comparative genomic analysis of primary versus metastasis in colorectal carcinomas evi vakiani,...
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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
Disclosure
• The authors have no financial conflicts of interest to disclose relevant to this study.
Metastatic colorectal carcinoma (CRC)A major cause of mortality
O’Connell et al JNCI 2004; 96: 1420
Majority of CRCs arise from adenomas
Adapted from Fearon and Vogelstein Cell 1990; 61: 759
Adenoma Primary invasive adenocarcinoma Metastasis
Targeted therapy of metastatic CRCThe EGFR pathway
P P
PP
Grb2Sos RasEGFR
Raf
MEK
ERK
PIK3CA
AKT
PTEN
SurvivalProliferationInvasion
TP53
Modified from Amado R G et al. JCO 2008;26:1626-1634
KRAS mutant
KRAS wild type
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
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
• KRAS (exons 2,3,4)
• NRAS (exons 2,3)
• BRAF (exon 15)
• PIK3CA (exons 5, 7, 9, 20)
• TP53 (all coding exons)
Study material
Sequence analysis
• Sequenom MALDI-TOF mass spectrometry-based assay
• Sanger
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)
Clinical characteristics of study group
Characteristic N=613
Median age, years (range)
62 (17–88)
Sex, %
Male 54.7
Female 45.3
Tumor Location, %
Right Colon 37.3
Left Colon 43.7
Rectun 18.9
Stage at presentation, %
I 9.7
II 18.1
III 23.2
IV 48.9
Incidence of RAS/BRAF mutations
N = 613
RAS/BRAF wt, 48.4%
G12C, 18G12A, 9
G12D, 75
G12R, 2
G12S, 13
G12V, 62
G12N, 1G13C, 4
G13D, 33
Q61H/R/L/E, 12
K117N, 3 A146T/V, 25
NRAS G12D/13D, 6
NRAS Q61K/R/L, 12 BRAF V600E/G, 39 BRAF K601E, 1
KRAS exon 2 mutations35.7%
RAS mutations outside exon 29.4%
BRAF mutations 6.5%
Incidence of PIK3CA mutations
% P
IK3
CA
mu
t ca
ses
p=0.001
11.7%
Incidence of TP53 mutations
% B
RA
F m
ut
case
s
p=0.002
Incidence of mutations in primary tumors vs metastasis
Mut
ant (
%)
p=0.01
p<0.001
p=0.01
p<0.001
BRAF mutations in metastatic CRC
p=0.01
p<0.0001
Concordance of mutational profile between matched primary tumor and metastasis
Frozen samples (N=84)
Gene Number of concordant cases
%
RAS/BRAF 78 92.8
PIK3CA 81 96.4
TP53 76 90.5
Total 70 83.3
Concordance of mutational profile between matched primary tumor and metastasis
Frozen samples After analysis of FFPE samples
Gene Number of concordant cases
% Number of concordant cases
%
RAS/BRAF 78 92.8 82 97.6
PIK3CA 81 96.4 83 98.8
TP53 76 90.5 80 95.2
Total 70 83.3 76 90.5
Concordance of mutational profile between matched metastatic foci
Frozen samples (N=31) After analysis of FFPE samples
Gene Number of concordant cases
% Number of concordant cases
%
RAS/BRAF 30 96.7 31 100
PIK3CA 31 100 31 100
TP53 29 93.5 30 96.7
Total 29 93.5 30 96.7
Concordance of mutational profile between matched primary tumor and metastasis
case 20 case 42
P
P
P
M
M
M
RAS/ BRAF
PIK3CA
TP53
KRAS G12 (G35A)
TP53 wtKRAS G12 (wt)
TP53 R248Q
KRAS discordant cases
20 PPrimary CRC in right colon
20 LLiver
metastasis
Clinical history of a primary sigmoid CRC
G
G A
KRAS A146 (G436A)
KRAS G12 (G35A)
KRAS G12 (wt)
KRAS A146 (wt)
42 PPrimary CRC
42 LLiver
metastasis
Different tumor morphologies
G
G
G
G
A
A
Different KRAS mutational profiles in a case of multiple primary CRC
Cecal CRC
Rectal CRC
Liver mets
KRAS G12V
KRAS A146T
KRAS A146T
TP53 wt
TP53 R306*
TP53 R306*
P
P
P
M
M
M
Concordance of mutational profile between matched primary tumor and metastasis
RAS/ BRAF
PIK3CA
TP53
Detection of a low frequency mutant allele in TP53 discordant cases
KRAS G12V (G35T)
TP53 R175H (G525A)
TP 53 wt
KRAS G12V (G35T)
2.2% R175H
79.5% R175H
TP 53 (454)
Primary CRC
Liver metastasis
P
P
P
M
M
M
Concordance of mutational profile between matched primary tumor and metastasis
RAS/ BRAF
PIK3CA
TP53
Hierarchical clustering
DNA copy number analysis of matched pairs of primary CRC and metastasis (n=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)
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 D’AngelicaYuman 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