Role of ihc on soft tissue tumours

IHC IN SOFT TISSUE TUMOURS

1. ROLE OF IHC ON SOFT TISSUE TUMOURS Dr.T.Arivazhagan Post graduate Department of Pathology

2. Introduction SOFT TISSUE( Mesoderm) Non epithelial tissue: Excludes: 1.Adipose tissue 1.Skeleton & joint 2.Skeletal & Smooth muscle 2.CNS 3.Blood & Lymph vessels 3.Hematopoietic 4.PNS 4.Lymphatic tissue

3. WHO classification ( Histogenesis) Adipocytic tumours Fibroblastic/ myofibroblastic tumour Fibrohistiocytic tumour Smooth & Skeletal muscle tumour Vascular tumour Pericytic/perivascular tumour Chondro-osseous tumour Tumours of uncertain differentiation

4. Ancillary techniques Histochemistry Electron microscopy Immunohistochemistry Cytogenetics & Molecular studies

5. IHC “ Immuno ” – Refers to antibodies “ Histo ” – Refers to tissue Albert Coons – First implemented (1941) Principle – Binding of specific antibody to the specific antigen

6. Definition Defined as companied process of anatomical, immunological and biochemical techniques to identify the discrete tissue components by interaction of target antigens with specific antibodies targeted with a visible label.

7. Uses Differentiate among histologically similar tumour Confirm the histological impression Support the diagnosis of rare tumour Support the diagnosis of unusual location or age

8. Protocol Tissue processing Antigen retrieval Inhibition of endogenous peroxidase Primary antibody incubation Secondary antibody incubation DAB staining Hematoxylin staining Dissolution & Clearing visualization

9. Markers for Fibroblastic/Myofibroblastic & Fibrohistiocytic tumour Intermediate filament Vimentin Keratin Glial fibrillary acid protein Actin Desmin CD 34 CD 68

10. Vimentin Cytoplasmic marker Expressed In all mesenchymal cells Fibrocyte Fibroblast Smooth muscle Endothelium Schwann cells Renal tubules Thyroid follicular cells

11. Used – Diagnosis of carcinomas of uncertain primary site Strong expression – Clue to Renal, Endometrial & Thyroid carcinoma Pitfall: Use of single marker is limited diagnostic value Because of co expression with other cytokeratins

12. Strong positive (>90%) Nodular fasciitis Fibrous histiocytoma Myofibroblastoma Angiomyxoid fibroma Giant cell angiofibroma Dermatomyofibroma Solitary fibrous tumour Congenital & infantile fibro sarcoma DFSP

13. Cytokeratin Cytoplasmic marker Epithelial cells Composed of > 20 isotypes Type I – Acidic – 9 to 20 Type II – Basic – 1 to 8 Expressed both carcinomas and sarcomas Different CK expressed in different epithelial types at different stage of differentiation

14. Sarcomas Usual Unusual Synovial sarcoma Neuroendocrine tumour Epithelioid sarcoma Metastatic melanomas Wilm’s tumour Rhabdomyosarcoma Pit falls: Also expressed various non epithelial tissue & tumours

15. Actin Cytoplasmic marker Seen in pure myogenic differentiation 6 isoforms Antibodies used – HHF35,1A4 Pit fall : +ve other than smooth muscle tumour Endometrial stromal tumour Synovial sarcoma GIST’s

16. Strong positive Myofibroblastoma Angiomyxoid fibroma Dermatomyofibroma Solitary myofibroma Angioleiomyoma Leiomyoma Leiomyosarcoma

17. Desmin Intermediate filament Present in cardiac , skeletal muscle & Smooth muscle Strong positive Myofibroblastoma Angiomyofibroblastoma Solitary myofibroma Rhabdomyoma Rhabdomyosarcoma Pit fall : +ve other than RMS like desmoplastic small round cell tumour

18. CD 68 Expressed only in multinucleated cells +ve: GCT of tendon sheath Tenosynovial giant cell tumour

19. MUSCLE DIFFERENTIATION

20. Actin Desmin Myogenic transcription factors Caldesmon Calponin Myoglobin Myosin

21. Myogenic transcription factor Role – Differentiation of mesenchymal progenitors to myogenic lineage Myo D1 & Myogenin – expressed only in fetal skeletal tissue Not in adult skeletal tissue Only nuclear positivity considered +ve: Embryonal & Alveolar RMS Pit falls: Also positive in non neoplastic myoblast,desmoid tumour, Wilm’s tumour

22. Caldesmon Cytoplasmic marker Calcium & Calmodulin binding protein Role – Contraction of smooth muscle 2 isoforms are available ( LMW & HMW) Expressed in Smooth muscle & Myoepithelial cells Not seen in myofibroblast +ve : LM,LMS,GIST’S, Glomus tumour -ve : RMS Pit fall: +ve in non smooth muscle lesion GIST

23. Calponin Cytoplasmic marker Actin , Tropomyosin & Calmodulin binding protein Role – Smooth muscle contraction Expressed in smooth muscle, myoepithelial cells, myofibroblast +ve : LM,LMS,NODULAR fasciitis -ve : GIST’s

24. Myoglobin Cytoplasmic marker Iron & o2 binding polypeptide Expressed in Skeletal muscle , Cardiac muscle, Rhabdomyoblast Lack of expression in Smooth muscle Pit fall : Also expressed in various other carcinomas ( BREAST,PROSTATE,COLON)

25. Markers for Adipocytes

26. S 100 MDM 2 CDK 4 P16

27. S 100 Calcium binding protein Acidic nature 3 isoforms Expressed in normal and neoplastic cells

28. Normal cell Neoplastic cell Melanocyte Nevi, Malignant melanoma Adipocyte Lipoma , Liposarcoma Schwann cell Schwannoma, Neurofibroma Cartilage Chondroma, chondrosarcoma Langerhans cells Langerhans cell histiocytosis

29. MDM 2 Murine double minute 2 Nuclear & Cytoplasmic maker + ve in Liposarcoma Pit fall: Also expressed in many sarcomas

30. CDK 4 Cyclin dependent kinase 4 Nuclear marker Role – Regulation of cell cycle Expressed in some Epithelial & Mesenchymal cells Pit fall: Over expression in Liposarcoma , Osteosarcoma

31. P 16 Cyclin dependent kinase inhibitor 2A Tumour suppressor protein Expressed in few carcinoma types & HPV associated SCC of different origin +ve : Well differentiated , Dedifferentiated Liposarcoma -ve : Normal fatty tissue & Lipoma

32. Vascular & Perivascular marker

33. CD 31 CD 34 Factor 8 Podoplanin Thrombomodulin CD 141 CD 105 VEGF-3

34. CD 31 Common vascular marker Platelet endothelial cell adhesion molecule 1 Most sensitive & specific marker Not seen in non endothelial cells Expressed Endothelial cell unction Platelet,monocyte,granulocyte +ve : Angiosarcoma, Haemangioendothelioma, Hemangioma,kaposi’s sarcoma

35. CD 34 Hematopoietic progenitor cell antigen Expressed in precursor of Myeloid, Lymphoid lineage & Mesenchymal cell Widely used But less specific than CD 31 +ve : Kaposi’s sarcoma, Angiosarcoma,DFSP, Solitary fibrous tumour, Neurofibroma Pit fall: Broad spectrum of expression used as screening marker

36. Factor 8 Cytoplasmic marker First specific marker for endothelium used in IHC Less sensitive +ve : Vascular tumour Pit fall: Also present in serum +ve in tumour necrosis & hemorrhage

37. Podoplanin Membranous / cytoplasmic markers Known as C2-40 Expressed in Fetal germ cells Membrane of several mature cells +ve : Lymphangioma, Mesothelioma, Adenomatoid tumour Pit fall: Broad spectrum of expression Used with other specific antibodies

38. Thrombomodulin Known as CD 141 Expressed in Endothelium, Trophoblast, Mesothelial cells +ve : Hemangioma Haemangioendothelioma Angiosarcoma Mesothelioma

39. VEGF-3 Transmembrane receptor tyrosine kinase specific protein Expressed in Endothelial & Trophoblast +ve : Kaposi’s sarcoma Angiosarcoma

40. Neural & Neuroendocrine markers

41. Synaptophysin Chromogranin Neuron specific enolase Neuro filament protein

42. Synaptophysin Membrane channel protein Expressed Neural cells Neuroendocrine cell +ve : Neuroblastoma Ganglioneuroma Medulloblastoma carcinoids

43. Chromogranin Calcium binding protein Expressed Neural cells Neuroendocrine cells +ve : Paragangliomas Pheochromocytoma Carcinoids -ve : PNET

44. Neuron specific enolase Expressed in neural & neuroendocrine cells +ve: Paragangliomas Carcinoids Melanomas Neuroblastoma Leiomyosarcoma Angiosarcoma

45. Neuro filament proteins Intermediate filaments of Neurons & Axons 3 types +ve : Neuroblastoma Merkel cell carcinoma Adrenal Pheochromocytoma

46. Neuroectodermal markers CD 99 CD 56

47. CD 99 CD 56 Transmembrane glycoprotein Neural cell adhesive molecules +ve Ewing’s PNET Lymphoma +ve Synovial sarcoma MPNST Schwannoma RMS,LMS -ve Neuroblastoma -ve Ewing’s / PNET

48. Osseous & Cartilaginous marker S 100 Ostocalnin Ostonectin Androgen receptor Pan cytokeratin

49. S 100 Ostonectin Soft tissue Chondroma Chondroblastoma Mesenchymal osteosarcoma Extra skeletal osteosarcoma

50. Melanocytic markers HMB-45 Throsinase Melan –A Microphthalmic transcription factor

51. HMB 45 Organelle specific marker Detect immature melanocytes +ve: Malignant melanoma Angiosarcoma Lymphomatosis -ve: Intradermal nevus

52. Thyrosinase Cytoplasmic marker Catalyze tyrosine – Incorporation into the melanin pigment Excellent for Malignant Melanoma

53. Melan -A Markers of melanosomes Not for melanocytes +ve : Epithelioid melanomas PECOMA’s Angiomyolipoma

54. Microphthalmic transcription factor Nuclear marker +ve: Primary melanomas Metastatic melanomas Clear cell sarcomas Leiomyosarcoma

55. Prognostic markers Ki 67 & analogs - Indicate distant metastasis P 53 - High grade tumour - Worst outcome P16 & P27

56. Cell type Tumour Diagnosis Round cell Rhabdomyosarcoma PNET Myo D1 CD 99 Spindle cell Schwannoma Neurofibroma Kaposi’s sarcoma DFSP MPNST S -100 S – 100 CD 34/CD 31 CD 34 S - 100 Epithelioid cell Angiosarcoma Clear cell sarcoma Epithelioid sarcoma Angiosarcoma CD 31/34 Melanoma markers CD 34 CD 31/34

57. Antibody Significance Actin Smooth muscle cell CD 31 Endothelial cells CD 34 Precursor of blood & Endothelial cell Cytokeratin Epithelial origin Desmin General marker for muscle cells HMB 45 Melanosomes Neurofilament Nervous tissue Neuron specific enolase Neuroendocrine cells S 100 Adipose cell, nervous cell, macrophage Vimentin Connective tissue

58. Sarcomas IHC Smooth , Skeletal, Endometrial stromal Desmin Vascular sarcomas CD 31/34 Sarcomas with NEURAL,LIPOMATOUS differentiation S 100 Synovial sarcomas Cytokeratin , CD 99 GIST CD 117 Chondro sarcoma CD57, Vimentin Ewing’s/ PNET CD 99, S100 Desmoplastic round cell tumour Cytokeratin , CD 99, Desmin Osteosarcoma CD 57, Vimentin, EMA