Rationale and design of the German-speaking myeloma multicenter group (GMMG) trial HD6: a randomized phase III trial on the effect of elotuzumab in VRD induction/consolidation and lenalidomide maintenance in patients with newly diagnosed myeloma.

BACKGROUND: Despite major advances in therapy, multiple myeloma is still an incurable malignancy in the majority of patients. To increase survival, deeper remissions (i.e. CR) translating into longer PFS need to be achieved. Incorporation of new drugs (i.e. bortezomib and lenalidomide) as induction and maintenance treatment in an intensified treatment concept, including high dose melphalan (200 mg/m2), has resulted in increased CR rates, and is considered the standard of care for younger patients. Elotuzumab in combination with lenalidomide and dexamethasone has given better results as lenalidomide and dexamethasone alone in a phase III trial. The GMMG-HD6 trial will be the first phase III trial investigating the role of elotuzumab in combination with bortezomib, lenalidomide and dexamethasone (VRD) induction/consolidation and lenalidomide maintenance within a high dose concept. METHODS: GMMG-HD6 is a randomized, open, multicenter phase III trial. The planned recruitment number is 564 NDMM patients. All patients will receive 4 VRD cycles as induction and undergo peripheral blood stem cell mobilization and harvesting. Thereafter they will be treated with high dose melphalan therapy plus autologous stem cell transplantation followed by 2 cycles of VRD consolidation and lenalidomide maintenance. Patients in arm B1 + B2 will additionally receive elotuzumab in the induction phase, whereas patients in A2 + B2 will be treated with elotuzumab added to consolidation and maintenance. The primary endpoint of the trial is PFS. Secondary objectives and endpoints are OS, CR rates after induction therapy comparing the two arms VRD (A1 + A2) vs VRD + elotuzumab (B1 + B2), CR rates after consolidation treatment, best response to treatment during the study, time to progression (TTP), duration of response (DOR), toxicity and quality of life. RESULTS: Since this is the publication of a study protocol of an ongoing study, no results can be presented. DISCUSSION: This phase III trial is designed to evaluate whether the addition of elotuzumab to an intensified treatment concept with high dose melphalan chemotherapy plus autologous stem cell transplantation and induction, consolidation and maintenance treatment with bortezomib and lenalidomide is able to improve PFS compared to the same concept without elotuzumab. TRIAL REGISTRATION: NCT02495922 on June 24th, 2015.

A novel component of the ubiquitin pathway, ubiquitin carboxyl extension protein 1 is overexpressed in prostate cancer.

Prostate cancer is among the most common tumors in industrialized nations. However, little is known about the molecular events underlying its development. In the present study we used suppressive subtraction hybridization (SSH) in combination with laser-assisted microdissection in order to compare gene expression between prostate carcinoma and the normal prostate proper. Both are mixed tissues which consist of an epithelial and a stromal compartment. We first compared mRNA (cDNA) expression by SSH and then used real-time quantitative RT-PCR analysis of microdissected tissue probes in order to verify differential expression of subtracted cDNA clones. We also used differentially expressed cDNAs for the synthesis of radiolabelled riboprobes in order to attribute differential expression to specific cell types in tissue sections by in situ hybridization. Using this approach we found an up-regulation of ubiquitin carboxyl extension protein 1 (UBCEP-1) mRNA in prostate carcinoma cells compared to the normal glandular epithelium of the prostate proper. UBCEP-1 mediated ubiquitin chain elongation may promote prostate carcinoma development by increasing via the proteasome pathway the degradation of proteins which are involved in growth inhibition or apoptosis.

Identification of p54(nrb) and the 14-3-3 Protein HS1 as TNF-alpha-inducible genes related to cell cycle control and apoptosis in human arterial endothelial cells.

TNF-alpha plays a pivotal role in inflammation processes which are mainly regulated by endothelial cells. While TNF-alpha induces apoptosis of several cell types like tumor cells, endothelial cells are resistant to TNFa mediated cell death. The cytotoxic effects of TNF-alpha on most cells are only evident if RNA or protein synthesis is inhibited, suggesting that de novo RNA or protein synthesis protect cells from TNF-alpha cytotoxicity, presumably by NF-kappaB mediated induction of protective genes. However, the cytoprotective genes involved in NF-kappaB dependent endothelial cell survival have not been sufficiently identified. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify rarely transcribed TNF-alpha inducible genes in human arterial endothelial cells related to cell survival and cell cycle. The TNF-alpha-induced expression of the RNA binding protein p54(nrb) and the 14-3-3 protein HS1 as shown here for the first time may contribute to the TNF-alpha mediated cell protection of endothelial cells. These genes have been shown to play pivotal roles in cell survival and cell cycle control in different experimental settings. The concerted expression of these genes together with other genes related to cell protection and cell cycle like DnaJ, p21(cip1) and the ubiquitin activating enzyme E1 demonstrates the identification of new genes in the context of TNF-alpha induced gene expression patterns mediating the prosurvival effect of TNF-alpha in endothelial cells.

Stromal cell-derived factor 1alpha (SDF-1alpha) induces gene-expression of early growth response-1 (Egr-1) and VEGF in human arterial endothelial cells and enhances VEGF induced cell proliferation.

Stromal cell-derived factor-1 (SDF-1), mainly known as a chemotactic factor for haematopoietic progenitor cells, also provides angiogenetic potency. Since the intracellular signalling of SDF-1-induced neovascularization remains unclear, we studied in human umbilical arterial endothelial cells (HUAEC) the influence of SDF-1alpha on induction of the genes of early growth response-1 (Egr-1) and VEGF, as well as the activation of extracellular regulated kinases (ERK) 1/2, which are all known to be involved in endothelial cell proliferation. We found a time-dependent induction of Egr-1 and VEGF mRNA expression and phosphorylation of ERK1/2 by SDF-1alpha. Furthermore, we demonstrated that Egr-1 expression is dependent on ERK 1/2 activation. Finally, we tried to confirm the relevance of the induced gene expression by detecting the [3H]thymidine incorporation as a marker for cell proliferation in HUAEC after stimulation with SDF-1alpha alone or together with VEGF. This particular test showed, that SDF-1alpha alone has no effect, but is able to significantly enhance VEGF induced DNA synthesis. In summary, SDF-1alpha is involved in different steps of endothelial cell proliferation, but, since Egr-1 and VEGF offer different functions, it may also play a so far undefined role on other conditions of the endothelium.

Association of cytochrome P450 2E1 polymorphisms and head and neck squamous cell cancer.

The development of head and neck squamous cell cancer (HNSCC) is known to be strongly associated with tobacco use. One of the main enzymes for bioactivation of tobacco-related substances is the cytochrome 450 (CYP)2E1, of which different genetic variants are described. Analyzing a correlation between certain neoplasia and alteration of the CYP2E1 gene, most studies focus on the polymorphisms -1053C>T and 7632T>A, but recently another polymorphism, named -71G>T, with enhanced transcriptional activity, has been identified. In the current case-control study we investigate the putative association of the mentioned CYP2E1 polymorphisms on the risk of HNSCC. Comparing 312 German individuals with HNSCC to 299 controls we found a significantly enhanced risk for the development of that neoplasia in smoking carriers of -71G>T heterozygosity, while in -1053C>T and 7632T>A polymorphisms a corresponding correlation was absent. Since a coincidence of an aberrant p53 gene and CYP2E1 mutations has been described, we choose a subgroup of 140 patients with HNSCC for analyzing an association of mutations in these two genes. However, no such association could be found in either of the mentioned polymorphisms. Further studies have to focus on the -71G>T polymorphism and its possible linkage to cancers, in which smoking is a known risk-factor, as well as its functional relevance concerning the bioactivation of tobacco-related substances.

Gemcitabine and paclitaxel in metastatic or recurrent squamous cell carcinoma of the head and neck: a phase I-II study.

The purpose of this study was to determine the maximum tolerated dose, toxicity profile and anti-tumor activity of paclitaxel in combination with gemcitabine when administered to patients with unresectable locally recurrent or metastatic squamous cell carcinoma of the head and the neck (SCCHN). Twenty-seven patients were treated in a phase I-II study with gemcitabine at a dose of 800 mg/m on days 1 and 8, escalating to a dose of 1,000 mg/m, plus escalating doses of paclitaxel (100, 135 and 175 mg/m) on day 2. Treatment consisted of 6 cycles repeated every 3 weeks. The main toxicity was myelosuppression. Other toxicities were mild and manageable. Due to grade 4 neutropenia at higher doses the recommended dose level of the gemcitabine/paclitaxel combination was 1,000/135 mg/m. Four patients achieved a partial response and no patient had a complete remission, giving an overall response rate of 14.8%. The median time of survival was 24 weeks. We conclude that the combination of paclitaxel and gemcitabine is tolerated, but shows insufficient clinical activity in patients with recurrent and/or metastatic SCCHN to warrant further testing.

The use of suppression subtractive hybridization for the study of SDF-1alpha induced gene-expression in human endothelial cells.

Stromal cell-derived factor-1 (SDF-1), the only ligand of the CXCR4 receptor, is mainly known as a chemotactic factor for hematopoietic progenitor cells. However, studies of knock-out mice have shown malformation of different organ-systems suggesting that SDF-1 may have a role in angiogenesis and cardiac and cerebral development. However, the underlying mechanisms of its action are largely unknown. Therefore, we performed suppression subtractive hybridization (SSH) in order to identify genes that are differentially expressed after stimulation of human arterial endothelial cells (HUAEC) with SDF-1. Using SSH we found ten genes, with varied functions, whose mRNA expression is induced by SDF-1alpha in HUAEC. We show that SSH is a reliable method for identifying differentially expressed genes and that SDF-1alpha may have more functions than previously reported.

Association between head and neck cancer and microsomal epoxide hydrolase genotypes.

Tobacco-associated carcinogens are catalyzed by microsomal epoxide hydrolase (mEH). Combinations of the Y113H and H139R polymorphic EPHX1 variants have been assumed to alter the enzyme activity and thus the risk of squamous cell head and neck cancer (SCCHN). Based on in vitro data, a putative low, medium and high mEH activity has been associated with combinations of these genotypes, and the respective activity categories have been frequently used in the estimation of risks for smoking-related cancers. We investigated the SCCHN risk for EPHX1 genotypes among 280 cases and 289 controls. We could not detect main effects of the EPHX1 genotypes, but a smaller risk of the 139HR genotype in smokers (odds ratio, OR, 0.57; 95% confidence interval, CI, 0.34-0.95). We could not confirm an increase of the SCCHN risk for genotype combinations according to a putative medium and high enzyme activity (OR 1.28, 95% CI 0.84-1.96; OR 0.98, 95% CI 0.58-1.64, respectively), but a significant heterogeneity of the estimated risks for the singular genotypes within these categories among smokers ( P=0.02). Further, p53 mutations among smoking cases were less frequent in the group with a putative high enzyme activity, although insignificant due to small numbers (OR 0.54, 95% CI 0.13-2.17). This supports uncertainties in categorizing genotypes with respect to limited enzyme activity data, especially when taken from in vitro experiments.