Autologous peripheral-blood progenitor-cell support following high-dosechemotherapy or chemoradiotherapy in patients with high-risk multiple myeloma.

PURPOSE: The aims of the current study were to evaluate in patients with high-risk multiple myeloma (MM) the feasibility and usefulness of high-dose chemotherapy or chemoradiotherapy followed by hematopoietic stem-cell support with autologous peripheral-blood progenitor cells (PBPC) harvested after high-dose cyclophosphamide (HDCYC). PATIENTS AND METHODS: Seventy-three patients with high-risk MM were entered onto the study. Before the procedure, all patients had received HDCYC to collect PBPC by leukapheresis. One patient died of infection after HDCYC. All other patients subsequently received high-dose melphalan (HDM) (140 mg/m2) either alone (n = 1) or associated with either busulfan (16 mg/kg; n = 4) or total-body irradiation (TBI) (8 to 15 Gy; n= 67). In addition, three of the latter patients received cyclophosphamide (120 mg/kg). Thereafter, PBPC were reinfused either alone in 61 patients or together with back-up bone marrow cells in 11 patients in whom the granulocyte-macrophage colony-forming unit (CFU-GM) cell content of the leukapheresis was low. RESULTS: One patient died of acute cardiac failure after reinfusion of PBPC; three patients did not respond after autologous blood progenitor cell transplantation (ABPCT), while the other 68 patients achieved either a complete response (CR; n = 32) or partial response (PR; n = 36). Thirty-six patients relapsed or progressed after a median response duration of 14.5 months (range, 3 to 43) and 19 of these subsequently died. Four other patients died while still responsive of lung cancer (n = 1) or infection (n = 3). The remaining 28 patients are currently alive and still responding with a median follow-up duration of 27 months (range, 6 to 66). The 3-year probability of survival was 66% +/- 12% (95% confidence interval [CI] after ABPCT and 77% +/- 51% (95% CI) from diagnosis. CONCLUSION: High-dose chemotherapy or chemoradiotherapy followed by autologous PBPC support in MM is feasible and efficient. Further studies are needed to confirm these encouraging, although preliminary, results and to compare this technique with other therapeutic strategies.

Long-term culture of peripheral blood stem cells: the effect of the addition of an irradiated stromal layer.

We have studied peripheral blood stem cells (PBSC) collected by cytapheresis following intensive chemotherapy, from 13 patients with acute leukemia, in long term culture (LTC). Peripheral blood was cultured with (n = 10) and without (n = 21) the addition of a preformed, irradiated stromal layer. In this latter LTC our results confirm that peripheral blood is capable of producing CFU-GM and nucleated cells in the absence of the formation of an adherent stromal layer. However, peripheral blood cultured in the presence of an irradiated stromal layer is capable of a significantly higher proliferative response (total production of CFU GM per flask - mean = 57529) than in the absence of an irradiated stromal layer (total production of CFU GM per flask - mean = 26739, p less than 0.03). Our results suggest that PBSC contain a primitive nonplastic adherent cell that requires the presence of a stromal layer for its expression. These findings provide further support for the use of peripheral blood stem cells for autologous transplantation.

Metabolic hydroxylation of the thiophene ring: isolation of 5-hydroxy-tienilic acid as the major urinary metabolite of tienilic acid in man and rat.

The metabolism of tienilic acid, a drug containing a thiophene ring, was reinvestigated in man, rat and dog. The major urinary metabolite in man and rat was isolated and completely characterized by comparison with a synthetic compound. This metabolite derives from the hydroxylation of the thiophene ring of tienilic acid in position 5. Its isomers, 3- and 4-hydroxy-tienilic acids, were synthetized but could be detected neither in man nor in rat urine. Because of its particular behaviour toward electrophiles, 5-hydroxy-tienilic acid was found to react with diazomethane with the formation of a complex mixture of methylated products. This made difficult its measurement by a previously described GLC technique, after acidic extraction and methylation by diazomethane. A new very simple assay using HPLC and direct injection of urine is described in this paper. This assay led to a very precise and reproductible determination of tienilic acid and its hydroxylated metabolite in urine. Up to 50% of tienilic acid is excreted in man or rat urine as 5-hydroxy-tienilic acid whereas this metabolite does not appear in dog urine. These data describe the first example of metabolic hydroxylation of the thiophene ring.

Collection of peripheral blood stem cells in multiple myeloma following single high-dose cyclophosphamide with and without recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF).

High-dose cyclophosphamide (HD-CY; 7 g/m2) was administered to patients suffering from high risk multiple myeloma (MM). The safety of this procedure, the recirculation and collection of peripheral blood stem cells (PBSC) and the effect of rhGM-CSF and HD-CY were studied. Group I patients (n = 21) were treated with HD-CY alone. Group II patients (n = 10) received 5 micrograms/kg/day rhGM-CSF iv after HD-CY. Neutropenia was shorter in group II (p = 0.01). In group II, the number of circulating colony forming units (CFU-GM) after 14 days was correlated with the number of circulating CFU-GM after 7 days (r = 0.85, p < 0.0001) and with the number of CD34+ cells (r = 0.839, p = 0.01). The total number of mononuclear cells (MNC) and CFU-GM collected per patient was two and seven-fold higher, respectively, in group II (p = 0.01 and p = 0.03). Recovered MNC and CFU-GM were 1.7 and 7-fold higher, respectively, in group II (p = 0.01 and p = 0.004). Our data show that HD-CY is an efficient means of collecting functional PBSC in MM. We suggest that rhGM-CSF is able to further enhance this yield in MM.

Indicators of haematopoietic recovery after bone marrow transplantation: the role of reticulocyte measurements.

The aim of this project was to study haematological recovery in patients following different types of bone marrow transplantation (BMT). Forty-three patients were analysed. The duration of haematopoietic suppression following conditioning regimens was much shorter in 12 peripheral blood stem cell transplantation patients than in 19 allogeneic BMT patients and in 12 autologous BMT patients. Among 12 different variables, the parameters with the highest specificity or predictive value for monitoring recovery in these patients were the absolute neutrophil count (ANC) at the classical cut-off point of 0.5 x 10(9)/l, an absolute reticulocyte count (RET) above 20 x 10(9)/l and a high fluorescent reticulocyte fraction (HFR) above 5%,. It is suggested that these parameters be used as the reference measurements for monitoring haematological recovery in similar studies. Among these variables, the HFR fraction was the earliest and most sensitive index of engraftment in 79.1% of patients, HFR recovery requiring a median time of 13 days after infusion, in comparison with a median period of 19 and 18 days, respectively, for RET and ANC (P<0.0001).

Reticulocytes in haematological disorders.

The increased precision of flow cytometric techniques permits the recognition of small differences even in the low or normal range of the reticulocyte count. Moreover, measurement of the RNA content of reticulocytes makes possible the identification of the youngest highly fluorescent macroreticulocytes (HFR) prematurely delivered from bone marrow in conditions of increased erythropoietic stimulation. The aim of this study was the definition, using the dedicated flow cytometers Sysmex R-1000 or R-3000 (Toa Medical Electronics Ltd, Kobe, Japan), of reticulocyte absolute number and HFR percentage in patients with haematological disorders prior to any treatment. Analysis of 54 healthy subjects and 100 untreated patients with five types of haematological disease is presented. In haemolytic anaemias (15 cases) both the reticulocyte count and HFR were greatly increased and the reticulocyte count was inversely correlated with Hb level, as in the reference population. In polycythaemia vera (20 cases) reticulocytes were moderately increased and directly correlated with Hb. In dyserythropoietic syndromes (20 cases) reticulocytes were low and HFR moderately increased; HFR showed an inverse correlation with Hb. In acute myeloid leukaemia (30 cases) reticulocytes were low and HFR increased; reticulocytes correlated with both HFR and Hb. In acute lymphoid leukaemia (15 cases), while the reticulocyte count did not differ from the reference group, the HFR was increased. These results provide reference values for the evaluation of reticulocyte counts and HFR in haematological diseases. From a physiopathological standpoint, they suggest that in anaemic patients the reticulocyte count directly reflects effective bone marrow erythrocyte production, while the proportion of circulating HFR more closely reflects the intensity of erythropoietic stimulation.

Automated reticulocyte counting for monitoring patients on chemotherapy for acute leukaemias and malignant lymphomas.

Flow cytometric reticulocyte counting including their maturation fractions was performed with a Sysmex R-3000 automated analyser during follow-up after induction and/or consolidation with combination chemotherapy in patients with acute leukaemias (AL, n = 39; 58 courses) and malignant lymphomas (ML, n = 21; 29 courses). The ML patients received granulocyte colony stimulating factor (G-CSF) in addition after chemotherapy. During the leucopenic phase only reticulocytes of low fluorescence ratio (LFR) at extremely low concentration (< 10 x 10(9)/l) were found. After a median interval of 17 days (range 8-43), the middle fluorescence fraction (MFR) began to rise, preceding high fluorescence ratio (HFR) reticulocytes by a median of 1 day in AL patients with complete or partial remission. In ML patients, MFR and HFR reticulocytes appeared more often simultaneously after a median interval of only 11 days (range 8-15) and increased faster during the first week of marrow recovery showing a pattern different from AL. Granulocytes passed the critical limit of 0.5 x 10(9)/l at a median of 5 days after appearance of MFR reticulocytes in AL but in ML on the same day as MFR and HFR (day 0). The absolute reticulocyte concentration reached the lower limit of the reference range after about 10 days in AL. Thus, finding MFR and, to a lesser extent, HFR at very low cell concentrations, may serve as sensitive early indicators of marrow recovery after chemotherapy and are much more sensitive parameters than the absolute reticulocyte concentration. The higher median values for reticulocytes (total, HFR and MFR) after G-CSF therapy suggests that G-CSF is not lineage specific and may also stimulate erythroid precursor cells.

Incomplete stroma formation after allogeneic marrow or autologous blood stem cell transplantation.

Long term and semi-solid culture techniques were used to evaluate the quality of stroma produced by bone marrow from 33 normal subjects and 57 patients (46 allogeneic bone marrow and 11 autologous blood stem cell transplant recipients). Bone marrow from transplant recipients was capable of sustained CFU-GM and nucleated cell production in long term culture. However, only 13% of the marrow investigated developed a complete, confluent stromal layer. These stromal abnormalities were observed in spite of complete hematopoietic reconstitution after transplantation and rarely improved with time. Our results suggest that the hematopoietic microenvironment is very fragile and susceptible to long term damage as a result of chemotherapy and the conditioning regimes used prior to transplantation.

Vitamin K dependent carboxylation: determination of the stereochemical course using 4-fluoroglutamyl-containing substrate.

The stereochemical course of the vitamin K dependent carboxylation has been elucidated using a (4S)-4-fluoroglutamyl-containing pentapeptide as a substrate. The absolute configuration of the [13C]-4-carboxy-4-fluoroglutamate obtained when the carboxylation was carried out with 13C-labeled sodium bicarbonate, was determined after reduction of the [13C]-4-carboxy-4-fluoroglutamyl residue into 4-fluoro-5,5'-dihydroxyleucine, hydrolysis, lactonization, and peracetylation. The absolute configuration at C-4 was determined to be S by locating the 13C label in the lactone ring of the trans isomeric lactone and in the hydroxymethyl group of the cis isomer following HPLC separation of both isomers and analysis by GC/MS/MS techniques. It follows that the vitamin K dependent carboxylation occurs with inversion of configuration.

Autologous blood progenitor cell transplantation in high-risk multiple myeloma.

The aim of the study was to evaluate the feasibility and the efficacy of high-dose chemoradiotherapy followed by autologous hematopoietic stem cell support with peripheral blood progenitor cells (PBPC) harvested after high-dose cyclophosphamide (HDCYC) treatment in patients with high-risk multiple myeloma (MM). Inclusion criteria were: age less than 65 years and high-risk MM defined as stage II MM, stage III MM, refractory or relapsed MM. The design of the study was: 1) HDCYC +/- hematopoietic growth factors followed by PBPC collection, and 2) high-dose melphalan combined with total body irradiation (or busulfan for previously irradiated patients) followed by PBPC reinfusion (ABPCT). All 60 patients completed the procedure except two who died from infection after HDCYC and another of acute cardiac failure after reinfusion of PBPC. Out of the 60 evaluable patients, three failed to respond while the other 57 achieved either a partial (n = 33) or complete (n = 24) response. Thirty-one patients progressed or relapsed after a median duration of response of 15 months (range: 3-43). The median follow-up for the other 26 responder patients was 24 months (range: 2-66). Twenty-one patients died, 18 of MM (2 after failure, 16 after relapse) and three responders of lung cancer (n = 1) and infection (n = 2). In conclusion, this study shows that this therapeutic approach is feasible and efficient.

Peripheral blood progenitor cell transplantation in 118 patients with hematological malignancies: analysis of factors affecting the rate of engraftment.

We retrospectively studied the factors affecting the rate of hematopoietic reconstitution (HR) in 118 patients with hematological malignancies who underwent peripheral blood progenitor cell (PBPC) transplantation at a single institution. The patients received a median number of 6.6 x 10(8) nucleated cells/kg corresponding to 9.5 x 10(4) (0.5-578) CFU-GM/kg and 6.8 x 10(6) (0.2-161) CD34-positive cells/kg. The median number of days to reach 500 polymorphonuclear cells/mm3 and 50,000 platelets/mm3 was 12.5 (6-93) and 14.5 (6-440) days, respectively. No patient died from infection during the aplastic phase. By multivariate analysis, we found that the dose of CFU-GM infused was the only factor that significantly affects the HR rate (p < 0.0001). Moreover, patients with acute myelogenous leukemia or those transplanted after busulfan or total-body irradiation conditioning regimens had a slower engraftment (p < 0.08). These results could lead to identifying patients who need growth factors posttransplantation and/or the reinfusion of "back-up" marrow together with PBPC.

[Collection of blood stem cells and therapeutic applications]. / Collection de cellules souches sanguines et applications thérapeutiques.

Peripheral blood stem cells were collected from 50 patients with different hematological malignancies. Leukaphereses (n = 263) were performed during the marrow regeneration phase following different types of chemotherapy. The mean number of CFU-GM collected per leukapheresis was 486 x 10(4), highest levels of CFU-GM being obtained in patients who received the most intensive chemotherapy and who had not been treated previously. Twenty-five patients underwent autologous blood stem cell transplantation. Hemopoietic recovery was complete and prompt in 24 patients. Only one patient had no megakaryocytic engraftment. These findings indicate that peripheral blood stem cells may be used for autologous transplantation in leukemic patients.