Characterization and mitigation of artifacts derived from NGS library preparation due to structure-specific sequences in the human genome.

BACKGROUND: Hybridization capture-based targeted next generation sequencing (NGS) is gaining importance in routine cancer clinical practice. DNA library preparation is a fundamental step to produce high-quality sequencing data. Numerous unexpected, low variant allele frequency calls were observed in libraries using sonication fragmentation and enzymatic fragmentation. In this study, we investigated the characteristics of the artifact reads induced by sonication and enzymatic fragmentation. We also developed a bioinformatic algorithm to filter these sequencing errors. RESULTS: We used pairwise comparisons of somatic single nucleotide variants (SNVs) and insertions and deletions (indels) of the same tumor DNA samples prepared using both ultrasonic and enzymatic fragmentation protocols. Our analysis revealed that the number of artifact variants was significantly greater in the samples generated using enzymatic fragmentation than using sonication. Most of the artifacts derived from the sonication-treated libraries were chimeric artifact reads containing both cis- and trans-inverted repeat sequences of the genomic DNA. In contrast, chimeric artifact reads of endonuclease-treated libraries contained palindromic sequences with mismatched bases. Based on these distinctive features, we proposed a mechanistic hypothesis model, PDSM (pairing of partial single strands derived from a similar molecule), by which these sequencing errors derive from ultrasonication and enzymatic fragmentation library preparation. We developed a bioinformatic algorithm to generate a custom mutation "blacklist" in the BED region to reduce errors in downstream analyses. CONCLUSIONS: We first proposed a mechanistic hypothesis model (PDSM) of sequencing errors caused by specific structures of inverted repeat sequences and palindromic sequences in the natural genome. This new hypothesis predicts the existence of chimeric reads that could not be explained by previous models, and provides a new direction for further improving NGS analysis accuracy. A bioinformatic algorithm, ArtifactsFinder, was developed and used to reduce the sequencing errors in libraries produced using sonication and enzymatic fragmentation.

PD-1 inhibitor combined with albumin paclitaxel and apatinib as second-line treatment for patients with metastatic gastric cancer: a single-center, single-arm, phase II study.

BACKGROUND: Immune checkpoint inhibitors have been approved for first- and third-line treatment of advanced gastric cancer. However, pembrolizumab alone in the second line did not improve overall survival compared to chemotherapy in the KEYNOTE-061 study. In this study, we aimed to explore the efficacy and safety of a three-drug regimen of PD-1 inhibitor combined with albumin paclitaxel and apatinib (a VEGFR inhibitor) for the second-line treatment of patients with metastatic gastric cancer (mGC). METHODS: This was a single-center, single-arm, phase II clinical study. Patients with mGC with stable microsatellite and negative HER-2 expression who failed first-line chemotherapy were enrolled. The enrolled patients were treated with PD-1 inhibitor (selected according to patients' requirements) in combination with albumin paclitaxel (125 mg/m2, intravenously, days 1 and 8, or 250 mg/m2, intravenously, day 1) and apatinib (250 or 500 mg, orally, days 1-21) every 3 weeks. The primary endpoint was progression-free survival (PFS), and the secondary endpoints were overall survival (OS), objective response rate (ORR), disease control rate (DCR), duration of response, and adverse events (AEs). RESULTS: From July 11, 2019, to October 13, 2022, a total of 43 patients were enrolled, of whom 10 were PD-L1 negative, 11 were PD-L1 positive, and 22 had unknown PD-L1 expression. As of the data cutoff on April 1st, 2023, nine patients had partial response, 29 had stable disease, and five experienced progressive disease, with the ORR of 20.9% and DCR of 88.3%. The median PFS was 6.2 months (95% CI, 3.9-9.3), and the median OS was 10.1 months (95% CI, 7.5-14.1). All patients suffered from alopecia and neurotoxicity. The other main AEs of grade 1 or 2 were bone marrow suppression (N = 21, 48.8%), hand-foot reaction (N = 19, 44.2%), hypertension (N = 18, 41.9%), hypothyroidism (N = 11, 25.6%), gastrointestinal bleeding (N = 3, 7.0%), and liver function damage (N = 5, 11.6%). Two patients reported grade 3-4 immune-related liver damage. CONCLUSION: Second-line PD-1 inhibitor combined with albumin paclitaxel and apatinib showed certain efficacy and safety in patients with mGC. TRIAL REGISTRATION: Clinical trials, NCT04182724. Registered 27 November 2019; retrospectively registered, https://clinicaltrials.gov/study/NCT04182724.

Complete Freund's adjuvant-induced acute inflammatory pain could be attenuated by triptolide via inhibiting spinal glia activation in rats.

BACKGROUND: Inflammatory pain is one of the most common clinical symptoms, mechanical allodynia and thermal hypersensitivities are associated with proinflammatory cytokines, and proinflammatory cytokine antagonists could alleviate the hypersensitivity. Previous studies showed that a traditional Chinese medicine ingredient, triptolide could inhibit inflammatory cytokines; however, it was still unknown whether triptolide had beneficial effects on treating inflammatory pain. MATERIALS AND METHODS: The effects of triptolide on Complete Freund's Adjuvant-induced acute inflammatory pain were investigated using behavioral tests. The activation of spinal glia was morphologically observed by immunofluorescent histochemistry. The levels of OX42, glia fibrillary acidic protein, and phosphorylated extracellular signal-regulated kinase in the spinal cord were detected by Western blot, and the messenger RNA levels of interleukin 1ß, interleukin 6, and tumor necrosis factor alpha were detected by real-time polymerase chain reaction. RESULTS: These results demonstrate that the triptolide effectively attenuates inflammatory pain induced by Complete Freund's Adjuvant, the underlying mechanism may regulate the phosphorylated extracellular signal-regulated kinase signaling pathway and inhibit the spinal glia activation, and then downregulate the proinflammatory cytokines; the triptolide may be clinically useful as a drug of anti-inflammatory pain. CONCLUSIONS: In the present study, we first reported that repeated systemic administration of triptolide could safely prevent and reverse inflammatory pain. The triptolide may serve as a new potential compound for developing safe therapeutics for patients suffering inflammatory pain.

Tumor reduction after SARS­CoV­2 infection in a patient with lung cancer: A case report.

Lung cancer is one of the most common malignancies worldwide. Since the global outbreak of the coronavirus disease 2019 (COVID-19) pandemic in 2020, the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on lung cancer has been extensively studied. Despite reports about SARS-CoV-2 infection inducing a significant increase in the number of medical visits for patients with cancer, the virus has also been reported to produce some unknown benefits. The present study reports the case of a patient with lung cancer whose tumor lesion was reduced in size after SARS-CoV-2 infection even though the therapeutic regimen remained unchanged. Although the mechanism involved is not yet understood, this case supports the novel idea of applying SARS-CoV-2 in oncolytic virotherapy.

Comprehensive application of modern technologies in precise liver resection.

BACKGROUND: Liver surgery has gone through the phases of wedge liver resection, regular resection of hepatic lobes, irregular and local resection, extracorporeal hepatectomy, hemi-extracorporeal hepatectomy and Da Vinci surgical system-assisted hepatectomy. Taking advantage of modern technologies, liver surgery is stepping into an age of precise liver resection. This review aimed to analyze the comprehensive application of modern technologies in precise liver resection. DATA SOURCE: PubMed search was carried out for English-language articles relevant to precise liver resection, liver anatomy, hepatic blood inflow blockage, parenchyma transection, and down-staging treatment. RESULTS: The 3D image system can imitate the liver operation procedures, conduct risk assessment, help to identify the operation feasibility and confirm the operation scheme. In addition, some techniques including puncture and injection of methylene blue into the target Glisson sheath help to precisely determine the resection. Alternative methods such as Pringle maneuver are helpful for hepatic blood inflow blockage in precise liver resection. Moreover, the use of exquisite equipment for liver parenchyma transection, such as cavitron ultrasonic surgical aspirator, ultrasonic scalpel, Ligasure and Tissue Link is also helpful to reduce hemorrhage in liver resection, or even operate exsanguinous liver resection without blocking hepatic blood flow. Furthermore, various down-staging therapies including transcatheter arterial chemoembolization and radio-frequency ablation were appropriate for unresectable cancer, which reverse the advanced tumor back to early phase by local or systemic treatment so that hepatectomy or liver transplantation is possible. CONCLUSIONS: Modern technologies mentioned in this paper are the key tool for achieving precise liver resection and can effectively lead to maximum preservation of anatomical structural integrity and functions of the remnant liver. In addition, large randomized trials are needed to evaluate the usefulness of these technologies in patients with hepatocellular carcinoma who have undergone precise liver resection.

OncoCTMiner: streamlining precision oncology trial matching via molecular profile analysis.

By establishing omics sequencing of patient tumors as a crucial element in cancer treatment, the extensive implementation of precision oncology necessitates effective and prompt execution of clinical studies for approving molecular-targeted therapies. However, the substantial volume of patient sequencing data, combined with strict clinical trial criteria, increasingly complicates the process of matching patients to precision oncology studies. To streamline enrollment in these studies, we developed OncoCTMiner, an automated pre-screening platform for molecular cancer clinical trials. Through manual tagging of eligibility criteria for 2227 oncology trials, we identified key bio-concepts such as cancer types, genes, alterations, drugs, biomarkers and therapies. Utilizing this manually annotated corpus along with open-source biomedical natural language processing tools, we trained multiple named entity recognition models specifically designed for precision oncology trials. These models analyzed 460 952 clinical trials, revealing 8.15 million precision medicine concepts, 9.32 million entity-criteria-trial triplets and a comprehensive precision oncology eligibility criteria database. Most significantly, we developed a patient-trial matching system based on cancer patients' clinical and genetic profiles, which can seamlessly integrate with the omics data analysis platform. This system expedites the pre-screening process for potentially suitable precision oncology trials, offering patients swifter access to promising treatment options. Database URL  https://oncoctminer.chosenmedinfo.com.

Postoperative transarterial chemoembolization benefits patients with high γ-glutamyl transferase levels after curative hepatectomy for hepatocellular carcinoma: a survival stratification analysis.

Hepatocellular carcinoma (HCC) is the most common liver tumor in Asian countries, and hepatectomy is currently regarded as the optimal curative treatment for HCC; however, the postoperative outcome remains unsatisfactory. Aiming at further clarification of prognostic factors after hepatectomy, we adopted a detailed stratification on survival periods. A total of 428 HCC patients undergoing curative hepatectomy were firstly divided into two groups using 2-year survival as cutoff point. Multivariate analysis showed that tumor-related factors, including vascular invasion (P < 0.001), high Edmondson grade (P < 0.001), large tumor size (P < 0.001) and high serum alpha-fetoprotein level (P = 0.001), were significant determinants for early death within 2 years, while postoperative transarterial chemoembolization (TACE) was demonstrated a protective factor (P = 0.013). Then the 281 patients with survival > 2 years were divided into two subgroups according to survival or death during follow-up to examine the late death related factors. We found that high serum γ-glutamyl transpeptidase (GGT), indicating severity of underlying liver disease, was significantly linked to death in this stage (P = 0.006). In further comparison of survival rates between subgroups stratified by early- and late-death indictors, we found the long-term outcomes of patients with high serum GGT were poor, regardless of the factors related with primary tumor. Furthermore, postoperative TACE decreased late death rate of patients with high GGT levels. In conclusion, despite the overwhelmed effects of primary tumor in the early stage after hepatectomy, postoperative TACE is beneficial for HCC patients with poor liver status.

Beneficial effects of splenectomy on liver regeneration in a rat model of massive hepatectomy.

BACKGROUND: Small-for-size syndrome is a widely recognized clinical complication after living donor liver transplantation or extended hepatectomy due to inadequate liver mass. The purpose of this study was to investigate the role of splenectomy in rats after massive hepatectomy, a surrogate model of small-for-size graft. METHODS: Rats were divided into eight groups, each with 20 animals: 50% hepatectomy (50% Hx), 50% hepatectomy+splenectomy (50% Hx+Sp), 60% Hx, 60% Hx+Sp, 70% Hx, 70% Hx+Sp, 90% Hx and 90% Hx+Sp. The following parameters were evaluated: liver function tests (ALT, AST and TBIL), liver regeneration ratio, DNA synthesis, proliferation cell nuclear antigen, hepatic oxygen delivery (HDO2) and hepatic oxygen consumption (HVO2). RESULTS: The liver regeneration ratio was enhanced in the Hx+Sp groups (P<0.05). In addition, compared with the Hx groups, the Hx+Sp groups had better liver functions (P<0.05). DNA synthesis and proliferation cell nuclear antigen were also increased in the Hx+Sp groups compared with the Hx groups (P<0.05). Furthermore, in the Hx+Sp groups, HDO2 and HVO2 were increased over those in the Hx groups (P<0.05), and were positively correlated with the liver regeneration ratio. CONCLUSIONS: Splenectomy significantly improved liver function, and enhanced DNA synthesis and proliferation cell nuclear antigen after massive hepatectomy in rats. This operation could be mediated through increased HDO2 and HVO2, which facilitate liver regeneration.

Fruquintinib in Combination With PD-1 Inhibitors in Patients With Refractory Non-MSI-H/pMMR Metastatic Colorectal Cancer: A Real-World Study in China.

Background: Fruquintinib, a vascular endothelial growth factor receptor inhibitor, is a new anticancer drug independently developed in China to treat refractory metastatic colorectal cancer (mCRC). In Japan, regorafenib combined with nivolumab has been demonstrated to be promising in patients with refractory mCRC. Here, in a real-world study, we were aimed to evaluate the efficacy of fruquintinib with various programmed death-1 (PD-1) inhibitors after standard treatment in Chinese non-microsatellite instability-high (MSI-H)/mismatch repair proficient mCRC patients. Methods: A total of 45 patients with refractory mCRC were involved in the study. They received fruquintinib (3 or 5 mg, orally administered once a day for 3 weeks followed by 1 week off in 4-week cycles) and a PD-1 inhibitor(200 mg pembrolizumab, 3 mg/kg nivolumab, 200 mg sintilimab or camrelizumab, intravenously administered on D1 once every 3 weeks). Progression-free survival (PFS), overall survival (OS), disease control rate (DCR), and objective response rate (ORR) were reviewed and evaluated. Results: Among the 45 patients, the median age was 54 years (29-85). The ORR was 11.1% (5/45), DCR 62.2% (28/45), median PFS equal 3.8 months, and median OS was 14.9 months. The response duration was 3.4 months. PFS between left and right primary tumors and PFS with or without lung metastases were both not significantly different (p > 0.05), which was inconsistent with the result of REGONIVO study. The multivariate analysis indicated no association of OS benefit in the specified subgroups. No adverse-effect-related deaths were reported. Conclusions: Fruquintinib, in combination with anti-PD-1, was observed to have clinical activity in a small population of patients with heavily pretreated mCRC in our center. Further studies are needed to verify this outcome in a large population.

Paired box 5 increases the chemosensitivity of esophageal squamous cell cancer cells by promoting p53 signaling activity.

BACKGROUND: Gene promoter methylation is a major epigenetic change in cancers, which plays critical roles in carcinogenesis. As a crucial regulator in the early stages of B-cell differentiation and embryonic neurodevelopment, the paired box 5 (PAX5) gene is downregulated by methylation in several kinds of tumors and the role of this downregulation in esophageal squamous cell carcinoma (ESCC) pathogenesis remains unclear. METHODS: To elucidate the role of PAX5 in ESCC, eight ESCC cell lines, 51 primary ESCC tissue samples, and eight normal esophageal mucosa samples were studied and The Cancer Genome Atlas (TCGA) was queried. PAX5 expression was examined by reverse transcription-polymerase chain reaction and western blotting. Cell apoptosis, proliferation, and chemosensitivity were detected by flow cytometry, colony formation assays, and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assays in ESCC cell lines with PAX5 overexpression or silencing. Tumor xenograft models were established for in vivo verification. RESULTS: PAX5 methylation was found in 37.3% (19/51) of primary ESCC samples, which was significantly associated with age (P = 0.007) and tumor-node-metastasis stage (P = 0.014). TCGA data analysis indicated that PAX5 expression was inversely correlated with promoter region methylation (r = -0.189, P = 0.011 for cg00464519 and r = -0.228, P = 0.002 for cg02538199). Restoration of PAX5 expression suppressed cell proliferation, promoted apoptosis, and inhibited tumor growth of ESCC cell lines, which was verified in xenografted mice. Ectopic PAX5 expression significantly increased p53 reporter luciferase activity and increased p53 messenger RNA and protein levels. A direct interaction of PAX5 with the p53 promoter region was confirmed by chromatin immunoprecipitation assays. Re-expression of PAX5 sensitized ESCC cell lines KYSE150 and KYSE30 to fluorouracil and docetaxel. Silencing of PAX5 induced resistance of KYSE450 cells to these drugs. CONCLUSIONS: As a tumor suppressor gene regulated by promoter region methylation in human ESCC, PAX5 inhibits proliferation, promotes apoptosis, and induces activation of p53 signaling. PAX5 may serve as a chemosensitive marker of ESCC.

Construction of a nomogram to predict the survival of metastatic gastric cancer patients that received immunotherapy.

Background: Immunotherapy has shown promising results for metastatic gastric cancer (MGC) patients. Nevertheless, not all patients can benefit from anti-PD-1 treatment. Thus, this study aimed to develop and validate a prognostic nomogram for MGC patients that received immunotherapy. Methods: Herein, MGC patients treated with anti-PD-1 between 1 October 2016 and 1 June 2022 at two separate Chinese PLA General Hospital centers were enrolled and randomly divided into training and validation sets (186 and 80 patients, respectively). The nomogram was constructed based on a multivariable Cox model using baseline variables from the training cohort. Its predictive accuracy was validated by the validation set. The consistency index (C-index) and calibration plots were used to evaluate the discriminative ability and accuracy of the nomogram. The net benefit of the nomogram was evaluated using decision curve analysis (DCA). Finally, we stratified patients by median total nomogram scores and performed Kaplan-Meier survival analyses. Results: We developed the nomogram based on the multivariate analysis of the training cohort, including four parameters: surgery history, treatment line, lung immune prognostic index (LIPI), and platelet-to-lymphocyte ratio (PLR). The C-index of the nomogram was 0.745 in the training set. The calibration curve for 1- and 2-year survival showed good agreement between nomogram predictions and actual observations. In the validation group, the calibration curves demonstrated good performance of the nomogram, with a C-index for overall survival (OS) prediction of 0.713. The OS of patients with a score greater than the median nomogram score was significantly longer than patients with a score lower or equal to the median (p < 0.001). Conclusion: We constructed a nomogram to predict the outcomes of MGC patients that received immunotherapy. This nomogram might facilitate individualized survival predictions and be helpful during clinical decision-making for MGC patients under anti-PD-1 therapy.

Hyperprogression under treatment with immune-checkpoint inhibitors in patients with gastrointestinal cancer: A natural process of advanced tumor progression?

Immunotherapy has shown great promise in treating various types of malignant tumors. However, some patients with gastrointestinal cancer have been known to experience rapid disease progression after treatment, a situation referred to as hyperprogressive disease (HPD). This minireview focuses on the definitions and potential mechanisms of HPD, natural disease progression in gastrointestinal malignancies, and tumor immunological microenvironment.

Evaluation of circulating tumor DNA as a prognostic biomarker for metastatic pancreatic adenocarcinoma.

Purpose: Pancreatic cancer is an aggressive solid tumor with a severe prognosis. Although tumor biomarkers are often used to identify advanced pancreatic cancer, this is not accurate, and the currently used biomarkers are not indicative of prognosis. The present study evaluated circulating tumor DNA (ctDNA) as a biomarker for prognosis prediction and disease monitoring in metastatic pancreatic adenocarcinoma (PAC). Methods: From 2017 to 2018, 40 patients with metastatic PAC were enrolled, and tumor tissue and blood samples were collected from 40 and 35 patients, respectively. CtDNA was sequenced by next-generation sequencing (NGS) with a 425-gene capture panel. The association of clinical characteristics, laboratory indicators, and dynamic ctDNA with patient outcomes was analyzed. Results: Mutations in KRAS (87.5%, N = 35) and TP53 (77.5%, N = 31) were most common in 40 tumor tissue. Patients' ECOG score, CA19-9, CEA, neutrophil-lymphocyte ratio (NLR), platelet- lymphocyte ratio (PLR) levels and mutations in ≥ 3 driver genes were strongly correlated with patients' overall survival (OS). Patients' gender, ECOG score, CA19-9, and CEA levels were associated with progression-free survival (PFS) (P<0.05). In 35 blood samples, univariate analysis showed a significant association between ECOG score, CA19-9, KRAS or CDKN2A mutation in ctDNA and OS and between CA19-9, CDKN2A or SMAD4 mutation in ctDNA and PFS. Cox hazard proportion model showed that patients' CDKN2A mutation in ctDNA (HR=16.1, 95% CI=4.4-59.1, P<0.001), ECOG score (HR=6.2, 95% CI=2.4-15.7, P<0.001) and tumor location (HR=0.4, 95% CI=0.1-0.9, P=0.027) were significantly associated with OS. Patients' CDKN2A mutation in ctDNA (HR=6.8, 95% CI=2.3-19.9, P=0.001), SMAD4 mutation in ctDNA (HR=3.0, 95% CI=1.1-7.9, P=0.031) and metastatic organ (HR=0.4, 95% CI=0.2-1.0, P=0.046) were significantly associated with PFS. Longitudinal changes in gene mutation allelic frequency (MAF) value were evaluated in 24 patients. Detection of progression disease (PD) by ctDNA was 0.9 months earlier than by radiological imaging (mean PFS: 4.6m vs 5.5m, P=0.004, paired t-test). Conclusions: The ctDNA has the potential as a specific survival predictive marker for metastatic PAC patients. Longitudinal ctDNA tracking could potentially help identify disease progression and be a valuable complement for routine clinical markers and imaging.

Analysis of a Systemic Inflammatory Biomarker in Advanced Bile Tract Carcinoma Treated with Anti-PD-1 Therapy: Prognostic and Predictive Significance of Lung Immune Prognostic Index Score.

Background: The application of immunotherapy is gradually increasing in advanced bile tract carcinoma (BTC), but only some patients could benefit from it. Validated biomarkers can screen out the beneficiaries. Therefore, the objective of this research is aimed at exploring the predictive value of lung immune prognostic index (LIPI) in advanced BTC patients receiving immunotherapy. Methods: This study was conducted on 110 BTC patients. The cut-off value of the derived neutrophil-to-lymphocyte (dNLR) ratio was obtained by the ROC curves to predict the tumor progression rate at the 6th month. The high levels of dNLR (≥the cut-off value) and lactate dehydrogenase (≥the upper limit of normal) were considered to be two risk factors for LIPI. Based on these two risk factors, patients were categorized into 3 groups based on risk factors: 0 for the good group, 1 for the intermediate group, and 2 for the poor group. Due to the limited number of patients in the poor group, it was integrated into the intermediate group to be the intermediate/poor group. Finally, the subjects were divided into two groups: LIPI-good and LIPI-intermediate/poor. Results: The results shed light on the 110 BTC patients' LIPI in advanced BTC patients receiving immunotherapy, indicating that the cut-off value of dNLR was 1.74. According to the risk stratification, 38 (34.5%) patients had a good LIPI score, whereas the LIPI score was intermediate/poor in 72 (65.5%). In addition, patients with good LIPI were related to longer progression-free survival (PFS) and overall survival (OS), compared to those with intermediate/poor LIPI (12.17 months vs. 3.17 months; 20.2 months vs. 8.7 months). According to multivariate analysis, the intermediate/poor LIPI group was independently correlated with over 2.3 times greater risk of tumor progression (HR = 2.301; 95% CI, 1.395-3.796; P = 0.001) and over 1.8 times greater risk of death (HR = 1.877; 95% CI, 1.076-3.275; P = 0.027) than the good group. Moreover, the result also revealed that there were significant differences of DCR for patients of the good group and the intermediate/poor group (86.8% vs. 65.3%; P = 0.012). Conclusion: Finally, this study verifies, for the first time, that LIPI is an independent factor affecting the survival and clinical efficacy of advanced BTC patients receiving immunotherapy. It may be difficult for patients with intermediate/poor LIPI to benefit from immunotherapy.

Retinoic Acid-Induced 2 (RAI2) Is a Novel Antagonist of Wnt/ß-Catenin Signaling Pathway and Potential Biomarker of Chemosensitivity in Colorectal Cancer.

Objective: Aberrant activation of Wnt/ß-catenin signaling contributes to the maintenance of cancer stem cells and chemoresistance in colorectal cancer (CRC). Retinoic acid-induced 2 (RAI2) was proved to be a tumor suppressor in CRC in our previous report. In this study, the role of RAI2 in Wnt/ß-catenin signaling was further investigated. Methods: As a transcriptional co-regulator, C-terminal Binding Protein 2 (CtBP2) was reported to be involved in Wnt signaling in multiple and complex ways. The correlation of RAI2 and CtBP2 in CRC was analyzed by TCGA dataset, and the interaction between RAI2 and CtBP2 was explored by co-immunoprecipitation (Co-IP) in CRC cells. The effect of RAI2 on the activity of Wnt signaling and the location of ß-catenin was detected by Dual-Luciferase reporter assay and Immunofluorescence respectively. Western blotting analysis was performed to detect the expression of target genes involved in Wnt signaling. Sphere formation assay was employed to detect the effect of RAI2 on stem cell like properties. Cell viability assay was used to detect the chemosensitivity of cells before and after transfection of RAI2. Results: The interaction between RAI2 and CtBP2 was confirmed by Co-IP in CRC cells. Besides, the negative correlation of RAI2 and CtBP2 in CRC was found by analyzing the TCGA dataset. Re-expression of RAI2 in human colon cancer cells (HCT116 and LoVo) suppressed the fluorescent activity of Wnt signaling, increased the phosphorylation and inhibited nuclear translocation of ß-catenin, with down-regulation of target genes like c-Myc, CyclinD1, ASCL2, and LGR5. In contrast, the mutated RAI2, which can't interact with CtBP2, has no above effects. We observed low expression of RAI2 in 33.89% (101/298) of CRC patients, which was significantly associated with reduced phosphorylation of ß-catenin (r=0.8866, P<0.0001), poor 5-year relapse-free survival (RFS) (P = 0.0029) and overall survival (OS) (P = 0.0102). Restoration of RAI2 in HCT116 and LoVo cells inhibited stem cell-like properties of CRC cells and increased chemosensitivity of these cells to oxaliplatin and fluorouracil. Conclusion: Low expression of RAI2 can serve as an independent poor prognostic marker. RAI2 inhibits Wnt signaling by interacting with or down-regulating CtBP2, resulting in repression of stem cell-like properties and increased chemosensitivity of CRC cells.

Targeted up-regulation of Drp1 in dorsal horn attenuates neuropathic pain hypersensitivity by increasing mitochondrial fission.

Mitochondria play an essential role in pathophysiology of both inflammatory and neuropathic pain (NP), but the mechanisms are not yet clear. Dynamin-related protein 1 (Drp1) is broadly expressed in the central nervous system and plays a role in the induction of mitochondrial fission process. Spared nerve injury (SNI), due to the dysfunction of the neurons within the spinal dorsal horn (SDH), is the most common NP model. We explored the neuroprotective role of Drp1 within SDH in SNI. SNI mice showed pain behavior and anxiety-like behavior, which was associated with elevation of Drp1, as well as increased density of mitochondria in SDH. Ultrastructural analysis showed SNI induced damaged mitochondria into smaller perimeter and area, tending to be circular. Characteristics of vacuole in the mitochondria further showed SNI induced the increased number of vacuole, widened vac-perimeter and vac-area. Stable overexpression of Drp1 via AAV under the control of the Drp1 promoter by intraspinal injection (Drp1 OE) attenuated abnormal gait and alleviated pain hypersensitivity of SNI mice. Mitochondrial ultrastructure analysis showed that the increased density of mitochondria induced by SNI was recovered by Drp1 OE which, however, did not change mitochondrial morphology and vacuole parameters within SDH. Contrary to Drp1 OE, down-regulation of Drp1 in the SDH by AAV-Drp1 shRNA (Drp1 RNAi) did not alter painful behavior induced by SNI. Ultrastructural analysis showed the treatment by combination of SNI and Drp1 RNAi (SNI + Drp1 RNAi) amplified the damages of mitochondria with the decreased distribution density, increased perimeter and area, as well as larger circularity tending to be more circular. Vacuole data showed SNI + Drp1 RNAi increased vacuole density, perimeter and area within the SDH mitochondria. Our results illustrate that mitochondria within the SDH are sensitive to NP, and targeted mitochondrial Drp1 overexpression attenuates pain hypersensitivity. Drp1 offers a novel therapeutic target for pain treatment.

Spinal toll like receptor 3 is involved in chronic pancreatitis-induced mechanical allodynia of rat.

BACKGROUND: Mechanisms underlying pain in chronic pancreatitis (CP) are incompletely understood. Our previous data showed that astrocytes were actively involved. However, it was unclear how astrocytic activation was induced in CP conditions. In the present study, we hypothesized that toll-like receptors (TLRs) were involved in astrocytic activation and pain behavior in CP-induced pain. RESULTS: To test our hypothesis, we first investigated the changes of TLR2-4 in the rat CP model induced by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Western blot showed that after TNBS infusion, TLR3, but not TLR2 or TLR4, was increased gradually and maintained at a very high level for up to 5 w, which correlated with the changing course of mechanical allodynia. Double immunostaining suggested that TLR3 was highly expressed on astrocytes. Infusion with TLR3 antisense oligodeoxynucleotide (ASO) dose-dependently attenuated CP-induced allodynia. CP-induced astrocytic activation in the spinal cord was also significantly suppressed by TLR3 ASO. Furthermore, real-time PCR showed that IL-1ß, TNF-α, IL-6 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in spinal cord of pancreatic rats. In addition, TLR3 ASO significantly attenuated CP-induced up-regulation of IL-1ß and MCP-1. CONCLUSIONS: These results suggest a probable "TLR3-astrocytes-IL-1ß/MCP-1" pathway as a positive feedback loop in the spinal dorsal horn in CP conditions. TLR3-mediated neuroimmune interactions could be new targets for treating persistent pain in CP patients.

Prognostic significance of tumor/stromal caveolin-1 expression in breast cancer patients.

Caveolin-1 (Cav-1) has been extensively characterized in cancer biological research. However, the role of Cav-1 in the interaction between tumor and stromal cells remains unclear. In the present study, we examined Cav-1 expression in tumor cells and stromal cells in breast cancer tissue by immunohistochemical analysis and evaluated its prognostic value in a training cohort. Immunohistochemical analysis of Cav-1 expression was scored as (++), (+) or (-) according to the proportion of positively stained tumor cells (T) and stromal cells (S). Correlation analysis between tumor/stromal Cav-1 expression and clinicopathological parameters revealed that only T(++) Cav-1 status was positively associated with tumor size and histological nodal status (P = 0.019 and 0.021, respectively). Univariate analysis revealed that combined T(++)/S(-) status was significantly correlated with unfavorable prognostic outcomes (P < 0.001). Multivariate analysis demonstrated that this combined status is an independent prognostic factor for primary breast cancer (P = 0.002). Clinical outcomes in different subgroups of breast cancer patients were also strictly dependent on this combined status (P < 0.05). The prognostic value of T(++)/S(-) Cav-1 status was also validated in the testing cohort. Collectively, our data indicate that high Cav-1 expression in tumor cells and lack of this expression in stromal cells could help identify a particular subgroup of breast cancer patients with potentially poor survival. Further studies are required to understand the regulatory mechanism of Cav-1 in the tumor microenvironment.

Clinical applications of sentinel lymph node biopsy in ductal carcinoma in situ of the breast: a dilemma.

Breast cancer is the fifth most common cause of cancer deaths in the world, which often spreads first to the axillary lymph nodes (ALN) from the primary tumor. ALN helps clinician stage breast cancer. In addition, it is one of the key prognostic factors for patients with invasive breast cancer. The sentinel lymph node (SLN) is defined as the first regional lymph node to receive lymphatic fluid from a malignant tumor. As a result, it seems possible to assess the complete nodal status with sentinel lymph node biopsy (SLNB), which is attractive and reliable approach for identifying lymph node metastasis. Ductal carcinoma in situ (DCIS) is the most common type of non-invasive breast cancer. However, the role of SLNB in DCIS is controversial because DCIS does not cause invasion and metastasis theoretically. In this review, clinical applications of SLNB in DCIS will be discussed. The potential benefit of accurately upstaging patients with DCIS and the minimal invasiveness of SLNB justify use of SLNB in selected high-risk DCIS patients. At least DSIS with microinvasion, have DCIS of sufficient extent on mammography or MRI, or indicated invasive or microinvasive focus by final histological examination, are recommended for SLNB. Moreover, large randomized trials to evaluate the usefulness of SLNB in DCIS patients after long-term follow-up on local control and survival are required for further evaluation.

Research on Mechanism of miR-214 Packaged with Lipidosome Nanoparticles on Prompting the Apoptosis of Intestinal Cancer Through Regulating p53 Pathway.

Our study aimed at studying mechanism of miR-214 packaged with lipidosome nanoparticles on prompting apoptosis of intestinal cancer through regulating p53 pathway. SW480 cells were divided into blank group, empty carrier group, agonist group and group with carrier and antagonist. The negative control group was set, and groups related to p53 pathway were set as agonist group, inhibitor group and group with antagonist and inhibitor. The effect of miR-214 packaged with lipidosome nanoparticles on proliferation and apoptosis of intestinal cancer cells and p53 pathway in intestinal cancer cells was observed. Expression level of miR-214 in group with carrier and antagonist was lower than in other groups. The proportion of active cells in the group with carrier and antagonist started to be reduced notably from the second day. There was no notable declining tendency active cells' proportion from other groups. The quantity of cell apoptosis in group with carrier and antagonist was higher than in the other groups. The expression level of cleaved Caspase-3 in the group with carrier and antagonist was notably higher than in the other groups. Moreover, expression of Bcl-2/Bax protein was reversed, while expression of p53 protein in the carrier and antagonist groups was notably higher than in the other groups. The antagonist of miR-214 packaged with lipidosome nanoparticles could target on p53 pathway. The activity of p53 pathway was reduced by miR-214, and expression of Bcl-2 was increased. The expressions levels of Bax and cleaved Caspase-3 were also reversed, and molecular mechanism was mainly related with restraining of p53 signal pathway.