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Quantification of Somatic Chromosomal Rearrangements in Circulating Cell- Free DNA from Ovarian Cancers
- 1. ©2012 MFMER | slide-1 CENTER FOR INDIVIDUALIZED MEDICINE Quantification of Somatic Chromosomal Rearrangements in Circulating Cell- Free DNA from Ovarian Cancers. Mayo Clinic George Vasmatzis, Ph.D. Director: Biomarker Discovery
- 2. ©2012 MFMER | slide-2 Center for INDIVIDUALIZED MEDICINE Science. 2005 Oct 28;310(5748):644-8. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Tomlins SA1, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, Varambally S, Cao X, Tchinda J, Kuefer R, Lee C, Montie JE, Shah RB, Pienta KJ, Rubin MA, Chinnaiyan AM. Recurrent chromosomal rearrangements have not been well characterized in common carcinomas. We used a bioinformatics approach to discover candidate oncogenic chromosomal aberrations on the basis of outlier gene expression. Two ETS transcription factors, ERG and ETV1, were identified as outliers in prostate cancer. We identified recurrent gene fusions of the 5' untranslated region of TMPRSS2 to ERG or ETV1 in prostate cancer tissues with outlier expression. By using fluorescence in situ hybridization, we demonstrated that 23 of 29 prostate cancer samples harbor rearrangements in ERG or ETV1. Cell line experiments suggest that the androgen-responsive promoter elements of TMPRSS2 mediate the overexpression of ETS family members in prostate cancer. These results have implications in the development of carcinomas and the molecular diagnosis and treatment of prostate cancer. TMPRSS2ERG TMPRSS2 ERG Chromosome 21
- 3. ©2012 MFMER | slide-3 Center for INDIVIDUALIZED MEDICINE
- 4. ©2012 MFMER | slide-4 Center for INDIVIDUALIZED MEDICINE Rearrangements in Oncology: What scheme to use? • Exome Sequencing SNV/LOH/Indels (need ref) $1500 ++ • Whole Genome Sequencing Complete $5,000 +++++ • MPseq Rearrangements/Fusions/CNVs $700 + Cost Data • RNAseq Fusions/Expression $500 + • Arrays CNVs/LOH $500 +
- 5. ©2012 MFMER | slide-5 Center for INDIVIDUALIZED MEDICINE DB_T_MP WSZ = 3e+05 FOLDER LU20241 1 5 9 13 18 23 28 33 38 43 48 53 58 63 68 73 78 83 88 93 98 103 109 115 121 127 133 139 145 151 157 163 169 175 181 187 193 199 205 211 217 223 229 235 241 247 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y UHMK1DDR2 GUK1OBSCN RYR2RYR2RYR2RYR2OBSCN RYR2RYR2RYR2RYR2 ALK EIF2AK2MAP4K3PKDCCEIF2AK2 PRKCEMAP4K3EIF2AK2ALKALK PRKCEPRKCEPRKCEEIF2AK2 TJP2TJP2 NTRK2NTRK2 ABL1CACNA1B SRCPIGU NCOA3SRCSRC PTPN1ZNF217ZMYND8ZMYND8ZNF217 ALK TGFA RYR2 AAK1 RYR2 EPHA4 ROR2ROR2 CACNA1BCACNA1B PIGU EIF2AK2EIF2AK2 ESR1 ROR2 C5 PIGU PTPN1 RXRAABL1
- 6. ©2012 MFMER | slide-6 Center for INDIVIDUALIZED MEDICINE
- 7. ©2012 MFMER | slide-7 Center for INDIVIDUALIZED MEDICINE Visualization
- 8. ©2012 MFMER | slide-8 Center for INDIVIDUALIZED MEDICINE SVAtoolsstructural variant analysis • NGS Quality Assessment • SV detection • SV evaluation • SV annotation / visualization readA position (bp) readBposition(bp) Chromosome 9 Potential chromosomal rearrangement
- 9. ©2012 MFMER | slide-9 Center for INDIVIDUALIZED MEDICINE Impact of masking vs. filtering average
- 10. ©2012 MFMER | slide-10 Center for INDIVIDUALIZED MEDICINE SVAtoolsstructural Copy number analysis
- 11. ©2012 MFMER | slide-11 Center for INDIVIDUALIZED MEDICINE Applications – Complex Events
- 12. ©2012 MFMER | slide-12 Center for INDIVIDUALIZED MEDICINE Applications – Complex Events
- 13. ©2012 MFMER | slide-13 Center for INDIVIDUALIZED MEDICINE Applications – Chromothripsis Every connection in even complex events are clear
- 14. ©2012 MFMER | slide-14 Center for INDIVIDUALIZED MEDICINE 15 Paired-End 500 bp fragments 15 Mate-Pair 2000 bp fragments bridged coverage 13x bridged coverage 5x Bridged coverage function of: fragment size, # of fragments large fragments more likely to span a given breakpointChr A Chr B
- 16. B3GALNT1 TP63 Fusion Junc1on 161Mb 189Mb • B3GALNT1/TP63 Fusion Gene • TP63 gene driven by B3GALNT1 Promoter Chromosome 3 P TP63 P B3GALNT1 Inversion involving TP63 and B3GALNT1 • Beta-1,3-Galactosyltransferase Gene Family • Catalyze transfer of sugar moie1es • Not implicated in Cancer B3GALNT1 Aubry et al Modern Pathology
- 17. Con B AD1 • Breakpoint confirmed by Sanger Sequencing PCR valida:on • Lung Tissue Valida1on: • Germ Line Evalua1on: PCR Primers: Chr3 Locus 2 Chr3 Locus 1 1. Map Reads to Hu Ref. Genome 2. PCR Primers: spanning breakpoint. 3. PCR / Agarose Gel 4. Excise DNA bands and Sanger seq. 5. Validate in pa1ent germ line DNA Con aN AD1 AD2 Control DNA Adjacent Normal Synchronous Lung Tumors TP63 B3GALNT1
- 18. ©2012 MFMER | slide-18 Center for INDIVIDUALIZED MEDICINE Scientific Advances as a Consequence of MPseq NGS for Test Development „ Constitutional genetics testing „ HemOnc (AML, etc) „ MPseq for solid tumors (lung, sarcoma, prostate, ovarian, etc) „ Junctions (PCR validation), Fusion genes, Rearrangements, CNVs, cnLOH „ Identification of targetable genomic changes in cancer. „ Monitoring for cancer recurrence „ Genomic relationship of independent primaries compared to primary-metastasis „ Genomic changes indicative of indolent and aggressive cancer „ Mutational screening on cytological biopsy specimens. „ Genomic characterization of Mouse Avatars „ Identification of viral integration sites
- 20. Figure 3. A. C. D. E. B. OC049
- 22. ©2012 MFMER | slide-22 050100150200250300350 0.0000.0100.0200.030 dx dy 010000200003000040000 1020 OC049 WSZ = 6e+05 gender= 0 COUNTSPERWINDOW 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 202122 23
- 23. ©2012 MFMER | slide-23 0 50 100 150 200 250 300 350 0.0000.0100.0200.030 dx dy 64 Ct+n (Ct+n = Cn + Ct ) Natural window count mode Mnat = 2*N + 2*T Deleted window count mode Mdel = 2*N + T tf = T/(T+N)=(Mnat - Mdel) / (Mnat/2) Mnat Mdel tf = T/(T+N) {T is the whole tumor and N is the Normal contamination} N T1 T2 T
- 25. | slide-25 Center for INDIVIDUALIZED MEDICINE Conclusions „ Many rearrangements can be observed in solid tumors with patient-specific junctions and can provide a personalized monitoring panel „ Personalized assays can be more sensitive that mutational panels „ Particular therapy can be monitored by choosing related junctions „ Particular clones can be monitored by choosing related junctions „ A major disadvantage is that we have to be able to develop patient specific assays on the fly „ There are several ongoing projects at Mayo „ Ovarian Cancer: Sensitive Detection of Relapse and Monitoring Therapy „ Sarcoma: Detection or Relapse „ Breast Cancer: Monitoring Therapy
- 26. ©2012 MFMER | slide-26 Center for INDIVIDUALIZED MEDICINE Molecular Biology Protocols Harris, Faye R. Kovtun, Irina V., Ph.D. Kosari, Farhad, Ph.D Murphy, Stephen J., Ph.D. Bruce Eckloff Halling Geoffrey C. Bioinformatics James Smadbeck Johnson, Sarah H. Drucker, Travis M. Zenka, Roman M. Gaitatzes, Athanasios (Saki) Vasmatzis, George Clinical Mariani, Andrea Block, Mathew Liu, Minetta Stephen Robinson Okuno, Scott