Hypodiploid Acute Lymphoblastic Leukemia Research Articles

Scylla and Charybdis: Unpalatable choices in managing hypodiploid acute lymphoblastic leukemia

Acquired TP53 alterations are present in > 90% of cases of paediatric low-hypodiploid acute lymphoblastic leukaemia (ALL), and ≈ 50% of patients with this subtype harbor germline pathogenic / likely pathogenic (P/LP) TP53 alterations. Despite dose intensified, conventional chemotherapy, survival in low-hypodiploid ALL remains dismal compared to other paediatric ALL. Individuals with underlying Li-Fraumeni Syndrome (LFS) are known to have increased sensitivity to genotoxic effect of chemotherapy and radiotherapy. Recent evidence shows a 25.1% 5-year cumulative incidence of SMNs post ALL therapy in an LFS population as compared to 0.7% in patients with either a wild type or VUS TP53. The parallel high risks of both relapse and SMN present unpalatable choices facing clinicians.

Venetoclax and dinaciclib elicit synergistic preclinical efficacy against hypodiploid acute lymphoblastic leukemia.

Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive blood cancer with a poor prognosis despite intensive chemotherapy or stem cell transplant. Children and adolescents with positive end-of-induction minimal residual disease have an overall survival lower than 30%. However, data regarding therapeutic alternatives for this disease is nearly nonexistent, emphasizing the critical need for new or adjunctive therapies that can improve outcomes. We previously reported on the therapeutic efficacy of venetoclax (ABT-199) in hypodiploid B-lineage ALL but with limitations as monotherapy. In this study, we set out to identify drugs enhancing the anti-leukemic effect of venetoclax in hypodiploid ALL. Using a highthroughput drug screen, we identified dinaciclib, a cyclin-dependent kinase inhibitor that worked synergistically with venetoclax to induce cell death in hypodiploid cell lines. This combination eradicated leukemic blasts within hypodiploid ALL patient-derived xenografts mice with low off-target toxicity. Our findings suggest that dual inhibition of BCL-2 (venetoclax) and CDK9/MCL-1 (dinaciclib) is a promising therapeutic approach in hypodiploid ALL, warranting further investigation to inform clinical trials in this high-risk patient population.

Prognostic factors and outcomes of infant acute lymphoblastic leukemia (ALL), hypodiploid ALL, and mixed-phenotype acute leukemia (MPAL) in Canada: a report from CYP-C

The epidemiology of infant acute lymphoblastic leukemia (ALL), hypodiploid ALL, and mixed-phenotype acute leukemia (MPAL) in Canada is unknown. The main objective was to describe the prevalence, prognostic factors, and outcomes of three rare and high-risk ALL subtypes in Canada. This is a retrospective study using the Cancer in Young People-Canada (CYP-C) database. Event-free survival (EFS) and overall survival (OS) were described by the Kaplan–Meier method and compared using the log-rank test. Among 2626 children aged 0–14 years diagnosed with B-cell acute lymphoblastic leukemia (B-ALL) between 2001 and 2018, 227 (8.6%) patients were identified to be infant ALL (n = 139), hypodiploid ALL (n = 43), or MPAL (n = 45). The 5-year EFS/OS was significantly worse in the infant ALL subgroup compared to that of hypodiploid ALL and MPAL. For the entire cohort, presenting White blood cells (WBCs) ≥50 × 109/L was independently associated with worse EFS/OS.

Nationwide Survey of Pediatric Hypodiploid Acute Lymphoblastic Leukemia in Japan

BackgroundPloidy is a highly significant prognostic factor in childhood acute lymphoblastic leukemia (ALL). Children with hypodiploid ALL continue to have inferior outcomes despite current intensive chemotherapy treatment. Hypodiploid ALL is divided into two groups based on genetic alterations. Near-haploid ALL with 24-31 chromosomes harbors alterations targeting receptor tyrosine kinase signaling, RAS signaling, and the lymphoid transcription factor gene IKZF3 and low-hypodiploid ALL with 32-39 chromosomes is characterized by alterations in TP53 that are commonly present in non-tumor cells, IKZF2 and RB1 . There is little information about children with hypodiploid ALL in Asia including Japan, although ethnic differences seem to exist. The aim of this study was to clarify the clinical characteristics and outcome of pediatric hypodiploid ALL in Japanese patients.MethodsPatients with fewer than 46 chromosomes were defined as hypodiploid ALL in this study. All reports of chromosomal analysis were re-reviewed by two independent investigators and 117 cases of hypodiploid ALL were identified out of 4261 patients who were registered in clinical trials between 1997 and 2012. A total of 64 patients were excluded due to the lack of follow-up data (n = 39), only a single cell exhibiting a chromosome number less than 46 in G-banding experiments (n = 18), incorrect diagnosis (n = 4), or congenital chromosomal aberrations such as Down syndrome (n = 1) and Robertson translocations (n = 2). Seventeen patients whose karyotypes at remission were not evaluable were also excluded.ResultsIn total 66 male (56.4%) and 51 female (43.6%) patients' data was evaluated with a median age of 6 years (range 1 to 15 years). There were 101, 8, 2, and 3 patients with chromosome numbers of 45, 44, 43, and 36, respectively. One patient exhibited each with 27, 28, and 29 chromosomes. No patients had a modal chromosome number of 30-35 or 37-42. Among the 117, 110 (94%) had B-precursor ALL and 105 (90%) had CD10 positivity. All T-ALL patients (n = 7) had 45 chromosomes. Out of 117 patients, 22 were TEL-AML1 fusion positive of which 19 had 45 chromosomes, 2 had 44 chromosomes, and 1 had 43 chromosomes. There were no patients with central nervous system (CNS) infiltration at the time of diagnosis. All but one patient who was diagnosed with T-ALL achieved a complete remission (CR) with a CR rate of 98.3%. Overall survival at 5 years varied significantly at 86.0%, 87.5%, and 62.5% in patients with 45, 44, and fewer than 44 chromosomes, respectively (P=0.037). There were no patients with CNS relapse in both the 44 and fewer than 44 chromosomes groups. Five patients out of 8 with fewer than 44 chromosomes had bone marrow relapse, even though 1 patient had undergone hematopoietic stem cell transplantation (HSCT) during his first CR. Four patients underwent HSCT after relapse, but only 1 was cured. In the 44 chromosome group, 2 of 8 patients underwent HSCT during their first CR and did not show relapse. However, one of them developed secondary renal cell carcinoma 10 years after treatment. One patient with 44 chromosomes underwent HSCT after relapse and failed to be salvaged. We also analyzed some variables that influenced event-free and overall survival in the 45 chromosome group: monosomy 7, sex chromosome abnormality, and 9p21.3 deletion. We were not able to identify any significant prognostic factor among patients with 45 chromosomes.ConclusionsAlthough the number of patients was small, patients with 44 chromosomes without HSCT showed better prognosis than those reported previously. However, the outcome of Japanese patients with fewer than 44 chromosomes was as poor as previously reported in other countries. The effect of HSCT in patients with hypodiploid ALL needs to be the subject of future studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TP53 Germline Variations Influence the Predisposition and Prognosis of Acute Lymphoblastic Leukemia in Children

Germline TP53 mutations is the genetic basis of Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in children with hypodiploid acute lymphoblastic leukemia (ALL) suggest that childhood leukemia is another manifestation of LFS. However, the pattern, prevalence, and clinical relevance of pathogenic variants in TP53 in childhood ALL remain unknown.To comprehensively characterize genetic variants in TP53 in childhood ALL, we performed targeted sequencing of TP53 coding regions in 3858 children from the Children's Oncology Group front-line ALL clinical trials (COG AALL0232 and P9900). In total, 49 different non-silent coding rare TP53 variants were identifiedin 77 (2.0%) patients sequenced, including 40 missense, 1 nonsense, 6 frameshift, and 2 inframe deletion variants. To determine which TP53 variants are potentially pathogenic and related to ALL risk, we examined their experimentally-validated p53 transcriptional activity, bioinformatically-predicted damaging effects on TP53 function, and also the variant frequency in non-ALL populations. Twenty-two TP53 variants were classified as ALL risk-related (VAR), including 7 protein-truncating and 15 missense variants, all of which were either absent or exceedingly rare in non-ALL populations (i.e., allele frequency < 0.006% in the ExAC dataset). Fourteen missense VAR reside in the p53 core DNA-binding domain, with two affecting residues essential for DNA contact and seven located at residues that are important for the overall architecture of the DNA-binding surface. The 27 remaining TP53 variants were classified as “variants of unknown significance” (VUS). Together, of 3858 children in this ALL cohort, 26 (0.67%) patients had a predicted VAR and 51 (1.3%) had a VUS in the TP53 gene.We next evaluated the association of germline TP53 variants with patient demographics and ALL tumor genetic characteristics. Children with a TP53 VAR were significantly older at diagnosis (P < 0.0001), and had significantly lower leukocyte count at presentation than those with a VUS or wild-type (WT) genotype (P= 0.006). Of 26 patients carrying a germline TP53 VAR, 17 (65.4%) exhibited hypodiploidy in ALL blasts (mostly low hypoploidy or masked hypoploidy). In contrast, hypodiploid ALL was only present in 3.9% and 1.2% of children with TP53 VUS or WT genotype, respectively. The prevalence of TP53 VAR did not differ by genetic ancestry (P= 0.9). We also examined the relationship between germline TP53 variants and treatment outcomes of ALL therapy. In the COG AALL0232 cohort, the presence of a TP53 VAR was associated with a significantly lower event free survival (EFS) and overall survival (OS) compared to patients without a risk variant (hazard ratio [HR] of 2.84, P= 0.0007 for EFS; and HR of 3.13, P= 0.003 for OS). In multivariate analyses, TP53 VAR remained prognostic after adjusting for ancestry, age, leukocyte count at diagnosis, and minimal residual disease at the end of remission induction therapy (HR of 3.36, P= 0.0002 for EFS; HR of 2.88, P= 0.007 for OS). Importantly, there was a markedly higher risk of second cancers among patients with TP53 VARs compared to those with TP53 VUS or WT TP53: 5-year cumulative incidence of 25.1% vs. 0.7%, P= 5.4 × 10-11. In the COG P9900 cohort, only 4 patients harbored TP53 VAR, of whom 3 cases experienced adverse events (one patient each with ALL relapse, second cancer, or death in remission), and both EFS and OS were significantly worse than patients with TP53 VUS or WT genotype (HR of 7.14, P= 0.0008 for EFS; HR of 14.19, P= 1.1 x 10-5 for OS). Taken together, 14 of 26 patients with a TP53 VAR experienced adverse events, with 5 ALL relapses (36% of all events) and 5 second cancers (36%). This pattern of adverse events was dramatically different from those in patients with WT TP53 or VUS, for whom ALL relapse accounted for 75% of all events with only 4% as second cancers (P= 1.2 × 10-7). In fact, even within hypodiploid ALL patients who experienced adverse events, the frequency of second cancer was significantly higher in those with a TP53 VAR than those without (50% vs 5%, P= 0.01), further suggesting that germline TP53 variation was the underlying cause of second cancers in these cases.In conclusion, our findings identified germline TP53 variants as an important determinant of ALL predisposition and adverse treatment outcomes, particularly the risk of second malignant neoplasms. DisclosuresBorowitz:Beckman Coulter: Honoraria; Becton-Dickinson Biosciences: Research Funding; HTG Molecular: Honoraria. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Hunger:Jazz Pharmaceuticals: Honoraria; Novartis: Consultancy; Erytech Pharmaceuticals: Consultancy; Amgen: Consultancy, Equity Ownership.

Clinical significance of novel subtypes of acute lymphoblastic leukemia in the context of minimal residual disease-directed therapy.

We evaluate clinical significance of recently identified subtypes of acute lymphoblastic leukemia (ALL) in 598 children treated with minimal residual disease (MRD)-directed therapy. Among the 16 B-ALL and 8 T-ALL subtypes identified by next generation sequencing, ETV6-RUNX1, high-hyperdiploid and DUX4-rearranged B-ALL had the best five-year event-free survival rates (95% to 98.4%); TCF3-PBX1, PAX5alt, T-cell, ETP, iAMP21, and hypodiploid ALL intermediate rates (80.0% to 88.2%); and BCR-ABL1, BCR-ABL1-like and ETV6-RUNX1-like and KMT2A-rearranged ALL the worst rates (64.1% to 76.2%). All but three of the 142 patients with day-8 blood MRD <0.01% remained in remission. Among new subtypes, intensified therapy based on day-15 MRD≥1% improved outcome of DUX4-rearranged, BCR-ABL1-like, and ZNF384-rearranged ALL, and achievement of day-42 MRD<0.01% did not preclude relapse of PAX5alt, MEF2D-rearranged and ETV6-RUNX1-like ALL. Thus, new subtypes including DUX4-rearranged, PAX5alt, BCR-ABL1-like, ETV6-RUNX1-like, MEF2D-rearranged and ZNF384-rearranged ALL have important prognostic and therapeutic implications.

Incidence and Therapeutic Implications of Germline TP53 Mutations in Hypodiploid Childhood Acute Lymphoblastic Leukemia: A Retrospective Analysis of the Italian Cohort

Background. Hypodiploid Acute Lymphoblastic Leukemia (ALL) is a rare subtype of childhood ALL known to be associated with a poor prognosis. Currently, intensive chemotherapy and haematopoietic stem cell transplantation are recommended as the main therapeutic strategy for these patients. Previous studies showed that low-hypodiploid ALL frequently carry TP53 variants, the majority of which are germline mutations. This finding suggests that hypodiploid ALL may be a manifestation of underlying Li-Fraumeni syndrome, with an associated high risk for secondary tumors. We analyzed TP53 variants in the Italian cohort of hypodiploid pediatric ALL patients diagnosed between 2000 and 2019, to explore frequency and characteristics, as well as the potential role of germline variants in the therapeutic strategy. Patients were treated according to the AIEOP-BFM ALL2000 (n=16), ALL2009 (n=16) and ALL2017 (n=8) protocols. Methods. We performed a targeted Next Generation Sequencing (NGS) Nextera Flex DNA panel of 40 genes, including TP53, in a retrospective series of hypodiploid paediatric ALL Italian patients enrolled in four nationwide frontline protocols. Ploidy was defined based on DNA index (DNAi) and/or cytogenetics/FISH, and cases with DNAi&amp;lt;0.8 were included in this study. Only TP53 variants with VF &amp;gt;5% and coverage 500X were considered. Bioinformatics analysis has been carried out by Sophia DDM software. NGS has been performed both in disease (hematopoietic) and germline (remission or buccal brush) tissues samples. Results. Nineteen TP53 variants were observed in 20/40 (50%) hypodiploid ALL patients, with 19/20 being low-hypodiploid ALL cases (DNAi between 0,6 and 0,8); 13/19 TP53 variants were known to be pathogenic whereas 6/19 were classified as Variant of Unknown Significance (VUS). Considering the VUS variants, 3/6 were missense, 1/6 frameshift and 2/6 in-frame ins/del. Notably, 13 patients out of 20 (65%) were found to carry a germline variant, while 5 patients presented a somatic variant; in 2 cases the remission sample was not available, but the VF&amp;lt;20% was indicative of a somatic variant. Among the germline variants, 1 was found to be a germline mosaicism, presenting a nonsense pathogenic TP53 variant both in remission (VF 10%) and buccal brush sample at remission (VF 8.5%). 16/19 variants reside in the p53 core DNA binding domain, known to be a fundamental site that mediates the transcription of p53 regulated genes, where most of the pathogenic mutations in cancer cells occur. The 3 variants outside the DNA binding domain were 1 nonsense, 1 frameshift and 1 missense, and were localized in the nuclear localization signal region (NLS) or in the oligomerization domain (OD). Considering the 32 patients with at least 5 years of follow-up, EFS was not significantly different between TP53 wild type (47.1% ± 12.1) vs TP53 variant (51.9% ± 17,8) subgroups. According to MRD stratification criteria, the patients carrying a TP53 variant were classified as medium risk in 14/20, standard risk in 5/20 and HR in 1 case, not statistically different from TP53 wild type. Collection of data on secondary neoplasms in patients and their relatives in ongoing. Conclusion. Our data represent the largest low hypodiploid patient cohort tested so far, confirming the high frequency of deleterious TP53 mutations. Mutational testing of TP53 in patients with hypodiploid ALL is warranted, in order to ensure a proper genetic counseling for patients with germline mutations and their families, with tailored clinical surveillance (according to Li-Fraumeni Syndrome guidelines). The best therapeutic regimen for hypodiploid patients is still a matter of debate, with MRD at the end of induction and TP53 germline mutations potentially indicating a highest relevance in the indication to hematopoietic stem cell transplantation. Chemotherapy-only treatment could be indicated for MRD-low patients and HSCT for MRD-High, potentially considering a reduced conditioning regimen for TP53 mutated cases. Disclosures Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Rizzari:Sobi: Consultancy, Other: Advisory Board.

Nationwide survey of pediatric hypodiploid acute lymphoblastic leukemia in Japan.

Ploidy is a highly significant prognostic factor for pediatric acute lymphoblastic leukemia (ALL). Children with hypodiploid ALL have poor outcomes despite current intensive chemotherapy. Little has been investigated with regard to hypodiploid ALL in Japanese children. We retrospectively collected clinical data on hypodiploid ALL cases from the registries of prospective multicenter trials conducted by the four independent clinical study groups in Japan between 1997 and 2012. A total of 117 ALL patients with hypodiploidy were analyzed in this study. There were 101, eight, and eight patients with 45, 44, and fewer than 44 chromosomes, respectively. The 5year overall survival rates differed significantly: 86.0%, 87.5%, and 62.5% for patients with 45, 44, and fewer than 44 chromosomes, respectively (P = 0.037). Of the eight patients with 44 chromosomes, seven were alive, including five patients who maintained complete remission without undergoing hematopoietic stem cell transplantation (HSCT). Of the eight patients with fewer than 44 chromosomes, six were good responders to prednisolone and none had induction failure, but the relapse rate was high (5/8). No patients had central nervous system relapse. Four patients underwent HSCT after relapse, but only one survived. Outcomes of Japanese ALL patients with fewer than 44 chromosomes were poor, as previously reported in other countries. Although the sample size was small, patients with 44 chromosomes had better prognoses than those previously reported. Further studies including international collaboration are needed to improve outcomes for pediatric ALL patients with fewer than 44 chromosomes.

Masked hypodiploidy: Hypodiploid acute lymphoblastic leukemia (ALL) mimicking hyperdiploid ALL in children: A report from the Children's Oncology Group

Hyperdiploidy with greater than 50 chromosomes is usually associated with favorable prognosis in pediatric acute lymphoblastic leukemia (ALL), whereas hypodiploidy with ≤43 chromosomes is associated with extremely poor prognosis. Sometimes, hypodiploidy is “masked” and patients do not have a karyotypically visible clone with ≤43 chromosomes. Instead, their abnormal karyotypes contain 50–78 or more chromosomes from doubling of previously hypodiploid cells. When the hypodiploid and doubled hyperdiploid clones are both present, patients can be identified by traditional test methods [karyotype, DNA Index (DI), fluorescence in situ hybridization (FISH)], but the incidence of masked hypodiploid cases in which only the doubled clone is visible is unknown. We analyzed 7013 patients with B-ALL enrolled in COG AALL03B1 (2003–2011) for whom chromosome studies were available. Of 115 patients with hypodiploidy (25–39 chromosomes), karyotypes of 40 showed only the hypodiploid clone, 47 showed mosaicism with both hypodiploid and hyperdiploid (doubled) karyotypes, and 28 with masked hypodiploidy showed only a hyperdiploid (doubled) clone. Unique karyotypic signatures were identified, and widespread loss of heterozygosity (LOH) was seen in the microsatellite panel for all patients with masked hypodiploidy. An increased awareness of the unusual karyotypic profile associated with a doubled hypodiploid clone and coordinated use of DI, FISH, and LOH studies when indicated can identify patients with masked hypodiploidy and allow appropriate treatment selection.

Hematologic malignancies and Li-Fraumeni syndrome.

Li–Fraumeni syndrome (LFS) is an autosomal dominant condition associated with a high risk of a broad range of childhood- and adult-onset cancers. LFS is related to germline mutations of the tumor-suppressor gene TP53. The most common reported leukemia associated with LFS is hypodiploid acute lymphoblastic leukemia, but myeloid malignancies including acute myeloid leukemia (AML), chronic myeloid leukemia, and myelodysplastic syndrome (MDS) are also reported, often in the setting of therapy-related disease. We reviewed the clinicopathologic characteristics including cytogenetics and molecular analysis for seven adult patients with LFS and hematologic malignancies evaluated at the Hereditary Hematologic Malignancy Clinic (HHMC) at MD Anderson Cancer Center. We present this LFS review series to increase awareness of LFS for the appropriate diagnosis of both patients and potentially affected relatives, as well as provide experience with patient outcomes in this difficult to treat population.

TP53 Germline Variations Influence the Predisposition and Prognosis of B-Cell Acute Lymphoblastic Leukemia in Children.

Purpose Germline TP53 variation is the genetic basis of Li-Fraumeni syndrome, a highly penetrant cancer predisposition condition. Recent reports of germline TP53 variants in childhood hypodiploid acute lymphoblastic leukemia (ALL) suggest that this type of leukemia is another manifestation of Li-Fraumeni syndrome; however, the pattern, prevalence, and clinical relevance of TP53 variants in childhood ALL remain unknown. Patients and Methods Targeted sequencing of TP53 coding regions was performed in 3,801 children from the Children's Oncology Group frontline ALL clinical trials, AALL0232 and P9900. TP53 variant pathogenicity was evaluated according to experimentally determined transcriptional activity, in silico prediction of damaging effects, and prevalence in non-ALL control populations. TP53 variants were analyzed for their association with ALL presenting features and treatment outcomes. Results We identified 49 unique nonsilent rare TP53 coding variants in 77 (2.0%) of 3,801 patients sequenced, of which 22 variants were classified as pathogenic. TP53 pathogenic variants were significantly over-represented in ALL compared with non-ALL controls (odds ratio, 5.2; P < .001). Children with TP53 pathogenic variants were significantly older at ALL diagnosis (median age, 15.5 years v 7.3 years; P < .001) and were more likely to have hypodiploid ALL (65.4% v 1.2%; P < .001). Carrying germline TP53 pathogenic variants was associated with inferior event-free survival and overall survival (hazard ratio, 4.2 and 3.9; P < .001 and .001, respectively). In particular, children with TP53 pathogenic variants were at a dramatically higher risk of second cancers than those without pathogenic variants, with 5-year cumulative incidence of 25.1% and 0.7% ( P < .001), respectively. Conclusion Loss-of-function germline TP53 variants predispose children to ALL and to adverse treatment outcomes with ALL therapy, particularly the risk of second malignant neoplasms.

TP53 Mutations in Hypodiploid Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemia (ALL) is an aggressive neoplasm of B- or T-lymphoid progenitors and is the commonest childhood tumor. ALL comprises multiple subtypes characterized by distinct genetic alterations, with stereotyped patterns of aneuploidy present in many cases. Although alterations of TP53 are common in many tumors, they are infrequent in ALL, with the exception of two ALL subtypes associated with poor outcome: relapsed disease and ALL with hypodiploidy. TP53 alterations are present in almost all cases of ALL with low hypodiploidy and are associated with alterations of the lymphoid transcription factor IKZF2 and the tumor-suppressor gene loci CDKN2A and CDKN2B. Remarkably, more than half of TP53 mutations in low-hypodiploid ALL in children are present in nontumor cells, indicating that low-hypodiploid ALL is a manifestation of Li-Fraumeni syndrome. These findings have profound implications for our understanding of the genetic pathogenesis of hypodiploid ALL, suggesting that alteration of TP53 function may promote the distinctive aneuploidy characteristic of hypodiploid ALL. Moreover, the identification of hypodiploidy mandates offering testing for TP53 mutational status to patients and their relatives, with appropriate counseling and disease surveillance.

PI3K and MEK Inhibition in Hypodiploid Acute Lymphoblastic Leukemia

Introduction: Hypodiploid acute lymphoblastic leukemia (ALL) is a rare, high-risk subtype of B-ALL associated with poor outcome. Genomic analysis of over 130 Hypodiploid ALL patients defined the distinct genomic landscape of this disease, defining two principal subtypes including Ras-activating alterations and IKZF3 loss in near haploid tumors (24-31 chromosomes) and mutation of TP53 coupled with homozygous IKZF2 loss in low hypodiploid tumors (32-39 chromosomes). New therapies are needed to improve outcome of this disease, necessitating preclinical studies in hypodiploid tumors in vitro, ex vivo and in vivo.Hypothesis: The observation of frequent Ras pathway activating mutations in hypodiploid ALL tumors, as well as increased phosphorylation of downstream Ras signaling targets, suggests that these tumors may be susceptible to PI3K/MEK inhibition. Ras signaling target activation was observed in hypodiploid tumors irrespective of mutational status. Here we used cell lines and representative xenograft models to explore the therapeutic efficacy of inhibiting PI3K/MAPK signaling in hypodiploid ALL.Methods: The near haploid cell lines NALM-16 and MHH-CALL-2 and eight patient-derived xenograft (PDX) models (four near haploid and four low hypodiploid) were selected for preclinical studies. Xenografts were representative of the most common genomic lesions observed in hypodiploid ALL patients, including deletion of NF1, PAG1 and IKZF3 in near haploid and mutation of TP53 and IKZF2 deletion in low hypodiploid (Holmfeldt et al. Nat Genet 2013; Mullighan et al. Blood 2015). Next-gen sequencing of xenograft and diagnosis tumor DNA confirmed the presence of key genetic alterations in the xenografts. A lentiviral vector was used for stable expression of luciferase in xenograft tumors for in vivo disease burden monitoring. Cell lines were treated in vitro with PI3K, mTOR, MEK, CDK4/6 and BET inhibitors either as single agents, inhibitor combinations or combined with chemotherapeutic agents. Xenografts were treated ex vivo with single agents and in vivo with PI3K and MEK inhibitors with or without chemotherapy.Results: NALM-16 and MHH-CALL-2 cell lines were most sensitive to PI3K, mTOR and BET inhibitors with IC50 values in the sub-micromolar range (Fig. 1).Treatment of xenografts ex vivo showed near haploid and low hypodiploid tumors to be four-fold more sensitive to pan-PI3K inhibitors and PI3K/mTOR dual inhibitors compared to PI3K α and d isoform-specific inhibitors. Near haploid and low hypodiploid xenografts were sensitive to MEK and BET inhibition ex vivo, but MEK inhibitors showed weak activity in the near haploid cell lines. Treatment with CDK4/6 inhibitors either as single agents or in combination with MEK inhibitors revealed minimal efficacy. Changes in cell signaling upon treatment was assayed by phosphoflow (Fig. 2). Akt and Erk1/2 phosphorylation was variable between tumors, whereas 4E-BP1 and S6 phosphorylation was consistently elevated, suggesting strong signaling through mTOR pathways. Pan-PI3K and PI3K/mTOR dual inhibitors showed potent activity against hypodiploid ALL tumors in vitro and ex vivo, however PI3K inhibition alone was insufficient to eliminate tumors in vivo. Substantial weight loss in mice treated with PI3K/mTOR dual inhibitors limited prolonged study in vivo. MAPK inhibition showed activity in both near haploid and low hypodiploid tumors ex vivo and displayed a modest reduction in tumor growth as a single agent in vivo. Combining the MEK inhibitor GDC-0973 with dexamethasone treatment increased efficacy, but induced significantly more weight loss than either monotherapy.Conclusion: Treatment of hypodiploid cell lines and xenografts in vitro and ex vivo, respectively, with PI3K and MEK inhibitors showed promising results, however the anti-tumor effect in vivo appeared mostly cytostatic and inhibitors were not sufficient as single agents to kill the tumor cells. MEK inhibitors showed stronger activity than PI3K inhibitors on xenografts in vivo and ex vivo, but this was not significantly increased when combined with dexamethasone treatment. The notable resistance of hypodiploid cells to dexamethasone may underlie this limited efficacy in addition to the increase in GDC-0973 clearance observed upon combination. Our findings also highlight the need to consider the PK properties of agents in the preclinical setting, particularly in combinations. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BCL-2, a Therapeutic Target for High Risk Hypodiploid B-Cell Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Specific genetic subsets, including hypodiploid ALL, are associated with particularly high rates of relapse. Despite the poor outcomes of hypodiploid B-ALL with traditional therapeutic approaches, there have been no known effective alternative therapies or novel candidates tested to improve outcome. We hypothesized that new therapeutic targets could by identified by integrated biochemical and genomic profiling, combined with functional drug assays in order to determine which pathways play an essential role in transformation. For biochemical profiling, we analyzed multiple pathways commonly deregulated in leukemias using phosphoflowcytometry (including receptor tyrosine kinases, JAK/STAT, MAPK, PI3K, PTEN, Bcl-2 survival and pro-apoptotic family members and p53). We subjected hypodiploid cell lines (NALM-16, MHH-CALL2) and patient derived xenograft samples in vitro to inhibitors against each of these pathways (PP2:Src family;Ruxolitinib: JAK/STAT; PD235901/CI1040: MAPK; GDC-0941, PI-90, PI-103, p110 (a, b, g, d): PI3K isoform specific; PP-242:mTOR; ABT-263/ABT-737: Bcl-2/Bcl-xl, and ABT-199: Bcl-2 specific). We found that the Bcl-2 inhibitors (ABT-263, ABT-737 and ABT-199) and to a lesser extent PI3K pathway inhibitors GDC-0941 and PP-242, but not the MAPK or RTK inhibitors, efficiently reduced proliferation of hypodiploid cells. However, only ABT-263/ABT-199 induced high levels of apoptosis at nanomolar concentrations. Based on the consistent efficacy observed with ABT-199 against hypodiploid patient-derived cells and cell lines in culture, we selected eight cryopreserved, previously xenografted (F3 generation) hypodiploid patient samples (4 low hypodiploid, chromosomal number between 32 and 39; and 4 Near Haploid, chromosomal number between 24 and 31) and three non-hypodiploid patient samples (Ph-positive,Ph-Like and Erg+) for a preclinical trial in immunodeficient mice. Each patient sample was engrafted into six mice, which were randomized to receive vehicle or ABT-199 daily over 60 days (Figure 1). Treatment started when the peripheral blood (PB) human CD45 count reached 15%. A rapid decrease in PB blasts was noted at 7 days (Figure 1). Eighty-five percent of the hypodiploid xenografts survived 60 days with either undetectable or low levels of leukemia in the PB. In contrastPh+ andPh-Like xenografts died within 10-20 days regardless of treatment. Importantly, hypodiploid leukemic blasts gradually emerged after discontinuing ABT-199 after 60 days. Additionally, despite low or undetectable levels of leukemic blasts in PB and reduced levels in bone marrow and spleen, all mice had high percentages of leukemic cells in the liver (Figure 2).In conclusion we have identified the survival protein Bcl-2 as a promising molecular target in hypodiploid B-ALL. ABT-199 for dramatically reduced leukemia cells in vitro and in vivo in patient-derived xenograft models of hypodiploid B-ALL. However, the liver represented a protective niche for these leukemias. In addition, our biochemical characterization of the organ infiltrating blasts collected from mice on trial indicate that the sensitivity of hypodiploid ALL to ABT-199 relies not only on high levels of Bcl-2 and deficiency for other survival proteins such as Bcl-xl but also on high levels of proapoptotic proteins, providing two different signatures that correlate with response to ABT-199. Using genome editing (CRISPR/Cas9) we interrogated the necessity for individual proapoptotic genes, including PUMA, NOXA, and BAD, for ABT-199-induced cell death. This study provides encouraging preclinical data that Bcl-2 may be a promising target for the treatment of hypodiploid B-ALL. Our studies identify signature biomarkers that correlate with drug response and identify essential proteins mediating ABT-199-induced cell death. Importantly, this report also identifies the limitations of using ABT-199 as single drug, and provides the rationale for using combinatorial therapies in order to improve the efficacy of the drug. [Display omitted] [Display omitted] DisclosuresMullighan:Loxo Oncology: Research Funding; Amgen: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees. Loh:Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding.

Pharmacological activation of wild-type p53 in the therapy of leukemia.

The tumor suppressor p53 is inactivated by mutations in the majority of human solid tumors. Conversely, p53 mutations are rare in leukemias and are only observed in a small fraction of the patient population, predominately in patients with complex karyotype acute myeloid leukemia or hypodiploid acute lymphoblastic leukemia. However, the loss of p53 function in leukemic cells is often caused by abnormalities in p53-regulatory proteins, including overexpression of MDM2/MDMX, deletion of CDKN2A/ARF, and alterations in ATM. For example, MDM2 inhibits p53-mediated transcription, promotes its nuclear export, and induces proteasome-dependent degradation. The MDM2 homolog MDMX is another direct regulator of p53 that inhibits p53-mediated transcription. Several small-molecule inhibitors and stapled peptides targeting MDM2 and MDMX have been developed and have recently entered clinical trials. The clinical trial results of the first clinically used MDM2 inhibitor, RG7112, illustrated promising p53 activation and apoptosis induction in leukemia cells as proof of concept. Side effects of RG7112 were most prominent in suppression of thrombopoiesis and gastrointestinal symptoms in leukemia patients. Predictive biomarkers for response to MDM2 inhibitors have been proposed, but they require further validation both invitro and invivo so that the accumulated knowledge concerning pathological p53 dysregulation in leukemia and novel molecular-targeted strategies to overcome this dysregulation can be translated safely and efficiently into novel clinical therapeutics.

Attenuation of Apoptosis-Stimulating Protein of p53-2 (ASPP2) Promotes IL-3-Independent Growth and Enhances Cytogenetic Abnormalities after Gamma-Irradiation in Mouse Hematopoietic Cells - Mimicking Early Leukemogenesis of Therapy-Associated Human AML

Inactivation of tp53 by chromosome/gene loss or loss-of function mutation is frequently observed in therapy-associated acute myeloid leukemia. Tp53 mutations associate with complex and monosomal karyotypes and provide the strongest predictive risk factor in this subgroup. ASPP2 is an independent tumor suppressor. We have previously shown that ASPP2 expression is attenuated in acute leukemia via methylation by unknown mechanisms. We now provide evidence that attenuation of ASPP2 expression may act as an early leukemia-initiating event - facilitating acquisition of structural chromosomal aberrations and loss of its binding partner p53.We found low ASPP2 mRNA and protein expression levels in complex karyotype AML, arguing for a role of ASPP2 loss in leukemogenesis. To evaluate for malignant transformation we engineered IL3-dependant murine Ba/F3 cells and stably silenced ASPP2 expression using retroviral-induced shRNA, Interestingly, only ASPP2(ko) cell strains could be successfully weaned from IL3 dependence - suggesting that loss of ASPP2 expression promoted cellular transformation. In contrast, empty vector control cells did not survive the weaning process. As expected, cellular proliferation was enhanced in the ASPP2(ko) cell lines as measured by standard XTT and CSFE protocols. To study how loss of ASPP2 could promote transformation, we performed cytogenetic analysis of the chromosome set of parental (+IL3) vs. gamma-irradiated empty vector controls (+IL3) and ASPP2(ko) cell strains (-IL3) with and without irradiation (+/- stress enforcement using gamma irradiation, 2 x 5Gy). Gamma irradiation did not significantly alter cytogenic analysis between the empty vector controls compared to the parental cells. Tantalizingly however, we found a consistent chromosomal loss of chr8, der8 and der19 - and, most intriguingly, loss of chromosome 11 (which locates tp53 in mice), and a monosomal karyotype in the ASPP2-attenuated cells. In addition, several marker chromosomes (6-8 in ASPP2i vs. 0-2 in empty vector controls were identified. We are now in the process of verifying our findings in knockout native human bone marrow blasts.Conclusions: Our findings provide novel evidence that loss of ASPP2 contributes to malignant transformation by promoting genomic instability as measured by increased cytogenetic abnormalities after gamma-irradiation. These findings may have important relevance for the pathogenesis of therapy-associated AML - but also of hypodiploid acute lymphoblastic leukemia, which is as well associated with tp53 loss-of-function and predicted for a dismal outcome. Studies are ongoing to elucidate the mechanisms of how loss of ASPP2 expression promotes human AML, as well as restore ASPP2 function as a novel therapeutic pathway to exploit in treating AML. DisclosuresNo relevant conflicts of interest to declare.

Genetic and epigenetic characterization of hypodiploid acute lymphoblastic leukemia.

To investigate the genetic and epigenetic landscape of hypodiploid (<45 chromosomes) acute lymphoblastic leukemia (ALL). Single nucleotide polymorphism array, whole exome sequencing, RNA sequencing, and methylation array analyses were performed on eleven hypodiploid ALL cases. In line with previous studies, mutations in IKZF3 and FLT3 were detected in near-haploid (25-30 chromosomes) cases. Low hypodiploidy (31-39 chromosomes) was associated with somatic TP53 mutations. Notably, mutations of this gene were also found in 3/3 high hypodiploid (40-44 chromosomes) cases, suggesting that the mutational patterns are similar in low hypodiploid and high hypodiploid ALL. The high hypodiploid ALLs frequently displayed substantial cell-to-cell variability in chromosomal content, indicative of chromosomal instability; a rare phenomenon in ALL. Gene expression analysis showed that genes on heterodisomic chromosomes were more highly expressed in hypodiploid cases. Cases clustered according to hypodiploid subtype in the unsupervised methylation analyses, but there was no association between chromosomal copy number and methylation levels. A comparison between samples obtained at diagnosis and relapse showed that the relapse did not arise from the major diagnostic clone in 3/4 cases. Taken together, our data support the conclusion that near-haploid and low hypodiploid ALL are different with regard to mutational profiles and also suggest that ALL cases with high hypodiploidy may harbor chromosomal instability.

Transplantation Outcomes for Children with Hypodiploid Acute Lymphoblastic Leukemia

Children with hypodiploid acute lymphoblastic leukemia (ALL) have inferior outcomes despite intensive risk-adapted chemotherapy regimens. We describe 78 children with hypodiploid ALL who underwent hematopoietic stem cell transplantation between 1990 and 2010. Thirty-nine (50%) patients had ≤ 43 chromosomes, 12 (15%) had 44 chromosomes, and 27 (35%) had 45 chromosomes. Forty-three (55%) patients underwent transplantation in first remission (CR1) and 35 (45%) underwent transplantation in ≥ second remission (CR2). Twenty-nine patients (37%) received a graft from a related donor and 49 (63%) from an unrelated donor. All patients received a myeloablative conditioning regimen. The 5-year probabilities of leukemia-free survival, overall survival, relapse, and treatment-related mortality for the entire cohort were 51%, 56%, 27%, and 22%, respectively. Multivariate analysis confirmed that mortality risks were higher for patients who underwent transplantation in CR2 (hazard ratio, 2.16; P = .05), with number of chromosomes ≤ 43 (hazard ratio, 2.15; P = .05), and for those who underwent transplantation in the first decade of the study period (hazard ratio, 2.60; P = .01). Similarly, treatment failure risks were higher with number of chromosomes ≤ 43 (hazard ratio, 2.28; P = .04) and the earlier transplantation period (hazard ratio, 2.51; P = .01). Although survival is better with advances in donor selection and supportive care, disease-related risk factors significantly influence transplantation outcomes.

Transplantation Outcomes for Children with Hypodiploid Acute Lymphoblastic Leukemia

Transplantation Outcomes for Children with Hypodiploid Acute Lymphoblastic Leukemia

Low Hypodiploid Acute Lymphoblastic Leukemia with Burkitt-like Morphology.

Low Hypodiploid Acute Lymphoblastic Leukemia with Burkitt-like Morphology.