Kourosh Lotfi

Activated innate lymphoid cells are associated with a reduced susceptibility to graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely used to treat hematopoietic cell disorders but is often complicated by graft-versus-host disease (GVHD), which causes severe epithelial damage. Here we have investigated longitudinally the effects of induction chemotherapy, conditioning radiochemotherapy, and allogeneic HSCT on composition, phenotype, and recovery of circulating innate lymphoid cells (ILCs) in 51 acute leukemia patients. We found that reconstitution of ILC1, ILC2, and NCR(-)ILC3 was slow compared with that of neutrophils and monocytes. NCR(+) ILC3 cells, which are not present in the circulation of healthy persons, appeared both after induction chemotherapy and after allogeneic HSCT. Circulating patient ILCs before transplantation, as well as donor ILCs after transplantation, expressed activation (CD69), proliferation (Ki-67), and tissue homing markers for gut (α4β7, CCR6) and skin (CCR10 and CLA). The proportion of ILCs expressing these markers was associated with a decreased susceptibility to therapy-induced mucositis and acute GVHD. Taken together, these data suggest that ILC recovery and treatment-related tissue damage are interrelated and affect the development of GVHD.

Musculoskeletal Pain in Patients With Chronic Myeloid Leukemia After Discontinuation of Imatinib: A Tyrosine Kinase Inhibitor Withdrawal Syndrome?

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

Comparison of idarubicin and daunorubicin regarding intracellular uptake, induction of apoptosis, and resistance

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.

Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia

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 naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ 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.

Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide

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 < 0·001) and β protein (P < 0·026), formed substantially lower amounts of the topo II–DNA complex, and had a markedly decreased level of Fas (CD95/APO‐1)‐ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P‐glycoprotein, multidrug resistance‐associated protein and the lung resistance‐related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara‐C, was the same for resistant and wild‐type cells. However, there was an increase in the activity of the cytosolic 5′‐nucleotidases (5′‐NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0·001) for the high Km and 134% (P < 0·331) for the low Km 5′‐NT in resistant cells. The high Km 5′‐NT is probably responsible for the decreased amount of the active metabolite CdA 5′‐triphosphate [40% decreased (P < 0·045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0·001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara‐C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5′‐NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide‐degrading enzymes and DNA topoisomerases, which may be altered in cross‐resistant cells.

Dasatinib induces fast and deep responses in newly diagnosed chronic myeloid leukaemia patients in chronic phase: clinical results from a randomised phase-2 study (NordCML006)

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.

Association of ABCB1 polymorphisms with survival and in vitro cytotoxicty in de novo acute myeloid leukemia with normal karyotype

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).

A prospective evaluation of patients' health-related quality of life during auto-SCT: a 3-year follow-up

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.

Pharmacological basis for cladribine resistance.

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.

Decreased survival in normal karyotype AML with single-nucleotide polymorphisms in genes encoding the AraC metabolizing enzymes cytidine deaminase and 5'-nucleotidase.

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 < 0.001 and 5 vs. 23 months, P = 0.015, respectively), and this was most pronounced in FLT3‐ITD‐positive/NPM1‐positive patients. We observed altered in vitro sensitivity to topoisomerase inhibitory drugs, but not to nucleoside analogues, and a decrease in global DNA methylation in cells carrying both CDA variant alleles. A shorter survival was also observed for the cN‐II variant allele, but only in FLT3‐ITD‐negative patients (25 vs. 31 months, P = 0.075). Our results indicate that polymorphisms in genes related to nucleoside analog drug metabolism may serve as prognostic markers in de novo NK‐AML. Am. J. Hematol. 88:1001–1006, 2013.