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Barbara DEWAELE, PhD Department of Human Genetics KU Leuven - UZ Leuven Leuven, Belgium

Identification of a novel , recurrent MBTD1-CXorf67 fusion in low grade endometrial stromal sarcoma. Barbara DEWAELE, PhD Department of Human Genetics KU Leuven - UZ Leuven Leuven, Belgium. November 16 th 2012 |CTOS 17th Annual Meeting | Hilton Prague in Prague, Czech Republic.

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Barbara DEWAELE, PhD Department of Human Genetics KU Leuven - UZ Leuven Leuven, Belgium

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  1. Identification of a novel, recurrentMBTD1-CXorf67fusion in low gradeendometrial stromal sarcoma Barbara DEWAELE, PhD Department of Human Genetics KU Leuven - UZ Leuven Leuven, Belgium November 16th 2012 |CTOS 17th Annual Meeting | Hilton Prague in Prague, CzechRepublic

  2. Endometrial stromal sarcomas (ESS) are rare mesenchymal tumors Non-pleomorphic Pleomorphic • Endometrialstromal tumors (EST) tissue architecture: • proliferativephaseendometrialstroma • EST represent a broad spectrum of tumors withvaryingdegrees of malignancy: • CurrentWHO classification: • BENIGN LOW-MALIGNANT MALIGNANT • ESN ESS UES ESS UES High-grade Low-grade JAZF1-SUZ12 JAZF1-PHF1 EPC1-PHF1 … YWHAE- FAM22 Complex karyotype Nuclearsize/irregularity in nuclear contour Mitoticactivity Tumor necrosis High mitoticactivity Tumor necrosis GoodprognosisIntermediateprognosis Poorprognosis AdaptedfromLee et al., Am. J. Surg. Pathol 2012: 36: 641-653.

  3. Aim: identification of novelfusions in ESS • Selection of two cases with t(X;17) byconventionalkaryotyping • Clinicallyandmorphologically: low grade ESS • Lackknownpathognomonic gene fusions of low grade ESS

  4. Clinicohistologicaland Immunohistochemical features of two t(X;17) ESS cases CD10 ER CD99

  5. Conventionalkaryotypingreveals the t(X;17) in two ESS cases dicentricchr. Case 2 Case 1 2 3 4 5 1 2 3 4 5 1 X X 6 7 8 9 10 11 12 6 7 8 9 10 11 12 13 14 15 16 17 18 13 14 15 16 17 18 19 20 21 22 19 20 21 22 45,X,t(X;17)(p11.2;q23),dic(4;22)(p15;q22) 46,X,t(X;17)(p11.2;q23)

  6. Conventionalkaryotypingreveals the t(X;17) in two ESS cases dicentricchr. Case 1 Case 2 1 2 3 4 5 1 2 3 4 5 X X 6 7 8 9 10 11 12 6 7 8 9 10 11 12 13 14 15 16 17 18 13 14 15 16 17 18 19 20 21 22 19 20 21 22 X derX 17 der17 X derX 17 der17

  7. Conventionalkaryotypingreveals the t(X;17) in two ESS cases andarray CGH indicatesthat the t(X;17) is balanced dicentricchr. Case 1 Case 2 2 3 4 5 1 2 3 4 5 X X 6 7 8 9 10 11 12 6 7 8 9 10 11 12 13 14 15 16 17 18 13 14 15 16 17 18 19 20 21 22 19 20 21 22 45,X,t(X;17)(p11.2;q23),dic(4;22)(p15;q22) 46,X,t(X;17)(p11.2;q23) Agilent 180K Case 1 Case 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

  8. Using RNA-seq, the MBTD1-CXorf67fusion was identified in the two ESS cases with the t(X;17) 17q21.33 MBTD1 (-) Chr. 17 • 2 ESS cases with t(X;17) • Paired-end RNA seq on Illumina platform • Case 1: 1,21 x 108 paired-end readsmappedto hg19 • Case 2: 6,29 x 107 • Analysis withArrayStudio and deFuse: • Case 1: 25 distinct and high-quality paired-end reads • Case 2: 2 link exon 16 of MBTD1 to exon 1 of CXorf67 q25.3 p13.3 Xp11.22 CXorf67 (+) Chr. X p22.33 q28 CXorf67 MBTD1 cDNA 1 16 1 Case 1 Reads 0 CXorf67 MBTD1 60 Case 2 0 16 1 3 5 7 13 17 1 84 kb 2,3 kb 40 Centromere Telomere

  9. Using RNA-seq, the MBTD1-CXorf67fusion was identified in the two ESS cases with the t(X;17) 17q21.33 MBTD1 (-) Chr. 17 • 2 ESS cases with t(X;17) • Paired-end RNA seq on Illumina platform • Case 1: 1,21 x 108 paired-end readsmappedto hg19 • Case 2: 6,29 x 107 • Analysis withArrayStudio and deFuse: • Case 1: 25 distinct and high-quality paired-end reads • Case 2: 2 link exon 16 of MBTD1 to exon 1 of CXorf67 q25.3 p13.3 Xp11.22 CXorf67 (+) Chr. X p22.33 q28 CXorf67 MBTD1 cDNA 1 16 1 Case 1 Reads CXorf67 MBTD1 60 Case 2 16 1 3 5 7 13 17 1 0 84 kb 2,3 kb 40 Centromere Telomere 0

  10. The MBTD1-CXorf67fusionupregulates the expression of the 3’ end of CXorf67 exon 1 control control control control MBTD1_exon 17 CXorf67 3’ end YWHAE-FAM22 YWHAE-FAM22 MBTD1_exon 15_16 CXorf67 5’ end JAZF1-SUZ12 JAZF1-SUZ12 MBTD1-CXorf67 MBTD1-CXorf67 Case 1 Case 1 MBTD1-CXorf67 MBTD1-CXorf67 Case 2 Case 2 Case 1 Reads CXorf67 MBTD1 60 Case 2 16 1 3 5 7 13 17 1 1 0 84 kb 2,3 kb 40 Centromere Telomere 0

  11. Using nested RT-PCR followedbySangersequencing, the MBTD1-CXorf67fusion was validated 141 245 351 456 1 FCS MBT1 MBT2 MBT3 MBT4 45 80 253 350 464 560 RNA Seq: control case 1 control case 2 200bp CXorf67 MBTD1 cDNA Case 2 503 MBTD1 exon 16 CXorf67 exon 1 Ser-rich ladder Case 1 K K S Q Q G N P A R L S P E Y H K 1 16 1 protein

  12. Expressionprofilingwithhierarchical clustering demonstrates clustering of JAZF1-SUZ12andMBTD1-CXorf67 low grade ESS 70 genes YWHAE-FAM22A JAZF1-SUZ12 JAZF1-SUZ12 YWHAE-FAM22A MBTD1-CXorf67 JAZF1-SUZ12 JAZF1-SUZ12 YWHAE-FAM22A MBTD1-CXorf67 ESS high ESS low UES

  13. One additional low grade ESS case positive for the MBTD1-CXorf67 fusion was identified using a specific FISH assay • Specific FISH assay todetect the t(X;17) translocation • 14 ESS (4 low grade ESS, 5 high grade ESS and 5 UES) • negative for JAZF1andYWHAErearrangements • paraffine tissues • identification of 1 additional case positive for MBTD1-CXorf67fusion

  14. Conclusions: • In thisstudy: • new gene fusion: MBTD1-CXorf67 • yet another cytogenetically distinct subgroup of ESS • based on clinico-histopathologic features and expression profiling: cluster together with • JAZF1-SUZ12 as low grade ESS • offers the opportunity to shed light on the functions of two poorly characterized genes • MBTD1 belongs to the Polycomb group (PcG) of proteins • CXorf67 : a yet uncharacterized protein • Future: • decipher downstream consequences of the fusion • evaluate the respective contributions of the MBTD1 and CXorf67 portions of the fusion • underlying oncogenic processes

  15. Acknowledgements: Department of Gynaecology: Frederic Amant InstitutBergonie, Bordeaux, France: Sabrina Croce The MSCMC and Institute of Oncology, Warsaw, Poland: Joanna Przybyl • Department of Human Genetics: Barbara Dewaele Maria Debiec-Rychter Jan Cools Peter Vandenberghe Vanessa Vanspauwen Quattrone Anna Julio Finalet Ferreiro Geerdens Ellen GianfeliciValentina KalenderZeynep • Departmentof Pathology: Raf Sciot Philippe Moerman • Department of Oncology: AgnieszkaWozniak

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