Single-cell transcriptomic analyses reveal distinct immune cell contributions to epithelial barrier dysfunction in checkpoint inhibitor colitis.

Immune checkpoint inhibitor (ICI) therapy has revolutionized oncology, but treatments are limited by immune-related adverse events, including checkpoint inhibitor colitis (irColitis). Little is understood about the pathogenic mechanisms driving irColitis, which does not readily occur in model organisms, such as mice. To define molecular drivers of irColitis, we used single-cell multi-omics to profile approximately 300,000 cells from the colon mucosa and blood of 13 patients with cancer who developed irColitis (nine on anti-PD-1 or anti-CTLA-4 monotherapy and four on dual ICI therapy; most patients had skin or lung cancer), eight controls on ICI therapy and eight healthy controls. Patients with irColitis showed expanded mucosal Tregs, ITGAEHi CD8 tissue-resident memory T cells expressing CXCL13 and Th17 gene programs and recirculating ITGB2Hi CD8 T cells. Cytotoxic GNLYHi CD4 T cells, recirculating ITGB2Hi CD8 T cells and endothelial cells expressing hypoxia gene programs were further expanded in colitis associated with anti-PD-1/CTLA-4 therapy compared to anti-PD-1 therapy. Luminal epithelial cells in patients with irColitis expressed PCSK9, PD-L1 and interferon-induced signatures associated with apoptosis, increased cell turnover and malabsorption. Together, these data suggest roles for circulating T cells and epithelial-immune crosstalk critical to PD-1/CTLA-4-dependent tolerance and barrier function and identify potential therapeutic targets for irColitis.

Human lung cancer harbors spatially organized stem-immunity hubs associated with response to immunotherapy.

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens and found an association with beneficial response to PD-1 blockade. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcome. This hub is distinct from mature tertiary lymphoid structures and is enriched for stem-like TCF7+PD-1+CD8+ T cells, activated CCR7+LAMP3+ dendritic cells and CCL19+ fibroblasts as well as chemokines that organize these cells. Within the stem-immunity hub, we find preferential interactions between CXCL10+ macrophages and TCF7-CD8+ T cells as well as between mature regulatory dendritic cells and TCF7+CD4+ and regulatory T cells. These results provide a picture of the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Spatial analysis of human lung cancer reveals organized immune hubs enriched for stem-like CD8 T cells and associated with immunotherapy response.

The organization of immune cells in human tumors is not well understood. Immunogenic tumors harbor spatially-localized multicellular 'immunity hubs' defined by expression of the T cell-attracting chemokines CXCL10/CXCL11 and abundant T cells. Here, we examined immunity hubs in human pre-immunotherapy lung cancer specimens, and found that they were associated with beneficial responses to PD-1-blockade. Immunity hubs were enriched for many interferon-stimulated genes, T cells in multiple differentiation states, and CXCL9/10/11 + macrophages that preferentially interact with CD8 T cells. Critically, we discovered the stem-immunity hub, a subtype of immunity hub strongly associated with favorable PD-1-blockade outcomes, distinct from mature tertiary lymphoid structures, and enriched for stem-like TCF7+PD-1+ CD8 T cells and activated CCR7 + LAMP3 + dendritic cells, as well as chemokines that organize these cells. These results elucidate the spatial organization of the human intratumoral immune response and its relevance to patient immunotherapy outcomes.

Combined PD-1, BRAF and MEK inhibition in BRAFV600E colorectal cancer: a phase 2 trial.

While BRAF inhibitor combinations with EGFR and/or MEK inhibitors have improved clinical efficacy in BRAFV600E colorectal cancer (CRC), response rates remain low and lack durability. Preclinical data suggest that BRAF/MAPK pathway inhibition may augment the tumor immune response. We performed a proof-of-concept single-arm phase 2 clinical trial of combined PD-1, BRAF and MEK inhibition with sparatlizumab (PDR001), dabrafenib and trametinib in 37 patients with BRAFV600E CRC. The primary end point was overall response rate, and the secondary end points were progression-free survival, disease control rate, duration of response and overall survival. The study met its primary end point with a confirmed response rate (24.3% in all patients; 25% in microsatellite stable patients) and durability that were favorable relative to historical controls of BRAF-targeted combinations alone. Single-cell RNA sequencing of 23 paired pretreatment and day 15 on-treatment tumor biopsies revealed greater induction of tumor cell-intrinsic immune programs and more complete MAPK inhibition in patients with better clinical outcome. Immune program induction in matched patient-derived organoids correlated with the degree of MAPK inhibition. These data suggest a potential tumor cell-intrinsic mechanism of cooperativity between MAPK inhibition and immune response, warranting further clinical evaluation of optimized targeted and immune combinations in CRC. ClinicalTrials.gov registration: NCT03668431.

Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.

Impact of Continuous Flow Left Ventricular Assist Device on Heart Transplant Candidates: A Multi-State Survival Analysis.

(1) Objectives: The aim of this study was to investigate the impact of the prolonged use of continuous-flow left ventricular assist devices (LVADs) on heart transplant (HTx) candidates. (2) Methods: Between January 2012 and December 2019, we included all consecutive patients diagnosed with end-stage heart failure considered for HTx at our institution, who were also eligible for LVAD therapy as a bridge to transplant (BTT). Patients were divided into two groups: those who received an LVAD as BTT (LVAD group) and those who were listed without durable support (No-LVAD group). (3) Results: A total of 250 patients were analyzed. Of these, 70 patients (28%) were directly implanted with an LVAD as BTT, 11 (4.4%) received delayed LVAD implantation, and 169 (67%) were never assisted with an implantable device. The mean follow-up time was 36 ± 29 months. In the multivariate analysis of survival before HTx, LVAD implantation showed a protective effect: LVAD vs. No-LVAD HR 0.01 (p < 0.01) and LVAD vs. LVAD delayed HR 0.13 (p = 0.02). Mortality and adverse events after HTx were similar between LVAD and No-LVAD (p = 0.65 and p = 0.39, respectively). The multi-state survival analysis showed a significantly higher probability of death for No-LVAD vs. LVAD patients with (p = 0.03) or without (p = 0.04) HTx. (4) Conclusions: The use of LVAD as a bridge to transplant was associated with an overall survival benefit, compared to patients listed without LVAD support.

Carpentier-Edwards Magna Ease bioprosthesis: a multicentre clinical experience and 12-year durability.

OBJECTIVES: The goal of this multicentre retrospective study was to compare long-term clinical and haemodynamic outcomes of the Carpentier-Edwards Magna Ease (CEME) bioprosthesis by patient age. METHODS: We included consecutive patients who underwent isolated and combined surgical aortic valve replacement (AVR) with CEME valve between January 2008 and March 2020 at 4 cardiac surgery centres in Italy. Survival distribution was evaluated at follow-up according to age and surgery type (combined or isolated AVR), together with freedom from structural valve deterioration (SVD), reoperation and combined events, i.e. SVD, reoperation, endocarditis and thromboembolic events. RESULTS: A total of 1027 isolated and 1121 combined AVR were included; 776 patients were younger than 65 years whereas 1372 were 65 years or older. The 30-day Valve-Academic-Research-Consortium mortality was 2% (<65 years) and 6% (≥ 65 years) (P < 0.001), whereas it was 3% for isolated AVR and 7% for combined AVR (P < 0.001). The 12-year survival was 81% for those younger than 65 years vs 45% for those equal to or older than 65 years (P < 0.001), whereas they were 61% vs 49% for isolated and combined AVR (P = 0.10). The 12-year freedom from combined events, excluding death, was 79% for those younger than 65 years vs 87% for those equal to or older than (P = 0.51), whereas they were 83% for isolated and 86% for combined AVR (P = 0.10). The 12-year freedom from SVD was 93% and 93% in patients younger than 65 and those equal to or older than 65 years (P = 0.63), and the results were comparable even in cases with isolated and combined AVR (92% vs 94%, P = 0.21). A multivariable Cox analysis including gender, presence of patient-prosthesis mismatch, isolated AVR and age showed that only the age was an independent risk factor for the incidence of SVD (P = 0.029). CONCLUSIONS: Outcomes from this large multicentre analysis demonstrated that a CEME bioprosthesis provides good clinical results and long-term durability even in patients younger than 65 years. Furthermore, the hazard for SVD has been shown to be lower for older age. CLINICAL TRIAL REGISTRATION NUMBER: 105n/AO/21.

Unilateral versus bilateral cerebral perfusion during aortic surgery for acute type A aortic dissection: a multicentre study.

OBJECTIVES: The aim of this retrospective multicentre study was to investigate and compare clinical outcomes of unilateral and bilateral antegrade cerebral perfusion (ACP) strategies on cerebral protection during surgery for type A aortic dissection. METHODS: Data from 646 patients who underwent surgical repair of thoracic type A aortic dissection using unilateral and bilateral ACP with moderate hypothermic circulatory arrest in 3 cardiac surgical institutions between 2008 and 2018 were analysed. Propensity matching was performed to assess which technique ensured better outcomes. RESULTS: Unilateral and bilateral ACP techniques were performed in 250 (39%) and in 396 (61%) patients, respectively. Propensity score analysis identified 189 matched pairs. In the matched cohort, the lowest core temperature was 27.5°C and 28°C in the bilateral and unilateral groups, respectively (P < 0.001). The unilateral technique required significantly shorter aortic cross-clamp and cardiopulmonary bypass times than bilateral technique [82 min vs 100 min (P < 0.001); 170 min vs 195 min (P < 0.001)]. The 30-day mortality was comparable (P = 0.325). The bilateral group reported a significantly higher incidence of permanent neurologic deficits (P < 0.001), left brain hemisphere stroke (P = 0.007) and all-combined complications (P < 0.001). Ten-year survival was comparable (P = 0.45). CONCLUSIONS: Unilateral and bilateral ACP are both valid brain protection strategies in the landscape of aortic arch surgery. While admitting all the study limitations, unilateral technique could offer some clinical advantages. CLINICAL REGISTRATION NUMBER: 76049.

Spatially organized multicellular immune hubs in human colorectal cancer.

Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.

Recurrent autoimmune myocarditis in a young woman during the coronavirus disease 2019 pandemic.

We report a unique case of a young woman with recurrent immune-mediated (virus-negative) lymphocytic fulminant myocarditis during the coronavirus disease 2019 pandemic. At the first endomyocardial biopsy (EMB)-proven episode, she had concomitant pneumonia, and a temporary biventricular assist device implant was followed by complete and long-lasting cardiac recovery. Five years later, she was re-admitted for relapsing cardiogenic shock with a recent history of pneumonia. She was treated with extracorporeal life support with apical venting for left ventricular unloading, and full recovery was achieved. Despite negative seriate nasopharyngeal swabs and EMB during hospitalization, an antibody positivity for severe acute respiratory syndrome coronavirus 2 was discovered after 4 weeks from discharge. This is the first report of an EMB-proven, immune-mediated (virus-negative) recurrence of fulminant myocarditis. We hypothesize that in patients with a predisposing immunogenetic background, autoimmune disease may be triggered or reactivated by major infections, for example, pneumonia, that may act as adjuvants leading to an immune-mediated hyper-response.

COVID-19 in Heart Transplant Recipients: A Multicenter Analysis of the Northern Italian Outbreak.

OBJECTIVES: The aim of this study was to assess the clinical course and outcomes of all heart transplant recipients affected by coronavirus disease-2019 (COVID-19) who were followed at the leading heart transplant centers of Northern Italy. BACKGROUND: The worldwide severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) pandemic has created unprecedented challenges for public health, demanding exceptional efforts for the successful management and treatment of affected patients. Heart transplant patients represent a unique cohort of chronically immunosuppressed subjects in which SARS-CoV-2 may stimulate an unpredictable clinical course of infection. METHODS: Since February 2020, we enrolled all 47 cases (79% male) in a first cohort of patients, with a mean age of 61.8 ± 14.5 years, who tested positive for SARS-CoV-2, out of 2,676 heart transplant recipients alive before the onset of the COVID-19 pandemic at 7 heart transplant centers in Northern Italy. RESULTS: To date, 38 patients required hospitalization while 9 remained self-home quarantined and 14 died. Compared to the general population, prevalence (18 vs. 7 cases per 1,000) and related case fatality rate (29.7% vs. 15.4%) in heart transplant recipients were doubled. Univariable analysis showed older age (p = 0.002), diabetes mellitus (p = 0.040), extracardiac arteriopathy (p = 0.040), previous PCI (p = 0.040), CAV score (p = 0.039), lower GFR (p = 0.004), and higher NYHA functional classes (p = 0.023) were all significantly associated with in-hospital mortality. During the follow-up two patients died and a third patient has prolonged viral-shedding alternating positive and negative swabs. Since July 1st, 2020, we had 6 new patients who tested positive for SARS-CoV-2, 5 patients asymptomatic were self-quarantined, while 1 is still hospitalized for pneumonia. A standard therapy was maintained for all, except for the hospitalized patient. CONCLUSIONS: The prevalence and mortality of SARS-CoV-2 should spur clinicians to immediately refer heart transplant recipients suspected as having SARS-CoV2 infection to centers specializing in the care of this vulnerable population.

Coronavirus disease 2019 (COVID-19) in the heart transplant population: a single-centre experience.

OBJECTIVES: Few anecdotal cases have been reported in the literature regarding heart transplant recipients and infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report our experience with 6 patients hospitalized in Northern Italy during the outbreak. METHODS: Of the 396 living heart transplant recipients from 1985 to 2020 included in the study, 6 patients developed the novel 2019 coronavirus disease. Risk factors, last follow-up characteristics, onset presentation, in-hospital course of disease and blood examinations data were collected for these patients. RESULTS: All patients were symptomatic and had positive results from a nasopharyngeal swab test for SARS-CoV-2. Of the 6 patients, 5 were hospitalized and 1 remained self-quarantined at home. Two patients died and 3 were discharged home. Two patients were admittted to the intensive care unit . Immunosuppressive therapy was modified with a median reduction comprising doses that were 50% cyclosporine and 50% mycophenolate. All patients received a medium-dose of corticosteroids as a bolus medication in addition to their therapy. All hospitalized patients received hydroxychloroquine; 2 patients received ritonavir/lopinavir. Broad-spectrum antibiotics for prophylaxis were administered to all. One patient had an ischaemic stroke and died of sepsis. CONCLUSIONS: In the absence of any strong evidence regarding the treatment of heart transplant recipients infected with SARS-CoV-2, we faced a new challenge in managing viral infection in an immunosuppressed population. Because immunomodulation interaction with the infection seems to be crucial for developing severe forms of the disease, we managed to reduce immunosuppressive therapy by adding medium doses of corticosteroids. Despite the limited number of affected patients, this report suggests that special considerations should be given to treating coronavirus disease in the heart transplant recipient population.

TAVR, SAVR and MI-AVR. Good Things Come to Those Who Wait.

Modern medicine uses a combination of advanced technology and established knowledge to reach its ultimate goal: healing patients while limiting risks and preventing disease [...].

Cotargeting among microRNAs in the brain.

MicroRNAs (miRNAs) play roles in diverse developmental and disease processes. Distinct miRNAs have hundreds to thousands of conserved mRNA binding sites but typically direct only modest repression via single sites. Cotargeting of individual mRNAs by different miRNAs could potentially achieve stronger and more complex patterns of repression. By comparing target sets of different miRNAs, we identified hundreds of pairs of miRNAs that share more mRNA targets than expected (often by twofold or more) relative to stringent controls. Genetic perturbations revealed a functional overlap in neuronal differentiation for the cotargeting pair miR-138/miR-137. Clustering of all cotargeting pairs revealed a group of nine predominantly brain-enriched miRNAs that share many targets. In reporter assays, subsets of these miRNAs together repressed gene expression by five- to 10-fold, often showing cooperative repression. Together, our results uncover an unexpected pattern in which combinations of miRNAs collaborate to robustly repress cotargets, and suggest important developmental roles for cotargeting.