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)
1. Adipocytic tumours
2. Fibroblastic/myofibroblastic tumour
3. Fibrohistiocytic tumour
4. Smooth & Skeletal muscle tumour
5. Vascular tumour
6. Pericytic/perivascular tumour
7. Chondro-osseous tumour
8. Tumours of uncertain differentiation

4. Ancillary techniques
1. Histochemistry
2. Electron microscopy
3. Immunohistochemistry
4. 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
1. Differentiate among histologically similar tumour
2. Confirm the histological impression
3. Support the diagnosis of rare tumour
4. 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
1. Intermediate filament
1. Vimentin
2. Keratin
3. Glial fibrillary acid protein
2. Actin
3. Desmin
4. CD 34
5. CD 68

10. Vimentin
• Cytoplasmic marker
• Expressed In all mesenchymal cells
1. Fibrocyte
2. Fibroblast
3. Smooth muscle
4. Endothelium
5. Schwann cells
6. Renal tubules
7. 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%)
1. Nodular fasciitis
2. Fibrous histiocytoma
3. Myofibroblastoma
4. Angiomyxoid fibroma
5. Giant cell angiofibroma
6. Dermatomyofibroma
7. Solitary fibrous tumour
8. Congenital & infantile fibro sarcoma
9. 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
1. Endometrial stromal tumour
2. Synovial sarcoma
3. GIST’s

16. Strong positive
1. Myofibroblastoma
2. Angiomyxoid fibroma
3. Dermatomyofibroma
4. Solitary myofibroma
5. Angioleiomyoma
6. Leiomyoma
7. Leiomyosarcoma

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

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

19. MUSCLE
DIFFERENTIATION

20. 1. Actin
2. Desmin
3. Myogenic transcription factors
4. Caldesmon
5. Calponin
6. Myoglobin
7. 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. 1. S 100
2. MDM 2
3. CDK 4
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. 1. CD 31
2. CD 34
3. Factor 8
4. Podoplanin
5. Thrombomodulin CD 141
6. CD 105
7. 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 :
1. Kaposi’s sarcoma
2. Angiosarcoma

40. Neural
&
Neuroendocrine
markers

41. 1. Synaptophysin
2. Chromogranin
3. Neuron specific enolase
4. Neuro filament protein

42. Synaptophysin
• Membrane channel protein
• Expressed
1. Neural cells
2. Neuroendocrine cell
+ve :
1. Neuroblastoma
2. Ganglioneuroma
3. Medulloblastoma
4. 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:
1. Paragangliomas
2. Carcinoids
3. Melanomas
4. Neuroblastoma
5. Leiomyosarcoma
6. Angiosarcoma

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

46. Neuroectodermal
markers
1. CD 99
2. 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 -ve

48. Osseous & Cartilaginous
marker
1. S 100
2. Ostocalnin
3. Ostonectin
4. Androgen receptor
5. Pan cytokeratin

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

50. Melanocytic markers
1. HMB-45
2. Throsinase
3. Melan –A
4. Microphthalmic transcription factor

51. HMB 45
• Organelle specific marker
• Detect immature melanocytes
• +ve:
1. Malignant melanoma
2. Angiosarcoma
3. Lymphomatosis
• -ve:
1. 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 :
1. Epithelioid melanomas
2. PECOMA’s
3. Angiomyolipoma

54. Microphthalmic
transcription factor
• Nuclear marker
• +ve:
1. Primary melanomas
2. Metastatic melanomas
3. Clear cell sarcomas
4. Leiomyosarcoma

55. Prognostic markers
1. Ki 67 & analogs
- Indicate distant metastasis
2. P 53
- High grade tumour
- Worst outcome
3. 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