Study Population
The study population comprised a prospective patient series with stage I-III colon cancer enrolled in a sentinel node project, as previously described (14). Briefly, all the patients underwent elective surgery with a curative intent at the Department of Surgery of Stavanger University Hospital in Stavanger, Norway, from June 2003 to February 2010. The patients were prospectively included after providing informed consent.
The Norwegian health system covers all medical expenses for diagnosis, management and surveillance, indicating no systematic selection bias in the cohort presented. Stavanger University Hospital provides all surgical services to a primarily Western population catchment area of approximately 330,000 inhabitants. Accordingly, the results should be representative for other Western populations.
Patients were excluded from the study cohort if any of the following occurred: noninvasive tumors (T0 and Tis), evidence of distant metastases (M+) at the time of surgery or on pre-op staging, preoperative chemotherapy, preoperative treatment with a self-expanding metal stent because of acute colon obstruction, missing tumor biopsies for DNA retrieval, missing or incomplete histopathology report (for node status, number and pN), or deviations from the SLN mapping protocol.
Radical surgical resection was applied to the specific tumor-bearing segment of the colon by using either an open or a laparoscopic technique following general surgical oncological principles. Tumors located proximal to the left flexure were defined as right-sided and tumors from the left flexure through the sigmoid colon as left-sided.
Tissue and LN Sampling
Ex vivo sentinel LN mapping was performed on resected specimens from all patients, as previously described (14). In the current study, the total numbers of histologically verified LNs were analyzed without separating the nodes into “sentinel” and “nonsentinel” categories.
The resected colonic segment was evaluated by gross and microscopic histopathological examination, including regional LN harvesting, following an institutional template established several years before the study (15) to determine the number of LNs present and the disease stage according to the tumor, node, metastasis (TNM) staging system of the Union for International Cancer Control. The extent of node metastasis was defined as pN0 for node-negative specimens, pN1 for one to three metastatic LNs and pN2 for three or more metastatic LNs in the specimen.
Outcome
The primary outcome of this study was the number of LNs harvested, reported either as the total number observed (that is, both sentinels and non-sentinels, with or without metastasis), and, alternatively, as the rate of appropriately harvested cases (that is, patients with ≥12 nodes per specimen, defined as an adequate harvest). Secondary outcomes were limited to number of metastatic LNs (pN+) and to the survival outcome at follow-up. Cancer-specific death was used as an endpoint. Patients alive at the end of follow-up were censored. Patients who died of other causes than cancer were censored at time of death. Follow-up was performed blinded to the patients’ clinical characteristics or molecular profile in the study.
Genetic Analyses
A sample from the tumor and the normal surrounding mucosa was collected from the resected colonic segment and was instantly frozen in liquid nitrogen. DNA was extracted from the tissue by using either a combination of the RNeasy Mini Kit and the DNeasy Mini Kit or the AllPrep DNA/RNA Mini Kit (all manufactured by Qiagen, Hilden, Germany) according to the manufacturer’s protocols.
The MSI status (microsatellite stable [MSS]; low frequency [MSI-L], or high frequency [MSI-H]) was determined by using Bethesda markers (BAT25, BAT26, D2S123, D5S346 and D17S250), as described (16). For MSI analyses, DNA from tumor and corresponding normal tissue were analyzed and compared. Analyses were run twice, and two people evaluated all results independently. Presence of two or more instable markers was defined as high-frequency MSI (MSI-H) and only one single unstable marker as low-frequency MSI (MSI-L). MSI-L was coded as MSS for the regression analysis in the current study, as per convention. Thus, MSI denotes MSI-H if not otherwise indicated. Mutations in codons 12 and 13 of the KRAS gene were detected by peptide nucleic acid clamp polymerase chain reaction (PCR) (17,18). The hot-spot mutation V600E in exon 15 of the BRAF gene was identified by using PCR and Sanger sequencing, as previously described (19).
For BRAF mutation analysis, all PCR products were sequenced in the 5′ direction, and electropherograms were scored both manually and semiautomatically by using Sequencing Analysis (version 5.3.1) and SeqScape software (version 2.5), respectively. The resulting sequence was compared with reference sequence NM_004333 (GenBank). For samples in which a mutation was found, a confirmatory sequencing reaction was performed in the 3′ direction.
Study Ethics
Written, informed consent was obtained from all the study subjects. This research project was approved by the Regional Committee for Medical Research Ethics (REK Vest 197.04, Biobank 15–10) according to national legislation.
Statistical Analyses
All statistical analyses were performed by using the software package IBM SPSS Statistics, version 20 (SPSS, Chicago, IL, USA). Descriptive data are presented as medians and ranges (or interquartile ranges) or as proportions, as appropriate. Dichotomous variables were tested by χ2 or Fisher exact test, as appropriate. Continuous variables were analyzed by using a nonparametric Mann-Whitney U test or the Kruskal-Wallis test for more than two groups. For variables associated with the number of LNs as a continuous outcome variable, a multiple linear regression adjusted for age and sex in a hierarchical mode was performed, as previously recommended (20,21). The variables were analyzed for normality, colinearity and interactions. The factors found to be associated with a sufficient (≥12 LNs) harvest were investigated by using univariate and multivariable analyses and are presented with odds ratios (ORs) and 95% confidence intervals (95% CIs). The multivariable model was adjusted for age and sex and included variables with P < 0.2. Goodness of fit was assessed with the Hosmer-Lemeshow test. Survival analyses were performed with the log-rank method by using Kaplan-Meier curves. All the tests were two-tailed, and the statistical significance was set at P < 0.05.