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BACKGROUNDRandomized trials of venetoclax plus anti-CD20 antibodies as first-line treatment in fit patients (i.e., those with a low burden of coexisting conditions) with advanced chronic lymphocytic leukemia (CLL) have been lacking.METHODSIn a phase 3, open-label trial, we randomly assigned, in a 1:1:1:1 ratio, fit patients with CLL who did not have TP53 aberrations to receive six cycles of chemoimmunotherapy (fludarabine-cyclophosphamide-rituximab or bendamustine-rituximab) or 12 cycles of venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. Ibrutinib was discontinued after two consecutive measurements of undetectable minimal residual disease or could be extended. The primary end points were undetectable minimal residual disease (sensitivity, RESULTSA total of 926 patients were assigned to one of the four treatment regimens (229 to chemoimmunotherapy, 237 to venetoclax-rituximab, 229 to venetoclax-obinutuzumab, and 231 to venetoclax-obinutuzumab-ibrutinib). At month 15, the percentage of patients with undetectable minimal residual disease was significantly higher in the venetoclax-obinutuzumab group (86.5%; 97.5% confidence interval [CI], 80.6 to 91.1) and the venetoclax-obinutuzumab-ibrutinib group (92.2%; 97.5% CI, 87.3 to 95.7) than in the chemoimmunotherapy group (52.0%; 97.5% CI, 44.4 to 59.5; PCONCLUSIONSVenetoclax-obinutuzumab with or without ibrutinib was superior to chemoimmunotherapy as first-line treatment in fit patients with CLL. (Funded by AbbVie and others; GAIA-CLL13 ClinicalTrials.gov number, NCT02950051; EudraCT number, 2015-004936-36.) BACKGROUNDRandomized trials of venetoclax plus anti-CD20 antibodies as first-line treatment in fit patients (i.e., those with a low burden of coexisting conditions) with advanced chronic lymphocytic leukemia (CLL) have been lacking.METHODSIn a phase 3, open-label trial, we randomly assigned, in a 1:1:1:1 ratio, fit patients with CLL who did not have TP53 aberrations to receive six cycles of chemoimmunotherapy (fludarabine-cyclophosphamide-rituximab or bendamustine-rituximab) or 12 cycles of venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. Ibrutinib was discontinued after two consecutive measurements of undetectable minimal residual disease or could be extended. The primary end points were undetectable minimal residual disease (sensitivity, RESULTSA total of 926 patients were assigned to one of the four treatment regimens (229 to chemoimmunotherapy, 237 to venetoclax-rituximab, 229 to venetoclax-obinutuzumab, and 231 to venetoclax-obinutuzumab-ibrutinib). At month 15, the percentage of patients with undetectable minimal residual disease was significantly higher in the venetoclax-obinutuzumab group (86.5%; 97.5% confidence interval [CI], 80.6 to 91.1) and the venetoclax-obinutuzumab-ibrutinib group (92.2%; 97.5% CI, 87.3 to 95.7) than in the chemoimmunotherapy group (52.0%; 97.5% CI, 44.4 to 59.5; PCONCLUSIONSVenetoclax-obinutuzumab with or without ibrutinib was superior to chemoimmunotherapy as first-line treatment in fit patients with CLL. (Funded by AbbVie and others; GAIA-CLL13 ClinicalTrials.gov number, NCT02950051; EudraCT number, 2015-004936-36.)

Background Fms-related tyrosine kinase 3 (FLT3) receptor serves as a prognostic marker and therapeutic target in acute myeloid leukemia (AML). Approximately one-third of AML patients carry mutation in FLT3, associated with unfavourable prognosis and high relapse rate. The multitargeted kinase inhibitor midostaurin (PKC412) in combination with standard chemotherapy (daunorubicin and cytarabine) was recently shown to increase overall survival of AML patients. For that reason, PKC412 has been approved for treatment of AML patients with FLT3-mutation. PKC412 synergizes with standard chemotherapy, but the mechanism involved is not fully understood and the risk of relapse is still highly problematic. Methods By utilizing the unique nature of mass cytometry for single cell multiparameter analysis, we have explored the proteomic effect and intracellular signaling response in individual leukemic cells with internal tandem duplication of FLT3 (FLT3-ITD) after midostaurin treatment in combination with daunorubicin or cytarabine. Results We have identified a synergistic inhibition of intracellular signaling proteins after PKC412 treatment in combination with daunorubicin. In contrast, cytarabine antagonized phosphorylation inhibition of PKC412. Moreover, we found elevated levels of FLT3 surface expression after cytarabine treatment. Interestingly, the surface localization of FLT3 receptor increased in vivo on the blast cell population of two AML patients during day 3 of induction therapy (daunorubicin; once/day from day 1–3 and cytarabine; twice/day from day 1–7). We found FLT3 receptor expression to correlate with intracellular cytarabine (AraC) response. AML cell line cultured with AraC with or without PKC412 had an antagonizing phosphorylation inhibition of pAKT (p = 0.042 and 0.0261, respectively) and pERK1/2 (0.0134 and 0.0096, respectively) in FLT3high compared to FLT3low expressing cell populations. Conclusions Our study provides insights into how conventional chemotherapy affects protein phosphorylation of vital signaling proteins in human leukemia cells. The results presented here support further investigation of novel strategies to treat FLT3-mutated AML patients with PKC412 in combination with chemotherapy agents and the potential development of novel treatment strategies.

Vitamin D (Vit-D), an essential nutrient, interacts with different drugs including chemotherapeutic agents like busulphan, an alkylating agent used for conditioning prior to stem cell transplantation. The correlation between Vit-D plasma levels and busulphan clearance was investigated in an uncontrolled prospective study in patients and mice. Plasma 25(OH)D levels were measured and busulphan pharmacokinetics calculated in 81 patients. Adults received oral busulphan (n = 34) while children received busulphan orally (n = 19) or intravenously (n = 28). Patients received no Vit-D supplementation. To confirm our findings, pharmacokinetics after a single dose of busulphan (oral or intravenous) were evaluated in two groups of mice (n = 60) receiving high or standard-level Vit-D supplementation. Both busulphan clearance (P < 0.0001) and 25(OH)D levels (P = 0.0004) were significantly higher in adults compared to children. A significant negative correlation (P = 0.041) was found between busulphan clearance and 25(OH)D levels in children treated orally. No such correlation was observed in adults or in children receiving intravenous busulphan. In addition, no significant effect of Vit-D levels on busulphan pharmacokinetics in mice regardless of the administration route. In conclusion, 25(OH)D can affect oral busulphan pharmacokinetics in children and its level should be considered when personalizing oral busulphan treatment. Further studies are warranted to confirm the underlying mechanisms.

Background Elucidation of the genetic mechanisms underlying treatment response to standard induction chemotherapy in AML patients is warranted, in order to aid in risk-adapted treatment decisions as novel treatments are emerging. In this pilot study, we explored the treatment-induced expression patterns in a small cohort of AML patients by analyzing differential gene expression (DGE) over the first 2 days of induction chemotherapy. Methods Blood samples were collected from ten AML patients at baseline (before treatment initiation) and during the first 2 days of treatment (Day 1; approximately 24 h, and Day 2; approximately 48 h after treatment initiation, respectively) and RNA was extracted for subsequent RNA sequencing. DGE between time points were assessed by pairwise analysis using the R package edgeR version 3.18.1 in all patients as well as in relation to treatment response (complete remission, CR, vs non-complete remission, nCR). Ingenuity Pathway Analysis (Qiagen) software was used for pathway analysis and visualization. Results After initial data quality control, two patients were excluded from further analysis, resulting in a final cohort of eight patients with data from all three timepoints. DGE analysis demonstrated activation of pathways with genes directly or indirectly associated with NF-κB signaling. Significant activation of the NF-κB pathway was seen in 50% of the patients 2 days after treatment start, while iNOS pathway effects could be identified already after 1 day. nCR patients displayed activation of pathways associated with cell cycle progression, oncogenesis and anti-apoptotic behavior, including the STAT3 pathway and Salvage pathways of pyrimidine ribonucleotides. Notably, a significant induction of cytidine deaminase, an enzyme responsible for the deamination of Ara-C, could be observed between baseline and Day 2 in the nCR patients but not in patients achieving CR. Conclusions In conclusion, we show that time-course analysis of gene expression represents a feasible approach to identify relevant pathways affected by standard induction chemotherapy in AML patients. This poses as a potential method for elucidating new drug targets and biomarkers for categorizing disease aggressiveness and evaluating treatment response. However, more studies on larger cohorts are warranted to elucidate the transcriptional basis for drug response.

Background: For fit CLL patients (pts), continuous BTK inhibitor treatment is replacing CIT as standard of care in frontline setting, particularly in pts with unfavorable prognostic factors. The time limited combinations venetoclax plus obinutuzumab (GVe) and venetoclax plus rituximab (RVe) have produced high rates of undetectable minimal residual disease (uMRD), which strongly associates with long progression-free survival (PFS) both in frontline and relapsed setting. For frontline therapy GVe is approved in this setting based on data from the CLL14 trial in an unfit population. However, data from a fit cohort are not yet available. The GAIA (CLL13) trial evaluated the efficacy and safety of three Ven+CD20 antibody-based regimens in comparison to CIT as a frontline treatment for fit pts with CLL and without TP53 mutation/deletion. Methods: Treatment-naïve fit (CIRS ≤6, normal creatinine clearance with ≥ 70ml/min) CLL pts requiring therapy were eligible. Based on known poor response to CIT, pts with TP53 aberrations were excluded. Pts were randomized in a 1:1:1:1 ratio to receive six courses of CIT (FCR for pt ≤65 years: fludarabine 25 mg/m² d1-3, cyclophosphamide 250 mg/m² d1-3, rituximab 375 mg/m² d1 cycle 1 and 500 mg/m² d1 cycle 2-6; BR for pt >65 years: bendamustine 90mg/m² d1-2, rituximab) or one of three venetoclax (V) combinations (standard ramp-up from cycle 1 d22, 400 mg/d cycle 2-12): V and rituximab (375/500mg/m² d1 cycle 1-6) [RVe], V and obinutuzumab (1000 mg d1, 8, 15 cycle 1 and d1 cycle 2-6) [GVe], or V, obinutuzumab and ibrutinib (420 mg/d cycle 1-12, if MRD-detectable continued until cycle 36) [GIVe] . Pts were stratified according to country, Binet stage and age (≤ 65/> 65 years). The co-primary endpoints of the trial are (1) the rate of uMRD ( Trial is registered at ClinicalTrials.gov (NCT02950051). Results: A total of 926 pts (CIT: 229 (150 FCR, 79 BR), RVe: 237, GVe: 229, GIVe: 231) with a median age of 61 years (range 27-84) were accrued between 12/2016 and 09/2019. The majority of pts were in advanced Binet stage (B: 37.8%, C: 35.6%) and unmutated IGHV status was present in 56%. Fourteen pts did not receive study treatment (13 FCR, 1 GVe) and were not included in the safety population. The data cut for the first co-primary endpoint analysis was February 28, 2021. The median observation time was 27.9 months. The co-primary endpoint uMRD in PB at MO15 was met as the rate of uMRD in ITT population was significantly higher in GVe compared to CIT: 86.5% (97.5% CI 80.6-91.1) vs 52.0% (CI 44.4-59.5; p The most common grade 3-5 treatment-emergent AE were neutropenia (50.5% of all pts), thrombocytopenia (12.2%), tumor lysis syndrome (7.5%), infusion-related reaction (7.2%), febrile neutropenia (6.5%) and pneumonia (5.3%)). Atrial fibrillation and bleeding events occurred more frequently in GIVe while infusion-related reactions were most common in the GVe arm (Table 1). The absolute numbers of second malignancies were 33, 19, 22 and 21 for CIT, RVe, GVe and GIVe. Fatal AEs occurred in 5, 7, 6 and 9 of the patients. Conclusions: The time-limited therapies of GVe and GIVe provided superior uMRD rates in PB at MO 15 compared to CIT. In addition, uMRD rates in BM and CRR were higher in GVe and GIVe in particular than in CIT. All arms showed a good safety profile in this fit pt population. Figure 1 Figure 1. Disclosures Eichhorst: AbbVie: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; BeiGene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AstraZeneca: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Speakers Bureau; Adaptive Biotechnologies: Speakers Bureau; Hexal: Speakers Bureau; ArQule: Membership on an entity's Board of Directors or advisory committees; Oxford Biomedica (UK): Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; Gilead: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Travel, accomodation, expenses, Research Funding, Speakers Bureau; Consultant Department I for Internal Medicine: Consultancy; University Hospital of Cologne: Current Employment. Kater: Genmab, LAVA: Other: Ad Board, Steering Committee; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; Abbvie: Honoraria, Other: Ad Board, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding. Von Tresckow: Celgene: Other: travel grant; AstraZeneca: Honoraria, Other; Roche: Honoraria, Other: Reasearch support, travel grant; Janssen: Honoraria, Other: Reasearch support, travel grant; AbbVie: Honoraria, Other: advisory board, travel grant. Staber: Roche: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Astra Zeneca: Consultancy, Honoraria; Takeda: Consultancy, Research Funding; MSD: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria. Tadmor: Janssen: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding. Poulsen: Janssen: Consultancy; Abbvie: Consultancy. Janssens: Sanofi: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Beigene, AstraZeneca: Consultancy, Speakers Bureau; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Trael Grant, Speakers Bureau; Abbvie, Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Noesslinger: Roche: Speakers Bureau; Abbvie,: Speakers Bureau; Janssen: Speakers Bureau; AstraZeneca: Honoraria; Gilead: Honoraria; Celgene: Honoraria. Jaeger: Norvartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: 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. Frederiksen: Abbvie: Research Funding; Gilead: Research Funding; Alexion: Research Funding; Novartis: Research Funding; Janssen Pharmaceuticals: Research Funding. Hebart: Roche: Honoraria; BMS: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria; Janssen: Honoraria. Simon: Gilead: Other: Travel support. Fink: AbbVie: Other: travel grant; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Celgene: Research Funding. Fischer: Abbvie: Honoraria; Roche: Honoraria, Other: Travel Grants. Kreuzer: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Mundipharma: Consultancy, Honoraria, Research Funding, Speakers Bureau. Ritgen: Abbvie: Consultancy, Other: Travel support, Research Funding; Chugai: Consultancy; MSD: Consultancy, Other: Travel support; Roche: Consultancy, Other: Travel support, Research Funding; Celgene: Other: Travel support. Brüggemann: Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Incyte: Other: Advisory Board; Janssen: Speakers Bureau. Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Stilgenbauer: AbbVie, Amgen, AstraZeneca, Celgene, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, Sunesis: Other: Research Support; AbbVie, Amgen, AstraZeneca, Celgene, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, Sunesis: Consultancy; AbbVie, Amgen, AstraZeneca, Celgene, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, Sunesis: Honoraria; AbbVie, Amgen, AstraZeneca, Celgene, Gilead, GSK, Hoffmann-La Roche, Janssen, Novartis, Sunesis: Research Funding. Hallek: Roche: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Research Funding, Speakers Bureau; Abbvie: Consultancy, Research Funding, Speakers Bureau. OffLabel Disclosure: Ibrutinib in combaintion with Venetoclax + Obinutuzumab is not approved.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors. Funding Agencies|Cancer Research Funds of Radiumhemmet [174283]; Thorsmans Stiftelse for Pre-leukemi Forskning; Gunnar Grimfors Gavofond for Hematologisk Forskning; Cathrine Everts Research Foundation; Lars Hierta Memorial Foundation; Emil Anderson Fund for Medical Research; Swedish Research CouncilSwedish Research CouncilEuropean Commission [2013-08807]; Karolinska Institute Foundation and Funds; Karolinska InstituteKarolinska Institutet

n/a Funding Agencies|Lund University; Skane University Hospital

Vitamin D (Vit-D), an essential nutrient, interacts with different drugs including chemotherapeutic agents like busulphan, an alkylating agent used for conditioning prior to stem cell transplantation. The correlation between Vit-D plasma levels and busulphan clearance was investigated in an uncontrolled prospective study in patients and mice. Plasma 25(OH)D levels were measured and busulphan pharmacokinetics calculated in 81 patients. Adults received oral busulphan (n = 34) while children received busulphan orally (n = 19) or intravenously (n = 28). Patients received no Vit-D supplementation. To confirm our findings, pharmacokinetics after a single dose of busulphan (oral or intravenous) were evaluated in two groups of mice (n = 60) receiving high or standard-level Vit-D supplementation. Both busulphan clearance (P 0.0001) and 25(OH)D levels (P = 0.0004) were significantly higher in adults compared to children. A significant negative correlation (P = 0.041) was found between busulphan clearance and 25(OH)D levels in children treated orally. No such correlation was observed in adults or in children receiving intravenous busulphan. In addition, no significant effect of Vit-D levels on busulphan pharmacokinetics in mice regardless of the administration route. In conclusion, 25(OH)D can affect oral busulphan pharmacokinetics in children and its level should be considered when personalizing oral busulphan treatment. Further studies are warranted to confirm the underlying mechanisms.

Purpose Despite therapeutic advances, patients with multiple myeloma (MM) continue to experience disease relapse and treatment resistance. The gene ABCB1 encodes the drug transporter P-glycoprotein, which confers resistance through drug extrusion across the cell membrane. Lenalidomide (Len) is excreted mainly via the kidneys, and, given the expression of P-gp in the renal tubuli, single-nucleotide polymorphisms (SNPs) in the ABCB1 gene may influence Len plasma concentrations and, subsequently, the outcome of treatment. We, therefore, investigated the influence of ABCB1 genetic variants on Len treatment outcomes and adverse events (AEs). Methods Ninety patients with relapsed or refractory MM, who received the second-line Len plus dexamethasone in the Rev II trial, were genotyped for the ABCB1 SNPs 1199G amp;gt; A (Ser400Asn, rs2229109), 1236C amp;gt; T (silent, rs1128503), 2677G amp;gt; T/A (Ala893Ser, rs2032582), and 3435C amp;gt; T (silent, rs1045642) using pyrosequencing, and correlations to response parameters, outcomes, and AEs were investigated. Results No significant associations were found between genotype and either best response rates or hematological AEs, and 1236C amp;gt; T, 2677G amp;gt; T or 3435C amp;gt; T genotypes had no impact on survival. There was a trend towards increased time to progression (TTP) in patients carrying the 1199A variant, and a significant difference in TTP between genotypes in patients with standard-risk cytogenetics. Conclusions Our findings show a limited influence of ABCB1 genotype on lenalidomide treatment efficacy and safety. The results suggest that 1199G amp;gt; A may be a marker of TTP following Len treatment in standard-risk patients; however, larger studies are needed to validate and clarify the relationship. Funding Agencies|Swedish Cancer Society; Swedish Research Council; AFA Insurance; Linkoping University; ALF Grants, Region Ostergotland; Celgene Corporation

Changes in the immune system induced by tyrosine kinase inhibitors (TKI) have been shown to positively correlate with therapy responses in chronic myeloid leukemia (CML). However, only a few longitudinal studies exist and no randomized comparisons between two TKIs have been reported. Therefore, we prospectively analyzed the immune system of newly diagnosed CML patients treated with imatinib (n = 20) or bosutinib (n = 13), that participated in the randomized BFORE trial (NCT02130557). Comprehensive immunophenotyping, plasma protein profiling, and functional assays to determine activation levels of T and NK cells were performed at diagnosis, 3, and 12 months after therapy start. All results were correlated with clinical parameters such as Sokal risk and BCR-ABL load measured according to IS%. At diagnosis, low Sokal risk CML patients had a higher frequency of cytotoxic cells (CD8 + T and NK cells), increased cytotoxic potential of NK cells and lower frequency of naïve and central memory CD4 + T cells. Further, soluble plasma protein profile divided patients into two distinct clusters with different disease burden at diagnosis. During treatment, BCR-ABL IS% correlated with immunological parameters such as plasma proteins, together with different memory subsets of CD4+ and CD8 + T cells. Interestingly, the proportion and cytotoxic potential of NK cells together with several soluble proteins increased during imatinib treatment. In contrast, no major immunological changes were observed during bosutinib treatment. In conclusion, imatinib and bosutinib were shown to have differential effects on the immune system in this randomized clinical trial. Increased number and function of NK cells were especially observed during imatinib therapy. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

Background: Non-HLA genes may contribute to the prognosis after hematopoietic stem cell transplantation. We investigated associations between single nucleotide polymorphisms in regions of MORC4, CD14, TLR4, NOD2, SLC22A4, SLC22A5, CARD8, NLRP3, and CLDN2 and the outcomes of patients undergoing allogeneic stem cell transplantation. Material/Methods: Single nucleotide polymorphisms in selected regions were determined and analyzed for putative associations with overall mortality and acute graft-versus-host disease. Significant associations were further explored by logistic regression, controlling for additional variables. Results: A significant association was identified between overall mortality among recipients and a nonsynonymous coding variant of MORC4 (rs6622126) in the recipient genetic makeup (P=0.029). Since MORC4 is located on the X-chromosome, the results were also analyzed separately for males and females. The association between overall mortality for recipients and the risk allele (rs6622126; A) was confirmed for males with respect to genetic makeup of recipients (P=0.012), donor genetic makeup (P=0.004), and the combined allele composition of the donor and recipient (P=0.001). A significant association was also identified between overall mortality and the recipient risk allele of CD14 (rs2569190; P=0.031), TLR4 (rs4986790; P=0.043), and NOD2 (carriage of at least 1 mutant allele of rs2066844, rs2066845, or rs2066847; P=0.048). Among the investigated genes, only the CD14 (rs2569190) recipient risk allele was significantly associated with acute graft-versus-host disease (P=0.023). Logistic regression models confirmed these findings, except for NOD2, and also identified a significant contribution by age at stem cell transplantation (MORC4, CD14, TLR4), diagnosis (CD14, TLR4), and prophylaxis (MORC4). Conclusions: Genetic variation in MORC4, CD14, and TLR4 may affect the outcome of allogeneic stem cell transplantation.

Circulating mitochondrial DNA (mtDNA) is receiving increasing attention as a danger-associated molecular pattern in conditions such as autoimmunity, cancer, and trauma. We report here that human lymphocytes [B cells, T cells, natural killer (NK) cells], monocytes, and neutrophils derived from healthy blood donors, as well as B cells from chronic lymphocytic leukemia patients, rapidly eject mtDNA as web filament structures upon recognition of CpG and non-CpG oligodeoxynucleotides of class C. The release was quenched by ZnCl2, independent of cell death (apoptosis, necrosis, necroptosis, autophagy), and continued in the presence of TLR9 signaling inhibitors. B-cell mtDNA webs were distinct from neutrophil extracellular traps concerning structure, reactive oxygen species (ROS) dependence, and were devoid of antibacterial proteins. mtDNA webs acted as rapid (within minutes) messengers, priming antiviral type I IFN production. In summary, our findings point at a previously unrecognized role for lymphocytes in antimicrobial defense, utilizing mtDNA webs as signals in synergy with cytokines and natural antibodies, and cast light on the interplay between mitochondria and the immune system. Funding agencies: Linkoping Medical Society; Linkoping University; ALF grants; Region Ostergotland, Sweden; Linkoping University Cancer; Ingrid Asp Foundation; Swedish Cancer Society

Background: Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with chronic myeloid leukaemia. Many patients have deep molecular responses, a prerequisite for TKI therapy discontinuation. We aimed to define precise conditions for stopping treatment. Methods: In this prospective, non-randomised trial, we enrolled patients with chronic myeloid leukaemia at 61 European centres in 11 countries. Eligible patients had chronic-phase chronic myeloid leukaemia, had received any TKI for at least 3 years (without treatment failure according to European LeukemiaNet [ELN] recommendations), and had a confirmed deep molecular response for at least 1 year. The primary endpoint was molecular relapse-free survival, defined by loss of major molecular response (MMR; >0·1% BCR-ABL1 on the International Scale) and assessed in all patients with at least one molecular result. Secondary endpoints were a prognostic analysis of factors affecting maintenance of MMR at 6 months in learning and validation samples and the cost impact of stopping TKI therapy. We considered loss of haematological response, progress to accelerated-phase chronic myeloid leukaemia, or blast crisis as serious adverse events. This study presents the results of the prespecified interim analysis, which was done after the 6-month molecular relapse-free survival status was known for 200 patients. The study is ongoing and is registered with ClinicalTrials.gov, number NCT01596114. Findings: Between May 30, 2012, and Dec 3, 2014, we assessed 868 patients with chronic myeloid leukaemia for eligibility, of whom 758 were enrolled. Median follow-up of the 755 patients evaluable for molecular response was 27 months (IQR 21–34). Molecular relapse-free survival for these patients was 61% (95% CI 57–64) at 6 months and 50% (46–54) at 24 months. Of these 755 patients, 371 (49%) lost MMR after TKI discontinuation, four (1%) died while in MMR for reasons unrelated to chronic myeloid leukaemia (myocardial infarction, lung cancer, renal cancer, and heart failure), and 13 (2%) restarted TKI therapy while in MMR. A further six (1%) patients died in chronic-phase chronic myeloid leukaemia after loss of MMR and re-initiation of TKI therapy for reasons unrelated to chronic myeloid leukaemia, and two (

Purpose Tyrosine kinase inhibitors (TKIs) have well-characterized immunomodulatory effects on T and NK cells, but the effects on the humoral immunity are less well known. In this project, we studied TKI-induced changes in B cell-mediated immunity.Methods We collected peripheral blood (PB) and bone marrow (BM) samples from chronic myeloid leukemia (CML) patients before and during first-line imatinib (n = 20), dasatinib (n = 16), nilotinib (n = 8), and bosutinib (n = 12) treatment. Plasma immunoglobulin levels were measured, and different B cell populations in PB and BM were analyzed with flow cytometry.Results Imatinib treatment decreased plasma IgA and IgG levels, while dasatinib reduced IgM levels. At diagnosis, the proportion of patients with IgA, IgG, and IgM levels below the lower limit of normal (LLN) was 0, 11, and 6% of all CML patients, respectively, whereas at 12 months timepoint the proportions were 6% (p = 0.13), 31% (p = 0.042) and 28% (p = 0.0078). Lower initial Ig levels predisposed to the development of hypogammaglobulinemia during TKI therapy. Decreased Ig levels in imatinibtreated patients were associated with higher percentages of immature BM B cells. The patients, who had low Ig levels during the TKI therapy, had significantly more frequent minor infections during the follow-up compared with the patients with normal Ig values (33% vs. 3%, p = 0.0016). No severe infections were reported, except recurrent upper respiratory tract infections in one imatinib-treated patient, who developed severe hypogammaglobulinemia.Conclusions TKI treatment decreases plasma Ig levels, which should be measured in patients with recurrent infections.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naive CD56(bright) NK cells had decreased relapse-free survival. In addition, the TNF-alpha/IFN-gamma cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents. Funding Agencies|Nordic Cancer Union; Finnish Society of Hematology; Biomedicum Helsinki Foundation; Research Foundation of Blood Diseases in Finland; Academy of Finland; Finnish Cancer Organizations; Signe and Ane Gyllenberg Foundation; Finnish Cancer Institute; Doctoral Programme in Clinical Research in the University of Helsinki

Introduction During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. Aim To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. Materials and Methods We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. Results At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n = 7). Dasatinib also decreased platelet aggregation (n = 4-6) and in vitro thrombus formation (n = 3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n = 7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients, see Figure 1. Download : Download high-res image (467KB) Download : Download full-size image Fig. 1 . A. Dasatinib (70 mg/day)-treated patient showing no pro-coagulant platelet formation (red arrow). B. Dasatinib (80 mg/day)-treated patient with pro-coagulant platelt formation (red arrow). Conclusions Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Background: Cytochrome P450 3A (CYP3A) isoenzyme metabolic activity varies between individuals and is therefore a possible candidate of influence on the therapeutic outcome of the tyrosine kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML). The aim of this study was to investigate the influence of CYP3A metabolic activity on the plasma concentration and outcome of imatinib in patients with CML. Methods: Forty-three patients with CML were phenotyped for CYP3A activity using quinine as a probe drug and evaluated for clinical response parameters. Plasma concentrations of imatinib and its main metabolite, CGP74588, were determined using liquid chromatography-mass spectrometry. Results: Patients with optimal response to imatinib after 12 months of therapy did not differ in CYP3A activity compared to nonoptimal responders (quinine metabolic ratio of 14.69 and 14.70, respectively; P = 0.966). Neither the imatinib plasma concentration nor the CGP74588/imatinib ratio was significantly associated with CYP3A activity. Conclusions: The CYP3A activity does not influence imatinib plasma concentrations or the therapeutic outcome. These results indicate that although imatinib is metabolized by CYP3A enzymes, this activity is not the rate-limiting step in imatinib metabolism and excretion. Future studies should focus on other pharmacokinetic processes so as to identify the major contributor to patient variability in imatinib plasma concentrations. Funding Agencies|Swedish Research Council; Swedish Cancer Society; Medical Research Council of Southeast Sweden; Novartis

Musculoskeletal pain in patients with chronic myeloid leukemia after discontinuation of Imatinib : a Tyrosine Kinase Inhibitor withdrawal syndrome?

Few studies have evaluated long-term health-related quality of life (HRQL) in patients during auto-SCT. This prospective study examined HRQL in 96 eligible patients before, during and up to 3 years after auto-SCT. The aim of the study was to make a comprehensive assessment of the frequency and severity of different symptoms in patients undergoing auto-SCT. The European Organization for Treatment and Research of Cancer Quality of Life Questionnaire (EORTC QLQ C-30) was administered 13 times. The second week during treatment was the period when patients had the lowest HRQL regarding both total quality of life and function and symptom scales. The patients recovered quickly and just two months after transplantation the baseline values were restored. Three years after transplantation most of the items in the questionnaire had stabilized, except role function and dyspnea, which had improved. There were significant differences between multiple myeloma (MM) and lymphoma patients' physical function, quality of life, fatigue and pain during week 2. At the 3-year follow-up, lymphoma patients indicated a better HRQL than MM patients. The quick recovery of patients after transplantation suggests that treatment is well tolerated; however, the supportive care could be improved at week 2, especially for the lymphoma patients.

In order to study nucleoside analog activation in the CEM cell line, a transfection protocol had to be optimized in order to silence an enzyme involved in nucleoside analog activation. Hematopoetic cell lines can be difficult to transfect with traditional lipid-based transfection, so the electroporation technique was used. Field strength, pulse length, temperature, electroporation media, siRNA concentration, among other conditions were tested in order to obtain approximately 70-80% mRNA and enzyme activity downregulation of the cytosolic enzyme deoxycytidine kinase (dCK), necessary for nucleoside analog activation. Downregulation was assessed at mRNA and enzyme activity levels. After optimizing the protocol, a microarray analysis was performed in order to investigate whether the downregulation was specific. Additionally two genes were differentially expressed besides the downregulation of dCK. These were however of unknown function. The leakage of intracellular nucleotides was also addressed in the electroporated cells since it can affect the DNA repair mechansism and the efficiency of nucleoside analogs. Three of these pools were increased compared to untreated, unelectroporated cells. The siRNA transfected cells with reduced dCK expression and activity showed reduced sensitivity to several nucleoside analogs as expected. The multidrug resistance to other drugs, as seen in nucleoside analog-induced resistant cells, was not seen with this model. The original publication is available at www.springerlink.com:Anna Fyrberg and Kourosh Lotfi, Optimization and evaluation of electroporation delivery of siRNA in the human leukemic CEM cell line, 2010, CYTOTECHNOLOGY, (62), 6, 497-507.http://dx.doi.org/10.1007/s10616-010-9309-6Copyright: Springer Science Business Mediahttp://www.springerlink.com/

Overexpression of the multi-drug transporter P-glycoprotein, encoded by the ABCB1 gene, is a clinically relevant problem in acute myeloid leukemia (AML). Polymorphisms in ABCB1 might contribute to cancer risk and therapeutic response. We therefore investigated the influence of polymorphisms G1199A, C1236T, G2677T/A and C3435T on cancer susceptibility, in vitro cytotoxicity and overall survival in 100 de novo AML patients with normal karyotype. Patients with 1236C/C or 2677G/G genotypes showed poorer survival than patients with other genotypes (P = 0.03 and P = 0.02, respectively). Both these genotypes were significant factors for survival in multivariate analysis, along with age, NPM1 and FLT3 mutation status. In vitro cytotoxicity studies demonstrated that leukemic cells from 1236T/T and 2677T/T patients were significantly more susceptible to mitoxantrone (P 0.02), and tended to be more susceptible to etoposide and daunorubicin (P = 0.07-0.09), but not to cytarabine. No significant difference in allele frequencies was found between patients and healthy volunteers (n = 400). Funding Agencies|Swedish Cancer Society||Swedish Research Council-Medicine||Cancer Society in Stockholm||Karolinska Institutet||County Council in Ostergotland

Mitochondrial deoxyguanosine kinase (dGK), is an enzyme responsible for activation of nucleoside analogs (NAs) to phosphorylated compounds which exert profound cytotoxicity, especially in hematological malignancies. Screening malignant melanoma cell lines against NAs revealed high sensitivity to several of them. This was believed to be due to the high levels of dGK expression in these cells. Downregulation of dGK in the melanoma cell line RaH5 using siRNA did not cause resistance to NAs as expected, but instead cells became more sensitive. This was probably partly due to the increased activity of another mitochondrial enzyme, thymidine kinase 2, seen in transfected cells.

Salinomycin has been used as treatment for malignant tumors in a small number of humans, causing far less side effects than standard chemotherapy. Several studies show that Salinomycin targets cancer-initiating cells (cancer stem cells, or CSC) resistant to conventional therapies. Numerous studies show that Salinomycin not only reduces tumor volume, but also decreases tumor recurrence when used as an adjuvant to standard treatments. In this study we show that starvation triggered different stress responses in cancer cells and primary normal cells, which further improved the preferential targeting of cancer cells by Salinomycin. Our in vitro studies further demonstrate that the combined use of 2-Fluoro 2-deoxy D-glucose, or 2-deoxy D-glucose with Salinomycin is lethal in cancer cells while the use of Oxamate does not improve cell death-inducing properties of Salinomycin. Furthermore, we show that treatment of cancer cells with Salinomycin under starvation conditions not only increases the apoptotic caspase activity, but also diminishes the protective autophagy normally triggered by the treatment with Salinomycin alone. Thus, this study underlines the potential use of Salinomycin as a cancer treatment, possibly in combination with short-term starvation or starvation-mimicking pharmacologic intervention.

Telomerase reverse transcriptase gene (TERT) promoter mutations are identified in many malignancies but not in hematological malignancies. Here we analyzed TERT and protection of telomeres 1 gene (POT1) mutations, and four different TERT SNVs in 226 acute myeloid leukemia (AML) patients and 806 healthy individuals in a case referent design, where also overall survival was assessed. A significant association for increased risk of AML was found for TERT SNVs, rs2853669 (OR = 2.45, p = 0.00015) and rs2736100 (OR = 1.5, p = 0.03). The overall survival for patients with CC genotype of rs2853669 was significantly shorter compared to those with TT or TC genotypes (p = 0.036 and 0.029 respectively). The influence of TERT rs2853669 CC on survival was confirmed in multivariable Cox regression analysis as an independent risk biomarker in addition to high risk group, higher age and treatment. No hot spot TERT promoter mutations at -228Cgreater thanT or -250Cgreater thanT or POT1 mutations could be identified in this AML cohort. We show that rs2853669 CC may be a risk factor for the development of AML that may also be used as a prognostic marker to identify high risk normal karyotype -AML (NK-AML) patients, for treatment guidance. Funding Agencies|Swedish Research Council; Swedish Cancer Society; County Council of Ostergotland; AFA Insurance; FORSS

We randomised 46 newly diagnosed patients with chronic myeloid leukaemia (median age 56) to receive dasatinib 100 mg QD or imatinib 400 mg QD and report outcome as an intention-to-treat analysis with 36 months follow-up. Early cytogenetic and molecular responses were superior in the dasatinib group, with a tendency that imatinib patients caught up with time. For instance, MR3.0 was reached at 3 months in 36% vs. 8% (P = 0.02), at 12 months in 81% vs. 46% (P = 0.02) and at 18 months in 73% vs. 65% (n.s.) of the patients in the two groups. In contrast, MR4.5 was consistently superior in the dasatinib group at all time points from 6 months onwards, reaching 61% vs. 21% (P < 0.05) at 36 months. Sixty-four vs. 71% of the patients in the dasatinib and imatinib arms, respectively, remained on assigned drug. Dasatinib dose was frequently reduced, but with maintained excellent effect. One imatinib patient progressed to blastic phase, but no CML-related deaths occurred. In conclusion, our data compare favourably with those of the dasatinib registration study, DASISION. The fast and deep molecular responses induced by dasatinib compared with imatinib may be exploited to increase the proportion of patients who can achieve a treatment-free remission after treatment discontinuation © 2014 The Authors. European Journal of Haematology Published by John Wiley & Sons Ltd. 243 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made

Health-related quality of life (HRQL) was evaluated in 94 patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative (MAC, n = 18) or reduced intensity conditioning (RIC, n = 76). HRQL was assessed with the EORTC QLQ C-30 during the inpatient period as well as during the following 3 years, i.e., at baseline and 12 times thereafter. Functional status and global quality of life decreased from baseline to weeks 2 and 3, especially role and social functions. Symptoms increased significantly during the first 3 weeks, particularly appetite loss, nausea and vomiting, diarrhoea and fatigue. It took at least 1 year for HRQL to return to the baseline level. The only function that improved significantly 3 years after HSCT was role function. Patients treated with MAC experienced significantly worse HRQL at baseline than patients treated with RIC, as well as more pain, sleep disturbance and appetite loss in weeks 3 and 4. Patients with extensive chronic graft-versus-host disease experienced reduced HRQL. These results provide a clinically useful overview of patients' HRQL during and after HSCT and indicate when they require increased support. The results demonstrate the importance of close follow-ups during the first year after HSCT to improve preventive or supportive interventions.

Deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK) catalyze the first step in the intracellular cascade of fludarabine (2-fluoroadenine-beta-D-arabinofuranoside) and cladribine (2-chlorodeoxyadenosine) phosphorylation, which leads to activation of these prodrugs, commonly used for treatment of chronic lymphocytic leukemia (CLL). Thus, resistance to nucleoside analogues may primarily be due to low levels of deoxynucleoside kinase activity. The purpose of this study was to investigate the activity profiles of dCK and dGK and characterize the possible relationship between the levels of dCK enzymatic activities and mRNA levels in B-CLL cells from untreated patient samples in an attempt to determine the best approach for predicting sensitivity to nucleoside analogues and thereby optimizing treatment of CLL. For this purpose, dCK and dGK analyses were done in blood cells from 59 untreated symptomatic patients with CLL. The dGK activity towards 2-chlorodeoxyadenosine was significantly lower than of dCK (median 73 pmol/mg protein/min (85-121, 95% CI) versus 353 pmol/mg protein/min (331-421)). The median dCK mRNA level was 0.107 (0.096-0.120, 95% CI). There was a lack of correlation between the activities of dCK and dGK, which indicates that these proteins are regulated independently. We also found that the dCK and dGK activity measurement towards their endogenous substrates were comparable to the nucleoside analogues tested. Such variations in enzyme activities and mRNA levels may well explain differences in clinical responses to treatment. There was no correlation between the levels of dCK mRNAs and enzymatic activities using a quantitative real-time PCR procedure. Sequencing of dCK mRNA did not reveal alternate splicing or mutations in the coding region. The relation between activity and mRNA levels was studied by short interfering RNA (siRNA) method, which showed that in the siRNA treated cells the down-regulation of dCK expression, and activity followed each other. However, in control cells the mRNA levels remained stable but the protein activity markedly decreased. These data demonstrate that the dCK activity is not reflected by dCK mRNA expression that indicates a post-translational mechanism(s).

Increased expression of P-glycoprotein encoded by the mdr-1 gene is a well-characterised mechanism for resistance to cancer chemotherapeutic drugs in cell lines. However, the P-glycoprotein expression after removal of the selection pressure has not fully been elucidated. The stability of P-glycoprotein expression in the presence (+) and absence (-) of vincristine (30 or 150 nM) was studied in multidrug resistant K562 cell lines (VCR30+, VCR150+, VCR30- and VCR150-) for 11 months. The P-glycoprotein protein and mdr-1 mRNA levels were determined at regular intervals using flow cytometry and real-time PCR, respectively. Chemosensitivity to a panel of antineoplastic drugs was measured using an MTT assay. The presence of vincristine (VCR30+ and VCR150+) resulted in high and stable levels of P-glycoprotein and mdr-1 mRNA during the whole period compared to wild type. As for the VCR30- and VCR150- subcultures, the expressions of P-glycoprotein and mdr-1 mRNA were stable for five months, and then the levels decreased rapidly. Concomitantly, the sensitivity to drugs known as P-glycoprotein substrates was restored. In conclusion, resistant cells growing in the presence of the inducing drug have a stable P-glycoprotein expression and resistance level, but removing the inducing drug may result in a sudden and rapid lowering of P-glycoprotein and mdr-1 mRNA levels as long as five months after drug withdrawal. The definitive version is available at www.blackwell-synergy.com:Henrik Green, Kourosh Lotfi, Anna Lena Zackrisson and Curt Peterson, Spontaneous Reversal of P-Glycoprotein Expression in Multidrug Resistant Cell Lines, 2003, Pharmacology and Toxicology, (93), 6, 297-304.http://dx.doi.org/10.1111/j.1600-0773.2003.pto930608.xPostprint available at: Linköping University Electronic Presshttp://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-46413

The inherent or acquired resistance of leukemic cells to cytostatic agents is a major clinical challenge. The purpose of this review was to elucidate and analyse the available data concerning mechanisms of resistance of cladribine with emphasis on recent advances in the characterization of activating and inactivating enzymes in the induction of resistance to cladribine. All available in vitro and clinical data on cladribine was undertaken. Cladribine, unlike many other drugs, is toxic to both dividing and indolent lymphoid malignancies. Cladribine is a prodrug and must be phosphorylated intracellularly to cladribine-monophosphate (MP) by the nuclear/cystosol enzyme deoxycytidine kinase (dCK) and the mitochondrial enzyme deoxyguanosine kinase. The cytotoxicity mainly depends on the accumulation of cladribine-triphosphates (TP) after phosphorylation of cladribine-MP by nucleoside monophosphate kinase and nucleoside diphosphate kinase. 5'-Nucleotidase (5'-NT) dephosphorylates cladribine-MP and the accumulation of cladribine-TP depends on the ratio of dCK and 5'-NT in the cells. The mechanisms underlying cladribine resistance are multifactorial, e.g. decreased nucleoside transport, decreased activity or deficiency of dCK, altered intracellular pools of competing nucleotides, altered regulation of ribonucleotide reductase and increased drug inactivation by 5'-NT. Finally, cladribine resistance may be a consequence of a defective induction of apoptosis. In spite of the fact that more than one mechanism can contribute to a cladribine resistance phenotype, a reduction in dCK activity is probably the major determinant of cladribine resistance. Insight into the mechanism of action and resistance to cladribine is crucial for its optimal use as well as for the development of newer analogues.

Anthracycline antibiotics are widely used as anticancer agents. Idarubicin (4-demethoxydaunorubicin; Ida), a semisynthetic derivative of daunorubicin (Dnr) is more potent than the parent compound in vitro and in vivo. The equitoxic dose of Ida in patients is about one-fourth of that of Dnr. We compared these drugs regarding cytotoxicity, apoptosis induction, and resistance mechanisms in human leukaemic cell lines. Cytotoxicity was studied by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide assay and drug-induced apoptosis by means of the Annexin V-fluorescein isothiocyanate method at similar intracellular concentrations (extracellular concentrations of 0.35 microM for Ida and 1 microM for Dnr). Ida was at least twice as potent as Dnr in MOLT-4, HL60, CEM, and K562 cell lines. It took 8 h for Ida to induce approximately 20% apoptosis, but at least 22 h for Dnr to reach 20% apoptosis at identical intracellular concentration. Ida induces a faster and higher apoptosis rate compared with Dnr. The human chronic myelogenous leukaemia cell line (K562) was selected for resistance to Dnr and Ida with and without verapamil (Ver). Continuous incubation with Dnr, but not with Ida, led to an increased mdr1 gene expression as assessed by real-time PCR. The development of mdr1 gene expression in Dnr-resistant cells could be reversed by the presence of Ver. Ver also reversed the cytotoxicity to Dnr, but not to Ida, in K562/Dnr cells. The results show that Ida is more effective than Dnr in inducing apoptosis and that there are differences in resistance mechanisms between the drugs.

Introduction: Multiple myeloma (MM) is an incurable plasma cell malignancy with high mortality rate. Treatment outcomes have improved since the introduction of new drugs such as the IMiD lenalidomide, but relapse rates and resistance is still a problem. The gene ABCB1 encodes the drug transporter p-glycoprotein (p-gp) which confers resistance through extrusion of drugs over the cell membrane. Lenalidomide is subject to limited metabolism and excreted mainly via the kidneys. In vitro studies have shown lenalidomide to be an ABCB1 substrate, and single nucleotide polymorphisms (SNPs) affecting gene expression, transporter function and/or activity may affect drug distribution and the subsequent outcome and risk of adverse events. However, in vivo studies of the effect of ABCB1 on lenalidomide pharmacokinetics are contradictory. Our aim was to investigate the influence of ABCB1 SNPs on lenalidomide treatment outcome and adverse events (AE). Materials & Methods: In the observational part of two connected studies, 133 Lenalidomide naïve patients at 1st relapse/refractory MM were treated with lenalidomide and dexamethasone for up to 9 cycles of 4 weeks. In the prospective 2nd part, 62 patients that had achieved at least partial response according to IMWG-criteria followed by at least two additional treatment cycles were randomized to either lenalidomide/dexamethasone or lenalidomide as a single drug. 90 patients (of which 47 was further randomized to the 2nd part) had samples available for genotyping of the ABCB1 SNPs 1199G>A (Ser400Asn, rs2229109), 1236C>T (rs1128503), 2677G>T/A (Ala893Ser, rs2032582) and 3435C>T (rs1045642) using Pyrosequencing. Correlations to overall survival, time to progression (TTP), response parameters and AE were investigated, and a p-value of 0.05 was considered significant. Results: No significant correlations to hematological AE or response rates were found, and no impact on survival for 1236C>T, 2677G>T/A or 3435C>T, neither in the whole population nor in patients randomized to the 2nd part. The results were similar also when risk (according to FISH) was considered. There was a trend towards improved TTP for patients carrying the 1199A variant; mean TTP 3.2 years (95%CI 2.3-4.1) vs 2.2 years (95%CI 1.8-2.6) for G/A and G/G, respectively (p=0.076). This trend was confirmed in the multivariable cox regression analysis; HR=0.280 (95%CI 0.74-1.054), p=0.06. The difference in TTP was significant in the non-high risk subgroup; mean TTP 4.3 years (95%CI 3.7-4.9) vs 2.3 years (95%CI 1.8-2.8), p=0.034, for G/A and G/G, respectively. Conclusion: No evidence was found for a large impact of 1236C>T, 2677G>T/A or 3435C>T on lenalidomide treatment outcome or risk of hematological AE. 1199G>A may be a potential marker of TTP in non-high risk MM but further studies in a larger cohort is needed to clarify the relationship and whether this is due to altered drug transport or efflux independent mechanisms. Citation Format: Ingrid Jakobsen Falk, Johan Lund, Henrik Gréen, Astrid Gruber, Evren Alici, Birgitta Lauri, Cecilie Blimark, Ulf-Henrik Mellqvist, Agneta Swedin, Karin Forsberg, Conny Carlsson, Mats Hardling, Lucia Ahlberg, Hareth Nahi, Kourosh Lotfi. The impact of ABCB1 single nucleotide polymorphisms on the outcome in lenalidomide treated multiple myeloma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2017-5030

Background The isocitrate dehydrogenase (IDH1/IDH2) genes are metabolic enzymes, which are frequently mutated in acute myeloid leukemia (AML). The enzymes acquire neomorphic enzymatic activity when they mutated. Methods We have investigated the frequency and outcome of the acquired IDH1/IDH2 mutations and the IDH1 SNP 105C > T (rs11554137) in 189 unselected de novo AML patients by polymerase chain reaction amplification followed by direct sequencing. The survival are presented in Kaplan Meier curves with log rank test. Multivariable survival analysis was conducted using Cox regression method, taking age, risk group, treatment, IDH1/2 mutations and IDH1 SNP105 genotype into account. Results Overall, IDH1/2 mutations were found in 41/187 (21.7%) of the AML patients. IDH1 codon 132 mutations were present in 7.9%, whereas IDH2 mutations were more frequent and mutations were identified in codon 140 and 172 in a frequency of 11.1% and 2.6%, respectively. The SNP 105C > T was present in 10.5% of the patients, similar to the normal population. A significantly reduced overall survival (OS) for patients carrying IDH2 codon 140 mutation compared with patients carrying wild-type IDH2 gene (p T variant can represent a new subgroup for risk stratification and may indicate new treatment options.

Drug resistance is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). We have previously reported a relationship between single nucleotide polymorphisms (SNPs) of ABCB1, encoding the multi-drug transporter P-glycoprotein, and overall survival (OS) in normal karyotype (NK)-AML. Here we extended this material, enabling subgroup analysis based on FLT3 and NPM1 status, to further elucidate the influence of ABCB1 SNPs. De novo NK-AML patients (n = 201) were analysed for 1199Ggreater thanA, 1236Cgreater thanT, 2677Ggreater thanT/A and 3435Cgreater thanT, and correlations to outcome were investigated. FLT3 wild-type 1236C/C patients have significantly shorter OS compared to patients carrying the variant allele; medians 20 vs. 49 months, respectively, P = 0.017. There was also an inferior outcome in FLT3 wild-type 2677G/G patients compared to patients carrying the variant allele, median OS 20 vs. 35 months, respectively, P = 0.039. This was confirmed in Cox regression analysis. Our results indicate that ABCB1 1236Cgreater thanT and 2677Ggreater thanT may be used as prognostic markers to distinguish relatively high risk patients in the intermediate risk FLT3 wild-type group, which may contribute to future individualizing of treatment strategies. Funding Agencies|Swedish Cancer Society; County Council of Ostergotland; AFA Insurance; Stockholm Cancer Society; Karolinska Institutet; Swedish Research Council

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIα (P

De novo acute myeloid leukemia with normal karyotype (NK-AML) comprises a large group of patients with no common cytogenetic alterations and with a large variation in treatment response. Single-nucleotide polymorphisms (SNPs) in genes related to the metabolism of the nucleoside analogue AraC, the backbone in AML treatment, might affect drug sensitivity and treatment outcome. Therefore, SNPs may serve as prognostic biomarkers aiding clinicians in individualized treatment decisions, with the aim of improving patient outcomes. We analyzed polymorphisms in genes encoding cytidine deaminase (CDA 79A>C rs2072671 and −451C>T rs532545), 5′-nucleotidase (cN-II 7A>G rs10883841), and deoxycytidine kinase (DCK 3′UTR 948T>C rs4643786) in 205 de novo NK-AML patients. In FLT3-internal tandem duplication (ITD)-positive patients, the CDA 79C/C and −451T/T genotypes were associated with shorter overall survival compared to other genotypes (5 vs. 24 months, P

Recent findings have indicated that tyrosine kinase inhibitors (TKIs) targeting the ERBB receptor family display anti-leukaemic effects, despite the lack of receptor expression on human leukaemic cells. The occurrence of activating mutations in the gene encoding FMS-like tyrosine kinase 3 (FLT3) in patients with acute myeloid leukaemia (AML) has rendered inhibition of this receptor a promising therapeutic target. Due to possibility of cross-reactivity, we investigated the effect of the irreversible pan-ERBB inhibitor canertinib (CI-1033) on leukaemic cells expressing FLT3. The drug had anti-proliferative and apoptotic effects on primary AML cells and human leukaemic cell lines expressing mutated FLT3. In several AML patient samples, a blast cell population expressing FLT3-internal tandem duplication (ITD) was eradicated by canertinib. Canertinib inhibited receptor autophosphorylation and kinase activity of both mutated and FLT3 ligand stimulated wildtype FLT3, leading to inhibition of the PI3-kinase and MAP kinase pathways. Apoptotic induction was dependent on pro-apoptotic BH3-only protein BCL2L11/BIM because siRNA silencing attenuated apoptosis. Moreover, the drug induced regression of cells expressing FLT3-ITD in a murine in vivo-transplantation model at previously described tolerated doses. These results indicate that canertinib, as an irreversible TKI, could constitute a novel treatment regimen in patients with mutated or overexpressed FLT3.

To characterize resistance mechanisms to the nucleoside analog 9-β-D-arabinofuranosylguanine (AraG) in the T-cell acute lymphoblastic leukemia cell line MOLT-4 and its AraG-resistant variant.A gene expression microarray analysis was performed, as well as gene expression and enzyme activity measurements of key enzymes in the activation of AraG. Cytotoxicity of AraG and cross-resistance to other compounds were evaluated using a standard cytotoxicity assay.Gene expression microarray analysis revealed that fetal hemoglobin genes and the multidrug resistance ABCB1 gene, encoding the drug efflux pump P-gp, were the most highly upregulated genes in the resistant cells, while genes traditionally associated with nucleoside analog resistance were not. Fetal hemoglobin and ABCB1 induction can be due to global DNA hypomethylation. This phenomenon was studied using AraG during a period of 4 weeks in MOLT-4 cells and the lung adenocarcinoma cell line A549, leading to up-regulation of hemoglobin gamma and ABCB1 as well as DNA hypomethylation. Inhibiting P-gp in the AraG-resistant MOLT-4 cells led to decreased proliferation, reduced hemoglobin expression, and highly induced ABCB1 expression.We show that AraG can cause hypomethylation of DNA and induce the expression of the fetal hemoglobin gamma gene and the ABCB1 gene. We speculate that the induction of ABCB1/P-gp may occur in order to help with excretion of hemoglobin degradation products that would otherwise be toxic to the cells, and we present data supporting our theory that P-gp may be linked to the induction of hemoglobin.

Essential thrombocythemia (ET) is characterized by high platelet counts and a slightly increased bleeding risk. Why severe hemorrhage does not occur more frequently is not known. Variations of platelet density (kg/l) depend mainly on cell organelle content in that high-density platelets contain more alpha and dense granules. This study compares ET patients (n = 2) and healthy volunteers (n = 2) with respect to platelet density subpopulations. A linear Percoll gradient containing prostaglandin E(1) was employed to separate platelets according to density. The platelet population was subsequently divided by density into 16 or 17 subpopulations. Determination of platelet counts was carried out. In each density fraction, platelet in vivo activity, i.e. platelet-bound fibrinogen, was measured using a flow cytometer. To further characterize platelet subpopulations, we determined intracellular concentrations of CD40 ligand (CD40L) and P-selectin in all fractions. Patients and controls demonstrated similar density distributions, i.e. 1 density peak. High-density platelets had more surface-bound fibrinogen in conjunction with signs of platelet release reactions, i.e. with few exceptions they contained less CD40L and P-selectin. Peak density platelets showed less surface-bound fibrinogen. These platelets contained less CD40L and P-selectin than nearby denser populations. The light platelets had more surface-bound fibrinogen than peak platelets together with elevated concentrations of CD40L. In ET, the malignant platelet production could exist together with platelets originating from normal megakaryocytes. It is also possible that clonal megakaryocytes produce platelets covering the entire density span. The 'normal' density distribution offers a tenable explanation as to why serious bleedings do not occur more frequently. Original Publication: Micha Milovanovic, Kourosh Lotfi, Tomas Lindahl, Claes Hallert and Petter Järemo, Platelet Density Distribution in Essential Thrombocythemia., Pathophysiology of Haemostasis and Thrombosis, 2010. http://dx.doi.org/10.1159/000314964 Copyright: Karger http://www.karger.com/

Imatinib is currently used for the treatment of chronic myeloid leukemia (CML). The main metabolite CGP74588 has similar potency to that of imatinib and is a product of CYP3A4 and CYP3A5 metabolism. However, the clinical significance of the metabolism on therapeutic response and pharmacokinetics is still unclear. We designed this study to investigate the role of the CYP3A activity in the response to imatinib therapy. Fourteen CML patients were phenotyped for in vivo CYP3A activity using quinine as a probe drug. The plasma concentration ratio of quinine and its CYP3A metabolite was used for assessing CYP3A activity. The patients were divided into complete molecular responders with undetectable levels of BCR-ABL transcripts after 12 months of therapy and into partial molecular responders who had failed to achieve a complete molecular response. Patients that achieved complete molecular response showed significantly (Mann-Whitney U-test, p = 0.013) higher in vivo CYP3A activity (median quinine metabolic ratio = 10.1) than patients achieving partial molecular response (median = 15.9). These results indicate a clinical significance of the CYP3A activity and its metabolic products in CML patients treated with imatinib. The original publication is available at www.springerlink.com: Henrik Green, Karin Skoglund, Franz Rommel, Rajaa A Mirghani and Kourosh Lotfi, CYP3A activity influences imatinib response in patients with chronic myeloid leukemia: a pilot study on in vivo CYP3A activity, 2010, EUROPEAN JOURNAL OF CLINICAL PHARMACOLOGY, (66), 4, 383-386. http://dx.doi.org/10.1007/s00228-009-0772-y Copyright: Springer Science Business Media http://www.springerlink.com/

Resistance toward nucleoside analogues is often due to decreased activities of the activating enzymes deoxycytidine kinase (dCK) and/or deoxyguanosine kinase (dGK). With small interfering RNA (siRNA), dCK and dGK were downregulated by approximately 70% in CEM cells and tested against six nucleoside analogues using the methyl thiazol tetrazolium assay. SiRNA-transfected cells reduced in dCK activity were 3- to 6-fold less sensitive to CdA, AraC, and CAFdA. The sensitivity to AraG and FaraA was unchanged, while the sensitivity toward gemcitabine was significantly increased. dGK depletion in cells resulted in lower sensitivity to FaraA, dFdC, CAFdA, and AraG, but slightly higher sensitivity to CdA and AraC.