Identification of Candidate Oncogenes and Chromosomal Breakpoint Sequencing By Targeted Locus Amplification in T-Cell Acute Lymphoblastic Leukemia
Authors: Vroegindeweij Eric M , Lammens Tim , Splinter Erik , Uyttebroeck Anne , Canté-Barrett Kirsten , Buijs-Gladdines Jessica GCAM , Smits Wilco K , Vandenberghe Peter , van Min Max J , Pieters Rob , Van Vlierberghe Pieter , Meijerink Jules PP 
Published: October 2019

Abstract
Background: T-cell acute lymphoblastic leukemia is characterized by clonal and mutual exclusive chromosomal rearrangements that recurrently activate TAL1, LMO2, TLX1, NKX2-1, TLX3, HOXA or MEF2C oncogenes. Most of these translocations or chromosomal rearrangements occur as erroneous D-J or V-DJ rearrangement attempts of T-cell receptor beta (TCRB) or TCR alpha/delta (TCRAD) genes, mostly positioning oncogenes under the transcriptional control of TCR enhancer elements. Alternatively, oncogenes can also be activated as consequence of BCL11B chromosomal rearrangements. Although many oncogenes are known in T-ALL, the driving oncogenic lesion in particular T-ALL cases remains unknown.
Aims: In this study, we aimed to clone reciprocal breakpoint sequences to elucidate cellular mechanisms that lead to recurrent BCL11B -TLX3 chromosomal translocations. Moreover, we want to identify oncogene candidates in various T-ALL patient samples with BCL11B-, TCRB- or TCRAD-translocations for which the candidate oncogene so far has not been identified.
Methods: We used Targeted Locus Amplification procedure, a recently developed method that relies on the crosslinking of DNA in live cells, DNA digestion and re-ligation to allow formation of circular DNA ligation fragments and inverted polymerase chain reaction amplification from specific view-point loci. Amplified DNA fragments are sequenced by next generation sequencing, allowing sequence identification in a region covering 2MB around selected regions of interest, including TLX3, TLX1, TAL1, LMO2, BCL11B, TCRAD (TRAJ61), TCRB (TCRBC2)
Results: TLA was successfully performed on 10 T-ALL patients having FISH validated TAL1 translocations (2 patients), LMO2 translocations (3 patients), TLX3 translocations (3 patients), TLX1 translocations (2 patients) or an inversion targeting NKX2.1 (1 patient). Analysis of both TAL1 translocated cases revealed a TAL1-TCRAD genomic fusion due to a classical t(1;14)(q32;q11) in 1 patient, but surprisingly reveal a TAL1-TCF7 genomic fusion due to a t(1;5)(q32;q31.1) chromosomal translocation in the second patient. For the LMO2 translocated cases, two patients showed classical LMO2-TCRAD (t(11;14)(p13;q11)) or LMO2-TCRB (t(7;11)(q35;p13) translocations, whereas the third patient presented with an unusual LMO2-BCL11B genomic fusion due to a t(11;14)(p13;q32). Two out of 3 TLX3-translocated patients had classical t(5;14)(q35;q32) translocations, whereas the TLX3 gene in the third patient was rearranged to the calcyphosine-like gene (CAPSL, which flanks the IL7Ra gene) on chromosomal 5p13.2 due to a t(5;5)( p13.2;q35) or an inv(5). One patient had an inversion on chromosome 14, i.e. inv(14)(q11;q13), that brings the NKX2.1 oncogene under the transcriptional control of the TCRAD enhancer. Finally, one TLX1-rearranged patient had a classical TLX1-TCRAD translocation, whereas the other presented with a chromosomal inversion involving the chromosomal band 10q24 (which included TLX1), but also revealed a novel translocation involving the centromere protein P gene (CENPP) on chromosome 9q22.31 with the TCRAD locus.
Summary/Conclusions: Targeted Locus Amplification identification of chromosomal rearrangements and genomic breakpoint sequences reveals novel complex translocations in 3 out of 10 T-ALL cases analyzed thus far, indicating higher complexity of chromosomal translocations of known T-ALL oncogenes as thus far anticipated. It further proved a useful tool to identify novel translocation partners from various loci such as the TCR or BCL11B genes that are recurrently involved in these chromosomal rearrangements in T-ALL. Cloning of molecular translocation breakpoints of diagnostic T-ALL patient samples may further provide excellent minimal residual disease markers for disease monitoring during the course of treatment.

Disclosures
Splinter: Cergentis BV: Employment. van Min:Cergentis BV: Employment.