Norman Lacayo - Profile on Academia.edu (original) (raw)
Papers by Norman Lacayo
Nature Communications, Feb 17, 2022
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to per... more The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre-and post normalization with CytofIn demonstrates effective batch correction while recapitulating the goldstandard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.
Supplementary Data from Epigenetic Targeting of <i>TERT</i>-Associated Gene Expression Signature in Human Neuroblastoma with <i>TERT</i> Overexpression
Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT... more Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT gene rearrangement. Supplementary Fig. S2. Effects of dinaciclib on genome-wide chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S3: C-Myc and MYCN protein expression in four NB cell lines. Supplementary Fig. S4: Effects of dinaciclib on the chromatin occupancy of epigenetic modulators at Dvl2, AURKA, and AURKB and in CLB-GA cells. Supplementary Fig. S5. Association of overexpression of FEN1, BIRC5, and UHRF1 with poor prognosis and effects of dinaciclib on chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S6. JQ1 sensitive Brd4 binding profiles in OCI-AML3 cells. Supplementary Fig. S7. JQ1 and dinaciclib are synergistic or additive in inducing cytotoxicity in NB cell lines. Supplementary Fig. S8. Combination index (CI) values for synergy experiment combining Dinaciclib with Brd4 inhibitor JQ1. Supplementary Fig. S9. Effects of dinaciclib on phosphorylation of Rb in CLB-GA and Kelly cells. Supplementary table S1. Primer sequences used in ChIP-qPCR and real-time RT-PCR analysis. Supplementary table 2. Gene sets enriched in NB patients with TERT gene rearrangement (n=32) versus without TERT gene rearrangement (n=278) within MYCN non-amplified NB of Tumor Neuroblastoma (TERT)-Fischer-394-custom. Supplementary table 3. Gene sets enriched in NB patients with high versus low TERT within Tumor Neuroblastoma Gene-TARGET-161. Supplementary table 4: Information on patient samples used in this study.
Journal for ImmunoTherapy of Cancer, Feb 1, 2023
Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric rela... more Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. Main body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 18Ffluorodeoxyglucose revealed diffuse leukemia with innumerable bone and lymph node lesions, interestingly sparing her sacrum, the site of her bone marrow aspirate sampling. Conclusions: We highlight this case as both peripheral blood MRD and 18F-fluorodeoxyglucose positron emission tomography imaging were more sensitive than standard bone marrow aspirate testing in detecting this patient's post-CAR relapse. Clinical/Biologic Insight: In the multiply relapsed B-ALL setting, where relapse patterns may include patchy medullary and/or EM disease, peripheral blood MRD and/or whole body imaging, may carry increased sensitivity at detecting relapse in patient subsets, as compared with standard bone marrow sampling. Contributors LS directed patient clinical care and was primary author of the manuscript. AW, CE, CB, KLD, SR, CA, NL, HRN, JO, and CM were involved in patient clinical care. All authors read and approved the final manuscript.
Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-X L / BCL2 inhi... more Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-X L / BCL2 inhibitor, may allow targeting of both BCL2 and BCL-X L without doselimiting thrombocytopenia associated with navitoclax monotherapy. The safety and preliminary efficacy of venetoclax with low-dose navitoclax and chemotherapy was assessed in this phase I doseescalation study (NCT03181126) in pediatric and adult patients with relapsed/refractory (R/R) acute lymphoblastic leukemia or lymphoblastic lymphoma. Forty-seven patients received treatment. A recommended phase II dose of 50 mg navitoclax for adults and 25 mg for patients <45 kg with 400 mg adult-equivalent venetoclax was identified. Delayed hematopoietic recovery was the primary safety finding. The complete remission rate was 60%, including responses in patients who had previously received hematopoietic cell transplantation or immunotherapy. Thirteen patients (28%) proceeded to transplantation or CAR T-cell therapy on study. Venetoclax with navitoclax and chemotherapy was well tolerated and had promising efficacy in this heavily pretreated patient population. In this phase I study, venetoclax with low-dose navitoclax and chemotherapy was well tolerated and had promising efficacy in patients with relapsed/refractory acute lymphoblastic leukemia or lymphoblastic lymphoma. Responses were observed in patients across histologic and genomic subtypes and in those who failed available therapies including stem cell transplant.
Oncotarget, Jun 23, 2020
The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although ... more The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.
Abstract 1509: Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach
Cancer Research, Apr 4, 2023
For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionabl... more For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionable results, partly due to the complex genomic alterations present. We hypothesized that an unbiased approach, combining whole-genome (WGS) and RNA sequencing (RNAseq), could overcome this and lead to a more comprehensive understanding of these diseases. While prior studies have evaluated WGS and RNAseq in pediatric cancers, few focused primarily on metastatic or relapsed disease. We also placed special focus on longitudinal profiling of patients, including with additional deep sequencing, to capture tumor evolution at the primary and metastatic sites, and to quantify the utility of resampling. We assembled a cohort of 191 high-risk pediatric oncology patients, including solid tumors, CNS tumors, and leukemias/lymphomas. We have representation of patients with relapsed/refractory disease (68), metastatic disease at diagnosis (10), rare diagnoses (19), prior cancer history, and estimated overall survival <50%. We characterized 280 samples with WGS (tumor ~60X; germline ~30X) and/or RNAseq (tumor, polyA selected, ≥20 million reads), including multiple samples taken from 85 patients at different time points (diagnosis, resection, relapse, etc.). Variants (SNVs), structural rearrangements (SVs), mutational signatures, and copy-number alterations (CNAs) were identified using WGS. RNAseq was used to profile gene expression outliers, gene fusions, and expression of variants identified by WGS. The integrated results were used to prioritize potentially actionable variants for each patient. For 20 patients (44 samples), we performed targeted deep sequencing of the DNA (~500X) to profile tumor evolution that cannot be captured by WGS. Multiple sampling from the same patient identified drastic spatial and temporal differences in the genomes and transcriptomes of these tumors. Using the Jaccard index as a measure of concordance between samples shows dynamic changes between samples collected at different time points across multiple modalities (range 0-1, 1 is identical); SNVs ranged from 0.01-0.79, SVs 0.01-0.73, major CNAs 0.07-0.99, minor CNAs 0.38-0.99, up expression outliers 0.12-0.56, down expression outliers 0.04-0.54, and fusions 0-1. Potentially biologically significant differences in therapy-induced mutations by platinum agents were also observed, highlighting the impact of therapy on tumor evolution. Clonal architectures were extracted from deep resequencing and show extensive spatial, temporal, and metastatic heterogeneity in these rare and highly aggressive malignancies that is not captured by WGS alone. Identifying clinically relevant evolution remains a challenge in most patients, but our results suggest that resampling of pediatric tumors at relapse or metastasis will be important for the effectiveness of targeted therapies in the future. Citation Format: Henry J. Martell, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, María V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, Alejandro Sweet-Cordero. Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1509.
Data from Epigenetic Targeting of <i>TERT</i>-Associated Gene Expression Signature in Human Neuroblastoma with <i>TERT</i> Overexpression
Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Part... more Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.Significance:Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.
Abstract B20: Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies
Poster Presentations - Proffered Abstracts, Jul 15, 2020
Targeted gene panel sequencing has become increasingly common in the management of pediatric canc... more Targeted gene panel sequencing has become increasingly common in the management of pediatric cancer patients. For some patients, these cancer gene panel tests have identified clinically actionable findings, but for many pediatric patients, no actionable alterations are identified. This is in part due to the low mutational burden of pediatric malignancies; thus, an unbiased approach may shed light on potentially actionable findings. To accomplish this, we examined the feasibility and utility of whole-genome sequencing (WGS) and RNA sequencing (RNAseq) in the management of high-risk pediatric oncology patients. We describe our experience with a cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and hematologic malignancies. The majority of patients were deemed high-risk due to relapsed/refractory disease. A second group of patients was defined as high-risk at time of initial diagnosis due to the presence of metastatic disease, an estimated overall survival of less than 50%, a rare tumor, an undifferentiated tumor, or prior history of another malignancy. When possible, multiple samples from an individual patient were collected (i.e., specimens at biopsy, resection, relapse, and/or from metastatic sites) to allow for evaluation of inter- and intratumoral heterogeneity. Close to 200 tumor samples were available for analysis using WGS and/or RNAseq analysis. Somatic DNA samples were sequenced to an average depth of 60X and germline samples to 30X. WGS samples were analyzed for SNVs, structural rearrangements (SVs), copy-number alterations (CNAs), and mutational signatures. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. These samples were analyzed to identify known and novel gene-fusions, measure allele specific expression of SNVs, and perform gene-expression outlier analysis. Expression of variants (SNV/SV) identified using WGS were confirmed using RNAseq. For gene expression outliers detected using RNAseq, the WGS data were used to predict possible mechanisms for the aberrant expression (such as CNA, gene fusions, or promoter hijacking). This analysis suggests that WGS and RNAseq analysis is feasible in a clinical setting and can reliably identify variants reported on gene panel tests. Furthermore, the use of WGS/RNAseq results in additional clinically informative findings while also enabling novel research to further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Avanthi T. Shah, Marcus R. Breese, Alex G. Lee, Henry J. Martell, Bogdan Tanasa, Stanley G. Leung, Aviv Spillingeer, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Florette K. Hazard, Soo-Jin Cho, Arun Rangaswami, Norman J. Lacayo, Sheri L. Spunt, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agarwaal, Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B20.
Molecular Cancer Therapeutics, Aug 5, 2021
Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric s... more Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.
Journal for ImmunoTherapy of Cancer
Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric rela... more Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. Main body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 1...
Nature Communications
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to per... more The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to int...
in childhood AML
incidence and clinical significance of nucleophosmin mutations
Gene Expression Analysis of CML Patients across the Age Spectrum
Blood, 2021
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 u...
Cancer Discovery, 2021
Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-XL/BCL2 inhibit... more Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-XL/BCL2 inhibitor, may allow targeting of both BCL2 and BCL-XL without dose-limiting thrombocytopenia associated with navitoclax monotherapy. The safety and preliminary efficacy of venetoclax with low-dose navitoclax and chemotherapy was assessed in this phase I dose-escalation study (NCT03181126) in pediatric and adult patients with relapsed/refractory (R/R) acute lymphoblastic leukemia or lymphoblastic lymphoma. Forty-seven patients received treatment. A recommended phase II dose of 50 mg navitoclax for adults and 25 mg for patients <45 kg with 400 mg adult-equivalent venetoclax was identified. Delayed hematopoietic recovery was the primary safety finding. The complete remission rate was 60%, including responses in patients who had previously received hematopoietic cell transplantation or immunotherapy. Thirteen patients (28%) proceeded to transplantation or CAR T-cell therapy on study. Venetoclax...
Haematologica, 2020
Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being develop... more Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations i...
Array CGH Discovers Novel Genomic Signatures in De Novo Acute Myeloid Leukemia (AML): Results of Children’s Oncology Group (COG) Study POG #9421
Blood, 2005
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 transi...
Molecular Inversion Probes (MIPs) Identify Novel Areas of Allelic Imbalance in Childhood Leukemia
Blood, 2007
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...
Adapting molecular inversion probe (MIP) technology for allele quantification in childhood leukemia
Journal of Clinical Oncology, 2007
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...
Cancer Research, 2020
Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Part... more Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of...
Comparison of the Transcriptomic Signature of Pediatric Vs. Adult CML and Normal Bone Marrow Stem Cells
Blood, 2018
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 stu...
Nature Communications, Feb 17, 2022
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to per... more The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre-and post normalization with CytofIn demonstrates effective batch correction while recapitulating the goldstandard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to integrate public datasets without necessitating identical control samples or bead standards for fast and robust analysis using CytofIn.
Supplementary Data from Epigenetic Targeting of <i>TERT</i>-Associated Gene Expression Signature in Human Neuroblastoma with <i>TERT</i> Overexpression
Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT... more Supplementary Fig. S1. Elevated mRNA expression of TERT and TERT-associated genes in NB with TERT gene rearrangement. Supplementary Fig. S2. Effects of dinaciclib on genome-wide chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S3: C-Myc and MYCN protein expression in four NB cell lines. Supplementary Fig. S4: Effects of dinaciclib on the chromatin occupancy of epigenetic modulators at Dvl2, AURKA, and AURKB and in CLB-GA cells. Supplementary Fig. S5. Association of overexpression of FEN1, BIRC5, and UHRF1 with poor prognosis and effects of dinaciclib on chromatin occupancy of epigenetic modulators in CLB-GA cells. Supplementary Fig. S6. JQ1 sensitive Brd4 binding profiles in OCI-AML3 cells. Supplementary Fig. S7. JQ1 and dinaciclib are synergistic or additive in inducing cytotoxicity in NB cell lines. Supplementary Fig. S8. Combination index (CI) values for synergy experiment combining Dinaciclib with Brd4 inhibitor JQ1. Supplementary Fig. S9. Effects of dinaciclib on phosphorylation of Rb in CLB-GA and Kelly cells. Supplementary table S1. Primer sequences used in ChIP-qPCR and real-time RT-PCR analysis. Supplementary table 2. Gene sets enriched in NB patients with TERT gene rearrangement (n=32) versus without TERT gene rearrangement (n=278) within MYCN non-amplified NB of Tumor Neuroblastoma (TERT)-Fischer-394-custom. Supplementary table 3. Gene sets enriched in NB patients with high versus low TERT within Tumor Neuroblastoma Gene-TARGET-161. Supplementary table 4: Information on patient samples used in this study.
Journal for ImmunoTherapy of Cancer, Feb 1, 2023
Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric rela... more Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. Main body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 18Ffluorodeoxyglucose revealed diffuse leukemia with innumerable bone and lymph node lesions, interestingly sparing her sacrum, the site of her bone marrow aspirate sampling. Conclusions: We highlight this case as both peripheral blood MRD and 18F-fluorodeoxyglucose positron emission tomography imaging were more sensitive than standard bone marrow aspirate testing in detecting this patient's post-CAR relapse. Clinical/Biologic Insight: In the multiply relapsed B-ALL setting, where relapse patterns may include patchy medullary and/or EM disease, peripheral blood MRD and/or whole body imaging, may carry increased sensitivity at detecting relapse in patient subsets, as compared with standard bone marrow sampling. Contributors LS directed patient clinical care and was primary author of the manuscript. AW, CE, CB, KLD, SR, CA, NL, HRN, JO, and CM were involved in patient clinical care. All authors read and approved the final manuscript.
Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-X L / BCL2 inhi... more Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-X L / BCL2 inhibitor, may allow targeting of both BCL2 and BCL-X L without doselimiting thrombocytopenia associated with navitoclax monotherapy. The safety and preliminary efficacy of venetoclax with low-dose navitoclax and chemotherapy was assessed in this phase I doseescalation study (NCT03181126) in pediatric and adult patients with relapsed/refractory (R/R) acute lymphoblastic leukemia or lymphoblastic lymphoma. Forty-seven patients received treatment. A recommended phase II dose of 50 mg navitoclax for adults and 25 mg for patients <45 kg with 400 mg adult-equivalent venetoclax was identified. Delayed hematopoietic recovery was the primary safety finding. The complete remission rate was 60%, including responses in patients who had previously received hematopoietic cell transplantation or immunotherapy. Thirteen patients (28%) proceeded to transplantation or CAR T-cell therapy on study. Venetoclax with navitoclax and chemotherapy was well tolerated and had promising efficacy in this heavily pretreated patient population. In this phase I study, venetoclax with low-dose navitoclax and chemotherapy was well tolerated and had promising efficacy in patients with relapsed/refractory acute lymphoblastic leukemia or lymphoblastic lymphoma. Responses were observed in patients across histologic and genomic subtypes and in those who failed available therapies including stem cell transplant.
Oncotarget, Jun 23, 2020
The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although ... more The 90 kDa Ribosomal S6 Kinase (RSK) drives cell proliferation and survival in cancers, although its oncogenic mechanism has not been well characterized. Phosphorylated level of RSK (T573) was increased in acute myeloid leukemia (AML) patients and associated with poor survival. To examine the role of RSK in AML, we analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in AML cell lines and patient AML cells. Characterization of mitotic phases showed that the metaphase/anaphase transition was significantly inhibited by BI-D1870. BI-D1870 treatment impeded the association of activator CDC20 with APC/C, but increased binding of inhibitor MAD2 to CDC20, preventing mitotic exit. Moreover, the inactivation of spindle assembly checkpoint or MAD2 knockdown released cells from BI-D1870-induced metaphase arrest. Therefore, we investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells.
Abstract 1509: Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach
Cancer Research, Apr 4, 2023
For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionabl... more For many pediatric cancer patients, commonly used gene-panel sequencing tests yield few actionable results, partly due to the complex genomic alterations present. We hypothesized that an unbiased approach, combining whole-genome (WGS) and RNA sequencing (RNAseq), could overcome this and lead to a more comprehensive understanding of these diseases. While prior studies have evaluated WGS and RNAseq in pediatric cancers, few focused primarily on metastatic or relapsed disease. We also placed special focus on longitudinal profiling of patients, including with additional deep sequencing, to capture tumor evolution at the primary and metastatic sites, and to quantify the utility of resampling. We assembled a cohort of 191 high-risk pediatric oncology patients, including solid tumors, CNS tumors, and leukemias/lymphomas. We have representation of patients with relapsed/refractory disease (68), metastatic disease at diagnosis (10), rare diagnoses (19), prior cancer history, and estimated overall survival <50%. We characterized 280 samples with WGS (tumor ~60X; germline ~30X) and/or RNAseq (tumor, polyA selected, ≥20 million reads), including multiple samples taken from 85 patients at different time points (diagnosis, resection, relapse, etc.). Variants (SNVs), structural rearrangements (SVs), mutational signatures, and copy-number alterations (CNAs) were identified using WGS. RNAseq was used to profile gene expression outliers, gene fusions, and expression of variants identified by WGS. The integrated results were used to prioritize potentially actionable variants for each patient. For 20 patients (44 samples), we performed targeted deep sequencing of the DNA (~500X) to profile tumor evolution that cannot be captured by WGS. Multiple sampling from the same patient identified drastic spatial and temporal differences in the genomes and transcriptomes of these tumors. Using the Jaccard index as a measure of concordance between samples shows dynamic changes between samples collected at different time points across multiple modalities (range 0-1, 1 is identical); SNVs ranged from 0.01-0.79, SVs 0.01-0.73, major CNAs 0.07-0.99, minor CNAs 0.38-0.99, up expression outliers 0.12-0.56, down expression outliers 0.04-0.54, and fusions 0-1. Potentially biologically significant differences in therapy-induced mutations by platinum agents were also observed, highlighting the impact of therapy on tumor evolution. Clonal architectures were extracted from deep resequencing and show extensive spatial, temporal, and metastatic heterogeneity in these rare and highly aggressive malignancies that is not captured by WGS alone. Identifying clinically relevant evolution remains a challenge in most patients, but our results suggest that resampling of pediatric tumors at relapse or metastasis will be important for the effectiveness of targeted therapies in the future. Citation Format: Henry J. Martell, Avanthi T. Shah, Alex G. Lee, Bogdan Tanasa, Stanley G. Leung, Aviv Spillinger, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, María V. Pons Ventura, Florette K. Hazard, Arun Rangaswami, Sheri L. Spunt, Norman J. Lacayo, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agrawal, Marcus R. Breese, Alejandro Sweet-Cordero. Longitudinal profiling of high-risk pediatric malignancies using a multiomics approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1509.
Data from Epigenetic Targeting of <i>TERT</i>-Associated Gene Expression Signature in Human Neuroblastoma with <i>TERT</i> Overexpression
Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Part... more Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of H3K36me3 in TERT and multiple TERT-associated genes. Brd4 and cyclin-dependent kinases (CDK) had critical regulatory roles in the expression and chromatin activation of TERT and multiple TERT-associated genes. Epigenetically targeting Brd4 or CDKs with their respective inhibitors suppressed the expression of TERT and multiple TERT-associated genes in neuroblastoma with TERT overexpression or MYCN amplification. ChIP-seq and ChIP-qPCR provided evidence that the CDK inhibitor directly inhibited Brd4 recruitment to activate chromatin globally. Therefore, inhibiting Brd4 and CDK concurrently with AZD5153 and dinaciclib would be most effective in tumor growth suppression, which we demonstrated in neuroblastoma cell lines, primary human cells, and xenografts. In summary, we describe a unique mechanism in neuroblastoma with TERT overexpression and an epigenetically targeted novel therapeutic strategy.Significance:Epigenetically cotargeting Brd4 and Cdks suppresses human neuroblastoma with TERT overexpression by inhibiting the TERT-associated gene expression networks.
Abstract B20: Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies
Poster Presentations - Proffered Abstracts, Jul 15, 2020
Targeted gene panel sequencing has become increasingly common in the management of pediatric canc... more Targeted gene panel sequencing has become increasingly common in the management of pediatric cancer patients. For some patients, these cancer gene panel tests have identified clinically actionable findings, but for many pediatric patients, no actionable alterations are identified. This is in part due to the low mutational burden of pediatric malignancies; thus, an unbiased approach may shed light on potentially actionable findings. To accomplish this, we examined the feasibility and utility of whole-genome sequencing (WGS) and RNA sequencing (RNAseq) in the management of high-risk pediatric oncology patients. We describe our experience with a cohort of over 100 high-risk pediatric oncology patients, with a combination of solid tumors, brain tumors, and hematologic malignancies. The majority of patients were deemed high-risk due to relapsed/refractory disease. A second group of patients was defined as high-risk at time of initial diagnosis due to the presence of metastatic disease, an estimated overall survival of less than 50%, a rare tumor, an undifferentiated tumor, or prior history of another malignancy. When possible, multiple samples from an individual patient were collected (i.e., specimens at biopsy, resection, relapse, and/or from metastatic sites) to allow for evaluation of inter- and intratumoral heterogeneity. Close to 200 tumor samples were available for analysis using WGS and/or RNAseq analysis. Somatic DNA samples were sequenced to an average depth of 60X and germline samples to 30X. WGS samples were analyzed for SNVs, structural rearrangements (SVs), copy-number alterations (CNAs), and mutational signatures. RNAseq was performed to a depth of at least 20 million paired-end reads for each sample. These samples were analyzed to identify known and novel gene-fusions, measure allele specific expression of SNVs, and perform gene-expression outlier analysis. Expression of variants (SNV/SV) identified using WGS were confirmed using RNAseq. For gene expression outliers detected using RNAseq, the WGS data were used to predict possible mechanisms for the aberrant expression (such as CNA, gene fusions, or promoter hijacking). This analysis suggests that WGS and RNAseq analysis is feasible in a clinical setting and can reliably identify variants reported on gene panel tests. Furthermore, the use of WGS/RNAseq results in additional clinically informative findings while also enabling novel research to further advance our understanding of these rare and highly aggressive pediatric malignancies. Citation Format: Avanthi T. Shah, Marcus R. Breese, Alex G. Lee, Henry J. Martell, Bogdan Tanasa, Stanley G. Leung, Aviv Spillingeer, Heng-Yi Liu, Inge Behroozfard, Phuong Dinh, Florette K. Hazard, Soo-Jin Cho, Arun Rangaswami, Norman J. Lacayo, Sheri L. Spunt, Tabitha Cooney, Jennifer G. Michlitsch, Anurag K. Agarwaal, Alejandro Sweet-Cordero. Integrative analysis of whole-genome and RNA sequencing in high-risk pediatric malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B20.
Molecular Cancer Therapeutics, Aug 5, 2021
Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric s... more Most circulating tumor DNA (ctDNA) assays are designed to detect recurrent mutations. Pediatric sarcomas share few recurrent mutations but rather are characterized by translocations and copy-number changes. We applied Cancer Personalized Profiling by deep Sequencing (CAPP-Seq) for detection of translocations found in the most common pediatric sarcomas. We also applied ichorCNA to the combined off-target reads from our hybrid capture to simultaneously detect copy-number alterations (CNA). We analyzed 64 prospectively collected plasma samples from 17 patients with pediatric sarcoma. Translocations were detected in the pretreatment plasma of 13 patients and were confirmed by tumor sequencing in 12 patients. Two of these patients had evidence of complex chromosomal rearrangements in their ctDNA. We also detected copy-number changes in the pretreatment plasma of 7 patients. We found that ctDNA levels correlated with metastatic status and clinical response. Furthermore, we detected rising ctDNA levels before relapse was clinically apparent, demonstrating the high sensitivity of our assay. This assay can be utilized for simultaneous detection of translocations and CNAs in the plasma of patients with pediatric sarcoma. While we describe our experience in pediatric sarcomas, this approach can be applied to other tumors that are driven by structural variants.
Journal for ImmunoTherapy of Cancer
Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric rela... more Chimeric antigen receptor (CAR) T cell therapy is an effective salvage therapy for pediatric relapsed B-cell acute lymphoblastic leukemia (B-ALL), yet is challenged by high rates of post-CAR relapse. Literature describing specific relapse patterns and extramedullary (EM) sites of involvement in the post-CAR setting remains limited, and a clinical standard for post-CAR disease surveillance has yet to be established. We highlight the importance of integrating peripheral blood minimal residual disease (MRD) testing and radiologic imaging into surveillance strategies, to effectively characterize and capture post-CAR relapse. Main body: Here, we describe the case of a child with multiply relapsed B-ALL who relapsed in the post-CAR setting with gross non-contiguous medullary and EM disease. Interestingly, her relapse was identified first from peripheral blood flow cytometry MRD surveillance, in context of a negative bone marrow aspirate (MRD <0.01%). Positron emission tomography with 1...
Nature Communications
The increasing use of mass cytometry for analyzing clinical samples offers the possibility to per... more The increasing use of mass cytometry for analyzing clinical samples offers the possibility to perform comparative analyses across public datasets. However, challenges in batch normalization and data integration limit the comparison of datasets not intended to be analyzed together. Here, we present a data integration strategy, CytofIn, using generalized anchors to integrate mass cytometry datasets from the public domain. We show that low-variance controls, such as healthy samples and stable channels, are inherently homogeneous, robust against stimulation, and can serve as generalized anchors for batch correction. Single-cell quantification comparing mass cytometry data from 989 leukemia files pre- and post normalization with CytofIn demonstrates effective batch correction while recapitulating the gold-standard bead normalization. CytofIn integration of public cancer datasets enabled the comparison of immune features across histologies and treatments. We demonstrate the ability to int...
in childhood AML
incidence and clinical significance of nucleophosmin mutations
Gene Expression Analysis of CML Patients across the Age Spectrum
Blood, 2021
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 u...
Cancer Discovery, 2021
Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-XL/BCL2 inhibit... more Combining venetoclax, a selective BCL2 inhibitor, with low-dose navitoclax, a BCL-XL/BCL2 inhibitor, may allow targeting of both BCL2 and BCL-XL without dose-limiting thrombocytopenia associated with navitoclax monotherapy. The safety and preliminary efficacy of venetoclax with low-dose navitoclax and chemotherapy was assessed in this phase I dose-escalation study (NCT03181126) in pediatric and adult patients with relapsed/refractory (R/R) acute lymphoblastic leukemia or lymphoblastic lymphoma. Forty-seven patients received treatment. A recommended phase II dose of 50 mg navitoclax for adults and 25 mg for patients <45 kg with 400 mg adult-equivalent venetoclax was identified. Delayed hematopoietic recovery was the primary safety finding. The complete remission rate was 60%, including responses in patients who had previously received hematopoietic cell transplantation or immunotherapy. Thirteen patients (28%) proceeded to transplantation or CAR T-cell therapy on study. Venetoclax...
Haematologica, 2020
Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being develop... more Type 1 regulatory (Tr1) T cells induced by enforced expression of IL-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft vs host disease while mediating graft vs leukemia (GvL) effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations i...
Array CGH Discovers Novel Genomic Signatures in De Novo Acute Myeloid Leukemia (AML): Results of Children’s Oncology Group (COG) Study POG #9421
Blood, 2005
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 transi...
Molecular Inversion Probes (MIPs) Identify Novel Areas of Allelic Imbalance in Childhood Leukemia
Blood, 2007
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...
Adapting molecular inversion probe (MIP) technology for allele quantification in childhood leukemia
Journal of Clinical Oncology, 2007
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...
Cancer Research, 2020
Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Part... more Neuroblastoma is a deadly pediatric solid tumor with infrequent recurrent somatic mutations. Particularly, the pathophysiology of tumors without MYCN amplification remains poorly defined. Utilizing an unbiased approach, we performed gene set enrichment analysis of RNA-sequencing data from 498 patients with neuroblastoma and revealed a differentially overexpressed gene signature in MYCN nonamplified neuroblastomas with telomerase reverse transcriptase (TERT) gene overexpression and coordinated activation of oncogenic signaling pathways, including E2Fs, Wnt, Myc, and the DNA repair pathway. Promoter rearrangement of the TERT gene juxtaposes the coding sequence to strong enhancer elements, leading to TERT overexpression and poor prognosis in neuroblastoma, but TERT-associated oncogenic signaling remains unclear. ChIP-seq analysis of the human CLB-GA neuroblastoma cells harboring TERT rearrangement uncovered genome-wide chromatin co-occupancy of Brd4 and H3K27Ac and robust enrichment of...
Comparison of the Transcriptomic Signature of Pediatric Vs. Adult CML and Normal Bone Marrow Stem Cells
Blood, 2018
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 stu...