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Background. The inclusion of gene mutations and chromosomal abnormalities in the 2022 WHO and ICC Classifications of MDS has enhanced diagnostic precision and is expected to improve clinical decision-making process. Although these two systems share similarities, clinically relevant discrepancies still exist and potentially cause inconsistency in their adoption in a clinical setting. In this study on behalf of the International Consortium for MDS (icMDS), we adopted a data-driven approach to provide a harmonization roadmap between the 2022 WHO and ICC classification for MDS. A modified Delphi Process consensus approach is currently ongoing among icMDS experts to finalize a harmonized MDS classification scheme. Methods. We analyzed retrospective international cohorts of patients with a diagnosis of MDS (n=7017) and AML (n=1002) according to WHO 2016 criteria. Hierarchical Dirichlet Processes were applied to define clusters capturing broad dependencies among all gene mutations and cytogenetic abnormalities. To investigate the features of importance and their impact on the clustering process, we employed the SHapley Additive exPlanations approach (SHAP). This allowed to define harmonized labels for each clinical entity. The clinical relevance of the unsupervised clustering was assessed through the analysis of phenotypic features and outcomes among each group. ( Blood 2022;140: 9828-9830) Results. Patients' characteristics are summarized in Table 1. We identified 9 clusters, defined by specific genomic features. The cluster of highest hierarchical importance was characterized by biallelic inactivation of TP53 (biTP53). According to SHAP analysis, bi TP53 was defined as 2 or more TP53 mutations, or 1 mutation with copy number loss or cnLOH. Most patients assigned to bi TP53 cluster had TP53 VAF>10% (77.9%) and complex karyotype (70.1%). Assignment to bi TP53 cluster was irrespective of blast count. Patients with monoallelic TP53 mutation segregated into other clusters. Hierarchically, the second cluster included patients with del(5q). SHAP analysis highlighted 5q deletion alone, or with one other chromosomal abnormality other than -7, and absence of bi TP53, as the most relevant features. Most of these patients had blast counts <5% (88.1%). The third distinct cluster included patients with SF3B1 mutations (in the absence of concurrent del(7q), abn3q26.2, complex karyotype or RUNX1 mutation). Most patients with MDS and SF3B1 mutation had <5% blasts (94.2%). Common co-mutated variants in the SF3B1 cluster included mutant DNMT3A (25.2%) and TET2 (38.3%). Morphologically defined MDS cases (i.e., not meeting criteria for bi TP53, del(5q) or SF3B1) were preferentially assigned to the following additional clusters: SF3B1 and concurrent higher-risk mutations (e.g., RUNX1 and ASXL1); SRSF2 and concomitant TET2 mutations; U2AF1 mutations with del(20q), del(7q) or -7; SRSF2 with TET2 mutations and co-mutational patterns including RUNX1 and ASXL1; and AML-like genomic signatures. Our analyses suggest that morphologically defined MDS is characterized by a large heterogeneity in terms of mutation profiles, not entirely captured by the presence of unilineage versus multilineage dysplasia, percentage of bone marrow blasts, and presence of hypocellularity and fibrosis. To better investigate the continuum between high risk MDS (i.e., patients with ≥10% blasts) and AML, an exploratory comparison was made using a cohort of AML (defined according to WHO 2016) patients analyzed using the same statistical methods. Only a partial overlap in genetic signatures was observed between MDS with ≥10% blasts and AML. However, similarities were observed between the AML-like MDS clusters (characterized by mutant NPM1, bZIP CEBPA, and Core Binding Factor abnormalities) and AML clusters defined by the same genetic signature, thus supporting the classification of these entities as AML, irrespective of blast count. Conclusion. Our study demonstrated the utility of a data-driven approach based on advanced statistical methods to generate a harmonized classification for MDS. Table 2 shows a provisional, hierarchical classification algorithm. Further refinement of entity labels and classification criteria is the subject of the ongoing modified Delphi Process consensus approach among icMDS experts.

Introduction: Brexucabtagene autoleucel (brexu-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T cell therapy approved for adults with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). Bridging therapy, defined as anti-leukemia therapy given between apheresis and lymphodepleting chemotherapy, was permitted on the pivotal ZUMA-3 trial. The optimal bridging therapy is unknown in this setting. Here we report patient and disease characteristics in those receiving bridging therapies prior to brexu-cel and the impact of bridging on clinical outcomes in a large multicenter cohort. Methods and Results: ROCCA includes 25 US institutions contributing retrospective data from 152 B-ALL patients treated with standard of care brexu-cel between 2021-2023. Ninety-nine patients (65%) received bridging therapy, while 53 patients (35%) received no bridging therapy. Bridging therapy consisted of cytotoxic chemotherapy ( n=65, 66%), immunotherapy ( n=26, 26%; 23 inotuzumab, 3 blinatumomab), tyrosine kinase inhibitors (TKI) ( n=20, 20%; 16 ponatinib), steroids only ( n=10, 10%), and intrathecal chemotherapy ( n=22, 22%). The median age of the cohort was 46 (IQR 32-61). Patients were heavily pre-treated with a median of 4 prior lines of therapy. The median days from apheresis to infusion was 33 (IQR 26-42). 119 patients had known pre-apheresis blast counts; the bridging group had higher pre-apheresis blast burden, with 21 of 76 patients (28%) having ≥ 50% blasts compared to only 5 of 43 patients (12%) in the no bridging group ( p=0.02). In addition, more patients who received bridging had pre-apheresis extramedullary disease (32% vs. 15%, p=0.02). All other baseline characteristics were similar between the two groups (Table 1). Fifty-four (54%) patients had response assessment after bridging therapy and prior to brexu-cel infusion. In patients who received inotuzumab for pre-apheresis active disease, defined as ≥ 5% blasts, 6 of 13 (46%) who had post-bridging assessment achieved a complete response (CR; 5 measurable residual disease (MRD)+, 1 MRD-), with a mean proportional reduction in blast burden of 46% from baseline. In contrast, after chemotherapy bridging, only 2 of 16 patients (13%) with pre-apheresis active disease had a CR. Patients receiving chemotherapy bridging had a mean proportional increase in blast burden of 4.4%. In recipients of TKI bridging, 2 of 4 patients (50%) with pre-apheresis active disease had a CR. Compared to no bridging, patients in the bridging group had similar rates of grade 3 or 4 CRS (9% bridging vs. 8% no bridging, p=0.73) and grade 3 or 4 ICANS (34% vs. 26%, p=0.32). In univariate analysis, there was no statistically significant difference in day 28 response with 63 patients (72%) achieving MRD- complete response in the bridging group as compared to 36 patients (78%) in the no bridging group ( p=0.60). Non-relapse mortality was similar between the two groups (17% bridging vs. 9% no bridging, p=0.20). Overall survival (OS) and progression-free survival (PFS) were superior in the no bridging group: 1-year OS 74.7% vs 56.8% (log-rank p=0.04), and 1-year PFS 57.3% vs 41.7% (log-rank p=0.04). In a multivariate analysis stratified by bridging and pre-apheresis disease burden, patients with pre-apheresis blasts ≥ 5% and bridging had worse survival than ≥ 5% blasts and no bridging: 1-year OS 45.3% vs. 87.5% and 1-year PFS 27.7% vs. 64.3% (Figure 1). However, patients with < 5% blasts had similar survival regardless of bridging. Conclusions: In our real-world analysis, adults with R/R B-ALL receiving bridging therapy had a higher disease burden at baseline. Despite greater baseline disease, there were no significant differences in toxicity and day 28 response in those receiving bridging therapy vs. not. While survival was inferior in patients with pre-apheresis active disease who received bridging, this may be confounded by the degree of tumor burden. In patients with < 5% blasts pre-apheresis, bridging did not impact survival. Furthermore, inotuzumab bridging demonstrated the most significant disease debulking. However, the comparisons in this study are limited by the variability in practice for post-bridging disease reassessment and the small proportion of patients with active disease who did not receive bridging. A more uniform approach to peri-CAR disease assessment would enable more robust study of optimal bridging strategies.

Abstract Introduction: Radiation therapy (RT) is the standard of care for central nervous system (CNS) cancers. Despite the success of RT in reducing tumor mass, RT-induced side effects such as ECM remodeling, vasculopathies and neuroinflammation are well documented that cause reduced neurogenecity and premature aging like complications during post-cancer survivorship. Since mesenchymal stromal cells (MSCs) are multipotent adult stem cells that support tissue integrity, homeostasis and repair through factors they release, we here investigated the potential of iPSC-derived MSC (iMSC)-secretome in immunomodulation and vasculature repair in response to radiation-induced alterations utilizing models of human monocytic and microglial cells (THP1, HMC-3) and human brain endothelial cells (HCMEC/D3). Approaches: (a) Investigate the impact of iMSC secretome on vasculature damage caused by radiation-induced cell injury and RT-induced inflammation, by evaluating its effects on (i) endothelial cell survival and morphogenesis (ii) activation of peripheral monocytes stimulated by X-ray radiation therapy (RT), RT-induced microglial injury, and vasculature injury mediated inflammation (b) Identify factors and pathways that may contribute to the observed phenotype. Results: Decline in intact angiogenic tube formation and less compact spheroid formation was observed for HCMEC/D3 cells after irradiation at doses (Gy): 2.5, 5, 10, 15, and 30, with maximal effect observed at 30Gy. Evaluating the effects of iMSC secretome on radiation induced endothelial cell damage and inflammation revealed increased cell viability with reduced apoptosis, higher cell adherance and better spheroid compactness in 5Gy irradiated HCMEC/D3 cells after iMSC-conditioned media (iMSC-CM) treatment. Reduction in NFκB promoter activity was observed in irradiated THP1- NFκB-Luc2 reporter line, with reduced levels of TNF-α release per unit cell viability in THP1 after iMSC-CM treatment. NFκB promoter activity was also reduced in effect of iMSC treatment in THP1- NFκB-Luc2 reporter cells subjected to irradiated vasculature cells (D3), with or without iMSC coculture and, irradiated microglial cells (HMC3), mimicking vasculature injury, microglial injury, and immune cell crosstalk, respectively. iMSC secretome facilitated a more networked tube formation in HCMEC/D3 cells in coculture of D3+iMSC cells that was longer retained. Cytokine arrays revealed iMSC-CM to have proangiogenic, immunomodulatory (immune suppressive) and chemotactic factors: MCP1, ANG (angiogenin), IL6, IL8, GRO, GROα, RANTES, and SDF1. Our initial pathway analysis using human phospho-proteome profiling showed iMSC secretome elevates levels of beta-catenin, and PRAS40 (AKT/mTOR modulator) and lowers level of pRSK1/2/3 in HCMEC/D3. Conclusions: Taken together, we demonstrated that iMSC-secretome complements the proangiogenic and immunosuppressive effects of iMSC during radiation induced injury. Further studies are required to validate the in vivo potential of iMSC-secretome as safe biological therapy in long-term cancer survivors. Citation Format: Kshama Gupta, Ralph B. Perkerson III, Tammee M Parsons, Prasanna Vibhute, Abba C. Zubair, Alfredo Quinones-Hinojosa, Takahisa Kanekiyo. Impact of human iPSC-derived MSC secretome in modulating radiation induced inflammation and brain vascular endothelial cell damage [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 C116.

Abstract Radiation therapy (RT) is an important non-surgical clinical treatment for glioblastoma (GBM) and brain metastases, but inherent radio-resistance can limit the efficacy in many patients. The Ataxia Telangiectasia Mutated (ATM) protein critically regulates radiotherapy-mediated DNA damage repair pathways, and defects in this kinase can lead to profound radiosensitivity. In this study, the efficacy of the highly potent ATM inhibitor WSD0628 was evaluated in GBM and melanoma brain metastasis patient derived xenografts (PDXs). In short term explant cultures of three PDXs, WSD0628 robustly suppressed RT-induced autophosphorylation of ATM at serine 1981 and ATM-mediated phosphorylation of Chk2 at threonine-68 and KAP1 at serine-824, with maximal inhibition at 100 nM. Similarly, RT-induced gH2AX foci were significantly reduced when combined with WSD0628 in the GBM43 cells. Consistent with the importance of ATM in the DNA damage response after radiation, WSD0628 significantly increased the radiosensitivity of U251 cells in clonogenic survival assays (0nM vs. 30nM at 5Gy, p<0.01). To evaluate the optimal duration of drug exposure, WSD0628 was removed from U251 cells at various intervals following irradiation with 5 Gy. Using clonogenic survival as a readout, a 10-fold increase in cytotoxicity was observed with an 8 hour drug exposure, with progressively increasing cytotoxicity gains with exposures up to 24 hours. In vivo efficacy of WSD0628 was evaluated in multiple intracranial PDX models. In an initial dose-ranging study, robust radiosensitization was observed in GBM43 treated with 2 Gy x 5 fractions combined with 5 mg/kg WSD0628 (20 day survival extension) or 10mg/kg WSD0628 (39 day with 10 mg/kg; p<0.01 for both dose levels). Moreover, a single dose of 12.5 Gy combined with 10 mg/kg WSD0628 had profound impact on treatment efficacy in the melanoma brain metastasis M12 PDX: at 180 days post-treatment, all mice in the combination group were electively euthanized, while median survival for sham or RT alone was 17d and 49d, respectively. Histologic analysis identified large intracranial tumors in all sham and RT-only treated mice, but only a small accumulation of melanotic cells without obvious tumor in the combination-treated mice. Immunohistochemical staining of NeuN and GFAP in the combination-treated mice showed preserved neuronal density at the 180-post treatment timepoint and minimal reactive gliosis within the ‘tumor scar’. An ongoing study is comparing this single-fraction 12.5 Gy regimen to a 2.4 Gy x 10 fraction regimen alone or in combination with WSD0628. Collectively, this study demonstrates the potential for profound radiosensitizing effects of WSD0628 in combination without obvious neuronal toxicity and has provided the scientific rational for the first-in-man study of this combination in recurrent GBM (NCT05917145) at Mayo Clinic. Citation Format: Zhiyi Xue, Ann C. Mladek, Sneha Rathi, Danielle M. Burgenske, Shiv K. Gupta, Brett L. Carlson, Zeng Hu, Lauren L Ott, Katrina K. Bakken, Rachael A. Vaubel, William F Elmquist, Wei Zhong, Jann N Sarkaria. The radiosensitizing effects of the novel brain penetrant and potent ATM inhibitor WSD0628 in glioblastoma and melanoma patient derived xenografts [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 A148.

Birt-Hogg-Dubé (BHD) syndrome is associated with an increased risk of multifocal renal tumors, including hybrid oncocytic tumor (HOT) and chromophobe renal cell carcinoma (chRCC). HOT exhibits heterogenous histologic features overlapping with chRCC and benign renal oncocytoma, posing challenges in diagnosis of HOT and renal tumor entities resembling HOT. In this study, we performed integrative analysis of bulk and single-cell RNA sequencing data from renal tumors and normal kidney tissues, and nominated candidate biomarkers of HOT, L1CAM, and LINC01187, which are also lineage-specific markers labeling the principal cell and intercalated cell lineages of the distal nephron, respectively. Our findings indicate the principal cell lineage marker L1CAM and intercalated cell lineage marker LINC01187 to be expressed mutually exclusively in a unique checkered pattern in BHD-associated HOTs, and these 2 lineage markers collectively capture the 2 distinct tumor epithelial populations seen to co-exist morphologically in HOTs. We further confirmed that the unique checkered expression pattern of L1CAM and LINC01187 distinguished HOT from chRCC, renal oncocytoma, and other major and rare renal cell carcinoma subtypes. We also characterized the histopathologic features and immunophenotypic features of oncocytosis in the background kidney of patients with BHD, as well as the intertumor and intratumor heterogeneity seen within HOT. We suggest that L1CAM and LINC01187 can serve as stand-alone diagnostic markers or as a panel for the diagnosis of HOT. These lineage markers will inform future studies on the evolution and interaction between the 2 transcriptionally distinct tumor epithelial populations in such tumors.

INTRODUCTION: A substantial proportion of patients with esophageal adenocarcinoma (EAC) do not report gastroesophageal reflux disease (GERD) symptoms. This study aimed to compare the risk factor profiles and cancer stage at presentation of patients with EAC with and without prior GERD. METHODS: In this retrospective cross-sectional study, patients with EAC were divided into 2 cohorts: (i) EAC with prior GERD: patients who reported typical GERD symptoms (heartburn or regurgitation) ≥1 year before cancer diagnosis and (ii) EAC without prior GERD: patients who did not report prior GERD symptoms or reported symptoms within 1 year of their cancer diagnosis. Baseline demographics, risk factors, and cancer stage at presentation were compared between the 2 cohorts. In addition, the distribution of patients based on numbers of BE/EAC–associated risk factors (1, 2, 3, 4, and 5 or more) was examined in the symptomatic and asymptomatic cohorts. RESULTS: Over 13 years, 388 patients with EAC with prior GERD and 245 patients with EAC without prior GERD were recruited. Both groups had similar baseline demographics and risk factors, but patients with EAC with prior GERD were more likely to have a history of BE. Asymptomatic patients had more advanced disease. Patients with 3 or more BE/EAC–related risk factors formed the largest proportion of patients in both the symptomatic and asymptomatic cohorts. DISCUSSION: Patients with EAC with and without prior GERD symptoms are phenotypically similar, suggesting that BE screening efforts to prevent or detect early EAC should not be restricted to just those with GERD.

Abstract Background Atherosclerotic cardiovascular disease (ASCVD), of which coronary artery disease (CAD) is the primary manifestation, is the leading cause of death worldwide. The AHA/ACC Pooled Cohort Equation (PCE) facilitates risk stratification and initiation of primary prevention of ASCVD, however their performance is suboptimal. Purpose We develop an artificial intelligence-based electrocardiogram (ECG) analysis (AI-ECG) algorithm to detect different stages of CAD and assessed the value of AI-ECG over PCE to predict incident myocardial infarction (MI) and survival over 3-years. Methods Using electronic health record data from approximately 7 million patients seen at over 70 hospitals and clinics across 5 states in the USA, we created independent AI-ECG models to identify elevated coronary artery calcium (CAC) by computer tomography, the presence of obstructive CAD by stress test or angiography, and left ventricular regional akinesis by echocardiogram, presumed to represent prior MI. Deep structured data, AI-enabled natural language processing of unstructured data were used to ensure cohort, and physician review of labeling were used to ensure cohort accuracy. These models were ensembled into a single model that was used to assess the utility of AI-ECG for predicting new occurrence MI in PCE- and age-stratified cohorts of patient with no prior history of ASCVD, over a 3-year timeframe using Cox’s proportional hazards analysis. Results A total of 6,080 patients (15,192 ECGs) were labeled positive for CAC ≥ 300, 12,561 patients (18,915 ECGs) positive for obstructive CAD, and 10,767 patients (23,870 ECGs) positive for regional akinesis. AI-ECG for identifying elevated CACS, obstructive CAD, and regional akinesis achieved AUROCs of 0.86, 0.80, and 0.93, respectively. All AI-ECG models identified increased 3-year risk for MI and death in AI-ECG positive versus AI-ECG negative patients and subgroups with 2 or 3 positive results had stepwise increased risk (Figure A). In a 3-year survival analysis of all-comer patients, the hazard ratio for those that tested positive on 1, 2, or 3 versus 0 positive disease-specific models were 1.61 (1.53-1.69). 2.40 (2.27-2.55), 3.42 (3.21-3.65), respectively. Risk stratification was also observed in cohorts without prior ASCVD stratified by low and high PCE-derived 10-year ASCVD risk (Figure B) and in cohorts stratified by age, including under the age of 40. Conclusions AI-ECG has potential to address unmet need for accessible risk stratification in patients in whom PCE under, over, or insufficiently estimate ASCVD risk, and in whom risk assessment over periods of time shorter than 10 years is desired.