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CHRONIC LYMPHOCYTIC
LEUKEMIA
DIAGNOSIS, TREATMENT OPTIONS
AND PROGNOSIS
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CANCER ETIOLOGY, DIAGNOSIS
AND TREATMENTS
CHRONIC LYMPHOCYTIC
LEUKEMIA
DIAGNOSIS, TREATMENT OPTIONS
AND PROGNOSIS
KIMBERLY RODRIQUEZ
EDITOR
New York
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Preface vii
Chapter 1 The Chemokine Receptor CCR3 in Chronic
Lymphocitic Leukemia: Possible Biological Role
in the Course of the Disease 1
R. Vladimirova, E. Vikentieva, D. Popova, I. Gigov,
J. Raynov, A. Mihova and M. Guenova
Chapter 2 New and Repositioning Approaches to the Treatment
of Chronic Lymphocytic Leukemia 15
Ida Franiak-Pietryga and Maria Bryszewska
Chapter 3 Prognostic and Predictive Indicators in Chronic
Lymphocytic Leukemia 77
Nili Saar, Pia Raanani and Uri Rozovski
Chapter 4 Molecular Cytogenetic Abnormalities in Chronic
Lymphocytic Leukemia: Prognostic Implications 115
Prabhjot Kaur
Index 149
PREFACE
(P < 0.001). CCR3 levels on B-cells didnt differ significantly among the risk
groups, while the expression on T-lymphocytes showed significant variations
(P = 0.020). A positive correlation between the levels of CCR3 on B-cells and
T-cells was found in the total group of patients, and particularly in stage Rai 0
(P < 0.001) and Rai I/II (P = 0.005). Cases with levels exceeding 10% of the
B-cells were characterized with higher WBC (P < 0.001), lymphocytes
(P = 0.003) and -cells absolute counts (P = 0.001). Positive correlations were
also found with -microglobulin (P = 0.027), Rai stage (P = 0.008) and risk
groups (P = 0.008). The higher the level of CCR3+ B-cells was detected, the
stronger the correlations were found.
These findings in regard to CCR3 in CLL-patients imply a possible
biological role of the receptor in the course of the disease. Besides, the high
levels of CCR3 on B-lymphocytes as well as on T-cells suggest a common
mechanism of activation of the two lymphocytic populations.
Chapter 2 Great progress has been made in the diagnosis and treatment
of Chronic Lymphocytic Leukemia (CLL), but this type of leukemia still
remains incurable, and the introduction of new drugs and new therapeutic
strategies are still desirable. For the last 20 years, significant progress in
molecular biology has resulted in better characterization and understanding of
the biology and prognosis of CLL. On the basis of recent research it has been
known that the critical role in the pathogenesis of CLL plays B-cell receptor
(BCR) activation and this provide new opportunities for the development of
innovative, more effective therapies. Recently, several small molecular kinase
inhibitors targeting the proximal BCR signaling pathway, including Brutons
tyrosine kinase (BTK) inhibitor (ibrutynib), and phosphatidylinositol 3-kinase
p110 (PI3Kp110) inhibitor (idelalisib), have been developed. These two
drugs are brand new, approved by US Food and Drug administration in 2014
and they are very promising in new personalized treatment strategy. Among
the new-targeted therapies, much attention is being paid to agents with a high
potential for triggering apoptosis in tumor cells. The PI3K/AKT signaling
pathway plays a crucial role in regulating survival of CLL B cells. The
inhibitors of this pathway, OSU-T315, MK2206 has also a strong antileukemic
activity in a very unique mechanism of action. Novel mechanism of abrogating
the AKT pathway and inhibition of BCR activity in CLL cells can be very
useful in the treatment of high risk leukemia, with del(17p13.1), unmutated
IGVH or those who may be resistant to ibrutinib.
Treatment approach in CLL has changed during last years. Purine
analogs (fludarabine, cladribine), pentostatins or other chemotherapeutics
(bendamustin) seem to be less effective and more toxic than monoclonal
Preface ix
In this chapter the authors review the various prognostic markers that help
clinicians predict the clinical course and response to therapy of treatment nave
CLL patients. They highlight IGHV mutation status as a predictive marker that
could assist in selecting treatment from the growing arsenal of available drugs
for CLL.
Chapter 4 CLL is the most common leukemia in the western world.
Mostly a disease of the elderly, this indolent disorder, has a very variable
clinical outcome and can evolve into an aggressive lymphoma. As indolent
disorders are considered incurable, treatment is delayed until the patient
progresses to high stage disease. These therapeutic decisions are based on
criteria devised by Kanti Rai and Jacques Louis Binet almost four decades
ago; this assessment requires only a physical examination and a complete
blood count. A significant change that has occurred in the past four decades is
the increased diagnosis of patients in low stage disease (during regular check-
ups for employment or other causes). In the 1970s, 40% of the patients were
diagnosed at stage I. In the 2000s, nearly 80% of the patients are diagnosed at
low stage. Although patients are diagnosed at an earlier stage, it is difficult to
determine the prognosis of Rai stage 0/1 patients. A subset of patients with
early stage CLL will rapidly evolve to a more aggressive and fatal disease, and
clinical staging, as it currently exists, fails to predict this progression.
Approximately 80% of individuals with CLL have acquired chromosomal
abnormalities within their malignant clone and can be categorized into five
prognostic groups accordingly: deletion 13q (median survival, 133 months);
deletion 11q (median survival, 79 months); trisomy 12 (median survival, 114
months); normal cytogenetics (median survival, 111 months); and deletion 17p
(median survival, 32 months). A complex cytogenetic karyotype can be
identified rarely and is commonly associated with poor prognostic features
including CD38 expression and unmutated IgHV. IGHV3-21 usage is an
independent poor prognostic factor. Novel mutation in CLL include the
NOTCH1 mutations, SF3B1 mutations, BIRC3 mutation among others. B cell
receptors play a unique role in the pathogenesis of the disease and has
prognostic and therapeutic implications. Stereotyped B cell receptors impact
outcome. New hierarchal classification is being suggested to include the
conventional cytogenetic markers and novel mutations. MicroRNAs (miRNA)
are short (~22nt in humans), endogenous non-coding ssRNA (single stranded)
molecules that regulate gene expression via translational repression or
transcript degradation. Recent evidence has shown that miRNAs are key to
gene regulation and have a global effect on various oncogenic, tumor
suppressor and cell survival pathways. Over the last decade several miRNAs
Preface xi
Chapter 1
ABSTRACT
Chemokines and their receptors play a crucial role in cell migration
in physiological and pathological settings. The study focuses on the
impact of the expression of the chemokine receptor CCR3 in patients
with chronic lymphocytic leukemia (CLL). CCR3 is expressed upon a
variety of immune cells as well as on anaplastic large cell lymphoma
cells. CCR3 expression on CLL-cells was reported in a small group of
patients, though the expression levels on B and T-cells and the clinical-
laboratory correlations remain unknown. Peripheral blood from a total of
91 untreated patients, staged according to Rai Staging System and
*
Corresponding author: Rositsa Vladimirova Andreeva, Department Cytogenetic and
immunology Military Medical Academy, Bulgaria 1606 Sofia 3 St. Georgi Sofiiski
Street, Tel: +359 888 351 777, e-mail: rossy_vladimirova@yahoo.com.
2 R. Vladimirova, E. Vikentieva, D. Popova et al.
INTRODUCTION
Chronic lymphocytic leukemia (CLL) is a neoplasm of B-lymphocytes
immunophenotypically characterized by the aberrant coexpression of CD5,
CD23 and forming proliferation centers in tissue infiltrates [1]. Although
generally with indolent clinical course, the disease poses serious medical,
pharmaco-economic and social problems in the clinical practice. CLL is a
heterogeneous disease at the clinical, molecular and cellular levels and
survival times vary widely depending on the clinical stage and risk group [2].
In recent years, intensive studies have been aimed at clarifying the main
pathogenetic mechanisms and therapeutic options. Chemokines and the
corresponding receptors play a crucial role in tumor progression and tumor
cell dissemination [3]. Up to now, several chemokines have been demonstrated
to be involved in the migration of CLL cells to lymph nodes, secondary
lymphoid organs and bone marrow. Besides, some chemokines are known to
have an anti-apoptotic effect and thus contribute to the survival of CLL cells
[4].
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 3
Flow Cytometry
Table 1. Distribution of patients by sex, age, Rai stage and risk groups
Statistical Analysis
Statistical analysis was carried out using the SPSS 21.0 software package
(SPSS Inc., Chicago, Illinois, USA). Continuous data parameters were
analyzed for normality using the W-ShapiroWilk test; data were presented as
median and (P10 P90), because variables had nonparametric distribution. A
nonparametric U-MannWhitney test and KruskalWallis test (for continuous
variables) were used. Spearmans rank correlation () was used to measure the
relationship between variables. Groups were assumed to differ significantly
when the P value was less than 0.05 and highly significant when the P value
was less than 0.001.
RESULTS
Flow cytometry allowed for detecting CCR3 both on normal and leukemic
B-cells as well as on T-cells in all samples of enrolled patients and healthy
controls though in a very wide range: 0.2 - 100% of B-cells and 0.1 - 100% of
T-cells. The percentage of positive B-cells was significantly higher in
leukemic samples compared with healthy controls median value and P10 -
P90 was 5% (0.7 40.6) vs. 0.5% (0.2 2.0), respectively (P < 0.001). The
same was found in regard to T-cells: the median value and P10 - P90 was
22.4% (1.5 98.0) in CLL vs. 1.4% (0.1 - 17.7) in the healthy control group
(P < 0.001) (Table 2). In addition, the highest levels of CCR3 expression on
B-cells as well as on T-cells were detected in high-risk patients, however,
being statically significant only in regard to the T-cell compartment
(P = 0.020) (Table 2).
Chemokine receptor CCR3 expressed by monoclonal B-lymphocytes
show positive correlation with the expression of CCR3 on T-cells in the total
group of patients with CLL (P < 0.001). The expression of CCR3 on T-
lymphocytes shows sufficient positive correlation with the expression levels of
CD49d (P = 0.006) (Table 3). Expression of CCR3 higher than 10% was
found in 28 patients throughout all risk groups. There were 10 cases in the low
risk group, 6 cases in the intermediate risk group (Rai I 4; Rai II 2) and
12 patients in the high-risk group (Rai III 5; Rai IV - 7). The CCR3 values in
these patients had positive correlations to the absolute count of leukocytes (P <
0.001), lymphocytes (P = 0.003), monoclonal B-cells (P = 0.001), to the
percentages of lymphocytes (P = 0.047) and monoclonal lymphocytes (P =
0.028), as well as to the 2-microglobulin concentration (P = 0.027). The
6 R. Vladimirova, E. Vikentieva, D. Popova et al.
positive correlations with the Rai stage (P = 0.008) and risk groups (P =
0.008) are moderate. The correlation between CCR3 and the haemoglobin
level was negative (P = 0.017) (Table 3). Expression of CCR3 higher than
20% was found in 18 patients, with 61% of them belonging to the high risk
group, 4 patients were in the low risk group, 3 patients in the intermediate
risk group (Rai I 1; Rai II 2) and 11 patients in the high risk group (Rai III
5 Rai IV - 6), in other words 7 patients were in the low and intermediate risk
groups, and 11 patients belonging to the high risk group, with 56 of the rest of
the patients being in the low and intermediate risk groups and 17 patients in
the high-risk group. In the case of this higher level of expression of the
receptor (%CCR3+ B-cells > 20%), the existing correlations with absolute
count of leukocytes, lymphocytes, monoclonal B-cells, with the percentage of
lymphocytes and B-lymphocytes as well as with 2-m concentration increase
their power (Table 3).
The correlations of the studied chemokine receptor changed within the
risk groups. The correlation between the expression of chemokine receptor on
the B- and T-cells is the strongest in the low risk group (P < 0.001), the
intensity of this correlation decreased in the intermediate risk group (P =
0.005) and was not found in high-risk patients. Aberrant expression of CCR3+
on monoclonal B-lymphocytes in the Rai 0 stage shows negative correlation to
platelets count (P = 0.022) and positive correlation to the -cells percentage
(P = 0.019) and the expression of Zap-70 (P = 0.010), while within the high-
risk group only the positive correlation to the expression of CD49d (P = 0.022)
was observed (Table 4). Inflammatory chemokine receptor CCR3 on T-
lymphocytes shown positive correlation with the levels of CD49d expressing
B-cells in the intermediate risk group (P = 0.014) (Table 4).
Rai Rai
Control CLL P Rai 0 P
I/II III/IV
(n = 17) (n = 91) value (n = 30) value
(n = 33) (n = 28)
%
0.5 5.0 5.6 3.7 7.7
CCR3+ <0.001 NS
(0.2 - 2.0) (0.7 - 40.6) (0.7 - 21.8) (1.0 - 22.2) (0.6 - 72.8)
B-cells
%
1.4 22.4 20.8 5.5 53.4
CCR3+ <0.001 0.020
(0.1 - 17.7) (1.5 - 98.0) (0.6 - 90.8) (0.62 - 90.8) (3.41 - 99.7)
T-cells
Median and (10 90); NS - not significant
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 7
DISCUSSION
Chemokines and their receptors play an important role in the tumour
development process, by directing cell traffic to the tumour microenvironment
or facilitating its exit from it. The general conclusion from the scientific
literature data brings to the fore the detection of aberrantly expressed
chemokine receptors from tumour cells [15].
Chemotaxic receptor CCR3 connects with a wide range of ligands -
CCL5, CCL2, CCL7, CCL11 (eotaxin), CCL13, CCL15, CCL24 (eotaxin - 2),
CCL26 (eotaxin - 3) and CCL28 with all bonds causing activation of the
receptor. The ligands of CCR3 bind it through a variable-strength connection,
with it being the strongest for CCL11, followed by CCL13 > CCL26 > CCL24
and CCL5 [10]. The binding of this eotaxin activates intracellular signals, the
final result being activation and migration.
A study of CCR3 expression in a small number of patients with CLL
found aberrant expression of the receptor on neoplastic cells and establishes
the higher migration activity of B-lymphocytes expressing CCR3 [11].
Therefore we aimed at investigating the chemokine receptor CCR3 (CD193)
expression on B- and T-lymphocytes in a larger cohort of CLL patients in
comparison to healthy controls and to correlate data with major clinical and
laboratory data in order to reveal a possible role for the biological course of
the disease.
In the present study we found increased levels of aberrant expression on
B-cells of patients with CLL compared to healthy controls, with the highest
levels of CCR3 reported in the high-risk group, as well as significantly higher
levels of positive T-lymphocytes. The proportion of positive monoclonal B-
cells showed a significant correlation with the CCR3-positive T-cells in CLL
patients which could suggest the presence of a common mechanism of
activation of the B- and T-cells through CCR3 in the case of disease. Cytokine
or chemokine stimulation in patients with CLL could probably be the reason
for the presence and the degree of this aberrant expression as there is at least
one in vitro study of isolated peripheral-blood and germinative B-cells that
proved the ability of B-lymphocytes to express CCR3 after stimulation with
IL-2 and IL-4 [16].
With the increase of CCR3 levels on B-lymphocytes an increasingly
strong connection between the receptor and adverse clinical factors such as
higher tumour burden, translated into higher leukocyte, lymphocyte and
monoclonal B-cell counts, 2-microglobulin levels, as well as decreased
haemoglobin levels and higher Rai stages, respectively could be found.
The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 9
CONCLUSION
The findings from the present study of the increased expression of CCR3
in patients with CLL and correlations with the disease characteristics clearly
demonstrate the role of this aberrant expression in the biological course of
disease. Further studies are warranted in order to elucidate the precise
mechanisms of possible common activation of the B- and T-cell compartments
and the pathogenetic consequences which might be the basis for investigating
novel therapeutic approaches.
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The Chemokine Receptor CCR3 in Chronic Lymphocitic Leukemia 11
BIOGRAPHICAL SKETCH
Name: Rositsa Vladimirova Andreeva
Affiliation: Member, European Research Initiative on CLL
Member, Bulgarian Medical Society of Hematology
Member, Bulgarian Association for Clinical Immunology
Education: 1990 Master of Biology - Sofia University St Kliment
Ohridski Faculty of Biology
2014 PhD Military Medical Academy, Sofia; Bulgaria; Hematology
Address: Bulgaria; 1606 Sofia,
Department Cytogenetic and Immunology
Military Medical Academy
3 St. Georgi Sofiiski Street
Research and Professional Experience: Flow cytometry in neoplastic
hematology; Clinical Immunology
12 R. Vladimirova, E. Vikentieva, D. Popova et al.
Chapter 2
ABSTRACT
Great progress has been made in the diagnosis and treatment of
Chronic Lymphocytic Leukemia (CLL), but this type of leukemia still
remains incurable, and the introduction of new drugs and new therapeutic
strategies are still desirable. For the last 20 years, significant progress
in molecular biology has resulted in better characterization and
understanding of the biology and prognosis of CLL. On the basis of
recent research it has been known that the critical role in the pathogenesis
of CLL plays B-cell receptor (BCR) activation and this provide new
opportunities for the development of innovative, more effective therapies.
Recently, several small molecular kinase inhibitors targeting the
proximal BCR signaling pathway, including Brutons tyrosine kinase
*
Corresponding Ida Franiak-Pietryga, Department of Clinical and Laboratory Genetics, Medical
University of Lodz, Pomorska 251, 92-213 Lodz, Poland; Telephone/Fax: +48 42 272 53
60; Email: ida.fp@interia.pl; ida.franiak-pietryga@umed.lodz.pl.
16 Ida Franiak-Pietryga and Maria Bryszewska
1. INTRODUCTION
Chronic lymphocytic leukemia (CLL) is a type of leukemia most
commonly diagnosed in Western Europe and North America, with an
incidence rate of 4.2/100,000 [1] Although the median age at initial diagnosis
is 67-72 years [2], with approximately 70% of patients being over 65, and a
male to female ratio of 1.7:1 [3], CLL is now increasingly affecting younger
patients between 20 and 60 years old. The only confirmed risk factor is a
family history of this disease. For relatives of CLL patients, the risk increases
by a factor of 2.5-7.5 [4].
The onset of the disease is usually asymptomatic; only abnormalities in
whole blood count such as leukocytosis with lymphocytosis are found.
Nowadays, CLL is diagnosed more often at an early, asymptomatic stage due
to more frequent routine blood tests. More advanced stages are characterized
by lymphadenopathy, hepatomegaly/splenomegaly, recurrent infections,
weakness, pallor and hemorrhagic diathesis, and general symptoms such as
weight loss, fever and night sweats are observed.
In recent years, there has been significant progress in the treatment
of CLL, firstly thanks to the introduction of immunochemotherapy with
monoclonal antibodies, and also through the use of small molecules, such as
tyrosine kinase inhibitors, targeting B-cell receptor signaling. Alkylating
agents and glucocorticoids were first used in the treatment of CLL in the
1950s [5, 6]. Purine analogues (cladribine, fludarabine and pentastatin) were
introduced in the 1980s. The development of rituximab by IDEC
pharmaceuticals and its subsequent FDA approval for treatment of non-
Hodgkin lymphoma in 1997 introduced chemoimmunotherapy regimens
which to this day remain the standard approach to initial therapy of younger
patients with CLL [7]. Interestingly, prednisone was the first agent that could
be regarded as an example of targeted therapy in CLL. Steroid hormones
modulate many intracellular pathways, two of which allow it to target
lymphoid malignancies. Firstly, glucocorticoid receptors interfere with the
activity of the nuclear factor B (NF-B) pathway; by inducing the
production of the inhibitory protein inhibitor B (IB), they promote
sequestration of NF-B in the cytoplasm [8]. This impairs transcription of NF-
B target genes and reduces cell proliferation and survival. Secondly,
glucocorticoids bind and inhibit AP-1 transcription factor activity, which is
necessary for cell proliferation [9].
Frontline therapies for CLL have been based on the administration of
cytostatic drugs (chlorambucil, fludarabine), which in many cases, control the
18 Ida Franiak-Pietryga and Maria Bryszewska
disease efficiently and are well tolerated [2]. However, patients carrying
certain prognostic markers, such as del(17p) or unmutated IGHV, do not
respond well to these therapies [10]. CLL treatment has greatly improved with
the development of more specific and targeted agents. Most of these new
agents are currently undergoing clinical trials or have already been approved,
following promising results in the majority of treated CLL cases. This review
first summarizes the available information of these novel agents in CLL, and
then discusses the future incorporation of other new compounds into the
therapeutic armamentarium.
2.1.1.1. Ibrutinib
PCI-32765 (Pharmacyclics) is a first-in-class oral covalent inhibitor of
Brutons tyrosine kinase that has been approved for the treatment of CLL
New and Repositioning Approaches to the Treatment 19
patients who have received at least one prior therapy, and as primary therapy
for those with a chromosome 17p13.1 deletion [16, 17]. This agent forms a
bond with the cysteine-481 of BTK [18]. It also inhibits several other kinases,
such as ITK (interleukin-2-inducible T-cell kinase), TEC, BMX, and EGFR.
Ibrutinib has demonstrated promising activity in studies involving relapsed
follicular lymphoma, CLL/SLL, diffuse large B-cell lymphoma and other non-
Hodgkin lymphoma subtypes, either as a single agent or in combination with
other drugs [19-23]. Byrd et al. [24] published results of 101 patients with
relapsed or refractory CLL (RESONATE trial) who received ibrutinib, 34% of
the patients had del(17p), and 78% had unmutated IGHV. The median number
of prior therapies was four, the median age was 64 years. The overall response
rate (ORR) was 90% with a 7% complete remission (CR) and 65% partial
remission (PR). The estimated progression-free survival (PFS) at 30 months
was 69%. For patients with del(17p) and del(11q), the median PFS was 28
months and 38.7 months, respectively, and was remarkably inferior to that of
patients without those aberrations [24]. In a pivotal phase 3 of this trial,
patients with relapsed or refractory CLL were randomized to receive ibrutinib
(n=195) or ofatumumab (n=196). The ibrutinib arm had much higher ORR and
superior PFS and overall survival (OS) as compared to ofatumumab arm [25].
In early-phase data from 31 previously untreated patients with CLL who were
65 years of age or older, ORR with ibrutinib was 84% (with CR in 23% of the
patients); the estimated rate of PFS at 30 months was 96%, and OS rate was
97%, with 81% of the patients continuing to take daily ibrutinib after three
years of follow-up [24]. In phase 3 of RESONATE-2 (NCT01722487), a
multicenter, open-label, randomized trial, the efficacy and safety of single-
agent ibrutinib was compared with those of chlorambucil in patients with
median age of 73 years with previously untreated CLL [26]. During a median
follow-up period of 18.4 months, ibrutinib resulted in significantly longer PFS
than chlorambucil (median not reached vs. 18.9 months), with a risk of
progression or death that was 84% lower with ibrutinib than with
chlorambucil. Ibrutinib significantly prolonged OS rate: at 24 months, the OS
was 98% vs. 85% with chlorambucil, and the OR with ibrutinib was 86% vs.
35% with chlorambucil [26].
It is important to note that most patients develop lymphocytosis after
initiating ibrutinib therefore a novel category of response - PR with
lymphocytosis - was reported. This is assumed to be due to trafficking of CLL
cells from the lymph nodes and other tumor sites into the peripheral blood,
likely due to inhibition of several molecular pathways involved in adhesion,
20 Ida Franiak-Pietryga and Maria Bryszewska
including CXCR4/5 [27, 28]. This problem generally resolves over the course
of between 6-9 months, but about 20% of patients have lyphocytosis over 12
months of ibrutinib treatment [29]. Persistent lymphocytosis is more frequent
in patients with mutated IGHV and in those with del(13q) [27]. Development
of lymphocytosis does not appear to be detrimental to long-term clinical
outcomes [27, 29, 30]. The high activity of ibrutinib in CLL with del(17p) is
remarkable and exceeds the efficacy of high-dose methylprednisone
combinations in this setting [24]. Ibrutinib induced responses in 68% of such
patients, whereas a PFS of 10 months was documented after treatment with
high dose steroids in combination with alemtuzumab [31]. The results of
combining ibrutinib with rituximab [32], ofatumumab [33], ublituximab [34]
or bendamustine [35] in patients with relapsed or refractory CLL high risk
disease including del(17p), and/or del(11q), and/or TP53 mutation look very
promising. However, some preclinical studies have reported antagonism
between ibrutinib and rituximab, probably due to the inhibition of ADCC by
ibrutinib [36-38], the data from these clinical trials (NCT02007044,
NCT02301156, NCT02264574, NCT02048813, NCT01886872) looks very
appealing. Other clinical trials with ibrutinib for treatment-nave and
previously treated CLL patients (RESONATE, HELIOS, RESONATE-2), for
CLL patients with del(17p) (RESONATE-17), for patients with mantle cell
lymphoma (SPARK, SHINE, RAIN), follicular lymphoma (DAWN) continue.
Resistance to ibrutinib has been under study. In several patients who
progress on ibrutinib, a mutation of BTK C481S and gain of function
mutations in PLC2, a signaling molecule causing a downregulation of BTK,
have been found [39]. This mutation reduces the binding affinity of ibrutinib
to BTK, and only allows for reversible BTK inhibition leading to transient
BTK inhibition [40].
2.1.1.2. Spebrutinib
CC-292 (AVL-292; Celgene) is a covalent small molecule Bruton's
tyrosine kinase (BTK) inhibitor and unlike ibrutinib, it does not inhibit the
SRC family kinase or ITK. This drug has indication to treatment not only in
CLL but also in B-cell Non-Hodgkins Lymphoma (B-NHL) and
Waldenstrms macroglobulinemia (WM). A total of 83 patients with relapsed
or refractory CLL were enrolled on a phase 1 study (NCT01351935) i
ntended to evaluate the safety and tolerability of AVL-292 as monotherapy
in subjects with relapsed or refractory CLL, B-NHL or WM [41]. The
most frequent grade adverse events were found to be neutropenia (21%),
New and Repositioning Approaches to the Treatment 21
2.1.1.3. Acalbrutinib
ACP-196 (Acerta Pharma) is a novel irreversible second generation BTK
inhibitor which is more selective for BTK than ibrutinib [43]. Acalabrutinib
binds covalently to Cys481 with improved selectivity and in vivo target
coverage compared to ibrutinib and CC-292 in CLL patients [43-45]. In
the in vitro signaling assay on primary human CLL cells, acalabrutinib
inhibited tyrosine phosphorylation of downstream targets of ERK, IKB, and
AKT [46]. Acalabrutinib demonstrated higher selectivity for BTK with IC50
determinations on nine kinases with a cysteine residue in the same position as
BTK [43]. Importantly, unlike ibrutinib, acalabrutinib did not inhibit EGFR,
ITK, or TEC [43, 46]. Compared with ibrutinib, acalabrutinib has much higher
IC50 (>1000 nM) or virtually no inhibition on kinase activities of ITK, EGFR,
ERBB2, ERBB4, JAK3, BLK, FGR, FYN, HCK, LCK, LYN, SRC or
YES1 [46]. The in vivo effects of acalabrutinib against CLL cells have been
demonstrated in a NSG mice model with xenografts of human CLL [47]. The
drug significantly inhibited proliferation of human CLL cells in the spleens of
NSG mice at all dose levels, as measured by the expression of Ki67. The
tumor burden decreased with the treatment in a dose-dependent manner.
Acalabrutinib inhibited BCR signaling by reducing the phosphorylation
of PLC2 and transiently increased CLL cell counts in the peripheral
blood [47]. To further assess the safety, efficacy, pharmacokinetics,
and pharmacodynamics of acalabrutinib in human CLL, a phase 1/2,
multicenter, open-label, and dose-escalation clinical trial has been ongoing
(NCT02029443). In the latest report, 61 patients with relapsed CLL were
enrolled [46]. These patients had relapsed or refractory CLL, 31 % of them
had del(17p) and 75 % had IGHV unmutated. In the phase 1 portion of this
study, patients were treated with acalabrutinib at an increasing dose of 100 to
400 mg once daily. In phase 2, the expansion phase, 100 mg was administered
twice daily. After a median follow-up of 14.3 months, the ORR was 95%, with
22 Ida Franiak-Pietryga and Maria Bryszewska
85% PR and 10% PR-L. The ORR was 100 % in those patients with
chromosome del(17p). The most frequent adverse events included headache,
diarrhea, and weight gain. There were no dose-limiting toxicities observed in
the phase 1 portion of the trial. Acalabrutinib does not inhibit EGFR, thus
could reduce the adverse events on skin rash and severe diarrhea [46].
At this time, a phase 3 study (NCT02477696) commenced comparing
acalabrutinib with ibrutinib in high-risk patients (del(17p), del(11q)) with
relapsed CLL. Additionally, studies in treatment-nave CLL are also being
conducted: (NCT02475681) (obinutuzumab + ACP-196 vs. obinutuzumab +
chlorambucil vs. ACP-196); and ACP-196 in combination with Obinutuzumab
in relapsed or refractory or untreated CLL/SLL/PLL (NCT02296918); and
ACP-196 in patients with relapsed or refractory and treatment-nave deletion
17p CLL/SLL (NCT02337829). A comparison of acalabrutinib with ibrutinib
and other BTK inhibitors will be the only way to confirm the advantages and
disadvantages of these agents.
2.1.1.4. ONO-4059
ONO-4059 (Ono Pharmaceuticals LTD/Gilead Sciences, Inc.) is a highly
potent and selective oral BTK inhibitor, which is more selective for BTK than
ibrutinib. This compound demonstrated anti-tumor activity in pre-clinical
models and in clinical trials in both CLL and NHL patients. ONO-4059
has a favorable safety profile along with promising efficacy over a long
duration in heavily pre-treated CLL, including refractory and currently poor
prognostic del(17p) patients [48]. In an ongoing phase 1 study, ONO-4059
was administered at doses ranging from 20 to 600mg in 25 heavily-pretreated
patients. The treatment was well tolerated, with mostly grade 1-2 adverse
events. Six patients had grade 3-4 neutropenia. The ORR was 84% with a
similar response rate in patients with del(17p); 17 patients had PR and 4 PR-L
for 21 responding patients [48].
In addition to the above mentioned new BTK inhibitors, a few more are
currently undergoing clinical trials: GDC-0834 [49, 50], CGI-560 [51], CGI-
1746 [51], HM-71224 [52], CNX-774 [53], LFM-A13 [54] and RN-486 [55]
(Table 1). All have been developed to target BTK and disrupt the survival of
normal B-cells and their malignant counterparts.
Table 1. Novel BTK inhibitors potentially effective in CLL
2.1.2.1. Idelalisib
GS-1101 (formerly CAL-101; Gilead Sciences) is an orally bioavailable,
small-molecule inhibitor of PI3K. Idelalisib has recently gained approval for
the treatment of relapsed/refractory CLL. It has been evaluated in a phase I
clinical trial in 54 CLL patients with relapsed/refractory disease; nodal
shrinkage and overall survival were obtained in 81% and 72% patients
respectively [60]. Idelalisib has shown considerable monotherapy activity
when given at dose levels of 100mg or higher twice daily in patients with
heavily pretreated indolent non-Hodgkins lymphoma. About one-fourth of the
patients had del(17p), and 91% had unmutated IGHV. The median number of
prior therapies was five. In the dose escalation phase, six dose levels of oral
idelalisib (50-350 mg once or twice daily) were tested. The ORR was 72%
(39% PR, 33% PR-L). The median PFS was 15.8 months. The most commonly
noted grade 3 adverse events were pneumonia (20%), neutropenic fever
28 Ida Franiak-Pietryga and Maria Bryszewska
(11%), and diarrhea (6%) [60]. Idelalisib was found to demonstrate substantial
activity in older, treatment-nave patients with CLL when used as a single
agent [61].
In a phase III clinical trial, idelalisib combined with the anti-CD20
antibody rituximab significantly improved progression-free survival (81%)
and overall survival (91%) in relapsed CLL patients (n = 220) compared to
placebo plus rituximab [62]. In this study, combination therapy achieved a
response and survival benefit compared with rituximab alone [62]. The
patients receiving idelalisib and rituximab demonstrated improved rates of OR
(81%) in comparison with those with rituximab monotherapy (13%). Median
PFS was 5.5 months in the rituximab arm, but was not reached in the idelalisib
combined with rituximab group. Moreover, OS at 12 months was 92% for
idelalisib combined with rituximab, compared to 80% for rituximab alone
[62]. Idelalisib combined with rituximab therapy is also very effective in
patients with high risk CLL (with del(17p), TP53 mutations, del(11q)) [63].
Another phase 3, randomized study evaluating the efficacy and safety of
idelalisib with ofatumumab for previously-treated CLL patients is in progress
(NCT01659021).
Idelalisib demonstrates a dual mechanism of action by inhibiting pro-
survival signaling pathways [57] and, like other kinase inhibitors, by blocking
ingress into and promoting egress out of the lymph node into the blood,
leading to the re-localisation of tumour cells. Release from the protective
lymph environment into the blood renders CLL cells more susceptible to
apoptosis. PI3K is expressed by all leucocytes including T cells, raising the
possibility that the therapeutic effect of idelalisib may, at least in part, be due
to effects on the surrounding immune cells in addition to direct effects on CLL
cells [64]. Intriguingly, IL-4 protects against idelalisib-induced apoptosis in
vitro [57], indicating that microenvironmental influences may protect CLL
cells against PI3K inhibitors and that co-inhibition of the function of
surrounding cells may be an important factor in successful treatment. Ongoing
clinical trials with idelalisib are examining the combination with other agents;
including rituximab, ofatumumab, obinutuzumab and bendamustine.
Furthermore, a recent publication has shown that a combination of idelalisib
with ibrutinib is synergistic, indicating potential benefit from combined or
sequential therapy [65]. In addition to idelalisib, the development of other
PI3K inhibitors for the treatment of lymphoid malignancies is ongoing: one
such example being TGR-1202, a novel PI3K inhibitor with significant
differences in its chemical structure compared to idelalisib and with lower
reported incidences of colitis in patients. TGR-1202 is currently in phase I
New and Repositioning Approach to Treatment 29
2.1.2.2. Duvelisib
IPI-145 (Infinity Pharmaceuticals, Inc) targets both PI3K and PI3K
isoforms [66] and induces apoptosis in CLL samples in vitro, abrogates bone
marrow stromal cell-mediated survival, and inhibits BCR-mediated signaling
and chemotaxis in response to CXCL12 [67]. It is very important that
duvelisib also killed CLL cells that were resistant to ibrutinib [68], so this may
hold true with other PI3K inhibitors, and could form an important strategy for
treating patients refractory to ibrutinib. In a phase 1 study, 55 patients with
relapsed or refractory CLL received duvelisib orally at doses from 8 mg to 75
mg twice daily. ORR was 58% with a CR rate of 2%. The PFS at 18 months
was 60%. In patients with del(17p), the median PFS was 14 months, while
89% of patients showed a reduction (50%) of enlarged lymph nodes [67, 68].
The most commonly noted grade 3-4 adverse event was neutropenia (42%).
Duvelisib is now in a number of clinical trials for CLL, including those
investigating its use in combination with anti-CD20 antibodies or
bendamustine, and in patients refractory to ibrutinib (NCT02292225,
NCT01871675, NCT02576275).
2.1.2.3. TGR-1202
TGR-1202 (formerly known as RP5264; TG Therapeutics) is an orally
available PI3K inhibitor with once-daily dosing, targeting the delta isoform
with nanomolar potency and several-fold selectivity over the alpha, beta, and
gamma isoforms of PI3K. Inhibition of PI3K delta signaling with TGR-1202
has demonstrated robust activity in numerous pre-clinical models and primary
cells from patients with hematologic malignancies. OConnor et al. [69] report
updated safety and efficacy results from a phase I study of TGR-1202 in
patients with relapsed and refractory CLL and lymphoma. TGR-1202 is
administered orally once-daily (QD) following a 3+3 dose escalation design.
Expansion cohorts were open at 800 mg, 1000 mg, and 1200 mg QD. Of 16
evaluable CLL patients, 15 (94%) achieved a nodal PR (median nodal of
76%), of which 10 (63%) achieved a PR according to the Hallek et al. [70]
criteria. Among the 32 evaluable NHL patients, 10 achieved an objective
response, including 3/11 evaluable patients with DLBCL, while responses
have been limited in patients with MCL (1/5) and HL (1/9). Of the 16
evaluable indolent NHL (FL & MZL) patients, 14 (88%) have achieved
reductions in tumor burden with six patients on study for over 12 cycles (and
30 Ida Franiak-Pietryga and Maria Bryszewska
durations upwards of 29+ cycles), with 5/12 FL and 1/4 MZL patients
achieving an objective response to date [69]. Burris et al. [71] reported the
data of 66 patients with NHL (20 CLL) enrolled to the study. The ORR in
patients with CLL was 63%. The most commonly noted grade 3-4 adverse
events were neutropenia (11%), anemia (8%), skin rush (5%), dyspnea (5%),
and fatigue (3%). TGR-1202 has been combined with ublituximab in patients
with relapse or refractory NHL, including CLL [72]. In total, 55 patients were
included to the trial. Infusion-related reaction was seen in 29% patients. ORR
in the CLL cohort was in 83% patients (all PR) was noted. In a preliminary
analysis of 16 patients with relapse and refractory NHL, the triplet
combination of TGR-1202, ublituximab and ibrutinib has been evaluated
(NCT02006485) and the triplet was safe with about 90% nodal response [73].
acute lymphoblastic leukemia (T-ALL) [78]. Although Pictilisib has not been
investigated in CLL yet, it has a great potential for antileukemic activity.
2.1.4.1. Fostamatinib
R788, an oral pro-drug of the active metabolite R406 (Rigel
Pharmaceuticals) is an ATP-competitive kinase inhibitor that also inhibits a
number of other kinases [89]. In vitro treatment of CLL cells with fostamatinib
inhibits BCR and integrin signaling, antagonizes the protective effect of
stromal cells, reduces migration to chemokines and adhesion to stromal
components, and induces a moderate degree of apoptosis [86]. Fostamatinib
prevents disease progression both in TCL1 transgenic mice, in which antigen-
dependent selection appears to play a similar role as in human CLL, and in a
non-Hodgkin lymphoma model that depends on cooperation between MYC
and BCR-derived signals [88, 90]. A group of 68 patients with relapsed and
refractory NHL and CLL were enrolled to a phase 1 study with fostamatinib as
monotherapy [91]. In this group, 11 patients had CLL and 55% achieved PR.
ORR of 10% was achieved in the follicular lymphoma cohort, and 11% in the
mantle cell lymphoma cohort. The dose-limiting toxicity was diarrhea,
neutropenia and thrombocytopenia. Further clinical phases in CLL had been
stopped due to negative phase 3 results in patients with rheumatoid arthritis.
This drug is currently being tested in immune thrombocytopenic purpura (ITP)
[92].
2.1.4.2. Entospletinib
GS-9973 (Gilead Sciences) is an oral inhibitor of SYK which is more
selective than R406 [93]. Entospletinib is currently being pursued in phase 2
32 Ida Franiak-Pietryga and Maria Bryszewska
clinical trial with patients with relapsed or refractory CLL (41 patients) and
NHL (145 patients). Patients received the drug 800 mg orally twice daily. In
the CLL cohort, ORR was achieved in 61% (all PR), with a median PFS of 14
months. Among adverse events, dyspnea, pneumonia, febrile neutropenia,
dehydration, and pyrexia were the most commonly noted [94].
2.1.5.1. Dasatynib
Dasatinib (Bristol-Myers Squibb) is an oral multikinase inhibitor targeting
SRC and ABL kinases which is approved for use in imatinib-resistant chronic
myeloid leukemia (CML). Recently, it has been shown that dasatinib not only
inhibits LYN but also BTK at low nanomolar concentrations [98]. Reports
from in vitro studies revealed that dasatinib induces apoptosis in CLL cells
with no correlation between response and inhibition of LYN phosphorylation.
However, induction of apoptosis in vitro was inversely correlated with drug-
induced inhibition of SYK phosphorylation. Dasatinib inhibited BCR
signaling, stromal cell contact and CD40 stimulation causing an antagonized
proapoptotic effect [99]. A group of 15 patients with relapsed or refractory
CLL were enrolled in a phase 2 study where they were administered of 140 mg
dasatinib once daily. The OR rate was 20% with a PFS of 7.5 months, and five
patients showed a reduction (50%) of enlarged lymph nodes.
Myelosuppression was the primary toxicity, with grade 4 neutropenia and
thrombocytopenia occurring in 40% and 13% of patients, respectively [100].
New and Repositioning Approach to Treatment 33
The BCL-2 family members are key regulators of the intrinsic apoptotic
pathway and are classified into three classes of proteins based on their
structural similarity and function: the anti-apoptotic proteins (BCL-2, BCL-xL,
MCL-1, BFL-1, and BCL-w) sequester the BH3-only proteins (BIM, BID,
PUMA, and NOXA) that in turn activate the pro-apoptotic proteins (BAX and
BAK). In some cases, anti-apoptotic BCL-2 proteins can also sequester pro-
apoptotic proteins. BAX/BAK oligomerization causes mitochondrial outer
membrane permeabilization, resulting in cytochrome c release and apoptosis
[101]. The dysregulation of BCL2 family proteins results in the inhibition of
apoptosis and uncontrolled proliferation of B-cells, leading to the development
of many hematological malignancies and contributing to the development of
resistance to chemotherapy. Therapeutic modulation of the BCL2 pathway
suggests the possibility of a promising new therapeutic strategy in diseases
with impaired mechanism of apoptosis, for example CLL and NHL [102].
2.2.1. Venetoclax
ABT-199 (GDC-0199, RG7601; Genentech/AbbVie) is a small-molecule
designed with greater selectivity for BCL2 [103]. The agent binds more avidly
to BCL2 than to BCL-XL by >3 orders of magnitude [104]. It binds to BCL-
XL and BCL-W but not to MCL1. The affinity of this agent for BCL2 is more
than 500 times higher than that of BCL-XL. Venetoclax is a very promising
agent in the future therapy of CLL. It induces apoptosis in CLL cells at
concentrations 10-fold lower than those required by navitoclax. However, a
200-fold higher concentration of venetoclax was required for induction of
platelet apoptosis compared with navitoclax. Unfortunately, venetoclax also
induces the tumor lysis syndrome (TLS), in which the debris of dying tumor
cells circulating in blood and affect other organs [105]. Preliminary reports
note an ORR of 77% and a CR rate of 23% in groups of patients with relapsed
or refractory CLL following venetoclax monotherapy [106]. The estimated
median PFS was 18 months. It is significant that the CR rate was higher than
that observed following the use of BCR inhibitors in relapsed or refractory
CLL. Venetoclax in combination with rituximab in 49 patients was associated
with a CR rate of 41%, with 50% of patients demonstrating MRD-negative
remission [107]. Two trials are ongoing: one is a randomized phase 3 study for
previously untreated CLL patients venetoclax + obinutuzumab vs.
chlorambucil + obinutuzumab (NCT02242942), and the other for relapsed or
34 Ida Franiak-Pietryga and Maria Bryszewska
2.2.2. Navitoclax
ABT-263 (Abbott Laboratories) is a first generation, NOXA-like BH3
mimetic developed for apoptosis-based therapy for CLL. It inhibits BCL2
family members and binds with high affinity to BCL-XL, BCL2 and BCL-W
but not MCL1 or A1 [109, 110]. During a phase 1 study including 26 CLL
patients with relapsed or refractory disease, 35% patients demonstrated PR and
an additional seven patients had stable disease for > 6 months. Median PFS
was calculated for 25 months. The benefits of navitoclax have been observed
in patients with fludarabine refractory CLL, bulky lymphandenopathy and
CLL with del(17p) [111]. The most frequent adverse event was reported as
thrombocytopenia due to BCL-XL inhibition.
2.2.3. Obatoclax
GX15-070 (Cephalon) is an inhibitor of the BCL2 family of proteins. This
inhibition induces apoptosis in cancer cells, preventing tumor growth. It is in
phase 2 clinical trials for the treatment of leukemia, lymphoma, myelofibrosis
and mastocytosis. OBrien et al. [112] reported a phase 1 study with obatoclax
mesylate (NCT00600964). The drug was administered to patients with
advanced CLL at doses ranging from 3.5 to 14 mg/m2 as a one-hour infusion
and from 20 to 40 mg/m2 as a three-hour infusion every three weeks. Twenty-
six patients received a total of 74 cycles. Dose-limiting reactions were
neurological (somnolence, euphoria, ataxia) and associated with the infusion.
The maximum tolerated dose (MTD) was 28 mg/m2 over three hours every
three weeks. One (4%) of the 26 patients achieved a partial response. Patients
with anemia (3/11) or thrombocytopenia (4/14) experienced improvements in
hemoglobin and platelet counts. Circulating lymphocyte counts were reduced
in 18 of 26 patients with a median reduction of 24%. Activation of Bax and
Bak was demonstrated in peripheral blood mononuclear cells, and induction of
apoptosis was related to overall obatoclax exposure, as monitored by the
plasma concentration of oligonucleosomal DNA/histone complexes.
Obatoclax mesylate has biological activity and modest single-agent activity in
heavily-pretreated patients with advanced CLL.
New and Repositioning Approach to Treatment 35
zone lymphoma (n = 9). The median follow-up time was 30 months (range 12-
48). The overall response rate was 70.0 %, with a CR rate of 31.7 %. The 2-
year OS and PFS of all patients were 75.0 and 41.0 %, respectively. RB
chemotherapy in patients with refractory or relapsed indolent B cell NHL was
effective with low toxicity [123].
Carfilzomib irreversibly blocks the release of NF-B to the nuclei thus
abrogating pathway activity [124]. This drug has demonstrated promising pre-
clinical activity in CLL and had little effect on baseline NF-B activity in
peripheral CLL cells [125]. However, NF-B is significantly more active in
the lymph node microenvironment [126]. The findings of a phase 1 study
indicate that carfilzomib provided stable disease in four patients with FL and
one patient with CLL/SLL [127].
To determine the safety and tolerability of carfilzomib in
relapsed/refractory CLL or small lymphocytic lymphoma (SLL), a phase 1
dose-escalation trial including nineteen patients treated with carfilzomib was
performed. The initial dose of 20 mg/m2 was escalated in four cohorts (27, 36,
45 and 56 mg/m2) on days 1, 2, 8, 9, 15 and 16 of 28-day cycles. Therapy was
generally well tolerated, and no dose limiting toxicities were observed. The
most common hematologic toxicities were thrombocytopenia and neutropenia.
All patients evaluable for response had stable disease, including patients with
del(17p) and fludarabine-resistant disease [128].
Other proteasome inhibitors such as MLN9708 (Millenium
Pharmaceuticals), CEP-18770 (Teva/Cephalon) and orpozomib (Onyx
Pharmaceuticals) are currently being investigated in phase 1 protocols in
patients with relapsed or refractory B-cell lymphomas.
The huge group of monoclonal antibodies (mAbs) offers promise. CD20 is
a well-established target for mAbs in CLL, and several other targets
including CD52, CD23, CD19 and CD37 have also been investigated. In
addition, further innovative new therapies are possible with incorporate
immunomodulatory drugs (lenalidomide), cyclin dependent kinase (CDK)
inhibitors (flavopiridol, dinaciclib, TG02), chimeric antigen receptor (CAR) T
cell therapy and novel strategies based on exportin-1 and selinexor. The use of
mAbs and other novel drugs in the treatment of CLL is so expansive that
deserves a separate publication.
Targeted therapies for CLL as outlined above have significantly changed
the treatment paradigm for patients with CLL. Several of these agents have
gained FDA approval and are commercially available. The greatest challenge
at this time is how best to incorporate these novel therapies in the clinical care
of patients.
New and Repositioning Approach to Treatment 37
3.1. Metformin
3.2. Statins
3.3. Danazol
Valproic acid (VPA) is a short-chain fatty acid that belongs to the group
of histone deacetylase inhibitors (HDAC-I), a relatively new class of agents
used for anticancer therapy. VPA has been used as an anticonvulsant and
mood-stabilizing drug for decades. Even when taken over a long time, VPA is
usually well-tolerated, although it is contraindicated during pregnancy because
of its teratogenic effects [161]. HDAC-Is have been found to exert pleiotropic
antitumor effects by inducing growth arrest, differentiation, and apoptosis,
under both in vitro and in vivo conditions [162]. In malignant cells, HDACIs
induce apoptosis by the upregulation of pro-apoptotic genes and repression of
antiapoptotic genes [163, 164]. Bokelmann and Mahlknecht [165] observed
that VPA, mediates apoptosis in CLL cells ex vivo by caspase activation via
both the extrinsic and the intrinsic apoptosis pathways, as indicated by the
activation of the caspase proteins 8 and 9, and cleavage of the proapoptotic
protein BID. VPA treatment reduced BCL2 mRNA levels, resulting in a lower
BCL2/BAX ratio. A year later, Bouzar et al. [166] reported that VPA acted in
a highly synergistic/additive manner with fludarabine (FA) and cladribine
(2CdA) to induce apoptosis of B-CLL cells. Moreover, VPA also restored
sensitivity to fludarabine in B cells from high-risk CLL patients.
The mechanism of apoptosis induced by VPA, either alone or combined
with FA or to 2CdA was caspase-dependent and involved the extrinsic
pathway. VPA stimulates hyperphosphorylation of p42/p44 ERK, cytochrome
c release and overexpression of BAX and FAS. Yoon et al. [167] confirm the
presence of a synergistic cathepsin B mediated activation of the apoptotic
response between VPA and FA in a CLL patient in vivo, leading to a decrease
in the anti-apoptotic proteins. They also found that the combination of FA and
VPA decreases the level of the anti-apoptotic proteins MCL1 and XIAP in
primary CLL cells. Moreover, the combined therapy resulted in reduced
lymphocyte count in five out of six patients, and reduced lymph node sizes in
four out of six. Karp et al. [168] report a VPA median cytotoxicity of 13.88%
(range 0-54.65%) in a study based on peripheral blood mononuclear cells
retrieved from 53 patients with CLL. It has been proved that the effect of VPA
on CLL cells is dependent on lactate dehydrogenase serum levels, but is
independent of all other prognostic markers. Considering resistance to therapy,
NOTCH1 mutations have been checked. It is belived that CD20 levels are low
in CLL, which may be responsible for the dysregulation of HDAC-mediated
epigenetic repression of CD20 expression [169]. This may prove to be
valuable information for future targeted therapy in CLL.
42 Ida Franiak-Pietryga and Maria Bryszewska
4.1. Dendrimers
CONCLUSION
Novel targeted therapies strongly suggest that the standard treatment
paradigm in CLL will change in the next few years. Particular attention should
be paid to BCR-, PI3K-, SYK-, LYN- and mTOR-targeting agents, as well as
BCL2-inhibitors, which already demonstrate encouraging activity both as
single agents and in combination with conventional chemotherapy across a
variety of B-cell neoplastic conditions, including unfavorable genetic features.
Meanwhile, as this process is very dynamic, it is without a doubt that the FDA
will approve new drugs and grant. As additional therapies are discovered and
optimized, the superiority of nanomedicines over current treatment options and
free drugs will continue to increase the efficiency of eradicating drug-resistant
cancers such as CLL. In addition, re-examining existing drugs as potential
complements for these therapies may identify very effective additive modes of
treatment. Novel and repositioning therapies continue to emerge in the
preclinical setting and will expand the armamentarium of drug combinations
for use in hematologic oncology in the coming decades.
44 Ida Franiak-Pietryga and Maria Bryszewska
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New and Repositioning Approach to Treatment 61
BIOGRAPHICAL SKETCHES
Name: Ida Franiak-Pietryga, Ph.D.
Summary
Affiliation
Department of Clinical and Laboratory Genetics,
Medical University of Lodz, Poland
Education
Professional experience:
Main accomplishments
Main responsibilities
Continuation of nanoparticle research Dendrimers as a potential new
drug for CLL; pre-clinical phase, in vivo studies.
Continuation of studies of CLL cells collected from patients before
and during therapy and after relapse from therapy to determine the
genetic and biologic features associated with tumor progression,
therapeutic response and/or resistance to therapy.
Discovery of features that distinguish CLL from their normal cell
counterparts to identify new targets for therapy and/or define
surrogate markers associated with more rapid rates of cancer
progression or resistance to standard therapies.
Research projects:
6. Fulbright
Patents:
P.401934 -- The application 4th generation poly(propylene imine)
dendrimer maltotriose modified PPI-G4-DS-Mal-III; [WIPO ST 10/C
PL401934], PCT/PL2013/000154
P.401936 -- The application 4th generation poly (propylene imine)
dendrimer maltotriose modified PPI-G4-OS-Mal-III; [WIPO ST 10/C
PL401936], PCT/PL2013/000163
P.416636 Medical application of 4th generation poly(propylene imine)
dendrimer maltotriose modified
Honors:
TOP500 Innovators in Poland (2015)
Fulbright Senior Award (2016)
Education:
- University of Lodz, Faculty of Mathematics, Physics and Chemistry,
M.Sc. in physics, 1973
- University of Lodz, Faculty of Biology and Earth Sciences,
Ph.D. in biophysics, 1982
- Postdoctoral position at the Department of Biochemistry,
McMaster University, Hamilton, Canada, 1987/1988 (15 months)
- University of Lodz, Faculty of Biology and Environmental Protection,
Habilitation in biophysics (D.Sc.), 1990
- University of Lodz, Professor title in biological sciences, 1996
Address: 141/143 Pomorska St., 90-236 Lodz, Poland
Research projects
Professional Appointments:
1998 till present Full Professor, University of Lodz,
Faculty of Biology and Environmental Protection,
Department of General Biophysics, Head of the Department
1990 - 1998 Associate Professor,
University of Lodz, Department of General Biophysics
1982 1990 Assistant Professor,
University of Lodz, Department of Thermobiology
1980 1982 Assistant, University of Lodz,
Department of Biophysics
1973 1980 Assistant, Medical Academy of Lodz,
Department of Biophysics
Honors:
Chapters in books
Books
Chapter 3
ABSTRACT
Several prognostic factors are available for patients with CLL who
require therapy. Of these, high levels of 2 microglobulin, adverse
cytogenetics and high expression levels of CD38 and ZAP70 surface
markers are well described and mark patients with unfavorable
prognosis [1]. In approximately 50% of patients, more than 2% of the
immunoglobulin variable heavy-chain gene (IGHV) nucleotide sequence
differs from that of germ-line non-clonal DNA [2]. Patients with mutated
IGHV respond better to chemo-immunotherapy [3], and patients with
unmutated IGHV have a shorter time to treatment, shorter time to next
treatment, inferior response to chemotherapy, higher rates of
chemotherapy resistance, and lower survival rates [4-6].
Until recently, CLL was considered an incurable disease. However,
two recent studies found that with the combination of fludarabine,
*
Corresponding Author: Uri Rozovski, MD; Institute of Hematology; Davidoff Cancer Center,
Beilinson Hospital; Petah-Tikva, Israel, 49100; Email: rozovski.uri@gmail.com.
78 Nili Saar, Pia Raanani and Uri Rozovski
HISTORICAL PERSPECTIVE
Chronic lymphocytic leukemia (CLL) is characterized by the gradual
accumulation of functionally incompetent, mature appearing lymphocytes, co-
expressing B-lymphocyte antigens (CD19, CD23) and aberrantly expressing
CD5 [9]. That the natural history of CLL is extremely variable was recognized
long ago. In the first case series published in 1924, Minot and Isaac reported
that approximately 30% of patients with CLL were long-term survivors.
However, most patients died few months to 5 years from diagnosis, usually
from CLL-related conditions [10] [Figure 1]. Staging systems developed
during the 1970's [11, 12] are still used today to delineate patients who require
therapy from those with indolent CLL, who are followed without treatment
[13]. In patients who require therapy, selection of first-line treatment is mainly
based on patient-related factors [13-15]. Disease-related factors are commonly
used to predict response to therapy and overall outcome, and only occasionally
to select a first line treatment modality [16-18]. In patients with relapsed
disease, the extent and duration of response to previous treatment are taken
into account, and specific abnormalities such as del(17p) or TP53 mutation
may guide therapy selection [13, 14].
Prognostic and Predictive Indicators in Chronic Lymphocytic 79
Adapted with permission from Figure 3 in Minot and Isaacs (1924) Chronic Lymphatic
Leukemia: Age Incidence. Duration and Benefit Derived from Irradiation, The
Boston Medical Journal, 191(1) @ The Massachusetts Medical Society.
most important outcome is that patients with early-stage disease need not be
treated [13, 14].
The Rai system is based on hierarchical grouping of disease
manifestations and assumes a gradual increase in the burden of leukemic
lymphocytes, starting in the blood and bone marrow (lymphocytosis),
progressively involving lymph nodes (lymphadenopathy), spleen and liver
(organomegaly), and eventually compromising bone marrow function (anemia
and/or thrombocytopenia) [11]. The original Rai system included 5 stages.
However, as there were only 3 actuarial survival curves, it was modified to
include 3 stages instead, corresponding with low, intermediate and high risk
features [20].
The Binnet system, commonly used in Europe, takes five potential sites
of involvement into consideration: cervical, axillary, and inguinal lymph
nodes, spleen, and liver. Patients are classified according to the number of
involved sites, plus the presence of anemia (hemoglobin <10 g/dL) and/or
thrombocytopenia (platelets <100,000/microL).
CYTOGENETIC ABNORMALITIES
Cytogenetic abnormalities are detected by fluorescence in situ
hybridization (FISH) in approximately 80% of patients with CLL, and are
independent predictors of disease progression and survival [21]. The most
commonly used panel in clinical practice includes del(13q), trisomy 12,
del(11q), and del(17p). The 'International Workshop for CLL' guidelines call
for FISH testing of all newly diagnosed patients using probes for 13q, T12,
11q, 6q, and 17p cytogenetic abnormalities [13], a model which was recently
validated in a large cohort of patients [22]. However, with the exception of
del(17p) testing, FISH analysis is not used for follow-up or therapy-related
decisions in current practice.
The most common genetic abnormality in CLL is deletion of 13q14,
which occurs in more than 50% of patients [23]. Individuals with 13q14
deletions have a relatively benign disease that usually manifests as stable or
slowly progressive isolated lymphocytosis. The common deleted region
includes two micro-RNA genes, miR-15a and miR-16-1 [23] that have been
shown to negatively regulate Bcl-2 expression, allowing CLL cells to evade
apoptosis [23-28].
Trisomy 12 is found in 15% of patients and confers an intermediate
prognostic risk [21, 29]. When compared to patients who were negative
Prognostic and Predictive Indicators in Chronic Lymphocytic 81
treatment the overall response rate in patients with U-CLL is even higher than
in M-CLL [76]. IGHV mutation status is one of the most important prognostic
factors used to stratify patients in clinical trials [77, 78]. However, because as
many as 50% of patients with M-CLL treated with FCR are likely to get cured
[7, 8], stratifying patients by IGHV mutation status should be considered
outside clinical trials, at least in patients who are candidates for treatment with
FCR.
SERUM MARKERS
Thymidine kinase (TK) is a cell cycle-dependent enzyme of the pyrimidine
salvage pathway [79, 80]. In order to be incorporated into the DNA, the
nucleoside thymidine must undergo phosphorylation, a step catalyzed by
thymidine kinase [81]. There exist at least two TK isoenzymes, which differ in
their properties and cellular distribution. Since TK1, the cytosolic isozyme, is
synthesized only in the G1/S phase of dividing cells, and is absent in resting
cells, it has been studied as a marker of proliferation [82-85].
Serum TK1 levels have been reported to have prognostic significance in
patients with low-grade non-Hodgkins lymphomas and CLL [86-92]. In CLL,
high levels of TK1 predict early progression [91], shorter survival, and
increased risk of Richter's transformation [92]. The cumbersome radioisotope
assay used to detect TK1 in serum hampered the application of TK1 as a
prognostic marker. With the introduction of new anti-TK1 antibody-based
assays, TK levels have become more clinically relevant [79]. Still, quantifying
serum TK1 is not in widespread use in routine practice.
Beta-2 microglobulin (B2M) is ubiquitously expressed in nucleated cells
and is an extracellular component of the MHC class 1 and other class 1-like
molecules [93]. Because CLL cells constitutively shed B2M [94], serum levels
are considered a reliable marker of tumor burden [95, 96] and correlate well
with disease stage and prognosis [97, 98]. B2M was deemed valuable even in
a subset of patients with early disease: patients with Binnet stage A CLL and
low levels of B2M were less likely to require therapy during a 3.5 year follow
up [96]. High levels of B2M are an independent adverse prognostic factor for
patients treated with FCR [99], and normalization of B2M during the first 6
months of treatment is predictive of superior progression free survival (PFS)
for patients treated with ibrutinib-based regimens [100].
CD23 is a B-lymphocyte specific antigen, constitutively expressed on
CLL cells [101]. When expressed on cell surface, CD23 functions as a low
84 Nili Saar, Pia Raanani and Uri Rozovski
affinity receptor for IgE [102]. After auto-proteolytic cleavage, soluble CD23
(sCD23) is detected in serum and functions as a potent mitogenic growth
factor which provokes entry of resting (Go) B cells into the G1/S phase [103].
Detection of CD23 on cell surface helps distinguish CLL (CD5+, CD23+)
from mantle cell lymphoma (CD5+, CD23-) [104]. High sCD23 in CLL
correlates with disease activity [105] and predicts shorter time to disease
progression and a shorter overall survival [106, 107].
SOMATIC MUTATIONS
Whole exome sequencing studies revealed relatively low mutation burden
[147-149], and only few recurrently mutated genes in CLL. Among 3334
patients tested for the 5/6 most frequently mutated genes in CLL, mutated
SF3B1 was detected in 11%, TP53 in 10%, NOTCH1 in 8%, and BIRC or
MYD88 were each detected in 2% of patients [150]. Mutations in ATM,
detected in a substantial minority of CLL patients across different series [151-
154], were not tested in this study. Based on their strong predictive power,
sometimes independent of other prognostic indicators, it has been suggested
that at least some of these mutations be integrated into the current hierarchical
model, based on FISH findings [155].
SF3B1: Mutated in CLL but also in myeloid malignancies, SF3B1 codes
for a protein that is part of the spliceosome, a complex of RNA and protein
subunits that takes part in alternative splicing [156], and other cellular
processes [157]. Mutations in SF3B1 confer an adverse prognosis, independent
of other prognostic markers in CLL [147, 148, 150], are more frequent in
patients with progressive disease [148, 150], and predict shorter time to first
treatment [147, 150, 158] as well as shorter PFS and OS [148, 158].
TP53: Located on the short arm of chromosome 17, the tumor suppressor
gene TP53 is the most frequently mutated gene in human cancers [159]. As a
primary gatekeeper, the p53 transcription factor will halt the cell cycle at G1
when damaged DNA is identified [160-165], and loss of p53 function will
cause genomic instability and accumulation of mutations [161]. In patients
with del(17p), mutated TP53 in the remaining allele occurs in 81-94% of
patients and predicts poor survival outcome [41, 43, 44, 166]. However,
whereas one prospective study did not find prognostic significance to the
presence of mutated TP53 in non-del(17p) CLL [44], most found that mutated
TP53 carries a poor prognosis independent of del(17p) [45, 166, 167]. Mutated
TP53 predicts resistance to standard chemo-immunotherapy [41, 43, 168-170],
early relapse [41, 168] and poor survival outcome [41, 43, 166, 168-170].
Even small mutated TP53 sub-clones found by sensitive next generation
sequencing in untreated CLL patients predict poor survival [167].
86 Nili Saar, Pia Raanani and Uri Rozovski
Some of the novel drugs that are currently available or tested in clinical
trials may be safe and effective even in patients with TP53 aberrations. For
example, overall response rate, PFS and OS were comparable in patients with
relapsed CLL and TP53 mutations treated with the oral immunomodulatory
agent lenalidomide [171]. Likewise, high response rates were observed in
treatment-nave and relapsed patients treated with the Bruton Tyrosine Kinase
(BTK) inhibitor ibrutinib [50, 172, 173]. It is therefore imperative to test for
TP53 mutations when treatment decisions are taken. The European Research
Initiative on CLL (ERIC) recommends TP53 mutation analysis in all CLL
patients who are candidates for treatment [174].
ATM: Located on the long arm of chromosome 11, the ataxia
telangiectasia-mutated (ATM) gene spans 146kd in 66 divided exons. Because
of its large size, it has been difficult to delineate polymorphisms from
pathogenic mutations, but alterations in ATM, including both mutations and
deletions, occur in 25% of patients with CLL at diagnosis [175]. Similar to
TP53, the ATM gene codes for a checkpoint inhibitor, and del11(q), detected
by FISH, is second only to del17(p) as the most unfavorable genetic
abnormality in CLL [176]. In patients with del11(q), ATM point mutations in
the second allele occur in 20% to 40% and predict worse outcome [175, 177].
NOTCH1: NOTCH1 encodes for a transmembrane protein that upon
ligand binding undergoes proteolytic cleavage, releasing an intracellular
domain which translocates to the nucleus and functions as a transcription
factor [178, 179]. In CLL, a frameshift mutation in codon 2515 results in a
truncated constitutively active intracellular protein. NOTCH1 mutations occur
more frequently with trisomy 12 [147, 180-182] and U-CLL [181-183].
Though not an independent prognostic indicator, they are associated with
advanced disease and poor prognosis [149, 181, 183-185].
BIRC3: BIRC3 encodes for a negative regulator of NF-B. Mutations in
BIRC3 lead to constitutive activation of NF-B, and have been associated with
U-CLL, advanced disease [150] and chemo-refractoriness to fludarabine-based
treatments [186].
MYD88: Mutations in this adaptor protein of the toll-like receptor-NF-B
pathway, are frequent in lymphoproliferative disorders, and the same leucine-
proline substitutions are found in lymphoplasmacytic leukemia (87% of
patients), diffuse large B-cell lymphoma (15% of patients) and CLL (3% of
patients), where it is associated with M-CLL and a favorable clinical course
[187].
Table 1. Prognostic and predictive indicators in treatment nave patients with CLL
Patient Staging Disease burden Disease Serum Cell Chromosomal IGHV Somatic
characteristics Kinetics markers surface aberrations status mutations
markers
Age 60 Rai / Binet Absolute Lymphocyte thymidine CD38 del(13q) Mutated TP53
lymphocyte doubling kinase vs. non-
Male sex count time ZAP-70 trisomy(12) mutated ATM
2-
ECOG 1 Marrow microglobulin CD49d del(11q) VH3.21 SF3B1
infiltration gene
pattern soluble CD23 del(17p) usage NOTCH1
(diffuse/non-
diffuse) del(6q) BIRC3
MICRORNA'S
MicroRNAs (miRNAs) are a class of small non-coding RNAs that
function as post-transcriptional regulators of gene expression [188]. The first
report linking microRNAs to cancer was in CLL, when a cluster of 2
microRNAs, miR-15-a/miR16-1 was found to reside in the minimally deleted
region of patients with CLL and 13q14 deletion [189]. This cluster is
downregulated in approximately 70% of patients with CLL and results in
increased levels of BCL2, which provide CLL cells relative protection from
apoptosis [190]. The miR-15-a/miR16-1 cluster is only one of many
microRNA clusters that are dysregulated in CLL [191]. MicroRNA profiles
have been found to correlate with other prognostic markers [192] as well
as with disease progression [193-198], response to treatment [199-201] and
survival outcome [192]. Plasma levels of miR155, for example, predicted
progression of monoclonal B-cell lymphocytosis to overt CLL [201], and
high levels predicted poor response to chemo-immunotherapy [201, 202].
Nonetheless, most expression signatures were not validated in separate cohorts
and different studies yielded inconsistent, sometimes contradictory results,
making it difficult to recommend any specific microRNA as a reliable
biomarker in clinical practice.
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100 Nili Saar, Pia Raanani and Uri Rozovski
Chapter 4
MOLECULAR CYTOGENETIC
ABNORMALITIES IN CHRONIC
LYMPHOCYTIC LEUKEMIA:
PROGNOSTIC IMPLICATIONS
Prabhjot Kaur, MD
Associate Professsor of Pathology, Department of Pathology,
Dartmouth Hitchcock Medical Center and
Geisel School of Medicine at Dartmouth 1 Medical Center Drive
Lebanon, NH, US
ABSTRACT
CLL is the most common leukemia in the western world. Mostly a
disease of the elderly, this indolent disorder, has a very variable clinical
outcome and can evolve into an aggressive lymphoma. As indolent
disorders are considered incurable, treatment is delayed until the patient
progresses to high stage disease. These therapeutic decisions are based on
criteria devised by Kanti Rai and Jacques Louis Binet almost four
decades ago; this assessment requires only a physical examination and a
complete blood count. A significant change that has occurred in the past
four decades is the increased diagnosis of patients in low stage disease
(during regular check-ups for employment or other causes). In the 1970s,
40% of the patients were diagnosed at stage I. In the 2000s, nearly 80%
of the patients are diagnosed at low stage. Although patients are
diagnosed at an earlier stage, it is difficult to determine the prognosis of
116 Prabhjot Kaur
Rai stage 0/1 patients. A subset of patients with early stage CLL will
rapidly evolve to a more aggressive and fatal disease, and clinical staging,
as it currently exists, fails to predict this progression.
Approximately 80% of individuals with CLL have acquired
chromosomal abnormalities within their malignant clone and can be
categorized into five prognostic groups accordingly: deletion 13q (median
survival, 133 months); deletion 11q (median survival, 79 months);
trisomy 12 (median survival, 114 months); normal cytogenetics (median
survival, 111 months); and deletion 17p (median survival, 32 months)
(Dhner et al., 2000). A complex cytogenetic karyotype can be identified
rarely and is commonly associated with poor prognostic features
including CD38 expression and unmutated IgHV (Haferlach et al., 2007).
IGHV3-21 usage is an independent poor prognostic factor. Novel
mutation in CLL include the NOTCH1 mutations (Puente et al., Nature
2011, Rossi et al., Blood 2012), SF3B1 mutations (Quesada et al., Nature
Gen 2012), BIRC3 mutation (Rossi et al., Blood 2012) among others. B
cell receptors play a unique role in the pathogenesis of the disease and
has prognostic and therapeutic implications. Stereotyped B cell receptors
impact outcome. New hierarchal classification is being suggested to
include the conventional cytogenetic markers and novel mutations.
MicroRNAs (miRNA) are short (~22nt in humans), endogenous non-
coding ssRNA (single stranded) molecules that regulate gene expression
via translational repression or transcript degradation. Recent evidence has
shown that miRNAs are key to gene regulation and have a global effect
on various oncogenic, tumor suppressor and cell survival pathways. Over
the last decade several miRNAs have been shown to be involved in the
pathogenesis of CLL. In this chapter, molecular cytogenetic prognostic
markers, BCRs, novel mutations, role of miRNA in the biology and
prognosis of this disease are discussed.
INTRODUCTION
Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in
the western world, seen mostly in the elderly age-group and has a very
variable clinical outcome. Traditionally considered an indolent, antigen
inexperienced leukemia of slowly accumulating cells that do not die, we now
acknowledge that CLL cells are highly proliferative, antigen experienced cells
that have a high cell turnover and a subset show an aggressive clinical course.
The aggressive behavior seen argues against the notion of a low proliferative
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 117
disease. In vivo studies using nonradioactive means suggest that CLL cells are
more dynamic than is usually appreciated. CLL cells have replication rates,
ranging from about 0.1 to more than 1.0 percent of the clone per day [1]. If the
total clonal burden of a typical patient with CLL is approximately 1012 cells,
these rates point to the daily production of some 109 to 1010 new leukemic
cells! [2]. Another traditional view that B-CLL is a disease primarily resulting
from the accumulation of clonal B lymphocytes that do not die has been
challenged by the evidence provided by telomere length which is an index of
cell replication. In a study by Damle et al. where telomere length was
quantified by a flow-FISH hybridization protocol, it was found that B-CLL
cells have shorter telomeres than normal age-matched B cells suggesting that
leukemic cells have extensive proliferative histories [3]. A significant change
that has occurred in the past four decades is the increased diagnosis of patients
in low stage disease mostly during regular check-ups for employment or other
causes. In the 1970s, 40% of the patients were diagnosed at stage I. In the
2000s, nearly 80% of the patients are diagnosed at low stage [4]. It is
especially difficult to determine the prognosis of Rai stage 0/1 patients. A
subset of patients with early stage CLL will rapidly evolve to a more
aggressive and fatal disease [5, 6]. As indolent disorders are considered
incurable, treatment is usually delayed until the patient progresses to high
stage disease. These therapeutic decisions, till recently, were based on criteria
devised by Kanti Rai and Jacques Louis Binet almost four decades ago; an
assessment that requires only a physical examination and a complete blood
count. Some patients may benefit from early treatment. However therapies are
not without toxicities and earlier clinical trials showed that treatment in early
stage disease did not improve survival [7]. Several prognostic markers based
on genetic, phenotypic, and molecular characteristics have emerged in the past
decade. The clinical utility of these newer prognostic indicators, alone or in
combination with each other and other clinical predictive systems are a subject
of active research. The cytogenetic evaluation has evolved tremendously since
the 1970s from conventional karyotyping on metaphase spreads, FISH analysis
on interphase chromosomes, microarrays, SNP arrays for copy number
measurement, DNA methylation to the new era of next generation sequencing
that encompasses whole genome/exome sequencing and targeted sequencing.
Indeed molecular genetics has traversed an exciting journey and Chronic
Lymphocytic Leukemia exemplifies it.
118 Prabhjot Kaur
del13q tumor burden where cases that showed greater than 65-85% of their
cells with the deletion showed worse prognosis. This was likely due to the
overexpression of genes involved in proliferation and underexpression of
genes involved in apoptosis in patients with more del (13q) cells [18-22].
Candidate genes in this region that were initially proposed included the
Retinoblastoma gene (RB1), Leu1, Leu2 and BrCA2 genes. The minimal
deleted region (MDR) includes the DLEU2/Mir15A/Mir16B loci, however
there is a large variation in the size of the deletion among patients. [23, 24]
[25-27]. MicroRNAs (miRs) will be addressed separately in this chapter.
Chromosome 11q and ATM as a candidate gene. 11q deletion is the
second most common aberration and reported in 20% of the patients [28-30].
It contains a minimal deleted region of 2-3 Mb at 11q22.3-11q23.1 with ATM,
the candidate tumor suppressor gene. The mutation has been associated with
defective DNA repair [31]. The Bcl-1 locus at 11q13 and the MLL gene at
11q23.3 are located outside the critical region. Patients with an 11q deletion
have a more rapid disease progression with shorter treatment-free periods and
inferior survival [9, 11, 28, 29, 32-35, 36].
The BCR includes sIg (IgM) and the Ig-/Ig- heterodimer (CD79a and
CD79b). The B-cell receptor (BCR) signaling pathway is critical to the
development and maturation of normal B cells [59] (See Figure 1). BCR
activation can result from chronic antigenic drive by microbial or viral
antigens to auto-stimulation of B-cells by self-antigens. The sIg subunits bind
antigen, resulting in receptor aggregation, while the / subunits transduce
signals to the cell interior. The BCR on antigenic stimulation leads to the
activation of ITAMs and SRC family kinases including spleen tyrosine
kinase (SYK) and LYN (Figure 1). These, along with adaptor proteins such
as CD19 and B-cell linker (BLNK), and signaling enzymes such as
phospholipase C-2 (PLC2), phosphatidalyinositol-3-kinase (PI3K)-together
called the signalosome - activate multiple signaling cascades. The downstream
effectors can be modulated toward the pro-apoptotic nuclear factor of activated
T cells (NFAT) arm or the pro-survival NF- kB arm depending on balancing
of the signaling cascades. These permit many distinct outcomes, including
survival, tolerance (anergy) or apoptosis, proliferation, and differentiation into
antibody-producing cells or memory B cells. B-cell maturation involves
somatic recombination and mutation of the IGHV genes that encode the
122 Prabhjot Kaur
antigen binding domains of the BCR [59-63]. CLL has distinct BCR signaling
compared with normal B cells [64-67]. CLL cells demonstrate anergy
(decreased or no response to BCR engagement) and this BCR signaling
capacity depends on the mutational status of the immunoglobulin heavy chain
variable (IG V) genes. CLL cells with mutated IGHV genes usually express
lower levels of surface IgM (sIgM) and present a weak response to BCR
engagement associated with indolent disease. In contrast, cells with unmutated
IGHV genes that predominantly express the ZAP-70 kinase, respond to BCR
stimulation and reflect an aggressive clinical behavior [66-69]. BCRs that can
react to multiple antigens (polyreactive) are associated with more aggressive
disease. CLL subsets that show strong intracellular responses demonstrate a
more aggressive clinical course. The microenvironment within a lymph node
supports strong co-stimulatory signals with stronger BCR signaling in contrast
to peripheral blood. The canonical NF-B pathway was significantly up-
regulated in lymph node samples, and the expression of the inhibitory I-Bs
was significantly lower [69-72]. These studies show that the BCR signaling
plays an important role in the pathogenesis of CLL. Several promising
therapeutic advances have been seen via targeting the molecules in the BCR
signaling pathway. Some examples of drugs that inhibit kinases within the
BCR signaling pathway are ibrutinib (an inhibitor of Brutons tyrosine kinase
(Btk)), fostamatinib (an inhibitor of Syk), idelalisib (an inhibitor of
phosphatidylinositol 3 kinase (PI3K)) and Everolimus (an mTOR inhibitor)
[61, 63, 73-79].
The IgHV mutation status. Damle et al. and Hamblin et al. described, in
landmark studies, the prognostic significance of the immunoglobulin heavy
chain variable region gene (IgHV) mutation status in CLL [80, 81]. Mutational
status is determined by comparing the sequence of the CLL cells to the germ
line sequence. A CLL cell is considered as mutated if the deviation is 2% or
otherwise <98% homologous to the corresponding germ line sequence. The
mutation status of the IgH variable region has been used to subdivide CLL into
two groups: one with a proposed pre-germinal center (unmutated) cell origin
and one with a post-germinal center (mutated) cell origin [80, 81]. The group
of CLL cases with unmutated IgH (U-CLL) genes showed rapid disease
progression and a short treatment-free survival [82]. The group with mutated
IgH genes (M-CLL) followed an indolent clinical course with long survival
probabilities. There have been significant differences in the distribution of
cytogenetic abnormalities based on mutation status. The overall incidence of
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 123
genomic aberrations (80%) are similar in the U-CLL and M-CLL subgroups.
The 13q14 deletion, the most common genetic abnormality in CLL, was found
more often in patients with M-CLL, a subset with a more favorable clinical
outcome [80, 81, 83]. High-risk genomic aberrations such as 17p- and 11q-
occurred almost exclusively in the U-CLL subgroup.
The analysis of DNA sequences to determine the status of
immunoglobulin V-gene mutations is laborious and not performed routinely in
clinical laboratories. There has been a search for surrogate markers that can be
studied by immunohistochemistry, flow cytometric analysis or PCR that can
be applied to daily use. CD38 expression, defined as greater than 30%
expression by neoplastic cells, was shown to be a surrogate marker for the
U-CLL genes [84-87]. CD38 promotes survival and proliferation of B cells on
their way to and after neoplastic transformation [88]. CD38 expression
predicts unfavorable prognosis, shorter time from diagnosis to therapy, and
worse overall survival. However, CD38 expression can change over time,
making it difficult to use it as a prognostic marker [85, 89].
used to define sets of similar rearranged VH DJH were as follows: (a) use of
the same VH, D, and JH germline genes; (b) use of the same D segment reading
frame and position relative to the VH, plus or minus one codon; and (c) an
amino acid identity within the HCDR3 of >=60% identity. More recently,
stereotyping criteria have become somewhat less stringent with respect to
amino acid identity and the IGHV gene used and more focused on amino acid
similarity (functional conservation) and lack of differences in CDR3 length.
Several studies showed the presence of such sterotyped Vh CDR3 sequences
among CLL BCRs and found that at least 30% of CLL cases could be
categorized into one of several subsets based largely on stereotyped amino
acid motifs. A growing body of evidence suggest that stereotyping may be
clinically relevant. For example, subsets of CLL cases with stereotyped BCRs
displayed distinctive features with regard to demographics, immunophenotype,
and outcome [98-101, 102].
IGHV3-21. Tobin et al., reported preferential use of the VH3-21 gene in
mutated CLL and showed that regardless of VH gene mutation status, the
VH3-21 cases showed overall poor survival. A large fraction of VH3-21 cases
also demonstrated shorter lengths of the third complementarity determining
region (CDR3) and noticeable similarities in both the nucleotide sequence and
thus amino acid composition. Further analysis of the light chain V gene use,
revealed a considerable restricted use of the Ig light chain gene V2-14 in the
majority of the VH3-21 cases. This suggest that a common antigen epitope is
recognized by the highly homologous VH3-21/V2-14 Ig molecules and
therefore antigen stimulation may be a promoting factor in the evolution of
certain CLL clones [103, 104, 105-107]. This finding is taken as evidence that
antigens and/or superantigens may be involved in lymphoma development by
stimulating proliferation and expansion of B cells expressing distinctive BCR.
This contradicted another time-held notion that CLL is a disease of antigen
inexperienced B cells [2].
in CLL without IGHV mutation [110]. These mutations were also found in
higher rates in patients that have chemo-refractory CLL [121].
MYD88 gene mutation, a critical adaptor molecule of the Toll-like
receptor (TLR) complex, is seen in 3-10% of CLL cases [31, 109]. The
recurrent MYD88 mutation in CLL (L265P) causes constitutive MYD88-
IRAK signaling, resulting in constitutive NF-B activity. MYD88 L265P
mutations have been found predominantly in M-CLL. This aberration is
potentially amenable to therapeutic targeting through direct inhibition of the
MYD88-IRAK complex, through proteasomal inhibition [122] or even
through the inhibition of Brutons tyrosine kinase (BTK) [123].
BIRC3 gene is a negative regulator of alternative NF-kB signaling
pathway. Its mutations or deletions, noted in less than 5-8% of cases, lead to
the activation of alternative NF-kB pathway [111]. ATM and the BIRC3 genes
are located on 11q. Initial studies speculated that BIRC3 mutations may
explain the poor prognosis of 11q deletion, however ATM mutation rather
than BIRC3 deletion and/or mutation predicts reduced survival in 11q-deleted
chronic lymphocytic leukemia [124]. Targeted sequencing of the BIRC3
coding sequence in CLL showed that BIRC3 inactivation is particularly
common in fludarabine-refractory patients (24%) [111]. BIRC3 disruptions
have been associated with unmutated IGHV gene configuration and 11q
deletion with an inferior progression-free survival [125].
rate given the known background mutation rate of the cancer. Studies have
followed patients over time and seen the differences in aberrations depending
on the time of evaluation, that is, whether the cytogenetic study was performed
at diagnosis, months after diagnosis, prior to treatment or following treatment.
The consensus has been that new aberrations are acquired over time and that
the acquisition of new genomic abnormalities usually portend a more
aggressive disease with decreased overall survival. Different cytogenetic
abnormalities show differing impact on clinical outcome. For example del
(17p) and/or del (11q) are associated with worse prognosis. The appearance of
new abnormalities is not restricted to cases that have undergone therapy [129],
though treatment can result in disease progression due to expansion of
previously present subclones [130-132]. SF3B1, not usually seen in
monoclonal B cell lymphocytosis (MBL) [133], a clonal condition that is
thought to precede CLL, may be an example of a driver mutation that evolves
from a minor sub-clone to a dominant sub-clone during disease progression or
relapse [74, 75] and therefore may have a role in clonal evolution in CLL [2,
134]. The incidence of TP53 mutations has been reported to increase as
the disease progresses; the incidence rises to 10%12% at first-line treatment
and approximately 40% at refractory CLL [5]. NOTCH1 and P53 are
mutually exclusive, whereas are seen in combination at the time of Richters
transformation of CLL. Emergence of NOTCH1, SF3B1, and BIRC3
abnormalities in addition to TP53 and 11q22-q23 lesions have been seen
changing prognostic categorization from lower to higher risk groups [135].
Clonal evolution can traverse different routes based on tumor heterogeneity or
acquisitions of new mutation during the wait and watch time period or some
driver mutations are positively selected post treatment and expand [129, 131,
136]. See Figure 2.
Hierarchal Classification
MicroRNAs
CONCLUSION
CLL represents a biologically dynamic and heterogeneous disease.
Advances in the basic sciences have led to a better understanding of this
disease process, better markers for prognosis and novel targets for
development of therapies. However development of prognostic models that
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 131
can be universally accepted and applied remain a challenge and are a subject
of ongoing research.
ACKNOWLEDGMENTS
I am grateful to Dr. G J Tsongalis (Professor, Dept of Molecular
Pathology, DHMC) for reviewing the manuscript. Dr. J Lefferts (Dept of
Pathology), DHMC, kindly assisted in editing the chapter.
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Den, Neste, E; Michaux, L; Heimann, P; Martiat, P; Bron, D; Lagneaux,
Molecular Cytogenetic Abnormalities in Chronic Lymphocytic 147
Belarus, 66 cell cycle, 35, 38, 55, 57, 58, 83, 85, 130,
benefits, 34 133
benign, 80 cell death, 38, 57
biological activity, 34 cell fate, 126
biological sciences, 65 cell lines, 25, 38, 43, 66, 69, 102
biomarkers, 111 cell signaling, 126
biomaterials, 75 cell surface, 18, 83, 84, 94
biomolecules, 75 CEP-18770, 36
blood, 1, 8, 17, 28, 33, 68, 70, 74, 80, 114, ceramide, 25
132, 140 CGI-1746, 22, 23
blood cultures, 132 CGI-560, 22, 23
bonds, 8, 42 chemical, 16, 28, 42
bone, 2, 10, 21, 27, 29, 40, 47, 80 chemokine receptor, 1, 3, 5, 6, 8, 106
bone marrow, 2, 10, 27, 29, 40, 80 chemokines, 2, 3, 10, 31
bortezomib, 35, 55, 144 Chemokines, 1, 2, 3, 8, 10, 11, 31
brain, 71 chemotaxis, 29, 30, 52
branching, 42 chemotherapeutic agent, 58
breast cancer, 25, 56, 58, 100 chemotherapy, 33, 36, 43, 51, 55, 58, 59,
Bulgaria, 1, 3, 11 78, 84, 90, 108, 109
cholesterol, 39, 58
cholesterol lowering agents, 39
C Chromosomal Aberrations, 118
chromosomal abnormalities, 95, 97, 116,
cancer, 3, 10, 11, 34, 35, 37, 39, 42, 46, 50,
129, 132
51, 53, 56, 57, 63, 66, 70, 72, 73, 90,
chromosome, 19, 22, 81, 85, 86, 89, 95,
101, 107, 108, 111, 112, 113, 127, 134,
112, 118, 120, 132, 133, 134, 135, 136,
137, 144
147
cancer cells, 34, 42, 53, 73
chromosome bands, 135
cancer progression, 63
classes, 27, 33
cancer therapy, 57
classification, 93, 108, 116, 129, 132, 133,
candidates, 83, 86, 112, 130, 134, 146
138
carbon, 42
cleavage, 41, 84, 86
carcinoma, 100
clinical oncology, 96, 99, 100, 103, 108
carfilzomib, 21, 35, 36, 47, 55, 56
clinical presentation, 90
cascades, 39, 121
clinical trials, 18, 20, 22, 28, 29, 30, 34, 37,
categorization, 128
38, 83, 86, 89, 117
CC-292, 20, 21, 47, 48
clone, 18, 116, 117, 127, 133
CCR, 7, 10
clusters, 11, 90
CCR3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11
CNX-774, 22, 24, 25
CCR3+ B-cells, 2, 6, 7
coding, 90, 111, 116, 127, 129, 143
CD193, 3, 4, 7, 8
codon, 86, 125, 126
cDNA, 64, 130
coenzyme, 39, 58
cell biology, 55, 105
colitis, 28
cell culture, 118
Index 151
collagen, 24, 48, 71 cytotoxicity, 39, 40, 41, 42, 47, 59, 66, 68,
colon, 58 81
colon cancer, 58
colorectal cancer, 145
combination therapy, 28, 47, 50
D
community, 45
Danazol, 16, 40, 59
comorbidity, 54
Dasatynib, 32
complementarity, 124, 125
data analysis, 43
complete blood count, 115, 117
DCA, 38
complexity, 90, 127, 129
defects, 44
composites, 75
degradation, 35, 43, 116, 126
composition, 58, 73, 125
dehydration, 32
compounds, 16, 18
Delta, 50, 144
conditioning, 98
Dendrimers, 42, 60, 61, 63, 64, 65, 66, 67,
configuration, 127
68, 69, 70, 71, 72, 73, 74, 75
consensus, 128
dendritic cell, 66
conservation, 125
deregulation, 112, 130, 146
control group, 2, 3, 5
detection, 8, 96, 97, 105, 118, 132, 137
cooperation, 31
deviation, 122
correlation, 3, 5, 6, 7, 8, 9, 32, 98, 135, 145,
diabetes, 16, 56
146
diabetic patients, 37
correlations, 1, 2, 5, 6, 9, 98, 135, 142
diarrhea, 22, 28, 31
corticosteroids, 40
discordance, 124, 141
cosmetic, 74
disease activity, 84, 103
covalent bond, 24
disease progression, 31, 38, 55, 80, 84, 90,
covering, 61
94, 95, 96, 101, 104, 105, 109, 110, 119,
creatinine, 102
122, 126, 128, 134, 137, 140
critical analysis, 93
diseases, 16, 23, 24, 25, 33, 37, 139, 144
Croatia, 75
disorder, 59, 115
crystallinity, 73
distribution, 5, 83, 122
cure, 61, 78, 79, 90
diversification, 143
cycles, 29, 34, 35, 36
DNA, 34, 40, 60, 66, 68, 77, 82, 83, 85,
cyclophosphamide, 44, 78, 81, 91, 92, 95,
117, 119, 121, 123, 124, 130, 137
102, 113
DNA damage, 66, 130, 137
cysteine, 19, 21
DNA repair, 119
cytochrome, 33, 41
DNA sequencing, 124
cytogenetics, 77, 97, 98, 116, 118, 129, 132,
DOI, 12, 63, 68, 69, 73, 74
146
dosing, 23, 29, 56
cytokines, 31
down-regulation, 38, 93, 111, 112, 147
cytometry, 4, 5, 11, 84, 105, 106
drug carriers, 74
cytoplasm, 17
drug delivery, 42, 75
cytostatic drugs, 17
drug design, 42
drug resistance, 39, 42, 49, 109, 137
152 Index
drugs, 15, 16, 19, 36, 37, 43, 48, 61, 62, 64, first generation, 34
66, 78, 81, 86, 122 flavonoids, 35, 55
DSC, 64 flavopiridol, 36, 81
Duvelisib, 29 fluorescence, 80, 93, 104, 133, 135, 136,
dysplasia, 40 146
dyspnea, 30, 32 Food and Drug Administration (FDA), 17,
26, 35, 36, 43
formation, 40, 60, 70
E Fostamatinib, 31, 52, 122
fragments, 121
elaboration, 23
frameshift mutation, 86
embryogenesis, 126
France, 9, 133
employment, 115, 117
FTIR, 74
endometriosis, 16, 40
functional changes, 139
England, 141, 143
Entospletinib, 31, 53
environment, 10, 28 G
enzyme, 23, 24, 27, 39, 74, 83, 121
eosinophils, 3, 10 GDC-0199, 33, 54
epigenetic silencing, 112 GDC-0834, 22, 23, 48
epilepsy, 16 GDC-094, 30, 51
ERA, 66 gene expression, 42, 49, 62, 90, 109, 110,
erythrocytes, 70, 74 116, 124, 129, 137, 139, 141
euphoria, 34 gene regulation, 116, 129
Europe, 61, 80 gene silencing, 66
evidence, 18, 37, 60, 106, 116, 117, 125, gene therapy, 74, 75
134, 135, 142 genes, 17, 41, 43, 49, 79, 80, 85, 91, 93, 99,
evolution, 94, 125, 127, 133, 134, 137, 145, 107, 111, 113, 118, 119, 121, 122, 123,
146 124, 125, 127, 130, 133, 134, 140, 141,
excision, 126 142, 143, 147
exons, 86, 121 genetic alteration, 125
exposure, 34 genetic defect, 144
extracellular matrix, 84 genetic mutations, 127
genetics, 61, 62, 97, 98, 107, 117, 129, 132,
146
F genome, 107, 108, 111, 117, 125, 126, 143
genomic instability, 85
family history, 17
genomics, 94, 144
family members, 33, 34, 35
germ line, 122
FAS, 41
gland, 40
FDA approval, 17, 36
glioblastoma, 57
fever, 17, 27
glucocorticoid receptor, 17
fibrillation, 69, 73
glucocorticoids, 17, 44
fibroblasts, 3, 38
glucose, 37, 56
fibrosis, 59
Index 153
inhibition, 16, 18, 19, 20, 21, 23, 28, 30, 32, ligand, 9, 24, 86, 110, 126, 141
33, 34, 35, 37, 44, 48, 50, 54, 57, 113, linear polymers, 60
127 liposomes, 42, 72, 74
inhibitor, 16, 17, 18, 20, 21, 22, 23, 24, 25, liver, 80
27, 28, 29, 30, 31, 32, 34, 45, 46, 47, 48, loci, 119, 135
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 82, locus, 118, 119, 134
86, 97, 122, 139 low risk, 3, 5, 6
initiation, 110, 129 LTD, 22, 30
inositol, 27 Luo, 100
institutions, 61 lymph, 2, 17, 19, 28, 29, 32, 36, 41, 56, 80,
insulin, 37, 69, 70, 72 81, 85, 89, 94, 106, 120, 122, 139
integration, 56 lymph node, 2, 19, 28, 29, 32, 36, 41, 56,
integrin, 31, 84, 106 80, 85, 89, 106, 122, 139
interface, 73 lymphadenopathy, 17, 80, 81, 94, 120
interphase, 117, 118, 135, 146 lymphocytes, 2, 3, 4, 5, 6, 8, 9, 38, 39, 55,
intervention, 126 57, 59, 78, 80, 91, 117, 126, 144
introns, 126 lymphocytosis, 17, 19, 46, 80, 90, 113, 128,
IPI-145, 29, 50 132, 145
irradiation, 92 lymphoid, 2, 17, 28, 46, 53, 54, 109, 133,
isozyme, 83, 101 135, 137, 138
lymphoid organs, 2
lymphoid tissue, 133
K lymphoma, 1, 3, 9, 10, 11, 17, 19, 20, 25,
26, 27, 29, 31, 34, 35, 36, 44, 45, 51, 52,
karyotype, 82, 96, 98, 116, 124, 137
53, 54, 55, 56, 84, 86, 94, 101, 108, 109,
karyotyping, 82, 98, 117, 118
115, 118, 125, 130, 134, 136, 137, 139,
kinase activity, 32
144, 145
kinetics, 23, 131
LYN Inhibition, 32
LYN kinase activity, 32
L lysine, 67
lysis, 33
lactate dehydrogenase, 41, 89
laser radiation, 70, 71
Leahy, 135 M
Lebanon, 115
mAb, 46
lesions, 128
macromolecules, 42, 60
leucine, 86
macrophages, 52
leukemia, 2, 9, 15, 17, 32, 34, 44, 46, 51,
magnesium, 68
61, 62, 63, 78, 86, 92, 93, 95, 96, 97,
magnetic resonance, 75
109, 115, 116, 130, 131, 133, 134, 135,
magnitude, 33
144, 145, 146
MAI, 104
leukocytes, 5
majority, 18, 118, 125
leukocytosis, 17
malignancy, 139
LFM-A13, 22, 25, 49
Index 155
propylene, 42, 61, 63, 65, 67, 68, 69, 70, 71, residue, 21, 24, 42
73, 74 resistance, 33, 41, 42, 44, 47, 51, 55, 58, 59,
prostate cancer, 58 63, 78, 84, 85, 109, 113
proteasome, 35, 36, 47, 54, 55, 56 respiration, 56
proteasome inhibitors, 35, 36, 54 response, 18, 19, 22, 26, 28, 29, 32, 34, 36,
protection, 39, 90 41, 46, 60, 63, 78, 79, 81, 82, 83, 86, 90,
protein kinases, 25 96, 102, 108, 122, 130, 132, 136, 137,
protein synthesis, 102 138, 145
proteins, 27, 33, 34, 35, 38, 40, 41, 42, 65, retinoblastoma, 133
66, 68, 73, 74, 121, 130 RG7601, 33
proteolysis, 40 rheumatoid arthritis, 26, 31
proteolytic enzyme, 66 risk, 2, 3, 4, 5, 6, 7, 8, 9, 16, 17, 19, 20, 22,
PUMA, 33 28, 37, 39, 41, 44, 45, 46, 47, 48, 51, 54,
pyrimidine, 51, 83 80, 83, 88, 89, 90, 95, 99, 101, 106, 112,
120, 123, 124, 128, 129, 141, 147
risk factors, 129
Q rituximab, 16, 17, 20, 28, 33, 35, 39, 44, 45,
46, 47, 49, 54, 62, 64, 78, 81, 92, 95, 96,
quantification, 105
99, 102, 113
quercetin, 35
RN-486, 22, 25
RNA, 80, 85, 90, 93, 111, 112, 126, 146
R rosiglitazone, 59
RP5264, 29
R788, 31, 52
radioisotope, 83
rash, 22 S
RB1, 119, 134
safety, 16, 19, 20, 21, 22, 24, 28, 29, 35, 36,
reactions, 34
37, 50, 51, 54, 90, 97
reactivity, 48
second generation, 21, 48, 82
receptor, 2, 5, 6, 7, 8, 9, 10, 15, 17, 18, 26,
secrete, 3
35, 36, 40, 44, 52, 53, 55, 56, 57, 60, 84,
secretion, 26
86, 94, 102, 104, 105, 121, 123, 127,
selectivity, 21, 23, 25, 29, 33, 47
137, 138, 139, 141, 142
senescence, 130
recognition, 139, 142
sensitivity, 25, 39, 41, 58, 118
recombination, 121, 124
sequencing, 85, 107, 117, 121, 125, 126,
recommendations, 100, 109, 137
127, 143
recovery, 69
serine, 51, 126
redistribution, 46
serum, 9, 41, 70, 72, 83, 84, 89, 101, 102,
regeneration, 69
103
relatives, 17
serum albumin, 70, 72
relevance, 71, 100, 101, 112, 129, 141, 146
sex, 4, 87, 88, 103
remission, 19, 33, 59, 81, 90
side effects, 37
replication, 117
signal transduction, 18, 32, 84, 105
repression, 41, 116
158 Index
signaling pathway, 15, 28, 32, 38, 43, 52, 78, 80, 81, 82, 83, 84, 85, 89, 90, 92, 93,
84, 121, 123, 127, 138, 139 95, 96, 98, 99, 101, 102, 103, 106, 107,
signalling, 53, 110, 138, 144 108, 109, 110, 111, 113, 114, 116, 117,
signals, 8, 31, 43, 49, 50, 53, 121 119, 120, 121, 122, 123, 124, 125, 126,
silica, 66 127, 128, 129, 134, 136, 137, 140, 144,
simvastatin, 39, 58, 59 147
siRNA, 66, 68, 70, 73, 75 survival rate, 78
SLE, 25, 26 survivors, 78
SNP, 117 susceptibility, 54
social problems, 2 Switzerland, 47, 51
sodium, 57 SYK Inhibition, 31
solid tumors, 30, 101 symptoms, 17
somatic mutations, 125 syndrome, 33, 59, 126
somnolence, 34 synthesis, 44, 58
Spebrutinib, 20, 21 systemic lupus erythematosus, 26
specialization, 62
spinal cord, 73
spinal cord injury, 73
T
spleen, 52, 53, 80, 121
T cell, 28, 30, 31, 36, 84, 105, 121
Spleen tyrosine kinase, 31, 52, 53
T cell receptor, 31
splenomegaly, 17
T lymphocytes, 10
squamous cell, 38, 57
tannins, 72
squamous cell carcinoma, 38, 57
target, 17, 18, 21, 22, 30, 31, 35, 36, 47, 48,
staging systems, 79
50, 52, 53, 55, 57, 59, 126, 139
stem cells, 57, 126
T-cell receptor, 84, 124
stereotyping, 124
TCR, 31, 104, 105
steroids, 20
techniques, 62, 118
stimulation, 8, 18, 32, 97, 118, 121, 125,
telangiectasia, 81, 86, 108
132, 135, 139
telomere, 108, 117
stratification, 44, 95, 99, 112, 147
teratogen, 59
stroke, 69
testing, 24, 37, 80, 114
stromal cells, 31
testosterone, 40
structure, 28, 37, 40, 42, 70, 71
TGR-1202, 28, 29, 50, 51
Styles, 114
therapeutic agents, 130
subgroups, 79, 94, 95, 123, 126, 129, 131,
therapeutic approaches, 9, 44
132, 145
therapeutic effect, 28
substitutions, 86
therapeutic goal, 91
substrate, 71
therapeutics, 100
suppression, 30, 43, 112, 126
therapy, 17, 19, 28, 33, 34, 36, 38, 41, 43,
surface modification, 42
44, 46, 59, 62, 63, 64, 77, 78, 79, 80, 81,
surveillance, 81
82, 83, 96, 99, 100, 111, 121, 123, 128,
survival, 2, 11, 16, 17, 19, 22, 27, 28, 29,
136, 139
30, 37, 43, 49, 50, 51, 52, 53, 55, 56, 58,
threonine, 126
Index 159