Tumor Markers

Objective
` ` `

Identify and select tumor markers for the diagnosis and management of cancer patients Understand the limitations of specific tumor markers Identify PSA as a screening test

Ideal Tumor Markers

` ` ` ` ` ` `

Be specific to the tumor Level should change in response to tumor size An abnormal level should be obtained in the presence of micrometastases The level should not have large fluctuations that are independent of changes in tumor size Levels in healthy individuals are at much lower concentrations than those found in cancer patients Predict recurrences before they are clinically detectable Test should be cost effective

Potential Uses of Tumor Markers

` ` ` ` ` ` ` `

Risk identification Screening Aiding diagnosis Assessing prognosis Predicting response to therapy Predicting severe toxicity due to therapy Postoperative surveillance Monitoring therapy in advanced disease

Common Tumor Markers

Analyte CEA CA-125 CA15-3, 27.29 CA 19-9 AFP PSA Free PSA HCG Hormone receptors NMP 22, BTA, FDP HER2/neu Cancer use Monitor colorectal, breast, lung cancer Ovarian cancer monitoring Monitor recurrences of breast cancer Pancreatic cancer Germ cell tumors, liver cancer Screen and monitor prostate cancer Distinguish prostate cancer from BPH Germ cell and trophoblastic tumors Breast cancer therapy Monitor recurrences of bladder cancer Breast cancer

Common Tumor Markers

Analyte CEA CA-125 CA15-3, 27.29 CA 19-9 AFP PSA Free PSA HCG Hormone receptors NMP 22, BTA, FDP HER2/neu Cancer use Monitor colorectal, breast, lung cancer Ovarian cancer monitoring Monitor recurrences of breast cancer Pancreatic cancer Germ cell tumors, liver cancer Screen and monitor prostate cancer Distinguish prostate cancer from BPH Germ cell and trophoblastic tumors Breast cancer therapy Monitor recurrences of bladder cancer Breast cancer

Three classes of tumor markers:

` ` `

Oncofetal antigens Proteins Polypeptide hormones

3 Classes of Tumor Markers

1.
`

Oncofetal antigens
such as AFP and CEA, which are normally expressed during fetal development but do not occur normally in the tissues or serum

2.
`

Proteins
occur in epithelial cells that become elevated in adeno- and squamous cell carcinomas, such as the CA 19-9, CA 125, and CA 15-3 proteins

3.
`

Polypeptide hormones
such as the chain of human chorionic gonadotropin ( hCG), and specific enzymes, such as the placental isoform of alkaline phosphatase, that become elevated in the serum of patients with specific tumors

Most tumor markers have been discovered in the sera of cohorts of patients with specific types of cancer Many are also found in the sera of patients with nonmalignant, e.g., inflammatory, conditions Most are NOT useful for screening
` `

Low sensitivities and specificities Prostate specific antigen (PSA) is the exception

Tumor markers are useful for monitoring specific cancers

` `

CEA is often elevated in the sera of patients with colon cancer if CEA serum levels elevated s/p resection of colon ca tumor recurrence

Oncoproteins are Markers for Cell Proliferation

`

Oncoproteins are proteins that are directly or indirectly involved in the control of mitosis and are altered such that they continuously signal the cell to divide These proteins
`

lie on signal transduction pathways carrying mitogenic signals from growth factors at the cell membrane to the nucleus are involved in the regulation of transcription, activating the genes ultimately causing cell growth and mitosis

HER2/Neu

Breast cancer

Often, these proteins can be detected in the serum of patients with abnormal cell growth, i.e., cancer or precancerous states

Cell Differentiation/Tumor Suppressors

`

` `

Proteins encoded by tumor suppressor genes are responsible for suppressing cell growth, either causing cell growth arrest in cell cycle or apoptosis Frequently, these suppressor genes undergo deletions or mutations Antioncogene protein p53
` ` `

It is involved in apoptosis, cell cycle arrest, cell senescence, and DNA damage response Deletions or mutations predispose cells to undergoing malignant transformation p53 mutations have been identified in nearly 50% of human malignancies

In breast cancer, several studies indicated that the presence of p53 Ab in the serum correlated with increased proliferation antigen (Ki-67) and lack of ER expression, indicating that it may serve as a breast cancer prognostic marker

Adhesion Molecules and Metastasis

`

Tumor metastases involve several major steps

` ` `

Tumor cells have to penetrate their adjacent surroundings, then invade vascular or lymphatic vessels The tumor cells are then carried to distant sites, until they are lodged in the venous or capillary beds In this new environment, these tumor cells must again penetrate the vascular walls in order to grow at the distant site

Cell adhesion molecules, include: integrins, selectins, cadherins, and cell adhesion molecules

Adhesion Molecules and Metastasis

`

Changes in levels of expression reflect the malignant behavior of cancer

`

Serum levels of E-selectin, intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) are increased in latestage breast cancer patients Elevated serum VCAM level may be used to predict a shorter survival

` `

One study indicated that post-chemotherapeutic levels of serum E-selectin and ICAM are associated with a response to treatment of patients with Hodgkin's disease Increased E-selectin, ICAM-1, and VCAM are suggested to be a prognostic factor for survival in patients with gastric cancer The appearance of cell adhesion molecules in serum might indicate the risk or occurrence of metastases or a poor prognosis

Other Markers become elevated due to high proliferation rate

` ` `

Hormones (hCG) Serum proteins Enzymes

` ` `

lactate dehydrogenase [LDH] alkaline phosphate [AP] metabolites

` ` `

vanillylmandelic acid [VMA] homovanillic acid [HVA] 5-hydroxyindoleacetic acid [5-HIAA]

Serum levels rise even higher when a benign tumor becomes malignant and metastasizes

Monoclonal-Antibody-Defined Tumor Markers

` `

Monoclonal antibodies have higher sensitivity and specificity than polyclonal antibodies For example, CA 19-9, CA 125, and CA 15-3 (monoclonal) are much more sensitive and specific than CEA (polyclonal) for pancreatic, ovarian, and breast carcinomas, respectively

Monoclonal-Antibody-Defined Tumor Markers

Clinical Applications of Tumor Markers

Clinical Applications of Tumor Markers

` ` ` ` ` ` ` `

Risk identification Screening Aiding diagnosis Assessing prognosis Predicting response to therapy Predicting severe toxicity due to therapy Postoperative surveillance and Monitoring therapy in advanced disease

Screening
` `

Use of screening tests should be confined to populations at risk for the disease Most tumor markers described are expressed both in neoplastic and benign conditions, their use is confined to monitoring tumor recurrence

Screening - PSA
`

Combination of the PSA and DRE provides the least costly approach to the early detection of prostate cancer
`

Especially recommended for African American men

` `

Incidence nearly twice 2xs that of the general population Death rate is up to 3xs higher

Screening permits the treatment of organ-confined, potentially curable prostate cancer discovered in men with a life expectancy of longer than 10 years

Screening - AFP
` `

Not approved for screening for hepatocellular carcinoma (HCC) in the US AFP is used to screen for HCC in China because of the high incidence of liver cancer

Screening CA 125
` `

The diagnosis of ovarian cancer has traditionally relied on imaging and discovery at surgery However, in most cases, at the time of detection, the tumor has advanced to a stage at which the possibility of cure is low The feasibility of screening for ovarian cancer in women by measuring serum CA 125 is still being investigated

Diagnosis
`

To improve the diagnostic yield of tumor markers:

` ` `

Multiple markers Specific patterns of multiple tumor markers seem to be associated with individual malignant diseases Measurement of the velocity (the rate of increase in PSA concentration over time) and the density

Prognosis: Recurrence, Metastasis and Survival

` `

Assessment of tumor aggressiveness helps to develop proper treatment The appearance of most of the circulating tumor markers has a lead time of several months (36 months) prior to the stage at which many of the physical procedures could be used for the detection of cancer The ease of drawing blood and the sensitivity of tumor marker tests make this noninvasive monitoring process now widely accepted

Prognosis: Recurrence, Metastasis and Survival

` ` `

Most tumor markers become increasingly elevated when the tumor metastasizes Unfortunately, very few tumor markers have a clear-cut boundary between benign and malignant stages Proteins that reflect the risk factors associated with the process of tumor metastases, such as proteases and adhesion molecules, are usually better markers for predicting prognosis
` `

However, most of these markers are still measured in tumor tissues and tissue cytosol In the near future, hope to measure these prognostic factors in the blood

Monitoring Treatment Response

` `

The serum level of tumor markers reflects the success of surgery or the efficacy of chemotherapy Detecting elevated levels of a tumor marker after surgery would indicate either incomplete removal of the tumor, recurrence, or the presence of metastases The measurement of serum tumor markers during chemotherapy also gives an indication of the effectiveness of the antitumor drug used and a guide for the selection of the most effective drug for each individual case

Recommendations for Ordering Tumor Marker Tests

`

Never Rely on the Result of a Single Test

`

Low specificity - Difficult to differentiate between malignant and benign diseases

` ` `

Most tumor marker elevations found in nonmalignant diseases may be transient, whereas with cancer the level often either remains elevated or rises continuously Ordering serial testing can help to detect falsely elevated levels due to transient elevation Order Serial Testing, be Certain to Order Every Test from the Same Laboratory Using the Same Assay Kit

Recommendations for Ordering Tumor Marker Tests

`

Be Certain that the Tumor Marker Selected for Monitoring Recurrence was Elevated in the Patient Prior to Surgery
`

Otherwise, multiple markers should be measured prior to the surgery in order to select the tumor marker showing the highest elevation as the marker for monitoring the disease activity

Multiple markers may be used to monitor the therapeutic effects for increased sensitivity

Recommendations for Ordering Tumor Marker Tests

`
`

Consider the Half-Life of the Tumor Marker when Interpreting the Test Result
Consider How the Tumor Marker is Removed from or Metabolized in the Blood Circulation ` benign liver diseases and disturbances in metabolism and excretion (AFP, CEA, CA 19-9, CA 15-3) ` disturbances of renal function (beta-2-microglobulin, calcitonin, PSA, CEA, CA 19-9, CA 15-3)

Recommendations for Ordering Tumor Marker Tests

`
`

Consider Ordering Multiple Markers to Improve Both the Sensitivity and the Specificity for Diagnosis
Be Aware of the Presence of Ectopic Tumor Markers
` ` `

For example, elevated AFP may be detected in metastatic lesions of the liver In other words, the appearance of ectopic tumor markers is associated with poor prognosis or metastases

Ectopic Tumor Markers

Individual Tumor Markers

-Fetoprotein (AFP)
` `

AFP is a major fetal serum protein and is also one of the major carcinoembryonic proteins In the fetus, AFP is synthesized by the yolk sac and the fetal hepatocytes and to a lesser extent by the fetal gastrointestinal tract and kidneys Elevated AFP
` `

HCC Yolk-sac-derived germ cell tumors

` `

Used for screening in China One study indicates that combined screening with AFP and ultrasonography results in increased sensitivity from 75% to near 100% in detecting HCC in hepatitis B and C

AFP
` `

Elevated in 50% of pts with HCC

`

often does not detect small lesions Yolk-sac tumors Cirrhosis Massive liver necrosis with regeneration Chronic hepatitis Normal pregnancy Fetal distress or death Fetal neural tube defects

False positive
` ` ` ` ` ` `

2-Microglobulin
`

` `

` ` `

is the constant light chain of the human histocompatibility locus antigen (HLA) expressed on the surface of most nucleated cells Elevated in solid tumors and lymphoproliferative diseases Serum concentration of 2M correlates with lymphocyte activity, making 2M a good marker for lymphoid malignancies of the B-cell line It has been used as an indicator of the patient's response to treatment CSF levels of 2M are useful for detecting metastases in the central nervous system (CNS) It is reported that serum 2M level is elevated in 75% of multiple myeloma patients and is useful in following the efficacy of the treatment of this disease

2M

Carcinoembryonic Antigen (CEA)

` ` ` `

CEA is the first of the carcinoembryonic proteins The most widely used tumor marker for gastrointestinal cancer today CEA is a nonspecific marker can be elevated in breast, lung, and liver cancers Elevated CEA levels prior to resection of colon ca. may suggest a worse prognosis

CEA
`

` `

The liver metabolizes CEA liver damage can impair CEA clearance and lead to increased levels in the blood circulation Increased CEA concentrations can be seen following radiation and chemotherapy It was recommended that CEA should form part of the AJCC staging system

CA 15-3 and CA 27.29

` `

These antigens correspond to sequences of mucins called polymorphic epithelial mucins (PEMs) Over-expressed on the cell surfaces of malignant glandular cells such as occur in breast cancer

CA 15-3
` ` ` ` ` `

Present in a variety of adenocarcinomas including breast, colon, lung, ovary, and pancreas More sensitive and specific marker for monitoring metastatic breast cancer Levels increase with higher stages of breast cancer Can be used to predict adverse outcomes in breast cancer patients Low sensitivity (23%) and specificity (69%) in detecting breast cancer CA 15-3 can also be elevated in chronic hepatitis, liver cirrhosis, sarcoidosis, tuberculosis, and systemic lupus erythematosus, and in smokers

CA 27.29
`

CA 27.29, another mucin marker MUC1-associated antigen, is a slightly more sensitive breast cancer marker than CA 15.3 The US Food and Drug Administration (FDA) has approved both CA 15-3 and CA 27.29 for monitoring therapy of advanced or recurrent breast cancer

CA 19-9
`

CA 19-9 is the first tumor marker of a group of new epitopes, including CA 125, CA 15-3, and CEA, defined by monoclonal antibodies CA 19-9 is related to Lewis blood group substances
`

Only serum antigen from cancer patients belonging to the Le ( +) or Le ( + ) blood group will be CA 19-9 positive

` ` `

Elevated in colorectal, gastric, and pancreatic cancer Also useful for monitoring therapy and detecting recurrence False positives may occur in patients with benign liver disease and may be due to cholestasis in these patients

CA 125
` `

` `

CA 125 is another antigenic determinant defined by a MAb and is associated with a mucin-like glycoprotein CA 125 is expressed by more than 80% of nonmucinous epithelial ovarian carcinomas and is found in most serous, endometrioid, and clear cell carcinomas of the ovary Pts on chemotherapy may show a false decline of CA 125 CA 125 is also used clinically for follow-up on uterine tumors (> 60% are elevated) and benign tumors, including endometriosis Recent studies showed greatly improved sensitivity of serum CA125 in combination with other markers

CA 72-4
`

` `

The CA 72-4 assay detects a mucin-like human adenocarcinoma-associated antigen, TAG-72, which is a highmolecular-weight mucin-like complex molecule Considered a carcinoembryonic protein - detected in both fetal epithelia and sera from pts with various carcinomas Currently, CA 72-4 is considered to be useful marker for the management of patients with gastric and colorectal carcinoma CA 72-4 has been proposed as a specific marker for tumor occurrence of resectable gastric cancer and a prognostic marker for survival CA 72-4 has been reported to be an independent prognostic marker for survival in colorectal cancer in multivariate analysis together with -hCG and CEA

Calcitonin
`

` `

Calcitonin is one of the circulating peptide hormones that may become elevated in patients with increased bone turnover rate associated with skeletal metastases Ectopically elevated in bronchogenic carcinomas Elevated in medullary carcinoma of the thyroid

Human Chorionic Gonadotropin

` `

` `

Synthesized and secreted by trophoblast cells of the placenta Both malignant and nonmalignant trophoblast cells synthesize and secrete not only the biologically active and -dimer but also the uncombined (or free) and subunits A free subunit of hCG has been detected in the serum of women during early pregnancy and in malignant tumors Measurement of the free -subunit is useful for the detection of recurrence or metastasis for choriocarcinoma when the intact hCG may remain normal More than 60% of patients with nonseminomatous germ cell tumors and 1030% with seminomas have elevated free hCG

HER2/neu (c-erbB-2) Oncoprotein

` `

The HER2/neu (c-erbB-2) oncogene is a transmembrane protein of the tyrosine kinase receptor family Elevated in the sera of pts with a number of different epithelial cell cancers, including breast, lung, colorectal, and ovarian cancers In breast cancer, serum HER2/neu is more important as a prognostic and predictive marker
`

At the time of initial diagnosis, serum HER2/neu is elevated in only 5 10% of breast cancer pts Higher levels predict shorter treatment response while low levels suggest longer or complete treatment responses

Serum levels correlate with response to chemotherapy

`

Also overexpressed in ovarian, prostate, gastric and bronchial carcinomas so it can be used as a therapeutic target in these cancers

p53
` `

p53 is a negative regulator of cell growth Functions as a tumor suppressor by inducing the expression of gene products that are responsible for inhibiting or arresting cell growth and proliferation The encoding gene for p53 has been found to be mutated in about 50% of almost all types of cancer

p53
` `

Because of its short half-life (20 minutes), the wild-type p53 protein in the blood circulation is not detectable The presence of p53 antibody in serum is associated with expression of p53 mutations and positively correlates with the degree of cancer malignancy

Prostate-Specific Antigen (PSA)

` ` `

PSA is unique to the prostate gland

`

Organ specific, not cancer specific

` `

Normal reference range is 04 ng/ml The cancer sensitivity and tissue specificity of PSA makes it the most useful tumor marker available for the screening and management of prostate cancer Lack of specificity in distinguishing prostate cancer and nonmalignant prostate lesions Benign conditions such as BPH, acute prostatitis, and infarction can also be correlated with elevated serum PSA levels

PSA
`

` `

Annual PSA screening is recommended both by the American Urological Association and the American Cancer Society for all men over the age of 50 Because of its tissue specificity, PSA is particularly useful for monitoring the success of surgical prostatectomy Complete removal of the prostate should result in an undetectable PSA level, while incomplete resection of the gland (not persistent disease) might result in measurable levels of PSA
` `

However, it should remain unchanged on extended follow-up Any increase in measurable PSA after a successful radical prostatectomy would indicate prostate cancer recurrence or metastasis

A transient and modest increase of PSA may occur during radiation therapy, which should not be misinterpreted as disease progression

PSA
` `

` ` `

Multiple studies suggest that > 80% of pts with prostate cancer are diagnosed from PSA > 4 ng/ml 50% of pts with PSA > 4 ng/ml do not have prostate cancer on biopsy even at significantly elevated levels, i.e., over 10 ng/ml PSA has a sensitivity of a minimum of 80% and a specificity of around 50% 20% of initially negative biopsies, when repeated over a 3year period, become positive Thus the specificity of PSA may be higher, assuming that the biopsies missed a malignant lesion and that a new malignancy did not occur over the 3 year time period

PSA
` `

The cutoff value of 4.0 ng/ml does not sufficiently distinguish pts with and without prostate cancer There are several approaches that have been developed both to increase the sensitivity and specificity of PSA in detecting prostate cancer
` ` `

Free PSA Complex PSA Percentage of free PSA

Free PSA
`

` `

Serum PSA exists in the serum largely (up to 90% of total PSA) in the form of a PSA-ACT (PSA 1-antichymotrypsin) complex In prostate cancer Increased complex PSA and decreased free PSA Percentage of free PSA = (fPSA/total PSA)/100
` ` `

Inverse relationship with prostate cancer risk (<6%) Free PSA ratio improves the specificity for prostate cancer detection in men with PSA between 410 ng/ml Free PSA >25% - usually associated with BPH Time interval from sample collection and assay should be less than 3 hours and the sample should be stored and shipped at 70rC

Free PSA is subject to quick degradation at 4rC or above

`

PSA Doubling Time, Velocity and Density

`

PSA doubling time

`

can predict recurrence after radical prostatectomy in androgenindependent prostate cancer patients PSA difference divided by the number of year(s) PSA velocity of 0.75 ng/year or above is a strong predictor of cancer with a specificity of 95% PSA velocity may be useful in predicting prostate cancer risk when PSA levels are between 2.0 and 4.0 ng/ml This is especially useful in predicting the risk of cancer and guiding the necessity of prostate biopsy in patients with low/normal PSA value (2.0 4.0 ng/ml)

PSA velocity rate of PSA increase over time

` ` ` `

PSA density ratio of total PSA to prostate gland volume as measured by transurethral ultrasonography
`

PSA densities of greater than 0.15 indicate an increased probability of prostate cancer as opposed to BPH

Conclusions and Directions for the Future

`

` `

In summary, a molecule, almost always a protein, could be used as a tumor marker as long as its changing concentration reflects tumor cell activity The assessment of clinical utility for an individual tumor marker is based on its sensitivity and specificity There has been a clear trend towards improving the test specificity and sensitivity by ordering multiple tumor markers PSA is useful in screening, diagnosis, and therapeutic monitoring
`

Free PSA >25%

lower risk of cancer

References
`

McPherson RA and Pincus MR. Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed. 2006 W.B. Saunders Company Djavan B, Eckersberger E, Finkelstein J, Sadri H, Taneja SS, Lepor H. Prostate-specific Antigen Testing and Prostate Cancer Screening. Prim Care Clin Office Pract. 2010. 37: 441459.

Tumor Markers in Breast Cancer

SERUM TUMOR MARKERS ` MUC-1 family: CA 15.3, MCA, CA 549, CA 27.29, CAM 26, CAM 29 ` CEA ` Oncoproteins (C-erbB-2) ` Glycolytic enzymes: LDH... ` Milk proteins: lactoalbumin ` Cytokeratins:TPA
` ` ` ` `

MUC-1 antigens : The most widely used Similar sensitivities and specificities The use of more than one MUC-1 antigen is unlikely to confer any advantage CEA measurement can provide additional complementary information One MUC-1 tumor marker and CEA

Tumor Markers in Localized Breast Cancer

`

Tumor Marker determination may complement patient staging:

`

CA 15.3 and CEA are related to tumor burden, with significantly higher values in node positive patients and in patients with larger tumors

` `

Preoperatively elevated levels of either CA 15.3 or CEA are associated with adverse outcome However, Most studies with large patient groups and long follow-up times conclude that pre-operative serum CA 15.3 and CEA concentrations are independent prognostic factors

Early Detection of Metastases

` `

` ` ` `

Increases in CA15.3 provide the first indication of recurrence, prior to clinical or radiological indication, in 40-55% of patients Additional CEA measurements can increase the sensitivity in the early detection of recurrence obtained with CA 15.3 by up to 5- 25%, Lead Time 2-18 months (mean 5.2 month) Specificity for the detection of recurrence: related to the cutpoint used CEA > 5 ng/ml : 5% of false positive results CA 15.3 > 35 U/ml: 6,5% of false positive results Using higher cut-off values (CA 15.3 60 U/ml, CEA 10 ng/ml) and at least two serial increases (>15%), specificity increased to almost 100%

Advanced Disease
` `

Tumor marker sensitivity is significantly higher than in loco-regional disease Combination of several markers (CA15.3, CEA, cytokeratins) increase the sensitivity to 90% in patients with distant metastases. Tumor marker sensitivity is related to the site of metastases

Therapy Monitoring
` `

The most important clinical application: Monitoring therapy response Patients in remission usually have decreasing marker levels, while those with progressive disease generally have increasing levels Biochemical changes often precede clinical or radiological signs of response or progression, potentially enabling earlier treatment decisions regarding continuation of ineffective therapy, change of therapy... Tumor markers should be measured prior to every chemotherapy course and at least three monthly for patients receiving hormone therapy

Estrogen and Progesterone Receptors as Tumor Markers

` `

` ` `

ER and PR are transcriptional factors which mediate the actions of estrogens and progesterone, respectively Both receptors are now known to exist in 2 different forms, ER-alpha and useful when deciding on adjuvant hormone treatment Not guarantee response, fails in 30~40% of patients to endocrine treatment As diagnostic marker when it is a primary unknown tumor Assay of hormone receptors is mandatory in the selection of patients with both, early and advanced breast cancer for treatment with hormone therapy

HER-2neu
` ` `

Oncogene present on chromosome 17 Gene amplified or over-expressed in 20-30% of invasive breast cancers It has been suggested that HER-2 over-expression is associated with relative resistance to hormone therapy, adjuvant therapy, but there are still conflicting reports Similar situation, controversy also exists on the association between overexpression and response to anthracycline-based adjuvant therapy.