Page 53 - Haematologica Vol. 107 - September 2022

ARTICLE - Genetic abnormalities in older ALL patients
T. Creasey et al.
tome, whole genome and exome sequencing and ident- ified 23 genetic subtypes.6 Despite this impressive co- hort, only 103 patients aged 60 years and over at diagnosis were included. Such efforts focused on older individuals are needed to improve prognostication and to identify novel therapeutic targets.7,8
A proportion of patients do not harbor a cytogenetically visible disease-defining lesion, but have a gene ex- pression profile similar to that of BCR-ABL1-positive dis- ease (Ph-like/BCR-ABL-like ALL).9,10 Approximately 50% of children and young adults with this entity have cytogen- etically-cryptic IGH-CRLF2 or P2RY8-CRLF2 rearrange- ments, which activate JAK-STAT signaling.11,12 Other recurrent gene rearrangements include ABL-class fusions (affecting ABL1, ABL2, PDGFRB or CSF1R) in 9-13% of Ph- like cases and the JAK-STAT pathway activating re- arrangements of JAK2 or EPOR in 7-10% and 3-6% of patients, respectively.11,12 To date, most studies have fo- cused on pediatric and young adult (<60 years) co- horts,11,13 although one study found the Ph-like signature in 24% of older BCP-ALL patients in a restricted subgroup lacking large scale aneuploidy.12 Separately, ZNF384 and MEF2D rearrangements have been reported in 2-6% of pediatric BCP-ALL cases, and form distinct clinical en- tities.14,15
Focal copy number abnormalities (CNA) frequently target genes that are involved in B-cell development or cell cycle regulation These secondary abnormalities drive transformation of a pre-leukemic clone into overt dis- ease and include deletions of EBF1 on 5q33.3, IKZF1 on 7p12.2, CDKN2A and CDKN2B on 9p21.3, PAX5 on 9p13.2, ETV6 on 12p13.2, BTG1 on 12q21.33 and RB1 on 13q14.2.16 Importantly, particular combinations of CNA have an im- pact on prognosis.17-19 The IKZF1plus profile is based on the co-occurrence of IKZF1 deletion with deletions of CDKN2A, CDKN2B, PAX5 or the pseudoautosomal region 1 (PAR1) on Xp22.33/Yp11.31 (resulting in P2RY8-CRLF2 fusion) in the absence of ERG deletion.17 This copy number profile is associated with a significantly poorer outcome in childhood ALL patients, highlighting the prognostic importance of large-scale copy number ana- lyses.
To date, profiling the genetic and genomic landscape of ALL has been primarily restricted to younger patients with few analyses focused on older individuals. Here, we applied cytogenetic, copy number and next-generation sequencing (NGS) techniques to investigate whether the primary and secondary genetic abnormalities of ALL in older adults are distinct from those encountered in their younger counterparts. We additionally sought to identify novel druggable targets, a particular priority for such pa- tients because of the high toxicity and low success rates of traditional chemotherapeutic approaches in this group.20
Methods
Patients and samples
Patients aged 60 years and over were identified for gen- etic profiling studies from two large UK-wide multi- center clinical trials (UKALL14 and UKALL60+). The UKALL14 study is registered as NCT01085617 (https://www.clinicaltrials.gov/ct2/show/NCT01085617), and the UKALL60+ study is registered as NCT01616238 https://clinicaltrials.gov/ct2/show/NCT01616238) (Online Supplementary Methods).
Baseline cytogenetic analyses, typically consisting of a di- agnostic karyotype and fluorescence in situ hybridization (FISH) for BCR-ABL1 fusion and KMT2A translocations, were performed in accredited diagnostic genetic labora- tories throughout the UK and then centrally reviewed and entered into the Leukaemia Research Cytogenetics Group database.
The study was approved by the institutional review board of each treatment center and all patients gave written in- formed consent for data collection and genetic studies as specified by the trials’ protocols.
Detection of primary genetic subgroups
Diagnostic karyotype and FISH results from the regional genetic centers were first examined and patients were coded into one of six subgroups: BCR-ABL1, TCF3-PBX1, KMT2A fusions, high hyperdiploidy (51-65 chromosomes), low hypodiploidy/near triploidy (HoTr) (30-39 or 60-78 chromosomes) or T-cell ALL (T-ALL). Reverse transcriptase polymerase chain reaction analysis was performed to identify the presence of Bcr-Abl p190, p210 and p230 on- coproteins in BCR-ABL1-positive cases. Next, all BCP-ALL cases lacking a primary chromosomal abnormality, here- after termed B-other ALL, were identified. B-other cases with available fixed cell samples were further investigated by the Leukaemia Research Cytogenetics Group to deter- mine the occurrence of cytogenetically cryptic abnormal- ities using dual color break-apart FISH probes for CRLF2, PDGFRB/CSF1R, ABL2, IGH, ZNF384, MEF2D (Cytocell, Cam- bridge, UK) and JAK2 (Kreatech Diagnostics, Amsterdam, the Netherlands). Separately, multiplex ligation-dependent probe amplification (MLPA) using the IKZF1-P335 kit (MRC Holland, the Netherlands) was performed on cases with available DNA as previously described21 and permitted the detection of the P2RY8-CRLF2 fusion that occurs through PAR1 deletion on Xp22.33/Yp11.31.
Copy number analysis
Single nucleotide polymorphism (SNP) arrays were per- formed on DNA extracted from diagnostic bone marrow samples obtained at trial enrolment. SNP arrays were per- formed at the Newcastle Genomics Centre, Newcastle- upon-Tyne Hospitals NHS Foundation Trust using the
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