ALBERT

Description: Background Measurable residual disease (MRD) is associated with inferior outcomes in patients with acute myeloid leukemia (AML). MRD monitoring enhances risk stratification and may guide therapeutic intervention. Post-induction MRD is frequently cleared with further therapy and the clearance may lead to better outcomes. In contrast, persistent MRD is associated with poor outcomes. At present it is not possible to predict which patients are likely to clear MRD with further therapy. Here we report a simple, objective, widely applicable and quantitative MFC approach using the ratio of blast/PDC to predict persistent MRD and poor outcomes in AML. Patients and Methods A cohort of 136 adult patients with a confirmed diagnosis of AML by WHO criteria who underwent standard induction therapy at a single center between 4/2014 and 9/2017 was initially included. 69 patients achieved complete morphologic remission (36 MRD-neg. and 33 MRD-pos.). MRD status was assessed by MFC using a different from normal (DfN) approach. PDC were quantified as the percent of total WBC by flow cytometry based on low side scatter, moderate CD45, CD303, bright CD123 and HLA-DR expression. Results The proportion of PDC was markedly decreased in patients with AML (≥20% blasts) (N=136) with a median of 0.016% (interquartile range IQR: 0.0019%-0.071%, Figure 1A), more than 10-fold lower than observed in normal controls (median 0.23%, IQR 0.17%-0.34%) (N=20). While there was no difference between MRD-neg. and normal control groups (median 0.31%, IQR: 0.17%-0.49%; vs. 0.28%, IQR: 0.17%-0.34%), MRD-pos. group had significantly reduced PDC proportion compared to the control (median 0.074%, IQR: 0.022%-0.33%, Wilcoxon rank sum, p=0.019). In an attempt to achieve better separation and to eliminate possible effects of hemodilution, the ratio of blast/PDC was calculated by using the proportions of blasts and PDCs out of total WBCs as quantitated by flow cytometry. A cut-off threshold of the blast/PDC ratio of 10 was chosen to separate each group (Figure 1B). Importantly, a ratio cut-off of 10 had a corresponding specificity of 97.4% for predicting MRD positivity status. MRD positivity was significantly associated with inferior overall survival (OS) and relapse-free survival (RFS) in our study cohort (OS HR 4.11 (95% CI: 1.30-13.03), p=0.016; RFS HR 4.20 (95% CI: 1.49-11.82), p=0.007, Figure 1C and D). The 2-year cumulative incidence of relapse in the MRD-neg. group compared to MRD-pos. group was 10% (95% CI: 2-24%) vs. 37% (95% CI: 18-56%, p=0.014). Importantly, blast/PDC ratio ≥10 was also strongly associated with inferior OS and RFS (OS HR 3.12 (95% CI: 1.13-8.60), p= 0.028; RFS HR 4.05 (95% CI: 1.63-10.11), p=0.003, Figure 1E and F), which is similar in magnitude to MRD positivity. Furthermore, MRD-pos. patients with blast/PDC ratio

Description: Background Minimal residual disease (MRD) negativity is a strong predictor for outcome in multiple myeloma. Next generation sequencing (NGS) for immunoglobulin heavy chain and kappa light chain VDJ rearrangements has become increasingly more common for MRD assessment. One of the known challenges with NGS for VDJ rearrangements is the vast diversity of sequences that are present, resulting in a need for a multiplex approach as common primers cannot be used to amplify all rearrangements. Also, somatic hypermutation may affect the annealing of primers and decrease the capture rate. The NGS VDJ assay developed by Adaptive Biotechnologies targets all theoretical combinations of VDJ sequences and has been used in several recent large randomized trials in multiple myeloma. The reported ~80% capture rate of the first version of the Sequenta/Adaptive 1.3 assay limited the ability to track MRD status post therapy. The assay has recently been updated and validated to increase resilience to somatic hypermutation. As there is no published reference data using this assay, we were motivated to assess VDJ capture in the clinical setting. Methods In total, 147 patients with newly diagnosed multiple myeloma (NDMM, n=101) or relapse/refractory multiple myeloma (RRMM, n=46) seen at Memorial Sloan Kettering Cancer Center were identified and included in the study. At bone marrow collection, patient samples were sorted for mononuclear cells and a subset of samples were sorted for CD138+ plasma cells. Stored bone marrow samples from these patients underwent DNA extraction and were sequenced with the Adaptive NGS VDJ assay. The same samples were also sequenced for genomic events using our internal NGS panel myTYPE. myTYPE is a custom capture panel targeting the most frequent multiple myeloma associated-somatic mutations, copy number alterations, and IGH translocations. Logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) of detection success in relation to clinical parameters such as age, gender, percent bone marrow plasma cells, as well as immunoglobulin heavy and light chain types, and myTYPE positivity. Results There overall capture rate for a unique VDJ sequence was 80%, 75% in NDMM samples and 89% in RRMM samples, respectively. The VDJ capture rate in samples that were myTYPE positive, e.g. samples with at least one genomic aberration detected by myTYPE, was 94%. In univariate analysis, the ORs of detecting a clonal VDJ sequence was 1.8 (95% CI 1.3-2.5) and 1.5 (1.2-1.9) for every 10% increase in plasma cells on bone marrow aspirate and biopsy, respectively. For every 1g/dL increase in M-spike, the OR of VDJ capture was 1.6 (1.2-2.2). Samples with at least one genomic aberration detected by myTYPE had a significantly higher detection rate of VDJ sequence, the OR of VDJ capture in myTYPE positive samples was 8.8 (3.2-31.3). Conversely, age, gender, type of immunoglobulin heavy chain (IgG or IgA), or light chain type (kappa or lambda) had no significant effect on the VDJ detection rate (Table). In multivariate analysis, myTYPE positivity was found to be an independent predictor of VDJ capture, with an OR of 4.9 (1.6-18.4, p=0.009) for myTYPE positive samples. The ORs were 1.4 (1.1-2.2, p=0.052) for an increase in 10% plasma cells on bone marrow aspirate and 1.5 (0.97-2.3, p=0.083) every 1g/dL increase in M-spike. Conclusion The VDJ capture rate using the updated Adaptive NGS VDJ assay was 94% in multiple myeloma samples of high quality as indicated by myTYPE positivity. The capture success rate was higher in samples with a greater disease burden. As expected, the assay was less sensitive in samples with insufficient DNA content. Our results are supportive of the use of this NGS VDJ in multiple myeloma, but also illustrate the importance of optimal sample ascertainment and processing. Disclosures Jacob: Adaptive Biotechnologies: Employment, Equity Ownership. Korde:Amgen: Research Funding. Mailankody:Juno: Research Funding; Physician Education Resource: Honoraria; Janssen: Research Funding; Takeda: Research Funding. Lesokhin:Serametrix, inc.: Patents & Royalties: Royalties; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Janssen: Research Funding; Genentech: Research Funding. Hassoun:Oncopeptides AB: Research Funding. Smith:Celgene: Consultancy, Patents & Royalties: CAR T cell therapies for MM, Research Funding. Landgren:Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding.

Description: Background: Solid tumor patients are at a heightened risk for developing therapy-related myeloid neoplasms (tMN). Recent studies show evidence of somatic mutations in leukemia-associated genes in normal healthy individuals, referred to as clonal hematopoiesis (CH). We and others have shown that clonal hematopoiesis (CH) is also frequent in cancer patients. A detailed characterization of the relationship between exposure to specific oncologic regimens, the molecular features of CH presentation and how these relate to tMN risk is warranted to inform treatment decisions, early detection and prevention strategies. Methods: To determine the relationship between CH and oncologic therapy, we performed a systematic interrogation of CH in a cohort of 17,478 solid tumor patients with clinical, outcome and molecular profiling by MSK-IMPACT. MSK-IMPACT is a targeted panel of cancer-associated mutations used to screen tumor samples against a blood control sample. Mutation detection was performed on blood derived sequencing data (median coverage at 600x) using the matched tumor as a comparator and accounted for background sequencing error rates. Results: Overall, 40% of the 17,478 patients were treatment naïve prior to IMPACT testing, 37% had received chemotherapy alone, 17% had received radiation therapy and 18% had received both. CH was identified in 4013 (23%) of patients, median VAF was 4% (range=1-80%). The vast majority (76%) had a single mutation whereas 9% had two and 5% had three or more. The number of mutations correlated with clone size (p-value=

Description: Introduction Minimal residual disease (MRD) negativity after initial therapy is a strong predictor of survival in multiple myeloma. Tracking of clonal immunoglobulin V(D)J rearrangements by next generation sequencing is highly sensitive for MRD and does not require immediate analysis of fresh samples. However, previous studies have found variable rates of baseline V(D)J sequence capture, which could limit tracking. In this study, we aimed to define the sample-related and disease-related factors that influence V(D)J capture. Methods We included 177 patients with plasma cell myeloma who had available stored mononuclear cells from a baseline bone marrow aspirate. Each sample was sequenced by two assays: The LymphoTrack® VDJ assays from Invivoscribe, and our in-house myeloma panel myTYPE, as a molecular control for detectable tumor derived DNA in the samples. MyTYPE positivity was defined by one or more single nucleotide variant, insertion, deletion, translocation or copy number variation that is known to occur in myeloma. Results and discussion The V(D)J capture rate in our whole cohort was 81 %, as compared with 95 % in the myTYPE positive samples, demonstrating the importance of tumor cell content for V(D)J capture. This was confirmed in multivariate logistic regression (Figure 1), where myTYPE positivity was a strong independent predictor of V(D)J capture success, with an odds ratio (OR) of 6.61 (95 % CI 2.22-24.81, p = 0.002). Plasma cell content estimated from bone marrow aspirate smears also contributed to the multivariate model, with an OR of 1.3 for each 10 % increase in plasma cell content (95 % CI 0.96-1.84, p=0.109), but this did not reach statistical significance after accounting for the strong effect of myTYPE. Finally, having lambda light chain restricted plasma cells was a strong predictor of V(D)J capture success (OR 6.91, 95 % CI 2.4-25.32, p = 0.001). Higher V(D)J capture rate in lambda-restricted myeloma as compared with kappa-restricted was mostly driven by a difference in immunoglobulin kappa gene (IGK) rearrangement capture (73 vs. 44 %, p 〈 0.001). As a potential explanation, we found up to 4 unique IGK rearrangements that are amenable to capture in lambda-restricted cases, as well as dramatically lower somatic hypermutation (SHM) of the IGK variable region in clonal rearrangements, as compared with kappa-restricted cases. SHM has previously been shown to cause V(D)J capture failure by interfering with PCR primer annealing. Both of these factors can be attributed to IGK inactivation by rearrangements involving the "kappa deleting element" region, affecting both IGK alleles in lambda-restricted plasma cells (Perfetti et al, Immunology, 2004). As an explanation for low plasma cell content in the samples used in this study, we describe how the tumor cell content of bone marrow aspirates decrease gradually in sequential pulls because of hemodilution: from the initial pull used for aspirate smear, to the final pull that is commonly used for research. Supporting the important role of hemodilution, we found V(D)J capture rates of 97 % in clinical samples (early pull aspirates) from our institution that were analyzed with the same NGS assays, as long as the bone marrow plasma cell infiltration was above 5 %. V(D)J capture probability appears to be determined by two factors: The abundance of clonal cells (i.e. tumor cell content), and the degree to which clonal sequences can be amplified by the assay (which is negatively affected by SHM). Thus, increasing the tumor cell content in samples as much as practically possible (i.e., optimal bone marrow aspirates and enrichment of CD138+ plasma cells) may compensate for SHM and improve V(D)J capture rates beyond 95 %. Conclusion V(D)J capture rates of at least 95 % are feasible in multiple myeloma using LymphoTrack® NGS assays, when the sample quality is good. The most important reason for V(D)J capture failure is low tumor cell content due to bone marrow aspirate hemodilution. Optimal performance depends on the use of early pull aspirates and/or subsequent tumor cell enrichment. Figure 1: Predicting V(D)J capture. Regression lines and individual data points are colored according to myTYPE status (red = positive; blue = negative); V(D)J capture probability on the y-axis (capture yes/no on the second y-axis) and bone marrow plasma cell percentage by aspirate smear on the x-axis; split into panels according to light chain restriction (right = lambda; left = kappa). Disclosures Ho: Invivoscribe, Inc.: Honoraria. Arcila:Invivoscribe, Inc.: Consultancy, Honoraria. Jacobsen:Invivoscribe, Inc.: Employment. Huang:Invivoscribe, Inc.: Employment. Miller:Invivoscribe, Inc.: Employment, Equity Ownership. Landgren:Pfizer: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction Multiple Myeloma (MM) pathogenesis is characterised by extensive genetic and clonal evolution with frequent on-treatment progression. To date, most studies have focused on single diagnostic or paired diagnostic relapse biopsies, and the molecular mechanisms eventually resulting in treatment failure are poorly understood. To determine the molecular underpinnings of disease in its most advanced stage, we performed comprehensive genome profiling of 4 patients with extra-medullary metastatic disease. Methods A total of 8 patients with extramedullary myeloma with 188 (median = 22) distinct metastatic lesions were enrolled as part of the medical donation program at MSK. Here, we present results from 4 patients. Patients 1 and 2 had an indolent disease with a total survival of ~10 years whereas patients 3 and 4 had very aggressive disease and 2-3 years survival. All patients had received a sequence of multi-modal myeloma treatments. Targeted gene sequencing using a myeloma specific targeted panel myTYPE was performed in 28 samples from all 4 patients to a median coverage of 667x. Additionally, 6 tumors from patients 1 and 2 were subject to WGS to a median coverage 92x. Results Driver events: Aberrations across all 28 samples sequenced using myTYPE were examined. We found t(4;14) in Patients 3 and 4 across all the metastatic lesions consistent with previous knowledge that these are early initiating events. Besides IGH translocations, we found copy number changes involving 1p-, 1q+ and 13q-. Patient 1 and 2 had 17p- and 8p- shared across all the metastatic lesions. All patients had RAS/RAF pathway mutations and additional mutations were found in FAM46C, TP53 and BIRC3. In WGS, we observed a median SNV, Indel & SV burden of 12150, 1196 & 70.5 respectively. This mutational load is greater than two-fold higher to previously published estimates derived from primary diagnostic samples. Clonal structure using WGS: Clonal phylogeny was constructed using nDirichlet process clustering. Evaluation of mutation and clonal spectra showed evidence of clonal diversification amongst sites but within each sample all mutations had fully clonal cancer cell fractions, i.e. there were no subclones. For Patient 1, the phylogenetic tree was dominated by 9,099 truncal mutations, and 150-462 site specific yet clonal mutations. For Patient 2, the tree was dominated by 8,540 truncal mutations and site specific clonal mutations (n= 356; 1,186). However, evaluation of copy number alterations showed evidence of subclonal emergence of copy number aberrations implicating chromosomes 5, 8,16,18, 20, 21. This suggests that in these patients late stage tumor development and metastatic dissemination is further shaped by accrual of CNAs. Mutational processes using WGS: Signature analysis was performed by deconvolution of observed WGS mutations on the set of mutational signatures reported by Alexandrov et al. Consistent with previous reports, Signature 9 was identified as the dominant mutation signature, contributing to a median of 27% of all mutations in our cohort. Signature 9 is related to AID and has been previously implicated in early myeloma pathogenesis. Whilst canonical IGH translocations were not identified in Patient 1 or 2, Patient 1 showed evidence for chromoplexy with closed chain translocations having breakpoints spanning chromosomes 1, 4, 11, 16, 17, 19 across all 4 sites. Patient 2 presented with localised hypermutation on chromosomes 1, 5 and 22 which are shared between the 2 sites. These results suggest that subsequent clonal sweeps have acted upon the genome since disease initiation. Conclusion Preliminary data from multi-region WGS of the evolutionary end-stage in MM shows a single dominant clone with known driver events in each patient. This is in contrast to the subclonal heterogeneity characteristic of early disease, and presents opportunities for targeted therapies. Our observations are consistent with convergent evolution, where selective pressure from many years of therapy results in a relatively homogenous genomic landscape. A larger cohort of samples will ascertain patterns of biological processes we present here. These early investigations provides new insights on MM pathogenesis and metastatic dissemination. Disclosures Landgren: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Consultancy; Pfizer: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Lesokhin:Genentech: Research Funding; Serametrix, inc.: Patents & Royalties: Royalties; Janssen: Research Funding; Takeda: Consultancy, Honoraria; Squibb: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

Description: Introduction At diagnosis, Multiple Myeloma (MM) is traditionally classified into two clinical and prognostic subgroups groups on the basis of initiating cytogenetic abnormalities: IGH translocations and hyperdiploidy. Currently, these events are clinically ascertained by Fluorescent In-Situ Hybridization (FISH). In recent years, comprehensive genome profiling studies have shown that MM pathogenesis is defined by a spectrum of acquired somatic lesions, many of which are biologically and clinically relevant. To this effect, targeted gene sequencing approaches are becoming routine in the upfront diagnostic settings. Here we present myTYPE, a MM-specific targeted next generation sequencing panel to identify germline and somatic substitutions, indels, Copy Number Aberrations (CNA) and IGH translocations. Methods A multiplex bait panel was designed to capture the exons of 120 genes implicated in MM pathogenesis, entire IGH locus as well as genome wide representation of single nucleotide polymorphisms (SNPs) (1 in 3Mb) to enable detection of arm level copy number events and recurrent focal events. These 120 genes were selected on the basis of 1) frequently mutated and driver genes in MM 2) genes in important signaling pathways, e.g the NFKB pathway 3) treatment targets and candidate genes for drug resistance, e.g. cereblon.To validate the efficacy of the assay, 16 constitutional bone marrow samples and 18 tumor samples were sequenced using myTYPE. For validation, 6/18 tumor/normal pairs sequenced using myTYPE were subject to WGS and remaining 12/18 tumor samples were subject to FISH. After sequencing, we obtained an overall median target coverage of 815x. Results After alignment, substitutions and indels were called using Caveman, Pindel and Strelka. CNAs were identified using Facets and IGH translocations were identified using Delly along with a modified version of BRASS. Below is a description of the genomic abnormalities captured by the myTYPE assay. SNVs and Indels For the 6 tumor/normal pairs sequenced using myTYPE and WGS, we obtained a total of 21 (median = 3) non-synonymous mutations using myTYPE. When limiting the WGS calls to myTYPE targets, we recovered 20/21 non-synonymous mutations identified by myTYPE. These involved SNVs and indels in key MM related drivers including NRAS, KRAS, FAM46C and TP53 among others. For the mutations identified by both myTYPE and WGS, there was a high correlation between the variant VAFs, R2= 0.99 and as expected is better in capturing subclonal mutations. IGH rearrangements and Copy Number Aberrations (CNA) Next we compared myTYPE and WGS results for recurrent CNAs in MM. We specifically looked at deletions of 1p, 13p, 16q, 17p and gains of 1q, 11q and found a 100% concordance of these aberrations identified by both assays. The remaining 12 samples sequenced using myTYPE also had orthogonal FISH. myTYPE identified a total of 7 IGH rearrangements, 4 of which are also reported by FISH. Three additional t(11;14) translocations were uniquely identified by myTYPE in cases that remained clinically uncharacterized. FISH was also used to probe deletions in 17q, 13q, 1p and 1q gain. All aberrations identified by FISH were also identified in myType. Additionally, 13q- in four samples and 1p- in one sample were uniquely identified by myTYPE. Conclusion In summary, we present a targeted assay capable of identifying somatic mutations, CNAs and IGH translocations of prognostic and diagnostic relevance in MM. When compared to conventional assays currently used in clinical practice, myTYPE identified at least one disease defining alterations in all samples screened. Evaluation of sensitivity and specificity will require larger clinical cohorts. Importantly, myTYPE enables comprehensive profiling, large sample multiplexing and short turn around times which renders it as an optimal assay for utilisation in the upfront clinical setting. Disclosures Korde: Amgen: Research Funding. Mailankody:Juno: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Physician Education Resource: Honoraria. Landgren:Pfizer: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Consultancy.

Description: Contact friction between moving components leads to severe wear and failure of engineering parts, resulting in large economic losses. The lubricating ability of the protic ionic liquid, tri-[bis(2-hydroxyethylammonium)] citrate (DCi), was studied as a neat lubricant and as an additive in a mineral oil (MO) at various sliding velocities and constant load on an aluminum–steel contact using a pin-on-disk tribometer. Tribological tests were also performed at different concentrations of DCi. When DCi was used as an additive in MO, friction coefficient and wear volume were reduced for each sliding velocity, with a maximum friction and wear reduction of 16% and 40%, respectively, when 2 wt % DCi was added to MO at a sliding velocity of 0.15 m/s. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) were also applied to analyze the wear mechanism of the interface lubricated by MO and DCi as additive.

Description: Background: The selective AURKA inhibitor alisertib (MLN8237) exhibits disease modifying activity in murine models of myelofibrosis by eradicating atypical megakaryocytes resulting in reduction of marrow fibrosis (Nat Med 2015). Here, we present long term follow-up results from the investigator initiated pilot study of alisertib in patients with myelofibrosis (clinical trials.gov Identifier NCT 02530619). Methods: 24 patients with DIPSS intermediate 1, intermediate-2, or high risk myelofibrosis who were in need of therapy, refractory/intolerant or unlikely to respond to JAK inhibitors with neutrophil count ≥ 1 x109/L, and platelet count ≥ 50 x109/L, received alisertib (provided by Millennium Pharmaceuticals Inc) at a dose of 50 mg twice daily for one week every 21 days. Toxicity assessment was performed by the standard common terminology criteria (Version 4.0). Response was assessed by the international working group for myelofibrosis research and treatment (IWGMRT) criteria. Correlative studies included assessments of JAK2V617F, CALR, and MPL mutant allele burden, degree of fibrosis and GATA1 expression in bone marrow samples obtained pre and post therapy. Results: We enrolled 17 patients with primary myelofibrosis, 4 with post essential thrombocythemia myelofibrosis and 3 with post polycythemia vera myelofibrosis. Median age was 72 years with 66% males. 79% of patients were DIPSS intermediate risk, and the remainder were high risk with 15 patients (62.5%) having received prior JAK inhibitor therapy. Driver mutational status was as follows; 58% JAK2V617F, 29% CALR, and 13% MPL mutated. At study entry, 54% of patients demonstrated palpable splenomegaly ≥ 5 cm below the left costal margin, 54% were transfusion dependent with all patients experiencing constitutional symptoms. At the time of data cut-off, patients received a median of 7.5 cycles (range; 1-29 cycles) of therapy. The 7 patients presently on study have received a median of 23 cycles (range; 8-29 cycles). Reasons for treatment discontinuation included progressive disease/lack of response in 11 (65%) patients, toxicity in 4 (24%) patients and refusal of further therapy in 2 (11%) patients.Safety and Efficacy assessments The most common treatment-emergent grade 3/4 adverse events included neutropenia (42%), thrombocytopenia (29%) and anemia (21%), with 4% each experiencing neutropenic fever, diarrhea, vertigo, elevated creatinine and elevated alanine aminotransferase. 22 patients were considered for response evaluation with 4 of 14 patients (29%) with palpable splenomegaly ≥ 5 cm achieving a spleen response, 1 of 13 patients (8%) becoming transfusion independent, and 5 of 22 patients (23%) experiencing symptom response with ≥ 50% reduction in the MPN-SAF total symptom score. However, when response assessment was restricted to 13 patients who had received a minimum of 5 cycles of therapy, spleen responses were observed in 4 of 7 (57%) patients, 1 of 5 (20%) achieved transfusion independence and 5 of 13 (38%) achieved symptom response. All patients presenting with leukocytosis (n=4) and thrombocytosis (n=2) had resolution with therapy. Of the 7 patients presently on study, four patients continue to demonstrate symptom response, two patients with both spleen and symptom response, and another patient with sustained anemia response. Correlative assessments We compared the intensity of staining of GATA1, a factor that is required for maturation, in sequential bone marrow biopsies from six patients at baseline and after a minimum of five cycles and observed a striking increase in the numbers of GATA1-positive megakaryocytes in five of six cases (Figure 1a). In addition, we observed a one grade reduction in marrow fibrosis in 4 of 6 paired samples (Figure 1b). This reduction in fibrosis was accompanied by sustained responses to the drug. Finally, we compared JAK2, MPL or CALR mutant allele burden in eight paired baseline and cycle 5 or 6 samples and observed decreases in 4 of 8 patients (Figure 1c). Conclusions: Alisertib is safe and well tolerated in patients with myelofibrosis with prolonged administration up to 1.7 years. In addition to providing clinical benefit, alisertib restored normal morphology and GATA1 expression in atypical megakaryocytes and reduced marrow fibrosis and mutant allele burdens. These findings demonstrate that AURKA inhibition should be further explored as a therapeutic option in myelofibrosis. Figure 1. Figure 1. Disclosures Swords: AbbVie: Employment. Watts:Jazz Pharma: Consultancy, Speakers Bureau; Takeda: Research Funding. Frankfurt:Celgene, Jazz, Agios: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees. Altman:Cyclacel: Other: payment to the institution to conduct clinical trial work; Epizyme: Other: payment to the institution to conduct clinical trial work; Ariad: Other: payment to the institution to conduct clinical trial work; Bayer: Other: payment to the institution to conduct clinical trial work; Celator: Other: payment to the institution to conduct clinical trial work; FujiFilm: Other: payment to the institution to conduct clinical trial work; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: payment to the institution to conduct clinical trial work; Agios: Other: Payment to the institution to conduct the trial ; Astellas Pharma: Other; Genetech: Other: Payment to the institution to conduct clinical trial work; Syros: Membership on an entity's Board of Directors or advisory committees; Incyte: Other: payment to the institution to conduct clinical trial work; GSK: Other: payment to the institution to conduct clinical trial work; Immune Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Boeringer Ingelheim: Other: payment to the institution to conduct clinical trial work; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: payment to the institution to conduct clinical trial work. Rampal:Celgene: Honoraria; Stemline: Research Funding; Incyte: Honoraria, Research Funding; Constellation: Research Funding; Jazz: Consultancy, Honoraria. Giles:Actuate Therapeutics Inc: Employment, Equity Ownership. Crispino:Forma Therapeutics: Research Funding; Scholar Rock: Research Funding.