Chronic Oxidative Stress Promotes Molecular Changes Associated with Epithelial Mesenchymal Transition, NRF2, and Breast Cancer Stem Cell Phenotype

Oxidative stress plays a role in carcinogenesis, but it also contributes to the modulation of tumor cells and microenvironment caused by chemotherapeutics. One of the consequences of oxidative stress is lipid peroxidation, which can, through reactive aldehydes such as 4-hydroxy-2-nonenal (HNE), affect cell signaling pathways. On the other hand, cancer stem cells (CSC) are now recognized as a major factor of malignancy by causing metastasis, relapse, and therapy resistance. Here, we evaluated whether oxidative stress and HNE modulation of the microenvironment can influence CSC growth, modifications of the epithelial to mesenchymal transition (EMT) markers, the antioxidant system, and the frequency of breast cancer stem cells (BCSC). Our results showed that oxidative changes in the microenvironment of BCSC and particularly chronic oxidative stress caused changes in the proliferation and growth of breast cancer cells. In addition, changes associated with EMT, increase in glutathione (GSH) and Nuclear factor erythroid 2-related factor 2 (NRF2) were observed in breast cancer cells grown on HNE pretreated collagen and under chronic oxidative stress. Our results suggest that chronic oxidative stress can be a bidirectional modulator of BCSC fate. Low levels of HNE can increase differentiation markers in BCSC, while higher levels increased GSH and NRF2 as well as certain EMT markers, thereby increasing therapy resistance

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary

Metastasis is responsible for the majority of cancer‐related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin‐fixed paraffin‐embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions

Comparison of three commercial decision support platforms for matching of next-generation sequencing results with therapies in patients with cancer

Objective Precision oncology depends on translating molecular data into therapy recommendations. However, with the growing complexity of next-generation sequencing-based tests, clinical interpretation of somatic genomic mutations has evolved into a formidable task. Here, we compared the performance of three commercial clinical decision support tools, that is, NAVIFY Mutation Profiler (NAVIFY; Roche), QIAGEN Clinical Insight (QCI) Interpret (QIAGEN) and CureMatch Bionov (CureMatch). Methods In order to obtain the current status of the respective tumour genome, we analysed cell-free DNA from patients with metastatic breast, colorectal or non-small cell lung cancer. We evaluated somatic copy number alterations and in parallel applied a 77-gene panel (AVENIO ctDNA Expanded Panel). We then assessed the concordance of tier classification approaches between NAVIFY and QCI and compared the strategies to determine actionability among all three platforms. Finally, we quantified the alignment of treatment suggestions across all decision tools. Results Each platform varied in its mode of variant classification and strategy for identifying druggable targets and clinical trials, which resulted in major discrepancies. Even the frequency of concordant actionable events for tier I-A or tier I-B classifications was only 4.3%, 9.5% and 28.4% when comparing NAVIFY with QCI, NAVIFY with CureMatch and CureMatch with QCI, respectively, and the obtained treatment recommendations differed drastically. Conclusions Treatment decisions based on molecular markers appear at present to be arbitrary and dependent on the chosen strategy. As a consequence, tumours with identical molecular profiles would be differently treated, which challenges the promising concepts of genome-informed medicine

FIV-ICSI et azoospermie (étude nancéienne et revue de la littérature)

NANCY1-SCD Medecine (545472101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Duplex reverse-hybridization assay for the simultaneous detection of KRAS/BRAF mutations in FFPE-extracted genomic DNA from colorectal cancer specimens

Abstract. We report the performance evaluation of a non-quantitative reverse-hybridization assay (KRAS-BRAF StripAssay) designed for the simultaneous detection of 10 mutations in codons 12 and 13 of the KRAS gene and BRAF mutation V600E. Dilution experiments using DNA from tumor cell lines or from formalin-fixed paraffin-embedded (FFPE) colorectal cancer (CRC) tissue were performed to assess assay sensitivity. Using 50 ng of total DNA (mutant and wild-type), the KRAS-BRAF StripAssay demonstrated a detection limit of 1% mutant sequence in a background of wild-type DNA. With respect to BRAF V600E, the KRAS-BRAF StripAssay was evaluated using 60 FFPE CRC samples previously analyzed by high resolution melting (HRM). Test strip hybridization identified 2/60 (3%) samples to carry the BRAF V600E mutation, and results were in agreement with those obtained by HRM analysis. This work demonstrates the KRAS-BRAF StripAssay to be a robust and sensitive method for the detection of common KRAS/BRAF mutations in genomic DNA isolated from FFPE tissue samples