Small Round Cell Tumors in Soft Tissue and Bone

Home , Fibrosarcoma, Histiocytoma, Mesoblastic nephroma, SATB2, Solitary fibrous tumor, Synovial sarcoma

4/13/2016

Ultrastructural Pathology Society, Seattle, March, 2016

Small Round Cell Tumors ACCME/Disclosure in

Soft Tissue and Bone Dr. Fisher has nothing to disclose

Cyril Fisher MA MD DSc FRCPath Professor of Tumor Pathology Royal Marsden Hospital and Institute of Cancer Research University of London UK

Adult Small Round Cell Tumors Ewing Sarcoma

• Ewing sarcoma • Any age • Ewing sarcoma-like tumors • mostly young adults • Desmoplastic small round cell tumor • Bone or soft tissue • Mesenchymal chondrosarcoma • Thorax, paravertebral • Small cell osteosarcoma • Abdomen • Poorly differentiated synovial sarcoma • Lung/chest wall • Small cell MPNST • Skin • Myoepithelial tumor • Aggressive • Clear cell sarcoma like tumor of GI tract • NE carcinoma, lymphoma, melanoma • Endometrial stromal sarcoma • Seminoma

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Ewing Sarcoma Ewing Sarcoma Ewing Sarcoma

• Small round cell • Atypical • CD99 + • large cell • CK ± • Adamantinoma-like • S100 pr ± • Clear cell • CD56 ± • Sclerosing CD99 CK • FLI1 ± • ERG ± • NKX2.2 +

Folpe 2005; Llombart-Bosch 2009

NKX2.2 Ewing Sarcoma: Ultrastructure

NK2 homeobox 2, at 20p11.22 (target of EWSR1-FLI1) • Ewing sarcoma >90% • Olfactory neuroblastoma 80-100% • Small cell carcinoma 25-30% • Mesenchymal chondrosarcoma 33-75% • PD synovial sarcoma 13% • Desmoplastic SRCT 0% • Merkel cell carcinoma 0%

*1 of CIC-DUX4 sarcoma, DSRCT, neuroblastoma, myoepithelial ca

Yoshida 2012;* Hung 2016

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Ewing Sarcoma Ewing Sarcoma and ES-like Tumors

• t(11;22)(q24;q12) EWSR1-FLI1 85% • Primitive cells • t(21;22)(q22;q12) EWSR1-ERG 10% • Scanty cytoplasm • t(2;22)(q35;q12) EWSR1-FEV • Few organelles • t(7;22)(p22;q12) EWSR1-ETV1 • Rare • t(17;22)(q12;q12) EWSR1-E1AF • inv(22)t(1;22)(q12;q12) EWSR1-ZSG • tonofilaments • t(2;22)(q31;q12) EWSR1-SP3 • junctions • t(20;22)(q13;q12) EWSR1-NFATC2 • neural • t(4;22)(q31;q12) EWSR1-SMARCA5 processes • t(16;21)(p11;q22) FUS-ERG • Glycogen • t(2;16)(q35;p11) FUS-FEV • t(4;19)(q35;q13) CIC-DUX4 • inv(X)(p11.4p11.22) BCOR-CCNB3 • t(X;19)(q13;q13) CIC-FOXO4

Ewing Sarcoma with FUS-ERG fusion S100 pr Ewing Sarcoma with FUS-ERG fusion SOX10 • F 27 • subcutaneous lesion thigh • circumscribed • 2 cm diameter • encapsulated • CD99 • ERG CD99 • S100 pr ERG • SOX10 • NKX.2.2

FUS-ERG

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‘Promiscuous’ Fusions Ewing Sarcoma and ES-like Tumors

• EWSR1-CREB1 angiomatoid FH, clear cell sarcoma, CCSLTGIT, PPMS • t(11;22)(q24;q12) EWSR1-FLI1 85% • EWSR1-ATF1 • t(21;22)(q22;q12) EWSR1-ERG 10% angiomatoid FH, clear cell sarcoma, CCSLTGIT • t(2;22)(q35;q12) EWSR1-FEV myoepithelial tumor, angiosarcoma, salivary HCCC, CCOC • ETV6-NTRK3 • t(7;22)(p22;q12) EWSR1-ETV1 infantile fibrosarcoma, inflammatory myofibroblastic tumor, • t(17;22)(q12;q12) EWSR1-E1AF mesoblastic nephroma, AML, secretory ca breast, mammary analogue secretory carcinoma of salivary glands, GIST • inv(22)t(1;22)(q12;q12) EWSR1-ZSG • ASPSCR1-TFE3 • t(2;22)(q31;q12) EWSR1-SP3 alveolar soft part sa, juvenile renal cell carcinoma • t(20;22)(q13;q12) EWSR1-NFATC2 • TMP3-ALK inflammatory myofibroblastic tumor, anaplastic large cell lymphoma • t(4;22)(q31;q12) EWSR1-SMARCA5 • YWHAE-NUTM22A/B • t(16;21)(p11;q22) FUS-ERG endometrial stromal sarcoma, clear cell sarcoma of kidney • t(2;16)(q35;p11) FUS-FEV • FUS-ERG Ewing sarcoma, AML • t(4;19)(q35;q13) CIC-DUX4 • BRD4-NUT • inv(X)(p11.4p11.22) BCOR-CCNB3 Ewing-like sarcoma, thymic & other carcinomas • t(X;19)(q13;q13) CIC-FOXO4

CIC-DUX4 Tumors with CIC-DUX4 fusion

• t(4;19)(q35;q13.1) • CD99+ • t(10;19)(q26.3;q13.1) (67% diffuse) • CK • Adult or pediatric • S100pr • Trunk, extremities • MUC4 • Mild pleomorphism, nucleoli • desmin • WT1 • Highly aggressive • Calretinin • FLI1± • ERG • c-MYC

Graham 2012; Italiano 2012; Choi 2013; Machado 2013; Specht 2014; Yoshida 2015 ; Smith 2015 Original images courtesy Dr EA Montgomery

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CIC-DUX4 vs Ewing sarcoma Tumors with BCOR-CCNB3 Fusion

CIC-DUX ES • t(X;X)(p11;p11) • Nuclear pleomorphism +/- - • M, 7-13 years, trunk, extremities • Nucleoli +/- - • Bone or soft tissue • Myxoid change + - • 5 year survival 75%, 10 year 56% • CD99 +/-focal uniform • Also BCOR-MAML3, BCOR-ZC3H7B • WT1, calretinin + + • NKX2.2 - +

Graham 2012; Italiano 2012; Choi 2013; Machado 2013; Smith 2014; Specht 2014; Yoshida 2015 Pierron 2012; Cohen-Gogo, 2014; Puls 2014; Peters 2015

BCOR-CCNB3 E/S Mesenchymal Chondrosarcoma

• CCNB3 • CD99+ (60%) CD99 • bcl-2 (90%)CCNB3 CD99 • CD117 (60%)

Original images courtesy Drs F Puls and V Sumathi

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Mesenchymal Chondrosarcoma Small Cell Osteosarcoma

Ultrastructure % • no external lamina • <1% of osteosarcoma • surface processes • Peak 3rd-4th decade • RER 37 • glycogen • Behavior like conventional 25 OS, but limited experience 12.5 Translocation

• t(8;8)(q21;q13.3) 12.5 • HEY1-NCOA2

12.5

Image courtesy of Dr R Tirabosco

Small Cell Osteosarcoma SATB2

Special AT-rich sequence binding protein 2 2q33 • Small cell osteosarcoma 100% Ultrastructure• CD99 - • Undifferentiated pleo. sarcoma (bone) 50% • •fibroblasticEWSR1 - • EWSR1-CREB3L1RER • Fibrosarcoma (bone) 45% • no external • FUSlamina - • Mes chondrosarcoma 23% • CIC-DUX - • glycogen • Ewing sarcoma 10% • •pre-osteoidSATB2+ • DLBC lymphoma 17%

Righi 2015; Davis 2015 Original images courtesy Dr R Tirabosco Debelenko 2011; Righi 2015, Davis 2015

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Synovial Sarcoma Synovial Sarcoma % 100

60 CK EMA 40

0 CK EMA bcl2 CD99 S100 CD34 Des SMA calret b-cat CD56

Synovial Sarcoma TLE1 % 100 • Transducin-like enhancer of Split • 9q21.32 80 • Groucho/TLE family – transcription inhibitors • Derived from profiling studies 60

• Santa Cruz Biotechnology M101 1:20 40 • Cell Marque Clone 1F5 1:200

0 CK EMA bcl2 CD99 S100 CD34 Des SMA calret b-cat CD56 TLE1

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TLE1 Synovial Sarcoma

• Synovial sarcoma 90-100% Monophasic SS • External lamina • Sometimes positive in • fragmented • MPNST • fibrosarcoma • Cell processes • solitary fibrous tumor MPNST • Junctions • mesothelioma

• sarcoma NOS Poorly Differentiated SS • Useful when negative

Fisher 1987 Kosemehmetoglu 2009; Jagdis 2009; Knosel 2009; Foo 2011

Synovial Sarcoma

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Malignant Peripheral Nerve Sheath Tumor Synovial Sarcoma

• SS18 (18q11.2) • SSX (Xp11.21-23) types 1, 2, 4 (of 5) • SS18/SSX fusion genes 60% SSX1 40% SSX2 <1% SSX4 • Fusion protein competes with wild type SS18 - loss of BAF47 > SOX2 activation > proliferation

Ladanyi 2002; Guillou 2004; Baird 2005; Storlazzi 2007; Amary 2007; Nakayama 2007; Kadoch 2013

MPNST: Small cell Malignant Peripheral Nerve Sheath Tumor % 100

SOX10S100 pr 80

0 S100 CD34 bcl2 GFAP PGP Des SMA CK EMA cat calret nestin TLE1 SOX10

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SOX10 Schwannoma MPNST • SRY-related HMG Box 10 (22q13.1) • Melanocytes, Schwann cells, myoepithelial cells, mast cells • BPNST • Schwannoma, neurofibroma, granular cell tumor Ultrastructure • MPNST 49% (SS 3%) • external lamina • more sensitive than S100 protein • interdigitating processes • pseudomesaxons • Clear cell sarcoma of soft tissue • Luse bodies • Myoepithelial tumors • Salivary gland tumors

Nonaka 2008; Ordonez 2013; Miettinen 2015

Malignant Peripheral Nerve Sheath Tumor Malignant Peripheral Nerve Sheath Tumor

Am J Surg Pathol. 2015 • 17p, 17q abnormalities • NF1 (17q11.2) inactivation

• sporadic and in NF1 rabbit monoclonal antibody, clone C36B11 (1:200 dilution; Cell Signaling Technology, Danvers, MA). • neurofibromin – Ras inactivation • 69% of all MPNST show complete or partial loss • TP53 (17p13) • 0% of epithelioid MPNST showed loss • mutations • CDKN2A (9p21) (p16INK4A) • mutations, deletion • EGFR (7p12) • Overexpression • Loss of H3K27me3 expression (1q42) (sporadic and postradiation) Carroll 2012; Prieto-Grenada 2015

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Malignant Myoepithelial Tumors Myoepithelial Tumors of ST/Bone

• M > F, 0-83 years (38) • Limbs, HN, trunk, lung • Subcutaneous > deep • Circumscribed • 42% myoepithelioma • 25% mixed tumors • 32% ca or malignant mixed tumors

Stout 1959; Kilpatrick 1997; Fernandez-Figueras 1998; Michal 1999; Kutzner 2001; Mentzel 2003; Hornick 2003; Flucke 2011; Agaram 2014; Puls 2014; Huang 2015

Myoepithelial Tumors of ST/Bone/Lung Myoepithelial Tumors of ST/Bone/Lung % 100 INI1EMA 90 • t(6;22)(p21;q12) EWSR1-POU5F1 (4/6 malignant) 80 • t(1;22)(q23;q12) EWSR1-PBX1 (3/7 mal) 70 • t(19:22(q13;q12) EWSR1-ZNF444 (2/2 mal) 60 • t(12;22)(q13;q12) EWSR1-ATF1 (0/1 mal) 50 40 • t(9;22)(q33;q12) EWSR1-PBX3 (0/3 mal) 30 • t(6;16)(p21;p11) FUS-POU5F1 (0/1 mal) 20 • t(1;22))(p34;q12) EWSR1-KLF17 (0/1 mal)* 10 0 • t(1;16)(p34;p11) FUS-KLF17 (1/5 mal) AE1/3 EMA S100 GFAP SMA Calpo Des p63 INI1 SOX10

Aggregated Data Brandal 2008; Brandal 2009; Antonescu 2010; Flucke 2012; Agaram 2014; Puls 2014; Huang 2015; Leduc 2016 *kidney 2015

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Myoepithelial Tumors of ST/Bone/Lung

• Morphology variable • IHC variable • EMA, S100 protein • Genetics variable • EWSR1, FUS • Many partners • Gold standard EM

CCS-like Tumor of GI Tract CCS-like Tumor of GI Tract

• About 40 cases • In bowel wall • M>F, median 41 years • Solid sheets • Ileum > jejunum > stomach • Discohesion • Metastasis to lymph node, liver • Mitoses, necrosis • Poor 2 year survival • O-L giant cells • Large size, necrosis are adverse • Aggressive

Alpers 1985; Donner 1998; Fukuda 2000; Pauwels 2002; Zambrano 2003; Friedrichs 2005; Venkataraman 2005; Huang 2006;; Antonescu 2006; Comin 2007; Joo 2009; Lagmay 2009; Terazawa 2009; Balkaransingh 2011; Shenjere 2012; Stockman 2012; Yang 2012; Thway 2014; Yegen 2015

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CCS-like Tumor of GI Tract CCS-like Tumor of GI Tract

GI tract Soft tissue • S100 pr 100% 100% • HMB45Uniform clear0 cells in nests 97% • Melan-A 0 72% • Sheets of relatively small cells • MiTF 0 80% • CKPseudoglandular/pseudopapillary0 10% • EMASpindled, plasmacytoid,0 36%rhabdoid • Osteoclast-like• (CD56,Clear SYN,secretory giantNF, NB84, vesiclescells SOX10) • Dense-core granules • Occasional gap junctions • No melanogenesis

Stockman 2012

CCS-like Tumor of GI Tract Tumors with EWSR1-ATF1 or EWSR1-CREB1 fusions

GI tract Soft tissue • Angiomatoid fibrous histiocytoma • S100 pr 100% 100% • Clear cell sarcoma of soft tissue • HMB45 0 97% • Uniform clear cells in nests • Clear cell sarcoma-like tumor of GIT • Melan-A 0 72% • Sheets of relatively small cells • MiTF 0 80% • Primary pulmonary myxoid sarcoma • CKPseudoglandular/pseudopapillary0 10% • Myoepithelial tumor of soft tissue • EMASpindled, plasmacytoid,0 36%rhabdoid • Angiosarcoma of salivary gland • Osteoclast-liket(2;22)(q33;q12)(CD56, SYN, giantNF, EWSR1-CREB1 NB84, cells SOX10) • Hyalinizing clear cell salivary carcinoma t(12;22)(q13;q12) EWSR1-ATF1 • Clear cell odontogenic carcinoma

Thway 2012; Gru 2013; Bilodeau 2013

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Endometrial Stromal Sarcoma Case 19 Seminoma

• Middle aged males • Midline in abdomen • CD10 • Primary or metastatic • ER • Lymphocytes/granulomas • PgR • SMA • Raised serum HCG • CK • H-caldesmon - Ultrastructure • Fibroblastic • RER, Golgi, few desmosomes, cilia JAZF1-SUZ12

Seminoma CK Small Round Cell Tumors

• Alveolar rhabdomyosarcoma desmin, myogenin • CD117 • Desmoplastic round cell tumor • OCT3/4 WT1, EMA, CK, desmin, NSE, NF, CD56 • SALL4 • Ewing sarcoma CD99, FLI1, ERG, CK, desmin • podoplanin CD117 OCT3/4 • CIC-DUX4 tumors • PLAP • CD99, ERG, MUC4, • CK • PD synovial sarcoma Ultrastructure• HCG TLE1, EMA, CK, CD99, CD56, bcl-2 • smooth nuclei • Mesenchymal chondrosarcoma • rope-like nucleoli,, CD99 • glycogen, few junctions • Small cell neuroendocrine carcinoma • annulate lamellae TTF1, CK, CD56, CG

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Small Round Cell Tumors Small Round Cell Tumors Ultrastructure • Alveolar rhabdomyosarcoma • Ewing sarcoma desmin, myogenin FOXO1 (+PCR) sparse organelles, glycogen, microtubules, tonofilaments • Desmoplastic round cell tumor • DSRCT WT1, EMA, CK, desmin, NSE, CD56 EWSR1 (+PCR) rare microtubules, DCG, glycogen, IF whorls • Ewing sarcoma • Poorly differentiated synovial sarcoma CD99, FLI1, ERG, CK, desmin EWSR1 (+PCR) processes, junctions, fragmented external lamina • CIC-DUX4 tumors • MPNST • CD99, ERG, MUC4, CIC-DUX4 processes, external lamina, Luse bodies • PD synovial sarcoma TLE1, EMA, CK, CD99, CD56, bcl-2 SS18 • Mesenchymal chondrosarcoma surface villi, scalloping, glycogen • Mesenchymal chondrosarcoma CD99 HEY-NCOA2 • E/s osteosarcoma • Small cell neuroendocrine carcinoma fibroblastic, RER, glycogen, pre-osteoid TTF1, CK, CD56, CG

Adjunctive Techniques in ST Pathology

1970 1980 1990 2000 2010

Personalisation Next Generation Sequencing

Prognosis Gene Expression Profiling Classification Diagnosis Molecular Diagnosis FISH, PCR, CGH THE END

Immunohistochemistry

Electron Microscopy

Light Microscopy