Tyrosine kinase inhibitors (TKIs) elicit high response rates among individuals with kinase-driven malignancies, including chronic myeloid leukemia (CML) and epidermal growth factor receptor-mutated non-small-cell lung cancer (EGFR NSCLC). However, the extent and duration of these responses are heterogeneous, suggesting the existence of genetic modifiers affecting an individual's response to TKIs. Using paired-end DNA sequencing, we discovered a common intronic deletion polymorphism in the gene encoding BCL2-like 11 (BIM). BIM is a pro-apoptotic member of the B-cell CLL/lymphoma 2 (BCL2) family of proteins, and its upregulation is required for TKIs to induce apoptosis in kinase-driven cancers. The polymorphism switched BIM splicing from exon 4 to exon 3, which resulted in expression of BIM isoforms lacking the pro-apoptotic BCL2-homology domain 3 (BH3). The polymorphism was sufficient to confer intrinsic TKI resistance in CML and EGFR NSCLC cell lines, but this resistance could be overcome with BH3-mimetic drugs. Notably, individuals with CML and EGFR NSCLC harboring the polymorphism experienced significantly inferior responses to TKIs than did individuals without the polymorphism (P = 0.02 for CML and P = 0.027 for EGFR NSCLC). Our results offer an explanation for the heterogeneity of TKI responses across individuals and suggest the possibility of personalizing therapy with BH3 mimetics to overcome BIM-polymorphism-associated TKI resistance.

Increasing awareness and availability of tests for anti-PF4/heparin complex antibodies has raised concerns about indiscriminate testing and inappropriate diagnosis of heparin-induced thrombocytopenia. A retrospective review of HITS testing at a large tertiary centre was performed to determine test patterns and incidence of HITS. Records of anti-PF4 tests over 4 years were reviewed. Positive results were matched against patient medical records and records of heparin utilisation for the diagnosis of HITS. Total of 33,308 patients (9.89 % of admissions) were exposed to at least 1 day of unfractionated heparin (UFH) or low molecular weight heparin (LMWH). Of 346 anti-PF4 antibody assays performed, 52 (15 %) were positive. Seventeen patients (4.9 % of total) were determined to have HITS. Of these, 13 cases (76 %) were patients who were initiated on haemodialysis via central venous catheters. Five patients (29 %) subsequently experienced thrombosis. The rate of HITS in patients given UFH was 0.33 %. HITS occurred in only one patient given LMWH. Of 1337 patients with chronic renal failure initiated on long term haemodialysis, the incidence of HITS was 0.97 %. Despite increased awareness of HITS, testing frequency remains conservative. An overwhelming majority of HITS cases were diagnosed among patients initiated on haemodialysis.

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1666 The use of the tyrosine kinase inhibitor (TKI), imatinib (IM), to target the oncogenic BCR-ABL kinase has resulted in profound responses in patients with chronic phase (CP) chronic myelogenous leukemia (CML). However, a subset of patients do not respond to TKIs, and are deemed to have primary resistance. Importantly, patients with European LeukemiaNet (ELN)-defined ‘failure’ or ‘suboptimal response’ are at increased risk of poorer long-term outcomes. Little is known about mechanisms underlying primary resistance, where only a minority of patients have BCR-ABL kinase domain (KD) mutations. Interestingly, East-Asian CML patients are reported to have lower complete cytogenetic response rates compared to the West (∼50 vs 80% respectively, Au et al. 2009). We used massively parallel DNA sequencing of paired-end ditags to identify genetic factors associated with resistance in CML patient samples. We discovered a novel deletion polymorphism in the BIM gene that correlated with resistance, and which represented a common polymorphism in normal East-Asian (12.3% carriers), but not African or Caucasian (0%), populations (n=2465). BIM is a pro-apoptotic BCL2 family member, and plays a central role in CML pathophysiology. Here, BCR-ABL suppresses FoxO3a-mediated BIM transcription to maintain a survival advantage, while preventing BIM expression following BCR-ABL inhibition results in TKI resistance. Inspection of BIM gene structure suggested the polymorphism would result in mutually exclusive splicing of exon 3 (E3) vs 4 (E4), leading to decreased expression of BIM transcripts encoding the pro-apoptotic BH3 domain (found only in E4). To test this hypothesis, we constructed a minigene to measure E3 vs E4 splicing, and found the polymorphism decreased splicing to E4 over E3 by >5-fold. Importantly, primary CML cells exhibited the same phenomenon, since polymorphism-containing samples expressed lower levels of E4- vs E3-containing transcripts (p=0.008), while general BIM transcription was unaffected. Our observations suggested a novel mechanism for intrinsic TKI resistance. Here, upon IM exposure, polymorphism-containing CML cells would favor induction of E3- vs E4-containing BIM transcripts, decreased expression of BH3-containing BIM isoforms, and impaired apoptosis. To facilitate these studies, we identified a Japanese CML cell line, KCL22, which contained the polymorphism, and confirmed it had a decreased E4/E3 transcript ratio compared to cells without the polymorphism. KCL22 cells also had decreased induction of E4-containing transcripts following IM, as well as lower levels of BIMEL protein, a major BH3-containing BIM isoform. Consistent with prior reports, KCL22 cells were resistant to IM, despite effective BCR-ABL inhibition, and had impaired apoptotic signalling upon IM exposure. Importantly, and as predicted by our model, pharmacologic restoration of BH3 activity (using the BH3-mimetic drug, ABT-737) sensitized cells to IM-induced death. Next, we used zinc finger nuclease-facilitated gene targeting to precisely create the polymorphism in the BIM gene of IM-sensitive K562 CML cells. We generated subclones that were heterozygous or homozygous for the polymorphism, and confirmed a decreased E4/E3 ratio in these cells in a polymorphism-dosage-dependent manner. Polymorphism-containing cells exhibited decreased induction of E4-containing transcripts following IM exposure, as well as impaired upregulation of BIMEL protein, and diminished apoptotic cell death. As in KCL22 cells, ABT-737 enhanced the ability of IM to activate apoptosis in polymorphism-containing cells. Using an expanded East-Asian CML cohort (n=203), we found the polymorphism correlated with TKI resistance (defined as ‘failure’ or ‘suboptimal’ per ELN criteria) in CP patients treated with 400 mg IM daily (p=0.02). Further, patients with the polymorphism were more likely to have resistance in the absence of a KD mutation than those without (OR=2.24, 95% CI of 1.22–4.12). In sum, we have found an East-Asian polymorphism in BIM that is associated with intrinsic resistance to TKIs. Screening for this polymorphism may be useful in identifying patients at risk of TKI resistance; a resistance we show can be overcome by BH3 mimetics. Our findings may also apply to other cancers and proliferative disorders in which drug-sensitivity is BIM-dependent. Disclosures: No relevant conflicts of interest to declare.

Chronic myelomonocytic leukemia (CMML) is a clonal disorder with a diagnostic challenge encountered frequently in routine hematology consultations, especially when few dysplastic features in bone marrow and/or no cytogenetics or molecular alterations are detected. Thus, easily applicable parameters in peripheral blood analysis are warranted to aid diagnosis. We studied the utility of flow cytometry analysis of peripheral blood (PB) for the diagnosis of CMML type I. PB samples from 16 CMML type I, age- matched normal (n=10) and reactive monocytosis (>1 x109/L) cases (n=9) were studied. A large panel of monoclonal antibodies was used ( FITC:CD36, CD35, CD15 and CD4, CD16,PCP5.5:CD34, CD163, CD13, PC7: CD117, CD38, CD2,CD7, APC: CD300, CD33, CD11B , CD11C ,CD56 , APCH7: CD14 , HLADR V450, CD64 PE and CD45 OC515) in an 8-color combination, including a backbone of 4 colors in all tubes. At least 1x 10(6) events were acquired by FACSCanto II (BD Biosciences, San Jose, USA). The data was analyzed with the Infinicyt software (Cytognos SL, Spain). Monocytes were selected on the automated population separator plot and confirmed by the expression of CD64 and HLADR. Lymphocytes were used as the internal control. As expected, CMML type 1 patients showed higher absolute monocyte counts in PB than reactive and normal cases (p=0.001), and higher percentage of monocytic cells by flow (p=0.001). CD64bright and CD163 expression were equally valid to identify monocytic population in all three groups (correlation r2 = 1). The majority of the monocytic population belonged to the late stages of maturation, being CD64+CD35+CD14+CD300+ in CMML as well as reactive and normal cases (p=0.14). CMML and reactive cases showed significantly lower levels of early stages of monocytic maturation (CD64+CD35-/+ CD14-CD300-) than normal controls ( p=0.04). The majority of type I CMML (69%) had more than 95% "classical" monocytes (CD14+CD16-) in the monocytic compartment, whereas only 22% of reactive cases and none of the normal cases had such condition (p=0.000). Statistically significant differences among the three groups were also found for "intermediate" (CD14+CD16+) and "non-classical" (CD14lowCD16+) monocytic populations, with the normal cases having higher percentage of "non classical" monocytes, and reactive cases higher "intermediate" (CD14+CD16+) monocytes (12% vs. 5% vs. 1%; -p <0.000-, and 2.6% vs. 3.7% vs. 1.4%; -p=0.01- for "non classical" and "intermediate" monocytes in normal, reactive and CMML cases, respectively). As for the aberrant antigenic expression, CD56 was most frequently expressed in monocytes of CMML (75% CMML with >20% CD56 positivity) followed by CD2 (31%) and CD7 (6%). CD56 could also be detected in reactive (22%) and normal (7%) cases, whereas CD2 and CD7 were always negative. Though useful, CD56 expression alone could overestimate the diagnosis of CMML type I whereas the utility of CD7 expression was doubtful due to its low frequency in CMMLs. When the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 and CD2 were considered, only CMML cases met both criteria (>95% of "classical" monocytes among the monocytic compartment in PB, and having an aberrant expression of CD56 or CD2 in >20% of monocytic cells), excluding all the reactive monocytosis and normal cases, although not all CMML (62%) met both criteria. Concerning the antigens analyzed on the monocytes, only CD163 and CD11b had a higher expression in CMML than in reactive or normal cases (p=0.01 & p=0.05). CD64, CD15, CD4, and CD45 were similarly expressed in CMML and reactive, but higher than in normal cases (p<0.03). CD35 was highest (p=0.01) and HLA-DR (p=0.01) and CD33 (p=0.02) lowest in reactive cases, compared to CMML or normal cases. No significant difference was found for the remaining antigens. In spite of these findings, in our opinion, its prospective utility to diagnose CMML or to distinguish it from reactive monocitosis was doubtful. Flow cytometry analysis can be useful to diagnose CMML type I in the routine laboratory by employing a few easily applicable parameters in PB (the pattern of distribution of "classical", "intermediate" and "non classical" monocytes together with the aberrant expression of CD56 or CD2), distinguishing it from reactive monocytosis, and helping to identify patients in whom other studies, as bone marrow assessment, should be performed. Disclosures Diez Campelo: Novartis: Research Funding, Speakers Bureau; Janssen: Research Funding; Celgene: Research Funding, Speakers Bureau. Puig:The Binding Site: Consultancy; Janssen: Consultancy.