title: Comprehensive Analysis of Disease-Related Genes in Chronic Lymphocytic Leukemia by Multiplex PCR-Based Next Generation Sequencing.
abstract: BACKGROUND
  High resolution molecular studies have demonstrated that the clonal acquisition of gene mutations is an important mechanism that may promote rapid disease progression and drug resistance in chronic lymphocytic leukemia (CLL). Therefore, the early and sensitive detection of such mutations is an important prerequisite for future predictive CLL diagnostics in the clinical setting.
  
  
  METHODS
  Here, we describe a novel, target-specific next generation sequencing (NGS) approach, which combines multiplex PCR-based target enrichment and library generation with ultra-deep high-throughput parallel sequencing using a MiSeq platform. We designed a CLL specific target panel, covering hotspots or complete coding regions of 15 genes known to be recurrently mutated and/or related to B-cell receptor signaling.
  
  
  RESULTS
  High-throughput sequencing was performed using as little as 40 ng of peripheral blood B-cell DNA from 136 CLL patients and a dilution series of two ATM- or TP53-mutated cell lines, the latter of which demonstrated a limit of mutation detection below 5%. Using a stringent functional assessment algorithm, 102 mutations in 8 genes were identified in CLL patients, including hotspot regions of TP53, SF3B1, NOTCH1, ATM, XPO1, MYD88, DDX3X and the B-cell receptor signaling regulator PTPN6. The presence of mutations was significantly associated with an advanced disease status und molecular markers of an inferior prognosis, such as an unmutated IGHV mutation status or positivity for ZAP70 by flow cytometry.
  
  
  CONCLUSIONS
  In summary, targeted sequencing using an amplicon based library technology allows a resource-efficient and sensitive mutation analysis for diagnostic or exploratory purposes and facilitates molecular subtyping of patient sets with adverse prognosis.
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Claudia
authors.initials: C
authors.lastname: Vollbrecht
authors.affiliation: Institute of Pathology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany.
authors.forename: Fabian Dominik
authors.initials: FD
authors.lastname: Mairinger
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Koitzsch
authors.affiliation: Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany; Department of Translational Genomics, Cologne Center of Genomics, University of Cologne, Cologne, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Peifer
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Katharina
authors.initials: K
authors.lastname: Koenig
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany.
authors.forename: Lukas Carl
authors.initials: LC
authors.lastname: Heukamp
authors.affiliation: Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany; Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Crispatzu
authors.affiliation: Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Wilden
authors.affiliation: Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Karl-Anton
authors.initials: KA
authors.lastname: Kreuzer
authors.affiliation: Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Hallek
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Margarete
authors.initials: M
authors.lastname: Odenthal
authors.affiliation: Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany.
authors.forename: Carmen Diana
authors.initials: CD
authors.lastname: Herling
authors.affiliation: Institute of Pathology, University Hospital Cologne, Cologne, Germany; Center for Integrated Oncology (CIO) Cologne-Bonn, University Hospital Cologne, Cologne, Germany; Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
authors.forename: Reinhard
authors.initials: R
authors.lastname: Buettner
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D015723:Gene Library; D056726:Genetic Association Studies; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D009154:Mutation; D011379:Prognosis; D012680:Sensitivity and Specificity