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Abstract Background Inflammatory regulatory T cells (Tregs) are one of the hallmarks of therapy resistance. Yet mechanisms leading to inflammatory Treg development and its contribution to inflammatory bowel disease (IBD) are unexplored. Methods Tregs derived from healthy PBMCS were characterized by immune and mitochondrial phenotyping. Transcripts were validated in dataset from IBD intestinal lesions. Wildtype (WT) and IL21 receptor-deleted (Il21r−/−) Tregs were examined in murine models of CD4+ T cell-induced colitis. Results Through in vitro cytokine challenge of Tregs and subsequent analysis, we found that IL21 induces Tregs co-expressing TNF, IFNγ and IL17. Transcriptomic and metabolomic integration revealed upregulation of mRNA and metabolites associated with glycolysis, and amino acid, nucleotide and lipid metabolism. Moreover, IL21-induced metabolic transcripts were more enriched in Treg cluster within scRNA-seq dataset from intestinal lesions of anti-TNF non-responders vs. non-responders. Mechanistically, cell imaging and real-time bioenergetic analysis revealed disruption to mitochondria-ER ultrastructure and function, coinciding with glycogen synthase kinase 3β (GSK3β) activation and glycolytic switch rather than pyruvate-mediated oxidative phosphorylation. GSK3β inhibition or methyl pyruvate supplementation rescued IL21-induced metabolic and inflammatory phenotypes. In contrast to WT Tregs, murine Il21r−/− Treg transfer efficiently prevented and rescued CD4+CD45Rbhigh T cell-induced colitis. Conclusions IL21-induced metabolic dysfunction can incapacitate Treg immunosuppressive function. Desensitizing Tregs to IL21 through various therapeutic approaches can alleviate IBD. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) K01DK124358, the Pilot & Feasibility Award by the Center for Cell Signaling in Gastroenterology (P30DK084567), and the Mayo Clinic Center for Biomedical Discovery Career Development Award, and David F. and Margaret T. Grohne Cancer Immunology and Immunotherapy (to AOB).

PURPOSE BMI affects breast cancer risk and prognosis. In contrast to cytotoxic chemotherapy, CDK4/6 inhibitors are given at a fixed dose, irrespective of BMI or weight. This preplanned analysis of the global randomized PALLAS trial investigates the impact of BMI on the side-effect profile, treatment adherence, and efficacy of palbociclib. METHODS Patients were categorized at baseline according to WHO BMI categories. Neutropenia rates were assessed with univariable and multivariable logistic regression. Time to early discontinuation of palbociclib was analyzed with Fine and Gray competing risk models. Unstratified Cox models were used to investigate the association between BMI category and time to invasive disease-free survival (iDFS). 95% CIs were derived. RESULTS Of 5,698 patients included in this analysis, 68 (1.2%) were underweight, 2,082 (36.5%) normal weight, 1,818 (31.9%) overweight, and 1,730 (30.4%) obese at baseline. In the palbociclib arm, higher BMI was associated with a significant decrease in neutropenia (unadjusted odds ratio for 1-unit change, 0.93; 95% CI, 0.91 to 0.94; adjusted for age, race ethnicity, region, chemotherapy use, and Eastern Cooperative Oncology Group at baseline, 0.93; 95% CI, 0.92 to 0.95). This translated into a significant decrease in treatment discontinuation rate with higher BMI (adjusted hazard ratio [HR] for 10-unit change, 0.75; 95% CI, 0.67 to 0.83). There was no significant improvement in iDFS with the addition of palbociclib to ET in any weight category (normal weight HR, 0.84; 95% CI, 0.63 to 1.12; overweight HR, 1.10; 95% CI, 0.82 to 1.49; and obese HR, 0.95; 95% CI, 0.69 to 1.30) in this analysis early in follow-up (31 months). CONCLUSION This preplanned analysis of the PALLAS trial demonstrates a significant impact of BMI on side effects, dose reductions, early treatment discontinuation, and relative dose intensity. Additional long-term follow-up will further evaluate whether BMI ultimately affects outcome.

AbstractIntroductionLeft bundle branch area pacing (LBBP) is a novel conduction system pacing method to achieve effective physiological pacing and an alternative to cardiac resynchronization therapy (CRT) with biventricular pacing (BVP) for patients with heart failure with reduced ejection fraction (HFrEF). We conduted this meta‐analysis and systemic review to review current data comparing BVP and LBBP in patients with HFrEF and indications for CRT.MethodsWe searched PubMed/Medline, Web of Science, and Cochrane Library from the inception of the database to November 2022. All studies that compared LBBP with BVP in patients with HFrEF and indications for CRT were included. Two reviewers performed study selection, data abstraction, and risk of bias assessment. We calculated risk ratios (RRs) with the Mantel–Haenszel method and mean difference (MD) with inverse variance using random effect models. We assessed heterogeneity using the I2 index, with I2 > 50% indicating significant heterogeneity.ResultsTen studies (9 observational studies and 1 randomized controlled trial; 616 patients; 15 centers) published between 2020 and 2022 were included. We observed a shorter fluoroscopy time (MD: 9.68, 95% confidence interval [CI]: 4.49–14.87, I2 = 95%, p < .01, minutes) as well as a shorter procedural time (MD 33.68, 95% CI: 17.80–49.55, I2 = 73%, p < .01, minutes) during the implantation of LBBP CRT compared to conventional BVP CRT. LBBP was shown to have a greater reduction in QRS duration (MD 25.13, 95% CI: 20.06–30.20, I2 = 51%, p < .01, milliseconds), a greater left ventricular ejection fraction improvement (MD: 5.80, 95% CI: 4.81–6.78, I2 = 0%, p < .01, percentage), and a greater left ventricular end‐diastolic diameter reduction (MD: 2.11, 95% CI: 0.12–4.10, I2 = 18%, p = .04, millimeter). There was a greater improvement in New York Heart Association function class with LBBP (MD: 0.37, 95% CI: 0.05–0.68, I2 = 61%, p = .02). LBBP was also associated with a lower risk of a composite of heart failure hospitalizations (HFH) and all‐cause mortality (RR: 0.48, 95% CI: 0.25–0.90, I2 = 0%, p = .02) driven by reduced HFH (RR: 0.39, 95% CI: 0.19–0.82, I2 = 0%, p = .01). However, all‐cause mortality rates were low in both groups (1.52% vs. 1.13%) and similar (RR: 0.98, 95% CI: 0.21–4.68, I2 = 0%, p = .87).ConclusionThis meta‐analysis of primarily nonrandomized studies suggests that LBBP is associated with a greater improvement in left ventricular systolic function and a lower rate of HFH compared to BVP. There was uniformity of these findings in all of the included studies. However, it would be premature to conclude based solely on the current meta‐analysis alone, given the limitations stated. Dedicated, well‐designed, randomized controlled trials and observational studies are needed to elucidate better the comparative long‐term efficacy and safety of LBBP CRT versus BIV CRT.

Abstract The Response Assessment in Neuro-Oncology (RANO) criteria for high-grade gliomas (RANO-HGG) and low-grade gliomas (RANO-LGG) were developed to improve the reliability of response assessment in glioma trials. Over time some limitations of these criteria were identified, and uncertainty emerged regarding integrating features of the modified RANO (mRANO) or the immunotherapy RANO (iRANO) criteria. Informed by data from a cohort of glioblastoma patients and other evaluations of RANO criteria that allowed evaluation of features of the different criteria, we propose updates to the RANO criteria (RANO 2.0). Based on the 2021 WHO classification of gliomas, we recommend a standard set of criteria for both high and low-grade gliomas, to be used for all trials regardless of the treatment modalities being evaluated. In the newly diagnosed setting, the post-radiotherapy MRI, rather than the post-surgical MRI, will be used as the baseline for future comparison. Since the incidence of pseudoprogression is high in the 12 weeks following radiotherapy, continuation of treatment and confirmation of progression during this period with a repeat MRI, or histopathologic evidence of unequivocal recurrent tumor, is required to define tumor progression. However, confirmation scans are not mandatory after this period nor for recurrent tumors. For treatments with a high likelihood of pseudoprogression, mandatory confirmation of progression with a repeat MRI is an option. The primary measurement remains the maximum cross-sectional area of tumor (2-dimensional) but volumetric measurements are an option. For IDH-wildtype glioblastoma, the non-enhancing disease will no longer be evaluated. In IDH-mutated tumors with a significant non-enhancing component, clinical trials may require evaluating both the enhancing and non-enhancing tumor components for response assessment.

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options, due in part to the low blood-brain-barrier (BBB) penetrance of many anti-cancer compounds. Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term cell cultures established from four patient-derived xenografts with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by RNA sequencing and mass spectrometry (proteomics) to characterize and compare cargo profiles for each condition. We found that depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Mass spectrometry of protein cargo further demonstrated both upregulated and downregulated peptide abundances with treatment compared to vehicle. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow for the selective interrogation of patient GBM-derived EVs, we have simultaneously pursued the identification of biomarkers to allow for immunoprecipitation of EVs from patient biofluids. Potential biomarkers, including PTPRZ1, B7H3, and IL13RA2, were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.

Background The tumor lysate, particle-loaded, dendritic cell (TLPLDC) vaccine is made by ex vivo priming matured autologous dendritic cells (DCs) with yeast cell wall particles (YCWPs) loaded with autologous tumor lysate (TL). The tumor lysate, particle only (TLPO) vaccine uses autologous TL-loaded YCWPs coated with silicate for in vivo DC loading. Here we report the 36-month prespecified analyses of this prospective, randomized, double-blind trial investigating the ability of the TLPO and TLPLDC (±granulocyte-colony stimulating factor (G-CSF)) vaccines to prevent melanoma recurrence in high-risk patients. Methods Patients with clinically disease-free stage III/IV melanoma were randomized 2:1 initially to TLPLDC versus placebo (n=124) and subsequently TLPO versus TLPLDC (n=63). All patients were randomized and blinded; however, the placebo control arm was replaced in the second randomization scheme with another novel vaccine; some analyses in this paper therefore reflect a combination of the two randomization schemes. Patients receiving the TLPLDC vaccine were further divided by their method of DC harvest (with or without G-CSF pretreatment); this was not randomized. The use of standard of care checkpoint inhibitors was not stratified between groups. Safety was assessed and Kaplan-Meier and log-rank analyses compared disease-free (DFS) and overall survival (OS). Results After combining the two randomization processes, a total of 187 patients were allocated between treatment arms: placebo (n=41), TLPLDC (n=103), or TLPO (n=43). The allocation among arms created by the addition of patients from the two separate randomization schemes does not reflect concurrent randomization among all treatment arms. TLPLDC was further divided by use of G-CSF in DC harvest: no G-CSF (TLPLDC) (n=47) and with G-CSF (TLPLDC+G) (n=56). Median follow-up was 35.8 months. Only two patients experienced a related adverse event ≥grade 3, one each in the TLPLDC+G and placebo arms. DFS was 27.2% (placebo), 55.4% (TLPLDC), 22.9% (TLPLDC+G), and 60.9% (TLPO) (p<0.001). OS was 62.5% (placebo), 93.6% (TLPLDC), 57.7% (TLPLDC+G), and 94.6% (TLPO) (p=0.002). Conclusions The TLPO and TLPLDC (without G-CSF) vaccines were associated with improved DFS and OS in this clinical trial. Given production and manufacturing advantages, the efficacy of the TLPO vaccine will be confirmed in a phase 3 trial. Trial registration number NCT02301611 .

AbstractBackgroundCOVID‐19 convalescent plasma (CCP) is an important therapeutic option for outpatients at high risk of hospitalization from SARS‐CoV‐2 infection. We assessed the safety of outpatient CCP transfusions administered during clinical trials.Study Design and MethodsWe analyzed data pertaining to transfusion‐related reactions from two randomized controlled trials in the U.S. that evaluated the efficacy of CCP versus control plasma in various ambulatory settings. Multivariable logistic regression was used to assess whether CCP was associated with transfusion reactions, after adjusting for potential confounders.ResultsThe combined study reported 79/1351 (5.9%) adverse events during the transfusion visit, with the majority 62/1351 (4.6%) characterized by mild, allergic‐type findings of urticaria, and/or pruritus consistent with minor allergic transfusion reactions; the other reported events were attributed to the patients' underlying disease, COVID‐19, or vasovagal in nature. We found no difference in the likelihood of allergic transfusion reactions between those receiving CCP versus control plasma (adjusted odds ratio [AOR], 0.75; 95% CI, 0.43–1.31). Risk of urticaria and/or pruritus increased with a pre‐existing diagnosis of asthma (AOR, 2.33; 95% CI, 1.16–4.67). We did not observe any CCP‐attributed antibody disease enhancement in participants with COVID‐19 or increased risk of infection. There were no life‐threatening severe transfusion reactions and no patients required hospitalization related to transfusion‐associated complications.DiscussionOutpatient plasma administration was safely performed for nearly 1400 participants. CCP is a safe therapeutic option for outpatients at risk of hospitalization from COVID‐19.

AbstractBackground and ObjectivesWe assessed the accuracy of preoperative gallium‐68 DOTA‐Tyr3‐octreotate (DOTATATE) positron emission tomography (PET) imaging in estimating multifocality and nodal metastases of small bowel neuroendocrine tumors (sbNETs).MethodsA multicenter analysis was performed on patients with sbNETs who underwent preoperative DOTATATE PET imaging and surgical resection, with manual palpation of the entire length of the small bowel, between January 2016 and August 2022. Preoperative imaging reports and blinded secondary imaging reviews were compared to the final postoperative pathology reports. Descriptive statistics were applied.ResultsOne‐hundred and four patients met inclusion criteria. Pathology showed 53 (51%) patients had multifocal sbNETs and 96 (92%) had nodal metastases. The original preoperative DOTATATE PET imaging identified multifocal sbNET in 28 (27%) patients and lymph node (LN) metastases in 80 (77%) patients. Based on original radiology reports, sensitivity for multifocal sbNET identification was 45%, specificity was 92%, positive predictive value (PPV) was 86%, and negative predictive value (NPV) was 62%. For the identification of LN metastases, sensitivity was 82%, specificity was 88%, PPV was 99%, and NPV was 29%.ConclusionsAlthough DOTATATE PET imaging is specific and relatively accurate, sensitivity and NPV are insufficient to guide surgical planning. Preoperative use should not replace open palpation to identify additional synchronous lesions or to omit regional lymphadenectomy.

Objectives: Augmentation of rotator cuff repair in patients at risk for retear has gained popularity. These techniques have generally consisted of allograft and xenograft patches. Recently, use of an autograft biceps from the normally discarded portion after biceps tenodesis was described as a potential patch for rotator cuff repair augmentation. As an autograft, it is free and readily available. The original description prepared the patch by placing it through a commercial skin graft preparation station. This technique results in a significant decrease in tenocyte viability, however, and results in a patch that is difficult to work with. Recently, an autograft compression plate has been employed to use as a point of care processor for adapting the discarded long head of the biceps after tenodesis into an autograft patch. The purpose of this study was to evaluate the feasibility of this point of care graft preparation method, and to specifically evaluate its effect on tenocyte viability in patients undergoing patch augmentation for rotator cuff repair. Methods: We reviewed the medical records of consecutive patients who underwent biceps tenodesis for either rotator cuff repair or anatomic shoulder arthroplasty. After tenodesis, and after 27 mm of the long head of the biceps was secured for compression into the patch, the remaining length of normally discarded tendon was longitudinally split, resulting in 2 equal lengths of remnant tendon from the same zone of the tendon. One sample was sent to pathology with no preparation, and the other was prepared as a biceps autograft patch using the autograft compression plate according to the manufacturer’s recommendations. Once prepared, this second biceps sample was sent to pathology along with the first. The request was to evaluate for tenocyte viability. Pathologists were not told which sample was compressed. Records were reviewed to determine if biceps tendon samples were viable at the time of harvest, and whether the compression process affected a change between the first and second specimen. Results: 20 consecutive patients were reviewed. All 20 biceps produced usable patches. The remnant biceps samples that were sent to pathology revealed the following: Sixteen of the 20 patients had biceps tenocyte viability that were read out as completely normal in both the compressed and non compressed samples. Of the 4 patients that did show some degree of tenocyte necrosis or death, 3 of the patients had identical findings between the compressed and non-compressed samples. Only 1/20 (5%) of the patients showed a difference between the compressed sample and the non-compressed sample, with the compressed sample demonstrating small areas of “patchy necrosis” while the non compressed sample showed normal tendon in that specimen. Conclusions: Compression of the long head of the biceps results in an autograft patch that is a promising option in rotator cuff augmentation. The method of compression plating to process the long head of the biceps remnant into an augmentation patch appears to have no significant effect on tenocyte viability, suggesting that the patch has live tenocytes at the time of point of care augmentation.