Pentostatin (Nipent) in T-cell malignancies. Leukemia Cooperative Group and the European Organization for Research and Treatment of Cancer.

Within this phase II trial of the European Organization for Research and Treatment of Cancer, we have investigated the safety and efficacy of pentostatin (Nipent; SuperGen, San Ramon, CA) in refractory lymphoid malignancies. Pentostatin was administered at a dosage of 4 mg/m2 every week for the first 3 weeks, then every 14 days, followed by maintenance therapy of 4 mg/m2 monthly for a maximum of 6 months. We have previously reported the results in T- and B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia, and hairy cell leukemia This report focuses on the outcome in T-cell malignancies: T-cell chronic lymphocytic leukemia, Sézary syndrome, mycosis fungoides, and T-zone lymphoma. Of 92 patients with these diagnoses enrolled, 76 were evaluable for response and toxicity, ie, 25 of 28 with T-cell chronic lymphocytic leukemia, 21 of 26 with Sézary syndrome, 22 of 26 with mycosis fungoides, and eight of 12 with T-zone lymphoma. All patients had progressive and advanced disease. Sixteen patients (21%) died during the first 9 weeks of treatment: 12 of progressive disease, two of infectious complications thought to be unrelated to treatment, one of myocardial infarction, and one of renal failure related to administration of intravenous contrast. Major toxicity (grades 3 and 4) included infection in 10.5% of patients, nausea/vomiting in 5%, and hepatotoxicity in 3%. One patient (1.3%) achieved a complete remission and 15 (19.7%) a partial remission. Better results were achieved in patients with Sézary syndrome or mycosis fungoides (complete remission + partial remission = 33.4% and 22.7%, respectively) than in patients with T-cell chronic lymphocytic leukemia (8%) or T-zone lymphoma (25%). We conclude that pentostatin is active in low-grade T-cell malignancies. Toxicities are mild to moderate at the dose schedule administered. Severe hematologic toxicity has not been observed. The efficacy at the present dose level is moderate. A higher dose might be necessary for some T-cell malignancies.

Pentostatin in T-cell malignancies--a phase II trial of the EORTC. Leukemia Cooperative Group.

PURPOSE: Within this phase II EORTC trial, we have investigated the safety and efficacy of pentostatin in lymphoid malignancies. We have previously reported the results in T- and B-cell prolymphocytic leukemia, B-cell chronic lymphocytic leukemia (B-CLL) and hairy cell leukemia. This report focuses on the outcome in T-cell malignancies: T-CLL, Sézary syndrome (Sézary), mycosis fungoides (MF) and T-zone lymphoma (TZL). PATIENTS AND METHODS: Of the 92 patients with these diagnoses enrolled, 76 were evaluable for response and toxicity, i.e., 25 of 28 with T-CLL, 21 of 26 with Sézary, 22 of 26 with MF, and 8 of 12 with TZL. All patients had progressive and advanced disease. Pentostatin was administered at a dosage of 4 mg/m2 every week for the first 3 weeks, then 4 mg/m2 every 14 days for another 6 weeks, followed by maintenance therapy of 4 mg/m2 monthly for a maximum of 6 months. RESULTS: Response rates (complete and partial responses) in patients with Sézary (n = 22) or MF (n = 21) were 33% and 23%, respectively, and in patients with T-CLL (n = 21) or TZL (n = 8) 8% and 25%, respectively. Sixteen (21%) patients died during the first ten weeks of treatment: twelve of progressive disease, two of infectious complications with progressive disease, one of myocard infarction and one of renal failure related to administration of i.v. contrast. Major toxicity (grade 3-4) included infection in 11% of patients, nausea/vomiting in 4%, diarrhea in 3%. Hematologic toxicity was mild to non-existent. CONCLUSIONS: We conclude that pentostatin is active in Sézary and MF but showed marginal activity in T-CLL or TZL. Toxicities are mild to moderate at the dose schedule administered. Due to its relatively specific lympholytic effect and its favorable toxicity spectrum, pentostatin might be especially useful for the palliative treatment of T-cell malignancies.

TGF-beta activity and expression of its receptors in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in Western countries and results from the accumulation of B-lymphocytes which are functionally abnormal and predominantly non-cycling in vivo. Consequently, it is important to understand why B-CLL cells accumulate in GO phase. Since TGF-beta is an important negative regulator of the immune system, a loss of responsiveness to this factor might provide a selective advantage to B-CLL cells. Here we review data on the role of TGF-beta in B-CLL. We show that the B-CLL cell response to TGF-beta signals is abnormal in vitro (inhibition of proliferation and induction of apoptosis). This lack of response of B-CLL cells to TGF-beta inhibition appears to be accompanied by a decrease or a loss of TGF-beta receptor expression.

Myeloid and lymphoid recovery following allogeneic bone marrow transplantation: a comparative study between related, unrelated bone marrow and allogeneic peripheral stem cell transplantation.

We studied myeloid and lymphoid recovery during a period of 12 months following HLA matched allogeneic bone marrow transplantation (BMT) in 15 patients. Patients were divided into three groups. Each group contained 5 patients according to the source of hematopoietic stem cell transplantation (HST): 1) related bone marrow transplantation (BMT), 2) allogeneic peripheral blood stem cell transplantation (PBSCT) and 3) matched unrelated donor transplantation (MUD). The rate and pattern of recovery of granulocytes, lymphocytes (T-cell subsets, B-cells, NK cells, subsets of CD45) were studied by cell counting and flow cytometry. Our results suggest faster recovery of PMN after PBSCT. Higher CD4 cell counts observed in the PBSCT group may have an impact on a lower incidence of opportunistic infections. Chronic GvHD mediated GvL effect seems to be more important in blood stem cell transplanted patients and this may have an influence on disease free survival.

Chronic lymphocytic leukemic B cells but not normal B cells are rescued from apoptosis by contact with normal bone marrow stromal cells.

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 +/- 3% and 31 +/- 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 +/- 2%, P < . 0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 +/- 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by beta1 and beta2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

Mitoxantrone versus daunorubicin in induction-consolidation chemotherapy--the value of low-dose cytarabine for maintenance of remission, and an assessment of prognostic factors in acute myeloid leukemia in the elderly: final report. European Organization for the Research and Treatment of Cancer and the Dutch-Belgian Hemato-Oncology Cooperative Hovon Group.

PURPOSE AND METHODS: Optimization of remission-induction and postremission therapy in elderly individuals with acute myeloid leukemia (AML) was the subject of a randomized study in patients older than 60 years. Remission-induction chemotherapy was compared between daunomycin (DNR) 30 mg/m2 on days 1, 2, and 3 versus mitoxantrone (MTZ) 8 mg/m2 on days 1, 2, and 3, both plus cytarabine (Ara-C) 100 mg/m2 on days 1 to 7. Following complete remission (CR), patients received one additional cycle of DNR or MTZ chemotherapy and were then eligible for a second randomization between eight cycles of low-dose (LD)-Ara-C 10 mg/m2 subcutaneously every 12 hours for 1 2 days every 6 weeks or no further treatment. RESULTS: A total of 242 patients was randomized to DNR and 247 to MTZ. Median age of both study groups was 68 years. Secondary AML was documented in 26% and 25% of patients in either arm. The probability of attaining CR was greater (P = .069) with MTZ (47%) than with DNR (38%). Median duration of neutropenia was 19 (DNR) and 22 days (MTZ). The greater response rate to MTZ therapy correlated with reduced occurrence of chemotherapy resistance (32% v 47%, P = .001). With a median follow-up of 6 years, 5-year disease-free survival (DFS) is 8% in each arm. Overall survival estimates are not different between the groups (6% v 9% at 5 yrs). Poor performance status at diagnosis, high WBC count, older age, secondary AML, and presence of cytogenetic abnormalities all had an adverse impact on survival. Secondary AML and abnormal cytogenetics predicted for shorter duration of CR. Among complete responders, 74 assessable patients were assigned to Ara-C and 73 to no further therapy. Actuarial DFS was significantly longer (P = .006) for Ara-C-treated (13% [SE = 4.0%] at 5 years) versus nontreated patients (7% [SE = 3%]), but overall survival was similar (P = .29): 18% (SE = 4.6%) versus 15% (SE = 4.3%). Meta-analysis on the value of Ara-C postremission therapy confirms these results. CONCLUSION: In previously untreated elderly patients with AML, MTZ induction therapy produces a slightly better CR rate than does a DNR-containing regimen, but it has no significant effect on remission duration and survival. Ara-C in maintenance may prolong DFS, but it did not improve survival.

Granulocyte colony-stimulating factor given in addition to interferon-alpha to mobilize peripheral blood stem cells for autologous transplantation in chronic myeloid leukaemia.

In order to potentially mobilize and harvest the Ph cells observed in most patients with chronic myeloid leukaemia (CML) during interferon-alpha (IF-alpha) therapy, G-CSF (filgrastim), 5 microg/kg/d, was administered subcutaneously together with IF-alpha to 30 CML patients in haematological remission but with various degrees of cytogenetic remission, after IF-alpha therapy. Peripheral blood stem cells (PBSC) were harvested using standard aphereses from day 5 of G-CSF Patients underwent one to four (median three) aphereses. Median total yields/kg were 7.6 (range 3.8-25) x 10(8) MNC, 3.4 (0-140) x 10(6) CD34+ cells, and 17 (1.1-107) x 10(4) CFU-GM. No patient had a significant increase in the percentage of Ph+ cells in the bone marrow under G-CSF therapy. The percentage of Ph+ cells in apheresis products tended to decrease between the first and the last apheresis (P = 0.05). 14 patients who were not responsive to IF-alpha were transplanted after conditioning with busulphan 16 mg/kg and melphalan 140 mg/m2. Median time to neutrophils > 0.5 x 10(9)/l was 20 d (16-114 d) and to platelets > 50 x 10(9)/l 18 d (12-149 d). Nine patients had a major cytogenetic response post graft, which correlated with the amount of Ph+ cells reinfused with the graft (P = 0.02). We conclude that this procedure is feasible, allowing the harvest of enough PBSC, some of them Ph- in patients who responded to IF-alpha, to allow autologous transplantation.

Use of recombinant GM-CSF during and after remission induction chemotherapy in patients aged 61 years and older with acute myeloid leukemia: final report of AML-11, a phase III randomized study of the Leukemia Cooperative Group of European Organisation for the Research and Treatment of Cancer and the Dutch Belgian Hemato-Oncology Cooperative Group.

We conducted a prospective randomized multicenter clinical trial comparing the effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an adjunct to intensive chemotherapy in patients of 61 years and older with untreated newly diagnosed acute myeloid leukemia (AML). Patients were randomized to either receive daunomycin-cytosine arabinoside with GM-CSF or daunomycin-cytosine arabinoside (control arm). Based on the rationale that GM-CSF might sensitize the leukemic cells to the cytotoxicity of chemotherapy as well as enhance white blood cell regeneration, GM-CSF was given during chemotherapy as well as after chemotherapy. Patients were treated with one, and in case of a partial response, with two remission induction cycles. When a complete remission was attained they received one additional cycle of consolidation therapy. Of 318 evaluable patients with a median age of 68 years, 157 were randomized to receive GM-CSF and 161 were assigned to control therapy. The effect of GM-CSF on treatment was evaluated according to intention-to-treat. Complete remission was achieved in 56% of the patients in the GM-CSF group and 55% of the control patients (P = .98). Recovery of neutrophils was significantly faster in GM-CSF-treated patients. The median time of recovery of neutrophils towards 0.5 x 10(9)/L was 23 days in the GM-CSF group versus 25 days in the control group (P = .0002) with the percentages of patients who recovered being 81% and 71%, respectively. With a median follow-up of 36 months, the probabilities of survival at 2 years after randomization were estimated at 22% for individuals assigned to the GM-CSF treatment as well as for control patients (P = .55). Disease-free survival at 2 years compared 15% and 19% for the two treatment groups (P = .69). The number of nights spent in the hospital, number of transfusions, and frequencies and types of hemorrhages and infections did not differ either. The cytogenetic results at diagnosis of this study in elderly AML shows that there is a relatively high numerical representation of patients with abnormal cytogenetics (55% of documented cases), who showed significantly inferior response rates and survival duration. We conclude that, except for a faster neutrophil recovery, GM-CSF during and after induction chemotherapy does not improve the clinical outcome of elderly patients with AML.

Quality of life in patients with acute myelogenous leukemia in prolonged first complete remission after bone marrow transplantation (allogeneic or autologous) or chemotherapy: a cross-sectional study of the EORTC-GIMEMA AML 8A trial.

A cross-sectional study of quality of life (QOL) was performed in 98 patients in continued first complete remission (CR) for 1-7.4 years, after inclusion in the AML 8A trial which prospectively compared allogeneic bone marrow transplantation (AlloBMT), autologous BMT (ABMT) and intensive consolidation chemotherapy. Several significant differences between the three treatment groups were observed, on the basis of patient self-reports, with regard to somatic symptoms (mouth sores, cough, hair loss, headache), repeated acute medical problems, physical functioning, role functioning, leisure activities and, above all, sexual functioning. There were also significant differences for overall physical condition, and overall quality of life. For all these parameters, the ranking was uniformly AlloBMT lower than ABMT lower than chemotherapy. These differences remain significant after adjustment for time interval between CR and QOL evaluation, sex or age. These results, confirming a higher risk of permanent impairment of QOL after BMT, may have an impact on medical decisions and warrant further studies.

Heterogenous response of B lymphocytes to transforming growth factor-beta in B-cell chronic lymphocytic leukaemia: correlation with the expression of TGF-beta receptors.

We investigated the potential role of transforming growth factor-beta (TGF-beta) on spontaneous and cytokine-induced proliferation of B-cell chronic lymphocytic leukaemia (B-CLL) cells in vitro. Purified B lymphocytes from 21 B-CLL patients were cultured for 5 d in the presence of medium alone, IL-2 and/or IL-10, in the presence or absence of TGF-beta, and proliferation was measured by 3H-thymidine incorporation. TGF-beta inhibited B-cell proliferation in the majority of patients (15/21) but no inhibition was detected in 6/21 patients whatever the type of stimulant used. Addition of neutralizing antibodies to TGF-beta increased spontaneous and cytokine-induced proliferation; this effect was dose dependent and specific because addition of an irrelevant chicken IgG had no effect on B-CLL proliferation. In resistant patients, neutralizing antibodies to TGF-beta did not increase the proliferation. The expression of TGF-beta receptors on B-CLL cells was significantly lower than the one observed on normal CD5+ B lymphocytes for which the sensitivity to TGF-beta inhibition was more marked than in CLL. In addition, we found a strong correlation between the response of leukaemic B cells to TGF-beta inhibitory action and the expression of TGF-beta receptors on these cells. In summary, TGF-beta appears to function in CLL as a negative regulator of B lymphocytes but loss of responsiveness to this factor accompanied by a decrease of TGF-beta receptor expression, might provide a selective advantage to B-CLL lymphocytes.

Ex vivo expansion of CD34 + CD38- cord blood cells.

CD34+ cord blood (CB) cells were expanded in stromal cell-free long-term culture (LTC), in the presence of various combinations of interleukin-3 (IL-3), stem cell factor (SCF), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and anti-transforming growth factor-beta (anti-TGF-beta) antibody. The progenitor cell expansion was evaluated by monitoring the increase of CD34+ and CD34 + CD38- cells over a period of 21 days. The expansion of immature (B1-CFC, HPP-CFC) and of more committed progenitors (CFU-GM, CFU-GEMM, BFU-E) was also evaluated in specific samples. Our results show that (a) CD34+ cell expansion is highly variable depending on the cord blood samples studied, (b) significant correlations between B1-CFC and CD34 + CD38- and between total CFU and CD34+ cell expansion are observed, (c) SCF in combination with IL-3 appears to expand cell subsets that continue to express their CD34 + CD38- phenotype and that generate both immature and committed progenitors, and (d) the addition of IL-6, GM-CSF, or anti-TGF-beta does not significantly improve these expansions.

A randomized phase II study on the effects of 5-Aza-2'-deoxycytidine combined with either amsacrine or idarubicin in patients with relapsed acute leukemia: an EORTC Leukemia Cooperative Group phase II study (06893).

5-Aza-2'-deoxycytidine combined with either amsacrine or idarubicin has been applied in a treatment protocol for patients with a relapse of acute myeloid or lymphocytic leukemia. Sixty-three patients received 5-Aza-2'-deoxycytidine 125 mg/m2 as a 6 h infusion every 12 h for 6 days in combination with either amsacrine 120 mg/m2 as a 1 h infusion on days 6 and 7 (n=30) or idarubicin 12 mg/m2 as a 15 min infusion on days 5, 6 and 7 (n = 33). Twenty-three patients (36.5%) obtained a complete remission (CR); eight of 30 patients treated with amsacrine and 15 of 33 treated with idarubicin. Patients with an interval of more than 1 year between initial diagnosis and start of the protocol achieved CR in 51.4%, compared to 15.4% for patients with an interval of less than 1 year. Patients with normal cytogenetics had a higher CR rate (61%) than those with abnormal cytogenetic findings (15.8%). Digestive tract and hematologic toxicity was prolonged, compared to standard induction schedules. Median disease-free survival was approximately 8 months, with only 20% of patients staying in remission for more than 1 year. 5-Aza-2'-deoxycytidine is a good antileukemic agent with considerable toxicity. Current results merit further investigations in previously untreated leukemia.

Complete remission following donor PBSC after low-dose cytarabine chemotherapy for early relapse of acute myelogenous leukemia after allogeneic stem cell transplantation.

A 55-year-old woman with chemotherapy-resistant acute myeloblastic leukemia (AML M2) relapsed 3 months after allogeneic PBSC transplant. The patient was treated with two cycles of low-dose cytarabine chemotherapy followed by G-CSF mobilized donor PBSC after cessation of all immunosuppressive treatment. Hematological and cytogenetic complete remission was observed after the first cycle. The patient had been previously treated for AGVHD after allogeneic PBSC transplantation and experienced a second AGVHD after the second cycles of cytoreductive treatment and donor PBSC infusion. Hematological recovery after donor PBSC infusion was faster than recovery after previous chemotherapy or high-dose chemotherapy. During treatment no febrile neutropenia was observed. This case shows that donor PBSC infusion cannot only provide prolonged complete hematological and cytogenetic remission but also seems to support accelerated hematopoietic recovery for some patients relapsing after allogeneic BMT.

Prognostic importance of serum soluble CD23 level in chronic lymphocytic leukemia.

Prognosis of B-cell chronic lymphocytic leukemia (CLL) is based on clinical staging whose limitation is the failure to assess whether the disease will progress or remain stable in early stage (Binet A, or Rai 0, I, II) patients. We previously reported that soluble CD23 (sCD23), a protein derived from the B-cell membrane CD23 Ag, is selectively elevated in the serum of CLL patients. This prospective study assessed the predictive value of serum sCD23 level measured at study entry on the overall survival of all CLL patients and on disease progression of stage Binet A patients. Prognostic value of repeated measurements of sCD23 over time in stage A patients was also analyzed. One hundred fifty-three CLL patients were prospectively followed with a median follow-up of 78 months. Eight clinical or biological parameters were collected from the date of the first sCD23 measurement. At study entry, by Cox model, Binet staging (P = .0001) and serum sCD23 level (P = .03) appeared as prognostic factors for survival. Patients with sCD23 level above median value (> 574 U/mL) had a significantly worse prognosis than those with lower values (median survival of 53 v 100+ months, P = .0001). During follow-up, sCD23 doubling time increased by 3.2 the risk of death (P = .001). Among stage A patients (n = 100), sCD23 determination at study entry was the sole variable predictive of disease progression, patients with sCD23 level above 574 U/mL had a median time progression of 42 months versus 88 months for those with lower levels (P = .0001). Stage A patients who doubled their sCD23 level exhibited a 15-fold increased risk of progression (P = .0001) and, in addition, the sCD23 increase preceded by 48 months disease progression. We conclude that in CLL patients, serum sCD23 level provides significant additional prognostic information in terms of overall survival. Most interestingly, among early stage patients, sCD23 determination at diagnosis and during the course of the disease may help to the early identification of patients who will rapidly progress to upper stages.

Imbalance in production of cytokines by bone marrow stromal cells following cytomegalovirus infection.

The effect of cytomegalovirus (CMV) infection on the production of growth factors and negative regulators in unstimulated or lipopolysaccharide-stimulated human bone marrow stromal cells was assessed. After 5 days, the constitutive and lipopolysaccharide-stimulated production of growth factors was significantly decreased in CMV-infected compared with uninfected stromal cells. This decrease was noted as early as 72 h after infection and appeared to be strictly related to viral replication. On the other hand, the production of inhibitory factors was increased after CMV infection, and this increased release was detectable as early as 24 h after infection. No modulation in the production of interleukin-10 was observed after CMV infection. These data suggest that CMV disturbs the balanced cytokine network, which controls proliferation and differentiation of hematopoietic progenitors. CMV-induced myelosuppression results from this lack of production of growth factors and excess production of inhibitory factors.

Granulocyte-macrophage colony-stimulating factor associated with induction treatment of acute myelogenous leukemia: a randomized trial by the European Organization for Research and Treatment of Cancer Leukemia Cooperative Group.

PURPOSE: To assess the value of granulocyte-macrophage colony-stimulating factor (GM-CSF) for induction treatment of acute myeloid leukemia (AML), both for priming of leukemic cells and for acceleration of hematopoietic recovery. PATIENTS AND METHODS: GM-CSF was administered 5 micrograms/kg/d by continuous intravenous (i.v.) infusion during induction therapy with daunorubicin (DNR) (days 1 to 3) and cytarabine (ARA-C) (days 1 to 7). A total of 102 patients were randomized onto four arms, as follows: (1) GM-CSF 24 hours before and during chemotherapy (arm +/-); (2) GM-CSF after chemotherapy until day 28 or recovery of polymorphonuclear leukocytes (PMNs) (arm -/+);(3) GM-CSF before, during, and after chemotherapy (arm +/+); or (4) no GM-CSF (arm -/-). Stopping rules were applied in case of an initial WBC count greater than 30 x 10(9)/L or a secondary increase of circulating blast cells. Analyses were performed according to the intention-to-treat principle. RESULTS: The complete remission (CR) rates were 77% (arm -/-), 72% (arm +/-), 48% (arm -/+), and 46% (arm +/+). Patients randomized to receive GM-CSF after induction (arms -/+ and +/+) had a significantly lower CR rate (P = .008) and a trend toward accelerated recovery of neutrophils, but no fewer infections or induction deaths. The lower CR rate appeared to be related to an increased resistance rate, with persistent leukemia. The main side effects of GM-CSF were fluid retention and hypotension. CONCLUSION: GM-CSF administered during induction treatment of AML with a DNR/Ara-C combination did not provide any clinical benefit. Furthermore, there was a significant decrease in the CR rate with more persistent leukemia when GM-CSF was administered during the hypoplastic phase after the chemotherapy courses.

Very low level of major BCR-ABL expression in blood of some healthy individuals.

The nested reverse transcriptase polymerase chain reaction (RT-PCR) provides a powerful tool for detection of minimal residual disease in CML. The RT-PCR used in the present study for detection of the major bcr-abl fusion gene, the hallmark and presumably the cause of CML, was optimized by: (a) increasing the amount of total RNA involved in the reverse transcription reaction to correspond to total RNA extracted from 10(8) cells; (b) using a specific abl primer in this reverse transcriptase reaction, and (c) reamplifying 10% of the RT-PCR product in a nested amplification. This optimized RT-PCR permitted to detect up to 1 copy of RNA bcr-abl synthesized in vitro, mixed with yeast RNA in a quantity equivalent to 10(8) white blood cells (WBC). Using the highly sensitive RT-PCR, a systematic study of the possible expression of bcr-abl RNA in WBC of healthy adults, children and umbilical cord blood (UCB) revealed the presence of bcr-abl transcripts in blood cells of 22/73 adults, 1/22 children but not in 22 samples of UCB. The comparison of these three groups indicated a significant tendency for the anomaly to increase in frequency with age.

Cellular uptake, cytotoxicity, and transport kinetics of anthracyclines in human sensitive and multidrug-resistant K562 cells.

Multidrug resistance in tumor cells is often associated with the presence of an approximately 170 kDa plasma membrane glycoprotein (Pgp) that acts as a drug-efflux pump and decreases intracellular antitumor drug concentration. We measured the uptake of seven anthracyclines (daunorubicin, doxorubicin, 4'-epi-doxorubicin, 4'-deoxy-doxorubicin, iododoxorubicin, 3'-(3-methoxymorpholino)-doxorubicin (FCE23762) and 4-demethoxy-daunorubicin) into K562 cells sensitive and resistant (K562/DNR) to daunorubicin. The K562/DNR subline expresses Pgp at the membrane surface, whereas its sensitive counterpart does not. Laser flow cytometry was used to quantitate intracellular anthracycline content. Uptake of daunorubicin, doxorubicin, 4'-epi-doxorubicin, and 4'-deoxy-doxorubicin was minimal in the K562/DNR subline as compared to their uptake in sensitive cells. On the contrary, iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin accumulate to nearly the same extent into sensitive and resistant K562 cells. Growth inhibition data indicated that the resistance factor for iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin is markedly decreased as compared to the other drugs. Fluorescence measurements were carried out to determine the kinetic parameters associated with the influx and efflux of the drugs into and out of K562 cells. Kinetic data indicated that iododoxorubicin, FCE23762, and 4-demethoxy-daunorubicin are not actively rejected from resistant cells, suggesting that they are poor substrates for Pgp-mediated transport. This observation is related to their ability to overcome the multidrug-resistant phenotype of K562/DNR cells in vitro.

Human cytomegalovirus increases constitutive production of interleukin-6 and leukemia inhibitory factor by bone marrow stromal cells.

Human cytomegalovirus (CMV) infection is often associated with myelosuppression and acute inflammatory reaction in immunocompromised patients. We have previously documented that CMV exposure of bone marrow (BM) stromal cells reduces the capacity of these cells to support hematopoiesis because of a decreased production of colony-stimulating factors. This study examines the potential role of CMV on constitutive and lipopolysaccharide (LPS)-stimulated production of cytokines involved in inflammatory reaction, interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) by BM stromal cells. The release of IL-6 was already detectable 2 hours post CMV-infection (2.5-fold increase in production) and the cumulative production of IL-6 after 5 days of infection was 23 +/- 1.2 ng/mL (ninefold increase in production). CMV was also able to induce a time-dependent production of LIF that was maximal 8 hours after CMV infection (2.5-fold increase in production). Concomitantly, there was no detectable release of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage CSF (GM-CSF) by CMV-infected stromal cells. The similar IL-6 and LIF production in the presence of polymyxin B ruled out the possibility that this increase could be caused by contamination of the viral stock by endotoxin. In addition, ultraviolet-inactivated virus behaved similarly to live virus and caused the release of IL-6 and LIF. However, heat-inactivated CMV was unable to induce IL-6 and LIF secretion by BM stromal cells. The production of IL-6 and LIF was also evaluated after stimulation by LPS. After 5 days of CMV exposure, the LPS-stimulated production of IL-6 and LIF was significantly lower than uninfected controls. This LPS-induced release of cytokine production was found to be dependent of viral replication. The experiments have shown that CMV is a potent inducer of IL-6 and LIF with differential effect on constitutive and LPS-stimulated cytokine production by stromal cells; we suggest that CMV induction of IL-6 and LIF during the first hours of infection could play a role in CMV-induced inflammatory reaction. Moreover, our results show that human CMV can disturb the balanced cytokine network involved in the regulation of hematopoiesis.