Relative Expression of Ikaros Isoforms Has a Prognostic Impact in Phildelphia-Negative Childhood Acute Lymphoblastic Leukemia.

Abstract Abstract 1282 Poster Board I-304 Ikaros, encoded by the IKZF1 gene, is a zinc-finger transcription factor crucial for normal differentiation of the lymphoid lineage. Multiple isoforms of Ikaros are generated by alternative splicing in lymphoid progenitors. Recent studies based primarily on high-risk (HR) patients with ALL, including Philadelphia-positive cases, have shown an inferior prognosis of patients with Ikaros alterations, leading in most cases to the expression of short, non-DNA binding isoforms of IKZF1. There is only a limited information about the overall frequency and prognostic impact of IKZF1 alterations in non-selected, BCR/ABL-negative ALL cohorts. Using a simplified yet efficient approach based on expression assay described by Iaccobucci et al. together with Agilent-on-chip semi-quantitative electrophoresis, we examined the expression of IKZF1 transcript variants in diagnostic bone marow (BM) samples from 94 children with Ph- ALL. The patients were diagnosed between November 2002 and December 2004 and treated by ALL IC-BFM 2002 protocol. Based on the analysis of peripheral blood from healthy donors and remission BM samples, we determined physiological range for relative expression of IKZF1 isoforms. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK8) and functional IK1 and IK2 isoforms was significantly elevated in 26 of 94 patients (28%). There were no associations between elevated short/long isoforms ratio and age, WBC, ALL IC risk group, TEL/AML1 or hyperdiploidy. Considering the key role of Ikaros in lymphoid lineage specification, we tested whether its altered expression was related to the expression of myeloid markers on leukemic blasts. Neither short/long isoforms ratio, nor single transcript variant expression had any relation to the level of myeloperoxidase (MPO), CD13, CD33, CD65, CD117, CD14 or CD15 expression estimated by flow cytometry. Patients having the short/long isoforms ratio more than 80% had a 5-year RFS 66.7±13.6% compared to 87.5±4.1% in other patients (p=0.04). The main difference between leukemic and normal samples was observed in the relative expression of IK6 dominant-negative isoform. Using a cut-off of 10%, 14 of 94 (15%) ALL samples had increased IK6 expression in relation to other isoforms. Only 2 of 94 patients (2%) expressed IK6 alone. Elevated relative IK6 expression in the range 10-20% (6 patients) had no prognostic impact. Using a cut-off of 20%, 5-year RFS survival was 90.2± 3.3% in the group with low IK6 expression compared to 37.5±17.1% in patients with the high expression (p<0.0001). With the cut-off of 50%, RFS was 20±17.9% in the IK6 high expression group compared to 89.4±3.3% in the group with low expression (p<0.0001). Of the 5 patients with IK6 expression higher than 50%, two were treated in ALL IC HR group (based on poor prednisone response), one in the intermediate risk and two in the standard risk group, based on WBC, age and BM status at day 15. Only 1 of 4 patients with available MRD would be classified as MRD-HR based on MRD higher than 10−3 at week 12. Surprisingly, elevated relative expression of IK4, IK4del, IK4A and IK8 (all non-DNA binding isoforms) had no prognostic impact. The absolute level of IKZF1 expression, which might have indicated IKZF1 haploinsufficiency, had no prognostic impact either. In conclusion, we showed that a substantial proportion of childhood Ph-negative ALL cases had an increased expression of short, non-DNA binding Ikaros transcripts. However, only elevated expression of the IK6 isoform had prognostic impact. Patients with IK6 expression higher than 50% (5% of all patients) had very poor prognosis. The method used in this study could serve as a rapid screening for a new subgroup of HR patients with ALL. Supported by GA UK 7393/2007, MSMT NPV 2B06064, VZ MSM 0021620813 and MZ 000064203. Disclosures No relevant conflicts of interest to declare.

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Prognostic Impact of Ikaros (IKZF1) Gene Alterations In Childhood ALL Treated with ALL IC-BFM 2002 Protocol: A Comparison of Gene Expression and Genomic-Based Methods.

Blood ◽

10.1182/blood.v116.21.1656.1656 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1656-1656

Author(s):

Jana Volejnikova ◽

Ester Mejstrikova ◽

Karel Svojgr ◽

Jan Stary ◽

Jan Trka ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Gene Deletion ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Treatment Protocol ◽

Induction Treatment ◽

Prognostic Impact ◽

Childhood All ◽

Gene Alterations

Abstract Abstract 1656 Introduction: Recently, Ikaros (IKZF1) gene alterations were found to predict poor prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, the implementation of IKZF1 status into the risk group stratification is discussed. So far, limited data are available concerning both IKZF1 importance in different treatment protocols for Ph-negative ALL and the choice of the best diagnostic method. In this study, we compared two methods based on either genomic DNA examination or gene expression analysis, and their prognostic impact within a treatment protocol for childhood ALL. Methods: Gene expression of functional (IK1, IK2) and non-DNA binding (IK4, IK6, IK8) IKZF1 isoforms was semi-quantitatively evaluated using Lab-on-a-chip (Agilent) electrophoresis and reported either as an absolute level or relatively to the total isoform signal. The thresholds for abnormal gene expression were set based on the analysis of peripheral blood (PB) of healthy donors, remission bone marrow (BM) samples of children with ALL, and sorted B- and T-cell precursor subpopulations. MLPA (multiplex ligation-dependent probe amplification) reaction including probes for Ikaros exons 1 to 8 was performed on BM DNA samples and the products were analyzed with the Coffalyser v9.4 software. Results: Results of both gene expression and MLPA analysis in the BM were available for 120 of 193 children diagnosed with ALL between 2002 and 2005. MLPA analysis revealed a deletion of at least one exon of IKZF1 gene in 17/120 (14%) patients. Of note, two patients with this deletion underwent a lineage switch (LS) from ALL to AML during the induction treatment. The entire IKZF1 gene was mononoallelically deleted in 3 patients. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK6, Ik6del, IK8) and functional (IK1 and IK2) isoform expression was significantly elevated (non-DNA binding isoforms>70%) in 21 of 120 (18%) patients. The expression of a dominant-negative IK6 isoform was significantly elevated (>20% of total) in 7 of 120 (6%) patients. Surprisingly, gene deletion on one allele was not accompanied by decrease of total IKZF1 gene expression. On the contrary, patients with IKZF1 deletion had higher total IKZF1 transcript level than those without deletion (p=0.008, Mann Whitney), but it was not possible to set any reliable expression threshold for the prediction of gene deletion. The change on a DNA level was not always reflected in relative gene expression: Of 17 patients with gene deletion, only 7 had significantly altered short/long isoform ratio and 5 patients had an increased expression of IK6. Conversely, of 7 patients with IK6 overexpression, two patients had no DNA alteration, suggesting a different mechanism of altered gene expression. We next evaluated the prognostic impact of IKZF1 alterations in 113 patients treated with ALL IC-BFM 2002 protocol (5 patients were excluded due to Ph-positive ALL with imatinib-based treatment and 2 patients due to treatment change after LS). Patients with IKZF1 gene deletion had significantly worse relapse-free survival (RFS) than other patients (5-year RFS 50.0±14.4% vs. 90.8±2.9%, p=0.0002). The presence of IKZF1 deletion did not correlate with minimal residual disease (MRD) during induction treatment (days 8, 15, 33), neither in BM nor in PB. Patients with IK6 overexpression had 5-year RFS 50.0±20.4% compared to 88.4± 3.2% in those with low IK6 expression (p=0.004). The elevated short/long isoform ratio (>70%) had no prognostic impact. The best prediction of relapse was achieved via combining two factors: the presence of IKZF1 deletion detected by MLPA or the relative IK6 overexpression (>50% of total isoform signal). The 5-year RFS was 50.0±13.4% for this group (14 pts, 7 relapses) compared to 91.6±2.8% for other patients (99 pts, 8 relapses, p<0.0001). Conclusion: This study confirmed that the presence of Ikaros gene alterations was connected with a high risk of relapse also in a BFM-based protocol for Ph-negative childhood ALL treatment. The deletion within IKZF1 locus did not necessarily correlate with an altered Ikaros gene expression. Ideally, both genomic and gene expression-based approach should be applied together for the evaluation of prognosis. However, if this is not possible, the examination of DNA changes by MLPA identifies more patients who subsequently relapse than the gene expression-based approach. Support: VZ MSM 0021620813, P301/10/1877, IGA NS/10472-3 Disclosures: No relevant conflicts of interest to declare.

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The Level of Histone Deacetylase 7 in Pediatric ALL and Its Effect on Survival

Blood ◽

10.1182/blood.v128.22.5099.5099 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5099-5099

Author(s):

Bei Hou ◽

Huyong Zheng

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Histone Deacetylase ◽

Conflicts Of Interest ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

Intermediate Risk ◽

Newly Diagnosed ◽

Altered Expression ◽

Expression Levels ◽

Pediatric All

Abstract Background Leukemia is the most common pediatric malignancy and a major cause of mortality and morbidity in children. Nearly 15,000 children are newly diagnosed with leukemia in China each year and among them, acute lymphoblastic leukemia (ALL) accounts for more than 75%. Altered expression of histone deacetylases (HDACs) is a common feature in several human malignancies and may represent an interesting target for cancer treatment . With increasing focus on precision medicine, HDACs have emerged as promising therapeutic target in pediatric ALL. Methods We detect the histone deacetylase 7(HDAC7) expression in the bone marrow samples of 254 children with newly diagnosed acute lymphoblastic leukemia (ALL) at Hematology Oncology Center of Beijing Children`s Hospital from January 2010 to the end of 2012 via qRT-PCR using the TaqMan gene expression probes. 3 patients that have been completely remission for 3 years are used as normal control. We find out that the expression levels of HDAC7 are associated with the unfavorable events (such as induction failure, relapse and/or death due to any cause ) occurence rates and 5-EFS. Results Here we find that the HDAC7 expression levels are associated with the unfavorable events occurence rates and 5-EFS. We find that in the intermediate risk group of 157 patients, the patients who have lower level of HDAC7 expression have higher unfavorable event occurence rates than the higher expression of HDAC7 group (p=0.001). The group of higher HDAC7 expression have higher 5-EFS than the lower group in both the intermediate risk group(p=0.001) and the T-ALL group(n=18). Conclusion We conclude that HDAC7 has a potent anti-oncogenic effect on specific pediatric B-cell leukemia, indicating that its deregulation may contribute to the pathogenesis of the disease. Disclosures No relevant conflicts of interest to declare.

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The Expression Pattern of Different Ikaros Isoforms In Adult B-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v116.21.4325.4325 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4325-4325

Author(s):

Li Yao ◽

Zi-Xing Chen ◽

Jian-nong Cen ◽

Su-ning Chen ◽

Jun He ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Dna Binding ◽

Expression Pattern ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Normal Karyotype ◽

Expression Level ◽

Wild Type ◽

Relative Expression ◽

Relative Expression Level

Abstract Abstract 4325 Ikaros plays an important role in the control of differentiation and proliferation of all lymphoid lineages. The multiple isoforms that lack different numbers of exons have different functions. The most previous studies focused on acute lymphoblastic leukemia(ALL) patients with Philadelphia chromosome. To investigate the expression pattern of different Ikaros isoforms in both BCR-ABL positive and BCR-ABL negative ALL, especially those in a normal karyotype. 114 adult B-ALL patients at diagnosis were involved in this study. 20 healthy normal volunteers and patients with leukemia in remission were also analyzed for Ikaros expression. The expression of different wild-type and aberrant Ikaros transcripts were detected and quantified by a fast, high-throughput capillary electrophoresis sizing method. The relative expression level of one isoform in a sample was calculated as the expression level of this isoform divided by the sum of expression level of all kinds of isoform in this sample. In 62 cases of BCR-ABL positive ALL, 32 patients(52%) expressed only non-DNA-binding isoforms, which consisted of Ik6(28/32,88%), Ik6+Ik6 ¢(3/32, 9%) and Ik6 ¢(1/32, 3%). 30 patients (49%) simultaneously expressed multiple Ikaros variants in a single sample corresponding to the wild-type Ik6 ¢ AIk6 AIk8 AIk4A AIk4 AIk5A AIk2 and aberrant Ik2(ins) AIk2(ins+del) AIk4(ins) and Ik4 (ins+del). The median value of relative expression level of each isoform was 0.01 A0.19 A0.07 A0.06 A0.22 A0.06 A0.23 A0.35 A0.11 A0.17 and 0.11, respectively. In 52 cases of BCR-ABL negative ALL, including 27 cases with a normal karyotype and 25 cases with recurring chromosomal abnormality other than t(9;22), 7 patients(13%) expressed only non-DNA-binding isoforms, which consisted of Ik6(2/7,29%) and Ik6+Ik6 ¢(5/7,71%). Notably, all of 7 patients have a normal karyotype. Therefore, the frequency of expression of only non-DNA-binding isoforms in patients with a normal karyotype was 39%(7/27), Whereas coexpression of multiple variants were identified in 45 patients (85%). The median value of relative expression level of each isoform was 0.01 A0.15 A0.11 A0.07 A0.25 A0.09 A0.25 A0.3 A0.16 A0.18 and 0.14 for Ik6 ¢ AIk6 AIk8 AIk4A AIk4 AIk5A AIk2 AIk2(ins) AIk2(ins+del) AIk4(ins) and Ik4 (ins+del), respectively. There was no statistical difference in relative expression level of different Ikaros variants between groups, except for Ik6. In normal donors, the median value of relative expression level of each isoform was 0 A0.09 A0.07 A0.06 A0.24 A0.12 A0.35 A0.49 A0.13 A0.18 and 0.07 for Ik6 ¢ AIk6 AIk8 AIk4A AIk4 AIk5A and Ik2 AIk2(ins) AIk2(ins+del) AIk4(ins) and Ik4 (ins+del),respectively. In comparison with normal donors, the expression of Ik2 AIk5A and Ik2(ins) are relatively lower, while the expression of Ik6 and Ik4(ins+del) are relatively higher in adult ALL patients. These results indicated that disproportional expression of different Ikaros isoforms could contribute to leukemogenesis. The overexpression of non-DNA- binding isoform, largely coexpression of Ik6 and Ik6 ¢, usually existed in patients with a normal karyotype, These information could help in the further classification of adult ALL subgroup and individualized treatment. Disclosures: No relevant conflicts of interest to declare.

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IKZF1 Deletion Is An Independent Predictor of Outcome In Pediatric Acute Lymphoblastic Leukemia Treated According to the ALL-BFM 2000 Protocol

Blood ◽

10.1182/blood.v116.21.409.409 ◽

2010 ◽

Vol 116 (21) ◽

pp. 409-409

Author(s):

Petra Breithaupt ◽

Barbara Meissner ◽

Martin Zimmermann ◽

Anja Möricke ◽

André Schrauder ◽

...

Keyword(s):

Treatment Outcome ◽

High Risk ◽

Cox Regression ◽

Conflicts Of Interest ◽

Residual Disease ◽

Risk Group ◽

Independent Predictor ◽

Lymphoblastic Leukemia ◽

Time Points ◽

Pediatric All

Abstract Abstract 409 Alteration of the IKZF1 gene – encoding the transcription factor IKAROS, a key player in lymphoid development and tumor suppression – has been reported to be associated with a poor outcome in pediatric precursor B-cell ALL, especially in cases positive for the BCR-ABL1 fusion gene. In order to assess the prognostic value of IKZF1 deletions in a representative cohort of pediatric ALL patients treated on the German ALL-BFM 2000 study protocol, we screened 409 patients by applying a multiplex ligation-dependent probe amplification (MLPA) assay covering all eight IKZF1 exons (P335-A3 ALL-IKZF1 probemix; MRC-Holland, Amsterdam, The Netherlands). In ALL-BFM 2000, risk group stratification (standard, SR; intermediate, MR; high, HR) was based on minimal residual disease (MRD) analysis at two different time points (TP) and required two MRD targets with sensitivities of ≤10−4 (Flohr et al. Leukemia 2008). SR patients were MRD-negative on treatment days 33 (TP1) and 78 (TP2). HR patients had residual disease (≥10−3) at TP2. MRD MR patients had positive MRD detection at either one and or both time points but at a level of <10−3 at TP2. Although MRD-based stratification criteria were introduced in ALL-BFM 2000, established high-risk parameters were also retained: patients with prednisone poor-response or ≥5% leukemic blasts in the bone marrow on day 33 or positivity for a t(9;22) or t(4;11) or their molecular equivalents (BCR/ABL1 or MLL/AF4 fusion RNA) were stratified into the high-risk group independent of their MRD results. First results on MRD and outcome were published earlier (Conter et al. Blood 2010). Out of the 409 patients analyzed in our study, 46 (11%) displayed a deletion in at least one of the eight IKZF1 exons. Forty-three out of the 46 cases showed heterozygous deletions, while 3 patients displayed homozygous loss of IKZF1 exons. MLPA results of 11 patients were validated with results derived from copy number/LOH analyses using Affymetrix SNP 6.0 arrays. IKZF1 deletion was significantly more common in precursor B compared to T cell ALL (13% vs. 4%, P = 0.03) and less frequent in TEL/AML1-positive ALL (3% vs. 13%, P = 0.004). Out of 11 BCR/ABL1-positive samples, only two were characterized by an IKZF1 deletion. Forty-four patients with IKZF1-deleted ALL had results of MRD analyses available for both informative time points (day 33 after induction and day 78 after consolidation). Despite a trend towards increasing incidence of IKZF1 deletion in patients with slow response, the distribution of IKZF1-deleted ALL patients over the risk groups was not significantly different from non-deleted ALL (SR: 40.9 vs. 41.9; MR: 45.5 vs. 52.3; HR: 13.6 vs. 5.7%; P = 0.153). Regarding treatment outcome, patients with an IKZF1 deletion had a significantly lower 5-year event-free survival (EFS) compared to non-deleted patients (0.78±0.06 vs. 0.86±0.02; P = 0.015). This result was due to a higher cumulative incidence of relapses in IKZF1-deleted patients (0.16±0.05 vs. 0.10±0.02; P = 0.031). In multivariate Cox regression analyses including known prognostic variables (gender, immunophenotype, WBC count at diagnosis, TEL/AML1 status, risk group criteria of ALL-BFM 2000), IKZF1 deletion conferred a risk of 2.16 (95% confidence interval 1.14 – 4.10; P = 0.018) for an event when compared to non-deleted patients. We conclude that IKZF1 deletion is an independent predictor of treatment outcome for patients enrolled on the ALL-BFM 2000 protocol and represents a candidate marker to be integrated in future algorithms for early risk stratification in pediatric ALL. Disclosures: No relevant conflicts of interest to declare.

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Relapsed Childhood High Hyperdiploid Acute Lymphoblastic Leukemia: Genome-Wide Screening Reveals the Presence of Preleukemic Ancestral Clones and the Secondary Nature of Microdeletions and RTK-RAS Mutations.

Blood ◽

10.1182/blood.v114.22.2591.2591 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2591-2591

Author(s):

Josef Davidsson ◽

Kajsa Paulsson ◽

David Lindgren ◽

Henrik Lilljebjörn ◽

Tracy Chaplin ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Structural Changes ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Snp Array ◽

Prognostic Impact ◽

Genetic Changes ◽

Ras Mutations ◽

Paired Samples

Abstract Abstract 2591 Poster Board II-567 Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% relapse. This makes it important to identify these patients already at diagnosis to ensure proper risk-stratification. To identify changes associated with relapse and ascertain the genetic evolution patterns, SNP array and mutation analyses of FLT3, KRAS, NRAS, and PTPN11 were performed on 11 paired diagnostic/relapse samples. The “triples trisomies” +4, +10, and +17 were detected in 64%, a frequency similar to the one generally observed at diagnosis, thus questioning their favorable prognostic impact. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. Based on the genetic relationship between the paired samples, three groups were delineated: 1) identical genetic changes at diagnosis and relapse (18%), 2) clonal evolution with all changes at diagnosis being present at relapse (18%), and 3) clonal evolution with some changes conserved, lost or gained (64%), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern and perhaps necessary for overt leukemia. Disclosures: No relevant conflicts of interest to declare.

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Outcome of Relapse After Allogeneic Transplantation for Childhood Acute Lymphoblastic Leukemia In Complete Remission. A Study of the Pediatric Diseases and Acute Leukemia Working Parties of the EBMT Group

Blood ◽

10.1182/blood.v116.21.1291.1291 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1291-1291

Author(s):

Adriana Balduzzi ◽

Myriam Labopin ◽

Vanderson Rocha ◽

Nabila Elarouci ◽

Giorgio Dini ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Hematopoietic Cell Transplantation ◽

Conflicts Of Interest ◽

Pediatric Population ◽

Lymphoblastic Leukemia ◽

Childhood Acute Lymphoblastic Leukemia ◽

Prognostic Impact ◽

Dismal Prognosis ◽

B Lineage ◽

Donor Lymphocyte Infusions

Abstract Abstract 1291 Introduction. Childhood acute lymphoblastic leukemia (ALL) relapse occurring after hematopoietic cell transplantation (HCT) has a very dismal prognosis. Its treatment is still controversial and ranges from palliative treatment or chemotherapy to donor lymphocyte infusions, second transplant or experimental approaches. Objectives. The aim of this study is to assess the actual outcome in a pediatric population. The primary endpoint of this study is the 2-year probability of survival of children with ALL relapsing after allogeneic HCT; the secondary endpoint is the relationship between outcome and time of relapse after transplant, for which the following categories were considered: <3, 3–6, 6–12, > 12 months. Patients. Patients younger than 18 years of age undergoing first HCT from any allogeneic donor for ALL in first (CR1) or second (CR2) remission between January 1st 1998 and December 31st 2007 reported to the EBMT were eligible for the study. Results. Out of 3628 transplanted children with ALL reported to the EBMT, 836 (median age 9 years, male 66%) relapsed at a median of 6 months (range 1–67; 25th, 75th 4, 12 months) after HCT. The HCT was performed in CR1 (60%) or CR2 (40%) for a B-lineage (60%) or T- (13%) or unknown (27%) immunophenotype ALL, from an HLA-matched related (44%), unrelated (59%) or mismatched related (7%) donor, with marrow (61%), peripheral (28%) or umbilical (11%) stem cells. Out of 836, 81% died at a median of 2 months (25th,75th centiles:1,7) and 19% were reported as alive at last follow-up at a median of 22 months after relapse (range: 1–130). The 3-year probability of overall survival (3y-OS) was 14% (SE 1). As to immunophenotype, disease phase and donor type, 3y-OS was 15% (SE 2) in B-lineage and 8% (SE 3) in T-ALL, 18% (SE 2) in patients transplanted in CR1 and 11% (SE 6) in CR2 and 17% (SE 2) in patients transplanted from an HLA-identical sibling and 12% (SE 2) from any other donor. According to time of relapse after transplant, 3-year OS was 6% (SE 2), 10% (SE 2), 15% (SE 2) and 27% (SE 4) in those who relapsed in the first quarter, second quarter, second semester or after the first year, respectively. Donor lymphocyte infusions were reported for 7% and a second HCT for 16% of the 836 relapsed children. The probability of undergoing a second HCT within 1 year after relapse was 17% (SE 1); this probability was 6% for relapses occurring <6 months and 25% for later relapses. 3y-OS of those who underwent a second HCT was 32% (SE 5). Conclusions. The multivariate analyses confirmed the prognostic role of disease phase and immunophenotype, but not of the type of donor, assessed the strong prognostic impact of the time elapsed in CR after HCT before relapse, being earlier relapses at worse outcome compared with later relapses, possibly due to the chance of undergoing a second HCT, which role per se was not statistically significant. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Impact of a Novel Biomarker, Serum Soluble LR11 on Acute Leukemia

Blood ◽

10.1182/blood.v116.21.2717.2717 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2717-2717

Author(s):

Chikako Ohwada ◽

Masahiro Takeuchi ◽

Shio Sakai ◽

Daijiro Abe ◽

Yusuke Takeda ◽

...

Keyword(s):

Treatment Outcome ◽

Acute Leukemia ◽

Myeloid Leukemia ◽

Hematological Malignancies ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Laboratory Data ◽

Type I ◽

Prognostic Impact ◽

Soluble Lr11

Abstract Abstract 2717 Introduction: LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding. LR11 plays a key role in the migration of undifferentiated vascular smooth muscle cells, and circulating sLR11 is known to be a biomarker of carotid intima-media thickness. Along with the fact that circulating sLR11 levels represent the accumulation of vascular immature cells, human CD34+CD38− immature hematopoietic precursors have been reported to express high levels of LR11 mRNA. We have recently found that LR11 is specifically and highly expressed on cell surface of acute leukemia cells in addition to normal leukocytes (unpublished data). These facts prompted us to evaluate the serum sLR11 level in patients with acute leukemia and other hematological malignancies to validate sLR11 as a novel circulating marker for treatment outcome and prognosis. Patients and Methods: Serum sLR11 levels were measured by ELISA method in 139 patients with acute leukemia and other hematological malignancies treated at a single institution from 1999 to 2010. Patients' laboratory data and treatment outcome were collected retrospectively in 43 acute myeloid leukemia (AML) and 23 acute lymphoblastic leukemia (ALL) patients. Results: sLR11 levels of acute leukemia patients were significantly increased [ALL, 73.5±93.5 ng mld−1 (range, 5.7–407.0), P<0.0001; AML, 26.8±29.1 ng ml−1 (range, 5.0–157.5), P<0.0001] in comparison to the control subjects (9.2±3.3 ng ml−1), while sLR11 levels in patients with chronic myeloid leukemia (17.9±11.1 ng ml−1), chronic lymphocytic leukemia (12.7±11.6 ng ml−1), multiple myeloma (10.5±4.8 ng ml−1), and POEMS syndrome (9.0±2.7 ng ml−1) were not significantly different from controls. sLR11 levels were significantly higher in ALL than those in other leukemias. Paired sample analysis of patients with AML and ALL at complete remission (CR) after chemotherapy showed significantly decreased sLR11 levels compared to the time of diagnosis (AML: 30.9±37.5 ng ml−1 vs. 10.4±4.3 ng ml−1, P=0.015, ALL: 39.1±126.0 ng ml−1 vs. 11.2±5.0 ng ml−1, P=0.0029). The multiple stepwise liner regression analysis showed that the peripheral blast proportion in both ALL and AML patients were independently associated with sLR11 at diagnosis (AML: r2= 0.21, P=0.0026, ALL: r2= 0.34, P=0.0043). Among 42 AML patients, sLR11 levels of subjects in the highest tertile of peripheral blast proportion (>67.5% of WBC) were 2.44- and 3.05-fold higher than those in the middle (23.0-64.0% of WBC) and lowest tertiles (<20.0% of WBC), respectively. Twenty out of 21 AML patients with <20 ng ml−1 sLR11 at diagnosis achieved CR after induction chemotherapy, and the CR rate was significantly higher in patients with <20 ng ml−1 sLR11 than in patients with ≥20 ng ml−1 (95.2% vs 65.5%, P=0.02). The probability of overall 5-year survival was significantly lower in AML patients with ≥20 ng ml−1 sLR11 at diagnosis than in those with <20 ng ml−1 [Figure1, 36.8% vs 63.7%, P = 0.04; hazard ratio (HR): 2.74; 95% confidence interval (CI): 1.04–8.01]. Conclusions: Serum sLR11 levels in patients with acute leukemia were significantly elevated and were associated with the peripheral blast population but not in other chronic proliferative hematological malignancies. These findings suggest that the serum sLR11 levels are predictive for pathogenic properties of immature blasts, including their migration and attachment activities, rather than simply associating with proliferating cell numbers. Especially in AML patients, serum sLR11 levels at diagnosis significantly affect CR rate and OS. Although larger scale studies including karyotype or FAB classification would be required for its patho-clinical significance, serum sLR11 is a promising novel biomarker for acute leukemia and it could play an important role as prognostic factor. Disclosures: No relevant conflicts of interest to declare.

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Identification of a Novel T-ALL Entity with NKX2-1/NKX2-2 Rearrangements

Blood ◽

10.1182/blood.v116.21.3139.3139 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3139-3139

Author(s):

Irene Homminga ◽

Rob Pieters ◽

Anton Langerak ◽

Johan de Rooi ◽

Andrew Stubbs ◽

...

Keyword(s):

T Cell ◽

Conflicts Of Interest ◽

Human Cancer ◽

Lymphoblastic Leukemia ◽

Cell Receptor ◽

High Expression ◽

Molecular Cytogenetic ◽

Oncogenic Pathways ◽

Cytogenetic Analyses ◽

On Chip

Abstract Abstract 3139 To identify novel oncogenic pathways in T-cell acute lymphoblastic leukemia (T-ALL), we combined expression profiling of 117 pediatric patient samples and detailed molecular cytogenetic analyses. Using unsupervised and supervised analyses, we identified a novel T-ALL entity that was associated with high expression of cell cycle genes and was accordingly denoted as the proliferative cluster. Patient samples in this cluster were devoid of known oncogenic rearrangements, and clustered together with TLX1-rearranged cases in unsupervised cluster analysis suggesting pathogenic mechanisms are conserved between these cases. Alike TLX1-rearranged cases, patients within this cluster were associated with cortical thymic arrest and expressed CD1 along with CD4 and CD8. Bioinformatic analyses identified NKX2-1 as strong outlier gene that was ectopically expressed in most patients samples belonging to this cluster. One patient sample had high expression of the homologous NKX2-2 gene. Molecular-cytogenetic analyses including the Chromatine Conformation Capture on Chip (4C) method revealed various rearrangement variants of the NKX2-1 locus at 14q13 in 6 patients, including inversions to the T-cell receptor alpha/delta locus (TRAD@) at 14q11, an inversion to the IgH@ locus at 14q32 and a translocation to the TRB@ locus at 7q34. A translocation of NKX2-2 to the TRAD@ locus was identified in the case that ectopically expressed NKX2-2. All these rearrangements were novel, and have not been observed before in human cancer. NKX2-1 rearrangements could be validated in an independent T-ALL cohort. Our data strongly imply that NKX2-1/NKX2-2 represent novel oncogenes for human T-ALL. Disclosures: No relevant conflicts of interest to declare.

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Immunoglobulin Heavy Chain Locus (IGH@) Translocations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Incidence and Risk Stratification

Blood ◽

10.1182/blood.v120.21.1274.1274 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1274-1274 ◽

Cited By ~ 1

Author(s):

Lisa J Russell ◽

Lisa Jones ◽

Amy Erhorn ◽

Amir Ensahei ◽

Dino Masic ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

White Cell Count ◽

Scanning System ◽

Childhood All ◽

True Incidence ◽

Partner Gene ◽

Automated Scanning

Abstract Abstract 1274 Translocations involving the IGH@ locus have recently been characterised as a novel cytogenetic subgroup in BCP-ALL, involving unique partner genes including: CEBP family of transcription factors; cytokine receptors, EPOR and CRLF2; and the inhibitory transcription factor, ID4. As for the mature B-cell malignancies, their expression is always deregulated by juxtaposition of transcriptional enhancers within the locus. We have developed a high throughput FISH screening approach to ascertain the true incidence of IGH@ translocations in BCP-ALL. We screened approximately 80% (2603/3194) of patients entered to the childhood clinical trial (UKALL2003) using an IGH@ break-apart rearrangement probe with automated scanning and capture (CytoVision scanning system, Leica Microsystems). We identified IGH@ translocations in 4% (104/2603) of patients tested. This included an incidence of 4% (96/2286) in BCP-ALL and the novel observation of 2% (8/317) in T-ALL patients. The median age at diagnosis of BCP- and T-ALL IGH@ translocation patients was significantly higher at 12 and 14 years, respectively, compared to the median age of 5 years for the whole trial. The median white cell count (WCC) was low in IGH@ positive BCP-ALL (median 9.05×109/L) and higher in T-ALL (median 72.8×109/L). This study confirmed CRLF2 to be the most frequent IGH@ partner gene, observed in 20% (22/104) of BCP-ALL patients. ID4 and the CEBP family were found in 8% (8/104) and 9% (10/104), respectively. CEBPD was most common (n=6), while CEBPA (n=2), CEBPE (n=1) and CEBPG (n=1) were rare. Single patients were identified with involvement of BCL2, IGF2BP1, IGK@ and the TCRA/D locus. Novel involvement of TAL1 was identified in one T-ALL patient. In 57% of children (59/104: BCP-ALL n=52, T-ALL n=7) the IGH@ translocation partner gene remains unknown. Patients with a known partner gene did not differ from those with an unknown partner gene with respect to gender, age, WCC and National Cancer Institute (NCI) risk. However, patients with a known partner gene were strongly associated with an intermediate cytogenetic risk group while patients with an unknown partner gene were mixed (good risk, 7% v 19%, intermediate risk, 93% v 69% and poor risk, 0% v 12% p=0.01). There was no difference seen for gender, age, WCC, cytogenetic risk and NCI risk when each partner gene was investigated independently, apart from those with CRLF2 involvement who had a higher WCC (<50×109/L 59% v 81% and >50×109/l 41% v 12%, p=0.005) and were solely within the intermediate cytogenetic risk group (100% v 74%, p=0.03). We investigated copy number aberrations of genes commonly altered in BCP-ALL by Multiplex Ligation-dependent Probe Amplification (MLPA) using the Salsa P335-A1 IKZF1 kit (MRC Holland) in 60 IGH@ translocation patients. The genes investigated were IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, as well as the rearrangement, P2RY8-CRLF2. Deletions of none (31%), one (18%), two (17%), three (22%) or four (8%) genes were found. Deletions of CDKN2A, CDKN2B and IKZF1 were the most frequent, in 18%, 21% and 20%, respectively. Whilst the incidence of CDKN2A/B deletions was similar to that seen in childhood BCP-ALL, IKZF1 deletions were more prevalent (20% v 14%) and PAX5 deletions occurred at a lower incidence in IGH@ rearranged patients (13% v 19%). In the two T-ALL patients with DNA available, both showed deletions of CDKN2A with one patient also showing deletion of CDKN2B. In conclusion, IGH@ translocations are present in 4% of childhood ALL with involvement in both B- and T-cell disease. Patients belong to the intermediate cytogenetic risk group with an increased incidence of IKZF1 deletions. This differs from our recent report on adult BCP-ALL, where patients with an IGH@ translocation were associated with a worse outcome, although the increased incidence of IKZF1 deletions remained consistent across all age ranges. In over 50% of IGH@ positive children, the translocation partner remains to be elucidated, indicating the presence of as yet unidentified cryptic rearrangements. Characterisation of these partners may identify additional oncogenes involved in leukemogenesis or provide potential novel therapeutic targets, as recently demonstrated for CRLF2 in BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

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Value of EVI1 Gene Expression Level in Adult Acute Lymphoblastic Leukemia (ALL): A Study from the Group for Research on Adult ALL (GRAALL)

Blood ◽

10.1182/blood.v124.21.1081.1081 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1081-1081

Author(s):

Claire Abbal ◽

Raouf Ben Abdelali ◽

Marina Lafage ◽

Françoise Huguet ◽

Thibaut Leguay ◽

...

Keyword(s):

Gene Expression ◽

Patient Outcome ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Expression Level ◽

Prognostic Impact ◽

Complex Karyotype ◽

Cell Precursor ◽

Genetic Profiles ◽

Lower Expression

Abstract Background: EVI1 gene overexpression is found in approximately 10% of acute myeloid leukemia (AML) patients, with a higher frequency seen in AML carrying chromosome 3q26 abnormality or MLL gene rearrangement, and associated with a dismal prognosis. Deregulation of EVI1 expression has also been reported in ALL, but its prognostic impact is unclear. Here, we retrospectively analyzed EVI1 expression in a large cohort of adult ALL patients, its correlation with ALL subsets, and its impact on patient outcome. Patients and Methods: EVI1 gene expression was measured by RQ-PCR detecting all splicing variants, with PBGD as control gene. We used dilutions of EVI1+ SKOV3 (kindly provided by Peter Valk, Rotterdam, The Netherlands) and EVI1-neg HL-60 cell line cDNA to build EVI1 and PBGD standard curves. Results were expressed as EVI1/PBGD ratio x 100. Blast samples from 354 patients treated in the GRAALL-2003/2005 and GRAAPH-2005 trials (191 B-cell precursor [BCP]-ALL, including 138 Ph-neg and 53 Ph+; 163 T-ALL) and 62 controls were analyzed. Immunophenotype results were centrally reviewed. In controls, median EVI1 expression level was 0.33% (Q1-Q3, 0.20-0.69). For prognostic analysis, we used the 1st and 99th percentiles of the controls (0.05% and 1.65%) to define patients with low and high EVI1 expression, respectively. Clinical endpoints were cumulative incidence of failure (CIF, failure meaning primary refractoriness or relapse) and event-free survival (EFS). Results: As illustrated in Figure 1, we observed that, as in one AML series, EVI1 expression may be up or down regulated in adult ALL. When compared to controls, the proportions of low and high EVI1 patients were 21 and 23% in Ph-neg BCP-ALL, 9 and 42% in Ph+ ALL, and 21 and 18% in T-ALL, respectively. In BCP-ALL patients, median EVI1 expression was similar to controls (0.53%; Q1-Q3, 0.11-1.88; p=0.15), but higher in the Ph+ as compared to the Ph-neg subgroup (0.93% versus 0.36%; p<0.001). In T-ALL patients, median expression tended to be lower than in controls (0.22%; Q1-Q3, 0.06-0.85; p=0.058). In these three ALL subgroups, EVI1 expression did not correlate with age or WBC. Among Ph-neg BCP-ALL patients, a lower expression was found in MLL-AF4+ t(4;11) cases (median, 0.04%; p<0.001), while no differences were observed for cases with t(1;19), an14q32, low hypodiploidy/near triploidy, complex karyotype, or IKZF1 deletion. Among T-ALL patients, a lower expression was found in cases with complex karyotype (median, 0.05%; p=0.03), while no differences were observed for cases with TLX1 overexpression, NOTCH1/FBXW7 mutation, N/K-RAS mutation or PTEN alteration. Only one patient had 3q26 abnormality (T-ALL with high EVI1 expression). Low or high EVI1 expression had no prognostic impact in Ph-neg as well as Ph+ BCP-ALL patients. In T-ALL, the proportion of patients with low EVI1 expression was more frequent in early and mature than in cortical T-ALL (33% and 33% vs 11%; p=0.002 and 0.028, respectively) and associated with a higher CIF (HR, 2.03; p=0.017) and shorter EFS (HR, 1.59; p=0.072). Low EVI1 expression was also significantly associated with an early T-cell precursor (ETP) phenotype (37.5% vs 18%; p=0.028). After adjustment on cortical and ETP phenotypes, as well as on 4-gene (NOTCH1/FBXW7/RAS/PTEN) genetic profiles, low EVI1 expression and high-risk genetic profiles remained independently associated with higher CIF (p=0.015 and <0.001, respectively) and shorter EFS (p=0.045 and 0.002, respectively). Conclusion: Overall, these results confirm that EVI1 gene expression is frequently deregulated in adult ALL. In BCP-ALL, down-regulation is observed in t(4;11) and up-regulation in BCR-ABL cases. Further studies are needed to confirm that, in T-ALL, a lower expression is associated with the early, mature and ETP phenotypes and independently predictive of a worse patient outcome. Figure 1 Figure 1. EVI1 gene expression in ALL and control samples. Disclosures No relevant conflicts of interest to declare.

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