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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., (© 2017 UICC.)

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., (© 2016 UICC.)

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., (© 2014 UICC.)

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., (Copyright © 2012 UICC.)

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., (Copyright © 2010 UICC.)