Transcriptomic Profiling Reveals Cancer-Associated Fibroblasts as Potential Targets for the Prognosis and Treatment of Cervical Squamous Cell Carcinoma

To understand the etiological, structural, and immunological characteristics of cervical squamous cell carcinoma (CSCC), we conducted single nucleus RNA sequencing (snRNA-seq) and spatial transcriptomics (ST) experiments for cervical samples from 20 individuals. When exploring the possible factors shaping the intra-individual immune heterogeneity in CSCC, we identified a cluster of cancer-associated fibroblasts (CAFs) enriched around some tumors, which highly expressed ACTA2, POSTN, ITGB4, and FAP. Results showed that the CAFs might support the growth and metastasis of tumors by inhibiting lymphocyte infiltration and remodeling the tumor extracellular matrix. Moreover, high CAF signals predicted poorer clinical outcomes for CSCC patients. Our data also revealed the infection profiles of HPV in tumors, the critical factors involved in the progression of cervical cancerous lesions, and the association between tumor metabolism and immune response intensity. Collectively, our findings may improve the prognostic and therapeutic methods for CSCC.

Primary Lymphoepithelioma-like Hepatocellular Carcinoma With Hepatic Cyst: a Case Report and Review of Literature

Background: Lymphoepithelioma-like hepatic carcinoma is a rare malignant tumor. It includes lymphoepithelioma-like hepatocellular carcinoma (LEL-HCC) and lymphoepithelioma-like intrahepatic cholangiocarcinoma (LEL-ICC). we report the first case of hepatic cyst and LEL-HCC, which is extremely rare among hepatocellular carcinoma.Case presentation: A 47-year-old female was admitted to our hospital with an unexpected health examination finding of liver nodules. Subsequently, the Computerized tomography (CT) revealed a tumor and a cyst. The Magnetic resonance imaging (MRI) found a 23×18 mm hepatic cyst in the right lobe of the liver and a 21×17 mm nodule in the left lateral lobe of the liver, and the nodule imaging was likely liver cancer. The patient underwent laparoscopic left lateral hepatectomy, and the histopathological examination revealed undifferentiated heterogeneous glandular epithelial cells with obvious lymphocyte infiltration. The Immunohistochemistry study CD3, CD20, Ki-67, CK, and CK19 results were positive, while CD10, P53, and CEA were negative in tumor tissues. Epstein-Barr virus (EBV) was negative in situ hybridization. For this patient, the LEL-HCC diagnostic was made according to the histopathological result. Surgical resection is the first choice for this kind of patients, but immunotherapy may be more promising for them in the future. Conclusion: LEL-HCC is a rare variant of HCC, characterized by dense lymphocyte infiltration and a good prognosis. We report the first patient with hepatic cysts and LEL-HCC, which is distinctly uncommon in the HCC patients. Because the hepatic cyst and tumor are similar in certain imaging studies, our work can provide information for clinical diagnosis.

CD8+ Lymphocyte Infiltration is a Specific Feature of Colitis Induced By Immune Checkpoint Inhibitors

Background Immune checkpoint inhibitors (ICPIs) have revolutionized cancer therapy, although immune-related adverse events (irAEs) remain a severe issue. The clinical characteristics of colitis induced by ICPIs are very similar to inflammatory bowel disease. Recently, CD8+ lymphocyte infiltration into organs has been associated with the onset of irAEs. The present study compared the histological infiltration of CD8+ lymphocytes in irAE colitis with that in other colitis. Methods Among 102 newly diagnosed and untreated patients, 12 with irAE colitis, 37 with ulcerative colitis (UC), 22 with Crohn's disease (CD), and 31 with ischemic colitis (IC) were retrospectively enrolled. Biopsy specimens were obtained from endoscopic areas of high inflammation for immunohistochemical analysis of the number of CD4+ and CD8+ lymphocytes in the most inflamed high-powered microscopic field. Results In irAE colitis, CD8+ lymphocyte infiltration was significantly greater than that of CD4+ lymphocytes (p < 0.01). The amount of CD8+ lymphocyte infiltration was significantly higher in irAE colitis than in UC (p < 0.05), CD (p < 0.05), and IC (p < 0.01). The CD8+/CD4+ ratio was also significantly higher in irAE colitis (p < 0.01 vs. UC, CD, and IC, respectively). The optimal cut-off CD8+/CD4+ ratio for diagnosing irAE colitis was 1.17 (sensitivity: 83%, specificity: 84%). The optimal cut-off the number of CD8+ lymphocytes for diagnosing irAE colitis was 102 cells/high-power field (sensitivity: 75%, specificity: 81%). Conclusions Greater CD8+ lymphocyte infiltration and a higher CD8+/CD4+ ratio may be simple and useful biomarkers to distinguish irAE colitis from other forms of colitis.

Early application of extracorporeal membrane oxygenation for myocarditis with shock: a case report

The current therapy for myocarditis is immunosuppressive therapy. However, in rare cases in which patients do not respond to intervention, their condition can rapidly deteriorate to myocarditis with shock, which is characterized by extensive and diffuse lymphocyte infiltration in the myocardium. Most cases of myocarditis are caused by virus-mediated damage of cardiomyocytes, and its clinical manifestations are ventricular arrhythmia and hemodynamic disturbances. Extracorporeal membrane oxygenation is an effective intervention, which regulates hemodynamic stability and avoids systemic hypoperfusion. This intervention has been used to sustain hemodynamic stability in patients with myocarditis and shock. We report here early application of extracorporeal membrane oxygenation for successful treatment of a patient with myocarditis and shock.

911 Immune profiling reveals enrichment of distinct immune signatures in high-risk oral potentially malignant disorders

BackgroundPatients with oral potentially malignant disorders (OPMD) having moderate or severe oral epithelial dysplasia (OED) have a greater risk of developing oral squamous cell carcinoma (OSCC) compared to mild OED with an odds ratio of 2.4.1 The involvement of specific immune cell types associated with malignant transformation have been reported, giving rise to clinical trials in immunoprevention. However, the immune landscape of OPMD remains understudied. In this study, we aimed to elucidate the immune landscape of high-risk OPMD by transcriptomic profiling for the identification of potential immunoprevention strategy.MethodsHistological evaluation was performed on hematoxylin and eosin (H&E)-stained tissues to investigate the differences of lymphocyte infiltration in benign lesions (n=16), high-risk OPMD consisted of moderate and severe OED (n=46) and early-stage OSCC (n=6). Formalin-fixed paraffin-embedded tissue sections of selected cases from each sample type were subjected to RNA sequencing. Weighted-gene-correlation network analysis (WGCNA) was used to identify key gene modules expressed in specific disease type.2 The immune landscape of high-risk OPMD was elucidated by the enrichment of immune signatures using single-sample gene set enrichment analysis.3–5 The response of high-risk OPMD to anti-PD1 treatment was predicted by the detection of T-cell-inflamed condition.6 Validation was performed by multiplex immunofluorescent (mIF) staining.ResultsOur H&E evaluation showed that lymphocyte infiltration into the epithelial was seen in 80% of high-risk OPMD and early-stage OSCC, compared to 9% of benign lesion. Gene modules identified from WGCNA analysis revealed that genes involved in immune-related pathways were overexpressed in high-risk OPMD and in early-stage OSCC when compared to benign lesion, but unchanged between high-risk OPMD and early-stage OSCC. We further demonstrated that immune signatures representing lymphocyte infiltration, MHC-I antigen presentation and cytotoxic immune responses were enriched in high-risk OPMD, indicating the presence of immune surveillance. High-risk OPMD can be grouped into the T-cell-inflamed and non-immune reactive subtypes. The T-cell-inflamed subtype is enriched with T cells, interferon signaling and PD-1/PD-L1 immune checkpoint proteins, suggesting that these lesions may be amenable to anti-PD1 treatment. Meanwhile, the non-immune reactive subtype demonstrated low enrichment in signatures for immune cell infiltration, indicating a need of intervention to induce lymphocyte infiltration. Using mIF staining, we observed an increase of CD45+ immune cell population expressing PD-L1 in high-risk OPMD.ConclusionsImmune surveillance is a prominent feature of high-risk OPMD. However, different subsets of high-risk OPMD exist, suggesting a need of different immunoprevention approaches to prevent disease progression which warrants further investigation.AcknowledgementsThis study was supported and funded by the Global Challenge Research Fund by the Medical Research Council, UK (MR/P024351/1) and Cancer Research Malaysia. We thank the Ong Heng Tiang & Ong Sek Pek Foundation for scholarship sponsorship.ReferencesIocca O, Sollecito TP, Alawi F, et al. Potentially malignant disorders of the oral cavity and oral dysplasia: a systematic review and meta-analysis of malignant transformation rate by subtype. Head Neck 2020;42:539–55.Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008;9:559.Subramanian A, Tamayo P, Mootha VK, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102:15545–50.Chen YP, Wang YQ, Lv JW, et al. Identification and validation of novel microenvironment-based immune molecular subgroups of head and neck squamous cell carcinoma: implications for immunotherapy. Ann Oncol 2019;30:68–75.Thorsson V, Gibbs DL, Brown SD, et al. The immune landscape of cancer. Immunity 2018;48:812–30.Ayers M, Lunceford J, Nebozhyn M, et al. IFN-gamma-related mRNA profile predicts clinical response to PD-1 blockade. J Clin Invest 2017;127:2930–40.Ethics ApprovalThe use of clinical specimens in this study has been approved by the Medical Ethics Committee, Faculty of Dentistry, University of Malaya [DF OS1624/0073(L)], and The National Medical Research Register, Malaysia [NMRR-16-1764-32566 (IIR)].

Melatonin Prevents T Lymphocyte Infiltration to the Kidneys of Hypertensive Rats, Induced by a High-Salt Diet, by Preventing the Expression of CXCR3 Ligand Chemokines

In a previous study, we demonstrated that melatonin prevents kidney damage in a salt-induced hypertension model by decreasing oxidative stress. We hypothesized that this effect involves melatonin’s immunomodulatory properties. In vivo Study-Dahl salt-sensitive (DSS) rats were fed normal chow, a high-salt diet (HSD), or a HSD and melatonin (30 mg/kg/day) in their water for eight weeks. Kidneys were harvested for immediate lymphocyte isolation and characterization by Flow cytometry (CD3+CD4+ and CD3+CD8+) and for lymphocyte chemoattractant (mainly CXCL chemokines) gene expression studies. In vitro study-rat mesangial cells (RMC) were cultured in a high-salt medium without and with melatonin. A HSD was associated with significant renal infiltration of CD4+ and CD8+ T lymphocytes compared to control. Melatonin significantly reduced renal lymphocyte infiltration. A HSD significantly increased mRNA expression of CXCL chemokines. Adding melatonin to the HSD abolished this effect. Treating RMC cells with salt increased the expression of CXCL10 and CXCL11 but not CXCL9. Adding melatonin to the culture media prevented this increase. Treating HSD-fed rats with melatonin decreased renal lymphocyte chemoattractant mRNA expression and is associated with significantly reducing renal T lymphocyte infiltration. Salt may have a direct effect on chemokine-producing renal cells, which is blunted by melatonin treatment.

Breast Cancer Consensus Subtypes: A system for subtyping breast cancer tumors based on gene expression

Breast cancer is heterogeneous in prognoses and drug responses. To organize breast cancers by gene expression independent of statistical methodology, we identified the Breast Cancer Consensus Subtypes (BCCS) as the consensus groupings of six different subtyping methods. Our classification software identified seven BCCS subtypes in a study cohort of publicly available data (n = 5950) including METABRIC, TCGA-BRCA, and data assayed by Affymetrix arrays. All samples were fresh-frozen from primary tumors. The estrogen receptor-positive (ER+) BCCS subtypes were: PCS1 (18%) good prognosis, stromal infiltration; PCS2 (15%) poor prognosis, highly proliferative; PCS3 (13%) poor prognosis, highly proliferative, activated IFN-gamma signaling, cytotoxic lymphocyte infiltration, high tumor mutation burden; PCS4 (18%) good prognosis, hormone response genes highly expressed. The ER− BCCS subtypes were: NCS1 (11%) basal; NCS2 (10%) elevated androgen response; NCS3 (5%) cytotoxic lymphocyte infiltration; unclassified tumors (9%). HER2+ tumors were heterogeneous with respect to BCCS.