SeekFusion - A Clinically Validated Fusion Transcript Detection Pipeline for PCR-Based Next-Generation Sequencing of RNA.

Detecting gene fusions involving driver oncogenes is pivotal in clinical diagnosis and treatment of cancer patients. Recent developments in next-generation sequencing (NGS) technologies have enabled improved assays for bioinformatics-based gene fusions detection. In clinical applications, where a small number of fusions are clinically actionable, targeted polymerase chain reaction (PCR)-based NGS chemistries, such as the QIAseq RNAscan assay, aim to improve accuracy compared to standard RNA sequencing. Existing informatics methods for gene fusion detection in NGS-based RNA sequencing assays traditionally use a transcriptome-based spliced alignment approach or a de-novo assembly approach. Transcriptome-based spliced alignment methods face challenges with short read mapping yielding low quality alignments. De-novo assembly-based methods yield longer contigs from short reads that can be more sensitive for genomic rearrangements, but face performance and scalability challenges. Consequently, there exists a need for a method to efficiently and accurately detect fusions in targeted PCR-based NGS chemistries. We describe SeekFusion, a highly accurate and computationally efficient pipeline enabling identification of gene fusions from PCR-based NGS chemistries. Utilizing biological samples processed with the QIAseq RNAscan assay and in-silico simulated data we demonstrate that SeekFusion gene fusion detection accuracy outperforms popular existing methods such as STAR-Fusion, TOPHAT-Fusion and JAFFA-hybrid. We also present results from 4,484 patient samples tested for neurological tumors and sarcoma, encompassing details on some novel fusions identified.

Genetic Variants in Host Protein Disulfide Isomerase 2 (PDIA2) are Associated with Susceptibility to Chlamydia Trachomatis Infection.

OBJECTIVES: Objective: Host genetics can influence susceptibility to Chlamydia trachomatis infection. This study examined two genetic variants in human protein disulfide isomerase A2 (PDIA2), a member of a family of protein chaperones that participate in the chlamydial life cycle. Methods: A total of 278 male and female subjects, positive or negative for C. trachomatis infection, were genotyped for PDIA2 polymorphisms (rs400037 and rs419949) using real-time PCR and pyrosequencing. Results: There was a significant odds ratio of 8.21 (95% CI: 1.77-38.16) for rs400037 and 9.89 (95% CI: 1.19-82.10) for rs419949, for the AA genotypes. Conclusion: This indicates that individuals with the PDIA2 AA genotypes have significantly increased susceptibility to C. trachomatis infection as compared to the other PDIA2 genotypes (GG, GA). This correlation may be explained by an interactive role of host protein disulfide isomerases in the attachment and entry of C. trachomatis into cells.

Desmoplastic Infantile Ganglioglioma: A MAPK Pathway-Driven and Microglia/Macrophage-Rich Neuroepithelial Tumor.

MAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%-27% variant allele frequency) and 1 showed a TPM3-NRTK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19-139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.