Differential Diagnosis of Paediatric Small Round and Blue Cell Tumors – An Update

Ivo Leuschner Kiel Paediatric Tumor Registry

Dept. of Paediatric Pathology

University Hospital Schleswig-Holstein

Campus Kiel UK SH

WHO - Classification of Soft Tissue Tumors (2013)

Tumor of the Paediatric Age Group Kiel Paediatric Tumor Registry

1995 (n) 1995 % 2011 (n)

Embryonal Tumors 4.060 15,2 8.676

Neuroblastoma 1.333 6,7 4.880

Nephroblastoma (1995 ‚with Variants‘) 1.282 6,4 3.337

Clear Cell Sarcoma of the Kidney 156

Malignant Rhabdoid Tumor of the Kidney 97

Hepatoblastoma 153 0,8 459

Sarcoma 1.755 14,0 8.482

Rhabdomyosarcoma 1.045 5,2 2.990

Ewing-Sarcoma-Family 830 4,2 1.233

Osteosarcoma 171 0,9

MPNST 177 0,9 513

Germ Cell Tumors 995 5,0 3.251

Vaskular Tumors 898 4,5 1.551

Langerhanszell Histiozytosis 529 2,7 1.114

Brain Tumors 453 2,3

Carcinoma 381 1,9 823

Mal. Lymphoma 2.667 13,4

others (mainly benigne Lesions) 8.206 43,8

total 19.944 44.638

Molecular Genetic Changes in Soft Tissue Tumors (1)

Tumor Entity chromosomal Aberration Molecular genetic change

myxoid/round cell Liposarcoma t(12;16)(q13;p11) t(12;22)(q13;q12)

FUS-DDIT3 (CHOP) EWSR1-DDIT3

inflammatory myofibroblastic Tumor (IMT) t(1;2)(q21;p23) t(2;2)(p23;q35) t(2,4)(p23;q21) t(2;11)(p23;p15)

TMP3-ALK RANBP2-ALK SEC31A-ALK CARS-ALK

infantile Fibrosarcoma t(12;15)(p13;q25) ETV6-NTRK3

'low-grade' fibromyxoid Sarcoma t(7;16)(q33-34;p11) t(11;16)(p11;p11)

FUS-BREB3l2 FUS-CREB3L1

Dermatofibrosarcoma protuberans Ring chromosome t(17;22)(q21;q13)

COL1A1-PDGFB

alveolar Rhabdomyosarcoma t(1;13)(p36;q14) t(2;13)(q36;q14) t(X;2)(q13;q36) t(2;2)(q35;p23) t(8;13;9)(p11.2;q14;9q32)

PAX7-FOXO1A (FKHR) PAX3-FOXO1A (FKHR) PAX3-FOXO4 (AFX) PAX3-NCOA1 FGFR1-FOXO1A (FKHR)

angiomatoid fibrous Histiocytoma t(2;22)(q33;q12) t(12;16)(q13;p11) t(12;22)(q13;q12)

EWSR1-CREB1 FUS-ATF1 EWSR1-ATF1

Synovial sarcoma t(X;18)(p11;q11) t(X;20)(p11;q13)

SS18-SSX1 (oder SSX2 or SSX4) SS18L1-SSX1

Clear cell sarcoma of Soft Tissues t(11;22)(q33;q12) t(12;22)(q13;q12)

EWSR1-CREB1 EWSR1-ATF1

Molecular Genetic Changes in Soft Tissue Tumors (2)

Tumor Entity chromosomal Aberration Molecular genetic chnage

DSRCT t(11;22)(q13;q12) EWSR1-WT1

extrask. myxoid Chondrosarcoma t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;15)(q22;q21) t(3;9)(q12;q23)

EWRS1-NR4A3 TAF15-NR4A3 TCF12-NR4A3 TGF-NR4A3

Alveolar Soft Part Sarcoma t(X;17)(p11;q25) ASPSCR1-TFE3

Ewing-Sarcoma-Family t(11;22)(q24;p12) t(21;22)q22.2;q24) t(7;22)(p22;q12) t(17;22)(q12;q12) t(2;22)(q33;q12) t(2;16)(q35;p11) t(16;21)(p11;q24)

EWSR1-FLI1 EWSR1-ERG EWSR1-ETV1 EWSR1-ETV4(E1AF) EWSR1-FEV FUS-FEV FUS-ERG

aggressive Fibromatosis of Desmoid type Mutation CTNNB1-Gen (beta-catenin)

Lipoblastoma t(8;8)(q12;q24.1) tT(8;7)(q12;q22) t(8;14)(q12;q24) t(8;2)(q12;q31) T(8;8)(q12;q12.1)

PLAG1-HAS2 PLAG1-COLIA2 PLAG1-RAD5ILI PLAG1-COL3AI PLAG1-RAB2A

Myxoma, intramusculär Mutation GNAS1-Gen

Ewing-Tumor-Family

Typical ‚small, round and blue cell‘ Tumor

Ewing-Sarcoma

Front page Time Magazine 1931

James Ewing, 1866 – 1943 Pathologist, Cornell University, New York Co-Founder of Sloan Kettering Cancer Centre

PAS

Retikulin Fasern

CD99

Ewing-Tumor- Family

HE, x400 CD 99 (MIC2)

PAS Recticulin fibers

Immunohistochemistry: CD99 (MIC2): 90%+

Ewing-Sarcoma-Variants

• Typical Ewing‘s sarcoma

• Adamantinoma-like Ewing Family tumor (EFT)

• Typical primitive neurectodermal tumor

• Spindle cell sarcoma-like EFT

• Sclerosing EFT

• Large cell EFT (atypical Ewing‘s sarcoma)

Folpe et al., 2005

Adamantinoma-like Ewing Family tumor (EFT)

Folpe et al., 2005

Spindle cell sarcoma-like EFT

Arnold et al., 2014

Large cell EFT (atypical Ewing‘s sarcoma)

Llombart-Bosch et al., et al., 2014

Chromosomal rearrangement Fusion gene Frequency

t(11;22)(q24;q12) FLI1-EWSR1 85

t(21;22)(q22;q12) ERG-EWSR1 5 - 15

t(7;22)(p22;q12) ETV1-EWSR1 <1

t(17;22)(q21;q12) ETV4-EWSR1 <1

t(17;22)(q12;q12 E1AF-EWSR1 <1

t(2;22)(q33;q12 FEV-EWSR1 <1

t(20;22)(q13;q12) NFATC2-EWSR1 <1

t(6;22)(p21;q12) POU5FI-EWSR1 <1

t(4;22)(q31;q12 SMARCA5-EWSR1 <1

inv(22)(q22q22) ZSG-EWSR1 <1

t(1;22)(p36.1;q12) ZNF278-EWSR1 <1

t(2;22)(q31;q12) SP3-EWSR1 <1

inv(22) in t(1;22)(p36.1;q12) PATZ-EWSR1 <1

t(16;21)(p11;q22) ERG-FUS <1

t(2;16)(q35;p11) FEV-FUS <1

t(4;19)(q35;q13) CIC-DUX4 <1

Inv(X)(p11) BCOR-CCNB3 <1

Genetic changes in Ewing-Sarcoma and ‚Ewing-like‘-Sarcoma

EWSR1 break-apart probe

Chromosomal rearrangement Fusion gene Frequency

t(11;22)(q24;q12) FLI1-EWSR1 85

t(21;22)(q22;q12) ERG-EWSR1 5 - 15

t(7;22)(p22;q12) ETV1-EWSR1 <1

t(17;22)(q21;q12) ETV4-EWSR1 <1

t(17;22)(q12;q12 E1AF-EWSR1 <1

t(2;22)(q33;q12 FEV-EWSR1 <1

t(20;22)(q13;q12) NFATC2-EWSR1 <1

t(6;22)(p21;q12) POU5FI-EWSR1 <1

t(4;22)(q31;q12 SMARCA5-EWSR1 <1

inv(22)(q22q22) ZSG-EWSR1 <1

t(1;22)(p36.1;q12) ZNF278-EWSR1 <1

t(2;22)(q31;q12) SP3-EWSR1 <1

inv(22) in t(1;22)(p36.1;q12) PATZ-EWSR1 <1

t(16;21)(p11;q22) ERG-FUS <1

t(2;16)(q35;p11) FEV-FUS <1

t(4;19)(q35;q13) CIC-DUX4 <1

Inv(X)(p11) BCOR-CCNB3 <1

Genetic changes in Ewing-Sarcoma and ‚Ewing-like‘-Sarcoma

Small round blue cell tumor with CIC-DUX4-translocation

Antonescu, 2013

CD99

Small round blue cell tumor with BCOR-CCNB3-translocation

Pierrone et al., 2012

Pathologists need a ‚thick skin‘ …

International Classification of Rhabdomyosarcoma (1995)

Favorable Prognosis Botryoid embryonal Rhabdomyosarcoma Spindle cell type of embryonal Rhabdomyosarcoma Intermediate Prognosis ‚Classical‘ embryonal Rhabdomyosarcoma (incl. pleomorphic embryonal RMS) Unfavorable Prognosis Alveolar Rhabdomyosarcoma

Newton et al., 1995

WHO Classification of Tumors of Soft Tissue and Bone (2013)

Embryonal rhabdomyosarcoma (ICD-O 8910/3)

(incl. botryoid variant) Alveolar rhabdomyosarcoma (ICD-O 8920/3)

Pleomorphic rhabdomyosarcoma (ICD-O 8901/3)

Spindle cell/sclerosing rhabdomyosarcoma (ICD-0 8912/3)

Alveolar Rhabdomyosarcoma

– about 20-30% of all Rhabdomyosarcoma

– Incidence equally distributed in children and adolescents

– Main sites: Extremities, Trunk

– high rate of Lymph node metastases

Embryonal Rhabdomyosarcoma ‚classical‘ type

Myogenin Expression in Rhabdomyosarcoma

• Embryonal RMS: low to moderate (up to 50% of tumor cells)

• Alveolar RMS: high (usually > 75% of tumor cells)

Myogenin expression in an alveolar RMS

Alveolar RMS Embryonal RMS

AP2 EGFR

P-cadherin Fibrillin

Diagnosis of alveolar RMS

• Based on:

– Cytology of cells: (usually) monomorphous small round blue cell tumor

– Reticulin fiber content: (Mostly) no fibers in tumor cell nests

– Immunohistochemistry:

• Myogenin: more than 75% of cells

• AP2 and/or p-cadherin expression

Incidence of alveolar RMS

Variabel incidence between Treatment-Studies/Countries:

• Newton 1995 (IRS; USA): 32 %

• COG D9803 Study (USA): 41 %

(after re-review) 25-30 %

• RMS 79 (Italy) 25.9 %

• CWS-81, -86, -91, -96 (Germany): 14.9 %

Incidence of alveolar RMS

Variabel incidence between Treatment-Studies/Countries:

• Newton 1995 (IRS; USA): 32 %

• COG D9803 Study (USA): 41 %

(after re-review) 25-30 %

• RMS 79 (Italy) 25.9 %

• CWS-81, -86, -91, -96 (Germany): 14.9 %

Diagnosis with H&E only

Molecular genetics of alveolar Rhabdomyosarcoma

– t(2;13)(q35;p14) Translocation (ca. 75 %)

• FOXO1A-PAX3 (FOXO1A: previous FKHR gene)

– t(1;13)(p36;q14) Translocation (5 bis 22 %)

• FOXO1A-PAX7

– t(13;8)(q14;p12) Translocation (rare)

• FOXO1A-FGFR1

– t(2;2)(q35;p23) Translocation (rare)

• PAX3-NCOA1

– t(2;X)(q35;q13) Translocation (rare)

• PAX3-AFX

Krams, 2002; Sorensen, 2002; Williamson, 2010, Wachtel 2004, Barr 2002

Incidence of ‚Fusion-negative‘ alveolar RMS

• COG: IRS III and IV: 20 - 25 %

later Studies: 40 - 45 %

• CWS: 17 % (2011)

• Aktuell: ca. 5 – 6% (recent data)

Incidence of ‚Fusion-negative‘ alveolar RMS

• COG: IRS III and IV: 20 - 25 %

later Studies: 40 - 45 %

• CWS: 17 % (2011)

• Aktuell: ca. 5 – 6% (recent data)

1 case every 2 to 3 years in Germany!

Molecular genetics of alveolar Rhabdomyosarcoma

• Genetic testing is mandatory in all alveolar RMS

Williamson et al., 2010 Davicione et al., 2009

Spindle cell differentiation in alveolar RMS

– Rarely a focal spindle cell differentiation can occur in alveolar RMS

• Especially in recurrences

Myogenin expression

P-cadherin expression

… sometimes pathologists just disappear

'Small round blue'-Tumors with loss of INI1-Expression

Tumors with Loss of INI1 Expression

Tumor type Loss of INI1 Expression (%)

Malignant Rhabdoid tumor of Soft Tissue of the Kidney in the Brain (ATRT) in the Liver (‚small cell undifferentiated Hepatoblastoma‘)

98

Medulläry Renal Cell Carcinoma 100*

Epitheloid Sarcoma 90

Epitheloid MPNST 50

Myoepithelial Carcinoma 40 (Pediatric cases) 10 (Adult cases)

Extraskeletal myxoid Chondrosarcoma 17

from: Hollmann 2011 * Very small numbers studied yet

Typical morphology of a malignant Rhabdoid Tumour

Small cell differentiation in INI1-negative Tumours

• A small cell differentiation is within the spectrum of rhabdoid tumours

• A genetic change in the SMARCA4 gene is found in a small subset of tumors instead of INI1 loss resulting in a loss of BRG1 expression

• SMARCA4 gene changes are typically found in ‚small cell carcinoma of the ovary

From: Kreiger et al., 2009

SMARCB1-(INI1) Gene

• Protein is part of the chromatin-remodeling complex

• Activates the transcription of genes

Malignant Rhabdoid Tumor of Soft Tissues

• Mean Age: 52 months (in comparison: mean age 16 months in renal MRT)

• Sex distribution m:w = 1,5:1

• Sites:

• Almost all sites of the body

Reinhard et al., 2008

INI-Protein

INI1 stain in small round blue cell tumors in patients below 5 years of age

Spectrum of genetic changes in the SMARCB1/INI1 gene Genetic Testing: • FISH: Deletions in 22q • Sequencing: SMARCB1 for mutations • MLPA: SMARCB1 In addition peripheral blood cells: 20 to 30% of patients have genetic germ line changes in the SMARB1/INI1 gene

aus: Eaton et al., 2011

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