Modeling Clonal Progression in SF3B1-Mutant Myelodysplastic Syndrome

SF3B1-mutant myelodysplastic syndrome (MDS) has recently been proposed as a distinct disorder characterized by ring sideroblasts, ineffective erythropoiesis and good prognosis. Selected co-occurring genetic abnormalities were reported associated with significantly worse outcome and suggested as exclusion criteria for the proposed entity. However, it remains unclear how a limited spectrum of co-occurring drivers affects SF3B1-mutant MDS biology to determine evolution from a relatively indolent condition to high risk malignancy. To gain a better insight into the clonal progression of SF3B1-mutant MDS, we analyzed SF3B1 co-mutations in a cohort of 176 SF3B1-mutated patients diagnosed with a myeloid neoplasm. RUNX1 and STAG2 were the only co-mutated genes found significantly associated with advanced disease phenotype (i.e. MDS with excess blasts and secondary acute myeloid leukemia) (OR=18.36 (2.18-862.91), P=0.001, and OR= Inf (3.57-Inf), P<.001, respectively), and reduced overall survival (P<.001). Based on these data, we hypothesized that acquisition of RUNX1 or STAG2 co-mutations in patients with SF3B1-mutant MDS drives progression to high-risk neoplasms. RUNX1 and STAG2 control hematopoietic stem and progenitor cell self-renewal and differentiation by regulation of gene expression. To explore the biological impact of RUNX1 or STAG2 loss in the context of SF3B1-mutant MDS, we disrupted RUNX1 or STAG2 using CRISPR/Cas9 in an induced pluripotent stem cell (iPSC) model of MDS-RS that we previously established by reprogramming of bone marrow cells from SF3B1-mutant individuals. This patient-derived system allows hematopoietic progenitor expansion through doxycycline-mediated expression of 5 transcription factors (5F-HPC) and multilineage differentiation upon doxycycline withdrawal. We asked how RUNX1 or STAG2 disruption affected SF3B1-mutant HPCs self-renewal and differentiation in our model of MDS-RS, and sought to define the underlying transcriptional changes. RUNX1-edited cells maintained significantly higher proportion of CD34 + HPCs, suggesting that RUNX1 mutation increases self-renewal capacity of SF3B1-mutant progenitors. Consistently, SF3B1/RUNX1 double-mutant 5F-HPCs showed positive enrichment of HSC-specific gene signatures. This was accompanied by broad upregulation of inflammatory programs, recapitulating the activated gene signatures we identified in SF3B1/RUNX1 co-mutated patients in our cohort. RUNX1 disruption promoted myeloid skewing at the expense of erythroid differentiation in SF3B1-mutant cells. Consistent with this, granulocyte-monocyte progenitors (GMP) and myelo-erythroid transcriptional programs were positively and negatively enriched, respectively. By contrast, in SF3B1/STAG2 double-mutant 5F-HPCs, both erythroid and myeloid populations were reduced compared to SF3B1 single mutant controls, suggesting a block in both myeloid and erythroid differentiation, despite the presence of pro-differentiation signals. This was supported by a profound down-regulation of genes involved in the response to external stimulus and suppression of GMP specific transcriptional signature. In summary, we identified RUNX1 and STAG2 mutations as main drivers of disease progression in SF3B1-mutant patients, and generated extensive cell line panels to interrogate their functional interaction with mutant SF3B1. By applying CRISPR/Cas9 editing to our MDS-RS model, we could overcome limited availability of primary MDS samples to show that RUNX1 or STAG2 co-mutations drive progression through distinct biological mechanisms in SF3B1-mutant HPCs. Disclosures No relevant conflicts of interest to declare.

Spectrum of Hematological Malignancies in 130 Patients with Germline Predisposition Syndromes - Mayo Clinic Germline Predisposition Study

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Molecular Characterization and Clinical Outcomes of Young Adult Patients (≤45 years old) with Philadelphia-Negative Myeloproliferative Neoplasms

Background: Polycythemia vera (PV), Essential Thrombocytosis (ET) and Primary Myelofibrosis (PMF) are Myeloproliferative Neoplasms (MPNs) with median age at diagnosis of ~56-70 years old. However, around 10%-15% of cases are diagnosed during young adulthood and there are scanty data about their molecular profile and its implications in clinical outcomes. Objective: To analyze the clinical and molecular characteristics of young adult patients (≤45y.o.) with MPNs (Y-MPN) and to correlate them with clinical features and outcomes. Material and Methods: This is a retrospective single-center study including MPN patients diagnosed below the age of 45 years. Molecular characterization was performed using DNA from granulocytes at diagnosis or before the start of cytoreductive therapy. JAK2V617F was assessed by quantitative allele-specific PCR and CALR mutations by fragment analysis of exon 9. Further molecular profiling was performed by next generation sequencing (NGS) with a custom panel of 25 myeloid-associated genes (ASXL1, CALR, CBL, CSF3R, DNMT3A, ETV6, EZH2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NRAS, PRPF8, RUNX1, SETPB1, SF3B1, S2HB3, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2) using Illumina technology. Pathogenic mutations in genes previously related with poor outcomes (ASXL1, EZH2, IDH1, IDH2, SRSF2 and U2AF1) were named as Mutations of Adverse Significance (MAS). Molecular alterations were correlated with diagnosis, progression to PV post ET (PVpET), progression to MF post PV/ET (MFpPV/ET), start of cytoreduction and major thrombotic events (MTE). Time to progression (TTP) and overall survival (OS) were calculated from diagnosis to progression (ELN criteria) and to last visit. Results: From 646 MPN followed in our clinic, 109 (17%) cases were Y-MPN; females 72 (66.1%). At diagnosis the median age was 35 y.o. (9-45). 23 patients (21%) were PV, 91% carried JAK2V617F, 4% (1) carried an exon-12 JAK2 mutation and 1 was JAK2V617F and exon-12 negative. 84 cases (77.1%) were ET, 53.5% (45) JAK2V617F, 25% (21) CALR, (52% type-1 mutation, see table) and 21.4% (18) triple negative (TN). There was 1 PMF with CALR type-1 mutation. No MPL canonical mutations were found. ET was predominantly diagnosed in females (M/F: 26/58). Regarding clinical variables, we found a high proportion of ET-JAK2V617F with high LDH values, higher platelet count for CALR-ET and ET-TN patients (967 and 978 vs 728 for ET-JAK2V617F, p=0.03) and higher frequency of MTE at or before diagnosis for JAKV617F cases (p=0.001). The mean follow-up was 152 months (SD +/-10.4); 16 progressions were registered (PFS 305 months); 8 patients to MFpPV/ET and 8 ET-JAK2V617F to PVpET. An increase in the VAF of JAK2V617F was observed at the time of progression either to PVpET or to MFpET/PV (see table). Seven MTE were registered during this time, 3 in JAK2V617F, 2 in CALR type-1, 1 in exon-12 and 1 in a TN case. 38 (34.8%) cases started cytoreduction, with median time to cytoreductive therapy of 251 (172-330) months; JAK2V617F cases started cytoreduction more often (p=0.04) than patients with other genotypes. No progression to AML nor deaths were recorded. The NGS panel was performed in 102 (93.5%) cases. Pathogenic mutations in non-driver genes were found in 41.2% (42) of cases, being TET2 (7%), ASXL1 (6%) and DNMT3A (5%) the most frequently mutated genes. Also, in 28.4% (29) variants of unknown significance (VUS) were found, involving TET2 (6%), SETBP1 (4%), SH2B3 (5%), and JAK2 (4%) among others. The mutations in SH2B3 (1 pathogenic, 5 VUS) were more frequent in JAK2V617F patients and those in DNMT3A were more common in PV patients. The presence of mutations in non-driver genes (pathogenic or VUS) did not correlate with MTE before or after diagnosis, the start of cytoreduction nor clonal progression. Regarding the 19 TN cases, in 7 (36.8%) one or more non-canonical pathogenic variants implicating MPL, JAK2 and TET2 genes were found. Finally, 8 patients (7.8%) harbored a MAS, of which 3 progressed to MF (2 CALR to MF and 1 ET-JAK2V617F to PVpET); TTP was similar to the rest of the cohort. Conclusions: Our data show that 41% of Y-MPN patients harbor pathogenic mutations in non-driver genes. There was no correlation between their presence and clonal progression, major thrombotic events or overall survival. Mutations of adverse significance did not predict major clinical outcomes. Monitoring of JAK2V617F allele-burden can help to predict progression to MFpPV/ET or PVpET. Disclosures Andrade-Campos: Sanofi-Genzyme: Consultancy, Speakers Bureau; Takeda-Shire: Speakers Bureau; Celgene-BMS: Consultancy. Fernández:Roche: Consultancy, Speakers Bureau. Salar:Janssen: Speakers Bureau; Roche: Speakers Bureau; Celgene: Speakers Bureau. Bellosillo:Qiagen: Consultancy, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau.

Evolutionary Dynamics of Non-Coding Regions in Pancreatic Ductal Adenocarcinoma

While the non-coding genome appears to play a role, the dynamic nature of noncoding alterations with respect to clonal progression of solid tumors remains unexplored. To address this gap in knowledge we performed multiregional whole genome sequencing and clonal analysis to elucidate the evolutionary dynamics of non-coding regions in pancreatic cancer relative to those of the coding genome. We find that the mutational burden of noncoding DNA is higher than coding DNA. However, when noncoding DNA was segregated into enhancer and non-enhancer regions, enhancers were more similar to coding DNA. Mutational signatures of noncoding and coding DNA further revealed the similar mutational spectra of enhancers to coding DNA whereas the mutational spectra of non-enhancer, noncoding DNA had an entirely different pattern. These findings shed light on the role of noncoding DNA in pancreatic cancer.

PML-RARA Fusion Transcripts Detectable 8 Months prior to Promyelocytic Blast Crisis in Chronic Myeloid Leukemia

Promyelocytic blast crisis arising from chronic myeloid leukemia (CML) is rare. We present a 40-year-old male who developed promyelocytic blast crisis 17 months after CML diagnosis, confirmed by the presence of the t(15;17) and t(9;22) translocations in the leukemic cells. Preserved nucleic acids from routine BCR-ABL1 testing provided a unique opportunity to evaluate clonal progression over time. Retrospective analysis demonstrated PML-RARA fusion transcripts were first detectable 8 months prior to blast crisis presentation. A review of 21 cases of promyelocytic blasts crisis published in the literature reveals a male predominance with earlier age at onset as compared to females. Interestingly, TKI therapy during chronic phase did not impact the time interval between diagnosis and promyelocytic blast crisis. Treatment with standard acute promyelocytic leukemia regimens provides more favorable outcomes with complete molecular remission. Although rare, it is important to consider a promyelocytic blast crisis when evaluating for transformation of CML due to its effective treatment with specific therapies.

Implications of Clonal Hematopoiesis for Precision Oncology

Clonal hematopoiesis (CH) is common in middle-aged and elderly populations and confers a risk of hematological malignancy and also death due to cardiovascular disease. Prior therapy with cytotoxic chemotherapy or radiation increases the risk of CH, especially that associated with TP53 or PPM1D mutations. CH can complicate interpretation of cell-free or circulating tumor DNA assays, since most blood DNA is derived from hematopoietic cells. The specific determinants of clonal progression are unclear, but the gene carrying the mutation, size of the mutant clone, and presence of multiple mutations appear to increase risk of evolution to myeloid leukemia. While CH is not yet modifiable, specific mutations such as TET2 or IDH1/IDH2 confer vulnerabilities to established drugs or developmental compounds, and investigators are developing clinical trials to try to exploit these vulnerabilities.

What To Tell Your Patient With Clonal Hematopoiesis And Why: Insights From Two Specialized Clinics

Acquired genetic mutations in hematopoietic stem or progenitor cells can lead to clonal expansion and imbalanced blood cell production. Clonal hematopoiesis is exceptionally common with human aging, confers a risk of evolution to overt hematologic malignancy, and also increases all-cause mortality and the risk of cardiovascular disease. The degree of risk depends on the specific mutation, number of mutations, mutant allele burden, and concomitant non-genetic risk factors (e.g., hypertension or cigarette smoking). People with clonal hematopoiesis may come to clinical attention in a variety of ways, including during the evaluation of a possible hematologic malignancy, as an incidental discovery during molecular analysis of a non-hematological neoplasm, after hematopoietic cell transplant, or as a result of germline testing for inherited variants. Even though the risk of clonal progression or a cardiovascular event in an individual patient may be low, the possibility of future clinical consequences may contribute to uncertainty and worry, since it is not yet known how to modify these risks. This review summarizes clinical considerations for patients with clonal hematopoiesis, including important points for hematologists to consider discussing with affected persons - individuals who may understandably be anxious about having a mutation in their blood that predisposes them to develop malignancy, but which is statistically more likely to result in a myocardial infarction or stroke. The increasing frequency with which people with clonal hematopoiesis are discovered and the need for counseling these patients is driving many institutions to create specialized clinics; we describe our own experience with forming such clinics.

Whole exome sequencing reveals the maintained polyclonal nature from primary to metastatic malignant peripheral nerve sheath tumor in two patients with NF1

Background Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas with high metastatic rates and poor overall patient survival. There are currently no effective therapies, underscoring the pressing need to define the molecular etiologies that underlie MPNST progression. The aim of this study was to examine clonal progression and identify the molecular events critical for MPNST spread. Methods In two patients with temporally and spatially distinct metastatic lesions, we employed whole exome sequencing (WES) to elucidate the genetic events of clonal progression, thus identifying the molecular events critical for MPNST spread. Results First, we demonstrated shared clonal origins for the metastatic lesions relative to the primary tumors, which were maintained throughout the course of MPNST progression, supporting the conclusion that cancer cells with metastatic potential already exist in the primary neoplasm. Second, we discovered TRIM23, a member of the Tripartite Motif family of proteins, as a regulator of MPNST lung metastatic spread in vivo. Conclusions The ability to track the genomic evolution from primary to metastatic MPNST offers new insights into the sequence of genetic events required for tumor progression and has identified TRIM23 as a novel target for future study in this rare cancer.

Clinical consequences of clonal hematopoiesis of indeterminate potential

Clonally restricted hematopoiesis is a common aging-associated biological state that predisposes to subsequent development of a hematological malignancy or cardiovascular death. Clonal expansion driven by leukemia-associated somatic mutations, such as DNMT3A, ASXL1, or TET2, is best characterized, but oligoclonality can also emerge without recognized leukemia-driver mutations, perhaps as a result of stochastic neutral drift. Murine models provide compelling evidence that a major mechanism of increased cardiovascular mortality in the context of clonal hematopoiesis is accelerated atherogenesis driven by inflammasome-mediated endothelial injury, resulting from proinflammatory interactions between endothelium and macrophages derived from circulating clonal monocytes. Altered inflammation likely influences other biological processes as well. The rate of development of overt neoplasia in patients with clonal hematopoiesis of indeterminate potential (CHIP), as currently defined, is 0.5% to 1% per year. Contributing factors to clonal progression other than acquisition of secondary mutations in hematopoietic cells (ie, stronger leukemia drivers) are incompletely understood. Disordered endogenous immunity in the context of increased proliferative pressure, short telomeres leading to chromosomal instability, an unhealthy marrow microenvironment that favors expansion of clonal stem cells and acquisition of new mutations while failing to support healthy hematopoiesis, and aging-associated changes in hematopoietic stem cells, including altered DNA damage response, an altered transcriptional program, and consequences of epigenetic alterations, are all potential contributors to clonal progression. Clinical management of patients with CHIP includes monitoring for hematological changes and reduction of modifiable cardiovascular risk factors; eventually, it will also likely include anti-inflammatory therapies and targeted approaches to prune emergent dangerous clones.

Clinical consequences of clonal hematopoiesis of indeterminate potential

Clonally restricted hematopoiesis is a common aging-associated biological state that predisposes to subsequent development of a hematological malignancy or cardiovascular death. Clonal expansion driven by leukemia-associated somatic mutations, such as DNMT3A, ASXL1, or TET2, is best characterized, but oligoclonality can also emerge without recognized leukemia-driver mutations, perhaps as a result of stochastic neutral drift. Murine models provide compelling evidence that a major mechanism of increased cardiovascular mortality in the context of clonal hematopoiesis is accelerated atherogenesis driven by inflammasome-mediated endothelial injury, resulting from proinflammatory interactions between endothelium and macrophages derived from circulating clonal monocytes. Altered inflammation likely influences other biological processes as well. The rate of development of overt neoplasia in patients with clonal hematopoiesis of indeterminate potential (CHIP), as currently defined, is 0.5% to 1% per year. Contributing factors to clonal progression other than acquisition of secondary mutations in hematopoietic cells (ie, stronger leukemia drivers) are incompletely understood. Disordered endogenous immunity in the context of increased proliferative pressure, short telomeres leading to chromosomal instability, an unhealthy marrow microenvironment that favors expansion of clonal stem cells and acquisition of new mutations while failing to support healthy hematopoiesis, and aging-associated changes in hematopoietic stem cells, including altered DNA damage response, an altered transcriptional program, and consequences of epigenetic alterations, are all potential contributors to clonal progression. Clinical management of patients with CHIP includes monitoring for hematological changes and reduction of modifiable cardiovascular risk factors; eventually, it will also likely include anti-inflammatory therapies and targeted approaches to prune emergent dangerous clones.