Daniela Damiani

A comparative analysis of the sensitivity of multidrug resistant (MDR) and non MDR cells to different anthracycline derivatives

Because of the fact that tumor cell sensitivity to cytotoxic agents may play a major role in cancer treatment, and several anthracyclines are widely used for first-line treatment of leukemia, lymphoma and other tumors, and since the overexpression of the mdr-1 gene-coded 170 Kd glycoprotein (P170) decreases cell sensitivity to anthracyclines, we investigated the relationship between P170 overexpression and the cytotoxicity of two classic anthracyclines (Daunorubicin or DNR and Doxorubicin or DX) and two lipophilic anthracycline derivatives (Idarubicin or IDA and Iododoxorubicin or IDX). For these purposes, we used multidrug resistant (MDR) and non-MDR tumor and leukemia cell lines and the MTT-microcultured tetrazolium colorimetric assay. We showed that mdr-1 gene overexpression was strongly associated with the development of a high level of resistance to DNR and DX, but not to the derivatives IDA and IDX. These data suggest that more lipophilic anthracycline derivatives may also be active in MDR cell systems.

The expression of the multidrug resistance-associated glycoprotein in B-cell chronic lymphocytic leukaemia

Summary. The expression of the multidrug resistance (MDR)‐associated 170 kDa glycoprotein (p170) was investigated in 63 cases of B‐cell chronic lymphocytic leukaemia (CLL), with two monoclonal antibodies (MRK‐16 and C‐219). By immunocytochemistry with MRK‐16 (63 cases), the great majority of the cells was positive, with a weak reaction in 61% of cases and a strong reaction in 39% of cases. By flow cytometry, the proportion of positive cells was 39±25% with MRK–16 (63 cases), and 23±22% with C–219 (36 cases). The expression of pl70 in leukaemic B‐lymphocytes suggests that also in B‐CLL the development of MDR can have some therapeutic relevance. By either method the proportion of positive cells was not related to prior treatment, time from diagnosis, absolute lymphocyte count, and clinical stage (Rais and workshop classifications), but 12 patients who were under treatment with α‐interferon had more positive cells than the other ones.

Adjuvant treatment with cyclosporin A increases the toxicity of chemotherapy for remission induction in acute non-lymphocytic leukemia

P-glycoprotein (Pgp)-related multidrug resistance (MDR) is frequently observed in acute non-lymphocytic leukemia (ANLL) and is associated with a poor response to standard chemotherapy. Cyclosporin A (CsA) is an effective downmodulator of Pgp-related MDR in vitro and has already been tested for that purpose in vivo also. Since Pgp is expressed in several normal cells and tissues, the modulation of Pgp can also modify total body exposure to antileukemic drugs and can alter and increase the toxicity of the antileukemic treatment. We report here the results of a study where 46 consecutive adult patients with ANLL were assigned to receive the same standard chemotherapy regimen of arabinosyl cytosine and idarubicin (IDA) for remission induction or consolidation, without or with CsA. Twenty-eight patients received 36 courses of chemotherapy without CsA and 18 patients received 32 courses of chemotherapy with CsA. CsA dose was 10–12.5u2009mg/kg/day and was given as a continuous i.v. infusion for 72u2009h. Whole blood CsA steady-state concentration ranged between 0.61 and 1.14u2009μM. The IDA area-under-the-curve was about twice as high in the cases that received CsA than in the other cases. CsA had no detectable effects on renal function and fluid balance, but significantly increased systemic blood diastolic pressure and conjugated bilirubine concentration. Furthermore, CsA-treated patients had greater, and more severe, oral and intestinal mucosal toxicity, with more severe adverse events, including more cases of gram-negative bacteremia, and with a delayed hemopoietic recovery. In conclusion, this study showed that an attempt at an effective downmodulation of Pgp-mediated MDR would substantially increase the hemopoietic and mucosal toxicity of antileukemic treatment and that the increase is accounted for, at least in part, by an increase of total body exposure to IDA.

ABCG2 overexpression in patients with acute myeloid leukemia: Impact on stem cell transplantation outcome

ABGG2 protein overexpression in acute myeloid leukemia (AML) has been associated with poor response to conventional chemotherapy and increased relapse risk. No data are available on the role of allogeneic stem cell transplantation (SCT) in reversing its negative prognostic role. We have reviewed the outcome of 142 patients with high risk AML who underwent allogeneic SCT in complete remission (n = 94) or with active disease (n = 48). Patients with ABCG2 overexpression at AML diagnosis have lower leukemia free survival (LFS) and increased cumulative incidence of relapse (CIR) compared with ABCG2− patients (5‐year LFS 50% vs. 65%, P = 0.01; 5‐year CIR 46% vs. 27%, P = 0.003). Five‐year overall survival was not significantly different between ABCG2+ and ABCG2− patients (39% vs. 51%, P = 0.1). However, if we consider only disease‐related deaths, ABCG2 maintains its negative role (64% vs. 78%, P = 0.018). The negative impact of ABCG2 overexpression was higher in patients undergoing SCT in CR compared with patients receiving transplant with active disease. Conditioning regimen did not abrogate the effect of ABCG2 overexpression, as CIR was higher in ABCG2+ patients receiving both myeloablative (44% vs. 22%, P = 0.018) or reduced intensity conditioning (50% vs. 32%, P = 0.03). In conclusion, ABCG2 overexpression at AML diagnosis identifies a subset of patients with poor outcome also after allogeneic SCT, mainly in terms of higher relapse rates. Prospective studies employing conditioning drugs or post‐transplant strategies able to target ABCG2 are needed to maximize the curative potential of stem cell transplantation.Am. J. Hematol. 90:784–789, 2015.

Cell kinetic effect of low dose arabinosyl cytosine

Summary. Low dose arabinosyl cytosine (ARA‐C) is effective for treatment of acute non‐lymphocytic leukaemia (ANLL). The mechanism of action is not clearly understood and it was suggested that low doses of the drug could induce leukaemic cells to differentiate. We investigated the effect of low dose ARA‐C (20 mg/m2/d, divided into two doses s.c. at 12 h intervals, × 20 d) on the cell cycle distribution of leukaemic cells in four cases of ANLL. By comparison, four other cases of ANLL were studied during treatment with standard dose ARA‐C (200 mg/m2/d as a continuous i.v. infusion × 7 d). Both treatments induced an accumulation of leukaemic cells in post G1 phases, at a variable extent and rate. During treatment by low dose ARA‐C, the mitotic index (MI) fell slowly to zero in two patients who achieved a complete remission (CR), while it fell but recovered during treatment in the patients who did not achieve a CR. The MI fell rapidly to zero in the four cases treated by standard dose, who achieved a CR. These data are consistent with the known cytotoxic activity of ARA‐C, via inhibition and slowing of DNA synthesis leading to defective cell proliferation and to cell death.

Granulocyte-macrophage colony-stimulating factor in acute non-lymphocytic leukemia before and after chemotherapy

SummaryThe introduction of hematopoietic growth factors into the management of leukemia can influence the outcome of treatment in several ways, depending on the sensitivity and the response of normal and leukemic cells. In this paper we report on the effects of the administration ofEscherichia coli-produced, human recombinant granulocyte-macrophage colony-stimulating factor (GMCSF) in 15 adult patients with acute nonlymphocytic leukemia (ANLL) resistant to first-line treatment or in relapse. GM-CSF was given at a dose of 5–10 μg/kg/day as a 6-h i.v. infusion, prior to chemotherapy (CHT) (for 7 days) and after CHT (until evidence of failure or of remission). In the pre-CHT period there was a clear trend towards an increase of circulating neutrophils (PMN) and/or blast cell count (median 0.3 vs. 1.0×109/l for PMN, and 0.5 vs. 2.3 for blast cells). After chemotherapy, in the patients who achieved complete remission (CR), the median time to a PMN count > 0.5×109/l and > 1×109/l was 16 days (range 13–27) and 19 days (range 13–42) respectively. The outcome of treatment was CR for 8/15 (53%), death during induction for 3/15 (20%), and failure for 4/15 (27%). All failures occurred in patients with an increase of blast cell count during pre-CHT GM-CSF administration. Toxicity and side effects were minor, apart from an acute respiratory syndrome that developed twice in the same patient, at doses of 10 and 3 μg/kg/day. These data suggest that investigation of GM-CSF in the treatment of ANLL is worth pursuing, with special attention to GM-CSF effects prior to chemotherapy.

ABCG2 overexpression and deoxyadenosine analogue activity in acute myeloid leukemia

We have read with great interest the article by Jabbour et al entitled “A Randomized Phase 2 Study of Idarubicin and Cytarabine With Clofarabine or Fludarabine in Patients With Newly Diagnosed Acute Myeloid Leukemia.” The authors reported that both clofarabine and fludarabine were associated with high complete remission rates and a 2-year overall survival rate higher than 50% and that, in comparison with treatment with idarubicin and cytarabine alone, fludarabine was associated with superior outcomes for younger patients. Despite these encouraging results, which are in line with what has been achieved in other studies using these 2 purine nucleoside analogues or cladribine, disease relapse is still the major cause of poor long-term outcomes for acute myeloid leukemia (AML) patients. Although disease-free survival is not clearly reported in Jabbour et al’s article, the median event-free survival with both clofarabine and fludarabine is approximately 12 months. Among the mechanisms of resistance of AML blasts to anticancer therapy, the role of multidrug resistance proteins is well established. In particular, the overexpression of ATP-binding cassette subfamily G member 2 (ABCG2) is associated with higher rates of failure to achieve remission and shorter disease-free survival. We have previously reported that fludarabinebased induction therapy does not overcome the negative impact of high ABCG2 levels on disease-free survival. To investigate the possible influence of ABCG2 on clofarabine activity, we tested clofarabine toxicity in 3 different cell lines: RPMI8266 (overexpressing ABCG2), A549 (heterozygous for the commonest amino acid variant in whites [Q141K] that alters protein function and modifies sensitivity to many anticancer drugs), and A498 (homozygous for the same polymorphism). HL60 (no ABC protein overexpression) and its ABCB1overexpressing subline HL60-DNR were used to assess the effect of clofarabine in the presence of classic P-glycoprotein–mediated multidrug resistance. In parallel, experiments in the same cellular settings were performed with fludarabine. Fludarabine fully overcame the negative effect of ABCB1 overexpression (with inhibition dose 50 [ID50] values of 30 and 24 ng/mL in HL60 and HL60-DNR, respectively). An almost equal effect was obtained with clofarabine (with ID50 values of 14 and 7.5 ng/mL in HL60 and HL60-DNR, respectively). Completely different was the impact of high levels of ABCG2 on the toxicity of the 2 drugs: in RPMI-8266, ID50 for fludarabine was 6000 ng/mL (ie, 200-fold higher than the value in HL60), and this supports our previous observation of a worse prognosis for ABCG2-overexpressing AML patients treated with fludarabine-based induction therapy. ID50 for clofarabine in ABCG2-overexpressing cells (650 ng/mL) was lower than the value for fludarabine but still almost 50 times higher than the value for the negative control. With respect to the ABCG2 polymorphism, no significant differences were observed for fludarabine, whereas in the presence of the Q141K variant, both in homozygosity and in heterozygosity, the negative impact on clofarabine was nearly completely overcome. In line with data published by Nagai et al, our findings confirm a possible role of ABCG2 overexpression as a cause of resistance to clofarabine, just as for fludarabine. ABCG2 seems to be responsible for an increased rate of AML relapse, maybe because of its expression in a blast cell population with a stemlike phenotype that could constitute a reservoir responsible for relapse. Deoxyadenosine analogues are certainly effective drugs for AML when they are used both in induction chemotherapy and in the setting of relapsed or refractory disease, but various mechanisms can be responsible for treatment failure. An evaluation of ABCG2 expression should be considered both at diagnosis and in the case of AML recurrence because ABCG2-negative patients could gain the maximal benefit from clofarabine and fludarabine.

ABCG2, Cytogenetics, and Age Predict Relapse after Allogeneic Stem Cell Transplantation for Acute Myeloid Leukemia in Complete Remission

Recent studies have shown that ABGG2 protein overexpression in acute myeloid leukemia (AML) may be associated with poor response to therapy and increased relapse risk. Few data are available in patients with AML undergoing allogeneic stem cell transplantation (SCT), particularly when in complete remission (CR). We analyzed 105 patients with AML who underwent allogeneic SCT in CR evaluating the role of ABCG2 and other pretransplantation features on subsequent transplantation outcomes. Factors negatively associated with leukemia-free survival (LFS) were unfavorable cytogenetics (3-year LFS 48% versus 80%, Pxa0= .0035) and ABCG2 positivity (65% versus 80%, Pxa0= .045). Three-year cumulative incidence of relapse (CIR) in the whole population was 20%; a higher incidence of relapse was associated with adverse cytogenetics (41% versus 16%, Pxa0= .018), ABCG2 overexpression (29% versus 15%, Pxa0= .04), and, marginally, agexa0>xa050xa0years (30% versus 14%, Pxa0= .06). We grouped patients according to the combination of these 3 risk factors: no patient relapsed within 3xa0years from SCT in the group without risk factors, whereas the 3-year CIR was 12% (95% confidence interval [CI], 2% to 25%) in the group with 1 risk factor and 47% (95% CI, 31% to 70%) in patients with 2 or 3 risk factors (Pxa0= .00005). In conclusion, allogeneic SCT does not seem to abrogate the negative prognosis associated with ABCG2 overexpression at diagnosis, specifically in terms of a higher relapse risk. ABCG2, age, and cytogenetics can predict AML relapse after SCT in patients who undergo transplantation while in CR.