PIK3CA Mutation Is Associated With Poor Prognosis Among Patients With Curatively Resected Colon Cancer

doi:10.1200/JCO.2008.18.6544

J Clin Oncol. 2009 March 20; 27(9): 1477–1484.

Published online 2009 February 23. doi: 10.1200/JCO.2008.18.6544

PMCID: PMC2659340

PIK3CA Mutation Is Associated With Poor Prognosis Among Patients With Curatively Resected Colon Cancer

Shuji Ogino, Katsuhiko Nosho, Gregory J. Kirkner, Kaori Shima, Natsumi Irahara, Shoko Kure, Andrew T. Chan, Jeffrey A. Engelman, Peter Kraft, Lewis C. Cantley, Edward L. Giovannucci, and Charles S. Fuchs

From the Department of Medical Oncology, Dana-Farber Cancer Institute; Departments of Pathology and Medicine, Brigham and Women's Hospital and Harvard Medical School; Departments of Epidemiology and Biostatistics, Harvard School of Public Health; Channing Laboratory, Gastrointestinal Unit, Massachusetts General Hospital; Massachusetts General Hospital Cancer Center; and Department of Systems Biology, Beth Israel Deaconess Medical Center, Boston, MA.

Corresponding author: Shuji Ogino, MD, PhD, Center for Molecular Oncologic Pathology, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, 44 Binney St, Room JF-215C, Boston, MA 02115 USA; e-mail: shuji_ogino/at/dfci.harvard.edu.

Received June 16, 2008; Accepted October 17, 2008.

This article has been cited by other articles in PMC.

Abstract

Purpose

PIK3CA mutation and subsequent activation of the AKT pathway play an important role in colorectal carcinogenesis. However, little is known about the prognostic role of PIK3CA mutation in colon cancer.

Patients and Methods

Using 450 resectable colon cancers (stage I to III) in two independent prospective cohorts, we detected PIK3CA mutation in 82 tumors (18%) by pyrosequencing. Cox proportional hazards models were used to calculate hazard ratios (HRs) of colon cancer–specific and overall mortalities, adjusted for patient characteristics and tumoral molecular features, including the CpG island methylation phenotype, microsatellite instability (MSI), LINE-1 hypomethylation, and p53, CIMP, KRAS and BRAF mutation.

Results

Compared with patients with PIK3CA wild-type tumors, those with PIK3CA-mutated tumors experienced an increase in colon cancer–specific mortality according to univariate analysis (HR = 1.64; 95% CI, 0.95 to 2.86), which persisted after adjusting for other known or potential risk factors for cancer recurrence (including MSI; multivariate HR = 2.23; 95% CI, 1.21 to 4.11). The effect of PIK3CA mutation on cancer survival seemed to differ according to KRAS mutational status. Among patients with KRAS wild-type tumors, the presence of PIK3CA mutation was associated with a significant increase in colon cancer–specific mortality (HR = 3.80; 95% CI, 1.56 to 9.27). In contrast, PIK3CA mutation conferred no significant effect on mortality among patients with KRAS-mutated tumors (HR = 1.25; 95% CI, 0.52 to 2.96).

Conclusion

Among patients who undergo a curative resection of colon cancer, PIK3CA mutation is associated with shorter cancer-specific survival. The adverse effect of PIK3CA mutation may be potentially limited to patients with KRAS wild-type tumors.

INTRODUCTION

Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is thought to play a critical role in the development of a variety of human malignancies, and several efforts are underway to define therapeutic inhibitors of the pathway.^1,2 PI3K interacts with phosphatidylinositol-3-phosphate at the membrane and catalyzes the phosphorylation of AKT, which activates the downstream signaling pathway.¹ The PIK3CA gene encodes the catalytic subunit p110 alpha of PI3K.^1,2 Mutant PIK3CA stimulates the AKT pathway and promotes cell growth in various cancers, including colon cancer.³ In addition, PI3K and AKT regulate the mTOR (FRAP1) pathway, which plays a pivotal role in the link between energy balance and tumor cell growth.^1–4

PIK3CA mutations have been described in 10% to 30% of colon cancers,^5–12 and are associated with KRAS mutation^8,13 and microsatellite instability (MSI).¹² In one small previous study of patients with colorectal cancer, PIK3CA mutation was associated with an inferior prognosis; however, a statistical power was limited in that analysis.¹¹ A second study in colorectal cancer suggested that, in aggregate, the presence of mutations in PIK3CA, KRAS, or BRAF conferred a worse patient outcome, but the effect of mutations in PIK3CA alone was not reported.⁸ Thus the relation of PIK3CA mutation to patient outcome in colon cancer still remains uncertain.

Among patients with curatively resected colon cancer who were participating in two large ongoing observational cohort studies, we examined the effect of PIK3CA mutations on patient survival. Because we concurrently assessed other tumoral molecular alterations, including p53, KRAS, and BRAF mutations, MSI, and the CpG island methylator phenotype (CIMP), we could evaluate the independent effect of PIK3CA mutation after controlling for these related molecular events.

PATIENTS AND METHODS

Study Population

We used the databases of two independent prospective cohort studies, the Nurses' Health Study (N = 121,700 women observed since 1976)^14,15 and the Health Professionals Follow-Up Study (N = 51,500 men observed since 1986).¹⁵ Every 2 years, participants have been sent follow-up questionnaires to update information on potential risk factors and to identify newly diagnosed cancer and other diseases. We calculated body mass index (BMI, kilograms per square meter) using self-reported height from the baseline questionnaire and weight from the biennial questionnaire that immediately preceded the diagnosis of colon cancer. In validation studies in both cohorts, self-reported anthropometric measures were well correlated with measurements by trained technicians (r > 0.96).¹⁶ On each biennial follow-up questionnaire, participants were asked whether they had a diagnosis of colon cancer during the previous 2 years. When a participant (or next of kin for decedents) reported colon cancer, we sought permission to obtain medical records. Study physicians, although blinded to exposure data, reviewed all records related to colon cancer, and recorded American Joint Committee on Cancer tumor stage and tumor location. For nonresponders, we searched the National Death Index to discover deaths and ascertain any diagnosis of colon cancer that contributed to death or was a secondary diagnosis. Approximately 96% of all incident colon cancer cases were identified through these methods. We collected paraffin-embedded tissue blocks from hospitals where patients with colon cancer underwent resections of primary tumors.¹⁵ Tissue sections from all colon cancer cases were reviewed and confirmed by a pathologist (S.O.). Tumor grade was categorized as high (≤ 50% glandular area) or low (> 50% glandular area).¹⁷ We excluded cases for which only biopsy tissue was available, because there was a small amount of tissue left after pathologic examination at original hospitals, and differential availability of results from biopsies might cause bias. On the basis of availability of tissue samples, we included a total of 450 stage I to III colon cancer cases (206 from the men's cohort and 244 from the women's cohort) diagnosed up to 2002. Written informed consent was obtained from all study subjects. This study was approved by the Human Subjects Committees at Brigham and Women's Hospital and the Harvard School of Public Health.

Measurement of Mortality

Patients were observed until death or June 2006, whichever came first. Ascertainment of deaths included reporting by the family or postal authorities. In addition, the names of persistent nonresponders were searched in the National Death Index. The cause of death was assigned by physicians blinded to information on lifestyle exposures and molecular changes in colon cancer. In patients who died as a result of colon cancer not previously reported, we obtained medical records with permission from next of kin. More than 98% of deaths in the cohorts were identified by these methods.¹⁸

Genomic DNA Extraction and Pyrosequencing of PIK3CA, KRAS, and BRAF

Genomic DNA from paraffin-embedded tissue was extracted, and whole genome amplification of genomic DNA was performed by polymerase chain reaction (PCR) using random 15-mer primers.¹⁹ PCR and pyrosequencing targeted for PIK3CA exons 9 and 20 (mutation hotspots; Fig 1), KRAS codons 12 and 13, and BRAF codon 600 were performed as previously described.^13,19,20

Fig 1.

PIK3CA mutations in colon cancer detected by pyrosequencing (antisense strand). (A) Wild-type exon 9 by the 9-RS2 primer. (B) The c.1633G>A mutation (arrow) causes a shift in reading frame and results in new peaks (arrowheads), which serves as (more ...)

Real-Time PCR to Determine CIMP Status

Sodium bisulfite treatment on tumor DNA and subsequent real-time PCR (MethyLight) assays were validated and performed as previously described.²¹ We quantified promoter methylation in eight CIMP-specific genes (CACNA1G, CDKN2A [p16], CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1).^22–24 The PCR condition was initial denaturation at 95°C for 10 minutes followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. CIMP-high was defined as six or more of eight methylated promoters using the eight-marker CIMP panel, CIMP-low was defined as one to five methylated promoters, and CIMP-0 was defined as 0 of eight methylated promoters, according to previously established criteria.²³

Pyrosequencing to Measure LINE-1 Methylation

To accurately quantify relatively high long interspersed nucleotide element-1(LINE-1) methylation levels, we used pyrosequencing as previously described.²⁵ LINE-1 methylation level measured by pyrosequencing has been shown to correlate well with overall 5-methylcytosine level (ie, global DNA methylation level) in tumor cells.^26,27

MSI Analysis

MSI status was determined as previously described.²³ In addition to the recommended MSI panel consisting of D2S123, D5S346, D17S250, BAT25, and BAT26,²⁸ we used BAT40, D18S55, D18S56, D18S67, and D18S487 (ie, 10-marker panel).²³ A high degree of MSI (MSI-high) was defined as the presence of instability in ≥ 30% of the markers, MSI-low was defined as the presence of instability in less than 30% of the markers, and microsatellite stability as no unstable marker.

Tissue Microarrays and Immunohistochemistry for p53

Tissue microarrays were constructed and immunohistochemistry was performed as previously described.^15,29 Appropriate positive and negative controls were included in each run of immunohistochemistry. All immunohistochemically stained slides were interpreted by a pathologist (S.O.) blinded from clinical and other laboratory data. A random sample of 118 tumors were re-examined for p53 by a second observer (K.N.) unaware of other data. The concordance between the two observers was 0.87 (κ = 0.75; P < .0001), indicating substantial agreement.

Statistical Analysis

We used Cox proportional hazards models to calculate hazard ratios (HRs) of death according to tumoral PIK3CA status, unadjusted and adjusted for age, sex, BMI, year of diagnosis, tumor location, stage, grade, and statuses of MSI, CIMP, LINE-1, KRAS, BRAF, and p53. For analyses of colon cancer–specific mortality, death as a result of colon cancer was the primary end point and deaths as a result of other causes were censored. To adjust for potential confounding, age and year of diagnosis were used as continuous variables, and all of the other covariates were used as categoric variables. We dichotomized tumor location (proximal v distal), BMI (< 30 v ≥ 30 kg/m²), tumor grade (high v low), CIMP (high v low/0), MSI (high v low/microsatellite stability), p53 (positive v negative), KRAS (mutated v wild-type), and BRAF (mutated v wild-type). There were three categories for LINE-1 methylation (≥ 70%, 55% to 70%, and < 55%). When there was missing information on tumor location (0.7% missing), tumor grade (0.7% missing), LINE-1 (2.7% missing), MSI (0.2% missing), p53 (0.9% missing), KRAS (0.4% missing), or BRAF (2.7% missing), we assigned a separate (“missing”) indicator variable and included those cases in the multivariate analysis models. We confirmed that excluding cases with a missing variable did not significantly alter results (data not shown). An interaction was assessed by including the cross-product of the PIK3CA variable and another variable of interest in a multivariate Cox model, and the likelihood ratio test was performed. To assess an interaction of PIK3CA and stage, we dichotomized tumor stage (I to II v III; P for interaction = .67), as well as treated stage as a linear ordinal variable (from I to III; P for interaction = .84), to confirm no significant interaction. The Kaplan-Meier method was used to describe the distribution of colon cancer–specific and overall survival time, and the log-rank test was performed. The χ² test was used to examine an association between categoric variables. The t test assuming unequal variances was performed to compare mean age and mean LINE-1 methylation level. All analyses used SAS version 9.1 (SAS Institute, Cary, NC), and all P values were two-sided.

RESULTS

PIK3CA Mutation in Colon Cancer

Among 450 patients who had undergone a curative resection of stage I to III colon cancer, we detected PIK3CA mutations in 82 (18%) by pyrosequencing technology (Fig 1). We assessed characteristics of clinical, pathologic, and molecular features of colon cancer cases, according to PIK3CA mutational status (Table 1). Compared with PIK3CA wild-type tumors, PIK3CA-mutated tumors were more likely to demonstrate KRAS mutation (P < .0001) and less likely to show p53 expression (P = .01). PIK3CA mutation was not significantly associated with any clinical features examined.

Table 1.

Clinical and Molecular Characteristics According to PIK3CA Mutational Status in Colon Cancer

PIK3CA Mutation and Patient Survival

We assessed the influence of PIK3CA mutation on patient survival in resectable colon cancer (stage I to III). Compared with patients with PIK3CA wild-type tumors, those with PIK3CA-mutated tumors experienced an increase in cancer-specific mortality (univariate hazard ratio [HR] = 1.64; 95% CI, 0.95 to 2.86; Table 2), although statistical significance was not reached. In the multivariate Cox model adjusting for potential predictors of patient outcome, PIK3CA mutation was associated with a significant increase in colon cancer–specific mortality (HR = 2.23; 95% CI, 1.21 to 4.11). The increase in the effect of PIK3CA mutation on survival in the multivariate analysis was mainly the result of adjusting for tumor stage, p53, and BMI; when we adjusted for tumor stage, p53, and BMI, the HR for colon cancer–specific mortality for COX-2–positive tumors was 2.03 (95% CI, 1.15 to 3.57). In contrast, PIK3CA mutation was not significantly associated with overall mortality in both univariate and multivariate analyses.

Table 2.

PIK3CA Mutation and Mortality in Stage I to III Colon Cancer

We further examined 5-year survival according to PIK3CA mutational status. Colon cancer–specific survival at 5 years was 90% for patients with PIK3CA wild-type tumors, as compared with 82% for those with PIK3CA-mutated tumors (log-rank P = .075; Fig 2). Overall survival was not significantly different according to PIK3CA status (84% in PIK3CA wild-type tumors v 78% in PIK3CA-mutated tumors; log-rank P = .97).

Fig 2.

Kaplan-Meier curves for (A) colon cancer–specific survival and (B) overall survival in stage I to III colon cancer according to PIK3CA status.

We also assessed PIK3CA exon 9 mutations and exon 20 mutations separately. There was, however, no significant difference in mortality between exon 9 and exon 20 mutations (data not shown).

Combined PIK3CA and KRAS Mutational Status and Prognosis

Given the potential interaction between the RAS and PI3K-AKT signaling pathways, we examined the influence of KRAS mutational status on the adverse prognostic effect of mutations in PIK3CA (Table 3). Among patients with KRAS wild-type tumors, the presence of PIK3CA mutation was associated with a significant increase in colon cancer–specific mortality (multivariate HR = 3.80; 95% CI, 1.56 to 9.27). In contrast, PIK3CA mutation conferred no significant effect on patient outcome among patients with KRAS-mutated tumors. However, a test for interaction between KRAS and PIK3CA did not reach statistical significance (P for interaction = .13).

Table 3.

Effect of PIK3CA Mutation on Mortality in Stage I to III Colon Cancer by Strata of KRAS Mutational Status

Stratified Analysis of PIK3CA Mutation and Prognosis

We further examined the influence of PIK3CA mutation on colon cancer–specific mortality across strata of other potential predictors of patient survival (Fig 3). The increased risk of cancer-specific death was apparent in all subgroups (although a wide 95% CI was noted in some subgroups). Notably, the effect of PIK3CA mutation was not significantly modified by age, sex, year of diagnosis, tumor location, stage, grade, BRAF, p53, MSI, LINE-1, or CIMP (all P for interaction ≥ .13).

Fig 3.

Stratified analysis of PIK3CA mutation and colon cancer–specific mortality. Log_e (adjusted HR) and 95% CI for PIK3CA-mutated tumors (v PIK3CA wild-type tumors) in various strata are shown. CIMP, CpG island methylator phenotype; HR, hazard ratio; (more ...)

DISCUSSION

PIK3CA mutation and subsequent activation of the AKT pathway are considered to play an important role in colon cancer development and clinical behavior.^1,5,8,11 When activated by these mechanisms, the PI3K/AKT pathway provides important downstream signals that promote cellular proliferation, survival, invasion, and neoangiogenesis.^1,2 In preclinical studies, inhibitors of PI3K and AKT have demonstrated antitumor activity in colon cancer cells, both alone and in combination with other agents, suggesting their possible utility in patients with colon cancer.^30,31 We therefore examined the prognostic significance of PIK3CA mutation among 450 patients who had undergone a curative resection of colon cancer. We found that tumoral PIK3CA mutation was associated with worse survival. The adverse effect of PIK3CA mutation on prognosis seemed to be robust and consistent across most strata of clinical and tumoral predictors of patient outcome. However, we did observe that the adverse effect of PIK3CA mutation was potentially limited to patients with KRAS wild-type tumors.

Detection of molecular aberrations in neoplastic lesions is important in cancer research,^32–37 and molecular classification is increasing important in colon cancer.^38,39 We used pyrosequencing assay to detect PIK3CA mutation in colon cancer, as we previously developed the assay.¹³ Pyrosequencing assay for PIK3CA mutation detection is certainly useful, because most activating PIK3CA mutations cluster in hotspots of exons 9 and 20, affecting the functionally important helical and kinase domains.^24,40 We also used pyrosequencing for KRAS and BRAF mutation detection and for quantification of LINE-1 methylation level, as we have previously described.^19,25 Pyrosequencing is a sensitive and quantitative sequencing assay and can reliably detect a mutant allele of low abundance (5% to 10% mutant) among wild-type alleles,¹⁹ which is a rather common situation in solid tumors. In fact, distribution and frequencies of various PIK3CA mutations in our sample are compatible with data in the previous studies.^5–8,11,12 Moreover, we have successfully demonstrated that PIK3CA mutation in colon cancer is associated with KRAS mutation and inversely with p53 expression.¹³

Little has been known regarding the impact of PIK3CA mutation in colon cancer on patient survival. One study has shown that PIK3CA mutation seems to predict shorter survival in colorectal cancers¹¹; however, there were only 18 PIK3CA-mutated tumors, and the number of deaths was not reported. Another recent study has shown that the presence of at least one mutation in PIK3CA, BRAF, or KRAS predicts poor survival in a population-based colon cancer sample⁸; however, the effect of PIK3CA mutation (by itself) on survival independent of clinical and other molecular predictors of outcome was not described. In contrast, using PIK3CA pyrosequencing technology, mutations in PIK3CA were detected in 82 (18%) of 450 patients and conferred an inferior survival, independent of clinical and other molecular predictors of patient outcome. In addition, we have also shown that the adverse effect of PIK3CA mutation on cancer mortality seemed to persist in KRAS wild-type tumors, whereas the effect of PIK3CA mutation was markedly attenuated among KRAS-mutated tumors. A growing body of evidence suggests that KRAS mutational status may define response to receptor tyrosine kinase inhibitors in a variety of human cancers, including colon cancer.^32,41–43 It is conceivable that the activation of the PI3K/AKT pathway may play much more important role in tumor aggressiveness in a background where the KRAS oncogene is not constitutively activated by a somatic mutation. Nonetheless, although our findings were generated from two prospective cohort studies, the results remain hypothesis-generating and require independent confirmation. Moreover, future clinical trials of therapeutic inhibitors of the PI3K/AKT pathway may consider KRAS mutational status as a predefined stratification factor.

In our cohorts, data on cancer treatment were limited. Nonetheless, it is unlikely that chemotherapy use differed according to tumoral PIK3CA status, because such data were not available to patients or treating physicians. In addition, beyond cause of mortality, data on cancer recurrences were not available in these cohorts. Nonetheless, given that the median survival for metastatic colon cancer was approximately 10 to 12 months during much of the time period of this study,⁴⁴ colon cancer–specific survival should be a reasonable surrogate for cancer-specific outcomes.

In summary, our large cohort study suggests that PIK3CA mutation is associated with poor survival in resectable stage I to III colon cancer. These findings may have considerable clinical implications. Considerable effort has been focused on defining therapeutic inhibitors of the PI3K/AKT pathway in colon cancer as well as other common malignancies. Future studies are needed to confirm this association as well as to elucidate exact mechanisms by which PIK3CA mutation affects tumor behavior.

Acknowledgment

We thank the Nurses' Health Study and Health Professionals Follow-Up Study cohort participants who have generously agreed to provide us with biologic specimens and information through responses to questionnaires; hospitals and pathology departments throughout the United States for providing us with tumor tissue materials; Janina Longtine for assistance in sequencing analysis; and Frank Speizer, Walter Willett, Graham Colditz, Susan Hankinson, Meir Stampfer, and many other staff members who implemented and have maintained the cohort studies.

Glossary Terms


AKT:	A transforming serine-threonine kinase involved in cell survival.

BRAF:	BRAF is an isoform of RAF. Raf proteins (Raf-1, A-Raf, B-Raf) are intermediate to Ras and MAPK in the cellular proliferative pathway. Raf proteins are typically activated by Ras via phosphorylation, and activated Raf proteins in turn activate MAPK via phosphorylation. However, Raf proteins may also be independently activated by other kinases.

KRAS:	The gene that encodes K-Ras, a protein that is a member of the small GTPase superfamily, in which a single amino acid substitution results in an activating mutation. Alternative splicing gives rise to variants encoding two isoforms that differ in the C-terminal region.

MSI (microsatellite instability):	Microsatellites are repeating units of 1-4 DNA base pairs that are distributed widely throughout the genome and have a high degree of repeat length variation in the population. Their length remains stable with cell division and inheritance so they may be used as molecular markers of cell lineage, in population genetic studies or paternity testing. Defects in the genes involved in DNA mismatch repair result in genomic instability that may be detected as MSI, an alteration in the length of the microsatellites from cell to cell.

mTOR:	The mammalian target of rapamycin belongs to a protein complex (along with raptor and GβL) that is used by cells to sense nutrients in the environment. mTOR is a serine/threonine kinase that is activated by Akt and regulates protein synthesis on the basis of nutrient availability. It was discovered when rapamycin, a drug used in transplantation, was shown to block cell growth presumably by blocking the action of mTOR.

PI3K:	Phosphatidylinositol-3 phosphate kinase (PI3K) adds a phosphate group to PI3, which is a downstream signaling molecule involved in survival/proliferative pathways mediated by growth factors such as the EGF and the PDGFs.

PIK3CA:	The catalytic subunit of phosphatidylinositol 3-kinase involved in the generation of PIP3 which, in turn, leads to the activation of Akt and other oncogenic kinases. Mutations in the PIK3CA gene have been found in a number of cancers, including ovarian, breast, colon, and lung carcinomas. (See PI3K and Akt/PKB).

CIMP:	The CpG island methylator phenotype is characterized by widespread, concordant promoter CpG island methylation, resulting in silencing of many tumor suppressor genes. CIMP-high (high degree of CIMP) in colorectal cancer is associated with old age, female sex, proximal colon, BRAF mutation, and MSI (microsatellite instability), and inversely with chromosomal instability, TP53 mutation, WNT/β-catenin (CTNNB1) activation, and genome-wide DNA hypomethylation.

CpG island methylator phenotype:	Also known as “CIMP.” CIMP is characterized by widespread, concordant promoter CpG island methylation, resulting in silencing of many tumor suppressor genes. CIMP-high (high degree of CIMP) in colorectal cancer is associated with old age, female sex, proximal colon, BRAF mutation, and MSI (microsatellite instability), and inversely with chromosomal instability, TP53 mutation, WNT/β-catenin (CTNNB1) activation, and genome-wide DNA hypomethylation.

FRAP1:	The HUGO Gene Nomenclature Committee (HGNC: http://www.genenames.org/)–approved official gene symbol for FK506 binding protein 12-rapamycin associated protein 1 or so-called mammalian target of rapamycin, or mTOR.

Footnotes

Supported by the National Institutes of Health Grants No. P01 CA87969, P01 CA55075, P50 CA127003, and K07 CA122826 (to S.O.); the Bennett Family Fund; and the Entertainment Industry Foundation. K.N. was supported by a fellowship grant from the Japanese Society for Promotion of Science.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health. Funding agencies did not have any role in the design of the study; the collection, analysis, or interpretation of the data; the decision to submit the manuscript for publication; or the writing of the manuscript.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

The author(s) indicated no potential conflicts of interest.

AUTHOR CONTRIBUTIONS

Conception and design: Shuji Ogino, Jeffrey A. Engelman, Lewis C. Cantley, Edward L. Giovannucci, Charles S. Fuchs

Financial support: Shuji Ogino, Katsuhiko Nosho, Charles S. Fuchs

Administrative support: Gregory J. Kirkner, Lewis C. Cantley, Edward L. Giovannucci, Charles S. Fuchs

Provision of study materials or patients: Shuji Ogino, Katsuhiko Nosho, Gregory J. Kirkner, Kaori Shima, Natsumi Irahara, Shoko Kure, Andrew T. Chan, Jeffrey A. Engelman, Charles S. Fuchs

Collection and assembly of data: Shuji Ogino, Katsuhiko Nosho, Gregory J. Kirkner, Kaori Shima, Natsumi Irahara, Shoko Kure, Andrew T. Chan, Jeffrey A. Engelman, Charles S. Fuchs

Data analysis and interpretation: Shuji Ogino, Katsuhiko Nosho, Gregory J. Kirkner, Kaori Shima, Natsumi Irahara, Shoko Kure, Andrew T. Chan, Jeffrey A. Engelman, Peter Kraft, Lewis C. Cantley, Edward L. Giovannucci, Charles S. Fuchs

Manuscript writing: Shuji Ogino, Katsuhiko Nosho, Peter Kraft, Edward L. Giovannucci, Charles S. Fuchs

Final approval of manuscript: Shuji Ogino, Katsuhiko Nosho, Gregory J. Kirkner, Kaori Shima, Natsumi Irahara, Shoko Kure, Andrew T. Chan, Jeffrey A. Engelman, Peter Kraft, Lewis C. Cantley, Edward L. Giovannucci, Charles S. Fuchs

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