PIK3CA mutation and methylation influences the outcome of colorectal cancer
- Authors:
- Published online on: December 29, 2011 https://doi.org/10.3892/ol.2011.544
- Pages: 565-570
Abstract
Introduction
Colorectal cancer (CRC) is among the most common types of cancer in both males and females and is associated with high mortality, particularly at advanced stages (1). Markers for defining individual risk signatures in CRC patients are of great clinical value, as they may allow for targeted therapies to improve the outcomes of CRC patients.
CRC arises through the accumulation of multiple genetic and epigenetic changes. Somatic mutations in APC, BRAF, KRAS, PIK3CA and TP53, as well as in other genes, have been frequently observed in CRC and are thought to drive colorectal tumorigenesis (2). We previously examined the mutation of genes in the EGFR pathway and found that the PIK3CA mutation is associated with poor prognosis in CRC patients (8,11).
In addition to genetic alterations, epigenetic silencing is a prevalent mechanism by which abnormal gene inactivation may occur in cancer and is involved in initiation and promotion (3). A predominant mode of epigenetic alteration in cancer is gene silencing via the hypermethylation of the CpG island promoter. Hypermethylation results in abnormal silencing of a number of tumor suppressors in numerous human malignancies including CRC (3).
It has been suggested that epigenetic abnormalities are able to cooperate with genetic alterations to cause aberrant gene function and result in cancer (4). Thus, in the present study, we examined KRAS and PIK3CA mutations and the methylation status of BNIP3, p16 and hMLH1 and analyzed the correlation between these molecular alterations and the clinicopathological characteristics of CRC. We aimed to clarify whether a combination of genetic and epigenetic alterations could be used to classify CRC patients in relation to their clinicopathological characteristics and outcomes.
Materials and methods
Patients and tissue samples
After providing informed consent, a total of 165 CRC patients who underwent surgical resection at Tokyo Medical and Dental University Hospital between March 2000 and April 2003 were recruited in this study. This study was approved by the institutional review board of the Tokyo Medical and Dental University. The study population (Table I) consisted of 62 females and 103 males, with a mean age of 64.5±10.3 years (range, 37–89). Sixty-two cancers were located in the proximal site and 103 were located in the distal site, including the rectum. Histological classification and tumor staging were performed according to the International Union Against Cancer Tumor-Node-Metastasis classification guidelines. No patient received preoperative chemotherapy or radiotherapy. Following surgery, patients with stage III CRC received oral or intravenous 5-fluorouracil (FU)-based adjuvant chemotherapy and patients with stage IV tumors received 5-FU-based systemic chemotherapy without radiotherapy. Patients were prospectively followed up following surgery for a median period of 64 months (range, 0.07–107.6). Resected specimens were fixed in 10% neutral buffered formalin and embedded in paraffin. For all cases, archival hematoxylin and eosin (H&E) slides of primary tumors were retrieved and reviewed to confirm pathological features.
 | Table IClinicopathological characteristics according to the mutation and methylation status of each gene. |
DNA extraction
Tissues were cut into 10-μm sections from paraffin-embedded blocks. After specimens were deparaffinized and washed, tumor tissue was manually dissected for comparison with H&E slides. The same amount of dissected tissue was used for each case. Genomic DNA was extracted using standard proteinase K (Invitrogen, Carlsbad, CA, USA) digestion, phenol/chloroform extraction, and ethanol precipitation, as previously described (5).
Mutation analysis
Exon 1 of the KRAS gene and exons 9 and 20 of the PIK3CA gene were selected for mutation analysis, since mutations are known to cluster in these regions (6,7). Primer sequences and PCR conditions have been reported previously (8). Following the purification of PCR products using a Microcon YM-100 Centrifugal Filter (Millipore Corporation, Billerica, MA, USA) and Centri-Sep Columns (Princeton Separations, Adelphia, NJ, USA), direct sequencing was carried out using the Big Dye Terminator Cycle Sequencing kit (Applied Biosystems, Foster City, CA, USA) and analyzed using an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems). Somatic mutations were further validated by independent PCR amplification and sequencing and matched with normal mucosa.
MethyLight analysis
Sodium bisulfite conversion and DNA recovery was performed using EpiTect Bisulfite (Qiagen, Hilden, Germany). Following sodium bisulfite conversion, genomic DNA was analyzed using the MethyLight technique, a fluorescence-based real time PCR (Q-PCR) assay (9) and the ABI Prism 7300 Real Time PCR system (Taqman; Applied Biosystems). Four sets of primers and probes designed specifically for bisulfite-converted DNA were used. Three sets were used to detect methylation in the gene of interest and the remaining set served as a reference for β-actin (ACTB) to normalize for input DNA. Reference primers and probes were designed in a region of the ACTB gene lacking CpG dinucleotides, thus allowing for equivalent amplification regardless of methylation levels. Primer and probe sequences were previously reported (10,11). SssI-treated HCT-15 DNA was used as a fully methylated positive control (100% methylation ratio). Parallel TaqMan PCR reactions were performed using primers specific for the bisulfite-converted methylated sequence of a particular locus and with ACTB reference primers. In each case, triplicate threshold cycle (Ct) values were obtained and averaged and expression levels were evaluated using the 2−ΔΔCt method. As an internal standard, each sample was normalized to its ACTB content and compared with the gene expression level of SssI-treated HCT-15 DNA as positive controls (calibration sample) as follows: 2ΔΔCt where ΔΔCt=(Ct-target-Ct-reference) treated sample - (Ct-target-Ct-reference) calibrator sample. We defined the percentage of the fully methylated reference (PMR) to be 2ΔΔCt ×100%. To define p16 or hMLH1 methylation status, a PMR cut-off value of 4 was used based on previously validated data (9). Based on the distribution of PMR values in normal colon epithelial tissue (10), a PMR cut-off value of >0 was used to define positive methylation status for BNIP3.
Statistical analysis
Statistical analysis was carried out using StatView Software (version 5.0). To estimate the differences among the groups, the Chi-square test, Fisher’s exact test, Student’s t-test and log-rank test were used where appropriate. The Kaplan-Meier method was used to estimate survival. Survival was calculated beginning from the date of surgery. Prognostic factors were estimated using multivariate analysis and the Cox proportional hazards model. P<0.05 was considered to indicate a statistically significant result.
Results
Mutation of KRAS and PIK3CA and methylation of BNIP3, p16 and hMLH1
KRAS mutations in exon 1 were observed in 32% of cases and PIK3CA mutations in exons 9 and 20 in 12% of cases. Methylation frequencies of the examined genes were 58% for BNIP3, 33% for p16 and 21% for hMLH1. Although hMLH1 methylation was significantly associated with distant metastasis (P=0.0005), other associations were not observed between clinicopathological characteristics and the mutation or methylation status of individual genes (Table I).
Correlation between mutation and methylation
Associations among gene mutations and gene methylations are shown in Table II. The KRAS mutation was significantly associated with BNIP3 (P<0.0001) and p16 methylation (P=0.04) as well as with the number of methylated genes (P=0.0082). p16 methylation was associated with BNIP3 methylation (P<0.0001) and hMLH1 methylation (P=0.03).
Correlation between molecular markers and outcomes
The correlation between the molecular parameters and disease-specific survival (DSS) were analyzed (Table III). Patients with hMLH1-methylated tumors had significantly shorter DSS compared with those without methylation [hazards ratio (HR), 2.231; P=0.05], but mutation or methylation of other single genes did not affect DSS. Having mutations in both the KRAS and PIK3CA genes did not correlate with DSS. By contrast, when cases were divided into 2 groups, namely a low methylation group (0 or 1 methylated genes) and a high methylation group (2 or 3 methylated genes), the high methylation group had significantly shorter DSS than the low methylation group (HR, 2.681; P=0.01). Integration of the KRAS mutation and methylation status of multiple genes did not reveal an association with DSS. However, CRCs were divided into 4 groups: CRC containing i) PIK3CA mutation and high methylation (5.2%); ii) PIK3CA mutation and low methylation (8.1%); iii) no mutation and high methylation (31.1%); and iv) no mutation and low methylation (55.6%). Patients with the PIK3CA mutation and/or high methylation (groups i, ii and iii) had a significantly poorer outcome in DSS compared with those with wild-type PIK3CA and low methylation (group iv; HR, 2.924; P=0.0087) (P=0.0059, log-rank test; Fig. 1).
Results of the univariate and multivariate analyses using the Cox proportional hazards model are shown in Table IV. Univariate analysis revealed that the PIK3CA mutation and/or high methylation was a significant prognostic factor for DSS, when used with the depth of the tumor, lymph node metastasis, distant metastasis, histological differentiation, lymphatic invasion and vessel invasion. Variables with P<0.05 in the univariate analysis were used for the multivariate analysis. PIK3CA mutation and/or high methylation, as well as distant metastasis and lymphatic invasion, were found to be independent and significant prognostic factors for DSS.
Discussion
In this study, we examined mutations in KRAS and PIK3CA, which comprise components of the EGFR pathway, and the methylation status of BNIP3, p16 and hMLH1 in CRCs to evaluate patients with the PIK3CA mutation and/or 2 or more methylations with a poorer prognosis compared with other patients. Our results suggest that a combination of epigenetic and genetic alterations is a potent biomarker for predicting CRC prognosis.
Integrated analysis of epigenetic and genetic alterations is increasingly significant in cancer research. Since CRC develops as a result of accumulating genetic and epigenetic alterations, the combination of genetic and epigenetic profiles could confer differential clinical phenotypes and potential variability in survival. Ward et al reported that although DNA methylation is associated with a worse outcome in CRC patients, this adverse prognostic effect is lost in methylated tumors with microsatellite instability (12). By integrating genetic and epigenetic analyses, Shen et al revealed that colon cancers correspond to 3 molecularly distinct subclasses of disease, each of which is relatively homogeneous (13). In the present study, CRCs were divided into 4 groups according to PIK3CA mutations and the number of methylated genes: CRCs with i) PIK3CA mutation and 2 or 3 methylations (5.2%); ii) PIK3CA mutation and less than 2 methylations (8.1%); iii) no mutation and 2 or 3 methylations (31.1%); and iv) no mutations and less than 2 methylations (55.6%). The last group was associated with better prognosis than the former 3 groups. CRC patients with the PIK3CA mutation and/or more than 1 methylation had worse outcomes compared with the other groups. This finding suggests that mutation analysis and determining methylation status may provide significant predictive information regarding tumor behavior and clinical outcome in CRC patients.
BNIP3 is a pro-apoptotic member of the Bcl-2 family (14). The expression of BNIP3 is induced by hypoxia, such as that which occurs during cardiac ischemia and in the hypoxic regions of tumors, and it acts against pro-survival proteins, including Bcl-2 and Bcl-xl (15–18). Transcriptional silencing of the BNIP3 gene has recently been observed in multiple human cancer cell lines, including colorectal, gastric, pancreatic, and hematopoietic tumors (19). Murai et al reported that aberrant hypermethylation of BNIP3 was detected in 66% of primary colorectal and 49% of primary gastric cancers, but not in normal tissue; these authors also revealed that BNIP3 is silenced by DNA methylation (20). Akada et al and Erkan et al revealed that loss of BNIP3 expression correlates with poor prognosis and increased chemoresistance in patients with pancreatic cancer (21,22). Manka et al demonstrated that BNIP3 silencing induces or increases the metastatic growth of breast cancer in distant organs (23). Moreover, our previous study also indicated that BNIP3 methylation is associated with poor clinical outcome and chemoresistance in primary CRC (10). These previous studies suggest that reduced BNIP3 expression contributes to carcinogenesis of the colon and rectum and may be a predictive factor for the prognosis of CRC. Therefore, the BNIP3 gene, as well as the hMLH1 and p16 genes, were tested in our methylated gene sets for predicting the outcome of CRC.
Activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway is thought to be critical in CRC development and clinical behavior (6,8,24,25). PIK3CA encodes the p110α catalytic subunit of PI3K and is mutated in 10–32% of CRCs (6,8,24,26–29). PIK3CA mutations elevate kinase activity, thereby activating the PI3K/AKT pathway and contributing to tumorigenesis through decreased apoptosis, loss of contact inhibition and increased tumor invasion (6,30); these factors have been associated with poor outcomes in CRC patients (8,24,31). Although previous studies have investigated the role of the PIK3CA mutation and gene methylation in CRC, the data are inconclusive. Certain studies reported a significantly higher frequency of the PIK3CA mutation in the CpG island methylator phenotype (CIMP)-high tumor compared with CIMP-negative tumors and a significant correlation between the PIK3CA mutation and RASSF2 gene methylation (29,32,33), whereas other studies failed to observe this correlation (11,34). In the present study, although CIMP status was not examined, the PIK3CA mutation did not exhibit an association with the methylation status of the genes examined. Moreover, few studies have described the effect of the combination of the PIK3CA mutation with gene methylation on patient outcome. Ogino et al demonstrated that the effect of the PIK3CA mutation on CRC patient survival is not significantly modified by CIMP (31). Their result was inconsistent with our finding that integration of the PIK3CA mutation and methylation status may be used to predict outcomes for CRC patients. It is possible that the methylated gene set used in this study was different from that used in the Ogino study. The BNIP gene included in our methylated set was not included in CIMP markers used to date. Moreover, there have been no studies regarding the influence of integrating the PIK3CA mutation and methylation status of the BNIP3 gene on outcomes of CRC. It would be useful to examine whether mutating other genes from the EGFR pathway also affects CRC prognosis in cooperation with BNIP3 methylation.
In conclusion, by integrating genetic and epigenetic alterations, we revealed that CRC patients with poor prognosis could be identified. Further studies are necessary to confirm our finding that a combination of the PIK3CA mutation with gene methylation may be useful for predicting outcomes of CRC patients and to elucidate the underlying mechanism behind these findings.
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