Deciphering the molecular complexity of the IKZF1plus genomic profile using Optical Genome Mapping

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. Genetic stratification is one of the hallmarks of the advancement of therapeutic intervention in pediatric B-cell precursor ALL (BCP-ALL) and led to an improved overall survival of >90%.^1-3 Despite these advancements, a proportion of patients still face challenges such as relapse or therapy-related toxicities.² Deletions targeting the lymphoid transcription factor IKAROS Family Zinc Finger 1 (IKZF1) occur in approximately 15% of pediatric B-ALL and have been associated with adverse outcome and as a predictor of relapse.^3-6 Moreover, when IKZF1 deletion co-occurred with at least one additional deletion in CDKN2A, CDKN2B (homozygous deletion only), PAX5, or PAR1 (P2RY8::CRLF2) in the absence of ERG deletion, so-called IKZF1^plus patients showed a very poor prognosis in the AIEOP-BFM ALL 2000 trial.⁷ The prognosis of IKZF1^plus was, however, dependent on measurable minimal residual disease (MRD), arguing for other leukemic drivers.⁷ To systematically assess the underlying genetic complexity of IKZF1-deleted BCP-ALL, we performed a retrospective genome-wide analysis utilizing Optical Genome Mapping (OGM), enabling the detection of all kinds of numerical and structural variants (SV) in one approach. Ultra-high molecular weight DNA was extracted from frozen peripheral blood or bone marrow cells from pediatric BCR::ABL1-negative BCP-ALL patients with either IKZF1 deletion (IKZF1^del) or IKZF1^plus profile within the AIEOPBFM ALL 2000 and 2009 trials (Online Supplementary Table S1). We show here that half of the patients displayed additional favorable or unfavorable prognostic markers, including ETV6::RUNX1, high hyperdiploidy (HeH), iAMP21, and gene fusions. Of interest, ETV6::RUNX1 and HeH were absent in IKZF1^plus patients. Importantly, when excluding patients positive for ETV6::RUNX1, HeH, or iAMP21, a similar event-free survival (EFS) was observed for both groups, questioning the prognostic relevance of the IKZF1^plus profile.

A total of 142 patients were included in this study and OGM (Bionano Genomics) was performed as previously described (Figure 1A).⁸ Informed consent was obtained from all patients involved in the study or from their legal representatives. The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by Hannover Medical School Ethics Committee (N. 8657_BO_K_2019, 11/03/2020). Conclusive results were obtained from 138 patients which were 98.2% (1,171/1,193) concordant to pre-existing multiplex ligation-dependent probe amplification (MLPA) data with respect to IKZF1, PAX5, CDKN2A, CDKN2B, PAR1, BTG1, EBF1, ETV6 and RB1 copy number status. For the detection of P2RY8::CRLF2, originating from an ~300 kbp deletion in the subtelomeric PAR1 region on chromosome X and Y, which is not completely resolved in the hg19 reference, the reference genomes hg38 or CHM13 (T2T) were used for mapping, resulting in improved resolution (Online Supplementary Figure S1). According to our data, 3 patients had to be excluded from further analysis: one due to a t(9;22)(q34.12;q11.23) resulting in BCR::ABL1 fusion, and 2 HeH patients for whom OGM did not confirm an IKZF1 deletion. Furthermore, 5 patients were reclassified due to the identification of an additional small deletion in PAX5 (exon 6-7), a previously undescribed intragenic ERG deletion, or because no deletion in PAX5, CDKN2A/B, or PAR1 could be detected. Overall, 94.9% (131/138) of the patients showed concordance regarding the IKZF1^del/plus definition. The final cohort consisted of 135 patients, including 71 classified as IKZF1^del and 64 classified as IKZF1^plus (Figure 1A).

In 52.6% (71/135) of the BCP-ALL patients in this study, genetic alterations with (putative) prognostic impact were present in addition to IKZF1^del/plus (Figure 1B). HeH, ETV6::RUNX1, or iAMP21 were detected in 18 patients. Patients with HeH and ETV6::RUNX1 were exclusively found in the IKZF1^del group. In 39.3% (53/135) of the cases, an additional alteration leading to a gene fusion was detected and validated on transcript level (Figure 1B; Online Supplementary Table S2). Gene fusions were found in both subgroups and grouped into ABL-class, CRLF2, JAK2, PAX5, ZNF384, and other fusions (Figure 1A). The CRLF2 group consisted only of P2RY8::CRLF2 fusions, which by definition allocated these patients to the IKZF1^plus subgroup. Five patients with PAX5::JAK2 fusions were allocated to the JAK2 group. We detected simple and complex SV that led gene fusions on a transcript level (Online Supplementary Table S1); for example, a simple inv(1)(q24.2q25.2) (Figure 2A), a derivative chromosome 1 including three inversions (Figure 2B), and a complex chromosome 1 involving a duplication and insertion (Figure 2C), that all led to RCSD1::ABL2 fusions. In the last case, an ~150 kbp segment containing exon 1-3 of RSCD1 was inserted in inverted orientation in between an ~150 kbp duplicated segment containing ABL2 exon 5-12. Similar to the findings by Stanulla et al.,⁷ a dismal 5-year EFS for patients with IKZF1^plus was observed compared to IKZF1^del and IKZF1^WT (Figure 3A). The cumulative incidence of relapse (CIR) was increased in IKZF1^plus patients (Figure 3B). Interestingly, the differences in 5-year EFS of IKZF1^plus and IKZF1^del were no longer seen when HeH, ETV6::RUNX1, and iAMP21 were excluded from the analysis (0.61±0.06 vs. 0.70±0.06) (Figure 3C). This can be explained by the fact that no patient with HeH or ETV6::RUNX1 experienced adverse events and these were found exclusively in the IKZF1^del group. However, IKZF1^plus patients were still more likely to experience a relapse (Figure 3D). In 45.3% (53/117) of patients without an established marker (HeH, ETV6::RUNX1, and iAMP21), a gene fusion was detected by OGM. Patients with a gene fusion had a statistically significant inferior 5-year EFS compared to patients without a fusion (0.56±0.07 vs. 0.73±0.06), while the CIR was just slightly increased (0.31±0.07 vs. 0.20±0.05) (Figure 2G, H). A particularly dismal EFS was observed in patients with ABL-class, PAX5, and CRLF2 fusions (Figure 3 I, J). Patients with other or no fusions had the most favorable outcomes. Due to the limited number of samples in each group, no conclusions could be drawn from the stratification of fusion subgroups for IKZF1^del and IKZF1^plus.

Figure 1.Overview and Optical Genome Mapping results of the analyzed cohort. (A) A total of 142 patients from the AIEOP-BFM ALL 2000/2009 trials were enrolled. Conclusive results were obtained from 138 patients; 3 patients were excluded. Based on the Optical Genome Mapping (OGM) findings, 5 patients were reclassified. An established prognostic marker (high hyperdiploidy, ETV6::RUNX1 or iAMP21) was identified in 18 patients and a gene fusion was discovered in 53 patients. (B) Heatmap of OGM results of the 135 analyzed IKZF1^del and IKZF1^plus patients. Patients were grouped into those with a prognostic marker (high hyperdiploidy, ETV6::RUNX1, iAMP21), IKZF1^del with or without gene fusion and IKZF1^plus with or without gene fusion. MRD: minimal residual disease; CNV: copy number variation; HR: high risk; MR: medium risk; SR: standard risk; n/a: not available; hom. del.: homozygous deletion; het. del.: heterozygous deletion; dup.: duplication; SV: structural variant.

Figure 2.RCSD1::ABL2 fusion resulting from three distinct structural variants detected by Optical Genome Mapping. (A) Simple inversion inv(1)(q24.2q25.2) with breakpoints in RCSD1 and ABL2. Upper track: cytobands. Second track: genes. Third track: reference genome chromosome 1q24.2-q25.2. Lower track: patient maps indicating inversion with breakpoints in q24.2 and q25.2. (B) Derivative chromosome 1 containing three inversions: der(1)inv(1)(q24.2q25.2)inv(1)(q24.2q41)inv(1)(q25.2q41). Upper track: cytobands. Second track: genes (orange) and segments according to genomic breakpoints (blue). Third track: reference genome chromosome 1q24.2-q41. Fourth track: patient maps indicating inversions. The bottom track shows a schematic representation of the inverted segments. (C) Complex event comprising an ~150 kbp duplication and an ~150 kbp insertion in inverted direction: der(1)dup(1) (q25.2q25.2)ins(1;1)(q25.2;q24.2q24.2). The references for chromosome 1q25.2 (upper bar) and 1q24.2 (lower bar) are shown in green and the corresponding patient maps in light blue. The duplicated region with a breakpoint in ABL2 is depicted in orange, while the inserted segment with a breakpoint in RCSD1 is represented in gray.

Figure 3.Kaplan-Meier plots for event-free survival and relapse incidence in IKZF1del and IKZF1^plus patients. (A) 5-year event-free survival (EFS) and (B) 5-year relapse incidence (CIR) of IKZF1^del and IKZF1^plus patients compared with 845 ALL-BFM 2000 patients with no IKZF1 deletion (IKZF1^WT). (C) 5-year EFS and (D) 5-year CIR of IKZF1del and IKZF1^plus patients when patients with high hyperdiploidy (HeH), ETV6::RUNX1, and iAMP21 were excluded from the analysis. (E) 5-year EFS and (F) 5-year CIR of IKZF1^del and IKZF1^plus patients when patients with HeH, ETV6::RUNX1, iAMP21 and gene fusions were excluded from the analysis. (G) 5-year EFS and (H) 5-year CIR of IKZF1^del/plus patients (without HeH, ETV6::RUNX1, iAMP21) with a gene fusion compared to patients without a gene fusion. (I) 5-year EFS and (J) 5-year CIR for the different fusion subgroups: ABL-class, CRLF2, JAK2, PAX5, ZNF384 and other fusions.

To evaluate the genuine prognostic relevance of the IKZF1^plus profile relative to patients solely harboring an IKZF1 deletion, we analyzed the outcome omitting those patients harboring HeH, ETV6::RUNX1, iAMP21, and gene fusions (Figure 3E, F). The disparity in 5-year EFS between IKZF1^plus and IKZF1^del was then negligible (0.68±0.09 vs. 0.77±0.09). In addition, the observed tendency for a higher likelihood of relapse in the IKZF1^plus group (0.25±0.09 vs. 0.15±0.06) did not reach statistical significance.

Among the 36 IKZF1^del and 28 IKZF1^plus patients without known prognostic markers (HeH, ETV6::RUNX1, iAMP21, gene fusion excluded), 4 IKZF1^del and 11 IKZF1^plus were categorized MRD standard risk, 25 IKZF1^del and 12 IKZF1^plus as medium risk, and 5 IKZF1^del and 4 IKZF1^plus as high risk. For 3 patients, MRD data was unavailable. Given the restricted sample size within each group, a statistical analysis of EFS and CIR was not conducted.

Consistent with recent studies comparing OGM to conventional cytogenetic techniques, a high overall concordance was achieved when comparing the OGM results to the existing MLPA data.^8,9 With the introduction of the IKZF-1^plus profile, the prognostic impact of the IKZF1 deletion and accompanying lesion in BCR::ABL1-negative BCP-ALL patients was further refined.⁷ In this study, we used OGM to perform a genome-wide evaluation of aneuploidies and SV in 97 patients from the AIEOP-BFM ALL 2000 trial, on which the IKZF1^plus profile had initially been established, and 45 patients with IKZF1^del/plus from the subsequent AIEOP-BFM ALL 2009 trial. Within this cohort, distinct classes of gene fusions were detected in 28.2% of IKZF1^del cases and 51.6% of IKZF1^plus cases. Consistent with existing literature, patients harboring a gene fusion, specifically BCR::ABL1-like fusions (ABL-class, CRLF2, and JAK2 fusions), exhibited an inferior 5-year EFS, suggesting their potential function as leukemic drivers.¹⁰ IKZF1 deletions are frequently observed in BCR::ABL1/-like B-ALL, which aligns with the finding that the majority of the detected fusions were classified as BCR::ABL1-like.^6,10,11 Interestingly, the 12 patients who carried favorable prognostic markers (HeH, ETV6::RUNX1) were exclusively found in the IKZF1^del subgroup and did not experience adverse events. It has recently been reported that an IKZF1 deletion negatively affected the prognosis in patients with HeH but not in those with ETV6::RUNX1.¹² When patients with HeH, ETV6::RUNX1, and also the unfavorable iAMP21 were excluded from the analysis, the poor prognostic value of the IKZF1 deletion became more pronounced, and the difference in EFS between IKZF1^del and IKZF1^plus diminished. Furthermore, when the newly detected gene fusions were excluded from the analysis as well, the prognostic value of IKZF1^plus and IKZF1^del became comparable but still inferior to IKZF1WT (EFS 0.68±0.09 vs. 0.77±0.07 vs. 0.87±0.01). IKZF1 lesions could have an independent prognostic effect, as it has been demonstrated that IKZF1 is implicated in multiple pathways essential for lymphoid cell differentiation and represents a potential therapeutic target.¹³ These findings align with previous studies, which have reported modest non-statistically significant differences between IKZF1^del and IKZF1^plus, but have confirmed the prognostic value of the deletion itself.^14,15 However, it is important to note that this cohort is limited, consisting of a small sample size, and only aneuploidies, CNV and SV were analyzed. Moreover, single nucleotide variants affecting, for example, the Ras and JAK-STAT pathways that are often subclonal were not investigated.^1,3 Nevertheless, the sample size of IKZF1^del/plus was comparable to that of the initial study.⁷ Our findings demonstrate that the prognostic relevance of the IKZF1^plus profile is not as pronounced as previously anticipated when the patients are genetically better characterized, but that the IKZF1 deletion itself still exerts a negative prognostic effect.

Footnotes

• Received August 18, 2023
• Accepted November 22, 2023

Correspondence

D. STEINEMANN - Steinemann.Doris@mh-hannover.de

Disclosures

No conflicts of interest to disclose.

Contributions

MSt and DS are responsible for study concept. JLL, MZ, WH, AKB, MSt and DS are responsible for study validation. JLL, MZ and DS are responsible for the formal analysis. JLL, MSt and DS are responsible for the investigation. MZ, WH, AKB, AM, GC, MSc, BS, MSt and DS provided resources. JLL, MZ, MSt and DS are responsible for data curation. JLL wrote the original draft. JLL, MZ, WH, MSc, BS, MSt and DS wrote, reviewed and edited the paper. JLL and MZ are responsible for visualization. MSt, BS and DS supervised the study. JLL and DS are responsible for project administration. MSt and DS are responsible for funding acquisition.