Phase II Trial of Sorafenib in Patients with Chemotherapy Refractory Metastatic Esophageal and Gastroesophageal (GE) Junction Cancer

Purpose

Vascular endothelial growth factor receptor (VEGFR2) directed therapies result in a modest survival benefit for patients with advanced esophageal and gastroesophageal (GE) junction cancer. Platelet-derived growth factor receptor (PDGFR) may contribute to escape from VEGFR2 inhibition. We evaluated the efficacy of sorafenib, a broad spectrum tyrosine kinase inhibitor targeting VEGFR2 and PDGFR as well as RET and RAF1, in patients with metastatic chemotherapy refractory esophageal and GE junction cancer.

Patients and Methods

This phase II trial of sorafenib 400 mg twice daily enrolled chemotherapy refractory patients with metastatic esophageal and GE junction cancer with primary endpoint of progression-free survival (PFS) rate at two months. Secondary endpoints included overall survival, objective response rate and toxicity.

Results

Among 34 patients, 8 week Kaplan-Meier estimated PFS was 61% (90%CI 45 to 73%). Median PFS is 3.6 months (95% CI 1.8 to 3.9 months), with median overall survival OS 9.7 months (95% CI 5.9 to 11.6 months). Grade 3 toxicities were uncommon and included hand foot skin reaction, rash, dehydration and fatigue. One patient (3%) with ongoing complete response and remains on trial for over 5 years. Whole exome sequencing of this tumor revealed mutations in many cancer-associated genes including ARID1A, PIK3CA, and TP53, and focal amplifications of HMGA2 and MET.

Conclusion

Sorafenib therapy results in disease stabilization and encouraging PFS in patients with refractory esophageal and GE junction cancer.

Trial Registration

ClinicalTrials.gov NCT00917462     

Polymorphism of the Thymidylate Synthase Gene and Thymidylate Synthase Levels in Colon Cancer Cell Lines and Different Tissues of Colorectal Cancer Patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Probing the Thymidylate Synthase Active site with Bisubstrate Analog Inhibitors

Abstract

Earlier reports on the enhancement of folate analog binding to thymidylate synthase (TS) by N¹⁰-propargylation prompted the synthesis of a bisubstrate analog containing an 8-deaza-N¹⁰-propargyl-5,6,7,8-tetrahydrofolate moiety coupled through a methylene linkage to 2′-deoxyuridylate.     

Optimal Duration of Fluorouracil-Based Adjuvant Chemotherapy for Patients with Resectable Gastric Cancer

Background

Although several clinical trials have suggested that postoperative adjuvant chemotherapy can improve survival of patients with gastric cancer, the optimal treatment duration has not been studied. This retrospective analysis evaluated the outcomes of patients with gastric cancer treated with six cycles of fluorouracil-based treatment compared with a cohort treated with four or eight cycles.

Methods

We retrospectively identified 237 patients with stage IB–IIIC gastric cancer who received four, six, or eight cycles of fluorouracil-based adjuvant chemotherapy administered every 3 weeks after radical gastrectomy. The endpoint was overall survival (OS). Factors associated with prognosis were also analyzed.

Results

The estimated 3-year OS rates for the four-, six-, and eight-cycle cohorts were 54.4%, 76.1%, and 68.9%, respectively; and the estimated 5-year OS rates were 41.2%, 74.0%, and 65.8%, respectively. Patients who received six cycles were more likely to have a better OS than those who received four cycles (P = 0.002). Eight cycles failed to show an additional survival benefit (P = 0.454). In the multivariate analysis, the number of chemotherapy cycles was associated with OS independent of clinical covariates (P<0.05). Subgroup analysis suggested that among patients in all age groups examined, male patients, and subgroups of fluorouracil plus oxaliplatin combined chemotherapy, stage III, poor differentiation, and gastrectomy with D2 lymphadenectomy, six cycles of adjuvant chemotherapy were associated with a statistically significant benefit of OS compared with four cycles (P<0.05).

Conclusions

Six cycles of adjuvant chemotherapy might lead to a favorable outcome for patients with gastric cancer, and two further cycles could not provide an additional clinical benefit.     

Computed Tomography (CT) Perfusion as an Early Predictive Marker for Treatment Response to Neoadjuvant Chemotherapy in Gastroesophageal Junction Cancer and Gastric Cancer - A Prospective Study

Objectives

To evaluate whether early reductions in CT perfusion parameters predict response to pre-operative chemotherapy prior to surgery for gastroesophageal junction (GEJ) and gastric cancer.

Materials and Methods

Twenty-eight patients with adenocarcinoma of the gastro-esophageal junction (GEJ) and stomach were included. Patients received three series of chemotherapy before surgery, each consisting of a 3-week cycle of intravenous epirubicin, cisplatin or oxaliplatin, concomitant with capecitabine peroral. The patients were evaluated with a CT perfusion scan prior to, after the first series of, and after three series of chemotherapy. The CT perfusion scans were performed using a 320-detector row scanner. Tumour volume and perfusion parameters (arterial flow, blood volume and permeability) were computed on a dedicated workstation with a consensus between two radiologists. Response to chemotherapy was evaluated by two measures. Clinical response was defined as a tumour size reduction of more than 50%. Histological response was evaluated based on residual tumour cells in the surgical specimen using the standardized Mandard Score 1 to 5, in which values of 1 and 2 were classified as responders, and 3 to 5 were classified as nonresponders.

Results

A decrease in tumour permeability after one series of chemotherapy was positively correlated with clinical response after three series of chemotherapy. Significant changes in permeability and tumour volume were apparent after three series of chemotherapy in both clinical and histological responders. A cut-off value of more than 25% reduction in tumour permeability yielded a sensitivity of 69% and a specificity of 58% for predicting clinical response.

Conclusion

Early decrease in permeability is correlated with the likelihood of clinical response to pre-operative chemotherapy in GEJ and gastric cancer. As a single diagnostic test, CT Perfusion only has moderate sensitivity and specificity in response assessment of pre-operative chemotherapy making it insufficient for clinical decision purposes.     

Molecular Marker Identification for Relapse Prediction in 5-FU-Based Adjuvant Chemotherapy in Gastric and Colorectal Cancers

To confirm the clinical significance of NF-κB and JNK protein expression from experimentally identified candidates for predicting prognosis for patients with 5-FU treatment, we evaluated the protein expression of surgically removed specimens. A total of 79 specimens were obtained from 30 gastric and 49 colorectal cancer patients who underwent R0 resection followed by postoperative 5-FU based adjuvant chemotherapy. Immunohistochemical examinations of NF-κB and JNK on tissue microarrays (TMAs) revealed that significantly shorter time-to-relapse (TTR) in both NF-κB(+) and JNK(−) subgroups in both gastric (NF-κB(+), p = 0.0002, HR11.7. 95%CI3 3.2–43.4; JNK(−), p = 0.0302, HR4.4, 95%CI 1.2–16.6) and colon (NF-κB(+), p = 0.0038, HR36.9, 95%CI 3.2–426.0; JNK(−), p = 0.0098, HR3.2, 95%CI 1.3–7.7) cancers. These protein expression patterns also show strong discriminately power in gastric cancer patients for overall survival rate, suggesting a potential utility as prognostic or chemosensitivity markers. Baseline expression of these proteins using gastric cancer cell lines demonstrated the reciprocal patterns between NF-κB and JNK, while 5-FU exposure of these cell lines only induced NF-κB, suggesting that NF-κB plays a dominant role in the response to 5-FU. Subsequent siRNA experiments confirmed that gene knockdown of NF-κB increased 5-FU-specific sensitivity, whereas that of JNK did not affect the chemosensitivity. These results suggest that the expression of these proteins may aid in the decisions involved with adjuvant chemotherapy for gastrointestinal tract cancers.     

Association of Thymidylate Synthase Polymorphisms with Acute Pancreatitis and/or Peripheral Neuropathy in HIV-Infected Patients on Stavudine-Based Therapy

Background

Low expression thymidylate synthase (TS) polymorphism has been associated with increased stavudine triphosphate intracellular (d4T-TP) levels and the lipodystrophy syndrome. The use of d4T has been associated with acute pancreatitis and peripheral neuropathy. However, no relationship has ever been proved between TS polymorphisms and pancreatitis and/or peripheral neuropathy.

Methods

We performed a case-control study to assess the relationship of TS and methylene-tetrahydrofolate reductase (MTHFR) gene polymorphisms with acute pancreatitis and/or peripheral neuropathy in patients exposed to d4T. Student’s t test, Pearson’s correlations, one-way ANOVA with Bonferroni correction and stepwise logistic regression analyses were done.

Results

Forty-three cases and 129 controls were studied. Eight patients (18.6%) had acute pancreatitis, and 35 (81.4%) had peripheral neuropathy. Prior AIDS was more frequent in cases than in controls (OR = 2.36; 95%CI 1.10–5.07, P = 0.0247). L7ow expression TS and MTHFR genotype associated with increased activity were more frequent in patients with acute pancreatitis and/or peripheral neuropathy than in controls (72.1% vs. 46.5%, OR = 2.97; 95%CI: 1.33–6.90, P = 0.0062, and 79.1% vs. 56.6%, OR = 2.90, 95%CI: 1.23–7.41, P = 0.0142, respectively). Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; OR_A+B = 0.34 [95%CI: 0.08 to 1.44], OR_B+A = 3.38 [95%CI: 1.33 to 8.57], OR_B+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm³ (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018).

Conclusions

Low expression TS plus a MTHFR genotype associated with increased activity is associated with the development of peripheral neuropathy in d4T-exposed patients.     

Difference in Ki67 and Thymidylate Synthase Expression in Primary Tumour Compared with Metastatic Nodes in Breast Cancer Patients

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.     

RNP免疫沉淀技术在斑马鱼胸苷酸合成酶基因翻译调控研究中的应用

为检测斑马鱼胸苷酸合成酶基因的表达产物TS蛋白在体内是否结合于自身的mRNA,并进一步检测TS蛋白与c-myc mRNA的结合情况。采用免疫沉淀结合得到相应的RNA,即RNP免疫沉淀技术,进一步结合RT-PCR技术分析与蛋白结合的核酸序列,并用Western蛋白印迹分析了TS蛋白。结果显示,人的TS106单克隆抗体可以免疫沉淀斑马鱼的TS蛋白,抗体免疫沉淀的核酸中含有TS mRNA和c-myc mRNA。在斑马鱼中,TS蛋白在体内结合于自身的TS mRNA,并能结合c-myc mRNA,参与基因的表达调控,阐明了TS的翻译调控机制在体内条件下的作用方式,同时也为构建斑马鱼抗肿瘤药理模型提供了理论基础。     

LC3B,mTOR, AMPK Are Molecular Targets for Neoadjuvant Chemotherapy in Gastric Cancers

Autophagy plays a dual role in oncogenesis processes. On one hand, autophagy enhances the cell resistance to oncogenic factors, and on the other hand, it participates in the tumor progression. The aim of the study was to find the associations between the effectiveness of the FLOT regimen in resectable gastric cancers (GCs) with the key autophagy-related proteins. Materials and Methods: The study included 34 patients with morphologically verified gastric cancer. All patients had FLOT neoadjunvant chemotherapy (NACT) (fluorouracil, leucovorin, oxaliplatin, and docetaxel) followed by gastrectomy. The studied tissue material was the non-transformed and tumor tissues obtained during diagnostic video gastroscopy in patients before the start of the combined treatment and after surgical treatment, frozen after collection. The LC3B, mTOR, and AMPK expression was determined by real-time PCR. The content of the LC3B protein was determined by Western blotting analysis. Results: The mRNA level and the content of the LC3B protein were associated with the tumor stage and the presence of signet ring cells. The AMPK mRNA level was increased in patients with the T4N0-2M0 stage by 37.7 and 7.33 times, which was consequently compared with patients with the T2N0M0 and T3N0-1M0 stages. The opposite changes in the mTOR and AMPK in the GCs before anti-cancer therapy were noted. The tumor size and regional lymph node affections were associated with a decrease in the mTOR mRNA level. A decrease in the mTOR expression was accompanied by an increase in the AMPK expression in the GCs. The mTOR expression was reduced in patients with a cancer spreading; in contrast, AMPK grew with the tumor size. There was an increase in the LC3B expression, which can probably determine the response to therapy. An increase in LC3B mRNA before the start of treatment and the protein content in cancers after NACT with a decrease in therapy effectiveness was recorded. There was an increase in the protein level in patients with partial regression and stabilization by 3.65 and 5.78 times, respectively, when compared with patients with complete tumor regression was noted. Conclusions: The anticancer effectiveness in GCS is down to the LC3B, mTOR, and AMPK expression. These were found to be entire molecular targets affecting the cancer progression and metastasis as well as the NACT effectiveness.     

Indirect Clinical Validation of a Programmed Death-Ligand 1 Laboratory-Developed Test for Gastric/Gastroesophageal Junction Adenocarcinoma with 22C3 Antibody Concentrate

Molecular Diagnosis & Therapy - The PD-L1 IHC 22C3 pharmDx used on the Dako Autostainer Link 48 (ASL48) staining platform is an established method for assessing programmed death-ligand 1...     

A Fatal Combination: A Thymidylate Synthase Inhibitor with DNA Damaging Activity

2′-deoxy-5-ethynyluridine (EdU) has been previously shown to be a cell poison whose toxicity depends on the particular cell line. The reason is not known. Our data indicates that different efficiency of EdU incorporation plays an important role. The EdU-mediated toxicity was elevated by the inhibition of 2′-deoxythymidine 5′-monophosphate synthesis. EdU incorporation resulted in abnormalities of the cell cycle including the slowdown of the S phase and a decrease in DNA synthesis. The slowdown but not the cessation of the first cell division after EdU administration was observed in all of the tested cell lines. In HeLa cells, a 10 μM EdU concentration led to the cell death in the 100% of cells probably due to the activation of an intra S phase checkpoint in the subsequent S phase. Our data also indicates that this EdU concentration induces interstrand DNA crosslinks in HeLa cells. We suppose that these crosslinks are the primary DNA damage resulting in cell death. According to our results, the EdU-mediated toxicity is further increased by the inhibition of thymidylate synthase by EdU itself at its higher concentrations.     

Thymidylate Synthase Polymorphism and Microsatellite Instability: Association in Colorectal Cancer

5‐Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5′ and 3′untraslated region (5′–3′ UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5′UTR) is more frequently associated with MSI + and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5′UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5′ UTR) genotypes with the MSI + and MSI ?, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI +, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

DNA甲基化转移酶抑制剂与胸苷酸合成酶抑制剂体外联合用药的抗肿瘤增效作用

通过在体外分别对DNA甲基化转移酶（DNA methyhransferase,DNMT）抑制剂5-氮杂胞苷（5-azacytidine,5-aza—C）和新型胸苷酸合成酶抑制剂盐酸洛拉曲克（nolatrexed dihydrochloride．Nolatrexed）联合用药于人大肠癌细胞LoVo和人肝癌细胞Hep3B的相互作用性质的观察,探讨DNMT抑制剂和胸苷酸合成酶抑制剂联合用药的可能性．使用MTT法测定二者单独用药或联合用药的抗肿瘤活性,用抑制浓度的分数之和（sum of fractional inhibitory concentration,SFIC）值及等效剂量分析方法（isobologram）评价联合用药的作用性质．结果显示,联合用药时其SFIC值均小于或等于1,由此得到的等效剂量曲线图形表现为凹形．可见,5-aza—C和Nolatrexed体外联合用药抗肿瘤相互作用性质为明显的增效作用,DNMT抑制剂和胸苷酸合成酶抑制剂联合用药达到抗肿瘤增效作用是可行的．     

晚期胃肠癌患者化疗后血红蛋白水平与临床疗效的相关性分析

目的:本研究通过回顾性分析晚期胃肠癌患者化疗后血红蛋白(Hb)水平与其临床疗效的关系,为临床治疗提供依据。方法:选择2009年1月~2014年12月我院收治的晚期胃肠癌患者共85例,采用回顾性调查方法,对患者进行1~5年的随访,分别对患者化疗前后贫血发生情况、Hb水平与临床疗效的关系、Hb水平与患者平均生存时间的关系进行分析。结果:化疗后患者发生贫血的比例(78.82%)明显高于化疗前(48.24%),差异有统计学意义(P0.05);PR组和SD组患者化疗前后Hb值的差异无统计学意义(P0.05);化疗前PD组患者Hb值显著高于化疗后,差异有统计学意义(P0.05);Hb水平越高,患者的平均生存时间越长,Hb的水平和患者的生存时间呈正相关。结论:晚期胃肠癌患者化疗后贫血发生率增加,化疗后Hb水平的变化与患者的预后和生存时间均存在相关关系,对患者进行常规治疗的同时采取相应措施纠正患者贫血症状,有利于改善晚期胃肠癌患者的预后,延长患者的生存时间。     

胃食管反流病患者餐后近端胃内酸分布及与食管酸暴露的关系

目的:探讨餐后胃食管反流病(GERD)病人近端胃内酸度的分布状态及其和食管酸暴露的相关性。方法:抽选我院12例GERD患者,应用3级锑电极对定位于LES上缘近侧5 cm(食管)和LES上缘远侧5 cm的贲门下(近端胃内)、LES上缘远侧10cm的近端胃远侧(近端胃内)进行pH监测,监测时间为空腹1 h和餐后4 h,同期抽选健康志愿者12例为对照组,计算两组患者食管酸暴露以及胃内整合酸度(IA)。结果:两组空腹时近端胃内IA和食管酸暴露比较无显著性差异(P0.05);对照组中,试检者餐后1、2、3、4 h贲门下IA均显著低于近端胃远侧部位(P0.05),但GERD组中IA部位差异不明显(P0.05);餐后2 h,两组近端胃内IA均有所回升,但是对照组未超过基线(P0.05),而GERD组明显高于基线水平(P0.05);两组食管酸暴露均主要在餐后2h发生,并且两组比较差异显著(P0.05);在餐后各时段,两组中食管酸暴露与IA均无显著相关性。结论:GERD餐后晚期近端胃酸分泌增高,扩大了酸性近端胃池,可部分解释GERD进食后食管过度酸暴露。     

A New Look at the Molecular Mechanism of Thymidylate Synthase and Its Interaction with Nucleotide Analogues

Abstract

The increasing availability of the crystal structures of a variety of complexes of thymidylate synthase¹ (TS) and its mutants with nucleotide and folate analogues, has greatly advanced our understanding of the mechanism of catalysis, and the detailed interaction of the enzyme with inhibitors.     

The Reliability of Endoscopic Biopsies in Assessing HER2 Status in Gastric and Gastroesophageal Junction Cancer: A Study Comparing Biopsies with Surgical Samples

AIM: The aim of this study is to validate the accuracy of HER2 assessment on biopsies by comparing matched biopsy/surgical material from the same patients. METHODS: HER2 status was evaluated by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in 103 cases of gastric and gastroesophageal junction cancers in coupled biopsy and surgical material. RESULT: Complete concordance between IHC and FISH results on biopsy versus surgical samples was noted in 80% and 95% of cases, respectively. At comprehensive comparison, including IHC and FISH data on biopsy and surgical samples, 89% of biopsies were predictive of HER2 status in surgical samples, whereas 11% showed variable inconsistencies. The majority of these (10 of 12 cases) showed IHC score 0/1+ on biopsy but were all IHC positive and amplified at surgery; in particular, three (3 of 35; 8.5%) IHC score 0 and four (4 of 16; 25%) IHC score 1+ cases were FISH amplified on biopsy material also, whereas the remaining three cases were FISH non-amplified on biopsy. The percentage of cases, which were FISH amplified with IHC score 1+ or 2+ on biopsies, were similar (25% and 33%, respectively) and they also shared a similar grade of amplification. These data suggest that both IHC score 1+ and 2+ on biopsy material represent “equivocal cases” that may merit further investigation. CONCLUSIONS: The predictive value of HER2 IHC in biopsies is high. FISH analysis should be considered for IHC score 2+ and 1+ biopsy cases. Approximately 8% of cases will not be accurately predicted by biopsy evaluation.     

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.