Development and validation of an RNA sequencing panel for gene fusions in soft tissue sarcoma.

Gene fusions are one of the most common genomic alterations in soft tissue sarcomas (STS), which contain more than 70 subtypes. In this study, a custom-designed RNA sequencing panel including 67 genes was developed and validated to identify gene fusions in STS. In total, 92 STS samples were analyzed using the RNA panel and 95.7% (88/92) successfully passed all the quality control parameters. Fusion transcripts were detected in 60.2% (53/88) of samples, including three novel fusions (MEG3-PLAG1, SH3BP1-NTRK1, and RPSAP52-HMGA2). The panel demonstrated excellent analytic accuracy, with 93.9% sensitivity and 100% specificity. The intra-assay, inter-assay, and personnel consistencies were all 100.0% in four samples and three replicates. In addition, different variants of ESWR1-FLI, COL1A1-PDGFB, NAB2-STAT6, and SS18-SSX were also identified in the corresponding subtypes of STS. In combination with histological and molecular diagnosis, 14.8% (13/88) patients finally changed preliminary histology-based classification. Collectively, this RNA panel developed in our study shows excellent performance on RNA from formalin-fixed, paraffin-embedded samples and can complement DNA-based assay, thereby facilitating precise diagnosis and novel fusion detection.

Identification of CXCL5 expression as a predictive biomarker associated with response and prognosis of immunotherapy in patients with non-small cell lung cancer.

BACKGROUND: The breakthrough of immunotherapy has revolutionized the treatment of non-small cell lung cancer (NSCLC). However, only a limited part of patients could derive clinical benefits. To study how immune microenvironment (IME) of patients could influence the therapeutic efficacy of immunotherapy, we evaluated the response patterns of NSCLC patients treated with PD-1 inhibitors and analyzed the molecules related to prognosis and efficacy of immunotherapy. METHODS: Tumor samples were collected from 47 NSCLC patients treated with PD-1 inhibitors. RNA expressions of tumor immune-related 289 genes were analyzed using NanoString nCounter. Immune infiltration and correlation between clinical information and expression of immune-related genes were assessed. RESULTS: Unsupervised clustering analysis revealed two groups infiltrated with different immune cells and differentially expressed genes (DEGs) including CXCL5, CXCL9, IDO1, and LAG3 were found between groups. Stratification based on DEGs indicated that the group with high expression of CXCL5 was characterized by neutrophils. Univariate and multivariate Cox analysis further demonstrated that CXCL5 mRNA expression was positively associated with worse progression free survival (PFS). Logistic analyses indicated high CXCL5 was associated with worse response to immunotherapy. CONCLUSIONS: CXCL5 may be a potential biomarker for prognosis and responsiveness to immunotherapy and may be a novel preventive and therapeutic target for NSCLC.

Major sperm protein BxMSP10 is required for reproduction and egg hatching in Bursaphelenchus xylophilus.

The pine wood nematode Bursaphelenchus xylophilus is a disastrous pathogen of pine forests in East Asia and Europe. Despite its decimating effect on pine forests, efficient and environmentally friendly methods available to control the pine wood nematode (PWN) are limited. The most abundant protein in nematode sperm, major sperm proteins (MSPs) have only been discovered in nematodes. In this study, phylogenetic analysis showed that BxMSP10 was highly conserved in the nematode and had a closer phylogenetic relationship with free-living nematodes than with plant-parasitic nematode species. BxMSP10 was specifically expressed in the seminal vesicle of male adults. dsRNA of BxMSP10 significantly decreased reproduction, egg hatching and population maintenance in B. xylophilus. These results indicated that BxMSP10 was a potential candidate for application in the control of B. xylophilus.

Specifically expressed genes of the nematode Bursaphelenchus xylophilus involved with early interactions with pine trees.

As the causal agent of pine wilt disease (PWD), the pine wood nematode (PWN), Bursaphelenchus xylophilus, causes huge economic losses by devastating pine forests worldwide. However, the pathogenesis-related genes of B. xylophilus are not well characterized. Thus, DNA microarrays were used to investigate differential gene expression in PWN where Pinus thunbergii was inoculated with nematodes, compared with those cultured on Botrytis cinerea. The microarrays comprised 31121 probes, 1310 (4.2%) of which were differentially regulated (changes of >2-fold, P < 0.01) in the two growth conditions. Of these 1310 genes, 633 genes were upregulated, whereas 677 genes were downregulated. Gene Ontology (GO) categories were assigned to the classes Cellular Component, Molecular Function, and Biological Process. The comparative gene expression analysis showed that a large number of the pathogenesis-related genes of B. xylophilus, such as pectate lyase genes, cytochrome P450s, UGTs, and ABC transporter genes, were highly expressed when B. xylophilus infected P. thunbergii. Annotation analysis indicated that these genes contributed to cell wall degradation, detoxification, and the reproduction process. The microarray results were validated using quantitative RT-PCR (qRT-PCR). The microarray data confirmed the specific expression of B. xylophilus genes during infection of P. thunbergii, which provides basic information that facilitates a better understanding of the molecular mechanism of PWD.