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Tumor mutational signatures have gained prominence in cancer research, yet the lack of standardized methods hinders reproducibility and robustness. Leveraging colorectal cancer (CRC) as a model, we explored the influence of computational parameters on mutational signature analyses across 230 CRC cell lines and 152 CRC patients. Results were validated in three independent datasets: 483 endometrial cancer patients stratified by mismatch repair (MMR) status, 35 lung cancer patients by smoking status and 12 patient-derived organoids (PDOs) annotated for colibactin exposure. Assessing various bioinformatic tools, reference datasets and input data sizes including whole genome sequencing, whole exome sequencing and a pan-cancer gene panel, we demonstrated significant variability in the results. We report that the use of distinct algorithms and references led to statistically different results, highlighting how arbitrary choices may induce variability in the mutational signature contributions. Furthermore, we found a differential contribution of mutational signatures between coding and intergenic regions and defined the minimum number of somatic variants required for reliable mutational signature assignment. To facilitate the identification of the most suitable workflows, we developed Comparative Mutational Signature analysis on Coding and Extragenic Regions (CoMSCER), a bioinformatic tool which allows researchers to easily perform comparative mutational signature analysis by coupling the results from several tools and public reference datasets and to assess mutational signature contributions in coding and non-coding genomic regions. In conclusion, our study provides a comparative framework to elucidate the impact of distinct computational workflows on mutational signatures. [ABSTRACT FROM AUTHOR]

Colorectal cancers (CRCs) evolve by a reiterative process of genetic diversification and clonal evolution. The molecular profile of CRC is routinely assessed in surgical or bioptic samples (1). Genotyping of CRC tissue has inherent limitations; a tissue sample represents a single snapshot in time, and it is subjected to spatial selection bias owing to tumor heterogeneity. Repeated tissue samples are difficult to obtain and cannot be used for dynamic monitoring of disease progression and response to therapy. We exploited circulating tumor DNA (ctDNA) to genotype colorectal tumors and track clonal evolution during treatment with the epidermal growth factor receptor (EGFR)-specific antibodies cetuximab or panitumumab. We identified alterations in ctDNA of patients with primary or acquired resistance to EGFR blockade in the following genes: KRAS, NRAS, MET, ERBB2, FLT3, EGFR and MAP2K1. Mutated KRAS clones, which emerge in blood during EGFR blockade, decline upon withdrawal of EGFR-specific antibodies, indicating that clonal evolution continues beyond clinical progression. Pharmacogenomic analysis of CRC cells that had acquired resistance to cetuximab reveals that upon antibody withdrawal KRAS clones decay, whereas the population regains drug sensitivity. ctDNA profiles of individuals who benefit from multiple challenges with anti-EGFR antibodies exhibit pulsatile levels of mutant KRAS. These results indicate that the CRC genome adapts dynamically to intermittent drug schedules and provide a molecular explanation for the efficacy of rechallenge therapies based on EGFR blockade., An improved understanding of the underlying molecular pathology of CRC has enabled tailored treatment regimens and helped optimize outcomes. CRC tissue is currently used to define the molecular status of [...]

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers (1). After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug (2). The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood (3-8). Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance., Defining the molecular bases of secondary resistance to anti-EGFR therapies is crucial to monitor, prevent and/or overcome drug refractoriness. To identify potential mechanisms of cetuximab resistance, we generated cetuximab-resistant variants [...]

Pooled data sets from patients with chemotherapy-refractory colorectal cancer treated with cetuximab between 2001 and 2008 was used to study the association between KRAS condon 13 mutations and response and survival after treatment with cetuximab compared to those observed with other KRAS mutations. Cetuximab was found to be associated with longer overall and progression-free survival among patients with chemotherapy-refractory colorectal cancer with p.G13D-mutated tumors than with other KRAS-mutated tumors and suggested a need for further randomized trials.

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, α polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors., Introduction The PI3K/PTEN/AKT signaling pathway is frequently deregulated in human cancer (1). This pathway plays a crucial role in cell growth, survival, and proliferation; hence, its components have recently emerged [...]

Systemic treatment of malignant pleural mesothelioma (MPM) is moderately active for the intrinsic pharmacological resistance of MPM cell and its ability to induce an immune suppressive environment. Here we showed that the expression of bromodomain (BRD) proteins BRD2, BRD4 and BRD9 was significantly higher in human primary MPM cells compared to normal mesothelial cells (HMC). Nanomolar concentrations of bromodomain inhibitors (BBIs) JQ1 or OTX015 impaired patient-derived MPM cell proliferation and induced cell-cycle arrest without affecting apoptosis. Importantly, BBIs primed MPM cells for immunogenic cell death, by increasing extracellular release of ATP and HMGB1, and by promoting membrane exposure of calreticulin and ERp57. Accordingly, BBIs activated dendritic cell (DC)-mediated phagocytosis and expansion of CD8C T-lymphocyte clones endorsed with antitumor cytotoxic activity. BBIs reduced the expression of the immune checkpoint ligand PD-L1 in MPM cells; while both CD8C and CD4C T-lymphocytes co-cultured with JQ1-treated MPM cells decreased PD-1 expression, suggesting a disruption of the immune-suppressive PD-L1/PD-1 axis. Additionally, BBIs reduced the expansion of myeloid-derived suppressor cells (MDSC) induced by MPM cells. Finally, a preclinical model of MPM confirmed that the anti-tumor efficacy of JQ1 was largely due to its ability to restore an immune-active environment, by increasing intra-tumor DC and CD8C T-lymphocytes, and decreasing MDSC. Thereby, we propose that, among novel drugs, BBIs should be investigated for MPM treatment for their combined activity on both tumor cells and surrounding immune-environment. [ABSTRACT FROM AUTHOR]

Background: COX-2 expression in tumour cells has been associated with poor prognosis in gastrointestinal and non-gastrointestinal cancers. The aim of our study was to test the hypothesis that higher levels of COX-2 expression are prognostically related to poor clinico-pathologic features in adenocarcinoma of the oesophagus. Methods: We reviewed the records of 100 consecutive patients undergoing resection for adenocarcinoma of the oesophagus to collect data on T-stage, N-stage, tumour recurrence and survival. T & N-stage was further confirmed by histological examination. COX-2 protein expression was assessed by immunohistochemistry in all patients and COX-2 m-RNA expression was measured by quantitative RT-PCR in a small group of patients. Results: Higher levels of COX-2 expression were associated with higher T stage (p = 0.008), higher N stage (p = 0.049), increased risk of tumour recurrence (p = 0.01) and poor survival (p = <0.001). A COX-2 score of >200 was associated with a median survival of 10 months compared to 26 months with a score of <200 (p = <0.001). Conclusion: Higher levels of COX-2 expression are associated with poor clinico-pathologic features and poor survival in patients with oesophageal adenocarcinoma. [ABSTRACT FROM AUTHOR]

An abstract of the article "Targeting oncogenic serine/threonine-protein kinase BRAF in cancer cells inhibits angiogenesis and abrogates hypoxia," by Alessia Bottos and colleagues.