https://orcid.org/0000-0003-2782-2752
Genome-wide technologies identified novel fusion genes in B-cell precursor acute lymphoblastic leukemia (B-ALL), refining the genetic characterization of ALL, particularly of the so called ‘B-other ALL’ [Citation1–4]. While the most relevant subsets is the Philadelphia (Ph)-like [Citation5], Gu et al. [Citation6] have described a detailed taxonomy of 23 subtypes, according to chromosomal rearrangements, mutations and genomic alterations: their detection is pivotal for refined diagnosis, stratification and identification of new therapeutic targets.
ZNF384 encodes a C2H2-type zinc finger protein that binds to promoters, regulates the transcription of the extracellular matrix genes, and has a high affinity for the transcriptional coactivator EP300 [Citation7]. Qian et al. [Citation8] reported an enrichment of ZNF384 binding sites, particularly in the promoter and/or enhancer regions of CLCF1 and BTLA, which were expressed at an average of 15.5- to 15.0-fold higher than wild-type ZNF384 cases, respectively. CLCF1 and BTLA are important for the activation of the JAK-STAT signaling pathway and B-cell differentiation, respectively.
Rearrangements involving ZNF384 (ZNF384-r) were described in B/myeloid mixed phenotype acute leukemia (B/MPAL) [Citation9] and in Ph- B-ALL; in the latter, at least 15 partner genes were identified (EWSR1, TAF15, TCF3, EP300, SYNRG, CREBBP, BMP2K, SMARCA2, CLLORF74, CCAR1, CLTC, DUX4, NIPBL, SEC24B and ARID1B) [Citation10,Citation11].
ZNF384-r were reported in 6% and 7% of childhood and adult B-ALL, respectively, with an enrichment in adolescents and young adults (15–24 years) [Citation10–13]. The most frequent ZNF384-r are TCF3-ZNF384 and EP300-ZNF384 in both cohorts. Activating mutations in RAS signaling pathway genes and higher FLT3 expression levels are frequently detected in patients with ZNF384-r. Furthermore, ALL cases with ZNF384-r exert a peculiar immunophenotype, with weak or negative expression of CD10, and aberrant expression of myeloid markers, such as CD13 and/or CD33 [Citation10].
We report the incidence, clinico-biologic features and prognosis—in terms of minimal residual disease (MRD) persistence and survival—of ZNF384-r cases in 185 newly-diagnosed B-other ALL patients (i.e. cases lacking major molecular transcripts, namely BCR/ABL1, TCF3/PBX1, KMT2A rearrangements and ETV6/RUNX1) enrolled in the GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto) LAL1913 [Citation14] (n = 83) and LAL2317 [Citation15] (n = 102) protocols designed for adult Ph-negative ALL, based on a pediatric-inspired and MRD-driven strategy, with the addition of blinatumomab in the latter.
All cases underwent, beyond cytomorphology and immunophenotype, a centralized comprehensive molecular screening: (i) ‘Ph-like predictor’ assay, (ii) Multiplex Ligation-dependent Probe Amplification (MLPA) to detect the most common copy number aberrations (CNA) using the SALSA MLPA kit P335 ALL-IKZF1 (MRC Holland, Amsterdam, The Netherlands), (iii) targeted RNA-sequencing, using the TruSight RNA Pan-Cancer Panel (Illumina, San Diego, CA) kit; these latter results were also used to determine the expression levels of FLT3, by means of fragments per kilobase of transcript per million mapped reads (FPKM); iv) sequencing of the JAK/STAT and RAS cascades using the Truseq custom amplicon kit (Illumina) or the SureSelect XT HS2 kit (Agilent, Santa Clara, CA); v) MRD analysis using IG-TR gene probes according to the EuroMRD consortium guidelines (Supplemental Material).
The median follow-up for the whole cohort is 22.5 months (range 0.56–74.2): 40 months (range 0.66–74.21) for patients in the GIMEMA LAL1913 trial and 11.2 months (range 0.56–27.4) for those in the LAL2317 trial, respectively.
We identified 17 (9.2%) patients with ZNF384-r, involving different partner genes: the most frequent fusion was ZNF384-EP300 (n = 9), followed by ZNF384-TAF15 (n = 4) ZNF384-TCF3 (n = 2) and ZNF384-ARID1B (n = 1). The results were confirmed by Sanger sequencing in all but 3 cases (lack of genomic material, Table S1 (Supplemental Material)). In addition, we identified a novel ZNF384-related fusion gene, ZNF384-SPI1 in a single patient. The clinical features of ZNF384-r patients were comparable to those of the whole cohort. The median age of ZNF384-r patients was 43 years (range 18–64), 47% were female and 53% male; the median WBC count was 4.82 × 109/L (range 1–54.9), while the median platelet count was significantly higher in ZNF-384-r patients compared to the remaining cases (176 × 109/L vs 49 × 109/L, p < .001) (Table S2, Supplemental Material). None of these patients carried PAX5 mutations, detected in 8 patients (4.3%).
Immunophenotype revealed that most ZNF-384-r patients were classified as B-common ALL (13/17), 3 cases as a pro-B ALL and a case as a pre-B ALL. In the 14 CD10+ ZNF384-r cases (13 common ALL and 1 pre-B ALL), CD10 expression was heterogeneous/weak (mean 53.4 ± 26.8; range ± 27.8; range 20–90%). Furthermore, 16/17 patients had myeloid antigens aberrant expression: CD13 was expressed in 9/17 cases (53%) (mean 64.4 ± 23.2%; range 20–90%) and CD33 in 16/17 cases (94%) (mean 62.6 ± 24.8%; range 20–90%). All ZNF384-r patients were classified as non-Ph-like by the BCR/ABL1-like predictor, thus making this genetic subset the most frequent among B-other non-Ph-like ALL (13%).
CNA analysis, carried out in 13/17 ZNF384-rearranged cases revealed CDKN2A/2B, ETV6 and IKZF1 deletions as the most frequent concomitant lesions, detected in 5 (38%), 5 (38%) and 3 (23%) cases; other sporadic deletions involved PAX5, BTG1 and RB1 identified in 1 patient (8%) each. Only 2 patients, both with ZNF384-TAF15, proved IKZF1plus (i.e. IKZF1 plus PAX5 and/or CDKN2A/B deletions).
Among the 13 ZNF384-r evaluable cases, we detected 8 RAS pathway mutations − 6 clonal and 2 subclonal—in 6 cases (46%). The most frequently mutated gene was PTPN11 affected by 3 mutations (D61N, D61V and T507K); FLT3 was affected by 2 mutations targeting M659I and D835Y, respectively. NRAS (G12D) and KRAS (K5N) were mutated in one case each. No TP53 mutations were identified. Finally, we evaluated FPKM FLT3 expression levels: the median FLT3 expression was significantly higher in ZNF384-r cases (803.027) compared to non ZNF384-r (302.8) cases (p < .0001) ( and Table S3, Supplemental Material). Overall, our findings are in line with a recent report in childhood ALL (11), showing that ZNF384-r cases are often enriched in ETV6 and CDKN2A/B deletions, are enriched in RAS pathway mutations, high FLT3 expression and display a peculiar phenotype.
Table 1. Clinical features of ZNF384-r cases.
Finally, MRD evaluation—feasible in 14 patients at time point (TP) 1, in 13 at TP2, in 11 at TP3 and in 8 at TP4—showed that 42.9%, 69.2%, 81.8% and 100% of ZNF384-r cases were negative or positive non-quantifiable (i.e. outside the quantitative range and/or with results not reproducible among replicates) at TP1, TP2, TP3 and TP4 respectively (Table S4, Supplemental Material). Indeed, disease-free survival (DFS), evaluated in the whole cohort and in each trial separately, was better in ZNF-384-r cases than in non-ZNF-384 r cases: in the whole cohort, the 24-months DFS was significantly better for ZN F-384-r cases than for the remaining patients (100%, CI:100–100 vs 65.4%, CI:56.5–75.8, p = .04); similar results were observed in both the LAL1913 (36-months DFS: 100%, CI: 100–100 vs 62.9%, CI:51.5–76.7 for ZNF-384 r vs non ZNF-384 r cases, p = .07) and LAL2317 (12-months DFS: 100%, CI:100–100 vs 74.2%, CI:62.8.5–87.67, p = .38) GIMEMA trials (). Statistical significance was not reached due to the small sample size. Comparable results were observed for overall survival (OS).
Figure 1. DFS of ZNF384-r vs non ZNF384-r cases. A. Estimated 24-months DFS of the whole cohort. B. Estimated 36-months DFS of patients enrolled in the GIMEMA LAL1913 trial. C. Estimated 12-months DFS of patients enrolled in the GIMEMA LAL2317 trial.
Overall, this cohort of adult ZNF384-r ALL patients appears to have a favorable prognostic likelihood. Although these cases are enriched for biological features which can be associated with an unfavorable outcome, such as FLT3 mutation/overexpression and CDKN2A/B deletions, it is intriguing to speculate that the concomitant presence of ZNF384-r abrogates this effect. Further investigation is warranted. Of the ZNF3824-r patients, 2 non-leukemia related deaths were recorded, whereas no relapses have so far been reported (median follow-up 18 months, range 4–62) with only 3 patients were allografted.
Lastly, with the limit of the small sample size, we evaluated if there were differences within the various fusion genes. EP300 rearranged cases (n = 9) had a relatively low median WBC count (3.9 × 109/L, range 1.3–54.9 × 109/L), median age was 44 years (19–50), all cases were B-common, no additional lesions were detected and FLT3 expression was 667.3. TAF15 rearranged cases (n = 4) were characterized by a higher WBC count (median 12.4 × 109/L, range 4.8–25.2 × 109/L), were younger (median age 19. 5 years, range 19–34), 2 were B-common ALL, 1 was a pro-B and 1 a pre-B and in 2 cases an IKZF1plus signature was detected; FLT3 expression levels were 1140.2, higher than in any other ZNF284-r subgroup. Finally, while 62.5% of EP300 rearranged cases became rapidly MRD-negative (at TP1), none of the TAF15 rearranged cases were negative at the first TP. Nevertheless, all evaluable patients, regardless of the partner gene, became MRD-negative at later TPs. Similar results were reported by Jeha et al. [Citation16], who correlated the genomic profile with MRD assessment in about 600 children.
In conclusion, our results suggest that ZNF384-r cases should be identified at the time of the diagnostic work-up adult ALL and should be regarded as a non-high risk group. These results appear superior to those reported by Paietta et al. [Citation3], who considered ZNF384/ZNF384-like as intermediate molecular risk; this might reflect the better overall outcome documented in the two GIMEMA trials, which in the adult population (≤55 years) exceeds 60% [Citation14,Citation15]. At variance, similar results in terms of overall good outcome have been reported by Moorman and colleagues [Citation4] and Qin and colleagues [Citation13]. Since in pediatric ZNF384-r patients late relapses were documented, a longer observation period is required. Finally, in case of relapse, a possible approach might be targeting FLT3.
Author contributions
SC performed experiments, designed research and wrote the manuscript, AT performed experiments and wrote the manuscript, IDS, MC, MA, MDP performed experiment, MM performed statistical analysis, OS, AS, LADN, DC performed experiments, MS performed bioinformatic analysis, VA performed statistical analysis, RB designed the trials, AG and RF designed research and critically revised the manuscript.
GLAL-2022-0785-File004.docx
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The authors wish to thank Associazione Italiana per la Ricerca sul Cancro (AIRC) 5 × 1000, Special Program Metastases (21198), Milan (Italy) to RF; Finanziamento Medi Progetti Universitari 2015 and 2021 to SC (Sapienza University of Rome), and PRIN 2017 (2017PPS2X4_002) to SC.
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References
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