Identification of mutations in patients with acquired pure red cell aplasia.

Idiopathic acquired pure red cell aplasia (PRCA) is a rare, autoimmune-related disease. This study aimed to describe the previously unidentified DNA alterations associated with PRCA. Here, next generation sequencing using a panel containing 295 critical genes was applied to detect potentially pathogenic mutations in four patients with PRCA. A total of 529 mutations were identified and further classified into three categories, namely, uncertain (n = 25), likely benign (n = 20) and benign (n = 484) mutations, based on the American College of Medical Genetics and Genomics (ACMG) 2015 guidelines and ClinVar database. The spatial proximity between two loci of the uncertain or benign mutations was evaluated using Hi-C datasets of KBM7 and K562 cell lines, respectively. Significant spatial proximity was observed in uncertain mutation pairs compared with benign mutation pairs. In addition, 17 variants were eventually identified after excluding those with mutant frequencies >0.001, including 7 newly identified variants. FANCF and LRP1B mutations existed twice in patients. FANCF and LRP1B mutations were likely to affect protein stability based on prediction analysis. Taken together, our data may provide valuable information about PRCA. FANCF and LRP1B mutations may be associated with acquired PRCA.

Expression of AE1/p16 promoted degradation of AE2 in gastric cancer cells.

BACKGROUND: Human anion exchanger 1 and 2 (AE1 and AE2) mediate the exchange of Cl(-)/HCO3 (-) across the plasma membrane and regulate intracellular pH (pHi). AE1 is specifically expressed on the surface of erythrocytes, while AE2 is widely expressed in most tissues, and is particularly abundant in parietal cells. Previous studies showed that an interaction between AE1 and p16 is a key event in gastric cancer (GC) progression, but the importance of AE2 in GC is unclear. METHODS: The relationship among AE1, AE2 and p16 in GC cells was characterized by molecular and cellular experiments. AE2 expression and pHi were measured after knockdown or forced expression of AE1 or p16 in GC cells. The effect of AE2 on GC growth and the correlation of AE2 expression with differentiation and prognosis of GC were also evaluated. The effect of gastrin on AE2 expression and GC growth was investigated in cellular experiments and mouse xenograft models. RESULTS: p16 binds to both AE1 and AE2 simultaneously. AE1 or p16 silencing elevated AE2 expression on the plasma membrane where it plays a role in pHi regulation and GC suppression. AE2 expression was decreased in GC tissue, and these decreased levels were correlated with poor differentiation and prognosis of GC. The low AE2 protein levels are due to rapid ubiquitin-mediated degradation that was facilitated in the presence of p16. Gastrin inhibited the growth of GC cells at least partially through up-regulation of AE2 expression. CONCLUSION: AE1/p16 expression promoted AE2 degradation in GC cells. Gastrin is a potential candidate drug for targeted therapies for AE1- and p16-positive GC.

Analysis on differential expressed genes of ovarian tissue between high- and low-yield laying hen.

In order to elucidate molecular genetic mechanism of laying hen reproduction at the transcriptional level and the structure of significantly differential genes, the mRNA differential display and reverse northern dot-blot were used to detect the differential expression of genes in the ovary tissue of low-yield laying hens and high-yield laying hens in the present study. Sixteen 32-week-old CAU-pink laying hens divided into two groups were used and the laying performance was measured. The results showed that only the egg numbers were significantly different between the two groups; and from 15 primer pairs, a total of 336 bands were displayed of which 59 cDNA bands were found to be differentially expressed in both high-yield and low-yield laying hen. The sequence analysis indicated that the expression of such bands as H-AP5, H-P5, and H-P4 was significantly potentiated in high-yield laying hen using primer pairs AP5/HT11G, P5/HT11G and P4/HT11G and these transcripts had high homology (98%) to HoxDb, HoxCa, and HoxBa, respectively. The differentially expressed gene fragments may be relevant to the progression of the high-yield hens to the egg-laying stage. And further study is required to elucidate the molecular function to improve the productivity of laying hens.

Prognostic and predictive values of the grading system of lymph node status in patients with advanced-stage gastric cancer.

Background: Lymph node metastasis is one of the most important prognostic factors of gastric cancer. However, the effect of germinal centers in lymph nodes on the prognosis of patients with gastric cancer has not been reported. This study aimed to investigate the contribution of germinal center generation to prognostic parameters and clinicopathological significance in gastric cancer. Methods: We retrospectively reviewed gastric cancer patients who underwent surgery from October 2012 to June 2022. We analyzed 5484 lymph nodes (210 patients) and calculated the lymph node metastasis rate (LNMR) and the proportion of non-metastatic lymph nodes containing three or more germinal centers (NML-GCP). Results: Using a grading system that incorporated LNMR and NML-GCP. The tumors were classified into three groups based on this system, which was found to be significantly associated with prognosis. The TNM stage and grading system of lymph node status were independent risk factors for overall survival (OS) and disease-free survival (DFS). The 5-year OS rates for patients with advanced gastric cancer were 85.07% (n=50), 58.34% (n=42), and 24.44% (n=21) for Grades 1, 2, and 3, respectively (p<0.0001). The 5-year DFS rates were 65.32% (n=58), 40.85% (n=51), and 5.88% (n=34), respectively (p<0.0001). Patients with Grade 1 advanced gastric cancer had higher 5-year OS and DFS rates compared to those with Grade 2 or 3 in TNM stage II and III. Furthermore, the 5-year OS and DFS rates differed significantly among patients with different grades of advanced gastric cancer who received chemotherapy (p<0.0001). Conclusion: These findings suggest that the grading system may be valuable for predicting prognosis and guiding clinical management in patients with gastric cancer, and provides good prognostic stratification for OS and DFS in patients with TNM stage II and III.

Inhibition of Heat Shock-Induced H3K9ac Reduction Sensitizes Cancer Cells to Hyperthermia.

Heat stress, clinically known as hyperthermia, is a promising adjunctive modality in cancer treatment. However, the efficacy of hyperthermia as a monotherapy is limited and the underlying mechanism remains poorly understood. Targeting histone modifications is an emerging strategy for cancer therapy, but little is known regarding the role of heat stress in altering these modifications. Here, we report that heat shock inhibits H3K9 acetylation (H3K9ac) via histone deacetylase 6 (HDAC6) regulation. Heat shock inhibits the interaction between HDAC6 and heat shock protein 90 (HSP90), enhances nuclear localization of HDAC6, and promotes HDAC6 phosphorylation, which is regulated by protein phosphatase 2A (PP2A). Combining hyperthermia with HDAC inhibitors vorinostat or panobinostat leads to better anti-cancer effects compared to monotherapy. KEAP1 and DPP7 as genes affected by heat-induced inhibition of H3K9ac, and combining them with hyperthermia can better induce apoptosis in tumor cells. This study reveals previously unknown mechanisms of H3K9ac decreased by heat shock in cancer cells and highlights a potential combinational therapy involving hyperthermia and targeting of these new mechanisms.

Prognostic and predictive values of the KIT11-mutated grading system in patients with gastrointestinal stromal tumors: a retrospective study.

The KIT11 mutation is the most frequent mutation pattern in gastrointestinal stromal tumors (GISTs). However, few studies have investigated the correlation between the KIT11-mutated grading system and imatinib mesylate (IM) sensitivity (the first choice for adjuvant treatment of GISTs). Here, we elucidated the clinical value of the KIT11-mutated grading system for prognostic prediction in patients with GISTs treated with IM. A total of 106 patients with GIST were treated with IM (8: intermediate-risk, 98: high-risk; 10: KIT9-mutated, 86: KIT11-mutated, 5: wild-type, and 5: other mutations). KIT11-mutated patients were divided into 3 grades based on the KIT11-mutated site and type. Clinical backgrounds and prognostic outcomes were retrospectively compared between the 3 groups. Of 86 KIT11-mutated patients treated with IM, 32 (37.21%) had grade 1 tumors, 37 (43.02%) had grade 2 tumors, and 17 (19.77%) had grade 3 tumors. The 5-year disease-free survival (DFS) was significantly worse in patients with grade 3 KIT11-mutated GISTs (41.96%, p = 0.001) than in those with grade 1 (93%) and grade 2 (70.64%) cases. The multivariable analysis suggested that the KIT11-mutated grading system was an independent risk factor for DFS in patients treated with IM (hazard risk, 2.512; 95% confidence interval, 1.370-4.607; p = 0.003). In conclusion, the KIT11-mutated grading system provides good prognostic stratification for DFS in patients treated with IM. Grade 1 tumors predict a favorable response to IM.

Gastrin suppresses the interdependent expression of p16 and anion exchanger 1 favoring growth inhibition of gastric cancer cells.

Our previous studies demonstrated that expression and interaction of p16 with anion exchanger 1 (AE1) in gastric cancer cells is correlated with progression and shorter survival of the cancer. In this article, the effects of gastrin on p16 and AE1 and its implication in prevention and treatment of gastric cancer were studied by molecular biology techniques, animal experiment and clinical analysis. The results showed that expression of p16 in human gastric body carcinoma was downregulated along with the progression of the cancer, suggesting the reverse correlations between gastrin and p16 in vivo. Further experiments indicated that gastrin suppressed the expression of p16 via the p16 promoter and thereafter resulted in the degradation of AE1 in gastric cancer cells. Silencing of AE1 or p16 significantly inhibited the proliferation of the cancer cells. Using a xenograft tumor model in nude mice, we showed that experimental systemic hypergastrinemia induced by the administration of omeprazole led to decreased expression of AE1 and p16 as well as to a marked growth inhibition of SGC7901 tumors. It is concluded that a moderate plasma gastrin level is beneficial to the growth inhibition of gastric cancer by suppressing the expression of AE1 and p16. This finding may have an important implication for the prevention and treatment of cancers arise in the gastric antrum.

Comparative analysis of colorectal mixed adenoneuroendocrine carcinoma and adenocarcinoma with neuroendocrine differentiation: a population-based study.

BACKGROUND: Colorectal mixed adenoneuroendocrine carcinoma (MANEC) and adenocarcinoma with neuroendocrine differentiation (ANED) are recognized as different tumors pathologically and clinically. In a population-based study, the clinicopathologic characteristics and treatment strategies of the two tumors were comparatively analyzed. METHODS: Patients with colorectal adenocarcinoma (ADEC), neuroendocrine carcinoma (NEC), MANEC and ANED were identified diagnosis from 2010 to 2014 using the Surveillance, Epidemiology, and End Results (SEER) database. The clinicopathologic data were analyzed by Chi-square test, univariable and multivariable Cox regression. Nomogram was performed to provide a prognostic evaluation for colorectal MANEC and ANED. RESULTS: Totally 82121 patients were recruited in this cohort. There was no difference between MANEC and ANED in clinicopathologic characteristics and prognosis (P>0.05). The survival data showed that 1-year and 3-year survival rates were 84.70% and 67.83% for ADEC, 66.83% and 51.98% for NEC, and 54.27% and 37.68% for MANEC and ANED, respectively. Stage and surgery were independent prognostic factors of colorectal MANEC/ANED. We also found that the prognosis was significantly different without vs with chemotherapy (P=0.000) in stage III colorectal MANEC/ANED; without vs with surgery (P=0.007), and without vs with chemotherapy (P=0.000) in stage IV colorectal MANEC/ANED. Radiation did nothing for improving the prognosis of colorectal MANEC/ANED in stage III and stage IV (P=0.557, 0.677). CONCLUSIONS: MANEC and ANED should be merged into the same category pathologically and clinically, and had the poorest prognosis. Stage and surgery were independent prognostic risk factors for colorectal MANEC/ANED. The prognosis of MANEC/ANED could not benefit from radiation.

Prognostic and predictive values of immune infiltrate in patients with head and neck squamous cell carcinoma.

This study sought to determine whether the in situ tumor-infiltrating immune lymphocytes, as a novel companion to the Immunoscore analysis, could be a promising, valuable prognostic and predictive marker in patients with head and neck squamous cell carcinoma (HNSCC). Total (CD3+) and cytotoxic (CD8+) T lymphocytes were assessed using immunohistochemistry in tumor nests and stroma obtained from patient surgical specimens. The "Immunoscore" methodology has been defined to quantify the amount of in situ immune infiltrate (from I0 to I4). Survival curves were measured using the Kaplan-Meier method, and differences in survival and response to therapy between the groups were estimated using the log-rank test. The prognostic value of the Immunoscore was determined using Cox multivariate analysis. The density and location of CD3+ and CD8+ lymphocytes and the associated Immunoscore correlated significantly with differences in disease-free survival (DFS) and overall survival (OS) (all P < .005). Compared with tumor-node-metastasis (TNM) staging, the Immunoscore was found to have an advantage in predicting survival (P = .000). In addition, a high Immunoscore was associated with the tumors of advanced-stage patients who underwent different treatment regimens. The Immunoscore could be a useful prognostic marker. The measurement of CD3+ and CD8+ cell infiltration may be beneficial in HNSCC patients and may help determine which patients may benefit most from definitive chemoradiotherapy.

Anti-tumour effects of small interfering RNA targeting anion exchanger 1 in experimental gastric cancer.

BACKGROUND AND PURPOSE: Anion exchanger 1 (AE1) is an integral membrane protein found in erythrocytes. Our previous studies have demonstrated that AE1 is expressed in human gastric cancer cells and may be involved in the carcinogenesis of cancer. In this study, we further investigated the role of AE1 in gastric carcinogenesis and the anti-tumour effects of AE1-targeted small interfering RNAs (siRNAs) in two experimental models of gastric cancer. EXPERIMENTAL APPROACH: Molecular and cellular experiments were performed to elucidate the role of AE1 in the malignant transformation of gastric epithelium and the effects of AE1-targeted siRNAs on gastric cancer cells. The anti-tumour effect of the siRNA was evaluated in vivo in two mouse models, nude mice implanted with human gastric cancer xenografts (Model I) and mice with gastric cancer induced by N-methyl-N-nitrosourea (MNU) and Helicobacter pylori (Model II). KEY RESULTS: AE1 was found to increase gastric carcinogenesis by promoting cell proliferation. AE1-targeted siRNA significantly suppressed AE1 expression and hindered tumour growth. Furthermore, the siRNA markedly decreased the detection rate of gastric cancer, in parallel with an increase in atypical hyperplasia at the end of the experiment in Model II. CONCLUSIONS AND IMPLICATIONS: Knockdown of AE1 expression in gastric mucosa by administration of synthetic siRNAs significantly inhibits the growth of gastric cancer and decreases the detection rate of this tumour in experimental mice. These results suggest that AE1 is potentially a key therapeutic target and the silencing of AE1 expression in gastric mucosa could provide a new therapeutic approach for treating gastric cancer.

Gastrin inhibits a novel, pathological colon cancer signaling pathway involving EGR1, AE2, and P-ERK.

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.

Expression of anion exchanger 1 is associated with tumor progress in human gastric cancer.

PURPOSE: Anion exchanger 1 (AE1) is a transmembrane glycoprotein which is abundantly expressed in erythrocyte plasma membrane and mediates the electroneutral exchange of Cl(-) and HCO(3) (-). We previously reported that the AE1 protein was unexpectedly expressed in the gastric and colonic cancer and take part in the carcinogenesis of the cancer cells. The aim of the present study is to determine the potential clinical implications of AE1 expression in gastric carcinoma. METHODS: Immunohistochemistry assay was used to determine the expression of AE1 protein. The expression of AE1 in normal and malignant tissues from 286 patients with early and advanced gastric carcinoma was examined. The correlations of AE1 expression with clinicopathological parameters, including age, tumor size, location and subtypes, expression frequency, survival period and lymph metastasis were assessed by Chi-squared test and t test analysis. RESULTS: AE1 immunoreactivity was negative in normal gastric tissue. Positive immunostaining of AE1 was detected in gastric carcinoma regardless of the location. AE1 was most frequently expressed in the gastric antrum carcinoma compared with gastric body cancer (P = 0.034). Expression of AE1 was significantly associated with bigger tumor size, deeper invasion, shorter survival period, and non-lymph metastasis. In para-cancer tissues of intestinal-type gastric cancer, the expression frequency of AE1 was higher than that in diffuse-type (P = 0.011). CONCLUSION: The results showed a strong association of AE1 expression with the onset and progression of the gastric cancer and that may be helpful for improving the tumor classification and the treatment of cancer.

Expression of cytoplasmic p16 and anion exchanger 1 is associated with the invasion and absence of lymph metastasis in gastric carcinoma.

The tumor suppressor p16 is a negative regulator of the cell cycle, commonly believed to act in the nucleus. We recently found that p16 protein is expressed in the cytoplasm of gastric cancer cells, concomitantly with anion exchanger 1 (AE1). The aim of this study was to analyze the significance of cytoplasmic p16 and its relationship to AE1 in the progression of gastric cancer. Expression of p16 and AE1 was examined by immunohistochemical analysis in 196 patients; 98 with early gastric cancer and 98 with advanced gastric cancer. The relationship between cytoplasmic p16 and clinicopathological features, and the relationship between cytoplasmic p16 and AE1, were analyzed statistically. Expression of p16 was observed in the nucleus in early stage gastric cancer, but was located mainly in the cytoplasm in advanced cancer cells. Furthermore, cytoplasmic expression of p16 was correlated with AE1 expression, and both were associated with the absence of lymph metastasis in gastric cancer. In conclusion, cytoplasmic immunoreactivity of p16 appears to be a good prognostic indicator in advanced gastric cancer. Co-localization of p16 and AE1 predicts a lack of metastasis in gastric cancer. The role of cytoplasmic p16 and AE1, and the mechanisms involved in the progression of gastric cancer, warrant further investigation.