Abstract
Abstract 144
PicoTiterPlate (PTP) pyrosequencing allows the detection of low-abundance oncogene aberrations in complex samples even with low tumor content. Here, we compared deep sequencing data of two Next-Generation Sequencing (NGS) assays to detect molecular mutations using a PCR-based strategy and, in addition, to uncover inversions, translocations, and insertions in a targeted sequence enrichment workflow (454 Life Sciences, Roche Diagnostics Corporation, Branford, CT). First, we studied 95 patients (CMML, n=81; AML, n=6; MDS, n=3; MPS, n=3; ET, n=2) using the amplicon approach and investigated seven candidate genes with relevance in oncogenesis of myeloid malignancies: TET2, RUNX1, JAK2, MPL, KRAS, NRAS, and CBL. 43 primer pairs were designed to cover the complete coding regions of TET2, RUNX1 (beta isoform), and hotspot regions of the latter genes. In total, 4128 individual PCR reactions were performed with DNA isolated from bone marrow mononuclear cells, followed by product purification, fluorometric quantitation, and equimolar pooling of the corresponding 43 amplicon products to generate one single sequence library per patient. For sequencing, a 454 8-lane PTP was used applying standard FLX chemistry and representing one patient per lane. The median number of base pairs sequenced per patient was 9.23 Mb. For each amplicon a median of 840 reads was generated (coverage range: 485–1929 reads). As initial proof-of-concept analysis 27 of the 95 patients with known mutations (n=32) as detected by conventional sequencing or melting curve analyses were investigated (range of cells carrying the respective mutation: 1.1% for JAK2 V617F to 98.14% for TET2 C1464X). In all cases, 454 NGS confirmed results from routine diagnostic methods (GS Amplicon Variant Analyzer software version 2.0.01). We then investigated the remaining 69 CMML patients: In median, 2 variances (range 1–8 variances), i.e. differences in comparison to the reference sequence, per patient were detected. These variances included both point mutations in all candidate genes and large deletions (12-19 bp) in CBL, RUNX1, and TET2. Only 20/81 patients of the CMML-cohort (24.69%) were without any detectable mutation. Secondly, in a cohort of six AML bone marrow specimens a custom NimbleGen array (385K format; Madison, WI) was used to perform a targeted DNA sequence enrichment procedure. In total, capture probes spanning 1.91 Mb were designed to represent all coding regions of 92 target genes (1559 exons) with relevance in hematological malignancies (e.g. KIT, NF1, TP53, BCR, ABL1, NPM1, or FLT3). In addition, the complete genomic regions were targeted for RUNX1, CBFB, and MLL. For sequencing, 454 Titanium chemistry was applied, loading three patients per lane on a 2-lane PTP including three molecular identifiers (MIDs) each. Data analysis was performed using the GS Reference Mapper software version 2.0.01. For the enrichment assay, the median enrichment of the targeted genomic loci was 207-fold, as assessed by ligation-mediated LM-PCR. Overall, 1,098,132 reads were generated in the two lanes, yielding a total sequence length of 386,097,740 bases. In median, 96.52% of the sequenced bases mapped against the human genome, and 66.0% were derived from the customized NimbleGen array capture probes, resulting in a median coverage of 18.7-fold . With this method it was possible to detect and confirm point mutations (KIT, FLT3-TKD, and KRAS) and insertions (FLT3-ITD). Moreover, by capturing chimeric DNA fragments and generating reads mapping to both fusion partners this approach detected balanced aberrations, i.e. inv(16)(p13q22) and the translocations t(8;21)(q22;q22) or t(9;11)(p22;q23). In conclusion, both assays to specifically sequence targeted regions with oncogenic relevance on a NGS platform demonstrated promising results and are feasible. The amplicon approach is more suitable for detection of mutations in a routine setting and is ideally suited for large genes such as TET2, ATM, and NF1, which are labor-intensive to sequence conventionally. The array-based capturing assay is characterized by a complex and time-consuming workflow with low-throughput. However, the ability to detect balanced genomic aberrations which are detectable thus far only by cytogenetics and FISH has the potential to become an important diagnostic assay, especially in tumors in which cytogenetics can not be applied successfully.
Disclosures:
Grossmann: MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Dicker:MLL Munich Leukemia Laboratory: Employment. Kazak:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.
Aging, Bone Marrow and Next-Generation Sequencing (NGS): Recent Advances and Future Perspectives