Inter-assay variability of next-generation sequencing-based gene panels

BACKGROUND: Tumor heterogeneity has been known to cause inter-assay discordance among next-generation sequencing (NGS) results. However, whether preclinical factors such as sample type, sample quality and analytical features of gene panel can affect the concordance between two different assays remains largely unexplored. METHODS: Replicate sets of DNA samples extracted from formalin-fixed paraffin-embedded tissues (FFPE) (n = 20) and fresh frozen (FF) tissues (n = 10) were herein analyzed using a tumor-only (TO) and paired tumor-normal (TN) gene panel in laboratories certified by the Clinical Laboratory Improvement Amendment. Reported variants from the TO and TN panels were then compared. Furthermore, additional FFPE samples were sequentially sliced from the same FFPE block and submitted to another TN panel assay. RESULTS: Substantial discordance (71.8%) was observed between the results of the two panels despite using identical DNA samples, with the discordance rate being significantly higher for FFPE samples (p < 0.05). Among the 99 variants reported only in the TO panel, 32.3% were consistent with germline variants, which were excluded in the TN panel, while 30.3% had an allele frequency of less than 5%, some of which were highly likely to be artificial calls. The comparison of two independent TN panel assay results from the same FFPE block also showed substantial discordance rate (55.3%). CONCLUSIONS: In the context of clinical settings, our comparative analysis revealed that inter-NGS assay discordance commonly occurred due to sample types and the different analytical features of each panel

Cisplatin, rather than oxaliplatin, increases paracellular permeability of LLC-PK1 cells via activating protein kinase C

The clinical use of cisplatin is limited by its adverse events, particularly serious nephrotoxicity. It was clarified that cisplatin is transported by a kidney-specific organic cation transporter (OCT2). OCT2 also mediates the uptake of oxaliplatin into renal proximal tubular cells; however, this agent does not lead nephrotoxicity. In the present study, we carried out comparative experiments with cisplatin and oxaliplatin using porcine kidney LLC-PK1 cell monolayers. In the fluorescein-labeled isothiocyanate-dextran flux assay, the basolateral application of cisplatin, but not oxaliplatin, resulted in an increase in the paracellular permeability of cell monolayers. Even though the cellular accumulation of platinum at 50 μM oxaliplatin could reach the same level at 30 μM cisplatin, oxaliplatin did not induce hyper-permeability in cell monolayers. Cisplatin, but not oxaliplatin, significantly activated PKC. In addition, the combination of PKC inhibitors recovered the increase in paracellular permeability. In conclusion, pharmacodynamic mechanisms via PKC could explain the difference in nephrotoxicity between cisplatin and oxaliplatin

Lactobacillus helveticus SBT2171 Attenuates Experimental Autoimmune Encephalomyelitis in Mice

We recently reported that Lactobacillus helveticus SBT2171 (LH2171) inhibited the proliferation and inflammatory cytokine production of primary immune cells in vitro, and alleviated collagen-induced arthritis (CIA) in mice, a model of human rheumatoid arthritis (RA). In this study, we newly investigated whether LH2171 could relieve the severity of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), which is an autoimmune disease, but develop the symptoms by different mechanisms from RA. In MS and EAE, main cause of the disease is the abnormality in CD4+ T cell immunity, whereas in RA and CIA, is that in antibody-mediated immunity. The intraperitoneal administration of LH2171 significantly decreased the incidence and clinical score of EAE in mice. LH2171 also reduced the numbers of pathogenic immune cells, especially Th17 cells, in the spinal cord at the peak stage of disease severity. Interestingly, before the onset of EAE, LH2171 administration remarkably decreased the ratio of Th17 cells to CD4+ T cells in the inguinal lymph nodes (LNs), where pathogenic immune cells are activated to infiltrate the central nervous system, including the spinal cord. Furthermore, the expression of interleukin (IL)-6, an inflammatory cytokine essential for Th17 differentiation, decreased in the LNs of LH2171-administered mice. Moreover, LH2171 significantly inhibited IL-6 production in vitro from both DC2.4 and RAW264.7 cells, model cell lines of antigen-presenting cells. These findings suggest that LH2171 might down-regulate IL-6 production and the subsequent Th17 differentiation and spinal cord infiltration, consequently alleviating EAE symptoms

Prognostic Impact of Tumor-Infiltrating Lymphocytes, Tertiary Lymphoid Structures, and Neutrophil-to-Lymphocyte Ratio in Pulmonary Metastases from Uterine Leiomyosarcoma

Background　The presence of tumor-infiltrating lymphocytes (TILs) and tertiary lymphoid structures (TLSs) in tumor tissue has been related to the prognosis in various malignancies. Meanwhile, neutrophil-to-lymphocyte ratio (NLR) as a systemic inflammation marker also has been associated with the prognosis in them. However, few reports have investigated the relationship between pulmonary metastases from sarcoma and these biomarkers. Methods　We retrospectively recruited 102 patients undergoing metastasectomy for pulmonary metastases from uterine leiomyosarcoma at Okayama University Hospital from January 2006 to December 2019. TILs and TLSs were evaluated by immunohistochemical staining of surgically resected specimens of pulmonary metastases using anti-CD3/CD8/CD103/Foxp3/CD20 antibodies. NLR was calculated from the blood examination immediately before the most recent pulmonary metastasectomy. We elucidated the relationship between the prognosis and these factors. Because we considered that the status of tumor tissue and systemic inflammation were equally valuable, we also assessed the impact of the combination of TILs or TLSs and NLR on the prognosis. Results　As for TILs, CD3-positive cells and CD8-positive cells were correlated with the prognosis. The prognosis was significantly better in patients with CD3-high group, CD8-high group, TLSs-high group, and NLR-low group, respectively. The prognosis of CD8-high/NLR-low group and TLSs-high/NLR-low group was significantly better than that of CD8-low/NLR-high group and TLSs-low/NLR-high group, respectively. Conclusions　CD3-positive TILs, CD8-positive TILs, TLSs, and NLR are correlated with the prognosis, respectively. The combination of CD8-positive TILs or TLSs and NLR may be the indicators to predict the prognosis of patients with pulmonary metastases from uterine leiomyosarcoma

Next-to-leading resummation of cosmological perturbations via the Lagrangian picture: 2-loop correction in real and redshift spaces

We present an improved prediction of the nonlinear perturbation theory (PT) via the Lagrangian picture, which was originally proposed by Matsubara (2008). Based on the relations between the power spectrum in standard PT and that in Lagrangian PT, we derive analytic expressions for the power spectrum in Lagrangian PT up to 2-loop order in both real and redshift spaces. Comparing the improved prediction of Lagrangian PT with $N$-body simulations in real space, we find that the 2-loop corrections can extend the valid range of wave numbers where we can predict the power spectrum within 1% accuracy by a factor of 1.0 ($z=0.5$), 1.3 (1), 1.6 (2) and 1.8 (3) vied with 1-loop Lagrangian PT results. On the other hand, in all redshift ranges, the higher-order corrections are shown to be less significant on the two-point correlation functions around the baryon acoustic peak, because the 1-loop Lagrangian PT is already accurate enough to explain the nonlinearity on those scales in $N$-body simulations.Comment: 18pages, 4 figure

Robust Dependency Parsing of Spontaneous Japanese Speech and Its Evaluation

Spontaneously spoken Japanese includes a lot of grammatically\ud ill-formed linguistic phenomena such as fillers,\ud hesitations, inversions, and so on, which do not appear in\ud written language. This paper proposes a method of robust\ud dependency parsing using a large-scale spoken language\ud corpus, and evaluates the availability and robustness\ud of the method using spontaneously spoken dialogue\ud sentences. By utilizing stochastic information about the\ud appearance of ill-formed phenomena, the method can robustly\ud parse spoken Japanese including fillers, inversions,\ud or dependencies over utterance units. As a result of an experiment,\ud the parsing accuracy provided 87.0%, and we\ud confirmed that it is effective to utilize the location information\ud of a bunsetsu, and the distance information between\ud bunsetsus as stochastic information

Effect of riboflavin deficiency on development of the cerebral cortex in Slc52a3 knockout mice

Riboflavin transporter 3 (RFVT3), encoded by the SLC52A3 gene, is important for riboflavin homeostasis in the small intestine, kidney, and placenta. Our previous study demonstrated that Slc52a3 knockout (Slc52a3−/−) mice exhibited neonatal lethality and metabolic disorder due to riboflavin deficiency. Here, we investigated the influence of Slc52a3 gene disruption on brain development using Slc52a3−/− embryos. Slc52a3−/− mice at postnatal day 0 showed hypoplasia of the brain and reduced thickness of cortical layers. At embryonic day 13.5, the formation of Tuj1+ neurons and Tbr2+ intermediate neural progenitors was significantly decreased; no significant difference was observed in the total number and proliferative rate of Pax6+ radial glia. Importantly, the hypoplastic phenotype was rescued upon riboflavin supplementation. Thus, it can be concluded that RFVT3 contributes to riboflavin homeostasis in embryos and that riboflavin itself is required during embryonic development of the cerebral cortex in mice

Laser-induced fluorescence imaging of plants using a liquid crystal tunable filter and charge coupled device imaging camera

ArticleReview of Scientific Instruments. 76, 106103 (2005)journal articl

Comprehensive genomic profiling for patients with chemotherapy‐naïve advanced cancer

Comprehensive genomic profiling (CGP) testing by next-generation sequencing has been introduced into clinical practice as part of precision cancer medicine to select effective targeted therapies. However, whether CGP testing at the time of first-line chemotherapy could be clinically useful is not clear. We conducted this single-center, prospective, observational study to investigate the feasibility of CGP testing for chemotherapy-naïve patients with stage III/IV gastrointestinal cancer, rare cancer, and cancer of unknown primary, using the FoundationOne® companion diagnostic (F1CDx) assay. The primary outcome was the detection rate of at least one actionable/druggable cancer genomic alteration. Actionable/druggable cancer genomic alterations were determined by the F1CDx report. An institutional molecular tumor board determined the molecular-based recommended therapies. A total of 197 patients were enrolled from October 2018 to June 2019. CGP success rate was 76.6% (151 of 197 patients), and median turnaround time was 19 days (range: 10-329 days). Actionable and druggable cancer genomic alterations were reported in 145 (73.6%) and 124 (62.9%) patients, respectively. The highest detection rate of druggable genomic alterations in gastrointestinal cancers was 80% in colorectal cancer (48 of 60 patients). Molecular-based recommended therapies were determined in 46 patients (23.4%). CGP testing would be a useful tool for the identification of a potentially effective first-line chemotherapy