Newborn rats chronically exposed to moderate hyperoxia (60% O2) exhibit abnormal respiratory control, including decreased eupneic ventilation. To further characterize this plasticity and explore its proximate mechanisms, rats were exposed to either 21% O2 (Control) or 60% O2 (Hyperoxia) from birth until studied at 3 – 14 days of age (P3 – P14). Normoxic ventilation was reduced in Hyperoxia rats when studied at P3, P4, and P6-7 and this was reflected in diminished arterial O2 saturations; eupneic ventilation spontaneously recovered by P13-14 despite continuous hyperoxia, or within 24 h when Hyperoxia rats were returned to room air. Normoxic metabolism was also reduced in Hyperoxia rats but could be increased by raising inspired O2 levels (to 60% O2) or by uncoupling oxidative phosphorylation within the mitochondrion (2, 4-dinitrophenol). In contrast, moderate increases in inspired O2 had no effect on sustained ventilation which indicates that hypoventilation can be dissociated from hypometabolism. The ventilatory response to abrupt O2 inhalation was diminished in Hyperoxia rats at P4 and P6-7, consistent with smaller contributions of peripheral chemoreceptors to eupneic ventilation at these ages. Finally, the spontaneous respiratory rhythm generated in isolated brainstem-spinal cord preparations was significantly slower and more variable in P3-4 Hyperoxia rats than in age-matched Controls. We conclude that developmental hyperoxia impairs both peripheral and central components of eupneic ventilatory drive. Although developmental hyperoxia diminishes metabolism as well, this appears to be a regulated hypometabolism and contributes little to the observed changes in ventilation.
Assessment of internal tandem duplications in FLT3 (FLT3-ITDs) and their allelic ratio (AR) is recommended by clinical guidelines for diagnostic workup of acute myeloid leukemia and traditionally performed through capillary electrophoresis (CE). Although significant progress has been made integrating FLT3-ITD detection within contemporary next-generation sequencing (NGS) panels, AR estimation is not routinely part of clinical NGS practice because of inherent biases and challenges. In this study, data from multiple NGS platformsdanchored multiplex PCR (AMP), amplicon [TruSeq Custom Amplicon (TSCA)], and hybrid-capturedwere analyzed through a custom algorithm, including platform-specific measures of AR. Sensitivity and specificity of NGS for FLT3-ITD status relative to CE were 100% (42/ 42) and 99.4% (1076/1083), respectively, by AMP on an unselected cohort and 98.1% (53/54) and 100% (48/48), respectively, by TSCA on a selected cohort. Primer analysis identified criteria for ITDs to escape detection by TSCA, estimated to occur in approximately 9% of unselected ITDs. Allelic fractions under AMP or TSCA were highly correlated to CE, with linear regression slopes near 1 for ITDs not duplicating primers, and systematically underestimated for ITDs duplicating a primer. Bias was alleviated in AMP through simple adjustments. This article provides an approach for targeted computational FLT3-ITD analysis for NGS data from multiple platforms; AMP was found capable of near perfect sensitivity and specificity with relatively accurate estimates of ARs.
Despite increased incidence of anal squamous cell carcinoma (ASCC), treatment recommendations have remained unchanged for the past 35 years. This article profiles the tumor microenvironment of patients with localized ASCC, examining CD8, PD‐1, PD‐L1, IDO1 and HLA class I expression and, specifically, characterizes expression of IDO1 in the context of several key components of the immune microenvironment.
AimsThe histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC.MethodsWe evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples).ResultsIn the retrospective cohort, 45% of ICCs and <1% of non-hepatic adenocarcinomas showed a cholangiolar pattern; albumin ISH was positive in 93% of ICCs with significant intratumorous heterogeneity. In the validation cohort, 29% of ICCs showed a cholangiolar pattern and 88% expressed albumin, while all metastatic non-hepatic neoplasms were negative (n=37) (sensitivity 88% and specificity 100%). Targetable genetic alterations (IDH mutations and FGFR2 fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay.ConclusionsThe cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations.
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