Objective:To analyze the expression of mismatch repair (MMR) proteins in colorectal cancers (CRC) and to evaluate the feasibility and potential pitfalls of immunohistochemistry (IHC) analysis for MMR.Methods:The IHC sections for MMR proteins were reviewed in 3 428 cases of resected CRC without neoadjuvant therapy at Tianjin Medical University Cancer Institute and Hospital from July 2014 to October 2018. For the cases with unclear MMR IHC results during the initial review, IHC staining was repeated and microsatellite instability (MSI) analysis was performed. Relationships between the expression of MMR proteins and MSI status as well as the clinicopathological parameters were analyzed.Results:IHC staining for MMR was repeated in 28 (0.8%) cases due to poor quality of original IHC sections. Inconsistent results between the original diagnosis and re-diagnosis were found in 119 (3.5%) cases, mainly resulting from PMS2 and MLH1. Finally, 261 (7.6%) cases of CRC showed mismatch repair deficiency (dMMR), mainly from the deficiency of both MLH1 and PMS2 (43.3%,113/261). In the 14 cases with MSI results, the concordant of MSI and MMR was 13 cases. In the 29 dMMR cases with next generation sequencing (NGS) results, the concordant of MSI-high and dMMR was 93.1%(27/29). The cases with inconsistent results between MSI and MMR showed negative expression of MSH6 or PMS2. Twenty-one CRC showed negative expression of MLH1 and partially positive (or weak positive) expression of PMS2, or negative expression of MSH2 and partially positive (or weak positive) expression of MSH6. Among the 19 cases with MSI results, 16 cases were MSI-high, two cases were MSI-low, and one case was microsatellite stable. Compared with mismatch repair proficiency (pMMR), dMMR was more frequently detected in female patients younger than 50 years old, with family history, at early stage (Ⅰ-Ⅱ) CRC, and in the tumors from right colon,with poor differentiation, or mucinous adenocarcinoma/signet ring cell carcinoma (all P<0.05). Conclusions:At present, IHC staining is a clinically effective and convenient method to detect MMR expression, but the operating process and result assessment remain variable and need to be standardized. MSI analysis can be performed in the difficult-to-evaluate cases for MMR to enhance prognostic evaluation and treatment option.

Objective:To explore the clinicopathological characteristics and prognostic factors of neuroendocrine neoplasms(NENs)at different sites in the digestive system.Methods:The clinicopathological parameters and follow-up data were collected from 284 pa-tients with NENs in the digestive system in Tianjin Medical University Cancer Institute and Hospital from March 2011 to December 2015.The incidence and clinicopathological features were compared between the cases of NENs at different sites and survival analysis was performed.Results:In this study,NENs were detected mostly frequently in the pancreas,followed by the colorectum and stom-ach.In the pancreas,neuroendocrine tumor(NET)G1(51.8%)and G2(35.8%)accounted for a large proportion of NENs.World Health Organization(WHO)grades were related to lymph node metastasis,adjacent organ invasion,and nerve invasion(P<0.05 for all)but were not associated with the overall survival time of the patients.The patients with pancreatic NENs with distant metastasis had poor overall survival(P<0.05).Regarding colorectal NENs,most patients had NET G1(82.5%),and the majority of patients were cured with endoscopic or transanal resection.Patients with NENs,lymph node metastasis,and distant metastasis had poor overall survival(P<0.05 for all).The ratio of male-to-female patients,proportion of patients aged older than 40 years,prevalence of neuroendocrine carci-nomas(NECs)and mixed adenoneuroendocrine carcinomas(MANECs),presence of lymph node and distant metastasis,and presence of advanced stage tumors were greater in patients with gastric NENs than in patients with pancreatic and colorectal NENs(P<0.05 for all).WHO grades and lymph node metastasis were associated with the overall survival time of patients with gastric NENs(P<0.05 for both).Conclusions:NENs in the digestive system are a group of heterogeneous tumors with different clinicopathological features at different sites.The distribution and clinicopathological features of Chinese patients with NENs in the digestive system are different from those of European and American patients.More multicenter studies with large sample sizes are still needed to understand the bi-ological behaviors and prognostic factors of NENs at different sites in the digestive system.

Objective:To explore the diagnostic value of ultrasound-guided fine needle aspiration biopsy(US-FNAB)for thyroid nod-ules.Methods:The clinical characteristics and cytopathological diagnosis of patients with thyroid nodules in Tianjin Medical University General Hospital were analyzed retrospectively;the results of the cytopathological and pathological diagnoses were compared and an-alyzed.Results:Of the 1,241 US-FNAB samples,the ratio of men to women with thyroid nodules was 1:3.83(257/984).The incidence of thyroid nodules gradually increased from the age of 20 years and declined after the age of 60 years.The nodules,which were less than or equal to 1.0 cm in size,accounted for 51.57%(640 cases),and Thyroid Imaging Reporting And Data System(TIRADS)classifica-tion 4 accounted for 86.38%(1 072 cases).Of cyto-pathological diagnoses,22.00%(273 cases)were non-diagnostic,9.75%(121 cases) were benign,30.62%(380 cases)were atypia with undetermined significance,32.15%(399 cases)were suspicious for malignancy,and 5.48%(68 cases)were malignant.In the 302 patients who underwent surgery,the number of cases of clear diagnoses,unavailable di-agnoses,and atypia of undetermined significance were 203,21,and 78,respectively.In the 203 cases of clear diagnoses,the sensitivi-ty,specificity,positive predictive value,negative predictive value,precision,and misdiagnoses following US-FNAB of thyroid nodules were 100.00%(201/201),50.00%(1/2),99.50%(201/202),100.00%(1/1),99.51%(202/203),and 0.49%(1/203),respectively.In the 78 cases that were atypia of undetermined significance,the malignancy rate was 70.51%.Whether the atypia of undetermined signifi-cance was malignant or not was related to the TIRADS classification(P<0.05),and not related to the age,sex,tumor size,or location of the nodules(P>0.05).Conclusions:US-FNAB has high diagnostic value for thyroid nodules and is worthy of being popularized widely.If it replaced some intra-operative frozen sectioning procedures,it may reduce intra-operative waiting time and financial burden of pa-tients.

Background and objective Progesterone and adiponectin receptor family member 3 (PAQR3) is a recently discovered tumor suppressor gene,which affects the development of a tumor by inhibiting cell proliferation,cell malignant transformation,angiogenesis,and tumor metastasis.This study investigates the expression of PAQR3 in lung cancer and its clinical significance.Methods A total of 106 patients with lung cancer received surgical treatment in hospital,and adjacent normal tissues of these patients were utilized as control group.The diagnosis of all patients was confirmed through clinical pathology.The expression of PAQR3 protein was detected by immunohistochemistry in lung cancer and adjacent normal tissues.The clinical significance of its expression was also investigated.Results The positive expression rate of PAQR3 protein in lung cancer was lower than that in adjacent normal tissues (P＜0.01).The positive expression rate of PAQR3 protein was unrelated to age,tumor size,and gender,but it exhibited a significant relationship with the pathological type and differentiation,TNM staging,and lymph node metastasis (P＜0.05).Kaplan-Meier survival analysis showed that the five-year survival rate of patients with PAQR3 protein positive expression was higher than that in patients with negative expression (P=0.026).Conclusion The expression of PAQR3 protein significantly decreased in lung cancer,indicating that PAQR3 protein plays an important role in the pathogenesis of PAQR3 in lung cancer.

Objective:This study aims to determine the suitable cell line to be used in isolating cancer stem cells by comparing the characteristics of tumor stem cells in renal cell carcinoma cell lines SN12C and 786-O. Methods:The rate of sphere formation in SN12C and 786-O cells was determined in serum-free medium (SFM). The expression levels of CD133, CD44, Nanog, and Oct3/4 were investi-gated through flow cytometry. Moreover, the tumorigenicity of spheroid cell that originated from SN12C and 786-O cells was investi-gated in vivo by using a tumor model. Results:The average time of sphere formation in SFM was shorter in SN12C than in 786-O (5 days vs. 7 days). Moreover, the expression levels of CD133, CD44, Nanog, and Oct3/4 in SN12C and 786-O significantly differed (P<0.05). When transplanted in nude mice, 786-O spheres were less tumorigenic than SN12C spheres. Conclusion:SN12C spheres possess the main defining characteristics of renal cancer stem cell;thus, SN12C is the more suitable cell line to be used to isolate cancer stem cells compared with 786-O.

Objective To explore whether hypoxia could promote epithelial-mesenchymal transition (EMT) in various differentiated colorectal cancer cells, and analyse the effect of hypoxia on invasion and migration of colorectal cancer cells. Methods HCT116 (poorly differentiated) and HT-29 (highly differentiated) colorectal adenocarcinoma cells were selected respectively. The morphological changes of two cell lines were observed after 0,10,25,50,100 and 150 mg/L cobalt chloride (CoCl2) treatment for 48 h. The expression of hypoxia-inducible factor-1α(HIF-1α) protein was analysed after 0, 10,25,50,100 and 150 mg/L CoCl2 treatment for 48 h. An optimal concentration of CoCl2 was then selected. Methylthiazolyl tetrazolium (MTT) assay was used to detect the proliferation of two kinds of colorectal cancer cells induced by CoCl 2 at different time points (0, 24, 48, 72 and 96 h), and to select an optimal time. Under the optimal concentration and time conditions, the HCT116 and HT-29 cells were processed by hypoxia (hypoxia group) and normoxia (normoxic group). Transwell invasion assay and Wound healing assay were used to detect cell invasion and migration in two groups. Western blot assay and RT-PCR were used to detect protein and mRNA expression levels of HIF-1α, E-cadherin and Vimentin in two groups. Results Two kinds of cells showed obvious morphological changes after 50 mg/L CoCl2 treatment for 48 h. HIF-1αprotein level first increased and then decreased in two groups of cells with the increased concentration of CoCl 2, and 50 mg/L CoCl2 was the optimal concentration (P<0.05). The cell proliferation showed a tendency to decrease after the increase in both kinds of cells with or without hypoxia for 0-96 h (P<0.05), and 48 h was the optimal time. The transmembrane number and cell migration rate were significantly more in hypoxia group than those of normoxic group (P<0.05). The protein and mRNA levels of HIF-1α and Vimentin were significantly higher in hypoxia group than those of normoxic group in HCT116 and HT-29 cell lines (P<0.05). E-cadherin protein and mRNA levels were significantly lower in hypoxia group than those of normoxic group (P<0.05). Conclusion Hypoxia can promote EMT in different differentiated colorectal cancer cells, and can enhance invasion and migration of two kinds of colorectal cancer cells.

Background:MicroRNA?506 (miR?506) has been reported to function in several tumors as a tumor suppressor gene or oncogene. However, the expression and role of miR?506 in pancreatic ductal adenocarcinoma (PDAC) remains unclear. In this study, we aimed to evaluate the phenotype of miR?506 in PDAC. Methods:Using miRNA insitu hybridization, we examined the expression of miR?506 in 113 PDACs and 87 paired normal pancreatic tissues. We evaluated miR?506 expression in PDAC cells, normal pancreatic ducts, and acinus/islands, and we analyzed the associations between miR?506 expression and the clinicopathologic characteristics of PDAC patients. Results:miR?506 expression was higher in PDAC than in matched normal pancreatic ductal cells (P<0.001). On the other hand, the combined group of well and moderately differentiated PDACs showed higher levels of miR?506 than the poorly differentiated ones (P=0.023). Moreover, miR?506 expression was negatively associated with pathologic T category (P=0.004) and lymph node metastasis (P=0.033), suggesting that miR?506 might inhibit the progression of PDAC. Conclusions:Our results suggest that miR?506 either plays a role as an oncogene in the tumorigenesis and a tumor suppressor in the progression or serves as a house?keeping, tumor?suppressing miRNA, whose expression can be activated by oncogenic signals in early development to hinder the progression of PDAC.

Objective:To investigate the clinical significance of epithelial-to-mesenchymal (EMT) in lung squamous cell carcino-ma (LSCC) and to examine the effect of EMT on the invasive and migration abilities of LSCC. Methods:Immunohistochemical stain-ing was performed to determine the expression of E-cadherin, Vimentin, and TGF-β1 in 79 LSCC patients, and the clinical significance was explored. SK-MES-1 lung squamous carcinoma cells were cultured in conditioned medium containing various concentrations of transforming growth factor-β1 (TGF-β1) for 5 and 10 days. The expression levels of E-cadherin and Vimentin were detected via West-ern blot and reverse transcription-polymerase chain reaction (RT-PCR). With different concentrations and induction times, invasion and wound healing assays were performed to evaluate the invasion and migration abilities. Results:E-cadherin expression was significantly lower, whereas Vimentin expression was significantly higher in LSCC with lymph node metastasis than in that without noda metastasis (P<0.05). In the tissues of 79 LSCC patients, TGF-β1 expression was significantly related to lymph node metastasis (P<0.05). Western blot showed that Vimentin expression was higher, whereas E-cadherin expression was lower in TGF-β1 inducing medium with 10 ng/mL SK-MES-1 cells than in the other media. RT-PCR showed similar results. Scratch test and invasion assay both showed that treat-ment of cells with cytokines markedly enhanced the migration and invasion of the cells. Conclusion:Lymph node metastasis of LSCC correlates with EMT. SK-MES-1 cells undergo EMT via TGF-β1 induction, which enhances invasion and migration.

Objective:To determine the expression of BMP4 in hepatocellular carcinoma (HCC) and to study the role of BMP4 in inducing epithelial-mesenchymal transition (EMT) to analyze the effect of BMP4 on the migration and invasion of HCC cells. Methods: The expression of BMP4 in HCC specimens was examined by immunohistochemistry staining, and the correlations were analyzed between the expression of BMP4 and clinicopathological data. The BMP4 expression plasmid was transfected into HepG2 cells to induce exogenous overexpression of BMP4 protein. The changes of HepG2 cell morphology were detected after BMP4 transfection by using a microscope; the changes of the expression of BMP4, EMT-related protein (E-cadherin, Vimentin) in HepG2 cells were detected by Western blot after transfection of BMP4;the wound healing assay in vitro was used to detect the effects of BMP4 gene transfection on the ability of migration of HepG2 cells;the invasion assay was used to determine the role of transfection of BMP4 on the invasive potential of HepG2 cells. Results: Immunohistochemistry staining method displayed that BMP4 expression was positively associated with age, histological differentiation, stage, and poor prognosis. After BMP4 overexpression, the morphology of HepG2 cells showed significant changes from a paving stone structure with cell-cell adhesion to a fibroblastic shape, which showed typical EMT change; Western blot exhibited that the expression of E-cadherin was downregulated and the Vimentin expression was upregulated in HepG2 cells;the wound healing and invasion assay showed that the migration and invasion potentials of HepG2 cells were significantly enhanced. Conclusion: BMP4, which displayed a high expression in HCC specimens, was closely associated with clinicopathologic data, and BMP4 may promote migration and invasion of HCC cells by inducing epithelial-mesenchymal transition.

Objective:To investigate the correlations of Lauren classification and world health organization (WHO) classification of gastric cancer (GC) with microvascular density (MVD), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and p53. Methods:The clinical data of 89 patients with GC were collected. The collected specimens were categorized on the basis of Lauren classification and WHO classification. CD34/periodic acid-Schiff (PAS) double staining was performed to validate MVD. Immunohistochemistry was conducted to investigate the expression levels of MMP-2, MMP-9, VEGF, VEGFR1, VEGFR2, and p53. Results:MVD was not correlated with Lauren classification or WHO classification (P>0.05). Lauren typing was associated with the expression levels of MMP-9, VEGFR1, and p53 (P<0.05). WHO classification was not related to any of the factors (P>0.05). Cox proportional hazards model revealed that Lauren classification and WHO classification were the prognostic factors of overall survival (P<0.05). Conclusion:This research on tumor related factors, angiogenesis, and different classifications of GC may provide new methods to treat this disease.