Search

Circulating cell-free DNA (ccfDNA) allows for noninvasive peripheral blood sampling of cancer-associated mutations and has established clinical utility in several solid tumors. We performed targeted next-generation sequencing of ccfDNA and bone marrow at the time of diagnosis and after achieving remission in 22 patients with acute myeloid leukemia (AML). Among 28 genes sequenced by both platforms, a total of 39 unique somatic mutations were detected. Five mutations (13%) were detected only in ccfDNA, and 15 (38%) were detected only in bone marrow. Among the 19 mutations detected in both sources, the concordance of variant allelic frequency (VAF) assessment by both methods was high (R2 = 0.849). Mutations detected in only 1 source generally had lower VAF than those detected in both sources, suggesting that either method may miss small subclonal populations. In 3 patients, sequencing of ccfDNA detected new or persistent leukemia-associated mutations during remission that appeared to herald overt relapse. Overall, this study demonstrates that sequencing of ccfDNA in patients with AML can identify clinically relevant mutations not detected in the bone marrow and may play a role in the assessment of measurable residual disease. However, mutations were missed by both ccfDNA and bone marrow analyses, particularly when the VAF was

Introduction: Liquid biopsy has been reported to be useful in predicting residual disease in patients with diffuse large B-cell lymphoma (DLBCL). Most of the studies focused on quantifying the level of circulating lymphoma-specific DNA. We explored the clinical relevance of the specific mutated genes in predicting progression in patients with DLBCL. Method: Peripheral blood samples were collected from patients with DLBCL based on their visit to clinic without other specific selection. Median age of patients is 69 (range 28-91), with 51% of the patients being male. These patients were treated on multiple protocols including R-CHOP, R-EPOCH, Magrath, HCVAD, CAR-T (#2 patients), and others. cfDNA was extracted and sequenced by next generation sequencing using 177 gene panel. The panel uses single primer extension (SPE) approach with UMI. Sequencing depth is increased to more than 2000X after removing duplicates. Low level mutations are confirmed by inspecting BAM file. Results: A total of 86 sample from 61 patients were collected post clinical remission at different time points (median 28 weeks, range: 1-994 weeks). Of these samples, 56 (65%) from 46 patients (75%) were positive. However, 6 of these samples from 4 patients had germline mutations or mutations in TET2, ASXL1, or DNMT3A that are consistent with CHIP (clonal hematopoiesis of indeterminate potential). The remaining 50 positive samples from 42 patients had 8 repeats on the same patients collected at different time points. Comparing the 19 negative patients with the 42 positive patients post-remission, patients with residual molecular disease were significantly older than patients without residual disease (P=0.01). However, there was no significant difference between the two groups in gender, ethnic background, LDH, cell of origin classification, or TP53 positivity by IHC. Patients with residual disease showed tendency for short progression-free survival (P=0.08). Focusing on patients with specific mutations detected in the cfDNA showed that 14 (23%) patients had mutations either in TP53 or MYD88. There was no significant difference in age between these two groups or any of the other clinical variables. However, patients with TP53/MYD88 mutations had significantly shorter survival (P=0.04). Conclusion: Post-remission residual disease as measured by circulating cfDNA is an independent predictor of potential relapse in patients with DLBCL. However, presence of it is important to determine the aggressiveness of the residual circulating clone. Residual circulating lymphoma DNA with TP53 or MYD88 mutations is a strong predictor of earlier relapse. Figure 1 Figure 1. Disclosures Pecora: Genetic testing cooperative: Other: equity investor; Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees. Feldman: Alexion, AstraZeneca Rare Disease: Honoraria, Other: Study investigator. Goy: Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Honoraria, Other; AbbVie/Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; Acerta: Consultancy, Research Funding; Elsevier's Practice Update Oncology, Intellisphere, LLC(Targeted Oncology): Consultancy; Celgene: Consultancy, Honoraria, Research Funding; Michael J Hennessey Associates INC: Consultancy; Elsevier PracticeUpdate: Oncology: Consultancy, Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Medscape: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Genentech/Hoffman la Roche: Research Funding; AbbVie/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; OncLive Peer Exchange: Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xcenda: Consultancy, Honoraria; Vincerx pharma: Membership on an entity's Board of Directors or advisory committees; Rosewell Park: Consultancy; LLC(Targeted Oncology): Consultancy; Genomic Testing Cooperative: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Xcenda: Consultancy; Hoffman la Roche: Consultancy; Incyte: Honoraria; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Infinity/Verastem: Research Funding; Janssen: Research Funding; Karyopharm: Research Funding; Vincerx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Physicians' Education Resource: Consultancy, Other: Meeting/travel support; COTA (Cancer Outcome Tracking Analysis): Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Phamacyclics: Research Funding; Constellation: Research Funding; Hackensack Meridian Health, Regional Cancer Care Associates/OMI: Current Employment.

Introduction: Acute graft-vs.-host disease (aGVHD) remains a major diagnostic and clinical problem in patients after allogenic hematopoietic stem cell transplant (HSCT). Finding biomarkers that play a role in aGVHD not only helps in predicting and diagnosing aGVHD, but might help in developing prophylaxis and therapeutic approaches. Using Next Generation Sequencing (NGS) and targeted RNA sequencing along with a machine learning approach to predict, we investigated the potential of discovering new biomarkers that can predict aGVHD. Methods: RNA extracted from bone marrow aspiration samples collected around day 90 post HSCT from 46 patients were sequenced using 1408 targeted genes. cDNA was first generated, then adapters were ligated. The coding regions of the expressed genes were captured from this library using sequence-specific probes to create the final library. Sequencing was performed using an Illumina NextSeq 550 platform. Ten million reads per sample in a single run were required. Read length was 2 × 150 bp. Expression profile was generated using Cufflinks. A machine learning system is developed to predict the GVHD cases and to discover the relevant genes. A subset of genes relevant to GVHD is automatically selected for the classification system, based on a k-fold cross-validation procedure (with k=10). For an individual gene, a Naïve Bayesian classifier was constructed on the training of k-1 subsets and tested on the other testing subset. To eliminate the underflow problem commonly associated with the standard Naïve Bayesian classifiers, we applied Geometric Mean Naïve Bayesian (GMNB) as the classifier to predict GVHD. The processes of gene selection and GVHD classification are applied iteratively to obtain an optimal classification system and a subset of genes relevant to GVHD. Results: The analyzed bone marrow samples included patients transplanted for aplastic anemia (#1), acute lymphoblastic leukemia (#9), acute myeloid leukemia (#16), mixed phenotype acute leukemia (#1), myelodysplastic syndrome (#10), chronic myelomonocytic leukemia (#5), and myeloproliferative neoplasm (#4). Of the 46 patients, 30 (65%) had a diagnosis of aGVHD (grade 2-4). The GMNB modified Bayesian model selected 7 genes as top classifiers. These top classifier genes included Class II Major Histocompatibility gene (CIITA), B-cell markers genes (CD19 and CD22), early T-cell related gene (TCL1A), hematopoietic-specific transcription factor (IKZF3), a gene involved in protein-protein interaction, and a gene involved in DNA helicase nucleotide excision repair (ERCC3). When these 7 genes were used in GMNB-modified classifier with 10-fold cross validation to predict aGVHD, the model classified 28 of the 30 positive cases accurately and 14 of the 16 negative cases accurately. The sensitivity was 93% (95% CI, 76%-99%). The specificity was 87.5% (95% CI: 60%-97%). The positive predictive value (PPV) was 93% (95% CI: 76%-99%) and the negative predictive value (NPV) was 87.5% (95% CI: 60%-98%). Conclusion: While most biomarker discovery has been focused on inflammatory cytokines, chemokines, and their receptors, our data suggest that hematopoietic proliferation and transcription regulators in bone marrow might provide important information for the diagnosis and prediction of aGVHD. This data suggests that biomarkers related to B-cell, T-cell, and MHC play a role in aGVHD at the bone marrow level. These findings also suggest that targeting these biomarkers in the bone marrow might be a realistic approach for prophylaxis and treatment that needs to be explored. Although further validation is needed, this study suggests that targeted RNA sequencing by NGS combined with machine learning algorithm can be a practical and cost-effective approach for the diagnosis and prediction of aGVHD. Figure 1 Figure 1. Disclosures Pecora: Genetic testing cooperative: Other: equity investor; Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees. Goy: Rosewell Park: Consultancy; Elsevier's Practice Update Oncology, Intellisphere, LLC(Targeted Oncology): Consultancy; Acerta: Consultancy, Research Funding; Genentech/Hoffman la Roche: Research Funding; Vincerx pharma: Membership on an entity's Board of Directors or advisory committees; Physicians' Education Resource: Consultancy, Other: Meeting/travel support; Vincerx: Honoraria, Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xcenda: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; OncLive Peer Exchange: Honoraria; Xcenda: Consultancy, Honoraria; AbbVie/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; COTA (Cancer Outcome Tracking Analysis): Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Elsevier PracticeUpdate: Oncology: Consultancy, Honoraria; Infinity/Verastem: Research Funding; Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; MorphoSys: Honoraria, Other; Genomic Testing Cooperative: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Hoffman la Roche: Consultancy; Michael J Hennessey Associates INC: Consultancy; LLC(Targeted Oncology): Consultancy; Medscape: Consultancy; Bristol Meyers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie/Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria; Constellation: Research Funding; Janssen: Research Funding; Karyopharm: Research Funding; Phamacyclics: Research Funding; Hackensack Meridian Health, Regional Cancer Care Associates/OMI: Current Employment. Rowley: ReAlta Life Sciences: Consultancy.

Introduction: Using next generation sequencing (NGS) in monitoring residual disease in patients with myeloid neoplasms is complicated by the significant heterogeneity in these diseases and the frequent presence of CHIP (clonal hematopoiesis of indeterminate potential) in patients with hematologic neoplasms on which these neoplasms arise. This is particularly relevant post hematopoietic stem cell transplant (HSCT). We explored the ability of using plasma cell-free DNA (cfDNA) in monitoring patients after HSCT and evaluated the potential of using liquid biopsy as a replacement for bone marrow biopsy. Method: cfDNA was isolated from 204 peripheral blood samples obtained from 75 patients, collected at various time points ranging from 27 days to 650 days (median 178 days) post-transplant. DNA from 102 bone marrow (BM) samples was extracted and sequenced using the same panel and approach as cfDNA. Diagnoses included 30 acute myeloid leukemia (AML), 2 chronic myelogenous leukemia (CML), 5 chronic myelomonocytic leukemia (CMML), 4 lymphoma, 10 myelodysplastic syndrome (MDS), 2 multiple myeloma (MM), 9 myeloproliferative neoplasm (MPN), 1 aplastic anemia, and 11 acute lymphoblastic leukemia. cfDNA was sequenced by NGS using 177 gene panel on Illumina platform. Single primer extension (SPE) approach with UMI was used. Sequencing depth was increased to more than 2000X after removing duplicates. Low-level mutations were confirmed by inspecting BAM file. Results: 156 cfDNA samples (76%) tested negative and 48 samples from 30 different patients were positive. The negative samples were collected from 28 days to 650 days post-transplant (median 277 days). The positive samples were collected from 27 days to 650 days post-transplant (median 188 days). One of these positive patients was in full clinical relapse at the time of testing. No negative patient who remained negative had clinical relapse. Five patients converted from negative to positive and 12 from positive to negative with subsequent testing. Three from the converted to positive patients developed clinical relapse. Patients who were positive without clinical relapse had median variant allele frequency (VAF) of 0.85% (range: 0.01-13.25) and typically one mutated gene. The mutated genes in this group were: JAK2, IDH2, ASXL1, TET2, DNMT3A, ASXL1, PTPN11, SF3B1, MPL, CEBPA1. Patients who had clinical relapse (#4) had median VAF of 16.33% (0.4%-57.63%) with multiple mutated genes. The mutated genes in this group were: TP53, FLT3, ASXL1, CEBPA, EZH1, NRAS, SETBP1, TET2. To evaluate relevance to BM testing, we compared BM samples with cfDNA samples collected within 120 days of each other. This showed 17 pairs with concordant negative results, 10 with concordant positive results, 5 pairs with positive by cfDNA but negative by BM cells, and one pair with positive by BM but negative by cfDNA. This BM positive sample was performed at 78 days after the cfDNA sample and showed mutation in DNMT3A gene at VAF of 0.63%. Four of the 5 pairs with positive cfDNA but negative BM were collected approximately 3 months after bone marrow and the 5th case was one month prior to BM sample. Conclusion: These data suggest that monitoring residual disease after HSCT using cfDNA and NGS is a reliable approach and may replace the need of bone marrow biopsy. However, low-level mutations should not be used as the sole criterion for determining relapse. Variant allele frequency and the mutated gene should be considered in evaluating actionable findings. Disclosures Pecora: Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees; Genetic testing cooperative: Other: equity investor. Rowley: ReAlta Life Sciences: Consultancy.

Introduction: Cytogenetic analysis is important for stratifying patients with various myeloid neoplasms. It has been reported that whole-genome sequencing can be used as an alternative to cytogenetic analysis in acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). With the increasing use of liquid biopsy in the diagnosis and monitoring of patients with various types of neoplasms, we explored the potential of using liquid biopsy and next generation sequencing (NGS) in detecting chromosomal structural abnormalities or copy number variation (CNV) in patients with myeloid neoplasms. For practical approach and for capturing single nucleotide variants (SNV) and to achieve enough depth in sequencing, we used targeted sequencing for determining the chromosomal structural abnormalities in cell-free DNA (cfDNA) in patients with myeloid neoplasms. Methods: Peripheral blood plasma samples from 144 patients with myeloid neoplasms were used to extract cfDNA for NGS testing. This included 49 patients with MDS, 31 with AML, and 64 patients with myeloproliferative neoplasms (MPN). The median age was 68.5 (range: 24-96); 56 (39%) were female. cfDNA was sequenced using 275 gene panel. The panel uses single primer extension (SPE) approach with UMI. Sequencing depth was increased to more than 1000X (after removing duplicates). CNVkit software was used for analyzing and visualizing copy number variations. All samples were confirmed to be diagnostic by showing mutations in diagnostic genes with variant allele frequency >20% or by showing diagnostic chromosomal structural abnormalities (e.g., 5q deletion in MDS, 5q- syndrome). Cytogenetic data on 35 corresponding bone marrow samples (18 AML and 17 MDS) were available for comparison. Results: Of the 144 samples, 47 (33%) showed chromosomal structural abnormalities. In the AML group, 20 of 31 (65%) showed cytogenetic abnormalities by cfDNA testing. Of these positive AML patients, 18 (90%) (58% of total AML) had poor-risk cytogenetics. Therefore, the AML patients with normal cytogenetics or cytogenetic abnormalities other than high-risk constituted 42% of total AML patients. Of the MDS group, 11 of 49 MDS patients (22%) showed cytogenetic abnormalities by cfDNA testing, 6 of whom (54.5%) had high-risk cytogenetics. Overall, 12% of all MDS had poor-risk cytogenetics by cfDNA testing. In the MPN group, 16 of 64 (25%) showed cytogenetic abnormalities, 2 of which (12.5%) had 7q deletion (3% of all MPN); the rest (87.5%) of cytogenetic-positive MPN (22% of total MPN) had other abnormalities including 20q-, +8, 12q, 17p-, 11q-, trisomy 9, trisomy 21 and others. To compare chromosomal abnormalities as detected by cfDNA NGS testing with conventional cytogenetic analysis of corresponding bone marrow samples, we classified cytogenetic findings based on risk stratification into either intermediate-risk or poor-risk. Of the 36 cases, there was 100% concordance between cfDNA data and cytogenetic data when findings were grouped based on risk classification. Two of the conventional cytogenetic samples showed no metaphases while one showed intermediate-risk abnormalities by cfDNA NGS analysis and the second showed poor-risk cytogenetic abnormalities by cfDNA NGS analysis. These 36 cases included 16 cases with normal cytogenetics. Simple abnormalities such as 5q-, 7q-, +8 were called in identical fashion, but some other abnormalities such as derivative chromosome and marker chromosome were resolved or interpreted differently by the cfDNA NGS analysis. The NGS panel design used in this study does not cover fusion genes or chromosomal translocation, and chromosomal translocations were missed at this time. Conclusions: This data shows that liquid biopsy using and targeted NGS is reliable in detecting chromosomal structural abnormalities in myeloid neoplasms and potentially can replace the need for conventional cytogenetic testing. While the current study was not designed to detect chromosomal translocations, a small, targeted panel of 275 genes is adequate for standard risk classification of myeloid neoplasms into intermediate or high-risk. Considering that in the same test complete mutation profiling can also be achieved along with chromosomal structural analysis, liquid biopsy in myeloid neoplasms might be considered as an efficient replacement to bone marrow biopsy in patients with myeloid neoplasms when fusion genes are not expected. Figure 1 Figure 1. Disclosures Goy: Kite Pharma: Membership on an entity's Board of Directors or advisory committees; Infinity/Verastem: Research Funding; COTA (Cancer Outcome Tracking Analysis): Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; OncLive Peer Exchange: Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Vincerx: Honoraria, Membership on an entity's Board of Directors or advisory committees; Elsevier PracticeUpdate: Oncology: Consultancy, Honoraria; AbbVie/Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Vincerx pharma: Membership on an entity's Board of Directors or advisory committees; LLC(Targeted Oncology): Consultancy; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Xcenda: Consultancy, Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy, Research Funding; Rosewell Park: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees; Elsevier's Practice Update Oncology, Intellisphere, LLC(Targeted Oncology): Consultancy; AbbVie/Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; MorphoSys: Honoraria, Other; Incyte: Honoraria; Novartis: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyers Squibb: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genomic Testing Cooperative: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Leadership role; Celgene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech/Hoffman la Roche: Research Funding; Janssen: Research Funding; Karyopharm: Research Funding; Michael J Hennessey Associates INC: Consultancy; Hoffman la Roche: Consultancy; Physicians' Education Resource: Consultancy, Other: Meeting/travel support; Medscape: Consultancy; Phamacyclics: Research Funding; Constellation: Research Funding; Xcenda: Consultancy; Hackensack Meridian Health, Regional Cancer Care Associates/OMI: Current Employment. Pecora: Genetic testing cooperative: Other: equity investor; Genetic testing cooperative: Membership on an entity's Board of Directors or advisory committees. Koprivnikar: Bristol Myers Squibb: Speakers Bureau. McCloskey: BMS: Honoraria, Speakers Bureau; COTA: Other: Equity Ownership; Takeda: Consultancy, Speakers Bureau; Pfizer: Consultancy; Novartis: Consultancy; Jazz: Consultancy, Speakers Bureau; Incyte: Speakers Bureau; Amgen: Speakers Bureau.

Introduction: Targeted RNA sequencing using Next Generation Sequencing (NGS) has significant advantages over transcriptome sequencing. In addition to information on mutations, fusion and alternative splicing, RNA quantification using targeted RNA sequencing is sensitive, reproducible and provides better dynamic range. We used targeted RNA sequencing for RNA profiling of diffuse large B-cell lymphoma (DLBCL) and explored its utility in the sub-classification of DLBC to ABC and GCB. Method: RNA extracted from 441 FFPE lymphnode samples with DLBC lymphoma and sequenced targeting 1408 genes. These cases were previously subclassified as ABC vs GCB using expression profiling and immunohistochemistry. We first normalized RNA expression data to PAX5 expression, then we tried to narrow down important markers using univariate significance tests. Setting the cutoff for false discovery rate at 0.0001, 48 variables remained significant, including 46 RNA levels and two genes (MYD88 and EZH2) mutation status. Using 60% of samples as training set, we used multiple statistical approaches for classification. Deep learning emerged as the best approach. We used autoencoder with 5 hidden layers and developed a model for classification of ABC vs GCB. To further improve on classification, we divided patients in each subgroup based on survival using simple tree model. In this tree model, expression level of CD58 emerged as a powerful prognostic marker for the ABC group and RLTPR expression in the GCB group. Results: Using probability of scoring developed based on deep learning and logestic regression, approximately 30% of the cases had a score between 0.5 and 0.75. For the remaining 70% of patients, the ABC vs GCB classification showed sensitivity and specificity of 96% and 97% for the testing set. We also applied the same approach to 60 independent cases classified using NanoString (Lymph2Cx). Our model showed sensitivity and specificity of 96% and 97% in the NanoString independent cases. Using the tree model for further classification of the ABC and GCB classes, CD58 mRNA levels separated the ABC group into two subgroups (ABC1 and ABC2) and RLTPR mRNA separated the GCB into two subgroups (GCB1 and GCB2) with significant difference in overall survival (P=0.0010) and progression-free survival (PFS) (P=0.0027). Conclusion: Targeted RNA sequencing is very reliable and practical for the subclassification of DLBCL and can provide clinical-grade reproducible test for prognostically subclassification of DLBCL. Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. De Dios:Genomic Testing Ccoperative: Employment. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2; Jazz: Consultancy. Ferreri:Roche: Research Funding; Celgene: Consultancy, Research Funding; Novartis: Consultancy; Kite: Consultancy. Piris:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding. Kantarjian:Ariad: Research Funding; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Research Funding; BMS: Research Funding; Takeda: Honoraria; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Cyclacel: Research Funding; Immunogen: Research Funding; Amgen: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Astex: Research Funding.

Introduction: The principle of precision cancer medicine is to customize therapy based on the genomic profiles of the cancer and the host constitution/response to the cancer. Since RNA expression is influence by many genetic mechanisms, RNA profiling may provide broader coverage of genomic changes and might be a better predictor of response to therapy. However, incorporating the many biological changes of the host and the cancer in the decision of selecting therapeutic approach is not practical without using computer-aided algorithms. This is particularly relevant when combination therapy is used. We explored the potential of developing algorithms for the prediction of complete response (CR) to novel combination therapy in patients with acute myeloid leukemia (AML) using targeted RNA expression profiling. Methods: RNA was extracted from the peripheral blood (PB) and bone marrow (BM) samples from patients with AML being treated on two different protocols: FLAG-IDA+venetoclax (F-I-V)( Abou Dalle I et al, ASH 2019; the NCT # is NCT03214562) and ivosidenib+venetoclax (I-V). In the initial study, 22 samples (9 PB and 13 BM) were used as training set. Subsequently 16 PB samples from the F-I-V arm and 4 from the I-V arm were collected and tested blindly as testing set after the development and locking of the algorithm. RNA was sequenced using NGS panel composed on compared of 1408 genes. The RNA sequencing is based on hybrid capture and the number of reads ranged from 5 to 10 million. RNA quantification was performed using Cufflinks. The RNA levels were normalized to ABL1 mRNA levels. Each gene is normalized by the mean and standard deviation of the gene. To develop a model for predicting CR, we used the training set in each arm and first evaluated the performance of each of the 1408 genes using receiver operating characteristic (ROC) curve. Then used the following mathematical methods for developing algorithms for predicting CR: Support Vector machine (SVM), Bayesian modeling with Gaussian Processes (GP), and Naïve Bayesian (NB). Results: In univariate analysis, multiple genes showed very high AUC. In the F-I-V arm, top genes in predicting CR were GLI3, SETBP1, SH3D19, ARHGAP20, ETS1, IKZF2, GNG4 and MAGEE1 with AUC ranged from 0.74 to 0.85. In the I-V arm, the top genes in predicting CR were STL, TNFRSF10D, PTGS2, RET, TFRC, NAV3, WSB1, and GAS1 with AUC varied from 0.91 to 0.96. Using the training samples we developed algorithms for predicting CR by SVM, NB and Bayesian GP. Upon testing these models using leave-one-out (LOO), the three algorithms performed similarly with AUC around 0.97 for the I-V arm and around 0.96 for the F-I-V arm. There was no difference between BM and PB in predicting CR. Therefore, we collected and sequenced only peripheral blood for blind testing. The three algorithms were tested using 16 PB samples from the F-I-V arm and 4 samples from the I-V arm. The SVM and NB algorithms predicted CR correctly in 15 of the 16 samples (94%) while Bayesian GP missed 4 of the 16 samples. As for the I-V arm, the NB predicted CR correctly in the 4 samples, while both SVM and Bayesian GP missed 3 of 4. Conclusions: While the data is limited and further validation is need, algorithms using RNA expression profiling of peripheral blood using targeted RNA NGS may provide an excellent tool for customizing therapeutic approach, especially in the age of combination therapy when number of cases for training can be limited. Furthermore, this study suggests that modeling using Nave Bayesian is reliable approach in developing prediction algorithms. Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Konopleva:Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Ablynx: Research Funding; Agios: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Astra Zeneca: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding. Loghavi:MDACC: Employment; AlphaSights: Consultancy; GLG Consultants: Consultancy. Takahashi:Symbio Pharmaceuticals: Consultancy. Kantarjian:Daiichi-Sankyo: Research Funding; Ariad: Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding; Takeda: Honoraria; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding. DiNardo:syros: Honoraria; daiichi sankyo: Honoraria; celgene: Consultancy, Honoraria; jazz: Honoraria; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees.

Cellular RNA levels are tightly regulated by very complex nuclear and cytoplasmic processes. The regulation of mutant mRNA in cancer cells is rarely studied. We explored the effects of mutations on mRNA levels in patients with diffuse large B-cell lymphoma (DLBCL). Using next generation sequencing (NGS) and variant allele frequency (VAF) of mutant RNA, we compared relative mutant mRNA or variant allele frequency (RNA-VAF) with variant allele frequency of mutant DNA (DNA-VAF) in the same samples from patients with DLBCL. Methods: RNA and DNA were extracted from 427 FFPE samples from patients with DLBCL. We sequenced the DNA using 177 gene panel and the RNA using 1408 gene panel. The DNA sequencing is based on Single Primer Extension (SPE) library preparation with unique molecular identifier (UMI) (Qiagen, Germantown, MD). The RNA sequencing is based on hybrid capture. Sequencing data of DNA is analyzed using the DRAGEN Platform. Sequence duplicates were removed before calculating VAF. The RNA sequencing data is analyzed using Illumina basespace. RNA VAF is calculated also after removing duplicates using Isaac variant caller. Only mutations detected by both DNA and RNA variant callers are compared. Results: A total of 1770 mutations were detected using the DNA panel and 2207 mutations were detected using the larger RNA sequencing panel. We focused on the most commonly mutated genes that included in both DNA and RNA panels and compared the VAF of the same mutations between DNA and RNA. The selected genes are: KMT2D, NOTCH2, CARD11, MYC, MYD88, EZH2, TP53, CD79B, BCL2, and TET2. The overall VAF in the RNA was significantly higher (P Figure Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Tam:Takeda: Consultancy; Paragon Genomics: Consultancy. Hsi:Abbvie: Research Funding; Jazz: Consultancy; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2. Piris:Nanostring: Membership on an entity's Board of Directors or advisory committees; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees; Kura: Research Funding; Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jansen: Membership on an entity's Board of Directors or advisory committees, Other: Lecture Fees. Kantarjian:Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; Novartis: Research Funding; Jazz Pharma: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; Astex: Research Funding; Takeda: Honoraria; BMS: Research Funding; Cyclacel: Research Funding; Ariad: Research Funding.

Background: Circulating cell-free DNA (ccfDNA) is highly fragmented DNA in plasma that is released by normal or tumor cells when they undergo apoptosis or necrosis. ccfDNA allows for non-invasive sampling of somatic genomic alterations and is informative in various solid tumors, including as a marker of measurable residual disease (MRD). We sought to assess the utility of baseline assessment and tracking of leukemia-associated mutations through peripheral blood sampling of ccfDNA in patients (pts) with acute leukemias. Methods: Plasma ccfDNA was isolated and analyzed using a next-generation sequencing (NGS) assay of 275 genes. This NGS analysis is based on Single Primer Extension library preparation with unique molecular identifier (Qiagen, Germantown, MD); a sequence coverage ≥ 100X (after removing duplicates) was required. Amplicon-based NGS was also performed on DNA extracted from the bone marrow (BM) in a CLIA-certified molecular diagnostics laboratory. This BM panel detects mutations in the coding sequence of 28 leukemia-associated genes, with an analytic sensitivity of 5-10%. The ccfDNA panel included all 28 genes evaluated on the BM NGS panel (ABL1, ASXL1, BRAF, DNMT3A, EGFR, EZH2, FLT3, GATA1, GATA2, HRAS, IDH1, IDH2, IKZF1, JAK2, KIT, KRAS, MDM2, MLL, MPL, MYD88, NOTCH1, NPM1, NRAS, PTPN11, RUNX1, TET2, TP53, WT1). Established bioinformatics pipelines were used to identify somatic variants. Results: Twenty-four pts (AML, n=22; ALL, n=2) underwent paired ccfDNA and BM sequencing at diagnosis prior to receiving frontline intensive chemotherapy. For baseline samples, ccfDNA was collected a median of 6 days after BM collection (range, 0-27 days) and a median of 0.5 days after start of induction chemotherapy (range, -7 to 7 days). Eleven pts (46%) also had ccfDNA collected at ≥1 time point during remission. Among the 28 genes of interest, the median number of mutations per pt detected in BM and in ccfDNA was 1 (range, 0-4) for both assays (P=0.39). A total of 40 mutations were detected: 18 mutations (45%) were detected by both methods, 7 (18%) were detected only in ccfDNA, and 15 (38%) were detected only in BM. Time from start of chemotherapy until ccfDNA collection did not appear to impact the concordance of ccfDNA and BM mutation analysis (P=0.87). Among mutations detected by ccfDNA in baseline samples, the median variant allelic frequency (VAF) was 33.7% (range, 2.7-90.8%). Among the 18 overlapping mutations, the concordance of VAF assessment by both methods was high (R2 = 0.849). Mutations detected by only one of the two methods were generally of lower VAF than those detected by both methods, suggesting that either method may miss small subclonal populations. The median VAF of mutations (as measured in ccfDNA) that were detected by both methods was higher than those detected only in ccfDNA (39.8% vs 25.2%, respectively; P=0.04); similarly, the median VAF of mutations (as measured in BM) that were detected by both methods was higher than those detected only in BM (40.2% vs 6.6%; P=0.001). Among the 7 mutations detected only by ccfDNA, ASXL1 was detected in 2 pts, WT1 in 1 pt, IDH1 in 1 pt, and BRAF and two EGFR mutations in 1 pt. Among the 5 pts in whom mutations were detected in ccfDNA but not BM, 2 eventually relapsed. In both pts, the discordant mutation (IDH1 and ASXL1) was detected in the relapse BM, suggesting that these were true mutations that were missed by NGS of the baseline BM. ccfDNA detected leukemia-associated mutations during remission that appeared to herald overt relapse (Figure 1). Two pts with t(8;21) AML developed new RUNX1 mutations detected by ccfDNA while in remission and subsequently relapsed 3 months and 14 months later. In both of these pts, the new RUNX1 mutation was confirmed in the BM at the time of morphological relapse. Another pt with AML had persistent TP53 and TET2 mutations detected by ccfDNA 1 month after allogeneic stem cell transplant and subsequently relapsed 1 month later. Conclusions: This study demonstrates that sequencing of ccfDNA can identify prognostic or targetable mutations not detected by BM NGS. However, true mutations were missed by both ccfDNA and BM analysis, suggesting that these methodologies may be complementary in the assessment and monitoring of pts with leukemia. The use of ccfDNA as a non-invasive method to detect mutations and track MRD in AML and other leukemias should be evaluated in larger, prospective cohorts. Disclosures Short: Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Jabbour:Amgen: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; AbbVie: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Garcia-Manero:Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Kantarjian:BMS: Research Funding; Amgen: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Immunogen: Research Funding; Takeda: Honoraria; Novartis: Research Funding; Ariad: Research Funding; Astex: Research Funding; Pfizer: Honoraria, Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Research Funding; Jazz Pharma: Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding. Ravandi:Macrogenix: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Xencor: Consultancy, Research Funding; Menarini Ricerche: Research Funding; Cyclacel LTD: Research Funding; Selvita: Research Funding.

Introduction: We previously reported a prognostic scoring system in CLL using pre-treatment factors in patients treated with ibrutinib [Ahn et al, 2016 ASH Annual Meeting]. Here we present long-term follow-up results and validation of the prognostic models in a large independent cohort of patients. We also determine the incidence of resistance-conferring mutations in BTK and PLCG2 genes in different clinical risk groups. Methods and Patients: The discovery cohort comprised 84 CLL patients on a phase II study with either TP53 aberration (deletion 17p or TP53 mutation) or age ≥65 years (NCT01500733). The validation cohort comprised 607 patients pooled from four phase II and III studies for ibrutinib in treatment-naïve or relapsed/refractory CLL (NCT01105247; NCT01578707; NCT01722487; NCT01744691). All patients received single-agent ibrutinib 420mg once daily. We used Cox regression models to identify independent predictors of PFS, Kaplan-Meier method to estimate probabilities of PFS, log-rank test to compare PFS, and Cochran-Armitage trend test to compare the incidence of mutation among subgroups. We used R version 3.5.0 or SAS® version 9.3 for statistical analyses. For biomarker correlation, we tested cellular DNA or cell-free DNA collected from patients in the discovery cohort with the targeted sequencing of BTK and PLCG2 genes. Result: At a median follow-up of 5.2 years, 28 (33.3%) of 84 patients in the discovery cohort progressed or died. 52 (61.9%) patients had treatment-naïve CLL. Independent factors of PFS on univariate analysis were; TP53 aberration, prior treatment, and β-2 microglobulin (B2M) >4mg/L (P4mg/L for Model 1, and TP53 aberration, advanced Rai stage, and relapsed/refractory CLL for Model 2 (Table 1). The high-risk group had all three adverse risk factors; the intermediate-risk group had two risk factors; and the low-risk group, none or one. The median PFS of the high-risk group was 38.9 months for Model 1 and 38.4 months for Model 2, and was significantly shorter than those of intermediate and low-risk groups. In the validation cohort, 254 (41.8%) of 607 patients progressed or died at a median follow-up of 4.2 years. 167 (27.5%) patients had treatment-naïve CLL. Both models showed statistically significant differences in PFS by risk groups (Table 1). For the high-risk group, 4-year PFS was 30.2% in Model 1 and 30.5% in Model 2, which were inferior to those of intermediate (53.4 and 52.4%) and low-risk groups (68.7 and 73.7%). Model 1 classified 20% of patients and Model 2 classified 28% of patients to the high-risk group. BTK and PLCG2 mutations are common genetic drivers of ibrutinib resistance in CLL. To determine whether the incidence of these mutations correlates with prognostic risk groups, we performed targeted sequencing of BTK and PLCG2 of samples collected from patients in the discovery cohort. We used cell-free DNA for patients who received long-term ibrutinib (≥3 years) and had low circulating tumor burden, and cellular DNA, for samples collected within 3 years on ibrutinib or at progression. Of 84 patients, 69 (82.1%) were tested at least once, and 37 (44.0%) were tested at least twice. The frequency of testing was similar across the risk groups by two models (P>0.05). The cumulative incidences of mutations at 5 years in the low-, intermediate-, and high-risk groups were: 21.4%, 44.8% and 50%, respectively, by Model 1 (P=0.02); and 22.6%, 41.4% and 66.7%, respectively, by Model 2 (P=0.01). Conclusion: We developed and validated prognostic models to predict the risk of disease progression or death in CLL patients treated with ibrutinib. Risk groups classified by three commonly available pre-treatment factors showed statistically significant differences in PFS. The clinically-defined high-risk disease was linked to higher propensity to develop clonal evolution with BTK and/or PLCG2 mutations, which heralded ibrutinib resistance. Disclosures Albitar: Neogenomics Laboratories: Employment. Ma:Neogenomics Laboratories: Employment. Ipe:Pharmacyclics, an AbbVie Company: Employment, Other: Travel; AbbVie: Equity Ownership. Tsao:Pharmacyclics LLC, an AbbVie Company: Employment. Cheng:Pharmacyclics LLC, an AbbVie Company: Employment. Dean:CTI BioPharma Corp.: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Employment, Equity Ownership. Wiestner:Pharmacyclics LLC, an AbbVie Company: Research Funding.

Background: The recent advances in molecular techniques and the adaptation of next generation sequencing (NGS) in routine clinical testing increased our ability to use molecular approaches in the diagnosis and classification of most hematologic diseases. Bone marrow aspiration and biopsy remains necessary for initial confirmation of diagnosis of neoplastic processes in bone marrow, but significant literature suggests that screening or monitoring patients by testing peripheral bloodcfDNAmight be a reliable alternative to marrow biopsy and might reduce the need for a painful bone marrow procedure. Here we report the results of routine clinical testing ofcfDNAthat is ordered by practicing hematologist in the context of the presence or the suspicion of the presence of hematologic neoplasm. Methods: A total of 227 peripheral blood samples were submitted for screeningcfDNAfor mutations in a 54 gene focusedMyeloidpanel using NGS sequencing. DNA was extracted from plasma usingNucliSenSEasyMAGautomated platform and then assayed using theTruSightMyeloid Sequencing Panel (Illumina; San Diego, CA) with an average sequencing depth of 10,000X. The average age patients was 71 (18-96) years. The reason for submitting samples wasruling out MDS in 199 and ruling out AML or other hematologic neoplasms in 28 samples. Of these samples, 12 patients had a follow up testing of bone marrow aspiration sample. Results: Of the 227 tested samples (Figure 1), 126 (55%) showed no evidence of mutation in any of the tested genes. Based on our previous data (see ASH abstract by Albitar et al, 2016), this suggested that MDS can be ruled out in these patients and bone marrow biopsy could be avoided and not recommended. In contrast, 101 (45%) had mutations in one or more genes. Twenty-nine (~12.8%) contained a mutation in a single gene with variant allele frequency (VAF) Conclusions:cfDNAtesting is reliable approach to screen for the presence of Hematologic neoplasm and potentially could avoid the need for bone marrow biopsy in almost half the patients expected to have MDS or otherhematopoeticneoplasms. Positive diagnosis can be confirmed in additional 45% of patients and only 12.8% of patients will be reported with questionable results. Except for those with TP53 mutations, the rest of the 12.8% cases can be classified as Clonal Hematopoiesis of Indeterminate Potential (CHIP). While bone marrow is still the gold standard, our real world experience shows liquid biopsies can be sensitive and non-invasive approach to rule out MDS or other hematological diseases. Disclosures Funari: NeoGenomics Laboratories: Employment. Ma:Neogenomics Laboratories: Employment. Thangavelu:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

The major complication of severe aplastic anemia is clonal evolution, defined as any new cytogenetic abnormality or progression to MDS/AML, which occurs in about 15% of SAA patients, usually many months to years after the diagnosis. Eltrombopag, a thrombopoietin receptor agonist, appears capable of stimulating hematopoietic stem cell proliferation in patients with bone marrow failure. Addition of eltrombopag to standard immunosuppressive treatment (IST) with horse antithymocyte globulin (hATG) and cyclosporine (CsA) markedly increases hematologic response rates in treatment-naive SAA, with overall response rates up to 90% and complete response rates approaching 60% (Townsley DM et al, ASH 2015, clinicaltrials.gov NCT01623167). In comparison, IST alone achieves 60% overall response rates, of which 10% are complete (Scheinberg Blood 2012). Somatic mutations in myeloid cancer candidate genes are present in one-third of patients after IST alone (Yoshizato T et al, NEJM 2015). Specific subsets of mutations were associated with clinical outcomes: a group including ASXL1 and DNMT3A with a poor response to IST, inferior survival, and clonal evolution, while BCOR and PIGA were associated with good response and favorable outcomes. Monosomy 7, the most prevalent cytogenetic abnormality defining clonal evolution, can develop in the absence of gene mutations, underscoring the non-determinative and complex role mutations play in clonal evolution (Dumitriu B et al, Blood 2015). Of patients with disease refractory to IST who were subsequently treated with single-agent eltrombopag, 19% (8/43) developed cytogenetic abnormalities, usually within the first year of treatment, but only rarely with morphologic changes consistent with MDS/AML (Desmond R et al, Blood 2014). The frequency of somatic mutations following treatment with eltrombopag added to IST in treatment-naïve SAA patients is unknown. We used amplicon-based next-generation sequencing to assess mutations in 54 candidate genes recurrently mutated in myeloid neoplasms. Bone marrow cells of 90 subjects who had been treated with IST/eltrombopag were obtained at 6 months following treatment initiation, or at the time of clonal evolution. At least one detectable mutation was identified in 21 (23%) subjects. All 21 patients had exhibited a hematologic response to treatment by 6 months; of those patients with somatic mutations, 14 of 19 (74%) had a complete hematologic response. In comparison, of the 69 patients who lacked mutations, 20 (29%) had a complete response. Nine different genes were mutated in total, with the most frequent genes being ASXL1 and BCOR. BCOR was associated with more robust responses (6 of 7 had a complete response) and younger age (range 12 - 49 years; Table). One subject with a longstanding history of JAK2-positive essential thrombocytosis and myelofibrosis at baseline had two additional mutations detectable following treatment, TET2 and ASXL1. With a median follow up of 21 months, clonal cytogenetic evolution occurred in 7/90 (8%) subjects. Three of seven patients also had a mutation in a myeloid cancer gene (two with DNMT3A and one with ASXL1/RUNX1); in the other four, no somatic mutation was detected, either at the 6-month time point or at time of cytogenetic evolution. Four patients had monosomy or partial deletion of chromosome 7: one patient had complex (t(3;3)(q21;q26), -7), one patient had deletion 13q that later disappeared, and one patient had trisomy 6 and trisomy 15 in 2 metaphases. The patient with complex cytogenetics did not have somatic mutations detected at evolution, and she later died due to relapsed AML following transplant. The rates of somatic mutations in myeloid cancer genes and of cytogenetic evolution in patients treated with IST/eltrombopag do not appear to be higher than we and others have reported in aplastic anemia treated with standard IST, without eltrombopag (Kulasekararaj AG et al, Blood 2014). The distribution of genes mutated and the allelic frequency of these mutations also were similar to patients treated with standard IST. These results suggest that the benefits of a higher response rate and quality of response associated with the addition of eltrombopag to IST for the initial treatment of SAA are not associated with a higher risk of clonal progression. Disclosures Townsley: GSK/Novartis: Research Funding. Cooper:GSK/Novartis: Research Funding. Winkler:GSK/Novartis: Research Funding. Scheinberg:Novartis: Consultancy, Speakers Bureau. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Desierto:GSK/Novartis: Research Funding. Fernandez Ibanez:GSK/Novartis: Research Funding. Dunbar:GSK/Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership. Young:GSK/Novartis: Research Funding.

Background: DNA methylation in AML/MDS plays a major role in the pathogenesis of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). The major genes involved in DNA methylation in AML/MDS are IDH1 and 2, TET2 and DNMT3A. Mutations in IDH1/2 result in the production of an aberrant metabolite, 2-hydroxyglutarate, which acts as a competitive inhibitor of a-ketoglutarate and inhibits TET2 oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Mutations in TET2 or IDH1/2 are associated with reduced levels of 5hmC and genomic hypermethylation. TET2 mutations and IDH1/IDH2 mutations are believed to be mutually exclusive. In addition, DNMT3A as a DNA methyltransferase enzyme is commonly mutated in AML/MDS and its mutation is believed to lead to hypomethylation. Understanding the interaction between these genes may influence therapy with IDH1/2 inhibitors. Toward better understanding of interaction between these genes, we analyzed the mutation profile of these genes in patients with AML/MDS. Methods: A total of 1182 bone marrow (BM) aspirate samples were tested by the commercially available TruSight Myeloid Next Generation Sequencing Panel (Illumina, San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. This NGS panel covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: IDH1/2 mutations were detected in 201 of the 1182 (17%). IDH1 was detected in 87 (7.4%) and IDH2 was detected in 120 (10.1%). This included 6 patients who had mutations in both IDH1 and IDH2. Variant (mutant) allele frequency (VAF) was significantly higher (P=0.01) in IDH2 as compared to IDH1 (median of 43.35% vs 35.0%, respectively). Thirteen patients (6.5%) had mutant VAF >50% suggesting homozygosity, 11 of which had IDH2 mutation. Two of the 6 patients with both IDH1 and IDH2 mutations had VAF 50% in 6 of these patient and in DNMT3A in 3 patients. Twenty four patients had TP53 mutation, of which 16 had IDH1 mutation and 8 had IDH2 mutation, which is disproportional with the prevalence of IDH1 mutation. There was no statistically significant difference in VAF between TP53 and IDH1/2, but 4 of these patients had both DNMT3 and IDH2 mutations and one had both IDH1 and IDH2 mutations. None of the patients with TP53 mutation had TET2 mutation. Conclusions: IDH2 mutations may coexist with IDH1 and TET2 mutations. This co-mutation appears to be in the same clone in some patients and in a separate clone in others. The presence of VAF>50% in 6.5% of patients, which suggests homozygosity, along with co-presence of IDH1 and IDH2 and TET2 mutations suggests possible dosage effects in the biology of MDS/AML. The high rate (29%) of co-presence of DNMT3A with IDH/1/2 mutations also suggests cooperation between the two mechanisms in influencing DNA methylation and leukemogenesis. The relatively high incidence of TP53 mutation in IDH1 patients suggests that IDH1 mutation might be associated with more aggressive disease than IDH2. This data suggests that there is interaction and significant interclonal and intraclonal heterogeneity in DNA methylation genes in AML/MDS. Complete profiling of these genes is necessary for better understanding and proper prediction of clinical behavior particularly when patients treated with DNA methylation inhibitors. Figure Figure. Disclosures Ma: Neogenomics Laboratories: Employment. Thangavelu:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Background:We have reported that peripheral blood cell-free DNA (cfDNA) is reliable for detecting bone marrow molecular abnormalities in patients with hematologic neoplasms. However, not clear is whether cfDNA is sufficient to detect mutations present at low variant allele frequency (VAF). Since patients with aplastic anemia (AA usually have relatively small clones in blood and bone marrow (BM), we compared mutations detected in BM cells with those detected in peripheral blood cfDNA from patientswith this disease. Methods: A total of 120 paired BM aspirate and PB plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA). We extracted DNA from bone marrow aspirate using the QIAamp DNA Mini Kit. We used NucliSenS EasyMAG automated platform for extracting total nucleic acid from PB plasma collected in EDTA. All paired BM and plasma samples were tested by the commercially available TruSight Myeloid Sequencing Panel (Illumina; San Diego, CA), which covers hot spot mutations in 54 genes. The average depth of sequencing was 10,000X. Results: One hundred twenty paired BM and cfDNA samples from 96 patients with aplastic anemia were tested. Of the 96 patients, 33 (34%; equivalent to 48 samples, 40%) had one or more mutations. We identified 54 different somatic mutations in these patients, of which 45 were unique. There was no significant difference (P=0.71, Sign test) in allele frequency between cfDNA and BM. The median mutant allele frequency was 10.9% in cfDNA and 12.6% in BM cells, and 40 of the 54 mutations had allele frequency ≤20% in BM cells, while 45 samples had allele frequency ≤20 in cfDNA. Six of the 33 patients with somatic mutations (18%) showed mutations in plasma cfDNA but not in BM. In contrast, 2 patients (6%) showed mutations in BM cells and not in cfDNA. One of these two patients had a mutation in ASXL1 gene detected in BM cells but not in cfDNA and a subsequent sample showed the same ASXL1mutation in BM cells and not in cfDNA, and a second clone with a different ASXL1 mutation detected in both BM cells and cfDNA. Overall concordance between BM cells and cfDNA in the 120 samples was 92% and there was no statistically significant difference between the two sample types (P=0.6). Summary and Conclusions: Seven samples (from 7 patients) of the 120 tested samples showed mutations in cfDNA and not in BM cells while 3 samples (from 2 patients) showed mutations in BM and not in cfDNA. VAF of mutations in cfDNA were similar to those in BM cells. Therefore, peripheral blood cfDNA should be tested in addition to BM cells for detecting mutations in patients with AA. Peripheral blood cfDNA can be used as a reliable means for monitoring patients with AA. cfDNA testing can be used as an alternative testing to bone marrow even when mutant allele frequency in bone marrow is Disclosures Albitar: Neogenomics Laboratories: Employment. Townsley:Novartis: Research Funding. Ma:Neogenomics Laboratories: Employment. De Dios:Neogenomics Laboratories: Employment. Funari:Neogenomics Laboratories: Employment. Young:GSK/Novartis: Research Funding. Albitar:Neogenomics Laboratories: Employment, Equity Ownership.

Introduction: Diagnosis of myelodysplastic syndrome (MDS) can be very difficult when blast count in bone marrow is Methodology: We analyzed Next Generation Sequencing (NGS) data from of 294 consecutive bone marrow samples referred to rule out MDS and were reported to be positive for mutation in one or more MDS-related genes. All samples were tested for mutations in the following genes: TET2, SF3B1, ASXL1, DNMT3A, SRSF2, RUNX1, NRAS, ZRSR2, EZH2, ETV6, TP53, CBL, NPM1, JAK2, U2AF1, IDH1, KRAS, IDH2, FLT3, PTPN11, SETBP1, and BCOR. The average depth of NGS testing in this targeted sequencing was approximately 10,000X. Results: Of the 294 MDS samples with mutations, 103 (35%) had blasts 20%. Four of the 11 patients (36%) with one gene mutation and Of the 92 cases with mutations in two genes or in one gene with allele frequency >20%, 26 patients (28%) had cytogenetic abnormalities confirming the diagnosis of MDS. In fact in this group of 26 patients with cytogenetic abnormalities, only one patient had mutations at 20%. There was no statistically significant difference in the degree of cytopenia between patients with 20%. There was no significant difference in the degree of cytopenia between the 36 patients with one gene mutation and 67 patients with more than one gene mutation. Conclusion: This data suggests that bone marrow samples from patients with peripheral cytopenia should be tested by cytogenetic and molecular profiling using NGS and the analysis of MDS-related genes. Our data suggests that when proper criteria are used, molecular profiling of bone marrow in the proper clinical presentation can help in confirming the diagnosis of MDS. Our data suggests that the presence of mutations in more than one gene and the detection of mutant allele frequency >20% may comprise reliable criteria for the diagnosis of MDS. The presence of mutation in 20% of DNA usually reflects mutation in 40% of the bone marrow cells. Patients with mutant allele frequency between 10% and 20% in the bone marrow and cytopenia most likely have MDS, but further studies are needed. Mutant allele frequency in bone marrow of Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is currently distinguished from myelodysplastic syndrome (MDS) based on the presence of 20% blasts in bone marrow, an arbitrary cut-off adopted by the WHO classification and replacing the 30% cut-off required by the older FAB (French, American and British) classification. Patients with t(15;17), t(8;21), or inversion 16 cytogenetic abnormalities are classified as having AML irrespective of the percentage of blasts. We explored the possibility that currently defined molecular abnormalities can distinguish AML from MDS without relying on an arbitrary percentage of blasts in the bone marrow. We compared the molecular profiles obtained by next generation sequencing (NGS) from consecutive patients with a clinical diagnosis of AML or MDS by WHO criteria. Methods: NGS data from 251 patients with the diagnosis of AML and 294 patients with the diagnosis of MDS was studied. All samples were analyzed using a panel of 25 genes including FLT3, NPM1 SF3B1, CBL, DNMT3A, ASXL1, BRAF, CEBPA, CSFR3, ETV6, EZH2, IDH1, IDH2, JAK2, c-KIT, KRAS, NRAS, PHF6, PTPN11, RUNX1, SETBP1, TET2, TP53, WT1, and ZRSR2. We compared the frequency of mutations in each gene between AML and MDS patients. Results: Mutations in FLT3 and NPM1 were uniquely and commonly detected in AML (27% and 22%, respectively). In contrast, mutations in SF3B1 gene were uniquely dominant (22%) in MDS and FLT3 and NPM1 mutations were rare (2% and 3%, respectively). SF3B1 mutations were extremely rare in AML (1%). Overall, 102 (41%) of all AML patients had mutations in either FLT3 or NPM1 and 8% of AML patients had mutations in both FLT3 and NPM1. In addition, WT1 gene was mutated in 8% of AML cases, but none of the MDS cases showed WT1 mutation. TET2 gene was commonly mutated in both AML and MDS (25% and 36%, respectively), but the frequency was significantly higher in MDS (P=0.003). IDH1, IDH2, NRAS, and PTPN11 were mutated slightly more often in AML than in MDS, while ASXL1, EZH2, and ZRSR2 were more frequently mutated in MDS than in AML. There was no statistically significant difference in mutation frequency between AML and MDS for the other genes analyzed. Conclusion: Mutations in FLT3, NPM1 and WT1 are molecular abnormalities characteristically detected in patients with AML and can be used as objective criteria for the classification of AML rather than blast count in bone marrow. These mutations are detected in 49% of AML patients. This suggests that approximately half of AML patients can be diagnosed based on the detection of molecular abnormalities, irrespective of bone marrow morphology. The presence of mutation in SF3B1 gene is also a characteristic molecular finding for MDS. Table. AML (No=251) MDS (No=294) P-Value No % No % FLT3 68 27 5 2 0.00001 NPM1 55 22 8 3 0.0001 SF3B1 3 1 66 22 0.00006 CBL 4 2 10 3 NS DNMT3A 51 20 51 17 0.07 ASXL1 44 18 75 26 0.01 BRAF 3 1 1 0 NS CEBPA 38 15 51 17 NS CSFR3 11 4 11 4 NS ETV6 3 1 6 2 NS EZH2 8 3 25 9 0.03 IDH1 20 8 7 2 0.03 IDH2 17 7 7 2 0.04 JAK2 4 2 10 3 NS KIT 2 1 0 0 NS KRAS 11 4 6 2 NS NRAS 34 14 18 6 0.01 PHF6 5 2 2 1 NS PTPN11 26 10 6 2 0.01 RUNX1 31 12 33 11 NS SETBP1 5 2 9 3 NS TET2 64 25 105 36 0.003 TP53 61 24 75 26 NS WT1 19 8 0 0 0.01 ZRSR2 7 3 30 10 0.02 Disclosures No relevant conflicts of interest to declare.

Background: Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton's tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). Mutations in BTK at the C481S hotspot alter the active site of the mutant BTK to the effect that Ibrutinib is reversibly bound. PLCγ2 is downstream of BTK in the B-cell signaling pathway; mutations in PLCγ2 at either of the R665W, L845F, or S707Y hotspots result in a constitutively activated PLCγ2. In order to better understand the development of these resistance mechanisms in patients with CLL, we developed a high sensitivity (HS) assay utilizing branched and locked nucleic acids (BNA and LNA, respectively). We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell free DNA (cf-DNA) from patients with CLL. Methods: Using custom BNA or LNA oligos in a wild-type blocking polymerase chain reaction, then sequencing by Sanger and NGS methods, we achieved 100x greater sensitivity than Sanger. Sanger sequencing was capable of detecting Results: No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. In contrast, all (N=7) tested patients with progressive disease on Ibrutinib showed one or more mutation in BTK or PLCγ2 using the HS method. Without the HS testing only 4 patients (57%) showed a mutation in BTK or PLCγ2. Two patients showed multiple mutant clones. One patient with double mutations in PLCγ2 (R665W and L845F) also showed triple independent mutations in BTK at codon C481 with HS testing. These mutations give rise to two distinct mutant proteins C481R (TGC>CGC) and C481S (TGC>AGC and TGC>TCC). NGS analysis confirmed that the the three BTK mutations are in three independent clones A second patient showed initially a mutation in BTK (C481S), but subsequent sample showed a mutation in PLCg2 (R665W), in addition to the BTK mutation. All mutations detected in the peripheral blood cells were also detectable in cell-free DNA (cfDNA) using HS testing. However, without using HS testing, a BTK mutation was detected in cfDNA from a patient and this mutation was not detectable when cellular DNA was used. Conclusions: Our data suggests that ibrutinib-naïve patients with CLL do not have BTK or PLCγ2 mutations even when a highly sensitive assay is used. Emerging BTK or PLCγ2 mutant clones can be seen after therapy with the possibility of multiple clones emerging at the same time and may involve both BTK and PLCγ2 genes in the same patient. Furthermore, testing cfDNA is not only as informative as cellular DNA, but might show mutations earlier than cellular DNA. This may have clinical relevance in patients with lymphoma when only few lymphoma cells are in circulation. Figure 1. Figure 1. Disclosures Wiestner: Pharmacyclics: Research Funding.

Introduction: Recent data suggest that MDS evolves by accumulating mutations. Early mutations may involve genes that require additional mutations prior to clinical manifestation as MDS. We explored if mutant allele burden and the relative mutation of one gene to another gene could provide information on the interclonal and intraclonal progression of MDS using next generation sequencing (NGS) in patients with early MDS. Methods: NGS data was generated from 96 patients diagnosed with MDS with marrow blast count 10%. A difference of 10% to 20% was considered mild, 20%-30% moderate, and >30% severe. A heat map reflecting these differences in mutant allele frequency was generated. Results: In this group of early MDS patients, 63 patients (66%) had more than one gene mutated and 38 (40%) had a significant (>10%) difference in allele frequency. The median number of genes mutated was 2 (range 1 to 5). Difference in mutant allele frequency was severe in 15 patients (16%), intermediate in 15 patients (16%), and mild in 13 patients (14%). TET2 was the most commonly mutated gene (43 patients, 45%) and was rarely the sole mutation with most cases exhibiting a mutation in a second gene (39 patients, 91%). The mutant allele burden was highest in TET2 in 26 of these 39 patients (67%), reflecting early event in the tumorigenic process. Of the 13 cases with TET2 mutation and allele burden less than the companion gene, 6 had a mutation in SF3B1, 3 had significant cytogenetic abnormalities (monosomy 5, del(7q), and trisomy 8), 2 had a mutation in SRSF2, 1 had a mutation in ZRSR2 and 1 had a mutation in ASXL1, which suggests that these abnormalities might be the initiating event. A second TET mutation (biallelic mutation) was detected in 16 of the 39 patients. SF3B1 was the most common gene having a solitary mutation (10% of all patients), although mutation in SF3B1 was detected in 27 patients (26% of all patients). All solitary SF3B1 mutations were associated with normal karyotypes, except for one patient with del(11q). JAK2 was mutated with SF3B1 in two cases diagnosed as RARS-T (refractory anemia with ring sideroblasts and thrombocytosis). In one case, the JAK2 and SF3B1 mutation allele frequencies were similar, but in the other, the JAK2 mutant allele frequency was 23% higher, suggesting that a myeloproliferative neoplasm was the initiating process. ASXL1 was mutated in 14 cases, 13 of which had additional mutations. DNMT3A gene was mutated in 18 cases, 5 of which were solitary; two of these five showed cytogenetic abnormalities. TP53 was mutated in 13 cases, but except for one case, all had either mutation in another gene or a cytogenetic abnormality. Conclusion: These data suggest that in patients with clinically confirmed early MDS, TET2 mutations are most likely the initiating oncogenic event, but mutations in other genes or cytogenetic abnormalities most likely lead to clinically confirmed MDS. In contrast, patients with SF3B1 mutation can have clinical disease without additional mutations. Our data suggest that SRSF2, ZRSR2, and ASXL1 may initiate mutagenesis in patients with MDS. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: In patients presenting with cytopenia, myelodysplastic syndrome (MDS) should be considered, but confirmation of diagnosis requires bone marrow biopsy and morphologic and cytogenetic evaluation. It is extremely difficult to rely on subjective morphologic features to confirm the diagnosis of MDS, when the karyotype is normal and blasts are not increased. Objective criteria for the diagnosis of MDS are needed in these cases. With recent advances in the characterization of molecular abnormalities in MDS, diagnosis of early MDS is becoming more objective by documenting the presence of MDS-specific molecular abnormalities in cases with appropriate clinical presentation. Since MDS is a disease of excessive apoptosis in bone marrow, DNA resulting from the apoptosis is abundant in circulation. We explored the potential of using cell free DNA in peripheral blood plasma using next generation sequencing (NGS) to confirm the diagnosis of early MDS without the need for marrow biopsy. Methods: Total nucleic acid was extracted from the plasma of 16 patients presenting with cytopenia and confirmed diagnosis of early MDS (blasts Results: Deep sequencing of cell free DNA in plasma from the 16 patients with early MDS showed at least one or more mutated gene confirming the diagnosis of MDS. Three patients (19%) showed mutation in one gene and the remaining 13 patients (81%) showed mutations in two or more genes. Cell free DNA in plasma from normal controls showed no evidence of mutations. When NGS data of cell free DNA from plasma was compared with Sanger sequencing data of DNA from cells in bone marrow, 10 of the 16 patients (63%) showed mutations in cell free DNA in plasma not detected by Sanger sequencing in DNA from cells in bone marrow. All mutations detected by NGS in cell free DNA in plasma were below the detection level of the Sanger technique and most likely represent subclones. NGS allowed the measurement of relative tumor load in plasma. Tumor load in plasma as detected by NGS was significantly (P=0.008) higher than that detected in cellular DNA, suggesting higher sensitivity of the former in detecting minimal residual disease and a better tool for monitoring therapy. Without exception, all detected mutations showed higher tumor load in plasma as compared with DNA from cells or whole blood, supporting the concept that plasma is enriched in tumor-specific DNA. Conclusion: NGS of cell free DNA in plasma using limited number of MDS-specific genes, when used in patients with cytopenia, presents an objective test for the confirmation of the diagnosis of MDS. Plasma is enriched in tumor-specific DNA in patients with MDS. Furthermore, mutation analysis of cell free DNA in plasma can detect subclones with mutations and can predict the emergence of new clones. Analysis of cell free DNA in plasma using NGS provides important data on tumor load, which can be used to monitor therapy, and predict progression, and also reduces the need for performing bone marrow biopsies. Disclosures No relevant conflicts of interest to declare.

Introduction: Diagnosis of myelodysplastic syndrome (MDS) based on bone marrow morphology can be very difficult, when blasts are not increased. The demonstration of cytogenetic abnormalities in these cases can confirm the diagnosis, providing cytopenia is documented. Cytopenia is usually the major reason for initiating work-up for myelodysplasia and , in general, cases with unicytopenia are the most difficult to make the diagnosis. In principle the recent characterization of the molecular abnormalities underlying the biology of MDS should provide objective biomarkers that can be used to confirm the diagnosis of MDS in the absence of cytogenetic abnormalities. Toward this goal, we developed a 14-gene panel to detect molecular abnormalities in patients referred to rule out MDS with blast count Method: Cytopenia is defined as having platelets Results: Fifty three of the 137 patients (39%) had a mutation in one or more genes. Of the 137 patients, three had tricytopenia, 14 had bicytopenia and 120 had unicytopenia. Two of the three with tricytopenia (66%) had mutations and nine of 14 with bicytopenia (64%) had mutations. In contrast 42 of the 120 patients with unicytopenia (35%) had mutation in one or more genes. Thirty of the 53 patients with mutation (57%) had one gene mutated and only 4 (13%) of these patients had bi- or tricytopenia. Of the remaining 23 patients with mutations in two or more genes a higher percentage (30%) of patients had bi- or tricytopenia. Compared to patients without mutations in the tested genes, those with mutation had significantly lower number of neutrophils (P=0.006), but higher percentage of monocytes (P=0.0002) and slightly higher percentage of lymphocytes (P=0.06). Twelve of 14 (86%) patients with cytogenetic abnormalities showed mutation in one or more genes and only three patients of the 14 (21%) had bi- or tricytopenia. Conclusion: Diagnosis of MDS at early stage of disease (blasts Disclosures No relevant conflicts of interest to declare.

Background: Refining risk stratification of acute myeloid leukemia (AML) using molecular profiling, especially those with intermediate cytogentic risk, is becoming standard of care. However, current WHO and ELN classifications are focused on few markers, mainly FLT3, NPM1, and CEBPA. While these abnormalities are relatively common, not all patients with AML and intermediate or normal cytogenetics will have abnormalities in these genes leaving large percentage of patients without refined risk stratification. We demonstrate that using 8 different AML-related genes are adequate to provide one or more molecular markers to further risk stratify patients with de novo AML. Method: Using direct sequencing we analyzed 211 samples referred from community practice with the diagnosis AML for molecular analysis. All samples were evaluated prospectively for mutations in FLT3, NPM1, IDH1, IDH2, CEBPA, WT1, RUNX1, and TP53 using direct sequencing. Fragment length analysis was used in addition to sequencing for FLT3 and NPM1. Available morphology, cytogenetics, and clinical data along with history were reviewed. Results: Of the 211 samples tested 103 (49%) had at least one or more molecular abnormality adequate for refining the risk classification. The mutations detected in these 103 patients were as follows: 27 (26%) FLT-ITD, 10 (10%) FLT3-TKD, 30 (29%) NPM1, 7 (7%) CEBPA, 14 (14%) IDH1, 13 (13%) IDH2, 10 (10%) WT1, 38 (37%) RUNX1, and 2 (2%) TP53. There was significant overlap and most patients had more than one mutation as illustrated in the graph below. However, if the testing was restricted to FLT3, NPM1, CEBPA and DNMT3A, only 56 (54%) would have had refined risk classification and 46% of patients would have remained without subclassification. The most striking finding was that all the remaining patients, who had no molecular abnormality detected in any of these 8 genes, had either history of MDS evolved to AML, therapy-related AML, or cytogenetic abnormalities other than intermediate (multiplex cytogenetic abnormalities or core-binding factor abnormality). Conclusion: Using FLT3, NPM1, CEBPA, and DNMT3A is inadequate for the molecular characterization of patients with AML. Patients with de novo AML and intermediate risk cytogenetics can be adequately prognostically subclassified and molecularly studied by testing only 8 genes. More importantly, this data confirms that the molecular biology driving de novo AML is significantly different from that driving MDS, AML with myelodysplasia-related changes, therapy-related AML, or AML with core binding factor or multiplex cytogenetics. Unlike de novo AML, these entities should be molecularly studied using MDS-specific driver genes. Furthermore, this data suggests that different therapeutic approaches should be developed for MDS and MDS-related AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

To the editor: We recently reported that using plasma rather than peripheral blood cells improves detection of the JAK2 V617F mutation,[1][1] which is the most important characteristic molecular abnormality in patients with nonchronic myeloid leukemia (non-CML)–myeloproliferative disease (MPD).[2

Abstract 995 Poster Board I-17 Several reports have suggested that mutations in the Wilms tumor 1 gene (WT1) represent an adverse prognostic factor in acute myeloid leukemia (AML). Here we examined the associations of WT1 mutations in exons 7 and 9 and the silent R301 single-nucleotide polymorphism (SNP) in exon 7 (A903G; NCBI dbSNP reference ID: rs16754) with outcome in AML patients treated at a single institution. Peripheral blood plasma and bone marrow samples from 174 newly diagnosed AML patients were tested for WT1 mutations in exons 7 and 9 by sequencing and fragment-length analysis for the detection of small deletions/insertions. Sequencing provided information on the specific genotype of the rs16754 SNP. The findings were correlated with outcome and other laboratory findings. WT1 mutation was detected in 7 of 50 (14%) AML patients 50. The silent R301 SNP was detected at frequencies of 4% for GG, 26% for GA, and 70% for AA genotypes, but there was no difference with age. A similar SNP genotype distribution was detected in normal control subjects. WT1 mutations were associated with higher white cell count (P=0.01) and higher percentage of blasts in bone marrow (P=0.03) and peripheral blood (P=0.009). In addition, WT1 mutation was significantly associated with FLT3 mutation (P=0.002) but not NPM1 mutation (P=0.8). WT1 mutation was also significantly associated with shorter survival (P=0.025), event-free survival (P=0.002), and complete remission duration (P=0.002) in patients Disclosures: No relevant conflicts of interest to declare.

Abstract 2161 Poster Board II-138 The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs). While other point mutations and small deletions and insertions in exons 12, 13, and 14 have been reported in the JAK2 JH2 domain, deletion of the entire exon 14 is rarely detected in patients with MPNs. In a series of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by direct sequencing of mRNA, we detected a complete exon 14 (88 bp) deletion mutation in 15% of total JAK2 transcript) in a small proportion of MPN cases, we decided to investigate the possibility it may be expressed at low levels (15% were confirmed by direct sequencing. The JAK2 exon 14 splice mutation was detected at low levels in 9 of the 61 MPN patients (15%), accounting for 3.96% to 33.85% (mean=12.04%) of JAK2 transcript in these individuals. Among the 183 suspected MPN patients, the exon 14 splice variant was detected in 20 of the 93 (21.5%) with V617F (mean expression level relative to wild type=5.41%, range=2.13%-26.22%) and 31 of the 90 (34.4%) without V617F (mean expression=3.88%; range=2.08%-12.22%). All 46 normal individuals were considered negative for the alternatively spliced transcript. In conclusion, the expression of an alternatively spliced JAK2 mRNA with deletion of exon 14, leading to a truncated JAK2 protein, is a common abnormality in patients with MPNs. This alternatively spliced transcript is more common in MPN patients without V617F mutation and might contribute to the leukemogenesis in these patients. Low levels of JAK2 exon 14 splice mutation may also contribute to the effects of the V617F mutation in patients with MPNs. However, this mutation is missed if DNA and not the RNA is used for testing for JAK2 mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract 1107 Poster Board I-129 The BCR-ABL1 tyrosine kinase is the target for kinase inhibitors in patients with chronic myeloid leukemia (CML). Multiple types of mutations in the BCR-ABL1 kinase domain have been reported. We previously reported a common alternatively spliced BCR-ABL mRNA with a 35-nucleotide insertion (35INS) between ABL1 kinase domain exons 8 and 9 that is expressed at various levels in CML patients with resistance to kinase inhibitors. Here we report 3 novel alternative splicing mutants expressed as the dominant transcripts in patient with CML and resistance to kinase inhibitors. We initially screened for ABL1 kinase domain mutations in approximately 200 patients with resistance to more than one of the three kinase inhibitors (imatinib, nilotinib, or dasatinib). Screening was conducted by first amplifying the fusion BCR-ABL1 transcript, to ensure that normal ABL1 was not amplified, then sequencing the ABL1 kinase domain coding region. We discovered 3 not previously described splice mutants, present in one patient each. All 3 showed >90% mutant transcript. The first resulted from the insertion of 79 nucleotides into the ABL1 exon 8-9 junction. The inserted sequence in fact contained a sequence from regions of intron 8, located 120 bp apart: the 35-nucleotide sequence previously described, and an additional 44-nucleotide segment downstream from 35INS. The combined 79-nucleotide insertion splice mutant showed the same protein change as 35INS (p C475YfsX11). The second splice mutation comprised an 84-nucleotide sequence from intron 7 inserted into the ABL1 exon7-8 junction, also causing a frameshift and protein truncation (p A424EfsX18). The third splice derived from a 231–nucleotide sequence from intron 4 retained in the ABL1 exon 4-5 junction. This insertion added 40 intron-encoded amino acids after K274, leading to a frameshift and early termination of BCR-ABL1 in the p-loop (p E275LfsX41). Exposure to kinase inhibitors may lead to the expression of alternatively spliced mRNA that is significantly elongated but causes a truncated fusion BCR-ABL1 that confers resistance to kinase inhibitors. These findings, when combined with the data on 35INS, support the concept that loss of the C-terminus of BCR-ABL1 is associated with significant resistance to kinase inhibitors; this mechanism appears to be a major source of resistance to kinase inhibitors. Disclosures No relevant conflicts of interest to declare.

Abstract 1261 Poster Board I-283 Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle except G0 and is widely used as a marker of cellular proliferation in human tumors. We recently demonstrated that levels of plasma circulating Ki-67 (cKi-67) are significantly higher in patients with newly diagnosed acute lymphoblastic leukemia (ALL) than in healthy control subjects, and that elevated levels of cKi-67 are associated with a shorter survival in ALL patients. Here we examined the associations of cKi-67 levels with laboratory and clinical variables in patients with chronic lymphocytic leukemia (CLL). The study included 194 patients with CLL and 96 healthy control subjects. The cKi-67 levels in plasma were determined using electro-chemiluminescence-based immunoassay using the Mesoscale Discovery platform. Since usually Ki-67 is used as an index of tumor cell proliferation, we took into account the lymphocyte count of the CLL patients in peripheral blood and normalized the levels of the cKi67 to the absolute number of lymphocytes in the peripheral blood establishing plasma cKI-67 index (cKi-67 level ng/1000 circulating lymphocytes/μL plasma). Median (range) levels of absolute cKi-67 were significantly higher in patients with CLL than in control subjects (914.65 [102.0-4975.12] ng/mL vs 353 [35.76-2830.65] ng/mL; P This data shows that there variability in proliferation between patients with CLL and those patients high relative proliferation (index) have more aggressive disease. Furthermore, plasma cKi-67 index and B2M levels are strong predictors of clinical behavior in CLL. Disclosures No relevant conflicts of interest to declare.

Abstract 3104 Poster Board III-41 Ki-67 and caspase-3 are widely accepted as proliferation and apoptosis markers in human tumors. We recently reported that Ki-67 can be detected as a circulating protein (cKi-67) in the plasma of patients with acute lymphoblastic leukemia (ALL), and that higher levels in plasma correlated with more aggressive disease. Here we investigated the levels of cKI-67 in patients with acute myeloid leukemia (AML). Since both apoptosis and proliferation are interconnected in defining the biology of a cancer, we also evaluated the level of apoptosis in AML as determined by caspase-3 activity in plasma. The study included a group of 106 newly diagnosed AML patients and 98 normal control subjects. We used the Meso Scale Discovery system for determining the levels of cKi-67. Apoptosis was determined by measuring caspase-3 activity (DEVD) in the plasma using a standard enzymatic fluorogenic assay. Median (range) levels of cKi-67 were significantly higher in patients with AML than in control subjects (1301 [0-6789] ng/mL vs 339 [35.76-2830.65] ng/mL; P70 years of age (n=84; P=0.02) and those with unfavorable cytogenetics (n=38; P=0.006). On the other hand, high levels of caspase-3 activity (above upper quartile of 14 pmol/min) were associated with shorter survival (P=0.02) irrespective of age or cytogenetic grouping. In conclusion, proliferation and apoptosis as determined by plasma cKi-67 level and caspase-3 activity are unusual in AML. In that, higher proliferation and lower apoptosis are associated with better survival, most likely reflecting that cells progressing through the cell cycle may respond better to chemotherapy. These measurements should be considered in new therapeutic approaches that target cell cycle. Disclosures No relevant conflicts of interest to declare.

Abstract 1891 Poster Board I-914 Myeloproliferative neoplasms (MPNs) are multipotent hematopoietic stem cell neoplasms characterized by excess production of various blood cells. Mutations in the thrombopoietin receptor gene (MPL) have been reported in JAK2 V617F-negative patients with MPNs. We evaluated the prevalence of MPL mutations relative to those of JAK2 mutations at V617 or in exons 12 through 15 in a large number of patients with suspected MPNs. A total of 2790 patients with suspected MPNs referred to our institution were first screened for JAK2 V617F and exon 12–15 mutations. Patients with no JAK2 mutations detected were then tested for MPL mutations in exons 10 and 11 by means of a sensitive MPL reverse-transcription-PCR-based assay with direct bidirectional sequencing. All JAK2 and MPL mutation assays were performed on plasma RNA, rather than DNA isolated from blood or bone marrow cells. Of the 2790 patients, 529 (18.96%) had a V617F mutation; 12 (0.43%) had small insertion/deletions in exon 12; and 7 (0.25%) had other JAK2 mutations, including point mutations in exons 13–15 and an exon 14 splice mutation. MPL mutations were identified in 66 of the 2242 (2.94%) JAK2 mutation-negative patients (2.37% of all tested patients). W515L was the dominant MPL mutant detected in 46 patients (70%) including two patients with homozygous W515L mutation. The other W515 variants (W515K, W515R, W515S, W515G, W515A, W515*) comprised 16% (N=11) of the MPL mutations. The remaining MPL mutations (n =9, 14%) were detected at other locations in exon 10 and 11. Two of these were novel exon 10 deletion/insertion mutations and two were unreported exon 11 point mutations. The exon 10 T496-A497ALVI (4AA) homozygous insertion was detected in one patient with a confirmed diagnosis of idiopathic myelofibrosis and the W515-P518 del/ins (KT) mutation was detected in another patient with unspecified MPN. The two novel MPL exon 11 point mutations were D545G and D545N. The S505N mutation (n=2) and V507I (n=1), R514K (n=1), and A519V (n=1) were also detected. Furthermore, three unreported silent polymorphisms/mutations (T496, L543, and D534) were detected. In conclusion, our results demonstrate that for every 100 V617F mutations detected in patients with MPNs, there are 2.3 JAK2 exon 12 mutations, 1.3 JAK2 exon 13–15 mutations, and 12.5 MPL mutations. Thus, MPL mutation detection should be performed on all JAK2 V617F-negative patients with suspected MPNs. In addition, our findings indicate that >20% of MPL mutations would have been missed if only W515L and W515K were analyzed; thus, sequencing of both exon 10 and 11 may be beneficial. Disclosures: No relevant conflicts of interest to declare.

Abstract 1240 Poster Board I-262 The ubiquitin-proteasome pathway is implicated in the pathogenesis of many hematological malignancies. We have reported that proteasome activities in plasma samples can be detected in patients with chronic lymphocytic leukaemia (CLL). Here we examined the correlation of plasma ubiquitin protein levels with clinical behavior in CLL patients. Using the Meso Scale Discovery (MSD) platform, we quantified poly-ubiquitin levels in plasma samples from 138 patients with CLL and 101 healthy control subjects, and examined the associations of ubiquitin levels with various clinical and laboratory variables and overall survival. Patients with CLL had significantly higher levels of poly-ubiquitin proteins (median=158.2; range=35.0-281.2) than did normal control subjects (median=57.3; range=22.0-160.2) (P193 ng/mL had significantly better survival than did those with lower levels. Using B2M and poly-ubiquitin in combination allowed us to stratify patients into 3 different groups : survival was greatest in patients with high poly-ubiquitin and low B2M levels, lowest in those with low poly-ubiquitin and high B2M levels, and intermediate in those with high levels of B2M and ubiquitin or low levels of both (P Disclosures No relevant conflicts of interest to declare.

Abstract 4249 Ki-67 and caspase-3 are widely accepted as proliferation and apoptosis markers. We recently reported that Ki-67 can be detected as a circulating protein (cKi-67) in the plasma of patients with acute lymphoblastic leukemia (ALL), and that higher levels correlated with more aggressive disease. Here we investigated the levels of cKi-67 in patients with chronic myeloid leukemia (CML). We also evaluated the level of apoptosis in CML as determined by plasma levels of caspase-3 activity. The study included 127 CML patients: 81 in chronic phase and 46 in accelerated phase/blast crisis. cKi-67 levels were determined with an electro-chemiluminescence-based immunoassay. Apoptosis was determined by measuring caspase-3 activity (DEVD) in the plasma using a standard enzymatic fluorogenic assay. Patients with CML had significantly (P354 ng/mL) had significantly longer survival than did those with lower levels (P=0.003); cKi-67 levels were not associated with outcome in accelerated/blast phase CML. Caspase-3 activity did not correlate with outcome in chronic or accelerated/blast phase patients. In conclusion, cell proliferation and apoptosis as determined by plasma cKi-67 level and caspase-3 activity are elevated in CML, but higher levels of cKi-67 unexpectedly correlated with longer survival in chronic phase CML. Although the cause for this is unknown, it is possible that higher levels of cKi-67 reflect that more stem cells are in cell cycle, and this may make them more susceptible to therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 4712 The ubiquitin-proteasome system (UPS) plays a major role in cell homeostasis in normal and neoplastic states. Expression and function of the UPS system vary with the specific characteristics of individual cell types, suggesting that determination of UPS “signatures” could be useful in identifying various cell populations. Since direct analysis of cancer cells is often problematic, even in hematologic diseases, we explored the potential of using UPS signatures in plasma to differentiate between various leukemias. We first analyzed plasma UPS profiles of patients with acute myeloid leukemia (AML; n=111), acute lymphoblastic leukemia (ALL; n=29), advanced myelodysplastic syndrome (MDS; n=20), chronic lymphocytic leukemia (CLL; n=118), or chronic myeloid leukemia (CML; n=128; 46 in accelerated/blast crisis [ACC/BL], 82 in chronic phase), and 85 healthy control subjects. Plasma levels of proteasome, ubiquitin (poly-ubiquitin), and the 3 proteasome enzymatic activities (chymotrypsin-like [Ch-L], caspase-like [Cas-L], trypsin-like [Tr-L]) were measured. Specific activities were calculated by normalizing each of the 3 enzyme activities to the levels of proteasome protein in plasma (Ch-L/p, Cas-L/p, and Tr-L/p). These 8 variables were used in multivariate logistic regression models to differentiate between leukemic processes. UPS signatures provided clear differentiation between patients with a leukemic process and normal controls (AUC=0.991), using 6 different variables (Tr-L/P, Ch-L, Ch-L/p, Cas-L, Cas-L/P, ubiquitin). Distinguishing between acute (AML, ALL, MDS) and chronic (CML, CLL) processes was less efficient (AUC=0.853 using Tr-L, Tr-L/P, Cas-L/P, Ch-L/P, proteasome, Ch-L), likely due to the high proportion (36%) of CML patients in ACC/BL phase. However, UPS signatures generally yielded powerful differentiation between individual leukemias (Table). MDS was not well differentiated from AML (AUC=0.791), reflecting the significant biological overlap of these diseases. These data support the potential usefulness of the UPS profile to aid in the differential diagnosis of various leukemias. Comparison Analytes N (Case/Control) AUC MDS vs Normal PS, UBT, C-L, Tr-L, C-LP Ch-P 105 (20/85) 0.98 ALL PS, UBT, Tr-L, C-LP 49 (20/29) 0.9845 AML PS, UBT, Ch-L, Tr-L, C-LP, Ch-LP, Tr-L/P 131 (20/111) 0.791 CLL C-L/P, Ch-LP 163 (27/136) 0.9909 CML UBT, C-LP, Tr-L 147 (20/127) 0.9496 CML (Chronic) UBT, C-LP, Tr-L, Tr-L/P 101 (20/81) 0.9608 CML (ACC/BL) UBT, C-LP, Tr-L, Tr-LP, Ch-L 66 (20/46) 0.9516 AML vs Normal UBT, C-L, Tr-L, Tr-LP, Ch-LP 196 (111/85) 0.9884 ALL C-LP, Ch-LP, UBT, Ch-LP, PS 140 (111/29) 0.9503 CLL C-LP, Ch-LP 283 (147/136) 0.9835 CML Tr-LP, Tr-L, UBT, C-LP, C-L, Ch-LP 238 (111/127) 0.8301 CML (Chronic) Tr-LP, Tr-L, UBT, C-LP, Ch-LP, PS 192 (111/81) 0.8396 CML (ACC/BL) Tr-LP, Tr-L, UBT, C-LP, Ch-LP, C-L 157 (111/46) 0.8239 ALL vs Normal UBT, Tr-L 114 (29/85) 0.9984 CLL UBT, Tr-L, Tr-LP, Ch-L, Ch-LP, C-LP 147 (29/118) 0.9031 CML no Tr-LP 156 (29/127) 0.9005 CML (Chronic) no Tr-L 110 (29/81) 0.9264 CML (ACC/BL) no Tr-L 75 (29/46) 0.8909 CLL vs Normal C-L/P, Ch-L, UBT 203 (118/85) 0.9978 CML C-LP, C-L, Ch-LP, Tr-L, UBT 245 (118/127) 0.9746 CML (Chronic) Cas-LP, C-L, Ch-LP, Tr-L UBT 199 (118/81) 0.9749 CML (ACC/BL) C-LP, C-L, Ch-LP, Tr-L, UBT 164 (118/46) 0.9746 CML vs Normal UBT, Tr-L 212 (127/85) 0.9986 Abbreviation: PS=proteasome protein Disclosures: No relevant conflicts of interest to declare.

Abstract 2179 Poster Board II-156 Point mutations in the BCR-ABL1 protein are the most commonly reported mechanism of resistance to imatinib mesylate (Gleevec or STI-571) treatment in patients with chronic myelogenous leukemia (CML). We have reported that some patients with imatinib-resistant CML express an alternatively spliced BCR-ABL1 mRNA with a 35-bp insertion (BCR-ABL135INS), but the prevalence and effect of this mutation have not been well delineated. We sought to improve understanding of this mutation in order to provide insight into mechanisms of resistance to tyrosine kinase inhibitors. Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in a group of 168 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot in patient samples. The effects of various kinase inhibitors on human K562 CML cells transfected with BCR-ABL135INS cDNA were tested by measuring cell survival, caspase-3 activation, and the BIM activation pathway. In our analysis of BCR-ABL1 genotypes in 168 imatinib-resistant chronic-phase CML patients, (56%) of patients expressed various levels of BCR-ABL135INS whereas only (25%) had detectable point mutations in BCR-ABL1. Expression of this truncated BCR-ABL1 fusion protein was also confirmed by immunoprecipitation at levels proportional to those predicted by mRNA quantification. In vitro, BCR-ABL135INS expression conferred resistance to the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib in a concentration-dependent fashion, but had no effect on the protein-synthesis inhibitor homoharringtonine (HHT or omacetaxine) or the aurora kinase inhibitor MK-0457 (VX-680). The combination of imatinib with nilotinib or HHT was synergistic in overcoming BCR-ABL135INS-induced resistance. Moreover, the resistance to imatinib induced by BCR-ABL135INS was associated with loss of BIM expression. This loss of BIM was overcome by pretreatment with HHT. In conclusion, these findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS found in imatinib-resistant CML patients. Optimal patient management in the future will likely require periodic testing for the expression of BCR-ABL-135INS mRNA during therapy. This may help in predicting various levels of resistance or the potential for eliminating the disease by adjusting the treatment strategy. Disclosures: No relevant conflicts of interest to declare.

Abstract 3825 Poster Board III-761 The risk of developing myelodysplastic syndrome (MDS) increases in the presence of a combination of genetic susceptibility factors and environmental carcinogenic factors. While numerous toxic agents have been described, genetic susceptibility factors for MDS are not well defined. A recent study showed that the TT genotype of the SNP rs1617640, located in the promoter of the eosinophile peroxidase (EPO) gene, is significantly associated with diabetic retinopathy and end-stage renal disease in patients with diabetes. Experimental models showed that the GG genotype is associated with several-fold lower erythropoietin expression than is the TT genotype. Since EPO plays a major role in the growth and differentiation of hematopoietic cells, we explored the association of the rs1617640 SNP genotype with MDS. We compared the EPO promoter SNP genotype in 187 MDS patients, 256 AML patients, and 95 normal control individuals. The GG genotype was significantly more common in patients with MDS (25.1%) than in normal control subjects (6.3%) and AML patients (12.5%) (P=0.0003); the GT genotype was present in 38.4% of MDS patients, 43.2% of AML patients, and 46.2% of normal control subjects. Combined analysis of data from MDS and control subjects indicated an elevated risk of MDS in individuals with the GG genotype relative to those with a non-GG genotype (odd ratio [OR] =4.98; 95% confidence interval [CI]=2.04-12.13). The GG (vs non-GG) genotype was also associated with elevated MDS risk when analysis was limited to the MDS and AML groups (OR =2.35; 95% CI=1.43-3.86). In contrast, analysis of AML and control group data showed a statistically insignificant elevated risk for AML in individuals with the GG genotype (OR=2.12; 95% CI=0.86-5.24). The odds of MDS and AML did not differ significantly with respect to GT (vs non-GT) or TT (vs non-TT) genotypes when compared with control subjects. Clinical and follow up data were available for 112 MDS patients and 186 AML patients. There was no correlation between EPO promoter genotype and response to therapy or overall survival in MDS or AML. In the MDS group, the GG genotype was significantly associated with shorter complete remission duration, as compared with the TT genotype (P=0.03). We found no correlation between EPO genotype and cytogenetic abnormalities, performance status, or other laboratory variables. In conclusion, the EPO promoter GG genotype shows strong association with MDS. Since MDS is well documented to develop after exposure to toxic agents, the clinical value of screening for the EPO promoter GG genotype in individuals at high risk due to exposure to toxic agents, then storing hematopoietic stem cells from those with the GG genotype for possible future use, should be investigated. Disclosures: No relevant conflicts of interest to declare.

Response Salama et al reported on the stability of JAK2V617F DNA (17 patients) and RNA (1 patient) in whole blood specimens from subjects with V617F-positive myeloproliferative neoplasms, specifically comparing the percentage of V617F relative to wild-type sequence in granulocytes and plasma at

The V617F point mutation in the JAK2 gene is considered the most important molecular abnormality that characterizes patients with non-chronic myeloid leukemia (CML)-myeloproliferative disease (MPD). While it is well-documented that the V617F mutation leads to continuous activation of the JAK2-STAT5 pathway, the levels of JAK2 mRNA expression is not well defined in myeloproliferative diseases. We quantified the levels total JAK2 mRNA as well as V617F mutant mRNA in the plasma of patients with non-CML MPD using RT/PCR, and compared the results with clinical findings and disease behavior. Patients with non-CML MPD (n = 175) had significantly higher levels (P

Nucleotides insertion in the nucleophosphamin (NPM1) gene has been reported in about one third of patients with acute myeloid leukemia (AML). Multiple studies showed that the presence of NPM1 mutations associated with better outcome in patients with AML. Studies reported to date have analyzed leukemic cells obtained from bone marrow or peripheral blood. We tested for mutations in the NPM1 gene using peripheral blood plasma and compared results with clinical outcome from a single institution. Analyzing plasma from 98 newly diagnosed patient with AML showed NPM1 mutation in 24 (23%) of patient while only one (4%) of 28 previously untreated patients with myelodysplastic syndrome (MDS) showed NPM1 mutation. Compared with peripheral blood cells, 2 (8%) of the 24 positive patients were negative by cells; none were positive by cells and negative by plasma. Most of the mutations detected (45%) were in patients with FAB classification M2, M4 and M5. In addition to the reported 4 bp insertion, we also detected 4 bp deletion in one patient in cells and plasma. Patients with NPM1 mutation had a significantly higher white blood cell count (P = 0.0009) and a higher blast count in peripheral blood (P = 0.002) and in bone marrow (P = 0.002). Blasts in patients with NPM1 mutant expressed lower levels of HLA-DR (P = 0.005), CD13 (P = 0.02) and CD34 (P < 0.0001), but higher CD33 levels (P = 0.0004). Patients with NPM1 mutation appear to have better chance of responding to standard therapy (P = 0.06). Event free survival of patients with NPM1 mutation was longer (P = 0.056) than in patients with intermediate cytogenetic abnormalities. The most striking difference in survival was in patients who required >35 days to respond to therapy (Figure). Survival was significantly longer in patients with NPM1 mutation requiring >35 days to respond (P = 0.027). This data not only support that NPM1 plays a significant role in the biology and clinical behavior of AML, but also show that plasma DNA is enriched with leukemia-specific DNA and is a reliable source for testing. Figure Figure

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many malignancies and is an important regulator of cell proliferation, apoptosis, DNA repair, and the stress response. Free circulating proteasomes have been reported in the plasma and sera of cancer patients. We measured proteasome peptidase activity in the plasma of patients with chronic myeloid leukemia (CML) and correlated these findings with clinical behaviour. Plasma samples from apparently healthy volunteers (n = 92) and patients in the chronic (n = 104) or accelerated/blast phase (n = 56) of CML were analysed with fluorogenic kinetic assays. Using peptide-AMC (7-amino-4-methylcoumaran) substrates, we measured the three reported proteasome enzymatic activities: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L). All proteasome activities were significantly higher in CML patients than in healthy volunteers. Extensive characterization of proteasome activities revealed correlations between levels or ratios of specific peptidase activities and disease phase, progression, aggressiveness, and survival. High Ch-L activity correlated with shorter survival in both CP (P=0.037) and Acc/Bl phase (P=0.047). Patients in CP and a ratio of Cas-L: Tr-L activity >1.48 had significantly shorter survival (P=0.03). Cas-L: Tr-L activity ratio was predictor of survival in Acc/Bl patients as a continuous variable. These findings suggest that proteasome activity in the plasma of CML patients reflects disease activity and can be used as a biomarker for predicting clinical behavior. Studies exploring the role of proteasome inhibitors in CML are therefore warranted.

Cytogenetic abnormalities are currently the most important predictors of response and clinical outcome for patients with acute myeloid leukemia (AML) or advanced-stage myelodysplastic syndrome (MDS). However, clinical outcomes of patients in each cytogenetic subgroup are very heterogeneous, and additional biological markers are needed to improve disease management. Most of the recently reported biomarkers, especially in patients with normal karyotypes, provide marginal value for predicting behavior or require significant and technically difficult procedures, such as gene expression profiling. We assessed the utility of measuring proteasome chymotrypsin-like (Ch-L) activity in plasma, using fluorogenic kinetic assays, to predict response and overall survival of patients with AML (N=174) and advanced-stage MDS (N=52); results from AML and MDS patients were pooled for all analyses after demonstrating similar findings. In a univariate logistic regression model, significant predictors of response were age group (< vs ≥70 years; odds ratio [OR]=1.92; 95% confidence interval [CI]=1.06, 3.48), cytogenetics (OR=2.20; 95% CI=1.28, 3.78), BUN as a continuous variable (OR=1.04, 95% CI=1.01, 1.07), and Ch-L activity as a continuous variable (OR=1.41, 95% CI=1.14, 1.76) (all P

The ubiquitin-proteasome pathway plays a major role in degrading proteins that regulate important cellular processes including cell cycle regulation, apoptosis, DNA repair, and stress response. Proteasomes have 2 active sites each for 3 different peptidase activities: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Different proteasome inhibitors affect each of the activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. We developed fluorogenic kinetic assays using peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities in peripheral blood plasma rather than cells. This approach allowed us to standardize measurements and express enzymatic activity as pmol AMC/sec/mL plasma. We tested Ch-L, Tr-L, and Cas-L activities in the plasma of 226 patients with chronic lymphocytic leukemia (CLL) and assessed their correlations with clinical behavior. Ch-L, Tr-L, and Cas-L activities were significantly (P

The ubiquitin-proteasome pathway plays a major role in regulating proteins involved in cell cycle regulation and apoptosis. Three different types of enzymatic activity have been reported for proteasomes: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Various proteasome inhibitors affect each of the three activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. We developed fluorogenic kinetic assays using peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities in cell-free plasma. We evaluated plasma activities in 57 patients with acute lymphoblastic leukemia (ALL) and assessed their correlations with clinical behavior. Significantly (P < 0.001) higher Ch-L, Tr-L, and Cas-L activities were detected in patients with ALL (medians: 1.40, 2.06, and 2.04 pmol AMC/sec/mL, respectively) than in healthy volunteers (n = 42) (medians: 0.80, 0.74, and 0.81 pmol AMC/sec/mL, respectively). While there was no significant difference between Ch-L and Cas-L activities in healthy controls, there was a significant difference between the 2 activities in patients with ALL. Cas-L, Tr-L, and Ch-L all correlated positively with lactic dehydrogenase (P

The ubiquitin-proteasome pathway is responsible for multiple pathways in cancer cells; proteasome inhibition causes rapid apoptosis of tumor cells. Three different types of peptidase activities have been reported for proteasomes: chymotrypsin-like (Ch-L), trypsin-like (Tr-L), and caspase-like (Cas-L) (postglutamyl peptide hydrolytic-like). Various proteasome inhibitors affect each of the 3 activities differently and at different concentrations. For example, NPI-0052 inhibits Ch-L and Tr-L activities at lower concentrations than does bortezomib, while bortezomib inhibits Cas-L at lower concentrations than does NPI-0052. These enzymatic activities are usually measured in normal or tumor cells to monitor therapy with proteasome inhibitors. Because rapidly proliferating leukemic cells pour their proteins, DNA, and RNA into the circulation, we developed fluorogenic kinetic assays using peripheral blood plasma. The assays used peptide-AMC (7-amino 4-methylcoumoran) substrates to measure Ch-L, Tr-L, and Cas-L activities. We measured proteasome activities in plasma from 188 patients with acute myeloid leukemia (AML) and 58 patients with myelodysplastic syndrome (MDS) and assessed their correlations with clinical behavior. Significantly (P < 0.001) higher Ch-L, Tr-L, and Cas-L activities were seen in AML patients (medians: 1.39, 1.51, and 2.40 pmol AMC/sec/mL, respectively) and MDS patients (medians: 1.16, 1.40, and 1.67 pmol AMC/sec/mL, respectively) than in healthy volunteers (n=42) (medians: 0.80, 0.74, and 0.81 pmol AMC/sec/mL, respectively). The difference in Cas-L activity between AML and MDS was significant (P 3 pmol AMC/sec/mL had significantly shorter survival (P = 0.04). Ch-L activity was also predictive of survival in AML independent of age and cytogenetic and performance status, but not independent of Cas-L. In MDS, higher levels of Cas-L, but not Ch-L, correlated with shorter survival and this was independent of cytogenetic abnormalities. The increased cell-free circulating proteasome activities most likely reflect the leukemic cells and may be a marker not only for disease, but also potentially for monitoring therapy. These data also suggest that patients with AML may benefit differentially from proteasome inhibitors depending on the specific therapeutic effect of the inhibitor.

Missense point mutations in the region encoding the ABL tyrosine kinase domain have been reported in approximately 35% of patients with imatinib-resistant chronic myeloid leukemia (CML). The reported mutations result in reactivation of the BCR-ABL tyrosine kinase. Screening patients with imatinib-resistant CML, we identified 42 different mutations in the ABL tyrosine kinase domain-encoding region, 2 of which were silent (no amino acid change): A864G and G909A. The A864G mutation was associated with a 54-nucleotide reduction in the length of the mRNA transcript, representing a loss of nucleotides 1089-1143; the G909A mutation was associated with a normal-length transcript. The nt1089-1143 transcript deletion represents a partial exon deletion in which the first half of exon 8 is skipped, suggesting that A864G leads to abnormal splicing. Splicing is regulated by 6- to 8-nucleotide exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs recognized by the SR proteins (a family of splicing factors). We therefore used ESEfinder to examine whether A864G or G909A alter ESE motifs, which could block the ability of SR proteins to recognize and bind. This search showed that A864G is at the 7th position of an AGCTGCAG ESE motif, a binding site for SR35, and is within 35 bp of the intron-exon junction. In total, ESEfinder predicted 18 putative SR35-binding ESEs within 50 bp of the intron-exon junction, covering 20% of the kinase domain. The AGCTGCAG motif is conserved in primates (chimpanzees and monkeys) but not in rodents, while A864G is found in mice Although similar links cannot be made with the G909A mutation, these data suggest that imatinib resistance may develop in some patients through alternative splicing and the expression of a truncated (or potentially elongated) protein.

The Val617Phe mutation in JAK2 (Janus kinase) has been reported in approximately 90% of patients with polycythemia vera (PV) and in almost 40% of patients with essential thrombocythemia (ET) or idiopathic myelofibrosis (IMF). JAK2 is a cytoplasmic tyrosine kinase that binds to growth factor receptors and phosphorylates several downstream proteins that are involved in cell proliferation and growth. The Val617Phe mutation can be detected in circulating granulocytes. Homozygous as well as hemizygous (deletion of 1 allele and mutation in the second) mutations have been reported. The clinical importance of hemi/homozygous as compared to heterozygous mutation is not known. Furthermore, the presence of normal lymphoid cells or non-neoplastic cells in samples makes it difficult to be certain whether the mutation is homozygous in a subpopulation of cells, or heterozygous. Since we have shown that the plasma of patients with leukemia is enriched with leukemia-specific DNA, RNA, and protein, we tested the possibility of detecting JAK2 mutations in patients with chronic myeloproliferative diseases using RNA from peripheral blood (PB) plasma rather than cells. In this study, PB plasma was obtained from 39 patients with IMF, 16 with PV, 8 with ET, and 23 with other chronic myeloproliferative disease not otherwise classified (MPD-NC). Bi-directional direct sequencing of RNA extracted from plasma was used to detect mutations in the JAK2 transcript. The Val617Phe mutation was detected in 88% of PV patients, of whom 50% (44% of all samples) were hemi/homozygous. This mutation was detected in 56% of IMF patients, 41% of whom were hemi/homozygous. The number of patients with ET was small, but 25% (2 patients) had hemi/homozygous mutation. 26% of the patients with MPD-NC had JAK2 mutation; of these, 33% had hemi/homozygous mutation. The plasma of 31 normal control individuals showed no evidence of mutation and mixing studies showed that the Val617Phe mutation was easily detectable when lysate from HEL cell line (homozygous for the mutation) were mixed with plasma down to 5 cell/1ml plasma. Patients with hemi/homozygous mutation had significantly larger spleens (P=0.0001), increased white cell counts (P=0.001), and increased monocyte counts in bone marrow (P=0.03). We cannot demonstrate statistically significant difference in survival between hemi/homozygous and heterozygous, however, the number of cases is too small. The presence of mutation (hemi/homozygous or heterozygous) was associated with better overall survival in patients

With the use of imatinib for treatment of chronic myeloid leukemia (CML), monitoring treatment response and quantifying leukemic cells in the body by specific detection of the bcr-abl fusion gene or its mRNA is becoming the standard of care. The high frequency of complete cytogenetic response in CML patients has led to reliance on quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) for detection of the bcr-abl mRNA when monitoring therapy. Peripheral blood (PB) and bone marrow (BM) specimens are usually used for qRT-PCR assays. However, standardization of the qRT-PCR assay and issues regarding sampling and the number of cells that need to be analyzed make reliance on such assays problematic. We previously showed that leukemic cells pour their RNA, DNA, and protein into circulation and, because of their high turnover rate, enrich the plasma with these components. We also hypothesize that plasma mRNA reflects disease activity in the entire body rather than the few cells in the analyzed sample, making plasma a more reliable source than PB cells. In this study we compared qRT-PCR results obtained from PB cells with those obtained from plasma in CML patients being treated with imatinib. PB cell and plasma samples obtained at baseline (n=67) and at 3 (n=43), 6 (n=22), 9 (n=19), and 12 (n=9) months of therapy were compared. The same qRT-PCR assay was used for cell- and plasma-based testing. However, the RNA extraction from plasma was standardized by using equal amounts from all samples and the RNA from 50μL plasma for each qRT-PCR assay. All plasma samples from CML patients were positive at baseline, whereas testing of more than 180 plasma samples from normal individuals or patients with leukemia other than CML showed no detectable bcr-abl transcript. In CML patients, the pattern of changes with therapy in the qRT-PCR in the plasma paralleled that obtained from cell-based testing. At 3 months, all patients who were negative by plasma-based testing were also negative by cell-based testing, whereas 6 of the 14 negative patients by cell-based testing were positive by plasma-based testing. At 6 months of therapy, 8 patients were negative by cell-based testing and all but 1 of these were negative by plasma; this patient was positive by plasma-based testing and became positive by cell-based testing at 9 and 12 months. 10 patients were negative by plasma-based testing at 6 months, 3 of whom were positive by cell-based testing. However, 2 of these 3 became negative by cell-based testing at 9 months and remained negative at 12 months. At 9 months of therapy, 9 patients were negative by cell-based testing, of whom 3 were positive by plasma-based testing; these 3 became positive by cell-based testing at 12 months. 8 patients were negative by plasma-based testing at 9 months, 1 of whom was positive by cell-based qRT-PCR. However, this patient became negative by cell-based testing at 12 months. This data show not only that plasma is a reliable source for testing and monitoring patients with CML, but that it is more reliable than PB cells for monitoring molecular response in CML. Furthermore, because plasma-based test results can be reported as mRNA copies/μL plasma, this platform has the potential to allow better standardization of testing among laboratories.

The mutation status of the immunoglobulin heavy chain variable region (IgVH) gene is an independent prognostic indicator in patients with chronic lymphocytic leukemia (CLL). Unmutated IgVH is associated with rapid disease progression and shorter survival. The assay for determining IgVH mutation status depends on specific amplification of the mRNA of the expressed clonal IgVH. The presence of more than 1 clone in patients with CLL has been speculated and can be documented when the 2 clones express different light chains. Determining the IgVH gene family that is expressed in subclones also allows the confirmation of the presence of two clones in some cases. We describe 3 patients with two clones of CLL discovered in the process of determining the IgVH mutation status. PCR amplification of the expressed IgVH mRNA yielded 2 distinct bands. Sequencing each band separately revealed 2 different clones in each patient. Interestingly, each clone had a different mutation profile. In 2 of the 3 patients, the 2 clones were from completely different IgVH gene families (VH1-2 with VH2-5 and VH4-34 with VH5-51); both of these patients showed kappa light chain restriction. In the third patient the expressed IgVH gene family was VH3-21 in both clones, but 1 was mutated (6.8%) and the second was unmutated. Two of the 3 patients had evidence of aggressive disease with hepatosplenomegaly and lymphadenopathy; 1 of these patients expressed the VH3-21 gene family, which is known to be associated with aggressive disease irrespective of its mutation status. The other patient had a 9-year history of indolent disease without therapy. The intensity of the expressed IgVH clones as determined by PCR indicated that the mutated clones were dominant in 2 patients (approximately 80%). In the patient with VH3-21 expression, the unmutated clone accounted for 80% of total expressed IgVH. These data suggest that the presence of more than 1 clone should be considered when testing for IgVH mutation status and, more importantly, that evolution of a more aggressive second clone should be suspected. The presence of 2 clones—1 arising in naïve cells and the second in memory cells—suggests the possibility that the first hit occurred very early in the ontogeny of lymphocytes and that a second hit occurred at a later stage. However, the presence of naïve and memory clones in the same family raises the possibility that the naïve leukemic cells might still go through the germinal center programming process.

The mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in the leukemic cells of patients with chronic lymphocytic leukemia (CLL) is an important prognostic marker. Lack of IgVH mutation is associated with rapid disease progression and shorter survival. The assay used to determine IgVH mutation status requires specific amplification of the mRNA of the expressed clonal IgVH gene in CLL. However, in some patients with CLL or lymphocytic lymphoma, the bulk of the disease resides in the lymph nodes or bone marrow, with few circulating leukemic cells. In addition, contamination of leukemic clonal cells by reactive polyclonal plasma cells can cause difficulty in obtaining homogenous IgVH mRNA for sequencing, and may therefore lead to failed sequencing or sequencing of the wrong IgVH mRNA. We have reported that plasma is enriched with leukemia-specific DNA, RNA, and protein because of the high turnover of leukemic cells relative to non-neoplastic cells. We thus reasoned that plasma might be a more reliable source of IgVH mRNA than cells from peripheral blood or bone marrow, where mixed populations of leukemic cells and non-neoplastic lymphocytes or plasma cells could hinder sequencing. We tested the plasma from 8 patients in whom routine (cell-based) testing for IgVH mutation status failed to yield results due to a paucity of leukemic cells (