Randomized phase II/III clinical trial of elpamotide for patients with advanced pancreatic cancer: PEGASUS‐PC Study

Gemcitabine is a key drug for the treatment of pancreatic cancer; however, with its limitation in clinical benefits, the development of another potent therapeutic is necessary. Vascular endothelial growth factor receptor 2 is an essential target for tumor angiogenesis, and we have conducted a phase I clinical trial using gemcitabine and vascular endothelial growth factor receptor 2 peptide (elpamotide). Based on the promising results of this phase I trial, a multicenter, randomized, placebo‐controlled, double‐blind phase II/III clinical trial has been carried out for pancreatic cancer. The eligibility criteria included locally advanced or metastatic pancreatic cancer. Patients were assigned to either the Active group (elpamotide + gemcitabine) or Placebo group (placebo + gemcitabine) in a 2:1 ratio by the dynamic allocation method. The primary endpoint was overall survival. The Harrington–Fleming test was applied to the statistical analysis in this study to evaluate the time‐lagged effect of immunotherapy appropriately. A total of 153 patients (Active group, n = 100; Placebo group, n = 53) were included in the analysis. No statistically significant differences were found between the two groups in the prolongation of overall survival (Harrington–Fleming P‐value, 0.918; log–rank P‐value, 0.897; hazard ratio, 0.87, 95% confidence interval [CI], 0.486–1.557). Median survival time was 8.36 months (95% CI, 7.46–10.18) for the Active group and 8.54 months (95% CI, 7.33–10.84) for the Placebo group. The toxicity observed in both groups was manageable. Combination therapy of elpamotide with gemcitabine was well tolerated. Despite the lack of benefit in overall survival, subgroup analysis suggested that the patients who experienced severe injection site reaction, such as ulceration and erosion, might have better survival.     

EGFR inhibitors prevent induction of cancer stem-like cells in esophageal squamous cell carcinoma by suppressing epithelial-mesenchymal transition

There exists a highly tumorigenic subset of esophageal squamous cell carcinoma (ESCC) cells defined by high expression of CD44. A novel therapy targeting these cancer stem-like cells (CSCs) is needed to improve prognosis of ESCC. CSCs of ESCC have a mesenchymal phenotype and epithelial-mesenchymal transition (EMT) is critical to enrich and maintain CSCs. EGFR, frequently overexpressed in ESCC, has pivotal roles in EMT induced by TGF-β in invasive fronts. Thus, EMT in invasive fronts of ESCC might be important for CSCs and EGFR could be a target of a novel therapy eliminating CSCs. However, effects of EGFR inhibitors on CSCs in ESCC have not been fully examined. EGFR inhibitors, erlotinib and cetuximab, significantly suppressed enrichment of CSCs via TGF-β1-mediated EMT. Importantly, EGFR inhibitors sharply suppressed ZEB1 that is essential for EMT in ESCC. Further, EGFR inhibitors activated Notch1 and Notch3, leading to squamous cell differentiation. EGFR inhibition may suppress expression of ZEB1 and induce differentiation, thereby blocking EMT-mediated enrichment of CSCs. In organotypic 3D culture, a form of human tissue engineering, tumor cells in invasive nests showed high expression of CD44. Erlotinib significantly blocked invasion into the matrix and CD44 high expressing CSCs were markedly suppressed by erlotinib in organotypic 3D culture. In conclusion, EMT is a critical process for generation of CSCs and the invasive front of ESCC, where EMT occurs, might form a CSC niche in ESCC. EGFR inhibitors could suppress EMT in invasive fronts and be one therapeutic option targeting against generation of CSCs in ESCC.     

An internally and externally validated nomogram for predicting the risk of irinotecan-induced severe neutropenia in advanced colorectal cancer patients

Background:

In Asians, the risk of irinotecan-induced severe toxicities is related in part to UGT1A1*6 (UGT, UDP glucuronosyltransferase) and UGT1A1*28, variant alleles that reduce the elimination of SN-38, the active metabolite of irinotecan. We prospectively studied the relation between the UGT1A1 genotype and the safety of irinotecan-based regimens in Japanese patients with advanced colorectal cancer, and then constructed a nomogram for predicting the risk of severe neutropenia in the first treatment cycle.

Methods:

Safety data were obtained from 1312 patients monitored during the first 3 cycles of irinotecan-based regimen in a prospective observational study. In development of the nomogram, multivariable logistic regression analysis was used to test the associations of candidate factors to severe neutropenia in the first cycle. The final nomogram based on the results of multivariable analysis was constructed and validated internally using a bootstrapping technique and externally in an independent data set (n=350).

Results:

The UGT1A1 genotype was confirmed to be associated with increased risks of irinotecan-induced grade 3 or 4 neutropenia and diarrhoea. The final nomogram included type of regimen, administered dose of irinotecan, gender, age, UGT1A1 genotype, Eastern Cooperative Oncology Group performance status, pre-treatment absolute neutrophil count, and total bilirubin level. The model was validated both internally (bootstrap-adjusted concordance index, 0.69) and externally (concordance index, 0.70).

Conclusions:

Our nomogram can be used before treatment to accurately predict the probability of irinotecan-induced severe neutropenia in the first cycle of therapy. Additional studies should evaluate the effect of nomogram-guided dosing on efficacy in patients receiving irinotecan.     

Overexpression of denticleless E3 ubiquitin protein ligase homolog (DTL) is related to poor outcome in gastric carcinoma

Background

Denticleless E3 ubiquitin protein ligase homolog (DTL) has been identified in amplified region (1q32) of several cancers and has an oncogenic function. In this study, we tested whether DTL acts as a cancer-promoting gene through its activation/overexpression in gastric cancer (GC).

Methods

We analyzed 7 GC cell lines and 100 primary tumors that were curatively resected in our hospital between 2001 and 2003.

Results

Overexpression of the DTL protein was detected in GC cell lines (4/7 cell lines; 57%) and primary GC tumor samples (42/100 cases; 42%). Knockdown of DTL using several specific siRNAs inhibited the proliferation, migration and invasion in a TP53 mutation-independent manner. Overexpression of the DTL was significantly correlated with lymphatic invasion, deeper tumor depth and higher recurrence rate. Patients with DTL-overexpressing tumors had a worse survival rate than those with non-expressing tumors in overall survival (P = 0.0498, log-rank test) and disease-free survival (P = 0.0324, log-rank test). In a multivariate analysis, DTL positivity was independently associated with a worse overall survival (P = 0.0104, hazard ratio 3.7 [1.36–10.1]) and disease-free survival (P = 0.0070 (hazard ratio, 3.9 (1.45–10.46)) following radical gastrectomy.

Conclusions

These findings suggest that DTL overexpression plays a crucial role in tumor cell proliferation and highlights its usefulness as a prognosticator and potential therapeutic target in gastric cancer.     

Novel 5-fluorouracil-resistant human esophageal squamous cell carcinoma cells with dihydropyrimidine dehydrogenase overexpression

5-Fluorouracil (5-FU) is a key drug for the treatment of esophageal squamous cell carcinoma (ESCC); however, resistance to it remains a critical limitation to its clinical use. To clarify the mechanisms of 5-FU resistance of ESCC, we originally established 5-FU-resistant ESCC cells, TE-5R, by step-wise treatment with continuously increasing concentrations of 5-FU. The half maximal inhibitory concentration of 5-FU showed that TE-5R cells were 15.6-fold more resistant to 5-FU in comparison with parental TE-5 cells. TE-5R cells showed regional copy number amplification of chromosome 1p including the DPYD gene, as well as high mRNA and protein expressions of dihydropyrimidine dehydrogenase (DPD), an enzyme involved in 5-FU degradation. 5-FU treatment resulted in a significant decrease of the intracellular 5-FU concentration and increase of the concentration of α-fluoro-ureidopropionic acid (FUPA), a metabolite of 5-FU, in TE-5R compared with TE-5 cells in vitro. Conversely, gimeracil, a DPD inhibitor, markedly increased the intracellular 5-FU concentration, decreased the intracellular FUPA concentration, and attenuated 5-FU resistance of TE-5R cells. These results indicate that 5-FU resistance of TE-5R cells is due to the rapid degradation of 5-FU by DPD overexpression. The investigation of 5-FU-resistant ESCC with DPYD gene copy number amplification and consequent DPD overexpression may generate novel biological evidence to explore strategies against ESCC with 5-FU resistance.     

Formation of microparticles from amylose-grafted poly(γ-glutamic acid) networks obtained by thermostable phosphorylase-catalyzed enzymatic polymerization

Amylose is a natural polysaccharide with helical conformation, which spontaneously forms water-insoluble assemblies, such as double helixes and inclusion complexes, at ambient temperatures in aqueous media, whereas it is synthesized as a water-soluble single chain by thermostable phosphorylase-catalyzed enzymatic polymerization at elevated temperatures in aqueous buffer solvents. In this study, we investigated the enzymatic polymerization at 80 °C using a primer-grafted poly(γ-glutamic acid) (PGA) in the presence or absence of poly(l-lactic acid) (PLLA) as a guest polymer for inclusion by amylose. Consequently, the produced amylose-grafted PGAs formed microparticles by cooling the mixtures at room temperature after the enzymatic polymerization in either the presence or the absence of PLLA. The particle sizes, which were evaluated by SEM measurement, were dependent on the feed ratios of PLLA. Based on the characterization results by the powder X-ray diffraction, IR, and dynamic light scattering measurements, a mechanism for the formation of the microparticles in the present system is proposed.

Thermostable phosphorylase-catalyzed enzymatic polymerization at 80 °C using a primer-grafted poly(γ-glutamic acid), followed by cooling at room temperature, induced the formation of microparticles.     

Whole-body distribution and radiation dosimetry of [11C]telmisartan as a biomarker for hepatic organic anion transporting polypeptide (OATP) 1B3

IntroductionTelmisartan, a nonpeptide angiotensin II AT1 receptor antagonist used as an antihypertensive drug, is specifically taken up by the liver through the OATP1B3. PET imaging with [¹¹C]telmisartan is expected to provide information about the whole body pharmacokinetics of telmisartan as well as its transport property by OATP1B3. The purpose of the study was to determine the biodistribution and radiation dosimetry of [¹¹C]telmisartan in humans.MethodsBiodistribution of [¹¹C]telmisartan was measured in three rats and six healthy male human volunteers. In the rat study, a dynamic emission scan was performed for 90 min. In the human study, dynamic whole-body PET images were acquired after intravenous injection of [¹¹C]telmisartan. ROIs were defined for source organs on the PET images to measure time-course of [¹¹C]telmisartan uptake as percentage injected dose and the number of disintegration for each organ. Radiation dosimetry was calculated with OLINDA/EXM.ResultsIn the rat study, most radioactivity was rapidly taken up by the liver and part of it was excreted into the biliary tract and intestine. Extrapolating from the rat data, the effective dose for the adult human being was estimated to be 3.65±0.01 microSv/MBq (n=3). In the human study, most of the tracer was taken up by the liver as well, although not as rapidly as in the rat. The activity in the gall bladder and intestine increased gradually. The effective dose for the adult human being was 4.24±0.09 microSv/MBq (n=6).Conclusions[¹¹C]Telmisartan is a safe PET tracer with a dosimetry profile comparable to other common ¹¹C PET tracers.     

A model to evaluate toxic factors influencing islets during collagenase digestion: the role of serine protease inhibitor in the protection of islets

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.