Methylation microarray-based detection of clinical copy-number aberrations in CLL benchmarked to standard FISH analysis.

Copy-number aberrations (CNAs) are assessed using FISH analysis in diagnostics of chronic lymphocytic leukemia (CLL), but CNAs can also be extrapolated from Illumina BeadChips developed for genome-wide methylation microarray screening. Increasing numbers of microarray data-sets are available from diagnostic samples, making it useful to assess the potential in CNA diagnostics. We benchmarked the limitations of CNA testing from two Illumina BeadChips (EPIC and 450k) and using two common packages for analysis (conumee and ChAMP) to FISH-based assessment of 11q, 13q, and 17p deletions in 202 CLL samples. Overall, the two packages predicted CNAs with similar accuracy regardless of the microarray type, but lower than FISH-based assessment. We showed that the bioinformatics analysis needs to be adjusted to the specific CNA, as no general settings were identified. Altogether, we were able to predict CNAs using methylation microarray data, however, with limited accuracy, making FISH-based assessment of deletions the superior diagnostic choice.

Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM).

Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays' specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.

Influence of Unequal Amplification of Methylated and Non-Methylated Template on Performance of Pyrosequencing.

Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.

IGHV-associated methylation signatures more accurately predict clinical outcomes of chronic lymphocytic leukemia patients than IGHV mutation load.

Currently, no molecular biomarker indices are used in standard care to make treatment decisions at diagnosis of chronic lymphocytic leukemia (CLL). We used Infinium MethylationEPIC array data from diagnostic blood samples of 114 CLL patients and developed a procedure to stratify patients based on methylation signatures associated with mutation load of the IGHV gene. This procedure allowed us to predict the time to treatment with a hazard ratio (HR) of 8.34 (95% confidence interval [CI]: 4.54-15.30), as opposed to a HR of 4.35 (95% CI: 2.60-7.28) using IGHV mutation status. Detailed evaluation of 17 cases for which the two classification procedures gave discrepant results showed that these cases were incorrectly classified using IGHV status. Moreover, methylation-based classification stratified patients with different overall survival (HR=1.82; 95% CI: 1.07-3.09), which was not possible using IGHV status. Furthermore, we assessed the performance of the developed classification procedure using published HumanMethylation450 array data for 159 patients for whom information on time to treatment, overall survival and relapse was available. Despite 450K array methylation data not containing all the biomarkers used in our classification procedure, methylation signatures again stratified patients with significantly better accuracy than did IGHV mutation load regarding all available clinical outcomes. Thus, stratification using IGHV-associated methylation signatures may provide better prognostic power than IGHV mutation status.

Bortezomib induces methylation changes in neuroblastoma cells that appear to play a significant role in resistance development to this compound.

The anticancer activity of bortezomib (BTZ) has been increasingly studied in a number of indications and promising results for the use of this treatment have been shown in neuroblastoma. As BTZ treatment is usually administered in cycles, the development of resistance and side effects in patients undergoing therapy with BTZ remains a major challenge for the clinical usage of this compound. Common resistance development also means that certain cells are able to survive BTZ treatment and bypass molecular mechanisms that render BTZ anticancer activity. We studied the methylome of neuroblastoma cells that survived BTZ treatment. Our results indicate that BTZ induces pronounced genome wide methylation changes in cells which recovered from the treatment. Functional analyses of identified methylation changes demonstrated they were involved in key cancer pathology pathways. These changes may allow the cells to bypass the primary anticancer activity of BTZ and develop a treatment resistant and proliferative phenotype. To study whether cells surviving BTZ treatment acquire a proliferative phenotype, we repeatedly treated cells which recovered from the first round of BTZ treatment. The repetitive treatment led to induction of the extraordinary proliferative potential of the cells, that increased with subsequent treatments. As we did not observe similar effects in cells that survived treatment with lenalidomide, and non-treated cells cultured under the same experimental conditions, this phenomenon seems to be BTZ specific. Overall, our results indicate that methylation changes may play major role in the development of BTZ resistance.

miR-151a enhances Slug dependent angiogenesis.

MicroRNAs (miRs) are small non-coding RNAs, that modulate cognate gene expression either by inducing mRNA degradation or by blocking translation, and play crucial and complex roles in tissue homeostasis and during disease initiation and progression. The sprouting of new blood vessels by angiogenesis is critical in vascular development and homeostasis and aberrant angiogenesis is associated with pathological conditions such as ischemia and cancer. We have previously established that miR-151a functions as an onco-miR in non-small cell lung cancer (NSCLC) cells by inducing partial EMT and enhancing tumor growth. Here, we identify anti-miR-151a as a molecule that promotes endothelial cell contacts and barrier properties, suggesting that miR-151a regulates cell-cell junctions. We find that induced miR-151a expression enhances endothelial cell motility and angiogenesis and these functions depend on miR-151a-induced Slug levels. Moreover, we show that miR-151a overexpression enhances tumor-associated angiogenesis in 3D vascularized tumor spheroid assays. Finally, we verify that miR-151a is expressed in the vasculature of normal lung and NSCLC tissue. Our results suggest that miR-151a plays multi-faceted roles in the lung, by regulating multiple functions (cell growth, motility, partial EMT and angiogenesis) in distinct cell types.

Publisher Correction: Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Chronic lymphocytic leukemia patients with heterogeneously or fully methylated LPL promotor display longer time to treatment.

AIM: We investigated whether DNA methylation regulates expression of LPL and PI3K complex genes in chronic lymphocytic leukemia (CLL) and evaluated the prognostic significance of LPL promoter methylation in CLL patients. Patients & methods: Methylation of LPL promoter was assessed in 112 patients using methylation-sensitive high-resolution melting (MS-HRM). RESULTS: Patients with a fully or heterogeneously methylated LPL promoter had significantly longer median time to treatment (p < 0.001) and 75% lower (hazard ratio: 0.25; 95% CI: 0.15-0.42; p < 0.001) risk of requirement for treatment as opposed to patients with nonmethylated promoter. Multivariate modeling confirmed independent prognostic value of these findings. CONCLUSION: Chronic lymphocytic leukemia patients with a fully or heterogeneously methylated LPL gene promoter display indolent disease course and acquisition of heterogeneous methylation of LPL promoter is insufficient to induce gene expression.

Methylation-Sensitive High Resolution Melting (MS-HRM).

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

Epigenetic changes in myelofibrosis: Distinct methylation changes in the myeloid compartments and in cases with ASXL1 mutations.

This is the first study to compare genome-wide DNA methylation profiles of sorted blood cells from myelofibrosis (MF) patients and healthy controls. We found that differentially methylated CpG sites located to genes involved in 'cancer' and 'embryonic development' in MF CD34+ cells, in 'inflammatory disease' in MF mononuclear cells, and in 'immunological diseases' in MF granulocytes. Only few differentially methylated CpG sites were common among the three cell populations. Mutations in the epigenetic regulators ASXL1 (47%) and TET2 (20%) were not associated with a specific DNA methylation pattern using an unsupervised approach. However, in a supervised analysis of ASXL1 mutated versus wild-type cases, differentially methylated CpG sites were enriched in regions marked by histone H3K4me1, histone H3K27me3, and the bivalent histone mark H3K27me3 + H3K4me3 in human CD34+ cells. Hypermethylation of selected CpG sites was confirmed in a separate validation cohort of 30 MF patients by pyrosequencing. Altogether, we show that individual MF cell populations have distinct differentially methylated genes relative to their normal counterparts, which likely contribute to the phenotypic characteristics of MF. Furthermore, differentially methylated CpG sites in ASXL1 mutated MF cases are found in regulatory regions that could be associated with aberrant gene expression of ASXL1 target genes.

The Common Follicle-Stimulating Hormone Receptor (FSHR) Promoter Polymorphism FSHR -29G > A Affects Androgen Production in Normal Human Small Antral Follicles.

Follicle-stimulating hormone receptors (FSHRs) are almost exclusively expressed on granulosa cells, and FSH action is probably most clearly reflected in intrafollicular hormone milieu of antral follicles. Little is known about the possible effects of the common single nucleotide polymorphism (SNP) FSHR -29G > A (rs1394205) on hormonal conditions in humsan small antral follicles (hSAFs) obtained from women in the natural menstrual cycle. This study investigated the follicle fluid (FF) concentrations of anti-Müllerian hormone, estradiol, progesterone, androstenedione, and testosterone in hSAF in relation to the different genotypes of FSHR -29G > A. FF from 362 follicles was collected in 95 women undergoing fertility preservation, who did not suffer from a disease that directly affected ovarian function. The testosterone levels of the minor A/A genotype were significantly increased compared to the A/G and the G/G genotype. Furthermore, significantly reduced androstenedione levels were observed for the G/G genotype, as compared to the A/G genotype, while the other hormones did not show statistical significant differences. In conclusion, the androgen levels of hSAF were significantly elevated in the minor SNP genotype in the FSHR promoter polymorphism FSHR -29G > A.

The association between miR-34 dysregulation and distant metastases formation in lung adenocarcinoma.

Lung cancer has the highest mortality rate amongst human cancers and the majority of deaths can be attributed to metastatic spread. The miR-34 family includes three tumor suppressive miRs: miR-34a, miR-34b and miR-34c. miR-34 downregulation is a frequent observation in human malignancies and is often attributed to hypermethylation of the miR-34a and miR-34b/c promoters. Here, the potential association between aberrant miR-34 expression and promoter methylation and distant metastases formation in lung adenocarcinoma (LAC) is investigated. The expression levels of miR-34a, miR-34b and miR-34c, as well as the methylation status of the miR-34a and miR-34b/c promoters were determined in a LAC patient cohort comprising 26 non-metastasizing and 26 metastasizing primary LACs, as well as 24 paired distant metastases and 25 tumor-adjacent normal lung samples using RT-qPCR and Methylation-Sensitive High Resolution Melting (MS-HRM) analysis. No difference in expression was observed for miR-34a when comparing metastasizing and non-metastasizing LACs (p=0.793). For both miR-34b and miR-34c, a significantly lower expression level was determined in metastasizing LACs compared to non-metastasizing LACs (p=0.0005 and p=0.002) with similarly decreased expression levels observed in the paired distant metastases. Hypermethylation was detected in 35/51 LACs compared to 0/25 tumor-adjacent normal lungs for the miR-34a promoter (p<0.0001). Similarly, 18/51 LACs compared to 1/25 tumor-adjacent normal lungs showed hypermethylation of the miR-34b/c promoter (p=0.003). No difference in methylation was observed between metastasizing and non-metastasizing LACs for neither the miR-34a (p=0.832) nor the miR-34b/c (p=0.900) promoter. In conclusion, miR-34a and miR-34b/c promoter hypermethylation is a frequent event in LAC occurring in 68.7% and 35.3% of tested cases (n=51), respectively. Low miR-34b and miR-34c expression was associated with distant metastases formation in LAC. These changes can be targeted as novel biomarkers in LAC.

Small RNA sequencing reveals metastasis-related microRNAs in lung adenocarcinoma.

The majority of lung cancer deaths are caused by metastatic disease. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and miRNA dysregulation can contribute to metastatic progression. Here, small RNA sequencing was used to profile the miRNA and piwi-interacting RNA (piRNA) transcriptomes in relation to lung cancer metastasis. RNA-seq was performed using RNA extracted from formalin-fixed paraffin embedded (FFPE) lung adenocarcinomas (LAC) and brain metastases from 8 patients, and LACs from 8 patients without detectable metastatic disease. Impact on miRNA and piRNA transcriptomes was subtle with 9 miRNAs and 8 piRNAs demonstrating differential expression between metastasizing and non-metastasizing LACs. For piRNAs, decreased expression of piR-57125 was the most significantly associated with distant metastasis. Validation by RT-qPCR in a LAC cohort comprising 52 patients confirmed that decreased expression of miR-30a-3p and increased expression of miR-210-3p were significantly associated with the presence of distant metastases. miR-210-3p tumor cell specificity was evaluated by in situ hybridization and its biomarker potential was confirmed by ROC curve analysis (AUC = 0.839). Lastly, agreement between miRNA-seq and RT-qPCR for FFPE-derived RNA was evaluated and a high level of concordance was determined. In conclusion, this study has identified and validated metastasis-related miRNAs in LAC.

Identification and validation of candidate epigenetic biomarkers in lung adenocarcinoma.

Lung cancer is the number one cause of cancer-related deaths worldwide. DNA methylation is an epigenetic mechanism that regulates gene expression, and disease-specific methylation changes can be targeted as biomarkers. We have compared the genome-wide methylation pattern in tumor and tumor-adjacent normal lung tissue from four lung adenocarcinoma (LAC) patients using DNA methylation microarrays and identified 74 differentially methylated regions (DMRs). Eighteen DMRs were selected for validation in a cohort comprising primary tumors from 52 LAC patients and tumor-adjacent normal lung tissue from 32 patients by methylation-sensitive high resolution melting (MS-HRM) analysis. Significant increases in methylation were confirmed for 15 DMRs associated with the genes and genomic regions: OSR1, SIM1, GHSR, OTX2, LOC648987, HIST1H3E, HIST1H3G/HIST1H2BI, HIST1H2AJ/HIST1H2BM, HOXD10, HOXD3, HOXB3/HOXB4, HOXA3, HOXA5, Chr1(q21.1).A, and Chr6(p22.1). In particular the OSR1, SIM1 and HOXB3/HOXB4 regions demonstrated high potential as biomarkers in LAC. For OSR1, hypermethylation was detected in 47/48 LAC cases compared to 1/31 tumor-adjacent normal lung samples. Similarly, 45/49 and 36/48 LAC cases compared to 3/31 and 0/31 tumor-adjacent normal lung samples showed hypermethylation of the SIM1 and HOXB3/HOXB4 regions, respectively. In conclusion, this study has identified and validated 15 DMRs that can be targeted as biomarkers in LAC.

Strategies for Integrated Analysis of Genetic, Epigenetic, and Gene Expression Variation in Cancer: Addressing the Challenges.

The development and progression of cancer, a collection of diseases with complex genetic architectures, is facilitated by the interplay of multiple etiological factors. This complexity challenges the traditional single-platform study design and calls for an integrated approach to data analysis. However, integration of heterogeneous measurements of biological variation is a non-trivial exercise due to the diversity of the human genome and the variety of output data formats and genome coverage obtained from the commonly used molecular platforms. This review article will provide an introduction to integration strategies used for analyzing genetic risk factors for cancer. We critically examine the ability of these strategies to handle the complexity of the human genome and also accommodate information about the biological and functional interactions between the elements that have been measured-making the assessment of disease risk against a composite genomic factor possible. The focus of this review is to provide an overview and introduction to the main strategies and to discuss where there is a need for further development.

The influence of DNA degradation in formalin-fixed, paraffin-embedded (FFPE) tissue on locus-specific methylation assessment by MS-HRM.

Readily accessible formalin-fixed paraffin embedded (FFPE) tissues are a highly valuable source of genetic material for molecular analyses in both research and in vitro diagnostics but frequently genetic material in those samples is highly degraded. With locus-specific methylation changes being widely investigated for use as biomarkers in various aspects of clinical disease management, we aimed to evaluate to what extent standard laboratory procedures can approximate the quality of the DNA extracted from FFPE samples prior to methylation analyses. DNA quality in 107 FFPE non-small cell lung cancer (NSCLC) samples was evaluated using spectrophotometry and gel electrophoresis. Subsequently, the quality assessment results were correlated with the results of locus specific methylation assessment with methylation sensitive high resolution melting (MS-HRM). The correlation of template quality with PCR amplification performance and HRM based methylation detection indicated a significant influence of DNA quality on PCR amplification but not on methylation assessment. In conclusion, standard laboratory procedures fairly well approximate DNA degradation of FFPE samples and DNA degradation does not seem to considerably affect locus-specific methylation assessment by MS-HRM.

Copy number variations alter methylation and parallel IGF2 overexpression in adrenal tumors.

Overexpression of insulin growth factor 2 (IGF2) is a hallmark of adrenocortical carcinomas and pheochromocytomas. Previous studies investigating the IGF2/H19 locus have mainly focused on a single molecular level such as genomic alterations or altered DNA methylation levels and the causal changes underlying IGF2 overexpression are still not fully established. In the current study, we analyzed 62 tumors of the adrenal gland from patients with Conn's adenoma (CA, n=12), pheochromocytomas (PCC, n=10), adrenocortical benign tumors (ACBT, n=20), and adrenocortical carcinomas (ACC, n=20). Gene expression, somatic copy number variation of chr11p15.5, and DNA methylation status of three differential methylated regions of the IGF2/H19 locus including the H19 imprinting control region were integratively analyzed. IGF2 overexpression was found in 85% of the ACCs and 100% of the PCCs compared to 23% observed in CAs and ACBTs. Copy number aberrations of chr11p15.5 were abundant in both PCCs and ACCs but while PCCs retained a diploid state, ACCs were frequently tetraploid (7/19). Loss of either a single allele or loss of two alleles of the same parental origin in tetraploid samples resulted in a uniparental disomy-like genotype. These copy number changes correlated with hypermethylation of the H19 ICR suggesting that the lost alleles were the unmethylated maternal alleles. Our data provide conclusive evidence that loss of the maternal allele correlates with IGF2 overexpression in adrenal tumors and that hypermethylation of the H19 ICR is a consequence thereof.

Improved reproducibility in genome-wide DNA methylation analysis for PAXgene-fixed samples compared with restored formalin-fixed and paraffin-embedded DNA.

Formalin fixation has been the standard method for conservation of clinical specimens for decades. However, a major drawback is the high degradation of nucleic acids, which complicates its use in genome-wide analyses. Unbiased identification of biomarkers, however, requires genome-wide studies, precluding the use of the valuable archives of specimens with long-term follow-up data. Therefore, restoration protocols for DNA from formalin-fixed and paraffin-embedded (FFPE) samples have been developed, although they are cost-intensive and time-consuming. An alternative to FFPE and snap-freezing is the PAXgene Tissue System, developed for simultaneous preservation of morphology, proteins, and nucleic acids. In the current study, we compared the performance of DNA from either PAXgene or formalin-fixed tissues to snap-frozen material for genome-wide DNA methylation analysis using the Illumina 450K BeadChip. Quantitative DNA methylation analysis demonstrated that the methylation profile in PAXgene-fixed tissues showed, in comparison with restored FFPE samples, a higher concordance with the profile detected in frozen samples. We demonstrate, for the first time, that DNA from PAXgene conserved tissue performs better compared with restored FFPE DNA in genome-wide DNA methylation analysis. In addition, DNA from PAXgene tissue can be directly used on the array without prior restoration, rendering the analytical process significantly more time- and cost-effective.

Genotyping common FSHR polymorphisms based on competitive amplification of differentially melting amplicons (CADMA).

PURPOSE: To provide an improved platform for simple, reliable, and cost-effective genotyping. BACKGROUND: Modern fertility treatments are becoming increasingly individualized in an attempt to optimise the follicular response and reproductive outcome, following controlled ovarian stimulation. As the field of pharmacogenetics evolve, genetic biomarkers such as polymorphisms of the follicle stimulating hormone receptor (FSHR) may be included as a predictive tool for individualized fertility treatment. However, the currently available genotyping methods are expensive, time-consuming or have a limited analytical sensitivity. Here, we present a novel version of "competitive amplification of differentially melting amplicons" (CADMA), providing an improved platform for simple, reliable, and cost-effective genotyping. METHODS: Two CADMA based assays were designed for the two common polymorphisms of the FSHR gene: rs6165 (c.919A > G, p. Thr307Ala, FSHR 307) and rs6166 (c.2039A > G, p. Asn680Ser, FSHR 680). To evaluate the reliability of the new CADMA-based assays, the genotyping results were compared with two conventional PCR based genotyping methods; allele-specific PCR (AS-PCR) and Sanger sequencing. RESULTS: The genotype frequencies for both polymorphisms were 35 % (TT), 42 % (CT), and 23 % (CC), respectively. A 100 % accordance was observed between the CADMA-based genotyping results and sequencing results, whereas 5 discrepancies were observed between the AS-PCR results and the CADMA-based genotyping results. Comparing the CADMA-based assays to (AS-PCR) and Sanger sequencing, the CADMA based assays showed an improved analytical sensitivity and a wider applicability. CONCLUSIONS: The new assays provide a reliable, fast and user-friendly genotyping method facilitating a wider implication in clinical practise.