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Comparative genomic analysis of primary versus metastasis in colorectal carcinomas

Comparative genomic analysis of primary versus metastasis in colorectal carcinomas. Evi Vakiani, Manickam Janakiraman, Rileen Sinha , Ronglai Shen, Zhaong Zeng, Andrea Cercek, Nancy Kemeny, Adriana Heguy, Philip Paty, Timothy Chan, Len Saltz, Martin Weiser, David Solit.

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Comparative genomic analysis of primary versus metastasis in colorectal carcinomas

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  1. Comparative genomic analysis of primary versus metastasis in colorectal carcinomas Evi Vakiani, Manickam Janakiraman, Rileen Sinha, Ronglai Shen, Zhaong Zeng, Andrea Cercek, Nancy Kemeny, Adriana Heguy, Philip Paty, Timothy Chan, Len Saltz, Martin Weiser, David Solit Human Oncogenesis and Pathogenesis Program & Departments of Pathology, Medicine, Surgery and Biostatistics Memorial Sloan-Kettering Cancer Center, New York, NY

  2. Disclosure • The authors have no financial conflicts of interest to disclose relevant to this study.

  3. Metastatic colorectal carcinoma (CRC)A major cause of mortality O’Connell et al JNCI 2004; 96: 1420

  4. Majority of CRCs arise from adenomas Adenoma Primary invasive adenocarcinoma Metastasis Adapted from Fearon and Vogelstein Cell 1990; 61: 759

  5. Targeted therapy of metastatic CRCThe EGFR pathway KRAS mutant EGFR Ras P P Sos Grb2 P P Raf PIK3CA PTEN MEK AKT KRAS wild type ERK Survival Proliferation Invasion TP53 Modified from Amado R G et al. JCO 2008;26:1626-1634

  6. Aims • Define the incidence of clinically relevant biological events in primary and metastatic CRC • Assess the concordance of such events between primary carcinomas and metastasis • Examine reasons for any observed discordances

  7. Study Design & Methods • 736 frozen tumors from 613 patients • 39 adenomas, 406 primary carcinomas, 291 metastases • 84 pairs of matched primary tumors and metastases • 31 pairs of matched metastases • Match confirmed by a fingerprinting assay Study material Sequence analysis • KRAS (exons 2,3,4) • NRAS (exons 2,3) • BRAF (exon 15) • PIK3CA (exons 5, 7, 9, 20) • TP53 (all coding exons) • Sequenom MALDI-TOF mass spectrometry-based assay • Sanger

  8. Study Design & Methods • In matched pairs with discordant mutational profiles • Analysis of formalin-fixed paraffin embedded tissue (FFPE) • Deep sequencing (454) (n=4) • DNA copy number analysis of matched primary tumors and metastasis • N=25 pairs • Array CGH (Agilent, 1M)

  9. Clinical characteristics of study group

  10. Incidence of RAS/BRAF mutations BRAF mutations 6.5% NRAS Q61K/R/L, 12 BRAF K601E, 1 BRAF V600E/G, 39 NRAS G12D/13D, 6 RAS mutations outside exon 2 9.4% A146T/V, 25 K117N, 3 Q61H/R/L/E, 12 G13D, 33 G13C, 4 G12N, 1 RAS/BRAF wt, 48.4% G12V, 62 KRAS exon 2 mutations 35.7% N = 613 G12S, 13 G12R, 2 G12D, 75 G12A, 9 G12C, 18

  11. Incidence of PIK3CA mutations 11.7% p=0.001 % PIK3CA mut cases

  12. Incidence of TP53 mutations p=0.002 % BRAF mut cases

  13. Incidence of mutations in primary tumors vs metastasis p=0.01 p<0.001 p<0.001 Mutant (%) p=0.01

  14. BRAF mutations in metastatic CRC p=0.01 p<0.0001

  15. Concordance of mutational profile between matched primary tumor and metastasis

  16. Concordance of mutational profile between matched primary tumor and metastasis

  17. Concordance of mutational profile between matched metastatic foci

  18. case 20 case 42 P RAS/ BRAF M P PIK3CA M P TP53 M Concordance of mutational profile between matched primary tumor and metastasis

  19. KRAS G12 (wt) TP53 wt 20 P Primary CRC in right colon G Clinical history of a primary sigmoid CRC KRAS G12 (G35A) TP53 R248Q 20 L Liver metastasis G A KRAS G12 (wt) KRAS A146 (G436A) G 42 P Primary CRC G A Different tumor morphologies KRAS discordant cases KRAS G12 (G35A) KRAS A146 (wt) 42 L Liver metastasis G G A

  20. KRAS G12V TP53 wt Cecal CRC KRAS A146T TP53 R306* Rectal CRC Different KRAS mutational profiles in a case of multiple primary CRC KRAS A146T TP53 R306* Liver mets

  21. RAS/ BRAF P M PIK3CA P M TP53 P M Concordance of mutational profile between matched primary tumor and metastasis

  22. KRAS G12V (G35T) TP 53 wt TP 53 (454) Primary CRC 2.2% R175H KRAS G12V (G35T) TP53 R175H (G525A) Liver metastasis Detection of a low frequency mutant allele in TP53 discordant cases 79.5% R175H

  23. RAS/ BRAF P M PIK3CA P M TP53 P M Concordance of mutational profile between matched primary tumor and metastasis

  24. Hierarchical clustering DNA copy number analysis of matched pairs of primary CRC and metastasis (n=25)

  25. Summary • KRAS/BRAF/PIK3CA mutations show a very high rate of concordance (>97%) between primary tumor and metastasis as well as between different metastatic foci • TP53 mutations show a high rate of concordance (>95%) between primary tumor and metastasis as well as between different metastatic foci • Reasons for observed discordances include cases of multiple primaries (KRAS), treatment effect (TP53) and presence of low level mutant alleles within primary tumor (TP53)

  26. Conclusions • Genotyping for KRAS/BRAF/PIK3CA mutations can be performed using primary tumor DNA. In cases of multiple primaries, metastasis should be tested (in the absence of DNA from metastasis consider testing all primaries) • Genotyping for TP53 mutations is more accurate using DNA from metastatic foci.

  27. Len Saltz Nancy Kemeny Diane Reidy Andrea Cercek Phil Paty Marty Weiser Zhaoshi Zeng Mike D’Angelica Yuman Fong Ronald Dematteo Peter Allen David Solit Manickam Janakiraman Aphrothiti Hanrahan Feng Xing Yogi Persaud Moriah Nissan Gopa Iyer Neal Rosen Charles Sawyers Tim Chan Adriana Heguy Kety Huberman Igor Dolgalev Sabrena Thomas Agnes Viale David Klimstra Jinru Shia Laura Tang Nora Katabi Ronglai Shen Rileen Sinha Nick Socci Cora Mariano Priscilla McNeil Katrina Allen Anas Idelbi

  28. Avg % aberration 29.8% 39.1% DNA copy number analysis in matched primary CRC and metastasis (n=25)

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