Quantification of cell-free mSHOX2 Plasma DNA for therapy monitoring in advanced stage non-small cell (NSCLC) and small-cell lung cancer (SCLC) patients.

PURPOSE: Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy regimens we need better methods for the assessment of a therapy response. MATERIAL AND METHODS: In a pilot study we prospectively enrolled 36 patients with advanced NSCLC and SCLC (34 stage IV, 2 stage IIIB) of whom 34 received standard platinum-based chemo/radiotherapy and two were treated with a tyrosine kinase inhibitor. We measured the levels of extracellular methylated SHOX2 DNA (mSHOX2) in plasma before and during therapy until re-staging. The mSHOX2 analysis was blinded with respect to the clinical data making it an observational study. RESULTS: According to the re-staging of 31 first-line patients, 19 patients were classified as non-responders while 12 patients were in the responder group. We observed a tight correlation between radiological data and the change of plasma mSHOX2 level as the equivalent for a therapy response. A ROC analysis showed a high discriminatory power for both patient groups already one week after therapy start (AUC 0.844). Additionally, a Kaplan-Meier and Cox Proportional Hazards analyses revealed a strong relationship between survival and plasma mSHOX2 value p ≤ 0.001 (hazard ratio 11.08) providing some evidence for mSHOX2 also being a predictive marker. CONCLUSION: The longitudinal measurement of extracellular plasma mSHOX2 DNA yields information about the response to cytotoxic treatment and allows an early assessment of treatment response for lung cancer patients. If confirmed in a larger study this would be a valuable tool for selecting and guiding a cytotoxic treatment.

Glyoxylate, a new marker metabolite of type 2 diabetes.

Type 2 diabetes (T2D) is characterized by a variety of metabolic impairments that are closely linked to nonenzymatic glycation reactions of proteins and peptides resulting in advanced glycation end-products (AGEs). Reactive aldehydes derived from sugars play an important role in the generation of AGEs. Using metabolite profiling to characterize human plasma from diabetic versus nondiabetic subjects we observed in a recent study that the reactive aldehyde glyoxylate was increased before high levels of plasma glucose, typical for a diabetic condition, could be measured. Following this observation, we explored the relevance of increased glyoxylate in diabetic subjects and in diabetic C57BLKS/J-Lepr (db/db (-/-)) mice in the pathophysiology of diabetes. A retrospective study using samples of long-term blood donors revealed that glyoxylate levels unlike glucose levels became significantly elevated up to 3 years prior to diabetes diagnosis (difference to control P = 0.034). Elevated glyoxylate levels impact on newly identified mechanisms linking hyperglycemia and AGE production with diabetes-associated complications such as diabetic nephropathy. Glyoxylate in its metabolic network may serve as an early marker in diabetes diagnosis with predictive qualities for associated complications and as potential to guide the development of new antidiabetic therapies.

A new metabolomic signature in type-2 diabetes mellitus and its pathophysiology.

OBJECTIVE: The objective of the current study was to find a metabolic signature associated with the early manifestations of type-2 diabetes mellitus. RESEARCH DESIGN AND METHOD: Modern metabolic profiling technology (MxP™ Broad Profiling) was applied to find early alterations in the plasma metabolome of type-2 diabetic patients. The results were validated in an independent study. Eicosanoid and single inon monitoring analysis (MxP™ Eicosanoid and MxP™ SIM analysis) were performed in subsets of samples. RESULTS: A metabolic signature including significantly increased levels of glyoxylate as a potential novel marker for early detection of type-2 diabetes mellitus was identified in an initial study (Study1). The signature was significantly altered in fasted diabetic and pre-diabetic subjects and in non-fasted subjects up to three years prior to the diagnosis of type-2 diabetes; most alterations were also consistently found in an independent patient group (Study 2). In Study 2 diabetic and most control subjects suffered from heart failure. In Study 1 a subgroup of diabetic subjects, with a history of use of anti-hypertensive medication further showed a more pronounced increase of glyoxylate levels, compared to a non-diabetic control group when tested in a hyperglycemic state. In the context of a prior history of anti-hypertensive medication, alterations in hexosamine and eicosanoid levels were also found. CONCLUSION: A metabolic signature including glyoxylate was associated with type-2 diabetes mellitus, independent of the fasting status and of occurrence of another major disease. The same signature was also found to be associated with pre-diabetic subjects. Glyoxylate levels further showed a specifically strong increase in a subgroup of diabetic subjects. It could represent a new marker for the detection of medical subgroups of diabetic subjects.

Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling.

BACKGROUND: Metabolomics is a valuable tool with applications in almost all life science areas. There is an increasing awareness of the essential need for high-quality biospecimens in studies applying omics technologies and biomarker research. Tools to detect effects of both blood and plasma processing are a key for assuring reproducible and credible results. We report on the response of the human plasma metabolome to common preanalytical variations in a comprehensive metabolomics analysis to reveal such high-quality markers. METHODS: Human EDTA blood was subjected to preanalytical variations while being processed to plasma: microclotting, prolonged processing times at different temperatures, hemolysis, and contamination with buffy layer. In a second experiment, EDTA plasma was incubated at different temperatures for up to 16 h. Samples were subjected to GC-MS and liquid chromatography-tandem mass spectrometry-based metabolite profiling (MxP™ Broad Profiling) complemented by targeted methods, i.e., sphingoids (as part of MxP™ Lipids), MxP™ Catecholamines, and MxP™ Eicosanoids. RESULTS: Short-term storage of blood, hemolysis, and short-term storage of noncooled plasma resulted in statistically significant increases of 4% to 19% and decreases of 8% to 12% of the metabolites. Microclotting, contamination of plasma with buffy layer, and short-term storage of cooled plasma were of less impact on the metabolome (0% to 11% of metabolites increased, 0% to 8% decreased). CONCLUSIONS: The response of the human plasma metabolome to preanalytical variation demands implementation of thorough quality assurance and QC measures to obtain reproducible and credible results from metabolomics studies. Metabolites identified as sensitive to preanalytics can be used to control for sample quality.

The role of DNA methylation as biomarkers in the clinical management of lung cancer.

It is now widely accepted that cancer is caused by complex interactions between genetic and epigenetic factors and the environment. Only in the last 20 years, DNA methylation has been recognized as an epigenetic mechanism, which plays a major role during the development and progression of cancers. Accordingly, DNA methylation profiling provides a useful source for biomarkers in distinct clinical questions; for example, risk stratification, diagnosis, staging, prognosis and therapy-response prediction. In the last 10 years, not only has an increase in the number of papers published on this subject been seen, but also an impressive technological advancement allowing for the highly sensitive and accurate quantification of DNA methylation biomarkers in challenging sample types. However, the development of a suitable biomarker with appropriate assay technology is not trivial. This is especially true for the choice of biomarkers used for the management of early diagnosis of lung cancer.

DNA methylation of the homeobox genes PITX2 and SHOX2 predicts outcome in non-small-cell lung cancer patients.

Biomarkers that facilitate prediction of disease progression in lung cancer patients might be clinically valuable in optimizing individualized therapy. In this study, the ability of the DNA methylation biomarkers PITX2 and SHOX2 to predict disease outcome in lung cancer patients has been evaluated. Quantitative, methylation-specific (HeavyMethyl), real-time polymerase chain reaction assays were used to measure DNA methylation of PITX2 and SHOX2 in bisulfite-converted DNA from formalin-fixed, paraffin-embedded tissues from 474 non-small-cell lung cancer patients. In univariate Cox Proportional Hazard analysis, high methylation of SHOX2 and PITX2 was a significant predictor of progression-free survival [SHOX2: n=465, hazard ratio (HR)=1.395 (1.130 to 1.721), P=0.002; PITX2: n=445, HR=1.312 (1.059 to 1.625), P=0.013]. Patients with low methylation of either PITX2 and/or SHOX2 (n=319) showed a significantly higher risk of disease progression as compared with patients with higher methylation of both genes [n=126; HR=1.555 (1.210 to 1.999), P=0.001]. This was particularly true for the subgroup of patients receiving no adjuvant radiotherapy or chemotherapy [n=258, HR=1.838 (1.252 to 2.698), P=0.002]. In multivariate analysis, both biomarkers added significant independent prognostic information to pT, pN, pM, and grade. Another interesting finding of this study was that SHOX2 and PITX2 DNA methylation was shown to be inversely correlated with TTF1 (also known as NKX2-1) expression (PITX2: P=0.018, SHOX2: P<0.001). TFF1 expression was previously found to be associated with improved survival in the same patient cohort. DNA methylation of PITX2 and SHOX2 is an independent prognostic biomarker for disease progression in non-small-cell lung cancer patients.

Performance evaluation of the DNA methylation biomarker SHOX2 for the aid in diagnosis of lung cancer based on the analysis of bronchial aspirates.

In the identification of subjects with lung cancer, increased DNA methylation of the SHOX2 gene locus in bronchial aspirates has previously been proven to be a clinically valuable biomarker. This is particularly true in cases where the cytological and histological results following bronchoscopy are undetermined. This previous case control study was conducted using research assay components and a complex work flow. To facilitate the use in a diagnostic setting, a CE marked in vitro diagnostic test kit to quantify SHOX2 DNA methylation in bronchial aspirates was developed and characterized. The presented assay for measuring SHOX2 DNA methylation in bronchial aspirates is based on two major steps: generation of bisulfite converted template DNA from patient samples followed by subsequent determination of SHOX2 biomarker methylation by real-time PCR. Individual kits for DNA preparation, real-time PCR analysis and work flow control were developed. This study describes the analytical performance (reproducibility, accuracy, interfering substances, cross-reactivity) of the in vitro diagnostic (IVD) test kit 'Epi proLung BL Reflex Assay'. In addition, the intended use of the test was validated in a clinical performance evaluation (case control) study comprised of 250 patients (125 cases, 125 controls). The results describe the test as a robust and reliable diagnostic tool for identifying patients with lung cancer using Saccomanno-fixed bronchial lavage specimens (AUC [95% confidence intervals] = 0.94 [0.91-0.98], sensitivity 78% [69-86]/specificity 96% [90-99]). This test may be used as a diagnostic adjunct to existing clinical and pathological investigations in lung cancer.

SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer in plasma.

INTRODUCTION: Recently, analysis of DNA methylation of the SHOX2 locus was shown to reliably identify lung cancer in bronchial aspirates of patients with disease. As a plasma-based assay would expand the possible applications of the SHOX2 biomarker, this study aimed to develop a modified SHOX2 assay for use in a blood-based test and to analyze the performance of this optimized SHOX2 methylation assay in plasma. METHODS: Quantitative real-time polymerase chain reaction was used to analyze DNA methylation of SHOX2 in plasma samples from 411 individuals. A training study (20 stage IV patients with lung cancer and 20 controls) was performed to show the feasibility of detecting the SHOX2 biomarker in blood and to determine a methylation cutoff for patient classification. The resulting cutoff was verified in a testing study composed of 371 plasma samples from patients with lung cancer and controls. RESULTS: DNA methylation of SHOX2 could be used as a biomarker to distinguish between malignant lung disease and controls at a sensitivity of 60% (95% confidence interval: 53-67%) and a specificity of 90% (95% confidence interval: 84-94%). Cancer in patients with stages II (72%), III (55%), and IV (83%) was detected at a higher sensitivity when compared with stage I patients. Small cell lung cancer (80%) and squamous cell carcinoma (63%) were detected at the highest sensitivity when compared with adenocarcinomas. CONCLUSIONS: SHOX2 DNA methylation is a biomarker for detecting the presence of malignant lung disease in blood plasma from patients with lung cancer.

Correlation of SHOX2 gene amplification and DNA methylation in lung cancer tumors.

BACKGROUND: DNA methylation in the SHOX2 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 SHOX2 gene expression and/or copy number alterations. An amplification of the SHOX2 gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples. METHODS: SHOX2 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 SHOX2 DNA methylation levels. SHOX2 expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH. RESULTS: A hypermethylation of the SHOX2 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 SHOX2 gene showed no difference. CONCLUSIONS: Frequent gene amplification correlated with hypermethylation of the SHOX2 gene locus. This concerted effect qualifies SHOX2 DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.

SHOX2 DNA methylation is a biomarker for the diagnosis of lung cancer based on bronchial aspirates.

BACKGROUND: 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. METHODS: 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. RESULTS: 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%]). CONCLUSIONS: 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.

A novel method for sensitive and specific detection of DNA methylation biomarkers based on DNA restriction during PCR cycling.

DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.

DNA methylation biomarkers of prostate cancer: confirmation of candidates and evidence urine is the most sensitive body fluid for non-invasive detection.

BACKGROUND: A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome-wide search. METHODS: Real-time PCR was used to measure four DNA methylation biomarkers: GSTP1 and three previously unreported candidates associated with the genes RASSF2, HIST1H4K, and TFAP2E in sodium bisulfite-modified DNA. Matched plasma and urine collected prospectively from 142 patients referred for prostate biopsy and 50 young asymptomatic males were analyzed. RESULTS: Analysis of all biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers discriminated PCa from biopsy negative patients with AUCs ranging from 0.64 for HIST1H4K (95% CI 0.55-0.72, P < 0.00001) to 0.69 for GSTP1 (95% CI 0.60-0.77, P < 0.00001). All biomarkers showed minimal correlation with PSA. Multivariate analysis did not yield a panel that significantly improved performance over that of single biomarkers. All biomarkers showed greater sensitivity for PCa in urine than in plasma DNA. CONCLUSIONS: Analysis of the biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers provided information independent of PSA and may warrant inclusion in nomograms for predicting prostate biopsy outcome. The biomarkers' PCa sensitivity was greater for urine than plasma DNA. The biomarker performances in urine DNA should next be validated in formal training and test studies.

Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue.

Regulatory T-cells (Treg) have been the focus of immunologic research due to their role in establishing tolerance for harmless antigens versus allowing immune responses against foes. Increased Treg frequencies measured by mRNA expression or protein synthesis of the Treg marker FOXP3 were found in various cancers, indicating that dysregulation of Treg levels contributes to tumor establishment. Furthermore, they constitute a key target of immunomodulatory therapies in cancer as well as transplantation settings. One core obstacle for understanding the role of Treg, thus far, is the inability of FOXP3 mRNA or protein detection methods to differentiate between Treg and activated T cells. These difficulties are aggravated by the technical demands of sample logistics and processing. Based on Treg-specific DNA demethylation within the FOXP3 locus, we present a novel method for monitoring Treg in human peripheral blood and solid tissues. We found that Treg numbers are significantly increased in the peripheral blood of patients with interleukin 2-treated melanoma and in formalin-fixed tissue from patients with lung and colon carcinomas. Conversely, we show that immunosuppressive therapy including therapeutic antibodies leads to a significant reduction of Treg from the peripheral blood of transplantation patients. In addition, Treg numbers are predictively elevated in the peripheral blood of patients with various solid tumors. Although our data generally correspond to data obtained with gene expression and protein-based methods, the results are less fluctuating and more specific to Treg. The assay presented here measures Treg robustly in blood and solid tissues regardless of conservation levels, promising fast screening of Treg in various clinical settings.

Sensitive detection of colorectal cancer in peripheral blood by septin 9 DNA methylation assay.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC. METHODOLOGY/PRINCIPAL FINDINGS: Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was approximately 20%. CONCLUSIONS/SIGNIFICANCE: Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.

DNA methylation biomarkers for blood-based colorectal cancer screening.

BACKGROUND: Sensitive, specific blood-based tests are difficult to develop unless steps are taken to maximize performance characteristics at every stage of marker discovery and development. We describe a sieving strategy for identifying high-performing marker assays that detect colorectal cancer (CRC)-specific methylated DNA in plasma. METHODS: We first used restriction enzyme-based discovery methods to identify marker candidates with obviously different methylation patterns in CRC tissue and nonpathologic tissue. We then used a selection process incorporating microarrays and/or real-time PCR analysis of tissue samples to further test marker candidates for maximum methylation in CRC tissue and minimum amplification in tissues from both healthy individuals and patients with other diseases. Real-time assays of 3 selected markers were validated with plasma samples from 133 CRC patients and 179 healthy control individuals in the same age range. RESULTS: Restriction enzyme-based testing identified 56 candidate markers. This group was reduced to 6 with microarray and real-time PCR testing. Three markers, TMEFF2, NGFR, and SEPT9, were tested with plasma samples. TMEFF2 methylation was detected in 65% [95% confidence interval, 56%-73%] of plasma samples from CRC patients and not detected in 69% (62%-76%) of the controls. The corresponding results for NGFR were 51% (42%-60%) and 84% (77%-89%); for SEPT9, the values were 69% (60%-77%) and 86% (80%-91%). CONCLUSIONS: The stringent criteria applied at all steps of the selection and validation process enabled successful identification and ranking of blood-based marker candidates.

Comparative DNA methylation analysis in normal and tumour tissues and in cancer cell lines using differential methylation hybridisation.

Immortalized human cancer cell lines are widely used as tools and model systems in cancer research but their authenticity with regard to primary tissues remains a matter of debate. We have used differential methylation hybridisation to obtain comparative methylation profiles from normal and tumour tissues of lung and colon, and permanent cancer cell lines originally derived from these tissues. Average methylation differences only larger than 25% between sample groups were considered for the profiles and with this criterion approximately 1000 probesets, around 2% of the sites represented on the array, indicated differential methylation between normal lung and primary lung cancer tissue, and approximately 700 probesets between normal colon and primary colon cancer tissue. Both hyper- and hypomethylation was found to differentiate normal tissue from cancer tissue. The profiles obtained from these tissue comparisons were found to correspond largely to those from the corresponding cancer cell lines, indicating that the cell lines represent the methylation pattern of the primary tissue rather well. Moreover, the cancer specific profiles were found to be very similar for the two tumour types studied. Tissue specific differential methylation between lung and colon tissues, in contrast, was found to be preserved to a larger extent only in the malignant tissue, but was not preserved well in the cancer cell lines studied. Overall, our data therefore provide further evidence that permanent cell lines are good model systems for cancer specific methylation patterns, but deviate with regard to tissue-specific methylation.