Chemoprotection and selection by chemotherapy of multidrug resistance-associated protein-1 (MRP1) transduced cells.

Utilization of chemotherapy for treatment of tumors is mainly limited by its hematological toxicity. Because of the low level expression of drug resistance genes, transduction of hematopoietic progenitors with multidrug resistance 1 (MDR1) or multidrug resistance-associated protein 1 (MRP1) genes should provide protection from chemotherapy toxicity. Successful transfer of drug resistance genes into hematopoietic cells might allow the administration of higher doses of chemotherapy and, therefore, increase regression of chemosensitive tumors. In addition, this approach can be used to select in vivo transduced cells by their enrichment after administration of cytotoxic drugs. Our group has studied the potential value of MRP1 to protect hematopoietic cells. The interest in the use of MRP1 as an alternative to MDR1 gene transfer for bone marrow protection lies in its different modulation. Indeed, classical P-gp reversal agents, tested in clinic to decrease MDR1 tumor resistance, have little or no effect on MRP1 function. This would allow, in the same patient, the use of reversal agents to decrease P-gp tumor resistance without reversing bone marrow protection of the transduced hematopoietic cells provided by MRP1. We constructed two different MRP1-containing vectors with either the Harvey retroviral long terminal repeat (LTR) or phosphoglycerate kinase (PGK) as promoters and generated ecotropic producer cells. MRP1 transduced fibroblasts were more resistant to doxorubicin, vincristine, and etoposide and their chemoprotection was increased after selection with chemotherapeutic agents in the presence of glutathione, a co-factor for MRP1 function. Lethally irradiated mice were engrafted with bone marrow (BM) cells transduced with MRP1 vectors (PGK promoter). We demonstrated that high expression of MRP1 in murine hematopoietic cells reduces doxorubicin-induced leukopenia and mortality. In addition, in vivo selection of MRP1-transduced BM cells was achieved following doxorubicin administration and allowed a better chemoprotection after the second chemotherapy cycle.

Pitfalls in the detection of disseminated non-hematological tumor cells.

There is not yet a consensus on the reliability of the methods that should be used for the detection of rare disseminated tumor cells from non-hematological malignancies. In this review, we will discuss the advantage and drawbacks of the classical approach of immunocytochemistry and the molecular detection by reverse transcriptase polymerase chain reaction (RT-PCR). The interpretation of the biological significance of circulating tumor cells and the pitfalls of the detection techniques are the main causes of discrepancy between the conclusions of different tumor-cell detection (TCD) studies.

Meropenem versus ceftazidime as empirical monotherapy for febrile neutropenic cancer patients.

A total of 101 cancer patients with 121 febrile neutropenia episodes were randomised to receive empirical treatment with i.v. meropenem (1g/8 h) or ceftazidime (2 g/8 h). After 3 days, 89% of patients were on unmodified therapy in the meropenem group, compared with 83% in the ceftazidime group. Of the evaluable episodes (n = 106), the success rate with unmodified empirical therapy until the end of the treatment course was slightly higher with meropenem than with ceftazidime (48% vs 38%, P=0.39). Furthermore, initial success with further infections was observed in 22% of episodes treated with meropenem and in 13% of episodes treated with ceftazidime. Glycopeptides were used as first modification in 28% and 39% of meropenem and ceftazidime recipients, respectively. Both treatments were well tolerated and there were no reports of drug-related nausea/vomiting or seizures. No significant differences in response rate or in tolerability were observed when analysing only the first febrile episodes. In conclusion, meropenem seems to be as efficacious and well tolerated as ceftazidime and may be associated with a lesser requirement for the addition of glycopeptides.

Highly purified CD34+ cells isolated using magnetically activated cell selection provide rapid engraftment following high-dose chemotherapy in breast cancer patients.

The primary objective of this study was to evaluate the safety of infusion of CD34+ cells, selected using a clinical scale magnetically activated cell sorting device, assessed by time to hematological engraftment and incidence of adverse events. Secondary objectives included evaluation of device performance in terms of purity and recovery of the CD34+ cell product. Breast cancer patients suitable for transplantation received cyclophosphamide and filgrastim for mobilisation, followed by three leukaphereses. The products of the first two leukaphereses underwent CD34+ cell selection. The product of the third leukapheresis was cryopreserved unmanipulated. Following high-dose cyclophosphamide, thiotepa and carboplatin, selected CD34+ cells were infused. In 54 patients who received selected cells only, the median time to platelet recovery and neutrophil recovery was 11 days (range 5-51) and 9 days (range 5-51), respectively. There were no adverse events associated with infusion of selected cells. A total of 126 leukapheresis samples was available before and after selection for central CD34+ analysis. The median purity was 96.1% (27.4-99.4) and the median recovery was 52. 3% (15.2-146.3). These data show that cells selected using magnetically activated cell selection provide safe and rapid engraftment after high-dose therapy. Bone Marrow Transplantation (2000) 25, 243-249.

Retroviral-mediated transfer of genes encoding interleukin-2 and interleukin-12 into fibroblasts increases host antitumor responsiveness.

The transfer of genes encoding cytokines into tumor cells has emerged as a new strategy to increase in vivo host reactivity to a variety of tumors. Because gene transfer into tumor cells cannot be easily applied in the clinical setting, we have developed an experimental model of gene transfer into fibroblasts and examined the capacity of these engineered cells to elicit an antitumor immune response. Interleukin-12 (IL-12) is a heterodimeric cytokine with pleiotropic activities presenting strong antitumor and antimetastatic effects in murine models. A bicistronic retroviral vector was constructed that contained the cDNAs encoding both chains (p40 and p35) of murine IL-12 separated by an internal ribosomal entry site sequence. Syngeneic cutaneous fibroblasts obtained from newborn mice and transduced to secrete either IL-12 or IL-2 were injected subcutaneously with B16F0 or B16F1 melanoma cells. The time of tumor occurrence and overall survival of mice were significantly prolonged when B16F1 cells were coinjected with cytokine-producing fibroblasts compared with B16F1 alone or B16F1 together with unmanipulated fibroblasts. Systemic effects were seen in the mice injected with either IL-2- or IL-12-secreting fibroblasts, with the highest proliferation capability and interferon-gamma production observed in vitro from splenocytes from recipients of IL-2-secreting fibroblasts. Injection of IL-2-secreting fibroblasts or coinjection of IL-2- and IL-12-producing fibroblasts resulted in a significant increase of survival in the B16F0 model; in some cases, complete disease eradication was observed. These results suggest that cutaneous fibroblasts represent a target of choice for gene transfer and would be useful in the treatment of minimal residual disease in humans.

Unreliability of carcinoembryonic antigen (CEA) reverse transcriptase-polymerase chain reaction (RT-PCR) in detecting contaminating breast cancer cells in peripheral blood stem cells due to induction of CEA by growth factors.

RT-PCR is increasingly used for the detection of minimal residual disease in solid tumors. Carcinoembryonic antigen (CEA) RT-PCR seemed to be highly specific for detection of tumor cells when tested on PBMC. A very high frequency of RT-PCR amplification product for CEA in PBSC from breast cancer patients mobilized with G-CSF was found. However, this result contrasted with tumor cell detection by immunocytochemistry (ICC) which showed no correlation with RT-PCR results. In addition, CEA mRNA was amplified in most G-CSF-mobilized PBSC samples derived from patients with hematological malignancies and from healthy donors of allogeneic stem cells, although no circulating epithelial cells could be demonstrated by ICC. CEA RT-PCR expression was observed in PBMC from healthy individuals incubated in vitro with G-CSF. These data suggest that CEA transcription can be induced by G-CSF, resulting in a loss of specificity of CEA RT-PCR for tumor cell detection in PBMC. We conclude, CEA RT-PCR may not be recommended to detect tumor cell contamination in peripheral blood from patients treated with G-CSF. This may have implications on tumor cell detection by RT-PCR in tissues where endogenous or exogenous growth factors may induce the transcription of CEA or other genes.

Clearance kinetics of CD34+ cells from peripheral blood: an independent predictor of hematologic recovery after high-dose chemotherapy and hematopoietic stem cell transplantation.

We measured the concentration of CD34+ cells in peripheral blood (PB) (1/2) h prior to and (1/2), 1, 3, 6, and 12 h following hematopoietic stem cell (HSC) infusion in 34 breast cancer patients treated with high-dose chemotherapy (HDC). The decrease in these concentrations over time enabled us to determine the clearance kinetics of CD34+ cells from PB. The absolute number of CD34+ cells in PB generally peaked at (1/2) h after infusion, then rapidly declined from 1 to 3 h post infusion and continued to fall until 12 h post transplant, but more slowly. In univariate analysis, CD34+cells/kg infused, CFU-GM/kg infused, the CD34+ count at (1/2) h, and the 12-h clearance of CD34+ cells from PB were predictors of hematologic recovery, as were each of the two phases of clearance when the slope was divided into rapid and slow phases (from (1/2) to 3 and from 3 to 12 h post transplant, respectively). We then stratified our population by the number of CD34+ cells/kg infused. In group 1, patients received 7.5 x 10(6) CD34+ cells/kg. After adjusting for CD34+ cells injected, age, and purged or unpurged graft in multivariate analysis, the 12 h clearance remained a predictor of hematologic recovery in group 1. In addition, the second phase of clearance (from 3 to 12 h after infusion) was an even better predictor than the 12 h clearance. In group 2, however, no statistically significant correlation was observed, even with the number of HSC injected. Results suggest that rapidity of clearance of CD34+cells from PB is an independent indicator of hematologic recovery in patients receiving lower doses of CD34+ cells. When the cell dose injected is over a threshold, PB clearance correlations with hematologic recovery are masked.

The administration of 10 microg/kg granulocyte colony-stimulating factor (G-CSF) alone results in a successful peripheral blood stem cell collection when previous mobilization with chemotherapy and hematopoietic growth factor failed.

Some heavily pretreated cancer patients fail to mobilize enough peripheral blood stem cells (PBSC) after stimulation with chemotherapy and hematopoietic growth factors. For these patients the best way to obtain an adequate PBSC collection is unknown. Here we report 6 heavily pretreated cancer patients who failed to mobilize sufficient PBSC after stimulation with chemotherapy and G-CSF 5 microg/kg/day. In these cases, we used G-CSF 10 microg/kg/day alone for six days at least 3 weeks after the last chemotherapy. After three consecutive leukaphereses starting on day 5, five patients had adequate PBSC collections. With 6 days of G-CSF 10 microg/kg/day alone, 2.8 x 10(6) (+/- 1) CD34+ cells/kg were collected. This was significantly higher than the number of CD34+ cells/kg collected after chemotherapy and G-CSF 5 microg/kg 0.3 x 10(6) (+/- 0.1) [P = 0.05]. Four patients received high-dose chemotherapy with PBSC support. Hematologic recovery observed in these patients was as expected. In conclusion, G-CSF 10 microg/kg alone can mobilize progenitor cells into peripheral blood when previous mobilization with chemotherapy and G-CSF 5 microg/kg fails.

Retrovirus-mediated gene transfer of the human multidrug resistance-associated protein into hematopoietic cells protects mice from chemotherapy-induced leukopenia.

Utilization of chemotherapy for the treatment of tumors is mainly limited by its hematological toxicity. Because of the low-level expression of drug resistance genes, transduction of hematopoietic progenitors with multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes should provide protection from chemotherapeutic agent toxicity. Successful transfer of drug resistance genes into hematopoietic cells may allow the administration of higher doses of chemotherapy and, thus, increase regression of chemosensitive tumors. The interest in the use of MRP as an alternative to MDR1 for bone marrow protection lies in its different modulation. This would allow, in the same patient, the use of MDR1 reversal agents to decrease MDR1 tumor resistance without reversing bone marrow (BM) protection of the MRP-transduced hematopoietic cells, since MRP expression is not reversed by these agents. We have constructed MRP-containing retroviral vectors using the phosphoglycerate kinase promoter and generated ecotropic producer cells. Lethally irradiated mice were engrafted with BM cells transduced by coculture with MRP producer cells. Evidence of long-term (9 months) gene transfer was provided by PCR of peripheral blood from MRP-transduced mice. Southern blot analysis confirmed the integrity of the provirus in the MRP-transduced mice. Long-term MRP expression (>5 months) was detected by RT-PCR and fluorescence-activated cell sorting of blood from living mice. High-level expression of MRP in murine hematopoietic cells reduces doxorubicin-induced leukopenia and mortality. Furthermore, we show in vivo selection of MRP-transduced cells following doxorubicin administration, with better and more significant chemoprotection after the second chemotherapy cycle. These data indicate that MRP retroviral gene transfer may be useful for chemoprotection and selection.

Clinical activity and benefit of irinotecan (CPT-11) in patients with colorectal cancer truly resistant to 5-fluorouracil (5-FU).

The aim of this prospective study was to assess the efficacy, clinical benefit and safety of CPT-11 (irinotecan) in patients with stringently-defined 5-fluorouracil-resistant metastatic colorectal cancer (CRC). 107 patients with documented progression of metastatic CRC during 5-FU were treated with CPT-11 350 mg/m2 once every 3 weeks in a multicentre phase II study. Tumour response and toxicity were assessed using WHO criteria. Changes in performance status (PS), weight and pain were also measured. The WHO response rate was 13/95 (13.7%, 95% CI 7.5% to 22.3%) eligible patients with a median duration of response of 8.5 months (37 weeks, range: 18-53+). There was also a high rate of disease stabilisation (44.2%) with a median duration of 4.8 months. The probability of being free of progression at 4 months was 50%. Median survival from first administration of CPT-11 was 10.4 months or 45 weeks (range: 3-66+ weeks). There was weight stabilisation or gain in 81% (73/90) of patients, a favourable outcome in PS in 91% (82/90) (improvement of WHO PS 2 or stabilisation of PS 0-1), and pain relief in 54% (26/48). There were no toxic deaths. Neutropenia was short-lasting and non-cumulative. Diarrhoea grade > or = 3 occurred in 7% of cycles and 28/107 (26%) of patients. CPT-11 350 mg/m2 once every 3 weeks has an encouraging degree of activity in progressive metastatic CRC truly resistant to 5-FU with a relatively high rate of tumour growth control translated into clinical benefit. The toxicity profile of CPT-11 is becoming better understood and has been considerably improved.

Interaction between protein kinase C and actin in megakaryocyte polyploidization.

Megakaryocytes undergo a peculiar and irreversible program by which they become polyploid through repeated cycles of DNA synthesis without concomitant cell division. In order to study the possible concomitant role of protein kinase C and actin in megakaryocyte polyploidization, three cell lines (DAMI, HEL and K562), expressing some properties of the megakaryocytic lineage and known to differentiate into the megakaryocytic pathway in the presence of phorbol esters, were cultivated in the presence of phorbol myristate acetate alone (PMA, 5 x 10(-9) M, activator of protein kinase C, PKC) or concomitantly with cytochalasin B (2 micrograms/ml, inhibitor of actin polymerization). We have previously shown that DAMI, HEL and K562 cells in which actin polymerization was inhibited by cytochalasin B, acquired megakaryocytic properties in the way that they became polyploid, acquired a megakaryocytic phenotype and arrested proliferation (4). After four days of culture in the presence of PMA and cytochalasin B, the number of polyploid cells (estimated by flow cytometry) increased in comparison with control or PMA-treated cells. However, it was lower than in cytochalasin B-treated cells. Indeed, control cells predominantly diploid (2N) became polyploid with the appearance of 8N, 16N and 32N cells after addition of PMA, cytochalasin B or PMA + cytochalasin B. The endomitotic index (EI, as described in 5) which corresponds to the mean of (¿log2 DNA content expressed in N¿-1) was 0.5 +/- 0.1, 0.7 +/- 0.1 and 0.3 +/- 0.1 in control DAMI, HEL and K562 cells, respectively. The EI increased to 0.9 +/- 0.2; 1.0 +/- 0.2 and 0.4 + 0.1 in cells treated with PMA and to 1.6 +/- 0.3; 1.4 +/- 0, and 0.9 +/- 0.2 when PMA was added concomitantly to cytochalasin B. Total DNA estimated from the cell content and the percentage of cells present at each ploidy stage did not change in cytochalasin B-treated cells in comparison to control conditions. However, treatment of DAMI, HEL and K562 cells with PMA alone or concomitantly with cytochalasin B revealed that the total DNA content significantly decreased in these conditions. At last, treatment of the three cell lines with PMA alone or concomitantly with cytochalasin B for 4 days caused a complete inhibition of proliferation. In conclusion, the concomitant addition of PMA and cytochalasin B to the three cell lines lead to an augmentation of cell ploidy and to a cessation of proliferation. However, we did not observe any synergistic effect of the two compounds. The possible interaction between actin and protein kinase C is discussed in the paper.

Human megakaryocyte polyploidization is associated with a decrease in GPIIIA expression.

Megakaryocytes are platelet forming cells and are characterized by polyploidization, a phenomenon by which nuclear division occurs without corresponding cytoplasmic separation. Among the markers allowing to identify megakaryocytes, glycoprotein (GP) IIIa with GPIb and GPIIb are the most important. Using GPIIIa as a marker to recognize megakaryocytes in the bone marrow, we have estimated GPIIIa expression by flow cytometry in megakaryocyte populations from normal individuals and from patients with chronic myelogenous leukemia, immune thrombocytopenic purpura or polycythemia vera. We showed that the expression of GPIIIa is decreasing during megakaryocyte polyploidization in normal and pathological situations.

Autologous peripheral blood stem cell transplantation for lung cancer.

Lung carcinoma, the most frequent cause of cancer-related death in both men and women, remains a difficult therapeutic problem. Small-cell lung carcinoma, despite its high response rate to chemotherapy, is associated with a rapid recurrence and ultimately limited overall survival. In attempts to exploit tumour chemosensitivity, high-dose chemotherapy (HDC) combining several active drugs has been studied to improve outcome. In addition, haematopoietic stem cell support has been used to allow dose escalation without major myelosuppression. In contrast to small-cell carcinoma, non-small-cell carcinoma of the lung is generally not very responsive to chemotherapy, and results with dose intensity in unresectable tumour have so far been very disappointing. We review the results of HDC in terms of response and survival, and discuss potential strategies to improve the effectiveness of dose intensity.

Immunological recovery and tumour-specific immunotherapeutic approaches to post-autologous haematopoietic stem cell transplantation.

Haematological recovery following autologous peripheral blood stem cell treatment is very rapid. Immune recovery is a more prolonged process requiring a year or more for full reconstitution. Because high-dose chemotherapy followed by autologous peripheral blood stem cell treatment results in (or is presumed to result in) minimal burden of residual malignant disease it provides a potentially ideal setting for immune-mediated anti-tumour therapy. The slow immune reconstitution following transplantation may however hamper the development of effective immunotherapy. Among different approaches proposed, we focus on the potential use of tumour antigens in conjunction with cellular therapy in an attempt to eliminate residual tumour following autologous transplantation.

Protein content and number of nucleolar organizer regions are enhanced during phorbol ester-induced differentiation of cultured human megakaryocytic cells.

In order to investigate the protein synthesis in megakaryocyte polyploidization, phorbol myristate acetate (PMA, 5 x 10(-9) M), a differentiation marker known to induce megakaryocyte polyploidization, was added to human megakaryocytic cell lines (DAMI, HEL and K562) and the expression of platelet/megakaryocytic integrins, the numbers of nucleolar organizer regions (AgNORs) and the total protein content were estimated. Following exposure of PMA, the expression of the platelet membrane glycoprotein GPIIIa and thrombospondin and transferrin receptors was augmented in the three cell lines. The number of AgNORs shifted from 16.4 +/- 4.3, 24.4 +/- 2.5 and 13.6 +/- 3.1 for unstimulated cells to 20.0 +/- 5.3, 38.7 +/- 7.9 and 16.8 +/- 2.3 for PMA-treated DAMI, HEL and K562 cells, respectively. Furthermore, after treatment with PMA, the numbers of AgNORs clusters or nucleoles increased significantly to 179%, 238% and 154% of controls in DAMI, HEL and K562 cell lines, respectively. Finally, addition of PMA culture for four days, significantly increased the protein contents to 153%, 171% and 254% of controls for DAMI, HEL and K562 cell lines, respectively (p < 0.05 by t-test). In conclusion, the increase in the total protein content and in the number of AgNORs by PMA, suggests that PMA-induced-megakaryocyte polyploidization occurs by enhanced protein production.

The G and F contents in megakaryocyte cell lines after stimulation with phorbol myristate acetate.

Megakaryocyte polyploidization responds to platelet demand and results from the lack of cytoplasmic separation while the nucleus keeps dividing. In order to investigate the role of actin in the megakaryocyte polyploidization, phorbol myristate acetate (PMA, 5 x 10(-9) M), a differentiation marker known to induce megakaryocyte polyploidization, was added to human megakaryocytic cell lines (DAMI and HEL) and G, F and total actins were estimated by DNase I inhibition. After four days of culture in the presence of PMA, G actin contents in pg per 10(6) cells were 13.0 pg +/- 2.8 and 1.0 pg +/- 0.1 for unstimulated DAMI and HEL cells. F actin contents per 10(6) cells were 5.8 pg +/- 1.5 and 0.1 pg +/- 0.0 for DAMI and HEL cells. Addition of PMA for four days to culture significantly increased G actin contents (235% and 268% of controls) and F actin contents (234% and 394%), for DAMI and HEL cell lines, respectively (p < 0.05 by t-test). In contrast, G/F actin ratio was not affected (p < 0.05 by t-test) by PMA. DAMI cells from each ploidy classes were then sorted on an ELITE Coulter and assayed for actin content. While total actin, G actin and F actin per cell increased in polyploid cells cultured with PMA, there was a reduction in G, F and total actin contents per diploid equivalent when cells became polyploid. In conclusion, megakaryocyte polyploidization of these cell lines is not related to an unbalance between G and F actins but would be rather due at least partly to a defect in total actin production that could lead to a prevention of the formation of the constriction ring in telophase.

Endomitotic index of megakaryocytes measured by flow cytometry helps to diagnose hematological disorders with abnormal platelet counts.

Platelet production is a regulated phenomenon. Indeed, megakaryocyte volume is inversely correlated to the platelet count not only in normal individuals and immune thrombocytopenic purpura patients, but also, and surprisingly, in chronic myeloid leukemia patients. Patients with polycythemia vera and essential thrombocythemia are located outside this regression line. Herein, we describe morphometrical data confirmed by the flow cytometric measurement of the megakaryocyte endomitotic index (EI). The EI is a value which reflects the mean ploidy of megakaryocytes and corresponds to the mean of (¿log2 DNA content expressed in N¿-1). In this study, the megakaryocyte endomitotic index of 14 normal individuals was compared to those of chronic myeloid leukemia (CML) patients (n = 16), immune thrombocytopenic purpura (ITP) patients (n = 11), essential thrombocythemia (ET) patients (n = 10) and polycythemia vera (PV) patients (n = 12). The megakaryocyte EI was significantly lower in CML patients than in normal individuals. In contrast, in ET, PV and ITP patients, megakaryocyte EI was higher than in normal individuals. An inverse relationship between the endomitotic index estimated by flow cytometry and the mean megakaryocyte volume performed by morphometry was observed in normal individuals, CML and ITP patients. In conclusion, the endomitotic index is higher in ITP, ET and PV patients and lower in CML patients when compared to normal individuals and is an interesting tool which can help to diagnose rapidly hematological disorders with abnormal platelet counts.