SummaryMultiple signatures of somatic mutations have been identified in cancer genomes. Exome sequences of 1,001 human cancer cell lines and 577 xenografts revealed most common mutational signatures, indicating past activity of the underlying processes, usually in appropriate cancer types. To investigate ongoing patterns of mutational-signature generation, cell lines were cultured for extended periods and subsequently DNA sequenced. Signatures of discontinued exposures, including tobacco smoke and ultraviolet light, were not generated in vitro. Signatures of normal and defective DNA repair and replication continued to be generated at roughly stable mutation rates. Signatures of APOBEC cytidine deaminase DNA-editing exhibited substantial fluctuations in mutation rate over time with episodic bursts of mutations. The initiating factors for the bursts are unclear, although retrotransposon mobilization may contribute. The examined cell lines constitute a resource of live experimental models of mutational processes, which potentially retain patterns of activity and regulation operative in primary human cancers.

BackgroundIt 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.MethodsWe 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.ResultsEvidence 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0442-0) contains supplementary material, which is available to authorized users.

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 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 recognised, especially now the extent of clonal heterogeneity in fully invasive disease is being realised. 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 metastasise.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 sub-clonal 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.

The use of the DNA dyes Hoechst (HO) and chromomycin A3 (CA3) has become the preferred combination for the bivariate analysis of chromosomes from both human and animals. This analysis requires a flow cytometer equipped with lasers of specific wavelength and of higher power than is typical on a conventional bench top flow cytometer. In this study, we have investigated the resolution of chromosome peaks in a human cell line with normal flow karyotype using different combinations of DNA dyes on a number of flow cytometers available in a flow cytometry core facility. Chromosomes were prepared from the human cell line using a modified polyamine isolation buffer. The bivariate flow karyotypes of different DNA dyes combination; 4′‐6‐diamidino‐2‐phenylindole (DAPI) or Hoechst with propidium iodide (PI), obtained from different flow cytometers were compared to the reference flow karyotype of DAPI or Hoechst with chromomycin A3, generated from a Mo‐Flo cell sorter using laser power settings of 300 mW each of UV and 457 nm. Good chromosome separation was observed in most of the flow cytometers used in the study. This study demonstrates that chromosome analysis and sorting can also be performed on benchtop flow cytometers equipped with the standard solid state 488 and 355 nm lasers, using a DNA dye combination of DAPI or Hoechst with PI. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

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Identification of genomic mutations by molecular testing plays an important role in diagnosis, prognosis, and treatment of myeloid neoplasms. Next-generation sequencing (NGS) is an efficient method for simultaneous detection of clinically significant genomic mutations with high sensitivity. Various NGS based in-house developed and commercial myeloid neoplasm panels have been integrated into routine clinical practice. However, some genes frequently mutated in myeloid malignancies are particularly difficult to sequence with NGS panels (e.g., CEBPA, CARL, and FLT3). We report development and validation of a 48-gene NGS panel that includes genes that are technically challenging for molecular profiling of myeloid neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN). Target regions were captured by hybridization with complementary biotinylated DNA baits, and NGS was performed on an Illumina NextSeq500 instrument. A bioinformatics pipeline that was developed in-house was used to detect single nucleotide variations (SNVs), insertions/deletions (indels), and FLT3 internal tandem duplications (FLT3-ITD). An analytical validation study was performed on 184 unique specimens for variants with allele frequencies ≥5%. Variants identified by the 48-gene panel were compared to those identified by a 35-gene hematologic neoplasms panel using an additional 137 unique specimens. The developed assay was applied to a large cohort (n = 2,053) of patients with suspected myeloid neoplasms. Analytical validation yielded 99.6% sensitivity (95% CI: 98.9–99.9%) and 100% specificity (95% CI: 100%). Concordance of variants detected by the 2 tested panels was 100%. Among patients with suspected myeloid neoplasms (n = 2,053), 54.5% patients harbored at least one clinically significant mutation: 77% in AML patients, 48% in MDS, and 45% in MPN. Together, these findings demonstrate that the assay can identify mutations associated with diagnosis, prognosis, and treatment options of myeloid neoplasms even in technically challenging genes.