Chongxu Han

Detection of apoptotic circulating tumor cells in advanced pancreatic cancer following 5-fluorouracil chemotherapy.

The objective of the present study was to evaluate CA19-9 and CK8/18 expression patterns in pancreatic cancer cell lines induced by 5-fluorouracil (5-Fu), and in circulating tumor cells (CTCs) in peripheral blood of patients previously untreated with advanced pancreatic cancer. Furthermore, the goal was to test the relationship of dynamic of CTCs with the effects of the first cycle of chemotherapy. To accomplish this study, CD45 antibody coated beads were used to discard white blood cells in peripheral blood. This was done in combination with CA19-9-Alexa488 and CK8/18-Alexa594 immunofluorescence staining to identify CTCs in circulation from 41 advanced pancreatic cancer patients, before and after chemotherapy. The PL45 pancreatic cancer cell line was incubated with 20 µmmol/L 5-Fu for 12 and 24 hours to induce apoptosis, and the expression patterns of CA19-9 and CK8/18 were measured, and the extent of apoptosis was evaluated. Subsequently, apoptotic cells and CTCs were measured. Of 41 patients with stage III and Ⅳ pancreatic cancer, 80.5% were detected with more than two CTCs in 7.5mL peripheral blood before any therapy and the median number of CTCs was 16.8±16.0 (0-59). After 7 days by the first cycle of 5-Fu chemotherapy, only 29.3% (12/41) of these patients were detected more than two CTCs in 7.5mL peripheral blood and the median number of CTCs was 3.8±7.8 (0-40) (P=0.000). And no CTCs were detected in 20 healthy donors from 7.5mL peripheral blood (P=0.000). Apoptotic CTCs were detected after advanced pancreatic patients were administered the first cycle of chemotherapy. Punctate, granular, and bubble-like morphologies with CA19-9 and CK8/18 staining were found and may reflect apoptosis in pancreatic cancer cells and CTCs. Apoptotic CTCs may indicate the efficacy of chemotherapy in pancreatic cancer patients. However, further studies are required to follow up these findings.

Expression and Prognostic Value of miR-486-5p in Patients with Gastric Adenocarcinoma

MicroRNA (miR)-486-5p expression is often reduced in human cancers. However, its expression in gastric carcinoma and its relation to clinicopathological features and prognosis are unclear. Tissue microarrays were constructed from 84 patients with gastric adenocarcinoma (GC) who were undergoing radical resection. miR-486-5p expression was detected by miRNA-locked nucleic acid in situ hybridization, and its correlations with clinicopathological features and overall survival were analyzed. Bioinformatic studies predict that fibroblast growth factor 9 (FGF9) is a potential target gene of miR-486-5p. miR-486-5p was mainly located in the cytoplasm of GC cells and neighboring normal tissues. Compared with paracancerous normal tissue, miR-486-5p expression was decreased in 63.1% (53/84) of the GC samples, increased in 32.1% (27/84) and unchanged in 4.8% (4/84). FGF9 expression was decreased in 69.0% (58/84) of GC samples and increased in 31.0% (26/84) compared with normal paracancerous tissues using immunohistochemical analysis. Low or unchanged miR-486-5p expression (P = 0.002), tumor stage (P = 0.001), tumor status (P = 0.001), node status (P = 0.001), tumor size (P = 0.004), and depth of tumor invasion (P = 0.013) were significant negative prognostic predictors for overall survival in patients with GC. After stratification according to American Joint Committee on Cancer (AJCC) stage, low/unchanged miR-486-5p expression remained a significant predictor of poor survival in stage II (P = 0.024) and stage III (P = 0.003). Cox regression analysis identified the following predictors of poor prognosis: tumor status (hazard ratio [HR], 7.19; 95% confidence interval [CI], 1.75–29.6; P = 0.006), stage (HR, 2.62; 95%CI, 1.50–4.59; P = 0.001), lymph node metastasis (HR, 2.52; 95% CI, 1.27–4.99; P = 0.008), low/unchanged miR-486-5p (HR, 2.47; 95% CI, 1.35–4.52; P = 0.003), high level of FGF9 (HR, 2.41; 95% CI, 1.42–4.09; P = 0.001) and tumor size (HR, 2.50; 95% CI, 1.30–4.82; P = 0.006). Low or unchanged expression of miR-486-5p compared with neighboring normal tissues was associated with a poor prognosis, while high expression was associated with a good prognosis in GC. miR-486-5p may thus be useful for evaluating prognosis and may provide a novel target treatment in patients with GC.

Population structure of clinical Vibrio parahaemolyticus from 17 coastal countries, determined through multilocus sequence analysis.

Vibrio parahaemolyticus is a leading cause of food-borne gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of clinical strains from worldwide collections remains largely undescribed, and the recorded outbreaks of V. parahaemolyticus gastroenteritis highlight the need for the subtyping of this species. We present a broad phylogenetic analysis of 490 clinical V. parahaemolyticus isolates from 17 coastal countries through multilocus sequence analysis (MLST). The 490 tested isolates fell into 161 sequence types (STs). The eBURST algorithm revealed that the 161 clinically relevant STs belonged to 8 clonal complexes, 11 doublets, and 94 singletons, showing a high level of genetic diversity. CC3 was found to be a global epidemic clone of V. parahaemolyticus, and ST-3 was the only ST with an international distribution. recA was observed to be evolving more rapidly, exhibiting the highest degree of nucleotide diversity (0.028) and the largest number of polymorphic nucleotide sites (177). We also found that the high variability of recA was an important cause of differences between the results of the eBURST and ME tree analyses, suggesting that recA has a much greater influence on the apparent evolutionary classification of V. parahaemolyticus based on the current MLST scheme. In conclusion, it is evident that a high degree of genetic diversity within the V. parahaemolyticus population and multiple sequence types are contributing to the burden of disease around the world. MLST, with a fully extractable database, is a powerful system for analysis of the clonal relationships of strains at a global scale. With the addition of more strains, the pubMLST database will provide more detailed and accurate information, which will be conducive to our future research on the population structure of V. parahaemolyticus.

The anti-apoptotic and prognostic value of fibroblast growth factor 9 in gastric cancer.

Fibroblast growth factor (FGF) 9 is a member of the FGF family, which promotes carcinogenesis in some solid tumours. However, its biological and prognostic significance in gastric cancer (GC) is unclear. We examined FGF9 expression in 180 GC and corresponding non-tumorous gastric tissue samples by immunohistochemistry and evaluated its role in predicting tumour prognosis. Knockdown of FGF9 by siRNA inhibited cell growth and induced apoptosis in GC cell lines. Fifty of the 180 GC specimens (27.8%) had high FGF9 protein expression, whereas decreased or unchanged expression was observed in 130 cases (72.2%). High FGF9 expression was a significant predictor of poor survival (28.1 vs. 55.8 months, P < 0.001). After stratification according to AJCC stage, FGF9 remained a significant predictor of shorter survival in stage II (30.6 vs. 64.9 months, P < 0.001) and stage III GC (29.7 vs. 58.9 months, P < 0.001). Multivariate and univariate analysis showed that higher expression of FGF9 can be used as a predictor for poor prognosis (HR, 2.95; 95% CI, 1.97–4.41; P < 0.001; and HR, 2.94; 95% CI, 2.01–4.31; P < 0.001, respectively). FGF9 may provide the anti-apoptotic function and be useful as a novel independent marker for evaluating GC prognosis

Circulating tumor cells in breast cancer beyond the genotype of primary tumor for tailored therapy

Although TNM staging based on tumor, node lymph status and metastasis status—is the most widely used method in the clinic to classify breast cancer (BC) and assess prognosis, it offers limited information for different BC subgroups. Circulating tumor cells (CTCs) are regarded as minimal residual disease and are proven to have a strong relationship with BC. Detection of ≥5 CTCs per 7.5 mL in peripheral blood predicts poor prognosis in metastatic BC irrespective of other clinical parameters, whereas, in early‐stage BC, detection of CK19+ CTCs are also associated with poor prognosis. Increasing data and clinical trials show that CTCs can improve prognostic accuracy and help tailor treatment for patients with BC. However, heterogeneous CTCs in the process of an epithelial‐mesenchymal transition (EMT) in BC makes it a challenge to detect these rare cells. Moreover, the genotypic and phenotypic features of CTCs are different from primary BC tumors. Molecular analysis of CTCs in BC may benefit patients by identifying those amenable to tailored therapy. We propose that CTCs should be used alongside the TNM staging system and the genotype of primary tumor to guide tailored BC diagnosis and treatment.

Sero-Prevalence and Genetic Diversity of Pandemic V. parahaemolyticus Strains Occurring at a Global Scale.

Pandemic Vibrio parahaemolyticus is an emerging public health concern as it has caused numerous gastroenteritis outbreaks worldwide. Currently, the absence of a global overview of the phenotypic and molecular characteristics of pandemic strains restricts our overall understanding of these strains, especially for environmental strains. To generate a global picture of the sero-prevalence and genetic diversity of pandemic V. parahaemolyticus, pandemic isolates from worldwide collections were selected and analyzed in this study. After a thorough analysis, we found that the pandemic isolates represented 49 serotypes, which are widely distributed in 22 countries across four continents (Asia, Europe, America and Africa). All of these serotypes were detected in clinical isolates but only nine in environmental isolates. O3:K6 was the most widely disseminated serotype, followed by O3:KUT, while the others were largely restricted to certain countries. The countries with the most abundant pandemic serotypes were China (26 serotypes), India (24 serotypes), Thailand (15 serotypes) and Vietnam (10 serotypes). Based on MLST analysis, 14 sequence types (STs) were identified among the pandemic strains, nine of which fell within clonal complex (CC) 3. ST3 and ST305 were the only two STs that have been reported in environmental pandemic strains. Pandemic ST3 has caused a wide range of infections in as many as 16 countries. Substantial serotypic diversity was mainly observed among isolates within pandemic ST3, including as many as 12 combinations of O/K serotypes. At the allele level, the dtdS and pntA, two loci that perfectly conserved in CC3, displayed a degree of polymorphism in some pandemic strains. In conclusion, we provide a comprehensive understanding of sero-prevalence and genetic differentiation of clinical and environmental pandemic isolates collected from around the world. Although, further studies are needed to delineate the specific mechanisms by which the pandemic strains evolve and spread, the findings in this study are helpful when seeking countermeasures to reduce the spread of V. parahaemolyticus in endemic areas.

Prevalence and genetic diversity of clinical Vibrio parahaemolyticus isolates from China, revealed by multilocus sequence typing scheme

The population structure of clinical Vibrio parahaemolyticus isolates spreading in China remains undefined. We brought 218 clinical isolates from the pubMLST database originating from different regions of China collected since the year of 1990, analyzed by multilocus sequence typing (MLST), to elucidate the prevalence and genetic diversity of V. parahaemolyticus circulating in Chinese population. The MLST scheme produced 137 sequence types (STs). These STs were clustered into six clonal complexes (CCs), six doublets, and 91 singletons, exhibiting a high level of genetic diversity. However, less diversity was displayed on the peptide level: only 46 different peptide sequence type (pST) were generated, with pST2 (44.0%, 96/218) and pST1 (15.1%, 33/218) the predominant. Further analysis confirmed all the pSTs belong to a single complex founded by pST1, pST2, pST3, and pST4. recA presented the highest degree of nucleotide diversity (0.026) and the largest number of variable sites (176) on the nucleotide level. pyrC was the most diverse locus on the peptide level, possessing the highest percentage of variable sites (9.2%, 15/163). Significant linkage disequilibrium with the alleles was detected when the Standardized Index of Association (ISA) was calculated both for the entire isolates collection (0.7169, P < 0.01) and for the 137 STs (ISA = 0.2648, P < 0.01). In conclusion, we provide an overview of prevalence and genetic diversity of clinical V. parahaemolyticus spreading in Chinese population using MLST analysis. The results would offer genetic evidences for uncovering the microevolution relationship of V. parahaemolyticus populations.

Unusual early-stage pancreatic sarcomatoid carcinoma.

Sarcomatoid carcinoma of the pancreas (SCP) is a very rare pathological type of carcinoma that usually has a poor prognosis. Its pathogenesis has not been elucidated. We herein report a case of an early-stage SCP involving successful treatment and a good prognosis. The patient was a 48-year-old Chinese man with a 5-mo history of vague abdominal pain. Ultrasonography revealed a 93 mm × 94 mm × 75 mm mass of mixed echogenicity in the tail of the pancreas. Laboratory test results were within the normal range, with the exception of an obviously increased pretreatment neuron-specific enolase level. The plasma transforming growth factor (TGF)β1 and interleukin-11 levels were obviously increased according to enzyme-linked immunosorbent assay. Microscopically, the excised tumor tissue comprised cancer cells and mesenchymal cells. Immunohistochemical analysis was positive for α-1-antichymotrypsin, pan-cytokeratin, cytokeratin 19, cytokeratin 8/18, and vimentin and negative for CD68 and lysozyme. The pathogenetic mechanism of this case shows that TGFβ1 may regulate the epithelial-to-mesenchymal transition in SCP. With early eradication of the tumor and systemic therapy, this patient has been alive for more than 3 years without tumor recurrence or distant metastasis. This case is also the first to show that TGFβ1 may regulate the epithelial-to-mesenchymal transition in early-stage SCP.

miR-486-5p expression pattern in esophageal squamous cell carcinoma, gastric cancer and its prognostic value

Micro RNA (miR)-486-5p is often aberrantly expressed in human cancers. The aim of this study was to identify the prognostic value of miR-486-5p expression in digestive system cancers. Tissue microarrays were constructed with 680 samples including 185 esophageal squamous cell carcinomas (ESCCs), 90 gastric adenocarcinomas (GCs), and 60 digestive system cancer tissues from 10 ESCC, 10 GC, 10 colon, 10 rectum, 10 liver, 10 pancreatic cancer, and corresponding normal tissues. Twenty normal digestive system mucosa tissues from healthy volunteers were included as normal controls. In GC, miR-486-5p expression was decreased in 62.8% of cases (59/94), increased in 33.0% (31/94), and unchanged in 4.2% (4/94); in ESCC its expression was decreased in 66.2% (129/195), increased in 32.3% (63/195), and unchanged in 1.5% (3/195). Expression of miR-486-5p was decreased in 12, and increased in 8, of 20 cases of colon or rectum cancer; decreased in 6, and increased in 4, of 10 cases of liver cancer; and decreased in 8, and increased in 2, of 10 cases of pancreatic cancer. Multivariate and univariate regression analysis demonstrated that low/unchanged miR-486-5p predicted poor prognosis in ESCC (hazard ratio [HR], 4.32; 95% confidence interval [CI], 2.62–7.14; P < 0.001; HR, 3.88; 95% CI, 2.43–6.22; P < 0.001, respectively) and GC (HR, 2.46; 95% CI, 1.35–4.50; P = 0.003; HR, 2.55; 95% CI, 1.39–4.69; P = 0.002, respectively). MiR-486-5p might therefore be an independent tumor marker for evaluating prognosis in patients with ESCC or GC.

Spreading of Pandemic Vibrio parahaemolyticus O3:K6 and Its Serovariants: A Re-analysis of Strains Isolated from Multiple Studies

In China, V. parahaemolyticus has been a leading cause of foodborne outbreaks and bacterial infectious diarrhea since the 1990s, and most infections have been associated with the pandemic V. parahaemolyticus O3:K6 and its serovariants. However, a comprehensive overview of the sero-prevalence and genetic diversity of the pandemic V. parahaemolyticus clone in China is lacking. To compensate for this deficiency, pandemic isolates in both clinical and environmental Chinese samples collected from multiple studies were analyzed in this study. Surprisingly, as many as 27 clinical pandemic serovariants were identified and were widely distributed across nine coastal provinces and two inland provinces (Beijing and Sichuan). O3:K6, O4:K68, and O1:KUT represented the predominant clinical serovars. Only four environmental pandemic serovariants had previously been reported, and they were spread throughout Shanghai (O1:KUT, O3:K6), Jiangsu (O3:K6, O4:K48), Zhejiang (O3:K6), and Guangdong (O4:K9). Notably, 24 pandemic serovariants were detected within a short time frame (from 2006 to 2012). The pandemic isolates were divided into 15 sequence types (STs), 10 of which fell within clonal complex (CC) 3. Only three STs (ST3, ST192, and ST305) were identified in environmental isolates. Substantial serotypic diversity was mainly observed among isolates within pandemic ST3, which comprised 21 combinations of O/K antigens. The pandemic O3:K6 serotype showed a high level of sequence diversity, which was shared by eight different STs (ST3, ST227, ST431, ST435, ST487, ST489, ST526, and ST672). Antimicrobial susceptibility testing revealed that most isolates shared similar antibiotic susceptibility profiles. They were resistant to ampicillin but sensitive to most other drugs that were tested. In conclusion, the high levels of serotypic and genetic diversity of the pandemic clone suggest that the involved regions are becoming important reservoirs for the emergence of novel pandemic strains. We underscore the need for routine monitoring to prevent pandemic V. parahaemolyticus infection, which includes monitoring antimicrobial responses to avoid excessive misuse of antibiotics. Further investigations are also needed to delineate the specific mechanisms underlying the possible seroconversion of pandemic isolates.