Submucosal gland differentiation and implications in esophageal basaloid squamous cell carcinomas.

Esophageal basaloid squamous cell carcinoma (BSCC) is a heterogenous entity with multilineage differentiation. It lacks systematical analysis on submucosal gland differentiation (SGD) due to the histological diversity and low incidence of esophageal BSCC. This study aims to find the correlation of SGD and clinicopathological features. A total of 152 esophageal BSCCs were separated into three histological groups: pure, mixed, and borderline group. The clinicopathological features were compared between different groups. The prevalence of SGD was also compared between cases of different groups. A panel of antibodies were used to identify SGD. The pure group differed from the mixed and borderline groups in many aspects, lymph node metastasis (LNM), cancer embolus, perineural invasion, and advanced stage occurred less frequently in the pure group (P<0.01). The pure group had a better but statistically insignificant overall survival (P=0.097). The squamous cell carcinoma (SCC) component or focal squamous differentiation was present in metastatic lymph nodes in almost all mixed BSCCs (95.7%, 22/23) with LNM. The LNM rate of superficial (T1b) BSCCs (17.6%, 6/34) was comparable to that of superficial (T1b) SCCs (18.5%, 57/308). However, LNM exclusively occurred in superficial mixed (3/5) and borderline (3/10) BSCCs. The IHC results demonstrated a prevalence of SGD in pure group (77%, 43/56). SGD is considered to be a favorable factor, while the squamous differentiation or invasive SCC component is an adverse factor in esophageal BSCCs. Refinement of classification is a promising way to improve patient management.

Tumor-Infiltrating Normal B Cells Revealed by Immunoglobulin Repertoire Clonotype Analysis Are Highly Prognostic and Crucial for Antitumor Immune Responses in DLBCL.

PURPOSE: Tumor-infiltrating B lymphocytes (TIL-B) have demonstrated prognostic and predictive significance in solid cancers. In this study, we aimed to distinguish TIL-Bs from malignant B-cells in diffuse large B-cell lymphoma (DLBCL) and determine the clinical and biological significance. EXPERIMENTAL DESIGN: A total of 269 patients with de novo DLBCL from the International DLBCL R-CHOP Consortium Program were studied. Ultra-deep sequencing of the immunoglobulin genes was performed to determine B-cell clonotypes. The frequencies and numbers of TIL-B clonotypes in individual repertoires were correlated with patient survival, gene expression profiling (GEP) data, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex IHC at single-cell resolution. RESULTS: TIL-B abundance, evaluated by frequencies of normal B-cell clonotypes in the immunoglobulin repertoires, remarkably showed positive associations with significantly better survival of patients in our sequenced cohorts. DLBCLs with high versus low TIL-B abundance displayed distinct GEP signatures, increased pre-memory B-cell state and naïve CD4 T-cell state fractions, and higher CD4+ T-cell infiltration. TIL-B frequency, as a new biomarker in DLBCL, outperformed the germinal center (GC) B-cell-like/activated B-cell-like classification and TIL-T frequency. The identified TIL-B-high GEP signature, including genes upregulated during T-dependent B-cell activation and those highly expressed in normal GC B cells and T cells, showed significant favorable prognostic effects in several external validation cohorts. CONCLUSIONS: TIL-B frequency is a significant prognostic factor in DLBCL and plays a crucial role in antitumor immune responses. This study provides novel insights into the prognostic determinants in DLBCL and TIL-B functions with important therapeutic implications.

Neoadjuvant sintilimab in combination with concurrent chemoradiotherapy for locally advanced gastric or gastroesophageal junction adenocarcinoma: a single-arm phase 2 trial.

In this multicenter, single-arm phase 2 trial (ChiCTR1900024428), patients with locally advanced gastric/gastroesophageal junction cancers receive one cycle of sintilimab (anti-PD1) and chemotherapy (S-1 and nab-paclitaxel), followed by 5 weeks of concurrent chemoradiotherapy and sintilimab, and another cycle of sintilimab and chemotherapy thereafter. Surgery is preferably scheduled within one to three weeks, and three cycles of adjuvant sintilimab and chemotherapy are administrated. The primary endpoint is the pathological complete response. Our results meet the pre-specified primary endpoint. Thirteen of 34 (38.2%) enrolled patients achieve pathological complete response (95% CI: 22.2-56.4). The secondary objectives include disease-free survival (DFS), major pathological response, R0 resection rate, overall survival (OS), event-free survival (EFS), and safety profile. The median DFS and EFS were 17.0 (95%CI: 11.1-20.9) and 21.1 (95%CI: 14.7-26.1) months, respectively, while the median OS was not reached, and the 1-year OS rate was 92.6% (95%CI: 50.1-99.5%). Seventeen patients (50.0%) have grade ≥3 adverse events during preoperative therapy. In prespecified exploratory biomarker analysis, CD3+ T cells, CD56+ NK cells, and the M1/M1 + M2-like macrophage infiltration at baseline are associated with pathological complete response. Here, we show the promising efficacy and manageable safety profile of sintilimab in combination with concurrent chemoradiotherapy for the perioperative treatment of locally advanced gastric/gastroesophageal junction adenocarcinoma.

NTRK gene alterations were enriched in hepatoid or enteroblastic differentiation type of gastric cancer.

AimsCurrently, the clinicopathological characteristics of gastric cancer (GC) with oncogenic NTRK alterations are not well known. Although NTRK fusion has been identified as prevalent in DNA mismatch repair protein deficient (dMMR) colorectal cancer (CRC), the relationship between NTRK alterations and dMMR protein expression in GC has not been previously explored. METHODS: Our study comprised 51 cases of EBV(Epstein-barr virus)-associated gastric carcinomas, 94 cases of dMMR GC, 90 cases of gastric adenocarcinoma with hepatoid or enteroblastic differentiation (GAHED) and 256 cases of conventional GC. Furthermore, to investigate the connection between NTRK fusion and dMMR proteins, we collected dMMR tumours of various types, including 21 cases of duodenal adenocarcinomas, 46 endometrioid carcinomas and 82 CRCs. NTRK fusion and amplification were screened in GC and various types of dMMR tumours using fluorescence in situ hybridisation (FISH), while cases positive for FISH translocation underwent next-generation sequencing testing. RESULTS: Our findings revealed the existence of two cases each of NTRK fusions and NTRK amplifications, which were all enriched in case of GAHED. Additionally, following an analysis of several types of cancers, we discovered that NTRK gene alterations were only present in dMMR CRC. CONCLUSIONS: Our results indicate that NTRK gene alterations are not enriched in GC with dMMR but are specifically enriched in cases of GAHED.

KIT A502_Y503 duplication mutation serves as a potential and universal target for neoantigen peptide in Chinese GIST patients.

BACKGROUND AND AIM: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor with high prevalence of KIT and PDGFRA mutations. Few effective treatments can be exploited in imatinib or sunitinib resistant cases. While in immunotherapy, application of the highly individualized cancer neoantigen vaccines is hampered due to high economic and time cost. In this study we identified the most frequent mutation in Chinese GIST patients and predicted candidate neopeptide by next generation sequencing (NGS). METHODS: Tumor tissues and matched blood samples of 116 Chinese GIST patients were collected. Genomic profile was detected through NGS, and 450 cancer genes were deeply sequenced. KIT mutations were identified, and long peptides containing the mutation were queried in NetMHCpan 4.0 tools to predict MHC class I binding of mutant peptides. RESULTS: The most frequent mutated genes in detected GIST patients were KIT (81.9%, 95/116), CDKN2A (18.97%, 22/116), and CDKN2B (15.52%, 18/116) in this cohort. The most common mutation of KIT was A502_Y503 duplication (15.93%, 18/113) in exon 9. Among the 116 cases, 103 were HLA I genotyped, and 101 were HLA II genotyped. In total, 16 samples with the mutation of KIT p.A502_Y503dup were identified to produce neoantigens with qualified HLA affinity. CONCLUSIONS: KIT hotspot mutation (p.A502_Y503dup) has the highest incidence, which may further eliminate the need for whole genome sequencing and patient-specific neoantigen prediction and synthesis. Therefore, for those carrying such mutation, accounting for around 16% of Chinese GIST patients and are usually less sensitive to imatinib, effective immunotherapies are in prospect.

Targeting WDxR motif reprograms immune microenvironment and inhibits hepatocellular carcinoma progression.

The WD-repeat (WDR) family affects carcinogenesis, but its role in the immune microenvironment is poorly characterized. Although functional loss or gain of WDR6 does not markedly change in vitro proliferative and invasive capacity of HCC cells, its deficiency in hepa1-6 cells drastically inhibits the growth and lung metastasis of orthotopically implanted tumors in immune-competent C57BL/6J mice. Mechanistically, WDR6 targets tumor suppressor UVRAG to the CUL4A-DDB1-ROC1 E3 ubiquitin ligase complex through a unique WDxR motif and promotes its degradation. This upregulates chromatin accessibility at the TNFα locus by blocking autophagic degradation of p65, elevates intratumoral myeloid-derived suppressor cell (MDSC) number, and reduces CD8+ T cell infiltration, thereby promoting HCC progression. These immunosuppressive effects are reversed by TNFα blockade. TNFα recruits NF-κB to activate the transcription of WDR6, establishing a WDR6-TNFα loop. Clinically, the WDR6/UVRAG/NF-κB pathway is hyperactivated in HCC, predicting a poor prognosis. Importantly, a WDxR-like peptide disrupts the WDR6/UVRAG complex and enhances the efficiency of anti-PD-L1 against HCC with WDR6 dysregulation.

Prevalence and Associations of Beta2-Microglobulin Mutations in MSI-H/dMMR Cancers.

Microsatellite instability (MSI) has emerged as an important predictor of sensitivity for immunotherapy-based strategies. ß-2-Microglobulin (B2M) contains microsatellites within the coding regions and is prone to somatic changes in MSI/mismatch repair deficiency (MSI/dMMR) tumors. To delineate prevalence and associations of B2M mutations in MSI-H/dMMR cancers, we investigated the mutational profile of B2M and clinical and pathological features in gastric cancer (GC), colorectal cancer (CRC), and endometrial cancer (EC) with a high incidence of microsatellite instability-high (MSI-H)/dMMR. Formalin-fixed paraffin-embedded (FFPE) tumor tissues along with matched normal tissues were collected from 108 MSI/dMMR patients with GC, CRC, and EC. Genomic profiling of tissue and blood samples were assessed next-generation sequencing (NGS). Immunohistochemistry (IHC) was used to examine the presence or absence of B2M protein. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. NGS assay revealed that genes involved in chromatin regulation, the PI3K pathway, the WNT pathway, and mismatch repair were extensively altered in the MSI-H cohort. Signature 6 and 26, 2 of 4 mutational signatures associated with defective DNA mismatch repair, featured with high numbers of small insertion/deletions (INDEL) dominated in all 3 types of cancer. Alternations in the exonic microsatellite regions of B2M were observed at various but high frequencies (57.5% in CRC, 23.9% in GC, and 13.6% in EC) and in different forms. Tumor mutational burden (TMB) was significantly higher in the patients carrying MSI-H/dMMR tumors with B2M mutation than that in patients with wild-type B2M (P = .026).The frame shift alteration occurring at the exonic microsatellite sties caused loss of function of B2M gene. In addition, a case with CRC carrying indels in B2M gene resisted the ICI treatment was reported. In conclusion, patients carrying MSI-H/dMMR tumors with B2M mutation showed significantly higher TMB. Prescription of ICIs should be thoroughly evaluated for these patients.

MIST: multiple instance learning network based on Swin Transformer for whole slide image classification of colorectal adenomas.

Colorectal adenoma is a recognized precancerous lesion of colorectal cancer (CRC), and at least 80% of colorectal cancers are malignantly transformed from it. Therefore, it is essential to distinguish benign from malignant adenomas in the early screening of colorectal cancer. Many deep learning computational pathology studies based on whole slide images (WSIs) have been proposed. Most approaches require manual annotation of lesion regions on WSIs, which is time-consuming and labor-intensive. This study proposes a new approach, MIST - Multiple Instance learning network based on the Swin Transformer, which can accurately classify colorectal adenoma WSIs only with slide-level labels. MIST uses the Swin Transformer as the backbone to extract features of images through self-supervised contrastive learning and uses a dual-stream multiple instance learning network to predict the class of slides. We trained and validated MIST on 666 WSIs collected from 480 colorectal adenoma patients in the Department of Pathology, The Affiliated Drum Tower Hospital of Nanjing University Medical School. These slides contained six common types of colorectal adenomas. The accuracy of external validation on 273 newly collected WSIs from Nanjing First Hospital was 0.784, which was superior to the existing methods and reached a level comparable to that of the local pathologist's accuracy of 0.806. Finally, we analyzed the interpretability of MIST and observed that the lesion areas of interest in MIST were generally consistent with those of interest to local pathologists. In conclusion, MIST is a low-burden, interpretable, and effective approach that can be used in colorectal cancer screening and may lead to a potential reduction in the mortality of CRC patients by assisting clinicians in the decision-making process. © 2022 The Pathological Society of Great Britain and Ireland.

The efficacy of additional surgical resection after endoscopic resection in pT1b esophageal squamous cell carcinoma: A multi-institutional retrospective study in China.

BACKGROUND: pT1b esophageal squamous cell carcinoma (ESCC) patients treated by endoscopic resection (ER) required additional treatment with surgical resection (SR) or chemoradiotherapy (CRT) according to 2020 Japan Gastroenterological Endoscopy Society (JGES) guideline. Given the evidences for this recommendation were largely based on small-size studies, our study collected 166 cases of ER-treated pT1b patients in order to investigate the efficacy of additional SR as compared to ER-alone treatment. METHODS: A multi-institutional retrospective study in China was conducted. The pT1b ESCC treated by ER + SR (n = 42) and ER-alone (n = 124) from 2007 to 2018 were recruited. Meanwhile, patients with positive lymphovascular invasion (LVI(+)) and/or with positive vertical margin (VM(+)) were put into high-risk group, and those with both VM(-) and LVI(-) were selected into low-risk group. The clinicopathological parameters, lymph node metastasis (LNM), and survival between ER + SR and ER-alone groups were analyzed. RESULTS: In high-risk group, concurrent LNM revealed in surgically resected specimens accounted for 52.6% cases in ER + SR group. After surgical removal, the incidence of post-resection LNM dropped down to 5.6%. However, in low-risk group, patients with ER + SR treatment did not exhibit any concurrent LNM in surgically resected specimens, and the incidence of their overall LNM was similar to that in ER-alone group (0% vs. 2.8%, p = 1.000). More importantly, these cases demonstrated significantly shorter overall survival (OS) than that in ER-alone group (81.8% and 100.0%, respectively, at 3 years; log-Rank: P = 0.010). CONCLUSIONS: For ER-treated pT1b patients in high-risk group, additional SR is strongly recommended. However, for those in low-risk group, additional SR does not generate much benefit for clearance of LNM, but brings harm to shorten their OS. Therefore, additional SR is not recommended for ER-treated pT1b patient in low-risk group.

HER2 overexpression/amplification status in colorectal cancer: a comparison between immunohistochemistry and fluorescence in situ hybridization using five different immunohistochemical scoring criteria.

OBJECTIVE: Although HER2 has gradually become an important therapeutic target for colorectal cancer (CRC), a unified and standard HER2 scoring system was still not established in CRC, and the debatable results of immunohistochemistry and fluorescence in situ hybridization (FISH) in CRC requires further exploration. METHODS: In this study, we use five immunohistochemical (IHC) scoring criteria (i.e., IRS-p, IRS-m, GEA-s, GEA-b and HERACLES) and two FISH criteria to evaluate HER2 status, and further evaluate the correlation between HER2 status and clinicopathological features, survival in a large, unselected Chinese cohort of 664 CRCs. RESULTS: Finally, we set HER2/CEP17 ratio ≥ 2.0, or an average HER2 copy number ≥ 6.0 as FISH-positive threshold and the amplification rate of HER2 gene was 7.08% (47/664).The HER2 positivity (IHC 3+) was 2.71%, 3.16%, 2.56%, 2.71% and 3.16%, according to the IHC scoring criteria of IRS-p, IRS-m, GEA-s, GEA-b and HERACLES, respectively. Set FISH results as the golden standard; receiver-operating characteristic analysis showed that IRS-p had both high sensitivity and specificity than other IHC scoring systems to evaluate HER2 status. Based on IRS-p criterion, There were significant differences in tumor differentiation (p = 0.038), lymphatic vascular invasion (p = 0.001), pN stage (p value = 0.043), and overall survival (p < 0.001) among IHC score 0-3 + groups. Meanwhile, there were significant differences in pT stage (p = 0.031), pN stage (p = 0.009) and overall survival (p < 0.001) among FISH subgroups. CONCLUSION: The IRS-p criterion was more suitable for assessing the HER2 status in CRC patients than other IHC criteria. Whereas for FISH scoring system, only HER2/CEP17 < 2.0, meanwhile HER2cn < 4.0 and HER2cn ≥ 6.0 were subgroups with unique clinicopathological characteristics.

Kruppel-like factor 13 inhibits cell proliferation of gastric cancer by inducing autophagic degradation of ß-catenin.

Zinc protein KLF13 is a tumor-suppressive member of Kruppel-like factors family, and yet the effect of KLF13 on gastric cancer has not been reported. Here, we aimed to investigate the role of KLF13 in gastric cancer and explored underlying molecular mechanisms. Firstly, it was found that KLF13 expression was significantly decreased in gastric cancer tissues and cancer cells compared with adjacent normal tissues and normal gastric epithelial cells, respectively. KEGG_Pathway and GO_BP analyses suggested that KLF13 was associated with CELL_CYCLE and CELL_PROLIFERATION. Then, our results further demonstrated that KLF13 could obviously inhibit gastric cancer proliferation and induce cell arrest at G2/M phase. Mechanistically, KLF13 decreased expressions of ß-catenin and its target genes, CCND1 and MYC, via triggering autophagic degradation of ß-catenin. KLF13 up-regulation facilitated co-localization and binding of ß-catenin with autophagy protein p62, and exogenous overexpression of ß-catenin or blocking autophagy process appeared to reverse KLF13-induced inhibition of gastric cancer proliferation. Furthermore, KLF13 overexpression promoted the expression of ubiquitin-conjugating enzyme E2, Ubc13 which is responsible for catalyzing the synthesis of 'Lys-63'-linked polyubiquitin chains and increased the binding of ß-catenin with E3 ubiquitin ligase, TRAF6. In vivo, KLF13 overexpression also suppressed xenograft tumor growth of gastric cancer and down-regulated expressions of Ki67, ß-catenin, Cyclin D1, and c-Myc in tumor tissues. Collectively, these data firstly demonstrated the involvement of KLF13 in inhibiting cell proliferation of gastric cancer through promoting autophagy-dependent degradation of ß-catenin, which reinforced the evidence for suppressive roles of KLF13 in human tumors.

A Retrospective Cohort Study of Factors Influencing Lymph Node Metastasis in Patients With Early Gastric Papillary Adenocarcinoma.

INTRODUCTION: High risk of lymph node metastasis (LNM) in gastric papillary adenocarcinoma causes endoscopists to worry about the suitability of endoscopic resection for early gastric papillary adenocarcinoma (EPAC). We compared risk factors and attempted to establish a scoring system to stratify LNM risk in patients with EPAC. METHODS: A retrospective analysis was performed on 2,513 patients with early gastric carcinoma (EGC) who underwent radical resection in 4 tertiary hospitals in China. Univariate and multivariate analyses were performed to compare the invasiveness in EPAC and other types of EGC and to evaluate potential factors in predicting LNM risk in EPAC groups. RESULTS: Three hundred thirty-five patients with EPAC were enrolled in our study, of which 62 patients were found to have LNM. After comparing clinicopathological characteristics of EPAC with and without LNM, the following factors were included in the risk scoring system: 1 point each for lower stomach location and tumor size >2.0 cm, 3 points for lymphovascular invasion, and 4 points for submucosal invasion; the risk scoring system was validated in a small internal validation set with an area under the curve of 0.844. DISCUSSION: Our results suggested that EPAC was highly invasive compared with other EGCs, especially differentiated EGC types, and need to be treated more rigorously. This proposed risk scoring system could stratify LNM risk in patients with EPAC, and endoscopic resection may only be performed safely on the groups with a low LNM rate.

Antigen-Specific T Cell Immunotherapy Targeting Claudin18.2 in Gastric Cancer.

T cell-based immunotherapy has led to many breakthroughs in the treatment of solid tumors. In this study, we found that membrane protein Claudin18.2 was a promising antigen in T cell-based immunotherapy for gastric cancer (GC). Firstly, we identified five HLA-A*0201- and seven HLA-A*1101-restricted T cell epitopes of Claudin18.2. Peripheral blood mononuclear cells (PBMCs) stimulated by Claudin18.2 peptides showed progressive anti-tumor ability and higher effective cytokine secretion than unstimulated PBMCs in vitro. In total, 81.8% of GC patients were Claudin18.2-positive by immunohistochemical (IHC) detection, and a positive correlation between Claudin18.2 expression and peptide reactivity (p = 0.002) was found. Clinicopathological features analyses demonstrated that Claudin18.2 expression did not correlate with gender, age, stage or Lauren classification. Survival analysis showed that a longer median progression-free survival (mPFS) was not related to peptide reactivity (p = 0.997), but related to a lower Claudin18.2 expression level (p = 0.047). These findings establish a foundation for the clinical application of Claudin18.2 targeted T cell-based immunotherapy in GC.

Efficacy and Safety of Sintilimab in Combination with Concurrent Chemoradiotherapy for Locally Advanced Gastric or Gastroesophageal Junction (GEJ) Adenocarcinoma (SHARED): Study Protocol of a Prospective, Multi-Center, Single-Arm Phase 2 Trial.

Purpose: Concurrent chemoradiotherapy (cCRT) is the mainstay therapy of locally advanced gastric (G) and gastroesophageal junction (GEJ) cancers with a poor prognosis. Programmed cell death receptor-1 (PD-1) inhibitor has been approved and recommended to treat ≥3 line G/GEJ patients. A significant clinical benefit of PD-1 inhibitors in addition to cCRT has been observed in locally advanced lung cancer. Sintilimab, a humanized IgG4 monoclonal antibody with high affinity and specificity for PD-1, has shown promising efficacy with an overall response rate of 85% in combination with chemotherapy in gastric cancer in a phase Ib study (NCT02937116). Patients and Methods: SHARED is a prospective, multicentre, single-arm Phase II trial in China, exploring the efficacy of sintilimab in combination with cCRT in locally advanced G/GEJ adenocarcinoma. According to a Simon optimal two-stage clinical design, 34 patients will be enrolled. All the eligible patients will receive one cycle of induction chemotherapy (S-1 plus nab-PTX) combined with sintilimab, followed by cCRT (radiotherapy plus nab-PTX) combined with sintilimab. Prior to the surgery, patients will receive another cycle of chemotherapy (S-1 plus nab-PTX) combined with sintilimab. In the adjuvant setting, all participants will be treated with 3 cycles of chemotherapy (S-1 plus nab-PTX) combined with sintilimab. The primary endpoint is the rate of pathological complete response (pCR). The secondary endpoints include disease-free survival (DFS), major pathological response (MPR), R0 resection rate, surgical AEs, overall survival (OS), event-free survival (EFS), and safety profile. Moreover, the prognostic value of tumor biomarkers and immune biomarkers will be explored. Conclusion: SHARED is designed to primally evaluate the efficacy and safety of sintilimab in combination with cCRT in locally advanced G/GEJ cancers and to prospectively validate the prognostic value of tumor biomarkers and immune biomarkers.

AKR1C3 regulated by NRF2/MAFG complex promotes proliferation via stabilizing PARP1 in hepatocellular carcinoma.

Aldo-keto reductase family 1 member C3 (AKR1C3) serves as a contributor to numerous kinds of tumors, and its expression is elevated in patients with hepatocellular carcinoma (HCC). However, the biological function of AKR1C3 in HCC remains unclear. Here we investigated the role of AKR1C3 in liver carcinogenesis using in vitro and in vivo models. We determined that AKR1C3 is frequently increased in HCC tissues with poor prognosis. Genetically manipulated cells with AKR1C3 construction were examined to highlight the pro-tumoral growth of both wild-type AKR1C3 and mutant in vitro and in vivo. We observed promising treatment effects of AKR1C3 shRNA by intratumoral injection in mice. Mechanically, we demonstrated that the transcription factor heterodimer NRF2/MAFG was able to bind directly to AKR1C3 promoter to activate its transcription. Further, AKR1C3 stabilized PARP1 by decreasing its ubiquitination, which resulted in HCC cell proliferation and low sensitivity of Cisplatin. Moreover, we discovered that the tumorigenic role of AKR1C3 was non-catalytic dependent and the NRF2/MAFG-AKR1C3-PARP1 axis might be one of the important proliferation pathways in HCC. In conclusion, blockage of AKR1C3 expression provides potential therapeutic benefits against HCC.

Prognostic Perspectives of STING and PD-L1 Expression and Correlation with the Prognosis of Epstein-Barr Virus-Associated Gastric Cancers.

Background/Aims: : Epstein-Barr virus-associated gastric cancers (EBVaGCs) have unique molecular and clinicopathological characteristics. The cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway is recently recognized as the critical innate immunity against pathogens and tumors. STING is also a master regulator in the cancer-immunity cycle and targeting STING could synergize with existing immune-checkpoint therapies. However, the role of STING in GC, especially in EBVaGC, and its correlation with programmed death-ligand 1 (PD-L1) remain largely unclear. Methods: We collected 78 cases of EBVaGCs and 210 cases of EBV-negative GC (EBVnGC) from a total of 1,443 cases of GC analyzed by EBV-encoded small RNA in situ hybridization. We investigated STING and PD-L1 expression and their concomitant prognostic value in EBVaGCs and EBVnGCs using tissue microarray and immunohistochemistry. The effects of STING and PD-L1 expression on the overall survival of patients with EBVaGC or EBVnGC were assessed by univariate and multivariate analysis. Results: We found that both STING and PD-L1 exhibited significantly higher expression in the EBVaGCs than that in the EBVnGCs. The expression of STING was positively correlated with that of PD-L1 in EBVaGCs. Simultaneous negative expression of STING and PD-L1, and positive expression of STING were independent prognostic risk factors for EBVaGC and EBVnGC, respectively. Conclusions: This is the first prognostic retrospective study of STING and PD-L1 expression and the prognosis among EBVaGC and EBVnGC. The expression and prognostic value of STING and PD-L1 are different in the two types of GCs. STING and PD-L1 are promising prognostic biomarkers and therapeutic targets for EBVaGC and EBVnGC.

Survival prediction on intrahepatic cholangiocarcinoma with histomorphological analysis on the whole slide images.

Intrahepatic cholangiocarcinoma (ICC) is cancer that originates from the liver's secondary ductal epithelium or branch. Due to the lack of early-stage clinical symptoms and very high mortality, the 5-year postoperative survival rate is only about 35%. A critical step to improve patients' survival is accurately predicting their survival status and giving appropriate treatment. The tumor microenvironment of ICC is the immediate environment on which the tumor cell growth depends. The differentiation of tumor glands, the stroma status, and the tumor-infiltrating lymphocytes in such environments are strictly related to the tumor progress. It is crucial to develop a computerized system for characterizing the tumor environment. This work aims to develop the quantitative histomorphological features that describe lymphocyte density distribution at the cell level and the different components at the tumor's tissue level in H&E-stained whole slide images (WSIs). The goal is to explore whether these features could stratify patients' survival. This study comprised of 127 patients diagnosed with ICC after surgery, where 78 cases were randomly chosen as the modeling set, and the rest of the 49 cases were testing set. Deep learning-based models were developed for tissue segmentation and lymphocyte detection in the WSIs. A total of 107-dimensional features, including different type of graph features on the WSIs were extracted by exploring the histomorphological patterns of these identified tumor tissue and lymphocytes. The top 3 discriminative features were chosen with the mRMR algorithm via 5-fold cross-validation to predict the patient's survival. The model's performance was evaluated on the independent testing set, which achieved an AUC of 0.6818 and the log-rank test p-value of 0.03. The Cox multivariable test was used to control the TNM staging, γ-Glutamytransferase, and the Peritumoral Glisson's Sheath Invasion. It showed that our model could independently predict survival risk with a p-value of 0.048 and HR (95% confidence interval) of 2.90 (1.01-8.32). These results indicated that the composition in tissue-level and global arrangement of lymphocytes in the cell-level could distinguish ICC patients' survival risk.

Combined assessment of tumour cell nest size and desmoplastic reaction as an excellent prognostic predictor in oesophageal squamous cell carcinoma.

AIMS: Tumour budding (TB) activity, cell nest size (CNS), and desmoplastic reaction (DR) have been confirmed to be significantly correlated with prognosis in oesophageal squamous cell cancer (ESCC) recently. However, there are limited data on the prognostic significance of combined assessment of cellular dissociation and tumour stroma in ESCC. METHODS: In all, 265 cases with resected ESCCs diagnosed between January 2018 and August 2019 were retrospectively reviewed. All slides were reviewed for assessing TB, CNS, and DR. The Cellular Dissociation Grading and our Combined CNS and DR (CNS/DR) Grading systems were adopted to re-grade ESCCs. RESULTS: High TB activity, small CNS, and immature DR had a strong association with shorter overall survival (OS) and progression-free survival (PFS) (P < 0.001, respectively) in ESCC. Combined assessment of CNS and DR in a 4-tiered grading system displayed a prognostic excellence for survival (P < 0.001), and outperformed the Cellular Dissociation Grading for both OS (area under the curve [AUC], 0.728 versus 0.644, P = 0.043) and PFS (AUC, 0.763 versus 0.667, P = 0.018) by receiver operator characteristic curves. Also, Combined CNS/DR Grading showed superiority in recognizing a G4 subgroup with the worst outcome in our cohort, to whom the most urgent attention needs to be called. CONCLUSIONS: This is the first study to propose a novel Combined Grading system based on CNS and DR in ESCC, which has been demonstrated to be relatively superior to Cellular Dissociation Grading in predicting prognosis. The findings shed new light on the histopathological grading of ESCC and facilitates identifying biologically aggressive ESCCs.

Methionine oxidation of CLK4 promotes the metabolic switch and redox homeostasis in esophageal carcinoma via inhibiting MITF selective autophagy.

BACKGROUND: Metabolic reprogramming and redox homeostasis contribute to esophageal squamous cell carcinoma (ESCC). CDC-like kinase 4 (CLK4) is a dual-specificity kinase that can phosphorylate substrates' tyrosine or serine/threonine residue. However, the role and mechanism of CLK4 in ESCC remain unknown. METHODS: CLK4 expression was analysed using publicly available datasets and confirmed in ESCC tissues and cell lines. The biological roles of CLK4 were studied with gain and loss-of-function experiments. Mass spectrometry was employed to examine the effects of CLK4 on metabolic profiling. In vitro kinase assay, co-immunoprecipitation, glutathione S-transferase pulldown, chromatin immunoprecipitation and luciferase reporter were used to elucidate the relationship among CLK4, microphthalmia-associated transcription factor (MITF), COP1 and ZRANB1. RESULTS: CLK4 down-regulation was observed in ESCC cell lines and clinical samples and associated with the methylation of its promoter. Low levels of CLK4 promoted ESCC development by affecting the purine synthesis pathway and nicotinamide adenine dinucleotide phosphate (NADPH)/nicotinamide adenine dinucleotide phosphate (NADP+ ) ratio. Interestingly, CLK4 inhibited ESCC development by blocking MITF-enhanced de novo purine synthesis and redox balance. Mechanistically, wild type CLK4 (WT-CLK4) but not kinase-dead CLK4-K189R mutant phosphorylated MITF at Y360. This modification promoted its interaction with E3 ligase COP1 and its K63-linked ubiquitination at K308/K372, leading to sequestosome 1 recognition and autophagic degradation. However, the deubiquitinase ZRANB1 rescued MITF ubiquitination and degradation. In turn, MITF bound to E- rather than M-boxes in CLK4 promoter and transcriptionally down-regulated its expression in ESCC. Clinically, the negative correlations were observed between CLK4, MITF, and purine metabolic markers, which predicts a poor clinical outcome of ESCC patients. Notably, CLK4 itself was a redox-sensitive kinase, and its methionine oxidation at M307 impaired kinase activity, enhanced mitochondria length and inhibited lipid peroxidation, contributing to ESCC. CONCLUSIONS: Our data highlight the potential role of CLK4 in modulating redox status and nucleotide metabolism, suggesting potential therapeutic targets in ESCC treatment.