ALBERT

Description: Key Points Integrated genomic profiling identifies high-risk adult T-ALL patients with poor response to intensified chemotherapy.

Description: Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genomic lesions. However, given recent recognition that multiple genomically distinct sub-clones can exist in AML, there is a risk that there may be selection for sub-clones from the transplanted sample that might not fully represent the patient’s disease. We transplanted 160 (70 T-ALL 56 AML, 32 B-ALL, 2 MLL) patient samples of which 120 engrafted into at least 1 irradiated NSG mouse. 45 AML samples engrafted with a median latency 107+/-41 days. Transplantation of 6 PDX AML samples resulted in immunophenotypically identical disease within 87+/-35 days. 2 MLL samples engrafted in 100% of mice with a median latency of 103+/-13 days. 25 B-ALL samples engrafted with a median latency of 95+/-44 days. Secondary transplantation of 3 PDX B-ALL samples resulted in engraftment of leukemia cells with an identical immunophenotype in 100% of transplanted mice within 52+/-3 days. 48 T-ALL samples engrafted in at least one mouse within 50 days. Secondary transplantation of a single T-ALL PDX sample resulted in 100% engraftment within 31+/-10 days. Genomic DNA and total RNA were isolated from 150 (AML: 16Pt+33PDX; MLL 2Pt+6PDX; B-ALL 17Pt+38PDX; T-ALL 19Pt+19PDX) samples. Adaptor ligated sequencing libraries were captured by solution hybridization using baitsets for 405 cancer-related genes and selected introns for 31 genes frequently rearranged for DNA-seq, and 405 cancer-related and 265 genes frequently rearranged for RNA-seq. All libraries were sequenced averaging 〉500x for DNA and 〉6M total pairs for RNA (HiSeq). We detected on average 23+/-12 including a mean 5+/-4 known pathogenic variants such as CDKN2A/B deletion (20/13); FLT3 (SNV & -ITD) and NOTCH (11 ea); WT1 and TP53 (10 ea); NRAS (9); PTPN11 (7); NPM1c, PTEN, and KRAS (6) DNMT3A, IDH1/2, and ASXL1 (5 ea); FBXW7, CEBPA, and TET2 (4 ea); PHF6 and NF1 (3 ea); IKZF1, ATM, and JAK2 (2 ea). Analyses of fusion RNA molecules detected known fusions: MLL-AF4 (4); MLL-AF9 (2), CRLF2-P2RY8, ETV6-RUNX1 or TEL-AML1, PBX1-TCF3 (2 ea); MLL-AF10, MLL-ELL, MLL-EP300, MLL-PTD, BCR-ABL, BCL2-IGK, MYH11-CBFB, along with novel fusions: TCF3-OAZ1, RB1-RCBTB2, PAX5-FLI1, and PAX5-MSI2. The mutations found in the 54 patient samples were consistently identified in the 96 PDX, however some cases showing variation in allele frequency between diagnostic and engrafted samples. Collectively, all 1420 and 288 disease relevant variant allele frequency (VAF) correlated significantly between patient and PDX samples (R2=0.55, R2=0.43), respectively. We then assessed VAF changes from diagnostic to PDX sample as a measure of clonal concordance. Diagnostic and PDX sample were considered discordant if at least one disease relevant VAF demonstrated significant variation between these samples, accounted for small variability of infrequent variances considering SD of sequencing detection. 31 samples were scored as concordant and 23 as discordant which were similarly distributed between disease lineages and did not correlate with diseases status, future relapse or overall survival. Using the same rules we further accessed concordance only between PDX samples in 23 cases when patient samples were transplanted into multiple mice. All 10 groups of PDX samples that were concordant with patient samples were also concordant within the groups. 5 groups of PDX samples that were discordant with patient samples were concordant within groups. 8 groups of PDX samples that were discordant with patient samples were also discordant within their groups. Overall 15 samples produced concordant engraftment in mice and 8 samples produced discordant engraftment. We hypothesized that specific genomic lesions in the 8 groups might underline this discordance. Mutations of FLT3, RAS, TP53, PTPN11 and NOTCH1 correlated with clonal discordance. These findings show that the leukemias that are engrafted in mice mirror the genomic diversity of primary leukemia samples, and that the majority of PDX samples have a genotype similar to that observed in the clinical isolate. More importantly, our data demonstrate the feasibility of developing a large, genetically annotated bank of PDX leukemia models that can be used to test and credential novel therapeutics that target driver mutations in different leukemia subsets. Disclosures Stein: Seattle Genetics, Inc.: Research Funding; Janssen Pharmaceuticals: Consultancy. Wang:Foundation Medicine Inc: Employment. Miller:Foundation Medicine: Employment. Armstrong:Epizyme: Consultancy.

Description: Key Points SH2B3 is a recessive tumor suppressor gene with germline and somatic mutations in ALL.

Description: Xenotransplantation of primary AML samples into immunodeficient mice (PDX models) represents a unique opportunity for pre-clinical testing on a group of primary human samples that possess defined genetic lesions. However, given our recent recognition that multiple genetically distinct subclones can exist in AML, there is a risk that there may be selection for sub-clones from the xenotransplanted sample that might not fully represent the patient’s disease. We sought to establish a collection of genetically defined AML samples capable of engraftment in immunodeficient mice. We transplanted 30 AML patient samples; within 150 days (median 91 days) post transplantation 12 samples produced human CD45+ CD33+ CD19- CD3- engraftment in one or multiple NSG mice. Median patient sample amplification in 25 mice was 21 fold. Genomic DNA and total RNA was isolated from 7 AML patient samples (3 diagnostic samples from patients who remain in remission; 2 diagnostic samples from patients who later relapsed, 2 diagnostic samples from patients with refractory disease) and 14 matched xenotransplanted samples (2 mice per patient sample). Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 272 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 〉499x for DNA and 〉20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The mutations found in the 7 diagnostic samples were consistently identified in the 14 engrafted AML samples, but with some cases showing variation in allele frequency between diagnostic and engrafted samples. This finding shows that the human disease that engrafted in mice mimics the genetic makeup of the disease found in patients. We then assessed for allele frequency (AF) changes from diagnostic to xenografted sample as a measure of clonal progression. Clonal progression was defined as emergence of a clone carrying a novel genetic variant in the xenografted sample as compared to the diagnostic patient sample. Five patient samples (from 10 mice) did not show emergence of novel genetic lesions. In this group 2 patients had refractory disease and 3 patients remain in remission. Two patient samples (from 4 mice) demonstrated apparent emergence of novel genetic lesions not detected in diagnostic patient samples. Both of these patients have relapsed since the diagnostic samples were acquired. In the first case, both xenotransplanted mice engrafted with disease carrying NRAS N12S mutation (AF 0.05 and 0.09), which subsequent evaluation revealed to be present below the limit-of-detection (AF 0.004) in the clinical isolate obtained from patient presentation. We are currently conducting the same analysis on the relapsed sample from this patient. In the second case, both mice engrafted with disease carrying PTPN11 E76V (AF 0.03 and 0.0016) while the patient diagnostic sample did not contain any evidence of the alteration at 718x unique sequence coverage. Of note, one xenografted sample had an IDH1 R132C and another had IDH2 R140Q mutation, both of which have previously been shown to play a role in AML pathogenesis. Available AML cell lines do not carry IDH1/2 mutations, making it challenging to test IDH1/2 inhibitors in pre-clinical settings. These xenografted samples offer an opportunity to test such inhibitors. Overall we conclude that the xenotransplanted samples possess the diversity of genetic abnormalities found in diagnostic AML samples and thus can be used to assess efficacy of novel targeted therapies. We would like to further investigate a model in which the absence of clonal progression in xenografted samples would predict a better patient outcome, while emergence of novel clones might indicate an increased potential for relapse. We are currently expanding the study to include more diagnostic, xenotransplanted and relapsed samples to assess the associations between the ability of a sample to engraft in mice with clinical outcome and genetic/epigenetic lesions. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.

Description: Abstract 471 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy associated with the activation of oncogenic transcription factors. Thus, 5% to 10% of pediatric and up to 30% of adult T-ALL patients show aberrant expression of the TLX1 transcription factor oncogene. Aberrant expression of TLX1 in Lck-TLX1 transgenics induces transformation of T-cell progenitors and the development of clonal T-cell lymphoblastic tumors with over 80% penetrance and a latency of 29 weeks. The prolonged latency before T-ALL development in TLX1 transgenic mice and the clonal nature of these tumors suggested the presence of cooperating mutations involved in the pathogenesis of these leukemias. Array Comparative Genomic Hybridization (aCGH) analysis identified several recurring chromosomal alterations in these tumors including three heterozygous deletions on chromosome 12, with a common deleted region containing only the Bcl11b gene. Moreover, DNA sequence analysis of Bcl11b showed the presence of Bcl11b mutations in 4/15 (27%) mouse TLX1 induced T-ALLs, which together with the 3 Bcl11b deletions identified in our aCGH analysis brings the prevalence of Bcl11b alterations in mouse TLX1 induced tumors to 7/15 (47%). Notably, mutation analysis of Bcl11b in a panel of 21 non TLX1-transgenic mouse T-ALL tumors failed to detect any Bcl11b mutations (P

Description: Abstract 294 T-cell acute lymphoblastic leukemia (T-ALL) is a highly aggressive hematologic tumor associated with poor prognosis. Over the last decade, microarray gene expression studies have shown that T-ALL encompasses distinct molecular groups defined by characteristic gene expression signatures and molecular studies have uncovered major mechanisms of T-cell transformation and numerous oncogenes and tumor suppressors involved in the pathogenesis of T-ALL. This molecular and genetic heterogeneity suggests that distinct groups of T-ALL may respond differently to chemotherapy. However, today there are no well established prognostic markers for patient stratification in this disease and all T-ALL patients are treated under the same therapeutic scheme. To overcome this obstacle here we performed a comprehensive analysis of the prognostic significance of molecular groups defined gene microarray expression profiling, copy number alterations identified by array-Comparative Genomic Hybridization, immunophenotypic markers and mutation analysis of all major adult T-ALL oncogenes (NOTCH1, IL7R, FLT3, NRAS) and tumor suppressor genes (FBXW7, PTEN, DNM2, PHF6, BCL11B, WT1, EZH2, ETV6, IDH1, IDH2, DNMT3A, GATA3 and RUNX1) in a clinical series of 53 primary leukemia samples uniformly treated according to the Eastern Cooperative Oncology Group (ECOG) E2993 protocol. Unsupervised analysis of gene expression oligonucleotide microarrays in this series revealed the presence of 2 stable gene expression clusters corresponding to early immature (n = 28) and cortical/mature (n = 25) adult T-ALLs respectively. Early immature T-ALLs show gene expression signatures related to hematopoietic stem cells and myeloid progenitors and are associated with poor prognosis and reduced overall survival compared with cortical/mature adult T-ALLs (P = 0.011). Immunophenotypic analysis showed that CD13 expression was strongly associated with poor survival in our patient series (P=0.002), whereas other early immature or myeloid antigens including CD34 and CD33 showed no clinical impact. Array comparative genomic hybridization (aCGH) analysis in this series identified absence of biallelic deletion of TCRγ, a cytogenetic feature associated with early induction failure and inferior overall survival rates in pediatric T-ALL (Gutierrez et al., JCO, 2010), in 27 out of 53 (51%) adult T-ALL samples analyzed. Notably, 22 of 27 T-ALLs with absence of biallelic deletion of TCRγ were found in the early immature adult T-ALL group, and as in pediatric T-ALL, this molecular marker was associated with inferior overall survival (P = 0.022). In addition, heterozygous deletions of the short arm of chromosome 17 encompassing the TP53 tumor suppressor gene predicted for worse clinical outcome (P = 0.0005); while, homozygous deletions of the CDKN2A/CDKN2B locus on the short arm of chromosome 9 was associated with favorable prognosis (P = 0.012). Most notably, the favorable prognostic effect of homozygous CDKN2A/CDKN2B deletions in our series was restricted to the good prognostic subtype of leukemia samples with a mature/cortical gene expression profile. Finally, comprehensive mutation analysis of adult T-ALL oncogenes and tumor suppressors demonstrated a favorable prognosis for patients with activation of NOTCH1 signaling as result of mutations in NOTCH1 and/or FBXW7 (P = 0.021) and for those harboring heterozygous inactivating mutations or deletions in the BCL11B tumor suppressor gene (P = 0.041). In contrast, somatic mutations in genes targeting the epigenetic regulators DNMT3A (P = 0.003) and IDH1/2 (P = 0.011) were associated with adverse prognosis. Multivariate analyses highlighted the association of NOTCH1 and/or FBXW7 mutations with favorable outcome and that of TP53 deletions and DNMT3A mutations with poor prognosis in adult T-ALL. Importantly, homozygous CDKN2A/CDKN2B deletions and CD13 expression may serve as prognostic markers to further stratify low-risk cortical/mature adult T-ALL leukemias, whereas DNMT3A mutations might be useful for risk stratification within high-risk early immature adult T-ALLs. Overall, our comprehensive analysis of molecular prognostic markers identify for the first time a subset of adult T-ALLs with very poor response to intensified chemotherapy, highlighting the need to introduce alternative therapies aiming to improve the therapeutic outcome in this group. Disclosures: No relevant conflicts of interest to declare.

Description: Cooperation between several epigenetic modulators defines MLL-rearranged leukemia as an epigenomic-driven cancer. Wild type MLL catalyzes trimethylation of lysine 4 on histone 3 from the methyl donor S-adenosylmethionine (SAM) at homeobox and other genes important for hematopoiesis, promoting their expression during development. However, in MLL-rearrangements, its methyltransferase domain is ubiquitously lost and replaced with 〉70 known fusion partners. Many of these fusion partners recruit DOT1L, the only known SAM-dependent lysine methyltransferase responsible for the methylation of lysine 79 of histone 3 (H3K79)—a mark associated with most actively transcribed genes. Therefore, the recruitment of DOT1L by MLL fusion partners to MLL-target genes leads to aberrant H3K79 hypermethylation at these loci, resulting in inappropriate gene expression and leukemogenesis. DOT1L as a therapeutic target in MLL has been genetically validated by several groups, leading to the development of SAM-competitive small molecule inhibitors of DOT1L. These inhibitors exhibit excellent biochemical activity and selectivity, yet have delayed cellular activity and needing relatively high doses, with viability effects requiring 7-10 days and EC50s for H3K79 methylation depletion of 1-3 μM in cell lines. In animal studies, this translates to a modest survival benefit while requiring high doses through continuous osmotic subcutaneous infusion. Further optimization of DOT1L inhibitors is therefore needed. To date, development of DOT1L inhibitors has been slow, perhaps related to inadequacy of discovery chemistry assay technologies. All biochemical assays are radioactivity-based and are not miniaturizeable; low-throughput and delayed cellular effects of DOT1L inhibition all hamper the discovery of improved inhibitors. Therefore a pressing need towards improved DOT1L inhibitor discovery is a robust, accessible, and rapid profiling platform. Toward this goal, we synthesized both FITC- and biotin-tagged DOT1L probe ligands. We confirmed by structural studies that binding of the probes were similar to our previously published inhibitor, depleted H3K79 methylation, and had antiproliferative effects in MLL-rearranged cell lines. We then utilized the probes to devise two non-radioactive, orthogonal biochemical assays to competitively profile putative inhibitors: one employing bead-based, proxmity fluorescence technology and the second using fluorescence polarization technology. These assays are robust and adaptable to high-throughput screening. We also designed a miniaturizable high-content imaging, immunofluorescence-based assay to assess the effect of DOT1L inhibitors on H3K79 methylation, reporting cellular IC50s after just four days of treatment. These three assays were validated against three known DOT1L inhibitors of different potencies, accurately differentiating between the compounds. Together, these orthogonal assays define an accessible platform capability to discover and optimize DOT1L inhibitors. Our platform rank-ordered a library of SAM derivatives that we synthesized, indicating that large substituents off the SAM base does not affect DOT1L binding. We also explored other features of the SAM core structure, identifying several chlorinated probes that had increased cellular potency (IC50 values ~10nM) relative to the initial compounds published, without losing specificity for DOT1L. The inhibitory effect on MLL-target gene expression correlated to the H3K79me2 decrease reported in high content assay, validating that our high-content assay accurately reports on downstream biology seen later in treatment. And as expected, the high-content potencies of our chlorinated DOT1L probes also correlated to increased anti-proliferative effect in MLL cells. Overall, we utilized chemistry, biology, and chemical biology tools to develop this profiling platform capability for more rapid discovery and optimization of small molecule DOT1L inhibitors. These assays can additionally be used to screen for non-SAM competitive inhibitors in high-throughput fashion. Furthermore, the DOT1L inhibitors and probes synthesized here (available as open-source tools) are useful in deeper mechanistic studies of the DOT1L complex and its role in MLL. Disclosures Armstrong: Epizyme: Consultancy.