Tumor Markers: Characteristics and Clinical Applications in Cancer Diagnosis

1. Pakistan Society Of Chemical PathologistsDistance Learning Programme In Chemical Pathology(DLP-2)Lesson No 19Tumour MarkersBy Col NaveedAsifConsultant Chemical Pathologist / Section Head Endocrinology and Tumour MarkersDepartment of Chemical Pathology and EndocrinologyAFIP Rawalpindi&Brig Aamir IjazMCPS, FCPS, FRCP (Edin), MCPS-HPEHOD and Professor of Pathology / AFIP Rawalpindi

2. Part IMCQs (One Best Type)

3. Q.1: Tumor markers that are to be put to clinical use should have certain characteristics that are applicable in all situations. An ideal marker has a number of characteristics. All the following are characteristics of an Ideal tumor marker EXCEPT: a. Have 100% accuracy in differentiating between healthy individuals and tumor patients b. Have a normal plasma level, urine level or both in the presence of micro metastasis c. Have high positive and negative predictive value d. Precede and predict recurrences before they are clinically detectable e. Provide a lead-time over clinical diagnosis b. Have a normal plasma level, urine level or both in the presence of micro metastasis

4. Tumor Marker • Substances present in, or produced by a tumor itself or produced by host in response to a tumor that can be used to differentiate a tumor from normal tissue or to determine the presence of a tumor based on measurements in blood or secretions • Such substances are found in cells, tissues or body fluids • Measured qualitatively or quantitatively by chemical, immunological or molecular biological methods • Some tumor markers represent re-expression of substances produced normally by embryonically closely related tissue e.g. CEA in Colon, stomach, liver, and pancreas

5. Q 2.Tumor markers are substances present in, or produced by, a tumor itself or by host. It can be detected in plasma or other body fluids including urine by different techniques. Which of the following markers is estimated in urine? a. Human kallikerin 2 b. Intercellular adhesion molecule-1 c. Lysophosphatidic acid d. Nuclear matrix proteins e. Urokinase-Plasminogen activator inhibitor d. Nuclear matrix proteins

6. Human Kallikerin 2 • Human glandular kallikrein 2 (hK2) is a prostate-specific kallikrein produced by the prostatic epithelium with approximately 80% DNA sequence homology with PSA • hK2 is a potent protease, with more than 20,000 times the activity of the relatively weak protease PSA • While PSA production is often decreased in poorly differentiated prostate cancers, hK2 production appears to be increased • In prostatism patients the ratio of hK2 to free PSA improves the discrimination between Prostate Cancer and Benign Hyperplasia within the diagnostic “Gray Zone” of total PSA 4 to 10 ng/ml • Monoclonal antibodies have been produced to detect hK2

7. Intercellular Adhesion Molecule-1 • ICAM-1 (Intercellular Adhesion Molecule 1) also known as CD54 (Cluster of Differentiation 54) • A member of the immunoglobulin superfamily Ig-like cell adhesion molecule expressed by several cell types including leukocytes and endothelial cells • Derangement of ICAM-1 expression contributes to the clinical manifestations of a variety of diseases, predominantly by interfering with normal immune function. • Among these are malignancies (e.g., melanoma and lymphomas), many inflammatory disorders (e.g., asthma and autoimmune disorders), atherosclerosis, ischemia, certain neurological disorders, and allogeneic organ transplantation.

8. Lysophosphatidic Acid • LPA is a phospholipid derivative, identical in structure to phosphatidic acid (PA) • Bulk of LPA production occurs in bodily fluids, outside the cell. From there, it can bind to, and activate, upwards of six different cell surface receptors, initiating a diverse range of signaling cascades resulting in cell proliferation • Dysregulation of LPA receptors can lead to hyperproliferation, which may contribute to oncogenesis and metastasis • Alongwith CA 125, plasma LPA level can be a useful marker for ovarian cancer, particularly in the early stages of disease

9. Nuclear Matrix Proteins • The nuclear matrix (NM) is a structure resulting from the aggregation of proteins and RNA in the nucleus of cells • Nuclear matrix proteins (NMPs) make up the internal structure of nucleus. They are associated with key reactions in nucleus like DNA replication and RNA synthesis • Expression pattern of NMP has become an important early indicator for numerous cancers/tumors • NMPs released by cancer cell are different from those in normal cell • Particular importance in bladder cancer patient, owing its excretion in urine

10. Urokinase Plasminogen Activator System • Urokinase plasminogen activator (uPA) is a serine protease with an important role in cancer invasion and metastases . • When bound to its receptor (uPAR), uPA converts plasminogen into plasmin and mediates degradation of the extracellular matrix during tumor cell invasion. • High levels of uPA and uPAR, as well as the plasminogen activator inhibitor -1 (PAI-1), have been associated with shorter survival in women with breast cancer; in contrast, high levels of PAI-2 appear to be associated with better outcomes .

11. Urokinase Plasminogen Activator System (cont) • One explanation is that tumor may be overproducing uPA, allowing cancer cells to spread beyond the tumor. High levels of PAI-1 may not be able to inhibit the growth of the tumor • uPA and PAI-1 can be measured by ELISAs on a minimum of 300 mg of fresh or frozen breast cancer tissue • Both are used for the determination of prognosis in patients with newly diagnosed, node-negative breast cancer • Overexpression of uPAand/or PAI-1 have been consistently related to poor prognosis • If a patient has high levels of uPA and PAI-1, risk of recurrence of disease is very high

12. Q. 3: Changes in concentration of tumor markers is used to describe the true status of a tumor. Different criteria/definitions have been devised based on various scientific facts. “A linear increase in the concentration of tumor marker in three consecutive samples on log scale provided no therapy is given”. This statement truly describes: a. Confirmation of diagnosis b. Partial remission of tumor c. Recurrence of tumor d. Relapse of tumor e. Screening of tumour c. Recurrence of tumor

13. Q.4:A 59 years male is a known patient of chronic liver disease. His recent result of Alpha Fetoprotein (AFP) is 197ng/ml. Now the major challenge for a Chemical Pathologist is to offer another biochemical test to rule out hepatocellular carcinoma in this patient. Many new candidate tumour markers have been suggested to be used alone or in combination with AFP. Which of the following biomarkers has the strongest evidence to be used in such patients? a. Alpha-L-fucosidase activity b. Human carboxylesterase 1 c. Lens culinaris agglutinin-reactive AFP d. Transforming growth factor-beta-1 e. Tumor-associated isoenzymes of gamma-glutamyltranspeptidase c. Lens culinarisagglutinin-reactive AFP

14. Lens Culinaris Agglutinin-reactive AFP(AFP-L3) • Lens culinaris agglutinin-reactive AFP (AFP-L3) is a fucosylated fraction of AFP that may be a helpful diagnostic and prognostic maker of hepatocellular carcinoma (HCC), particularly in patients with low serum AFP levels. • The sensitivity and specificity of AFP-L3 assay (using a cut-off of ≥5 percent) for HCC were found to be 42 and 85 percent, respectively. • In addition, patients with high AFP-L3 levels using the highly sensitive assay had lower survival rates than patients with AFP-L3 levels of less than 5 percent.

15. Alpha-L-Fucosidase Activity • The lysosomal hydrolase, alpha-L-fucosidase (alpha-L- fucosidefucohydrolase; (AFU), is present in many mammalian tissues including humans where it degrades fucose-containing glycoconjugates. • Deficiency of AFU results in a rare neurovisceral storage disease known as fucosidosis • Women with low serum activity of the enzyme may be prone to ovarian carcinoma. • Raised serum concentrations of AFU have been described in patients with a variety of benign diseases, including diabetes, hyperthyroidism and cirrhosis, alcoholic hepatitis and acute viral hepatitis. • Increased AFU activity has been found in patients with carcinoma of the lung, breast, stomach, ovary, uterus and hepatocellular carcinoma • AFU is both less sensitive and less specific than alpha-fetoprotein as a serum marker of hepatocellular carcinoma.

16. Human Carboxylesterase 1 • Liver carboxylesterase 1 (CES1, hCE-1 or CES1A1) is an enzyme, also historically known as serine esterase 1 (SES1), monocyte esterase and cholesterol ester hydrolase (CEH) • It is involved in both drug metabolism and activation, as well as other biological processes including • Detoxification of xenobiotics • Involvement in cholesterol metabolism • Catalyses the hydrolysis of heroin and cocaine • Activation of many prodrugs such as angiotensin-converting enzyme (ACE) inhibitors, Carboxylesterase 1 deficiency may be associated with non-Hodgkin lymphoma or B-cell lymphocytic leukemia

17. Transforming Growth Factor-beta-1 • Transforming growth factor beta 1 or TGF-β1 is a polypeptide member of the transforming growth factor beta superfamily of cytokines • It is a secreted protein that performs many cellular functions, including the control of cell growth, cell proliferation, cell differentiation and apoptosis • Heterozygous mutations in TGFB1 gene result in a rare-Camurati-Engelmann disease type I (CED) with characteristic anomalies in the skeleton. It is a form of dysplasia • Some TGFB1 gene mutations are acquired. The TGFβ-1 overexpression occurs in certain types of prostate cancers, breast, colon, lung, and bladder cancers

18. Tumor-associated Isoenzymes Of Gamma-glutamylTranspeptidase • γ-glutamyltranspeptidase is a membrane-bound enzyme which hydrolyzes γ-glutamyl • y-GTactivity is high in foetal liver, in hepatocellular carcinoma (HCC) and in the preneoplastic lesions which precede these tumours, but is low in adult liver tissue • In damaged hepatocytes, particularly in hepatocarcinogenesis, GGT is significantly released into the blood from hepatic tissues • However the total activity of GGT has a significant overlap with various liver diseases which limits its value in diagnosis • However sensitivity and specificity of GGT is increased in HCC when combined with AFP

19. Q: 5. A 35 years old lady presented with five years history of pelvic mass. On laparotomy it turned out to be of ovarian origin. Histopathology revealed carcinoma ovary. Blood sample was sent for CA 125 level and result was 20 IU/ml. What could be the most likely cause of normal CA 125 level? a. Endometrial carcinoma alongwith carcinoma ovary b. Haemolysed sample c. Mucinous type of carcinoma ovary d. Multi-loculated cysts in ovaries e. Photometric method of analysis c. Mucinous type of carcinoma ovary

20. CA 125 • Cancer Antigen (CA) 125 is a high molecular weight glycoprotein expressed by epithelial ovarian tumors and other pathologic and normal tissues of mullerian duct origin • CA 125 is a most promising marker for ovarian cancer • About 80% of non-mucinous epithelial ovarian cancers have raised CA 125 levels, while normal levels are seen in 75% of mucinous tumors like Brenner, sex cord and germ cell tumors • CA 125 is used for monitoring response to therapy, for detecting residual disease following initial therapy and for detection of recurrent metastasis in ovarian cancer. • However it has limitations in early detection of ovarian cancer due to its low sensitivity and low specificity

21. Benign conditions with raised CA 125 levels

22. Malignant conditions with raised CA 125levels

23. Q. 6: HyperglycosylatedhCG (hCG-H) is a glycoprotein with the same polypeptide structure as hCG with higher molecular weight and much larger N- and O-linked oligosaccharides. It has some important clinical applications. hCG–H is useful in all these conditions EXCEPT: a. Detecting non-seminomatous testicular tumors b. Monitoring placental implantation in pregnancy c. Predicting down syndrome pregnancies d. Predicting eclampsia during pregnancy e. Predicting pregnancy outcome after in-vitro fertilization a. Detecting non-seminomatous testicular tumors

24. HyperglycosylatedhCG (hCG-H) • HyperglycosylatedhCG (hCG-H) is a major glycosylation variant of hCG which has a different 3dimensional structure, is also produced by placenta • It is made by extravillouscytotrophoblastcells of placenta • It promotes trophoblast invasion during choriocarcinoma, growth of cytotrophoblast cells and placental implantation in pregnancy • hCG-H is the principal form of total hCG made in early pregnancy. In serum it accounts for 90 +/- 11% of total hCG in the 3rd complete week of gestation and 54 +/- 7% of total hCGduring the 4th complete week of gestation. Its level decreases in remaining pregnancy

25. Clinical Applications of HCG-H • Gestational trophoblastic diseases are governed and regulated by the presence of hCG-H • Management of quiescent gestational trophoblastic diseases • Predicting down syndrome pregnancies- Triple test (hCG/hCG-H, a-fetoprotein, unconjugated estriol, inhibin) • Predicting hypertensive disorders • To differentiate pregnancies that will miscarry and pregnancies that will go to term • To test for early pregnancy in in-vitro fertilized and infertility clinic cases

26. Q. 7: A new tumour marker is being evaluated in a Chemical Pathology lab for the diagnosis of a tumour. At a serum cut off level of 2.5 ng/ml, the sensitivity and specificity of the tumour marker is 94% and 56%, respectively. Increasing the level to 8.0 ng/ml the sensitivity and specificity become 51% and 93%, respectively. The Chemical Pathologist is in search of a cut-off value with optimum sensitivity and specificity. The most appropriate statistical procedure for this purpose would be: a. Chi-square test b. Kaplan–Meier survival estimator c. Pearson` correlation coefficient d. Receiver operating curve e. Student`s t test d. Receiver operating curve

27. Q. 8. A 62 year man has Serum PSA level of 6.9 ng/ml. According to the available evidence, the most promising method of PSA testing to avoid unnecessary prostatic biopsy in this patient is: a. Free to total PSA percentage b. PSA assay with age related cut-off values c. PSA Density d. PSA velocity e. Serum isoform [-2]proPSA e. Serum isoform [-2]proPSA

28. Improving the Accuracy of PSA • Numerous strategies have been proposed to improve the diagnostic performance of PSA when levels are less than 10.0 ng/ml • These strategies include • Measuring PSA velocity • PSA density • Free PSA • Complexed PSA • Using age- and race-specific reference ranges • Serum isoform [-2]proPSA

29. Free to total PSA percentage • The ratio of free-to-total PSA is reduced in men with prostate cancer • Biopsies should be performed only in men with lower ratios. • An optimal cutoff selected for biposy is 25 % • Men with a normal free-to-total PSA ratio still had an 8% probability of having cancer

30. PSA density: PSA concentration / prostatic volume • It is determined by trans-rectal ultrasonography • PSA density measurements better discriminates between cancer and non-cancer groups than PSA levels alone

31. PSA velocity • It is the rate of PSA increase as a function of time • A baseline concentration of PSA in each patient is established, the rate of increase of PSA is then calculated • Men with a PSA velocity > 0.75 ng/ml/year are at increased risk of being diagnosed with prostate cancer

32. PSA assay with age related cut-off values AGE (in years) CUTOFF 40 to 49 0 to 2.5 ng/ml 50 to 59 0 to 3.5 ng/ml 60 to 69 0 to 4.5ng/ml 70 to 79 0 to 6.5 ng/ml

33. Serum isoform [-2]proPSA •  It is also known as P2PSA • Is a specific isoform of the PSA proenzymeproPSA • Increases the detection of prostate cancer for men with PSA values between 2.0 to 10.0 ng/ml • Reduces the number of unnecessary biopsies by 7.6 % with sensitivity of 95 % for detecting prostate cancer

34. Q. 9: In the last a few decades cancer research has resulted in discovery of many new tumour markers e.g. Osteopontin and human epididymis protein 4 (HE4). Which of the following laboratory techniques is most helpful in the discovery of these tumour markers through their genetic over-expression : a. Chemiluminescence b. DNA sequencing c. Mass spectrometry d. Microarray e. PCR d. Microarray

35. Q. 10: A 32 y male has a unilateral swelling of his left testis and symptoms of hyperthyroidism. His thyroid profile was as following: • Serum Free T3 4.12 ng/ml (1.60-4.20) • Serum T4 2.18 pg/ml (0.70-1.68) • Serum TSH 0.14 mIU/L (0.30-4.0) His physician has sought your advice regarding the diagnosis of testicular swelling in this patient. The most probable testicular tumour you would like to exclude is: a. Embryonal carcinoma b. Granulosa cell tumour c. Leydig cell tumour d. Sertoli cell tumour e. Unclassified tumour a. Embryonalcarcinoma

36. Hyperthyroidism Associated with Testicular Tumor • Germ cell tumors are divided into seminomatousor non-seminomatoustypes • Ninety percent of non-seminomatous tumors express either alphafetoprotein or hCG • Intact hCG consists of two subunits. The α subunit is identical to the α subunit of the pituitary gonadotrophinsand thyroid-stimulating hormone (TSH). Β subunit is unique to hCG • hCGcan activate the TSH receptor when present in excess and induce thyrotoxicosis.

37. Part IIShort Answer Questions:

38. Q.11: A 32 years old lady presented in surgical OPD with lump in her left breast for last six months. On examination there was thickness, swelling and redness of skin with nipple retraction and bloody discharge. Later on her mastectomy was done and specimen was sent for histopathology. Her laboratory tests revealed following results: • CEA : 52 ng/ml (< 2.5) • CA 15-3 : 86 U/ml (30) • Estrogen receptor (ER) : Negative in breast tissue by IHC* • Progesterone receptor (PR) : Negative in breast tissue by IHC • HER2/neu : Negative in breast tissue by IHC Please answer following questions   • What is name of breast cancer she is suffering from? • Can ER, PR and HER2/neu be assayed in serum? If yes, please write name(s) of assay which can be used for analyses in serum.

39. Q.11: a. What is name of breast cancer she is suffering from? Triple-negative breast cancer b. Can ER, PR and HER2/neu be assayed in serum? If yes, please write name(s) of assay which can be used for analyses in serum. • No serum assay is available for ER and PR. • Only Her2/neu can be assayed in serum by following technique • Enzyme immunoassay • Chemiluminescent assay

40. Q.12: A 40 years old female has five years history of iron deficiency anaemia and constipation off and on for same duration. She never consulted doctor for these complaints. Later on she developed severe pain in right iliac fossa and was operated upon for Acute Appendicitis. During closing of abdomen surgeon found abnormal small nodular growth on omentum. On further exploration likewise growth was found in both ovaries. Tissue was taken and sent for histopathology. IHC was done on tumor tissue which revealed CK7 negative and CK20 positive in tumor cells. Other laboratory tests were also advised. Their results revealed: • CEA: 25 U/l (less than 2.5) • CA 19.9: 111 U/ml (less than 37) • CA 242: 55 U/ml (less than 20) • Stool for occult blood is equivocal Please answer following questions   • What type of cancer she is having? • Name a single tumor marker emerging as a reliable screening test for this tumor. What is the most suitable sample for its detection? Comment in not more than one line about its sensitivity and specificity in this cancer.

41. Q.12: • What type of cancer she is having? Colorectal adenocarcinoma with ovarian metastasis b. Name a single tumor marker emerging as a reliable screening test for this tumor. What is the most suitable sample for its detection? Comment in not more than one line about its sensitivity and specificity in this cancer. (1) Increased stool (fecal) levels of Tumor M2-Pyruvate Kinase (TM2-PK) an excellent method of screening for colorectal tumors. Sample required for its detection is stool. It is a tumor marker with high sensitivity and high specificity with no false negative, but false positive may be occurring. When measured in feces with a cutoff value of 4 U/ml, its sensitivity has been estimated to be 85% for colon cancer and 56% for rectal cancer. Its specificity is 95%. (2) Fecal DNA testing for which stool sample (collection of one entire bowel movement) is required. Its sensitivity for detection of adenocarcinoma is 72-77% and Specificity is 95.2%.

42. Q.13: A 39 years old lady reported to a private Gynae clinic with full term pregnancy. She gave birth to a baby boy through normal vaginal, but obstructed delivery. After about one month same lady ended up in the emergency in critical condition with abdominal pain, vaginal bleeding, cough, difficulty in breathing and fits. Please answer following questions a. What is most likely diagnosis? b. Name TWO biochemical tests which can be helpful to confirm the diagnosis. Write in not more than TWO lines importance and interpretation of the test

43. Q.13: a. What is most likely diagnosis? Choriocarcinoma or gestational trophoblastic neoplasm b. Name TWO biochemical tests which can be helpful to confirm the diagnosis. Write in not more than TWO lines importance and interpretation of the test 1. Serum β-hCG level – it becomes normal within 2-4 weeks after a normal delivery. So persistent elevation after a nonmolar pregnancy is indicative of GTD. 2. Serum HyperglycosylatedhCG (hCG-H)- it is a very sensitive marker to differentiate active from quiescent GTD. If hCG-H is >40% of total hCG or > 3000 IU/L, it is indicative of active GTD and interventions such as hysterectomy or chemotherapy should be done 3. CSF (cerebrospinal fluid) to serum hGCratio: Normal CSF (cerebrospinal fluid) to serum hGC ratio is 1:60, levels greater than 1:60 indicate cerebral metastases

44. Q.14: Currently a number of tumor markers are available for ovarian cancer. CA125 is the only marker that can be recommended for use. New ovarian cancer markers offer promise, however, their contribution to the current standard of care is unknown and further clinical trials are needed. CA 125 lacks sensitivity and specificity particularly in early diagnosis of ovarian cancer. Many strategies have been proposed to improve the diagnostic accuracy of CA 125 for ovarian cancer, though there is no consensus about acceptance of these modifications. Please answer following questions (One mark each): a. Name FOUR strategies proposed for improvement of diagnostic performance of CA 125. b. Write brief description of THREE of these strategies (not more than 3-4 lines for each).

45. Q.14: a. Name FOUR strategies proposed for improvement of diagnostic performance of CA 125. • Risk of malignancy index (RMI) • Risk of ovarian malignancy algorithm. (ROMA) • OVA1 test • OVASure test b. Write brief description of THREE of these strategies (Please see next a few slides).

46. Risk of malignancy index (RMI) • RMI combines three pre-surgical features: serum CA125 (CA125), menopausal status (M) and ultrasound score (U). The RMI is a product of the ultrasound scan score, the menopausal status and the serum CA125 level (IU/ml). • RMI = U x M x CA125 • The ultrasound result is scored 1 point for each of the following characteristics: multilocular cysts, solid areas, metastases, ascites and bilateral lesions. U = 0 (for an ultrasound score of 0), U = 1 (for an ultrasound score of 1), U = 3 (for an ultrasound score of 2–5). • The menopausal status is scored as 1 = pre-menopausal and 3 = post-menopausal • The classification of 'post-menopausal' is a woman who has had no period for more than 1 year or a woman over 50 who has had a hysterectomy. • Serum CA125 is measured in IU/ml and can vary between 0 and hundreds or even thousands of units.

47. Risk of ovarian malignancy algorithm. (ROMA) • Risk of ovarian malignancy algorithm is a qualitative serum test that combines results of HE4, CA 125 and menopausal status into a numerical score • ROMA is intended to aid in assessing whether a premenopausal or postmenopausal woman who presents with an ovarian adnexal mass is at high or low likelihood of finding malignancy on surgery. ROMA must be interpreted in conjunction with an independent clinical and radiological assessment. The test is not intended as a screening or stand-alone diagnostic assay. • ROMA (HE4 + CA125) should not be used without an independent clinical/radiological evaluation • ROMA is determined using the following equation: • ROMA (%) = exp (PI)/[1 – exp(PI)]*100. • 13.1% and 27.7% as the cutoff points for pre- and postmenopausal patients, respectively, and predictive index =(PI)

48. OVA1 Test • OVA1 test is a qualitative serum test that combines the result of five immunoassays into a single numeric score. Five markers are; CA 125, Prealbumin (transthyretin), apolipoprotein A1, transferrin and beta 2 microglobulin • Its a proprietary algorithm (i.e., OvaCalc) to determine the likelihood of malignancy in women with pelvic mass for whom surgery is planned • It is indicated for women who meet the following criteria i.e. age over 18, ovarian adnexal mass present for which surgery is planned, and not yet referred to an oncologist. • OVA1 score has values between 0 and 10.

49. Q.15: Cancer is caused by the accumulation of genetic and epigenetic mutations that normally play a role in the regulation of cell proliferation, thus leading to uncontrolled cell growth. Depending on how they affect each process, these genes can be grouped into two general categories: tumor suppressor genes (growth inhibitory) and proto-oncogenes (growth promoting). Mutant alleles of proto-oncogenes are called oncogenes. Below is a list of different body tumors. You are required to write ONE oncogene and ONE tumor suppressor gene associated with each tumor: a. Colorectal cancer: b. Renal cancer c. Medullary thyroid carcinoma d. Lung cancer:

50. Q.15: a. Colorectal cancer: • K- ras mutation • APC mutation • The protein product of the normal KRAS gene is a GTPase and is an early player in many signal transduction pathways necessary for the propagation of growth • Adenomatous polyposis coli (APC) also known as deleted in polyposis 2.5 (DP2.5) is a protein that in humans is encoded by the APC gene. • The APC protein is a negative regulator that controls Beta-catenin concentrations and interacts with E-cadherin, which are involved in normal cell adhesion