Laboratory Diagnosis of Cancer 1.Histological methods 2.Cytopathology - FNAC/Exfoliative 3.Immunohistochemistry/ EM 4.Molecular diagnosis 5.Tumor Markers

Histological methods - Frozen Section Used per-operatively To determine nature of mass lesion - whether inflammatory / benign / malignant To evaluate sentinel node status, margins of excised cancer

Cytological Methods - Fine Needle Aspiration Cytology Using regular small bore needle : readily palpable sites – breast, lymph node, thyroid Using special needle: prostate Using ultrasound/CT guidance: deep seated structures also

Cytological Methods - Exfoliative Cytology Pap smears : Carcinoma cervix Ascitic / pleural fluid cytology Cerebrospinal fluid

Immunohistochemistry Categorizing undifferentiated malignancies Categorizing leukemia / lymphomas Determining site of origin of metastasis Detecting molecules of prognostic / therapeutic significance

Immunohistochemical stains Carcinomas: Cytokeratin, EMA, CEA Sarcomas: Vimentin Muscle derived sarcomas: Desmin, Actin Tumors of endothelium: Factor VIII, CD31 Leukocyte malignancies 1.CD 45: LCA (Leukocyte common antigen) - almost all except mature RBCs 2.CD 19, 20: B Cell Lymphoma 3.CD 3, 5, 7, 4, 8: T cell Lymphoma 4.CD 15+, CD30+ : Hodgkin’s Lymphoma Neural crest derived tumors: S100 +ve 1. Neuroendocrine tumors(carcinoid) : S 100 & Chromogranin 2. Neural tumors (neuroblastoma) : S-100 & NF +ve 3. Melanoma : S-100 & HMB45 +ve 4. Small cell carcinoma : S100 & NSE Miscellaneous: Mesothelioma: Calretinin, Medullary carcinoma of thyroid: Calcitonin, Myeloma: kappa/lambda light chains, Prostate carcinoma: PSA, Hepatocellular carcinoma: AFP, Trophoblastic tumors: HCG, Gastrointestinal stromal tumors: CD117 (c-Kit), Mantle cell lymphoma: Cyclin D1

7 CD 19 1.Lymphoma 2.Neuroblastoma 3.Rhabdomyosarcoma 4.Melanoma

A 49-year-old man complains of pain in his left thigh for 3 months. On physical examination his thigh is increased in size, compared to the right. A plain film radiograph reveals the presence of a 15 cm solid mass that does not appear to arise from bone, but it does have infiltrative margins. A biopsy of this mass is taken, and on microscopic examination the mass is composed of highly pleomorphic spindle cells. Which of the following immunohistochemical markers is most likely to be demonstrated in the cells of this mass? A Cytokeratin B Factor VIII C Alpha fetoprotein D Lambda light chain E Vimentin

9 S100 If also positive for 1.NF - ? 2.Chromogranin - ? 3.HMB45 - ?

Molecular diagnosis Cytogenetic analysis PCR assays ( polymerase chain reaction) FISH ( fluorescent in-situ hybridisation) Tissue arrays Useful in diagnosis, prognosis Minimal residual disease, To detect hereditary predisposition to cancer

Flow Cytometry Rapid quantitative analysis of cell features like membrane antigens, DNA content Identification of cell surface antigens in leukemias/lymphomas Ploidy analysis in solid tumours

TUMOR MARKERASSOCIATED CANCER AFPHepatocellular carcinoma, yolk sac tumor (endodermal sinus tumor) of ovary or testis Bence Jones protein Multiple myeloma, Waldenström's macroglobulinemia (represent light chains in urine) CA 15-3Breast carcinoma CA 19-9Pancreatic, colorectal carcinomas CA 125Surface-derived ovarian cancer (e.g., serous cystadenocarcinoma; helpful in distinguishing benign from malignant tumors) CEAColorectal and pancreatic carcinomas (monitor for recurrences) LDHMalignant lymphoma (prognostic factor for response to standard therapy) PSAProstate carcinoma (also increased in prostate hyperplasia)

CEA (Carcinoembryonic Antigen) Glycoprotein, produced by embryonic tissue of gut/pancreas/liver Increased in Cancer of Colon, Pancreas, Breast Also increased in benign disorders – alcoholic cirrhosis, hepatitis, ulcerative colitis Useful in postoperative detection of residual disease / recurrence of colon cancer, monitoring treatment of breast cancer

AFP (Alpha Feto-Protein) Glycoprotein, produced by yolk sac/fetal liver/GIT Increased in Cancer of liver / germ cell tumors of testes Also increased in benign disorders – alcoholic cirrhosis, hepatitis, toxic liver injury, pregnancy Useful in post-therapy measurement – index of response to therapy / recurrence

Electron microscopy in cancer Carcinomas: Tonofilaments, desmosomes Neuroendocrine tumors: Secretory granules Malignant melanoma: Melanosomes Langerhan cell histiocytosis: Birbeck granules Insulinoma: Crystal containing granules

Required reading: Robbins Basic Pathology 9E, p Recommended reading: Robbins and Cotran Pathologic Basis of Disease 8E, p