Effect and mechanism of the Twist gene on invasion and metastasis of gastric carcinoma cells

AIM: To study the effect of the transfected Twist gene on invasion and metastasis of gastric carcinoma cells and the possible mechanisms involved. METHODS: Human gastric carcinoma MKN28 cells were stably transfected with Twist sense plasmid, and MKN45 cells were stably transfected with Twist antisense plasmid using the lipofectamine transfection technique. RT-PCR, Western blotting, EMSA, gelatin zymography assay, and in vitro invasion and migration assays were performed. Nude mice metastasis models were established by the abdominal cavity transfer method. RESULTS: Cell models (TwistS-MKN28) that steadily expressed high Twist protein were obtained. Compared with MKN28 and pcDNA3-MKN28 cells, adherence, migration and invasion ability of TwistS -MKN28 cells were clearly raised. The number of cancer nodules was increased significantly in the abdominal cavity and liver of nude mice inoculated with TwistS-MKN28 cells. Overexpression of Twist in MKN28 cells increased Tcf-4/ Lef DNA binding activity, and promoted expression of Tcf-4’s downstream target genes cyclin D1 and MMP-2. However, suppression of Twist (TwistAS-MKN45) inhibited MKN45 cell invasion and the expression of cyclin D1 was reduced. The activity of MMP-2 was also decreased. CONCLUSION: These results indicate that Twist promotes gastric cancer cell migration, invasion and metastasis, and Twist may play an important role in Wnt/ Tcf-4 signaling.

Chloride intracellular channel 1 regulates colon cancer cell migration and invasion through ROS/ERK pathway

AIM: To investigate the mechanisms of chloride intracellular channel 1 (CLIC1) in the metastasis of colon cancer under hypoxia-reoxygenation (H-R) conditions.

Individualized prediction of perineural invasion in colorectal cancer: development and validation of a radiomics prediction model

Objective： To develop and validate a radiomics prediction model for individualized prediction of perineural invasion（PNI） in colorectal cancer（CRC）.Methods： After computed tomography（CT） radiomics features extraction, a radiomics signature was constructed in derivation cohort（346 CRC patients）. A prediction model was developed to integrate the radiomics signature and clinical candidate predictors [age, sex, tumor location, and carcinoembryonic antigen（CEA） level]. Apparent prediction performance was assessed. After internal validation, independent temporal validation（separate from the cohort used to build the model） was then conducted in 217 CRC patients. The final model was converted to an easy-to-use nomogram.Results： The developed radiomics nomogram that integrated the radiomics signature and CEA level showed good calibration and discrimination performance [Harrell＇s concordance index（c-index）： 0.817; 95% confidence interval（95% CI）： 0.811–0.823]. Application of the nomogram in validation cohort gave a comparable calibration and discrimination（c-index： 0.803; 95% CI： 0.794–0.812）.Conclusions： Integrating the radiomics signature and CEA level into a radiomics prediction model enables easy and effective risk assessment of PNI in CRC. This stratification of patients according to their PNI status may provide a basis for individualized auxiliary treatment.

High level of urokinase plasminogen activator contributes to cholangiocarcinoma invasion and metastasis

AIM: To investigate the role of urokinase plasminogen activator (uPA) in cholangiocarcinoma (CCA) invasion and its correlation with clinicopathological parameters. METHODS: uPA expression in CCA tissue was determined by immunohistochemistry. The level of uPA from two CCA cell lines (HuCCA-1 and KKU-M213) and a noncancer immortalized cholangiocyte cell line (H69) was monitored by plasminogen-gelatin zymography and western blotting, whereas that of plasminogen activator inhibitor type 1 (PAI-1) protein and uPA receptor (uPAR)mRNA was monitored by western blotting and quantitative real-time reverse transcriptase polymerase chain reaction, respectively. Two independent methods were employed to suppress uPA function: a synthetic uPA inhibitor (B428) and silencing of uPA gene expression using siRNA. In vitro invasion of the uPA-disrupted cells was assessed by Matrigel-coated Transwell assay. RESULTS: The immunohistochemical study showed that 75.3% (131/174) of CCA tissues expressed uPA. High uPA expression was correlated with lymphatic invasion and metastasis of CCA patients. Plasminogen-gelatin zymography of the conditioned media and cell-surface eluates showed that both CCA cell lines, but not H69, expressed both secreted and membrane-bound forms of uPA. Although the two CCA cell lines, HuCCA-1 and KKU-M213, expressed a relatively high level of uPA and uPAR, the latter exhibited a much lower degree of in vitro invasiveness, correlating with a high expression of PAI-1 in the latter, but not in the former. Suppressing uPA function with a specific uPA inhibitor, B428, or with siRNA against uPA reduced in vitro invasiveness of KKU-M213 cells, demonstrating the requirement for uPA in the invasiveness of CCA cells. Therefore, our in vivo and in vitro studies suggest that uPA is an important requirement for the invasion process of CCA. CONCLUSION: uPA expression correlates with lymphatic invasion and metastasis in vivo and is required for CCA cell invasion in vitro , suggesting its potential as a therapeutic target.

Inhibitory activities of plumbagin on cell migration and invasion and inducing activity on cholangiocarcinoma cell apoptosis

Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma(CCA) cell line(CL-6) in comparison with human embryonic fibroblast cell line(OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and Cell Event? Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration(IC_(50), Mean±SD) values of plumbagin and the standard drug 5-fluorouracil were(24.00±3.33) and(1 036.00±137.77) μmol/L, respectively. The corresponding values for OUMS cells were(57.00±5.23) and(2 147.00±209.98) μmol/L, respectively. The selectivity index was 2.28. The inhibitory activities of plumbagin on cell migration and invasion were potent and concentration-dependent with IC_(50) of 25.0 μmol/L and complete inhibition at 25.0 μmol/L. Flow cytometry analysis showed that plumbagin at 12.5 μmol/L(half IC_(50)) induced CL-6 cell apoptosis(43.24% of control) through stimulation of caspase 3/7 activities. Complete cell apoptosis was observed at 12.5 μmol/L. Conclusions: The cytotoxic activity and inhibition of migration and invasion including apoptosis induction in the human CCA cell line(CL-6) suggest that plumbagin could be a promising candidate for CCA chemotherapeutics. However, its relatively low selective cytotoxic effect on CCA cells is a major concern.

A 3D biophysical model for cancer spheroid cell-enhanced invasion in collagen-oriented fiber microenvironment

The process of in situ tumors developing into malignant tumors and exhibiting invasive behavior is extremely complicated.From a biophysical point of view,it is a phase change process affected by many factors,including cell-to-cell,cell-to-chemical material,cell-to-environment interaction,etc.In this study,we constructed spheroids based on green fluorescence metastatic breast cancer cells MDA-MB-231 to simulate malignant tumors in vitro,while constructed a three-dimensional(3D)biochip to simulate a micro-environment for the growth and invasion of spheroids.In the experiment,the 3D spheroid was implanted into the chip,and the oriented collagen fibers controlled by collagen concentration and injection rate could guide the MDA-MB-231 cells in the spheroid to undergo directional invasion.The experiment showed that the oriented fibers greatly accelerated the invasion speed of MDA-MB-231 cells compared with the traditional uniform tumor micro-environment,namely obvious invasive branches appeared on the spheroids within 24 hours.In order to analyze this interesting phenomenon,we have developed a quantitative analyzing approach to explore strong angle correlation between the orientation of collagen fibers and invasive direction of cancer cell.The results showed that the oriented collagen fibers produced by the chip can greatly stimulate the invasion potential of cancer cells.This biochip is not only conducive to modeling cancer cell metastasis and studying cell invasion mechanisms,but also has the potential to build a quantitative evaluation platform that can be used in future chemical drug treatments.

Effect of hypoxia-inducible factor-1α on proliferation and invasion of prostate cancer PC-3 cell in hypoxic situation

Objective We transfected recombinant expression plasmid of pcDNA3. 1-HIF-1α into prostate cancer cells, to research effect of HIF-1α on proliferation of prostate cancer cell PC-3. Methods We selected a stable expression cell line with G418 we selected by transfection

Histologic subtypes of non-muscle invasive bladder cancer

The majority of bladder cancers(BCs)are non-muscle invasive BCs(NMIBCs)and show the morphology of a conventional urothelial carcinoma(UC).Aberrant morphology is rare but can be observed.The classification and characterization of histologic subtypes(HS)in UC in BC have mainly been described in muscle in-vasive bladder cancer(MIBC).However,the currently used classification is ap-plied for invasive urothelial neoplasm and therefore,also valid for a subset of NMIBC.The standard transurethral diagnostic work-up misses the presence of HS in NMIBC in a considerable percentage of patients and the real prevalence is not known.HS in NMIBC are associated with an aggressive phenotype.Conse-quently,clinical guidelines categorize HS of NMIBC as“(very)high-risk”tumors and recommend offering radical cystectomy to these patients.Alternative strategies for bladder preservation can only be offered to highly selected patients and ideally within clinical trials.Novel treatment strategies and biomarkers have been established MIBC and NMIBC but have not been comprehensively invest-igated in the context of HS in NMIBC.Further evaluation prior to implementation into clinical practice is needed.

BMP-6 inhibits microRNA-21 expression in breast cancer through repressing 6EF1 and AP-1

MicroRNAs （miRNAs）, which are small noncoding RNA molecules, play important roles in the post-transcriptional regulation process. The microRNA-21 gene （miR-21） has been reported to be highly expressed in various solid tumors, including breast cancer. Bone morphogenetic protein-6 （BMP-6） has been identified as an inhibitor of breast cancer epithelial-mesenchymal transition （EMT） through rescuing E-cadherin expression. We initiated experi- ments to identify the relationships between miR-21 and BMP-6 in breast cancer progression. Real-time PCR analysis showed that miR-21 expression was very high in MDA-MB-231 cells that expressed little BMP-6. A reverse correla- tion between BMP-6 and miR-21 was also determined in breast cancer tissue samples. Moreover, BMP-6 inhibited miR-21 transcription in MDA-MB-231 cells. In order to investigate how BMP-6 inhibited the miR-21 promoter （miPPR-21）, we constructed a series of miPPR-21 reporters. Luciferase assay results indicated that BMP-6 inhibited miPPR-21 activity through the E2-box and AP-l-binding sites. We also demonstrated that both δEF1 and TPA in- duced miR-21 expression. Using site-directed mutation and CHIP assay, we found that δEF1 induced miPPR-21 ac- tivity by binding to the E2-box on miPPR-21. Moreover, TPA triggered miPPR-21 activity through the AP-I binding sites. BMP-6 treatment significantly reduced the binding of these factors to miPPR-21 by decreasing the expression of δEF1 and c-Fos/c-Jun. We also demonstrated that BMP-6-induced downregulation of miR-21 modified the activ- ity of PDCD4 3＇UTR and inhibited MDA-MB-231 cell invasion. δEF1 overexpression and TPA induction blocked this inhibitory effect of BMP-6. In conclusion, BMP-6-induced inhibition of miR-21 suggests that BMP-6 may function as an anti-metastasis factor by a mechanism involving transcriptional repression of miR-21 in breast cancer.

Nestin in gastrointestinal and other cancers: Effects on cells and tumor angiogenesis

Nestin is a class Ⅵ intermediate filament protein that was originally described as a neuronal stem cell marker during central nervous system (CNS) development, and is currently widely used in that capacity. Nestin is also expressed in non-neuronal immature or progenitor cells in normal tissues. Under pathological conditions, nestin is expressed in repair processes in the CNS, muscle, liver, and infarcted myocardium. Furthermore, increased nestin expression has been reported in various tumor cells, including CNS tumors, gastrointestinal stromal tumors, pancreatic cancer, prostate cancer, breast cancer, malignant melanoma, dermatofibrosarcoma protuberances, and thyroid tumors. Nestin is reported to correlate with aggressive growth, metastasis, and poor prognosis in some tumors; however, the roles of nestin in cancer cells have not been well characterized. Furthermore, nestin is more specifically expressed in proliferating small-sized tumor vessels in glioblastoma and gastric, colorectal, and prostate cancers than are other tumor vessel markers. These findings indicate that nestin may be a marker for newly synthesized tumor vessels and a therapeutic target for tumor angiogenesis. It has received a lot of attention recently as a cancer stem cell marker in various cancer cells including brain tumors, malignant rhabdoid tumors, and uterine, cervical, prostate, bladder, head and neck, ovarian, testicular, and pancreatic cancers. The purpose of this review is to clarify the roles of nestin in cancer cells and in tumor angiogenesis, and to examine the association between nestin and cancer stem cells. Nestin has the potential to serve as a molecular target for cancers with nestin-positive cancer cells and nestin-positive tumor vasculature.

Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45

AIM：To clarify the role of activated Notch2 in the invasiveness of gastric cancer.METHODS：To investigate the invasiveness of silencing Notch2 gene expression,we established a Notch2small interfering RNA（siRNA） transfected cell line using the MKN-45 gastric cancer cell line.After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction（RT-PCR） and Western blotting,migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer.RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9（MMP9）,Akt,p-Akt.To confirm the relationship between PI3KAkt and MMP9,the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS：Notch2 expression was dramatically decreased after Notch2 siRNA transfection（100.00% ± 9.74% vs 11.61% ± 3.85%,P 〈 0.01 by qRT-PCR）.There was also a marked reduction of Notch target gene Hes1（100.00% ± 4.74% vs 61.61% ± 3.58%,P 〈 0.05） at the mRNA,indicating an inhibition of Notch signaling.Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels（100.00% ± 9.74% vs 65.61% ± 7.58%,P 〈 0.05）.Down-regulation of Notch2 by siRNA enhanced tumor cell invasion（100.00% ± 21.64% vs 162.22% ± 16.84%,P 〈 0.05） and expression of MMP9（1.56 fold,P 〈 0.05）,and activated the pro-MMP9 protein to its active form（1.48 fold,P 〈 0.05）.There was no significant difference in the protein levels of Akt between the two groups（100.00% ± 10.87% vs 96.61% ± 7.33%,P 〉 0.05）,while down-regulation of Notch2 elevated p-Akt expression（100.00% ± 9.87% vs 154.61% ± 13.10%,P 〈 0.05）.Furthermore,p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002（p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%,P 〈 0.05;MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%,P 〈 0.05）.CONCLUSION：Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway

Accessory Breast Cancer Occurring Concurrently with Bilateral Primary Invasive Breast Carcinomas:A Report of Two Cases and Literature Review

The development of accessory breast tissue,which is found anywhere along the milk line,is attributed to the failure of milk line remnants to regress during embryogenesis.Primary tumors may arise from any ectopic breast tissue.Accessory breast cancer occurring concurrently with primary invasive breast cancer is extremely rare.Two such cases were reported in this article.One was a 43-year-old Chinese female who exhibited bilateral breast cancer(invasive ductal carcinoma,not otherwise specified,IDC-NOS) and an accessory breast carcinoma(IDC-NOS) incidentally identified in her left axilla.The ectopic breast tissue in her right axilla presented with adenosis.The patient was surgically treated,followed by postoperative docetaxel epirubicin(TE) chemotherapy.The second case was a 53-year-old Chinese female with bilateral breast cancer(apocrine carcinoma) accompanied by an accessory breast carcinoma(IDC-NOS) in her right axilla that was also incidentally identified.The patient was surgically treated after three doses of cyclophosphamide epirubicin docetaxel(CET) neoadjuvant chemotherapy,followed by adjuvant chemotherapy of the same regimen.

Lack of CD44 variant 6 expression in rectal cancer invasive front associates with early recurrence

AIM: To investigate the prognostic value of CD44 variant 6 (CD44v6), a membranous adhesion molecule, in rectal cancer. METHODS: Altogether, 210 rectal cancer samples from 214 patients treated with short-course radiotherapy (RT, n = 90), long-course (chemo) RT (n = 53) or surgery alone (n = 71) were studied with immunohistochemistry for CD44v6. The extent and intensity of membranous and cytoplasmic CD44v6 staining, and the intratumoral membranous staining pattern, were analyzed.RESULTS: Membranous CD44v6 expression was seen in 84% and cytoplasmic expression in 81% of the cases. In 59% of the tumors with membranous CD44v6 expression, the staining pattern in the invasive front was determined as "front-positive" and in 41% as "front-negative". The latter pattern was associated with narrower circumferential margin (P = 0.01), infiltrative growth pattern (P < 0.001), and shorter disease-free survival in univariate survival analysis (P = 0.022) when compared to the "front-positive" tumors. CONCLUSION: The lack of membranous CD44v6 in the rectal cancer invasive front could be used as a method to identify patients at increased risk for recurrent disease.

Suppression of colorectal cancer metastasis by nigericin through inhibition of epithelial-mesenchymal transition

AIM: To evaluate the effect of nigericin on colorectal cancer and to explore its possible mechanism. METHODS: The human colorectal cancer (CRC) cell lines HT29 and SW480 were treated with nigericin or oxaliplatin under the conditions specified. Cell viability assay and invasion and metastasis assay were performed to evaluate the effect of nigericin on CRC cells. Sphereforming assay and soft agar colony-forming assay were implemented to assess the action of nigericin on the cancer stem cell properties of CRC cells undergone epithelial-mesenchymal transition (EMT). RESULTS: Compared with oxaliplatin, nigericin showed more toxicity for the HT29 cell line (IC50, 12.92 ± 0.25 μmol vs 37.68 ± 0.34 μmol). A similar result was also obtained with the SW116 cell line (IC50, 15.86 ± 0.18 μmol vs 41.02 ± 0.23 μmol). A Boyden chamber assay indicated that a significant decrease in the number of HT29 cells migrating through polyvinylidene fluoride membrane was observed in the nigericin-treated group, relative to the vehicle-treated group [11 ± 2 cells per high-power field (HPF) vs 19.33 ± 1.52 cells per HPF, P < 0.05]. Compared to the control group, the numbers of HT29 cells invading through the Matrigel-coated membrane also decreased in the nigericin-treated group (6.66 ± 1.52 cells per HPF vs 14.66 ± 1.52 cells per HPF, P < 0.05). Nigericin also reduced the proportion of CD133+ cells from 83.57% to 63.93%, relative to the control group (P < 0.05). Nigericin decreased the number of spheres relative to the control group (0.14 ± 0.01 vs 0.35 ± 0.01, P < 0.05), while oxaliplatin increased the number of spheres relative to the control group (0.75 ± 0.02 vs 0.35 ± 0.01; P < 0.05). Nigericin also showed a decreased ability to form colonies under anchorage-independent conditions in a standard soft agar assay after 14 d in culture, relative to the control group (1.66 ± 0.57 vs 7 ± 1.15, P < 0.05), whereas the colony numbers were higher in the oxaliplatin group relative to the vehicle-treated controls (14.33 ± 0.57 vs 7 ± 1.15, P < 0.05). We further detected the expression of E-cadherin and vimentin in cells treated with nigericin and oxaliplatin. The results showed that HT29 cells treated with nigericin induced an increase in E-cadherin expression and a decrease in the vimentin expression relative to vehicle controls. In contrast, oxaliplatin downregulated the expression of E-cadherin and upregulated the expression of vimentin in HT29 cells relative to vehicle controls. CONCLUSION: This study demonstrated that nigericin could partly reverse the EMT process during cell invasion and metastasis.

Treatment and surveillance for non-muscle-invasive bladder cancer:a clinical practice guideline(2021 edition)

Non-muscle invasive bladder cancer(NMIBC)is a major type of bladder cancer with a high incidence worldwide,resulting in a great disease burden.Treatment and surveillance are the most important part of NIMBC management.In 2018,we issued“Treatment and surveillance for non-muscle-invasive bladder cancer in China:an evidencebased clinical practice guideline”.Since then,various studies on the treatment and surveillance of NMIBC have been published.There is a need to incorporate these materials and also to take into account the relatively limited medical resources in primary medical institutions in China.Developing a version of guideline which takes these two issues into account to promote the management of NMIBC is therefore indicated.We formed a working group of clinical experts and methodologists.Through questionnaire investigation of clinicians including primary medical institutions,24 clinically concerned issues,involving transurethral resection of bladder tumor(TURBT),intravesical chemotherapy and intravesical immunotherapy of NMIBC,and follow-up and surveillance of the NMIBC patients,were determined for this guideline.Researches and recommendations on the management of NMIBC in databases,guideline development professional societies and monographs were referred to,and the European Association of Urology was used to assess the certainty of generated recommendations.Finally,we issued 29 statements,among which 22 were strong recommendations,and 7 were weak recommendations.These recommendations cover the topics of TURBT,postoperative chemotherapy after TURBT,Bacillus Calmette–Guérin(BCG)immunotherapy after TURBT,combination treatment of BCG and chemotherapy after TURBT,treatment of carcinoma in situ,radical cystectomy,treatment of NMIBC recurrence,and follow-up and surveillance.We hope these recommendations can help promote the treatment and surveillance of NMIBC in China,especially for the primary medical institutions.

Intravesical bacillus Calmette-Guerin(BCG)in treating non-muscle invasive bladder cancer—analysis of adverse effects and effectiveness of two strains of BCG(Danish 1331 and Moscow-I)

Objective:To compare the differences in adverse effects and efficacy profile between bacillus Calmette-Guerin(BCG)Danish 1331 and BCG Moscow-I strain in management of non-muscle invasive bladder cancer.Methods:Clinical data of 188 cases of non-muscle invasive bladder cancer treated with BCG between January 2008 and December 2018 in our institute were collected prospectively and analysed retrospectively,and 114 patients who completed a minimum of 12 months of follow-up were analysed.Patient and tumor characteristics,strain of BCG,adverse effects,and tumor progression were included for analysis.Intravesical BCG was instilled in intermediate-and high-risk patients.Six weeks of induction BCG,followed by three weekly maintenance BCG at 3,6,12,18,and 24 months was advised in high-risk patients.Results:Overall 68 patients received BCG Danish 1331 strain and 46 patients received Moscow-I strain.Patient and tumor characteristics were well balanced between the two groups.The median follow-up period was 42.5 months and 34.5 months in Danish 1331 and Moscow-I groups,respectively.Adverse events like dropout rate,antitubercular treatment requirement,and need of cystectomy were higher in Moscow-I group(n=31,67.4%)when compared to Danish 1331 strain(n=33,48.5%)(p=0.046).On direct comparison between Danish 1331 and Moscow-I strain,there was similar 3-year recurrence-free survival(80.0%vs.72.9%)and 3-year progression-free survival(96.5%vs.97.8%).Conclusion:Study results suggest no significant differences between Danish 1331 and Moscow-I strain in recurrence-free survival and progression-free survival,but a significantly higher incidence of moderate to severe adverse events in BCG Moscow-I strain.

Narrow band imaging for bladder cancer

Narrow band imaging(NBI)is a newly developed technology aiming to provide additional endoscopic information for patients with bladder cancer.This review focuses on the diagnostic accuracy and treatment outcome using NBI cystoscopy for the treatment of nonmuscle invasive bladder cancer.Current results showed improved sensitivity of NBI cystoscopy compared to conventional white light cystoscopy,although lower specificity and increased false-positive results were reported using NBI cystoscopy.The treatment outcome using NBI technology in transurethral resection of bladder tumor had a positive impact while decreased number of residual tumors and tumor recurrence at follow-up were reported.In the future,the application of NBI technology might refine the treatment and follow-up protocol in patients with non-muscle invasive bladder cancer.However,this large scale prospective studies are required to confirm the real cost-effectiveness of this new technology.

Does immunohistochemical staining have a clinical impact in early gastric cancer conducted endoscopic submucosal dissection?

AIM: To evaluate clinicopathologic parameters and the clinical significance related lymphovascular invasion (LVI) by immunohistochemical staining (IHCS) in endoscopic submucosal dissection (ESD). METHODS: Between May 2005 and May 2010, a total of 348 lesions from 321 patients (mean age 63 ± 10 years, men 74.6%) with early gastric cancer (EGC) who met indication criteria after ESD were analyzed retrospectively. The 348 lesions were divided into the absolute (n = 100, differentiated mucosal cancer without ulcer ≤ 20 mm) and expanded (n = 248) indica-tion groups after ESD. The 248 lesions were divided into four subgroups according to the expanded ESD indication. The presence of LVI was determined by factor Ⅷ-related antigen and D2-40 assessment. We compared LVI IHCS-negative group with LVI IHCSpositive in each group. RESULTS: LVI by hematoxylin-eosin staining (HES) and IHCS were all negative in the absolute group, while was observed in only the expanded groups. The positive rate of LVI by IHCS was higher than that of LVI by HES (n = 1, 0.4% vs n = 11, 4.4%, P = 0.044). LVI IHCS-positivity was observed when the cancer invaded to the mucosa 3 (M3) or submucosa 1 (SM1) levels, with a predominance of 63.6% in the subgroup that included only SM1 cancer (P < 0.01). In a univariate analysis, M3 or SM1 invasion by the tumor was significantly associated with a higher rate of LVI by IHCS, but no factor was significant in a multivariate analysis. There were no cases of tumor recurrence or metastasis during the median 26 mo follow-up. CONCLUSION: EGCs of the absolute group are immunohistochemically stable. The presence of LVI may be carefully examined by IHCS in an ESD expanded indication group with an invasion depth of M3 or greater.

THE FURTHER STUDY ON BIOLOGICAL BEHAVIOR OF COLORECTAL ADENOCARCINOMA:THE EXTRACELLULAR PROTEOGLYCAN DEGRADED BY ARYLSULFATASE B

The fresh tissues were obtained from 64 colorectal adenocarcinoma (43 well-differentiated and moderately-differentiated adenocarcinoma, 12 poorly- differentiated adenocarcinoma and 9 mutinous cell carcinoma including signet ring cell carcinoma) during surgical operation. The resected edge of each specimen was used for control group. The arylsulfatase B was studied by histochentical staining in different types of colorectal adenocarcinoma, among which 19 cases were investigated by electronhistochemical staining so as to observe the Ruthenium Red granules alteration which represented the extracellular proteoglycan changes and ultrastructure of cancer cells.The results showed that the mutinous cell carcinoma was of the most Intensive arylsulfatase B activity and has a lot of secretory granules with various electron densities in the cytoplasm. The Ruthenium Red granules close to the cancer cell disappeared, a part of remainders changed into the lowered electron density and indistinct shape. In contrast, the other types adenocarcinoma revealed less enzyme activity and a fewer secretory granules. The Ruthenium Red granules near the cancer nest showed that their electron density and size were identical with those of the control group. All of these mentioned above indicate that mucinouscell carcinoma may release hydrolase into pericancerous matrix to degrade the proteoglycans. In view of the network structure formed by proteoglycan in the connective tissue, network has ability to hinder the cancer cell spreading. Because the arylsulfatase B is able to degrade the dermatan sulfate proteoglycan which is component of proteoglycans in the extracellular matrix of human colon. We consider that the arylsulfatase B may lead to destruction of the network barriers in the connective tissue in favour of cancer cell invasion. So the mucinous cell carcinoma is more malignant than those of other colorectal adenocarcinoma.

A Th2-score in the tumor microenvironment as a predictive biomarker of response to Bacillus Calmette Guérin in patients with non-muscle invasive bladder carcinoma:A retrospective study

Intravesical Bacillus Calmette Guerin(BCG)is the gold standard therapy for intermediate/high-risk nonmuscle invasive bladder cancer(NMIBC).However,the response rate is~60%,and 50%of non-responders will progress to muscle-invasive disease.BCG induces massive local infiltration of inflammatory cells(Th1)and ultimately cytotoxic tumor elimination.We searched for predictive biomarker of BCG response by analyzing tumor-infiltrating lymphocyte(TIL)polarization in the tumor microenvironment(TME)in pre-treatment biopsies.Pre-treatment biopsies from patients with NMIBC who received adequate intravesical instillation of BCG(n=32)were evaluated retrospectively by immunohistochemistry.TME polarization was assessed by quantifying the T-Bet+(Th1)and GATA-3+(Th2)lymphocyte ratio(G/T),and the density and degranulation of EPX+eosinophils.In addition,PD-1/PD-L1 staining was quantified.The results correlated with BCG response.In most non-responders,Th1/Th2 markers were compared in pre-and post-BCG biopsies.ORR was 65.6%in the study population.BCG responders had a higher G/T ratio and a greater number of degranulated EPX+cells.Variables combined into a Th2-score showed a significant association with higher scores in responders(p=0.027).A Th2-score cut-off value>48.1 allowed discrimination of responders with 91%sensitivity but lower specificity.Relapse-free survival was significantly associated with the Th2-score(p=0.007).In post-BCG biopsies from recurring patients,TILs increased Th2-polarization,probably reflecting BCG failure to induce a pro-inflammatory status and,thus,a lack of response.PD-L1/PD-1 expression was not associated with the response to BCG.Our results support the hypothesis that a preexisting Th2-polarized TME predicts a better response to BCG,assuming a reversion to Th1 polarization and antitumor activity.