Citations

Aims Remote monitoring of pulmonary artery pressure has reduced heart failure (HF) hospitalizations in chronic HF as elevation of pulmonary artery pressure provides information that can guide treatment. The venous system is characterized by high capacitance, thus substantial increases in intravascular volume can occur before filling pressures increase. The inferior vena cava (IVC) is a highly compliant venous conduit and thus a candidate for early detection of change in intravascular volume. We aimed to compare IVC cross‐sectional area using a novel sensor with cardiac filling pressures during experimental manipulation of volume status, vascular tone, and cardiac function. Methods and results Experiments were conducted in sheep to manipulate volume status (colloid infusion), vascular tone (nitroglycerin infusion) and cardiac function (rapid cardiac pacing). A wireless implantable IVC sensor was validated ex‐vivo and in‐vivo , and then used to measure the cross‐sectional area of the IVC. Right‐ and left‐sided cardiac filling pressures were obtained via right heart catheterization. The IVC sensor provided highly accurate and precise measurements of cross‐sectional area in ex‐vivo and in‐vivo validation. IVC area changes were more sensitive than the corresponding changes in cardiac filling pressures during colloid infusion ( p < 0.001), vasodilatation ( p < 0.001) and cardiac dysfunction induced by rapid pacing ( p ≤ 0.02). Conclusions Inferior vena cava area can be remotely and accurately measured in real time with a wireless implantable sensor. Changes in IVC area are more sensitive than corresponding changes in filling pressures following experimental volume loading and fluid redistribution. Additional research is warranted to understand if remote monitoring of the IVC may have advantages over pressure‐based monitors in HF.

Abstract Background: SARS-Cov-2 infections are associated with increased mortality and morbidity, largely due to inflammatory cascades and cytokine release syndrome (CRS). Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence and subsequent expansion of somatic, leukemia-associated driver mutations in apparently healthy individuals with normal blood counts. CHIP has been associated with increased inflammation, with cytokines such as IL1-b, IL6 and TNF-a being elevated at baseline in affected individuals. We hypothesized that the presence of CHIP in patients with COVID-19 would result in excessive inflammation-related mortality and morbidity. Methods: We used the Mayo Clinic COVID-19 database to identify patients with COVID-19 on whom peripheral blood mononuclear cells (PBMC) were available for research use (IRB approved). We carried out target-capture next generation sequencing for 220 CH related genes, by previously described methods (1000 x coverage, variant allele fraction/VAF detection limit &gt;0.5%; Kusne Y et al AJH 2021). CHIP was defined by the presence of a CH mutation with a VAF&gt;1% in an individual with normal baseline blood counts. Demographics, blood counts, and inflammatory markers (CRP and cytokine levels- ELISA assay) at COVID-19 diagnosis and during follow-up (as clinically indicated) were collected. COVID-19 disease severity was classified based on the presence and severity of CRS, graded using the Penn Grading Scale (Porter et. al., 2018), and the WHO ordinal scale (WHO Blueprint, 2020). We used Fisher's exact test and the Wilcoxon rank sum test to compare categorical and continuous variables. Survival analysis was performed using the Kaplan-Meier method. We accounted for differences in age and sex using multivariable-adjusted proportional hazards regression models. Results: Seventy-two CHIP mutations were detected in 56 (25%) of the 227 patients with COVID-19 that had PBMC available; median age 69 years (range; 42-99 years), 61% male. Fifteen (26%) patients had 2 CHIP mutations, while 1 patient had 3 CHIP mutations. Common mutations encountered included DNMT3A (32%) , TET2 (19%) , SF3B1 (8%) , ASXL1 (6%) , MPL (5%) , and TP53 (5%; Figure 1A). COVID-19 patients with CHIP were older in age (median 69 vs 57 years; p&lt;0.0001) and had higher baseline MCP-1 (p=0.04) levels. However, there were no differences in sex, comorbidities, blood counts, IL1-b, IL6 and TNF-a levels between the two groups. The median follow-up for the entire cohort was 9 months. The relative change from baseline in blood counts and inflammatory markers (CRP and cytokines) during follow-up was similar in CHIP and non-CHIP patients, with the exception that COVID-19-onset neutropenia was more common in CHIP patients (8% vs 1%; p=0.017) compared to those without CHIP. At last follow up neutropenia had resolved in all patients. Both groups had comparable number of patients with CRS (61% CHIP vs 53% non-CHIP patients, p=0.354, Figure 1B), however, CHIP patients had more severe CRS (median Penn Grade 3 versus 2 in non-CHIP, p=0.018, Figure 1C). Based on the WHO ordinal scale, CHIP patients were more likely to experience hospitalization with severe disease and death (61% versus 45% in non-CHIP, p = 0.049). Moreover, COVID-19 CHIP patients experienced worse overall survival in comparison to patients without CHIP (median 13.1 months vs not reached, p&lt;0.001, Figure 1D). This association remained consistent after adjusting for age and sex at the time of COVID-19 diagnosis (HR 3.15, 95% 1.32 - 7.55, p = 0.010). At last follow-up, 22 deaths were documented: 13 (23%) in patients with CHIP and 9 (5%) in the non-CHIP group (p=0.02), with the primary cause for mortality being hypoxic respiratory failure (62% in CHIP vs 44% non-CHIP, p=0.04). Conclusions: In this study, we observe an age-independent impact of CHIP on COVID-19 associated inflammatory morbidity (CRS) and mortality (hypoxemic respiratory failure). We are currently carrying out detailed single cell (ssDNA, RNA and ATAC-seq) and proteomic studies (O-link PEA assays) to better elucidate this pathophysiology. Figure 1 Figure 1. Disclosures Patnaik: Kura Oncology: Research Funding; StemLine: Research Funding.

Abstract Background: Chronic myelomonocytic leukemia (CMML) is an aging disease that arises on the background of clonal hematopoiesis, commonly involving multiple TET2 mutations or TET2/SRSF2 co-mutations. In a prior study of 1084 CMML patients (Leukemia 2020; 34:1407-1421), we demonstrated a high prevalence of TET2 mutations (56%) in CMML, with approximately 30% of patients having &gt;1 TET2 mutation. The cell-type and allele specific distribution of these mutations remains of great interest and are thus far poorly defined. To visualize the genotypic and immunophenotypic correlates, we performed single-cell DNA analysis and simultaneous protein profiling on 2 CMML patients with multiple TET2 mutations. Methods: After Mayo Clinic IRB approval, two CMML patients with dual TET2 mutations and available peripheral blood mononuclear cells were included in the study. Patient samples were labeled with TotalSeq TM -D Heme Oncology Cocktail (BioLegend, San Diego, CA) consisting of 45 unique oligo-conjugated cell surface markers. The samples were then washed and loaded into a Tapestri microfluidics cartridge, where single cells (3,500 cells/µL) were encapsulated, lysed and barcoded using the MissionBio Myeloid custom amplicon panel consisting of 312 amplicons in 45 genes relevant to myeloid disorders (MissionBio, San Francisco, CA). The individual barcoded DNA and protein for each cell was purified, recovered, and labeled with Illumina i5/i7 indices, quanitifed by Agilent Bioanalyzer and pooled for sequencing. The libraries were sequenced via Illumina NovaSeq by the Mayo Genomics Core, and data was recovered and analyzed using MissionBio Tapestri Insights v3.0.2. Results: Two dual TET2 mutated CMML patients were included in the study, both male, ages 79 and 83, and both with CMML-0. Case 1 had TET2 p.G1172Efs*53 (variant allele fraction/VAF-6%) and p.H1732Tfs*13 (VAF-13%) mutations, while case 2 had TET2 p.Y620Ffs* (VAF-85%), p.G898* (VAF-82%), and SRSF2 p.95H (VAF-45%) mutations, respectively. In both patients, TET2 mutations were found to be preferentially distributed in monocytes (CD11b+, CD14+, CD64+, CD13+, CD33+, HLA-DR+), with very few classically defined granulocyte (CD16+,CD13+.CD33+, HLA-DR neg), B (CD19+,CD22+)/T (CD2+,CD4+,CD5+,CD7+) lymphocytes, NK cells (CD16+/CD56+/HLA-DR neg) and plasma cells (CD38+/CD138+), demonstrating mutant TET2. Among monocytes, TET2 mutations were enriched in classical/M01 (CD11b+, CD14+,CD16-) and intermediate/M02 monocytes and were infrequent in the MO3 fraction, commensurate with monocyte repartitioning seen in CMML. Interestingly, a large number of single cells with TET2 mutations also demonstrated an aberrant surface phenotype, with universal expression of CD14+ and CD11b+, along with a mixture of other markers (CD163+, CD11c, CD4+, CD2+, and/or CD56+). With regards to allelic distribution, while several permutations and combination were seen involving heterozygosity and homozygosity for the TET2 mutations identified on bulk sequencing, data was corroborative for both TET2 mutations being located on trans alleles. Conclusions: In this study, we demonstrate that TET2 mutations in CMML are highly enriched in classical circulating monocytes and infrequently occur in classical granulocytes, B/T and NK cells; with multiple TET2 mutations most commonly co-existing on trans alleles. Analyses on additional samples along with copy number variation assessments are ongoing. Figure 1: Lollipop plot of TET2 exons with dual TET2 mutations labeled above and oxygenase domain labeled in green; below are number of cells identified with each genotype for Case 1 (A) and Case 2 (B). Mutations are labeled proximal (yellow) and distal (red) and alleles are in grey. The directionality of the arrows to different genotypes is based on decreasing cell number and genotype. Uniform manifold approximation and projection (UMAP) plot of Case 1 (C) and Case 2 (D) with cells clustered by immunophenotype and labeled by genotype (non TET-2 mutated purple, heterozygous TET2 in green, homozygous TET2 in yellow). Relative protein expression of T-cell phenotype (CD14-, CD3+, CD4+/-,CD8+/-, CD5+, CD7+, CD2+), or classical monocytes (CD14+/CD16-) for Case 1 (E & F) and Case 2 (G & H) overlayed on UMAP. Royal blue represents non TET2-mutated cells, and mutated cells are labeled in pastel blue, green and light orange by mutation type. Figure 1 Figure 1. Disclosures Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Patnaik: StemLine: Research Funding; Kura Oncology: Research Funding.

Abstract Alcohol‐associated liver disease (ALD) is emerging worldwide as the leading cause of liver‐related morbidity, mortality, and indication for liver transplantation. The ALD Special Interest Group and the Clinical Research Committee at the digital American Association for the Study of Liver Diseases meeting in November 2020 held the scientific sessions to identify clinical unmet needs in ALD, and addressing these needs using clinical research methodologies. Of several research methodologies, the sessions were focused on (a) studying disease burden of ALD using large administrative databases, (b) developing biomarkers for noninvasive diagnosis of alcohol‐associated hepatitis (AH) and estimation of disease prognosis, (c) identifying therapeutic targets for ALD and AH, (d) deriving accurate models to predict prognosis or posttransplant alcohol relapse as a basis for developing treatment algorithm and a uniform protocol on patient‐selection criteria for liver transplantation, and (e) examining qualitative research methodologies in studying the barriers to implementation of multidisciplinary integrated care model by hepatology and addiction teams for the management of dual pathology of liver disease and of alcohol use disorder. Prospective multicenter studies are required to address many of these clinical unmet needs. Further, multidisciplinary care models are needed to improve long‐term outcomes in patients with ALD.

PLEXIND1 is upregulated in several cancers, including pancreatic ductal adenocarcinoma (PDAC). It is an established mediator of semaphorin signaling, and neuropilins are its known coreceptors. Herein, we report data to support the proposal that PLEXIND1 acts as a transforming growth factor beta (TGFβ) coreceptor, modulating cell growth through SMAD3 signaling. Our findings demonstrate that PLEXIND1 plays a pro-tumorigenic role in PDAC cells with oncogenic KRAS (KRASmut). We show in KRASmut PDAC cell lines (PANC-1, AsPC-1,4535) PLEXIND1 downregulation results in decreased cell viability (in vitro) and reduced tumor growth (in vivo). Conversely, PLEXIND1 acts as a tumor suppressor in the PDAC cell line (BxPC-3) with wild-type KRAS (KRASwt), as its reduced expression results in higher cell viability (in-vitro) and tumor growth (in vivo). Additionally, we demonstrate that PLEXIND1-mediated interactions can be selectively disrupted using a peptide based on its C-terminal sequence (a PDZ domain-binding motif), an outcome that may possess significant therapeutic implications. To our knowledge, this is the first report showing that (1) PLEXIND1 acts as a TGFβ coreceptor and mediates SMAD3 signaling, and (2) differential roles of PLEXIND1 in PDAC cell lines correlate with KRASmut and KRASwt status.

Abstract Large cell carcinoma (LCC) is an aggressive lung cancer subtype with poor prognosis and no targeted therapies. We previously reported that tumor-associated fibroblasts (TAFs) derived from LCC tumors exhibit premature senescence, and coculture of pulmonary fibroblasts with LCC cell lines selectively induces fibroblast senescence, which in turn drives LCC cell growth and invasion. Here we report that MMP1 is overexpressed specifically in LCC cell lines. Notably, silencing MMP1 expression in LCC cancer cell lines using shRNA revealed that MMP1 expression by LCC cells is necessary for induction of fibroblast senescence in coculture experiments with normal pulmonary fibroblasts, as revealed by the analysis of a panel of standard senescence markers, including β-galactosidase (SA-βgal) staining, permanent growth arrest and expression of senescence-associated secretory factors. Injecting control (shScr) or shMMP1 LCC cells into immunodeficient nude mice revealed that tumor growth, tumor take and cancer cell dissemination to the lung were reduced in shMMP1 H460 tumors compared to control tumors. We also observed fewer senescent fibroblasts in tumors from shMMP1 H460 cells using Sentragor staining, which allows identification of senescent cells in paraffin embedded tissues. Moreover, we found that recombinant active MMP1 in combination with TGF-β1 were sufficient to induce normal fibroblast senescence. In terms of the potential underlying mechanisms, treatment with the antioxidant n-acetyl cysteine (NAC) significantly attenuated the increase in SA-βgal+ fibroblasts elicited by co-stimulation with rMMP1 and TGF-β1, and its corresponding conditioned medium elicited a significantly lower growth and invasion in LCC cancer cells, revealing the oxidative stress implication in fibroblast senescence induction and associated pro-tumorigenic secretome. In summary, our results establish a new role for MMP1 in cancer and support that LCC cells elicit a tumor-supporting niche through the aberrant secretion of MMP1 and TGF-β1 to induce senescence in adjacent fibroblasts. Furthermore, we implicate oxidative stress in MMP1/TGF-β1-induced TAF senescence and support a novel therapeutic strategy in LCC based on targeting senescent TAFs. Citation Format: Marta Gabasa, Evette S. Radisky, Rafael Ikemori, Giulia Bertolini, Marselina Arshakyan, Alexandra Hockla, Paula Duch, Ornella Rondinone, Alejandro Llorente, Maria Maqueda, Alexandre Perera, Noemí Reguart, Luca Roz, Derek C. Radisky, Jordi Alcaraz. MMP1 and TGF-β1 cooperate to drive tumor progression in large cell carcinoma of the lung through fibroblast senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2515.

Background and Aims Recurrent primary sclerosing cholangitis (rPSC) following liver transplant (LT) has a negative impact on graft and patient survival; little is known about risk factors for rPSC or disease course in children. Approach and Results We retrospectively evaluated risk factors for rPSC in 140 children from the Pediatric PSC Consortium, a multicenter international registry. Recipients underwent LT for PSC and had >90 days of follow‐up. The primary outcome, rPSC, was defined using Graziadei criteria. Median follow‐up after LT was 3 years (interquartile range 1.1‐6.1). rPSC occurred in 36 children, representing 10% and 27% of the subjects at 2 years and 5 years following LT, respectively. Subjects with rPSC were younger at LT (12.9 vs. 16.2 years), had faster progression from PSC diagnosis to LT (2.5 vs. 4.1 years), and had higher alanine aminotransferase (112 vs. 66 IU/L) at LT (all P < 0.01). Inflammatory bowel disease was more prevalent in the rPSC group (86% vs. 66%; P = 0.025). After LT, rPSC subjects had more episodes of biopsy‐proved acute rejection (mean 3 vs. 1; P < 0.001), and higher prevalence of steroid‐refractory rejection (41% vs. 20%; P = 0.04). In those with rPSC, 43% developed complications of portal hypertension, were relisted for LT, or died within 2 years of the diagnosis. Mortality was higher in the rPSC group (11.1% vs. 2.9%; P = 0.05). Conclusions The incidence of rPSC in this cohort was higher than previously reported, and was associated with increased morbidity and mortality. Patients with rPSC appeared to have a more aggressive, immune‐reactive phenotype. These findings underscore the need to understand the immune mechanisms of rPSC, to lay the foundation for developing new therapies and improve outcomes in this challenging population.

Abstract Considerable efforts have been made to characterize active enhancer elements, which can be annotated by accessible chromatin and H3 lysine 27 acetylation (H3K27ac). However, apart from poised enhancers that are observed in early stages of development and putative silencers, the functional significance of cis-regulatory elements lacking H3K27ac is poorly understood. Here we show that macroH2A histone variants mark a subset of enhancers in normal and cancer cells, which we coined ‘macroH2A-Bound Enhancers’, that negatively modulate enhancer activity. We find macroH2A variants enriched at enhancer elements that are devoid of H3K27ac in a cell type-specific manner, indicating a role for macroH2A at inactive enhancers to maintain cell identity. In following, reactivation of macro-bound enhancers is associated with oncogenic programs in breast cancer and its repressive role is correlated with the activity of macroH2A2 as a negative regulator of BRD4 chromatin occupancy. Finally, through single cell epigenomic profiling, we show that the loss of macroH2A2 leads to increased cellular heterogeneity that may help to explain the role of macroH2A variants in defining oncogenic transcriptional dependencies.