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Targeted Next-Generation Sequencing Detects Point Mutations, Insertions

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Targeted Next-Generation Sequencing Detects Point Mutations, Insertions Leukemia (2011) 25, 671–680 & 2011 Macmillan Publishers Limited All rights reserved 0887-6924/11 www.nature.com/leu ORIGINAL ARTICLE Targeted next-generation sequencing detects point mutations, insertions, deletions and balanced chromosomal rearrangements as well as identifies novel leukemia-specific fusion genes in a single procedure V Grossmann1,3, A Kohlmann1,3, H-U Klein2, S Schindela1, S Schnittger1, F Dicker1, M Dugas2, W Kern1, T Haferlach1 and C Haferlach1 1MLL Munich Leukemia Laboratory, Munich, Germany and 2Department of Medical Informatics and Biomathematics, University of Mu¨nster, Mu¨nster, Germany DNA sequence enrichment from complex genomic samples (RT-PCR) or direct sequencing, not only allow stratification of using microarrays enables targeted next-generation sequen- patients into distinct prognostic risk groups,5,6 but also serve as cing (NGS). In this study, we combined 454 shotgun pyro- molecular markers to monitor minimal residual disease.7–9 sequencing with long oligonucleotide sequence capture arrays. We demonstrate the detection of mutations including point In this study, we combined 454 PicoTiterPlate (PTP) mutations, deletions and insertions in a cohort of 22 patients pyrosequencing with long oligonucleotide sequence capture presenting with acute leukemias and myeloid neoplasms. arrays to evaluate whether this technique permits a compre- Importantly, this one-step methodological procedure also hensive genetic characterization of a cancer genome. In allowed the detection of balanced chromosomal aberrations, particular, we addressed the question whether this combination including translocations and inversions. Moreover, the geno- of methods would detect not only point mutations, as well as mic representation of only one of the partner genes of a chimeric fusion on the capture platform also permitted deletions and insertions, but also capture target sequences that identification of the novel fusion partner genes. Using acute would reveal balanced chromosomal aberrations in a one-step myeloid leukemias harboring RUNX1 abnormalities as a model procedure. This principle is proven by investigating the complex system, three novel chromosomal fusion sequences and recombinome of leukemias harboring alterations in RUNX1,10–13 KCNMA1 as a novel RUNX1 fusion partner gene were detected. PDGFRB14 and MLL.15 At present, 32 chromosomal partner This assay has the strong potential to become an important regions have been described for RUNX1 translocations, but the method for the comprehensive genetic characterization of corresponding partner gene has only been identified in 17 particular leukemias and other malignancies harboring 12 complex genomes. translocations. Similarly, 104 partner regions have been Leukemia (2011) 25, 671–680; doi:10.1038/leu.2010.309; described for the MLL gene, but only 64 partner genes are published online 21 January 2011 molecularly characterized.15 Keywords: targeted next-generation sequencing; balanced In this study, we further demonstrate that the contiguous chromosomal rearrangements; fusion genes genomic representation of only one of the partner genes of a chimeric fusion on the capture microarray assay was sufficient to identify also any potentially unknown partner gene from a balanced chromosomal aberration by subsequent shotgun sequencing. Introduction DNA sequence enrichment from complex genomic samples has Patients and methods been proposed to enable a targeted next-generation sequencing (NGS) workflow. Several methods for massively parallel enrich- Patient cohort ment of the sequencing templates exist. Hybridization to In this study, we analyzed 19 acute leukemia cases (16 acute customized microarrays containing synthetic oligonucleotides myeloid leukemias (AMLs), 3 acute lymphoblastic leukemias) that match the target sequence allows capturing templates from and 3 patients with a myeloproliferative neoplasm sent to the randomly sheared, adaptor-ligated genomic DNA with high MLL Munich Leukemia Laboratory for diagnostic procedures specificity.1 Other methods are based on biotinylated RNA between October 2005 and September 2008 (Table 1). All capture probes to capture size-selected genomic DNA in samples in this study were obtained from untreated patients solution2 or allow a simultaneous amplification of up to 4000 at the time of diagnosis. The study design adhered to the tenets targeted sequences using microdroplet technology.3 of the Declaration of Helsinki and was approved by the Today, the genetic characterization necessary for optimal institutional review board before its initiation. Patients were treatment of leukemias requires a combination of different labor- 16 diagnosed using cytomorphology, chromosome banding intensive methods, such as chromosome banding analysis and 17 18 analysis, FISH, molecular genetics and flow cytometry. fluorescence in situ hybridization (FISH).4 Characteristic leukemia- specific fusion genes, detected by reverse transcriptase-PCR Molecular genetics Correspondence: Dr C Haferlach, MLL Munich Leukemia Laboratory, Standard mutational analysis by Sanger sequencing was Max-Lebsche-Platz 31, Munich 81377, Germany. performed on the purified fraction of mononuclear cells after E-mail: [email protected] 3These authors contributed equally to this work Ficoll density centrifugation. Isolation of mononuclear cells, Received 21 May 2010; revised 16 September 2010; accepted 15 genomic DNA (QIAamp DNA Mini kit, Qiagen, Hilden, November 2010; published online 21 January 2011 Germany), mRNA (MagNA Pure LC mRNA HS Kit, Targeted next-generation sequencing in leukemia V Grossmann et al 672 Table 1 Patients with chromosomal abnormalities Case Diagnosis Fusion genes Gender Sample type Cytogenetic correlate of fusions N01 AML M4eo CBFB–MYH11a Female Bone marrow inv(16)(p13q22) N03 AML M5a MLL–MLLT3 (AF9)a Female Bone marrow t(9;11)(p22;q23) N04 AML M2 RUNX1–RUNX1T1a Male Bone marrow t(8;21)(q22;q22) N05 AML M5b MLL–ELL, –SFRS14 Female Bone marrow t(11;19)(q23;p13) N14 AML MLL–MLLT10 (AF10) Male Bone marrow t(10;11)(p12;q23) N16 AML M4 MLL–MLLT6 (AF17) Female Bone marrow t(11;17)(q23;q12) N17 AML M5a MLL–MLLT10 (AF10) Male Bone marrow der(10)t(10;11)(p12;q22)inv(11)(q22q23), der(11)t(10;11)(p12;q22) N20 t-AML M5a MLL–MLLT1 (ENL) Male Bone marrow t(11;19)(q23;p13.3) N21 Pro-B-ALL MLL–AFF1 (AF4) Female Bone marrow t(4;11)(q21;q23) N38 AML M5a MLL–MLLT10 (AF10)a Male Bone marrow der(10)t(10;11)(p12;q11)inv(11)(q11q23), der(11)t(10;11)(p12;q23) N39 AML M5b MLL–MLLT4 (AF6)a Male Bone marrow t(6;11)(q27;q23) N40 t-Pro-B-ALL MLL–AFF1 (AF4)a Female Bone marrow t(4;11)(q21;q23) N41 t-AML MLL–ELLa Female Bone marrow t(11;19)(q23;p13.1) N42 AML M0 MLL–MLLT1 (ENL)a Female Peripheral blood t(11;19)(q23;p13.3) N27 AML M1 RUNX1–chr. 17 Female Bone marrow t(7;11;17;21)(p22;q11;q21;q22) N28 AML RUNX1–KCNMA1 Male Peripheral blood t(10;21)(q22;q22) N29 CMML-2 RUNX1–chr. 5 Male Peripheral blood t(5;21)(q11;q22) N30 AML M3v RUNX1–chr. 10 Female Bone marrow t(10;21)(q21;q22) N33 c-ALL ETV6–RUNX1a Female Bone marrow t(12;21)(p13;q22) N36 HES/CEL PDGFRB–DTD1a Male Peripheral blood t(5;20)(q33;p12) N37 HES PDGFRB–MYO18Aa Male Bone marrow t(5;17)(q33;q11.2) Abbreviations: AML, acute myeloid leukemia; CEL, chronic eosinophilic leukemia; CMML, chronic myelomonocytic leukemia; HES, hypereosinophilic syndrome. aKnown from standard routine procedures or previous observations. Note: case N06 not displayed (AML with a normal karyotype). Roche Applied Science, Penzberg, Germany) and random To quantify enrichment of the genomic DNA, four regions primed complementary DNA synthesis was performed as were selected for quantitative PCR measuring SYBR green described previously.19 The analysis of KRAS mutations in fluorescence according to the manufacturer’s protocols codons 12, 13 and 61 was carried out as previously described.20 (Supplementary Figure 2). The enriched and ligation-mediated Exons 2 and 3 were amplified by PCR, and PCR products were PCR-amplified samples were compared against the non-en- analyzed using the BigDye terminator v1.1 cycle sequencing kit riched and ligation-mediated PCR amplified samples, that is, not (Applied Biosystems, Darmstadt, Germany). Analyses for NPM1 hybridized to a capture array, using a LightCycler LC480 mutations, FLT3 internal tandem duplications, FLT3 tyrosine real-time PCR system (Roche Applied Science). kinase domain mutations, KITD816 mutations and KIT exon 8 mutations were performed as described previously.21–25 Microarray designs A high-density oligonucleotide microarray representing capture Targeted sequence capture microarray assay probes covering 1.91 Mb of genomic sequences was synthesized Briefly, 20 mg genomic DNA was fragmented by nebulization to according to a standard microarray manufacturing protocol small sizes of 300–500 bp to generate blunt-ended fragments. (Roche NimbleGen 385K format). Overlapping microarray The DNA was quantified and the fragment size population was probes of more than 60 bases each on the array spanned each assessed by electrophoresis (Agilent Bioanalyzer 2100 DNA target genome region, with a probe positioned every 10 bases Chip 7500, Agilent, Bo¨blingen, Germany). The fragmented DNA for the forward strand of the genome. was then processed according to the recommended NimbleGen A first array captured short segments corresponding to all protocol (Roche Applied Science, User Guide 3.1; July 2008). In exon regions of 92 distinct target genes (genome build hg18). brief, linkers were ligated to the polished fragments in the library
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