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

DisclosureThe authors have no financial conflicts of interest to disclose relevant to this study.

Metastatic colorectal carcinoma (CRC)A major cause of mortality

OConnell et al JNCI 2004; 96: 1420

Majority of CRCs arise from adenomasAdenomaPrimary invasive adenocarcinomaMetastasis

Targeted therapy of metastatic CRCThe EGFR pathway

AimsDefine 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

Study Design & Methods736 frozen tumors from 613 patients39 adenomas, 406 primary carcinomas, 291 metastases84 pairs of matched primary tumors and metastases31 pairs of matched metastasesMatch confirmed by a fingerprinting assay

KRAS (exons 2,3,4)NRAS (exons 2,3)BRAF (exon 15) PIK3CA (exons 5, 7, 9, 20)TP53 (all coding exons)Study materialSequence analysis Sequenom MALDI-TOF mass spectrometry-based assay

Sanger

Study Design & MethodsIn matched pairs with discordant mutational profilesAnalysis of formalin-fixed paraffin embedded tissue (FFPE)Deep sequencing (454) (n=4)

DNA copy number analysis of matched primary tumors and metastasis N=25 pairsArray CGH (Agilent, 1M)

Clinical characteristics of study group

CharacteristicN=613Median age, years (range)62 (1788)Sex, % Male54.7 Female45.3Tumor Location, % Right Colon37.3 Left Colon43.7 Rectun18.9Stage at presentation, % I9.7 II18.1 III23.2 IV48.9

Incidence of RAS/BRAF mutationsN = 613RAS/BRAF wt, 48.4%G12C, 18G12A, 9G12D, 75G12R, 2G12S, 13G12V, 62G12N, 1G13C, 4G13D, 33Q61H/R/L/E, 12K117N, 3 A146T/V, 25NRAS G12D/13D, 6 NRAS Q61K/R/L, 12 BRAF V600E/G, 39 BRAF K601E, 1KRAS exon 2 mutations35.7%RAS mutations outside exon 29.4%BRAF mutations 6.5%

Incidence of PIK3CA mutations% PIK3CA mut casesp=0.00111.7%

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exon 9, 7.3%45

exon 20, 3.4%21

other, 1%6

wild type, 88.3%541

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Incidence of TP53 mutations% BRAF mut casesp=0.002

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247

366

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TP53 mutant247

TP53 wt366

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Incidence of mutations in primary tumors vs metastasisMutant (%)p=0.01p

BRAF mutations in metastatic CRCp=0.01p

Concordance of mutational profile between matched primary tumor and metastasis

Frozen samples (N=84)GeneNumber of concordant cases%RAS/BRAF7892.8PIK3CA8196.4TP537690.5

Total7083.3

Concordance of mutational profile between matched primary tumor and metastasis

Frozen samplesAfter analysis of FFPE samplesGeneNumber of concordant cases%Number of concordant cases%RAS/BRAF7892.88297.6PIK3CA8196.48398.8TP537690.58095.2

Total7083.37690.5

Concordance of mutational profile between matched metastatic foci

Frozen samples (N=31)After analysis of FFPE samplesGeneNumber of concordant cases%Number of concordant cases%RAS/BRAF3096.731100PIK3CA3110031100TP532993.53096.7

Total2993.53096.7

Concordance of mutational profile between matched primary tumor and metastasisRAS/ BRAFPIK3CATP53

KRAS G12 (G35A)TP53 wtKRAS G12 (wt)TP53 R248QKRAS discordant cases20 PPrimary CRC in right colon20 LLiver metastasisClinical history of a primary sigmoid CRC GGA

Different KRAS mutational profiles in a case of multiple primary CRCCecal CRCRectal CRCLiver metsKRAS G12VKRAS A146TKRAS A146TTP53 wtTP53 R306*TP53 R306*

Concordance of mutational profile between matched primary tumor and metastasisRAS/ BRAFPIK3CATP53

Detection of a low frequency mutant allele in TP53 discordant casesKRAS G12V (G35T)TP53 R175H (G525A)TP 53 wtKRAS G12V (G35T)2.2% R175H79.5% R175HTP 53 (454)Primary CRCLiver metastasis

Concordance of mutational profile between matched primary tumor and metastasisRAS/ BRAFPIK3CATP53

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

SummaryKRAS/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)

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.

David Solit Manickam JanakiramanAphrothiti HanrahanFeng XingYogi PersaudMoriah NissanGopa Iyer

Neal Rosen

Charles SawyersTim Chan

Adriana HeguyKety HubermanIgor DolgalevSabrena Thomas

Agnes Viale

Len SaltzNancy KemenyDiane ReidyAndrea Cercek

Phil PatyMarty WeiserZhaoshi Zeng

Mike DAngelicaYuman FongRonald DematteoPeter Allen

David KlimstraJinru ShiaLaura TangNora Katabi

Ronglai ShenRileen SinhaNick Socci

Cora MarianoPriscilla McNeilKatrina AllenAnas Idelbi

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

Approximately 50,000 patients died of CRC in 2009*Mutually exclusive*Mutually exclusive*Mutually exclusive*

Primary % aberration 30%Liver mets % aberration 39% (p=0.13)

8 metachronous resection17 synchronous*