Citations

We previously showed that both reactive oxygen species-inducing chemotherapy and some genetic subtypes of B precursor acute lymphoblastic leukaemia (ALL) can induce bone marrow (BM) mesenchymal stromal cells (MSC) to become cancer associated fibroblasts (CAFs). Exposure of MSC to ALL-cell conditioned media (CM) is sufficient to generate CAFs - direct cell-cell contact is not required (Burt, Dey et al, 2024). We showed that CAFs can lead to chemoresistance via transfer of mitochondria to ALL cells through tunnelling nanotubes (Burt, Dey et al 2019). We noted that CAFs in the ALL BM had features of senescence, prompting the current study in which we aimed to investigate the link between senescence and CAFs in ALL. We used 3 genetic subtypes of ALL; BCR::ABL1 (SD1 cells, representing “adult” ALL) , ETV6::RUNX1 (REH cells, representing “childhood” ALL) and KMT2A::AFF1 (SEM cells, known from our previous work NOT to induce CAFs). We exposed HS27a stromal cells to the ALL CM at a number of timepoints over a period of three weeks and characterised senescence using qPCR, cytochemical B-galactosidase assays, flow cytometry and immunofluorescence (IF). Cytokine arrays were performed to quantify the senescence associated secretory phenotype (SASP) components and assess the complexity of SASP components in all three ALL subtypes. A more prolonged exposure (> 10 days) to CM was required for senescence to develop in contrast to CAF formation which occurs early (2-3 days) in HS27a MSC cells. Senescence marker expression peaked at around day 11 to day 14 of CM exposure before subsiding to lower levels around day 21. On day 14, 70% of HS27a cells treated with SD1 CM were positive for beta galactosidase compared to below 25% positive for REH and SEM cells. Cell cycle arrest was demonstrated by P16 and Ki67 IF. 95% of SD1 CM treated HS27a cells were positive for P16 compared to less than 10% in RPMI control. Likewise, only 29% of HS27a exposed to SD1 CM were Ki67 positive compared to 66% in the RPMI control. Expression levels of other senescence-associated markers such as SERPINE1, SERPINB2, SOD2, NFKB1 were quantified by qPCR and were all significantly (p<0.001) upregulated in SD1 CM treated HS27a (2 to 40 fold) compared to SEM CM (-2 to 5 fold) and REH CM (-0.5 to 1.0) treated HS27a. SASP associated cytokines IL6 (1000-5000pg/ml), IL8 (2500-5000pg/ml), CXCL10 (500-1500pg/ml), CCL5 (1500-2250pg/ml), IL10 (25-50pg/ml), were significantly (p<0.0001) upregulated in the SD1 CM treated HS27a compared to controls and other ALL cell line exposed HS27a. Finally, HMGB2 and LMNB1 genes, loss of which is associated with senescence were significantly (p<0.001) downregulated in the SD1 CM (-2.5 to 0.5 fold) treated HS27a compared to controls. In view of the prominence of senescence in SD1-exposed HS27a cells, we tested the effects of two different senolytic drugs venetoclax (BCL2i) and navitoclax (BCL2i/BCLXLi) in vitro. Briefly, HS27a cells were exposed to SD1 CM for 11 days, and after washing, increasing concentrations of the drugs were added for up to 72 hrs followed by washing, fixing and DAPI-staining and counting. HS27a exposed to SD1 CM was highly sensitive to low dose treatment of navitoclax (0.5uM) with 80% cell death 48 hrs post treatment. HS27a cells exposed to SD1 CM were less sensitive to venetoclax with only 50% cell death observed at higher doses (5uM) even after 72 hrs post treatment. Further senolytic drug screening experiments are ongoing. Our data suggest that BCR::ABL1 but not ETV6::RUNX1 or KMT2A::AFF1 ALL cells, can induce paracrine senescence in HS27a MSC cells. We suggest that paracrine senescence in the stromal microenvironment may be a component of treatment response in B-ALL. ^^ and ** contributed equally

Introduction: Hyponatremia is a common electrolyte abnormality that has been associated with poor outcomes in several conditions including acute myocardial infarction (AMI). However, those studies were performed in the era before percutaneous coronary intervention (PCI), focused mostly on ST-elevation myocardial infarction (STEMI), and sodium levels up to 72 h of admission. Aim: The purpose of this study was to identify the prognostic importance of hyponatremia in patients with acute myocardial infarction. Methods: We performed a cross sectional and retrospective analysis of 100 patients with a diagnosis of non-ST-elevation myocardial infarction (NSTEMI) and STEMI presenting at PSH, Vadodara, Gujarat, India from January 2022 to May 2024. Our independent variables were sodium levels on the day of admission. Dependent variables were in-hospital mortality, 30-day mortality, length of hospital stay, intensive care admission, new heart failure diagnosis, and ejection fraction. Infarct size was determined by echocardiographic examination that was performed on admission, day 2 or 3 of hospitalization Results: Hyponatremia, defined as a plasma sodium level <135 mmol/L (<135 mEq/L). 32% of patients had hyponatremia up to 7 days of admission. Intensive care admission was higher in patients with hyponatremia up to7 days (70% vs 383%), they had worse 30-day mortality (15% vs to 3%), in hospital mortality (12% vs 2%), and new heart failure diagnosis (32% vs 16%). Hyponatremia on admission was associated with in-hospital mortality (15% vs 4%), 30-day mortality (19% vs 6 %). Conclusions: This study suggests that hyponatremia on admission and at any point during the first seven days of hospitalization are associated with in-hospital and 30-day mortality and prognosis worsens with the severity of hyponatremia. Further studies are required to determine if plasma sodium levels may serve as a simple marker to identify patients at high risk.

Introduction: Stroke-related mortality poses significant challenges in the US. Increased at-home deaths since COVID-19 pandemic prompted changes in the provision of end-of-life care. Question: What were the settings of stroke deaths in the US during COVID-19 pandemic? Methods: Decedent-level mortality data from death certificates in CDC repository were obtained for the year 2020 (pandemic) and 2019 (comparison). Demographic data include age, sex, race/ethnicity, education, marital status, and place of stroke death, including inpatient, outpatient/emergency room (ER), hospice/nursing facilities (H/NF), and at-home. Multivariable logistic regression models assessed demographic impact on stroke mortality by place-of-death, yielding odds ratios (OR) with significance threshold of p<0.05. Results: A total of 5,400 and 5,712 stroke-related deaths occurred in 2019 and 2020, respectively. During COVID-19, decedents >65 years were more likely to die in H/NF (OR 10.05, p<0.001) but less likely in outpatient/ER (OR 0.32, p<0.001) or at home (OR 0.57, p=0.007) compared to those <35 years. High education level did not significantly influence the place of death than having high school diploma or less (p>0.05). Males were more likely to die at home (OR 1.38, p<0.001) but less likely as inpatient (OR 0.79, p<0.001) than females. Hispanic decedents had a higher likelihood of inpatient death (OR 1.22, p=0.041) but less likely to die in H/NF (OR 0.55, p<0.001) compared to non-Hispanic decedents. Compared to White decedents, Black decedents were more likely to die in outpatient/ER (OR 1.44, p=0.018), while American Indian decedents were more likely to die as inpatient (OR 3.12, p=0.005). Asian/Pacific Islander decedents had a higher chance of inpatient death (OR 1.47, p=0.002) but a lower likelihood of dying in H/NF (OR 0.61, p=0.010). Married decedents were more likely than singles to die as inpatient (OR 1.37, p=0.001) or in outpatient/ER (OR 1.99, p<0.001), but less likely to die in H/NF (OR 0.61, p=0.002) or at home (OR 0.56, p<0.001). Conclusion: Our analysis revealed the influence of demographics on the settings of stroke fatalities during the COVID-19 pandemic. Further investigation is needed to determine contributing factors.

Abstract Lymphopenia following chemoradiation for high grade gliomas (HGG) is common and is associated with decreased survival. NT-I7 (efineptakin alfa) is a long-acting analog of the lymphocyte pro-survival cytokine, interleukin-7. Our Phase I dose-escalation study of 19 patients with HGG demonstrated early elevations in CD4+ and CD8+ T cells in the first 4 weeks after administration of NT-I7. In this study, we explored the diversity and potential functionality of the NT-I7-expanded T cell population. A subset of 4 subjects with glioblastoma multiforme (GBM) treated at 720 µg/kg of NT-I7 during adjuvant temozolomide in our study (NCT03687957) underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of peripheral blood. CITE-seq with TCR clonotype sequencing (10x Genomics 5’) permitted simultaneous tracking of the T cell phenotype at the protein level, individual T cell clones, and gene expression dynamics between 4 timepoints: pre-treatment, and post-treatment weeks 1, 2, and 13. Here, we report the results of differential expression analyses between timepoints and patient-specific differences in T cell clonotype expansions. Both CD4+ and CD8+ T cells in our study patients had increased expression of T cell activation markers LTB, CISH, and CD74. CD8+ T cell subsets demonstrated additional elevations in genes important in cytotoxic function (granzymes (GZMA, GZMB) and perforin (PRF1)), MHC Class II expression (HLA-DRA, HLA-DRB1), and AP-1 transcription factor subunits (JUND, JUN, JUNB, FOS, FOSB). After excluding known viral antigen clonotypes, 2 of 4 patients demonstrated TCR clonotype expansion. Notably, nearly all the expanded TCR clonotypes were found within CD8+ T cells, but not CD4+ T cells. NT-I7 promotes activation, cytotoxic function, and selective clonotype expansion of CD8+ T cells in patients with HGG. This, combined with its role in increased CD4+ T cell counts, suggests NT-I7 may be a powerful tool to restore immune function after treatment-associated lymphopenia in HGG.

Abstract BACKGROUND Lymphopenia, a common finding in patients with high-grade gliomas (HGGs), is associated with poor outcomes including worse survival, fatigue, and in some cases, increased opportunistic infections. Interleukin-7 (IL-7) is a cytokine crucial for lymphocyte survival and proliferation. We previously demonstrated long-acting IL-7 (rhIL-7-hyFc, efineptakin alfa, NT-I7) increases absolute lymphocyte counts (ALCs) and extends survival in preclinical murine models of HGG. Here, we report results using rhIL-7-hyFc to effectively reduce lymphopenia in patients with HGG. METHODS We performed a phase II randomized, placebo-controlled, double-blind study of rhIL-7-hyFc in patients with newly diagnosed HGGs (82% IDH-wildtype glioblastoma in both groups). Patients received rhIL-7-hyFc (720mcg/kg) or placebo one-week post-radiation and temozolomide, with subsequent doses at weeks 13, 25, and 37. The primary endpoint was ALC four weeks post-initial administration; exploratory endpoints included CD4 count, progression-free survival (PFS), and overall survival (OS). RESULTS We enrolled 22 patients (median age 59.5 years, range 33-74; 40.9% female) with HGG. Both groups exhibited comparable demographics, including MGMT methylation status, and received a median of 2 doses of rhIL-7-hyFc or placebo. At 4 weeks, the pre-specified primary endpoint, ALC significantly increased relative to baseline in the treatment group, compared to placebo (median percent change=148% vs. 16.7%, p=0.007), as did CD4 count (121% vs. 10.5%, p=0.011). There were no treatment-related grade 4/5 adverse events. Exploratory endpoints of PFS and OS will be reported at time of conference. CONCLUSIONS rhIL-7-hyFc is safe and effective in improving ALC and CD4 count in patients with newly diagnosed HGG undergoing temozolomide and concurrent radiation therapy. This study was powered to meet the primary endpoint; larger trials will likely be required to demonstrate clinical efficacy. Nevertheless, these data support the continued exploration of rhIL-7-hyFc as an adjunct to standard therapy to mitigate lymphopenia and potentially extend survival for patients with HGG.

Abstract BACKGROUND Glioblastoma (GBM), one of the deadliest primary tumors of the central nervous system, is characterized by a highly immunosuppressive tumor microenvironment (TME). Tumor-associated macrophages and microglia (TAMs) are a dominant population of immune cells in the GBM TME and substantially contribute to immunosuppression, tumor progression, and treatment resistance. Elucidating the molecular mechanisms underlying TAM behavior is crucial to the development of effective immuno-therapeutic strategies for treating GBM. The adaptor protein, Caspase Recruitment Domain-containing protein 9 (CARD9) is primarily expressed in myeloid cells and its activation triggers assembly of the CARD9-BCL10-MALT1 (CBM) complex, which then promotes activation of NF-κB and/or MAPK pathways. The aim of this study was to investigate the role of the CBM complex in the GBM TME and assess its potential as a therapeutic target. METHODS/RESULTS In silico analyses revealed that GBM tumors demonstrate increased CARD9 and MALT1 expression in comparison to non-tumor brain tissue, with CARD9 expressed only within the myeloid compartment. Using co-culture, scratch wound, and transwell assays, we found that GBM tumor cells induce macrophage M2 polarization and migration via a mechanism that is dependent on CARD9/MALT1 signaling within macrophages. scRNA-Seq and flow-cytometric analysis of orthotopic syngeneic immunocompetent GBM tumor models revealed that the GBM TME is dominated by macrophages. Disruption of MALT1 protease activity within the TME leads to reduced tumor infiltration by immunosuppressive TAMs, remodeling of TAMs toward an immunoreactive anti-tumor phenotype, and improved survival of GBM-bearing mice. Additionally, we found that tumor growth was abrogated when GBM-bearing mice were treated with either a MALT1-protease inhibitor or a CARD9 inhibitor. CONCLUSIONS Our study suggests that blockade of the CARD9-MALT1 signaling axis impairs GBM-induced macrophage recruitment and M2 polarization and inhibits tumor progression. These findings nominate the CARD9-MALT1 signaling axis as a new target for TAM-directed immunotherapy for the treatment of GBM.

Context.— Cardiac and pulmonary allograft recipients represent a unique population, frequently interacting with support groups and exhibiting intense curiosity about their pathology. Like other solid organ transplant patients, they have enduring and frequent interaction with the laboratory for routine allograft surveillance. Objective.— To address patient requests to understand what happens to their explanted organ and to better understand their disease while simultaneously improving awareness of pathologists’ roles in their continuing care. Design.— At routine follow-up appointments, transplant nurse coordinators offer each allograft recipient the opportunity to interact with a pathologist in our “ On My Path ” program. Organ viewing occurs in a private setting, in a specialized room. Relevant pathology is discussed, and questions are answered, with documentation in the medical record. The patient is subsequently gifted a 3-dimensional model of their explanted organ. Transplant coordinators were surveyed for their feedback on the experience. Results.— One hundred fifty-eight interactions have been documented (2017–2022) at our institution, including patients who underwent cardiac transplant (96, 61%), single or bilateral lung transplant (54, 34%), or combination lung and heart transplant (8, 5%). Transplant coordinators reported an increase in patient understanding of their disease and emotional closure related to the disease through the On My Path program. Conclusions.— Pathologists providing direct patient care is a feasible model that addresses currently unmet desires of the transplant population to better understand their pathology. Providing a 3-dimensional model helps to empower patients and drives satisfaction. These interactions also improve awareness about pathology as a discipline and its importance in the continued care of transplant recipients.

ABSTRACT Background Nab ‐sirolimus (ABI‐009, nab ‐rapamycin; Aadi Bioscience Inc. [Aadi]) is a human albumin‐bound form of sirolimus nanoparticles, a potent mTOR inhibitor. This phase I trial was conducted to define dose‐limiting toxicities (DLT), maximum tolerated or recommended phase II dose (MTD/RP2D), and pharmacokinetics of Nab ‐sirolimus in combination with temozolomide and irinotecan. Methods Using a rolling 6 design, Nab ‐sirolimus was administered intravenously (IV) on days (D) 1 and 8 of cycle (C) 1. In subsequent cycles, Nab ‐sirolimus was administered D1 and D8 in combination with temozolomide (125 mg/m 2 /dose, maximum 250 mg/dose) and irinotecan (90 mg/m 2 /dose) orally, daily on D1–5. Cycle duration was 21 days. Three dose levels (DL) of Nab ‐sirolimus were investigated (DL1: 35 mg/m 2 /dose, DL‐1: 20 mg/m 2 /dose, and DL‐2: 15 mg/m 2 /dose). The observation period for estimating the MTD/RP2D was defined by cycles 1 and 2. Results Thirty‐three patients were enrolled, 32 were eligible. Dose determination included 17 evaluable patients, median (range) age 12 (2–20) years and six additional patients were enrolled (four evaluable for toxicity) on a pharmacokinetic cohort. C1 or C2 DLTs were primarily thrombocytopenia including 2/5 patients at DL1, 2/6 patients at DL‐1, and 1/6 patients at DL‐2. One patient (DL1) with Ewing Sarcoma had a partial response and remained on study for 35 cycles. Rapamycin clearance was dose dependent. Irinotecan clearance and its active metabolite SN‐38 exposure were not affected by coadministration with Nab ‐sirolimus. Conclusion The MTD for Nab ‐sirolimus was 15 mg/m 2 /dose IV on D1 and D8 in combination with temozolomide 125 mg/m 2 /dose and oral irinotecan 90 mg/m 2 /dose daily for 5 days during 21D cycles. Trial Registration ClinicalTrials.gov identifier NCT02975882

Background/Objectives: Our objective was to describe the use of intraoperative radiation therapy (IORT) for the treatment of recurrent/persistent cervical or endometrial cancer and assess predictors of postoperative complications and 3-year mortality. Methods: In this multi-site retrospective study, data were abstracted for recurrent/persistent endometrial or cervical cancer patients who underwent IORT from June 2004 to May 2021. Complications were graded on the six-point Accordion scale. Variables associated with complications were analyzed with univariate logistic regression, while variables associated with death within 3 years were analyzed with Cox proportional hazards modeling. Survival was analyzed with the Kaplan–Meier method. Results: Eighty patients had planned IORT for recurrent/persistent endometrial (n = 35) or cervical cancer (n = 45). The mean age of the cohort was 56.8 years (SD = 13.7), and the median disease-free interval from primary disease to recurrence was 20.0 months (IQR 10.0–63.1). The overall survival at 3 years was 48.6% (95% CI: 38.3–61.6%) with a median survival of 2.8 years. Within 30 days postoperative, 16 patients (20.1%) had grade 3–5 complications and one death (1.3%) occurred. Factors associated with grade 3+ complication included ECOG PS 2–3 (OR 18.00, p = 0.04), neoadjuvant chemotherapy and/or immunotherapy (OR 6.98, p < 0.01), and pelvic sidewall involvement (OR 8.80, p = 0.04). Factors associated with death within 3 years of surgery included ECOG PS 2–3 (HR 8.97, p < 0.01), neoadjuvant chemotherapy and/or immunotherapy (HR 2.34, p = 0.03), whether exenteration was performed (HR 2.64, p = 0.01), and positive resection margin (HR 3.37, p < 0.01). Conclusions: In well-selected patients, IORT is a feasible and safe option for the treatment of recurrent/persistent gynecologic malignancy with an appreciable survival benefit.