Characterisation of cells markers associated with IKZF1plus in BCP-ALL.

The presence of IKZF1 deletions has been associated with an increased relapse rate in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). There is a particular subset of IKZF1del cases called IKZF1plus (defined by the co-occurrence of IKZF1del and deletions in CDKN2A/B, PAX5, or the PAR1 region, in the absence of ERG deletions), which is also associated with worse prognosis, but some recent studies have not found major differences between the IKZF1del and IKZF1plus groups. Therefore, the IKZF1plus group still needs further comprehension and our study aims to characterise the molecular heterogeneity and identify molecular markers exclusively associated with IKZF1plus. Two independent series of cases (TARGET, n = 125 and GenLAb, n = 60) were evaluated by segregating patients into 3 groups: IKZF1plus, IKZF1del, and IKZF1wild. Differential expression analyses showed that the membrane protein-coding genes most associated with the IKZF1plus group were: KCNA5, GREB1, EPOR, SDK1, and PTPRB. Notably, KCNA5 and GREB1 differential expression levels were validated in the GenLAb validation series. Regarding copy number alterations, we observed a high frequency of VPREB1 deletions in the IKZF1plus group, as well as additional exclusive deletions in the CD200 and BTLA genes. Recent research suggests that the importance of the IKZF1plus profile varies depending on the genetic subgroup. In this scenario, we found associations between IKZF1plus and certain genes in BCP-ALL, being KCNA5 and GREB1 the most promising biomarkers for predicting IKZF1plus. A deeper understanding of these genetic profiles will allow a better risk assessment and offer precise rationale for therapeutic strategies in BCP-ALL.

IKZF1 and BTG1 silencing reduces glucocorticoid response in B-cell precursor acute leukemia cell line.

INTRODUCTION: Secondary genetic alterations, which contribute to the dysregulation of cell cycle progression and lymphoid specialization, are frequently observed in B-cell precursor acute lymphoblastic leukemia (B-ALL). As IKZF1 and BTG1 deletions are associated with a worse outcome in B-ALL, this study aimed to address whether they synergistically promote glucocorticoid resistance. METHODS: Small interfering RNA was used to downregulate either IKZF1, or BTG1, or both genes in the 207 B-ALL cell line. Cell viability was investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays. The expression levels of IKZF1, BTG1 and glucocorticoid-responsive genes (DUSP1, SGK1, FBXW7 and NR3C1) were evaluated by real time quantitative real time polymerase chain reaction (PCR). RESULTS: Isolated silencing of BTG1, IKZF1, or both genes in combination under dexamethasone treatment increased cell viability by 24%, 40% and 84%, respectively. Although BTG1 silencing did not alter the expression of glucocorticoid-responsive genes, IKZF1 knockdown decreased the transcript levels of DUSP1 (2.6-fold), SGK1 (1.8-fold), FBXW7 (2.2-fold) and NR3C1 (1.7-fold). The expression of glucocorticoid-responsive genes reached even lower levels (reducing 2.4-4 fold) when IKZF1 and BTG1 silencing occurred in combination. CONCLUSIONS: IKZF1 silencing impairs the transcription of glucocorticoid-responsive genes; this effect is enhanced by concomitant loss of BTG1. These results demonstrate the molecular mechanism by which the combination of both genetic deletions might contribute to higher relapse rates in B-ALL.

IKZF1plus is a frequent biomarker of adverse prognosis in Mexican pediatric patients with B-acute lymphoblastic leukemia.

Background: Recurrent genetic alterations contributing to leukemogenesis have been identified in pediatric B-cell Acute Lymphoblastic Leukemia (B-ALL), and some are useful for refining classification, prognosis, and treatment selection. IKZF1plus is a complex biomarker associated with a poor prognosis. It is characterized by IKZF1 deletion coexisting with PAX5, CDKN2A/2B, or PAR1 region deletions. The mutational spectrum and clinical impact of these alterations have scarcely been explored in Mexican pediatric patients with B-ALL. Here, we report the frequency of the IKZF1plus profile and the mutational spectrum of IKZF1, PAX5, CDKN2A/2B, and ERG genes and evaluate their impact on overall survival (OS) in a group of patients with B-ALL. Methods: A total of 206 pediatric patients with de novo B-ALL were included. DNA was obtained from bone marrow samples at diagnosis before treatment initiation. A custom-designed next-generation sequencing panel was used for mutational analysis. Kaplan-Meier analysis was used for OS estimation. Results: We identified the IKZF1plus profile in 21.8% of patients, which was higher than that previously reported in other studies. A significantly older age (p=0.04), a trend toward high-risk stratification (p=0.06), and a decrease in 5-year Overall Survival (OS) (p=0.009) were observed, although heterogeneous treatment protocols in our cohort would have impacted OS. A mutation frequency higher than that reported was found for IKZF1 (35.9%) and CDKN2A/2B (35.9%) but lower for PAX5 (26.6%). IKZF1MUT group was older at diagnosis (p=0.0002), and most of them were classified as high-risk (73.8%, p=0.02), while patients with CDKN2A/2BMUT had a higher leukocyte count (p=0.01) and a tendency toward a higher percentage of blasts (98.6%, >50% blasts, p=0.05) than the non-mutated patients. A decrease in OS was found in IKZF1MUT and CDKN2A/2BMUT patients, but the significance was lost after IKZF1plus was removed. Discussion: Our findings demonstrated that Mexican patients with B-ALL have a higher prevalence of genetic markers associated with poor outcomes. Incorporating genomic methodologies into the diagnostic process, a significant unmet need in low- and mid-income countries, will allow a comprehensive identification of relevant alterations, improving disease classification, treatment selection, and the general outcome.

Impact of IKZF1 Deletions in the Prognosis of Childhood Acute Lymphoblastic Leukemia in Argentina.

An association of deletions in the IKZF1 gene (IKZF1del) with poor prognosis in acute lymphoblastic leukemia (ALL) has been demonstrated. Additional deletions in other genes (IKZF1plus) define different IKZF1del subsets. We analyzed the influence of IKZF1del and/or IKZF1plus in the survival of children with ALL. From October 2009 to July 2021, 1055 bone marrow samples from patients with ALL were processed by Multiplex ligation-dependent probe amplification (MLPA). Of them, 28 patients died during induction and 4 were lost-in-follow-up, resulting in an eligible 1023 cases. All patients were treated according to ALLIC-BFM-2009-protocol. Patients were classified into three subsets: IKZF1not-deleted (IKZFF1not-del), IKZF1deleted (IKZF1del) and IKZF1del plus deletion of PAX5, CDKN2A, CDKN2B and/or alterations in CRLF2 with ERG-not-deleted (IKZF1plus). The LFSp and SE were calculated with the Kaplan−Meier calculation and compared with a log-rank test. From the 1023 eligible patients, 835 (81.6%) were defined as IKZF1not-del, 94 (9.2%) as IKZF1del and 94 (9.2%) as IKZF1plus. Of them, 100 (9.8%) corresponded to Standard-Risk (SRG), 629 (61.5%) to Intermediate-Risk (IRG) and 294 (28.7%) to High-Risk (HRG) groups. LFSp(SE) was 7 5(2)% for IKZF1not-del, 51 (6)% for IKZF1del and 48 (6)% for IKZF1plus (p-value < 0.00001). LFSp(SE) according to the risk groups was: in SRG, 91 (4)% for IKZF1not-del, 50 (35)% IKZF1del and 100% IKZF1plus (p-value = ns); in IRG, 77 (2)% IKZF1not-del, 61 (10)% IKZF1del and 54 (7)% IKZF1plus (p-value = 0.0005) and in HRG, 61 (4)% IKZF1not-del, 38 (8)% IKZF1del and 35 (9)% IKZF1plus (p-value = 0.0102). The IKZF1 status defines a population of patients with a poor outcome, mainly in IRG. No differences were observed between IKZF1del versus IKZF1plus. MLPA studies should be incorporated into the risk-group stratification of pediatric ALL.

IKZF1 deletions associate with CRLF2 overexpression leading to a poor prognosis in B-cell precursor acute lymphoblastic leukaemia.

Cytokine Receptor-Like Factor 2 (CRLF2) overexpression occurs in 5-15% of B-cell precursor acute lymphoblastic leukaemia (B-ALL). In ∼50% of these cases, the mechanisms underlying this dysregulation are unknown. IKAROS Family Zinc Finger 1 (IKZF1) is a possible candidate to play a role in this dysregulation since it binds to the CRLF2 promoter region and suppresses its expression. We hypothesised that IKZF1 loss of function, caused by deletions or its short isoforms expression, could be associated with CRLF2 overexpression in B-ALL. A total of 131 paediatric and adult patients and 7 B-ALL cell lines were analysed to investigate the presence of IKZF1 deletions and its splicing isoforms expression levels, the presence of CRLF2 rearrangements or mutations, CRLF2 expression and JAK2 mutations. Overall survival analyses were performed according to the CRLF2 and IKZF1 subgroups. Our analyses showed that 25.2% of patients exhibited CRLF2 overexpression (CRLF2-high). CRLF2-high was associated with the presence of IKZF1 deletions (IKZF1del, p = 0.001), particularly with those resulting in dominant-negative isoforms (p = 0.006). Moreover, CRLF2 expression was higher in paediatric samples with high loads of the short isoform IK4 (p = 0.011). It was also associated with the occurrence of the IKZF1 plus subgroup (p = 0.004). Furthermore, patients with CRLF2-high/IKZF1del had a poorer prognosis in the RELLA05 protocol (p = 0.067, 36.1 months, 95%CI 0.0-85.9) and adult cohort (p = 0.094, 29.7 months, 95%CI 11.8-47.5). In this study, we show that IKZF1 status is associated with CRLF2-high and dismal outcomes in B-ALL patients regardless of age.

Philadelphia-like acute lymphoblastic leukemia: Characterization in a pediatric cohort in a referral center in Colombia.

BACKGROUND: Philadelphia-like (Ph-like) acute lymphoblastic leukemia (ALL) is a subtype of pediatric leukemia with high risk factors and poor outcome. There are few reports of its prevalence in Latin America. AIM: This study evaluated the frequency and clinical and biological characteristics of Ph-like ALL in a pediatric cancer center in Colombia. METHODS: The Ph-like genetic profile was analyzed by a low-density array (LDA). Samples from patients with Ph-like ALL were analyzed by fluorescent in situ hybridization for cytokine receptor like factor 2 (CRLF2) and ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) rearrangements. Copy number variations were assessed by multiplex ligation probe amplification. RESULTS: Data from 121 patients were analyzed. Fifteen patients (12.4%) had Ph-like ALL, and these patients had significantly higher leukocyte counts at diagnosis and higher levels of minimal residual disease on days 15 and 33 of induction than patients without the Ph-like subtype. There were no significant differences in sex, age, or response to prednisone at day 8 between the two groups. CRLF2 rearrangements were identified in eight patients, and ABL1 rearrangements were identified in two patients. Other genetic alterations alone or in combination were identified in 77% of patients, including deletions in cyclin dependent kinase inhibitor 2 A/B (46.2%), IKAROS family zinc finger 1 (38.3%), and paired box 5 (30.8%). CONCLUSIONS: Ph-like ALL had a 12.4% prevalence in our cohort of patients with pediatric ALL. The identification of this group of patients has importance for risk stratification and future targeted therapy.

CRLF2 and IKZF1 abnormalities in Mexican children with acute lymphoblastic leukemia and recurrent gene fusions: exploring surrogate markers of signaling pathways.

The gene fusions BCR-ABL1, TCF3-PBX1, and ETV6-RUNX1 are recurrent in B-cell acute lymphoblastic leukemia (B-ALL) and are found with low frequency in coexistence with CRLF2 (cytokine receptor-like factor 2) rearrangements and overexpression. There is limited information regarding the CRLF2 abnormalities and dominant-negative IKZF1 isoforms associated with surrogate markers of Jak2, ABL, and Ras signaling pathways. To assess this, we evaluated 24 Mexican children with B-ALL positive for recurrent gene fusions at diagnosis. We found CRLF2 rearrangements and/or overexpression, dominant-negative IKZF1 isoforms, and surrogate phosphorylated markers of signaling pathways coexisting with recurrent gene fusions. All the BCR-ABL1 patients expressed CRLF2 and were positive for pCrkl (ABL); most of them were also positive for pStat5 (Jak2/Stat5) and negative for pErk (Ras). TCF3-PBX1 patients with CRLF2 abnormalities were positive for pStat5, most of them were also positive for pCrkl, and two patients were also positive for pErk. One patient with ETV6-RUNX1 and intracellular CRLF2 protein expressed pCrkl. In some cases, the activated signaling pathways were reverted in vitro by specific inhibitors. We further analyzed a TCF3-PBX1 patient at relapse, identifying a clone with the recurrent gene fusion, P2RY8-CRLF2, rearrangement, and phosphorylation of the three surrogate markers that we studied. These results agree with the previous reports regarding resistance to treatment observed in patients with recurrent gene fusions and coexisting CRLF2 gene abnormalities. A marker phosphorylation signature was identified in BCR-ABL1 and TCF3-PBX1 patients. To obtain useful information for the assessment of treatment in B-ALL patients with recurrent gene fusions, we suggest that they should be evaluated at diagnosis for CRLF2 gene abnormalities and dominant-negative IKZF1 isoforms, in addition to the analyses of activation and inhibition of signaling pathways.

High frequency of the risk allele of rs4132601 and rs11978267 from the IKZF1 gene in indigenous Mexican population.

BACKGROUND: IKZF1 is a relevant gene associated with the pathogenesis of acute lymphoblastic leukemia, and the rs4132601 (T>G) and rs11978267 (A>G) polymorphisms have been associated with the development of this disease in several populations. The aim of this study was to determine the allelic and genotypic frequencies of the rs4132601 and rs11978267 polymorphisms in two indigenous Mexican groups (Cora and Huichol) and Mestizo populations from Nayarit, Mexico, and compare them with the frequencies of both polymorphisms in other populations of the world. METHODS: One hundred, 116, and 100 subjects from the Mestizo, Huichol, and Cora populations, respectively, all of them residents of the state of Nayarit, Mexico, were analyzed. The frequencies of rs4132601 and rs11978267 were determined by allelic discrimination using TaqMan assays. RESULTS: The allelic frequencies of rs4132601 were as follows: Mestizo group T = 0.74, G = 0.26; Cora T = 0.745, G = 0.255; and Huichol T = 0.47, G = 0.53. In the case of the rs11978267 polymorphism, the allelic frequencies were Mestizo A = 0.745, G = 0.255; Cora A = 0.735, G = 0.265; and Huichol A = 0.457, G = 0.543. For each population, both polymorphisms were in Hardy-Weinberg equilibrium. CONCLUSION: The Huichol population from Nayarit presented the highest frequencies of the risk allele reported to date in the whole world for both rs4132601 and rs11978267 polymorphisms.

Integrated Genomic Analysis of Chromosomal Alterations and Mutations in B-Cell Acute Lymphoblastic Leukemia Reveals Distinct Genetic Profiles at Relapse.

The clonal basis of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is complex and not fully understood. Next-generation sequencing (NGS), array comparative genomic hybridization (aCGH), and multiplex ligation-dependent probe amplification (MLPA) were carried out in matched diagnosis-relapse samples from 13 BCP-ALL patients to identify patterns of genetic evolution that could account for the phenotypic changes associated with disease relapse. The integrative genomic analysis of aCGH, MLPA and NGS revealed that 100% of the BCP-ALL patients showed at least one genetic alteration at diagnosis and relapse. In addition, there was a significant increase in the frequency of chromosomal lesions at the time of relapse (p = 0.019). MLPA and aCGH techniques showed that IKZF1 was the most frequently deleted gene. TP53 was the most frequently mutated gene at relapse. Two TP53 mutations were detected only at relapse, whereas the three others showed an increase in their mutational burden at relapse. Clonal evolution patterns were heterogeneous, involving the acquisition, loss and maintenance of lesions at relapse. Therefore, this study provides additional evidence that BCP-ALL is a genetically dynamic disease with distinct genetic profiles at diagnosis and relapse. Integrative NGS, aCGH and MLPA analysis enables better molecular characterization of the genetic profile in BCP-ALL patients during the evolution from diagnosis to relapse.

IKZF1 Gene Deletion in Pediatric Patients Diagnosed with Acute Lymphoblastic Leukemia in Mexico.

The IKZF1 gene is formed by 8 exons and encodes IKAROS, a transcription factor that regulates the expression of genes that control cell cycle progression and cell survival. In general, 15-20% of the patients with preB acute lymphoblastic leukemia (preB ALL) harbor IKZF1 deletions, and the frequency of these deletions increases in BCR-ABL1 or Ph-like subgroups. These deletions have been associated with poor treatment response and the risk of relapse. The aim of this descriptive study was to determine the frequency of IKZF1 deletions and the success of an induction therapy response in Mexican pediatric patients diagnosed with preB ALL in 2 hospitals from 2017 to August 2018. Thirty-six bone marrow samples from patients at the Instituto Nacional de Pediatría in Mexico City and the Centro Estatal de Cancerología in Tepic were analyzed. The IKZF1 deletion was identified by MLPA using the SALSA MLPA P335 ALL-IKZF1 probemix. Deletions of at least 1 IKZF1 exon were observed in 7/34 samples (20.6%): 3 with 1 exon deleted; 1 with 2 exons, 1 with 5 exons, 1 with 6 exons, and 1 patient with a complete IKZF1 deletion. This study was descriptive in nature; we calculated the frequency of the IKZF1 gene deletion in a Mexican pediatric population with preB ALL as 20.6%.

IKZF1 deletion and co-occurrence with other aberrations in a child with chronic myeloid leukemia progressing to acute lymphoblastic leukemia.

Chronic myeloid leukemia (CML) is a rare disease in children. Different from that in adults, childhood CML involves transformative events occurring over a short time period. CML transformation to lymphoid blast phase (BP) is associated with copy number abnormalities, characteristic of BCR-ABL1 positive acute lymphoblastic leukemia, but not of CML in the chronic phase. Here, we present an unusual case of CML progressing to BP in a 1.6-year-old child, harboring IKZF1, PAX5, CDKN2A, and ETV6 deletions at diagnosis. It remains to be addressed whether distinct mechanisms might account for CML pathogenesis in early childhood.

CRLF2 expression associates with ICN1 stabilization in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematopoietic malignancy with few molecular alterations showing a consensual prognostic value. CRLF2 overexpression was recently identified in high-risk T-ALL patients. For these cases, no genomic abnormality was found to be associated with CRLF2 overexpression. IKZF1 has been recently shown to be a direct transcriptional regulator of CRLF2 expression. Moreover, it is known that NOTCH1 antagonizes IKZF1 in T-ALL. In light of these pieces of evidence, we reasoned that IKZF1 binding perturbation and CRLF2 upregulation could be associated in T-ALL. We evaluated two independent series of pediatric T-ALL cases (PHOP, n = 57 and TARGET, n = 264) for the presence of common T-ALL molecular abnormalities, such as NOTCH1/FBXW7 mutations. We also assessed CRLF2 and IKZF1 gene expression. CRLF2 overexpression was observed in 14% (PHOP) and 16% (TARGET) of T-ALL patients. No correlation was found between mRNA expression of CRLF2 and IKZF1 in both cohorts. Interestingly, we show that patients with mutations affecting NOTCH1-PEST domain and/or FBXW7 had higher CRLF2 expression (P = .04). In summary, we demonstrate for the first time that only mutations resulting in ICN1 (intracellular domain of NOTCH1) stabilization are associated with CRLF2 overexpression.

COBL is a novel hotspot for IKZF1 deletions in childhood acute lymphoblastic leukemia.

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in childhood B-cell precursor acute lymphoblastic leukemia. Because of its clinical importance, we previously mapped breakpoints of intragenic deletions and developed a multiplex PCR assay to detect recurrent intragenic ΔIKZF1. Since the multiplex PCR was not able to detect complete deletions (IKZF1 Δ1-8), which account for ~30% of all ΔIKZF1, we aimed at investigating the genomic scenery of IKZF1 Δ1-8. Six samples of cases with IKZF1 Δ1-8 were analyzed by microarray assay, which identified monosomy 7, isochromosome 7q, and large interstitial deletions presenting breakpoints within COBL gene. Then, we established a multiplex ligation-probe amplification (MLPA) assay and screened copy number alterations within chromosome 7 in 43 diagnostic samples with IKZF1 Δ1-8. Our results revealed that monosomy and large interstitial deletions within chromosome 7 are the main causes of IKZF1 Δ1-8. Detailed analysis using long distance inverse PCR showed that six patients (16%) had large interstitial deletions starting within intronic regions of COBL at diagnosis, which is ~611 Kb downstream of IKZF1, suggesting that COBL is a hotspot for ΔIKZF1. We also investigated a series of 25 intragenic deletions (Δ2-8, Δ3-8 or Δ4-8) and 24 relapsed samples, and found one IKZF1-COBL tail-to-tail fusion, thus supporting that COBL is a novel hotspot for ΔIKZF1. Finally, using RIC score methodology, we show that breakpoint sequences of IKZF1 Δ1-8 are not analog to RAG-recognition sites, suggesting a different mechanism of error promotion than that suggested for intragenic ΔIKZF1.

Frequency of copy number abnormalities in common genes associated with B-cell precursor acute lymphoblastic leukemia cytogenetic subtypes in Brazilian children.

Copy number alterations (CNAs) in genes committed to B-cell precursors have been associated with poor survival in subgroups of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated submicroscopic alterations in a series of 274 Brazilian children with BCP-ALL by multiplex ligation-dependent probe amplification and evaluated their correlation with clinical and laboratory features. The relevance of overlapping CNA abnormalities was also explored. Deletions/amplifications in at least one gene were identified in 83% of the total series. In children older than 2 years, there was a predominance of CNAs involving deletions in IKZF1, CDKN2A, and CDKN2B, whereas the pseudoautosomal region 1 (PAR1) had deletions that were found more frequently in infants (P <0.05). Based on the cytogenetic subgroups, favorable cytogenetic subgroups showed more deletions than other subgroups that occurred simultaneously, specifically ETV6 deletions (P <0.05). TCF3-PBX1 was frequently deleted in RB1, and an absence of deletions was observed in IKZF1 and genes localized to the PAR1 region. The results corroborate with previous genome-wide studies and aggregate new markers for risk stratification of BCP-ALL in Brazil.