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Treatment of hematologic malignancies with patient-derived anti-CD19 chimeric antigen receptor (CAR) T-cells has demonstrated long-term remissions for patients with otherwise treatment-refractory advanced leukemia and lymphoma. Conversely, CAR T-cell treatment of solid tumors, including advanced gastric cancer (GC), has proven more challenging due to on-target off-tumor toxicities, poor tumor T-cell infiltration, inefficient CAR T-cell expansion, immunosuppressive tumor microenvironments, and demanding preconditioning regimens. We report the exceptional results of autologous Claudin18.2-targeted CAR T cells (CT041) in a patient with metastatic GC, who had progressed on four lines of combined systemic chemotherapy and immunotherapy. After two CT041 infusions, the patient had target lesion complete response and sustained an 8-month overall partial response with only minimal ascites. Moreover, tumor-informed circulating tumor DNA (ctDNA) reductions coincided with rapid CAR T-cell expansion and radiologic response. No severe toxicities occurred, and the patient\'s quality of life significantly improved. This experience supports targeting Claudin18.2-positive GC with CAR T-cell therapy and helps to validate ctDNA as a biomarker in CAR T-cell therapy. Clinical Insight: Claudin18.2-targeted CAR T cells can safely provide complete objective and ctDNA response in salvage metastatic GC.

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OBJECTIVE: To establish and characterize a diverse library of head and neck squamous cell cancer (HNSCC) cultures using conditional reprogramming (CR).
METHODS: Patients enrolled on an IRB-approved protocol to generate tumor cell cultures using CR methods. Tumor and blood samples were collected and clinical information was recorded. Successful CR cultures were validated against banked reference tumors with short tandem repeat genotyping. Cell morphology was archived with photodocumentation. Clinical and demographic factors were evaluated for associations with successful establishment of CR culture. Human papilloma virus (HPV) genotyping, clonogenic survival, MTT assays, spheroid growth, and whole exome sequencing were carried out in selected cultures.
RESULTS: Forty four patients were enrolled, with 31 (70%) successful CR cultures, 32% derived from patients who identified as Black and 61% as Hispanic. All major head and neck disease sites were represented, including 15 (48%) oral cavity and 8 (26%) p16-positive oropharynx cancers. Hispanic ethnicity and first primary tumors (vs. second primary or recurrent tumors) were significantly associated with successful CR culture. HPV expression was conserved in CR cultures, including CR-024, which carried a novel HPV-69 serotype. CR cultures were used to test cisplatin responses using MTT assays. Previous work has also demonstrated these models can be used to assess response to radiation and can be engrafted in mouse models. Whole exome sequencing demonstrated that CR cultures preserved tumor mutation burden and driver mutations.
CONCLUSIONS: CR culture is highly successful in propagating HNSCC cells. This study included a high proportion of patients from underrepresented minority groups.
METHODS: Not Applicable Laryngoscope, 134:2748-2756, 2024.

Targeted immunotherapy with monoclonal antibodies (mAbs) is an effective and safe method for the treatment of malignancies. Development of mAbs with improved cytotoxicity, targeting new and known tumor-associated antigens, therefore continues to be an active research area. We reported that Dickkopf-1 (DKK1) is a good target for immunotherapy of human cancers based on its wide expression in different cancers but not in normal tissues. As DKK1 is a secreted protein, mAbs binding directly to DKK1 have limited effects on cancer cells in vivo.
The specificity and antibody-binding capacity of DKK1-A2 mAbs were determined using indirect ELISA, confocal imaging, QIFIKIT antibody-binding capacity and cell surface binding assays. The affinity of mAbs was determined using a surface plasmon resonance biosensor. A flow cytometry-based cell death was performed to detect tumor cell apoptosis. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays were used to evaluate the ability of DKK1-A2 mAbs to mediate ADCC and CDC activities against tumor cells in vitro. Flow cytometry data were collected with an FACSymphony A3 cell analyzer and analyzed with FlowJo V.10.1 software. Human cancer xenograft mouse models were used to determine the in vivo therapeutic efficacy and the potential safety and toxicity of DKK1-A2 mAbs. In situ TUNEL assay was performed to detect apoptosis in tumors and mouse organs.
We generated novel DKK1-A2 mAbs that recognize the DKK1 P20 peptide presented by human HLA-A*0201 (HLA-A2) molecules (DKK1-A2 complexes) that are naturally expressed by HLA-A2+DKK1+ cancer cells. These mAbs directly induced apoptosis in HLA-A2+DKK1+ hematologic and solid cancer cells by activating the caspase-9 cascade, effectively lysed the cancer cells in vitro by mediating CDC and ADCC and were therapeutic against established cancers in their xenograft mouse models. As DKK1 is not detected in most human tissues, DKK1-A2 mAbs neither bound to or killed HLA-A2+ blood cells in vitro nor caused tissue damage in tumor-free or tumor-bearing HLA-A2-transgenic mice.
Our study suggests that DKK1-A2 mAbs may be a promising therapeutic agent to treat human cancers.

OBJECTIVE: Open science is a movement and set of practices to conduct research more transparently. Implementing open science will significantly improve public access and supports equity. It also has the potential to foster innovation and reduce duplication through data and materials sharing. Here, we survey an international group of researchers publishing in cardiovascular journals regarding their perceptions and practices related to open science.
METHODS: We identified the top 100 \'Cardiology and Cardiovascular Medicine\' subject category journals from the SCImago journal ranking platform. This is a publicly available portal that draws from Scopus. We then extracted the corresponding author\'s name and email from all articles published in these journals between 1 March 2021 and 1 March 2022. Participants were sent a purpose-built survey about open science. The survey contained primarily multiple choice and scale-based questions for which we report count data and percentages. For the few text-based responses we conducted thematic content analysis.
RESULTS: 198 participants responded to our survey. Participants had a mean response of 6.8 (N=197, SD=1.8) on a 9-point scale with endpoints, not at all familiar (1) and extremely familiar (9), when indicating how familiar they were with open science. When asked about where they obtained open science training, most participants indicated this was done on the job self-initiated while conducting research (n=103, 52%), or that they had no formal training with respect to open science (n=72, 36%). More than half of the participants indicated they would benefit from practical support from their institution on how to perform open science practices (N=106, 54%). A diversity of barriers to each of the open science practices presented to participants were acknowledged. Participants indicated that funding was the most essential incentive to adopt open science.
CONCLUSIONS: It is clear that policy alone will not lead to the effective implementation of open science. This survey serves as a baseline for the cardiovascular research community\'s open science performance and perception and can be used to inform future interventions and monitoring.

Standard of care treatment of non-muscle invasive bladder cancer (NMIBC) with intravesical Bacillus Calmette Guérin (BCG) is associated with side effects, disease recurrence/progression and supply shortages. We recently showed in a phase I trial (NCT03421236) that intravesical instillation in patients with NMIBC with the maximal tolerated dose of Ty21a/Vivotif, the oral vaccine against typhoid fever, might have a better safety profile. In the present report, we assessed the immunogenicity of intravesical Ty21a in patients of the clinical trial that had received the maximal tolerated dose and compared it with data obtained in patients that had received standard BCG.
Urinary cytokines and immune cells of patients with NMIBC treated with intravesical instillations of Ty21a (n=13, groups A and F in NCT03421236) or with standard BCG in a concomitant observational study (n=12, UROV1) were determined by Luminex and flow cytometry, respectively. Serum anti-lipopolysaccharide Typhi antibodies and circulating Ty21a-specific T-cell responses were also determined in the Ty21a patients. Multiple comparisons of different paired variables were performed with a mixed-effect analysis, followed by Sidak post-test. Single comparisons were performed with a paired or an unpaired Student\'s t-test.
As compared with BCG, Ty21a induced lower levels of inflammatory urinary cytokines, which correlated to the milder adverse events (AEs) observed in Ty21a patients. However, both Ty21a and BCG induced a Th1 tumor environment. Peripheral Ty21a-specific T-cell responses and/or antibodies were observed in most Ty21a patients, pointing the bladder as an efficient local immune inductive site. Besides, Ty21a-mediated stimulation of unconventional Vδ2 T cells was also observed, which turned out more efficient than BCG. Finally, few Ty21a instillations were sufficient for increasing urinary infiltration of dendritic cells and T cells, which were previously associated with therapeutic efficacy in the orthotopic mouse model of NMIBC.
Ty21a immunotherapy of patient with NMIBC is promising with fewer inflammatory cytokines and mild AE, but induction of immune responses with possible antitumor potentials. Future phase II clinical trials are necessary to explore possible efficacy of intravesical Ty21a.

Although the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combinations are effective in advanced melanoma, it remains unclear whether their mechanisms of action overlap.
We used single cell (sc) RNA-seq, flow cytometry and IHC analysis of responding SM1, D4M-UV2 and B16 melanoma flank tumors and SM1 brain metastases to explore the mechanism of action of the anti-PD-1+LAG-3 and the anti-PD-1+CTLA-4 combination. CD4+ and CD8+ T cell depletion, tetramer binding assays and ELISPOT assays were used to demonstrate the unique role of CD4+T cell help in the antitumor effects of the anti-PD-1+LAG-3 combination.
The anti-PD-1+CTLA-4 combination was associated with the infiltration of FOXP3+regulatory CD4+ cells (Tregs), fewer activated CD4+T cells and the accumulation of a subset of IFNγ secreting cytotoxic CD8+T cells, whereas the anti-PD-1+LAG-3 combination led to the accumulation of CD4+T helper cells that expressed CXCR4, TNFSF8, IL21R and a subset of CD8+T cells with reduced expression of cytotoxic markers. T cell depletion studies showed a requirement for CD4+T cells for the anti-PD-1+LAG-3 combination, but not the PD-1-CTLA-4 combination at both flank and brain tumor sites. In anti-PD-1+LAG-3 treated tumors, CD4+T cell depletion was associated with fewer activated (CD69+) CD8+T cells and impaired IFNγ release but, conversely, increased numbers of activated CD8+T cells and IFNγ release in anti-PD-1+CTLA-4 treated tumors.
Together these studies suggest that these two clinically relevant immune checkpoint inhibitor (ICI) combinations have differential effects on CD4+T cell polarization, which in turn, impacted cytotoxic CD8+T cell function. Further insights into the mechanisms of action/resistance of these clinically-relevant ICI combinations will allow therapy to be further personalized.

Tertiary lymphoid structures (TLS) are organized aggregates of immune cells that develop postnatally in non-lymphoid tissues and are associated with pathological conditions. TLS typically comprise B-cell follicles containing and are encompassed by T- cell zones and dendritic cells. The prognostic and predictive value of TLS in the tumor microenvironment (TME) as potential mediators of antitumor immunity have gained interest. However, the precise relationship between localization and maturation of TLS and the clinical outcome of their presence in clear cell renal cell carcinoma (ccRCC) is yet to be elucidated.
Immunohistochemistry and multispectral fluorescence were used to evaluate the TLS heterogeneity along with TME cell-infiltrating characterizations. A thorough investigation of the prognostic implications of the TLS heterogeneity in 395 patients with ccRCC from two independent cohorts was conducted. Associations between TLS heterogeneity and immunologic activity were assessed by quantifying the immune cell infiltration.
Infiltrated TLS were identified in 34.2% of the ccRCC samples (N=395). These TLS were found to be tumor-proximal, tumor-distal, or both in 37.8%, 74.1%, and 11.9% of the TLS-positive cases, respectively. A higher proportion of early TLS was found in tumor-distal TLS (p=0.016), while tumor-proximal TLS primarily comprised secondary follicle-like structures (p=0.004). In the main study cohort (Fudan University Shanghai Cancer Center, N=290), Kaplan-Meier analyses revealed a significant correlation between the presence of tumor-proximal TLS and improved progression-free survival (PFS, p<0.001) and overall survival (OS, p=0.002). Conversely, the presence of tumor-distal TLS was associated with poor PFS (p=0.02) and OS (p=0.021). These findings were further validated in an external validation set of 105 patients with ccRCC. Notably, the presence of mature TLS (namely secondary follicle-like TLS, with CD23+ germinal center) was significantly associated with better clinical outcomes in patients with ccRCC. Furthermore, novel nomograms incorporating the presence of tumor-proximal TLS demonstrated remarkable predictability for the 8-year outcomes of resected ccRCC (area under the curve >0.80). Additionally, ccRCC samples with tumor-distal TLS enriched with primary follicle-like TLS exhibited higher programmed death-ligand 1 tumor-associated macrophages levels and regulatory T cells infiltration in the tumor-distal region, indicative of a suppressive TME.
This study for the first time elucidates the impact of TLS localization and maturation heterogeneities on the divergent clinical outcomes of ccRCC. The findings reveal that most TLS in ccRCC are located in the tumor-distal area and are associated with immature, immunosuppressive characterizations. Furthermore, our findings corroborate previous research demonstrating that tumor-proximal TLS were associated with favorable clinical outcomes.

OBJECTIVE: Tricuspid regurgitation (TR) is a prevalent valve disease associated with significant morbidity and mortality. We aimed to apply machine learning (ML) to assess risk stratification in patients with ≥moderate TR.
METHODS: Patients with ≥moderate TR on echocardiogram between January 2005 and December 2016 were retrospectively included. We used 70% of data to train ML-based survival models including 27 clinical and echocardiographic features to predict mortality over a 3-year period on an independent test set (30%). To account for differences in baseline comorbidities, prediction was performed in groups stratified by increasing Charlson Comorbidity Index (CCI). Permutation feature importance was calculated using the best-performing model separately in these groups.
RESULTS: Of 13 312 patients, mean age 72 ± 13 years and 7406 (55%) women, 7409 (56%) had moderate, 2646 (20%) had moderate-severe and 3257 (24%) had severe TR. The overall performance for 1-year mortality by 3 ML models was good, c-statistic 0.74-0.75. Interestingly, performance varied between CCI groups, (c-statistic = 0.774 in lowest CCI group and 0.661 in highest CCI group). The performance decreased over 3-year follow-up (average c-index 0.78). Furthermore, the top 10 features contributing to these predictions varied slightly with the CCI group, the top features included heart rate, right ventricular systolic pressure, blood pressure, diuretic use and age.
CONCLUSIONS: Machine learning of common clinical and echocardiographic features can evaluate mortality risk in patients with TR. Further refinement of models and validation in prospective studies are needed before incorporation into the clinical practice.