Whole-Genome Mate Pair Sequencing Transforms Cytogenetic Analysis of Hematologic Malignancies: Development of a Targeted Panel to Detect Diagnostic/Prognostic Chromosomal Rearrangements and Copy Number Changes in AML

Acute myeloid leukemia (AML) is the most common form of acute leukemia affecting both children and adults. Once recognized as a single disease, the World Health Organization now groups AML into ten subtypes based on genetic abnormality. This is based on differences among recurrent genetic abnormalities with respect to response to therapy, relapse risk and overall survival. Currently most testing of AML patients occurs by karyotype analysis, FISH or RT-PCR. Here we describe a next generation sequencing approach to better classify and accurately detect abnormalities associated with AML.

For the validation of whole genome mate pair sequencing (MPseq) targeted panel to test patients with AML, twenty-five abnormal and ten normal samples extracted from each sample type (peripheral blood (PB) and bone marrow (BM)) were run with the MPseq assay. Samples were chosen based on reported abnormalities previously tested by conventional karyotyping and/or FISH. The mate pair panel is designed to detect rearrangements involving genes ABL1, BCR, CBFB, CREBBP, DEK, KAT6A(MYST3), MECOM(EVI1), MLF1, MLL(KMT2A), MYH11, NPM1, NUP214(CAN), NUP98, PML, RARA, RPN1, RUNX1(AML1), RUNX1T1(ETO) and copy number changes on chromosomes 5, 7, 8, 13, 17, and 20. DNA was processed using the Illumina Nextera Mate Pair library preparation kit, multiplexed at 2 samples/lane and sequenced on the Illumina HiSeq. Data was aligned to the reference genome (GRCh38) using BIMA and abnormalities were identified using SVAtools, both in-house developed tools. A targeted analysis approach was utilized using a well-defined list of recurrent chromosomal abnormalities associated with known diagnostic and prognostic significance as well as targeted therapies in AML. The karyotype, FISH and MPseq results were compared to determine concordance.

To test the limit of detection based on percent tumor, samples previously tested with FISH were diluted. Tumor percentage was determined by the abnormal FISH result and was diluted with a normal sample to mimic various percentages (100%, 50%, 25%, 10%, 5%, 2%). For both PB and BM specimens, the rearrangements were detected down to the 10% dilution and the copy number changes were detected down to 25%. Based on these cutoffs, all 50 abnormal samples (25 PB, 25 BM) used for the validation were 100% concordant with abnormalities targeted by the panel that were previously detected by FISH and/or karyotype. In addition, MPseq identified additional abnormalities not detected by karyotype or FISH and further characterized several complex abnormalities. One example includes a case with MLLT10/MLL(KMT2A) fusion detected by MPseq which was missed by the AML FISH panel because the rearrangement involved an insertional translocation. Another example is the identification of BCR/FGFR1 fusion in a case when AML FISH identified three copies of BCR.

In conclusion, we validated a whole genome mate pair sequencing targeted panel to detect diagnostic/prognostic chromosomal rearrangements and copy number changes in patients with AML. Due to the limitations in detecting very low level abnormalities, MPseq would not be recommended for follow-up post therapy or minimal residual disease testing unless the coverage is increased with deeper sequencing. However, our validation study demonstrate the clinical utility of MPseq as a potential replacement assay to conventional FISH on diagnostic AML samples and highlight the increased diagnostic yield. Thus, MPseq represents a next generation sequencing technology that has the potential to revolutionize the diagnosis of hematologic malignancies and provide an opportunity to advance precision medicine.