SCPA607 Biological markers for cancer diagnosis
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1 SCPA607 Biological markers for cancer diagnosis Associate Professor Dr. Wannee Jiraungkoorskul Department of Pathobiology, Faculty of Science, Mahidol University Tel: , 1
2 Objectives Describe the definition, purpose of use, and classification of tumor marker. Describe the most common tumor marker used in various cancers. 2
3 What is a biomarker? Biomarker is a substance used as an indicator of a biologic state or particular disease state. Biomarker should analyze organ function, diagnosis diseases in a non-invasive way. Biomarker can be any molecule such as protein, carbohydrate, nucleic acids, enzyme, hormone, etc. Biomarker can be test from blood, urine, body fluid, tissue, etc. 3
4 Biomarker in organ disease 4
5 Biomarker in disease 5
6 Biomarker: Purpose, Specimen 6
7 First tumor marker The first tumor marker Bence Jones Protein was identified in 1846 by Henry Bence Jones, who detected a heat precipitate in samples of acidified urine from patients suffering from multiple myeloma. This protein is not present in healthy urine sample. Multiple myeloma is a type of bone marrow cancer that is most common in people who are older than 60 years. Henry Bence Jones English physician ( ) 7
8 Brief history of tumor markers 8
9 Cancer biomarker or Tumor biomarker : Definition It is a biomarker found in blood, urine or body tissue that can be elevated by the presence of one or more types of cancer. 9
10 What makes an ideal tumor marker? 10
11 Screening Diagnosis Prognosis Monitoring of disease, Response to treatment, Detection of recurrence 11
12 Tumor Marker : Screening Screening asymptomatic individual Use of tumor markers has generally not been an effective strategy due to: Most of the clinically used tumor markers are found in normal cell and benign conditions in addition to cancer cells. The relatively low prevalence of individual cancer types. The screening role is generally very useful if restricted to groups with risk factors as: Family history Environmental exposure geographic prevalence Clinical profile 12
13 Tumor Marker : Screening High risk groups: are individuals at increased risk for malignancy Tumor markers used for screening of high risk individuals: Alpha fetoprotein (AFP) For patients with liver cirrhosis or chronic hepatitis who are at risk for development of hepatocellular carcinoma Prostate specific antigen (PSA) For men older than 50 years in conjunction with a physical examination or clinical profile for early detection of cancer prostate Calcitonin For first degree relatives of a patient or family history with medullary carcinoma 13
14 Tumor Marker : Screening Sensitivity & Specificity of screening tumor markers: Ideal tumor marker for screening asymptomatic population should be: 100% sensitive: Always positive in patients with the disease 100% specific: Always negative in individuals who do not have the disease For examples: If a test gives positive results in 99 patients out of 100 patients: its sensitivity is 99% If a test gives negative results in 90% normal individuals out of 100 normal individual. Its speciificity is 90% 14
15 Tumor Marker : Screening In reality, an ideal tumor marker which gives 100% specificity and 100% sensitivity does not exit. To increase sensitivity and specificity of a tumor marker: Combination of multiple tumor markers Combination of tumor markers with other procedures e.g. combination of Carbohydrate Antigen 125 (CA125) with ultrasonography for early detection of ovarian malignancy 15
16 Tumor Marker : Prognosis For cancer patients, determination of prognosis is based on determination of aggressiveness of tumor, which, in turn determines how a patient should be treated (surgery, chemotherapy, radiotherapy, etc) As the serum concentrations of tumor markers increases with progression and usually reaches the highest levels when tumors become metastasized, the serum levels at diagnosis are likely to reflect the aggressiveness of the tumor and predict the outcome. High levels of serum tumor marker measured during diagnosis would indicate the presence of a malignant or metastatic tumor associated with poor prognosis. 16
17 Tumor Marker : Monitoring of disease, Response of treatment & Detection of recurrence Markers usually increase with progressive disease, decrease with remission and do not change significantly with stable disease. After treatment, tumor markers are routinely followed serially to monitor the response to treatment and recurrence. It is desirable to monitor the patient using a highly sensitive tumor marker test to detect recurrence as early as possible. The appearance of most of the circulating tumor markers have a lead time of several month (3-6 months) prior to the stage at which many of the physical procedures can be used for detection of the cancer. So rising tumor marker levels may detect recurrence of disease before any clinical or radiological evidence of disease is apparent (biochemical recurrence). 17
18 Example of tumor markers are used for screening, prognosis, treatment monitoring, and detecting recurrence of several types of cancer. Whereas few markers are used for screening, many are used to monitor therapy. Endocrine and hormone metabolite markers are often used to aid in diagnosis of secreting tumors. 18
19 Type of tumor marker: Specimen 1. Cellular or Tissue tumor markers: measured in tissues, markers which can be detected in the tissue by immunological tests are called immunohistochemical (IHC) markers. 2. Humoral or Serological tumor markers: measured in blood and other body fluids. 19
20 Immunohistochemical markers Immunohistochemistry (IHC) is the process of selectively imaging antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. IHC takes its name from the roots "immuno", in reference to antibodies used in the procedure, and "histo," meaning tissue. Albert Coons conceptualized and first implemented the procedure in Albert Coons American physician 20
21 Immunohistochemical tumor markers list 21
22 Cytokeratin in squamous cell carcinoma Squamous cell carcinoma (SCC) is a malignant neoplasm arising from the squamous epithelium of the oral cavity most commonly from the lip, then tongue, floor of mouth, gingiva, palate, and buccal mucosa. a) Squamous cell carcinoma (H&E). b) Cytokeratin stains in SCC. 22
23 Cytokeratin in papillary thyroid carcinoma athology/thyroid/papillary-carcinoma-thyroid-[7- th07g].jpeg?width=600&height=450&format=4 23
24 HMB45 in melanoma Malignant mucosal melanoma (MMM) is a neural crest derived neoplasm originating from melanocytes and demonstrating melanocytic differentiation. a) Mucosal melanoma (H&E) b) IHC staining for HMB
25 S100 in adenoid cystic carcinoma Several members of the S100 protein family are useful as markers for certain tumors and epidermal differentiation. It can be found in melanomas, schwannomas, neurofibromas. Adenoid cystic carcinoma of salivary gland tumors, are composed of epithelial and myoepithelial cells. 25 a) polymorphous low-grade adenocarcinoma b) S-100 protein
26 GFAP in canalicular adenoma Canalicular adenoma is a benign epithelial salivary gland neoplasm characterized by chains of columnar cells and preference for the minor salivary glands. Glial fibrillary acidic protein (GFAP) is expressed by numerous cell types of the central nervous system (CNS) including astrocytes and ependymal cells. GFAP has also been found to be expressed in glomeruli and peritubular fibroblasts taken from rat kidneys Leydig cells, keratinocytes, osteocytes and chondrocytes. a) Canalicular adenoma, b) GFAP staining is positive 26
27 Desmin in alveolar rhadomyosarcoma Desmin is an intermediate filament that is expressed in both smooth and skeletal muscle myocytes. It is also expressed in neoplasms with smooth or skeletal muscle differentiation yosarcoma_macro_2711_2.jpg abdosarcoznerdesmin.jpg 27
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29 Classification of tumor marker 29
30 Type of tumor marker: 1. Cancer antigen Oncofetal antigen is normally produced during the embryonic development. In adults, its production is limited or completely absent. Elevated concentrations in adults result from reactivation of certain genes that control cellular growth and are directly connected to malignant process. 1. Alfa-fetoprotein (AFP) : Liver and germ cell tumors (testis and ovary used together with ß-hCG). 2. Carcino-embryonic antigen (CEA) : Advanced adenocarcinomas, breast, liver and lung cancers. 30
31 Alpha fetoprotein (AFP) Alpha-fetoprotein (alpha-fetoglobulin, or alpha fetal protein) is a protein that in humans is encoded by the AFP gene, is located on the q arm of chromosome 4 (4q25). Measurement of AFP is generally used in two clinical contexts. First, it is measured in pregnant women through the analysis of maternal blood or amniotic fluid as a screening test for certain developmental abnormalities. Second, serum AFP level is elevated in people with tumors. Normal levels are <15 ng/ml, Half life 3-5 days 31
32 AFP test kit This AFP test utilizes a combination of colloidal gold conjugate and monoclonal antibodies to selectively detect elevated levels of AFP in serum or plasma. The test has a cut-off value of 20ng/ml. 32
33 AFP ELISA kit Materials Provided with AFP ELISA Kit: 1. Microtiter wells coated with AFP ELISA Antibody 2. Zero buffer 3. Reference standard set: 0, 5, 20, 50, 150 & 300 ng/ml 4. AFP EIA Kit Enzyme Conjugate Reagent 5. Tetramethylbenzidine Substrate 6. AFP Elisa Test Stop Solution 7. Wash Buffer Concentrate 50X 33
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37 Carcinoembryonic antigen (CEA) High molecular weight glycoprotein Normal value <2.5ng/ml, Half life 3-13 days Expressed during fetal development then re-expressed in tumor growth Clinical use to detect colorectal, lung, breast ovarian, and GI cancers 37
38 Carcinoembryonic antigen (CEA) In colon cancer: After complete removal of colon cancer, the levels should fall to normal in 6-12 weeks. CEA has sensitivity of 97% in detecting recurrence in patients with preoperative elevated levels. Increase concentration indicates poor prognosis within a given stage. High level correlate with metastasis; 80% patient of colon cancer with level > 20ng/ml have recurrence in 14 months. Concentrations < 5ng/ml before therapy correlate with localized disease and good prognosis. 38
39 CEA test kit 39
40 CEA ELISA kit Material Provided with CEA ELISA Kit: 1. Microtiter wells coated with Antibody 2. CEA standards containing; 0, 3, 12, 30, 60, and 120 ng/ml 3. Enzyme Conjugate Reagent 4. Substrate TMB 5. Stop Solution 6. Wash Buffer Concentrate 50X) 7. Control set 40
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42 Type of tumor marker: 1. Cancer antigen This is a heterogeneous group of more specific markers that comprises various membrane structures of tumor cells. 42
43 Cancer antigen 125 (CA-125) It is a mucin glycoprotein that encoded by MUC16 gene. Normal value <35 U/ml, Half life 3-5 days Interpretation: Normal concentration does not exclude tumor. Correlates with poorer prognosis for ovarian cancer if elevated 3-6 weeks after surgery Rising levels during chemotherapy is associated with tumor progression and fall to normal is associated with response. Values > 65 U/ml correlate with peritoneal involvement. 43
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45 Type of tumor marker : 2. Enzymes Certain enzymes that are produced more in-tensely if a malignant process is occurring in the organism can also be used as tumor markers. EA enzyme assay IA immunoassay IHC immunohistochemistry RIA radioimmunoassay 45
46 Prostate Specific Antigen (PSA) Normal value < 4ng/ml, Half life 4 days. Produced by the epithelial cells of the prostatic ducts and also by periurethral gland, pancrease, salivary gland and breast PSA is elevated in prostate infection, irritation and benign prostate enlargement. 46
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49 PSA test kit 49
50 PSA test kit Material Provided with PSA ELISA Kit: 1. Microtiter wells coated with PSA ELISA Test Antibody 2. PSA EIA Test Kit Zero buffer 3. PSA EIA Reference standard containing 0, 2, 4, 15, 50 & 100 ng/ml 4. PSA ELISA Test Enzyme Conjugate Reagent 5. PSA Elisa TMB Substrate 6. Stop Solution 7. PSA Test Kit Wash Buffer Concentrate 50X 50
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52 Type of tumor marker : 3. Hormone Malignant formations can alter the synthesis and secretion of various hormones. Quantitative and qualitative alterations of the synthesis and hormone secretion can therefore be the indicators of a malignant process and can be monitored as tumor markers. ACTH, adrenocorticotropic hormone ADH, antidiuretic hormone GH, growth hormone HVA, homovanillic acid 5-HIAA, hydroxyindoleacetic acid PTH, parathyroid hormone PRL, prolactin VMA, vanillylmandelic acid ELISA, enzyme-linked immunosorbent assay HPLC, high-performance liquid chromatography IA, immunoassay LC-MS/MS, liquid chromatography mass spectrometry MTC, medullary thyroid carcinoma RIA, radioimmunoassay SIADH, syndrome of inappropriate antidiuretic hormone secretion 52
53 Type of tumor marker : 3. Hormone 53
54 Human chorionic gonadotropin (hcg) A glycoprotein hormone synthesized by placenta. Molecule consists of two subunits: alpha & beta Normal value of beta hcg < 5mIU/ml, Half life hrs. Used as a routine pregnancy test. Diagnosis and monitor course and evaluate prognosis of gestational trophoblastic tumors, choriocarcinoma and germ cell tumors of the ovary and testis. 54
55 hcg test kit Material Provided with free beta hcg ELISA Kit: 1. Antibody-coated microtiter wells. 2. Reference standards 0, 2.5, 5, 10, 25, 50 miu/ml 3. Zero Buffer (Sample diluent) 4. Enzyme Conjugate Reagent 5. TMB Substrate 6. Wash Buffer Concentrate 50X 7. Stop Solution 55
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57 Type of tumor marker : 4. Oncogene Oncogenes (also called: proto-oncogenes) are normal cellular genes which on being activated found to be associated with cancer. They are involved in normal cellular processes such as differentiation and growth factors signaling. C-myc C-erbB2 C-abl/bcr N-myc 57
58 Type of tumor marker : 5. Tumor suppressor gene Tumor suppressor genes are normal genes that slow down cell division, repair DNA mistakes, or tell cells when to die (a process known as apoptosis or programmed cell death). When tumor suppressor genes don't work properly, cells can grow out of control, which can lead to cancer. Retinoblastoma (Rb) p53 APC BRCA-1, -2 58
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63 Case: The Angelina Jolie Effect Angelina Jolie, inherited breast cancer and the BRCA1 gene 63
64 Type of tumor marker: 6. Receptor 64
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68 Case study A 50 years old female suffered from cancer breast 5 years ago, and underwent radical surgical procedure. She did not have any family history for cancer breast. Recently, metastases were detected in her liver. Which one of the following tumor markers is the best for diagnosis, prognosis, & monitoring therapeutic intervention of this case? A. BRCA1 B. BRCA2 C. Alpha fetoprotein (AFP) D. CA
69 Factors that affect serum concentrations of tumor markers False positive results occur with: Inflammatory conditions Benign condition Presence of liver disease causes disturbances in metabolism and excretion of some tumor markers Disturbances of renal function affects levels of some tumor markers Consequences of diagnostic and therapeutic procedures: digitorectal examination, mammography, surgery, radio and chemotherapy As a consequence of different physiological conditions: as in pregnancy may affect ß-hCG, AFP 69
70 Some benign conditions associated with rise in tumor markers 70
71 Factors that affect serum concentrations of tumor markers False negative results occur with: Insufficient expression of the marker or production of the marker in some of the tumor cells only Insufficient blood circulation in the tumor Production of autoantibodies against the marker Rapid degradation and clearance of the marker 71
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73 Common serum markers for cancer diagnosis/prognosis AFP CEA CA15-3 CA19-9 CA125 PSA PSAf PAP htg HCGb Ferr NSE B2M A2M Lung x x x x x x x Pancreas x x x x x Kidney x x x x Breast x x x Ovarian x x x x x x Cervical x x Uterine x x x x Prostate x x x x x Liver x x x x x Gastro x x x Colon x x x x x Bladder x Brain x Leukemia x x x Myeloma x Thyroid x x Testicular x x x x
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78 Things to remember No ideal tumor marker is known so far. Therefore, the best approach is: Take a good history, Perform thorough physical examination. Use a battery of markers (>1 marker/tumor) Use confirmatory investigations: Histopathology, ultrasonography, per rectal examination, x rays 78
79 Cancer Biomarkers. by Alexandros G Georgakilas 2012 Cancer Biomarkers: Minimal and Noninvasive Early Diagnosis and Prognosis. by Debmalya Barh Angelo Carpi Mukesh Verma Mehmet Gunduz 2017 Biomarkers in Cancer. by Victor R Preedy Vinood Patel
80 SCPA607 Biological markers for cancer diagnosis 80
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