PIK3CA and PTEN gene and exon mutation-specific clinicopathologic and molecular associations in colorectal cancer.

PURPOSE
PIK3CA and PTEN mutations are prevalent in colorectal cancer and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. Relationships between phosphoinositide 3-kinase (PI3K) pathway mutation, clinicopathologic characteristics, molecular features, and prognosis remain controversial.


EXPERIMENTAL DESIGN
A total of 1,093 stage I-IV colorectal cancers were screened for PIK3CA (exons 9 and 20), KRAS (codons 12-13), BRAF (codon 600) mutations, and microsatellite instability (MSI). PTEN (exons 3-8) and CpG island methylator phenotype (CIMP) status were determined in 744 and 489 cases. PIK3CA data were integrated with 17 previous reports (n = 5,594).


RESULTS
PIK3CA and PTEN mutations were identified in 11.9% and 5.8% of colorectal cancers. PTEN mutation was associated with proximal tumors, mucinous histology, MSI-high (MSI-H), CIMP-high (CIMP-H), and BRAF mutation (P < 0.02). PIK3CA mutation was related to older age, proximal tumors, mucinous histology, and KRAS mutation (P < 0.04). In integrated cohort analysis, PIK3CA exon 9 and 20 mutations were overrepresented in proximal, CIMP-low (CIMP-L), and KRAS-mutated cancers (P ≤ 0.011). Comparing PIK3CA exonic mutants, exon 20 mutation was associated with MSI-H, CIMP-H, and BRAF mutation, and exon 9 mutation was associated with KRAS mutation (P ≤ 0.027). Disease-free survival for stage II/III colorectal cancers did not differ by PI3K pathway status.


CONCLUSION
PI3K pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-H/CIMP-H/BRAF(mut)), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the traditional serrated pathway (CIMP-L/KRAS(mut)) of tumorigenesis. Our data highlight the PI3K pathway as a therapeutic target in distinct colorectal cancer subtypes.

PIK3CA and PTEN status are implicated as markers of colorectal cancer response to mitogen-activated protein/ extracellular signal-regulated kinase (MEK) inhibitors and anti-EGF receptor (EGFR) antibody therapy (cetuximab or panitumumab). Some but not all studies of anti-EGFR antibody treatment in metastatic colorectal cancer have reported resistance for tumors with PIK3CA mutation or PTEN loss (9)(10)(11), and similar results have been obtained in preclinical studies of MEK inhibition for KRAS-mutated colorectal cancer cell lines (12). Conversely, KRAS mutation has been associated with resistance to single agent PI3K inhibitors (13), but such cell lines may respond to concomitant inhibition of the PI3K and mitogen-activated protein kinase (MAPK) pathways (14). On the basis of these and other findings, drug combination studies of MAP2K and PI3K or mTOR inhibitors have been proposed tailored to tumor PI3K and MAPK pathway mutation status (15).
Emerging data indicate that the conflicting PIK3CA association data may in part be explained by intrinsic differences between exon 9 and 20 mutated cancers. Two recent studies on stage I-IV colorectal cancers have suggested that exon 20 mutations correlate more strongly with BRAF mutation, CIMP-high/-low (CIMP-H/-L) and MSI-H status (31,32), and one reported that exon 9 mutations may more closely associate with KRAS mutation (31,32). The latter has further been observed in a study on metastatic colorectal cancer (30). However, these suggestions have been based on pairwise comparisons between PIK3CA exonic mutant and wild-type tumors, rather than direct comparisons between exon 9 and 20 mutated cases required to substantiate such relationships.
The prognostic value of PIK3CA mutation may also vary by exon mutation status, with one study reporting an association of exon 20 but not exon 9 mutations with worse outcome in stage III tumors (33). However, another large study of patients with stage I-IV colorectal cancer found no effect of either exon 9 or 20 mutations on survival; instead, a small proportion of tumors with concomitant exon 9 and 20 mutations were reported to show poor outcomes (32). In addition, exonic location of PIK3CA mutation may influence response to anti-EGFR antibody therapy, with a recent analysis of multiple cohorts suggesting that mutations in PIK3CA exon 20 but not exon 9 are predictive of tumor resistance (34).
To comprehensively define the characteristics of PIK3CAand PTEN-mutated colorectal cancer, we have analyzed 1,093 stage I-IV cancers for gene and exon mutation-specific relationships with patient clinicopathologic and tumor molecular features including KRAS, BRAF, MSI, and CIMP status. Associations with prognosis were investigated for persons with resected stage II/III cancer. To refine results on PIK3CA exon 9 and 20 mutation-specific associations, our data were integrated with 17 published cohorts comprising a total of 5,594 patients with colorectal cancer.

Patients
A total of 1,093 patients with colorectal cancer were recruited from the Royal Melbourne Hospital (Parkville, VIC, Australia), Western Hospital Footscray (Footscray, VIC, Australia), St Vincent's Hospital Sydney (Darlinghurst, NSW, Australia), and the Royal Adelaide Hospital (Adelaide, SA, Australia). The study was approved by ethics committee, and patients gave informed consent. Fresh-frozen (n ¼ 744) or formalin-fixed paraffin-embedded (n ¼ 349) tumor and matched normal specimens were sourced from hospital Translational Relevance PIK3CA and PTEN mutations are common in colorectal cancer, and potential markers of response to mitogen-activated protein/extracellular signal-regulated kinase inhibitors and anti-EGF receptor antibody therapy. This cohort study and integrated analysis with 17 previous reports defines the characteristics of PIK3CAand PTEN-mutated colorectal cancers. We show that phosphoinositide 3-kinase (PI3K) pathway mutation is prominent in proximal colon cancers, with PIK3CA exon 20 and PTEN mutations associated with features of the sessile-serrated pathway (MSI-high/CIMP-high/ BRAF mutation), and PIK3CA exon 9 (and to a lesser extent exon 20) mutation associated with features of the so-called traditional serrated pathway of tumorigenesis (CIMP-low/KRAS mutation). Our data highlight the PI3K pathway as a preferential therapeutic target in distinct colorectal cancer subtypes, and establish the prevalence of compound PI3K/MAPK pathway genotypes relevant to the development of combination therapies. Our results clarify reports on the prognostic values of PIK3CA and PTEN mutations, showing that these do not predict relapse in stage II/III colorectal cancer. tissue banks. Ninety-four cancers were stage I, 278 stage II,  525 stage III, and 196 stage IV. Four hundred and forty-four  cancers were from the proximal colon (cecum to transverse  colon), 342 from the distal colon (splenic flexure to sigmoid colon), and 306 from the rectum (unavailable for 1 case). None of the patients had clinical features of familial adenomatous polyposis, Lynch, or other familial cancer syndromes. Treatment and follow-up data were prospectively recorded. Patient characteristics are summarized in Table 1.

Microsatellite instability analysis
Hematoxylin and eosin (H&E)-stained tissue sections were reviewed, and for tumor samples areas with more than 60% neoplastic cells were macrodissected. DNA was extracted using standard protocols and PCR-amplified for the Bethesda consensus panel of microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250) using fluorescently labeled primers (35). Reaction products were analyzed on a 3130xl Genetic Analyzer (Applied Biosystems). MSI-H was diagnosed if instability was evident at 2 or more markers.

CIMP marker analysis
Tumor CIMP status was determined using MethyLight real-time PCR for the Weisenberger and colleagues 5 marker panel (IGF2, SOCS1, RUNX3, CACNA1G, and NGN1) and the reference gene ALU (C-4; ref. 36). The percentage of methylated reference (PMR) was calculated for GENE:ALU ratio of template amount in a sample against GENE:ALU ratio of template amount in methylated reference DNA. Tumors with a PMR more than 10 for 3 to 5 CIMP markers were classified as CIMP-H, those with 1 to 2 methylated markers as CIMP-L, and 0 methylated markers as CIMP-0.

LOH analysis
LOH at the PTEN locus was determined from singlenucleotide polymorphism (SNP) array data for tumor and matched normal samples (Human610-Quad BeadChip arrays; Illumina) using OncoSNP software (Isis Innovations) as described previously (37). SNP call rates for normal samples were more than 98% and for tumor samples were more than 97%.

Statistical analyses
Statistical analyses were conducted using the statistical computing software R (R Development Core Team, 2011). For univariate analyses, differences between groups were assessed using Fisher exact test for categorical variables and Kruskal-Wallis test for continuous variables. Multivariate analyses for association between gene mutation and clinicopathologic or molecular features were conducted using logistic regression. Integrated multicohort analysis for PIK3CA exon mutation-specific associations was conducted using the DerSimonian-Laird random effects pooling method (ref. 38; rmeta R package version 2.16) and mixed effects logistic regression with the association of interest treated as fixed effect and the study as random effect. Interstudy heterogeneity was assessed using Woolf test. Changes in proportions of tumor MSI/CIMP/KRAS/BRAF genotypes according to PIK3CA exon 9 or 20 mutation status were estimated using mixed effects logistic regression and a 3-stage hierarchical multinomial-Dirichlet model (Supplementary Methods). Outcome analyses for patients with resected stage II/III colorectal cancer were conducted for 5-year disease-free survival (DFS). DFS was defined as time from surgery to the first confirmed relapse, with censoring done when a patient died or was alive without recurrence at last contact. Survival curves were generated according to the method of Kaplan and Meier. Univariate survival distributions were compared using the log-rank test, and multivariate analyses used Cox proportional hazards models. All statistical analyses were two-sided and considered significant if P < 0.05.

PIK3CA and PTEN mutation prevalence and spectra
A population-based series of 1,093 patients with stage I-IV colorectal cancers was screened for somatic mutations in  showed mutations in either PI3K pathway member. A subset of 1.2% (n ¼ 9) of tumors harbored mutations in both genes, perhaps suggesting synergy, although this was only borderline statistically significant (P ¼ 0.092).
Clinicopathologic and molecular associations of PIK3CA mutation Clinicopathologic and molecular correlates of PIK3CA mutation were evaluated overall and according to exonic location. Overall, PIK3CA gene mutation was significantly associated with older age at diagnosis, proximal tumor site, mucinous histology, and presence of KRAS mutation (P 0.037 for all comparisons), but not with patient gender, tumor stage, differentiation, BRAF mutation, MSI, or CIMP status (Table 1). In analyses stratified by MSI status, the relationships with older age, proximal tumor site, and KRAS mutation remained significant for MSS cancers (P 0.010 for all comparisons), whereas the relationship with mucinous histology was observed for MSI-H cancers only (P ¼ 0.004; Supplementary Table S4).
Although some of these associations were evident for both exon 9 and 20 mutations separately, others seemed to be exon-specific (Table 2). Compared with PIK3CA wildtype status, mutation of either exon 9 or 20 alone was significantly associated with proximal tumor location (P 0.002 for both comparisons). In contrast, mucinous histology was specific to exon 20-mutated tumors when compared with both wild-type and exon 9-mutated tumors (P 0.045 for both comparisons). The significant relationship with older age at diagnosis and KRAS mutation was observed for exon 9 but not exon 20 mutation as compared with wild-type (age: P ¼ 0.023 vs. P ¼ 0.139; KRAS: P < 0.001 vs. P ¼ 0.318), although the direct comparisons between exon 9-and 20-mutated cases did not reach statistical significance (age, P ¼ 0.810; KRAS, P ¼ 0.095). Common and differential relationships by exon mutation status remained apparent in analyses stratified by MSI status. In MSS cancers, exon 9 and 20 mutations were both associated with proximal tumor location (P 0.024 for both comparisons), whereas associations with older age and KRAS mutation were significant only for exon 9 (age, P ¼ 0.014; KRAS, P < 0.001). In MSI-H cancers, exon 9 mutation occurred with KRAS mutation (P ¼ 0.042), and exon 20 mutation with mucinous histology (P ¼ 0.009; Supplementary Table S5).
In multivariate logistic regression analysis to assess for independent relationships between PIK3CA mutation and clinicopathologic or molecular features, proximal tumor location was significantly associated with overall PIK3CA gene mutation (P ¼ 0.002; Supplementary Table S6). For a model excluding CIMP status, which was only available for a limited subset of patients, KRAS mutation was further independently associated with overall PIK3CA gene mutation (P ¼ 0.034; Supplementary Table S6). In multivariate analyses by exonic site, a significant independent association with proximal location was observed for either exon 9 or 20 mutation compared with PIK3CA wild-type (P 0.028 for both comparisons), whereas an independent relationship with KRAS mutation was observed only for exon 9-mutated tumors (P ¼ 0.01; Supplementary Tables  S7 and S8). Direct comparison of exon 9 and 20 mutations provided suggestive evidence of an overrepresentation of KRAS mutation in exon 9-mutated tumors (P ¼ 0.029), although this was not found in the model excluding CIMP status (P ¼ 0.247; Supplementary Table S9).

Clinicopathologic and molecular associations of PTEN mutation
In 744 patients with colorectal cancer evaluated for PTEN, presence of mutation was significantly associated with proximal tumor location, mucinous histology, BRAF mutation, MSI-H, and CIMP-H status (P 0.014 for all comparisons), but unrelated to patient age, gender, tumor stage, differentiation, and KRAS mutation (Table 1). When considering MSS cases only, no significant associations were observed, but for MSI-H cases, the positive association between PTEN and BRAF mutation remained significant (P ¼ 0.019; Supplementary Table S10). Consistent with defective DNA mismatch repair, MSI-H cancers showed an overrepresentation of frameshift mutations in 2 poly-adenine tracts located in PTEN exons 7 and 8 as compared with MSS cancers (MSI-H: 46.2%, 12 of 26; MSS: 3.4%, 1 of 29; P < 0.001).
In multivariate logistic regression analysis including all clinicopathologic and molecular features, MSI-H remained independently associated with PTEN mutation (P < 0.001; Supplementary Table S11). For a model excluding CIMP status, BRAF mutation additionally reached statistical significance for independent association (P ¼ 0.037; Supplementary Table S11).

PIK3CA or PTEN mutation and outcome in stage II and III colorectal cancer
For patients with resected stage II or III colorectal cancer, the influence of PI3K pathway mutation status on DFS was assessed using Cox proportional hazards analysis. Clinical follow-up information was available for 585 patients analyzed for PIK3CA mutation and 381 patients analyzed for both PIK3CA and PTEN mutation. The median duration of follow-up was 32 months for the former and 33 months for the latter patient group.
In both univariate and multivariate analyses adjusted for age at diagnosis, gender, tumor location, stage, differentiation, MSI status, and adjuvant treatment (Table 3), neither PIK3CA nor PTEN mutation were associated with the risk of recurrence [PIK3CA mutation: multivariate HR, 0.79, 95% confidence interval (CI), 0.50-1.25; PTEN mutation: multivariate HR, 0.78, 95% CI, 0.32-1.86). There was no evidence for differential outcomes when classifying PIK3CA mutations into those affecting exon 9 or 20 (exon 9 vs. wildtype: multivariate HR, 0.87, 95% CI, 0.52-1.46; exon 20 vs. wild-type: multivariate HR, 0.64, 95% CI, 0.28-1.47; Supplementary Table S12). Results were similar when separating patients into groups who did and did not receive adjuvant chemotherapy (Supplementary Tables S13-S15). Accordingly, overall PI3K pathway mutation status, defined as either PIK3CA mutation, PTEN mutation, or Integrated analysis of PIK3CA exon 9 and 20 mutations for clinicopathologic and molecular associations To further refine our analysis of the clinicopathologic and molecular associations of PIK3CA exon 9-or 20-mutated tumors, we identified previous publications reporting such details using the PubMed literature search engine (www. ncbi.nlm.nih.gov/pubmed), the COSMIC database (www. sanger.ac.uk/genetics/CGP/cosmic) and cross-referenced citations. A total of 17 eligible cohorts were identified and data retrieved for patient gender, tumor stage, location, MSI status, CIMP status, BRAF mutation, and KRAS mutation (Supplementary Table S17). Tumors with mutations in both PIK3CA exons 9 and 20 were excluded. To maintain consistency for evaluation of KRAS and BRAF, mutation data were limited to KRAS codons 12 and 13 and the BRAF codon 600 (V600E). ORs for integrated studies were calculated for PIK3CA exon 9 or 20 mutation versus wild-type,  and exon 20 versus exon 9 mutation using 2 alternative approaches, the DerSimonian-Laird random effects pooling method and mixed effects logistic regression.
Analyses excluding data from this study reproduced these results, although in the direct comparison between exon 20and exon 9-mutated cancers, the exon 20 mutation association with KRAS wild-type reached only borderline statistical significance (Supplementary Fig. S3).
Differential tumor MSI/CIMP/KRAS/BRAF genotypes according to PIK3CA exon 9 or 20 mutation status To further define the differential associations of PIK3CA exon 9 or 20 mutation with tumor location, MSI, CIMP, KRAS, and BRAF mutation, combined analysis was conducted for cohorts with available complete patient data to test for differences in proportions of tumor compound genotypes [n ¼ 1,318; this study, The Cancer Genome Atlas (TCGA; ref. 39) and Whitehall and colleagues (31)]. The 8 most numerous states plus an omnibus state consisting of the remaining genotypes were modeled using mixed effects logistic regression or, alternatively, a 3-stage hierarchical multinomial-Dirichlet model.
associations were replicated using the multinomial-Dirichlet model (Supplementary Fig. S4). Direct comparisons between PIK3CA exonic mutants did not reach statistical significance, although power to detect such differences was limited because of small sample sizes.

Discussion
This study presents the first substantial survey of somatic PTEN mutations in sporadic colorectal cancer and is the most comprehensive analysis to date of PIK3CA exon mutation-specific associations with patient characteristics, tumor molecular features and outcome. Our findings show significant gene and exon mutation-specific differences in clinicopathologic and molecular associations for PIK3CA and PTEN and clarify previous data on the prognostic value of these changes in resected stage II/III colorectal cancer.
Somatic mutations in PTEN exons 3 to 8 were detected in 6% of sporadic colorectal cancers, with approximately half localized in the phosphatase domain and half localized in the C2 tensin-type domain of the protein. Although there was a significant overrepresentation of tumors with "two hits," consistent with a classical tumor suppressor role, 47% of PTEN mutated tumors had only one detected mutation and lacked LOH. Although some of these tumors may carry mutations in exons not evaluated in our screen or may have lost expression of the wild-type protein, a potential role for PTEN haploinsufficiency has been suggested in previous functional studies (40). Consistent with existing reports, PTEN mutations were significantly more common in MSI-H tumors as compared with MSS tumors (16)(17)(18)(19)(20). In keeping with the characteristics of the MSI-H/sessile-serrated path-way of colorectal tumorigenesis (41,42), PTEN mutation was further associated with proximal tumor location, mucinous histology, BRAF mutation, and CIMP-H status in our cohort. Notably, within MSI-H cancers, the positive association between PTEN and BRAF mutation remained significant, suggesting selection for comutation of these PI3K and MAPK pathway members in this colorectal cancer subtype.
Consistent with previous studies, PIK3CA mutations in exon 9 or 20 were detected in 12% of sporadic colorectal cancers with mutation of the former exon about twice as frequent as the latter (2)(3)(4)(5). Approximately, 0.3% of colorectal cancers had 2 detected mutations as reported by others (2,11,26,30,32). Overall, PIK3CA gene mutation was significantly associated with older age at diagnosis, proximal tumor location, mucinous histology, and presence of KRAS mutation, relationships which have been described in a number of reports (5,26,(28)(29)(30)(31)(32). However, when stratified by exonic location, significant differences were evident between PIK3CA exon 9 and 20 mutation in our cohort. Compared with both wild-type and exon 9mutated tumors, the relationship with mucinous histology was specific to exon 20 mutation. Furthermore, the associations with older age at diagnosis and KRAS mutation seemed stronger for exon 9-mutated tumors, although the direct comparison with exon 20-mutated tumors did not reach statistical significance. A PIK3CA exon 9 mutationspecific association with KRAS mutation has been suggested by others, but a statistical difference as compared with exon 20 mutation was also not formally shown in these reports (30)(31)(32). In an analysis stratified by MSI status, the association between PIK3CA exon 9 mutation and KRAS mutation Figure 2. Integrated multicohort analysis for differences in proportions for tumor MSI/CIMP/KRAS/BRAF genotypes according to PIK3CA exon 9 or 20 mutation status. Estimates and 95% CIs were calculated using a mixed effects logistic regression model; only the 8 most numerous tumor states were modeled, with the remaining states combined into an "other classes" category. This analysis pooled data from 3 data sources: this study, the TCGA (39), and Whitehall and colleagues (31). Ã , P < 0.05; ex9, exon 9; ex20, exon 20; wt, wild-type. was observed for both MSS and MSI-H cases, but the association between PIK3CA exon 20 mutation and mucinous histology was evident for MSI-H cases only. Given the limited power to show PIK3CA exon mutationspecific clinicopathologic and molecular associations in our and previous studies, we integrated our results with findings from 17 published cohorts, together encompassing 5,594 patients. In the combined analysis, PIK3CA exon 9 or 20 mutation were both significantly associated with proximal tumor location, CIMP-L and KRAS mutation, in both instances showing a corresponding underrepresentation in distal tumors with MSS/CIMP-0/KRAS wt /BRAF wt genotype. Directly comparing PIK3CA exonic mutants, exon 20 mutation was specifically associated with features of the MSI-H/ sessile-serrated pathway including MSI-H, CIMP-H, and BRAF mutation. The association of exon 20 mutation with mucinous histology observed in our cohort and the suggestive decrease in stage III as compared with stage I/II cancers in the combined cohorts are further consistent with the established characteristics of this pathway (41,42). In contrast, PIK3CA exon 9 mutation was more strongly associated with KRAS mutation, and showed overrepresentation in proximal tumors with MSS/CIMP-0/KRAS mut /BRAF wt or MSS/CIMP-L/KRAS mut /BRAF wt genotype. The molecular basis for these differential exon-specific associations remains to be elucidated, but may be related to these 2 classes of mutations causing gain of p110a function through different mechanisms. Exon 9-mutated p110a has been shown to induce cell transformation independently of binding to p85 but requires interaction with RAS-GTP, whereas exon 20-mutated p110a is active in the absence of RAS-GTP binding but highly dependent on the interaction with p85 (43).
Preclinical and clinical studies suggest that PIK3CA mutation and PTEN loss of protein are important predictors of resistance to MEK inhibitors and anti-EGFR antibody therapy (12)(13)(14), and drug combination studies are proposed on the basis of PI3K and MAPK pathway mutation status. The latter include combinations of MAP2K and PI3K or mTOR inhibitors for KRAS/PIK3CA, KRAS/PTEN, and BRAF/PIK3CA double-mutant tumors, MAP2K or BRAF and PI3K or mTOR inhibitors for BRAF/PTEN double-mutant tumors, and PI3K or mTOR inhibitors for PIK3CA/PTEN double-mutant tumors (15). Optimization of the design of such clinical trials for colorectal cancer requires a detailed knowledge of the prevalence of these respective mutation genotypes. Here, we estimate that approximately 6.0% of sporadic colorectal cancers are KRAS/PIK3CA and 1.5% BRAF/PIK3CA double-mutant (integrated analysis), and 1.5% are KRAS/PTEN, 1.9% BRAF/PTEN, and 1.2% PIK3CA/PTEN double-mutant (this study). The low frequencies of these double-mutant cases underscore the need for collaborative international efforts to undertake such drug combination studies.
In our cohort, PIK3CA, PTEN, or overall PI3K pathway mutation status were not associated with prognosis of resected stage II/III colorectal cancer, and similar results were obtained when analyzing PIK3CA exon 9 or 20 status separately. Findings were similar when separating patients into groups who did and did not receive adjuvant chemotherapy. Our results for overall PIK3CA mutation contrast with some smaller previous studies reporting shorter RFS for PIK3CA-mutated stage II/III colorectal cancer (27) and inferior time to local failure for stage I-III rectal cancer (44). However, in line with our findings a recent analysis of 1,170 patients with stage I-III colorectal cancer did not find an association between PIK3CA mutation status and cancer-specific survival, either overall or for exon 9 or 20 alone, although adjuvant treatment details were not available (32). Taken together, the current evidence does not support a role for PIK3CA and/or PTEN mutation as a prognostic biomarker for colorectal cancer. Potential caveats to our findings include that mutation screening was limited to PIK3CA mutation hotspots and certain PTEN exons. Although the selected regions have been shown to comprise the majority of mutations in colorectal cancer (www.sanger.ac.uk/genetics/CGP/cosmic), this may have impacted on the power to detect significant clinicopathologic, molecular, and outcome associations. Furthermore, loss of PTEN protein expression, observed in approximately 30% of colorectal cancers (6)(7)(8), is a documented alternative to mutation, and our study will therefore have underestimated the overall frequency of PTEN inactivation. Although no significant interstudy heterogeneity was observed in our integrated multicohort analysis for molecular variables, some heterogeneity was evident for patient gender.
In conclusion, our data suggest a model in which PIK3CA exon 20 and PTEN mutation are both associated with the sessile-serrated pathway of tumorigenesis (proximal/ MSI-H/CIMP-H/BRAF mut ), whereas exon 9 (and to a lesser extent exon 20) mutation seems to be associated with the so-called traditional serrated pathway (proximal/CIMP-L/ KRAS mut ; Fig. 3). These findings highlight the PI3K pathway as a preferential therapeutic target in these distinct colorectal cancer subtypes, a contention supported by drug treatment studies in colorectal cancer cell lines showing increased sensitivity of MSI-H cases to the PI3K pathway inhibitors rapamycin and LY-294002 (45). Our results underscore the importance of considering mutation-associated phenotypic and molecular heterogeneity, both between and within individual cancer genes, in the development of molecularly targeted therapies.

Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.