Norman Lacayo | Stanford University (original) (raw)

Papers by Norman Lacayo

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid le... more Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood 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 patients are due to unique molecular characteristics that differ from 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 by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. 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 (TMM) 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 (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion 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 particular biology of pediatric CML might impact the treatment of those patients in the future. Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

Poster Presentations - Proffered Abstracts

Molecular Genetics and Metabolism

Blood

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...

Blood

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...

Blood

The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine... more The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine (A), daunorubicin (D), and etoposide (E) as Induction I; all subsequent therapy is risk-adapted. Pharmacokinetic, pharmacodynamic, and gene expression studies are performed after exposure to high-dose or low-dose cytarabine. Patients with no response (NR, ≥ 25% bone marrow blasts) to Induction I receive low-dose ADE + gemtuzumab ozogamicin (GO) as Induction II, whereas all others receive ADE. Minimal residual disease (MRD) is monitored by flow cytometry and is defined as ≥ 0.1% cells with leukemia-associated immunophenotypes among bone marrow mononuclear cells. Patients who are MRD+ after induction therapy receive GO as a single agent before consolidation chemotherapy or stem cell transplantation. Among the 112 patients enrolled since October 2002, 37 had -7, FAB M6 or M7, FLT3 internal tandem duplication (ITD), or MDS or treatment-related AML and were classified as high-risk (HR); 32 h...

Cancers

Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid p... more Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid progenitor cells that affects patients of all ages. Despite decades of research and improvement in overall outcomes, standard therapy remains ineffective for certain subtypes of AML. Current treatment is intensive and leads to a number of secondary effects with varying results by patient population. Due to the high cost of discovery and an unmet need for new targeted therapies that are well tolerated, alternative drug development strategies have become increasingly attractive. Repurposing existing drugs is one approach to identify new therapies with fewer financial and regulatory hurdles. In this review, we provide an overview of previously U.S. Food and Drug Administration (FDA) approved non-chemotherapy drugs under investigation for the treatment of AML.

Journal of the National Comprehensive Cancer Network

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Advancements in techn... more Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Advancements in technology that enhance our understanding of the biology of the disease, risk-adapted therapy, and enhanced supportive care have contributed to improved survival rates. However, additional clinical management is needed to improve outcomes for patients classified as high risk at presentation (eg, T-ALL, infant ALL) and who experience relapse. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for pediatric ALL provide recommendations on the workup, diagnostic evaluation, and treatment of the disease, including guidance on supportive care, hematopoietic stem cell transplantation, and pharmacogenomics. This portion of the NCCN Guidelines focuses on the frontline and relapsed/refractory management of pediatric ALL.

Blood

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and morta... more Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture t...

Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the pred... more Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) based on small molecule inhibitors has been hindered. This may be due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mechanisms that underlie this successful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, we report the development of two erythroleukemia variants, RVC and RDC, which were derived by step-wise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC, which retained P-g...

JAMA Network Open

IMPORTANCE Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expressio... more IMPORTANCE Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expression information is necessary for a complete understanding of the tumorigenic processes. OBJECTIVE To evaluate the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer. DESIGN, SETTING, AND PARTICIPANTS This cohort study was conducted as a consortium between the University of California, Santa Cruz (UCSC) Treehouse Childhood Cancer Initiative and clinical genomic trials. RNA sequencing (RNA-Seq) data were obtained from the following 4 clinical sites and analyzed at UCSC: British Columbia Children's Hospital (n = 31), Lucile Packard Children's Hospital at Stanford University (n = 80), CHOC Children's Hospital and Hyundai Cancer Institute (n = 46), and the Pacific Pediatric Neuro-Oncology Consortium (n = 24). The study dates were

Molecular Case Studies

Gliomatosis peritonei is a rare pathologic finding that is associated with ovarian teratomas and ... more Gliomatosis peritonei is a rare pathologic finding that is associated with ovarian teratomas and malignant mixed germ cell tumors. The occurrence of gliomatosis as a mature glial implant can impart an improved prognosis to patients with immature ovarian teratoma, making prompt and accurate diagnosis important. We describe a case of recurrent immature teratoma in a 10-yr-old female patient, in which comparative analysis of the RNA sequencing gene expression data from the patient's tumor was used effectively to aid in the diagnosis of gliomatosis peritonei.

Blood

1436 Background: The use of flow cytometry or real-time PCR-based methods to detect minimal resid... more 1436 Background: The use of flow cytometry or real-time PCR-based methods to detect minimal residual disease (MRD) in children with Acute Lymphoblastic Leukemia (ALL) is a powerful tool for risk-adapted therapy stratification. However, current protocols for MRD detection incorrectly anticipate leukemia-free survival in 20–30% of low and intermediate risk patients. In this study, we present a new method for MRD detection using next-generation sequencing of the variable region of the immunoglobulin heavy chain (IgH) gene that can overcome two important limitations of current approaches as: (1) it detects lower levels of leukemia cells and (2) it identifies multiple evolved clones. Methods: To capture IgH sequences, we developed a set of multiplexed primers that allow the amplification of all known alleles of each V and J segment. We optimized the protocol to minimize amplification bias between primers. The products were then sequenced using the Illumina platform to obtain >1 millio...

Blood

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. ...

Blood

954 Background: About 90% of pediatric patients with newly diagnosed acute myeloid leukemia (AML)... more 954 Background: About 90% of pediatric patients with newly diagnosed acute myeloid leukemia (AML) have disease responsive to chemotherapy and achieve an initial complete response (CR) with anthracycline/cytarabine-based induction chemotherapy. However, 30–70% of patients of varying risks relapse within 4–5 years. Prospective identification of patients unlikely to benefit from induction therapy or likely to relapse could spare patients from treatment-related toxicities and allow consideration of alternative therapies. Current prognostic factors (e.g., cytogenetics and FLT3 ITD) of value at the population level are imperfect at the individual level. SCNP is a tool used to measure the effects of multiple modulators (including drugs) on signaling pathways at both the single-cell and individual patient level. A set of classifiers has been developed using SCNP technology that predicts the likelihood of response to anthracycline/cytarabine-based induction in adult patients with newly diagn...

Blood

4843 Background: All major molecular technology platforms have used cryopreserved samples as a so... more 4843 Background: All major molecular technology platforms have used cryopreserved samples as a source in biomarker development studies. Such samples enable the conduct of prospectively designed studies using banked samples with relevant clinical annotations, thus potentially reducing the duration of biomarker development. Single Cell Network Profiling (SCNP) is a new technology platform requiring viable cells. It uses multi-parametric flow cytometry to measure biological pathways focusing on intra-cellular signaling and post-translational modulations in response to a variety of modulators (growth factors, cytokines, drugs, etc.). As part of a collaboration between industry, academic institutions, and cooperative groups to improve acute myeloid leukemia (AML) management, we are developing clinical tests based on SCNP to predict induction chemotherapy response or risk of relapse. To date, all the development studies have been performed on DMSO cryopreserved specimens of ficoll fractio...

Blood

Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia ... more Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012). Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy ...

Blood

We previously reported a 36% event-free survival (EFS) estimate for patients with normal karyotyp... more We previously reported a 36% event-free survival (EFS) estimate for patients with normal karyotype (NK) on the COG study POG #9421 (n=144). In addition, we hypothesized that gene expression profiling would identify signatures linked to clinical outcome and useful for retrospective risk determination. Bone marrows in a subset of patients with NK (n=58) were analyzed using a 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. Prediction analysis for microarrays (PAM) was used to find genes that identified samples associated-with and unassociated-with events (relapse or death); after analyzing 28,711 genes with PAM we chose a 727-gene cluster that differentiated patients with NK on the basis of clinical outcome (cumulative classification error rate 19%). The analysis was biased for a larger number of genes in order to obtain a more biologically informative gene pathways ana...

Blood

Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein a... more Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein and occur in 25–35% of adult AML. NPM-mu are most commonly found in cases with normal karyotype, and are frequently associated with FLT3/ITD mutations. NPM-mu have been associated with high remission induction rates and improved survival, especially in patients with normal karyotype that lack FLT3/ITD mutations. The incidence and clinical significance of NPM-mu in childhood AML are less well-characterized. The AIEOP in Italy reported NPM-mu in 7 of 107 (6.5%) children treated on its AML02 protocol, and a Taiwanese group reported NPM-mu in 1 of 47 (2.1%) of children. The prognostic significance of NPM-mu in childhood AML is not known. The purpose of this study was to determine the incidence and clinical significance of NPM-mu in two large cohorts of children with newly-diagnosed AML treated on U.S. cooperative group phase III clinical trials (CCG-2961 and POG-9421). Criteria for selection ...

Blood

1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy... more 1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we comp...

Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid le... more Introduction Pediatric chronic myeloid leukemia (CML) accounts for 10-15% of pediatric myeloid leukemias and 2-9% of all pediatric leukemias. There are several unique characteristics of CML diagnosed in children, adolescents, and young adults, compared to adults. They present with higher white blood 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 patients are due to unique molecular characteristics that differ from 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 by FACS from pediatric CML (n=9), adult CML (n=10), pediatric normal (n=10) and adult normal (n=10) bone marrow samples. Total RNA was isolated from cells, and cDNA libraries were generated. 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 (TMM) 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 (LogFC). We performed three comparisons: (1) Pediatric CML vs Normal, (2) Adult CML vs Normal, and (3) Pediatric CML vs Adult CML. A False Discovery Rate (FDR) of £ .05 and absolute log2 fold-change > 1 was used to define differentially expressed genes in each comparison. Over-representation analysis was used to identify potentially unique pathways based on differentially expressed genes. Clinical and demographic features at diagnosis were extracted for pediatric and adult CML patients and compared using Fisher's exact test (categorical variables) or Wilcoxon rank sum test (continuous variables). Results Pediatric patients were diagnosed with CML at a median of 11 years (interquartile range (IQR): 10-14) compared to 54 years (IQR: 33-62) for adult patients. At diagnosis, pediatric patients had higher platelet counts (p=0.001) and larger spleen sizes (p=0.010) than adult patients, whereas the white blood cell count and phase at diagnosis did not differ. We found 606 genes (210 up- and 396 down-regulated) differentially expressed in pediatric CML CD34+ cells compared to pediatric normal controls. Interestingly, transcriptional regulators involved in blood cell differentiation including GATA1, TAL1, and KLF1 were differentially enriched in pediatric CML. In comparing adult CML patients to normal adult CD34+ cells, we found 920 genes (379 up- and 541 down-regulated) differentially expressed. Among all dysregulated genes we identified (1352 genes), 174 genes (54 up- and 120-down-regulated) overlapped when comparing pediatric and adult CML patients. Significantly enriched pathways in both adult and pediatric CML cells included PI3K/AKT signaling, MAPK signaling, and Notch/Wnt signaling, which have been previously reported. We found 437 unique genes that were dysregulated only in pediatric CML (270 up- and 167 down-regulated). Notch/Wnt signaling and Rho signaling pathways were significantly enriched. DLC1, a tumor suppressor gene that encodes a RhoGTPase-activating protein, has been known to be downregulated in solid tumors and hematologic malignancies. Interestingly, our data showed that DLC1 is significantly upregulated by 3-fold (p=0.0238) in pediatric CML, but not adult CML CD34+ cells. In addition, we observed that ABR, an inducer of C/EBPa that encodes an activator of RhoGEF and GTPase, was significantly downregulated by 2-fold (p=0.0119) in pediatric but not in adult CML CD34+ cells. Conclusion 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 particular biology of pediatric CML might impact the treatment of those patients in the future. Gotlib: Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: co-chair of the Study Steering Committee and Research Funding; Blueprint Medicines Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair of the Response Adjudication Committee and Research Funding, Research Funding.

Poster Presentations - Proffered Abstracts

Molecular Genetics and Metabolism

Blood

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...

Blood

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...

Blood

The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine... more The multicenter AML02 trial randomly assigns patients to receive high-dose or low-dose cytarabine (A), daunorubicin (D), and etoposide (E) as Induction I; all subsequent therapy is risk-adapted. Pharmacokinetic, pharmacodynamic, and gene expression studies are performed after exposure to high-dose or low-dose cytarabine. Patients with no response (NR, ≥ 25% bone marrow blasts) to Induction I receive low-dose ADE + gemtuzumab ozogamicin (GO) as Induction II, whereas all others receive ADE. Minimal residual disease (MRD) is monitored by flow cytometry and is defined as ≥ 0.1% cells with leukemia-associated immunophenotypes among bone marrow mononuclear cells. Patients who are MRD+ after induction therapy receive GO as a single agent before consolidation chemotherapy or stem cell transplantation. Among the 112 patients enrolled since October 2002, 37 had -7, FAB M6 or M7, FLT3 internal tandem duplication (ITD), or MDS or treatment-related AML and were classified as high-risk (HR); 32 h...

Cancers

Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid p... more Acute myeloid leukemia (AML) is a clinically and genetically heterogenous malignancy of myeloid progenitor cells that affects patients of all ages. Despite decades of research and improvement in overall outcomes, standard therapy remains ineffective for certain subtypes of AML. Current treatment is intensive and leads to a number of secondary effects with varying results by patient population. Due to the high cost of discovery and an unmet need for new targeted therapies that are well tolerated, alternative drug development strategies have become increasingly attractive. Repurposing existing drugs is one approach to identify new therapies with fewer financial and regulatory hurdles. In this review, we provide an overview of previously U.S. Food and Drug Administration (FDA) approved non-chemotherapy drugs under investigation for the treatment of AML.

Journal of the National Comprehensive Cancer Network

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Advancements in techn... more Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Advancements in technology that enhance our understanding of the biology of the disease, risk-adapted therapy, and enhanced supportive care have contributed to improved survival rates. However, additional clinical management is needed to improve outcomes for patients classified as high risk at presentation (eg, T-ALL, infant ALL) and who experience relapse. The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for pediatric ALL provide recommendations on the workup, diagnostic evaluation, and treatment of the disease, including guidance on supportive care, hematopoietic stem cell transplantation, and pharmacogenomics. This portion of the NCCN Guidelines focuses on the frontline and relapsed/refractory management of pediatric ALL.

Blood

Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and morta... more Introduction: In children diagnosed with leukemia, relapse and its associated morbidity and mortality remain the most dreaded consequences of the disease. Therefore, the discovery and implementation of novel and broadly applicable therapeutic strategies for these patients are urgently needed. Currently, a number of precision therapeutic approaches have been formulated where molecular analyses of the malignant cells have been used to inform, often multiple, probable targets and potential therapeutic agents. However, a common drawback of this approach has been the uncertainty involved in selecting the drug with the most and clinically relevant cytotoxic potential. In vitro xenograft approaches, although can provide key information on drug activity and side effects, are time consuming and impractical and cumbersome in most cases. We have recently demonstrated the ability of a bone marrow stromal derived cell line to sustain the growth and survival of patient leukemic cells in culture t...

Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the pred... more Despite considerable efforts in reversing clinical multidrug resistance (MDR), targeting the predominant multidrug transporter ABCB1/P-glycoprotein (P-gp) based on small molecule inhibitors has been hindered. This may be due to the emergence of alternative drug resistance mechanisms. However, the non-specific P-gp inhibitor cyclosporine (CsA) showed significant clinical benefits in patients with acute myeloid leukemia (AML), which likely represents the only proof-of-principle clinical trial using several generations of MDR inhibitors. Nevertheless, the mechanisms that underlie this successful MDR modulation by CsA are not elucidated because of the absence of CsA-relevant cellular models. In this study, we report the development of two erythroleukemia variants, RVC and RDC, which were derived by step-wise co-selection of K562/R7 drug-resistant leukemia cells with the etoposide-CsA and doxorubicin-CsA drug combinations, respectively. Interestingly, both RVC and RDC, which retained P-g...

JAMA Network Open

IMPORTANCE Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expressio... more IMPORTANCE Pediatric cancers are epigenetic diseases; therefore, considering tumor gene expression information is necessary for a complete understanding of the tumorigenic processes. OBJECTIVE To evaluate the feasibility and utility of incorporating comparative gene expression information into the precision medicine framework for difficult-to-treat pediatric and young adult patients with cancer. DESIGN, SETTING, AND PARTICIPANTS This cohort study was conducted as a consortium between the University of California, Santa Cruz (UCSC) Treehouse Childhood Cancer Initiative and clinical genomic trials. RNA sequencing (RNA-Seq) data were obtained from the following 4 clinical sites and analyzed at UCSC: British Columbia Children's Hospital (n = 31), Lucile Packard Children's Hospital at Stanford University (n = 80), CHOC Children's Hospital and Hyundai Cancer Institute (n = 46), and the Pacific Pediatric Neuro-Oncology Consortium (n = 24). The study dates were

Molecular Case Studies

Gliomatosis peritonei is a rare pathologic finding that is associated with ovarian teratomas and ... more Gliomatosis peritonei is a rare pathologic finding that is associated with ovarian teratomas and malignant mixed germ cell tumors. The occurrence of gliomatosis as a mature glial implant can impart an improved prognosis to patients with immature ovarian teratoma, making prompt and accurate diagnosis important. We describe a case of recurrent immature teratoma in a 10-yr-old female patient, in which comparative analysis of the RNA sequencing gene expression data from the patient's tumor was used effectively to aid in the diagnosis of gliomatosis peritonei.

Blood

1436 Background: The use of flow cytometry or real-time PCR-based methods to detect minimal resid... more 1436 Background: The use of flow cytometry or real-time PCR-based methods to detect minimal residual disease (MRD) in children with Acute Lymphoblastic Leukemia (ALL) is a powerful tool for risk-adapted therapy stratification. However, current protocols for MRD detection incorrectly anticipate leukemia-free survival in 20–30% of low and intermediate risk patients. In this study, we present a new method for MRD detection using next-generation sequencing of the variable region of the immunoglobulin heavy chain (IgH) gene that can overcome two important limitations of current approaches as: (1) it detects lower levels of leukemia cells and (2) it identifies multiple evolved clones. Methods: To capture IgH sequences, we developed a set of multiplexed primers that allow the amplification of all known alleles of each V and J segment. We optimized the protocol to minimize amplification bias between primers. The products were then sequenced using the Illumina platform to obtain >1 millio...

Blood

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. ...

Blood

954 Background: About 90% of pediatric patients with newly diagnosed acute myeloid leukemia (AML)... more 954 Background: About 90% of pediatric patients with newly diagnosed acute myeloid leukemia (AML) have disease responsive to chemotherapy and achieve an initial complete response (CR) with anthracycline/cytarabine-based induction chemotherapy. However, 30–70% of patients of varying risks relapse within 4–5 years. Prospective identification of patients unlikely to benefit from induction therapy or likely to relapse could spare patients from treatment-related toxicities and allow consideration of alternative therapies. Current prognostic factors (e.g., cytogenetics and FLT3 ITD) of value at the population level are imperfect at the individual level. SCNP is a tool used to measure the effects of multiple modulators (including drugs) on signaling pathways at both the single-cell and individual patient level. A set of classifiers has been developed using SCNP technology that predicts the likelihood of response to anthracycline/cytarabine-based induction in adult patients with newly diagn...

Blood

4843 Background: All major molecular technology platforms have used cryopreserved samples as a so... more 4843 Background: All major molecular technology platforms have used cryopreserved samples as a source in biomarker development studies. Such samples enable the conduct of prospectively designed studies using banked samples with relevant clinical annotations, thus potentially reducing the duration of biomarker development. Single Cell Network Profiling (SCNP) is a new technology platform requiring viable cells. It uses multi-parametric flow cytometry to measure biological pathways focusing on intra-cellular signaling and post-translational modulations in response to a variety of modulators (growth factors, cytokines, drugs, etc.). As part of a collaboration between industry, academic institutions, and cooperative groups to improve acute myeloid leukemia (AML) management, we are developing clinical tests based on SCNP to predict induction chemotherapy response or risk of relapse. To date, all the development studies have been performed on DMSO cryopreserved specimens of ficoll fractio...

Blood

Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia ... more Background Detection of minimal residual disease (MRD) in pediatric acute lymphoblastic leukemia (ALL) is a strong predictor of outcome. In addition, MRD testing prior to stem cell transplant for ALL can inform on the risk of relapse. The ClonoSIGHT test uses deep sequencing of immunoglobulin and T-cell receptors to identify and monitor MRD. In retrospective cohorts, we have previously shown this technology is highly correlated with flow cytometry and PCR-based MRD methods, but has even greater sensitivity than both technologies (Faham et al, Blood 2012; Gawad et al, Blood 2012). Here we report on four clinical cases where we used the ClonoSIGHT assay to prospectively monitor MRD, in both the medullary and extramedullary compartments, to demonstrate the feasibility of this technology for MRD monitoring of children with relapsed ALL. Methods Universal primer sets were used to amplify rearranged variable (V), diversity (D), and joining (J) gene segments from the immunoglobulin heavy ...

Blood

We previously reported a 36% event-free survival (EFS) estimate for patients with normal karyotyp... more We previously reported a 36% event-free survival (EFS) estimate for patients with normal karyotype (NK) on the COG study POG #9421 (n=144). In addition, we hypothesized that gene expression profiling would identify signatures linked to clinical outcome and useful for retrospective risk determination. Bone marrows in a subset of patients with NK (n=58) were analyzed using a 43,760-element spotted arrays containing 41,751 unique genes and expressed sequence tags; arrays were obtained from the Stanford University Microarray Core Facility. Prediction analysis for microarrays (PAM) was used to find genes that identified samples associated-with and unassociated-with events (relapse or death); after analyzing 28,711 genes with PAM we chose a 727-gene cluster that differentiated patients with NK on the basis of clinical outcome (cumulative classification error rate 19%). The analysis was biased for a larger number of genes in order to obtain a more biologically informative gene pathways ana...

Blood

Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein a... more Nucleophosmin mutations (NPM-mu) result in aberrant cytoplasmic localization of the NPM protein and occur in 25–35% of adult AML. NPM-mu are most commonly found in cases with normal karyotype, and are frequently associated with FLT3/ITD mutations. NPM-mu have been associated with high remission induction rates and improved survival, especially in patients with normal karyotype that lack FLT3/ITD mutations. The incidence and clinical significance of NPM-mu in childhood AML are less well-characterized. The AIEOP in Italy reported NPM-mu in 7 of 107 (6.5%) children treated on its AML02 protocol, and a Taiwanese group reported NPM-mu in 1 of 47 (2.1%) of children. The prognostic significance of NPM-mu in childhood AML is not known. The purpose of this study was to determine the incidence and clinical significance of NPM-mu in two large cohorts of children with newly-diagnosed AML treated on U.S. cooperative group phase III clinical trials (CCG-2961 and POG-9421). Criteria for selection ...

Blood

1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy... more 1440 Background: Measurement of minimal residual disease (MRD) during and after induction therapy has emerged as the most important predictor of outcome in pediatric acute lymphoblastic leukemia (ALL). Despite this, over 1/3 of relapses occur in patients who are MRD negative. In addition, ∼50% of children that have detectable MRD do not relapse. The Children Oncology Group (COG) trials use flow cytometry (FC) with a sensitivity of 10−4 for MRD detection and subsequent intensification of therapy in MRD+ patients. A more sensitive tool for monitoring MRD could lead to the identification of more patients who are likely to relapse, while a more specific assay could prevent unwarranted therapy intensification. To this end, we are employing the LymphoSIGHT platform developed by Sequenta Inc., which utilizes high-throughput sequencing for identification of clonal gene rearrangements in the B-cell repertoire and subsequent MRD measurement. In this blinded pilot study (COG AALL12B1), we comp...