Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer death worldwide. PDAC is an aggressive disease with an 11-month median overall survival and a five-year survival of less than 5%. Incidence of PDAC is constantly increasing and is predicted to become the second leading cause of cancer in Western countries within a decade. Despite research and therapeutic development, current knowledge about PDAC molecular mechanisms still needs improvements and it seems crucial to identify novel therapeutic targets. Genomic analyses of PDAC revealed that transforming growth factor β (TGFβ) signaling pathways are modified and the SMAD4 gene is altered in 47% and 60% of cases, respectively, highlighting their major roles in PDAC development. TGFβ can play a dual role in malignancy depending on the context, sometimes as an inhibitor and sometimes as an inducer of tumor progression. TGFβ signaling was identified as a potent inducer of epithelial-to-mesenchymal transition (EMT), a process that confers migratory and invasive properties to epithelial cells during cancer. Therefore, aberrant TGFβ signaling and EMT are linked to promoting PDAC aggressiveness. TGFβ and SMAD pathways were extensively studied but the mechanisms leading to cancer promotion and development still remain unclear. This review aims to describe the complex role of SMAD4 in the TGFβ pathway in patients with PDAC.

Microsatellite instability (MSI) status is routinely assessed in patients with colorectal and endometrial cancers as it contributes to Lynch syndrome initial screening, tumour prognosis and selecting patients for immunotherapy. Currently, standard reference methods recommended for MSI/dMMR (deficient MisMatch Repair) testing consist of immunohistochemistry and pentaplex PCR-based assays, however, novel molecular-based techniques are emerging. Here, we aimed to evaluate the performance of a custom capture-based NGS method and the Bio-Rad ddPCR and Idylla approaches for the determination of MSI status for theranostic purposes in 30 formalin-fixed paraffin embedded (FFPE) tissue samples from patients with endometrial (n = 15) and colorectal (n = 15) cancers. All samples were previously characterised using IHC and Promega MSI Analysis System and these assays set as golden standard. Overall agreement, sensitivity and specificity of our custom-built NGS panel were 93.30%, 93.75% and 92.86% respectively. Overall agreement, sensitivity and specificity were 100% with the Idylla MSI system. The Bio-Rad ddPCR MSI assay showed a 100% concordance, sensitivity and specificity. The custom capture-based NGS, Bio-Rad ddPCR and Idylla approaches represent viable and complementary options to IHC and Promega MSI Analysis System for the detection of MSI. Bio-Rad ddPCR and Idylla MSI assays accounts for easy and fast screening assays while the NGS approach offers the advantages to simultaneously detect MSI and clinically relevant genomic alterations.

Microsatellite instability (MSI) is a molecular scar resulting from a defective mismatch repair system (dMMR) and associated with various malignancies. MSI tumours are characterized by the accumulation of mutations throughout the genome and particularly clustered in highly repetitive microsatellite (MS) regions. MSI/dMMR status is routinely assessed in solid tumours for the initial screening of Lynch syndrome, the evaluation of cancer prognosis, and treatment decision-making. Currently, pentaplex PCR-based methods and MMR immunohistochemistry on tumour tissue samples are the standard diagnostic methods for MSI/dMMR. Other tissue methods such as next-generation sequencing or real-time PCR-based systems have emerged and represent viable alternatives to standard MSI testing in specific settings. The evolution of the standard molecular techniques has offered the opportunity to extend MSI determination to liquid biopsy based on the analysis of cell-free DNA (cfDNA) in plasma. This review aims at synthetizing the standard and emerging techniques used on tumour tissue samples for MSI/dMMR determination. We also provide insights into the MSI molecular techniques compatible with liquid biopsy and the potential clinical consequences for patients with solid cancers.

Damage-specific DNA-binding protein 2 (DDB2) was originally identified as a DNA damage recognition factor that facilitates global genomic nucleotide excision repair (GG-NER) in human cells. DDB2 also contributes to other essential biological processes such as chromatin remodeling, gene transcription, cell cycle regulation, and protein decay. Recently, the potential of DDB2 in the development and progression of various cancers has been described. DDB2 activity occurs at several stages of carcinogenesis including cancer cell proliferation, survival, epithelial to mesenchymal transition, migration and invasion, angiogenesis, and cancer stem cell formation. In this review, we focus on the current state of scientific knowledge regarding DDB2 biological effects in tumor development and the underlying molecular mechanisms. We also provide insights into the clinical consequences of DDB2 activity in cancers.

Human solid malignancies harbour a heterogeneous set of cells with distinct genotypes and phenotypes. This heterogeneity is installed at multiple levels. A biological diversity is commonly observed between tumours from different patients (inter-tumour heterogeneity) and cannot be fully captured by the current consensus molecular classifications for specific cancers. To extend the complexity in cancer, there are substantial differences from cell to cell within an individual tumour (intra-tumour heterogeneity, ITH) and the features of cancer cells evolve in space and time. Currently, treatment-decision making usually relies on the molecular characteristics of a limited tumour tissue sample at the time of diagnosis or disease progression but does not take into account the complexity of the bulk tumours and their constant evolution over time. In this review, we explore the extent of tumour heterogeneity with an emphasis on ITH and report the mechanisms that promote and sustain this diversity in cancers. We summarise the clinical strikes of ITH in the management of patients with cancer. Finally, we discuss the current material and technological approaches that are relevant to adequately appreciate ITH.

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Specific HPV genotypes have been recognized as risk factors inducing head and neck cancers (HNC). The aim of this study was to validate a real-time PCR assay to detect accurately High Risk HPV DNA in Formalin Fixed Paraffin Embedded (FFPE) and oral cytobrush samples and compare the results with conventional PCR. Repeatability, reproducibility and limit of detection of Cobas assay were estimated for oral cytobrush and FFPE samples of patients with HNC. 53 samples of patients with a HNC were then used for assay comparison with conventional PCR. Finally, 26 samples of patients with anogenital neoplasia cancer were analyzed as control and assays comparison. Among the 53 samples of patients with HNC, 12 (26.7%) were HPV positive, 33 (73.3%) were HPV negative and 8 (15.1%) were non contributive with the Cobas assay. Among the 26 samples of patients with anogenital neoplasia, 15 (57.7%) were HPV positive and 11 were HPV negative (42.3%). One sample was found with an HPV 16 and HPV 18 co-infection. Only 3 samples were found with discrepant results. Cobas assay was found suitable for routine HPV detection with a very good repeatability and reproducibility for all HPV genotypes (CV < 0.6% and <0.4% respectively). Sensitivity and specificity for Cobas assay were 91.7% [61.5%;99.8%] and 96.9% [83.8%;99.9%] respectively. Ten nanograms of DNA were sufficient for the detection of HPV 16, HPV 18 and HPV in FFPE and oral cytobrush samples. Cobas assay was found comparable to conventional PCR and can detect accurately and rapidly HPV DNA in FFPE and oral cytobrush samples for the management of HNC and other types of HPV-associated neoplasia.

Background Assessment of KRAS , NRAS ( RAS ) and BRAF mutations is a standard in the management of patients with metastatic colorectal cancer (mCRC). Mutations could be assessed using next-generation sequencing (NGS) or real-time PCR-based assays. Times to results are 1 to 2 weeks for NGS and 1 to 3 days for real-time PCR-based assays. Using NGS can delay first-line treatment commencement and using PCR-based assays is limited by the number of possible analysed targets. The Idylla system is a real-time PCR cartridge-based assay, able to analyse hotspots mutations using one section of FFPE tumour tissue sample. To combine short delays and analysis of a large gene-panel, we propose here a laboratory workflow combining the Idylla system and NGS and compatible with FFPE samples with low tissue quantity. In this study we evaluated and validated the Idylla system for the analysis of RAS and BRAF mutations by pipetting directly DNA in the cartridge instead of FFPE section as recommended by the manufacturer. Materials and methods DNA extracted from 29 FFPE samples from mCRC patients with NGS-characterized RAS and BRAF mutations were tested with the Idylla KRAS and the Idylla NRAS-BRAF mutation tests to assess sensitivity, specificity, reproducibility and limit of detection of each test. Results A 100% concordance was found between NGS and Idylla results for the determination of KRAS (12/12) , NRAS (12/12) and BRAF (11/11) mutations with a sensitivity and a specificity of 100%. The system showed a good reproducibility with CV inferior to 3%. LOD was reached with 2.5 ng of DNA for KRAS and NRAS mutations and 5 ng of DNA for BRAF mutations. Conclusions The analysis of RAS and BRAF mutations using DNA pipetted directly in the cartridge of the Idylla system showed a good sensitivity, specificity, reproducibility and LOD, and can be integrated in a laboratory workflow for samples with few tissue without compromising a further complete tumour characterization using NGS.