A custom Genexus myeloid assay (CMA) underwent a technical evaluation for detection of variants from both DNA and RNA in a single assay format. The custom assay was initially verified with commercial DNA and RNA controls containing known myeloid variants. Seventy-five patient specimens with various DNA and RNA variants were selected for replicate testing. The CMA generated consistent data on two Genexus sequencers, with occasional samples failing quality metrics randomly. All 22 control DNA variants were detected, with 95% reported as key. Sensitivity and positive predictive value for clinical variants were 95.60% and 97.60%, respectively, increasing to 98.63% and 99.65%, respectively, for allele frequencies ≥5%. FLT3 duplications were consistently detected at lower expected frequencies. Low-level, false-positive key variants were mostly recurrent and filterable. Fusion calling sensitivity was 100% for control and clinical RNA samples, with 97.09% of replicates reporting expected fusions. The CMA reliably detects key DNA and RNA variants in myeloid specimens within 24 hours on the Genexus platform. Variants and fusion transcripts were identified with high sensitivity and minimal nucleic acid input, providing a rapid and precise workflow for reporting clinically relevant myeloid disease variants.

IDH-mutant astrocytomas maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, producing extreme inter-arm telomere length heterogeneity, yet how this heterogeneity shapes structural genome evolution remains unknown. Using Oxford Nanopore long-read sequencing of 20 IDH-mutant astrocytomas, we profiled structural variants (SVs), copy number variants, extrachromosomal DNA (ecDNA) and measured allele-specific telomere lengths from individual long reads. We identified pervasive complex rearrangements, including chromothripsis and foldback events consistent with breakage-fusion-bridge cycles, and widespread ecDNAs. SV breakpoints were enriched at telomeric and centromeric regions regardless of local telomere length, revealing constitutive structural fragility. Arm-level telomere length analysis uncovered a dual-mode model: arms with short telomeres preferentially harbored breakage-associated events, while arms with long ALT-maintained telomeres were enriched for ecDNA and amplification-associated events. These findings identify chromosome-arm-specific telomere length as a determinant of structural genome evolution in ALT-driven tumors.

High-grade gliomas (HGGs) are aggressive tumors with poor outcomes and limited treatment options. Here, we combined genomic and transcriptomic tumor profiling with drug testing in a patient-derived 3-dimensional culture model to identify individualized treatments and predictive biomarkers. Activity of single agents targeting frequently dysregulated glioma pathways was relatively poor ex vivo and generally reflected historical patient data. However, compounds targeting PI3K, epigenetic, and survival/senescence signaling were effective in some cases. Drug sensitivity correlated with transcriptional rather than genomic features and suggested heterogeneity as a resistance mechanism. Bromodomain and extraterminal domain inhibition was particularly effective in tumors enriched in the mesenchymal transcriptional subtype, promoted proneural transition, and was overcome by upregulated PI3K signaling. Notably, combinations were largely effective, with 6 strategies exhibiting stronger efficacy than corresponding single agents in most cases (58-77%). This study identifies HGG vulnerabilities and associated biomarkers, resistance mechanisms, and effective combination strategies that warrant further clinical validation.

Introduction Although circulating tumor DNA (ctDNA) has emerged as a promising prognostic tool in various malignancies, evidence in endometrial cancer at high risk of recurrence is limited. This study evaluated the association of pre- and post-surgical ctDNA with advanced-stage disease, disease-free and overall survival in endometrial cancer with high-risk features. Material and Methods This prospective observational study was conducted at Mayo Clinic (7/2016-6/2021). Patients with endometrial cancer at preoperative biopsy, confirmed by final pathology, were included. Blood samples were collected before and 10 weeks after surgery. Tumor-specific junctions identified in pathology specimens and blood samples were used to detect ctDNA. Associations between pre- and post-surgical ctDNA and advanced-stage disease, recurrence, and death were evaluated using logistic regression [odds ratio (OR) and 95% confidence interval] and Cox proportional hazards [hazard ratio (HR) and 95% confidence interval]. Results Thirty-six patients were included: 6 (16.7%) intermediate risk, 1 (2.8%) high-intermediate risk, 28 (77.8%) high risk, and 1 (2.8%) advanced metastatic. Detection of pre- or post-surgical ctDNA was not significantly associated with advanced disease (pre-surgical OR 5.69 [0.88-66.02]; post-surgical OR 5.86 [0.83-72.68]). Pre-surgical ctDNA did not significantly predict recurrence (HR 0.99 [0.30-3.23]) or death (HR 3.23 [0.40-25.91]). In contrast, post-surgical ctDNA was associated with increased risk of recurrence (HR 3.32 [1.05-10.51]) and death (HR 5.97 [1.11-36.08]). Conclusion Post-surgical ctDNA detection was associated with poor outcomes in patients with endometrial cancer. These findings support the potential of ctDNA as a biomarker to personalize surveillance and guide post-surgical treatment strategies.

OBJECTIVES:Sarcomatoid urothelial carcinoma (sUC) is associated with poor clinical outcomes. Herein, we evaluated clinicopathologic features (including PD-L1 expression) for institutional cases. METHODS:Sixty-five patients with sUC treated by radical cystectomy were matched for age, sex, and pathologic stage to 190 patients with pure urothelial carcinoma (UC). Outcomes were evaluated using the Kaplan-Meier method. Immunohistochemistry (IHC) results for tumor-specific PD-L1 expression in sUC were quantified using H-scores (range, 0-300). Cases with constitutive PD-L1 expression (cPD-L1; H-score ≥240) were evaluated for amplification events using fluorescence in situ hybridization (n = 28), mate-pair sequencing (Mpseq; n = 6), RNA sequencing (n = 5), and targeted next-generation sequencing (n = 8). PD-L1 regulatory pathways were evaluated using gene set enrichment analysis in the Cancer Hallmark dataset. RESULTS:Most patients with sUC had advanced disease (≥pT2, 62/65, 95%), without significant differences in overall and cancer-specific survival, when matched to pure UC. Median PD-L1 H-scores using different clones were 100 (SP142), 120 (SP263), and 195 (22C3). cPD-L1 was seen in 43% (28/65) of cases of sUC that were enriched for TP53, TERT, and CDKN2A/B alterations. PD-L1 amplification was identified in 7 (of 28, 25%) of these cases. Bionformatics analysis consistently identified IL6-JAK-STAT3 and interferon α/γ signaling in sUC with cPD-L1 expression. CONCLUSIONS:Our results suggest that cPD-L1 expression in most sUC occurs secondary to IL6-JAK-STAT3 and interferon α/γ signaling. This provides a biologic rationale for evaluating response to immunotherapy in this patient population.

PURPOSE:Constitutional MLH1 promoter hypermethylation (CMPH) is a relatively rare cause of Lynch syndrome. While most cases appear to be sporadic, some result from secondary epimutations, mainly caused by germline variants in the MLH1 promoter region. This study describes the clinical phenotype and genetic etiology of CMPH in the largest clinical cohort to date. METHODS:A retrospective analysis was conducted for 422 individuals who underwent clinical CMPH testing. Promoter sequencing was used to identify the underlying variants. Long-read sequencing further characterized MLH1 promoter methylation. RESULTS:CMPH was identified in 15.6% of the study cohort participants. Of these, 63 exhibited clinical features consistent with Lynch syndrome. The most common associated cancers were colorectal cancer, followed by endometrial cancer, breast cancer, and sebaceous neoplasms. Mendelian inheritance of CMPH was observed in 5 families in the study cohort, indicating secondary epimutations. Promoter sequencing identified 8 unique germline variants, including 3 novel variants. Methylation analysis by long-read sequencing revealed mutant allele-specific promoter methylation for these variants. CONCLUSION:Our findings provide the most comprehensive review of the clinical phenotype associated with CMPH and highlight the significant contribution of promoter variants to its etiology. These results underscore the need to include the assessment of constitutional MLH1 promoter methylation for Lynch syndrome diagnosis.

How much we sleep at night is believed to impact next-day emotional experiences. Yet, theexisting research is encumbered by methodological limitations. To address this issue we harnessedexperience sampling data (68,232 observations across 10,905 days) from 1,415 Belgianparticipants to examine whether normal variations in sleep duration linearly or nonlinearlyinfluence next-day fatigue, stress, happiness, anxiety, despondence, and calmness. We tested8,960 models as part of a multiverse analyses in this non-pre-registered study. Findings indicateeven small increases in sleep duration positively associate with next day emotional experiences,that effects are generally stronger in the period after waking relative to later in the day, and thateffect magnitudes differ markedly across emotions. Analyses indicating some sleep-emotioneffects are stronger at higher absolute sleep amounts should be cautiously interpreted given thisstudy’s observational and exploratory nature.

OBJECTIVE:We investigated the clinical validity and feasibility of a DNA junction-based quantitative polymerase chain reaction assay to detect and quantitate circulating tumor DNA (ctDNA) in the blood of high-risk patients with endometrial cancer. METHODS:Whole genome sequencing tumor data was analyzed from 36 patients to select prominent somatic tumor-specific DNA junctions. Personalized quantitative polymerase chain reaction assays were developed for each junction and applied to available blood specimens to measure levels of ctDNA. RESULTS:Pre-surgical blood ctDNA was detected in 71% of the cases tested (56% early-stage vs 88% advanced-stage). In patients with available pre-amplified pre-surgical ctDNA (n = 15), pre-surgical ctDNA levels were elevated in patients with vital status or "alive with disease" or "died of disease" compared to patients with "no evidence of disease" (p < .005). Among 17 patients followed serially, ctDNA detection preceded clinical detection of recurrence in 5 of 8 cases and was concurrent in 1, with the caveat that imaging was rarely concurrent with blood draw timing. CONCLUSIONS:Personalized junction-based measurement of ctDNA demonstrated promising clinical validity in pre-surgical prognosis and relapse prediction.

High copy-number endometrial cancers (HCNECs) are dominated by excessive duplications scattered across the genome, termed here as the hyperduplication genomophenotype (HDGP). We identified locations and sizes of duplications in 171 endometrial cancer cases and designated 71 HCNEC cases as HDGP. We also investigated the response to the pan-ERBB inhibitor afatinib in a subset of HDGP-EC cases with ERBB2/ERBB3 duplications using a patient-derived three-dimensional culture model. Our analysis demonstrates that beyond tandem duplications there is a more general pattern involving coordinated duplication of multiple distant regions of the genome, demonstrating preferential selectivity to overexpressed potential oncogenes within a broad network. This suggests that HDGP increases tumor fitness and resistance to therapy by perturbing important gene networks in concert rather than only driver genes, suggesting a mechanistic basis for the ineffectiveness of targeted drugs in these patients and highlighting the need for combination therapies in these highly aggressive cases.

PURPOSE:Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications. EXPERIMENTAL DESIGN:Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase-mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples. RESULTS:Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression. CONCLUSIONS:In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications.Experimental Design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase-mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples.Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression.Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

PURPOSE:The pathogenicity of intragenic duplications depends on their structural configuration. Tandem duplications often disrupt reading frames and cause gene loss of function, whereas interspersed (nontandem) duplications are largely benign. When the configuration cannot be determined, current guidelines presume a tandem structure, leading to some laboratories automatically classifying such variants as likely pathogenic or pathogenic. This study evaluates the validity of this presumption for DMD, in patients with and without clinical indications of dystrophinopathy. METHODS:We performed high-coverage long-read genome sequencing on 15 patients with intragenic DMD duplications. A total of 4 patients had clinically indicated dystrophinopathy testing, whereas in the remaining 11 patients, the duplications were detected without clear indications of dystrophinopathy (eg, through carrier screening). RESULTS:All 4 patients with clinical indications had tandem duplications. In contrast, 64% (7/11) of the cases without such indications had interspersed duplications, with 4 subsequently reclassified as likely benign, 2 (likely) pathogenic, and 1 uncertain. These duplications were often complex, involving coduplications or codeletions with other regions. CONCLUSION:Our findings challenge the presumption that intragenic DMD duplications are predominantly in tandem. This highlights the need for a cautious variant interpretation approach, particularly in carrier screening and other settings in which variants are identified without indications of dystrophinopathy.

Background The COVID-19 pandemic emphasized an urgent need for devices used in the self-collection of biospecimens in an evolving patient care system. The mailing of biospecimen self-collection kits to patients, with samples returned via mail, provides a more convenient testing regimen, but could also impart patient sampling variabilities. User compliance with device directions is central to downstream testing of collected biospecimens and clear instructions are central to this goal.Methods Here, we performed an evaluation of 10 oral DNA collection devices involving either swab or saliva self-collection and analyzed ease of use and comfort level with a device, as well as DNA recovery quantity/quality and sample stability.Results We show that while these DNA quality/quantity metrics are comparable between devices, users prefer direct saliva collection over swab-based devices.Conclusions This information is useful in guiding future experiments including their use in human RNA, microbial, or viral sample collection/recovery and their use in clinical testing.

Abstract Accurate response assessment in patients with high-grade gliomas (HGGs) is complicated by post-treatment radiographic changes that are equivocal for treatment effect versus progression. Plasma-based liquid biopsy assays for circulating tumor DNA (ctDNA) have had limited sensitivity to date, due in part to the presence of the blood-brain barrier that limits the diffusion of tumor-derived analytes out of the central nervous system. To address this challenge, we developed a ctDNA plasma-based assay to detect individualized tumor-derived amplified junctions in patients with HGGs. These junctions occur due to large-scale chromosomal rearrangements, such as those associated with epidermal growth factor receptor (EGFR) or cyclin-dependent kinase 4 (CDK4) amplification. To identify these amplified junctions, whole genome sequencing was performed on resected tissue. Primers were designed to flank junction breakpoints and were then utilized on each patient’s plasma samples to detect the amplified junction via qPCR. Tumor-specific amplified junctions were robustly detected in plasma from 14/14 (100%) patients with IDH-mutant or wild-type grade 4 astrocytomas whose tumor genomes demonstrated amplification-related junctions, located at chromosomes 7, 11, or 12. In comparison, ctDNA could not be detected in three control cases that did not have an amplification or did not have high copy number junctions. Importantly, serial plasma samples obtained prior to and at disease recurrence in 4 patients demonstrated an increasing abundance of amplified junctions that accurately correlated with disease progression in 4/4 patients (100%). In two of these patients, increasing abundance of amplified junctions preceded radiographic changes compatible with disease progression by approximately 2 or 4 months, respectively. In conclusion, tumor-specific amplified junctions could reliably be detected in plasma from patients with IDH-mutant or wild-type grade 4 astrocytomas and correlated with changes in disease burden. Further work is currently ongoing to evaluate these promising results in a larger cohort of patients with serial plasma samples.

This mixed-methods study investigates whether online vigilance promotes mental fatigue, and whether this effect is greater when under pressure to be available online. Additionally, it examines whether passively sensed smartphone behavior can serve as a digital proxy for online vigilance. Data were collected from 1,315 adult participants, who received 84 experience sampling questionnaires over 14 days, providing 67,762 usable datapoints on individuals’ perceptions of momentary online vigilance, mental fatigue, and availability pressure. Additionally, the smartphone use of 834 participants was passively monitored. Findings revealed both a momentary and lagged association between self-reported online vigilance and self-reported mental fatigue. Availability pressure was not a significant moderator, but did predict mental fatigue directly and indirectly, by promoting online vigilance. We found behavioral smartphone use features were weakly associated with self-reported online vigilance and mental fatigue. Overall, this study provides initial support that online vigilance may play a role in the development of mental health conditions such as burnout via its tendency to promote one of its precursors, mental fatigue.

Hedonic overconsumption (e.g., overconsumption of gratifying behaviors, e.g., eating, gaming) is common in daily life and often problematic, pointing to the need for adequate behavioral models. In this article, we develop a self-regulatory framework proposing that when an actual consumption experience falls short of hedonic expectations - such as when being distracted - people will want to consume more to compensate for the shortfall. In a preliminary meta-analysis, a small-scale field experiment on distraction during lunch and subsequent afternoon snacking (Study 1), and a pre-registered experience sampling (ESM) study (Study 2) involving more than 6,000 consumption episodes in everyday life, across multiple consumption domains, we investigated the predictions from our Hedonic Compensation Model. There was clear and consistent evidence across studies and analyses for the prediction that distraction during consumption compromises the actual enjoyment of a given consumption experience. Both empirical studies yielded consistent evidence for a negative effect of actual enjoyment on consumption satisfaction but inconsistent and weaker evidence for the expected role of actual-expected enjoyment discrepancies for this part of the model. There was also consistent evidence for the expected negative association between consumption satisfaction and the need for further gratification. Finally, there was moderate and inconsistent support linking need for further gratification to subsequent consumption across Study 1 (amount and frequency of snacking in the afternoon) and Study 2 (shorter duration to subsequent consumption). Taken together, the present framework provides a good starting point for studying the links among compromising consumption contexts, consumption enjoyment, and subsequent hedonic compensation.

Supplemental Table 1 and Figures 1-5 combined in single PDF file. Supplemental Table 1: Mate Pair sequencing data statistics. Supplemental Figure 1: KRAS Mutations in PDAC tumors. Supplemental Figure 2: Frequency copy number plots at the SMAD4 locus (A), chromosome 18 (B), FHIT (C), ZNF521 (D) loci in the primary PDAC tumors and for SMAD4, FHIT and ZNF521 in the PDX tumors (E). Supplemental Figure 3: Expression level changes of commonly hit genes. Supplemental Figure 4: TGFbeta responsive genes. Supplemental Figure 5: FHIT pathway genes and signaling networks.

Supplemental Figure 2 presents genome coverage plots for four example cases illustrating complex rearrangements between chromosomes (A-D) plus a schematic of the complex TMPRSS2-ERG rearrangement in PR084. Supplemental Figure 3 illustrates the number of breakpoints from genomic rearrangements hitting chromosome 21q. Supplemental Figure 4 illustrates survival analysis from data from Fraser et al 2017 comparing non-indolent prostate cancers with TMPRSS2-ERG fusions with interstitial deletion or retention.