Splicing modulators impair DNA damage response and induce killing of cohesin-mutant MDS and AML.

Splicing modulation is a promising treatment strategy pursued to date only in splicing factor-mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we showed that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing factor 3B subunit 1 (SF3B1) H3B-8800 and E-7107. We identified drug-induced alterations in splicing, and corresponding reduced gene expression, of a number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We found that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators due to their long length and large number of exons per transcript. Furthermore, we demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators not only resulted in impaired DNA damage response and accumulation of DNA damage, but it sensitized cells to subsequent killing by poly(ADP-ribose) polymerase (PARP) inhibitors and chemotherapy and led to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML.

Research Strategies for Low-Survival Cancers.

While substantial progress has been made to improve the diagnosis, prognosis, and survivorship of patients with cancer, certain cancer types, along with metastatic and refractory disease, remain clinical challenges. To improve patient outcomes, ultimately, the cancer research community must meet and overcome these challenges, leading to improved approaches to treat the most difficult cancers. Here, we discuss research progress aimed at gaining a better understanding of the molecular and cellular changes in tumor cells and the surrounding stroma, presented at the 56th Irish Association for Cancer Research (IACR) Annual Conference. With a focus on poor prognosis cancers, such as esophageal and chemo-resistant colorectal cancers, we highlight how detailed molecular knowledge of tumor and stromal biology can provide windows of opportunity for biomarker discovery and therapeutic targets. Even with previously characterized targets, such as phosphoinositide 3-kinase (PI3K), one of the most altered proteins in all human cancers, new insights into how this protein may be more effectively inhibited through novel combination therapies is presented.

Clinical and Laboratory Associations with Methotrexate Metabolism Gene Polymorphisms in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease that causes loss of joint function and significantly reduces quality of life. Plasma metabolite concentrations of disease-modifying anti-rheumatic drugs (DMARDs) can influence treatment efficacy and toxicity. This study explored the relationship between DMARD-metabolising gene variants and plasma metabolite levels in RA patients. DMARD metabolite concentrations were determined by tandem mass-spectrometry in plasma samples from 100 RA patients with actively flaring disease collected at two intervals. Taqman probes were used to discriminate single-nucleotide polymorphism (SNP) genotypes in cohort genomic DNA: rs246240 (ABCC1), rs1476413 (MTHFR), rs2231142 (ABCG2), rs3740065 (ABCC2), rs4149081 (SLCO1B1), rs4846051 (MTHFR), rs10280623 (ABCB1), rs16853826 (ATIC), rs17421511 (MTHFR) and rs717620 (ABCC2). Mean plasma concentrations of methotrexate (MTX) and MTX-7-OH metabolites were higher (p < 0.05) at baseline in rs4149081 GA genotype patients. Patients with rs1476413 SNP TT or CT alleles have significantly higher (p < 0.001) plasma poly-glutamate metabolites at both study time points and correspondingly elevated disease activity scores. Patients with the rs17421511 SNP AA allele reported significantly lower pain scores (p < 0.05) at both study intervals. Genotyping strategies could help prioritise treatments to RA patients most likely to gain clinical benefit whilst minimizing toxicity.

Mutation-Independent Allele-Specific Editing by CRISPR-Cas9, a Novel Approach to Treat Autosomal Dominant Disease.

CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor ß-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner.

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.

Towards Stratified Medicine in Plasma Cell Myeloma.

Plasma cell myeloma is a clinically heterogeneous malignancy accounting for approximately one to 2% of newly diagnosed cases of cancer worldwide. Treatment options, in addition to long-established cytotoxic drugs, include autologous stem cell transplant, immune modulators, proteasome inhibitors and monoclonal antibodies, plus further targeted therapies currently in clinical trials. Whilst treatment decisions are mostly based on a patient's age, fitness, including the presence of co-morbidities, and tumour burden, significant scope exists for better risk stratification, sub-classification of disease, and predictors of response to specific therapies. Clinical staging, recurring acquired cytogenetic aberrations, and serum biomarkers such as ß-2 microglobulin, and free light chains are in widespread use but often fail to predict the disease progression or inform treatment decision making. Recent scientific advances have provided considerable insight into the biology of myeloma. For example, gene expression profiling is already making a contribution to enhanced understanding of the biology of the disease whilst Next Generation Sequencing has revealed great genomic complexity and heterogeneity. Pathways involved in the oncogenesis, proliferation of the tumour and its resistance to apoptosis are being unravelled. Furthermore, knowledge of the tumour cell surface and its interactions with bystander cells and the bone marrow stroma enhance this understanding and provide novel targets for cell and antibody-based therapies. This review will discuss the development in understanding of the biology of the tumour cell and its environment in the bone marrow, the implementation of new therapeutic options contributing to significantly improved outcomes, and the progression towards more personalised medicine in this disorder.

Elucidating drivers of oral epithelial dysplasia formation and malignant transformation to cancer using RNAseq.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.

The clonal relationships between pre-cancer and cancer revealed by ultra-deep sequencing.

The study of the relationships between pre-cancer and cancer and identification of early driver mutations is becoming increasingly important as the value of molecular markers of early disease and personalised drug targets is recognized, especially now the extent of clonal heterogeneity in fully invasive disease is being realized. It has been assumed that pre-cancerous lesions exhibit a fairly passive progression to invasive disease; the degree to which they, too, are heterogeneous is unknown. We performed ultra-deep sequencing of thousands of selected mutations, together with copy number analysis, from multiple, matched pre-invasive lesions, primary tumours and metastases from five patients with oral cancer, some with multiple primary tumours presenting either synchronously or metachronously, totalling 75 samples. This allowed the clonal relationships between the samples to be observed for each patient. We expose for the first time the unexpected variety and complexity of the relationships between this group of oral dysplasias and their associated carcinomas and, ultimately, the diversity of processes by which tumours are initiated, spread and metastasize. Instead of a series of genomic precursors of their adjacent invasive disease, we have shown dysplasia to be a distinct dynamic entity, refuting the belief that pre-cancer and invasive tumours with a close spatial relationship always have linearly related genomes. We show that oral pre-cancer exhibits considerable subclonal heterogeneity in its own right, that mutational changes in pre-cancer do not predict the onset of invasion, and that the genomic pathway to invasion is neither unified nor predictable. Sequence data from this study have been deposited in the European Nucleotide Archive, Accession No. PRJEB6588.

A novel genomic signature reclassifies an oral cancer subtype.

Verrucous carcinoma of the oral cavity (OVC) is considered a subtype of classical oral squamous cell carcinoma (OSCC). Diagnosis is problematic, and additional biomarkers are needed to better stratify patients. To investigate their molecular signature, we performed low-coverage copy number (CN) sequencing on 57 OVC and exome and RNA sequencing on a subset of these and compared the data to the same OSCC parameters. CN results showed that OVC lacked any of the classical OSCC patterns such as gain of 3q and loss of 3p and demonstrated considerably fewer genomic rearrangements compared to the OSCC cohort. OVC and OSCC samples could be clearly differentiated. Exome sequencing showed that OVC samples lacked mutations in genes commonly associated with OSCC (TP53, NOTCH1, NOTCH2, CDKN2A and FAT1). RNA sequencing identified genes that were differentially expressed between the groups. In silico functional analysis showed that the mutated and differentially expressed genes in OVC samples were involved in cell adhesion and keratinocyte proliferation, while those in the OSCC cohort were enriched for cell death and apoptosis pathways. This is the largest and most detailed genomic and transcriptomic analysis yet performed on this tumour type, which, as an example of non-metastatic cancer, may shed light on the nature of metastases. These three independent investigations consistently show substantial differences between the cohorts. Taken together, they lead to the conclusion that OVC is not a subtype of OSCC, but should be classified as a distinct entity.

Clinical validation of controlled grass pollen challenge in the Environmental Exposure Unit (EEU).

RATIONALE: The Environmental Exposure Unit (EEU), a controlled allergen exposure model of allergic rhinitis (AR), has traditionally utilized ragweed pollen. We sought to clinically validate the use of grass pollen in the EEU. METHODS: Healthy volunteers with a history of AR symptoms during grass pollen season and supportive skin test responses attended the EEU for 3 hrs of rye grass pollen exposure (Lolium Perenne). Non-atopic controls were also recruited. Participants assessed individual rhinoconjunctivitis symptoms to generate Total Nasal Symptom Score (TNSS; max 12) and Total Symptom Score (TSS; max 24) and recorded Peak Nasal Inspiratory Flow (PNIF) q30min while in the EEU. Participants returned the following day for an additional 3 hrs of pollen exposure. Two separate groups allowed for the exploration of lower vs. higher pollen concentrations and subsequent effects on symptoms. RESULTS: 78 participants were screened, of whom 39 were eligible and attended the 2x3h EEU visits, plus 8 non-atopic controls. Mean TSS, TNSS and PNIF values amongst participants in the higher pollen concentration group (target 3500 grains/m3) after the first 3 hr exposure were 18.9, 9.7 and 68 L/min, respectively. In comparison, mean TSS, TNSS and PNIF values in the lower pollen concentration (2500 grains/m3) group were only 13.3, 7.6, and 82 L/min, respectively. The subsequent day of pollen exposure did not appreciably alter the maximal TSS/TNSSs, but rather resulted in a more rapid onset of symptomatology, with higher mean scores at the 30 min, 60 min and 90 min timepoints. The non-atopic controls remained asymptomatic. CONCLUSIONS: This study provides clinical validation of the ability to generate allergic rhinoconjunctivitis symptoms amongst grass-allergic individuals in the EEU.

The clinicopathological and gene expression patterns associated with ulceration of primary melanoma.

Ulceration of primary melanomas is associated with poor prognosis yet is reported to predict benefit from adjuvant interferon. To better understand the biological processes involved, clinicopathological factors associated with ulceration were determined in 1804 patients. From this cohort, 348 primary tumor blocks were sampled to generate gene expression data using a 502-gene cancer panel and 195 blocks were used for immunohistochemistry to detect macrophage infiltration and vessel density. Gene expression results were validated using a whole genome array in two independent sample sets. Ulceration of primary melanomas was associated with more proliferative tumors, tumor vessel invasion, and increased microvessel density. Infiltration of tumors with greater number of macrophages and gene expression pathways associated with wound healing and up-regulation of pro-inflammatory cytokines suggests that ulceration is associated with tumor-related inflammation. The relative benefit from interferon reported in patients with ulcerated tumors may reflect modification of signaling pathways involved in inflammation.

Next-generation sequencing analysis for detecting human papillomavirus in oral verrucous carcinoma.

OBJECTIVE: The etiology of oral verrucous carcinoma is unknown, and human papillomavirus 'involvement' remains contentious. The uncertainty can be attributed to varied detection procedures and difficulties in defining 'gold-standard' histologic criteria for diagnosing 'verrucous' lesions. Their paucity also hampers investigation. We aimed to analyze oral verrucous lesions for human papillomavirus (HPV) subtype genomes. STUDY DESIGN: We used next-generation sequencing for the detection of papillomavirus sequences, identifying subtypes and computing viral loads. We identified a total of 78 oral verrucous cases (62 carcinomas and 16 hyperplasias). DNA was extracted from all and sequenced at a coverage between 2.5% and 13%. RESULTS: An HPV-16 sequence was detected in 1 carcinoma and 1 hyperplasia, and an HPV-2 sequence was detected in 1 carcinoma out of the 78 cases, with viral loads of 2.24, 8.16, and 0.33 viral genomes per cell, respectively. CONCLUSIONS: Our results indicate no conclusive human papillomavirus involvement in oral verrucous carcinoma or hyperplasia.

An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing.

Squamous cell carcinoma (SCC) of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding) of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.

The transcriptional consequences of somatic amplifications, deletions, and rearrangements in a human lung squamous cell carcinoma.

Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.

Next-generation sequencing for simultaneous determination of human papillomavirus load, subtype, and associated genomic copy number changes in tumors.

Human papillomavirus (HPV) infection in cases of squamous cell carcinoma of the oropharynx is a powerful predictive and prognostic biomarker. We describe how the use of next-generation sequencing can provide a novel method for the detection of HPV in DNA isolated from formalin-fixed paraffin-embedded tissues. Using this methodology in a cohort of 44 head and neck tumors, we identified the samples that contained HPV sequences, the viral subtype involved, and a direct readout of viral load. Specificity of HPV detection by sequencing compared to traditional detection methods using either PCR or p16 immunohistochemistry was 100%. Sensitivity was 50% when either compared to PCR [confidence interval (CI) = 29% to 71%] or 75% when compared to p16 (CI = 47% to 91%). In addition, we demonstrate the ability of next-generation sequencing to detect other HPV subtypes that would not have been detected by traditional methods, and we demonstrated the ability to apply this method to any tumor and any virus in a panel of eight human cancer cell lines. This methodology also provides a tumor genomic copy number karyogram, and in the samples analyzed here, a lower level of chromosome instability was detected in HPV-positive tumors compared to HPV-negative tumors, as observed in previous studies. Thus, the use of next-generation sequencing for the detection of HPV provides a multiplicity of data with clinical significance in a single test.

A computational index derived from whole-genome copy number analysis is a novel tool for prognosis in early stage lung squamous cell carcinoma.

Squamous cell carcinoma of the lung is remarkable for the extent to which the same chromosomal abnormalities are detected in individual tumours. We have used next generation sequencing at low coverage to produce high resolution copy number karyograms of a series of 89 non-small cell lung tumours specifically of the squamous cell subtype. Because this methodology is able to create karyograms from formalin-fixed paraffin-embedded material, we were able to use archival stored samples for which survival data were available and correlate frequently occurring copy number changes with disease outcome. No single region of genomic change showed significant correlation with survival. However, adopting a whole-genome approach, we devised an algorithm that relates to total genomic damage, specifically the relative ratios of copy number states across the genome. This algorithm generated a novel index, which is an independent prognostic indicator in early stage squamous cell carcinoma of the lung.

Patterns of expression of DNA repair genes and relapse from melanoma.

PURPOSE: To use gene expression profiling of formalin-fixed primary melanoma samples to detect expression patterns that are predictive of relapse and response to chemotherapy. EXPERIMENTAL DESIGN: Gene expression profiles were identified in samples from two studies (472 tumors). Gene expression data for 502 cancer-related genes from these studies were combined for analysis. RESULTS: Increased expression of DNA repair genes most strongly predicted relapse and was associated with thicker tumors. Increased expression of RAD51 was the most predictive of relapse-free survival in unadjusted analysis (hazard ratio, 2.98; P = 8.80 × 10(-6)). RAD52 (hazard ratio, 4.73; P = 0.0004) and TOP2A (hazard ratio, 3.06; P = 0.009) were independent predictors of relapse-free survival in multivariable analysis. These associations persisted when the analysis was further adjusted for demographic and histologic features of prognostic importance (RAD52 P = 0.01; TOP2A P = 0.02). Using principal component analysis, expression of DNA repair genes was summarized into one variable. Genes whose expression correlated with this variable were predominantly associated with the cell cycle and DNA repair. In 42 patients treated with chemotherapy, DNA repair gene expression was greater in tumors from patients who progressed on treatment. Further data supportive of a role for increased expression of DNA repair genes as predictive biomarkers are reported, which were generated using multiplex PCR. CONCLUSIONS: Overexpression of DNA repair genes (predominantly those involved in double-strand break repair) was associated with relapse. These data support the hypothesis that melanoma progression requires maintenance of genetic stability and give insight into mechanisms of melanoma drug resistance and potential therapies.

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens.

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material.