He, Rong

Abstract Heterozygous germline variants in DDX41 are identified in ~5% of patients with Myelodysplastic Syndrome (MDS) or Acute Myeloid Leukemia (AML). Recognition of the hereditary nature of such cancers and a better understanding of the factors that affect progression to malignancy has deep implications for health surveillance of patients and relatives that share a pathogenic DNA variant. DDX41 has a role in ribosome biogenesis and in opposing R-loop accumulation at promoters, but the mechanisms through which DDX41 loss drives cancer development are poorly understood. In a collaboration between Cambridge, UK and Mayo Clinic, USA we identified 85 patients with germline DDX41 variants [73/85 with germline pathogenic variants (GPV) and 12 with variants of uncertain significance (VUS)]. The majority were identified through targeted myeloid NGS upon presentation with AML (30/85) or MDS (44/85). Average age at diagnosis was 65 (35-93) with male predominance (57/85). The most common GPV was the NM_016222.4(DDX41):c.415_418dup; p.D140Gfs*2 (15/85). The majority (53.4% 39/73), also acquired a somatic variants in the other DDX41 allele (referred to as GPV+Som). The most common somatic variant (82% 32/39) was the R525H, a variant predicted to reduce the ATPase function of the helicase domain. We hypothesise that the markedly reduced DDX41 activity in cells with biallelic variants, is a key cause of progression to overt malignancy. In our cohort, we observed that somatic variants occurred at a similar frequency in association with start loss, truncating, splice, in-frame deletion, and missense GPV. However, whereas MDS was the predominant diagnosis within GPV (67.6% 23/34, AML 26.4% 9/34), AML was the most frequent diagnosis within GPV+Som (AML 51%.2 20/39, MDS 38.4% 15/39). Overall, there was a significantly higher likelihood of AML in the presence of a somatic variant (p<0.05), suggesting that there might be a dosage effect on the phenotype. This pattern was observed across all the GPV, with the exception of missense, suggesting that many might be benign polymorphysms. In addition, although somatic variants are typically subclonal (overall mean variant allele fraction VAF=9.87%, 3-27.8%), we observed a higher somatic VAF in AML over MDS cases (12.4% vs 7.7% respectively p<0.05). A lower bone marrow cellularity was also characteristic of GPV+Som vs GPV alone (mean observed/expected cellularity for age 0.9 vs 1.32 respectively p<0.05). In two cases, serial bone marrow morphology and NGS data were available, and we observed marked reduction in bone marrow cellularity upon acquisition of the R525H clone. The mechanisms of AML evolution appear different in DDX41 HMMS compared to sporadic cases. Interestingly, over half of our patients (21/34 GPV and 23/39 GPV+Som) did not exhibit additional somatic drivers. Our work suggests that acquisition of a somatic variant is a critical event in disease progression. Furthermore, the near universally subclonal nature of somatic variants suggest that they may affect disease phenotype through non-cell autonomous mechanisms. Citation Format: Ludovica Marando, Talha Badar, Yael Kusne, Audrey Morris, Terra Lasho, Abhishek Mangaonkar, Christy Finke, Carles Crawley, James M. Foran, Aref Al-Kali, Andrew King, Hassan Alkhateeb, Naseema Gangat, Chi Wong, Rong He, David Viswanatha, Faisal Basheer, Mark Litzow, Alejandro Ferrer, George Vassiliou, Mrinal Patnaik. Investigating the role of the somatic DDX41 variant in the context of DDX41 hereditary myeloid malignancy syndromes (HMMS) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A037.

Abstract Objectives VEXAS syndrome is an adult-onset autoinflammatory disease caused by a somatic pathogenic mutation in the UBA1 (ubiquitin-like modifier activating enzyme 1) gene. Patients present with rheumatologic manifestations and cytopenias and may have an increased predisposition to myelodysplastic syndrome (MDS) and plasma cell neoplasms. Prior studies have reported on the peripheral blood and bone marrow findings in patients with VEXAS syndrome. Due to the protean clinical presentation and lack of specificity of morphologic features (eg, vacuoles in early erythroid and granulocytic precursors), an optimal screening methodology to identify these patients in a timely fashion is desirable. Methods To further evaluate and describe the salient diagnostic morphologic features in VEXAS syndrome, we carried out a comprehensive study of the largest single-institution cohort to date. Diagnostic and follow-up bone marrow biopsy specimens from 52 male patients with molecularly identified VEXAS syndrome underwent central review. Results Cytopenias were common in all cases, primarily macrocytic anemia, monocytopenia, and thrombocytopenia. Bone marrow aspirate and biopsy were often hypercellular, with an increased myeloid/erythroid ratio, granulocytic hyperplasia with left shift, erythroid left shift, and megakaryocyte hyperplasia, which exhibited a range of striking morphologic findings. Distinctly vacuolated myeloid and erythroid precursors were seen in more than 95% of cases. Conclusions Our data reveal potential novel diagnostic features, such as a high incidence of monocytopenia and distinct patterns of atypical megakaryopoiesis, that appear different from dysmegakaryopoiesis typically associated with MDS. In our experience, those findings are suggestive of VEXAS, in the appropriate clinical context.

SummaryAmong 210 patients with myelodysplastic syndromes (MDSs) with del(5q), molecular information was available at diagnosis or at least 3 months before leukaemic transformation in 146 cases. Multivariate analysis identified therapy‐related setting (p = 0.02; HR 2.3) and TP53 variant allele frequency (VAF) ≥22% (p < 0.01; HR 2.8), but not SF3B1 mutation (p = 0.65), as independent risk factors for survival. Median survival was 11.7 versus 4 years (5/10‐year survival 73%/52% vs. 42%/14%) in the absence (N = 112) versus presence (N = 34) of ≥1 risk factors; leukaemia‐free survival was affected by TP53 VAF ≥22% (p < 0.01). Such information might inform treatment decision‐making in MDS‐del(5q) regarding allogeneic stem cell transplant.

INTRODUCTION: The Stromal Antigen 2 ( STAG2), on Xq25, is the most common cohesin complex gene mutated in myeloid neoplasms (MN) (5-10%). STAG2 mutations ( STAG2m) were incorporated in the ELN2022 as AML-MDS defining genetic mutations inferring poor prognosis and is a genetic marker in IPSS-M risk stratification. The PHD-finger protein 6 ( PHF6) gene, on Xq26.3, is a tumor suppressor gene, mutated in 3% of MN, and is more common in T-ALL. PHF6m is believed to be a late event in MN with little data on its prognostic implications in MN. Both STAG2m and PHF6m are more prevalent in males, but their implications on the clinical presentation of MN have not been compared prior. METHOD: Our study is a multicenter study at Mayo Clinic (Rochester, Florida, Arizona). After IRB approval, we retrospectively analyzed charts of 7935 patients who had NGS between 2016-2022. STAG2m was found in 96 patients, and PHF6m in 116 (including 3 with concurrent STAG2m and PHF6m). We excluded 4 PHF6m patients who had T-ALL. Data was captured at time of NGS. MDS risk stratification was by IPSS-M and AML by ELN2022. Overall survival (OS) was calculated from date of NGS to date of last follow-up. BlueSky Software was used for statistical analysis. RESULTS: Characteristics: We compared 96 STAG2m vs 112 PHF6m MN patients. Of STAG2m cases, 78% were male vs 76% in PHF6m MN. Median age was 72 vs 73 years, respectively. Diagnoses included AML (29% of STAG2m vs 34% of PHF6m MN), MDS (54% vs 28%), MDS/MPN (10% vs 21%) and MPN (3% vs 9%). MDS was more common among STAG2m MN (p&lt;.001) while MDS/MPN was more common among PHF6m MN (=.05). MDS-IB was the most common subtype in both (69% vs 52% in STAG2m vs PHF6m MDS). MDS-LB and 5q subtypes were only found in PHF6m MDS, and there was more PHF6m MDS-RS (13% vs 2%). Median IPSS-M score was 0.9 vs 0.8 in STAG2m vs PHF6m MDS (both high-risk). Majority of STAG2m AML were adverse risk (96%) while PHF6m AML were intermediate (79%) (p&lt;.001). AML cases included 43% vs 30% secondary AML (sAML) in STAG2m vs PHF6m AML. Among STAG2m and PHF6m MN, 17% of each were tMN. Cytogenetic abnormalities were more common in PHF6m MN (50% vs 31%, p=.005). The median bone marrow (BM) blasts count among STAG2m MN was 7% vs 4% in PHF6m MN (p=.03). Genetic characteristics: STAG2m were nonsense, frameshift and splice site (54%, 36% and 11%). PHF6m were nonsense, frameshift, splice site, missense and start loss variant mutations (44%, 33%, 12%, 10% and 1%). The median variant allele frequency (VAF) of STAG2m MN was 50% compared to 32% in PHF6m MN (p=.008). Co-mutations: The median number of co-mutations was 3 in STAG2m and 4 in PHF6m MN (p=.04). The most common were TET2m (37% in STAG2m MN vs 45% in PHF6m MN), RUNX1m (31% in both), SRSF2m (39% vs 20%), ASXL1m (66% vs 38%), BCORm (19% vs 6%), U2AF1m (13% vs 14%) and IDH2m (18% vs 7%). ASXL1m, SRSF2m, BCORm, IDH2m and SF3B1m were more common among STAG2m MN (p=&lt;.001, .003, .006, .02, .09). JAK2m, IDH1m are more common among PHF6m MN (p=.02, .06). Survival: Of STAG2m and PHF6m MN, 70% and 72% of patients received treatment. HSCT was performed in 23 (24%) and 23 (21%) of STAG2m and PHF6m patients, including AML (n=9, n=10), MDS (n=12, n=8), MDS/MPN (n=2, n=2). OS among STAG2m and PHF6m MN was 20.5 and 22.1 months, respectively. OS among AML cases was 13.5 vs 21 months (p=.6) in STAG2m vs PHF6m, was NR vs 22 months (p=.4) in MDS, 11.7 vs 17.6 months in MDS/MPN (p=.4), and 10.4 vs 27.4 months (p=.5) in MPN. There was no difference between males and females in either STAG2m or PHF6m MN. HSCT led to longer OS among both STAG2m (NR vs 19.9 months, p=.01) and PHF6m MN patients (NR vs 17.6 months, p=.07). Among AML patients, HSCT did not significantly impact OS (18 vs 9.6 months, p=.3 and 25.1 vs 21 months, p=.2, in STAG2m and PHF6m). HSCT improved OS in STAG2m MDS (NR vs 20.5 months, p=.03). In PHF6m MDS, OS was 22.1 vs 13.1 months in HSCT vs non-HSCT (p=.3). CONCLUSION: STAG2m and PHF6m are 2 X-linked mutations that occur in MN. Both commonly occur in elderly males. MDS was the most common diagnosis in STAG2m MN. STAG2m co-occurred particularly with SRSF2m ASXL1m and BCORm, which are associated with poor prognosis. In our cohort, STAG2m AML carried a poorer prognosis than PHF6m AML. Our study confirms the ELN2022 AML risk stratification. MDS risk classification and OS did not differ between STAG2m and PHF6m in our cohort. A larger cohort is required to confirm our findings and may provide stronger validation.

BACKGROUND: Recent World Health Organization (WHO) classifications (5 th edition), International Consensus Classification (ICC) recognized TP53-mutated ( TP53mut) myeloid neoplasms (MNs) as a distinct subcategory, acknowledging their uniformly poor outcomes. However, there are critical differences between the two guidelines in how TP53 allelic status is assigned. Both WHO-5 and ICC acknowledged the poor outcome of multi-hit TP53mut but excluded single-hit TP53mut. These changes are driven by a pivotal study demonstrating favourable outcome of single-hit TP53mut, however this study predominantly included de novo myelodysplastic syndromes (MDS) cases (Bernard et al. Nat Med 2020). In contrast, we previously reported poor outcome of TP53mut therapy-related myeloid neoplasm (t-MN) irrespective of the allelic status of TP53 (Hiwase et al Blood 2022andShah et al BCJ 2023). Finally, the ICC removed the subcategory of “therapy-related,” substituting it with diagnostic qualifiers instead. The WHO has grouped t-MN with secondary MN and renamed it as “myeloid neoplasm post cytotoxic therapy,” with the assertion that only multi-hit T P53mut MDS occurring post-cytotoxic therapy have poor outcome compared with single-hit. METHODS: We analyzed one of the largest international cohorts of TP53mut MN (n=544) with full characterization of TP53mut allele status utilizing uniform ICC criteria and similar large numbers of de novo and t-MN cases managed at Mayo Clinic Enterprise (USA) and South Australian MDS Registry, Adelaide (Australia) with clinical, integrated genomic profiling, and long-term follow-up. RESULTS: In our cohort of TP53mut MN (n=544), 475 (87.3%) MN had TP53mut variant allele frequency (VAF) of ≥10% and thus satisfied ICC criteria for TP53mut MN. Majority of TP53mut t-MN presented with MDS (bone marrow (BM) blasts 0-9%) compared de novo MN (62% vs. 48%; P=0.0012). While acute myeloid leukemia (AML) (BM blasts ≥20%) were more prevalent in TP53mutde novo MN compared to t-MN (38% vs. 24%; P= 0.001) and there was no difference in frequency of MDS/AML (BM blasts 10-19%) between the two cohorts (14% vs. 14%; P=0.8) ( Figure 1A). Majority of TP53mut were observed in the DNA-binding domain in t-MN and de novo MN (95% vs. 96.2%; P=0.48). However, mutations in Tetramer Domain were more frequent in t-MN (4.3% vs. 0.3%; P=0.002), whereas those in the TAD2 domain were more frequent in de novo TP53mut MN (2.4 vs. 0%; P =0.006). Integrating the mutation, conventional G-banding cytogenetic, copy number variation (CNV), FISH, and SNP array analysis, TP53mut MDS (BM blast 0-9%; n=260) were classified as multi-hit (n=179, 68.9%) or multi-equivalent (n=35, 13.5%) and single-hit (n=46, 17.7%) (Arber et al. Blood 2022). Striking enrichment of multi-hit/multi-hit equivalent was observed in TP53mut t-MDS compared to de novo MDS (91% vs. 72%; P&lt;0.0001). While single-hit TP53mut were prevalent in de novo MDS compared to t-MDS (28% vs. 9%; P&lt;0.0001). Importantly, median overall survival (OS) of single-hit was significantly longer than multi-hit de novo MDS (25.4 vs. 7.2 months; P=0.0002). In contrast to this we did not observe OS difference between single-hit and multi-hit TP53mut t-MDS (11.6 vs. 10.9 months; P =0.73) ( Figure 1B). CONCLUSION: Our study based on a large international cohort of T53mut MN provides compelling evidence that in contrast to de novo MDS, single hit TP53mut t-MDS is associated with poor outcome. Our data supports the consideration of single hit t-MDS as a multi-hit equivalent.

Background: Clonal cytopenia of undetermined significance (CCUS) is characterized by the presence of a clone with somatic mutations in genes associated with myeloid neoplasms (MN) or abnormal cytogenetics, along with unexplained and persistent cytopenias in the absence of significant marrow dysplasia. CCUS is considered a premalignant state with a risk of progression to MN. The clinical outcomes of patients (pts) with high risk CCUS (Weeks et al, NEJM Evidence 2023) may be similar that of some patients with myelodysplastic syndromes (MDS). This study aims to predict the outcomes of CCUS patients using the molecular international prognostic scoring system (IPSS-M) developed for MDS. Methods: A retrospective analysis was conducted in patients diagnosed with CCUS between 2015 and 2023, we included patients harboring pathogenic mutations (MUT) identified through next-generation sequencing (NGS) testing at two institutions: Mayo Clinic and the University of Texas Southwestern Medical Center, Dallas, TX, USA were included. Progression was considered present if patients with CCUS progressed into myeloid neoplasm at follow up. Statistical analysis was performed using JMP® 17.1.0 Software. IPSS-M risk scores were calculated via the IPSS-M Risk Calculator (mds-risk-model.com). Results: A total of 103 patients were included in the study. The median age was 72 years, the majority were male patients (73%), and the median bone marrow blast % at the time of NGS was 1%. Forty-five % had an isolated mutation in a single gene, and 55% had co-mutations in ≥1 gene. The median number of mutations was 2 (range 1 - 5), occurring across 43 different genes. The most common mutations were in TET2 (N=28; 27%), ASXL1 (N=21; 20%), SRSF2 (N=21; 20%), U2AF1 (N=18; 17%), DNMT3A (N=16; 16%), and TP53 (N=12; 12%). Additionally, 25% of patients had an abnormal karyotype. Hematologic and genetic characteristics are summarized in (Table 1A). The median IPSS-M score was -0.9 (-3 to +2.34) and the median IPSS- Revised (R) score was 2 (0 - 6). The majority of patients had low (L) IPSS-M risk category (53%), followed by moderate low (ML) (19%) and very low (VL) (17%). Per IPSS-R, the majority had low-risk category (47%), followed by VL (38%) and intermediate (14%). Overall, 26 (25%) pt died after a median follow-up of 34 months and 21% progressed to MN (MDS 14 & CMML 8), and 1 patient with MDS and 1 with CMML progressed to secondary AML (2%). The median overall survival (mOS) was not reached, with a 2-year estimated (YE) OS and leukemia-free survival (LFS) of 80%. Predictive Power: When utilizing the IPSS-M risk category ( L, M, H) * by combining the risk categories into L *(L+VL), M * (ML+MH), and H *(H+VH) to overcome the low sample size limitation, it significantly predicted OS (p &lt; .0001) ( Figure 1A) and median MN-free survival (FS) (p &lt; .0001) ( Figure 1B). The 2YE-OS rates by IPSS-M risk category were as follows: VL - 93%, L - 87%, ML - 75%, high (H) - 25%, very high (VH) - 0% and the 2YE-OS rates by IPSS-M ( L, M, H) are summarized in Table 1B. IPSS-M hazard ratio (HR) for OS was 1.6 for ( M*) compared to 25 for ( H *) risk category (p &lt; .0001). The ( L *) category, being the largest, was used as the reference level. Conclusion: This study provides the first real-world experience of using the IPSS-M and a modified version of the IPSS-M ( L, M, H) * risk category model to predict survival and progression into MN in CCUS (MUT) pt. The IPSS-M risk category was found to be a significant predictor of OS and MN-free survival. We propose that larger cohorts are needed to validate our findings.

Background: According to the international consensus classification (ICC), diagnosis of myelodysplastic syndromes (MDS) with del(5q) requires presence of i) del5q, alone or associated with no more than one other cytogenetic abnormality, exclusive of -7/del(7q), ii) &lt;5% bone marrow (BM) and &lt;2% circulating blasts, and iii) the absence of “multi-hit” TP53 ( Arber et al. Blood 2022;140: 1200). We have recently reported on TP53 variant allele frequency (VAF) ≥22% being the sole genetic predictor of inferior survival in MDS-del(5q) (Fleti. et al. AJH 2023;98:E76). In the current study, we examined the additional prognostic impact of therapy-related setting, in the context of TP53 VAF and other risk factors. Methods : The current study included patients with MDS-del(5q) who were seen at the Mayo Clinic or Moffitt Cancer Center, USA. Diagnosis of MDS-del(5q) and therapy-related qualification was according to ICC criteria ( Arber et al. Blood 2022;140: 1200), with the exception that patients with biallelic or multi-hit TP53 were included to allow unbiased analysis of impact on prognosis. Patient selection was based on availability of mutation information from next-generation sequencing (NGS). The latter was assessed either at diagnosis or at different time points during the disease course, but at least 3 months before leukemic transformation. Otherwise, all analyses, including estimation of overall and leukemia-free survival, were based on variables collected at time of initial diagnosis of MDS-del(5q). TP53 mutation VAF threshold of 22% was considered based on our previously published report cited above. Age cutoff at 67 years was determined by ROC analysis. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results: A total of 210 patients (median age 72 years; 65% females), meeting the above-stipulated criteria for MDS-del(5q), were considered: 172 primary and 38 therapy-related. Table 1 outlines clinical and laboratory characteristics obtained at the time of diagnosis: 42% were transfusion-dependent, 69% had hemoglobin level &lt;10 g/dL, 30% platelet count &lt;150 x 10(9)/L, and 15% carried additional cytogenetic abnormality. NGS data performed either at diagnosis or at least 3 months prior to leukemic transformation was available in 146 patients; the most frequent mutations were TP53 (21%), SF3B1 (16%), DNMT3A (12%), TET2 (12%), ASXL1 (10%), and JAK2 (7%) (Table 1). At a median follow-up of 3.9 years (range 0.002-18.5) for living patients, 95 (45%) deaths and 34 (16%) leukemic transformations were recorded; treatment undertaken included lenalidomide in 162 (77%) cases. NGS data-exclusive multivariable analysis that included all 210 patients identified therapy-related setting (p&lt;0.01; HR 2.9; Figure 1a), age &gt;67 years (p=0.01; HR 1.8), transfusion-dependency (p=0.04; HR 1.5), and platelet count &lt;150 x 10(9)/L (p=0.03; HR 1.6), as independent predictors of inferior survival. The significant effect of age and platelet count was sustained when analysis was limited to patients with primary MDS-del(5q) but lost when applied to therapy-related disease. NGS data-inclusive multivariable analysis, which was applicable to the subset of 146 patients, identified, as risk factors, therapy-related setting (p&lt;0.01; HR 2.3) and TP53 VAF ≥22% (p&lt;0.01; HR 2.4; Figure 1b) while age &gt;67 years sustained borderline significance (p=0.07; HR 1.8). Figure 1c depicts survival data based on the presence or absence of TP53 VAF ≥22% and therapy-related qualification; low-risk with no risk factors (N=112; median survival 11.7 years; 5/10-year survival 73%/52%) and high-risk with one or both risk factors (N=34; median survival 4 years; 5/10-year survival 42%/14%); leukemia-free survival was affected by TP53 VAF ≥22% (p&lt;0.01) but not therapy-related setting (p=0.13). Conclusions : The current study highlights TP53 VAF and therapy-related setting as the most prominent risk factors for survival in MDS-del(5q) and proposes a simple risk model that distinguishes patients with a projected 10-year survival that exceeds 50%, in the absence of both risk factors, and plummets to 14%, in the presence of one of the two risk factors. Accordingly, allogeneic stem cell transplant might be deferred in the former instance while it should be pursued sooner than later, in the latter instance.

Background: Biallelic TP53 alterations result from either sequence variations or deletions involving TP53 and are not necessarily synonymous with “multi-hit TP53”. According to the 2022 international consensus classification (ICC) for myeloid neoplasms (MN), multi-hit TP53 signifies i) the presence of two or more distinct TP53 mutations, each with variant allele frequency (VAF) ≥10%, ii) a single TP53 mutation with VAF ≥50%, or iii) a single TP53 mutation with VAF ≥10% accompanied by a cytogenetically-apparent del(17p13.1), copy-neutral loss of heterozygosity (LOH) at the 17p TP53 locus, or, in the absence of LOH information, complex karyotype ( Arber et al. Blood 2022;140: 1200). The current study is focused on MN with multi-hit TP53 and examines the additional prognostic impact of morphologic subtype designation, bone marrow (BM) or peripheral blood (PB) blast percentage, TP53 VAF, and MN with diagnostic qualifiers (i.e., therapy-related, or secondary progressing from myelodysplastic/myeloproliferative/overlap syndromes/neoplasms). Methods : The current study was conducted under an institutional review board approved minimum risk protocol that allowed retrospective collection and analysis of data from Mayo Clinic patient records. Multi-hit TP53 was defined as per ICC criteria as outlined above ( Arber et al. Blood 2022;140: 1200). Morphologic subtype designations of MN were according to ICC criteria and assigned at the time of TP53 detection. NGS and cytogenetic information was available in all study patients. Survival analyses were calculated from time of TP53 mutation detection. Conventional statistical methods were applied using JMP Pro 16.0.0 software (SAS Institute, Cary, NC, USA). Results : Initial screening flagged 143 patients with biallelic TP53 abnormalities derived from formal laboratory reports of NGS data and cytogenetic studies; of these, 130 met ICC criteria for multi-hit TP53: pure erythroid leukemia (PEL; N=24), acute myeloid leukemia (AML)-not PEL (N=54), myelodysplastic syndromes (MDS; N=36), MDS/AML (N=11), and other MN (N=5). Of these 130 informative cases, 128 (98%) harbored complex/monosomal karyotype (CK/MK). Further analysis excluded patients with “other” MN (N=5) and those without CK/MK (N=2), the latter to mitigate the confounding effect of CK/MK on survival and the former because of small sample size and disease heterogeneity. Clinical and laboratory characteristics of the 124 study patients are outlined in table 1. Survival analysis stratified by ICC-defined MN subtypes revealed the prognostic relevance of morphological distinction between PEL vs “AML-not PEL” (p&lt;0.01; HR 2.4) and PEL vs “ TP53-muated MDS/AML” (p=0.02; HR 2.3) while survival was similar between “AML-not PEL” and “ TP53-mutated MDS/AML” (p=0.9; Figure 1a). Survival in “ TP53-mutated MDS” was significantly longer, compared to PEL (p&lt;0.01; HR 0.2), “ TP53-mutated AML-not PEL” (p=0.01; HR 0.5), and “ TP53-mutated MDS/AML” (p=0.07; HR 0.5), the latter with borderline significance (Figure 1a). Multivariable analysis confirmed the independent prognostic relevance of ICC subtype designation (p&lt;0.01) and also revealed additional negative prognostic contribution from advanced age (p=0.02), male gender (p=0.02), MN, secondary (p&lt;0.01), MN, therapy-related (p=0.03), and DNMT3A mutation (p=0.02). Significance was retained in all instances, with the exception of MN, therapy-related (p=0.15), when analysis was repeated after excluding PEL cases (Figure 1b). Conclusions : The current study confirms the prognostic validity of the ICC morphologic subtype designation and TP53 VAF classification threshold, in the context of multi-hit TP53. The study also highlights the prognostic distinction between PEL and “ TP53-mutated AML-not PEL” and the prognostic alignment between the latter and “ TP53-muated MDS/AML”, both of which displayed inferior survival, compared to “ TP53-mutated MDS”. The study also suggests additional prognostic contribution from secondary and therapy-related qualification while the observation regarding DNMT3A mutation requires validation with higher number of informative cases.

Introduction: Germline DEAD-box RNA helicase-1 mutant (DDX41) predisposition syndrome ( DDX41-MT-GPS) is one of the most frequent hereditary predisposition syndromes in adults with myeloid neoplasms (MN-2-5%). Somatic mosaicism involving the unmutated DDX41 allele (e.g., R525H) as a potential mechanism for myeloid evolution has been reported in 39-50% of patients (pts). In addition, given the later age of onset, somatic mosaic states composed of age-related clonal hematopoiesis (ARCH) (e.g., DNMT3A, ASXL1, TET2, and TP53), with reported frequencies less than expected in MDS and AML, have been documented. We conducted this study to define the somatic mosaic landscape in DDX41-MT-GPS . Methods: After approval from the Mayo Clinic IRB the prospective GPS database was queried for pts with DDX41-MT GPS. Germline confirmation was conducted on skin fibroblast-, or hair follicle-derived DNA. Somatic CH testing was conducted using a targeted NGS panel as previously described. Given the high frequency of variants of undetermined significance (VUS), comparisons were made between pathogenic ( DDX41path) and DDX41VUS. Results: We identified 106 pts with DDX41-MT-GPS, of which 83 (78%) met criteria for MN, 16 (15%) for CH/CCUS (clonal cytopenias of undetermined significance), while 7 (6%) were unaffected carriers. Somatic mosaicism in DDX41-GPS patients without MN Sixteen DDX41-MT pts without MN met criteria for CH/CCUS ( DDX41path [n=4] and DDX41VUS [n=12]; median age 69 years (yrs) (range [R], 19-78) ( DDX41path [69 yrs] and DDX41VUS [67 yrs], p=0.85), with a male predilection (75%). Four (25%) pts had somatic DDX41 MT (R525H [n=3] and A376T [n=1]) with median variant allele frequency (VAF) 10.5% (R, 5-27); 2 (50%) pts with germline DDX41path had R525H (VAF; 5% and 13%) and 1 (8%) pt each with germline DDX41VUS had R525H (VAF 5%) and A376T (VAF 27%) somatic variants, respectively. Abnormal cytogenetics (CG) were observed in 2/13 evaluable (15%) pts ([+8 and del20q]) in the germline DDX41path group only (p= 0.05). ARCH co-mutations were observed in 11 (69%) pts, not significantly (NS) different between DDX41path (75%) and DDX41VUS (75%), p= 0.66. Individual co-mutations: DNMT3A (25% vs 16%, p= &gt; 0.99), ASXL1 (25% vs 8%, p=0.71), TET2 (25% vs 8%, p= &gt;0.99), and JAK2 (0% vs 8%, p= &gt;0.99) were NS different between the two groups. Somatic mosaicism in DDX41-GPS patients with MN Eight-three pts met criteria for MN ( DDX41path [n=43] and DDX41VUS [n=40]; median age 67 yrs (range [R], 27-92), with a male predilection (58%). Fifty-four (65%) pts had MDS ( DDX41path [55%] and DDX41VUS [45%], p=0.55) and 29 (35%) had AML ( DDX41path [41%] and DDX41VUS [59%], p=0.65). Overall, 15 (18%) pts had somatic DDX41 MT; 12 (80%) pts with DDX41path and 3 (20%) pts with DDX41VUS (p= 0.003). The median VAF of somatic DDX41 MT was 12% (R, 5-37), NS different between DDX41path (10.5%) and DDX41VUS (12%) (p=0.08). The most common somatic DDX41 MT was R525H (66% [n=10]), significantly associated with DDX41path (9/10 [90%]), p=0.01. ARCH was observed in 17 (20%) pts, significantly higher in DDX41path (76%) compared to DDX41VUS (24%), p= 0.03. Abnormal CG were observed in 10/73 evaluable (14%) pts (-Y [n=2], del 20 [n=3], del 5q [n=2], del 7q [n=1], t (3;8) [n=1] and complex CG [n=1]), higher in DDX41path (70%) compared to DDX41VUS (30%) with p value &gt;0.99. We compared the spectrum of CH between DDX41 MT CH/CCUS and MN pts ( Table 1) and found no difference in the proportion of pts with somatic DDX41 MT (p= 0.52) and ARCH/myeloid MT (p= 0.56). The individual occurrences of DNMT3A (p= 0.49), ASXL1 (p= 0.71), TET2 (p=0.59), JAK2 (p&gt;0.99) and TP53 (p= 0.56) mutations were NS different between the two groups ( Figure 1). Conclusion: We define the somatic mosaic landscape in DDX41-MT GPS and demonstrate that somatic mutations involving the other allele, especially R525H, are seen in ~20% of pts (&gt;80% of R525H present in MN). The frequency and composition of ARCH and myeloid driver mutations was lower than expected for the age of presentation, in comparison to de novo and secondary MDS/AML and occurred more frequently in DDX41path compared to DDX41VUS. Forty-two % (n=41), including 37% with MN, did not have somatic mosaicism, underscoring the fact that mechanisms of leukemia progression remain to be defined in subsets of affected pts.

BACKGROUND: Recently proposed World Health Organization (WHO, 5 th edition) and International Consensus Classification (ICC) recognized myeloid neoplasms (MN) harboring TP53-mutated ( TP53mut) as a separate entity. However, there are critical differences between the two classifications regarding allelic-status, variance allele frequency (VAF) cut-off and blast categories, that may lead to under- or overestimation of the risk. METHODS: We analyzed MN harboring TP53mut (VAF ≥2%, n=536) managed at Mayo Clinic (USA) and South Australia Health Network (Australia). ICC (Arber et al, Blood 2022) and WHO (Bluebook, Beta v2) definitions were used to stratify MN harboring TP53mut. RESULTS: Of 536 cases, 379 (71%) had myelodysplastic syndromes (MDS) (&lt;20% blasts) and 157 (29%) had acute myeloid leukemia (AML). Using WHO, 160 (42%) of 379 were classified as MDS-bi TP53 as they harbored 2 TP53mut (n=115; 30%) or 1 TP53mut plus confirmed loss of heterozygosity (LOH) or copy neutral LOH (cnLOH) of TP53 locus (n=45; 12%). Sixty-two (16%) TP53mut MDS with VAF &gt;49% were classified as presumptive MDS-bi TP53. Of the 157 cases with 1 TP53mut VAF ≤49%, 17 confirmed to have an intact wild-type TP53 copy and were classified as monoallelic. While 140 (89%) cases did not have copy number variation (CNV) analysis. We next evaluated the number of TP53mut cases classified differently by WHO and ICC. As WHO does not recognize TP53mut AML as a separate entity, 157 (29%) TP53mut AML were not classifiable. 46% (174/379) of the TP53mut MDS including 48 (30%), 12 (19%) and 79 (65%) of bi TP53, presumptive bi TP53 and monoallelic TP53mut MDS according to WHO were classified differently by ICC ( Figure 1A). Discordance between the two classifications were predominantly driven by: (i) The difference in VAF cut-off. Sixty patients (11%) with TP53mut VAF &lt;10% were excluded from the ICC classification of TP53mut MN as it mandates TP53mut VAF cut-off ≥10%. 11 (18%) and 41 (68%) of these had bi TP53, monoallelic TP53mut MDS by WHO criteria while 8 (13%) had AML. Importantly, within the VAF &lt;10% cohort, median overall survival (OS) of 2 TP53mut or 1 TP53mut with 17p13.1 (hereafter 17p) deletion or complex karyotype (CK, n=30) was significantly poor compared to 1 TP53mut VAF &lt;10% (11.6 vs 32.4 months; P =0.002, Figure 1B), suggesting that ICC may underestimate the poor prognosis of multi-hit or multi-hit equivalent TP53mut MN with VAF &lt;10%. (ii) The difference in bone marrow blast cut-off. The underlying assumption by WHO is that prognosis of bi TP53mut MDS is poor irrespective of blast percentage category (0-9% vs 10-19%). In contrast, ICC considers that TP53mut MDS/AML (blasts 10-19%) is associated with poor prognosis irrespective of TP53 allelic status. Median OS of TP53mut MDS with 10-19% blasts was significantly poor compared to blast 0-9% (6.4 vs 11.6 months, P &lt;0.0001). Second, median OS of bi TP53mut was significantly poor than monoallelic TP53mut in MDS- blast 0-9% (10.1 vs 15.9 months; P =0.0001) but not in blasts 10-19% (6.8 vs 6.2 months; P =0.19). Hence, exclusion of monoallelic TP53mut MDS with blast 10-19% as TP53mut MN from the WHO classification may underestimate the poor prognosis. (iii) The difference in the definition of biallelic and multi-hit TP53 loss. 35 cases with 1 TP53mut VAF ≤49%, had 17p deletion but did not have CNV analysis to confirm the loss of TP53 locus and could not be classified further using WHO. ICC classified 18 (50%) of these cases as multi-hit. Median OS of 1 TP53mut VAF ≤49%, and 17p deletion on karyotype was significantly poor than 1 TP53mut (VAF ≤49%without 17p deletion on karyotype (6.4 vs 14.6 months, P =0.001). (iv) The difference in the definition of multi-hit equivalent. 32% (33/104) WHO defined monoallelic cases without available CNV data, were classified as multi-hit-equivalent by ICC as they harbored CK. However, WHO does not consider CK as presumptive evidence of bi TP53. Median OS of 1 TP53mut VAF ≤49% with CK was significantly poor compared to 1 TP53mut without CK (8.2 vs 26.9 months, P =0.0008). CONCLUSION: Our study provides evidence that poor prognosis of ‘multi-hit’ TP53 with VAF &lt;10% is underestimated by ICC. On the other hand, poor prognosis of monoallelic TP53 MDS with 10-19% blasts as well as 1 TP53mut VAF ≤49% in the presence of CK is underestimated by WHO. These data suggest that both classifications can be further refined.