Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34(+) progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2(mut)) cells, whereas ASXL1(mut) as well as DNMT3A(mut) cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells

Frequent ZNF217 mutations lead to transcriptional deregulation of interferon signal transduction via altered chromatin accessibility in B cell lymphoma

Recent exome-wide studies discovered frequent somatic mutations in the epigenetic modifier ZNF217 in primary mediastinal B cell lymphoma (PMBCL) and related disorders. As functional consequences of ZNF217 alterations remain unknown, we comprehensively evaluated their impact in PMBCL. Targeted sequencing identified genetic lesions affecting ZNF217 in 33% of 157 PMBCL patients. Subsequent gene expression profiling (n = 120) revealed changes in cytokine and interferon signal transduction in ZNF217-aberrant PMBCL cases. In vitro, knockout of ZNF217 led to changes in chromatin accessibility interfering with binding motifs for crucial lymphoma-associated transcription factors. This led to disturbed expression of interferon-responsive and inflammation-associated genes, altered cell behavior, and aberrant differentiation. Mass spectrometry demonstrates that ZNF217 acts within a histone modifier complex containing LSD1, CoREST and HDAC and interferes with H3K4 methylation and H3K27 acetylation. Concluding, our data suggest non-catalytic activity of ZNF217, which directs histone modifier complex function and controls B cell differentiation-associated patterns of chromatin structure

Associations of clonal hematopoiesis with recurrent vascular events and death in patients with incident ischemic stroke

Clonal hematopoiesis (CH) is common among older people and associated with an increased risk of atherosclerosis, inflammation, and shorter overall survival. Age and inflammation are major risk factors for ischemic stroke, yet the association of CH with risk of secondary vascular events and death is unknown. We investigated CH in peripheral blood DNA from 581 patients with first-ever ischemic stroke from the Prospective Cohort with Incident Stroke-Berlin study (PROSCIS-B) using error-corrected targeted sequencing. The primary composite endpoint (CEP) consisted of recurrent stroke, myocardial infarction, and all-cause mortality. 348 somatic mutations with a variant allele frequency ≥ 1% were identified in 236/581 patients (41%). CH was associated with large-artery atherosclerosis stroke (P = 0.01) and white matter lesion (P < 0.001). CH-positive patients showed increased levels of pro-inflammatory cytokines such as IL-6, IFN-γ, hsCRP, and VCAM-1. CH-positive patients had a higher risk for the primary CEP (HR: 1.55, 95%-CI 1.04 - 2.31, P = 0.03), which was more pronounced in patients with larger clones. CH clone size remained an independent risk factor (HR 1.30, 95%-CI 1.04 - 1.62, P = 0.022) in multivariable Cox regression. While our data show that in particular larger and TET2- or PPM1D-mutated clones are associated with increased risk of recurrent vascular events and death, this risk is partially mitigated by a common germline variant of the IL-6 receptor (IL-6R p.D358A). The CH mutation profile is accompanied by a pro-inflammatory profile opening new avenues for preventive precision medicine approaches to resolve the self-perpetuating cycle of inflammation and clonal expansion

Dynamics of clonal hematopoiesis under DNA-damaging treatment in patients with ovarian cancer

Clonal hematopoiesis (CH) driven by mutations in the DNA damage response (DDR) pathway is frequent in patients with cancer and is associated with a higher risk of therapy-related myeloid neoplasms (t-MNs). Here, we analyzed 423 serial whole blood and plasma samples from 103 patients with relapsed high-grade ovarian cancer receiving carboplatin, poly(ADP-ribose) polymerase inhibitor (PARPi) and heat shock protein 90 inhibitor (HSP90i) treatment within the phase II EUDARIO trial using error-corrected sequencing of 72 genes. DDR-driven CH was detected in 35% of patients and was associated with longer duration of prior PARPi treatment. TP53- and PPM1D-mutated clones exhibited substantially higher clonal expansion rates than DNMT3A- or TET2-mutated clones during treatment. Expansion of DDR clones correlated with HSP90i exposure across the three study arms and was partially abrogated by the presence of germline mutations related to homologous recombination deficiency. Single-cell DNA sequencing of selected samples revealed clonal exclusivity of DDR mutations, and identified DDR-mutated clones as the origin of t-MN in two investigated cases. Together, these results provide unique insights into the architecture and the preferential selection of DDRmutated hematopoietic clones under intense DNA-damaging treatment. Specifically, PARPi and HSP90i therapies pose an independent risk for the expansion of DDR-CH in a dose-dependent manner

A genetically defined signature of responsiveness to erlotinib in early-stage pancreatic cancer patients: Results from the CONKO-005 trial

Background: high recurrence rates of up to 75% within 2 years in pancreatic ductal adenocarcinoma (PDAC) patients resected for cure indicate a high medical need for clinical prediction tools and patient specific treat-ment approaches. Addition of the EGFR inhibitor erlotinib to adjuvant chemotherapy failed to improve out-come but its efficacy in some patients warrants predictors of responsiveness. Patients and Methods: we analysed tumour samples from 293 R0-resected patients from the randomized, multicentre phase III CONKO-005 trial (gemcitabine +/- erlotinib) with targeted sequencing, copy number, and RNA expression analyses. Findings: a total of 1086 mutations and 4157 copy-number aberrations (CNAs) with a mean of 17.9 /tumour were identified. Main pathways affected by genetic aberrations were the MAPK-pathway (99%), cell cycle control (92%), TGFb signalling (77%), chromatin remodelling (71%), and the PI3K/AKT pathway (65%). Based on genetic signatures extracted with non-negative matrix factorization we could define five patient clusters, which differed in mutation patterns, gene expression profiles, and survival. In multivariable Cox regression analysis, SMAD4 aberrations were identified as a negative prognostic marker in the gemcitabine arm, an effect that was counteracted when treated with erlotinib (DFS: HR=1.59, p = 0.016, and OS: HR = 1.67, p = 0.014). Integration of differential gene expression analysis established SMAD4 alterations with low MAPK9 expression (n = 91) as a predictive biomarker for longer DFS (HR=0.49; test for interaction, p = 0.02) and OS (HR = 0.32; test for interaction, p = 0.001). Interpretation: this study identified five biologically distinct patient clusters with different actionable lesions and unravelled a previously unappreciated association of SMAD4 alteration status with erlotinib effective-ness. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that SMAD4 status might guide addition of erlotinib treatment in early-stage PDAC patients. (C) 2021 The Author(s). Published by Elsevier B.V

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients

Acute myeloid leukemia with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40% of patients. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least 1 mutation, in addition to t(8;21), was identified in 95%, with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes: , , and Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTK patients, we identified multiple mutations in the same pathway. Deep sequencing (∼42 000×) of 126 mutations in 62 complete remission samples from 56 patients identified 16 persisting mutations in 12 patients, of whom 5 lacked in quantitative polymerase chain reaction analysis. mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (hazard ratio, 1.96; 95% confidence interval, 1.22-3.15; = .005). Together with age and white blood cell counts, , -internal tandem duplication, and mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n = 13) and unstable (n = 6) subgroups could be identified

Genomic landscape and clonal evolution of acute myeloid leukemia with t(8;21): an international study on 331 patients

Acute myeloid leukemia (AML) with t(8;21)(q22;q22) is characterized by considerable clinical and biological heterogeneity leading to relapse in up to 40%. We sequenced coding regions or hotspot areas of 66 recurrently mutated genes in a cohort of 331 t(8;21) patients. At least one mutation in addition to t(8;21) was identified in 95% with a mean of 2.2 driver mutations per patient. Recurrent mutations occurred in genes related to RAS/RTK signaling (63.4%), epigenetic regulators (45%), cohesin complex (13.6%), MYC signaling (10.3%), and the spliceosome (7.9%). Our study identified mutations in previously unappreciated genes GIGY2F, DHX15, and G2E3. Based on high mutant levels, pairwise precedence, and stability at relapse, epigenetic regulator mutations were likely to occur before signaling mutations. In 34% of RAS/RTK^{mutated} patients, we identified multiple mutations in the same pathway. Deep sequencing (≈42,000x) of 126 mutations in 62 CR samples from 56 patients identified 16 persisting mutations in 12 patients, of whom five lacked RUNX1-RUNX1T1 in qPCR analysis. KIT^{high} mutations defined by a mutant level ≥25% were associated with inferior relapse-free survival (HR=1.96; 95%-CI 1.22-3.15; P=.005). Together with age and white blood cell counts, JAK2, FLT3-ITD^{high}, and KIT^{high} mutations were identified as significant prognostic factors for overall survival in multivariate analysis. Whole-exome sequencing was performed on 19 paired diagnosis, remission, and relapse trios. Exome-wide analysis showed an average of 16 mutations with signs of substantial clonal evolution. Based on the resemblance of diagnosis and relapse pairs, genetically stable (n=13) and unstable (n=6) subgroups could be identified

Role of donor clonal hematopoiesis in allogeneic hematopoietic stem-cell transplantation

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age = 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P = .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk