Histopathological samples are a treasure-trove of DNA for clinical research. However, the quality of DNA can vary depending on the source or extraction method applied. Thus a standardized and cost-effective workflow for the qualification of DNA preparations is essential to guarantee interlaboratory reproducible results. The qualification process consists of the quantification of double strand DNA (dsDNA) and the assessment of its suitability for downstream applications, such as high-throughput next-generation sequencing. We tested the two most frequently used instrumentations to define their role in this process: NanoDrop, based on UV spectroscopy, and Qubit 2.0, which uses fluorochromes specifically binding dsDNA. Quantitative PCR (qPCR) was used as the reference technique as it simultaneously assesses DNA concentration and suitability for PCR amplification. We used 17 genomic DNAs from 6 fresh-frozen (FF) tissues, 6 formalin-fixed paraffin-embedded (FFPE) tissues, 3 cell lines, and 2 commercial preparations. Intra- and inter-operator variability was negligible, and intra-methodology variability was minimal, while consistent inter-methodology divergences were observed. In fact, NanoDrop measured DNA concentrations higher than Qubit and its consistency with dsDNA quantification by qPCR was limited to high molecular weight DNA from FF samples and cell lines, where total DNA and dsDNA quantity virtually coincide. In partially degraded DNA from FFPE samples, only Qubit proved highly reproducible and consistent with qPCR measurements. Multiplex PCR amplifying 191 regions of 46 cancer-related genes was designated the downstream application, using 40 ng dsDNA from FFPE samples calculated by Qubit. All but one sample produced amplicon libraries suitable for next-generation sequencing. NanoDrop UV-spectrum verified contamination of the unsuccessful sample. In conclusion, as qPCR has high costs and is labor intensive, an alternative effective standard workflow for qualification of DNA preparations should include the sequential combination of NanoDrop and Qubit to assess the purity and quantity of dsDNA, respectively.

Epidermal growth factor receptor-1 (EGFR-1/HER-1/ErbB-1) regulates proliferation and cell fate during epidermal development. HER-1 is activated by several EGF-family ligands including heparin-binding epidermal growth factor–like growth factor (HB-EGF), a mitogenic and chemotactic molecule that participates in tissue repair, tumor growth, and other tissue-modeling phenomena, such as angiogenesis and fibrogenesis. We found that mesenchymal stem cells (MSCs), the precursors of different mesenchymal tissues with a role in processes in which HB-EGF is often involved, normally express HER-1, but not HB-EGF itself. Under the effect of HB-EGF, MSCs proliferate more rapidly and persistently, without undergoing spontaneous differentiation. This effect occurs in a dose-dependent fashion, and is specific, direct, and HER-1 mediated, as it is inhibited by anti–HER-1 and anti–HB-EGF blocking antibodies. Moreover, HB-EGF reversibly prevents adipogenic, osteogenic, and chondrogenic differentiation induced with specific media. These data show that HB-EGF/HER-1 signaling is relevant to MSC biology, by regulating both proliferation and differentiation.

One-hundred-fifty-three biliary cancers, including 70 intrahepatic cholangiocarcinomas (ICC), 57 extrahepatic cholangiocarcinomas (ECC) and 26 gallbladder carcinomas (GBC) were assessed for mutations in 56 genes using multigene next-generation sequencing. Expression of EGFR and mTOR pathway genes was investigated by immunohistochemistry. At least one mutated gene was observed in 118/153 (77%) cancers. The genes most frequently involved were KRAS (28%), TP53 (18%), ARID1A (12%), IDH1/2 (9%), PBRM1 (9%), BAP1 (7%), and PIK3CA (7%). IDH1/2 (p=0.0005) and BAP1 (p=0.0097) mutations were characteristic of ICC, while KRAS (p=0.0019) and TP53 (p=0.0019) were more frequent in ECC and GBC. Multivariate analysis identified tumour stage and TP53 mutations as independent predictors of survival. Alterations in chromatin remodeling genes (ARID1A, BAP1, PBRM1, SMARCB1) were seen in 31% of cases. Potentially actionable mutations were seen in 104/153 (68%) cancers: i) KRAS/NRAS/BRAF mutations were found in 34% of cancers; ii) mTOR pathway activation was documented by immunohistochemistry in 51% of cases and by mutations in mTOR pathway genes in 19% of cancers; iii) TGF-ß/Smad signaling was altered in 10.5% cancers; iv) mutations in tyrosine kinase receptors were found in 9% cases. Our study identified molecular subgroups of cholangiocarcinomas that can be explored for specific drug targeting in clinical trials.

Epithelial-to-mesenchymal transition (EMT) has a central role in cancer progression and metastatic dissemination and may be induced by local inflammation. We asked whether the inflammation-induced acquisition of mesenchymal phenotype by neoplastic epithelial cells is associated with the onset of mesenchymal stromal cell-like immune-regulatory properties that may enhance tumour immune escape. Cell lines of lung adenocarcinoma (A549), breast cancer (MCF7) and hepatocellular carcinoma (HepG2) were co-cultured with T, B and NK cells before and after EMT induction by either the supernatant of mixed-lymphocyte reactions or inflammatory cytokines. EMT occurrence following inflammatory priming elicited multiple immune-regulatory effects in cancer cells resulting in NK and T-cell apoptosis, inhibition of lymphocyte proliferation and stimulation of regulatory T and B cells. Indoleamine 2,3-dioxygenase, but not Fas ligand pathway, was involved at least in part in these effects, as shown by the use of specific inhibitors. EMT induced by inflammatory stimuli confers to cancer cells some mesenchymal stromal cell-like immune-modulatory properties, which could be a cue for cancer progression and metastatic dissemination by favouring immune escape.

Patients with sickle-cell disease (SCD) suffer from tissue damage and life-threatening complications caused by vasoocclusive crisis (VOC). Endothelin receptors (ETRs) are mediators of one of the most potent vasoconstrictor pathways in mammals, but the relationship between vasoconstriction and VOC is not well understood. We report here that pharmacological inhibition of ETRs prevented hypoxia-induced acute VOC and organ damage in a mouse model of SCD. An in vivo ultrasonographic study of renal hemodynamics showed a substantial increase in endothelin-mediated vascular resistance during hypoxia/reoxygenation-induced VOC. This increase was reversed by administration of the dual ETR antagonist (ETRA) bosentan, which had pleiotropic beneficial effects in vivo. It prevented renal and pulmonary microvascular congestion, systemic inflammation, dense rbc formation, and infiltration of activated neutrophils into tissues with subsequent nitrative stress. Bosentan also prevented death of sickle-cell mice exposed to a severe hypoxic challenge. These findings in mice suggest that ETRA could be a potential new therapy for SCD, as it may prevent acute VOC and limit organ damage in sickle-cell patients.

Although many literature data are available on the role of Notch signaling in T-cell acute lymphoblastic leukemia (ALL) biology, the importance of this molecular pathway in the development of B-lineage ALL (B-ALL) cells in the BM microenvironment is unknown so far. In this study, we used anti-Notch molecules neutralizing Abs and γ-secretase inhibitor (GSI) XII to investigate the role of the Notch signaling pathway in the promotion of human B-ALL cell survival in presence of stromal cell support. The treatment with combinations of anti-Notch molecule neutralizing Abs resulted in the decrease of B-ALL cell survival, either cultured alone or cocultured in presence of stromal cells from normal donors and B-ALL patients. Interestingly, the inhibition of Notch-3 and -4 or Jagged-1/-2 and DLL-1 resulted in a dramatic increase of apoptotic B-ALL cells by 3 days, similar to what is obtained by blocking all Notch signaling with the GSI XII. Our data suggest that the stromal cell–mediated antiapoptotic effect on B- ALL cells is mediated by Notch-3 and -4 or Jagged-1/-2 and DLL-1 in a synergistic manner.