Consensus Recommendations to Optimize the Detection and Reporting of NTRK Gene Fusions by RNA-Based Next-Generation Sequencing.

The detection of gene fusions by RNA-based next-generation sequencing (NGS) is an emerging method in clinical genetic laboratories for oncology biomarker testing to direct targeted therapy selections. A recent Canadian study (CANTRK study) comparing the detection of NTRK gene fusions on different NGS assays to determine subjects' eligibility for tyrosine kinase TRK inhibitor therapy identified the need for recommendations for best practices for laboratory testing to optimize RNA-based NGS gene fusion detection. To develop consensus recommendations, representatives from 17 Canadian genetic laboratories participated in working group discussions and the completion of survey questions about RNA-based NGS. Consensus recommendations are presented for pre-analytic, analytic and reporting aspects of gene fusion detection by RNA-based NGS.

CANTRK: A Canadian Ring Study to Optimize Detection of NTRK Gene Fusions by Next-Generation RNA Sequencing.

The Canadian NTRK (CANTRK) study is an interlaboratory comparison ring study to optimize testing for neurotrophic receptor tyrosine kinase (NTRK) fusions in Canadian laboratories. Sixteen diagnostic laboratories used next-generation sequencing (NGS) for NTRK1, NTRK2, or NTRK3 fusions. Each laboratory received 12 formalin-fixed, paraffin-embedded tumor samples with unique NTRK fusions and two control non-NTRK fusion samples (one ALK and one ROS1). Laboratories used validated protocols for NGS fusion detection. Panels included Oncomine Comprehensive Assay v3, Oncomine Focus Assay, Oncomine Precision Assay, AmpliSeq for Illumina Focus, TruSight RNA Pan-Cancer Panel, FusionPlex Lung, and QIAseq Multimodal Lung. One sample was withdrawn from analysis because of sample quality issues. Of the remaining 13 samples, 6 of 11 NTRK fusions and both control fusions were detected by all laboratories. Two fusions, WNK2::NTRK2 and STRN3::NTRK2, were not detected by 10 laboratories using the Oncomine Comprehensive or Focus panels, due to absence of WNK2 and STRN3 in panel designs. Two fusions, TPM3::NTRK1 and LMNA::NTRK1, were challenging to detect on the AmpliSeq for Illumina Focus panel because of bioinformatics issues. One ETV6::NTRK3 fusion at low levels was not detected by two laboratories using the TruSight Pan-Cancer Panel. Panels detecting all fusions included FusionPlex Lung, Oncomine Precision, and QIAseq Multimodal Lung. The CANTRK study showed competency in detection of NTRK fusions by NGS across different panels in 16 Canadian laboratories and identified key test issues as targets for improvements.

Natural spring water gargle samples as an alternative to nasopharyngeal swabs for SARS-CoV-2 detection using a laboratory-developed test.

The objective of this study was to validate the use of spring water gargle (SWG) as an alternative to oral and nasopharyngeal swab (ONPS) for SARS-CoV-2 detection with a laboratory-developed test. Healthcare workers and adults from the general population, presenting to one of two COVID-19 screening clinics in Montréal and Québec City, were prospectively recruited to provide a gargle sample in addition to the standard ONPS. The paired specimens were analyzed using thermal lysis followed by a laboratory-developed nucleic acid amplification test (LD-NAAT) to detect SARS-CoV-2, and comparative performance analysis was performed. An individual was considered infected if a positive result was obtained on either sample. A total of 1297 adult participants were recruited. Invalid results (n = 18) were excluded from the analysis. SARS-CoV-2 was detected in 144/1279 (11.3%) participants: 126 from both samples, 15 only from ONPS, and 3 only from SWG. Overall, the sensitivity was 97.9% (95% CI: 93.7-99.3) for ONPS and 89.6% (95% CI: 83.4-93.6; p = 0.005) for SWG. The mean ONPS cycle threshold (Ct ) value was significantly lower for the concordant paired samples as compared to discordant ones (22.9 vs. 32.1; p < 0.001). In conclusion, using an LD-NAAT with thermal lysis, SWG is a less sensitive sampling method than the ONPS. However, the higher acceptability of SWG might enable a higher rate of detection from a population-based perspective. Nonetheless, in patients with a high clinical suspicion of COVID-19, a repeated analysis with ONPS should be considered. The sensitivity of SWG using NAAT preceded by chemical extraction should be evaluated.

DNMT3A and TET2 dominate clonal hematopoiesis and demonstrate benign phenotypes and different genetic predispositions.

Age-associated clonal hematopoiesis caused by acquired mutations in myeloid cancer-associated genes is highly prevalent in the normal population. Its etiology, biological impact on hematopoiesis, and oncogenic risk is poorly defined at this time. To gain insight into this phenomenon, we analyzed a cohort of 2530 related and unrelated hematologically normal individuals (ages 55 to 101 years). We used a sensitive gene-targeted deep sequencing approach to gain precision on the exact prevalence of driver mutations and the proportions of affected genes. Mutational status was correlated with biological parameters. We report a higher overall prevalence of driver mutations (13.7%), which occurred mostly (93%) in DNMT3A or TET2 and were highly age-correlated. Mutation in these 2 genes had some distinctive effects on end points. TET2 mutations were more age-dependent, associated with a modest neutropenic effect (9%, P = .012), demonstrated familial aggregation, and associated with chronic obstructive pulmonary disease. Mutations in DNMT3A had no impact on blood counts or indices. Mutational burden of both genes correlated with X-inactivation skewing but no significant association with age-adjusted telomere length reduction was documented. The discordance between the high prevalence of mutations in these 2 genes and their limited biological impact raise the question of the potential role of dysregulated epigenetic modifiers in normal aging hematopoiesis, which may include support to failing hematopoiesis.

Comparative xylose metabolism among the Ascomycetes C. albicans, S. stipitis and S. cerevisiae.

The ascomycetes Candida albicans, Saccharomyces cerevisiae and Scheffersomyces stipitis metabolize the pentose sugar xylose very differently. S. cerevisiae fails to grow on xylose, while C. albicans can grow, and S. stipitis can both grow and ferment xylose to ethanol. However, all three species contain highly similar genes that encode potential xylose reductases and xylitol dehydrogenases required to convert xylose to xylulose, and xylulose supports the growth of all three fungi. We have created C. albicans strains deleted for the xylose reductase gene GRE3, the xylitol dehydrogenase gene XYL2, as well as the gre3 xyl2 double mutant. As expected, all the mutant strains cannot grow on xylose, while the single gre3 mutant can grow on xylitol. The gre3 and xyl2 mutants are efficiently complemented by the XYL1 and XYL2 from S. stipitis. Intriguingly, the S. cerevisiae GRE3 gene can complement the Cagre3 mutant, while the ScSOR1 gene can complement the Caxyl2 mutant, showing that S. cerevisiae contains the enzymatic capacity for converting xylose to xylulose. In addition, the gre3 xyl2 double mutant of C. albicans is effectively rescued by the xylose isomerase (XI) gene of either Piromyces or Orpinomyces, suggesting that the XI provides an alternative to the missing oxido-reductase functions in the mutant required for the xylose-xylulose conversion. Overall this work suggests that C. albicans strains engineered to lack essential steps for xylose metabolism can provide a platform for the analysis of xylose metabolism enzymes from a variety of species, and confirms that S. cerevisiae has the genetic potential to convert xylose to xylulose, although non-engineered strains cannot proliferate on xylose as the sole carbon source.

Modulation of histone H3 lysine 56 acetylation as an antifungal therapeutic strategy.

Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections.

Forward genetics in Candida albicans that reveals the Arp2/3 complex is required for hyphal formation, but not endocytosis.

Candida albicans is a diploid fungal pathogen lacking a defined complete sexual cycle, and thus has been refractory to standard forward genetic analysis. Instead, transcription profiling and reverse genetic strategies based on Saccharomyces cerevisiae have typically been used to link genes to functions. To overcome restrictions inherent in such indirect approaches, we have investigated a forward genetic mutagenesis strategy based on the UAU1 technology. We screened 4700 random insertion mutants for defects in hyphal development and linked two new genes (ARP2 and VPS52) to hyphal growth. Deleting ARP2 abolished hyphal formation, generated round and swollen yeast phase cells, disrupted cortical actin patches and blocked virulence in mice. The mutants also showed a global lack of induction of hyphae-specific genes upon the yeast-to-hyphae switch. Surprisingly, both arp2 Delta/Delta and arp2 Delta/Delta arp3 Delta/Delta mutants were still able to endocytose FM4-64 and Lucifer Yellow, although as shown by time-lapse movies internalization of FM4-64 was somewhat delayed in mutant cells. Thus the non-essential role of the Arp2/3 complex discovered by forward genetic screening in C. albicans showed that uptake of membrane components from the plasma membrane to vacuolar structures is not dependent on this actin nucleating machinery.

Lethal photosensitization of periodontal pathogens by a red-filtered Xenon lamp in vitro.

BACKGROUND: The ability of Helium-Neon (He-Ne) laser irradiation of a photosensitizer to induce localized phototoxic effects that kill periodontal pathogens is well documented and is termed photodynamic therapy (PDT). OBJECTIVES: We investigated the potential of a conventional light source (red-filtered Xenon lamp) to activate toluidine blue O (TBO) in vitro and determined in vitro model parameters that may be used in future in vivo trials. MATERIALS AND METHODS: Porphyromonas gingivalis 381 was used as the primary test bacterium. RESULTS: Treatment with a 2.2 J/cm2 light dose and 50 micro g/ml TBO concentration resulted in a bacterial kill of 2.43 +/- 0.39 logs with the He-Ne laser control and 3.34 +/- 0.24 logs with the lamp, a near 10-fold increase (p = 0.028). Increases in light intensity produced significantly higher killing (p = 0.012) that plateaued at 25 mW/cm2. There was a linear relationship between light dose and bacterial killing (r2 = 0.916); as light dose was increased bacterial survival decreased. No such relationship was found for the drug concentrations tested. Addition of serum or blood at 50% v/v to the P. gingivalis suspension prior to irradiation diminished killing from approximately 5 logs to 3 logs at 10 J/cm2. When serum was washed off, killing returned to 5 logs for all species tested except Bacteroides forsythus (3.92 +/- 0.68 logs kill). CONCLUSIONS: The data indicate that PDT utilizing a conventional light source is at least as effective as laser-induced treatment in vitro. Furthermore, PDT achieves significant bactericidal activity in the presence of serum and blood when used with the set parameters of 10 J/cm2, 100 mW/cm2 and 12.5 micro g/ml TBO.