Sreeharsha TumourMarkers

Tumour markers : 

Tumour markers Dr Sreeharsha

What are they? : 

What are they? Tumour markers are substances, usually proteins, that are produced by the body in response to cancer growth or by the cancer tissue itself. Synthesized in excess concentration Endogenous products or products of newly switched on genes

IDEAL TUMOR MARKER : 

IDEAL TUMOR MARKER • 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

Why are they done? : 

Why are they done? The goal is to be able to screen for and diagnose cancer early, when it is the most treatable and before it has had a chance to grow and spread. So far, no tumour marker has gained acceptance as a general screen The markers are either not specific enough (too many false positives, leading to expensive and unnecessary follow-up testing) or they are not elevated early enough in the disease process.

Why are they done?..... : 

Why are they done?..... Tumour markers are not diagnostic in themselves. A definitive diagnosis of cancer is made by looking at biopsy specimens (e.g., of tissue ) under a microscope. However, tumour markers provide information that can be used to:

Importance of tumor marker detection for : 

• Screening • Diagnosis • Stage • Prognosis • Guide treatment • Monitor treatment • Determine recurrence Importance of tumor marker detection for

Slide 7: 

In 1968 the world Health Organisation recommended ten principles be followed when countries consider developing national screening programmes. The essence of these is that the disease should be important, well understood and be able to be recognized and tested for at an early stage. Medical support and treatment must be available and be more beneficial if given at an early stage. The health benefits must be greater than any harm done by the screening process which itself must be cost effective.

How are they identified : 

How are they identified ELISA (EIA) for detection of proteins (enzyme linked immunosorbent assay) ELISA is the most widely used immunnodiagnostic technique in many different clinical applications, including diagnosis of HIV, Hepatitis and others as well as hormones, tumor markers or the screening of blood banks etc. The most commonly used format is the microplate ELISA commonly known as „micro-ELISA“. • first published in 1971 – by Engvail & Perimann (Sweden) – by van Weemen & Schuurs (Netherlands) • first commercial one in 1976 – to detect Hepatitis B surface antigen

ELISA : 

ELISA microplate 8 x 12 matrix of 96 wells (each 1 cm high, 0.7 cm diameter) up to 70 μl per well next steps in case of positive results • re-testing • Western-blot The dark blue spot represents a positive clone.The light blue spots are positive controls, the next two wells to the right are negative controls.

Slide 10: 

Sensitivity of markers determined by The ability of the assay to reliably detect it in a biological sample Critical tumour mass present when it can be detected Percentage of tumours expressing the same marker Specificity of a tumour markers determined by Fidelity with which the assay detects only the marker Percentage of other tumours that test positive for the same marker Other non malignant conditions in which the marker is elevated

PITFALLS IN IMMUNOASSAYS : 

PITFALLS IN IMMUNOASSAYS HETEROPHILE ANTIBODIES HIGH DOSE HOOK EFFECT – Definition: A sample with an extremely high analyte concentration that produces a result below that of the highest calibrator. Occurs with hCG, Prolactin, LH, FSH, PSA, AFP and CA-125 assays. • Measured analyte levels are significantly lower than expected. The Hook Effect can be eliminated by development of a two step immunochemical assay. The laboratory should have a dilution protocol in place to test for the Hook Effect NON-IMMUNOREACTIVE HORMONE ISOFORMS CROSS-REACTING SUBSTANCES

Slide 12: 

If repeated measurements of tumor marker are needed, some clinical testing laboratories provide a special reporting mechanism, a serial monitor, that links test results and other data pertaining to the person being tested. This requires a unique identifier for the person. In the United States commonly a Social Security number & Civil Personal Record (CPR) in Bahrainare used for this. One important function of this mechanism is to ensure that each test is performed using the same assay kit. For example, for AFP many different commercial assay kits, based on different technologies, are available. AFP measurements obtained using different assay kits are not comparable unless special calculations are performed.

PCR : 

PCR It’s a means of selectively amplifying a particular segment of DNA. The segment may represent a small part of a large and complex mixture of DNAs: e.g.a specific exon of a human gene. It can be thought of as a molecular photocopier

CLASSIFICATION : 

CLASSIFICATION 1.Oncofetal antigens e.g. AFP, CEA, Pancreatic oncofetal antigen, fetal sulfoglycoprotein. 2. Tumor associated antigens Cancer Antigens e.g. CA125, CA19-9, CA15-3, CA72-4 CA50 etc. 3.Hormones e.g.β-hCG, Calcitonin 4. Hormone receptors e.g. estrogen and progesterone receptors 5. Enzymes and Isoenzymes -PSA, PAP, NSE, PALP, TDT, Lysozyme, alpha amylase 6. Serum and tissue proteins β-2microglobulin,GFAP, protein S-100, ferritin, fibrinogen degradation products 7. Other biomolecules e.g. polyamines

Classification : 

Classification Cancer-specific markers: Cancer-specific markers are related to the presence of certain cancerous tissue. Because there is a large overlap between the many different tumor tissue types and the markers produced these markers might not be specific in making a diagnosis. They can, however, be useful in the follow-up of treated patients to describe progress of the disease or response to treatment. A few examples of these markers are CEA, CA19-9, CA125 tissue-specific markers Tissue-specific markers are related to specific tissues which have developed cancer. These substances are not specifically related to the tumor, and may be present at elevated levels when no cancer is present. But unlike the previous group, elevated levels point to a specific tissue being at fault. Examples include PSA, beta-HCG - (Human chorionic gonadotropin), AFP - (Alpha-fetoprotein), AFP-L3 - (a lectin-reactive AFP) and Thyroglobulin

Common : 

Common

Less common : 

Less common

COMMON TUMOR MARKERS : 

COMMON TUMOR MARKERS Analyte Cancer Use CEA Monitor colorectal, breast, lung cancer CA-125 Ovarian cancer monitoring CA15-3, 27. 29 Monitor recurrences of breast cancer AFP Germ cell tumors, liver cancer Total PSA Screen and monitor prostate cancer Free PSA Distinguish prostate cancer from BPH HCG Germ cell and trophoblastic tumors Hormone receptors Breast cancer therapy NMP 22, BTA ,FDP Monitor recurrences of bladder canc

CEA : 

CEA Described by Gold and Freedman in1965 as a marker for Colorectal Cancer Molecular mass of approximately 200 kDa(kilo dalton) Glycoprotein with a carbohydrate composition ranging from 50 - 85% of molecular mas fetal glycoprotein found on cell surfaces, produced by fetal GI tract, liver, and pancreas CEA levels 5 - 10 times upper limit of normal suggests colon cancer CEA is not used to screen for colon cancer

CEA : 

CEA Found also in 30~50% of breast cancer, small cell lung cancer, mucinous cystadenocarcinoma of ovary, adenocarcinoma of cervix Elevation (<10 ng/ml) in smokers, COPD, inflammatory bowel disease, liver inflammation or cirrhosis, renal failure, fibrocystic breast disease

CEA Distribution In Healthy Individuals andPatients with Non-Malignant Conditions : 

CEA Distribution In Healthy Individuals andPatients with Non-Malignant Conditions Gastrointestinal

CEA Distribution In PatientsWith Malignant Disease : 

CEA Distribution In PatientsWith Malignant Disease

CEA as a Marker forColorectal Cancer(ASCO) : 

CEA as a Marker forColorectal Cancer(ASCO)

Prostate specific antigen(PSA) : 

Prostate specific antigen(PSA) PSA is a neutral protease consisting of a 34-kilodalton single-chain glycoprotein of 240 amino acid residues with a carbohydrate side chain. It is related to the kallikrein family Tissue specific antigen, produced by prostatic alveolar and ductal epithelial cells , a serine protease, t 1/2：2~3 days 3 BIOCHEMICAL FORMS free form – smallest proportion largest form - bound to á1-antichymotrypsin third form - bound to á2-macroglobulin Half life – 2 to 3 days

Factors increasing PSA levels : 

Factors increasing PSA levels all lower gi tract procedures Trans perineal and trans rectal prostate bx. a/c urinary retention Prostatic massage Bicycling After sexual activity a/c prost

PSA : 

PSA Screening - ref value 0-4 ng/ml Staging – localised cancer capsular penetaration on histologically > 10ng/ml Monitoring of Rx – levels fall after resection of organ confined cancers (undectectable after 2 – 4 weeks) Detection of recurrence

Improving performance of PSA as a tumour marker : 

Improving performance of PSA as a tumour marker age-specific serum PSA cut-off levels PSA velocity - in excess of 0.75 ng/ml/year was predictive of cancer free to total PSA ratio- -An ↑in ratio of free/total PSA is associated with increased probability of prostate cancer PSA density Cost – Rs.350

Acid Phosphatase : 

Acid Phosphatase This enzyme is found in high concentraitions in the normal prostate as well as in Primary and metastatic prostate cancers. Acid Phosphatase may also originate from other tissues. The more sensitive immunological test (RIA or counterimmunoelectrophoresis for prostatic acid phosphatase, wich are specific for the enzyme of prostate tissue) often gives positive results in the early stages of disease. Because of the reliability of acid phosphatase as a marker in early disease, it was hoped that the test could be used as a screening tool. However, it may also be elevated in up to 6 percent of cases of benign prostatic hypertrophy and other conditions. Thus, its predictive value is low on theoretical grounds. So, acid phosphatase is useful in following patients with advanced disease.

CA-125 : 

CA-125 CA-125 glycoprotein molecular weight 200-1,000 kda Introduced in 1983 by Bast for ovarian cancer In the US, in 1998 25,400 new cases will be diagnosed and 14,500 women will die as a result of this disease 70% of the women with ovarian cancer are over the age of 50 One half of patients with stage 1 ovarian cancer have elevated CA-125 levels and a five year survival rate of 90%. In late stage disease, the five year survival rate is from 4-30% Worldwide incidence is highest in industrialized countries and lowest in Japan and India Mucin like molecule procuced by mesothelial cells of the peritoneum and other tissues of mullerian origin Shed in body fluids and makes its way to blood stream

CA-125 Distribution In Healthy Subjects and Patients withNon-Malignant Conditions : 

CA-125 Distribution In Healthy Subjects and Patients withNon-Malignant Conditions % Distribution of CA-125 <35 35-65 >65 u/mL u/mL u/mL Healthy Individuals 98 1.7 1.3 Non-Malignant Conditions Pregnancy 73 22 5 Cirrhosis 30 13 57 Pulmonary Disease 94 0 6 Pelvic Inflammatory Disease 76 3 21 Endometriosis 86 11 3 Ovarian Cysts 90 7 3 Uterine Fibroids 77 13 10

CA-125 Distribution In Patients WithMalignant Disease : 

CA-125 Distribution In Patients WithMalignant Disease % Distribution of CA-125 Cancers <35 35-65 >65 u/mL u/mL u/mL Ovarian 14 9 77 Lung 56 19 25 Breast 82 8 10 Endometrial 70 8 22 Cervical 66 15 19 Colorectal 76 11 12

Usefulness of CA 125 in CA ovary : 

Usefulness of CA 125 in CA ovary - screening – shouldn’t be used; normal levels doesn’t exclude malignancy - diagnosis – poor specificity - prognosis – valuable in assesing progressive disease and tumour response to chemotherapy - detection of early recurrence - cost Rs.600/-

Alpha-Fetoprotein : 

Alpha-Fetoprotein Glycoprotein, found in fetal liver, yolk sac, GI tract, biochemically related to albumin in adults 60 KD molecular wt half-life：4~6 days Normal serum levels： 12~15th gestational week 30~40ng/ml At birth 30ng/ml >1 years old (adult) <20 ng

Higher levels seen in : 

Higher levels seen in 1o liver cancer, hepatoblastomas, germ cell tumours (yolk sac tumors, embryonal cell carcinoma), gastric, colorectal, biliary, lung malignancy and pancreatis Transient rise may occur in - liver regeneration , hepatitis ,CLD , pregnancy, NTD.

Monitoring response to treatment : 

Monitoring response to treatment after surgical resection S.AFP elvels fall with in a reference range i.e <10 ng/ml in 5-6 days if resection appears complete but AFP levels does not fall to the reference range, residual tumour is invariably present. Cost – Rs.350

Human Chorionic gonadotrophin : 

Human Chorionic gonadotrophin GP contains Galactose and hexosamine Alpha and beta subunits Secreted by syncytiotrophoblast Serum and urine HCG ↑in early gestation and peak in the first trimester (60~90 days) T ½: 1.25 days, ~30 hours Elevated in：gestational trophoblastic disease (a progressive rise in after 90 days of gestation → highly suggestive), choriocarcinoma Also rised in Germ cell tumours – teratoma seminoma, dysgerminoma

Slide 38: 

Usefullness in choriocarcinoma - diagnosis- serum and urine HCG - prognosis – determines the chemo. regimens, poor prognosis >40 000u/l Monitoring response to Rx.- monthly determination of HCG is recommended until the levels are normal for 12 months. In Germ Cell Tumors Detection Monitor treatment response (ex. C/T)：production of BHCG ceases on commencement of tx, rising or persistently elevated levels are diagnostic of resistance to C/T evaluate radicality of the surgery: ex. In testicular cancer, the presence of β-HCG after orchiectomy → residual cancer and needs further treatment Monitor relapse Cost is 400/-

CA 19-9 : 

CA 19-9 a mucin which reacts with monoclonal antibody 111 6 NS 19-9. It is believed to be involved in cell adhesion and was originally discovered in human colorectal carcinoma cell lines. Normal - < 37

Serum CA 19-9 levels may be raised in : 

Serum CA 19-9 levels may be raised in pancreatic carcinomas (70-100% ) hepatocellular carcinoma (22-51%) gastric carcinoma (42%) and colorectal carcinoma (20%) Benign conditions acute and chronic pancreatitis, cholestasis, cirrhosis, acute cholangitis and cystic fibrosis

Slide 41: 

The elevation of CA 19-9 in benign pancreatic disease is lower than with carcinoma of pancreas and usually does not exceed 120 U/ml. CA 19-9 is most useful in pancreatic cancer. Cost – Rs. 600/-

CA 19-9 as a Marker forPancreatic Cancer(ASCO) : 

CA 19-9 as a Marker forPancreatic Cancer(ASCO)

CA15-3 : 

CA15-3 Normal：<31 U/ml ↑in 20% with localized breast cancer, ~80% with metastatic disease, esp. if with bone involvment Specificity of 86%, sensitivity of 30% Also increased in gastric, pancreatic, cervical lung cancer CA 15-3 is a mucin-like membrane glycoprotein. Conditions asso with high levels - normal healthy individuals 5 – 6 % - benign disease of hepatic origin - CA Breast, Ovarian, Lung and liver CA

Usefulness in CA Breast : 

Usefulness in CA Breast Prognosis - levels are realated to tumour stage - significantly higher levels seen in nodal involvement and larger tumours monitoring response to treatment - in patients with CA Breast with high levels it can be used to monitor response to Rx. along with clinical evaluation and imaging. Cost- Rs.700/-

Estrogen receptor : 

Estrogen receptor ER is a protein found in the nucleus of breast and uterine tissues. The level of ER in the tissue is used to determine whether a person with breast cancer is likely to respond to estrogen therapy with tamoxifen, which binds to the receptors blocking the action of estrogen. 2 isoforms：ERa + ERb ERa → better prognosis, predictor of relapse 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 ERb → distinct biological roles and ligand binding specificity, good prognostic factor, correlate with low grade and (-) axillary LN status

Slide 46: 

Women who are ER-negative have a greater risk of recurrence than women who are ER-positive HER-2/neu oncogene (using monoclonal antibody)：overexpression related to poor prognosis in breast cancer Oncogene c-erbB-2 gene：overexpressed in 30% of breast cancers, correlation between c-erbB-2 gene positivity, positive axillary node status, reduced time to relapse and reduced overall survival BRCA1 gene on chromosome 17q：familial breast-ovarian cancer syndrome, and breast cancer in early-onset breast cancer families → high risk screening

Progesterone receptor : 

Progesterone receptor PR consists of two proteins, like the estrogen receptor, which are located in the nuclei of both breast and uterine tissues. Persons who test negative for both ER and PR have less than a 5% chance of responding to endocrine therapy Those who test positive for both markers have greater than a 60% chance of tumor shrinkage when treated with hormone therapy.

Nuclear matrix protein (NMP22) and bladder tumor-associated analytes (BTA) : 

Nuclear matrix protein (NMP22) and bladder tumor-associated analytes (BTA) NMP22 is a structural nuclear protein that is released into the urine when bladder carcinoma cells die. Approximately 70% of bladder carcinomas are positive for NMP22. BTA is comprised of type IV collagen, fibronectin, laminin, and proteoglycan, which are components of the basement membrane.

Slide 49: 

These released into the urine when bladder tumor cells attach to the basement membrane of the bladder wall BTA is elevated in about 30% of persons with low-grade bladder tumors and over 60% of persons with high-grade tumors

Neuron Specific Enolase : 

Neuron Specific Enolase Neuron specific enolase is an isozyme of the glycolytic pathway that is found only in brain and neuroendocrine tissue. Its an immunohistochemical marker for tumors of the central nervous system, neuroblastomas, and APUD tumors. Use of NSE has been evaluated in lung cancer and neuroblastoma.

Thyroid Cancer : 

Thyroid Cancer Thyroglobulin： Tissue-specific, glycoprotein produced by thyroid follicular cells normal: <60 ug/L Also increased in breast or lung cancer Thyrocalcitonin： Produced by thyroid C cells and medullary thyroid cancer normal: <100 ng/L or <29 p mole/L Effective in screen patients with 1st degree relatives affected by medullary thyroid cancer and multiple endocrine neoplasia type 2

Melanoma : 

Melanoma Tyrosinase Use PCR to detect hematogenous spread of melanoma cells from a solid tumor in peripheral blood S100B protein For confirmation of amelanotic malignant melanoma in immunohistology ↑in 70% with stage IV metastasized melanoma

THANK YOU : 

THANK YOU