Sabine Weickmann

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.

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

BackgroundThis 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.MethodsMarker 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.ResultsValid 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%]).ConclusionsHypermethylation 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.

Methods for isolation of cell-free plasma DNA strongly affect DNA yield.

Extracellular nucleic acids are present in plasma, serum, and other body fluids and their analysis has gained increasing attention during recent years. Because of the small quantity and highly fragmented nature of cell-free DNA in plasma and serum, a fast, efficient, and reliable isolation method is still a problem and so far there is no agreement on a standardized method. We used spin columns from commercial suppliers (QIAamp DNA Blood Midi Kit from Qiagen; NucleoSpin Kit from Macherey-Nagel; MagNA Pure isolation system from Roche Diagnostics) to isolate DNA from 44 plasma samples in parallel at laboratories in Berlin and Munich. DNA in all samples was quantified by real-time PCR on a LightCycler 480 using three different targets (GAPDH, ß-globin, ERV). The quantities of cell-free DNA for the different isolation methods and genes varied between medians of 1.6 ng/mL and 28.1 ng/mL. This considerable variation of absolute DNA values was mainly caused by the use of different isolation methods (p<0.0001). Comparable results were achieved by the use of the genes GAPDH and ERV while higher values were obtained by use of ß-globin. The laboratory site had only minor influence on DNA yield when manual extraction methods were used.

Integrity of Cell‐Free Plasma DNA in Patients with Lung Cancer and Nonmalignant Lung Disease

In several papers an increased quantity of cell‐free plasma DNA as well as the presence of long DNA fragments in cell‐free plasma and serum has been described. We isolated cell‐free DNA from plasma, serum, and bronchial lavage supernatants from 33 lung cancer patients and 27 patients with a benign lung disease. The DNA was amplified by real‐time PCR, and the quantity as well as the DNA integrity was determined. We did not find significant differences between the patient populations. Our results led us to conclude that this method is not useful in a diagnostic setting and is not able to differentiate between lung cancer patients and patients with a benign lung disease.

An Improved Method for the Isolation of Free‐Circulating Plasma DNA and Cell‐Free DNA from Other Body Fluids

Cell‐free DNA is of increasing interest in different fields, such as cancer research, prenatal diagnosis, and the diagnosis of nonmalignant diseases. The quantity of free‐circulating DNA in plasma, serum, and other body fluids is usually low and its isolation is still a challenge. Here we evaluate the application of the new commercially available NucleoSpin Plasma XS Kit for the isolation of cell‐free DNA from plasma, serum, and cell‐free bronchial lavage samples. The NucleoSpin columns proved superior to the QIAamp system in terms of DNA yield, purity, and retrieval of small DNA fragments. The procedure is fast and easy to standardize and might be valuable for laboratories working in the field of CNAPS.

miR‐199a‐3p and miR‐214‐3p improve the overall survival prediction of muscle‐invasive bladder cancer patients after radical cystectomy

To improve the clinical decision‐making regarding further treatment management and follow‐up scheduling for patients with muscle‐invasive bladder cancer (MIBC) after radical cystectomy (RC), a better prediction accuracy of prognosis for these patients is urgently needed. The objective of this study was to evaluate the validity of differentially expressed microRNAs (miRNAs) based on a previous study as prognostic markers for overall survival (OS) after RC in models combined with clinicopathological data. The expression of six miRNAs (miR‐100‐5p, miR‐130b‐3p, miR‐141‐3p, miR‐199a‐3p, miR‐205‐5p, and miR‐214‐3p) was measured by RT‐qPCR in formalin‐fixed, paraffin‐embedded tissue samples from 156 MIBC patients who received RC in three urological centers. Samples from 2000 to 2013 were used according to their tissue availability, with follow‐up until June 2016. The patient cohort was randomly divided into a training (n = 100) and test set (n = 56). Seventy‐three samples from adjacent normal tissue were used as controls. Kaplan–Meier, univariate and multivariate Cox regression, and decision curve analyses were carried out to assess the association of clinicopathological variables and miRNAs to OS. Both increased (miR‐130b‐3p and miR‐141‐3p) and reduced (miR‐100‐5p, miR‐199a‐3p, and miR‐214‐3p) miRNA expressions were found in MIBC samples in comparison to nonmalignant tissue samples (P < 0.0001). miR‐199a‐3p and miR‐214‐3p were independent markers of OS in Cox regression models with the significant clinicopathological variables age, tumor status, and lymph node status. The prediction model with the clinicopathological variables was improved by these two miRNAs in both sets. The predictive benefit was confirmed by decision curve analysis. In conclusion, the inclusion of both miRNAs into models based on clinical data for the outcome prediction of MIBC patients after RC could be a valuable approach to improve prognostic accuracy.

Detection of tumor-specific mRNA in cell-free bronchial lavage supernatant in patients with lung cancer.

Abstract: Bronchoscopy is a standard procedure in the workup of patients with suspicious pulmonary lesions. We wondered whether it is possible to isolate malignancy‐associated mRNA from cell‐free lavage supernatant. Extracellular mRNA from cell‐free lavage supernatant of 25 patients with lung cancer (23 with non‐small cell lung cancer, 2 with small cell lung cancer) was isolated, reverse‐transcribed, and amplified by reverse transcriptase polymerase chain reaction. The quantity and quality of the isolated RNA were checked after cDNA synthesis by amplification with β‐actin‐specific primers. Afterwards, a panel of eight genes known to be expressed in lung tumors was used for the detection of tumor‐associated mRNA expression in lavage supernatant and serum. mRNA coding for β‐actin could be isolated from lavage supernatant of all 25 patients. In addition, the expression of at least one tumor‐associated gene was detectable in all patients. These results show that intact mRNA can be isolated from cell‐free lavage supernatant and that its quantity and quality are sufficient for the detection of tumor‐associated gene expression alterations. This may open new possibilities for the diagnosis of lung cancer.

Tissue-Based MicroRNAs as Predictors of Biochemical Recurrence after Radical Prostatectomy: What Can We Learn from Past Studies?

With the increasing understanding of the molecular mechanism of the microRNAs (miRNAs) in prostate cancer (PCa), the predictive potential of miRNAs has received more attention by clinicians and laboratory scientists. Compared with the traditional prognostic tools based on clinicopathological variables, including the prostate-specific antigen, miRNAs may be helpful novel molecular biomarkers of biochemical recurrence for a more accurate risk stratification of PCa patients after radical prostatectomy and may contribute to personalized treatment. Tissue samples from prostatectomy specimens are easily available for miRNA isolation. Numerous studies from different countries have investigated the role of tissue-miRNAs as independent predictors of disease recurrence, either alone or in combination with other clinicopathological factors. For this purpose, a PubMed search was performed for articles published between 2008 and 2017. We compiled a profile of dysregulated miRNAs as potential predictors of biochemical recurrence and discussed their current clinical relevance. Because of differences in analytics, insufficient power and the heterogeneity of studies, and different statistical evaluation methods, limited consistency in results was obvious. Prospective multi-institutional studies with larger sample sizes, harmonized analytics, well-structured external validations, and reasonable study designs are necessary to assess the real prognostic information of miRNAs, in combination with conventional clinicopathological factors, as predictors of biochemical recurrence.

Comparison of Nucleosomes and Quantitative PCR Using Diverse DNA Isolation Methods

Reliable standardization for the isolation and detection methods prior to quantification of cell-free DNA are still lacking. QIAamp DNA Blood Midi Kit (Q), the NucleoSpin-Kit (MN), and the MagNA-Pure isolation system (RD) were compared on the amount of DNA isolated versus the immunological quantification of circulating nucleosomes using the Cell Death Detection ELISA plus (RD). Forty-four plasma samples were used with each system in parallel at laboratories in Berlin and Munich. Subsequently, DNA was quantified by real-time PCR on a LightCycler 480 (RD) in Berlin using targets for s-globin. Correlations of plasma nucleosomes and cell-free DNA measured by real-time PCR ranged between R = 0.32 and R = 0.64 and were comparable between the two laboratories. Large differences in the amounts of cell-free DNA were observed between the diverse isolation methods: the RD isolation system gave the highest DNA yield followed by MN and Q systems. Most comparable results with nucleosome ELISA were achieved using the Roche system.

miR-9-5p in Nephrectomy Specimens is a Potential Predictor of Primary Resistance to First-Line Treatment with Tyrosine Kinase Inhibitors in Patients with Metastatic Renal Cell Carcinoma

Approximately 20–30% of patients with metastatic renal cell carcinoma (mRCC) in first-line treatment with tyrosine kinase inhibitors (TKIs) do not respond due to primary resistance to this drug. At present, suitable robust biomarkers for prediction of a response are not available. Therefore, the aim of this study was to evaluate a panel of microRNAs (miRNAs) in nephrectomy specimens for use as predictive biomarkers for TKI resistance. Archived formalin-fixed, paraffin embedded nephrectomy samples from 60 mRCC patients treated with first-line TKIs (sunitinib, n = 51; pazopanib, n = 6; sorafenib, n = 3) were categorized into responders and non-responders. Using the standard Response Evaluation Criteria in Solid Tumors, patients with progressive disease within 3 months after the start of treatment with TKI were considered as non-responders and those patients with stable disease and complete or partial response under the TKI treatment for at least 6 months as responders. Based on a miRNA microarray expression profile in the two stratified groups of patients, seven differentially expressed miRNAs were validated using droplet digital reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) assays in the two groups. Receiver operating characteristic curve analysis and binary logistic regression of response prediction were performed. MiR-9-5p and miR-489-3p were able to discriminate between the two groups. MiR-9-5p, as the most significant miRNA, improved the correct prediction of primary resistance against TKIs in comparison to that of conventional clinicopathological variables. The results of the decision curve analyses, Kaplan-Meier analyses and Cox regression analyses confirmed the potential of miR-9-5p in the prediction of response to TKIs and the prediction of progression-free survival after the initiation of TKI treatment.