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Sequenom Inc. Message Board

  • viorel263 viorel263 Nov 11, 2012 6:25 PM Flag

    incredible, what's your take on this: (Re:Massarray4) and HH traveling to LONDON (Nov 15)

    Multi-Purpose Utility of Circulating Plasma DNA Testing in Patients with Advanced Cancers

    1 Division of Clinical Studies, The Institute of Cancer Research, Sutton, Surrey, United Kingdom,

    2 Drug Development Unit, Royal Marsden NHS Foundation Trust, Sutton, Surrey, United Kingdom

    Published: November 7, 2012 (in PLOS ONE)


    Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5–1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical ‘hot-spot’ mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

    Mass Spectrometry TypePLEX technology and OncoCarta panel (v1.0)

    The OncoCarta panel (v1.0) consists of 24 pools of primer pairs and extension primers, and has the capacity to detect 238 mutations in 19 genes. The protocol provided by Sequenom (San Diego, CA) was followed with minor modifications. The amount of DNA added to the polymerase chain reaction (PCR) was 20 ng per reaction for FFPE DNA samples. For plasma DNA samples, 30 µl of DNA were added to 30 µl of pure water, and used for the OncoCarta panel (v1.0) processing. DNA was amplified using the OncoCarta PCR primer pools, unincorporated nucleotides were inactivated by shrimp alkaline phosphatase (SAP), and a single base extension reaction was performed using extension primers that hybridize immediately adjacent to the mutations and a custom mixture of nucleotides. Salts were removed by the addition of a cation exchange resin. Multiplexed reactions were spotted onto SpectroCHIP II arrays, and DNA fragments were resolved by MALDI-TOF on the Compact Mass Spectrometer (Sequenom, San Diego, CA).

    Data analysis

    Data analysis was performed using MassArray Typer Analyzer software (Sequenom), which facilitates visualization of data patterns and the raw spectra. Typer automates the identification of mutants by comparing ratios of the wild type peak to that of all suspected mutants and generates an OncoMutation report detailing specific mutations and the ratios of wildtype and mutation peaks. All mutations from the Oncomutation report were reviewed manually by 2 blinded operators, with selected reviewed mutations from the OncoMutation report compared and confirmed to be concordant. Manual review of mutations on all OncoCarta spectra was performed to identify “real” mutant peaks from salt peaks or other background peaks. Statistical analyses are detailed in the Supplemental Methods S1


    The Sequenom OncoCarta panel has also enabled us to analyze more than 230 known mutation ‘hot-spots’ mutations in over a hundred patients in a high throughput fashion. The OncoCarta panel covers a large and increasing number of oncogenes and can be adapted to include additional genes of interest. It allows tumor mutation detection even with minimal amounts of tumor DNA, poor tissue preservation and the presence of significant amounts of normal DNA. Next generation sequencing technology will allow more DNA coverage and data acquisition, allowing the sequencing of hundreds of full length genes, which will be critical to the study of genes where mutations can be found in multiple disparate locations, as is the case for many tumor suppressor genes such as BRCA1, BRCA2, p53 and PTEN.

    In conclusion, we envision that biomarker studies such as that described above can have a potential impact on molecular stratification and patient care. We therefore recommend a concerted effort by the cancer community in order to develop analytically validated assays on cpDNA that can be clinically qualified for more broad utilization [2], [3]. WE ENVISION THAT THE ANALYSES OF cpDNA WILL BECOME PART OF CANCER PATIENT STANDART OF CARE(emphasis mine). Finally, with whole exome and cancer genome analyses becoming increasingly feasible and affordable, studies to analyze the feasibility of a deep sequencing approach from the plasma of cancer patients are now warranted [28].


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