AbstractAcute Lymphoblastic Leukemia Research Articles

Abstract 4595: Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL.

Abstract Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Despite intensive chemotherapy, 20% of pediatric and over 50% of adult ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making disease relapse and resistance to therapy the most significant challenge in the treatment of this disease. Using whole exome sequencing, here we identify mutations in NT5C2, a 5’-nucleotidase enzyme responsible for inactivation of nucleoside analog chemotherapy drugs, in 20/103 (19%) relapse T-ALLs and in 1/35 (3%) relapse B-precursor ALLs analyzed. NT5C2 mutant proteins show increased nucleotidase activity in vitro and conferred resistance to chemotherapy with 6-mercaptopurine and 6-thioguanine when expressed in ALL lymphoblasts. These results support a prominent role for activating mutations in NT5C2 and increased nucleoside analog metabolism in disease progression and chemotherapy resistance in ALL. Citation Format: Gannie Tzoneva, Arianne Perez-Garcia, Zachary Carpenter, Hossein Khiabanian, Valeria Tosello, Maddalena Allegretta, Elisabeth Paietta, Janis Racevkis, Jakob M. Rowe, Martin S. Tallman, Maddalena Paganin, Jana Hoff, Renate Kirschner-Schwabe, Teresa Palomero, Raul Rabadan, Adolfo Ferrando. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4595. doi:10.1158/1538-7445.AM2013-4595

Abstract 4550: High throughput screen to identify compounds that reverse glucocorticoid resistance in pediatric leukemia.

Abstract Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although cure rates are approaching 85%, it remains one of the most common causes of death from disease in children. Glucocorticoids (e.g. dexamethasone) form a critical component of chemotherapy regimens for pediatric ALL and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. We have previously established a clinically relevant ALL xenograft model, consisting of primary pediatric ALL biopsies engrafted in immune-deficient mice, in which in vitro and in vivo dexamethasone sensitivity significantly correlate with patient outcome. In this study we used a high throughput screen (HTS) to identify novel small compounds that reverse glucocorticoid resistance. A xenograft (ALL-19) derived from an aggressive and chemoresistant pediatric ALL that induced early fatality in the patient, and is representative of the most common pediatric ALL subtype (B-cell precursor, BCP-ALL, or c-ALL) was used to identify compounds that potentially will have wide applicability. Cells were exposed in vitro to dexamethasone at a concentration ineffective in cell killing against this xenograft (1 μM), but which is around 100-fold greater than the IC50 for other (sensitive) xenografts, simultaneously with one of 40,000 HTS library compounds (10 μM). Effective compounds were then tested alone and in the presence of dexamethasone, to identify those that sensitized the xenograft to glucocorticoids, rather than those that were toxic alone. The compound 2-(4-chlorophenoxy)-2-methyl-N-(2-(piperidin-1-yl)phenyl)propanamide showed little activity alone (IC50 22 μM), but when combined with dexamethasone caused a marked decrease in cell viability. Pre-incubating ALL-19 xenograft cells with either the compound or dexamethasone did not increase the sensitizing effect. Fixed-ratio combination assays were performed against a broad panel of dexamethasone-resistant and -sensitive xenografts representative of BCP-ALL, T-ALL and MLL-rearranged ALL, in order to determine whether the compound was broadly active. CalcuSyn software was employed to determine combination indices, and synergy was observed in all 4 BCP-ALL xenografts tested, which included a dexamethasone-sensitive BCP-ALL and a dexamethasone-resistant Philadelphia chromosome-positive ALL. Synergy was also observed in dexamethasone-resistant MLL-rearranged ALL xenograft cells. Additionally, despite an antagonistic effect observed in a dexamethasone-resistant T-ALL xenograft, the compound potentiated the effects of dexamethasone in a glucocorticoid-sensitive T-ALL. In conclusion, using a clinically relevant xenograft model and a HTS, this study has identified a novel compound that reverses dexamethasone resistance in BCP-ALL xenografts, and may have applications in other leukemia subtypes. Citation Format: Cara Toscan, Tim Failes, Greg Arndt, Richard Lock. High throughput screen to identify compounds that reverse glucocorticoid resistance in pediatric leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4550. doi:10.1158/1538-7445.AM2013-4550

The Association of Glutathione S-transferase Gene Mutations (including GSTT1 and GSTM1) with the Prognostic Factors and Relapse in Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. It accounts for one fourth of all childhood cancers and approximately 75% of all childhood leukemias. Some prognostic factors determine the outcome of therapy [e.g. age, sex, initial white blood cell count (WBC), etc.]; however, it is believed that other mechanisms such as glutathione S-transferase (GST) gene mutation, the expression of lung resistance protein (LRP), and multidrug resistance-associated protein (MRP) also plays a role in treatment failure. In this study, GST gene mutations including GSTM1 and GSTT1 were evaluated in patients with leukemia. Thirty newly diagnosed ALL patients younger than 15 years of age participated in the present study. Bone marrow aspiration and biopsy were evaluated for immune phenotyping and DNA was extracted for GST genotyping. All data plus sex, age, initial WBC count, central nervous system (CNS) or testicular involvement, immune phenotype, and outcome (relapse or not) were analyzed statistically. Genotyping showed that 46% were double null, 50% were M1 null and 93.3% were T1 null for GST mutations. There was no statistically significant relationship between GSTT1 and GSTM1 mutations, or between double null status, prognostic factors and relapse (P > .05). So, although the results of GST mutations were consistent, it seems that these mutations are not statistically significant.

Moderate dose cranial radiotherapy causes central adrenal insufficiency in long-term survivors of childhood leukaemia

Acute lymphoblastic leukaemia (ALL) is the most common childhood malignancy. The survival rate in the Scandinavian countries is now around 85%. ALL patients treated with cranial radiotherapy (CRT) are at risk for growth hormone deficiency (GHD), but little is known about other pituitary insufficiencies, e.g. ACTH. Adult ALL patients (median age at study 25years), treated with 24Gy (18-30) of CRT during childhood were investigated. We performed an insulin tolerance test (ITT) to evaluate cortisol secretion. We measured basal serum ACTH and cortisol levels before and after 5years of GH therapy. 14 out of 37 (38%) ALL patients had a subnormal cortisol response to an ITT (257-478nmol/L) while there was no significant difference in basal cortisol levels between 44 patients and controls (P>0.3). Female, but not male ALL patients had significantly lower ACTH levels compared to controls (P=0.03). After 5years of GH therapy only male ALL patients had significantly lowered basal plasma cortisol (P=0.02). ALL survivors, treated with a moderate dose CRT, have a central adrenal insufficiency 20years after diagnosis. An increased awareness of the risk for an adrenal insufficiency is of importance and life-long surveillance of the entire hypothalamic-pituitary axis is recommended in these patients.

Precursor B-Cell Acute Lymphoblastic Leukemia/Lymphoma with L3 Morphology, Philadelphia Chromosome, MYC Gene Translocation, and Coexpression of TdT and Surface Light Chains: A Case Report

Acute lymphoblastic leukemia is predominantly found in children. It is a neoplasm of precursor cells or lymphoblasts committed to either a B- or T-cell lineage. The immature cells in B-acute lymphoblastic leukemia/lymphoma can be small or medium sized with scant or moderate cytoplasm and typically express B-cell markers such as CD19, cytoplasmic CD79a, and TdT without surface light chains. These markers, along with cytogenetic studies, are vital to the diagnosis, classification, and treatment of these neoplasms. We present an unusual case of a precursor B-cell ALL, in an 82-year-old woman, who presented with pancytopenia and widespread lymphadenopathy. The cells show L3 morphology (Burkitt-like lymphoma) with coexpression of TdT and surface light chains in addition to an MYC gene translocation and Philadelphia chromosome.

In search of Pinkel’s children: unravelling the biological heterogeneity of childhood acute lymphoblastic leukaemia by genotype and treatment molecular response

Acute lymphoblastic leukaemia (ALL), the commonest childhood malignancy has seen remarkable progress since the 1960s with cure rates now approaching 85%. To achieve this patients undergo intensive treatment that usually takes 2.5-3.5 years involving on average 15 different chemotherapeutic drugs. In 1971, Donald Pinkel reported Total Therapy-Protocol V that used 5 drugs and cranial radiation therapy over a similar time period. Today, one half of these patients (Pinkel's children) remain alive and free of leukaemia. The aim of this study was to evaluate the impact post-induction minimal residual disease (MRD) levels had on survival and its relationship with the more established clinical and biological prognostic predictors of outcome in the hope of identifying a subgroup of patients that are at very low risk of failure. A retrospective review of 250 Irish children with ALL was carried out. MRD status after 28 days of induction chemotherapy and other known predictors of outcome were correlated with 5 year event-free survival (EFS). MRD status was the strongest predictor of outcome with 5 year EFS rates greater that 90% seen in those patients with low-risk MRD and this was associated with TEL/AML1 rearrangement, high hyperdiploidy (HH) karyotype and female gender. Both MRD and karyotype are powerful determinants of outcome in childhood ALL. Therefore, it is reasonable to conclude that the majority of children cured by Pinkel et al. in the late 1960s were most likely composed of low-risk MRD, TEL/AML1 and HH patients.

Where do we stand in the treatment of relapsed acute lymphoblastic leukemia?

Abstract Acute lymphoblastic leukemia (ALL) is the most common and one of the most treatable cancers in children. Although the majority of children with ALL are now cured, 10%-20% of patients are predicted to relapse and outcomes with salvage therapy have been disappointing, with approximately only one-third of children surviving long-term after disease recurrence. Several prognostic factors have been identified, with timing of recurrence relative to diagnosis and site of relapse emerging as the most important variables. Despite heterogeneity in the elements of salvage therapy that are delivered in trials conducted internationally, outcomes have been remarkably similar and have remained static. Because most intensive salvage regimens have reached the limit of tolerability, current strategies are focusing on identifying new agents tailored to the unique biology of relapsed disease and identifying methods to develop these agents efficiently for clinical use. Recently, high-resolution genomic analyses of matched pairs of diagnostic and relapse bone marrow samples are emerging as a promising tool for identifying pathways that impart chemoresistance.

Pharmacotherapeutic advances in the treatment of acute lymphoblastic leukaemia in adults.

Acute lymphoblastic leukaemia (ALL) in adults is a challenging malignancy in that many patients will show evidence of initial chemotherapy responsiveness but will subsequently relapse. The disease is heterogeneous and outcomes vary dramatically depending on the prognostic factors present in an individual patient. An important determinant of outcome is the age of the patient. The stunning success of therapy in paediatric ALL has led to the use of intensive paediatric regimens in adolescents and young adults with what appear to be improved outcomes. For patients who relapse or have high-risk features, blood and marrow transplantation (BMT) continues to play an important role in the therapeutic armamentarium. The use of reduced-intensity conditioning regimens for allogeneic BMT suggests that outcomes may be improved by this approach. Monoclonal antibodies are showing benefit as single agents in the relapsed setting or in combination with chemotherapy in newly diagnosed patients. In recent years, several new chemotherapeutic agents have shown promise as single agents and are being incorporated into multi-agent chemotherapy. The development of tyrosine kinase inhibitors for Philadelphia chromosome-positive leukaemias has significantly improved outcomes. The molecular revolution has led to the identification of new aberrant molecular pathways in the pathogenesis of ALL, and drugs targeting these aberrancies are in various stages of development preclinically and clinically. These developments bring the hope that therapeutic outcomes in adult ALL can begin to approach those seen in the paediatric setting.

Abstract A102: Longer formula feeding and later age at introduction of solids increase the odds ratio of pediatric acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The literature suggests multiple points of interaction between infant feeding patterns and leukemogenesis whereby diet may influence normal development of the immune system and levels of insulin-like growth factor 1 (IGF-1) in the serum. Thus the intent of the current study is to examine the association between infant feeding practices and age at introduction of solids, on risk of ALL. Incident cases of infant and childhood ALL (N=142, ages 0 to 14 years) were approached and enrolled in a case-control study at Texas Children's Cancer Center (TCCC). Controls (N=284) were recruited at TCCC and satellite clinics, as well as from participants in formative research for the National Children's Study in Houston, San Antonio and Austin, Texas. Cases and controls were frequency matched on age, sex, race and ethnicity. Differences in proportions of breast and bottle feeding between cases and controls were assessed by chi square test. Differences in the mean durations of feeding practices and age at introduction of solids were assessed by Student's t test. The odds ratios (OR) of ALL were calculated using multiple logistic regression analysis in two models with the infant feeding group (independent variables) characterized as follows and a reference group of exclusively breastfed: in the first model, ever formula fed; in the second model, exclusively fed formula or fed both breast milk and formula. A third addressed the effects of durations of breast and formula feeding and age at the introduction of solid foods on the odds of ALL. Each model was calculated before and after adjustment for race, ethnicity, child's age and maternal smoking during pregnancy. Cases started solid foods significantly later than controls. More mothers of cases than controls smoked during pregnancy. Compared to the controls, cases had longer duration of formula feeding, and upon analysis of the exclusive formula versus mixed breast and formula fed, mixed feeding groups had a longer duration of formula intake. In a preliminary multivariate model, each additional month of formula feeding was associated with a 16% (OR: 1.16, 95% CI 1.08-1.25) increased odds of ALL; and likewise each additional month of delaying the age at introduction of solids was also associated with a 14% (OR1.14, 95% CI: 1.04 -1.26) increased odds of ALL after adjustment for covariates. In this ethnically diverse population, duration of formula feeding and age at introduction of solid foods were directly associated with increased odds of ALL. Further research needs to address the factors influencing duration of formula feeding and delay in introduction of solids. Our results highlight the role of energy balance in early life as critical contributors to risk for pediatric ALL. Citation Format: Jeremy Schraw, Yong Q. Dong, Michael E. Scheurer, Steven Hirschfeld, M Fatih Okcu, Michele R. Forman. Longer formula feeding and later age at introduction of solids increase the odds ratio of pediatric acute lymphoblastic leukemia. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr A102.

Successful engraftment of human acute lymphoblastic leukemia cells in NOD/SCID mice via intrasplenic inoculation

Acute lymphoblastic leukemia (ALL) is a heterogeneous disorder, and primary drug resistance and relapse are thought to be the main causes for treatment failure in ALL patients. For these refractory or relapsed patients, there is an increasing demand to identify novel therapeutic approaches, which will highly rely on the use of xenotransplantation models in translational research. Given the critical role that the spleen plays in the hematopoiesis and lymphopoiesis in adult mice, intrasplenic inoculation of ALL cells into immunodeficient mice may represent a feasible route for leukemic xenotransplantation. In the present study, engraftments via intrasplenic inoculation in anti-mCD122 mAb conditioned NOD/SCID mice were achieved in 5 out of 11 cases, and the engrafted cells reconstituted a complete leukemic phenotype. The engrafted cells sustained the self-renewal capacity of leukemia-initiating cells as tested by serial xenotransplantation and can be used for evaluation of antileukemic drugs. These data suggest that the combination of intrasplenic inoculation and the targeted depletion of CD122+ cells could provide a novel approach for the xenotransplantation of ALL cells in NOD/SCID mice. Furthermore, this model can be used for stem cell research, long-term analysis of engraftment kinetics and in vivo drug tests.

A Case Report of Simultaneous Occurrence of Wilms Tumor and Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most and Wilms tumor is the sixth most common malignancy in childhood. Genetic changes in WT1 or WT2 in Wilms tumor and a wide range of chromosomal mutations are reported in ALL that may predispose them to other congenital anomalies especially in Wilms tumor patients. Hospitalized patient was a 3-year-old girl with high-grade fever, petechia, and solid abdominal mass. The patient had anemia, thrombocytopenia, and leukocytosis in her initial homogram, and a large renal mass originated from left kidney in abdominal imagings. Bone marrow aspiration revealed ALL, and simultaneous kidney biopsy uncovered her Wilms tumor. Leukemia and Wilms tumor are 2 independent malignancies but occurred together in this patient. It is reasonable to investigate cellular and genetic relation of these 2 independent malignancies.

Screening Survivors of Childhood Acute Lymphoblastic Leukemia for Obesity, Metabolic Syndrome, and Insulin Resistance

Acute lymphoblastic leukemia (ALL) survivors were screened for risk factors of cardiovascular disease. Forty-four ALL survivors in first remission were enrolled. Twenty-six also received 12–18 Gy cranial radiotherapy (RT). Patients’ body mass indexes (BMIs) at dignosis and during the study were compared. Metabolic syndrome (MS) evaluation was performed in patients, parents, and siblings older than 6 years. Homeostasis Model Assessment (HOMA) index of the survivors was also calculated. In survivors with impaired fasting glucose levels, oral glucose tolerance test (OGTT) was performed. Thyroid functions and IGF-1 and/or IGFBP-3 levels of the survivors who received cranial RT were evaluated. Median age of the survivors was 11.5 years (6–23). At diagnosis, mean BMI percentile was 46.7 (3–95) and mean z-score was −0.09 ± 1.14; during the study, these values rose to 71.1 ± 25.6 (3–100) and 0.8 ± 0.94, respectively (P < .001). One patient (2.2%) and nine survivors (20%) were obese at diagnosis and during the study, respectively (P = .005). Survivors had significantly higher BMI percentile and BMI z-score compared to their siblings (P = .006 and P = .011, respectively). The study group was small and we could not show a correlation of the patients’ obesity with RT, thyroid functions, IGF-1, and IGFBP-3 levels. In three survivors (6.8%), there was MS. Maternal and paternal MS was not found as a risk factor for MS of the survivors (P = .1, P = .5, respectively). The HOMA index revealed insulin resistance (IR) in 12 (27.2%) of the survivors, whereas OGTT revealed abnormal glucose regulation and/or IR in four. As a conclusion, ALL survivors have high risk for obesity and MS.

Brown macroalgae produce anti-leukemia compounds

Acute lymphoblastic leukemia (ALL) is a fast-growing cancer in which the body produces a large number of immature white blood cells (lymphocytes). Although treatable in children with fairly good results, in adults complete remission rates are low and unlikely to improve by any significant degree. The challenge is to maintain the remissions and, for those who develop recurrent disease, to provide effective therapy. To this end, it is imperative to identify new and active leukemia-specific drugs. In this sense, we evaluated the in vitro anti-proliferative effect of extracts from brown algae, namely Halopteris scoparia, Cladostephus spongiosus, Cystoseira tamariscifolia and Cystoseira nodicaulis. MTT assays, DAPI staining and flow cytometry led us to conclude that Cystoseira extracts selectively inhibited proliferation and induced apoptosis in several human leukemia cell lines (SUP-T1, PF-382 and THP-1), but not in non-tumoral S17 cells. Further studies have demonstrated the anti-proliferative activity of Cystoseira associated with a clear arrest in the G0/G1 phase, indicating that the cell cycle status of leukemic cells plays an important role in the response to treatment. Though the active compounds remain to be identified, Cystoseira extracts proved to be a promising source of anti-leukemia compounds.

Sidney Farber and the Treatment of Childhood Acute Lymphoblastic Leukemia with a Chemotherapeutic Agent

Acute lymphoblastic leukemia (ALL) is the most common malignancy affecting children accounting for approximately 30% of childhood cancers, which was uniformly fatal before the advent of effective chemotherapy. In this historical note, the fundamental contribution of Sidney Farber to the treatment of ALL by using, for the first time, aminopterin—a chemotherapeutic agent—has been recognized.

Abstract 4783: Proteomic analysis of acute lymphoblastic leukemia: de novo versus relapse

Abstract Acute lymphoblastic leukemia (ALL) is a heterogeneous disease with distinct biological and prognostic grouping. The initial response to induction chemotherapy is the most important prognostic factor in patients with acute lymphoblastic leukemia. Patients with rapid early response (RER &amp;lt; 5% of blast in bone marrow at day 7 after induction chemotherapy) have better event-free survival compared to slow early responders (SER ≤ 25% of blast). However, no predictive marker for relapse or treatment failure is currently available. This study aimed to find plausible biomarkers of drug sensitivity in bone marrow mononuclear cells (BMMNCs) of childhood ALL cases using 2-dimensional gel electrophoresis and subsequent MALDI-TOF/mass spectrometry. The 714 spots in RER and 555 spots in SER were visualized in gels. Of these 28 up-regulated and 25 down-regulated proteins in SER_as compared to RER_were identified. The detected proteins were 60-kDa heat shock protein, actin, lamin-B1, heterogeneous nuclear ribonucleoprotein (hnRNP) K, and so on. HnRNPK, down-regulated in SER was further verified by Western blot analysis. In future studies, the roles of HnRNPK in drug sensitivity/resistance are undergoing to elucidate as a potential biomarker for ALL therapy. [Funding source: NRF grant (R13-2005-012-01003-0)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4783. doi:1538-7445.AM2012-4783

Abstract 4879: Distinct roles of p53 and p19ARF in MYC-dependent tumor oncogene addiction

Abstract Acute T-cell lymphoblastic leukemia (T-ALL) is a pediatric malignancy in which the MYC oncogene is frequently overexpressed. Although, T-ALL responds to standard therapies, in 20% of cases, treatment fails to induce sustained remission, resulting in reduced patient survival. Inhibition or restoration of the mutant gene products that drive tumorigenesis can reverse the aberrant cell growth and result in elimination of tumor cells through the phenomenon of oncogene addiction. In order to recapitulate tumor regression and recurrence, we have developed conditional mouse models of MYC-dependent T-ALL, thereby allowing for the investigation of therapeutic resistance. Recently, we have shown oncogene addiction in our MYC-driven murine model of T-ALL occurs through both tumor cell-intrinsic mechanisms, such as cellular senescence and apoptosis, and tumor cell-extrinsic, host-dependent mechanisms, including inhibition of angiogenesis, immune system-dependent tumor clearance, and autocrine/paracrine survival signaling. We now present evidence that loss of either p53 or p19ARF facilitates tumor recurrence through distinct mechanisms. p53 utilizes host-dependent mechanisms by allowing tumors to maintain angiogenesis, while p19ARF employs tumor-cell intrinsic mechanisms to circumvent senescence upon oncogene inactivation in vivo. To further interrogate the mechanisms that underlie p53- and p19ARF-dependent tumor recurrence, we performed gene expression analysis on MYC wild-type, MYC/p53-/-, and MYC/p19-/- tumors before and after MYC inactivation. Utilizing novel computational techniques to compare gene expression from our T-ALL cells to gene expression of normal hematopoietic differentiation from the Immunologic Genome Project, we have delineated the regulatory pathways underlying the effects of tumor suppressor loss in human MYC-driven lymphoma cells in the presence of targeted MYC-inactivation. We have also utilized expression analysis to establish gene signatures from unique to our MYC/p53-/- and MYC/p19-/- murine T-ALL models and shown them to be prognostic for specific human T-ALL subtypes, as well as patient relapse-free survival. Overall, we have utilized whole transcriptomic analysis to further define the underlying cell signaling by which loss of p53 and p19 facilitate tumor recurrence and demonstrated that loss of these tumor suppressors can predict outcome in human patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4879. doi:1538-7445.AM2012-4879

Abstract 2484: Whole-exome sequencing of a rare case of familial childhood acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer in children, accounting for approximately 25% of all pediatric cancer cases. However, familial childhood ALL is extremely rare. Few families with multiple non-twinned siblings diagnosed with childhood ALL have been reported, and to date no highly penetrant leukemia susceptibility gene(s) has been identified to explain this uncommon occurrence. We postulated that pure (nonsyndromic) familial childhood ALL could result from the accumulation of disadvantaging rare DNA variants in predisposing genes or biological pathways. To address this hypothesis, we used next-generation sequencing technologies to capture and re-sequence the whole-exomes of a family comprising the mother, father and two male non-twinned affected siblings (sibling A and sibling B). Both brothers were diagnosed with the identical ALL subtype, pre-B hyperdiploid childhood ALL, three years apart. The similar clinical and molecular characteristics of the siblings suggest shared etiologic factors. Using the Agilent SureSelect All Human Exon 38 Mb Kit and the SOLiD 3 Plus system, we captured and sequenced a total of 17.5 Gb of sequence for the entire family, with a mean coverage of 47X. For each individual, approximately 96% or 36.4 Mb of the targeted bases were covered α1X and 70% of the targeted bases or 26.4 Mb passed our thresholds for variant calling. We identified 52,038 positions at which the called allele(s) differed from the reference genome in at least one of the four family members. In total, we identified 19,096 germline variants in sibling A and 28,061 in sibling B, of which 2,355 (12.3%) and 2,125 (7.6%), respectively, were previously undiscovered in dbSNP. We investigated non-synonymous homozygous variant and compound heterozygous positions shared between the siblings, as well as genes/pathways with increased burden of rare non-synonymous variants. Based on several criteria (PolyPhen annotation, known allele frequency, etc.), we identified variants that are strong functional candidates to explain this case of pure familial childhood ALL. In parallel, high-density genotyping was also performed (Illumina Omni 2.5M) for quality control and structural variant detection, allowing the identification of putatively shared copy number variants that may also be involved in leukemogenesis. Though independent validation and functional assessment is required, this is the first study to identify genetic factors involved in pure familial childhood ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2484. doi:1538-7445.AM2012-2484

Abstract 2736: PIM2 is up-regulated in response to metformin-induced cell death triggered by ER stress/UPR in ALL lymphoblasts

Abstract Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, and despite significant overall improvements in cure rates, outcome for patients diagnosed with resistant phenotypes or those who relapse is dismal. We investigated the mechanism of cell death induced by metformin (MET) in ALL cell models. We showed that MET induced significant growth inhibition and apoptosis in CCRF-CEM (T-ALL) and NALM6 (Bp-ALL) cells. Western blots revealed that MET activated p-AMPK, p-ACC, p-Akt, and p-4EBP1. The latter suggested that regulation of protein translation may be an important determinant in MET-induced cell death. Indeed, inhibition of mTOR/protein translation with rapamycin (RAPA) rescued MET-induced cell death in ALL cells. In addition, knockdown of AMPK expression in ALL cells using shRNA (shAMPK) abrogated MET-induced apoptosis as compared to control cells expressing scramble shRNA (shCTRL), indicating that AMPK mediated MET's cytotoxicity. Western blots demonstrated that shAMPK cells expressed lower level of total AMPK, p-p38MAPK, p-mTOR, and p-4EBP1 compared to shCTRL cells, indicating that AMPK and protein translation are critical in MET sensitivity. Indeed, pulse labeled 35S-methionine experiments demonstrated increase incorporation confirming the importance of protein synthesis is MET-induced cytotoxicity. More important, we uncovered that MET-induced apoptosis correlated with induction of ER stress as evidenced by up-regulation of IRE1α and CHOP although GRP78 decreased significantly. We further demonstrated that RAPA rescued MET-treated cells by relieving ER stress/UPR mediated cell death. We previously showed that the unfolded protein response (UPR) in ALL cells is regulated by the contextual crosstalk between AMPK and Akt (Mol Cancer Ther 10:437, 2011). Therefore, we evaluated the effects of co-targeting Akt and AMPK using the Akt inhibitor X/perifosine + MET and found these combinations to be synergistic. To further investigate the relationship between protein translation and ER stress/UPR in MET-induced cell death, we examined the role of PIM1/2 kinases. We found that expression of PIM2 was increased in MET-treated ALL with concomitant decreased in the expression of IRE1α, ATF6, and CHOP, suggesting that PIM2 maybe up-regulated as a compensatory survival mechanism aimed at relieving MET-induced ER stress/UPR mediated cell death. To test this hypothesis, we co-treated ALL cells with a PIM1/2 kinase inhibitor + MET and found that PIM2 inhibition synergistically sensitized ALL cells to MET (CI=0.28). Taken together, our data indicate that PIM2 plays a role in buffering cell death in MET-treated cells, and that regulation of protein translation modulates ER stress/UPR induced apoptosis in ALL cells. Consequently, our data support strategies targeting these synthetic lethal interactions as suitable for clinical translation in patients with ALL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2736. doi:1538-7445.AM2012-2736

Abstract 5221: A fusion toxin for the treatment of acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the most common cancer of childhood. Two major challenges in the therapy of B-lineage ALL are overcoming acquired drug resistance and treatment of relapse. The interaction of ALL cells with the bone marrow microenvironment is thought to assist the development of resistance to therapeutic drugs. Therefore, we are focusing on the bone marrow microenvironment as a possible target for treatment by investigating the interactions between cell surface receptors on the ALL cells that are engaged by factors produced in the bone marrow. Recently, we were the first to report that pre-B ALL cells express the B cell-activating factor receptor (BAFF-R). In the current study, we used a fusion protein, consisting of the toxin Gelonin (a plant toxin which inhibits protein synthesis by inactivating ribosomes) and the ligand for the BAFF-R, BAFF/BLys for selective killing of ALL cells. Gelonin cannot enter the cells by itself since it lacks the ability to bind to the cell surface. Here, we demonstrate that rGel/BLyS binds to ALL cells expressing BAFF-R and, upon internalization, it induces apoptosis of these cells and causes down-regulation of survival genes even in the presence of stromal protection. Further, we transplanted human leukemic cells into NSG mice and allowed the cells to proliferate for 6 days to form an appreciable tumor burden. Then 3.75 mg/kg rGel/BLyS was administered bi-weekly for 3 weeks. Mice not receiving treatment had to be sacrificed at around day 34 whereas mice treated with rGel/BLyS survived significantly (p&amp;lt;0.002) longer. Remarkably, FACS analysis of peripheral blood from mice treated with rGel/BLyS, using human CD19 and CD10 antibodies, showed that the treatment had eliminated virtually all circulating leukemia cells. However, the bone marrow (BM) of these mice clearly contained leukemic cells. We used AMD3100, a CXCR4 antagonist, to mobilize the ALL cells protected in the bone marrow microenvironment, in combination with rGel/BLyS. In the combination treatment group, mice were first treated with rGel/BLyS alone to reduce the overall leukemia burden. Then the combination treatment was initiated, with AMD3100 injected 5-6 hours before the rGel/BLyS was administered. This strategy was used to ensure the presence of ALL cells in circulation at the time when the toxin would also be in the circulation. The combination treatment resulted in a significant reduction of the tumor load in the bone marrow and complete eradication of ALL cells from the circulation. To conclude, rGel/BLyS is a very promising therapeutic agent for ALL with selective cytotoxicity mediated by its fusion to the ligand for the BAFF-R. Moreover, by combining this selective but toxic fusion protein with a non-toxic ALL mobilizing agent, we were able to significantly deplete the pool of malignant lymphoblasts in vivo that could form the basis for relapse in the bone marrow. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5221. doi:1538-7445.AM2012-5221

Abstract SY25-04: Sequencing the genome of acute lymphoblastic leukemia

Abstract Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy, and remains the leading cause of non-traumatic death in children and young adults. Current treatment of ALL utilizes combination systemic and intrathecal therapy with mostly non-targeted chemotherapeutic agents, and further escalation of treatment intensity is limited by unacceptable short- and long term toxicity. Targeted therapy with tyrosine kinase inhibitors in BCR-ABL1 positive leukemia has been dramatically successful, and this observation has stimulated intense interest in identifying rational targets for novel therapies in additional patients with ALL. Moreover, ALL has long been known to consist of a number of subtypes characterized by distinct constellations of genetic alterations defined by karyotypic analysis, microarray profiling and candidate gene sequencing, including structural chromosomal rearrangements, DNA copy number alterations and DNA sequence mutations. While these approaches have been informative in identifying novel somatic genetic alterations in ALL, they are inherently incapable of comprehensively identifying all and somatic genetic alterations driving leukemogenesis and influencing treatment outcome. In addition, these approaches have limited ability to fully characterize the nature of intratumoral clonal heterogeneity and genetic diversity, which is now recognized to be a hallmark of ALL, and an important determinant of treatment failure and relapse. Several groups are now utilizing next-generation sequencing to provide a comprehensive portrait of the nature and frequency of inherited and somatic genetic alterations in ALL. These projects, including the St Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project, and the National Cancer Institute - Children's Oncology Group TARGET (Therapeutically Applicable Research to Generate Effective Therapies) Initiative multi-institutional collaborative efforts that are using transcriptome sequencing (RNA-seq), exome sequencing and whole genome sequencing (WGS) of large numbers of ALL samples. Results and future directions for several of these projects will be described, and include: (1) Early T-cell precursor (ETP) ALL: WGS of this aggressive leukemia has shown that ETP is characterized by frequent mutations in multiple critical cellular pathways, including hematopoetic development, cytokine receptor and Ras signaling, and epigenetic regulation of gene expression. These findings have identified several potential new approaches for therapy in ETP ALL. (2) High risk “BCR-ABL1-like” B-progenitor ALL: This form of leukemia exhibits many features similar to BCR-ABL1 positive ALL, yet lacks expression of the BCR-ABL1 chimeric fusion protein. RNA-seq and WGS have identified a range of novel rearrangements, structural variations and sequence mutations that activate cytokine receptor and tyrosine kinase signaling. Importantly, these alterations are transforming, and potentially druggable with currently available tyrosine kinase inhibitors. (3) Infant leukemia. Infant ALL is highly aggressive and commonly accompanied by rearrangement of the mixed lineage leukemia (MLL) gene. WGS of MLLr infant leukemia extends the findings of pre-WGS genomic profiling studies by showing that very few additional somatic genetic alterations are present in this form of ALL. These studies have also stimulated the development of novel, sensitive and specific algorithms for the detection of structural genetic alterations described by Dr Zhang in this session, and notably, have shown that WGS and RNA-seq may provide complementary genetic information. Also, these studies have also shown that in addition to comprehensive characterization of “discovery” cohorts of leukemia genomes, detailed analysis of mutational frequency in extended recurrence cohorts, and complementary profiling of gene expression is required to accurately define the nature and frequency of genetic alterations across the range of ALL subtypes, and to assess their role in leukemogenesis. Importantly, these studies have largely focused on somatic genetic variation affecting coding genes, and a key goal of ongoing studies is the investigation of the role of inherited and somatic non-coding variation in leukemogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY25-04. doi:1538-7445.AM2012-SY25-04