DNA In Peripheral Blood Mononuclear Cells Research Articles

Clinical validation of peripheral blood mononuclear cell DNA methylation markers for accurate early detection of hepatocellular carcinoma in Asian patients

BackgroundHepatocellular carcinoma (HCC), a leading cause of cancer-related deaths globally, poses significant challenges in early detection. Improved diagnostic accuracy can drastically influence patient outcomes, emphasizing the need for innovative, non-invasive biomarkers.Methods:This study utilized a cohort of 402 participants, including healthy controls, chronic hepatitis patients, and HCC patients from Bangladesh, to evaluate DNA methylation signatures in peripheral blood mononuclear cells (PBMC). We performed targeted next-generation sequencing on selected genes previously identified to assess their methylation dynamics. The development of M8 and M4 scores was based on these dynamics, using Receiver Operating Characteristic (ROC) analysis to determine their effectiveness in detecting early-stage HCC alongside existing markers such as epiLiver and alpha-fetoprotein (AFP).Results:Integration of M8 and M4 scores with epiLiver and AFP significantly enhances diagnostic sensitivity for early-stage HCC. The M4+epiLiver score achieves a sensitivity of 79.4% in Stage A HCC, while combining M4 with AFP increases sensitivity to 88.2–95.7% across all stages, indicating a superior diagnostic performance compared to each marker used alone.Conclusions:Our study confirms that combining gene methylation profiles with established diagnostic markers substantially improves the sensitivity of detecting early-stage HCC. This integrated diagnostic approach holds promise for advancing non-invasive cancer diagnostics, potentially leading to earlier treatment interventions and improved survival rates for high-risk patients.

Transcriptome- and DNA methylation-based cell-type deconvolutions produce similar estimates of differential gene expression and differential methylation

BackgroundChanging cell-type proportions can confound studies of differential gene expression or DNA methylation (DNAm) from peripheral blood mononuclear cells (PBMCs). We examined how cell-type proportions derived from the transcriptome versus the methylome (DNAm) influence estimates of differentially expressed genes (DEGs) and differentially methylated positions (DMPs).MethodsTranscriptome and DNAm data were obtained from PBMC RNA and DNA of Kenyan children (n = 8) before, during, and 6 weeks following uncomplicated malaria. DEGs and DMPs between time points were detected using cell-type adjusted modeling with Cibersortx or IDOL, respectively.ResultsMost major cell types and principal components had moderate to high correlation between the two deconvolution methods (r = 0.60–0.96). Estimates of cell-type proportions and DEGs or DMPs were largely unaffected by the method, with the greatest discrepancy in the estimation of neutrophils.ConclusionVariation in cell-type proportions is captured similarly by both transcriptomic and methylome deconvolution methods for most major cell types.

Low-level viremia episodes appear to affect the provirus composition of the circulating cellular HIV reservoir during antiretroviral therapy.

Low-level viremia (LLV) ranging from 50 to 1,000 copies/ml is common in most HIV-1-infected patients receiving antiretroviral therapy (ART). However, the source of LLV and the impact of LLV on the HIV-1 reservoir during ART remain uncertain. We hypothesized that LLV may arise from the HIV reservoir and its occurrence affect the composition of the reservoir after LLV episodes. Accordingly, we investigated the genetic linkage of sequences obtained from plasma at LLV and pre-ART time points and from peripheral blood mononuclear cells (PBMCs) at pre-ART, pre-LLV, LLV, and post-LLV time points. We found that LLV sequences were populated with a predominant viral quasispecies that accounted for 67.29%∼100% of all sequences. Two episodes of LLV in subject 1, spaced 6 months apart, appeared to have originated from the stochastic reactivation of latently HIV-1-infected cells. Moreover, 3.77% of pre-ART plasma sequences were identical to 67.29% of LLV-3 plasma sequences in subject 1, suggesting that LLV may have arisen from a subset of cells that were infected before ART was initiated. No direct evidence of sequence linkage was found between LLV viruses and circulating cellular reservoirs in all subjects. The reservoir size, diversity, and divergence of the PBMC DNA did not differ significantly between the pre- and post-LLV sampling points (P > 0.05), but the composition of viral reservoir quasispecies shifted markedly before and after LLV episodes. Indeed, subjects with LLV had a higher total PBMC DNA level, greater viral diversity, a lower proportion of variants with identical sequences detected at two or more time points, and a shorter variant duration during ART compared with subjects without LLV. Overall, our findings suggested that LLV viruses may stem from an unidentified source other than circulating cellular reservoirs. LLV episodes may introduce great complexity into the HIV reservoir, which brings challenges to the development of treatment strategies.

The identification of intact HIV proviral DNA from human cerebrospinal fluid

We evaluated the HIV-1 DNA reservoir in peripheral blood mononuclear cells (PBMC) and cerebrospinal fluid (CSF) in people with HIV (PWH) and associations to cognitive dysfunction. Using the intact proviral DNA assay (IPDA), an emerging technique to identify provirus that may be the source of viral rebound, we assessed HIV DNA in CSF and PBMC in PWH regardless of antiretroviral therapy (ART). CSF was used as a sampling surrogate for the central nervous system (CNS) as opposed to tissue. IDPA results (3′ defective, 5′ defective, and intact HIV DNA) were analyzed by compartment (Wilcoxon signed rank; matched and unmatched pairs). Cognitive performance, measured via a battery of nine neuropsychological (NP) tests, were analyzed for correlation to HIV DNA (Spearman's rho). 11 CSF and 8 PBMC samples from PWH were evaluated both unmatched and matched. Total CSF HIV DNA was detectable in all participants and was significantly higher than in matched PBMCs (p ​= ​0.0039). Intact CSF HIV DNA was detected in 7/11 participants and correlated closely with those in PBMCs but tended to be higher in CSF than in PBMC. CSF HIV DNA did not correlate with global NP performance, but higher values did correlate with worse executive function (p ​= ​0.0440). Intact HIV DNA is frequently present in the CSF of PWH regardless of ART. This further supports the presence of an HIV CNS reservoir and provides a method to study CNS reservoirs during HIV cure studies. Larger studies are needed to evaluate relationships with CNS clinical outcomes.

Abstract 791: Evaluation of BCR spike-in controls using DriverMap adaptive immune receptor (AIR) profiling technology

Abstract The DriverMap™ Adaptive Immune Receptor (AIR) repertoire profiling assay employs multiplex RT-PCR for comprehensive profiling of CDR3 or full-length receptor profiling of all variable heavy and light chains of T-cell receptors (TCR) and B-cell receptors (BCR) from either RNA or DNA. In this study, initiated by a U.S. Food and Drug Administration (FDA) consortium to compare performance of different BCR repertoire profiling technologies, we tested spike-ins in peripheral blood mononuclear cell (PBMC) RNA and DNA control mixtures prepared from nine different B-cell lines. We measured the sensitivity, linearity and accuracy of AIR RNA and DNA BCR profiling for the IGH, IGK and IGL chains of the spike-in controls. Our comprehensive analysis, encompassing CDR3 profiling and full-length receptor profiling (CDR1, CDR2, and CDR3) sequencing, revealed a 100X increase in sensitivity of detection of spike-in controls in RNA compared to the DNA assay. The high sensitivity of the full-length DriverMap AIR RNA assay allowed the detection of controls from all nine cell lines, with 8 out of 9 cell lines detected in the DNA assay, which demonstrates the robustness of the DriverMap AIR profiling technology. This study highlights the remarkable sensitivity of the AIR RNA assay in quantifying transcripts. This approach enables the detection of low-frequency BCR clonotypes, which is particularly advantageous when working with samples containing low numbers of B cells. Furthermore, our AIR DNA assay in combination with spike-in controls offers a quantitative tool for minimal residual disease (MRD) applications, providing accurate insight into cell numbers, which then facilitates tracking clonal expansion of immune cells. Citation Format: Alex Chenchik, Tianbing Liu, Mikhail Makhanov, Dongfang Hu, Khadija Ghias, Paul Diehl. Evaluation of BCR spike-in controls using DriverMap adaptive immune receptor (AIR) profiling technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 791.

COMPARISON OF DNA METHYLATION IN SALIVA, BUFFY COAT AND PBMC SAMPLES IN CATSLIFE

Abstract The design of the Colorado Adoption/Twin Study of Lifespan behavioral development and cognitive aging (CATSLife) allows for separation of genetic and environmental factors affecting health and cognitive aging. Currently, this sample is transitioning through midlife. One goal of this study is to identify epigenetic changes associated with aging, however methylation profiles differ across tissue types. Therefore, we describe here the analysis of DNA methylation in three CATSLife sample types: saliva, buffy coat, and peripheral blood mononuclear cells (PBMCs). Obtaining saliva is least invasive, although it may be less informative about brain and cognitive aging processes. In contrast, while buffy coats and PBMCs may be more informative, obtaining blood is more invasive. Buffy coats are less expensive to obtain than PBMCs, however they contain more cell types which may complicate analysis. Our analyses of 91 participants, suggested that saliva samples were less likely to pass initial quality control (< 96% CpGs detected). Unsurprisingly, the buffy coat and PBMC DNA methylation signatures are more similar to each other (r=0.97) than either is to saliva (rs=0.88—0.92). In initial analyses, each sample type demonstrated expected familial/heritable patterns of similarity across sites (MZ twins > DZ twins > biological & adoptive siblings). Given the additional cost of isolating PBMCs and computational methods to account for complex cell mixtures, the use of buffy coat DNA increases accessibility to analyze epigenetic changes longitudinally, enabling the early identification of epigenetic biomarkers of cognitive decline and the effect of both genes and environments on this process.

Digital PCR Assay for Evaluation of CD19-CAR-T Cells In Vivo Expansion in Aggressive B-Cell Lymphoma: Impact on Clinical Outcome

Background: Chimeric antigen receptor (CAR)-T cells therapy has revolutionized hematological treatment mainly in non-Hodgkin lymphoma (NHL) demonstrating unprecedented clinical responses when directed against CD19 combining the antigen-binding properties of antibodies with the effector functions of T cells. In particular, CAR-T cell peak concentrations and area under the curve in the first 28 days after Axicabtagene Ciloleucel (Axi-cel) infusion have been associated with long-term efficacy. Based on a digital PCR assay for sensitive CAR-T cells detection, we aimed to detect the CAR-T product in patients (pts) and associate its kinetics with patient outcome. Methods: A retrospective analysis was conducted involving 28 patients diagnosed with NHL treated with Axi-cel CAR-T products (2019-2023) in the Department of Hematology of the Clinical University Hospital of Valencia, Spain according to the Declaration of Helsinki ethical criteria. 97 peripheral blood mononuclear cell (PBMC) DNA samples were extracted from 3-12 ml EDTA tubes. We performed serial measurements of CAR-T-cell blood levels (7,14, 28 days post-infusion) using a newly developed axi-cel-specific dPCR assay (Bio-Rad,USA) with QIAcuity Digital PCR System and QIAcuitySoftware for data analysis (QIAGEN, Germany). The peak CAR-T-cell blood concentrations (CAR-T-Cmax) and the cumulative CAR-T-cell levels over the first 28 days in PB by area under the curve (AUC CAR-T) were calculated based on CAR-T dPCR values (copies/μL). Response assessment was performed per institutional practice and based on Lugano Criteria. Statistical analyses were performed using SPSS Software (IBM, Germany, v.22). Logistic regression was used to evaluate the relationship between covariate and outcome. Kruskal-Wallis and Dunn's tests were used when 3 subgroups were involved. Survival analyses were conducted according to CAR-T dPCR values. Results: A total of 28 pts (16 [57.1%] females) who received CAR-T cell therapy were included [25 (89%) DLBCL; 3 (11%) PMBCL]. The patient's best overall response assignment (partial or complete) were observed in twenty-two (78,4%) named as responders (R), otherwise six (21,4%) nonresponders (NR) around day 30 of evaluation. CAR-T-CMax ranged from 2.30 to 176.10 (median, 40.16) CAR-T copies/μL, 7 days later of majority expansion day post infusion: R pts had a higher peak expansion of 66.38 copies/µL compared to 32.43 copies/µL although this difference did not reach statistical significance ( p=018). Expansión was faster in R pts (median 7.64 days pot-infusion) versus NR pts (median 12.83, p=0,10) . Quantitative 7-d CAR-T copies/μL, and AUC value from days 0 to 7, showed statistical significance differences post-infusion between R and NR (52.08±32.48 vs 15.51±20.51, p<0.005; 3603.88±2520.02 vs 1291.74±1948.77, p<0.037). As for response assessment, 7-d CAR-T values were able to successfully predict the development of NR pts with an area under the curve of 0.841 (CI 95% 0.66-1.00), a sensitivity of 0.950 and a specificity of 0.625 (Fig 1.). Eleven pts (39.3%) died at a median of 3.6 months (1.7-6.1 months) post-infusion. For survival analyses, a cut-off value of 18.35 7-d CAR-T copies/μL that maximized the Log-Rank statistic, was selected to divide pts into R and NR categories. Median overall survival (OS) was 23.9 [16.3-NA, 95% CI] months and 13.2 [3.30-3.96, 95% CI] months for R and NR according to CAR-T copies/μL 7 days, respectively (Fig.2). Conclusions: Digital PCR for CAR-T evaluation shows that quantitative CAR-T copies/μL could be a potential predictor of short-term response and survival at first 7 days post-infusion value in pts with DLBCL or PMBCL treated with CAR-T cell therapy.

B-381 A Multiplex Blood-based Assay Targeting DNA methylation in Peripheral Blood Mononuclear Cells Enables Early Detection of Breast Cancer

Abstract Background The immune system can monitor the development of tumors, and the DNA methylation is involved in the body immune response to tumors. Therefore, the DNA from peripheral blood mononuclear cell (PBMC) that carries cancer-specific epigenetic aberrations may serve as a non-invasive marker for early detection of breast cancer (BC). Methods Genome-wide DNA methylation profiling on PBMC DNA from BC patients and healthy controls (Cohort I, n = 80) was performed in the discovery phase by Infinium 850K BeadChips. The candidate CpG sites were selected and validated in a multistep validation phase (Cohort II, n = 200) via pyrosequencing and targeted bisulfite sequencing. After that, the multiplex quantitative methylation-specific PCR assay (mBC-MSP) was established to detect BC, and the performance of this diagnostic method was analyzed in a multicenter cohort (Cohort III, n = 501). Results A total of 289 differentially methylated CpG positions (DMPs) between BC patients and healthy controls were identified. Among them, eight significant DMPs markers were selected and further validated using pyrosequencing and targeted bisulfite sequencing, and four hyper-methylated DMPs (cg18637238, cg16652347, cg13828440, cg11754974) passed the final validation. Then, we developed a mBC-MSP assay based on the four methylated markers. This assay exhibited excellent performance in distinguishing BC patients, with an AUC of 0.925, sensitivity of 83.1%, and specificity of 90.4%. More importantly, compared with CA153, CA125 and CEA, the mBC-MSP assay had higher sensitivity for early-stage BC (stage 0, 88.2% vs 0%, 0%, 5.9%) and minimal tumor with diameter ≤1.5 cm (91.7% vs 0%, 2.1%, 0%). Conclusions The mBC-MSP assay based on the four identified DNA methylation markers from PBMC can serve as a noninvasive, accurate, rapid, and high-throughput method for early diagnosis of BC, and it is more sensitive than traditional tumor markers for detecting early-stage BC and minimal tumor.

The role and mechanism of myeloperoxidase in dermatomyositis

ObjectiveDermatomyositis (DM) is the best known subtype of idiopathic inflammatory myopathies. The hallmarks of DM muscle pathology including microangiopathy, inflammatory infiltration, and perifascicular atrophy. Recent findings have revealed pathogenetic effects of myeloperoxidase (MPO) by causing oxidative damage and regulating abnormal immunity in multiple disease conditions. In this study, we aimed to explore the role of MPO in the pathogenesis of DM. MethodsThe peripheral blood mononuclear cell (PBMC) mRNA expression and DNA methylation of MPO were verified using real-time qPCR and bisulfite pyrosequencing, respectively. Plasma MPO levels were measured with enzyme-linked immunosorbent assay, and their relationships with clinical characteristics were analyzed. The expression and distribution of MPO in muscle were tested by immunofluorescence. Purified human native MPO protein was used to stimulate human dermal microvascular endothelial cells (HDMECs) and skeletal muscle myotubes. The cell viability, tube forming capacity, permeability, adhesion molecule expressions in HDMECs, and atrophy and programmed cell death pathways in myotubes were then observed. ResultsMPO gene methylation was decreased, while mRNA expression and plasma levels were increased in DM. Plasma MPO of DM patients was positively correlated with serum creatine kinase (CK). MPO mainly distributed around endomysia capillaries and perifascicular atrophy in DM muscle biopsies, and was co-localized with CD4+, CD8+ T cells and CD19+ B cells. MPO not only could influence the cell viability, tube forming capacity, permeability and expression of adhesion molecules (including ICAM 1, VCAM 1 and E-selectin) of HDMECs, but also could cause atrophy of myotubes. ConclusionsOur study disclosed, for the first time, that MPO plays an important role in promoting inflammatory infiltration and inducing muscle damage in DM patients. MPO may be a potential biomarker for DM muscle involvement and MPO targeted drugs may be promising in DM treatment.

DNA methylation-based testing in peripheral blood mononuclear cells enables accurate and early detection of colorectal cancer.

An effective blood-based method for the diagnosis of colorectal cancer (CRC) has not yet been developed. Molecular alterations of immune cells occur early in tumorigenesis, providing the theoretical underpinning for early cancer diagnosis based on immune cell profiling. Therefore, we aimed to develop an effective detection method based on peripheral blood mononuclear cells (PBMCs) to improve the diagnosis of CRC. Analysis of the genome-wide methylation landscape of PBMCs from CRC patients and healthy controls by microarray, pyrosequencing, and targeted bisulfite sequencing revealed five DNA methylation markers for CRC diagnosis, especially early-stage CRC (eCRC). A single-tube multiple methylation-specific quantitative PCR assay (multi-msqPCR) for simultaneous detection of five methylation markers was established, which allowed quantitative analysis of samples with as little as 0.1% PBMC DNA and had better discriminative performance than single-molecule detection. Then, a CRC diagnostic model (CDM) based on methylation markers and the multi-msqPCR method was constructed that achieved high accuracy for eCRC (AUC=0.91; sensitivity=81.18%; specificity=89.39%), which was improved compared to CEA (AUC=0.79). The CDM also enabled a high degree of discrimination for advanced adenoma (AA) cases (AUC=0.85, sensitivity=63.04%). Follow-up data also demonstrated that the CDM could identify CRC potential up to 2 years before currently used diagnostic methods. In conclusion, the approach constructed in this study based on PBMC-derived DNA methylation markers and a multi-msqPCR method is a promising and easily implementable diagnostic method for eCRC.

Significant hypomethylation of MMP9 gene promoter in patients with systemic lupus erythematosus.

Scholars are exploring novel diagnostic and prognostic biomarkers with higher sensitivity and specificity for systemic lupus erythematosus (SLE). In this regard, DNA methylation alterations have aroused attention. The association between the dysfunction of MMP9 and TNFAIP3 genes and SLE has been previously demonstrated. Therefore, in this study, we investigated the methylation level of MMP9 and TNFAIP3 promoters in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls. Eighty Iranian SLE patients and 77 healthy individuals were enrolled. The methylation quantification endonuclease-resistant DNA (MethyQESD) method was used to assess methylation levels of MMP9 and TNFAIP3 in extracted DNA of PBMCs. To quantify the diagnostic utility of the promoter methylation level of these genes, the receiver operating characteristic (ROC) curve was constructed. MMP9 promoter was significantly hypomethylated in SLE patients compared with healthy people (p < 0.001), while there was no significant difference in terms of TNFAIP3 promoter methylation levels (p = 0.167). Also, this differential MMP9 methylation was observed in patients with renal involvement and patients without renal involvement (42.07 ± 25.73 vs 56.74 ± 29.71, p = 0.007). ROC analyses indicated that the diagnostic power of the MMP9 promoter methylation level for SLE was 0.839 [95% CI (0.781-0.911)]. Moreover, MMP9 methylation level was negatively correlated with creatinine and anti-dsDNA concentration and positively correlated with C3 and C4 levels. The results of this study highlight the application of MMP9 methylation level in PBMCs of SLE patients as a diagnostic biomarker.

The Effects of a Multi-Ingredient Supplement Containing Wasabia Japonica Extract, Theacrine, and Copper (I) Niacin Chelate on Peripheral Blood Mononuclear Cell DNA Methylation, Transcriptomics, and Sirtuin Activity

Herein, we determined if a multi-ingredient supplement (NAD3; 312 mg of combined Wasabia japonica extract, theacrine, and copper (I)niacin chelate) versus a placebo (CTL) affected peripheral blood mononuclear (PMBC) transcriptomic, DNA methylation, and sirtuin activity profiles in middle-aged adults after 12 weeks of supplementation. Several mRNAs demonstrated interactions (n = 148 at ±1.5-fold change, p &lt; 0.01), and more stringent filtering indicated that 25 mRNAs were upregulated and 29 were downregulated in the NAD3 versus CTL group. Bioinformatics on these 64 mRNAs suggested that DNA conformational alterations may have been promoted with NAD3 supplementation, and this was corroborated with more CpG sites being hypermethylated (p &lt; 0.001) in the CTL versus the NAD3 group when examining pre- to post-intervention changes (369 versus 35). PBMC SIRT activity decreased in CTL participants (p &lt; 0.001), but not in NAD3 participants (p = 0.289), and values at 12 weeks trended higher in NAD3 participants (p = 0.057). Interestingly, the pre- to post- changes in SIRT activity values significantly correlated with changes in PBMC NAD+: NADH values obtained from a previous investigation in these participants (r = 0.534, p = 0.015). In conclusion, the current mRNA and DNA methylation data indirectly suggest that NAD3 supplementation may affect PBMC DNA conformation, while other direct assays suggest that NAD3 supplementation maintains SIRT activity through the potential maintenance of NAD+: NADH levels. However, these results are preliminary due to limited n-sizes and the study being performed in middle-aged adults.

Abstract 2299: Misclassification of clonal hematopoietic mutations as tumor-derived confound accurate ctDNA-response assessment in patients with metastatic non-small-cell lung cancer: analyses from the phase 3 MYSTIC trial

Abstract Introduction: Circulating tumor DNA (ctDNA) can act as a molecular response (MR) biomarker. Non-tumor informed, plasma-only ctDNA assays are an accessible and rapid mechanism to assess ctDNA levels in patients undergoing treatment for lung cancer but may be impacted by clonal hematopoietic (CH) mutations misclassified as tumor derived. Here, we assess the impact of CH on MR measurement in patients receiving durvalumab, with or without tremelimumab, versus chemotherapy in the Phase III MYSTIC trial. Methods: Using a non-tumor informed, mutation based, ctDNA assay that assesses 500-genes (Guardant Health [GH] Omni, subset to the GH360 genomic coordinates [74-genes] for additional analyses) we calculated plasma-only molecular response (MR) based on change in ctDNA levels between baseline and a 4 or 6-week on-treatment timepoint in 713 patients. FoundationOne CDx tissue data subset to GH Omni genomic coordinates enabled “in-silico” tumor-informed MR analysis where only mutations detectable in tumor tissue were followed as response indicators, in 273 of 713 patients. Targeted duplex-sequencing of peripheral blood mononuclear cell (PBMC) DNA was performed to assess the presence of CH mutations misclassified as tumor derived through plasma-only analyses. Results: Plasma-only MR assessment associated with survival outcomes in immunotherapy treatment arms but not the chemotherapy arm; tumor shrinkage on imaging at 6-week radiological evaluation also predicted survival outcomes only in the immunotherapy arms, suggesting that early tumor response is a stronger predictor of outcome for patients treated with immunotherapy. In-silico tumor-informed MR analyses led to an improved association between MR and outcome across all treatment arms and an increased rate in observed ctDNA clearance. Early tumor-informed ctDNA clearance strongly indicated survival benefit in patients receiving durvalumab and tremelimumab. Targeted analyses of mutations categorized as tumor derived in plasma-only analyses, but absent from tumor tissue sequencing analyses in the same patient, revealed that 87/154 (57%) were PBMC-derived highlighting CH as capable of confounding plasma-only analyses. Conclusion: These exploratory analyses from a phase III randomized study support early-ctDNA kinetics as predictive of long-term benefit in metastatic NSCLC patients and support technical optimization of CH-subtraction approaches to maximize utility of ctDNA as a response biomarker in patients with NSCLC. Citation Format: Chris Abbosh, Michael Kuziora, Daniel Stetson, Zhongwu Lai, Yashaswi Shrestha, Paul Labrousse, Xiaojin Shi, Helen Mann, Diana Merino Vega, Matthew D. Hellmann, Solange Peters, J. Carl Barrett, Darren Hodgson. Misclassification of clonal hematopoietic mutations as tumor-derived confound accurate ctDNA-response assessment in patients with metastatic non-small-cell lung cancer: analyses from the phase 3 MYSTIC trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2299.

PD46-01 DETECTION OF INDIVIDUALIZED MUTATIONS AND MONITORING OF POSTOPERATIVE RECURRENCE USING CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyCME1 Apr 2023PD46-01 DETECTION OF INDIVIDUALIZED MUTATIONS AND MONITORING OF POSTOPERATIVE RECURRENCE USING CIRCULATING TUMOR DNA IN PATIENTS WITH UPPER TRACT UROTHELIAL CARCINOMA Daichi Tamura, Masakazu Abe, Daiki Ikarashi, Renpei Kato, Yoichiro Kato, Shigekatsu Maekawa, Mitsugu Kanehira, Ryo Takata, Yutaka Suzuki, Hidewaki Nakagawa, Satoshi Nishizuka, and Wataru Obara Daichi TamuraDaichi Tamura More articles by this author , Masakazu AbeMasakazu Abe More articles by this author , Daiki IkarashiDaiki Ikarashi More articles by this author , Renpei KatoRenpei Kato More articles by this author , Yoichiro KatoYoichiro Kato More articles by this author , Shigekatsu MaekawaShigekatsu Maekawa More articles by this author , Mitsugu KanehiraMitsugu Kanehira More articles by this author , Ryo TakataRyo Takata More articles by this author , Yutaka SuzukiYutaka Suzuki More articles by this author , Hidewaki NakagawaHidewaki Nakagawa More articles by this author , Satoshi NishizukaSatoshi Nishizuka More articles by this author , and Wataru ObaraWataru Obara More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003359.01AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Upper tract urothelial carcinoma (UTUC) is an aggressive disease which have high recurrence after surgery. Current standard methods for the diagnosis and detection of recurrence are invasive or low sensitivity. Circulating tumor DNA (ctDNA) analysis using digital polymerase chain reaction (dPCR) has demonstrated promising results for monitoring as recurrence in several cancers. The aim of this study was to evaluate the validity of plasma and urinary ctDNA as a tumor biomarker for UTUC among a perioperative period. METHODS: This study included 23 patients who underwent radical nephroureterectomy (RNU) diagnosed as UTUC from January 2019 to December 2020. In each patient, whole exome sequencing (WES) of tumor DNA and corresponding peripheral blood mononuclear cell (PBMC) DNA were performed by next generation sequencer (NGS). To investigate ctDNA by dPCR analysis, we selected tumor specific gene mutations included in our original primer-probe library. We also collected plasma and urine ctDNA from each patient. Longitudinal variant allele frequency (VAF) of ctDNA were plot on a pre- and post-operative period. RESULTS: The median age was 71 years old (range 57-82). 13 cases were renal pelvis cancer, and 10 cases were ureteral cancer. Most frequently mutated genes revealed by tumor DNA analysis using NGS were TERT (43%), KMT2D (35%), TTN (30%), FGFR3 (26%), and TP53 (26%). 17 cases (74%) could be performed ctDNA monitoring by our primer-probe library. Tumor specific mutations were detected in preoperative samples in 15 cases. Tumor specific mutations were not detected in two cases who were ypT0 due to neoadjuvant chemotherapy. Six cases experienced recurrence after RNU, five of which showed increase of VAFs of ctDNA. CONCLUSIONS: ctDNA analysis using dPCR could detect tumor specific mutations in majority of patients with UTUC. Individualized ctDNA monitoring could be a useful biomarker for postoperative recurrence. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1169 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daichi Tamura More articles by this author Masakazu Abe More articles by this author Daiki Ikarashi More articles by this author Renpei Kato More articles by this author Yoichiro Kato More articles by this author Shigekatsu Maekawa More articles by this author Mitsugu Kanehira More articles by this author Ryo Takata More articles by this author Yutaka Suzuki More articles by this author Hidewaki Nakagawa More articles by this author Satoshi Nishizuka More articles by this author Wataru Obara More articles by this author Expand All Advertisement PDF downloadLoading ...

New findings on the immune response following corneal transplantation

AbstractWe performed a multi‐centre, cross‐sectional analysis of the current immunological status of human full thickness (FT) and posterior lamellar (PL) corneal transplant (CT) recipients and control subjects using multiple technology platforms. The primary goal of this study was to define pathways associated with adverse immune responses to corneal transplantation and to generate and compare comprehensive, pathway‐based profiles of immune activity in CT acute rejection (AR). The secondary goal was to identify a composite biomarker of CT AR. Clinical details, including a comprehensive clinical examination, and blood samples, enabling analysis of peripheral blood mononuclear cell (PBMC) DNA, RNA and phenotype, were collected from adults with clinically diagnosed AR of FL‐CT (n = 35) and PL‐CT (n = 21) along with Stable CT recipients (n = 177) and adults with non‐transplanted corneal disease (n = 40). For those with AR, additional samples were collected 3 months later. CT acute rejection was clinically diagnosed and defined as precipitates on the corneal graft but not on the peripheral recipient cornea, either scattered or in the form of a Khodadoust line along with an increase in central corneal thickness. Immune cell analysis was performed by whole‐genome microarrays (whole blood) and high dimensional multi‐colour flow cytometry (peripheral blood mononuclear cells). For both, no activation signature was identified within the B cell and T cell repertoire at the time of AR diagnosis. Nonetheless, in FT‐ but not PL‐CT recipients, AR was associated with differences in B cell maturity and regulatory CD4+ T‐cell frequency compared to stable allografts. Our results suggest that, in contrast to solid organ transplants, genetic or cellular assays of peripheral blood are unlikely to be clinically exploitable for prediction or diagnosis of AR. However, further investigation of circulating B cell and T cell subpopulations may provide insights into the regulation of anti‐donor immune response in human CT recipients with differing AR risk.

A Noncoding Germline Variant Creates a Gain-of-Function De Novo Enhancer That up-Regulates IL5 Transcription to Cause Familial Hypereosinophilia, Revealing a Previously Undescribed Mechanism for Human Disease

Introduction: Familial eosinophilia (FE) is a rare inherited condition characterized by lifelong idiopathic hypereosinophilia (absolute eosinophil count >1.5 x 109/L) in multiple family members with or without clinical manifestations. Although the gene underlying FE in one large kindred was mapped to chromosome 5q31-33 using genomewide linkage analysis, targeted sequencing of genes in this region, including interleukin 5 (IL5), was unrevealing. Despite more recent studies demonstrating selective dysregulation of IL5 transcription, the underlying genetic abnormality in FE has remained elusive to date. Methods: Whole genome sequencing (WGS) was performed using peripheral blood mononuclear cell (PBMC) DNA from 5 affected and 1 unaffected family members of the above-described kindred followed by targeted Sanger sequencing of candidate variants. After identifying a single nucleotide variant common to all affected family members in a noncoding region upstream of IL5, RNA sequencing (RNAseq), including long-read PacBio sequencing, was performed to examine IL5 gene expression and to identify IL5 isoforms. To predict potential transcription factor binding sites, we applied motif analyses around the variant and performed chromatin immunoprecipitation (ChIP)-PCR to assess chromatin occupancy of the predicted transcriptional regulators (HMGA1, FOXO3) and of RNA Polymerase II (Poll II). The variant of interest was introduced into a B-lymphoblast cell line (NCI-BL1184) via CRISPR/Cas9, after which clones derived from single cells were compared with respect to IL5 transcription (via quantitative PCR) and chromatin occupancy of HMGA1, FOXO3, and Pol II (by ChIP-PCR). Chromatin marks associated with active gene expression at enhancers (H3K4me1, H3K27Ac) and promoters (H3K4me3) were assessed by ChIP-PCR. Integration of genomewide ChIP-Seq, ATAC-Seq and gene expression analyses are underway to further assess the role of the variant as the primary driver of aberrant IL5 expression. Results: WGS revealed a novel single nucleotide variant in all 5 affected family members located 13.19 kb from the transcription start site (TSS) of the IL5 gene and 250 bp from the TSS of the RAD50 gene. Targeted Sanger sequencing confirmed the presence of this variant in 4/4 affected and 0/4 unaffected family members. Bulk RNA-seq revealed abnormally long transcripts spanning the entire intergenic region between the variant and the IL5 TSS in affected PBMCs, while long-read RNA-seq identified novel full-length IL5 isoforms, including one containing all 5 exons of the IL5 gene. By contrast, RAD50 transcription was unaffected by the variant. In silico motif analysis predicted that the variant generates potential binding sites for transcriptional regulators, including the High Mobility Group A1 (HMGA1) chromatin regulator and the forkhead transcription factor, FOXO3. ChIP-PCR confirmed robust chromatin binding of both HMGA1 and FOXO3 at the variant site in PBMCs from affected family members, but not in PBMCs lacking the variant. Introduction of the single nucleotide variant into B-lymphoblast cells via CRISPR/Cas9 recapitulates the findings in affected family members, with up-regulation of IL5 expression and protein levels and no effects on IL4, IL13, or RAD50 gene expression. In edited lymphoblasts with the variant, HMGA1 chromatin binding and that of H3K4me3/H3K27Ac are enriched in the 200 bp region encompassing the variant, although FOXO3 occupancy was not enriched in this setting nor were there changes in occupancy of control histone H3 in that region. Conclusions: We describe a previously unknown mechanism of human disease: a non-coding single nucleotide germline variant that results in a de novo enhancer and up-regulation of IL5 production in a family with autosomal dominant hypereosinophilia. Orthogonal methods indicate that this variant enables HMGA1 to bind DNA, recruit active histone marks, and induce IL5 expression from a de novo TSS. This finding was validated by CRISPR/Cas9-mediated introduction of the variant into a B cell lymphoblast cell line that does not normally produce IL-5. This mechanism highlights the importance of assessing non-coding regions in the study of Mendelian disorders and may be relevant to other human diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical value of plasma and peripheral blood mononuclear cells Epstein-Barr Virus DNA dynamics on prognosis of allogeneic stem cell transplantation.

The application of intracellular and extracellular Epstein–Barr virus (EBV) DNA in allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been poorly characterized. We conducted a combined prospective-retrospective study of 300 patients who underwent allo-HSCT between 2016 to 2019 in our center and monitored for EBV DNA within the first year after HSCT. Combining the optimal cut-off value of EBV DNA load (7.3×104 copies/106 cells) in peripheral blood mononuclear cells (PBMCs) and qualitative detection in plasma (400 copies/mL) allowed for the better differentiation of EBV-related posttransplant lymphoproliferative disorders (EBV-PTLD), with increased sensitivity (100%) and specificity (86%), and provided the effective risk stratification of EBV DNA level according to their impact on transplant outcomes. By multivariate analysis, patients with intermediate-level of EBV DNA load (low EBV DNA load in PBMCs or high load in PBMCs but negative in plasma) was associated with superior overall survival (HR 1.92, 95% CI 1.03-3.57, p=0.039) and lower transplant-related mortality (HR 3.35, 95% CI 1.31-8.58, p=0.012) compared to those with high-level (high load in PBMCs and positive in plasma). Notably, high EBV-level group had poor reconstitution of CD4+ and CD8+T cells, and both low and high EBV-level groups showed abnormally increase in IL-10 level within one year. Additionally, patients with peak EBV DNA load in PBMCs during 3-12 months had a higher incidence of chronic graft versus host disease (GVHD) than those within 3 months post transplantation (17.4% vs 13.7%, p=0.029). Collectively, EBV DNA in PBMCs can synergistically predict the risk of EBV-PTLD and GVHD. The intermediate-level of EBV DNA presented in plasma and PBMCs might contribute to a better reconstitution of T cells associated with favorable prognosis of allo-HSCT.

Identification of Novel Loci Involved in Adalimumab Response in Crohn's Disease Patients Using Integration of Genome Profiling and Isoform-Level Immune-Cell Deconvoluted Transcriptome Profiling of Colon Tissue.

Crohn’s disease is a consequence of dysregulated inflammatory response to the host’s microbiota. Although anti-TNF treatment improves the quality of the patient’s life, a large proportion of patients lose response to the treatment. The past decade of research has led to a continuum of studies showcasing the heterogeneity of anti-TNF response; thus, the aim of the present study was to dissect transcriptome-wide findings to transcript isoform specific levels and combine the analyses with refined information of immune cell landscapes in colon tissue, and subsequently select promising candidates using gene ontology and genomic integration. We enrolled Slovenian Crohn’s disease patients who were naïve with respect to adalimumab treatment. We performed colon tissue RNA sequencing and peripheral blood mononuclear cell DNA genotyping with a subsequent contemporary integrative approach to combine immune cell deconvoluted isoform transcript specific transcriptome analysis, gene ontology layering and genomic data. We identified nine genes (MACF1, CTSE, HDLBP, HSPA9, HLA-DMB, TAP2, LGMN, ANAPC11, ACP5) with 15 transcripts and 16 variants involved in the adalimumab response. Our study identified loci, some of which were previously shown to contribute to inflammatory bowel disease susceptibility, as novel loci involved in adalimumab response in Crohn’s disease patients.

Identification of an Epigenetic Signature for Coronary Heart Disease in Postmenopausal Women's PBMC DNA.

Menopause is accompanied with an increased risk of cardiovascular disease. DNA methylation may have a significant impact on postmenopausal women's development of coronary heart disease. DNA methylation alterations in peripheral blood mononuclear cells (PBMCs) from women with coronary heart disease and healthy controls were detected using the Illumina Infinium MethylationEPIC BeadChip platform in this work. We employed Sangerbox technology and the GO and KEGG databases to further study the pathogenesis of coronary heart disease in postmenopausal women. After that, we used functional epigenetic module analysis and Cytoscape to remove the hub genes from the protein–protein interaction networks. Five genes (FOXA2, PTRD, CREB1, CTNAP2, and FBN2) were the hub genes. Lipid accumulation, endothelial cell failure, inflammatory responses, monocyte recruitment and aggregation, and other critical biological processes were all influenced by these genes. Finally, we employed methylation-specific PCR to demonstrate that FOXA2 was methylated at a high level in postmenopausal women with coronary heart disease. To better understand coronary heart disease in postmenopausal women's molecular mechanisms, our study examine the major factors contributing to the state of DNA methylation modification, which will help discover novel diagnostic tools and treatment options.

Associations of HIV persistence, cigarette smoking, inflammation, and pulmonary dysfunction in people with HIV on antiretroviral therapy.

We aimed to investigate the relationship between measures of HIV persistence with antiretroviral therapy (ART) and cigarette smoking, systemic markers of inflammation, and pulmonary function.Retrospective study of 82 people with HIV (PWH) on ART for a median of 6.9 years (5.6–7.8) and plasma HIV RNA levels <50 copies/mL.HIV DNA and cell-associated HIV RNA (CA-RNA) were measured in peripheral blood mononuclear cells (PBMC) and plasma HIV RNA was measured by single-copy assay (SCA). Plasma levels of 17 inflammatory mediators were measured by Bio-Plex, and standard pulmonary function tests (PFT) were performed in all participants.Median age was 52 years and 41% were women. Most had preserved CD4+ T cell counts (median (IQR) 580 (361–895) cells/mm3). Median plasma HIV RNA was 1.3 (0.7–4.6) copies/mL, and median levels of HIV DNA and CA-RNA in PBMC were 346 (140–541) copies and 19 (3.7–49) copies per 1 million PBMC, respectively. HIV DNA was higher in smokers than in nonsmokers (R = 0.3, P < 0.05), and smoking pack-years positively correlated with HIV DNA and CA-RNA (R = 0.3, P < 0.05 and R = 0.4, P < 0.01, respectively). HIV DNA, CA-RNA, and plasma HIV RNA were not significantly associated with any measure of pulmonary function or inflammation.Cigarette smoking was associated with HIV DNA and CA-RNA levels in blood, but measures of HIV persistence were not associated with pulmonary function or inflammation.