Journal of Hematology & Oncology

Patients with multiple myeloma (MM), an age-dependent neoplasm of antibody-producing plasma cells, have compromised immune systems and might be at increased risk for severe COVID-19 outcomes. This study characterizes risk factors associated with clinical indicators of COVID-19 severity and all-cause mortality in myeloma patients utilizing NCATS National COVID Cohort Collaborative (N3C) database. The N3C consortium is a large, centralized data resource representing the largest multi-center cohort of COVID-19 cases and controls nationwide (>16 million total patients, and >6 million confirmed COVID-19+ cases to date). Our cohort included myeloma patients (both inpatients and outpatients) within the N3C consortium who have been diagnosed with COVID-19 based on positive PCR or antigen tests or ICD-10-CM diagnosis code. The outcomes of interest include all-cause mortality (including discharge to hospice) during the index encounter and clinical indicators of severity (i.e., hospitalization/emergency department/ED visit, use of mechanical ventilation, or extracorporeal membrane oxygenation (ECMO)). Finally, causal inference analysis was performed using the propensity score matching (PSM) method. As of 05/16/2022, the N3C consortium included 1,061,748 cancer patients, out of which 26,064 were MM patients (8,588 were COVID-19 positive). The mean age at COVID-19 diagnosis was 65.89 years, 46.8% were females, and 20.2% were of black race. 4.47% of patients died within 30 days of COVID-19 hospitalization. Overall, the survival probability was 90.7% across the course of the study. Multivariate logistic regression analysis showed histories of pulmonary and renal disease, dexamethasone, proteasome inhibitor/PI, immunomodulatory/IMiD therapies, and severe Charlson Comorbidity Index/CCI were significantly associated with higher risks of severe COVID-19 outcomes. Protective associations were observed with blood-or-marrow transplant/BMT and COVID-19 vaccination. Further, multivariate cox proportional hazard analysis showed that high and moderate CCI levels, International Staging System (ISS) moderate or severe stage, and PI therapy were associated with worse survival, while BMT and COVID-19 vaccination were associated with lower risk of death. Finally, matched sample average treatment effect on the treated (SATT) confirmed the causal effect of BMT and vaccination status as top protective factors associated with COVID-19 risk among US patients suffering from multiple myeloma. To the best of our knowledge, this is the largest nationwide study on myeloma patients with COVID-19.

BackgroundCD19-directed chimeric antigen receptor (CAR) T-cell therapies have transformed the treatment landscape for pediatric B-cell acute lymphoblastic leukemia (B-ALL), yet relapse driven by antigen escape remains a major limitation. Dual-targeting CAR approaches recognizing CD19 and CD22 have shown promising clinical activity. However, sustained remissions are limited by insufficient CAR T-cell persistence. MethodsCAR22.19, a fully human tandem CD19/CD22 CAR, was developed and clinically applied within a named-patient program in nine heavily pretreated pediatric patients with relapsed/refractory B-ALL. Treatment indications were CD19-negative blast population (n=5), relapse after CD19 CAR T (n=3) and/or restricted access to approved CAR T-cell products (n=3). Autologous and donor-derived CAR22.19 T-cells (CART22.19) were manufactured using a GMP-compliant, semi-automated fresh in fresh out process. Safety and efficacy were assessed through standardized clinical monitoring, measurable residual disease analysis, and CAR T-cell kinetics. ResultsPreclinical validation demonstrated antigen-specific cytotoxicity and dual-antigen activity. Clinically, CART22.19 were well tolerated, with no treatment-related deaths and no grade [≥]3 neurotoxicity, while grade [≥]3 cytokine release syndrome occurred in 38.5% (5/13) of infusions and resolved with standard interventions. An initial complete molecular remission was achieved in 78% (7/9) of patients, with a 12-month overall survival rate of 53.3% (95% CI, 17.7-79.6%). Sustained treatment response in CD19-CD22 cases underscore the functional contribution of the CD22-targeting domain. In contrast, all patients refractory to prior CD19 CAR T-cell therapies relapsed early with retained CD19CD22 expression. Limited in vivo persistence was found to be a key mechanism of treatment failure. Notably, durable remission and sustained functional persistence of CART22.19 was achieved in one patient refractory to autologous CART22.19 following infusion of donor-derived CART22.19 after reduced-intensity conditioning (RIC) allogeneic hematopoietic stem cell transplantation (alloHSCT) in non-remission. ConclusionsCART22.19 therapy demonstrated a favorable safety profile and promising clinical activity in a high-risk pediatric population, with dual targeting enabling disease control in CD19-negative disease. However, limited CAR T-cell persistence remains a major obstacle to sustained remission. Our findings support further clinical development of CART22.19 and highlight the potential of donor-derived CAR T-cells following RIC alloHSCT as a novel therapeutic strategy to enhance persistence and improve outcomes in heavily pretreated pediatric patients.

Belantamab mafodotin, an antibody-drug conjugate targeting B-cell maturation antigen, has demonstrated significant clinical efficacy in combination therapies for relapsed/refractory multiple myeloma. Belantamab mafodotin exerts therapeutic effects through cytotoxicity of its payload, monomethyl auristatin F, and through mediation of antibody-induced cell death. Long-term clinical responses were observed with monotherapy treatment, despite dose holds, suggesting adaptive immune system involvement. Here, we show that belantamab mafodotin induces markers of immunogenic and inflammatory cell death in vitro and ex vivo. Belantamab mafodotin monotherapy treatment triggers acute inflammation detectable in patient serum within 24 hours, with increases in granzyme B, CXCL9, CCL3, and CCL4 linked to response depth achieved. High expression of LRP1 and TLR2, receptors that mediate immunogenic cell death on patients monocytoid (monocyte/macrophage) cells, suggests an important function of monocytoid cells to mediate the inflammation and immunogenic cell death cascades. Inflammation is followed by remodeling of the innate and adaptive immune system, a reduction in immune inhibitory signaling and the emergence of CD4 granzyme B-expressing cells in patients in remission vs those that relapse. Belantamab mafodotins ability to promote adaptive immune responses and its cytotoxic activity may help explain the durable responses observed in treated patients, despite dose and schedule modifications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/25335116v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f541eforg.highwire.dtl.DTLVardef@bbef70org.highwire.dtl.DTLVardef@64a584org.highwire.dtl.DTLVardef@1ae8bac_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chimeric antigen receptor T-cell (CAR-T) therapy targeting CD19 has transformed outcomes for children with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL), yet the influence of molecular subtype on outcomes remains unclear. We evaluated the impact of cytogenetic and molecular signatures on complete response (CR), overall survival (OS), and leukemia-free survival (LFS) after CD19 CAR-T therapy in eighty-six pediatric patients with R/R B-ALL treated with tisagenlecleucel. CR was assessed 30 days after infusion. Cytogenetic data were available for 84 patients and molecular profiling for 62. Survival analyses included 72 patients who received CD19 CAR-T as the sole cellular therapy. Seventy-seven patients achieved CR (89.5%). Pre-infusion bone marrow blasts of [&ge;]20% were associated with lower CR rates (53.8% vs 95.9%, p<0.0001) and significantly reduced OS and LFS (both p<0.0001). Among molecular markers, RAS mutations correlated with inferior OS (p=0.0222) and LFS (0.0402). In multivariate analysis, bone marrow blasts >20% and RAS mutations independently predicted inferior OS. Post CAR-T, CD19 negative relapses showed almost twice higher prevalence of RAS mutations (66% vs 37.5%). These findings highlight RAS mutations as a key molecular predictor of outcome after CD19 CAR-T therapy and suggest emergence of unique risk stratification for patients receiving CD19-targeting therapy.

Reovirus is an oncolytic virus with natural tropism for cancer cells. We previously showed that reovirus intravenous administration in myeloma patients was safe, but disease control associated with viral replication in the cancer cells was not observed. Here we show that ex vivo proteasome inhibitors (PIs) potentiate reovirus replication in circulating classical monocytes, increasing viral delivery to myeloma cells. We found that the anti-viral signals in monocytes primarily rely on the NF-kB activation and that this effect is impaired by the addition of PIs. Conversely, PIs improved reovirus-induced monocyte and T cell activation against cancer cells. Based on these preclinical data, we conducted a phase 1b trial of the reovirus Pelareorep together with the PI carfilzomib in 13 heavily pretreated bortezomib-resistant MM patients. Objective responses associated with reovirus active replication in MM cells, T cell activation and monocytic expansion were noted in 70% of patients.

Acute myeloid leukemia (AML) is characterized by malignant myeloid precursors that span a cellular hierarchy from dedifferentiated leukemic stem cells to mature blasts. While the diagnostic and prognostic importance of AML blast maturation is increasingly recognized, personalized therapies are currently not tailored to a patients individual makeup of this cellular hierarchy. In this study, we use multiplexed image-based ex vivo drug screening (pharmacoscopy) to systematically quantify the drug sensitivity across the cellular hierarchy of AML patients. We analyzed 174 prospective and longitudinal patient samples from 44 newly diagnosed AML patients, which indicated that differences in the AML hierarchy significantly identified poor responses to first-line therapy, outperforming European LeukemiaNet (ELN) criteria. Critically, drug response profiling across the AML hierarchy of each patient improved the accuracy of predicting patient response to first-line therapy (AUC 0.91), and revealed alternative individualized treatment options targeting the complete AML hierarchy of non-responding patients. We confirmed these findings in an independent cohort of 26 relapsed/refractory AML patients, for whom pan-hierarchy response profiling improved response predictions post hoc. Overall, our results quantify the clinical importance of therapeutically targeting the complete cellular hierarchy of newly diagnosed AML, and identify multiplexed image-based ex vivo drug screening to enable quantification and targeting of the AML maturation hierarchy for improved personalized treatment.

Non-ICANS neurotoxicities (NINTs) are serious, atypical toxicities associated with ciltacabtagene autoleucel, a commercial chimeric antigen receptor (CAR) T cell therapy approved for relapsed/refractory multiple myeloma. Risk factors contributing to the development of NINTs are poorly understood. In a cohort of 109 patients, we identify predisposing risk factors and propose strategies to mitigate NINTs. We show that high peak absolute lymphocyte count is a strong NINT predictor which directly correlates with flow cytometry-based peripheral blood CAR T cell quantitation. The observed CAR lymphocytosis was polyclonal with a bias towards CD4+ CAR T cells rich in memory marker expression. We then identified CAR lymphocytosis associated CD4+ CAR T cell populations which exhibited increased inflammatory pathway gene expression. Finally, we characterize NINT associated CD4+ CAR T cell populations which are potential therapeutic targets for future exploration. One Sentence SummaryCiltacabtagene autoleucel associated non-ICANS neurotoxicities are driven by high CD4+ CAR T cell expansion exhibiting memory marker expression and upregulated inflammatory gene signaling pathways.

Children with acute lymphoblastic leukemia (ALL) are treated with multiagent chemotherapy that causes profound changes to the immune system. There are limited data on how disease and therapy impact antigen-specific immune memory, leading to inconsistent guidelines on best practices for revaccination of this population. Here, to inform vaccine guidance, we investigated whether immunity derived from routine childhood measles and varicella zoster virus (VZV) vaccines is maintained during and after therapy for childhood ALL. We report that antibodies against measles and VZV were significantly reduced in children with ALL (n=45) compared to healthy controls (n=13), particularly in older children in whom a longer time had passed since their most recent vaccine dose. However, the avidity of the measles and VZV-specific antibodies was indistinguishable between groups. Despite changes to the composition of the T cell compartment, both overall and antigen-specific T cell function were preserved in children with ALL. These data provide compelling evidence for revaccination of children following ALL treatment. Intact T cell responses suggest that post-treatment revaccination would be effective.

Background.CAR T-cell therapy is the standard of care for R/R DLBCL, but more than half of patients fail to achieve long-term remission. Identification of cellular biomarkers in CAR T- cell infusion products (IPs) that predict complete remission beyond six months may guide the development of strategies to improve outcomes. Methods.IPs from 13 R/R DLBCL patients were analyzed using a 39-marker mass cytometry panel, comparing cell populations between long-term responders (R) and non-responders (NR). Both unsupervised and supervised analyses were performed. Longitudinal blood samples were analyzed for 30 days to track CAR T-cell subpopulation dynamics. Results.At a median follow-up of 13.5 months, median progression-free survival (PFS) was 13.3 months (95% CI: 9.7-24.3) in R (n=8) versus 3.5 months (95% CI: 0.5-5.4) in NR (n=5). The HR for PFS was 56.67 (95% CI: 7.3-439.3; P=0.0001). A subset of CD3+CXCR3+CD7+ CAR T-cells found in both CD4+ and CD8+ populations was significantly enriched in R and expressed higher levels of perforin, granzyme B, and NKG2D (restricted to CD8+). NR had more CXCR3+CD7+LAG3+ CAR T-cells. CD3, CD7, CXCR3, and NKG2D cell surface levels were higher in R, whereas LAG3, Ki67, and CD71 were elevated in NR. A predictive cut-off ratio of CD3+CXCR3+CD7+LAG3+CAR+ T-cells <0.83 and CD3+CXCR3+CD7+NKG2D+CAR+ T-cells >1.034 yielded a predictive accuracy of 0.92. Serum CXCL9 and CXCL10 levels were not different between groups. Conclusions.Increased frequency of CAR T-cells expressing CD7, CXCR3 and NKG2D in R versus LAG3 and CD71 in NR emerged as strong correlates of therapeutic outcome.

Immune checkpoint inhibitor immunotherapy has revolutionized the treatment of non-small cell lung cancer (NSCLC). Despite the immense success, still a significant proportion of patients do not develop durable responses, allowing disease progression accompanied by high mortality rates. Therefore, there is an imperative need for identification of reliable non-invasive predictive biomarkers to guide therapeutic decisions. Herein, we constructed a blood immunomap in NSCLC patients with metastatic disease, using a high-dimensional mass cytometry approach. Assessment of clinical responses to aPD1 immunotherapy revealed, among others, a significant expansion of CD8+PD-L1+ T cells in individuals not responding to immunotherapy. Of interest, CD8+PD-L1+ T cells were enriched in tumor biopsies and bronchoalveolar lavage of NSCLC individuals at early stages of disease as well as in pleural infusions of individuals with thoracic malignancies. Transcriptomic analysis revealed that CD8+PD-L1+ T cells exhibited a regulatory/exhausted phenotype, while various transcripts associated with the overall survival of NSCLC individuals, were mapped. Overall, our findings define an immunomap in the early stage and advanced NSCLC patients and identify immune-related events which may benefit the quest for identification of predictive biomarkers of immunotherapy responses.

The European LeukemiaNet (ELN) risk stratification of acute myeloid leukemia (AML) uses genetic and molecular markers to categorize patients. However, disease heterogeneity, particularly in the intermediate-risk group, complicates stratification. Ideker et al. developed the Network-Based Stratification (NBS) method, combining protein network analysis and mutation profiling via machine learning. We applied NBS to intermediate- risk AML patients to refine prognosis and identify distinct molecular subtypes compared to the 2022 ELN scheme. We selected 170 intermediate-risk AML patients based on the 2022 ELN classification from TCGA (n=58), BEAT AML (n=87), and FIMM (n=25) datasets. Using NBS, we analyzed 3,108 genes from WGS or WES data, mapping them onto a cancer-specific protein network for clustering based on network-propagated mutation profiles. We conducted 200 iterations of sub-sampling, considering patients with at least 3 mutated genes and using consensus clustering for robust stratification, assessing associations with clinical and transcriptomic features. NBS identified five distinct molecular subgroups characterized by unique mutation patterns: IDH1-dominant (Cluster 1), DNMT3A-dominant (Cluster 2), low-frequency multi-mutated (Cluster 3), FLT3/NPM1/DNMT3A co- mutated (Cluster 4), and FLT3-dominant (Cluster 5). Cluster 4 showed significantly worse overall survival (HR = 1.81; p = 0.05). In addition, ex vivo drug sensitivity and transcriptomic analyses revealed significant variation in therapeutic response and pathway activation across clusters. These findings underscore the power of machine learning-driven approaches like NBS to uncover hidden molecular structure within intermediate-risk AML groups, enabling more precise prognostication and potentially informing personalized therapeutic strategies. Key pointsO_LIML-based NBS stratification reveals distinct subgroups within intermediate-risk AML with unique molecular and clinical profiles. C_LIO_LIAML with NPM1/FLT3-ITD/DNMT3A mutations define a high-risk group with distinct drug sensitivities, including FLT3 inhibitors. C_LI

Lirilumab is a fully human monoclonal antibody designed to block killer inhibitory receptors (KIR), which are major immune checkpoints involved in the regulation of NK cell-mediated killing of HLA-I-expressing tumors. EFFIKIR is a multicenter randomized double-blind 3-arm placebo-controlled phase II trial with lirilumab as single-agent as maintenance therapy of elderly patients with AML in first complete remission (NCT01687387). Two dose schedules led to either continuous or intermittent KIR occupancy. 153 patients were randomized and 152 patients were treated after 3+7 induction therapy. The median follow-up was 36.6 months. Lirilumab was well tolerated, with no significant hematological toxicity. The median LFS were 17.6, 6.7 and 13.9 months in the 0.1mg/kg arm, 1mg/kg arm and placebo arm, respectively. An excess in early relapse led to early termination of treatment in the 1mg/kg arm. Extensive analysis of immune cell fate following KIR blockade evidenced a decrease of KIR+ NK cell absolute counts following KIR blockade, associated with a decrease of Bcl-2. Lirilumab also bound antigen-experienced CD8+ T cells, and induced a transient decrease of CD69 expression. Besides, lirilumab bound v{delta}2+ {gamma}{delta}T cells with a high cytotoxic potential, and induced a decrease of DNAM-1 and Bcl-2, the latter being associated with a decrease of KIR+ {gamma}{delta}T cell, and with a drastic reduction of time to relapse. Overall, the potentially deleterious effects on immune effectors may have resulted in the impairment of immune surveillance associated with an unexpected high rate of early relapse in the group of patients exposed to prolonged full KIR blockade. KEY POINTSO_LIProlonged full KIR blockade leads to potentially deleterious effects on NK cells, CD8+ T cells and v{delta}2+ {gamma}{delta}T cells C_LIO_LICombined inhibitory effects of KIR blockade may have resulted in the impairment of immunosurveillance associated with high rate of relapse C_LI

Ide-cel and cilta-cel are the two FDA-approved anti-BCMA CAR T cell therapies for the treatment of relapsed/refractory multiple myeloma. Here, we studied a patient who was initially treated with ide-cel with progressive disease and subsequently treated with cilta-cel with a complete response. To elucidate the underlying mechanisms underpinning the distinct clinical outcomes, we conducted multimodal, cross-tissue, and longitudinal single-cell analyses. This enabled us to directly compare the specific cellular and molecular factors distinguishing these two CAR therapies including their cell phenotypes, post-infusion kinetics, and endogenous immune landscapes. We found that the ide-cel infusion product was dominated by CD4+ CAR T cells, upregulated a terminal effector phenotype, and exhibited elevated activation signatures. Post-infusion, ide-cel CAR T cells failed to proliferate, sustain cytotoxicity, or migrate into the bone marrow, resulting in persistent myeloma cells and dysregulated monocytes and natural killer cells. In contrast, the cilta-cel infusion product exhibited a balanced ratio of CD4+ and CD8+ CAR T cells, upregulated a resident memory-like signature, and displayed signatures of IL-1 and IL-2 family cytokine signaling. Post-infusion, cilta-cel CAR T cells retained their resident memory-like profile, were durably retained in the peripheral blood, and successfully infiltrated the bone marrow, leading to effective tumor clearance and reestablishment of immune homeostasis. Our results present important clinical evidence that cilta-cel can serve as an effective salvage treatment following ide-cel failure. By providing a direct patient-matched comparison between two CAR therapies, our study uncovers important insights into both CAR T-cell intrinsic properties and immune environmental factors that contribute to effective BCMA CAR T-cell treatment.

BackgroundWhile hypomethylating agents (HMA) are currently used to treat myelodysplastic syndrome (MDS) and patients with cancer, their effects on reactivation and/or upregulation of oncogenes are generally not well elucidated. SALL4 is a known oncogene that plays an important role in MDS. In this study, we examined the impact of HMA on SALL4 methylation and expression. MethodsPaired bone marrow samples from a cohort of MDS patients on the BMT-AZA trial, collected before and after four cycles of azacytidine (AZA) treatment, were used to explore the relationship between changes in SALL4 expression, treatment response and clinical outcome with a follow-up of up to 40 months. No/low-SALL4 expressing leukemic cell lines were used to study the relationship between SALL4 methylation and expression. A novel locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island critical for SALL4 expression. ResultsIn MDS patients, we noted SALL4 upregulation after AZA treatment in 40% of the cases. Significantly, patients with SALL4 upregulation had a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a 500bp CpG island within the 5UTR-Exon1-Intron1 region was critical for SALL4 expression. Importantly, in cell lines and patients, we confirmed that HMA treatment led to demethylation of the same CpG region and upregulation of SALL4 expression. ConclusionsCRISPR-DiR was useful to define the critical region important for gene activation. Along with analysis of patient samples, we demonstrated that demethylation and upregulation of an oncogene after HMA treatment can indeed occur and should be further studied.

BackgroundNovel drugs are dynamically changing current treatment regimens for multiple myeloma (MM). Novel drugs have improved prognosis of MM patients in clinical studies but are expensive. Little is known about up-to-date real-world application and costs. MethodsWe performed a retrospective observational cohort analysis (cost-outcome description; 2012-2017) in a claims database of a major Swiss health insurance company which covers 14% of the Swiss population (Helsana Versicherungen AG).We used primary (MM diagnoses via ICD-10) and secondary features (prescribed MM-specific drugs) as inclusion criteria and defined a hierarchy of drug regimens to classify treatments as: 1) proteasome inhibitor (PI)-based regimen (e.g. bortezomib); 2) IMID-based regimen (e.g. lenalidomide); 3) chemotherapy (CHEMO)-based regimen (e.g. bendamustin); 4) monoclonal antibody (MAB)-based regimen (e.g. daratunumab). Direct medical costs of mandatory health insurance were analysed in 2017 Swiss Francs (CHF; third party payer perspective). ResultsOverall, we identified n=1054 prevalent MM patients (2012-2017) and n=378 incident MM patients (2015-2017; men: 47.1%; age group <=75 years: 48.7%). The number of prevalent patients per year increased over time (from n=314 in 2012 to n=645 in 2017). PI-based regimens were the most frequent first line approach for incident patients (76.0%), followed by IMID-based (21.9%) and CHEMO-based regimens (2.1%). Only four patients were treated with MAB-drugs. For later lines, IMID-based regimens were most often used (2nd line: 56.4%; 3rd line: 2 of 3 patients), followed by PI-based regimens (43.6% and 1 of 3 patients, respectively). 161 of 1054 prevalent MM patients (15.3%) were treated with autologous hematopoietic stem cell transplantation (HSCT), 4 patients with allogeneic HSCT. Average costs per patient per treatment line varied considerably (reliable data available from 2012 to 2014; mean duration of lines between 112 and 388 days): PI-based regimens: CHF 81352; IMID-based: CHF 73495; CHEMO-based: CHF 683. Mean daily costs under MM treatment stepwise increased from CHF 209 in 2012 to CHF 254 in 2017 (relative increase: 21.5%). Annual direct medical costs in Switzerland for seven novel MM drugs were extrapolated to be 60.1 Mio CHF in 2012 and 118.6 Mio CHF in 2017 (relative increase: 97.3%), corresponding to mean annual outpatient MM drug costs per patient of CHF 28000 in 2017. Annual death rates decreased systematically from 18.6% in 2012 to 15.5% in 2017 (p for trend: 0.03). No statistically significant difference in death rates emerged for 2017 compared with 2012 (risk ratio: 0.83; 95%-CI: 0.63 to 1.10; absolute risk reduction: 3.1%). ConclusionsCurrent treatment patterns for MM patients in Switzerland show variation concerning applied drug regimens as well as costs. An increasing prevalent population of MM patients in combination with increasing costs per day under treatment lead to a substantial and growing budget impact for the Swiss social insurance system.

BackgroundIndividualized treatment decisions for patients with multiple myeloma (MM) requires accurate risk stratification that takes into account patient-specific consequences of genetic abnormalities and tumor microenvironment on disease outcome and therapy responsiveness. MethodsPreviously, SYstems Genetic Network AnaLysis (SYGNAL) of multi-omics tumor profiles from 881 MM patients generated the mmSYGNAL network, which uncovered different causal and mechanistic drivers of genetic programs associated with disease progression across MM subtypes. Here, we have trained a machine learning (ML) algorithm on activities of mmSYGNAL programs within individual patient tumor samples to develop a risk classification scheme for MM that significantly outperformed cytogenetics, International Staging System, and multi-gene biomarker panels in predicting risk of PFS across four independent patient cohorts. ResultsWe demonstrate that, unlike other tests, mmSYGNAL can accurately predict disease progression risk at primary diagnosis, pre- and post-transplant and even after multiple relapses, making it useful for individualized dynamic risk assessment throughout the disease trajectory. ConclusionmmSYGNAL provides improved individualized risk stratification that accounts for a patients distinct set of genetic abnormalities and can monitor risk longitudinally as each patients disease characteristics change.

Targeted immunotherapies against B-cell maturation antigen (BCMA) have transformed the treatment landscape of Multiple Myeloma (MM). Fc receptor-like 5 (FCRL5) has emerged as an alternative target. However, resistance frequently emerges within months, posing a significant clinical challenge. Structural alterations and mutations in BCMA only account for the minority of cases and insights into BCMA antigen escape remain largely unknown. This study investigates novel (epi)genetic mechanisms of antigen escape through comprehensive multi-omic Oxford Nanopore profiling of sequential pre-treatment and relapse samples. We identify acquired DNA-hypermethylation across the entire BCMA gene, and hypermethylation of the FCRL5 promoter, both resulting in epigenetic gene silencing as novel resistance mechanisms through which MM cells evade therapy. These findings underscore the dynamic clonal evolution of MM under therapeutic pressure and highlight the critical role of epigenetic modifications in resistance. Furthermore, we demonstrate the potential of advanced sequencing technologies for capturing epigenetic and complex genomic alterations in clinical settings, paving the way for personalized treatment strategies and predictive biomarkers for early resistance detection. Statement of significanceAcquired DNA hypermethylation of BCMA and FCRL5 regulatory regions, leading to gene expression downregulation, represent novel epigenetic resistance mechanisms to anti-BCMA and anti-FCRL5 immunotherapies. Furthermore, DNA methylation marks serve as a molecular memory of therapeutic pressure, capturing the treatment history of cancer cells.

In recent years, chimeric antigen receptor (CAR) T cell therapy has shown great promise in treating hematological malignancies. However, using virus in manufacture of CAR T cells brings about several problems. The application of CRISPR/Cas9 genome editing technology emerges in constructing novel CAR T cells by disrupting endogenous genes. Here we successfully develop a two-in-one approach to generate non-viral genome specific targeted CAR T cells through CRISPR/Cas9. By targeting a CAR in AAVS1 safe harbor locus, we demonstrated that these CAR T cells behave comparable to those conventionally produced by lentivirus. Furthermore, PD1-knockin anti-CD19 CAR T cells show a superior ability to eradicate tumor cells with high PD-L1 expression. In the adoptive therapy for relapsed/refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL), we observed durable responses without serious adverse events and complete remission (CR) in patients treated with these PD1 knockout CAR T cells. Collectively, our results prove the safety and feasibility of non-viral genome specific integrated CAR T cells, thus providing a new potential strategy for cancer treatment using these novel CAR T cells.

Leveraging endogenous tumor-resident T-cells for immunotherapy using bispecific antibodies (BsAb) targeting CD20 and CD3 has emerged as a promising therapeutic strategy for patients with B-cell non-Hodgkin lymphomas. However, features associated with treatment response or resistance are unknown. To this end, we analyzed data from patients treated with epcoritamab-containing regimens in the EPCORE NHL-2 trial (NCT04663347). We observed downregulation of CD20 expression on B-cells following treatment initiation both in progressing patients and in patients achieving durable complete responses (CR), suggesting that CD20 downregulation does not universally predict resistance to BsAb-based therapy. Single-cell immune profiling of tumor biopsies obtained following one cycle of therapy revealed substantial clonal expansion of cytotoxic CD4+ and CD8+ T-cells in patients achieving CR, and an expansion of follicular helper and regulatory CD4+ T-cells in patients whose disease progressed. These results identify distinct tumor-resident T-cell profiles associated with response or resistance to BsAb therapy.

Acute leukemias (ALs) are complex hematological disorders, and accurate diagnosis is crucial for guiding treatment decisions and predicting patient outcomes. While changes in cell marker levels are well documented, the impact of these changes on marker relationships through an integrative systems approach remains uncharacterized. To address this gap, we conducted a 12-year study investigating 41 markers, including ontogenic markers and those used to diagnose both common and rare leukemia types, using immunophenotyping flow cytometry (IFC) data from 1,069 leukocyte samples obtained from peripheral blood (PB) or bone marrow (BM) aspirates of patients with suspected ALs. Machine learning techniques, such as principal component analysis (PCA) and random forest (RF) classification, demonstrated the stratification power of the cellular markers. Hierarchical clustering analysis of leukocyte ontogenetic markers revealed disease-specific clusters, irrespective of sex or sample type (PB or BM). Additionally, we found that patients with acute myeloid leukemia (AML) showed mild disruption in cell marker correlations, whereas the most significant dysregulation was observed in patients with T-cell acute lymphoblastic leukemia (T-ALL). Importantly, we identified ontogenic correlation changes indicating clusters of immature versus mature leukocyte markers, as well as cell lineage-specific markers influencing cellular relationships. These findings underscore the value of integrating systems strategies into conventional IFC analyses to enhance synthetic diagnosis and deepen our understanding of ALs pathophysiology.