Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

Human mast cells express leptin and leptin receptors

Mast cells are immune cells that produce and secrete a variety of mediators and cytokines that influence various inflammatory and immune processes. Leptin is a cytokine regulating metabolic, endocrine as well as immune functions via the leptin receptor which is expressed by many immune cells. However, there are no data about leptin receptor expression in mast cells. Immunohistochemical and immunofluorescent double stainings showed the expression of leptin and leptin receptors in mast cells in human skin and several parts of the respiratory, gastrointestinal and urogenital tract. Leptin was expressed in mast cells expressing the classification marker chymase, whereas a variable expression was observed in tryptase positive mast cells. For leptin receptors, the expression pattern was tissue dependent and not related to tryptase or chymase expression. Our results demonstrate the expression of leptin and leptin receptors on mast cells, suggesting paracrine and/or autocrine immunomodulatory effects of leptin on mast cells.     

methBLAST and methPrimerDB: web-tools for PCR based methylation analysis

Background  

DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

ChIP-seq is a powerful method for obtaining genome-wide maps of protein-DNA interactions and epigenetic modifications. CHANCE (CHip-seq ANalytics and Confidence Estimation) is a standalone package for ChIP-seq quality control and protocol optimization. Our user-friendly graphical software quickly estimates the strength and quality of immunoprecipitations, identifies biases, compares the user's data with ENCODE's large collection of published datasets, performs multi-sample normalization, checks against quantitative PCR-validated control regions, and produces informative graphical reports. CHANCE is available at https://github.com/songlab/chance.     

Therapeutic Efficacy Assessment of CK6, a Monoclonal KIT Antibody,in a Panel of Gastrointestinal Stromal Tumor Xenograft Models

We evaluated the efficacy of CK6, a KIT monoclonal antibody, in a panel of human gastrointestinal stromal tumor (GIST) xenograft models. Nude mice were bilaterally transplanted with human GIST xenografts (four patient derived and two cell line derived), treated for 3 weeks, and grouped as follows: control (untreated); CK6 (40 mg/kg, 3 × weekly); imatinib (50 mg/kg, twice daily); sunitinib (40 mg/kg, once daily); imatinib + CK6; sunitinib + CK6 (same doses and schedules as in the single-agent treatments). Tumor volume assessment, Western blot analysis, and histopathology were used for evaluation of efficacy. Statistical analysis was performed using Mann-Whitney U (MWU) and Wilcoxon matched-pairs tests. CK6 as a single agent only reduced tumor growth rate in the UZLX-GIST3 model (P = .053, MWU compared to control), while in none of the other GIST models an effect on tumor growth rate was observed. CK6 did not result in significant anti-proliferative or pro-apoptotic effects in any of the GIST models, and moreover, CK6 did not induce a remarkable inhibition of KIT activation. Furthermore, no synergistic effect of combining CK6 with tyrosine kinase inhibitors (TKIs) was observed. Conversely, in certain GIST xenografts, anti-tumor effects seemed to be inferior under combination treatment compared to single-agent TKI treatment. In the GIST xenografts tested, the anti-tumor efficacy of CK6 was limited. No synergy was observed on combination of CK6 with TKIs in these GIST models. Our findings highlight the importance of using relevant in vivo human tumor xenograft models in the preclinical assessment of drug combination strategies.     

Pazopanib,a Receptor Tyrosine Kinase Inhibitor,Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.