ALBERT

Description: Abstract 8 T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy of immature T cell progenitors in which activating mutations of NOTCH1 occur in over 50% of cases and loss-of-function mutations in FBW7, which degrades activated NOTCH1, occur in 8-16% of cases. We and others have characterized c-MYC as a critical downstream transcriptional target of Notch signaling in this context, and loss of FBW7 also contributes here as intact FBW7 marks c-MYC protein for proteosomal degradation. Inhibition of Notch signaling in T-ALL cells by various means including small molecule gamma-secretase inhibitors (GSI) causes cell cycle/growth arrest and in some cases apoptosis, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor PTEN is also mutated or lost in up to 20% of T-ALL cases. PTEN activity converts PIP3 to PIP2, and thus loss of PTEN potentiates PI3K signaling. It was observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition with GSI, leading to the idea that constitutive PI3K/AKT signaling relieves T-ALL cells of their “addiction” to oncogenic Notch signaling. Since GSI and other NOTCH1 signaling inhibitors are presently under investigation as targeted therapeutic agents for T-ALL and other cancers including breast, brain, colorectal, pancreatic, and melanoma, this idea that PTEN loss could confer resistance to NOTCH1 inhibition raises concern that patients with PTEN-negative (or presumably PIK3CA-mutated) diseases may fail Notch inhibitor therapy. As the studies linking GSI-resistance/Notch-independence to PTEN loss were limited to established cell lines, we sought to address this issue using a well established and genetically defined mouse retroviral transduction/bone marrow transplantation model. Briefly, we generated primary murine T-cell acute leukemias using mutated NOTCH1 retroviruses on both wild-type and PTEN-null backgrounds and tested these tumors for response to GSI. Unexpectedly, we observed these primary murine leukemias to undergo growth arrest and cell size reduction with GSI treatment on both wild-type and PTEN-null backgrounds. We even observed wild-type background tumors which had lost PTEN spontaneously after either serial transplantation or extended culture in vitro to remain sensitive to GSI. Given that most cases of human T-ALL have also lost expression of p16INK4A/p14ARF tumor suppressors either by deletion or silencing, and since p16INK4A/p14ARF are critical regulators at the G1/S cell cycle checkpoint, we also generated primary murine tumors on a Pten, Ink4a/Arf double-null background. Even when both PTEN and Ink4a/Arf were deleted, we still observed the murine tumors to remain sensitive to GSI. To confirm these findings were not unique to murine tumors, we examined a panel of 13 primary human T-ALL cases including 7 PTEN-positive cases (6 of which were NOTCH1 mutated) and 6 PTEN-negative cases (4 of which were NOTCH1 mutated). We found 11 of these 13 cases to be GSI sensitive (6 PTEN-positive, 5 PTEN-negative) as illustrated by decreased proliferation and reduction in cell size. We found only 2 cases to be GSI resistant, including 1 PTEN-positive and 1 PTEN-negative. Our panel contained only one case with FBW7 mutation; this case was PTEN-positive and GSI-sensitive. Thus, in contrast to previous studies with established cell lines, we find in this study using primary human T-ALL samples and genetically defined primary murine leukemias that PTEN loss indeed does not confer resistance to Notch inhibition. Of note, other groups have postulated a role for FBW7 mutation in conferring resistance to Notch inhibition; however, we encountered only 2 GSI-resistant cases in our panel of primary human samples, neither of which was FBW7 mutated. In generating the murine Notch leukemias, we also had the opportunity to compare disease penetrance/latency and clonality among the various genetic backgrounds. We noted that PTEN loss was associated with accelerated disease onset and multiclonal tumors as compared to wild-type, whereas INK4A/ARF loss alone had no such effect. These findings suggest NOTCH1 activation and PTEN loss may collaborate in leukemia induction; however, technical caveats related to potentially enhanced retroviral transduction efficiency of PTEN-null bone marrow progenitors limit this conclusion, and thus more definitive experiments to address this issue are needed. Disclosures: No relevant conflicts of interest to declare.

Description: Pathologic germ line mutations that predispose patients to cancer are estimated to occur in 4-30% of all pediatric oncology cases. In addition to leukemia specific familial predisposition syndromes, children with rare constitutional syndromes, heterogeneous dysmorphic syndromes, and multiple-cancer hereditary predisposition syndromes are all at an increased risk for hematologic malignancies. However, to date no genome-wide analysis has been done to define the range of germ line mutations that occur in pediatric patients with hematological malignancies. To determine the frequency of pediatric cancer patients that have germ line variants of pathological significance in genes that predisposed to cancer, we analyzed the germ line and tumor DNA from 1120 pediatric cancer patients that were enrolled in the St. Jude – Washington University Pediatric Cancer Genome Project (PCGP). Samples were analyzed by whole-genome sequencing (n = 595), whole-exome sequencing (n = 456), or both (n = 69). Single nucleotide variants (SNVs), insertions/deletions (indels), structural variations (SV) and copy number alterations (CNAs) were detected using our analytical pipeline and all single nucleotide polymorphisms (SNPs) previously identified in non-cancer populations were filtered out. Our analysis then focused on the 23 cancer predisposition genes recently recommended for germ line analysis by the American College of Genetics and Genomics, along with an additional 8 genes that have been previously shown to predispose to pediatric cancer at a high penetrance. All variants in these 31 genes were classified as pathologic, likely pathologic, uncertain significance, likely benign, and benign based on literature review and in-silico predictions on the effect of novel mutations. An expanded analysis including a total of 565 genes known to play a role in oncogenesis was also evaluated. Pathologic or likely pathologic germ line variants in one of the 31 genes were detected in 8% (90/1120) of patients, including: 16% (46/287) of patients with solid tumors, 8.6% (21/245) with brain tumors, and 3.9% (23/588) with leukemia. Expanding this analysis to 565 cancer gene resulted in only a slight increase, with a pathologic or likely pathologic variant being detected in 8.6% (97/1120) of patients. The most frequently effected genes included TP53 (n=48), APC (n=7) and BRCA2(n=6). Importantly, in 〉50% of these patients, analysis of their tumor DNA revealed the absence of a wild type allele for the cancer predisposition gene that was altered in the germ line. The 588 pediatric patients with leukemia included 116 acute myeloid leukemias (AMLs: FAB M7 n=20; Core Binding Factor leukemias n=86; MLL-R n=10) and 472 acute lymphoblastic leukemias (ALLs: E2A-PBX1 n=53; ERG-R n=39; TEL-AML1 n=53; Hyperdiploid n=69; Hypodiploid n=47; BCR-ABL1 n=40; T-ALL n=32; MLL-R n=40; BCR-ABL-like n=31; and Other n=68). Across this cohort, 3.9% (23/588) of leukemia patients harbored a pathologic germ line mutations in one of the 31 cancer pre-disposing genes. This number increased to 4.6% (27/588; 28 mutations) when the expanded gene list was evaluated. TP53 (n=10) was the most frequently altered germ line gene in pediatric leukemia patients and was found predominantly in low-hypodiploid ALL, as previously reported. Germ line pathologic variants were also identified in KRAS, RUNX1, APC, BRCA2, and RET (2 cases each), and NRAS, SH2B3, BRCA1, MUTYH, PTCH1, SDHA,VHL, and NF2 (1 case each). Although germ line mutations in RUNX1 and SH2B3are typically associated with myeloid neoplasms, we identified these lesions in 3 cases of B lineage ALL suggesting an association with a wider spectrum of leukemia. In conclusion, a small but significant proportion of pediatric patients with leukemia carry a germ line variant of pathologic significance in a cancer predisposition gene. These results suggest that these germ line lesions likely play a direct role in the pathogenesis of the patient’s presenting leukemia. Moreover, our results suggest that these patients would benefit from future clinical surveillance for the development of a second cancer. Lastly, these data demonstrate the power of comprehensive next generation DNA/RNA sequencing for the identification of pediatric patients who carry a germ line pathologic variant in a cancer predisposition gene. Disclosures No relevant conflicts of interest to declare.

Description: Relapse following remission induction chemotherapy remains the major challenge in the successful treatment of childhood T cell acute lymphoblastic leukemia (T-ALL). Relapse often results from the outgrowth of residual leukemia cells that are present below the limit of detection or involves a new therapy-related secondary leukemia. Individualization of treatment might improve the outcome and long-term quality of life for T-ALL patients. Molecular genetic markers represent clinically useful factors which predict responses to therapy. T-cell receptor gamma (TCRG) gene rearrangements occur in more than 90% of T-ALL and provide markers of lymphoblast clonality. Determining rearrangements in the TCRG could be critical to the diagnosis and treatment of T-ALL in children and adults. Mutations in the NOTCH1, FBW7, and PTEN genes have been identified at high frequencies in pediatric T-ALL cases. Activating NOTCH1 mutations have been found in more than 50% of ALL patients, resulting in constitutive NOTCH1 signalling, whereas PTEN mutations are inactivating, resulting in increased PI3K/AKT signalling. FBW7 has been identified as an important tumor suppressor. Several studies reported that frequent mutations in the substrate binding domain (e.g. Arg465, Arg479, Arg505) for FBW7 in T-ALL cell lines and primary T-ALL specimens result in sustained NOTCH1 levels and downstream signalling and gamma secretase inhibitor resistance, suggesting an alternate mechanism for NOTCH1 deregulation. To investigate the mechanism of T-ALL relapse, we analyzed the TCRG gene rearrangements and mutational status of the NOTCH1, FBW7, and PTEN genes by comparing sequences in paired diagnostic and relapsed T-ALL samples from 11 children to evaluate their stabilities throughout disease progression and association with treatment failure. The age distribution of 11 patients ranged from four years to fifteen years. Original TCRG sequence (a measure of leukemia clonality) was fully preserved at relapse in 3 (27.3%) patients. Clonal evolution was identified in 8 (72.7%) patients, reflected in changes in TCRG sequence. In 3 patients at diagnosis, NOTCH1 mutations were detected. At relapse, the major leukemia clones exhibited different NOTCH1 mutations. For another patient, a NOTCH1 mutation was detected at relapse but not at diagnosis. No FBW7 mutations were detected either at diagnosis or relapse. In 5 patients at diagnosis, PTEN mutations were detected and at relapse, 2 preserved the same mutation and 2 lost their mutations, while the additional sample harbored a different PTEN mutation. Our comparative sequence analysis of pediatric T-ALL samples provided detailed insight in the stabilities and changes of TCRG rearrangements and NOTCH1, FBW7 and PTEN mutation status during disease development. Re-emergence of the initial ALL clone or the occurrence of a secondary ALL clone may be clinically important to guide subsequent therapy. Collectively, our results suggest that for the majority of cases, relapse is associated with appearance of a new leukemic clone. For a subset of these cases, this is accompanied by a distinct subset of NOTCH1 mutations and, to a lesser extent, PTEN mutations. FBW7 mutations are rare. Better understanding of the changes in oncogenes and tumor suppressor genes with progression of T-ALL may identify new targets for therapy and facilitate the design of individualized therapy for this disease. Further study is needed to determine whether the newly identified relapse ALL clones were present at diagnosis as minor subclinical populations.

Description: Acute megakaryoblastic leukemia (AMKL) accounts for ~10% of childhood AML. AMKL patients without Down syndrome have a poor outcome with a 3 year survival of less than 40%. To gain insight into the biology of this disease, we previously performed transcriptome sequencing on diagnostic blasts from a discovery cohort of 14 pediatric cases and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult samples. This analysis identified novel fusion transcripts restricted to pediatric AMKL including CBFA2T3-GLIS2,GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9. To confirm their role in oncogenesis and gain insight into the mechanism whereby these fusions promote disease, we introduced each of them into murine hematopoietic cells and assessed their effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a control retrovirus failed to form colonies after the second replating. By contrast, expression of each of the fusion genes resulted in a marked increase in self-renewal capacity, with colony formation persisting through 10 replatings. Immunophenotypic analysis revealed evidence of megakaryocytic differentiation in CBFA2T3-GLIS2 and MN1-FLI1 cohorts, whereas NIPBL-HOXB9 and GATA2-HOXA9 cells carried markers consistent with myeloid progenitors. Transplantation of fusion gene modified bone marrow cells into syngeneic recipients induced overt leukemia in all cohorts with the exception of CBFA2T3-GLIS2, suggesting an essential requirement for cooperative mutation(s) in cases expressing this chimeric gene. To assess self-renewal activity of the leukemia generated in our murine models, we conducted secondary transplants for all cohorts. In all cases, the leukemia was transplantable with a shorter latency than in the primary transplant setting. To characterize the tumors at the molecular level, 5 samples from each of the 3 fusions underwent array comparative genomic hybridization, transcriptome, and whole exome sequencing. Samples demonstrated a small number of cooperating mutations with 1.5 copy number alterations (range 0-6) and 6.4 single nucleotide variations (range 2-13) per case. Overall, cases carried an average of 7.9 mutations (range 2-14). Despite the low number of lesions, recurrently mutated genes were identified. These include activating mutations in Flt3, Kras, and cMet, as well as loss of function mutations in the tumor suppressors Phactr4, Wt1, and Tet2. A comparison between fusion subtypes did not reveal any statistically significant differences, although there was a trend towards a greater number of mutations in the GATA2-HOXA9 cohort. Transcriptome sequencing of cohorts, along with normal hematopoietic progenitor subsets, confirmed unique gene expression patterns between each of the fusions. Consistent with immunophenotyping, MN1-FLI1 demonstrated enrichment of the MEP signature while NIPBL-HOXB9 and GATA2-HOXA9 were enriched for CMP and monocyte precursor signatures respectively. ChIP-seq analysis of each of the fusions is underway to definitively identify the genomic targets whose expression is directly altered by their binding. A common characteristic between all fusions is the presence of protein interaction domains contributed by the N term partner, and DNA binding domains contributed by the C term partner. To determine if these fusions have a novel gain of function distinct from their independent counterparts, we introduced each partner gene into murine bone marrow cells for transplantation experiments. As previously described, introduction of MN1 into hematopoietic cells led to a highly penetrant leukemia. In contrast, HOXA9, HOXB9, and FLI1 all had 〉75% disease free survival with few myeloid leukemias resulting from their over expression, while GATA2 failed to induce any disease at all. NIPBL’s size precluded transplant assays. Therefore, to evaluate its contribution we introduced a point mutation previously shown to disrupt binding of NIPBL to the cohesion component MAU2. This alteration abrogated the ability of the fusion to induce leukemia in our transplant model, demonstrating the importance of this interaction in the pathogenesis of disease. In conclusion, our data confirms a pathogenic role for GATA2-HOXA9, MN1-FLI1, and NIPBL-HOXB9 in AMKL. Further studies delineating the cooperating mutations required for CBFA2T3-GLIS2 are indicated. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 757 Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%). To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. *TAG and ALG contributed equally to this work. Disclosures: Biondi: BMS, Novartis, Micromed: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding. Doehner:Hoffmann La Roche: Honoraria.

Description: Abstract 69 Infant (〈 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing. Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining. An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p

Description: NOTCH1 is activated by mutation in more than 50% of human T-cell acute lymphoblastic leukemias (T-ALLs) and inhibition of Notch signaling causes cell-cycle/growth arrest, providing rationale for NOTCH1 as a therapeutic target. The tumor suppressor phosphatase and tensin homolog (PTEN) is also mutated or lost in up to 20% of cases. It was recently observed among human T-ALL cell lines that PTEN loss correlated with resistance to Notch inhibition, raising concern that patients with PTEN-negative disease may fail Notch inhibitor therapy. As these studies were limited to established cell lines, we addressed this issue using a genetically defined mouse retroviral transduction/bone marrow transplantation model and observed primary murine leukemias to remain dependent on NOTCH1 signaling despite Pten loss, with or without additional deletion of p16Ink4a/p19Arf. We also examined 13 primary human T-ALL samples obtained at diagnosis and found no correlation between PTEN status and resistance to Notch inhibition. Furthermore, we noted in the mouse model that Pten loss accelerated disease onset and produced multiclonal tumors, suggesting NOTCH1 activation and Pten loss may collaborate in leukemia induction. Thus, in contrast to previous findings with established cell lines, these results indicate PTEN loss does not relieve primary T-ALL cells of their “addiction” to Notch signaling.