The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine-based adjuvant chemotherapy

In order to compare methylation profiles according to MSI status, 49 MSI-high carcinomas and 196 microsatellite stable (MSS) or MSI-low carcinomas were selected from archived samples. All 245 selected patients had undergone their first surgical resection for CRC at Samsung Medical Center (n = 132; random selection after acquisition of 40 MSI-high carcinomas) or Seoul National University Hospital (n = 113; consecutively selected) between July 2003 and October 2006.

Patients with inflammatory bowel disease, a family history of Lynch syndrome or familial adenomatous polyposis, or synchronous multiple colorectal cancers were excluded. Surgical resection techniques were standardized in the respective hospitals during the study period. Tumor stage and depth of invasion were defined according to the tumor, node, metastasis (TNM) classification developed by the American Joint Committee on Cancer [28]. Stage I was assigned in 27, II in 91, III in 82 and IV in 45 cases. None of the patients had received chemotherapy or radiotherapy prior to surgery. During study period, adjuvant chemotherapy regimen for stage II and III CRCs was mainly based on fluoropyrimidines (5-FU or capecitabine). Mayo Clinic regimen was used for administration of 5-FU: six cycles of rapid intravenous infusion of 20 mg/m² leucovorin, followed immediately by an intravenous bolus of 425 mg/m² fluorouracil, on days 1 to 5 every four weeks. The palliative chemotherapy regimen for stage IV CRCs was based on fluoropyrimidines, oxaliplatin, or irinotecan.

Formalin-fixed, paraffin-embedded tissues were used for DNA extraction. DNA was extracted from 10 μm sections stained with 0.1% methylene blue by manual microdissection under microscopy using 20-gauge needles and proteinase K solution as described previously [29]. Clinicopathologic data were collected by review of medical records and pathology slides. In the present study, tumors located from the cecum to the transverse colon were classified as proximal and those from the splenic flexure to the rectum were classified as distal.

All patients gave informed consent prior to specimen collection according to our institutional guidelines. This study was carried out in accordance with the Declaration of Helsinki. The institutional review boards at Samsung Medical Center and Seoul National University Hospital approved the study protocol.

RFS data for patients with stage I, II or III CRCs was obtained by review of medical records using the intranet resources at both hospitals. RFS was measured from the date of resection to the date of the first recurrence documented by imaging or pathologic confirmation or until the censoring date of May 31, 2010.

We performed a subgroup analysis of RFS to determine the impact of fluoropyrimidine-based adjuvant chemotherapy on clinical outcome in patients with stage II and stage III CRCs with respect to CIMP status. Among the 173 patients with stage II or stage III CRCs, we excluded from analysis 20 patients who received oxaliplatin-based adjuvant chemotherapy, 15 patients with rectal cancers who received concurrent chemo-radiation therapy and 14 patients who did not undergo any follow-up examinations. A total of 124 patients with stage II or stage III CRCs were ultimately analyzed. Among them, 46 cases had undergone surgery without subsequent adjuvant chemotherapy and 78 received fluoropyrimidine-based adjuvant chemotherapy after surgery. The median follow-up time for these patients was 44.5 months (range, 6-75 months).

Five widely used methylation markers that allow for excellent discrimination of CIMP status were employed in this study. This panel of markers consists of the CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1 genes [20]. The MethyLight assay (Applied Biosystems) was applied to quantitatively determine methylation status as previously described [20]. Briefly, two sets of primers and probes designed specifically for bisulfite-converted DNA were used. Reaction specificity for methylated DNA was confirmed separately using human DNA treated with CpG methyltransferase SssI (New England Biolabs). In addition to CIMP markers, methylation of O ⁶ -methylguanine-DNA methyltransferase (MGMT) gene was also analyzed by methylation-specific polymerase chain reaction due to its importance in KRAS mutations [30‐32] and clinical significance in survival [33]. CIMP classification was based on the numbers of methylated genes in each panel. Tumors were classified as CIMP-high if three or more markers were methylated, CIMP-low if one or two markers were methylated, and CIMP-negative if a methylated marker was not observed [14, 20].

We used five microsatellite markers recommended by the National Cancer Institute Workshop on MSI to determine MSI status [34]. PCR analyses were performed using a DNA autosequencer (Applied Biosystems 373A sequencer; Applied Biosystems, CA, USA). The mobility shift of PCR products from the tumor DNA was compared to that from corresponding normal colonic mucosa. Tumors were classified as MSI-high if band shifts were observed in two or more markers compared to the control, MSI-low if shifts were observed in one marker, and MSS if no shift was observed [34]. BRAF mutations in exon 11 and exon 15 and KRAS mutations in codons 12 and 13 in exon 2 were analyzed by PCR and automated sequencing as previously described [29].

Patient clinicopathologic features were compared using the x ² test and Student's t-test, with p < 0.05 considered significant. Survival curves were analyzed by the Kaplan-Meier method, and differences between individual curves were evaluated by the log-rank test. Multivariate analysis using the Cox proportional hazards model was performed to explore the potential association between clinicopathologic and molecular parameters and RFS.

The characteristics of CIMP-high and CIMP-low/negative CRCs are summarized in Table 1. CIMP-high carcinomas were found in 34 cases (13.9%) and were more frequently associated with proximal tumor locations, poorly differentiated histology, mucinous histology, BRAF mutation, MGMT methylation, and MSI-high compared to CIMP-low/negative carcinomas (P < 0.05).

All BRAF mutations were missense mutations in codon 600 of exon 15 (V600E) and BRAF and KRAS mutations were mutually exclusive. BRAF mutations were found more frequently in CIMP-high (26.5%) than in CIMP-low/negative carcinomas (0.9%). When stratified by MSI status, BRAF mutation rates were 12.2% for MSI-high CRCs and 2.6% for MSI-low/MSS CRCs (P = 0.010). The rates of KRAS mutations were comparable in CIMP-high (35.3%) and CIMP-low/negative carcinomas (34.1%). When stratified by MSI status, KRAS mutation rates were 28.6% for MSI-high CRCs and 35.7% for MSI-low/MSS CRCs (P = 0.346). KRAS mutation was significantly more prevalent in carcinomas with MGMT methylation (46.3%) than in cases without MGMT methylation (46.3% vs. 28.5%; P = 0.006).

There were a total of 200 patients with stage I, II, and III CRCs, and the three-year RFS survival for each stage was 91.6%, 88.0%, and 71.6%, respectively. In these patients, RFS did not differ significantly between CIMP-high and CIMP-low/negative groups (three-year RFS: 89.0% vs. 80.6%; P = 0.274). Regarding MSI, patients with MSI-high CRCs showed longer RFS than those with MSI-low or MSS CRCs, although this difference did not reach statistical significance (three-year RFS: 90.6% vs. 79.2%; P = 0.069).

On multivariate analysis of RFS of patients with stage I, II or stage III CRCs, TNM stage was an independent predictor of recurrence (Table 2). We performed an additional multivariate analysis of RFS for the 124 patients with stage II or stage III CRCs who had undergone surgery only or received fluoropyrimidine-based adjuvant chemotherapy after surgery. The following variables were included in the multivariate model: age, gender, TNM stage, histological differentiation, CIMP status, MSI status, treatment modality (surgery alone versus surgery with chemotherapy), BRAF mutation, KRAS mutation and MGMT methylation. Only TNM stage was identified as an independent predictor of RFS in these patients (hazard ratio: 5.963, 95% CI: 1.351-26.314). As no recurrence occurred in patients with CIMP-high CRCs treated with fluoropyrimidine-based adjuvant chemotherapy, the effect of an interaction between CIMP status and treatment modality in these patients could not be analyzed using the Cox proportional hazards model.

To explore the impact of fluoropyrimidine-based adjuvant chemotherapy for stage II and stage III CRCs according to CIMP status, we analyzed the RFS of 124 patients with stage II or stage III CRCs. The RFS was not significantly different between CIMP-high group and CIMP-low/negative group (three-year RFS: 91.7% vs. 84.0%; P = 0.31). In addition, RFS was not significantly affected by treatment modality. The three-year RFS for patients who received surgery alone and those who underwent surgery followed by fluoropyrimidine-based adjuvant chemotherapy was 84.1% and 86.5%, respectively (P = 0.387). Patients with MSI-high CRCs experienced longer RFS compared to those with MSI-low or MSS CRCs, although this difference did not reach statistical significance (three-year RFS: 94.0% vs. 82.3%; P = 0.088).

Figure 1 shows RFS according to CIMP status in patients who underwent surgery alone (Figure 1A) and in patients who received fluoropyrimidine-based adjuvant chemotherapy after surgery (Figure 1B) for stage II or III CRCs. In patients treated with surgery alone (Figure 1A), RFS did not differ significantly between CIMP-high and CIMP-low/negative groups. However, in patients treated with surgery and fluoropyrimidine-based adjuvant chemotherapy (Figure 1B), CIMP-high group had a longer RFS than the CIMP-low/negative group although this difference did not reach statistical significance (three-year RFS: 100% vs. 82.4%; P = 0.073).

Since our results suggested an interaction between CIMP-high status and fluoropyrimidine-based chemotherapy in stage II or III CRCs, we performed survival analyses according to treatment modality in patients with CIMP-low/negative (Figure 2A) and CIMP-high CRCs (Figure 2B). The characteristics of these patients according to CIMP status and treatment modality are summarized in Table 3. In both patients with CIMP-low/negative and patients with CIMP-high CRCs, those undergoing surgery followed by chemotherapy showed a higher LN metastasis rate and consequently a more advanced TNM stage than those undergoing surgery alone (P < 0.001 for CIMP-low/negative CRCs; P = 0.352 for CIMP-high CRCs). In patients with stage II or III CIMP-low/negative CRCs (Figure 2A), RFS was not significantly different between those undergoing surgery alone and those receiving surgery with adjuvant chemotherapy. When stratified according to TNM stage, a similar trend was observed for both stage II and stage III CRCs (Figure 3A and 3C). However, in patients with stage II or III CIMP-high CRCs (Figure 2B), the RFS of those treated with surgery and fluoropyrimidine-based adjuvant chemotherapy was significantly higher than that of patients treated with surgery alone (three-year RFS: 100% vs. 71.4%; P = 0.022). Figures 3B and 3D show the RFS for patients with stage II and stage III CIMP-high CRCs, respectively. In both stages, patients receiving adjuvant chemotherapy demonstrated longer RFS than those receiving surgery alone.