ihc in diagnosis of soft tissue tumor

1. IMMUNOHISTOCHEMISTRY IN
DIAGNOSIS OF SOFT TISSUE
TUMOURS
DR. PANNAGA. P. KUMAR
MODERATOR- DR. SHWETHA

2. INTRODUCTION
• Soft tissue – defined as non epithelial tissue excluding the skeleton,
joints, CNS, hematopoietic and lymphoid tissues.
• It is represented by muscles, fat, fibrous tissue along with vessels
serving these tissues and peripheral nervous system.
• Embryologically it is derived from mesoderm with some contribution
from neuroectoderm.
• Soft tissue tumors are highly heterogeneous group of tumors that are
classified on histogenic basis according to adult tissue they resemble.

3. WHO CLASSIFICATION
Adipocytic tumors
 Fibroblastic/myofibroblastic tumors
 Fibrohistiocytic tumors
 Smooth muscle tumors
 Pericytic/Perivascular tumors
 Skeletal muscle tumors
 Vascular tumors
 Chondro-osseous tumors
 Tumors ‘‘Of uncertain differentiation’’ category

4. ANCILLARY TECHNIQUES IN SOFT TISSUE
TUMOURS
• HISTOCHEMISTRY
• ELECTRON MICROSCOPY
• IMMUNOHISTOCHEMISTRY
• CYTOGENETICS AND MOLECULAR STUDIES

5. IMMUNOHISTOCHEMISTRY
• Immunohistochemistry combines anatomical, immunological and
biochemical techniques to identify discrete tissue components by
interaction of target antigens with specific antibodies tagged with a
visible label.
• It is the most practical way to evaluate the presence of certain protein
and carbohydrate epitopes on tissue sections, and evaluation of cell or
tumor specific or cell cycle related markers.

7. APPLICATIONS OF IHC
• The applications of immunohistochemistry fall into 3 main categories:
- Identification of rare/ “atypical” benign pseudosarcomatous tumors
- Exclusion of non-sarcomatous neoplasms
- Classification of sarcomas
• Others:
- Distinguish among histologically similar tumours.
- Confirms histologic impression.
- Support diagnosis of rare tumour type.
- Support diagnosis when tumour arises in unusual location or age

8. GROUPS OF IHC MARKERS
• Intermediate filament proteins
• Markers of muscle differentiation
• Neural and neuro endocrine specific markers
• Melanocytic differentiation markers
• Markers of endothelial differentiation
• Epithelial markers
• Prognostic markers
• Markers for proteins indicative of fusion gene

9. INTERMEDIATE FILAMENT PROTEINS
• Vimentin
• Keratin
• Glial fibrillary acid proteins

10. VIMENTIN
• Cytoplasmic marker.
• Expressed in all mesenchymal cells.
• Expressed by sarcomatoid carcinoma at any site.
• Marker of tissue preservation.
• Greatest utility is in the diagnosis of carcinomas of uncertain primary
site, where strong co-expression may be a clue to renal, endometrial,
and thyroid carcinomas.

11. • In some mesenchymal tissues
vimentin is typically co-expressed
along with the type-specific
intermediate filaments
e.g. Desmin and Vimentin co-
expression in muscle cells
Vimentin and GFAP in some
Schwann cells
• Absence may be clue to rare tumors
like alveolar soft part sarcoma and
perivascular epitheloid cell neoplasm.
SMALL CELL OVARIAN CARCINOMA

12. CYTOKERATINS
• Cytoplasmic marker.
• Most complex member of intermediate filament family, found in
epithelial cells.
• Grouped according to their molecular weight.
• Over the past decade its clear that cytokeratin expression is not
restricted to carcinomas.

13. • Among sarcomas there are two patterns of expression
Usual expression – Synovial sarcoma, Epitheloid sarcoma
Anomalous expression – Neuroendocrine tumors, metastatic
melanomas, epitheloid hemangioendothelioma, epitheloid angio
sarcoma, PNET, Wilm’s tumor, rhabdomyosarcoma.
SYNOVIAL SARCOMA

14. GLIAL FIBRILLARY ACID PROTEIN
• Intermediate filaments of glial cells especially astrocytes and
ependymal cells.
• Also present in myoepithelial cells of salivary glands, breast.
• Marker for ependymal and glial tumors, especially myxopappillary
ependymomas, GI schwannomas, myoepitheliomas of salivary glands
and skin adnexa.
• Absent in MPNST and GIST.

15. MARKERS OF MUSCLE DIFFERENTIATION
• Actin
• Desmin
• Myogenic transcription factors
• Caldesmon
• Calponin
• Myoglobin
• Myosin

16. ACTIN
• Cytoplasmic marker
• Seen in tissues with pure myogenic differentiation.
• Six isoforms – Skeletal muscle alpha, Smooth muscle alpha & gamma,
Cardiac muscle alpha and nonmyogenous beta & gamma actins
• ANTIBODIES used- HHF35 , 1A4 and Asr-1
• HHF35- Muscle specific actin : myofibroblastic leiomyoma,
leiomyosarcoma, rhabdomyoma, rhabdomyosarcoma.
• 1A4- Smooth muscle actin: myofibroblastic leiomyoma,
leiomyosarcoma.

17. DESMIN
• Intermediate filament which binds the myofilaments as bundles.
• Present in cardiac and skeletal muscle cells, parenchymal smooth
muscle cells and some vascular smooth muscle cells.
• Positivity is seen in rhabdomyosarcoma, endometrial carcinosarcoma,
dedifferentiated liposarcoma, 70% of leiomyomas,
angiomyofibroblastoma, aggressive angiomyxoma.

18. MYOGENIC TRANSCRIPTION FACTORS
• Myogenic regulatory proteins play a crucial role in commitment and
differentiation of mesenchymal progenitor cells to myogenic lineage.
• MyoD1 and myogenin- expressed in nuclei of fetal and regenerating
but not in adult skeletal muscle cells or mesenchymal cells.
• Only nuclear positivity should be considered.
• Both are expressed in embryonal and alveolar rhabdomyosarcoma.
• Also in rare tumors with rhabdomyoblastic differentiation.

20. CALDESMON
• Actin, calcium and calmodulin binding cytoskeleton associated protein
• Involved in regulation of smooth muscle contraction
• Highly expressed in smooth muscle and myoepithelial cells but not
myofibroblasts
• Positive- leiomyomas, glomus tumors, leiomyosarcomas, GISTs
• Negative- rhabdomyosarcoma

21. CALPONIN
• Actin and tropomysin binding cytoskeleton associated protein
• Important in regulation of smooth muscle contraction
• Expressed in smooth muscle, myoepithelial cells and myofibroblast.
• Positive – leiomyomas, leiomyosarcoma, nodular fasciitis
• Negative - GIST

22. NEURAL AND NEURO ENDOCRINE SPECIFIC
MARKERS
• Synaptophysin
• Chromogranin
• Neuron specific enolase
• Neuro filament proteins

23. SYNAPTOPHYSIN
• Membrane channel protein
• Expressed in neural and neuroendocrine
cells such as ganglion cells, axons,
paraganglia.
• Positive- neuroblastomas,
ganglioneuromas, medulloblastoma,
chief cells of paragangliomas, carcinoids.
Olfactory neuroblastoma

24. CHROMOGRANIN
• Calcium binding protein in neural
and neuroendocrine cells.
• Positive- paragangliomas,
pheochromocytoma, metastatic
neuroendocrine tumors, carcinoids
• Negative in PNET
Pancreatic islet

25. NEURON SPECIFIC ENOLASE
• Expressed in most neural and neuroendocrine cells.
• Positive- paragangliomas, carcinoids, melanomas, neuroblastomas,
Ewing family of tumors
• Also seen in fibroadenoma, ductal carcinoma of breast,
leiomyosarcoma, angiosarcoma

26. NEURO FILAMENT PROTEINS
• Intermediate filaments of neurons
and their axons
• 3 types- NFL, NFM, NFH.
• Neurofilaments are present only in
neurons and adrenal medulla
• Positive- neuroblastoma, adrenal
pheochromocytoma, Merkel cell
carcinoma
Ganglioneuroblastoma

27. MARKERS OF NERVE SHEATH
DIFFERENTIATION
• S-100 protein
• Claudin-1
• CD-57
• Glut-1
• p75NTR

28. S-100 PROTEIN
• Acidic, calcium binding protein
• 3 types-
Alpha- alpha : myocardium, skeletal muscle, neurons
Alpha- beta : melanocytes, glial cells, chondrocytes, skin adnexae
Beta- beta : Langerhans cells, schwann cells

29. DISTRIBUTION OF S-100 PROTEIN IN
NONNEOPLASTIC AND NEOPLASTIC
Normal Cell Tumor
Melanocyte Nevi, malignant melanoma, all types
Schwann cell Schwannoma, neurofibroma, true nerve sheath
myxoma
Cartilage Chondroma (Extraskeletal myxoid
chondrosarcoma < 30%)
Adipocyte Liposarcoma (variable)
Regenerating skeletal muscle Rhabdomyosarcoma (variable)
Myoepithelial cells Myoepithelioma, mixed tumor
Langerhans cells / Interdigitating reticulum
cell
Rosai-Dorfman disease, Langerhans cell
histocytosis, Interdigitating reticulum cell
sarcoma
Tumors with unknown normal cell counterpart Ossifying fibromyxoid tumor (>50%) synovial
sarcoma (20-30%)

30. S100
Schwannoma MPNST

31. CLAUDIN-1
• Help to determine tight
junction structure and
permeability
• Expressed in perineural cells
• Positive in perineuromas
Perineuroma

32. CD-57
• Myelin associated glycoprotein
• Leu7 and HNK1 antibody is used to detect it
• Expressed in oligodendroglia and schwann cells
• Positive- MPNST, a percentage of synovial sarcoma and
leiomyosarcoma.

33. NERVE GROWTH FACTOR RECEPTOR P75
• Expressed on neuronal axons, schwann cells, perineural cells,
perivascular fibroblast, myoepithelium
• Positive- schwannoma, neurofibroma, MPNST, synovial sarcoma,
rhabdomyosarcoma, malignant melanoma.

34. NEUROECTODERMAL MARKERS
• CD99
• CD56

35. CD99
• Transmembrane glycoprotein, exact function is
unknown
• Plays a role in cell adhesion and regulation of
cellular proliferation
• Expressed in nearly all human tissues
• Important in Ewing’s/ PNET (>90%), Lymphoblastic
lymphoma (>90%), poorly differentiated synovial
sarcoma (>75%)
• Negative in neuroblastoma
Ewing’s sarcoma

36. CD56
• Neural cell adhesion molecule
• Positive- synovial sarcoma, MPNST,
schwannoma, rhabdomyosarcoma,
leiomyosarcoma, leiomyoma,
chondrosarcoma, osteosarcoma
• Small, blue, round cell tumors
Negative- Ewing’s/ PNET
Positive- alveolar and primitive
rhabdomyosarcoma, small cell carcinoma,
Wilm’s tumor Small cell carcinoma- lung

37. MELANOCYTIC DIFFERENTIATION
MARKERS
• HMB-45
• Tyrosinase
• Melan-A
• Microphthalmia transcription factor

38. HMB-45
• Its an antibody which detects oncofetal
glycoprotein which is present in
immature but not mature melanosomes.
• Its organelle specific.
• Positive- malignant melanoma,
angiomyolipoma, clear cell tumor of lung,
lymphangiomatosis, Perivascular
epitheloid cell tumors
• Negative in intradermal nevus.
Malignant melanoma

39. TYROSINASE
• Catalyses tyrosine incorporation into
melanin pigment
• Target for melanoma therapy
• Cytoplasmic marker
• Excellent marker for metastatic
melanoma
melanoma

40. MELAN-A
• Product of MART-1 gene (melanoma antigen
recognised by T cells)
• Function- unknown
• Marker of melanosomes and not melanomas
• Positive- epitheloid melanomas, PEComas,
angiomyolipoma.
Angiomyolipoma

41. MICROPHTHALMIA TRANSCRIPTION
FACTOR
• Nuclear protein, transcriptional regulator.
• Critical for melanocyte development
• Positive – primary melanomas, metastatic melanoma, clear cell
sarcoma, osteoclastic giant cells, epitheloid histiocytes.
• Also in leiomyosarcomas, atypical fibroxanthomas, atypical
lipomatous neoplasms.

42. MARKERS OF ENDOTHELIAL
DIFFERENTIATION
• von Willebrand Factor
• CD-31
• CD-34
• Fli-1
• Ulex lectin
• VEGFR-3
• Podoplanin
• Thrombomodulin

43. VON WILLEBRAND FACTOR
• First endothelium specific marker employed in IHC studies
• Least sensitive of vascular markers
• Positive in 50-75% of vascular tumors
• It is not only produced by endothelial cells but circulates in serum,
therefore found in zones of tumor necrosis and haemorrhage.

44. CD-31
• Newest of commonly used vascular markers
• Platelet-endothelial cell adhesion molecule-
1(PECAM-1)
• Most sensitive and specific
• Not seen in non-endothelial tissue/ tumors
except macrophages and platelets
• Intense cytoplasmic staining in endothelial
cells
• Positive- angiosarcomas,
hemangioendothelioma, hemangiomas,
Kaposi’s sarcoma Angiosarcoma

45. CD-34
• Hematopoietic progenitor cell antigen
• Expressed in early hematopoietic blasts,
all endothelial cells ,subsets of fibroblasts,
interstitial cells of Cajal, nerve sheath
• Cytoplasmic staining- spindle cells
Distinct membranous staining- large
cytoplasmic cells
• Positive- Kaposi sarcoma, (50%)
angiosarcoma, dermatofibrosarcoma
protruberans, solitary fibrous tumors,
hemangiopericytomas, neurofibromas,
GISTs.
Solitary fibrous tumor

46. FLI-1
• Freund’s leukemia site gene.
• Only available nuclear marker of endothelial differentiation
• Positive- >95% of endothelial neoplasms of all types and degrees of
malignancy, including hemangiomas, hemangioendothelioma,
angiosarcomas and Kaposi’s sarcoma
• Not expressed in epitheloid sarcomas.

47. ULEX LECTIN
• Was a popular alternative marker of endothelial cells and tumors.
• But its now known to be present in a wide range of epiyhelial tumors,
which limits its diagnostic utility.

48. VEGFR-3
• Vascular endothelial growth factor receptor- 3
• Transmemberane receptor tyrosine kinase specific for subsets of
endothelia and trophoblast
• Plays a role in lymphatic endothelial proliferation and lymphatic
vessel formation.
• Positive in (95%) Kaposi’s sarcoma, (50%) angiosarcoma
• Not seen in non-endothelial neoplasms

49. THROMBOMODULIN
• CD141, thrombin binding and thrombolysis activating antithrombotic
protein
• Expressed in endothelia, trophoblast and mesothelial cells
• Positive- inconsistently in hemangiomas, hemangioendothelioma and
angiosarcoma
• Also present in mesothelioma.

50. HUMAN HERPES VIRUS 8 LATENCY
ASSOCIATED NUCLEAR ANTIGEN
• HHV8 – causative agent of Kaposi’s sarcoma
• Positive in >90% of Kaposi’s sarcoma
• LANA expression is not seen in non Kaposi’s sarcoma, except primary
effusion lymphoma and Castleman disease

51. PROGNOSTIC MARKERS
• Ki67 and analogs
• p53
• p16 and p27

52. KI-67 AND ANALOGS
• Encode by single gene on chromosome 10
• Expression confined to late G1, S, M and G2 growth phases
• Appears to be localized to nucleolus
• Ki-67 labelling index of >20% is an independent predictor of distant
metastases and tumor mortality

53. P53
• Cell cycle regulator, nuclear protein.
• Arrests cells with damaged DNA in G1 phase
• P53 over expression – high grade tumor, worst outcome

54. P16 AND P27
• Cyclin dependent kinase inhibitors
• p16 loss of expression is seen in MPNST but not in neurofibromas.
• p27 loss of expression is seen in malignant transformation of
neurofibromas.

55. MARKERS FOR PROTEINS INDICATIVE OF
FUSION GENE
• FLI-1
• WT-1
• TFE-3
• INI-1

56. FLI-1
• Translocation: t(11;22)
• Fusion gene: EWS-FLI1
• Protein: FLI1(nuclear)
• FLI1 also in endothelial cells and
tumors, T cells.
• Positive- Ewing’s sarcoma,
lymphoblastic lymphoma.

57. WT-1
• Marker of t(11;22)(13;q24)
• Fusion of EWS and WT1 genes
• Nuclear positivity- desmoplastic small round cell tumor
• Cytoplasmic positivity- rhabdomyosarcoma, Wilm’s tumor

58. TFE-3
• der(17)t(X;17)(p11;q25)
• Translocation of alveolar soft part
sarcoma
• Fusion of TFE3 gene to ASPL gene
• Low levels of TFE3 is present in all
normal tissues
• Strong nuclear expression is seen in
alveolar soft part sarcoma, granular cell
tumors, rare pediatric renal carcinomas
Alveolar soft part sarcoma

59. INI-1
• Deletions of hSNF5/INI-1/SMARCB1/BAF47 gene on chromosome 22
• INI1 – tumor suppressor, present in all normal tissues.
• Implicated in pathogenesis of atypical teratoid / rhabdoid tumor of
CNS and extrarenal rhabdoid tumours.

60. IHC IN DIAGNOSIS OF SARCOMAS
• Common histologic scenarios in which immunohistochemistry can
provide valuable clues to correct diagnosis are
-undifferentiated round cell tumours
-monomorphic spindle cell tumours
-poorly differentiated epitheloid tumours
-pleomorphic sarcomas

65. CONCLUSION
• After ∼30 years of widespread usage, immunohistochemistry (IHC)
has become a standard method of diagnosis for surgical pathology.
• Because of the plethora of diagnoses and often subtle nature of
diagnostic criteria, IHC finds particular utility in soft tissue tumors.
The use of progressively small amounts of tissue for diagnosis
highlights the importance of this method.
• The sensitivity and crispness of IHC stains have progressively
improved with the advent of new techniques.

66. • Traditionally, IHC detects cell-typic markers that characterize cell
phenotypes like myogenin for skeletal muscle, and cytokeratin for
epithelium.
• However, the advent of genetic discoveries have led to IHC testing for
detection of fusion gene products or overexpressed oncogenes
associated with deletions and mutations.

67. • Proliferation-based markers such as Ki-67 can also be used for
prognosis and grading, but more standardization is needed.
• Development of monoclonal antibody-based pharmaceuticals, such as
imatinib or crizotinib, holds the promise of tailored anticancer
therapy. IHC thus has assumed importance not only for diagnosis but
also for guidance of personalized medicine.

68. REFERENCES
• Gown MA, Folpe L.A. Immunohistochemistry for analysis of Soft Tissue Tumor. In
Enzinger and Weiss’ Soft Tissue Tumors. 5th Edition; China; Mosby Elsevier 2008.
129-174
• Kumar V Abbas A.K., Aster C.J. Robins & Cotran Pathologic Basis of Disease Vol 1.
9th ed; Haryana; Elsevier 2014.
• Yohe L.S, Hall H.J. Application of Immunohistochemistry to Soft Tissue Neoplasms
Arch Pathol Lab-Med; March 2002;
• Somerhausen A S N. Immunohistochemistry in the diagnosis of Soft Tissue Tumors