Clinicopathological features in metastatic colorectal cancer patients with KRAS wild type versus codon 12 and codon 13 mutant: Results from a multicenter, cross-sectional study by the Japan Study Group of KRAS Mutation in Colorectal Cancer Takayuki Yoshino,1 Toshiaki Watanabe,2 Kentaro Yamazaki,3 Hiroyuki Uetake,4 Megumi Ishiguro,4 Kenichi Sugihara,4 Yasuo Ohashi5 1National Cancer Center Hospital East, Chiba, Japan, 2Teikyo University School of Medicine, Tokyo, Japan, 3Shizuoka Cancer Center, Shizuoka, Japan, 4Tokyo Medical and Dental University, Graduate School, Tokyo, Japan, 5Public Health Research Foundation, Tokyo, Japan ASCO GI 2011 #407 Background Study profile Results Discussions The KRAS mutation mainly located in the codon 12 and 13 indicates patients with metastatic colorectal cancer (CRC) lack of response to anti-epidermal growth factor receptor (EGFR) antibodies. Many studies have reported that approximately 30-40% of CRC patients have KRAS mutations.1-3 We have previously reported the correlation between KRAS mutation rate and sample backgrounds;4 however, the clinicopathological features of KRAS mutant CRC have not been fully clarified. Recently, some analysis showed improved outcomes for association of KRAS p.G13D mutation in patients with chemotherapy-refractory metastatic colorectal cancer treated with cetuximab.5-6 KRAS WT, KRAS MT KRAS WT, KRAS MT(p.G13D), KRAS MT(others) 　 Sample registration, n=5,887 　 from 389 facilities From Oct. 2009 to Mar. 2010 Cut-off: Apr. 2010 Gender We found the independent factors correlated with KRAS mutation were gender, age, date of sample obtained and primary tumor site. It suggests that some correlations exist between KRAS mutation and gender or primary tumor site since these factors were consistent with previous reports Although there is no consistent trend regarding age in the previous reports, this large cohort showed significantly more frequent KRAS mutation as the age was higher. Some reports showed “Adenomatous polyps increase with age”;15 while, another report showed “adenomatous polyps with KRAS mutation decreased with age”.16 Thus, it remains unknown why more frequent KRAS mutation occurred with age. There is no clear reason why KRAS mutation rate was lower in older samples. Since the concordance rate was very high between primary and metastases in this study, although these were not obtained from the same patients, it would be better to obtain new specimen if possible. Excluded, n=97 　 Cancelation 14 　 Ineligible 1 　 Uncollected 82　 Clinicopathological features: WT vs. MT Clinicopathological features: WT, MT(p.G13D), MT(others) n KRAS　MT P value Ferreira C.G. et al. 20107 Male Female 1,566 1,563 515 (32.9%) 589 (37.7%) 0.005 Andreyev HJN. et al. 19988 1,187 1,009 430 (36.2%) 399 (39.5%) 0.11 Roth A.D. et al. 20109 749 550 280 (37.4%) 201 (36.5%) 0.80 Abubaker J. et al. 200910 135 150 37 (27.4%) 43 (28.7%) 0.8132 Bennani B. et al. 201011 31 7 (22.6%) 11 (35.5%) 0.263 The present study 3,475 2,257 1,232 (35.5%) 923 (40.9%) <0.0001 n KRAS　WT KRAS　MT P value Gender Male 3,475 (60.6%) 2,243 (64.6%) 1,232 (35.5%) <0.0001 Female 2,257 (39.4%) 1,334 (59.1%) 923 (40.9%) Age < 50 560 ( 9.8%) 389 (69.5%) 171 (30.5%) 0.0007 50-59 1,258 (21.9%) 798 (63.4%) 460 (36.6%) 60-69 2,081 (36.3%) 1,289 (61.9%) 792 (38.1%) 70 =< 1,833 (32.0%) 1,101 (60.1%) 732 (39.9%) Year Surgically resected < 2006 748 (13.0%) 497 (66.4%) 251 (33.6%) 0.0285 2006 445 ( 7.8%) 282 (63.4%) 163 (36.6%) 2007 =< 4,171 (72.8%) 2,561 (61.4%) 1,610 (38.6%) Biopsy < 2009 110 ( 1.9%) 79 (71.8%) 31 (28.2%) 0.0524 2009 =< 258 ( 4.5%) 158 (61.2%) 100 (38.8%) Primary tumor site appendix 25 ( 0.4%) 9 (36.0%) 16 (64.0%) right-sided colon 1,713 (29.9%) 887 (51.8%) 826 (48.2%) left-sided colon 2,160 (37.7%) 1,528 (70.7%) 632 (29.3%) rectum 1,817 (31.7%) 1,142 (62.9%) 675 (37.1%) others* 17 ( 0.3%) 11 (64.7%) 6 (35.3%) n KRAS　WT KRAS　MT (p.G13D) KRAS　MT (others) Gender Male 3,475 2,243 (64.5%) 222 ( 6.4%) 1,010 (29.1%) Female 2,257 1,334 (59.1%) 198 ( 8.8%) 725 (32.1%) Age < 50 560 389 (69.5%) 42 ( 7.5%) 129 (23.0%) 50-59 1,258 798 (63.4%) 91 ( 7.2%) 369 (29.3%) 60-69 2,081 1,289 (61.9%) 140 ( 6.7%) 652 (31.3%) 70 =< 1,833 1,101 (60.1%) 147 ( 8.0%) 585 (31.9%) Primary tumor site appendix 25 9 (36.0%) 2 ( 8.0%) 14 (56.0%) right-sided colon 1,713 887 (51.8%) 169 ( 9.9%) 657 (38.4%) left-sided colon 2,160 1,528 (70.7%) 102 ( 4.7%) 530 (24.5%) rectum 1,817 1,142 (62.9%) 147 ( 8.1%) 528 (29.1%) others* 17 11 (64.7%) 0 ( 0.0%) 6 (35.3%) Site of the sample obtained Primary tumor 5,258 3,281 (62.4%) 385 ( 7.3%) 1,592 (30.0%) Metastasis 474 296 (62.4%) 35 ( 7.4%) 143 (30.2%) liver 216 146 (67.6%) 13 ( 6.0%) 57 (26.4%) lung 74 48 (64.9%) 7 ( 9.5%) 19 (25.7%) lymph node 37 22 (59.5%) 2 ( 5.4%) 13 (35.1%) local 45 29 (64.4%) 3 ( 6.7%) 13 (28.9%) dissemination 70 34 (48.6%) 6 ( 8.6%) 30 (42.9%) 32 17 (53.1%) 4 (12.5%) 11 (34.4%) KRAS WT vs. KRAS MT(p.G13D) KRAS MT(p.G13D) vs. KRAS MT(others) Odds-ratio 95%CI P value 1.500 1.224 - 1.838 <0.0001 0.805 0.650 - 0.997 0.0465 0.667 0.544 - 0.817 1.243 1.003 - 1.539 1.098 0.785 - 1.536 0.3549 1.383 0.960 - 1.994 0.1992 1.038 0.813 - 1.327 1.024 0.790 - 1.327 1.127 0.910 - 1.396 0.831 0.663 - 1.040 0.826 0.668 - 1.022 1.059 0.846 - 1.324 0.527 0.114 - 2.448 0.588 0.133 - 2.598 0.0558 0.490 0.397 - 0.604 1.105 0.889 - 1.374 2.325 1.842 - 2.934 0.729 0.571 - 0.932 0.871 0.704 - 1.077 1.231 0.983 - 1.542 - 1.008 0.699 - 1.453 0.9673 0.988 0.672 - 1.454 0.9514 0.992 0.688 - 1.431 1.012 0.688 - 1.489 1.332 0.749 - 2.371 0.5733 0.940 0.510 - 1.734 0.8520 0.803 0.361 - 1.785 1.531 0.639 - 3.666 1.293 0.303 - 5.520 0.634 0.143 - 2.819 1.136 0.345 - 3.746 0.953 0.270 - 3.359 0.662 0.276 - 1.587 0.824 0.341 - 1.992 0.497 0.166 - 1.483 1.507 0.478 - 4.756 　 KRAS test, n=5,790 　　 Direct sequencing 5,479 　　 Luminex 311 Undetectable samples, n=58 　 Direct sequencing 56 　 Luminex 2　 KRAS test detectable, n=5,732 　　 Direct sequencing 5,423 　　 Luminex 309 Results Sample Background Age Objectives n Gender Male 3,475 (60.6%) Female 2,257 (39.4%) Age < 50 560 ( 9.8%) Median:65 （12-92） 50-59 1,258 (21.9%) 60-69 2,081 (36.3%) 70 =< 1,833 (32.0%) Type of sample Surgically resected 5,364 (93.6%) Biopsy 368 ( 6.4%) Year < 2006 748 (13.0%) 2006 445 ( 7.8%) 2007 761 (13.3%) 2008 1,255 (21.9%) 2009 1,843 (32.2%) 2010 312 ( 5.4%) < 2009 110 ( 1.9%) 2009 =< 258 ( 4.5%) Stage I 166 ( 2.9%) II 814 (14.2%) III 1,765 (30.8%) IV 2,805 (48.9%) recurrence 152 ( 2.7%) unknown 30 ( 0.5%) * not included in the logistic regression n KRAS　MT P value Roth A.D. et al. 20109 =< 60 60 < 644 655 227 (35.2%) 254 (38.8%) 0.21 Abubaker J. et al. 200910 =< 50 50 < 87 198 29 (33.3%) 51 (25.8%) 0.1940 Bennani B. et al. 201011 =< 40 41-60 　　16 　　30 5 (31.3%) 13 (43.3%) 0 ( 0.0%) NS Barault L. et al. 200812 =< 65 66-74 75 =< 　149 　160 　267 54 (36.2%) 55 (34.4%) 89 (33.3%) 0.836 The present study < 50 50-59 60-69 70 =< 560 1,258 2,081 1,833 171 (30.5%) 460 (36.6%) 792 (38.1%) 732 (39.9%) 0.0007 Logistic regression: WT vs. MT This study aimed to clarify the clinicopathological features of KRAS mutant CRC in comparison with KRAS wild type in large-scale Japanese population. reference odds-ratio 95% CI Gender Female vs. Male 1.210 1.082 - 1.354 Age 50 – 59 < 50 1.308 1.053 - 1.624 60 – 69 1.365 1.113 - 1.674 70 =< 1.399 1.137 - 1.720 Year Surgically resected (2006) Surgically resected (<2006) 1.150 0.896 - 1.474 Surgically resected (2007=<) 1.188 1.005 - 1.403 Biopsy (<2009) 1.003 0.751 - 1.341 Biopsy(2009=<) Biopsy (<2009) 1.482 0.907 - 2.423 Primary tumor site right-sided colon left-sided colon 2.272 1.972 - 2.625 rectum 1.570 1.379 - 1.789 * not included in the logistic regression Conclusions Logistic regression: WT vs. MT(p.G13D) Logistic regression: MT(p.G13D) vs. MT(others) To clarify the clinicopathological features between KRAS codon 12 and 13 mutant CRC. To examine whether the clinicopathological features of KRAS p.G13D mutation is more similar to those of KRAS wild type or other KRAS mutations. As far as we know, this is one of the largest observational study of KRAS mutational status in CRC. The frequency of KRAS mutation (37.6%) in Japanese CRC patients is similar to those reported in previous studies from other countries. Independent factors correlated with KRAS mutation were gender, age, date of sample obtained and primary tumor site. Clinicopathological features were similar between KRAS codon 12 and 13 mutant CRC patients. KRAS p.G13D mutation was remarkably higher in female and right-sided colon; however, it was not influenced by age. Another possible finding was that this mutation may be higher in lung metastasis. However, this study suggested that clinicopathological features were not similar between KRAS wild type, KRAS p.G13D and other mutations. It suggests a possibility that KRAS p.G13D mutation might be a different tumor than other KRAS mutations, since some different trends were seen between those mutations. Presently, any KRAS mutation is regarded to be the same; however, this study suggests that it might be an aggregation from different tumors. reference odds-ratio 95% CI Gender Female vs. Male 0.710 0.576 - 0.876 Age 50 – 59 < 50 0.919 0.620 - 1.361 60 – 69 0.980 0.676 - 1.421 70 =< 0.837 0.577 - 1.214 Primary tumor site right-sided colon left-sided colon 0.365 0.281 - 0.475 rectum 0.727 0.569 - 0.930 reference odds-ratio 95% CI Gender Female vs. Male 0.799 0.643 - 0.955 Age 50 – 59 < 50 1.338 0.875 - 2.046 60 – 69 1.549 1.037 - 2.313 70 =< 1.288 0.862 - 1.924 Primary tumor site right-sided colon left-sided colon 0.750 0.570 - 0.986 rectum 1.104 0.855 - 1.425 Methods Key eligibility criteria Histologically confirmed colorectal adenocarcinoma Adequate tumor samples Gender Age [Odds ratio of biopsy （2009=<） against surgery specimen （<2006）is estimated as (1.003×1.482)=1.486] KRAS WT KRAS MT(p.G13D) KRAS MT(others) KRAS WT KRAS MT(p.G13D) KRAS MT(others) There were the significant difference of the frequency of KRAS mutation Female (40.9%) and male (35.5%) (Odds-ratio 1.210) As the age was higher, more frequent KRAS mutation (Odds-ratio 1.308-1.399) Date of the sample obtained： after 2007 (38.6％), 2006 (36.6％), before 2005 (33.6％) (Odds-ratio 1.118)　 Right-sided colon (48.2%) and left-sided colon (29.3%) (Odd ratio 2.272) 59.1% 64.5% 8.8% 6.4 % 32.1% 29.1% 60.1% 61.9% 63.4% 69.5% 8.0% 6.7% 7.2% 7.5% 31.9% 31.3% 29.3% 23.0% P=0.0001* P=0.5285* P=0.0002* Sample size: 5,000 Data collection: Sample for KRAS test Surgically resected specimen or biopsy from primary tumor or metastases Paraffin-embedded tumor blocks or thinly sliced tumor sections Patients and sample backgrounds Gender, age Primary tumor site Type of sample (surgically resected / biopsy) Date of the sample obtained Site of the sample obtained (primary tumor / metastases) Stage (l / ll / lll / IV / recurrence / unknown) Duration of formalin fixation (<24h / 24-48h / 48h< / unknown) Formalin concentration (10% / 20% / unknown) <50 (n=560) male (n=3,475) Primary tumor site Primary tumor site appendix 25 ( 0.4%) right-sided colon 1,713 (29.9%) left-sided colon 2,160 (37.7%) rectum 1,817 (31.7%) others 17 ( 0.3%) Site of the sample obtained Primary tumor 5,258 (91.7%) Metastasis 474 ( 8.2%) liver 216 (45.6%) lung 74 (15.6%) lymph node 37 ( 7.8%) local 45 ( 9.5%) dissemination 70 (14.8%) 32 ( 6.8%) n KRAS　MT P value Roth A.D. et al. 20109 right left 516 783 207 (40.1%) 274 (35.0%) 0.070 Abubaker J. et al. 200910 right colon left colon 44 241 18 (40.9%) 62 (25.7%) 0.0454 Bennani B. et al. 201011 proximal colon distal colon rectum 14 　 20 28 4 (28.6%) 4 (20.0%) 10 (35.7%) NS Barault L. et al 200812 244 331 89 (36.5%) 109 (32.9%) 0.377 Tie J. et al 201013 right-sided colon left-sided colon 185 153 172 70 (38%) 43 (28%) 54 (31%) 0.07 The present study appendix others 25 1,713 2,160 1,817 17 16 (64.0%) 826 (48.2%) 632 (29.3%) 675 (37.1%) 6 (35.3%) <0.0001 50-59 (n=1,258) 60-69 (n=2,081) female (n=2,257) KRAS MT(codon 12), KRAS MT(codon 13) 70=< (n=1,833) Clinicopathological features: MT(codon 12) vs. MT(codon 13) *Cochran-Armitage trend test n KRAS　MT (codon 12) (codon 13) P value Gender Male 3,475 996 (28.7%) 236 ( 6.8%) 0.0820 Female 2,257 718 (31.8%) 205 ( 9.1%) Age < 50 560 129 (23.0%) 42 ( 7.5%) 0.3496 50-59 1,258 363 (28.9%) 97 ( 7.7%) 60-69 2,081 643 (30.9%) 149 ( 7.2%) 70 =< 1,833 579 (31.6%) 153 ( 8.3%) Primary tumor site appendix 25 14 (56.0%) 2 ( 8.0%) 0.1710 right-sided colon 1,713 650 (37.9%) 176 (10.3%) left-sided colon 2,160 519 (24.0%) 113 ( 5.2%) rectum 1,817 525 (28.9%) 150 ( 8.3%) others* 17 6 (35.3%) 0 ( 0.0%) Primary tumor site Site of the sample obtained References KRAS mutation rate KRAS WT KRAS MT(p.G13D) KRAS MT(others) KRAS WT KRAS MT(p.G13D) KRAS MT(others) No. of sample KRAS　WT KRAS　MT n % 95%CI All 5,732 3,577 62.4 61.1-63.7 2,155 37.6 36.3-38.9 　 Codon12 　 Codon13 1,714 441 29.9 7.7 28.7-31.1 7.0-8.4 Primary tumor (n=5,258) 1. Karapetis CS. et al. N Engl J Med. 2008;359:1757-1765. 2. Amado RG. et al. J Clin Oncol. 2008;26:1626-1634. 3. Van Cutsem E. et al. N Engl J Med. 2009;360:1408-1417. 4. Yamazaki K. et al. ESMO 2010 Abst.595P 5. De Roock W. et al. JAMA. 2010;304:1812-1820. 6. Bando H., Yoshino T. et al. ASCO-GI 2011 #448 7. Ferreira C.G. et al. J Clin Oncol 2010;28,suppl:3614. 8. Andreyev HJN. et al. J Natl Cancer Inst 1998;90:675-684. 9. Roth A.D. et al. J clin oncol 2010;28:466-474. 10. Abubaker J. et al. J Pathology 2009;219:435-445. 11. Bennani B. et al. Int J Biol Markers 2010;25:179-84. 12. Barault L. et al. Int J Cancer 2008;122:2255-9. 13. Tie J. et al. Int J Cancer. 2010 Jul 15. [online ahead of print] 14. Andreyev HJN. Br J Cancer 2001;85:692-696. 15. Winawer S.J. et al. N Engl J Med. 1993;328:901-906. 16. Rashid A. et al. Gut. 1999;44:826-33. appendix (n=25) 62.4% 7.3% 30.3% 36.0% 8.0% 56.0% Metastasis (n=474) Send formalin-fixed paraffin-embedded tumor blocks or thinly sliced tumor sections to commercial laboratories （10µm 5 slices and 3µm 1 slice for HE staining） Investigate KRAS point mutations in the codon 12 and 13 by following laboratories’ SOP 62.4% 7.4% 30.2% Hospitals Laboratories Data center 1. Sample registration 2. Enrollment confirmation 6. Result 3. Sample 2’. Registration information 5. Result 4. KRAS test right-sided colon (n=1,713) liver (n=216) 51.8% 9.9% 38.4% 67.6% 6.0% 26.4% lung (n=74) 64.9% 9.5% 25.7% KRAS mutation rate in codon 12 and 13 n % Codon12 1,714 AGT CGT GAT GCT GTT TGT others 120 15 814 107 493 162 3 7.0 0.9 47.5 6.2 28.8 9.5 0.1 Codon13 441 CGC GAC GAG TGC 6 420 1 11 1.4 95.2 0.2 2.5 0.7 left-sided colon (n=2,160) 70.7% 4.7 % 24.5% lymph node (n=37) n Codon 12 Codon 13 Andreyev HJN. et al. 200114 3,439 900 (26.2%) 297 (8.6%) Ferreira C.G. et al. 20107 3,129 933 (29.8%) 171 (5.5%) Andreyev HJN. et al. 19988 2,721 755 (27.7%)* 146 (6.6%) Roth A.D. et al. 20109 1,299 372 (28.6%) 102 (7.9%) The present study 5,732 1,714 (29.9%) 441 (7.7%) others 30.3％ (n=1,735) * not included in the logistic regression 59.5% 5.4% 35.1% Logistic regression: MT(codon 12) vs. MT(codon 13) local (n=45) 64.4% 6.7% 28.9% reference odds-ratio 95% CI Gender Female vs. Male 0.823 0.665 - 1.018 Age 50-59 <50 1.243 0.820 - 1.885 60-69 1.422 0.959 - 2.108 70=< 1.252 0.844 - 1.857 Primary tumor site right-sided colon left-sided colon 0.806 0.619 - 1.050 appendix 0.473 0.106 - 2.114 rectum 1.081 0.841 - 1.389 rectum (n=1,817) 62.9% 8.1% 29.1% KRAS MT 37.6% (n=2,155) dissemination (n=70) 48.6% 8.6% 42.9% KRAS p.G13D mutation was remarkably higher in female. KRAS p.G13D mutation occurred at a constant rate regardless of age (P=0.5285); however, KRAS other mutations increased with age (P=0.0002). In contrast, KRAS wild type decreased with age (P=0.0001). KRAS p.G13D mutation seemed remarkably higher in right-sided colon than left-sided colon. (right：9.9%, left：4.7%) KRAS p.G13D mutation was tend to be higher in lung metastasis (9.5%) compared to other metastatic sites; however, this is uncertain because the sample size has varied through the metastatic sites. KRAS WT 62.4% (n=3,577) Acknowledgement This study was funded by Comprehensive Support Project for Oncology Research (CSPOR) of Public Health Research Foundation. The research institutes are Tokyo Medical and Dental University, Teikyo University School, and National Cancer Center Hospital East. We would like to thank the 389 sample providing hospitals. p.G13D 7.3％ (n=420) Clinicopathological features of KRAS codon 13 mutation were similar to those of KRAS codon 12 mutation * 755/2,214