Peripheral Blood Cell DNA Research Articles

Peripheral Blood Cell DNA Methylation Can Predict Steroid Response in Nephrotic Syndrome

Peripheral Blood Cell DNA Methylation Can Predict Steroid Response in Nephrotic Syndrome

Evaluation of cytotoxicity, acute toxicity, genotoxicity and antioxidant and antigenotoxicity activities of the sarcotesta fraction of punica granatum L. rich in lectin (PgTel)

ABSTRACT Punica granatum, popularly known as pomegranate, is a fruit tree with wide worldwide distribution, containing numerous phytochemicals of great medicinal value. The aim of the present study was to determine the phytochemical profile and antioxidant potential of a protein fraction (PF) derived from P. granatum sarcotesta which is rich in lectin. In addition, the acute oral toxicity, genotoxicity and antigenotoxicity of this protein fraction (PF) from P. granatum sarcotesta was measured. The phytochemical profile of PF was determined using HPLC. The in vitro antioxidant effect was assessed using the methods of total antioxidant capacity (TAC) and DPPH and ABTS+ radical scavenging. Acute oral toxicity was determined in female Swiss mice administered a single dose of 2000 mg/kg. This PF was examined for genotoxicity and antigenotoxicity at doses of 500, 1000 and 2000 mg/kg, utilizing mouse peripheral blood cells. Phytochemical characterization detected a high content of ellagic acid and antioxidant capacity similar to that of ascorbic acid (positive control). PF was not toxic (LD50 >2000 mg/kg) and did not exert a genotoxic effect in mice. PF protected the DNA of peripheral blood cells against damage induced by cyclophosphamide. In conclusion, this PF fraction exhibited significant antioxidant activity without initiating toxic or genotoxic responses in mice.

Mosaic loss of Y chromosome in monocytes is associated with lower survival after transcatheter aortic valve replacement.

Mosaic loss of Y chromosome (LOY) in blood cells is the most common acquired mutation, increases with age, and is related to cardiovascular disease. Loss of Y chromosome induces cardiac fibrosis in murine experiments mimicking the consequences of aortic valve stenosis, the prototypical age-related disease. Cardiac fibrosis is the major determinant of mortality even after transcatheter aortic valve replacement (TAVR). It was hypothesized that LOY affects long-term outcome in men undergoing TAVR. Using digital PCR in DNA of peripheral blood cells, LOY (Y/X ratio) was assessed by targeting a 6 bp sequence difference between AMELX and AMELY genes using TaqMan. The genetic signature of monocytes lacking the Y chromosome was deciphered by scRNAseq. In 362 men with advanced aortic valve stenosis undergoing successful TAVR, LOY ranged from -4% to 83.4%, and was >10% in 48% of patients. Three-year mortality increased with LOY. Receiver operating characteristic (ROC) curve analysis revealed an optimal cut-off of LOY >17% to predict mortality. In multivariate analysis, LOY remained a significant (P < 0.001) independent predictor of death during follow-up. scRNAseq disclosed a pro-fibrotic gene signature with LOY monocytes displaying increased expression of transforming growth factor (TGF) β-associated signaling, while expression of TGFβ-inhibiting pathways was down-regulated. This is the first study to demonstrate that LOY in blood cells is associated with profoundly impaired long-term survival even after successful TAVR. Mechanistically, the pro-fibrotic gene signature sensitizing the patient-derived circulating LOY monocytes for the TGFβ signaling pathways supports a prominent role of cardiac fibrosis in contributing to the effects of LOY observed in men undergoing TAVR.

Differences in Peripheral Blood Cell DNA Methylation Between Nephrotic Syndrome Subgroups

Differences in Peripheral Blood Cell DNA Methylation Between Nephrotic Syndrome Subgroups

OS05.6.A Modification of the tumor microenvironment in patients with glioblastoma using autologous, genetically modified, hematopoietic stem cell-based therapy: the TEM-GBM STUDY (NCT03866109)

Abstract Background Bone marrow-derived macrophages account for substantial GBM tumor volume and contribute to the local inflammatory tumor microenvironment, disease progression and treatment response. Material and Methods We have developed a genetically modified, autologous hematopoietic stem cell-based platform designed to deliver Interferon-alpha (IFNa), thanks to a transcriptional and post-transcriptional control mechanism mediated by miRNA target sequences, specifically into the tumor microenvironment via Tie-2 expressing monocytes (Temferon). Results As of Feb 2022, 3 escalating doses of Temferon (from 0.5 to 2.0x106/kg) were tested across 15 patients with newly diagnosed, unmethylated MGMT glioblastoma (GBM) assigned to 5 cohorts. The duration of follow-up from surgery is 6 - 28 mo (2 - 25 mo after Temferon). To date, no dose limiting toxicities have been identified. As expected, one month after the administration of the highest tested dose, the hematopoietic system of Temferon-treated patients was composed of up to 30% of CD14+ genetically modified cells, as determined by the presence of vector genomes in the DNA in peripheral blood and bone marrow cells. Temferon-derived progeny persisted, albeit at lower levels, up to 18 months (longest time of analysis). Despite the substantial proportion of engineered cells, very low median concentrations of IFNα were detected in the plasma (D+30, 5.9; D+90, 8.8pg/mL) and in the CSF (D+30, 1.5; D+90, 2.4pg/mL), indicating tight regulation of transgene expression. SAEs were mostly attributed to conditioning chemotherapy (e.g. infections) or disease progression (e.g. seizures). 1 SUSAR (persistent GGT elevation) has occurred. Median OS is 15 mo from surgery (range 6.1-28.4 mo; 10.8 mo post Temferon). Of the 15 pts treated so far, 4 pts belonging to low dose cohorts underwent 2nd surgery. Homing of transduced cells from BM to the tumor site was demonstrated by the presence of gene-marked cells in the specimens collected from 3 of the 4 analyzed pts. Single-cell RNA seq performed on CD45+ cells purified from the TME of Temferon-treated pts compared to recurrent tumors belonging to GBM pts treated as per the current standard of care, highlighted a Temferon signature defined by the induction of markers of IFNa responses and macrophage repolarization. Potential long-term benefit with Temferon was identified in a patient from cohort 3, who had disease progression at D+120 with two distant enhancing lesions, and increased tumor necrosis. One year following Temferon, with no 2nd line therapy added, there was approximately 40% reduction in enhancing tumor volume compared to D+180 with a stable clinical and imaging picture thereafter. Conclusion The results provide initial evidence of Temferon’s potential to modulate the TME of GBM patients, and anecdotal evidence for long lasting effects of Temferon in prevention of disease progression.

PERIPHERAL BLOOD DNA METHYLATION-BASED MACHINE LEARNING MODELS ARE PREDICTIVE OF FUTURE KNEE OA PROGRESSION: BIOSPECIMENS AND DATA FROM THE OSTEOARTHRITIS INITIATIVE AND JOHNSTON COUNTY OSTEOARTHRITIS PROJECT

Purpose: Knee osteoarthritis (OA) is a heterogeneous disease characterized by a variety of clinical and molecular phenotypes, for which there exist no widely-available biomarkers. We have previously published a pilot analysis of baseline peripheral blood cell DNA methylation patterns from a small group of patients from the Osteoarthritis Initiative (OAI), demonstrating that methylation-based models could distinguish future radiographic progressors from nonprogressors. In the current study, we apply this method to the FNIH OsteoArthritis Biomarkers Consortium (OABC) subcohort of the OAI that included pain-, radiographic-, and dual- (both pain and radiographic) progressors compared to nonprogressors and to similarly constructed groups in the Johnston County Osteoarthritis Project (JoCoOA) and an independent OAI cohort from our prior work.

TEM-GBM: An Open-Label, Phase I/IIa Dose-Escalation Study Evaluating the Safety and Efficacy of Genetically Modified Tie-2 Expressing Monocytes to Deliver IFN-α within Glioblastoma Tumor Microenvironment

Background: We developed a macrophage-based treatment relying on ex vivo transduction of autologous hematopoietic stem and progenitor cells (HSPC) to express immune-payloads within the TME. Our ATMP (Temferon) targets IFN-a, an immune-modulatory molecule counteracting also neo-angiogenesis and tumor growth, to a subset of Tie2-expressing, tumor-infiltrating macrophages known as TEMs.Materials and Methods: TEM-GBM is an open-label, Phase I/IIa dose-escalation study evaluating safety and efficacy of Temferon in up to 21 newly diagnosed glioblastoma patients with unmethylated MGMT promoter. Key eligibility criteria include age 18-70 years, ECOG 0-1 and KPS >70%, and adequate cardiac, renal, hepatic and pulmonary function. Important exclusion criteria include the presence of active autoimmune disease or receipt of any oral or parenteral chemotherapy or immunotherapy within 2 years of screening. Autologous CD34+ HSPC are mobilized with lenograstim and plerixafor, collected by apheresis, purified and transduced ex vivo with a 3 rd generation lentiviral vector encoding for IFN-a2. Transgene expression is confined to TEMs by the Tie2 promoter and post-transcriptional regulation by microRNA-126 thus achieving tumor specificity. The study evaluates safety and biological activity of Temferon in 7 cohorts of three patients each, where escalating doses of Temferon are co-administered with a fixed CD34+ cell dose of non-manipulated supporter cells following a sub-myeloablative conditioning regimen (Thiotepa + BCNU or + Busulfan).The primary endpoints for this study are: •Engraftment of Temferon over the first 90 Days•The proportion of patients achieving hematologic recovery by Day +30 from ASCT•Short-term tolerability of Temferon; stable blood counts and absence of cytopenias, absence of significant organ toxicities (> grade 2); absence of Replication Competent LentivirusThe figure below reports the TEM-GBM study design.Results: As of 28th June 2021, 18 patients have been enrolled; 15 received Temferon (D+0) with follow-up of 30 - 697 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil and platelet engraftment occurred at D+13 and D+12 for patients in cohort 1-3 and D+16 and D+15 for patients assigned to cohort 4 and 5, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted subsequently, albeit at lower levels (up to 18 months). Very low concentrations of IFNa were detected in the plasma (average 7.8 pg/ml at D+30; baseline < LLOQ) and in the cerebrospinal fluid (average 1.6 pg/ml at D+30; baseline < LLOQ), suggesting tight regulation of transgene expression. Seven deaths occurred: six at D+241, +322, +340, +402, +478, +646 after Temferon administration due to disease progression, and one at D+60 due to complications following the conditioning regimen. Nine patients had progressive disease (PD; range D-12 to +239). SAEs include infections, venous thromboembolism, brain abscess, hemiparesis, GGT elevation and poor performance status compatible with autologous stem cell transplantation, concomitant medications and PD. Four patients underwent second surgery. These recurrent tumors had gene-marked cells present and increased expression of IFN-responsive gene signatures compared to diagnosis, indicative of local IFNa release by TEMs. In one patient, a stable lesion (as defined by MRI) had a higher proportion of T cells and TEMs within the myeloid infiltrate and an increased IFN-response signature than in a progressing lesion. The T-cell immune repertoire changed with evidence for expansion of tumor-associated clones. Tumor microenvironment characterization by scRNA and TCR sequencing is ongoing.Conclusion: These interim results show that Temferon is generally well tolerated by patients, with no dose limiting toxicities identified to date. The results provide initial evidence of Temferon's potential to activate the immune system and reprogram the tumor microenvironment (TME), as predicted by preclinical studies. [Display omitted] DisclosuresNaldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

Associations of maternal exposure to 41 metals/metalloids during early pregnancy with the risk of spontaneous preterm birth: Does oxidative stress or DNA methylation play a crucial role?

BackgroundFew studies have explored the effects of multiple types of metals/metalloids on spontaneous preterm birth (SPB). A nested case–control study was conducted in Shanxi Province to investigate the associations between maternal exposure to 41 metals/metalloids during early pregnancy and the risk of SPB, and to clarify the underlying mechanisms of oxidative stress and DNA methylation. MethodsA total of 74 controls with full-term delivery and 74 cases with SPB were included in the nested case–control study. The metals/metalloids in serum and the DNA adducts in peripheral blood cell DNA were determined using ICP-MS and UPLC-QqQ-MS/MS, respectively. Unconditional logistic regression models were employed to estimate the associations of the risk of SPB with the metal concentrations, as well as with the levels of oxidative stress/DNA methylation. In addition, linear regression models were used to investigate the associations between the metal/metalloid concentrations and the levels of oxidative stress/DNA methylation. ResultsAfter adjusting for potential confounders, the concentrations of Mn, Fe, Cu, Nd, Hg, and Pb in maternal serum during early pregnancy were positively associated with the risk of SPB. Compared with the lowest levels (Quartile 1) of Mn, Fe, Cu, Nd, Hg, and Pb, the odds ratios of SPB increased to 5.21 (95% CI: 1.63, 16.68), 3.47 (95% CI: 1.07, 11.21), 16.23 (95% CI: 3.86, 68.18), 10.54 (95% CI: 2.79, 39.86), 5.88 (95% CI: 1.72, 20.11), and 4.09 (95% CI: 1.31, 12.77) in the highest levels (Quartile 4), respectively. A significant increase in 8-OHdG was associated with the increased exposure to Fe, Pr, Eu, Er, and Lu. The levels of 5-MdC, 5-HmdC, and N6-MdA—the indicators of DNA methylation—were associated with exposure to multiple metals/metalloids. However, no significant associations were observed between the levels of oxidative stress or DNA methylation and the risk of SPB. ConclusionsExposure to multiple types of metals/metalloids during early pregnancy is positively associated with the risk of SPB. Oxidative stress and DNA methylation are significantly associated with exposure to multiple metals/metalloids. Systemic oxidative stress and DNA methylation have not been proven to be the mediating mechanisms of metals increasing the risk of SPB.

379TiP TEM-GBM: A phase I-IIa clinical study of genetically modified Tie-2-expressing monocytes in patients with glioblastoma multiforme

We developed a cell-based treatment, Temferon, relying on ex-vivo transduction of autologous HSPCs to express IFNa within the tumor microenvironment by Tie-2-expressing macrophages (TEMs). As of 8th April 2021, 18 patients have been enrolled; 12 received Temferon (D+0) with follow-up of 42 – 640 days. There was rapid engraftment and hematological recovery after the conditioning regimen. Median neutrophil & platelet engraftment occurred at D+13 and D+12, respectively. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA of peripheral blood and bone marrow cells, were found within 14 days post treatment and persisted albeit at lower levels up to 18 months.

Clonal Hematopoiesis of Indeterminate Potential (CHIP) in Patients with Coronary Artery Disease and in Centenarians. Further Clues Linking Chip with Cardiovascular Risk

Background. Age-related clonal hematopoiesis of indeterminate potential (CHIP), a relatively common condition in elderly people, has been associated with an increased risk of hematological neoplasms, and, to a greater extent, of overall mortality. Earlier this year, Jaiswal and colleagues have suggested that excess mortality in subjects with CHIP is related to an increased risk of cardiovascular diseases, and provided a convincing model linking TET2 mutations to a pro-inflammatory state favoring the development of atherosclerotic plaques.Aims. We sought to replicate the recent findings of an increased prevalence of CHIP in patients with Coronary Artery Disease (CAD), by studying patients enrolled in the Verona Heart Study. We were also interested in studying a group of selected ultra-centenarians (mean age 105.6 ± 2.0 years), as the prevalence of CHIP has been previously reported to increase linearly with age until near 90 years, but no data have been produced until now in such a peculiar population of “super-controls”.Methods. We analyzed whole-exome sequencing (WES) data from peripheral-blood cells DNA in 99 patients with angiographically proven severe CAD, and 79 centenarians. The mean coverage varied from 30x to 100x (maximum in centenarians). We focused on somatic mutations predicted to alter the protein function in 6 genes (TET2, ASXL1, DNMT3A, JAK2, PPM1D, TP53) ,known to be key players in the development of CHIP.Results. The prevalence of CHIP in CAD patients was 18.2%, a figure reproducing the increased prevalence in other CAD populations recently reported by Jaiswal and colleagues. By contrast, the prevalence of CHIP in centenarians was only 2.5%, a figure in clear countertrend with respect to the linear increase of CHIP observed until the age of 90 years in previous studies. This suggested a kind of “survival bias” indirectly supporting the pathogenic role of age-related CHIP. Interestingly, the majority (85%) of the driver mutations in CAD patients occurred in TET2, consistently with the pro-atherosclerotic role of this gene in mouse models.Conclusions. Our results add further to the recent hypothesis that links CHIP to an increased risk of cardiovascular disease. DisclosuresMartinelli:CELGENE: Consultancy; PFIZER: Consultancy; ROCHE: Consultancy; JOHNSON & JOHNSON: Consultancy; ARIAD/INCYTE: Consultancy; AMGEN: Consultancy.

Subclinical Cytomegalovirus and Epstein-Barr Virus Shedding Is Associated with Increasing HIV DNA Molecular Diversity in Peripheral Blood during Suppressive Antiretroviral Therapy.

Cytomegalovirus (CMV) almost universally infects persons with HIV (PWH), and it is a driver of persistent inflammation and HIV persistence. The mechanisms underlying the association between CMV (and possibly other herpesviruses) and HIV persistence are unclear. Serially collected blood samples were obtained from men who have sex with men (MSM) who started antiretroviral therapy (ART) within 1 year of their estimated date of HIV infection (EDI). Total CMV and Epstein-Barr virus (EBV) DNA were quantified in peripheral blood mononuclear cells by droplet digital PCR (ddPCR). Deep sequencing of the HIV DNA partial env gene was performed, and the dynamics of viral diversity over time were analyzed in relation to CMV and EBV shedding status. In total, 37 MSM PWH were included and followed for a median of 23 months (IQR, 22 to 28). Participants started ART within a median of 3.1 months (IQR, 1.5 to 6.5) after EDI and remained virally suppressed thereafter. A total of 18 participants (48.6%) were classified as high EBV shedders, while 19 (51.4%) were classified as CMV shedders. In longitudinal analyses, normalized molecular diversity levels tended to increase over time among participants with detectable CMV and high EBV DNA (0.03 ± 0.02, P = 0.08), while they significantly declined among participants with no/low viral shedding (-0.04 ± 0.02, P = 0.047, interaction P < 0.01). Subclinical CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART. Whether persistent CMV/EBV replication could be targeted as a strategy to reduce the size of the latent HIV reservoir is an avenue that should be explored.IMPORTANCE As part of this study, we evaluated the molecular characteristics of the HIV DNA reservoir over time during antiretroviral treatment (ART) in relation to those of other chronic viral infections (i.e., cytomegalovirus [CMV] and Epstein-Barr virus [EBV]). We demonstrated that the presence of CMV and high-level EBV DNA in peripheral blood cells was associated with changes in HIV DNA molecular diversity. Specifically, HIV DNA molecular diversity increased over time among participants with detectable CMV and high-level EBV DNA, while it significantly declined among participants with no/low viral shedding. Although the current study design does not allow causality to be inferred, it does support the theory that persistent CMV and EBV shedding could contribute to the dynamics of the HIV DNA reservoir during suppressive ART, even when ART is initiated during the earliest phases of HIV infection.

Clinical significance of clonal hematopoiesis in the interpretation of blood liquid biopsy.

As the use of next‐generation sequencing (NGS) for plasma cell‐free DNA (cfDNA) continues to expand in clinical settings, accurate identification of circulating tumor DNA mutations is important to validate its use in the clinical management for cancer patients. Here, we aimed to characterize mutations including clonal hematopoiesis (CH)‐related mutations in plasma cfDNA and tumor tissues using the same ultradeep NGS assay and evaluate the clinical significance of CH‐related mutations on the interpretation of liquid biopsy results. Ultradeep targeted NGS using Oncomine Pan‐Cancer Panel was performed on matched surgically resected tumor tissues, peripheral blood cells (PBCs), and 120 plasma cfDNA samples from 38 colorectal cancer patients. The clinical significance of the CH‐related mutations in plasma cfDNA was evaluated by longitudinal monitoring of the postoperative plasma samples. Among the 38 patients, 74 nonsynonymous mutations were identified from tumor tissues and 64 mutations from the preoperative plasma samples. Eleven (17%) of the 64 mutations identified in plasma cfDNA were also detected in PBC DNA and were identified to be CH‐related mutations. Overall, 11 of 38 (29%) patients in this cohort harbored at least one CH‐related mutation in plasma cfDNA. These CH‐related mutations were continuously detected in subsequent postoperative plasma samples from three patients which could be misinterpreted as the presence of residual disease or as lack of treatment response. Our results indicated that it is essential to integrate the mutational information of PBCs to differentiate tumor‐derived from CH‐related mutations in liquid biopsy analysis. This would prevent the misinterpretation of results to avoid misinformed clinical management for cancer patients.

Epigenetic mechanism in search for the pathomechanism of diabetic neuropathy development in diabetes mellitus type 1 (T1DM).

The aim of this study was to check the hypothesis concerning the crucial role of DNA methylation (one of the epigenetic mechanisms) within selected genes related to the destruction and regeneration of neural cells and its input in the pathogenesis of diabetic neuropathy, using a model of the DNA in peripheral blood cells. A cross-sectional, case-control study was conducted, consisting of 24 adult Type 1 Diabetes Melitus (T1DM) patients with autonomic neuropathy (CAN), 25 T1DM patients without neuropathy and 25 matched, healthy adults acting as a control (Ctrl). The Ewing's tests, using the ProSciCard apparatus (Mewicon CATEEM-Tec GmbH), was employed to assess the severity of the patients' symptoms of autonomic neuropathy. For DNA methylation analysis, DNA material of each sample DNA after bisulfite conversion was used for the hybridization of BeadChips (Infinium Methylation EPIC Kit, Illumina), and imaged on the Illumina HiScan. The changes in the expression of selected genes were examined using real-time PCR. Probes were labeled using fluorescein amidite, FAM (Thermo Fisher Scientific). Amplification was performed using the continuous fluorescence detection 7900 HT Fast Real-Time PCR system (Thermo Fisher Scientific). The expression ratio of the target mRNA was normalized to the level of 18s RNA and compared with the control. Statistical analysis was performed using Statistica version 13.1. The statistically significant results were recognized, with a value of p < 0.05. Clinical analysis of the investigated groups revealed a significantly higher percentage of personal insulin pump users in the group without neuropathy. The glucose metabolic control, based on the HbA1c level analysis, was also significantly better in T1DM patients without CAN. The Bumphunter method for DNA methylation analysis showed statistically significant regions related to the genes involved in nerve regeneration ninjurin 2 (NINJ2) and functionality (BR serine/threonine kinase 2 BRSK2, claudin 4 CLDN4). When compared with T1DM patients without neuropathy, T1DM patients with neuropathy showed significantly increased methylation in the first NINJ2 axon, and a lower level of DNA methylation in the region of the first intron of BRSK2, as well as the CLDN4 5'UTR regions. The qRT-PCR results confirmed the decreased expression of NINJ2 and CLDN4 genes in patients with T1DM with CAN. The different DNA methylation profiles, correlating with the expression of genes related to nervous tissue development and regeneration in patients with T1DM with autonomic neuropathy provide evidence for the role of epigenetic mechanisms promoting the development of CAN, a chronic complication of T1DM.

PF535 CLINICAL AND MOLECULAR CHARACTERISTICS OF SRSF2‐MUTATED NEOPLASMS

Background:Splicing factors are the most frequently mutated genes in myeloid neoplasms. SF3B1‐mutated myelodysplastic syndromes (MDS) have been recently recognized as a distinct subtype strongly associated with ring sideroblasts. Conversely, SRSF2 has been found to be recurrently mutated in chronic myelomonocytic leukemia (CMML), and predicting worse prognosis in MDS, myelofibrosis (MF) and acute myeloid leukemia (AML).Aims:Hence, SRSF2‐mutated neoplasms constitute an heterogenous molecular‐defined subgroup, whose clinical characteristics have not been systematically characterized. We aimed at a comprehensive characterization of clinical features and co‐mutational determinants of SRSF2‐mutated neoplasms across WHO classification boundaries.Methods:We analyzed clinical and molecular characteristics of SRSF2‐mutated myeloid neoplasms diagnosed at 4 referral centers. All clinical information was prospectively annotated in clinical chart records and a common panel of 23 recurrently mutated genes in myeloid neoplasms were analysed using Illumina HiSeq sequencing of peripheral blood cell DNA. Multivariate analyses (MVA) accounted for left censoring at mutation assessment time and right‐censoring in case of disease‐modifying treatments.Results:Overall, 279 patients carrying SRSF2‐mutation were identified, including 84 MDS (49 blast excess, 21 multi‐lineage dysplasia, 7 ring sideroblasts, 7 unclassified), 76 MPN (64 primary‐MF [PMF], 5 secondary‐MF, 3 polycytemia vera, 2 essential thrombocytemia, 2 chronic neutrophilic leukemia), 71 MDS/MPN (59 CMML, 12 MDS/MPN unclassified), 47 AML (38 with MDS‐related changes, 9 NOS), 1 blastic plasmacytoid dendritic cell neoplasm. No difference in age was found between WHO subgroups. Male/female ratio was 3.3. Median overall survival (mOS) was 27.3 months (95%CI: 19.9–34.7). The median number of co‐mutated genes was 3 (range 0–7). SRSF2 mutation was the unique driver mutation detected in 13 cases. The most frequently co‐mutated genes were ASXL1 (38%), TET2 (35%), JAK2 (23%), RUNX1 (17%), IDH2 (15%), CBL (10%), STAG2 (10%), NRAS (8%). We used sequencing data to identify the order of acquisition of SRSF2 mutations in double‐mutant patients and found that SRSF2 mutation was acquired first in 80 (28.7%) cases, sub‐clonal in 111 (39.8%). Mutational order could not be assessed in 88 (31.5%) cases. In MVA, AML phenotype was independently predicted by RUNX1 (OR 4.2, p &lt; .001), IDH2 (OR 4.3, p = .001), STAG2 mutation (OR 3.2, p = .02). Similarly, co‐occurring mutation in JAK2 (OR 77.5, p &lt; .001), MPL (OR 194, p &lt; .001) and ASXL1 (OR 3.1, p = .01) predicted MPN phenotype. MDS/MPN was predicted by TET2 (OR 7.2, p &lt; .001), SETBP1 (OR 4.3, p = .02) mutation and SRSF2‐first mutation (OR 3.1, P = .004). Factors predicting OS were blast count (p &lt; .001), white blood cells (p = .009), TET2 (HR. 54, p &lt; .001), IDH2 (HR. 5, p = .021), NRAS (HR 2.23, P = .017) mutation. Notably, fibrosis, cytogenetics, WHO categories were not associated with different survival in MVA.Summary/Conclusion:In conclusion, SRSF2‐mutated myeloid neoplasms are a subset of myeloid neoplasms characterized by bone marrow dysplasia and overall dismal prognosis, whose fibrotic or proliferative phenotype results from a restricted co‐mutation pattern. Among MPN, SRSF2 mutation is almost invariably associated with MF, further supporting the concept that PMF is an MDS/MPN disorder. Co‐mutational pattern is the best predictor of clinical phenotype, suggesting that recognition of a distinct entity with a unique molecular basis may significantly improve classification and treatment of patients with these disorders.

Significantly altered peripheral blood cell DNA methylation profile as a result of immediate effect of metformin use in healthy individuals

BackgroundMetformin is a widely prescribed antihyperglycemic agent that has been also associated with multiple therapeutic effects in various diseases, including several types of malignancies. There is growing evidence regarding the contribution of the epigenetic mechanisms in reaching metformin’s therapeutic goals; however, the effect of metformin on human cells in vivo is not comprehensively studied. The aim of our study was to examine metformin-induced alterations of DNA methylation profiles in white blood cells of healthy volunteers, employing a longitudinal study design.ResultsTwelve healthy metformin-naïve individuals where enrolled in the study. Genome-wide DNA methylation pattern was estimated at baseline, 10 h and 7 days after the start of metformin administration. The whole-genome DNA methylation analysis in total revealed 125 differentially methylated CpGs, of which 11 CpGs and their associated genes with the most consistent changes in the DNA methylation profile were selected: POFUT2, CAMKK1, EML3, KIAA1614, UPF1, MUC4, LOC727982, SIX3, ADAM8, SNORD12B, VPS8, and several differentially methylated regions as novel potential epigenetic targets of metformin. The main functions of the majority of top-ranked differentially methylated loci and their representative cell signaling pathways were linked to the well-known metformin therapy targets: regulatory processes of energy homeostasis, inflammatory responses, tumorigenesis, and neurodegenerative diseases.ConclusionsHere we demonstrate for the first time the immediate effect of short-term metformin administration at therapeutic doses on epigenetic regulation in human white blood cells. These findings suggest the DNA methylation process as one of the mechanisms involved in the action of metformin, thereby revealing novel targets and directions of the molecular mechanisms underlying the various beneficial effects of metformin.Trial registrationEU Clinical Trials Register, 2016-001092-74. Registered 23 March 2017, https://www.clinicaltrialsregister.eu/ctr-search/trial/2016-001092-74/LV.

False-Positive Plasma Genotyping Due to Clonal Hematopoiesis.

Purpose: Plasma cell-free DNA (cfDNA) genotyping is increasingly used in cancer care, but assay accuracy has been debated. Because most cfDNA is derived from peripheral blood cells (PBC), we hypothesized that nonmalignant mutations harbored by hematopoietic cells (clonal hematopoiesis, CH) could be a cause of false-positive plasma genotyping.Experimental Design: We identified patients with advanced non-small cell lung cancer (NSCLC) with KRAS, JAK2, or TP53 mutations identified in cfDNA. With consent, PBC DNA was tested using droplet digital PCR (ddPCR) or next-generation sequencing (NGS) to test for CH-derived mutations.Results: We first studied plasma ddPCR results from 58 patients with EGFR-mutant NSCLC. Two had KRAS G12X detected in cfDNA, and both were present in PBC, including one where the KRAS mutation was detected serially for 20 months. We then studied 143 plasma NGS results from 122 patients with NSCLC and identified 5 JAK2 V617F mutations derived from PBC. In addition, 108 TP53 mutations were detected in cfDNA; for 33 of the TP53 mutations, PBC and tumor NGS were available for comparison, and 5 were present in PBC but absent in tumor, consistent with CH.Conclusions: We find that most JAK2 mutations, some TP53 mutations, and rare KRAS mutations detected in cfDNA are derived from CH not tumor. Clinicians ordering plasma genotyping must be prepared for the possibility that mutations detected in plasma, particularly in genes mutated in CH, may not represent true tumor genotype. Efforts to use plasma genotyping for cancer detection may need paired PBC genotyping so that CH-derived mutations are not misdiagnosed as occult malignancy. Clin Cancer Res; 24(18); 4437-43. ©2018 AACRSee related commentary by Bauml and Levy, p. 4352.

Abstract 1229: Clonal hematopoiesis of indeterminate potential (CHIP), centenarians and age-related cardiovascular risk: Is TET2 the culprit

Abstract Background. Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related hematological condition, that has been associated with an increased risk of hematological malignancies and of overall mortality. This excess mortality in subjects with CHIP seems to be related to an increased risk of cardiovascular atherosclerotic diseases (Jaiswal S, NEJM 2014). Our aim was to analyze the prevalence of CHIP in patients with Coronary Artery Disease (CAD) and in a group of selected ultra-centenarians ((age &amp;gt;105 years), since no data hasve been produced until now in such a peculiar population of “super-controls” (Garagnani P, Aging 2013). Methods. We performed whole-exome sequencing (WES) from peripheral-blood cells DNA in 99 patients with angiographically proven severe CAD from the Verona Heart Study (Girelli D, NEJM 2000;), and 79 semisupercentenarians (age &amp;gt;105 years). The mean coverage varied from 30x to 100x (higher in centenarians). We analyzed somatic mutations predicted to alter function in 6 key genes (TET2, ASXL1, DNMT3A, JAK2, PPM1D, TP53), selected as the most frequently detected in CHIP according to literature. Results. The prevalence of CHIP in CAD patients was 18.2%, similarly to the increased prevalence detected in other CAD populations (Jaiswal S, NEJM 2017). On the other hand, CHIP was rare in centenarians (prevalence of 2.5%), a result that contradicts the expected exponential increase of CHIP observed after the age of 70 years in previous studies. This result suggested a kind of “survival bias”, that indirectly supports the pathogenic role of age-related CHIP. An utterly fascinating finding was in the frequency of driver mutations in CAD subjects with CHIP: the majority (85%) of CAD patients presented mutations in TET2. Conclusions. Our outcomes add further insights to the nature of CHIP, which could be seen as a preleukemic condition that also leads to an intrinsic odd of cardiovascular events. The Our outcomes add further insights to the recent hypothesis that links CHIP to an increased risk of cardiovascular disease. The study results raise the question over a possible many-sided clinical and biological significance of CHIP that might depend on the driver mutation. In particular, we propose that TET2-driven CHIP is the most responsible for the higher cardiovascular risk, consistently with the pro-atherosclerotic role of this gene in mouse models (Fuster JJ, Science 2017, Jaiswal S, NEJM 2017). Citation Format: Luca Bertamini, Claudia Sala, Nicola Martinelli, Cristina Papayannidis, Cristina Giuliani, Giovanni Malerba, Paolo Garagnani, Maria Abbondanza Pantaleo, Michele Cavo, Oliviero Olivieri, Giovanni Martinelli, Claudio Franceschi, Domenico Girelli. Clonal hematopoiesis of indeterminate potential (CHIP), centenarians and age-related cardiovascular risk: Is TET2 the culprit [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1229.

Mendelian Randomization Suggests Causal Influence of Glycemic Traits on DNA Methylation

Type 2 diabetes (T2D) is caused by interactions between genetic predisposition and environmental exposure. DNA methylation, a key epigenetic mark, is one measure of the molecular impact of environmental factors on DNA. We tested for associations between glycemic traits and peripheral blood cell DNA methylation in 473,669 CpG sites across the genome in individuals from multiple ancestries, and tested for causal relationships at these loci. We conducted an epigenome-wide fixed effects meta-analysis for 3 glycemic traits-fasting glucose (FG), log fasting insulin (FI), and HbA1c—in 13,543 nondiabetic individuals of African (N=3,746), European (N=9,254), and Hispanic (N=543) ancestry across 16 cohorts. All analyses were adjusted for age, sex, smoking status, cell types, BMI, and technical covariates. We used Mendelian randomization (MR) analyses to determine whether glycemic traits causally influence methylation at the CpG sites identified in the meta-analysis. We performed MR analyses in Framingham Heart Study individuals (N=6,472), using separate genetic risk scores (GRS) as instruments for each of the 3 glycemic traits. In the epigenome-wide meta-analysis of all ancestries, there were 43 CpG sites associated with FG, 89 with FI, and 54 with HbA1c, using a Bonferroni threshold of P&amp;lt;1.1×10-7. In addition to detecting many novel candidate loci for each trait, we found associations with all 3 glycemic traits at previously reported T2D methylated loci SREBF1, LINC00649, and ABCG1. In MR analyses, we showed that a higher GRS for FI was associated with greater methylation at cg17058475 in CPT1A (P=4×10-4) and higher GRS for HbA1c with greater methylation at cg05779219 near THBD (P=4×10-4). We identified many novel candidate methylation loci associated with glycemic traits and 2 CpG sites where MR analysis indicated that methylation is causally influenced by glycemic traits. These findings support the hypothesis that variation in glycemia influences peripheral blood DNA methylation. Disclosure D.A. DiCorpo: None. S. Lent: None. W. Guan: None. M. Hivert: None. J.S. Pankow: None.

Association between DSCR4 gene methylation in plasma in early pregnancy and Down's syndrome.

Down's syndrome (DS), a chromosomal abnormal genetic disease caused by a local or total copy of chromosome 21, leads to patients suffering from delayed body growth, special facies, mild to moderate mental retardation and other symptoms, seriously affecting the life of patients. The aim of the present study was to examine the association between Down's syndrome critical region 4 (DSCR4) gene methylation in plasma in high-risk pregnant women with DS in early pregnancy (hereinafter referred to as pregnant women in early pregnancy) and DS, in order to screen new epigenetic markers for the clinical diagnosis of DS. DNA in peripheral blood cells and plasma in pregnant women in early pregnancy were treated with hydrosulphite. DSCR4 genes with different methylation levels were amplified by methylation-specific polymerase chain reaction (PCR), and the methylation difference of the CpG site of the DSCR4 amplification product in peripheral blood DNA was verified via restriction endonuclease analysis. The expression of DSCR4 with different methylation levels in peripheral blood of pregnant women in early pregnancy were detected via reverse transcriptase-quantitative PCR (RT-qPCR), and the DSCR4 gene functions were studied via the intervention in DSCR4 expression with small interfering RNA (siRNA). Methylation-specific PCR and restriction endonuclease analysis revealed that DSCR4 genes were differentially methylated in peripheral blood DNA in pregnant women in early pregnancy. Additionally, DSCR4 showed a low methylation status in plasma but a high methylation status in peripheral blood cells. RT-qPCR revealed that non-methylated DSCR4 was highly expressed in the peripheral blood of pregnant women in early pregnancy, and thus was an epigenetic marker of fetal DS. siRNA results showed that the downregulation of DSCR4 inhibited cell migration and invasion, but had no effect on cell proliferation. The results suggest that the DSCR4 gene was differentially methylated in peripheral blood DNA in pregnant women in early pregnancy. Furthermore, DSCR4 exists in a non-methylated state in plasma and in a hyper-methylated state in blood cells. DSCR4 can therefore promote the migration and invasion of trophocytes and serve as an epigenetic marker of non invasive clinical diagnosis of DS.

PCR for equine infectious anemia virus detection in whole blood from naturally infected horses in Colombia

The equine infectious anemia virus is associated with a variety of clinical symptoms that include anorexia, weight loss, pirexia, and anemia. Serological tests and reverse transcriptase nested polymerase chain reaction (RT-nPCR) have been used to detect EIAV in infected horses. In the present study, the use of non-nested PCR for the detection of EIAV gag gene sequences in peripheral blood cells DNA from horses bred in farms around Medellín (Colombia) is reported. EIAV DNA was detected by PCR in blood cells from fifteen horses, which had been found positive in an agar gel immunodiffusion test, despite the absence of clinical symptoms of expression. An amplified product to about 1.300 bp was obtained with a round of PCR without requiring nested PCR or RT-PCR.