ALBERT

Description: Introduction - AML is a complex group of malignancies, with heterogeneity in morphology, cytogenetics, molecular characteristics, aggressiveness and importantly, in its response to treatment and survival outcomes. Next generation sequencing by the Cancer Genome Atlas Research Network analysed 200 primary AML cases and identified 23 genes that display recurrent somatic mutations at varying frequency in AML (NEJM 368(22):2059-2074). Defects in DNA repair are frequently identified in treatment-related AML and inherited mutations in genes of DNA repair pathways predispose patients to myeloid malignancies. For example, biallelic mutations in FANC genes, which cause the recessive heritable bone marrow failure syndrome Fanconi Anaemia (FA) are associated with high risk of progression to AML and other cancers (Kutler et al.Blood, 101:1249-1256), suggesting a potential involvement of FANC gene mutations in AML pathogenesis. Methods - In this study we present a two-stage approach to gene discovery in AML: initial unbiased whole genome sequence (WGS) and whole exome sequence (WES) analysis of tumour DNA from a cytogenetically normal AML case at diagnosis and relapse, and corresponding germ-line DNA (prepared from mesenchymal stromal cells). Potential oncogenic mutations and changes associated with disease progression were identified. WES of a further 96 diagnostic AML samples further defined recurrent mutations and allowed identification of affected functional groups and networks in AML. Results – WGS and WES were performed on diagnosis, non-haematopoietic and relapse samples from an index AML patient. Somatic SNVs and indels unique to the tumour samples include a number of variants in genes previously reported as recurrently somatically mutated in AML including FLT3, WT1 and IDH2. Somatic mutations in genes not previously associated with AML were also identified including a mutation in FANCD2 (p.S1412N) present in the index AML tumour DNA at diagnosis and at relapse. Variants in genes recurrently mutated at low frequency in AML can also be disease drivers, however separating such genes from the background level of mutation in AML requires analysis across multiple samples, and sequencing studies to determine recurrence and/or mutations in proteins involved in the same functional pathway or complex. STRING-db v9.05 (Franceschini et al. NAR, 2013(41), Database issue) was used to identify a larger network of proteins, including and associated with the FANC genes, involved in homologous recombination-mediated DNA repair. Known somatic mutations from other AML studies were mapped onto this network; as shown in Figure 1 multiple genes in this extended network are affected by somatic mutation in AML suggesting a potential role in pathogenesis. Analysis of our WES data from diagnosis samples from a further 96 Australian AML cases identified an additional two somatic mutations in genes from the extended STRING-db v9.05 FANC network. In total we identified 18 mutations in the 16 classified FANC genes and 8 variants in the BLM complex as shown in Figure 2. Two of the germline FANC gene mutations, FANCM-Q13333fs and FANCD2-R926X, are known pathogenic mutations in FA. Patients with mutations in the 8 FANC genes of the core complex form a distinct subset from those with mutations in the other 8 FANC genes. 5 of the 8 patients with mutations in the BLM complex also form a separate group while BLM complex mutations are present in 2 patients that also have FANC mutations. For the two patients with acquired changes the allele frequency for these FANC mutations is greater than 25% suggesting an early origin in disease. Discussion. Our findings suggest that germline and somatic mutations affecting function of the FANC DNA repair pathway may be a recurrent abnormality in AML, potentially contributing to leukaemogenesis. FANC/BLM gene mutations frequently co-exist with mutations in DNMT3A and DNMT1; 46% of the patients with DNMT3A/DNMT1 mutations are also mutant for FANC or BLM complex genes representing significant over-representation (p = 0.021). Within the group of FANC and BLM patients there is also significant under-representation of FLT3-ITD mutations and mutations in N-RAS and K-RAS (p = 0.051), raising the possibility that defects in homologous DNA repair may favour cooperation with alternative signalling pathways. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Abstract LBA-3 Mutations in the transcription factor genes, RUNX1 and CEBPA, can lead to an autosomal dominant familial predisposition to MDS/AML. Using a candidate gene approach, we have detected domain specific heterozygous mutations in the GATA2 gene in 4 MDS/AML families which predispose to MDS/AML. The same novel heterozygous T354M missense mutation was observed in 3 families and a 355delT mutation in 1 family, all with multigenerational transmission of MDS and/or MDS/AML. Importantly, these genetic variants segregate with all affected members in each of the families. The 2 mutated threonine residues are in 5 consecutive highly conserved threonine residues at the DNA-binding, protein-protein interacting second zinc finger (ZF2) of GATA2. Neither these mutations, nor any other variants in the GATA2 coding sequence, were seen in a population screen of 695 normal individuals. Haplotype analysis suggests that the T354M mutation has multiple ancestral origins. While mutations in RUNX1 and CEBPA, can also lead to familial predisposition to MDS/AML, these patients with GATA2 mutations are unique in that there is no obvious pre-MDS or pre-leukaemic phenotype such as thrombocytopenia (RUNX1) and eosinophilia (CEBPA) in predisposed carriers. Most patients in these families have had a rapid disease course “appearing out of the blue” leading to death, with a variety of ages of onset from teenagers to early 40s. Yet remarkably, there are still asymptomatic carriers in their 60s. One of these carriers, and his 2 children, has had bone marrow prophylactically stored over 15 years ago in case of disease onset. No pathogenic GATA2 coding sequence changes were found in 268 sporadic MDS/AML patient samples. Additionally, GATA2 mutations were not found in germline samples from 35 other families predisposed to AML and various other hematological malignancies. Both the T354M and 355delT mutants appear to localize appropriately to the nucleus and maintain at least some DNA binding in electrophoretic mobility shift assays. We used the known murine Gata3 ZF2 structure bound to DNA to model the effects of the observed mutations and demonstrated that the T354 residue does not contact DNA but makes polar contact with the adjacent threonines, and via its amino group, with C349 which coordinates the zinc atom. Replacement of the T354 side-chain with the bulky methionine moiety may affect the zinc contacts and is predicted to alter the overall structure of this ZF2. In contrast, 355delT will shorten the conserved threonine string which is predicted to impact on the orientation and position of L359 which directly contacts DNA. Thus, 355delT is likely to have an effect on DNA binding. Luciferase reporter assays indicate that T354M and 355delT greatly reduce the transactivation ability of GATA2 on multiple response elements, impacting on downstream target genes such as RUNX1 and CD34. Of note, T354M shows a markedly lesser synergistic effect than wildtype (WT) GATA2 with PU.1 on the CSF1R promoter. Competition assays show that these mutations may be acting in a dominant negative fashion in some biological contexts. In stable promyelocytic HL-60 cell lines expressing regulatable GATA2 (WT or T354M), T354M allows proliferation to proceed even under stimulus to differentiate with all-trans retinoic acid. Microarray studies indicated that the down regulation of proapoptotic BCL-xS by T354M, but not WT, may be responsible for this phenotype. GATA2 is considered to be a hematopoietic “stemness” gene, highly expressed in haematopoietic stem cells and is required for megakaryocyte and mast cell production. GATA2 is down regulated during myeloid differentiation and forced overexpression prevents such differentiation. Discovery of GATA2 mutants in MDS/AML predisposed families provides new tools for probing the mechanism of GATA2 induced leukemogenesis, and possibly also for clarifying its role in maintenance of stemness. Our findings highlight the power of investigating familial predispositions to cancer identifying specific mutations with unique biological effects. They have immediate implications for diagnostic genetic testing, and longer term therapeutic implications through identification of drugable biological pathways such as apoptosis. The poor outcome associated with these mutations may suggest that an aggressive strategy is appropriate in the treatment of affected individuals in families found to be carrying GATA2 mutations. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: Prognosis for multiple myeloma (MM) patients has improved dramatically since the introduction of novel therapies. However, prognosis in the t(4;14) subgroup, characterized by expression of the histone methyltransferase MMSET, remains relatively poor due to the acquisition of a highly aggressive, motile and invasive phenotype and a generally modest response to therapy. We have previously demonstrated that MMSET promotes an epithelial-to-mesenchymal transition (EMT) in prostate cancer1. While the term EMT is not commonly used to describe MM, we hypothesise that an EMT-like process plays a critical role in t(4;14)­-positive MM disease pathogenesis. In this study, we conducted a comprehensive evaluation of the association between t(4;14) and an EMT-related gene expression signature in MM and identify N-cadherin as a therapeutically-targetable EMT-related gene in t(4;14)-positive MM. Methods and results: Expression of a core EMT-related signature, comprising 169 mesenchymal genes and 49 epithelial genes, was assessed in CD138-selected MM plasma cells from newly-diagnosed MM patients in four independent microarray datasets (E-GEOD-19784 [n = 327 MM patients], E-GEOD-26863 [n = 304], E-MTAB-317 [n = 226] and E-MTAB-363 [n = 155]), accessed through ArrayExpress (EMBL-EBI). In each dataset, gene expression was compared in t(4;14)-positive and t(4;14)-negative patients using linear models for microarray data (LIMMA). Twenty six mesenchymal genes were found to be upregulated in t(4;14)-positive patients across the 4 datasets (p 〈 0.05, Fisher’s method). A general loss of epithelial genes was not observed, likely due to the haematopoietic origins of MM. Upregulated genes included key EMT drivers (TWIST1, SOX9, TCF4) and genes associated with the cytoskeleton (VIM), adhesion and migration (CDH2, ITGB1, NCAM1) and signalling pathways involved in EMT (BMPR1A, IL6R, TGFB2). The t(4;14)-mediated regulation of EMT-related genes including TWIST1, CDH2, BMPR1A and ITGB1 was confirmed by assessing the effects of MMSET knockdown, knockout and add-back in the t(4;14)-positive human myeloma cell line KMS-11. We have previously demonstrated that N-cadherin (CDH2) expression is elevated in plasma cells from approximately 50% of newly diagnosed multiple myeloma (MM) patients and that elevated serum N-cadherin is associated with poor prognosis2. In order to identify whether N-cadherin is a potential therapeutic target in t(4;14)-positive myeloma, the effects of an N-cadherin peptide inhibitor ADH-1 (Exherin™) or shRNA-mediated N-cadherin knock-down were assessed on MM PC adhesion and proliferation in vivo and in vitro. The role of N-cadherin in MM tumour establishment and intramedullary growth was investigated using the C57BL/KalwRijHsd mouse model of MM. In this model, intravenously injected luciferase-expressing mouse MM PC cells (5TGM1-SFG) home to the bone marrow and initiate systemic MM disease. C57BL/KalwRijHsd mice bearing 5TGM1-SFG N-cadherin knock-down cells had significantly reduced tumour burden, as assessed by bioluminescent imaging and serum paraprotein levels, after 4 weeks compared with mice bearing control 5TGM1-SFG cells. Furthermore, daily intraperitoneal ADH-1 administration (100 mg/kg/day) to 5TGM1-SFG cell-bearing mice significantly significantly decreased tumour burden when administered at the time of tumour inoculation, but had no effect on established tumours. These results suggest that N-cadherin plays a role in extravasation, homing and/or tumour establishment in vivo. Conclusion: This study identified an extensive EMT-like gene expression signature driven by MMSET in t(4;14)-positive MM patients. This provides insight as to how the t(4;14) translocation leads poor prognostic outcomes in up to 20% of MM patients. Furthermore, these studies demonstrate a potential role for N-cadherin in MM tumour dissemination in t(4;14)-positive MM and suggest that N-cadherin represents a novel candidate for therapeutic targeting in these patients. 1Ezponda et al. Oncogene, 2013 2Vandyke et al. Br J Haematol, 2013 Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Early molecular response (EMR, BCR-ABL (IS) ≤ 10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early 3 months may be too late for effective therapeutic intervention. Thus, alternative approaches are required to identify poor responders at the time of diagnosis. The aim of this study was to identify plasma biomarkers at diagnosis that will predict for subsequent EMR failure, early transformation or the development of BCR-ABL1 kinase domain mutations. Cytokine profiling has proven valuable in identifying prognostic factors in myelofibrosis and myelodysplastic syndromes; however, similar comprehensive studies are lacking to date in CML. Methods: Plasma samples from CP-CML patients enrolled to the TIDEL II trial were collected prior to starting imatinib treatment (n=186) and after 6 months on TKI (n=17); and compared to those of healthy donors (n=19). The levels of 39 cytokines, chemokines and growth factors (CC&GF) were measured using a Luminex multiplex assay. To identify potential biomarkers to predict EMR failure, random forest analysis and recursive partitioning techniques in R were applied as statistical methods. Results: Plasma concentrations of 13/39 CC&GF were significantly elevated at CP-CML diagnosis compared to healthy donor samples. Most (EGF, bFGF, VEGF, TGF-α, CXCL1, CCL4, sCD40L and IL-4) werenormalized after 6 months of TKI treatment while others (TNF-α, sIL-2Ra, IL-8, IL-10, IL-1a) remained at higher levels, possibly reflecting persistent disease-induced alterations within the microenvironment. A third subset of CC&GF, such as CCL2, CCL3 and CCL22, showed higher circulation levels only in TKI-treated patients but not at diagnosis, suggesting that changes in these CC&GF could be treatment-related. 183/186 patients had BCR-ABL1 assessments available at 3 months, and 23/183 (13%) did not achieve EMR. Random forest analysis identified TGF-α, IL-6 and IFN-α as the most important CC&GF associated with EMR failure. Recursive partitioning incorporating these three variables produced a classification tree based only on TGF-α and IL-6, and demonstrated that 12/20 (60%) of patients who were TGF-αhi/IL-6hi failed to achieve EMR (Table 1). Importantly, this group contained 3/3 (100%) patients who transformed within the first 12 months of TKI treatment. Both TGF-α (7.99 vs. 60.57 pg/ml, p

Description: Abstract 3540 Background: GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress, coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses. GADD45A expression is normally rapidly induced in response to radiation and cytotoxic drugs1 and ectopic expression of GADD45A in the M1 leukemic cell line sensitises cells to stress-induced apoptosis in response to a range of genotoxic agents.2 Silencing of GADD45A by promoter methylation is a hallmark of many tumors,3–5 however to date there have been no investigations to determine whether this is associated with response to therapy. In AML, we have shown GADD45A expression is broadly down-regulated6 and here we investigate the mechanism of GADD45A repression in AML and its clinical significance. METHODS: We analysed 131 diagnostic bone marrow mononuclear cell samples from a retrospective cohort of patients with de novo AML. 93 of these patients were treated with induction chemotherapy. Patients 60 years or under were treated with chemotherapy regimens containing idarubicin and high dose cytarabine, and patients older than 60 received idarubicin and standard dose cytarabine. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (Sequenom MassARRAY) to analyze methylation of 4 GADD45A promoter CpG dinucleotides previously shown to be associated with silencing of GADD45A in breast and prostate cancer.4,5 We determined association of CpG hyper-methylation with outcome in our treated patient cohort. For AML cells with GADD45A hyper-methylation, and for those with normal levels of GADD45A promoter methylation, we also determined the response to cytotoxic agents in vitro in the presence and absence of hypo-methylating agents. RESULTS: We observed hyper-methylation of the 4 CpG residues in the proximal promoter of GADD45A in 49 of 131 (37%) de novo AML patients and in 6 AML cell lines. Multivariable analysis showed that methylation of a single CpG (CpG1) was an independent predictor of poor survival in AML overall (median survival 281 days versus 794 days, HR 2.25, p=0.009), in normal karyotype AML (NK-AML) (median survival of 281 versus 793 days, HR 5.77, p=0.03, 〉250 days), and in the elderly patient group (〉60 years, median survival of 218 versus 392 days, HR 2.64, p=0.03)(see Figure 1). Additionally, treatment of AML cell lines and patient blasts with decitabine resulted in selective induction of GADD45A mRNA in samples with GADD45A hyper-methylation, and this was associated with increased sensitivity to daunorubicin. CONCLUSIONS: DNA methylation of the GADD45A proximal promoter is an independent predictor of poor outcome particularly in AML patients with normal karyotype and in the elderly group. Our biological data shows that induction of GADD45A mRNA expression with decitabine in hyper-methylated samples is associated with increased response to cytotoxic agents. Thus GADD45A promoter CpG methylation represents a new biomarker that may provide prognostic information in the heterogeneous NK-AML group, and in elderly patients. Given that recent trials are combining Azacitidine with chemotherapy and other agents for initial induction treatment of AML9, this may represent a marker to help define patients that will benefit from this approach. Elderly patients with GADD45A hyper-methylation may benefit from treatment with hypo-methylating agents which are associated with less toxicity (see Refs 7,8). Disclosures: Wei: Celgene: Honoraria, Research Funding.

Description: Abstract 2396 Background: Despite recent advances in understanding the key molecular mechanisms of leukemogenesis, the outcome for patients with Acute Myeloid Leukemia, particularly with a normal karyotype, remains poor. For this large group of patients, genetic alterations in genes such as FLT3, NPM1, CEBPA, IDH1/2, and DNMT3A provide useful prognostic information. However, risk stratification of this group remains only partially resolved and markers of response that can be therapeutically targeted would likely improve outcome for these patients. GADD45A is a tumor suppressor gene that plays cell-type dependent roles in cellular stress coordinating DNA repair and de-methylation, cell cycle arrest, and pro-apoptotic or pro-survival responses (Cancer Ther. 2009;7:268). Methylation of four discrete CpG residues in the proximal promoter of GADD45A is a hallmark of many solid tumours and has been associated with impaired cell stress signalling and reduced drug response (Cancer Res. 2009;69:1527; Oncogene. 2005;24:2705). In AML, GADD45A expression is broadly down-regulated both in normal karyotype and other cytogenetic classes. Down-regulation of GADD45A in AML has been associated with FLT3-ITD (Leukemia. 2009;23:729) and RUNX1 mutations (Satoh et al, Leukemia. 2011;Epub). For those patients without these mutations, the mechanism of GADD45A down-regulation and its prognostic significance remains unknown. We hypothesised that the promoter of GADD45A is methylated in AML and that this methylation is functionally important in patient response. Methods: Using the Sequenom MassARRAY methodology we screened for methylation of four GADD45A promoter CpG dinucleotides (CpG1–4) previously shown to be associated with silencing of GADD45A in breast and prostate cancer, in a retrospective cohort of 222 AML patients collected at diagnosis from the Royal Adelaide Hospital. We then determined association of CpG methylation with outcome and mutation status in our treated patient cohort of 167 patients. In AML cell lines and in primary patient samples we also determined the response to cytotoxic agents in vitro in the presence and absence of demethylating agents. Results: We observed hypermethylation of the CpG1–4 in the proximal promoter of GADD45A in 93 of 222 (42%) of AML patients and in 6 AML cell lines. In the 167 patients treated with standard induction chemotherapy regimes, 61 patients showed methylation of the GADD45A proximal promoter. Of the four CpG residues, methylation of CpG1 was associated with poor overall and event-free survival, in AML overall (Figure 1A) and in normal karyotype AML (Figure 1B). GADD45A CpG1 methylation was significantly associated with IDH1 and IDH2 mutations (p

Description: Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34+CD38−CD123+ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

Description: Introduction: Tyrosine kinase inhibitor (TKI) resistance remains a major impediment to successful treatment of patients with chronic myeloid leukemia (CML). The 3rd generation TKI ponatinib (Pon), has been demonstrated to successfully overcome BCR-ABL1 kinase domain (KD) mutation based resistance including BCR-ABL1T315I, which inhibits the binding of all other available TKIs. Pon has demonstrated efficacy in CML patients who acquired resistance to other TKIs. However, little is known about the mechanisms which result in resistance to Pon. Here, modes of Pon resistance have been investigated invitro in both TKI naïve and treated BCR-ABL1+ cell lines. Methodology and Results: Pon resistance was generated in 4 BCR-ABL1+ cell lines 1) K562, 2) K562 DOX (increased ABCB1) 3) K562 DOX 55D –resistant to 55 nM dasatinib (Das) and 4) K562 T315I – T315I mutation developed in the presence of Das. Both Das treated cell lines have 3-4 fold higher expression of BCR-ABL1 mRNA compared to control lines. The K562 T315I line contains 44% T315I mutation whereas no KD mutation was found in the other lines. Parental Pon naive controls were maintained in parallel. Resistant cell lines (Table 1) were established by exposing the above cell lines to increasing concentrations of Pon for 660-900 days. The K562 T315IR resistant line demonstrated a 〉17 fold increase in IC50Pon compared to naïve control. While no expression of ABCB1 or ABCG2 was observed, BCR-ABL1 transcript level was raised 7 fold with a concomitant 4 fold increase in pBcr-Abl by western blot. In addition, the level of T315I in the resistant line increased from 44% to 81%, suggesting that increasing T315I% together with Bcr-Abl upregulation were the major resistant mechanisms in this line. The K562 DOX 55DR line demonstrated transient increases in BCR-ABL1 that reduced to basal levels concomitant with the development of the compound mutations G250E (71%) and E255K (64%). The IC50Pon was increased 47 fold compared to control, and high Das and imatinib (Im) IC50s confirmed the resistance to other TKIs (IC50Das: 1350 nM and IC50Im: 260 μM). In contrast, the Das naïve K562R and K562 DOXR resistant lines did not have demonstrable KD mutations. While 7AAD and annexin V staining confirmed Pon resistance, the IC50Pon was not higher in K562R and only 2 fold higher in K562 DOXR compared to controls, suggesting resistance to Pon was likely mediated by a Bcr-Abl independent mechanism. This was supported by a 2 fold reduction in pBcr-Abl and pSTAT5 in the Pon resistant lines compared to controls. Therefore, the expression levels of alternative pathway molecules were examined to determine possible mechanisms. Axl, a receptor tyrosine kinase has been previously associated with TKI resistance. A 7 fold increase in AXL mRNA was found in K562R line, and the K562 DOXR line demonstrated increased Axl protein. Importantly, after 72 hours incubation with Pon and the Axl inhibitor, R428, the viability of the two resistant lines were significantly reduced (Fig 1), suggesting Axl is likely critical in mediating Pon resistance. Furthermore, increased Axl was associated with increased cell adhesion in both lines, and adherence is a hallmark of Axl expression. Conclusion: As Pon binds to native Bcr-Abl stronger than to Bcr-AblT315I, an increased proportion of T315I reduced sensitivity to Pon in vitro. Pon exposure in a cell line predisposed to KD mutations resulted in the development of the compound mutations G250E and E255K, which rendered the cells insensitive to all tested TKIs. Interestingly the mode of resistance in the two Das naïve lines appears Bcr-Abl independent, and is likely mediated by Axl. While limited to in vitro studies these findings suggest that in the setting of prior TKI exposure, mutations are likely to be a primary form of Pon resistance. In the TKI naïve setting Bcr-Abl independent modes of resistance may be more likely suggesting combination therapeutic approaches may be warranted to overcome or prevent Pon resistance in the high risk setting. Abstract 4515. Table 1: Summary of resistance mechanisms detected in 4 cell lines. Pon culture condition (nM) Pon resistance Bcr-Abl dependent/ independent BCR-ABL1 mRNA(fold ↑ ) pBcr-Abl & pSTAT5(fold ↑ ) KD mutation IC50Pon(fold ↑ ) AXL mRNA(fold ↑ ) ↑ cell surface Axl Adherence K562 T315IR 100 DP 7 4 T315I 〉17 5 ✖ ✖ K562 DOX 55DR 200 DP 1 1 G250E E255K 47 ✖ ✖ ✖ K562R 200 IDP 1 0.5 ✖ ✖ 7 ✖ ✓ K562 DOXR 200 IDP 3 0.5 ✖ 2 ✖ ✓ ✓ ✓= yes; x = no; ↑=increased; DP = dependent; IDP = independent. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. White:Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Ariad: Research Funding.

Description: Multiple myeloma (MM) is a malignant neoplasm of plasma cells in bone marrow. Daratumumab (Dara) is a CD38-directed antibody which is Food and Drug Administration (FDA) approved for treatment of patients with MM. Dara is recommended by the National Comprehensive Cancer Network (NCCN) guidelines, which many community oncologist use as the standard of care guidelines. Therefore, we consider it important to analyze Dara use and effects in the real-world community Oncology clinic setting. We evaluated the survival outcomes, both progression-free survival (PFS) and overall survival (OS), as well as the time to progression (TTP) of MM and common adverse events (ADEs) seen in patients at a single site, Monter Cancer Center in the Northwell Health (NH) system. This is a single group study evaluating outcomes of patients with MM who received Dara at NH. Primary objectives include evaluating OS, defined as time from initiating Dara therapy to time of death from any cause. Secondary objectives include PFS, time from initiation of Dara therapy to documented disease progression. We also plan to evaluate time to next treatment (TTNT), TTP and rate of ADEs within 30 days of initiating Dara. This is a retrospective study of patients with MM who received Dara at Monter Cancer Center, Northwell Health Cancer Institute. Basic demographics; gender, age at diagnosis, body mass index (BMI), and ethnicity, were collected. Treatment information collected included, previous number of therapies, dose of Dara in total and per body weight in kg, number of doses of Dara received. Data was collected using electronic medical records from January 2015 through December 2019. Patient charts were reviewed, collecting data from the time of diagnosis and initiation of Dara to date of last known follow-up, or death. Progression of disease information and initiation of new therapy was collected during this time frame. Standard methods of survival analysis were used to analyze the primary objectives. Kaplan-Meier estimate was used to analyze PFS and OS at 6, 12, 24, and 36 months. TTNT was defined as length of time from the date of initiation of Dara to the date the patient was started on a new therapeutic regimen. TTP was defined as the date the patient started Dara therapy to the date of documentation of disease progression or relapse. Cumulative incidence rates were calculated to determine the rates of incidence of next treatment in the presence of competing risk of death, and rate of ADEs in patients during this time frame. Rate of ADEs were reported with corresponding 95% confidence intervals using a binomial confidence interval. A total of 50 patients were included in the analysis. Baseline demographics are in line with published data, mean age was 68.8 years, with a non-Caucasian predominance of 68% to 32% Caucasian. The gender profile was 42% male. Prior mean number of therapies was 2.9, with a median of 2. Dose of Dara used was in line with the recommended dosing with mean 16.3mg/kg dosing. The mean number of doses received were 17. Similar to prior published data the predominant heavy chain MM phenotype was IgG at 48%. The OS, at 36 months was 54%, where 23 patients (46%) had documented date of death during the 5-year time span reviewed. Thirty-one patients (62%) experienced disease progression (TTP), ranging from 28 to 1047 days from start of Dara treatment, with a median TTP of 317 days. The median PFS was 436 days (95% CI: 334-676 days). There were 46% patients that continued on another line of therapy post Dara , with the TTNT ranging from 25 to 541 days, median 175 days from initial Dara therapy. Among patients who had at least one adverse event, the most frequently occurring pattern was fatigue, pyrexia, and chills at 22%, followed by infusion related reactions at 20%. Dara has shown strong clinical efficacy clinical trials. The patients in clinical trials are often not reflective of real-world clinical patients. This study evaluates patients at a single cancer center in the NH system. The data reinforces the efficacy and tolerability of Dara in the real-world setting. It also depicts the difference in OS, PFS, TTP, TTNT and ADEs in the real-world setting. The data suggests Dara outcomes in community-based oncology centers vary from clinical trial data. However, it does indicate similar improved benefits in OS, PFS and TTNT compared with other retrospective studies at other centers. Further prospective studies would be beneficial in evaluating this real-world impact. Disclosures No relevant conflicts of interest to declare.