The utility of the lineage specific immunohistochemical stains SATB2, CDX2, and villin, and the mucin glycoproteins MUC2, MUC5AC, and MUC6 to distinguish pulmonary invasive mucinous adenocarcinoma from metastatic colorectal carcinoma.

CONTEXT: The lungs are a common site of tumor metastasis. While morphology and immunophenotype can help differentiate primary from metastatic tumors, distinguishing pulmonary invasive mucinous adenocarcinoma (PIMA) from metastatic colorectal adenocarcinoma (CRC) may occasionally be challenging due to overlapping morphological and immunohistochemical features. Lineage-specific markers such as CDX2, TTF-1, and napsin A are helpful with pulmonary non-mucinous adenocarcinoma (PNMA), however they are non-specific and insensitive when applied to PIMA. SATB2 is a newer marker that distinguishes CRC from upper gastrointestinal and pancreaticobiliary tumors; its utility in distinguishing CRC from PIMA has not been fully elucidated. OBJECTIVE: To evaluate the performance of lineage-specific and mucin glycoprotein immunostains in distinguishing PIMA and CRC. DESIGN: We stained tissue microarrays comprising 34 PNMA, 31 PIMA, and 32 CRC with CK7, CK20, SATB2, CDX2, villin, TTF-1, napsin A, and gel-forming mucins MUC2, MUC5AC, and MUC6. RESULTS: PIMA showed significant (>50% of cells) expression of SATB2 (6%), CDX2 (6%), villin (74%), TTF-1 (13%), and napsin A (23%). However, significant CK7 expression was seen in nearly all PIMA (30/31) and none of the metastatic CRC. CONCLUSION: Our results suggest that CK7 remains one of the most useful markers for distinguishing primary PIMA from metastatic CRC. Expression of the mucin glycoproteins MUC5AC and MUC6 and lack of expression of MUC2 favored a diagnosis of PIMA, but expression of these markers was too heterogeneous to be of clinical utility. To our knowledge this is the only study comparing the immunohistochemical profile of PIMA and metastatic CRC in lung metastasectomy specimens.

Ultrastructural cardiac pathology: the wide (yet so very small) world of cardiac electron microscopy.

Electron microscopy (EM) was a popular diagnostic tool in the 1970s and early 80s. With the adoption of newer, less expensive techniques, such as immunohistochemistry, the role of EM in diagnostic surgical pathology has dwindled substantially. Nowadays, even in academic centers, EM interpretation is relegated to renal pathologists and the handful of (aging) pathologists with experience using the technique. As such, EM interpretation is truly arcane-understood by few and mysterious to many. Nevertheless, there remain situations in which EM is the best or only ancillary test to ascertain a specific diagnosis. Thus, there remains a critical need for the younger generation of surgical pathologists to learn EM interpretation. Recognizing this need, cardiac EM was made the theme of the Cardiovascular Evening Specialty Conference at the 2023 United States and Canadian Academy of Pathology (USCAP) annual meeting in New Orleans, Louisiana. Each of the speakers contributed their part to this article, the purpose of which is to review EM as it pertains to myocardial tissue and provide illustrative examples of the spectrum of ultrastructural cardiac pathology seen in storage/metabolic diseases, cardiomyopathies, infiltrative disorders, and cardiotoxicities.

RV-specific Targeting of Snai1 Rescues Pulmonary Hypertension-induced Right Ventricular Failure by Inhibiting EndMT and Fibrosis via LOXL2 Mediated Mechanism.

Background: Pulmonary hypertension (PH)-induced right ventricular (RV) failure (PH-RVF) is a significant prognostic determinant of mortality and is characterized by RV hypertrophy, endothelial-to-mesenchymal transition (EndMT), fibroblast-to-myofibroblast transition (FMT), fibrosis, and extracellular matrix (ECM)-remodeling. Despite the importance of RV function in PH, the mechanistic details of PH-RVF, especially the regulatory control of RV EndMT, FMT, and fibrosis, remain unclear. The action of transcription factor Snai1 is shown to be mediated through LOXL2 recruitment, and their co-translocation to the nucleus, during EndMT progression. We hypothesize that RV EndMT and fibrosis in PH-RVF are governed by the TGFß1-Snai1-LOXL2 axis. Furthermore, targeting Snai1 could serve as a novel therapeutic strategy for PH-RVF. Methods: Adult male Sprague Dawley rats (250-300g) received either a single subcutaneous injection of Monocrotaline (MCT, 60mg/kg, n=9; followed for 30-days) or Sugen (SU5416 20mg/kg, n=9; 10% O 2 hypoxia for 3-weeks followed by normoxia for 2-weeks) or PBS (CTRL, n=9). We performed secondary bioinformatics analysis on the RV bulk RNA-Seq data from MCT, SuHx, and PAB rats and human PH-PVF. We validated EndMT and FMT and their association with Snai1 and LOXL2 in the RVs of MCT and SuHx rat models and human PH-RVF using immunofluorescence, qPCR, and Western blots. For in vivo Snai1 knockdown (Snai1-KD), MCT-rats either received Snai1-siRNA (n=7; 5nM/injection every 3-4 days; 4-injections) or scramble (SCRM-KD; n=7) through tail vein from day 14-30 after MCT. Echocardiography and catheterization were performed terminally. Bulk RNASeq and differential expression analysis were performed on Snai1- and SCRM-KD rat RVs. In vitro Snai1-KD was performed on human coronary artery endothelial cells (HCAECs) and human cardiac fibroblasts (HCFs) under hypoxia+TGFß1 for 72-hrs. Results: PH-RVF had increased RVSP and Fulton index and decreased RV fractional area change (RVFAC %). RV RNASeq demonstrated EndMT as the common top-upregulated pathway between rat (MCT, SuHx, and PAB) and human PH-RVF. Immunofluorescence using EndMT- and FMT-specific markers demonstrated increased EndMT and FMT in RV of MCT and SuHx rats and PH-RVF patients. Further, RV expression of TGFß1, Snai1, and LOXL2 was increased in MCT and SuHx. Nuclear co-localization and increased immunoreactivity, transcript, and protein levels of Snai1 and LOXL2 were observed in MCT and SuHx rats and human RVs. MCT rats treated with Snai1-siRNA demonstrated decreased Snai1 expression, RVSP, Fulton index, and increased RVFAC. Snai1-KD resulted in decreased RV-EndMT, FMT, and fibrosis via a LOXL2-dependent manner. Further, Snai1-KD inhibited hypoxia+TGFß1-induced EndMT in HCAECs and FMT in HCFs in vitro by decreasing perinuclear/nuclear Snai1+LOXL2 expression and co-localization. Conclusions: RV-specific targeting of Snai1 rescues PH-RVF by inhibiting EndMT and Fibrosis via a LOXL2-mediated mechanism.

Human leukocyte antigen class I antibody-activated endothelium promotes CD206+ M2 macrophage polarization and MMP9 secretion through TLR4 signaling and P-selectin in a model of antibody-mediated rejection and allograft vasculopathy.

HLA donor-specific antibodies (DSA) elicit alloimmune responses against the graft vasculature, leading to endothelial cell (EC) activation and monocyte infiltration during antibody-mediated rejection (AMR). AMR promotes chronic inflammation and remodeling, leading to thickening of the arterial intima termed transplant vasculopathy or cardiac allograft vasculopathy (CAV) in heart transplants. Intragraft-recipient macrophages serve as a diagnostic marker in AMR; however, their polarization and function remain unclear. In this study, we utilized an in vitro Transwell coculture system to explore the mechanisms of monocyte-to-macrophage polarization induced by HLA I DSA-activated ECs. Anti-HLA I (IgG or F(ab')2) antibody-activated ECs induced the polarization of M2 macrophages with increased CD206 expression and MMP9 secretion. However, inhibition of TLR4 signaling or PSGL-1-P-selectin interactions significantly decreased both CD206 and MMP9. Monocyte adherence to Fc-P-selectin coated plates induced M2 macrophages with increased CD206 and MMP9. Moreover, Fc-receptor and IgG interactions synergistically enhanced active-MMP9 in conjunction with P-selectin. Transcriptomic analysis of arteries from DSA+CAV+ rejected cardiac allografts and multiplex-immunofluorescent staining illustrated the expression of CD68+CD206+CD163+MMP9+ M2 macrophages within the neointima of CAV-affected lesions. These findings reveal a novel mechanism linking HLA I antibody-activated endothelium to the generation of M2 macrophages which secrete vascular remodeling proteins contributing to AMR and CAV pathogenesis.

Extent of Surgery for Medullary Thyroid Cancer and Prevalence of Occult Contralateral Foci.

Importance: Standard treatment for patients with medullary thyroid cancer (MTC) consists of total thyroidectomy with central neck dissection, but the rationale for bilateral surgery in patients with unilateral disease on ultrasonography remains unclear. Objective: To determine the presence of occult contralateral disease (lesions not seen on preoperative ultrasonography) in patients with MTC as a rationale for total thyroidectomy. Design, Setting, and Participants: This multi-institutional, retrospective cohort study was conducted from September 1998 to April 2022 in academic medical centers and included patients with MTC who underwent thyroidectomy with preoperative imaging. Main Outcomes and Measures: The primary end point was the prevalence of sonographically occult foci of MTC in the contralateral lobe among patients with sporadic MTC. Results: The cohort comprised 176 patients with a median age at diagnosis of 55 years (range, 2-87 years), 69 (57.6%) of whom were female. Genetic testing was performed in 109 patients (61.9%), 48 (27.5%) of whom carried germline RET variants. Initial surgical management consisted of total thyroidectomy (161 [91.0%]), lobectomy followed by completion thyroidectomy (7 [4.0%]), and lobectomy alone (8 [4.5%]). Central and lateral neck dissections were performed as part of initial therapy for 146 patients (83.1%). In the entire cohort of 176 patients, 46 (26.0%) had contralateral foci disease and 9 (5.1%) had occult contralateral foci that were not identified on preoperative ultrasonography. Among 109 patients who underwent genetic testing, 38 (34.9%) had contralateral disease, 8 (7.3%) of whom had occult contralateral disease not seen on preoperative ultrasonography. Patients with sporadic MTC experienced a 95.7% reduction in the odds of having a focus of MTC in the contralateral lobe compared with patients with a germline RET variant (odds ratio, 0.043; 95% CI, 0.013-0.123). When adjusting for age, sex, tumor size, and lymph node involvement, the odds ratio of having contralateral MTC in patients with sporadic disease was 0.034 (95% CI, 0.007-0.116). Among patients who underwent lobectomy alone with postoperative calcitonin levels, 5 of 12 (41.7%) achieved undetectable calcitonin levels (<2.0 pg/mL; to convert to pmol/L, multiply by 0.292). Conclusions and Relevance: The results of this cohort study suggest that a staged approach involving initial thyroid lobectomy could be considered in patients with sporadic MTC and no contralateral ultrasonography findings, with no further surgery if calcitonin levels became undetectable. Further work using prospective randomized clinical trials to evaluate lobectomy as a biochemical cure in patients presenting with unilateral disease is warranted.

Spatial multiomics of arterial regions from cardiac allograft vasculopathy rejected grafts reveal novel insights into the pathogenesis of chronic antibody-mediated rejection.

Cardiac allograft vasculopathy (CAV) causes late graft failure and mortality after heart transplantation. Donor-specific antibodies (DSAs) lead to chronic endothelial cell injury, inflammation, and arterial intimal thickening. In this study, GeoMx digital spatial profiling was used to analyze arterial areas of interest (AOIs) from CAV+DSA+ rejected cardiac allografts (N = 3; 22 AOIs total). AOIs were categorized based on CAV neointimal thickening and underwent whole transcriptome and protein profiling. By comparing our transcriptomic data with that of healthy control vessels of rapid autopsy myocardial tissue, we pinpointed specific pathways and transcripts indicative of heightened inflammatory profiles in CAV lesions. Moreover, we identified protein and transcriptomic signatures distinguishing CAV lesions exhibiting low and high neointimal lesions. AOIs with low neointima showed increased markers for activated inflammatory infiltrates, endothelial cell activation transcripts, and gene modules involved in metalloproteinase activation and TP53 regulation of caspases. Inflammatory and apoptotic proteins correlated with inflammatory modules in low neointima AOIs. High neointima AOIs exhibited elevated TGFß-regulated transcripts and modules enriched for platelet activation/aggregation. Proteins associated with growth factors/survival correlated with modules enriched for proliferation/repair in high neointima AOIs. Our findings reveal novel insight into immunological mechanisms mediating CAV pathogenesis.

Severe Acute Respiratory Syndrome Coronavirus 2 Infection Alters Mediators of Lung Tissue Remodeling In Vitro and In Vivo.

BACKGROUND: Altered mediators of airway tissue remodeling such as matrix metalloproteinases (MMPs) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may contribute to morbidity in coronavirus disease 2019 (COVID-19); however, the differential impact of SARS-CoV-2 variants of concern (VOCs) on MMPs is unknown. METHODS: Using both in vitro human airway cell culture model and in vivo transgenic mouse model of SARS-CoV-2 infection, we studied the differential effect of SARS-CoV-2 VOCs on expression of key MMPs and inflammatory mediators in airway cells and tissues. RESULTS: The most consistent findings with all SARS-CoV-2 variants in infected compared to uninfected human bronchial epithelial cell air-liquid interface cultures were the SARS-CoV-2-induced increases in MMP-12 and tissue inhibitor of MMPs. Infection with both SARS-CoV-2 wild type and SARS-CoV-2 Delta variant over 3 days postinfection (dpi) and with Beta variant over 7 dpi increased lung tissue levels of MMP-9 compared to uninfected mice. Overall, SARS-CoV-2 variants had differential dose-dependent impact on secretion of MMP-1, MMP-2, MMP-9, and MMP-12 that varied at the protein versus the gene level and in the early noninflammatory compared to late inflammatory phase of infection. CONCLUSIONS: We provide novel mechanistic insight that the differential impact of SARS-CoV-2 variants on severity of COVID-19 may partially be attributed to unique changes in MMPs.