Somatic NGS Analysis of DNA Damage Response (DDR) Genes ATM, MRE11A, RAD50, NBN, and ATR in Locally Advanced Rectal Cancer Treated with Neoadjuvant Chemo-Radiotherapy.

BACKGROUND: Neoadjuvant chemo-radiotherapy (nCRT) represents the standard of care for locally advanced rectal cancer (LARC); however, there exists no biomarker that can predict the cancer's response to treatment as less than 20% of patients experience pathological complete response (pCR). Ionizing radiations induce double strand breaks (DSBs) and trigger a DNA damage response (DDR) involving ATM, ATR, and the MRN complex (MRE11, Rad50, and NBS1). In this study, we performed an extensive mutational analysis of the genes involved in the DDR pathway in LARC patients who have undergone nCRT. METHODS: 13 LARC patients with pCR and 11 LARC patients with partial response (pPR) were investigated using a NGS dedicated panel, designed for formalin-fixed paraffin-embedded (FFPE) samples, containing ATR, ATM, and MRE11-RAD50-NBN genes. The identified variants were classified according to guidelines' recommendations. RESULTS: Eight non-benign variants, six of which were observed in 3 (23%) out of 13 pCR patients, were identified. In particular, a pCR patient carried out a pathogenetic frameshift mutation in exon 21 of the RAD50 gene. The two remaining non-benign missense variants were found in 2 (18%) out of 11 patients in the pPR group. CONCLUSIONS: Our data show that the genes involved in the Homologous Recombination (HR) pathway are rarely mutated in LARC; however, given the identification of a missense mutation in RAD 50 in one case of pCR, it could be worth exploring its potential role as a biomarker in larger series.

SARS-CoV-2 Transmission Control Measures in the Emergency Department: The Role of Rapid Antigenic Testing in Asymptomatic Subjects.

Limiting transmission of SARS-CoV-2 from asymptomatic people assumes the paramount importance of keeping fragile subjects protected. We evaluated the utility of rapid SARS-CoV-2 antigen testing in asymptomatic subjects attending emergency departments in non-COVID-19 areas, using a single nasopharyngeal swab specimen collected in universal transport medium to perform both rapid antigen testing and rRT-PCR (used as reference standard) in a cohort of 899 patients. In the overall sample, the rapid antigen test had 43.9% sensitivity, 100% specificity, 100% positive predictive value, 93.6% negative predictive value. Considering subjects with rRT-PCR cycle threshold ≤30, the test had 80.4% sensitivity, 100% specificity, 100% positive predictive value, 98.8% negative predictive value. Considering subjects with rRT-PCR cycle threshold ≤25, the test had 94.7% sensitivity, 100% specificity, 100% positive predictive value and 99.7% negative predictive value. Despite low sensitivity, routine application of rapid antigen testing in the emergency department can lead to isolation in less than 30 min of about a half of asymptomatic COVID-19 subjects assigned to non-COVID-19 areas by clinical triage. The rapid test correctly identified 94.7% of asymptomatic patients with cycle threshold ≤ 25 that are supposed to be more infective; thus, it could be a useful measure to contain viral transmission in non-COVID-19 areas.

The Potential Role of Genomic Signature in Stage II Relapsed Colorectal Cancer (CRC) Patients: A Mono-Institutional Study.

Purpose: The absolute benefit of adjuvant chemotherapy in stage II CRC is only 3-4%. The identification of biomarkers through molecular profiling could identify patients who will more benefit from adjuvant chemotherapy. Patients and Methods: This retrospective analysis examined tissue blocks from 17 patients affected by relapsed stage II CRC, whose comprehensive genomic profiling of tumors was conducted through next-generation sequencing (NGS) via Roche-FoundationOne®. Results: Mutations were found in APC (76.5%), TP53 (58.8%) and KRAS (52.9%). Only KRAS wild-type samples showed FBXW7. APC frameshift mutations and MLH1 splice variant were conversely significant correlated (7% v 93%, P = 0.014). The median number of gene mutations reported was 6 (range 2-14). The TP53 mutation was associated most frequently with lung metastasis (P = 0.07) and high tumor budding (P = 0.03). Despite no statistical significance, lung recurrence, LVI/Pni, MSI and more than 6 genetic mutations were correlated to worse DFS and OS. Patients carried co-mutations of TP53-FBXW7 reported the worse DFS (4 v 14 months) and OS (4 v 65 months) compared to the other patients. Conclusion: According to the present analysis, the setting of relapsed CRC emerges as one of the fields of greatest utility for NGS, looking at personalized cancer care.

Histological growth patterns and molecular analysis of resected colorectal lung metastases.

Lung is the site of metastasis in about 15-25 % of colorectal cancer (CRC) patients. Lung metastasectomy of CRC represents a standard therapy in patients with resectable metastases. In this study we investigated both histological patterns of metastases and mutations in MAPkinase pathway genes and their relationship to prognosis. The study included 74 patients that underwent metastasectomy of colorectal lung metastasis (CLM). In patients that underwent surgical resection of more than one metastasis in the same operation the largest was chosen. In patients that had undergone multiple lung metastasectomy only the sample from the first metastasectomy was included. Histologically metastases were scored according to amount and distribution of necrosis and fibrosis and three patterns were identified: "pattern A", metastasis with extensive, confluent central necrosis surrounded by a rim of neoplastic glands; "pattern B", metastasis characterized by a proliferation of neoplastic glands in a dense stroma with focal necrosis mainly intraglandular; "pattern C", metastasis with a mixed A and B morphology. In all samples direct sequencing of exon 2 of KRAS and NRAS genes and exon 15 of BRAF genes was carried out.Histological patterns weren't related to metastasis size or other clinical features however pattern C metastases showed a significant worst disease free survival (DFS). KRAS mutations were observed in 39 % of patients. Mutations in KRAS codon 13 resulted significantly associated with synchronous metastasis and poor prognosis. No mutations were identified in exon 2 NRAS gene whilst 1.4 % harboured a mutation in BRAF. To our knowledge this is the first study that investigates in a large series of CLM histological growth patterns, molecular alterations and their relationship to prognosis. Our data suggest a prognostic role in CLM of KRAS specific mutations and histopathological patterns.

A 17-Gene Expression Signature for Early Identification of Poor Prognosis in Clear Cell Renal Cell Carcinoma.

The Identification of reliable Biomarkers able to predict the outcome after nephrectomy of patients with clear cell renal cell carcinoma (ccRCC) is an unmet need. The gene expression analysis in tumor tissues represents a promising tool for better stratification of ccRCC subtypes and patients' evaluation. METHODS: In our study we retrospectively analyzed using Next-Generation expression analysis (NanoString), the expression of a gene panel in tumor tissue from 46 consecutive patients treated with nephrectomy for non-metastatic ccRCC at two Italian Oncological Centres. Significant differences in expression levels of selected genes was sought. Additionally, we performed a univariate and a multivariate analysis on overall survival according to Cox regression model. RESULTS: A 17-gene expression signature of patients with a recurrence-free survival (RFS) < 1 year (unfavorable genomic signature (UGS)) and of patients with a RFS > 5 years (favorable genomic signature (FGS)) was identified and resulted in being significantly correlated with overall survival of the patients included in this analysis (HR 51.37, p < 0.0001). CONCLUSIONS: The identified Genomic Signatures may serve as potential biomarkers for prognosis prediction of non-metastatic RCC and could drive both follow-up and treatment personalization in RCC management.

MYC Upregulation Confers Resistance to Everolimus and Establishes Vulnerability to Cyclin-Dependent Kinase Inhibitors in Pancreatic Neuroendocrine Neoplasm Cells.

INTRODUCTION: Dysregulation of the mechanistic target of rapamycin complex 1 (mTORC1)-dependent pathways in pancreatic neuroendocrine neoplasms (PanNENs) underlies the introduction of the mTORC1 inhibitor everolimus as treatment of advanced progressive PanNENs. Although everolimus significantly increases progression-free survival, most patients acquire secondary resistance to the drug. This study aimed at identifying mechanisms involved in acquisition of resistance to everolimus. METHODS: BON-1 and everolimus-resistant (ER) BON-1 cells were used as in vitro system of sensitivity and acquired resistance. Transcriptome changes occurring in BON-1 and ER-BON-1 were investigated by RNA sequencing and validated by quantitative PCR analysis. RNA extracted from patients' biopsies was used to validate MYC upregulation. Drug screening and functional assays were performed using ER-BON-1 cells. Cell cycle progression was evaluated by FACS analysis. RESULTS: Our results show that MYC overexpression is a key event in the development of secondary resistance to everolimus in PanNEN cell lines and in metastatic lesions from neuroendocrine neoplasm patients. MYC knockdown restored ER-BON-1 sensitivity to everolimus. Pharmacological inhibition of MYC mediated by the cyclin-dependent kinase inhibitor dinaciclib strongly reduced viability of ER-BON-1. Dinaciclib synergized with everolimus and inhibited ER-BON-1 cell cycle progression. DISCUSSION: Our findings suggest that MYC upregulation drives the development of secondary resistance to everolimus in PanNENs and that its inhibition is an exploitable vulnerability. Indeed, our results indicate that combined treatments with cyclin-dependent kinase and mTOR inhibitors may counteract secondary resistance to everolimus in PanNENs and may pave the ground for new therapeutic regimens for these tumors.

Short tandem repeat profiling for the authentication of cancer stem-like cells.

Colorectal and glioblastoma cancer stem-like cells (CSCs) are essential for translational research. Cell line authentication by short tandem repeat (STR) profiling ensures reproducibility of results in oncology research. This technique enables to identify mislabeling or cross-contamination of cell lines. In our study, we provide a reference dataset for a panel of colorectal and glioblastoma CSCs that allows authentication. Each cell line was entered into the cell Line Integrated Molecular Authentication database 2.1 to be compared to the STR profiles of 4485 tumor cell lines. This article also provides clinical data of patients from whom CSCs arose and data on the parent tumor stage and mutations. STR profiles and information of our CSCs are also available in the Cellosaurus database (ExPASy) as identified by unique research resource identifier codes.

The RNA-binding protein MEX3A is a prognostic factor and regulator of resistance to gemcitabine in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer. Most patients present with advanced disease at diagnosis, which only permits palliative chemotherapeutic treatments. RNA dysregulation is a hallmark of most human cancers, including PDAC. To test the impact of RNA processing dysregulation on PDAC pathology, we performed a bioinformatics analysis to identify RNA-binding proteins (RBPs) associated with prognosis. Among the 12 RBPs associated with progression-free survival, we focused on MEX3A because it was recently shown to mark an intestinal stem cell population that is refractory to chemotherapeutic treatments, a typical feature of PDAC. Increased expression of MEX3A was correlated with higher disease stage in PDAC patients and with tumor development in a mouse model of PDAC. Depletion of MEX3A in PDAC cells enhanced sensitivity to chemotherapeutic treatment with gemcitabine, whereas its expression was increased in PDAC cells selected upon chronic exposure to the drug. RNA-sequencing analyses highlighted hundreds of genes whose expression is sensitive to MEX3A expression, with significant enrichment in cell cycle genes. MEX3A binds to its target mRNAs, like cyclin-dependent kinase 6 (CDK6), and promotes their stability. Accordingly, knockdown of MEX3A caused a significant reduction in PDAC cell proliferation and in progression to the S phase of the cell cycle. These findings uncover a novel role for MEX3A in the acquisition and maintenance of chemoresistance by PDAC cells, suggesting that it may represent a novel therapeutic target for PDAC.

Evaluation of Prognostic Factors for Survival in Transverse Colon Cancer.

Background: Although most of the analyses included transverse colon cancers (TCC) among right colon cancer (RCC), it is not completely clear if they present total similarities with RCC or if they have their specific features. Therefore, we present an observational study to evaluate clinicopathological characteristics and survival data of patients with TCC. Methods: We retrospectively reviewed 450 RCC, of whom 97 stages I-IV TCC were included in this multicenter study; clinicopathological and molecular parameters were analyzed to identify prognostic factors for disease-free survival (DFS) and overall survival (OS). Results: Most of TCC cases were male (61%), with ≤70 years old (62%), and good performance status (ECOG PS 0, 68%). According to WHO classification, 41 (49%) and 40 (48%) tumors were classified as well to moderate and poorly/undifferentiated respectively, regardless of mucinous component (30%). About molecular data, 8 (26%), 45 (63%), and 14 (24%) were MSI-H, KRAS wild-type, and BRAF V600E mutant, respectively. With a median follow-up of 34 months, there were 29 and 50 disease recurrences and deaths respectively. Charlson comorbidity index ≥5 was a significant prognostic factor for DFS (HR = 7.67, 95% CI 2.27-25.92). Colon obstruction/perforation (HR = 2.65, 95% CI 1.01-7.01), and BRAF mutant (HR = 3.03, 95% CI 0.97-9.50) cases showed a worst, despite not statistically significant, DFS. Whereas for OS, at the multivariate model, only tumor grade differentiation (HR = 5.26, 95% CI 1.98-14.01) and BRAF mutation status (3.71, 95% CI 1.07-12.89) were independent prognostic factors. Conclusions: Poorly/undifferentiated tumor grade and BRAF V600E mutation are independent prognostic factors for OS in TCC. Further prospective clinical trials are needed to better define TCC treatment in order to improve patient outcome.

The treatment paradigm of right-sided metastatic colon cancer: harboring BRAF mutation makes the difference.

PURPOSE: BRAF mutations represent the main negative prognostic factor for metastatic colorectal cancer and a supposed negative predictive factor of response to standard chemotherapy. We have explored survival difference in right-sided colon cancer (RCC) patients according to BRAF mutations, with the aim to identify any predictive factors of response to targeted-based therapy. METHODS: A retrospective study of RCC patients, with BRAF known mutation status, treated with chemotherapy (CT) from October 2008 to June 2019 in 5 Italian centers, was conducted. RESULTS: We identified 207 advanced RCC patients: 20.3% BRAF mutant and 79.7% BRAF wild type (wt). BRAF-mutant cancers were more likely to be pT4 (50.0% v 25.7%, p = 0.016), undifferentiated (71.4% v 44.0%, p = 0.004), KRAS wt (90.5% v 38.2%, p < 0.001), and MSI-H (41.7% v 16.2%, p = 0.019) tumors, with synchronous (52.4% v 31.5%, p = 0.018) and peritoneal metastases (38.1% v 22.4%, p = 0.003). Median overall survival (OS) was 16 v 27 months in BRAF mutant and BRAF wt (P = 0.020). In first-line setting, BRAF-mutant showed a 2ys OS of 80% in clinical trials, 32% in anti-VEGF, 14% in epidermial growth factor receptor (EGFR), and 0% in chemotherapy alone regimens (P = 0.009). BRAF-mutant patients demonstrated worse survival, regardless of targeted therapy administered. However, survival difference was statistically significant in the anti-EGFR-treated subgroup (16 v 28 months, P = 0.005 in BRAF mutant v BRAF wt, respectively). CONCLUSIONS: Our study demonstrated that BRAF status makes the difference in treatment's outcome. Therefore, the anti-EGFR should not be excluded in all advanced RCC but considered on a case-by-case basis.