SOME TUMOUR MARKERS CHEMICAL PATHOLOGY.pptx

Tumours markers

1. MUSA DUNGUS TUMOUR MARKERS

2. OUTLINE Introduction Historical background Clinical applications Classification Analytical methodology Summary and conclusion

3. Introduction Definition: A tumour marker is a substance produced by a tumour, or by the host in response to a tumour, that is found in blood, body fluids or tissue that may be used to predict the tumours presence, size and response to therapy.

4. Such substances are found in cells,tissues,or body fluids and are measured qualitatively or quantitatively by chemical,immunological ,or molecular biological methods .

5. Tumour markers, by broad definition, are biochemical analytes that are useful for cancer detection, tumour growth prediction , or progression of the illness . They are most commonly used in diagnosis of lymphoma, leukaemia, and colorectal, pulmonary, gastric, pancreatic, breast, liver, or prostatic carcinomas.

6. Historical background Historical perspective The first tumour marker reported was the Bence Jones protein . Since its discovery in 1847 by precipitataion of a protein in acidified urine, its measurement has been a diagnostic test for multiple myeloma.

7. History cont’d Bence J ones protein was identified as the monoclonal light chain of immunoglobulin secreted by plasma cells. The second era of tumour markers, from 1928 to 1963 , included the discovery of hormones , enzymes , isoenzymes , and proteins and their application to the diagnosis of cancer and the beginning of the chromosomal analysis of tumours. The third era was heralded by the discovery of AFP in 1963 and CEA in 1965 . The fourth era started in 1975 with the development of monoclonal antibodies and their subsequent use to detect oncofetal antigen . Examples are carbohydrate antigens such as CA 125 , CA 15.3 , and CA 27.29 .

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9. What is an ideal Tumour Marker? An ideal tumour marker is: detectable only when malignancy is present. specific for the type and site of malignancy. correlates with the amount of malignant tissue present. responds rapidly to a change in tumour size. easy and cheap to measure, from a laboratory point of view. At present, no tumour marker fulfils all of the above criteria.

10. Tumour markers are used: Screening for Cancers e.g. PSA Monitoring treatment Diagnosis (when biopsy is not feasible) Determine prognosis Detecting recurrence

11. POTENTIAL USES OF TUMOUR MARKERS 12 Screening in general population. Differential diagnosis in symptomatic individuals Prognostic indicator of disease progression. Clinical staging of cancer (evaluating tumour burden) Estimation of tumour value

12. s 13 Evaluation of success of treatment . Detection of recurrence of cancer Monitoring of response to therapy Determination of direction for immunotherapy Radioimmunolocalization of tumour masses

13. Classes of tumour markers Oncofoetal and carbohydrate antigens Enzymes Hormones Proteins and Tissue Receptors Genetic markers/ Oncogenes Blood group antigens Cytokeratins Miscellaneous markers

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15. ONCOFOETAL ANTIGENS Oncofetal antigens are proteins made by genes that are very active during fetal development but function at a very low level after birth. The genes become activated when a malignant tumor arises and produce large amounts of protein.

16. Oncofoetal antigen Antigens comprise the largest class of tumor marker and include the tumor-associated glycoprotein antigens. Important tumor markers in this class are: alpha-fetoprotein ( AFP ), carcinoembryonic antigen ( CEA ), prostate specific antigen ( PSA ), CA-125 , CA-19-9 , CA-15-3 , and bladder tumor -associated antigen( BTA ).

17. 18 A ONCOFETAL ANTIGEN - Carcinoembryonic antigen ( CEA ) - Alpha fetoprotein ( AFP ) - Human chorionic gonadotropin ( HCG ) - Tissue polypeptide antigen ( TPA ) - Tennesse antigen ( TENAGEN ) - Fetal sulfoglycoprotein ( FSA ) - Pancreatic oncofetal antigen ( POA ) - Breast cancer associated markers

18. Oncofoetal antigen Carcinoembryonic antigen(CEA) Carcinoembryonic antigen (CEA) is a protein found in many types of cells but associated with tumours and the developing foetus. CEA is tested in blood. The normal range is <2.5 ng/ml in an adult non-smoker and <5.0 ng/ml in a smoker.

19. CEA cont’d The CEA was one of the first oncofoetal antigens to be described and exploited clinically. It is a complex glycoprotein of molecular weight 20,000 , that is associated with the plasma membrane of tumour cells, from which it may be released into the blood. Elevated CEA levels are found in a variety of cancers other than colonic , including pancreatic , gastric , lung , and breast . It is also detected in benign conditions including cirrhosis , inflammatory bowel disease , chronic lung disease , and pancreatitis . The CEA was found to be elevated in up to 19 percent of smokers and in 3 percent of a healthy control population.

20. CEA cont’d As a screening test, the CEA is also inadequate . Since cancer prevalence in a healthy population is low, an elevated CEA has an unacceptably low positive predictive value, with excess false positives. Also, since elevated CEA occurs in the advanced stage of incurable cancer but is low in the early, curable disease, the likelihood of a positive result affecting a patient's survival is diminished. The CEA has been suggested as having prognostic value for patients with colon cancer. Preoperative CEA values have been positively correlated with stage and negatively correlated with disease free survival

21. CEA cont’d Although not satisfactory for screening a healthy population, CEA has been used to monitor recurrence. The CEA is of some use as a monitor in treatment . Usually the CEA returns to normal within 1 to 2 months of surgery, but if it returns elevated persistent disease may be indicated. Early data suggested that CEA preceded clinical relapse by several months. Determinations of CEA should be done frequently: at a minimum of every 3 months.

22. AFP Alpha-fetoprotein(AFP) Alpha-Fetoprotein is a normal fetal serum protein synthesized by the liver, yolk sac, and gastrointestinal tract that shares sequence homology with albumin. It is a major component of foetal plasma, reaching a peak concentration of 3 mg/ml at 12 weeks of gestation. Following birth, it clears rapidly from the circulation, having a half life of 3.5 days, and its concentration in adult serum is less than 20 ng/ml . AFP is of importance in diagnosing hepatocellular carcinoma and may be useful in screening procedures.

23. AFP AFP elevation is more common in areas where hepatocellular carcinoma is endemic, such as Africa and in patients who are HBsAg positive AFP is a marker for hepatocellular and germ cell tumours with elevation of > 500ng/ml . It is a glycoprotein produced in large amounts during fetal life and is homologous to albumin. In healthy adults, less than 10 µg/L of AFP is found in the circulation. AFP is elevated in normal pregnancy, benign liver disease (hepatitis, cirrhosis), as well as in cancer.

24. AFP AFP is elevated in testicular germ cell tumors containing embryonal or endodermal sinus elements. A definitive positive marker value is highly sensitive in indicating relapse or response to treatment. The AFP is less frequently elevated in other malignancies such as pancreatic cancers, gastric cancers , colonic cancers, and bronchogenic cancers. This elevation was not necessarily associated with liver metastases.

25. PSA Prostate-specific antigen(PSA) The most commonly tested tumour marker for the prostate gland is prostate specific antigen. It is normally present in low levels in the blood of all adult men. The normal range is 0 to 4 ng/ml. PSA is prostate-specific, not cancer-specific. A variety of conditions can raise PSA levels: prostatitis, BPH, and prostate cancer. PSA levels can also be influenced by a number of other things. Some prostate glands normally produce more PSA than others. PSA levels tend to increase with age. And, PSA levels can vary with race : African Americans often have higher PSA levels; Asian men often have lower PSA levels.

26. PSA PSA seems to have the capability of achieving at least one of the characteristics of an ideal tumour marker- tissue specificity; it is found in normal prostatic epithelium and secretions but not in other tissues. It is a glycoprotein , whose function may be to lyse the seminal clot. PSA is highly sensitive for the presence of prostatic cancer. The elevation correlated with stage and tumor volume. It is predictive of recurrence and response to treatment. Finally, the antigen has prognostic value in patients with very high values prior to surgery are likely to relapse.

27. The PSA level can be elevated in prostate cancer, prostatitis, benign prostatic hypertrophy, and prostatic trauma, as well as after ejaculation. In prostate cancer, the positive predictive value of PSA levels greater than 4 ng per mL is 20 to 30 percent and rises to 50 percent when PSA levels exceed 10 ng per mL. Nevertheless, 20 to 30 percent of men with prostate cancer have PSA levels within normal ranges.

28. CA 125 CA 125 CA125 is an antigen present on 80 percent of nonmucinous ovarian carcinomas . It is defined by a monoclonal antibody ( OC125 ) that was generated by immunizing laboratory mice with a cell line established from human ovarian carcinoma. CA125 is often elevated in patients with ovarian cancer, its level following the patient's clinical course. The CA125 is elevated in other cancers including endometrial , pancreatic , lung , breast , and colon cancer , and in menstruation , pregnancy , endometriosis , and other gynaecologic and non gynaecologic conditions.

29. CA 19-9 CA19-9 CA19-9 is a monoclonal antibody generated against a colon carcinoma cell line to detect a monosialoganglioside found in patients with gastrointestinal adenocarcinoma . It is found to be elevated in 21 to 42 percent of cases of gastric cancer, 20 to 40 percent of colon cancer, and 71 to 93 percent of pancreatic cancer, and has been proposed to differentiate benign from malignant pancreatic disease.

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31. enzymes ENZYMES Many enzymes that occur in certain tissues are found in blood plasma at higher levels when the cancer involves that tissue. Enzymes are usually measured by determining the rate at which they convert a substrate to an end product, while most tumour markers of other types are measured by a test called an immunoassay. Some examples of enzymes whose levels rise in cases of malignant diseases are acid phosphatase , alkaline phosphatase , amylase , creatine kinase , gamma glutamyl transferase , lactate dehydrogenase , and terminal deoxynucleotidyl transferase

32. Prostatic acid phosphatase Acid phosphatase This enzyme is found in high concentrations 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 immunoelectrophoresis for prostatic acid phosphatase, which are specific for the enzyme of prostate tissue) often gives positive results in the early stages of disease.

33. 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(such as small cell lung cancer, pheochromocytoma, carcinoid, melanoma, medullary thyroid carcinoma). Galactosyl Transferase II Galactosyl Transferase II, an isozyme of galactosyl transferase, has been shown to be elevated in a variety of malignancies, predominantly gastrointestinal . In colon cancer its level correlated with the extent of disease and disease progression; in pancreatic cancer it was more sensitive and specific in distinguishing benign from malignant disease than CEA and other tests

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35. hormones HORMONES Hormones are produced by many tumours. The hormone may be a natural product of its associated organ or represent abnormal synthesis reflecting unregulated cancer cell metabolism. Examples include insulin production by islet cell tumor, calcitonin by medullary thyroid carcinoma, ACTH and ADH by lung and adrenal cancers, and catecholamines by pheochromocytoma. Human Chorionic Gonadotropin HCG is a glycoprotein consisting of subunits a and b, which are nonconvalently linked. The hormone is normally produced by the syncytiotrophoblastic cells of the placenta and is elevated in pregnancy. Its most important uses as a tumor marker are in gestationa l trophoblastic disease and germ cell tumors .

36. hCG All gestational trophoblastic tumors produce HCG, and it is a valuable marker in these tumors, screening reliably in all cases and indicating poor responses to treatment. The level correlates with tumor mass and thus has prognostic value . HCG is extremely sensitive , being elevated in women with minute amounts of tumor. The patient is followed weekly during treatment, and at the completion of treatment indefinite follow up is advised to detect recurrence. HCG is essential in managing trophoblastic neoplasms. The level of HCG is occasionally elevated in other cancers including those of breast, lung, and gastrointestinal tract , but in these diseases it has found little clinical application.

37. Protein and tissue receptors PROTEINS and TISSUE RECEPTORS Tissue receptors, which are proteins associated with the cell membrane, are another type of tumor marker. These substances bind to hormones and growth factors, and therefore affect the rate of tumor growth. Some tissue receptors must be measured in tissue samples removed for a biopsy, while others are secreted into the extracellular fluid and may be measured in the blood. Some important receptor tumor markers are oestrogen receptor , progesterone receptor , interleukin-2 receptor , and epidermal growth factor receptor . Immunoglobulins Production of a monoclonal immunoglobulin molecule is characteristic of multiple myeloma. These paraproteins are usually complete antibody molecules but may be isolated light chains or, rarely, heavy chains. They may be lambda or kappa light chains.

38. Protein and tissue receptors cont’d S-100 proteins The S-100 proteins are a group of 19 related calcium-binding proteins. Some members such as S-100A4, S-100A2, and S-100b are associated with cancer progression. The expression of S-100A4 in breast, oesophageal and gastric cancers correlates with worse outcome and more aggressive disease. RECEPTORS AS TUMOUR MARKERS Estrogen receptor (ER): 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.

39. . Women who are ER-negative have a greater risk of recurrence than women who are ER-positive. Tissue levels are measured using one of two methods. The tissue can be homogenized into a cytosol, and an immunoassay used to measure the concentration of ER receptor protein. Alternatively, the tissue is frozen and thin-sectioned. An immunoperoxidase stain is used to detect and measure the estrogen receptors in the tissue. Progesterone receptor (PR): PR consists of two proteins, like the estrogen receptor, which are located in the nuclei of both breast and uterine tissues. PR has the same prognostic value as ER, and is measured by similar methods. Tissue that does not express the PR receptors is less likely to bind estrogen analogs used to treat the tumor.

40. Tissue receptors as tumour markers 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. Epidermal growth factor receptor(EGFR) This is a prototype of a family of tyrosine kinase receptors. In cancerous tissue, these growth factors can promote growth both in a autocrine and paracrine fashion. The EGFR was found to be a strong prognostic indicator in head and neck, ovarian, cervical, bladder and oesophageal cancers. Patients with elevated EGFR showed reduced overall survival.

41. Genetic markers GENETIC MARKERS/ONCOGENES Cancerous growth is an inheritable characteristic of cells and is thought to be the outcome of genetic changes. Some tumor markers are the product of oncogenes. Some important oncogenes are BRAC-1 , myc , p53 , RB , and Ph . BRCA1 and BRCA2 In 5-10% of breast cancer, there is a strong inherited familial risk. These highly penetrant and autosomal dominant genes , BRCA1 and BRCA2, account for most cases of familial breast cancer. They are found in 60-80% of familial breast cancer. K- ras gene This binds to guanine nucleotide and functions as a molecular switch that regulates mitogenic signals from growth factors to nucleus.

42. Mutated K- ras is present in 95% of pancreatic cancers, 40% of colon cancers, and 30% of lung and bladder cancers. Activated ras is detected by expression of the ras gene product, p21 in cancer tissue.

43. Analytical methodology quality requirements relevant to all tumor marker measurements may be viewed under the following broad headings: • Pre-analytical requirements - choice of tumor marker, specimen type, specimen timing, sample handling. • Analytical requirements – assay standardisation, internal and external quality control, interferences. • Post-analytical requirements – reference intervals, interpretation and reporting of tumor marker results.

44. Samples used for TMs estimations 1- Blood, plasma, and Serum. 2- Pleural fluid, ascetic fluid, or pericardial fluid. 3- Urine. 4- CSF.

45. Analytical methodology Enzyme assay:measurement of enzyme activity using spectrophotometric determinations. Immunoassay : using radioimmunoassay(RIA) and enzyme linked immunosorbent assay(ELISA). Immunohistochemistry : for measurement of markers directly in the tissues e.g. cathepsin concs,genetic markers, etc.

46. Immunocytochemical assays : used to measure steroid hormone receptors . This assay is cheaper and simpler,require less time, and can be performed using less tissue as compared to classic quantitative biochemical method for assaying steroid receptors in tumour tissue specimens(titration assay) and enzyme immunoassays which are now both obsolete.

47. RECOMMENDATIONS Never rely on the result of a single test. When ordering serial testing, be certain to order every test from the same laboratory using the same assay kit. Be certain that the tumour marker selected for monitoring recurrence was elevated in the patient prior to surgery. Consider the half-life of the tumour marker when interpreting the test result. Consider how the tumour marker is removed or metabolized from the blood circulation. Consider ordering multiple markers to improve both the sensitivity and the specificity for diagnosis Be aware of the presence of ectopic tumour markers

48. Summary and conclusion A tumour marker is a substance produced by a tumour, or by the host in response to a tumour, that is used to differentiate a tumour from a normal tissue. Although there is no ideal tumour marker at the moment, tumour markers can be used for the following purposes: screening, making a diagnosis of cancer, determining the prognosis and monitoring the course in a patient in remission or therapy. The major classes of tumor markers are:Oncofoetal and carbohydrate antigens, Enzymes, Hormones, Proteins and Tissue Receptors, and Genetic markers/ Oncogenes .

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50. Corrections Stool: may be used for p53 and APC gene assay. hCG ; not HCG Can tumour markers be used alone for diagnosis?no has to be in conjunction with other factors eg histology,clinical state of d pt and other markers High dose hook effect Why smoking cause increase in conc of CEA(mechanism) Definition of terms: -specificity -sensitivity -+ ve predictive value -negative predictive value -diagnostic accuracy

51. Commonly assayed markers in this centre - bence jones proteins -PSA -enzymes(AST,ALT,ALP,GGT,NSE,LDH) -hormones( hCG , Methods to increase specificity and sensitivity -tumour volume and conc of marker - density - velocity -