The molecular diagnostics laboratory processed 2228 unique clinical samples in 2012 from NCI/NIH patients, and performed over 9300 tests. Another 1000+ tests were performed to support a variety of collaborative translational research projects. The majority of the laboratory's tests are conventional PCR assays, including both DNA and RT-PCR assays, however mutation-based testing is rapidly increasing in volume. The assays performed by the molecular diagnostics laboratory include assays for B and T-cell clonality, for lymphoma related translocations, for cancer associated viruses (EBV, HTLV, HHV-8), and RT-PCR assays for translocations associated with pediatric sarcomas. Quantitative PCR is performed for HTLV 1/2 to follow viral load for the Metabolism Branch's ATL clinical trials, and for EGFRvIII to assess expression of this cancer-specific target transcript. KRAS, BRAF, and EGFR mutation analyses are performed to assess eligibility and response to EGFR-targeted therapies for several Medicine Branch clinical trials. BRAF mutation analysis is provided for eligibility for novel RAF inhibitors, and to a novel Surgery Branch trial combining targeted immunotherapy with the BRAF inhibitor vemurafinib. We continued to offer a multiplexed assay panel (TOP) for the Thoracic Oncology Groups Molecular profiling Protocol, which stratified patients to one of 6 treatment arms based on the mutational composition of the patient's tumor. This panel includes the above mentioned mutations plus NRAS, AKT1, PIK3CA and ERBB2. MGMT methylation analysis is performed to determine eligibility for Neuro-Oncology branch protocols and predict response to temozolomide, and IDH1/2 is performed to assist in glioma diagnosis and as a prognostic marker. The laboratory also has developed a qRT-PCR assay to assess the presence of the tumor specific variant EGFRvIII, which is required for eligibility to a novel immunotherapy protocol targeting this EGFR variant. Assays are performed on materials derived from a variety of sources.Paraffin fixed tissue specimens account for about 50% of the samples, blood bone marrow, 45%. The remaining 5% of samples are derived from fresh or frozen tissue, cerebrospinal fluid, and other body fluids. The laboratory's developmental research effort is focused on the application of multiplexing technologies for clinical purposes, especially next generation sequencing technologies, and on developing clinical assays for circulating tumor DNA. The laboratory is completing validation of a flexible 50 gene Next Generation Sequencing (NGS) based targeted mutation panel that will be available in 2013 for use in multiple clinical oncology protocols, and has initiated a program with Surgery branch to follow patient responses to immunotherapy, by measuring changes in circulating DNA targets. A high percentage of the paraffin section studies are complex and labor intensive, requiring macro or microdissection of specific components of lesional tissues, and are performed under the guidance of a fellow or staff pathologist. The molecular laboratory is particularly expert in extracting nucleic acids from small fragments of archival paraffin-embedded tissues, and our expertise in this area has been widely sought out in a number of laboratories within and outside of NIH. The laboratory is actively involved in identifying and developing methodologies to improve mutation detection and other types of biomarkers in small biopsy samples, including cytology samples. This includes incorporation of a variety of laser capture microdissection technologies. The laboratory maintains 4 Perkin-Elmer 9700 PCR platforms, one ABI 7900HT real-time PCR machine, one ABI 3130xl capillary electrophoresis unit, one Pyromark 24 pyrosequencing instrument. one Fluidigm Biomark System, 1 Roche 454 Junior, and 1 Ion Torrent personal Genome Analyzer. The molecular diagnostics laboratory supports translational research of both NCI and NIH researchers. Among the NCI and NIH tenured investigators and clinicians that have utilized the laboratory's resources in 2012 are: Dr. Austin Barrett (NHLBI) Dr. Susan Bates (NCI) Dr. John Bennett (NIAID) Dr. Michael Bishop (NCI) Dr. Howard Fine (NCI) Dr. Daniel Fowler (NCI) Dr. William Gahl (NHGRI) Dr. Giuseppe Giaccone (NCI) Dr. Steven Holland (NIAID) Dr. Elaine Jaffe (NCI) Dr. Su Young Kim (NCI) Dr. Daniel Kastner (NHGRI) Dr. Amy Klion (NHLBI) Dr. Robert Kreitman (NCI) Dr. Shivaani Kummar (NCI) Dr. Gerald Marti (FDA) Dr. Dean Metcalfe (NIAID) Dr. Kenneth Olivier (NIAID) Dr. Stefania Pittaluga (NCI) Dr. Martha Quezado (NCI) Dr. Koneti Rao (NIAID) Dr. Steven Rosenberg (NCI) Dr. Claude Sportes (NCI) Dr. Helen Su (NHLBI) Dr. Maria Turner (NCI) Dr. Thomas Uldrick (NCI) Dr. Gulbu Uzel (NIAID) Dr. Katherine Warren (NCI) Dr. Samuel Wells (NCI) Dr. Wyndham Wilson (NCI) Dr. James Yang (NCI) Dr. Neil Young (NHLBI)
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