CDO1 Promoter Methylation is a Biomarker for Outcome Prediction of Anthracycline Treated, Estrogen Receptor-Positive, Lymph Node-Positive Breast Cancer Patients

<p>Abstract</p> <p>Background</p> <p>Various biomarkers for prediction of distant metastasis in lymph-node negative breast cancer have been described; however, predictive biomarkers for patients with lymph-node positive (LNP) disease in the context of distinct systemic therapies are still very much needed. DNA methylation is aberrant in breast cancer and is likely to play a major role in disease progression. In this study, the DNA methylation status of 202 candidate loci was screened to identify those loci that may predict outcome in LNP/estrogen receptor-positive (ER+) breast cancer patients with adjuvant anthracycline-based chemotherapy.</p> <p>Methods</p> <p>Quantitative bisulfite sequencing was used to analyze DNA methylation biomarker candidates in a retrospective cohort of 162 LNP/ER+ breast cancer patients, who received adjuvant anthracycline-based chemotherapy. First, twelve breast cancer specimens were analyzed for all 202 candidate loci to exclude genes that showed no differential methylation. To identify genes that predict distant metastasis, the remaining loci were analyzed in 84 selected cases, including the 12 initial ones. Significant loci were analyzed in the remaining 78 independent cases. Metastasis-free survival analysis was conducted by using Cox regression, time-dependent ROC analysis, and the Kaplan-Meier method. Pairwise multivariate regression analysis was performed by linear Cox Proportional Hazard models, testing the association between methylation scores and clinical parameters with respect to metastasis-free survival.</p> <p>Results</p> <p>Of the 202 loci analysed, 37 showed some indication of differential DNA methylation among the initial 12 patient samples tested. Of those, 6 loci were associated with outcome in the initial cohort (n = 84, log rank test, p < 0.05).</p> <p>Promoter DNA methylation of cysteine dioxygenase 1 (CDO1) was confirmed in univariate and in pairwise multivariate analysis adjusting for age at surgery, pathological T stage, progesterone receptor status, grade, and endocrine therapy as a strong and independent biomarker for outcome prediction in the independent validation set (log rank test p-value = 0.0010).</p> <p>Conclusions</p> <p>CDO1 methylation was shown to be a strong predictor for distant metastasis in retrospective cohorts of LNP/ER+ breast cancer patients, who had received adjuvant anthracycline-based chemotherapy.</p

Free-Circulating Methylated DNA in Blood for Diagnosis, Staging, Prognosis, and Monitoring of Head and Neck Squamous Cell Carcinoma Patients: An Observational Prospective Cohort Study

Abstract BACKGROUND Circulating cell-free DNA methylation testing in blood has recently received regulatory approval for screening of colorectal cancer. Its application in other clinical settings, including staging, prognosis, prediction, and recurrence monitoring is highly promising, and of particular interest in head and neck squamous cell carcinomas (HNSCCs) that represent a heterogeneous group of cancers with unsatisfactory treatment guidelines. METHODS Short stature homeobox 2 (SHOX2) and septin 9 (SEPT9) DNA methylation in plasma from 649 prospectively enrolled patients (training study: 284 HNSCC/122 control patients; testing study: 141 HNSCC/102 control patients) was quantified before treatment and longitudinally during surveillance. RESULTS In the training study, 59% of HNSCC patients were methylation-positive at 96% specificity. Methylation levels correlated with tumor and nodal category (P &amp;lt; 0.001). Initially increased methylation levels were associated with a higher risk of death [SEPT9: hazard ratio (HR) = 5.27, P = 0.001; SHOX2: HR = 2.32, P = 0.024]. Disease recurrence/metastases were detected in 47% of patients up to 377 days earlier compared to current clinical practice. The onset of second cancers was detected up to 343 days earlier. In the testing study, sensitivity (52%), specificity (95%), prediction of overall survival (SEPT9: HR = 2.78, P = 0.022; SHOX2: HR = 2.50, P = 0.026), and correlation with tumor and nodal category (P &amp;lt;0.001) were successfully validated. CONCLUSIONS Methylation testing in plasma is a powerful diagnostic tool for molecular disease staging, risk stratification, and disease monitoring. Patients with initially high biomarker levels might benefit from intensified treatment and posttherapeutic surveillance. The early detection of a recurrent/metastatic disease or a second malignancy could lead to an earlier consecutive treatment, thereby improving patients' outcomes. </jats:sec

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

<p>Abstract</p> <p>Background</p> <p>DNA methylation in the <it>SHOX2 </it>locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with <it>SHOX2 </it>gene expression and/or copy number alterations. An amplification of the <it>SHOX2 </it>gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.</p> <p>Methods</p> <p><it>SHOX2 </it>expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect <it>SHOX2 </it>DNA methylation levels. <it>SHOX2 </it>expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.</p> <p>Results</p> <p>A hypermethylation of the <it>SHOX2 </it>locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the <it>SHOX2 </it>gene showed no difference.</p> <p>Conclusions</p> <p>Frequent gene amplification correlated with hypermethylation of the <it>SHOX2 </it>gene locus. This concerted effect qualifies <it>SHOX2 </it>DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.</p

SHOX2 DNA Methylation is a Biomarker for the diagnosis of lung cancer based on bronchial aspirates

<p>Abstract</p> <p>Background</p> <p>This study aimed to show that SHOX2 DNA methylation is a tumor marker in patients with suspected lung cancer by using bronchial fluid aspirated during bronchoscopy. Such a biomarker would be clinically valuable, especially when, following the first bronchoscopy, a final diagnosis cannot be established by histology or cytology. A test with a low false positive rate can reduce the need for further invasive and costly procedures and ensure early treatment.</p> <p>Methods</p> <p>Marker discovery was carried out by differential methylation hybridization (DMH) and real-time PCR. The real-time PCR based HeavyMethyl technology was used for quantitative analysis of DNA methylation of SHOX2 using bronchial aspirates from two clinical centres in a case-control study. Fresh-frozen and Saccomanno-fixed samples were used to show the tumor marker performance in different sample types of clinical relevance.</p> <p>Results</p> <p>Valid measurements were obtained from a total of 523 patient samples (242 controls, 281 cases). DNA methylation of SHOX2 allowed to distinguish between malignant and benign lung disease, i.e. abscesses, infections, obstructive lung diseases, sarcoidosis, scleroderma, stenoses, at high specificity (68% sensitivity [95% CI 62-73%], 95% specificity [95% CI 91-97%]).</p> <p>Conclusions</p> <p>Hypermethylation of SHOX2 in bronchial aspirates appears to be a clinically useful tumor marker for identifying subjects with lung carcinoma, especially if histological and cytological findings after bronchoscopy are ambiguous.</p

Characterization and Identification of Epigenetic Breast Cancer Biomarkers

Biomarker, die eine Diagnose der Brustkrebserkrankung zu einem frühen Stadium ermöglichen, die eine Prognose des Ausgangs der Erkrankung oder die das Ansprechen auf eine bestimmte Behandlung erlauben, sind für die Verbesserung des Managements der Erkrankung von großem klinischen Interesse. DNA Methylierung spielt eine wesentliche Rolle bei der Karzinogenese und stellt damit eine wertvolle Quelle für Brustkrebsbiomarker dar. Ziel dieser Arbeit war die Charakterisierung bekannter Methylierungsbiomarker, wie der prognostischen Biomarker TFF1, PITX2 und uPA und der diagnostischen Biomarker RASSF1A, LIMK1, SLIT2, SLITRK1 und HS3ST2. Zusätzlich wurden Kandidatengene analysiert, um neue Biomarker zu identifizieren. Das PITX2 Gen kodiert für drei Transkripte: PITX2 A, B and C. Die Isoform C wird durch einen alternativen Promotor reguliert. Eine Analyse von Brustkrebszelllinien zeigte eine Korrelation der Methylierung der Promotorregionen mit der Expression der entsprechenden Transkripte. Es konnte gezeigt werden, dass Promotormethylierung der Transkriptvarianten A und B ein stärkerer prognostischer Biomarker verglichen mit der Promotormethylierung der Variante C ist. Die Analyse von Tumoren von 384 Brustkrebspatientinnen im Rahmen einer DNA Mikroarraystudie führte zu der Identifizierung von BMP4, NR5A1, BARX1 und FGF4 als Methylierungsbiomarker, welche eine Vorhersage des Ausgangs der Krankheit bei Lymphknoten-positiven, Steroidhormonrezeptor-positiven Brustkrebspatientinnen erlaubt, die eine adjuvante Anthrazyklin-basierte Chemotherapie erhalten haben. Bis heute ist nicht klar, wie die verschiedenen Komponenten des Tumors zu der gemessenen Methylierung beitragen. Die Lasermikrodissektion ist eine Technologie, die die Isolierung bestimmter Zellen ermöglicht aber nicht genügend Material für die meisten Standardmethoden zur Methylierungsmessung liefert. Eine zusätzliche Herausforderung tritt bei Proben von Formalin-fixierten und Paraffin-eingebetteten Geweben auf, welche nur fragmentierte DNA niedriger Qualität enthält. Deshalb wurde eine Methode zur simultanen Analyse mehrerer Gene bei einer limitierten Anzahl von dissezierten Zellen aus Formalin-fixierten und Paraffin-eingebetteten Geweben entwickelt. Diese Methode umfasst: 1. ein optimiertes, für die Konversion von DNA aus einigen wenigen Zellen geeignetes, Bisulfitbehandlungsprotokoll, 2. sequentielle PCR Amplifikationen und 3. eine neue Methode zur quantitativen Bisulfitsequenzierung basierend auf der Einführung eines Normalisierungssignals in das PCR Produkt. Diese Methode wurde verwendet, um die Methylierung der Gene PITX2, RASSF1A, uPA, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2 und TFF1 bei drei ausgewählten, invasiven duktalen Brustkrebsfällen zu analysieren. Mikrodissezierte Proben wurden von intraduktalem und invasivem Karzinom, Stroma, Tumor-infiltrierenden Lymphozyten, Fettgewebe, Brustwandmuskel, Adenose und normalem Drüsenepithel gewonnen. Alle analysierten Gene zeigten Hypermethylierung in invasiven und intraduktalen Krebszellen verglichen mit den anderen Gewebekomponenten, mit Ausnahme von TFF1, welches eine umgekehrte Methylierung aufwies. Einige Gene zeigten geringe Methylierung im normalen anliegenden Brustgewebe und in Tumor-infiltrierenden Lymphozyten. Die Methylierung variierte wenig innerhalb eines Gewebetypes und zwischen verschiedenen Paraffinblöcken eines Brustkrebsfalls. Die entwickelte Methode ist ein leistungsstarkes Werkzeug für die Methylierungsanalyse von multiplen Markern in verschiedenen mikrodissezierten Gewebekomponenten und kann hilfreich für die Analyse der verschiedenen Übergangsstadien von Brust und anderen Krebsformen sein. Diese Methode ist außerdem für viele Anwendungen neben der Analyse mikrodissezierter Zellen geeignet, wie z.B. Methylierungsanalyse mehrerer Gene in Körperflüssigkeiten (Serum, Plasma, Urin), Spülflüssigkeiten, disseminierten Tumorzellen, embryonalen Zellen, isolierten Stammzellen, etc.Biomarkers that enable the diagnosis of breast cancer at an early state, which allow for a prognosis of the outcome of the disease, and which are suitable for predicting the response to a certain treatment are highly desired for clinical use to improve patient management. DNA methylation has been shown to play a major role in carcinogenesis, suggesting that DNA methylation analysis may be a valuable source for breast cancer biomarkers. This work aimed to characterize known methylation biomarkers, such as the prognostic biomarkers: TFF1, PITX2 and uPA and the diagnostic biomarkers: RASSF1A, LIMK1, SLIT2, SLITRK1 and HS3ST2. In addition, candidate genes were analyzed in order to identify novel biomarkers. The PITX2 gene drives three transcripts: PITX2 A, B and C. The isoform C is regulated by an alternative promoter. The analysis of breast cancer cell lines reveal a correlation of methylation of the promoter regions and the expression of the respective transcripts. It has been shown that promoter methylation of the transcript variants A and B is a stronger prognostic biomarker as compared with the methylation of the promoter of the variant C. The analysis of tumors from 384 breast cancer patients in the course of a DNA microarray study led to the identification of promoter methylation of the genes BMP4, NR5A1, BARX1 and FGF4 as biomarkers, which allow for a prediction of disease outcome in lymph node positive, steroid-hormone receptor-positive breast cancer patients, who have received adjuvant anthracycline-based chemotherapy. Up to now, it has not been clear how the different components of the cancerous tissue contribute to the observed methylation. Laser microdissection is a technology which allows for the isolation of distinct cells but does not provide sufficient material for most standard methylation assays. An additional challenge has been encountered for samples from formalin-fixed and paraffin-embedded tissues, which contain only fragmented DNA of poor quality. Therefore, a method to analyze several genes simultaneously in a limited number of cells dissected from sections from formalin-fixed and paraffin-embedded tissues was developed. This method comprises: 1. an optimized bisulfite treatment protocol suitable for the conversion of DNA obtained from a few cells, 2. sequential PCR amplifications, and 3. a novel method for quantitative bisulfite sequencing based on the incorporation of a normalization domain into the PCR product. The method was used to analyze the methylation of the genes PITX2, RASSF1A, uPA, LHX3, PITX3, LIMK1, SLITRK1, SLIT2, HS3ST2 and TFF1 in three selected cases of invasive ductal carcinoma of the breast. The microdissected samples were obtained from intraductal and invasive carcinoma, stroma, tumor infiltrating lymphocytes, adipose tissue, chest wall muscle, adenosis, and normal ductal epithelia. All analyzed genes showed hypermethylation in invasive and intraductal carcinoma cells as compared to other tissue components with the exception of TFF1, which showed inverse methylation. Some genes showed low level methylation in normal adjacent breast tissue and tumor infiltrating lymphocytes. The methylation of the individual genes varied little within one tissue type and between different blocks from one case of breast cancer. The developed method is a powerful new tool for analyzing the methylation of multiple markers in different microdissected tissue components and may help to investigate different transitional states of breast and other cancers. However, this method is suitable for many applications beyond laser microdissection analysis, such as methylation analysis of several genes in body fluids (serum, plasma, urine), lavages, disseminated cancer cell, embryonic cells, isolated stem cells and so forth

Rapid analysis of CpG methylation patterns using RNase T1 cleavage and MALDI-TOF

Here, we introduce a method for the fast and accurate analysis of DNA methylation based on bisulfite-treated DNA. The target region is PCR amplified using a T7 RNA polymerase promoter-tagged primer. A subsequent in vitro transcription leads to a transcript which contains guanosine residues only at sites that contained methylated cytosines before bisulfite treatment. In a single tube reaction using guanosine-specific cleavage by RNase T1, a specific pattern of RNA fragments is formed. This pattern directly represents the methylation state of the sample DNA and is analyzed using matrix-assisted laser desorption ionization time-of-flight technology. This method was successfully applied to the analysis of artificially methylated and unmethylated DNA, mixtures thereof and colon DNA samples. The applicability for the analysis of both PCR products and cloned PCR products is demonstrated. The observed methylation patterns were confirmed by bisulfite sequencing

Fibroblast growth factor (FGF), FGF receptor (FGFR), and cyclin D1 (CCND1) DNA methylation in head and neck squamous cell carcinomas is associated with transcriptional activity, gene amplification, human papillomavirus (HPV) status, and sensitivity to tyrosine kinase inhibitors

Background!#!Dysregulation of fibroblast growth factor receptor (FGFR) signaling pathway has been observed in head and neck squamous cell carcinoma (HNSCC) and is a promising therapeutic target for selective tyrosine kinase inhibitors (TKIs). Potential predictive biomarkers for response to FGFR-targeted therapies are urgently needed. Understanding the epigenetic regulation of FGF pathway related genes, i.e. FGFRs, FGFs, and CCND1, could enlighten the way towards biomarker-selected FGFR-targeted therapies.!##!Methods!#!We performed DNA methylation analysis of the encoding genes FGFR1, FGFR2, FGFR3, FGFR4, FGF1-14, FGF16-23, and CCND1 at single CpG site resolution (840 CpG sites) employing The Cancer Genome Research Atlas (TCGA) HNSCC cohort comprising N = 530 tumor tissue and N = 50 normal adjacent tissue samples. We correlated DNA methylation to mRNA expression with regard to human papilloma virus (HPV) and gene amplification status. Moreover, we investigated the correlation of methylation with sensitivity to the selective FGFR inhibitors PD 173074 and AZD4547 in N = 40 HPV(-) HNSCC cell lines.!##!Results!#!We found sequence-contextually nuanced CpG methylation patterns in concordance with epigenetically regulated genes. High methylation levels were predominantly found in the promoter flank and gene body region, while low methylation levels were present in the central promoter region for most of the analyzed CpG sites. FGFRs, FGFs, and CCND1 methylation differed significantly between tumor and normal adjacent tissue and was associated with HPV and gene amplification status. CCND1 promoter methylation correlated with CCND1 amplification. For most of the analyzed CpG sites, methylation levels correlated to mRNA expression in tumor tissue. Furthermore, we found significant correlations of DNA methylation of specific CpG sites with response to the FGFR1/3-selective inhibitors PD 173074 and AZD4547, predominantly within the transcription start site of CCND1.!##!Conclusions!#!Our results suggest an epigenetic regulation of CCND1, FGFRs, and FGFs via DNA methylation in HNSCC and warrants further investigation of DNA methylation as a potential predictive biomarker for response to selective FGFR inhibitors in clinical trials

Clinical performance validation of PITX2 DNA methylation as prognostic biomarker in patients with head and neck squamous cell carcinoma.

Despite advances in combined modality therapy, outcomes in head and neck squamous cell cancer (HNSCC) remain dismal with five-year overall survival rates of less than 50%. Prognostic biomarkers are urgently needed to identify patients with a high risk of death after initial curative treatment. Methylation status of the paired-like homeodomain transcription factor 2 (PITX2) has recently emerged as a powerful prognostic biomarker in various cancers. In the present study, the clinical performance of PITX2 methylation was validated in a HNSCC cohort by means of an independent analytical platform (Infinium HumanMethylation450 BeadChip, Illumina, Inc.).A total of 528 HNSCC patients from The Cancer Genome Atlas (TCGA) were included in the study. Death was defined as primary endpoint. PITX2 methylation was correlated with overall survival and clinicopathological parameters.PITX2 methylation was significantly associated with sex, tumor site, p16 status, and grade. In univariate Cox proportional hazards analysis, PITX2 hypermethylation analyzed as continuous and dichotomized variable was significantly associated with prolonged overall survival of HNSCC patients (continuous: hazard ratio (HR) = 0.19 [95%CI: 0.04-0.88], p = 0.034; dichotomized: HR = 0.52 [95%CI: 0.33-0.84], p = 0.007). In multivariate Cox analysis including established clinicopathological parameters, PITX2 promoter methylation was confirmed as prognostic factor (HR = 0.28 [95%CI: 0.09-0.84], p = 0.023).Using an independent analytical platform, PITX2 methylation was validated as a prognostic biomarker in HNSCC patients, identifying patients that potentially benefit from intensified surveillance and/or administration of adjuvant/neodjuvant treatment, i.e. immunotherapy

PD-1 (PDCD1) Promoter Methylation Is a Prognostic Factor in Patients With Diffuse Lower-Grade Gliomas Harboring Isocitrate Dehydrogenase (IDH) Mutations

Immune checkpoints are important targets for immunotherapies. However, knowledge on the epigenetic modification of immune checkpoint genes is sparse. In the present study, we investigated promoter methylation of CTLA4, PD-L1, PD-L2, and PD-1 in diffuse lower-grade gliomas (LGG) harboring isocitrate dehydrogenase (IDH) mutations with regard to mRNA expression levels, clinicopathological parameters, previously established methylation subtypes, immune cell infiltrates, and survival in a cohort of 419 patients with IDH-mutated LGG provided by The Cancer Genome Atlas. PD-L1, PD-L2, and CTLA-4 mRNA expression levels showed a significant inverse correlation with promoter methylation (PD-L1: p=0.005; PD-L2: p < 0.001; CTLA-4: p b 0.001). Furthermore, immune checkpoint methylation was significantly associated with age (PD-L2: p = 0.003; PD-1: p = 0.015), molecular alterations, i.e. MGMT methylation (PD-L1: p < 0.001; PD-L2: p < 0.001), ATRX mutations (PD-L2: p < 0.001, PD-1: p = 0.001), and TERT mutations (PD-L1: p= 0.035, PD-L2: p < 0.001, PD-1: p b 0.001, CTLA4: p < 0.001) as well a smethylation subgroups and immune cell infiltrates. In multivariate Cox proportional hazard analysis, PD-1 methylation qualified as strong prognostic factor (HR = 0.51 [ 0.34-0.76], p=0.001). Our findings suggest an epigenetic regulation of immune checkpoint genes via DNA methylation in LGG. PD-1 methylation may assist the identification of patients that might benefit from an alternative treatment, particularly in the context of emerging immunotherapies. (c) 2018 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY licens