Investigating genomic, proteomic, and post-transcriptional regulation profiles in colorectal cancer: a comparative study between primary tumors and associated metastases.

INTRODUCTION: Approximately 50% of patients with primary colorectal carcinoma develop liver metastases. This study investigates the possible molecular discrepancies between primary colorectal cancer (pCRC) and their respective metastases. METHODS: A total of 22 pairs of pCRC and metastases were tested. Mutation profiling of 26 cancer-associated genes was undertaken in 22/22primary-metastasis tumour pairs using next-generation sequencing, whilst the expression of a panel of six microRNAs (miRNAs) was investigated using qPCRin 21/22 pairs and 22 protein biomarkers was tested using Reverse Phase Protein Array (RPPA)in 20/22 patients' tumour pairs. RESULTS: Among the primary and metastatic tumours the mutation rates for the individual genes are as follows:TP53 (86%), APC (44%), KRAS (36%), PIK3CA (9%), SMAD4 (9%), NRAS (9%) and 4% for FBXW7, BRAF, GNAS and CDH1. The primary-metastasis tumour mutation status was identical in 54/60 (90%) loci. However, there was discordance in heterogeneity status in 40/58 genetic loci (z-score = 6.246, difference = 0.3793, P < 0.0001). Furthermore, there was loss of concordance in miRNA expression status between primary and metastatic tumours, and 57.14-80.95% of the primary-metastases tumour pairs showed altered primary-metastasis relative expression in all the miRNAs tested. Moreover, 16 of 20 (80%) tumour pairs showed alteration in at least 3 of 6 (50%) of the protein biomarker pathways analysed. CONCLUSION: The molecular alterations of primary colorectal tumours differ significantly from those of their matched metastases. These differences have profound implications for patients' prognoses and response to therapy.

Targeted Next-Generation Sequencing Validates the Use of Diagnostic Biopsies as a Suitable Alternative to Resection Material for Mutation Screening in Colorectal Cancer.

BACKGROUND: Mutation testing in the context of neoadjuvant therapy must be performed on biopsy samples. Given the issue of tumour heterogeneity, this raises the question of whether the biopsies are representative of the whole tumour. Here we have compared the mutation profiles of colorectal biopsies with their matched resection specimens. METHODS: We performed next-generation sequencing (NGS) analysis on 25 paired formalin-fixed, paraffin-embedded colorectal cancer biopsy and primary resection samples. DNA was extracted and analysed using the TruSight tumour kit, allowing the interrogation of 26 cancer driver genes. Samples were run on an Illumina MiSeq. Mutations were validated using quick-multiplex-consensus (QMC)-polymerase chain reaction (PCR) in conjunction with high resolution melting (HRM). The paired biopsy and resection tumour samples were assessed for presence or absence of mutations, mutant allele frequency ratios, and allelic imbalance status. RESULTS: A total of 81 mutations were detected, in ten of the 26 genes in the TruSight kit. Two of the 25 paired cases were wild-type across all genes. The mutational profiles, allelic imbalance status, and mutant allele frequency ratios of the paired biopsy and resection samples were highly concordant (88.75-98.85%), with all but three (3.7%) of the mutations identified in the resection specimens also being present in the biopsy specimens. All 81 mutations were confirmed by QMC-PCR and HRM analysis, although four low-level mutations required a co-amplification at lower denaturation temperature (COLD)-PCR protocol to enrich for the mutant alleles. CONCLUSIONS: Diagnostic biopsies are adequate and reliable materials for molecular testing by NGS. The use of biopsies for molecular screening will enhance targeted neoadjuvant therapy.

Targeted next generation sequencing reveals a common genetic pathway for colorectal cancers with chromosomal instability and those with microsatellite and chromosome stability.

INTRODUCTION: Microsatellite stable sporadic colorectal cancers (CRCs) can be classified as either tumours with chromosomal instability (CIN+) or tumours that are 'Microsatellite and Chromosomal Stable' (MACS). The CIN + tumours are aneuploid whilst MACS are near-diploid; little else is known about their differences. We compared the mutation profiles of CIN + and MACS CRCs. METHOD: Targeted Next Generation Sequencing for mutation in 26 driver genes (TruSight-26 kit) was undertaken in 46 CIN + and 35 MACSCRCs. Tumours were compared for mutation frequency, allelic imbalance and clonal heterogeneity. RESULTS: Mutations were detected in 58% genes and, overall, mutation in driver genes was at expected frequencies. Comparison of classes revealed similar mutation frequencies in most genes and allelic imbalance atAPC and TP53. Differences were seen in mutation frequency in KRAS (41% CIN+ vs 68% MACS, p = 0.015) and GNAS (0% CIN+ vs 12% MACS, p = 0.032). Twenty percent CIN + CRCs harboured mutations only in TP53 - a profile not seen in the MACS tumours (p = 0.009). None of the differences were significant after multiple testing corrections. CONCLUSIONS: The mutation profiles of CIN and MACS CRCs are similar. The events allowing aneuploidy (or forcing retention of diploidy) remain unknown.

N_LyST: a simple and rapid screening test for Lynch syndrome.

AIMS: We sought to use PCR followed by high-resolution melting analysis to develop a single closed-tube screening panel to screen for Lynch syndrome. This comprises tests for microsatellite instability (MSI), MLH1 methylation promoter and BRAF mutation. METHODS: For MSI testing, five mononucleotide markers (BAT25, BAT26, BCAT25, MYB, EWSR1) were developed. In addition, primers were designed to interrogate Region C of the MLH1 promoter for methylation (using bisulphite-modified DNA) and to test for mutations in codon 600 of BRAF. Two separate cohorts from Nottingham (n=99, 46 with MSI, 53 being microsatellite stable (MSS)) and Edinburgh (n=88, 45 MSI, 43 MSS) were tested. RESULTS: All the cases (n=187) were blind tested for MSI and all were correctly characterised by our panel. The MLH1 promoter and BRAF were tested only in the Nottingham cohort. Successful blinded analysis was performed on the MLH1 promoter in 97 cases. All MSS cases showed a pattern of non-methylation while 41/44 cases with MSI showed full methylation. The three cases with MSI and a non-methylated pattern had aberrations in MSH2 and MSH6 expression. BRAF mutation was detected in 61% of MSI cases and 11% of MSS cases.Finally, 12 cases were blind screened by using the whole panel as a single test. Of these, five were identified as MSS, four as MSI/non-LS and three as MSI/possible LS. These results were concordant with the previous data. CONCLUSION: We describe the Nottingham Lynch Syndrome Test (N_LyST). This is a quick, simple and cheap method for screening for Lynch syndrome.

QMC-PCRx: a novel method for rapid mutation detection.

AIMS: We previously described the quick multiplex consensus PCR (QMC-PCR) as a method for rapid mutation screening in low-quality template. QMC-PCR has two-stages: a prediagnostic multiplex (PDM) reaction followed by a single specific diagnostic reaction with high-resolution melting (HRM) analysis. We aimed to develop QMC-PCRx in which second stage was multiplexed to allow testing of multiple targets. METHODS: The PDM reaction was retained without change. For the second stage, in silico design was used to identify targets amenable to a multiplex specific diagnostic reaction and multiplex HRM (mHRM) analysis. Following optimisation, 17 colorectal cancers were tested for mutation in five hotspots. For QMC-PCR, each target was tested individually. For QMC-PCRx, the targets were tested in the following combinations (i) KRAS exon 3/PIK3CA exon 20/PTEN exon 3 in triplex and (ii) PTEN exon 7/NRAS exon 2 in duplex. The degree of agreement between the novel QMC-PCRx and the standard QMC-PCR was tested by the percentage concordance. RESULTS: Optimisation of mHRM showed that peaks needed to be separated (without overlap) and the optimal number was three targets per test. Our experimental design produced distinct and widely separated peaks for the individual targets although one of the primers needed a GC-tail. A total of 85 individual targets were tested; this required 85 second-stage PCR/HRM tests by QMC-PCR versus 34 second-stage tests by QMC-PCRx. The percentage concordance between the singleplex and multiplex methodologies was 100%. CONCLUSIONS: A multiplexed analysis using HRM is possible without loss of diagnostic accuracy. The novel QMC-PCRx protocol can significantly reduce workload and costs of mutation screening.

"Squirrel" Primer-Based PCR Assay for Direct and Targeted Sanger Sequencing of Short Genomic Segments.

Currently, short DNA segments of sub-100 bp can be sequenced either directly by next-generation sequencing and pyrosequencing, which are expensive, or indirectly, via Sanger sequencing combined with the cumbersome and failure-prone plasmid cloning. To circumvent these issues, we have generated a novel sequencing-purposed PCR assay using long-tailed primers (squirrel primers) to Sanger sequence directly sub-100 bp genomic amplicons. Squirrel primers, 40-65 nt in length, were used to amplify 51-93 bp long genomic sequences of KRAS exons 2 and 3, BRAF exon 15, PI3K catalytic subunit alpha exon 20, and phosphatase and tensin homolog exon 3 from colorectal cancer (CRC) cell lines and preamplified clinical CRC samples with known mutation status by PCR. Following this, a short second pair of primers that bind at the 5' region of the long tails was used for sequencing on the 3130 × l ABI Prism Genetic Analyzer. The sequencing data were analyzed via FinchTV software. High-quality sequencing data were obtained from 51 to 93 bp long genomic sequences with our novel PCR assay, with capture of all of the target sequences in all of the samples in both the forward and reverse directions and confirmation of the mutation status of the CRC samples. Whereas the sequencing quality was independent of the template type, it showed a squirrel primer tail length-dependent pattern. Our novel PCR assay for direct and targeted Sanger sequencing of short genomic segments has potential applications in focused molecular/genetic profiling of cancer in research and diagnostics fields in which fragmented DNA, such as circulating tumor DNA and archival tissue DNA, are used as starting templates.