肿瘤抗原致敏树突状细胞协同CD3AK细胞的抗肿瘤作用

目的:观察雷公藤内酯醇是否能抑制肿瘤细胞增殖,并探讨其是否通过诱导肿瘤细胞凋亡发挥抗肿瘤作用.方法:选择人宫颈癌细胞株HeLa为研究对象,以MTr实验检测肿瘤细胞的增殖,以AnnexinV/PI染色检测细胞的凋亡,R123染色检测线粒体膜电势的变化.Hoechst 33258荧光染色法观察细胞形态学改变,琼脂糖凝胶电泳检测染色体DNA断裂.结果:雷公藤内酯醇能明显抑制HeLa细胞的增殖;AnnexinV/PI染色结果证明雷公藤内酯醇能诱导HeLa细胞凋亡,R123染色结果说明雷公藤内酯醇诱导的细胞凋亡与线粒体膜电势的变化有关.雷公藤内酯醇处理的HeLa细胞可见凋亡特有的形态学及生物化学改变,DNA电泳呈梯状条带.结论:雷公藤内酯醇可以通过诱导肿瘤细胞凋亡而发挥抗肿瘤作用.     

The design and optimization of RNA trans-splicing molecules for skin cancer therapy

Targeting tumor marker genes by RNA trans-splicing is a promising means to induce tumor cell-specific death. Using a screening system we designed RNA trans-splicing molecules (RTM) specifically binding the pre-mRNA of SLCO1B3, a marker gene in epidermolysis bullosa associated squamous cell carcinoma (EB-SCC). Specific trans-splicing, results in the fusion of the endogenous target mRNA of SLCO1B3 and the coding sequence of the suicide gene, provided by the RTM. SLCO1B3-specific RTMs containing HSV-tk were analyzed regarding their trans-splicing potential in a heterologous context using a SLCO1B3 expressing minigene (SLCO1B3-MG). Expression of the chimeric SLCO1B3-tk was detected by semi-quantitative RT-PCR and Western blot analysis. Cell viability and apoptosis assays confirmed that the RTMs induced suicide gene-mediated apoptosis in SLCO1B3-MG expressing cells. The lead RTM also showed its potential to facilitate a trans-splicing reaction into the endogenous SLCO1B3 pre-mRNA in EB-SCC cells resulting in tk-mediated apoptosis. We assume that the pre-selection of RTMs by our inducible cell-death system accelerates the design of optimal RTMs capable to induce tumor specific cell death in skin cancer cells.     

Analysing the mutational status of PIK3CA in circulating tumor cells from metastatic breast cancer patients

The frequently altered phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in the regulation of cellular processes required for breast carcinogenesis. The aim of the project was to develop a method to identify hotspot mutations in the PIK3CA gene in circulating tumor cells (CTCs) of metastatic breast cancer (metBC) patients.From 44 enrolled CTC-positive metBC patients a total number of 57 peripheral blood samples were analysed by CellSearch^®. Genomic DNA of enriched CTCs was isolated, amplified and analyzed for PIK3CA mutations in exons 9 and 20 which lead to E542K, E545K or H1047R amino acid changes and result in increased PI3K activity. The mutations were detected by using SNaPshot-methodology comprising PCR amplification and single nucleotide primer extension.SNaPshot analysis was established using genomic DNA from different breast cancer cell lines and then successfully transferred to investigate blood samples and single cells. Overall, twelve hotspot mutations in either exon 9/E545K (6/12, 50%) or exon 20/H1047R (6/12, 50%) could be determined within 9 out of 57 (15.8%) blood samples from 7 out of 44 (15.9%) patients; CTC counts ranged from 1 to 9748. PIK3CA variants E542K, E545G and E545A were not detected.Analysing the PIK3CA genotype of CTCs has clinical relevance with respect to drug resistance, e.g. against HER2-targeted therapy. The herein described approach including SNaPshot technology provides a simple method to characterize hotspot mutations within CTCs enriched from peripheral blood and can be easily adopted for analysing further therapeutically relevant SNPs.     

Tumor differentiation is not a risk factor for lymph node metastasis in elderly patients with early gastric cancer

BackgroundThe aim of this study was to identify risk factors for lymph node metastasis in elderly patients (70 years or more) with early gastric cancer.MethodsWe reviewed the prospectively collected database of 6893 patients with early gastric cancer who had undergone curative gastrectomy in 3 tertiary cancer centers between January 2003 and December 2009 in Korea. Patients were sorted into 4 groups according to age: less than 50, fifties, sixties, and 70 years or more. Risk factors for lymph node metastasis in early gastric cancer were analyzed.ResultsOne thousand and thirty five patients (15.0%) were 70 years or more. As age increased, the frequency of large differentiated tumor, lymphatic and submucosa invasion increased. Old age was associated with a lower risk for lymph node metastasis in patients with early gastric cancer (Odds ratio [OR], OR, 0.622; 95% CI, 0.5466–0.830, P = 0.010). Ulceration or differentiation of tumor was not associated with lymph node metastasis in elderly patients with early gastric cancer.ConclusionsElderly patients with undifferentiated type histology early gastric cancer without other risk factors for lymph node metastasis may be candidates for endoscopic resection.     

The repressive effect of green tea ingredients on amyloid precursor protein (APP) expression in oral carcinoma cells in vitro and in vivo

In a hamster model of N-methyl-N-benzylnitrosamine (MBN)-induced oral carcinogenesis, the incidence of buccal pouch (HBP) carcinomas in MBN-treated hamsters (17.8+/-7.5) was significantly higher than MBN-treated hamsters given tea (10.8+/-3.9) (P<0.05). Amyloid precursor protein (APP) expression was also significantly increased in MBN-induced HBP carcinomas but was significantly reduced by tea intake (P<0.0001). Furthermore, APP expression and secretion by OECM-1 oral squamous cell carcinoma cells was inhibited by a major polyphenolic ingredient of green tea, (-)-epigallocatechin gallate, in a dose-dependent manner. Thus, APP might promote oral carcinogenesis, whereas green tea ingredients might diminish it by down-regulating APP.     

Referral patterns in advanced non-small cell lung cancer: Impact on delivery of treatment and survival in a contemporary population based cohort

IntroductionChemotherapy improves overall survival (OS) in advanced non-small cell lung cancer (NSCLC), yet low rates of chemotherapy utilization have been observed. We sought to characterize the clinical effectiveness of chemotherapy in the general population by evaluating referral patterns, predictors of chemotherapy receipt and outcomes.MethodsAll referred cases of stage IIIB/IV NSCLC in British Columbia from January 1 to December 31, 2009 were retrospectively reviewed. Patient demographics, tumor characteristics and treatments were extracted. OS was estimated using the Kaplan–Meier method. Cox Proportional Hazards modeling was used to control for confounding variables. Multiple logistic regression was used to assess factors that predicted for chemotherapy treatment.Results1373 patients were identified. Median age 70 years, 53% male, 37% ECOG ≥ 3. Histology: 34% non-squamous, 21% squamous and 46% NOS. 748 (54%) patients were assessed by medical oncology and 417 (30%) received chemotherapy. Predictors of chemotherapy treatment were younger age, ECOG 0–2, living in a rural area and not receiving radiotherapy. There was an improvement in OS in patients who received chemotherapy at 13.1 months versus best supportive care 5.4 months (p < 0.0001). This remained statistically significant when controlling for ECOG, sex, age, histology (HR 0.68, CI 0.59–0.78).ConclusionsIn this population-based setting, 37% of patients had an ECOG ≥ 3 at the time of referral, 54% were assessed by a medical oncologist and only 30% received chemotherapy. This is despite the awareness that chemotherapy significantly improves survival. Strategies to optimize appropriate referral such that patients do not miss out on life-prolonging therapy should be evaluated.     

What to do for the incidental pancreatic cystic lesion?

BackgroundIncidental pancreatic cysts are identified in 1% of all patients undergoing CT scans of the abdomen for whatever reason. The aim of this review was to provide an overview of the current evidence relating to the investigation and management of these lesions.MethodsPubMed was searched to identify relevant studies relating to the investigation and management of incidentally discovered pancreatic cystic lesions.ResultsInitial investigation of incidentally discovered pancreatic cysts should be with either specific pancreas protocol CT or contrast enhanced MRI with MRCP. The diagnostic yield of these investigations can be increased with the addition of EUS/FNA and cyst fluid analysis in appropriately selected patients. Surgical intervention may be indicated in otherwise fit patients who are identified as having mucinous neoplasms.ConclusionApplying a systematic approach to the investigation of incidentally discovered pancreatic cysts means that in the majority of cases cyst aetiology can be accurately determined and appropriate management plans developed.     

Characterization of a clonal isolate of an oxaliplatin resistant ovarian carcinoma cell line A2780/C10

A single cell clonal sub-line A2780/C10B that is 18-fold resistant to oxaliplatin and approximately threefold cross-resistant to cisplatin and exhibiting a metastasis associated cellular phenotype was characterized for mechanisms of resistance. The cell line exhibited a 50% reduction in the accumulation of both oxaliplatin and cisplatin relative to the parent line, while extensive decline in Pt-DNA adduct levels occurred only following oxaliplatin treatment. The basal GSH levels were fivefold higher in A2780/C10B compared to A2780 and had a fivefold elevation in gamma-GT suggesting this may be the mechanism involved in GSH elevation. The basal levels of ERCC-1, XPA and MRP-2 mRNA levels in A2780/C10B were not higher than those in A2780. The highly reduced Pt-DNA adduct formation only for oxaliplatin, but not cisplatin may be a reflection of the fact that at equimolar concentrations oxaliplatin makes fewer Pt-DNA adducts than cisplatin. The data indicate that multiple lesions occur in a single cell to produce the resistant phenotype.     

The XPD Asp312Asn and Lys751Gln polymorphisms, corresponding haplotype, and pancreatic cancer risk

We evaluated the association between the XPD exon 10 Asp(312)Asn and exon 23 Lys(751)Gln polymorphisms and the risk of pancreatic cancer in a hospital-based study of 344 patients and 386 controls frequency matched by age, gender, and race. Stratified analyses showed ever smokers carrying the Asn(312)Asn genotype had a significantly reduced risk when compared with those carrying the (312)Asp allele (OR=0.46, 95% confidence interval 0.24-0.88) (P for interaction=0.03). The (312)Asp-(751)Gln was identified as the putative at risk haplotype. Our study shows that the XPD gene polymorphism could be a genetic risk modifier for smoking-related pancreatic cancer.     

Tumour heterogeneity and the evolution of polyclonal drug resistance

Cancer drug resistance is a major problem, with the majority of patients with metastatic disease ultimately developing multidrug resistance and succumbing to their disease. Our understanding of molecular events underpinning treatment failure has been enhanced by new genomic technologies and pre-clinical studies. Intratumour genetic heterogeneity (ITH) is a prominent contributor to therapeutic failure, and it is becoming increasingly apparent that individual tumours may achieve resistance via multiple routes simultaneously – termed polyclonal resistance. Efforts to target single resistance mechanisms to overcome therapeutic failure may therefore yield only limited success. Clinical studies with sequential analysis of tumour material are needed to enhance our understanding of inter-clonal functional relationships and tumour evolution during therapy, and to improve drug development strategies in cancer medicine.     

Unraveling molecular pathways of poorly differentiated neuroendocrine carcinomas of the gastroenteropancreatic system: A systematic review

BackgroundPoorly differentiated neuroendocrine carcinomas (NECs) are rare and aggressive tumors. Their molecular pathogenesis is still largely unknown, and consequently, the best therapeutic management also remains to be determined. We conducted a systematic review on molecular alterations found in gastroenteropancreatic NECs (GEP-NECs) and discuss potential applications of targeted therapies in setting.Materials and methodsSystematic review of studies about molecular features in tumor tissues of patients with GEP-NECs. The Medline, Lilacs, Embase, Cochrane, Scopus and Opengrey databases were sought, without time, study design or language restrictions.ResultsOf the 1.564 studies retrieved, 41 were eligible: 33 were retrospective studies and eight were case reports. The studies spanned the years 1997–2017 and involved mostly colorectal, stomach and pancreas primary tumors. Molecular alterations in the TP53 gene and the p53 protein expression were the most commonly observed, regardless of the primary site. Other consistently found molecular alterations were microsatellite instability (MSI) in approximately 10% of gastric and colorectal NEC, and altered signaling cascades of p16/Rb/cyclin D1, Hedgehog and Notch pathways, and somatic mutations in KRAS, BRAF, RB1 and Bcl2. In studies of mixed adeno-neuroendocrine carcinomas (MANECs) the molecular features of GEP-NEC largely resemble their carcinoma/adenocarcinomas tumor counterparts.ConclusionsDespite the paucity of data about the molecular drivers associated with GEP-NEC, some alterations may be potentially targeted with new cancer-directed therapies. Collaborative clinical trials for patients with advanced GEP-NEC are urgently needed.     

Influence of DNA copy number and mRNA levels on the expression of breast cancer related proteins

For a panel of cancer related proteins, the aim was to shed light on which molecular level the expression of each protein was mainly regulated in breast tumors, and to investigate whether differences in regulation were reflected in different molecular subtypes. DNA, mRNA and protein lysates from 251 breast tumor specimens were analyzed using appropriate microarray technologies. Data from all three levels were available for 52 proteins selected for their known involvement in cancer, primarily through the PI3K/Akt pathway. For every protein, in cis Spearman rank correlations between the three molecular levels were calculated across all samples and within each intrinsic gene expression subtype, enabling 63 comparisons altogether due to multiple gene probes matching to single proteins. Subtype-specific relationships between the three molecular levels were studied by calculating the variance of subtype-specific correlation and differences between overall and average subtype-specific correlation. The findings were validated in an external dataset comprising 703 breast tumor specimens. The proteins were sorted into four groups based on the calculated rank correlation values between the three molecular levels. Group A consisted of eight proteins with significant correlation between DNA copy number levels and mRNA expression, and between mRNA expression and protein expression (Bonferroni adjusted p < 0.05). Group B consisted of 14 proteins with significant correlation between mRNA expression and protein expression. Group C consisted of 15 proteins with significant correlation between copy number levels and mRNA expression. For the remaining 25 proteins (group D), no significant correlations was observed. Stratification of tumors according to intrinsic subtype enabled identification of positive correlations between copy number levels, mRNA and protein expression that were undetectable when considering the entire sample set. Protein pairings that either demonstrated high variance in correlation values between subtypes, or between subtypes and the total dataset were studied in particular. The protein expression of cleaved caspase 7 was most highly expressed, and correlated highest to CASP7 gene expression within the basal-like subtype, accompanied by the lowest amounts of hsa-miR-29c. Luminal A-like subtype demonstrated highest amounts of hsa-miR-29c (a miRNA with a putative target sequence in CASP7 mRNA), low expression of cleaved caspase 7 and low correlation to CASP7 gene expression. Such pattern might be an indication of hsa-miR-29c miRNA functioning as a repressor of translation of CASP7 within the luminal-A subtype. Across the entire cohort no correlation was found between CCNB1 copy number and gene expression. However, within most gene intrinsic subtypes, mRNA and protein expression of cyclin B1 was found positively correlated to copy number data, suggesting that copy number can affect the overall expression of this protein. Aberrations of cyclin B1 copy number also identified patients with reduced overall survival within each subtype. Based on correlation between the three molecular levels, genes and their products could be sorted into four groups for which the expression was likely to be regulated at different molecular levels. Further stratification suggested subtype-specific regulation that was not evident across the entire sample set.     

Antitumor activity in advanced cancer patients with thymic malignancies enrolled in early clinical drug development programs (Phase I trials) at Gustave Roussy

ObjectivesThymic epithelial neoplasms (TENs) represent a rare entity with poor prognosis and limited systemic treatment options. The aim of this study was to assess the clinical benefit, the efficacy and toxicities of agents for patients with TEN enrolled in Phase I trials.Materials and methodsWe reviewed retrospectively patients with advanced TEN enrolled in Phase I trials at Gustave Roussy (DITEP) between 1994 and 2012. Efficacy was assessed using RECIST version 1.1.ResultsTwenty-two treated patients were enrolled (15 with thymic carcinoma, 7 with thymoma). The median number of prior systemic therapies was 2 (0–8). The median age was 50 years (range 23–72), and 4 females were treated. Treatments received encompassed mTOR inhibitor (mTORi) in 4 of patients, antiangiogenic agents (AA) in 11 patients, and other targeted therapies in 7 patients. 18% had grade 3-4 toxicity, 85% all grade toxicity and no toxic death was reported. One patient experienced a complete response (CR) and 3 a partial response (PR); 16 patients had stable disease (median 6.6 months; range 1.0–30.7) and 2 had a progressive disease. The median overall survival was 54.5 months (95% CI 25–75.50). The median progression free survival (PFS) was 6.6 months (95% CI 1.35–11.59). Median PFS was 11.6 months for mTORi, 6.9 for AA, and 6.6 for other targeted therapies.ConclusionPhase I trials appear as a sound therapeutic option in TENs pts progressing after standard treatments. Use of AA and mTORi seem to yield a good clinical response and warrant further investigation.     

Transfection of chimeric anti-CD138 gene enhances natural killer cell activation and killing of multiple myeloma cells

Reprogramming of NK cells with a chimeric antigen receptor (CAR) proved an effective strategy to increase NK cell reactivity and recognition specificity toward tumor cells. To enhance the cytotoxicity of NK cells against CD138-positive multiple myeloma (MM) cells, we generated genetically modified NK-92MI cells carrying a CAR that consists of an anti-CD138 single-chain variable fragment (scFv) fused to the CD3ζ chain as a signaling moiety. The genetic modification through a lentiviral vector did not affect the intrinsic cytolytic activity of NK-92MI toward human erythroleukemic cell line K562 cells or CD138-negative targets. However, these retargeted NK-92MI (NK-92MI-scFv) displayed markedly enhanced cytotoxicity against CD138-positive human MM cell lines (RPMI8226, U266 and NCI-H929) and primary MM cells at various effector-to-target ratios (E:T) as compared to the empty vector-transfected NK-92MI (NK-92MI-mock). In line with the enhanced cytotoxicity of NK-92MI-scFv, significant elevations in the secretion of granzyme B, interferon-γ and proportion of CD107a expression were also found in NK-92MI-scFv in response to CD138-positive targets compared with NK-92MI-mock. Most importantly, the enhancement in the cytotoxicity of NK-92MI-scFv did not attenuate with 10Gy-irradiation that sufficiently blocked cell proliferation. Moreover, the irradiated NK-92MI-scFv exerted definitely intensified anti-tumor activity toward CD138-positive MM cells than NK-92MI-mock in the xenograft NOD-SCID mouse model. This study provides the rationale and feasibility for adoptive immunotherapy with CD138-specific CAR-modified NK cells in CD138-positive plasmacytic malignancies, which potentially further improves remission quality and prolongs the remission duration of patients with MM after upfront chemotherapy.     

肝癌围手术期肝脏功能检测方法

肝切除术后肝衰竭是肝癌患者围手术期重要的并发症和致死原因。目前有多种检测方法（包括常规肝功能检测和肝储备功能定量检测）,综合应用能评价肝脏功能,预测肝衰竭。     

Enrichment map profiling of the cancer invasion front suggests regulation of colorectal cancer progression by the bone morphogenetic protein antagonist,gremlin-1

The cancer invasion front (CIF), a spatially-recognized area due to the frequent presence of peritumoral desmoplastic reaction, represents a cancer site where many hallmarks of cancer metastasis occur. It is now strongly suggested that the desmoplastic microenvironment holds crucial information for determining tumor development and progression. Despite extensive research on tumor-host cell interactions at CIFs, the exact paracrine molecular network that is hardwired into the proteome of the stromal and cancer subpopulations remains partially understood. Here, we interrogated the signaling pathways and the molecular functional signatures across the proteome of a desmoplastic coculture model system of colorectal cancer progression. We discovered a group of bone morphogenetic protein (BMP) antagonists that coordinates major biological programs in CIFs, including cell proliferation, invasion, migration and differentiation processes. Using a mathematical model of cancer cell progression, coupled to in vitro cell migration assays, we demonstrated that the prominent BMP antagonist gremlin-1 (GREM1) may trigger motility of cancer cell cohorts. Our data collectively demonstrate that the desmoplastic CIFs deploy a microenvironmental signature, based on BMP antagonism, in order to regulate the motogenic fates of cancer cell cohorts invading the adjacent stroma.