TGFBR2 and BAX Mononucleotide Tract Mutations, Microsatellite Instability, and Prognosis in 1072 Colorectal Cancers

Study group

We utilized the database of two prospective cohort studies, the Nurses' Health Study (N = 121,701 women followed since 1976) and the Health Professionals Follow-up Study (N = 51,529 men followed since 1986) [26], [27]. Participants have been sent biennial questionnaires to update information on potential risk factors and to identify newly diagnosed cancers in themselves and their first degree relatives. We collected paraffin-embedded tumor tissue blocks of incident colorectal cancers from hospitals throughout the U.S. where participants with colorectal cancer underwent tumor resection [26], [27]. Clinical characteristics of the cases are described in Table 2 (on the left, under the column heading “All cases”). There was no significant difference in demographic features between cases with tissue available and those without available tissue among our cohort studies [26]. A majority of cases have previously been characterized for statuses of TGFBR2, MSI, CIMP, KRAS, BRAF, PIK3CA and LINE-1 methylation [28], [29], [30], [31]. However, none of our previous studies have analyzed the prognostic significance of mononucleotide mutation of TGFBR2 or BAX. BAX mutation has not been analyzed in any of our previous studies. Thus, this study represents a new study utilizing a resource of the existing materials and database, analogous to novel studies using well-described cell lines (e.g., SW480 cell line) or mouse models (e.g., Apc min mouse model).

Hematoxylin and eosin stained tissue sections from all colorectal cancer cases were reviewed by a pathologist (S.O.) unaware of other data. Tumor differentiation was categorized as well-moderate vs. poor (>50% vs. ≤50% glandular areas). We excluded cases which were preoperatively treated. Based on the availability of adequate follow-up and tumor tissue data, 1072 stage I–IV colorectal cancer cases diagnosed up to 2004 were included in the current study ( Figure 1 ). Patients were observed until death or June 30 2009, whichever came first. Death of a participant was confirmed by the National Death Index. Returning questionnaire indicated informed consent from all study subjects. Informed consent was obtained from all study subjects. Tissue collection and analyses were approved by the Human Subjects Committees at Harvard School of Public Health and Brigham and Women's Hospital.

Microsatellite instability (MSI) analysis and detection of TGFBR2 and BAX mononucleotide tract mutations

DNA was extracted from paraffin embedded tissue. MSI analysis was performed using 10 microsatellite markers (D2S123, D5S346, D17S250, BAT25, BAT26, BAT40, D18S55, D18S56, D18S67 and D18S487) [30]. MSI-high was defined as the presence of instability in ≥30% of the markers, and MSI-low/microsatellite stable (MSS) as instability in 0–29% of markers [30]. Mononucleotide tract mutations of TGFBR2 and BAX were examined in MSI-high tumors. Primers and PCR conditions for TGFBR2 were previously described [28]. Primer sequences for BAX were; 5′-(FAM) ATCCAGGATCGAGCAGGGCG-3′ and 5′-ACTCGCTCAGCTTCTTGGTG-3′. PCR condition was preheat at 95°C for 5 min, 45 cycles (at 94-55-72°C for 30-30-30 sec), and extension at 72°C for 2 min. PCR products were electrophoresed and analyzed by ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA).

Pyrosequencing of KRAS, BRAF and PIK3CA

PCR and Pyrosequencing targeted for KRAS (codons 12 and 13) [32], BRAF (codon 600) [33]and PIK3CA (exons 9 and 20) were performed as previously described [29].

Methylation analyses for CpG islands and LINE-1

Sodium bisulfite treatment and subsequent real-time PCR (MethyLight [34]) were previously validated [35], and performed to quantify promoter methylation in eight CpG islands (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) [36], [37], [38]. CIMP-high was defined as the presence of ≥6/8 methylated markers, CIMP-low as the presence of 1/8 to 5/8 methylated markers, and CIMP-0 as the absence (0/8) of methylated markers [37], [39]. LINE-1 methylation levels were quantified by PCR-Pyrosequencing [40], [41].

Statistical analysis

We used SAS program (Version 9.1, SAS Institute, Cary, NC) for all statistical analyses. All p values were two-sided. When we perform multiple hypothesis testing (i.e., analyses of molecular correlates and interactions), a p value for statistical significance was adjusted to p = 0.0038 ( = 0.05/13) by Bonferroni correction. The chi-square test (or Fisher's exact test) was performed for categorical variables. The t test assuming unequal variances was done to compare mean age and mean LINE-1 methylation level. For survival analysis, the Kaplan-Meier method and log-rank test were used. For analyses of colorectal cancer-specific mortality, deaths as a result of causes other than colorectal cancer were censored. To control for confounding, we used multivariate stage-matched (stratified) Cox proportional hazards model to compute hazard ratio (HR) of death. To avoid residual confounding and overfitting, disease stage (I, II, III, IV, unknown) was used as a stratifying variable, utilizing the “strata” option in the SAS “proc phreg” command. The multivariate model initially included age at diagnosis (continuous), sex, year of diagnosis (continuous), body mass index (BMI; <30 vs. ≥30 kg/m²), family history of colorectal cancer in any first degree relative (present vs. absent), tumor location (proximal vs. distal), tumor differentiation (well-moderate vs. poor), CIMP (high vs. low/CIMP-0), LINE-1 methylation (continuous), KRAS, BRAF and PIK3CA. A backward elimination method with a threshold of p = 0.20 was used to limit the number of variables in the final model and avoid overfitting. For cases with missing information in any of the categorical variables [BMI (0.1%), tumor location (1.0%), tumor grade (0.6%), CIMP (2.5%), KRAS (0.5%), BRAF (0.7%) and PIK3CA (8.8%)], we included those cases in a majority category of a given covariate to avoid overfitting. We confirmed that excluding cases with missing information in any of the covariates did not substantially alter results (data not shown).

A multivariate logistic regression analysis was performed to examine an independent relationship of each covariate with TGFBR2 mutation (as an outcome variable). The multivariate model initially included a similar, but not the same set of the covariates as the initial Cox model, considering possible cause-effect relationship with TGFBR2 mutation. Specifically, disease stage and tumor differentiation were likely consequences (rather than causes) of TGFBR2 mutation. Thus, those variables were not included in the logistic regression model. A backward elimination with a threshold of p = 0.10 was used to select variables in the final model and avoid overfitting.

Mononucleotide mutations of TGFBR2 and BAX in MSI-high colorectal cancers

Among 1072 colorectal cancers in the two prospective cohort studies, MSI-high was observed in 162 (15%) tumors. TGFBR2 and BAX mononucleotide tract mutations were detected in 117 (of 159, 74%) and 48 (of 158, 30%) MSI-high tumors, respectively. Among MSI-high tumors, TGFBR2 mutation was significantly associated with CIMP-high (p = 0.0010) ( Table 2 ).

Multivariate analysis to assess independent relations with TGFBR2 mutation

We performed multivariate logistic regression analysis to examine whether TGFBR2 mutation was independently associated with any clinical, pathologic and other molecular variables. In MSI-high tumors, TGFBR2 mutation was independently associated with CIMP-high [multivariate odds ratio (OR), 3.57; 95% confidence interval (CI), 1.66–7.66; p = 0.0011].

Mononucleotide mutations of TGFBR2 and BAX and colorectal cancer prognosis

During adequate follow-up (11.6 years of median follow-up of censored cases), there were 505 deaths including 302 colorectal cancer-specific deaths. Among all cases, MSI-high was significantly associated with longer colorectal cancer-specific survival compared to MSS/MSI-low cancers by log-rank test (p<0.0001), univariate and multivariate Cox regression analysis (adjusted HR, 0.34; 95% CI, 0.20–0.57; p<0.0001) ( Table 3 ). When we separately examined TGFBR2-mutated MSI-high cases and TGFBR2-wildtype MSI-high cases, both groups showed significantly longer colorectal cancer-specific survival compared to MSS/MSI-low cases ( Figure 2 , Table 3 ). When we separately examined BAX-mutated MSI-high cases and BAX-wildtype MSI-high cases, both groups showed significantly longer colorectal cancer-specific survival compared to MSS/MSI-low cases ( Figure 2 , Table 3 ). In overall mortality analyses, although somewhat attenuated, results showed similar trends ( Table 3 ). Among MSI-high cases, patient survival did not significantly differ by TGFBR2 or BAX mutation status.

We compared colorectal cancer specific and overall survival between TGFBR2-mutated MSI-high cases and TGFBR2-wildtype MSI-high cases (or between BAX-mutated MSI-high cases and BAX-wild type MSI-high cases). There was no significant difference between the two groups ( Table 1 ).