Single-cell analysis of treatment-resistant prostate cancer: Implications of cell state changes for cell surface antigen-targeted therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)-a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single-cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to current and future antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

The Asian Fabry Cardiomyopathy High-Risk Screening Study 2 (ASIAN-FAME-2): Prevalence of Fabry Disease in Patients with Left Ventricular Hypertrophy.

Background: Fabry disease (FD) is a rare X-linked lysosomal storage disorder that commonly manifests cardiovascular complications. We aimed to assess the prevalence of FD in a Chinese population with left ventricular hypertrophy (LVH) whilst implementing a gender-specific screening approach. Methods: Patients with LVH, defined as a maximum thickness of the left ventricular septal/posterior wall ≥ 13 mm, were considered eligible. All patients with hypertrophic cardiomyopathy (HCM) were excluded. Plasma α-galactosidase (α-GLA) enzyme activity was assessed using a dried blood spot test. Males with low enzyme activity underwent genetic testing to confirm a diagnosis of FD whereas females were screened for both α-GLA and globotriaosylsphingosine concentration and underwent genetic analysis of the GLA gene only if testing positive for ≥1 parameter. Results: 426 unrelated patients (age = 64.6 ± 13.0 years; female: male = 113:313) were evaluated. FD was diagnosed in 3 unrelated patients (age = 69.0 ± 3.5 years, female: male = 1:2) and 1 related female subject (age = 43 years). Genetic analyses confirmed the late-onset cardiac variant GLA c.640-801G>A (n = 3) and the missense variant c.869T>C associated with classic FD (n = 1). Cardiac complications were the only significant findings associated with the late-onset c.640-801G>A mutation, manifesting as mild or severe concentric LVH. In contrast, the classic c.869T>C mutation FD exhibited multisystemic manifestations in addition to severe concentric LVH. Conclusions: The prevalence of FD is lower in Chinese patients with LVH when HCM is excluded. The pathological variant c.640-801G>A remains the most common cause of late-onset FD, while the detection of FD in females can be improved by utilizing a gender-specific screening method.

Early LAAO device migration after combined atrial fibrillation ablation and left atrial appendage occlusion.

The combined procedure of catheter ablation and percutaneous left atrial appendage occlusion for patients with atrial fibrillation has been shown to be safe and feasible using radiofrequency energy or cryoballoon.

Swallowed topical steroid therapy for eosinophilic oesophagitis in children: practical, evidence-based guidance by the BSPGHAN Eosinophilic Oesophagitis Working Group.

OBJECTIVE: To develop evidence-based guidance for topical steroid use in paediatric eosinophilic oesophagitis (pEoE) in the UK for both induction and maintenance treatment. METHODS: A systematic literature review using Cochrane guidance was carried out by the British Society of Paediatric Gastroenterology, Hepatology and Nutrition (BSPGHAN) Eosinophilic Oesophagitis (EoE) Working Group (WG) and research leads to determine the evidence base for preparation, dosing and duration of use of swallowed topical steroid (STS) formulations in EoE. Seven themes relating to pEoE were reviewed by the WG, alongside the Cochrane review this formed the evidence base for consensus recommendations for pEoE in the UK. We provide an overview of practical considerations including treatment regimen and dosing. Oral viscous budesonide (OVB) and, if agreed by local regulatory committees, orodispersible budesonide (budesonide 1 mg tablets) were selected for ease of use and with most improvement in histology. A practical 'how to prepare and use' OVB appendix is included. Side effects identified included candidiasis and adrenal gland suppression. The use of oral systemic steroids in strictures is discussed briefly. RESULTS: 2638 citations were identified and 18 randomised controlled trials were included. Evidence exists for the use of STS for induction and maintenance therapy in EoE, especially regarding histological improvement. Using the Appraisal of Guidelines, Research and Evaluation criteria, dosing of steroids by age (0.5 mg two times per day <10 years and 1 mg two times per day ≥10 years) for induction of at least 3 months was suggested based on evidence and practical consideration. Once histological remission is achieved, maintenance dosing of steroids appears to reduce the frequency and severity of relapse, as such a maintenance weaning regimen is proposed. CONCLUSION: A practical, evidence-based flow chart and guidance recommendations with consensus from the EoE WG and education and research representatives of BSPGHAN were developed with detailed practical considerations for use in the UK.

Olfactory neuroblastoma mimics molecular heterogeneity and lineage trajectories of small-cell lung cancer.

The olfactory epithelium undergoes neuronal regeneration from basal stem cells and is susceptible to olfactory neuroblastoma (ONB), a rare tumor of unclear origins. Employing alterations in Rb1/Trp53/Myc (RPM), we establish a genetically engineered mouse model of high-grade metastatic ONB exhibiting a NEUROD1+ immature neuronal phenotype. We demonstrate that globose basal cells (GBCs) are a permissive cell of origin for ONB and that ONBs exhibit cell fate heterogeneity that mimics normal GBC developmental trajectories. ASCL1 loss in RPM ONB leads to emergence of non-neuronal histopathologies, including a POU2F3+ microvillar-like state. Similar to small-cell lung cancer (SCLC), mouse and human ONBs exhibit mutually exclusive NEUROD1 and POU2F3-like states, an immune-cold tumor microenvironment, intratumoral cell fate heterogeneity comprising neuronal and non-neuronal lineages, and cell fate plasticity-evidenced by barcode-based lineage tracing and single-cell transcriptomics. Collectively, our findings highlight conserved similarities between ONB and neuroendocrine tumors with significant implications for ONB classification and treatment.

Single Cell Analysis of Treatment-Resistant Prostate Cancer: Implications of Cell State Changes for Cell Surface Antigen Targeted Therapies.

Targeting cell surface molecules using radioligand and antibody-based therapies has yielded considerable success across cancers. However, it remains unclear how the expression of putative lineage markers, particularly cell surface molecules, varies in the process of lineage plasticity, wherein tumor cells alter their identity and acquire new oncogenic properties. A notable example of lineage plasticity is the transformation of prostate adenocarcinoma (PRAD) to neuroendocrine prostate cancer (NEPC)--a growing resistance mechanism that results in the loss of responsiveness to androgen blockade and portends dismal patient survival. To understand how lineage markers vary across the evolution of lineage plasticity in prostate cancer, we applied single cell analyses to 21 human prostate tumor biopsies and two genetically engineered mouse models, together with tissue microarray analysis (TMA) on 131 tumor samples. Not only did we observe a higher degree of phenotypic heterogeneity in castrate-resistant PRAD and NEPC than previously anticipated, but also found that the expression of molecules targeted therapeutically, namely PSMA, STEAP1, STEAP2, TROP2, CEACAM5, and DLL3, varied within a subset of gene-regulatory networks (GRNs). We also noted that NEPC and small cell lung cancer (SCLC) subtypes shared a set of GRNs, indicative of conserved biologic pathways that may be exploited therapeutically across tumor types. While this extreme level of transcriptional heterogeneity, particularly in cell surface marker expression, may mitigate the durability of clinical responses to novel antigen-directed therapies, its delineation may yield signatures for patient selection in clinical trials, potentially across distinct cancer types.

Aveir VR real-world performance and chronic pacing threshold prediction using mapping and fixation electrical data.

AIMS: Aveir VR performance and predictors for its pacing threshold (PCT) in a real-world cohort were investigated. METHODS: Electrical measurements at various stages of an Aveir VR implant were prospectively collected. Predictors for 3-month PCT were studied. A retrospective cohort of consecutive 139 Micra implants was used to compare the PCT evolution. High PCT was defined as ≥1.5 V, using a pulse width of 0.4 ms for Aveir and 0.24 ms for Micra. Excellent PCT was defined as ≤0.5 V at the respective pulse width. RESULTS: Among the 123 consecutive Aveir VR implant attempts, 122 (99.2%) were successful. The majority were of advanced age (mean 79.7) and small body size (mean BSA 1.60). Two patients (1.6%) experienced complications, including one pericardial effusion after device reposition and one intraoperative device dislodgement. Eighty-eight patients reached a 3-month follow-up. Aveir 3-month PCT was correlated with impedance at mapping (P = 0.015), tether mode (P < 0.001), end-of-procedure (P < 0.001), and mapping PCT (P = 0.035), but not with PCTs after fixation (P > 0.05). Tether mode impedance >470 ohms had 88% sensitivity and 71% specificity in predicting excellent 3-month PCT. Although it is more common for Aveir to have high PCT at end of procedure (11.5% for Aveir and 2.2% for Micra, P = 0.004), the rate at 3 months was similar (2.3% for Aveir and 3.1% for Micra, P = 1.000). CONCLUSION: Aveir VR demonstrated satisfactory performance in this high-risk cohort. Pacing thresholds tend to improve to a greater extent than Micra after implantation. The PCT after fixation, even after a waiting period, has limited predictive value for the chronic threshold. Low-mapping PCT and high intraoperative impedance predict chronic low PCT.

Hypofractionated re-irradiation with bevacizumab for relapsed chemorefractory glioblastoma after prior high dose radiotherapy: a feasible option for patients with large-volume relapse.

PURPOSE: There remains no standard of care for patients with recurrent and chemorefractory glioblastoma. Re-irradiation (reRT) provides an additional management option. However, published series predominantly focus on small reRT volumes utilizing stereotactic hypofractionated regimens. Concerns regarding toxicity have limited utilisation of reRT for larger recurrences, however this may be mitigated with use of bevacizumab (BEV). METHODS AND MATERIALS: A prospective database of patients managed with the EORTC-NCIC (Stupp) protocol 60 Gy chemoradiotherapy protocol for glioblastoma between 2007 and 2021 was reviewed for those patients receiving reRT for chemorefractory relapse. Serial MRI and PET were used to establish true progression and exclude patients with pseudoprogression or radionecrosis from reRT. The primary endpoint was overall survival (OS) from date of reRT. Prognostic factors were also assessed. RESULTS: 447 patients managed for glioblastoma under the Stupp protocol were identified, of which 372 had relapsed and were thus eligible for reRT. 71 patients underwent reRT. Median relapse-free survival from diagnosis for the reRT and overall cohorts were similar at 11.6 months (95%CI:9.4-14.2) and 11.8 months (95%CI:9.4-14.2) respectively. 60/71 (85%) reRT patients had received BEV prior to reRT and continued concurrent BEV during reRT. Of the 11 patients not managed with BEV during reRT, 10 required subsequent salvage BEV. ReRT patients were younger (median 53 vs. 59 years, p < 0.001), had better performance status (86% vs. 69% ECOG 0-1, p = 0.002) and more commonly had MGMT promoter-methylated tumours (54% vs. 40%, p = 0.083) compared to non-reRT patients. Median reRT PTV volume was 135cm3 (IQR: 69-207cm3). Median OS from reRT to death was 7.1 months (95%CI:6.3-7.9). Patients aged < 50, 50-70 and > 70 years had post-reRT median OS of 7.7, 6.4 and 6.0 months respectively (p = 0.021). Median post-reRT survival was longer for patients with ECOG performance status 0-1 compared to 2-3 (8.1 vs. 6.3 months, p = 0.039). PTV volume, site of relapse, MGMT promoter-methylation status and extent of initial surgical resection were not associated with post-reRT survival. ReRT was well-tolerated. Out of the 6 patients (8%) admitted to hospital after reRT, only one was for reRT toxicity. This was a CTCAE grade 3 radiation necrosis event in a patient managed without prior BEV. CONCLUSION: Patients with recurrent glioblastoma who have been previously treated with 60 Gy radiotherapy have a meaningful survival benefit from large volume re-irradiation which is well tolerated. ReRT should not be ignored as a salvage treatment option in patients with chemorefractory progressive disease.

Role of CD38 in anti-tumor immunity of small cell lung cancer.

Introduction: Immune checkpoint blockade (ICB) with or without chemotherapy has a very modest benefit in patients with small cell lung cancer (SCLC). SCLC tumors are characterized by high tumor mutation burden (TMB) and low PD-L1 expression. Therefore, TMB and PD-L1 do not serve as biomarkers of ICB response in SCLC. CD38, a transmembrane glycoprotein, mediates immunosuppression in non-small cell lung cancer (NSCLC). In this brief report, we highlight the potential role of CD38 as a probable biomarker of immunotherapy response in SCLC. Methods: We evaluated the role of CD38 as a determinant of tumor immune microenvironment in SCLC with bulk and single-cell transcriptomic analyses and protein assessments of clinical samples and preclinical models, including CD38 in vivo blockade. Results: In SCLC clinical samples, CD38 levels were significantly correlated with the gene expression of the immunosuppressive markers FOXP3, PD-1 and CTLA-4. CD38 expression was significantly enhanced after chemotherapy and ICB treatment in SCLC preclinical models and clinical samples. A combination of cisplatin/etoposide, ICB, and CD38 blockade delayed tumor growth compared to cisplatin/etoposide. Conclusion: Our study provides a preliminary but important direction toward exploring CD38 as a potential biomarker of ICB response and CD38 blockade as a combination strategy for chemo-immunotherapy in SCLC.

Functional Characterisation of the ATOH1 Molecular Subtype Indicates a Pro-Metastatic Role in Small Cell Lung Cancer.

Molecular subtypes of Small Cell Lung Cancer (SCLC) have been described based on differential expression of transcription factors (TFs) ASCL1, NEUROD1, POU2F3 and immune-related genes. We previously reported an additional subtype based on expression of the neurogenic TF ATOH1 within our SCLC Circulating tumour cell-Derived eXplant (CDX) model biobank. Here we show that ATOH1 protein was detected in 7/81 preclinical models and 16/102 clinical samples of SCLC. In CDX models, ATOH1 directly regulated neurogenesis and differentiation programs consistent with roles in normal tissues. In ex vivo cultures of ATOH1-positive CDX, ATOH1 was required for cell survival. In vivo, ATOH1 depletion slowed tumour growth and suppressed liver metastasis. Our data validate ATOH1 as a bona fide oncogenic driver of SCLC with tumour cell survival and pro-metastatic functions. Further investigation to explore ATOH1 driven vulnerabilities for targeted treatment with predictive biomarkers is warranted.

Immune heterogeneity in small-cell lung cancer and vulnerability to immune checkpoint blockade.

Atezolizumab (anti-PD-L1), combined with carboplatin and etoposide (CE), is now a standard of care for extensive-stage small-cell lung cancer (ES-SCLC). A clearer understanding of therapeutically relevant SCLC subsets could identify rational combination strategies and improve outcomes. We conduct transcriptomic analyses and non-negative matrix factorization on 271 pre-treatment patient tumor samples from IMpower133 and identify four subsets with general concordance to previously reported SCLC subtypes (SCLC-A, -N, -P, and -I). Deeper investigation into the immune heterogeneity uncovers two subsets with differing neuroendocrine (NE) versus non-neuroendocrine (non-NE) phenotypes, demonstrating immune cell infiltration hallmarks. The NE tumors with low tumor-associated macrophage (TAM) but high T-effector signals demonstrate longer overall survival with PD-L1 blockade and CE versus CE alone than non-NE tumors with high TAM and high T-effector signal. Our study offers a clinically relevant approach to discriminate SCLC patients likely benefitting most from immunotherapies and highlights the complex mechanisms underlying immunotherapy responses.

Personalized cardiac resynchronization therapy guided by real-time electrocardiographic imaging for patients with non-left bundle branch block.

BACKGROUND: Patients with heart failure and a non-left bundle branch block (non-LBBB) QRS pattern have a limited response to biventricular pacing (BVP). OBJECTIVE: A personalized cardiac resynchronization therapy (CRT) implantation approach guided by real-time electrocardiographic imaging (ECGi) was studied. METHODS: Twenty patients with left ventricular ejection fraction (LVEF) ≤ 35%, QRS duration ≥ 120 ms, and non-LBBB [13 (65%) with right bundle branch block and 7 (35%) with intraventricular conduction delay] were recruited. During CRT implantation, right atrial, right ventricular, coronary sinus, His-bundle, and/or left bundle leads were inserted. The total activation time (TAT) with different pacing combinations were measured in real time during implantation by ECGi. The configuration producing the shortest TAT was chosen. Clinical response was defined as ≥1 New York Heart Association class improvement. Echocardiographic response was defined as left ventricular end-systolic volume reduction ≥ 15% and/or LVEF improvement ≥ 10% at 6 months. RESULTS: After ECGi-guided CRT implantation, LVEF improved from 26% ± 6% to 34% ± 11% (P < .01) and New York Heart Association class improved from 3.0 ± 0.5 to 2.0 ± 0.6 (P < .01). Both clinical and echocardiographic response rates were 70%. The ECGi approach resulted in better acute electrical resynchronization over BVP as measured by TAT reduction (40% vs 14%; P < .01). The percentage of TAT reduction was found to be a strong predictor for echocardiographic response (area under the curve for the receiver operating characteristic curve 0.91; 95% confidence interval 0.78-1.00). A strong positive correlation between percentage TAT reduction and percentage LVEF improvement (Pearson R = 0.70; P = .001) was found. CONCLUSION: ECGi-guided CRT implantation in patients with non-LBBB generates superior acute electrical resynchronization compared with BVP and is associated with favorable clinical and echocardiographic outcomes.

Robustness of magnetic resonance imaging and positron emission tomography radiomic features in prostate cancer: Impact on recurrence prediction after radiation therapy.

Background and purpose: Radiomic features from MRI and PET are an emerging tool with potential to improve prostate cancer outcomes. However, feature robustness due to image segmentation variations is currently unknown. Therefore, this study aimed to evaluate the robustness of radiomic features with segmentation variations and their impact on predicting biochemical recurrence (BCR). Materials and methods: Multi-scanner, pre-radiation therapy imaging from 142 patients with localised prostate cancer was used. Imaging included T2-weighted (T2), apparent diffusion coefficient (ADC) MRI, and prostate-specific membrane antigen (PSMA)-PET. The prostate gland and intraprostatic tumours were manually and automatically segmented, and differences were quantified using Dice Coefficient (DC). Radiomic features including shape, first-order, and texture features were extracted for each segmentation from original and filtered images. Intraclass Correlation Coefficient (ICC) and Mean Absolute Percentage Difference (MAPD) were used to assess feature robustness. Random forest (RF) models were developed for each segmentation using robust features to predict BCR. Results: Prostate gland segmentations were more consistent (mean DC = 0.78) than tumour segmentations (mean DC = 0.46). 112 (3.6 %) radiomic features demonstrated 'excellent' robustness (ICC > 0.9 and MAPD < 1 %), and 480 features (15.4 %) demonstrated 'good' robustness (ICC > 0.75 and MAPD < 5 %). PET imaging provided more features with excellent robustness than T2 and ADC. RF models showed strong predictive power for BCR with a mean area under the receiver-operator-characteristics curve (AUC) of 0.89 (range 0.85-0.93). Conclusion: When using radiomic features for predictive modelling, segmentation variability should be considered. To develop BCR predictive models, radiomic features from the entire prostate gland are preferable over tumour segmentation-based features.

Automatic algorithmic driven monitoring of atrioventricular nodal re-entrant tachycardia ablation to improve procedural safety.

Background: During slow pathway modification for atrioventricular nodal reentrant tachycardia, heart block may occur if ablation cannot be stopped in time in response to high risk electrogram features (HREF). Objectives: To develop an automatic algorithm to monitor HREF and terminate ablation earlier than human reaction. Methods: Digital electrogram data from 332 ablation runs from February 2020 to June 2022 were included. They were divided into training and validation sets which contained 126 and 206 ablation runs respectively. HREF in training set was measured. Then a program was developed with cutoff values decided from training set to capture all these HREF. Simulation ablation videos were rendered using validation set electrogram data. The videos were played to three independent electrophysiologists who each determined when to stop ablation. Timing of ablation termination, sensitivity, and specificity were compared between human and program. Results: Reasons for ablation termination in the training set include short AA time, short VV time, AV block and VA block. Cutoffs for the program were set to maximize program sensitivity. Sensitivity and specificity for the program in the validation set were 95.2% and 91.1% respectively, which were comparable to that of human performance at 93.5% and 95.4%. If HREF were recognized by both human and program, ablations were terminated earlier by the program 90.2% of times, by a median of 574 ms (interquartile range 412-807 ms, p < 0.001). Conclusion: Algorithmic-driven monitoring of slow pathway modification can supplement human judgement to improve ablation safety.

Investigation of momentary negative symptoms in patients with early psychosis in daily life: An experience sampling study.

Negative symptoms are a key therapeutic target in promoting functional recovery in early psychosis intervention, but momentary negative symptom manifestations remain understudied in the early stage of illness. We employed an experience-sampling methodology (ESM) to evaluate momentary affective experiences, hedonic capacity for an event recalled, current activities and social interactions, and associated appraisals for 6 consecutive days in 33 clinically-stable early psychosis patients (within 3 years of treatment for first-episode psychosis) and 35 demographically-matched healthy controls. Adjusted multilevel linear-mixed models revealed higher intensity and variability of negative affect in patients than controls, but no group difference in affect instability as well as positive affect intensity and variability. Patients demonstrated no significantly greater anhedonia for event, activity or social interactions relative to controls. Higher preference for company (when alone) and to be alone (when in company) was observed in patients than controls. No significant group difference in pleasantness to be alone or proportion of time being alone. Our results indicate no evidence for blunting of affective experiences, anhedonia (social and non-social) and asociality in early psychosis. Future research complementing ESM with multiple digital phenotyping measures will facilitate more refined negative symptom assessment in the daily life of patients with early psychosis.

Transcatheter Treatment of Native Coarctation of the Aorta in a Child Using a Balloon-Expandable Endoprosthesis.

A 7-year-old child with native coarctation of the aorta was treated by transcatheter therapy using a 29-mm balloon-expandable stent. The procedure was successful and free of complications, and the patient was discharged home the same day. This stent has several features making it uniquely advantageous for treating this condition. (Level of Difficulty: Advanced.).

ETV4 mediates dosage-dependent prostate tumor initiation and cooperates with p53 loss to generate prostate cancer.

The mechanisms underlying ETS-driven prostate cancer initiation and progression remain poorly understood due to a lack of model systems that recapitulate this phenotype. We generated a genetically engineered mouse with prostate-specific expression of the ETS factor, ETV4, at lower and higher protein dosage through mutation of its degron. Lower-level expression of ETV4 caused mild luminal cell expansion without histologic abnormalities, and higher-level expression of stabilized ETV4 caused prostatic intraepithelial neoplasia (mPIN) with 100% penetrance within 1 week. Tumor progression was limited by p53-mediated senescence and Trp53 deletion cooperated with stabilized ETV4. The neoplastic cells expressed differentiation markers such as Nkx3.1 recapitulating luminal gene expression features of untreated human prostate cancer. Single-cell and bulk RNA sequencing showed that stabilized ETV4 induced a previously unidentified luminal-derived expression cluster with signatures of cell cycle, senescence, and epithelial-to-mesenchymal transition. These data suggest that ETS overexpression alone, at sufficient dosage, can initiate prostate neoplasia.

Lineage tracing reveals clonal progenitors and long-term persistence of tumor-specific T cells during immune checkpoint blockade.

Paired single-cell RNA and T cell receptor sequencing (scRNA/TCR-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scRNA/TCR-seq analysis of 187,650 T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients with non-small cell lung cancer after immune checkpoint blockade (ICB). Regions with viable cancer cells are enriched for exhausted CD8+ T cells, regulatory CD4+ T cells (Treg), and follicular helper CD4+ T cells (TFH). Tracking T cell clonotypes across tissues, combined with neoantigen specificity assays, reveals that TFH and tumor-specific exhausted CD8+ T cells are clonally linked to TCF7+SELL+ progenitors in tumor draining LNs, and progressive exhaustion trajectories of CD8+ T, Treg, and TFH cells with proximity to the tumor microenvironment. Finally, longitudinal tracking of tumor-specific CD8+ and CD4+ T cell clones reveals persistence in the peripheral blood for years after ICB therapy.

CRACD loss promotes small cell lung cancer tumorigenesis via EZH2-mediated immune evasion.

The mechanisms underlying immune evasion and immunotherapy resistance in small cell lung cancer (SCLC) remain unclear. Herein, we investigate the role of CRACD tumor suppressor in SCLC. We found that CRACD is frequently inactivated in SCLC, and Cracd knockout (KO) significantly accelerates SCLC development driven by loss of Rb1, Trp53, and Rbl2. Notably, the Cracd-deficient SCLC tumors display CD8+ T cell depletion and suppression of antigen presentation pathway. Mechanistically, CRACD loss silences the MHC-I pathway through EZH2. EZH2 blockade is sufficient to restore the MHC-I pathway and inhibit CRACD loss-associated SCLC tumorigenesis. Unsupervised single-cell transcriptomic analysis identifies SCLC patient tumors with concomitant inactivation of CRACD, impairment of tumor antigen presentation, and downregulation of EZH2 target genes. Our findings define CRACD loss as a new molecular signature associated with immune evasion of SCLC cells and proposed EZH2 blockade as a viable option for CRACD-negative SCLC treatment.

The association between altered intestinal microbiome, impaired systemic and ocular surface immunity, and impaired wound healing response after corneal alkaline-chemical injury in diabetic mice.

Purpose: We aim to investigate the effect of sustained hyperglycemia on corneal epithelial wound healing, ocular surface and systemic immune response, and microbiome indices in diabetic mice compared to controls after alkaline chemical injury of the eye. Methods: Corneal alkaline injury was induced in the right eye of Ins2Akita (Akita) mice and wild-type mice. The groups were observed at baseline and subsequently days 0, 3, and 7 after injury. Corneal re-epithelialization was observed under slit lamp with fluorescein staining using a cobalt blue light filter. Enucleated cornea specimens were compared at baseline and after injury for changes in cornea thickness under hematoxylin and eosin staining. Tear cytokine and growth factor levels were measured using protein microarray assay and compared between groups and time points. Flow cytometry was conducted on peripheral blood and ocular surface samples to determine CD3+CD4+ cell count. Fecal samples were collected, and gut microbiota composition and diversity pattern were measured using shotgun sequencing. Results: Akita mice had significantly delayed corneal wound healing compared to controls. This was associated with a reduction in tear levels of vascular endothelial growth factor A, angiopoietin 2, and insulin growth factor 1 on days 0, 3, and 7 after injury. Furthermore, there was a distinct lack of upregulation of peripheral blood and ocular surface CD3+CD4+ cell counts in response to injury in Akita mice compared to controls. This was associated with a reduction in intestinal microbiome diversity indices in Akita mice compared to controls after injury. Specifically, there was a lower abundance of Firmicutes bacterium M10-2 in Akita mice compared to controls after injury. Conclusion: In diabetic mice, impaired cornea wound healing was associated with an inability to mount systemic and local immune response to ocular chemical injury. Baseline and post-injury differences in intestinal microbial diversity and abundance patterns between diabetic mice and controls may potentially play a role in this altered response.