A computational pipeline for identifying gene targets and signalling pathways in cancer cells to improve lymphocyte infiltration and immune checkpoint therapy efficacy.

BACKGROUND: Tumour-infiltrating lymphocytes (TILs) are crucial for effective immune checkpoint blockade (ICB) therapy in solid tumours. However, ∼70% of these tumours exhibit poor lymphocyte infiltration, rendering ICB therapies less effective. METHODS: We developed a bioinformatics pipeline integrating multiple previously unconsidered factors or datasets, including tumour cell immune-related pathways, copy number variation (CNV), and single tumour cell sequencing data, as well as tumour mRNA-seq data and patient survival data, to identify targets that can potentially improve T cell infiltration and enhance ICB efficacy. Furthermore, we conducted wet-lab experiments and successfully validated one of the top-identified genes. FINDINGS: We applied this pipeline in solid tumours of the Cancer Genome Atlas (TCGA) and identified a set of genes in 18 cancer types that might potentially improve lymphocyte infiltration and ICB efficacy, providing a valuable drug target resource to be further explored. Importantly, we experimentally validated SUN1, which had not been linked to T cell infiltration and ICB therapy previously, but was one of the top-identified gene targets among 3 cancer types based on the pipeline, in a mouse colon cancer syngeneic model. We showed that Sun1 KO could significantly enhance antigen presentation, increase T-cell infiltration, and improve anti-PD1 treatment efficacy. Moreover, with a single-cell multiome analysis, we identified subgene regulatory networks (sub-GRNs) showing Stat proteins play important roles in enhancing the immune-related pathways in Sun1-KO cancer cells. INTERPRETATION: This study not only established a computational pipeline for discovering new gene targets and signalling pathways in cancer cells that block T-cell infiltration, but also provided a gene target pool for further exploration in improving lymphocyte infiltration and ICB efficacy in solid tumours. FUNDING: A full list of funding bodies that contributed to this study can be found in the Acknowledgements section.

Algorithm Training and Testing for a Nonendoscopic Barrett's Esophagus Detection Test in Prospective Multicenter Cohorts.

BACKGROUND & AIMS: Endoscopic Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC) detection is invasive and expensive. Nonendoscopic BE/EAC detection tools are guideline-endorsed alternatives. We previously described a 5-methylated DNA marker (MDM) panel assayed on encapsulated sponge cell collection device (CCD) specimens. We aimed to train a new algorithm using a 3-MDM panel and test its performance in an independent cohort. METHODS: Algorithm training and test samples were from 2 prospective multicenter cohorts. All BE cases had esophageal intestinal metaplasia (with or without dysplasia/EAC); control subjects had no endoscopic evidence of BE. The CCD procedure was followed by endoscopy. From CCD cell lysates, DNA was extracted, bisulfite treated, and MDMs were blindly assayed. The algorithm was set and locked using cross-validated logistic regression (training set) and its performance was assessed in an independent test set. RESULTS: Training (N = 352) and test (N = 125) set clinical characteristics were comparable. The final panel included 3 MDMs (NDRG4, VAV3, ZNF682). Overall sensitivity was 82% (95% CI, 68%-94%) at 90% (79%-98%) specificity and 88% (78%-94%) sensitivity at 84% (70%-93%) specificity in training and test sets, respectively. Sensitivity was 90% and 68% for all long- and short-segment BE, respectively. Sensitivity for BE with high-grade dysplasia and EAC was 100% in training and test sets. Overall sensitivity for nondysplastic BE was 82%. Areas under the receiver operating characteristic curves for BE detection were 0.92 and 0.94 in the training and test sets, respectively. CONCLUSIONS: A locked 3-MDM panel algorithm for BE/EAC detection using a nonendoscopic CCD demonstrated excellent sensitivity for high-risk BE cases in independent validation samples. (Clinical trials.gov: NCT02560623, NCT03060642.).

High-titer manufacturing of SARS-CoV-2 Spike-pseudotyped VSV in stirred-tank bioreactors.

The severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic highlighted the importance of vaccine innovation in public health. Hundreds of vaccines built on numerous technology platforms have been rapidly developed against SARS-CoV-2 since 2020. Like all vaccine platforms, an important bottleneck to viral-vectored vaccine development is manufacturing. Here, we describe a scalable manufacturing protocol for replication-competent SARS-CoV-2 Spike-pseudotyped vesicular stomatitis virus (S-VSV)-vectored vaccines using Vero cells grown on microcarriers in a stirred-tank bioreactor. Using Cytodex 1 microcarriers over 6 days of fed-batch culture, Vero cells grew to a density of 3.95 ± 0.42 ×106 cells/mL in 1-L stirred-tank bioreactors. Ancestral strain S-VSV reached a peak titer of 2.05 ± 0.58 ×108 plaque-forming units (PFUs)/mL at 3 days postinfection. When compared to growth in plate-based cultures, this was a 29-fold increase in virus production, meaning a 1-L bioreactor produces the same amount of virus as 1,284 plates of 15 cm. In addition, the omicron BA.1 S-VSV reached a peak titer of 5.58 ± 0.35 × 106 PFU/mL. Quality control testing showed plate- and bioreactor-produced S-VSV had similar particle-to-PFU ratios and elicited comparable levels of neutralizing antibodies in immunized hamsters. This method should enhance preclinical and clinical development of pseudotyped VSV-vectored vaccines in future pandemics.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.

The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.

Next-generation Multi-target Stool DNA Panel Accurately Detects Colorectal Cancer and Advanced Precancerous Lesions.

The multi-target stool DNA (mt-sDNA) test screens for colorectal cancer by analyzing DNA methylation/mutation and hemoglobin markers to algorithmically derive a qualitative result. A new panel of highly discriminant candidate methylated DNA markers (MDM) was recently developed. Performance of the novel MDM panel, with hemoglobin, was evaluated in a simulated screening population using archived stool samples weighted to early-stage colorectal cancer and prospectively collected advanced precancerous lesions (APL). Marker selection study (MSS) and separate preliminary independent verification studies (VS) were conducted utilizing samples from multi-center, case-control studies. Sample processing included targeted MDM capture, bisulfite conversion, and MDM quantitation. Fecal hemoglobin was quantified using ELISA. Samples were stratified into 75%/25% training-testing sets; model outcomes were cross-validated 1,000 times. All laboratory operators were blinded. The MSS included 232 cases (120 colorectal cancer/112 APLs) and 490 controls. The VS featured 210 cases (112 colorectal cancer/98 APLs) and 567 controls; APLs were 86.7% adenomas and 13.3% sessile serrated lesions (SSL). Average age was 65.5 (cases) and 63.2 (controls) years. Mean sensitivity in the VS from cross-validation was 95.2% for colorectal cancer and 57.2% for APLs, with specificities of 89.8% (no CRC/APLs) and 92.4% (no neoplasia). Subgroup analyses showed colorectal cancer sensitivities of 93.4% (stage I) and 94.2% (stage II). APL sensitivity was 82.9% for high-grade dysplasia, 73.4% for villous lesions, 49.8% for tubular lesions, and 30.2% for SSLs. These data support high sensitivity and specificity for a next-generation mt-sDNA test panel. Further evaluation of assay performance will be characterized in a prospective, multi-center clinical validation study (NCT04144738). PREVENTION RELEVANCE: This study highlights performance of the next-generation mt-sDNA test, which exhibits high sensitivity and specificity for detecting colorectal cancer and APLs. This noninvasive option has potential to increase screening participation and clinical outcomes. A multi-center, clinical validation trial is underway. See related commentary by Bresalier, p. 93.

Plasma Assay of Cell-Free Methylated DNA Markers of Colorectal Cancer: A Tumor-Agnostic Approach to Monitor Recurrence and Response to Anticancer Therapies.

BACKGROUND: Radiographic surveillance of colorectal cancer (CRC) after curative-intent therapy is costly and unreliable. Methylated DNA markers (MDMs) detected primary CRC and metastatic recurrence with high sensitivity and specificity in cross-sectional studies. This study evaluated using serial MDMs to detect recurrence and monitor the treatment response to anti-cancer therapies. METHODS: A nested case-control study was drawn from a prospective cohort of patients with CRC who completed curative-intent therapy for CRC of all stages. Plasma MDMs were assayed vis target enrichment long-probe quantitative-amplified signal assays, normalized to B3GALT6, and analyzed in combination with serum carcinoembryonic antigen to yield an MDM score. Clinical information, including treatment and radiographic measurements of the tumor burden, were longitudinally collected. RESULTS: Of the 35 patients, 18 had recurrence and 17 had no evidence of disease during the study period. The MDM score was positive in 16 out of 18 patients who recurred and only 2 of the 17 patients without recurrence. The MDM score detected recurrence in 12 patients preceding clinical or radiographic detection of recurrent CRC by a median of 106 days (range 90-232 days). CONCLUSIONS: Plasma MDMs can detect recurrent CRC prior to radiographic detection; this tumor-agnostic liquid biopsy approach may assist cancer surveillance and monitoring.

Utilising the intrinsic fluorescence of pomalidomide for imaging applications.

Optimisation of protein degraders requires balancing multiple factors including potency, cell permeability and solubility. Here we show that the fluorescence of pomalidomide can be used in high-throughput screening assays to rapidly assess cellular penetration of degrader candidates. In addition, this technique can be paired with endocytosis inhibitors to gain insight into potential mechanisms of candidates entering a target cell. A model library of pomalidomide conjugates was synthesised and evaluated using high-throughput fluorescence microscopy. This technique based on intrinsic fluorescence can be used to guide rational design of pomalidomide conjugates without the need for additional labels or tags.

ASPSCR1-TFE3 reprograms transcription by organizing enhancer loops around hexameric VCP/p97.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Positive Predictive Value for Multitarget Stool DNA After Bariatric and Metabolic Surgery.

BACKGROUND AND AIMS: Bariatric and metabolic surgery (BMS) may adversely affect noninvasive stool tests for colorectal cancer (CRC) screening through several mechanisms. Multitarget stool DNA (mt-sDNA) is approved for CRC screening; however, performance in post-BMS patients is unknown. As the rates of BMS are anticipated to increase with rising incidence of obesity, it is important to evaluate mt-sDNA test performance among these patients. METHODS: In a multisite academic and community-based practice, we obtained mt-sDNA results from 10/2014 to 12/2019 through electronic records and an institutional BMS registry. Average CRC risk patients with BMS prior to a positive mt-sDNA underwent a detailed chart review. Follow-up colonoscopy findings were compared to those among BMS patients screened with colonoscopy alone and a historical cohort of patients without BMS, screened by mt-sDNA. The primary study endpoint was the positive predictive value (PPV) for advanced colorectal neoplasia. RESULTS: Among 336 average-risk patients who had mt-sDNA after BMS, mt-sDNA was positive in 49 (14.6%), 47/49 (96%) underwent follow-up colonoscopy, and the PPV for advanced neoplasia was 12/47 (25.5%). This is similar to the PPV for advanced colorectal neoplasia (425/1542, 28%) in a historical cohort of persons without prior BMS, screened by mt-sDNA at our center (P = .86). Among those who had prior BMS, the rate of advanced neoplasia was higher after mt-sDNA compared to screening colonoscopy alone. CONCLUSION: Despite anatomic and physiologic mechanisms that could alter blood or DNA content in stool, BMS does not appear to adversely affect the PPV of mt-sDNA.

Plasma Methylated DNA Markers for Melanoma Surveillance.

PURPOSE: Surveillance after primary melanoma treatment aims to detect early signs of low-volume systemic disease. The current standard of care, surveillance imaging, is costly and difficult to access. We therefore sought to develop methylated DNA markers (MDMs) as promising alternatives for disease surveillance. METHODS: We used reduced representation bisulfite sequencing (RRBS) to identify MDMs in DNA samples obtained from metastatic melanoma, benign nevi, and normal skin tissues. The identified MDMs underwent validation in an independent cohort of tissue and buffy coat DNA samples. Subsequently, we tested the validated MDMs in the plasma DNA of patients with metastatic melanoma undergoing surveillance with total body imaging and compared them with cancer-free controls. To estimate the overall predictive accuracy of the MDMs, we used random forest modeling with bootstrap cross-validation. RESULTS: Forty MDMs demonstrated discrimination between melanoma cases and controls consisting of benign nevi and normal skin. Nine MDMs passing biological validation in tissue were run on 77 plasma samples from individuals with a history of metastatic melanoma, 49 of whom had evidence of disease detected by imaging at the time of blood draw, and 100 cancer-free controls. The cross-validated sensitivity of the panel for imaging-positive disease was 80% with a specificity of 100% in cancer-free controls, resulting in an overall AUC of 0.88 (95% CI, 0.81 to 0.96). The survival estimates for patients with melanoma who tested positive for the panel at 6 months and 1 year were 67% and 56%, respectively, while those who tested negative had survival rates of 100% and 92%. CONCLUSION: MDMs identified by RRBS demonstrate a high degree of concordance with imaging results in the plasma of patients with metastatic melanoma. Further prospective studies in larger intended use cohorts are needed to confirm these findings.

Assessing the capacity of methylated DNA markers of cervical squamous cell carcinoma to discriminate oropharyngeal squamous cell carcinoma in human papillomavirus mediated disease.

OBJECTIVE: Early identification of human papillomavirus associated oropharyngeal squamous cell carcinoma (HPV(+)OPSCC) is challenging and novel biomarkers are needed. We hypothesized that a panel of methylated DNA markers (MDMs) found in HPV(+) cervical squamous cell carcinoma (CSCC) will have similar discrimination in HPV(+)OPSCC tissues. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissues were obtained from patients with primary HPV(+)OPSCC or HPV(+)CSCC; control tissues included normal oropharynx palatine tonsil (NOP) and cervix (NCS). Using a methylation-specific polymerase chain reaction, 21 previously validated cervical MDMs were evaluated on tissue-extracted DNA. Discrimination between case and control cervical and oropharynx tissue was assessed using area under the curve (AUC). RESULTS: 34 HPV(+)OPSCC, 36 HPV(+)CSCC, 26 NOP, and 24 NCS patients met inclusion criteria. Within HPV(+)CSCC, 18/21 (86%) of MDMs achieved an AUC ≥ 0.9 and all MDMs exhibited better than chance classifications relative to control cervical tissue (all p < 0.001). In contrast, within HPV(+)OPSCC only 5/21 (24%) MDMs achieved an AUC ≥ 0.90 but 19/21 (90%) exhibited better than chance classifications relative to control tonsil tissue (all p < 0.001). Overall, 13/21 MDMs had statistically significant lower AUCs in the oropharyngeal cohort compared to the cervical cohort, and only 1 MDM exhibited a statistically significant increase in AUC. CONCLUSIONS: Previously validated MDMs exhibited robust performance in independent HPV(+)CSCC patients. However, most of these MDMs exhibited higher discrimination for HPV(+)CSCC than for HPV(+)OPSCC. This suggests that each SCC subtype requires a unique set of MDMs for optimal discrimination. Future studies are necessary to establish an MDM panel for HPV(+)OPSCC.

Methylated DNA Markers for Sporadic Colorectal and Endometrial Cancer Are Strongly Associated with Lynch Syndrome Cancers.

Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgical prophylaxis.To validate a panel of methylated DNA markers (MDM) previously identified in sporadic colorectal cancer and endometrial cancer for discrimination of these cancers in LS.In a case-control design, previously identified MDMs for the detection of colorectal cancer and endometrial cancer were assayed by qMSP on tissue-extracted DNA. Results were normalized to ACTB values within each sample. Least absolute shrinkage and selection operator models to classify colorectal cancer and endometrial cancer were trained on sporadic cases and controls and then applied to classify colorectal cancer and endometrial cancer, in those with LS, and cross-validated.We identified colorectal cancer cases (23 with LS, 48 sporadic), colorectal controls (32 LS, 48 sporadic), endometrial cancer cases (30 LS, 48 sporadic), and endometrial controls (29 LS, 37 sporadic). A 3-MDM panel (LASS4, LRRC4, and PPP2R5C) classified LS-CRC from LS controls with an AUC of 0.92 (0.84-0.99); results were similar for sporadic colorectal cancer. A 6-MDM panel (SFMBT2, MPZ, CYTH2, DIDO1, chr10.4479, and EMX2OS) discriminated LS-EC from LS controls with an AUC of 0.92 (0.83-1.0); the AUC for sporadic endometrial cancer versus sporadic controls was nominally higher, 0.99 (0.96-1.0).MDMs previously identified in sporadic endometrial cancer and colorectal cancer discriminate between endometrial cancer and benign endometrium and colorectal cancer and benign colorectum in LS. This supports the inclusion of patients with LS within future prospective clinical trials evaluating endometrial cancer and colorectal cancer MDMs and may provide a new avenue for cancer screening or surveillance in this high-risk population. PREVENTION RELEVANCE: Lynch syndrome (LS) markedly increases risks of colorectal and endometrial cancers. Early detection biomarkers for LS cancers could reduce the needs for invasive screening and surgery. Methylated DNA markers previously identified in sporadic endometrial cancer and colorectal cancer discriminate between benign and cancer tissue in LS.

Neoplasia Diagnosis After Multi-target Stool DNA Is Enhanced Among Lowest Baseline Detectors.

BACKGROUND AND AIMS: Variation in colorectal neoplasia detection limits the effectiveness of screening colonoscopy. By evaluating neoplasia detection rates of individual colonoscopists, we aimed to quantify the effects of pre-procedural knowledge of a positive (+) multi-target stool DNA (mt-sDNA) on colonoscopy quality metrics. METHODS: We retrospectively identified physicians who performed a high volume of + mt-sDNA colonoscopies; colorectal neoplasia at post-mt-sDNA colonoscopy was recorded. These colonoscopists were stratified into quartiles based on baseline adenoma detection rates. Baseline colonoscopy adenoma detection rates and sessile serrated lesion detection rates were compared to post-mt-sDNA colonoscopy neoplasia diagnosis rates among each quartile. Withdrawal times were measured from negative exams. RESULTS: During the study period (2014-17) the highest quartile of physicians by volume of post-mt-sDNA colonoscopies were evaluated. Among thirty-five gastroenterologists, their median screening colonoscopy adenoma detection rate was 32% (IQR, 28-39%) and serrated lesion detection rate was 13% (8-15%). After + mt-sDNA, adenoma diagnosis increased to 47% (36-56%) and serrated lesion diagnosis increased to 31% (17-42%) (both p < 0.0001). Median withdrawal time increased from 10 (7-13) to 12 (10-17) minutes (p < 0.0001) and was proportionate across quartiles. After + mt-sDNA, lower baseline detectors had disproportionately higher rates of adenoma diagnosis in female versus male patients (p = 0.048) and higher serrated neoplasia diagnosis rates among all patients (p = 0.0092). CONCLUSIONS: Knowledge of + mt-sDNA enriches neoplasia diagnosis compared to average risk screening exams. Adenomatous and serrated lesion diagnosis was magnified among those with lower adenoma detection rates. Awareness of the mt-sDNA result may increase physician attention during colonoscopy. Pre-procedure knowledge of a positive mt-sDNA test improves neoplasia diagnosis rates among colonoscopists with lower baseline adenoma detection rates, independent of withdrawal time.

Targeting Potential of Innate Lymphoid Cells in Melanoma and Other Cancers.

Reinvigorating the killing function of tumor-infiltrating immune cells through the targeting of regulatory molecules expressed on lymphocytes has markedly improved the prognosis of cancer patients, particularly in melanoma. While initially thought to solely strengthen adaptive T lymphocyte anti-tumor activity, recent investigations suggest that other immune cell subsets, particularly tissue-resident innate lymphoid cells (ILCs), may benefit from immunotherapy treatment. Here, we describe the recent findings showing immune checkpoint expression on tissue-resident and tumor-infiltrating ILCs and how their effector function is modulated by checkpoint blockade-based therapies in cancer. We discuss the therapeutic potential of ILCs beyond the classical PD-1 and CTLA-4 regulatory molecules, exploring other possibilities to manipulate ILC effector function to further impede tumor growth and quench disease progression.

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma.

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients.

Detection of endometrial cancer using tampon-based collection and methylated DNA markers.

OBJECTIVE: Alterations in DNA methylation are early events in endometrial cancer (EC) development and may have utility in EC detection via tampon-collected vaginal fluid. METHODS: For discovery, DNA from frozen EC, benign endometrium (BE), and benign cervicovaginal (BCV) tissues underwent reduced representation bisulfite sequencing (RRBS) to identify differentially methylated regions (DMRs). Candidate DMRs were selected based on receiver operating characteristic (ROC) discrimination, methylation level fold-change between cancers and controls, and absence of background CpG methylation. Methylated DNA marker (MDM) validation was performed using qMSP on DNA from independent EC and BE FFPE tissue sets. Women ≥45 years of age with abnormal uterine bleeding (AUB) or postmenopausal bleeding (PMB) or any age with biopsy-proven EC self-collected vaginal fluid using a tampon prior to clinically indicated endometrial sampling or hysterectomy. Vaginal fluid DNA was assayed by qMSP for EC-associated MDMs. Random forest modeling analysis was performed to generate predictive probability of underlying disease; results were 500-fold in-silico cross-validated. RESULTS: Thirty-three candidate MDMs met performance criteria in tissue. For the tampon pilot, 100 EC cases were frequency matched by menopausal status and tampon collection date to 92 BE controls. A 28-MDM panel highly discriminated between EC and BE (96% (95%CI 89-99%) specificity; 76% (66-84%) sensitivity (AUC 0.88). In PBS/EDTA tampon buffer, the panel yielded 96% (95% CI 87-99%) specificity and 82% (70-91%) sensitivity (AUC 0.91). CONCLUSION: Next generation methylome sequencing, stringent filtering criteria, and independent validation yielded excellent candidate MDMs for EC. EC-associated MDMs performed with promisingly high sensitivity and specificity in tampon-collected vaginal fluid; PBS-based tampon buffer with added EDTA improved sensitivity. Larger tampon-based EC MDM testing studies are warranted.

Adverse Events and Serological Responses After SARS-CoV-2 Vaccination in Individuals With Inflammatory Bowel Disease.

INTRODUCTION: We determined adverse events after 4 doses of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccine in those with inflammatory bowel disease (IBD), associations between antibodies and injection site reactions (ISR), and risk of IBD flare. METHODS: Individuals with IBD were interviewed for adverse events to SARS-CoV-2 vaccine. Multivariable linear regression assessed the association between antibody titers and ISR. RESULTS: Severe adverse events occurred in 0.03%. ISR were significantly associated with antibody levels after the fourth dose (geometric mean ratio = 2.56; 95% confidence interval 1.18-5.57). No cases of IBD flare occurred. DISCUSSION: SARS-CoV-2 vaccines are safe for those with IBD. ISR after the fourth dose may indicate increased antibodies.

Multitarget Stool DNA Testing Has High Positive Predictive Value for Colorectal Neoplasia on the Second Round of Testing.

BACKGROUND & AIMS: Multitarget stool DNA (mt-sDNA) testing is a stool-based screening test for colorectal cancer (CRC). In a single instance of testing, the pivotal Food and Drug Administration-approval study (NCT01397747) found that 16% of mt-sDNA tests were positive, and the positive predictive value (PPV) for CRC or advanced precursor lesions (APL) was 27.3%. We aimed to examine real-world longitudinal performance by determining the test-positive rate and PPV of mt-sDNA on the second round of testing. METHODS: Colonoscopy and pathology reports were reviewed retrospectively for patients with a negative mt-sDNA on the first round of screening and a positive mt-sDNA on the second round. The test-positivity rate and PPV for CRC, APL, and any colorectal neoplasia were calculated for the second mt-sDNA and compared with baseline PPVs from a previously published cohort of patients from our institution who tested positive on the first round of screening. RESULTS: A total of 2758 patients completed a second test at a median of 3.2 years after the first test. Of these, 422 (15%) had a positive second mt-sDNA. The PPV was 0.25% for CRC, 24% for APL, and 67% for any colorectal neoplasia. There was no significant difference in PPV on the second mt-sDNA test compared with the first round (24% vs 28% for APL; P = .12). CONCLUSIONS: mt-sDNA test positive rate and PPV were similar between the first and second rounds of screening. These observations confirm the utility of a second round of mt-sDNA screening and may inform estimates of mt-sDNA effectiveness for CRC screening.