Comparative Epigenetic Profiling Reveals Distinct Features of Mucosal Melanomas Associated with Immune Cell Infiltration and Their Clinical Implications.

Mucosal melanoma exhibits limited responsiveness to anti-PD-1 therapy. However, a subgroup of mucosal melanomas, particularly those situated at specific anatomic sites like primary malignant melanoma of the esophagus (PMME), display remarkable sensitivity to anti-PD-1 treatment. The underlying mechanisms driving this superior response and the DNA methylation patterns in mucosal melanoma have not been thoroughly investigated. We collected tumor samples from 50 patients with mucosal melanoma, including 31 PMME and 19 non-esophageal mucosal melanoma (NEMM). Targeted bisulfite sequencing was conducted to characterize the DNA methylation landscape of mucosal melanoma and explore the epigenetic profiling differences between PMME and NEMM. Bulk RNA sequencing and multiplex immunofluorescence staining were performed to confirm the impact of methylation on gene expression and immune microenvironment. Our analysis revealed distinct epigenetic signatures that distinguish mucosal melanomas of different origins. Notably, PMME exhibited distinct epigenetic profiling characterized by a global hypermethylation alteration compared with NEMM. The prognostic model based on the methylation scores of a 7-DMR panel could effectively predict the overall survival of patients with PMME and potentially serve as a prognostic factor. PMME displayed a substantial enrichment of immune-activating cells in contrast to NEMM. Furthermore, we observed hypermethylation of the TERT promoter in PMME, which correlated with heightened CD8+ T-cell infiltration, and patients with hypermethylated TERT were likely to have improved responses to immunotherapy. Our results indicated that PMME shows a distinct methylation landscape compared with NEMM, and the epigenetic status of TERT might be used to estimate prognosis and direct anti-PD-1 treatment for mucosal melanoma. SIGNIFICANCE: This study investigated the intricate epigenetic factor of mucosal melanomas contributed to the differential immune checkpoint inhibitor response, and found that PMME exhibited a global hypermethylation pattern and lower gene expression in comparison to NEMM. TERT hypermethylation may contribute to the favorable responses observed in patients with mucosal melanoma undergoing immunotherapy.

Design Principles and Routes for Calcium Alkoxyaluminate Electrolytes.

Multivalent-ion battery technologies are increasingly attractive options for meeting diverse energy storage needs. Calcium ion batteries (CIB) are particularly appealing candidates for their earthly abundance, high theoretical volumetric energy density, and relative safety advantages. At present, only a few Ca-ion electrolyte systems are reported to reversibly plate at room temperature: for example, aluminates and borates, including Ca[TPFA]2, where [TPFA]- = [Al(OC(CF3)3)4]- and Ca[B(hfip)4]2, [B(hfip)4]2- = [B(OCH(CF3)2)4]-. Analyzing the structure of these salts reveals a common theme: the prevalent use of a weakly coordinating anion (WCA) consisting of a tetracoordinate aluminum/boron (Al/B) center with fluorinated alkoxides. Leveraging the concept of theory-aided design, we report an innovative, one-pot synthesis of two new calcium-ion electrolyte salts (Ca[Al(tftb)4]2, Ca[Al(hftb)4]2) and two reported salts (Ca[Al(hfip)4]2 and Ca[TPFA]2) where hfip = (-OCH(CF3)2), tftb = (-OC(CF3)(Me)2), hftb = (-OC(CF3)2(Me)), [TPFA]- = [Al(OC(CF3)3)4]-. We also reveal the dependence of Coulombic efficiency on their inherent propensity for cation-anion coordination.

Proficiency testing of diagnosis in histopathology and immunohistochemistry of breast pathology in China: results from a pilot work of National Single Disease Quality Control Program for breast cancer.

AIM: Pathologists are currently supposed to be aware of both domestic and international guidelines for breast cancer diagnosis, but it is unclear how successfully these guidelines have been integrated into routine clinical practice in China. Thus, this national proficiency testing (PT) scheme for breast pathology was set up to conduct a baseline assessment of the diagnostic capability of pathologists in China. METHODS: This national PT plan is designed and implemented according to the "Conformity assessment-General requirements for proficiency testing" (GB/T27043-2012/ISO/IEC 17043:2010). Five cases of breast cancer with six key items, including histologic type, grade, ER, PR, HER2, and Ki67, were selected for testing among 96 participants. The final PT results were published on the website of the National Quality Control Center for Cancer ( http://117.133.40.88:3927/cn/col22/362 ). RESULTS: Our study demonstrated that the median PT score was 89.5 (54-100). Two institutions with scores < 67 were deemed unacceptable. The accuracy of histologic type, ER, PR, HER2, and Ki67 was satisfactory (all > 86%). However, the histologic grade showed low accuracy (74.0%). The unacceptable results mainly included incorrect evaluation of histologic grade (36.7%), inaccurate evaluation of ER/PR/HER2/Ki67 (28.2%), incorrect identification of C-AD as IBC-NST (15.7%), inappropriate use of 1+/2+/3+ rather than staining percentage for ER/PR (6.1%), misclassification of ER/PR < 1% weak expression as positive staining (1.4%), and no evaluation of histologic grade in ILC, MC, and IMC (5.8%). CONCLUSIONS: our nationwide PT program exhibited a satisfactory baseline assessment of the diagnostic capability of pathologists in China. More importantly, we identify some areas for further improvement.

Role of immunophenotypic characterisation in prognostic subtyping of intrahepatic cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (iCCA) is classified by the 5th WHO classification of tumours of the digestive system as large duct type (LDT) and small duct type (SDT), based on the anatomical location, morphological appearances, immunophenotype, and gene events. We evaluated the subtyping system using real-world data and established a supplementary method using immunohistochemical (IHC) detection. We retrospectively investigated 190 cases of surgically resected iCCA and classified them according to histological evaluations and gene detection. The prognostic value of the IHC markers were evaluated according to the relapse-free survival (RFS) and overall survival (OS). Basic histological classification was insufficient, with 61 cases classified as uncertain. This method showed no prognostic value for RFS or OS. The four-marker IHC detection, including EMA, S100P, N-cadherin, and CRP, which classified 68 cases as LDT, 108 cases as SDT, and 14 cases as uncertain, was highly efficient in subtyping and prognosis. The seven-marker method, including CD56, MUC5AC and MUC6, was consistent with the four-marker method. FGFR2 gene fusion was exclusively detected in 20 cases of SDT iCCA, according to the four- and seven-marker IHC detection. This novel method of iCCA classification exhibited diagnostic, prognostic and therapeutic value in clinical practice.

Multiple primary lung cancer: Updates of clinical management and genomic features.

In recent decades, multiple primary lung cancer (MPLC) has been increasingly prevalent in clinical practice. However, many details about MPLC have not been completely settled, such as understanding the driving force, clinical management, pathological mechanisms, and genomic architectures of this disease. From the perspective of diagnosis and treatment, distinguishing MPLC from lung cancer intrapulmonary metastasis (IPM) has been a clinical hotpot for years. Besides, compared to patients with single lung lesion, the treatment for MPLC patients is more individualized, and non-operative therapies, such as ablation and stereotactic ablative radiotherapy (SABR), are prevailing. The emergence of next-generation sequencing has fueled a wave of research about the molecular features of MPLC and advanced the NCCN guidelines. In this review, we generalized the latest updates on MPLC from definition, etiology and epidemiology, clinical management, and genomic updates. We summarized the different perspectives and aimed to offer novel insights into the management of MPLC.

HER2 expression and relevant clinicopathological features in esophageal squamous cell carcinoma in a Chinese population.

BACKGROUND: Despite great progress in surgery and other treatments, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still very poor. HER2 has strong therapeutic implications in certain cancers, such as breast cancer and gastric cancer. However, literature on the frequency of HER2 expression in ESCC is scarce. In the present study, HER2 protein expression, HER2 gene amplification and the relationship between HER2 status and clinicopathological characteristics were evaluated in a large cohort of Chinese ESCC patients. METHODS: A total of 857 consecutive ESCC patients who received radical esophagectomy without neoadjuvant therapy between January 2014 and October 2015 were included in this retrospective study. HER2 protein expression was analyzed by immunohistochemistry (IHC), and its correlation with clinicopathological parameters was assessed. In addition, 65 cases, including 13 HER2 overexpression (3+) cases and 52 HER2 equivocal (2+) cases from the 857-case cohort, and another 104 ESCC cases, including 1 HER2 overexpression (3+) case, 3 HER2 equivocal (2+) cases and 100 HER2 negative (1+/0) cases, were selected to construct tissue microarrays (TMAs). Dual-color in situ hybridization (DISH) was performed on the TMAs to assess HER2 gene amplification and the relationship with clinicopathological parameters. RESULTS: We found HER2 overexpression (3+) status in 1.5% (13/857) of cases and HER2 equivocal (2+) status in 6.1% (52/857) of cases. HER2 IHC expression was significantly associated with gender (P = 0.028). However, there were no significant correlations between HER2 IHC expression and age, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05). Regarding the 169 cases analyzed by DISH, 14 (of 14, 100%) HER2 overexpression (3+) cases, 10 (of 55, 18.2%) HER2 equivocal (2+) cases, and 0 (of 100, 0%) HER2 negative (1+/0) cases showed HER2 gene amplification. HER2 gene amplification was not significantly associated with clinicopathological characteristics such as age, gender, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05). CONCLUSIONS: Approximately 1.5% of the Chinese ESCC patients had HER2 overexpression based on IHC. IHC and DISH had a high concordance rate. These results provide valuable insight for the future treatment of ESCC.

Major challenges in accurate mutation detection of multifocal lung adenocarcinoma by next-generation sequencing.

Background: Many patients with advanced non-small cell lung cancer manifested with metastasis, and molecular heterogeneity may exhibit between primary and metastatic tumors. We sought to investigate the clinical detection strategy of primary and metastatic tumors in Chinese patients with NSCLC.Methods: Here, 77 paired tumors of Chinese patients with lung adenocarcinoma were analyzed, and 1836 mutation in hotspot regions of 22 genes were identified by next-generation sequencing. The expression of ALK in these paired tumors was also detected by immunohistochemistry.Results: The results showed that the concordance rate in multiple pulmonary nodules, primary-LN metastasis pairs and primary-distant metastasis pairs was 67.7%, 94.1% and 86.7%, respectively. In multiple pulmonary nodules, the concordance rate was 100% when the pathologic diagnosis was intrapulmonary metastasis, whereas the concordance rate was 23.1% when the pathologic diagnosis was multiple primary tumors. TP53 and CTNNB1 mutations were detected as the recurrent alterations in LN metastasis. Moreover, the concordance of ALK status was observed in these pairs.Conclusions: Our data suggested that hotspot mutations and ALK status in the primary-metastasis pairs had a high concordance in lung adenocarcinoma. Clinical detection of one lesion may be enough to identify the key alterations except that patients are diagnosed with multiple primary tumors or have disease progression after benefiting from target therapy.

Correlation analysis of mesenchymal-epithelial transition factor protein and human epidermal growth receptor 2 protein expression in 1479 cases of lung adenocarcinoma in China.

BACKGROUND: To investigate the correlation between mesenchymal-epithelial transition factor (C-Met) and human epidermal growth receptor 2 (HER2) protein expression in primary lung adenocarcinoma tissues. METHOD: A total of 1479 resected primary lung adenocarcinoma patients were enrolled in the present study for detecting of C-Met and HER2 protein by immunohistochemistry, and correlation analysis was made between the above two biomarkers and related clinicopathological features. RESULT: Both C-Met and HER2 proteins were found to stain highly positive in lung adenocarcinomas, and a positive correlation was found between them (χ2 = 118.5, P = 2.707 × 10-21 ). In addition, HER2 protein expression was correlated with sex, pathological stage, lymph node metastasis, and major subtypes; and C-Met was correlated with sex (P < 0.05). CONCLUSION: The expression of C-Met and HER2 protein in lung adenocarcinoma is highly correlated, and whether it is synergistic in the targeted therapy of lung adenocarcinoma deserves further study.

Distinct MET Protein Localization Associated With MET Exon 14 Mutation Types in Patients With Non-small-cell Lung Cancer.

BACKGROUND: The MET gene has been recognized as a potential important therapeutic target in non-small-cell lung cancer (NSCLC). We sought to investigate the MET exon 14 mutations in a cohort of Chinese patients with NSCLC. METHODS: We tested 461 NSCLCs for MET exon 14 mutations by sequencing whole exon 14 and its flanking introns. The protein expression was determined by immunohistochemical analysis. RESULTS: In this study, we identified MET exon 14 mutations in 9 (2.0%) of 461 NSCLCs. Of these 9 mutations, 7 (77.8%) were located in the splice sites of MET exon 14, with MET overexpression in 6. One point mutation c.3010C>T (p.Arg1004Ter) was nonsense mutation with no MET expression. One insertion mutation was within exon 14 of MET with MET overexpression. MET protein localization in tumor cells with MET exon 14 mutations was different between mutation types. Three point mutations that disrupted the splice donor site of intron 14 were membranous staining, whereas the other mutations were cytoplasmic staining. Patients with MET exon 14 splice site mutations were significantly older. The incidence of MET exon 14 mutations in sarcomatoid carcinoma was significantly higher than in other histologic types (P = .034). CONCLUSION: Distinct MET protein localization is associated with MET exon 14 mutation types in patients with NSCLC. Different MET exon 14 mutation types were identified in a subset of Chinese patients with NSCLC who could possibly benefit from MET targeted therapy.

PIK3CA mutations and downstream effector p-mTOR expression: implication for prognostic factors and therapeutic targets in triple negative breast cancer.

Although PIK3CA mutations and phosphorylated mTOR (p-mTOR) expression are two main events on PI3K/Akt/mTOR pathway, limited data has reported their roles in triple negative breast cancer (TNBC). Thus, we evaluated the associations of these two biomarkers and clinicopathological characteristics and prognosis in large Chinese TNBC patients. Immunohistochemistry (IHC) analysis was used to assess p-mTOR expression level in 218 TNBC patients. Direct sequencing was applied to detect the most important hotspot regions in exons 9 and 20 of PIK3CA gene. In the TNBC cohort, mutations were identified in 11.5% cases which were associated with basal-like subtype. The somatic point mutations were independently associated with worse overall survival (HR=0.400, 95% CI: 0.193-0.830, P=0.014). As for p-mTOR expression, 47.7% of the tumors were positive and the staining was shown in cytoplasm, nuclear and perinuclear areas. There were significant differences observed in tumor size, lymph node status, and clinical stage between p-mTOR positive and p-mTOR negative cases. Notably, we found a significant association between p-mTOR expression and PIK3CA mutations. Patients with p-mTOR staining also demonstrated shorter overall survival (HR=0.710, 95% CI: 0.514-0.980, P=0.037). Therefore, PIK3CA mutations and its downstream effector p-mTOR expression were two important regulators for activating the PI3K/Akt/mTOR pathway. Both of them could be served as adverse prognostic biomarkers and may contribute to the targeted therapy for TNBC patients with poor outcome.

Detection of ALK translocation in non-small cell lung carcinoma (NSCLC) and its clinicopathological significance using the Ventana immunohistochemical staining method: a single-center large-scale investigation of 1, 504 Chinese Han patients.

OBJECTIVE: The novel fully automated immunohistochemistry (IHC) assay-Ventana anaplastic lymphoma kinase (ALK)-D5F3 for screening ALK rearrangements has been approved by China's Food and Drug Administration in 2013, our previous study disclosed a highly specificity and sensitivity nearly 100%, and its efficacy needs to be evaluated in a large cohort of primary lung adenocarcinoma patients, and to compare clinicopathological features with ALK (+) and ALK (-) lung adenocarcinoma. METHODS: A total of 1,504 consecutive surgical lung adenocarcinoma cases of Chinese Han population were collected and re-diagnosed according to the 2011 multidisciplinary classification of lung adenocarcinoma. Fully automated Ventana ALK-D5F3 IHC staining with a binary scoring was adopted to evaluate staining and correlated with clinicopathological characters, including age, sex, differentiation degree, histological subtype, lymph node metastasis, and clinical staging. ALK (+) patients were followed-up, and targeted therapy of ALK-inhibitors was adopted and observed in patients with stage IV according to the NCCN guideline. RESULTS: ALK positive adenocarcinomas were identified in 6.6% of the surgically resected 1,504 NSCLCs, and significantly younger than the negative group (P<0.05).Mucinous adenocarcinoma (28.2%) was determined to be predominant in ALK (+) cases, followed by the solid type (11.7%), specific type (6.8%), papillary type (5.6%), acinar type (5.5%), and lepidic type (3.1%), and the differences were statistically significant (χ2=42.011, P<0.05). ALK (+) adenocarcinoma with lymph node metastasis (10.8%) were significantly higher than that without lymph node metastasis (4.5%) (χ2=19.809, P<0.05); and ALK (+) in phase IV (20%) was significantly higher than phase III (12.9%), phase II (4.2%), phase I (4.5%), and phase 0 (0) (χ2=36.068, P<0.05). Multivariate logistic regression disclosed that patient age, AJCC staging, and histological mucinous subtype were correlated with ALK positive staining (OR=0.959, 1.578, 5.036, respectively). Sixty eight patients had followed-up results, five patients out of which primarily diagnosed or progressed into Stage IV benefited well from targeted therapy with Crizotinib. CONCLUSIONS: The ALK fusion protein was seen in 6.6% Chinese NSCLC patients, and mostly seen in younger, clinically higher staging, mucinous and solid predominant adenocarcinoma. Clinical trials in patients of Stage IV confirmed that ALK-D5F3 Ventana IHC is serviceable in screening ALK-positive candidates for molecular targeted therapy.

Prevalence and Clinicopathological Characteristics of HER2 and BRAF Mutation in Chinese Patients with Lung Adenocarcinoma.

AIMS: To determine the prevalence and clinicopathological characteristics of BRAF V600E mutation and HER2 exon 20 insertions in Chinese lung adenocarcinoma (ADC) patients. METHODS: Given the fact that the driver mutations are mutually exclusive in lung ADCs, 204 EGFR/KRAS wild-type cases were enrolled in this study. Direct Sanger sequencing was performed to examine BRAF V600E and HER2 exon 20 mutations. The association of BRAF and HER2 mutations with clinicopathological characteristics was statistically analyzed. RESULTS: Among the 204 lung ADCs tested, 11 cases (5.4%) carried HER2 exon 20 insertions and 4 cases (2.0%) had BRAF V600E mutation. HER2 mutation status was identified to be associated with a non-smoking history (p<0.05). HER2 mutation occurs in 9.4% of never smokers (10/106), 8.7% of female (8/92) and 2.7% of male (3/112) in this selected cohort. All four BRAF mutated patients were women and three of them were never-smokers. No HER2 mutant patients harbor BRAF mutation. CONCLUSIONS: HER2 and BRAF mutations identify a distinct subset of lung ADCs. Given the high prevalence of lung cancer and the availability of targeted therapy, Chinese lung ADC patients without EGFR and KRAS mutations are recommended for HER2 and BRAF mutations detection, especially for those never smokers.

Morphological changes of cortical pyramidal neurons in hepatic encephalopathy.

BACKGROUND: Hepatic encephalopathy (HE) is a reversible neuropsychiatric syndrome associated with acute and chronic liver diseases. It includes a number of neuropsychiatric disturbances including impaired motor activity and coordination, intellectual and cognitive function. RESULTS: In the present study, we used a chronic rat HE model by ligation of the bile duct (BDL) for 4 weeks. These rats showed increased plasma ammonia level, bile duct hyperplasia and impaired spatial learning memory and motor coordination when tested with Rota-rod and Morris water maze tests, respectively. By immunohistochemistry, the cerebral cortex showed swelling of astrocytes and microglia activation. To gain a better understanding of the effect of HE on the brain, the dendritic arbors of layer V cortical pyramidal neurons and hippocampal CA1 pyramidal neurons were revealed by an intracellular dye injection combined with a 3-dimensional reconstruction. Although the dendritic arbors remained unaltered, the dendritic spine density on these neurons was significantly reduced. It was suggested that the reduction of dendritic spines may be the underlying cause for increased motor evoked potential threshold and prolonged central motor conduction time in clinical finding in cirrhosis. CONCLUSIONS: We found that HE perturbs CNS functions by altering the dendritic morphology of cortical and hippocampal pyramidal neurons, which may be the underlying cause for the motor and intellectual impairments associated with HE patients.