1. Soft Tissue Tumors Academy of Pathology and Laboratory Medicine of Puerto Rico April 2013 Bruce Horten, M.D. Medical Director Integrated Oncology, New York

2. Part I Sarcomas In My Consult Practice  MFH Evaluation IHC Molecular  FISH  RT∙PCR Part II  GIST IHC Molecular  Small Round Cell Tumors IHC Molecular Brain Tumor Evaluation

3. Explosion of Interest in Soft Tissue Tumors WHO Classification of Tumors of Soft Tissue and Bone 4 th Edition 2013 Editors include C. Fletcher, J. Bridge USCAP Annual Meeting, Baltimore 2013 * Long Course Soft Tissue Tumors C. Fletcher, C. Antonescu * Special Courses Practical Guide to Molecular Testing in Cancer J. Bridge Advanced Molecular Pathology F. Barr New York Pathological Society, Saturday May 4 President’s All Day Symposium: Soft Tissue Neoplasms C. Antonescu, C. Fletcher, M. Miettinen

4. Most Common Sarcoma Diagnoses Currently a very elusive statistic  Dependant on a site’s approach to malignant fibrous histiocytoma.  As a result, very few current texts offer such a statistic. Enzinger/Weiss or Rosai  In adults, spindle cell sarcomas dominate. 35-45% MFH and liposarcoma.  In childhood, round cell sarcomas dominate. Rhabdomyosarcoma, neuroblastoma, PNET / Ewing’s. Current WHO (2013) on sarcomas  75% include undifferentiated pleomorphic sarcoma, liposarcoma, leiomyo-, myxofibro-, synovial sarcoma and MPNST.  75% highly malignant  75% in extremities (esp. thigh)

5. Malignant Fibrous Histiocytoma (MFH) Now considered a diagnosis of exclusion. The most high grade, poorly differentiated myofibroblastic or fibroblastic sarcoma. To properly evaluate such a tumor, it is now considered essential to exclude (usually via immunohistochemistry) more specific entities. If all investigations come up negative, the new term of choice is: “Undifferentiated Pleomorphic Sarcoma (UPS)”

6. What Has Become of MFH? Fletcher Study (AJSP 1992;16:213-228)  61% Pleomorphic sarcoma with specific differentiation  13% Pseudosarcoma  26% Remain MFH though now termed UPS

7. What Are The Specific Subtypes (61%)? About 1/3 are myogenic including:  Leiomyo- and rhabdomyo- sarcoma  However the rhabdo subtype is very rare Another 1/3 are liposarcomas including:  Dedifferentiated (abdomen) and pleomorphic A final 1/3 includes:  Myxofibrosarcoma and  Malignant peripheral nerve sheath tumor

8. Do These Subtypes Really Matter? The myoid subtypes are very aggressive with a highly metastatic rate (70% to >90%). 5 yr. The liposarcoma subtypes are less aggressive with a 5 yr. metastatic rate of 15% (dedifferentiated) to 40% (pleomorphic).

9. Pseudosarcomas (13%) Sarcomatoid carcinoma. Most common. Melanoma Anaplastic lymphoma

10. How Are These MFH-like Entities Recognized? Principally By Immunohistochemistry MyoidLipoidMPNST Smooth Muscle ActinDedifferentiated: CD34, MDM2/CDK4 S∙100 SMMHCPleomorphic: Lipoblasts CD34 CaldesmonS∙100 Calponin Desmin Myogenin

11. But Also By Using Molecular Techniques Above all gene fusions  Translocations  Inversions  Deletions  Duplications Examples  DDIT3 (CHOP) FISH break-apart probe Myxoid liposarcoma  FOX01 (FKHR) FISH break-apart probe Alveolar rhabdomyosarcoma

12. How Are Pseudosarcomas Recognized? By Immunohistochemistry Sarcomatoid Carcinoma MelanomaAnaplastic Lymphoma CAM 5.2S∙100CD30 AE1/AE3HMB45ALK-1 CK903Melan A CK5/6MiTF1 p63

13. And What of the Remaining Undifferentiated Pleomorphic Sarcoma? By immunohistochemistry, focal smooth muscle actin may be seen but no desmin reactivity. The pattern for myofibroblasts. By molecular techniques, largely nonspecific. BUT by CGH (Comparative Genomic Hybridization) some shared factors with pleomorphic leiomyosarcoma.

14. Part II Gastrointestinal Stromal Tumors  Most common mesenchymal tumor of the gastrointestinal tract  At least ½ in the stomach of which ¼ of these gastric tumors are malignant

15. Gastrointestinal Stromal Tumors Cell of origin: Interstitial cells of Cajal Immunohistochemistry:  DOG1 – Most sensitive marker. Also ANO-1.  CD117 (KIT)  CD34  With exclusions S100 and SMA Prognostic Factors:  Tumor size: 2,5,10,>10 cm.  Mitotic activity: ≤ or > 5 per 50 hpf  Anatomic site: gastric vs. small intestinal

16. Mutations in GIST KIT 75% Exon 9 8% Exon 11 65% Exon 13 1% Exon 17 1% PDGFRA 10% Exon 12 2% Exon 14 Rare Exon 18 8% Includes D842V Wild Type 15%

17. Predictive Value of Mutations Exon 11 mutation KIT (65%)  Favorable survival and response to Imatinib (Gleevec) Exon 9 mutation KIT (8%)  Best response to Imatinib if dose is doubled to 800 mg. Exon 18 (D842V) PDGFRA (8%)  Resistent to Imatinib Wild type (15%)  Regular dose of Imatinib but response variable

18. Small Round Cell Tumors of Childhood Neuroblastoma Ewing’s sarcoma/primitive neuroectodermal tumor (ES/PNET) Rhabdomyosarcoma Desmoplastic small round cell tumor

19. IHC Analysis of Small Round Cell Tumors NERD AE1/AE3 e DDesmin RD Vimentin ERDCD45 Synaptophysin N eCD56 N e Chromogranin N e DS100 N CD99 EEMA D FLI1 EMyogenin R WT1(c) DNSE NE D

20. Neuroblastoma The most common of the small round cell tumors outside the CNS. Grading systems  At least 5 are proposed.  Differentiation is key. 5%: Neuropil with ganglion cells  Age: 2 yrs.  Mitotic rate: 10/10 hpf Other prognostic factors  IHC: CD45, S∙100 favorable CD44, BCL2, P-glyoprotein unfavorable  Flow: Aneuploid and 100% S, G 2, M phases favorable  FISH: NMYC Amplification in 25%. Unfavorable.

21. Ewing’s Sarcoma/ Primitive Neuroectodermal Tumor Older and broader age range than neuroblastoma. Also broader sites of origin from bone to soft tissues. 95% of cases feature the fusion of the EWS gene at 22q12 with another gene especially FLI-1 (80-90%).  Translocation: t(11;22)  Fusion gene: EWSR1-FLI-1  Protein: FLI-1 (nuclear) IHC CD99 and FLI-1 versus NSE in neuroblastoma

22. EWSR1 And Its Partners At Least 9 Different Partners In Ewing’s/PNET 3 in myoepithelial tumor of soft tissue 2 in clear cell sarcoma 1 in extraskeletal myxoid chondro sarcoma 1 in myxoid/round cell liposarcoma 1 in desmoplastic small round cell sarcoma

23. Rhabdomyosarcoma Embryonal versus Alveolar  Sites: Head/neck and urogenital vs. extremities  Cytology/Histology  Prognosis  IHC: Myogenin, desmin, sarcomeric actin

24. Molecular Analysis of Rhabdomyosarcoma Embryonal  No distinctive genetic features Alveolar  FOXO1 (FKHR)  Translocations 2;13 (PAX3-FOX) more aggressive than 1;13 (PAX 7-FOX)  NMYC in about 50% of alveolar subtype

25. Brain Tumor Evaluation Immunohistochemistry CAM 5.2EMACHROMOGRANIN VimentinPRGH GFAPS∙100Prolactin Neu∙NACTH Ki67LH TSH FSH FISH 1p/19q deletion