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.

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.

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.

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.

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.

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.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis.

Competitive amplification of differentially melting amplicons (CADMA) improves KRAS hotspot mutation testing in colorectal cancer.

BACKGROUND: Cancer is an extremely heterogeneous group of diseases traditionally categorized according to tissue of origin. However, even among patients with the same cancer subtype the cellular alterations at the molecular level are often very different. Several new therapies targeting specific molecular changes found in individual patients have initiated the era of personalized therapy and significantly improved patient care. In metastatic colorectal cancer (mCRC) a selected group of patients with wild-type KRAS respond to antibodies against the epidermal growth factor receptor (EGFR). Testing for KRAS mutations is now required prior to anti-EGFR treatment, however, less sensitive methods based on conventional PCR regularly fail to detect KRAS mutations in clinical samples. METHODS: We have developed sensitive and specific assays for detection of the seven most common KRAS mutations based on a novel methodology named Competitive Amplification of Differentially Melting Amplicons (CADMA). The clinical applicability of these assays was assessed by analyzing 100 colorectal cancer samples, for which KRAS mutation status has been evaluated by the commercially available TheraScreen® KRAS mutation kit. RESULTS: The CADMA assays were sensitive to at least 0.5% mutant alleles in a wild-type background when using 50 nanograms of DNA in the reactions. Consensus between CADMA and the TheraScreen kit was observed in 96% of the colorectal cancer samples. In cases where disagreement was observed the CADMA result could be confirmed by a previously published assay based on TaqMan probes and by fast COLD-PCR followed by Sanger sequencing. CONCLUSIONS: The high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

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.

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.

Identification and validation of highly frequent CpG island hypermethylation in colorectal adenomas and carcinomas.

In our study, whole-genome methylation arrays were applied to identify novel genes with tumor specific DNA methylation of promoter CpG islands in pre-malignant and malignant colorectal lesions. Using a combination of Illumina HumanMethylation27 beadchips, Methylation-Sensitive High Resolution Melting (MS-HRM) analysis, and Exon arrays (Affymetrix) the DNA methylation pattern of ∼14,000 genes and their transcript levels were investigated in six normal mucosas, six adenomas and 30 MSI and MSS carcinomas. Sixty eight genes with tumor-specific hypermethylation were identified (p < 0.005). Identified hypermethylated sites were validated in an independent sample set of eight normal mucosas, 12 adenomas, 40 MSS and nine MSI cancer samples. The methylation patterns of 15 selected genes, hypermethylated in adenomas and carcinomas (FLI1, ST6GALNAC5, TWIST1, ADHFE1, JAM2, IRF4, CNRIP1, NRG1 and EYA4), in carcinomas only (ABHD9, AOX1 and RERG), or in MSI but not MSS carcinomas (RAMP2, DSC3 and MLH1) were validated using MS-HRM. Four of these genes (MLH1, AOX1, EYA4 and TWIST1) had previously been reported to be hypermethylated in CRC. Eleven genes, not previously known to be affected by CRC specific hypermethylation, were identified and validated. Inverse correlation to gene expression was observed for six of the 15 genes with Spearman correlation coefficients ranging from -0.39 to -0.60. For six of these genes the altered methylation patterns had a profound transcriptional association, indicating that methylation of these genes may play a direct regulatory role. The hypermethylation changes often occurred already in adenomas, indicating that they may be used as biomarkers for early detection of CRC.

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.

Increased sensitivity of KRAS mutation detection by high-resolution melting analysis of COLD-PCR products.

Considerable effort has been invested in the development of sophisticated technologies enabling detection of clinically significant low-level tumor specific KRAS mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation-containing templates based on the lower melting temperature of mutant homoduplexes versus wild-type homoduplexes. We have developed a fast COLD-PCR and high-resolution melting (HRM) protocol to increase the sensitivity of KRAS mutation detection. The clinical applicability of COLD-PCR for KRAS mutation detection was assessed by analyzing 61 colorectal cancer specimens, for which KRAS mutation status has been evaluated by the FDA approved TheraScreen(®) KRAS mutation kit. The sensitivity was increased by 5- to 100-fold for melting temperature decreasing mutations when using COLD-PCR compared to standard PCR. Mutations, undetectable by the TheraScreen(®) kit in clinical samples, were detected by COLD-PCR followed by HRM and verified by sequencing. Finally, we have observed a previously undescribed low prevalence synonymous mutation (KRAS c.39C>T, codon 13) in colorectal cancer specimens and in the peripheral blood from an unaffected individual. In conclusion, COLD-PCR combined with HRM, is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments.

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.

PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment.

BACKGROUND: DNA methylation is a highly characterized epigenetic modification of the human genome that is implicated in cancer. The altered DNA methylation patterns found in cancer cells include not only global hypomethylation but also discrete hypermethylation of specific genes. In particular, numerous tumor suppressor genes undergo epigenetic silencing because of hypermethylated promoter regions. Some of these genes are considered promising DNA methylation biomarkers for early cancer diagnostics, and some have been shown to be valuable for predicting prognosis or the response to therapy. CONTENT: PCR-based methods that use sodium bisulfite-treated DNA as a template are generally accepted as the most analytically sensitive and specific techniques for analyzing DNA methylation at single loci. A number of new methods, such as methylation-specific fluorescent amplicon generation (MS-FLAG), methylation-sensitive high-resolution melting (MS-HRM), and sensitive melting analysis after real-time methylation-specific PCR (SMART-MSP), now complement the traditional PCR-based methods and promise to be valuable diagnostic tools. In particular, the HRM technique shows great potential as a diagnostic tool because of its closed-tube format and cost-effectiveness. SUMMARY: Numerous traditional and new PCR-based methods have been developed for detecting DNA methylation at single loci. All have characteristic advantages and disadvantages, particularly with regard to use in clinical settings.

Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM.

BACKGROUND: The High Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. METHODS: Here, we have assessed the quality of DNA extracted from up to 30 years old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five years intervals. For each sample, the methylation levels of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation levels estimated by the two methods for each sample. RESULTS: CDKN2A promoter methylation levels were successfully determined by SMART-MSP and MS-HRM in all 54 samples. Identical methylation estimates were obtained by the two methods in 46 of the samples. The methylation levels of the RARB promoter were successfully determined by SMART-MSP in all samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Twenty-seven of the remaining 34 samples, for which the methylation level could be estimated, gave the same result as observed when using SMART-MSP. CONCLUSION: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies using DNA derived from up to 30 years old FFPE tissue. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions.

A fragile site within the HPC1 region at 1q25.3 affecting RGS16, RGSL1, and RGSL2 in human breast carcinomas.

Genomic alterations affecting chromosome arm 1q are considered to be an early event in breast carcinogenesis and are correlated with good prognosis for the patients. In the search for new breast cancer susceptibility genes, we focused on three genes from the Regulator of G protein Signaling family clustered on 1q25.3 within the HPC1 region. RGS16, RGSL2, and RGSL1 encode proteins interacting with G proteins and accelerating termination of the G protein signaling. To evaluate the implication of these genes in somatic breast cancer, we analyzed a 154-kb segment at 1q25.3 using allelic imbalance (AI) mapping. A panel of 222 patients diagnosed with primary breast cancer was analyzed using newly identified, intragenic short tandem repeats (STRs). A high rate of AI (>50%) was found across the region and led to the identification of internal chromosomal breakpoints. A detailed mapping of the breakpoints revealed intragenic microdeletions affecting the coding regions of RGSL2, RGSL1, and the regulatory region of RGS16. The promoter region of RGS16 was found to be methylated in 10% of the tumors. A decrease in the RGS16 expression was found in tumors with chromosomal breakpoints, AI, and aberrant methylation. We found a significant association between AI of RGSL2 and localized disease, which correlated with good prognosis for patients with breast cancer. In conclusion, we found the 1q25.3 region to be highly unstable in breast tumors comprising a cluster of chromosomal breakpoints, intragenic microdeletions, frequent allelic imbalance correlating with long metastasis-free survival, and RGS16 promoter methylation affecting the protein expression.

Familial cancer associated with a polymorphism in ARLTS1.

BACKGROUND: The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene. METHODS: We studied samples from 216 patients with various types of sporadic tumors or idiopathic pancytopenia, peripheral-blood samples from 109 patients with familial cancer or multiple cancers, and control blood samples from 475 healthy people or patients with diseases other than cancer. We performed functional studies of cell lines lacking ARLTS1 expression with the use of both the full-length ARLTS1 gene and a truncated variant. RESULTS: We found a gene at 13q14, ARLTS1, a member of the ADP-ribosylation factor family, with properties of a tumor-suppressor gene. We analyzed 800 DNA samples from tumors and blood cells from patients with sporadic or familial cancer and controls and found that the frequency of a nonsense polymorphism, G446A (Trp149Stop), was similar in controls and patients with sporadic tumors but was significantly more common among patients with familial cancer than among those in the other two groups (P=0.02; odds ratio, 5.7; 95 percent confidence interval, 1.3 to 24.8). ARLTS1 was down-regulated by promoter methylation in 25 percent of the primary tumors we analyzed. Transfection of wild-type ARLTS1 into A549 lung-cancer cells suppressed tumor formation in immunodeficient mice and induced apoptosis, whereas transfection of truncated ARLTS1 had a limited effect on apoptosis and tumor suppression. Microarray analysis revealed that the wild-type and Trp149Stop-transfected clones had different expression profiles. CONCLUSIONS: A genetic variant of ARLTS1 predisposes patients to familial cancer.

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.

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.