Abstract: 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.

Abstract: 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.

Abstract: Cancer development is an evolutionary genomic process with parallels to Darwinian selection. It requires acquisition of multiple somatic mutations that collectively cause a malignant phenotype and continuous clonal evolution is often linked to tumor progression. Here, we show the clonal evolution structure in 15 myelofibrosis (MF) patients while receiving treatment with JAK inhibitors (mean follow-up 3.9 years). Whole-exome sequencing at multiple time points reveal acquisition of somatic mutations and copy number aberrations over time. While JAK inhibition therapy does not seem to create a clear evolutionary bottleneck, we observe a more complex clonal architecture over time, and appearance of unrelated clones. Disease progression associates with increased genetic heterogeneity and gain of RAS/RTK pathway mutations. Clonal diversity results in clone-specific expansion within different myeloid cell lineages. Single-cell genotyping of circulating CD34 + progenitor cells allows the reconstruction of MF phylogeny demonstrating loss of heterozygosity and parallel evolution as recurrent events.

Abstract: Background: Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of hematologic cancer associated mutations in the peripheral blood (PB) of at least 10% of elderly people without history of hematologic disorders (Genovese et al ., NEJM, 2014; Jaiswal et al ., NEJM, 2014). At present, caution is needed when predicting clinical consequences from CHIP in healthy people. An essential step towards a better understanding of CHIP requires identification of the cell of origin, clonal expansion patterns within the hematopoietic differentiation tree, and its dynamic behavior under stress scenarios (e.g. chemotherapy). Methods: PB and bone marrow (BM) samples were collected from 437 donors ≥ 55 years without known hematologic disease including a sub-cohort of 72 patients with newly diagnosed non-hematologic cancer requiring chemotherapy. Whole blood DNA was screened for CHIP with a 54 gene panel. A total of 63 PB and 9 BM samples were flow-sorted and variant allele frequencies (VAFs) were quantified in the hematopoietic fractions. In the cancer cohort, 32 clonal mutations were studied at 110 time points to investigate clonal dynamics under chemotherapy. Results: We identified 168 confirmed variants in 121 patients. 34 patients (28.1%) had 2 or more mutations (Fig. 1A). Presence of ≥ 2 mutations was significantly associated with peripheral artery disease (P=.002), diabetes (P=.04), and hyperlipoproteinaemia (P= .047). The most frequent combination was DNMT3A / TET2 (n=10) followed by DNMT3A/DNMT3A and TET2/TET2 in four cases each. TET2 mutations were significantly associated with DNMT3A (P=.015) and ASXL1 (P=.046) (Fig. 1B). Allelic burden of 91 mutations in 63 patients was determined in CD34+ progenitors, monocytes, granulocytes, NK-, B-, and T-cells (median VAFs: 5.1%, 7.1%, 6.3%, 6.0%, 1.9%, and 0.5%). B- and T-cells showed significantly lower VAFs when compared to WB or any other sorted cell fraction (P & lt;.001 for each comparison). NK-cells showed significantly higher VAFs than T- and B-cells (P & lt;.001), reaching comparable VAFs of myeloid cell fractions (Fig. 1C). Next, we compared mutation-specific effects on allelic burden within the cellular subfractions for DNMT3A, TET2, ASXL1, SF3B1, and TP53. No differences were observed except for a higher VAF in T-cells of DNMT3A -mutated individuals compared to other CHIP positive patients (P & lt;.001), indicating an involvement of very early hematopoietic stem cells (HSCs). Next, we tracked individual mutations in flow-sorted stem and precursor cells in the BM of 9 CHIP patients (example in Fig. 1D). In all cases, we were able to identify the mutation in the Lin-CD34+CD38- HSC fraction. Although mutations showed different expansion profiles [expansion ratio (ER)=VAF(monocytes)/VAF(HSCs) or ER=VAF(granulocytes)/VAF(HSCs)], the biggest expansion proportion always occurred in the stem cell compartment indicative for early clonal dominance. In patients with more than one clonal mutation, the repartition of patient specific mutations showed similar patterns in most cases, suggesting that mutations were acquired within one clone. However, in some cases differential outgrowth of mutations was observed, indicating oligoclonality (examples in Fig. 1E). In the cancer cohort, CHIP was significantly associated with chemotherapy dose reduction due to hematotoxicity (P=.028). When following 32 mutations at 110 time points, we observed 3 different patterns of VAF dynamics: 1) increasing, 2) decreasing, and 3) no major changes (example for each in Fig. 1F). We categorized the 3 groups by defining a VAF change of at least +/- 50 % in ≥ 2 time points as cut-off. Only one of the 13 DNMT3A mutations showed VAF dynamics (1/13=7.7%), in the remaining 19 clonal mutations other than DNMT3A, VAF dynamics were observed in 13 clones (68.4% vs. 7.7%; P & lt;.001). When excluding the 7 solely DNMT3A- mutated cases, we observed significantly lower hemoglobin levels prior to cycles 5 and 7 (P=.049 and P=.02) and an elevated red cell transfusion necessity (P=.013). Conclusion: CHIP derives from somatic mutations in Lin-CD34+CD38- HSCs and leads to preferential expansion in myeloid and NK-cell fractions. Clonal dynamics during chemotherapy lead to higher rates of red blood cell transfusions and dose reductions. Larger prospective studies in homogenously treated cancer patients are now warranted to verify the impact of CHIP during chemotherapy applications. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction Large-scale sequencing studies have unraveled the mutational landscape of myelofibrosis (MF), demonstrating clonal heterogeneity and importance of genetically defined subgroups in disease prognosis and progression. In order to elucidate the genetics of MF progression and its molecular drivers during JAK inhibition therapy, we performed in-depth genetic studies on longitudinal blood samples from 15 MF patients covering a disease span of 3 to 5 years after initiation of ruxolitinib. Methods Sequential samples from 15 MF patients (PMF n=8; post-ET/PV-MF n=7) accounting for a total of 42 time points representing 58.5 years of ruxolitinib treatment were investigated by whole-exome sequencing (WES). Additionally, we performed targeted deep sequencing of patient-specific mutations in flow-sorted cell fractions to study clonal repartition within the hematopoietic differentiation tree. Finally, we genotyped more than 5000 Lin-CD34+ progenitor cells using a single-cell multiplexed qPCR approach on a micro-fluidic platform (Fluidigm) to infer MF phylogeny. Results WES identified a median of 14 non-silent somatic mutations per patient at initiation of ruxolitinib treatment (=baseline WES; Figure 1A). When comparing mutations between first and last investigated time points, the majority of baseline mutations (162/201=81%) could be detected also at a later disease stage. A total of 39 mutations were lost and 80 new mutations were detected at the last time point. All patients showed at least one gained/ lost mutation in sequential samples. We noted frequent acquisition of mutations in genes of the RAS/RTK pathways in one third of patients. Two patients with a JAK2 V617F mutation achieved a molecular remission at a level of persisting residual disease of 1x10-3 with ruxolitinib therapy. In one of them, a total of 13 mutations were detected at baseline. In the second sample, taken three years later, a completely different set of mutations was identified and at the last time point, four years after initiation of therapy, none of the mutations were detected. This likely represents genetic drift during neutral evolution as a consequence of a rapid expansion after JAK inhibition. All other 13 patients showed only a modest - if any - decrease of 10-20% JAK2/CALR allele burden which was often accompanied with the expansion of JAK2/CALR-wildtype clones due to positive selection and/or freed clonal space under treatment. However, in some patients with durable response to ruxolitinib, we noted opposing dynamics of clones questioning a common origin. The three patients who progressed to leukemia showed a higher number of mutations at baseline and all of them acquired mutations in KRAS or NRAS over time. As one example, MPN18 harbored mutations in ASXL1, ETV6, and SRSF2 at baseline. Thereafter, and in addition to other driver genes (IDH2,KRAS) a second JAK2 Mutation at codon R867 was acquired, which has been reported to confer treatment resistance to JAK Inhibitors (Marty, Blood 2014). Mutation analysis in flow-sorted cell fractions showed a higher allelic mutation load in the myeloid compared to the lymphoid compartment with only few mutations being detected at low allele frequency in lymphocytes. Interestingly, some patients showed evidence of differential expansion among different myeloid cell lineages (Figure 1B). Next, we sorted 480 CD34+ single-cells per sample from 12 time points from 8 patients which allowed identification of subclones at ≥2% frequency based on priori power calculations. Sorting errors (e.g.cell doublets, empty wells)determined the mean cell sorting failure rate to be 12.5%. We employed a heuristic search algorithm to select a phylogenetic tree with Maximum Likelihood under a finite site model of evolution. Loss of heterozygosity (LOH) events were found in 7/8 patients and were not restricted to the JAK2 locus. In some patients, LOH of JAK2 occurred independently in two subclones, a phenomenon of convergent evolution (Figure 1C). We also noted cases with multiple 9pUPDs, of which one got selected during therapy. LOH events gave rise to both, a mutant homozygous but also reversion to a wildtype genotype. Conclusions Comprehensive serial genotyping of MF patients treated with ruxolitinib revealed heterogeneous patterns of clonal composition and evolution. Our data support LOH as a major determination factor for clonal diversification in MF. EM, KY, and MF contributed equally Figure 1 Disclosures Zenz: Abbvie: Consultancy, Honoraria, Other: Travel support; Roche: Consultancy, Other: Travel support; Janssen: Consultancy; Takeda: Consultancy; Gilead: Honoraria. Bullinger:Pfizer: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Abbvie: Honoraria; Bayer: Other: Financing of scientific research; Seattle Genetics: Honoraria; Sanofi: Honoraria; Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria. Le Coutre:Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau. Ogawa:Kan Research Laboratory, Inc.: Consultancy; Asahi Genomics: Equity Ownership; Qiagen Corporation: Patents & Royalties; RegCell Corporation: Equity Ownership; ChordiaTherapeutics, Inc.: Consultancy, Equity Ownership; Dainippon-Sumitomo Pharmaceutical, Inc.: Research Funding. Damm:Novartis: Research Funding; AbbVie: Other: Travel support.

Abstract: We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia ( & lt;2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%] ). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P = .022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL.