Abstract: Background : Infectious disease plays a central role in malignancy, with up to one in six cancers having a microbial association (Parkin Int. J. Cancer 2006). Lymphomas in particular are associated with multiple viral pathogens, including Epstein Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV), and HIV. Sequencing of cell-free DNA (cfDNA) is an emerging technique in the diagnosis and surveillance of cancer. While studies to date have focused primarily on tumor-associated somatic variants, cfDNA may also provide insight into the infectious and immune state of cancer patients. We examined cfDNA from lymphoma patients of multiple histologic subtypes to characterize viral detection and dynamics. Methods: Plasma from 360 pre-treatment patients with various lymphoma histologies was analyzed along with that of 69 healthy adults. Multiple samples per patient were included when available. All samples underwent deep sequencing with error correction by CAPP-Seq (Newman Nat Biotech 2016). Reads were filtered for homology to the human genome and endogenous retroviruses, mapped to NCBI consensus genomes for human-hosted viral species, and filtered by breadth of genomic coverage. Viral read count was normalized by total sequencing depth to determine viral read fraction (VRF). EBV fragment size was assessed via single-read BLAST alignment length considering reads with expect value 〈 1E-5. Integration sites were assessed with the VirusClip package (Ho Oncotarget 2015). Results: Patients with most lymphoma histologic subtypes had viral loads not significantly different from those of healthy adults. However, post-transplant lymphoproliferative disorder (PTLD) patients receiving immunosuppression for solid organ transplants had significantly increased total viremia (Fig 1A) and EBV levels (Fig 1B) when compared to healthy adults and non-transplant DLBCL patients. EBER+ classical Hodgkin lymphoma (cHL) displayed no difference in total viremia but had significantly elevated EBV. In an EBV-positive PTLD patient, cfDNA viral levels tracked both clinical viral qPCR and circulating tumor DNA (ctDNA) levels in serial samples leading to diagnosis (Fig 1C). Elevated EBV levels were also present in a subset of non-transplant DLBCL. In a cohort of DLBCL patients treated with frontline R-CHOP-like chemotherapy (n=152), individuals with pre-treatment EBV frequency greater than VRF 1E-7 had significantly higher risk of disease progression at three years (HR 1.8, CI 1.0-3.4, p=0.013) (Fig 1D). Immunosuppression in transplant patients is associated with the expansion of the endogenous anellovirus family (De Vlaminck Cell 2013). Accordingly, anellovirus was detected significantly more often in PTLD patients (91% of samples) compared to DLBCL NOS (2.8%) and controls (1.4%) (Fig 1E, p 〈 0.0001). As the standard-of-care R-CHOP regimen for DLBCL has activity against both B- and T- lymphocytes, we hypothesized that an immunosuppressive effect might be observed. In non-transplant DLBCL patients receiving R-CHOP (n=31), we detected anellovirus in 6% of samples at the time of first chemotherapy infusion, 16% immediately before cycle 2, but in no samples from post-treatment patients in complete response (Fig 1F). Viral integration into the host genome is associated with malignant transformation. We profiled a cohort of EBER+ cHL (n=8) and found circulating EBV/human chimeric reads suggesting integration in all cases. Viral fragment size distribution also distinguishes integrated DNA from shorter free episomes and may increase cancer screening performance (Lam PNAS 2018). We profiled EBV fragment sizes in cHL and PTLD patients grouped by EBER positivity. Plasma from EBER+ cHL and PTLD patients was significantly enriched in longer fragments (Fig 1G), suggesting nucleosomal protection of EBV integrated within tumor genomes but not their benign episomal counterparts. Conclusions: Viral infection in lymphoma has diagnostic and prognostic significance: elevated circulating EBV levels are associated with active PTLD (Kanakry Blood 2016) and poor outcomes in advanced HL (Kanakry Blood 2013) and DLBCL (Tisi Leuk & Lymph 2015). Our work demonstrates the utility of cfDNA sequencing for simultaneous characterization of malignancy, infection, and immunosuppression. The integration of viral dynamics into cfDNA analysis may assist in risk stratification and treatment monitoring in lymphoma patients. Disclosures Dührsen: Amgen: Research Funding; Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Honoraria. Hüttmann:Celgene: Other: Travel expenses; Roche: Other: Travel expenses. Meignan:F. Hoffman-La Roche Ltd: Honoraria. Casasnovas:Janssen: Consultancy; Takeda: Honoraria; Janssen: Honoraria; MSD: Honoraria; Merck: Honoraria; Gilead Sciences: Honoraria; Celgene: Honoraria; Roche: Consultancy; Roche: Research Funding; takeda: Consultancy; Gilead Sciences: Consultancy; Roche: Honoraria; Gilead Sciences: Research Funding; merck: Consultancy; MSD: Consultancy. Westin:Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Apotex: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees; Celgen: Membership on an entity's Board of Directors or advisory committees. Gaidano:Amgen: Consultancy, Honoraria; Morphosys: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Advani:Bayer: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Agensys: Research Funding; Infinity: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Merck: Research Funding; Janssen: Research Funding; Cell Medica: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Kyowa: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding; Celgene: Research Funding; Kura: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Regeneron: Research Funding; Autolus: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Gilead/Kite: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Forty Seven Inc.: Research Funding; Celgene: Research Funding.

Abstract: Introduction: Cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) have an emerging diagnostic role in multiple malignancies including in lymphomas (Kurtz et al ASH 2017). In classical Hodgkin Lymphoma (cHL), malignant Reed Sternberg (RS) cells are rare, requiring laser capture microdissection from archival tissues or flow sorting from viable tumor cell suspensions for genotyping. We profiled ctDNA in cHL to assess the utility of ctDNA in the noninvasive evaluation of somatic single nucleotide variants (SNVs), somatic copy number alterations (SCNAs), and tumor EBV status. Methods: A total of 53 subjects with HL (29 with early stage and 24 with advanced disease) were studied encompassing a total of 95 blood and tissue samples (72 from Stanford, 23 from UZ Leuven). Plasma samples were sequenced with CAPP-Seq (Newman et al Nat Biotech 2016), using a panel informed by the genotyping of primary tumor biopsies. The genotypes of cHL patients were compared to that of 189 patients with other B-cell malignancies. Given the thoracic distribution of most cHL, we also compared ctDNA levels to that of 55 lung carcinomas. ctDNA levels were calculated as the product of the cfDNA concentration and the mean allelic fraction of somatic mutations. Results: The median pretreatment ctDNA level in cHL was 125 hGE/mL (15 - 5277 hGE/mL), corresponding to a median variant allelic fraction (VAF) of 3.2% (0.3 - 13.9%) (Fig 1A). Pretreatment ctDNA burden was greater in cHL cases than in follicular lymphoma (FL) cases (p = 0.002), but was not significantly different from that of diffuse large B-cell lymphoma (DLBCL) (p = 0.26). Plasma genotyping in cHL and DLBCL also identified similar numbers of SNVs, recovering a median of 108 mutations in cHL and 117 mutations in DLBCL (p = 0.53). In samples with available diagnostic PET/CT, pre-treatment ctDNA levels in cHL were significantly correlated with total metabolic tumor volume (MTV) (Spearman ρ = 0.615, p = 0.006) (Fig 1B), but not with diagnostic PET/CT SUVmax, stage, bulky status ( 〉 10 cm), B-symptoms, or presence of extranodal disease. Surprisingly, despite the lower tumor purity of RS cells in cHL tumor masses than that of malignant B-cells in DLBCL, the relationship between ctDNA and PET/CT estimates of disease burden in cHL was highly similar to that of DLBCL. Specifically, cHL and DLBCL were statistically indistinguishable for the ratio between ctDNA levels and MTV (mean ctDNA/MTV of 2.1 vs 1.5 hGE/mL per cm3 tumor, p = 0.38), and both were significantly higher than that of non small cell lung carcinoma (NSCLC) (p 〈 0.0001) (Fig 1C). In patients with available mid-treatment cfDNA (n = 10), we monitored ctDNA concentrations and observed that circulating tumor burden falls rapidly, with a third of our patients reaching undetectable levels within the first month after start of therapy. PD-L1 copy number gains, previously shown to be prognostic for survival in cHL treated with checkpoint inhibitors, were observed in 42% of cHL patients with ctDNA VAFs above our SCNA limit of detection (1%) and were genotyped significantly more frequently than in other non-PMBCL B-cell malignancies (42% vs 18%, p = 0.005) (Fig 1D). Coding SNVs in the most commonly mutated genes involved STAT6 (24%), SOCS1 (20%), GNA13 (20%), TNFAIP3 (18%), and B2M (16%) while noncoding SNVs in IGK and IGH were more abundant in cHL and DLBCL respectively (Fig 1E). EBV tumor cell presence has previously been shown to be prognostic in cHL (Keegan et al JCO 2005). Prior to therapy, EBV cfDNA constituted a significantly larger fraction of total cfDNA in patients confirmed by EBER ISH to have EBV+ cHL than in either EBER-negative cHL patients or healthy controls (p 〈 0.0001) (Fig 1F). Conclusions: Levels of ctDNA in cHL are higher than might be expected based on tumor purity, with pre-treatment levels similar to DLBCL and higher than FL. ctDNA allows for reliable noninvasive genotyping of cHL at diagnosis, encompassing coding and non-coding SNVs and additional clinically significant factors such as tumor EBV status and SCNAs. Additional cases are currently being profiled and expanded analyses of genotyping and monitoring will also be presented at the meeting. Disclosures Dührsen: Celgene: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Roche: Honoraria, Research Funding; Janssen: Honoraria; Amgen: Research Funding; Gilead: Consultancy, Honoraria. Hüttmann:Roche: Other: Travel expenses; Celgene: Other: Travel expenses. Gaidano:Gilead: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Morphosys: Honoraria; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria. Westin:Apotex: Membership on an entity's Board of Directors or advisory committees; Celgen: Membership on an entity's Board of Directors or advisory committees; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees. Advani:Regeneron: Research Funding; Kyowa: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Infinity: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Millenium: Research Funding; Cell Medica: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Agensys: Research Funding; Forty Seven Inc.: Research Funding; Celgene: Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Astra Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Gilead/Kite: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Autolus: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Celgene: Research Funding; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Other: Participated in an advisory board; Merck: Research Funding.

Abstract: To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements. Patients and Methods We identified 286 patients with AML with t(11q23) enrolled onto German-Austrian Acute Myeloid Leukemia Study Group and Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group prospective treatment trials. Material was available from 177 AML patients for EVI1 expression analysis. Results We divided 286 MLL-rearranged AMLs into three subgroups: t(9;11)(p22;q23) (44.8%), t(6;11)(q27;q23) (14.7%), and t(v;11q23) (40.5%). EVI1 overexpression (EVI1 + ) was found in 45.8% of all patients with t(11q23), with t(6;11) showing the highest frequency (83.9%), followed by t(9;11) at 40.0%, and t(v;11q23) at 34.8%. Concurrent gene mutations were rare or absent in all three subgroups. Within all t(11q23) AMLs, EVI1 + was the sole prognostic factor, predicting for inferior overall survival (OS; hazard ratio [HR], 2.06; P = .003), relapse-free survival (HR, 2.28; P = .002), and event-free survival (HR, 1.79; P = .009). EVI1 + AMLs with t(11q23) in first complete remission (CR) had a significantly better outcome after allogeneic transplantation compared with other consolidation therapies (5-year OS, 54.7% v 0%; Mantel-Byar, P = .0006). EVI1 − t(9;11) AMLs had lower WBC counts, more commonly FAB M5 morphology, and frequently had additional trisomy 8 (39.6%; P 〈 .001). Among t(9;11) AMLs, EVI1 + again was the sole independent adverse prognostic factor for survival. Conclusion Deregulated EVI1 expression defines poor prognostic subsets among AML with t(11q23) and AML with t(9;11)(p22;q23). Patients with EVI1 + MLL-rearranged AML seem to benefit from allogeneic transplantation in first CR.

Abstract: BCR-ABL1 inhibitors have revolutionized treatment of CML patients. However several drawbacks remain, including therapy resistance of T315I-mutated CML and incapability of current drugs to eliminate quiescent CML stem cells warranting development of novel therapies. In addition, drugs with the potential to enhance efficacy of BCR-ABL1 targeting agents could improve treatment of CML patients. Members of the Tyro3, Axl, Mer receptor (TAMR) tyrosine kinase family are abundantly expressed in physiological and malignant hematopoiesis and their ligand Gas6 can support hematopoietic (progenitor) cells. Evidence in the literature indicates that Axl is upregulated upon treatment with imatinib (IM). In this study, we investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 (former designation R428) in primary CML (stem cell) samples, cell lines and preclinical models. In a first step we quantified Axl-expressing cells by flow cytometry in chronic phase (CP) CML bone marrow at primary diagnosis and healthy bone marrow donors. Here, we found higher numbers of Axl-positive cells in CML bone marrow compared to controls (11.42±0.42% (n=5) vs. 0.65±0.10% (n=6), respectively; p=0.0015). In addition, we determined Gas6 plasma levels by ELISA in healthy controls and CML patients in CP and blast crisis (BC). These analyses revealed that Gas6 plasma levels were upregulated in a stage specific manner (plasma levels of Gas6: healthy controls 1290±684 pg/ml (n=14), CP 3465±405 pg/ml (n=50), BC 10940±3868 pg/ml (n=7); p=0.0001). Thus, the Gas6-Axl axis represents a potential therapeutic target in CML patients. Based on this finding we analyzed efficacy of BGB324 in Axl-expressing BV173, KCL22, K562, BaF3_BCR-ABL1-wt and BaF3_BCR-ABL1-T315I cell lines in vitro. We found inhibition of proliferation in all analyzed cell lines with IC50 values ranging from 500-3000 nM. Combination experiments with the IC50 dose of BGB324 and IM revealed additive effects of both treatments in all cell lines. Dose finding experiments with BGB324 in sorted CD34+ primary CML cells grown in the presence of physiological growth factors yielded a mean IC50 of 1.1 ± 0.3 mM (n=3), which was similar to nilotinib. BGB324 did not accumulate CD34+CFSEmax (undivided) cells any more than nilotinib but enhanced apoptosis of CD34+ cells in combination with nilotinib. Notably, there was a consistent inhibitory effect of 3 mM BGB324 alone or in combination with nilotinib against colony forming cells (CFC) with the more primitive BFU-E and GEMM being most sensitive to inhibition (n=4). Interestingly, the Ph- lymphocytes (confirmed by FISH) sorted simultaneously with Ph+ CD34+ cells from the same CML patient were unaffected in terms of viability when treated with Axl inhibitor; thus the BGB324’s activity is cell context specific. Encouraged by these data we analyzed efficacy of BGB324 in an aggressive preclinical CML model in which bone marrow cells were retrovirally transduced with constructs containing T315I-mutated BCR-ABL1. Transduced cells were subsequently i.v. transplanted into sublethally irradiated recipient mice, who rapidly developed blast crisis CML. Mice were treated with 25 mg BGB324 or vehicle BID by oral gavage starting from day 3 after transplantation when homing of transduced cells to the bone marrow is completed. In this model we found a significant prolongation of survival upon treatment with BGB324 (Figure 1). Analyses of the leukemia phenotype by differential blood counts and flow cytometry revealed that BGB324 reduced leukemia cell burden (WBC 138±20x103/µl (n=14) vs. 60x103±21 k/µl WBC in the BGB324 treated group (n=13); p=0.0078). Taken together, these data suggest that the Gas6-Axl axis represents a therapeutic target and that BGB324 is a potent molecule effective against T315I-mutated and wt CML alone and in combination with TKI. Furthermore, BGB324 induces apoptosis of quiescent Ph+ CML stem/progenitor cells. Thus BGB324 might open up novel therapeutic avenues in CML patients. Disclosures: Loges: BerGenBio: research support Other.

Abstract: Abstract 2742 Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10−5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10−3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10−5, 10−4, 10−3, and 10−2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10−5, 10−4, and 10−3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10−5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 – 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 – 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 – 1.138)) laboratory results (Fig. 1–3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing. Disclosure: Sher: Invivoscribe Technologies Inc: Employment. Miller:Invivoscribe Technologies Inc: Employment. Huang:Invivoscribe Technologies Inc: Employment. Shaw:Invivoscribe Technologies Inc: Employment.

Abstract: Abstract 1665 Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. Thus far, limited data is available on the technical performance in a clinical diagnostic setting. Here, we investigated - as an international consortium - the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 8 laboratories from 6 countries. As a first candidate gene we selected TET2, a frequently mutated gene in myeloproliferative neoplasms. In total, 31 primer pairs including a 10-base molecular barcode sequence were designed and evaluated: All coding exons of TET2 were represented by 27 amplicons. In addition, 2 primer pairs were amplifying hotspot regions to characterize the RING finger domain and linker sequence for CBL and 2 amplicons covered KRAS exons 2 and 3. To execute our study, we used the small volume Titanium emulsion PCR setup (454 Life Sciences, Branford, CT). A cohort of 18 chronic myelomonocytic leukemia (CMML) patient samples were centrally collected by the Munich Leukemia Laboratory and characterized by conventional sequencing for mutations in TET2, CBL, and KRAS. In this selected cohort 33 distinct mutations in TET2, 7 mutations in CBL, and 3 mutations in KRAS, respectively, were detected by Sanger sequencing (plus 10 SNPs and one silent mutation). Each of the participating laboratories received anonymized aliquots of 1.6 μg of genomic DNA to be processed for the generation of PCR amplicons suitable for 454 deep-sequencing. In detail, a total of 31 × 18 (n=558) PCRs were locally performed at each laboratory, i.e. a total of 4464 PCR reactions across 8 centers. Subsequently, at each site each PCR product was individually purified and quantified and corresponding pools were generated by combining 31 amplicons in an equimolar ratio for each patient sample. After processing the samples using the 454 workflow, 3 patients each were loaded per lane on an 8-lane PicoTiterPlate on the GS FLX sequencer instrument. Overall, each of the 8 participating laboratories generated in median 432,606 reads across the 31 PCR amplicons (“Passed Filter Wells”). The median coverage per amplicon was 713-fold, ranging from 553-fold to 878-fold. Dropouts of single amplicons with no coverage obtained were observed in 4/8 laboratories in 61 of 4464 PCR products (1.4%). After alignment of the obtained sequences against the reference genome a total of 92 variants (44 distinct mutations and 10 SNPs) were observed across 22 amplicons. For this analysis, a given variant was scored if, in median, both forward and reverse reads were harboring the variant in at least 20% of reads, i.e. in line with the Sanger sequencing detection limit (GS FLX Amplicon Variant Analysis software v.2.3). In comparison to data available from Sanger sequencing, 454 amplicon deep-sequencing detected all mutations and SNPs that were previously known (few comparisons not possible due to single amplicon dropouts). In 90/92 variant comparisons all eight laboratories consistently detected the variant (two KRAS mutations being detected with a range from 18.0% - 22.6% of reads carrying the mutation). We did not observe a considerable bias in the measurements of the 92 variants between any two centers. Based on paired t-tests for equivalence, with equivalence limits for the standardized expected differences between two centers of -+ε (ε=0.5), the null hypothesis of dissimilar measurements was rejected for all pairs of centers (alpha=0.05). The estimated standard deviation of the measurements across centers was 3.1% (95% CI: [2.9%, 3.2%] ), demonstrating the high precision of 454 sequencing to detect mutations. Additionally, we took advantage of the high sensitivity of deep-sequencing. As such, we observed 7 distinct novel mutations (n=2 TET2, n=3 CBL, n=2 KRAS) with frequencies below the Sanger sequencing cut-off value of 20% (median values ranging from 2.8% - 12.6%). These low-level mutations were consistently detected in all laboratories (one CBL mutation with 〈 3% frequency detected in only 5/8 centers). In conclusion, we here demonstrate in a multicenter analysis that amplicon-based deep-sequencing is technically feasible, achieves a high concordance across multiple laboratories, and therefore allows a broad and in-depth molecular characterization of hematological malignancies with high diagnostic sensitivity. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Garicochea:Roche Diagnostics: Research Funding. Grossmann:MLL Munich Leukemia Laboratory: Employment. Hanczaruk:454 Life Sciences: Employment. Jansen:Roche Diagnostics: Research Funding. te Kronnie:Roche Diagnostics: Research Funding. Martinelli:Roche Diagnostics: Research Funding. McGowan:454 Life Sciences: Employment. Stabentheiner:Roche Diagnostics: Research Funding. Timmermann:Roche Diagnostics: Research Funding. Vandenberghe:Roche Diagnostics: Research Funding. Young:Roche Diagnostics: Research Funding. Dugas:Roche Diagnostics: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Roche Diagnostics: Research Funding.

Abstract: Abstract 1399 Introduction: Massively parallel pyrosequencing in picoliter-sized wells is an innovative technique and allows highly-sensitive deep-sequencing to detect molecular aberrations. As an international consortium we had investigated previously the robustness, precision, and reproducibility of 454 amplicon next-generation sequencing (NGS) across 10 laboratories from 8 countries (Kohlmann et al., Leukemia, 2011;25:1840–8). Aims: In Phase II of the study we now established distinct working groups for various hematological malignancies, i.e. acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphatic leukemia (CLL), chronic myelogenous leukemia (CML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPN). 26 laboratories from 13 countries are currently part of the research consortium. Each working group selected gene targets and developed amplicons of interest to be studied in various hematological malignancies by deep-sequencing (454 Life Sciences, Branford, CT). Results: In total, 74 genes were identified by the study centers to be of interest for mutational screenings in the respective scientific working groups. Overall, 1146 primer sequences resulting in 573 amplicons were designed and tested. Where appropriate, individual genes were combined into panels and validated designs were set up as standardized preconfigured oligonucleotide primer plates. So far, in AML 679 cases had been screened for CEBPA mutations. RUNX1 mutations were analyzed in 864 cases applying the deep-sequencing read counts to study the stability of such mutations at relapse and the utility of this marker to detect minimal residual disease. Analyses on DNMT3A (n=126) and BCOR (n=83) were focused to perform landscape analyses and to investigate the prognostic utility of these markers. Additionally, this working group is focusing on TET2, ASXL1, and TP53 (n=195) analyses. A novel prognostic model is being developed allowing to stratify AML into prognostic subgroups based on molecular markers only. In ALL, 236 pediatric and adult cases have been screened for TP53 mutations both at diagnosis and relapse of ALL. Pediatric and adult leukemia expert labs developed content to study the mutation incidence of other B and T lineage markers such as IKZF1, JAK2, IL7R, PAX5, LEF1, CRLF2, PHF6, WT1, JAK1, PTEN, AKT1, IL7R; NOTCH1, or FBXW7. Interestingly, the molecular landscape of CLL is changing rapidly. As such, a separate working group focused on analyses including NOTCH1, SF3B3, MYD88, XPO1, FBXW7 and BIRC3. 541 cases were screened already to investigate the range of mutational burden of NOTCH1 mutations for their prognostic relevance in a large unselected cohort of adult CLL. In MDS, RUNX1 mutation analyses were performed in 898 cases. Further, the prognostic relevance of TP53 mutations in MDS with isolated deletions of chromosome 5q was studied in a cohort including 105 MDS 5q- cases. Additional content was developed targeting genes of the cellular splicing component, e.g. SF3B1, SRSF2, SF1, U2AF1, ZRSR2. In BCR-ABL-negative MPN, 10 genes of interest (JAK2, MPL, EZH2, IDH1, IDH2, TET2, CBL, IKZF1, SH2B3, ASXL1) have been analyzed in a cohort of 170 cases searching for novel somatic mutations addressing their relevance for disease progression and leukemia transformation. Moreover, an assay was developed and applied to 10 CMML cases allowing the simultaneous analysis of 25 leukemia-associated target genes in a single sequencing using just 20 ng of starting DNA. A group of laboratories focused on ultra-deep sequencing analyses of the BCR-ABL tyrosine kinase domain. Analyses were performed so far on 106 cases to study the dynamics of expansion of mutated clones under various tyrosine kinase inhibitors. Conclusion: A comprehensive molecular characterization of hematological malignancies today requires high diagnostic sensitivity and specificity. As part of the IRON-II study, a network of laboratories studied a variety of hematological diseases applying standardized amplicon-based deep-sequencing assays. Distinct working groups have been forged to address scientific questions and in total 4013 cases had been analyzed thus far. Disclosures: Kohlmann: Roche Diagnostics: Honoraria; MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership; Roche Diagnostics: Research Funding.

Abstract: BCR-ABL1 inhibitors have revolutionized the treatment of CML patients. However several drawbacks remain, including clinical resistance of T315I-mutated CML. Further, the clinical success of ponatinib, a selective inhibitor of T315I-mutated BCR-ABL1 is hampered by vascular side effects. Therefore, novel treatment strategies are warranted especially in T315I-mutated CML. We investigated the relevance of the Gas6-Axl axis in CML patients and the therapeutic potential of the clinically applicable small molecule Axl inhibitor BGB324 in primary CML (stem cell) samples, cell lines and preclinical models. We previously found that Gas6 and Axl represent potential novel targets in this disease and that BGB324 inhibits CML growth in vitro and in vivo (Erdmann et al., ASH meeting 2013, New Orleans, #1469). We next wished to confirm Axl as specific therapeutic target and therefore down regulated its expression in KCL-22 and K562 cells by means of shRNA. In these experiments blockade of Axl inhibited CML cell proliferation in comparison to control-transduced cells, thereby confirming that Axl promotes CML growth. Subsequently, we added BGB324 to shAxl- and shcontrol-transduced cells. Surprisingly BGB324 inhibited cell growth significantly more in shAxl-transduced cells in comparison to control-treated shAxl-transduced cells (p 〈 0.05). These experiments indicated that BGB324 was inhibiting an additional target which supported CML cell proliferation, besides Axl. In order to identify this target we carried out a Kinome Scan revealing that BGB324 binds to native and mutated ABL1. Interestingly, the affinity of BGB324 for ABL1 carrying different mutations including T315I was 5 to 50 fold higher compared to unmutated ABL1 (Table 1). Next, we incubated BaF3 cells stably transfected with BCR-ABL1p210, BCR-ABL1T315I, BCR-ABL1M351T and BCR-ABL1E255K with various concentrations of BGB324 in order to determine its IC50 in the different cell lines. These experiments showed in concordance with the Kinome Scan that BGB324 was more potently inhibiting growth of mutated BCR-ABL1 compared to BCR-ABL1p210 (IC50 BCR-ABL1p210 1266 ± 126 nM; BCR-ABL1T315I 726 ± 194 nM; BCR-ABL1M351T 847 ± 10 nM and BCR-ABL1E255K 794 ± 39 nM; n=2-3; p 〈 0.05 compared to BCR-ABL1p210). Notably, further experiments revealed that BGB324 inhibited KCL-22 cells and K526 cells to a similar extent compared to the combination of imatinib (IM) and shAxl. Thus, BGB324 is a dual inhibitor of BCR-ABL1 and Axl. As the inhibition of BCR-ABL1T315I is of special clinical interest we wished to confirm this finding further in vivo. Therefore we inoculated BCR-ABL1p210 and BCR-ABL1T315I cells subcutaneously into NSG mice. After the tumors reached a size of 80-100 mm3 mice were randomized to receive either placebo control or 50 mg/kg BGB324 delivered twice daily by oral gavage. This experiment showed potent inhibition of tumor growth after 12 days with higher activity of BGB324 in mice bearing BCR-ABL1T315I tumors (placebo: 1751 ± 606 mm3, BGB324: 614 ± 224 mm3; p=0.001) compared to mice bearing BCR-ABL1p210 tumors (placebo: 1432 ± 403 mm3; BGB324: 632 ± 229 mm3; p=0.05) (Figure 1). Subsequently, tissue harvested at end-stage was subjected to immunohistochemical staining for the proliferation marker phospho-histone H3 and Western Blot analyses of cleaved caspase 3 in order to determine whether reduced proliferation and/or increased apoptosis was responsible for reduced growth of BCR-ABL1T315I tumors upon treatment with BGB324. These analyses revealed that proliferation as determined by histomorphometric analysis of phosho-histone H3 was reduced while cleaved caspase 3 levels were unchanged. These data were further corroborated by the finding that treatment with BGB324 reduced the level of phosphorylated MapK as determined by immunoblotting and densitometry. Thus, BGB324 inhibits proliferation of BCR-ABL1T315I cells in vivo. Altogether, our findings show that BGB324 represents a dual inhibitor of Axl and ABL kinase with therapeutic potential in CML, in particular in BCR-ABL1T315I disease. As BGB324 was shown to be well tolerated in healthy volunteers (Wnuk-Lipinska et al., AACR meeting 2013 San Diego #1747), our findings pave the way for clinical investigation of BGB324 in (T315I-mutated) CML. Table 1 Gene KinomeScan Kd (nM) KinaseProfiler IC50 (nM) Axl 0.4 3 ABL1 51.88 51 ABL1(E255K) 1.15 n/a ABL1(T315I) 10.13 4 ABL1(Y253F) 18.19 26 Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Loges: BerGenBio: Research Funding, travel support, advisory boards Other.