Recent Advances In Select Round Cell Sarcomas
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1 Recent Advances In Select Round Cell Sarcomas Rajiv M. Patel, M.D. Associate Professor of Pathology & Dermatology University of Michigan, Ann Arbor, MI 1
2 Translocation Associated Sarcomas Monomorphic histomorphology Simple karyotype Distinctive clinical features 2
3 SRCS in Younger Patients Significant subset are small round cell sarcomas ARMS Myxoid/round cell liposarcoma DSRCT PDSS Ewing family tumors (EFT) Small Round Blue Cell Tumor IHC AB SCCA MM ML Ewing PNET RMS PDSS DRCT PANK + +/ +/ Rare + + S / Rare +/ CD45 + TdT + Desmin +/ Rare + + CD99 +/ + +/ + Rare AB, antibody; SCCA, small cell carcinoma; MM, melanoma; ML, lymphoma; PNET, primitive neuroectodermal tumor; RMS, rhabdomyosarcoma; PDSS, poorly differentiated synovial sarcoma; DRCT, desmoplastic small round cell tumor CD99 3
4 Ewing Sarcoma/PNET Ewing Family Tumors (EFT) Rare highly malignant SRBCT of bone or ST Large rapidly growing mass Children & adolescents, but occurs at any age Extraskeletal ES/PNET most common in deep ST of extremities, but any site including superficial 10 year SR 60% with multimodality Tx ES 4
5 ES/PNET 5
6 PAS PAS-D CD99 CD99 6
7 CD99 EWS (22q12) Break apart probe t(11;22), t(21;22) EWS/PNET (FLI1/EWS, ERG/EWS) t(11;22), t(21;22) DSRCT (WT 1/EWS, ERG/EWS) t(12;22) Clear Cell Sarcoma (ATF1/EWS) t(9;22) ES Myxoid Chondrosarcoma (CHN/EWS) t(16;22) Myxoid/Round Cell Liposarcoma (CHOP/EWS) 7
8 Primary Cutaneous Ewing Sarcoma Pan-CK pck EMA 8
9 S100p 9
10 22q12 (EWS gene) - Normal 22q12 (EWS gene) Positive break-apart Difficult To Classify SRCSTS Group of poorly characterized small round cell soft tissue sarcoma (SRCSTS) Subset with t(4;19)(q35;q13.1) translocation (CIC DUX4) 10
11 Ewing & Ewing like family tumors translocations Gene fusions, chromosomal rkaryotype Anatomic location Morphology expression markers references Ewing sarcoma EWSR1-FLI1 t(11;22)(q24;q12) Bone or soft tissues Ewing sarcoma +++ FLI1 Delattre et or al. (1994) or cutaneous so-called 'atypical Ewing sarcoma' EWSR1-ERG t(21;22)(q22;q12) +++ ERG Sorensen et al. (1994) EWSR1-ETV1 t(7;22)(p22;q12) Bone or soft tissues +++ NSE, S100, DES, EMA Jeon al. (1995) EWSR1-ETV4 t(7;22)(p21;q12) Extraosseous +++ Kaneko et al. (1996) EWSR1-FEV t(2;22)(q35;q12) Extraosseous +++ Peter et al. (1997) Round cell sarcoma with EWSR1 -non ETS rearrangement EWSR1-NFATC2 t(20;22)(q13;q12) Bone So-called 'atypical +++ None Szuhai et al. (2009) Ewing sarcoma' EWSR1-SP3 t(2;22)(q31;q12) Bone or soft tissues So-called 'atypical +++ NSE Wang et al. (2007) Ewing sarcoma' EWSR1-PATZ1 inv(22) in t(1;22) Chest wall PNET +++ DES, keratins Mastrangelo et al. (2000) EWSR1-SMARCA5 t(4;22)(q31;q12) Lumbar spine So-called 'atypical +++ NSE, Sumegi et al. (2011) Ewing sarcoma' Synaptophysi Round cell sarcoma with non EWSR1-ETS rearrangement FUS-ERG t(16;21)(p11;q22) Chest wall Ewing sarcoma +++ ERG, NSE Shing et al. (2003) FUS-FEV t(2;16)(q35;p11) Bone (clavicle) Ewing sarcoma +++ None Ng et al. (2007) Round cell sarcoma with CIC-DUX4 rearrangement CIC-DUX4 t(4;19)(q35;q13) Soft tissues URCS or so-called Weak focal WT1 'atypical Ewing sarcoma' t(10;19)(q26;q13) ETV4 Kawamura-Saito et al. (2006) Round cell sarcoma with BCOR-CCNB3 rearrangement BCOR-CCNB3 inv(x)(p11) Bone URCS 50%+++ CCNB3 Pierron et al. (2012) Undifferentiated round cell sarcoma None (yet!) Non recurrent Variable URCS - Variable Fletcher (2008) Abbreviations: PNET, peripheral neuroectodermal tumor: URCS, undifferentiated round cell saroma, +++, strong membranous expression; -, no expression. Undifferentiated / unclassified sarcomas 2013 Undifferentiated spindle cell sarcoma Undifferentiated pleomorphic sarcoma Undifferentiated round cell sarcoma Undifferentiated epithelioid sarcoma Undifferentiated sarcoma NOS Undifferentiated / unclassified sarcomas Previously included in the fibrohistiocytic tumours, namely malignant fibrous histiocytomas Lack identifiable line of differentiationwhen characterized by presently available technology Dedifferentiated types of specific sarcomas are not included in this category Lack distinct clinical or morphological characteristics Genetic subgroups are emerging within this family stay tuned 11
12 Undifferentiated round cell and spindle cell sarcoma EWSR1 involved in non ETS fusions with: PATZ1, POU5F1, SMARCA5, NFATC2 or SP3 Another recurrent rearrangement involves CIC DUX4 fusion gene/protein upregulates genes of the PEA3 subclass of ETS family BCOR CCNB1 fusions One or more separate entities, or best classified as variants of Ewing sarcoma? Cancer Genetics and Cytogenetics 1996; 87(1):
13 CIC DUX4 oncogene N terminus CIC DUX4 C terminus CIC (loc: 19q13.2) SOX related HMG subfamily Intact HMG box DNA binding domain DUX4 (loc: 4q35.2) DUX family, 2 homeobox domains Loss of DNA binding homeodomains Transcription factor: Upregulate expression PEA3 family genes ETV1, ETV4, ETV5 (ERM) (may be translocation partners for EWSR1) Kawamura Saito M, Yamazaki Y, et al. Hum Mol Genet 2006 t(4;19) CIC DUX4 Reported Cases Article Pediatric Adult Richkind KE, Romansky SG, et al. Cancer Genet Cytogenet Somers GR, Shago M, et al. Pediatr Dev Pathol Kawamura Saito M, Yamazaki Y, et al. Hum Mol Genet Rakheja D, Goldman S, et al. Pediatr Dev Pathol Yoshimoto M, Graham C, et al. Cancer Genet Cytogenet Graham C, Chilton Macneill S, et al. Hum Pathol Italiano A, Sung YS, et al. Genes Chromosomes Cancer Total # cases:
14 Purpose Further characterize CIC DUX4 SRCSTS Clinicopathologic features Cytogenetic Molecular Immunohistochemical Database Search U of M & William Beaumont Hospital Small round cell soft tissue sarcomas Difficult to precisely classify Lacked molecular or cytogenetic evidence of known sarcoma associated translocations 20 candidates with FFPET and/or FFT CIC DUX4 fusion + Histologic, IHC & clinical features evaluated 14
15 Study Design RT PCR RNA isolated from FFPE & FFT Reverse transcriptase Nested PCR for CIC DUX4 transcript Amplicons identified by AGE + rxns Sanger sequenced Study Design RT PCR Assays to exclude other SRCSTS EWS FLI1 (exons 7/exon 6; exon 7/exon 9) EWS ERG (exon 7/ exon 6) EWS WT1 SYT SSX1 and SYT SSX2 GAPDH (house keeping gene) Interphase FISH Translocation Probes Fusion Probes Single or dual fusion (increased specificity) Break Apart Probes Multiple fusion partners with common breakpoint 15
16 FISH Enumeration Probes Peri Centromeric Repetitive (alphoid) sequences Locus Specific Specific gene loci * INTERPHASE FISH EWS break apart SYT break apart Study Design Negative EWSR1 Break apart FISH POSIDON PROBES CIC DUX4 positive case 16
17 Negative SYT Break apart FISH POSIDON PROBES CIC DUX4 positive case Study Design TWO INTERPHASE FISH STRATEGIES 1. CIC DUX4 fusion RP11 317E13 (FITC centromeric to CIC) RP11 521G19 (TRITC telomeric to DUX4) + t4;19 Fusion FISH CIC DUX4 fusion + = yellow fusion signal in majority of nuclei examined 17
18 CIC DUX4 Fusion FISH RP11 317E13 Chr 19 RP11 521G19 Chr 4 CIC DUX4 fusion + = yellow fusion signal in majority of nuclei examined Study Design TWO INTERPHASE FISH STRATEGIES 2. CIC rearrangement (#chrm 19 homologs) RP11 46I12 (FITC near chrm 19 centromere) RP11 569M1 (TRITC spans entire CIC) Chr. 19 Break apart FISH CIC rearrangement = 3 TRITC (red) and 2 FITC (green control ) signals 18
19 CIC Rearrangement FISH Chr 19 RP11 569M1 RP11 46I12 CIC rearrangement = 3 TRITC (red) and 2 FITC (green control ) signals RT PCR Study Design CIC DUX4, nested RT PCR Exclusion EWS, Synovial Sarcoma, DSRCT EWS FLI1 (exon 7/exon 6, exon 7/exon 9) EWS ERG (exon7/exon 6) EWS WT1 SYT SSX1 SYT SSX2 Conventional Cytogenetics Immunohistochemistry CD99 INI1 S100 Desmin Myogenin TLE1 Pan cytokeratin Study Design 19
20 Results: CIC DUX4 positive cases Case Cytogenetics RT PCR FISH CIC DUX4 EWS SYT DSRCT CIC DUX4 CIC EWS SYT 16 t(4;19) t(4;19) N/A N/A excluded of which 9 had EWS FLI1 and 7 negative by all assays Case 17 Pre treatment Karyotype 4 19 Case ,XX,add(2)(p16),t(3;7)(p24.2;p13),t(4;19)(q35;q13.1),+8,t(11;17)(q13;q25),add(16)(p13.1), +mar2[cp6] DUX4 CIC Normal t(4;19)(q35;q13.1) 20
![3q14), 14[cp17] RT PCR for CIC DUX4 Fusion EWS FLI1 CIC DUX4 EWS Fli1 control](/docs-images/87/96199913/images/21-3.jpg "plasmid courtesy of Dr. Elizabeth Lawlor CIC DUX4 control plasmid courtesy of Dr.")
21 Case ,X,t(X;1)(q11.2;p34),del(1)(p21p36),+del(1)(p22p36), t(3;20)(p21;q13.3),t(4;19)(q35;q13.1),+8,del(13)(q12.3q14), 14[cp17] RT PCR for CIC DUX4 Fusion EWS FLI1 CIC DUX4 EWS Fli1 control plasmid courtesy of Dr. Elizabeth Lawlor CIC DUX4 control plasmid courtesy of Dr. Takuro Nakamura Sanger Sequencing Fusion Variant 1 21
22 Results: CIC DUX4 positive cases Case Age Sex Primary tumor location Size (cm) F Pelvis (R. perineum/gluteal) F Extremity (R. calf) M Extremity (L. knee) F Extremity (L. shoulder) 6.0 Case 17 R 11 cm 0.8 cm Case 17 Right Calf, 20 cm 22
23 Case 21 23
24 Case 16 Case 17 Case 23 24
25 Case 17 Case 23 Lobular growth pattern Geographic necrosis Closely spaced round cells Slight nuclear pleomorphism Course chromatin Large nucleoli High mitotic rate Indistinct cytoplasm Focal cytoplasmic clearing Focal myxoid matrix Immunohistochemistry Case CD99 Pan CK TLE1 S100 Desmin Myogenin INI ** ** ** + ** * + ** + *focal ** very focal 25
26 CD99 (case 16) 26
27 27
28 Clinical Outcome Case Recurrence Metastasis Treatment Outcome Survival (mo) 16 Yes Lung s Chemotherapy Surgical resection Radiotherapy DOD No Lung s Brain m Chemotherapy Surgical resection DOD No Pelvis m Surgical resection Radiotherapy DOD No Lung s Chemotherapy Surgical resection Radiotherapy DOD 16.8 s, synchronous m, metachronous CIC DUX4 Sarcoma 19 Cases 8 Pediatric : 11 Adult Age 20 years (6 62 years). Sex 11 Females : 8 Males Size 8.4 cm ( cm) Location Soft tissue: 8 Extremity / 4 Trunk / 5Pelvis / 2 H&N Metastases 11 cases: 10 Lung / 2 Brain / 2 Pelvis / 1 Bone Histology Small round cell histology CD99 12 weak focal / 4 positive / 2 negative / 1 not done Outcome 8 DOD avg 12 (9 17) months 1 AWD 24 months 4 NED 6, 7, 7, 30 months 6 Lost or NA CIC DUX4 Sarcoma Karyotype Case 16 Case 17 Somers 2004 Kawamura Saito 2006 Kawamura Saito 2006 Rakheja 2008 Richkind ,X,t(X;1)(q11.2;p34),del(1)(p21p36),+del(1)(p22p36), t(3;20)(p21;q13.3),t(4;19)(q35;q13.1),+8,del(13)(q12.3q14), 14[cp17] 45 47,XX,add(2)(p16),t(3;7)(p24.2;p13),t(4;19)(q35;q13.1), +8,t(11;17)(q13;q25),add(16)(p13.1),+mar2[cp6] 47,XX,i(1)(q10),der(4)t(4;19)(q33 q35;q13.1),+8, t(15,17)(q24;p11.2 p12),der(19)t(19;20)(q13.1;p11.2), der(22)t(20;22)(q13;q13)[10] 53 55,XX,+2,t(4;19)(q35;q13),+6,+8,+13,+16,+17,del(19)(q13), , XY,t(4;19)(q35;q13),+6,+16 46,XY,t(4;19)(q35;q13.1)[11] 46,XY,t(4;19)(q35;q13.1)[16] 28
29 UNDIFFERENTIATED ROUND CELL SARCOMA WITH BCOR CCNB3 29
30 30
31 CCNB3 Spc CIC DUX ES 31
32 32
33 33
34 Conclusions CIC DUX4 SRCSTS is a novel recurrent translocationassociated sarcoma with a distinctive histopathology Studies have confirmed their aggressive clinical behavior and rapid development of resistance to conventional therapy BCOR CCNB3 (BCS) have histologic, IHC and gene expression profiles distinct from other round cell sarcomas such as ES and CIC DUX4 tumors Most BCS appear to be chemosensitive to ES based therapy protocols The OS of BCS seems more favorable than CIC rearranged sarcomas, but similar to ES and SS 34
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