B-precursor Acute Lymphoblastic Leukemia Patients Research Articles

CD22 Exon 12 Deletion as an Independent Predictor of Poor Treatment Outcomes in B-ALL.

We previously reported a splicing defect (CD22ΔE12) associated with the deletion of exon 12 of the inhibitory co-receptor CD22 (Siglec-2) in leukemia cells from patients with CD19+ B-precursor acute lymphoblastic leukemia (B-ALL). CD22ΔE12 causes a truncating frameshift mutation and yields a dysfunctional CD22 protein that lacks most of the cytoplasmic domain required for its inhibitory function, and it is associated with aggressive in vivo growth of human B-ALL cells in mouse xenograft models. Although CD22ΔE12 with selective reduction of CD22 exon 12 (CD22E12) levels was detected in a high percentage of newly diagnosed as well as relapsed B-ALL patients, its clinical significance remains unknown. We hypothesized that B-ALL patients with very low levels of wildtype CD22 would exhibit a more aggressive disease with a worse prognosis because the missing inhibitory function of the truncated CD22 molecules could not be adequately compensated by competing wildtype CD22. Here, we demonstrate that newly diagnosed B-ALL patients with very low levels of residual wildtype CD22 ("CD22E12low"), as measured by RNAseq-based CD22E12 mRNA levels, have significantly worse leukemia-free survival (LFS) as well as overall survival (OS) than other B-ALL patients. CD22E12low status was identified as a poor prognostic indicator in both univariate and multivariate Cox proportional hazards models. CD22E12low status at presentation shows clinical potential as a poor prognostic biomarker that may guide the early allocation of risk-adjusted, patient-tailored treatment regimens and refine risk classification in high-risk B-ALL.

CD9 Blockade Effectively Suppresses Disease Progression of Pediatric B-Precursor Acute Lymphoblastic Leukemia and Enhances Chemosensitivity

Despite major advances in risk-directed multi-agent chemotherapy, specific subtypes of high-risk and relapsed/refractory pediatric B-precursor acute lymphoblastic leukemia (B-ALL) remain associated with poor clinical outcomes, highlighting the need for development of novel and effective therapeutic strategies. CD9, a tetraspanin family protein, has been associated with metastasis and progression of various types of malignancies, but its prognostic relevance and therapeutic potential in pediatric B-ALL remain largely unknown. In a cohort of pediatric B-ALL cases, we identified that CD9+ patients had significantly lower 5-year overall (OS) and relapse-free survival (RFS) rates when compared with CD9- cases (OS: 81.5% vs 100%, P= 0.022; RFS: 70.7% vs 92.3%, P= 0.029). Subgroup analysis revealed remarkably poorer outcomes in CD9+ patients of the high-risk group (OS: 61.5% vs 100%, P= 0.042; RFS: 46.2% vs 88.9%, P= 0.045). A similar trend was also observed in patients of the intermediate-risk group but not in the standard-risk group. In univariate analysis, CD9 positivity, age <1 year, white cell count ≥100 × 10^9/L and poor prednisone response were associated with lower RFS rate. In multivariate analysis, CD9 positivity (HR = 6.0; P= 0.019) remained as an independent prognostic factor for lower RFS rate. The efficacy of targeting CD9 for treatment of pediatric B-ALL was evaluated in the NOD/SCID mouse xenograft model. Our results showed that administration of CD9 neutralizing antibody as a single agent substantially reduced multi-organ leukemic burden by >90% (P ≤ 0.037) and significantly prolonged survival (P ≤ 0.007) of animals xenografted with the intermediate-risk 697 (TCF3-PBX1+) and high-risk RS4;11 (MLL-AF4+), but not the standard-risk Reh (ETV6-RUNX1+) cell lines. Similarly, CD9 antibody treatment significantly decreased B-ALL progression in patient-derived xenografts with a wide spectrum of genetic and disease features, including high-risk infantile MLL-AF4+ cases as well as those with relapsed/refractory diseases (P ≤ 0.009). Importantly, CD9 antibody in combination with conventional chemotherapy consisting of vincristine, dexamethasone and L-asparaginase further prolonged animal survival, when compared to animals treated with CD9 antibody or chemotherapy alone (P ≤ 0.006). In vitro, CD9 antibody significantly reduced proliferation of B-ALL cells (P= 0.004) and markedly enhanced vincristine-, dexamethasone- and L-asparaginase-induced apoptosis (P ≤ 0.044). Mechanistic studies revealed that CD9 was involved in B-ALL migration and stromal adhesion by modulating the affinity of integrin very late antigen-4. Our results collectively suggest that expression of CD9 in pediatric B-ALL patients was associated with adverse survival outcomes and that CD9 blockade, in adjunct to chemotherapy, could be a promising strategy for treatment of high-risk and relapsed/refractory pediatric B-ALL, possibly mediated by disruption of leukemia-stroma interaction in the bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Leukemia-induced dysfunctional TIM-3+CD4+ bone marrow T cells increase risk of relapse in pediatric B-precursor ALL patients.

Interaction of malignancies with tissue-specific immune cells has gained interest for prognosis and intervention of emerging immunotherapies. We analyzed bone marrow T cells (bmT) as tumor-infiltrating lymphocytes in pediatric precursor-B cell acute lymphoblastic leukemia (ALL). Based on data from 100 patients, we show that ALL is associated with late-stage CD4+ phenotype and loss of early CD8+ T cells. The inhibitory exhaustion marker TIM-3 on CD4+ bmT increased relapse risk (RFS = 94.6/70.3%) confirmed by multivariate analysis. The hazard ratio of TIM-3 expression nearly reached the hazard ratio of MRD (7.1 vs. 8.0) indicating that patients with a high frequency of TIM-3+CD4+ bone marrow T cells at initial diagnosis have a 7.1-fold increased risk to develop ALL relapse. Comparison of wild type primary T cells to CRISPR/Cas9-mediated TIM-3 knockout and TIM-3 overexpression confirmed the negative effect of TIM-3 on T cell responses against ALL. TIM-3+CD4+ bmT are increased in ALL overexpressing CD200, that leads to dysfunctional antileukemic T cell responses. In conclusion, TIM-3-mediated interaction between bmT and leukemia cells is shown as a strong risk factor for relapse in pediatric B-lineage ALL. CD200/TIM-3-signaling, rather than PD-1/PD-L1, is uncovered as a mechanism of T cell dysfunction in ALL with major implication for future immunotherapies.

Identification and targeting of CD22ΔE12 as a molecular RNAi target to overcome drug resistance in high-risk B-lineage leukemias and lymphomas.

AimCD22ΔE12 as an oncogenic driver lesion in aggressive and drug-resistant B-precursor acute lymphoblastic leukemia (BPL) cells. The purpose of the present study was to identify the CD22ΔE12-specific signature transcriptome in human BPL cells and evaluate the clinical potential of a nanoscale formulation of CD22ΔE12-siRNA as an RNAi therapeutic against drug-resistant BPL. CD22ΔE12-siRNA nanoparticles significantly improved the event-free survival (EFS) outcome of NOD/SCID (NS) mice challenged with human BPL xenograft cells.MethodsGene expression and translational bioinformatics methods were applied to examine the expression of the CD22ΔE12-specific signature transcriptome in human BPL cells in subsets of BPL patients. Survival analysis for mice challenged with BPL cells and treated with CD22ΔE12 siRNA was performed using standard methods.ResultsLeukemia cells from CD22ΔE12-Tg mice exhibit gene and protein expression profiles consistent with constitutive activation of multiple signaling networks, mimicking the profiles of relapsed BPL patients as well as newly diagnosed high-risk patients with BCR-ABL+/Philadelphia chromosome (Ph)+ BPL as well as Ph-like BPL. A nanoscale formulation of CD22ΔE12-siRNA abrogated the in vivo clonogenicity of the leukemia-initiating leukemic cell fraction in xenograft specimens derived from patients with relapsed BPL and significantly improved the EFS outcome of NS mice challenged with drug-resistant human BPL xenograft cells.ConclusionThe CD22-RNAi technology is applicable to all BPL patients both high risk and standard risk. That is because CD22ΔE12 is a characteristic feature of drug-resistant leukemic clones that escape chemotherapy and cause relapse in both high risk and low risk subgroups of patients. The technology therefore has the potential (1) for prevention of relapses by selectively killing the clones that are most likely to escape chemotherapy and cause relapse as well (2) for treatment of relapses in BPL. This research project may also lead to innovative salvage regimens against other forms of CD22ΔE12-positive relapsed B-lineage leukemias and lymphomas.

Nanoparticle Formulation to Improve the Efficacy of Radiation Therapy Against Radiation-resistant Leukemia

Patients with B-precursor acute lymphoblastic leukemia (BPL) are often treated with TBI-based conditioning, followed by hematopoietic stem-cell transplantation (HSCT). However, the leukemic relapse in high-risk BPL patients remained the major cause of patient mortality (Gaynon et al., 2006, Balduzzi et al., 2014). The incidence of post-HSTC relapse correlated with the level of residual leukemia burden prior to total body irradiation (TBI), indicating the radiation-resistant population of BPL cells may have contributed to the relapse. STAT-3 signaling pathway, along with PI3-K and NFkB pathways, regulates the cell survival after exposure to radiation-induced oxidative stress (Uckun et al., 2010), therefore contributes to the radiation resistance of BPL cells. Spleen tyrosine kinase (SYK), a key regulator of STAT-3, was chosen as the main target of reducing the radiation resistance (Mocsai et al., 2010). The authors identified C61, a small-molecule chemical compound inhibitor for SYK phosphorylation, which sensitized the resistant BPL cells toward radiation.

Liposomal Nanoparticles of a Spleen Tyrosine Kinase P-Site Inhibitor Amplify the Potency of Low Dose Total Body Irradiation Against Aggressive B-Precursor Leukemia and Yield Superior Survival Outcomes in Mice

This study was designed to improve the efficacy of radiation therapy against radiation-resistant leukemia. We report that the potency of low dose radiation therapy against B-precursor acute lymphoblastic leukemia (BPL) can be markedly enhanced by combining radiation with a liposomal nanoparticle (LNP) formulation of the SYK-P-site inhibitor C61 (“C61-LNP”). C61-LNP plus low dose total body irradiation (TBI) was substantially more effective than TBI alone or C61-LNP alone in improving the event-free survival outcome NOD/SCID mice challenged with an otherwise invariably fatal dose of human ALL xenograft cells derived from relapsed BPL patients. C61-LNP plus low dose TBI also yielded progression-free survival, tumor-free survival and overall survival outcomes in CD22ΔE12×BCR–ABL double transgenic mice with advanced stage, radiation-resistant BPL with lymphomatous features that were significantly superior to those of mice treated with TBI alone or C61-LNP alone.

Low Dose Total Body Irradiation Combined With Recombinant CD19-Ligand × Soluble TRAIL Fusion Protein is Highly Effective Against Radiation-resistant B-precursor Acute Lymphoblastic Leukemia in Mice

In high-risk remission B-precursor acute lymphoblastic leukemia (BPL) patients, relapse rates have remained high post-hematopoietic stem cell transplantation (HSCT) even after the use of very intensive total body irradiation (TBI)-based conditioning regimens, especially in patients with a high “minimal residual disease” (MRD) burden. New agents capable of killing radiation-resistant BPL cells and selectively augmenting their radiation sensitivity are therefore urgently needed. We report preclinical proof-of-principle that the potency of radiation therapy against BPL can be augmented by combining radiation with recombinant human CD19-Ligand×soluble TRAIL (“CD19L–sTRAIL”) fusion protein. CD19L–sTRAIL consistently killed radiation-resistant primary leukemia cells from BPL patients as well as BPL xenograft cells and their leukemia-initiating in vivo clonogenic fraction. Low dose total body irradiation (TBI) combined with CD19L–sTRAIL was highly effective against (1) xenografted CD19+ radiochemotherapy-resistant human BPL in NOD/SCID (NS) mice challenged with an otherwise invariably fatal dose of xenograft cells derived from relapsed BPL patients as well as (2) radiation-resistant advanced stage CD19+ murine BPL with lymphomatous features in CD22ΔE12xBCR-ABL double transgenic mice. We hypothesize that the incorporation of CD19L–sTRAIL into the pre-transplant TBI regimens of patients with very high-risk BPL will improve their survival outcome after HSCT.

CD22ΔE12 as a molecular target for corrective repair using RNA trans-splicing: anti-leukemic activity of a rationally designed RNA trans-splicing molecule.

Our recent studies have demonstrated that the CD22 exon 12 deletion (CD22ΔE12) is a characteristic genetic defect of therapy-refractory clones in pediatric B-precursor acute lymphoblastic leukemia (BPL) and implicated the CD22ΔE12 genetic defect in the aggressive biology of relapsed or therapy-refractory pediatric BPL. The purpose of the present study was to further evaluate the biologic significance of the CD22ΔE12 molecular lesion and determine if it could serve as a molecular target for corrective repair using RNA trans-splicing therapy. We show that both pediatric and adult B-lineage lymphoid malignancies are characterized by a very high incidence of the CD22ΔE12 genetic defect. We provide experimental evidence that the correction of the CD22ΔE12 genetic defect in human CD22ΔE12(+) BPL cells using a rationally designed CD22 RNA trans-splicing molecule (RTM) caused a pronounced reduction of their clonogenicity. The RTM-mediated correction replaced the downstream mutation-rich segment of Intron 12 and remaining segments of the mutant CD22 pre-mRNA with wildtype CD22 exons 10-14, thereby preventing the generation of the cis-spliced aberrant CD22ΔE12 product. The anti-leukemic activity of this RTM against BPL xenograft clones derived from CD22ΔE12(+) leukemia patients provides the preclinical proof-of-concept that correcting the CD22ΔE12 defect with rationally designed CD22 RTMs may provide the foundation for therapeutic innovations that are needed for successful treatment of high-risk and relapsed BPL patients.

BAALC Gene Expression in Adult B-precursor Acute Lymphoblastic Leukemia: Impact on Prognosis

Background: Adult B-precursor acute lymphoblastic leukemia (ALL) remains a major therapeutic challenge. Various molecular markers have extensively been investigated to improve its risk profile characterization, disease progression and resistance to treatment. Aim: To analyze the brain and acute leukemia, cytoplasmic (BAALC) gene expression and to assess its prognostic impact in B-precursor ALL. Subjects and Methods: BAALC mRNA expression was analyzed using real time PCR in 200 primary adult B-precursor ALL patients. Patients were grouped into 2 groups according to median BAALC expression. Results: High BAALC expression was associated with older age (P=0.037), higher white blood cell count (P =0.019), LDH concentration (p=0.007), higher incidence of positive CD34 (P =0.011) and positive BCR-ABL (P=0.011). High BAALC expression was associated with primary therapy resistance in the overall cohort (P = 0.001), in BCR-ABL− and BCR-ABL+ subgroups (P = 0.039, 0.003 respectively). Multivariate analysis showed that BAALC expression was an independent risk factor for chemotherapy resistance in the overall cohort ( =0.003, OR=3.133, 95% CI=1.482-6.623) and in both BCR-ABL- (p=0.049, OR=2.359, 95%CI=1.004-5.538), and BCRABL+ subgroups (p=0.014, OR=2.672, 95%CI=1.824-3.326). Higher BAALC expression was associated with a shorter overall survival (OS) (p= 0.010) and disease free survival (DFS) (p<0.001) in the overall cohort, and DFS in BCR-ABL- subgroup (p<0.001). Multivariate analysis showed that higher BAALC expression independently predicted OS and DFS in the overall cohort (P = 0.039, 0.002 respectively) and DFS in BCR-ABL- subgroup (p=0.001). Conclusion: High BAALC expression is associated with refractory disease in adult B-precursor ALL, and predicts shorter OS and DFS. Determination of BAALC expression may contribute to risk stratification of adult B-precursor ALL, and may improve the currently disappointing cure rate.

SYK as a New Therapeutic Target in B-Cell Precursor Acute Lymphoblastic Leukemia

The identification of SYK as a master regulator of apoptosis controlling the activation of the PI3-K/AKT, NFκB, and STAT3 pathways—three major anti-apoptotic signaling pathways in B-lineage leukemia/lymphoma cells—prompts the hypothesis that rationally designed inhibitors targeting SYK may overcome the resistance of malignant B-lineage lymphoid cells to apoptosis and thereby provide the foundation for more effective multi-modality treatment regimens for poor prognosis B-precursor acute lymphoblastic leukemia (BPL). In recent preclinical proof-of-concept studies, a liposomal nanoparticle (LNP) formulation of a SYK substrate-binding site inhibitor, known as C61, has been developed as a nanomedicine candidate against poor prognosis and relapsed BPL. This nanoscale formulation of C61 exhibited a uniquely favorable pharmacokinetics and safety profile in mice, induced apoptosis in radiation-resistant primary leukemic cells taken directly from BPL patients as well as in vivo clonogenic BPL xenograft cells, destroyed the leukemic stem cell fraction of BPL blasts, and exhibited potent in vivo anti-leukemic activity in xenograft models of aggressive BPL. Further development of C61-LNP may provide the foundation for new and effective treatment strategies against therapy-refractory BPL.

Constitutive Function of the Ikaros Transcription Factor in Primary Leukemia Cells from Pediatric Newly Diagnosed High-Risk and Relapsed B-precursor ALL Patients

We examined the constitutive function of the Ikaros (IK) transcription factor in blast cells from pediatric B-precursor acute lymphoblastic leukemia (BPL) patients using multiple assay platforms and bioinformatics tools. We found no evidence of diminished IK expression or function for primary cells from high-risk BPL patients including a Philadelphia chromosome (Ph)+ subset. Relapse clones as well as very aggressive in vivo clonogenic leukemic B-cell precursors isolated from spleens of xenografted NOD/SCID mice that developed overt leukemia after inoculation with primary leukemic cells of patients with BPL invariably and abundantly expressed intact IK protein. These results demonstrate that a lost or diminished IK function is not a characteristic feature of leukemic cells in Ph+ or Ph- high-risk BPL.

Outcome Of Standard Risk B-Precursor Acute Lymphoblastic Leukemia Treated Following The Children's Cancer Group-1991 (CCG-1991) Study: Experience From a Single Center In Saudi Arabia

BackgroundRemarkable improvements in the treatment of childhood acute lymphoblastic leukemia (ALL) have been achieved primarily because of the work of cooperative groups such as the Children's Oncology Group. The objective of the present study was to compare survival outcomes among children with standard-risk (SR) B-precursor acute lymphoblastic leukemia (ALL) treated at Princess Norah Oncology Center, Jeddah, Saudi Arabia, with survival outcomes of the Children's Cancer Group-1991 (CCG-1991). MethodsA retrospective study of newly diagnosed SR B-precursor ALL children during the period January 2000 to December 2007 and treated on CCG-1991 protocol following the oral methotrexate/double delayed intensification arm (regimen OD). Medical records of patients were reviewed. Data collected include: age, sex, WBC, date of: diagnosis, remission, induction, relapse, death, last follow-up; cytogenetics, phenotype at diagnosis, CNS status, toxicity, and outcome. Statistical AnalysisOverall survival (OS) was calculated from the time of diagnosis to the time of death from any cause or until the last follow-up for surviving patients. Event-free survival (EFS) was measured from the date of diagnosis until the occurrence of first event (failure to achieve remission, relapse, death from any cause, or until the last follow-up). Kaplan-Meier method was used to estimate survival rates. ResultsA total of 123 SR B-precursor ALL patients were treated following CCG-1991 (regimen OD). The median duration of follow up was 7 (range: 0.03 – 11.7) years. Median age was 3.9 (range: 1.5 – 9.6) years and the median WBC count was 5.6 (range: 0.9 – 16.7) x 109/L. There were 67 male patients accounting for 54.5% compared to 56 (45.5%) females. The 5-year EFS and OS for SR B-precursor ALL patients were 79.4%±3.7% and 86%±3.2%, respectively. In total, 19 (15.4%) patients had relapse as first event. Death as first event occurred in 6 (4.9%) patients. Of this, 3 (2.4%) occurred during induction, and 3 (2.4%) occurred while in remission.Comparison of treatment outcomes revealed that our patients had inferior 5-year EFS and OS compared to those reported in the CCG-1991 study (EFS and OS rates of 90.7%±0.9% and 96.2%±0.6%; respectively). The difference is significant and may have been due to treatment regimen, as all our patients were treated with regimen OD. However, when comparison was limited to patients treated with regimen OD in the CCG-1991 study (EFS and OS rates of 88.7%±1.4% and 96%±0.9%; respectively), the difference in survival outcome remained significant. ConclusionWe postulate that clinical and biological factors may have contributed for the outcome to vary between the present and the CCG-1991 study. The high incidence of death as first event in our patient population may be related to these factors and deserves further exploration. The fact that our patients were not on clinical trial and were treated following a standard protocol may have contributed to this variation. This underscores the need to further refine risk stratification. Disclosures:No relevant conflicts of interest to declare.

Molecular Basis Of Minimal Residual Disease In Subtypes Of Pediatric B Cell Acute Lymphoblastic Leukemia

IntroductionDespite advances in diagnosis and treatment, B-precursor acute lymphoblastic leukemia (B-ALL) remains the most common childhood cancer and one of the leading causes of cancer-related death in children and adolescents. Although B-ALL is highly curable, approximately 10 - 20% of children diagnosed with B-ALL still do not respond to the current treatment protocols. Minimal residual disease (MRD) at the end of induction of remission is strongly associated with prognosis. Therefore there is an urgent need to understand the molecular mechanisms underpinning MRD and to identify biomarkers for the development of novel and more effective therapeutic strategies. This project was undertaken to determine whether molecular perturbation in patients with positive MRD at day 46 differs from those with negative MRD in different subtypes of B-ALL and to identify biological pathways dysregulated. We hypothesized that gene expression profiles differ significantly between patients with positive MRD at day 46 and patients with negative MRD. MethodsWe analyzed publicly available gene expression data derived from samples obtained from 189 patients with B-ALL (47 with positive MRD at day 46 and 142 with negative MRD). The data was downloaded from the NCBI's Gene Expression Omnibus (GEO) database under accession number GSE33315. Patients were classified into seven subtypes of B-ALL which are hyperdiploid, ETV6-RUNX1, MLL rearrangement, hypodiploid, BCR-ABL1, TCF3-PBX1 and others (no detectable recurring genetic abnormalities). Samples from patients with BCR-ABL1 were excluded due to a different prognosis and treatment approach. Patients with TCF3-PBX1 were excluded due to the small sample size; leaving 165 patients in the analysis (35 with positive MRD at day 46 and 130 with negative MRD). We analyzed gene expression data using both supervised and unsupervised analysis. Supervised analysis was performed between patients with positive MRD and negative MRD for each subtype of B-ALL. Unsupervised analysis using hierarchical clustering was performed on significantly differently expressed genes (P < 0.005) to identify functionally related genes with similar patterns of expression profiles. Pathway analysis was performed using the Ingenuity Pathways Analysis (IPA) system to identify biological pathways that are dysregulated in response to positive MRD in different subtypes of B-ALL. ResultComparison of gene expression profiles between positive MRD and negative MRD revealed significantly differentially expressed genes between the two groups. The numbers of significantly (P < 0.005) differentially expressed genes for hyperdiploid, ETV6-RUNX1, MLL rearrangement, hypodiploid and others were 93, 82, 87, 140 and 289 genes; respectively. The identified genes included BCL2, BECN1, CBFB, IKZF1, PAX5, SH2B3 and TOX which are known to be associated with B-ALL. Unsupervised analysis using hierarchical clustering and GO analysis revealed similarity in patterns of gene expression within subtypes of B-ALL and functional relationships among the identified genes. Among the identified genes included genes involved in cell death and survival, cellular development and DNA replication, recombination, and repair. Network and Pathway analysis revealed multi-gene regulatory networks and key biological pathways including granzyme B signaling, TCA cycle II and B cell receptor signaling. Pathway analysis also revealed upstream regulators including RB1, CDKN2A and TP53 which have been reported to be involved in the hypodiploid subtype, a subtype characterized with poorer prognosis. ConclusionAlthough the sample size is small, our analysis demonstrates that molecular perturbation significantly differs between pediatric B-ALL patients with positive MRD and those with negative MRD, and that these differences are subtype-specific. The results further demonstrate that biological pathways are dysregulated in response to MRD status and that use of gene expression analysis has the promise to stratify patients on the basis of MRD status and to identify potential biomarkers. Disclosures:No relevant conflicts of interest to declare.

Absence of Genomic &lt;i&gt;Ikaros/IKZF1&lt;/i&gt; Deletions in Pediatric B-Precursor Acute Lymphoblastic Leukemia

Here we report the results of gene expression analyses using multiple probesets aimed at determining the incidence of Ikaros/IKZF1 deletions in pediatric B-precursor acute lymphoblastic leukemia (BPL). Primary leukemia cells from 122 Philadelphia chromosome (Ph)+ BPL patients and 237 Ph- BPL patients as well as normal hematopoietic cells from 74 normal non-leukemic bone marrow specimens were organized according to expression levels of IKZF1 transcripts utilizing two-way hierarchical clustering technique to identify specimens with low IKZF1 expression for the 10 probesets interrogating Exons 1 through 4 and Exon 8. Our analysis demonstrated no changes in expression that would be expected from homozygous or heterozygous deletions of IKZF1 in primary leukemic cells. Similar results were obtained in gene expression analysis of primary leukemic cells from 20 Ph+ positive and 155 Ph- BPL patients in a validation dataset. Taken together, our gene expression analyses in 534 pediatric BPL cases, including 142 cases with Ph+ BPL, contradict previous reports that were based on SNP array data and suggested that Ph+ pediatric BPL is characterized by a high frequency of homozygous or heterozygous IKZF1 deletions. Further, exon-specific genomic PCR analysis of primary leukemia cells from 21 high-risk pediatric BPL patients and 11 standard-risk pediatric BPL patients, and 8 patients with infant BPL did not show any evidence for homozygous IKZF1 locus deletions. Nor was there any evidence for homozygous or heterozygous intragenic IKZF1 deletions.

Analysis of Patterns of Gene Expression Variation within and between Ethnic Populations in Pediatric B-ALL

B-Precursor acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer. Although 80% of B-ALL patients are able to be cured, significant challenges persist. Significant disparities in clinical outcomes and mortality rates exist between racial/ethnic populations. The objective of this study was to determine whether gene expression levels significantly differ between ethnic populations. We compared gene expression levels between four ethnic populations (Whites, Blacks, Hispanics, and Asians) in the United States. Additionally, we performed network and pathway analysis to identify gene networks and pathways. Gene expression data involved 198 samples distributed as follows: 126 Whites, 51 Hispanics, 13 Blacks, and 8 Asians. We identified 300 highly significantly (P < 0.001) differentially expressed genes between the four ethnic populations. Among the identified genes included the genes PHF6, BRD3, CRLF2, and RNF135 which have been implicated in pediatric B-ALL. We identified key pathways implicated in B-ALL including the PDGF, PI3/AKT, ERBB2-ERBB3, and IL-15 signaling pathways.

Triple Intrathecal Therapy Alone Was Delayed until the Disappearance of Blasts From Peripheral Blood in Children with Acute Lymphoblastic Leukemia: The Experience of a Single Hospital in Taiwan

AbstractAbstract ▪3564▪This icon denotes a clinically relevant abstract BackgroundThe introduction of CNS-directed therapy had successfully improved the outcome of children with acute lymphoblastic leukemia (ALL). Pui et al. (N Engl J Med, 2009) demonstrated that with effective risk-adjusted systemic chemotherapy and intrathecal therapy initiated at diagnosis (Dx), cranial irradiation can be safely omitted from CNS-directed treatment in all children with newly diagnosed ALL. Manabe et al. (J Clin Oncol, 2001) reported a delay of diagnostic lumbar puncture (LP) and triple intrathecal therapy (TIT) after 1 week of prednisolone monotherapy. Starting 1999, in order to eliminate the adverse prognostic impact of traumatic LP with blasts, we conducted a prospective study of delayed TIT after the disappearance of blasts from peripheral blood (PB) on around 10 days of multi-drug induction therapy. MethodsSince June 1999, children with ALL were treated with TIT alone without cranial irradiation, and chemotherapy with TPOG-ALL-93 or TPOG-ALL-2002 (activated in January 2002) protocols. These protocols had been reported previously (Leukemia, 2010). B-precursor ALL patients were stratified into standard risk (SR), high risk (HR) and very high risk (VHR) groups, according to age, WBC count, cytogenetics and molecular analysis at Dx. T-cell ALL was designated as VHR. The first TIT was performed until the disappearance of blasts from PB. If patients with persistent PB blasts on the treatment day 10 (D10), the TIT was planned. Furthermore, SR patient with detectable blasts on D10, the treatment was upgraded to HR group. Cranial irradiation was totally omitted in all patients. For patients with CNS-1 status, SR group received TIT 20 times in TPOG-ALL-93 and 14 times in TPOG-ALL-2002 protocols, respectively. HR patients with CNS-1 status received 23 and 17 times of TIT in TPOG-ALL-93 and 2002 protocols, respectively. In VHR group, TIT was administered 19 times for CNS-1. Additional TIT doses were given if patients with CNS-2 or CNS-3. ResultsFrom June 1999 to August 2010, a total of 158 ALL patients were consecutively diagnosed at our hospital. There were 86 patients in SR group, 41 in HR and 31 in VHR, respectively. Six patients were excluded: 2 refusals to further treatment and 4 protocol violations. There were 86 boys and 66 girls enrolled. The median age at Dx was 4.1 years (range, 0.3–17.7 years) with 7 infants. The median WBC count at Dx was 12,100/μL (range, 800–596,000/μL). Fourteen had counts >100,000/μL. There were 141 patients of B-precursor ALL and 11 of T-cell ALL. At Dx, 17 patients (16 in SR and 1 in VHR) did not have detectable blasts on PB. In the remaining 135 patients, the blasts of 101 patients (74.8%) can be eliminated from PB after 10-day induction therapy, those included 77.9% of SR, 75.6% of HR and 65.4% of VHR, respectively. Six patients of SR group were upgraded to HR protocol since the detection of PB blasts on D10. Bleeding into the CSF at the time of LP was apparent in 4 patients (2.6%), but no leukemic blasts were identified in any patients.As of July 31, 2012, the follow-up duration of 133 surviving patients ranged from 24 to 153 months (median, 88 months). The 7-year event-free survival and overall survival rates were 83.0 ± 3.2% (± SE) and 86.5 ± 3.1%, respectively. No isolated CNS relapse occurred. One infant with MLL rearrangement experienced combined CNS, BM and testicular relapses and one child with t(12;21) had combined CNS and molecular BM relapses. The cumulative risk of any CNS relapse was 1.5 ± 1.0% at 7 years. ConclusionsA delay of TIT after disappearance of PB blasts with multi-drug induction therapy can effectively prevent CNS disease in children with ALL. Cranial irradiation can be completely omitted for CNS prophylaxis in our treatment protocols. Disclosures:No relevant conflicts of interest to declare.

Overexpression of LEF1 predicts unfavorable outcome in adult patients with B-precursor acute lymphoblastic leukemia

AbstractAberrant activation of the Wnt pathway plays a pathogenetic role in various tumors and has been associated with adverse outcome in acute lymphoblastic leukemia (ALL). LEF1, a key mediator of Wnt signaling, has been linked to leukemic transformation, and recurrent mutations of LEF1 have been identified in pediatric T-ALL. Here we evaluated the prognostic significance of LEF1 expression in B-precursor ALL patients. LEF1 expression was determined by quantitative real-time RT-PCR in 282 adult B-precursor ALL patients treated on 06/99 and 07/03 GMALL trials. Patients were grouped into quartiles (Q1-Q4) according to LEF1 expression levels (LEF1 high, Q4; n = 71; LEF1 low, Q1-Q3; n = 211). Patients with high LEF1 expression had a significantly shorter relapse-free survival (RFS) compared with low LEF1 expressers (5-year RFS: LEF1 high, 27%; LEF1 low, 47%; P = .05). Importantly, high LEF1 expression was also associated with inferior RFS in standard-risk patients and was independently predictive for RFS (P = .02) in multivariate analyses for this subgroup. Thus, high LEF1 expression identifies B-precursor ALL patients with inferior RFS, supporting a pathogenetic role of Wnt signaling in ALL. Standard-risk patients with high LEF1 expression might benefit from early treatment modifications and new molecular therapies, including agents targeting the Wnt pathway.

ETV6-RUNX1-positive childhood acute lymphoblastic leukemia: improved outcome with contemporary therapy

ETV6-RUNX1 fusion is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL). To evaluate whether outcomes for this drug-sensitive leukemia are improved by contemporary risk-directed therapy, we studied clinical features, response and adverse events of 168 children with newly diagnosed ETV6-RUNX1-positive ALL on St Jude Total Therapy studies XIIIA (N=36), XIIIB (N=38) and XV (N=94). Results were compared with 494 ETV6-RUNX1-negative B-precursor ALL patients. ETV6-RUNX1 was associated with age 1-9 years, pre-treatment classification as low risk and lower levels of minimal residual disease (MRD) on day 19 of therapy (P<0.001). Event-free survival (EFS) or overall survival (OS) did not differ between patients with or without ETV6-RUNX1 in Total XIIIA or XIIIB. By contrast, in Total XV, patients with ETV6-RUNX1 had significantly better EFS (P=0.04; 5-year estimate, 96.8±2.4% versus 88.3±2.5%) and OS (P=0.04; 98.9±1.4% versus 93.7±1.8%) than those without ETV6-RUNX1. Within the ETV6-RUNX1 group, the only significant prognostic factor associated with higher OS was the treatment protocol Total XV (versus XIIIA or XIIIB) (P=0.01). Thus, the MRD-guided treatment schema including intensive asparaginase and high-dose methotrexate in the Total XV study produced significantly better outcomes than previous regimens and demonstrated that nearly all children with ETV6-RUNX1 ALL can be cured.

Down syndrome childhood acute lymphoblastic leukemia has a unique spectrum of sentinel cytogenetic lesions that influences treatment outcome: a report from the Children's Oncology Group

AbstractChildren with Down syndrome (DS) have an increased risk of acute lymphoblastic leukemia (ALL) and an inferior outcome. We reviewed data from 2811 children with ALL enrolled in Children's Oncology Group P9900, which included prospective testing for the major cytogenetic lesions in childhood ALL: ETV6-RUNX1, TCF3-PBX1, BCR-ABL1, and MLL translocations and trisomies of chromosomes 4 and 10. Eighty (3%) B-precursor ALL patients had DS. Age, sex, white blood cell count, and risk group were similar between DS-ALL and non–DS-ALL but significantly more patients with DS-ALL were white (91.2% vs 76.4%, P = .001). Children with DS-ALL had lower rates of the favorable cytogenetic lesions ETV6-RUNX1 (2.5% vs 24%, P < .001) and trisomies 4 and 10 (7.7% vs 24%, P < .001). Five-year event-free (EFS) and overall survival (OS) were inferior in children with DS-ALL: 69.9% ± 8.6% versus 78.1% ± 1.2% (P = .078), and 85.8% ± 6.5% versus 90.0% ± 0.9% (P = .033). However, when children with MLL translocations, BCR-ABL1, ETV6-RUNX1, and trisomies 4 and 10 were excluded, the EFS and OS were similar for children with and without DS (EFS 68.0 %± 9.3% vs 70.5% ± 1.9%, P = .817; and OS 86.7% ± 6.7% vs 85.4% ± 1.5%; P = .852), both overall and adjusted for race. DS-ALL displays a unique spectrum of biologic subtypes with different frequencies of sentinel cytogenetic lesions having a large influence on outcome.

High throughput transcriptome sequencing of pediatric relapsed acute lymphoblastic leukemia (ALL).

9521 Background: Relapsed ALL carries a very poor prognosis despite intensive therapy, indicating the need for new insights into disease mechanisms. We have previously used global gene expression profiling and copy number analysis in diagnosis/relapse pairs to better understand the biologic mechanisms leading to relapse. To create an integrated genomic profile we have now focused on high throughput RNA sequencing to detect mutations, insertions, deletions and fusion transcripts. Methods: To date we have sequenced 4 pediatric matched diagnosis/relapse pairs (i.e. 8 marrow samples) from B-precursor ALL patients. RNA sequencing was performed using the Illumina GAIIx Analyzer. Each sample was sequenced in 7 lanes using single-read 54 base pair sequencing to cover 52% of genes at 5X and 34% of genes at 20X. BWA (v0.5.5) and Samtools (v1.7) were used to align the reads to the human genome and predict single nucleotide variants (SNVs) and insertion/deletions (indels) across the genome. Known single nucleotide polymorphisms (SNPs) were filtered out by comparison to diagnostic samples and through use of dbSNP (r130) and 1,000 Genomes Project databases. We have focused our initial analysis on relapse specific lesions shared by samples presumably indicating selection for common chemoresistance pathways. Results: We found 232 variants that were relapse specific and shared among all 4 patients. Sixty-seven of the variants were within exons: 14 deletions, 6 indels, 34 insertions and 13 SNVs. There were 2,454 variants that were relapse specific and shared among 3 of 4 patients. Of these, 766 variants were within exons: 144 deletions, 69 indels, 344 insertions and 209 SNVs. There were 16,100 relapse specific variants found in 2 of 4 patients. Of these, 5,184 were within exons: 1,058 deletions, 300 indels, 2,146 insertions and 1,680 SNVs. Validation of a subset of these variants in a larger cohort of patients using Taqman assays and Sanger sequencing is underway. Conclusions: RNA sequencing in relapsed pediatric ALL reveals many novel relapse specific variants that are shared among patients. Furthur analysis of these variants will be necessary to determine their functional significance and potential therapeutic relevance. No significant financial relationships to disclose.