Thymidylate synthase gene polymorphism predicts response to capecitabine in advanced colorectal cancer

Thymidylate synthase is a target enzyme for chemotherapeutic agents such as 5-fluorouracil and capecitabine, its oral prodrug. The human thymidylate synthase gene promoter is polymorphic, having either double or triple repeats of a 28-bp sequence. It has previously been shown that colorectal cancer patients who are homozygous for the triple tandem repeats (L/L) have significantly higher thymidylate synthase mRNA expression than those homozygous for the double repeat variant (S/S). Capecitabine is converted to 5-fluorouracil by a sequential triple enzyme pathway, with the last step catalyzed by the tumor-associated angiogenic factor thymidine phosphorylase. We have recently shown that individuals with metastatic colorectal cancer treated with 5-fluorouracil have a higher response rate if they are homozygous for the genotype S/S as opposed to S/L or L/L. Our hypothesis is that individuals homozygous for the double repeat variant (S/S) should have a better response to capecitabine than their counterparts with S/L or L/L. In this retrospective pilot study we assessed the thymidylate synthase polymorphic status of 24 patients with metastatic colorectal cancer and determined their response to capecitabine. We found that 75% (3/4) of individuals with the S/S variant responded to capecitabine, compared to 8% (1/12) and 25% (2/8) of those with the S/L and L/L variants, respectively. Our data suggest that genotyping patients for the thymidylate synthase polymorphism would be useful in identifying patients who are more likely to respond to capecitabine treatment for advanced colorectal cancer.     

Thymidylate synthase and thymidine phosphorylase gene expression as predictive parameters for the efficacy of 5-fluorouracil-based adjuvant chemotherapy for gastric cancer

AIM: To investigate the prognostic role of thymidylate synthase (TS) and thymidine phosphorylase (TP) mRNA levels in T3 or T4 gastric cancer treated with 5-fluorouraci-based adjuvant chemotherapy. METHODS: Fifty-one patients with T3 or T4 gastric cancer received systemic 5-fluorouraci-based adjuvant chemotherapy, and intratumoral expression of TS and TP in 51 gastric cancer tissue samples was tested by real- time quantitative PCR. RESULTS: The median disease-free survival (DFS) time was 10.2 mo in the patients. There were no significant differences in DFS between the groups with high and low levels of TP. However, the group with low level of TS had a longer DFS (14.4 mo vs 8.3 mo, P = 0.017). The median overall survival (OS) time was 18.5 mo, and there were significant differences in OS between the groups with high and low levels of TS or TP (for TS, 17.0 mo vs 21.3 mo, P = 0.010; for TP, 16.6 mo vs 22.5 mo, P = 0.009). Moreover, the coupled low expression of these two genes was strongly associated with a longer survival time of patients as compared with that of a single gene. CONCLUSION: Expression of TS and TP mRNA is a useful predictive parameter for the survival of postoperative gastric cancer patients after 5-fluorouracil- based adjuvant chemotherapy.     

Analysis of gene amplification in human tumor cell lines.

Oncogene amplification has been observed in various primary tumors and tumor-derived cell lines. In several types of cancer, amplification of specific oncogenes is correlated with the stage of tumor progression. To estimate the frequency of gene amplification in other tumor types and to determine whether the ability to grow in vivo is associated with gene amplification in tumor cell lines, we have developed a modified version of the in-gel renaturation assay that detects human DNA sequences of unknown nature amplified as little as 7- to 8-fold. This assay was used to screen 16 cell lines derived from various solid tumors and leukemias. Amplified DNA sequences were detected in only one cell line, Calu-3 lung adenocarcinoma. This cell line was found to contain coamplified NGL (formerly termed neu) and ERBA1 oncogenes. However, when one of the amplification-negative cell lines, PC-3 prostatic carcinoma, was selected for in vivo growth in nude mice, amplified DNA sequences became detectable in these cells. The amplified sequences included the MYC oncogene, which showed no amplification in the parental cell line but was amplified 10- to 12-fold in the in vivo-selected cells. MYC amplification may, therefore, provide tumor cells with a selective advantage specific for in vivo growth.     

胸苷酸合成酶基因多态性与晚期非小细胞肺癌化疗敏感性的关系

目的探讨胸苷酸合成酶（TS）基因多态性与晚期非小细胞肺癌（NSCLC）患者对铂类为基础的化疗敏感性的关系。方法 101例晚期NSCLC患者采用顺铂或卡铂为主的方案化疗,2~3个周期后进行临床疗效评价。应用PCR方法分析患者TS 5′-非翻译区（UTR）基因多态性,以非条件logistic回归模型分析不同基因型与化疗敏感性的关系。结果 101例患者的总有效率为30.7%。携带TS 5′-UTR 2R等位基因患者的化疗敏感性是携带TS 5′-UTR 3R/3R基因型患者的1.54倍（95%CI=0.65~3.68,P=0.331）,但无统计学差异。携带3R/3R基因型患者粒细胞减少和恶心、呕吐的化疗毒性反应程度低于3R/2R和2R/2R基因型患者（P〈0.05）。结论 TS 5′-UTR基因可能不是NSCLC铂类药物化疗的独立敏感性预测因子。     

抑瘤基因NGX6对结肠癌血管形成的影响

目的:探讨抑瘤基因NGX6对结肠癌血管形成的影响.方法:将HT-29细胞分为3组:pcDNA3.1(+)/NGX6/HT-29组(稳定转染并表达NGX6基因的HT-29细胞)、pcDNA3.1(+)/HT-29组(转染空白质粒的HT-29细胞)及HT-29对照组.通过鸡胚绒毛尿囊膜血管形成实验检测结肠癌细胞接种鸡胚血管形成情况.裸鼠成瘤实验观测种植瘤及其对裸鼠的影响,然后对种植瘤进行免疫组织化学实验检测其内部血管生成.RT-PCR检测NGX6基因及VEGF基因在各组细胞中的表达.结果:pcDNA3.1(+)/NGX6/HT-29组鸡胚血管形成平均数量较HT-29与pcDNA3.1(+)/HT-29组明显减少(6.2±0.2 vs 8.6±0.2,8.4±0.3,P<0.05):HT-29组裸鼠明显消瘦且PcDNA3.1(+)/NGX6/H T-29组与HT-29和pcDNA3.1(+)/HT-29组种植瘤平均质量、种植瘤微血管密度明显降低(1.83±0.25 g vs 4.06±0.20 g,4.16±0.4 g;1.83±0.52 vs 7.36±1.12 vs 6.96±1.43,均P<0.05); VEGF在pcDNA3.1(+)/NGX6/HT-29细胞及种植瘤中较其余两组明显降低,而pcDNA3.1(+)/HT-29细胞与未转染的HT-29细胞与种植瘤中呈高表达;且HE染色切片显示HT-29组及PcDNA3.1(+)/HT-29组种植瘤血管内都有癌栓,而PcDNA3.1(+)/NGX6/HT-29组种植瘤内未见有癌栓形成.结论:NGX6基因能抑制结肠癌细胞HT-29血管的形成,抑瘤基因NGX6的抑癌能力部分是通过抑制血管形成来实现的.     

A tumor hypoxic niche protects human colon cancer stem cells from chemotherapy

Purpose

Hypoxia has been found to play an important role in regulating the biological characteristics of cancer stem cells (cCSCs). In this study, we tested whether a tumor hypoxic niche serves to the chemotherapeutic resistance of colon cCSCs.

Methods

Each of 23 fresh samples of human colon adenocarcinoma was transplanted into nude mice. The tumor-bearing mice randomly and equally received (A) saline, (B) 5-fluorouracil (15 mg/kg), (C) oxaliplatin (10 mg/kg), and (D) oxaliplatin plus 5-fluorouracil when xenografts reached 250 mm³ (n = 10). After 2-week treatment, tumor cells were quantified by flow cytometry for expression of CD133 and the hypoxic proportion of CD133⁺ and CD133^? cells which were also sorted and detected for ki67 and pimonidazole via immunofluorescence.

Results

The hypoxic subpopulation of CD133⁺ and CD133^? cells was 66.5 and 26.4 %, respectively. Although there was no marked change for the hypoxic subpopulation of CD133⁺ cells after treatment, the hypoxic fraction of proliferative CD133⁺ cells was increased by 14.62, 16.45, and 20.46 % in groups B, C, and D, respectively. Furthermore, proliferative cells in CD133⁺ and CD133^? cells were reduced by 29.93 and 62.5 % in group C, and by 25.26 and 68.22 % in group D; in group B, however, the proliferative CD133⁺ cells were increased by 37.09 %; the CD133^? cells were unchanged.

Conclusions

Most CD133⁺ cCSCs are located in a hypoxic niche, where cCSCs are better at retaining proliferating property under chemotherapy. Oxaliplatin, rather than 5-FU, inhibits proliferation of cCSCs, which may be the mechanism underlying a better outcome by oxaliplatin in colon cancer patients.     

Ectopic fascioliasis mimicking a colon tumor

Fasciola hepatica, a leaf shaped trematode that is common in cattle, sheep and goats, is acquired by eating raw water plants like watercress or drinking water infected with the encysted form of the parasite. The varied clinical presentations of fascioliasis still make a high index of suspicion mandatory. Besides having a wide spectrum of hepatobiliary symptoms like obstructive jaundice, cholangitis and liver cirrhosis, the parasitic infection also has extrabiliary manifestations. Until recently, extrahepatic fascioliasis has been reported in the subcutaneous tissue, brain, lungs, epididymis, inguinal lymph nodes, stomach and the cecum. In this report, a strange manifestation of the fasciola infection in a site other than the liver, a colonic fascioliasis, is presented.     

Thymidylate synthase expression in primary colorectal tumours is correlated with its expression in metastases

Objective. Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides and as such a critical target for fluoropyrimidines, which are widely used in the treatment of colorectal cancer (CRC). The purpose of this study was to investigate TS expression in the primary tumours (PTs) and their metastases (M) in advanced CRC. Material and methods. TS expression was determined immunohistochemically in paraffin-embedded biopsies of PT-M pairs in 39 CRC patients, as related to the clinical data. Results. There was no difference in the mean TS index of PTs compared with that of M, 1.25 and 1.14, respectively (p=0.12). TS expression of PTs was above the mean more often than that of M (61.5% and 41.0%, respectively, p=0.035). High TS expression in PTs was significantly related to high expression in M (the Fisher exact test, p=0.001). Using the absolute index values, TS expression in PT and M was significantly correlated (Pearson R=0.501, p=0.001). In 29/39 (74.3%) pairs, PT and M had concordant expression levels (Cohen's kappa 0.508, 95% CI 0.260–0.756, p=0.001; intraclass correlation coefficient (ICC) = 0.679, 95% CI 0.358–0.836, p=0.0001). No significant association was found between TS expression and any of the clinicopathological variables, disease outcome (DFS, DSS) or its response to treatment in univariate or multivariate analysis. Conclusions. Albeit usually higher, TS expression in PT was closely correlated with TS expression in M. This suggests that measurement of TS in primary CRC accurately predicts TS expression in subsequent metastases, which may help in selecting those patients most likely to respond to 5-FU-based regimens.     

Polymorphism of thymidylate synthase gene associated with its protein expression in human colon cancer

AIM： To correlate the polymorphisms in the 5＇-untranslated region with thymidylate synthase （TS） protein expression in Han Chinese colonic neoplasms. METHODS： Adenocarcinoma samples were from 68 patients who received no treatment before surgery. Tandem repeat length of TS gene was determined by PCR amplification of genomic DNA. Intratumoral TS protein expression was studied immunohistochemically in corresponding sections from paraffin-embedded primary loci. Immunoreactivity was semiquantitatively evaluated by immunoreactivity score （IRS）. RESULTS： Double-（2R） and triple-repeated （3R） sequences of the TS gene were found in the cancer tissues. Three genotypes of TS were found： 2R/2R （n = 6）, 2R/3R （n = 22） and 3R/3R （n = 40）. Patients who were homozygous for triple-repeated （3R/3R） sequences showed significantly higher IRS of TS than patients who were homozygous for double-repeated （2R/2R） sequences or heterozygous patients （2R/3R）： 5.73 ±3.25 vs 2.17 ± 1.47 or 3.77 ±2.64, P = 0.008 or P = 0.015. But no statistical significance of IRS in cancer tissues was observed between 2R/3R genotype and 2R/2R genotype. CONCLUSION： There is a relationship between TS genotype and TS protein expression in clinical specimens. The data might offer an advantage for selection of Chinese cancer patients to receive fluoropyrimidines treatment.     

Thymidylate synthase expression in primary colorectal tumours is correlated with its expression in metastases

OBJECTIVE: Thymidylate synthase (TS) is the rate-limiting enzyme in the synthesis of pyrimidine nucleotides and as such a critical target for fluoropyrimidines, which are widely used in the treatment of colorectal cancer (CRC). The purpose of this study was to investigate TS expression in the primary tumours (PTs) and their metastases (M) in advanced CRC. MATERIAL AND METHODS: TS expression was determined immunohistochemically in paraffin-embedded biopsies of PT-M pairs in 39 CRC patients, as related to the clinical data. RESULTS: There was no difference in the mean TS index of PTs compared with that of M, 1.25 and 1.14, respectively (p=0.12). TS expression of PTs was above the mean more often than that of M (61.5% and 41.0%, respectively, p=0.035). High TS expression in PTs was significantly related to high expression in M (the Fisher exact test, p=0.001). Using the absolute index values, TS expression in PT and M was significantly correlated (Pearson R=0.501, p=0.001). In 29/39 (74.3%) pairs, PT and M had concordant expression levels (Cohen's kappa 0.508, 95% CI 0.260-0.756, p=0.001; intraclass correlation coefficient (ICC) = 0.679, 95% CI 0.358-0.836, p=0.0001). No significant association was found between TS expression and any of the clinicopathological variables, disease outcome (DFS, DSS) or its response to treatment in univariate or multivariate analysis. CONCLUSIONS: Albeit usually higher, TS expression in PT was closely correlated with TS expression in M. This suggests that measurement of TS in primary CRC accurately predicts TS expression in subsequent metastases, which may help in selecting those patients most likely to respond to 5-FU-based regimens.     

Molecular analysis of sentinel lymph node in colon carcinomas by one-step nucleic acid amplification (OSNA) reduces time to adjuvant chemotherapy interval

BackgroundThe interval between surgery and adjuvant chemotherapy (AC) is a predictive factor of survival in high-risk colon cancer (CC). This study aimed to evaluate the impact of intraoperative sentinel lymph node (SLN) analysis using the one-step nucleic acid amplification (OSNA) technique on the time interval between surgery and AC.MethodsWe performed a prospective study analyzing 56 consecutive patients who had surgery for CC between July 2012 and October 2014, including 20 patients needing AC. SLN status was determined intraoperatively in 17 patients in the OSNA group; when positive, a portacath (PAC) was placed during the procedure for upcoming AC. In the remaining patients, we proceeded without SLN status determination and the PAC was installed after definitive histopathological analysis of the specimen if needed.ResultsThere was no difference between the groups regarding cancer staging, duration of hospitalization (7.5 days in the OSNA group and 10 days in the control group, p = 0.43) and major complications (20% vs 30% respectively, p = 0.55). The time interval between surgery and adjuvant chemotherapy was significantly shorter in the OSNA group at 35 (±8) days vs 67 (±36) days (p = 0.021).ConclusionSLN status determination by the OSNA technique is safe, feasible and could significantly reduce time between surgery and adjuvant chemotherapy in a pilot study.     

New perspectives in colon cancer chemotherapy

Camptothecin and some of its derivatives, all inhibitors of topoisomerase I, have been found to inhibit growth and induce regression of human colon carcinoma xenografts in nude mice. Some clinical trials of these compounds have been already completed, many more are being held. The mother compound, camptothecin, which is water insoluble, has been administered orally in a Phase I clinical trial. Main toxicity encountered has been diarrhea with minimal leukocytopenia. Camptothecin is now in Phase 2 clinical trials. 9-Aminocamptothecin, a water-insoluble derivative, is now in Phase I trials. Topotecan and iridotecan, two water-soluble derivatives, have undergone Phase I trials, showing mostly intestinal toxicity, followed by bone marrow depression.     

Squamous-cell carcinoma of the colon responsive to combination chemotherapy

PURPOSE: The majority of colorectal neoplasms diagnosed are adenocarcinomas. Other histologies such as squamous, adenosquamous, carcinoid tumors, or lymphoid tumors are occasionally identified. Given the rarity of squamous-cell tumors, it is very difficult to study their natural course and response to therapy. An attempt is made to describe the frequency, anatomic location, and response to therapy with a review of the literature. METHODS: From the Cancer Registry at the University of Missouri-Columbia Ellis Fischel Cancer Center, tumors of the colon identified above the dentate line were selected for chart review. Data were extracted from cases between the years 1940 and 1996. The key terms used to identify cases were epidermoid, squamous cell, and cancer of the rectum or colon. Using this approach, forty patients were identified and each record was reviewed. RESULTS: The majority of these cases were anal cancers with proximal extension into the rectum and were excluded. Of 4,561 cases of epithelial colon and rectal cancers identified, only one additional case of squamouscell cancer could be verified. In this report we describe a patient with a primary squamous-cell carcinoma of the sigmoid colon with metastatic disease to the liver at diagnosis who responded to systemic chemotherapy. We believe this to be the first reported case of this rare tumor type in which the patient's tumor responded to systemic chemotherapy. Two cases with a thorough review of literature are presented. CONCLUSIONS: Primary squamous-cell carcinoma of the colon is a rare malignancy of unknown cause and pathogenesis. Metastatic tumors to the colon should be ruled out in all cases before therapy. Early detection and surgery remain the main therapeutic options, but as presented in our case, response to chemotherapy in advanced disease is encouraging.     

Mammalian cells resistant to tumor suppressor genes.

Expression of p53 causes growth arrest or apoptosis in many normal and neoplastic cell types, but the relationship between these two effects has remained obscure. To begin to dissect the underlying mechanisms at a genetic level, we have generated mutant cells resistant to the action of wild-type p53. Rat embryo fibroblasts transformed with ras and a temperature-sensitive p53 (tsp53(135val)) gene were chemically mutagenized and selected for growth at a temperature at which p53 adopts a wild-type conformation (31.5 degrees C). Clones that grew exponentially at 31.5 degrees C were selected. Cell fusion experiments demonstrated that the mutations conferring resistance to p53-mediated growth arrest were dominant. The mutagenized clones were resistant not only to p53-mediated growth arrest, but also to the apoptosis induced by E1A in conjunction with p53, and partially resistant to the retinoblastoma tumor suppressor, pRB. The results suggest that a single downstream pathway can control the induction of growth arrest and apoptosis, and that both p53 and RB function through this pathway.     

化疗对结肠癌Th1和Th2类细胞因子基因表达的影响及意义

目的探讨结肠癌患者体内Th1和Th2类细胞因子的基因表达水平及化疗对其表达的影响。方法收集67例结肠癌患者、35例正常人和30例结肠息肉患者血清。采用RT-PCR技术,检测3组及结肠癌组化疗后外周血单个核细胞(PBMC)Th1和Th2类细胞因子mRNA表达水平。结果正常对照及结肠息肉组Th1和Th2类细胞因子基因表达呈阴性或基本不表达。结肠癌组IL-4、IL-6、IL-10mRNA表达水平显著高于正常对照及结肠息肉组(P<0.05),且与结肠癌的分化程度及临床分期相关(P<0.05);IFN-γ无阳性表达(0/67),IL-2则仅有3例阳性表达,其表达与结肠癌的分化程度及临床分期无相关性(P>0.05)。化疗后Th1类细胞因子表达增强,Th2类细胞因子的表达则减弱(P<0.05)。结论结肠癌患者Th1和Th2类细胞因子表达失衡,细胞免疫受抑,化疗可使结肠癌患者Th2类细胞因子的强势表达向Th1类逆转。     

Rapid PCR amplification of minimal enediyne polyketide synthase cassettes leads to a predictive familial classification model

A universal PCR method for the rapid amplification of minimal enediyne polyketide synthase (PKS) genes and the application of this methodology to clone remaining prototypical genes from producers of structurally determined enediynes in both family types are presented. A phylogenetic analysis of the new pool of bona fide enediyne PKS genes, consisting of three from 9-membered producers (neocarzinostatin, C1027, and maduropeptin) and three from 10-membered producers (calicheamicin, dynemicin, and esperamicin), reveals a clear genotypic distinction between the two structural families from which to form a predictive model. The results from this study support the postulation that the minimal enediyne PKS helps define the structural divergence of the enediyne core and provides the key tools for generating enediyne hybrid genes/molecular scaffolds; by using the model, a classification is also provided for the unknown enediyne PKS genes previously identified via genome scanning.     

肿瘤易感基因101在胃癌耐药细胞中的表达及作用

目的 探讨肿瘤易感基因(TSG)101在多药耐药胃癌细胞中的表达和作用。方法 应用半定量RT-PCR和Western blot方法观察TSG101在胃癌细胞SGC7901及其长春新碱(VCR)耐药亚系SGC7901／VCR中的表达。将TSG101真核表达载体转染SGC7901细胞，通过Western blot方法鉴定转染细胞TSG101蛋白的表达。用流式细胞仪检测细胞周期的变化和细胞内阿霉素(ADR)的平均荧光强度。用MTT法检测细胞生长曲线和对VCR、ADR的药敏性。结果 胃癌耐药细胞SGC7901／VCR与亲本细胞SGC7901相比，TSG101的表达明显增高。SGC7901细胞转染TSG101真核表达载体后其表达较对照细胞明显增高，G1期细胞比例减少而S期比例增加，细胞增殖加快，且对VCR、ADR的敏感性减低，细胞内阿霉素蓄积和潴留减少。结论 TSG101真核表达载体转染SGC7901后其耐药性明显增强，提示TSG101在胃癌多药耐药中发挥了一定的作用。