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  • The study cohort is derived from a consecutive series of 213 stage I-III surgically treated colon cancers at Stavanger University Hospital during a 4 year period, with details as described previously [32]. The study was approved by the regional ethics committee (#197.04) and all patients gave informed consent prior to inclusion. All patients underwent surgery with curative intent. Patients with lymph node positive (pN+) stage III disease who were physiologically fit (e.g ECOG performance status 0–1), were offered adjuvant therapy according to national guidelines at the time. For the current project, a sub-cohort of patients with colon cancer stage II and III were recruited. Patients who had either a follow-up of at least 36 months at the time of selection (= 3 years, by which time >90% of recurrences occur) or who had died of CRC at commencement of the current project were eligible. A total of 64 patients fulfilled these criteria and were included for MLPA analysis. Follow up was performed using the 11-digit social security number and access to electronic patient files for any sign of recurrent disease or a fatal event related to colon cancer. The selection was based on the information available at 3 years follow up, thus excluding any patient with shorter follow-up at commencement of the study. Recurrence was defined as any locoregional or systemic relapse of the disease at time of follow up. DNA from the 64 patients’ tumor samples was extracted and isolated from fresh frozen tissue using DNeasy Mini kit or AllPrep DNA&RNA Mini Kit (Qiagen, Hilden, Germany), as described previously [33]. Multiplex ligation-dependent probe amplification (MLPA): The multiplex ligation-dependent probe amplification procedure, using the SALSA MS-MLPA kit ME042-B1 CIMP probe mix (for detecting methylation status in the promoter regions of 8 different genes; RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) as given in Table 1, were performed according to the vendors recommendations (MRC-Holland, Amsterdam, the Netherlands), and as previously described [34]. As an activating mutation of BRAFV600E is tightly associated with CIMP positivity [35], a mutation specific probe is included in the SALSA MS-MLPA kit that detects the V600E (1799T>A) somatic mutation if this is present in the sample. Table data removed from full text. Table identifier and caption: 10.1371/journal.pone.0122391.t001 Sequences, chromosomal location, and fragment lengths for the investigated positions/probes in the MLPA method. §Refers to hg18 From all 64 DNA samples, 100 ng DNA was heat-denatured in a total volume of 5 μl Tris-EDTA buffer, and further performed as recommended by the vendor. Briefly, a mixture of probe-mix (ME042, MRC-Holland, Amsterdam, the Netherlands) and buffer were added to the denatured DNA, and probes were allowed to hybridize to the DNA at 60C for 16 hours. Each sample was divided into two tubes: one of which was ligated, while the other was ligated and then digested using the methylation-sensitive restriction enzyme HhaI. Both reactions were then subjected to a PCR reaction using a thermal cycler (GeneAmp 2700, Applied Biosystems, Foster City, CA, USA), and fragment analysis performed on a capillary sequencer (ABI 3130xl, Applied Biosystems, Foster City, CA, USA). Extracted DNA from normal colonic mucosa was used as a normal reference (n = 12 samples). Additionally, the colorectal cancer commercial cell-lines Caco-2 and HT-29 were used as cancer controls (see supporting information, S1 Data file, and S1 and S2 Figs). The raw data from the analysis were analyzed using Coffalyser.NET (beta version, MRC-Holland, Amsterdam, the Netherlands). Criteria for scoring of CIMP phenotype were more than 20% methylation of a minimum of one third of the probes within at least three of the five genes in the Weisenberger panel[35], as previously described [34]. For CNV phenotype scoring, all eight genes investigated were used. Copy number variation (CNV) was used as a proxy for chromosomal instability (CIN) in the study. Loss and gain were defined using the relative ratio of sample vs reference for a probe were less than 0.7, or higher than 1.3, respectively. For region analysis, aberration in one or more probes was defined as CNV of the region, and a high CNV phenotype if five or more of the eight regions (1p36.11, 3p22.2, 5q31.1, 9p21.3, 11p15.5, 15q24.2, 16p13.13 and 17q21.33) were aberrant. The MSI phenotype was investigated based on the Bethesda panel of genes using fragment analysis, as previously described [36]. Furthermore, methylation status of the MLH1 gene using MLPA was used to correlate the methylation status in relation to MSI. In the following, MSI status refers to the results from the Bethesda panel, whereas MLH1 investigated by the MLPA method is referred to as MLH1 methylation. KRAS mutational analyses were performed as previously described in [37]. BRAF mutation status was determined based on two different methods. One, the MLPA method [34], in which the BRAF mutation specific probe generated a signal if the V600E mutation was present, and; second, by using conventional PCR and sequencing analysis, as described previously [38].
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