Endomyocardial biopsy in clinical practice: the diagnostic yield and insights from a 5-year single-center experience.

OBJECTIVES: Endomyocardial biopsy (EMB) is a diagnostic tool for evaluating various cardiac conditions, such as myocarditis and myocardial infiltrative diseases. It is also the gold standard screening technique for detecting allograft rejection after heart transplantation. Despite advances in noninvasive imaging modalities for myocardial tissue characterization, EMB is still necessary for making a definitive diagnosis and determining treatment for certain conditions. Herein, we report our recent experience using EMB and its diagnostic yield. METHODS AND RESULTS: We retrospectively reviewed EMBs performed at our institution from March 2018 through March 2023. Clinical data, including patient characteristics, indication and diagnostic yield of EMB, and procedure-related complications, were collected. Histopathological findings of the biopsies were recorded and classified based on the degree to which they matched the clinical diagnosis and cardiac magnetic resonance imaging (CMR) findings. A total of 212 EMBs obtained in 178 consecutive patients were retrospectively analyzed, with 42 biopsies performed for allograft rejection surveillance (10 patients) and the remaining performed for presumptive diagnosis of acute myocarditis or unexplained cardiomyopathy. Among the non-heart transplant cases, 54.7% of EMBs provided a clear diagnosis. The most common diagnosis was myocarditis (69%), followed by cardiac amyloidosis (CA) (26%). EMB was also helpful in detecting several rare cardiac conditions, such as eosinophilic granulomatosis with polyangiitis (EGPA), Fabry disease, and cardiac sarcoidosis. In a cohort of 101 patients who underwent both CMR and EMB, the results were concordant in 66% of cases. However, in 24.7% of patients, EMB was able to identify pathological conditions where CMR results were inconclusive, highlighting its complementary role in determining an accurate diagnosis. No complications were reported in any of the 212 EMBs performed. CONCLUSIONS: With advances in cardiac imaging modalities, EMB is not routinely indicated for the diagnosis of cardiomyopathy. However, EMB is still an important tool for diagnosing specific cardiac diseases and could be crucial for confirming the diagnosis. EMB is generally safe if performed at experienced centers.

Mapping cell-to-tissue graphs across human placenta histology whole slide images using deep learning with HAPPY.

Accurate placenta pathology assessment is essential for managing maternal and newborn health, but the placenta's heterogeneity and temporal variability pose challenges for histology analysis. To address this issue, we developed the 'Histology Analysis Pipeline.PY' (HAPPY), a deep learning hierarchical method for quantifying the variability of cells and micro-anatomical tissue structures across placenta histology whole slide images. HAPPY differs from patch-based features or segmentation approaches by following an interpretable biological hierarchy, representing cells and cellular communities within tissues at a single-cell resolution across whole slide images. We present a set of quantitative metrics from healthy term placentas as a baseline for future assessments of placenta health and we show how these metrics deviate in placentas with clinically significant placental infarction. HAPPY's cell and tissue predictions closely replicate those from independent clinical experts and placental biology literature.

RNA binding protein IGF2BP2 expression is induced by stress in the heart and mediates dilated cardiomyopathy.

The IGF2BP family of RNA binding proteins consists of three paralogs that regulate intracellular RNA localization, RNA stability, and translational control. Although IGF2BP1 and 3 are oncofetal proteins, IGF2BP2 expression is maintained in many tissues, including the heart, into adulthood. IGF2BP2 is upregulated in cardiomyocytes during cardiac stress and remodeling and returns to normal levels in recovering hearts. We wondered whether IGF2BP2 might play an adaptive role during cardiac stress and recovery. Enhanced expression of an IGF2BP2 transgene in a conditional, inducible mouse line leads to dilated cardiomyopathy (DCM) and death within 3-4 weeks in newborn or adult hearts. Downregulation of the transgene after 2 weeks, however, rescues these mice, with complete recovery by 12 weeks. Hearts overexpressing IGF2BP2 downregulate sarcomeric and mitochondrial proteins and have fragmented mitochondria and elongated, thinner sarcomeres. IGF2BP2 is also upregulated in DCM or myocardial infarction patients. These results suggest that IGF2BP2 may be an attractive target for therapeutic intervention in cardiomyopathies.

A universal cell-free DNA approach for response prediction to preoperative chemoradiation in rectal cancer.

The standard treatment approach for stage II/III rectal cancer is neoadjuvant chemoradiation therapy (nCRT) followed by surgery. In recent years, new treatment approaches have led to higher rates of complete tumor eradication combined with organ-preservation strategies. However, better tools are still needed to personalize therapy for the individual patient. In this prospective observational study, we analyzed colon-derived cell-free (cf)DNA (c-cfDNA) using a tissue-specific DNA methylation signature, and its association with therapy outcomes. Analyzing plasma samples (n = 303) collected during nCRT from 37 patients with locally advanced rectal cancer (LARC), we identified colon-specific methylation markers that discriminated healthy individuals from patients with untreated LARC (area under the curve, 0.81; 95% confidence interval, 0.70-0.92; P < .0001). Baseline c-cfDNA predicted tumor response, with increased levels linked to larger residual cancer. c-cfDNA measured after the first week of therapy identified patients with maximal response and complete cancer eradication, who had significantly lower c-cfDNA compared with those who had residual disease (8.6 vs 57.7 average copies/ml, respectively; P = .013). Increased c-cfDNA after 1 week of therapy was also associated with disease recurrence. Methylation-based liquid biopsy can predict nCRT outcomes and facilitate patient selection for escalation and de-escalation strategies.

A case series of eosinophilic myocarditis: different faces of the same coin.

Background: Eosinophilic myocarditis (EM) is a rare form of myocarditis with various aetiologies and dire consequences if not diagnosed and treated expeditiously. Case summary: We report three cases of EM at different stages of the disease with differing clinical manifestations. We highlight the diagnostic workup including the role of multimodality imaging and endomyocardial biopsy (EMB), and the treatment strategies. Discussion: EM is an underdiagnosed and potentially life-threatening disease. Therefore, a high clinical suspicion for EM should arise when patients with signs and symptoms of cardiovascular disease develop hypereosinophilia or vice versa. Early identification of this condition using multimodality imaging and EMB is of paramount importance as the disease may progress to the irreversible late fibrotic stage if treatment is delayed.

Establishment of a controlled slow freezing-based approach for experimental clinical cryopreservation of human prepubertal testicular tissues.

Objective: To develop an efficient, clinical-grade, freezing protocol toward experimental clinical cryopreservation of testicular tissues in prepubertal boys suffering from cancer. Design: Experimental cryopreservation of testicular tissue. Setting: University Medical Center. Patients: Adult patients undergoing orchiectomy for various tumors and prepubertal boys scheduled for gonadotoxic treatment. Interventions: None. Main Outcome Measures: Histopathological analysis of tissue architecture, structural integrity, and cellular morphology was performed for control and frozen-thawed cryopreserved tissues.The number of seminiferous tubules per testicular section was calculated. The survival of spermatogonial stem cells (SSCs) and Sertoli cells of the control and frozen-thawed cryopreserved tissues was analyzed by immunofluorescence staining. Results: Uncontrolled Slow Freezing, Controlled slow freezing, and vitrification similarly preserved the integrity of the adult testicular tissues and the survival of SSCs and Sertoli cells. Controlled slow freezing of prepubertal testicular tissues effectively preserved their architecture, the number of tubules, SSCs, and Sertoli cells. In addition, we observed SSC loss after chemotherapy in prepubertal boys, reemphasizing the importance of fertility preservation before gonadotoxic treatment. Conclusions: Future fertility restoration for male survivors of pediatric cancers depends on the development of an optimal prepubertal testicular tissue cryopreservation method. Our findings demonstrate the effectiveness of controlled slow freezing for cryopreservation of human prepubertal testicular tissues and may contribute to more effective banking of these tissues and potential fertility restoration. Clinical Trial Registration Number: NIH research clinical trials number: NCT02529826.

Senolytic elimination of Cox2-expressing senescent cells inhibits the growth of premalignant pancreatic lesions.

OBJECTIVE: Cellular senescence limits tumourigenesis by blocking the proliferation of premalignant cells. Additionally, however, senescent cells can exert paracrine effects influencing tumour growth. Senescent cells are present in premalignant pancreatic intraepithelial neoplasia (PanIN) lesions, yet their effects on the disease are poorly characterised. It is currently unknown whether senolytic drugs, aimed at eliminating senescent cells from lesions, could be beneficial in blocking tumour development. DESIGN: To uncover the functions of senescent cells and their potential contribution to early pancreatic tumourigenesis, we isolated and characterised senescent cells from PanINs formed in a Kras-driven mouse model, and tested the consequences of their targeted elimination through senolytic treatment. RESULTS: We found that senescent PanIN cells exert a tumour-promoting effect through expression of a proinflammatory signature that includes high Cox2 levels. Senolytic treatment with the Bcl2-family inhibitor ABT-737 eliminated Cox2-expressing senescent cells, and an intermittent short-duration treatment course dramatically reduced PanIN development and progression to pancreatic ductal adenocarcinoma. CONCLUSIONS: These findings reveal that senescent PanIN cells support tumour growth and progression, and provide a first indication that elimination of senescent cells may be effective as preventive therapy for the progression of precancerous lesions.

Safety and Efficacy of Combined Low-Dose Lithium and Low-Dose Aspirin: A Pharmacological and Behavioral Proof-of-Concept Study in Rats.

Despite established efficacy in bipolar disorder patients, lithium (Li) therapy has serious side effects, particularly chronic kidney disease. We examined the safety and behavioral effects of combined chronic low-dose aspirin plus low-dose Li in rats to explore the toxicity and therapeutic potential of this treatment. Rats were fed regular or Li-containing food (0.1% [low-dose, LLD-Li] or 0.2% [standard-dose, STD-Li]) for six weeks. Low-dose aspirin (1 mg/kg) was administered alone or together with Li. Renal function and gastric mucosal integrity were assessed. The effects of the combination treatment were evaluated in depression-like and anxiety-like behavioral models. Co-treatment with aspirin did not alter plasma Li levels. Chronic STD-Li treatment resulted in significant polyuria and polydipsia, elevated blood levels of creatinine and cystatin C, and increased levels of kidney nephrin and podocin-all suggestive of impaired renal function. Aspirin co-treatment significantly damped STD-Li-induced impairments in kidney parameters. There were no gastric ulcers or blood loss in any treatment group. Combined aspirin and LLD-Li resulted in a significant increase in sucrose consumption, and in the time spent in the open arms of an elevated plus-maze compared with the LLD-Li only group, suggestive of antidepressant-like and anxiolytic-like effects, respectively. Thus, we demonstrate that low-dose aspirin mitigated the typical renal side effects of STD-Li dose and enhanced the beneficial behavioral effects of LLD-Li therapy without aggravating its toxicity.

Myocarditis after BNT162b2 mRNA Vaccine against Covid-19 in Israel.

BACKGROUND: Approximately 5.1 million Israelis had been fully immunized against coronavirus disease 2019 (Covid-19) after receiving two doses of the BNT162b2 messenger RNA vaccine (Pfizer-BioNTech) by May 31, 2021. After early reports of myocarditis during adverse events monitoring, the Israeli Ministry of Health initiated active surveillance. METHODS: We retrospectively reviewed data obtained from December 20, 2020, to May 31, 2021, regarding all cases of myocarditis and categorized the information using the Brighton Collaboration definition. We analyzed the occurrence of myocarditis by computing the risk difference for the comparison of the incidence after the first and second vaccine doses (21 days apart); by calculating the standardized incidence ratio of the observed-to-expected incidence within 21 days after the first dose and 30 days after the second dose, independent of certainty of diagnosis; and by calculating the rate ratio 30 days after the second dose as compared with unvaccinated persons. RESULTS: Among 304 persons with symptoms of myocarditis, 21 had received an alternative diagnosis. Of the remaining 283 cases, 142 occurred after receipt of the BNT162b2 vaccine; of these cases, 136 diagnoses were definitive or probable. The clinical presentation was judged to be mild in 129 recipients (95%); one fulminant case was fatal. The overall risk difference between the first and second doses was 1.76 per 100,000 persons (95% confidence interval [CI], 1.33 to 2.19), with the largest difference among male recipients between the ages of 16 and 19 years (difference, 13.73 per 100,000 persons; 95% CI, 8.11 to 19.46). As compared with the expected incidence based on historical data, the standardized incidence ratio was 5.34 (95% CI, 4.48 to 6.40) and was highest after the second dose in male recipients between the ages of 16 and 19 years (13.60; 95% CI, 9.30 to 19.20). The rate ratio 30 days after the second vaccine dose in fully vaccinated recipients, as compared with unvaccinated persons, was 2.35 (95% CI, 1.10 to 5.02); the rate ratio was again highest in male recipients between the ages of 16 and 19 years (8.96; 95% CI, 4.50 to 17.83), with a ratio of 1 in 6637. CONCLUSIONS: The incidence of myocarditis, although low, increased after the receipt of the BNT162b2 vaccine, particularly after the second dose among young male recipients. The clinical presentation of myocarditis after vaccination was usually mild.

Real-world Israeli single institution experience with PET-PSMA for staging of patients with clinically staged localized prostate carcinoma.

BACKGROUND: A recent prospective trial, the proPSMA study, showed superior specificity and sensitivity of Positron emission tomography (PET) - Prostate-specific membrane antigen (PSMA) imaging compared standard Computerized tomography (CT) and bone scan for staging of recently diagnosed high-risk local prostate carcinoma for curative intent treatment. AIM: To share our experience with false-positive PET PSMA scans in newly diagnosed intermediate-risk prostate cancer. METHODS AND RESULTS: Here, we report a series of eight patients who underwent systemic staging using PET-PSMA with false-positive results who were ultimately treated with definitive radiation or surgery. Of the eight patients, two patients were diagnosed with favorable intermediate disease, four with unfavorable intermediate risk, and two with high-risk disease. Seven of eight were shown to have false-positive bone uptake, one patient had uptake in lung nodules. Three patients underwent bone biopsy and proven benign. The rest of the patients were proven as non-metastatic radiologically by repeat PSMA, CT, or Magnetic resonance imaging (MRI). All subsequently preceded to definitive localized treatment and remain disease free as of this study. CONCLUSION: This study emphasizes the importance of prudent clinical judgment when utilizing this highly sensitive imaging technique.

Spatial Intratumoral Heterogeneity Expression of PD-L1 Antigen in Head and Neck Squamous Cell Carcinoma.

INTRODUCTION: Immune-checkpoint inhibitors have demonstrated a significant survival benefit in metastatic and non-resectable head and neck squamous cell carcinoma (HNSCC). Patients with a combined positivity score (CPS) of 20 and higher benefit the most from therapy. Inaccurate definition of the CPS category might lead to the incorrect stratification of patients to immunotherapy. This study's main aim was to investigate programmed death-ligand 1 (PD-L1) antigen expression in HNSCC in diverse clinical situations and histological settings. MATERIALS AND METHODS: This is a prospective cohort study conducted in a tertiary referral medical center. Tissues were investigated for PD-L1 expression using the FDA-approved 22C3 immunohistochemistry assay (Dako). We analyzed potential associations between the CPS category and meaningful demographic, clinical, and outcome metrics. Furthermore, we investigated morphologically separate sites for CPS scores in whole surgical tissue specimens and matched preoperative biopsies. RESULTS: We analyzed 36 patients, of whom 26 had oral cavity SCC and 10 had laryngeal SCC. The overall, disease-specific, and progression-free survival of the HNSCC group of patients were not associated with the CPS category (p = 0.45, p = 0.31, and p = 0.88, respectively). There was a significant (18%, 95% CI 0.65-0.9) inconsistency between the CPS category determined in biopsies versus whole carcinoma analyses. We also found an uneven distribution of whole-tumor CPS attributed to spatial carcinoma invasiveness, tumor differentiation, and inflammatory cell infiltration heterogeneity. DISCUSSION AND CONCLUSIONS: Our data suggest that careful selection of tumor area for CPS analysis is important. PD-L1 antigen expression, clinically represented by CPS, may be up- or down-categorized in different clinical and pathological circumstances. The high whole-tissue CPS category scatter may clinically result in potential treatment modifications. We argue that CPS analysis requires not only adequacy (at least 100 viable tumor cells), but also correct representation of the tumor microenvironment.

Neonatal thrombotic microangiopathy secondary to factor I variant with Hirschsprung disease.

Neonatal thrombotic microangiopathy (TMA) is a rare and severe disease characterized by a triad of non-immune hemolytic anemia, thrombocytopenia, and organ dysfunction in neonates. We describe herein an early-term infant who underwent hemicolectomy at 4 days of age due to intestinal perforation. Following surgery, the patient had recurrent bouts of vomiting and abdominal distention, together with acute kidney injury, non-immune hemolytic anemia, and severe thrombocytopenia. Low complement levels raised the possibility of complement-mediated neonatal TMA. Finally, genetic tests identified a heterozygous mutation in the complement factor I gene. Anti-C5 monoclonal antibody therapy led to complete cessation of the hematological and renal manifestations, but symptoms of intestinal obstruction recurred. Intestinal biopsy demonstrated aganglionosis, compatible with Hirschsprung disease. This presentation is the first known case of neonatal complement-mediated TMA associated with Hirschsprung disease. Moreover, it highlights the importance of considering a diagnosis of TMA in cases of atypical neonatal infectious presentation.

Human Nasal Turbinate Tissues in Organ Culture as a Model for Human Cytomegalovirus Infection at the Mucosal Entry Site.

The initial events of viral infection at the primary mucosal entry site following horizontal person-to-person transmission have remained ill defined. Our limited understanding is further underscored by the absence of animal models in the case of human-restricted viruses, such as human cytomegalovirus (HCMV), a leading cause of congenital infection and a major pathogen in immunocompromised individuals. Here, we established a novel ex vivo model of HCMV infection in native human nasal turbinate tissues. Nasal turbinate tissue viability and physiological functionality were preserved for at least 7 days in culture. We found that nasal mucosal tissues were susceptible to HCMV infection, with predominant infection of ciliated respiratory epithelial cells. A limited viral spread was demonstrated, involving mainly stromal and vascular endothelial cells within the tissue. Importantly, functional antiviral and proleukocyte chemotactic signaling pathways were significantly upregulated in the nasal mucosa in response to infection. Conversely, HCMV downregulated the expression of nasal epithelial cell-related genes. We further revealed tissue-specific innate immune response patterns to HCMV, comparing infected human nasal mucosal and placental tissues, representing the viral entry and the maternal-to-fetal transmission sites, respectively. Taken together, our studies provide insights into the earliest stages of HCMV infection. Studies in this model could help evaluate new interventions against the horizontal transmission of HCMV.IMPORTANCE HCMV is a ubiquitous human pathogen causing neurodevelopmental disabilities in congenitally infected children and severe disease in immunocompromised patients. The earliest stages of HCMV infection in the human host have remained elusive in the absence of a model for the viral entry site. Here, we describe the establishment and use of a novel nasal turbinate organ culture to study the initial steps of viral infection and the consequent innate immune responses within the natural complexity and the full cellular repertoire of human nasal mucosal tissues. This model can be applied to examine new antiviral interventions against the horizontal transmission of HCMV and potentially that of other viruses.

Rapamycin and dexamethasone during pregnancy prevent tuberous sclerosis complex-associated cystic kidney disease.

Chronic kidney disease is the main cause of mortality in patients with tuberous sclerosis complex (TSC) disease. The mechanisms underlying TSC cystic kidney disease remain unclear, with no available interventions to prevent cyst formation. Using targeted deletion of TSC1 in nephron progenitor cells, we showed that cysts in TSC1-null embryonic kidneys originate from injured proximal tubular cells with high mTOR complex 1 activity. Injection of rapamycin to pregnant mice inhibited the mTOR pathway and tubular cell proliferation in kidneys of TSC1-null offspring. Rapamycin also prevented renal cystogenesis and prolonged the life span of TSC newborns. Gene expression analysis of proximal tubule cells identified sets of genes and pathways that were modified secondary to TSC1 deletion and rescued by rapamycin administration during nephrogenesis. Inflammation with mononuclear infiltration was observed in the cystic areas of TSC1-null kidneys. Dexamethasone administration during pregnancy decreased cyst formation by not only inhibiting the inflammatory response, but also interfering with the mTORC1 pathway. These results reveal mechanisms of cystogenesis in TSC disease and suggest interventions before birth to ameliorate cystic disease in offspring.

Differential microRNAs expression in calcified versus rheumatic aortic valve disease.

BACKGROUND: The aortic valve (AV) is the most commonly affected valve in valvular heart diseases (VHDs). The objective of the study is to identify microRNA (miRNA) molecules expressed in VHDs and the differential expression patterns of miRNA in AVs with either calcification or rheumatism etiologies. METHODS: Human AVs were collected during valve replacement surgery. RNA was extracted and miRNA containing libraries were prepared and sequenced using the next generation sequencing (NGS) approach. miRNAs identified as differentially expressed between the two etiologies were validated by quantitative real-time polymerase chain reaction (qPCR). The receiver operating characteristic (ROC) curve analysis was performed to examine the ability of relevant miRNA to differentiate between calcification and rheumatism etiologies. RESULTS: Rheumatic and calcified AV samples were prepared for the NGS and were successfully sequenced. The expression was validated by the qPCR approach in 46 AVs, 13 rheumatic, and 33 calcified AVs, confirming that miR-145-5p, miR-199a-5p, and miR-5701 were significantly higher in rheumatic AVs as compared with calcified AVs. ROC curve analysis revealed that miR-145-5p had a sensitivity of 76.92% and a specificity of 94.12%, area under the curve (AUC) = 0.88 (P = .0001), and miR-5701 had a sensitivity of 84.62% and a specificity of 76.47%, AUC = 0.78 (P = .0001), whereas miR-199a-5p had a sensitivity of 84.62%, and a specificity of 57.58%, AUC = 0.73 (P = .0083). CONCLUSION: We documented differential miRNA expression between AV disease etiologies. The miRNAs identified in this study advance our understanding of the mechanisms underlining AV disease.

Chronic expression of p16INK4a in the epidermis induces Wnt-mediated hyperplasia and promotes tumor initiation.

p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.

Author Correction: Clinical Implications of Sub-grouping HER2 Positive Tumors by Amplicon Structure and Co-amplified Genes.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.