Patnaik, Mrinal M.

Abstract Background: Germline predisposition states (GPS), including inherited bone marrow failure syndromes (IBMFS) are seen in 5-10% of individuals with myeloid neoplasms (MN). Given overlap with somatic driver genes, blood, saliva, and buccal swabs are suboptimal specimens for GPS testing in MN; the latter two due to contamination with blood cells(Jerez, 2024). Skin biopsy with fibroblast culture-derived DNA remains the gold standard, but the process is expensive, takes 6-8 weeks to complete, and fibroblast culture failure can occur in some patients with telomere biology disorders. Given the need to rapidly screen donors for HSCT and adjust therapy/conditioning in certain IBMFS, we developed a rapid hematologic germline testing algorithm using hair follicle-derived DNA. Methods The study was conducted after IRB approval through the Mayo Clinic GPS/IBMFS clinic. To perform PCR-based hair follicle testing, we obtained 15-25 hair follicles (bulbs) per patient. The bulbs were placed in a microcentrifuge tube and subjected to a 24-hour digestion and extraction protocol using a modified version of the QIAGEN DNA Purification from Dried Blood Spots protocol. DNA quality was assessed by Nanodrop and Qubit.Testing included either validation of known or suspected germline variants (e.g., RUNX1, DDX41, ETV6), or panel testing (e.g., GPS or IBMFS). Pathogenic variants were validated by repeat testing in a CAP/CLIA certified lab prior to medical decision making. 23 patients underwent whole exome sequencing (WES) in a CAP/CLIA lab, and 13 patients had concurrent germline testing using alternate DNA sources (8 skin biopsies, 1 saliva, 4 buccal swabs). Results Ninety-three patients (49 male, 53%) underwent hair follicle-derived germline DNA testing (median age 63 years). Indications for germline testing included the presence of a suspected variant on somatic testing with variant allele frequency (VAF) between 35%-60% ( n= 66, 71%), patient age <50 years (n=26, 28%), antecedent thrombocytopenia (n=15, 16%), presence of syndromic features (n=4, 4%), patient history of multiple malignancies (n=4, 4%), and positive family history (n=15, 16%). Time to reporting variants after receipt of hair follicle-derived DNA was <14 days. There were zero complications associated with the procedure. DNA quality as assessed by Nanodrop revealed a median nucleic acid concentration of 25.64 ng/μL. The median A260/A280 ratio was 1.86 (range 0.701-2.133) and the median A260/A230 ratio was 1.27 (range -8.193-9.223). DNA quality was insufficient for chromosomal microarray (CMA) assessments. Of 93 patients evaluated, 67 (72%) had ≥1 germline variant, with 3 having >1 germline variant for a total of 72 variants. Pathogenic/likely pathogenic variants identified included: DDX41 (30, 41.7%), TP53 (4, 5.6%), ANKRD26 (4, 5.6%), RUNX1 (4, 5.6%), GATA2 (3, 4.2%), TERT (3, 4.2%), JAK3 (2, 2.8%), SAMD9/SAMD9L (2, 2.8%), ATM (2, 2.8%), and 1 each (1.4%) in BCL2, BCOR, BPGM, CDKN2A, CEBPA, CHEK2, CSF3R, CXCR4, DNMT3A, ETV6, GATA1, MPL, MPO, POT1, PTPN11, RPL5, and SH2B3. In the 13 patients that had concurrent germline testing, results were identical in all cases, except for 1 false negative in a buccal specimen (PTPN11) and 1 case of germline mosaicism, where the TP53 variant was identified in the blood and hair follicle DNA but not in skin fibroblast DNA. After testing, 30 (44.7%) patients had a change in their management plan: 8 (27%) with DDX41-GPS were observed due to known latency of DDX41-mutant MN, 6 (20%) were referred for HSCT based on the germline variant (CEBPA, DDX41, GATA1, GATA2, RPL5, TP53), 6 (20%) were placed on an enhanced surveillance plan for cancer screening (1 ANKRD26, 3 TERT, 2 TP53), 4 (13%) patients undergoing HSCT had related donors removed from consideration as they were carriers (2 DDX41, 1 ANKRD26, and 1 SAMD9), 2 (7%) patients with neutropenia and underlying germline variants (1 ATM, 1 DDX41) had G-CSF therapy discontinued due to concerns for clonal stimulation, and 1 (3%) patient with a germline CDKN2A mutation (hematological diagnosis- CMML) was recommended to avoid radiation therapy for prostate cancer. Conclusion In summary, we validate a rapid and simple hair follicle-derived DNA based germline assessment for patients with hematological disorders and demonstrate a positive impact on clinical care in 44% of patients. We are currently optimizing DNA quality so that germline copy number alterations can be better detected using CMA.

Abstract Heterozygous germline variants of DDX41 occur in 2-4% of adult Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) cases, making them the most prevalent cause of inherited predisposition to these diseases. Homozygous deleterious variants are never observed in the germline, and loss of both DDX41 alleles causes apoptosis of proliferative cells, consistent with essentiality. Most disease-associated germline variants cause loss of full-length protein expression (truncating). However, 30-40% are missense (non-truncating) and their role in disease risk compared to truncating variants remains in question. Previous reports have shown that both truncating and non-truncating Pathogenic/Likely Pathogenic (P/LP) variants (defined by ACMG scoring) are associated with DDX41-specific disease features, including co-mutation of the contralateral allele of DDX41. In contrast, variants of unknown significance (VUS) are less associated with these features. Acquired co-mutations of DDX41, of which p.R525H accounts for 50-70%, are restricted to DDX41-mutated patients and are always missense. Our previous studies indicated that p.R525H lacks the essential DDX41 function and cannot support hematopoietic progenitor proliferation. However, the frequency with which this mutation occurs and the absence of acquired truncating mutations suggest that p.R525H has a specific effect on DDX41 function that is distinct from complete loss and promotes pathogenesis specifically in the context of existing P/LP DDX41 mutation. To understand how germline non-truncating variants predispose to disease, we characterized the effect of six recurrent non-truncating P/LP variants (p.G173R, p.R219H, p.P258L, p.Y259C, p.K331del, and p.I396T) and three VUS (p.K9E, p.Y33H, and p.M155H) on DDX41 function. We utilized a genetic complementation approach in murine lineage-negative (Linneg) bone marrow cells with inducible deletion of DDX41. We found that all non-truncating P/LP and VUS forms of DDX41 could fully rescue the colony forming potential of Ddx41-deficient cells to a similar level as wild-type protein, whereas the p.R525H variant completely failed to rescue. To determine the effect of the non-truncating variants on DDX41 function at endogenous expression levels, we utilized CRISPR to modify the DDX41 alleles in MOLM13 human AML cells. We produced viable, proliferating cell lines with homozygous mutations for all 3 VUS and 4/6 P/LP variants. For p.P258L and p.K331del, like p.R525H, we could not produce homozygous mutant cell lines despite screening hundreds of clones and finding several with heterozygosity. To further study the P/LP variants that were functional in the cell line, we generated C57Bl/6 mouse lines harboring p.G173R, p.Y259C, and p.I396T mutations in the endogenous Ddx41 alleles. Heterozygous mutant mice were phenotypically normal in all three strains. However, we could not obtain homozygous mutant mice for p.G173R or p.Y259C, indicating these cannot support embryonic/fetal development. In contrast, p.I396T-homozygous mutant mice were born at Mendelian ratios and were phenotypically normal until at least 15 months of age. Since p.R525H mutation is common in patients with non-truncating P/LP variants, we sought to model the combined effect of these mutations. We utilized the Linneg cells with inducible knockout of Ddx41, co-expressed each non-truncating variant with p.R525H by viral transduction, and monitored the colony formation potential. Remarkably, cultures expressing P/LP variants or M155I combined with p.R525H had dramatically reduced colony formation, while wild-type DDX41 and the other 2 VUS supported normal colony formation when co-expressed with p.R525H. Thus, we conclude p.R525H has a dominant-negative effect on certain non-truncating DDX41 variants. This is consistent with the emerging idea that the presence of an acquired hotspot DDX41 co-mutation is sufficient to classify a non-truncating DDX41 variant as pathogenic.Our results demonstrate that many DDX41 missense variants retain protein function when overexpressed. However, at endogenous expression levels, some P/LP variants are unable to support cell proliferation, indicative of hypomorphic effects on protein function. In patients, acquisition of the p.R525H mutation in the contralateral allele disrupts the functionality of P/LP variants through dominant-negative effects, leading to hematopoietic progenitor defects that are hallmark of the DDX41 pathogenesis.

Abstract Background Extramedullary disease (EMD) in multiple myeloma (MM) represents an aggressive phenotype with poor outcomes. We performed single-cell RNA sequencing (scRNA-seq) on clonal plasma cells (CPCs) from EMD tissue, solitary plasmacytomas (SPM), and bone marrow aspirates (BMAs) from patients with MM to identify transcriptional states unique to EMD. Methods We processed FFPE tumor samples from EMD and SPMs and cryopreserved CD138+ BMAs from MM, using the 10x Genomics FLEX platform. Cells/nuclei were fixed, hybridized with Human WTA probes, pooled, and sequenced (10,000–40,000 cells/library) on NovaSeq X. After removing low quality cells, data was normalized and integrated using RPCA in Seurat. Plasma cell identity was assigned via Cellarium and canonical markers. Clonality was inferred by light chain restrictions and refined via random forest modeling. Cell cycle effects were regressed out to reduce proliferation bias. Results We analyzed 128, 908 CPCs from 16 samples [8 EMD (62, 669 CPCs), 4 SPM (20, 607 CPCs), 4 BMA (45, 632 CPCs)] and identified eight transcriptionally distinct cell clusters. Differential expression and pathway comparisons were performed between EMD/SPM/BMA within each of these clusters. Integration of gene expression and pathway analyses revealed consistent upregulation of cell cycle and E2F pathways in EMD. We identified two highly proliferative cell cycle clusters (Cluster 2 with S-phase genes and Cluster 4 with a G2/M transcriptional program) that were expanded in EMD compared to BMA and SPM. Cluster 0 comprised a quiescent CPC population enriched for RNA processing pathways alongside downregulation of ER stress and autophagy. EMD cells demonstrated suppressed interleukin signaling [fold change (FC) –33.8), and class I MHC antigen processing (FC –23.5) relative to BMA, suggestive of an immune suppressive transcriptional program. Angiogenesis-related genes [VEGFA (log2FC 2.4), ADM2 (log2FC 2.2)] and EZH2 [log2FC 2.1; q<0.0001)] were upregulated in EMD. Compared to SPM, EMD plasma cells in this cluster demonstrated marked downregulation of CD74 (log2FC –2.6; q<0.0001), a key modulator of MHC class II antigen presentation. Cluster 1 comprised of metabolically stressed secretory CPCs and displayed downregulation of early growth response pathways in EMD compared to BMA, concurrent with upregulation of E2F signaling. Suppression of IL-2/STAT5, IL-6/JAK/STAT3, and TGF-β pathways in EMD suggests reduced reliance on bone marrow–derived cytokines and impaired immunoregulatory signaling. CD44 was upregulated in EMD versus BMA (log2FC 3.2), consistent with its role in EMD spread and marrow independence. Cluster 3 was enriched for extracellular matrix–interacting plasma cells (COL1A1, MMP2) with upregulated epithelial–mesenchymal transition (EMT) pathways in EMD compared to both BMA (FC 7.5) and SPM (FC 7.07). Cluster 6 was defined by a migratory, motility-primed plasma cell population (CD44, AHNAK, LMNA and S100A10). Notably, IL32 was overexpressed in EMD compared to BMA (log2FC 5.1; adj. p = 2.02×10⁻¹⁴), a finding linked to hypoxia-driven proliferation. Compared to SPM, EMD cells also had elevated MYC/E2F activity and DNA repair, hallmarks of a proliferative phenotype. Cluster 5 displayed a strong interferon-stimulated gene signature (ISG15, IFIT1, TNFSF10), with enrichment of type I IFN, JAK–STAT, and TNF pathways. Within this inflammatory cluster, EMD exhibited marked suppression of immune signaling, metabolism (TCA, OXPHOS), and TP53 programs, consistent with an immune-evasive, metabolically suppressed, and deregulated state. Finally, within Cluster 7, a smaller subset defined by NFκB–driven anti-apoptotic transcriptional activity, EMD exhibited significant downregulation of KLF2 (log2FC –3.7; adj. p = 1.77×10⁻²⁷), likely reflecting disruption of the KDM3A–KLF2–IRF4 axis, a transcriptional network critical for bone marrow homing and plasma cell survival. We consistently observed downregulation of the MAPK pathway in EMD compared to BMA, aligning with prior bulk RNA-seq data and contrasting with the near-universal presence of MAPK mutations, highlighting a disconnect between mutational status and pathway activation. Conclusion In the largest yet reported single-cell transcriptional atlas of EMD and SPM, we demonstrate EMD to be highly proliferative with metabolic reprogramming, EMT, and downregulation of key immunologic signaling pathways, indicating immune escape and microenvironmental independence.

Abstract BACKGROUND Ivosidenib is an oral inhibitor of mutant isocitrate dehydrogenase type I (IDH1) currently approved for the treatment of IDH1 mutated acute myeloid leukemia and myelodysplastic syndrome (MDS). Hematopoietic stem and progenitor cells carrying preleukemic mutations, referred to as clonal hematopoiesis (CH), are the origin of myeloid neoplasms (MN). Individuals who have cytopenias and CH, but do not meet diagnostic criteria for a hematologic malignancy, are classified as having Clonal Cytopenias of Undetermined Significance (CCUS), a condition associated with a high risk of progression to MN. While CCUS patients (pts) can have multiple mutations, they show lower mutational burden on average compared to MN. Thus, we hypothesized that treatment with ivosidenib would improve cytopenias in IDH1-mutant CCUS. METHODS; We performed a decentralized phase II single-arm study to characterize the safety and efficacy of ivosidenib in IDH1-mutant CCUS (NCT0503044). Drug was shipped to pts with local oncologists performing standard-of-care clinical exams supplemented by study team lead interviews for adverse event monitoring. Research labs were drawn by local providers or through Quest. QT corrected interval (QTc) was monitored through a mobile electrocardiogram device (AliveCor). Eligible patients were aged ≥ 18 years and had confirmed IDH1-mutant CCUS with at least 6 months of unexplained cytopenias (hemoglobin<10 g/dL, platelets<100 x109/L or absolute neutrophil count <1.8 x109/L). The primary endpoint was hematologic response of at least 8 weeks in duration based on the IWG 2006 criteria for MDS. RESULTS 20 pts with IDH1-mutant CCUS have been enrolled. Most were male (70%) with a median age of 66 (range 41-79). Overall, 74% had a self-reported race of non-Hispanic white, 11% of Hispanic white, 5% of Asian and 11% of black or African American. Most pts presented with neutropenia (95%) including some with co-occurring thrombocytopenia (21%). One pt presented with anemia and thrombocytopenia without neutropenia. Prior to treatment, the average number of co-mutations beyond IDH1 was 2 (range 0-3), with most pts (74%) harboring a co-occurring spliceosome gene mutation. The IDH1 variant allele fraction (VAF) was lower in the blood (mean=19%, range 3-35%) than in the bone marrow (mean=31%, range 18-43%) in most (93%) patients. Out of the 20 enrolled pts, 17 received at least 8 weeks of therapy at the time of our data cutoff (7/29/2025) and were included in the efficacy analysis. Grade ≥3 adverse events (AEs) occurred in five patients (26%) including two with grade ≥3 treatment-related adverse events (pancytopenia and sepsis in the first pt, rash in the second pt). The most common all-grade treatment-related adverse events were nausea (n=5, 26%), prolonged QTc (n=3, 16%), anemia (n=3, 16%), and diarrhea (n=3, 16%). There were no instances of reported differentiation syndrome. Overall, 82% of pts achieved a hematologic response (14/17) including improvement in neutropenia (88%) and thrombocytopenia (75%). With a median follow-up of 16 months (range 0.6-24 months), the median duration of hematologic response was not reached. Among responders, 86% had a continued response at one year. Two pts progressed to MDS after 4 and 17 months of treatment characterized by recurrence of cytopenias and the emergence of new clones. A surveillance bone marrow biopsy was performed at 18 cycles for the 8 pts who remained on treatment with none showing disease progression. Most pts (90%, N=9/10) treated for at least 12 months had a decrease in IDH1-mutant CH VAF over time and 40% (N=4/10) showed IDH1 mutation clearance (VAF <2%) with a median time to clearance of 9 months. Among those with decreasing IDH1 clonal burden, all had a corresponding decrease in other co-occurring mutations present at baseline with one responder showing emergence of a newly detectable CHEK2 mutation without disease progression. CONCLUSIONS Decentralized trials represent a feasible solution for the conduct of clinical trials in rare hematologic neoplasms and precursor conditions. Ivosidenib was well-tolerated and induced durable hematologic remissions in IDH1-mutant CCUS. Most pts showed a reduction in clonal burden with mutation clearance commonly seen with long-term treatment demonstrating that ivosidenib modifies the natural history of IDH1-mutant CCUS.

Abstract Background Mosaic chromosomal alterations (mCAs) are large-scale somatic gains, losses, or copy-neutral loss of heterozygosity (CNLOH) detectable by genotyping of peripheral blood DNA. They represent a form of clonal hematopoiesis and are observed in 0.5-5% of cancer-free individuals. The presence of any autosomal mCA has been associated with >3-fold risk of hematological malignancies. In particular, mCAs occurring in regions related to lymphoid malignancies have an 11-fold increased risk of lymphoid malignancies and a 68.6-fold risk for chronic lymphocytic leukemia (CLL). Monoclonal B-cell lymphocytosis (MBL), the precursor state to CLL, is defined by <5×10⁹/L clonal B cells in otherwise healthy individuals and is classified as low-count (<0.5x109/L) or high-count (0.5-5x10⁹/L) based on clonal B-cell count. MBL is also associated with increased risk of lymphoid malignancy (beyond CLL), with 4.3-fold and 74-fold risk in individuals with low- and high-count MBL, respectively. We previously reported a significant enrichment of autosomal mCAs in MBL, demonstrating a 1.7-fold association in those with low-count MBL and a 50.5-fold association in those with high-count MBL. Whether mCAs independently predict lymphoid malignancy risk after accounting for MBL status is unknown. Here, we evaluated the impact of mCAs on risk of incident lymphoid malignancies in individuals with MBL and in individuals screened negative for MBL. Methods CLL-type MBL (CD5 positive) was identified using flow cytometry assay on peripheral blood. Participants were age ≥40, had no prior hematologic malignancy, and were enrolled through the Mayo Clinic Biobank or the Division of Hematology at Mayo Clinic, Rochester. Participants were followed for incident lymphoid malignancy, including CLL, with diagnoses confirmed by medical record review. mCAs with cell fraction >1% were detected from array-based genotyping of whole blood DNA. Cox proportional hazards models, adjusted for age and sex, estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for risk of developing an incident lymphoid malignancy in individuals with autosomal mCA versus those without an autosomal mCA. Results In 4,331 participants with available MBL and mCA status, the median age was 67 years, and 45% were male. Flow cytometry identified 3,231 individuals who screened negative for MBL, 754 individuals with low-count MBL, and 346 individuals with high-count MBL. Autosomal mCAs were present in 6.5% of individuals without MBL, 7.6% with low-count MBL, and 71.1% with high-count MBL. Over a median follow-up of 6.9 years, 237 individuals developed an incident lymphoid malignancy. The most common types were CLL, multiple myeloma, and diffuse large B-cell lymphoma. Ignoring MBL status, the presence of an autosomal mCA was associated with increased risk of lymphoid malignancy (HR = 2.65; 95% CI: 1.45–4.86; P = 0.002), replicating previous results. However, after excluding individuals with MBL, this association attenuated; those with mCA alone did not have a significantly elevated risk (HR = 1.60; 95% CI: 0.57-4.52; P = 0.38) compared to individuals without MBL or mCA. We next examined the effect of mCAs among individuals with MBL. In those with low-count MBL, the most frequent mCA events were 13q loss (2.1%), 12 gain (1.9%), and 4q CNLOH (1.2%), and individuals with an mCA had a borderline significant risk of developing a lymphoid malignancy (HR = 2.78; 95% CI: 1.01–7.67; P = 0.049) compared to those without an mCA. Similarly, in those with high-count MBL, the most frequent mCA events were 13q loss/CNLOH (31.2%), 12 gain (13.0%), and 11q loss (4.9%), and the presence of an mCA conferred an increased risk compared to those without mCA (HR = 1.51; 95% CI: 1.09-2.08; P = 0.012). Conclusions Autosomal mCAs were more frequent in individuals with high-count MBL compared to those with low-count or no MBL. mCAs showed limited predictive value for lymphoid malignancy among individuals without MBL. In contrast, in individuals with low-count MBL, autosomal mCAs emerged as a potential biomarker for identifying those at risk of developing a lymphoid malignancy. In high-count MBL, mCAs were also a biomarker, but were driven by known chromosomal abnormalities identified through clinical testing. These findings suggest that the utility of mCAs as a predictive marker for lymphoid malignancy is contingent on the presence of MBL and may inform risk stratification and surveillance strategies in this population.

Abstract Introduction: TERT promoter (pTERT) variants are among the most frequent genetic alterations in human cancers, where they drive oncogenesis through telomerase reactivation. However, somatic, pathogenic pTERT variants have also been seen in non-cancerous conditions such as telomere biology disorders (TBD), where they attempt to rescue the bone marrow failure phenotype (adaptive compensation). We conducted this study to assess the phenotype and outcomes of pTERT mutations in TBD patients. Methods: After IRB approval, the Mayo Clinic institutional TBD database was queried to identify all patients with somatic pTERT mutations. We have designed a customized error corrected sequencing panel that covers somatic mutations (52 genes) associated with adaptive and maladaptive outcomes in TBD patients. This covers the promoter region for TERT and can detect variants down to 0.5% variant allele fraction (VAF) with 10,000 X sequencing depth. Clinical data was retrospectively abstracted. Results: We identified 7 pTERT variants (3 hotspots) in 6 (two females (33%), mean age 61 years, 4 alive). The three hotspots were: (1) c.-57A>C (2 individuals, mean VAF 17.1%; range 11.6%-22.6%), (2) c.-124C>T (4 individuals, mean VAF 12.9%; range 1.9%-41.3%), and (3) c.-146C>T (1 individual, VAF 1.2%). One individual had both the c.-124C>T (VAF 41.3%) and the c.-146C>T somatic pTERT variants, occurring in the context of a germline TERT variant. The median VAF for all 7 pTERT variants was 6.1% (range 1.2%-41.3%). The hotspot, c.-57A>C, occurred in the context of pathogenic germline TERT (VAF 49.9%) variants with pathogenic somatic co-variants in PPM1D (avg. VAF 1.5%). The second hotspot, c.-124C>T, occurred in the context of germline TERT (avg. VAF 49.6%) and RTEL1 (VAF 47.3%) variants with pathogenic somatic co-variants including ASXL1 (VAF 1%), DNMT3A (VAF 8%), and PPM1D (VAF 4%). Five of six assessable patients with pTERT variants had shortened telomere lengths (TL), with results pending for one individual. Among those with the c.-57A>C variant, granulocyte TLs were at or below the 10th percentile, while lymphocyte TLs were below the 10th percentile in one patient and below the 1st percentile in another. For those with the c.-124C>T variant, all granulocyte TLs were below the 1st percentile. Lymphocyte TLs in this group included one at the 10th percentile and two at or below the 1st percentile. The median follow up for all patients was 2 years. Hotspot 1 (c.-57A>C) variants were associated with normocellular bone marrow biopsies with one patient having normal blood counts and the other having chronic macrocytic anemia (grade 2) and thrombocytopenia (< grade 1). In patients with hotspot 2 and 3 somatic variants, bone marrow cellularity ranged from 15–50% (1 patient had grade 2-3 hypocellularity), with normal bone marrow morphology, except for polytypic plasma cells (10–15%) identified in a patient with IgG kappa multiple myeloma (currently MRD negative on maintenance therapy). This patient also had a history of head and neck cancer and follicular lymphoma. Of these four patients, only one had normal blood counts (co-occurring c.-146C>T variant); the rest had cytopenias (n=3, the worst being grade 2 thrombocytopenia), progressive macrocytosis (n=3), or eosinophilia (n=1). Comorbidities encountered in these patients included myasthenia gravis (1), hypogonadism (1), and sarcoidosis (1). Moreover, four patients (2 each for hotspots 1 and 2) presented with idiopathic pulmonary fibrosis, while liver fibrosis was seen in 4 patients (also 2 each for hotspots 1 and 2). At last follow up, none of these patients had documented progression to myelodysplastic syndrome or acute myeloid leukemia. Conclusions: We describe the spectrum and clinical features of somatic pTERT mutations in TBD patients. pTERT mutations involved the following hotspot regions, c.-124C>T, c.-146C>T, and c.-57A>C, and co-occurred with other somatic variants (i.e., PPM1D and DNMT3A). None of the 6 patients with pTERT mutations had a severe bone marrow failure phenotype or developed myeloid neoplasms at last follow up, exemplifying a unique mechanism of adaptive somatic rescue.

Abstract Prognosis in acute myeloid leukemia (AML) patients is influenced by various factors, most importantly, patient clinical status and cytogenetic/molecular abnormalities. Within those factors, elevated lactate dehydrogenase (LDH) is considered a poor prognostic indicator in AML patients. Our study aims at exploring the different factors associated with elevated LDH and their impact on overall survival (OS). After obtaining IRB approval, we reviewed the charts of 381 AML patients diagnosed at Mayo Clinic, Rochester, between the years of 2016 and 2023. All data was recorded at the time of AML diagnosis. Patients with no cytogenetic and molecular data were excluded from this study. LDH levels were recorded if measured within 10 days of diagnosis and treatment for AML was not yet given. Both risk stratification and response evaluation were done following the 2022 European LeukemiaNet (ELN) guidelines. OS was calculated from the date of AML diagnosis to the date of last follow-up or death. We used BlueSky Statistics V 10.3.4 for statistical analysis. We identified 299 patients (78.4%) who had elevated LDH levels (normal ≤ 222 U/L) at the time of diagnosis with a median of 417 U/L (range, 223-7989). The median age for those patients was 68 years (range, 18-94), with 78 (26.1%) of them having a prior myeloid disease (MDS, MPN, or MDS/MPN) before AML diagnosis. By risk stratification, more than half of the patients had an adverse risk profile (n=171, 57.2%), while 71 (23.7%) and 57 (19.1%) had an intermediate and favorable risk, respectively. Most of those patients had an abnormal karyotype (n=204, 68.7%), with complex karyotype (n=103, 34.7%) being the most common abnormality, followed by monosomy 7 (n=50, 16.8%) and trisomy 8 (n=46, 15.5%). Next-generation sequencing (NGS) was performed on 268 (89.6%) patients with elevated LDH, and the most common mutations were TP53 (n=60, 22.3%), FLT3-ITD (n=59, 21.1%), and NPM1 (n=51, 18.5%). TP53 mutations were more common in patients with normal/low white blood cell count (WBC) in contrast with FLT3 and NPM1, which were the most common mutations in patients with high WBC. After grouping the patients according to their LDH levels (group 1: 223-444 U/L, group 2: 444-666 U/L, group 3: > 666 U/L), we found that FLT3-ITD mutations were more likely to occur at higher LDH levels i.e. groups 2 and 3 (p= 0.002). The majority of patients with elevated LDH (n=262, 87.6%) received therapy following diagnosis, with the most common treatment regimens being a combination of a hypomethylating agent and venetoclax (HMA+VEN) (n=97, 37%) and a (7+3)-based induction therapy (n=77, 29.4%). When comparing patients with elevated LDH (n=299, 78.4%) to patients who had non-elevated LDH (n=82, 21.5%), no difference in both median age at diagnosis and risk stratification was found. We noticed an increase in WBC (median, 7.7 vs. 2.1) (p<0.001) along with an increase in peripheral blasts (median, 28% vs. 7%) (p<0.001) in elevated LDH patients. Among patients with non-elevated LDH, 21% (n=17) had a complex karyotype compared to 34.7% (n=103) in the elevated LDH group (p=0.022). The molecular profile was different between the 2 groups, where the most common mutations in patients with non-elevated LDH were IDH2 (n=19, 25.3%), RUNX1 (n=17, 23%), and DNMT3A (n=16, 21.6%). All patients (n=82, 100%) in the non-elevated LDH group received therapy after diagnosis. There was no difference in terms of treatment regimens used between the 2 groups. Moreover, no statistical difference in remission rates after one line of therapy was found between those groups where non-elevated LDH patients had a 65.9% remission rate compared to the 55.3% remission rate in patients with elevated LDH (p=0.098). Patients with elevated LDH had a shorter OS than patients with non-elevated LDH (9.5 vs. 26.4 months, p=0.0008). However, median OS was similar across the different groups of elevated LDH. Using univariate analysis in our cohort (n=381), age, HSCT, risk stratification, FLT3 mutation, and LDH elevation all had an impact on OS. However, when comparing those variables using multivariate analysis, age and FLT3 mutation lost their significance (p-value of 0.25 and 0.84, respectively).Complex karyotype and FLT3 mutations were more common in patients with elevated LDH. These patients tend to have higher WBC and peripheral blasts. Elevated LDH had a negative impact on patients' OS regardless of patients' risk classification.

ABSTRACT Background Chronic myelomonocytic leukemia (CMML) is largely a disease of individuals older than 50 years; its occurrence in patients aged 50 years and younger is rare. The authors sought to characterize the clinicopathologic features and genetic profile of young‐onset (age 50 years and younger) CMML (y‐CMML) compared with older onset (older than 50 years) CMML (o‐CMML). Methods The authors conducted a multi‐institutional study of 32 patients with y‐CMML and 119 patients with o‐CMML, analyzing clinical, morphologic, cytogenetic, and molecular data. Results Patients who had y‐CMML frequently presented with splenomegaly (59% vs. 11%; p < .001) and hepatomegaly (25% vs. 2%; p < .001) compared with those who had o‐CMML. Although the overall incidence of cytogenetic abnormalities was similar, patients who had y‐CMML exhibited distinct mutational patterns. PTPN11 mutations were significantly more common in y‐CMML (13% vs. 3%; p = .034) than in o‐CMML, whereas TET2 (6% vs. 57%; p < .001) and SRSF2 (16% vs. 46%; p = .002) mutations were significantly less frequent. In addition, multi‐hit TET2 (6% vs. 34%; p = .002) and TET2‐SRSF2 (3% vs. 29%; p = .003) co‐mutations were rare in y‐CMML. However, the molecular differences did not affect the CMML‐specific risk‐stratification categories because the clinical outcomes, including overall survival and leukemia‐free survival, did not differ between the two groups. Conclusions CMML in patients aged 50 years and younger is rare and presents with unique clinical and mutational features, suggesting a distinct pathogenesis.

Summary Chronic myelomonocytic leukaemia (CMML) is a morphological hybrid between myelodysplastic and myeloproliferative neoplasms and is characterized by clonal monocytosis. Myeloid neoplasm‐associated somatic mutations are present in >90% of patients with CMML but are not diagnostically specific but a subset are prognostically relevant. Current drug therapy in CMML is mostly palliative and not disease‐modifying. Allogeneic haematopoietic stem cell transplantation is a potentially curative treatment modality in CMML and its timing is determined by formal prognostic models including the recently developed BLAST and BLAST‐molecular risk models.