ALBERT

Description: BACKGROUND: Mixed phenotype acute leukemia (MPAL) is a rare form of leukemia in children. Current evidence supports the use of acute lymphoblastic leukemia (ALL)-directed regimens as initial therapy for MPAL; Children's Oncology Group (COG) ALL regimens are commonly used for pediatric ALL in the United States. Data for the predictive value of minimal residual disease (MRD) to risk-stratify therapy for pediatric MPAL is sparse overall and currently unknown in the context of COG ALL regimens. The primary objective for this study was to therefore examine the predictive value of MRD for event-free and overall survival (EFS, OS) in a centrally-reviewed, strictly-defined pediatric MPAL cohort treated according to COG ALL regimens. METHODS: A retrospective cohort of pediatric MPAL treated from 2008-2016 was assembled from six institutions in the United States. All sites submitted primary diagnostic flow cytometry for central review. Two independent hematopathologists blinded to clinical outcomes confirmed the diagnosis of MPAL according to the WHO2008 and/or WHO2016 classification and assigned a MPAL phenotype. MRD was assessed by multi-parameter flow cytometry ("negative" defined as

Description: Background: Mixed phenotype acute leukemia (MPAL) is a high risk leukemia with features of acute myeloid (AML) and acute lymphoblastic leukemia (ALL), either due to co-expression of antigens of multiple lineages, or the presence of multiple immunophenotypically distinct populations. WHO 2008 classifies MPAL as T/myeloid (T/M), B/myeloid (B/M), MLL rearranged (MLL) MPAL, BCR-ABL1 (Ph+) MPAL, and MPAL not otherwise specified (NOS). Patients are managed with divergent chemotherapeutic approaches with survival estimates of 50-70%. Apart from Ph+ and MLL rearrangement, the genetic basis of MPAL is poorly defined. Our goal was to define the molecular basis of MPAL, and to compare with potentially related forms of leukemia (AML, T-ALL and early T-cell precursor (ETP) ALL) as a rational foundation for future trials. Furthermore, we examined whether multi-lineal cases harbor genetically distinct subclones, or arise from the acquisition of founding alterations in a multi-lineage hematopoietic progenitor. Methods: 155 cases of pediatric leukemia initially diagnosed as MPAL were studied by central pathology review and/or central flow cytometry (134 cases), confirming the diagnosis according to WHO criteria in 115 cases (fig. 1). Median age was 7 years (0-18) with 52 T/M, 37 B/M, 15 MLL, 8 NOS, and 2 Ph+ (fig. 2). Samples were studied by whole genome and/or exome, RNA sequencing, and SNP array analysis. 44 multi-lineal samples were flow sorted into 2-4 lymphoid, myeloid, and ambiguous subpopulations (15 T/M, 19 B/M, 7 MLL, 1 Ph+, 2 NOS) and subjected to exome sequencing and SNP array. Mutational data were compared to data from 196 AML, 39 ETP-ALL, and 245 T-ALL cases. Results: We identified 35 recurrently mutated genes, the most common of which were WT1 (21%), FLT3 (18%), NRAS (16%), JAK3 (11%), RUNX1 (11%), KMT2D (9%), PTPN11 (9%), ASXL1 (7%), and CREBBP (7%). T/M and B/M subtypes are characterized by distinct patterns of genomic alteration. 48% of T/M cases harbored in-frame chimeric fusion, several of which are described in T-ALL, including ETV6-NCOA2 and ZEB2-BCL11B, NUP214-ABL1 and PICALM-MLLT10, and novel fusions involving hematopoietic regulators (e.g. ETV6-MAML and MNX1-IKZF1). 42% of B/M cases had in-frame fusions of ZNF384 with CREBBP, EP300, and TCF3, while we also identified isolated fusions involving ERG and NF1. Mutations of Ras signaling genes were present in 50% of B/M cases, in contrast to 10% of T/M cases. Epigenetic modifying genes, including CREBBP, SETD2, KMT2D, EZH2 and SUZ12 were mutated in 45% of the combined T/M and B/M cohorts. Cases with MLL gene rearrangements had few sequence alterations. In comparison to other subtypes of leukemia, the mutational spectrum of T/M MPAL, with alterations in transcription factors (60% cases), epigenetic genes (50%) and JAK-STAT signaling (35%) was more similar to ETP-ALL (64%, 72%, 44%) and T-ALL (49%, 60%, 21%) than to AML (19%, 21%, 11%). Similarly, B/M cases have increased alterations in these pathways (42%, 42%, 25%) compared to AML. Sequencing of MPAL subpopulations revealed that 27% of cases had the same SNVs/indels in each subpopulation, and 47% of cases had at least two-thirds of mutations present in each subpopulation. All multi-lineal cases with alterations of regulators WT1 and RUNX1 showed similar allele frequencies of these mutations in all populations. Alternatively, cases with mutations in signaling (FLT3, NRAS, KRAS, PTPN11) or epigenetic regulatory genes (CREBBP, KMT2D, SETD2) only showed consistent presence of alterations across each subpopulation in 60% of the cases. Conclusions: Our analysis has shown that T/M and B/M MPAL are distinct subtypes of leukemia. B/M MPAL is characterized by frequent RAS pathway mutations and ZNF384 fusions with multiple different fusion partners, suggesting that this gene plays a critical role in hematopoietic development for progenitor cells with B lymphoid and myeloid potential. The findings of mutational similarity to ETP ALL, and sharing of genomic lesions between subclones in the majority of cases strongly suggests that MPAL represents part of a spectrum of immature leukemias that arise in a hematopoietic progenitors that may propagate multiple immunophenotypic populations. These results will guide the design of therapeutic strategies for each subtype of MPAL and ETP ALL, and xenografts representative of each subtype are being used to examine sensitivity to therapeutic agents. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loh: Abbvie: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Zwaan:Pfizer: Research Funding; Pfizer: Consultancy. Reinhardt:Pfizer: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Other: Travel Accomodation. Inaba:Arog: Research Funding. Mullighan:Loxo Oncology: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau.

Description: Introduction Off-target effects of induction therapy to treat Acute Lymphoblastic Leukemia (ALL) can lead to hepatotoxicity, cardiotoxicity, and neuropathy, particularly in patients with previous co-morbidities. Incorporation of pegylated L-asparaginase (L-ASP) into pediatric and adult ALL induction regimens has increased remission rate but results in severe hepatotoxicity. Levocarnitine (L-carnitine), a small amino-acid derivative, has been reported in small case series in children and adults with ALL to reduce asparaginase-induced hepatotoxicity. L-carnitine functions as a transport molecule and facilitates long-chain free fatty acid beta-oxidation; exogenous supplementation with L-carnitine may therefore protect hepatocytes through facilitation of fatty acid transport and maintenance of antioxidant balance in mitochondria. However, the effects of L-carnitine on ALL cell survival and possible contributions of L-carnitine to chemotherapy resistance in ALL are not known. Given the ability of L-carnitine to enhance energy production, we sought to investigate whether L-carnitine could compromise the efficacy of common induction chemotherapy agents against ALL cells. Methods Human ALL cells, BV173 and RS4;11 were treated with L-carnitine and either daunorubicin (DNR) or vincristine (VCR) for 72 hours (n=4-6). RS4;11 cells were additionally treated with L-ASP (n=4), and current testing to assess dexamethasone in RS4;11 is in progress. Experiments were performed in 96-well culture plates with a seeding density of 90,000-120,000 cells/well (~5 x105 cells/mL). Growth media (RPMI 1640, 1% sodium pyruvate, 1% glutamine, 10% fetal bovine serum) was supplemented to physiologic (50 μM) and supraphysiologic (100-200 μM) levels of L-carnitine (normal reference range: 25-70 μM). Chemotherapy doses were chosen to represent ~EC90 concentrations (BV173: 2 nM VCR and 25 nM DNR; RS4;11: 2 nM VCR, 20 nM DNR, and 0.1 IU/mL L-ASP). After 72 hours, cell viability was measured by trypan blue exclusion and confirmed with spectrophotometric analysis with the Alamar Blue cell viability assay. Data were analyzed using one-way analysis of variance (ANOVA) with significance defined as p

Description: Key Points Obesity is associated with increased risk for persistent minimal residual disease after induction therapy for pediatric BP-ALL.

Description: INTRODUCTION: Mixed phenotype acute leukemia (MPAL) is a category of acute leukemia established in the World Health Organization (WHO) 2001 classification, significantly modified in WHO2008, and again refined in the most recent WHO2016 update. The current WHO2016 iteration conceptualizes MPAL as a stem cell disorder whereby most cases will manifest heterogeneity of lineage-specific antigen expression by multi-parameter flow cytometry. The WHO2016 definition also urges caution for cases otherwise consistent with B-cell acute lymphoblastic leukemia (B-ALL) that express myeloperoxidase (MPO) as the sole representation of myeloid lineage. These cases met the WHO2008 definition but may not meet the newer WHO2016 criteria. There is limited data on the clinical impact of these recent changes in the WHO classification for MPAL. METHODS: Six institutions identified cases diagnosed as MPAL between 2008 and 2016 according to WHO criteria. The diagnostic flow cytometry was then reanalyzed by two independent hematopathologists blinded to clinical outcomes. The cases were evaluated as to whether they met criteria for WHO2008 MPAL and/or WHO2016 MPAL. Cases of WHO2008 MPAL were further subdivided into those that otherwise met criteria for B-ALL (including non-lineage specific expression of ±CD13, ±CD15, ±CD33) but qualified as MPAL due to MPO expression (MPO+MPAL) and all remaining cases of MPAL that demonstrated additional myeloid lineage specificity as described by the WHO criteria (MLS+MPAL). Data for a distinct cohort of pediatric B-ALL without significant MPO expression diagnosed during the same study period was submitted from one participating site to serve as a reference cohort (n=258). Endpoints of interest to evaluate clinical outcomes according to the WHO classification were event-free and overall survival (EFS, OS). All statistical tests were two-sided with significance set at p

Description: Resistance to multimodal chemotherapy continues to limit the prognosis of acute lymphoblastic leukemia (ALL). This occurs in part through a process called adhesion-mediated drug resistance, which depends on ALL cell adhesion to the stroma through adhesion molecules, including integrins. Integrin α6 has been implicated in minimal residual disease in ALL and in the migration of ALL cells to the central nervous system. However, it has not been evaluated in the context of chemotherapeutic resistance. Here, we show that the anti-human α6-blocking Ab P5G10 induces apoptosis in primary ALL cells in vitro and sensitizes primary ALL cells to chemotherapy or tyrosine kinase inhibition in vitro and in vivo. We further analyzed the underlying mechanism of α6-associated apoptosis using a conditional knockout model of α6 in murine BCR-ABL1+ B-cell ALL cells and showed that α6-deficient ALL cells underwent apoptosis. In vivo deletion of α6 in combination with tyrosine kinase inhibitor (TKI) treatment was more effective in eradicating ALL than treatment with a TKI (nilotinib) alone. Proteomic analysis revealed that α6 deletion in murine ALL was associated with changes in Src signaling, including the upregulation of phosphorylated Lyn (pTyr507) and Fyn (pTyr530). Thus, our data support α6 as a novel therapeutic target for ALL.

Description: Abstract 3574 Intensification of therapy for acute lymphoblastic leukemia (ALL) has improved long-term survival but is associated with considerable treatment-related toxicity (TRT). While obesity has been shown to affect event-free survival (EFS) and overall survival (OS) in ALL, little information exists as to the effect of extremes of weight (underweight or obese) on morbidity due to TRT. To this end, we conducted a secondary analysis of 1,902 children treated on the Children's Oncology Group study CCG-1961 to determine whether weight extremes influence development of TRT and EFS/OS in children with higher risk ALL. CCG-1961 was a randomized study for patients 1–21 years old with National Cancer Institute High-Risk ALL (HR-ALL) that evaluated marrow response at day 7 of Induction and compared regimens of differing intensity and/or duration (for Rapid Early Responders, RER) or anthracycline used in Delayed Intensification (for Slow Early Responders, SER). Weight category was determined using the United States Centers for Disease Control and Prevention Z-scores for body mass index (BMI) by age (age ≥ 2 yrs) and weight by length (age 95th percentile). At diagnosis, 107 patients (5.6%) were underweight and 264 were obese (13.9%). Multivariate linear and logistic regression analyses controlled for age, gender, race/ethnicity, payer type (as a surrogate for socioeconomic status), initial white blood cell count, presence of central nervous system disease, RER/SER status, and phase of treatment independent of regimen. All of the preceding predictors were independently significantly associated with development of grade 3/4 non-hematological toxicity in multivariate analysis (p

Description: BACKGROUND : Mixed phenotype acute leukemia (MPAL) as defined according to the World Health Organization 2016 guidelines (WHO2016) comprises approximately 2-3% of acute leukemia. In other acute leukemias, minimal residual disease (MRD) has emerged as the predominant predictor of relapse. MRD at end of Induction (EOI) is therefore a key risk-stratifying feature for lymphoid and myeloid disease regimens. To our knowledge, no studies to date have reported on the predictive value of MRD for treatment of MPAL. Although the absence of randomized trials for MPAL precludes a clear determination of optimal regimen, acute lymphoblastic leukemia (ALL) directed-therapy is increasingly considered frontline treatment internationally. The primary objective for this study was to evaluate the association of EOI MRD with event free survival (EFS) from ALL regimens used for treatment of MPAL. METHODS : Flow cytometry data for all acute leukemias diagnosed 2008-2015 and treated with Children's Oncology Group regimens for ALL were examined by two flow cytometrists blinded to clinical outcomes (MO, SL). Data was analyzed using CellQuestPro and FACSDiva software (BD Biosciences, San Jose, CA). A comprehensive panel of 21 B-, T-, and myeloid markers were evaluated in all cases. A diagnosis of MPAL was strictly established according to the WHO2016 definition. EOI MRD was performed routinely by flow cytometry using a "difference from normal" approach. Cases with insufficient flow cytometry to either (1) establish the diagnosis of MPAL or (2) evaluate the presence and quantity of MRD were excluded. Due to the rarity of the T/myeloid phenotype in the dataset (n=1), the analysis was restricted to B/Myeloid. The primary endpoint was EFS; secondary outcomes explored predictors of EOI MRD positivity and the prevalence of recurrent cytogenetic abnormalities. MRD was categorized as a binomial (negative

Description: Introduction Obesity is associated with increased morbidity and mortality in children diagnosed with high-risk acute lymphoblastic leukemia (HR-ALL). The mechanism of this adverse influence is thought to be due to adipose tissue and adipokines affecting immune function and/or decreasing chemotherapy efficacy. Adiposity, and not body weight, is therefore the central feature for exploring the influence of obesity on outcome in HR-ALL. To date, multiple international consortia have investigated this association using the surrogate measure of Body Mass Index (BMI). BMI estimates body composition based on height and weight and is therefore not able to distinguish between fat mass, bone density, and lean muscle. While BMI is generally a good indicator of overall adiposity, changes in weight that may occur over the course of therapy reflect not only a change in fat mass, but also changes in other components of body composition. We therefore hypothesized that BMI may not be a sensitive measure for assessing change in adiposity during therapy. Methods Body composition was assessed serially by BMI and the gold-standard Dual-energy X-ray Absorptiometry (DXA) as an ancillary aim in children with newly diagnosed HR-ALL enrolled on a prospective clinical trial investigating bone health. Children were treated as per Children’s Oncology Group HR-ALL regimens (CCG1961, AALL0232, AALL1131). Body composition was compared at three time-points (TP): within 24 hours of diagnosis (TP1), 28 days later (TP2, end of induction), and at the end of delayed intensification (TP3, mean interval of 8.2 months in the cohort). Age and tanner stage at diagnosis, gender, and ethnicity were collected. DXA was used to analyze body composition as defined by total mass of “body minus head” and separated into respective percentages of lean muscle, fat mass, and bone mineral content. BMI was converted to a percentage per Center for Disease Control and Prevention age- and gender- population norms. BMI percentage was compared to body fat percentage (BF%) overall and individually at each TP. Changes in body composition across TPs were evaluated by DXA. IRB approval was obtained and informed consent documented for all subjects. Results Of 51 subjects enrolled in the trial, a sub-cohort of 34 (66.6%) had sufficient DXA data for analysis. Of these, 85% (29/34) had DXA and BMI data at all 3 TPs, while 5/34 were too ill at diagnosis to complete the imaging. There were no significant differences in age, gender, and tanner stage between those included in the DXA sub-cohort and excluded; the cohort with DXA data consisted of a significantly higher prevalence of self-identified Hispanic subjects (31/34, 91% vs 11/17 65%). BF% by DXA was significantly correlated with BMI when observations from all TPs were combined (n=96, Spearman rho 0.6, p=0.002) and at each TP (TP1 rho=0.6, p=0.002; TP2 rho =0.7, p