BCOR Mutations Research Articles

What Is the Optimal Treatment Modality in Molecularly Defined Secondary AML? a Multicenter Cohort Study

Introduction Secondary ontogeny acute myeloid leukemia (AML), defined by the presence of mutations in ASXL1, BCOR, EZH2, SF2B1, SRSF2, STAG2, U2AF1 or ZRSR2 was recently integrated into both the ICC and WHO diagnostic criteria, as well as into the ELN 2022 risk classification as adverse risk. However, it is unknown whether cytarabine + anthracycline (7+3), liposomal cytarabine and daunorubicin (CPX-351) or hypomethylating agent + venetoclax (HMA+VEN) is the optimal frontline treatment for these patients (pts). We performed the largest multicenter retrospective cohort study to date, dedicated to pts with molecular-defined secondary ontogeny AML, comparing treatment modalities and assessing the impact of clinical and molecular characteristics on response and survival. Methods We included pts with newly diagnosed (ND) AML who were treated at 4 large academical centers with 7+3, CPX-351 or HMA+VEN. Secondary ontogeny was defined as absence of TP53 mutation and presence of at least one of the aforementioned mutations. Composite complete response (cCR) was defined as complete response (CR) + CR with incomplete count recovery (CRi). Logistic regression was fitted to evaluate odds ratio (OR) for cCR. Overall survival (OS) was compared between groups by log-rank test. COX regression for OS were fitted to evaluate effect of secondary/co-mutations within each treatment group, as well as the effect of treatment and allogeneic stem cell transplantation (SCT) as a time-varying covariate. Results Out of 1132 ND AML pts in the entire cohort, 401 (35%) were molecularly defined as secondary ontogeny and included in the final analysis. Initial treatment was 7+3 in 173 (43%), HMA+VEN in 162 (40%) and CPX-351 in 66 pts (17%). Pts in the HMA+VEN group were older (median age 74 years[yrs]; CPX351 66 yrs; 7+3 63 yrs, p<0.001), while pts in the CPX351 group were more likely to have prior myeloid neoplasm (n=52 [79%]; HMA+VEN n=72 [44%]; 7+3 n=48 [28%], p<0.001) and prior HMA exposure (n=27 [41%]; HMA+VEN n=23 [14%]; 7+3 n=28 [16%], p<0.001). The cCR rates were 56% (97/173), 56% (90/162) and 44% (29/66) in 7+3, HMA+VEN and CPX-351, respectively (p=0.21). In the 7+3 group, the presence of RUNX1 co-mutation was associated with lower cCR rate (OR 0.8, 95% confidence interval [CI] 0.7-0.95, p=0.01), while PTPN11 was associated with higher cCR rate (OR 1.4, 95% CI 1.1-1.8, p=0.011). In the HMA+VEN group, monosomal karyotype (OR 0.7, 95% CI 0.5-0.9, p=0.002), NRAS co-mutation (OR 0.6, 95% CI 0.5-0.8, p<.001) and SF3B1 mutation (OR 0.76, 95% CI 0.6-0.9, p=0.008) were associated with lower cCR rate and FLT3-TKD with higher cCR rate (OR 1.5, 95% CI 1.1-2.1, p=0.01). In the CPX-351 group, NRAS co-mutation was associated with lower cCR rate (OR 0.65, 95% CI 0.5-0.9, p=0.005). As most younger pts (aged ≤ 60 yrs, n=85) were treated with 7+3 (n=64) or CPX-351 (n=13) and almost exclusively older pts (age >75, n=78) were treated with HMA+VEN (n=74), we performed age-group based analyses. In pts aged > 60 yrs (n=318), the median OS (mOS) was comparable between groups (7+3: 16 months [mo], 95% CI 13-27; HMA+VEN 15 mo, 95% CI 13-19; CPX-351 11 mo, 95% CI 9-28 p=0.57). Similar results were seen in pts 60-75 yrs (n=238): 7+3 mOS 16 mo, 95% CI 13-27; HMA+VEN mOS 16 mo, 95% CI 13-26; CPX-351 mOS 10 mo, 95% CI 8-28, p=0.74. The effect of secondary/co-mutations on survival differ between groups: For 7+3 splicing mutations were associated with worse OS, whereas IDH1/2 were associated with improved OS ( Figure). Conversely, in HMA+VEN and CPX351 groups K/NRAS co-mutations were associated with worse survival. NPM1 co-mutation did not affect survival in any treatment group. In multivariable analysis, age, prior myeloid disease, K/NRAS co-mutations and monosomal karyotype were associated with worse OS ( Table). Conversely, BCOR mutation, IDH1/2 co-mutation and SCT were associated with improved OS. Treatment with HMA+VEN or CPX351 were not statistically different vs 7+3, although there was a trend for better OS with HMA+VEN vs 7+3 (HR 0.68, 95% CI 0.44-1.03, p=0.069). Conclusion In pts with ND secondary ontogeny AML the OS with HMA+VEN was at least comparable to 7+3 or CPX351 when adjusted for multiple covariates. The effect of co-mutations on response and OS differed between treatment modalities and may aid in optimal treatment selection in this population. Prospective trials should evaluate the potential superiority of HMA+VEN and effect of co-mutations in secondary ontogeny AML.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Clonal Evolution of Der(1;7)(q10;p10) Myeloid Neoplasms

Background der(1;7)(q10;p10) (der(1;7)) is a recurrent unbalanced translocation found in 1.5~6% of MDS and AML patients. It is caused by rearrangement of centromere alpha satellites in chromosomes 1 and 7 resulting in an amplification of 1q and a deletion of 7q. Previous studies comparing der(1;7)(+) cases to those with other lesions of 7q loss (-7/del(7q)) demonstrated a higher frequency of RUNX1 mutations, no TP53 mutations and a better prognosis for der(1;7) cases suggesting that der(1;7)(+) may define a unique entity of myeloid neoplasms (MN). However, with the lack of comprehensive molecular characterization using unbiased platforms such as whole genome/exome sequencing, the entire picture of der(1;7)(+) MN still remains unclear. Methods We enrolled a total of 148 MN cases harboring der(1;7) and an additional 3,238 der(1;7)(−) MN cases: -7/del(7q), +1q and ‘OTHER’. We first analyzed paired tumor/normal DNA from 26 der(1;7) cases using whole exome sequencing (WES), followed by targeted-capture sequencing of 329 genes, including those commonly mutated in MN and others newly identified in der(1;7) cases in WES. To investigate the origin of der(1;7)(+) MN, we also enrolled 11,234 healthy individuals to evaluate der(1;7) in clonal hematopoiesis, in which der(1;7) was detected on based on SNP array-based karyotyping. Results In accordance with previous reports, der(1;7) MDS cases showed an improved overall survival (OS) compared to -7/del(7q) MDS cases (Hazard Ratio (HR) =0.71, p=0.098), which however, was shorter than OS in +1q (HR=1.36, p=0.11) and ‘OTHER’ (HR=1.8, p<0.001) MDS cases. der(1;7)(+) cases were characterized by very high mortality from infection (45.5%), while mortality from disease progression was less common (36.4%), showing a sharp contrast to -7/del(7q) and ‘OTHER’ MDS cases which had 13.9% and 10.8% infection-related deaths, and 72.3% and 76.9% disease progression, respectively (Figure 1A.). WES in 26 der(1;7)(+) cases revealed a novel transcription factor, MYB in 2 cases, which was found in 18 (12%) of 148 der(1;7) cases using targeted-capture sequencing. der(1;7)(+) cases were characterized by high frequency of mutations in transcription factors, including RUNX1 (37.8%), GATA2 (12%), ETV6 (13%), and BCOR (14%) (Figure 1B.), which were significantly more frequent in der(1;7) cases than other sub-types (Odds Ratio (OR)<6)(Figure 1B.). Very high frequency of ETNK1 mutations is highly unique to der(1;7)(+) cases (18.9%) compared to that in other cases (<10%). High frequency of mutations in transcription factors seems to be a unique feature of der(1;7)(+) cases. Trisomy 8 and del(20q) CNAs were also common in der(1;7) cases (16% and 21%, respectively). Together with DNMT3A and del(20q), der(1;7) showed the highest variant allele frequency (VAF), suggesting the early origin of these lesions. Additionally, we identified der(1;7) in 6 out of 11,234 healthy individuals using SNP array-based highly sensitive copy number detection, of which 4 died from MDS, suggesting that der(1;7)(+) clonal hematopoiesis might be the origin of MDS in these cases. Moreover, we also analyzed longitudinal samples from 9 der(1;7)(+) cases using duplex-sequencing, der(1;7) showed consistently high VAF, further supporting that der(1;7) tended to represent the major clone. Conclusion Showing a unique set of co-mutation pattern and clinical features, der(1;7) MN defines a distinct subset of myeloid neoplasms, which may originate from der(1;7)(+) clonal hematopoiesis. der(1;7)(+) patients were at higher risk of life-threatening infection, which needs to be carefully prevented and monitored during their clinical course.

Distinct Immune and Molecular Profiles of Hepatitis-Associated Aplastic Anemia

Background: Hepatitis-associated aplastic anemia (HAAA) is characterized by the development of hematopoietic failure within months following an acute episode of non-infectious hepatitis. Although disease presentation with pancytopenia and a hypocellular marrow in HAAA is similar to non-hepatitis immune AA (non-HAAA), the immune and molecular profile in HAAA has not been completely elucidated. Here, we aimed to assess HAAA patients for cytokines and presence of clonal events linked to AA, the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones and somatic mutations in peripheral blood mononuclear cells (PBMC) subpopulations, and compared to a non-HAAA cohort. Methods: A cohort of HAAA patients (n=30), age and sex-matched non-HAAA patients (n=24) and healthy controls (n=23) seen at the National Institutes of Health since 2003 were retrospectively screened for a panel of inflammatory cytokines and growth factors by Luminex, and somatic mutations in sorted CD3-, CD3+CD4+, and CD3+CD8+ PBMC fractions by error-corrected DNA sequencing. A customized panel with 42 myeloid-related and 49 autoimmunity-related genes at minimum limited of detection of 0.1% were used. All samples were before any immunosuppressive treatment (IST). Cytokine levels and frequencies of PNH clones and clonal hematopoiesis (CH) in different fractions were compared among groups. Results: HAAA patients were in average young (median age=23); 37% were at age < 18 (Table 1). Although disease severity and blood counts were similar to the non-HAAA cohort, immune and molecular markers of immune AA were distinct among cohorts. The frequency of PNH clones was significantly lower in HAAA compared to the non-HAAA cohort (15% v 58%, p=0.0008). The median size of PNH clones in HAAA and non-HAAA was 1.4% and 7.4%, respectively (p =0.03). The cytokine profiles performed in the sera of 22 HAAA before IST were marked by significantly higher levels of IL-1ra (p=0.0008), CXCL-10 (p=0.0038), and VEGF (p=0.0023) in comparison to 31 non-HAAA assessed patients; IL-6 (p=0.0002) and CD40L (p=0.0001) levels were significantly lower than in non-HAAA. As reported in immune AA, HAAA patients had higher levels of TPO (p=0.0001), G-CSF (p=0.0001), and EPO (p=0.0001) than controls; lower levels of CCL-5 (p=0.0001) and CCL-11 (p=0.0025) were seen in the HAAA. FLT3-L levels were markedly elevated in both AA cohorts when compared to HC. The clonal landscape was also strikingly different between HAAA and non-HAAA in all fractions evaluated (Figure 1). In HAAA, 3 out of 21 assessed patients (14%) had CH in the CD3- fraction at maximum variant allele frequency (VAF) of 1.8%; one patient had two RUNX1 mutations while a JAK2 and DNMT3A mutation were found in the other two patients. No mutations were found in CD4+ and CD8+ compartments in subjects with HAAA. In contrast, 13 of 24 non-HAAA patients (54%) had CH in the CD3- populations, which was dominated by PIGA, BCOR, and DNMT3A clones, a pattern consistent with the known clonal landscape of immune AA. The frequency of non-HAAA with CH in the CD4+ and CD8+ compartments was 7/24 (29%) and 8/24 (30%) patients, respectively; CH in these fractions was similar to those found in CD3- dominated by mutations in PIGA, BCOR, and DNMT3A at VAFs ranging from 0.2% to 15% (Figure 1). CH in lymphoid-related genes was only found in a single patient with a STAT3 p.S614Rmutation at VAF of 3% exclusive to the CD8+ fraction. Conclusion: HAAA has a distinct immunologic and molecular profile than non-HAAA patients. PNH clones were less frequent in HAAA and patterns of cytokines were significantly different than the non-HAAA cohort. CH in PIGA and BCOR, highly associated with AA, were not present in our small HAAA cohort. Our results indicate a distinct underlying pathophysiology that initiates the immune destruction resulting in marrow failure in HAAA patients. Confirming these findings in a larger cohort and correlating with clinical outcomes is ongoing.

Genetic Evolution from Chronic Myeloproliferative Neoplasms to Acute Myeloid Leukemia: An Analysis of Forty-Six Paired Samples

Background: Secondary acute myeloid leukemia (sAML) may arise from chronic myeloproliferative neoplasm (MPN). The genetic evolution pattern may be understood by paired sample analysis. Knowledge of genetic change in MPN/sAML is still limited and more extensive studies should be done. Patients and Methods: Forty-six paired samples (27 males) at diagnosis of MPN (12 polycythemia vera, 6 essential thrombocythemia, 25 primary myelofibrosis and 3 MPN-unclassified) and sAML transformation were available. Mutational analyses of 27 genes ( JAK2V617F, CALR, MPL, ASXL1, IDH1, IDH2, TET2, DNMT3A, EZH2, SRSF2, U2AF1, SF3B1, ZRSR2, SMC1, STAG2, SMC3, RUNX1, SETBP1, IKZF1, WT1, K-RAS, N-RAS, C-CBL, TP53, RAD21, BCOR and BCORL1) were performed. Mutations and their variant allele frequencies (VAF) were measured by next generation sequencing. Results: At baseline (MPN), the mutation number was 2 (maximal 5). Forty-five samples harbored driver gene mutations (31 JAK2V617F, 13 CALR and 1 MPL). Twenty-eight samples harbored at least one epigenetic regulator mutations (11 TET2, 8 DNMT3A,7 ASXL1, 5 EZH2, 2 IDH2, 1 IDH1). Twelve samples harbored spliceosome mutations which were mutually exclusive (6 SRSF2, 3 SF3B1, 2 U2AF1 1 ZRSR2). Other mutations were RUNX1, BCORL1 (n=2 each), CBL and BCOR (n=1 each). In sAML, the median mutation number was 3 (maximal 7). Loss of mutation was found for JAK2V617F (9/31), TET2 (1/11) and SRSF2 (1/6). Acquisition was most common for RUNX1 (n=9), followed by TP53, ASXL1 (n=5 each), K-RAS (n=4) , ZRSR2, STAG2 (n=3 each). The median number of acquired mutation was 1 (maximal 5). The frequencies of gene mutations are summarized in Figure 1A. The baseline VAF of JAK2V617F was high (>35%) in 80.6% (25/31). In progression to sAML, VAF was constant in 29.0% (9/31, VAF changes <10%), increased in 20.0% (9/31, increased VAF> 10%), decreased in 41.9% (13/31, decreased VAF>10%) (Figure 1B). The baseline VAF in CALR mutation was high (>35%) in 91.3% (12/13). VAF remained stable in 76.9% (10/13), and loss of CALR mutation was not observed (Figure 1B). TET2 mutations tended to be stable (7/16), but clonal expansion (5/16 of all mutation events at MPN), or acquisition (n=2) could be found during transformation to sAML. DNMT3A mutations remained stable (5/8), exhibited clonal expansion (2/8), or acquisition (n=1) in sAML progression. ASXL1 mutations remained stable (6/7), with clonal expansion (1/7), or acquisition (n=5) during MPN/sAML progression. All EZH2 mutants had clonal expansion (5/5) and one acquired in sAML (Figure 1B). We found that CALR mutationwas often the founding clone with high VAF at MPN phase, stable clone during sAML, and might acquire mutations in signaling pathways (1 CBL, 1 KRAS), epigenetic regulators (2 ASXL1, 1 IDH2), transcription factors (2 RUNX1, 1 BCOR), spliceosome (2 ZRSR2, 1 SF3B1), and cohesin complex (1 STAG2). JAK2 mutationwas often preceded by other ancestral mutations such as TET2, ASXL1, DNMT3A, EZH2, IDH2, SRSF2 or ZRSR2. During evolution, leukemia arose from non- JAK2 mutated clones with mutations in EZH2, DNMT3A, RUNX1, EZH2, SETBP1, or acquisition of TP53, SF3B1, BCOR and STAG2 mutations) in sAML. In outcome, the median time from MPN to sAML was 82 (range 3.7 to 338.5) months. Female gender, loss of JAK2V617F, presence of U2AF1 and absence of IDH1 were associated with shorter time to sAML transformation. No other genetic mutation in our gene panel or the number of mutations harbored by patients in our cohort had significant impact on time to sAML. Marked increase of VAF was observed for JAK2 (7/22) , DNMT3A (1/7) , IDH1 (2/3), IDH2 (1/3), TET2 (3/10) , EZH2 (4/5), C-CBL (1/1) and SRSF2 (1/4). Marked decrease of VAF was observed for JAK2 (4/22), MPL (1/1), DNMT3A (1/7) , ASXL1 (1/8), SF3B1 (2/3), SRSF2 (1/4) and U2AF1 (1/2). Multiple mutational clones were detected for TET2 (two clones in 4 samples and 3 clones in 1). Clonal expansion and reduction were observed in two pairs of samples upon sAML transformation. Conclusions: At the MPN stage, most patients had driver mutations with frequent co-mutations of epigenetic modifier or spliceosome genes. CALR mutant clones were stable while for JAK2, sAML often arose from clones without JAK2V617F which acquired RUNX1, TP53, ASXL1 and K-RAS. Research funding:Ministryof Science and Technology Council,Taiwan (NSTC 112-2314-B-182-055) and Ministry of Health and Welfare, Taiwan (112-TDU-B-222-124001)

Pre-Transplant Somatic Co-Occurring Mutations (by next generation sequencing) in Acute Myeloid Leukemia: Frequency and Impact on Clinical Outcomes after Allogeneic Hematopoietic Cell Transplantation - a Large Study on Behalf of the EBMT Acute Leukemia Working Party

Background: Acute myeloid leukemia (AML) is a very heterogeneous hematological malignancy, which includes numerous genetically defined subsets. The genomic classification of AML with the identification of mutations in transcription factors, epigenetic modifiers, spliceosome, cohesin complex, and signaling pathways has led to a more accurate risk stratification model. The main genetic aberrations included in the European LeukemiaNet (ELN) 2022 classification are NPM1 (risk group according to karyotype and FLT3-ITD status), FLT3-ITD (intermediate risk), bZIP in-frame mutated CEBPA (favorable risk), and RUNX1, ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations (all belonging to the adverse risk group). In the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT), the frequency and prognostic value of different gene-gene interactions has not been studied and may differ from that of patients treated with chemotherapy alone. We aimed at evaluating, through the European Society for Blood and Marrow Transplantation (EBMT) database, the frequency and impact of different recurrent somatic mutations, either alone or in association, on prediction of relapse and survival in patients receiving allo-HSCT. Methods: This is a retrospective, registry-based, multicenter analysis from the EBMT with the approval of the EBMT Acute Leukemia Working Party. Adult patients aged more than 18 years with a diagnosis ofAML who received an allo-HSCT between 2013-2022, with an available genetic profile determined at diagnosis by next generation sequencing (NGS) were included. Results: We identified 836 allografted AML patients who had NGS performed at diagnosis. Most of these patients had de novo AML (88%), with a median age of 53 years (range: 18-78 years). Karyotype was favorable in 7%, intermediate in 69% and adverse in 24% of patients. At transplant, 74% of patients were in first remission (CR1) and 13% in CR2. The most frequent detectable mutations by frequency were DNMT3A (33%), FLT3 (29%), NPM1 (29%), TET2 (28%), NRAS (23%), RUNX1 (22%), WT1 (22%), BCOR (19%), ASXL1 (17%), IDH2 (17%), IDH1 (15%), SRSF2 (13%), STAG2 (12%), CEBPA (11%), TP53 (10%), KRAS (10%), and PTPN11 (10%) . By multiple correspondence analysis, two independent groups of co-occurring mutations were identified, the first group included DNMT3A, NPM1 and FLT3, the second group included ASXL1, SRSF2 and RUNX1. Outcome analysis was performed on the subset of 298 patients allografted in CR1 with available data for the aforementioned six genes ( DNMT3A, NPM1, FLT3, ASXL1, SRSF2 and RUNX1). Most of these patients had de novo AML (90%), with a median age of 53 years (range: 19-75 years). Patients received primarily reduced intensity conditioning (58%) and peripheral blood stem cells (93%) from matched sibling donors (35%), matched unrelated (28%), and haploidentical donors (21%). Seventy percent of these patients had intermediate-risk cytogenetics, while 27% were classified as adverse-risk. Median follow up calculated by the reverse Kaplan-Meier method was 2.5 years. Overall, the 2-year relapse incidence (RI), leukemia-free survival (LFS) and overall survival (OS) were 24%, 62% and 69%, respectively. When outcome analysis was performed according to the presence or absence of single mutations, none of the six mutations significantly affected RI or LFS. The 2-year OS was positively affected by the presence of NPM1 mutation (78% vs 66%; p=0.02) and FLT3 (79% vs 65%, p=0.01) but not significantly affected by the other 4 mutations. When mutations were investigated in groups, the 2-year RI, LFS and OS were 24%, 70% and 78%, respectively, for patients with NPM1 mutation regardless of other co-mutations, 35%, 56% and 69% for patients with FLT3-ITDand/or DNMT3A mutation, wild type NPM1, regardless of other co-mutations, 17%, 70% and 74% for patients with RUNX1 and/or ASXL1 and/or SRSF2 mutation without FLT3-ITD and with wild type NPM1 and wild type DNMT3A and 20%, 56% and 61% for patients with all six genes unmutated. Conclusion: NGS at diagnosis can be extremely useful in risk stratification of AML patients undergoing allo-HSCT, potentially allowing adequate post-transplant interventions. Notably, the 2-year LFS of 70% for patients harboring RUNX1 and/or ASXL1 and/or SRSF2 mutation indicates that allo-HSCT can overcome the adverse risk associated with these somatic mutations at diagnosis.

Common Pathways for Acute Myeloid Leukemia Progression in Systemic Mastocytosis with Associated Hematologic Neoplasm

Background: Mastocytosis is a heterogenous disease of mast cell (MC) clonal proliferation with pathologic accumulation in organ systems such as but not limited to skin, bone marrow, gastrointestinal tract, liver and spleen. Mast cells are CD34+, CD117+ (stem cell factor, KIT) with greater than 90% of people with mastocytosis harboring KIT D816V activating mutations which allows for increased proliferation and accumulation of neoplastic mast cells leading to downstream mast cell activation and intolerable symptoms. Advanced systemic mastocytosis (advSM) is categorized into three major subtypes, aggressive SM, MC leukemia and SM with an associated hematologic neoplasm (SM-AHN). SM-AHN is the most common advSM subtype accounting for about 75% of advSM cases and typically includes myeloid neoplasms such as MDS, CMML, or MPN. In most pts with SM-AHN, clinical symptoms is driven by the MC component and the goal of treatment with KIT inhibitors is to decrease the MC burden, thus improving quality of life and overall survival (Nat Med refs), However, the median survival of SM-AHN remains poor at 2-3.5 years with many pts transforming to advanced myeloid neoplasms especially an acute myeloid leukemia (AML). The use of next generation sequencing has helped to prognosticate patients with AdvSM. Patients with advSM typically exhibit multiple somatic mutations other than KIT D816V We describe the molecular signature of patients with SM-AHN who transformed to AML. Commonly involved genes are TET2, SRSF2, ASXL1, RUNX1, and CBL with inferior OS and adverse clinical characteristic associated with SRSF2/ ASXL1/ RUNX1 (S/A/R). To better prognosticate, Jawhar and colleagues introduced the MARS score. This incorporated molecular findings in AdvSM to help estimate overall survival which identified 3 risk categories (low, intermediate, and high). The overall survival in low, intermediate and high were not reached, 4.3 years, and 1.9 respectively for patients with AdvSM. While mutational findings in the MARS score can help prognosticate overall survival in these patients, the molecular footprint of progression or transformation to acute myeloid leukemia has not been established. We describe the molecular signature of patients with SM-AHN who transformed to AML. Methods: We analyzed 10 pts with SM-AHN who transformed to AML from 2000-2022. Of those patients, 7 were found to have full next generation sequencing at the time of initial consultation and transformation. These data were collected and descriptive analysis was performed. Results: We identified 7 pts with SM-AHN who progressed to AML. At SM-AHN diagnosis all pts had at least 2 additional co-mutations with a maximum of 7 co-mutations along with KIT D817V. The most common co-mutations were splicing factor mutations ( SRSF2 and SF3B1), TET2, RASEZH2, CUX1 and CBL. Six of the seven received treatment for SM-AHN. These patients received treatment with multikinase inhibitors or hypomethylating agents. The acquired driver mutations were NPM1, FLT3-ITD, RUNX1, and RAS pathway. In patient 1, a new FLT3-ITD and RUNX1 was acquired with an increased VAF of a RAS pathway mutation ( PTPN1) which was originally present in the SM-AHN diagnosis. Patient 3 also developed a new RUNX1 mutation at AML transformation. Patients 4 and 5 had new NPM1 mutations and patients 6 and 7 had a new RAS pathway mutations . Patient 2 acquired new BCOR, RIT1 and CBL mutations. Upon transformation to AML, 5/7 patients had resolution or a decrease of greater than 10% of KIT D816V VAF. One of the seven had no change in KIT D816V VAF at the time of transformation, this was also the only patient to receive no prior therapy. Only 1/7 had a VAF increase of greater than 20% in KIT D817V at transformation. The MARS score was calculated at the time of diagnosis of SM-AHN which showed that only 4 of 7 had a high MARS score. Time from SM-AHN diagnosis to AML progression ranged from 4-31 months. Survival was calculated from time of SM-AHN diagnosis to death which ranged from 6-47 months. Conclusion: Acquisition of known AML driver mutations (RAS, RUNX1, NPM1, FLT3-ITD) are commonly found in SM-AHN patients at time of progression to AML. The MARS score calculated in our patients did not correlate with time to transition to AML.

Single-Cell Long-Read Sequencing Enables the Dissection of BTK Subclonal Dynamics in CLL Treatment

Introduction: BTK inhibition is an effective frontline therapy for patients with CLL. However, patients who develop BTK C481S mutations relapse quickly. We recently published a longitudinal study investigating subclonal evolution in 38 patients receiving BTKi treatment for CLL. Of the 38 patients, 6 developed a subclone containing a BTK C481S mutation identified in the whole-exome sequencing (WES) data at the time of relapse. To understand whether a BTK C481S mutation alone can drive relapse or if an additional CLL-relevant mutation is required, we investigate these subclones on a single-cell level using PacBio Multiplexed Arrays Sequencing (MAS-seq). MAS-seq generates single-cell, long-read RNA sequencing data, which is highly accurate and provides coverage across the entire mRNA transcript. Methods: For each patient, B cells were isolated from a pre-BTKi treatment sample and a sample taken at the time of relapse. Single-cell cDNA library preps were generated using the 10X Genomics Chromium platform. The cDNA was then sequenced using MAS-Seq. After processing the MAS-seq data using PacBio's Isoseq pipeline, Seurat was used to perform a single-cell analysis for each sample. We used scBayes, a Bayesian-statistical approach developed in our lab (Qiao et al., Genome Research, in review), to assign subclone identity to individual cells based on the expression of previously identified somatic mutations. The transcript-wide coverage of MAS-seq increases the likelihood of coverage at the somatic mutation sites compared to traditional single-cell RNA sequencing methods. Results: We have sequenced and analyzed samples from 4 patients in this study (pt 1-4). In the pretreatment sample of pt 1, two subclones containing different TP53 mutations were present. At the time of relapse, a BTK C481S subclone evolved from one of these TP53 subclones, becoming the dominant clonal population. No other CLL-relevant mutations were detected in this patient. Pts 2-4 had an additional CLL-relevant mutation evolve with the BTK C481S mutation in the dominant subclone at the time of relapse. In pt 2, a subclone with an NFKBIE and the BTK mutation evolved from a cell population where BRAF, BCOR, and EGFR2 mutations were present. The BTK C481S subclone in Pt 3 also had new KMT2D and CREBBP mutations. This subclone evolved from a cell population with an ASXL1 and SF3B1 mutation. In pts 1-3, the BTK subclone was only seen at the time of relapse. Pt 4 did not have any CLL-relevant mutations identified before treatment. In a sample taken 6 months before relapse, a BTK C481S subclone is visible at a low frequency with no other CLL-relevant mutations. At the time of relapse, a DICER1 variant, a gene recently implicated in CLL (Knisbacher et al., Nature Genetics, 2022), evolved from the BTK subclone and is seen at the time of relapse as the dominant subclone. Furthermore, the full-transcript sequencing at the single-cell level enabled us to identify a second BTK C481S subclone present at the time of relapse and determine that it was independent of the subclone containing both the BTK C481S and DICER1 mutations. This second BTK subclone had no other CLL-relevant mutations and remained at a very low frequency. Conclusion: Single-cell long-read RNA sequencing via MAS-seq provides transcript-wide coverage, which enables genotyping at somatic mutation sites. Using this method, we found that the dominant relapse clones all contained a BTK C481S mutation, which either co-evolved with additional CLL-relevant mutations (3/4 patients) or occurred on the background of existing TP53 mutations (1/4 patients).

NGS Profile and the Mathematical Prediction Model for Venetoclax Combination Therapy in HM-Screen-Japan 02 Study

[Introduction] Azacitidine and venetoclax combination therapy (Aza/Ven) is a novel strategy for acute myeloid leukemia (AML). Although this regimen is widely used as a first line treatment not only for unfit/elderly patients but also for fit/young patients in the real-world situation, appropriate patient selection is an important issue. HM-SCREEN-Japan 02 (UMIN-CTR UMIN000046371) is a Japanese multicenter prospective observational study to evaluate the clinical utility of targeted sequencing. In this study, we performed the sub-analysis of newly diagnosed AML patients who received Aza/Ven as a 1 st line treatment to elucidate the genetic background of AML. Furthermore, we proposed a novel mathematical model to predict the response of Aza/Ven. [Methods] AmoyDx® Myeloid Blood Cancer Panel for acute myeloid leukemia was used for the targeted sequencing. We performed linear discriminant analysis to predict complete response patients by Aza/Ven based on clinical information before treatment. We utilized three clinical quantities for the analysis: Wilms Tumor-1 (WT1) mRNA expression level, the number of “favorable mutations”, and that of “unfavorable mutations”. Favorable and unfavorable mutations were defined as IDH1, IDH2, NPM1, and CEBPA mutations, and ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2 and TP53 mutations, respectively, based on the previous reports (NEJM 2019, Blood 2022). The data were standardized and utilized for the linear discriminant analysis. Statistical analyses were performed using R version 4.2.3 software (R Foundation for Statistical Computation, Vienna, Austria). [Results] Total 158 patients were enrolled in our study. Among 48 patients who received Aza/Ven as a 1 st line therapy, NGS analyses were performed prior to treatment in 38 patients. Their median age was 70.5 (39-92) years old. AML with myelodysplasia-related changes was the major subtype (71%) and 37 percent of the cases had high risk cytogenetic abnormalities at diagnosis. After 1 st line Aza/Ven treatment, complete remission (CR)/ CR with incomplete blood count recovery (CRi) was achieved in 63% of the cases while the remaining 37% were non-CR. There was a trend that patients with NPM1 and IDH1/2 mutations achieved CR/CRi, while patients with RUNX1 mutation were not tended to achieve CR (Figure 1). Next, we performed linear discriminant analysis of treatment response using three pretreatment clinical factors. Linear discriminant analysis found a linear combination of prognostic factors that best separates two classes in the group of patients. Thirty-seven patients were employed to determine the discriminant function D, given by D = 0.638 × x − 0.76 × y + 0.68 × z + 0.145. Here x, y, and z represented the WT1 quantity in peripheral blood, the number of “favorable mutations”, and that of “unfavorable mutations” after standardization, respectively. Using this model, 81.1% of cases were correctly classified, and so were 75.7% of cases with leave-one-out cross validation method (Table 1). [Discussions] As previously reported (NEJM 2019, Blood adv 2020), IDH1/2 and NPM1 mutations tended to be sensitive to Aza/Ven in our cohort. However, it would be hard to predict the response of Aza/Ven by a single gene mutation because multiple gene mutations were co-existed in most of the cases (Figure 1) and clinical factors including tumor markers might also affect the outcome. Our mathematical model, involving gene mutations and WT1, could efficiently predict the response of Aza/Ven, which may support the selection of 1 st line treatment. In conclusion, our study revealed the genetic landscape of real-world Aza/Ven therapy and provided a potential prognostic model.

Characterization of Cases with the Rare Cytogenetic Abnormality I(7)(p10) Reveals an Association with IDH2 Mutated Acute Myeloid Leukemia

Background: The chromosomal alteration i(7)(p10) is a rare abnormality in hematological malignancies. It leads to a loss of the long arm (7q) and duplication of the short arm (7p) of chromosome 7. However, the presence, frequency and clinical implications of i(7)(p10) in hematological neoplasms has not been analyzed systematically to date. Aim: (1) Analysis of the frequencyand occurrence of i(7)(p10) in hematological malignancies. (2) Characterization of additional cytogenetic alterations and co-occurring mutations. (3) Analysis of potential clinical implications. Patients and methods: All samples sent for diagnosis of suspected hematologic neoplasms between 08/2005 - 05/2023 were used for detection of i(7)(p10). Presence of i(7)(p10) was analyzed using chromosome banding analysis (CBA). In cases with available molecular genetic analyses the following genes were analyzed: ASXL1, BCOR, CEBPA, DNMT3A, EZH2, FLT3-ITD, IDH1, IDH2, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1 and ZRSR2. Variants of unknown significance were excluded from statistical analysis. All reported p-values are two-sided and were considered significant at p<0.05. Results: In 34 cases an i(7)(p10) was detected. Regarding specific entities, i(7)(p10) was mainly found in AML (23/34, 68%) and rarely in other entities (mature B-cell neoplasms (B-NHL): 6/34, 18%; MDS: 4/34, 12%; ALL: 1/34, 3%). In 20/34 cases, i(7)(p10) comprised the sole cytogenetic abnormality, including 19 AML cases and 1 MDS case. In 10/34 cases, additional chromosomal alterations were detected, leading to a complex karyotype in 5 of these cases. Of note, all cases with a complex karyotype were B-NHL. Additionally, in 4 cases i(7)(p10) was present only in a subclone (1 AML, 1 B-NHL, 2 MDS cases) (Figure A). In 18/34 cases, molecular genetic data was available, revealing DNMT3A (15/18), IDH2 (14/18) and BCOR (7/18) as most frequently mutated genes. As these 18 cases comprised n=15 cases diagnosed as AML, further analysis focused on these patients only. 14/15 cases harbored a mutation in IDH2, the remaining case showed an IDH1 mutation. Further, in 14/15, 7/15 and 4/15 cases, mutations in DNMT3A, BCOR and EZH2 were detected, respectively. By contrast, mutations that are known to frequently co-occur with IDH2 mutations in AML ( SRSF2, ASXL1, NPM1, STAG2, RUNX1, NRAS) were absent or only very rarely detected (Figure B). 12/14 cases with IDH2 mutations showed the mutation site p.Arg172Lys, 2/14 cases p.Arg140Leu, which is known to be the most frequent IDH2 mutation site. This is corroborated by analysis of a control cohort of n=60 IDH2 mutated AML cases without i(7)(p10), in which only 8/60 cases with p.Arg172Lys were detected (86% vs. 13%, p<0.001). Further, comparison of variant allele frequency (VAF) of IDH2 with the VAF of co-mutations suggested the presence of IDH2 in the main clone of all cases, although IDH2 VAF of cases with i(7)(p10) was slightly but not significantly lower than the IDH2 VAF of AML cases without i(7)(p10) (38.3 vs. 45.9). Moreover, comparison of IDH2 mutated AML with and without i(7)(p10) revealed a predominance of females (13/14, 93% vs. 29/60, 48%, p<0.001) and of younger age (63 vs. 72 years, p=0.015) in cases with i(7)(p10). Analysis of overall survival (OS) did not reveal a significant difference between IDH2 mutated AML cases with and without i(7)(p10), although cases with i(7)(p10) showed a trend towards a better outcome (37 vs. 14 months). Conclusions: (1) The rare but recurrent cytogenetic aberration i(7)(p10) is mainly found in AML, where associations with mutations in IDH2, DNMT3A and BCOR were observed. By contrast, other AML-defining mutations ( NPM1, FLT3-ITD, CEPBA) were largely absent, further no defining fusions/rearrangements or a complex karyotype were detected. (2) The vast majority of patients were female and younger than patients with IDH2 mutation without i(7)(p10). (3) Almost all cases showed the rare hotspot p.Arg172Lys and i(7)(p10) as sole aberration, thus it can be speculated that these cases define a distinct subgroup. (4) OS seems to be slightly better than in IDH2 mutated AML without i(7)(p10), although this difference was not statistically significant. (5) Taking into account that the IDH2 mutation was always detected in the main clone and that no other targetable genes/aberrations were observed, these patients are potentially suitable for targeted therapy with IDH inhibitors.

Acute Myeloid Leukemia (AML) Patients with Myelodysplasia (MDS) Related-Gene Mutations in First Complete Remission (CR1) Found to Have Benefit from Allogeneic Hematopoietic Stem Cell Transplant (HCT)

Introduction: Acute myeloid leukemia (AML) with myelodysplasia-related gene mutations is a new disease entity introduced in the International Consensus Classification of Myeloid Neoplasms 2022 (ICC 2022). Myelodysplasia-related gene mutations include ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 . Allogeneic hematopoietic stem cell transplant (HCT) represents a curative option for subgroups of AML patients, but not for all. In a prior study (Song et al.) reported the survival benefit of allogeneic HCT in 59 patients with AML with secondary type mutations (STM) including SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2. Allogeneic HCT could abrogate adverse prognosis in AML with STMs, and reduce its mortality risk by 58% (Song et al. BMT 2022). In the present study, we aim to evaluate the frequency of this new disease entity of AML with MDS-related mutations and evaluate the prognostic impact of allogeneic HCT in patients with MDS-related mutations achieving CR1. For comparison of the benefit from HCT, the mantel-Byar test was applied to take into account time-dependent covariate of HCT in CR1 to avoid immortal bias. Patients and Methods: We conducted a retrospective study that included 1,228 adult patients who were diagnosed with AML from January 1997 to January 2020and achieved CR1 in 5 centers (Toronto, Vancouver, and Montreal, Canada; Hwasun and Seoul, South Korea). Next-generation sequencing was performed as their standard clinical practice using targeted sequencing panel in each institution. As defined in ICC 2022, AML with myelodysplasia-related gene mutations was defined as the case of AML with ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 mutation(s). We considered allogeneic HCT in CR1 as a time-dependent covariate and used a Mantel-Byar test to avoid immortal bias. We identified 278 pts (22.6%) who had at least one MDS-related gene mutation at diagnosis. Relapse-free survival (RFS) at 2 years was selected for the primary endpoint, defined as the time from CR1 to relapse or death from any cause. EZR software version 1.41 was used throughout the study for statistical analysis. Results: The median follow-up duration among survivors after achieving CR1 was 16.5 months (mo) (Range: 1.3-217.9 mo). Between the HCT vs non-HCT group, there were no differences in sex, cytogenetic risk or other somatic mutation except NPM1 mutation which was more frequently detected in non-HCT group (p <0.001). As expected, younger pts were more frequently noted in HCT group, p=0.001). First, considering HCT as non-time dependent covariate, we have compared RFS and OS between HCT vs non-HCT groups. The 1 year RFS rate was superior in the HCT group (77.7%) to that in non-HCT group (49.9%, p<0.0001; Fig.1A). A similar trend was noted for the 1-year OS rate presenting higher OS rate in the HCT group (83.8%) than in the non-HCT group (72.7%, p=0.0003). Next, when HCT in CR1 was taken into account as a time-dependent (td) covariate, Mantel-Byar analysis showed a consistent finding of superior RFS in HCT group to non-HCT group: time dependent covariate of HCT in CR1 was found to be favorable (Hazard ratio [(HR) of 0.45, (95% C.I. [0.31-0.65]; p=0.00003)] as shown in Fig.1B which is a Simon-Makuch plot. In multivariate analysis (MVA) for RFS, time-dependent covariate of HCT in CR1 retained its statistical significance, thus concluding that HCT in CR1 is an independent prognostic factor for RFS (HR: 0.44, 95% CI [0.29-0.68], p =0.0002) together with other variables found to be adverse on RFS including monosomal karyotype (p=0.03), IDH1 mutation (p=0.01), and age > 60 years (p=0.002); while CEBPA (p=0.02) and NPM1 (p=0.049) mutations were found to be favorable on RFS in MVA. Conclusion: In our cohort of 1228 AML patients, we have identified 278 AML patients with myelodysplasia-related gene mutations (22.6%). We have confirmed that allogeneic HCT in CR1 is strongly recommended in a subgroup of AML with myelodysplasia-related gene mutations based on the favorable impact of HCT in CR1 on RFS from the present study. This result supports the urgent referral of this patient group for allogeneic HCT in CR1.

Involvement of the JAK-STAT Pathway in the Molecular Landscape of Fusion-Free Myeloid Neoplasms with Eosinophilia

Introduction. Gene fusions leading to the constitutive activation of tyrosine kinases (TK) such as PDGFRA, PDGFRB or FGFR1 have been the first recurrent genetic defects involved in myeloid hypereosinophilic syndromes (HES). Although activation of the Janus kinase (JAK)/Signal Transducer and Activator of Transcription(STAT) pathway is critical for eosinophil production and survival, genes involved in the JAK/STAT pathway are not included in most next-generation sequencing (NGS) panels used for the etiological workup of hypereosinophilia (HE). Methods. A custom 149-gene NGS panel including subunits of the IL3/IL5/GM-CSF receptors, TK ( PDGFRA/B, FGFR1, ABL1, FLT3, KIT), intracellular proteins of the JAK-STAT and RAS-MAPK pathways was performed in 64 consecutive adult patients (experimental group) referred between March 2012 to June 2023 to the French Reference Center for Hypereosinophilic Syndromes (CEREO) for HE/HES displaying at least one clinic-biological feature suggestive of myeloid neoplasm ( i.e. splenomegaly, other unexplained CBC abnormality besides HE, increased serum tryptase and/or vitamin B12 levels, corticosteroid-refractory HE and/or sensitivity to either TK inhibitors or JAK inhibitors). All of them were negative by PCR and/or FISH analyses for gene rearrangements involving PDGFRA, PDGFRB or FGFR1. Patients with lymphocytic HES (n=7), idiopathic HES (n=26) and HE of undetermined significance (n=11) were used as controls (total, n=44). Results. Concordant with the latest recommendations of the international cooperative working group on eosinophil-associated disorders, at least one mutation was reported in 50/64 (78%) patients of the experimental group versus 8/44 (18%) patients in the control group (p<0.001) when applying a threshold of at least 3% of variant allele frequency (VAF). Mutations in the control group implied genes involved in age-related clonal hematopoiesis ( e.g. DNMT3A, TET2), with low VAF in almost all cases. All 35 patients with at least one mutation involving the JAK/STAT pathway belonged to the experimental group, among whom all 22 patients treated with steroids were refractory to therapy. Eighteen patients had STAT5B mutations, including 13 (72%) with the somatic N642H mutational hotspot. Two patients harbored JAK2Ex13InDel mutations, including one with eosinophilia and erythrocytosis. Previously unreported molecular alterations were also evidenced, including seven patients with JAK1 mutations and three STAT5A-mutated patients who shared common features i.e. the co-occurrence of BCOR mutations, high hemoglobin levels and eosinophil hyperplasia. Of note, the latter mutations were not reported both in a public database (GnomAD) as well as in a second cohort of 613 samples referred for suspicion of myeloid malignancies (yet without HE) studied with the same NGS workflow, thereby strongly supporting their association with myeloid HES. Deciphering the data from bulk sequencing also suggests that JAK-STAT mutations were frequently preceded by (or associated with) myelodysplasia-related gene mutations, with SF3B1 (12/36) and ASXL1 (10/36) mutations being the most common. Overall, both KIT D816V (n=2) and RAS/MAPK pathway activation mutations (n=3) were rare. No mutation of either PDGFRA/B, FGFR1 or IL3/IL5/GM-CSF-receptor genes was evidenced.In the experimental group, 17/18 (94%) patients (including 12 with JAK-STAT mutations) treated with ruxolitinib and with > 3 months of follow-up responded to treatment (12 complete and 5 partial hematological responses). Discussion. These dataemphasize the usefulness of NGS in daily practice for the workup of fusion-free HES patients harboring features suggestive of myeloid neoplasms. In such patients, druggable mutations involving the JAK-STAT pathway (including yet unidentified STAT5A mutations) are frequent. Most JAK/STAT mutations occur in the setting of a preexisting myelodysplastic or myelodysplastic/myeloproliferative disease harboring mutations in RNA-splicing genes or chromatin modifiers. These findings provide a rationale for refining treatment algorithms in fusion-free myeloid HES patients, supporting the use of JAK inhibitors as frontline therapy. More data are warranted to assess whether JAK inhibition enables sustained molecular remission in all disease subtypes.

208P Identification of BCOR mutation as a novel predictor of immunotherapy efficacy in gastrointestinal tumors

208P Identification of BCOR mutation as a novel predictor of immunotherapy efficacy in gastrointestinal tumors

Validation of the molecular international prognostic scoring system in patients with myelodysplastic syndromes defined by international consensus classification

Myelodysplastic syndromes (MDS) have varied prognoses and require a risk-adapted treatment strategy for treatment optimization. Recently, a molecular prognostic model (Molecular International Prognostic Scoring System [IPSS-M]) that combines clinical parameters, cytogenetic abnormalities, and mutation topography was proposed. This study validated the IPSS-M in 649 patients with primary MDS (based on the 2022 International Consensus Classification [ICC]) and compared its prognostic power to those of the IPSS and revised IPSS (IPSS-R). Overall, 42.5% of the patients were reclassified and 29.3% were up-staged from the IPSS-R. After the reclassification, 16.9% of the patients may receive different treatment strategies. The IPSS-M had greater discriminative potential than the IPSS-R and IPSS. Patients with high, or very high-risk IPSS-M might benefit from allogeneic hematopoietic stem cell transplantation. IPSS-M, age, ferritin level, and the 2022 ICC categorization predicted outcomes independently. After analyzing demographic and genetic features, complementary genetic analyses, including KMT2A-PTD, were suggested for accurate IPSS-M categorization of patients with ASXL1, TET2, STAG2, RUNX1, SF3B1, SRSF2, DNMT3A, U2AF1, and BCOR mutations and those classified as MDS, not otherwise specified with single lineage dysplasia/multi-lineage dysplasia based on the 2022 ICC. This study confirmed that the IPSS-M can better risk-stratified MDS patients for optimized therapeutic decision-making.

Association of social deprivation with survival in younger adult patients with AML: an Alliance study

Survival of patients with acute myeloid leukemia (AML) is influenced by genetic factors, age, and race. Social deprivation is increasingly recognized as an important contributor to disparities in cancer outcomes, but studies of adult AML are lacking. We analyzed associations between social deprivation index (SDI) and outcome in 1,893 patients with AML treated on Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology frontline protocols. Patients with low (first quartile, lowest deprivation) and high (quartiles 2-4) SDI were analyzed for associations with baseline clinical, cytogenetic and molecular features, and outcomes. Except for racial-ethnic identity, SDI was not associated with baseline clinical characteristics. Patients aged <60 years with high SDI harbored BCOR (3% vs 7%, P=.03), IDH1 (6% vs 11%, P=.03) and STAG2 (2% vs 5%, P=.02) mutations less often than patients with low SDI, suggesting possible differences in the underlying disease biology. High SDI patients had shorter disease-free (median: 1.5 vs 2.8 years, P=.02) and overall survival (OS; median: 1.9 vs 3.0 years, P=.005) than low SDI patients. In multivariable analysis, high SDI was associated with worse OS (HR: 1.28, 95% CI: 1.06-1.55, P=.001). Survival differences occurred mainly in the 2017 European LeukemiaNet favorable genetic-risk group. There were no significant associations between SDI and molecular features or survival among patients aged ≥60 years. We conclude that high SDI associated with worse survival, independent from other pretreatment prognostic factors, in younger AML patients. The role of socioeconomic factors in AML should be further investigated to overcome potentially avoidable survival disparities.

Abstract 5026: Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies

Abstract Background: Ptpn1 is a member of Protein Tyrosine Phosphatase family that dephosphorylates tyrosine residues. We previously reported that an Mcm2 hypomorphic mouse (designated Mcm Cre/Cre) develops T cell acute lymphoblastic leukemia (T-ALL), due to acquisition of 100-1000 kb interstitial deletions involving tumor suppressor genes. When crossed to mice that express a NUP98-HOXD13 (NHD13) fusion gene, NHD13+;Mcm2Cre/Cre mice develop B-cell precursor ALL (BCP-ALL). Sparse whole genome sequencing revealed somatic copy number variants (CNV) involving Pax5 and Ptpn1. The role of Pax5 in B-cell development, differentiation, and leukemia has been widely studied. However, little is known about deletions of Ptpn1 and its role in BCP-ALL. Objective: The principal objective is to investigate how deletion of Ptpn1 impacts BCP-ALL development in mouse models. Methods: Mice expressing NHD13 fusion (NHD13+) were crossed with Ptpn1 knockout mice. The F2 cross generated 6 genotypes that were positive or negative for NHD13 and wild type/heterozygous/homozygous for Ptpn1. Mice were studied for incidence of BCP-ALL, which was characterized by whole-exome sequencing, molecular pathway analysis and transcriptome-sequencing. Results: NHD13+;Ptpn1-/- mice developed BCP-ALL, characterized by hyperleukocytosis, variable anemia and thrombocytopenia, expression of B220 and/or CD19, and invasion of non-hematopoietic tissues (liver, kidney, lung). In addition, NHD13+;Ptpn1+/- mice that developed BCP-ALL lost the wild-type (WT) Ptpn1 allele in the leukemic cells. Similar to human BCP-ALL, NHD13+;Ptpn1-/- mice that developed BCP-ALL showed presence of clonal TCR-delta rearrangements as well as IGH rearrangements in the leukemic cells. Whole exome sequencing showed presence of acquired mutations in genes known to be involved in B-cell differentiation, such as Pax5 or Bcor, as well as genes involved in B cell signaling pathways, such as Jak3, Jak1 and Flt3 and Western blots demonstrated accumulation of phosphorylated STAT3 protein. Conclusion: This study demonstrates that Ptpn1 loss along with expression of an NHD13 fusion gene leads to a highly penetrant BCP ALL in mice, suggesting a role for Ptpn1 in preventing malignant transformation. Taken together, these findings are consistent with a collaborative model for BCP ALL in which the NHD13 transgene leads to increased stem cell self-renewal, somatic Bcor or Pax5 mutations block normal B cell differentiation, and somatic signaling mutations (Jak1/3, Flt3) lead to hyperproliferation, which is potentiated by Ptpn1 deficiency. Citation Format: Toshihiro Matsukawa, Nupur Nigam, Ryan Bertoli, Michel L. Tremblay, Mianmian Yin, Peter D. Aplan. Protein tyrosine phosphatase Ptpn1 knockout in mouse models drives B-cell hematological malignancies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5026.

Alterations of cohesin complex genes in acute myeloid leukemia: differential co-mutations, clinical presentation and impact on outcome

Functional perturbations of the cohesin complex with subsequent changes in chromatin structure and replication are reported in a multitude of cancers including acute myeloid leukemia (AML). Mutations of its STAG2 subunit may predict unfavorable risk as recognized by the 2022 European Leukemia Net recommendations, but the underlying evidence is limited by small sample sizes and conflicting observations regarding clinical outcomes, as well as scarce information on other cohesion complex subunits. We retrospectively analyzed data from a multi-center cohort of 1615 intensively treated AML patients and identified distinct co-mutational patters for mutations of STAG2, which were associated with normal karyotypes (NK) and concomitant mutations in IDH2, RUNX1, BCOR, ASXL1, and SRSF2. Mutated RAD21 was associated with NK, mutated EZH2, KRAS, CBL, and NPM1. Patients harboring mutated STAG2 were older and presented with decreased white blood cell, bone marrow and peripheral blood blast counts. Overall, neither mutated STAG2, RAD21, SMC1A nor SMC3 displayed any significant, independent effect on clinical outcomes defined as complete remission, event-free, relapse-free or overall survival. However, we found almost complete mutual exclusivity of genetic alterations of individual cohesin subunits. This mutual exclusivity may be the basis for therapeutic strategies via synthetic lethality in cohesin mutated AML.

Gene fusions are frequent in ACTH-secreting neuroendocrine neoplasms of the pancreas, but not in their non-pancreatic counterparts

Ectopic Cushing syndrome is a rare clinical disorder resulting from excessive adrenocorticotrophic hormone (ACTH) produced by non-pituitary neoplasms, mainly neuroendocrine neoplasms (NENs) of the lung, pancreas, and gastrointestinal tract, and other less common sites. The genetic background of ACTH-producing NENs has not been well studied. Inspired by an index case of ACTH-producing pancreatic NEN carrying a gene fusion, we postulated that ACTH-producing NENs might be enriched for gene fusions. We herein examined 21 ACTH-secreting NENs of the pancreas (10), lung (9), thymus (1), and kidney (1) using targeted RNA sequencing. The tumors were classified according to the most recent WHO classification as NET-G1/typical carcinoid (n = 4), NETG-2/atypical carcinoid (n = 14), and NET-G3 (n = 3). Overall, targeted RNA sequencing was successful in 11 cases (4 of 10 pancreatic tumors, 5 of 9 pulmonary tumors, and in the one renal and one thymic tumor). All four successfully tested pancreatic tumors revealed a gene fusion: two had a EWSR1::BEND2 and one case each had a KMT2A::BCOR and a TFG::ADGRG7 fusion, respectively. EWSR1 rearrangements were confirmed in both tumors with a EWSR1::BEND2 by FISH. Gene fusions were mutually exclusive with ATRX, DAXX, and MEN1 mutations (the most frequently mutated genes in NETs) in all four cases. Using RNA-based variant assessment (n = 16) or via the TSO500 panel (n = 5), no pathogenic BCOR mutations were detected in any of the cases. Taken together, gene fusions were detected in 4/4 (100%) pancreatic versus 0/7 (0%) non-pancreatic tumors, respectively. These results suggest a potential role for gene fusions in triggering the ACTH production in pancreatic NENs presenting with ectopic Cushing syndrome. While the exact mechanisms responsible for the ectopic ACTH secretion are beyond the scope of this study, overexpressed fusion proteins might be involved in promoter-mediated overexpression of pre-ACTH precursors in analogy to the mechanisms postulated for EWSR1::CREB1-mediated paraneoplastic phenomena in certain mesenchymal neoplasms. The genetic background of the ACTH-producing non-pancreatic NENs remains to be further studied.

Clinical and molecular spectrum and prognostic outcomes of U2AF1 mutant clonal hematopoiesis- a prospective mayo clinic cohort study

• U2AF1 mutant CH is infrequent in the spectrum of genes mutated in age-related CH (<10%) and commonly presents as clonal cytopenias of undetermined significance (CCUS). • U2AF1 CH is more common in males (67%) and frequently co-occurs with truncating BCOR mutations (29%; with 86% occurring in males). • U2AF1 mutant CH is associated with a high rate (25%) and short latency (17.5 months) for progression to myeloid neoplasms. • While U2AF1 mutant CH usually occurs in the elderly (median age 71 years), the context specific occurrence in younger patients with telomere biology disorders needs further assessment.

X-linked BCOR variants identified in Chinese Han patients with congenital heart disease.

Congenital heart disease (CHD) frequently manifests as a complex phenotype and approximately one-third of cases may be caused by genetic factors. BCOR, an X-linked gene encoding the corepressor of BCL6, has been demonstrated to be closely involved in human heart development. However, whether BCOR variants represent the genetic etiology underlying CHD needs further investigation. We performed whole exome sequencing on CHD nuclear families and identified a candidate gene, BCOR, by robust bioinformatic analysis and medical literature searches. Targeted DNA sequencing of the candidate gene was conducted and then the association between variants and the risk of developing CHD was analyzed. The effects of BCOR mutations on gene expression, localization, protein interaction, and signaling pathways were evaluated in vitro. We identified a BCOR hemizygous missense variant (c.1448C>T, p.Pro483Leu) in a male proband presented with CHD/heterotaxy. Sanger sequencing confirmed that this variant was inherited from his asymptomatic mother. Interestingly, through literature searches, we observed another novel BCOR hemizygous missense variant (c.1619G>A, p.Arg540Gln) in a CHD patient with heterotaxy, supporting the pathogenic evidence of BCOR variants. Functional experiments conducted in vitro revealed that the variant p.Pro483Leu altered the subcellular localization of BCOR protein, disrupted its interaction with BCL6, and significantly promoted cell proliferation, whereas the variant p.Arg540Gln displayed no obvious effects. Nevertheless, transcriptional analysis revealed that down-regulation of BCOR substantially enhanced the activities of mitogen-activated protein and phosphoinositide 3-kinase-AKT signaling pathways, which are closely attributed to heart development. Targeted sequencing of 932 sporadic CHD patients enriched nine variants of BCOR predicted as likely rare and damaging and a septal defect was present in 81.8% (9/11) of them, including the two probands, which was consistent with the possible phenotype caused by BCOR defects. The findings of the present study indicate that variants in BCOR may predispose individuals to CHD in the Chinese Han population.