Loss Of Heterozygosity Research Articles

Classification of Philadelphia-Negative MPN As Low Risk and High Risk MPN Based on Peripheral Blood Values and Molecular Genetics Only

Background: Philadelphia-negative myeloproliferative neoplasms (Ph -MPN) are characterized by overproduction of differentiated hematopoietic cells mediated in the majority of cases by mutations in JAK2, MPL or CALR (MPN driver genes). Ph - MPN include polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF) and MPN, unclassifiable (MPN-U) which differ in clinical presentation, prognosis, fibrosis and leukemic transformation rate as well as therapeutic options. However, WHO based classification requires experienced pathologists, in particular to diagnose prefibrotic PMF as high risk MPN, and thus remains observer dependent. Aim: Observer independent classification of Ph - MPN solely based on molecular genetics and peripheral blood (PB) parameters. Patients and Methods: We analyzed 267 Ph - MPN cases diagnosed as ET (n=76), PV (n=74), PMF (n=68) or MPN-U (n=49) following the WHO4R classification including only patients with an MPN driver mutation (median age: 68 years [22-91]; female: 41%; median follow-up: 5 years). All samples were analyzed by cytomorphology and whole genome sequencing (median coverage 100x). Mutation status of 20 genes associated with myeloid malignancies were analyzed in detail. Results: Genomic landscape of the MPN cohort showed mutations in JAK2 in 222 (83%) (n=132: single mutation; n=90: either mutation and copy neutral loss of heterozygosity or mutation and 9p gain), in CALR in 36 (14%), and in MPL in 9 (3%) patients. The median number of mutations per patient was two (range: 1-6). Within the cohort, 141 patients (53%) harbored MPN driver mutations only, while in 126 (47%) one to 5 additional mutations were found. The most frequent additional mutations were ASXL1 (n=50), TET2 (n=49; thereof 15 biallelic), SRSF2 (n=26), DNMT3A (n=18), U2AF1 (n=11) and TP53 (n=9; thereof 3 biallelic). Regarding PB parameters, high WBC (≥11×10 9/ L) were detected in 127 patients (48%), high PLT (≥450×10 9/ L) in 116 (43%) and anemia (HB <♂12 or ♀11 g/dL) in 50 patients (19%). We then performed Cox regression analyses for overall survival (OS) including only genetic abnormalities and PB values. In univariate analyses, biallelic TET2 inactivation ( TET2bi: hazard ratio/HR: 3.789), RUNX1 (n=4; HR: 13.479), ASXL1 (HR: 1.886), SRSF2 (HR: 3.218) mutations as well as high PLT (HR: 0.440), high WBC (HR: 1.998) and PB blasts >1% (n=57; HR: 1.904) showed significant associations with OS (for all p<0.02), while no effect was observed for MPN driver mutations, DNMT3A and monoallelic TET2 mutations. In addition, anemia showed a trend towards poor prognosis (HR: 1.595; p=0.079). Following this, we determined high and low risk MPN. In the absence of thrombocytosis, high risk MPN was defined if any of the four following risk factors were present: i) mutation in ASXL1 or SRSF2 or RUNX1 or TET2bi, ii) PB blasts >1%, iii) high WBC or iv) anemia. In the presence of thrombocytosis, at least two of these risk factors were required (Figure 1). Survival analysis revealed a significantly worse median OS of high risk (n=137; 51%) compared to low risk MPN (n=130; 49%) (8 years vs. not reached; p<0.001). This proposed novel stratification reflected prognosis better than WHO entities (median OS: MPN-U: 7 years; MF: 8 years; PV/ ET not reached; overall p=0.001) (Figure 1A). All 76 ET cases (100%) and 44/74 PV cases (59%) were classified as low risk MPN, while 63/68 PMF cases (93%) and 44/49 MPN-U cases (90%) were classified as high risk MPN (Figure 1B). Within low risk and high risk MPN, no significant OS differences were observed between the WHO entities ( p=0.114 and p=0.355, respectively). Conclusions: JAK2-pathway mutated Ph - MPN can be categorized into low and high risk MPN based on molecular genetics and PB values only. The presence of these objectively assessable risk factors in a patient with ET or PV warrants for a profound reevaluation of bone marrow histology (eventually by a reference pathologist for MPN) and clinical course before a high risk MPN (including prefibrotic PMF) can be ruled out. Whether the predictive value of low risk MPN based on molecular genetics and PB values only could be sufficient to omit BM biopsy in selected patients, needs to be evaluated in further prospective studies.

Clinical Implications of TP53 Mutations/Allelic State in Patients (Pts) with Myelodysplastic Syndromes/Neoplasms (MDS) Treated with Hypomethylating Agents (HMA)- a Multicenter, Retrospective Analysis from the Validate Database

TK and JPB Co-first; RK, DS, and AMZ are Co-senior authors. Introduction MDS with TP53 mutations ( TP53 MT) are associated with high-risk disease features and rapid progression to AML. Effective treatment for this aggressive subtype is still not available. As compared to monoallelic TP53 MT, biallelic MT have been associated with adverse outcomes and worse overall survival (OS), but the impact of allelic status on treatment response and OS after HMA therapy are not well established. In this study, we analyzed TP53 allelic state for a large well annotated cohort of pts with MDS who were treated with HMAs. Methods The VALIDATE database includes pts with MDS treated with frontline HMA from 14 specialized centers. Pts were considered to have biallelic TP53 MT according to the ICC definition (Arber et al. 2022, Blood) if they meet any of the following: a. two TP53 MT (variant allelic frequency [VAF]>10% for each) b. single TP53 MT with copy neutral loss of heterozygosity (CN-LOH, available for 308 pts) c. TP53 MT with del17p and d. Single TP53 MT with VAF >50%. Time to event analyses were estimated using Kaplan-Meier estimator and groups were compared by the log-rank test from the time of HMA initiation. Only higher risk (HR)-MDS pts (IPSS>1 or IPSS-R>3.5) with BM biopsy within 180 days after HMA initiation were assessed for complete response (CR) and overall response rate (ORR) according to the IWG 2023 criteria (Zeidan et al, Blood 2023) (n=480). OS was calculated from time of HMA therapy initiation. This study was supported by an independent research grant from AbbVie. Results A total of 816 pts met eligibility and were included in this analysis. Median age was 68 years (IQR: 61-74) and 64% were men. MDS excess blast (EB) 1/2 was the most common MDS subtype (62%). Overall, 78% of the pts were treated with HMA monotherapy (26% decitabine [DEC] and 74% azacitidine [AZA]) and 22% received HMA as part of combination therapy. In total, 40% of the pts underwent allogenic HSCT. Median HMA treatment time was 5 months (mo), IQR:3-11. Median follow up time was 17 mo (IQR:9-31). TP53 MT were detected in 253 pts (31%) at time of MDS diagnosis, of whom 153 (60%) pts had biallelic TP53 MT and 100 (40%) pts had monoallelic TP53 MT. Missense MTs were identified in 80% of pts with monoallelic TP53 MT. TP53 MT/del17p (33%), TP53 MT/CN-LOH (28%), and multiple TP53 MT (25%) were the most common combinations in pts with biallelic TP53 MT. Pts with TP53 MT had higher rates of MDS-EB (74% vs. 61%, p<0.001). Complex karyotype was observed in 112 (73%) pts with biallelic TP53 MT and 81 (81%) pts with monoallelic TP53 MT (p=0.154). Median number of HMA cycles among pts with TP53 MT was 8 (IQR:5-12) and higher compared to pts without TP53 MT (5 cycles, IQR:3-9), p=0.032 ( Panel-A). In a logistic regression model for CR, TP53 MT was not associated with probability of achieving CR (1.4, 0.89-2.29). CR rates for pts with biallelic TP53 MT (18%) and monoallelic TP53 MT (14%) were not significantly different, p=0.37. There were also no significant differences in ORR observed between biallelic TP53 MT (59%) and monoallelic TP53 MT (54%) pts, p=0.39. Among TP53 MTpts treated with HMA monotherapy, pts treated with AZA compared to DEC had similar odds for ORR (OR:1.1, 95%CI: 0.7-1.6)/CR (OR: 0.8, 95%CI: 0.4-1.3) and hazard for OS (HR: 1.1, 0.9-1.4). As expected, the median OS (mo, 95%CI) for pts with TP53 MT (12, 10-13) was significantly lower than pts without TP53 MT (28, 25-31), p<0.001. Importantly, pts with biallelic TP53 MT (11, 9-14) had no significant difference in median OS compared to monoallelic TP53 MT (13, 11-17), p=0.268 (Panel-B). In a multivariable cox proportional hazard regression model for OS (hazards ratio, 95%CI) adjusted for age (1.0, 0.9-1.0), HSCT (0.4, 0.3-0.4), IPSS-M score (1.2, 1.1-1.3), and complex karyotype (1.4, 1.1-1.8), TP53 MT status (1.8, 1.4-2.3) was still associated with OS. However, biallelic TP53 MT had comparable risk to monoallelic TP53 MT (1.2, 0.9-1.7). Conclusions To our knowledge, VALIDATE is the largest database of MDS pts with TP53 MT who were treated with HMA. We confirm that TP53 MT have significant negative impact on survival, but not on response to HMA therapy. Importantly, we did not observe OS differences between monoallelic and biallelic TP53 MT. We also show that the type of HMA monotherapy used does not impact outcome of these pts. Overall, pts with TP53 MT have very poor outcomes and are in desperate need of new, effective therapies.

Pediatric Burkitt Leukemia with Underlying MSH2, MUTYH and POT1 Germline Variants: The Complexities and Relevance of Understanding Cancer Predisposition

Monoallelic germline variants in POT1 can lead to POT1-tumor predisposition (POT1-TPD), an autosomal dominant disorder leading to a spectrum of neoplasia, predominantly, melanoma, glioma, angiosarcoma and chronic lymphocytic leukemia (CLL). Recently, germline POT1 variants have been described to be associated with familial clonal hematopoiesis syndrome.The POT1 protein forms a part of shelterin complex, which has been implicated in DNA damage protection, telomere length regulation and chromosomal stability. The proposed mechanism of oncogenesis by POT1 variants is presumed to be secondary to increased telomere length causing genomic instability and cellular immortality. While POT1-TPD can cause CLL in adulthood, childhood leukemias have not been commonly observed. Here, we present a case of a previously healthy 12-year-old male diagnosed with Burkitt leukemia. He presented with leukocytosis and tumor lysis syndrome. Bone marrow evaluation revealed hypercellular marrow involved by Burkitt lymphoma, comprising over 90% marrow cellularity. Flow cytometry showed an abnormal lambda monotypic B-lymphoid population positive for CD10, CD19, CD20, CD22 and CD45. FISH studies demonstrated low-level MYC rearrangement (9%); negative for BCL2 and BCL6 rearrangement. EBV staining was negative. Acute leukemia NGS panel (63 genes) revealed a Tier 1 somatic variant in PHF6. RNA sequencing did not detect additional fusions. His chemotherapy course was complicated by significant toxicities including prolonged count recovery, severe peripheral neuropathy, neurotoxicity, kidney injury and several infectious complications. Given extensive family history of cancers on the maternal side including colorectal, duodenal, brain and skin cancers, a germline cancer predisposition NGS panel was sent from cultured skin fibroblast and identified two heterozygous pathogenic variants in MSH2 (c.942+3A>T) and MUTYH (c.1187G>A) and a heterozygous likely pathogenic variant in POT1 (c.233T>C). To delineate potential roles of these germline variants in oncogenesis, additional testing was performed. Immunohistochemical staining of bone marrow revealed intact nuclear expression of MLH1, PMS2, MSH2,and MSH6,suggesting microsatellite stability. Extended tumor profiling through whole exome sequencing did not identify additional somatic variants or copy loss of heterozygosity in MSH2, MUTYH and POT1 but additional somatic loss of function variants were seen in CCND3, CASP8, ARID1, and PTPRD.To assess potential functional consequences of the POT1 germline variant on DNA double-strand break (DSB) damage repair, the patient lymphocytes were exposed to 2 Gy ionizing radiation and mobilization and kinetics of DNA DSB repair was assessed at 1h and 24h post-irradiation (IR) using phosphorylation (p) of ATM, SMC1 and H2AX (gamma), and cell death and apoptosis was also assessed at 24h post-irradiation by flow cytometry. There was normal mobilization and repair of DNA DSBs in T, B and NK cells although there was a subset of CD19dim B cells that showed constitutive DNA DSBs at1h with partial repair at 24h. It is not evident that this defect can be attributed to the germline defects as they are also observed in several other clinical contexts, including post-chemotherapy. Telomere length assay from peripheral blood demonstrated telomere length at 50 th percentile in both lymphocytes and granulocytes. Children in families with adult-onset cancer predisposition syndromes may present with cancers but the potential contribution of these germline variants to these childhood cancers is unknown. Our molecular and functional studies on both germline and tumor tissues did not suggest direct contribution of these germline variants on lymphomagenesis, however there might be other mechanism of inactivation not identified from this study. This case highlights the complexities of understanding genetic susceptibility in childhood cancers. We propose a multifactorial oligogenic inheritance of cancer susceptibility in this patient through several mechanisms of genomic instability.

Heterogeneous patterns of heterozygosity loss in isolated populations of the threatened eastern barred bandicoot (Perameles gunnii).

Identifying and analysing isolated populations is critical for conservation. Isolation can make populations vulnerable to local extinction due to increased genetic drift and inbreeding, both of which should leave imprints of decreased genome-wide heterozygosity. While decreases in heterozygosity among populations are frequently investigated, fewer studies have analysed how heterozygosity varies among individuals, including whether heterozygosity varies geographically along lines of discrete population structure or with continuous patterns analogous to isolation by distance. Here we explore geographical patterns of differentiation and individual heterozygosity in the threatened eastern barred bandicoot (Perameles gunnii) in Tasmania, Australia, using genomic data from 85 samples collected between 2008 and 2011. Our analyses identified two isolated demes undergoing significant genetic drift, and several areas of fine-scale differentiation across Tasmania. We observed discrete genetic structures across geographical barriers and continuous patterns of isolation by distance, with little evidence of recent or historical migration. Using a recently developed analytical pipeline for estimating autosomal heterozygosity, we found individual heterozygosities varied within demes by up to a factor of two, and demes with low-heterozygosity individuals also still contained those with high heterozygosity. Spatial interpolation of heterozygosity scores clarified these patterns and identified the isolated Tasman Peninsula as a location where low-heterozygosity individuals were more common than elsewhere. Our results provide novel insights into the relationship between isolation-driven genetic structure and local heterozygosity patterns. These may help improve translocation efforts, by identifying populations in need of assistance, and by providing an individualised metric for identifying source animals for translocation.

SNP array genomic analysis of matched pairs of brain and liver metastases in primary colorectal cancer

PurposeBrain metastasis formation is a rare and late event in colorectal cancer (CRC) patients and associated with poor survival. In contrast to other metastatic sites, the knowledge on chromosomal aberrations in brain metastases is very limited.MethodsTherefore, we carried out single nucleotide polymorphism (SNP) array analyses on matched primary CRC and brain metastases of four patients as well as on liver metastases of three patients.ResultsBrain metastases showed more chromosomal aberrations than primary tumors or liver metastases. Commonly occurring aberrations were gain of 8q11.1-q24.3 (primary CRC), gain of 13q12.13-q12.3 (liver metastases), and gain of 20q11.1-q13.33 (brain metastases). Furthermore, we found one copy-neutral loss of heterozygosity (cn-LOH) region on chromosome 3 in primary CRC, three cn-LOH regions in liver metastases and 23 cn-LOH regions in brain metastases, comprising 26 previously undescribed sites.ConclusionThe more frequent occurrence of cn-LOHs and subsequently affected genes in brain metastases shed light on the pathophysiology of brain metastasis formation. Further pairwise genetic analyses between primary tumors and their metastases will help to define the role of affected genes in cn-LOH regions.

Application of array comparative genomic hybridization in clinical diagnostics of intellectual disability/developmental delay in children

Background: This study was designed to analyze and evaluate the potential pathogenic genomic imbalance in children with unexplained intellectual disability (ID) and/or developmental delay (DD) and its association with phenotypes, and to investigate the value of array-based comparative genomic hybridization (array-CGH). Methods: A total of 72 Children with ID/DD were evaluated by array-CGH for detection of genomic copy number variations (CNVs). Results: The results of the array-CGH revealed that 10(14%) of the 72 patients had pathogenic CNVs, in that six cases had pathogenic CNV in a single chromosome, 2 cases had multiple microdeletions and 2 cases had combined microdeletion and microduplication, 2 cases had pathogenic CNVs in chromosome 1p36 and Xq28 region. One case had variation of unknown significance in chromosome region 15q11.2. Large bands of copy neutral loss of heterozygosity were detected in 2 cases comprising more than 10% of genome. Conclusions: Array-CGH being a high-throughput and rapid tool, allows for the etiological diagnosis in some of the children with unexplained ID/DD.

ERG Is a New Predisposition Gene for Bone Marrow Failure and Hematological Malignancy

There remain gaps in our knowledge of hereditary and sporadic causes of hematological malignancy (HM) and bone marrow failure (BMF) that prevent optimal diagnosis, disease surveillance and treatment. Here we report the discovery of ERG as a novel predisposition gene for BMF and HM. ERG is a known oncogene, typically via gene-fusions, leading to dysregulated ERG overexpression in blood and solid cancers. We identified a germline ERG ETS domain variant p.Y373C segregating with thrombocytopenia in a mother, who progressed to AML (27 yr) and then therapy-related MDS (35 yr), and in her 2 sons. All three showed copy neutral loss of heterozygosity of all or part of chromosome 21q, including the ERG locus, with the oldest son showing at least 2 somatic genetic rescue (SGR) events. The possibility of causal RUNX1 variants were ruled out, with the smallest somatic cnLOH event beginning within the RUNX1 gene, but not encompassing the RUNT domain where the majority of pathogenic missense variants are located. ERG, a highly constrained gene (LOEUF <0.33), is critical for definitive hematopoiesis, adult hematopoietic stem cell (HSC) function and platelet maintenance. An identical corresponding heterozygous germline variant (p.Y343C) in ERG’s closest gene by homology, FLI1, causes platelet-type bleeding disorder-21 (BDPLT21, OMIM #617443). Through global collaborations, we have identified 15 heterozygous variants in the ERG gene, 13 of which are missense and 2 truncating variants, in 17 individuals with cytopenia and/or HM (mainly myeloid) or lymphedema (Table). Onset of hematological symptoms ranged from birth to 38 years for truncating and constrained ETS domain variants. Of these 15 variants, 12 have been confirmed germline including 2 de novo. Only 4 meiotic transmissions are observed. None of the missense variants in the highly conserved ETS domain of ERG which mediates DNA binding, protein-protein interactions and nuclear localization, are present in gnomAD. We have functionally characterized 19 ERG variants, 12 potentially pathogenic, 1 known mouse pathogenic variant and 3 population controls demonstrating that most ETS domain missense variants display loss-of-function (LOF) characteristics disrupting transcriptional transactivation (Figure), DNA-binding and/or nuclear localization in vitro. Robust preliminary data from ex vivo models of ERG overexpression in mouse fetal liver cells in tissue culture (cytokine-independence), a mouse transplant assay and previous germline mutant Erg mouse models are concordant with ETS domain missense variants being LOF compared to wildtype ERG and benign controls. Together, these data provide clinical, in vitro and ex vivo functional studies implicating LOF variants in hematological disease predisposition. LOF ERG mutations also occur in sporadic cases of HM. Recently, as part of a Genomics England Research Consortium population study, 4 truncating ERG variants were described in 7 individuals across 4 families with 3 meiotic transmissions and a de novo case with primary lymphedema (1) and we add 2 novel missense variants here. One patient showed SGR across the ERG locus in blood. Blood phenotypes were not described. Our results demonstrate that germline ERG variants predispose to diverse cytopenia, BMF and HM in both children and adults. In our family mentioned above, the mother received an unrelated alloHSCT due to t-MDS while her 2 sons with cytopenias continue to be monitored. The natural history of this new syndrome will require careful identification of germline lesions with additional longitudinal studies in more patients and families needed. This ERG syndrome parallels GATA2 deficiency syndrome (HM and lymphedema) and RUNX1 Familial Platelet disorder-myeloid malignancy (thrombocytopenia and HM). Like the well-known disease genes GATA2 and RUNX1, ERG is also a member of the transcription factor heptad involved in HSC maintenance and differentiation. ERG adds to a growing list of genes whose unregulated expression contributes to HM and other cancers. Identification of causal germline ERG variants like those outlined in this study, has direct clinical implications for patient and family management including diagnosis, counselling, surveillance and treatment strategies such as selection of bone marrow transplant donors and potential for targeted therapies including gene and cell therapy. Reference 1. Greene D et al. Nat.Med. 29:679-688 2023

Homozygous gene disruption in diploid yeast through a single transformation

As industrial shochu yeast is a diploid strain, obtaining a strain with mutations in both allelic genes was considered difficult. We investigated a method for disrupting two copies of a homozygous gene with a single transformation. We designed a disruption cassette containing an intact LYS5 flanked by nonfunctional ura3 gene fragments divided into the 5'- and 3'-regions. These fragments had overlapping sequences that enabled LYS5 removal as well as URA3 regeneration through loop-out. Furthermore, both ends of the disruption cassette had an additional repeat sequence that allowed the cassette to be removed from the chromosome through loop-out. First, 45 bases of 5'- and 3'-regions of target gene sequences were added on both ends of this cassette using polymerase chain reaction; the resultant disruption cassette was introduced into a shochu yeast strain (ura3/ura3 lys5/lys5); then, single allele disrupted strains were selected on Lys drop-out plates; and after cultivation in YPD medium, double-disrupted strains, in which replacement of another allelic gene with disruption cassette by loss of heterozygosity and regeneration of URA3 in one of the cassettes by loop-out, were obtained by selection on Ura and Lys drop-out plates. The disruption cassettes were removed from the double-disrupted strain via loop-out between repeat sequences in the disruption cassette. The strains that lost either URA3 or LYS5 were counter-selected on 5-fluoroorotic acid or α-amino adipic acid plates, respectively. Using this method, we obtained leu2/leu2 and leu2/leu2 his3/his3 strains in shochu yeast, demonstrating the effectiveness and repeatability of this gene disruption technique in diploid yeast Saccharomyces cerevisiae.

The loss of neoantigens is an important reason for immune escape in multiple myeloma patients with high intratumor heterogeneity.

Intratumor heterogeneity (ITH) is an important factor for clinical outcomes in patients with multiple myeloma (MM). High ITH has been proven to be a key reason for tumor immune escape and treatment resistance. Neoantigens are thought to be associated with ITH, but the specific correlation and functional basis for this remains unclear. We study this question through the whole-exome sequencing (WES) data from 43 high ITH newly diagnosed MM patients in our center. Mutant allele tumor heterogeneity (MATH) was conducted to quantify ITH. The cutoff value for high intratumor heterogeneity was determined by comparing MATH of different kinds of tumors. NeoPredPipe was performed to predict neoantigens and binding affinity. Compared to other tumors, MM has a relatively low tumor mutation burden but a high ITH. Patients with high MATH had significantly shorter progression-free survival times than those with low MATH (p = 0.001). In high ITH samples, there is a decrease in strong-binding neoantigens (p = 0.019). The loss of strong-binding neoantigens is a key factor for insensitivity to therapy (p = 0.015). Loss of heterozygosity in HLA was not observed. In addition, patients with fewer neoantigens loss had higher rates of disease remission (p = 0.047). CD8 + T cells (p = 0.012) and NK cells (p = 0.011) decreased significantly in patients with high neoantigens loss rate. A prediction model based on neoantigens was built to evaluate the strength of immune escape. The loss of strong-binding neoantigens explains why tumors with high ITH have a higher degree of immune escape and may be feasible for deciding the clinical treatment of MM.

HLA Class I Allele Loss and Bone Marrow Transplantation Outcomes in Immune Aplastic Anemia

In patients with immune-mediated acquired aplastic anemia (AA), HLA class I alleles often disappear from the surface of hematopoietic progenitor cells, potentially enabling evasion from cytotoxic T lymphocyte-mediated pathogenesis. Although HLA class I allele loss has been studied in AA patients treated with immunosuppressive therapy (IST), its impact on allogeneic bone marrow transplantation (BMT) has not been thoroughly investigated. The purpose of this study was to evaluate the clinical implications of HLA class I allele loss in patients with acquired AA undergoing allogeneic BMT. The study enrolled acquired AA patients who underwent initial BMT from unrelated donors through the Japan Marrow Donor Program between 1993 and 2011. The presence of HLA class I allele loss due to loss of heterozygosity (HLA-LOH) was assessed using pretransplantation blood DNA and correlated with clinical data obtained from the Japanese Transplant Registry Unified Management Program. A total of 432 patients with acquired AA were included in the study, and HLA-LOH was detected in 20 of the 178 patients (11%) available for analysis. Patients with HLA-LOH typically presented with more severe AA at diagnosis (P=.017) and underwent BMT earlier (P < .0001) compared to those without HLA-LOH. They also showed a slight but significant recovery in platelet count from the time of diagnosis to BMT (P=.00085). However, HLA-LOH status had no significant effect on survival, engraftment, graft failure, chimerism status, graft-versus-host disease, or other complications following BMT, even when the 20 HLA-LOH+ patients were compared with the 40 propensity score-matched HLA-LOH- patients. Nevertheless, patients lacking HLA-A*02:06 or HLA-B*40:02, the alleles most frequently lost and associated with a better IST response, showed higher survival rates compared to those lacking other alleles, with estimated 5-year overall survival (OS) rates of 100% and 44%, respectively (P=.0042). In addition, in a specific subset of HLA-LOH- patients showing clinical features similar to HLA-LOH+ patients, the HLA-A*02:06 and HLA-B*40:02 allele genotypes correlated with better survival rates compared with other allele genotypes, with estimated 5-year OS rates of 100% and 43%, respectively (P=.0096). However, this genotype correlation did not extend to all patients, suggesting that immunopathogenic mechanisms linked to the loss of certain HLA alleles, rather than the HLA genotypes themselves, influence survival outcomes. The survival benefit associated with the loss of these two alleles was confirmed in a multivariable Cox regression model. The observed correlations between HLA loss and the pretransplantation clinical manifestations and between loss of specific HLA class I alleles and survival outcomes in AA patients may improve patient selection for unrelated BMT and facilitate further investigations into the immune pathophysiology of the disease.

A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

PATH-31. RECURRENT COPY NUMBER CHANGES IN GLIOBLASTOMA, IDH-WILDTYPE: ARE OTHER +7/-10 PATTERNS CLINICALLY RELEVANT?

Abstract Glioblastoma (GBM), IDH-wildtype (WT) is currently defined as an IDH-WT and H3-WT diffuse astrocytic glioma with molecular diagnostic features including whole +7/−10, TERT promoter (TERTp) mutation, and EGFR amplification and/or morphological diagnostic features such as necrosis and microvascular proliferation. To further understand the diversity of copy number signatures of GBM, IDHWT and evaluate the diagnostic utility of other recurrent copy number changes, we analyzed paired chromosomal microarray and next-generation sequencing mutational profiling data from 418 adult IDH-WT and H3-WT tumors with GBM morphological diagnostic features. The most common molecular features were TERTp mutations (85%), CDKN2A/B heterozygous/homozygous deletion (78%/66%), +7/-10 (63%), EGFR amplification (41%), +19 (36%), PTEN mutations (35%), partial -13 (33%), partial -14 (33%), +20 (32%), TP53 mutations (32%), and partial -22 (29%). At least one molecular diagnostic biomarker was present in 92% (n=385) of cases, and 29% (n=121) of cases had all three. The +7/-10 biomarker was the most frequent chromosomes 7/10 copy number change (68%, n=265). Other +7/-10 patterns were seen in 30% (n=125) of cases and included isolated +7 or -10, combinations of +7 and -10 with partial +7, partial -10 or 10 copy neutral loss of heterozygosity (cnLOH). Overall, 93% (390/418) of cases had whole and other chromosome 7/10 copy number changes. In comparison to tumors with +7/-10, tumors with other +7/-10 patterns were significantly enriched for partial 13 loss (Bonferroni adjusted p=0.04) but were not associated with significant difference in patient overall survival (p=0.30, 158/418 had available survival data). Our findings reiterate and expand the spectrum of copy number changes associated with GBM, IDH-WT. The similar overall survival rates between cases with the +7/-10 biomarker and those with other +7/-10 patterns suggest that other +7/-10 patterns may be functionally equivalent to +7/-10 and diagnostically relevant as a molecular diagnostic biomarker for this tumor type.

EPCO-44. RHABDOID TUMOR PREDISPOSITION SYNDROME DUE TO A GERMLINE SMARCB1 MUTATION IN TWO INFANT SIBLINGS

Abstract INTRODUCTION Rhabdoid tumors (RTs) are highly aggressive malignancies caused by a loss–of–function mutation in the SMARCB1 tumor suppressor gene on chromosome 22 that makes up the SW1/SNF chromatin remodeling complex involved in gene transcription. In rhabdoid tumor predisposition syndrome (RTPS), a biallelic germline SMARCB1 mutation markedly increases the risk of affected individuals developing early, aggressive, and rapidly progressing RTs, most commonly in the CNS, referred to as atypical teratoid rhabdoid tumors (AT/RT), and the kidneys, but oftentimes presents with synchronous lesions. In this case series, we present a family affected by RTPS with a recurrence of AT/RT in two infant siblings with the same germline SMARCB1 mutation. CASE SERIES DESCRIPTION An 8-month-old female was brought to the emergency department for 3 months of vomiting, developmental regression, and CN VI palsy. Brain imaging revealed a posterior fossa mass, acquired obstructive hydrocephalus, and spinal metastasis. Four years later, her 3-month-old sister was also brought in for evaluation for 2 months of vomiting, irritability, increasing head circumference, and vertical gaze palsy. Her brain imaging similarly showed a posterior fossa mass and acquired obstructive hydrocephalus, but no metastasis. Both siblings underwent subtotal tumor resections and ventriculoperitoneal shunt placement followed by chemotherapy. While on active treatment, they experienced rapid disease progression and ultimately succumbed to their illness. Genetic testing on the family identified the same in-frame deletion in the SNF5 homology domain of SMARCB1 and loss of heterozygosity of chromosome 22 in the two sisters; their mother was found to be a carrier. CONCLUSION This case series describes a family affected by RTPS, demonstrating the risk of familial recurrence in SMARCB1 germline mutations and emphasizing the need for genetic testing and counseling for these families should they wish to have additional children.

Unexpectedly high prevalence of structural genomic variations in the Long QT Syndrome: evidence for broader clinical uptake

Abstract Background Long QT syndrome (LQTS) is an inherited channelopathy predisposing young and otherwise healthy individuals to ventricular arrhythmias causing syncope or sudden cardiac death. Point mutations in the KCNQ1, KCNH2, and SCN5A genes are the most common causes of LQTS, and up to 75% of patients can be genotyped by standard DNA sequencing. However, standard genetic testing fails to detect structural genomic rearrangements - including large deletions or duplications of DNA segments - known as copy number variants (CNVs). While previous studies suggested that CNVs account for less than 5% of genotype-negative cases(1-2), no recent data have been reported. Purpose The study aimed to determine the prevalence and spectrum of CNVs in LQTS susceptibility genes in unrelated patients who tested negative for point mutations in a panel of 16 genes associated with LQTS. Methods Next-generation sequencing (NGS) was used to screen CNVs in unrelated, clinically diagnosed LQTS patients without point mutations in 16 genes associated with LQTS. Loss of heterozygosity in LQTS-associated genes was used to assess the presence of CNVs using standard commercial software. The multiplex ligation-dependent probe amplification (MLPA) assay was used to confirm the CNVs identified by NGS. Results The cohort included 48 patients (63% women; mean age 31±19 years, range 3 years to 68 years) with definitive clinical diagnosis of LQTS (mean QTc 490±33 ms) according to the 2022 ESC guidelines(3). Overall, 7/48 patients (15%, 95% CI 6-28%) had CNVs. Six of the seven CNVs involved KCNQ1 in the heterozygous state, including three large deletions (exons 3-6, exons 12-15, and exon 16) and three duplications (exon 2, exons 3-6, and exons 3-15), whereas the remaining patient had a complete KCNH2 deletion. MLPA confirmed all CNVs identified by NGS. Of note, no CNVs were identified in LQTS-related genes whose pathogenicity has been questioned. Conclusions Our study highlights the unexpectedly high prevalence of structural genomic rearrangements in the KCNQ1 and KCNH2 genes in LQTS patients. Routine screening for copy number variants in LQTS patients is critical for accurate diagnosis, risk stratification, and personalized treatment strategies.Figure 1

Region-specific Wnt signaling responses promote gastric polyp formation in patients with familial adenomatous polyposis.

Germline adenomatous polyposis coli (APC) mutation in patients with familial adenomatous polyposis (FAP) promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding nonpolyp corpus biopsies and organoids from patients with FAP for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss of heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in the antrum. Our findings suggest that heterozygous APC mutation in patients with FAP may be sufficient to drive polyp formation in the corpus region while subsequent loss of heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared with the rare, yet adenomatous polyps in the antrum.

Bomedemstat (IMG-7289), an LSD1 Inhibitor, Manages the Signs and Symptoms of Essential Thrombocythemia (ET) While Reducing the Burden of Cells Homozygous for Driver Mutations

The ultimate goal in the treatment of ET is to eliminate mutant cells with the capacity to both proliferate and self-renew. Lysine-specific demethylase-1, LSD1, is an enzyme critical for regulating the proliferation of hematopoietic stem cells and the maturation of progenitors (Sprussel et al. 2012). Bomedemstat is an orally active LSD1 inhibitor that reduces platelets, inflammatory cytokines, and the mutant cell burden in mouse models of myeloproliferative neoplasms (Jutzi et. al., 2019). All current treatments - hydroxyurea, interferon, or anagrelide - have their limitations and do not substantially affect the natural history of ET. IMG-7289-CTP-201 was a global, open-label, Phase 2b study of bomedemstat (IMG-7289, MK-3543) taken once daily for 24+ weeks in patients with ET resistant to or intolerant of at least one standard treatment (NCT04254978). The primary endpoint was reduction of platelets to &amp;lt;400 x 10 9/L in the absence of new thromboembolic events. Exploratory endpoints included changes in frequencies of mutant alleles (VAF) assessed by deep sequencing (median exonic depth =1784 reads) of 261 genes of germline and somatic DNA. Homozygosity of mutant alleles was imputed when VAF &amp;gt;50% and/or loss-of-heterozygosity (LOH) was evident based on the difference between the minor allele frequency of flanking single nucleotide polymorphisms (“MAF method”) in germline versus granulocyteDNA. Single cell genotypes (scDNA) (Tapestri ®) were determined in stem/progenitor (CD34+) and monocytes (CD14+) cells in patients with LOH (using the MAF method) showing a decrease in LOH in whom follow-up samples were available. For patients treated ≥24 wks, 95% (61/64) had reduced platelet counts to ≤400 x 10 9/L without new thromboembolic events, in a median time of 10 wks. In the first 48 weeks, four patients experienced a thromboembolic event ( per PI) (1 PE, 2 TIAs, and 1 transient global amnesia). Of the 14/73 (19%) patients with Day 1 WBC count &amp;gt;10 x 10 9/L, 100% treated for ≥24 wks had reduced the WBC count to ≤10 x 10 9/L; mean Hb remained stable. In patients with baseline MPN-SAF TSS &amp;gt;20 (28/73), at Wk 24, 78% (18/23) experienced an improvement with 52% (12/23) improving ≥10 points. The median baseline score for worst fatigue was 4.0 (N=73); by Wk 24, the score had improved to 3.0 (N=62). Among all patients (N=73), the most common AEs regardless of causality were dysgeusia (55%), constipation (38%), thrombocytopenia (34%), and arthralgia (27%). Twenty-four patients reported serious AEs (SAE), with related events ( per PI) in 4 patients. Twenty-one patients discontinued treatment, 11 due to AEs (3 for dysgeusia), 7 subject's decisions, 1 PI decision, 1 disease progression, and 1 unrelated death due to aspiration pneumonia. There have been no safety signals or deaths related to drug in patients treated for up to 979 days. At Wk 24, 86% patients remained on study and 75% at Wk 48. At ~Wk 24, 39 of 46 (85%) of evaluable patients had a decrease in driver VAF (Fig. 1). The greatest changes in VAF occurred in patients with cells homozygous for a driver mutation (Fig. 1). Nine of ten patients with homozygous driver clones had follow-up data: by bulk sequencing of germline+granulocyte DNA, 7 showed a reduction in the proportion of homozygous cells. In all cases tested, scDNA of CD14+ (monocytes) and CD34+ (stem/progenitor) cells were qualitatively similar to results obtained with bulk granulocyte sequencing (Fig.2). By bulk sequencing, the proportion of homozygous cells dropped by a mean of 66% (-4% to -100% (-100% = homozygosity not detectable)). Decreases in LOH by bulk and sc sequencing during treatment with bomedemstat were identified in patients with homozygosity at JAK2, CALR, and MPL. This study demonstrates that treatment with bomedemstat results in a consistent hematologic response in the majority of patients with ET. Further, deep bulk sequencing of germline+granulocyte DNA reveals LOH ( i.e., homozygosity) events with high accuracy that enables determination of the distribution of homozygote, heterozygote and wild-type genotypes. Furthermore, this study shows that sequencing germline+granulocyte DNA provides a reliable qualitative view of the impact of treatment on the genotypic distribution among CD34+ cells as revealed by scDNA genotyping. Finally, bomedemstat showed activity reducing stem/progenitor cells homozygous for mutations in JAK2, CALR, and MPL.

TP53 mutations and Their Impact on Survival in Patients with Myeloproliferative Neoplasms

Introduction: TP53 mutations in patients with myeloproliferative neoplasms (MPN) are associated with poor prognosis, including progression to blast phase MPN. However, low variant allele fraction (VAF) TP53 mutations have been reported to remain stable over years in chronic phase MPN. A major unmet clinical need in MPN is the ability to discriminate patients with TP53-mutant MPN who are at high-risk of secondary AML (sAML) and warrant immediate intervention from those who are at lower risk of sAML in whom active surveillance can be employed. Therefore, we sought to identify parameters associated with leukemic transformation and overall survival in the context of MPN with genetic aberrations in TP53. Materials and Methods: We retrospectively analyzed a cohort of 947 MPN patients from the Dana-Farber Cancer Institute Hematologic Malignancies Data Repository (HMDR) with at least one clinical next-generation sequencing (NGS) panel performed. Patient characteristics such as age at MPN diagnosis, gender, MPN subtype and driver mutations were recorded. Furthermore, information about the course of disease was extracted including occurrence of sAML and overall survival (Figure 1). We also analyzed type and number of additional mutations as well as cytogenetics. With respect to TP53-specific parameters, we evaluated the number of TP53 mutations, TP53 VAF, loss of heterozygosity (LOH) at the TP53 locus, phenotypic annotations of TP53 (i.e. PHANTM score) and 17p deletion. We defined “multi-hit” TP53 as the presence of two or more TP53 mutations, TP53 VAF higher than 50%, TP53 mutation plus 17p deletionor TP53 mutation and documented LOH. Results: A total of 947 patients were analyzed, of which 40 harbored at least one detectable TP53 mutation. A total of 13 patients were found to have a multi-hit TP53 mutations defined by &amp;gt; 50% VAF in 6 patients, two or more TP53 mutations in 5 patients and TP53 mutation + 17p deletion in 5 patients. The MPN diagnosis at time of TP53 mutation detection was post ET/PV myelofibrosis (secondary MF) (n=23, 58%), primary myelofibrosis (MF) (n=7, 18%), pre-fibrotic MF (n=2, 5%), essential thrombocythemia (ET) (n=6, 15%) and polycythemia vera (PV) (n=2, 5%). Two patients with ET and one patient with PV did not have a concurrent in-house bone marrow biopsy performed at the time the TP53 mutation was detected. Two patients with ET developed sAML within 12 months of TP53 mutation detection, without prior mention of fibrosis. Age at first MPN diagnosis was not significantly different between patients with or without TP53 mutation. The average time from initial MPN diagnosis to detection of the first TP53 mutation was 9 years (range: 0-33 years). The most common MPN driver mutation among patients with TP53 mutations was JAK2 (75%), followed by CALR (13%)and MPL (5%). Out of all TP53-mutated patients, 8% showed a triple negative status. The most frequent additional mutations among patients with TP53 mutations were TET2 (25%), U2AF1 (15%), ASXL1 (13%), and DNMT3A (10%). There was no significant difference between single-hit and multi-hit TP53 status regarding MPN subtype, driver mutations and co-mutations (Table 1). Seven patients (single-hit: 15%, multi-hit: 23%) with a TP53 mutation developed sAML during the course of their disease, compared with only 3% of all patients without a TP53 mutation and 50% (single-hit: 41%, multi-hit: 69%) were deceased at the time of the last follow-up compared to 18% of all patients without a TP53 mutation. We focused on overall survival from the initial MPN diagnosis and considered whether patients developed bone marrow fibrosis during their disease course (Figure 1). Survival did not differ significantly between single-hit TP53 and patients with multi-hit TP53 (p=0.2), but survival did differ significantly between multi-hit TP53 patients and TP53 wildtype patients with MF/prefibrotic MF/Secondary MF (p=0.02) as well as compared to all MPN patients without a TP53 mutation (p&amp;lt;0.001). Survival was not significantly different between single-hit TP53 and TP53 wildtype MF/prefibrotic MF/Secondary MF patients (p=0.4). Conclusions: In a large cohort of 947 molecularly characterized MPN patients, 4% of the cohort developed a TP53 mutation during their course of disease. 18% of all TP53-mutant patients developed sAML with an adverse effect on overall survival for patients with multi-hit but not single-hit TP53 mutations.

Clonal Hematopoiesis and Venous Thromboembolism in the UK Biobank

Background: Patients with myeloid malignancies have an increased risk of thrombosis, but less is known about the risk of thrombosis in clonal hematopoiesis of indeterminate potential (CHIP). We evaluated genotype-specific associations between CHIP and both prevalent and incident venous thromboembolism (VTE) in the largest such analysis to date of the UK Biobank. Methods: We examined 425,573 individuals without a prevalent hematologic malignancy diagnosis from the UK Biobank. Whole exome sequencing and single nucleotide polymorphism array data were analyzed to identify individuals with CHIP and/or mosaic chromosomal alterations (mCA), respectively. Prevalent and incident VTE cases were ascertained with respect to the time of DNA sampling. VTE events were identified based on ICD10 codes, including but not limited to, pulmonary embolism, phlebitis/thrombophlebitis of deep veins of lower veins, portal vein thrombosis, and Budd Chiari syndrome. Median follow-up time for incident VTE was 11.8 years. Associations of CHIP and VTE were assessed using logistic regression for prevalent analyses and Cox proportional hazards for incident analyses. The association analyses were adjusted for age at the time of DNA sampling, sex, Caucasian ancestry, genetic principal components, ever smoked status, and body mass index (BMI) as covariates. Results: Prevalent VTE and CHIP: While CHIP overall was not associated with an increased risk of prevalent VTE (odds ratio (OR) 1.06; 95% CI 0.87-1.28, P= 0.56) (Figure 1), JAK2-mutant CHIP and JAK2 CHIP with loss of heterozygosity at the JAK2 locus (LOH 9p) were powerfully associated with increased risk of thrombosis, OR = 7.49 (95% CI 3.02-18.56, P=1.41e-05) and 6.74 (95% CI 1.62-28.08; P=8.71e-03). Having both CHIP and autosomal mCA was associated with a nominally increased risk of VTE (OR 1.76; 95% CI 1.02-3.06, P= 0.04). Otherwise, non- JAK2 CHIP , ASXL1 CHIP , TET2 CHIP , DNMT3A CHIP were not associated with increased prevalent VTE, regardless of clone size. Incident VTE and CHIP: CHIP was modestly associated with incident VTE with a hazard ratio of 1.15 (1.03-1.28; P=0.01), and CHIP with a larger clone size (VAF &amp;gt; 10%) was associated with a hazard ratio of 1.22 (95% CI 1.08-1.39; P= 0.001) (Figure 2). Consistent with our analysis of prevalent VTE, JAK2-CHIP and JAK2-CHIP with LOH 9p were strongly associated with incident VTE with hazard ratios of 3.76 (95% CI 1.79-7.9, P= 0.0004) and 4.89 (95% CI 1.84-13.04; P=0.0015). Other associations, including overall CHIP, CHIP with VAF &amp;gt;10%, CHIP together with autosomal mCA, autosomal mCA, non- JAK2 CHIP, TET2 CHIP with VAF &amp;gt;10%, showed modest increased hazard ratios between 1 and 2. Conclusions: In this study of risk of thrombosis in the UK Biobank, CHIP was not significantly associated with prevalent VTE and was modestly associated incident VTE. Among CHIP genotypes analyzed, only JAK2 mutations were significantly associated with both prevalent and incident VTE risk (OR 7.49 and HR 3.76, respectively). JAK2-mutant CHIP may be an undetected contributor to cases of VTE and may have therapeutic implications.

The Temporal Evolution of Chromosome 1q Gain and Hyperdiploidy and Its Impact on Clinical Outcomes in Multiple Myeloma

INTRODUCTION: Multiple myeloma (MM) evolution is shaped by the acquisition and selection of distinct genomic events over time. While various temporal patterns have been identified, the precise chronological order in which key clonal genomic defining events are acquired, and their specific impact on clinical outcomes remain to be fully elucidated. METHODS: To decipher the genomic life history of MM and its impact on clinical outcomes, we interrogated the high coverage (80X) whole genome sequencing (WGS) of 378 MM patients. Overall, 319 and 59 WGS were generated from samples collected at diagnosis and at relapse, respectively. Newly diagnosed MM (NDMM) patients were treated upfront with proteosome inhibitors, lenalidomide, dexamethasone, +/- monoclonal antibodies. To estimate the relative and absolute temporal relationship among clonal events, we leveraged the molecular time approach, based on the corrected ratio between duplicated and non-duplicated single nucleotide variants (SNV). RESULTS: Overall, 300/378 (79%) patients had at least one large chromosomal gain defined as &amp;gt;1Mb with &amp;gt;50 clonal SNV eligible for molecular time analysis, for a total of 1493 events tested. Consistent with recent findings, using clock-like mutational signatures SBS1-SBS5, we estimated that the first gain in MM is typically acquired around the second or third decade of life, with a median time lag of approximately 33 years between acquisition and diagnosis. In 158/178 (89%) of hyperdiploid (HRD) patients, all trisomies of odd-numbered chromosomes were acquired simultaneously. Among patients with canonical IGH translocations and HRD, the molecular time when trisomies were acquired was usually late compared to HRD without canonical IGH translocations (p&amp;lt;0.0001). Next, we examined the temporal relationship between IGH canonical translocations and gains linked to the same IGH canonical translocation in 29 patients. We consistently observed that when IGH canonical translocations co-occurred with HRD (n=7), and/or 1q gain (n=8) in the same patient, the canonical IGH translocation was always found to be the initiating event, occurring earlier than the chromosomal gains. Interestingly, in 104/178 (58.4%) HRD patients, the earliest multi-gain event was not always restricted to odd chromosomes, but in involved other non-HRD chromosomes such as: 6p (n=43) and 1q (n=32), demonstrating that gain 1q, which is assumed to occur at progression, can also be acquired very early in disease life history. A total of 92 and 21 patients with 1q gain and 1q amp (&amp;gt;3 copies) were identified, respectively. In 14/21 (66%) with 1q amp, the first and second 1q extra copies were acquired in two distinct and independent time windows. Interestingly, patients with early 1q gain showed the same clinical outcomes as 1q amp, and inferior progression free survival outcomes compared to late 1q gain (p=0.01; Figure 1). In 18/21 1q amp the first 1q duplication was acquired early, suggesting that the adverse prognostic effect of 1q gain/amp in NDMM may be more dependent on the timing of its acquisition rather than the number of extra copies gained. To investigate late temporal patterns and differentiate selection versus acquisition of large gains at relapse after autologous stem cell transplant (ASCT), we interrogated 25/59 (42%) RRMM patients exposed to melphalan with detectable SBS-MM1. To establish the temporal order of events, we employed the melphalan mutational signature, SBS-MM1, as a unique temporal barcode (Diamond et al. Blood 2023). Overall, we observed that 21% of all loss of heterozygosity (LOH) events and 15% of all gains were acquired after ASCT (i.e., SBS-MM1 mutations were only detectable in the pre-gain/duplicated SNV). Notably, among these events, 1q gain was acquired after ASCT in 3/4 patients, indicating that the acquisition of 1q gain can occur even after administration of first-line therapy. CONCLUSIONS: In our study, we have uncovered distinct temporal patterns in the acquisition of early genomic drivers in MM. These patterns contribute to the reconstruction and better understanding of the life history of MM and may hold potential clinical and prognostic implications.

Venetoclax Has Potent Efficacy in NPM1 mutated AML with Acquired Resistance Associated with Either Perturbed Pro-Survival Signalling or NPM1 wild-Type Populations

Background We have previously reported outcomes for patients (pts) receiving venetoclax (VEN) combined with intensive chemotherapy in fit older pts ≥65 years with newly diagnosed AML (Chua et al, JCO 2020). In contrast to younger populations, NPM1(mut) does confer favorable prognosis in older AML populations. Our prior studies indicate that VEN-based therapies in older pts have promising efficacy in NPM1(mut) AML (DiNardo et al, Blood 2020). To characterise this further, we performed a posthoc analysis of the efficacy of single agent VEN and in combination with chemotherapy in NPM1(mut) AML in the CAVEAT study and explored mechanisms of treatment failure using a multi-omic approach. Methods NPM1 MRD was performed using RT-qPCR (sensitivity 10 -6). Integrated analysis from bulk sequencing (targeted 42-gene NGS, whole exome/genome sequencing (WES/WGS), RNAseq), single cell (sc) proteogenomic and transcriptomic assessments (scDNA plus surface protein, Tapestri MissioBio; scRNAseq, 10x Genomics) were performed. Flow cytometry included analysis of BCL-2, BCL-XL, MCL1 and BFL1 expression. Results Of 85 pts enrolled on the CAVEAT study, 20 had NPM1(mut). Median age of NPM1(mut) vs (wt) was 71 vs 70 years. At a median follow-up of 38.6 months, CR/CRi rates and median OS for NPM1(mut) vs (wt) was 85% vs 72% (p=0.37), and 43.9 vs 15.1 months (p=0.04), respectively. Pts received a 7-day VEN monotherapy pre-phase prior to CAVEAT induction with paired bone marrow (BM) assessments pre- and at day 8 to evaluate VEN sensitivity. NPM1(mut) pts had a greater median relative blast reduction after 7-day VEN at -66% (range -7 to -98%), compared to -37% (range +51 to -94%) for NPM1(wt) (p=0.03). 18/20 NPM1(mut) sub-group pts completed induction and 17 had serial NPM1 MRD assessments. The NPM1(mut) level fell a median of 4.55-log 10 following induction, with 6 (35%) pts achieving MRD negative (MRDneg) status. After all consolidation, 12/16 (75%) were MRDneg. 12/12 (100%) pts in long-term follow up remain MRDneg. We investigated the mechanisms of resistance in the 5/20 pts with NPM1(mut) whose disease was primary refractory (RD; n=1) or had acquired secondary resistance (Fig). Two patients (CAL-045, CAL-021) had shown BM blast reductions of ≥45% during VEN pre-phase. Subsequently, at the time of clinical treatment failure, both were NPM1(mut) MRD negative indicating eradication of the starting NPM1(mut) clone and early evolution of a new NPM1(wt) population as the mechanism of resistance. Detailed genomic analysis of CAL-045 showed that a TET2/CBL/NPM1(mut) clone was eradicated, with persistence of TET2 and TET2/CBL(mut) clones lacking NPM1(mut) at the time of refractory disease. scRNAseq analysis of these NPM1(wt) clones revealed a pro-inflammatory signature with elevation in BFL1, a pro-survival gene not targeted by VEN (Fig). In patient CAL-021, NPM1(mut) was also absent with the emergence of nonsense/frameshift BAX variants at relapse as previously described (Moujalled et al, Blood 2022). BAX deficiency is associated with resistance to BH3-mimetics. Two other cases (CAL-014, CAL-020) showed BM blast reductions of &amp;lt;45% during VEN pre-phase. In CAL-014, the expansion of an NPM1 subclone was associated with FLT3-ITD. WES revealed evolution of FLT3-ITD loss of heterozygosity at relapse. Flow cytometric analysis demonstrated disproportionate upregulation of intracellular MCL-1 (Fig). In case CAL-020, no obvious genetic determinants of resistance were identified at relapse, with persistence of a DNMT3A/ KIT/ NPM1(mut) clone (VAF~50%). Whole transcriptome analysis revealed skewed expression of a KIT(mut)allele (from 45% to 88%) at relapse, and protein assessment by flow cytometry revealed marked upregulation of BCL-XL (Fig). Conclusion VEN in combination with intensive chemotherapy for NPM1(mut) AML pts ≥65 years resulted in rapid and deep responses ( NPM1(mut) MRD-negativity) that are associated with prolonged remissions and OS. Mechanisms of treatment failure among pts with NPM1(mut) are diverse and may include kinase-linked upregulation of alternative BCL-2 family pro-survival members or disruption of downstream pro-apoptotic BAX.