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Papers by Volker Liebenberg
Journal of Thoracic Oncology, Oct 1, 2011
Introduction: Recently, analysis of DNA methylation of the SHOX2 locus was shown to reliably iden... more 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.
Certains aspects de cette invention concernent des compositions et des procedes destines a fourni... more Certains aspects de cette invention concernent des compositions et des procedes destines a fournir des fragments d'ADN a partir d'un echantillon a distance. Dans des aspects particuliers de cette invention, un echantillon a distance contient de l'ADN, l'ADN est isole de l'echantillon a distance, puis traite de maniere a permettre la differenciation de cytosine methylee et non methylee. En outre, des modes de realisation particuliers concernent des compositions et des procedes d'analyse de methylation d'ADN derive d'un echantillon a distance. D'autres aspects concernent des compositions et des procedes d'amplification de genome entier d'ADN traite au bisulfite.
International Journal of Cancer, 1993
The tumor-associated CD30 antigen is presently under study as a target for active specific immuno... more The tumor-associated CD30 antigen is presently under study as a target for active specific immunotherapy of Hodgkin's lymphoma with anti-idiotypic antibodies. Internal image antibodies (Ab2 beta) 9G10 and 14G9 against the CD30-specific antibody HRS-4 (Ab1) have been described, which induce a CD30-specific T- and B-cell response in BALB/c mice and New Zealand white rabbits. In extension of this work, murine monoclonal anti-idiotypic Ab2 beta 9G10, mimicking structures of the nominal CD30 antigen, was used to generate monoclonal Ab3 in mice and polyclonal Ab3 in rabbits with specificity for CD30. The Ab2 beta 9G10-specific murine monoclonal Ab3 4A4 bound specifically to the 120-kDa band of CD30 present on Hodgkin cell lines and Hodgkin tumor tissue, and effectively inhibited binding of Ab1 HRS-4 to Ab2 9G10 as well as to CD30+ cells. Monoclonal Ab3 4A4 was cytotoxic for CD30+ cell lines in vitro and effectively prevented the s.c. growth of L540 cell tumors after passive i.v. administration in a SCID mouse tumor model. While this cytotoxic effect of the IgM subclass monoclonal Ab3 4A4 was due to complement activation, the murine monoclonal Ab1 HRS-4 and a polyclonal Ab3 preparation of IgG-subclass from New Zealand white rabbits were cytotoxic by an antibody-dependent cell-mediated mechanism in vitro. In conclusion, Ab2 beta 9G10 is able to induce a CD30-specific cytotoxic IgG and IgM response. Cytotoxicity was shown to be mediated by complement activation and antibody-dependent cell-mediated cytotoxicity in vitro and in vivo and across species barriers. Thus, the CD30-like Ab2 beta 9G10 may hold promise for effective active specific immunotherapy of human Hodgkin's lymphoma.
Copyright © 2014 Victoria J. Nikiforova et al. This is an open access article distributed under t... more Copyright © 2014 Victoria J. Nikiforova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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
Cancer Research, 2009
Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a surv... more Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a survival rate of over 90% when detected early. Currently, less than half of guideline eligible individuals are screened regularly for CRC, highlighting a need for improved non-invasive methods of early detection. We previously identified and validated a blood-based biomarker, Septin 9, which identifies methylated DNA in plasma from all stages of CRC. We have now conducted a genome-wide screen to identify additional blood-based biomarkers that either augment the performance of Septin 9 for CRC detection or perform individually as well as Septin 9. Methods: Differential methylation hybridization (DMH) via customized high-density microarrays with >50,000 features was used to explore methylation differences between Septin 9 (-) CRC (n = 13), Septin 9 (+) CRC (n = 14), normal adjacent to tumor (NAT, n = 11) and whole blood (n = 8) DNA. Significant probesets were identified using a Benjamin-Hoc...
Cancer Research, 2009
AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The workup of patients with suspected lung canc... more AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The workup of patients with suspected lung cancer aims to establish a definitive diagnosis with the least invasive diagnostic procedures. Confirming malignancy often remains a challenge, because the tumor may not be accessible for biopsy by bronchoscopy and cytology is inconclusive. This potentially triggers additional diagnostic procedures bearing risks for the patient and eventually causing a delay in diagnosis. This retrospective clinical study aims to demonstrate that a recently developed Heavy Methyl (HM) sensitive detection methylation assay of the SHOX2 gene can be translated into clinical practice as a marker to diagnose bronchial carcinoma in patients undergoing workup for suspected lung cancer. 423 patients with suspected lung cancer consisting of 237 lung cancer Cases and 186 Controls with benign lung disease had flexible bronchoscopy with bronchial lavage specimens taken from suspicious regions. 272 Saccomanno fixed bronc...
European Respiratory Journal, Sep 1, 2011
Background & objectives: SHOX2 DNA methylation ( m SHOX2) has been shown previously to identi... more Background & objectives: SHOX2 DNA methylation ( m SHOX2) has been shown previously to identify lung cancer in bronchial aspirates and a test for m SHOX2 is available in Europe as an IVD test to aid pathologists in the diagnosis of lung cancer. DNA methylation biomarkers can also be used to detect tumor-derived circulating DNA in blood. The objective of the present study was to develop a modified assay for detection of m SHOX2 in plasma and to evaluate the clinical performance in patients. Methods: A real time PCR duplex assay originally developed for quantification of m SHOX2 in a high background of unmethylated DNA in bronchial aspirates was modified for the unique requirements of plasma. Following assay optimization, quantitative real-time PCR was used to analyze m SHOX2 in plasma samples (n = 411). A training study was performed to determine a cut-off for patient classification (n = 20 lung cancer patients, n = 20 controls) and the resulting cut-off was verified in a testing study (n = 202 stages I – IV lung cancer patients, n = 169 controls, including patients with other cancers like e.g. of prostate). Results: The assay reliably detected 15 pg of methylated DNA in a background of 50,000 pg unmethylated DNA. The m SHOX2 assay differentiated lung cancer patients from controls with a sensitivity of 60% and a specificity of 90%. Patients with stages II (72%), III (55%) and IV (83%) lung cancer were detected at a higher sensitivity as compared with stage I patients (27%). Small-cell lung cancer (80%) and squamous cell carcinoma (63%) were detected at higher sensitivity than adenocarcinoma (39%). Conclusions: m SHOX2 is a promising biomarker for detection of malignant lung disease in plasma.
PloS one, 2015
Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy... more 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. 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. 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...
International journal of oncology, 2012
In the identification of subjects with lung cancer, increased DNA methylation of the SHOX2 gene l... more 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 perfo...
Zeitschrift für Gastroenterologie, 2014
Cancer & Metabolism, 2014
Mayerle et al.: Metabolic biomarkers for the differential diagnosis of pancreatic ductal adenocar... more Mayerle et al.: Metabolic biomarkers for the differential diagnosis of pancreatic ductal adenocarcinoma vs. chronic pancreatitis. Cancer & Metabolism 2014 2(Suppl 1):P60.
Journal of Thoracic Oncology, Oct 1, 2011
Introduction: Recently, analysis of DNA methylation of the SHOX2 locus was shown to reliably iden... more 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.
Certains aspects de cette invention concernent des compositions et des procedes destines a fourni... more Certains aspects de cette invention concernent des compositions et des procedes destines a fournir des fragments d'ADN a partir d'un echantillon a distance. Dans des aspects particuliers de cette invention, un echantillon a distance contient de l'ADN, l'ADN est isole de l'echantillon a distance, puis traite de maniere a permettre la differenciation de cytosine methylee et non methylee. En outre, des modes de realisation particuliers concernent des compositions et des procedes d'analyse de methylation d'ADN derive d'un echantillon a distance. D'autres aspects concernent des compositions et des procedes d'amplification de genome entier d'ADN traite au bisulfite.
International Journal of Cancer, 1993
The tumor-associated CD30 antigen is presently under study as a target for active specific immuno... more The tumor-associated CD30 antigen is presently under study as a target for active specific immunotherapy of Hodgkin's lymphoma with anti-idiotypic antibodies. Internal image antibodies (Ab2 beta) 9G10 and 14G9 against the CD30-specific antibody HRS-4 (Ab1) have been described, which induce a CD30-specific T- and B-cell response in BALB/c mice and New Zealand white rabbits. In extension of this work, murine monoclonal anti-idiotypic Ab2 beta 9G10, mimicking structures of the nominal CD30 antigen, was used to generate monoclonal Ab3 in mice and polyclonal Ab3 in rabbits with specificity for CD30. The Ab2 beta 9G10-specific murine monoclonal Ab3 4A4 bound specifically to the 120-kDa band of CD30 present on Hodgkin cell lines and Hodgkin tumor tissue, and effectively inhibited binding of Ab1 HRS-4 to Ab2 9G10 as well as to CD30+ cells. Monoclonal Ab3 4A4 was cytotoxic for CD30+ cell lines in vitro and effectively prevented the s.c. growth of L540 cell tumors after passive i.v. administration in a SCID mouse tumor model. While this cytotoxic effect of the IgM subclass monoclonal Ab3 4A4 was due to complement activation, the murine monoclonal Ab1 HRS-4 and a polyclonal Ab3 preparation of IgG-subclass from New Zealand white rabbits were cytotoxic by an antibody-dependent cell-mediated mechanism in vitro. In conclusion, Ab2 beta 9G10 is able to induce a CD30-specific cytotoxic IgG and IgM response. Cytotoxicity was shown to be mediated by complement activation and antibody-dependent cell-mediated cytotoxicity in vitro and in vivo and across species barriers. Thus, the CD30-like Ab2 beta 9G10 may hold promise for effective active specific immunotherapy of human Hodgkin's lymphoma.
Copyright © 2014 Victoria J. Nikiforova et al. This is an open access article distributed under t... more Copyright © 2014 Victoria J. Nikiforova et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 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
Cancer Research, 2009
Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a surv... more Introduction: Colorectal cancer (CRC) is the second leading cause of cancer deaths despite a survival rate of over 90% when detected early. Currently, less than half of guideline eligible individuals are screened regularly for CRC, highlighting a need for improved non-invasive methods of early detection. We previously identified and validated a blood-based biomarker, Septin 9, which identifies methylated DNA in plasma from all stages of CRC. We have now conducted a genome-wide screen to identify additional blood-based biomarkers that either augment the performance of Septin 9 for CRC detection or perform individually as well as Septin 9. Methods: Differential methylation hybridization (DMH) via customized high-density microarrays with >50,000 features was used to explore methylation differences between Septin 9 (-) CRC (n = 13), Septin 9 (+) CRC (n = 14), normal adjacent to tumor (NAT, n = 11) and whole blood (n = 8) DNA. Significant probesets were identified using a Benjamin-Hoc...
Cancer Research, 2009
AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The workup of patients with suspected lung canc... more AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO The workup of patients with suspected lung cancer aims to establish a definitive diagnosis with the least invasive diagnostic procedures. Confirming malignancy often remains a challenge, because the tumor may not be accessible for biopsy by bronchoscopy and cytology is inconclusive. This potentially triggers additional diagnostic procedures bearing risks for the patient and eventually causing a delay in diagnosis. This retrospective clinical study aims to demonstrate that a recently developed Heavy Methyl (HM) sensitive detection methylation assay of the SHOX2 gene can be translated into clinical practice as a marker to diagnose bronchial carcinoma in patients undergoing workup for suspected lung cancer. 423 patients with suspected lung cancer consisting of 237 lung cancer Cases and 186 Controls with benign lung disease had flexible bronchoscopy with bronchial lavage specimens taken from suspicious regions. 272 Saccomanno fixed bronc...
European Respiratory Journal, Sep 1, 2011
Background & objectives: SHOX2 DNA methylation ( m SHOX2) has been shown previously to identi... more Background & objectives: SHOX2 DNA methylation ( m SHOX2) has been shown previously to identify lung cancer in bronchial aspirates and a test for m SHOX2 is available in Europe as an IVD test to aid pathologists in the diagnosis of lung cancer. DNA methylation biomarkers can also be used to detect tumor-derived circulating DNA in blood. The objective of the present study was to develop a modified assay for detection of m SHOX2 in plasma and to evaluate the clinical performance in patients. Methods: A real time PCR duplex assay originally developed for quantification of m SHOX2 in a high background of unmethylated DNA in bronchial aspirates was modified for the unique requirements of plasma. Following assay optimization, quantitative real-time PCR was used to analyze m SHOX2 in plasma samples (n = 411). A training study was performed to determine a cut-off for patient classification (n = 20 lung cancer patients, n = 20 controls) and the resulting cut-off was verified in a testing study (n = 202 stages I – IV lung cancer patients, n = 169 controls, including patients with other cancers like e.g. of prostate). Results: The assay reliably detected 15 pg of methylated DNA in a background of 50,000 pg unmethylated DNA. The m SHOX2 assay differentiated lung cancer patients from controls with a sensitivity of 60% and a specificity of 90%. Patients with stages II (72%), III (55%) and IV (83%) lung cancer were detected at a higher sensitivity as compared with stage I patients (27%). Small-cell lung cancer (80%) and squamous cell carcinoma (63%) were detected at higher sensitivity than adenocarcinoma (39%). Conclusions: m SHOX2 is a promising biomarker for detection of malignant lung disease in plasma.
PloS one, 2015
Most patients suffering from advanced lung cancer die within a few months. To exploit new therapy... more 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. 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. 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...
International journal of oncology, 2012
In the identification of subjects with lung cancer, increased DNA methylation of the SHOX2 gene l... more 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 perfo...
Zeitschrift für Gastroenterologie, 2014
Cancer & Metabolism, 2014
Mayerle et al.: Metabolic biomarkers for the differential diagnosis of pancreatic ductal adenocar... more Mayerle et al.: Metabolic biomarkers for the differential diagnosis of pancreatic ductal adenocarcinoma vs. chronic pancreatitis. Cancer & Metabolism 2014 2(Suppl 1):P60.