Whole-Genome Mate Pair Sequencing Reflex Test to Characterize Chromosome Rearrangements in Hematologic Neoplasia

Chromosomal alterations such as translocations, inversions and deletions involving one or more breaks are common in hematologic malignancies. Mate pair sequencing (MPseq) is highly informative when a chromosome rearrangement of uncertain significance is identified by conventional chromosome or fluorescence in situ hybridization (FISH) studies and characterization of the genes affected could be helpful, but an alternative clinical assay is not available for this purpose. MPseq utilizes a modified library preparation method to generate paired end sequencing from long fragments of genomic DNA, resulting in higher sensitivity and lower cost compared to conventional Next Generation Sequencing (NGS) for detecting structural variation. Precise molecular characterization by MPseq of clinically significant genes at or near the breakpoints of chromosome rearrangements can provide tremendous diagnostic, prognostic and predictive value, and this assay has already demonstrated its potential to offer targeted therapeutic treatment opportunities for patients with hematologic malignancies.

Of the 33 MPseq tests that have been performed following abnormal chromosome or FISH studies of a hematologic malignancy, 15 were for lymphoid and 18 were for myeloid disorders. Each case had a chromosome alteration detected by routine chromosome or by FISH studies. In all cases, multiple partner genes or just one partner gene was known and the MPseq analysis was limited to the region targeted by the rearrangement. Sanger sequencing confirmed each abnormal result identified by MPseq. Of these 33 samples, 26 were found to have direct concordance between MPseq and the targeted rearrangement, 4 demonstrated concordance but the rearrangement was found to be more complex than appreciated from conventional cytogenetic studies and in 3 cases MPseq did not identify the abnormality likely due to the presence of a low level clone and/or as a result of the repetitive nature of the DNA near the rearrangement. Of the 30 concordant cases, MPseq identified in 21 of these a clinically relevant gene at the breakpoint resulting in identification of a fusion gene or deletion of a potentially significant gene within the rearrangement. In 5 cases, the gene at the breakpoint was predicted to be influenced by a position effect such as translocation to an immunoglobulin locus. In all 26 cases where a clinically significant gene was identified, MPseq provided diagnostic, prognostic and/or predictive value which had not been appreciated with conventional cytogenetic studies alone.

MPseq was critical for the identification of fusion genes allowing for the classification of Philadelphia Chromosome (Ph)-Like B-ALL. In one example of adult B-ALL, an inv(1)(q25q41) was identified by chromosomes and subsequent MPseq confirmed a chimeric gene comprised of the 5' end of RCSD1 fused to the tyrosine kinase domain of ABL2 resulting in an RCSD1-ABL2 fusion. A second example of relapsed pediatric B-ALL identified an ETV6 rearrangement by FISH and subsequent MPseq confirmed a chimeric gene composed of exons 1-4 of ETV6 and exons 15-20 including the tyrosine kinase domain of NTRK3 . Each fusion identified may be targetable by tyrosine kinase inhibitors. MPseq was also valuable in identification of fusion genes in patients with myeloid malignancies. In one example of infant AML, FISH studies identified a cryptic NUP98 rearrangement and subsequent MPseq confirmed a chimeric gene comprised on exons 1-13 of NUP98 fused to exons 25-28 of the KDM5A gene. This fusion is a known recurrent rearrangement associated with non-Down syndrome acute megakaryocytic leukemia. In summary, by presenting our initial clinical experience along with case examples, we demonstrate that MPseq represents an NGS technology that has the potential to revolutionize the diagnosis of hematologic malignancies and provide an opportunity to advance precision medicine.