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Small Round Cell Tumors in

Soft Tissue and Bone

Ultrastructural Pathology Society, Seattle, March, 2016

Cyril Fisher MA MD DSc FRCPathProfessor of Tumor Pathology

Royal Marsden Hospital and Institute of Cancer Research University of London

UK

ACCME/Disclosure

Dr. Fisher has nothing to disclose

Adult Small Round Cell Tumors

• Ewing sarcoma• Ewing sarcoma-like tumors• Desmoplastic small round cell tumor• Mesenchymal chondrosarcoma• Small cell osteosarcoma • Poorly differentiated synovial sarcoma• Small cell MPNST• Myoepithelial tumor • Clear cell sarcoma like tumor of GI tract• NE carcinoma, lymphoma, melanoma• Endometrial stromal sarcoma• Seminoma

Ewing Sarcoma

• Any age• mostly young adults

• Bone or soft tissue • Thorax, paravertebral• Abdomen• Lung/chest wall• Skin• Aggressive

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Ewing Sarcoma

• Small round cell• Atypical

• large cell• Adamantinoma-like• Clear cell• Sclerosing

Folpe 2005; Llombart-Bosch 2009

Ewing Sarcoma Ewing Sarcoma

CD99 CK

• CD99 +

• CK ±

• S100 pr ±

• CD56 ±

• FLI1 ±

• ERG ±

• NKX2.2 +

NKX2.2

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

Ewing Sarcoma: Ultrastructure

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Ewing Sarcoma

• Primitive cells• Scanty cytoplasm• Few organelles• Rare

• tonofilaments• junctions• neural

processes• Glycogen

Ewing Sarcoma and ES-like Tumors• t(11;22)(q24;q12)• t(21;22)(q22;q12)• t(2;22)(q35;q12)• t(7;22)(p22;q12)• t(17;22)(q12;q12) • inv(22)t(1;22)(q12;q12)• t(2;22)(q31;q12)• t(20;22)(q13;q12)• t(4;22)(q31;q12)• t(16;21)(p11;q22)• t(2;16)(q35;p11)• t(4;19)(q35;q13)• inv(X)(p11.4p11.22)• t(X;19)(q13;q13)

EWSR1-FLI1 85%EWSR1-ERG 10% EWSR1-FEV EWSR1-ETV1EWSR1-E1AF EWSR1-ZSGEWSR1-SP3EWSR1-NFATC2EWSR1-SMARCA5FUS-ERGFUS-FEVCIC-DUX4BCOR-CCNB3CIC-FOXO4

• F 27 • subcutaneous lesion thigh• circumscribed• 2 cm diameter• encapsulated

Ewing Sarcoma with FUS-ERG fusion S100 pr Ewing Sarcoma with FUS-ERG fusion SOX10

ERG

• CD99• ERG• S100 pr• SOX10• NKX.2.2

CD99

FUS-ERG

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‘Promiscuous’ Fusions• EWSR1-CREB1

angiomatoid FH, clear cell sarcoma, CCSLTGIT, PPMS• EWSR1-ATF1

angiomatoid FH, clear cell sarcoma, CCSLTGITmyoepithelial tumor, angiosarcoma, salivary HCCC, CCOC

• ETV6-NTRK3infantile fibrosarcoma, inflammatory myofibroblastic tumor, mesoblastic nephroma, AML, secretory ca breast,mammary analogue secretory carcinoma of salivary glands, GIST

• ASPSCR1-TFE3alveolar soft part sa, juvenile renal cell carcinoma

• TMP3-ALKinflammatory myofibroblastic tumor, anaplastic large cell lymphoma

• YWHAE-NUTM22A/Bendometrial stromal sarcoma, clear cell sarcoma of kidney

• FUS-ERGEwing sarcoma, AML

• BRD4-NUTEwing-like sarcoma, thymic & other carcinomas

Ewing Sarcoma and ES-like Tumors• t(11;22)(q24;q12)• t(21;22)(q22;q12)• t(2;22)(q35;q12)• t(7;22)(p22;q12)• t(17;22)(q12;q12) • inv(22)t(1;22)(q12;q12)• t(2;22)(q31;q12)• t(20;22)(q13;q12)• t(4;22)(q31;q12)• t(16;21)(p11;q22)• t(2;16)(q35;p11)• t(4;19)(q35;q13)• inv(X)(p11.4p11.22)• t(X;19)(q13;q13)

EWSR1-FLI1 85%EWSR1-ERG 10% EWSR1-FEV EWSR1-ETV1EWSR1-E1AF EWSR1-ZSGEWSR1-SP3EWSR1-NFATC2EWSR1-SMARCA5FUS-ERGFUS-FEVCIC-DUX4BCOR-CCNB3CIC-FOXO4

Tumors with CIC-DUX4 fusion

• t(4;19)(q35;q13.1)

• t(10;19)(q26.3;q13.1)

• Adult or pediatric

• Trunk, extremities

• Mild pleomorphism, nucleoli

• Highly aggressive

Graham 2012; Italiano 2012; Choi 2013; Machado 2013; Specht 2014; Yoshida 2015 ; Smith 2015

CIC-DUX4

• CD99+ (67% diffuse)

• CK• S100pr• MUC4• desmin• WT1• Calretinin• FLI1±• ERG• c-MYC

Original images courtesy Dr EA Montgomery

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CIC-DUX4 vs Ewing sarcoma

CIC-DUX ES

• Nuclear pleomorphism +/- -

• Nucleoli +/- -

• Myxoid change + -

• CD99 +/-focal uniform

• WT1, calretinin + +

• NKX2.2 - +

Graham 2012; Italiano 2012; Choi 2013; Machado 2013; Smith 2014; Specht 2014; Yoshida 2015

Tumors with BCOR-CCNB3 Fusion

• t(X;X)(p11;p11)

• M, 7-13 years, trunk, extremities

• Bone or soft tissue

• 5 year survival 75%, 10 year 56%

• Also BCOR-MAML3, BCOR-ZC3H7B

Pierron 2012; Cohen-Gogo, 2014; Puls 2014; Peters 2015

BCOR-CCNB3

CD99 CCNB3

Original images courtesy Drs F Puls and V Sumathi

• CCNB3

• CD99+ (60%)

• bcl-2 (90%)

• CD117 (60%)

CD99

E/S Mesenchymal Chondrosarcoma

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Mesenchymal Chondrosarcoma

Ultrastructure• no external lamina• surface processes• RER• glycogen

Translocation• t(8;8)(q21;q13.3)• HEY1-NCOA2

Small Cell Osteosarcoma

• <1% of osteosarcoma

• Peak 3rd-4th decade

• Behavior like conventionalOS, but limited experience 12.5

%

12.5

37

12.5

25

Image courtesy of Dr R Tirabosco

Small Cell Osteosarcoma

Original images courtesy Dr R Tirabosco

Ultrastructure• fibroblastic

• RER• no external

lamina• glycogen• pre-osteoid

Debelenko 2011; Righi 2015, Davis 2015

• CD99 -

• EWSR1 -EWSR1-CREB3L1

• FUS -

• CIC-DUX -

• SATB2+

SATB2

Special AT-rich sequence binding protein 2 2q33

• Small cell osteosarcoma 100%

• Undifferentiated pleo. sarcoma (bone) 50%

• Fibrosarcoma (bone) 45%

• Mes chondrosarcoma 23%

• Ewing sarcoma 10%

• DLBC lymphoma 17%

Righi 2015; Davis 2015

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Synovial Sarcoma

CK EMA

0

20

40

60

80

100

Synovial Sarcoma

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

%

0

20

40

60

80

100

Synovial Sarcoma

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

%TLE1

• Transducin-like enhancer of Split• 9q21.32• Groucho/TLE family – transcription inhibitors• Derived from profiling studies

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

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TLE1

• Synovial sarcoma 90-100% • Sometimes positive in

• MPNST• fibrosarcoma • solitary fibrous tumor • mesothelioma • sarcoma NOS

• Useful when negative

Kosemehmetoglu 2009; Jagdis 2009; Knosel 2009; Foo 2011

Monophasic SS

Poorly Differentiated SS

MPNST

Synovial Sarcoma

• External lamina

• fragmented

• Cell processes

• Junctions

Fisher 1987

Synovial Sarcoma

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Synovial Sarcoma

• SS18 (18q11.2)

• SSX (Xp11.21-23) types 1, 2, 4 (of 5)

• SS18/SSX fusion genes60% SSX140% 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

Malignant Peripheral Nerve Sheath Tumor

MPNST: Small cell

0

20

40

60

80

100

Malignant Peripheral Nerve Sheath Tumor

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

%

S100 prSOX10

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SOX10

• SRY-related HMG Box 10 (22q13.1)• Melanocytes, Schwann cells, myoepithelial cells, mast cells

• BPNST• Schwannoma, neurofibroma, granular cell tumor

• MPNST 49% (SS 3%)• more sensitive than S100 protein

• Clear cell sarcoma of soft tissue• Myoepithelial tumors • Salivary gland tumors

Nonaka 2008; Ordonez 2013; Miettinen 2015

Schwannoma MPNST

Ultrastructure• external lamina• interdigitating processes

• pseudomesaxons• Luse bodies

Malignant Peripheral Nerve Sheath Tumor

• 17p, 17q abnormalities• NF1 (17q11.2) inactivation

• sporadic and in NF1• neurofibromin

– Ras inactivation

• TP53 (17p13)

• mutations

• CDKN2A (9p21) (p16INK4A)

• mutations, deletion

• EGFR (7p12)

• Overexpression

• Loss of H3K27me3 expression (1q42)(sporadic and postradiation)

Carroll 2012; Prieto-Grenada 2015

Malignant Peripheral Nerve Sheath Tumor

rabbit monoclonal antibody, clone C36B11 (1:200 dilution; Cell Signaling Technology, Danvers, MA). 

• 69% of all MPNST show complete or partial loss• 0% of epithelioid MPNST showed loss

Am J Surg Pathol. 2015

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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

Malignant Myoepithelial Tumors

0

10

20

30

40

50

60

70

80

90

100

Myoepithelial Tumors of ST/Bone/Lung

AE1/3 EMA S100 GFAP SMA Calpo Des p63 INI1 SOX10

%

Aggregated Data

EMAINI1

Myoepithelial Tumors of ST/Bone/Lung

• t(6;22)(p21;q12) EWSR1-POU5F1 (4/6 malignant)

• t(1;22)(q23;q12) EWSR1-PBX1 (3/7 mal)

• t(19:22(q13;q12) EWSR1-ZNF444 (2/2 mal)

• t(12;22)(q13;q12) EWSR1-ATF1 (0/1 mal)

• t(9;22)(q33;q12) EWSR1-PBX3 (0/3 mal)

• t(6;16)(p21;p11) FUS-POU5F1 (0/1 mal)

• t(1;22))(p34;q12) EWSR1-KLF17 (0/1 mal)*

• t(1;16)(p34;p11) FUS-KLF17 (1/5 mal)

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

• About 40 cases• M>F, median 41 years• Ileum > jejunum > stomach• Metastasis to lymph node, liver• Poor 2 year survival• Large size, necrosis are adverse

Alpers 1985; Donner 1998; Fukuda 2000; Pauwels 2002; Zambrano 2003; Friedrichs 2005; Venkataraman2005; Huang 2006;; Antonescu 2006; Comin 2007; Joo 2009; Lagmay 2009; Terazawa 2009; Balkaransingh 2011; Shenjere 2012; Stockman 2012; Yang 2012; Thway 2014; Yegen 2015

CCS-like Tumor of GI Tract

• In bowel wall

• Solid sheets

• Discohesion

• Mitoses, necrosis

• O-L giant cells

• Aggressive

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

• Uniform clear cells in nests • Sheets of relatively small cells• Pseudoglandular/pseudopapillary• Spindled, plasmacytoid, rhabdoid• Osteoclast-like giant cells

GI tract Soft tissue• S100 pr 100% 100%• HMB45 0 97%• Melan-A 0 72%• MiTF 0 80%• CK 0 10%• EMA 0 36%

(CD56, SYN, NF, NB84, SOX10)

Stockman 2012

• Clear secretory vesicles

• Dense-core granules

• Occasional gap junctions

• No melanogenesis

CCS-like Tumor of GI Tract

• Uniform clear cells in nests • Sheets of relatively small cells• Pseudoglandular/pseudopapillary• Spindled, plasmacytoid, rhabdoid• Osteoclast-like giant cells

GI tract Soft tissue• S100 pr 100% 100%• HMB45 0 97%• Melan-A 0 72%• MiTF 0 80%• CK 0 10%• EMA 0 36%

(CD56, SYN, NF, NB84, SOX10)t(2;22)(q33;q12) EWSR1-CREB1t(12;22)(q13;q12) EWSR1-ATF1

Tumors withEWSR1-ATF1 or EWSR1-CREB1 fusions

• Angiomatoid fibrous histiocytoma• Clear cell sarcoma of soft tissue• Clear cell sarcoma-like tumor of GIT• Primary pulmonary myxoid sarcoma• Myoepithelial tumor of soft tissue• Angiosarcoma of salivary gland• Hyalinizing clear cell salivary carcinoma• Clear cell odontogenic carcinoma

Thway 2012; Gru 2013; Bilodeau 2013

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Case 19 Endometrial Stromal Sarcoma

• CD10• ER• PgR• SMA• CK• H-caldesmon -

JAZF1-SUZ12

Ultrastructure • Fibroblastic

• RER, Golgi, few desmosomes, cilia

Seminoma

• Middle aged males

• Midline in abdomen

• Primary or metastatic

• Lymphocytes/granulomas

• Raised serum HCG

CK

OCT3/4CD117

Seminoma

• CD117• OCT3/4• SALL4• podoplanin• PLAP• CK• HCGUltrastructure

• smooth nuclei• rope-like nucleoli,, • glycogen, few junctions• annulate lamellae

Small Round Cell Tumors

• Alveolar rhabdomyosarcoma desmin, myogenin

• Desmoplastic round cell tumorWT1, EMA, CK, desmin, NSE, NF, CD56

• Ewing sarcoma CD99, FLI1, ERG, CK, desmin

• CIC-DUX4 tumors• CD99, ERG, MUC4,• PD synovial sarcoma

TLE1, EMA, CK, CD99, CD56, bcl-2 • Mesenchymal chondrosarcoma

CD99• Small cell neuroendocrine carcinoma

TTF1, CK, CD56, CG

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Small Round Cell Tumors

• Alveolar rhabdomyosarcoma desmin, myogenin FOXO1 (+PCR)

• Desmoplastic round cell tumorWT1, EMA, CK, desmin, NSE, CD56 EWSR1 (+PCR)

• Ewing sarcoma CD99, FLI1, ERG, CK, desmin EWSR1 (+PCR)

• CIC-DUX4 tumors• CD99, ERG, MUC4, CIC-DUX4• PD synovial sarcoma

TLE1, EMA, CK, CD99, CD56, bcl-2 SS18• Mesenchymal chondrosarcoma

CD99 HEY-NCOA2• Small cell neuroendocrine carcinoma

TTF1, CK, CD56, CG

Small Round Cell TumorsUltrastructure

• Ewing sarcomasparse organelles, glycogen, microtubules, tonofilaments

• DSRCT rare microtubules, DCG, glycogen, IF whorls

• Poorly differentiated synovial sarcoma processes, junctions, fragmented external lamina

• MPNST processes, external lamina, Luse bodies

• Mesenchymal chondrosarcomasurface villi, scalloping, glycogen

• E/s osteosarcoma fibroblastic, RER, glycogen, pre-osteoid

Light Microscopy

Electron Microscopy

Immunohistochemistry

Molecular Diagnosis FISH, PCR, CGH

1970 1980 1990 2000 2010

Next Generation Sequencing

Adjunctive Techniques in ST Pathology

Gene Expression Profiling

Personalisation

Prognosis Classification

Diagnosis THE END