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Purpose: We consider an existing clinical cohort with events but limited resources for the investigation of a further potentially expensive marker. Biological material of the patients is stored in a biobank, but only a limited number of samples can be analyzed with respect to the marker. The question arises as to which patients to sample, if the number of events preclude standard sampling designs. Methods: Modifications of the nested case‐control and the case‐cohort design for the proportional hazards model are applied, that allow efficient sampling in situations where standard nested case‐control and case‐cohort are not feasible. These sampling designs are compared to simple random sampling and extreme group sampling, the latter including only patients with extreme outcomes, ie either with an event early in time or without an event until at least a point later in time. Results: The modified nested case‐control design and the modified case‐cohort design provide powerful methods for sampling in a clinical cohort with many events. The simple random sampling usually is less efficient. If focus is on precise estimation of a potential effect in terms of a hazard ratio, extreme group sampling is not competitive. If focus is on screening for important biomarkers, extreme group sampling markedly outperforms the other sampling designs. Conclusions: When it is not feasible to sample all events, a modified nested case‐control design or case‐cohort design leads to efficient effect estimates in the proportional hazards model. If screening for important biomarkers is the primary objective, extreme group sampling is preferable. [ABSTRACT FROM AUTHOR]

Abstract: The widespread use of high‐dose therapy and autologous stem cell transplantation (ASCT) as well as the introduction of novel agents have significantly improved outcomes in multiple myeloma (MM) enabling long‐term survival. We here analyze factors influencing survival in 865 newly diagnosed MM patients who underwent first‐line ASCT at our center between 1993 and 2014. Relative survival and conditional survival were assessed to further characterize long‐term survivors. Achievement of complete response (CR) post‐ASCT was associated with prolonged progression‐free survival (PFS) in the whole cohort and with significantly superior overall survival (OS) in the subgroup of patients receiving novel agent‐based induction therapy. Landmark analyses performed at 1, 3, and 5 years post‐ASCT revealed that sustainment of any response had a highly significant influence on survival with no significant differences between sustained CR and sustained inferior responses. Furthermore, outcome was independently improved by administration of maintenance therapy. A subset of patients did experience long‐term survival >15 years. However, conditional survival demonstrated a persistent risk of myeloma‐associated death and cumulative relative survival curves did not show development of a clear plateau, even in prognostically advantageous groups. In conclusion, in this large retrospective study, sustained response after first‐line ASCT was found to be a major prognostic factor for OS independent of depth of sustained response. Administration of maintenance therapy further improved outcome, supporting the hypothesis that interventions to prolong responses achieved post‐ASCT may be essential to reach long‐term survival, especially in the setting of persisting residual disease. [ABSTRACT FROM AUTHOR]

B cell receptor (BCR) signaling is a key for survival of chronic lymphocytic leukemia (CLL) cells, and BCR signaling inhibitors are clinically active. However, relapse and resistance to treatment require novel treatment options. To detect novel candidate therapeutic targets, we performed a genome-wide DNA methylation screen with custom arrays and identified aberrant promoter DNA methylation in 2,192 genes. The transcription factor NFATC1 that is a downstream effector of BCR signaling was among the top hypomethylated genes and was concomitantly transcriptionally upregulated in CLL. Intriguingly, NFATC1 promoter DNA hypomethylation levels were significantly variant in clinical trial cohorts from different disease progression stages and furthermore correlated with Binet disease staging and thymidine kinase levels, strongly suggesting a central role of NFATC1 in CLL development. Functionally, DNA hypomethylation at NFATC1 promoter inversely correlated with RNA levels of NFATC1 and dysregulation correlated with expression of target genes BCL-2, CCND1 and CCR7. The inhibition of the NFAT regulator calcineurin with tacrolimus and cyclosporin A and the BCR signaling inhibitor ibrutinib significantly reduced NFAT activity in leukemic cell lines, and NFAT inhibition resulted in increased apoptosis of primary CLL cells. In summary, our results indicate that the aberrant activation of NFATC1 by DNA hypomethylation and BCR signaling plays a major role in the pathomechanism of CLL. [ABSTRACT FROM AUTHOR]

Objective Analysis of the efficiency and toxicity of cyclophosphamide-based stem cell mobilization in patients with relapsed multiple myeloma ( RMM). Methods Peripheral blood stem cells ( PBSCs) were mobilized with high dose cyclophosphamide (2 g/m2 daily on days 1 and 2) and G- CSF plus pre-emptive/rescue plerixafor in RMM patients (first to third relapse) treated within the Re LApsE trial of the German-Speaking Myeloma Multicenter Group ( GMMG). Results Mobilization was initiated with high-dose cyclophosphamide (HD-CY) and G- CSF in 30 patients. Fifteen patients received additional pre-emptive/rescue administration of plerixafor. Stem cell collection was successful (≥2×106 CD34+ cells per kg bw) in 77% (23/30 patients). Patients with prior high-dose melphalan collected a significantly lower median total number of PBSCs than patients without prior high-dose melphalan (3.3×106 vs 17×106 CD34+ cells/kg bw). Toxicity of HD-CY was frequent with 12 serious adverse events ( SAE) in 37% of patients (11/30 patients). Infections accounted for the majority of SAE reports. In two patients, SAEs were lethal (septic shock). Conclusions These data proof feasibility of PBSC collection at relapse but emphasize the importance of collection and storage of additional PBSC transplants during first-line treatment when mobilization is more efficient and less toxic. [ABSTRACT FROM AUTHOR]

Aims Deleted in malignant brain tumours 1 ( DMBT1) exerts functions in the regulation of epithelial differentiation and inflammation and has been proposed as a tumour suppressor. Because chronic inflammation is a hallmark of cholangiocarcinogenesis, the aim of this study was to investigate the expression of DMBT1 in biliary tract cancer ( BTC) and to correlate this expression with clinicopathological data. Methods and results The expression of DMBT1 protein was examined immunohistochemically in 157 BTC patients [41 intrahepatic ( ICC), 60 extrahepatic cholangiocarcinomas ( ECC) and 56 adenocarcinomas of the gallbladder ( GBAC)]. Additionally, 56 samples of high-grade biliary intraepithelial neoplasia (Bil IN 3) and 92 corresponding samples of histological non-neoplastic biliary tract tissues were included. DMBT1 expression was increased significantly in Bil IN 3 compared to normal tissue ( P < 0.0001) and BTC ( P < 0.0001). BTC showed no significant difference in DMBT1 expression compared to non-neoplastic biliary tissue ( P = 0.315). Absent DMBT1 expression in non-neoplastic biliary tissue of BTC patients was associated with poorer survival ( P = 0.027). DMBT1 expression was correlated significantly with patients' age ( P < 0.001). Conclusion DMBT1 is expressed differently in cholangiocarcinogenesis and poorer patients' survival rates are associated with absent DMBT1 expression in non-neoplastic biliary tissue, suggesting a tumour-suppressive role of DMBT1 in early cholangiocarcinogenesis. [ABSTRACT FROM AUTHOR]

Therapeutic options in heavily pretreated relapsed/refractory multiple myeloma patients are often very limited because of impaired bone marrow function. Bendamustine is effective in multiple myeloma and has a favourable toxicity profile. We hypothesized that dose-intensified bendamustine (180 mg/m2 , day 1 and 2) followed by autologous blood stem cell support (ASCS) would improve bone marrow function with low post-transplant toxicity in patients with severely impaired haematopoiesis. We analyzed 28 consecutive myeloma patients, with a median of three prior lines of therapy (range 2-7), who had relapsed from the last treatment with very limited bone marrow function and were therefore ineligible for conventional chemotherapy, novel agents or trial enrolment. Dose-intensified bendamustine with ASCS improved haematopoiesis as reflected by increased platelet counts (median 40/nl vs 94/nl, p = 0.0004) and white blood cell counts (3.0/nl vs 4.8/nl, p = 0.02) at day +100. The median time until engraftment of platelets (>50/nl) was 11 days (0-24 days) and of white cell counts (>1.0/nl) 0 days (0-24 days). At least, a minimal response was achieved in 36% of patients. The disease stabilization rate was 50% while the median progression-free survival rate was limited to 2.14 months. Most importantly, patients were once again eligible for alternative treatments including enrolment into clinical trials. We conclude that dose-intensified bendamustine followed by ASCS is safe and feasible for multiple myeloma patients with very limited bone marrow reserve. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Oxaliplatin is frequently used as part of a chemotherapeutic regimen with 5-fluorouracil in the treatment of colorectal cancer (CRC). The cellular availability of oxaliplatin is dependent on metabolic and transporter enzymes. Variants in genes encoding these enzymes may cause variation in response to oxaliplatin and could be potential predictive markers. Therefore, we used a two-step procedure to comprehensively investigate 1,444 single nucleotide polymorphisms (SNPs) from these pathways for their potential as predictive markers for oxaliplatin treatment, using 623 stage II-IV CRC patients (of whom 201 patients received oxaliplatin) from a German prospective patient cohort treated with adjuvant or palliative chemotherapy. First, all genes were screened using the global test that evaluated SNP*oxaliplatin interaction terms per gene. Second, one model was created by backward elimination on all SNP*oxaliplatin interactions of the selected genes. The statistical procedure was evaluated using bootstrap analyses. Nine genes differentially associated with overall survival according to oxaliplatin treatment (unadjusted p values < 0.05) were selected. Model selection resulted in the inclusion of 14 SNPs from eight genes (six transporter genes, ABCA9, ABCB11, ABCC10, ATP1A1, ATP1B2, ATP8B3, and two metabolism genes GSTM5, GRHPR), which significantly improved model fit. Using bootstrap analysis we show an improvement of the prediction error of 3.7% in patients treated with oxaliplatin. Several variants in genes involved in metabolism and transport could thus be potential predictive markers for oxaliplatin treatment in CRC patients. If confirmed, inclusion of these variants in a predictive test could identify patients who are more likely to benefit from treatment with oxaliplatin. [ABSTRACT FROM AUTHOR]

For better lung cancer diagnosis and therapy, early detection markers of tumor dissemination are urgently needed, as most lung cancers do not show symptoms until extensive metastasis formation has already taken place. Our previous studies showed that in non-small cell lung cancer (NSCLC) early tumor dissemination is associated with a loss of chromosome 4q12-q32 and the presence of disseminated tumor cells (DTC) in the bone marrow. In order to identify the potential target gene in this region, a screen for methylation-dependent expression was performed. Lung cancer cell lines showing a loss of 4q as well as a normal bronchial epithelial cell line as control were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) followed by expression profiling. Seven genes within the 4q target region, which have been associated with a positive DTC status before were found to be regulated by hypermethylation. QRT-PCR in an independent sample set identified HERC5 as a potential target gene. Quantitative methylation analysis of these lung tissue samples revealed that HERC5 promoter hypermethylation was significantly associated with positive DTC status ( p = 0.020) and occurrence of brain metastases ( p = 0.015). In addition, hypermethylation of the HERC5 promoter in NSCLC was identified as a predictor for poor survival for Stage I adenocarcinoma patients ( p = 0.022) and also for poor overall survival in metastatic lung cancer patients ( p = 0.028). In conclusion, HERC5 may function as a prognostic marker and is associated with tumor dissemination in lung cancer. [ABSTRACT FROM AUTHOR]

Breast cancer is a heterogeneous disease at both the clinical and molecular levels. This heterogeneity may give rise to different therapy responses. Molecular profiling has facilitated identification of signatures for stratifying patients who would potentially benefit from given therapies. Previously, we reported on a subset of genes with the potential for predicting response of primary breast cancer to neoadjuvant chemotherapy. Herein, we report that patients with luminal (estrogen receptor α [ERα]-expressing) breast cancer were enriched for nonresponders. To identify novel factors that contribute to the survival of breast cancer cells, a loss-of-function screen was performed with a subset of genes overexpressed in patients with disease resistant to chemotherapy. This approach led us to identify protein phosphatase 1, regulatory subunit 15B (PPP1R15B) as a factor with a potentially essential role in the survival of ERα-positive breast cancer cells. Functional analyses showed that PPP1R15B depletion results in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle, and apoptosis induction. Moreover, our data revealed a regulatory role for PPP1R15B in activating ERα. Furthermore, a high level of PPP1R15B mRNA expression was associated with poor outcome following tamoxifen-based therapy. Accordingly, knockdown of PPP1R15B expression sensitized tamoxifen-resistant MCF-7 breast cancer cells to tamoxifen while reducing ERα abundance in these cells. Our findings reveal a novel role for PPP1R15B in the survival and therapy response of ERα-positive breast cancer and may open new avenues for tumor subtype-specific therapeutic strategies in the era of personalized medicine. [ABSTRACT FROM AUTHOR]

In various types of cancers including glioblastoma, accumulating evidence show the existence of cancer stem-like cells ( CSCs), characterized by stem cell marker expression, capability of differentiation and self-renewal, and high potential for tumor propagation in vivo. LGR5, whose expression is positively regulated by the Wnt signaling pathway, is a stem cell marker in intestinal mucosa and hair follicle in the skin. As Wnt signaling is also involved in brain development, the function of LGR5 in the maintenance of brain CSCs is to be assessed. Our study showed that the LGR5 transcript level was increased in CSCs. Co-immunofluorescence staining demonstrated the co-localization of CD133- and LGR5-positive cells in glioblastoma tissue sections. Functionally, silencing of LGR5 by lentiviral shRNA-mediated knockdown induced apoptosis in brain CSCs. Moreover, LGR5 depletion led to a downregulation of L1 cell adhesion molecule expression. In line with an important function in glioma tumorigenesis, LGR5 expression increased with glioma progression and correlated with an adverse outcome. Our findings suggest that LGR5 plays a role in maintenance and/or survival of brain CSCs. [ABSTRACT FROM AUTHOR]

To identify novel glioma-associated pathomechanisms and molecular markers, we performed an array-based comparative genomic hybridization analysis of 131 diffuse astrocytic gliomas, including 87 primary glioblastomas (pGBIV), 13 secondary glioblastomas (sGBIV), 19 anaplastic astrocytomas (AAIII) and 12 diffuse astrocytomas (AII). All tumors were additionally screened for IDH1 and IDH2 mutations. Expression profiling was performed for 74 tumors (42 pGBIV, 11 sGBIV, 13 AAIII, 8 AII). Unsupervised and supervised bioinformatic analyses revealed distinct genomic and expression profiles separating pGBIV from the other entities. Classifier expression signatures were strongly associated with the IDH1 gene mutation status. Within pGBIV, the rare subtype of IDH1 mutant tumors shared expression profiles with IDH1 mutant sGBIV and was associated with longer overall survival compared with IDH1 wild-type tumors. In patients with IDH1 wild-type pGBIV, PDGFRA gain or amplification as well as 19q gain were associated with patient outcome. Array-CGH analysis additionally revealed homozygous deletions of the FGFR2 gene at 10q26.13 in 2 pGBIV, with reduced FGFR2 mRNA levels being frequent in pGBIV and linked to poor outcome. In conclusion, we report that diffuse astrocytic gliomas can be separated into 2 major molecular groups with distinct genomic and mRNA profiles as well as IDH1 gene mutation status. In addition, our results suggest FGFR2 as a novel glioma-associated candidate tumor suppressor gene on the long arm of chromosome 10. [ABSTRACT FROM AUTHOR]

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca2+ homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where β is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity. [ABSTRACT FROM AUTHOR]