Cancer and ihc markers

oncology

1. Cancer and IHC markers

2. Immunohistochemistry In Tumor Diagnosis IHC needed in 10-25% of malignant tumors for Narrowing of possibilities Specific diagnosis Unsuspected diagnosis A correct histopathological diagnosis saves time, money and inconvenience for the patients and clinicians.

3. Planning of Diagnostic I mmunohistochemistry Likelihood of a given diagnosis. Relevant differential diagnoses. Optimal selection of antibodies for a diagnostic algorithm Primary antibody panels and secondary antibody panels Panel should include not only antibodies that would expect to be positive in a given tumor , but also antibodies that would be expected to be negative .

4. IHC has revolutionized the field of histopathology . Today IHC markers of almost every tumor has been defined. IHC still has the long way to know the histogenesis of yet unknown lesions.

5. Definitions Immunohistochemistry is a technique for identifying tissue constituents (antigens) by means of antigen-antibody interactions. The site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary antibody labeling method.

6. IMMUNOHSITOCHEMISTRY STEPS Slide 3 of 23 Tissue sections Antigen retrieval Blocking endogenous enzymes Secondary antibody Primary antibody Microscopy Observation Blocking background staining Chromogen Substrate Counterstain Mounting

7. Steps of Immunohistochemistry Tissue sections are incubated with primary antibodies against the target antigen. The specimens are then washed and a secondary antibody against primary antibody is added. The secondary antibodies are often biotinylated , that is, conjugated to biotin, a vitamin with an extremely high affinity for the protein streptavidine .

8. A complex made of streptavidin and the enzyme peroxidase is used for staining. The addition of a chromogenic substrate such as diaminobenzidine (DAB) or aminoethylcarbazole (AEC), leads to a color staining reaction that accurately reflects the distribution of the target antigen in the tissue sample.

9. Unmasking of Antigen Sites Tissue fixation on routinely histotechnique can masked antigens because of formalin cross-linking or even destroys some antigenic epitopes . The retrieval techniques of unmasked antigens : (1) proteolytic enzyme digestion (2) microwave (3) microwave and trypsin (4) pressure cooker

10. Antigen Recognition 1’Ab : primary antibody 2’Ab : secondary antibody B : biotin SA : streptavidine HRP : horseradish peroxidase Target is antigen (protein argets ) Blocking reagent : BSA (bovine serum albumin) The sandwich of antigen recognition on tissue samples.

11. The methods of Identifying Tissue Antigens Direct labeling of antibody Secondary antibody labeling

12. Labels Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels

13. Enzyme Labels Horseradish peroxidase Alkaline phosphatase Bacterial-derived beta-D- galactosidase

14. POTENTIALLY MORPHOLOGICALLY-INDETERMINATE TUMORS GENERIC CATEGORIES Round-cell tumors Undifferentiated tumors Metastatic tumors Spindle-cell tumors Hematopoietic tumors

15. POTENTIALLY UNDIFFERENTIATED ROUND CELL TUMORS Small cell carcinoma Malignant melanoma Lymphoma Neuroblastoma Ewing sarcoma/Primitive neuroectodermaltumor (ES/PNET) Rhabdomyosarcoma (RMS ) Poorly differentiated synovial sarcoma( PDSS) Desmoplasticround cell tumor ( DRCT)

16. ANTIBODIES APPLIED TO THE STUDY OF ROUND-CELL TUMORS KERATIN S100 EMA HMB45 CD45 MART-1 CD15 FLI-1 VIMENTIN SYN DES CD56 MSA CD99 MYOGENIN

17. APPROACH TO ROUND CELL TUMOR N0N SARCOMA NS NS SARCOMA S S S S Small cell carcinoma melanoma lymphoma neuroblastoma rhabdomyosarcoma ES/ PNET PDSS DRCT CK + +/- - - RARE +/- + + S -100 - + - + - +/- +/- - Desmin - +/- - - + - - + CD99 ( Mic 2) - - +/- - -/+ + + RARE LCA (CD 45) - - + - - - - -

18. APPROACH TO UNDIFFERENTIATED MALIGNANT TUMOR Lymphoma carcinoma melanoma sarcoma Mesothelioma glioma LCA (CD -45) + - - - - - CK - + - - + - S-100 - -/+ + -/+ - - Vimentin -/+ -/+ + + + + GFAP - - - - - + HMB 45 - - + - - -

19. LYMPHOMA WHICH CAN BE LCA (CD 45) -NEGATIVE Lymphoblastic Lymphoma Anaplastic large cell Lymphoma Plasmablastic Lymphoma Peripheral T-cell type ( Some cases) Lymphoma Classic Hodgkin lymphoma ( RS cells) LYMPHOMA LCA (CD 45) + CK - S -100 - Vimentin -/+ GFAP - HMB 45 -

20. CARCINOMAS WHICH CAN BE LMW-CK NEGETIVE Renal cell carcinoma Adrenal cortical carcinoma Small cell carcinoma CARCINOMA LCA (CD 45) - CK + S-100 -/+ Vimentin -/+ GFAP - HMB-45 -

21. SARCOMA WHICH CAN BE CK-POSITIVE Synovial sarcoma Epitheloid sarcoma Malignant rhabdoid tumor Myogenic tumor (some) Angio sarcoma(some ) sarcoma LCA (CD45) - CK - S-100 -/+ Vimentin + GFAP - HMB 45 -

22. If LCA ,CK and S-100 all are negative Possibilities are Angiosarcoma (add CD 31) Anaplasticplasmacytoma ( add CD79a & Kappa / lambda) Hodgkin lymphoma Germinoma Various sarcomas

23. D/D OF MALIGNANT MESOTHELIOMA EMA Desmin p53 Reactive mesothelial cells - or focal + + - Malignant mesothelioma + (membranous) - Or focal + +

24. D/D OF MALIGNANT MESOTHELIOMA Msothelioma Adenocarcinoma Calretinin + (nuclear) - CK 5/6 + - WT – 1 + (nuclear) - CEA - + MOC – 31 - + B 72.3 - +

25. METASTATIC CARCINOMA OF UNKNOWN ORIGIN

26. ANTIBODIES USED IN EVALUATION OF METASTATIC CARCINOMAS OF UNKNOWN ORIGIN CK & EMA S100 protein CD15 PSA Thyroglobulin TTF-1 CEA CA19.9 CA-125 Placental alkaline phosphatase ER , PR

27. APPROACH TO METASTATIC ADENOCARCINOMA

28. CK 7 + VE ADENOCA CK 20-VE ADENOCA

29. Approach To Metastatic Squamous cell carcinoma Prediction of primary site of a squamouscell carcinoma is a difficult task. In some cases the following can be done : CK20 + ( CK7 +) : Transitional carcinoma of urinary bladder EBV + : Nasopharyngeal carcinoma / lymphoepitheliomalike carcinoma Mediastinaltumor : CD 5 : positive = primary thymiccarcinoma negative = metastatic carcinoma

30. Poorly Differentiated Carcinoma CYTOKERATINPOSITIVE

31. SPINDLE CELL TUMORS

32. POTENTIALLY UNDIFFERENTIATED SPINDLE-CELL TUMORS Synovial sarcoma Fibrosarcoma Malignant peripheral nerve sheath tumor(MPNST) Leiomyosarcoma Solitary fibrous tumor

33. PANEL APPLIED TO THE STUDY OF SPINDLE-CELL TUMORS KERATIN S100 PROTEIN ACTIN CD34 EMA CD56 VIMENTIN CD31 DESMIN CD57 THROMBOMODULIN CD99

34. SCREENING PANEL FOR SPINDLE CELL TUMORS Synovial sarcoma MPNST Fibro sarcoma Leiomyo sarcoma Solitary fibrous tumor CK + - - Rare Rare S-100 +/- + - Rare - CD 34 - +/- - Rare + Smooth muscle actin - - +/- ( myofibrosarcoma ) + -

35. MALIGNANT PERIPHERAL NERVE SHEATH TUMOR(MPNST ) S-100

36. PANEL APPLIED TO THE STUDY OF HEMATOPOIETIC TUMORS CD15 CD79A CD20 LYSOZYME CD30 MYELOPEROXIDASE CD43 CD3 CD45 CD5 CD68 CD163

37. APPROACH TO LYMPHOID LESIONS LCA (CD 45) : to differentiate lymphoid from other non-lymphoid lesions Bcl-2 : + in follicular lymphomas-in follicular hyperplasia Kappa & lambda chain restriction : monoclonal neoplastic proliferation from polyclonal reactive plasma cell proliferation

38. APPROACH TO LYMPHOID LESIONS CD 45 CD3 CD45 RO CD20 CD15 CD30 CD68 CD99 B cell lymphoma + - - + - +/- - - T cell + + + - - + - - Hodgkin’s dis. +/- - - - + + - - lymphoblastic +/- - - - - - - + histiocytic +/- - - - - - + -

39. D/D OF HODGKIN LYMPHOMA NLPHL ClassicalHL LCA (CD 45) + _ CD20 + +/_ CD30 _ + CD 15 _ + EMA + _ CD- 57 Many small lymphocytes form rosettes the L &H cells Few CD 57+ small lymhocytes Bcl – 6 + in L & H cells _ in RS cells EBER _ Commonly + nt (60%)

40. NHL IHC PANEL IN SMALLB-CELL LYMPHOMA CD-5 CD-23 IgD CyclinD1 Bcl-6 B- Chroniclymphocytic leukemia / small lyphocyticlymphoma + + + _ _ Mantle cell lymphoma + _ + + _ Lowgrade B-cell lymphoma _ _ _ _ _ Follicular center celllymphoma + +/_ _ _ +

41. SYSTEMIC APPROACH FOR IMMUNOHISTOCHEMISRY

42. BREAST CARCINOMA Minimum markers are : ER, PR & c-erbB2 ER + ,PR+ tumors are a/w better prognosis Show better response to hormonal therapy ER positive in invasive lobular carcinoma of breast

43. PANEL FOR GASTROINTESTINAL SYSTEM Gastric carcinoma Keratin, EMA , CEA + CK 7 + , CK20 + Carcinoidtumor ACTH , NSE , Chromogranin + Synaptophysin , keratin + Colorectal Carcinoma CK20 + , CK 7 –( D/D lung & ovarian adenocarcinoma GIST CD117 + (100%) Cytoplasmic & membrane ( D/D Fibromatosis )

44. PANEL FOR MALE GENITAL SYSTEM Tumor Antibody panel Prostate Carcinoma PAP & PSA + 34BE12 –(d/d benign lesion) P504S + Seminoma PLAP , CD117 , LDH , Vimentin + EMA , CD 30 , HMWK – EmbryonalCa EMA ,CD30 , HMWK + Choriocarcinoma hCG + Yolk sac tumor AFP + Leydingcell tumor Inhibin +

45. PANEL FOR HEPATOBILIARY SYSTEM Tumor Antibody panel Hepatocellularcarcinoma AFP , Keratin , EMA + Hep-par1 + CEA - Cholangiocarcinoma EMA , CEA,Keratin + GB Carcinoma CK 7+ & CK20 + (d/d cholangiocarcinoma) Pancreaticcarcinoma Keratin , EMA , CEA , CD 19-9+

46. PANEL FOR URINARY SYSTEM Tumor Antibody panel Renalcell carcinoma Coexpressionof keratin + vimentin A103 , inhibin –(d/d adrenocorticalca ) CA125 -(d/d ovarian ca) TTF -1 , thyroglobulin –( d/d thymiccarcinoma ) Transitional carcinoma of bladder CK 7+ & CK20 + CEA , cathepsinB +

47. PANEL FOR ADRENAL GLAND Tumor Antibody panel Pheochromocytoma Catecholamine, neuronal marker + S-100 + Neuroblastoma NSE , neurophilaments + Chromogranin , Synaptophysin + Adrenocorticalneoplasm Vimentin , Synaptophysin + I nhibin , A103 + Chromogranin

48. PANEL FOR SKELETAL SYSTEM Tumor Antibody panel Osteoid Osteonectin , osteopontin + Osteocalcin + Cartilage S-100 + Ewing’ ssarcoma CD99 , vimentin + NSE , neurophilaments +

49. PANEL FOR THYROID Tumor Antibody panel Papillary carcinoma CK 7 + , CK 20 -, CK 19 + HMWK + Follicular carcinoma Thyroglobulin , TTF –1+ LMWK , EMA + Medullarycarcinoma Calcitonin , CEA + Chromogranin +

50. PANEL FOR FEMALE GENITAL SYSTEM Tumor Antibody panel Adenocarcinomacervix Keratin , EMA , CEA , HPV + Endometrial carcinoma ER , PR + HER2/ neu , GLUT 1 + Ovarian carcinoma CK 7 + , CK 20 -, CA 125+ Mesothelioma Calretinin , thrombomodulin + CK 5/6 +

51. PANEL FOR CENTRAL NERVOUS SYSTEM Tumor Antibody panel Astrocyticneoplasm GFAP + AE 1/3CK cocktail - Oligodendroglioma S100 , Leu7+ Ependymoma GFAP , S100 , vimentin + Meningioma EMA , S 100 + CK 20-

52. PANEL FOR SALIVARY GLAND Tumor Antibody panel MucoepidermoidCarcinoma CK 7 , CK 14 + Aciniccell carcinoma Keratin , amylase , IgA + Transferrin , lactoferrin + Adenoid cystic carcinoma Keratin, CEA,S100 , CD117 + Lysosome , lactoferrin + Myoepithelioma Keratin , S100 + Vimentin , actin , myosin+

53. PANEL FOR THYMUS Tumor Antibody panel Thymic carcinoma CD5 , CD 70 + TTF -1 -(d/d from lung carcinoma) Thymoma Keratin , EMA , CEA+ CD 99, CD 1a + S100 +

54. ANTIBODY TO SPECIFICITY CK Carcinoma ( epithelial tumors) Vimentin Sarcoma ( mesenchymaltumors ) GFAP Gliomas HMB -45 Melanoma S-100 Neoplasm of neural origin CD 68 Proliferationof mast cell CD 45 Neoplasm of lymphoid origin CD 45 RO Neoplasm of T cell origin CD 3 Neoplasm of T cell origin CD 20 Neoplasm of B cell origin CD 15 Hodgkin’s disease CD117 GIST CD5 Mantle cell lymphoma NB -84 Neuroblastoma Chromogranin Neuroendocrinetumors ( pan-endocrine marker)

55. THANKS