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PDF File - (58K) - Secondary screen identified 168 GSC -effective compounds with selective inhibition profile

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

PDF File - (32K) - GSC-enriched GBM samples form tumors after transplantation into immunodeficient mice

PDF File - (874K) - A - Representative images demonstrating tumor formation success in an animal transplanted with serum (I) and sphere (II) derived GBM146 Ki67 and Human Nuclei (HuNu) immunostaining shows that virtually all transplanted cells died off in the animal transplanted with serum derived cells while an infiltrating tumor mass is visible in the animal transplanted with sphere derived cells of the same GBM sample (i - injection site, V- ventricle, SVZ - subventricular zone). B - Immunohistological sections for Human Nuclei (HuNu) and Ki67 demonstrating good survival of transplanted cells in animals that were sacrificed on the same day as well as 48h after transplantation (IT- injection tract, ST-striatum).

PDF File - (67K) - Effects of OLDA, PALDA and NADA are not mediated via cannabinoid, vanilloid or dopamine receptors A - Exposure of GSC -enriched GBM 157 cells to CB1, CB2, TRPV1 receptor agonists or their combination, does not reproduce the phenotype seen after exposure to OLDA, PALDA or NADA. Graph showing ATP level expressed as % of DMSO treated controls. B - Exposure of OLDA, PALDA or NADA-pretreated GSC -enriched GBM 157 cells (at IC50 concentration) to antagonists of CB, TRPV and D receptors does not rescue the phenotype caused by OLDA, PALDA or NADA (B, C and D, respectively). The graphs are showing ATP levels expressed as % of the DMSO treated controls (100%). SE shown.

PDF File - (61K) - OLDA, Emetine, 5256360 and 5560509 decrease clonal sphere formation in low passage GBM312 Graphs show a concentration-dependent reduction of clonal neurosphere size, neurosphere number as well as total cell mass. Error bars represent SEM.

PDF File - (39K) - Limiting dilution transplantation of GBM157 cells treated with OLDA, 5256360 or 5560509 shows decreased tumor formation frequency

PDF File - (523K) - OLDA, Emetine, 5256360 and 5560509 inhibit proliferation of sphere cultured GBM312 cells, using CFSE (carboxyfluorescein diacetate, succinimidyl ester) cell tracing experiments Proliferative inhibition is graphically represented by an increase in fluorescence or a shift to the right, away from that of the control. A proliferation index (PI) is a numerical value generated for each sample that measures the total increase in cell number divided by the original parent number. PI's are expressed as a percent of control: Emetine (0.2�M)- 18.6%, OLDA (4�M)- 16.0%, Compound 33 (2�M)- 31%, and Compound 62 (2�M)- 24.3%.

PDF File - (40K) - Growth kinetics of serum and sphere derived cells in screening (sphere) media reveals only minor differences in the degree of growth/proliferation between sphere and serum derived cultures of the same cell type Graph shows results of an ATP-based proliferation assay as measured 3 days after cell plating. Error bars depict SEM.

PDF File - (55K) - Sensitivity of GBM cells to OLDA, PALDA and NADA changes as a function of time, for which the cells were cultured in GSC -enriching media GBM107 cells were exposed to GSC -enriching media for increasing amounts of time and were then screened for sensitivity to dopaminergic cannabinoid compounds. While screening in serum-containing media requires a higher compound concentration due to the protective effect of serum in general, serum-derived cells become more and more sensitive towards GSC -specific compounds over time after transfer into sphere media (A, B, C). Such a difference is not present using non-GSC selective chemotherapeutics (D) or non-GBM control cells (E, F). ('ser in ser': serum derived cells screened in serum media; 'ser in sph': serum derived cells screened in sphere media; '1d sph': serum derived cells were cultured in sphere media for one day before the screen, etc.)

PDF file - 59K, Amino acid and ammonia changes with ASNase treatment in media and cell supernatants.

Oncometabolism can be targeted for the development of less myelotoxic oncotherapeutics. Lactate dehydrogenase A (LDHA) is central to the Warburg effect, a potential oncometabolic shift in neuroblastoma (NBL). Advanced surgical, cytotoxic and cell-differentiating therapies improved survival of children with NBL. Anti-GD2 monoclonal antibodies (mAb) effectively targeting NBL are also incorporated into complex therapies. However, poor clinical outcomes of high-risk NBL require improvements. Here, we verified the pre-reported prognostic value of LDHA expression in NBL using the R2 onco-genomics platform. Kaplan–Meier curves re-demonstrated that higher tumor LDHA expression correlates with worse survival. Multivariate statistics confirmed LDHA is independent from age, stage, and MYCN amplification. In conclusion, a molecular construct is proposed with anti-GD2 mAbs utilized for the targeted delivery of liposomes containing an LDHA inhibitor, Oxamate. Development and preclinical testing of this immunoliposome may validate targeted inhibition of the Warburg effect for NBL. Impact: Development of therapeutics against oncometabolism.Targeted specified drug-delivery with mAb.Sparing normal tissues from profound LDHA inhibition.Immunoliposome loaded with an anti-metabolite.If preclinically successful, has translational potential.

Background/Aim: Hydration and hydroxyurea (HU) can modify sickle cell disease (SCD) severity. Optimal nutrition and L-glutamine (Gln) may provide further amelioration. Patients and Methods: Reviews of medical records and nutrition surveys were used to investigate severity of pediatric patients with SCD in relation to nutrition, growth, hematologic parameters, and disease-modifying agents. Results: Among 25 females and 25 males (9.1±7 years), beta-globin genotypes were: HbSS/Sβ(0), 60%; HbSC, 32%; HbSβ(+), 8%. The mean number of annual pain crises (APC) was 0.97±1.1. APCs increased ≥2-fold as HbF dropped to

The Ewing sarcoma family of tumors (ESFT) are high-grade small round blue cell malignancies traditionally presenting in children and adolescents. The most common site of primary disease is bone, though extraskeletal primary sites are well-recognized.() We present six cases of primary ESFT of the kidney and one case of the adrenal gland. Patients were 11–18 years of age at diagnosis. Metastases at diagnosis were present in most cases (n=6). All patients underwent surgery, and most received radiation (n=5). Five patients relapsed after initial remission. Comprehensive review of the primary renal ESFT literature was used to analyze various factors, including age, gender, disease metrics, metastases at diagnoses, and overall survival in a total of 362 cases. Notably, while the general ESFT population has reported rates of metastasis at diagnosis of 20–25%,(2) this rate in the renal ESFT population was 53% with a rate of 59% in adolescent and young-adult (AYA) patients (11–24 years). Nodal disease at diagnosis was present in 24% of renal ESFT cases compared with 3.2% in patients with primary skeletal ESFT.(3) While this malignant process may share histologic and molecular features with its bone and soft tissue counterparts, primary renal ESFT presentations appear to be more aggressive and have worse outcomes.

In the age of ever-increasing developments in targeted cancer treatments, new immune-based approaches for brain tumor therapy represent an attractive avenue. Despite encouraging pre-clinical data, results in patients have been sub-optimal, likely due to tumor-induced immune suppression and intrinsic resistance to immune attack. Chemotherapy and biologic agents may be able to disrupt these mechanisms and restore tumor sensitivity to immune attack. In this study, we explore whether a combination of gemcitabine and rapamycin can sensitize medulloblastoma cells to immunotherapy in vitro and in vivo.With the commercial medulloblastoma cell line, Daoy, we explored the concentrations of combinations of Gemcitabine with rapamycin needed to induce cytotoxicity. Next, we used flow cytometry to assess the cytotoxicity of chemotherapy-treated Daoy cells with the addition of anti-tumor T-cells, generated from naive T-cells stimulated in the presence of Daoy lysate-pulsed dendritic cells. Then, we examined the efficacy of chemotherapy alone versus chemotherapy plus immunotherapy in tumor growth inhibition of subcutaneous medulloblastoma xenografts.Rapamycin alone at1,000 nM had moderate activity against Daoy cells in vitro and ICCombining immunotherapy and chemo-biologic therapy inhibit medulloblastoma cell and xenograft growth, and may offer an effective treatment for patients with medulloblastoma.

Severe neutropenia is defined as an absolute neutrophil count (ANC) of less than 0.5 × 109/L. Severe congenital neutropenia (SCN) is an inborn disorder with maturation arrest of granulocytes due to various genetic abnormalities, which may lead to immunodeficiency. Among several associated genetic mutations, the variants or heterozygous mutations of the ELANE gene coding neutrophil elastase comprise approximately 50% of the genetic causes of SCN. We present a newborn (male) with severe neutropenia due to a novel ELANE gene mutation. The newborn was born at 386/7 weeks gestation to a 25-year-old mother with hypertension and morbid obesity. Pregnancy and delivery were uncomplicated but the baby obtained a complete blood count (CBC) on day of life 2 for a work up of hyperbilirubinemia. He was noted to initially have an ANC of 0.2 × 109/L and 0 on subsequent blood counts. A bone marrow biopsy showed a left shift and consistent with myeloid maturation arrest. In direct DNA sequencing analysis, we found an ELANE gene mutation (Val119Glu, V119E), which may be a new gene mutation to cause SCN. The diagnosis of SCN in newborns is usually based on neutropenia identified on a routine CBC. Sufficient awareness and high suspicion of this rare disease can prevent missed or delayed diagnosis of SCN. Our analysis also suggests a new pathological mutation in the ELANE gene and supports the important role of molecular testing in SCN.

Background Oxidative stress contributes to the complex pathophysiology of sickle cell disease. Oral therapy with pharmaceutical-grade l-glutamine (USAN, glutamine) has been shown to increa...

Langerhans cell histiocytosis (LCH) is the most common histiocytic disorder, characterized by the pathologic clonal proliferation and accumulation of immature Langerhans cells within organs. Multiple organ systems can be affected, resulting in a spectrum of clinical manifestations. Isolated gastrointestinal involvement in LCH is rare and usually presents in childhood as a multisystem disease and usually has poor outcomes. We describe a 20-year-old Hispanic female with multifocal, single-system gastrointestinal LCH. Initially diagnosed from a CD1a, S100, and CD207 (Langerin) positive appendix tissue after an appendectomy and confirmed multifocal with an endoscopy. She had a full clinical and endoscopic resolution of disease with cytarabine therapy.

Glutamine, glutamate, asparagine, and aspartate are involved in an enzyme-network that controls nitrogen metabolism. Branched-chain-amino-acid aminotransferase-1 (BCAT1) promotes proliferation of gliomas with wild-type IDH1 and is closely connected to the network. We hypothesized that metabolism of asparagine, glutamine, and branched-chain-amino-acids is associated with progression of malignant gliomas. Gene expression for asparagine synthetase (ASNS), glutaminase (GLS), and BCAT1 were analyzed in 164 gliomas from 156 patients [33-anaplastic gliomas (AG) and 131-glioblastomas (GBM), 64 of which were recurrent GBMs]. ASNS and GLS were twofold higher in GBMs versus AGs. BCAT1 was also higher in GBMs. ASNS expression was twofold higher in recurrent versus new GBMs. Five patients had serial samples: 4-showed higher ASNS and 3-higher GLS at recurrence. We analyzed grade and treatment in 4 groups: (1) low ASNS, GLS, and BCAT1 (n=96); (2) low ASNS and GLS, but high BCAT1 (n=26); (3) high ASNS or GLS, but low BCAT1 (n=25); and (4) high ASNS or GLS and high BCAT1 (n=17). Ninety-one % of patients (29/32) with grade-III lesions were in group 1. In contrast, 95% of patients (62/65) in groups 2-4 had GBMs. Treatment was similar in 4 groups (radiotherapy-80%; temozolomide-30%; other chemotherapy-50%). High expression of ASNS, GLS, and BCAT1 were each associated with poor survival in the entire group. The combination of lower ASNS, GLS, and BCAT1 levels correlated with better survival for newly diagnosed GBMs (66 patients; P=0.0039). Only tumors with lower enzymes showed improved outcome with temozolomide. IDH1(WT) gliomas had higher expression of these genes. Manipulation of amino acid metabolism in malignant gliomas may be further studied for therapeutics development.

Background.Sickle cell disease (SCD) affects millions of people worldwide, including >100,000 Americans, and is often complicated by acute crises. Complex pathophysiology makes it challenging to ameliorate SCD severity, which can be influenced by hydration and disease-modifying agents such as hydroxyurea (HU). Due to the increased metabolic demands of chronic illness, optimal nutrition and presumably antioxidant L-glutamine (Gln) may further contribute to disease control. Thus, we hypothesized that better nutrition and Gln adherence may reduce occurrences of pain crises in SCD. Methods.An IRB-approved retrospective review of pediatric patients with SCD ≥1 y of age was conducted at our institution, along with voluntary nutrition surveys. Evaluations were carried out for associations between disease severity and clinical/laboratory variables representing nutrition, growth, hematologic parameters [fetal hemoglobin (HbF), mean corpuscular volume (MCV)], and treatment compliance (with HU and/or Gln). Results.Preliminary analyses included data from 50 SCD patients (25 F/25 M), with a mean age of 9.1 ±7 y and a mean BMI of 56.4 ±32.6% (±SDEV). Sixty percent of the patients had HbSS/Sß0 (32%-HbSC and 8%-HbSß+). The average number of annual pain crises (APC, 0.97 ±1.14) requiring hospital visits or admissions per patient rose with age. HbF expectedly dropped with age. HbF levels - 0.87 ±1.1 for well hydrated/well nourished (n=14, 3.1 ±2.3 y) - 1.4 ±2 for well hydrated/poorly nourished (n=10, 11 ±6.4 y) - 1.97 ±2 for poorly hydrated/poorly nourished (n=8, 13 ±5.4 y). There were no correlations between caloric intake and BMI. However, 20% of surveyed patients (12 ±7.8 y) had Z-scores lower than -1 SD for both height and weight. Their mean APC was significantly higher (2.5 ±2.5vs.1 ±1.3,p=0.03 byt-test). Their prealbumin levels trended lower than normal for age, and were more diminished in older patients. Nearly 60% of our patients were on HU (80% with HbSS/Sß0, age 10.8 ±7.4 y). Good HU adherence was reported by 22 patients, typically ≥90%, which was associated with higher MCVs (92 ±9.6 vs. 74 ±10 f/L,p Conclusions.Well-nourished/hydrated patients were younger and had a milder disease phenotype, whereas more severe disease was seen in poorly nourished older patients, often with suboptimal compliance. L-Glutamine can be a clinically meaningful addition to HU and should be further studied in pediatric SCD. Disclosures No relevant conflicts of interest to declare.

Background: Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors. Materials and Methods: We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas. Results. Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (IC₅₀s) of 0.12->10 IU/ml. Erwinaze at

Anti-metabolites are less-myelosuppressive than DNA-damaging anticancer drugs and may be useful against brain tumors.We evaluated the asparagine/glutamine-deaminating agent Erwinaze with/without temozolomide against brain tumor cells and mouse medulloblastomas.Erwinaze treatment of cell lines and neurospheres led to dose-dependent reductions of cells (reversible by L-glutamine), with half maximal inhibitory concentrations (ICErwinaze enhances cytotoxicity of temozolomide in vitro, and improves survival in SMO/SMO mice, likely by reducing cerebrospinal fluid glutamine. Temozolomide-associated toxicity prevented demonstration of any potential combinatorial advantage with Erwinaze in vivo.

BACKGROUND The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance. METHODS Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients. RESULTS During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P

Objectives: To present a clinicocytopathologic correlation of an atypical case of cat scratch disease (CSD) involving retroperitoneal lymph nodes, with emphasis on communication between service teams for managing lymphadenopathy of unknown origin. We consider clinical and cytologic differential diagnoses and review the literature on atypical cases of CSD, with emphasis on abdominal presentation and cytologic findings. Methods: Clinical services met with the cytology service to review clinical and pathologic features. Literature was reviewed via PubMed search (Harbor-UCLA subscriptions). Immunohistochemistry and Steiner silver stains were performed by Harbor-UCLA Department of Pathology. Enzyme-linked immunosorbent assay IgG and IgM Bartonella henselae titers were carried out by Quest Nichols Institute. Results: Fine-needle aspirate Diff-Quik and Papanicolaou smears and H&E-stained cell block showed abundant histiocytes, monocytoid B cells, and numerous neutrophils associated with necrosis corresponding to a late stage of CSD infection. Silver stain was positive for clumps of pleomorphic organisms. IgM and IgG antibody titers were elevated. Conclusions: The cytologic findings of CSD in an atypical abdominal presentation are similar to those of a classic presentation. Laboratory workup for atypical CSD should include at least two other modalities aside from cytomorphologic features. Close clinical and cytologic correlation avoided potentially unnecessary and harmful surgery and enabled timely treatment.

Primary non-Hodgkin’s lymphoma of the bone (PLB) is extremely rare in the pediatric population with less than 100 cases reported in the English literature. Most commonly, patients present with atraumatic bone pain and grossly normal radiographic findings. PLB is in the histopathological class of “small round cell tumors of bone”, as with most common bone tumors. The diagnosis is confirmed by immunohistochemical or flow cytometry based detection of tumor-specific proteins. We present a case of stage IV PLB of B-lymphoblastic type with an excellent response to chemotherapy to increase awareness among general pediatricians and pathologists about the importance of making the correct diagnosis, given the excellent prognosis for this disease.

Background Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Acute myeloid leukemia or myelodysplastic syndrome during the course of ALL is a rare entity. Some of these cases are therapy-related while the others occur due to lineage switch. The correct diagnosis relies on comparing the immunophenotypes and cytogenetic/molecular alterations of the myeloid neoplasm and the ALL. We present the clinical, pathologic and cytogenetic features of a case of an 18-year-old male with prior treatment for B-lymphoblastic leukemia (B-ALL) who developed therapy-related myeloid neoplasm (t-MN) 4–5 years after his initial diagnosis of B-ALL. Case presentation A 13-year-old boy with no significant past medical history presented to Harbor-UCLA Medical Center (HUMC) in November 2012 with night sweats, fevers and chills, nausea, vomiting, diarrhea, fatigue, weakness, and weight loss. Peripheral blood flow cytometric analysis disclosed B-ALL. The blasts expressed CD10, CD19, CD22 (dim), CD34, CD38, HLA-DR, and TdT, and were negative for CD20, CD13, CD33, CD117, and cytoplasmic MPO. Chromosomal analysis and a supplemental fluorescence in situ hybridization (FISH) study performed on the bone marrow aspirate showed an abnormal karyotype (47,XY,+X,del(9)(p21p21)[4]/46,XY[16]). He achieved remission after induction chemotherapy and remained in remission until March 2016 when bilateral testicular masses were noted. Biopsy of the left testicular mass showed relapsed B-ALL. Cerebrospinal fluid (CSF) contained rare TdT-positive blasts, suggestive of minimal/early involvement by B-ALL. However, there was no evidence of acute leukemia in his bone marrow at this time. He was then treated with COG protocol AALL1331 randomized to blinatumomab arm and achieved second remission. In June 2017, the patient's peripheral blood smear showed 11% circulating monoblasts. By flow cytometry, the blasts expressed CD4, CD11b, CD13, CD15, CD33, CD38, CD56, and CD64. In addition, a few TdT-positive blasts were seen in his CSF cytospin smear. Bone marrow biopsy was subsequently performed which was consistent with evolving acute myeloid leukemia. A diagnosis of myeloid neoplasm, consistent with t-MN was made. Chromosomal analysis and FISH studies performed on his bone marrow aspirate showed normal karyotype (46,XY[20]), negative FISH result for deletion 9p21 locus, and positive KMT2A (MLL) rearrangement, respectively. Despite of chemotherapy, the patient died within one month after diagnosis. Discussion and conclusion Diagnosis of t-MN should be suspected in patients with a history of receiving cytotoxic agents and/or irradiation. In this case study, we diagnosed t-MN with KMT2A rearrangement in a patient with history of B-ALL with 9p deletion and gain of X chromosome. Unusual features associated with this case are discussed.

Background Amino acid (AA) pathways may contain druggable targets for glioblastoma (GBM). Literature reviews and GBM database (http://r2.amc.nl) analyses were carried out to screen for such targets among 95 AA related enzymes. Methods First, we identified the genes that were differentially expressed in GBMs (3 datasets) compared to non-GBM brain tissues (5 datasets), or were associated with survival differences. Further, protein expression for these enzymes was also analyzed in high grade gliomas (HGGs) (proteinatlas.org). Finally, AA enzyme and gene expression were compared among the 4 TCGA (The Cancer Genome Atlas) subtypes of GBMs. Results We detected differences in enzymes involved in glutamate and urea cycle metabolism in GBM. For example, expression levels of BCAT1 (branched chain amino acid transferase 1) and ASL (argininosuccinate lyase) were high, but ASS1 (argininosuccinate synthase 1) was low in GBM. Proneural and neural TCGA subtypes had low expression of all three. High expression of all three correlated with worse outcome. ASL and ASS1 protein levels were mostly undetected in high grade gliomas, whereas BCAT1 was high. GSS (glutathione synthetase) was not differentially expressed, but higher levels were linked to poor progression free survival. ASPA (aspartoacylase) and GOT1 (glutamic-oxaloacetic transaminase 1) had lower expression in GBM (associated with poor outcomes). All three GABA related genes -- glutamate decarboxylase 1 (GAD1) and 2 (GAD2) and 4-aminobutyrate aminotransferase (ABAT) -- were lower in mesenchymal tumors, which in contrast showed higher IDO1 (indoleamine 2, 3-dioxygenase 1) and TDO2 (tryptophan 2, 3-diaxygenase). Expression of PRODH (proline dehydrogenase), a putative tumor suppressor, was lower in GBM. Higher levels predicted poor survival. Conclusions Several AA-metabolizing enzymes that are higher in GBM, are also linked to poor outcome (such as BCAT1), which makes them potential targets for therapeutic inhibition. Moreover, existing drugs that deplete asparagine and arginine may be effective against brain tumors, and should be studied in conjunction with chemotherapy. Last, AA metabolism is heterogeneous in TCGA subtypes of GBM (as well as medulloblastomas and other pediatric tumors), which may translate to variable responses to AA targeted therapies. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3148-1) contains supplementary material, which is available to authorized users.

Acute promyelocytic leukemia (APL) with t(15;17)(q22;q21)/PML-RARα is a subtype of acute myeloid leukemia (AML) with distinct morphologic and immunophenotypic characteristics. It is a highly aggressive disease that requires rapid diagnosis and early intervention. In addition to morphologic evaluation, flow cytometry has been widely used to facilitate prompt diagnosis of this disease. Compared with other types of AML, APL typically displays a triad of absent or weak CD34, absent HLA-DR, and positive CD117. HLA-DR positive APL is extremely rare and its clinical and pathological features have not been reported. A total of 45 cases of APL with t(15,17)/PML-RARα were diagnosed at Harbor-UCLA Medical Center from year 2006 to 2015. Among them, only two cases were positive for HLA-DR by flow cytometry immunophenotyping. Here we describe the clinical, morphologic, immunophenotypic, and cytogenetic features of these two cases.

BACKGROUND: Anti-metabolites are less myelosuppressive than DNA-damaging drugs used for brain tumors, and can be tested for efficacy and less overlapping toxicity if combined. We studied if anti-leukemia agent Erwinaze (which depletes asparagine and glutamine) has antitumor effect with or without temozolomide (TMZ) against brain tumors. METHODS: Erwinaze, TMZ, and their combinations were tested against malignant glioma and medulloblastoma cells, neurospheres, and against SMO/SMO mouse model (typically succumbing from spontaneous medulloblastoma). MTT cell viability assays were used for dose-response curves. Mice survival and weight loss were used as efficacy and toxicity surrogates, respectively. RESULTS: Erwinaze monotherapy showed dose-dependent cytotoxicity against 4 tested cell lines with range of IC50s 0.1->10 IU/ml. Erwinaze at

Glioblastoma (GBM) is among the most lethal of all cancers. GBM consist of a heterogeneous population of tumor cells among which a tumor-initiating and treatment-resistant subpopulation, here termed GBM stem cells, have been identified as primary therapeutic targets. Here, we describe a high-throughput small molecule screening approach that enables the identification and characterization of chemical compounds that are effective against GBM stem cells. The paradigm uses a tissue culture model to enrich for GBM stem cells derived from human GBM resections and combines a phenotype-based screen with gene target-specific screens for compound identification. We used 31,624 small molecules from 7 chemical libraries that we characterized and ranked based on their effect on a panel of GBM stem cell-enriched cultures and their effect on the expression of a module of genes whose expression negatively correlates with clinical outcome: MELK, ASPM, TOP2A, and FOXM1b. Of the 11 compounds meeting criteria for exerting differential effects across cell types used, 4 compounds showed selectivity by inhibiting multiple GBM stem cells-enriched cultures compared with nonenriched cultures: emetine, n-arachidonoyl dopamine, n-oleoyldopamine (OLDA), and n-palmitoyl dopamine. ChemBridge compounds #5560509 and #5256360 inhibited the expression of the 4 mitotic module genes. OLDA, emetine, and compounds #5560509 and #5256360 were chosen for more detailed study and inhibited GBM stem cells in self-renewal assays in vitro and in a xenograft model in vivo. These studies show that our screening strategy provides potential candidates and a blueprint for lead compound identification in larger scale screens or screens involving other cancer types. Mol Cancer Ther; 10(10); 1818–28. ©2011 AACR.

Background: A Phase 3, double-blind, randomized, placebo-controlled trial of L-glutamine in 230 patients (ages 5-58) over 48 weeks was conducted at 31 centers in the United States. Twice daily oral L-glutamine or placebo was administered, based on body weight approximating 0.3 g/kg/dose. The primary endpoint was the number of sickle cell crises pain (SCCs) during 48 weeks of treatment. The treatment effect measured was the difference of the median number of SCCs between two arms at week 48. To qualify as an SCC, the patient had to have received treatment with I.V. narcotics/ketorolac at an emergency department or during hospitalization in patients with a history of ≥2 crisis from the previous year. Splenic sequestration, acute chest syndrome and priapism were also considered to be SCCs. Patients stabilized on hydroxyurea (HU) treatment for at least 3 months prior to study screening (66% in both arms of the study) remained on HU for the duration of the study. The primary analysis via a Cochran-Mantel-Haenszel (CMH) test statistic showed a highly significant treatment effect (p=0.005). There was a median of 3 crises for the L-glutamine treatment arm and 4 for the placebo arm (25% difference). There were no concerning adverse events from the treatment compared with the placebo arm. In this abstract, we reported the results from additional analyses of times to first and second crisis as well as cumulative recurrent events, which further demonstrated the clinically meaningful treatment effect of L-glutamine. Although not pre-specified as part of the study nor the drug approval process, external and internal reviewers of the data were interested in examining any differential effects of L-glutamine on patients that had either low, moderate or high frequencies of SCCs prior to study screening. Methods: An evaluation of the data was performed based on patients by three categories of crises; 2 crises (low), 3 - 5 (moderate) and ≥6 crises (high) as collected from their medical records at entry into the phase 3 study. The negative binomial regression (NBR) model was used to ascertain the rate ratios (rate of crises of L-glutamine assignment arm divided by the rate of crises on placebo). The NBR model was utilized to generate an estimate of treatment effect and treatment by subgroup interactions for the three subgroups. A rate ratio < 1.0 favored L-glutamine treatment effect. A significant interaction could be seen if the treatment arm is better than placebo for one category but worse than placebo for another. Subgroups with corresponding sample sizes were as follows: 2 SCCs in year prior: L-glutamine (n = 53) and placebo (n = 26). 3-5 SCCs in year prior: L-glutamine (n = 76) and placebo (n = 36). ≥6 SCCs in year prior: L-glutamine (n = 22) and placebo (n = 16). Results: Rate ratios (bolded) as shown in Table 1 were similar between all categories (2 crises, 3 to 5 crises, and ≥ 6 crises; 0.87, 0.74, and 0.82, respectively). There was no difference in treatment effect among the categories. These findings are confirmed by the lack of treatment by the prior year SCCs interaction; p = 0.8397. Numerically and graphically (Figure 1), there is an apparent lower rate ratio in those that had 3 to 5 SCCs in the year prior to screening. Discussion: The number of crises in the year prior to study screening is viewed as an indicator of disease severity, although this may not always be true due to the intra-patient variability in disease presentation. The apparent lower rate ratio in those that had 3 to 5 SCCs in the year prior to screening may be due to having the largest sample size of the three categories., The analysis indicates that the benefit of L-glutamine treatment observed in the phase 3 trial is consistent regardless of the history (one year) of SCCs prior to the initiation of L-glutamine therapy. Reference:A Phase 3 Trial of l-Glutamine in Sickle Cell Disease | NEJM. New England Journal of Medicine. https://www.nejm.org/doi/full/10.1056/NEJMoa1715971. Published 2018. Accessed July 24, 2018. Disclosures Niihara: Emmaus Medical, Inc: Employment, Equity Ownership. Stark:Emmaus Medical, Inc: Employment, Equity Ownership. Razon:Emmaus Medical, Inc: Employment, Equity Ownership. Tran:Emmaus Medical, Inc: Employment, Equity Ownership. Becerra:Emmaus Medical, Inc: Employment.

Background. Cultured brain tumors can form neurospheres harboring tumorigenic cells with self renewal and differentiation capacities. Renewable neurosphere formation has clinical predictive value in adult malignant gliomas, yet its prognostic role for pediatric brain tumors is unknown. Methods. Established neurosphere conditions were used for culturing samples from glial, embryonal and mixed glioneuronal tumors from 56 pediatric patients. Potential associations between neurosphere formation and clinical outcome were analyzed retrospectively. Results. Thirty-seven percent of all samples formed renewable neurospheres. Analysis of available clinical outcome data from 51 patients demonstrated significantly increased hazard ratios (HR) for both disease progression (HR = 9.9, P < 0.001) and death (HR = 16.6, P < 0.01) in the neurosphere forming group. Furthermore, neurosphere formation correlated with adverse progression free survival (PFS) in glial and embryonal tumors, but not in mixed glioneuronal tumors. Overall survival (OS) was significantly worse for neurosphere-forming patients with embryonal tumors, as a group and amongst the subgroup with medulloblastoma, but not in the glial group. Multivariate analysis showed that neurosphere formation was associated with diminished PFS and OS independent of age, gender, or treatment. Neurosphere formation was an independent predictor of diminished PFS of glial tumors after adjusting for grade. Multivariate analysis, adjusting for both Ki67 staining and neurosphere formation, demonstrated that neurosphere formation remained predictive of progression whereas Ki67 did not. Conclusions. Neurosphere formation is more predictive of pediatric brain tumor progression than semi-quantitative Ki67 staining. Pediatric brain tumor derived neurospheres may provide a predictive model for preclinical explorations. Pediatr Blood Cancer. 2010;55:644–651. © 2010 Wiley-Liss, Inc.

The objectives of this study were to assess the incidence of clinical allergy and end-induction antiasparaginase (anti-ASNase) antibodies in children with high-risk acute lymphoblastic leukemia treated with pegylated (PEG) Escherichia coli ASNase and to determine whether they carry any prognostic significance.Of 2057 eligible patients, 1155 were allocated to augmented arms in which PEG ASNase replaced native ASNase postinduction. Erwinia chrysanthemi (Erwinia) ASNase could be used to replace native ASNase after allergy, if available. Allergy and survival data were complete for 990 patients. End-induction antibody titers were available for 600 patients.During the consolidation phase, 289 of 990 patients (29.2%) had an allergic reaction. There were fewer allergic reactions to Erwinia ASNase than to native ASNase (odds ratio, 4.33; P .0001) or PEG ASNase (odds ratio, 3.08; P .0001) only during phase 1 of interim maintenance. There was no significant difference in 5-year event-free survival (EFS) between patients who received PEG ASNase throughout the entire study postinduction versus those who developed an allergic reaction to PEG ASNase during consolidation phase and subsequently received Erwinia ASNase (80.8% ± 2.8% and 81.6% ± 3.8%, respectively; P = .66). Patients who had positive antibody titers postinduction were more likely to have an allergic reaction to PEG ASNase (odds ratio, 2.4; P .001). The 5-year EFS rate between patients who had negative versus positive antibody titers (80% ± 2.6% and 77.7% ± 4.3%, respectively; P = .68) and between patients who did not receive any ASNase postconsolidation and those who received PEG ASNase throughout the study (P = .22) were significantly different.The current results demonstrate differences in the incidence rates of toxicity between ASNase preparations but not in EFS. The presence of anti-ASNase antibodies did not affect EFS.

Purpose: Many molecular pathways, including cell cycle control, angiogenesis, and drug resistance, mediate tumor growth and survival. Vascular endothelial growth factor-A (VEGF-A) serum levels 100 pg/mL have been associated with good and poor prognoses, respectively. Experimental Design: The hypothesis was that serum VEGF-A levels in standard-risk acute lymphoblastic leukemia pediatric patients at induction are predictive of event-free survival (EFS). One hundred seventeen patients were entered in CCG-1962 study and randomized into the native and polyethylene glycolated asparaginase arms. VEGF-A levels were quantified by an ELISA assay. Results: All patients had a decrease in VEGF-A levels by day 14 of induction, but they later dichotomized; EFS group levels remained low and event group levels increased. A correlation exists between high VEGF-A levels at entry to induction and time to event. Moreover, 6-year EFS patients have lower end of induction VEGF-A levels (28 ± 6 pg/mL) than event patients (>100 pg/mL; P < 0.01). Kaplan-Meier curves using various VEGF-A values were produced; with ≤30 at entry into induction (day 0) and ≤60 pg/mL at the end of induction (day 28), patients with low VEGF-A levels had superior EFS (P < 1e−4). Furthermore, patients who had an increase in VEGF-A during induction (ΔVEGF-positive, days 0-28) were more likely to have an event (P < 1e−4). Bifurcation by asparaginase treatment arm did not alter these results. Conclusions: These observations strongly support that high VEGF-A levels in induction are an asparaginase treatment–independent predictive marker for EFS. Hence, an anti-VEGF-A therapy should be tested in acute lymphoblastic leukemia.

We investigated the anti-asparaginase antibody (Ab) and asparaginase enzymatic activity in the sera of 1,001 patients (CCG-1961) with high-risk acute lymphoblastic leukemia (HR-ALL). Patients received nine doses of native Escherichia coli asparaginase during induction. Half of rapid early r

Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner—as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors. Implications: Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy. Mol Cancer Res; 12(5); 694–702. ©2014 AACR.

Immunotherapy has the potential to improve clinical outcomes with little toxicity for pediatric patients with brain tumors. We conducted a pilot feasibility study of tumor lysate-pulsed dendritic cell (DC) vaccination in pediatric patients (1 to 18 years old) with newly diagnosed or recurrent high-grade glioma (HGG). A total of nine DC vaccine doses, each containing 1 × 10(6) cells per dose were administered to three out of the seven originally enrolled patients. Toxicities were limited to mild side-effects, except in one case of elevated alkaline phosphatase, which resolved without clinical consequences. Two patients with primary lesions amongst the three vaccinated were alive at the time of writing, both without evidence of disease. Pre- and post-vaccination tumor samples from a patient with an anaplastic oligoastrocytoma that recurred failed to demonstrate immune cell infiltration by immunohistochemistry. Peripheral cytokine levels were evaluated in one patient following DC vaccination and demonstrated some changes in relation to vaccination. DC vaccine is tolerable and feasible with some limitations for pediatric patients with HGG. Dendritic cell based immunotherapy may provide some clinical benefit in pediatric patients with glioma, especially for patients with minimal residual disease, but further investigation of this modality is required.

The clinical practice of oncology is being transformed by molecular diagnostics that will enable predictive and personalized medicine. Current technologies for quantitation of the cancer proteome are either qualitative (e.g., immunohistochemistry) or require large sample sizes (e.g., flow cytometry). Here, we report a microfluidic platform—microfluidic image cytometry (MIC)—capable of quantitative, single-cell proteomic analysis of multiple signaling molecules using only 1,000 to 2,800 cells. Using cultured cell lines, we show simultaneous measurement of four critical signaling proteins (EGFR, PTEN, phospho-Akt, and phospho-S6) within the oncogenic phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway. To show the clinical application of the MIC platform to solid tumors, we analyzed a panel of 19 human brain tumor biopsies, including glioblastomas. Our MIC measurements were validated by clinical immunohistochemistry and confirmed the striking intertumoral and intratumoral heterogeneity characteristic of glioblastoma. To interpret the multiparameter, single-cell MIC measurements, we adapted bioinformatic methods including self-organizing maps that stratify patients into clusters that predict tumor progression and patient survival. Together with bioinformatic analysis, the MIC platform represents a robust, enabling in vitro molecular diagnostic technology for systems pathology analysis and personalized medicine. Cancer Res; 70(15); 6128–38. ©2010 AACR.

The discovery of the tumour-inhibitory properties of asparaginase began 50 years ago with the observation that guinea-pig serum-treated lymphoma-bearing mice underwent rapid and often complete regression. Soon afterwards, the asparaginase of bacterial origin was isolated. The asparaginases of bacterial origin induce anti-asparaginase neutralising antibodies in a large proportion of patients (44-60%), thus negating the specific enzymatic activity and resulting in failure of the target amino acid deamination in serum. There is immunological cross-reaction between the antibodies against various formulations of native Escherichia coli-asparaginase and polyethylene glycol (PEG)-asparaginases, but not to Erwinia asparaginase, as suggested by laboratory preclinical findings. This evidence was strongly inferred from the interim analyses in the Children's Cancer Group (CCG)-1961 study. Thus, anti-E. coli or PEG-asparaginase antibodies seropositive patients may benefit from the Erwinia asparaginase. The inter-relationships between asparaginase activity, asparagine (ASN) and glutamine deamination remain largely unexplored in patients. Studies have shown that ASN depletion is insufficient to induce apoptosis in T lymphoblasts in vitro and that the inhibitory concentration of CEM T-cell line is correlated with the asparaginase concentration responsible for 50% glutamine deamination. The optimal catalysis of ASN and glutamine deamination in serum by asparaginase induces apoptosis of leukaemic lymphoblasts. The percentage of ASN and glutamine deamination was predicted by asparaginase activity. Asparaginase activity of 0.1 IU/mL provided insufficient depletion of both amino acids in high-risk acute lymphoblastic leukaemia (ALL) patients. With increasing glutamine deamination, mean asparaginase activities and percentages of post-treatment samples with effective ASN depletion (3 micromol/L) increase. Both glutamine and ASN deamination are predicted by asparaginase activity. Further population analyses resulted in identification of sigmoid relationships between asparaginase levels and post-treatment glutamine and ASN deamination.Furthermore, pharmacodynamic analyses strongly suggested that/=90% deamination of glutamine must occur before optimal ASN deamination takes place, due to the de novo ASN biosynthesis by the liver. These pharmacodynamic results from the best-fit population pharmacokinetic/pharmacodynamic model obtained from nonlinear mixed effects model pharmacodynamic analyses for standard-risk ALL patients are similar. These analyses produced the following results: (i) asparaginase activity/=0.4 IU/mL provided insufficient deamination of ASN, whereas0.4-0.7 IU/mL was required for optimal (90%) ASN and glutamine deamination; and (ii) deamination of glutamine is dependent on asparaginase activity and it correlates with enhanced serum ASN deamination. Thus, glutamine deamination enhances asparaginase efficacy in ALL patients. Deamination of ASN/=90% of control or ASN concentration3 micromol/L may be associated with improved survival in this subset of patients. Our findings support the pharmacodynamic mechanism of PEG-asparaginase for disease control in ALL patients. These results taken together strongly support new experimental approaches for application of population pharmacokinetic/pharmacodynamic analyses to further enhance survival of leukaemia patients.