Patients
Rectal cancer patients from the Radboud University Medical Center, Nijmegen, the Netherlands and diagnosed between 2010 and 2012 with a biopsy confirmed rectal adenocarcinoma were retrospectively included in this study. To prevent any influence of neoadjuvant therapy on the results, patients undergoing surgical resection of the primary tumor without neoadjuvant chemo- and/or radiotherapy were included.
Patient characteristics were obtained from medical records, including age, gender, clinical- and pathological characteristics. This project was conducted in accordance with the Declaration of Helsinki, and did not require approval of the local IRB according to local WMO regulations.
Tumor identification and DNA isolation
For each patient, five tissue samples were obtained from representative formalin-fixed paraffin-embedded (FFPE) tumor blocks containing material of 1 preoperative diagnostic biopsy, 2 superficial tumor tissue samples and 2 deep (central) tumor tissues samples of the resected specimen. Optimal FFPE blocks (with adequate tumor cellularity of ≥20% from full samples, and >10% in biopsy samples) for smMIP analysis were identified and marked by an expert pathologist (I.N.) on representative hematoxylin and eosin (H&E) stained slides. When inadequate tumor cell percentages were obtained, this was determined as insufficient reading depth and the sample was not further analysed. To obtain sufficient genomic DNA, marked tumor areas were cut out from 10 sequential (non-stained) slides (each 6 µm thick). DNA was isolated at 56 °C for 1 hour using TET-lysis buffer with 5% Chelex-100 (Bio-Rad, Hercules, USA) and 400µg proteinase K (Qiagen, Valencia, USA), followed by inactivation at 95°C during 10 minutes (29). The DNA concentration was determined using the Qubit High Sensitivity Kit (Invitrogen, Carlsbad, USA) per manufacturer’s protocol.
smMIP sequencing
A panel of 911 smMIPs was used to detect variants in 31 cancer-related genes, as displayed in Table 1. To provide gender control, smMIPs targeting AMELX and AMELY were
included. The smMIP sequencing protocol has previously been clinically validated and used in the Radboud University Medical Center (29). One hundred nanogram of isolated DNA was included per sample. After sample preparation, manual library preparation was performed (29). The purified libraries were diluted. Sequencing was performed using the NextSeq500 (Illumina, San Diego, USA) per manufacturer's protocol (300 cycles High Output sequencing Kit, Illumina, San Diego, USA), resulting in 2 x 150 bp paired end reads.
Sequence data analysis
Sequence data was generated from the NextSeq500, after which Bcl to FASTQ conversion and demultiplexing of barcoded reads was automatically performed. Sequence Pilot software (JSI Medical Systems GmbH, Ettenheim, Germany) was used for generating consensus reads and variant identification, with settings as previously described (29). Variants found in samples passing gender control and exceeding an average minimum reading depth of 180 were automatically filtered with an in-house Python script, as depicted in Figure 1. This threshold excludes, with a certainty of >95%, the presence of a mutation at minimally 10% mutant allele frequency within covered regions. As SOX9 and SEC63 have many pseudogenes resulting in uncertainty about found mutations, we have excluded these from these analysis. Due to a technical sequencing artifact (in all samples), PTEN mutation c.407G>A was excluded from the analysis.
Statistical analysis
Statistical analysis was performed using SPSS version 23 (SPSS, Inc., Chicago, USA). Numerical data is presented as mean (standard deviation) or median (interquartile range) based on distribution. Categorical data is presented as frequencies and percentages. In order to quantify tumor heterogeneity, differences in mutational status between biopsy, deep and superficial tumor samples were analyzed by calculating the percentages of concordance and disconcordance. Concordance was defined as all five samples (1 biopsy, 2 deep samples, and 2 superficial samples) showing identical (or no) mutations. Disconcordance was defined as ≥1 mutation(s) in either of the 5 samples, which was not found in (one of) the other samples. For all tests performed, P<0.05 was considered statistically significant.