Abstract: Purpose: Classical introduction therapy of etoposide combined with cytarabine and daunorubicin (DAE) is commonly applied in childhood acute myeloid leukemia (AML), but etoposide has an increasing risk of secondary cancer. In this study the non-inferiority effect of homoharringtonine (H) versus Etoposide was compared in induction phase for Chinese childhood AML treated by CCLG-AML 2015 protocol. Patients and Methods: The total of 818 childhood AML patients (median age of 80 months; range from 1 to 193 months) from CCLG-AML 2015 study group (40 centers) were randomly allocated to two induction arms of DAE and DAH. During the course of induction I, 467 patients in DAE group received daunorubicin and cytarabine (DA) plus etoposide (D: 40 mg/m2 per day on days 1, 3 and 5; A: 100 mg/m2 every 12 hours from day 1 to 7; E: 100 mg/m2 per day from days 1 to 5), and 351 patients in DAH group received the same DA does plus homoharringtonine ( H: 3 mg/m2 per day from days 1 to 5). During the course of induction II, Idarubicin (10 mg/m2 per day on days 1, 3 and 5) was used to instead of daunorubicin, and patients accepted corresponding IAE or IAH treatment. All patients were divided into standard, intermediate or high risk group (SR, IR or HR group) according to CCLG-AML 2015 regimen (table 1). They were assessed by bone marrow (BM) aspiration and morphologically defined complete remission (CR: blasts ≤5%), partial remission (PR: blasts between 6~19%), or non-remission (NR: blasts ≥20%) on days 28 of induction. Results: DAH/IAH group showed non-inferiority for remission rates both in induction Ⅰ (DAE 70.2% vs DAH 76.6%, P = 0.041) and induction Ⅱ (IAE 79.4% vs IAH 87.7%, P = 0.016). Total CR rate at end of induction Ⅰ reached 73.0% and it didn't differ between DAE and DAH group for IR or HR group (IR group: DAE, 73.9% vs. DAH, 77.3%, P = 0.529; HR group: DAE, 53.9% vs. DAH, 62.6%, P = 0.128). But for SR group, CR rate of DAH group is significantly higher than DAE group (DAE, 85.1% vs. DAH, 95.1%, P = 0.013). It has similar results after induction Ⅱ. Total CR rate reached 83.1% and all patients has almost gained CR/PR for SR or IR group, only 2 patients still couldn't obtain remission. There was no significant difference in SR or IR group between two arms, but for HR group, CR rate significantly increased in those who accepted IAH chemotherapy (SR group: IAE, 91.2% vs. IAH, 95.0%, P = 0.398; IR group: IAE, 87.1% vs. IAH, 92.5%, P = 0.275; HR group: IAE, 66.1% vs. IAH, 78.8%, P = 0.050). Conclusion: Homoharringtonine is an effective cytotoxic drug and DAH regimen showed non-inferiority induction effect compared with classical DAE regimen in childhood AML, especially for patients of standard risk group. Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction: Bruton's tyrosine kinase (BTK) is a downstream intermediary of B cell receptor (BCR) signaling. As revealed by ibrutinib, disruption of BCR signaling results in significant anti-tumor activity in various B cell malignancies. BGB-3111 is a potent, specific and irreversible BTK inhibitor. In biochemical and cellular assays, BGB-3111 was more selective than ibrutinib for BTK vs. EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, BLK and TEC. Activity against these other kinases is implicated in ibrutinib-associated toxicities such as diarrhea, bleeding, and atrial fibrillation. In preclinical animal studies, BGB-3111 demonstrated superior oral bioavailability, achieving higher exposure and more complete target inhibition in the tissues than ibrutinib. We report here the initial results of an ongoing phase 1 trial of BGB-3111 in patients (pts) with advanced B cell malignancies. Patients/Methods: This first-in-human, open label phase 1 study comprised a dose-escalation (DE) component, followed by an ongoing safety, schedule and efficacy expansion component. The results of the planned interim analysis performed at the end of DE are reported here. During DE, pts with relapsed or refractory World Health Organization (WHO) classification defined B-lymphoid malignancies were enrolled to 1 of 5 dose cohorts of BGB-3111 (40, 80, 160, 320mg PO QD; 160mg PO BID) in a modified 3+3 dose escalation design. Adverse events (AEs) were reported per CTCAE v4.03 (patients with baseline cytopenias remained evaluable for neutropenia and thrombocytopenia) and responses per histology-specific standard criteria (NHL IWG criteria 2014; modified CLL IWG criteria 2015; WM IWWM criteria 2013). BGB-3111 pharmacokinetics (PK) was analyzed by dose level, and BTK occupancy determined using an irreversible binding assay in PBMCs. Results: 25 pts were enrolled in DE: 40mg (n=4), 80mg (n=5), 160mg (n=6) and 320mg (n=6) QD, and 160mg BID (n=4). Pts had received a median 2 (range: 1-7) prior therapies, for diagnoses listed in Table 1. As of 30 July 2015, all were evaluable for AE and response. BGB-3111 exposure increased in a dose-proportional manner from 40mg to 320mg daily. The Cmax and AUC0-24h of BGB-3111 at 80mg QD was comparable to that reported for ibrutinib at 560mg QD, and the free drug concentration of BGB-3111 at 40mg QD was comparable for that reported for ibrutinib 560mg QD. Sustained 24 hour BTK occupancy in PBMCs was demonstrated in all pts at 40mg QD (24 hour BTK occupancy 98.6 +/-1.1%), and at all higher dose levels. No dose limiting toxicities (DLT) were encountered, and the maximum tolerated dose (MTD) was not reached. The recommended phase 2 dose (320mg daily) was determined based on the pharmacokinetics, pharmacodynamics, safety and efficacy of BGB-3111. Three pts discontinued BGB-3111 due to disease progression. There were no drug-related SAEs, AEs leading to drug discontinuation, or AE-related deaths. Of 21 ≥grade 3 AEs, 3 were assessed by investigators as potentially drug-related - all were self-limiting neutropenia in CLL pts, two of whom had neutropenia at baseline. No G3/4 bleeding events were recorded. Four pts had a baseline history of atrial fibrillation/flutter (AF); no exacerbation or new event of AF was reported. 16 responses, including 1 complete remission, have been observed. Response by histology is summarized in Table 1 and Figure 1. 22/25 pts remain on study treatment, free of progression, at a median of 204 days (range 138-321). Table. Response by Histology to BGB-3111 Histology n Objective Response (no. in CR) SD Continuing Treatment (as of 30th July 2015) Days on treatment (median; range) CLL 8 6 (0) 2 8 199 (140-252) MCL 6 4 (1) 1 5 227 (165-315) Waldenström's 6 5 (0) 0 5 232 (152-321) DLBCL 2 0 (0) 1 1 159 FL 1 0 (0) 1 1 194 MZL 1 0 (0) 1 1 138 HCL 1 1 (0) 0 1 285 Conclusions: These preliminary Phase 1 results suggest that the selective BTK inhibitor BGB-3111 is safe and highly clinically active. Complete blockade of BTK in PBMC at low doses and excellent tolerability at higher doses raises the possibility that BTK blockade in deep tissue sites will be complete. This question is currently being explored in the expansion phase. Figure 1. Best response in 22 pts evaluated by CT scan (SPD, CLL and NHL) or IgM levels (WM). Not included here are 1 responder with HCL (PR), and 2 pts who progressed before restaging. Figure 1. Best response in 22 pts evaluated by CT scan (SPD, CLL and NHL) or IgM levels (WM). Not included here are 1 responder with HCL (PR), and 2 pts who progressed before restaging. Disclosures Tam: Janssen: Consultancy, Honoraria, Research Funding. Off Label Use: BGB-3111 is not licensed for treatment of B-cell malignancies. Grigg:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Merck: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees. Opat:Janssen Pharmaceuticals: Other: Provision of subsidized medications only. Seymour:Phebra: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding, Speakers Bureau; Infinity: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hedrick:Beigene: Employment. Yang:Beigene: Employment, Equity Ownership. Wang:Beigene: Employment, Equity Ownership. Luo:Beigene: Employment, Equity Ownership. Xue:Beigene: Employment, Equity Ownership. Roberts:Walter and Eliza Hall Institute of Medical Research: Employment; AbbVie: Research Funding; Servier: Research Funding; Genentech: Research Funding.

Abstract: Introduction: Through previous studies using Sanger sequencing and targeted gene sequencing, we found that the frequencies of certain genes in Chinese patients with chronic lymphocytic leukemia (CLL) were different from those in CLL patients from the western countries. However, until now, there are no published whole-exome sequencing (WES) studies in Chinese CLL patients. To better define the genomic landscape of CLL cases in China, we conducted WES studies of CLL cases in our center. Methods: This study include 43 CLL patients, of whom 40 were untreated at the time of sampling. The median age of the 43 patients were 61 years, the male-to-female ratio was 2:1. We isolated peripheral blood mononuclear cells, then CD19 positive cells were purified by using magnetic microbeads in some cases. The buccal cells or the leukocytes that were depleted of CD19 positive cells were used as germline controls. We purified genomic DNA and used the purified DNA for library construction, after which we sequenced the library using the Illumina HiSeq2500 platform. After sequencing, software including Burrows-Wheeler Aligner (BWA) and GATK were used for the bioinformatic analysis. Results: In the 43 Chinese patients with CLL, the median number of somatic mutations was 67 (range:10-128) and the median number of nonsynonymous mutations was 26 (range: 5-55). We found that the number of mutations in patients older than 60 was significantly higher than that in the younger patients. Somatic mutations were detected across 991 different genes, of which 24 genes were identified as drivers in the study from the Dana-Faber Cancer Institute (DFCI). The 24 genes included NOTCH1, SF3B1, MYD88, POT1, TP53 and so on. Seventy-nine genes were recurrently mutated and 13 genes were found to be mutated in 3 or more cases. The most frequently mutated gene was NOCTH1 (6 cases, 14.0%). FBXW7, another gene that is involved in the regulation of the NOTCH1 pathway, was also mutated in 3 cases. IGLL5, MUC4 and MYD88 were also among the most frequently mutated genes, with each of them being mutated in 5 cases (11.6%). We found that the frequencies of mutations in IGLL5 (11.6% vs. 2.2%, P=0.0056), MYD88 (11.6% vs. 3.0%, P=0.0147), BCOR (9.3% vs. 2.2%, P=0.0246), and SF3B1 (4.7% vs. 21.0%, P=0.0084) were significantly different between Chinese CLL patients and patients from western countries. BCOR-mutated patients had more frequent aberrations involving NOTCH1 pathway (NOTCH1 and/or FBXW7 mutation) than BCOR-unmutated patients (3/4 [75.0%] vs. 6/39 [15.4%] , P=0.0242) (Figure 1A). Analysis of 10967 tumor samples from the Cancer Genome Atlas (TCGA) program revealed that tumors with BCOR mutations had a higher frequency of NOTCH1 aberrations (NOTCH1 and/or FBXW7 mutation) than those without BCOR mutations (116/349 [33.2%] vs. 722/10618 [6.8%] ; odds ratio: 6.824, 95%CI: 5.393-8.634, P 〈 0.0001) (Figure 1B). These data suggest BCOR mutations and NOTCH1 aberration may cooperate in the pathogenesis of CLL and other types of tumors. NFKBIA mutations were identified in 2 cases (Figure 1C) and we found that the mRNA level of NFKBIA was significantly lower in the NFKBIA mutated CLL cells than in the normal B cells (Figure 1D-E); in the MEC-1 cell line, significant activation of NF-кB pathway was observed after knockdown of NFKBIA using siRNA (Figure 1F). Conclusion: Patients with CLL in China and patients with CLL in the western countries have different mutational landscapes. BCOR gene mutations may cooperate with aberrations involving the NOTCH1 pathway in the pathogenesis of CLL. NFKBIA could be a potential tumor suppressor in the pathogenesis of CLL. Figure 1 Disclosures No relevant conflicts of interest to declare.

Abstract: Introduction : BGB-3111 is a potent, highly specific and irreversible Bruton tyrosine kinase (BTK) inhibitor, with greater selectivity than ibrutinib (IB) for BTK vs. other TEC- and HER-family kinases. We previously reported that BGB-3111 320mg daily (the RP2D, given as a single or split dose) achieved plasma concentrations 6- to10-fold higher than that of IB 560mg QD. Complete BTK occupancy in peripheral blood mononuclear cells (PBMCs) was observed in all pts treated in the dose-escalation (DEsc) component of the Phase 1 trial, and preliminary data suggested that 〉 90% blockade was achievable in lymph nodes (LN). We now report the results of BTK occupancy analyses in LN specimens from a dedicated pharmacodynamics (PD) cohort and update safety and efficacy in patients with CLL/SLL. Aims: (1)To determine BTK occupancy in LN samples from pts receiving either a daily or twice-daily regimen; and (2) to define the safety profile and activity of BGB-3111 in pts with relapsed/refractory (R/R) or previously untreated CLL/SLL. Methods: This Phase 1 trial included a DEsc component in pts with R/R B-cell malignancies, and disease-specific expansion cohorts (ECs), including CLL/SLL, at the RP2D (320mg daily, given either QD or as a split BID dose). Additionally, in a PD cohort, pts with R/R B-cell malignancies were assigned to BGB-3111 160mg BID vs 320mg QD, with paired LN biopsies at baseline and at day 3 (pre-dose), in order to determine BTK occupancy in LN at the time of trough drug exposure. Adverse events (AEs) are reported per CTCAE v4.03, and response according to the 2012 clarification of the International Workshop on CLL criteria (for CLL pts) or the 2014 Lugano Classification (for SLL pts). BTK occupancy was determined by competitive fluorescent probe assay. The data cut-off for this report was 10 June 2016. Results: BTK Occupancy: 30 pts were evaluated for LN BTK occupancy (CLL, n=9; DLBCL, n=3; FL, n=5; MCL, n=6; MZL, n=3; WM, n=4), 23 pts were enrolled in the PD cohort; 7 patients in other ECs (160mg BID) consented to optional paired LN biopsies. BTK occupancy in LN by dose/schedule is shown in Figure 1. Median occupancy was 99.5% (160mg BID, n=18) vs 94.4% (320mg QD, n=12) (p=0.002, Wilcoxon). The proportion of pts with 〉 90% occupancy was 94% (160mg BID) vs 58% (320mg QD) (p=0.027, Fisher exact). Occupancy did not appear to differ amongst histologic subtypes. CLL/SLL Safety and Activity: As of 10 Jun 2016, 45 pts with CLL (n=42) or SLL (n=3) have been enrolled: 8 pts in DEsc (80mg QD [n=1], 160mg QD [n=2] , 160mg BID [n=2], and 320mg QD [n=3] ), and 37 in either the PD cohort or CLL/SLL EC (160mg BID, n=19; 320mg QD, n=18). 29 CLL/SLL pts are included in this analysis; 11 pts were excluded because of short ( 〈 12 weeks) follow-up, and 5 pts accrued at a single study site were excluded because of insufficient study documentation at baseline. Demographic and disease characteristics are shown in Table 1. BGB-3111 was well tolerated with 69% subjects reporting no drug related AE 〉 Gr 1 severity within the first 12 weeks of therapy. The most frequent AEs of any attribution were petechiae/ bruising (38%), upper respiratory tract infection (31%, all Gr 1/2), diarrhea (28%, all Gr 1/2), fatigue (24%, all Gr 1/2), and cough (21%, all Gr 1/2). Three SAEs were assessed as possibly related to BGB-3111 (Gr 2 cardiac failure, Gr 2 pleural effusion and Gr 3 purpura, all n=1). The case of Gr 3 purpura was the only major bleeding event reported. Atrial fibrillation (Gr 2) occurred in one pt. Three pts had temporary dose interruption for AE, and one pt discontinued BGB-3111 for AE. After a median follow-up of 7.5 months (2.9-17.3 months), the response rate is 90% (26/29), with PR in 79% (23/29) and, PR-L in 10% (3/29), SD in 7% (2/29), and non-evaluable response in one pt who discontinued treatment prior to week 12. No instances of disease progression or Richter transformation have occurred. Conclusions: BGB-3111 is well-tolerated and highly active in R/R CLL/SLL. While trough BTK occupancy in lymph nodes was robust with either 320mg QD or 160mg BID dosing, complete and continuous occupancy (median 99.5%) was more frequently achieved with the 160mg PO BID regimen. Late stage clinical trials will determine if the optimized BTK occupancy with this regimen translates into improvements in disease control and reduced drug resistance. Figure. Figure. Disclosures Tam: janssen: Honoraria, Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Opat:Roche: Consultancy, Honoraria, Other: Provision of subsidised drugs, Research Funding. Gottlieb:Indee: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees. Simpson:Celgene, Roche, Janssen: Honoraria; Amgen Pharmaceuticals: Research Funding. Anderson:Walter and Eliza Hall Institute of Medical Research: Other: Walter and Eliza Hall Institute of Medical Research receives milestone payments for the development of venetoclax. Kirschbaum:Beigene: Employment. Wang:Beigene: Employment. Xue:Beigene: Employment. Yang:BeiGene: Employment. Hedrick:Beigene: Employment. Seymour:Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Roberts:Genentech: Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone payments related to venetoclax; Janssen: Research Funding; Genentech: Research Funding; AbbVie: Research Funding; Servier: Research Funding.

Abstract: Background ENKTL is a subtype of mature T cell and NK cell lymphoma, with a higher incidence in Asia than in Europe and North America. Pts with rr-ENKTL have a poor prognosis after failing an L-asparaginase-based regimen, and lack effective treatment. Epstein-Barr virus (EBV) infection is one of the mechanisms and characteristics of ENKTL, which induces immune tolerance of tumor by upgrading PD-L1 expression in tumor cells. Blocking the PD-1 pathway could, therefore, be an effective treatment for ENKTL. A Phase 2 trial is being conducted to evaluate CS1001, a first full-length, fully human immunoglobulin G4 (IgG4) mAb directed against PD-L1, in pts with rr-ENKTL. Method This is a multicenter, single-arm, Phase 2 clinical trial to evaluate CS1001 monotherapy in rr-ENKTL. Pts aged 18-75 years with rr-ENKTL after failing prior asparaginase-based chemotherapy or chemoradiotherapy were eligible if they had ECOG performance status (PS) ≤ 1, adequate organ function and bone marrow function, and at least one measurable or evaluable lesion. All the pts will receive CS1001 1200 mg intravenously every 3 weeks for up to 2 years, until disease progression, intolerance, etc. The primary endpoint was objective response rate (ORR) assessed by an independent radiological review committee (IRRC) per Lugano 2014 criteria. Key secondary endpoints included ORR by investigators, complete and partial response rate, time to response, duration of response (DoR) and progression-free survival, overall survival and safety. Adverse events (AEs) were summarized according to NCI CTCAE v4.03. Result As of June 17, 2019, 29 pts (male: 16, 55.2%; median age: 44 years, range 30-74) were enrolled. Twenty (20, 69.0%) pts entering the study had an ECOG PS of 1. Twenty-two (22, 75.9%) pts had Stage IV of disease at screening. Eight (8, 27.6%) pts received 2 lines of prior systemic therapy and 6 (20.7%) pts received ≥3 lines of prior systemic therapy. The median duration of follow-up was 5.55 (range, 0.69-12.19) months. Among the 22 efficacy-evaluable pts, the investigator-assessed ORR was 40.9%. A total of 7 (31.8%) pts achieved complete response (CR), and 2 (9.1%) pts had partial response (PR); 1 additional pt achieved PR after pseudo-progression. The DoR ranged from 0.03+ to 8.61+ months; median DoR was not achieved. All CR pts were still in remission. The IRRC assessments were not available at the time of data cut-off. The median duration of treatment was 11.7 (range, 2.9 to 53.0) weeks. Fifteen (15, 51.7%) pts remained on treatment and 14 (48.3%) had discontinued from study treatment (12 due to progressive disease, 2 due to AEs). The majority of pts (25, 86.2%) had treatment-emergent AEs (TEAEs), of whom 21 (72.4%) pts reported treatment-related AEs (TRAEs). The most frequently reported (≥10%) TRAEs were pyrexia (6, 20.7%), blood thyroid stimulating hormone increased (4, 13.8%), white blood cell count decreased (4, 13.8%), and rash (3, 10.3%). Three (3, 10.3%) pts reported Grade (G) ≥3 TRAEs. Two (2) G5 AEs (death and haemophagocytic lymphohistiocytosis) were reported in 2 pts; none was assessed as related to CS1001 by the investigators. Serious AEs were observed in 5 (17.2%) pts, and 1 of them (sinus node dysfunction) was assessed as related to CS1001 by the investigator. Immune-related AEs (irAEs) were reported in 5 pts, including a G3 rash and the remaining irAEs were G1. TEAEs that led to permanent treatment discontinuation occurred in 2 (6.9%) pts, which included haemophagocytic lymphohistiocytosis (G5) and facial nerve disorder (G2). No death or permanent discontinuation due to AEs were assessed as related to the study drug. After data cut-off date, 3 additional pts reached response assessment time point, of which 2 pts achieved CR, resulting in ORR of 44% (11/25) and a CR rate of 36% (9/25). The updated data will be reported at the ASH conference. Conclusion In this study, CS1001 was shown to be active with a high CR rate and durable response, and was generally well-tolerated in pts with rr-ENKTL. The preliminary safety and efficacy profile of CS1001 support further exploration and development of CS1001 in rr-ENKTL pts. Disclosures No relevant conflicts of interest to declare.

Abstract: Background Circular RNAs (circRNAs), a novel family of non-coding RNAs, have crucial roles in cancer progression. Conventional research is mainly focus on nuclear genome derived circRNAs (nu-circRNAs). The biological and clinical characteristics of mitochondrial genome derived-circRNAs (mt-circRNA) remains largely unknown, especially in chronic lymphocytic leukemia (CLL), the most prevalent incurable B-cell neoplasm in western countries. Lack of convenient and reliable clinical biomarkers is a hindrance in monitoring the progression of CLL. It is in urgent need of screening effective new biomarkers and exploring potential therapeutic targets associated with CLL initiation and progression. Recent studies have shown that circRNAs are enriched and stable in serum exosomes. However, the biological mechanisms of exosomal circRNAs remain unclear. In this study, we attempted to identify the novel characteristics of a mt-circRNA mc-COX2, which was abundant in plasma exosomes and could be involved in the progression of CLL. Since CLL patients have specific expression features of exosomal circRNAs in plasma, the function of the circRNAs and their clinical significance is urged to be explored. Methods Firstly, to unveil circRNAs expression profiles in CLL, plasma samples from five treatment-naive CLL patients and five age-/sex-matched healthy donors (HDs) were collected for circRNA microarray analyses. Northern blot and RNA- FISH were conducted to verify the existence of circRNAs in mitochondria. qPCR and other functional analysis such as RNase R, actinomycin D and RIP experiments were carried out to demonstrated the clinical and biological characteristics of mc-COX2, one of the mt-circRNAs. Cell apoptosis ability was determined by FCM. Moreover, electron microscope, partical size analysis, FCM and Western blot were used to explore the existence of exosomes and q-PCR analysis was performed to detect the expression of mc-COX2. Results Mt-circRNAs were highly expressed in CLL patients plasma (Figure A, B). Herein, we reported a novel circRNA named as mc-COX2 which was generated from the COX2 gene on mitochondrial genome by back-splicing and closely related to prognosis of CLL patients (Figure C). The enrichment of mc-COX2 in the mitochondria was further confirmed by RNA-FISH (Figure D). Northern blot was performed using head-to-tail probe of mc-COX2 and the results showed that mc-COX2 was detectable within the splice sites (Figure E). Notably, obviously different from nu-circRNAs, mc-COX2 showed lower stability with lower tolerance to RNase R and actinomycin D, but it was much more stable compared with linear RNAs (Figure G, F). And mc-COX2 cannot bind to AGO2 protein, suggesting that it probably function via other mechanisms instead of acting as ceRNA (Figure H). Furthermore, the up-regulated expression of mc-COX2 was positively associated with leukemogenesis and worse survival of CLL patients (Figure I, J). CLL patients with TP53 deletion rather than mutation displayed higher expression of mc-COX2 (Figure K). The endogenous reduction of mc-COX2 could induce cell apoptosis (Figure L). In addition, we indicated that mc-COX2 was highly enriched in exosomes, by which circRNAs could enter the circulation and be readily measured in the serum (Figure M). Moreover, the existence of mc-COX2 in plasma suggests that mc-COX2 may serve as a potentially novel prognosis biomarker for CLL patients, guiding targeted therapy in clinic. Conclusions In summary, we demonstrated the existence and clinical significance of mc-COX2, a novel class of circRNA species abundant in CLL plasma exosomes for the first time, which was distinct from nu-circRNAs. Furthermore, the specific high expression of mc-COX2 in CLL plasma which was strongly correlated with P53 deletion, can indicate worse prognosis of CLL patients. Taken together, our study not only identifies a novel circRNA which may serve as a new "liquid biopsy" biomarker for CLL prognosis but also expands the current knowledge regarding molecular mechanisms of circRNAs, providing potential diagnostic and therapeutic implications for CLL. It would be of great interest to explore the biogenesis of mt-circRNAs and their impact on mitochondrial function in future studies. Figure Disclosures No relevant conflicts of interest to declare.

Abstract: Disintegrin is a kind of small native peptides derived from the snake venom, containing RGD sequences. Its interaction with integrins, such as aIIbb3, anb3 and a5b1, can inhibit platelet aggregation. hinder the process of angiogenesis and tumor metastasis. In order to further investigate the function of disintegrin in platelet aggregation, cell adhesion and angiogenesis, the sequence encoding the disintegrin domain of agacutin was fused with enhanced green fluorencent protein(eGFP) and inserted in plasmid pQE-30. The recombinant protein was expressed in E.Coli M15 after induction by IPTG, amounting to 38% of the total bacterial protein. The cells expressing integrin such as breast cancer cell line MDA-MB-231 and endothelial cell line EA.hy926 can specifically bind to the recombinant GFP-disintegrin. Flow cytometry showed that the recombinant protein could bind to platelet specfically and could compete with anti-b3 integrin monoclonal antibody CD61. Moreover, the recombinant protein could inhibit platelet aggregation induced by collagen and ADP in a dose-dependent manner, but had no ability to impede the platelet aggregation induced by Ristocetin. Meanwhile, it could inhibit cell (MDA-MB-231 and EA.hy926) adhesion to fibronectin with inhibition rate of 69% and 48%, respectively. Finally, it could induce apoptosis of EA.hy926 endothelial cells and inhibit the angiogenesis on chicken chorioallantoic membrane mode. As a result, it is demonstrated that GFP-disintegrin has the combined quality of its disparate components and can serve as a novel tool for study of tumor and angiogenesis. In addition, the recombinant protein can efficiently inhibit platelet aggregation and angiogenesis, which will be useful for designing the potential drug for anti-thrombosis and anti-tumor metastasis.

Abstract: Introduction: The BTK inhibitor ibrutinib (IB) is highly active in WM, achieving major responses (CR+VGPR+PR) in approximately 70% of pts. However, VGPR is infrequent, with rates ≤15% in reported series (Treon NEJM 2015, Dimopoulos EHA 2016). BGB-3111 is a potent, highly-specific and irreversible BTK inhibitor, with greater selectivity than IB for BTK vs. other TEC- and HER-family kinases, and superior bioavailability. We previously reported that the recommended phase 2 dose of BGB-3111 is 320mg daily (in single or split dose) in pts with advanced B cell malignancies. This achieves plasma levels equivalent to 6-10 fold that of IB, and 〉 90% continuous inhibition of BTK in lymph node biopsies. We specifically investigated the safety and efficacy of BGB-3111 in pts with WM in an expansion cohort of the initial Phase 1 trial. Reported here are updated results of this study, including data from WM specific expansion cohorts. Aims: To define the safety profile, and clinical activity of BGB-3111 in pts with WM. Methods: These results are from a pre-specified a component of a Phase 1 study (Part 1: dose escalation [DEsc] in pts with R/R B cell malignancies, Part 2: disease-specific dose expansion cohorts [EC] at the recommended Phase 2 dose that included patients with relapsed / refractory or previously untreated WM). Adverse events (AEs) were reported per CTCAE v4.03. Responses were determined according to the modified Sixth International Workshop on WM (IWWM) criteria. The data cut-off is 10 June 2016. Results: As of 10 June 2016, 31 pts with WM have been enrolled; 6 pts in DEsc (40mg [n=2], 80mg [n=2] , 160mg QD [n=1], and 320mg QD [n=1] ), and 25 in the WM EC (160mg BID [n=18], 320mg QD [n=7] ). Three pts in DEsc were increased to 160mg QD after analysis of DEsc data, as allowed by protocol. Twenty-four pts are included in this analysis; 5 pts were excluded because of short ( 〈 60 day) follow-up for safety and efficacy, and 2 pts accrued at a single site were excluded because of insufficient documentation at baseline. Patient demographics, disease characteristics, and prior treatment history are summarized in Table 1. BGB-3111 was well tolerated with 71% reporting no drug related AE 〉 Gr 1 severity within the first 12 weeks of therapy. The most frequent AEs ( 〉 20%) of any attribution (all Gr 1/2) were upper respiratory infection (25%), diarrhea (25%), and nausea (21%). There were 2 SAEs assessed as possibly related to BGB-3111 (Gr 2 atrial fibrillation, Gr 3 cryptococcal meningitis); in both cases, BGB-3111 was temporarily held but safely resumed. In total, 2 pts developed AF (one Gr 1, one Gr 2). No serious hemorrhage (≥Gr 3 or CNS hemorrhage of any grade) was reported. After a median follow-up of 7.6 months (2-21 months), the response rate was 92% (22/24). The major response rate was 83% (20/24), with VGPR ( 〉 90% reduction in IgM and reduction in extramedullary disease) in 33% (8/24) and PR (50-90% reduction in IgM and reduction in extramedullary disease) in 50% (12/24) pts. Pts with WM refractory to their last therapy were equally responsive: major response 77% [10/13], VGPR 31% [4/13] , PR 46% [6/13]. Median time to initial response and major response were 29 days and 34 days, respectively. IgM decreased from a median of 29.9g/l at baseline to 3.0g/l; hemoglobin increased from a median of 10.1 g/dl at baseline to 13.5g/l. Two of 3 pts who had a dose increase after reaching a stable IgM plateau had further falls in IgM levels after dose escalation. Kinetics of IgM and hemoglobin response are presented in Figure 1. Lymphadenopathy was present in 8 pts at baseline; serial CT demonstrated reduction or resolution in all 8 pts (27%-100% reduction in SPD). Only one patient discontinued BGB-3111, due to exacerbation of pre-existing bronchiectasis while in VGPR. There have been no cases of disease progression. Analysis of response by genomic characteristics (including MYD88 and CXCR4 mutational status) is ongoing. Conclusions: BGB-3111 is well-tolerated and highly active in WM. The depth and quality of responses, as reflected by the VGPR rate of 33%, warrant a randomized comparison against ibrutinib in pts with WM. Table 1. Table 1. Figure 1. Figure 1. Disclosures Tam: Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; janssen: Honoraria, Research Funding. Opat:Roche: Consultancy, Honoraria, Other: Provision of subsidised drugs, Research Funding. Simpson:Amgen Pharmaceuticals: Research Funding; Celgene, Roche, Janssen: Honoraria. Anderson:Walter and Eliza Hall Institute of Medical Research: Other: Walter and Eliza Hall Institute of Medical Research receives milestone payments for the development of venetoclax. Kirschbaum:Beigene: Employment. Wang:Beigene: Employment. Xue:Beigene: Employment. Yang:BeiGene: Employment. Hedrick:Beigene: Employment. Seymour:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Roberts:AbbVie: Research Funding; Servier: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Genentech: Patents & Royalties: Employee of Walter and Eliza Hall Institute of Medical Research which receives milestone payments related to venetoclax.

Abstract: Abstract 3142 The Warburg effect describes a pro-oncogenic metabolic switch in which cancer cells, including leukemia cells, take up more glucose than normal tissue, yet use less glucose for oxidative phosphorylation and favor glycolysis even in the presence of oxygen (aerobic glycolysis). However, the molecular mechanisms underlying the Warburg effect remain unclear. Growth factor (GF) receptors are believed to play a key role in programming cancer cell metabolism. These GF receptors are expressed in many hematopoietic malignancies as constitutively activated tyrosine kinase mutants. Thus, we examinined whether tyrosine kinase signaling — commonly upregulated in hematopoietic malignancies — regulates the Warburg effect to contribute to leukemogenesis and disease progression. We performed phospho-proteomics studies and found that pyruvate kinase M2 isoform (PKM2), which is a rate-limiting enzyme of glycolysis, is tyrosine phosphorylated in leukemia cells expressing FGFR1 fusion tyrosine kinases, which are associated with 8p11 leukemia/lymphoma syndrome. We also found that 8p11 leukemogenic FGFR1 directly phosphorylates and inhibits PKM2. Recent seminal studies from Dr. Lew Cantley's group demonstrated that the enzymatic activity of PKM2 is inhibited by phosphotyrosine binding; PKM2 expression is important for aerobic glycolysis and provides a growth advantage to tumors. However, it remains unclear which dedicated tyrosine kinase pathways are physiologically responsible for this regulation and whether PKM2 itself is tyrosine phosphorylated to achieve inhibition of PKM2 in cancer cells. Here we report that FGFR1 inhibits PKM2 by direct phosphorylation at Y105. This consequently inhibits the formation of tetrameric, active PKM2 by disrupting cofactor fructose-1,6-bisphosphate (FBP) binding in a putative “inter-molecule manner”, where one molecule in an active PKM2 tetramer, when phosphorylated, may function as an inhibitory binding partner to the other sister molecules. In addition, phosphorylation of PKM2 at Y105 is common in many human leukemia cell lines expressing oncogenic tyrosine kinases such as BCR-ABL, FLT3-ITD, and JAK2V617F. Furthermore, expression of the PKM2 Y105F mutant in cancer cells following RNAi-mediated knockdown of endogenous PKM2 leads to decreased cell proliferation under hypoxia, increased oxidative phosphorylation with reduced lactate production, and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation regulates PKM2 to program cancer cell metabolism and promote tumor growth. This may represent a common, acute molecular mechanism to regulate the Warburg effect, in addition to the chronic changes that are believed to be regulated by hypoxia inducible factor 1 and Myc. Disclosures: No relevant conflicts of interest to declare.

Abstract: Vascular endothelial growth factor (VEGF) is a crucial mediator of angiogenesis, and plays an important role in pathogenesis of leukemia. However, the importance of VEGF in differentiation or apoptosis of leukemic cells remains to be evaluated. A competitor DNA fragment template of VEGF gene mimic was constructed with the method of gene recombinant technologies, and a competitive quantitative reverse transcriptase-polymerase chain reaction (cQRT-PCR) for analyzing VEGF gene expression was performed to assess the regulation of VEGF gene expression in the process of all-trans retinoic acid (ATRA)-induced differentiation of an acute promyelocytic leukemia cell line NB4. In construction of a standard curve from which the amount of target cDNA was derived, serial dilutions of the target were co-amplified with a constant amount of mimic, and the intensities of bands corresponding to the target and the mimic were measured. CD11b antigen and nitroblue tetrazolium (NBT) reduction rate of NB4 cells were also assayed at different time points. cQRT-PCR was a sensitive and reliable tool for analysis of VEGF gene expression, with a detectable range from 1 to 2 times 10 the fifth power molecules. The number of VEGF gene transcripts detected by means of cQRT-PCR assay was 42.3, 12.6, 3.6, and less than 1.0 times 10 the fifth powder per microgram of NB4 total RNA at 0, 12, 24 and 48 hours after ATRA treatment, respectively. The rapid down-regulation of VEGF gene expression during ATRA-induced NB4 cell differentiation was accompanied by an upregulation of CD11b expression and an increased NBT reduction rate. In conclusion, cQRT-PCR could be used as an efficient method of qualitative analysis of VEGF gene expression. ATRA significantly depresses VEGF expression and its antileukemic effect can be brought through the two ways of differentiation induction and angiogenesis inhibition.