Design of a NGS Platform for the Integrated Analysis of Genetic Mutations, Copy Number Alteration and Somatic Hypermutation of IGHV in CLL
Authors: Alcoceba Miguel , Garcia-Alvarez Maria , Puiggros Anna , Espinosa-Hevia Luis , Gómez-Llonín Andrea , Navarro Almudena , Bastida Jose Maria , Gutierrez Norma C. , Garcia-Marco Jose A. , Espinet Blanca , García-Sanz Ramón , González Marcos 
Published: October 2019

Abstract
INTRODUCTION
The use of new drugs in chronic lymphocytic leukemia (CLL) has allowed to revert the poor prognosis conferred by both the alterations of TP53 (deletion and mutation) and the absence of somatic hypermutation (HMS) of the heavy chain gene of immunoglobulins (IGHV). These alterations are part of the essential pre-treatment tests of the CLL according to the International Workshop on CLL Guidelines (iwCLL, Hallek et al, 2018). Three independent methodologies are required for these analyses, consequently time- and cost-consuming. Next-generation sequencing (NGS) could integrate all three types of studies into one.
AIM
To design a NGS panel for the detection of genetic alterations with diagnostic, prognostic and predictive value in CLL, as well as other alterations with clinical relevance.
PATIENTS AND METHODS
A capture panel (SureSelect QXT, Agilent) of 170Kb was designed covering the coding region of 17 genes, 11 regions of copy number alteration (CNAs), 2 translocations and the HMS-IGHV. A total of 83 samples of patients with CLL from three centers were sequenced. The effectiveness of the panel to detect already known alterations was evaluated in two groups of samples: 46 samples that presented any essential alteration of the iwCLL, including mutations of TP53, del (11q), +12, del (13q), del (17p), and the HMS status of the IGHV (group 1), and in 19 other samples with ≥1 different known cytogenetic / molecular alteration (group 2). Four healthy individuals were included as control. The samples were sequenced in a MiSeq (Illumina, average coverage of 600x, paired-end reading of 126bp). The analysis was carried out using a specific algorithm developed by Dreamgenics bioinformaticians, confirming the alterations with Integrative Genome Viewer (IGV).
RESULTS
The NGS panel allowed the identification of 214 mutations, 92 CNA regions and 83 IGHV genes. The concordance of results between the NGS panel and conventional methods was 100% (78/78) for mutations, 92% for CNAs (172/187) and 90% for use of IGHV (57/63). Considering the alterations of group 1, the agreement was 100% in mutations of TP53 (26/26), 100% of del (11q) (41/41), 98% +12 (41/42), 98% of del (17p) (42/43), and 79% of del (13q) (33/42). Two of the discrepancies in CNAs were cases identified by NGS but FISH negative [del (17p) and del13q)], probably because they are small deletions (<100kb). There was a case with +12 in 14% of cells and 8 cases with (13q) not identified by NGS, probably due to poor coverage of the region. The panel allowed identifying CNAs present in 25% of the samples and mutations in 10%.
CONCLUSIONS
This panel of NGS allows to detect> 93% of the essential genetic alterations of the iwCLL, and> 94% of the total genetic alterations analyzed. The results are preliminary but promising, although validation of the methodology is required.

Disclosures
No relevant conflicts of interest to declare.