Ferrer, Alejandro

Background: Telomeres are repetitive sequences at the end of linear chromosomes that preserve genetic information. Patients with telomere biology disorders (TBDs) are subject to multiorgan manifestations, which include the liver. However, due to the rare nature of the disorder, these manifestations are not fully characterized. Methods: Here, we describe the hepatic manifestations of patients with TBD at a large tertiary center within a specialized TBD clinic; 121 consecutive patients with TBD seen from 2017 to 2024 were included. The median age of the TBD diagnosis was 43 years old, with 60% male. The most common pathogenic/likely pathogenic variant was TERT (25%), followed by RTEL1 (10%) and TERC (7%). Results: Evidence of hepatic involvement in TBD was observed in 60% of patients (73 of 121), as determined by imaging studies or abnormal liver chemistries. Hepatic steatosis was the most prevalent imaging finding (56%), while a cholestatic pattern of liver injury was the predominant laboratory abnormality (73%). Biopsy-proven portosinusoidal vascular disorder was present in 4 patients. Findings of portal hypertension were observed in 25 patients (34%), with decompensation observed in 12 patients (16%). Traditional liver imaging modalities (computer tomography, ultrasound, and MRI) were compared with magnetic resonance elastography in 41 patients. Of these, 7 patients had abnormal magnetic resonance elastography with normal traditional imaging (17%), indicating higher sensitivity of magnetic resonance elastography to detect liver involvement. Conclusions: This study describes the liver manifestations within a cohort of patients with TBD with findings of portosinusoidal vascular disorder and hepatopulmonary syndrome similar to other cohorts and a noted higher prevalence of hepatic steatosis as compared with the general population. Liver stiffness measurements, integrated with imaging and laboratory testing, further improve characterization of hepatic manifestations in TBD.

Abstract Germline variants in DDX41 are the most frequent genetic predisposition to adult hematologic malignancies. The most common variants are truncating, implicating loss of function in the pathogenesis. However, non-truncating variants account for 30-40% of cases, and their impact on essential DDX41 functions remains unknown. We utilized a genetic complementation assay to assess the functionality of 10 recurrent germline non-truncating variants of DDX41. All variants restored viability to Ddx41 -deficient hematopoietic progenitor cells at exogenous expression levels. In contrast, the hotspot mutant p.R525H, which is somatically acquired at disease onset in >50% of patients, failed to restore viability. CRISPR-based modeling in cell lines and mice revealed heterogeneity: some variants were non-functional at endogenous expression levels whereas others maintained complete functionality, supporting normal cell proliferation and even lifelong hematopoiesis in a homozygous setting. Notably, co-expression of p.R525H with some variants caused impaired hematopoietic progenitor cell viability, indicating a dominant-negative effect of p.R525H. In contrast, other variants, all classified as variants of unknown significance, were unaffected by the presence of p.R525H. A screen of 100 disease-associated variants confirmed that many non-truncating germline variants are susceptible to p.R525H-mediated dominant-negative effects, whereas wild-type DDX41 is not. These findings indicate that DDX41 variant curation is complicated by variable effects on functionality and variant-specific interactions with somatically-acquired DDX41 mutations. The dominant-negative effect of p.R525H provides a mechanistic basis for the conclusion of recent patient cohort analyses that co-occurrence with a somatic hotspot mutation is a reliable indicator of DDX41-driven disease in carriers of non-truncating variants.

Abstract Background Telomere maintenance and surfactant-related genetic mutations are increasingly recognized as contributing factors in familial pulmonary fibrosis (PF). Pathogenic variants in the SFTPA2 gene have been associated with early-onset pulmonary fibrosis and an elevated risk of developing lung cancer. Currently, PF lacks a definitive cure beyond lung transplantation, and treatment options remain limited. Understanding the prevalence and clinical implications of pathogenic SFTPA2 variants is critical. This study aims to assess the prevalence of PF, lung cancer, and other co-morbidities among individuals carrying pathogenic SFTPA2 variants. Ultimately, this study seeks a comprehensive understanding of the natural history and potential screening needs for SFTPA2 variants. Methods Individuals with pathogenic SFTPA2 variants were identified in the Tapestry genomic database at our academic medical center. Electronic medical records (EMR) were reviewed for personal or family histories of PF, lung cancer, and other significant comorbidities. Imaging reports were evaluated for radiographic evidence of PF or lung cancer. Studies were approved by the local IRB. Results Out of 98,109 participants in the Tapestry database, 18 individuals were identified as carriers of SFTPA2 variants. The variants (NM_001098668.4) included c.460G>C - p.(Ala154Pro) in 13 individuals, c.557A>G - p.(Tyr186Cys) in 1 individual, and c.532G>A - p.(Val178Met) in 4 individuals. The mean age was 60.5 years, and 67% were female. No participants had a formal diagnosis of PF; however, five (28%) had prior chest CTs showing ground-glass opacities, fibrosis, or irregular nodularity. EMR review did not confirm clinically significant PF. Notably, 44% reported a family history of primary lung cancer, though no familial PF was documented. Comorbidities included inflammatory bowel disease (22%, with a tapestry cohort prevalence of 9%) and a personal history of cancer in 44%, encompassing nine distinct malignancies such as metastatic colon adenocarcinoma (1), prostate cancer (1), basal cell carcinoma of the skin (1), endometrial cancer (1), breast cancer (2), melanoma (1), and squamous cell carcinoma of the skin (1). Conclusions In this cohort of individuals with SFTPA2 variants, no cases of clinically confirmed PF or lung cancer were identified. However, there was a notable prevalence of abnormal parenchymal chest CT findings, inflammatory bowel disease, personal cancer history, and family history of lung cancer. These findings underscore the variable penetrance of SFTPA2 variants and highlight the need for longitudinal studies to elucidate the genetic, environmental, and immunologic factors that modulate disease expression. Improved understanding may inform screening strategies and therapeutic interventions for at-risk individuals. This abstract is funded by: None

Abstract While missense somatic mutations in the serine/arginine rich splicing factor 2 (SRSF2) occur early in the clonal development of myeloid neoplasms, they are uniquely enriched in chronic myelomonocytic leukemia (CMML), an MDS/MPN overlap neoplasm, where their estimated prevalence is ∼50%. Short-read RNA-sequencing experiments have demonstrated that missense alterations of the proline 95 residue hotspot (SRSF2P95) globally dysregulate exon inclusion, though the full transcript structures induced by such alternative splicing events remain uncharacterized. Given the striking enrichment of SRSF2 mutations in CMML, we hypothesized that there exist unique splicing alterations contributing to the biology of this disease, and sought to profile the landscape of alternative transcription induced by SRSF2 mutations in CMML using long-read RNA-Seq. We conducted bulk long-read RNA-Seq on bone marrow mononuclear cells from 5 CMML patients with SRSF2P95H (n=3) and SRSF2P95L (n=2) mutations, with VAFs ranging 20-48%, along with 5 CMML splicing factor wildtype patients. Libraries were prepared with the Pacbio Kinnex full-length RNA kit and sequenced on a Revio SPRQ sequencer. Reads were processed and aligned using the IsoSeq pipeline, and isoform collapsing, quality control, reference comparison, and quantification were performed with IsoQuant and SQANTI3. Differential transcript and gene expression was assessed with DESeq2, while differential transcript usage was assessed with the DRIM-Seq and stageR packages. After long-read collapsing and quality control, we identified 255,505 unique transcript isoforms, of which 14,845 (∼5.8%) were novel to the reference transcriptome annotation (GENCODE v39). We identified 23 transcripts differentially expressed in SRSF2-mutant samples, including 9 novel transcripts, the majority of which (n=7) were upregulated in SRSF2-mutant cases. A higher number of transcripts (n=80) and genes (n=65) demonstrated significant changes in proportional transcript usage. Among transcripts with increased expression and usage in SRSF2-mutant cases, we identified a novel transcript of the gene YBX1, which encodes an RNA- and DNA-binding transcription factor required for cell survival and self-renewal in myeloid neoplasms. This novel transcript, which we termed YBX1ΔEx4, has an in-frame loss of exon 4, and is predicted to encode a full-length protein missing a 30 amino acid region of the cold-shock (DNA-binding) domain. Using short read RNA-Seq data from an additional 29 CMML patients, as well as an independent dataset (GSE251806) comprising 43 blast-phase CMML patients, we confirmed increased exclusion of YBX1 exon 4 in SRSF2-mutant cases, with an even greater loss of exon 4 in blast-phase SRSF2-mutant cases. We hypothesize an important role of this isoform in CMML disease progression and are currently performing functional studies to characterize the effect of YBX1ΔEx4. Citation Format: Nickolas Steinauer, Terra Lasho, Christy Finke, Pankaj Pradeep, Jenna Fernandez, Alejandro Ferrer, Moritz Binder, Abhishek Mangaonkar, Mrinal M. Patnaik, . Long-read RNA-Seq reveals a novel isoform landscape of SRSF2-mutant chronic myelomonocytic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1497.

Abstract Introduction Heterozygous germline pathogenic variants (PVs) in the 5' untranslated region of ANKRD26 cause inherited thrombocytopenia and predisposition to hematologic malignancy (HM). The estimated lifetime HM risk is ~10% based on families ascertained from an inherited thrombocytopenia cohort. However, the malignancy spectrum, molecular features, precursor states, and overall clinical outcomes remain poorly defined. Methods We established an international, multicenter cohort of individuals with ANKRD26-related thrombocytopenia diagnosed after undergoing genetic evaluation due to a personal and/or family history of thrombocytopenia and/or HM. Clinical, pathologic, and molecular data were collected for 27 individuals with germline ANKRD26 PVs from 21 unrelated families treated at 11 institutions across 5 countries. Statistical analyses were performed using R (v4.4.2). Results Patients were predominantly male (18/27) and white (25/27). Thrombocytopenia was typically identified in childhood (median age 10 years, range 0-48) with a genetic diagnosis made later (median age 53 years, range 2-80). The mean (+/- SD) pre-malignancy platelet count was 45 +/- 24 K/uL (range 5-87). ANKRD26 PVs observed in each family included: c.-126T>C (n=9), c.-128G>A (n=3), c.-134G>A (n=3), c.-116C>T (n=3), c.-118C>T (n=2), and c.-118C>A (n=1). Thirteen HMs were diagnosed in 11 of 27 (41%) patients, including myelodysplastic neoplasm/syndrome (MDS, n=5), acute myeloid leukemia (AML, n=3), chronic myeloid leukemia (CML, n=2), MDS/myeloproliferative neoplasm (MPN) overlap (n=2), and Langerhans cell histiocytosis (n=1). The median age at first HM diagnosis was 63 years (range 49-80). Recurrent somatic features in myeloid malignancies included complex karyotype (4/11, 36%) and TP53 mutations (5/10, 50%). Of the 3 patients with AML, 1 achieved a partial remission (PR) with venetoclax combined with cladribine, idarubicin, and cytarabine but relapsed, 1 achieved a PR with azacitidine but relapsed <100 days after haploidentical allogeneic stem cell transplantation (alloSCT), and 1 had primary refractory disease. Among 5 patients with MDS, 1 was refractory to decitabine, 1 underwent alloSCT but relapsed 2 years post-transplant, 1 remains in remission post-alloSCT with 1 year of follow-up, 1 is undergoing alloSCT evaluation, and 1 was lost to follow-up. Both patients with CML achieved durable major molecular remissions with imatinib. To better estimate HM risk, we assumed 100% penetrance of thrombocytopenia for ANKRD26 PV carriers and included family members with chronic thrombocytopenia who had not had germline genetic testing. In this expanded series (n=62), 16 individuals developed a HM (26%). Additional diagnoses included MDS with progression to AML (n=1), AML (n=1), CML (n=1), and leukemia of unknown type (n=2). Cumulative incidence of HM was 0% by age 40 years, 17% by age 60 years, and 60% by age 80 years. Somatic sequencing of peripheral blood was performed in 5 HM-unaffected ANKRD26 PV carriers. One individual was found to have clonal hematopoiesis driven by TET2 (c.1648C>T, p.Arg550*) with a variant allele frequency (VAF) of 9% at age 55. Bone marrow biopsy showed mild megakaryocytic atypia and 4% blasts without overt evidence of malignancy. To assess the prevalence of ANKRD26-related HM, we assessed the frequency of ANKRD26 PVs in 2 large MDS/AML cohorts. In the ARUP database (9662 AML, 6397 MDS), 2 individuals (0.01%) had an ANKRD26 PV with a VAF consistent with germline origin. In the MSK-IMPACT Heme Germline MDS/AML cohort (n = 284), no ANKRD26 P/LP variants were identified. Conclusions Germline ANKRD26 PVs underlie <1% of MDS/AML diagnoses but may represent a high-risk subtype characterized by complex karyotype, TP53 mutations, and high risk of relapse. Among our series, the incidence of HM was low early in life but increased substantially with age, reaching 60% by 80 years. This exceeds prior estimates, which were derived from individuals ascertained from thrombocytopenia-based registries. Together, our findings suggest that ANKRD26-associated HM may represent a high-risk clinical entity, and that age and family history should inform surveillance strategies. More studies are needed to define genetic and environmental modifiers of HM risk as well as precursor states to enable early detection and disease interception.

Abstract Introduction: DDX41 (DEAD-box helicase 41) mutations (mt) represent the most prevalent germline predisposition syndrome (GPS) in adult patients (pts) with myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). This molecular subtype is typically associated with late-onset disease, runs an indolent clinical course, with a relative absence of high-risk co-mutations. While several retrospective studies have reported favorable responses to venetoclax (VEN)-based therapies, anecdotal observations suggest variable outcomes. We aimed to evaluate the clinical characteristics and treatment outcomes of patients with DDX41-mt MDS/AML treated at the Mayo Clinic. Methods: We conducted a retrospective study of pts diagnosed with DDX41-mt germline predisposition syndrome with concurrent myeloid neoplasms at our institution. Germline testing was conducted under an IRB approved protocol (Mayo Clinic IRB# 16-004173; clinical-trials gov. Identifier: NCT02958462). Of 198 pts with DDX41-mt myeloid neoplasms, 77 had high-risk disease—43 with MDS with increased blasts (MDS-IB1, n = 11; IB2, n = 32) and 34 with AML (WHO 2022). We assessed how clinical, and disease features influenced outcomes in this subgroup. Among these pts, 69 (90%) pts had likely pathogenic/pathogenic variant (DDX41path) variants and 8 (10%) pts had variant of undetermined significance (DDX41VUS) according to variant classification criteria from the American College of Medical Genetics/ the Association of Medical Genetics/ the Association for Molecular Pathology (ACMG/AMP) guidelines. Results: The median age was 68 years (yrs) (range [R],34-92), 70% were male and the median bone marrow blast % was 15 (R,5-60). The most common DDX41 germlinevariants (> 5% cases) included p.D140fs (26%), p.M1I (22%), and p.P258L (6.5%). 74% had DDX41 truncating variants. Thirty-one (40%) acquired a somatic DDX41-mt (R525H [81%]) and 43 (56%) had other somatic mutations. Among these, the most frequent were ASXL1 (n= 9, 21%), TP53 (n= 8, 10%), splicing factor (n= 7, 9%), and DNMT3A (n= 5, 6%). Twenty-six, (34%), 26 (34%), 15 (19%) and 9 (12%) pts were treated with hypomethylating agents (HMA), HMA+VEN, intensive chemotherapy (IC) and active surveillance/supportive care, respectively. Overall, the composite complete remission rate (cCR) in high-risk MDS and AML was 56%. The cCR rates (41%, 59% and 68.7%, p= 0.10) and median duration of response (DOR; 43.7, 44.9 and 28.9 months [mo], p= 0.46) were not significantly different with HMA, HMA+VEN, and IC induction. Thirty-one (40%) pts received allogeneic stem cell transplantation (allo-HCT); 4 (36%), 15 (47%) and 12 (35%) in MDS-IB1, MDS-IB2 and AML groups, respectively (p= 0.56). With a median follow up of 34.7 mo (R, 0.7-136.7), the median overall survival (OS) from the time of diagnosis was 56.9 mo (95% CI, 50.7-63.0) and was not significantly (NS) different between MDS-IB1 (not reached, 70% at 3 yrs), MDS-IB2 (56.9 mo, 86% at 3 yrs) and AML (55.7 mo, 60% at 3 yrs) groups, p= 0.16). The median OS was NS different between DDX41path and DDX41VUS (58.6 vs 55.7 mo, p= 0.50), respectively. We conducted a multivariable analysis for OS using variables that showed significance or trend towards significance in univariable analysis. Concurrent splicing factor mutations (HR; 5.29, 95% CI: 1.29-21.6, p= 0.02) remained negatively prognostic, while there was a trend towards better survival outcomes with allo-HCT (HR; 0.48, 95% CI: 0.21-1.07, p= 0.07). Bone marrow blast ≥ 20% (HR; 1.87, p= 0.12) and TP53-mt (HR; 2.14, p= 0.12) did not retain significance. Conclusion: In our cohort of patients with germline DDX41-mt high-risk MDS and AML, VEN-based regimens were not superior to HMA alone or IC, in terms of response rates and outcomes. On multivariable analysis, the presence of concurrent splicing factor mutations independently and negatively impacted survival with a trend towards better outcomes with allo-HCT. These findings highlight the heterogeneity within DDX41-mt myeloid neoplasms and underscore the need for future studies in larger cohorts to better define predictors of response and survival.

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.