of oropharyngeal cancers among the HIV-seropositive patients.

AUTHOR
Fariba S. Younai, DDS, is
a professor of clinical
dentistry in the section of
oral medicine and
orofacial pain in the
division of oral biology and
medicine at the University
of California, Los Angeles,
School of Dentistry, where
she currently serves as the
vice chair of the division of
oral biology and medicine.
Dr. Younai earned her
dental degree from the
School of Dental Medicine
at the State University of
New York at Stony Brook
and completed her training
in hospital dentistry at Long
Island Jewish Medical
Center in New York.
Conict of Interest
Disclosure: None reported.

new global pattern of

oropharyngeal cancer
epidemiology started to
emerge in the 1990s.
An increased incidence
of malignant lesions, specifically
squamous cell carcinomas, was being
noted to occur mostly in the posterior
region of the oropharynx and among
younger individuals who did not have
the traditional oral cancer risks such
as smoking and excess alcohol use.1
Since then, the scientific evidence has
increasingly shown that this subset
of head and neck cancers is a distinct
entity connected to an infection with
human papillomavirus (HPV) and, in
fact, exhibits better overall survival
rates and response to treatment.2-6 This
association between HPV and oral
malignancy is even more pronounced
among individuals seropositive for
the human immunodeficiency virus

(HIV) who, in recent years, have also

started to present with an increased
incidence of head and neck and
oropharyngeal squamous cell carcinomas.3

Oropharyngeal Cancer Epidemiology

Every year, more than 600,000 new
cases of head and neck cancer (HNC) are
reported worldwide.7 As the sixth most
common type of cancer, the incidence
rates of HNC have great variations across
the globe it is the most common form
of cancer in India while its incidence
rates in the United States and in northern
Europe are lower than in countries in
Latin America.8 The majority of head
and neck cancers are of the squamous
cell carcinoma (SCC) type involving the
oral cavity, oropharynx, hypopharynx,
larynx, sinonasal tract and nasopharynx.7
The term oral cavity cancer (OCC)
typically refers to a malignant lesion
in the anterior part of the oral cavity,
F E B R U A R Y 2 0 1 6 93

oropharyngeal cancer
C D A J O U R N A L , V O L 4 4 , N 2

including gingivae, floor of the mouth,

buccal mucosa, retromolar trigone, hard
palate and the anterior two-thirds of
the tongue, while the oropharyngeal
cancer (OPC) designation is for those
lesions located in any of four distinct
subsites: the posterior pharyngeal wall,
the soft palate, the tonsillar complex
and the base of the tongue.9,10
Several studies have provided
overwhelming evidence for the increasing
incidence of oropharyngeal cancer
worldwide. In the U.S., an assessment of
the incidence data from the Surveillance,
Epidemiology and End Results (SEER)
Program of the National Cancer Institute
(NCI) showed that between 1973 and
1995, the incidence of tonsillar SCC
among men younger than 60 years of age
increased by about 2 to 3 percent per year.11
Another analysis of the data from the
SEER Program and the mortality data from
the National Center for Health Statistics
(NCHS) further revealed that from the
late 1980s to 2001, the risk for tonsillar
SCC increased for males age 40-64 years
while the incidence for this type of cancer
and progression to death decreased for
those age 65 years and older.12 A parallel
pattern was reported from Sweden and
Finland during the same approximate
time period.13,14 Looking at the global
statistics, one study using data from the
Cancer Incidence in Five Continents
database, maintained by the International
Agency for Research on Cancer (IARC),
constructed age-period-cohort models
to examine the incidence trends for
OPC and OCC across 23 countries
around the world.15 This comprehensive
analysis showed that between 1983
and 2002, significant increases in OPC
incidence were observed among both
men and women, predominantly among
individuals younger than 60 years and in
economically developed countries such
as the U.S., Australia, Canada, Japan
94F E B R U A R Y 2 01 6

and Slovakia.15 The striking finding in

this analysis was that the increased OPC
incidence among men corresponded
with a reduction in the incidence of
both OCC and lung cancer, but among
women, a reverse pattern was observed
where the increased OPC incidence
occurred with concomitant increases in the
incidence of OCC and lung cancer. These
findings suggested significant differences
between the OPC risk factors and those
of OCC and lung cancer, differences
that may be more influential in cancer
development among men than women.

Several studies have

provided overwhelming
evidence for the increasing
incidence of oropharyngeal
cancer worldwide.

After the introduction of effective

combination antiretroviral therapies
(ART or cART) in the mid-1990s, the
incident rates for the AIDS-defining
cancers, such as Kaposis sarcoma, was
dramatically reduced, while a trend toward
increased incidence of other types of
cancer, like lung cancer, invasive anal
SCC, head/neck cancers and Hodgkin
lymphoma, started to emerge.16-18 Pooled
data from the North American AIDS
Cohort Collaboration on Research and
Design (NA-ACCORD) studies between
1996 and 2009 showed that the HIVinfected individuals risk of development
of HNC, specifically SCC, was threefold
higher than that of the general U.S.
population.19 This report also showed a
modest role for immunosuppression (low
CD4 count) prior to cancer diagnosis.

It is not entirely clear if the increase

in the incidence of head and neck
cancers among HIV-infected individuals
is related to behavioral factors such
as tobacco and alcohol use, immune
suppression, long-term use of cART,
other infectious agents or results from
a combination of all these factors.

HPV Infection and Oropharyngeal

Cancer
Globally, smoking, alcohol
consumption and betel nut chewing
are traditional risk factors for the SCC
type of HNC and OCC.20 In fact, aside
from cases of oral dysplasia seen related
to betel nut chewing that is common
in Asia and the tropical Pacific region,
traditional patients with OCC have been
older men with a significant smoking
and drinking history. As noted before,
there has been a significant reduction
in the rates of OCC among older male
smokers, mostly due to reductions in the
global smoking and alcohol consumption
rates.21,22 At the same time, there has
been a dramatic increase in incidence
rates of oropharyngeal cancer among
middle-aged white men who are often
nonsmokers or former/light smokers, a
phenomenon now attributed to infection
with HPV.23-26 In the U.S., by 2010,
the rate of OPC among men became
higher than the rate of cervical cancer
in women and if this trend continues,
by the year 2020 the annual number
of HPV-positive OPC will surpass the
annual number of cervical cancers.27 An
analysis of the SEER data from three
regions (Hawaii, Iowa and Los Angeles)
showed that while the incidence rate for
HPV-negative OPC dropped from 2.0
per 100,000 in 1988 to 1.0 per 100,000
in 2004, the rates for HPV-positive
tumors increased by more than threefold,
from 0.8 per 100,000 in 1988 to 2.6
per 100,000 in 2004 (F I G U R E 1 ).27

C D A J O U R N A L , V O L 4 4 , N 2

4
3.5

Rates per 100,000

3
2.5
2
1.5
1

Oropharynx (overall)
HPV-negative oropharynx
HPV-positive oropharynx

.5
0

19881990

19911992

19951996

19992000

20032004

Surveillance Period
FIGURE 1. Incidence rates for overall HPV-positive and HPV-negative OPC (SEER data from Hawaii, Iowa and

Los Angeles 1988-2004). Adopted from J Clinical Oncology 2011;29(32):4294-4301.

HPV-positive squamous cell

carcinoma is a genetically, clinically
and epidemiologically distinct subtype
of HNC with a predilection for the
oropharyngeal tissues, referred to by
many as HPV-associated oropharyngeal
squamous cell carcinoma (OPSCC).28
The first reports of a potential role for
HPV in the HNC development were
published almost 35 years ago.29-31
However, it was not until the surge in the
HNC incidence that the role of HPV in
OPC carcinogenesis became subject to
intense scientific research for its natural
history, transmission patterns and primary
and secondary prevention approaches.
Human papillomavirus has more
than 320 different subtypes found in
many vertebrae, of which more than 200
genotypes have been fully sequenced
in humans.32 HPV has five different
genera (alpha, beta, gamma, mu and nu)
and although each group has a specific
anatomic site predilection, they all share
great epithelial tropism and are found in
either skin, mucosa or both, even when
nonpathogenic.33 In their pathogenic form
in the skin, the anogenital tissues and
the tracheobronchial and oral mucosa,
several HPV subtypes lead to benign
hyperproliferative epithelial lesions that

include cutaneous and genital warts and

recurrent respiratory papillomatosis.34 In
the anogenital tissues specifically, a number
of HPV genotypes are connected to
premalignant and malignant lesions such as
squamous intraepithelial lesions (SILs) and
anal, vaginal and cervical cancers. Of more
than 15 high-risk HPV subtypes, HPV
16 and 18 are best known for their role
in anogenital cancers and were initially
targeted for vaccine development.35

The HPV Life Cycle

Human papillomavirus is a small, nonencapsulated virus consisting of a core with
an 8,000 base pair long circular DNA and
an outer capsid that contains two main
proteins, L1 and L2.34 The L1 protein is the
highly conserved main capsid component,
and because of an ability to elicit virusneutralizing antibodies in humans, it has
been the basis for the currently available
HPV vaccines; the L2 protein along with
L1 interacts with a number of cellular
proteins during the viral entry process
into the host cell.35 The virus has six main
nonstructural regulatory proteins, E1, E2,
E4, E5, E6 and E7, each with a specific
function related to viral replication and/
or oncogenesis.34 The E and L designations
are based on the order of expression of

these viral gene products, early or late

in the transcription process during viral
replication. The HPV genome contains
segments referred to as open reading frames
(ORF codons or sequence of nucleotides
with the potential to be transcribed into
RNA) that encode early proteins E1
through E8 and late proteins L1 and L2.34
Of all the regulatory proteins, E1 and E2
are necessary for viral replication and,
therefore, the genes encoding for these
as well as the capsid proteins L1 and L2
are called the core genes, while the
genes encoding for the other proteins that
mainly affect the cellular environment of
the virus are called accessory genes.34,35
The viral infection of the epithelial
basal cells and HPV entry into the cells
nucleus is thought to ensue a series of
steps that include: a) microtrauma to the
epithelium, b) an interaction between
the viral L1 capsid protein and the
extracellular matrix (ECM) or the cell
surface of basal layer keratinocytes and
c) an interaction between the L2 capsid
protein and the host lysosomal membrane
for viral endocytosis.34-37 After entry into
the host cell, papillomaviruses replicate
and assemble exclusively in the nucleus
in a fashion that is regulated by the
patterns of keratinocyte maturation and
differentiation across the epithelial cell
layers. For instance, the expression of the
early regulatory proteins occurs in the
lower undifferentiated or intermediately
differentiated keratinocytes while
the transcription of the late capsid
proteins takes place in the upper layer
of keratinocytes undergoing terminal
differentiation.36 Of all the viral proteins,
E1 and E2 actively function in viral DNA
replication,36 E4 facilitates virion release
into the environment,38 and E5, E6 and E7
facilitate the transformation of regularly
maturing keratinocytes into malignant
cells. More specifically, E5 protein activates
the cell growth-promoting signaling of
F E B R U A R Y 2 0 1 6 95

oropharyngeal cancer
C D A J O U R N A L , V O L 4 4 , N 2

TABLE

TNM Staging for Tumors of the Oropharynx

factors such as platelet-derived growth

factor (PDGF) and epidermal growth
factor (EGF). The other viral proteins
E6 and E7 interfere with epithelial cell
cycle regulatory mechanisms. E6 promotes
uncontrolled cell growth by mainly
mediating the degradation of p53, the
protein product of the tumor suppressor
gene TP53; in humans, this gene has a
gatekeeper function, conserving genetic
stability, preventing genome mutations
and inducing death (apoptosis) in cells
that have damaged DNA. E7 inactivates
members of the retinoblastoma protein
(pRb) family of tumor suppressor proteins
produced by tumor suppressor gene
RB; normally, these proteins prevent
excessive cell growth by inhibiting cell
cycle progression until a cell is ready to
divide.39,40 Taken together, these viral
proteins and the genes that encode for
them (referred to as oncogenes) serve to
prevent apoptosis and promote cell cycle
progression with enhanced uncontrolled
viral DNA replication. Because the
expression of E6 and E7 oncogenes
occurs in the malignant phenotype in
HPV-induced cancers, their transcription
products are currently targeted for
therapeutic HPV vaccine development.41
After viral DNA replication is
complete, the new infectious progeny
virus accumulates in the keratinocytes
in the outermost layers of the skin or
mucosal surface. The assembled viruses
may remain dormant in the tissues
and not produce a clinical disease. In
these chronic infections, the release
of the new virions occurs through
normal epithelial desquamation process
from the surface and therefore is not
associated with host cell lysis and an
inflammatory response. This is one
mechanism that HPV uses to evade the
host immune response, others include a
life cycle that is exclusively intraepithelial
and without viremia, a lack of any
96F E B R U A R Y 2 01 6

TNM

Denitions

TX

Primary tumor cannot be assessed

T0

No evidence of primary tumor

Tis

Carcinoma in situ

T1

Tumor 2 cm or less in greatest dimension

T2

Tumor more than 2 cm but not more than 4 cm in greatest dimension

T3

Tumor more than 4 cm in greatest dimension

T4a

Tumor invades the larynx, deep/extrinsic muscle of tongue, medial pterygoid, hard
palate or mandible

T4b

Tumor invades lateral pterygoid muscle, pterygoid plates, lateral nasopharynx or

skull base or encases carotid artery

Nx

Regional lymph nodes cannot be assessed

N0

No regional lymph node metastasis

N1

Metastasis in a single ipsilateral lymph node, 3 cm or less in greatest dimension

N2

Metastasis in a single ipsilateral lymph node, more than 3 cm but not more than 6
cm in greatest dimension; or in multiple ipsilateral lymph nodes, none more than 6
cm in greatest dimension; or in bilateral or contralateral lymph nodes, none more
than 6 cm in greatest dimension
N2a

Metastasis in a single ipsilateral lymph node more than 3 cm but not more than 6
cm in greatest dimension

N2b

Metastasis in multiple ipsilateral lymph nodes, none more than 6 cm in greatest

dimension

N2c

Metastasis in a lymph more than 6 cm in greatest dimension

N3
Mx

Distant metastasis cannot be assessed

M0

No distant metastasis

M1

Distant metastasis

Adopted from CA Cancer J Clin 2005;55;242-258. American Cancer Society.

proinflammatory cytokine production

and no activation of Langerhans or
dendritic cell signaling.34 The HPV
infection can be immunologically
cleared or remain dormant and not
produce a high number of viral copies
until immunologic suppression.34
It should be noted that much of the
HPV life cycle mechanisms described
are based on studies of anogenital
infections as the natural history of oral
and oropharyngeal HPV is still under
investigation. Preliminary studies suggest
that like anogenital infections, oral HPV
clears within one or two years, mostly
because of oral mucosal immune responses,
but the rate of clearance is dependent upon

the oral viral load.42 It is estimated that

about 7 percent of the U.S. population
(men and women aged 14 to 69 years)
have oral HPV, 1 percent carrying the
HPV type 16, with peak prevalence among
individuals aged 30 to 34 years,43 while
among HIV-seropositive patients this rate
may be as high as 12.3 percent.44 In one
study, the rate of HPV carriage among
HIV-negative men who have sex with men
(MSM) attending a sexually transmitted
disease (STD) clinic was 13.7 percent, 5.9
percent for high-risk HPV, not correlating
with the rate in the anogenital samples,
suggesting a different natural history of
the virus in the oropharyngeal tissues.45
Interestingly, the level of oral HPV carriage

C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 2 . A ginigival SCC in an HIV-seropositive

patient.

seems to correlate with sexual behaviors.

Indeed, having a higher number of oral
sex and rimming partners, recently or
over a lifetime, is associated with increased
odds of oral HPV prevalence.43,46-50
As data support a lower rate of oral
HPV clearance among HIV-seropositive
individuals, especially those with lower
CD4 counts,50 a higher risk of oral cancer
development,51 higher mortality rates for
oral cancer than many other cancers,52 and
considering the high rate of HPV (and
high-risk-HPV) carriage in the oral tissues
of both HIV-seropositive and HIV-negative
high-risk individuals, especially correlated
with sexual behaviors as described
above, cancer prevention strategies
must therefore include targeted patient
education as well as clinical vigilance,
especially by oral health care providers.

Clinical Considerations
As previously described, the great
majority of oropharyngeal cancers are
squamous cell carcinomas that are found,
for the most part, in the tonsillar complex
as well as the base of the tongue, the
soft palate and the posterior pharyngeal
wall. In clinical dental practice, a careful
visual assessment of the oral/pharyngeal
structures and the regional lymph nodes
with digital palpation is critical to
detecting cancerous lesions. In addition,
alarm symptoms such as trismus, dysphagia,
changes in tongue mobility and otalgia
should be further investigated for lesions
that may have penetrated deeper tissues in

the region.53 Other diagnostic modalities

such as computed tomography (CT) and
magnetic resonance imaging (MRI) or
positron emission tomography are helpful
in more accurate assessment of the extent
of OPC. Traditionally, the TNM staging,
based on the tumor size (T), regional nodal
involvement (N) and level of distant
metastasis (M), has been used to classify
cancers into stages 1 to 4 in order to guide
treatment decisions and to predict the
treatment outcomes. The TABLE shows
the definitions for the TNM staging of
oropharyngeal cancers according to the
Fourth Collaborative Staging between the
American Joint Committee on Cancer
(AJCC) and the International Union
Against Cancer (UICC).54 Currently, the
major clinical outcome of oropharyngeal
cancer treatment is to preserve patients
function; therefore, nonsurgical treatment
options using chemotherapy and radiation
therapy are preferred whenever possible.54
However, organ preservation therapy
may not be possible for all patients and
surgical resection may be the only option.
In patients who require surgical excision,
the pathologic stage of the tumor (pTNM)
derived from histopathologic examination
of the tumor and/or regional lymph nodes
is useful in selecting postoperative adjuvant
therapy and for estimating prognosis.54
The histologic classification of
OPSCC consists of well-differentiated,
moderately differentiated and poorly
differentiated squamous cell carcinomas.53
The significance of this classification is
that when the level of maturation of the
cells and their organization in the tumor
tissue are close to normal cells, referred
to as well-differentiated, the rate of
growth and spread of the tumor is slower
than when tumors are poorly or even
moderately differentiated. For advanced
stage disease where tumor resection is
not an option, induction chemotherapy
with cisplatin in combination with

5-fluorouracil and/or docetaxel has

shown improved patient survival.55-58
Other possible agents under investigation
for use in induction chemotherapy
include the biological agents, among
which anti-epidermal growth factor
receptor-targeted (EGFR) monoclonal
antibody may provide additional
benefits to standard chemotherapy.59
It has not been determined if the
treatment outcomes for the HIVseropositive patients are any different
from the outcomes for patients without
HIV infection. FIGURE 2 shows a gingival
SCC in an HIV-positive patient who was
also diagnosed with anal intraepithelial
neoplasia and SCC for the preceding
four years; both cancers were positive for
HPV 16. The initial treatment of this
gingival lesion consisted of only surgical
resection with no radiation therapy and
the lesion recurred in less than three
months, requiring a marginal resection of
the jaw. One retrospective study of 73 HIV
patients with HNSCC suggested that HIV
infection may negatively impact patients
locoregional control (presence or absence of
recurrence or second primary tumors) and
overall survival.60 This study had certain
limitations though, it included a variety of
cancer types and locations and the patients
had varying degrees of immune suppression
and cART use and received a combination
of treatment approaches including
radiation and/or chemotherapy. Clearly,
more robust studies with adequate patient
stratification and longer periods of followup are necessary to truly demonstrate the
impact of HIV infection in HNC survival.

Molecular Studies
As mentioned earlier, HPV-associated
OPSCC has better clinical prognosis, both
in terms of progression rate and response
to treatment. Although the exact reasons
for this phenomenon remain elusive,
among the proposed mechanisms are the
F E B R U A R Y 2 0 1 6 97

oropharyngeal cancer
C D A J O U R N A L , V O L 4 4 , N 2

younger age of individuals at diagnosis,

lower rates of smoking and alcoholrelated morbidities and more sensitivity
to treatment because of HPV-induced
P53 dysfunction and better apoptotic
response of cancer cells to radiation and
chemotherapy.61 In addition to these
factors, HPV-positive OPSCC has a more
indolent clinical behavior with a clinical
staging that is in an earlier T-category and
a more advanced N-category but lymph
node metastasis that is often cystic in
nature.62 Based on these properties inferred
by HPV infection, HPV testing of the
cancer tissues is becoming the standard
of care at many cancer treatment centers.
Molecular detection of HPV DNA, the
gold standard for the identification of
HPV in tissues, can be accomplished
by methods such as Southern transfer
hybridization, dot blot hybridization, in
situ hybridization (ISH), hybrid capture
and polymerase chain reaction (PCR).63,64
Although identifying the presence
of HPV in the cancer tissue samples is
important to predicting its prognosis,
it has become exceedingly clear that
even among HPV-positive OPSCC
lesions, there is a great degree of
heterogeneity that is driven by the level
of HPV biological activity, offering
an opportunity for specific prognostic
molecular markers that may further
enhance outcomes of these cancers in
the future.59 For instance, the level of
expression of p16INK4a (a cyclin-dependent
kinase inhibitor that promotes cell cycle
arrest) in the HPV-positive SCC tissues
may be a good molecular proxy for this
level of activity. It turns out that the
suppressive effect of HPV E7 protein on
pRb leads to an over expression of the
INK4a, a tumor suppressor gene and
its protein product p16INK4A and hence
improves the overall cancer prognosis.
Therefore, immunostaining for p16INK4A
in conjunction with HPV DNA testing
98F E B R U A R Y 2 01 6

of the tissue can be a good prognostic

marker for the level of HPV biological
activity.65 The clinical relevance of this
was shown in one study that compared
the prognostic value of HPV-16 DNA
viral load with p16 protein expression,
showing the most favorable prognosis
for patients whose tumors were HPV16-positive/p16 expressive.66 It should
be noted that continuous smoking and
alcohol use can change the behavior of
HPV-positive tumors, as both smoking
and alcohol can induce mutational
loss of p53 and p16INK4A.59 Currently,

Based on these properties

inferred by HPV infection,
HPV testing of the cancer
tissues is becoming the
standard of care at many
cancer treatment centers.

several other molecular mechanisms

are being investigated for their possible
contributions to the overall prognosis
of HPV-positive tumors. Some of these
include the role of HPV in the Wnt
and Notch signaling pathways involved
in keratinocyte differentiation, the
role of epigenetic factors such as the
differences in the methylation profile
of HPV-positive and HPV-negative
tumors and the contribution of
angiogenesis factors such as vascular
endothelial growth factor (VEGF) and
endothelin-1 in tumor progression.67-69

Prevention Strategies
With an estimated life-long risk
of cervical HPV infection up to
80 percent among sexually active
women, HPV is considered the most

common sexually transmitted infection

worldwide.70Although HPV is known
to be transmitted through direct skin
or mucosa contact, its transmission
patterns and the exact determinants
of susceptibility and infectivity are less
established. In one recent study, a perperson transmission probability of about
20 percent during a six-month period was
shown among discordant couples.71 In this
study, the transmission rates from women
to men and men to women were the same,
varied little with the circumcision status
of the men and showed a trend toward
higher rates for HPV 16. Because of its
relationship with cancer development
in cervical tissues, HPV malignant
subtypes have been targeted for vaccine
development. In the U.S., a quadrivalent
HPV vaccine (4vHPV) that targets HPV
6, 11, 16 and 18 has been available since
2006 and a 9-valent vaccine (9vHPV)
targeting additional HPV subtypes 31,
33, 45, 52 and 58 was licensed for use in
females and males in December 2014.72
In addition, a bivalent HPV vaccine
against HPV subtypes 16 and 18 has also
been available since 2009.73 According
to the U.S. Advisory Committee on
Immunization Practices (ACIP), the
HPV vaccines are recommended to be
used routinely for females and males
at age 11 or 12 years (can be started
beginning at age 9) up to age 26 years.74
Each HPV vaccine is delivered through
a series of three intramuscular injections
over a six-month period and is not
recommended for pregnant women;
if the vaccine schedule is interrupted,
the vaccination series does not need to
be restarted and there are no booster
doses recommended.74 These vaccines
are now available in most countries
in the world and are recommended
by the World Health Organization to
be used in young women after age 9
and in two doses six months apart.75

C D A J O U R N A L , V O L 4 4 , N 2

There is no doubt that vaccination

of females, despite low vaccine coverage,
has contributed to a reduction in the
prevalence of cervical infections of the
targeted HPV subtypes. One U.S. study
showed an HPV positivity rate of 5.1
percent of cervicovaginal specimens from
2007 to 2010 compared to 11.5 percent
of the specimen in 2003-2006.76 What
is not clear is the extent to which HPV
vaccination has impacted the incidence
of cervical cancer, and even less is
known about its impact on other types of
HPV-related cancers. A recent Centers
for Disease Control and Prevention
study used about 2,700 cancer archival
tissues collected from 1993 to 2005
from seven U.S. cancer registries and,
after HPV DNA testing of the samples,
provided estimates for the impact of HPV
vaccines in reducing the HPV-related
cancers.77 The study estimated that the
quadrivalent vaccine can potentially
prevent the majority of invasive cervical,
anal, oropharyngeal and vaginal cancers,
almost 25,000 cases annually, and the
9-valent vaccine has the potential to
prevent an additional 4,000 cases.77
These estimates are very encouraging
and call for improved vaccination
coverage for both young men and women,
before they become sexually active.

Conclusion
HPV-related oropharyngeal
cancers are increasing, and while HPV
vaccination can reduce the incidence
of these cancers, early clinical detection
and lower cancer staging is key to
patient survival after diagnosis. Focused
scientific research has started to produce
reliable prognostic molecular markers
and novel treatment approaches that,
once clinically validated and adopted in
everyday practice, can forever transform
the landscape of oropharyngeal cancer
detection, staging and treatment.

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59. Wirth LJ, Allen AM, Posner MR, et al. Phase I dosending study of paclitaxel with panitumumab, carboplatin
and intensity-modulated radiotherapy in patients with locally
advanced squamous cell cancer of the head and neck. Ann
Oncol 2010;21(2):342-7.
60. Mourad WF, Hu KS, Shasha D, et al. Long-term outcome
of seropositive HIV patients with head and neck squamous cell
carcinoma treated with radiation therapy and chemotherapy.
Anticancer Res 2013;33(12):5511-6.
61. Guihard S, Ramolu L, Macabre C, et al. The NEDD8
conjugation pathway regulates p53 transcriptional activity and
head and neck cancer cell sensitivity to ionizing radiation. Int J
Oncol 2012 Oct;41(4):1531-40.
62. Goldenberg D, Begum S, Westra WH, et al. Cystic
lymph node metastasis in patients with head and neck
cancer: An HPV-associated phenomenon. Head Neck 2008
Jul;30(7):898-903.
63. Zaravinos A. An updated overview of HPV-associated
head and neck carcinomas. Oncotarget 2014;5(12):3956-69.
64. Zaravinos A, Mammas IN, Sourvinos G, Spandidos DA.
Molecular detection methods of human papillomavirus (HPV).
Int J Biol Markers 2009;24(4):215-22.
65. Liang C, Marsit CJ, McClean MD, et al. Biomarkers of
HPV in head and neck squamous cell carcinoma. Cancer Res
2012;72(19):5004-13.
66. Weinberger PM, Yu Z, Haty BG, et al. Molecular
classication identies a subset of human papillomavirus
associated oropharyngeal cancers with favorable prognosis. J
Clin Oncol 2006;24(5):736-47.
67. Rampias T, Sasaki C, Psyrri A. Oral Oncol
2014;50(5):356-63.
68. van Kempen PM, Noorlag R, Braunius WW, et al.
Dierences in methylation proles between HPV-positive
and HPV-negative oropharynx squamous cell carcinoma: A
systematic review. Epigenetics 2014;9(2):194-203.
69. Baruah P, Lee M, Wilson PO, et al. Impact of p16 status on
pro- and anti-angiogenesis factors in head and neck cancers.
Br J Cancer 2015;113(4):653-659.
70. Veldhuijzen NJ, Snijders PJ, Reiss P, et al. Factors aecting
transmission of mucosal human papillomavirus. Lancet Infect
Dis 2010 Dec;10(12):862-74.
71. Burchell AN, Coutle F, Tellier PP, et al. Genital transmission
of human papillomavirus in recently formed heterosexual
couples. J Infect Dis 2011;204:1723.
72. Food and Drug Administration. Highlights of prescribing
information. Gardasil 9 (human papillomavirus 9-valent vaccine,
recombinant). Silver Spring, Md.: U.S. Department of Health
and Human Services, Food and Drug Administration; 2014.
www.fda.gov/downloads/BiologicsBloodVaccines/Vaccines/
ApprovedProducts/UCM426457.pdf. Accessed Aug. 12, 2015.

73. FDA Licensure of Bivalent Human Papillomavirus

Vaccine (HPV2, Cervarix) for Use in Females and Updated
HPV Vaccination Recommendations from the Advisory
Committee on Immunization Practices (ACIP). MMWR
2010;59(20):626-629.
74. Petrosky E, Bocchini Jr. JA, Hariri S, et al. Use of 9-Valent
Human Papillomavirus (HPV) Vaccine: Updated HPV
Vaccination Recommendations of the Advisory Committee on
Immunization Practices. MMWR 2015;64(11):300-304.
75. World Health Organization. Weekly epidemiological
record. Human papillomavirus vaccines. WHO position paper
2014;89:465-492.
76. Markowitz LE, Hariri S, Lin C, et al. Reduction in human
papillomavirus (HPV) prevalence among young women
following HPV vaccine introduction in the United States,
National Health and Nutrition Examination Surveys, 20032010. J Infect Dis 2013;208(3):385-93.
77. Saraiya M, Unger ER, Thompson TD, et al. HPV Typing of
Cancers Workgroup. U.S. assessment of HPV types in cancers:
Implications for current and 9-valent HPV vaccines. J Natl
Cancer Inst 2015;107(6):djv086.
THE AUTHOR,

Fariba S. Younai, DDS, can be reached at

fyounai@dentistry.ucla.edu.

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

Oral Cancer
Chemoprevention: Current
Status and Future Direction
Diana V. Messadi, DDS, MMSc, DMSc, and Kazumichi Sato, DDS, PhD

A B S T R A C T The aim of this study is to review the current status of cancer

chemoprevention and its effectiveness in treatment of oral premalignant lesions

and prevention of their progression to oral cancer. The challenges encountered in
the different oral cancer chemoprevention clinical trials, including lack of surrogate
endpoints, reversal of histologic premalignant changes as study endpoints, tobacco
use, human papillomavirus, delivery system, adverse effects and risk of bias in
clinical studies, are presented.

AUTHORS
Diana V. Messadi, DDS,
MMSc, DMSc, is a
professor and chair of the
section of oral medicine
and orofacial pain and the
associate dean for
education and faculty
development at the
University of California, Los
Angeles, School of
Dentistry.
Conict of Interest
Disclosure: None reported.

Kazumichi Sato, DDS,

PhD, is an associate
professor in the department
of oral medicine and oral
and maxillofacial surgery
at Tokyo Dental College in
Sugano, Ichikawa, Chiba,
Japan. During the
preparation of this
manuscript he was a
research scholar in the
section of oral medicine
and orofacial pain at the
University of California, Los
Angeles, School of
Dentistry.
Conict of Interest
Disclosure: None reported.

ancer chemoprevention
is defined as the use
of natural, synthetic
or biological chemical
agents to suppress,
reduce or prevent the progression of
carcinogenesis. The term was first
coined by Sporn et al.1 It is defined as
drugs that have an effect of slowing
or stopping cancer development and
preventing malignant transformation
of premalignant lesions. Clinicians
have considered that the best
target populations for oral cancer
chemoprevention are patients with
oral premalignant lesions (OPLs) and/
or postoperative oral cancer patients
without tumor bearing for prevention
of secondary primary tumors.2-4
One of the major obstacles in
chemoprevention studies to date has
been the inability to identify oral
lesions with a high risk of malignant

transformation and the difficulty in

patient recruitment. The most common
OPLs are leukoplakia, erythroplakia
and erosive lichen planus. These lesions
are very heterogeneous, varying in
color, size, texture and anatomic site,
with studies showing a wide percentage
progressing to malignancy ranging from
1.1 to 25 percent. This variability arises
partly because of the differences in study
populations (i.e., clinic-based versus
population-based), tobacco history and
durations of follow-up after leukoplakia
diagnosis.5-6 Oral leukoplakia, the most
common OPL, is clinically defined as a
white patch or plaque on the oral mucosa
that cannot be removed by scraping
and cannot be classified clinically or
microscopically as another disease entity.
Biopsy and histopathologic analysis
is required to definitively diagnose as
leukoplakia and erythroplakia lesions.
Under the microscope, these lesions
F E B R U A R Y 2 0 1 6 101

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

FIGURE . A 54-year-old male with a leukoplakia

lesion on the right lateral border of the tongue
measured by an intraoral ruler for size documentation
before administering the chemopreventive agent.

demonstrate a spectrum of possible

histopathological features ranging
from hyperkeratosis to hyperplasia and
various grades of dysplasia including
mild, moderate and severe dysplasia to
carcinoma in situ (CIS) and squamous
cell carcinoma (SCC).7-9 These lesions
are considered the perfect model to
study the effect of chemopreventive
agents on cancer progression due to
their easy accessibility for both direct
visualization (F I G U R E 1 ), sample
acquisitions and monitoring of clinical,
histologic and molecular responses to
any given preventive intervention.
Most chemopreventive trials use a
combination of clinical change and
histology to identify eligible patients and
to monitor the impact of drugs. In most
studies, the inclusion criteria for patients
with OPL include histologic diagnosis
of dysplasia, mainly mild, moderate or
severe, and severe hyperkeratosis in a
high-risk location (i.e., soft palate, floor
of mouth, ventral tongue and alveolar
ridge). If a diagnosis of CIS or SCC is
rendered, then patients undergo the
routine treatment of surgical excision
and are excluded from the studies.9-13
Although numerous clinical trials have
been performed based on objective
scientific data, most reviews indicate that
these studies need larger cohort groups to
be successfully validated. The Cochrane
Collaboration states that there are no
significant studies that demonstrated
prevention of malignant transformation
102F E B R U A R Y 2 01 6

of OPL.13-14 Needless to say, promising

chemopreventive agents are needed as
a treatment option for OPL, especially
in lesions that are diffuse, with multiple
occurrences such as proliferative
verrucous leukoplakia (PVL) or in
elderly people who may have difficulties
undergoing extensive surgeries.

Methods: Search Strategy and Data

Collection
The following databases were searched:
Medline, PubMed and Clinicaltrials.gov.
The search was restricted to randomized
clinical trials for chemoprevention of OPL
and development of secondary primary
tumors after head and neck cancer therapy.
The data of each clinical trial, such as
study design, administration method,
effects of each endpoint, side effects
and risk of bias evaluated as a reference
of assessment tools, were extracted and
are summarized in TABLES 13 .15-24

Current Status of Chemopreventive

Agents for Oral Precancer Lesions
Vitamin A (retinol), 13-cis-retinoic
Acid (isotretinoin) and Beta-Carotene
Vitamin A (retinol) and retinoic acid
(teretinoin, alitretinoin, isotretinoin, etc.),
also known as retinoids, as well as betacarotene of provitamin A, have been used
in several clinical trials because of their
effect on specific biological processes such
as epithelial development, differentiation,
proliferation and apoptosis (TA BLE 1 ).1519
Extensive studies of retinoids and
beta-carotene have shown that their
administration period and evaluation
points are characteristically longer than
in other chemopreventive agents such
as cyclooxygenase-2 inhibitor, epidermal
growth factor receptor-targeted therapy
and some natural compounds.25 Lippman
et al. conducted a phase I controlled study
where leukoplakia patients were randomly

selected and underwent induction

therapy with a high-dose (1.5mg/kg/day)
of isotretinoin for three months; in the
second phase, patients were randomly
assigned to maintenance therapy with
either beta-carotene (30mg/day) or
low-dose (0.5mg/kg/day) isotretinoin for
nine months.26 The authors reported that
the rate of clinical disease progression
(increase of lesion size or appearance
of any new lesion) in the isotretinoin
group was significantly lower than the
rate in the beta-carotene group. Side
effects included dry skin, cheilitis and
conjunctivitis, which were minimal
during the administration of low-doses
of isotretinoin compared to high-doses
of isotretinoin. Long-term follow-up
(66 months) was reported and showed
no significant differences regarding
malignant tumor transformation.26 In
addition, studies of vitamin A and betacarotene treatments were found to have
no effect after several years following
the end of a one-year treatment period
(TA BLE 2 ).15,19 Another study by Lippman
et al., with a long-term intervention
period of low-dose isotretinoin in a
large sample group of patients with
history of oral cancer, showed that
isotretinoin did not reduce the rate
of second primary tumors or increase
survival compared with a placebo.16
Studies by Papadimitrakopoulou et
al. on a small cohort of patients with
OPL defined these precancerous lesions
as clinical and histologic evidence
of measurable or assessable OPLs
(leukoplakia and/or erythroplakia),
histologic examination showing dysplasia
or extensive leukoplakia with hyperplasia
and symptoms (e.g., pain), cosmetic cases
(e.g., leukoplakia of the lips) or high-risk
location (i.e., soft palate, floor of mouth,
ventral tongue and alveolar ridge).17
Their results showed no effect (clinical
and histological response) in long-term

C D A J O U R N A L , V O L 4 4 , N 2

intervention periods when administrating

low-dose isotretinoin.17,19 In general,
the balance of dose and side effects has
been important for retinoids and betacarotene when studying their potential
use as chemopreventive agents for oral
cancer (TA BLE 3 ). Although clinical
or histological regression were seen in
short-term periods of observation, the
response was reversed when the treatment
was terminated and this resulted in
posttreatment lesional recurrences.17,27

Green Tea
Similar to retinoids and betacarotene, green tea has been studied as a
chemopreventive agent for an extensive
period. The background for its use stems
from the idea that drinking green tea
reduces cancer risk.28 In Japan, with its
large population of tobacco users (around
20 percent of adults) and drinking green
tea as part of its culture, reports show that
oral cancer patients are about 1 percent
of all cancer patients and the prevalence
of OPL is 2.5 percent, which is lower than
other countries.25,30 The leading compound
in the mechanism of green tea cancer
chemoprevention is a type of polyphenol,
specifically, epigallocatechin-3-gallate
(EGCG). Studies showed that multiple
signal pathways are involved in the
inhibitory effect of polyphenols on cancer
cell growth.29-32 A phase II study reported
by Tsao et al. used three green tea dosages
including a 1,000 mg/m2 of green tea
extract based on a previous phase I study.20
The clinical response in this phase II study
did not reach statistical significance in
all green tea extract arms versus placebo.
However, the two higher-dose green tea
extract arms had higher responses (clinical
response), confirming dose-response effect
(TA BLE 1 ). Although some side effects
were observed, the therapy was well
tolerated and safe. They concluded that
these promising results supported longer-

term clinical testing of green tea extract

for oral cancer prevention. On the other
hand, a cross over clinical trial of green
tea was reported with a focus on dropout
cases. The study showed that the rates of
drop out were significantly higher among
smokers than nonsmokers.31 Only a few
of the dropout cases were interviewed,
with bitter taste and teeth staining as the
main reasons for their drop out. Green
tea extract does not cause bitter taste and
teeth staining, but absorption of caffeine
should be considered according to patient
preference. Studies are being conducted

This potential benet is still

not strong enough for a
universal recommendation
to drink green tea in order
to prevent oral cancer.

to enhance the bioavailability and

potency of green tea by manufacturing a
prodrug formulation of EGCG. Another
promising approach is to formulate
nanoparticles for effective delivery.32-33
Despite the promising evidence on
the benefits of green tea in preventing
oral and other cancers in animal models
and cell cultures, this potential benefit
is still not strong enough for a universal
recommendation to drink green tea
in order to prevent oral cancer.33

Cyclooxygenase-2 (COX-2) Inhibitor

The anticarcinogenic properties of
COX-2 inhibitor have been thoroughly
investigated in multiple experimental
and clinical studies.34-37 In case of OPL,
Mulshine et al. reported the use of the
cyclooxygenase inhibitor ketorolac as an

oral rinse in oropharyngeal leukoplakia,

unfortunately, their study demonstrated
that ketorolac did not cause leukoplakia
regression as compared to placebo.37
Similarly, Papadimitrakopoulou et al.
also reported no effective results of COX-2
inhibitor as a chemopreventive agents in
OPL (TABLES 1 and 2 ).21 They also
reported some adverse reactions observed
by patients such as dizziness and oral pain,
including one patient with a grade three
cerebrovascular accident (cerebral
infarction) (TABLE 3 ). They concluded that
these results discouraged their
pursuit of this agent as an oral cancer
chemopreventive agent. These studies
confirm that using COX-2 inhibitor solely
as a chemopreventive agent for oral cancer
will unlikely be available in clinical practice.

Bowman-Birk Inhibitor Concentrate

Bowman-Birk inhibitor concentrate
(BBIC) is defined as a serine protease
inhibitor isolated from soybeans possessing
domains with trypsin and chymotrypsin
inhibitory activity.38 Although BBIC has
been shown in some cell culture and animal
studies to inhibit oral carcinogenesis,
the specific inhibitory mechanism is still
unknown.39-42 It is considered that BBIC
may be acting as a tumor suppressor
similar to the serine protease inhibitors.
It also has anti-inflammatory properties,
inhibiting free radical production and
altering the levels of several oncogenes
(TABLES 1 and 2 ).22 BBIC has few side
effects because it is prepared from natural
food and is a common ingredient in
Japanese foods (soybean-based foods
such as miso and tofu). Therefore, BBIC,
just like green tea, was expected to be
an effective chemopreventive agent for
oral cancer. Recently, Armstrong et al.
performed a phase IIb double-blinded
placebo controlled study that showed no
difference between the placebo and the
BBIC arm in oral cancer prevention.22, 42-43
F E B R U A R Y 2 0 1 6 103

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

TABLE 1

Summary of the Nine Studies (eligible participants, study design, control, location of study, sample size, registration period) 1524
Summary of the study
Author
Year

Agents

Eligible participants

Study design
(RCT: randomized
control trial)

Control

Location

Jyothirmayi
1996

Vitamin A

Head and neck cancer treated with surgery

and/or radiation therapy (disease free)

RCT
two-arm

Placebo

India

Khuri
2006

Isotretinoin
(13-cis retinoic acid)

Head and neck cancer treated with surgery

and/or radiation therapy (stage 1 or 2)

RCT (pilot phase III)

two-arm
multicenter

Placebo

USA

Papadimitrakopoulou
2009

Isotretinoin
(13-cis retinoic acid)

Premalignant lesions

two-arm (open-label
trial) single-center

Beta-carotene + retinyl
palmitate
or retinyl palmitate
alone

USA

Nagao
2014

Beta-carotene

Leukoplakia
(never smoked or ex-smokers)

RCT
two-arm
multicenter

Placebo (50mg/day
vitamin c)

Japan

Tsao
2009

Green tea extract

Premalignant lesions

RCT (phase II)

four-arm
single-center

Placebo

USA

Papadimitrakopoulou
2008

Cyclooxygenase-2
(COX-2)

Premalignant lesions

RCT (pilot phase II)

three-arm
multicenter

Placebo

USA

Armstrong
2013

Bowman-Birk inhibitor
concentrate

Leukoplakia, erythroplakia

RCT (phase IIb) two-arm Placebo

multicenter

USA

Mallery
2014

Freeze-dried black
raspberry
(black raspberry: BRB)

Premalignant lesions
(no tobacco use)

RCT
two-arm
multicenter

Placebo

USA

Sun
2010

ZengShengPing
(a mixture of
medicinal herbs)

Leukoplakia

RCT
two-arm
single-center

Placebo

China

Freeze-Dried Black Raspberry and

ZengShengPing
Freeze-dried black raspberry (BRB)
contains natural ingredients such as
vitamin A, vitamin E, beta-carotene,
multiple anthocyanins and phytosterols.
These ingredients have been shown to
exhibit a chemopreventive effect both in
an in vitro model using oral cancer cell
lines and in vivo in a pilot study with oral
leukoplakia patients.44-47 BRB has many
functions, including the suppression of
redox-mediated intracellular signaling,
reducing production of pro-angiogenic
cytokines and stimulating an apoptotic
104F E B R U A R Y 2 01 6

pathway.46 A clinical study using a

topical 10% BRB gel applied daily for a
three-month period demonstrated
statistical significant regression of OPL,
where 41 percent of participants achieved
a decrease in lesional dysplastic grade
when compared to patients using placebo23
(TABLE 1 ). The authors attributed the
BRBs chemopreventive effect to its
primary phenolic compounds,
anthocyanins. In addition, the comparison
of the histological grade and loss of
heterozygosity (LOH) in the pretreatment
versus posttreatment tissues demonstrated
that application of BRB resulted in a

statistical decrease of histologic grading,

whereas placebo gel application did not
have a significant impact.23
ZengShengPing (ZSP) is a registered
trademark and herbal mixture composed
of six herbs (Sophora tonkinensis,
Polygonum bistorta, Prunella vulgaris,
Sonchus brachyotus, Dictamnus dasycarpus
and Dioscorea bulbifera). Although the
effective compounds of ZSP have not
yet been identified, individual herbs of
ZSP are known to contain bioactive
chemicals with anti-inflammatory
and anticancer activities.48 Preclinical
studies have shown that ZSP inhibited

C D A J O U R N A L , V O L 4 4 , N 2

Sample size

Registration
period
(enrolled period)

106 [agents 56, control 50]

(oral cancer patients: 77.4%)

1992

1190 [agents 590, control 600]

(oral cancer patients: 30.1%)

19911999

162 [Isotretinoin 81, beta-carotene

+ retinyl palmitate 45, retinyl

1992 2001

regressing leukoplakia lesions was still

present after three months following
cessation of ZSP treatment (TA B L E 3 ).
These two compounds were investigated
in small pilot projects; larger cohort groups
need to be studied in order to successfully
validate these products as chemopreventive
agents for oral precancerous conditions.

Epidermal Growth Factor ReceptorTargeted Therapy

palmitate alone 36]

(167: randomized patients)
46 [agents 23, control 23]

no mention

39 patients completed
[agents 28, control 11]
(41: randomized patients)

20022008

50 patients completed
[agents 32, control 18]
(56: randomized patients)

20002004

89 patients completed
[agents 43, control 46]
(132: randomized patients)

19992009

40 patients
[agents 22, control 18]

no mention

112 patients completed)

[agents 59, control 53]
(120: randomized patients)

19982001

inflammation and inflammationassociated carcinogenesis in animal

models.24 Clinical trials in China from
the late 1970s to early 1990s confirmed
that oral intake of ZSP had reduced
the progression of histopathologically
confirmed precancerous esophageal
squamous dysplasia to carcinoma by 48
to 52 percent compared to placebo.48
Sun et al. used ZSP in a short-term
randomized placebo-controlled clinical
trial on patients with leukoplakia lesions
after demonstrating its preventive
effect in two animal models.24 Their
study showed that clinical responses in

Oral premalignant lesions have

increased epidermal growth factor
receptor-targeted (EGFR) protein
expression and increased EGRF gene
copy number compared to normal
mucosa. Oral premalignant lesions
with overexpression of EGFR or EGFR
gene copy number gain are at higher
risk for malignant transformation.49
EGFRtargeted therapy has already
been FDA approved and widely used
as a chemotherapeutic drug for several
types of solid tumors including oral,
lung and breast cancer. Several studies
have demonstrated the role of EGFR
dysregulation as a molecular marker
of cancer risk and a key player in the
process of malignant transformation.49
Clinical trials with EGFR targeted
agents, including cetuximab, erlotinib
and vandetanib, are currently
underway to evaluate the efficacy
of EGFR inhibitors for oral cancer
chemoprevention. The major one is the
multi-institutional study known as the
Erlotinib Prevention of Oral Cancer
(EPOC) study (NCT00402779).49

Curcumin and Other

Chemopreventive Agents
Curcumin is the primary component in
the spice turmeric. The anticancer effects
of curcumin mainly result from multiple
biochemical mechanisms that are involved
in the regulation of programmed cell
death and survival signals. The curcumin

targets that are involved in signaling

pathways include transcription factors,
growth factors, inflammatory cytokines
and reactive oxidative stress.50-51 Its role as
a promising new chemopreventive agent
for oral cancer has been widely received.
Cheng et al. reported in their phase I
clinical trial of curcumin in patients with
systemic high-risk conditions, including
oral leukoplakia, that curcumin is nontoxic
for human use when taken by mouth for
three months for up to 8,000 mg/day.52
Additionally, histologic regression was
observed in two out of seven patients with
oral leukoplakia, where dysplasia regressed
to hyperkeratosis. Unfortunately, one
patient with oral leukoplakia developed
oral cancer in spite of curcumin treatment.
Another pilot study was conducted
with head and neck cancer patients.
These patients were given chewable 1
gram (gm) of curcumin gelatin capsules
for 18 months, and then their saliva was
collected and incubated with oral cancer
cells in culture. The authors found that
saliva containing curcumin inhibited
IKKb (a transcription factor) kinase
activity in these cells. IKKb is a known
transcription factor involved in the
carcinogenesis process, thus indicating that
IKKb kinase could be a useful biomarker
for detecting the effect of curcumin
in head and neck cancer patients.53
Curcumin has unfavorable
characteristics such as its hydrophobicity
and rapid metabolic rate. Therefore, drug
delivery systems such as nanoparticles,
liposomes, microemulsions and polymeric
implantable devices have been examined
as one of the viable alternatives to deliver
therapeutic concentrations of curcumin.54
Other chemopreventive agents that
have recently been investigated as new
potential chemopreventive agents for oral
precancer conditions include Pioglitazone,
Akt pathway inhibitor (SR13668)
and lyophilized strawberries.55-59
F E B R U A R Y 2 0 1 6 105

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

TABLE 2

Administration Method (period, route of administration); Effects (evaluation point, endpoints, results of endpoints) in Nine Studies 1524
Administration method

Eects

Author
Year

Period

Route of administration

Evaluation point

Endpoints

Jyothirmayi
1996

1 year

Oral administration
200,000 IU/week

3 years

#0 Recurrence: including regional recurrence

#1 Second primary tumors

Khuri
2006

3 years

Oral administration
30mg/day (3x daily)

7 years

#0 Rate of second primary tumors (primary endpoint)

#1 Rate of smoking-associated cancers
#2 Primary tumor recurrence
#3 Smoking-associated diseasefree survival
#4 Overall survival

Papadimitrakopoulou
2009

3 years

Oral administration
0.5mg/kg/day (1 year)
0.25mg/kg/day (2 years)

5 years

#0 Clinical response at 3 months (primary endpoint)

#1 Toxicity
#2 Improvement in histology
#3 Oral cancer free survival

Nagao
2014

1 year

Oral administration
10mg/day with 500mg/day
vitamin C

5 years

#0 Role in clinical remission (primary endpoint)

#1 Malignant transformation

Tsao
2009

12 weeks

Oral administration
500mg/m2, 750mg/m2,
1000mg/m2
(3x daily)

12 weeks

#0 Clinical and histological response (primary endpoint)

#1 Qualitative and quantitative toxicities of GTE
#2 Eects of treatment on the expression of biomarkers
(VEGF, p53, Ki-67, Cyclin D1, The p16 promoter methylation)
#3 Any potential correlation between treatment ecacy and/
or toxicity with plasma concentrations of EGCG or caeine

Papadimitrakopoulou
2008

12 weeks

Oral administration
100mg or 200mg

12 weeks

#0 Clinical response rate (primary endpoint)

#1 Histological improvement rate

Armstrong
2013

6 months
(PR,CR ) add 12
months

Oral administration
(powder + water)
600 C.I. units of BBIC/day
(2x daily)

6 months

#0 Percent change in total lesion area (primary endpoint)

#1 Change in clinical impression
#2 Central pathology review
#3 Change in buccal-cell Neu protein
#4 Change in serum Neu protein
#5 Change in buccal cell protease activity

Mallery
2014

3 months

Application
(4x daily)

3 months

#0 Change in lesional size

#1 Change in histological grade
#2 Change in LOH events
#3 Cumulative responsiveness score

Sun
2010

812 months

Oral administration 3.6mg

(3x daily)

3 months
(after cessation of
treatment)

#0 clinical response (primary endpoint)

#1 AgNOR and PCNA - labeling index

vit A: Vitamin A; N.S: not statistically signicant; GTE: Green tea extract; EGCG: epigallocatechin 3-gallate; PR: partial response; CR: complete response;
BBIC: Bowman-Birk inhibitor concentrate; LOH: loss of heterozygosity; BRB: black raspberry

106F E B R U A R Y 2 01 6

C D A J O U R N A L , V O L 4 4 , N 2

Results of endpoints
#0 vit A 11/56, placebo 5/50
#1 vit A 0/56, placebo 2/50
#0 N.S (Isotretinoin 130/590, placebo 131/600)
#1 N.S (Isotretinoin 84/590, placebo 98/600)
#2 N.S (recurrence-free survival P=0.21)
#3 N.S (P=0.15)
#4 N.S (P=0.73)
#0 N.S (P=0.29)
#1 refer to Table 2 side eects and adverse eects section
#2 N.S
#3 N.S
#0 N.S (P=0.346: 1 year observation period)
#1 Cases of malignant transformation: experimental
2/23, placebo 3/23
(60 months of the median duration of follow-up)
#0 Clinical response: versus placebo (P=0.09),
histological response: versus placebo (P=0.65)
#1 The incidence of insomnia and nervousness
was higher in patients receiving GTE at 750 and
1,000 mg/m2
#2 No mention of treatment side eects
#3 1,000 mg/m2 dosing was most likely the reason for
the increased insomnia and nervousness
#0 N.S (P=1.0)
#1 N.S (P=0.71)
#0 N.S (P>0.94)
#1 No mention
#2 N.S (n=88)
#3 N.S (n=45)
#4 N.S (n=45)
#5 N.S (n=41)
#0 BRB versus Control P<0.01
#1 BRB (pre versus post P<0.05), Control (pre versus
post P=0.496)
#2 BRB (pre versus post P<0.005), Control (pre versus
post P=0.160)
#3 BRB versus Control P<0.005
#0 P<0.01
#1 P<0.05

Discussion
Surrogate Endpoint and Biomarkers
The endpoint of any clinical trial
addressing oral cancer chemoprevention
in OPL is prevention of malignant
transformation. Among nine studies
summarized in TA B L E 2 , four studies
using retinoids set carcinogenesis as
the endpoint.16-19 However, when
duration and frequency of malignant
transformation are considered, surrogate
endpoints have to be set. The National
Institutes of Health (NIH) defines
surrogate endpoint as a biomarker
intended to substitute for a clinical
endpoint. Some studies establish
surrogate endpoint according to the
mechanisms of chemoprevention agents,
such as LOH, DNA hypermethylation
and NF-kB signaling.4 Martinez et al.60
emphasized the importance of LOH on
chromosomes 3p and 9p as a surrogate
biomarker and using it as one of the
inclusion criteria for eligible lesions
having a high possibility of malignant
transformation. A study by Mao et al.61
reported that seven out of 19 patients
with an OPL and LOH at 3p14 and/or
9p21 developed head and neck squamous
cell carcinoma (HNSCC) whereas one
of 18 patients with an OPL without LOH
at 3p14 and/or 9p21 developed HNSCC.
The EPOC study set eligible participants
as those who demonstrated LOH at
3p14 and/or 9p21; so one of the key
features of EPOC study is recognized to
confirm molecular risk assessment of oral
cancer.49 Studies for green tea, presented
in this review, examined the expression
of vascular endothelial growth factor
(VEGF), p53, Ki-67, Cyclin D1 and the
p16 promoter methylation as surrogate
endpoints using green tea extract as a
chemopreventive agent. They showed no
difference in biomarker expression in spite
of the presence of a clinical response.20,29-31

These studies demonstrate that

there is a dire need to identify novel
prognostic markers for oral precancer
conditions. These biomarkers should
not only predict the risk for oral cancer
progression, but also serve as targets
for pharmacologic interventions.

Factors Influencing the Effects of

Chemopreventive Agents
Tobacco Cessation. Tobacco use is
recognized as one of the most common
risk factor for oral cancer and OPL
development. Tobacco cessation can
reduce oral precancer incidence and
development of secondary primary
tumors.62,63 In a 10-year follow up
study in India, the cessation of tobacco
use led to a substantial fall in the
incidence rate of oral leukoplakia
in individuals that quit tobacco
use compared to the nonquitters.63
Almost all of the studies presented in
this review showed the participants
tobacco consumption data, but none
mentioned whether any tobacco
cessation programs were offered to
these patients. In designing clinical
trials for OPL chemoprevention,
role of tobacco cessation should be
considered as one of the variables
in leukoplakia regression.
Human Papillomavirus (HPV) Vaccine
as Chemopreventive Agent. Several
reports confirmed that HPV infection
plays a role in the development of
HNSCC with a higher incidence
in oropharyngeal tissues versus oral
mucous membrane.64-68 HPV vaccines
have been administered for prevention
of cervical cancer in more than 120
countries, with high effectiveness
and few side effects reported.
Currently, there are no reports on
the effectiveness of HPV vaccines for
oral cancer prevention. Multicentric
longitudinal studies should be initiated
F E B R U A R Y 2 0 1 6 107

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

TABLE 3

Effects (side effects and adverse effects); Qualities of the Nine Studies 1524
Eects (results)

Qualities of study

Author
Year

Side eects
Adverse eects

Limitation (risk of bias)

Jyothirmayi
1996

No clinical obvious side eects of

vitamin A.

* High risk of selection bias

* High risk of performance bias
* Low risk of detection bias
* Low risk of attrition bias
* Low risk of reporting bias

Khuri
2006

Grade II, III and IV toxicities were experienced

by 28.0%, 6.4%, and 0.3% of patients
in the isotretinoin arm and by 7.5%, 2.2% and
0.8% of those in the placebo arm, respectively
(P<.001).

* High risk of selection bias

* Low risk of performance bias
* Unclear risk of detection bias
* Low risk of attrition bias
* Low risk of reporting bias

Papadimitrakopoulou
2009

Cheilitis, conjunctivitis and skin reaction were

signicantly more common in the 13cRA arm
(P<0.0001).
Grade 2 or higher toxicities were also signicantly
higher in the 13cRA arm (P<0.0001).

* High risk of selection bias

* High risk of performance bias
* Low risk of detection bias
* Low risk of attrition bias
* Low risk of reporting bias

Nagao
2014

No toxicities due to the supplements were

identied.

* Low risk of selection bias

* Low risk of performance bias
* Low risk of detection bias
* Low risk attrition bias
* Low risk of reporting bias

*Company participation.
(No mention of conict of interest.)

Tsao
2009

The incidence of insomnia and nervousness was

higher in patients receiving GTE at 750 and
1,000 mg/m2.
No grade 4 or 5 adverse events.

* Low risk of selection bias

* Low risk of performance bias
* Unclear risk of detection bias
* Low risk of attrition bias
* High risk of reporting bias

* Company funded trial.

* Lack of description of tobacco use during
the study.

Papadimitrakopoulou
2008

Some adverse events were observed (no grade

4 toxicities) including grade 3 of ischemic
cerebrovascular incidence.

* High risk of selection bias

* Low risk of performance bias
* Unclear risk of detection bias
* Low risk of attrition bias
* Low risk of reporting bias

Armstrong
2013

No signicant adverse events related to

administration.

* Low risk of selection bias

* Low risk of performance bias
* Low risk of detection bias
* Unclear risk of attrition bias
* High risk of reporting bias

*Company participation in the beginning

of the study.
*Lack of description of tobacco use during
the study.

Mallery
2014

No deleterious eects were observed in either

BRB or placebo gel.

* High risk of selection bias

* Low risk of performance bias
* Unclear risk of detection bias
* Low risk of attrition bias
* Low risk of reporting bias

* Company participation and one author

had ownership interest (including patency).

Sun
2010

Drug toxicity was not monitored.

* High risk of selection bias

* High risk of performance bias
* Unclear risk of detection bias
* Unclear risk of attrition bias
* Low risk of reporting bias

* Lack of description of tobacco use

during the study.
* ZengShengPing is a registered
trademark, but authors did not
show conict of interest statement.

13cRA: 13-cis retinoic acid; GTE: green tea extract; BRB: black raspberry
108F E B R U A R Y 2 01 6

Other bias

C D A J O U R N A L , V O L 4 4 , N 2

to examine the incidence of oral cancer

in patients who received the HPV
vaccine. Unfortunately, none of the
studies presented in this review had
HPV vaccines as one of the variables.
Limitations (Risk of Bias) and
Intervention of Pharmaceutical Companies.
Risk of bias was evaluated for each
mentioned study. These biases
included selection bias, performance
bias, detection bias, attrition bias and
reporting bias. Although most of them
had no serious problems with regard to
risk of bias, some studies showed selection
bias, in regard to bias of allocation
and concealment of participants and
six of nine studies did not mention
the method of randomization.
TA B L E 3 illustrates companysponsored clinical trials for
commercialized chemopreventive
agents and agents under production.
In general, companies provide the
pharmaceutical products, so it is
imperative for these studies to show
conflict of interest and independent
third-party assessments for the
purpose of publication bias.69

Drug Delivery System

Drug delivery systems for
chemopreventive agents have recently
evolved as an important factor in
chemoprevention success. The
experiment using solid lipid nanoparticles
encapsulated curcumin and aspirin for
chemoprevention of pancreatic cancer
in a carcinogen-induced hamster model
is recognized as the new starting point
for cancer chemoprevention.70-72 This
administration method changed not
only the property of absorption and
metabolism of agents, but also the side
effects due to dose reduction. The limited
efficacy of several chemopreventive
agents in preclinical and clinical
studies is attributed largely to their

low bioavailability, which results in

subtherapeutic concentrations at the
target site. To overcome the bioavailability
issues, advanced drug delivery systems,
designed to provide localized or targeted
delivery of these agents, may represent
a more viable therapeutic option.
Nanoparticles has emerged as one of
the viable delivery systems but caution
should be taken when using large amounts
of these particles as neurotoxicity
induced by solid lipid nanoparticles
due to their lipophilicity, may result in
crossing of the blood-brain barrier.73

Drug delivery systems for

chemopreventive agents
have recently evolved as
an important factor in
chemoprevention success.

Among nine studies summarized

in TA B L E 2 , the study by Mallery et
al.23 is the only clinical trial that
used local application as opposed
to oral administration. Based on
the characteristics of each agent, it
is important to consider the agent
concentration in tissue and duration
of agents action when choosing an
administration route. For patients
with OPL, topical application has
an advantage in that patients can
apply the agent directly. However,
some agents have a bad taste and
unpleasant stickiness, so patients
deliberately forget to apply them to
avoid the unpleasant taste. Still some
studies consider topical application
as a better alternative due to the
direct effect on the oral mucosa.74

Side Eects and Adverse Eects

It is well documented that not all
OPL lesions progress to oral squamous cell
carcinoma. Therefore, the justification to
use chemopreventive agents in all patients
with OPL is controversial, as these
treatments will be unnecessary for more
than half of the affected patients. This
is the reason that all chemopreventive
agents should have minimal side effects
and adverse reactions. One of the most
severe side effects was reported by
Papadimitrakopoulou et al. using COX-2
inhibitor as chemopreventive agents in
which they observed cerebral infarction
during the clinical study (TA BLE 3 ).19,27
They also mentioned that subsequent
data from other studies indicated adverse
cardiovascular effects of the same agent,
which raised the possibility that it was
drug related.19 In addition, EGFRtargeted
therapy has been shown to include several
side effects, such as severe acneiform
exanthema or seborrheic dermatitis,
and interstitial pneumonia.49 This is
why most investigators prefer the use
of well-tolerated natural compounds
in chemopreventive agents.76

Best Approach for Designing Clinical

Trials, Systematic Review and Clinical
Practice Guidelines
In designing clinical practice
guidelines, systematic review along with
documentation of undesirable side effects,
gender, adverse reactions, costs and
patient preference should be considered.
Age should also be considered as well,
because the administration period and
desirable control period of malignant
transformation differ in elderly people who
may have difficulty in undergoing surgical
intervention than in middle-aged people
who have OPL with wide distribution or
multiple occurrences. It is also important to
prepare the answers for patients inquiries
such as Will I continue to take this
F E B R U A R Y 2 0 1 6 109

chemoprevention
C D A J O U R N A L , V O L 4 4 , N 2

agent for the rest of my life? In addition,

food culture and luxury items including
tobacco and alcohol vary according to
each country, so the interpretation of
chemopreventive agents in OPL should
be analyzed differently for each country.

Conclusions and Future Developments

Quality of life is an important
parameter in treatment design, and
maintenance of oral function is considered
as a major factor in living a healthy life.
Developing the ideal drug is, of course, the
holy grail of chemoprevention. It has long
been presumed that a chemopreventive
agent must have a tolerable side effect
profile in order to be feasible for use in an
otherwise healthy premalignant population.
The presence of chemopreventive agents
as a treatment option could play a major
role in the maintenance of oral function
and sustaining a good quality of life.
We have focused on clinical studies and
discussed the different pros and cons of
chemopreventive agents. Unfortunately,
there is no scientific evidence at this time
to support the use of chemopreventive
agents as a standard of care in treating
patients with OPL. At present, the
consensus for any OPL management is
surgery and long term follow-up. Although
progress toward chemoprevention of oral
cancer has been made, this field is still
in its earliest stages of development and
remains investigational. It is also time to
capitalize on prevention strategies already
poised for execution, such as HPV targeting
and tobacco cessation. The rapidly
evolving understanding of the molecular
mechanism of oral carcinogenesis offers
new promises for innovation in developing
chemopreventive agents that are safe and
effective. The ultimate goal is to develop
chemopreventive agents that can be
easily given to the population at high risk
for oral cancer, as fluoridated drinking
water is used to prevent dental caries.
110F E B R U A R Y 2 01 6

ACKNOWLEDGMENT

The authors would like to acknowledge Mr. Michael Gordon

for his editorial contribution.
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doi:10.1111/odi.12269.
66. Friedman JM, Stavas MJ, Cmelak AJ. Clinical and scientic
impact of human papillomavirus on head and neck cancer.
World J Clin Oncol 2014; 10:781-791.
67. Beachler DC, Abraham AG, Silverberg MJ, et al. Incidence
and risk factors of PV-related and HPV-unrelated Head and
Neck Squamous Cell Carcinoma in HIV-infected individuals.
Oral Oncol 2014; 50:1169-1176.
68. Zaravinos A. An updated overview of HPV-associated
head and neck carcinomas. Oncotarget 2014; 30:39563969.
69. Mulshine JL, Ondrey FG. Not signicant but important.
Cancer Prev Res 2013; 6:371-374.
70. Grandhi BK, Thakkar A, Wang J, et al. A novel
combinatorial nanotechnology-based oral chemopreventive

regimen demonstrates signicant suppression of pancreatic

cancer neoplastic lesions. Cancer Prev Res (Phila) 2013;
6:1015-1025.
71. Yang CS, Wang H, Hu B. Combination of chemopreventive
agents in nanoparticles for cancer prevention. Cancer Prev Res
(Phila) 2013; 61011-1014.
72. Sharma D, Sukumar S. Big punches come in nanosizes for
chemoprevention. Cancer Prev Res (Phila) 2013; 6:10071010.
73. Helson L. Curcumin (diferuloylmethane) delivery methods:
A review. Biofactors 2013; 39:21-26.
74. Brown RS, Edwards D, Walsh-Chocolaad T, et al. Topical
tacrolimus with custom trays in the treatment of severe oral
chronic graft-versus-host disease refractory to a potent topical
steroid therapy: A case report. Oral Surg Oral Med Oral
Pathol Oral Radiol 2013; 115:e26-e30.
75. Iqbal Z, Jain N, Jain GK, et al. Dental therapeutic systems.
Recent Pat Deliv Formul 2008; 2:58-67.
76. Saba NF, Haigentz M Jr, Vermorken JB, et al. Prevention
of head and neck squamous cell carcinoma: Removing the
chemo from chemoprevention. Oral Oncol 2015; 51:112118.
THE CORRESPONDING AUTHOR, Diana V. Messadi, DDS, MMSc,
DMSc, can be reached at dmessadi@dentistry.ucla.edu.

F E B R U A R Y 2 0 1 6 111

stem cells
C D A J O U R N A L , V O L 4 4 , N 2

Targeting Cancer Stem

Cells in Oral Cancer
Qilin Xu, MD, PhD, and Anh D. Le, DDS, PhD

A B S T R A C T The concept of tumor development driven by a unique subpopulation

of cancer stem cells (CSCs), or the CSCs hypothesis, may help to explain the high
mortality, low response to treatment and tendency of developing multiple tumors in
oral cancer. We will review current knowledge of the CSCs hypothesis in oral cancer
and the traits displayed by CSCs, focusing on the resistance to therapy and attempts
being made to treat oral cancer by specifically targeting CSCs.

AUTHORS
Qilin Xu, MD, PhD, is a
clinician scientist who
studies stem cells and
cancer. She has extensive
expertise in tumor
microenvironment, cancer
metastasis and cancer stem
cells. Dr. Xu currently
directs independent
research projects in the oral
and maxillofacial surgery
research lab at the
University of Pennsylvania
School of Dentistry.
Conict of Interest
Disclosure: None reported.

112F E B R U A R Y 2 01 6

Anh D. Le, DDS, PhD, is

the Norman Vine professor
and chair of the
department of oral and
maxillofacial surgery at the
University of Pennsylvania
School of Dentistry. She is
a diplomate of the
American Board of Oral
and Maxillofacial Surgery
and fellow of the American
Association of Oral and
Maxillofacial Surgeons.
Conict of Interest
Disclosure: None reported.

ancer that forms in the

tissues of the oral cavity or
the oropharynx is referred
to as oral cancer, which is
among the 10 most common
cancers worldwide. It was estimated
that in 2014 there would be 45,780 new
cases affecting the oral cavity of both
sexes with an estimated death rate of
8,390 in the U.S. alone.1 Among all oral
mucosal origins, about 90 percent are
oral squamous cell carcinoma (OSCC).2
OSCC occurs most commonly on the soft
palate, roof or floor of the mouth, gingiva,
tongue and other areas of the oral cavity.
Despite advances in cancer treatment,
many patients still fail in therapy, resulting
in disease progression, recurrence and
reduced overall survival. Historically,
a lot of attention has been directed
toward understanding the genetic and
biochemical mechanisms that cause
resistance to chemo- and radiotherapies.

However, cancer is widely understood to

be a heterogeneous disease and there is
increasing awareness that intratumoral
heterogeneity contributes to treatment
failure and disease progression.3 It is
becoming increasingly clear that cancer
cells display features of normal tissue
organization, whereby cancer stem cells
(CSCs) can drive tumor growth. Like
other forms of cancer, oral cancer also
exhibits the property of heterogeneity
and majorly comprises three different
types of tumor cells, including bulk tumor
cells with a high degree of differentiation,
transit amplifying cells with maximum
proliferation and a small population of
cells with elevated plasticity (self-renewal
capacity) that may contribute to the
tumor diversity.4-6 The growth and spread
of oral cancer is driven by these so-called
cancer stem-like cells or tumor-initiating
cells (TICs), which are capable of longterm self-renewal and generation of the

C D A J O U R N A L , V O L 4 4 , N 2

The Stochastic Model

The Cancer Stem Cell Model

Normal cells

Cancer stem
cells (CSC)

Self-renewal

Major genetic/epigenetic event

Transient
amplifying cells

Clonal selection factors

Dierentiated tumor cells

FIGURE 1A . The stochastic model indicates a clonal evolution of cancer and

postulates that cancer cell phenotypes are primarily dened by intrinsic factors,
in particular, driver mutations.

FIGURE 1B. The cancer stem cell model postulates that cancer is organized in
a hierarchical structure that, at least in part, resembles that of the tissue of origin.
The tumorigenic potential is limited to the cancer stem cell subpopulation and its
cellular heterogeneity is a product of multipotent cancer stem cells.

FIGURES 1. Models of tumor heterogeneity.

phenotypically diverse populations of

tumor cells. Accumulating evidence has
implicated an important role of CSCs
in cancer progression, invasion, distant
metastasis and recurrence, the reasons
behind the high morbidity and mortality of
the majority of patients with oral cancer.7-9
In this narrative review, we will discuss
the CSCs hypothesis in oral cancer and
the traits displayed by CSCs. Specifically,
we will focus on the markers of CSCs and
how they are linked to metastasis and
resistance to chemo- and radiotherapies.
Finally, we will discuss the novel approach
for developing new strategies to overcome
therapeutic resistance through specific
elimination of CSCs in oral cancer.

CSCs Hypothesis in Oral Cancer

There are two models that account
for heterogeneity and inherent
differences in tumor-regenerating
capacity: the stochastic or clonal
evolution and the hierarchy or cancer
stem cell model (FIGURE 1 ).10-12
In the stochastic model, malignant
transformation arises from a random
mutation that can affect any cell. Cancers
evolve by a reiterative process of clonal
expansion of mutant cell progeny
with proliferative advantage, genetic
diversification and clonal selection due to
intrinsic (e.g., activated signaling pathways,
etc.) and extrinsic factors (e.g., hypoxia,
stress, drug treatment, etc.), and that

these states are, at least in part, reversible.

Intrinsic properties and extrinsic selection
pressure allow some mutant subclones with
survival advantages to become dominant
while others become extinct or remain
dormant (FIGURE 1A ).5,10,11,13 Based on
the stochastic model and the assumption
that the vast majority of tumor cells
are able to propagate and drive tumor
growth, the goal of cancer treatment has
traditionally been to kill all cancerous cells.
This theory has been challenged
recently by the CSCs hypothesis, which
proposes that, similar to the hematopoietic
system, only one cell type, the CSCs, can
initiate tumors.5 The CSC model posits that
some cancers are organized into a hierarchy
F E B R U A R Y 2 0 1 6 113

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C D A J O U R N A L , V O L 4 4 , N 2

of subpopulations of tumorigenic CSCs

and their nontumorigenic progeny. And
cellular heterogeneity of the cancer is a
product of multipotent CSCs (FIGURE 1B ).
CSCs exhibit self-renewal and multilineage
differentiation potentials, which are
achieved by symmetric and asymmetric
divisions, respectively. The asymmetric
division leads to a generation of transient
amplifying cells with more restricted lineage
potential, which eventually produces
the most differentiated progenies. Tumor
cells lacking stem cell properties, or
nontumorigenic cancer cells, will not be
able to initiate self-propagating tumors,
regardless of their differentiation status
or proliferative capacity. In these cases,
CSCs are thought to drive tumor growth
and disease progression perhaps because
of their intrinsic capabilities of metastasis
and resistance to therapies.14,15,16 However,
the cellular and molecular mechanisms
that govern the dynamic transition from
CSCs to transient amplifying cells and
vice versa are still poorly understood,
although some key embryonic and
developmental pathways, such as Hedgehog
or Wnt pathways that are active in
noncancerous stem cells, have also been
demonstrated to be active in CSCs.17
The paradigm of tumor heterogeneity
and CSCs hypothesis also apply to oral
cancer.8,18 The existence of CSCs in
head and neck squamous cell carcinoma
(HNSCC) was first described in 2007
by Prince et al., which were isolated and
identified according to the expression of the
cell surface marker CD44.19 A subsequent
study on OSCC reported that about 60
percent of cells were positive for stem cell
markers Oct-4, Nanog, CD133, CD117 and
ABCG2.20 Later on, other studies attempted
to isolate CSCs from oral squamous cell
carcinomas according to the expression
of distinct stem cell-related markers like
CD44, CD133, CD117, OCT-4 and
aldehyde dehydrogenase.21,22 The idea that
114F E B R U A R Y 2 01 6

CSCs are responsible for maintaining tumor

growth is also based on the findings that
CSCs transplanted into nonobese diabetic/
severe combined immunodeficiency (NOD/
SCID) mice not only displayed significant
self-renewal ability but also promoted
tumor growth through their aberrant
differentiation capacity, thus reproducing
the heterogeneity of the original tumor.19 In
vitro culture of purified CD133+ oral CSCs
in a serum-free culture condition generated
only about 60 percent CD133+ cells, also
indicating that CSC-formed sphere-like
bodies are still heterogeneous by nature.20

The identication and

formulation of new
methods for CSCs in oral
cancer remains a challenge
for targeting oral cancer.

Identication of CSCs
By definition, both CSCs and normal
tissue stem cells possess self-renewal capacity;
however, self-renewal is typically deregulated
in CSCs. For many cancers, CSCs represent
a distinct population that can be prospectively isolated from the remainder of the
tumor cells and can be shown to have
long-term clonal repopulation and selfrenewal capacity the defining features of a
CSC. CSCs are defined by their ability to:
Generate a xenograft that is
representative of the parent tumor.
Self-renew as demonstrated by
serial passages in a xenograft
assay at clonal cell doses.
Give rise to daughter cells that may
possess proliferative capacity but are
unable to establish or maintain the
tumor clone upon serial passages.23

The identification and formulation

of new methods for CSCs in oral cancer
remains a challenge for targeting oral
cancer. CSCs in OSCCs have been
defined by diverse methodologies using
cell lines, primary tumor specimens and
patient-derived xenografts (PDX).24
A number of assays, such as sphere
formation, side population (SP) by
Hoechst dye exclusion, Aldefluor and
the expression of different cell surface
markers (e.g., CD44, CD133) have been
used to identify, isolate and subsequently
characterize CSCs populations in
squamous cell carcinomas. Specific
markers like aldehyde dehydrogenase
1 (ALDH1), CD44 and Bmi-1 have
shown early promising results both in
CSCs detection and in guiding treatment
protocols. Long-surviving cells in the oral
mucosa can be identified through labeling
and flow cytometry. Interestingly, oral
CSCs populations show upregulation of
the stem cell-related genes such as Oct-4,
Nanog, Nestin, CK19, BMI-1, CD117
(c-kit), CD44 and CD133 with depressed
expression of genes associated with
differentiation.25 Moreover, the CD117
(c-kit), CD44 and CD133 are highly
expressed within the poorly differentiated
metastatic stages of oral cancer
patients, thus contributing to invasion,
malignancy and worse prognosis.21
Once the unique subpopulation of
tumor cells has been isolated, functional
xenotransplantation assay, the gold
standard for CSCs identification, is used to
assess the tumorigenicity and self-renewal
potential of putative CSCs populations in
vivo.25,26 Interestingly, the CD44+ CSC-like
oral cancer cells are more commonly found
to be less than 10 percent and have the
potential to form tumors when transplanted
into NOD/SCID immunocompromised
mice.19 These tumors are responsible for
a tumors cellular heterogeneity and can
be serially subcultured while maintaining

C D A J O U R N A L , V O L 4 4 , N 2

the properties of CSCs. In contrast, the

CD44 counterparts are incapable of
inducing tumor formation. In certain tumor
cases, a small subset of cancer cells that
are CD133+ possess higher clonogenicity,
invasiveness and increased in vivo
tumorigenicity as compared to CD133
counterparts. Meanwhile, these CD133+
CSC-like cells displayed significantly
increased resistance to conventional
chemotherapy both in vitro and in vivo.27

CSCs and Field Cancerization

OSCC has a high propensity for
local failure, which is attributed to
recurrence at the primary site or the
development of second primary tumors
(SPT). Field cancerization that refers to
the existence of transformed cells in areas
adjacent to the primary tumor has been
one of the probable reasons underlying
disease relapse. The concept of field
cancerization, coined by Slaughter in
1953, proposes that the normal tissue
adjacent to the tumor harbors certain
preneoplastic genetic fingerprints, which
can eventually lead to development
of local recurrence or second primary
tumors. Slaughter and his group based this
concept on the following observations:
Tumor-adjacent mucosa being
molecularly abnormal.
Multifocal areas of precancerous
changes develop due to a
prolonged and widespread
exposure to carcinogens.
Oral cancer often consists of
multiple independent lesions
that sometimes coalesce.
The formation of second
primary tumors and recurrences
can be explained by the
presence of residual abnormal
tissue after surgery.28,29
Even though the concept of the
CSCs potentially being responsible for
field cancerization in OSCC has not

been explored extensively, the detection

of CSC markers in the tumor-adjacent
normal mucosa has provided evidence
to support their possible role in field
cancerization. Recent studies revealed that
tumor-adjacent normal tissues showed an
increased expression of the CSC markers
ATR and ABCG1.30,31 Studies in rat oral
carcinogenesis models have also shown
expression of CSC-related markers,
OCT4 and SOX2, in the clinically
normal and dysplastic oral mucosa.32
These results were further validated by
the findings that these CSC-related

Recent studies revealed

that tumor-adjacent normal
tissues showed an increased
expression of the CSC
markers ATR and ABCG1.

markers were also highly expressed in

the clinically normal mucosa adjacent to
tumors and in the precancerous lesions
of oral cancer patients.32 Apart from the
possible role of CSCs in the development
of the neoplastic field associated with
oral carcinogenesis, the expression of
these markers (OCT4 and SOX2) in the
normal tissues adjacent to tumors also
suggests a de-differentiation-mediated
origin of these CSCs. Taken together,
establishment and experimental validation
of this concept will provide new insights
into the mechanism of loco-regional
recurrence and development of SPTs.
Identification of CSC-specific molecules
that drive the filed cancerization process
may be useful for early diagnosis, prognosis
and development of novel targets for
the prevention and treatment of SPTs.

CSCs and Metastasis

Mortality in oral cancer often results
from metastasis to regional lymph
nodes and distant organs, and CSCs
may be critical players of metastasis.
Because CSCs have been isolated from
primary tumors as well as metastatic
sites, an important question is whether
there are distinct subsets of CSCs that
are either stationary or migratory.33 If
these are two distinct pools, it will be
necessary to therapeutically target both.
Preliminary work in HNSCC cell lines
shows that the highly metastatic cell
lines have almost 20 times more SP cells
as compared to their weakly metastatic
parental cell line.34 However, additional
work is required to assess the in vivo
metastatic ability of these populations and
answer the questions mentioned above.
These SP populations did show stemlike characteristics of chemoresistance,
increased in vitro invasiveness and an
activation of signaling pathways that
are involved in stem cell self-renewal,
as compared to non-SP cells.34
Epithelial-mesenchymal transition
(EMT) is a developmental program
that, when activated in cancer cells,
increases invasiveness and motility.35
Given the importance of EMT in
facilitating dissemination of cancer
cells, a relevant question appears as to
what is the contribution of EMT to the
process of self-renewal of metastatic
CSCs. The answer to this important
question comes from the landmark
work by Mani and colleagues, who
showed that the induction of the EMT
program in immortalized mammary
epithelial cells caused them to acquire
stem-like properties in addition to the
mesenchymal phenotype.16 Subsequently,
their work cemented this connection
by showing the EMT promoter ZEB1
also promotes stemness by repressing the
miRNAs that target stemness factors.36
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C D A J O U R N A L , V O L 4 4 , N 2

The connection between EMT and

self-renewal has also been explored in oral
cancer. CSCs isolated from tumors of oral
cancer patients have features consistent
with EMT, namely loss of expression of
epithelial markers like E-cadherin and
acquisition of mesenchymal markers
such as Vimentin and Snail.37 Snail is a
master regulator of EMT and controls
invasiveness and metastasis in many
cancers.38 The stem-like properties of these
CSCs are dependent on Snail expression,
suggesting a causal link between EMT
and stemness.37,39 In a recent study, it was
proven that the EMT regulator, Twist,
directly regulates the stemness factor Bmi1
in head and neck cancer.40 These studies
further demonstrate the link between EMT
and stemness in head and neck cancer.

HPV and Its Role in CSCs

Human papillomavirus (HPV), a
sexually transmitted infection, has been
identified as an etiologic agent for an
overwhelming majority of OSCCs in
the U.S.41 With the decline of tobacco
use in the U.S., smoking-related (HPVnegative) oropharyngeal malignancy
cases have decreased.42 The proportion
of oropharyngeal cancers attributable to
HPV (HPV-positive OSCC) has risen
substantially in the U.S. Indeed, while
only 16 percent of OSCCs in the 1980s
were HPV-positive, approximately 73
percent of tumors in the 2000s were
HPV-positive.42 Patients with HPV-OSCC
have significantly better prognosis than
those with HPV-negative OSCC.43-45
A significant carcinogenesis has been
associated with high-risk HPV 16 and
18 infections to oral epithelium. During
skin abrasions, infection occurs with
basal stem cells, which is the only site
of HPV infection, in the host through a
disturbed epithelial wall.46 After infection,
the viral DNA is retained in basal stem
cells. These HPV-positive stem cells then
116F E B R U A R Y 2 01 6

divide again and finally differentiate to

become proliferating cells in order to form
the epithelium. Studies showed that the
HPV-positive cells can express oncogenic
proteins E6 and E7, which target important
proteins including p53, pRb, Notch-1,
p16, cyclinD1 and EGFR.41,47 A positive
correlation has also been established
between HPV infection and carcinoma
of the tonsil and oral tongue cancer. HPV
initially produces a premalignant lesion
of heterogeneous cells with different
states of the viral genome and persistent
infection then progresses toward malignant

With the decline of tobacco

use in the U.S., smokingrelated (HPV-negative)
oropharyngeal malignancy
cases have decreased.

transformation. A study showed that

HPV E6 and E7 activated Wnt signaling
pathway in HPV16-positive oropharyngeal
squamous carcinoma cells and might be
involved in de-differentiation to CSCs
from oral cancer cells.48 Moreover, recently
a new HPV-positive UM-SCC-104
HNSCC cell line has been identified that
has a subpopulation of ALDH+ CSCs,
which is highly tumorigenic, that can
self-renew and has the capacity to recreate
tumor heterogeneity.49 However, it is not
clear whether the CSCs are originated
from infected epithelial stem cells that
have transformed to become CSCs or
from differentiated epithelial cells that
will subsequently give rise to new CSCs.
To date, the effects of HPV16 on
CSCs are not well understood. One
idea in the field was that patients with

HPV-positive HNSCC responded more

favorably to treatment than did patients
with HPV-negative HNSCC because
HPV-positive tumors might harbor fewer
CSCs. Nevertheless, in vivo limiting
cell dilution experiments revealed that
CSC frequency is greater in HPV-positive
tumors than in HPV-negative tumors.50
Indeed, data from a large cohort of patients
indicates that HPV16-positive HNSCC
is associated with increased ALDH1
staining in tumor cells.50 Therefore,
this study suggested that patients with
HPV-positive HNSCC have a better
prognosis and respond more favorably to
chemotherapy and radiation therapy than
do patients with HPV-negative HNSCC,
likely due to CSC phenotype or quality.51
In addition, CSCs from HPV-positive and
HPV-negative HNSCC cell lines are very
resistant to cisplatin therapy.52 Moreover,
in vivo studies indicated that cells on
the bulk of HPV-positive HNSCC might
respond better to platinum-based cancer
therapy.52 Further studies are warranted to
elucidate the relation of HPV infection
and CSC formation in HNSCC.

CSCs and Resistance to Therapy

As for most types of cancers,
conventional treatment for oral
cancer includes surgery, radiation and
chemotherapy, which can be used alone
or in combination. Chemotherapy, apart
from antibody treatment, remains the
only systemic treatment option that can
at anytime be combined with surgery and
radiation. Clinically, the use of radiation
and high-dose chemotherapy, in many
instances, results in good initial responses
of tumors, unless the dosage is limited
by co-morbidities. Unfortunately, the
frequent development of unresponsiveness
to further treatment raises the question
pertaining to the underlying causes.
Advances in CSC research may provide
some explanation to these phenomena.

C D A J O U R N A L , V O L 4 4 , N 2

Tumor
degenerate
Tumorigenesis

CSC-targeted
therapy

Tumor regression
Elimination of CSCs

Heterogeneous
tumor cells

Cancer stem cell (CSC)

Conventional
cancer therapy

CSCs lead to tumor regrowth/metastasis

Transient amplifying cell

Recurring tumor/
disseminated malignancy

Dierentiated tumor cell

FIGURE 2. Therapeutic implications of cancer stem cells. Cancer stem cells (red) self-renew and dierentiate within the tumors to form additional cancer stem cells as well as transient

amplifying cells (green) and dierentiated tumor cells (blue), which have limited proliferative potential. Conventional therapy that kills primarily nontumorigenic cancer cells can shrink
tumor size, but is not able to eliminate cancer stem cells (CSCs), which are the potential players in recurring tumor and metastasis. Targeting CSCs can lead to tumor regression.

In the past few years, studies have

begun to investigate the role of CSCs
in the therapeutic resistance of cancers.
In these studies, cell surface markers
were used to identify and purify CSCs
from tumors. These studies showed that
the fraction of CSCs is enriched in
tumor samples or cancer cell cultures
after treatment with radiation or
chemotherapeutic drugs, and it was
therefore proposed that CSCs are
particularly resistant to radiotherapy37,53
and chemotherapy.54,55,56 This resistance
could then possibly contribute to
treatment failure and, consequently,
CSCs could represent a novel target for
therapeutic treatment.57,58 The therapy
response and sensitivity of oral CSCs
is measured through sphere-forming
capacity, SP status and ALDH activity to
a variety of chemotherapeutic drugs and
radiations. For example, cells from tumor
spheres of esophageal cancer (EC) showed
greater resistance to cisplatin than to
EC cells grown under differentiating
conditions.54 Other studies have shown
therapy resistance in CSCs defined
by cell surface markers. A CD44high
subpopulation from oral cancer cells
showed increased viability and survival

relative to CD44low cells after treatment

with a panel of cytotoxic drugs including
5-FU, docetaxel, paclitaxel, cisplatin
and carboplatin as well as radiation.55
Cisplatin-resistant HNSCC cells express
high levels of stem cell surface markers
(CD133 and c-Kit) as well as stemness
markers Oct4, Nanog, Nestin and Bmi1.56
Likely, radiation resistance has been
shown to increase CSC subpopulations
in SCC cell lines and primary tumors.37,53
Most recently, a study showed that oral
CSCs undergoing EMT represent the
majority of invasive cells and are more
radioresistant than any other populations
in reconstructed 3-D tissues.59
Acquired therapy resistance in
CSCs can arise by different mechanisms
that confer survival advantage during
treatment. The DNA protection
and damage repair pathways lead to
momentous resistance to radiation and
chemotherapy, which has been shown
in glioblastoma.57,60 Another mechanism
of CSC radioresistance is increased free
radical scavenging and reduced levels of
reactive oxygen species (ROS), which has
been shown in breast cancer.61 Whether
radioresistance of oral cancers is mediated
through similar molecular mechanisms

is unknown, but it does hold promise for

novel therapeutic strategies. Oral CSCs
also express high levels of ATP binding
cassette (ABC) transporters that can
actively efflux drugs and shield them from
the adverse effects of chemotherapeutic
insult. For example, oral CSCs upregulate
different types of ABC transporters and
confer chemoresistance and the detail
mechanism needs to be explored.27,62,63
In addition to an increased drug efflux
capacity, CSCs also exhibit intrinsic
resistance to apoptosis and play an
important role in therapy resistance in
oral cancer. For example, the CD44high/
EGFRlow population exhibits an EMT
phenotype and resistance to radiotherapy,
cisplatin and EGFR-targeted therapies.64
Survivin, a member of the inhibitor
of apoptosis proteins (IAP) family,
has been identified as one of the most
crucial biomarkers in the recognition of
drug resistance. Knockdown of survivin
by siRNA could induce apoptosis and
enhance radiosensitivity in OSCC cells
in vitro and in vivo.65 Livin, one of the
most important members of the IAP
family, is associated with invasive and
oncogenic phenotypes such as tumor
cell invasion, tumor cell migration,
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C D A J O U R N A L , V O L 4 4 , N 2

tumor cell proliferation and resistance

to apoptosis in human OSCC cells.66
Recent studies showed that increased
expression of survivin in breast and
colon CSCs represent one of the major
factors involved in increased resistance to
apoptosis and chemotherapy.67,68 Future
studies are required to investigate whether
oral CSCs have an increased expression
of survivin that may also contribute
to their increased resistance to both
chemotherapy and radiation therapy.

Targeting CSCs in Current Cancer

Treatment
The increasing knowledge of the
existence and biology of CSCs has led
to the study of their specific elimination,
which can be of clinical benefit because
an eradication of the root of cancer
would stop tumor growth and ultimately
lead to tumor involution as bulk tumor
cells die off or succumb to therapy
(FIGURE 2 ). The characteristics of a
given CSC population for their marker
gene expression and their proliferative
state or drug resistance may support the
decision for certain treatment options.
As CSCs can be identified based on
their cell surface molecules, developing
specific antibodies/immunotoxins against
these antigens will be a useful way to
eradicate CSCs selectively. Akin to many
solid tumors, OSCCs are histologically
heterogeneous for the expression of the
CD44 marker. CD44v6 antibodies either
radiolabeled or coupled with a cytotoxic
drug entered phase I clinical testing in
head and neck cancer patients. However,
the use of CD44 as a specific marker to
identify CSCs in head and neck cancer
remains controversial. In a phase I doseescalation study, the treatment with a
radiolabeled antibody showed promising
antitumor effects.69 One patient, however,
developed toxic epidermal necrolysis
and died, indicating that perhaps anti118F E B R U A R Y 2 01 6

CD44v6 was not exclusively targeting the

CSCs. In later dose-escalation studies,
the antibody was coupled to mertansine,
a cytotoxic drug, but toxic effects were
severe,70,71 indicating that the drug may
not be selective enough for this approach.
This observation is experimentally
supported by immunohistologic studies
showing that the expression of CD44v6 is
not exclusively restricted to the tumor.72
Swaminathan and colleagues developed
polymeric nanoparticles (CD133NPs)
consisting of a monoclonal antibody
against CD133, paclitaxel (a conventional

These strategies have

resulted in various degrees
of improved prognosis and
survival but have not yet
displayed equal success
in regards to a cure.

chemo-agent) and a fluorescence probe.73

The effectiveness of CD133NPs as
anti-CSC agents was demonstrated in
a remarkable reduction in the ability of
mammosphere and colony formation as
compared to the untreated control or
paclitaxel-only treated cells. Also, in vivo
tumor growth was reduced significantly in
the CD133NP-treated animal groups.73
Several lines of evidence have
shown that different mechanisms can
protect CSCs from T-cell-mediated
immunosurveillance and NK-cellmediated cytotoxicity.74-76 Therefore,
immunotherapies specifically targeting
CSCs are very promising approaches to
overcome the therapeutic resistance.
CSCs can be eliminated selectively by
developing antitumor T-cell vaccines.
One potential target in HNSCC is the

recently described CD8 defined T-cell

epitope of ALDH177 or the development
of a CSC-dendritic cell vaccine.78 Success
of these potential therapies will depend
on how well immunological responses
to CSCs can be modulated, for example,
by vaccine adjuvants upregulating
antigen-processing and presentation.
An alternative immunotherapeutic
approach to target OSCC is the use
of monoclonal antibodies. Different
strategies have thus far entered the clinic:
Antibodies directed against
tumor surface antigens trigger
immune effector cells that
cause tumor cell death.
Antibodies that are conjugated
to cytotoxins or radiation
emitters causing cell damage
directly upon binding.
Antibodies blocking or inhibiting
cellular pathways after binding
to the respective receptor.
These strategies have resulted
in various degrees of improved
prognosis and survival but have not
yet displayed equal success in regards
to a cure. This variable success can be
explained by tumor immune-escape
(e.g., downregulation of the target)
and a heterogeneous expression of
the antibody targets in the tumor.
A number of studies investigating
the use of antibodies to target CSCs
of solid cancers are underway.79,80
Heterogeneous signals from tumor
microenvironments nurture cancer stem
cells.81 Pharmacological interruptions of
these paracrine-signaling systems thus
have therapeutic potential to eradicate
CSCs in the tumor bulk. Epidermal
growth factor receptor (EGFR) is
overexpressed in approximately 90
percent of tumors. Recent research
results implicated a possible role of
EGFR in regulating OSCC-CSCs.82
Several EGFR-targeted drugs are FDA

C D A J O U R N A L , V O L 4 4 , N 2

approved for clinical use, including the

antibodies cetuximab and panitumumab
and small molecule inhibitors erlotinib
and afatinib. A specific affinity of
these antibodies toward CSCs has
not been described in in vitro testing;
however, it has been revealed that the
EGFR-targeting monoclonal antibody,
cetuximab, offers clinical benefits for
patients with head and neck tumors.83
Moreover, other anti-EGFR therapies
based on tyrosine kinase inhibitors have
been designed to bind covalently and
irreversibly to their targets. Afatinib, an
oral small molecule ErbB family blocker
that irreversibly binds to ErbB1 (EGFR),
ErbB2 (HER2) and ErbB4 (HER4),84 is
being investigated in HNSCC treatment
with encouraging phase II results and
several ongoing phase III trials.
It is becoming increasingly clear that
the CSC population consists of not just
one particular CSC phenotype but a
plasticity of interchangeable states and a
variety of different clones. Therefore, it
should be taken into consideration that
even though certain drugs can eradicate
a certain cohort of cancer stem-like cells,
a variety of CSC clones with different
properties within the same tumor as well
as from different patients with the same
cancer type may not be treatable with the
same compound. Therefore, considerable
attention has to be paid to the selection
of drug compounds, or a combination,
to effectively target the CSC pool,
including quiescent cells, which appear
to be able to escape most treatments.
A deeper understanding of molecular
mechanisms governing the biology
of tumorigenic cells should also be
considered a priority for optimal
development of innovative antiCSC drugs. It is conceivable that
pharmacological inhibition of paracrineacting pathways results in cytostatic
effects correlated with the disruption

of the tumor-microenvironment
interplay. Taking into account the
complex modalities of activation of these
pathways, the identification of predictive
biomarkers remains a challenge, ideally
requiring biomarker measurement within
a microenvironmental context. It is also
worth considering that the therapeutic
potential of self-renewal pathway
antagonists could be counterbalanced by
potential serious adverse events because
of interference with crucial mechanisms
of tissue homeostasis. Therefore, a
deeper understanding of adult stem
cell biology is required to determine a
therapeutic window for anti-CSC agents.

Conclusion
The cancer stem cells hypothesis
opens up a wide field for future research
and further validation is needed to obtain
better understanding. An important
aspect is the reliability of identifying and
characterizing CSCs based on the cell
surface markers. This would offer a more
precise knowledge of the type of cells that
generate a tumor, their tissue distribution,
the relationships with their progenies
and the implications of their proliferative
activity and invasive capacity for
the prognosis of cancer patients. A
greater understanding of the biological
properties of CSCs could also lead to
the development of novel antitumor
drugs that can specifically target these
cells. The optimal therapy should aim at
targeting not only those cells undergoing
differentiation (bulk tumor population)
but also those resistant CSCs and
concomitantly destroying the CSC niche
for the survival. Thus, it is hoped that
this novel strategy will result in a rapid
exclusion of all tumor cell subpopulations
and avoid the possible repopulation of
the tumor mass by tumor-initiating cells
or by originally differentiated tumor cells
that have regained renewal activity.

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THE CORRESPONDING AUTHOR, Anh D. Le, DDS, PhD, can be reached

at anhle@dental.upenn.edu.

nanodiamonds
C D A J O U R N A L , V O L 4 4 , N 2

A Chemopreventive
Nanodiamond Platform
for Oral Cancer Treatment
Albert Yen, BS; Kangyi Zhang, PhD; Giulia Daneshgaran, BS; Ho-Joong Kim, PhD;
and Dean Ho, PhD

A B S T R A C T Standard oral cancer therapy generally includes a combination of

surgery with chemotherapy and/or radiotherapy. This treatment paradigm has not
changed in some time. In this paper, we propose a chemopreventive nanodiamond
platform for the delivery of celecoxib (Celebrex) to oral cancer lesions. This
innovative platform allows for sustained drug release under physiological conditions,
potentially enhancing chemopreventive efficacy of celecoxib without the physical
and toxicological damage associated with conventional means of drug delivery.

AUTHORS
Albert Yen, BS, is in the
department of
bioengineering, Henry
Samueli School of
Engineering and Applied
Science at the University of
California, Los Angeles.
Conict of Interest
Disclosure: None reported.
Kangyi Zhang, PhD, is in
the division of oral biology
and medicine at the
University of California, Los
Angeles, School of
Dentistry.
Conict of Interest
Disclosure: None reported.
Giulia Daneshgaran,
BS, is in the department of
integrative biology and
physiology at the University
of California, Los Angeles.
Conict of Interest
Disclosure: None reported.

Ho-Joong Kim, PhD, is in

the department of
chemistry at Chosun
University in Gwangju,
Republic of Korea.
Conict of Interest
Disclosure: None reported.
Dean Ho, PhD, is a
professor in the division of
oral biology and medicine
and department of
bioengineering and is
co-director of the Jane and
Jerry Weintraub Center for
Reconstructive
Biotechnology at the
University of California, Los
Angeles.
Conict of Interest
Disclosure: None reported.

pproximately 45,780
Americans were diagnosed
with oral cancer in 2015.1
Treating oral cancer usually
requires surgical resection
with postoperative chemotherapy and/
or radiotherapy.2,3 Although the fiveyear survival rate from 2004 to 2010
for oral cancer patients was 66 percent,
modern treatments for oral cancer
are not without obstacles.1 Surgery is
physically traumatic, and radiotherapy
often leads to adverse physiological
effects, including infection and oral
mucositis.3 Chemotherapy subjects
patients to drug toxicity, and the
effectiveness of chemotherapy is often
hampered by poor drug bioavailability,
drug resistance and limited cellular
uptake. Furthermore, close to onethird of oral cancer patients undergo
a disease relapse after treatment.2

Recent advances in nanomedicine

have driven the development of
novel treatment modalities for a
multitude of diseases, including oral
cancer. Among these advances are
drug-loaded nanoparticles designed to
mitigate the shortcomings associated
with chemotherapy. Though such
nanoparticles come in all sizes, shapes
and compositions, their overarching
purpose is the same: sustained and
targeted delivery of therapeutic agents
to a tumor site.4 By modulating their
material properties, nanoparticles can
be designed to release their therapeutic
payload over a long period of time,
eliminating toxicity associated with bolus
injections.4 Nanoparticle drug carriers
are also passively targeted to tumors
because of the enhanced permeability
and retention (EPR) effect, augmenting
treatment specificity and improving
F E B R U A R Y 2 0 1 6 121

nanodiamonds
C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 1. A transmission emission

micrograph (TEM) of a single

nanodiamond reveals a highly
ordered diamond core and a faceted
diamond surface. A fast Fourier
transform of the TEM is contained in
the inset. This fast Fourier transform
further highlights the ordered
diamond core of the nanodiamond.
(Reprinted with permission from the
American Chemical Society, 2011.)

therapeutic efficacy.4 Functionalization

of nanoparticles with targeting moieties
can further enhance this specifity.4
One such nanoparticle drug carrier
is the nanodiamond (ND), a truncated
octahedral nanocarbon that is 4-5 nm in
diameter (FIGURE 1 ).5-7 NDs are usually
generated as a byproduct of industrial
detonation processes involving carbonbased materials.6,7 Because NDs are
a waste byproduct of these industrial
processes, ND synthesis is highly scalable.
The surface composition of the ND
is also variable and easily modified,
serving as a foundation for electrostatic
adsorption or chemical conjugation of
small-molecule drugs, genes, targeting
motifs and other therapeutic compounds
(FIGURE 2 ).6-15 Anthracylines are one
class of drug that bind potently but
reversibly to NDs through electrostatic
interactions and pi stacking, allowing
for the synthesis of ND-anthracycline
complexes that release their payload over
time.11-13 Because NDs are endocytosed
into cells via macropinocytosis and
clathrin-mediated endocytosis, any
attached therapeutic payload will readily
enter a target cell along with its ND
122F E B R U A R Y 2 01 6

FIGURE 2 . Carboxylated nanodiamond (highlighted in green) serves as a common foundation for a

wide variety of nanodiamond surface modications. These surface modications facilitate the adsorption
or chemical conjugation of a broad range of molecular therapeutics. (Reprinted with permission from
Nature Publishing Group, 2012.)

carrier.10,16 NDs also contain nitrogen

vacancy (NV) centers, which occur
as natural defects or are introduced by
chemical modification.17,18 These NV
centers imbue NDs with fluorescent
properties, making NDs not only a drug
carrier but also a biological probe.17,18
Although the scalability and versatility
of NDs render them a suitable candidate
for various clinical applications, the
most critical indicator of NDs clinical
potential is their biocompatibility. A
series of in vitro and in vivo studies have
demonstrated the biocompatibility of NDs
in their functional form, be it that of an
imaging agent or a drug carrier.12,19,20 Both
fluorescent and detonation NDs were
proven to be nontoxic toward HepG2
and HeLa cell lines.20 Furthermore,
an in vivo study conducted on grafted
chemoresistant mammary mice tumors
showed that mice could tolerate a
lethal dose of doxorubicin delivered
by NDs, and that NDs alone induced
no systemic inflammatory response.12
Taken together, the advantageous
therapeutic and diagnostic properties
of NDs motivate the development
of an ND platform for drug delivery

to oral cancer lesions. One drug that

could benefit from the implementation
of this platform is celecoxib (Cxb), a
commonly prescribed nonsteroidal antiinflammatory drug. Cxb is a selective
inhibitor of cyclooxygenase-2 (COX-2),
an enzyme implicated in tumorigenesis
and cancer progression.21,22 COX-2 has
been found to be overexpressed in oral
squamous cell carcinomas (OSCCs) and
is a promising target for chemopreventive
Cxb therapies.23-26 However, a randomized
phase II clinical trial of Cxb in patients
with oral premalignant lesions returned
inconclusive results, as only a small
fraction of patients had a partial or
complete response to Cxb treatment.27
In this paper, ND-polyethyleneiminepoly(ethylene glycol) conjugate
(ND-PEI-PEG) was synthesized as
a proof-of-concept drug carrier for
Cxb (FIGURE 3A ). This ND-polymer
conjugate sequestered and released Cxb
in a sustained, pH-dependent manner.
Arginylglyclaspartic acid (RGD), a
tripeptide that binds preferentially
to integrins often overexpressed on
OSCCs, can also be conjugated to NDPEI-PEG to create an actively targeted

C D A J O U R N A L , V O L 4 4 , N 2

ND-Cxb with RGD targeting

functionality
Carboxylated ND

Polyethyleneimine
800

Poly(ethylene
glycol) Diacid 600

Neutral
charge

Positive
charge

Arginylglycylaspartic
acid

Celecoxib

Hydrodynamic size (nm) and -potential (mV)

FIGURE 3A .
FIGURES 3. Carboxylated nanodiamond (NDCOOH) (3A ) is functionalized with polyethyleneimine

250
200
150
100
50
0
Hydrodynamic size (nm)
-potential (mV)

-50
ND-COOH

ND-PEI

ND-PEI-PEG

ND-PEI-PEGRGD

ND-Cxb
(no RGD)

ND-Cxb
(with RGD)

800 (PEI) and poly(ethylene glycol) 600 diacid

(PEG) to generate a ND-polymer conjugate, ND-PEIPEG. The polymer network formed by PEI and PEG
sequesters Cxb. The conjugation of targeting motif
arginylglyclaspartic acid (RGD) to PEG is not necessary
for Cxb loading, but grants active targeting functionality
to the ND-Cxb complex. The average hydrodynamic
size (shown in blue) (3B ) increases upon conjugation of
each polymer component (n 3, error bars = standard
deviation). RGD functionalization and Cxb incorporation
further increases the average hydrodynamic size.
The average -potential also changes upon polymer
functionalization and celecoxib loading, suggesting that
the outermost chemical groups are replaced and/or
modied throughout ND-Cxb synthesis (n 3, error bars
= standard deviation).

FIGURE 3B .

variant, ND-PEI-PEG-RGD, without

compromising Cxb loading and release.28,29
This multimodal ND-Cxb platform has
the potential to enhance cellular uptake
of drug while prolonging therapeutic
release, enhancing the chemopreventive
effects of Cxb without compromising
the drugs therapeutic window.

Materials and Methods

Synthesis of ND-PEI-PEG. All
chemical reagents were purchased
from Sigma-Aldrich (St. Louis). ND
hydrogel was purchased from Nanocarbon
Institute Co. Ltd. (Nagano, Japan) and
lyophilized to obtain stock ND powder.
Carboxylated ND (ND-COOH) was

generated from stock ND after heattreating at 475 degrees Celsius for three
hours and resuspended in deionized
water. Coupling reagents 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide
(EDC) and N-hydroxysuccinimide
(NHS) were used to facilitate a
series of two consecutive EDC/NHS
coupling reactions, performed in
water at pH 5.5-6.0: conjugation of
polyethyleneimine 800 (PEI) to carboxyl
groups on the surface of ND-COOH
and conjugation of poly(ethylene
glycol) diacid 600 (PEG) to terminal
amine groups on PEI. A water wash
was performed between each coupling
reaction to remove excess reagents.

RGD Functionalization for Active

Targeting. To confer active targeting
functionality to ND-PEI-PEG, an
additional EDC/NHS coupling reaction
was used to conjugate RGD (SigmaAldrich, St. Louis) onto the terminal
carboxylic groups of PEG. The reaction was
performed in water at pH 5.5-6.0. A water
wash was performed after the coupling
reaction to remove excess reagents.
Qualitative Model of Drug Loading and
Release. Fluorescein diacetate (SigmaAldrich, St. Louis) was solvated in
acetonitrile (Sigma-Aldrich, St. Louis)
at a concentration of 1 mg/mL. The
fluorescein diacetate solution was then
added to an equal volume of 10 mg/mL
F E B R U A R Y 2 0 1 6 123

nanodiamonds
C D A J O U R N A L , V O L 4 4 , N 2

FIGURE 4 . Successful incorporation of

uorescein diacetate (FDA) with ND-PEI-PEGRGD (4A ) is validated by monitoring FDA

release under pH 4 (left) and pH 7.5 (right)
conditions. Because FDA uoresces when
excited by ultraviolet (UV) light, the presence
of a uorescent signature against a UV light
background conrms the loading and release
of FDA. After 24 hours, the uorescent signature
of the pH 4 sample is greater than that of the
pH 7.5 sample, suggesting a pH-dependent
release mechanism. FDA is also successfully
incorporated into ND-PEI-PEG (no RGD) (4B ).
After 30 days, the uorescent signatures of
the pH 4 sample (left) and pH 7.5 sample
(right) are indistinguishable, indicating that the
entire payload is gradually released over an
extended period of time, regardless of pH.

Celecoxib concentration (g/mL)

50
45
40
35
30

25
20
15

10
5
0
0

pH5
pH1

200

400

600

800
1000
Time (minutes)

1200

1400

1600

FIGURE 5A .

Neutral charge

Positive charge

Celecoxib

FIGURE 5B .

ND-PEI-PEG (or ND-PEI-PEG-RGD).

The mixture was probe sonicated for
30 minutes and spun down at 18,000 g.
The precipitate was then resuspended in
aqueous buffer solutions of pH 4 and pH
7.5. Fluorescein diacetate release under
differing pH conditions was observed for
30 days by illuminating the pH 4 and pH
7.5 solutions under ultraviolet light.
Loading of Celecoxib Onto Nanodiamond
Carriers. Cxb (Sigma-Aldrich, St. Louis)
and ND-PEI-PEG (or ND-PEI-PEGRGD) was solvated in dimethyl sulfoxide
(DMSO) at a 1:5 mass ratio of Cxb to
ND carrier. The DMSO suspension was
added drop-wise to twice the volume of
deionized water. The resultant mixture
was shaken on a rotator for one hour and
then centrifuged for 20 minutes at 14,000
rpm. After removing the supernatant,
the ND-Cxb precipitate was probe
sonicated in 1 mL of DMSO at high
amplitude to dissociate the ND-Cxb
complex, releasing Cxb into the DMSO.
124F E B R U A R Y 2 01 6

FIGURES 5 . Cxb release from ND-Cxb complexes (no RGD) (5A ) is measured over a period of 24 hours (1,440
minutes) under pH 1 (red) and pH 5 (blue) conditions. A greater initial burst release is observed under pH 1 conditions,
but average Cxb concentrations gradually decrease to similar baseline levels (n = 4, error bars = standard deviation). The
high pKa of the amine groups on PEI (5B ) may lead to preferential celecoxib release from the ND-Cxb at low pH, possibly
triggering the proton sponge eect. Under acidic conditions like those found in the cell endosome, the amine groups
on PEI are protonated, dissociating the ND-Cxb complex due to electrostatic repulsion. The dissociation of the ND-Cxb
complex subsequently releases Cxb into the endosome. The PEI also acts as a proton sponge, removing protons from the
endosome and generating a negative proton gradient. This negative proton gradient could initiate an inux of protons (and
therefore intracellular medium) from the intracellular space, rupturing the endosome and releasing Cxb into the cell.

The ND carrier was separated from

the DMSO solution by centrifuging for
20 minutes at 14,000 rpm and a UV-Vis
spectrophotometer was used to measure
the absorbance of the Cxb-containing
supernatant at 260 nm. The concentration
of loaded Cxb was determined by
converting the final supernatant
absorbance value to a concentration
value with a Cxb standard curve.
Characterization of ND-Cxb. Carrier
intermediates were solvated in deionized
water and the hydrodynamic size and
-potential measurements were taken

with a Zetasizer Nano-ZS (Malvern

Instruments, Worcestershire, United
Kingdom). The hydrodynamic size and
-potential of loaded ND-Cxb complexes
were also measured in this manner.
Quantitative Evaluation of Celecoxib
Release From ND-Cxb. Cxb was loaded
onto ND-PEI-PEG as previously
described. The ND-Cxb complexes were
resuspended in 1 mL of pH 1 aqueous
buffer solution and 1 mL of pH 5 aqueous
buffer solution (four replicates for each
pH). The ND-Cxb solutions were rotated
continuously over a period of 24 hours.

C D A J O U R N A L , V O L 4 4 , N 2

PBS
Dox 100 g
NDX 100 g
Dox 200 g
NDX 200 g

12

100
Survival probability (%)

Relative tumor volume (V/Vo)

9
6
3
0

9 12 15 18

40
20

6 12 18 24 30 36 42 48 54 60
Days after implantation

FIGURE 6A . A chemoresistant mammary tumor

strain was xenografted into the mammary glands of
female mice. The black arrows represent days on
which the mice were injected with therapy. NDX was
found to inhibit tumor growth in these mice to a greater
degree than doxorubicin treatment alone. (Reprinted
with permission from the American Association for the
Advancement of Science, 2011.)

FIGURE 6B . All xenografted mice succumbed to

cancer before the conclusion of the 60-day study when
treated with free doxorubicin. In fact, a double dose
of doxorubicin (200 g) led to a quicker death than
a standard dose of doxorubicin (100 g) due to drug
toxicity. In contrast, xenografted mice treated with NDX at
equivalent doxorubicin dosages of 100 g and 200 g
survived signicantly longer than their doxorubicin-treated
counterparts did. The otherwise lethal 200 g dosage
of doxorubicin was well tolerated by the mice when
delivered by ND, and all mice that received this double
dosage via ND delivery survived the entire course of
treatment. (Reprinted with permission from the American
Association for the Advancement of Science, 2011.)

Dox (Day 45)

ND-Dox (Day 70)

TUNEL

H&E (200x)

H&E

NDs (Day 28)

Treatment
Dox 100 g
Dox 200 g
NDX 100 g
NDX 200 g
PBS

60

Days after injections

(6 days after implantation)

CON (Day 25)

80

FIGURE 6C . The delivery of NDX to brain tumors with a convection-enhanced

delivery system was in a glioma mice model. Hematoxylin and eosin (H&E) stains (top
and middle row) of treated glioma tissue showed that NDX treatment led to signicant
tumor regression compared to doxorubicin, ND and PBS controls. The H&E stains were
validated with a TUNEL stain, which stains apoptotic cells green (bottom row). (Reprinted
with permission from Elsevier, 2014.)

At select time points over this 24-hour

period, the ND-Cxb suspensions were
spun down at 14,000 rpm for 20 minutes
and the aqueous supernatant was removed.
This aqueous supernatant was diluted
with 1 mL of dimethyl sulfoxide and a
UV-Vis spectrophotometer was used to
read the supernatant absorbance at 260
nm. Cxb concentrations at each time
point were determined by converting
absorbance values to concentration
values with a Cxb standard curve.

Results and Discussion

Synthesis and Characterization of
Nanodiamond Carrier. Preliminary
drug loading attempts indicated that
Cxb adsorbed poorly to the surface of
unmodified NDs and ND-COOH. In order
to improve Cxb sequestration while still
allowing for release of drug, ND-COOH is
polymer-functionalized through sequential
conjugation of PEI and PEG, generating
the ND-polymer conjugate, ND-PEI-PEG
(FIGURE 3A ). This PEI-PEG mesh serves
to entrap Cxb molecules and ensure drug
release after cellular uptake of ND-Cxb
via the PEI-induced proton sponge
effect.30 The terminal PEG moieties
on ND-PEI-PEG provide free carboxyl
groups for conjugation of targeting ligand
RGD, should active targeting be desired.
In addition, a drug loading approach
as described avoids the need for direct
conjugation of the drug to the ND
surface that can preclude drug elution.
Polymer conjugation to NDCOOH is monitored by measuring the
hydrodynamic size and -potential of
carrier intermediates in deionized water
after the conclusion of each coupling
reaction. There is a marked increase in
hydrodynamic size after the addition
of each polymer component, from an
average diameter of 49.11 26.54 nm
for ND-COOH to a final diameter of
129.2 53.9 nm for ND-PEI-PEG-RGD
F E B R U A R Y 2 0 1 6 125

nanodiamonds
C D A J O U R N A L , V O L 4 4 , N 2

Anti-EGFRNDLP-Epi

NDLP-Epi

Epi

PBS

Wk1 Wk2 Wk3 Wk4 Wk5 Wk6 Wk7

FIGURE 7. The ecacy of nanodiamond-lipid

hybrids loaded with epirubicin (NDLP-Epi) was

evaluated using a triple negative breast cancer mice
model. The ecacy of a targeted NDLP-Epi variant
functionalized with antibodies against epidermal
growth factor receptor (anti-EGFR-NDLP-Epi) was
also investigated in this study. Tumor growth was
visualized weekly by measuring the luciferase signal.
Mice treated with epirubicin alone succumbed
to drug toxicity after week three (second row),
while mice treated with a PBS control showed little
inhibition of tumor growth (rst row). Treatment with
untargeted NDLP-Epi slowed tumor growth relative
to the PBS control (third row), but near-complete
tumor regression was achieved with anti-EGFRNDLP-Epi treatment (bottom row). (Reprinted with
permission from Wiley-VCH, 2013.)

(FIGURE 3B ). An additional size increase

is observed upon Cxb incorporation,
indicating successful drug loading. The
-potential also fluctuates throughout the
synthesis procedure, reflecting changes
in the surface charge of the ND (FIGURE
3B ). For instance, ND-COOH exhibits a
negative -potential ( 46.70 6.83 mV)
due to the deprotonated carboxyl groups
on its surface. Upon PEI conjugation,
the -potential becomes positive (52.70
4.45 mV), indicating that the surface
carboxyl groups were replaced by
protonated amine groups found on PEI.
Cxb incorporation into either NDPEI-PEG or ND-PEI-PEG-RGD also
gives rise to a change in -potential.
Validation of Carrier Loading and Release
of Payload. To determine whether the
completed ND carrier could sequester
hydrophobic molecules like Cxb, NDPEI-PEG (or ND-PEI-PEG-RGD) was
126F E B R U A R Y 2 01 6

incorporated with fluorescein diacetate

(FDA), a hydrophobic fluorophore, by first
solvating FDA in acetonitrile and mixing
this water-miscible organic phase with an
aqueous phase containing the ND carrier.
Because of its hydrophobicity, FDA is
thought to preferentially complex with the
ND carrier upon mixing with the aqueous
phase, becoming embedded in the polymer
network on the surface of the ND carrier.
The ND carriers were subsequently
suspended in pH 4 and pH 7.5 buffer
solutions. The fluorescent signature of
the pH 4 buffer solution increased more
quickly than that of the pH 7.5 buffer
solution, indicating a pH-dependent
release mechanism that favors FDA
release at lower pH (FIGURE 4A ). After
30 days, the fluorescent signature of both
buffer solutions were identical, confirming
that FDA was loaded onto the ND carriers
and gradually released over a month-long
period, albeit, more quickly under acidic
conditions (FIGURE 4B ). Cxb loading was
achieved with a similar loading method.
pH-Dependent Celecoxib Release.
After loading ND-PEI-PEG with Cxb,
the resultant ND-Cxb complexes were
suspended in pH 1 and pH 5 buffer
solutions. Again, a pH-dependent
release mechanism is observed, with
greater initial burst release of Cxb
at pH 1 (FIGURE 5A ). After 1,440
minutes (24 hours), Cxb levels in both
pH 1 and pH 5 solutions decrease to
similar baseline concentrations.
The pH-dependent release mechanism
of both FDA and Cxb from the ND
carrier may be attributed to the high pKa
of amine groups found in PEI. Under
acidic conditions, the proton transfer
equilibrium shifts in favor of amine
protonation (NH2 to NH3+). The greater
number of positively charged species
in the PEI layer of ND-Cxb (or NDFDA) generates electrostatic repulsions
that dissociate the ND-Cxb complex

(FIGURE 5B ). Hence, payload release

is achieved under acidic conditions.
The preferential release of Cxb from
ND-Cxb under acidic conditions is
critical for its therapeutic efficacy. Upon
endocytosis by OSCCs, ND-Cxb will enter
the acidic cell endosome, triggering Cxb
release via protonation of amine groups
on PEI.31 The rapid protonation of these
amine groups removes protons from the
endosome, generating a negative proton
gradient that induces an influx of protons
from the intracellular space. This influx of
intracellular fluid lyses the endosome, thus
releasing Cxb into intracellular medium.
In this way, PEI is not only utilized as
a pH-sensitive trigger for Cxb release,
but it also acts as a proton sponge that
creates the proton gradient necessary for
endosomal rupture and subsequent release
of Cxb throughout the target cell.30

Therapeutic Outlook
Preclinical studies conducted with
similar ND-based delivery systems have
already returned promising results. The first
of these preclinical studies involved the
treatment of highly chemoresistant mice
mammary tumors with ND-doxorubicin
conjugates (NDX).12 NDX was able
to overcome drug efflux transporters
commonly found in chemoresistant tumor
strains, ensuring drug retention, whereas
free drug would be effluxed out of the cell.12
Mice treated with NDX were also able
to tolerate a double dose of doxorubicin,
showing significantly improved tumor
regression compared to a free doxorubicin
control (FIGURE 6A ).12 In fact, when
delivered in its free form, this double dose
of doxorubicin killed the mice before the
end of the treatment period (FIGURE 6B ).
The efficacy of NDX was also
studied in a mice model of glioblastoma
multiforme, an aggressive brain cancer
with high mortality rates.32 NDX and
free doxorubicin were delivered directly

C D A J O U R N A L , V O L 4 4 , N 2

to mice gliomas through a convectionenhanced delivery system. On average,

mice treated with NDX survived almost
20 days longer than mice treated with free
doxorubicin. NDX also induced greater
tumor regression and apoptotic cancer cell
death than free doxorubicin (FIGURE 6C ).32
Targeted variants of ND-drug
conjugates have also been implemented
successfully in preclinical mice models.
The efficacy of an epirubicin-loaded
ND-liposome hybrid (NDLP-Epi) was
compared to that of anti-EGFR-NDLPEpi, an NDLP variant functionalized
with antibodies targeted to epidermal
growth factor receptor (EGFR).33 Both the
untargeted and targeted variants were used
to treat mice models of triple negative
breast cancers, which often overexpress
EGFR. Free epirubicin killed the mice
after three weeks of treatment (FIGURE
7 ).33 Although mice treated with NDLPEpi survived the seven-week treatment
period, tumor growth was only impeded
to some degree (FIGURE 7 ). In contrast,
anti-EGFR-NDLP-Epi treatment resulted
in almost full tumor regression by the
end of the treatment period (FIGURE 7 ).
Comprehensive in vitro and in
vivo studies are required to validate
the safety and therapeutic efficacy of
ND-Cxb. Nonetheless, our current
data highlights a proof-of-concept ND
platform for chemopreventive oral
cancer applications. Given the prior
success of ND-drug systems, NDs may
soon carve out their own niche amongst
modern oral cancer treatments.
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cyclooxygenases in inammation, cancer and development.
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22. Greenhough A, Smartt HJM, Moore AE, et al. The COX-2/
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2009;30(3):377-386.
23. McCormick DL, Phillips JM, Horn TL, Johnson WD, Steele
VE, Lubet RA. Overexpression of cyclooxygenase-2 in rat
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expression in oral squamous cell carcinoma. Int J

Immunopathol Pharmacol 2004;17(3):273-282.
25. Kim Y-Y, Lee E-J, Kim Y-K, et al. Anti-cancer eects
of celecoxib in head and neck carcinoma. Mol Cells.
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26. Feng L, Wang Z. Chemopreventive eect of
celecoxib in oral precancers and cancers. Laryngoscope
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27. Papadimitrakopoulou VA, William WN, Dannenberg AJ,
et al. Pilot randomized phase II study of celecoxib in oral
premalignant lesions. Clin Cancer Res 2008;14(7):20952101.
28. Thomas GJ, Nystrm ML, Marshall JF. v6 integrin in
wound healing and cancer of the oral cavity. J Oral Pathol
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29. Ruoslahti E. RGD and other recognition sequences for
integrins. Annu Rev Cell Dev Biol 1996;12:697-715.
30. Behr J. The proton sponge: A trick to enter cells the viruses
did not exploit. Chim Int J Chem 1997;2(1):34-36.
31. Murphy RF, Powers S, Cantor CR. Endosome pH measured
in single cells by dual uorescence ow cytometry: Rapid
acidication of insulin to pH 6. J Cell Biol 1984;98(5):17571762.
32. Xi G, Robinson E, Mania-Farnell B, et al. Convectionenhanced delivery of nanodiamond drug delivery platforms for
intracranial tumor treatment. Nanomedicine 2014;10(2):381391.
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THE CORRESPONDING AUTHOR,

Dean Ho, PhD, can be reached at

dean.ho@ucla.edu.

F E B R U A R Y 2 0 1 6 127

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RM Matters

C D A J O U R N A L , V O L 4 4 , N 2

Getting the All-Clear Signal: Medical Clearance

Forms and Follow-Up
CDA Risk Management Sta

ts been a long day, but a good day, at

your practice. When you finally head
home for dinner, its getting dark and
traffic is not on your side. Red lights
flash ahead. Two cars speed through
the crossing before the gates come down,
but you slow to a stop and wait. The bell
rings loudly, the striped gate arm lowers
and the warning light glows. Theres no
mistaking that a train is coming. And,
once it passes, the ringing stops, the
gates rise and the light goes out. You
have the all-clear signal to drive ahead.
Wouldnt it be nice if these
signals were as clear in dentistry?
In a perfect world, all the elements
for a patients total health would come
together seamlessly to make care easy
and stress-free. In the real world,
though, it takes thoughtful coordination
between the dentist and patients
physician prior to dental treatment,
especially when it comes to medically
compromised patients. And the key to
getting the all-clear to proceed with
treatment is a medical clearance form.
Take, for instance, the case of a
66-year-old patient whose medical
history included open-heart surgery
and high blood pressure. His dentist
started the medical clearance process
the right way she sent a form
requesting more information to the
patients physician. The physician
shared that the patient was taking
Coumadin for atrial fibrillation and
was, therefore, subject to bleeding.
Unfortunately, heres where
signals got crossed. Based on the form
response, and without seeking further
clarification from the physician, the
dentist instructed her patient to
discontinue taking Coumadin five
days prior to any treatment. Over

It takes thoughtful
coordination between
the dentist and patients
physician prior to dental
treatment, especially when
it comes to medically
compromised patients.

the course of four years, the patient

suspended his anticoagulant regimen
before every dental appointment.
Following this protocol, the patient
had stopped taking his Coumadin
prior to a recall appointment at which
the dentist diagnosed a needed root
canal and crown. The procedure was
scheduled one week after, which resulted
in the patient resuming Coumadin for
only three days and then suspending it
again for another five. At the time the

You are not a statistic.

You are also not a sales goal or a market segment. You are a dentist.
And we are The Dentists Insurance Company, TDIC. Its been 35 years
since a small group of dentists founded our company. And, while times
may have changed, our promises remain the same: to only protect
dentists, to protect them better than any other insurance company and to
be there when they need us. At TDIC, we look forward to delivering on
these promises as we innovate and grow.
Contact the Risk Management Advice Line at 800.733.0634.

Protecting dentists. Its all we do.

800.733.0634 | thedentists.com | CA Insurance Lic. #0652783

F E B R U A R Y 2 0 1 6 129

F E B . 2 0 16

RM MAT TERS
C D A J O U R N A L , V O L 4 4 , N 2

patient presented for treatment, local

anesthetic was administered and he
appeared to fall asleep. However, when
the dentist attempted to wake him, he
was unresponsive. Paramedics transported
the patient to the hospital and it was
determined he had suffered a massive
stroke during the dental procedure.
Medical experts who reviewed the
case stated Coumadin takes time to reach
therapeutic levels in the bloodstream.
By following the dentists instruction to
stop the regimen five days prior to any
treatment, the patient was placed at
increased risk of stroke. Once therapeutic
levels of anticoagulant were decreased, a
stroke could have occurred at any time.
Starting and stopping Coumadin in
succession for two dental appointments
likely increased the patients risk.

According to the ADA, there is

strong evidence that typical dental
patients do not need to discontinue
anticoagulant use; local measures can
be used to control bleeding. For patients
with a higher risk of bleeding, any
suggested modification to the medication
regimen prior to dental treatment
should only be done with advice from
the patients physician. In the event a
physician refuses to provide medical
clearance for dental treatment, The
Dentists Insurance Company (TDIC)
recommends getting the patient involved
in contacting his or her physician directly.
The dentist in this case should
have explored the options thoroughly
with the patients physician and
discussed alternatives to stopping
the anticoagulation regimen.

Further clarification prior to treatment

and clear communication between dentists
and physicians can truly save lives.

What is your role in the medical

clearance process?
The following preventive measures can
protect your practice and your patients:
1. Obtain health history.
Every new patient should complete a
health history form. The form should
be reviewed, signed and dated by the
patient at each appointment, as well
as initialed and dated by the dentist.
Both signatures serve as evidence
that the information is current and
the patients health was discussed.
Additionally, TDIC recommends
checking with your carrier or local
CONTINUED ON 132

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130F E B R U A R Y 2 01 6

. . . . to the right buyer!

Knowing how, means doing all of the following - with precision:

1.
2.
3.
4.
5.
6.

Valid practice appraisal.

Contract preparation and negotiations, including critical tax allocation
consideration.
Bank financing or Seller financing, with proper agreements to adequately protect
the Seller and make the deal close - realistically and expeditiously.
Performance of due diligence
requirements, to prevent later problems.
Preparation of all documentation
for stock sale, when applicable.
Lease negotiations.

All six of these

services costs no more.
Maybe even less!

Lee Skarin & Associates

has scores of Buyers in their
database. The Buyers profiles
personal desires and financial
ability have been categorized to
expertly select the right Buyer
for your practice. Expert Buyer
selection solidifies a deal.
Lee Skarin & Associates services
all of Southern California.

LEE SKARIN
& ASSOCIATES INC.

SELL YOUR PRACTICE . . . . .

Lee Skarin & Associates is Californias leading Dental Practice

Broker. Their in-house attorney, Kurt Skarin, PhD., J.D.,
specializes in these matters. He
does all of the above, and more.
He is the catalytic agent that
makes the sale happen - quickly and smoothly.

Dental Practice Brokers

CA DRE #00863149

Your calls are invited. Put our thirty years of experience to work for you!
Visit our website for current listings: www.LeeSkarinandAssociates.com

2IFHV

805.777.7707
818.991.6552
800.752.7461

F E B . 2 0 16

RM MAT TERS
C D A J O U R N A L , V O L 4 4 , N 2

CONTINUED FROM 130

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Weve got new shirts in the CDA Store, online at cda.org/store.

Also new to the store:

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dental society every two years for any

form updates. TDIC policyholders
have online access to sample health
history forms in English and Spanish.
2. Request medical clearance.
Be sure to describe the treatment
plan in detail, including both
anticipated prescriptions and
over-the-counter medications. For
medically compromised patients, it
is important to speak directly with
the treating physician. However,
a conversation is not a substitute
for a signed medical clearance
form. TDIC also offers a newly
updated sample medical clearance
for dental treatment form that can
be customized to include specific
questions for the physician.
3. Proceed with treatment.
Once the medical clearance form
has been returned, review it for
a qualified signature and clear
responses. Nurse practitioners are
registered nurses with advanced
training in diagnosing and
treating illnesses, so a medical
clearance form signed by a
nurse practitioner following a
standardized procedure, rather than
a physician, is acceptable. The
most important steps of the process
are verifying and clarifying any
medical issues causing concern.
As health care becomes increasingly
integrated, opportunities to collaborate
for the best patient experiences increase.
Take note of medical compromise warning
lights. And take time to coordinate your
treatment with the patients physician to
ensure a safe and effective outcome.
TDICs Risk Management Advice Line at
800.733.0634 is staffed with trained analysts
who can answer medical clearance and other
questions related to dental practice.

132F E B R U A R Y 2 01 6

Specialists in the Sale and Appraisal of Dental Practices

How much is your practice worth??

Selling or Buying, Call PPS today!

2016 marks PPS 50th Year Anniversary

of Serving California Dentists

Practices
Wanted

SOUTHERN CALIF
CALIFORNIA

NORTHERN
NORT
RN CALIFORNIA
(415) 899-8580 (800) 422-2818
Raymond and Edna Irving
Ray@PPSsellsDDS.com
www.PPSsellsDDS.com

(714) 832-0230 (800) 695-2732

Thomas Fitterer and Dean George
PPSincnet@aol.com
www.PPSDental.com

California DRE License 1422122

California DRE License 324962

6097 SAN FRANCISCOS PARKSIDE DISTRICT Part-time

practice collected $130,000+ in 2015. 2-ops with separate digital
x-ray room. 3rd op available. Great opportunity for someone
looking to enter private practice with low start-up costs.
6096 FRESNO Long established. 4-days per week of hygiene.
Realized collections of $450,000 in 2015. 4-ops.
6095 WEST CONTRA COSTA COUNTYS PINOLE Located
off Interstate 80 in Dental Village alongside Appian Way. 3-days of
Hygiene. 2015 tracking $425,000 in collections. Delivery systems
in ops were upgraded 11-years ago.
6094 PERIO PRACTICE - SAN FRANCISCO BAY AREA
This offering shall appeal to the Periodontist who wants a high-end
practice in aQH[WUHPHO\desirable area. 2015 trending $700,000 in
Available Profits.
6093 CENTRAL MARIN COUNTY Located in hub of Marin
County. Consistent $700,000+ per
year performer with strong
SOLDoffice.
Profits. 3-days of hygiene. Digital
6092 ROSEVILLE 2015 trending $350,000. 3-ops with 4th
available. Convenient locationOon
Douglas Boulevard. End cap
S LD
suite in strip center with fantastic exposure.
6089 MOUNT SHASTA Small town living renowned for outdoor
living. Perfect escape from Rat Race and corporate intrusion. On
3-day week, 2015 trending $850,000 with $450,000 in Profits.
6088 SANTA CRUZ Well established, lots of patients. Strong
Hygiene Department with 6.5 days
Dof hygiene per week. Collected
SOL$675,000+.
$600,000 in 2014. 2015 trending
6087 LAKE TAHOE - NEVADA'S STATELINE
"Fee-for-Service as practice is "out-of-network with insurance
D $600,000 with Profits of
companies. Collections lastSyear
OLtopped
$220,000. 3.5 days of hygiene per week. Nevada State Board of
Dental Examiners accepts the Western Boards.
6081 SANTA CLARA El Camino Real location. 2015 tracking
$775,000 with Profits of $325,000. Management is on "cruise
control." New Doc who is ambitious and extends hours shall push
practice over the $1 Million bar very quickly. 5-ops in 1,700 sq.ft.
6080 SAN RAMON 8+ days of Hygiene per week. $450,000
invested in 6-Op office. Consistent
SOLD$900,000+ per year performer.
Attractive transition arrangements available.
6071 CHICO Strength is 4-day Hygiene schedule. Retiring DDS
focuses on restorative. Endo, O
Perio & Pedo referred. 2014
S OS,LD
collected $450,000. Beautiful 4-Op office. Full Price $150,000.
6070 VISALIA Strong foundation and well-positioned for
successor. Strong Hygiene Department, beautiful facility, well
equipped. Digital throughout. Not a Delta Premiere practice.
Revenues trending $750,000 for 2015 on part-time schedule.
Extend hours and be busier. Best location!

ARROWHEAD Absentee Owner. Grosses $450,000. Hi Identity

Lake Drive Building. Practice $350,000. RE $250,000.
DANA POINT Grossed $950 in 2013 with ortho. No longer
doing ortho. Absentee Owner. Full price $650,000
EAST LOS ANGELES - EMERGENCY SALE Huge Latino
population within 3 radial miles. Long established. Million Dollar
Potential Hi Identity office. Full Price $330,000.
GRANADA HILLS Location. Seeks Specialists.
HEMET Grosses $850,000 with opportunity to increase substantially.
Seller will work back as will associates. Great opportunity for Oral
Surgeon or Corporate Group.
INDIO DENTAL OFFICE Next to City Hall. 2 ops in 4,000 sq.ft. hi
identity building includes real estate. Asking $650,000. Make Offer.
,59,1( Great location. Busy Lady DDS will Solo Group her 5 ops or
partner into acquiring building with Specialists.
LOS ANGELES HMO Grossing $4 Million. In Escrow. Back-Up
Offers requested.
MISSION VIEJO Freeway location. Solo Group. New DDS with
patients will be paid 40%. Join Million Dollar state-of-art office.
Membership $30,000.
NORCO - CORONA Recently renovated. Gorgeous 8 ops and digital
includes cone beam. Grossing near $100K/month. Hi identity building
apprx 3,000 sq.ft. Great for Absentee Buyer or Specialist as Seller will
work back on contract. Full Price for practice $1.1 Million and Building
$900,000.
REDLANDS Full Price $50,000 Established 27 years. Connect with
3,000 employee Employer. Rent $850/mth year one, $1,250/mth next
5-years and then $1,450/mth 5 more years.
REDLANDS 5 Ops and digital. Rent $2,400. 900 Patients. Absentee
Owner. Full Price $250,000
RIVERSIDE Divorce Sale. Full Price $31,000 includes 4 ops in
historic professional location. Includes all charts seen prior to 2014.
Rent negotiable.
SAN FERNANDO VALLEY Grossed $550,000 in 2014. Will do
$650,000 in 2015. Full Price $550,000.
SAN FERNANDO VALLEY Long established. Renovated in 2010 at
cost of $350,000+. Gorgeous 2,000 sq.ft. 6 ops. Digital includes
Panorex. Full Price $500,000.
TUSTIN BUILDING 2,000 sq.ft. available at best intersection.
60,000 autos pass daily.
TUSTIN DENTAL BUILDING 5 ops in 1,875 sq.ft. office in Tustin
Hills. $1.4 Million.
VALENCIA - SANTA CLARITA 60,000-to-70,000 autos pass this
intersection daily. 8 ops plumbed, 4 equipped. 2,000 sq.ft. Full Price
$220,000.
YUCCA VALLEY Small dental building on .4 acres. Land Value
$150,000.

**FOUNDERS OF PRACTICE SALES**

Wall Street Investor seeks small Groups to manage. PPS is banding

practices together of Owners retiring in near future. Cash in and work
back for growing Group. Enjoy working with team spirit. Register your
interest with Tom Fitterer.

120+ years of combined expertise and experience!

3,000+ Sales - - 10,000+ Appraisals
**CONFIDENTIAL**
PPS Representatives do not give our business name when returning your calls.

DENTAL PRACTICE BROKERAGE

Making your transition a reality.

Dr. Lee
Maddox
LIC #01801165

Dr. Thomas
Wagner
LIC #01418359

Dr. Dennis
Hoover
LIC #0123804

Dr. Russell
Okihara
LIC #01886221

Jim
Engel
LIC #01898522

Kerri
McCullough
LIC #01382259

Mario
Molina
LIC #01423762

Jaci
Hardison
LIC #01927713

Steve
Caudill
LIC #00411157

Thinh
Tran
LIC #01863784

(949) 675-5578
25 Years in Business

(916) 812-3255
40 Years in Business

(209) 605-9039
36 Years in Business

(619) 694-7077
33 Years in Business

(925) 330-2207
42 Years in Business

(949) 566-3056
35 Years in Business

(949) 675-5578
35 Years in Business

(949) 675-5578
26 Years in Business

(951) 314-5542
25 Years in Business

(949) 675-5578
11 Years in Business

PRACTICE SALES PARTNERSHIPS MERGERS VALUATIONS/APPRAISALS ASSOCIATESHIPS CONTINUING EDUCATION

NORTHERN CALIFORNIA
BENICIA: Practice & Building. 1,545 sq. ft.,
4 Ops, Open-Dental software, Digital X-ray
& Pan, Laser. 2014 GR $550K. #CA298
FOLSOM: FACILITY ONLY 1,200 sq. ft.
w/3 Ops, Digital & Pano, new compressor
#CA209
GREATER SACRAMENTO: 7 Ops,
3,079 sq. ft. (Shared w/2nd DDS Separate
Practices), 2013 GR $974K. #CA140
LIVERMORE: Practice & Building. 1,100
sq. ft., 4 Ops, Digital Pan & X-ray, Laser,
Dentrix. 2014 GR $740K. #CA300
MARIN COUNTY: Mill Valley 1,260 sq.
ft. 3 Ops, 1 addl Plumbed. Dentrix, Digital,
Intra-Oral. #CA224
MARIN COUNTY: 3 Op practice w/views
of Corte Madera Creek. 2014 GR$315K.
Paperless charts, Schick, Eaglesoft. #CA286
MARIN COUNTY: 1,250 sq. ft.
3 Ops. Fee for service 2014 GR $370k. On
pace for same in 2015. #CA302
MILLBRAE:VTIWOHDVHGRIFH
with 5 Ops, 1 addl Plumbed, state-of-the-art
Equipment. 2014 GR $670K. #CA262
N. COAST: Endo Practice. 6 Ops,
5 Plumbed 3,300 sq. ft. Digital, Microscopes,
EndoVision. #CA214
N. EAST OF SACRAMENTO: 2,500 sq.
ft, 7 Ops with intra-oral, Digital X-ray, and
Eaglesoft. 2014 GR $1.5MM. #CA268
NORTHERN CALIFORNIA: Endo
Practice. 3 Ops, 1 Plumbed, 1,200 sq. ft.
2 Microscopes, Digital. 2013 GR $319K+
#CA158
NORTHERN CALIFORNIA: Perio Practice,
Partnership Position. 6 Ops, 1,500 sq. ft.
Dentrix. Owner Financing Available. #CA168
N. OF SACRAMENTO: 1,750 sq. ft. w/4
Ops. Intra-Oral, Digital, Pano, Laser, CAD/
CAM, Dentrix. 2014 GR $1MM. #CA260

OROVILLE: 1,000 sq. ft. Dentrix &

Dentrix software, Digital Pan & X-rays,
Laser, Intra-Oral. 2014 GR $512K. #CA287

BANNING: 6+ Ops. Paperless, Digital,

EagleSoft. 8 Days Hyg./Week. 2014 GR
$1.4MM+. #CA183 - IN ESCROW!

OROVILLE: 3 Ops, recently remodeled.

Great satellite or startup practice. Owner
retiring. #CA288

BEVERLY HILLS: Small boutique

practice, 2 Ops, 1 Equipped, Open Dental,
Digital, 2014 GR $120K on 3 days/wk.
#CA215

PINOLE:2SV(QGRRIFHZ'LJLWDO;UD\
Microscopes, and PBS Endo in approx. 1,200
sq. ft. 2014 GR $672K. #CA284
ROSEVILLE/GRANITE BAY: 1,320
sq. ft., 3 equip. Ops, 1 addl Plumbed. Dexis
Digital X-ray. 2014 GR $434K. #CA296
SACRAMENTO: 1,684 sq. ft., 6 equip.
stations in bay, 2 addl Plumbed. 2014 GR
$590K. #CA269
SACRAMENTO: Russian-speaking. 4 Ops,
1,500 sq. ft.. All new Equipment, practice
started recently. $1M over last 12 months.
#CA290
SACRAMENTO: 2,500 sq. ft., 6 equip. Ops,
1 addl Plumbed. Dentrix software, Digital
X-ray, Pano. 2014 GR $788K. #CA297
SAN FRANCISCO: Periodontal Practice
& Condo Unit. 1,160 sq. ft. w/4 Ops, 2014
GR $714K w/$363K adj. net. #CA274
SAN RAMON: FACILITY ONLY, 1,654 sq.
ft., 4 Ops. Pelton & Crane Equipment, Digital
X-ray, Digital Pan, I.O. cameras. #CA306
SANTA ROSA: General Dentistry &
Building. 3 Ops. 2013 GR $542K w/Adj. Net
$182K. #CA200

CARSON: 3 Ops. Paperless, EagleSoft,

Digital, Pano. All equip. <3 yrs. old. 2014
GR $143K. #CA280
CYPRESS: 5 Ops, 35 yrs. of Goodwill.
7 days Hygiene per week. $948K GR.
#CA257 - IN ESCROW!
GREATER LOS ANGELES: Perio
Practice. 5 Ops, 34 Yrs. of Goodwill. Dentrix,
Digital, Laser, great referral base. #CA173
HACIENDA HEIGHTS: General Dentistry,
5 Ops, 3 Equipped, retail center, Digital,
Pano. PPO. #CA295
HUNTINGTON BEACH: 5 Ops, 28 yrs.
of Goodwill, Digital, Pano, Laser, 12 days
of hyg./wk. GR of $1.1MM+. #CA263
INLAND EMPIRE: Endo Practice. 4 Ops,
3-yr. new equip., Digital, Cone Beam CT.
2014 GR $739K with low overhead. #CA281

SONOMA:6WDQGDORQHVTIWRIFH
w/4 Ops. Digital X-rays, Lasers, CAD/CAM.
2014 GR $675K on 3 day/week. #CA270

INLAND EMPIRE: 7 Ops, Dentrix, Digital,

Pano, 30 yrs. goodwill. 4 days
of Hygiene. #CA283

YOLO COUNTY: Pediatric practice, 2

equip. Ops, 1,000 sq. ft., Intra-oral, Open
Dental software. 2014 GR $245K. #CA301

INLAND EMPIRE: General Dentistry,

4 Ops, Camera, Digital, Pano, 2014 GR
$534K, Adj. Net $196K. #CA285

CENTRAL CALIFORNIA

LAGUNA BEACH: New Listing! General

Dentistry. 5 Ops, 3 Equipped. Great Location.
2014 GR $503K. #CA303

CALAVERAS COUNTY: 4 Ops, 1,752

sq. ft., Dexis Digital X-ray, Eaglesoft, Cerec,
Pano. Av. GR last 3 years $450K. #CA294
CENTRAL COAST: General Dentistry.
6 Ops, 8 days Hygiene/wk. GR over $2MM
for last 3 yrs. Est. 30+ yrs. #CA208

N. OF SACRAMENTO:VTIWRIFH
w/4 Ops, Dentrix, Pano. Owner worked 39
weeks in 2014. #CA267

KINGS COUNTY: General Dentistry.

4 Ops, Pano, established for 50+ yrs. GR
of $246K in 2014. #CA265

N. OF SACRAMENTO: 1,800 sq. ft., 4

Ops, Eaglesoft software, laser. 2014 GR
$289K. #CA307

MADERA: Building & practice. 6 Ops,

3000 sq. ft., Dentrix software, Dexis software,
Pano. 2014 GR $850K. #CA289

NORTH SACRAMENTO: 3 Ops in a

leased space with <1,000 sq. ft., PPO, 2 days
Hygiene, Digital, Easy Dental. #CA266

PORTERVILLE: General Dentistry, 6 Ops.

2014 GR $555K, 7 year old Equipment, retail
center. #CA223

NORTH SACRAMENTO: Practice &

condo. 2,500 sq. ft., 5 Ops, Dexis Digital
X-ray, Pano, Laser, Dentrix. 2014 GR $673K.
#CA305

ANAHEIM HILLS: with 4 Ops, est. for

34 yrs. Dentrix, 6 days Hygiene per week.
#CA279

OAKLAND: Appx. 1,500 sq. ft. w/4 Ops,

Dentrix software, Dexis Digital X-ray. 2014
GR $869K, adj. net $370K. #CA293

BAKERSFIELD: General Dentistry with

4 Ops, Dentrix, Dexis, Pano, PPO/FFS. 2014
GR $329K #CA299 - IN ESCROW!

SOUTHERN CALIFORNIA

OLD

NORTHERN CALIFORNIA OFFICE

1.800.519.3458

BEVERLY HILLS: 5 Ops. EagleSoft,

Digital, CEREC. Long-term staff, newer
Equipment. 2014 GR 1.07MM, Adj. Net
of $406K. #CA210

Henry Schein Corporate Broker #01230466

LOS ANGELES: General Dentistry, 6 Ops,

5 Equipped, Est. 50+ yrs., SoftDent, Digital.
2014 GR $591K. #CA255 - IN ESCROW!
PALM DESERT: 5 Ops, Est. for 32 yrs.,
6 days of Hygiene/week. GR of $824K and
$339K adj. net. #CA245 - IN ESCROW!

SOUTH PASADENA: General Dentistry.

4 Ops, 3 Equipped, paperless, Digital, est. 37
yrs. 2014 GR $856K with $271K adj. net.
#CA244 - IN ESCROW!
VICTORVILLE: 3 Ops, 3 Plumbed. 2,150
sq. ft. Est. 34 yrs., SoftDent. 2014 GR $273K.
#CA149

OLD
S
WHITTIER: General Dentistry. 4 Ops,

WEST HOLLYWOOD: 4 Ops, Intra-Oral

Camera, Digital, Laser, 5 yr. old equip. 2014
GR of $613K . #CA212
3 Equipped. Dentrix, Dexis. Est for 50+ yrs.
on main street. 2014 GR $217K. #CA276

SAN DIEGO
CHULA VISTA: Est. 50+ yrs. 4 Ops, 3
days of Hygiene, Dentrix. $493K GR in 2013.
#CA109
CHULA VISTA EAST: New Listing!
General Dentistry. 3 Ops. Est. 19 years.
Professional bldg. 2014 GR $454K. #CA304
COLLEGE AREA: Very busy 6 Ops with
room to expand to 9 Ops. PPO, Dentrix,
Digital. 2014 GR $1.7MM #CA231
DOWNTOWN: LEASEHOLD SALE.
0RGHUQDQGFKLFGRZQWRZQRIFHLQSULPH
location. 3 Ops + room to expand. #CA232

SOL

ESCONDIDO: 4 Ops, 3 Chairs, Central

Escondido, Doctor Retiring Excellent
Opportunity to merge/grow. #CA292
LA JOLLA: 3 Ops, FFS and Delta Premier.
2014 GR of $559K. Owner retiring. #CA278
MID-TOWN: New Listing! 4 Ops+ 1
surgical Op, Modern & Bright. HMO/
PPO/FFS, Digital Pan/Ceph, Convenient
Location. #CA309

SOL

NORTH COUNTY INLAND: General

Dentistry, 6 Ops, FFS/PPO. Free-standing
bldg. also available to buy. #CA271
NORTH COUNTY, VISTA: New
Listing! Beautiful and modern 5 Ops
FFS/PPO, Digital X-rays & Pano, Most
Specialty work referred out. Seller retiring
due to health. #CA308

S. BAY AREA, SAN DIEGO: General

Dentistry, 3 Ops, 4 days hyg/wk. Retail
center, Dentrix, Digital Pano, PPO & FFS.
GR 2014 $524K. #CA206

SOL

PASADENA AREA: General Dentistry.

3 Ops, Dentrix, Dexis, CEREC, established
for 50+ yrs. #CA282

SOUTH BAY AREA: New Listing! 4

Ops, PPO/FFS, EagleSoft, Digital, Modern
and Spacious. Practice & Building for Sale.
#CA313

PICO RIVERA: General Dentistry,

6 Ops, Est. in 1960. DentiSoft, Pano, 4
days of Hygiene per week. 2014 GR of
$690K. #CA258- IN ESCROW!

OUT OF CALIFORNIA

S. ORANGE COUNTY: Pedo Practice with

4 Ops, 1 year new Equipment, Digital, Pano.
$236K GR with room to grow. #CA222
SANTA BARBARA: New Listing!
General Dentistry. 4 Ops. Est. 40+ yrs. 8
days hyg./wk. Long term staff. GR of $827K.
#CA291

www.henryscheinppt.com

MAUI, HAWAII: New Listing! 7 Ops,

5 Equipped, Modern Design, CEREC. 2014
GR $1.4MM+. #HI101
CENTRAL OAHU, HAWAII:
New Listing! General Dentistry. 3 Ops in
Central Oahu. Dentrix, Dexis, Pano. 2014
GR $454K #HI102
HONOLULU, HAWAII:New Listing!
3 Ops, Dentrix, Digital, FFS/PPO. #HI100

SOUTHERN CALIFORNIA OFFICE

1.888.685.8100

16PT3352J

Regulatory Compliance

C D A J O U R N A L , V O L 4 4 , N 2

Infection Control Q-and-A

CDA Practice Support
Following are answers to questions
asked in recent months by dental
practices about infection control.
A new employee is joining our staff.
She used to work at another dental
practice. What is our practices obligation
with regard to providing the hepatitis B
vaccination to the new employee?
Cal/OSHA requires an employer to
offer the hepatitis B (HBV) vaccination
to employees who are occupationally
exposed to blood-borne pathogens. The
offer must be made within 10 working
days of initial assignment to the job
and after the employer has provided
the employee with information on the
vaccines efficacy, safety, method of
administration and the benefits of being
vaccinated. An employer need not
make the HBV vaccination available to
employees who have previously received
the vaccination series, who are already
immune as revealed by appropriate tests
for HBV antibodies or who are prohibited
from receiving the vaccine for medical
reasons. However, an employer may not
make any of these three exemptions a
condition of employment. The HBV
series received as a child does not count
for the purpose of this requirement.
Employers can request documentation of
previous vaccination from employees.
The employer must provide for
postvaccination testing one to two
months after the last shot and another
vaccination series and testing for the
employee if the postvaccination test
results are negative. If the second
postvaccination test is negative, the
employer must refer the employee
to a health care provider for
counseling on infection prevention.
An employee who tests negative
need not be excluded from work.
If an employee refuses the offer
of vaccination, the employee must
sign a declination, the wording of

which is mandated by Cal/OSHA.

A declination form and additional
information on HBV vaccination,
including recommendations, is
available on cda.org/practicesupport.
Staff attended different infection
control courses recently and heard
conflicting information about scrubs.
What are Cal/OSHAs rules?
The blood-borne pathogens
regulation does not address the use of
scrubs or even uniforms. The regulation
requires the use of personal protective
equipment (PPE). Consider this
question: In your office, are scrubs
used as PPE or are they uniforms? If
they are uniforms, then appropriate

PPE must be worn over the scrubs

when treating patients. In responding
to the question, What type of PPE
should be used by employees in a
dental office? Cal/OSHA states:
PPE will be considered
appropriate only if it does not
permit blood or other potentially
infectious material (OPIM)
to pass through employees
underlying garments, or to
reach the skin, eyes, mouth or
other mucous membranes under
normal conditions of use. PPE
must retain this capability during
the entire course of its use by
the employee. This allows the

When Looking To Invest In Professional

Dental Space Dental Professionals Choose

Linda Brown
30 Years of Experience Serving
the Dental Community Proven
Record of Performance

For your next move,

contact: LINDA BROWN
Direct: (818) 466-0221
Office: (818) 593-3800
Email: LindaB@TOLD.COM
Web: www.TOLD.com
Cal BRE: 01465757

Dental Office Leasing and Sales

Investment Properties
Owner/User Properties
Locations Throughout
Southern California

F E B R U A R Y 2 0 1 6 135

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INVENTORY IS LOW!
ITS A SELLERS MARKET!

Paul Maimone
Broker/Owner

ARCADIA (4) op comput G.P. Located in a well known Prof. Bldg. on a main thoroughfare.
Cash/Ins/PPO pt base. Annual Gross Collect $300K+ on a (3) day week.
BAKERSFIELD #29 - (4) op comput G.P. (3) ops eqtd, (1) add. plumbed. Located in a free
stand bldg. Cash/Ins/PPO. Digital x-rays. Annual Gross Collect $300K+ p.t. Seller moving.
BAKERSFIELD #31 - Free Stand. Bldg. & Pract. (4) op comput G.P. w excell. exposure &
signage. (3) ops eqt./4th plumbed. Annual Gross Collect $325K+ Cash/Ins/PPO. NEW
CAMARILLO (3) op compt. G.P. 2 eqtd./3rd has a Pano. Cash/PPO. $120K Gross p.t. NEW
GROVER BEACH - (3) op Turnkey Office w included charts (included, but not guaranteed). (2)
ops eqtd w newer eqt. 3rd plmbed. Networked, digital Pano & x-ray. Dentrix. In a strip ctr. NEW
MONTEBELLO - (4) comput G.P. (2) ops eqtd. Located in a busy shop. ctr. w exposure &
visibility. Annual Gross Collect. $200K on a p.t. schedule. Cash/Ins/PPO. Seller retiring. NEW
MONTEREY PARK (6) op comput G.P. located in a street front suite on a main thoroughfare
w exposure/visibility. Cash/Ins/PPO/Small % Denti-Cal. Gross Collect $250K+ p.t. REDUCED
MURRIETA (5) op comput. G.P. (4) ops eqtd/(1) add. plumb. Cash/PPO patients. No HMO
or Denti-Cal. Located in a condo (also available), in a smaller prof. bldg.. Gross Collect approx..
$400K/yr. Seller is giving up private practice for a Govt. position. Motivated!
OXNARD (4) op comput. G.P. in a prof. bldg. Gross Collect ~ $250K/yr on (3) days. Digital xrays. Dentrix. Cash/Ins/PPO/HMO/Denti-Cal. Refers diff Endo/O.S./Perio & Ortho. NEW
SANTA BARBARA COUNTY (3) op comput G.P. & a 1,900 sq ft Bldg. that houses the
practice & a residential unit that can be rented or lived in. Fee for Service. No PPO, HMO or
Denti-Cal. 2015 Gross Collections ~ $275K on a relaxed 3 day week. Seller refers all O.S.,
Perio, Ortho, Endo & implant placement. Seller retiring but will assist w transition. NEW
SHERMAN OAKS (3) op comput G.P. in a well known Med/Dental bldg. Fee for Service.
Annual Gross Collect $160K+ p.t. Great Starter or Satellite. Seller retiring. REDUCED
So. KERN COUNTY (6) op comput. G.P. located in a Bakersfield suburb in a small strip ctr. w
exposure/visibility. Pano eqtd. Limited competition. Cash/Ins/PPO pts. Annual Gross Collect.
Approx. $350K p.t. Used to do $1M+ f.t.. Seller is moving and is motivated. REDUCED
SAN FERNANDO VALLEY - (8) op comput. G.P. w modern equipt. Located in a prof. bldg. on
a main thoroughfare. Cash/Ins/PPO/HMO. Cap Ck approx $7K/mos. Collect $1.3M+/yr. NEW
SANTA ANA - absentee owned (6) op fully eqtd G.P. First floor street front location on a main
thoroughfare. Exposure/visibility/signage. Cash/Ins/PPO. No HMO & No Denti-Cal. Pano eqtd
& Computerized. Annual Gross Collect. ~ $525K on a (3) to (4) day Associate run week.
TUSTIN - (4) op comput. G.P. (3) ops eqtd/4th plumbed. Located in a busy shop ctr. on a main
thoroughfare. Excellent exposure, visibility, signage & parking. Digital x-rays. Turnkey Office
w some pt. charts included but not guaranteed. SOLD
THOUSAND OAKS (4) ops/(2) eqtd comput. Turnkey Office with included charts. Chart
included but not guaranteed. Located in a condo in a Prof. Bldg. on a main thoroughfare. NEW
UPCOMING PRACTICES: Bakersfield, Beverly Hills, Central Coast, Covina, Downey,
Duarte, Goleta, Oxnard, Pomona, San Gabriel, Torrance, Van Nuys, Visalia & West L.A..
D&M SERVICES:
Q Practice Sales and Appraisals
Q Practice Search & Matching Services
Q Practice and Equipment Financing Q Locate and Negotiate Dental Lease Space
Q Expert Witness Court Testimony
Q Medical/Dental Bldg. Sales & Leasing
Q Pre - Death and Disability Planning Q Pre - Sale Planning

P.O. Box #6681, WOODLAND HILLS, CA. 91365

Toll Free 866.425.1877 Outside So. CA or 818.591.1401 www.dmpractice.com
Serving CA Since 1994 CA BRE Broker License # 01172430

CA Representative for the National Association of Practice Brokers (NAPB)

employer to select PPE based

on the type of exposure and
the quantity of blood or OPIM
reasonably anticipated to be
encountered during performance
of a task or procedure.
An employer must provide,
repair, launder and replace PPE.
Must staff have Cal/OSHA training
every year? I thought it was only required
every other year for license renewal.
Both Cal/OSHA and the Dental
Board of California have regulations
governing infection control in dental
settings. Both agencies require training
that covers similar subjects but each
has slightly different requirements.
The Dental Board requires licensed
professionals to take a two-hour infection
control course every license renewal
period. The course must be approved
by the dental board and it must be
provided by a dental board-approved
continuing education provider. The
course content must relate to the dental
boards infection control regulations.
Cal/OSHA requires employers to
provide blood-borne pathogens training
to occupationally exposed employees
upon hire, whenever a change in
procedures may lead to increased
exposure, and at least annually. The
training must be offered during work
hours at no cost to the employee and
be provided by a knowledgeable trainer.
There is no minimum time requirement
for the training, but it must cover:
1. Epidemiology and symptoms
of blood-borne diseases.
2. Modes of transmission of
blood-borne pathogens.
3. The dental practices exposure
control plan, the blood-borne
pathogens regulation and means
to obtain a copy of both.
4. How to recognize tasks that
may involve exposure.
CONTINUES ON 138

136F E B R U A R Y 2 01 6

CARROLL

Matching the Right Dentist

to the Right Practice

& C O M P A N Y

Complete Evaluation of Dental Practices & All Aspects of Buying and Selling Transactions

4085 SANTA ROSA GP & BUILDING

Practice and real estate are offered for sale in a well-established
condominiumized medical/dental complex conveniently located
in the heart of Santa Rosa, near Memorial Hospital. This 1,200
sq. ft. single story office is centrally located in a desirable, mixed
use commercial and residential corridor known as Doctors Row.
The office comes equipped, furnished and ready for you to
continue your professional career with established and new
patients (approx. 750 active patients).
Tastefully decorated with a homey dcor, the practice has 3 fully
equipped ops, reception area, private office, staff lounge, etc.
Seller is retiring after almost 20 years but will assist for a smooth
transition. Average Gross Receipts of $256K with adj. net of
approx. $110K. Asking price $160K for the practice, and $270K
for the real estate.
4091 HOLLISTER GP & PEDIATRIC
Country living at its best ~ small town community feel with
affordable housing, in quaint bedroom community to Silicon
Valley. General and Pediatric practice located in corner
professional building on well-travelled street near Hazel Hawkins
Hospital. Fully-equipped 1,600 sq. ft. office with 2 enclosed adult
ops and 3 open pedo ops. Great opportunity for a turn key
practice with trained staff and approximately 700 active patients.
2014 GR $228K. Seller is relocating out of the area, but will help
for a smooth transition. Asking price only $125K.
4090 SANTA CLARA DENTAL FACILITY
Turn-key dental facility in highly visible 30 year old modern,
commercial & professional mix building with large daytime
business draw & large residential population, in a well-travelled
area. Dental practice has been at this location for 30 years. Seller
has relocated & is offering 6 fully furnished & functional
operatories. Office remodeled in 2010. 2,240 sq. ft. suite
includes large waiting room, large front office, central lab, 2
private offices, break room & bathroom. Existing lease has 4
options to renew for 5 years.
4089 SUNNYVALE GP
Extremely desirable location on the corner of two well known/
major cross streets with easy freeway access. Beautifully
appointed practice
4 fully equipped ops. Lots
ce with 5 plumbed,
p um
pl
u
G ssetting. Seller is offering 12 years
of natural light and
nd a park
par
arkkIN
llilike
ike
D
Nox
of goodwill and P
aapprox.
appr
ap
pEpr
pro
ox 500 active patients. Average gross
ox.
receipts (last 3 yyears)
ears) $328K with an adjusted average net of
$122K. Seller will help for a smooth transition. Asking price
$250K.
4084 SAN BRUNO GP
Located in Tanforan Shopping Center. 2014 Gross Receipts
$279K. Convenient, spacious design, 5 op.& private office.
Asking $175K.

www.carrollandco.info dental@carrollandco.info

4010 SF GP
State-of-the-art, modern dental practice in gorgeous facility with
recently upgraded reception, business and private office in
approx. 3,200 sq. ft. office
ce
e with
Gw 6 fully equipped ops. Located
IpNme
close to downtown.
wn.. Equipment
Equ
qD
uip
men
e includes Inter-oral camera, laser,
N
digital x-ray, air aabrasive,
Omnicam, Cerec, and implant system.
bPas
br
aEsiv
asi
iv Omn
ive,
2014 Gross Receipts
i t over $1.3
$1 Million. 2015 on schedule for
$1.6 Million. Asking $1.1 Million.
4086 SILICON VALLEY PERIO
Well-established Perio practice in prime San Jose location with
referral sources nearby. Located in a commercial & residential
mix neighborhood with a large daytime business draw. Approx.
1,100 sq. ft. office with 4 fully-equipped ops. Well trained
dedicated staff, seller retiring and willing to help for smooth
transition. 2014 GR $482K+, 2015 on schedule for $539K+ as
of August. Asking $295K.
4092 SAN JOSE GP
Well-established practice offering over 40 years of
goodwill. Fabulous location conveniently situated at two major
cross streets, right
ght offff Hwyy 280.
28 4 fully equipped ops in 1,400
sq. ft.Practice average
gross
ge
eg
ro
oss
sGs receipts $665K+ with average adj.
N
I
N+D 7 ddays of hygiene and 1,300-1,400
net income of $2
$223K+
2P23
2E
23K+
3K+
K
K+
active patients, alll fee-for-service
fee for servic (Delta Premier Provider only, no
other PPOs accepted). Seller is willing to help Buyer for a
smooth transition. This opportunity wont last long. Asking
$550K.
4088 NEWARK/FREMONT DENTAL FACILITY
1,400 sq. ft. facility with 4 fully-equipped operatories setup for
right-handed delivery, reception area, private office, consult room,
staff lounge, lab area, sterilization area, storage area, 2
bathrooms, common area and plenty of parking. Located in mall
close to new housing. Lease expires in 3 years with 5 year
option to renew. Landlord willing to negotiate new 10 year lease
at a fair market rate. Equipment list available. Asking $80K.
SF DENTAL FACILITY
Facility only in the Sunset district, located on Ocean Avenue. 2
fully equipped ops with room for a 3rd op. Asking price $85K.
Lease is transferable to Buyer.
Carroll & Company
2055 Woodside Road, Ste 160
Redwood City, CA 94061
P (650) 362-7004
F (650) 362-7007
dental@carrollandco.info
www.carrollandco.info
CA DRE #00777682
Mike Carroll

Pamela Carroll-Gardiner

P (650) 362-7004 F (650) 362-7007

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CONTINUED FROM 136

5. Use and limitations of engineering

controls, work practices and
personal protective equipment.
6. Type, use, location, handling,
decontamination and disposal of
personal protective equipment.
7. Explanation of basis for selection
of personal protective equipment.
8. HBV vaccine information:
efficacy, safety, method of
administration, benefits, offered
at no cost to employees.
9. Actions to take and who
to contact in an emergency
involving infectious materials.
10.Post-exposure procedures including
reporting, medical follow-up and
sharps injury log recording.

11. Employers requirements for postexposure evaluation and follow-up.

12. Signs and labels.
Is it true that the motors of lowspeed handpieces must be sterilized?
The subject is addressed in the CDC
2003 Guidelines for Infection Control
in Dental Health-Care Settings. All
handpieces and attachments, including
motors, must be heat sterilized between
patients unless they are single-use disposable
items. This includes, but is not limited to, all
handpiece attachments, handpiece motors,
reusable prophy angles, reusable air and
water syringe tips and ultrasonic scaler tips.
CDA contacted the CDC Oral Health
Division, which provided additional
information. Recent studies have shown

Free eDelivery.

the internal portions of some low-speed

handpiece motors have the potential to
become contaminated when used with
both disposable and reusable prophylaxis
angles. The studies also have shown
that there is the potential for internal
contamination to be released through
the prophylaxis angle into the mouth
of a patient during subsequent uses.
In summary, unless all components of
handpieces are properly heat sterilized
between patients, there is the potential
for microorganisms to enter, remain and
then be released during use on patients.
The 2003 CDC guidelines provide
the following recommendations for
dental handpieces and other devices
attached to air and waterlines:
1. Clean and heat sterilize handpieces
and other intraoral instruments
that can be removed from the
air and waterlines of dental
units between patients.
2. Follow the manufacturers
instructions for cleaning, lubricating
and sterilizing handpieces and
other intraoral instruments that
can be removed from the air
and waterlines of dental units.
3. Do not surface disinfect, use
liquid chemical sterilants or
ethylene oxide on handpieces
and other intraoral instruments
that can be removed from the air
and waterlines of dental units.
Low-speed motors can be removed
from air and waterlines, and should
therefore be heat sterilized.
RESOURCES

Available for iPad, iPhone, Android or Kindle Fire.

Check it out at cda.org/apps.

138F E B R U A R Y 2 01 6

CDC. Guidelines for infection control in dental health-care

settings 2003. MMWR 2003; 52 (No. RR-17):1-66. www.
cdc.gov/mmwr/PDF/rr/rr5217.pdf.
Chin JR, Miller CH, Palenik CJ. Internal contamination of airdriven low-speed handpieces and attached prophy angles. J
Am Dent Assoc 2006;137:12751280.
Chin JR, Westerman AE, Palenik CJ, Eckert SG. Contamination
of handpieces during pulpotomy therapy on primary teeth.
Pediatr Dent 2009;31:7175.
Herd S, Chin J, Palenik CJ, Ofner S. The in vivo contamination
of air-driven low-speed handpieces with prophylaxis angles. J
Am Dent Assoc 2007;138:13601365.

Periscope

C D A J O U R N A L , V O L 4 4 , N 2

Periscope oers synopses of current ndings in

dental research, technology and related elds

PERIODONTICS

DENTAL MATERIALS

Nonsurgical periodontal therapy

Flowable dental composite

Slot DE, Jorritsma KH, Cobb CM, Van der Weijden FA. The eect
of the thermal diode laser (wavelength 808-980 nm) in nonsurgical periodontal therapy: A systematic review and meta-analysis. J
Clin Periodontol 2014 Jul;41(7):681-92.

Shouha P, Swain M, Ellakwa A. The eect of ber aspect ratio and

volume loading on the exural properties of owable dental composite. Dent Mater 2014 Nov;30(11):1234-44.

Clinical question: Is there an adjunctive eect of a diode

laser (DL) following nonsurgical periodontal debridement
(SRP) during the initial phase of periodontal therapy on
the clinical parameters of periodontal disease?
Material and methods: The MEDLINE-PubMed, CochraneCentral Register of Controlled Trials and EMBASE databases
were searched up to September 2013. Probing pocket
depth (PPD) and clinical attachment loss (CAL) were
selected as outcome variables. In addition, plaque scores
(PS), bleeding scores (BS) and the Gingival Index (GI) were
considered outcome measures. Data were extracted and a
meta-analysis (MA) was performed where appropriate.
Results: Independent screening of 416 unique papers resulted
in nine eligible publications. The MA evaluating PPD, CAL and
PS showed no signicant eect. A favorable adjunctive use of
the DL was observed for the outcome parameters GI and BS.
Conclusion: For PPD and CAL, the collective evidence
regarding adjunctive use of the DL with SRP indicates that the
combined treatment provides an eect comparable to that of
SRP alone. With respect to BS, the results showed a small but
signicant eect favoring the DL, however, the clinical relevance
of this dierence remains a question. This systematic review
questions the adjunctive use of DL with traditional mechanical
modalities of periodontal therapy in patients with periodontitis.
Bottom line: The adjunctive benet of using the diode laser with
traditional periodontal scaling and root planing is questionable.
Gerald Drury, DDS

Purpose: The objective of this study was to evaluate the ecacy

on exural properties of owable dental resin composite reinforced
with short glass ber of dierent aspect ratios (ARs) and volume
percent loadings. It is hypothesized that with the addition of
randomly oriented bers it is possible to signicantly improve
exural strength and modulus while maintaining owability.
Methods: Ten groups of samples with varying glass ber volume
loads (0, 5%, 10%, 20%, 40% and 60%) and three dierent
ARs (5.2, 68 and 640) were tested in three point bending to
fracture according to ISO 4049. A owable resin composite
was used as the control and as the lled resin composite that
was subsequently reinforced with bers. Load deection results
were used to calculate exural strength and exural modulus.
Scanning electron microscope images were used to determine the
mode(s) of failure, to describe surface features of reinforcement
and were correlated with force displacement graphs.
Results: When compared to the sculptable control (68.6 vol%
ller loaded) results for exural strength varied from a mean
reduction of 42 percent (p > 0.05) for the low AR group to an
increase of 77 percent (p < 0.001) for the high AR samples.
Flexural modulus results varied from a low of 6.6 [0.67] GPa for the
nonreinforced spatulated control to 20.3 [1.31] GPa (p < 0.001) for
the 60 percent loaded low AR group. The low ber loaded mid AR
group was still owable with 49 percent total loading (5% ber/44%
ller) but gave strength values (181.2 [33.5] MPa) 30 percent higher
than the sculptable control (p>0.05) and comparable modulus.
Clinical implications: This study shows that short and very short
glass bers can signicantly reinforce owable dental composite.
The bers aspect ratio was shown to be more important than volume
loading for exural strength. It appears possible to produce a light
cured short glass ber reinforced owable material with superior
exural properties compared to conventional universal composites.
Karen Schulze, DDS, PhD

F E B R U A R Y 2 0 1 6 139

Largest
Broker in
Northern
California
Extensive Buyer
Database &
Unsurpassed
Exposure allows
us to offer you

BAY AREA

BAY AREA CONTINUED

AC-335 SAN FRANCISCO: Great Practice! 2100

sf, 8ops in desirable location. Call for Details
$475k!
AG-511 SAN FRANCISCO: Trendy, tony West
Portal neighborhood. 800+ sf w 3 ops $315k
AN-490 SAN FRANCISCO: This is an opportunity
of a lifetime! 1,000 sf w/ 4 ops. $795k
AN-514 SAN FRANCISCO Facility: Located in the
bustling financial district! 1,007 sf w/4 ops.
$150k
BN-183 HAYWARD: Kick it up a notch by increasing the current very relaxed work schedule! 1,300 sf w/ 3 ops $150k
BN-279 CONTRA COSTA COUNTY: Excellent
Merger Opportunity! 2-story. 1,350 sf w/ 3
ops +1 addl $60k
BC-361 OAKLAND: Established for over 23+
years! 2,200 sf w/ 7 ops. Now Only: $330k
BC-381 PLEASANT HILL Facility: Open Floor
Plan! 1,852 sf w/ 6 equipped ops! $80k
BC-509 SAN LEANDRO: Facility Only, 800 sf,
3ops w/ xray in each op. Call for Details $60k
BG-407 SAN LEANDRO: Prof bldg. Great signage! 1200 sf w/ 3 ops $125k
BN-426 BERKELEY: Step into this quality practice and youll know you belong here! 1,384
sf w/ 3 ops. $495k
BC-432 PITTSBURG: Own this family-oriented
Practice! 1,640 sf w/ 6 ops. $350k
BN-463 FREMONT: Gross Revenues Exceeded
$590k in 2014 on 4 day work week! 1,720sf
w/ 3op + 4 addl. Now Only $435k!
BN-504 RICHMOND: Established Practice and
Real Estate! 1,450 sf w/ 2 ops + 2 addl
$100k /RE $700k
CC-456 SOLANO COUNTY: 2,997 sf w/ 6 Dr
ops + 2 Hyg ops +1 addl $850k
CN-482 SANTA ROSA: Rare Opportunity in
highly desirable area. 1050 sf w 3 ops $150k
DC-476 DUBLIN: Shared Facility. Great for Specialist - Endo, Pedo or Ortho. 1100 sf w/ 2
ops+1 addl $125k

DC-406 SAN JOSE: Amazing opportunity in

Westgate Shopping Center. 6 ops + 80 mall
hours per week $400k
DC-522 PLEASANTON: Location, Location, Location! Do not pass this opportunity! 2ops in
712sf office $149k
DG-499 SARATOGA Facility: 2 fully equipped
ops & room for 1 addl w 1,178sf . Move-In
Ready NOW ONLY $99.5k
DN-447 SUNNYVALE: Quality, family-oriented
opportunity awaits your talent and skill. 1,200
sf w/ 3 ops + 1 addl. $395k
DN-467 GILROY Facility: This traditionally
styled practice is perfectly situated! 1,325 sf
w/ 3 ops + 1 addl. $75k
DN-497 PLEASANTON Facility: Great Location!
870 sf w/ 3 ops + 1 addl. Owner Financing
w/10% Down! Reduced! $95k
DG-519 SANTA CLARA Facility: Move In Ready!
2240 sf w 6 fully equipped ops $225k

800.641.4179

NORTHERN CALIFORNIA
EN-340 SACRAMENTO: Large HMO practice!
3,400 sf w/ 10 ops and Plumbed for 1 addl
$950k
EN-378 LINCOLN: quality practice with a wonderful patient base! 1,369 sf w/ 2 op + 3 addl.
$170k
EN-379 ROSEVILLE: An amazing opportunity in
the location of your dreams! 1,040 sf w/ 3ops.
$295k
EN-464 ROCKLIN Facility: Dont miss out on
this remarkable opportunity! 2,150 sf w/ 4
ops. $150k
EN-475 ROSEVILLE Facility: Hesitate and you
might miss out on this opportunity! 875 sf
w/ 2 ops + 2 addl. $49.5k
EG-479 FOLSOM: History is alive here with
tributes to the past! 1,600 sf w/ 3ops.. $225k
EG-496 AUBURN: Associate-Driven HMO practice in Downtown. 3 fully equipped ops $315k

WPS@SUCCEED.NET

Timothy Giroux, DDS

Jon B. Noble, MBA

Mona Chang, DDS

John M. Cahill, MBA

Edmond P. Cahill, JD

NORTHERN CALIFORNIA CONTINUED

NORTHERN CALIFORNIA CONTINUED

EN-484 FOLSOM Facility: Come live, practice and grow here!

1,934 sf w/ 4 Ops. $150k
EN-503 FOLSOM Facility: Take a close look at this opportunity!
2,150 sf w/5 ops. Reasonable Offers Considered!
EG-508 FOLSOM Facility: Youll want to spend your days here!
1,500 sf w/ 4 ops + 1 addl. $60k
EN-516 CITRUS HEIGHTS: well-established, quality prac ce is loaded w/30+ years of goodwill. 1,358 sf w/ 3 ops + 2 addl $140k
EG-521 FOLSOM Facility: Stands out above the rest! Dont Miss
this one! 1,200 sf w 3 ops. Well Equipped! $115k
EG-523 SACRAMENTO: Spacious practice and Real Estate Available
NOW! Call for Details!
FN-299 FERNDALE: Live and practice on the beautiful North
Coast! 1,300 sf w/ 3 ops $195k (Real Estate: $309k)
FC-334 NORTHERN CA: Emphasis on prevention. 1,200 sf w/ 4 ops
$480k / Real Estate Also Available!
FC-415 FT. BRAGG: Excellent practice in peaceful, family-oriented
community! 1,800 sf w/ 5 ops + 1 hyg. Op. $425k
GC-472 ORLAND: Live & Practice in charming small town community.
1,000 sf w/ 2ops. Seller Retiring. $160k
GG-386 REDDING: Amazing Practice. Lease or Buy Real Estate!
2,860 sf w/ 4 ops. Plumbed for 2 addl! ONLY $275k
GG-453 CHICO: 5,000 sf 7 ops Perfect for 1 or more dentists! $395k
GG-454 PARADISE: ~2,550 sf w 9 ops. 40 yrs goodwill! Amazing Opportunity! $595k
GN-201 CHICO: Beautiful practice, major thoroughfare, stellar
reputation! 1,400 sf w/ 4 ops & room for another $425k
GN-244 OROVILLE: Must See! Gorgeous, Spacious. 2,500 sf w/5
ops! Collections over $450k in 2013. Only $315k
GN-258 REDDING: Pristine and attractive! Conveniently located!
2,100 sf w/ 3 ops + 2 addl. Now Only $300k!
GN-399 REDDING: Loyal patient base and relaxed workweek schedule. 1,440 sf w/3 ops. $150k
GN-418 REDDING: Goodwill Galore! Established for ~37 years. Seller
is retiring! 3,200 sf w/6 ops +2 addl. $495k
GN-507 CHICO: It just doesnt get any better than this! 3,000 sf w/
7ops. Practice $535k Real Estate $750k
HC-461 SONORA: In the beautiful Sierra Foothills, 4ops, 1350sf, free
-standing bldg.. Practice $700k & Real Estate Also Available!
HG-298 REDDING FOOTHILLS: HEALTH FORCES SALE! Includes Cerec!
2,000 sf w/ 5 ops. Practice $75k & Real Estate Also Available!
HN-213 ALTURAS: Close to Oregon Border. FFS practice is 2,200
sf w/ 3ops +1 addl $115k

HN-280 NO EAST CA: Only Practice in Town 900 sf w/ 2 ops $110k

HN-290 PLACERVILLE: Excellent Merger Op! FFS. 1,400 sf w/ 4 ops
$210k
HG-448 LAKE TAHOE AREA: Upscale Family Practice. 3400sf w 6 ops
$725k
CENTRAL VALLEY
IC-468 SAN JOAQUIN VLY: 2500+sf, 6 ops, Mo vated Seller, Price
Reduced. All oers considered! $350k
IG-367 MERCED: Newly Remodeled, Paperless. 1,550 sf w/4 ops
REDUCED! $305k
IN-397 FRESNO/MADERA: Focused on quality dental care & patient comfort! 2,000 sf w/5ops. Seller Motivated: $440k
IN-429 TRACY Facility: Move-in ready Hesitate and you might
miss out! 2,488 sf, 5 ops $245k/RE: $650k
IN-474 STOCKTON: Too good to be true? Absolutely not! 1,600 sf
w/ 3 ops. $95k
IC-468 SAN JOAQUIN VALLEY: High-End Restorative Practice! Dont
miss out! 2,500 sf w/ 6ops. $425k
IN-506 TURLOCK: Practice in the heart of the Central Valley!
2,000 sf w/ 5ops + 1 addl. $425k
IN-512 Merced: This immaculate practice is an absolute jewel!
1,200 sf w/ 4ops + 1 addl. $140k
JC-349 FRESNO Facility: Mo vated Seller re ring! Step right in and
make yours! Call for Details!
JG-491 FRESNO: Well-established. 40-50 new Pt/mo. 1,452 sf w/
4 fully equipped ops $425k
SPECIALTY PRACTICES
I-9461 CENTRAL VALLEY Ortho: 1,650 sf w/5 chairs/bays & plumbed
for 2 addl $180k
CC-346 SO MARIN CO Perio: 1,142 sf w/ 3 ops. Meticulously maintained! No reasonable oer will be refused! REDUCED! $199k
CG-424 NAPA Prostho: Office has Digital X-ray & NEW 3D Imaging
Unit! Ready for Experienced, high-end Prosthodontist! On track to
collect just under $1m $725k
CC-405 SOLANO CO. Endo: Endodontic Practice in a vibrant community! 1,250 sf w/ 4 ops. $485k
DC-459 SF PENINSULA Perio) 50% Partnership Buy In! Call for Details! $580k
CG-481 S SONOMA CO Ortho: 2070 sf w 7 chairs + 1 exam in Med/
Prof Plaza $295k
BG-517 NORTH EAST BAY Endo: Coming Soon! Call for Details!

ASK THE BROKER CAN NOW BE FOUND AT

WWW.WESTERNPRACTICESALES.COM

Tech Trends

C D A J O U R N A L , V O L 4 4 , N 2

A look into the latest dental and

general technology on the market

Articial Intelligence

Interpreting Text Messages

With the advent of products like Amazons Echo, the voice-activated

personal assistant for the home, it is looking more likely that some form
of articial intelligence will become commonplace in our lives in the
near future. In fact, a new study conducted by Ericsson ConsumerLab
Insights has found that 50 percent of smartphone users believe
smartphones will be replaced by articial intelligence within ve years.
This is Ericssons fth edition of its annual trend report, which also
found that eight out of 10 consumers would like to use technology
to enhance their vision, memory and hearing and 55 percent of
smartphone owners believe bricks used to build homes could include
sensors that monitor mold, leakage and electricity issues within the next
ve years.

If it wasnt already too dicult to interpret communication via text

message, a new study by Binghamton University has made matters
worse. The study revealed that ending a text message with a period
is perceived to be less sincere than those that dont end with a
period. The university surveyed 126 undergraduates for its study. The
participants read a series of exchanges that appeared either as text
messages or as handwritten notes. Particularly, one-word responses
such as Okay, Sure and Yeah that included a period were rated as
less sincere. The lead researcher was quoted as saying, Texting
is lacking many of the social cues used in actual face-to-face
conversations. When speaking, people easily convey social and
emotional information with eye gaze, facial expressions, tone of voice,
pauses and so on Thus, it makes sense that texters rely on what they
have available to them emoticons, deliberate misspellings that mimic
speech sounds and, according to our data, punctuation.

Blake Ellington, Tech Trends editor

Adobe Post (Adobe Systems Inc., Free)

Blake Ellington, Tech Trends editor

Social media is the perfect place to capture the attention of followers

with stunning graphics. With Adobe Post for iPhone, users can create
and share captivating visual presentations with no prior experience
needed. An Adobe ID or Facebook account is required to use this app.

Hue (Phillips, Hue white E26 starter kit, $79.95; Hue white

Post starts with a series of premade templates called Remix Posts

that users can edit and customize according to their needs. Users can
also create new posts from scratch without any templates. Any text
can be sized or oriented using touch screen gestures. Options for text
include adjustments for color, font, background shape, alignment,
opacity and spacing. Photos can be obtained from the iOS camera or
photo library in addition to online sources from the public domain or
Adobe Lightroom and Creative Cloud. Color palettes and photo lters
can be dynamically applied and changed when creating the post.
Users share their posts on Facebook, Twitter and Instagram through
the Share Sheets feature for iOS apps. In addition, users can also
save their posts to the iOS camera roll.
With the ability to create social media graphics like an expert
designer, Adobe Post for iPhone empowers novice users, especially
those with small businesses under minimal budgets, to create visually
impactful presentations that deliver powerful messages.
Blake Ellington, Tech Trends editor

E26 bulb, $14.95)

When considering the so-called Internet of Things, in which myriad
devices are connected and controllable via the Internet, light bulbs
may not be the rst thing users would think of wanting to control
via their smart device. However, Philips has managed to deliver
surprisingly useful functionality by way of its Hue lighting system.
The bulbs themselves are energy-ecient LED lights that can be
controlled (and synchronized) to radiate the full spectrum of colors,
as well as dim, ash, pulse and more and are available in a variety
of shapes, sizes and models (bulbs, lighting strips, etc.).
The brain of the system is the Hue bridge, which connects your smart
device to the Philips Hue lights. Linked to Wi-Fi via your router, the
bridge allows for the control of up to 50 lights and accessories and is
accessible and controllable via the Internet for out-of-home control.
In practice, the Hue lighting system is both impressive and fun with
dozens of pre-created scenes that are accessible via the Hue app.
Plus, the system is also Apple HomeKit compatible, allowing for direct
control via Siri on iOS devices.
Blaine Wasylkiw, CDA director of online services

142F E B R U A R Y 2 01 6

What will you discover in Anaheim?

Community. Foster fantastic teamwork

through shared experiences at CDA
Presents. Get connected to your team,
and collaborate with your colleagues and
mentors, during interactive workshops,
networking events and after-hours fun.

Thurs.Sat.
May 1214,
2016

Anaheim
Convention
Center

Register today
cdapresents.com

The Art
and Science
of Dentistry

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