Genomic mutations caused by cytotoxic agents used in cancer chemotherapy may cause secondary malignancies as well as contribute to the evolution of treatment-resistant tumour cells. The stable ...diploid genome of the chicken DT40 lymphoblast cell line, an established DNA repair model system, is well suited to accurately assay genomic mutations.
We use whole genome sequencing of multiple DT40 clones to determine the mutagenic effect of eight common cytotoxics used for the treatment of millions of patients worldwide. We determine the spontaneous mutagenesis rate at 2.3 × 10(-10) per base per cell division and find that cisplatin, cyclophosphamide and etoposide induce extra base substitutions with distinct spectra. After four cycles of exposure, cisplatin induces 0.8 mutations per Mb, equivalent to the median mutational burden in common leukaemias. Cisplatin-induced mutations, including short insertions and deletions, are mainly located at sites of putative intrastrand crosslinks. We find two of the newly defined cisplatin-specific mutation types as causes of the reversion of BRCA2 mutations in emerging cisplatin-resistant tumours or cell clones. Gemcitabine, 5-fluorouracil, hydroxyurea, doxorubicin and paclitaxel have no measurable mutagenic effect. The cisplatin-induced mutation spectrum shows good correlation with cancer mutation signatures attributed to smoking and other sources of guanine-directed base damage.
This study provides support for the use of cell line mutagenesis assays to validate or predict the mutagenic effect of environmental and iatrogenic exposures. Our results suggest genetic reversion due to cisplatin-induced mutations as a distinct mechanism for developing resistance.
The first genomic scar-based homologous recombination deficiency (HRD) measures were produced using SNP arrays. As array-based technology has been largely replaced by next generation sequencing ...approaches, it has become important to develop algorithms that derive the same type of genomic scar scores from next generation sequencing (whole exome "WXS", whole genome "WGS") data. In order to perform this analysis, we introduce here the scarHRD R package and show that using this method the SNP array-based and next generation sequencing-based derivation of HRD scores show good correlation (Pearson correlation between 0.73 and 0.87 depending on the actual HRD measure) and that the NGS-based HRD scores distinguish similarly well between BRCA mutant and BRCA wild-type cases in a cohort of triple-negative breast cancer patients of the TCGA data set.
The aim of the present study was to analyse the preoperative platelet count and the platelet-lymphocyte ratio (PLR) in patients with colorectal cancer (CRC) of different stages and with hepatic ...metastasis of CRC (mCRC) and to compare these factors as potential prognostic markers. Clinicopathological data of 10 years were collected retrospectively from 336 patients with CRC and 118 patients with mCRC. Both in the CRC and the mCRC group overall survival (OS) was significantly worse in patients who had elevated platelet count (hazard ratio HR = 2.2, p < 0.001 and HR = 2.9, p = 0.018, respectively). Multivariate analysis indicated that elevated platelet count was an independent prognostic factor of CRC (HR = 1.7, p = 0.035) and mCRC (HR = 3.1, p = 0.017). Disease-free survival (DFS) was significantly worse in patients with elevated platelet count in the CRC group (HR = 2.0, p = 0.011). In the multivariate analysis the PLR was not a prognostic factor in either of the two cohorts (HR = 0.92, p < 0.001 and HR = 0.89, p = 0.789, respectively). The platelet count is a valuable prognostic marker for the survival in patients both with CRC and mCRC while the PLR is not prognostic in either group.
PARP inhibitors are approved for the treatment of solid tumor types that frequently harbor alterations in the key homologous recombination (HR) genes, BRCA1/2. Other tumor types, such as lung cancer, ...may also be HR deficient, but the frequency of such cases is less well characterized. Specific DNA aberration profiles (mutational signatures) are induced by homologous recombination deficiency (HRD) and their presence can be used to assess the presence or absence of HR deficiency in a given tumor biopsy even in the absence of an observed alteration of an HR gene. We derived various HRD-associated mutational signatures from whole-genome and whole-exome sequencing data in the lung adenocarcinoma and lung squamous carcinoma cases from TCGA, and in a patient of ours with stage IVA lung cancer with exceptionally good response to platinum-based therapy, and in lung cancer cell lines. We found that a subset of the investigated cases, both with and without biallelic loss of BRCA1 or BRCA2, showed robust signs of HR deficiency. The extreme platinum responder case also showed a robust HRD-associated genomic mutational profile. HRD-associated mutational signatures were also associated with PARP inhibitor sensitivity in lung cancer cell lines. Consequently, lung cancer cases with HRD, as identified by diagnostic mutational signatures, may benefit from PARP inhibitor therapy.
Basal-like and luminal breast tumors have distinct clinical behavior and molecular profiles, yet the underlying mechanisms are poorly defined. To interrogate processes that determine these distinct ...phenotypes and their inheritance pattern, we generated somatic cell fusions and performed integrated genetic and epigenetic (DNA methylation and chromatin) profiling. We found that the basal-like trait is generally dominant and is largely defined by epigenetic repression of luminal transcription factors. Definition of super-enhancers highlighted a core program common in luminal cells but a high degree of heterogeneity in basal-like breast cancers that correlates with clinical outcome. We also found that protein extracts of basal-like cells are sufficient to induce a luminal-to-basal phenotypic switch, implying a trigger of basal-like autoregulatory circuits. We determined that KDM6A might be required for luminal-basal fusions, and we identified EN1, TBX18, and TCF4 as candidate transcriptional regulators of the luminal-to-basal switch. Our findings highlight the remarkable epigenetic plasticity of breast cancer cells.
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•The basal-like trait is generally dominant and defined by epigenetic mechanisms•Common core epigenetic programs in luminal tumors are defined by FOXA1 super-enhancers•Basal tumors show repression of luminal factors and high epigenetic heterogeneity•Nuclear extracts or transcription factors can reprogram breast cancer cells
Luminal and basal-like breast tumors are clinically distinct. Using somatic cell fusions, Su et al. demonstrate the general dominance of the basal-like phenotype. Epigenomic profiling of parental luminal and basal-like cells and heterofusions define common luminal programs but a high degree of heterogeneity in basal-like cancer cells.
Gene expression profiles of clinical cohorts can be used to identify genes that are correlated with a clinical variable of interest such as patient outcome or response to a particular drug. However, ...expression measurements are susceptible to technical bias caused by variation in extraneous factors such as RNA quality and array hybridization conditions. If such technical bias is correlated with the clinical variable of interest, the likelihood of identifying false positive genes is increased. Here we describe a method to visualize an expression matrix as a projection of all genes onto a plane defined by a clinical variable and a technical nuisance variable. The resulting plot indicates the extent to which each gene is correlated with the clinical variable or the technical variable. We demonstrate this method by applying it to three clinical trial microarray data sets, one of which identified genes that may have been driven by a confounding technical variable. This approach can be used as a quality control step to identify data sets that are likely to yield false positive results.
The ability to produce apolipoprotein (apo) B-containing lipoproteins enables hepatocytes, enterocytes, and cardiomyocytes to export triglycerides. In this study, we examined secretion of ...apoB-containing lipoproteins from mouse kidney and its putative impact on triglyceride accumulation in the tubular epithelium. Mouse kidney expressed both the apoB and microsomal triglyceride transfer protein genes, which permit lipoprotein formation. To examine de novo lipoprotein secretion, kidneys from human apoB-transgenic mice were minced and placed in medium with 35S-amino acids. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB immunoprecipitation. 35S-Labeled apoB100 was recovered in ∼1.03–1.04 g/ml lipoproteins (i.e. similar to the density of plasma low density lipoproteins). Immunohistochemistry of kidney sections suggested that apoB mainly is produced by tubular epithelial cells. ApoB expression in the kidney cortex was reduced ∼90% in vivo by treating wild type mice with apoB-antisense locked nucleic acid oligonucleotide. Inhibition of apoB expression increased fasting-induced triglyceride accumulation in the kidney cortex by 20–25% (p = 0.008). Cholesterol stores were unaffected. Treatment with control oligonucleotides with 1 or 4 mismatching base pairs affected neither the triglyceride nor the cholesterol content of the kidney cortex. The results suggest that mammalian kidney secretes apoB100-containing lipoproteins. One biological effect may be to dampen excess storage of triglycerides in proximal tubule cells.
Thrombocytosis portends adverse prognostic significance in many types of cancers including ovarian and lung carcinoma. In this study, we determined the prevalence and prognostic significance of ...thrombocytosis (defined as platelet count in excess of 400 × 10
3/μl) in patients with colorectal cancer. We performed a retrospective analysis of 310 consecutive patients diagnosed at our Institution between 2004 and 2013. The patients (48.7% male and 51.3% female) had a mean age of 69.9 years (+/- 12.7 years) at diagnosis. Thrombocytosis was found in a total of 25 patients, with a higher incidence in those with stage III and IV disease (14.4% of patients). Although the mean platelet count increased with the depth of tumor invasion (pT), its values remained within normal limits in the whole patient cohort. No patient with stage I cancer (n=57) had elevated platelet count at diagnosis. By contrast, five of the 78 patients (6.4%) with stage II cancer showed thrombocytosis, and four of these patients showed early recurrence and/or metastatic disease, resulting in shortened survival (they died within one year after surgery). The incidence of thrombocytosis increased to 12.2% and 20.6%, respectively, in patients with stage III and IV disease. The overall survival rate of patients with thrombocytosis was lower than those without thrombocytosis in the stage II and III disease groups, but this difference disappeared in patients with stage IV cancer who did poorly regardless of their platelet count. We concluded that thrombocytosis at diagnosis indicates adverse clinical outcome in colorectal cancer patients with stage II or III disease. This observation is especially intriguing in stage II patients because the clinical management of these patients is controversial. If our data are confirmed in larger studies, stage II colon cancer patients with thrombocytosis may be considered for adjuvant chemotherapy.
The chicken DT40 cell line is a widely used model system in the study of multiple cellular processes due to the efficiency of homologous gene targeting. The cell line was derived from a bursal ...lymphoma induced by avian leukosis virus infection. In this study we characterized the genome of the cell line using whole genome shotgun sequencing and single nucleotide polymorphism array hybridization. The results indicate that wild-type DT40 has a relatively normal karyotype, except for whole chromosome copy number gains, and no karyotype variability within stocks. In a comparison to two domestic chicken genomes and the Gallus gallus reference genome, we found no unique mutational processes shaping the DT40 genome except for a mild increase in insertion and deletion events, particularly deletions at tandem repeats. We mapped coding sequence mutations that are unique to the DT40 genome; mutations inactivating the PIK3R1 and ATRX genes likely contributed to the oncogenic transformation. In addition to a known avian leukosis virus integration in the MYC gene, we detected further integration sites that are likely to de-regulate gene expression. The new findings support the hypothesis that DT40 is a typical transformed cell line with a relatively intact genome; therefore, it is well-suited to the role of a model system for DNA repair and related processes. The sequence data generated by this study, including a searchable de novo genome assembly and annotated lists of mutated genes, will support future research using this cell line.
The evolutionary features of clear-cell renal cell carcinoma (ccRCC) have not been systematically studied to date. We analyzed 1,206 primary tumor regions from 101 patients recruited into the ...multi-center prospective study, TRACERx Renal. We observe up to 30 driver events per tumor and show that subclonal diversification is associated with known prognostic parameters. By resolving the patterns of driver event ordering, co-occurrence, and mutual exclusivity at clone level, we show the deterministic nature of clonal evolution. ccRCC can be grouped into seven evolutionary subtypes, ranging from tumors characterized by early fixation of multiple mutational and copy number drivers and rapid metastases to highly branched tumors with >10 subclonal drivers and extensive parallel evolution associated with attenuated progression. We identify genetic diversity and chromosomal complexity as determinants of patient outcome. Our insights reconcile the variable clinical behavior of ccRCC and suggest evolutionary potential as a biomarker for both intervention and surveillance.
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•ccRCC evolutionary subtypes correlate with clinical phenotypes•Genetic diversity and chromosome complexity contribute to patient outcomes•Early fixation of multiple driver events leads to rapid growth and metastases•Subclonal diversification is linked with slower growth and attenuated metastases
A multi-center prospective study on 101 patients with clear-cell renal cell carcinoma resolves the evolutionary features and subtypes underpinning the diverse clinical phenotypes of the disease and suggests these features as potential biomarkers for guiding intervention and surveillance.
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