Detection of FLT3 Internal Tandem Duplication in Targeted, Short-Read-Length, Next-Generation Sequencing Data

A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays. A recurrent somatic mutation frequently found in cytogenetically normal acute myeloid leukemia (AML) is internal tandem duplication (ITD) in the fms-related tyrosine kinase 3 gene (FLT3). This mutation is generally detected in the clinical laboratory by PCR and electrophoresis-based product sizing. As the number of clinically relevant somatic mutations in AML increases, it becomes increasingly attractive to incorporate FLT3 ITD testing into multiplex assays for many somatic mutations simultaneously, using next-generation sequencing (NGS). However, the performance of most NGS analysis tools for identifying medium-size insertions such as FLT3 ITD mutations is largely unknown. We used a multigene, targeted NGS assay to obtain deep sequence coverage (>1000-fold) of FLT3 and 26 other genes from 22 FLT3 ITD-positive and 29 ITD-negative specimens to examine the performance of several commonly used NGS analysis tools for identifying FLT3 ITD mutations. ITD mutations were present in hybridization-capture sequencing data, and Pindel was the only tool out of the seven tested that reliably detected these insertions. Pindel had 100% sensitivity (95% CI = 83% to 100%) and 100% specificity (95% CI = 88% to 100%) in our samples; Pindel provided accurate ITD insertion sizes and was able to detect ITD alleles present at estimated frequencies as low as 1%. These data demonstrate that FLT3 ITDs can be reliably detected in panel-based, next-generation sequencing assays. See related Commentary on page 27 See related Commentary on page 27 CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Recurrent somatic mutations play an important role in diagnosis, treatment, and prognosis in acute myeloid leukemia (AML). These mutations include not only the cytogenetic aberrations that define current classification schemes in AML, but also gene-level mutations that are valuable prognostic markers and help to guide complex treatment decisions. Such prognostic markers are especially important in cytogenetically normal AML, in which clinical outcomes are heterogeneous but risk stratification of patients based on prognosis is challenging.1Walter M.J. Graubert T.A. Dipersio J.F. Mardis E.R. Wilson R.K. Ley T.J. Next-generation sequencing of cancer genomes: back to the future.Per Med. 2009; 6: 653Crossref PubMed Scopus (20) Google Scholar Among the most common of these gene mutations are internal tandem duplication mutations, ranging in size from 15 bp to approximately 300 bp, in the juxtamembrane domain region of the fms-related tyrosine kinase 3 gene (FLT3); these mutations occur in approximately 20% to 30% of AML patients and have been associated with increased relapse risk and decreased overall survival in patients with cytogenetically normal AML.2Nakao M. Yokota S. Iwai T. Kaneko H. Horiike S. Kashima K. Sonoda Y. Fujimoto T. Misawa S. Internal tandem duplication of the flt3 gene found in acute myeloid leukemia.Leukemia. 1996; 10: 1911-1918PubMed Google Scholar, 3Abu-Duhier F.M. Goodeve A.C. Wilson G.A. Care R.S. Peake I.R. Reilly J.T. Genomic structure of human FLT3: implications for mutational analysis.Br J Haematol. 2001; 113: 1076-1077Crossref PubMed Scopus (77) Google Scholar, 4Kottaridis P.D. Gale R.E. Frew M.E. Harrison G. Langabeer S.E. Belton A.A. Walker H. Wheatley K. Bowen D.T. Burnett A.K. Goldstone A.H. Linch D.C. The presence of a FLT3 internal tandem duplication in patients with acute myeloid leukemia (AML) adds important prognostic information to cytogenetic risk group and response to the first cycle of chemotherapy: analysis of 854 patients from the United Kingdom Medical Research Council AML and 2001; PubMed Scopus Google Scholar As a treatment is in AML patients with a normal and FLT3 ITD myeloid on diagnosis, risk and J PubMed Scopus Google Scholar testing for these mutations is frequently of the for patients with a of AML and a normal CME Accreditation Statement: This activity (“JMD 2013 CME Program in Molecular Diagnostics”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for The ASCP designates this journal-based CME activity (“JMD 2013 CME Program in Molecular Diagnostics”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to have that recurrent mutations in several other genes be prognostic markers in mutations in the and genes have been found to occur in a of AML and their presence be associated with in myeloid on diagnosis, risk and J PubMed Scopus Google Scholar, mutations, but not mutations, define a of acute myeloid leukemia with a gene that is associated with a 2009; 113: PubMed Scopus Google Scholar, T.J. M.J. T. mutations in acute myeloid J Med. PubMed Scopus Google Scholar, M. mutation in patients with acute myeloid and of into a survival PubMed Scopus Google Scholar, E.R. K. mutations found by sequencing an acute myeloid leukemia J Med. 2009; PubMed Scopus Google Scholar, K. M. H. K. mutations in acute myeloid from a and clinical analysis from the AML PubMed Scopus Google Scholar, Y. H. mutation and their prognostic in a of patients with acute myeloid PubMed Scopus Google Scholar, K. K. S. H. of tandem of the gene in patients to with acute myeloid leukemia and normal a of the PubMed Scopus Google Scholar, T. S. of mutations in a of PubMed Scopus Google Scholar testing of and for mutations in of these genes is the number of genes and the of mutation in AML it and to testing with current targeted next-generation sequencing can provide mutational of many genes with a and for sequencing of clinically gene mutations and in PubMed Scopus Google Scholar a to these is the performance of these in identifying the of mutation in cancer from NGS E.R. The the Med. PubMed Scopus Google Scholar are of identifying of mutations, the most common NGS analysis tools have been only for their in for and mutations not the for a clinical have not been the of to reliably detect medium-size insertions to 300 such as FLT3 is largely unknown. FLT3 ITD mutations is for multiplex testing in AML, FLT3 and 26 other genes from ITD-positive and ITD-negative cancer specimens to FLT3 ITD mutations can be in NGS to targeted sequencing and with sequencing is an for sequencing FLT3 ITD insertion and also to a tool for these mutations that can be into the analysis of a for somatic mutations in that NGS can be used to detect FLT3 ITD mutations, and that of tools to the FLT3 ITD can in an accurate to PCR and in the of a for mutational in The in the present were from a of specimens from AML patients to for FLT3 ITD 15 specimens from AML patients from the Molecular and specimens from the and human were and were for in the present by the for and at was from and specimens using a to the a from and specimens was using and and of the were using a and a for samples; from specimens was for size using a PCR of and specimens and of 1 in with of to and of from specimens of of at bp in to a PCR M. M. M. M. M. and of PCR and for of and gene in of the PubMed Scopus Google Scholar in the present were tested for FLT3 ITD mutations by PCR and at the for Molecular at was with the juxtamembrane domain region of by on an data were using with the product size were with an ITD insertion of the size by the The ITD was using by S. M. K. M. FLT3 internal tandem duplication in with acute myeloid prognostic and to PubMed Scopus Google Scholar and by S. T.A. M. M. 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T. and PCR in sequencing PubMed Scopus Google Scholar A of our sequencing is in were by for and to bp of sequence for gene in the of the to were with and in multiplex on an to obtain sequencing The size of for of to obtain an coverage of approximately We used the to sequence a of specimens from AML and cancer and D.C. acute leukemia with the and Google Scholar, T. 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These were by only a of the at these that the were a to with ITD by NGS tool not the insertion size that of an insertion were the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in 1 the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in and were FLT3 ITD was not in the Molecular by these were to a laboratory the of FLT3 ITD were by and were FLT3 ITD was not in the Molecular by these were to a laboratory the of FLT3 ITD were by in which an FLT3 ITD was detected are in ITD insertion size in not The tool not the insertion size that of an insertion were the performance of Genomic and in the first of these tools were not tested on the of As and the are commonly used analysis were in and were FLT3 ITD was not in the Molecular by these were to a laboratory the of FLT3 ITD were by in a in which an FLT3 ITD was detected are in ITD insertion size in not the Pindel were generally with PCR and in the of insertions not Pindel insertions and only insertions and were from PCR and data of the for this a with a which the Pindel with ITD alleles in the the other only insertion was by ITD alleles were by PCR The insertion was present at a to PCR and that other insertions with size and were detected by this insertion was is the performance of Pindel in this of were in other that also of and to detect FLT3 ITD insertions. this used the a to at the FLT3 ITD and and insertions in the from with FLT3 ITD from the human We ITD-positive was in these with coverage of and used the size as the ITD size and This was to the with ITD mutations that were and it detected insertions in the with ITD alleles The of insertion mutations were generally with and in most insertion sizes from PCR by only a detected the insertion in that was by PCR but by for this overall with the other in an and in a the insertion sizes by bp and bp from PCR and Pindel as these in 100% sensitivity (95% CI = to 100%) for ITD-positive which for that into and in the of FLT3 ITD insertions. We used the tools from the first data and to an 15 AML specimens and 26 and specimens for FLT3 ITD mutations. The AML specimens in this found to be ITD-positive by our analysis was to FLT3 ITD Pindel and insertions in of the FLT3 ITD-positive AML in data and detected insertions in with by PCR were no FLT3 ITDs by Pindel in 26 ITD-negative specimens were in which Pindel detected ITD insertions but that had been ITD-negative by These were found to ITD alleles These data that Pindel and have a specificity to the FLT3 ITD in data it only ITDs in data The for this low sensitivity is but be related to the overall of coverage in data with the from our Pindel and ITD sizes as alleles with a size of Pindel of 26 ITD alleles by PCR and CI = to 100%) and 22 of the 26 CI = to tools ITD insertions in with the PCR as the the overall sensitivity for identifying an ITD-positive of insertion in an ITD-positive was 100% CI = 83% to 100%) for Pindel and and had 100% specificity ITDs detected in 29 ITD-negative CI = 88% to This of our for clinical mutation not and is to on the of using M. K. K. S. M. The a for next-generation sequencing PubMed Scopus Google Scholar, J.F. T. and mutational analysis using PubMed Scopus Google Scholar the of ITD with PCR and a were We in ITD size and number to be these be to However, were in which AML to be ITD-negative by PCR and were by Pindel and detected a in the other but not detected a the of the insertion was Pindel for and only for with a coverage of for the FLT3 ITD in This that the of these insertions was to the with ITD alleles that were by these ITDs be but the of of of the data that had to insertions of the size as detected in NGS from these was to for using a based on PCR and which the presence of FLT3 ITDs to the ITD size by NGS This that that Pindel be to NGS have ITD and insertion size with in Gale R.E. R.K. M. Burnett A.K. Linch D.C. data on the prognostic of mutations in acute myeloid leukemia be related to the of PubMed Scopus Google Scholar, R.E. Burnett A.K. R.K. Linch D.C. Medical Research Council of FLT3 internal tandem duplication and with mutations in a of patients with acute myeloid PubMed Scopus Google Scholar these from the Pindel and with from PCR and to the the was generally in ITD size and PCR but of the insertions detected by Pindel was 3 bp of the and of the 26 ITD insertions by were However, in the sizes by from the size with ITD alleles insertions that by bp and bp from the in with a ITD the size by ITD insertions are of in tandem is in the and of sequence this for of these of the sequence in the with the FLT3 juxtamembrane region that only a of the insertion of with tandem as a for the these using the ITD size from Pindel and were largely with PCR a of ITD We also the ITD from Pindel and to the from from PCR and NGS data can provide a of the of and this is for the FLT3 ITD insertion mutations, which ITD from our data, used the number of by Pindel and the coverage in the the that to the for were by the in the FLT3 and 15 region to at an The from had with from PCR and The from Pindel were with the PCR = CI = to = this was to an with a ITD that had many of size and in an of the The from had with = CI = to = and were to ITD our used coverage the and was to the of the These that ITD from NGS be for a in a with the ITD alleles be to FLT3 ITD the ITD with We the presence of the ITD in a We also the frequencies of the FLT3 of these were present at frequencies with A were present at other of number in a but this also was of the FLT3 ITD the and sequencing to the used in the present our sequencing data to and to in FLT3 ITD insertions. were to Pindel using the in which as an We and using data from the of by the the using Pindel was to these data insertions were detected in only of the for the and with of for the data of the in which insertions were most were ITD alleles and insertions were largely not to be a FLT3 ITD with and that the to detect ITD insertions with be by to the of the of on the to detect the FLT3 ITD insertions. We sequence data from the ITD-positive using and with for and the using Pindel as in and are in the ITD-positive from data FLT3 ITD insertions were in from the data and in of using the The in the data were bp, bp, the ITD in the bp, was detected in and the data to the ITD the present used and with sequencing to sequence FLT3 and 26 other to FLT3 ITD mutations can be from NGS We approximately coverage of FLT3 from and the to be and 29 ITD-negative specimens used as and AML PCR tested as ITD but to have low ITDs by NGS and We that ITD insertions were in these data, and of these mutations were in NGS an of with and with that not be to the these as of the presence of insertions in NGS data, that be to in the clinical of other in the recurrent insertion mutations are to We tested the of several NGS analysis tools to FLT3 ITD insertions and found that only of the reliably detected the ITD mutations. Pindel was 100% for ITD insertions in the ITD-positive specimens in the present and in no in 29 specimens to be ITD-negative by PCR of ITD alleles was in most with alleles by in which the ITD was as low as 1%. This sensitivity for mutations was also by the of ITD insertions in that were ITD-negative by but for which of the the presence of ITD We also that of from of to FLT3 and 15 was as Pindel at identifying these mutations. The that Pindel and ITD insertions that were by several other tools in common can be to in the The of the tools are able to only insertions that are the that are approximately 15 to bp and that are of the other tools are to detect mutations from with sizes that a with the Pindel and include that are with the These are for identifying medium-size such as in FLT3 ITD mutations, this size is to be detected by and for by analysis of the of such mutations to and as NGS Pindel and to be for insertion mutations in other in which analysis of can mutation Pindel and at FLT3 ITD insertions in the present it is important to that the sensitivity and specificity be this We Pindel and to a only sequence mutations are to be of Pindel to and especially to are to in the of a number of many of which be of the sequence at for other and of insertion mutations as and can in the performance of can be only using a of from NGS to in cancer have of their to sequence for such increased sensitivity to a of mutations, with current testing which are targeted to mutational that include a are many to NGS and of tools for accurate of mutation in have that accurate of FLT3 ITD mutations is in the of deep sequencing with a targeted using These are a in the of targeted sequencing as a for testing of and of cancer in We for with and for their in using the performance and the for and sequencing and the Pathology for sequencing and for of this with of the ITD based on PCR and of the with FLT3 with of AML for FLT3 ITD at the of FLT3 ITDs by NGS a that to the ITD detected by Pindel and a which to the ITD by such were not in other FLT3 ITD-negative with and FLT3 for in the frequencies were for the FLT3 gene were in frequencies of to mutations and frequencies of 100% to mutations. The FLT3 was by PCR and in and is by the The was from have of A with gene no was the frequencies of FLT3 and FLT3 ITD with in data and data FLT3 ITD by in the region of the 3 ITD that on the presence of as not as with to have increased sensitivity with data with with with with with Molecular of a of Molecular the article by that a next-generation for the of FLT3 mutations.