Cell Precursor Acute Lymphoblastic Leukemia Research Articles

The choroid plexus stroma constitutes a sanctuary for paediatric B-cell precursor acute lymphoblastic leukaemia in the central nervous system.

Despite current central nervous system‐directed therapies for childhood B‐cell precursor acute lymphoblastic leukaemia, relapse at this anatomical site still remains a challenging issue. Few reports have addressed the study of the specific cellular microenvironments which can promote the survival, quiescence, and therefore chemoresistance of B‐cell precursor acute lymphoblastic leukaemia cells in the central nervous system. Herein, we showed by immunofluorescence and electron microscopy that in xenotransplanted mice, leukaemic cells infiltrate the connective tissue stroma of the choroid plexus, the brain structure responsible for the production of cerebrospinal fluid. The ultrastructural study also showed that leukaemia cells are able to migrate through blood vessels located in the choroid plexus stroma. In short‐term co‐cultures, leukaemic cells established strong interactions with human choroid plexus fibroblasts, mediated by an increased expression of ITGA4 (VLA‐4)/ITGAL (LFA‐1) and their ligands VCAM1/ICAM1. Upon contact with leukaemia cells, human choroid plexus fibroblasts acquired a cancer‐associated fibroblast phenotype, with an increased expression of α‐SMA and vimentin as well as pro‐inflammatory factors. Human choroid plexus fibroblasts also have the capacity to reduce the proliferative index of leukaemic blasts and promote their survival and chemoresistance to methotrexate and cytarabine. The inhibition of VLA‐4/VCAM‐1 interactions using anti‐VLA‐4 antibodies, and the blockade of Notch signalling pathway by using a γ‐secretase inhibitor partially restored chemotherapy sensitivity of leukaemia cells. We propose that the choroid plexus stroma constitutes a sanctuary for B‐cell precursor acute lymphoblastic leukaemia cells in the central nervous system. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Low expression of CTBP2 and CASP8AP2 predicts risk of relapse in childhood B-cell precursor acute lymphoblastic leukemia: a retrospective cohort study

CtBP is a known corepressor abundantly expressed in cancer and regulates genes involved in cancer initiation, progression, and metastasis. This study aimed to investigate the prognostic significance of CTBP2 expression in a cohort of pediatric patients with B cell precursor acute lymphoblastic leukemia (BCP-ALL). It further evaluated the role of combined CTBP2 and CASP8AP2 expression in risk of relapse of BCP-ALL. The expression of CTBP2 mRNA was retrospectively detected by a qRT-PCR approach in bone marrow samples from 104 children with newly diagnosed BCP-ALL. CASP8AP2 was assessed simultaneously in the 100 patients included in this study. The receiver operating characteristic (ROC) curve analysis determined the cut off levels for CTBP2 and CASP8AP2 expression with good predictive significance for relapse of BCP-ALL. Patients with low CTBP2 expression had inferior relapse-free survival (RFS) and event-free survival (EFS) when compared to patients with high-CTBP2 expression. The expression level of CTBP2 was significantly associated with CASP8AP2 expression (r = 0.449, P < 0.001). Patients were stratified into three groups according to the combined evaluation of the two gene expression, and patients with simultaneous low-expression had the worst outcome (6-year RFS: 64.6%±12.8%, P < 0.001). Multivariate analysis demonstrated the expression of CTBP2 and CASP8AP2, minimal residual disease (MRD) at day 33 remained as independent prognostic factors for RFS. Based on the final Cox hazards model, we proposed an algorithm to calculate the risk index, which was more precise for predicting relapse. In conclusion, low expression of CTBP2 and CASP8AP2 correlated with poor outcome and predicted risk of relapse in pediatric BCP-ALL.

Chimeric Antigen Receptor T Cell Therapy in Patients with Multiply Relapsed or Refractory Extramedullary Leukemia.

Autologous CD19-directed chimeric antigen receptor T lymphocyte (CAR-T) therapy is an approved and effective treatment for the management of patients with refractory and multiply relapsed B cell precursor acute lymphoblastic leukemia (B-ALL). Experience using this therapy in pediatric patients with extramedullary (EM) disease is limited, in part because these patients have frequently been excluded from clinical trials owing to concerns for an increased risk of immune effector cell-associated neurotoxicity syndrome (ICANS). We infused 7 patients with refractory or multiply relapsed B-ALL who presented with isolated EM relapse with tisagenlecleucel. Six patients had isolated central nervous system (CNS) leukemia, and 1 patient had an isolated testicular relapse. An initial complete response was seen in all patients, with 5 patients remaining in CAR-T-induced remission at a median of 18 months from first infusion. Reversible ICANS was seen in 1 patient with CNS leukemia. Durable B cell aplasia occurred in 3 patients, with a median time to B cell recovery of 6.5 months in the other patients. These data suggest that CAR-T therapy has promising safety and efficacy in treating EM leukemia, although definitive conclusions are limited by the small size of the cohort and limited follow-up period.

Outcome of CD20-positive Adult B-cell Acute Lymphoblastic Leukemia and the Impact of Rituximab Therapy

BackgroundIn adult B cell precursor acute lymphoblastic leukemia (BCP-ALL), CD20 expression has generally been associated with an adverse prognosis. Incorporating rituximab to standard of care is found to improve the outcome of CD20+ BCP-ALL. The aim of this study is to estimate the prognostic effect of CD20 expression and the impact of rituximab in BCP-ALL in Saudi Arabia. Patients and MethodsWe performed a retrospective study of 55 Saudi adult patients with BCP-ALL in King Fahad Specialist Hospital in Dammam from 2008 to 2017. ResultsThe proportion of CD20+ cases was approximately 55%. Excluding rituximab-treated patients, the 5-year overall survival (OS) rate of CD20+ patients was lower than CD20− patients (56% vs. 66%; P = .62). Among CD20+ patients, the proportion that received rituximab was approximately 27%. Comparing CD20+ patients with and without rituximab, all patients who received rituximab achieved complete remission (CR) 4 weeks post-induction. The 3-year OS rate (88% vs. 63%; P = .35) and the 2-year event-free survival rate (70% vs. 68%; P = .75) were in favor of rituximab. In univariate and multivariate analyses, CR 4 weeks post-induction is recognized as an independent predictor of outcome. However, differences in survival rates did not have a statistical significance. ConclusionCD20 expression in adult patients with BCP-ALL seems to be higher in Saudi Arabians than in Caucasians, and it seems to have a tendency towards an inferior outcome in terms of OS. Incorporating rituximab to standard of care seems to improve the outcome in terms of CR, OS, and event-free survival.

Blinatumomab and inotuzumab for B cell precursor acute lymphoblastic leukaemia in children: a retrospective study from the Leukemia Working Group of the Spanish Society of Pediatric Hematology and Oncology (SEHOP).

Blinatumomab and inotuzumab ozogamycin represent promising alternatives to conventional chemotherapy in acute lymphoblastic leukaemia (ALL). We analysed data from 29 children with ALL treated under compassionate use with blinatumomab, inotuzumab or both. The complete remission (CR) rate in a heavily pretreated population with overt relapse was 47·6%. At earlier stages (first/second CR), both antibodies represented a useful tool to reduce minimal residual disease, and/or avoid further toxic chemotherapy until stem cell transplantation. Six patients developed grade 3 reversible non-haematological toxicity. The 12-month overall survival and event-free survival rates were 50·8±26·4% and 38·9±25·3% with blinatumomab, 45·8±26% and 27·5±25% with inotuzumab.

Immunophenotypic modulation in pediatric B lymphoblastic leukemia and its implications in MRD detection

Minimal/Measurable residual disease (MRD) is the most reliable and powerful indicator of prognosis in B cell precursor acute lymphoblastic leukemia (BCP-ALL). Modulation of antigenic expression in leukemic cells is known to occur postchemotherapy and thus carries a potential risk of false MRD quantification by flowcytometry. We studied the immunophenotypic modulation of nine antigens in residual leukemic cells at postinduction MRD assessment in 31 BCP - ALL children. We found significant downmodulation of CD10, CD38, CD58, CD81 and upmodulation of CD19, CD45 in leukemic cells. Downmodulation of CD34 was observed but was not statistically significant. Expression of CD20 and CD22 remained stable in most of the cases. MRD-positive cases showed loss of diagnostic LAIP and some showed gain of new LAIP compared to baseline. Various combination of antibodies including the novel markers should be incorporated into the panel to increase the sensitivity of MRD detection.

Implementation of RNA sequencing and array CGH in the diagnostic workflow of the AIEOP-BFM ALL 2017 trial on acute lymphoblastic leukemia

Risk-adapted therapy has significantly contributed to improved survival rates in pediatric acute lymphoblastic leukemia (ALL) and reliable detection of chromosomal aberrations is mandatory for risk group stratification. This study evaluated the applicability of panel-based RNA sequencing and array CGH within the diagnostic workflow of the German study group of the international AIEOP-BFM ALL 2017 trial. In a consecutive cohort of 117 children with B cell precursor (BCP) ALL, array analysis identified twelve cases with an IKZF1plus profile of gene deletions and one case of masked hypodiploidy. Genetic markers BCR-ABL1 (n = 1), ETV6-RUNX1 (n = 25), and rearrangements involving KMT2A (n = 3) or TCF3 (n = 3) were assessed by established conventional techniques such as karyotyping, FISH, and RT-PCR. Comparison of these results with RNA sequencing analysis revealed overall consistency in n=115/117 cases, albeit with one undetected AFF1-KMT2A fusion in RNA sequencing and one undetected ETV6-RUNX1 fusion in conventional analyses. The combined application of RNA sequencing, FISH, and CGH+SNP array reliably detected all genetic markers necessary for risk stratification and will be used as the diagnostic standard workflow for BCP-ALL patients enrolled in the AIEOP-BFM ALL 2017 study. Prospectively, consistent collection of genome-wide CGH+SNP array as well as RNA sequencing data will be a valuable source to elucidate new prognostic lesions beyond established markers of pediatric ALL. In this respect, RNA sequencing identified various gene fusions in up to half of the IKZF1plus (n = 6/12) and B-other (n = 19/36) cases but not in cases with hyperdiploid karyotypes (n = 35). Among these fusions, this study reports several previously undescribed in frame PAX5 fusions, including PAX5-MYO1G and PAX5-NCOA6.

Modifiers of Endothelial Permeability in the Setting of CAR-t Therapy Related Immune Cell Associated Neurotoxicity Syndrome

Background Chimeric Antigen Receptor T-cell therapy (CAR-t) for patients with relapsed or refractory B cell precursor acute lymphoblastic leukemia (ALL) is associated with high overall remission rates but also with significant complications. Cytokine release syndrome (CRS) and Immune Cell Associated Neurotoxicity Syndrome (ICANS) are life-threatening sequelae that may occur in CAR-t recipients. ICANS presents with varying symptoms ranging from confusion to seizure and cerebral edema. There is no consensus on specific biological events that incite ICANS. Blood brain barrier (BBB) permeability is altered in CD19 CAR-t therapy, but the cause of altered BBB permeability is not clear. IL6 targeted interventions that control symptoms of CRS are ineffective against ICANS, suggesting these entities have separate pathologic mechanisms. Criteria for ICANS severity grading and guidelines for supportive care management are published, but there is no universal predictor of ICANS. Identification of manipulable biologic factors to predict the onset of ICANS would represent significant progress for clinical management of ICANS. Methods We collected plasma samples from 12 adult patients with varying ICANS severity following CAR-t therapy, aged 24-74 years. Diagnoses were diffuse large B cell lymphoma (DLBCL), transformed follicular lymphoma (TFL), and primary mediastinal B cell lymphoma (PMBCL) in varying stages of progression or relapse. Pre-CAR-t, midpoint and 7 or 8 day post CAR-t samples were evaluated with multiplex cytokine arrays to quantify relative levels of 105 angiogenic cytokines. Results were compared between two groups: patients who did not develop ICANS (n=5), and patients who developed any grade of ICANS (n=7; 2 patients grade 0-2, 5 patients grade 3-4). Results There was a trend toward higher baseline relative levels of two angiogenic factors, ApoA1 and Angiogenin, in patients with ICANS relative to those without. Interestingly, ApoA1 increased over time in patients who did not develop ICANS; a 320 +/- 56 % increase in ApoA1 occurred from baseline to day 7/8. In contrast, ApoA1 decreased to 88 +/- 25% of baseline by day 7/8 in patients who developed ICANS (No ICANS vs ICANS; p=0.003). Similarly, Angiogenin increased by 289 +/- 77% over baseline by day 7/8 in patients who did not develop ICANS, while it decreased to 98 +/- 24 % of baseline by day 7/8 in patients who developed ICANS (No ICANS vs ICANS, p = 0.03). Conclusion Our data suggests that ApoA1 and Angiogenin levels at baseline, and changes in ApoA1 or Angiogenin over time, may indicate risk of ICANS. This correlation requires validation in a larger patient population as well as quantification of absolute ApoA1 and Angiogenin concentrations in blood. If ApoA1 and Angiogenin baseline levels are indeed correlated with the risk of ICANS, this may represent major progress in clinical management of ICANS.

Multimodal Therapeutic Approach of Cytokine Release Syndrome Developing in a Child Given Chimeric Antigen Receptor-Modified T Cell Infusion

Objectives:To describe a pediatric case of cytokine release syndrome secondary to chimeric antigen receptor-modified T cells associated with acute respiratory distress syndrome.Design:Case report.Setting:PICU.Patients:A 14-year-old boy with refractory B cell precursor acute lymphoblastic leukemia given chimeric antigen receptor cells developed severe cytokine release syndrome 7 days after the drug product infusion with progressive respiratory failure. He was admitted to PICU with a clinical picture of acute respiratory distress syndrome, requiring mechanical ventilation, and secondary hemophagocytic lymphohistiocytosis.Interventions:Hemoadsorption with cartridge column (Cytosorb) in combination with continuous renal replacement therapy was associated to the anti-cytokine therapy (tocilizumab, a monoclonal antibody targeting interleukin-6 receptor).Measurements and Main Results:Decrease of the inflammatory biomarkers (ferritin, interleukin-6, interleukin-10) in the first 96 hours associated with a progressive improvement of acute respiratory distress syndrome (Pao2/Fio2 ratio) 7 day after the start of the multimodal treatment.Conclusions:This case suggests that hemoadsorption with cartridge column in combination with continuous renal replacement therapy and tocilizumab is safe and potentially effective in pediatric patients with severe cytokine release syndrome.

A unique mutator phenotype reveals complementary oncogenic lesions leading to acute leukemia.

Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4-32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.

Prediction of Patients at Risk of CD19Neg Relapse Following CD19-Directed CAR T Cell Therapy in B Cell Precursor Acute Lymphoblastic Leukemia

CD19-directed chimeric antigen receptor T cell (CAR-T) therapy has shown impressive results in children and adults with relapsed or refractory B-ALL or diffuse large B-cell lymphoma. However, 30 - 70% of initial responders will eventually relapse with CD19 antigen loss (CD19Neg) (Maude SL, et al. N Engl J Med. 2018). To avoid CD19Neg relapse, patients may undergo a hematopoietic stem cell transplant (HSCT). HSCT is an expensive and often morbid procedure that many physicians would prefer to avoid. The development of tools to accurately predict which patients are at risk for CD19Neg relapse would guide treatment decisions regarding HSCT or alternative therapies. Since CD19Neg relapses also occur in patients treated with other CD19-directed immunotherapies, like blinatumomab (Mejstríková E, et al. Blood Cancer J. 2017), a predictive model to detect patients at risk of CD19Neg relapse would have broader therapeutic impact. To address this problem, we performed CyTOF and RNA-seq analysis from paired patient samples collected before CD19-directed CAR-T administration and after CD19Neg relapse. High dimensional phenotyping by CyTOF clustered patient samples based on their mechanism of CD19 expression loss (frameshift mutation versus expression of intracellular isoforms), even before CAR-T administration. In addition, we identified identical immunoglobulin heavy and light chain RNA sequences before CAR-T administration and after CD19Neg relapse, suggesting that the clones destined to cause relapse are present at the time of CAR-T administration. Altogether, these results support our hypothesis that resistant tumor cells are present before CAR-T administration and could be discovered and interrogated for CD19Neg relapse prediction. To identify cell subpopulations responsible for driving CD19Neg relapse, we used the B cell developmental classifier previously developed in our lab (Good Z, et al. Nat Med. 2018). We observed a significant increase in the Early-non-BI population (CD38Pos CD24Pos CD19Neg CD20Neg CD3Neg CD16Neg CD61Neg cells) after CD19Neg relapse, suggesting that CD19 loss is associated with the loss of other B cell features. Since our classifier relies on CD19 to classify cells, we compared the resulting classification of cells when CD19 was included or excluded in the classifier. This change had minimal impact in cell classification from healthy bone marrow controls. However, when applied to the samples collected before CAR-T administration, we found a subpopulation of CD19Pos Pro-B cells that classified as Early-non-BI cells when CD19 was excluded from the classification. We hypothesize that these Pro-B "discordant" cells are those that lose CD19 expression to escape the immune pressure exerted by the CD19-directed CAR-T and mediate CD19Neg relapse. Further, we found Pro-B "discordant" cells in 77% of independent cohort of 22 B-ALL samples collected at the time of diagnosis, suggesting these cells exist in de novo B-ALL. We likewise identified a CD19Neg IgMPos Early-non-BI subpopulation in 4 healthy bone marrow and further studies are ongoing to characterize these cells. We continue to interrogate this candidate population as that responsible for CD19Neg relapse after CAR-T cell therapy. In addition, we performed differential expression analysis between paired samples collected before (CD19Pos) and after (CD19Neg) CAR-T therapy. Through the application of the developmental classifier, we identified that CD19 loss is associated with upregulation of key B cell transcription factors IKAROS, PAX5 and glucocorticoid receptor in the pre-pro-B to Pre-B stages. Moreover, after CD19 loss, there are also increases in levels of phosphorylated proteins pSYK, pSRC and pSTAT5, involved in IL7 receptor and pre-BCR signaling pathways, essential for B cell development. This suggests that CD19Neg cells activate unique tumorigenic pathways that may provide novel therapeutic opportunities. Exploration and validation of these therapeutic targets could significantly improve clinical outcome and care of patients with CD19Neg B-ALL. In conclusion, these results support the feasibility to predict patients at risk for CD19Neg relapse together with the mechanism behind it. Future studies will be conducted to confirm unique tumorigenic pathways in CD19Neg B cells and determine their therapeutic potential. Disclosures Mullighan: Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel; Amgen: Honoraria, Other: speaker, sponsored travel; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Loxo Oncology: Research Funding. Grupp:Humanigen: Consultancy; CBMG: Consultancy; Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy.

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.

Efficacy and Safety Outcomes in the Phase 3 INO-Vate Trial By Baseline CD22 Positivity Assessed By Local Laboratories

Efficacy and Safety Outcomes in the Phase 3 INO-Vate Trial By Baseline CD22 Positivity Assessed By Local Laboratories

Gene expression of ASNS, LGMN and CTSB is elevated in a subgroup of childhood BCP-ALL with PAX5 deletion.

Resistance to L-asparaginase (L-asp) is a major contributor to poor treatment outcomes of several subtypes of childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL). Asparagine synthetase (ASNS), legumain (LGMN) and cathepsin B (CTSB) serve a key role in L-asp resistance. The association between genetic subtypes of BCP-ALL and the expression of ASNS, LGMN and CTSB may elucidate the mechanisms of treatment failure. Bone marrow samples of 52 children newly diagnosed with BCP-ALL were screened for major genetic abnormalities and ASNS, LGMN and CTSB gene expression levels. The cohort was further divided into groups corresponding to the key genetic aberrations occurring in BCP-ALL: Breakpoint cluster region and Abelson murine leukemia viral oncogene homolog 1 fusion; hyperdiploidy, hypodiploidy, ETS variant 6 and runt-related transcription factor 1 fusion and other BCP-ALL with no primary genetic aberration identified. A subgroup analysis based on the differences in copy number variations demonstrated a significant increase of ASNS, LGMN and CTSB median expression in other BCP-ALL cases with paired box 5 (PAX5) deletion (P=0.0117; P=0.0036; P<0.0001, respectively) compared with those with wild-type PAX5. Patients with high ASNS expression exhibited longer relapse-free survival (RFS) compared with those with low ASNS levels (P=0.0315; HR, 0.19; 95% CI, 0.04-0.86); the 5-year RFS for patients in the high ASNS expression group was 90.15% (95% CI, 87.90-92.40%). Despite the impact on ASNS, LGMN and CTSB expression, PAX5 deletion did not influence RFS in the other BCP-ALL group (P=0.6839). Therefore, the results of the present study revealed high levels of ASNS, LGMN and CTSB expression in the other BCP-ALL group with concomitant PAX5 deletion and no subsequent deterioration in 5-year RFS. High ASNS expression level, as a single factor, was strongly associated with an improved outcome.

Synergism Through WEE1 and CHK1 Inhibition in Acute Lymphoblastic Leukemia.

Introduction: Screening for synthetic lethality markers has demonstrated that the inhibition of the cell cycle checkpoint kinases WEE1 together with CHK1 drastically affects stability of the cell cycle and induces cell death in rapidly proliferating cells. Exploiting this finding for a possible therapeutic approach has showed efficacy in various solid and hematologic tumors, though not specifically tested in acute lymphoblastic leukemia. Methods: The efficacy of the combination between WEE1 and CHK1 inhibitors in B and T cell precursor acute lymphoblastic leukemia (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the therapeutic strategy was tested in terms of cytotoxicity, induction of apoptosis, and changes in cell cycle profile and protein expression using B/T-ALL cell lines. In addition, the efficacy of the drug combination was studied in primary B-ALL blasts using clonogenic assays. Results: This study reports, for the first time, the efficacy of the concomitant inhibition of CHK1/CHK2 and WEE1 in ALL cell lines and primary leukemic B-ALL cells using two selective inhibitors: PF-0047736 (CHK1/CHK2 inhibitor) and AZD-1775 (WEE1 inhibitor). We showed strong synergism in the reduction of cell viability, proliferation and induction of apoptosis. The efficacy of the combination was related to the induction of early S-phase arrest and to the induction of DNA damage, ultimately triggering cell death. We reported evidence that the efficacy of the combination treatment is independent from the activation of the p53-p21 pathway. Moreover, gene expression analysis on B-ALL primary samples showed that Chek1 and Wee1 are significantly co-expressed in samples at diagnosis (Pearson r = 0.5770, p = 0.0001) and relapse (Pearson r= 0.8919; p = 0.0001). Finally, the efficacy of the combination was confirmed by the reduction in clonogenic survival of primary leukemic B-ALL cells. Conclusion: Our findings suggest that the combination of CHK1 and WEE1 inhibitors may be a promising therapeutic strategy to be tested in clinical trials for adult ALL.

Clinicians' Perspectives on Cure in Adult Patients withAcute Lymphoblastic Leukemia with Minimal Residual Disease: A Delphi Study.

Hematologic complete remission (CR) is achievable for most adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL). However, minimal residual disease (MRD) in patients with hematologic CR is associated with increased risk of relapse, shorter survival, and poorer transplantation outcomes. This study explored the concept of cure in adults with Philadelphia chromosome-negative (Ph−) BCP-ALL by MRD status at first hematologic CR (CR1) to inform evaluation of the clinical and economic benefits of new agents, where the concept of cure is important but long-term data are not available. The study used modified Delphi methodology involving clinicians experienced in the treatment of adult ALL. Participants completed a questionnaire, which was followed by country-specific panel discussions to discuss results and identify consensus on concepts and definitions. Clinicians from France (n = 4), Germany (n = 4), and the UK (n = 5) took part. Participants described cure in terms of the probability of future relapse. Relapse-free survival (RFS) was the preferred outcome measure to describe cure for the three patient groups considered (patients with MRD at CR1; patients who become negative for MRD after further treatment; patients who continue to have MRD). Consensus was reached on definitions of cure: that cure would begin to be considered at 3 years’ RFS and/or would be highly likely at 5 years’ RFS. Participants agreed that patients with MRD should usually undergo hematopoietic stem cell transplantation to have the best chance of survival; consensus was reached that alternatives are required when transplantation is not an option. Panels agreed that patients who achieve cure have a higher mortality rate and lower health-related quality of life than the general population. This study provides quantitative and qualitative information on the concept of cure in Ph− BCP-ALL in CR by MRD status applicable to interpreting the value of new therapies.Funding: Amgen.Plain Language Summary: Plain language summary available for this article.

The prognostic significance of Wilms’ tumor gene 1 (WT1) expression at diagnosis in adults with Ph-negative B cell precursor acute lymphoblastic leukemia

The prognostic significance of Wilms' tumor gene 1 (WT1) expression at diagnosis in adults with B cell precursor acute lymphoblastic leukemia (BCP-ALL) remains poorly understood. A total of 257 adults with Ph-negative BCP-ALL who were consecutively diagnosed and received at least 1 course of induction therapy at our institute were retrospectively analyzed. The WT1 expression patterns were significantly different among the molecularly and cytogenetically defined groups (E2A-PBX1, TEL-AML1, and MLL rearrangements; high hyperdiploidy and B-other). By considering the WT1 expression pattern and the relapse status, 2 cutoff values, 1.8% and 7.2%, were arbitrarily selected to place patients into WT1-low, WT1-inter, and WT1-high groups. In the B-other patients who achieved complete remission (CR), WT1-low and WT1-high patients had similar 3-year relapse-free survival (RFS), disease-free survival (DFS), and overall survival (OS) rates, which were all significantly lower than those of WT1-inter patients. The combined WT1-low/high expression group (n = 132) had significantly lower 3-year RFS, DFS, and OS rates compared with the WT1-inter group (n = 63) of B-other patients (RFS and DFS all P < 0.0001; OS P = 0.0018 and 0.0008). WT1 low/high expression as well as treating with chemotherapy only was independent poor prognostic factors for RFS, DFS, and OS in the B-other patients who achieved CR. Therefore, the molecularly and cytogenetically defined adult Ph-negative BCP-ALL groups have characteristic WT1 expression patterns, and WT1 low/high expression at diagnosis predicts poor outcome in B-other patients.

MLPA as a complementary tool for diagnosis of chromosome 21 aberrations in childhood BCP-ALL

Chromosome 21 abnormalities are the most frequent genetic findings in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cases. Majority of patients are effectively diagnosed with fluorescence in situ hybridization (FISH) and karyotyping; however, some cases may require additional tools to be used. Bone marrow samples of 373 childhood BCP-ALL patients were tested for chromosome 21 copy number variations (CNVs) with Multiplex Ligation-dependent Probe Amplification (MLPA) P327 array. Results from MLPA and cytogenetics were compared between groups according to the type of abnormality found on chromosome 21. Out the group of 235 patients, chromosome 21 multiplication was found by FISH assay in 56 cases (23.81%), ETV6-RUNX1 fusion in 34 (14.47%) and iAMP21 in 3 (1.28%) children, remaining 142 (60.43%) patients had no known chromosome 21 aberration. Median peak ratios of all tested probes in MLPA in aforementioned groups were 1.47 (IQR 1.28–1.77) vs. 1.00 (IQR 1.00–1.09) vs. 2.79 (IQR 1.97–2.83) vs. 1.00 (1.00–1.11), respectively. Aforementioned peak ratio of ETV6-RUNX1 fusion group was similar with patients of no known chromosome 21 aberration (p = 0.71). Interestingly, both groups differed from patients with chromosome 21 multiplication (p < 10−5) and with iAMP21 (p < 10−5). All cases of iAMP21 were correctly recognized by MLPA. MLPA seems to be good additional tool in the diagnostic process of chromosome 21 CNVs, especially in cases with iAMP21.

PF176 MONOCYTIC SWITCH AND DISCREPANCY BETWEEN FLOW CYTOMETRIC AND MOLECULAR MINIMAL RESIDUAL DISEASE ARE FREQUENT IN DUX4 REARRANGED AND PAX5‐P80R MUTATED B‐CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:In 2014, we used flow‐cytometry (FC) to describe a subtype of pediatric B cell precursor acute lymphoblastic leukemia (BCP ALL) with a lineage switch of leukemic blasts into monocytoid cells (swALL) during early treatment (Slamova et al., 2014). Although part of the swALL cases harbored deletion of ERG (ERGdel) and/or IKZF1 alterations, the genetic background remained unknown in the majority of cases. Recently, DUX4‐rearranged (DUX4r) ALL was described as a new subset of BCP ALL. Based on its association with ERGdel, we hypothesized that DUX4r ALL may represent a BCP ALL subtype highly prone to swALL. After the lineage switch, the monocytoid cells lose BCP markers (typically CD19) while they retain Immunoglobulin and T‐cell receptor gene rearrangements (IG/TR), which results in a discrepancy between BCP‐immunophenotype oriented FC‐based and IG/TR‐oriented quantitative (q) PCR‐based minimal residual disease (MRD) detection.Aims:In the current study we aimed to describe the genotypic composition of swALL. Next, we wanted to evaluate in detail the impact of swALL phenomenon on discrepancies between FC‐based and PCR‐based MRD assessment during the induction and consolidation phases of treatment and its homogeneity across genetic/biological ALL subtypes.Methods:Occurrence of swALL phenomenon was assessed in 738 patients consecutively diagnosed between 09/2007‐02/2019 via 8‐color FC using a panel of monoclonal antibodies against BCP and myelomonocytic markers as described previously (Slamova et al., 2014). RNA sequencing (RNAseq) was performed in 126 ALL negative for routinely assessed (cyto)genetic markers in order to classify them into additional genetic/biological subtypes. MRD was assessed via FC and qPCR in peripheral blood at day (d) 8 and in bone marrow at d15, d33 and week (w) 12.Results:We identified swALLs in 59/738 (8%) of ALL cases. The frequency of swALL ranged from 0 to 100% across individual genetic/biological subtypes (Table 1). Compared to non‐swALL, swALL was enriched mainly for DUX4r, PAX5 P80R and ZNF384 subtypes (Figure 1).We observed a poorer MRD correlation in swALL compared to non swALL at d8 PB (0.55 vs 0.81, respectively), d15 BM (0.61 vs 0.9, respectively) and d33 BM (0.35 vs 0.69, respectively). Concordance between MRD results at d15 at the level of 1e‐3 was 97% in DUX4r swALL, but only 17% in PAX5‐P80R swALL. The main cause of discordance was the underestimation of MRD by FC compared to qPCR. The poor correlation of both methods at d15 in PAX5‐P80R indicates rapid loss of B cell phenotype. Correlation of both methods was uneven across ALL subtypes. At d33, 41% and 33% of patients with DUX4r and PAX5‐P80R swALL, respectively, were discordantly called to have MRD ≥ 10‐3 by PCR but not by BCP FC (which could theoretically lead to discordant risk stratification). On the contrary, the MRD categorization using the same cut‐off level at d15 was fully concordant in high‐hyperdiploid and ZNF384r swALLs.Summary/Conclusion:We identified DUX4r and PAX5‐P80R ALL as the most prevalent subtypes among swALL. We showed that the correlation between FC‐ and qPCR‐based MRD is influenced by the treatment time point and genetic background of ALL. Blasts in PAX5‐P80R mutated swALL cases lose B cell antigens early and already at day 15 often majority of blasts is of monocytic phenotype. Loss of B cell phenotype in DUX4r cases is more gradual and at day 15 we observe blasts with decreased expression of CD19 but still clearly positive.imageSupported by NV18‐03‐00343, NV18‐07‐00430, 16‐32568A, UNCE/MED/015

Establishment and characterization of a novel childhood acute lymphoblastic leukemia cell line, HXEX-ALL1, with chromosome 9p and 17p deletions

BackgroundAlthough contemporary chemotherapy has improved the cure rate of childhood acute lymphoblastic leukemia (ALL) to nearly 90%, relapsed/refractory ALL is still a leading cause of tumor-related death in children. To clarify the underlying mechanisms of relapsed/refractory childhood ALL, researchers urgently need to establish novel cell models from patients with relapsed ALL after treatment with contemporary chemotherapy.MethodsCell culture technique was used to establish the HXEX-ALL1 cell line from primary B cell precursor ALL (BCP-ALL) cells. Molecular and cellular biological techniques including flow cytometry, polymerase chain reaction (PCR), short tandem repeat (STR) analysis, conventional cytogenetics, and chromosomal microarray analysis (CMA) were used to characterize the HXEX-ALL1 cell line. Nude mice were used for xenograft studies.ResultsA stable ALL cell line, HXEX-ALL1, derived from a 6-year-old boy of Han nationality with BCP-ALL at the second relapse, was established and maintained in culture for more than 18 months. The HXEX-ALL1 cell line was authenticated as being derived from primary leukemia cells based on morphologic, immunophenotypic, cytogenetic and STR analyses and demonstrated tumorigenicity in nude mice. WGS data showed that there were 27,006 novel single nucleotide polymorphisms (SNPs) and 193,951 novel insertion/deletions (InDels) in HXEX-ALL1 cells. Compared with the other BCP-ALL cell lines in use, the HXEX-ALL1 cells have a special karyotype represented by trisomy 8 and 9p and 17p deletions with a multidrug resistance phenotype, especially highly resistant to asparaginase.ConclusionsThe HXEX-ALL1 cell line may prove to be a useful model for the study of relapsed/refractory childhood ALL, particularly for the researches on asparaginase resistance.