Michael Fleischhacker

Cell-free DNA in the blood as a solid tumor biomarker--a critical appraisal of the literature.

Circulating cell-free DNA (cfDNA) has been suggested as a cancer biomarker. Several studies assessed the usefulness of quantitative and qualitative tumor-specific alterations of cfDNA, such as DNA strand integrity, frequency of mutations, abnormalities of microsatellites, and methylation of genes, as diagnostic, prognostic, and monitoring markers in cancer patients. Most of the papers that could be evaluated in this review resulted in a positive conclusion. However, methodical diversity without the traceability of data and differently designed and often underpowered studies resulted in divergent results between studies. In addition, the limited diagnostic sensitivity and specificity of cfDNA alterations temper the effusive hope of novel tumor markers, raising similar issues as those for other tumor markers. To validate the actual clinical validity of various cfDNA alterations as potential cancer biomarkers in practice for individual tumor types, the main problems of the observed uncertainties must be considered in future studies. These include methodical harmonization concerning sample collection, processing, and analysis with the traceability of measurement results as well as the realization of well-designed prospective studies based on power analysis and sample size calculations.

Changes in Concentration of DNA in Serum and Plasma during Storage of Blood Samples

Cell-free DNA in serum and plasma has been suggested to have diagnostic potential because associations between DNA concentrations and several disorders have been described (1). The concentration of cell-free DNA circulating in plasma and serum has been analyzed in several studies and used as an interchangeable index of the quantity of circulating DNA in blood (2)(3)(4). However, it is known that the DNA concentration in serum is ∼3- to 24-fold higher than in plasma (3)(4)(5)(6). Recently published articles in this journal showed that various preanalytical factors of blood sampling and processing can affect the DNA concentration in plasma (5)(7)(8), but these findings do not explain the difference between plasma and serum concentrations of DNA mentioned above. Comparative investigations of the preanalytical conditions influencing the DNA concentration in serum and plasma are lacking. In addition, reference intervals for the concentration of cell-free DNA in serum were established without considering these factors (9). Thus, to complement the data of Lui et al. (5), we analyzed the influence of time delay in blood processing for plasma and serum at room temperature and at 4 °C. Venous blood samples from 10 healthy volunteers (5 females and 5 males; mean age, 42 years) were simultaneously collected in …

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.

Detection of Amplifiable Messenger RNA in the Serum of Patients with Lung Cancer

Abstract: Recently, in addition to the detection of circulating tumor cells in peripheral blood of patients with solid tumors, the presence of free circulating nucleic acids in the plasma and serum has also been described. We have focused on the possibility of isolating and amplifying intact extracellular, tumor‐related mRNA from the plasma/serum of patients with lung cancer. For this purpose, we established several RT‐PCR‐based amplification systems for the detection of a panel of five different genes. The expression of these genes was either shown to be restricted to lung tissue or associated with malignancy. We examined two small groups of 18 patients with lung cancer before and during chemotherapy, respectively. The message for β‐actin (control for integrity of the RNA) was detected in all of the analyzed sera from the control group and patients with lung cancer. Analysis of CK‐19 expression was positive in the majority of tumor patients, but positive results were also shown in all of the control sera. The expression of MAGE‐2 and TTF‐1 genes was not observed in any of the patients in either the lymphocyte preparations or serum samples. Expression of the PGP 9.5 gene was observed in the cells of all 18 patients, but mRNA in the serum was only detectable in one case. The hnRNP‐B1 mRNA was detectable in 14/18 sera, and Her2/neu‐specific mRNA could be amplified from the serum of 7/18 patients. Combining the last two markers, we were able to detect all patients with a malignant lung tumor.

Detection of Microsatellite Alterations in the DNA Isolated from Tumor Cells and from Plasma DNA of Patients with Lung Cancer

Abstract: In this paper, we show that the same panel of three microsatellite markers is useful for the detection of alterations in the DNA of tumor cells and plasma from patients diagnosed with SCLC and NSCLC. In 31% of the SCLC patients, we detected a microsatellite alteration(s) or LOH in at least one locus. In the group of patients diagnosed with NSCLC, a microsatellite alteration or LOH was detected in at least one locus in 33% of the patients. In all but 2 patients, the identical alteration observed in the DNA from tumor cells was also detected in the DNA isolated from blood plasma. This work confirms the results described by other groups and it extends the diagnostic possibilities of finding tumor cell‐specific DNA alterations also in the DNA freely circulating in plasma and serum of patients with cancer.

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.

Cell-free DNA resuscitated for tumor testing

Extracellular DNA floating around in blood plasma provides an accessible template for detecting mutations associated with tumors. A new technique is able to quantify such mutated DNA and predict relapse in individuals with colorectal cancer. The technique complements other approaches, such as the analysis of tumor cells in the plasma (pages 985–990).