Spontaneous remission in a secondary acute myelogenous leukaemia following invasive pulmonary aspergillosis.

Spontaneous remission of adult acute myelogenous leukaemia (AML) represents a rare event. We report a 60-year-old female patient suffering from secondary AML M1 and severe invasive pulmonary hyalohyphomycosis highly suggestive of aspergillosis. Two months after the diagnosis of leukaemia, she achieved a spontaneous remission lasting 3 months, although neither cytostatic drugs nor corticoids were administered because of a septic condition. At the time of remission, a chronic hepatitis C virus infection and a polyclonal hypergammaglobulinaemia were present, and the patient received granulocyte colony-stimulating factor once. This report represents the first documentation of a spontaneous remission in AML following invasive pulmonary hyalohyphomycosis. Possible mechanisms of this phenomenon are discussed.

Phenotypic and functional lymphocyte recovery after CD34+-enriched versus non-T cell-depleted autologous peripheral blood stem cell transplantation.

To determine the effect of CD34+ selection on immune recovery after high-dose chemo/radiotherapy in the setting of autologous stem cell transplantation (ASCT), we analyzed quantitative and qualitative lymphocyte reconstitution for up to 1 year post-transplantation in 27 consecutive adult patients receiving either CD34+-enriched or unmanipulated autologous stem cell (SC) grafts. Pretransplant immunological parameters were identical for both treatment groups. Total lymphocyte counts as well as CD3+ T cells provided a similar course of recovery in both cohorts, returning to baseline values within the first 3 months. There were no significant differences in the reconstitution kinetics of CD4+, CD8+, CD45RA+, and CD45RO+ T cells. CD4+ and CD45RA+ T cells between the two groups were significantly decreased within the first 6 months, returning to pretransplant baseline values by 1 year. Although within the first 3 months the majority of CD3+ cells were activated as demonstrated by expression of HLA-DR, we observed a significant loss of CD25+ T cells in both groups within the first 6 months. B cell numbers returned to baseline values within 3 months but in vivo B cell function measured by serum immunoglobulin M (IgM) and IgA levels did not recover as early as 6 months post-transplantation. T cell function measured by proliferation in response to the lectins phytohemagglutinin (PHA) and Concanavalin A (ConA) and to alloantigens in the mixed lymphocyte reaction (MLR) was significantly impaired, but tended to return to pretransplant baseline values by 1 year. Although preliminary, our results provide strong evidence that T cell depletion (TCD) by CD34+ enrichment using the CellPro device does not result in delayed phenotypic immune reconstitution after autologous peripheral blood stem cell transplantation (PB-SCT). Even in the absence of a high thymic T cell regenerative capacity in adults, T cell numbers and subset distributions were restored within the time frame studied. T and B cell function, however, remained significantly impaired for a prolonged period of time (>6 months after SCT) with a more profound defect in patients autografted with CD34+-enriched SC.

Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells.

In order to examine the regulatory effects of major Th1-derived cytokines, such as IL-12, and Th2 cytokines, IL-4 and IL-10, on the formation of neopterin and degradation of tryptophan, two metabolic pathways induced by interferon-gamma (IFN-gamma) in human monocytes/macrophages, we investigated the human monocytic cell line THP-1, primary human macrophages, and peripheral blood mononuclear cells (PBMC). Neopterin formation and tryptophan degradation were induced similarly by IFN-gamma in all three cell types investigated, but the effects of interleukins were different between THP-1, primary macrophages and PBMC. In PBMC, but not in THP-1 cells and primary macrophages, IL-12 was found to be additive to the effects of IFN-gamma to superinduce neopterin formation and tryptophan degradation. IL-4 and IL-10 reduced the effects of IFN-gamma on monocytic cells, and both cytokines were additively antagonistic to IFN-gamma in PBMC and THP-1 cells. Finally, on preincubation, but not on addition of IL-12, the effects of IL-4 and IL-10 on PBMC could be abrogated, whereas no such effect was seen in THP-1 cells. The results show that IL-12 up-regulates neopterin formation and tryptophan degradation by inducing additional IFN-gamma production by Th1 cells, while a direct effect of IL-12 on monocytes/macrophages appears to be absent. Similarly, IL-4 and IL-10 inhibit neopterin production and tryptophan degradation in PBMC by down-regulating Th1-type cytokine production and possibly also via direct deactivation of IFN-gamma effects towards monocytes/macrophages. The results clearly show how Th1 cell-mediated immunity may be up- or down-regulated by endogenous cytokine production.

Differential activity of P-glycoprotein in normal blood lymphocyte subsets.

To better understand the phenomenon of P-glycoprotein (P-170) expression we investigated lymphocyte subpopulations for P-170 function in healthy volunteers. Studies were based on three-colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P-170. Marked Rh123 efflux was detected in CD8+ T lymphocytes with CD8+/CD45RA+ T cells (naive cells) showing significantly higher P-170 activity as compared with CD8+/CD45RA- cells (P<0.04). Vice versa, CD8+/CD45RO+ T cells (memory cells) demonstrated less P-170 activity than CD8+/CD45RO- cells (P<0.04). P-170 function was less prominent in CD4+ T cells, however, Rh123 efflux was higher in the CD4+/CD45RA+ and CD4+/CD45RO- subpopulations (P<0.025) corresponding to the CD8+ results. Dye efflux differed significantly between activated and non-activated CD8+ and CD4+ as well as CD8+/CD11b+ and CD8+/CD11b- T lymphocytes. Since CD16+ natural killer cells (NK) expressed the highest level of P-170, the NK cytotoxicity against 51Cr-labelled K562 target cells was assayed in the presence or absence of P-170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R-verapamil and dexnigaldipine-HCP in a dose-dependent manner. The differential expression of P-170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR-modulators suggest a physiological role of P-170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.

Reversal of multidrug resistance by the staurosporine derivatives CGP 41251 and CGP 42700.

It has been shown previously that the staurosporine derivative CGP 41251, a specific inhibitor of protein kinase C (IC50 = 50 nM), exhibits antitumor activity and reverses mdr1 mediated multidrug resistance. At present, the compound is evaluated as an anticancer drug in clinical phase I trials. We compared the effects of CGP 41251 with CGP 42700, another staurosporine derivative, which exhibits low protein kinase C inhibiting activity (IC50 = > 100 microM). We found that in contrast to CGP 41251, CGP 42700 does not show antiproliferative activity in HeLa and KB cells in tissue culture (up to a concentration of 10 microM). We compared both compounds for their ability to reverse mdr1-mediated resistance in KB-C1 and in HeLa-MDR1 cells (transfected with the mdr1 gene). CGP 42700 is able to reverse mdr1-mediated resistance to a similar extent as CGP 41251. The intracellular accumulation of rhodamine 123 in KB-C1 cells following pretreatment with CGP 41251 for 30 min was higher than that following treatment with CGP 42700 if determined in medium without serum. However, quantitation of rhodamine efflux in an ex vivo assay using human CD8+ cells in serum showed that CGP 42700 is more effective in inhibiting the efflux of rhodamine 123 than CGP 41251. We conclude from our results that (1) CGP 42700 is more effective in reversal of multidrug resistance in serum than CGP 41251, indicating that the compound may be useful for treatment of patients, and (2) CGP 42700 does not inhibit protein kinase C and cell proliferation and, therefore, may be less toxic and elicit less side effects in humans than other chemosensitizers.

Cytokine priming of the granulocyte respiratory burst in myelodysplastic syndromes.

Increased susceptibility to infections in patients with myelodysplastic syndromes (MDS) is thought to be due to neutropenia as well as functional abnormalities of neutrophils. In the present study we examined the effect of two different stimulants (fMLP, PMA) and three cytokines (alphaTNF, G-CSF and GM-CSF), both singly and in combination on granulocyte (RB) in 25 MDS patients compared to seven healthy controls. Single fMLP and PMA-stimulation showed similar results for both groups. Preincubation with cytokines enhanced fMLP-stimulated RB in most MDS patients and controls, but in patients to a significantly lesser extent when compared to the control group (p < or = 0,05). Combinations of alphaTNF + GM-CSF and alphaTNF + G-CSF were highly synergistic in priming fMLP-stimulated burst in both groups. But again, as with the single cytokine priming this effect was markedly reduced in MDS patients compared to controls (p < or = 0,05). A specific priming defect for one of the cytokines or a cytokine combination could not be demonstrated. Serum alphaTNF levels were measured in 18 and neutrophil alkaline phosphatase (NAP) index in 23 patients. Results did not correlate with variations of the RB in MDS patients. We conclude that reduced alphaTNF, GM-CSF and G-CSF priming of granulocyte RB is a frequent finding in MDS and may contribute to the enhanced susceptibility to bacterial infections.

CD34+-selected autologous peripheral blood stem cell transplantation (PBSCT) in patients with poor-risk hematological malignancies and solid tumors. A single-centre experience.

Between July 1994 and December 1996, PBSC were mobilized in 28 patients with poor-risk hematological malignancies and solid tumors. CD34+ cells were positively immunoselected using the Ceprate CS System. By December 1996, 22 patients had been reinfused with a median of 3.325 (0.078-9.5) x 10(6)/kg CD34+ cells. In three patients unselected back-up PBSC had to be transfused along with selected CD34+ cells because of a CD34+ cell number <0.5 x 10(6)/kg. G-CSF (10 microg/kg) was started on day +1 and all patients engrafted within a median day number of 12 (range, 10-22) until leukocytes >1.0 x 10(9)/l and a median day number of 56 (range, 10-180) until platelets >20.0 x 10(9)/l (ie platelet transfusion independence). Time to leukocyte and platelet recovery was significantly shorter in patients receiving >2.0 x 10(6)/kg purified CD34+ cells as compared to patients reinfused with <2.0 x 10(6)/kg CD34+ cells. The hematopoietic recovery time was similar to that of 18 historical control patients treated with unseparated ABMT +/- PBSCT with the exception of a significantly faster leukocyte engraftment in patients receiving >2.0 x 10(6)/kg CD34+ cells and a significantly delayed platelet recovery time in patients receiving <2.0 x 10(6)/kg purified CD34+ cells. There was a trend for a better overall survival and a lower probability of progression/relapse as compared to the historical controls. We observed five episodes of serious opportunistic infections (three pulmonary fungal infections, two cases of cryptosporidiosis) after the take. Four of these patients had been reinfused with <2.0 x 10(6)/kg CD34+ cells probably indicating a delayed immune reconstitution after CD34+-selected PBSCT.

Paclitaxel- and docetaxel-dependent activation of CA-125 expression in human ovarian carcinoma cells.

Taxanes represent a new class of antineoplastic agents that are being evaluated in several malignant tumors; they have been shown to induce a high remission rate and to prolong survival in ovarian cancer patients. However, CA-125 has been suggested to be an unreliable marker for monitoring response to paclitaxel therapy. Therefore, we were interested in whether taxanes may directly modulate CA-125 expression. Human ovarian carcinoma cell lines OVCAR-3, HOC-7, SKOV-6, 2780, 2774, and HTB-77 were treated with paclitaxel or docetaxel. Secreted, surface-associated, and cytosolic CA-125 were estimated by means of a sandwich solid-phase RIA or by immuno-flow cytometry. In addition to in vitro antiproliferative activity, paclitaxel and docetaxel augmented the expression of the tumor marker CA-125 in the three ovarian carcinoma cell lines, OVCAR-3, HOC-7, and SKOV-6, constitutively expressing this tumor marker. The three CA-125-negative cell lines, 2780, 2774, and HTB-77, did not respond to taxane treatment by expressing this tumor marker, although their proliferation was markedly inhibited. The taxane-mediated induction of CA-125 was found to be dependent on intact protein and RNA biosynthesis. However, CA-125 concentration was increased in the supernatant medium only and not on cell surface or cytosol. Our results demonstrate an in vitro activation of ovarian carcinoma cells in terms of CA-125 secretion by taxanes. This may explain the CA-125 fluctuations observed in vivo under paclitaxel treatment and may indicate that CA-125 is not a reliable tumor marker during taxane chemotherapy.

Multidrug resistance in acute leukemia: a comparison of different diagnostic methods.

Accurate measurement of P-glycoprotein (P-170) expression in clinical samples still remains a controversial issue. In this study tumor cell P-170 expression was assessed in 29 patients suffering from acute leukemia (17 acute myeloid leukemia (AML) and 12 acute lymphoblastic leukemia (ALL)) using three different techniques: flow cytometry measuring rhodamine 123 (Rh123) efflux (functional level), immunocytochemistry (protein level) and RT-PCR (mRNA level). Rh123 efflux was detectable in 10/29 (34%) of all cases, in 9/17 (53%) of AML and in 1/12 (8%) of ALL samples. In AML patients a significant association of CD34 expression and P-170 activity was observed (P < 0.02). All AML patients with the FAB subtype M5 were Rh123 negative (P < 0.007). Cytospin preparations were analyzed for staining with monoclonal antibodies JSB1 and MM4.17. Eight of 16 (50%) AML and 0/9 (0%) ALL cases expressed the multidrug resistance (MDR) protein assessed by JSB1. With MM4.17 87% of AML and 50% of ALL patients were scored positive. Agreement between both antibodies was found in only 13/23 (57%) samples. Extracted RNA from 12 patients was analyzed by RT-PCR to evaluate the expression of MDR1 and multidrug resistance-associated protein (MRP) mRNA. An increased level of MDR1 mRNA was detectable in 4/7 AML and 0/5 ALL cases. MRP expression was found in 3/7 AML and 0/5 ALL patients. Comparison of Rh123 assay and immunocytochemistry revealed a very good correlation when using MoAb JSB1 (P < 0.004) but not with MM4.17 (not significant (NS)). JSB1 also showed a much better association with the PCR results (P < 0.05) than MM4.17 (NS). Finally, we compared the results of the functional Rh123 assay and RT-PCR and observed a high correlation for Rh123/MDR1 (r = 0.819, P < 0.001) but low for Rh123/MRP (r = 0.562, NS). We conclude that measurement of Rh123 efflux and immunocytochemical staining of cytospin preparations with JSB1 allows the accurate monitoring of P-170 expression in acute leukemia. The simplicity of these two MDR assays suggests their use for routine MDR screening.

Effect of cyclosporin A in a patient with refractory nephrotic syndrome associated with B chronic lymphocytic leukemia.

We report on a patient with an almost 20-year history of B chronic lymphocytic leukemia (B-CLL). During the last 2 years, the patient developed nephrotic syndrome (NS) due to membranous glomerulonephritis (MN), refractory to standard therapeutic regimens. Neither NS nor B-CLL responded objectively to systemic administration of two different combinations of corticosteroids and alkylating agents (chlorambucil, cyclophosphamide). Third-line therapy with cyclosporin A resulted in reduction of proteinuria and improvement of leukemia. Withdrawal of the drug led to an increase in leukocyte count.

Decreased potency of MDR-modulators under serum conditions determined by a functional assay.

A variety of agents are capable of overcoming P-glycoprotein-mediated multidrug resistance (MDR) in vitro. However, the clinical potential of these compounds is often limited due to high plasma protein binding. We compared the efficacy of several MDR-reversing compounds in serum-free culture medium and under serum conditions by means of a functional assay. Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rh123) was measured from normal peripheral blood CD8+ T-lymphocytes which express low levels of P-glycoprotein. Inhibition of Rh123 efflux by R-verapamil, dexnigludipine-HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum-free medium and in serum at concentrations from 0.1 to 50 mumol/l. With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P-glycoprotein under serum conditions at concentrations achievable in vivo. The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5 mumol/l a sufficient inhibitory effect was observed. Subsequently this approach was applied to patients suffering from B-cell chronic lymphocytic leukaemia (B-CLL; n = 3) and acute myeloid leukaemia (AML; n = 2) which were positive in the Rh123 efflux assay. As for normal CD8+ T-lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rh123 efflux from the leukaemic cells. Thus the interpretation of results of clinical 'modulator' trials should consider the decreased bioavailability of MDR-reversing agents.

Identification of a favorable subgroup of patients with generalized immunocytomas by follicular dendritic cells.

In the present study, 89 patients with generalized immunocytoma were analyzed retrospectively for the prognostic influence of clinical features and of immunophenotype using bone marrow biopsies. Univariate analysis selected the following variables as significant for survival: age over 60 years (p = 0.021), Rai and Binet stage (p = 0.008 and 0.004, respectively), presence or absence of follicular dendritic cells (FDC, p = 0.036), presence or absence of anemia (p = 0.037). Multivariate regression analysis showed independent prognostic significance of age (p = 0.001), Rai stage (p = 0.003), and the presence of follicular dendritic cells (p = 0.010). Comparing CD5 positive and CD5 negative immunocytomas, no survival advantage for one of the two groups was seen. Presence of follicular dendritic cells clearly identified a favorable prognostic subgroup: of the 89 patients presented, only three of 18 (16.6%) with detectable FDC in bone marrow died within the observation period, as compared to 34 of 71 (52.1%) patients without FDC. Multivariate analysis emphasized the independent prognostic value of the presence of FDC. The high predictive capacity of follicular dendritic cells could add useful information to the commonly used Rai and Binet staging systems for immunocytoma.

Low frequency of activity of P-glycoprotein (P-170) in acute lymphoblastic leukemia compared to acute myeloid leukemia.

The purpose of our investigations was to measure P-glycoprotein (P-170) activity in blast cells of 35 adults with acute myeloid leukemia (AML), and 24 children and adults with acute lymphoblastic leukemia (ALL) at time of diagnosis. Studies were based on a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123 (Rh123), which is transported from the cell by the P-170 pump. Dual-fluorescence staining with Rh123 and phycoerythrin-labeled monoclonal antibodies allowed selective measurement of Rh123 efflux in blast cells. Samples were scored positive when the fraction of blast cells showing Rh123 efflux exceeded 10% after a 120-min incubation. Activity of P-170 was observed in 19 (54%) of the 35 AML cases and was completely blocked in the presence of multidrug resistance inhibitors. Efflux activity was significantly higher in CD34-positive AML samples (p < 0.02). All AML patients with the FAB-subtype M5 (n = 5) lacked Rh123 pumping activity (p < 0.03). The complete remission rate in response to induction chemotherapy was significantly higher for Rh123-negative (11/13, 85%) than for Rh 123-positive AML patients (4/15, 27%) (p < 0.007). At a median follow-up of 9 months overall survival was significantly shorter for Rh123-positive than for Rh123-negative patients (p < 0.05). In contrast to AML, we could detect Rh123 efflux in only two (8%) out of 24 ALL cases. The immunological subtypes of these two positive cases was of B-ALL and pre-T-ALL. Bone marrow cryostat sections from 13 AML and five ALL patients were further analyzed for staining with monoclonal antibodies MM4.17 and JSB1. Ten of 13 AML and two of five ALL cases expressed the MDR protein. Our results indicate that there is a rather low frequency of P-170 pumping activity in ALL compared with AML. Further, functional activity of P-170 contributes to chemoresistance in de novo AML.

Treatment of relapsed and refractory acute myelogenous leukaemia with aclacinomycin A (ACA) and etoposide (VP-16).

Ten patients with refractory and relapsed acute myelogenous leukaemia (AML) and one patient with CML in blast crisis were treated with aclacinomycin A (ACA, 60 mg/m2/day for 5 days) and etoposide (VP-16, 100 mg/m2/day for 5 days) and analysed retrospectively. Of 11 patients, seven (63%) achieved an objective response including four CRs (36%). Most impressively, two patients experienced consecutive CRs (second, third, and fourth) following relapses. Only two patients (18%) were primary resistant with persisting leukaemia. In conclusion, the combination of ACA and VP-16 is an active regimen in refractory and relapsed AML with a toxicity comparable with other salvage regimens.

Bone marrow lymphocyte subsets in myelodysplastic syndromes.

AIM: To examine lymphocyte subsets in patients with myelodysplastic syndromes (MDS); and to correlate immunohistological variables with prognosis. METHODS: Bone marrow trephine biopsy specimens from 65 patients with MDS were immunophenotyped using a panel of antibodies. A minimum of 1000 cells from representative areas of marrow sections were counted at light microscopy. The association between immunohistological variables and prognosis was assessed. RESULTS: Compared with normal control marrows (n = 23) no major abnormalities of T cells (CD3), T cell subsets (CD4, CD8, CD25, TCR gamma/delta) or natural killer cells (CD56, CD57) were seen in the 65 patients. In high risk MDS (RAEB, RAEB-t) 19% of the cases showed increased numbers of B lymphocytes compared with none in the low risk group (RA, RARS) (p < 0.0090). Only percentages of B cells above 3% significantly correlated with poor survival (p = 0.0121 for CD19, p = 0.046 for CD22). CONCLUSIONS: The deviations in T lymphocyte counts seen in peripheral blood and in bone marrow aspirates could not be verified in bone marrow biopsy specimens.

[Functional detection of P-glycoprotein expressing cells with flow cytometry]. / Funktionelle Erfassung P-Glykoprotein-exprimierender Zellen mittels Durchflusszytometrie.

Classical multidrug resistance (MDR) is associated with the overexpression of a membrane glycoprotein termed P-glycoprotein (P-gp) that transports a variety of apparently unrelated anti-cancer drugs out of cells. Based on the fluorescent properties of the dye rhodamine 123 (Rh 123) we aimed to develop a functional flow cytometric assay for the detection of MDR-expressing cells. Using drug sensitive cell lines (KB-3-1) and MDR mutants (KB-8-5, KB-C1) experimental conditions were established enabling demonstration of significant differences in Rh 123 accumulation/efflux. Using two-colour flow cytometry we subsequently analysed 42 consecutive patients suffering from B-cell chronic lymphocytic leukemia (B-CLL). 34/42 (81%) cases showed a marked Rh 123 efflux which was completely abolished in the presence of the MDR inhibitor verapamil. The percentage of Rh 123 effluxing cells ranged from 10 to 70% with a median of 32%. In 26 cases extracted RNA was analysed by quantitative polymerase chain reaction to evaluate the expression of MDR 1 mRNA. In 25 of 26 (96%) cases MDR 1 mRNA was detectable. The levels of MDR 1 mRNA expression correlated well with the results of the Rh 123 efflux assay (r = 0.72; p < 0.0001). In addition, 37 cases of acute myeloid leukemia were analysed and demonstrated Rh 123 efflux in 18/37 (49%) cases. In this entity the percentage of Rh 123 effluxing cells ranged from 12 to 80% (median 45%). Additionally, we evaluated the Rh 123 efflux/retention in peripheral blood cells of healthy volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of P-glycoprotein in B-cell chronic lymphocytic leukemia determined by a flow cytometric assay.

BACKGROUND: Chemoresistance in some hematologic malignancies has been associated with overexpression of P-glycoprotein, which is encoded by the MDR1 gene (also known as PGY1). However, inconsistencies in data on frequency and clinical relevance of multidrug resistance in B-cell chronic lymphocytic leukemia (B-CLL) may reflect a need for improved techniques to detect this overexpression. PURPOSE: Our purpose was to measure P-glycoprotein activity in peripheral blood cells of B-CLL patients and to analyze possible clinical correlations (disease duration, prior treatment, Rai disease stage, lymphocyte counts, and disease progression). METHODS: P-glycoprotein activity was assayed in peripheral blood cells of 42 consecutive B-CLL patients (22 treated and 20 untreated). We used dual fluorescence in a flow cytometric assay that detects efflux of the fluorescent dye rhodamine 123, which is transported from the cell by the P-glyprotein pump. Leukemia cells were costained with monoclonal antibody Leu12/CD19, and rhodamine 123 efflux was measured. Expression of MDR1 and MDR3 (also known as PGY3) messenger RNA (mRNA) was quantitatively evaluated by polymerase chain reaction (PCR) in 26 cases. RESULTS: Marked rhodamine 123 efflux was observed in 34 (81%) of the 42 cases and was abolished in the presence of multidrug resistance inhibitors. Rhodamine 123 efflux was not associated with Rai stage, lymphocyte counts, duration of disease, or disease progression. Although rhodamine 123-negative cases were about equally distributed among untreated and previously treated patients, the percentage of cells with rhodamine 123 efflux was significantly lower for untreated patients than for those treated with chemotherapy regimens including at least one multidrug resistance-associated drug. MDR1 mRNA was detected in 25 of 26 cases and MDR3 mRNA in all 26. MDR1 mRNA expression was significantly correlated with rhodamine 123 efflux, whereas MDR3 mRNA expression was not significantly correlated; MDR1 and MDR3 mRNA expression was not significantly associated with Rai stage, prior treatment, or disease progresssion. CONCLUSIONS: These findings suggest that P-glycoprotein overexpression in B-CLL is intrinsic rather than acquired and that P-glycoprotein activity is enhanced after exposure to multidrug resistance-associated drugs. This enhanced activity does not seem to be associated with more aggressive disease. Our results also indicate that an assay of P-glycoprotein function combined with PCR is suitable for clinical multidrug resistance screening. IMPLICATIONS: Additional studies are needed to determine whether functional activity of P-glycoprotein, measured by rhodamine 123 efflux, is directly related to clinical drug resistance.