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Depuis plus de 60 ans, les Centres de lutte contre le cancer (CLCC) participent au service public hospitalier et sont exclusivement dédiés à la lutte contre le cancer. Cet article revient sur leur histoire, leur modèle, leur stratégie groupe avec la création d’Unicancer, la place qu’ils occupent dans la cancérologie française aujourd’hui, leur contribution aux plans cancer et les défis auxquels ils seront confrontés dans les dix prochaines années.

Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA) induces clinical remission in APL patients by enhancing the rapid differentiation of APL cells and the clearance of PML-RARα, APL’s hallmark oncoprotein. In the present study, we demonstrated that both autophagy and Beclin 1, an autophagic protein, are upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of an APL-derived cell line named NB4 cells. This induction of autophagy is associated with downregulation of Bcl-2 and inhibition of mTOR activity. Small interfering RNA-mediated knockdown of BECN1expression enhances apoptosis triggered by ATRA in NB4 cells but does not affect the differentiation process. These results provide evidence that the upregulation of Beclin 1 by ATRA constitutes an anti-apoptotic signal for maintaining the viability of mature APL cells, but has no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.

The human p53gene is a tumor suppressor mutated in half of colon cancers. Although p53 function appears important for proliferation arrest and apoptosis induced by cancer therapeutics, the prognostic significance of p53mutations remains elusive. This suggests that p53 function is modulated at a posttranslational level and that dysfunctions affecting its modulators can have a prognostic impact. Among p53 modulators, homeodomain interacting protein kinase (HIPK) 2 emerges as a candidate “switch” governing p53 transition from a cytostatic to a proapoptotic function. Thus, we investigated the possible prognostic role of HIPK2 on a retrospective series of 80 colon cancer cases by setting up a multiplexed cytometric approach capable of exploring correlative protein expression at the single tumor cell level on TMA. Crossing the data with quantitative PCR and p53gene sequencing and p53 functional assays, we observed the following: despite a strong impact on p21 transcription, the presence of disabling p53mutations has no prognostic value, and the increased expression of the HIPK2 protein in tumor cells compared with paired normal tissue cells has a strong impact on survival. Unexpectedly, HIPK2 effect does not appear to be mediated by p53 function because it is also observed in p53-disabling mutated backgrounds. Thus, our results point to a prominent and p53-independent role of HIPK2 in colon cancer survival.

The stem cell source for autologous transplantation has shifted from bone marrow to peripheral blood (PB). We previously showed that relapse incidence in patients with acute myelocytic leukemia autografted in first remission (CR1) was greater with PB than bone marrow, and a poorer outcome was associated with a shorter CR1 to PB transplantation interval (≤ 80 days). Leukemic and normal progenitors are CD34+and can be concomitantly mobilized; we assessed whether an association exists between the infused CD34+cell dose and outcome. The infused CD34+cell doses were available for 772 patients autografted more than 80 days after CR1 and were categorized by percentiles. We selected the highest quintile (> 7.16 × 106/kg) as the cutoff point. By multivariate analysis, relapse was more probable in patients who received the highest dose (hazard ratio = 1.48; 95% confidence interval, 1.12-1.95; P= .005), and leukemia-free survival was worse (hazard ratio = 0.72; 95% confidence interval, 0.55-0.93; P= .01). In conclusion, in patients autografted in first remission, relapse was higher and leukemia-free survival lower for those who received the highest CD34+PB doses.

The stem cell source for autologous transplantation has shifted from bone marrow to peripheral blood (PB). We previously showed that relapse incidence in patients with acute myelocytic leukemia autografted in first remission (CR1) was greater with PB than bone marrow, and a poorer outcome was associated with a shorter CR1 to PB transplantation interval (≤ 80 days). Leukemic and normal progenitors are CD34+ and can be concomitantly mobilized; we assessed whether an association exists between the infused CD34+ cell dose and outcome. The infused CD34+ cell doses were available for 772 patients autografted more than 80 days after CR1 and were categorized by percentiles. We selected the highest quintile (> 7.16 × 106/kg) as the cutoff point. By multivariate analysis, relapse was more probable in patients who received the highest dose (hazard ratio = 1.48; 95% confidence interval, 1.12-1.95; P = .005), and leukemia-free survival was worse (hazard ratio = 0.72; 95% confidence interval, 0.55-0.93; P = .01). In conclusion, in patients autografted in first remission, relapse was higher and leukemia-free survival lower for those who received the highest CD34+ PB doses.

Despite the excellent efficacy of imatinib in chronic myeloid leukemia (CML), the response in patients is heterogeneous, which may in part be caused by pharmacogenetic variability. Imatinib has been reported to be a substrate of the P-glycoprotein pump. In the current study, we focused on the ABCB1 (MDR1) genotype. We analyzed the 3 most relevant single nucleotide polymorphisms of MDR1 in 90 CML patients treated with imatinib. Among the patients homozygous for allele 1236T, 85% achieved a major molecular response versus 47.7% for the other genotypes (P = .003). For the 2677G>T/A polymorphism, the presence of G allele was associated with worse response (77.8%, TT/TA; vs 47.1%, GG/GA/GT; P = .018). Patients with 1236TT genotype had higher imatinib concentrations. One of the haplotypes (1236C-2677G-3435C) was statistically linked to less frequent major molecular response (70% vs 44.6%; P = .021). Hence, we demonstrated the usefulness of these single nucleotide polymorphisms in the identification of CML who may or may not respond optimally to imatinib.

Despite the excellent efficacy of imatinib in chronic myeloid leukemia (CML), the response in patients is heterogeneous, which may in part be caused by pharmacogenetic variability. Imatinib has been reported to be a substrate of the P-glycoprotein pump. In the current study, we focused on the ABCB1(MDR1) genotype. We analyzed the 3 most relevant single nucleotide polymorphisms of MDR1in 90 CML patients treated with imatinib. Among the patients homozygous for allele 1236T, 85% achieved a major molecular response versus 47.7% for the other genotypes (P= .003). For the 2677G>T/A polymorphism, the presence of G allele was associated with worse response (77.8%, TT/TA; vs 47.1%, GG/GA/GT; P= .018). Patients with 1236TT genotype had higher imatinib concentrations. One of the haplotypes (1236C-2677G-3435C) was statistically linked to less frequent major molecular response (70% vs 44.6%; P= .021). Hence, we demonstrated the usefulness of these single nucleotide polymorphisms in the identification of CML who may or may not respond optimally to imatinib.

This phase 2 study evaluated the efficacy and safety of the oral farnesyltransferase inhibitor tipifarnib in adults with refractory or relapsed acute myeloid leukemia (AML). Patients (n = 252) received tipifarnib 600 mg twice a day for 21 days in 28-day cycles. Median age was 62 years; 99 (39%) patients were 65 years or older. Eleven (4%) of 252 patients achieved complete remission (CR) or complete remission with incomplete platelet recovery (CRp; 9 CR and 2 CRp). Nineteen patients (8%), including those who achieved CR/CRp, achieved a reduction in bone marrow blasts to less than 5% blasts. Bone marrow blasts were reduced more than 50% in an additional 8 patients (total = 27; 11%). Median survival was 369 days for patients who achieved CR/CRp. Myelosuppression was the most common adverse event. The most common nonhematologic toxicities were fever, nausea, and hypokalemia. Single-agent treatment with tipifarnib induced durable CR/CRp, which was associated with prolonged survival, in some patients with refractory or relapsed AML. The response rate observed in this heavily pretreated group of patients suggests the requirement to enhance the response rate either by combining tipifarnib with other active agents or determining factors that are predictive of response.

This phase 2 study evaluated the efficacy and safety of the oral farnesyltransferase inhibitor tipifarnib in adults with refractory or relapsed acute myeloid leukemia (AML). Patients (n = 252) received tipifarnib 600 mg twice a day for 21 days in 28-day cycles. Median age was 62 years; 99 (39%) patients were 65 years or older. Eleven (4%) of 252 patients achieved complete remission (CR) or complete remission with incomplete platelet recovery (CRp; 9 CR and 2 CRp). Nineteen patients (8%), including those who achieved CR/CRp, achieved a reduction in bone marrow blasts to less than 5% blasts. Bone marrow blasts were reduced more than 50% in an additional 8 patients (total = 27; 11%). Median survival was 369 days for patients who achieved CR/CRp. Myelosuppression was the most common adverse event. The most common nonhematologic toxicities were fever, nausea, and hypokalemia. Single-agent treatment with tipifarnib induced durable CR/CRp, which was associated with prolonged survival, in some patients with refractory or relapsed AML. The response rate observed in this heavily pretreated group of patients suggests the requirement to enhance the response rate either by combining tipifarnib with other active agents or determining factors that are predictive of response.

Using high-performance liquid chromatography–tandem mass spectrometry, we assessed trough imatinib plasma levels in 68 patients with chronic myeloid leukemia (CML) who responded or not to standard-dose imatinib, after at least 12 months' treatment. Mean trough imatinib plasma levels were significantly higher in the group with complete cytogenetic response (56 patients) than in the group without (12 patients; P = .03) and higher in the group with major molecular response (MMR) than in the group without (34 patients [1452 ± 649 ng/mL] versus 34 patients [869 ± 427 ng/mL]; P < .001). Regarding trough imatinib plasma levels and their discrimination potential for MMR, the area under receiver operating characteristic curve was 0.775, with best sensitivity (77%) and specificity (71%) at a plasma threshold of 1002 ng/mL. Therefore, monitoring of imatinib plasma levels could be very useful for the management of patients with CML or should at least be checked in the case of treatment failure or suboptimal response.

Using high-performance liquid chromatography–tandem mass spectrometry, we assessed trough imatinib plasma levels in 68 patients with chronic myeloid leukemia (CML) who responded or not to standard-dose imatinib, after at least 12 months' treatment. Mean trough imatinib plasma levels were significantly higher in the group with complete cytogenetic response (56 patients) than in the group without (12 patients; P= .03) and higher in the group with major molecular response (MMR) than in the group without (34 patients [1452 ± 649 ng/mL] versus 34 patients [869 ± 427 ng/mL]; P< .001). Regarding trough imatinib plasma levels and their discrimination potential for MMR, the area under receiver operating characteristic curve was 0.775, with best sensitivity (77%) and specificity (71%) at a plasma threshold of 1002 ng/mL. Therefore, monitoring of imatinib plasma levels could be very useful for the management of patients with CML or should at least be checked in the case of treatment failure or suboptimal response.

Imatinib mesylate, a BCR‐ABL tyrosine kinase inhibitor, induces apoptosis in chronic myeloid leukemia cells. Resistance to imatinib is currently the most important concern of this treatment. One of the main mechanisms of this resistance is overexpression of BCR‐ABL.In the current study, the authors investigated the correlation between BCR‐ABL overexpression and apoptosis in BaF/BCR‐ABL and LAMA84 cell lines resistant to imatinib suddenly deprived of the inhibitor, and compared with their sensitive counterpart.Removal of imatinib from culture medium led to a decrease in Bcr‐Abl protein expression by Day 5, which was sustained for ≥ 3 weeks of imatinib deprivation. Apoptosis was observed after 3 days of imatinib deprivation in resistant lines accompanied by caspase activation, loss of membrane asymmetry (annexin V staining), and alteration of mitochondrial potential (dihexyloxacarbocyanine iodide [DiOC6]). Transient activation of the STAT5/Bcl‐xL pathway and Akt kinase activity preceded these responses.Thus, imatinib removal led to apoptosis of BCR‐ABL–overexpressing leukemic cells, a phenomenon that could be exploited to sensitize imatinib‐resistant cells to the cytotoxic effect of other drugs. Cancer 2005. © 2004 American Cancer Society.

Several studies have compared bone marrow (BM) and peripheral blood (PB) as stem cell sources in patients receiving allografts, but the cell doses infused have not been considered, especially for BM. Using the ALWP/EBMT registry, we retrospectively studied 881 adult patients with acute myelocytic leukemia (AML), who received a non–T-depleted allogeneic BM (n = 515) or mobilized PB (n = 366) standard transplant, in first remission (CR1), from an HLA-identical sibling, over a 5-year period from January 1994. The BM cell dose ranged from 0.17 to 29 × 108/kg with a median of 2.7 × 108/kg. The PB cell dose ranged from 0.02 to 77 × 108/kg with a median of 9.3 × 108/kg. The median dose for patients receiving BM (2.7 × 108/kg) gave the greatest discrimination. In multivariate analyses, high-dose BM compared to PB was associated with lower transplant-related mortality (RR = 0.61; 95% CI, 0.39-0.98; P= .04), better leukemia-free survival (RR = 0.65; 95% CI, 0.46-0.91; P= .013), and better overall survival (RR = 0.64; 95% CI, 0.44-0.92; P= .016). The present study in patients with AML receiving allografts in first remission indicates a better outcome with BM as compared to PB, when the dose of BM infused is rich.

Several studies have compared bone marrow (BM) and peripheral blood (PB) as stem cell sources in patients receiving allografts, but the cell doses infused have not been considered, especially for BM. Using the ALWP/EBMT registry, we retrospectively studied 881 adult patients with acute myelocytic leukemia (AML), who received a non–T-depleted allogeneic BM (n = 515) or mobilized PB (n = 366) standard transplant, in first remission (CR1), from an HLA-identical sibling, over a 5-year period from January 1994. The BM cell dose ranged from 0.17 to 29 × 108/kg with a median of 2.7 × 108/kg. The PB cell dose ranged from 0.02 to 77 × 108/kg with a median of 9.3 × 108/kg. The median dose for patients receiving BM (2.7 × 108/kg) gave the greatest discrimination. In multivariate analyses, high-dose BM compared to PB was associated with lower transplant-related mortality (RR = 0.61; 95% CI, 0.39-0.98; P = .04), better leukemia-free survival (RR = 0.65; 95% CI, 0.46-0.91; P = .013), and better overall survival (RR = 0.64; 95% CI, 0.44-0.92; P = .016). The present study in patients with AML receiving allografts in first remission indicates a better outcome with BM as compared to PB, when the dose of BM infused is rich.

Acute myeloid leukemias (AMLs) carrying inv(16)/t(16;16) chromosomal abnormalities are associated with a good prognosis. However, studies of this AML subtype have been hampered by the few number of patients reported, frequently collectively considered with those with AML carrying the t(8;21) translocation. We performed a retrospective study in 110 patients with inv(16)/t(16;16) AML (median age, 34 years) prospectively enrolled in 6 trials conducted in France between 1987 and 1998, with the aim to investigate prognostic factors for complete remission (CR) achievement and outcome of CR patients in this AML subtype. CR rate was 93%. Bad-prognosis factors for CR achievement were higher white blood cell count (WBC) and lower platelet count (optimal cutpoints at 120 and 30 × 109/L, respectively). At 3 years, estimated overall survival, disease-free survival (DFS), and cumulative incidence of relapse were 58%, 48%, and 42%, respectively. In multivariate analysis, (1) advanced age (optimal cutpoint, 35 years) was the only factor for shorter DFS and (2) advanced age and low platelet count were the 2 factors for shorter survival of CR patients. Outcome of CR patients (1) was not influenced by WBC and cytogenetic findings and (2) was similar among patients allocated to receive allogeneic transplantation, high-dose, or intermediate-dose cytarabine. Interestingly, advanced age was associated with a trend for more frequent additional chromosome abnormalities and predictive of higher cumulative incidence of relapse rather than death in first CR. These results markedly contrast with those reported in patients with t(8;21) AML in whom WBC, and not age, was the main high-risk factor for relapse, DFS, and survival.

Acute myeloid leukemias (AMLs) carrying inv(16)/t(16;16) chromosomal abnormalities are associated with a good prognosis. However, studies of this AML subtype have been hampered by the few number of patients reported, frequently collectively considered with those with AML carrying the t(8;21) translocation. We performed a retrospective study in 110 patients with inv(16)/t(16;16) AML (median age, 34 years) prospectively enrolled in 6 trials conducted in France between 1987 and 1998, with the aim to investigate prognostic factors for complete remission (CR) achievement and outcome of CR patients in this AML subtype. CR rate was 93%. Bad-prognosis factors for CR achievement were higher white blood cell count (WBC) and lower platelet count (optimal cutpoints at 120 and 30 × 109/L, respectively). At 3 years, estimated overall survival, disease-free survival (DFS), and cumulative incidence of relapse were 58%, 48%, and 42%, respectively. In multivariate analysis, (1) advanced age (optimal cutpoint, 35 years) was the only factor for shorter DFS and (2) advanced age and low platelet count were the 2 factors for shorter survival of CR patients. Outcome of CR patients (1) was not influenced by WBC and cytogenetic findings and (2) was similar among patients allocated to receive allogeneic transplantation, high-dose, or intermediate-dose cytarabine. Interestingly, advanced age was associated with a trend for more frequent additional chromosome abnormalities and predictive of higher cumulative incidence of relapse rather than death in first CR. These results markedly contrast with those reported in patients with t(8;21) AML in whom WBC, and not age, was the main high-risk factor for relapse, DFS, and survival.

Inappropriate expression of the multidrug resistance(MDR1) gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1 gene overexpression, grew continuously in the presence of 1 μM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC50) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 μM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of theBCR-ABL+ AR230 cell line with theMDR1 gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.

Inappropriate expression of the multidrug resistance(MDR1)gene encoding the P-glycoprotein (Pgp) has been frequently implicated in resistance to different chemotherapeutic drugs. We have previously generated chronic myeloid leukemia (CML) cell lines resistant to the tyrosine kinase inhibitor imatinib mesylate (STI571), and one line (LAMA84-r) showed overexpression not only of the Bcr-Abl protein but also of Pgp. In the present study, we investigated this phenomenon in other cell lines overexpressing exclusively Pgp. Thus, cells from the K562/DOX line, described as resistant to doxorubicin due to MDR1gene overexpression, grew continuously in the presence of 1 μM imatinib, but died in 4 to 5 days if the Pgp pump modulators verapamil or PSC833 were added to the imatinib-treated culture. Analysis of cell proliferation by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay confirmed the differential sensitivity of K562/DOX to imatinib, which was also reversed by verapamil or PSC833. Flow cytometric analysis of the total phosphotyrosine content by intracytoplasmic staining after a 2-hour incubation with escalating doses of imatinib showed that the inhibitory concentrations of 50% (IC50) for inhibition of cellular protein tyrosine phosphorylation were 15, 10, and 5 μM for K562/DOX, K562/DOX plus verapamil, and K562, respectively. Retroviral-mediated transfection of theBCR-ABL+AR230 cell line with theMDR1gene decreased its sensitivity to imatinib, an effect that was also reversed by verapamil. The possible role of MDR overexpression in clinical resistance to imatinib remains to be defined. We therefore confirm that imatinib should be added to the extensive list of drugs that can be affected by the MDR phenomenon.

De novo erythroleukemia (EL) is a rare disease. Reported median survival are poor and vary from 4 to 14 months. The value of hematopoietic stem cell transplantation (HSCT) for EL is unknown. This EBMT registry study reports on the largest series of patients with EL treated with HSCT in first complete remission—103 autologous and 104 HLA identical sibling allogeneic HSCT. Outcome and identification of prognostic factors for each type of transplantation were evaluated. For autologous HSCT, outcome at 5 years showed a leukemia-free survival (LFS) of 26% ± 5%, a relapse incidence (RI) of 70% ± 6%, and a transplant-related mortality (TRM) of 13% ± 4%. By multivariate analysis, the only prognostic factor was age. For allogeneic HSCT, outcome at 5 years showed an LFS of 57% ± 5%, an RI of 21% ± 5%, and a TRM of 27% ± 5%. By multivariate analysis, prognostic factors were graft-versus-host disease and age. This study represents the largest series of de novo EL treated with HSCT and shows that allogeneic HSCT is by far the most effective treatment.

De novo erythroleukemia (EL) is a rare disease. Reported median survival are poor and vary from 4 to 14 months. The value of hematopoietic stem cell transplantation (HSCT) for EL is unknown. This EBMT registry study reports on the largest series of patients with EL treated with HSCT in first complete remission—103 autologous and 104 HLA identical sibling allogeneic HSCT. Outcome and identification of prognostic factors for each type of transplantation were evaluated. For autologous HSCT, outcome at 5 years showed a leukemia-free survival (LFS) of 26% ± 5%, a relapse incidence (RI) of 70% ± 6%, and a transplant-related mortality (TRM) of 13% ± 4%. By multivariate analysis, the only prognostic factor was age. For allogeneic HSCT, outcome at 5 years showed an LFS of 57% ± 5%, an RI of 21% ± 5%, and a TRM of 27% ± 5%. By multivariate analysis, prognostic factors were graft-versus-host disease and age. This study represents the largest series of de novo EL treated with HSCT and shows that allogeneic HSCT is by far the most effective treatment.

It is often postulated that trans-3,4’,5-trihydroxystilbene (resveratrol, RES) exhibits cell growth regulatory and chemopreventive activities. However, mechanisms by which this polyphenol inhibits tumor cell growth, and its therapeutic potential are poorly understood. Using various human leukemia cells, we have first defined the anti-tumoral doses of this compound. RES inhibited the proliferation and induced the apoptosis of all tested lymphoid and myeloid leukemia cells with IC50 = 5–43 μM. Prior to apoptosis, RES-induced caspase activity in a dose-dependent manner and cell cycle arrest in G2/M-phase, correlating with a significant accumulation of cyclins A and B. Leukemia cell death with RES required both caspase-dependent and -independent proteases, as it was significantly inhibited by simultaneous addition of Z-VAD-FMK and leupeptin to these cultures. While RES did not affect non-activated normal lymphocytes, this agent decreased the growth and induced the apoptosis of cycling normal human peripheral blood lymphocytes at lower concentrations (IC50 <8 μM) than those required for most leukemia cells. RES also induced the apoptosis of early normal human CD34+ cells and decreased the number of colonies generated by these precursor cells in a dose-dependent manner (IC50 = 60 μM). Together, the data point to the complexity of RES-mediated signaling pathways and revealed the high anti-proliferative and proapoptotic activities of RES in normal cycling hemopoietic cells.

It is often postulated that trans-3,4',5-trihydroxystilbene (resveratrol, RES) exhibits cell growth regulatory and chemopreventive activities. However, mechanisms by which this polyphenol inhibits tumor cell growth, and its therapeutic potential are poorly understood. Using various human leukemia cells, we have first defined the anti-tumoral doses of this compound. RES inhibited the proliferation and induced the apoptosis of all tested lymphoid and myeloid leukemia cells with IC(50) = 5-43 microM. Prior to apoptosis, RES-induced caspase activity in a dose-dependent manner and cell cycle arrest in G(2)/M-phase, correlating with a significant accumulation of cyclins A and B. Leukemia cell death with RES required both caspase-dependent and -independent proteases, as it was significantly inhibited by simultaneous addition of Z-VAD-FMK and leupeptin to these cultures. While RES did not affect non-activated normal lymphocytes, this agent decreased the growth and induced the apoptosis of cycling normal human peripheral blood lymphocytes at lower concentrations (IC(50) <8 microM) than those required for most leukemia cells. RES also induced the apoptosis of early normal human CD34(+) cells and decreased the number of colonies generated by these precursor cells in a dose-dependent manner (IC(50) = 60 microM). Together, the data point to the complexity of RES-mediated signaling pathways and revealed the high anti-proliferative and proapoptotic activities of RES in normal cycling hemopoietic cells.

While the t(8;21) translocation is one of the most recurrent chromosomal abnormalities in acute myeloid leukemia, prognostic studies have been hampered by the relatively few number of patients reported. We thus performed a large retrospective study in 161 adults and children with t(8;21) acute myeloid leukemia, all prospectively enrolled in 6 different trials conducted in France between 1987 and 1998 (median follow-up 4.9 years). Prognostic studies were performed in the 154 patients who achieved a complete remission. Individual data were registered, including sex, age, blood and marrow counts, extramedullary disease, and cytogenetics. The value of allogeneic stem cell transplantation versus chemotherapy as postremission therapy was evaluated according to the intent-to-treat principle. Estimated 5-year disease-free survival (DFS) and overall survival were 52% and 59%, respectively. Outcome was not significantly better in patients from the stem cell transplantation group (estimated 5-year DFS and survival, 56% vs 52% and 67% vs 57%; P = .55 and .64, respectively). White blood cell count (WBC) was the only identified prognostic factor. To further take into account the spontaneous differentiation potential of the leukemic clone, a WBC index was derived as the product of WBC by the ratio of marrow blast. This WBC index was a more powerful factor than the original WBC, allowing us to distinguish 3 subgroups of patients with different outcomes (low index, < 2.5; intermediate index, 2.5-20; high index, 20 or more). In multivariate analysis, the WBC index was the only prognostic factor for DFS (P = .003), complete remission duration (P = .002), and overall survival (P = .04).

While the t(8;21) translocation is one of the most recurrent chromosomal abnormalities in acute myeloid leukemia, prognostic studies have been hampered by the relatively few number of patients reported. We thus performed a large retrospective study in 161 adults and children with t(8;21) acute myeloid leukemia, all prospectively enrolled in 6 different trials conducted in France between 1987 and 1998 (median follow-up 4.9 years). Prognostic studies were performed in the 154 patients who achieved a complete remission. Individual data were registered, including sex, age, blood and marrow counts, extramedullary disease, and cytogenetics. The value of allogeneic stem cell transplantation versus chemotherapy as postremission therapy was evaluated according to the intent-to-treat principle. Estimated 5-year disease-free survival (DFS) and overall survival were 52% and 59%, respectively. Outcome was not significantly better in patients from the stem cell transplantation group (estimated 5-year DFS and survival, 56% vs 52% and 67% vs 57%; P= .55 and .64, respectively). White blood cell count (WBC) was the only identified prognostic factor. To further take into account the spontaneous differentiation potential of the leukemic clone, a WBC index was derived as the product of WBC by the ratio of marrow blast. This WBC index was a more powerful factor than the original WBC, allowing us to distinguish 3 subgroups of patients with different outcomes (low index, < 2.5; intermediate index, 2.5-20; high index, 20 or more). In multivariate analysis, the WBC index was the only prognostic factor for DFS (P= .003), complete remission duration (P= .002), and overall survival (P= .04).

Achieving a complete cytogenetic response (CCgR) is a major target in the treatment of chronic myeloid leukemia (CML) with interferon-α (IFN-α), but CCgRs are rare. The mean CCgR rate is 13%, in a range of 5% to 33%. A collaborative study of 9 European Union countries has led to the collection of data on 317 patients who were first seen between 1983 and 1997 and achieved CCgRs with IFN-α alone or in combination with hydroxyurea. The median time to first CCgR was 19 months (95% CI, 17-21; range, 3-84 months). At last contact, 212 patients were still alive and in continuous CCgR; 105 patients had lost CCgR, but 53% of them were still alive and in chronic phase. IFN-α treatment was discontinued permanently in 23 cases for response loss, in 36 cases for chronic toxicity (15 are still in unmaintained continuous CCgR), and in 8 cases because it was believed that treatment was no longer necessary (7 of these 8 patients are still in unmaintained continuous CCgR). The 10-year survival rate from first CCgR is 72% (95% CI, 62%-82%) and is related to the risk profile. High-risk patients lost CCgR more frequently and more rapidly and none survived more than 10 years. Low-risk patients survived much longer (10-year survival probability 89% for Sokal low risk and 81% for Euro low risk). These data point out that a substantial long-term survival in CCgRs is restricted mainly to low-risk and possibly intermediate-risk patients and occurs significantly less often in high-risk patients.

Achieving a complete cytogenetic response (CCgR) is a major target in the treatment of chronic myeloid leukemia (CML) with interferon-α (IFN-α), but CCgRs are rare. The mean CCgR rate is 13%, in a range of 5% to 33%. A collaborative study of 9 European Union countries has led to the collection of data on 317 patients who were first seen between 1983 and 1997 and achieved CCgRs with IFN-α alone or in combination with hydroxyurea. The median time to first CCgR was 19 months (95% CI, 17-21; range, 3-84 months). At last contact, 212 patients were still alive and in continuous CCgR; 105 patients had lost CCgR, but 53% of them were still alive and in chronic phase. IFN-α treatment was discontinued permanently in 23 cases for response loss, in 36 cases for chronic toxicity (15 are still in unmaintained continuous CCgR), and in 8 cases because it was believed that treatment was no longer necessary (7 of these 8 patients are still in unmaintained continuous CCgR). The 10-year survival rate from first CCgR is 72% (95% CI, 62%-82%) and is related to the risk profile. High-risk patients lost CCgR more frequently and more rapidly and none survived more than 10 years. Low-risk patients survived much longer (10-year survival probability 89% for Sokal low risk and 81% for Euro low risk). These data point out that a substantial long-term survival in CCgRs is restricted mainly to low-risk and possibly intermediate-risk patients and occurs significantly less often in high-risk patients.

For the management of chronic myeloid leukemia (CML), prediction or early determination of the response to interferon-alpha (IFN-α) treatment is important for identifying nonresponder patients to whom alternative therapy may be proposed. In this study, the levels of expression of both BCR-ABL and subunit 2c of IFN-α receptor (IFN-αR2c) genes were analyzed at diagnosis in 74 patients with chronic phase CML treated with an IFN-α monotherapy. By using blood samples, real-time quantitative polymerase chain reaction was performed to quantify BCR-ABL, IFN-αR2c, and G6PDH mRNA as external control. The results were compared with hematologic and cytogenetic responses to IFN-α. A wide variation in the BCR-ABL/G6PDH ratio was observed at diagnosis (median, 6.68%; range, 0.18%-41.31%), but no significant association with response to IFN-α was observed. In contrast, the variation of IFN-αR2c/G6PDH ratio at diagnosis was significantly associated with the achievement of major cytogenetic response (MCR; 34% or lower Ph+metaphases). Median values of IFN-αR2c/G6PDH ratio for patients achieving MCR and for those who did not achieve it were 110.75% (range, 9.47%-612.30%) and 64.42% (range, 5.96%-425.40%), respectively (P = .037). In addition, this novel molecular factor, combined with the achievement of complete hematologic response at 3 months, makes it possible to predict MCR achievement with high probability by Kaplan-Meier analysis (91% ± 17% at 24 months; P = .0001).

For the management of chronic myeloid leukemia (CML), prediction or early determination of the response to interferon-alpha (IFN-α) treatment is important for identifying nonresponder patients to whom alternative therapy may be proposed. In this study, the levels of expression of both BCR-ABLand subunit 2c of IFN-α receptor (IFN-αR2c) genes were analyzed at diagnosis in 74 patients with chronic phase CML treated with an IFN-α monotherapy. By using blood samples, real-time quantitative polymerase chain reaction was performed to quantify BCR-ABL, IFN-αR2c, and G6PDH mRNA as external control. The results were compared with hematologic and cytogenetic responses to IFN-α. A wide variation in the BCR-ABL/G6PDH ratio was observed at diagnosis (median, 6.68%; range, 0.18%-41.31%), but no significant association with response to IFN-α was observed. In contrast, the variation of IFN-αR2c/G6PDH ratio at diagnosis was significantly associated with the achievement of major cytogenetic response (MCR; 34% or lower Ph+metaphases). Median values of IFN-αR2c/G6PDH ratio for patients achieving MCR and for those who did not achieve it were 110.75% (range, 9.47%-612.30%) and 64.42% (range, 5.96%-425.40%), respectively (P= .037). In addition, this novel molecular factor, combined with the achievement of complete hematologic response at 3 months, makes it possible to predict MCR achievement with high probability by Kaplan-Meier analysis (91% ± 17% at 24 months; P= .0001).

Targeting the tyrosine kinase activity of Bcr-Abl with STI571 is an attractive therapeutic strategy in chronic myelogenous leukemia (CML). A few CML cell lines and primary progenitors are, however, resistant to this compound. We investigated the mechanism of this resistance in clones of the murine BaF/3 cells transfected with BCR-ABL and in 4 human cell lines from which sensitive (s) and resistant (r) clones were generated by various methods. Although the resistant cells were able to survive in the presence of STI571, their proliferation was approximately 30% lower than that of their sensitive counterparts in the absence of the compound. The concentration of STI571 needed for a 50% reduction in viable cells after a 3-day exposure was on average 10 times higher in the resistant (2-3 µmol/L) than in the sensitive (0.2-0.25 µmol/L) clones. The mechanism of resistance to STI571 varied among the cell lines. Thus, in Baf/BCR-ABL-r, LAMA84-r, and AR230-r, there was up-regulation of the Bcr-Abl protein associated with amplification of the BCR-ABL gene. In K562-r, there was no Bcr-Abl overexpression, but the IC50 for the inhibition of Bcr-Abl autophosphorylation was increased in the resistant clones. Sequencing of the Abl kinase domain revealed no mutations. The multidrug resistance P-glycoprotein (Pgp) was overexpressed in LAMA84-r, indicating that at least 2 mechanisms of resistance operate in this cell line. KCL22-r showed neither Bcr-Abl up-regulation nor a higher threshold for tyrosine kinase inhibition by STI571. We conclude that BCR-ABL–positive cells can evade the inhibitory effect of STI571 by different mechanisms, such as Bcr-Abl overexpression, reduced intake mediated by Pgp, and, possibly, acquisition of compensatory mutations in genes other than BCR-ABL.

CD40 ligand (CD40L)/CD40 interactions play a central role in T-cell–dependent B-cell activation as previously shown by in vitro studies, the phenotype of CD40L knockout mice and the defective expression of CD40L in patients who have X-linked immunodeficiency with hyper-IgM. The distribution of CD40 in cells other than of myeloid and lymphoid lineages has suggested additional functions for this receptor/ligand couple. Here we show that CD40L stimulates myelopoiesis with a noticeable effect on megakaryocytopoiesis in cocultures of hematopoietic progenitor cells and bone marrow stromal cells. These results suggest a mechanism by which T-cell or platelet-associated or soluble CD40L may regulate myelopoiesis.

Interferon (IFN) is an effective treatment for chronic myeloid leukemia (CML) in chronic phases, and a number of in vitro antileukemic effects of IFN on CML cells have been reported. The transfer of cytokine genes into tumor cells is reportedly a valuable approach to improve the antitumor activity of cytokines in various models. We first investigated the possibility of transducing CML cells with the retroviral vectors LIα2SN and LIγSN, encoding the IFN-α2 and IFN-γ genes, respectively, and with the bicistronic vector LIα2IrIγSN coexpressing the IFN-α 2 and IFN-γ genes. We then analyzed the effects of IFN-α2 and IFN-γ produced alone or simultaneously on the proliferation of CML cells. We optimized the transduction efficiency by using the CML-derived K562 cell line. We then introduced IFN genes into CML CD34+ cells. Secretion of IFN-α and IFN-γ was demonstrated in K562 and CML CD34+ cells transduced with the different vectors. The MHC class I antigens were overexpressed in both K562 and CML CD34+ transduced cells. Inhibition of the proliferation of LIα2IrIγSN-transduced CML cells was greater than with the LI alpha 2SN and the LIγSN-transduced CML cells. We demonstrate an additive effect of IFN-α and IFN-γ on the inhibition of K562 and CML CD34+ cell proliferation.

In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle (Viallard et al., Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. Cell synchronization was performed in G1 with sodium n-butyrate, at the G1/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G2/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G1/S boundary by thymidine or inside the G1 phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G1/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle showed that cyclin B1 was present in the G1 phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G1 phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G2/M phases and independent from the DNA replication cycle. Cytometry 39:117–125, 2000 © 2000 Wiley-Liss, Inc.

MATWIN (Maturation and Accelerating Translation With INdustry) is part of the nationwide effort to support cancer innovation. This unique program is willing to support innovative research projects providing tools, resources, and staff dedicated to project leaders wishing to optimize the industrial attractiveness of their project. The overall objective is clear: fight cancer always more effectively.

Stem cell factor (SCF) has been shown to synergize with filgrastim to mobilize CD34+ cells into the peripheral blood. To determine if addition of SCF to chemotherapy and filgrastim reduces the number of leukaphereses required to achieve a target yield of 5 × 106 CD34+ cells/kg, 102 patients with multiple myeloma were randomized to receive mobilization chemotherapy with cyclophosphamide (4 g/m2) and either SCF (20 µg/kg/d) combined with filgrastim (5 µg/kg/d) or filgrastim alone (5 µg/kg/d), administered daily until leukaphereses were completed. After collection, patients were treated with myeloablative therapy supported by autologous peripheral blood progenitor cell (PBPC) infusion and filgrastim (5 µg/kg/d). There was a significant difference between the treatment groups in the number of leukaphereses required to collect 5 × 106 CD34+ cells/kg (median of 1 v 2 for SCF + filgrastim and filgrastim alone, respectively, P = .008). Patients receiving the combination of SCF plus filgrastim had a 3-fold greater chance of reaching 5 × 106 CD34+ cells/kg in a single leukapheresis compared with patients mobilized with filgrastim alone. The median CD34+ cell yield was significantly increased for the SCF group in the first leukapheresis (11.3 v 4.0 × 106/kg, P = .003) and all leukaphereses (12.4v 8.2 × 106/kg, P = .007). Total colony-forming unit–granulocyte-macrophage (CFU-GM) and mononuclear cell counts were also significantly higher in the SCF group in the first leukapheresis and in all leukaphereses. As expected for patients mobilized to an optimal CD34+ cell yield, the time to engraftment was similar between the 2 treatment groups. Cells mobilized with the combination of SCF plus filgrastim were thus considered effective and safe for achieving rapid engraftment. Treatment with SCF plus filgrastim was well tolerated, with mild to moderate injection site reactions being the most frequently reported adverse events. There were no serious allergic-like reactions to SCF. The addition of SCF to filgrastim after cyclophosphamide for PBPC mobilization resulted in a significant increase in CD34+cell yield and a concomitant reduction in the number of leukaphereses required to collect an optimal harvest of 5 × 106CD34+ cells/kg.

Gene transduction into immature human hematopoietic cells collected from umbilical cord blood, bone marrow, or mobilized peripheral blood cells could be useful for the treatment of genetic and acquired disorders of the hematopoietic system. Immunodeficient mouse models have been used frequently as recipients to assay the growth and differentiation of human hematopoietic stem/progenitor cells. Indeed, high levels of human cell engraftment were first reported in human/murine chimeras using NOD/SCID mice, which now are considered as the standard for these types of experiments. However, NOD/SCID mice have some clear disadvantages (including spontaneous tumor formation) that limit their general use. We have developed a new immunodeficient mouse model by combining recombinase activating gene-2 (RAG2) and common cytokine receptor gamma chain (gamma c) mutations. The RAG2-/-/gamma c-double mutant mice are completely alymphoid (T-, B-, NK-), show no spontaneous tumor formation, and exhibit normal hematopoietic parameters. Interestingly, human cord blood cell engraftment in RAG2-/-/gamma c- mice was greatly enhanced by the exogenous administration of human cytokines interleukin-(IL-3) granulocyte-macrophage colony-stimulating factor, (GM-CSF), and erythropoietin in contrast to the NOD/SC ID model. This unique feature of the RAG2-/-/gamma c-mouse model should be particularly well suited for assessing the role of different cytokines in human lymphopoiesis and stem/progenitor cell function in vivo .

The expression of neurotrophins and their receptors, the low-affinity nerve growth factor receptor (p75LNGFR) and the Trk receptors (TrkA, TrkB, and TrkC), was investigated in human bone marrow from 16 weeks fetal age to adulthood. Using reverse transcription-polymerase chain reaction, all transcripts encoding for catalytic and truncated human TrkB or TrkC receptors were detected together with trkAI transcripts, whereas trkAII transcripts were found only in control nerve tissues. Transcripts for the homologue of the rat truncated TrkC(ic113) receptor were identified for the first time in human tissue. Stromal adventitial reticular cells were found immunoreactive for all neutrophin receptors. In contrast, hematopoietic cell types were not immunoreactive for p75LNGFRbut showed immunoreactivity for one or several Trk receptors. TrkA immunoreactivity was found in immature erythroblasts. Catalytic TrkB immunoreactivity was observed in eosinophilic metamyelocytes and polymorphonuclear cells. Truncated TrkB immunoreactivity was found in erythroblasts and megacaryocytes. Immunoreactivity for both catalytic and truncated TrkC receptor was observed in promyelocytes, myelocytes, some polymorphonuclear cells and megacaryocytes. Neutrophin transcript levels appeared higher at fetal than at adult stages, no variation in Trk family transcript levels was observed. The local expression of neurotrophin genes suggests a wide range of paracrine and/or autocrine mode of action through their corresponding receptors within the bone marrow.

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine which is involved in the regulation of the immune response and haematopoiesis. The authors investigated the regulation of the expression of LIF by glucocorticosteroids (GC). Endothelial cells (EC) constitutively produce LIF and this production is enhanced by interleukin 1 (IL-1). GC were found to inhibit the basal production of LIF by EC and to suppress its IL-1-induced augmentation. Whether corticosteroids suppress LIF production by blocking transcription of LIF mRNA, or by blocking LIF synthesis at a post-transcriptional level was examined. Northern blot hybridization analysis demonstrated that GC act mainly by decreasing the LIF mRNA level. In the presence of translation inhibitors a superinduction of LIF mRNA was observed. Dexamethasone (DEX) at a concentration of 1 μM was responsible for a rapid increase in the degradation rate of LIF mRNA which resulted in reducing its level by more than 50% within 2 h, whereas the transcription rate of LIF gene was not significantly altered in these conditions. These results demonstrated that GC inhibit LIF mRNA expression mainly by increasing the turnover rate of the LIF mRNA. The early LIF mRNA destabilizing activity of GC was translation dependent as shown by experiments with protein translation inhibitors. The results indicate that corticosteroids are inhibitors of LIF expression and that this inhibition mainly occurs through post-transcriptional mechanisms.

Stem cell factor (SCF) has been shown to synergize with filgrastim to mobilize CD34+cells into the peripheral blood. To determine if addition of SCF to chemotherapy and filgrastim reduces the number of leukaphereses required to achieve a target yield of 5 × 106CD34+cells/kg, 102 patients with multiple myeloma were randomized to receive mobilization chemotherapy with cyclophosphamide (4 g/m2) and either SCF (20 μg/kg/d) combined with filgrastim (5 μg/kg/d) or filgrastim alone (5 μg/kg/d), administered daily until leukaphereses were completed. After collection, patients were treated with myeloablative therapy supported by autologous peripheral blood progenitor cell (PBPC) infusion and filgrastim (5 μg/kg/d). There was a significant difference between the treatment groups in the number of leukaphereses required to collect 5 × 106CD34+cells/kg (median of 1 v2 for SCF + filgrastim and filgrastim alone, respectively, P= .008). Patients receiving the combination of SCF plus filgrastim had a 3-fold greater chance of reaching 5 × 106CD34+cells/kg in a single leukapheresis compared with patients mobilized with filgrastim alone. The median CD34+cell yield was significantly increased for the SCF group in the first leukapheresis (11.3 v4.0 × 106/kg, P= .003) and all leukaphereses (12.4v8.2 × 106/kg, P= .007). Total colony-forming unit–granulocyte-macrophage (CFU-GM) and mononuclear cell counts were also significantly higher in the SCF group in the first leukapheresis and in all leukaphereses. As expected for patients mobilized to an optimal CD34+cell yield, the time to engraftment was similar between the 2 treatment groups. Cells mobilized with the combination of SCF plus filgrastim were thus considered effective and safe for achieving rapid engraftment. Treatment with SCF plus filgrastim was well tolerated, with mild to moderate injection site reactions being the most frequently reported adverse events. There were no serious allergic-like reactions to SCF. The addition of SCF to filgrastim after cyclophosphamide for PBPC mobilization resulted in a significant increase in CD34+cell yield and a concomitant reduction in the number of leukaphereses required to collect an optimal harvest of 5 × 106CD34+cells/kg.

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1phase by sodiumn-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodiumn-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.

Antisense oligonucleotides were used to determine the role of the BCR-ABL gene in the proliferation of chronic myeloid leukaemia (CML) clonogenic cells. Peripheral blood Philadelphia chromosome positive cells were obtained from eight CML patients at diagnosis (chronic phase = 7; accelerared phase = 1). Mononuclear cells were incubated with synthetic antisense 18-mer oligonucleotides complementary to the two different junctions b2a2 or b3a2. The type of junction (b2a2 or b3a2) was previously determined by RT-PCR techniques. Cells incubated for 12 to 14 hours with or without sense oligonucleotides served as controls. After incubation with oligonucleotides, the cell DNA synthesis was analysed by flow cytometry using the BrdUrd/DNA method and, the cell plating efficiency in methylcellulose was determined. In six of the seven patients in chronic phase, there was a significant inhibition of CFU-GM production which was only 68.4 ± 19%; (p <.01) of that found in controls. The S phase index, which depends upon the percentage of S phase cells ax well as the fluorescence intensity, was 48 ± 29% (p <.01) of the control values for the seven patients in chronic phase. Interestingly, for the only CML patient in accelerated phase, antisense oligomers had no inhibitory effect on either the production of CFU-GM or the number of S phase cells. In improving the specificity of oligomers, it might be usefull for gene-targeted anti-leukemic therapy and/or bone marrow purging.

Advanced low-grade lymphomas are usually incurable with conventional-dose chemotherapy. It is uncertain whether cures are possible with high-dose therapy and bone marrow transplant from a human leukocyte antigen (HLA)-identical sibling. We sought to determine the outcome of HLA-identical sibling bone marrow transplants in advanced low-grade lymphoma in an observational study of 113 patients conducted at 50 centers participating in the International Bone Marrow Transplant Registry (IBMTR). The median patient age was 38 years (range, 15 to 61). Eighty percent had stage IV disease at the time of transplantation. The median number of prior chemotherapy regimens was two (range, 0 to 5). Thirty-eight percent had refractory disease and 29% a Karnofsky performance score (KPS) less than 80%. All patients underwent allogeneic bone marrow transplantation from a HLA-identical sibling donor. The conditioning regimen included total-body irradiation (TBI) in 82% of patients; cyclosporine was used for graft-versus-host disease prophylaxis in 74%. Survival, disease-free survival, recurrence rate, treatment-related mortality, and causes of death were determined. Three-year probabilities of recurrence, survival, and disease-free survival were 16% (95% confidence interval [CI], 9% to 27%), 49% (95% CI, 39% to 60%), and 49% (95% CI, 39% to 59%), respectively. Higher survival was associated with pretransplant KPS =90%, chemotherapy-sensitive disease, use of a TBI-containing conditioning regimen, and age less than 40 years. We conclude that high-dose therapy followed by transplantation from a HLA-identical sibling leads to prolonged survival in some patients with advanced low-grade lymphoma. Most mortality is treatment-related, and recurrences are rare. © 1998 by The American Society of Hematology.

In this study, we investigated the impact of recombinant interleukin-2 (rIL-2) after high dose chemotherapy and autologous bone marrow transplantation (ABMT) in 25 patients with refractory or relapsed Hodgkin's disease (HD) (11 patients) and non Hodgkin's lymphoma (NHL) (14 patients). 48% of patients had resistant disease, 84% achieved complete remission after ABMT. rIL-2 was started at a median of 54 days post-transplant and consisted of a first cycle of 5 days followed by 4 cycles of 2 days every other week. Patients received a mean of 160 ×× 106IU/m2rIL-2 and hematological toxicity was moderate and transient. None of the 5 evaluable patients with measurable disease responded to rIL-2. After a 5 year median follow-up, the probability of survival and DFS is 72% (HD: 73% and NHL: 70%, p = NS) and 45% (HD: 36% and NHL: 48%, p = NS) respectively. These somewhat encouraging results warrant further evaluation of rIL-2 after ABMT in controlled studies, especially in NHL patients stratified for previous chemosensitivity.

Endothelial cells (EC) may represent a major source of cytokines in the bone marrow. In this study we have examined the production and the regulation of the production of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) by EC. Human umbilical vein endothelial cells (HUVEC) were chosen as a working model as they are a well known source of cytokines. These cells secrete LIF/HILDA (90 pg/mL/106cells/48 h) in basal conditions. This secretion is profoundly altered by interleukin-1α(IL-1α). Secretion of LIF/HILDA is increased threefold on stimulation with IL-1αat a concentration of 100 lU/mL. The secreted protein is bioactive as demonstrated by its proliferative effects on DA1a cells. Modulation of the production of LIF/HILDA by glucocorticoids (GC) was also examined. In striking contrast to what was observed for IL-1α, the synthetic GC dexamethasone (DXM) at a concentration of 10–6mol/L consistently inhibited the basal secretion of LIF/HILDA by an average of threefold and suppressed the IL-1α-induced increase of the secretion of this cytokine by HUVEC. In an effort to extend results obtained with HUVEC to the bone marrow endothelium, we have also examined the production of LIF/HILDA by human bone marrow endothelial cells (HBMEC). Our study shows that HBMEC are quantitatively a very important source of this cytokine (above 7.25 ng/mL/106cells/48 h) suggesting that they are a major source of LIF/HILDA in the bone marrow. Again, IL-1αproved to be a very potent stimulus for the secretion of LIF/HILDA and synthetic GC such as DXM when used at a concentration of 10–6mol/L inhibited by an average of threefold the basal secretion of LIF/HILDA and had a suppressive effect on the IL-1α-induced increase of this secretion. The downregulation of LIF/HILDA production in the bone marrow by GC may be important to understand the effects of GC on hematopoiesis.

Among patients included in the multicentric trial ALL87, 95 patients were randomly allocated to purged ABMT arm for post-remission therapy. Immunological phenotyping performed in patients at diagnosis allowed to assigned 51 patients (54%) to the B lineage and 34 patients (36%) to the T lineage. Only 64 patients (67%) actually received ABMT mainly because of early relapses. Fifty-two patients were depleted according to the protocol: 25 B-ALL were depleted with CD10+CD19 mAbs, 19 T-ALL with CD2+CD5+CD7 mAbs and 8 patients received an Asta-Z purged BMT. Among the 12 remaining patients, 4 received Asta-Z purged BMT and 8 an unpurged one. Using an intention to treat analysis, overall survival and event free survival were similar to results observed in chemotherapy group. This study emphasizes the importance of a precise phenotyping at ALL diagnosis which allows specific immunologic bone marrow purging for ABMT.

Recombinant human erythropoietin (rHuEPO) stimulates erythropoietic bone marrow cells and increases erythrocyte production. This prospective study was designed to evaluate the effects of rHuEPO on regeneration of erythropoiesis after allogeneic or autologous bone marrow transplantation (BMT). Seventeen centers participated in this randomized, double-blind, placebo-controlled multicenter trial. The randomization was performed centrally for each center and stratified according to allogeneic or autologous BMT and major ABO-blood group incompatibility. One hundred and six patients received rHuEPO after allogeneic BMT and 109 patients received placebo. After autologous BMT, 57 patients were treated with rHuEPO and 57 with placebo. Patients received either 150 IU/kg/day C127 mouse-cell-derived rHuEPO or placebo as continuous intravenous infusion. Therapy started after bone marrow infusion and lasted until independence from erythrocyte transfusions for 7 consecutive days with stable hemoglobin levels ≥9 g/100 mL or until day 41. After allogeneic BMT, the reticulocyte counts were significantly higher with rHuEPO from day 21 to day 42 after BMT. The median time (95% confidence intervals) to erythrocyte transfusion independence was 19 days (range, 16.3 to 21.6) with rHuEPO and 27 days (range, 22.3 to >42) with placebo (P <.003). The mean (±SD) numbers of erythrocyte transfusions until day 20 after BMT were 6.6 ± 4.8 with rHuEPO and 6.0 ± 3.8 with placebo. However, from day 21 to day 41, the rHuEPO-treated patients received 1.4 ± 2.5 (median, 0) transfusions and the control group received 2.7 ± 4.0 (median, 2) transfusions (P =.004). In the follow-up period from day 42 up to day 100, 2.4 ± 5.6 transfusions were required with rHuEPO and 4.5 ± 9.6 were required with placebo (P= .075). A multivariate analysis (ANOVA) showed that acute graft-versus-host disease (GVHD), major ABO-blood group incompatibility, age greater than 35 years, and hemorrhage significantly increased the number of transfusions. However, after day 20, rHuEPO significantly reduced the number of erythrocyte transfusions in these patient groups, as well as reducing incompatibility in the major ABO-blood group. For the whole study period, rHuEPO reduced the transfusion requirements in GVHD III and IV from 18.4 ± 8.6 to 8.5 ± 6.8 U (P= .05). After autologous BMT, there was no difference in the time to independence from erythrocyte transfusions and in the regeneration of reticulocytes. Marrow purging strongly increased the requirement for transfusions as well as the time to transfusion independence. rHuEPO had no relevant effect on regeneration of thrombopoiesis after allogeneic or autologous BMT. Overall, no major differences in side effects or complications between rHuEPO-treated and placebo-treated patients occurred. After allogeneic BMT, rHuEPO significantly accelerates the reconstitution of erythropoiesis and reduces the number of erythrocyte transfusions after day 20. The strongest effect occurs in patients with acute GVHD. After autologous BMT, rHuEPO has no clinically relevant effect on regeneration of erythropoiesis.

We report four cases of polysomy 8 (one tetrasomy and three pentasomies) observed in acute monocytic leukemia (FAB M4 and M5). Three of them showed a rearrangement of 11q23 identified by conventional cytogenetic analysis and/or chromosome painting. Our cases as well as a review of the literature, suggest that polysomy 8 is preferentially associated with monocytic differentiation (24/31). These polysomies have been observed in 21 de novo leukemias and in 10 secondary hematological disorders. A 11q23 rearrangement has been detected in 9 out of 32 patients, by conventional cytogenetic techniques in 7 and by FISH in 2. We suggest that these cases should be analysed by FISH and molecular studies in order to detect a rearrangement of MLL/11q23. Monocytic differentiation is often associated with a change of the MLL gene and the polysomy 8 might be a particular clonal evolution secondary to 11q23 abnormality.