BCP Acute Lymphoblastic Leukemia Research Articles

RISK FACTORS AND EARLY SIGNS OF CRITICAL CONDITIONS IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA ADMITTED TO THE INTENSIVE CARE UNIT

Relevance: Acute lymphoblastic leukemia (ALL) is the most common cancer among children, accounting for nearly a quarter of all childhood cancers.
 The study aimed to determine the risk factors and signs of critical conditions in children with acute lymphoblastic leukemia admitted to an intensive care unit (ICU).
 Methods: The approach used was a systematic review. Data was collected from sources published in 2019-2023. Four cohort studies, four retrospective analyses, two literature reviews, one case-control study, and one case study were included in this systematic review.
 Results: The prognosis in pediatric ALL depends on the initial number of blast cells in the peripheral blood. Patients with B-cell precursor acute lymphoblastic leukemia (BCP ALL) and low blast cell numbers survived better than patients with T-cell acute lymphoblastic leukemia (T-ALL) and low cell count. IL1B and NLRP1 genetic polymorphisms enhanced ALL risk and reduced infectious comorbidity. However, these gene polymorphisms must be confirmed in juvenile leukemia. KRAS, FLT3, NRAS, PTPN11, KMT2D, PTEN, and NOTCH1 gene mutations affected pediatric ALL patient features and treatment results. These mutations demonstrate the relevance of genetic profiling in risk classification and tailored management. Gene variations and availability of effective medication contributed. Pediatric BCP-ALL patients with the PAX5P80R mutation had worse 5-year overall survival, higher white blood cell counts, male preponderance, and more genetic abnormalities. Pediatric BCP-ALL focused on genetic analysis and risk stratification. Children of African American and European American ancestry showed varied incidence, recurrence, and outcome rates for ALL. African American children exhibited lower incidence but greater recurrence rates and poorer prognosis than European American children.
 Conclusion: Risk factors for these patients’ admission to ICU include comorbidities, infectious diseases, hypoxia, and hemodynamic instability, as well as age and baseline white blood cell count at diagnosis.

P362: MINIMAL-LOSS-CYTOMETRY FOR EVALUATION OF CEREBROSPINAL FLUID IN PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA. SINGLE CENTER STUDY.

Background: Central nervous system (CNS) involvement at diagnosis of acute lymphoblastic leukemia (ALL) represents a risk factor for CNS relapse. Currently, examination of the cerebrospinal fluid (CSF) is performed and evaluated quantitatively using a counting chamber and qualitatively using cytomorphological analysis of cytospin. Recently, flow cytometry (FC) evaluation of CSF was shown to provide more reliable results and to predict relapse risk (de Haas et al., 2021, Thastrup et al., 2020). Independently of these studies, we applied a lyse-no wash technique aiming at minimizing cell loss during preparation and at improved cell concentration estimate. Aims: Our aim was to compare our findings with morphological criteria and published data and correlate them with clinical outcome in pediatric patients with ALL. Methods: We included pediatric patients with newly diagnosed ALL (04/2014 – 01/2022), whose CSF examination was performed in CLIP laboratory (n=208). Patients were classified as B cell precursor ALL (BCP ALL; 85%) and T ALL (15%) and treated according to following protocols: AIEOP-BFM ALL 2009 (n=118), AIEOP-BFM ALL 2017 (n=71), EsPhALL(n=8), Interfant06 (n=5), off-protocol (n=6). During follow up, 14 patients relapsed (5 CNS incl. combined, 9 non-CNS). CSF (drawn into tube with no fixative agent and processed within 2 hours) was concentrated by centrifugation and incubated with combination of antibodies (CD20/CD10/CD45/CD34/CD19/CD3/Syto41 in BCP ALL and CD4/CD99/CD5/CD3/CD7/CD16 + 56/CD8/CD45/Syto41 in T ALL). Residual erythrocytes were lysed with NH4Cl solution, followed by immediate acquisition on BD FACS Lyric or BD LSRII cytometers. Blasts were quantified to initial CSF volume. Cluster of 20 events was required to assign samples as FC positive (FC+). In parallel, CNS status was concluded according to morphological criteria, being classified as CNS1 (no blasts), CNS2a/b (≤5 WBC/μL with blasts), CNS2c/CNS3 (≥5 WBC/μL with blasts) according to treatment protocol guidelines. Continuous variables were compared with the Mann–Whitney test, survival data were analyzed using Mantel-Cox test. P values<0.05 were considered as significant. Results: Using FC, we identified atypical blasts in 50 out of the 208 analyzed samples (24%). Median of analyzed CSF volume was 367µL (range 143-1033µL), which enabled median sensitivity 0.054 events/µL (range 0.02-0.14ev/µL). Mean blast concentration was 0.47ev/µL (range 0-25ev/µL) in all samples and 2 ev/µL (range 0.01-25ev/µL, median 0.53ev/µL) in FC+ samples. We observed significantly higher blast concentration in patients with T ALL, in patients with CNS2 and CNS3 and those who subsequently relapsed. Patients with FC+ had significantly lower relapse-free survival than FC- patients (55% vs 94%), which provided better separation than CNS status (68% vs.85% for CNS2/3 vs. CNS1, respectively). Image:Summary/Conclusion: Using fresh CSF sample with lyse-no wash protocol enabled us to detect approximately 10x higher blast count (0.24ev/µL in FC+CNS1 and 1.25ev/µL in FC+CNS2/3) than was recently published (0.015ev/µL in FC+CNS1 and 0.073ev/µL in FC+CNS2/3 in Thastrup et al., 2020), and thus it better correlates to cytomorphological data. Interestingly, the proportion of FC+ patients in our cohort is comparable to (24% vs 25%), showing that although in our method we lose fewer cells, we identify the same proportion of FC+ patients. The presented study shows a higher impact of FC positivity on relapse risk than described previously. The disadvantage of our method is a need for a fresh sample, which is investigated locally. Supported by UNCE/MED/015, NU20J-07-00028.

Prognostic Relevance of Structural Maintenance of Chromosomes 4 Overexpression in Pediatric Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Abnormal expression of structural maintenance of chromosomes (SMC) 4 has been observed in multiple tumors and plays a vital role in cancer development. However, the association between SMC4 expression and clinical characteristics in Chinese childhood patients with ALL is unknown. Thus, this study aimed to investigate the relationship between SMC4 expression and clinical features and prognosis in these patients. Real-time quantitative polymerase chain reaction (PCR) was performed to detect the expression of SMC4 in Chinese pediatric ALL patients and in patients achieving complete remission (CR). Then, the relationships between SMC4 expression and clinical features, such as gender, age, white blood cell (WBC) count, French-American-British (FAB) classification, immunophenotype, fusion gene, prednisone response, and minimal residual disease (MRD) were determined. Furthermore, survival and prognostic factor analyses were carried out to examine the prognostic value of SMC4 expression. The expression level of SMC4 was significantly higher in bone morrow cells of newly diagnosed pediatric ALL patients than in those of healthy controls (p = 0.006), especially in B-cell precursor ALL (BCP ALL). Moreover, SMC4 expression was correlated with different clinical parameters. Furthermore, a decrease of SMC4 expression was detected in BCP ALL patients achieving CR. The high SMC4 expression group had both worse event-free survival rate and poorer overall survival rate. However, multivariate analysis showed that SMC4 expression was not an independently predictive factor of BCP ALL outcome. These results revealed that SMC4 expression in BM was associated with various clinical outcomes in pediatric patients with ALL, although it was not an independent outcome factor.

Clinical features and outcome of pediatric acute lymphoblastic leukemia with low peripheral blood blast cell count at diagnosis.

Peripheral blood (PB) blast cell count on day 8 of prednisone therapy has been considered one of the strongest predictors of outcome in children with acute lymphoblastic leukemia (ALL). However, little is known about the clinical features and prognostic impact of PB blast cell count at diagnosis in these patients. The aim of this study was to evaluate the relationship between initial PB blast cell count and clinical prognosis of pediatric ALL.The study comprised 367 patients with ALL, aged 0 to 14 years, enrolled and treated using the Chinese Children's Leukemia Group-ALL 2008 protocol between 2011 and 2015. The majority (91.6%) of patients were B-cell precursor ALL (BCP ALL), and 8.4% were T-cell ALL (T-ALL).Patients with BCP ALL in the low PB blast cell count group (<1 × 109/L) had significantly superior survival rates to those in the high count group (≥30 × 109/L). In T-ALL, the low count group showed significantly inferior survival rates compared to both the intermediate count group (1–29.9 × 109/L) and high count group. Multivariate analysis revealed that the initial white blood cell count and minimal residual disease at the end of induction therapy were independently predictive of BCP ALL outcome, while risk stratification was shown to be an independent prognostic factor for T-ALL outcome.These results indicated that low blast cell count in PB at diagnosis was associated with different clinical outcomes in patients with BCP ALL and T-ALL, although it was not an independent outcome predictor by multivariate analysis.

Transient Switch to Myeloid Lineage in Acute Lymphoblastic Leukemia during Induction Therapy: The Role of CD371 Expression and Implication for Minimal Residual Disease Detection

Background Multiparametric flow cytometry (MFC) is critical in the diagnosis and management of pediatric acute lymphoblastic leukemia (ALL), through immunophenotyping (IP), and minimal residual disease (MFC MRD) analysis. The aberrant expression of myeloid markers in B- and T-lineage ALL is a well-known phenomenon. It may be the result of an adaptive mechanism by lineage switch (SW), defined as any variation of blast IP over time. CD371 is a transmembrane glycoprotein usually expressed on normal myeloid cells and most of the myeloid blasts. Aberrant expression of CD371 was observed in DUX4-rearranged B cell precursor ALL (BCP ALL). Aims To investigate the clinical and biological features of CD371 positive (CD371+) pediatric BCP ALL, pointing out its potential implication in MFC MRD monitoring on Day 15. Materials and Methods From June 2014 to January 2017, 862 children with newly diagnosed t(9;22)(q34.1;11.2);BCR-ABL1 negative BCP ALL, were consecutively enrolled in the AIEOP BFM ALL 2009 study by AIEOP centers. Peripheral blood (PB) and bone marrow (BM) samples (SMPs) were processed and analyzed in the Laboratory of Diagnosis and Research of Pediatric Hematology-Oncology, University of Padua, Italy, according to standardized operating protocols designed by the AIEOP BFM Flow Network. At diagnosis, 9 combinations of 8 monoclonal antibodies (MoAbs) were used for IP. MFC MRD (Day 8 on PB, Day 15, 33, and 78 on BM) was performed with 2 combinations of 8 MoAbs from June 2014 to May 2016. Later, a dry 10 colors preformulated tube was adopted for MFC MRD monitoring. Results At diagnosis, CD371 expression was assessed in 823 of 862 (95.5%) SMPs by as many patients (pts; age: 1-17 years; male/female: 446/377). Of those, CD371 was positive in 75 of 823 SMPs (9.1%). CD371 positivity was associated with older age, euploidy, a more immature immunophenotype (B-I as per EGIL classification), and the aberrant expression of CD2 antigen, as well as at least one myeloid marker (Table). CD371+ SMPs showed a stronger expression of CD34, CD45, and CD58 antigens than CD371 negative SMPs (Table). We performed MFC MRD analysis on 207 SMPs of CD371+ BCP ALL (42 on Day 8, 72 on Day 15, 40 on Day 33, and 53 on Day 78). During the first 15 days of Induction therapy, a monocytoid population appeared in 76 SMPs [26 of 42 (61.9%) on Day 8 and 50 of 72 (69.4%) on Day 15]. It was characterized by a strong expression of CD34, CD58 and CD45, reduced expression of CD19, and high SSC. We interpreted that phenomenon as an SW to the myelomonocytic lineage, as previously described in a subtype of BCP ALL expressing CD2 at diagnosis. Myelomonocytic SW displayed 2 different patterns: (a) a single population of blasts with heterogeneous expression of CD19 (strong to dim/negative); (b) 2 distinct populations: the first one with the IP of diagnosis, the second one showing a downregulation of CD19 and CD34, an intensification of CD45, and an increase of SSC (Figure). At the same time points, a clear monocytoid population was visible on May-Grunwald-Giemsa stained smears. The comparison between MFC MRD and PCR MRD data showed a higher concordance when both the populations were included in the final amount of blasts on Day 15 (concordance rate: 89% vs. 82%). SW was transient and disappeared after Day 15, even though chemotherapy was always carried on as per therapeutic ALL protocol. CD371 antigen was an accurate marker of SW in our cohort [sensitivity = 0.93 (95% CI ± 0.06), specificity = 0.98 (95% CI ± 0.005), accuracy = 0.98]. Finally, CD371 positivity was associated with a worse response to Induction therapy, showing a higher proportion of pts enrolled in the high therapeutic risk group of the trial, most frequently due to a slow early response according to PCR MRD on Day 33 and 78 (Table). Conclusions We described a new subtype of pediatric BCP ALL, characterized by the aberrant expression of CD371 and a potential myelomonocytic SW during the first phase of Induction Therapy. Accurate identification of the lineage SW is mandatory to properly assess MFC MRD on Day 15 in these pts, avoiding an underestimation of blast cells. This is particularly important, considering that CD371+ BCP ALL was associated with a poor response to Induction therapy. Even in presence of a prevalent monocytoid population, chemotherapy should be carried on according to a therapeutic protocol for ALL. CD371 antigen should be part of IP diagnosis panel for ALL. A multicenter study of AIEOP BFM Flow Network centers is ongoing. Disclosures Brüggemann: Amgen: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy.

Coagulation FXIII-a Protein Expression Defines Three Novel Sub-Populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized By Distinct Gene Expression Signatures

Background Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). FXIII-A expression determined by flow cytometry (FC) exhibited characteristically distinct expression patterns in subgroups of lymphoblasts. The FXIII-A negative subgroup was significantly associated with the 'B-other' genetic category and had an unfavorable disease outcome. Methods RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology (GO) analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed (DE) genes were validated by RT-Q-PCR. Results We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the expression of FXIII-A protein, as determined by FC. We observed three well-defined categories of FXIII-A protein expression: FXIII-A negative (&lt;20% FXIII-A positive blasts), FXIII-A dim (20-80% FXIII-A positive blasts), and FXIII-A bright (&gt;80% FXIII-A positive blasts). The intensity of the FXIII-A expression increased continuously, which excluded the existence of a distinct FXIII-A negative and a FXIII-A bright subpopulation within the FXIII-A dim subgroup (Image 1/A). These three subgroups defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. There were 26 DE genes found when comparing the FXIII-A negative with the FXIII-A bright subgroup. The FXIII-A dim vs. bright comparison resulted in 155 DE genes and there were 88 DE genes identified between the FXIII-A negative and dim subgroups (Image 1/B). Expression of F13A1 gene was detected and readily validated by RT-Q-PCR in every sample. Intensity of gene expression; however, was characteristically different among samples of the three different FXIII-A protein expression subgroups with an increasing intensity in terms of relative fold-changes measured by RT-Q-PCR from the FXIII-A negative, through dim to bright subgroups. We selected 13/45 genes based on fold-change results detected by microarray, whereas an additional 32/45 genes were selected according to enriched functional categories of potential interest as defined by the GO analysis. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2, validated by RT-Q-PCR according to the FXIII-A status, followed the pattern of intensity of the expression of the F13A1 gene. Of these genes, ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 appear to have a role in leukemia and other forms of cancer. NUP43 has not yet been shown to be associated with any forms of human cancer in contrast to other members of the NUP gene family. PLAC8 which is a trophoblast lineage marker was most intensively expressed in the FXIII-A dim subgroup. This gene has been shown to be aberrantly activated in various types of cancer arising in mammals and mammalian cancer cell lines, but not in any subtype of human ALL (Image 1/C). Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of 'B-other' samples. We identified 14 DE genes by microarray overlapping with DE genes of the 'B-other' subgroup defined by the COALL Group. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the 'B-other' subgroup. Conclusions We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL. Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Validated genes proved biologically and clinically relevant. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment. Figure Disclosures No relevant conflicts of interest to declare.

Coagulation FXIII-A Protein Expression Defines Three Novel Sub-populations in Pediatric B-Cell Progenitor Acute Lymphoblastic Leukemia Characterized by Distinct Gene Expression Signatures.

Background: Leukemic B-cell precursor (BCP) lymphoblasts were identified as a novel expression site for coagulation factor XIII subunit A (FXIII-A). Flow cytometry (FC) revealed three distinct expression patterns, i.e., FXIII-A negative, FXIII-A dim, and FXIII-A bright subgroups. The FXIII-A negative subgroup was significantly associated with the “B-other” genetic category and had an unfavorable disease outcome.Methods: RNA was extracted from bone marrow lymphoblasts of 42 pediatric patients with BCP-acute lymphoblastic leukemia (ALL). FXIII-A expression was determined by multiparameter FC. Genetic diagnosis was based on conventional cytogenetic method and fluorescence in situ hybridization. Affymetrix GeneChip Human Primeview array was used to analyze global expression pattern of 28,869 well-annotated genes. Microarray data were analyzed by Genespring GX14.9.1 software. Gene Ontology analysis was performed using Cytoscape 3.4.0 software with ClueGO application. Selected differentially expressed genes were validated by RT-Q-PCR.Results: We demonstrated, for the first time, the general expression of F13A1 gene in pediatric BCP-ALL samples. The intensity of F13A1 expression corresponded to the FXIII-A protein expression subgroups which defined three characteristic and distinct gene expression signatures detected by Affymetrix oligonucleotide microarrays. Relative gene expression intensity of ANGPTL2, EHMT1 FOXO1, HAP1, NUCKS1, NUP43, PIK3CG, RAPGEF5, SEMA6A, SPIN1, TRH, and WASF2 followed the pattern of change in the intensity of the expression of the F13A1 gene. Common enhancer elements of these genes revealed by in silico analysis suggest that common transcription factors may regulate the expression of these genes in a similar fashion. PLAC8 was downregulated in the FXIII-A bright subgroup. Gene expression signature of the FXIII-A negative subgroup showed an overlap with the signature of “B-other” samples. DFFA, GIGYF1, GIGYF2, and INTS3 were upregulated and CD3G was downregulated in the “B-other” subgroup. Validated genes proved biologically and clinically relevant. We described differential expression of genes not shown previously to be associated with pediatric BCP-ALL.Conclusions: Gene expression signature according to FXIII-A protein expression status defined three novel subgroups of pediatric BCP-ALL. Multiparameter FC appears to be an easy-to-use and affordable method to help in selecting FXIII-A negative patients who require a more elaborate and expensive molecular genetic investigation to design precision treatment.

Whole-genome DNA methylation characteristics in pediatric precursor B cell acute lymphoblastic leukemia (BCP ALL).

In addition to genetic alterations, epigenetic abnormalities have been shown to underlie the pathogenesis of acute lymphoblastic leukemia (ALL)—the most common pediatric cancer. The purpose of this study was to characterize the whole genome DNA methylation profile in children with precursor B-cell ALL (BCP ALL) and to compare this profile with methylation observed in normal bone marrow samples. Additional efforts were made to correlate the observed methylation patterns with selected clinical features. We assessed DNA methylation from bone marrow samples obtained from 38 children with BCP ALL at the time of diagnosis along with 4 samples of normal bone marrow cells as controls using Infinium MethylationEPIC BeadChip Array. Patients were diagnosed and stratified into prognosis groups according to the BFM ALL IC 2009 protocol. The analysis of differentially methylated sites across the genome as well as promoter methylation profiles allowed clear separation of the leukemic and control samples into two clusters. 86.6% of the promoter-associated differentially methylated sites were hypermethylated in BCP ALL. Seven sites were found to correlate with the BFM ALL IC 2009 high risk group. Amongst these, one was located within the gene body of the MBP gene and another was within the promoter region- PSMF1 gene. Differentially methylated sites that were significantly related with subsets of patients with ETV6-RUNX1 fusion and hyperdiploidy. The analyzed translocations and change of genes’ sequence context does not affect methylation and methylation seems not to be a mechanism for the regulation of expression of the resulting fusion genes.

Research progress of Ph-like acute lymphoblastic leukemia

Ph-like acute lymphoblastic leukemia (ALL) is a subtype of B cell precursor acute lymphoblastic leukemia (BCP ALL) with poor outcome that exhibits a gene expression profile similar to breakpoint cluster region-Abelson leukemia virus (BCR-ABL)1-positive ALL but lacks the BCR-ABL1 fusion protein. The gene abnormalities involve of cytokine signaling pathway, kinase signaling pathway, and B cell developmental transcription factors. Protein kinase inhibitors combined with chemotherapy might be applied in Ph-like ALL. The investigation of Ph-like ALL will contribute to precise risk stratification of BCP-ALL and targeted therapy. The research progress of Ph-like ALL will be introduced in this article, including epidemiology, susceptibility locus, gene variants, diagnosis, treatment and prognosis. Key words: Precursor B-cell lymphoblastic leukemia-lymphoma; Gene expression profiling; Protein kinase inhibitors; Prognosis

ANGIOPOIETIN1 - a Novel Factor Implicated In MLL-Rearranged Acute Lymphoblastic Leukemia and Regulated In a Fusion Gene-Dependent Manner

AbstractAbstract 66 Background:MLL-rearranged acute lymphoblastic leukemia (ALL) is prevalent in infants, constituting 70% of the cases. The preferred MLL translocation partner is the gene AF4, resulting in t(4;11)(q21;q23), which arises in 50% of infant ALL patients. This translocation generates the fusion genes MLL/AF4 and AF4/MLL, and is associated with an aggressive clinical presentation and poor outcome. Biologically, cells expressing MLL/AF4 show resistance to stress- and chemotherapy-related apoptosis. Concordantly, we have previously shown that RNAi-mediated depletion of MLL/AF4 in the t(4;11)-positive ALL cell line SEM results in induction of cell death and impaired both clonogenicity and in vivo engraftment. In order to characterize this phenotype on a molecular level, we have performed gene expression profiling of SEM cells depleted of MLL/AF4 and corresponding controls. Expression of >1000 genes was affected, including a subset of angiogenic genes, most prominently ANGIOPOIETIN1 (ANGPT1), a proangiogenic cytokine reported to play a role in acute myeloid leukemia (AML), hematopoietic stem cell (HSC) quiescence and bone marrow (BM) niche maintenance, but to date not implicated in ALL. Here we report a novel link between ANGPT1 expression and MLL-rearranged ALL. Methods:Gene expression profiling was performed using the Illumina HT-12 platform and data processed using BeadStudio and Genespring software suites. ANGPT1 expression was analyzed by real-time RT-PCR (qRT-PCR), and ANGPT1 protein secretion determined using enzyme-linked immunosorbent assay (ELISA). The MLL/AF4 status of cells was modulated with fusion transcript-specific siRNAs and knockdown monitored by qRT-PCR. RNAi-mediated depletion of ANGPT1 was achieved using siRNA or lentiviral shRNA constructs, and validated on transcript and protein level. Effects on cell cycle progression and proliferation in response to ANGPT1 knockdown in t(4;11)-positive cells were assessed by flow cytometry and trypan blue exclusion assay, respectively. For in vivo studies, SEM cells were sequentially transduced to express both luciferase and either non-target control shRNA (shNTC) or shANGPT1. Doubly transduced cells were selected for and FACS-sorted prior to intrafemoral transplantation into immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Disease progression was monitored using bioluminescence imaging and engraftment assessed by flow cytometry at the terminal timepoint. Results:ANGPT1 expression was screened in a MLL-rearranged ALL patient cohort (n=35), comprising t(4;11)-positive (n=20), t(11;19)-positive (n=10) and t(9;11)-positive patients (n=5), and in a MLL-wildtype BCP ALL patient cohort (n=8). MLL-rearranged patients showed ANGPT1 upregulation, t(4;11)-positive patients having the strongest overexpression by 232-fold when compared to ANGPT1 levels in CD19+ peripheral blood (PB) cells. A 27-fold and 13-fold upregulation was detected in t(11;19)- and t(9;11)-positive patients, respectively. Conversely, MLL-wildtype BCP ALL patients had similar ANGPT1 levels as CD19+ PB cells, with only a 2-fold increase. In addition to its high expression in t(4;11)-positive ALL, ANGPT1 levels were shown to be dependent on MLL/AF4; a reduction of ANGPT1 mRNA and protein correlated with siRNA-mediated MLL/AF4 depletion in a time-dependent manner in both cell lines and primary patient samples. This was concordant with expression array data, which indicated an up to 4-fold decrease of ANGPT1 in response to MLL/AF4 depletion. The functional role of ANGPT1 in t(4;11)-positive ALL was assessed by RNAi; sustained depletion of ANGPT1 in SEM cells resulted in cell cycle arrest and a marked decrease in proliferation. In vivo, mice transplanted with shANGPT1 expressing SEM cells showed reduced splenic infiltration and development of solid tumours at the injection site, as opposed to a systemic spread of the disease and massive splenomegaly in mice injected with shNTC expressing SEM cells. Conclusions:In this study we have identified ANGPT1 as a novel player in t(4;11)-positive ALL, as defined by overexpression, MLL/AF4-dependent regulation and functional consequences in vivo and in vitro. Currently we are investigating ANGPT1-mediated signalling in t(4;11) ALL cells, as it represents an attractive potential therapeutic target. Disclosures:No relevant conflicts of interest to declare.

Bioluminescent Tracking of Human and Mouse Acute Lymphoblastic Leukemia Reveals Potent Immunogenicity of Luciferase In Some Preclinical Models of Leukemia

AbstractAbstract 2140Xenograft models have quickly become the preferred methodology for the preclinical evaluation of treatments for acute lymphoblastic leukemia (ALL). The efficient engraftments in immune-deficient mice achieved with both primary ALL samples and cell lines have facilitated identification of the anti-ALL activity of a wide variety of agents. Despite widespread usage, however, little is known about the early ALL localization and engraftment kinetics in this model, limiting experimental read-outs primarily to survival and end-point analysis at high disease burden. In this study, we have developed bioluminescent imaging of ALL cells to provide a noninvasive, longitudinal measure of leukemia burden that will enhance the sensitivity of preclinical models. Three human precursor B cell (BCP) ALL lines (Nalm-6, RS-4-11 and 380) and two murine BCP ALL lines (289 and 309) were stably tranduced with a lentiviral vector conferring expression of both green fluorescent protein (GFP) and firefly luciferase (ffLuc). Non-obese diabetic/severe combined immunodeficient/IL2Rgamma null (NSG) mice were injected intravenously with 1×106 ALL cells via the lateral tail vein and imaged daily for the first 7 days, then twice weekly thereafter. Animals were also monitored weekly for peripheral leukemia burden by flow cytometric detection of GFP positive cells in blood. Each human ALL line was readily detectable by bioluminescence within 48 hours of injection, providing a measure of disease burden at least one week earlier than can be achieved by peripheral disease monitoring. The human ALL lines Nalm-6 and RS-4-11 initially concentrated in the liver and bone marrow of NSG mice, only appearing in the spleen after 1–2 weeks, while 380 first localized to bone marrow only. In contrast, the murine ALL lines were rapidly detectable in spleen and bone marrow but did not accumulate in the liver. For both murine and human ALL, the initial localization was followed by in situ expansion and subsequent seeding of peripheral sites, with disease burden correlating to increasing bioluminescence intensity. This study, therefore, reveals significant cell line- and species-related differences in leukemia migration, especially early in expansion, which may confound observations between various leukemia models. Furthermore, in a pilot study we demonstrate that this in vivo imaging approach is feasible for primary human ALL samples. To evaluate the utility of bioluminescence in an immune competent leukemia model, we compared the engraftment of ffLuc/GFP+ mouse ALL in syngeneic wild-type (wt) and immune-deficient mice. In contrast to the unhindered rapid expansion of disease in NSG and syngeneic (H-2d) gc-/- (lymphocyte deficient) mice (median survival 21 days, p<0.05 versus wt), wild-type mice sustained a low level of disease for the first 7 days that was subsequently eliminated. Unlabeled and GFP-only+ ALL cells engraft and expand rapidly in wt mice (median survival 25 and 18 days, respectively), and NK-replete/T and B cell-deficient mice engraft with ffLuc/GFP+ ALL cells after an initial delay in expansion (median survival 25 days), indicating that ffLuc is the target of an immune response. This is further supported by a competitive repopulation experiment in which wt mice received 1×106 mixed population cells (95% ffLuc/GFP+ cells and 5% unlabeled leukemia); no mice developed ffLuc/GFP+ disease, while 4/9 eventually developed unlabeled disease. Overall this study demonstrates the increased sensitivity and potential for standardization that in vivo bioluminescent imaging confers on xenograft ALL models. The application of this bioluminescence approach, however, will be limited in immune competent ALL models by the strong immune-mediated clearance of ffLuc+ cells. Disclosures:No relevant conflicts of interest to declare.

Increased ICOS Ligand mRNA Expression Is Associated with Very Early Relapse in Pediatric Acute Lymphoblastic Leukemia (ALL) Patients.

The cure rate in pediatric acute lymphoblastic leukemia (ALL) is about 85%. Therapy failures are mainly due to relapse. Relapse rates could possibly be influenced by the anti-leukemic activity of the immune system, e.g. by the Th1/Th2 balance. It would be desirable to increase the cure rate if the immune system could be induced to participate in the elimination of leukemia cells. However, leukemia cells, especially ALL cells are poor stimulators for T cells and do not induce a Th1 response believed to be necessary for tumor cell elimination. Missing costimulatory molecules on the leukemia cells, especially CD80 and CD86, are commonly accepted as the main reason for their poor immunostimulatory activity. On the other hand, efficient costimulation does not guarantee improved cure rates. On the contrary, in a mouse model inoculation of CD86 transfected lymphoma cells led to tumor progression possibly by interaction with CD152 or induction of a Th2 response. Indeed, in an earlier study, we examined the mRNA expression of CD80 and CD86 in marrow samples of ALL patients and found an increased expression of CD86 and IL-4 in patients with a late leukemic relapse raising the possibility of a Th2 shift predisposing to relapse (Stachel et al, Eur J Med Res, 2006). It becomes increasingly clear that members of the costimulatory family other than CD80 and CD86 also influence the immune response. ICOS/ICOS ligand and PD-1/PD1 ligand both play an important role in the second step of Th2 induction. To determine whether increased or decreased expression of costimulatory molecules play a role in the pathogenesis of relapse in pediatric ALL we examined the expression of various costimulatory molecules in leukemic marrow samples in a prospective study. Samples from 49 consecutive pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP ALL) were analyzed by semiquantitative RT-PCR for CD28, CD152 (CTLA-4), ICOS and ICOS ligand (B7RP-1).A total of 29 patients (60.4 %) remained relapse free and 19 (39.6 %) relapsed after a median follow up of 24.5 months (range: 6 to 50 months). Of those relapsing five patients suffered a very early relapse (VER, within 18 months from diagnosis), seven an early relapse (ER, between 18 and 30 months from diagnosis) and seven patients a late relapse (LR, later than 30 months from diagnosis). We found that in the subgroup of ALL patients experiencing a VER expression of mRNA ICOS ligand was significantly increased (2.0 +/− 0.7 (mean +/− SD relative intensity units) compared to non-relapsing patients (1.1 +/− 0.8, p=0.02). This trend was also visible in the late relapsing patient group (1.4 +/− 0.8 vs 0.9 +/− 0.6, ns). By comparative densitometry we estimated the copy number of ICOS ligand in the VER group to be 500,000 per 12,500 BM cells vs 350,000 per 12,500 BM cells in non-relapsing ALL patients.Since ICOS ligation on T-cells by ICOS ligand on B-cells appears to favor a Th2 polarization of the immune system our results could provide further evidence that a Th2 shift induced by leukemic blasts could confer an increased risk for very early ALL relapse. These data could provide a rationale for immunotherapeutic strategies aimed at strengthening a Th1 polarized immune response in the treatment for childhood BCP ALL.

In vitro profiling of the sensitivity of pediatric leukemia cells to tipifarnib: identification of T-cell ALL and FAB M5 AML as the most sensitive subsets

AbstractAlthough the prognosis of pediatric leukemias has improved considerably, many patients still have relapses. Tipifarnib, a farnesyl transferase inhibitor (FTI), was developed to target malignancies with activated RAS, including leukemia. We tested 52 pediatric acute myeloid leukemia (AML) and 36 pediatric acute lymphoblastic leukemia (ALL) samples for in vitro sensitivity to tipifarnib using a total cell-kill assay and compared these results to those obtained with normal bone marrow (N BM) samples (n = 25). AML samples were significantly more sensitive to tipifarnib compared to B-cell precursor ALL (BCP ALL) or N BM samples. Within AML, French-American-British (FAB) M5 samples were most sensitive to tipifarnib. T-cell ALL samples were significantly more sensitive than BCP ALL and N BM samples. In AML there was a marked correlation between tipifarnib resistance and daunorubicin or etoposide resistance, but not to cytarabine or 6-thioguanine. RAS mutations were present in 32% of AML and 18% of ALL samples, but there was no correlation between RAS mutational status and sensitivity to tipifarnib. Future studies are needed to identify biomarkers predictive of tipifarnib sensitivity. In addition, clinical studies, especially in T-cell ALL, seem warranted.

Detection of Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia by 4color Cytometry in International Multicenter Trial Minimini.

FC is still not employed in MRD based treatment protocols. One problem is lack of standardization suitable for prospective trials involving multiple clinical centers and FC laboratories. Therefore, we established a MiniMini Project, an international collateral study within the ALL IC BFM 2002 treatment protocol for childhood acute lymphoblastic leukemia (ALL). The MiniMini Project provides a mainframe of minimal panel of monoclonal antibody combinations to evaluate MRD by FC. Patients (pts) are stratified according non-MRD criteria (prednisone response at day 8 in peripheral blood (PB), percentage of blasts at day 15 and day 33 in bone marrow (BM), leukocytosis, and age at diagnosis and presence of BCR/ABL or MLL/AF4 fusions). Identical immunophenotypic populations are reported in all pts regardless presenting phenotype. Each laboratory investigates at least 2 pts with B lineage ALL by the T ALL combinations and vice versa. These “cross-lineage controls” together with data on subpopulations that are negative at diagnosis were used to set the specificity cutoff values at each time point (diagnosis, day 8 BM and PB, day 15, day 33 day 52 BM). MRD levels obtained by Ig/TCR rearrangements RQ-PCR in 32 pts (24 pts BCP ALL, 8 pts T ALL) were used to define specificity thresholds. 185 pts were investigated in the participating laboratories. We used data from first Czech cohort of pts (92 pts in total, 16 pts T lineage, 74 pts B lineage, in standard risk group (SRG), n=36, IRG, n=40 and HRG, n=16) in whom clinical data as well as standard FC analysis results were available. We compared morphological percentage of blasts (used for stratification) to a level of residual disease by FC. There was high concordance in SRG of both methods, except 1 patient redirected into IRG group (M3 BM vs. only 14% of blasts by FC). In IRG, concordance was in 92.5% of pts, 3 pts should be placed in HRG group according FC. 98.9% of pts morphologically in complete remission at day 33 were confirmed by FC. Although FC data confirm a significant difference between PGR and PPR in PB specimens at day 8 (p=0.0014), there is an overlap in percentage of leukemic cells between these categories. In total, MRD level above 0.1% was observed in BM of 100, 99, 84, 32 and 3.5 % pts in days 0, 8, 15, 33 and 52, respectively and in PB of 95% pts at day 8. Our first results show feasibility of FC standardization. The choice of subpopulations and the cutoff points will be validated in an independent cohort within the same Project.

The effect of cytokines, including IL4, IL7, stem cell factor, insulin-like growth factor on childhood acute lymphoblastic leukemia

We have investigated the effects of some interleukins, such as interleukin (IL) 4, IL7, stem cell factor (SCF) and insulin-like growth factor (IGF-1), known to be involved in human lymphopoiesis, on proliferation, clonal growth and differentiation of cells from two acute lymphoblastic leukemia (ALL) derived pre-B cell lines, that is, Nalm 1, Nalm 6 and purified blasts from 37 childhood ALL. IL4 did not display any promoting activity, an inhibitory effect being observed in two patients. IL7 showed an heterogeneous responsiveness, not related to immunophenotype or cytogenetic features, proliferation and clonal growth being observed in a minority of ALL. In other patients no or even inhibitory effects on proliferation were observed. In one case this inhibition of DNA synthesis was accompanied by maturation of the cells, as demonstrated by the induced expression of surface immunoglobulins (sIg); other IL7 treated samples failed to express sIg, but showed a decreased expression of terminal deoxynucleotidyl transferase and cALL antigen, suggesting that the cells have a potential of limited maturation by IL7. SCF, known to synergize with IL7 in the most primitive stages of normal B cell development, did not enhance the IL7 response in B cell precursor ALL. Finally IGF-1 failed to induce a proliferative response and clonal growth in BCP ALL either alone or in combination with IL7.