Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 1 Supplementary Table 1. PIK3CA mutation in colorectal cancer Exon Domain Nucleotide change* Amino acid change* cases 9 Helical c.1621t>a p.e541t 1 9 Helical c.1624g>a p.e542k 40 # 9 Helical c.1631c>a p.t544n 3 # 9 Helical c.1631c>t p.t544i 1 9 Helical c.1633g>a p.e545k 57 9 Helical c.1634a>c p.e545a 1 9 Helical c.1634a>g p.e545g 1 9 Helical c.1636c>a p.q546k 14 # 20 Kinase c.3129g>t p.m1043i 18 20 Kinase c.3133g>a p.d1045n 1 20 Kinase c.3136g>a p.a1046t 2 20 Kinase c.3137c>a p.a1046e 1 20 Kinase c.3139c>t p.h1047y 5 20 Kinase c.3140a>c p.h1047p 1 20 Kinase c.3140a>g p.h1047r 41 20 Kinase c.3140a>t p.h1047l 11 # 20 Kinase c.3142c>t p.h1048y 1 # *Nomenclature of mutation follows the recommendations by the Human Genome Variation Society (http://www.hgvs.org/mutnomen/). # One case had both c.1636c>a and c.1631c>a mutations, another had c.3140a>t and c.3142c>t mutations and another had c.1624g>a and c.1631c>a mutations.
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 2 Supplementary Table 2. Clinical, pathological and molecular features of colorectal cancer according to PIK3CA mutation status in the Nurses Health Study PIK3CA wildtype Only in exon 9 PIK3CA mutation present P (exon 9 Only in exon vs. 20 exon 20) Feature No. No. No. No. No. No. 634 542 53 37 2 In both exon 9 and exon 20 P (across all categories) Mean age at diagnosis (years) ± SD 66.7 ± 8.3 66.7 ± 8.3 67.2 ± 8.6 66.5 ± 8.6 64.3 ± 5.4 0.96 Year of diagnosis 0.74 0.55 Prior to 1997 264 42% 232 43% 19 36% 12 32% 1 50% 1997 or after 370 58% 310 57% 34 64% 25 68% 1 50% Family history of colorectal cancer in first degree relatives 0.39 0.0048 Absent 514 81% 441 81% 44 83% 28 76% 1 50% Present 120 19% 101 19% 9 17% 9 24% 1 50% Tumor location 0.70 0.87 Rectum 132 21% 117 22% 10 19% 5 14% 0 0 Distal colon 190 30% 161 30% 15 28% 13 35% 1 50% Proximal colon 310 49% 262 49% 28 53% 19 51% 1 50% Disease stage 0.41 0.59 I 146 23% 123 23% 17 32% 6 16% 0 0 II 196 31% 165 30% 16 30% 14 38% 1 50% III 176 28% 151 28% 13 25% 11 30% 1 50% IV 85 13% 72 13% 7 13% 6 16% 0 0 Unknown 31 5% 31 6% 0 0 0 0% 0 0
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 3 Tumor grade 0.10 0.41 Low 557 88% 474 88% 50 94% 31 84% 2 100% High 73 12% 64 12% 3 6% 6 16% 0 0 MSI status 0.17 0.51 MSI-low/MSS 508 81% 432 81% 46 87% 28 76% 2 100% MSI-high 116 19% 100 19% 7 13% 9 24% 0 0 CIMP status 0.067 0.21 CIMP-low/0 488 79% 415 79% 45 88% 27 73% 1 50% CIMP-high 129 21% 112 21% 6 12% 10 27% 1 50% BRAF status 0.091 0.28 Wild-type 501 80% 425 79% 47 89% 27 75% 2 100% Mutant 128 20% 113 21% 6 11% 9 25% 0 0 KRAS status 0.30 0.0031 Wild-type 429 68% 382 70% 25 47% 21 58 % 1 50% Mutant 204 32% 160 30% 28 53% 15 42% 1 50% Mean LINE-1 methylation level (%) ± SD 63.1 ± 9.4 62.8 ± 9.4 65.5 ± 9.7 63.1 ± 9.0 58.4 ± 12.2 0.94 TP53 expression 0.68 0.062 Negative 319 58% 265 56% 32 74% 21 70% 1 50% Positive 229 42% 208 44% 11 26% 9 30% 1 50%
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 4 Supplementary Table 3. Clinical, pathological and molecular features of colorectal cancer according to PIK3CA mutation status in the Health Professionals Follow-Up Study PIK3CA mutation present PIK3CA wildtype exon 9 (exon 9 vs. exon 20 9 and exon 20 Only in P Only in In both exon Feature No. No. No. exon 20) No. No. No. 536 439 56 36 5 P (across all categories) Mean age at diagnosis (years) ± SD 71.1 ± 8.7 71.0 ± 8.6 71.5 ± 8.9 70.2 ± 9.2 80.2 ± 7.1 0.83 Year of diagnosis 0.35 0.76 Prior to 1997 237 44% 192 44% 24 43% 19 53% 2 40% 1997 or after 299 56% 247 56% 32 57% 17 47% 3 60% Family history of colorectal cancer in first degree relatives 0.26 0.042 Absent 437 82% 363 83% 46 82% 26 72% 2 40% Present 99 18% 76 17% 10 18% 10 28% 3 60% Tumor location 0.92 0.16 Rectum 126 24% 113 26% 7 13% 5 14% 1 20% Distal colon 169 32% 139 32% 17 30% 12 33% 1 20% Proximal colon 236 44% 182 42% 32 57% 19 53% 3 60% Disease stage 0.18 0.15 I 136 25% 108 25% 16 29% 9 25% 3 60% II 131 24% 105 24% 12 21% 14 39% 0 0 III 132 25% 113 26% 11 20% 8 22% 0 0 IV 66 12% 52 12% 8 14% 4 11% 2 40% Unknown 71 13% 61 14% 9 16% 1 3% 0 0
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 5 Tumor grade 0.32 0.69 Low 495 93% 406 93% 52 95% 32 89% 5 100% High 38 7% 31 7% 3 5% 4 11% 0 0 MSI status 0.0002 0.0004 MSI-low/MSS 470 89% 388 89% 54 96% 24 69% 4 80% MSI-high 60 11% 46 11% 2 4% 11 31% 1 20% CIMP status 0.15 0.0093 CIMP-low/0 418 88% 347 90% 43 86% 25 74% 3 60% CIMP-high 58 12% 40 10% 7 14% 9 26% 2 40% BRAF status 0.15 0.17 Wild-type 492 92% 406 93% 52 93% 30 83% 4 80% Mutant 43 8% 32 7% 4 7% 6 17% 1 20% KRAS status 0.56 0.0009 Wild-type 318 60% 277 64% 23 41% 17 47% 1 20% Mutant 214 40% 158 36% 33 59% 19 58% 4 80% Mean LINE-1 methylation level (%) ± SD 62.4 ± 9.6 62.1 ± 9.8 63.2 ± 9.4 64.7 ± 8.8 63.9 ± 6.0 0.64 TP53 expression 0.87 0.24 Negative 201 56% 157 54% 24 69% 18 67% 2 67% Positive 156 44% 135 46% 11 31% 9 33% 1 33%
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 6 Supplementary Table 4. Clinical, pathological and molecular features of colorectal cancer according to overall PIK3CA mutation status PIK3CA wildtype mutant PIK3CA P Feature No. No. No. No. 1170 981 189 Sex 0.097 Male 536 46% 439 45% 97 51% Female 634 54% 542 55% 92 49% Mean age (years) ± SD 68.7 ± 8.7 68.6 ± 8.7 69.2 ± 9.1 0.42 Year of diagnosis 0.53 Prior to 1997 501 43% 424 43% 77 41% 1997 or after 669 57% 557 57% 112 59% Family history of colorectal cancer in first degree relatives 0.18 Absent 951 81% 804 82% 147 78% Present 219 19% 177 18% 42 22% Tumor location 0.019 Rectum 258 22% 230 24% 28 15% Distal colon 359 31% 300 31% 59 31% Proximal colon 546 47% 444 45% 102 54% Disease stage 0.25 I 282 24% 231 24% 51 27% II 327 28% 270 28% 57 30% III 308 26% 264 27% 44 23% IV 151 13% 124 13% 27 14% Unknown 102 9% 92 9% 10 6% Tumor grade 0.60 Low 1052 91% 880 90% 172 91% High 111 9% 95 10% 16 9% MSI status 0.77 MSI-low/MSS 978 85% 820 85% 158 84% MSI-high 176 15% 146 15% 30 16%
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 7 CIMP status 0.34 CIMP-low/0 906 83% 762 83% 144 80% CIMP-high 187 17% 152 17% 35 20% BRAF status 0.72 Wild-type 993 85% 831 85% 162 86% Mutant 171 15% 145 15% 26 14% KRAS status <0.0001 Wild-type 747 64% 659 67% 88 47% Mutant 418 36% 318 33% 100 53% Mean LINE-1 methylation level (%) ± SD 62.8 ± 9.5 62.5 ± 9.6 64.1 ± 9.2 0.53 TP53 expression 0.0011 Negative 520 57% 422 55% 98 70% Positive 385 43% 343 46% 42 30%
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 8 Supplementary Table 5. analysis of the relations between KRAS mutation and PIK3CA mutation in colorectal cancers Variable independently associated with PIK3CA OR P KRAS mutation vs wild-type 2.65 (1.89-3.73) <0.0001 Other variables in the model CIMP high vs low/0 1.65 (1.07-2.54) 0.024 The multivariate logistic regression model initially included age, sex, year of diagnosis, tumor location, microsatellite instability, CpG island methylator phenotype, KRAS, BRAF and LINE-1 methylation. A backward elimination with threshold of p=0.05 was used to select variables in the final models. CI, confidence interval; OR, odds ratio.
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 9 Supplementary Table 6. PIK3CA exon 9 or exon 20 mutation in colorectal cancer and patient mortality PIK3CA status No. Colorectal cancer-specific mortality Overall mortality Stagestratified (95% Stagestratified (95% Wild-type 981 277 1 (referent) 1 (referent) 1 (referent) 467 1 (referent) 1 (referent) 1 (referent) Exon 9 mutant (regardless of exon 20 status) Exon 20 mutant (regardless of exon 9 status) 116 33 80 22 1.03 (0.71-1.47) 0.96 (0.62-1.48) 1.15 (0.80-1.65) 1.01 (0.65-1.56) 1.15 (0.79-1.66) 1.11 (0.71-1.71) 55 37 1.02 (0.77-1.36) 0.90 (0.65-1.26) 1.09 (0.82-1.44) 0.94 (0.67-1.31) 0.98 (0.74-1.31) 0.92 (0.66-1.29) The multivariate, stage-stratified Cox regression model initially included age, sex, year of diagnosis, tumor location, tumor grade, microsatellite instability, CpG island methylator phenotype, KRAS mutation, BRAF mutation, LINE-1 methylation and PIK3CA mutation status in the other exon. A backward elimination with a threshold of P = 0.05 was used to select variables in the final models. CI, confidence interval;, hazard ratio.
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 10 Supplementary Table 7. PIK3CA mutation in colorectal cancer and patient mortality according to cohort (gender) Colorectal cancer-specific mortality Overall mortality PIK3CA status No. Stagestratified (95% Stagestratified (95% Men (HPFS) Women (NHS) Wild-type 439 120 1 (referent) 1 (referent) 1 (referent) 227 1 (referent) 1 (referent) 1 (referent) Mutant 97 26 0.99 (0.65-1.51) 1.07 (0.70-1.65) 1.01 (0.66-1.55) 51 0.97 (0.72-1.32) 1.03 (0.76-1.40) 0.93 (0.69-1.27) Wild-type 542 157 1 (referent) 1 (referent) 1 (referent) 240 1 (referent) 1 (referent) 1 (referent) Mutant 92 25 0.92 (0.60-1.40) 1.00 (0.65-1.52) 1.11 (0.72-1.70) 34 0.81 (0.57-1.16) 0.85 (0.60-1.23) 0.88 (0.62-1.27) The multivariate, stage-stratified Cox regression model initially included age, sex, year of diagnosis, tumor location, tumor grade, microsatellite instability, CpG island methylator phenotype, KRAS mutation, BRAF mutation and LINE-1 methylation. A backward elimination with a threshold of P = 0.05 was used to select variables in the final models. CI, confidence interval; HPFS, Health Professionals Follow-Up Study;, hazard ratio; NHS, Nurses Health Study.
Liao X et al. PIK3CA Mutation in Colorectal Cancer. Page 11 Supplementary Table 8. Combined PIK3CA and BRAF mutation status in colorectal cancer and patient mortality PIK3CA BRAF No. Colorectal cancer-specific mortality Overall mortality Stagestratified stratified Stage- (95% (95% Wild-type Wild-type 831 234 1 (referent) 1 (referent) 1 (referent) 392 1 (referent) 1 (referent) 1 (referent) Mutant Wild-type 162 43 Wild-type Mutant 145 41 Mutant Mutant 26 8 0.93 (0.67-1.29) 1.09 (0.78-1.52) 1.24 (0.61-2.52) 0.82 (0.59-1.14) 1.11 (0.79-1.56) 1.19 (0.58-2.44) 0.80 (0.57-1.11) 1.37 (0.95-1.97) 2.40 (1.12-5.16) 73 71 12 0.93 (0.72-1.19) 1.17 (0.91-1.50) 1.06 (0.60-1.89) 0.88 (0.68-1.14) 1.21 (0.94-1.57) 1.07 (0.60-1.92) 0.80 (0.62-1.03) 1.34 (1.0-1.78) 1.48 (0.81-2.73) PIK3CA KRAS Wild-type Wild-type 659 167 1 (referent) 1 (referent) 1 (referent) 294 1 (referent) 1 (referent) 1 (referent) Mutant Wild-type 88 21 Wild-type Mutant 318 109 Mutant Mutant 100 30 0.97 (0.61-1.52) 1.42 (1.12-1.81) 1.18 (0.80-1.74) 0.97 (0.61-1.54) 1.19 (0.93-1.52) 0.87 (0.59-1.29) 1.0 (0.63-1.59) 1.17 (0.91-1.49) 0.85 (0.57-1.27) 35 170 50 0.88 (0.62-1.25) 1.27 (1.05-1.54) 1.11 (0.82-1.50) 0.91 (0.64-1.30) 1.15 (0.95-1.39) 0.93 (0.69-1.27) 0.89 (0.62-1.27) 1.09 (0.90-1.33) 0.84 (0.62-1.15) The multivariate, stage-stratified Cox regression model initially included age, sex, year of diagnosis, tumor location, tumor grade, microsatellite instability, CpG island methylator phenotype, KRAS mutation and LINE-1 methylation. A backward elimination with a threshold of P = 0.05 was used to select variables in the final models. CI, confidence interval;, hazard ratio.