The ATOM-Seq sequence capture panel can accurately predict microsatellite instability status in formalin-fixed tumour samples, alongside routine gene mutation testing.

Microsatellite instability (MSI) occurs across a number of cancers and is associated with different clinical characteristics when compared to microsatellite stable (MSS) cancers. As MSI cancers have different characteristics, routine MSI testing is now recommended for a number of cancer types including colorectal cancer (CRC). Using gene panels for sequencing of known cancer mutations is routinely performed to guide treatment decisions. By adding a number of MSI regions to a small gene panel, the efficacy of simultaneous MSI detection in a series of CRCs was tested. Tumour DNA from formalin-fixed, paraffin-embedded (FFPE) tumours was sequenced using a 23-gene panel kit (ATOM-Seq) provided by GeneFirst. The mismatch repair (MMR) status was obtained for each patient from their routine pathology reports, and compared to MSI predictions from the sequencing data. By testing 29 microsatellite regions in 335 samples the MSI status was correctly classified in 314/319 samples (98.4% concordance), with sixteen failures. By reducing the number of regions in silico, comparable performance could be reached with as few as eight MSI marker positions. This test represents a quick, and accurate means of determining MSI status in FFPE CRC samples, as part of a routine gene mutation assay, and can easily be incorporated into a research or diagnostic setting. This could replace separate mutation and MSI tests with no loss of accuracy, thus improving testing efficiency.

The genomic landscape of 2,023 colorectal cancers.

Colorectal carcinoma (CRC) is a common cause of mortality1, but a comprehensive description of its genomic landscape is lacking2-9. Here we perform whole-genome sequencing of 2,023 CRC samples from participants in the UK 100,000 Genomes Project, thereby providing a highly detailed somatic mutational landscape of this cancer. Integrated analyses identify more than 250 putative CRC driver genes, many not previously implicated in CRC or other cancers, including several recurrent changes outside the coding genome. We extend the molecular pathways involved in CRC development, define four new common subgroups of microsatellite-stable CRC based on genomic features and show that these groups have independent prognostic associations. We also characterize several rare molecular CRC subgroups, some with potential clinical relevance, including cancers with both microsatellite and chromosomal instability. We demonstrate a spectrum of mutational profiles across the colorectum, which reflect aetiological differences. These include the role of Escherichia colipks+ colibactin in rectal cancers10 and the importance of the SBS93 signature11-13, which suggests that diet or smoking is a risk factor. Immune-escape driver mutations14 are near-ubiquitous in hypermutant tumours and occur in about half of microsatellite-stable CRCs, often in the form of HLA copy number changes. Many driver mutations are actionable, including those associated with rare subgroups (for example, BRCA1 and IDH1), highlighting the role of whole-genome sequencing in optimizing patient care.

Associations between AI-Assisted Tumor Amphiregulin and Epiregulin IHC and Outcomes from Anti-EGFR Therapy in the Routine Management of Metastatic Colorectal Cancer.

PURPOSE: High tumor production of the EGFR ligands, amphiregulin (AREG) and epiregulin (EREG), predicted benefit from anti-EGFR therapy for metastatic colorectal cancer (mCRC) in a retrospective analysis of clinical trial data. Here, AREG/EREG IHC was analyzed in a cohort of patients who received anti-EGFR therapy as part of routine care, including key clinical contexts not investigated in the previous analysis. EXPERIMENTAL DESIGN: Patients who received panitumumab or cetuximab ± chemotherapy for treatment of RAS wild-type mCRC at eight UK cancer centers were eligible. Archival formalin-fixed paraffin-embedded tumor tissue was analyzed for AREG and EREG IHC in six regional laboratories using previously developed artificial intelligence technologies. Primary endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: A total of 494 of 541 patients (91.3%) had adequate tissue for analysis. A total of 45 were excluded after central extended RAS testing, leaving 449 patients in the primary analysis population. After adjustment for additional prognostic factors, high AREG/EREG expression (n = 360; 80.2%) was associated with significantly prolonged PFS [median: 8.5 vs. 4.4 months; HR, 0.73; 95% confidence interval (CI), 0.56-0.95; P = 0.02] and OS [median: 16.4 vs. 8.9 months; HR, 0.66 95% CI, 0.50-0.86; P = 0.002]. The significant OS benefit was maintained among patients with right primary tumor location (PTL), those receiving cetuximab or panitumumab, those with an oxaliplatin- or irinotecan-based chemotherapy backbone, and those with tumor tissue obtained by biopsy or surgical resection. CONCLUSIONS: High tumor AREG/EREG expression was associated with superior survival outcomes from anti-EGFR therapy in mCRC, including in right PTL disease. AREG/EREG IHC assessment could aid therapeutic decisions in routine practice. See related commentary by Randon and Pietrantonio, p. 4021.

Analysis of an Indian colorectal cancer faecal microbiome collection demonstrates universal colorectal cancer-associated patterns, but closest correlation with other Indian cohorts.

It is increasingly being recognised that changes in the gut microbiome have either a causative or associative relationship with colorectal cancer (CRC). However, most of this research has been carried out in a small number of developed countries with high CRC incidence. It is unknown if lower incidence countries such as India have similar microbial associations.Having previously established protocols to facilitate microbiome research in regions with developing research infrastructure, we have now collected and sequenced microbial samples from a larger cohort study of 46 Indian CRC patients and 43 healthy volunteers.When comparing to previous global collections, these samples resemble other Asian samples, with relatively high levels of Prevotella. Predicting cancer status between cohorts shows good concordance. When compared to a previous collection of Indian CRC patients, there was similar concordance, despite different sequencing technologies between cohorts.These results show that there does seem to be a global CRC microbiome, and that some inference between studies is reasonable. However, we also demonstrate that there is definite regional variation, with more similarities between location-matched comparisons. This emphasises the importance of developing protocols and advancing infrastructure to allow as many countries as possible to contribute to microbiome studies of their own populations.

The risk of colorectal cancer in individuals with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene: An English population-based study.

BACKGROUND: Studies have demonstrated a higher risk of developing colorectal cancer (CRC) in individuals with Cystic Fibrosis (CF), and also a potentially increased risk in carriers of cystic fibrosis transmembrane conductance regulator (CFTR) mutations. Life expectancy for those with CF is rising, increasing the number at risk of developing CRC. METHODS: The incidence of CRC amongst individuals with CF was calculated using data from CORECT-R and linked UK CF Registry and Secondary User Services (SUS) data. Crude, age-specific and age-standardised rates were compared to those without CF. The presence of CFTR mutations in individuals with CRC was assessed using 100,000 Genomes Project data. FINDINGS: The crude incidence rate of CRC in the CF population was 0.29 per 1,000 person-years (28 cases). The CF population were significantly younger than those without (median age at CRC diagnosis 52 years versus 73 years; p<0·01). When age-adjusted, there was a 5-fold increased CRC incidence amongst individuals with CF compared to those without (SIR 5.0 95%CI 3.2-6.9). When compared to other population studies the overall prevalence of CFTR mutations in the CRC population was significantly higher than expected (p<0·01). INTERPRETATION: CF is linked to an increased risk of CRC. The incidence of CFTR mutations in the CRC population is higher than would be expected, suggesting an association between CFTR function and CRC risk. Further research is needed to develop effective screening strategies for these populations. FUNDING: Cancer Research UK (grants C23434/A23706 & C10674/A27140).

Sparse modelling of cancer patients' survival based on genomic copy number alterations.

Copy number alterations (CNA) are structural variation in the genome, in which some regions exhibit more or less than the normal two chromosomal copies. This genomic CNA profile provides critical information in tumour progression and is therefore informative for patients' survival. It is currently a statistical challenge to model patients' survival using their genomic CNA profiles while at the same time identify regions in the genome that are associated with patients' survival. Some methods have been proposed, including Cox proportional hazard (PH) model with ridge, lasso, or elastic net penalties. However, these methods do not take the general dependencies between genomic regions into account and produce results that are difficult to interpret. In this paper, we extend the elastic net penalty by introducing additional penalty that takes into account general dependencies between genomic regions. This new model produces smooth parameter estimates while simultaneously performs variable selection via sparse solution. The results indicate that the proposed method shows a better prediction performance than other models in our simulation study, while enabling us to investigate regions in the genome that are associated with the patients' survival with sensible interpretation. We illustrate the method using a real dataset from a lung cancer cohort and simulated data.

Correction: Developing a Raman spectroscopy-based tool to stratify patient response to pre-operative radiotherapy in rectal cancer.

Correction for 'Developing a Raman spectroscopy-based tool to stratify patient response to pre-operative radiotherapy in rectal cancer' by Chloe J. Kirkby et al., Analyst, 2021, 146, 581-589, DOI: .

Characterisation of dysplastic liver nodules using low-pass DNA sequencing and detection of chromosome arm-level abnormalities in blood-derived cell-free DNA.

High-grade dysplasia carries significant risk of transformation to hepatocellular carcinoma (HCC). Despite this, at the current standard of care, all non-malignant hepatic nodules including high-grade dysplastic nodules are managed similarly. This is partly related to difficulties in distinguishing high-risk pathology in the liver. We aimed to identify chromosome arm-level somatic copy number alterations (SCNAs) that characterise the transition of liver nodules along the cirrhosis-dysplasia-carcinoma axis. We validated our findings on an independent cohort using blood-derived cell-free DNA. A repository of non-cancer DNA sequences obtained from patients with HCC (n = 389) was analysed to generate cut-off thresholds aiming to minimise false-positive SCNAs. Tissue samples representing stages from the multistep process of hepatocarcinogenesis (n = 184) were subjected to low-pass whole genome sequencing. Chromosome arm-level SCNAs were identified in liver cirrhosis, dysplastic nodules, and HCC to assess their discriminative capacity. Samples positive for 1q+ or 8q+ arm-level duplications were likely to be either HCC or high-grade dysplastic nodules as opposed to low-grade dysplastic nodules or cirrhotic tissue with an odds ratio (OR) of 35.5 (95% CI 11.5-110) and 16 (95% CI 6.4-40.2), respectively (p < 0.0001). In an independent cohort of patients recruited from Nottingham, UK, at least two out of four alterations (1q+, 4q-, 8p-, and 8q+) were detectable in blood-derived cell-free DNA of patients with HCC (n = 22) but none of the control patients with liver cirrhosis (n = 9). Arm-level SCNAs on 1q+ or 8q+ are associated with high-risk liver pathology. These can be detected using low-pass sequencing of cell-free DNA isolated from blood, which may be a future early cancer screening tool for patients with liver cirrhosis. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Luminal Bioavailability of Orally Administered ω-3 PUFAs in the Distal Small Intestine, and Associated Changes to the Ileal Microbiome, in Humans with a Temporary Ileostomy.

BACKGROUND: Oral administration of purified omega-3 (ω-3) PUFAs is associated with changes to the fecal microbiome. However, it is not known whether this effect is associated with increased PUFA concentrations in the gut. OBJECTIVES: We investigated the luminal bioavailability of oral ω-3 PUFAs (daily dose 1 g EPA and 1g DHA free fatty acid equivalents as triglycerides in soft-gel capsules, twice daily) and changes to the gut microbiome, in the ileum. METHODS: Ileostomy fluid (IF) and blood were obtained at baseline, after first capsule dosing (median 2 h), and at a similar time after final dosing on day 28, in 11 individuals (median age 63 y) with a temporary ileostomy. Fatty acids were measured by LC-tandem MS. The ileal microbiome was characterized by 16S rRNA PCR and Illumina sequencing. RESULTS: There was a mean 6.0 ± 9.8-fold and 6.6 ± 9.6-fold increase in ileal EPA and DHA concentrations (primary outcome), respectively, at 28 d, which was associated with increased RBC ω-3 PUFA content (P ≤ 0.05). The first oral dose did not increase the ileal ω-3 PUFA concentration except in 4 individuals, who displayed high luminal EPA and DHA concentrations, which reduced to concentrations similar to the overall study population at day 28, suggesting physiological adaptation. Bacteroides, Clostridium, and Streptococcus were abundant bacterial genera in the ileum. Ileal microbiome variability over time and between individuals was large, with no consistent change associated with acute ω-3 PUFA dosing. However, high concentrations of EPA and DHA in IF on day 28 were associated with higher abundance of Bacteroides (r2 > 0.86, P < 0.05) and reduced abundance of other genera, including Actinomyces (r2 > 0.94, P < 0.05). CONCLUSIONS: Oral administration of ω-3 PUFAs leads to increased luminal ω-3 PUFA concentrations and changes to the microbiome, in the ileum of individuals with a temporary ileostomy. This study is registered on the ISRCTN registry as ISRCTN14530452.

Microbiome Analysis of More Than 2,000 NHS Bowel Cancer Screening Programme Samples Shows the Potential to Improve Screening Accuracy.

PURPOSE: There is potential for fecal microbiome profiling to improve colorectal cancer screening. This has been demonstrated by research studies, but it has not been quantified at scale using samples collected and processed routinely by a national screening program. EXPERIMENTAL DESIGN: Between 2016 and 2019, the largest of the NHS Bowel Cancer Screening Programme hubs prospectively collected processed guaiac fecal occult blood test (gFOBT) samples with subsequent colonoscopy outcomes: blood-negative [n = 491 (22%)]; colorectal cancer [n = 430 (19%)]; adenoma [n = 665 (30%)]; colonoscopy-normal [n = 300 (13%)]; nonneoplastic [n = 366 (16%)]. Samples were transported and stored at room temperature. DNA underwent 16S rRNA gene V4 amplicon sequencing. Taxonomic profiling was performed to provide features for classification via random forests (RF). RESULTS: Samples provided 16S amplicon-based microbial profiles, which confirmed previously described colorectal cancer-microbiome associations. Microbiome-based RF models showed potential as a first-tier screen, distinguishing colorectal cancer or neoplasm (colorectal cancer or adenoma) from blood-negative with AUC 0.86 (0.82-0.89) and AUC 0.78 (0.74-0.82), respectively. Microbiome-based models also showed potential as a second-tier screen, distinguishing from among gFOBT blood-positive samples, colorectal cancer or neoplasm from colonoscopy-normal with AUC 0.79 (0.74-0.83) and AUC 0.73 (0.68-0.77), respectively. Models remained robust when restricted to 15 taxa, and performed similarly during external validation with metagenomic datasets. CONCLUSIONS: Microbiome features can be assessed using gFOBT samples collected and processed routinely by a national colorectal cancer screening program to improve accuracy as a first- or second-tier screen. The models required as few as 15 taxa, raising the potential of an inexpensive qPCR test. This could reduce the number of colonoscopies in countries that use fecal occult blood test screening.

The colorectal cancer-associated faecal microbiome of developing countries resembles that of developed countries.

BACKGROUND: The incidence of colorectal cancer (CRC) is increasing in developing countries, yet limited research on the CRC- associated microbiota has been conducted in these areas, in part due to scarce resources, facilities, and the difficulty of fresh or frozen stool storage/transport. Here, we aimed (1) to establish a broad representation of diverse developing countries (Argentina, Chile, India, and Vietnam); (2) to validate a 'resource-light' sample-collection protocol translatable in these settings using guaiac faecal occult blood test (gFOBT) cards stored and, importantly, shipped internationally at room temperature; (3) to perform initial profiling of the collective CRC-associated microbiome of these developing countries; and (4) to compare this quantitatively with established CRC biomarkers from developed countries. METHODS: We assessed the effect of international storage and transport at room temperature by replicating gFOBT from five UK volunteers, storing two in the UK, and sending replicates to institutes in the four countries. Next, to determine the effect of prolonged UK storage, DNA extraction replicates for a subset of samples were performed up to 252 days apart. To profile the CRC-associated microbiome of developing countries, gFOBT were collected from 41 treatment-naïve CRC patients and 40 non-CRC controls from across the four institutes, and V4 16S rRNA gene sequencing was performed. Finally, we constructed a random forest (RF) model that was trained and tested against existing datasets from developed countries. RESULTS: The microbiome was stably assayed when samples were stored/transported at room temperature and after prolonged UK storage. Large-scale microbiome structure was separated by country and continent, with a smaller effect from CRC. Importantly, the RF model performed similarly to models trained using external datasets and identified similar taxa of importance (Parvimonas, Peptostreptococcus, Fusobacterium, Alistipes, and Escherichia). CONCLUSIONS: This study demonstrates that gFOBT, stored and transported at room temperature, represents a suitable method of faecal sample collection for amplicon-based microbiome biomarkers in developing countries and suggests a CRC-faecal microbiome association that is consistent between developed and developing countries.

Adaptor Template Oligo-Mediated Sequencing (ATOM-Seq) is a new ultra-sensitive UMI-based NGS library preparation technology for use with cfDNA and cfRNA.

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20-25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq's suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

Prediction of tumour pathological subtype from genomic profile using sparse logistic regression with random effects.

The purpose of this study is to highlight the application of sparse logistic regression models in dealing with prediction of tumour pathological subtypes based on lung cancer patients' genomic information. We consider sparse logistic regression models to deal with the high dimensionality and correlation between genomic regions. In a hierarchical likelihood (HL) method, it is assumed that the random effects follow a normal distribution and its variance is assumed to follow a gamma distribution. This formulation considers ridge and lasso penalties as special cases. We extend the HL penalty to include a ridge penalty (called 'HLnet') in a similar principle of the elastic net penalty, which is constructed from lasso penalty. The results indicate that the HL penalty creates more sparse estimates than lasso penalty with comparable prediction performance, while HLnet and elastic net penalties have the best prediction performance in real data. We illustrate the methods in a lung cancer study.

Developing a Raman spectroscopy-based tool to stratify patient response to pre-operative radiotherapy in rectal cancer.

Rectal cancer patients frequently receive pre-operative radiotherapy (RT), prior to surgical resection. However, colorectal cancer is heterogeneous and the degree of tumour response to pre-operative RT is highly variable. There are currently no clinically approved methods of predicting response to RT, and a significant proportion of patients will show no clinical benefit, despite enduring the side-effects. We evaluated the use of Raman spectroscopy (RS), a non-destructive technique able to provide the unique chemical fingerprint of tissues, as a potential tool to stratify patient response to pre-operative RT. Raman measurements were obtained from the formalin-fixed, paraffin-embedded (FFPE) pre-treatment biopsy specimens of 20 rectal cancer patients who received pre-operative RT. A principal component analysis and linear discriminant analysis algorithm was able to classify patient response to pre-operative RT as good or poor, with an accuracy of 86.04 ± 0.14% (standard error). Patients with a good response to RT showed greater contributions from protein-associated peaks, whereas patients who responded poorly showed greater lipid contributions. These results demonstrate that RS is able to reliably classify tumour response to pre-operative RT from FFPE biopsies and highlights its potential to guide personalised cancer patient treatment.

Mutational signature in colorectal cancer caused by genotoxic pks+ E. coli.

Various species of the intestinal microbiota have been associated with the development of colorectal cancer1,2, but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin3. This compound is believed to alkylate DNA on adenine residues4,5 and induces double-strand breaks in cultured cells3. Here we expose human intestinal organoids to genotoxic pks+ E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

No Significant Association Between the Fecal Microbiome and the Presence of Irritable Bowel Syndrome-type Symptoms in Patients with Quiescent Inflammatory Bowel Disease.

Background: The microbiome is implicated in the pathogenesis of inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). Whether a distinct microbiome profile is associated with the reporting of IBS-type symptoms in IBD patients is uncertain. We aimed to resolve this issue using a cross-sectional study design. Methods: Using clinical disease activity indices, the Rome III criteria for IBS and fecal calprotectin levels, we divided IBD patients into 4 groups: IBS-type symptoms, quiescent disease, occult inflammation, and active disease. A16S rRNA microbiome analysis was performed to determine whether any taxa were differentially abundant, and whether there were any differences in alpha or beta diversity in patients reporting IBS-type symptoms compared with those in the other 3 groups. Results: Of 270 patients included, 70 (25.9%) had IBS-type symptoms, 81 (30.0%) quiescent IBD, 66 (24.4%) occult inflammation, and 53 (19.6%) active IBD. At phylum level, there was a nonsignificant increase in the abundance of Actinobacteria in patients reporting IBS-type symptoms, but no other differences at any taxonomic level. When compared with patients reporting IBS-type symptoms, mean alpha diversity was greater in patients with quiescent disease, although this was nonsignificant (28.6 vs 31.7, P = 0.33), and similar to those with occult inflammation and active disease. Beta diversity variation among the 4 groups was significant for unweighted (P = 0.002) but not weighted (P = 0.21) UniFrac analysis. Conclusions: Reporting IBS-type symptoms was not associated with distinct microbiome alterations. Unmeasured confounding could have impacted the significance of our findings.

A randomised trial of the effect of omega-3 polyunsaturated fatty acid supplements on the human intestinal microbiota.

OBJECTIVE: Omega-3 polyunsaturated fatty acids (PUFAs) have anticolorectal cancer (CRC) activity. The intestinal microbiota has been implicated in colorectal carcinogenesis. Dietary omega-3 PUFAs alter the mouse intestinal microbiome compatible with antineoplastic activity. Therefore, we investigated the effect of omega-3 PUFA supplements on the faecal microbiome in middle-aged, healthy volunteers (n=22). DESIGN: A randomised, open-label, cross-over trial of 8 weeks' treatment with 4 g mixed eicosapentaenoic acid/docosahexaenoic acid in two formulations (soft-gel capsules and Smartfish drinks), separated by a 12-week 'washout' period. Faecal samples were collected at five time-points for microbiome analysis by 16S ribosomal RNA PCR and Illumina MiSeq sequencing. Red blood cell (RBC) fatty acid analysis was performed by liquid chromatography tandem mass spectrometry. RESULTS: Both omega-3 PUFA formulations induced similar changes in RBC fatty acid content, except that drinks were associated with a larger, and more prolonged, decrease in omega-6 PUFA arachidonic acid than the capsule intervention (p=0.02). There were no significant changes in α or ß diversity, or phyla composition, associated with omega-3 PUFA supplementation. However, a reversible increased abundance of several genera, including Bifidobacterium, Roseburia and Lactobacillus was observed with one or both omega-3 PUFA interventions. Microbiome changes did not correlate with RBC omega-3 PUFA incorporation or development of omega-3 PUFA-induced diarrhoea. There were no treatment order effects. CONCLUSION: Omega-3 PUFA supplementation induces a reversible increase in several short-chain fatty acid-producing bacteria, independently of the method of administration. There is no simple relationship between the intestinal microbiome and systemic omega-3 PUFA exposure. TRIAL REGISTRATION NUMBER: ISRCTN18662143.

Survival of Head and Neck Cancer Cells Relies upon LZK Kinase-Mediated Stabilization of Mutant p53.

Head and neck squamous cell carcinoma (HNSCC) includes epithelial cancers of the oral and nasal cavity, larynx, and pharynx and accounts for ∼350,000 deaths per year worldwide. Smoking-related HNSCC is associated with few targetable mutations but is defined by frequent copy-number alteration, the most common of which is gain at 3q. Critical 3q target genes have not been conclusively determined for HNSCC. Here, we present data indicating that MAP3K13 (encoding LZK) is an amplified driver gene in HNSCC. Copy-number gain at 3q resulted in increased MAP3K13 mRNA in HNSCC tumor samples and cell lines. Silencing LZK reduced cell viability and proliferation of HNSCC cells with 3q gain but not control cell lines. Inducible silencing of LZK caused near-complete loss of colony-forming ability in cells harboring 3q gain. These results were validated in vivo by evidence that LZK silencing was sufficient to reduce tumor growth in a xenograft model of HNSCC. Our results establish LZK as critical for maintaining expression of mutant stabilized p53. Cancer Res; 77(18); 4961-72. ©2017 AACR.

The genomic road to invasion-examining the similarities and differences in the genomes of associated oral pre-cancer and cancer samples.

BACKGROUND: It is frequently assumed that pre-invasive lesions are simpler precursors of cancer and will contain a limited subset of the genomic changes seen in their associated invasive disease. Driver mutations are thought to occur early, but it is not known how many of these are present in pre-invasive lesions. These assumptions need to be tested with the increasing focus on both personalised cancer treatments and early detection methodologies. METHODS: We examined genomic copy number changes in 256 pre-invasive and invasive samples from 69 oral cancer patients. Forty-eight samples from 16 patients were further examined using exome sequencing. RESULTS: Evidence of a shared ancestor of both dysplasia and carcinoma was seen in all but one patient. One-third of dysplasias showed independent copy number events. The remainder had a copy number pattern that was similar to or simpler than that of the carcinoma. All dysplasias examined contained somatic mutations absent in the related carcinoma. Previously observed copy number changes and TP53 mutations were very frequently observed, and almost always shared between dysplasia and carcinoma. Other gene changes were more sporadic. Pathway analysis confirmed that each patient's disease developed in a different way. Examining the numbers of shared mutations and the rate of accumulation of mutations showed evidence that all samples contain a population of sub-clones, with little evidence of selective advantage of a subset of these. CONCLUSIONS: These findings suggest that most of the genomic changes driving oral cancer occur in the pre-cancerous state by way of gradual random accumulation rather than a dramatic single event.

Comparing mutation calls in fixed tumour samples between the affymetrix OncoScan® array and PCR based next-generation sequencing.

BACKGROUND: The importance of accurate and affordable mutation calling in fixed pathology samples is becoming increasingly important as we move into the era of personalised medicine. The Affymetrix OncoScan® Array platform is designed to produce actionable mutation calls in archival material. METHODS: We compared calls made using the OncoScan platform with calls made using a custom designed PCR panel followed by next-generation sequencing (NGS), in order to benchmark the sensitivity and specificity of the OncoScan calls in a large cohort of fixed tumour samples. 392 fixed, clinical samples were sequenced, encompassing 641 PCR regions, 403 putative positive calls and 1528 putative negative calls. RESULTS: A small number of mutations could not be validated, either due to large indels or pseudogenes impairing parts of the NGS pipeline. For the remainder, if calls were filtered according to simple quality metrics, both sensitivity and specificity for the OncoScan platform were over 98%. This applied even to samples with poorer sample quality and lower variant allele frequency (5-10%) than product claims indicated. CONCLUSIONS: This benchmarking study will be useful to users and potential users of this platform, who wish to compare technologies or interpret their own results.