Tina Kjeldsen

Investigation of MGMT and DAPK1 methylation patterns in diffuse large B-cell lymphoma using allelic MSP-pyrosequencing

The tumor suppressor genes MGMT and DAPK1 become methylated in several cancers including diffuse large B-cell lymphoma (DLBCL). However, allelic methylation patterns have not been investigated in DLBCL. We developed a fast and cost-efficient method for the analysis of allelic methylation based on pyrosequencing of methylation specific PCR (MSP) products including a SNP. Allelic methylation patterns were reliably analyzed in standards of known allelic methylation status even when diluted in unmethylated DNA to below 1% methylation. When studying 148 DLBCL patients MGMT and DAPK1 methylation was observed in 19% and 89%, respectively, and among methylated and heterozygous patients 29% and 55%, respectively, were biallelically methylated. An association between the T-allele of the rs16906252 SNP and MGMT methylation was observed (p-value = 0.04), and DAPK1 methylation of the A-allele was associated with shorter overall survival (p-value = 0.006). In future cancer research allelic MSP-pyrosequencing may be used to study a wide range of other loci.

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

BackgroundCancer 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.MethodsWe 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.ResultsThe 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.ConclusionsThe high analytical sensitivity and specificity of CADMA may increase diagnostic sensitivity and specificity of KRAS mutation testing in mCRC patients.

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.

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.

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.

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.

Abstract B28: Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay

MGMT is a tumor suppressor gene, which becomes methylated in many different cancers including diffuse large B-cell lymphoma (DLBCL). Methylation of MGMT has been associated with the T-allele of the rs16906252 SNP found in the MGMT promoter. However, allelic MGMT methylation patterns and a possible association with the T-allele of this SNP have not been investigated in DLBCL. Today, standard treatment for DLBCL patients is rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), and methylation of MGMT has been shown to predict chemosensitivity to the alkylating agent cyclophosphamide in the era before rituximab. We developed a novel method for the analysis of allelic MGMT methylation patterns based on pyrosequencing of antisense methylation specific PCR (MSP) products including the rs16906252 SNP. Samples from 148 DLBCL patients, of which 75 received R-CHOP, were studied using this assay. Standard bisulfite pyrosequencing and bisulfite sequencing of single clones were used to confirm data obtained by allelic MSP pyrosequencing. The allelic MSP pyrosequencing assay could reliably analyze allelic methylation patterns in standards of known allelic methylation status even when diluted in unmethylated DNA down to 1.25% methylation. MGMT methylation was observed for 28 patients of which seven were heterozygous. Thus, an association between the T-allele and MGMT methylation was observed (p-value =0.04). Among the seven methylated and heterozygous patients, two were methylated at both alleles, four were methylated only at the T-allele, and one only at the C-allele. Finally, we found no statistical significant associations between MGMT methylation or SNP genotypes with clinical parameters among the R-CHOP treated patients. In conclusion, allelic MSP pyrosequencing is a fast and cost-efficient method for evaluation of allelic methylation patterns, which may have wide implications for locus specific methylation analysis of tumor suppressor genes in future cancer research. Citation Format: Lasse Sommer Kristensen, Marianne Bach Treppendahl, Mia Seremet Girkov, Fazila Asmar, Helene Myrtue Nielsen, Tina Kjeldsen, Elisabeth Ralfkiaer, Lise Lotte Hansen, Kirsten Gronbaek. Investigation of MGMT methylation patterns in diffuse large B-cell lymphoma using a novel allelic methylation-specific PCR pyrosequencing assay. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B28.

Abstract 2115: Increased sensitivity of KRAS mutation detection by High-Resolution Melting analysis of COLD-PCR products

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Much effort has been put into the development of sophisticated technologies enabling the detection of clinically significant low-level tumor specific KRAS mutations. The constitutively active KRAS oncogene harbors a mutation hotspot in codon 12 and 13 of exon 2. These mutations are predictors of response to treatment with the monoclonal antibodies panitumumab and cetuximab targeting the epidermal growth factor receptor (EGFR). Therefore, it is crucial to develop robust and sensitive assays targeting these mutations. Coamplification at lower denaturation temperature-PCR (COLD-PCR) is a new form of PCR that selectively amplifies mutation containing amplicons based on the lower melting temperature of heteroduplexes versus homoduplexes. We have developed an assay based on real-time COLD-PCR and high-resolution melting (HRM) targeting codon 12 and 13 for simultaneous detection of the various mutations found in this mutation hotspot. By using mutation containing cell-lines serially diluted into wild-type DNA we have compared the sensitivity of HRM after standard PCR with the sensitivity of HRM after COLD-PCR. We have also analyzed 60 colorectal cancer samples for which KRAS mutation status previously has been evaluated by the DxS kit which is approved by the U.S. Food and Drug Administration. Replacing standard PCR by COLD-PCR increased the sensitivity of the assay by up to 50 fold depending on the specific mutation. Furthermore, we have obtained very similar results when using our COLD-PCR assay as we obtained when using the DxS kit and confirmed the HRM results with Sanger sequencing. In conclusion, applying COLD-PCR to HRM analysis is a simple way of increasing the sensitivity of KRAS mutation detection without adding to the complexity and cost of the experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2115.

Abstract LB-61: Comparative study of different diagnostic techniques (AI, MLPA, FISH) in breast cancer patients with 1q25.3 alterations

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Breast cancer is the most frequently diagnosed malignancy among women and is the leading cause of cancer related death worldwide. Molecular genetic studies have revealed many subgroups of breast cancer within which the genomic alterations affecting chromosome arm 1q are considered to be an early event in breast carcinogenesis and are correlated with favorable prognosis for the patients. We have found a high percentage of concordance between the AI and MLPA assays pointing towards gain of the 1q25.3 region in breast cancer. Aims: Our main objective was to compare the sensitivity and specificity of AI and synthetic probe-based MLPA with other cytogenetic method (FISH) in breast cancer patients with or without previously confirmed alterations within 1q25.3. Methods: FISH was performed on formalin-fixed paraffin-embedded tumor material in order to verify previous findings and assess the level of genetic alterations of 1q25.3 in breast cancer. The 1q25.3 test and 1p35 reference probes labeled with different fluorochromes were utilized for analysis. Results: A total of 70 nuclei from each breast cancer case were examined and scored for the percentage of 1q25.3 alterations. The non-tumorigenic nuclei obtained from healthy individuals served as adequate cut-off for the 1q25.3-specific changes. The overall FISH results are consistent with results obtained from previous analysis in the majority of analysed cases. Furthermore, FISH resolved the level of 1q25.3 alterations in few cases that were uncertain by AI and MLPA analysis. Conclusion: This study shows that both AI and MLPA assays are able to map regions of copy number changes in the cancer genome and are in concordance with molecular cytogenetics. These three techniques proved to be an efficient tool for diagnosis of 1q25.3 alterations, however, confusing results in AI and MLPA analysis might be obtained while using non-microdissected tumor DNA as a template. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-61.