An Oncogenic Alteration Creates a Microenvironment that Promotes Tumor Progression by Conferring a Metabolic Advantage to Regulatory T Cells.

Only a small percentage of patients afflicted with gastric cancer (GC) respond to immune checkpoint blockade (ICB). To study the mechanisms underlying this resistance, we examined the immune landscape of GC. A subset of these tumors was characterized by high frequencies of regulatory T (Treg) cells and low numbers of effector T cells. Genomic analyses revealed that these tumors bore mutations in RHOA that are known to drive tumor progression. RHOA mutations in cancer cells activated the PI3K-AKT-mTOR signaling pathway, increasing production of free fatty acids that are more effectively consumed by Treg cells than effector T cells. RHOA mutant tumors were resistant to PD-1 blockade but responded to combination of PD-1 blockade with inhibitors of the PI3K pathway or therapies targeting Treg cells. We propose that the metabolic advantage conferred by RHOA mutations enables Treg cell accumulation within GC tumors, generating an immunosuppressive TME that underlies resistance to ICB.

Elevated Myl9 reflects the Myl9-containing microthrombi in SARS-CoV-2-induced lung exudative vasculitis and predicts COVID-19 severity.

The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.

Cervical mucus can be used for metabolite screening in cervical cancer.

Approximately 660,000 women are diagnosed with cervical cancer annually. Current screening options such as cytology or human papillomavirus testing have limitations, creating a need to identify more effective ancillary biomarkers for triage. Here, we evaluated whether metabolomic analysis of cervical mucus metabolism could be used to identify biomarkers of cervical intraepithelial neoplasia (CIN) and cervical cancer. The case-control group consisted of 181 CIN, 69 squamous cell carcinoma (SCC) patients, and 48 healthy controls in the primary cohort. We undertook metabolomic analyses using ultra-HPLC-tandem mass spectrometry. Univariate and multivariate analyses were carried out to profile metabolite characteristics, and receiver operating characteristic (ROC) analysis identified biomarker candidates. Five metabolites conferred the highest discriminatory power for SCC: oxidized glutathione (GSSG) (area under the ROC curve, 0.924; 95% confidence interval, 0.877-0.971), malic acid (0.914, 0.859-0.968), kynurenine (0.884, 0.823-0.945), GSSG/glutathione (GSH) (0.936, 0.892-0.979), and kynurenine/tryptophan (0.909, 0.856-0.961). Malic acid was the best marker for detection of CIN2 or worse (0.858, 0.793-0.922) and was a clinically useful metabolite. We confirmed the reproducibility of the results by validation cohort. Additionally, metabolomic analyses revealed eight pathways strongly associated with cervical neoplasia. Of these, only the tricarboxylic acid cycle was strongly associated with all CINs and cancer, indicating active energy production. Aberrant arginine metabolism by decreasing arginine and increasing citrulline might reduce tumor immunity. Changes in cysteine-methionine and GSH pathways might drive the initiation and progression of cervical cancer. These results suggest that metabolic analysis can identify ancillary biomarkers and could improve our understanding of the pathophysiological mechanisms underlying cervical neoplasia.

Performance of an ancillary test for cervical cancer that measures miRNAs and cytokines in serum and cervical mucus.

Currently, human papillomavirus tests and cytology are used to screen for cervical cancer. However, more accurate ancillary screening tests are needed. MicroRNAs (miRNAs) and cytokines are promising biomarkers that are aberrantly expressed in cervical cancer. Therefore, the potential of developing new screening markers based on the levels of miRNAs and cytokines in serum and local mucus samples from the same patients with cervical neoplasia was investigated. miRNA screening was performed by microarray and measurement using real-time reverse-transcriptase PCR. Cytokine were measured using multiplex bead assay, and changes in expressions were analyzed based on disease severity. As lesions progressed, miR-20b-5p, -155-5p, -144-3p, -451a, and -126-3p expression levels were increased in mucus, and miR-16-5p, -223-3p, and -451a expression levels were decreased in serum. Regarding cytokines, IL-6, IL-8, monocyte chemoattractant protein-1, Eotaxin, interferon-γ, and RANTES were increased, whereas granulocyte-colony-stimulating factor (G-CSF) was significantly decreased in mucus. miRNAs and cytokines in serum did not have high diagnostic accuracy. However, a combination of miR-20b-5p, -451a, -126-3p, Eotaxin, as well as G-CSF in mucus samples, had high diagnostic accuracy with an area under the receiver operating characteristic curve of 0.989 (0.979-0.999). Our results suggest that using mucus for this ancillary test is more beneficial than serum.

Synthetic retinoid-mediated preconditioning of cancer-associated fibroblasts and macrophages improves cancer response to immune checkpoint blockade.

BACKGROUND: The proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs. METHODS: We investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models. RESULTS: High infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs. CONCLUSION: Our data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.

Helicobacter pylori infection associated DNA methylation in primary gastric cancer significantly correlates with specific molecular and clinicopathological features.

Helicobacter pylori induces DNA methylation in gastric mucosa, which links to gastric cancer (GC) risk. In contrast, CpG island methylator phenotype (CIMP) is defined as high levels of cancer-specific methylation and provides distinct molecular and clinicopathological features of GC. The association between those two types of methylation in GC remains unclear. We examined DNA methylation of well-validated H. pylori infection associated genes in GC and its adjacent mucosa and investigated its association with CIMP, various molecular subtypes and clinical features. We studied 50 candidate loci in 24 gastric samples to identify H. pylori infection associated genes. Identified loci were further examined in 624 gastric tissue from 217 primary GC, 217 adjacent mucosa, and 190 mucosae from cancer-free subjects. We identified five genes (IGF2, SLC16A2, SOX11, P2RX7, and MYOD1) as hypermethylated in H. pylori infected gastric mucosa. In non-neoplastic mucosa, methylation of H. pylori infection associated genes was higher in patients with GC than those without. In primary GC tissues, higher methylation of H. pylori infection associated genes correlated with CIMP-positive and its related features, such as MLH1 methylated cases. On the other hand, GC with lower methylation of these genes presented aggressive clinicopathological features including undifferentiated histopathology, advanced stage at diagnosis. H. pylori infection associated DNA methylation is correlated with CIMP, specific molecular and clinicopathological features in GC, supporting its utility as promising biomarker in this tumor type.

The extracellular matrix protein EMILIN-1 impacts on the microenvironment by hampering gastric cancer development and progression.

BACKGROUND: The contribution of the tumor microenvironment and extracellular matrix to the aggressive biology of Gastric Cancer (GC) has been recently characterized; however, the role of EMILIN-1 in this context is unknown. EMILIN-1 is an essential structural element for the maintenance of lymphatic vessel (LV) integrity and displays anti-proliferative properties as demonstrated in skin and colon cancer. Given the key role of LVs in GC progression, the aim of this study was to investigate the role of EMILIN-1 in GC mouse models. METHODS: We used the syngeneic YTN16 cells which were injected subcutaneously and intraperitoneally in genetically modified EMILIN-1 mice. In alternative, carcinogenesis was induced using N-Methyl-N-nitrosourea (MNU). Mouse-derived samples and human biopsies were analyzed by IHC and IF to the possible correlation between EMILIN-1 expression and LV pattern. RESULTS: Transgenic mice developed tumors earlier compared to WT animals. 20 days post-injection tumors developed in EMILIN-1 mutant mice were larger and displayed a significant increase of lymphangiogenesis. Treatment of transgenic mice with MNU associated with an increased number of tumors, exacerbated aggressive lesions and higher levels of LV abnormalities. A significant correlation between the levels of EMILIN-1 and podoplanin was detected also in human samples, confirming the results obtained with the pre-clinical models. CONCLUSIONS: This study demonstrates for the first time that loss of EMILIN-1 in GC leads to lymphatic dysfunction and proliferative advantages that sustain tumorigenesis, and assess the use of our animal model as a valuable tool to verify the fate of GC upon loss of EMILIN-1.

A novel support vector machine-based 1-day, single-dose prediction model of genotoxic hepatocarcinogenicity in rats.

The development of a rapid and accurate model for determining the genotoxicity and carcinogenicity of chemicals is crucial for effective cancer risk assessment. This study aims to develop a 1-day, single-dose model for identifying genotoxic hepatocarcinogens (GHCs) in rats. Microarray gene expression data from the livers of rats administered a single dose of 58 compounds, including 5 GHCs, was obtained from the Open TG-GATEs database and used for the identification of marker genes and the construction of a predictive classifier to identify GHCs in rats. We identified 10 gene markers commonly responsive to all 5 GHCs and used them to construct a support vector machine-based predictive classifier. In the silico validation using the expression data of the Open TG-GATEs database indicates that this classifier distinguishes GHCs from other compounds with high accuracy. To further assess the model's effectiveness and reliability, we conducted multi-institutional 1-day single oral administration studies on rats. These studies examined 64 compounds, including 23 GHCs, with gene expression data of the marker genes obtained via quantitative PCR 24 h after a single oral administration. Our results demonstrate that qPCR analysis is an effective alternative to microarray analysis. The GHC predictive model showed high accuracy and reliability, achieving a sensitivity of 91% (21/23) and a specificity of 93% (38/41) across multiple validation studies in three institutions. In conclusion, the present 1-day single oral administration model proves to be a reliable and highly sensitive tool for identifying GHCs and is anticipated to be a valuable tool in identifying and screening potential GHCs.

Lower accuracy of cytological screening for high-grade squamous intraepithelial neoplasia in women over 50 years of age in Japan.

BACKGROUND: As the population ages in developed countries, the number of Pap smears for cervical cancer screening of older women is increasing. There is concern that cervical atrophy may cause misinterpretation of results for this segment of the population. The present study evaluated the accuracy of screening for high-grade intraepithelial lesions (HSILs) in women younger or older than 50 years, to determine whether aging affects cytological interpretation. METHODS: Patients with HSIL cytology (N = 1565) were dichotomized into those aged 20-49 years or aged ≥ 50 years. Association between histology results and age was examined. Pearson's chi-squared test and Cochran-Armitage trend test were used for statistical analysis. RESULTS: The positive predictive value (PPV) for cervical intraepithelial neoplasia (CIN)2 and worse was 65.2% (62/95) in older women but 87.3% (482/552) in younger women (p < 0.001). Older patients had a significantly lower PPV (p = 1.69 × 10-8). Separately analyzing chronic cervicitis, CIN1 and overt cancer grouped together, compared with another group composed of CIN2 and CIN3, we found that the PPV for CIN2 and CIN3 was lower in older than in younger women [44.2% (42/95)-vs-82.4% (455/552), p < 0.001], respectively. CONCLUSIONS: HSILs are associated with a wide range of disease categories as age increases, and the accuracy of HSIL interpretation is lower in older women.

γH2AX, a DNA Double-Strand Break Marker, Correlates with PD-L1 Expression in Smoking-Related Lung Adenocarcinoma.

In recent years, the choice of immune checkpoint inhibitors (ICIs) as a treatment based on high expression of programmed death-ligand 1 (PD-L1) in lung cancers has been increasing in prevalence. The high expression of PD-L1 could be a predictor of ICI efficacy as well as high tumor mutation burden (TMB), which is determined using next-generation sequencing (NGS). However, a great deal of effort is required to perform NGS to determine TMB. The present study focused on γH2AX, a double-strand DNA break marker, and the suspected positive relation between TMB and γH2AX was investigated. We assessed the possibility of γH2AX being an alternative marker of TMB or PD-L1. One hundred formalin-fixed, paraffin-embedded specimens of lung cancer were examined. All of the patients in the study received thoracic surgery, having been diagnosed with lung adenocarcinoma or squamous cell carcinoma. The expressions of γH2AX and PD-L1 (clone: SP142) were evaluated immunohistochemically. Other immunohistochemical indicators, p53 and Ki-67, were also used to estimate the relationships of γH2AX. Positive relationships between γH2AX and PD-L1 were proven, especially in lung adenocarcinoma. Tobacco consumption was associated with higher expression of γH2AX, PD-L1, Ki-67, and p53. In conclusion, the immunoexpression of γH2AX could be a predictor for the adaptation of ICIs as well of as PD-L1 and TMB.