Multidrug resistance (MDR) is a phenomenon by which cells become resistant to unrelated chemotherapeutic agents. The prognostic value that lung resistance protein (LRP) and multidrug resistance-related protein 1 (MRP1) have in the setting of pediatric acute lymphoblastic leukemia (ALL) is controversial. The aim of this study was to investigate the expression of LRP and MRP1 and effect on clinical outcome and prognosis. The mRNA expression of LRP and MRP1 were analyzed in leukemic blasts of 34 pediatric ALL patients. LRP and MRP1 mRNA expression were detected in 41.2% and 35.3%, respectively. Eleven (91.7%) of 12 patients without LRP achieved CR compared with 9 (50.0%) of 18 with LRP expression. Similarly, 11 (100%) of 11 patients without MRP1 expression achieved CR compared with 9 (47.4%) of 19 with MRP1 expression and higher LRP expression rate or MRP1 expression rate was present in patients with relapse than MDR genes negative patients. The expression of either of two genes was associated with poorer 2-year survival. Also, patients expressing both genes had poorer outcomes and had worse 2-year survival. We suggest that MDR expression affects complete remission and survival rates in ALL patients. Thus, diagnosis appears to provide prognostic information for pediatric ALL.

Acute leukemias are caused by genetic and epigenetic mechanisms involving tumor suppressor genes and oncogenes. Aberrant DNA methylation patterns are the most frequent molecular alterations detected in acute myeloid leukemia (AML). Gravin is down-regulated in several solid tumors and is implicated in tumorigenesis. To explore its role in the molecular pathogenesis and its possible prognostic importance in AML, we have evaluated the expression levels of the gravin gene in 83 acute myeloid leukemia patients as compared with controls using quantitative real-time polymerase chain reaction (qRT-PCR). Mean gravin expression was 0.53 ± 1.34 and 8.81 ± 11.6 for patients and controls, respectively, and was found to be about 16-fold lower than controls. Gravin gene expression was lower than controls in 83.1 % (69/83) and was similar to controls in 16.9 % (14/83) of cases (p < 0.0001). It was found that there was no significant correlation between gravin expression and laboratory prognostic markers (p > 0.05). Gravin expression was highest in complete remission (1.065 ± 1.79) and lowest in relapse (0.019 ± 0.03) with a statistical difference (p = 0.004). Patients with gravin expression below median level had higher risk to develop relapse (OR = 8.689, 95 % CI = 2.464-30.638; p < 0.0001). No statistical correlation was reported between gravin expression and survival times (OS, DFS) (p = 0.482, 0.409, respectively), and this was confirmed in multivariate analysis. Gravin gene expression was found to be decreased in acute myeloid leukemia, and the degree of its decreased expression has been found to be correlated with poor prognosis.

Introduction Chronic lymphocytic leukemia (CLL) is one of the most common B cell malignancies in the western world. The importance of B cell receptor (BCR) signaling in CLL pathology has led to the emergence of a number of targeted therapies specific to kinases in this pathway. Ibrutinib, a potent irreversible Bruton's Tyrosine Kinase (BTK) inhibitor, is highly effective in CLL with minimal side effects. However, resistance to Ibrutinib is now emerging among patients in several clinical trials. Aims/Methods To understand the molecular mechanisms underlying ibrutinib resistance to aid in early detection of resistant clones and to inform further treatment choices, we have developed an in vitro ibrutinib resistant model using BCR signalling dependent B cell lymphoma lines (Ramos and Namalwa). These cells have been conditioned by exposure to escalating doses of ibrutinib - starting from 0.001μM and increased over time to a lethal dose of 0.5μM in order to select drug-resistant cells. Single cell clones have been isolated from both the naïve and the resistant cells. The growth kinetics of 7 Ramos and 5 Namalwa resistant clones has reached the same pattern as the parental cells. Additionally, 3 naïve clones were isolated from each sensitive parental cell line. Dysregulation of the BCR signaling pathway was characterized by both flow cytometry, using specific anti-phosphokinase antibodies, and by Western blotting. In addition, RNA from these clones was isolated and analyzed on an Affymetrix HTA2.0 gene expression array (n=6 from two cell lines paralleled to one naïve clone from each cell line). Results In Ramos (n=3), gene expression profiling revealed significant upregulation of BCL2 and interferon response genes including interferon gamma (IFNG). However, in Namalwa (n=3), in addition to BCL2 upregulation, the expression arrays showed the overexpression of BCR signaling pathway genes including BTK itself, CD79B, LCK and GAB2 (Growth factor receptor-bound protein 2- associated-binder 2), suggesting hyper-activation of BCR pathway and constitutive activation of BTK and downstream kinases. Moreover, initial screening showed that not all of the resistant clones behave similarly. In Ramos, one clone apparently escapes ibrutinib inhibition by modifying different pathways compared to the other analysed clones, suggesting the potential for multiple mechanisms by which the malignant B cell clones can evade the toxic activity of targeted therapy. Results of this analysis were validated by qPCR and additional resistant clones were tested for BCL2 and IFNG expression. In Ramos, 7 resistant clones showed significant upregulation of BCL2 (mean 9.7-fold increase, range 2.1- 31.8, SEM 4.5) and IFNG(mean increase 8.3, range 2.6-19.1, SEM 2.2). IFNγ, is a known immune modulator linked to various stress signals indicating one possible resistance mechanism of malignant cells to anti-cancer therapies. In Namalwa, qPCR of 5 resistant clones showed significant upregulation of BTK (mean increase 10.3, range 2- 31: SEM: 5.4) and confirmed at the protein expression level by Western blotting as well; where the resistant cells over-express BTK protein compared to their respective naïve cells (n=6, mean increase 1.6, range 1.1- 1.9, SEM 0.16). Moreover, CD79B was over-expressed (mean over-expression 12.4, range 1.5- 22.2, SEM: 4.3) and 6-fold overexpression of LCK (mean increase 6, range 3.1- 9.5, SEM: 1.4) compared to the analysed naïve clones. Furthermore, validation with qPCR showed 12-fold upregulation of the GAB2gene (mean upregulation 12.7, SEM: 4.8), an adaptor protein which transmits crucial signals to activate downstream PI3K signaling with links to leukemogenesis. Conclusion Our results demonstrate that common mechanisms of resistance to BCR inhibition include both the up-regulation of B-cell receptor signaling activity and the up-regulation of anti-apoptotic molecules, particularly BCL2. More potent and more target-specific BTK inhibitors may overcome the hyperactivity of the BCR pathway. In contrast, inhibition of BCL2 with venetoclax may be beneficial in overcoming the emergent resistance to ibrutinib as these cells become more sensitive to BCL2 inhibition. These initial results support the combination of ibrutinib with the pro-apoptotic BCL2 inhibitor, venetoclax, which we are currently studying in the Bloodwise TAP CLARITY study. Disclosures Hillmen: Pharmacyclics: Research Funding; Janssen: Honoraria, Research Funding; Roche: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Abbvie: Research Funding.

The study was based on 58 (40 males, 18 females) consecutive, previously untreated CLL, 7 HCL (5 males, 2 females), 23 CD5 negative LPD (15 males, 8 females) and 12 multiple myeloma (8 males, 4 females). Mean age of CLL, HCL, CD5 negative LPD and multiple Abstract CD26/dipeptidyl peptidase IV (DPPIV) is a multifunctional membrane protein and it is strongly upregulated in activated B-cells. We aimed to evaluate CD26 expression in mature B cell neoplasms, and its prognostic role in B cell chronic lymphocytic leukaemias (B-CLL). CD26 expression was evaluated by flow cytometry in various B cell neoplasms. CD26 expression was high in MMs and HCLs, variable in B-CLLs and in CD5neg B-CLPDs. Kaplan-Meier curves revealed a significantly shorter progression free survival (PFS), and lymphocytic doubling time (LDT) in the CD26 high expression group (p=0.014, 0.024 respectively). High CD26, CD38 and/or ZAP70 showed significantly shorter PFS, (p=0.020, 0.022 respectively) and LDT (p=0.024, 0.024 respectively) when compared to both low expression CD26, CD38 and/or ZAP70. CD26 expression may identify subsets of B-CLL patients with an unfavorable clinical outcome, thus suggesting its potential role as a marker in a future routine cytofluorimetric panel for B-CLLs.