Page 54 - Haematologica Vol. 107 - September 2022

ARTICLE - Genetic abnormalities in older ALL patients
T. Creasey et al.
Affymetrix Cytoscan HD (Affymetrix, Santa Clara, CA, USA) or Illumina CytoSNP 850k (Illumina, San Diego, CA, USA) arrays according to the manufacturers’ protocols. Deletions in IKZF1, CDKN2A, CDKN2B, PAX5, RB1, ETV6, EBF1 and BTG1 (‘driver genes’) were specifically identified from the SNP array results, with loss of any part of the gene considered significant. All arm-level and focal CNA were then examined to detect recurrent abnormalities.
Next-generation sequencing
Separately, targeted NGS analyses were performed on se- lected samples using a custom SureSelect XT2 target en- richment kit (Agilent, Santa Clara, CA, USA). Samples were selected based on availability of DNA and the presence of CNA by SNP array. The capture library was designed to tar- get either the coding regions or full sequence of 44 genes that are well known to be implicated in ALL (Online Sup- plementary Table S1). Libraries were prepared in accord- ance with the manufacturer’s protocol and sequenced on the NextSeq 550 (Illumina, San Diego, CA, USA) using 100 bp paired-end chemistry (Online Supplementary Methods).
Survival analysis
Survival analysis was restricted to patients enrolled in UKALL14 because all these patients received similar in- tensive treatment with curative intent.22 Patients were grouped according to primary chromosomal abnormalities as described previously.23 Briefly, patients with complex karyotypes, HoTr or JAK-STAT activating rearrangements were classed as very high risk; patients with any KMT2A fusions were classed as high risk; patients with BCR-ABL1 and other kinase-activating fusions were classed as having tyrosine kinase-activating (TKA) abnormalities; all other BCP-ALL patients were classed as standard risk; and T- ALL patients were analyzed separately. All P-values were two-sided and, because of multiple testing, values <0.01 were considered statistically significant. All analyses were performed using Intercooled Stata (StataCorp, College Station, TX, USA) and R version 3.4.3 (http://www.R-pro- ject.org).
Results
Patients’ demographics and baseline cytogenetics
We identified a total of 210 patients aged ≥60 years from the UKALL14 (n=95) and UKALL60+ (n=115) clinical trials. The median age of the patients was 64 years (range, 60- 83) and 24% (n=50) were over 70 years at diagnosis. The male:female ratio was 1:1. In total, 90% (n=189) had con- firmed BCP-ALL and 5% (n=11) had T-cell disease. The re- maining 5% (n=10) did not have a diagnostic immunophenotype centrally recorded. Numbers of pa- tients decreased with advancing age but no significant dif-
ference was seen in the genetic subgroups represented in different age groups (P=0.47) (Figure 1A, B). The most prevalent abnormality was BCR-ABL1, present in 28% (55/200) of evaluable patients. Of these, the p210, p190 and p230 isoforms were present in 40% (22/55), 33% (18/55) and <1% (1/55) of patients, respectively. Two pa- tients had both p190 and p210 isoforms identified and the Bcr-Abl isoform was unknown in the remaining 22% (12/55) patients. Low hypodiploidy/near triploidy (HoTr) was the second most prevalent primary chromosomal ab- normality and was identified in 14% (28/200) of patients (Online Supplementary Table S2) and KMT2A-v rearrange- ments were discovered in a further 6% (12/200) of pa- tients. Of the patients with BCP-ALL, 47% (88/189) did not have a primary chromosomal abnormality identified by routine cytogenetic and FISH analyses performed in re- gional cytogenetic centers (B-other ALL).
Among the 11 patients with T-ALL, TLX1 (n=1) and TLX3 (n=1) rearrangements were identified. The other nine pa- tients either had no rearrangements identified (n=5) or were not tested (n=4).
Individual patients’ demographic, clinical and genetic data are shown in Online Supplementary Table S3.
Gene rearrangements in patients with B-other acute lymphoblastic leukemia
Patients with B-other ALL included those with normal (n=21), failed (n=25) or complex karyotypes (n=5). Patients with dic(9;12) (n=2), IGH translocation (n=5) or other non- subgroup-defining chromosomal abnormalities (n=30) were also included in the B-other category. Fixed cell samples were available for 74% (65/88) of B-other patients and gene rearrangements were identified in 21% (19/65) (Table 2). Not all samples could be screened for all abnor- malities due to lack of availability of material for multiple FISH experiments.
CRLF2 rearrangements were identified in 17% (8/48) of successfully screened cases. The CRLF2 rearrangement partners were IGH (n=5), P2RY8 (n=2) and unknown (n=1). Two additional patients had P2RY8-CRLF2 fusion identified by MLPA, through the presence of PAR1 deletion (Table 1). IGH translocations were present in 26% (14/53) of B-other samples tested. Of these, five cases accounted for pa- tients with IGH-CRLF2 translocations detailed above, and three cases had separate primary genetic abnormalities identified (one ZNF384 translocation and two P2RY8- CRLF2 fusions). In the remaining six samples, the IGH partners were CEBPA (n=1), CEBPD (n=1), CEBPE (n=1), BCL2 (n=1) and unknown (n=2). ZNF384 and MEF2D rearrange- ments were each identified in 8% (3/40) and 3% (1/39) of screened B-other cases, respectively.
In total, CRLF2, IGH, and ZNF384 rearrangements were present in 5%, 3% and 1% of the complete patient cohort, respectively (Figure 1C). No variant ABL1 (0/83), PDGFRB
Haematologica | 107 September 2022
2053