Angela Michelutti

P-glycoprotein (PGP) and lung resistance-related protein (LRP) expression and function in leukaemic blast cells

P‐glycoprotein (PGP) lung resistance protein (LRP) and multidrug resistance associated protein (MRP) expressions and function were evaluated by flow cytometry in 65 leukaemic patients (38 acute non‐lymphocytic leukaemias, eight acute lymphocytic leukaemias, 19 Ph‐positive chronic myeloid leukaemias in blastic phase). By using the MRK‐16, the LRP‐56 and the MRPm6 MoAbs, 34% of the cases did not over‐express any proteins (−); 24.5% over‐expressed (+) only PGP, 11% only LRP, 1.5% only MRP, 24.5% both PGP and LRP, and 4.5% both PGP and MRP. The mean intracellular daunorubicin accumulation (IDA) and rhodamine 123 (Rh123) retention in the presence or absence of the reversal agent SDZ PSC 833 (PSC) of the PGP−/LRP−/MRP− cases were comparable to the ones observed in normal leucocytes. With respect to the non‐over‐expressing cases, the PGP−/LRP+/MRP− cases showed only an impaired IDA (mean 204 ± 29; P < 0.001). The PGP+/LRP+/MRP− cases had a defect both in IDA (mean 166 ± 47, P < 0.001) and Rh123 retention (mean 0.42 ±0.14; P < 0.001), which were both corrected by PSC. All the PGP+/LRP+/MRP− cases had a defect in IDA (mean daunorubicin (DNR) accumulation 192 ± 44; P < 0.001. However, only in 8/16 of them an evident defect in Rh123 retention was found. In conclusion, both PGP and LRP over‐expression were common in leukaemia. An impaired IDA was found in all cases over‐expressing PGP, LRP or both. The study of Rh123 retention could give incorrect information about the blast cells’ ability to accumulate cytotoxic drugs in patients over‐expressing both PGP and LRP.

Quantitative assessment of WT1 gene expression after allogeneic stem cell transplantation is a useful tool for monitoring minimal residual disease in acute myeloid leukemia.

Introduction:  WT1 overexpression is described in several oncological diseases including acute myeloid leukemia (AML). Quantification of WT1 in bone marrow samples may be useful as a marker of minimal residual disease (MRD) and may predict the relapse of AML after allogeneic hematopoietic stem cell transplant (HSCT).

Effects of homoharringtonine alone and in combination with alpha interferon and cytosine arabinoside on 'in vitro' growth and induction of apoptosis in chronic myeloid leukemia and normal hematopoietic progenitors

Homoharringtonine (HHT) is a cephalotaxine alkaloid that showed clinical efficacy in the chronic phase of chronic myeloid leukemia (Ph1+CML). As a single agent, it resulted in effectively controlling leukocytosis and in producing sporadic karyotypic conversions; its clinical use in combination with interferon (IFN-α) for the treatment of CML could thus be considered. In this study we evaluated the growth inhibition and the induction of apoptosis determined by HHT alone and in combination with IFN-α and cytosine arabinoside (Ara-C) on normal and CML (both in chronic, CML-CP and in blastic phase; CML-BP) hematopoietic progenitors. HHT is able to determine a dose-dependent cell growth inhibition; evaluation of cytotoxic activity on semisolid cultures showed an activity significantly higher on CML-CP than on normal cells (P = 0.02 for HHT 50 ng/ml and P = 0.01 for HHT 200 ng/ml). HHT exerted a synergistic effect with IFN-α, Ara-C and IFN-α + Ara-C in inhibiting CML-CP colony growth; the same activity was demonstrated by the combination of HHT with Ara-C and by the triple combination, but not by HHT + IFN-α, on normal myeloid progenitors. The triple combination only was able to exert a synergistic effect in CML-BP. The induction of apoptosis resulted HHT dose-dependent in CML-CP and normals; at higher drug concentrations (100–200–1000 ng/ml), HHT induced a significant increase of apoptotic cells (for normals: P = 0.04, P = 0.02 and P = 0.04; for CML-CP: P = 0.01, P = 0.01 and P = 0.04, respectively); no significant changes were observed in CML-BP. In conclusion, the differences in cytotoxic effect and apoptosis induction observed, depending on the various phases of CML, add experimental evidence to the different clinical results between the chronic phase, where the clone is responsive to HHT, and the acute phase, where the drug is ineffective. The in vitro synergism of HHT with Ara-C and IFN-α in CML-CP suggests further evaluation in the clinical setting.

Overexpression of multidrug resistance‐associated p170‐glycoprotein in acute non‐lymphocytic leukemia

Abstract: Resistance to several cytotoxic agents, including anthracyclines, vinca alkaloids and epipodophylline derivatives (multidrug resistance, or MDR) can develop in tumor cells by overexpression of a 170‐kd glycoprotein (p170) which is an essential component of a membrane transport system leading to increased drug efflux and decreased intracellular drug concentration. By means of a p170‐directed monoclonal antibody (MRK‐16) and immunocytochemistry (alkaline phosphatase anti‐alkaline phosphatase technique), we investigated the expression of p170 in marrow blast cells of 59 cases (38 at diagnosis and 21 in relapse) of acute‐non‐lymphocytic leukemia (ANLL). The proportion of strongly MDR‐positive cells was higher in relapse that at diagnosis (median 15.5% vs 1.5%). Out of 31 patients who were evaluable for the results of first remission induction, failure of first‐line treatment (including Daunorubicin, standard‐dose and high‐dose Arabinosyl Cytosine, and sometimes also Mitoxantrone) occurred in 8/22 MDR‐positive cases and in 1/9 MDR‐negative ones (p = 0.21). Failure of first‐line treatment was always associated with a progressive increase of p170 expression. Total failures (no remission plus early relapse) were more frequent (p = 0.001) among MDR‐positive cases (16/22) than among the others (2/9). These data show that MDR is very frequent in ANLL also at diagnosis and suggest that MDR can contribute to early failure of standard treatment.

Caspofungin as first line therapy of pulmonary invasive fungal infections in 32 immunocompromised patients with hematologic malignancies

Invasive Fungal Infections (IFI) remain a severe and major complication among patients with hematologic diseases, but the recent availability of new antifungal agents (echinocandins and new azoles) have improved the chance of cure. Caspofungin (Cancidas‐Merck) is a large lipopeptide molecule able to inhibit the enzyme complex 1,3‐d‐glucan synthetase; this action specifically damages the fungal cell wall. Caspofungin (CAS) is active, in vitro and in vivo, against most Candida species and Aspergillus species. We report on our experience with this drug as first‐line therapy for proven or probable pulmonary IFI in immunocompromised patients with hematologic malignancies. Thirty‐two consecutive patients (20 males and 12 females, with a median age of 52 yr) have been treated with CAS (27 acute leukemias, 1 chronic leukemia, 3 lymphomas and 1 multiple myeloma). Sixteen patients (50%) had a relapsed or resistant hematologic disease, while 12 patients were in complete remission and 4 were at onset of disease; 8/32 (25%) developed IFI after a hematopoietic stem cell transplant (HSCT) procedure. Seven out of 32 patients (22%) had a proven pulmonary IFI (7/7 Aspergillosis) and 25 (78%) had a probable IFI with pulmonary localization as defined according to international consensus. Thirty‐one patients (97%) had less than 1000 granulocytes/mL at onset of infection and at the start of CAS therapy. The CAS was given at the dose of 70 mg on day 1, followed by 50 mg/day. Median duration of CAS therapy was 20 d (range 8–64); all the 31 neutropenic patients received concomitant granulocyte colony‐stimulating factor (G‐CSF). The overall response rate was 56% (18/32) with 12/18 complete responses and 6/18 partial responses; two patients (6%) had a stable disease. Twelve out of 32 (38%) did not respond and seven died of mycotic infection. Univariate analysis showed that granulocytes recovery (>500/mL vs. <500/mL) and status of hematologic disease (remission/onset vs. refractory/relapsed) were significantly associated to favourable outcome. No clinical adverse events (AE) were reported and only a grades I and II transient increase of serum alkaline phosphatase and/or transaminases occurred in 4/32 (12%) patients. After CAS therapy six non‐responders and six cases with a partial or stable response were rescued with voriconazole. Two out of six patients (33%) in the former group and 6/6 (100%) in the latter obtained a complete resolution of IFI. Our experience suggests an efficacy of CAS, in combination with G‐CSF, as first‐line treatment of proven or probable IFI with pulmonary localization. The drug was well tolerated and there were no significant hepatic AE even in patients receiving CAS with cyclosporine after a HSCT. A significant proportion of non‐responders or partial responders to CAS can be rescued with a subsequent voriconazole‐based therapy.

Liposome-encapsulated daunorubicin for PGP-related multidrug resistance.

The possibility that Daunoxome (DNX), a combination of daunorubicin (DNR) with a liposomal targeting system, escapes PGP was tested. Two pairs of leukaemic cell lines, each consisting of the parental non‐multidrug resistance (MDR) line and of a MDR variant, were studied for cytotoxicity (MTT test) and for cellular DNR kinetic and accumulation (flow cytometry). DNX and free DNR were equally toxic against non‐MDR cells, whereas the liposomal anthracycline was more toxic than the free drug against the MDR variant. Non‐MDR cells accumulated DNR more rapidly when they were exposed to free DNR than to DNX, but MDR cells accumulated more DNR when they were exposed to DNX. The kinetics of DNX and free DNR were also studied in the blast cells of 41 cases of acute leukaemia and they were found to be related to blast cell PGP expression. In 15 cases with a low PGP expression intracellular DNR accumulation was faster and higher with free DNR than with DNX. In 26 cases with a high PGP expression the area under the curve was similar with DNX and free DNR, but the kinetics of intracellular DNR accumulation showed an early low plateau with free DNR and a slow and continuous increase with DNX. In MDR cell lines the ratio was more favourable to DNX than to free DNR. We conclude that liposome encapsulated DNR is partially protected from PGP and that it is worth testing for the treatment of PGP‐positive acute leukaemia.

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.

P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression in acute promyelocytic leukaemia

We analysed the expression of three drug transporter proteins [p‐glycoprotein (PGP), lung resistance‐related protein (LRP) and multidrug resistance‐associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor‐risk adult acute non‐lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non‐multidrug‐resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two‐ to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.

Kinetics of Th1/Th2 cytokines and lymphocyte subsets to predict chronic GVHD after allo-SCT: results of a prospective study.

The role of different cytokines and cells of immune system in the pathogenesis of chronic GVHD (cGVHD) is still controversial. Earlier studies, which were either retrospective or analysed one or a few factors, did not show unequivocal results. We prospectively evaluated cytokine levels and lymphocyte subsets in 30 patients who underwent Allo-SCT to investigate their possible correlation with cGVHD. Levels of IL-4, IL-6, IL-10, IFN-γ, tumour necrosis factor-alpha (TNF-α) and its soluble receptors were assessed by ELISA in 30 patients at different times after SCT. Lymphocyte subsets were evaluated by flow cytometry in peripheral blood at the same times as cytokines. A multivariate analysis was performed using principal component analysis and multi-factor ANOVA (analysis of variance). Eighteen patients developed cGVHD at a median time of 6 months (range, 5–9) after SCT. In multivariate analysis, we observed a correlation between cGVHD and clusters of cytokines and lymphocyte subsets from the third to the sixth month after SCT. These clusters changed their composition over time, but they constantly included natural killer (NK) and CD152+ T cells as negative predictors of cGVHD. TNF-α prevailed among other cytokines before the onset of cGVHD. This prevalence could be related partly to the defect of immunoregulatory cells.

P-glycoprotein, lung resistance-related protein and multidrug resistance-associated protein in de novo adult acute lymphoblastic leukaemia

Summary.  P‐glycoprotein (P‐gp), lung resistance‐related protein (LRP) and multidrug resistance‐associated protein (MRP) expression, and blast cell intracellular daunorubicin accumulation (IDA) were evaluated in 95 previously untreated cases of adult acute lymphoblastic leukaemia (ALL) using flow cytometry. Forty‐five out of 95 (47%) patients were P‐gp positive (+), 12/66 (18%) were LRP+ and 11/66 (17%) were MRP+. Eighteen out of 66 (28%) patients showed a simultaneous multidrug resistance (MDR)‐related protein expression higher than controls for more than one protein, while 24/66 (36%) cases did not overexpress any protein. Twenty‐one out of 24 (87%) cases overexpressing at least one MDR‐related protein had a defect in accumulating daunorubicin into their blast cells, while only 4/24 (16%) cases who did not overexpress any protein had similar features. The complete remission rates were similar in MDR‐positive and ‐negative (–) patients but relapses within 6 months were more frequent in P‐gp+ cases, and therefore the disease‐free survival duration was shorter in P‐gp+ than in P‐gp– patients (P = 0·01). The number of MRP+ and/or LRP+ cases was too small to be able to draw any conclusion on their role in affecting or predicting therapy outcome. In conclusion, P‐gp overexpression associated with a defect in daunorubicin accumulation is a frequent feature in adult ALL at onset and seems to be related to poorer therapy outcome and, consequently, a shorter disease‐free survival. LRP and MRP overexpression seems to be a rare event and no conclusion can be drawn on its prognostic role.