Recurrent Mutations Research Articles

Recurring EPHB1 mutations in human cancers alter receptor signalling and compartmentalisation of colorectal cancer cells

BackgroundEphrin (EPH) receptors have been implicated in tumorigenesis and metastasis, but the functional understanding of mutations observed in human cancers is limited. We previously demonstrated reduced cell compartmentalisation for somatic EPHB1 mutations found in metastatic colorectal cancer cases. We therefore integrated pan-cancer and pan-EPH mutational data to prioritise recurrent EPHB1 mutations for functional studies to understand their contribution to cancer development and metastasis.MethodsHere, 79,151 somatic mutations in 9,898 samples of 33 different tumour types were analysed with a bioinformatic pipeline to find 3D-mutated cluster pairs and hotspot mutations in EPH receptors. From these, 15 recurring EPHB1 mutations were stably expressed in colorectal cancer followed by confocal microscopy based in vitro compartmentalisation assays and phospho-proteome analysis.ResultsThe 3D-protein structure-based bioinformatics analysis resulted in 63% EPHB1 mutants with compartmentalisation phenotypes vs 43% for hotspot mutations. Whereas the ligand-binding domain mutations C61Y, R90C, and R170W, the fibronectin domain mutation R351L, and the kinase domain mutation D762N displayed reduced to strongly compromised cell compartmentalisation, the kinase domain mutations R743W and G821R enhanced this phenotype. While mutants with reduced compartmentalisation also had reduced ligand induced receptor phosphorylation, the enhanced compartmentalisation was not linked to receptor phosphorylation level. Phosphoproteome mapping pinpointed the PI3K pathway and PIK3C2B phosphorylation in cells harbouring mutants with reduced compartmentalisation.ConclusionsThis is the first integrative study of pan-cancer EPH receptor mutations followed by in vitro validation, a robust way to identify cancer-causing mutations, uncovering EPHB1 mutation phenotypes and demonstrating the utility of protein structure-based mutation analysis in characterization of novel cancer genes.BS_YKV7jikZ2WYCZfZ4ZjJVideo

Recognizing puzzling PD1 + infiltrates in marginal zone lymphoma by integrating clonal and mutational findings: pitfalls in both nodal and transformed splenic cases

BackgroundA marked increase in PD1-positive TFH cells in nodal MZL cases (NMZL) was reported previously and could prompt suspicion for a diagnosis of peripheral T-cell lymphoma (PTCL), especially angioimmunoblastic T-cell lymphoma (AITL).Case presentationTo demonstrate that the pitfall might exist not only in NMZL but also in transformed splenic MZL (tSMZL), two NMZL cases (70 y/o female with enlarged left cervical lymph node and 75 y/o male with generalized lymphadenopathy) and one case of tSMZL (47 y/o male with nodal and extranodal involvement) with obvious PD1-positive T-cell hyperplasia were described here. Although all their initial diagnoses were prompted to be AITL, they were comprehensively characterized by clinical features, morphologic, immunophenotypic, clonality, and targeted exosome sequencing (TES) findings. Case 1 and Case 2 were NMZL with increased PD1 + T cells in the “peripheral pattern” or “mixed peripheral and central pattern”, and Case 3 was SMZL with abundant PD1-positive T cells in the “nodular pattern” that transformed to tSMZL (DLBCL) with PD1-positive T cells distributed in the “diffuse pattern.” In addition to the monoclonal IG rearrangement and polyclonal TCR rearrangement results, TES demonstrated enriched and recurrent mutations in MZLs and failed to find aberrations described in AITL- or TFH-derived lymphomas.ConclusionsIt is important to realize that this pitfall can also occur in more diagnostically difficult tSMZL cases; the integration of histopathology with clonality and mutation studies is also highlighted.

Polyhexamethylene biguanide promotes adaptive cross-resistance to gentamicin in Escherichia coli biofilms.

Antimicrobial resistance is a critical public health issue that requires a thorough understanding of the factors that influence the selection and spread of antibiotic-resistant bacteria. Biocides, which are widely used in cleaning and disinfection procedures in a variety of settings, may contribute to this resistance by inducing similar defense mechanisms in bacteria against both biocides and antibiotics. However, the strategies used by bacteria to adapt and develop cross-resistance remain poorly understood, particularly within biofilms -a widespread bacterial habitat that significantly influences bacterial tolerance and adaptive strategies. Using a combination of adaptive laboratory evolution experiments, genomic and RT-qPCR analyses, and biofilm structural characterization using confocal microscopy, we investigated in this study how Escherichia coli biofilms adapted after 28 days of exposure to three biocidal active substances and the effects on cross-resistance to antibiotics. Interestingly, polyhexamethylene biguanide (PHMB) exposure led to an increase of gentamicin resistance (GenR) phenotypes in biofilms formed by most of the seven E. coli strains tested. Nevertheless, most variants that emerged under biocidal conditions did not retain the GenR phenotype after removal of antimicrobial stress, suggesting a transient adaptation (adaptive resistance). The whole genome sequencing of variants with stable GenR phenotypes revealed recurrent mutations in genes associated with cellular respiration, including cytochrome oxidase (cydA, cyoC) and ATP synthase (atpG). RT-qPCR analysis revealed an induction of gene expression associated with biofilm matrix production (especially curli synthesis), stress responses, active and passive transport and cell respiration during PHMB exposure, providing insight into potential physiological responses associated with adaptive crossresistance. In addition, confocal laser scanning microscopy (CLSM) observations demonstrated a global effect of PHMB on biofilm architectures and compositions formed by most E. coli strains, with the appearance of dense cellular clusters after a 24h-exposure. In conclusion, our results showed that the PHMB exposure stimulated the emergence of an adaptive cross-resistance to gentamicin in biofilms, likely induced through the activation of physiological responses and biofilm structural modulations altering gradients and microenvironmental conditions in the biological edifice.

Recurrent Mutations in Refractory/Relapsed Diffuse Large B-Cell Lymphoma by Targeted Gene Sequencing

Introduction: Whole-genome sequencing of diffuse large B-cell lymphoma (DLBCL) has identified recurrent mutations involved in pathogenesis and potentially affecting response to therapy. In this pilot study, a targeted gene panel was created to identify mutations associated with relapse/refractoriness. Material and Methods: A 14-gene targeted panel was designed to sequence thirteen patients who were in remission and 8 cases that had relapsed/refractory to treatment. A paired diagnostic biopsy and a relapse biopsy were sequenced to find genes repeatedly altered in relapse. Results: A total of 751 nonsynonymous and truncating mutations were identified. Truncated mutations in NOTCH1, TNFAIP3, and CD58 were associated with poor treatment outcomes. In cases that did not respond to treatment, a high number of mutations were found in the EZH2 gene, followed by the DNA-binding domain of TP53 and MYD88. Termination mutations in the intracellular domain of NOTCH were found in 75% of non-responsive cases. Co-occurrence of loss of function mutations of TNFAIP3 and missense mutations in MYD88 was associated with a non-responsive cohort. Discussion: The study highlights mutations associated with chemotherapeutic response in DLBCL with implications for initial diagnostic biopsy response prediction.

A recurrent pathogenic splicing mutation of Colony-stimulating factor 1 receptor is responsible for a Chinese family with diffuse leukoencephalopathy.

A recurrent pathogenic splicing mutation of Colony-stimulating factor 1 receptor is responsible for a Chinese family with diffuse leukoencephalopathy.

Colloid Pattern of Salivary Mucinous Adenocarcinomas With Recurrent BRAF V600E Mutations.

The relationship between various patterns of mucin-producing salivary adenocarcinomas, including invasive salivary adenocarcinomas with mucinous differentiation, such as colloid and papillary carcinomas, remains unclear. Herein, we aimed to describe the clinicopathologic characteristics, immunophenotypes, molecular underpinnings, and clinical behavior of salivary mucinous adenocarcinomas (MA) to clarify their classification. We described a broad series of colloid and papillary patterns of MAs, indicating that papillary pattern presented papillary cystic proliferation of mucinous columnar cells as salivary intraductal papillary mucinous neoplasms with recurrent AKT1 E17K mutations, whereas colloid adenocarcinomas containing large mucinous pools or lakes around the malignant epithelial nests or islands harbored BRAF V600E mutations with worse prognosis. Typical morphologic structures, CK7(+), CK20(-), CDX2(-), p63(-), p40(-), MAML2 fluorescence in situ hybridization (-), AR(-), TTF-1(-), S100(-), mammaglobin(-), or S100/mammaglobin(+) with ETV6 fluorescence in situ hybridization (-) immunophenotype, and recurrent AKT1 E17K or BRAF V600E mutations may be defined. To our knowledge, this small series represents the first genetic study on a typical colloid pattern of MA, and our study with the spectrum documentation for MA in clinicopathologic characteristics, histologic and immunophenotypes, molecular features, and clinical behavior will allow for a better understanding of these rare but distinctive tumors.

Dark kinase annotation, mining, and visualization using the Protein Kinase Ontology.

The Protein Kinase Ontology (ProKinO) is an integrated knowledge graph that conceptualizes the complex relationships among protein kinase sequence, structure, function, and disease in a human and machine-readable format. In this study, we have significantly expanded ProKinO by incorporating additional data on expression patterns and drug interactions. Furthermore, we have developed a completely new browser from the ground up to render the knowledge graph visible and interactive on the web. We have enriched ProKinO with new classes and relationships that capture information on kinase ligand binding sites, expression patterns, and functional features. These additions extend ProKinO's capabilities as a discovery tool, enabling it to uncover novel insights about understudied members of the protein kinase family. We next demonstrate the application of ProKinO. Specifically, through graph mining and aggregate SPARQL queries, we identify the p21-activated protein kinase 5 (PAK5) as one of the most frequently mutated dark kinases in human cancers with abnormal expression in multiple cancers, including a previously unappreciated role in acute myeloid leukemia. We have identified recurrent oncogenic mutations in the PAK5 activation loop predicted to alter substrate binding and phosphorylation. Additionally, we have identified common ligand/drug binding residues in PAK family kinases, underscoring ProKinO's potential application in drug discovery. The updated ontology browser and the addition of a web component, ProtVista, which enables interactive mining of kinase sequence annotations in 3D structures and Alphafold models, provide a valuable resource for the signaling community. The updated ProKinO database is accessible at https://prokino.uga.edu.

Novel Biomarkers and Molecular Targets in ALL.

Acute lymphoblastic leukemia (ALL) is a widely heterogeneous disease in terms of genomic alterations, treatment options, and prognosis. While ALL is considered largely curable in children, adults tend to have higher risk disease subtypes and do not respond as favorably to conventional chemotherapy. Identifying genomic drivers of leukemogenesis and applying targeted therapies in an effort to improve disease outcomes is an exciting focus of current ALL research. Here, we review recent updates in ALL targeted therapy and present promising opportunities for future research. With the utilization of next-generation sequencing techniques, the genomic landscape of ALL has greatly expanded to encompass novel subtypes characterized by recurrent chromosomal rearrangements, gene fusions, sequence mutations, and distinct gene expression profiles. The evolution of small molecule inhibitors and immunotherapies, and the exploration of unique therapy combinations are some examples of recent advancements in the field. Targeted therapies are becoming increasingly important in the treatment landscape of ALL to improve outcomes and minimize toxicity. Significant recent advancements have been made in the detection of susceptible genomic drivers and the use of novel therapies to target them.

Discovery of U2AF1 neoantigens in myeloid neoplasms

BackgroundMyelodysplastic syndromes (MDS) arise from somatic mutations acquired in hematopoietic stem and progenitor cells, causing cytopenias and predisposing to transformation into secondary acute myeloid leukemia (sAML). Recurrent mutations in spliceosome...

Abstract A160: The FGFR axis is a potential radiosensitization target

Abstract Background: Establishing methods that sensitize cancers to photons is important for improving the treatment outcome of radiotherapy. Although sequencing has been widely used to identify druggable mutations in various cancers, this strategy have not been used extensively to identify new targets for radiotherapy sensitization. Thus, we performed clinical sequencing of radiotherapy-treated patient samples and investigated whether inhibition of the identified candidate targets resulted in radiosensitization in vitro and in vivo. Material and Methods: Ninety-eight patients with newly diagnosed and pathologically confirmed cervical cancer who had been treated with definitive radiotherapy were analyzed. DNA was extracted from formalin fixed and paraffin embedded pre-treatment tumor biopsy specimens. The exons of 409 cancer-related genes were sequenced using a next generation sequencer. Identified mutations were analyzed for correlations with clinical outcome. The radiosensitization induced by inhibition of the candidate target was investigated in in vitro clonogenic assays and nude mouse xenograft models using radioresistant cancer cell lines and small molecule inhibitors. The in vitro sensitizer enhancement ratio (SER) was calculated as the ratio of radiation dose required to reduce clonogenic cell survival by 50% without drug to that with drug. Results: Recurrent mutations were identified in PIK3CA (35.7%), ARID1A (25.5%), NOTCH1 (19.4%), FGFR3 (16.3%), FBXW7 (19.4%), TP53 (13.3%), EP300 (12.2%), and FGFR4 (10.2%). The prevalence of mutations in FGFR family genes combined (24.5%) was almost as high as that for well-studied PIK3CA and ARID1A. Fifty-five percent (21/38) of the identified FGFR mutations were located in the tyrosine kinase domain. Five-year progression-free survival (PFS) rates for FGFR mutation-positive patients (n = 24) were significantly worse than those for FGFR mutation-negative patients (n = 74) (43.9% vs. 68.5%). Multivariate analysis identified FGFR mutations as significant predictors of worse 5 year PFS, independent of clinicopathological variables. To investigate whether the FGFR axis can be targeted by drugs to increase radiosensitization, the radiosensitizing effect of a human-administrable selective pan-FGFR inhibitor LY2874455 was investigated. LY2874455 (at 100 nM) showed a potent in vitro radiosensitizing effect (SER: 1.62, 1.53, and 1.31 for A549, H1299, and H1703 cells, respectively). In A549 xenograft models, the combination of X-rays (10 Gy on day 1) and LY2874455 (3 mg/kg body weight, once daily, on Day 1–7) led to significantly greater suppression of tumor growth than either treatment with X-rays or LY2874455 alone, with no evidence of toxicities. LY2874455 treatment inhibited ERK signaling, the main target of the FGFR axis, both in vitro and in vivo. Conclusion: These data indicate that targeting the FGFR axis with LY2874455 could potentially be used to sensitize cancers to radiotherapy, which warrants clinical validation. Citation Format: Takahiro Oike, Eisuke Horigome, Akiko Adachi, Narisa Dewi Maulany Darwis, Atsushi Shibata, Tatsuya Ohno. The FGFR axis is a potential radiosensitization target [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A160.

Abstract C171: Immune desert microenvironment is associated with poor outcome in Wilms tumors

Abstract Background: Presence of anaplasia is a potent marker for adverse outcome in patients with Wilms tumors with diffuse anaplasia (DAWT). Patients with WT with focal anaplasia (FAWT) are considered as intermediate risk. Besides TP53 mutations thought to arise in anaplastic components, the crosstalk between genomic alterations and immune landscape remains unknown. Methods: We performed whole-exome and/or RNA sequencing on genomic DNA derived from 12 matched tumor-normal WTs and extended analysis in an independent dataset of 9 cases. TP53 mutations and tumor infiltrating lymphocytes (TILs: CD3, CD4 and CD8) were also assessed. Validation was performed using TARGET-WT (n=96) and GEO-WT (n=12) cohorts. Prognostic role of TILs was investigated in another 55 pre-treated WTs. Results: TP53 (50%) and DROSHA (33.3%) were the sole recurrent mutations identified. TP53 mutations occurred in mutually exclusive manner with chromatin-modifying genes which were altered in both FAWT (100%) and DAWT (33.3%). Microenvironment analysis revealed a reproducible immune desert WTs with poor outcome that enriched in DAWT cases and TP53 mutation, which mirrored the inversion of CD4:CD8 ratio in TP53-mutated comparing to TP53-wild tumors (P=0.003). Immune desert WTs were also characterized by overexpression of DNA repair pathways, PRC2, HDCA, and polycomb targets of EZH2, leading to higher sensitivity to HDCA inhibitor of vorinostat. Additionally, the immunophenotypes were tightly associated with replication stress subtypes; WTs with high replication stress were susceptible to ATR and WEE1 inhibitors. High ratio of CD4/CD8 was considered as independent recurrence-free and overall survival factors for WTs (both P<0.05). Conclusions: Our results reveal that WT with anaplastic features are characterized by either TP53 alterations or chromatin-modifying genes. These data implicate TP53 as a tumor cell-intrinsic driver of an immune desert phenotype, in contrast with adult TP53 tumors. TP53 inactivation might therefore causes epigenetic deregulation across cancer sites, and has implications for immunotherapy. Citation Format: Xiaoping Su, Gabriel Malouf. Immune desert microenvironment is associated with poor outcome in Wilms tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C171.

Characterization of Comparative Clinical Outcomes and Molecular Features of Black Patients with Acute Promyelocytic Leukemia (APL) Reveals Similar Survival and Distinct Genomic Profiles

Introduction: Although APL, a subtype of acute myeloid leukemia (AML), is an aggressive disease, its treatment was revolutionized with combination all- trans retinoic acid (ATRA) and arsenic trioxide (ATO), making APL a highly curable malignancy. Molecularly, APL is characterized by t(15;17) resulting in PML:: RARA gene fusion as disease-initiating event and by several recurrent gene mutations (eg, FLT3) identified in previous studies. In AML, prior studies have established inferior overall survival (OS) of Black versus White AML patients (pts) in both population-based analyses and clinical trials, found a negative prognostic impact of higher social deprivation (SDI), and characterized differences in frequencies and impact of disease-associated gene mutations. Herein, we compared OS of Black and White APL pts and evaluated genomic landscape of Black pts, which has not been comprehensively assessed to date. Methods: For nationwide population analysis, 2 sets of cancer registries were included in the Surveillance Epidemiology End Results (SEER) Program of the National Cancer Institute to identify 829 adults diagnosed with de novo APL (confirmed by cytogenetic and/or molecular findings) in 1995-2019. To assess survival in the setting of clinical trials, we analyzed 249 pts treated on historic frontline Cancer and Leukemia Group B (CALGB)/Alliance for Clinical Trials in Oncology (Alliance) trials with or without inclusion of ATRA (CALGB 9191, 1992-1995; CALGB 9710, 1999-2005). For Alliance pts, neighborhood SDI was assigned based on pt-reported residence zip code and classified as low (1-25, n=33) or high deprivation (26-100, n=95). OS was analyzed separately for pts diagnosed before and after 2012 to account for treatment advances with ATRA/ATO. To assess molecular features, we performed integrated genomic profiling (paired tumor/normal whole-exome and transcriptome sequencing) on 31 Black APL Alliance pts (9/31 completed, with complete results to be presented at the Annual Meeting). Results: On a population level using SEER data, 3-year (3y) OS significantly improved from 65% for cases diagnosed in 1995-2012 to 75% in those diagnosed after 2012 (p=<0.001), concordant with ATRA/ATO use. Notably, OS of Black and White pts did not differ significantly before or after 2012. This held true in race-specific findings for median household income, Yost index and urban/rural status; none of which significantly associated with OS. Older age was the only parameter associated with OS (3y rates, 1995-2012: <40y, 79%; 40-59y, 71%; ≥60y, 44%; and 2012-2019: <40y, 92%; 40-59y, 80%; ≥60y, 56%). Again, OS of Black and White pts did not differ within age groups. Likewise, OS of Black and White APL pts treated in a relatively controlled setting of clinical trials was not significantly different. As in population-based findings, OS improved over time and with ATRA addition (3y OS rates, 1992-1995, 66%; 1999-2005, 85%), and older age was a strong negative OS predictor (p<0.001). When comparing OS of pts treated on clinical trials with population-based OS, trial pts had a longer 3y OS (84% vs 64%, p<0.001). Notably, pts with SDI >25 (n=95) had a worse OS than pts with SDI <25 (n=33, p=0.03). On a molecular level, preliminary results of whole-exome sequencing (finalized for 9/31 Black pts) showed frequent mutations in FLT3 (5/9 pts), a known APL-associated event, but also revealed several recurrent gene mutations not reported in prior APL sequencing efforts, which were almost exclusively based on White pts, including mutations in DPP6, GOLA6L, NLGN2 and SP8 (found in ≥2 pts). Intriguingly, CALR mutations were found in 3/9 pts. Mutations in FLT3 and CALR were mutually exclusive and found in 8/9 pts, with the remaining pt harboring a SORBS2 mutation. Conclusions: In contrast to most cancer types, including AML, there was no OS disparity with respect to race in APL pts, both in population-based analyses and in the setting of historic clinical trials. Older age and residence with high SDI (>25) were negative OS predictors. Molecularly, our pilot data reveal existence of ancestry-associated differences in driver mutations, suggesting that FLT3 and CALR mutations represent necessary proliferative signals in addition to PML:: RARA-driven differentiation arrest. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov IDs: NCT00048958, NCT00899223, NCT00900224; https://acknowledgments.alliancefound.org

Development of Circulating Tumor DNA (ctDNA) for Molecular Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML)

Introduction: Despite advances in therapeutic options, the majority of AML patients experience relapsed or refractory disease. Standard-of-care (SOC) methods for MRD detection (multiparametric flow cytometry and single gene or structural variant molecular assays) are hampered by (1) inadequate lower limits of detection (LOD), (2) paucity of recurrent mutations across AML genomes, (3) insufficient numbers of identified mutations to characterize clonal evolution, and/or (4) need for invasive bone marrow biopsy. Herein we develop and apply non-invasive circulating tumor DNA (ctDNA) liquid biopsies using a customized Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) approach to simultaneously quantify MRD and characterize clonal dynamics in AML. Methods: To increase the number of tumor-specific mutations per case for MRD detection and improve the LOD for ctDNA-MRD, we performed whole exome sequencing (WES) on purified blasts and paired T cells (latter as germline control) from 24 patients with AML (n = 20 previously untreated, n = 4 with relapsed/refractory disease) and designed personalized hybrid capture panels. We combined the personalized panels with a shared hybrid capture panel of 58 recurrently mutated (‘canonical‘) AML genes covering 225kB as a composite approach to generate patient-specific AML-CAPP-Seq assays. We applied AML-CAPP-Seq to track single nucleotide variants (SNVs) in 468 biological samples from the 24 AML patients and 21 healthy control subjects. We compared the performance of MRD detection in cellular (bone marrow mononuclear cell/BMMC, peripheral blood mononuclear cell/PBMC) and cell-free (BM-ctDNA, PB-ctDNA) compartments against SOC clinical MRD assays as well as patient specific clinical outcomes. Results: We observed a median of 30 SNVs (IQR 16.5-40.25) per patient from WES, compared to only 2 (IQR 1-3.25) if using the AML-focused canonical panel alone (p < 0.0001) ( Fig. 1A). This number of variants enabled sensitive molecular disease monitoring in 96% (23/24) of patients with a LOD of < 0.01% tumor fraction from typical quantities of cell-free DNA. In contrast, when using the canonical panel alone, only 83% (20/24) of patients had 1 or more SNV to allow MRD detection. Notably, only 12.5% (3/24) of patients in this cohort had a feature amenable to SOC molecular MRD methods. Upon comparing quantitative levels of MRD in each biological compartment, we found that tumor burden in PB-ctDNA, BM-ctDNA and BMMC samples were highly correlated (r > 0.92, p < 0.0001) with similar amounts of disease. Interestingly, PB-ctDNA contained quantitatively more tumor DNA than corresponding PBMC samples (p = 0.0002), most pronounced at low tumor burdens (median 8.2-fold enrichment when mean tumor allele fraction in PBMCs was < 1%). These results suggest that PB-ctDNA effectively captures total body BM disease and mutational burden in AML and serves as a superior analyte for MRD monitoring compared to PBMCs. When evaluating the performance of AML-CAPP-Seq against clinical SOC methods, our assay correctly detected molecular disease in 99.6% (237/238) of samples known to contain measurable disease, including 98.2% (54/55) of samples at timepoints of clinical MRD positivity during clinical remission (CR). However, restricting trackable SNVs to only ‘canonical’ genes led to significant degradation of performance. Specifically, only 69.1% (38/55) of samples with positive MRD during CR by clinical SOC assay were detected by ctDNA if canonical genes alone were employed (p = 0.0001). Intriguingly, when using the complete gene panel, AML-CAPP-Seq also identified molecular MRD in 37.5% (30/80) of samples with undetectable MRD by SOC assays. Finally, stratifying patients who achieved CR after 1st treatment line based on PB-ctDNA MRD significantly predicted relapse free survival (p = 0.046, hazard ratio (HR) = 4.5). Moreover, attaining an MRD negative CR in PB-ctDNA at any timepoint was strongly prognostic of overall survival (p = 0.0004, HR = 8.5) ( Fig. 1B). Conclusions: ctDNA is a promising biomarker for ultrasensitive detection and tracking of MRD in AML without need for invasive bone marrow assessment. Tracking mutations beyond canonical AML genes improves performance of such assays. ctDNA analysis has numerous areas of clinical utility, including response and relapse prediction, evaluation of clonal dynamics, and exploration of mechanisms of therapy resistance.

An In Vivo PiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88 L265P

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Genetic Mutations in Primary Refractory Diffuse Large B-Cell Lymphoma: Next Generation Sequencing Analysis

Introduction Diffuse large B-cell lymphoma (DLBCL) accounts for 30-40% of newly diagnosed adult non-Hodgkin lymphoma. About 60-70% of DLBCL patients could be cured with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisolone (R-CHOP), while the other patients show chemo-refractory or relapse of disease and have poor prognosis. The prognostic difference between DLBCL patients is not fully understood, and many attempts to classify DLBCL have been made. Recently, with the development of the next generation sequencing (NGS) technique, Schmitz et al. identified four prominent genetic subtypes named MCD, BN2, N1, EZB. In this study, we performed targeted deep sequencing in DLBCL patients and analyzed whether there is any genetic difference between the patients who are primary refractory disease and the patients who are not. Methods Totally, 77 patients who was diagnosed with DLBCL and treated with R-CHOP from January 2021 to April 2023 were included in this analysis. Formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained at diagnosis were archived. Genomic DNA was extracted using QIAmp DNA FFPE tissue kit according to the manufacturer's protocol. Genetic profiling included the targeted deep sequencing of 424 genes, which had been selected based on recurrent mutations from previous studies, and sequenced using an Illumina Nextseq. The threshold of mutation positivity as a variant allele frequency ≥2% for SNV/Indel and as a copy number ≥4.0(gain) for CNV. Patients were assigned as germinal center B-cell (GCB) or non-GCB subtype based on the immunohistochemistry (IHC) algorithm. BCL-2 and MYC FISH were conducted in patients with double-expressor DLBCL with non-GCB subtype. Results Median age of the patients were 66 years (24-86 years), and 49 patients (63.6%) were male. Forty-five patients (58.5%) were advanced stage. Twenty-four patients (31.2%) were GCB subtype and 51 patients (66.2%) were non-GCB subtype. Among the GCB type DLBCL patients, 10 patients were double expressor DLBCL, and there was no case of double hit lymphoma. As a result of targeted deep sequencing, 317 mutations were found in at least 1 patient, and the patients had a median number of 16 mutations (4-50). KMT2D was the most commonly detected mutation (22/77, 29.3%), followed by MYD88 (19/77, 25.3%), PCLO (19/77, 25.3%), PIM1 (19/77, 25.3%), TP53 (18/77, 24.0%). According to NGS analysis, 26 patients (33.85%) were assigned to MCD subgroup, 13 patients (16.9%) to BN2, 11 patients (14.3%) to N1, 10 patients (13.0%) to EZB, and 17 patients (17%) were not assigned to any subrgroup. Median follow-up of the patients was 13.8 months (0.4-22.8 months), and 12 patients (15.6%) relapsed and 5 patients (6.5%) died. Median PFS and OS were not reached. Among relapsed patients, 7 patients were primary refractory to R-CHOP. In primary refractory DLBCL patients, TP53 (3/7, 42.9%) and CIITA (3/7, 42.9%) were most commonly detected. CIITA is known to be associated with immune escape and treatment resistance. CIITA, RASSF54, STAT6, SYK significantly more mutated in the primary refractory patients than the other ( CIITA, 42.9% vs. 8.6%, p=0.031; RASSF54, 28.6% vs. 2.9%, p=0.039; STAT6, 28.6% vs. 1.4%, p=0.021; SYK, 28.6% vs. 2.9%, p=0.039) (Table 1). Conclusion CIITA, RASSF54, STAT6, SYK were frequently mutated in primary refractory DLBCL in this study. Because of the small sample size, however, the prognostic significance of these mutations needed to be studied in further researches.

Genetic Subtyping Using Whole Exome Sequencing across Large B Cell Lymphoma Entities in a Series of 109 DLBCL and Plasmablastic Lymphoma Cases

Background: Diffuse Large B Cell Lymphomas are still a significantly heterogeneous disease with few molecularly defined entities. Recently genomic based algorithms have been proposed for DBCL NOS disease classification, including the Lymphgen tool 1. Other large B cell lymphoma entities, including High Grade BCL Dual Hit or plasmablastic lymphoma remain poorly characterized according to this molecular based clustering methods. Here we sought to apply this tool for disease classification in a series of 109 large B cell lymphoma cases derived from GELTAMO clinical trials (NCT01848132 and NCT2015-005390-21) and retrospective multicentric series to confirm its application across different large B cell lymphoma entities. Methods: We retrieved FFPE and liquid biopsy diagnostic samples from 109 cases and performed histopathological confirmation, FISH testing and whole exome sequencing (Agilent Sureselect v6) by NGS. Sequencing data and available fusion data for BCL2 and BCL6 were used to classify the cases according to Lymphgen (https://llmpp.nih.gov/lymphgen). Results: 109 cases were classified as different large B cell lymphoma entities, including 31 DLBCL NOS GCB type, 8 HGBCL/DLBCL DH/NOS, 41 DLBCL NOS non-GCB type, 22 plasmablastic lymphoma, 3 THRLBCL, 1 PMBL, 2 DLBCL NOS and 1 3B Follicular Lymphoma. After WES median Tumor Mutation Burden was 972. Overall genetic classification by Lymphgen was obtained in 53 cases (49%) with the remaining 56 cases considered unclassified (molecular subtype other/NOS). Of the genetically identified cases, 18 were classified as ST2 (33%), 15 were considered EZB (28%), 5 BN2 (10%) and 3 MCD (5%). Interestingly 12 (22%, overall, 11%) were genetically composite, including 8, ST2/EZB; 1, ST2/BN2; 1, ST2/MCD and 2 ST2/EZB/MCD. According to histopathological subtype 17 GCB type DLBCL were classified as EZB (8, 47%), ST2 (5, 30%) and BN2 (1, 6%). 3 cases (17%) were genetically composite. 6 out of 8 cases of HGBCL/DLBCL DH/NOS were classified as either EZB (2), ST2 (2) or composite EZB/ST2 (2). 19 non-GCB type DLBCL were classified as ST2 (5, 26%), BN2 (4, 21 %), EZB (3, 16 %) and MCD (3, 16%). 4 cases were genetically composite (21%). Interestingly, plasmablastic lymphomas showed ST2 genetic features, found in 5 out of 22 (23%) and composite ST2/EZB in 2 cases. Genes overrepresented in the ST2 cluster include TET2, PRRC2C, STAT3,DOCK8 and CLTC. Plasmablastic Lymphomas classified as other showed recurrent mutations in PRRC2C, PABPC1, TRRAP, PRKCB, EP300 and TP53. Conclusions: ST2 and EZB genetic groups stand as the most frequent genetic subtypes of DLBCL NOS. While ST2 is found in either GCB and non-GCB subtypes, EZB is found preferentially in cases with the GCB phenotype, including GCB type DLBCL and HGBCL/DLBCL DH. In comparison with already published series we found a relatively higher prevalence of the ST2 genetic subtype among DLBCL NOS. MCD and BN2 genetic subtypes are restricted to the non-GCB subtype, but its prevalence is relatively low. Importantly, a significant fraction of cases (~50%) is still genetically unclassified (molecular subtype NOS) using WES and FISH-derived fusion data. Proper identification of the A53 cluster and relevant dysregulated pathways in the molecular NOS group require more research. Specific large B cell lymphoma entities such as plasmablastic lymphoma show features of ST2 genetic subtype in a significant fraction of tumors. These features include JAK/STAT3 pathway activation related genes and fit well with already published data 2,3. In addition, recurrent mutations in TET2, involved in epigenetic regulation of gene expression and TRRAP, target of C-MYC transcriptional activation, were enriched in this molecular subtype of plasmablastic lymphoma. Importantly ~70% remained unclassified. Mutations in this group are recurrently found in PRRC2C as well as PABPC1, both involved in VEGFA-VEGFR2 signaling. In summary, DLBCL NOS molecular subtyping supplies exact molecular classification in ~50% of our cohort. ST2 and EZB subtypes constitute ~60% of the cases with a fraction of ~15% of composite ST2/EZB. Application of the molecular classifier to plasmablastic lymphomas shows intraspecific genetic diversity and points toward dysregulation of JAK/STAT, MAPK/Erk and VEGFA-VEGFR2 signaling as well as MYC transcriptional activation. REFERENCES 1. PMID: 32289277 2. PMID: 33225311 3. PMID: 32273478

Quantum-First Trial: FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD)-Specific Measurable Residual Disease (MRD) Clearance Assessed through Induction (IND) and Consolidation (CONS) Is Associated with Improved Overall Survival (OS) in Newly Diagnosed (nd) FLT3-ITD+ AML Patients (pts)

Background Detection of MRD by flow cytometry or gene fusion transcript quantitation increasingly is used to guide treatment decisions in AML. Molecular monitoring of recurrent gene mutations for MRD detection is an alternative strategy but, other than for nucleophosmin 1 ( NPM1) gene quantitation, remains controversial. FLT3-ITD is among the most common recurrent mutations in AML and confers a worse prognosis vs FLT3-wild-type and FLT3-TKD+ AML. Routine use of FLT3-ITD detection in remission as a predictor of relapse risk or OS has been limited by the low sensitivity of both conventional PCR-based detection methods and broad NGS platforms in clinical practice settings. More recently, FLT3-ITD-specific PCR-NGS assays such as getITD (PMID: 31089248) show greater sensitivity and ease of interpretation that point to eventual routine clinical application. However, the clinical value of these measurements has not been evaluated prospectively in large scale randomized controlled trials of FLT3 inhibitors. The phase 3 QuANTUM-First study (NCT02668653) evaluated the novel, potent, and highly selective type II FLT3 inhibitor quizartinib (Quiz) in nd FLT3-ITD+ AML pts and demonstrated that Quiz added to intensive IND and CONS, ± transplant, followed by single-agent continuation (CONT) therapy (Tx) resulted in a significant improvement in OS (PMID: 37116523). We analyzed if FLT3-ITD-specific MRD in QuANTUM-First pts impacted the clinical outcome or the benefits provided by Quiz in nd FLT3-ITD+ AML pts. Methods Genomic DNA, isolated from bone marrow aspirates or peripheral blood from pts after achievement of remission after 1 or 2 courses of IND and at end of CONS (prior to transplant or CONT cycle 1 day 1 [C1D1] for transplant pts and prior to CONT C1D1 for non-transplant pts), was analyzed with a FLT3-ITD PCR-NGS assay specifically developed for this trial (PMID: 31722002). ITD mutations detected after IND were cross-validated against the ITD detected at enrollment for each patient. Using a custom bioinformatics program, ITD mutations were identified, and variant allele frequencies (VAFs) were calculated with a sensitivity of 10 −5. MRD was classified as undetectable (using the 0 cutoff) or detectable above or below a 10 −4 predefined cutoff (based on lower limit of quantification for the assay). Comparisons of complete response (CR), composite complete response (CRc=CR+CRi), and the rate of pts achieving CRc during IND with no MRD between arms were made using a stratified Cochran-Mantel-Haenszel test. Comparison of FLT3-ITD VAF during IND and during CONS between arms was made using Wilcoxon rank sum test. All P-values were not adjusted for multiplicity. Results In QuANTUM-First, 539 nd FLT3-ITD+ AML pts were randomized to Quiz (n=268) or placebo (PBO; n=271). Of the 539 randomized pts, 368 (68.3%) achieved CRc after 1 or 2 courses of IND, and MRD analysis was performed on 321 (87.2%) of these pts (162 pts in Quiz and 159 pts in PBO) using samples collected at the time of response assessment during IND, before further Tx. MRD was also assessed in 337 pts (172 pts in Quiz and 165 pts in PBO) by end of up to 4 cycles of CONS prior to CONT/maintenance Tx. Of these pts, 166 additionally received transplant (87 in Quiz, 79 in PBO). The % of pts in CRc at end of IND with FLT3-ITD MRD of <10 −4 was similar between study arms (25.4% Quiz vs 21.8% PBO, nominal P = 0.3430), however, a greater % of pts in CRc had undetectable MRD with Quiz than PBO (12.3% vs 7.0%, nominal P = 0.0403). Among pts with CRc at end of IND, the median best FLT3-ITD VAF by end of CONS was lower (0% versus 0.0017%; nominal P = 0.0006) with Quiz vs PBO (Fig 1) and, using the 0 cutoff at end of IND, a longer OS was observed with Quiz vs PBO, regardless of MRD status, with a greater effect with Quiz (HR, 0.789 in MRD− pts; HR, 0.749 in MRD+ pts; Fig 2). In MRD+ pts, the median OS was not reached with Quiz and 35.4 months with PBO (Fig 2). Performing this analysis with an MRD cutoff of 10 ‒4 yielded similar findings (HR, 0.696 in MRD− pts; HR, 0.800 in MRD+ pts). Additional data about MRD impact on pts ± transplant in this trial will be presented. Conclusions These findings demonstrate the potential prognostic utility of FLT3-ITD-specific MRD measurements in the clinical management of pts with FLT3-ITD+ AML. Our data suggest that long-term OS benefits conferred by Quiz in part derive from a deep and sustained reduction of the FLT3-ITD+ leukemia burden.

Signaling Pathway Gene Set Enrichment Analysis in Whole Exome Sequencing Data from Discordant Identical Twins Unveils Relevant Pathways As a Clue to Understand Somatic Mutation Acquisition and Clonal Evolution in Myeloproliferative Neoplasms

Although the role of classic driver mutations (i.e., JAK2, MPL, and CALR) in myeloproliferative neoplasm (MPN) development is well characterized, additional somatic mutations likely modulate the phenotype. Recurrent somatic mutations, such as those in TET2, DNMT3A, ASXL1 genes are commonly identified in MPN using targeted myeloid panels, however identification of somatic mutations using an unbiased approach may unveil novel pathways involved in MPN pathogenesis. We performed whole exome sequencing of blood samples of three sets of discordant identical twins in which one twin has MPN and the other twin is unaffected. The twin sets included all MPN subtypes (1 polycythemia vera, 1 essential thrombocythemia, 1 primary myelofibrosis). We also performed whole exome sequencing on additional family members, including the mother and father of the PV twin and the brother of the ET twin. Sequencing information from the discordant identical twins is a unique opportunity to identify somatic mutations in the affected twin that may contribute to MPN pathogenesis, and also to potentially identify clonal hematopoiesis in the unaffected twin. We mapped all the identified genetic variants to the affected genes, and we filtered all the genes mapped to those variants depending on whether they were present either in the MPN-affected twin or in the unaffected counterpart for further gene set enrichment pathway analysis. The gene set enriched signaling pathways from identified genetic variants for each condition-exclusive (either MPN or unaffected twin) allows us to identify and prioritize key pathways in MPN development in terms of somatic mutations acquisition. Furthermore, besides performing the analysis within each single MPN set, we analyzed the gene sets resulting from combining the different families allowing us not only to identify pathways at each single MPN type level but also among the different MPN diseases (PV, ET, and MF). We divided all the enrichment analyses into coding and silent variants to obtain a higher degree of pathway refinement. In single discordant twin sets we identified several pathways that may play an important role within MPN pathogenesis such as defective mismatch DNA repair, facilitated mitotic progression, extracellular matrix degradation, MAPK signaling, FGFR signaling, O-glycosylation of proteins, collagen metabolism and laminin interactions, neutrophil degranulation, interleukin-33 signaling, Rho GTPases and cGMP regulation, and nuclear pore complex (NPC) assembly. Interestingly, only when we analyze all the MPN-types together all the statistically significant pathways resulting from coding variants correspond only to DNA repair, specifically defective mismatch repair (PMS2, MLH1, MutSα, MutSβ, and TP53). Our findings reveal that DNA repair genes might occupy a central role within MPN acquisition and development, whereas at the same time each single MPN clinical entity might possess its own distinct mechanisms differentiating each one from the others.

Single-Cell Proteomic Analysis Reveals Menin Inhibition-Induced Proteomic Alterations in AML Patients Treated with Revumenib

Menin inhibition is a promising therapeutic approach for acute myeloid leukemia (AML) with certain genetic alterations including KMT2A rearrangements or NPM1 mutations. The first-in-human Phase 1 clinical trial of revumenib (SNDX-5613) in patients with R/R AML showed promising results and induced complete remission in patients who refractory to multiple lines to therapy. These findings suggest that revumenib could provide a new therapeutic option for patients with AML with KMT2A and NPM1 genetic alterations. AML is characterized by the existence of distinct differentiation hierarchies, a phenomenon substantially contributing to disease heterogeneity. AML hierarchies exhibit distinct expressions of phenotypic profiles, apoptotic networks, signaling pathways, and metabolic profiles, leading to varying sensitivity towards targeted therapies, resulting in differential sensitivity to targeted therapies. Revumenib induces AML differentiation and thus shift the balance between AML hierarchies, resulting in drastic alterations in the overall AML proteomic profiles and enrichment of distinct leukemia phenotypes with differential sensitivity to targeted therapies. Detailed interrogation of revumenib-induced proteomic alterations could permit precise targeting of persistent cells and development of data-driven combinatorial approaches to achieve higher response rates. Moreover, the distinct combinations of genetic alterations can impact the efficacy of revumenib-induced differentiation and therapeutic outcomes. Thus, to better understand these complexities we performed multimodal single-cell (sc) analysis of AML landscape in R/R AML treated with revumenib (n=4) in Phase 1. We utilized two leukemia-focused 51-parameter CyTOF panels to analyze pre and post-treatment samples from R/R AML patients treated with revumenib. Sc proteogenomic profiling was performed using MissionBio platform using a panel covering 37 genes with recurrent AML-relevant mutations. UMAP and clustering analyses of CyTOF data identified AML blasts, unveiled quantitative changes in cell composition, and delineated proteomic shifts induced under revumenib therapy pressure. We observed enrichment of CD11b+/-CD68+, more differentiated monocytic cells situated at close proximity to AML blasts and depletion of immature AML blasts. Through unsupervised analysis, we discovered discrete AML differentiation hierarchies, delineated by a differentiation continuum that initiates with immature AML blasts (CD34+/-c-Kit+/-), progresses through intermediate AML blasts (CD68+/CD11b-/CD14-), and culminates with more differentiated monocytic cells (CD68++/CD11b+/CD14+/-). Notably, intermediate AML blasts and monocytic cells were enriched in post-treatment samples. Simultaneous ScDNA+protein analysis showed enrichment of differentiated AML blasts with monocytic/dendritic cell phenotype and harboring AML mutations. Differentiated AML blasts expressed higher levels of MCL1 and BCL-xL and upon differentiation we observed gradual downregulation of BCL2. Importantly, intermediate and more differentiated AML blasts under revumenib therapy expressed high levels of p53 protein, a feature that can partly attributed to NPM1 mutations. Sc proteomic analysis delineated proteomic shifts elicited by revumenib and identified potential therapeutic partners, by targeting MCL1, BCL-xL and MDM2, that could be used in combination with revumenib to improve therapeutic efficacy. We next performed differential expression analysis by comparing the proteomic profiles of pre- and post-therapy samples. We observed suppression of less differentiated AML phenotypic profiles (c-Kit, CD123, CD34) along with MEIS1 and PBX3. Importantly, we also observed significant alterations in immune landscape, suggesting that revumenib could rewire Myeloid-to-T and NK cell interaction/communication by inducing proteomic shifts and driving AML blast differentiation. Further analysis of an expanded cohort of R/R AML patients is currently underway. In conclusion, multimodal sc analysis revealed profound proteomic alterations induced by revumenib and identified potential therapeutic targets. These findings present new avenues for optimizing combinatorial treatment regimens, enhancing revumenib's efficacy, and illuminating survival mechanisms in persistent, differentiated AML blasts.

Germline Predisposition in Myeloid Neoplasms Aged ≥50: A Novel Approach for Allogeneic Stem Cell -Transplantation Decision-Making

Introduction Patients aged 50 or older (≥50) with myeloid neoplasms (MNs), non-affected relatives, and no previous platelet or organ disorder are routinely not tested for germline predisposition. However, approximately 70% of myelodysplastic neoplasms (MDS) patients undergoing an allogeneic hematopoietic stem cell transplantation (alloHSCT) from familial donors, in 2021, were ≥50 years old (y.o.) (EBMT). Given the significant frequency of pathogenic/likely pathogenic (P/LP) germline variants in these patients (about 8%) along with the risk of transplanting cells with the same variant, it seems reasonable to perform universal germline testing in hematological neoplasms although challenging due to labor and cost constraints. Aims To address this, we developed a pragmatic approach incorporating a specific gene list to the diagnostic myeloid somatic panel to identify patients and exclude relatives with shared germline variants. To do that we: (1) Conducted a literature review to assess the gene-disease association's validity in this specific context. (2) Designed a germline-augmented virtual somatic panel (GASP) based on our findings. (3) Tested the virtual panel's performance in 133 MNs cases, aged ≥50, with matched germline-tumor exome sequencing, and no prior organ or platelet disorders. Methods We considered those genes included in the WHO Classification and NCCN Clinical Practice Guidelines as myeloid predisposition genes. For autosomal recessive disorders, heterozygous carriers were not considered. We analyzed the relation of these genes with MNs diagnosed ≥50 y.o. without prior organ or platelet dysfunction: To establish a valid association for a gene, we required the presence of P/LP variants in at least two peer-reviewed studies in the subset of interest. GASP combined genes with recurrent somatic mutations in myeloid disorders, as proposed by Duncavage et al. (Blood 2022), and those associated with the specific cohort of interest. WES was performed on paired tumoral-germline samples on a HiSeq 2000/Novaseq6000 instrument (Illumina Inc) with a target of 100x depth coverage. Results From 2018 to 2023, we collected samples from 133 patients (121 MDS and 12 CMML cases -FAB myelodysplastic variant) diagnosed with WHO-established MDS, aged ≥50, without prior organ or platelet disorders. Among the main characteristics at baseline, we highlight a median age at diagnosis of 57 years old (range, 50-88), and the presence of a first-degree relative with a myeloid neoplasm diagnosis in 7% of the cases. The literature search revealed 11 genes associated with germline predisposition within the cohort of interest. Seven were WHO/NCCN genes: DDX41, TERT, GATA2, CEBPA, SAMD9, ERCC6L2, and TP53. Additionally, four “recently associated genes” were incorporated based on recent descriptions in germline MNs ≥50 y.o. and reported at least in two peer-reviewed articles: CHEK2, DNAH9, ATM, and SH2B3 (Table 1). Next, we tested the accuracy of both the GASP and Duncavage's recommended somatic panel in our cohort of 133 myeloid neoplasms characterized by WES. Among these cases, we identified 15 (11%) P/LP germline variants in both WHO/NCCN and recently associated genes. Duncavage's somatic panel could only detect 8 out of 15 (53%) cases, while the expanded GASP detected 13 out of 15 (87%) variants (Figure 1). The GASP showed a C-index of 0.933, outperforming Duncavage's panel, which scored 0.767. Conclusions Eleven genes have been found to harbor P/LP germline variants in patients with MNs diagnosed ≥50 y.o. and no previous organ or platelet disorder. In our cohort, 11% of patients carried a P/LP germline variant in a MNs predisposition gene. Such a finding would have gone undetected as germline predisposition testing is not typically advised for these patients. Expanding the myeloid somatic panel (GASP) allows the identification of 87% of P/LP variants during the diagnosis process, enabling clinicians to exclude carrier relatives as potential allo-SCT donors.