Page 132 - 2019_07 resto del Mondo-web

M. Zaliova et al.
different type of ERGdel was found in four of 16 positive patients (ERGdel-diff) (Table 1 and Figure 1). Using PCR, we found ERGdel in 33 of 50 (66%) DUX4r-ALL. As expected, all 12 patients with IntERGdel detected by SNP array were positive by PCR; surprisingly, also three of four patients with ERGdel-diff detected by SNP array were positive by PCR, demonstrating co-existence of var- ious deletion types, each in a different proportion of cells. Importantly, PCR revealed IntERGdel in 18 of 31 patients negative by SNP array. Using AmpliSeq, ERGdel was detected in 39 of 50 (78%) of patients with DUX4r-ALL, including all 33 PCR-positive and an additional six of 17 PCR-negative patients.
In non-DUX4r-ALL, all 63 and 68 patients tested by SNP array and PCR, respectively, were ERGdel-negative. No ERGdel was found in nine non-DUX4r patients tested by AmpliSeq at higher coverage setting.
Prognostic impact of DUX4r and ERGdel
Neither the whole cohort (n=118) nor its consecutively
analyzed part (n=88) showed a significant difference in EFS or OS between the DUX4r-ALL patients and the non- DUX4r B-other ALL (Online Supplementary Figure S5). Similarly, despite the fact that, according to all the analy- ses, patients with ERGdel fared better than patients lack- ing the deletion, the difference between ERGdel-positive and ERGdel-negative patients within the whole B-other ALL cohort did not reach a statistical significance, whatev- er method for the ERGdel detection was used (SNP array, PCR, AmpliSeq or combination of all) (Online Supplementary Figure S6).
When the prognostic impact of the ERGdel was ana- lyzed only within DUX4r-ALL, its favorable effect on out- come was again statistically non-significant when SNP array results were taken into account (combining both IntERGdel and ERGdel-diff patients; P>0.3). When PCR or AmpliSeq results were used, the positive prognostic impact of ERGdel was statistically significant in the whole DUX4r-ALL cohort (n=50) (PCR: EFS, P=0.022; OS, P=0.029. AmpliSeq: EFS, P=0.099; OS, P=0.032) and for AmpliSeq also in its consecutive part (n=27) (EFS, P=0.020; OS, P=0.016) (Figure 2).
Expression of DUX4, RAG1 and RAG2 and ERGalt Similarly to other recurrent deletions in BCP-ALL, ERGdel is thought to represent a result of illegitimate RAG1/RAG2-mediated V-(D)-J recombination. This theory is strongly supported by the presence of sequence motifs
highly homologous to recombination signal sequences in close proximity to ERGdel breakpoints.3 It has also been suggested that DUX4 may facilitate ERGdel by increasing accessibility of ERG gene locus.9 We analyzed expression levels of DUX4, RAG1 and RAG2 in 44 DUX4r-ALL with available RNAseq data and did not find any difference between patients stratified by ERGdel presence based on results of any of the detection methods used (data not shown).
We analyzed the association between ERGdel and expression of ERGalt transcripts. In total, 41 of 44 DUX4r ALL cases expressed ERGalt. The expression levels varied substantially, and in some DUX4r ALL cases, the levels were very low and undistinguishable from those found in non-DUX4r ALL (Online Supplementary Figure S4). Interestingly, the expression of ERGalt was significantly higher in patients with versus patients without ERGdel detected by PCR/AmpliSeq, but not between patients with versus patients without ERGdel detected by SNP array (Figure 3A-C). There was no significant correlation of DUX4 and ERGalt expression levels in DUX4r cases (data not shown).
Frequency of copy number aberrations in DUX4r-ALL stratified by ERGdel
We analyzed the total number of copy number aberra- tions (CNA) in 47 DUX4r-ALLs with available SNP array data. We found a significantly higher number of CNA in patients positive compared to negative for ERGdel, inde- pendently of the ERGdel screening method used (Figure 4). The most common CNA were deletions (del) of the CDKN2A/B, IKZF1 and PAX5 genes (found in 43%, 23% and 20% of DUX4r-ALL patients, respectively) (Online Supplementary Table S2). Deletions of CDKN2A/B were evenly distributed among ERGdel-positive and ERGdel- negative patients. The frequency of IKZF1del was identi- cal in the two groups when the ERGdel was determined by SNP array; however, when PCR results were used for the ERGdel assessment, the ERGdel-positive patients had a significantly higher percentage of IKZF1del compared to ERGdel-negative patients (33% vs. 0% of IKZF1del-posi- tivity, respectively; P=0.02). Frequency of PAX5del was lower in ERGdel-positive compared to negative patients but the statistical significance was reached only on ERGdel stratification according to SNP array (0% vs. 29% PAX5del-positive patients; P=0.04). Simultaneous deletion of IKZF1 and CDKN2A/B or PAX5 (no PAR1 was found in DUX4r-ALL) was found in six DUX4r-ALL patients. All
Table 1. Results of ERGdel screening by three different methods in 50 DUX4r-acute lymphoblastic leukemia.
SNP array Result
IntERGdel ERGdel-diff
No ERGdel
ND
PCR (IntERGdel)
AmpliSeq (IntERGdel) N. of
N. of Result patients
12 Positive
4 Positive
Negative
31 Positive Negative
3 Negative
N. of patients
12
3
1
18 13
3
Result
Positive
Positive
Negative
Positive Positive Negative
Positive
Negative
patients
12
3
1
18 4 9
2
1
SNP: single nucleotide polymorphism; PCR: polymerase chain reaction; AmpliSeq: amplicon sequencing method; N: numbers; ND: not done.
1410
haematologica | 2019; 104(7)