Validation and clinical application of a targeted next-generation sequencing gene panel for solid and hematologic malignancies.

BACKGROUND: Next-generation sequencing (NGS) is a high-throughput technology that has become widely integrated in molecular diagnostics laboratories. Among the large diversity of NGS-based panels, the Trusight Tumor 26 (TsT26) enables the detection of low-frequency variants across 26 genes using the MiSeq platform. METHODS: We describe the inter-laboratory validation and subsequent clinical application of the panel in 399 patients presenting a range of tumor types, including gastrointestinal (GI, 29%), hematologic (18%), lung (13%), gynecological and breast (8% each), among others. RESULTS: The panel is highly accurate with a test sensitivity of 92%, and demonstrated high specificity and positive predictive values (95% and 96%, respectively). Sequencing testing was successful in two-thirds of patients, while the remaining third failed due to unsuccessful quality-control filtering. Most detected variants were observed in the TP53 (28%), KRAS (16%), APC (10%) and PIK3CA (8%) genes. Overall, 372 variants were identified, primarily distributed as missense (81%), stop gain (9%) and frameshift (7%) altered sequences and mostly reported as pathogenic (78%) and variants of uncertain significance (19%). Only 14% of patients received targeted treatment based on the variant determined by the panel. The variants most frequently observed in GI and lung tumors were: KRAS c.35G > A (p.G12D), c.35G > T (p.G12V) and c.34G > T (p.G12C). CONCLUSIONS: Prior panel validation allowed its use in the laboratory daily practice by providing several relevant and potentially targetable variants across multiple tumors. However, this study is limited by high sample inadequacy rate, raising doubts as to continuity in the clinical setting.

Genome sequence of OXA-48 carbapenemase-producing Klebsiella pneumoniae KpO3210.

Klebsiella pneumoniae KpO3210 is a OXA-48 carbapenemase-producing isolate obtained from a blood culture in the context of an outbreak in Hospital Universitario La Paz (Madrid, Spain) in 2010. It belongs to the major clone detected during the outbreak and is resistant to all beta-lactams and to several other antibiotics.

Adenovirus E1a protein enhances the cytotoxic effects of the herpes thymidine kinase-ganciclovir system.

Cancer gene therapy based on the use of suicide genes, such as the thymidine kinase gene, is not producing satisfactory results. Several approaches have been delineated to enhance the therapeutic responses, including augmentation of the bystander effect, the combination of the herpes simplex virus thymidine kinase-ganciclovir (HSVTK-GCV) system into replication competent adenoviruses and others. Moreover, because usually less than 20% of human malignant cells are in S-phase, the HSVTK-GCV system is not as efficient as expected. To increase the cytotoxic effects of the HSVTK-GCV system, we hypothesized that concomitant expression of E1a protein, which drives cells to proliferation and S-phase, could increase the effects of the HSVTK-GCV system. Several retroviruses were constructed carrying bicistronic sequences of TK and E1a 12S genes under the control of the CMV promoter. The constructions were tested in murine (NIH-3T3, MSC11A5) and human cells (IMR90, HeLa, MDA-MB435). A clear increase of the HSVTK-GCV system killing effect in nonconfluent cells was observed in the cells studied, especially in NIH-3T3, MSC11A5, IMR90, and MDA-MB435 expressing cells. In confluence, the NIH3T3 and IMR90 E1a-TK-expressing cells were also very sensitive and most malignant E1a-TK-expressing cells showed an irreversible G2-M cell cycle arrest. Moreover, the concomitant expression of adenovirus E1a and the HSVTK-GCV system increased the sensitivity to anticancer agents such as cisplatin. These results show that adenovirus E1a protein expression clearly enhances the cytotoxic effects of the HSVTK-GCV system and the response to treatment with cisplatin.

Modulation of PI3K/Akt pathway by E1a mediates sensitivity to cisplatin.

In order to investigate the molecular mechanisms implicated in the induction of chemo sensitivity by adenovirus E1a gene expression, we decided to investigate which signal transduction pathways could be affected by the E1a gene in Human Normal Fibroblast (IMR90). No effect was observed in SAPK pathways (p38MAPK and JNK), but E1a was able to affect the Akt activation mediated by insulin. This result was confirmed by transient transfection experiments performed in Cos-7 cells and also observed in other transformed cell lines such as A431. Furthermore, E1a expression induces a decrease in the basal status of Akt activity. Finally we demonstrated that E1a is able to block the Akt activation mediated by cisplatin and correlates with a sensitive phenotype. In summary, our data demonstrate that specific inhibition of the PI3K/Akt pathway mediates some of the biological properties of E1a such as induction of chemosensitivity.

Inconclusive presence of adenovirus sequences in human leukemias and lymphomas.

Adenoviruses are ubiquitous viruses related to human mild upper tract respiratory infections. Murine cells are semi-permissive to adenovirus replication, and persistent or abortive infections have been associated with tumorigenic potential. Given that only human lymphoid cells are semi-permissive and abortive infections have been described, we hypothesized that adenovirus could be related to the transformation of human haematopoietic cells. We studied 30 lymphomas, 46 leukemias, 10 reactive lymphadenopathies and 40 normal human spleens. The presence of adenovirus sequences and proteins were studied using PCR, southern-blot, slot-blot, in situ hybridization, immunohistochemistry, and western-blot techniques. By using nested PCR, adenovirus sequences were detectable in about 30% of lymphomas, but in less than 10% of leukemias, reactive lymphadenopathies and normal spleens. In no case were we able to demonstrate adenovirus products by in situ hybridization, immunohistochemistry or western-blot. These results indicate that adenovirus sequences are present in a significant number of human lymphomas, but that the number of positive cells is extremely low and no protein expression could be detected. Therefore, we are unable to conclude that persistent infections of human lymphoid cells by adenovirus is related to a higher risk of developing malignant lymphomas or leukemias.