Therapy‐related myeloid neoplasms (t‐MN) are a distinct subgroup of myeloid malignancies with a p... more Therapy‐related myeloid neoplasms (t‐MN) are a distinct subgroup of myeloid malignancies with a poor prognosis that include cases of therapy‐related myelodysplastic syndrome (t‐MDS), therapy‐related myeloproliferative neoplasms (t‐MPN) and therapy‐related acute myeloid leukemia (t‐AML). Here, we report a series of patients with clinical features consistent with juvenile myelomonocytic leukemia (JMML), an overlap syndrome of MDS and myeloproliferative neoplasms that developed after treatment for another malignancy.
Hispanic children have a 10-30% greater incidence rate of ALL than non-Hispanic whites, and nearl... more Hispanic children have a 10-30% greater incidence rate of ALL than non-Hispanic whites, and nearly double the rate observed in African-Americans. 1 Ethnic differences in ALL incidence may be explained by population-level differences in the frequency of genetic risk factors, including those first discovered in genome-wide association studies of Europeanancestry populations. As Hispanics are an admixed population with European, African and Native American ancestry, differences in ALL incidence observed in Hispanics may be attributable to genetic risk factors associated with Native American ancestry. Increased Native American ancestry has been linked to increased risk of relapse among Hispanic children with ALL, 6 but no study has yet investigated the contribution of genomewide Native American ancestry to ALL incidence. Using genome-wide SNP data from 298 Hispanic children with B cell ALL and 456 matched controls from the California Childhood Leukemia Study (CCLS), we investigated whether genome-wide Native-American ancestry was associated with increased risk of B-cell ALL. Additionally, we assessed whether the risk alleles at loci identified in genome-wide association studies of European-ancestry populations (IKZF1, CDKN2A, PIP4K2A, ARID5B, CEBPE) were more common in individuals with greater levels of Native American ancestry. Finally, we quantified the contribution of these validated risk loci to the increased ALL incidence observed in Hispanics relative to populations of European or African ancestry.
• CMV is prevalent in pretreatment bone marrow from childhood ALL and not in acute myeloid leukem... more • CMV is prevalent in pretreatment bone marrow from childhood ALL and not in acute myeloid leukemia. • In utero infection with CMV is a risk factor for ALL (OR 5 3.71, P 5 .0016) and is more pronounced in Hispanics (OR 5 5.90, P 5 .006). It is widely suspected, yet controversial, that infection plays an etiologic role in the development of acute lymphoblastic leukemia (ALL), the most common childhood cancer and a disease with a confirmed prenatal origin in most cases. We investigated infections at diagnosis and then assessed the timing of infection at birth in children with ALL and age, gender, and ethnicity matched controls to identify potential causal initiating infections. Comprehensive untargeted virome and bacterial analyses of pretreatment bone marrow specimens (n 5 127 ALL in comparison with 38 acute myeloid leukemia cases in a comparison group) revealed prevalent cytomegalovirus (CMV) infection at diagnosis in childhood ALL, demonstrating active viral transcription in leukemia blasts as well as intact virions in serum. Screening of newborn blood samples revealed a significantly higher prevalence of in utero CMV infection in ALL cases (n 5 268) than healthy controls (n 5 270) (odds ratio [OR], 3.71, confidence interval [CI], 1.56-7.92, P 5 .0016). Risk was more pronounced in Hispanics (OR55.90, CI51.89-25.96) than in non-Hispanic whites (OR52.10 CI5 0.69-7.13). This is the first study to suggest that congenital CMV infection is a risk factor for childhood ALL and is more prominent in Hispanic children. Further investigation of CMV as an etiologic agent for ALL is warranted.
Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and oc... more Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p<0.0001), and 3.09-fold (p<0.0001), respectively, compared to pediatric CML CD34+ cells. Furthermore, DLC1, which is known as a negative regulator of Rho pathway, was significantly upregulated in pediatric CML CD34+ cells by 2.47-fold (p=0.0493) compared to adult CML CD34+ cells. These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.
Background-Older age has historically been an adverse prognostic factor in pediatric acute myeloi... more Background-Older age has historically been an adverse prognostic factor in pediatric acute myeloid leukemia (AML). The impact of age relative to that of other prognostic factors on the outcome of patients treated in recent trials is unknown. Methods-Clinical outcome and causes of treatment failure of 351 patients enrolled on three consecutive protocols for childhood AML between 1991 and 2008 were analyzed according to age and protocol. Results-The more recent protocol (AML02) produced improved outcomes for 10-to 21-yearold patients compared to 2 earlier studies (AML91 and 97), with 3-year rates of event-free survival (EFS), overall survival (OS) and cumulative incidence of refractory leukemia or relapse (CIR) for this group similar to those of 0-to 9-year old patients: EFS, 58.3% ± 5.4% vs. 66.6% ± 4.9%, P=.20; OS, 68.9% ± 5.1% vs. 75.1% ± 4.5%, P=.36; cumulative incidence of refractory leukemia or relapse, 21.9% ± 4.4%; vs. 25.3% ± 4.1%, P=.59. EFS and OS estimates for 10-15year-old patients overlapped those for 16-21-year-old patients. However, the cumulative incidence of toxic death was significantly higher for 10-to 21-year-old patients compared to younger patients (13.2% ± 3.6 vs. 4.5% ± 2.0%, P=.028). Conclusion-The survival rate for older children with AML has improved on our recent trial and is now similar to that of younger patients. However, deaths from toxicity remain a significant problem in the older age group. Future trials should focus on improving supportive care while striving to develop more effective antileukemic therapy.
BACKGROUND-The effect of body mass index (BMI) on treatment outcome of children with acute myeloi... more BACKGROUND-The effect of body mass index (BMI) on treatment outcome of children with acute myeloid leukemia (AML) is unclear and needs further evaluation. METHODS-Children with AML (n=314) enrolled in 4 consecutive St. Jude protocols were grouped according to BMI (underweight, <5 th percentile; healthy weight, 5 th to 85 th percentile; and overweight/obese, ≥ 85 th percentile). RESULTS-Twenty-five (8.0%) patients were underweight, 86 (27.4%) overweight/obese, and 203 (64.6%) had healthy weight. Five-year overall survival of overweight/obese patients (46.5±7.3%) was lower than that of patients with healthy weight (67.1±4.3%, P < .001); underweight patients also tended to have lower survival rates (50.6±10.7%, P = .18). In a multivariable analysis adjusting for age, leukocyte count, FAB type, and study protocols, patients with healthy weight had the best survival rate among the 3 groups (P = .01). When BMI was considered as continuous variable, patients with lower or higher BMI percentiles had worse survival (P = .03). There was no difference in the occurrence of induction failure or relapse among BMI groups but underweight and overweight/obese patients had a significantly higher cumulative incidence of treatment-related mortality, especially due to infection (P = .009). CONCLUSIONS-An unhealthy BMI is associated with worse survival and more treatmentrelated mortality in children with AML. Meticulous supportive care, with nutritional support and
9530 Background: Leukemia accounts for ∼40% of newly diagnosed pediatric malignancies, and relaps... more 9530 Background: Leukemia accounts for ∼40% of newly diagnosed pediatric malignancies, and relapsed leukemia is the leading cause of death in childhood cancer. Genomic instability events contribute to neoplastic development and have been used to classify and risk stratify non-leukemic adult and pediatric tumors. Analyzing leukemic blasts for gene copy changes with advanced molecular techniques could prove useful in further risk stratifying and developing new treatment strategies for pediatric leukemia. Methods: Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel with high specificity and sensitivity at the highest genomic resolution, and were originally designed for single nucleotide genotyping. The MIP assay was adapted to analyze both gene copy number and loss of heterozygosity (LOH) events in pediatric leukemia samples (pre-B ALL, T-ALL, AML). DNA was extracted (100 ng) from paired bone marrow (diagnosis) and peripheral blood (remission) samples (n = 40). The MIP assay was run with a customized Affymetrix 20K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). Gene copy number changes were identified by comparing probe signal intensity between leukemia samples and normal cell-lines. LOH events were determined by identifying genotype changes between matched leukemic and remission samples. Results: Each sample had unique patterns of multiple gene copy changes and LOH events distributed across all chromosomes. Additionally, samples were found to have overlapping copy number changes and LOH regardless of leukemia type. AML samples had fewer LOH events and could be separated by unsupervised clustering from the other leukemia samples. Conclusions: MIPs represent novel genotyping technology that can be adapted for gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of LOH and deleted genes may represent a common molecular mechanism that requires further investigation. No significant financial relationships to disclose.
Treatment-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/t-AML) is a devastating ... more Treatment-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/t-AML) is a devastating complication for pediatric patients with osteosarcoma. Even with intensive chemotherapy, overall survival ranges from 10-26%. These regimens cause significant morbidity, poor quality of life and increased toxicity, at times precluding the ultimate goal of hematopoietic cell transplant (HCT). Chromatin remodeling therapy with decitabine and vorinostat has shown some promise in adults with relapsed/refractory AML and MDS. The synergistic combination of methotrexate and asparaginase, also referred to as &amp;#39;Capizzi methotrexate&amp;#39;, is not generally part of upfront treatment for AML but does have some activity in relapsed/refractory disease. Using this rationale, we report the use of this combination regimen in 2 children with t-AML where chromatin remodeling therapy with subsequent Capizzi methotrexate successfully bridged both patients to HCT. Patients were treated at Lucile Packard Children&amp;#39;s Hospital Stanford between 2010 and 2018 for osteosarcoma and subsequently for t-AML. An overview of the treatments used is described in Table 1. An adolescent female was treated for metastatic osteosarcoma. She developed an isolated pulmonary recurrence 5 months after completing therapy. She underwent resection of her pulmonary nodule and then was enrolled on a clinical trial. Four months later, she developed another pulmonary recurrence. At this time, she began treatment with ifosfamide and etoposide. After the sixth cycle, she was noted to have blasts in her blood, and a bone marrow evaluation confirmed t-MDS. Cytogenetics were negative for deletion of 5q, 7q or MLL rearrangement. Repeat bone marrow evaluation two months later revealed progression to t-AML. She initially received 5 days of high dose cytarabine without response and was then transitioned to Treatments A and B. Bone marrow evaluation on day 30 of Treatment B showed morphologic remission with 11% MRD. She underwent Treatment C and then proceeded to a matched sibling HCT. She had no detectable osteosarcoma at the time of her transplant. The patient remained in remission from t-AML but subsequently died from recurrent osteosarcoma more than 2 years after HCT. An adolescent male was treated for non-metastatic osteosarcoma of the left distal femur with methotrexate, adriamycin, cisplatin, ifosfamide and etoposide. Six years after completing treatment, he developed weight loss, epistaxis and pancytopenia. Bone marrow aspiration revealed a hypercellular marrow with 18% blasts, increased marrow fibrosis and cytogenetics with a complex del(7q)/+8 clone, consistent with t-MDS. He received Treatment B, and a bone marrow evaluation on day 25 showed a hemodilute specimen with MRD &amp;amp;lt;0.1%. He then received vincristine and methotrexate on day 35 followed by erwinia on day 36. Repeat bone marrow evaluation on day 42 again showed a hypocellular marrow with MRD of &amp;amp;lt;0.1%. He went on to receive another cycle of vincristine and methotrexate followed by days 1 through 5 of Treatment B. Bone marrow evaluation on day 29 showed a hypocellular marrow with insufficient viable cells for MRD analysis. The patient received an alpha/beta T cell depleted haploidentical transplant. He remains in morphologic remission with no evidence of disease more than 3 months after HCT. Treatment-related MDS/AML remains a devastating complication of osteosarcoma treatment with no standard treatment. The two cases presented here demonstrated good response to a novel combination of therapy with decitabine/vorinostat followed by Capizzi methotrexate. Given the significant toxicity associated with most regimens for t-MDS/t-AML, this regimen should be considered when first line salvage chemotherapy has failed. The precise regimen as well as the optimal number of cycles prior to stem cell transplant should be evaluated in the context of a clinical trial. Disclosures No relevant conflicts of interest to declare.
Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the ... more Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the leading cause of death for children with cancer. Genomic instability events contribute to tumorigenesis and have been used to classify and risk stratify adult and pediatric cancers. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution, and can detect both gene copy number and loss of heterozygosity (LOH) events in clinical samples. Studying pediatric leukemia samples with MIP technology may identify new molecular alterations that could prove useful in risk stratification and discovery of new therapeutic targets for childhood leukemia. Objective: To use MIP technology to identify novel areas of allelic imbalance in childhood leukemia. Methods: DNA was extracted from leukemia blasts at diagnosis (n=45, 23 pre-B ALL, 14 AML, 7 pre-T ALL, 1 Burkitt’s). DNA was also extracted from normal peripheral blood collected at remission to use as paired germline controls. The MIP assay was run with a customized Affymetrix 24K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). DNA required for this assay was limited to 75 ng per sample. Copy number changes and LOH were identified by comparing probe signal intensity between leukemia and normal germline samples. Clinical cytogenetic data (karyotype and FISH analysis) was used as a control to confirm findings from known areas of allelic imbalance. Results: Each leukemia sample had unique patterns of allelic imbalance distributed across all chromosomes. MIPs identified all clinically reported cytogenetic copy number changes for each sample, in addition to areas of allelic imbalance not clinically reported. Samples had recurring areas of copy number changes and LOH events shared by all leukemia types. MIPs detected areas of allelic imbalance in both previously described and novel genes. Areas of recurring genomic deletions included: ATR (3q23), TLX3 (5q35.1), ADRB3 (8p12), CDKN2A (9p21.3), DOCK8 (9p24.3), PAX5 (9p13.2), PTPN11 (12q24.13), C3AR1 (12p13.31), TCRA (14q11.2), AKT1 (q14q32.33). Areas of recurring genomic amplification included: SLC2A9 (4p16.1), RAI14 (5p13.2), CDH12 (5p14.3), PMCHL1(5p14.3), AURKB (17p13.1). These findings are being validated with Quantitative Real-Time PCR. Conclusions: MIPs represent a novel genomic technology that can identify previously unreported gene copy number and LOH events in childhood leukemia. Unique and overlapping areas of allelic imbalance were found in both childhood ALL and AML clinical samples. The shared genomic regions of allelic imbalance between different leukemia types may represent a common molecular mechanism of leukemogenesis that warrants further investigation. Analysis of more childhood leukemia samples through Pediatric Cooperative Groups may help to determine how common these areas of allelic imbalance are in children and whether they are of prognostic significance. Further exploration of these copy number and LOH events may help us to better understand the biology of childhood leukemia and its clinical behavior.
Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subt... more Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subtype, FLT3 status and response to induction therapy are determinants of outcome with current therapies. We hypothesize that array comparative genomic hybridization (CGH) will identify gene copy number changes that are determinants of outcome. Array CGH was performed on diagnostic bone marrow samples from patients on the COG study POG #9421. In order to determine regions of altered gene copy number, labeled genomic DNA samples were hybridized together with sex-matching normal human reference DNA to cDNA microarrays with 41,751 features (corresponding to 24,473 unique Unigene cluster IDs), arrays were obtained from the Stanford University Microarray Core Facility. Control hybridizations were performed to assess intra- and inter-experimental variability. We studied 70 samples with adequate high-quality DNA. Circular binary segmentation was used to distinguish discrete gene copy number transition points from chance noise events and to transform primary clone-by-clone data into genomic regions of equal copy number. Using gain/loss threshold, based on two-standard deviation range of control self-to-self distribution, novel gene amplifications and deletions were found in profiled samples. The highest alteration recurrence was observed for gains of chromosome 8 (21%) and losses of chromosome 6 (29%). The area of chromosome 8 which was found to be gained is notable for the presence of potential oncogenes such as ERK8. The deleted area of chromosome 6 is notable for the presence of potential regulators of oncogenesis: MDC1, DDR1, NFKBIL1, TNF, and BRD2. In summary, array CGH has identified novel areas of gene copy number gain and loss in this population of pediatric de novo AML patients. Further studies are needed to assess whether these genes are associated with outcome, known risk factors and whether they will provide insight into the heterogeneity of de novo AML.
Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,... more Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays as an in vitro test for sensitivity to these agents, investigators have found that leukemia cells from pediatric AML patients with Down Syndrome, which are more sensitive to cytarabine than blasts from non-Down Syndrome AML patients, have been associated with an increased ratio of deoxycytidine kinase to cytidine deaminase and an increased level of cystathionine-beta-synthetase [Ge, Jensen, Stout et al. Cancer Res2004; 64, 728–35]. Looking for related findings in non-Down syndrome AML, leukemic blasts from non-Down syndrome children with AML were examined utilizing gene expression profiling and MTT assays. Expression signatures from cDNA arrays were related to the drug sensitivity results to daunorubicin and cytarabine by supervised clustering. These results will then be analyzed to look for relationships to the patient’s clinical outcomes and identify genes that may be potential therapeutic targets for drug resistance reversal. Bone marrow or blood specimens from 103 patients registered on the Pediatric Oncology Group (POG) protocol 9421 were studied. The diagnostic specimens were tested using MTT assays to measure the IC50 (drug concentration that causes 50% of the cells to die) for both daunorubicin and cytarabine. Samples from ninety-three de novo childhood AML patients were analyzed with a 43,760 element spotted array (containing 41,751 unique genes and expression sequence tags [ESTs]) from the Stanford University Microarray Core Facility. We selected the gene expression profile from 10 samples with the highest and lowest IC50’s for each drug. Then, we used Significance Analysis of Microarrays (SAM) to find significant differences in the gene expression between samples sensitive and resistant to each drug. A cluster of 118 overexpressed genes was associated with resistance to daunorubicin (e.g., BCL6, BAPX1, BCL2A1, MBD2, RAB31 and CDKN1A). A cluster of 24 overexpressed genes was associated with resistance to cytarabine (e.g., RIT1, SH3BP2, BCL2A1, NFKBIA, and PTPRE). In summary, MTT assays and gene expression profiling have identified genes that correlate with daunorubicin and cytarabine resistance. Study of the relationship among drug resistance patterns, gene expression profiles and clinical outcome may allow us to better predict patients’ prognoses at diagnosis and also can be used to identify novel targets for drug resistance reversal.
Abstract 482 Background: Acute megakaryoblastic leukemia (AMKL) in children without Down syndrome... more Abstract 482 Background: Acute megakaryoblastic leukemia (AMKL) in children without Down syndrome represents approximately 10% of pediatric acute myeloid leukemia (AML). Published reports regarding prognosis of non-Down syndrome children with AMKL vary; hence, some groups treat these patients as high-risk while others as standard-risk. As a result, the optimal treatment strategy, including the role of hematopoeitic stem cell transplant (HSCT), remains unclear. Methods: We reviewed the treatment and outcomes of patients with AMKL treated on two pediatric AML protocols. Pediatric Oncology Group (POG) 9421 (1995-99) included two cycles of induction chemotherapy with randomization to standard or high-dose DAT (daunorubicin, cytarabine, 6-thioguanine) followed by HSCT for children in remission with matched sibling donors or three cycles of consolidation chemotherapy with or without cyclosporine for those without suitable donors. St. Jude protocol AML02 (2002-2008) randomized patients during induction to receive high- or low-dose cytarabine with daunorubicin and etoposide (ADE). Patients without complete remission (CR) received gemtuzumab ozogamicin (GO) with induction 2 chemotherapy (ADE). After induction, AMKL patients with availabel donor proceeded to HSCT while those without donors received three cycles of consolidation chemotherapy including mitoxantrone and cytarabine. Comparisons of event-free survival (EFS) and overall survival (OS) were performed by Mantel-Haenszel log-rank test. Independent effect of HSCT on EFS or OS were analyzed by Cox proportional hazard model, with HSCT as time-dependent variable. Results: The 49 patients with AMKL treated on POG 9421 had a median age at diagnosis of 1.8 years (range 0.13-16.2 years), and a median leukocyte count of 13.5 × 109/L (range 0.3-98.4). The patient with the t(1;22) translocation remains in CR. 39 patients had CR after induction, 5 partial response and 4 no response with no difference between standard- and high-dose DAT. The 5-year EFS for all AMKL patients was 34.7% ± 7.5%. Of the 39 patients in CR, 6 underwent matched sibling HSCT, of whom 4 remain in CR, and the other 33 patients received consolidation chemotherapy, of whom 15 remain in continuous remission. The 5-year OS for patients undergoing HSCT was 66.7% ± 19.3% compared to 36.1% ± 8.4% for those receiving consolidation chemotherapy (p = 0.2). The 26 patients with AMKL treated on SJCRH AML02 had a median age at diagnosis of 1.2 years (range 0.21-11.2 years), and a median leukocyte count of 11.8 × 109/L (range 2.3-72.9). All 5 patients with the t(1;22) translocation achieved CR after one induction cycle, with negative minimal residual disease, and remain in CR after treatment with chemotherapy only. The 3-year EFS was 35.9% ± 12.9% for the other AMKL patients, significantly lower than the 61.5% ± 4.8% for non-AMKL patients (p = 0.026). The 3-years OS estimates are 100% for patients with t(1;22), 48% ± 14.1% for other AMKL patients, and 71.8% ± 4.4% for non-AMKL patients (p = 0.03). Of the 21 patients without t(1;22), 14 received HSCT with 7 survivors (3-year EFS 46.3 ± 17%) while 7 received chemotherapy with only one long-term survivor (3-year EFS 14.3 ± 9.4%) (p=0.55). Conclusions: AMKL patients with the t(1;22) translocation have excellent treatment outcome with chemotherapy alone. In contrast, AMKL patients without t(1;22) fared poorly, especially when they were treated with only chemotherapy. Additional studies of larger number of patients are needed to determine if they would benefit from HSCT. Disclosures: No relevant conflicts of interest to declare.
Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with mye... more Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison. Results Pediatric CML vs Adult CML We found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis. Adult CML vs Adult Normal We found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described. Pediatric CML vs Pediatric Normal We found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism. Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.
IntroductionEx vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstra... more IntroductionEx vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. MethodsWe compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associ...
Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a vi... more Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing–based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.
Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte ... more Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation u...
Transcriptional repression by chimeric transcription factors is emerging as a common theme in leu... more Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most si...
The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #... more The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #9421 (n=144) was 36%. We previously reported a subgroup of patients (n=68) with AML and NK that could be divided into 2 groups whose clinical outcomes correlated with abnormalities of FLT3 [internal tandem duplications (ITD) or activating loop mutations]. EFS estimates were 13% for patients with mutant FLT3 and 61% for children with wild-type FLT3 (P=0.01). We hypothesized that gene expression profiling would identify signatures that are linked to clinical outcome and can be used for risk determination. Cytogenetic testing was carried out in clinical laboratories at the institutions in which AML was diagnosed and then centrally reviewed. We analyzed bone marrow from 45 patients with NK on 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. FLT3 status (mutant or wild type) wa...
The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;... more The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome. ...
Therapy‐related myeloid neoplasms (t‐MN) are a distinct subgroup of myeloid malignancies with a p... more Therapy‐related myeloid neoplasms (t‐MN) are a distinct subgroup of myeloid malignancies with a poor prognosis that include cases of therapy‐related myelodysplastic syndrome (t‐MDS), therapy‐related myeloproliferative neoplasms (t‐MPN) and therapy‐related acute myeloid leukemia (t‐AML). Here, we report a series of patients with clinical features consistent with juvenile myelomonocytic leukemia (JMML), an overlap syndrome of MDS and myeloproliferative neoplasms that developed after treatment for another malignancy.
Hispanic children have a 10-30% greater incidence rate of ALL than non-Hispanic whites, and nearl... more Hispanic children have a 10-30% greater incidence rate of ALL than non-Hispanic whites, and nearly double the rate observed in African-Americans. 1 Ethnic differences in ALL incidence may be explained by population-level differences in the frequency of genetic risk factors, including those first discovered in genome-wide association studies of Europeanancestry populations. As Hispanics are an admixed population with European, African and Native American ancestry, differences in ALL incidence observed in Hispanics may be attributable to genetic risk factors associated with Native American ancestry. Increased Native American ancestry has been linked to increased risk of relapse among Hispanic children with ALL, 6 but no study has yet investigated the contribution of genomewide Native American ancestry to ALL incidence. Using genome-wide SNP data from 298 Hispanic children with B cell ALL and 456 matched controls from the California Childhood Leukemia Study (CCLS), we investigated whether genome-wide Native-American ancestry was associated with increased risk of B-cell ALL. Additionally, we assessed whether the risk alleles at loci identified in genome-wide association studies of European-ancestry populations (IKZF1, CDKN2A, PIP4K2A, ARID5B, CEBPE) were more common in individuals with greater levels of Native American ancestry. Finally, we quantified the contribution of these validated risk loci to the increased ALL incidence observed in Hispanics relative to populations of European or African ancestry.
• CMV is prevalent in pretreatment bone marrow from childhood ALL and not in acute myeloid leukem... more • CMV is prevalent in pretreatment bone marrow from childhood ALL and not in acute myeloid leukemia. • In utero infection with CMV is a risk factor for ALL (OR 5 3.71, P 5 .0016) and is more pronounced in Hispanics (OR 5 5.90, P 5 .006). It is widely suspected, yet controversial, that infection plays an etiologic role in the development of acute lymphoblastic leukemia (ALL), the most common childhood cancer and a disease with a confirmed prenatal origin in most cases. We investigated infections at diagnosis and then assessed the timing of infection at birth in children with ALL and age, gender, and ethnicity matched controls to identify potential causal initiating infections. Comprehensive untargeted virome and bacterial analyses of pretreatment bone marrow specimens (n 5 127 ALL in comparison with 38 acute myeloid leukemia cases in a comparison group) revealed prevalent cytomegalovirus (CMV) infection at diagnosis in childhood ALL, demonstrating active viral transcription in leukemia blasts as well as intact virions in serum. Screening of newborn blood samples revealed a significantly higher prevalence of in utero CMV infection in ALL cases (n 5 268) than healthy controls (n 5 270) (odds ratio [OR], 3.71, confidence interval [CI], 1.56-7.92, P 5 .0016). Risk was more pronounced in Hispanics (OR55.90, CI51.89-25.96) than in non-Hispanic whites (OR52.10 CI5 0.69-7.13). This is the first study to suggest that congenital CMV infection is a risk factor for childhood ALL and is more prominent in Hispanic children. Further investigation of CMV as an etiologic agent for ALL is warranted.
Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and oc... more Chronic myeloid leukemia (CML) accounts for 2-9% of leukemias in children and adolescents, and occurs with much greater frequency in adults. Compared to adults, children with CML tend to present with higher white blood cell counts and larger spleens, suggesting that the biology of pediatric CML is different from adult CML. We hypothesize that the differences in clinical presentation of pediatric CML are due to unique molecular characteristics that differ from adult CML. To test this hypothesis, we compared the transcriptomic signature of pediatric and adult CML CD34+ cells and healthy age-matched CD34+ cells. CD34+ cells were isolated by FACS from pediatric CML (n=9), adult CML (n=10), pediatric healthy (n=10), and adult healthy (n=10) bone marrow samples. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. Raw sequences were trimmed and aligned to the hg38 reference genome with STAR/2.5.1b aligner. Gene level counts were determined with STAR -quantMode option using gene annotations from GENCODE (p5). Differential gene expression and pathway analysis were conducted with R/3.5.3. Counts were normalized with trimmed mean of M-values from the EdgeR/ 3.24.3 package and further transformed with VOOM from the Limma/ 3.38.3 package. A linear model using the empirical Bayes analysis pipeline also from Limma was then used to obtain p-values, adjusted p-values and log-fold changes. Four comparisons were performed: (1) pediatric CML vs pediatric healthy, (2) adult CML vs adult healthy, (3) pediatric CML vs adult CML, and (4) pediatric healthy vs adult healthy. A False Discovery Rate of ≤ .05 and absolute log2 fold-change &amp;amp;gt; 1 was used to define differentially expressed genes (DEGs) in each comparison. To identify potentially unique pathways based on DEG, pathway over-representation was calculated with either goana from the limma package or clueGO. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients. Median WBC counts were 273,000 and 143,000 in pediatric and adult patients respectively. A total of 1352 genes were differentially expressed in either adult or pediatric CML CD34+ cells compared to healthy CD34+ cells, 174 of which were expressed similarly in pediatric and adult CML CD34+ cells (54 up- and 120 down-regulated). There were 746 differentially expressed genes (325 up- and 421 down-regulated) in adult CML CD34+ cells compared to adult healthy CD34+ cells, and 432 differentially expressed genes (156 up- and 276 down-regulated) in pediatric CML CD34+ cells compared to pediatric healthy CD34+ cells. In direct comparison of pediatric and adult CML CD34+ cells, 446 genes (270 up and 176 down) were dysregulated in pediatric CML CD34+ cells. Pathway analysis showed that Rho signaling pathway was downregulated in pediatric CML CD34+ cells and several genes in Rho pathway were uniquely dysregulated. ARHGAP27 and VAV2 were significantly upregulated in adult CML CD34+ cells by 3.7-fold (p=0.0453) and 11-fold (p=0.0072), respectively, compared to pediatric CML CD34+ cells. In addition, several genes involved in the NADPH oxidase pathway, one of the best-characterized Rho GTPase-regulated systems, were differently expressed in CML. NCF1, CYBB, and S100A8 were significantly upregulated in adult CML CD34+ cells by 4-fold (p=0.0045), 3.26-fold (p&amp;amp;lt;0.0001), and 3.09-fold (p&amp;amp;lt;0.0001), respectively, compared to pediatric CML CD34+ cells. Furthermore, DLC1, which is known as a negative regulator of Rho pathway, was significantly upregulated in pediatric CML CD34+ cells by 2.47-fold (p=0.0493) compared to adult CML CD34+ cells. These results demonstrate unique molecular characteristics of pediatric CML that may contribute to the clinical differences at presentation between adult and pediatric disease. A better understanding of the molecular biology of CML across the ages will provide new insights into the pathogenesis of pediatric CML and potentially inform future treatment decisions. Disclosures Davis: Jazz Pharmaceuticals: Research Funding; Novartis Pharmaceuticals: Honoraria. Hijiya: Novartis: Consultancy; Stemline Therapeutics: Consultancy.
Background-Older age has historically been an adverse prognostic factor in pediatric acute myeloi... more Background-Older age has historically been an adverse prognostic factor in pediatric acute myeloid leukemia (AML). The impact of age relative to that of other prognostic factors on the outcome of patients treated in recent trials is unknown. Methods-Clinical outcome and causes of treatment failure of 351 patients enrolled on three consecutive protocols for childhood AML between 1991 and 2008 were analyzed according to age and protocol. Results-The more recent protocol (AML02) produced improved outcomes for 10-to 21-yearold patients compared to 2 earlier studies (AML91 and 97), with 3-year rates of event-free survival (EFS), overall survival (OS) and cumulative incidence of refractory leukemia or relapse (CIR) for this group similar to those of 0-to 9-year old patients: EFS, 58.3% ± 5.4% vs. 66.6% ± 4.9%, P=.20; OS, 68.9% ± 5.1% vs. 75.1% ± 4.5%, P=.36; cumulative incidence of refractory leukemia or relapse, 21.9% ± 4.4%; vs. 25.3% ± 4.1%, P=.59. EFS and OS estimates for 10-15year-old patients overlapped those for 16-21-year-old patients. However, the cumulative incidence of toxic death was significantly higher for 10-to 21-year-old patients compared to younger patients (13.2% ± 3.6 vs. 4.5% ± 2.0%, P=.028). Conclusion-The survival rate for older children with AML has improved on our recent trial and is now similar to that of younger patients. However, deaths from toxicity remain a significant problem in the older age group. Future trials should focus on improving supportive care while striving to develop more effective antileukemic therapy.
BACKGROUND-The effect of body mass index (BMI) on treatment outcome of children with acute myeloi... more BACKGROUND-The effect of body mass index (BMI) on treatment outcome of children with acute myeloid leukemia (AML) is unclear and needs further evaluation. METHODS-Children with AML (n=314) enrolled in 4 consecutive St. Jude protocols were grouped according to BMI (underweight, <5 th percentile; healthy weight, 5 th to 85 th percentile; and overweight/obese, ≥ 85 th percentile). RESULTS-Twenty-five (8.0%) patients were underweight, 86 (27.4%) overweight/obese, and 203 (64.6%) had healthy weight. Five-year overall survival of overweight/obese patients (46.5±7.3%) was lower than that of patients with healthy weight (67.1±4.3%, P < .001); underweight patients also tended to have lower survival rates (50.6±10.7%, P = .18). In a multivariable analysis adjusting for age, leukocyte count, FAB type, and study protocols, patients with healthy weight had the best survival rate among the 3 groups (P = .01). When BMI was considered as continuous variable, patients with lower or higher BMI percentiles had worse survival (P = .03). There was no difference in the occurrence of induction failure or relapse among BMI groups but underweight and overweight/obese patients had a significantly higher cumulative incidence of treatment-related mortality, especially due to infection (P = .009). CONCLUSIONS-An unhealthy BMI is associated with worse survival and more treatmentrelated mortality in children with AML. Meticulous supportive care, with nutritional support and
9530 Background: Leukemia accounts for ∼40% of newly diagnosed pediatric malignancies, and relaps... more 9530 Background: Leukemia accounts for ∼40% of newly diagnosed pediatric malignancies, and relapsed leukemia is the leading cause of death in childhood cancer. Genomic instability events contribute to neoplastic development and have been used to classify and risk stratify non-leukemic adult and pediatric tumors. Analyzing leukemic blasts for gene copy changes with advanced molecular techniques could prove useful in further risk stratifying and developing new treatment strategies for pediatric leukemia. Methods: Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel with high specificity and sensitivity at the highest genomic resolution, and were originally designed for single nucleotide genotyping. The MIP assay was adapted to analyze both gene copy number and loss of heterozygosity (LOH) events in pediatric leukemia samples (pre-B ALL, T-ALL, AML). DNA was extracted (100 ng) from paired bone marrow (diagnosis) and peripheral blood (remission) samples (n = 40). The MIP assay was run with a customized Affymetrix 20K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). Gene copy number changes were identified by comparing probe signal intensity between leukemia samples and normal cell-lines. LOH events were determined by identifying genotype changes between matched leukemic and remission samples. Results: Each sample had unique patterns of multiple gene copy changes and LOH events distributed across all chromosomes. Additionally, samples were found to have overlapping copy number changes and LOH regardless of leukemia type. AML samples had fewer LOH events and could be separated by unsupervised clustering from the other leukemia samples. Conclusions: MIPs represent novel genotyping technology that can be adapted for gene copy analysis of childhood leukemia. Unique and distinguishing signatures of allelic imbalance can be determined between ALL and AML clinical samples using MIP technology. The unexpected overlap of LOH and deleted genes may represent a common molecular mechanism that requires further investigation. No significant financial relationships to disclose.
Treatment-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/t-AML) is a devastating ... more Treatment-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/t-AML) is a devastating complication for pediatric patients with osteosarcoma. Even with intensive chemotherapy, overall survival ranges from 10-26%. These regimens cause significant morbidity, poor quality of life and increased toxicity, at times precluding the ultimate goal of hematopoietic cell transplant (HCT). Chromatin remodeling therapy with decitabine and vorinostat has shown some promise in adults with relapsed/refractory AML and MDS. The synergistic combination of methotrexate and asparaginase, also referred to as &amp;#39;Capizzi methotrexate&amp;#39;, is not generally part of upfront treatment for AML but does have some activity in relapsed/refractory disease. Using this rationale, we report the use of this combination regimen in 2 children with t-AML where chromatin remodeling therapy with subsequent Capizzi methotrexate successfully bridged both patients to HCT. Patients were treated at Lucile Packard Children&amp;#39;s Hospital Stanford between 2010 and 2018 for osteosarcoma and subsequently for t-AML. An overview of the treatments used is described in Table 1. An adolescent female was treated for metastatic osteosarcoma. She developed an isolated pulmonary recurrence 5 months after completing therapy. She underwent resection of her pulmonary nodule and then was enrolled on a clinical trial. Four months later, she developed another pulmonary recurrence. At this time, she began treatment with ifosfamide and etoposide. After the sixth cycle, she was noted to have blasts in her blood, and a bone marrow evaluation confirmed t-MDS. Cytogenetics were negative for deletion of 5q, 7q or MLL rearrangement. Repeat bone marrow evaluation two months later revealed progression to t-AML. She initially received 5 days of high dose cytarabine without response and was then transitioned to Treatments A and B. Bone marrow evaluation on day 30 of Treatment B showed morphologic remission with 11% MRD. She underwent Treatment C and then proceeded to a matched sibling HCT. She had no detectable osteosarcoma at the time of her transplant. The patient remained in remission from t-AML but subsequently died from recurrent osteosarcoma more than 2 years after HCT. An adolescent male was treated for non-metastatic osteosarcoma of the left distal femur with methotrexate, adriamycin, cisplatin, ifosfamide and etoposide. Six years after completing treatment, he developed weight loss, epistaxis and pancytopenia. Bone marrow aspiration revealed a hypercellular marrow with 18% blasts, increased marrow fibrosis and cytogenetics with a complex del(7q)/+8 clone, consistent with t-MDS. He received Treatment B, and a bone marrow evaluation on day 25 showed a hemodilute specimen with MRD &amp;amp;lt;0.1%. He then received vincristine and methotrexate on day 35 followed by erwinia on day 36. Repeat bone marrow evaluation on day 42 again showed a hypocellular marrow with MRD of &amp;amp;lt;0.1%. He went on to receive another cycle of vincristine and methotrexate followed by days 1 through 5 of Treatment B. Bone marrow evaluation on day 29 showed a hypocellular marrow with insufficient viable cells for MRD analysis. The patient received an alpha/beta T cell depleted haploidentical transplant. He remains in morphologic remission with no evidence of disease more than 3 months after HCT. Treatment-related MDS/AML remains a devastating complication of osteosarcoma treatment with no standard treatment. The two cases presented here demonstrated good response to a novel combination of therapy with decitabine/vorinostat followed by Capizzi methotrexate. Given the significant toxicity associated with most regimens for t-MDS/t-AML, this regimen should be considered when first line salvage chemotherapy has failed. The precise regimen as well as the optimal number of cycles prior to stem cell transplant should be evaluated in the context of a clinical trial. Disclosures No relevant conflicts of interest to declare.
Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the ... more Background: Leukemia accounts for over 30% of newly diagnosed childhood malignancies, and is the leading cause of death for children with cancer. Genomic instability events contribute to tumorigenesis and have been used to classify and risk stratify adult and pediatric cancers. Molecular Inversion Probes (MIPs) analyze genetic target sequences in parallel at the highest genomic resolution, and can detect both gene copy number and loss of heterozygosity (LOH) events in clinical samples. Studying pediatric leukemia samples with MIP technology may identify new molecular alterations that could prove useful in risk stratification and discovery of new therapeutic targets for childhood leukemia. Objective: To use MIP technology to identify novel areas of allelic imbalance in childhood leukemia. Methods: DNA was extracted from leukemia blasts at diagnosis (n=45, 23 pre-B ALL, 14 AML, 7 pre-T ALL, 1 Burkitt’s). DNA was also extracted from normal peripheral blood collected at remission to use as paired germline controls. The MIP assay was run with a customized Affymetrix 24K Cancer Panel (representing oncogenes, tumor suppressor, DNA repair, cell growth, and metabolism genes). DNA required for this assay was limited to 75 ng per sample. Copy number changes and LOH were identified by comparing probe signal intensity between leukemia and normal germline samples. Clinical cytogenetic data (karyotype and FISH analysis) was used as a control to confirm findings from known areas of allelic imbalance. Results: Each leukemia sample had unique patterns of allelic imbalance distributed across all chromosomes. MIPs identified all clinically reported cytogenetic copy number changes for each sample, in addition to areas of allelic imbalance not clinically reported. Samples had recurring areas of copy number changes and LOH events shared by all leukemia types. MIPs detected areas of allelic imbalance in both previously described and novel genes. Areas of recurring genomic deletions included: ATR (3q23), TLX3 (5q35.1), ADRB3 (8p12), CDKN2A (9p21.3), DOCK8 (9p24.3), PAX5 (9p13.2), PTPN11 (12q24.13), C3AR1 (12p13.31), TCRA (14q11.2), AKT1 (q14q32.33). Areas of recurring genomic amplification included: SLC2A9 (4p16.1), RAI14 (5p13.2), CDH12 (5p14.3), PMCHL1(5p14.3), AURKB (17p13.1). These findings are being validated with Quantitative Real-Time PCR. Conclusions: MIPs represent a novel genomic technology that can identify previously unreported gene copy number and LOH events in childhood leukemia. Unique and overlapping areas of allelic imbalance were found in both childhood ALL and AML clinical samples. The shared genomic regions of allelic imbalance between different leukemia types may represent a common molecular mechanism of leukemogenesis that warrants further investigation. Analysis of more childhood leukemia samples through Pediatric Cooperative Groups may help to determine how common these areas of allelic imbalance are in children and whether they are of prognostic significance. Further exploration of these copy number and LOH events may help us to better understand the biology of childhood leukemia and its clinical behavior.
Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subt... more Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subtype, FLT3 status and response to induction therapy are determinants of outcome with current therapies. We hypothesize that array comparative genomic hybridization (CGH) will identify gene copy number changes that are determinants of outcome. Array CGH was performed on diagnostic bone marrow samples from patients on the COG study POG #9421. In order to determine regions of altered gene copy number, labeled genomic DNA samples were hybridized together with sex-matching normal human reference DNA to cDNA microarrays with 41,751 features (corresponding to 24,473 unique Unigene cluster IDs), arrays were obtained from the Stanford University Microarray Core Facility. Control hybridizations were performed to assess intra- and inter-experimental variability. We studied 70 samples with adequate high-quality DNA. Circular binary segmentation was used to distinguish discrete gene copy number transition points from chance noise events and to transform primary clone-by-clone data into genomic regions of equal copy number. Using gain/loss threshold, based on two-standard deviation range of control self-to-self distribution, novel gene amplifications and deletions were found in profiled samples. The highest alteration recurrence was observed for gains of chromosome 8 (21%) and losses of chromosome 6 (29%). The area of chromosome 8 which was found to be gained is notable for the presence of potential oncogenes such as ERK8. The deleted area of chromosome 6 is notable for the presence of potential regulators of oncogenesis: MDC1, DDR1, NFKBIL1, TNF, and BRD2. In summary, array CGH has identified novel areas of gene copy number gain and loss in this population of pediatric de novo AML patients. Further studies are needed to assess whether these genes are associated with outcome, known risk factors and whether they will provide insight into the heterogeneity of de novo AML.
Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,... more Daunorubicin and cytarabine are the most effective chemotherapeutic agents for AML. Using MTT (3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays as an in vitro test for sensitivity to these agents, investigators have found that leukemia cells from pediatric AML patients with Down Syndrome, which are more sensitive to cytarabine than blasts from non-Down Syndrome AML patients, have been associated with an increased ratio of deoxycytidine kinase to cytidine deaminase and an increased level of cystathionine-beta-synthetase [Ge, Jensen, Stout et al. Cancer Res2004; 64, 728–35]. Looking for related findings in non-Down syndrome AML, leukemic blasts from non-Down syndrome children with AML were examined utilizing gene expression profiling and MTT assays. Expression signatures from cDNA arrays were related to the drug sensitivity results to daunorubicin and cytarabine by supervised clustering. These results will then be analyzed to look for relationships to the patient’s clinical outcomes and identify genes that may be potential therapeutic targets for drug resistance reversal. Bone marrow or blood specimens from 103 patients registered on the Pediatric Oncology Group (POG) protocol 9421 were studied. The diagnostic specimens were tested using MTT assays to measure the IC50 (drug concentration that causes 50% of the cells to die) for both daunorubicin and cytarabine. Samples from ninety-three de novo childhood AML patients were analyzed with a 43,760 element spotted array (containing 41,751 unique genes and expression sequence tags [ESTs]) from the Stanford University Microarray Core Facility. We selected the gene expression profile from 10 samples with the highest and lowest IC50’s for each drug. Then, we used Significance Analysis of Microarrays (SAM) to find significant differences in the gene expression between samples sensitive and resistant to each drug. A cluster of 118 overexpressed genes was associated with resistance to daunorubicin (e.g., BCL6, BAPX1, BCL2A1, MBD2, RAB31 and CDKN1A). A cluster of 24 overexpressed genes was associated with resistance to cytarabine (e.g., RIT1, SH3BP2, BCL2A1, NFKBIA, and PTPRE). In summary, MTT assays and gene expression profiling have identified genes that correlate with daunorubicin and cytarabine resistance. Study of the relationship among drug resistance patterns, gene expression profiles and clinical outcome may allow us to better predict patients’ prognoses at diagnosis and also can be used to identify novel targets for drug resistance reversal.
Abstract 482 Background: Acute megakaryoblastic leukemia (AMKL) in children without Down syndrome... more Abstract 482 Background: Acute megakaryoblastic leukemia (AMKL) in children without Down syndrome represents approximately 10% of pediatric acute myeloid leukemia (AML). Published reports regarding prognosis of non-Down syndrome children with AMKL vary; hence, some groups treat these patients as high-risk while others as standard-risk. As a result, the optimal treatment strategy, including the role of hematopoeitic stem cell transplant (HSCT), remains unclear. Methods: We reviewed the treatment and outcomes of patients with AMKL treated on two pediatric AML protocols. Pediatric Oncology Group (POG) 9421 (1995-99) included two cycles of induction chemotherapy with randomization to standard or high-dose DAT (daunorubicin, cytarabine, 6-thioguanine) followed by HSCT for children in remission with matched sibling donors or three cycles of consolidation chemotherapy with or without cyclosporine for those without suitable donors. St. Jude protocol AML02 (2002-2008) randomized patients during induction to receive high- or low-dose cytarabine with daunorubicin and etoposide (ADE). Patients without complete remission (CR) received gemtuzumab ozogamicin (GO) with induction 2 chemotherapy (ADE). After induction, AMKL patients with availabel donor proceeded to HSCT while those without donors received three cycles of consolidation chemotherapy including mitoxantrone and cytarabine. Comparisons of event-free survival (EFS) and overall survival (OS) were performed by Mantel-Haenszel log-rank test. Independent effect of HSCT on EFS or OS were analyzed by Cox proportional hazard model, with HSCT as time-dependent variable. Results: The 49 patients with AMKL treated on POG 9421 had a median age at diagnosis of 1.8 years (range 0.13-16.2 years), and a median leukocyte count of 13.5 × 109/L (range 0.3-98.4). The patient with the t(1;22) translocation remains in CR. 39 patients had CR after induction, 5 partial response and 4 no response with no difference between standard- and high-dose DAT. The 5-year EFS for all AMKL patients was 34.7% ± 7.5%. Of the 39 patients in CR, 6 underwent matched sibling HSCT, of whom 4 remain in CR, and the other 33 patients received consolidation chemotherapy, of whom 15 remain in continuous remission. The 5-year OS for patients undergoing HSCT was 66.7% ± 19.3% compared to 36.1% ± 8.4% for those receiving consolidation chemotherapy (p = 0.2). The 26 patients with AMKL treated on SJCRH AML02 had a median age at diagnosis of 1.2 years (range 0.21-11.2 years), and a median leukocyte count of 11.8 × 109/L (range 2.3-72.9). All 5 patients with the t(1;22) translocation achieved CR after one induction cycle, with negative minimal residual disease, and remain in CR after treatment with chemotherapy only. The 3-year EFS was 35.9% ± 12.9% for the other AMKL patients, significantly lower than the 61.5% ± 4.8% for non-AMKL patients (p = 0.026). The 3-years OS estimates are 100% for patients with t(1;22), 48% ± 14.1% for other AMKL patients, and 71.8% ± 4.4% for non-AMKL patients (p = 0.03). Of the 21 patients without t(1;22), 14 received HSCT with 7 survivors (3-year EFS 46.3 ± 17%) while 7 received chemotherapy with only one long-term survivor (3-year EFS 14.3 ± 9.4%) (p=0.55). Conclusions: AMKL patients with the t(1;22) translocation have excellent treatment outcome with chemotherapy alone. In contrast, AMKL patients without t(1;22) fared poorly, especially when they were treated with only chemotherapy. Additional studies of larger number of patients are needed to determine if they would benefit from HSCT. Disclosures: No relevant conflicts of interest to declare.
Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with mye... more Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10 to 15% of children with myeloid leukemia and 2 to 9% of all pediatric leukemias. Prior to the discovery of tyrosine kinase inhibitors (TKI) such as imatinib, stem cell transplantation was the only curative treatment for both adults and children with CML. However, due to the small numbers of patients, standardized treatment approaches for pediatric CML have not been established. There are several unique characteristics of CML diagnosed in children and adolescents, and young adults (AYA; 16-29 years), compared to adults. Children and AYA with CML present with a higher white blood count and have larger spleens, higher peripheral blast counts, and lower hemoglobin levels, suggesting that the biology of pediatric CML is different than adult CML. In addition, potential side effects of TKIs unique to pediatric CML patients include impaired bone growth, fertility and immune function, however none have been extensively studied. We hypothesize that the differences in clinical presentation of pediatric CML patients are due to unique molecular characteristics that are absent in adult CML patients. To test this hypothesis, we studied the transcriptomic signature of pediatric CD34+ CML cells compared to adult CML and normal age-matched bone marrow CD34+ cells. Methods CD34+ cells were isolated from pediatric CML (n=7), adult CML (n=8), pediatric normal (n=2) and adult normal (n=3) bone marrow samples. Total RNA was isolated from cells, and then cDNA libraries were generated. Prepared libraries were sequenced on the Illumina HiSeq 4000 instrument. We aligned reads using the HISAT2 alignment software, and mapped to genes with HT-Seq. We removed genes that had zero reads across all the samples, resulting in a set of 4,696 genes that were detected in one or more samples. In case of technical replicates, we used mean of replicates. We performed three differential expression comparisons with edgeR: (1) Pediatric CML vs Adult CML, (2) Adult CML vs Adult Normal, and (3) Pediatric CML vs Pediatric Normal. We used a False Discovery Rate (FDR) of £ 20% and absolute log2 fold-change ³ 1 for selecting differentially expressed genes in each comparison. We used Fisher's exact test to identify significant KEGG pathways for the differentially expressed genes in each comparison. Results Pediatric CML vs Adult CML We found 24 differentially expressed genes (15 over- and 9 under-expressed). Though no pathway was found to be significant at the false discovery rate (FDR) £ 20%, we identified a number of sub-pathways that are relevant. For example, the Chemokine Signaling pathway shows at the top of the list (ordered by raw p-value) because of two genes, XCR1 and HCK, associated with VEGF and MAPK pathways involved in cell proliferation, angiogenesis, DNA repair, and cancer pathogenesis. Adult CML vs Adult Normal We found 60 genes (30 over- and 30 under-expressed) differentially expressed when comparing adult CML patients to normal adults. Ten genes overlapped with 24 genes we identified when comparing pediatric and adult CML patients. We found 11 pathways as significant at FDR £ 10%. Multiple pathways, including Cell adhesion, allograft rejection, Graft versus Host Disease, and Type I diabetes pathways, showed downregulation of MHC, with subsequent downstream reduction in expression of apoptosis-related genes. The IL-17 pathway makes sense, as MAPK, well-known to be associated with various cancers, is down-regulated. Lastly, in the NK pathway the gene DAP12 is up-regulated. This gene is known as a tyrosine kinase binding protein, and although tyrosine kinase inhibitors are the standard treatment for CML, the role of DAP12 in relation to leukemia has not yet been described. Pediatric CML vs Pediatric Normal We found 509 genes (350 over- and 159 under-expressed) differentially expressed in pediatric CML patients compared to normal. Interestingly, transcriptional regulators are differentially enriched in the hematopoietic stem cell differentiation function group including GATA1, GATA2, KLF1 and KLF2. RFC is down-regulated. RFC is a mismatch repair gene known to be involved in colorectal cancer. Many of the significant pathways are involved in glucose and fatty acid metabolism. Our pilot study identified novel molecular features of pediatric CML bone marrow stem cells, providing new insights into the novel biomarkers and pathogenesis of pediatric CML. Disclosures Gotlib: Blueprint Medicines: Consultancy, Honoraria, Research Funding; Promedior: Research Funding; Deciphera: Consultancy, Honoraria, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Kartos: Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.
IntroductionEx vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstra... more IntroductionEx vivo gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. MethodsWe compare the two approaches for integration of IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)-IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associ...
Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a vi... more Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing–based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.
Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte ... more Granulocytes from patients with chronic granulomatous disease (CGD) have dysfunctional phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that fails to generate sufficient antimicrobial reactive oxidative species. CGD patients with severe persistent fungal or bacterial infection who do not respond to antibiotic therapy may be given apheresis-derived allogeneic granulocyte transfusions from healthy volunteers to improve clearance of intractable infections. Allogeneic granulocyte donors are not HLA matched, so patients who receive the donor granulocyte products may develop anti-HLA alloimmunity. This not only precludes future use of allogeneic granulocytes in an alloimmunized CGD recipient, but increases the risk of graft failure of those recipients who go on to need an allogeneic bone marrow transplant. Here, we provide the first demonstration of efficient functional restoration of CGD patient apheresis granulocytes by messenger RNA (mRNA) electroporation u...
Transcriptional repression by chimeric transcription factors is emerging as a common theme in leu... more Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most si...
The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #... more The event-free survival (EFS) estimate for patients with normal karyotype (NK) on COG study POG #9421 (n=144) was 36%. We previously reported a subgroup of patients (n=68) with AML and NK that could be divided into 2 groups whose clinical outcomes correlated with abnormalities of FLT3 [internal tandem duplications (ITD) or activating loop mutations]. EFS estimates were 13% for patients with mutant FLT3 and 61% for children with wild-type FLT3 (P=0.01). We hypothesized that gene expression profiling would identify signatures that are linked to clinical outcome and can be used for risk determination. Cytogenetic testing was carried out in clinical laboratories at the institutions in which AML was diagnosed and then centrally reviewed. We analyzed bone marrow from 45 patients with NK on 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. FLT3 status (mutant or wild type) wa...
The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;... more The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome. ...
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