Laboratory Diagnosis of Biochemical Tumor Markers.pptx
- 1. Laboratory Diagnosis of BiochemicalTumor Markers Moderator – Prof. Wahid Ali Presenter – Dr. Rameez Uddin Nayak
- 2. Definition of aTumor Marker • A tumor marker is a substance produced by a tumor or by the host in response to a tumor, which is used to differentiate a tumor from normal tissue, or to detect the presence of a tumor based on measurements in the blood or secretions. • Such substances are found in cells, tissues, or body fluids and are measured qualitatively or quantitatively by chemical, immunologic, or molecular biological methods. • Tumor markers are are grouped under the general categories of (1) enzymes, (2) hormones, (3) oncofetal antigens, (4) carbohydrate markers, (5) blood group antigens, (6) proteins, (7) receptors, and (8) genes. • Ideal tumor marker- Concentrations usually increase with progressive disease, decrease with remission, and do not change significantly with stable disease.
- 3. Brief History ofTumor Markers • In 1847, Bence Jones protein was the first ever reported tumor marker discovered by precipitation of proteins in acidified boiled urine. • More than 100 years after its discovery, the Nobel Prize–winning studies of Porter, Edelman and Poulik identified the Bence Jones protein as the monoclonal light chain of immunoglobulin secreted by tumor plasma cells. • General application of markers for cancer monitoring started with the discovery of α-fetoprotein (AFP) in 1963 and carcinoembryonic antigen (CEA) in 1965. • Since 1975 ,monoclonal antibodies have been used to detect oncofetal antigens and antigens derived from tumor cell lines. • Advancements in molecular genetics has been applied for detection of mutated ras oncogene in sloughed cellular DNA in fecal material and thus can be used to detect colon cancer.
- 4. Current Applications ofTumor Markers and Their Limitations For Screening for Cancers – Limited Utility For screening purpose, a marker should be elevated at early stage of disease, when the disease is localized and potentially curable. In screening, diagnostic method should be able to separate true positives from false positives. Most circulating cancer markers only are elevated markedly in the late stages of disease (with the exception of PSA). With the exception of PSA, most cancer markers are not specific for a particular tissue, and elevations may be due to diseases of other tissue, including benign and inflammatory diseases. Hence there is a limited role of tumor markers for
- 5. For Diagnosing Cancer– Limited Utility Low diagnostic sensitivity and specificity. Only useful for diagnosis if a particular type of cancer is more prevalent in a certain population. For Prediction of Therapeutic response – Important A direct correlation has been noted in between the therapeutic response and corresponding reduction in the level of tumor maker associated with that particular cancer. This includes HERr-2/neu amplification for predicting response to Herceptin in breast cancer patients.
- 6. CLASSIFICATION • Hormones • Oncofetalantigens • Enzymes • Tumor- associated proteins • Mucin and other glycoproteins • Monoclonal antibody defined tumor markers
- 7. ENZYMES Alkaline Phosphatase (ALP) •Arises from liver, bone, or placenta • Elevated alkaline phosphatase is seen in primary or secondary liver cancers. • Greatest elevations are seen in patients with osteoblastic lesions, such as in those with prostatic cancer with bone metastases. • Minimal elevations are seen in patients with osteolytic lesions, such as those with breast cancer with bone metastases • May also get elevated in other malignancies such as leukemia, sarcoma • Placental alkaline phosphatase (PALP) , synthesized by trophoblast ,may be elevated malignancies of ovary, lung, trophoblastic, and gastrointestinal origin.
- 8. Creatine Kinase (CK) •Creatine kinase catalyzes the phosphorylation of creatine by adenosine triphosphate. • CK is a dimer consisting of two subunits—M (muscle) and B (brain)—and three isoenzymes—CK1 (BB), CK2 (MB), and CK3 (MM). • CK1 is present in the (1) brain, (2) prostate gland, (3) gastrointestinal tract, (4) lung, (5) bladder, (6) uterus, and (7) placenta. • As a tumor marker, elevations in CK1 have been observed in prostate cancer and small cell carcinoma of the lung.
- 9. Lactate Dehydrogenase (LDH) •Lactate dehydrogenase (LD) is an enzyme in the glycolytic pathway that is released as the result of cell damage. • Elevation of LD in malignancy is rather nonspecific. • It has been demonstrated in a variety of cancers, including liver cancer, non- Hodgkin’s lymphoma, acute leukemia, non-seminomatous germ cell testicular cancer, seminoma, neuroblastoma, and other carcinomas, such as breast, colon, stomach, and lung cancer. • Elevation of LD5 in the spinal fluid may be an early indicator of central nervous system metastases.
- 10. Neuron-Specific Enolase • Thisenzyme is found in neuronal tissue and cells of the diffuse neuroendocrine system. • NSE is found in tumors associated with neuroendocrine origin, including small cell lung cancer(SCLC), neuroblastoma, carcinoid, pheochromocytoma, medullary carcinoma of the thyroid, melanoma, and pancreatic endocrine tumor. • More than 90% advanced neuroblastoma have been reported to have elevated serum concentrations of NSE and elevated NSE levels are associated with poor prognosis.
- 11. PROSTATE SPECIFIC ANTIGEN(PSA) • Functionally, PSA is a serine protease of the kallikrein family. • It is produced exclusively by epithelial cells of the acini and ducts of the prostate gland. • PSA is an extremely useful tumor marker and is used to detect and monitor treatment of prostate cancer. • Increased in : Malignancy- Prostate cancer Benign Conditions- Benign Prostatic Hyperplasia (BPH), Prostatitis.
- 12. • Unfortunately, thereoccurs a significant overlap of PSA concentrations between these Prostate cancer and BPH, particularly between 4 and 10 ng/L. • Hence selecting an optimum cutoff of PSA for the recommendation of a prostate biopsy is almost impossible. • Clinical uses of PSA : 1. Prostate cancer screening, diagnosis, prediction of cancer risk and recurrence. 2. For Screening of Prostate cancer- Serum PSA with digital rectal examination (DRE) increases sensitivity and specificity of screening process. 3. To correlate with the clinical stage of prostate cancer- higher PSA concentrations is associated with advanced stages.
- 13. Complexed PSA- PSA is capableof complexing with various endogenous protease inhibitors, α-1 antichymotripsin (ACT), α2- macroglobulin and α-protease inhibitor (API), known as complexed PSA (cPSA). Serum PSA exists in the serum largely (up to 90% of total PSA) in the form of a PSA-ACT complex which is readily detectable by most immunoassays, whereas complexes with α2- macroglobulin escape detection by commercial PSA. The noncomplexed forms, known as free PSA (fPSA), are unreactive with plasma protease inhibitors.
- 15. • In prostatecancer, there is generally an increase in the serum concentration of complexed PSA and a corresponding decrease in unbound or free PSA . • In BPH, the relative amount of free PSA is higher than complexed PSA. • Thus immunoassays have been developed that quantify either cPSA or fPSA.
- 16. • PSA doublingtime The time required for the PSA value to double. It can predict recurrence after radical prostatectomy in androgen-independent prostate cancer patient. • PSA velocity and density PSA velocity defines rate at which PSA levels change in blood over time. A rise of 0.75ng/ml/year or more is strong predictor of cancer with specificity of 95%. more useful when PSA values are between 2 to 4ng/ml. PSA density >0.15 indicate probability of prostate cancer rather than BPH.
- 17. BETA-2 MICROGLOBULIN • Itis the constant light chain of the human histocompatibility locus antigen expressed on the surface of most nucleated cells. • Increased in Malignancies- Multiple myeloma Chronic lymphocytic Leukemia Some lymphomas Benign condition – Renal disease
- 18. • Serum concentrationof β2M correlates with lymphocyte activity, making β2M 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.
- 19. CSF levels ofβ2M are useful for detecting metastases in the central nervous system. Beta 2 macroglobulin level is helpful in determining prognosis. Patient with higher level of beta 2 microglobulin usually have poorer prognosis.
- 20. The Urokinase-Plasminogen ActivatorSystem • Consists of three main components: urokinase-plasminogen activator (uPA, a serine protease), the uPA membrane-bound receptor (uPAR), and the uPA inhibitors, PAI-1 and PAI-2. • uPA has been used as a prognostic marker in breast cancer majorly and some other cancers. • Clinically, as a prognostic marker, uPA in breast cancer helps predicting survival and aiding treatment decisions, particularly in node-negative patients. • Preliminary studies have implicated uPA as a prognostic marker in (1) ovarian (2) renal, (3) hepatocellular, (4) pancreatic, (5) urinary bladder (6) lung (adenocarcinoma), and (7) cervical cancers and (8) gliomas.
- 21. Cathepsins • Cathepsins, agroup of proteases that play a role in ECM degradation and growth factor release, contributing to tumor invasion and progression. • CB and CD expression is upregulated in Stromal and tumor cells, suggesting stromal involvement in matrix degradation. • CB serves as a prognostic marker in breast cancer. • High concentration of CL from tumor correlates with a decrease in relapse-free survival
- 22. Telomerase • Telomeres arespecialized structures at the termini of eukaryotic chromosomes. • Telomeres are composed of hexanucleotide repeats (TTAGGG) that signal the end of a chromosome and inhibit the DNA repair mechanism from joining the ends of chromosome together. • Telomerases is a ribonucleoprotein complex that prevents telomeres from shortening that maintains chromosome stability and cellular proliferation. • Telomerase is normally active during embryogenesis but is repressed in most somatic cells before or shortly after birth. • Germline cells, activated lymphocytes, and other immortal cells, like tumor cell show no shortening of telomere length and possess telomerase activity.
- 23. • Telomerase activityhas been detected in more than 80% of cancers, including (1) lung, (2) breast, (3) pancreatic, (4) bladder, including many other cancers • Its activity is not necessary for cancer development, but correlate with tumor progression and prognosis. • In pancreatic secretion samples, which contain freshly exfoliated cells, detection of telomerase activity is associated with cancer detection.
- 24. HORMONES Adrenocorticotropic Hormone (ACTH) Majorlyelevated in I. Cushing’s syndrome, II. Small Cell Carcinoma of Lung III. Pancreatic, breast, gastric, and colon cancer, and benign conditions, such as chronic obstructive pulmonary disease, mental depression, obesity, hypertension, diabetes mellitus, and stress • Elevated plasma concentrations of ACTH could be the result of pituitary or ectopic production. • High concentration of ACTH (>200 ng/L) is suggestive of ectopic origin. • Failure of dexamethasone to suppress cortisol is also indicative of ectopic production.
- 25. Calcitonin • It isa 32 amino acid polypeptide produced by the C cells of the thyroid. • Normally secreted in response to serum calcium and it inhibit the release of calcium from bone and thus lowers the serum calcium concentration. • Malignancy most commonly and strongly associated with increased calcitonin level is Medullary carcinoma of the thyroid • Calcitonin levels correlates with tumor size and metastasis of tumor. • Also useful in monitoring treatment of recurrence of tumor. • Calcitonin concentrations are also elevated in some patients with carcinoid tumors and cancers of the lung, breast, kidney, and liver and some non neoplastic conditions such as (1) pulmonary disease, (2) pancreatitis, (3) hyperparathyroidism, (4) pernicious anemia, (5) Paget’s disease of bone,
- 26. Human Chorionic Gonadotropin(HCG) • Human chorionic gonadotropin is a glycoprotein secreted by the syncytiotrophoblastic cells of the normal placenta. • hCG consists of α and β subunits. The β subunit is unique to hCG while the α subunit is common to several other hormones: (LH), (FSH), and (TSH) and hence the β sunbunit is tested in serum and urine routinely. Normal range - <5.0 IU/L. Half life- 10-20 hr. • Clinical Applications 1. Elevated in Trophoblastic tumors eg. Choriocarcinoms (>1 million IU/L). 2. Elevated in >90% of non seminonomatous germ cell tumor. 3. Elevated serum concentrations of hCG are also found in 45 to 60% of biliary and pancreatic cancers and in 10 to 30% of bladder, renal, and prostate cancers.
- 27. ONCOFETAL ANTIGENS • Oncofetalantigens are proteins produced during fetal life. • Present in high concentration in the sera of fetuses and decrease to low concentration or disappear after birth. • In cancer patients, these proteins often reappear, revealing that certain genes are reactivated as the result of the malignant transformation of cells.
- 28. α-Fetoprotein • AFP isa marker for hepatocellular and germ cell carcinoma. (nonseminomas eg. Embryonal Carcinoma, Yolk Sac tumors) • Structurally resembles Albumin and its concentration decreases progressively during intra-uterine life. • The normal serum AFP concentration is < 10 μg/L. • During pregnancy, maternal AFP concentration starts increasing around 12 weeks and peaks during third trimester (around 500 μg/L). • Clinical application 1. Elevated AFP levels (>1000 μg/L) are strongly indicative of cancer, particularly hepatocellular carcinoma, where early detection is critical for resectable tumors
- 29. 2. Screening andDiagnosis: AFP screening with ultrasonography, has improved early detection of hepatocellular carcinoma in high-risk areas. For prognosis >10 μg/L correlate with poorer survival. 3. AFP is elevated in yolk sac tumors, while hCG is associated with choriocarcinoma 4. AFP is a key marker for monitoring cancer therapy efficacy.
- 30. Carcinoembryonic Antigen (CEA) •CEA is a glycoprotein and the domain structures of CEA and the heavy chain of IgG are very similar and hence it belongs to Immunoglobulin gene superfamily. • Because of high false positive and false negative results, it is not routinely used for screening. • Normal Value- 3 μg/L for nonsmokers, and 5 μg/L for smokers • The CEA concentration is elevated in a variety of cancers, such as colorectal, lung, gastric, breast, pancreatic, ovarian, and uterine carcinomas. • In colorectal cancer, CEA levels correlate with disease stage, predict metastasis, and track therapy success. • It disappears after resection of tumor and reappears in the event of recurrences and metastasis.
- 31. CARBOHYDRATE MARKERS Carbohydrate-related tumormarkers may be: 1. antigens on the tumor cell surface 2. secreted by the tumor cells Commonly used examples include CA 15-3, CA 125, CA27.29. CA 15-3 • CA 15-3 is a marker for breast carcinoma. • CA 15-3 has an upper normal limit of 25 kU/L • Elevated levels found in 23% of primary and 69% of metastatic breast cancer. • Effective for monitoring therapy and disease progression in metastatic breast cancer
- 32. CA 27.29 • CA27.29 is a marker for breast carcinoma. • CA 27.29 is FDA-approved for detecting recurrent breast cancer in stage II/III patients and monitoring therapy in stage IV cases. • Major role in detecting recurrence of breast cancer with a high specificity of 97.9% and high positive predictive value of 83.3% and a high negative predictive value of 92.6%. • Better than CA 15-3 for detecting recurrence.
- 33. CA 125 • CA125 is a marker for monitoring ovarian cancer. • Normal upper limit of CA 125 is 35 kU/L. • Primarily used to monitor therapy response in patients with epithelial ovarian cancer and detect residual or recurrent disease after first-line therapy • Elevated in various cancers, including ovarian, endometrial, pancreatic, lung, and colorectal, and benign conditions like cirrhosis, hepatitis, and endometriosis. • In ovarian carcinoma, higher CA 125 levels correlate with tumor size, stage, and prognosis, with higher concentrations indicating more advanced disease.
- 34. CA-125 A high molecularweight mucin (>200 kDa) like glycoprotien Normal value <35U/ml Half life- 3to 5 days Elevated in more than 80% of non-mucinous ovarian cancers.
- 35. Other Ovarian CancerBiomarkers • HE4 is a marker for ovarian cancer. • It has a high negative predictive value for Ovarian cancer. • Also elevated in other cancers, including breast, endometrial, gastrointestinal, and lung cancer. • HE4 is FDA-approved for monitoring recurrence or progression of epithelial ovarian cancer. • Combining HE4 with CA 125 in an algorithm called the Risk of Malignancy Index (ROMA), improves prediction accuracy for ovarian cancer.
- 36. BLOOD GROUP ANTIGENS CA19-9 • Upper reference limit is 37 kU/L. • CA 19-9 is used primarily for monitoring progression of pancreatic, colorectal, and gastric cancers. • Elevated levels are seen in various cancers such as pancreatic (80%), hepatobiliary, and colorectal. • Higher concentrations are found in patients with unresectable pancreatic cancer. • Also predictive of prognosis, indicating respectability and survival rates for pancreatic cancer
- 37. Proteins as TumorMarkers
- 38. Urinary Bladder TumorMarkers • Nuclear Matrix Protein (NMP22) Nuclear matrix proteins (NMPs) make up the internal structure of the nucleus and aid in DNA replication and RNA synthesis. Measured by nuclear mitotic apparatus protein (NuMa) in urine. • Bladder Tumor–Associated (BTA) Analytes Tested in urine as surveillance with cytology and cystoscopy of patients already known to have bladder cancer
- 39. RECEPTORS AND OTHERMARKERS *VMA – Vanillylmandelic acid *HVA – Homovanillic acid *PCA3- Prostate Cancer Antigen 3
- 40. Estrogen and ProgesteroneReceptors (ER & PR) • Used in breast cancer as indicators for hormonal therapy. • Patients with positive estrogen and progesterone receptors tend to respond to hormonal treatment. • Also serve as prognostic factors in breast cancer as patients positive for hormone receptors tend to survive longer. • ER-positive tumors are 7–8 times more likely to respond to hormonal treatments compared to ER-negative tumors. • Response rates to hormonal therapy are higher in tumors that are both ER- and PR-positive (75%) compared to ER-positive/PR-negative tumors (40%). • False-Negatives: Occasionally, patients with ER-negative results may still respond to hormonal therapy
- 41. GENETIC AND MOLECULARMARKERS Oncogenes • RAS Genes RAS proteins, located in cellular membranes, act as molecular switches by transmitting mitogenic signals from growth factors to the nucleus via signal transduction pathways. Mutations in RAS genes (e.g., KRAS and NRAS) is found neuroblastomas, acute myeloid leukemia (AML), pancreatic cancer (95%), colon cancer (40%), and lung and bladder cancers (30%). KRAS mutation testing is vital for metastatic colorectal cancer patients, as tumors with wild-type KRAS respond better to monoclonal antibody therapies targeting the EGFR pathway (e.g., cetuximab, panitumumab).
- 42. c-myc • Activation ofthe c-myc gene is linked to various cancers, including B- and T-cell lymphomas, sarcomas, and endotheliomas. • Amplification of c-myc is linked to clinical aggressiveness in lung carcinomas and gliomas. • c-myc expression in colon cancer is 5–40 times higher than in normal tissue, but this increase does not correlate with disease progression • A decrease in c-myc expression post-chemotherapy is indicative of a favorable treatment response.
- 43. Her-2/neu (ERBB2) • Her-2/neu,also known as ERBB2 amplification is found in breast, ovarian, and gastrointestinal cancers, with strong prognostic value in breast cancer. • Among the oncogenes ERBB2, RAS, and MYC, ERBB2 has the strongest prognostic value for breast cancer. • Herceptin treatment is given only to breast cancer patients with HER2/neu amplification. • Elevated HER2/neu concentrations in breast cancer correlate with larger tumors, lymph node positivity and a high-grade cancer.
- 44. Tumor Suppressor Genes P53 •p53 gene (TP53) controls cell division by regulating entry into the S phase of the cell cycle. Loss of its function through deletion or mutation leads to uncontrolled cell growth and cancer progression • 75-80% of colon carcinomas have TP53 deletions or mutations, and up to 70% of breast cancers show TP53 mutations. • Monoclonal antibodies are used to detect mutant p53 proteins, which accumulate in cancer cells, particularly in colorectal, breast, and small cell lung cancers. • Overexpression of mutant p53 in cancers, especially breast and small cell lung cancer, is linked to poor prognosis.
- 45. BRCA1 and BRCA2 •BRCA1 on chromosome 17q and BRCA2 on chromosome 13q inherit a genetic predisposition to both breast and ovarian cancer. • BRCA1 encodes a protein that may function as a transcription factor, and mutations in BRCA1 and BRCA2 can be detected in the germline. • Carriers of BRCA1 mutations have an 85% risk of breast cancer and a 45% risk of ovarian cancer by age 85.
- 46. Genes Indication Source MLH1,MSH2, MSH6, PMS2 Colon, NSCLC, esophagogastric, HNSCC Tumor BRAF Colon, NSCLC, melanoma, papillary thyroid, leukemia, glioma Tumor PD-L1 Colon, NSCLC, soft tissue sarcoma Tumor c KIT/ CD117 GIST, soft tissue sarcoma Tumor PDGFRA GIST, soft tissue sarcoma Tumor BCR-ABL1 Leukemia Blood, marrow FLT3 Leukemia Blood, marrow Other genes acting as tumor markers
- 47. Cell Free DNA •Also known as Liquid Biopsy • Cell-free DNA (cfDNA) is DNA that's not contained within cells and is present in bodily fluids like blood, saliva, and urine. • cfDNA is released into the bloodstream through a natural process of cell death • Circulating cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) are increasingly used in liquid biopsy (LB) analyses to aid in prognosis, identify tumor-specific genomic alterations, select targeted therapies, and monitor treatment efficacy. • Tumor-specific genomic alterations in cfDNA are detectable using techniques such as PCR, next-generation sequencing (NGS), Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq), and Tagged-Amplicon Deep Sequencing (TAm-Seq).
- 48. • Possible sourceof cell free DNA are thought to be due to Necrosis, Apoptosis and active secretion by tumor cells. • Non invasive approach for tumor detection and prenatal diagnosis. • Recently used for monitoring progression and treatment response in Breast and Ovarian cancer.
- 50. Hook Effect inImmunoassay • The hook effect refers to the prozone phenomenon, also known as antibody excess, or the postzone phenomenon, also known as antigen excess • The hook effect in immunoassays is a phenomenon that can lead to falsely low results, particularly when the concentration of the analyte being measured is extremely high • The hook effect occurs when an excessive amount of the target analyte in a sample interferes with the immunoassay, leading to a false-negative or falsely low result. • It happens when there is too much analyte for the antibodies to bind effectively, resulting in incomplete complex formation, which reduces the signal detected by the assay • The hook effect can be clinically significant in detecting high levels of hormones (like hCG in pregnancy tests or prolactin), where false-negative results can lead to misdiagnosis.
- 52. ANALYTICAL METHODS Radioimmunoassay (RIA) •Radioimmunoassay (RIA) is a type of immunoassay that uses radioactively labeled molecules to detect and quantify the presence of a specific substance (antigen) in a sample. • RIA relies on the competition between a radioactively labeled antigen (tracer) and an unlabeled antigen (sample) for binding to a specific antibody • The amount of radioactivity measured is inversely proportional to the amount of antigen in the sample. • The antigen of interest is labeled with a radioactive isotope (commonly iodine-125 or iodine-131), which emits radiation detectable by a scintillation counter or gamma counter. • The labeled antigen competes with the unlabeled antigen in the sample for binding sites on a specific antibody
- 53. • After theantigen-antibody binding, the bound fraction (complex) and free fraction (unbound antigen) are separated, usually by a physical method such as precipitation or filtration. • The radioactivity of the bound antigen is measured, and this signal is used to calculate the concentration of the antigen in the sample. • RIA is used in clinical diagnostics to measure hormones (e.g., thyroid hormones, insulin, hCG), drugs (e.g., therapeutic drug monitoring),
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- 56. Monoclonal Antibody Coatedwell Test Specimen (Serum) HRP labelled Antibody Conjugate Test Antigen: Sandwich between solid phase Ab and Enzyme labelled Ab Incubate for 1 hour at 37 degree celcius Remove Unbound Enzyme labelled Antibody Chemiluminescence Reagent added Read reactive light unit with Luminometer