Reclassification of RAS/BRAF allele mutations predicts the survival benefit of triplet chemotherapy in metastatic colorectal cancer.

Background: Different RAS/BRAF allele mutations imply distinct biological properties in various solid tumors. Recently, several studies have focused on the predictive and prognostic roles of various RAS/BRAF allele mutations in colorectal cancer (CRC) but the results remain controversial. Methods: Between March 2017 and September 2022, the patients diagnosed as stages I-IV CRC with detailed medical records including next-generation sequencing (NGS) data and clinicopathological follow-up information available at our center were enrolled. Survival data were estimated using the Kaplan-Meier method, and the difference was tested in a log-rank test. Multivariate tests were carried out using Cox models. Results: A total of 1029 CRC patients were included, and the incidence of RAS/BRAF mutation was 58.4%. The hypermutated cohort was defined as patients with microsatellite instability-H or POLE/D mutation. In the non-hypermutational cohort, only KRAS G13D mutation was associated with a higher incidence and inferior disease-free survival in patients with stage I-III CRC. In the cohort of patients with non-hypermutated metastatic colorectal cancer (mCRC), we assessed the risk of various RAS/BRAF allele mutations and subsequently reclassified patients into four groups based on first-line median progression-free survival: wild type (group 1), low-risk RAS/BRAF mutation (group 2, RAS/BRAF mutations other than KRAS G13D/G12V/G12C or BRAF V600E), high-risk RAS mutation (group 3, KRAS G13D/G12V/G12C), and BRAF V600E mutation (group 4). mCRC patients with high-risk RAS mutation could significantly benefit from intensive triplet chemotherapy (hazard ratio, 2.54; 95% confidence interval, 1.36-5.12; p = 0.0091). Conclusion: In the non-hypermutated CRC cohort, the prognostic risk of various RAS/BRAF allele mutations varied between local and metastatic CRC. KRAS G13D mutation tended to be the only prognostic marker for stages I-III CRC; however, KRAS G13D/G12V/G12C mutations collectively defined a high-risk subgroup of mCRC patients with poor prognosis, who would benefit from intensive triplet chemotherapy.

TAK-242 protects against oxygen-glucose deprivation and reoxygenation-induced injury in brain microvascular endothelial cells and alters the expression pattern of lncRNAs.

Background: Deep hypothermic circulatory arrest (DHCA) is a technique used during the surgical treatment of aneurysms of the thoracic aorta in adult patients, and complex congenital heart disease in neonates. And brain microvascular endothelial cells (BMECs) are essential components of the cerebrovascular network and participate in maintaining the blood-brain barrier (BBB) and brain function. In our previous study, we found that oxygen-glucose deprivation and reoxygenation (OGD/R) activated Toll-like receptor 4 (TLR4) signaling in BMECs, and induced pyroptosis and inflammation. In this study, we further investigated the potential mechanism of ethyl(6R)-6-[N-(2-Chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1-carboxylate (TAK-242) on BMECs under OGD/R, as in patients with sepsis, the TAK-242 was tested in clinical trials. Methods: To confirm the function of TAK-242 on BMECs under OGD/R, cell viability, inflammatory factors, inflammation-associated pyroptosis, and nuclear factor-κB (NF-κB) signaling were determined using Cell Counting Kit-8 (CCK-8) assay, enzyme-linked immunosorbent assay (ELISA), and western blotting, respectively. To investigate the lncRNAs associated with TLR4 during OGD/R, long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) expression patterns were profiled with RNA deep sequencing. Moreover, to confirm whether lncRNA-encoded short peptides, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used. Results: Relative control group, OGD/R inhibited the cell viability, increased the section of inflammatory factors secretion, including IL-1ß, IL-6, and TNF-α, and promoted the pathways of TLR4/NLRP3/Caspase-1 and TLR4/NF-κB. However, TAK-242 + OGD/R group promoted OGD/R cell viability, decreased OGD/R-induced inflammatory factors secretion, and inhibited the pathways of TLR4/NLRP3/Caspase-1 and TLR4/NF-κB. In addition, AABR07000411.1, AABR070006957.1, and AABR070008256.1 were decreased in OGD/R cells compared with controls, but TAK-242 restored their expression under OGD/R condition. AABR07000473.1, AC130862.4, and LOC10254972.6 were induced by OGD/R, but were suppressed in TAK-242 + OGD/R cells compared with OGD/R. Moreover, AABR07049961.1, AC127076.2, AABR07066020.1, and AABR07025303.1-encoded short peptides were dysregulated in OGD/R cells, and TAK-242 attenuated the dysregulation of AABR07049961.1, AC127076.2, and AABR07066020.1-encoded short peptides. Conclusions: TAK-242 alters the expression pattern of lncRNAs in OGD/R cells, and differently expressed lncRNAs may exert a protective effect against OGD/R injury through a mechanism of competing endogenous RNA (ceRNA) and encoding short peptides. These findings maybe provide a new theory basis for the treatment of DHCA.

Circuit- and laminar-specific regulation of medial prefrontal neurons by chronic stress.

BACKGROUND: Chronic stress exposure increases the risk of mental health problems such as anxiety and depression. The medial prefrontal cortex (mPFC) is a hub for controlling stress responses through communicating with multiple limbic structures, including the basolateral amygdala (BLA) and nucleus accumbens (NAc). However, considering the complex topographical organization of the mPFC neurons in different subregions (dmPFC vs. vmPFC) and across multiple layers (Layer II/III vs. Layer V), the exact effects of chronic stress on these distinct mPFC output neurons remain largely unknown. RESULTS: We first characterized the topographical organization of mPFC neurons projecting to BLA and NAc. Then, by using a typical mouse model of chronic restraint stress (CRS), we investigated the effects of chronic stress on the synaptic activity and intrinsic properties of the two mPFC neuronal populations. Our results showed that there was limited collateralization of the BLA- and NAc-projecting pyramidal neurons, regardless of the subregion or layer they were situated in. CRS significantly reduced the inhibitory synaptic transmission onto the BLA-projecting neurons in dmPFC layer V without any effect on the excitatory synaptic transmission, thus leading to a shift of the excitation-inhibition (E-I) balance toward excitation. However, CRS did not affect the E-I balance in NAc-projecting neurons in any subregions or layers of mPFC. Moreover, CRS also preferentially increased the intrinsic excitability of the BLA-projecting neurons in dmPFC layer V. By contrast, it even caused a decreasing tendency in the excitability of NAc-projecting neurons in vmPFC layer II/III. CONCLUSION: Our findings indicate that chronic stress exposure preferentially modulates the activity of the mPFC-BLA circuit in a subregion (dmPFC) and laminar (layer V) -dependent manner.

Metastatic sites and lesion numbers cooperated to predict efficacy of PD-1 inhibitor-based combination therapy for patients with metastatic colorectal cancer.

BACKGROUND: Limited data have been used to evaluate the efficacy of immunotherapy in metastatic colorectal cancer (mCRC). Furthermore, potential markers that can be used to identify responding patients and to further improve efficacy have not been fully explored. METHODS AND RESULTS: In our study, we included a total of 97 patients with mCRC, who each received programmed death-1 (PD-1) inhibitor-based combination therapy at our center. All 12 hypermutated patients benefited from immunotherapy, with median progression-free survival (mPFS) reaching 28.3 months, regardless of liver metastasis. The objective response rate (ORR) of non-hypermutated patients was 16.5% (14/85), with an mPFS of 4.0 months. For non-hypermutated patients, multivariate analysis revealed that the combination of liver metastasis and baseline lesion number significantly stratified response and survival. The lesion-based analysis indicated that the lymph node was the most responsive, followed by the peritoneum and lung, with liver metastasis being the least responsive. None of the patients (0/7) with negative programmed ligand-1 (PD-L1) expression responded, and positive PD-L1 expression may serve as a biomarker (mPFS 5.7 vs. 2.2 months, p = 0.002) that can be used to further guide treatment in non-hypermutated mCRC with liver metastasis (CRLMs). CONCLUSION: Patients with hypermutated mCRC benefited significantly from immunotherapy, whereas the non-hypermutated cohort with liver metastasis and numerous lesions showed less benefit. The lesion sites reflected varying levels of efficacy, among which PD-L1 potentially cooperated to guide the immunotherapy of CRLMs.

APOC1 predicts a worse prognosis for esophageal squamous cell carcinoma and is associated with tumor immune infiltration during tumorigenesis.

Background: Esophageal carcinoma (ESCA), a common malignant tumor of the digestive tract with insidious onset, is a serious threat to human health. Despite multiple treatment modalities for patients with ESCA, the overall prognosis remains poor. Apolipoprotein C1 (APOC1) is involved in tumorigenesis as an inflammation-related molecule, and its role in esophageal cancer is still unknown. Methods: We downloaded documents and clinical data using The Cancer Genome Atlas (TCGA)and Gene Expression Omnibus (GEO) databases. We also conducted bioinformatics studies on the diagnostic value, prognostic value, and correlation between APOC1 and immune infiltrating cells in ESCA through STRING (https://cn.string-db.org/), the TISIDB (http://cis.hku.hk/TISIDB/) website, and various other analysis tools. Results: In patients with ESCA, APOC1 was significantly more highly expressed in tumor tissues than in normal tissues (p < 0.001). APOC1 could diagnose ESCA more accurately and determine the TNM stage and disease classification with high accuracy (area under the curve, AUC≥0.807). The results of the Kaplan-Meier curve analysis showed that APOC1 has prognostic value for esophageal squamous carcinoma (ESCC) (p = 0.043). Univariate analysis showed that high APOC1 expression in ESCC was significantly associated with worse overall survival (OS) (p = 0.043), and multivariate analysis shows that high APOC1 expression was an independent risk factor for the OS of patients with ESCC (p = 0.030). In addition, the GO (gene ontology)/KEGG (Kyoto encyclopedia of genes and genomes) analysis showed a concentration of gene enrichment in the regulation of T-cell activation, cornification, cytolysis, external side of the plasma membrane, MHC protein complex, MHC class II protein complex, serine-type peptidase activity, serine-type endopeptidase activity, Staphylococcus aureus infection, antigen processing and presentation, and graft-versus-host disease (all p < 0.001). GSEA (gene set enrichment analysis) showed that enrichment pathways such as immunoregulatory-interactions between a lymphoid and non-lymphoid cell (NES = 1.493, p. adj = 0.023, FDR = 0.017) and FCERI-mediated NF-KB activation (NES = 1.437, p. adj = 0.023, FDR = 0.017) were significantly enriched in APOC1-related phenotypes. In addition, APOC1 was significantly associated with tumor immune infiltrating cells and immune chemokines. Conclusion: APOC1 can be used as a prognostic biomarker for esophageal cancer. Furthermore, as a novel prognostic marker for patients with ESCC, it may have potential value for further investigation regarding the diagnosis and treatment of this group of patients.

Soluble PD-L1: a potential dynamic predictive biomarker for immunotherapy in patients with proficient mismatch repair colorectal cancer.

BACKGROUND: Circulating soluble programmed death ligand 1 (sPD-L1) can negatively regulate T-cell function and serve as a prognostic or predictive marker in a variety of cancers. However, rare studies have evaluated the potential roles of sPD-L1, and no study has estimated its predictive value for the efficacy of immune treatment in colorectal cancer (CRC). METHODS: Plasma samples from 192 CRC patients were used to estimate correlations between clinicopathological features and sPD-L1, secreted PD-L1 (secPD-L1) and exosomal PD-L1 (exoPD-L1). Baseline and posttreatment sPD-L1 levels were also investigated in 55 patients with metastatic CRC (mCRC) treated with chemotherapy ± targeted therapy and 40 patients with proficient mismatch repair (pMMR) mCRC treated with combination immunotherapy. Both sPD-L1 and secPD-L1 were quantified by enzyme-linked immunosorbent assay, while exoPD-L1 was analyzed using flow cytometry. RESULTS: secPD-L1 was the major component and positively correlated with sPD-L1 in CRC, while exoPD-L1 was almost undetectable. Higher levels of sPD-L1 were detected in patients with distant metastasis, especially those with distant lymph node metastasis and tissue combined positive score (CPS) instead of tumor proportion score (TPS). Chemotherapy or targeted therapy did not significantly impact sPD-L1 concentration. Progressive disease on combination immunotherapy was associated with an increase in sPD-L1 level, whereas no significant change was observed in patients with durable clinical benefit. CONCLUSION: sPD-L1 mainly consisted of secPD-L1, and its level was higher in patients with distant metastasis, especially distant lymph node metastasis and positive CPS. sPD-L1 is a potential dynamic marker to identify rapid progression on combination immunotherapy and avoid ineffective treatment for pMMR CRC.

Coexistence of anaplastic lymphoma kinase rearrangement in lung adenocarcinoma harbouring epidermal growth factor receptor mutation: A single-center study.

BACKGROUND: Accumulating evidences confirm that epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement have coexisted in lung adenocarcinoma (LUAD). However, Its biological mechanism, clinicopathological features, and optimization of targeted drugs have not yet been completely elucidated. AIM: To explore the clinical profile of LUAD patients with co-mutations of EGFR and ALK genes, with hopes of scientifically guiding similar patients towards selected, targeted drugs. METHODS: Two hundred and thirty-seven LUAD patients were enrolled. EGFR mutations were detected by the amplification refractory mutation system-peptide nucleic acid technique, while the expression of ALK rearrangement was screened by the 5'/3' imbalance strategy for reverse transcription followed by quantitative polymerase chain reaction analysis. The clinicopathological features of these patients were analysed retrospectively, and the follow-up data were collected. RESULTS: There were six cases with co-mutations of EGFR and ALK genes, which were more common in women, non-smoking and stage IV LUAD patients with bone metastasis, hence a positive rate of 2.53% (6/237). EGFR-tyrosine kinase inhibitors (EGFR-TKIs) were their preferred drugs for targeted therapy in these patients, with progression-free survival ranging from two months to six months. CONCLUSION: In Gannan region, the positive rate of co-mutations of EGFR and ALK genes in LUAD patients is relatively high, and the co-mutations are more common in women, non-smoking and stage IV patients with bone metastasis. These patients prefer EGFR-TKIs as their preferred targeted drugs, but the therapeutic effect is not good. EGFR/ALK dual-TKIs may be more effective targeted drugs, which needs further study.

Development of a Humanized VHH Based Recombinant Antibody Targeting Claudin 18.2 Positive Cancers.

Claudin 18.2 (CLDN18.2), a tight junction (TJ) family protein controlling molecule exchange between cells, is frequently over-expressed in gastric cancer, pancreatic adenocarcinomas and in a fraction of non-small cell lung cancer cases. The tumor properties indicate that CLDN18.2 could be an attractive drug target for gastric and pancreatic cancers. In this study, we present effective strategies for developing anti-CLDN18.2 therapeutic candidates, based on variable domain of heavy chain of heavy chain antibodies (VHHs). CLDN18.2-specific VHHs were isolated by panning a phage display library from an alpaca immunized with a stable cell line highly expressing CLDN18.2. Humanized VHHs fused with human IgG1 Fc, as potential therapeutic candidates, exhibited desirable binding specificity and affinity to CLDN18.2. In vitro experiments showed that hu7v3-Fc was capable of eliciting both antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) on CLDN18.2 positive tumor cells. In the mouse xenograft model, the anti-tumor efficacy of hu7v3-Fc was significantly more potent than Zolbetuximab, the benchmark anti-CLDN18.2 monoclonal antibody. Moreover, in vivo biodistribution using zirconium-89 (89Zr) labeled antibodies demonstrated that hu7v3-Fc (89Zr-hu7v3-Fc) exhibited a better tumor penetration and a faster tumor uptake than Zolbetuximab (89Zr-Zolbetuximab), which might be attributed to its smaller size and higher affinity. Taken together, anti-CDLN18.2 hu7v3-Fc is a promising therapeutic agent for human CLDN18.2 positive cancers. Furthermore, hu7v3 has emerged as a potential module for novel CLDN18.2 related therapeutics.

miR-181a-5p restrains non-small cell lung cancer cell invasion and migration by regulating the GTSE1/p53/NF-κB axis.

microRNAs (miRNAs) are relevant to metastasis and invasion of non-small cell lung cancer (NSCLC). This study investigated the role of miR-181a-5p in lung cancer. Expression patterns of miR-181a-5p and GTSE1 in the human NSCLC cell line A549 and normal lung epithelial cell line BASE-2B were detected. miR-181a-5p mimic was delivered into A549 cells utilizing Lipofectamine 2000 to overexpress miR-181a-5p, followed by analysis of cell viability, proliferation, apoptosis, invasion, and migration. GTSE1 (G2 and S phase-expressed-1) was predicted as the downstream target gene of miR-181a-5p using bioinformatics analysis software. Targeting relationship between miR-181a-5p and GTSE1 was validated via dual-luciferase assay, RIP assay, and RNA pull-down. Activation of the p53/NF-κB pathway was determined. miR-181a-5p was weakly-expressed in NSCLC cells relative to normal lung epithelial cells. miR-181a-5p overexpression prevented NSCLC cell proliferation, migration, and invasion. Mechanically, miR-181a-5p targeted GTSE1. GTSE1 overexpression partly annulled repression of miR-181a-5p overexpression on NSCLC cell malignant behavior. miR-181a-5p activated the p53 pathway and inhibited the NF-κB pathway by targeting GTSE1. Overall, this study for the first time validated that miR-181a-5p impeded NSCLC cell invasion and migration through activation of the p53 pathway and inhibition of the NF-κB pathway by targeting GTSE1, which may provide a potential novel insight into NSCLC treatment.

Low expression of miR-27b in serum exosomes of non-small cell lung cancer facilitates its progression by affecting EGFR.

Non-small cell lung cancer (NSCLC) is a malignant tumor. Serum exosomal miR-27b is related to tumor diagnosis. We explored the roles of serum exosomal miR-27b in NSCLC. NSCLC patients were assigned to NSCLC-early/terminal groups, with healthy subjects as controls. miR-27b expression was assessed using reverse transcription-quantitative polymerase chain reaction, and its diagnostic efficiency was analyzed using the receiver operating characteristic curve. The correlation between serum exosomal miR-27b expression and tumor markers carcinoembryonic antigen 125 (CA125), carcinoembryonic antigen (CEA), and cytokeratin 19-soluble fragment (CYFRA21-1) was analyzed using the Pearson analysis. The downstream target genes were predicted. Epidermal growth factor receptor (EGFR) level was assessed using enzyme-linked immunosorbent assay. Correlations of miR-27b expression with serum EGFR level and CA125, CEA, and CYFRA21-1 levels were analyzed using the Pearson analysis. Serum exosomal miR-27b was diminished in NSCLC and was further decreased in the NSCLS-terminal group. The sensitivity of miR-27b < 0.8150 for NSCLC diagnosis was 76.64%, and the specificity was 83.33%. Serum exosomal miR-27b was negatively correlated with CA125, CEA, and CYFRA21-1. miR-27b targeted EGFR. Serum EGFR was raised in NSCLC and was further elevated in the NSCLS-terminal group. miR-27b expression was negatively correlated with EGFR level. EGFR level was positively correlated with CA125, CEA, and CYFRA21-1 levels. Collectively, low expression of miR-27b assisted NSCLC diagnosis, and miR-27b exerted effects on NSCLC through EGFR.

Genomic and transcriptomic analysis of MSI-H colorectal cancer patients with targetable alterations identifies clinical implications for immunotherapy.

Introduction: Targetable alterations such as BRAFV600E mutation and NTRK fusion are enriched in microsatellite instability-high (MSI-H) colorectal cancer (CRC). MSI-H with targetable alterations (MSI-H altered) might present unique opportunities for both targeted therapy and immunotherapy. We systematically evaluated the molecular characteristics and immune-related features of MSI-H altered and MSI-H without targetable alterations (MSI-H wt) CRC patients in our study. Methods: Among 1938 continuously enrolled CRC patients, 126 patients with MSI-H status (6.50%) were included in this retrospective study. Genomic and transcriptomic data were investigated by next-generation sequencing (NGS) and gene expression profiling (GEP), respectively. Results: BRAFV600E, NTRK1, and FGFR2 mutations were the most frequent targetable alterations in MSI-H CRC patients. The MSI-H altered phenotype was significantly associated with older age (p< 0.001), right side (p=0.024) and females (p= 0.036). No lynch syndrome (LS) patients were identified in MSI-H altered group. The tumor mutational burden (TMB), and tumor neoantigen burden (TNB) of MSI-H altered and wt subgroups were comparable (p<0.05). Subsequently, transcriptomic study analysis further revealed MSI-H altered CRC patients were linked to an immune-active tumor microenvironment with higher levels of Teff IFN-gamma, CYT, and MERCK 18 signatures, and lower levels of the IPRES gene signature, EMT and TGF Beta signatures. In addition, case study supported MSI-H CRC patient harboring targetable alterations might also achieved a long-term disease-free survival benefit from immunotherapy. Discussion: Our study preliminary revealed MSI-H altered as a novel subtype of MSI-H CRC patients with unique molecular signatures and immune-active tumor microenvironment. Given the accessibility of immune checkpoint inhibitors (ICIs) treatment, our results might provide clinical evidence for immunotherapy in MSI-H CRC patients with targetable alterations.

Development of a Deep Learning Model to Assist With Diagnosis of Hepatocellular Carcinoma.

BACKGROUND: An accurate pathological diagnosis of hepatocellular carcinoma (HCC), one of the malignant tumors with the highest mortality rate, is time-consuming and heavily reliant on the experience of a pathologist. In this report, we proposed a deep learning model that required minimal noise reduction or manual annotation by an experienced pathologist for HCC diagnosis and classification. METHODS: We collected a whole-slide image of hematoxylin and eosin-stained pathological slides from 592 HCC patients at the First Affiliated Hospital, College of Medicine, Zhejiang University between 2015 and 2020. We propose a noise-specific deep learning model. The model was trained initially with 137 cases cropped into multiple-scaled datasets. Patch screening and dynamic label smoothing strategies are adopted to handle the histopathological liver image with noise annotation from the perspective of input and output. The model was then tested in an independent cohort of 455 cases with comparable tumor types and differentiations. RESULTS: Exhaustive experiments demonstrated that our two-step method achieved 87.81% pixel-level accuracy and 98.77% slide-level accuracy in the test dataset. Furthermore, the generalization performance of our model was also verified using The Cancer Genome Atlas dataset, which contains 157 HCC pathological slides, and achieved an accuracy of 87.90%. CONCLUSIONS: The noise-specific histopathological classification model of HCC based on deep learning is effective for the dataset with noisy annotation, and it significantly improved the pixel-level accuracy of the regular convolutional neural network (CNN) model. Moreover, the model also has an advantage in detecting well-differentiated HCC and microvascular invasion.

Usefulness of diffusion derived vessel density computed from a simplified IVIM imaging protocol: An experimental study with rat biliary duct blockage induced liver fibrosis.

OBJECTIVES: Liver vessel density can be evaluated by DDVD (diffusion derived vessel density): DDVD(b0b1) = Sb0/ROIarea0 - Sb1/ROIarea1, where Sb0 and Sb1 refer to the liver signal when b is 0 or 1 s/mm2. Sb1 and ROIarea1 may be replaced by other b-values. With a rat biliary duct ligation (BDL) model, this study assesses the usefulness of liver DDVD computed from a simplified IVIM imaging protocol using b = 25 and b = 50 to replace b = 1 s/mm2, alone and in combination with other IVIM parameters. METHODS: Male Sprague-Dawley rats were used. The rat number was 5, 5, 5, and 3 respectively, for the timepoints of 7, 14, 21, 28 days post-BDL surgery. 12 rats had partial biliary duct recanalization performed after the rats had BDL for 7 days and then again followed-up for a mean of 14 days. Liver diffusion MRIs were acquired at 3.0 T with a b-value distribution of 0, 25, 50, 75, 100, 150, 300, 700, 1000 s/mm2. DDVDmean (control rats n = 6) was the mean of DDVD(b0b25) and DDVD(b0b50). IVIM fitting started from b = 0 s/mm2 with segmented fitting and a threshold b of 50 s/mm2 (n = 5 for control rats). Three 3-D spaces were constructed using a combination of the four diffusion parameters. RESULTS: The control rats and BDL rats (n = 18) had a liver DDVDmean of 84.0 ± 26.2 and 44.7 ± 14.4 au/pixel (p < 0.001). All 3-D spaces totally separated healthy livers and all fibrotic livers (n = 30, BDL rats and recanalization rats). The mean relative distance between healthy liver cluster and fibrotic liver cluster was 0.331 for PF, Dslow, and Dfast; 0.381 for PF, Dfast, and DDVDmean; and 0.384 for PF, Dslow, and DDVDmean. CONCLUSION: A combination of PF, Dslow, and Dfast allows total separation of healthy livers and fibrotic livers and the integration of DDVD improved the separation.

SARS-CoV-2 Host Receptor ACE2 Protein Expression Atlas in Human Gastrointestinal Tract.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through interactions with its receptor, Angiotensin-converting enzyme 2 (ACE2), causing severe acute respiratory syndrome and death in a considerable proportion of people. Patients infected with SARS-CoV-2 experience digestive symptoms. However, the precise protein expression atlas of ACE2 in the gastrointestinal tract remains unclear. In this study, we aimed to explore the ACE2 protein expression pattern and the underlying function of ACE2 in the gastrointestinal tract, including the colon, stomach, liver, and pancreas. METHODS: We measured the protein expression of ACE2 in the gastrointestinal tract using immunohistochemical (IHC) staining with an ACE2-specific antibody of paraffin-embedded colon, stomach, liver, and pancreatic tissues. The correlation between the protein expression of ACE2 and the prognosis of patients with gastrointestinal cancers was analyzed by the log-rank (Mantel-Cox) test. The influence of ACE2 on colon, stomach, liver, and pancreatic tumor cell line proliferation was tested using a Cell Counting Kit 8 (CCK-8) assay. RESULTS: ACE2 presented heterogeneous expression patterns in the gastrointestinal tract, and it showed a punctate distribution in hepatic cells. Compared to that in parallel adjacent non-tumor tissues, the protein expression of ACE2 was significantly increased in colon cancer, stomach cancer, and pancreatic cancer tissues but dramatically decreased in liver cancer tissues. However, the expression level of the ACE2 protein was not correlated with the survival of patients with gastrointestinal cancers. Consistently, ACE2 did not affect the proliferation of gastrointestinal cancer cells in vitro. CONCLUSION: The ACE2 protein is widely expressed in the gastrointestinal tract, and its expression is significantly altered in gastrointestinal tumor tissues. ACE2 is not an independent prognostic marker of gastrointestinal cancers.

Collagen deposition in the liver is strongly and positively associated with T1rho elongation while fat deposition is associated with T1rho shortening: an experimental study of methionine and choline-deficient (MCD) diet rat model.

BACKGROUND: A number of questions concerning the histological mechanism of elongated T1rho in liver fibrosis remain unanswered. Using a rat model of non-alcoholic fatty liver disease (NAFLD) induced with methionine and choline-deficient (MCD) diet, the primary aim of this study is to clarify whether collagen deposition per se causes liver T1rho elongation. METHODS: There were 45 rats in the NAFLD model group and 8 rats in the control group. NAFLD model rats were fed MCD diet for 1, 2, 4, 6, 8, or 10 weeks, respectively. At the endpoint, the rats had in vivo MRI at 3.0 T and followed by histology. For T1rho data acquisition, a rotary echo spin-lock pulse was implemented in a three-dimensional fast field echo sequence with frequency selective fat suppression. The spin-lock frequency was set to 500 Hz, and the spin-lock times of 5, 10, 40, and 50 ms were used. Liver specimens were processed with hematoxylin-eosin staining for steatosis and inflammation evaluation, and Masson's trichrome staining for collagen visualization. The semiquantitative histopathological evaluation was based on NASH Clinical Research Network criteria. Histomorphometric analysis calculated percentages of fat and collagen accumulations in the livers. RESULTS: A strong (r=0.82) and significant (P<0.0001) positive correlation between liver collagen content and liver T1rho was observed. Rats with no or minimal inflammation could have very long T1rho value. Among experimental rats without a positive fibrosis grading, five rats did not have an inflammation score (i.e., had minimal inflammation or no inflammation) while four had a positive inflammation score; the difference in liver T1rho between these two types of rats was minimal. Eight control rat livers and 15 stage-1 fibrosis rat livers were separated by liver T1rho completely. When four subgroups of experiment rats were selected where the liver collagen had a very narrow range within these subgroups, all these four subgroups showed a trend of negative correlation between liver fat and liver T1rho. CONCLUSIONS: Collagen deposition in the live strongly contributes to liver T1rho elongation, while fat deposition contributes to T1rho shortening. In a well-controlled experimental setting, T1rho measure alone allows separation of healthy livers and stage-1 liver fibrosis in the MCD rat liver model.

Exhausted CD8+T Cells in the Tumor Immune Microenvironment: New Pathways to Therapy.

Tumor-specific CD8+T cells are exposed to persistent antigenic stimulation which induces a dysfunctional state called "exhaustion." Though functioning to limit damage caused by immune response, T cell exhaustion leads to attenuated effector function whereby cytotoxic CD8+T cells fail to control tumor progression in the late stage. This pathway is a dynamic process from activation to "progenitor exhaustion" through to "terminally exhaustion" with distinct properties. With the rapid development of immunotherapy via enhancing T cell function, new studies are dissecting the mechanisms and identifying specific biomarkers of dynamic differentiation during the process of exhaustion. Further, although immune checkpoint inhibitors (ICIs) have achieved great success in clinical practice, most patients still show limited efficacy to ICIs. The expansion and differentiation of progenitor exhausted T cells explained the success of ICIs while the depletion of the progenitor T cell pool and the transient effector function of terminally exhausted T cells accounted for the failure of immune monotherapy in the context of exorbitant tumor burden. Thus, combination strategies are urgent to be utilized based on the reduction of tumor burden or the expansion of the progenitor T cell pool. In this review, we aim to introduce the concept of homeostasis of the activated and exhausted status of CD8+T cells in the tumor immune microenvironment, and present recent findings on dynamic differentiation process during T cell exhaustion and the implications for combination strategies in immune therapy.

Blockage of ETS homologous factor inhibits the proliferation and invasion of gastric cancer cells through the c-Met pathway.

BACKGROUND: Gastric cancer (GC) is one of the most common and deadliest types of cancer worldwide due to its delayed diagnosis and high metastatic frequency, but its exact pathogenesis has not been fully elucidated. ETS homologous factor (EHF) is an important member of the ETS family and contributes to the pathogenesis of multiple malignant tumors. To date, whether EHF participates in the development of GC via the c-Met signaling pathway remains unclear. AIM: To investigate the role and mechanism of EHF in the occurrence and development of GC. METHODS: The expression of EHF mRNA in GC tissues and cell lines was measured by quantitative PCR. Western blotting was performed to determine the protein expression of EHF, c-Met, and its downstream signal molecules. The EHF expression in GC tissues was further detected by immunohistochemical staining. To investigate the role of EHF in GC oncogenesis, small interfering RNA (siRNA) against EHF was transfected into GC cells. The cell proliferation of GC cells was determined by Cell Counting Kit-8 and colony formation assays. Flow cytometry was performed following Annexin V/propidium iodide (PI) to identify apoptotic cells and PI staining to analyze the cell cycle. Cell migration and invasion were assessed by transwell assays. RESULTS: The data showed that EHF was upregulated in GC tissues and cell lines in which increased expression of c-Met was also observed. Silencing of EHF by siRNA reduced the proliferation of GC cells. Inhibition of EHF induced significant apoptosis and cell cycle arrest in GC cells. Cell migration and invasion were significantly inhibited. EHF silencing led to c-Met downregulation and further blocked the Ras/c-Raf/extracellular signal-related kinase 1/2 (Erk1/2) pathway. Additionally, phosphatase and tensin homolog was upregulated and glycogen synthase kinase 3 beta was deactivated. Moreover, inactivation of signal transducer and activator of transcription 3 was detected following EHF inhibition, leading to inhibition of the epithelial-to-mesenchymal transition (EMT). CONCLUSION: These results suggest that EHF plays a key role in cell proliferation, invasion, apoptosis, the cell cycle and EMT via the c-Met pathway. Therefore, EHF may serve as an antineoplastic target for the diagnosis and treatment of GC.

90-gene signature assay for tissue origin diagnosis of brain metastases.

BACKGROUND: Brain metastases (BM) are the most common intracranial tumors. 2-14% of BM patients present with unknown primary site despite intensive evaluations. This study aims to evaluate the performance of a 90-gene expression signature in determining the primary sites for BM samples. METHODS: The sequence-based gene expression profiles of 708 primary brain tumors (PBT) collected from The Cancer Genome Atlas (TCGA) database were analyzed by the 90-gene expression signature, with a similarity score for each of 21 common tumor types. We then used Optimal Binning algorithm to generate a threshold for separating PBT from BM. Eighteen PBT samples were analyzed to substantiate the reliability of the threshold. In addition, the performance of the 90-gene expression signature for molecular classification of metastatic brain tumors was validated in a cohort of 48 BM samples with the known origin. For each BM sample, the tumor type with the highest similarity score was considered tissue of origin. When a sample was diagnosed as PBT, but the similarity score below the threshold, the second prediction was considered as the primary site. RESULTS: A threshold of the similarity score, 70, was identified to discriminate PBT from BM (PBT: > 70, BM: ≤ 70) with an accuracy of 99% (703/708, 95% CI 98-100%). The 90-gene expression signature was further validated with 18 PBT and 44 BM samples. The results of 18 PBT samples matched reference diagnosis with a concordance rate of 100%, and all similarity scores were above the threshold. Of 44 BM samples, the 90-gene expression signature accurately predicted primary sites in 89% (39/44, 95% CI 75-96%) of the cases. CONCLUSIONS: Our findings demonstrated the potential that the 90-gene expression signature could serve as a powerful tool for accurately identifying the primary sites of metastatic brain tumors.