Molecular pathology/genetics of sarcomas Gunhild Mechtersheimer Institute of Pathology, University of Heidelberg Sarkomkonferenz: 17.03.2011 Berlin
Characterization of soft tissue sarcomas / STS (~ 1% of all malignancies in adulthood, ~ 55 subtypes) - the pathologist s tasks - Tumor classification Tumor grading Tumor staging Further parameters of prognostic relevance Pathogenetic principles Targets for molecular therapy => Stratification of prognosis and therapy Sarkomkonferenz: 17.03.2011 Berlin
Recurrent and tumor-subtype specific balanced chromosomal translocations in soft tissue sarcomas - First description in 1986 in myxoid liosarcomas - Sarkomkonferenz: 17.03.2011 Berlin
Genomic dichotomy of soft tissue sarcomas Simple karyotypes with specific translocations resulting in fusion genes, with amplifications, or with mutations. E.g.: - t(11;22)(q24;q12) - EWS/FLI-1 in Ewing s sarcomas - MDM2-amplification in well-differentiated liposarcomas and in dedifferentiated liposarcomas - KIT or PDGFR- mutations in GIST Complex, unbalanced karyotypes with numerous chromosomal aberrations E.g.: - undifferentiated pleomorphic high-grade sarcomas (MFH) - leiomyosaracomas -MPNST Sarkomkonferenz: 17.03.2011 Berlin
Soft tissue sarcomas with specific chromosomal translocations (approximately 20% of soft tissue sarcomas) Riggi et al.: J Pathol 213: 4-20, 2007 Sarkomkonferenz: 17.03.2011 Berlin
Chromosomal translocations and most important consequences CML t(9;22)(q34;q11) 9 22 der (9) der (22) Burkitt s lymphoma t(8;14)(q24;q32) 8 14 der (8) der (14) BCR BCR ABL ABL MYC IGH IGH MYC 5 5 3 3 5 5 1 2 3 3 3 C1 C2 C3 5 Fusion gene 3 5 3 2 C1 C2 C3 3 8,5 kb BCR/ABL mrna myc IgH P210 fusion protein Constitutively active tyrosine kinase Upregulated expression of structurally normal MYC protein
Recurrent translocations in STS (ca. 20%): Chimeric fusion genes FUS EWS FKHR SYT DDIT3 FLI1 ERG WT1 ATF1 CHN PAX3 PAX7 SSX1 SSX2 SSX4 Myxoid/round cell liposarcoma Ewing s sarcoma/pnet Desmoplastic small, round cell tumor Clear cell sarcoma of soft tissue Extraskeletal myxoid chondrosarcoma Alveolar rhabdomyosarcoma Synovial sarcoma Transkription factor gene Gene with RNA / DNA-binding domain Activated transcription / Deregulated control of proliferation Sarkomkonferenz: 17.03.2011 Berlin
Impact of translocations in soft tssue sarcomas Diagnosis Pathogenesis Prognosis? Therapy?
Differential diagnosis of spindele cell sarcomas Leiomyosarcoma / Desmin GIST / CD117 (KIT) SySa mf / CD56 (N-CAM) MPNST / CD56 (N-CAM)
Synovial sarcoma versus MPNST Translocation t(x;18)(p11;q11) in synovial sarcoma
Molecular detection of translocations Reverse transcriptase-pcr (RT-PCR) Detection of fusion transcripts Fluorescence in situ-hybridization (FISH) Fusion assay: Detection of the fusion of two chromosomal subregions using dual colour FISH Break apart assay: Detection of a chromosomal breakpoint using dual colour FISH Immunhistochemistry Detection of oncogenic fusion proteins
Synovial sarcoma versus MPNST -> Translocation t(x;18)(p11;q11) in syonvial sarcoma SS18(SYT)-SSX1/SSX2/SSX4 SSX1/SSX2/SSX4 fusion gene SS18-SSX1/SSX2/SSX4 SSX1/SSX2/SSX4 fusion transcript Chimeric SS18-SSX1/SSX2/SSX4 SSX1/SSX2/SSX4 protein Capillary electrophoretically mediated detection of a SS18(SYT)-SSX1 fusion transcript by RT-multiplex PCR in a case of monophasic synovial sarcoma (FFPE) SS18-SSX1 (147 bp) ß-Aktin (190 bp)
Differential diagnosis of myxoid sarcomas Myxoid liposarcoma: t(12;16)(q13;p11) -> FUS-DDIT3 Myxoid liposarcoma (LS) Myxofibrosarcoma (MFS) Dedifferentiated LS/MFS-like
Detection of a DDIT3(CHOP)-translocation in a myxoid liposarcoma using dual colour break apart-fish - Normal signal Break within the DDIT3-locus (12q13)
Immunhistochemical detection of oncogenic fusion proteins Inflammatory myofibroblastic tumor, e.g. t(1;2)(q25;p23) ALK Ewing s sarcoma / PNET t(11;22)(q24;q12) FLI1 Alveolar sarcoma of soft parts t(x;17)(p11.2;q25) TFE3 Synovial sarcoma t(x;18)(p11;q11) SYT Desmoplastic small, round cell tumor t(11;22)(q22;q12) WT1
Pathogenetic impact of translocations in sarcomas FUS-CHOP (DDIT3) fusion in myxoid liposarxomas (MLS) FUS-CHOP protein expression in MPC und MLS Tumor growth in SCID mice T1-2 T1-2 Human MRLS grams T1-1 MPC-FUS-CHOP FUS-CHOP expression -> initiating event in the pathogenesis of Mesenchymal progentitor cells (MPC) -> precursors of MLS
High-grade STS with complex karyotypes - Differential diagnoses - Myxofibrosarcoma Leiomyosarcoma Pleomorphic rhabdomyosarcoma Pleomorphic liposarcoma MPNST UPHGS/MFH AJSP 1992 Dedifferentaiated liposarcoma Melanoma Carcinoma Lymphoma
Karyotype of a leiomyosarcoma Nilbert et al.: Cancer Genet Cytogenet 1990
High-grade STS with complex karyotypes - Molecular characterization - Comparative genomic hybrodization (CGH) Array-CGH (cdna-arrays) Expression profiling (oligonucleotid-arrays) Proteomics UPHGS/MFH Pleomorphic LS
CGH-profile: Undifferentiated pleomorpgic high-grade sarcoma (MFH)
Chromosomal imbalances detected by CGH in 158 high grade STS - unsupervised hierarchical cluster analysis - MFH LMS MFH LMS MFH LMS MFH MFH MPNST LS-DD MFH LS-PL MPNST LS-PL LS-DD are genetically different from other subtypes of high-grade STS Subgroups of MFH are genetically similar to either LMS or LS-PL or MPNST => MFH are not a distinct entity + 12q13-15 + 5p13-15 + 6q22-q24-11q22-q24-10q23-q25
Chromosome 5p array-cgh Control-DNA Tumor-DNA Contig comprising 416 5p-BACs, 100 control-bacs (resolution approx. 50 kb) Matrix of genomic fragments (BAC- or PAC-clonees = Gain in tumor genome = Loss in tumor genome = Balanced status Adamovics et al., Genes Chromosom Cancer 2006
Chromosome 5p-DNA array: Analysed sarcomas Chromsomal status of 5p Balanced (n = 18) Low level gain (n = 9) Distinct amplification (n = 7) Sarcoma subtype MFH (n = 4) MPNST (n = 7) LS-PL (n = 4) LS-DD (n = 3) MFH (n = 1) MPNST (n = 1) LS-PL (n = 6) LS-DD (n = 1) MFH (n = 1) MPNST (n = 2) LS-PL (n = 2) LS-DD (n = 2) Adamowicz et al.: Genes Chrosmosomes Cancer, 2006
Distinct chromosome 5p-amplifications in STS (7/34) and candidate genes => 4 highly amplified consensus regions 5,0 4,0 3,0 2,0 1,0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 5.0 4.0 3.0 2.0 1.0 Candidate genes involved in proliferation or differenetiation: (1): NKD2, htert, SLC6A3, IRX2; (2): POLS (3): TRIO (4): SKP2, FYB, FGF10, PTGER4, FBOX32 Strict correlation between gene copy number and mrna-expression (critical target genes) ->TRIO, NKD2 und IRX2 5pter 5pcen (1) (2) (3) (4) Adamovics et al., Genes Chromosom Cancer 2006
Expression Profiling Unsupervised clustering Marcus Renner, Heidelberg (KO.SAR)
Morphological liposarcoma subtypes Well-differentiated Myxoid Round cell Dedifferentuated Dedifferentiated Pleomorphic
Chromosomal imbalances detected by CGH in 73/79 liposarcomas - unsupervised hierarchical cluster analysis (Eisen et al., 1998) - WD-LS DD-LS MR-LS PL-LS + 5p13-p15 + 1q21-q24 + 12q13-q15-13q14-q21
Dedifferentiated liposarcomas are genetically different from pleomorphic and myxoid/round cell liposarcomas Cluster 1: + 12q13-q15* Cluster 2: + 1p12-p21*. + 1q21-q24*, + 5p13-p15*, + 17p11.2-p12*, - 2q33-q36* *p < 0.005 Cluster 1 Cluster 2 Liposarcomas of cluster 2 show significant associations with higher numbers of chromosomal gains and losses high grade malignancy (G2/G3 vs. G1) tumor site (lower extremity vs. retroperitoneum)
Array-CGH of a dedifferentiated liposarcoma 6q Oncogene-Array (188 BAC/PAC Clones) 12q Chrom. 12q Array (112 BAC Clones) 3,75 3,5 3,25 3 2,75 2,5 2,25 2 1,75 1,5 1,25 1 0,75 0,5 0,25 0-0,25-0,5 MDM2 MDM2 Distinct amplifications: Cyclin D2 (12q13), MDM2 (12q14.3-q15), CDK4 (12q14), GLI (12q13.2-q13.3), DLK (12q13), MYB (6q22), Cyclin D1 (11q13) Fritz et al.: Cancer Res, 2002
Dedifferentiated (DDLS) and pleomorphic LS (PLLS) - Differentially expressed genes - PLLS DDLS CDK4 (12q14) MDM2 (12q14.3-q15) Marcus Renner, Heidelberg (KO.SAR)
MDM2 as a target for the MDM2 antagonist nutlin-3a in dedifferentiated liposarcoma (LS) cells with MDM2 overexpression Nutlin-3a (5 mol/l) induces considerable inhibition of proliferation, apoptosis, and G2 cell cycle arrest in dedifferentiated LS cells compared with normal adipocytes. Nutlin-3a induces activation of the p53 pathway in dedifferentiated LS cells. Proliferation following Nutlin-3a treatment Proliferation (% control) Increased p53, p21 and MDM2 protein expression following nutlin-3a treatment % Apoptosis / Annexin Aopotosis following Nutlin3a treatment NADIP DDLS LS141 Singer et al.: Cancer Res 2007
Molecular pathology/genetics of soft tissue sarcomas - perspectives - Definition of biological tumor entities Identification of pathogenetically and biologically relevant genes Detection of molecular targets Basis for new therapeutic strategies
Dedifferentiated liposarcomas: Genetically separate subgroups Subgroup 1: + 12q13-q15 Subgroup 2: + 12q13-q15, + 1q21-q24* Subgroup 3: + 12q13-q15, + 6q22-q24*, + 12q24*, + 20q13*, - 11q22-q23*, - 13q14-q21* *p < 0.005 Subgroup 1 Subgroup 2 Subgroup 3 SG 2 vs. SG 1: Trend to more chromosomal gains (p = 0.0508) SG 3 vs. SG 1: Significantly more chromosomal gains (p = 0.0198) and chromosomal losses (p = 0.0025) No correlation with tumor s grade, stage (primary vs. recurrent) or site
Potential therapeutic target: MAP3K5 (6q22.33) CGH: Frequent 6q22-q24 amplifications in high-grade liposarcomas (PLLS, DDLS) and MFHs Array-CGH: Chromosome 6q22.33 MAP3K5 Mitogen-activated proteinkinase kinase kinase 5 (ASK1, MEKK5) Thioredoxin TNFR1 MAP3K5 MAP2K 4/7 + JUNK - PPAR + Loss of adipogenic differentiation Chibon et al.: Genes Chromosomes Cancer 2004
Differentiation of the MFH cell line TSL Lipid accumulation under Thioredoxin und N-Acetyl-Cystein (NAC) Untreated NAC Thioredoxin Troglitazon (PPAR- L) Chibon et al.: Genes Chromosomes Cancer 2004
Molecular pathology/genetics of soft tissue sarcomas - perspectives - Definition of biological tumor entities Identification of pathogenetically and biologically relevant genes Detection of molecular targets Basis for new therapeutic strategies