Differentiating between Hyperdiploidy and Pseudo-Hyperdiploidy in B-Lymphoblastic Leukemia Utilizing Low-Coverage Mate-Pair Sequencing

Purpose: B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/LBL) represents the most common childhood malignancy. Classification of recurrent genetic abnormalities in B-ALL/LBL is essential for risk stratification and patient management. Observed in approximately 5% of B-ALL/LBL cases, hypodiploidy is considered a poor prognostic finding and can be further characterized as near-haploidy (24-31 chromosomes), low hypodiploidy (32-39 chromosomes) and high hypodiploidy (40-43 chromosomes). Importantly, masked hypodiploid clones that have undergone endoreduplication can be mischaracterized as hyperdiploidy, a favorable prognostic finding in B-ALL/LBL. These "doubled" hypodiploid clones are termed pseudo-hyperdiploid and their accurate detection is critical. Mate-pair sequencing (MPseq) is a next-generation sequencing (NGS) technology designed to sequence larger DNA fragments (2000-5000bp) than paired-end sequencing. This allows for the detection of large genetic rearrangements (e.g. translocations, inversions, etc.) and copy number variants (CNVs) at a high resolution with low base coverage. It has demonstrated particular clinical utility for genetic characterization of hematologic malignancies. To expand the role of MPseq in the evaluation of hematologic neoplasms, we aimed to improve the analysis of MPseq results to allow for the detection of copy-neutral loss of heterozygosity (cnLOH) that would enable the differentiation between hyperdiploid and pseudo-hyperdiploid clones. Methods: A new algorithm was applied that statistically analyzes common SNP locations in MPseq data in order to detect both cnLOH regions, and provides global heterozygosity levels at a resolution that allows for the detection of hypodiploidy. This method accounts for the low base-coverage of MPseq data (<10x) in order to provide the highest possible accuracy and precision without increasing cost by requiring changes to the input or sequencing requirements. The algorithm was applied to 17 B-ALL samples classified by conventional chromosome analysis, FISH, and/or chromosomal microarray as either pseudo-hyperdiploidy (n=8) or hyperdiploidy (n=9). The cases were sequenced using MPseq with 101bp read lengths across a range of base coverages (1-10x; 30-135 million fragments) and tumor percentages (50-100%). Results: We demonstrate the method's ability to differentiate between hyperdiploidy and pseudo-hyperdiploidy in all cases without any changes to the MPseq input or sequencing requirements. We detected an average of 11.5 whole-chromosome cnLOH (range 8-20) in the pseudo-hyperdiploidy cases, compared to an average of 0.67 whole-chromosome cnLOH (range 0-2) in the hyperdiploidy cases. Additionally, the method allows for the detection of cnLOH regions across the genome. For example, we observed a recurrent cnLOH of chromosome 9 in the hyperdiploid subtype (3/9 hyperdiploid cases), which is a recurrent abnormality in the disease. The method provides an easy to read visual output that compares favorably to the output of chromosomal microarray, increasing its utility for clinical application. Conclusions: This advancement in the analysis of low-coverage sequencing data makes MPseq available for the genetic characterization of B-ALL/LBL, where hypodiploidy is a primary cytogenetic abnormality and is sometimes missed by conventional cytogenetic techniques. Additionally, the algorithm allows for the detection of cnLOH regions across the genome, which is important across many disease types for diagnosis, prognosis, and the detection of potential therapeutic targets. The addition of the algorithm to the MPseq analysis pipeline makes it a more complete genetic characterization tool and brings it closer to its promise as a potential single assay replacement for conventional cytogenetic techniques. Disclosures No relevant conflicts of interest to declare.

A small survey of UV-bright stars around the northern ecliptic pole: seeking new p-mode sdB variables for the TESS mission

Starting in 2019, the TESS mission will monitor the northern ecliptic pole for 1 year. Data will be collected at 30-minute and 2-minute cadences, and only a limited amount of slots will be reserved for targets requiring a 20- second cadence. Only the 20-second cadence is sufficient to sample p-mode oscillations in sdB stars. From the seismic measurements obtained with the Kepler spacecraft we have gained a wealth of new insights in structural and rotational aspects of mainly g-mode variable sdB stars. Unfortunately only one traditional p-mode sdB variable was found in the main Kepler field. The TESS mission offers the opportunity to obtain more long-time-base coverage of p-mode sdB variables, especially at the ecliptic poles where the time-base will be longest. Thus far, there were only two known (p-mode) sdBVs around the northern ecliptic pole (β > 78): LS Dra and V366 Dra. In this paper we describe our efforts to find more.We compiled a new sample of 76 sdB candidates around the northern ecliptic pole, based on GALEX and optical colours, and we used low-resolution Balmer-line spectroscopy for classification. We identified 39 new sdB stars, of which 29 have characteristics (Teff > 28000 K or a composite spectrum) that may put them in the p-mode instability strip.With our 39 new sdB stars, we augmented the number of known sdB stars in the the northern ecliptic pole area (β> 73) by 46%. Besides these sdB stars, among our spectral classifications are various sdO stars, He-sdB stars, blue horizontal-branch stars, white dwarfs, cataclysmic variables and main sequence B stars. We obtained time-resolved photometry of most of the p-mode sdB candidates, and found one new sdBV, J19384+5824, with a moderately high pulsation amplitude of ≥ 9 mmag.