CHIP Mutations Research Articles

Clonal Hematopoiesis of Indeterminate Potential and Long-term Outcomes in Heart Transplantation

BackgroundClonal hematopoiesis of indeterminate potential (CHIP) mutations, an aging trait, has been associated with progression of cardiovascular disease and development of malignancy. Uncertainty prevails regarding a robust association between CHIP and heart transplantation outcomes. ObjectivesTo determine prevalence of CHIP mutations in heart transplantation and their association with long-term outcomes including cardiac allograft vasculopathy (CAV), graft failure, malignancy, and all-cause mortality. MethodsWe conducted a mixed retrospective-prospective observational study of heart transplant recipients with targeted sequencing for CHIP mutations (Variant allele frequency (VAF) of ≥2%). The primary composite outcome was first occurrence of CAV grade ≥2, graft failure, malignancy, cardiac re-transplantation, or all-cause death. Secondary outcomes were the individual components of the composite primary outcome. Sensitivity analyses with base-case and extreme scenarios were performed. ResultsAmong 95 HT recipients, 30 had CHIP mutations (31.6%). DNMT3A mutations were most common (44.7%) followed by PPM1D (13.2%), SF3B1 (10.5%), TET2 (7.9%) and TP53 (7.9%). The only significant independent predictor of CHIP was age at enrollment or age at transplantation. After multivariable adjustment, CHIP was not associated with the primary outcome which occurred in 44 (46.3%) patients (HR=0.487; 95%CI:0.197-1.204; p=0.119), malignancy alone or death. ConclusionWe demonstrated no association between CHIP mutations and post-transplant outcomes including CAV, graft failure, malignancy, and all-cause mortality. In line with previously published data, our analysis provides additional evidence on the lack of clinical value of using CHIP mutations as a biomarker for surveillance in outcomes after heart transplantation.

Abstract 2300: AI enabled whole exome& transcriptome liquid biopsy addressing MCED, MRD, and therapy selection on a single platform

Abstract Effective clinical management of cancer patients requires an accurate and early diagnosis, highly sensitive monitoring of minimal residual disease (MRD), and precise therapy selection. There are multiple tests available that attempt to address each of these needs independently with varying degrees of clinical utility. Caris Assure is a proprietary circulating nucleic acid sequencing platform that couples whole exome and transcriptome (WES/WTS) sequencing on white blood cells and plasma with advanced machine learning techniques to satisfy all three testing needs on one platform. This test detects SNVs, INDELs, structural variants, copy number, gene expression, tumor mutational burden (TMB), microsatellite instability (MSI), fragment length, and aneuploidy of both somatic (tumor and clonal hematopoiesis) and germline origin. Caris’ extensive database of over 350,000 tissue WES/WTS from solid malignancies was used to train deep learning neural networks to identify the molecular underpinnings of cancer. These networks were then deployed on WES/WTS data from plasma and buffy coat in pursuit of signals that can inform early detection, MRD and therapy selection. Validation studies were performed to characterize the analytic and clinical performance of Assure on over 3000 patient blood samples. These samples include ~1000 non-cancer patients (controls), ~1700 newly diagnosed patients where blood was collected at surgery (early detection), ~500 early-stage patients during adjuvant therapy at multiple time points (MRD), and ~200 locally advanced/metastatic patients where matched tissue testing was also performed (therapy selection). For early detection, stratification of blood samples from patients with stage I-IV cancer versus those with no reported cancer resulted in an AUC > 0.99 and included over 30 types of solid tumors. Notably, at 99.5% specificity, the sensitivities for stages I-IV (n= 119, 54, 50, 27) were 73%, 80%, 76%, and 89%. In the MRD setting for high-risk patients, the disease-free survival of patients whose cancers were predicted to recur was significantly shorter (39.6 mo) than those predicted not to recur (93.4 mo) (HR: 5.18, 95%CI: 2.94-9.09, p<.00001). This performance was observed across multiple lineages of cancer including but not limited to breast, colon, lung, and bladder. Lastly, for therapy selection, detection of driver mutations where blood was collected within 30 days of matched tissue demonstrated high concordance with a PPA of 93.8% and PPV of 96.8%. CHIP correction proved to be essential as ~35% percent of patients had CHIP mutations, including KRAS, BRAF, ATM, BRCA1/2, findings that could lead to improper therapy selection. Herein, we demonstrate for the first time a single liquid biopsy assay that addresses the entire continuum of care in clinical oncology with optimal diagnostic, prognostic, and predictive utility for patients and physicians. Citation Format: Jim Abraham, Valeriy Domenyuk, Maria Perdigones Borderias, Takayuki Yoshino, Elisabeth I. Heath, Emil Lou, Stephen Liu, John Marshall, Wafik S. El-Deiry, Anthony Shields, Martin Dietrich, Yoshiaki Nakamura, Takao Fujisawa, David D. Halbert, Dominic Sacchetti, Seth Stahl, Adam Stark, Sergey Klimov, Sourabh Antani, Chadi Nabhan, Jeffrey Swensen, George Poste, Matt Oberley, Milan Radovich, George W. Sledge, David Spetzler. AI enabled whole exome& transcriptome liquid biopsy addressing MCED, MRD, and therapy selection on a single platform [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 2300.

Driver mutation zygosity is a critical factor in predicting clonal hematopoiesis transformation risk

Clonal hematopoiesis (CH) can be caused by either single gene mutations (eg point mutations in JAK2 causing CHIP) or mosaic chromosomal alterations (e.g., loss of heterozygosity at chromosome 9p). CH is associated with a significantly increased risk of hematologic malignancies. However, the absolute rate of transformation on an annualized basis is low. Improved prognostication of transformation risk is urgently needed for routine clinical practice. We hypothesized that the co-occurrence of CHIP and mCAs at the same locus (e.g., transforming a heterozygous JAK2 CHIP mutation into a homozygous mutation through concomitant loss of heterozygosity at chromosome 9) might have important prognostic implications for malignancy transformation risk. We tested this hypothesis using our discovery cohort, the UK Biobank (n = 451,180), and subsequently validated it in the BioVU cohort (n = 91,335). We find that individuals with a concurrent somatic mutation and mCA were at significantly increased risk of hematologic malignancy (for example, In BioVU cohort incidence of hematologic malignancies is higher in individuals with co-occurring JAK2 V617F and 9p CN-LOH; HR = 54.76, 95% CI = 33.92–88.41, P < 0.001 vs. JAK2 V617F alone; HR = 44.05, 95% CI = 35.06–55.35, P < 0.001). Currently, the ‘zygosity’ of the CHIP mutation is not routinely reported in clinical assays or considered in prognosticating CHIP transformation risk. Based on these observations, we propose that clinical reports should include ‘zygosity’ status of CHIP mutations and that future prognostication systems should take mutation ‘zygosity’ into account.

A Retrospective Review Assessing the Development of Myelodysplastic Syndromes in Patients with Plasma Cell Dyscrasias

Introduction Myelodysplastic syndrome (MDS) are hematopoietic disorders defined by dysplasia of the myeloid lineage accompanied by persistent cytopenias, and recurrent cytogenetic abnormalities (MDS). Whilst therapy related myeloid disorders in patients with Multiple Myeloma have been well described, there is lack of information regarding co-occurrence of these disease states in early plasma cell disorders as well as multiple myeloma at diagnosis which may also then allude to whether there is a founder effect to the clones. Recent advances in commercial sequencing assays of genomic data have allowed for identification of relevant gene mutations in patients with hematologic malignancies, such that the presence of these gene mutations have been incorporated into classification systems. In our study, we aimed to assess the occurrence of reported MDS in patients with plasma cell disorders i.e. Monoclonal Gammopathy of unknown significance (MGUS), Smoldering myeloma (SM) and Multiple myeloma (MM) as well as relevant gene mutations that would be implicated in the pathogenesis of MDS even in those patients who do not meet the full criteria for MDS (clonal hematopoiesis of indeterminate potential; CHIP mutations). Methods In this retrospective review, clinical and bone marrow based genomic data (Tempus xT ® 684 gene DNA-based NGS platform) was gathered on 57 patients with a diagnosis of MGUS, SM, and/or MM. Pathogenic gene mutations that are frequently associated with MDS were reported in our patient population. Bone marrow morphologic assessments for MDS related changes were also collected at the time of genomic data abstraction. Those patients who had defined MDS were further subclassified using the World Health Organization 2022 fifth edition classification system. Those observed as having subtle MDS findings on bone marrow biopsy, but with identified CHIP mutations, not meeting criteria for MDS, were also identified. Results Among 57 patients with MGUS (28), SM(9), and MM(20), 2 had met criteria for defined MDS at the time of their assessment. An additional 5 had bone marrow morphologic findings concerning for MDS, but did not meet criteria to be fully defined. A total of 14 myeloid disorder related pathogenic mutations were identified in our patient population (6 in MM at disease diagnosis, 7 in MGUS, and 1 in SM). Overall, the most frequently observed recurring mutations in the cohort were that of DNMT3A (57.1%, mean VAF 4.45%), TET2 (14.3%, mean VAF 4.0%), and SF3B1 (14.3%, mean VAF 39.2%). Patients with defined MDS had recurring pathogenic mutations in genes SF3B1, ASXL1 and DNMT3A. Discussion In our cohort, we identified several patients with features both diagnostic and suggestive of myeloid disorders (MDS) during work up for a plasma cell disorder, in particular MGUS, SM, and/or MM prior to therapeutic intervention. Molecular work up also identified gene mutation subsets in plasma cell disorder patients that have otherwise been implicated in the pathogenesis of myeloid disorders. The identification of both morphologic and molecular abnormalities in our cohort may inform therapeutic considerations as well as vigilance for the discovery or evolution of myeloid disorders during follow-up , in particular when unexpected cytopenias or new clinical concerns emerge. We expect to design future prospective studies, based on our observations, to further inform of the risk of myeloid disorders in our plasma cell disorder patient cohorts, as well as understand its impact on therapies such as immunomodulatory agents, alkylators and/or autologous stem cell transplant.

Can Emergency Hematopoiesis Due to Sepsis Lead to Clonal Hematopoiesis? Implications for Understanding the Pathogenesis of Clonal Evolution

O.O &amp; T.K are co-first authors. Clonal (myelo) hematopoiesis of indeterminate potential (CHIP) arises from somatic mutations in myeloid genes, providing a proliferative and survival advantage to the affected “CHIP-initiating” early hematopoietic precursor cells. Understanding the conditions that lead to the emergence of detectable clones (early seeding of myeloid neoplasia-MN) is of great interest due to its clinical implications. Among CH facilitating states, aging has been extensively studied, but a clear distinction between the impact of chronological versus biological aging remains challenging. Additionally, other factors contributing to CHIP development may include chemotherapy, genetic predisposition, bone marrow failure states, or immunodeficiency. However, not all CHIP-promoting factors have been identified because they are subtle, pathophysiologically complex, and thus remain cryptic. We hypothesized that episodes of emergency hematopoiesis (EH), such as those caused by acute infections, could potentially result in acquisition of CHIP with repeated episodes of EH resulting in clonal expansion. If this hypothesis is confirmed, one could envision that both instances of significant hematopoietic expansion (high leukocytosis) and repeated cycles of EH could be at play in conjunction with chronological factors. For instance, the older an individual is, the greater is the number of EH episodes possible. To examine our theory, we prospectively collected blood samples from patients admitted at the intensive care unit (ICU) of Cleveland Clinic and Yale University. Inclusion criteria required a white blood cell (WBC) count ≥ 15K/uL. Patients with known or prior malignancies and those exposed to chemotherapy were excluded. For relevant comparisons, we assessed CH incidence in our cohort against a group of patients with solid tumors screened for CH (n=107) but without acute infections, along with historical CHIP cohorts. CH screening employed a targeted NGS myeloid gene panel with a reported VAF cut-off of 2%. A total of 78 individuals admitted to the ICU with possible infections and leukocytosis were enrolled in this study. The median age was 70 years (IQR: 61-77), with a M:F ratio of 1.7 (Table 1). Time from leukocytosis onset to sampling was 3 days (IQR: 1-5), the median WBC at ICU admission was 18 K/uL (IQR: 16-21), and the median absolute neutrophil count (ANC) was 16 K/uL (IQR: 12-20). Overall, 94 variants were identified including 16 (median VAF 50.1) variants of potentially germline origin, and 4 variants of unknown significance. We observed 74 CHIP-associated mutations in 37 (47%) patients at a median VAF of 14% (4-47%). The landscape of CHIP mutations included alterations in DNMT3A (20%), TET2 (12%), ASXL1 (12%), SMC1A, TP53, and PPM1D genes (6% each). Although higher rates of CHIP were expected in the cancer cohort, no difference was found between the two groups (40% vs. 47% in the ICU, p=0.3). DNMT3A, ASXL1 and PPM1D mutations were more frequently encountered in the cancer cohort (Table 1). The prevalence of CHIP in the ICU cohort was higher in older age groups: 30-39 (29%), 50-69 (49%), and ≥70 (51%), compared to the expected frequency in comparable age ranges (and median range) in a historical healthy population, which was 1%, 4%, and 10%, respectively (&amp;lt;0.00001; Fig.1). Notably, CHIP was more prevalent among smokers (57%; p=0.003). No significant differences were found when comparing CHIP + and CHIP - patients of the ICU cohort with regards to WBC (median 18K/uL both). Similarly, CHIP prevalence did not change according to the degree of leukocytosis in an analysis where patients were ranked by WBC (≥18K/uL at 44% vs. &amp;lt;18K/uL at 46%). When looking at the impact of CHIP on critical care outcomes, a logistic regression model accounting for age, gender, and mutation type revealed that the presence of DNMT3A mutations was associated with a higher risk of diagnosis of overt septic shock (log OR= 2.4, 95% CI 0.19-5.4, p&amp;lt;0.05). In summary, our prospective observational study has shown that sepsis is associated with preferential mutant clone expansion, primarily due to EH rather than the extent of leukocytosis and may be contributing to the acquisition of new clones. Whether such episodes result in permanent acquisition of new CHIP mutations with potential for MN progression will be the subject of longitudinal analysis of these samples, and findings will be presented at the ASH meeting.

Parallel Monitoring of Measurable Residual Disease and Chimerism By NGS in Myelodysplastic Syndrome after Allogenic STEM Cell Transplant

Background: Minimal Residual Disease (MRD) and chimerism monitoring in patients with Myelodysplastic Syndrome (MDS) is essential to track and quantify the proportion of donor and recipient cells to assess engraftment and treatment efficacy. Next Generation Sequencing (NGS) is rapidly evolving, can identify several somatic mutations simultaneously, quantifies allele burden, and is a suitable technology for this task. Traditionally its main limitations have been high single base error rates, which limited the sensibility to 0.1-1% Variant Allele Frequency (VAF). However, sensitivities up to 10 -4 VAF can be achieved by applying novel ultrasensitive personalized sequencing techniques. Aims: This study explores the applicability of PhasedSNP, a deep sequencing approach that quantifies chimerism in parallel to MRD in MDS patients after Allogenic Stem Cell Transplantation (ASCT). Methods: We included 24 patients older than 18 years who underwent ASCT between 2015 and 2022 at four third-level hospitals. We excluded patients with missing follow-up due to procedure-related mortality or relapse before day 100 and patients with unavailable samples. Fifty-three post-transplant samples were sequenced (six at day +30; two at +60; 21 at +100; 21 at +180, and three at day +360). Seven additional pre-transplant samples were included to confirm the donor and receptor signal switch. The genomic DNA of the donor and receptor was sequenced with an in-house PhasedSNP panel that covers 58 regions with paired polymorphisms in less than 15bp. Receptor DNA was also sequenced with a 42-gene panel (Ion Torrent TM) to identify somatic mutations used as biomarkers in follow-up samples to quantify MRD as previously described in ( Onecha E. et al. Haematologica 2019). Briefly, an amplicon-multiplexed mini-panel was defined for every patient to detect the selected MRD biomarkers and donor/receptor phased SNPs identified at diagnosis. The mini-panel included molecular-tagged primer pairs to amplify every selected marker (PhasedSNPs and somatic mutations) in three biological replicates. Final libraries were sequenced on the Ion S5 System platform with a median coverage of 211,190 reads per amplicon. A median of 558 ng of genomic DNA (range 73.44 - 858.6 ng) was sequenced in follow-up cases. Results: After excluding potential CHIP and Germline mutations, the most frequently mutated genes in the cohort were STAG2 (21%), SETBP1TP53RUNX1 andETV6 (16% each one). The baseline genotyping permitted to identify at least one somatic MRD biomarker in 79% of the patients (19/24), and 50% (12/24) presented more than one somatic mutation. MRD could be calculated for 42 of 53 samples. Thirteen patients presented sample at day +180 and clinical information. Four of them progressed, being three of them MRD positive. At this time-point the sensitivity of the test was 75% and the negative predictive value of 86%. The baseline PhasedSNP panel identified chimerism biomarkers (donor and receptor) for all patients. The PhasedSNP chimerism test presented a dynamic detection range below 10 -5 in serial dilutions. The test was positive for 48/53 samples using a threshold of 10 -4, with an average signal of 7·10 -3 (range 2·10 -5 to 4·10 -2). The correlation between PhasedSNP chimerism and MRD was high (R=0.921, p&amp;lt;0.001). However, the chimerism signal was consistently above the MRD signal, most likely due to the detection of healthy receptor cells with the PhasedSNP test. This can be seen in the two examples presented in the figure, with one patient ( MDS7, Figure, top) with an equivalent signal of chimerism at days +30, +100, and +180, but with MRD negativity at day +420 and two logarithms reduction in chimerism compared the highest value (day 100). Regarding the other patient, MDS13 ( Figure, bottom), chimerism and MRD dynamics evolve parallel but with more than two logarithmic differences between both values. This patient was MRD negative at day +100, but positive at day +180, and relapses one year later. Summary/Conclusion: NGS-based parallel detection of chimerism and MRD in MDS is feasible and has high applicability and sensitivity, with several advantages compared to traditional methods. Nevertheless, further research and a larger cohort is necessary to draw meaningful conclusions.

Multi-Omic Profiling of Macrophages Lacking Tet2 or Dnmt3a Reveals Mechanisms of Hyper-Inflammation in Clonal Hematopoiesis

Introduction Clonal Hematopoiesis of Indeterminate Potential (CHIP) is a source of age-associated inflammation, caused by somatic mutations in bone marrow stem cells that promote clonal expansion. The functional impact of CHIP mutations on macrophages derived from these stem cells is linked to increased cardiovascular disease risk among other diseases. Interestingly, CHIP-associated mutations frequently occur in genes with opposing functions: DNMT3A and TET2, enzymes responsible for DNA methylation and demethylation, respectively. While it is known that DNMT3A KO and TET2 KO in macrophages lead to hyperinflammatory responses, the underlying mechanism remains poorly understood how opposing mutations lead to a similar phenotype. Methods To evaluate how DNMT3A and TET2 contribute to inflammatory responses in the context of CHIP, we exposed bone marrow derived macrophages from Dnmt3a KO, Tet2 KO, and C57BL/6 Vav1-Cre wild type (WT) control mice to 0.5 ng/mL of the inflammatory stimuli lipopolysaccharide (LPS). We then measured gene responses with RNA-seq replicates across 12 time points from 0 to 24 hours, paired with single cell RNA-seq (scRNA-seq), ATAC-seq, and 50x-coverage whole genome enzymatic methyl-seq (EM-seq) for multiple time points. Results Although the composition and kinetics of the LPS-induced expression program were similar in all genotypes, Dnmt3a KO and Tet2 KO had significantly greater changes in gene expression and chromatin accessibility by 2 hours compared to WT, particularly for a large subset of inflammatory response genes, like Il1b, Il6, Cxcl1, Tnfsf9, and Il23a. The promoter regions of the 278 genes significantly overexpressed (adjusted p &amp;lt; 0.05) for both KOs at 2 hours compared to WT were enriched with binding sites of the inflammatory pioneer factor AP-1 (p = 0.001). Inflammatory signals were observed even before stimulation, suggesting altered DNA methylation in each KO predisposed cells to hyperinflammatory states. Bulk RNA-seq found the gene set ‘IFNA response’ from MSigDB Hallmarks collection to be most enriched at baseline in both KOs compared to WT (FDR &amp;lt; 0.0007). At baseline Dnmt3a KO and Tet2 KO had 1,566 and 441 over-expressed genes, respectively (adjusted p &amp;lt; 0.05). scRNA-seq similarly showed a baseline inflammatory population enriched with type 1 IFN response gene expression that contained more cells with measurable baseline expression of inflammatory genes for both KOs. ATAC-seq identified highly significant enrichment of AP-1 binding sites at baseline and at 2 hours in the 2,270 regions more accessible in Dnmt3a KO compared to WT (p = 1x10 -40), as well as the 2,148 regions less accessible in Tet2 KO compared to WT (p = 1x10 -41). Although LPS stimulation did not lead to sites with altered CpG methylation using a genome-wide significance threshold, methylation globally differed between genotypes at baseline especially for promoter and enhancer regions, and this differential methylation directly explained some of the hyper-responsive chromatin accessibility and gene expression. For example, the promoter region of Cxcl1 for both KOs was significantly hypomethylated and more accessible at baseline, with these chromatin accessibility and associated expression differences becoming even larger at 2 hours of LPS stimulation. In general, DNA methylation and chromatin accessibility were rarely concordantly altered in both KOs at regions near genes with differential expression. For adjusted p-value &amp;lt; 0.05, Dnmt3a KO had 236,062 differentially hypomethylated CpG segments and 1,023 hypermethylated, while Tet2 KO had 7,488 hypomethylated and 4,482 hypermethylated segments. Conclusions Our integrative multi-omic analysis demonstrates that Dnmt3a and Tet2 modulate DNA methylation to limit inflammatory gene expression in healthy macrophages. We find loss of Dnmt3a or Tet2 activity in macrophages alters baseline DNA methylation at different regions, separately influencing the transcription factor binding landscape of AP-1, and inducing similar baseline over-expression of type 1 interferon response genes. Upon stimulation, these differences lead to higher inflammatory gene expression, possibly through more cooperation with transcription factors NF-κB and IRFs. Targeting AP-1 and type 1 interferon may be a therapeutic avenue for blocking the pathogenic role of CHIP for many inflammatory diseases of aging.

Non-preferential, but detrimental accumulation of macrophages with clonal hematopoiesis-driver mutations in cardiovascular tissues

Abstract Background Clonal hematopoiesis of indeterminate potential (CHIP) is an acquired genetic risk factor of cardiovascular (CV) disease transmitted by myeloid cells. Harboring CHIP is associated with higher risk of coronary artery disease and worse prognosis of congestive heart failure. Studies show that circulating monocytes of CHIP carriers are more pro-inflammatory than those of non-carriers, but to what extend mutant monocytes and their progenies accumulate in the tissue of CV lesions remains unknown. First, we aimed to identify the frequencies of mutant macrophages in human carotid plaque and heart tissues relative to those of mutant monocytes in the patients’ blood. Second, we compared the gene expression profiles of circulating and tissue myeloid cells of CHIP carriers with those from non-carriers. Methods We recruited patients undergoing carotid endarterectomy and aortic valve replacement. Blood and CV tissue (carotid plaque or right atrium appendix) of patients who provided study consents were retrieved during the surgeries and immediately processed for the isolation of lymphocytes and myeloid cells. Whole blood DNA was subjected to CHIP mutation screening. DNA and RNA of eight DNMT3A or TET2 mutation carriers and eight matched non-carriers were isolated from sorted cells. We measured the frequencies of CHIP mutations in each sorted cell type using a droplet-digital PCR method. RNA of the sorted myeloid cells from the carriers and matched non-carriers were subjected to RNA sequencing. Results Our data showed that CHIP-mutant monocytes in the blood is similar to that of CHIP-mutant macrophages in both the plaque (ratio versus monocytes =1.2, p=0.44) and the heart (ratio versus monocytes=0.89, p=0.47), suggesting a comparable ability of mutant and non-mutant monocytes to accumulate in the cardiovascular lesions. Near absence of CHIP clones in lymphocytes (ratio versus monocytes=0.06, p&amp;lt;0.0001) confirmed the myeloid skewing driven by DNMT3A or TET2 mutations. Unexpectedly, we detected similar size of CHIP clones in CCR2-positive and CCR2-negative cardiac macrophages (ratio=0.95, p=0.77), challenging the current paradigm that CCR2-negative cardiac macrophages represent a resident population with minimal monocyte contribution. Transcriptomic profiles of blood and tissue monocytes/macrophages from these carriers were distinct from those of eight matched non-carriers, featuring differentially up-regulated cytokines (eg. IL1B, S100A9) and chemokines (eg. CXCL1, CCL3) in cells from the carriers. Myeloid cells from the carriers up-regulated genes enriching pathways related to immune activation and inflammation. Conclusion Our study manifested the presence of pro-inflammatory CHIP-mutant myeloid cells in human plaque and heart tissues. The equivalent size of CHIP clones in the circulation and tissue suggests a comparable ability of mutant and non-mutant monocytes to accumulate in CV lesions, but inflammation may mediate CHIP-associated CV diseases.

Identification of cell-intrinsic effects of mutated circulating cells due to clonal hematopoiesis in patients with heart failure

Abstract Clonal hematopoiesis of indeterminate potential (CHIP) is caused by somatic mutations in hematopoietic stem cells and is associated with worse prognosis in patients with heart failure with reduced ejection fraction (HFrEF). Circulating immune cells derived from patients with HFrEF harboring CHIP mutations show enhanced proinflammatory signatures. However, it is unclear, whether these signatures are derived from the relatively low number of cells harboring CHIP-driver mutations, or are indicators of a systemic pro-inflammatory activation associated with CHIP. Methods and Results We assessed the cell-intrinsic effects of the most prevalent CHIP-driver gene (DNMT3A) mutant-carrying circulating immune cells in a total of 5 patients with HFrEF. Using an improved protocol to detect mutant cells on a single cell level (MutDetect-Seq), as illustrated in figure 1A, we show that CHIP mutations can be detected in all major immune cell types (e.g. T cells, monocytes, B cells, NK cells). DNMT3A mutant monocytes demonstrated the highest number of significantly upregulated genes relative to other cell types. Specifically, DNMT3A mutant monocytes exhibit altered gene expression profiles associated with inflammation and phagolysosome function (figure 1B). In addition, DNMT3A mutant carrying CD4+, but not CD8+ T cells, showed significant changes in gene expression patterns towards effector cells and away from a naïve T cell state. Finally, increased paracrine signaling pathways involved in amplifying inflammatory circuits are predicted between mutated and non-mutated monocytes. Conclusion This is the first study to decipher the genetic signature of DNMT3A-CHIP-driver mutation carrying circulating immune cells, thereby providing first evidence for the cell intrinsic effects of DNMT3A mutations on peripheral blood cell subsets in patients with HFrEF. The altered gene expression profiles associated with inflammation and phagolysosome function of mutant monocyte as well as the skewing of mutated T cells towards effector cells provide novel insights into how CHIP might promote worse prognosis in heart failure patients.MutDetect-seq

Genomic Landscape of Ibrutinib- and/or Acalabrutinib-intolerant Patients with B-cell Malignancies Treated with Zanubrutinib in a Phase 2 Study

Introduction:Bruton tyrosine kinase inhibitors (BTKi) have shown remarkable utility in the treatment of B-cell malignancies; yet some patients have experienced toxicities to the BTKis, ibrutinib and acalabrutinib, which lead to dose reduction or treatment discontinuation. Zanubrutinib is a potent and selective next-generation BTKi. BGB-3111-215 (NCT04116437) is an ongoing, phase 2 study of the safety and efficacy of zanubrutinib monotherapy in patients with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL) who discontinued ibrutinib, acalabrutinib, or acalabrutinib and ibrutinib because of intolerance. Methods:Because the genomic profile of patients with B-cell malignancies who are intolerant to ibrutinib and/or acalabrutinib has not been extensively studied, mutations were assessed using a 106 gene-targeted next-generation sequencing panel (PredicineHEME TM, Predicine) on baseline and disease progression samples. Samples were sequenced to a median depth of &amp;gt;20,000 reads, with a validated sensitivity of 0.25% mutant allele frequency for all genomic regions, and 0.1% for mutational hotspots. Variant allele frequency (VAF) &amp;lt;0.1% for hotspot mutations and &amp;lt;0.25% for non-hotspot mutations were excluded from analysis; germline and CHIP mutations were also excluded from analysis. The association between gene mutations and progression-free survival (PFS, defined as time from the date of first zanubrutinib dose to date of first PD or death) was quantified by the log rank test and the hazard ratio and summarized by the Kaplan-Meier method. Results:As of June 6, 2022, baseline peripheral blood samples were collected from 71 (95.9%) of the 74 patients enrolled in the study. There were 46 patients with CLL, 11 with WM, eight with SLL, three with MCL, and three with MZL. A total of 56 patients were intolerant to ibrutinib only; 15 were intolerant to acalabrutinib or acalabrutinib and ibrutinib. At baseline, high mutation frequencies were observed in TP53, SF3B1, ATM, and NOTCH1 across disease indications ( Figure). The majority of TP53 mutations were missense mutations and located in the DNA binding domain. Nineteen different mutations were identified in ATM and were distributed across the whole gene with a majority clustered in the 3' regions encoding the FAT and PI3 kinase domains. All mutations (n=21) in SF3B1 were missense and 38% (n=8/21) of the mutations occurred at p.K700E/c.A2098G. In NOTCH1, the most common mutation (n=8/14, 57.1%) was a two base pair deletion (c.7541_7542delCT) in the PEST domain of exon 34. In patients with CLL/SLL with TP53, SF3B1, or ATM mutations at baseline, shorter progression-free survival was observed compared to patients without these mutations, suggesting that mutations in DNA damage response and RNA splicing/metabolism pathways are associated with less favorable prognosis. BTK mutations were detected in 4.2% (n=3/71) of baseline samples and in 57.1% (n=4/7) of samples available at disease progression (PD). All BTK mutations were at the C481 BTKi binding site. Two patients with baseline BTK mutations had PD and increased VAF of BTK mutants were detected for both patients at PD. A third patient died due to illness unrelated to treatment. Two patients acquired new BTKC481 mutations at PD. Conclusion: This is the first study to describe the genomic landscape of patients with B-cell malignancies who were intolerant to ibrutinib and/or acalabrutinib. Here we show that the gene mutational profile of these patients at baseline or at/after disease progression is comparable with patients with relapse/refractory disease who tolerate ibrutinib and, consistent with other studies, patients with mutations in TP53, SF3B1 or ATM genes had less favorable prognosis on BTKi. Further, intolerant patients who progressed on zanubrutinib acquired new BTK mutations and/or had an increase in the frequency of BTK mutations. These data contribute to the growing understanding of the mutational profiles of patients with B-cell malignancies, particularly those who were intolerant to BTKi, and further explores the association between mutations and response to subsequent zanubrutinib treatment. This study may provide supporting information to inform treatment guidelines for patients who become intolerant to BTKi.

A practical approach to curate clonal hematopoiesis of indeterminate potential in human genetic data sets

AbstractClonal hematopoiesis of indeterminate potential (CHIP) is a common form of age-related somatic mosaicism that is associated with significant morbidity and mortality. CHIP mutations can be identified in peripheral blood samples that are sequenced using approaches that cover the whole genome, the whole exome, or targeted genetic regions; however, differentiating true CHIP mutations from sequencing artifacts and germ line variants is a considerable bioinformatic challenge. We present a stepwise method that combines filtering based on sequencing metrics, variant annotation, and population-based associations to increase the accuracy of CHIP calls. We apply this approach to ascertain CHIP in ∼550 000 individuals in the UK Biobank complete whole exome cohort and the All of Us Research Program initial whole genome release cohort. CHIP ascertainment on this scale unmasks recurrent artifactual variants and highlights the importance of specialized filtering approaches for several genes, including TET2 and ASXL1. We show how small changes in filtering parameters can considerably increase CHIP misclassification and reduce the effect size of epidemiological associations. Our high-fidelity call set refines previous population-based associations of CHIP with incident outcomes. For example, the annualized incidence of myeloid malignancy in individuals with small CHIP clones is 0.03% per year, which increases to 0.5% per year among individuals with very large CHIP clones. We also find a significantly lower prevalence of CHIP in individuals of self-reported Latino or Hispanic ethnicity in All of Us, highlighting the importance of including diverse populations. The standardization of CHIP calling will increase the fidelity of CHIP epidemiological work and is required for clinical CHIP diagnostic assays.

Integrating Mosaic Chromosomal Alterations and Single-Nucleotide Variants to Predict Evolution of Clonal Hematopoiesis

Background: Clonal hematopoiesis (CH) is an age-related expansion of mutated hematopoietic stem cells. Multiple studies have previously described distinct types of CH, CHIP (mutations in genes commonly associated with myeloid malignancies) as well as mosaic chromosomal alterations (mCA), that have been associated with increased risk of hematologic malignancies and cardiovascular morbidity. We hypothesized that the presence of both mCA and CHIP mutations may have an additive impact vis-à-vis risk of hematologic malignancy. Methods: Data on 91,335 patients from the BioVU repository, a Vanderbilt University Medical Center biobank with linked de-identified EHRs, spanning 2006 to 2021 were included in the analysis. Peripheral blood-derived DNA of 91,335 subjects from BioVU were genotyped on the Illumina Multi-Ethnic Global Array (MEGAEX) platform, which contains more than 2 million SNPs. CHIP mutation calls (DNMT3A R882/JAK2 V617F) were made by deconvoluting the MEGAEX array intensity data. Predetermined array intensity scores greater than a defined threshold for DNMT3A R882 and JAK2 V617F mutations were highly predictive of the corresponding mutation, so these mutations were included in the analysis. Individuals with a diagnosis of hematologic malignancy (HM) prior to blood draw were excluded, while those that went on to develop HM post-blood draw were included in the analyses. The relationship between independent variables and disease progression and death was assessed using Cox proportional hazards model. All statistical analyses were performed using R. Results: In this large cohort (n=91,335), CH is strongly age-related with an increased frequency in the elderly, as has been previously shown (eg., Bick et al, Nature 2021; Jaiswal et al NEJM 2014). In total, we identified mCA in 765 (0.8%) patients. DNMT3A R882 and JAK2 V617F SNV/indels were identified in 1167 (1.3%) and 347 (0.4%) patients, respectively. Most frequent mCA detected in this cohort include 9p CN-LOH (n=105, 6.9%), del (5q) (n=99, 6.5%), del (20q) (n=98, 6.4%), del (7q) (n=94, 6.2%), and del (13q) (n=67, 4.4%). Combined mCA and DNMT3A R882/JAK2 V617F mutations were reported in 147 (0.2%). Co-occurring JAK2 and 9p CN-LOH (n= 100, OR=5658, q<0.001) are consistent with the well-known mechanisms of biallelic alterations involved in the pathogenesis of HM. There were significant correlations between abnormal blood counts and CH lesions, for example, high platelet count had a higher frequency of JAK2 V617F and 9p CN-LOH (OR=2.57, q=3.41e-141). Individuals with mCA are associated with lower WBC and platelet count, and any blood count abnormality (Figure 1A). Finally, we looked at how presence of both mCA and JAK2/DNMT3A impact the risk of HM. In total, 1615 cases in the entire BioVU cohort were reported to develop HM: 947 myeloid, AML (n=210), CML (n=73), MDS (n=315), MPN (n=349) and 668 lymphoid, ALL (n=72), B-NHL (n=252), T-NHL (n=22), MM (n=322). The presence of mCA was associated with a higher incidence of HM (hazard ratio (HR) = 6.85, 95% confidence interval (CI) = 5.9-7.95, P < 0.001), and DNMT3A/JAK2 was associated with higher incidence of HM ( HR=5.7, 95% CI= 4.75-6.85, P<0.001), and presence of both (mCA and DNMT3A/JAK2) were significantly associated with higher incidence of HM (HR=26.67, 95% CI= 19.48-36.5, P<0.001) than observed in no CH patients (Figure 1B). Conclusion: In this large cohort of patients, the presence of CH (both mCA and DNMT3A/JAK2) in apparently healthy individuals positively correlated with blood count abnormalities and increased incidence of hematologic malignancy. The results from this large cohort of patients emphasize the significance of detecting both mCA and SNV/indels in clonal hematopoiesis. These results should be validated in large datasets to determine if detecting both mCAs and CH can augment clinical care and outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Application of a Flow Cytometric Core Marker Set in the Diagnostic Workup of Patients with Suspected Myelodysplastic Syndromes

Background Flow cytometry (FC) performed according to ELN 2012 criteria (Westers et al., 2012) is recommended by ELN guidelines for diagnostic workup in suspected myelodysplastic syndromes (MDS, Malcovati et al., 2013). Recently, the ELN working group on FC in MDS identified 17 core markers independently related to MDS as well as >3% myeloid progenitor cells (MPC) to be strongly related to MDS. In parallel, during the last decade next generation sequencing (NGS) became increasingly relevant for MDS diagnostics. Aim To validate the new ELN criteria (ELN 2022) in comparison to cytomorphology (CM) in an independent cohort of patients prospectively undergoing diagnostics for suspected MDS and their correlation with MDS specific variants as detected by NGS. Methods Bone marrow samples of 4658 patients (female/male, 1833/2825; median age 72 years, range 18-96) with suspected MDS were analyzed in parallel by CM, FC and NGS (73 gene panel). Cases with non-MDS neoplasms were excluded. Results CM and FC (ELN 2012) revealed concordant diagnostic results in 80%. 1342 (29%) cases were diagnosed MDS by both CM and FC (CM+FC+), 2384 (51%) were CM-FC-. Discordant cases were 329 (7%) CM+FC- and 603 (13%) CM-FC+, respectively. Application of ELN 2022 criteria for the diagnostic of MDS by FC (either at least 3 out of 17 markers present or more than 3% MPC) resulted in a similar degree of concordance (78%). 1016 (22%) were CM+FC+ and 2626 (56%) were CM-FC-. Discordant cases were 655 (14%) CM+FC- and 361 (8%) CM-FC+, respectively. Next, we determined the portion of cases with at least one MDS-related gene mutation as analyzed by NGS in cases discordant between CM and FC. Regarding FC ELN 2012 criteria 248/329 (75%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 352/603 (58%). Considering ELN 2022 criteria 527/655 (81%) of CM+FC- cases had at least one mutation, while in CM-FC+ cases the rate was 236/361 (65%). Focusing on mutations in detail revealed similar frequencies of CHIP mutations (ASXL1, DNMT3A, TET2) regardless of CM and FC results. Concordant CM and FC cases (ELN 2012) harbor at least one CHIP mutation in 38% (CM+FC+ 506/1341, 38%; CM-FC- 901/2384, 38%). In CM+FC- cases 137/329 (39%) had at least one CHIP mutation, while in CM-FC+ cases the rate was 220/603 (37%). Application of ELN 2022 criteria resulted in a similar distribution (CM+FC+ 379/1016, 37%; CM-FC- 983/2626, 37%; CM+FC- 254/655, 39%; CM-FC+ 138/361, 38%). Analyzing cases discordant between CM and FC regarding non CHIP mutations showed a strong correlation between CM+FC- cases and SF3B1 mutations (ELN 2012: 102/324, 32%; ELN 2022: 190/651, 29%). In contrast, in CM-FC+ cases just 13/597 (2%, ELN 2012) and 11/357 (3%, ELN 2022), respectively, had an SF3B1 mutation. In line with this correlation, CM+FC- cases (ELN 2012) with mutated SF3B1 had ≥5-14% ring sideroblasts (RS) in 17/102 (17%) and ≥15% RS in 78/102 (77%). The respective figure for CM+FC- cases (ELN 2022) were ≥5-14% RS in 22/190 (12%) and ≥15% RS in 160/190 (84%). On the other hand SRSF2 mutations were found in discordant cases slightly more frequently in CM-FC+ cases compared to CM+FC- cases (ELN 2012: 95/557 (17%) of CM-FC+ and 40/315 (13%) of CM+FC- cases). This correlation was even clearer with ELN 2022 criteria (75/334 (23%) of CM-FM+ and 101/628 (16%) of CM+FC- cases). Evaluating remaining cases discordant between CM and FC after excluding those with CHIP mutations, SF3B1 mutations, and SRSF2 mutations revealed no major differences regarding presence of at least one of the remaining mutations (ELN 2012: 32/107 (30%) of CM+FC- cases and 62/275 (23%) of CM-FC+ cases; ELN 2022: 59/175 (34%) of CM+FC- cases and 43/146 (30%) of CM-FC+ cases). Conclusion FC has already proven its power in MDS diagnostics. We here confirm the newly defined criteria of ELN for diagnosing MDS (ELN 2022) to reveal results comparable to ELN 2012 criteria. Thus, a reduced core set of markers is already sufficient for MDS diagnosis by FC without loss in accuracy. Furthermore, cases with results discordant between CM and FC are associated with MDS-related mutations with particular sensitivity of CM in SF3B1 mutated cases and of FC in SRSF2 mutated cases. The higher frequency of mutations in cases discordant between CM and FC applying ELN 2022 criteria as compared to ELN 2012 criteria argues in favor of a higher specificity of the ELN 2022 criteria. Follow-up analyses are indicated to reveal the significance of discordant findings.

Abstract 12418: Prevalence and Clinical Characteristics of Patients With Decompensated Heart Failure and Clonal Haematopoiesis of Indeterminate Potential

Introduction: CHIP is associated with inflammation, atherogenesis and poor outcomes in patients with HFrEF. Although inflammation may be important in the pathophysiology of HFpEF, CHIP has not previously been assessed in patients with HFpEF. We examined the prevalence of CHIP and its associated clinical characteristics in patients with HFpEF or HFrEF. Methods: Error-corrected targeting sequencing of 75 known haematopoietic driver genes was performed on peripheral blood DNA from patients admitted to hospital with acute HF (HFrEF [LVEF&lt;40%] and HFpEF [LVEF≥40%]). CHIP mutations and their associated clonal population size were analysed and associations with clinical phenotypes and inflammatory markers were assessed. Results: 96 patients were enrolled; 48 had HFrEF and 48 had HFpEF. CHIP mutations with VAF ≥2% were detected in 5 patients with HFrEF (10%) and 8 patients with HFpEF (17%). CHIP mutations with VAF ≥1% were detected in 25 patients with HFrEF (52%) and 21 patients with HFpEF (44%). HFrEF patients with CHIP were a similar age to those without CHIP (71.1 ± 14.2yrs CHIP vs 68.9 ± 13.0yrs without CHIP; p=0.58) but HFpEF patients with CHIP were older than those without CHIP (80.1 ± 8.8yrs CHIP vs 70.0 ± 12.3yrs no CHIP; p=0.002). There was an age-dependent rise in CHIP prevalence which appeared greater in patients with HFpEF than HFrEF. DNMT3A was the most commonly mutated gene. Patients with CHIP had higher concentrations of pro-inflammatory interleukins but CHIP was not associated with previous MI or NT-proBNP. Conclusions: There is a substantial prevalence of CHIP in patients admitted to hospital with HF. In this cohort, CHIP was uncommon in patients with HFpEF under the age of 70 years but was frequent in older patients. The association between age and CHIP was stronger in HFpEF than HFrEF. CHIP was associated with inflammation. Its potential role in the pathogenesis of HF warrants further exploration in larger groups, including in elderly patients with HFpEF.

Clonal Hematopoiesis Analyses in Clinical, Epidemiologic, and Genetic Aging Studies to Unravel Underlying Mechanisms of Age-Related Dysfunction in Humans.

Aging is characterized by increased mortality, functional decline, and exponential increases in the incidence of diseases such as cancer, stroke, cardiovascular disease, neurological disease, respiratory disease, etc. Though the role of aging in these diseases is widely accepted and considered to be a common denominator, the underlying mechanisms are largely unknown. A significant age-related feature observed in many population cohorts is somatic mosaicism, the detectable accumulation of somatic mutations in multiple cell types and tissues, particularly those with high rates of cell turnover (e.g., skin, liver, and hematopoietic cells). Somatic mosaicism can lead to the development of cellular clones that expand with age in otherwise normal tissues. In the hematopoietic system, this phenomenon has generally been referred to as “clonal hematopoiesis of indeterminate potential” (CHIP) when it applies to a subset of clones in which mutations in driver genes of hematologic malignancies are found. Other mechanisms of clonal hematopoiesis, including large chromosomal alterations, can also give rise to clonal expansion in the absence of conventional CHIP driver gene mutations. Both types of clonal hematopoiesis (CH) have been observed in studies of animal models and humans in association with altered immune responses, increased mortality, and disease risk. Studies in murine models have found that some of these clonal events are involved in abnormal inflammatory and metabolic changes, altered DNA damage repair and epigenetic changes. Studies in long-lived individuals also show the accumulation of somatic mutations, yet at this advanced age, carriership of somatic mutations is no longer associated with an increased risk of mortality. While it remains to be elucidated what factors modify this genotype-phenotype association, i.e., compensatory germline genetics, cellular context of the mutations, protective effects to diseases at exceptional age, it points out that the exceptionally long-lived are key to understand the phenotypic consequences of CHIP mutations. Assessment of the clinical significance of somatic mutations occurring in blood cell types for age-related outcomes in human populations of varied life and health span, environmental exposures, and germline genetic risk factors will be valuable in the development of personalized strategies tailored to specific somatic mutations for healthy aging.

Thrombotic and Hemorrhagic Issues Associated with Myeloproliferative Neoplasms.

Thrombotic and hemorrhagic complications are related to a significant rate of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs), they are therefore called “thrombohemorrhagic” syndromes. Several clinical factors, such as age and presence of cardiovascular comorbidities are responsible for thrombotic complications. High blood counts, platelet alterations, presence of JAK2 mutation and possibly of other CHIP mutations such as TET2, DNMT3A, and ASXL1, procoagulant microparticles, NETs formation, endothelial activation and neo-angiogenesis are some of the parameters accounting for hypercoagulability in patients with myeloproliferative neoplasms. Bleeding complications emerge as a result of platelet exhaustion. They can be also linked to a functional deficiency of von Willebrand factor, when platelet counts rise above 1000G/L. The mainstay of management consists on preventing hemostatic complications, by antiplatelet and/or anticoagulant treatment and myelosuppressive agents in high-risk patients.Circumstances related to a high thrombohemorrhagic risk, such as pregnancy and the perioperative period, prompt for specific management with regards to anticoagulation and myelosuppression treatment type. In order to apply a patient-specific treatment strategy, there is a need for a risk score assessment tool encompassing clinical parameters and hemostasis biomarkers.

Graft-versus-host disease after liver transplantation is associated with bone marrow failure, hemophagocytosis, and DNMT3A mutations

Graft-versus-host disease after liver transplantation (LT-GVHD) is rare, frequently fatal, and associated with bone marrow failure (BMF), cytopenias, and hyperferritinemia. Given hyperferritinemia and cytopenias are present in hemophagocytic lymphohistiocytosis (HLH), and somatic mutations in hematopoietic cells are associated with hyperinflammatory responses (clonal hematopoiesis of indeterminate potential, CHIP), we identified the frequency of hemophagocytosis and CHIP mutations in LT-GVHD. We reviewed bone marrow aspirates and biopsies, quantified blood/marrow chimerism, and performed next-generation sequencing (NGS) with a targeted panel of genes relevant to myeloid malignancies, CHIP, and BMF. In all, 12 marrows were reviewed from 9 LT-GVHD patients. In all, 10 aspirates were evaluable for hemophagocytosis; 7 had adequate DNA for NGS. NGS was also performed on marrow from an LT cohort (n=6) without GVHD. Nine of 10 aspirates in LT-GVHD patients showed increased hemophagocytosis. Five (71%) of 7 with LT-GVHD had DNMT3A mutations; only 1 of 6 in the non-GVHD LT cohort demonstrated DNMT3A mutation (p=.04). Only 1 LT-GVHD patient survived. BMF with HLH features was associated with poor hematopoietic recovery, and DNMT3A mutations were over-represented, in LT-GVHD patients. Identification of HLH features may guide prognosis and therapeutics. Further studies are needed to clarify the origin and impact of CHIP mutations on the hyperinflammatory state.

Age-Related Changes in Human HSC Methylomes Affect Gene Regulatory Elements Critical to HSC Function and Reveal Novel HSC Aging Candidate Genes

Age-Related Changes in Human HSC Methylomes Affect Gene Regulatory Elements Critical to HSC Function and Reveal Novel HSC Aging Candidate Genes

MM-205: Clonal Hematopoiesis in Multiple Myeloma, Relations with Treatment Toxicity

Context Multiple myeloma (MM) is a clonal plasma cell neoplasm with a mean age of diagnosis of 68. The main targets in the treatment are immunomodulatory therapies and ASCT. CHIP mutations are common in plasma cell neoplasms. CHIP have been shown to be associated with previous cytotoxic treatment. Objective To detect accompanying somatic mutations, dysplasia, and presence of CHIP in MM patients. To evaluate in terms of treatment-related toxicities (TrTs) in patients with CHIP. Design The data of 45 MM patients who underwent bone marrow biopsy and studied NGS between February 2020 and January 2021 at Trakya University Hospital were retrospectively re-evaluated. Patients or Other Participants The mean age of the patients was 63.67; 24 of the patients were female and 21 were male. Bone marrow biopsy was performed in all patients and NGS was studied. The treatments received by the patients were retrospectively analyzed and recorded. Interventions The patients were diagnosed with MM using the WHO 2016 Myeloid Neoplasm Classification. Somatic mutation results were analyzed by the QIAact Myeloid DNA NGS Panel molecular analysis. Hematologic TrTs National Cancer Institute’s common nomenclature criteria was used for adverse events. Results When the data of 45 MM patients were analyzed, CHIP was detected in 35 patients. Mutational profile showed DNMT3A mutation in 11 patients, TET2 in 13 patients, RAS in 10 patients and other mutations including ASXL1, CSFR3R or RUNX1 in 11 patients. Though anemia is an end organ damage of MM, DNMT3A positivity was related with initial presentation of anemia (p=0.043). Thrombocytopenia was observed at the time of diagnosis in two patients with positive RAS (p=0.006). We found that patients using maintenance lenalidomide or bortezomib after ASCT were associated with the development of anemia in DNMT3A positive patients, but not with the development of anemia in TET2 positive patients. Neutropenia after treatment was observed in 22 patients, and in 20 of these patients, CHIP was present (p=0.039). Anemia after treatment was observed in 32 patients and 27 of these patients had CHIP (p=0.009). Conclusions Patients with CHIP are more prone to develop cytopenia and other toxicities with treatment. Patients cannot receive optimal myeloma treatment by delays or dose reductions in treatment. Investigation of CHIP in MM patients is necessary in treatment planning and follow-up.

Indeterminate and oncogenic potential: CHIP vs CHOP mutations in AML with NPM1 alteration

In AML patients, recurrent mutations were shown to persist in remission, however, only some have a prognostic value and persistent mutations might therefore reflect a re-established premalignant state or truly active disease causing relapse. We aimed to dissect the nature of co-mutations in NPM1 mutated AML where the detection of NPM1 transcripts allows highly specific and sensitive detection of complete molecular remission (CMR). We analysed 150 consecutive patients who achieved CMR following intensive treatment by next generation sequencing on paired samples at diagnosis, CMR and relapse (38/150 patients). Patients with persistence or the acquisition of non-DTA (DNMT3A, TET2, ASXL1) mutations at CMR (23/150 patients, 15%) have a significantly worse prognosis (EFS HR = 2.7, p = 0.003; OS HR = 3.6, p = 0.012). Based on clonal evolution analysis of diagnostic, CMR and relapse samples, we redefine pre-malignant mutations and include IDH1, IDH2 and SRSF2 with the DTA genes in this newly defined group. Only the persistence or acquisition of CHOP-like (clonal hematopoiesis of oncogenic potential) mutations was significantly associated with an inferior outcome (EFS HR = 4.5, p = 0.0002; OS HR = 5.5, p = 0.002). Moreover, the detection of CHOP-like mutations at relapse was detrimental (HR = 4.5, p = 0.01). We confirmed these findings in a second independent whole genome sequencing cohort.