Round cell tumours

pathology of round cell tumours of osseo articular system like ewings sarcoma, mesenchymal chondrosarcoma,small cell osteosarcoma, plasma cell neoplasms and other hematopoietic malignancies. how immunochemistry os playing pivotal role in differential diagnosis.

1. CLASSIFICATION, GRADING,IHC, MOLECULAR ANALYSIS OF ROUND CELL NEOPLASM OF OSTEOARTICULAR SYSTEM BY – Dr. KANWALPREET KAUR MODERATOR- Dr. KARUNA GUPTA

2. ROUND CELL NEOPLASM   Heterogeneous group of neoplasms are characterised by the sheets of poorly differentiated cells: Small (similar to lymphocyte in size) Round (round nuclei and scanty cytoplasm) Blue (blue staining due to high nuclear/cytoplasmic ratios

3. OSSEO ARTICULAR TUMORS IN THIS CATEGORY E wings sarcoma/Primitive neuroectodermal tumour(ES/PNET Hematopoietic malignancies Plasma cell neoplasms Small cell osteosarcoma Mesenchymal chondrosarcoma Poorly differentiated small cell synovial sarcoma Rhabdomyosarcoma ( embryonal and alveolar subtypes)

4. GRADING AND CLASSIFICATION Histological grading of bone sarcomas is an attempt to predict the biological behaviour of a malignant tumour Criteria used— 1. C ellularity i.e ., the relative amount of cells compared to matrix 2 .Nuclear features of the tumour cells Tumours which are monomorphic, such as small cell malignancies (Ewing sarcoma, malignant lymphoma and myeloma), do not lend themselves to histological grading . Mesenchymal chondrosarcomas and dedifferentiated chondrosarcomas are always high grade

5. EWINGS SARCOMA/PNET R ound cell sarcomas that show varying degrees of neuroectodermal differentiation  Ewing’s sarcoma - limited neural differentiation  PNET - more neural features AGE- 5-20 years (commonly) infancy or adulthood rarely SEX PREDILITION- males:females =1.4:1

6. SITE: Ewings sarcoma/PNET

7. RADIOGRAPH : Ewings sarcoma/PNET OSTEOLYTIC LESION LAMELLATED PERIOSTEAL REACTION SUNBURST PERIOSTEAL REACTION

8. GROSS Generally arises in medullary cavity of shaft from which it permeates cortex and invades soft tissue Rarely, periosteal Soft, tan-white, areas of haemorrhage and necrosis

9. MICROSCOPY:Ewings sarcoma/PNET 1. Classic or conventional (typical) Ewing sarcoma 2. P rimitive neuroectodermal tumor (PNET) 3. A typical Ewing sarcoma Same immunohistochemical and molecular features, differing only in the extent of neural differentiation. Each subtype is considered a high-grade tumor .

10. MICROSCOPY : classic Solid sheets of cells divided into irregular masses by fibrous strands Cells- small uniform round nucleus frequent indentations small nuleoli scanty cytoplasm indistinct cell outlines Necrosis is common

11. EWINGS TUMOUR: classic type sheets of uniform population of cells; round nuclei ; finely granular chromatin; indistinct cell borders; necrosis common; mitotic activity usually not prominent

12. EWINGS TUMOUR: PNET Homer- wright rosette

13. 3. ATYPICAL - most difficult group to recognize G reat degree of cytologic variability and/or unusual growth patterns e.g. large nuclei with irregular nuclear membranes and prominent nucleoli; abundant eosinophilic cytoplasm imparting a rhabdoid appearance.

14. HISTOCHEMISTRY The cells of ES/PNET usually contain large amounts of cytoplasmic glycogen, as demonstrated by a PAS stain with diastase control

15. IMMUNOCHEMISTRY:Ewings sarcoma/PNET Reactivity for vimentin , CD 99 FLI-1 And variable immunohistochemical reactivity for neuron specific enolase , CD57, synaptophysin and cytokeratin Non reactive for chromogranin , cytokeratin, glial fibrillary protein, desmin , muscle specific actin, myogenin , CD 31 and CD45

16. IMMUNOHISTOGRAM OF EWINGS SARCOMA/PNET Non reactive for chromogranin , glial fibrillary protein, desmin , muscle specific actin, myogenin and CD45. Keratin is positive in 20% to 30% of cases, usually with a focal distribution.

17. CD 99 reactivity FLI-1 Protein expression

18. CD99 - strong, diffuse membranous staining pattern 84-100 % sensitive. If a tumor is negative for CD99 , it is highly unlikely to be Ewing sarcoma NOT A SPECIFIC MARKER C an be seen in RMS, glial tumours, neuroendocrine tumours, lymphoblastic lymphoma , WT, clear cell sarcoma of kidney, teratoma , synovial sarcoma, osteosarcoma and mesenchymal chondrosarcoma CD99 is important for distinction between Ewings sarcoma/PNET and metastatic neuroblastoma

19. FLI1 - Only nuclear staining is considered positive NOT SPECIFIC MARKER B ut also positive in lymphoblastic lymphoma, m yeloid neoplasms, DSRCT, Malignant melanoma, merkel cell carcinoma,synovial sarcoma, and some vascular neoplasms.

20. MOLECULAR GENETIC FEATURES R eciprocal translocation t(11;22)(q24;q12) Fusion of EWSR1 gene(encodes for RNA binding protein) at 22q12 with FLI1 gene(member of ETS family of transcription factors)   The t(21;22)(q12;q12) translocation involves the gene  ERG , which is located on chromosome 21   t(7;22)(p22;q12) translocation involves a gene known as  ETV1  at 7p22. Recently a translocation involving chromosomes 4 and 9 with CIC and DUX4 gene has been identified

21. Rearrangements of EWSR1 with non–ETS-family genes—including NFATC2 , POU5F1 , SMARCA5 , ZSG, and SP3— are also rarely identified

22. FISH for EWSR1 genomic rearrangements is highly sensitive (>95%) but nonspecific because other tumours may show rearrangement of this locus . RT-PCR for EWSR1 fusion genes - highly sensitive (> 95%) and specific (100 %).

23. Differential diagnosis… From lymphomas- EWINGS SARCOMA LYMPHOMAS negative for CD3 and CD 20 Negative for CD99 except lymphoblastic lymphoma TdT , CD34,CD43,CD10, CD79a and genetic rearrangement studies- distinguish lymphoblastic lymphoma from EWS/PNET From metastatic neuroblastoma ; Metastatic neuroblastoma occurs in children<2years very uncommon age group for Ewings sarcoma CD 99 helps in distinction

24. Differential diagnosis… Metastatic rhabdomyosarcoma Muscle markers are used for differential diagnosis Ewing sarcoma is typically positive for FLI1, CD99 ( diffuse membranous) both are non specific. A differential panel should also include: 1. muscle markers, 2. leukocyte common antigen 3.terminal deoxynucleotidyl transferase

25. MALIGNANT LYMPHOMA Primary Lymphoma- Originates in bone with no evidence of extraskeletal disease or disseminated bone marrow involvement. Rare Secondary skeletal involvement by a primary extraosseous lymphoma is much more common than primary bone lymphoma AGE-usually affects 40-60 years Lymphoma however can involve children; though it is much less common than Ewing sarcoma in this age group SITE- meta- diaphyses of long bones (femur, humerus , and tibia) and axial skeleton (pelvis and vertebrae)

26. RADIOGRAPHY: Extensive involment with permeative destructive process. Shaft is usually involved Radiographs appear as if there are multiple small holes with intervening normal bone Lesion shows mixture of lysis and sclerosis Periosteal new bone formation is unusual Bone scan- positive Positive bone scan or lesion on MRI + normal radiograhs = suggest malignant lymphoma

27. GROSS: fish-flesh appearance

28. MICROSCOPY ->Under low power, lesion is visible between normal bony trabeculae and in marrow fat ->Nodular growth pattern- distinctly uncommon ->Fine fibrosis may be evident ->Most lymphomas show polymorphic infiltrate -> Nuclei are not uniform in shape and size This lack of uniformity is helpful in distinguishing lymphoma from Ewings sarcoma of bone

29. MICROSCOPY UNUSUAL FEATURES- because of fibrosis cells may appear spindled Cells may have storiform pattern- lead mistaken diagnosis of malignant fibrous histiocytoma Cells may have clustering growth pattern- metastatic carcinoma

30. MICROSCOPY B-cell lymphomas , most commonly diffuse large B-cell lymphoma T-cell lymphomas of bone- Anaplastic large cell lymphoma Lymphoblastic lymphoma N on-Hodgkin B-cell lymphomas , including follicular lymphoma, marginal zone lymphoma , mantle cell lymphoma, and small lymphocytic lymphoma Hodgkin lymphoma- late stages M yeloid sarcoma (granulocytic sarcoma)

31. IMMUNOHISTOCHEMISTRY TUMOUR MARKERS B-cell lymphomas CD45, pan B-cell markers (CD19, CD20 , CD79a,PAX-5) Negative for CD3, CD5 Anaplastic large cell lymphoma CD30 ,CD3, CD4, Lymphoblastic lymphoma terminal deoxynucleotidyl transferase . CD10, CD43, CD99, FLI-1, CD45 - weakly positive or negative Hodgkin’s lymphoma CD30, PAX-5, CD15. Myeloid sarcoma (granulocytic sarcoma) Positive- myeloperoxidase, lysozyme, and CD43,CD45 Negative for CD20 and CD3 Most of lymphomas except lymphoblastic lymphoma are negative for CD99

32. A potential diagnostic pitfall… Lymphoblastic lymphoma may be positive for CD99 and negative for CD45 , an immunoprofile that overlaps with that of Ewing sarcoma TdT , CD34,CD43,CD10, CD79a and genetic rearrangement studies- distinguish lymphoblastic lymphoma from EWS/PNET

33. MOLECULAR GENETICS Specific studies on primary lymphomas of bone are lacking.

34. PLASMA CELL NEOPLASMS malignant proliferation of monoclonal plasma cells can present as a solitary lesions (solitary plasmacytoma ) or more commonly as part of widespread disease (multiple myeloma) AGE-50-70years rare below 40 years SITE-: vertebrae, ribs, skull, pelvis, femur , clavicle and scapula

35. RADIOGRAPHY: Multiple punched out lesions Purely lytic Rarely associated with sclerosis Purely lytic lesion; Well defined margins

36. GROSS Usually has a soft red-brown appearance . However, some myelomas show the fish-flesh appearance typical of malignant lymphoma .

37. MICROSCOPY Sheets of plasma cells- abundant cytoplasm eccentric nucleus nucleus round and clumped chromatin Rarely, cytoplasm contains crystalline inclusions Binuleated cells are commonly found Plasma cells – cytologic atypia Occasionally, nuclei are so pleomorphic that plasma cell nature of cells is obscured- DD involves malignant lymphoma Highly vascular 10-15% cases- amyloid deposits- walls of vessels or between tumor cells

38. MICROSCOPY WELL DIFFERNTIATED MYELOMA . POORLY DIFFERNTIATED MYELOMA

39. HISTOLOGICAL DIFFERENTIAL DIAGNOSIS INCLUDES: 1.Lymphoma 2.malignant carcinoma 3.occasionally chronic osteomyelitis Immunohistochemitry plays an important role here

40. IMMUNOHISTOCHEMISTRY Myeloma cells positive- CD38 CD138 Multiple myeloma oncogene 1 ( MUM-1) Characteristically express monotypic cytoplasmic Ig and lacks surface Ig The monotypic expression of kappa or lambda immunoglobulin by the tumour cells establishes the diagnosis of malignancy

41. IMMUNOHISTOCHEMISTRY Myeloma cells negative  CD19,CD20,CD27,CD22 Normal plasma cells  CD 27+ , CD 19 + Myeloma cells frequently express the natural killer antigen CD56/58 which is not expressed in reactive plasma cells

42. Differential diagnosis… Myeloma cells  weakly positive or negative for CD45,CD20 most B-cell lymphomas  strong staining for both markers . Both myeloma and carcinoma are positive for EMA Keratin markers are more reliable in ruling out carcinoma Myeloma cells  positive for CD138 Multiple myeloma oncogene 1 (MUM-1 ) Some carcinomas occasionally express CD138 MUM-1 is helpful in making distinction

43. MESENCHYMAL CHONDROSARCOMA Aggressive cartilaginous neoplasm AGE-20-60 year BONES COMMONLY AFFECTED- jaw bones ribs vertebrae pelvis USUAL LOCATION WITHIN LONG BONE- diaphysis (cortex or medulla)

44. MICROSCOPY: Dimorphic pattern-areas of well differentiated cartilage alternate with undifferntiated stroma . Boundaries between two components is abrupt Undifferentiated element is composed of small cells and can be confused with ewings sarcoma/PNET, malignant lymphoma and hemangiopericytoma Hemangiopericytoid vasculature- another salient feature

45. MICROSCOPY Hemangiopericytomatous pattern malignant cartilaginous nodule ; hypercellular region of small cells

46. IMMUNOHISTOCHEMISTRY Vimentin S-100 protein staining- limited to chondroblastic islands. lacking in small cell component CD57 CD99- limited to small cell component Nuclear immunoreactivity – Sox9 and osteocalcin Focal reactivity for actin,desmin,myogenin,NSE MOLECULAR GENETICS HEY1 – NCOA2 gene fusion complex cytogenetic alterations, including identical Robertsonian translocation t(13;21 )( q10;q10)

47. Differntial diagnosis Hematopoietic stains will rule out lymphoma Both mesenchymal chondrosarcoma and Ewings sarcoma/PNET share immunoreactivity for CD99 FLI-1 - positive in 75% cases of Ewings sarcoma Sox9- sensitive and specific marker for mesenchymal chondrosarcoma

48. SMALL CELL OSTEOSARCOMA Osteosarcoma is the most common nonhematopoietic primary malignant bone tumor . But small cell osteosarcoma ( histological variant) is an extremely uncommon tumor with a poor prognosis AGE- 10-25 years ; rare in preschool children another peak age incidence- after 40 SEX- M:F=3:2 SITE- metaphyseal area of long bones lower end of femur upper end of tibia upper end of humerus

49. Small cell osteosarcoma is a rare histological variant MICROSCOPY- Small size and uniformity of tumour cells and diffuse pattern of growth is seen – simulating appearance of Ewing sarcoma/PNET and malignant lymphoma Some cases- these cells are spindle rather than round Focal production of osteoid – distinguishing feature Areas of cartilage production can be seen Difficult to distinguish small cell osteosarcoma from ewings sarcoma/PNET when osteoid is not present

50. IMMUNOHISTOCHEMISTRY- N o specific immunophenotype for osteosarcoma Immunohistochemically heterogenous Positive for vimentin , desmin , smooth muscle actin, cytokeratin epithelial membrane antigen, S-100 type I collagen, type II collagen, type IV collagen proteins associated with bone metabolism like- osteonectin,osteocalcin , osteopontin . CD 99

51. Differential diagnosis Hematopoietic immunostains will distinguish lymphoma from small cell osteosarcoma Small cell osteosarcoma can be immunoreactive with CD99 , so it is not a useful stain in ruling out Ewing sarcoma FLI-1 is a better marker most Ewing sarcomas are positive but small cell osteosarcomas are negative .

52. The stromal component of small cell osteosarcoma may resemble mesenchymal chondrosarcoma . both can show reactivity to CD99 - Careful attention paid to the type of matrix production— osteoid in osteosarcoma and cartilage in mesenchymal chondrosarcoma is the best way to make the distinction

53. POORLY DIFFERTIATED SMALL CELL SYNOVIAL SARCOMA Small cell variant of poorly differentiated synovial sarcoma is easily misdiagnosed for other small round cell tumours IMMUNOCHEMISTRY- positive for keratin 7,13 and 19 EMA, vimentin , CD99 MOLECULAR GENETICS- t(x;18)(p11.2;q11.2) fusion of SS18( chr 18) with SSX1 ( chr X)

54. RHABDOMYOSARCOMA Only two categories show small round cell picture on histology: 1.Embryonal rhabdomyosarcoma 2.Alveolar rhabdomyosarcoma

55. EMBRYONAL RHABDOMYOSARCOMA Arises from unsegmented and undifferientiated mesoderm SITE- head and neck region- orbit, nasopharynx , middle ear retroperitoneum bile ducts urogenital tract AGE- 3-12 years GROSS- poorly circumscribed, white, soft ALVEOLAR RHABDOMYOSARCOMA AGE - 10- 25 years SITE- Extremities- forearm,arms , perirectal, perineal regions Most common sites of metastatic involvement  bone marrow , lungs, soft tissues, lymph nodes

56. MICROSCOPY: Embryonal type Perivascular pseudorosette Small round cells