Glioblastoma with high O6-methyl-guanine DNA methyltransferase expression are more immunologically active than tumors with low MGMT expression.

Background: Glioblastoma (GBM) is a highly lethal brain tumor. The effectiveness of temozolomide (TMZ) treatment in GBM is linked to the methylation status of O6-methyl-guanine DNA methyltransferase (MGMT) promoter. Patients with unmethylated MGMT promoter have limited treatment options available. Consequently, there is a pressing need for alternative therapeutic strategies for such patients. Methods: Data, including transcriptomic and clinical information, as well as information on MGMT promoter methylation status in primary GBM, were obtained from The Cancer Genome Atlas (TCGA) (n=121) and Chinese Glioma Genome Atlas (CGGA) (n=83) datasets. Samples were categorized into high and low MGMT expression groups, MGMT-high (MGMT-H) and MGMT-low (MGMT-L) tumors. A comprehensive transcriptome analysis was conducted to explore the tumor-immune microenvironment. Furthermore, we integrated transcriptome data from 13 GBM patients operated at our institution with findings from tumor-infiltrating lymphocyte (TIL) cultures, specifically investigating their response to autologous tumors. Results: Gene signatures associated with various immune cells, including CD8 T cells, helper T cells, B cells, and macrophages, were noted in MGMT-H tumors. Pathway analysis confirmed the enrichment of immune cell-related pathways. Additionally, biological processes involved in the activation of monocytes and lymphocytes were observed in MGMT-H tumors. Furthermore, TIL culture experiments showed a greater presence of tumor-reactive T cells in MGMT-H tumors compared to MGMT-L tumors. These findings suggest that MGMT-H tumors has a potential for enhanced immune response against tumors mediated by CD8 T cells. Conclusion: Our study provides novel insights into the immune cell composition of MGMT-H tumors, which is characterized by the infiltration of type 1 helper T cells and activated B cells, and also the presence of tumor-reactive T cells evidenced by TIL culture. These findings contribute to a better understanding of the immune response in MGMT-H tumors, emphasizing their potential for immunotherapy. Further studies are warranted to investigate on the mechanisms of MGMT expression and antitumor immunity.

Immuno-genomic analysis reveals eosinophilic feature and favorable prognosis of female non-smoking esophageal squamous cell carcinomas.

Most of esophageal squamous cell carcinoma (ESCC) develop in smoking males in Japan, but the genomic etiology and immunological characteristics of rare non-smoking female ECSS remain unclear. To elucidate the genomic and immunological features of ESCC in non-smoking females, we analyzed whole-genome or transcriptome sequencing data from 94 ESCCs, including 20 rare non-smoking female cases. In addition, 31,611 immune cells were extracted from four ESCC tissues and subject to single-cell RNA-seq. We compared their immuno-genomic and microbiome profiles between non-smoking female and smoking ESCCs. Non-smoking females showed much better prognosis. Whole-genome sequencing analysis showed no significant differences in driver genes or copy number alterations depending on smoking status. The mutational signatures specifically observed in non-smoking females ESCC could be attributed to aging. Immune profiling from RNA-seq revealed that ESCC in non-smoking females had high tumor microenvironment signatures and a high abundance of eosinophils with a favorable prognosis. Single-cell RNA-sequencing of intratumor immune cells revealed gender differences of eosinophils and their activation in female cases. ESCCs in non-smoking females have age-related mutational signatures and gender-specific tumor immune environment with eosinophils, which is likely to contribute to their favorable prognosis.

Alterations in Intratumoral Immune Response before and during Early-On Nivolumab Treatment for Unresectable Advanced or Recurrent Gastric Cancer.

We investigated the tumor immune response in gastric cancer patients receiving third-line nivolumab monotherapy to identify immune-related biomarkers for better patient selection. Nineteen patients (10 males, median age 67 years) who received nivolumab as a third- or later-line therapy were enrolled. We analyzed the tumor immune response in durable clinical benefit (DCB) and non-DCB patients. Pre-treatment and early-on-treatment tumor transcriptomes were examined, and gene expression profiles, immunograms, and T cell receptor (TCR) repertoire were analyzed. DCB was observed in 15.8% of patients, with comparable secondary endpoints (ORR; objective response rate, OS; overall survival, PFS; progression-free survival) to previous trials. The immunograms of individual subjects displayed no significant changes before or early in the treatment, except for the regulatory T cell (Treg) score. Moreover, there were no consistent alterations observed among cases experiencing DCB. The intratumoral immune response was suppressed by previous treatments in most third- or later-line nivolumab recipients. TCR repertoire analysis revealed newly emerged clonotypes in early-on-treatment tumors, but clonal replacement did not impact efficacy. High T cell/Treg ratios and a low UV-radiation-response gene signature were linked to DCB and treatment response. This study emphasizes the tumor immune response's importance in nivolumab efficacy for gastric cancer. High T cell/Treg ratios and specific gene expression signatures show promise as potential biomarkers for treatment response. The tumor-infiltrating immune response was compromised by prior treatments in third-line therapy, implying that, to enhance immunotherapeutic outcomes, commencing treatment at an earlier stage might be preferable. Larger cohort validation is crucial to optimize immune-checkpoint inhibitors in gastric cancer treatment.

Managing a case of Breus' mole with severe fetal growth restriction via sequential ultrasonographic imaging and MRI.

A Breus' mole is a massive subchorionic thrombohematoma that arises below the chorionic plate on the fetal side of the placenta. It requires careful perinatal management because of the associated high incidence of severe fetal growth restriction and intrauterine fetal demise. However, the mechanism of its development remains unclear, and there are no reports examining the continuous changes in the hematomas. Herein, we report a case of a Breus' mole in which ultrasonographic massive subchorionic thrombohematoma changes were observed during pregnancy. A 40-year-old pregnant patient presented with fetal growth restriction, a hematoma with a highly echoic lesion, and an extremely thickened placenta. The clinical picture of massive subchorionic thrombohematoma gradually changed from a high-echoic phase with a 7-cm thick placenta to a high- and low-echoic mixed phase to a completely low-echoic phase with a prominent atrophic placenta (placental thickness = 3.5 cm) in almost 8 weeks. At 34 weeks of gestation, a male infant was delivered via cesarean section due to its nonreassuring fetal status with extremely low birth weight (1230 g). Postpartum histological findings revealed the presence of a Breus' mole. In conclusion, we observed the ultrasonographic changes of the massive subchorionic thrombohematoma that were detected as a placental hemorrhagic infarction by magnetic resonance imaging, from a high- to low-echoic area. The clinical course from massive subchorionic thrombohematoma to Breus' mole may be a prominent atrophic change in the placental tissue during pregnancy. These sequential ultrasonographic findings could be a key factor in understanding the pathophysiology of Breus' moles.

Epicardial fat and Stage B heart failure among overweight/obese and normal weight individuals with diabetes mellitus.

PURPOSE: Although up to 20% of people with type 2 diabetes (DM) have normal BMI (< 25 kg/m2), it remains unclear whether there is a difference in the development of cardiac dysfunction between those with normal and higher BMI. Furthermore, little is known about the relationship of visceral fat with BMI or fitness in asymptomatic patients with DM. METHODS: We prospectively enrolled asymptomatic patients with DM and divided into two groups: BMI ≥ 25kg/m2 (overweight/obese group) versus < 25kg/m2(normal-weight group). Resting echocardiogram followed by exercise stress echocardiogram and exercise gas exchange analysis (in a subgroup) was performed. Cardiac function was evaluated using left ventricular longitudinal strain (LVLS), E/e', and relative wall thickness (RWT). In addition, epicardial fat thickness (EFT) was measured to estimate visceral fat. RESULTS: Normal-weight patients with DM had more EFT compared with overweight/obese patients (0.66 ± 0.17 cm vs. 0.59 ± 0.22 cm, p < 0.05), despite the overlap between the groups. There was no significant difference in the prevalence of LV remodeling (p = 0.49), impaired LVLS (p = 0.22), or increased E/e' (p = 0.26), and these were consistently observed when matched for race. The majority of patients (63%) achieved ≥ 85% of percent peak-predicted VO2. At peak, there was no significant difference in peak VO2 normalized by eLBM (36.4 ± 7.7 vs. 37.8 ± 7.1 ml/kg eLBM/min, p = 0.43) while VO2 normalized by weight (23.6 ± 6.5 vs. 29.6 ± 6.7 ml/kg/min, p < 0.001) and VO2 ratio (5.7 ± 1.7 vs. 7.3 ± 2.4 METs, p = 0.001) were significantly lower in patients with obese/overweight group. There was no significant difference between patients with higher and lower EFT. CONCLUSIONS: Patients with DM and normal BMI have excess epicardial fat compared to those with overweight/obese. Epicardial fat was not directly linked to prevalence of subclinical dysfunction.

Strength training is more effective than aerobic exercise for improving glycaemic control and body composition in people with normal-weight type 2 diabetes: a randomised controlled trial.

AIMS/HYPOTHESIS: Type 2 diabetes in people in the healthy weight BMI category (<25 kg/m2), herein defined as 'normal-weight type 2 diabetes', is associated with sarcopenia (low muscle mass). Given this unique body composition, the optimal exercise regimen for this population is unknown. METHODS: We conducted a parallel-group RCT in individuals with type 2 diabetes (age 18-80 years, HbA1c 47.5-118.56 mmol/mol [6.5-13.0%]) and BMI <25 kg/m2). Participants were recruited in outpatient clinics or through advertisements and randomly assigned to a 9 month exercise programme of strength training alone (ST), aerobic training alone (AER) or both interventions combined (COMB). We used stratified block randomisation with a randomly selected block size. Researchers and caregivers were blinded to participants' treatment group; however, participants themselves were not. Exercise interventions were conducted at community-based fitness centres. The primary outcome was absolute change in HbA1c level within and across the three groups at 3, 6 and 9 months. Secondary outcomes included changes in body composition at 9 months. Per adherence to recommended exercise protocol (PP) analysis included participants who completed at least 50% of the sessions. RESULTS: Among 186 individuals (ST, n=63; AER, n=58; COMB, n=65) analysed, the median (IQR) age was 59 (53-66) years, 60% were men and 83% were Asian. The mean (SD) HbA1c level at baseline was 59.6 (13.1) mmol/mol (7.6% [1.2%]). In intention-to-treat analysis, the ST group showed a significant decrease in HbA1c levels (mean [95% CI] -0.44 percentage points [-0.78, -0.12], p=0.002), while no significant change was observed in either the COMB group (-0.35 percentage points, p=0.13) or the AER group (-0.24 percentage points, p=0.10). The ST group had a greater improvement in HbA1c levels than the AER group (p=0.01). Appendicular lean mass relative to fat mass increased only in the ST group (p=0.0008), which was an independent predictor of HbA1c change (beta coefficient -7.16, p=0.01). Similar results were observed in PP analysis. Only one adverse event, in the COMB group, was considered to be possibly associated with the exercise intervention. CONCLUSIONS/INTERPRETATION: In normal-weight type 2 diabetes, strength training was superior to aerobic training alone, while no significant difference was observed between strength training and combination training for HbA1c reduction. Increased lean mass relative to decreased fat mass was an independent predictor of reduction in HbA1c level. TRIAL REGISTRATION: ClinicalTrials.gov NCT02448498. FUNDING: This study was funded by the National Institutes of Health (NIH; R01DK081371).

Clinical and Echocardiographic Diversity Associated With Physical Fitness in the Project Baseline Health Study: Implications for Heart Failure Staging.

BACKGROUND: Clinical and echocardiographic features may carry diverse information about the development of heart failure (HF). Therefore, we determined heterogeneity in clinical and echocardiographic phenotypes and its association with exercise capacity. METHODS: In 2036 community-dwelling individuals, we defined echocardiographic profiles of left and right heart remodeling and dysfunction. We subdivided the cohort based on presence (+) or absence (-) of HF risk factors (RFs) and echocardiographic abnormalities (RF-/Echo-, RF-/Echo+, RF+/Echo-, RF+/Echo+). Multivariable-adjusted associations between subgroups and physical performance metrics from 6-minute walk and treadmill exercise testing were assessed. RESULTS: The prevalence was 35.3% for RF-/Echo-, 4.7% for RF-/Echo+, 39.3% for RF+/Echo-, and 20.6% for RF+/Echo+. We observed large diversity in echocardiographic profiles in the Echo+ group. Participants with RF-/Echo+ (18.6% of Echo+) had predominantly echocardiographic abnormalities other than left ventricular (LV) diastolic dysfunction, hypertrophy and reduced ejection fraction, whereas their physical performance was similar to RF-/Echo-. In contrast, participants with RF+/Echo+ presented primarily with LV hypertrophy or dysfunction, features that related to lower 6-minute walking distance and lower exercise capacity. CONCLUSIONS: Subclinical echocardiographic abnormalities suggest HF pathogenesis, but the presence of HF risk factors and type of echo abnormality should be considered so as to distinguish adverse from benign adaptation and to stratify HF risk.

Defining left ventricular remodeling using lean body mass allometry: a UK Biobank study.

PURPOSE: The geometric patterns of ventricular remodeling are determined using indexed left ventricular mass (LVM), end-diastolic volume (LVEDV) and concentricity, most often measured using the mass-to-volume ratio (MVR). The aims of this study were to validate lean body mass (LBM)-based allometric coefficients for scaling and to determine an index of concentricity that is independent of both volume and LBM. METHODS: Participants from the UK Biobank who underwent both CMR and dual-energy X-ray absorptiometry (DXA) during 2014-2015 were considered (n = 5064). We excluded participants aged ≥ 70 years or those with cardiometabolic risk factors. We determined allometric coefficients for scaling using linear regression of the logarithmically transformed ventricular remodeling parameters. We further defined a multiplicative allometric relationship for LV concentricity (LVC) adjusting for both LVEDV and LBM. RESULTS: A total of 1638 individuals (1057 female) were included. In subjects with lower body fat percentage (< 25% in males, < 35% in females, n = 644), the LBM allometric coefficients for scaling LVM and LVEDV were 0.85 ± 0.06 and 0.85 ± 0.03 respectively (R2 = 0.61 and 0.57, P < 0.001), with no evidence of sex-allometry interaction. While the MVR was independent of LBM, it demonstrated a negative association with LVEDV in (females: r = - 0.44, P < 0.001; males: - 0.38, P < 0.001). In contrast, LVC was independent of both LVEDV and LBM [LVC = LVM/(LVEDV0.40 × LBM0.50)] leading to increased overlap between LV hypertrophy and higher concentricity. CONCLUSIONS: We validated allometric coefficients for LBM-based scaling for CMR indexed parameters relevant for classifying geometric patterns of ventricular remodeling.

Immunotherapies targeting neoantigens are effective in PD-1 blockade-resistant tumors.

Only a small fraction of tumor-infiltrating lymphocytes can specifically recognize and attack cancer cells in PD-1/PD-L1 blockade therapy. Here, we investigate approaches to expand the neoantigen-specific CD8+ T cells to overcome the difficulties in treating PD-1/PD-L1 blockade-resistant tumors. Mutation-associated neoepitopes of murine nonsmall cell lung cancer ASB-XIV were estimated by whole-exome and RNA sequencing and predicted by MHC-I binding affinity (FPKM >1) in silico. Using ASB-XIV-specific CD8+ T cells, we screened a panel of 257 neoepitope peptides derived from ASB-XIV missense and indel mutations. Mutated Phf3 peptide (mPhf3) was successfully identified as an immunogenic neoepitope. Prophylactic mPhf3-DC vaccination inhibited ASB-XIV tumor growth through CD8+ T cell-mediated antitumor immunity. Combining the mPhf3-DC vaccine and anti-PD-1 treatment elicited robust antitumor activity through the induction of mPhf3-specific CD8+ T cells in the tumor microenvironment. Furthermore, the adoptive transfer of mPhf3-specific CD8+ T cells eradicated ASB-XIV tumors. Likewise, the combination of mutated Cdt1 peptide (mCdt1)-DC vaccine and anti-PD-1 treatment or adoptive transfer of mCdt1-specific CD8+ T cells also led to significant regression of PD-1 blockade-resistant murine gastric YTN16 tumors. In conclusion, a novel immunogenic neoepitope of ASB-XIV was identified for immunotherapy targeting neoantigens. Identification of immunogenic neoantigens can extend the therapeutic strategies by increasing the frequency of neoantigen-specific T cells, even for PD-1/PD-L1 blockade-resistant tumors.

Effectiveness of a Community-Based Structured Physical Activity Program for Adults With Type 2 Diabetes: A Randomized Clinical Trial.

Importance: The efficacy of physical activity interventions among individuals with type 2 diabetes has been established; however, practical approaches to translate and extend these findings into community settings have not been well explored. Objective: To test the effectiveness of providing varying frequencies of weekly structured exercise sessions to improve diabetes control. Design, Setting, and Participants: The IMPACT (Initiate and Maintain Physical Activity in Communities Trial) study was a controlled randomized clinical trial (randomization occurred from October 2016 to April 2019) that included a 6-month, structured exercise intervention either once or thrice weekly vs usual care (UC; advice only). The exercise intervention was conducted at community-based fitness centers. Follow-up visits were conducted in a university research clinic. Participants included adults with type 2 diabetes (hemoglobin A1c [HbA1c] 6.5%-13.0%, not taking insulin, and no precluding health issues). Data analysis was performed from January to April 2022. Interventions: A once-weekly structured exercise group, a thrice-weekly structured exercise group, or UC. Main Outcomes and Measures: The primary outcome was HbA1c at 6 months. Results: A total of 357 participants (143 women [40.1%]) with a mean (SD) age of 57.4 (11.1) years were randomized (119 each to the UC, once-weekly exercise, and thrice-weekly exercise groups). There was no significant difference in HbA1c change by study group in the intention-to-treat analysis at 6 months. Specifically, HbA1c changed by -0.23% (95% CI, -0.48% to 0.01%) in the thrice-weekly exercise group and by -0.16% (95% CI, -0.41% to 0.09%) in the once-weekly exercise group. A total of 62 participants (52.1%) in the once-weekly exercise group and 56 participants (47.1%) in the thrice-weekly exercise group were at least 50% adherent to the assigned structured exercise regimen and were included in the per-protocol analysis. Per-protocol analysis showed that HbA1c changed by -0.35% (95% CI, -0.60% to -0.10%; P = .005) at 3 months and by -0.38% (95% CI, -0.65% to -0.12%; P = .005) at 6 months in the thrice-weekly exercise group compared with UC. There was no significant decrease in HbA1c in the once-weekly exercise group. The exercise intervention was effective in improving self-reported minutes of metabolic equivalent tasks per week for participants in the thrice-weekly exercise group (both overall and per protocol). Conclusions and Relevance: Although the intervention was not effective in the intention-to-treat analysis, participants in the thrice-weekly exercise group who attended at least 50% of the sessions during the 6-month exercise intervention program improved HbA1c levels at 6 months. Future efforts should focus on improving adherence to thrice-weekly structured exercise programs to meet exercise guidelines. Trial Registration: ClinicalTrials.gov Identifier: NCT02061579.

Novel missense COL2A1 variant in a fetus with achondrogenesis type II.

Achondrogenesis type II (ACG2) is a lethal skeletal disorder caused by pathogenic variants in COL2A1. We present a fetus with cystic hygroma and severe shortening of the limbs at 14 weeks of gestation. We performed postnatal genetic analysis of the parents and fetus to diagnose the disease. A novel missense variant of COL2A1 [NM_001844.5: c.2987G>A, (p. Gly996Asp)] was identified, which led to the ACG2 diagnosis.

Association of left ventricular diastolic function with coronary artery calcium score: A Project Baseline Health Study.

BACKGROUND: Coronary artery calcium (CAC) and left ventricular diastolic dysfunction (LVDD) are strong predictors of cardiovascular events and share common risk factors. However, their independent association remains unclear. METHODS: In the Project Baseline Health Study (PBHS), 2082 participants underwent cardiac-gated, non-contrast chest computed tomography (CT) and echocardiography. The association between left ventricular (LV) diastolic function and CAC was assessed using multidimensional network and multivariable-adjusted regression analyses. Multivariable analysis was conducted on continuous LV diastolic parameters and categorical classification of LVDD and adjusted for traditional cardiometabolic risk factors. LVDD was defined using reference limits from a low-risk reference group without established cardiovascular disease, cardiovascular risk factors or evidence of CAC, (n â€‹= â€‹560). We also classified LVDD using the American Society of Echocardiography recommendations. RESULTS: The mean age of the participants was 51 â€‹± â€‹17 years with 56.6% female and 62.6% non-Hispanic White. Overall, 38.1% had hypertension; 13.7% had diabetes; and 39.9% had CAC >0. An intertwined network was observed between diastolic parameters, CAC score, age, LV mass index, and pulse pressure. In the multivariable-adjusted analysis, e', E/e', and LV mass index were independently associated with CAC after adjustment for traditional risk factors. For both e' and E/e', the effect size and statistical significance were higher across increasing CAC tertiles. Other independent correlates of e' and E/e' included age, female sex, Black race, height, weight, pulse pressure, hemoglobin A1C, and HDL cholesterol. The independent association with CAC was confirmed using categorical analysis of LVDD, which occurred in 554 participants (26.6%) using population-derived thresholds. CONCLUSION: In the PBHS study, the subclinical coronary atherosclerotic disease burden detected using CAC scoring was independently associated with diastolic function. GOV IDENTIFIER: NCT03154346.

Clinical outcomes in pregnant women with coronavirus disease 2019 in a perinatal medical centre in Japan: a retrospective study of the first 1 year of the pandemic.

In this retrospective study, we analysed clinical and demographic data from the medical records of 31 pregnant women with coronavirus disease 2019 (COVID-19) who were treated at our hospital between April 2020 and April 2021. The most common symptom was a fever; ∼10% of patients were asymptomatic. One patient with rapidly worsening pneumonia needed a Caesarean Section at 30 weeks and was admitted for intensive care. Twelve patients received perinatal care in our hospital (10 live births, one stillbirth, and one artificial abortion). Six patients delivered vaginally; the others delivered via caesarean section. Two patients had complications, including severe hypertensive disorders and preeclampsia. All patients recovered from COVID-19. Severe acute respiratory syndrome coronavirus 2 was not detected in the placenta, umbilical cord, cord blood, amniotic fluid, vaginal fluid, or breast milk in any patient. There were no neonatal adverse outcomes. The possibility of transmitting the coronavirus to pregnancy-related samples was low.IMPACT STATEMENTWhat is already known on the subject? COVID-19 has been affecting different countries in diverse ways, and the incidence, mortality, and morbidity rates of patients with COVID-19 vary widely by country or region and race. These differences in results may reflect racial differences and differences in national health care systems. Moreover, the information about the perinatal outcomes of pregnant women with COVID-19 and their newborns from Japan is limited.What do the results of this study add to what is known? We described the perinatal outcomes of 31 Japanese pregnant women with COVID-19 who were managed safely in a perinatal medical centre in Tokyo Japan, during the first 1 year of the pandemic.What are the implications of these findings for clinical practice and/or further research? Severe pneumonia and perinatal complications may occur, although no maternal and neonatal deaths were observed for COVID-19-positive pregnant women in our facility. Therefore, it is important to prevent this infection during pregnancy with the provision of effective medical care.

Principal component analysis of early immune cell dynamics during pembrolizumab treatment of advanced urothelial carcinoma.

Immune checkpoint inhibitors have been approved as second-line therapy for patients with advanced urothelial carcinoma (UC). However, which patients will obtain clinical benefit remains to be determined. To identify predictive biomarkers for the pembrolizumab (PEM) response early during treatment, the present study investigated 31 patients with chemotherapy-resistant recurrent or metastatic UC who received 200 mg PEM intravenously every 3 weeks. Blood was taken just before the first dose and again before the second dose, and the peripheral blood mononuclear cells of all 31 pairs of blood samples were immune phenotyped by flow cytometry. Data were assessed by principal component analysis (PCA), correlation analysis and Cox proportional hazards modeling in order to comprehensively determine the effects of PEM on peripheral mononuclear immune cells. Absolute counts of CD45RA+CD27-CCR7- terminally differentiated CD8+ T cells and KLRG1+CD57+ senescent CD8+ T cells were significantly increased after PEM administration (P=0.042 and P=0.043, respectively). Senescent and exhausted CD4+ and CD8+ T cell dynamics were strongly associated with each other. By contrast, counts of monocytic myeloid-derived suppressor cells (mMDSCs) were not associated with other immune cell phenotypes. The results of PCA and non-hierarchical clustering of patients suggested that excessive T cell senescence and differentiation early during treatment were not necessarily associated with a survival benefit. However, decreased mMDSC counts after PEM were associated with improved overall survival. In conclusion, early on-treatment peripheral T cell status was associated with response to PEM; however, it was not associated with clinical benefit. By contrast, decreased peripheral mMDSC counts did predict improved overall survival.

Selection of RNA-based evaluation methods for tumor microenvironment by comparing with histochemical and flow cytometric analyses in gastric cancer.

Understanding the tumor microenvironment (TME) and anti-tumor immune responses in gastric cancer are required for precision immune-oncology. Taking advantage of next-generation sequencing technology, the feasibility and reliability of transcriptome-based TME analysis were investigated. TME of 30 surgically resected gastric cancer tissues was analyzed by RNA-Seq, immunohistochemistry (IHC) and flow cytometry (FCM). RNA-Seq of bulk gastric cancer tissues was computationally analyzed to evaluate TME. Computationally analyzed immune cell composition was validated by comparison with cell densities established by IHC and FCM from the same tumor tissue. Immune cell infiltration and cellular function were also validated with IHC and FCM. Cell proliferation and cell death in the tumor as assessed by RNA-Seq and IHC were compared. Computational tools and gene set analysis for quantifying CD8+ T cells, regulatory T cells and B cells, T cell infiltration and functional status, and cell proliferation and cell death status yielded an excellent correlation with IHC and FCM data. Using these validated transcriptome-based analyses, the immunological status of gastric cancer could be classified into immune-rich and immune-poor subtypes. Transcriptome-based TME analysis is feasible and is valuable for further understanding the immunological status of gastric cancer.

Association between effector-type regulatory T cells and immune checkpoint expression on CD8+ T cells in malignant ascites from epithelial ovarian cancer.

BACKGROUND: Regulatory T cells (Tregs) play an important role in the antitumor immune response in epithelial ovarian cancer (EOC). To understand the immune-inhibitory networks of EOC, we addressed the association between Tregs and immune checkpoint expression on T cells in the tumor microenvironment of EOC. METHODS: A total of 41 patients with stage IIIC and IV EOC were included in the analysis. We harvested cells from malignant ascites and investigated them using multi-color flow cytometry. We categorized the Tregs into 3 groups: effector-type Tregs, naïve Tregs and non-Tregs, based on the expression patterns of CD45RA and Foxp3 in CD4+ T cells. Furthermore, the relationships between the expression of various immune checkpoint molecules, such as PD-1, on CD8+ T cells and each of the Treg subtypes was also evaluated. RESULTS: The median frequency of naïve Tregs, effector-type Tregs and non-Tregs were 0.2% (0-0.8), 2.0% (0-11.4) and 1.5% (0.1-6.3) in CD4+ T cells of malignant ascites from EOC patients, respectively. A high frequency of effector-type Tregs was associated with high-grade serous carcinoma compared with the other histotypes. Patients with higher proportions of effector-type Tregs showed a trend towards increased progression-free survival. We also demonstrated a correlation between a higher proportion of effector-type Tregs and increased PD-1 expression on CD8+ T cells. In addition, C-C chemokine receptor 4 expression was also observed in effector-type Tregs. CONCLUSION: These data suggest that multiple immune-inhibitory networks exist in malignant ascites from EOC patients, suggesting an approach towards combinational immunotherapies for advanced EOC patients.

Evaluation of diastole by echocardiography for detecting early cardiac dysfunction: an outcome study.

AIMS: Timely detection of subclinical left ventricular diastolic dysfunction (LVDDF) is of importance for precise risk stratification of asymptomatic subjects. Here, we evaluated the prevalence of LVDDF and its prognostic significance in the general population using two grading approaches: the 2016 ASE/EACVI recommendations and population-derived, age-specific criteria. METHODS AND RESULTS: We randomly recruited 1407 community-dwelling participants (mean age, 51.2 years; 51.1% women; 53.5% with cardiovascular risk factors). We measured left heart dimensions, strain, tricuspid regurgitation, transmitral blood flow, and mitral annular tissue velocities using conventional echocardiography and Doppler imaging. We utilized these measurements to grade of LVDDF according to the 2016 recommendations and population-derived, age-specific approach. According to the 2016 recommendations, 26 subjects (1.85%) were classified as having the advanced stage (Grade 2), whereas in 109 participants (7.75%) diastolic function was indeterminate. When applying the population-derived criteria, the prevalence of advanced LVDDF was 17.9% (n = 252). During the follow-up period (8.4 years), 100 participants experienced adverse cardiac events. After full adjustment, we did not observe any significant differences in the risk of events between subjects with indeterminate or any grade of LVDDF and subjects with normal diastolic function when classified according to the 2016 recommendation (P ≥ 0.25). In contrast, the adjusted risks of adverse cardiac events (HR = 1.28; P = 0.0045) were significantly elevated in participants with LVDDF when classified according to the population-derived criteria. CONCLUSIONS: Our study underscored the importance of considering age- and population-derived thresholds in LVDDF grading in subjects at high cardiovascular risk which led to a better risk stratification and outcome prediction.

Two distinct phenotypes of immunologically hot gastric cancer subtypes.

An in-depth understanding of the tumor microenvironment (TME) is required for the development of improved combination immunotherapies for gastric cancer. Recently, we classified these cancers into four main types defined by their immunological attributes, namely Hot 1, Hot 2, Intermediate and Cold. Of these, the T cell-inflamed "Hot" tumors were further divided into Hot 1 and Hot 2 with different clinical outcomes. Thus, overall survival and progression-free survival of patients with Hot 1 tumors were shorter than with Hot 2. In the present study, we re-evaluated RNA-Seq data of 6 Hot 1 and 6 Hot 2 gastric cancers to elucidate the underlying reason for the poor prognosis and T cell dysfunction in the former. In addition, 56 Hot 1 and 27 Hot 2 tumors in The Cancer Genome Atlas (TCGA) were analyzed. We report that single sample Gene Set Enrichment Analysis (ssGSEA) and differential gene expression analysis identified differences between Hot 1 and Hot 2 tumors involved in metabolism and cell adhesion pathways. Therefore, it is suggested that strategies to modulate active metabolism in Hot 1 tumors should be integrated into the treatment of these gastric cancers.

Modification of improved-genome editing via oviductal nucleic acids delivery (i-GONAD)-mediated knock-in in rats.

BACKGROUND: Improved genome-editing via oviductal nucleic acids delivery (i-GONAD) is a new technology that facilitates in situ genome-editing of mammalian zygotes exiting the oviductal lumen. The i-GONAD technology has been developed for use in mice, rats, and hamsters; however, oligonucleotide (ODN)-based knock-in (KI) is more inefficient in rats than mice. To improve the efficiency of i-GONAD in rats we examined KI efficiency using three guide RNAs (gRNA), crRNA1, crRNA2 and crRNA3. These gRNAs recognize different portions of the target locus, but also overlap each other in the target locus. We also examined the effects of commercially available KI -enhancing drugs (including SCR7, L755,507, RS-1, and HDR enhancer) on i-GONAD-mediated KI efficiency. RESULTS: The KI efficiency in rat fetuses generated after i-GONAD with crRNA2 and single-stranded ODN was significantly higher (24%) than crRNA1 (5%; p < 0.05) or crRNA3 (0%; p < 0.01). The KI efficiency of i-GONAD with triple gRNAs was 11%. These findings suggest that KI efficiency largely depends on the type of gRNA used. Furthermore, the KI efficiency drugs, SCR7, L755,507 and HDR enhancer, all of which are known to enhance KI efficiency, increased KI efficiency using the i-GONAD with crRNA1 protocol. In contrast, only L755,507 (15 µM) increased KI efficiency using the i-GONAD with crRNA2 protocol. None of them were significantly different. CONCLUSIONS: We attempted to improve the KI efficiency of i-GONAD in rats. We demonstrated that the choice of gRNA is important for determining KI efficiency and insertion and deletion rates. Some drugs (e.g. SCR7, L755,507 and HDR enhancer) that are known to increase KI efficiency in culture cells were found to be effective in i-GONAD in rats, but their effects were limited.