Childhood pre-B Acute Lymphoblastic Leukemia Research Articles

Transcriptional signatures associated with persisting CD19 CAR-T cells in children with leukemia

In the context of relapsed and refractory childhood pre-B cell acute lymphoblastic leukemia (R/R B-ALL), CD19-targeting chimeric antigen receptor (CAR)-T cells often induce durable remissions, which requires the persistence of CAR-T cells. In this study, we systematically analyzed CD19 CAR-T cells of 10 children with R/R B-ALL enrolled in the CARPALL trial via high-throughput single-cell gene expression and T cell receptor sequencing of infusion products and serial blood and bone marrow samples up to 5 years after infusion. We show that long-lived CAR-T cells developed a CD4/CD8 double-negative phenotype with an exhausted-like memory state and distinct transcriptional signature. This persistence signature was dominant among circulating CAR-T cells in all children with a long-lived treatment response for which sequencing data were sufficient (4/4, 100%). The signature was also present across T cell subsets and clonotypes, indicating that persisting CAR-T cells converge transcriptionally. This persistence signature was also detected in two adult patients with chronic lymphocytic leukemia with decade-long remissions who received a different CD19 CAR-T cell product. Examination of single T cell transcriptomes from a wide range of healthy and diseased tissues across children and adults indicated that the persistence signature may be specific to long-lived CAR-T cells. These findings raise the possibility that a universal transcriptional signature of clinically effective, persistent CD19 CAR-T cells exists.

Very long intergenic non-coding RNA transcripts and expression profiles are associated to specific childhood acute lymphoblastic leukemia subtypes.

Very long intergenic non-coding RNAs (vlincRNAs) are a novel class of long transcripts (~50 kb to 1 Mb) with cell type- or cancer-specific expression. We report the discovery and characterization of 256 vlincRNAs from a cohort of 64 primary childhood pre-B and pre-T acute lymphoblastic leukemia (cALL) samples, of which 61% are novel and specifically expressed in cALL. Validation was performed in 35 pre-B and pre-T cALL primary samples. We show that their expression is cALL immunophenotype and molecular subtype-specific and correlated with epigenetic modifications on their promoters, much like protein-coding genes. While the biological functions of these vlincRNAs are still unknown, our results suggest they could play a role in cALL etiology or progression.

Childhood pre-B cell acute lymphoblastic leukemia with translocation t(1;19)(q21.1;p13.3) and two additional chromosomal aberrations involving chromosomes 1, 6, and 13: a case report

BackgroundThe translocation t(1;19)(q23;p13), which results in the TCF3-PBX1 chimeric gene, is one of the most frequent rearrangements observed in B cell acute lymphoblastic leukemia. It appears in both adult and pediatric patients with B cell acute lymphoblastic leukemia at an overall frequency of 3 to 5%. Most cases of pre-B cell acute lymphoblastic leukemia carrying the translocation t(1;19) have a typical immunophenotype with homogeneous expression of CD19, CD10, CD9, complete absence of CD34, and at least diminished CD20. Moreover, the translocation t(1;19) correlates with known clinical high risk factors, such as elevated white blood cell count, high serum lactate dehydrogenase levels, and central nervous system involvement; early reports indicated that patients with translocation t(1;19) had a poor outcome under standard treatment.Case presentationWe report the case of a 15-year-old Syrian boy with pre-B cell acute lymphoblastic leukemia with abnormal karyotype with a der(19)t(1;19)(q21.1;p13.3) and two yet unreported chromosomal aberrations: an interstitial deletion 6q12 to 6q26 and a der(13)t(1;13)(q21.1;p13). According to the literature, cases who are translocation t(1;19)-positive have a significantly higher incidence of central nervous system relapse than patients with acute lymphoblastic leukemia without the translocation. Of interest, central nervous system involvement was also seen in our patient. ConclusionsTo the best of our knowledge, this is the first case of childhood pre-B cell acute lymphoblastic leukemia with an unbalanced translocation t(1;19) with two additional chromosomal aberrations, del(6)(q12q26) and t(1;13)(q21.3;p13), which seem to be recurrent and could influence clinical outcome. Also the present case confirms the impact of the translocation t(1;19) on central nervous system relapse, which should be studied for underlying mechanisms in future.

Novel transcriptional targets of ETV6, a transcription factor frequently altered in childhood pre-B acute lymphoblastic leukemia

Novel transcriptional targets of ETV6, a transcription factor frequently altered in childhood pre-B acute lymphoblastic leukemia

Macrophage colony-stimulating factor receptor marks and regulates a fetal myeloid-primed B-cell progenitor in mice

AbstractAlthough it is well established that unique B-cell lineages develop through distinct regulatory mechanisms during embryonic development, much less is understood about the differences between embryonic and adult B-cell progenitor cells, likely to underpin the genetics and biology of infant and childhood PreB acute lymphoblastic leukemia (PreB-ALL), initiated by distinct leukemia-initiating translocations during embryonic development. Herein, we establish that a distinct subset of the earliest CD19+ B-cell progenitors emerging in the E13.5 mouse fetal liver express the colony-stimulating factor-1 receptor (CSF1R), previously thought to be expressed, and play a lineage-restricted role in development of myeloid lineages, and macrophages in particular. These early embryonic CSF1R+CD19+ ProB cells also express multiple other myeloid genes and, in line with this, possess residual myeloid as well as B-cell, but not T-cell lineage potential. Notably, these CSF1R+ myeloid-primed ProB cells are uniquely present in a narrow window of embryonic fetal liver hematopoiesis and do not persist in adult bone marrow. Moreover, analysis of CSF1R-deficient mice establishes a distinct role of CSF1R in fetal B-lymphopoiesis. CSF1R+ myeloid-primed embryonic ProB cells are relevant for infant and childhood PreB-ALLs, which frequently have a bi-phenotypic B-myeloid phenotype, and in which CSF1R-rearrangements have recently been reported.

Childhood Pre-B acute lymphoblastic leukemia and glutathione S-transferase omega 1 and 2 polymorphisms.

Acute lymphoblastic leukemia (ALL) is the most prevalent malignancy among children and makes up 23% of total childhood cancers worldwide. Pre-B ALL is one of the most common ALLs, comprising about 80% of childhood cases. A variety of genes are involved in metabolizing carcinogens. These gene polymorphisms can result in less efficient or overly-down metabolic pathways, which may contribute to the susceptibility to develop cancer. Glutathione S-transferase omega (GSTO) is a new known class among GSTs superfamily. GSTO1 and GSTO2 polymorphisms have been reported to be related to several types of disease. We assessed the association between GSTO1 and GSTO2 polymorphisms and childhood pre-B ALL risk in Iran. This case-control study analyzed GSTO1 A140D (rs. 4925) and GSTO2 N142D (rs. 156697) gene polymorphisms using a polymerase chain reaction-restriction fragment length polymorphism method, in 100 patients and 120 healthy controls. The genotype frequencies were not significantly different between patients and healthy controls. Odds ratio (95% confidence intervals) for mutant homozygotes were 1.54 (0.628-3.778) and 0.791 (0.349-1.793) for GSTO1 A140D and GSTO2 N142D, respectively. This study found no significant association between Pre-B ALL and GSTO1 A140D and GSTO2 N142D polymorphisms.

Clinical Features and Prognostic Significance of TCF3-PBX1 Fusion Gene in Chinese Children with Acute Lymphoblastic Leukemia by Using a Modified ALL-BFM-95 Protocol

For children with precursor B (pre-B) acute lymphoblastic leukemia (ALL) with TCF3-PBX1 fusion gene, their prognosis has been a controversial topic. From January 2008 to December 2012 in our hospital, 450 patients were diagnosed as ALL. Clinical characteristics of 20 patients with TCF3-PBX1 fusion gene were analyzed retrospectively, which were classified to the intermediate-risk (IR) group according to Chinese Children Leukemia Group-2008 (CCLG-2008) risk-stratification criteria and protocol based on the backbone of BFM 95 trails. Eighty five cases without TCF3-PBX1 in the same IR group were regarded as the comparison group. There were no differences in age, gender, initial white blood cell (WBC) count, status of central nerves system (CNS) at diagnosis and complete remission (CR) rates of bone marrow (BM) between the two groups (P > .05). The 5-year probability of event-free survival (EFS) rates were 84.4 ± 15.6% and 73.5 ± 15.6% in the TCF3-PBX1 group and the comparison group (P = .35), respectively. The 5-year probability of overall survival (OS) rates were 86.0 ± 17.6% and 81.8 ± 17.6% (P = .46), respectively. Relapse rates were 10.5% and 12.9% (P = 1.00), respectively. There were not cases with CNS relapse in the TCF3-PBX1 group. When intensive chemotherapy was used, the TCF3-PBX1 was associated with a favorable outcome in childhood pre-B ALL.

Mechanisms of Clonal Evolution of Pre-Leukemic Clones in Childhood Pre-B Acute Lymphoblastic Leukemia

Background and hypothesis: Childhood pre-B acute lymphoblastic leukemia (ALL) can frequently be retraced to a pre-leukemic clone carrying a prenatally acquired genetic lesion (e.g. ETV6-RUNX1gene rearrangement). After birth, pre-leukemic clones can acquire secondary mutations and, hence, evolve towards overt leukemia. While this concept is well established, the mechanism(s) driving clonal evolution are not known. Epidemiological findings hint to a role of delayed childhood infections and chronic inflammation as etiologic factors of childhood ALL, but do not illuminate mechanism of clonal evolution of pre-leukemic cells. In this study, we demonstrate that cooperation between the AID cytosine deaminase and the RAG1/RAG2 V(D)J recombinase promotes acquisition of secondary genetic lesions that promote progress of pre-leukemic B cell precursors towards full-blown leukemia.Results: The enzymatic activity of RAG1/RAG2 (VDJ recombination) and AID (somatic hypermutation, class-switch recombination) are strictly segregated to early and late stages of B cell development, respectively. While RAG1 and RAG2 are actively expressed at stages of early B cell development (bone marrow and fetal liver) that give rise to pre-B ALL, little is known about the function of AID in early B-lymphopoesis. As the involvement of AID in pre-B leukemic clonal evolution is incumbent on its expression during early stages of B-lymphopoesis, we tested CD19+ pre-B cells isolated from human bone marrow (BM) for indicators of AID activity, namely, somatic hypermutation (SHM) and class switch recombination (CSR). Interestingly, most pre-B cell clones carry rearranged Ig VH region genes that are mutated at low levels (average mutation frequency 26 x 10-3 bp). Likewise, pre-B cells isolated from fetal liver tissues (three donors; 10-19 weeks of gestation) carried Ig VH region genes mutated at low levels (average mutation frequency 14 x 10-3 bp). In addition, about one third of fetal liver pre-B cells had undergone CSR to Cγ3, Cγ1 and Cα regions. These findings highlight the previously unknown function of AID in two important sites of early human B-lymphopoesis. Based on these results, we hypothesized that a specific B cell subset during early pro- and pre-B cell differentiation can concomitantly express both AID and the RAGs and, hence, would be particularly susceptible to clonal evolution of cells that carry a pre-leukemic lesion.Our subsequent studies identified late pre-B cells (Fraction D) as a natural subset of increased genetic vulnerability. Late pre-B cells downregulate IL7 receptor/Stat5 signaling, which enables expression of RAG1 and RAG2 and immunoglobulin light chain gene rearrangement. Loss of IL7 receptor/Stat5 signaling also removes an important safeguard against premature expression of AID. Therefore, late pre-B cells are poised to express AID at high levels in response to inflammatory stimuli (e.g. LPS) in concurrence with RAG1 and RAG2. Studying clonal evolution of patient-derived pre-B ALL cells, we found evidence for concomitant AID and RAG1/RAG2 activity. Further studying a genetic mouse model for pre-leukemic pre-B cells carrying ETV6-RUNX1, we found that repeated exposure to LPS can cause overt leukemia but not in the absence of either AID or Rag1. Additionally, whole exome sequencing of human B cell clones that were engineered to express AID, RAG1/RAG2 alone or in combination revealed that concurrent expression of AID with RAG1/RAG2 dramatically increased the frequency of structural chromosomal lesions.Conclusion: Consistent with epidemiological findings on the etiology of childhood ALL, we conclude that reduced cytokine signaling (here, IL7R) in late pre-B cells renders pre-leukemic clones distinctively vulnerable to genetic lesions that can be acquired in the context of repeated exposure to inflammatory stimuli (e.g. chronic and recurrent infections during childhood). Our results support a role for AID and RAGs cooperation for the generation of secondary lesions in leukemia subgroups that require additional leukemogenic events, and therefore, provide the genetic and molecular basis to support the Delayed Infections Hypothesis for leukemia progression in children. DisclosuresNo relevant conflicts of interest to declare.

Chemosensitivity and angiogenic potency of pre-B acute lymphoblastic leukemia cell line Sup-B15

Objective It has been reported that Ik6 overexpression is associated with poor outcome of childhood pre-B acute lymphoblastic leukemia and Sup-B15 cell line is Ik6 positive. There were fewer studies on the Sup-B15 in the country, so the aim of this research is to explore chemosensitivity and angiogenic potency of pre-B acute lymphoblastic leukemia cell line Sup-B15 associated with Ik6 positive expression through making a comparison between Sup-B15 and Nalm-6 cell line. Methods The Ik6 mRNA and protein expression were detected by nested reverse transcription polymearse chain reaction and Western blot.The effect of vincristine, daunorubicin, L-asparaginase and dexa-methasone on the proliferation of cells was measured by using CCK-8 assay.The migration and invasion of cells were detected by transwell assay and mRNA expression of angiogenesis genes(VEGF, Flt-1, PlGF, IGF-1, Ang1 and Ang2) was measured by real-time PCR. Results No expression of Ik6 was detected in Nalm-6 cell line.Compared with Nalm-6, Sup-B15 cell line had a lower rate of growth.The 4 drugs could inhibit the proliferation of cells in a dose-and time-depen-dence manner.Sup-B15 was more sensitive to daunorubicin, L-asparaginase and dexamethasone, and the folds of respective IC50 value were 9.5, 1.1 and 1.5.But Sup-B15 was more resistant to vincristine than Nalm-6.The result of transwell assay showed that less Sup-B15 cells transmigrated to the lower chamber.A significant lower expression level of angiogenesis genes was detected in Sup-B15 cells. Conclusion These findings suggest that Sup-B15 is more sensitive to chemotherapy and less angiogenic compared with Nalm-6. Key words: Sup-B15; Nalm-6; Chemosensitivity; Invasiveness

Cooperation Between Aid and the Rag1/Rag2 V(D)J Recombinase Drives Clonal Evolution of Childhood Acute Lymphoblastic Leukemia

AbstractAbstract ▪519▪This icon denotes a clinically relevant abstract Background and Rationale:In many cases, childhood acute lymphoblastic leukemia (ALL) can be retraced to a recurrent genetic lesion in utero which establishes a pre-leukemic clone. The TEL-AML1 fusion gene for instance, arises prenatally and defines the most frequent subtype of childhood ALL. Strikingly, ∼1 of 100 healthy newborns carry a TEL-AML1 pre-leukemic clone, but only less than 1% of these children eventually develop leukemia. Encounter of infectious antigen leads to activation of the mutator enzyme AID in mature B cells. While AID is required for somatic hypermutation and class switching during late stages of B cell development, its pre-mature activation may be deleterious. The underlying questions for this project were: (1) how are B cells safeguarded from pre-mature AID expression during their early development and (2) whether pre-mature AID expression during early B cell development is deleterious in the sense that it promotes the clonal evolution of a pre-leukemic B cell clone in the bone marrow. Results:Studying gene expression in a clinical trial for children with high risk pre-B ALL (COG P9906; n=207), we found that high expression levels of AID at the time of diagnosis is predictive of poor overall survival and a higher frequency of leukemia relapse. These findings suggest that AID may be a contributing factor to the clonal evolution of childhood pre-B ALL. Previous work by Michael Lieber's group proposed cooperation of AID and the V(D)J recombinase Rag1/Rag2 as a key mechanism leading to the acquisition of chromosomal translocations in human B cell malignancies (Tsai et al., 2008). Activity of Rag1/Rag2 V(D)J recombinase and AID is segregated to early and late stages of B cell development, respectively. However, we found that experimental withdrawal of IL7 receptor (IL7R) signaling in pre-B cells not only activates Rag1/Rag2 expression and V(D)J recombinase but also rendered pre-B cells responsive to antigen (LPS) encounter with strong upregulation of AID. We found that upon withdrawal of IL7, transcription of AID and Rag1/Rag2 is activated by the same elements through a Pten/FoxO-dependent pathway. To test whether IL7R signaling also negatively regulates AID activation in human pre-B cells, we performed a comprehensive analysis of human B cell development in bone marrow samples from two children carrying deleterious mutations of the IL7RA and IL2RG genes encoding the two chains of the human IL7R. As opposed to normal human pre-B cells, pre-B cells from IL7RA and IL2RG-mutant patients carried somatically mutated immunoglobulin genes consistent with aberrant expression of AID in these cells. Based on these observations, we propose that Fraction D pre-B cells represent a subset of increased genetic vulnerability owing to concomitant expression of both AID and Rag1/Rag2.To test whether the vulnerability of Fraction D pre-B cells is relevant in the clonal evolution of childhood ALL, we challenged pre-B cells carrying the TEL-AML1 fusion gene with 5 consecutive cycles of IL7 withdrawal (−IL7) and LPS stimulation (+LPS). To distinguish between the potential contribution of AID and Rag1/Rag2 to secondary genetic lesions, -IL7/+LPS-challenges were performed with wildtype pre-B cells, AID−/−, Rag1−/− and AID−/− Rag1−/− double knockout pre-B cells. TEL-AML1-bearing pre-B cells were labeled with firefly luciferase and then 25 million cells were injected into 7 recipient animals per group. While wildtype TEL-AML1 pre-B cells that went through 5 rounds of -IL7/+LPS-challenge caused leukemia in all recipient mice, TEL-AML1 pre-B cells lacking either AID or Rag1 failed to give rise to full-blown leukemia in transplant recipients. Conclusion:While one in 100 newborns carry the TEL-AML1 fusion molecule, the mechanisms that lead to the acquisition of critical secondary genetic lesions are not known. Here, we report a novel, IL7R/Stat5-dependent mechanism by which pre-B cells are rendered non-responsive to LPS-dependent upregulation of AID. We propose that Fraction D pre-B cells represent a subset of increased natural genetic vulnerability in the context of concomitant activativity of AID and Rag1/Rag2. Frequent exposure to infectious antigens (e.g. LPS) in this constellation may propagate clonal evolution towards full-blown leukemia. Disclosures:No relevant conflicts of interest to declare.

In Vivo Tungsten Exposure Alters B-Cell Development and Increases DNA Damage in Murine Bone Marrow

High environmental tungsten levels were identified near the site of a childhood pre-B acute lymphoblastic leukemia cluster; however, a causal link between tungsten and leukemogenesis has not been established. The major site of tungsten deposition is bone, the site of B-cell development. In addition, our in vitro data suggest that developing B lymphocytes are susceptible to tungsten-induced DNA damage and growth inhibition. To extend these results, we assessed whether tungsten exposure altered B-cell development and induced DNA damage in vivo. Wild-type mice were exposed to tungsten in their drinking water for up to 16 weeks. Tungsten concentration in bone was analyzed by inductively coupled plasma mass spectrometry and correlated with B-cell development and DNA damage within the bone marrow. Tungsten exposure resulted in a rapid deposition within the bone following 1 week, and tungsten continued to accumulate thereafter albeit at a decreased rate. Flow cytometric analyses revealed a transient increase in mature IgD(+) B cells in the first 8 weeks of treatment, in animals of the highest and intermediate exposure groups. Following 16 weeks of exposure, all tungsten groups had a significantly greater percentage of cells in the late pro-/large pre-B developmental stages. DNA damage was increased in both whole marrow and isolated B cells, most notably at the lowest tungsten concentration tested. These findings confirm an immunological effect of tungsten exposure and suggest that tungsten could act as a tumor promoter, providing leukemic "hits" in multiple forms to developing B lymphocytes within the bone marrow.

Abstract 4335: The genomic landscape of childhood pre-B acute lymphoblastic leukemia

Abstract Precursor B-cell acute lymphoblastic leukemia (pre-B ALL) is the most frequent pediatric cancer. Increased understanding of the pathobiology of B-cell ALL has led to risk-targeted treatment regimens and increased survival rates. However, the underlying causes of this pediatric cancer are still unclear. We are using next-generation sequencing technology to better understand the genomic landscape of pre-B ALL and to build a catalogue of variations involved in pediatric ALL onset and/or progression. Using a unique “quartet” design that involves matched tumor (at diagnosis) and normal (remission) samples, as well as DNA from both parents, we will be able to identify somatic mutations driving the leukemic process. Here, we report the deep-sequencing of the whole exomes, and the partial miRNomes, of over 60 childhood B-cell ALL quartets. Using ABI SOLiD technology, we captured over 4.0 Gb of sequence on average per sample with a mean coverage of 40X. Genome-wide genotyping (Illumina's Omni 2.5 array) was also for quality control and structural variant identification. For each individual, approximately 97% of the targeted region was covered α1X and 80% of the targeted bases passed our thresholds for variant calling (≥ 5X coverage, MQV ≤ 20). According to these criteria, about 25,000 SNPs were found per individual. Using this quartet design we were able to incorporate parental sequence information to reduce sequencing errors and facilitate the identification of true variants within a given family, and of leukemia-specific variants within the ALL cohort. We investigated the somatic mutation profiles of the ALL genomes and identified both recurrent and private leukemia-specific mutations, and highlighted genes/pathways with an increased burden of somatic loss of function variants. Our goal now is to validate potential driver mutations that could play a direct role in leukemogenesis through functional assessment. Ultimately, this work will provide invaluable insights to understand the genetic mechanisms underlying pediatric ALL which could lead to the development of powerful clinical tools to improve detection, diagnosis and treatment of this childhood cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4335. doi:1538-7445.AM2012-4335

5T4 oncofetal antigen is expressed in high risk of relapse childhood pre-B acute lymphoblastic leukemia and is associated with a more invasive and chemotactic phenotype

Although the overall prognosis in childhood acute lymphoblastic leukaemia (ALL) is good, outcome after relapse is poor. Recurrence is frequently characterised by the occurrence of disease at extramedullary sites such as the central nervous system and testes. Subpopulations of blasts able to migrate to such areas may have a survival advantage and give rise to disease recurrence. Gene expression profiling of 85 diagnostic pre-B-ALL bone marrow samples revealed higher 5T4 oncofoetal antigen transcript levels in cytogenetic high-risk subgroups of patients (p < 0.001). Flow cytometric analysis determined that bone marrow from relapse patients have a significantly higher percentage of 5T4 positive leukemic blasts than healthy donors (p = 0.005). The high-risk Sup-B15 pre-B-ALL line showed heterogeneity in 5T4 expression, and the derived, 5T4+ (Sup5T4) and 5T4− (Sup) subline cells, displayed differential spread to the omentum and ovaries following intraperitoneal inoculation of immunocompromised mice. Consistent with this, Sup5T4 compared to Sup cells show increased invasion in vitro concordant with increased LFA-1 and VLA-4 integrin expression, adhesion to extracellular matrix and secretion of matrix metalloproteases (MMP-2/-9). We also show that 5T4 positive Sup-B15 cells are susceptible to 5T4 specific superantigen antibody-dependent cellular toxicity providing support for targeted immunotherapy in high risk pre-B-ALL.

Evaluation of CD9 as a Candidate Leukemia Stem Cell Marker In Childhood Precursor B Cell Acute Lymphoblastic Leukemia

AbstractAbstract 3241Leukemia cells are believed to arise from leukemia stem cells (LSC). It is also known that LSC are responsible for relapse in certain types of leukemia, such as acute myeloid leukemia (AML). However, the existence and role of LSC in acute lymphoblastic leukemia (ALL) is unclear. CD9 was reported to be a marker for LSC in B-ALL using cell lines (Nishida H. et al., 2009). CD9 is a tetraspanin and is believed to be involved in cell adhesion, motility, and signaling events. It is also involved in metastasis; however, the mechanisms are unknown. Since childhood ALL is a heterogeneous group of diseases and cell lines can be different from primary leukemia cells, we tested the role of CD9 as a candidate LSC marker using primary precursor B (preB) ALL cells from pediatric patients. Two methods, Raman spectroscopy and serial transplantation of sorted leukemia cells in NOD/SCID/IL2R g null (NSG) mice, were used to confirm LSC. Raman spectroscopy is a laser-based technique for the single cell analysis of intrinsic molecular vibrations reflecting cellular biochemical information. It can provide a quantitative assessment of the levels of DNA, RNA, proteins, lipids, and carbohydrates in the cell, as well as molecular-level conformational changes. Previous studies by our group showed that unique Raman fingerprints were identified in normal blood cells, ALL cells, and stem cells, including hematopoietic stem cells and embryonic stem cells. Four preB ALL samples were stained for CD9 and sorted by flow cytometry. ALL samples were obtained from patients at diagnosis or freshly harvested from NSG mice engrafted with primary leukemia samples. All samples showed heterogeneous expression of CD9. CD9 high-positive cells and negative cells were flow sorted. Raman spectra of freshly sorted CD9 high-positive and negative cells were obtained. 10 to 20 cells were analyzed in each sample. CD34 positive cells, which were isolated from normal donors, were also analyzed by Raman spectroscopy as a control. No unique Raman fingerprints were identified to separate CD9 high-positive cells from negative cells using Principal Component Analysis (PCA). Furthermore, CD9 high-positive and negative cells from three preB ALL samples were transplanted into NSG mice via intra-bone marrow injection. Equal cell numbers (5×105 to 1.5×106 cells) were used for positive and negative samples in each injection. The majority of the mice from both groups (transplanted with CD9 high-positive or negative cells) developed leukemia 3 to 4 months after injection. Leukemia phenotype was confirmed to be the same as the original leukemia. In conclusion, although CD9 was shown to be a marker for LSC in B-ALL cell lines, it does not appear to be an LSC marker in primary preB ALL. Since childhood preB ALL is a heterogeneous group of diseases, larger cohorts are necessary to confirm our findings. Raman spectroscopy may be a useful screening tool for analysis of cellular intrinsic markers. Disclosures:No relevant conflicts of interest to declare.

Detection of Fetomaternal Genotype Associations in Early-Onset Disorders: Evaluation of Different Methods and Their Application to Childhood Leukemia

Several designs and analytical approaches have been proposed to dissect offspring from maternal genetic contributions to early-onset diseases. However, lack of parental controls halts the direct verification of the assumption of mating symmetry (MS) required to assess maternally-mediated effects. In this study, we used simulations to investigate the performance of existing methods under mating asymmetry (MA) when parents of controls are missing. Our results show that the log-linear, likelihood-based framework using a case-triad/case-control hybrid design provides valid tests for maternal genetic effects even under MA. Using this approach, we examined fetomaternal associations between 29 SNPs in 12 cell-cycle genes and childhood pre-B acute lymphoblastic leukemia (ALL). We identified putative fetomaternal effects at loci CDKN2A rs36228834 (P = .017) and CDKN2B rs36229158 (P = .022) that modulate the risk of childhood ALL. These data further corroborate the importance of the mother's genotype on the susceptibility to early-onset diseases.

The RAC specific guanine nucleotide exchange factor Asef functions downstream from TEL-AML1 to promote leukaemic transformation

TEL-AML1 is an oncogenic fusion protein associated with childhood pre-B acute lymphoblastic leukaemia. From published microarray datasets we identified the Rho Guanine Nucleotide Exchange Factor (RhoGEF) Asef to be associated with TEL-AML1 leukaemia. However, the Asef gene is not a direct target of TEL-AML1 transcriptional control. Forced expression of Asef in vitro induced and maintained proliferation of haemaopoietic progenitor cells and co-operated with TEL-AML1 greatly to enhance proliferation and haemopoietic colony size. In haemopoietic transplantation reconstitution assays Asef and TEL-AML1 together failed to induce a leukaemic phenotype.

No evidence for association between TGFB1 promoter SNPs and the risk of childhood pre-B acute lymphoblastic leukemia among French Canadians

TGFB1 is a potent growth inhibitor to a wide variety of cell types including hematopoietic cells and deregulation of the TGFB1 signaling pathway has been implicated in the development of several cancers.[1][1] Functional regulatory polymorphisms of the TGFB1 gene have been directly associated with

Garlic compounds selectively kill childhood pre-B acute lymphoblastic leukemia cells in vitro without reducing T-cell function: Potential therapeutic use in the treatment of ALL

Drugs used for remission induction therapy for childhood precursor-B acute lymphoblastic leukemia (ALL) are nonselective for malignant cells. Several garlic compounds have been shown to induce apoptosis of cancer cells and to alter lymphocyte function. To investigate the effect of garlic on the apoptosis of ALL cells and lymphocyte immune function, cells from newly diagnosed childhood ALL patients were cultured with several commonly used chemotherapeutic agents and several garlic compounds. Apoptosis, lymphocyte proliferation and T-cell cytokine production were determined using multiparameter flow cytometry. At concentrations of garlic compounds that did not result in significant increases in Annexin V and 7-AAD staining of normal lymphocytes, there was a significant increase in apoptosis of ALL cells with no alteration of T-cell proliferation as determined by CD25/CD69 upregulation or interferongamma, interleukin-2 or tumor necrosis factor-alpha intracellular cytokine production. In contrast, the presence of chemotherapeutic agents resulted in nonselective increases in both lymphocyte and ALL apoptosis and a decrease in T-cell proliferation and cytokine production. In conclusion, we show selective apoptosis of malignant cells by garlic compounds that do not alter T-cell immune function and indicate the potential therapeutic benefit of garlic compounds in the treatment of childhood ALL.

Promoter SNPs in G1/S checkpoint regulators and their impact on the susceptibility to childhood leukemia

AbstractMutations leading to the alteration of cell-cycle checkpoint functions are a common feature of most cancers. Because of the highly regulated nature of the cell cycle, it seems likely that variation in gene dosage of key components due to functional regulatory polymorphisms could play an important role in cancer development. Here we provide evidence of the involvement of promoter single-nucleotide polymorphisms (pSNPs) in the cyclin-dependent–kinase inhibitor genes CDKN2A, CDKN2B, CDKN1A, and CDKN1B in the etiology of childhood pre-B acute lymphoblastic leukemia (ALL). A case-control study, conducted in 240 patients with pre-B ALL and 277 healthy controls, combined with a family-based analysis using 135 parental trios, all of French-Canadian origin, were used to evaluate single-site genotypic as well as multilocus haplotypic associations for a total of 10 pSNPs. Using both study designs, we showed evidence of association between variants CDKN2A −222A, CDKN2B −593A, and CDKN1B −1608A, and an increased risk of ALL. These findings suggest that variable expression levels of cell-cycle inhibitor genes CDKN2A, CDKN2B, and CDKN1B due to regulatory polymorphisms could indeed influence the risk of childhood pre-B ALL and contribute to carcinogenesis.

Identification of transcripts modulated by ETV6 expression

Deletions at chromosome 12p12-13 are observed in 26-47% of childhood pre-B acute lymphoblastic leukaemia (ALL) cases, suggesting the presence of a tumour suppressor gene (TSG). Accumulating genetic and functional evidence points to ETV6 as being the most probable TSG targeted by the deletions. ETV6 is a ubiquitously expressed transcription factor of the ETS family with very few known targets. To understand its function and to elucidate the impact of its absence in leukaemia, we conducted a study to identify targeted genes. Following the induction of ETV6 expression, global expression was evaluated at different time points. We identified 87 modulated genes, of which 10 (AKR1C1, AKR1C3, IL18, LUM, PHLDA1, PTGER4, PTGS2, SPHK1, TP53 and VEGF) were validated by real-time quantitative reverse transcription-polymerase chain reaction. To assess the significance of the validated candidate genes in leukaemia, their expression patterns were determined, as well as that of ETV6, in pre-B ALL patients. The expression of IL18, LUM, PTGER4, SPHK1 and TP53 was significantly correlated with that of ETV6, further suggesting that ETV6 could regulate the expression of these genes in leukaemia. This work constitutes another step towards the understanding of the functions of ETV6 and the impact of its inactivation in childhood leukaemia.