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Keywords:

  • CML;
  • Ph-negative cell collection;
  • lenograstim;
  • autografting;
  • leukapheresis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

This study aimed to demonstrate that sufficient Ph-negative blood progenitors could be collected following administration of glycosylated rhG-CSF (lenograstim) to patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) who responded to recombinant alpha-interferon (α-IFN) (Ph-positive marrow metaphases < 35%). 23 patients received lenograstim (150 μg/m2) once daily for a median of 9 d. Peak circulating numbers of white blood cells (36.4 × 109/l), CD34+ cells (24/μl) and colony-forming unit-granulocyte-macrophage (CFU-GM; 1346.5/ml) occurred at a median of day 8, day 8 and day 7, respectively. Two to six (median three) leukaphereses (LK) were performed from days 5 to 12. The median number of mononuclear cells (MNC), CD34+ cells and CFU-GM collected per patient was 7.35 × 108/kg, 2.72 × 106/kg and 10.23 × 104/kg, respectively. 22/23 patients had LK which contained either 104 CFU-GM/kg and/or 106 CD34+ cells/kg; 47 LK (from 20/22 patients) were evaluable for cytogenetics. The median percentage of Ph-positive cells was 0, and 43/47 LK (91%) contained < 35% Ph-positive cells; 25 (53%) were entirely negative. Sixteen of 20 evaluable patients had one or more LK with < 35% Ph-positive cells, and 12 had at least one 100% Ph-negative LK. Mobilization and collection of Ph-negative cells were not influenced by the dose or duration of α-IFN administration before (or during) lenograstim administration or by the quality of cytogenetic response (complete v major) during lenograstim administration. No significant side-effects were observed. Thus, lenograstim administration can result in satisfactory Ph-negative blood progenitor cell collection. Autologous transplantation of such cells may be used when indicated.

Chronic myeloid leukaemia (CML) is an acquired clonal haemopoietic stem cell disorder characterized by the presence of a chromosomal marker, the Philadelphia (Ph) chromosome, which results from a reciprocal translocation t(9,22) (q34;q11) and is present in all cells of the clone ( De Klein et al, 1982 ). This translocation transposes the c-abl proto-oncogene from its normal position on chromosome 9 to a new position on chromosome 22, in proximity to the BCR gene ( Groffen et al, 1984 ). The BCR-ABL hybrid gene or the generated fusion proteins can be detected and analysed using different molecular techniques.

Allogeneic bone marrow transplantation (alloBMT), which is the only curative treatment for CML, is restricted to a minority of patients who have both a human leucocyte antigen identical donor and are < 50 years of age ( Thomas et al, 1986 ). We, and others, have reported that recombinant alpha-interferon (α-IFN) can induce complete (cytogenetic) responses in about 30% of patients who, if not cured, may still have a significantly prolonged survival ( Montastruc et al, 1995 ; Kantarjian et al, 1995 ; The Italian Cooperative Study Group on Chronic Myeloid Leukemia, 1994). Thus, for the majority of patients who either cannot benefit from alloBMT or do not respond to IFN, alternative treatments such as autologous stem cell transplantation (ASCT) must be considered ( Reiffers et al, 1996 ; Goldman, 1990). Previous studies have indicated that ASCT must be performed during the chronic phase as the results observed in patients transplanted during transformation are very poor ( McGlave et al, 1994 ). The different sources of stem cells used to date do not seem to significantly influence the outcome following transplantation; there are no studies showing a significant difference between bone marrow and peripheral blood progenitor cell (PBPC) transplantation, or between purged or unpurged stem cell transplantation, on patient survival ( Reiffers et al, 1994 , 1996; McGlave et al, 1994 ). However, as for patients undergoing ASCT for acute leukaemia, it has been recently reported that, using gene marking techniques, leukaemic progenitor cells (i.e. Ph-positive or BCR-ABL-positive cells) could contribute to, or perhaps cause, relapse following transplantation ( Deisseroth et al, 1994 ). This observation has led investigators to experiment with different techniques of stem cell purging.

Existing ex-vivo purging techniques treat haemopoietic stem cells either with chemotherapeutic agents, e.g. cyclophosphamide derivatives or tyrosine kinase inhibitors ( Carlo-Stella et al, 1991 ), interferons ( McGlave et al, 1990 ), antisense oligonucleotides targeted to different genes (e.g. myc, myb or the BCR-ABL gene) ( De Fabritiis et al, 1995 ; Ratajczak et al, 1992 ), or expand the normal progenitor cell population using long-term cultures ( Barnett et al, 1994 ). Although these techniques have permitted efficient in vitro suppression of leukaemic cell growth, when applied clinically they do not appear to induce a longer duration of Ph-negativity than that observed without purging ( Carlo-Stella et al, 1991 ; McGlave et al, 1990 ; De Fabritiis et al, 1995 ; Ratajczak et al, 1992 ; Barnett et al, 1994 ). In vivo techniques, pioneered as early as 1978 ( Körbling et al, 1981 ), have developed significantly since growth factors (mainly granulocyte-colony stimulating factor, G-CSF) have become available. The Genova group has shown that PBPC mobilized with both intensive chemotherapy (similar to induction chemotherapy used for acute myeloid leukaemia) and G-CSF contain a high proportion of Ph-negative progenitor cells ( Carella et al, 1993 ); this proportion seems to be particularly high (reaching 100%) when mobilization is performed in patients during the early chronic phase.

As recombinant α-IFN can induce complete responses in patients with CML, it could therefore be used for in vivo purging. In a recent study ( Italian Cooperative Study Group, 1993), patients in whom α-IFN induced a cytogenetic response were proposed for bone marrow harvesting; in most cases a high proportion of Ph-negative cells was collected, but the number of progenitor cells was very low (because of the toxicity of α-IFN), indicating that this technique would be difficult to apply to autologous transplantation. Based upon these results, and the capacity of G-CSF to mobilize PBPC in healthy individuals and in patients with solid tumours or haematological malignancies, we conducted a phase II study to examine whether rhG-CSF (lenograstim 150 μg/m2/d for 6–12 d) could mobilize a sufficient amount of normal PBPC in patients with CML who respond following treatment with α-IFN (defined as < 35% Ph-positive metaphases in the marrow).

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. References

Patients

23 patients (median age 42 years, range 22–62) with Ph-positive CML entered the study ( Table I), of whom nine had achieved a major cytogenetic response (MCR, defined as marrow Ph-positive metaphases < 35%) and 14 had achieved a complete cytogenetic response (CCR, defined as no detectable marrow Ph-positive metaphases) following treatment with α-IFN according to criteria defined by Kantarjian et al (1995 ). The median duration of α-IFN administration prior to study entry was 17.2 months (1–73) and the median daily dose of α-IFN at inclusion was 4 million units/m2 (0.6–10). One patient had achieved a cytogenetic response before commencing treatment with α-IFN, and was still at that stage when entered into the study 1 month later; for the remaining 22 patients the duration of α-IFN treatment was > 6 months. No patient had received prior haemopoietic growth factors.

Table 1. Table I. Baseline patient characteristics at study entry.Thumbnail image of
  • a

    Abbreviations: WBC, white blood cell; MCR, major cytogenetic response; CCR, complete cytogenetic response. Group A: α-IFN discontinued the day before lenograstim administration; group B: α-IFN maintained during lenograstim administration.

  • Treatment

    All patients received lenograstim (glycosylated recombinant human G-CSF, supplied by Chugaï-Rhône Poulenc, France) in four haematology departments in France and Australia. Vials containing 263 μg of lyophilized lenograstim were reconstituted with 1 ml of sterile water for injection, and 150 μg/m2 was administered daily by subcutaneous injection. Lenograstim treatment was planned to be given for 12 consecutive days (days 1–12) but was terminated in the event of hyperleucocytosis (≥70 × 109/l) or when leukaphereses (LK) were completed.

    For the first 11 patients (group A), α-IFN was discontinued the day before lenograstim administration (day 0); the remaining 12 patients (group B) continued to receive α-IFN during treatment with lenograstim.

    This study was approved by the Ethical committees of Hôpital Saint-Louis (Paris, France) and Mater Misericordiae Hospital (Brisbane, Australia).

    Collection

    It was initially planned to perform LK following 8, 10 or 12 d of treatment with lenograstim. However, as progenitor cell mobilization was achieved in most patients after only 5 d of treatment with lenograstim, it was subsequently decided to perform LK between days 5 and 12 according to the use in each participating centre. The decision to commence LK did not depend on CD34 cell evaluation. LK were performed according to the established local protocols, and could be stopped when ≥106/kg CD34+ cells had been collected.

    Evaluation

    Prestudy screen included a complete medical history and physical examination. Complete blood counts (CBC), with differential and marrow cytogenetic evaluation, were performed before commencing lenograstim treatment (day 0) and daily CBCs, with differentials, were subsequently performed on days 1–15. Blood colony-forming unit-granulocyte macrophage (CFU-GM) assays and CD34+ cell evaluations were performed before lenograstim treatment (day 0) and on days 5, 7, 11 and 13. Prior to freezing each LK product, the numbers of MNC, CD34+ cells and CFU-GM were evaluated. Karyotype analysis was performed on each LK and considered evaluable when 10 or more metaphases were available.

    The procedure was considered successful when ≥104/kg CFU-GM and/or 106/kg CD34+ cells had been collected. Complete success was defined as a successful collection with no Ph-positive cell contamination in at least one LK, whereas partial success was defined as successful collection with Ph-positive metaphases of 1–35%.

    Data were processed and analysed using the statistical analysis system (SAS) version 6.08. Percentages and median values (for quantitative variables) were compared using Fisher's exact test and the Student's test, respectively. Correlations were analysed using correlation and regression SAS procedures.

    Safety

    Patients were evaluated at each visit for adverse events using clinical and laboratory parameters. To detect eventual disease progression following lenograstim administration, patients were followed up every 3 months for 1 year and marrow karyotypes were performed at the end of the follow-up period.

    RESULTS

    1. Top of page
    2. Abstract
    3. MATERIALS AND METHODS
    4. RESULTS
    5. DISCUSSION
    6. Acknowledgements
    7. References

    Mobilization

    For the entire study population, the peak WBC (36.4 × 109/l; range 16.4–59.4) occurred on median day 8 (5–13); the peak was higher in group A (39 × 109/l; 26.1–59.4) than in group B (26.9 × 109/l; 16.4–50) but the difference was not statistically significant ( Table II). The peak circulating number of CD34+ cells (median 24/μl; 2–52) also occurred on median day 8 (5–12) and there was no statistically significant difference between groups A and B. The peak circulating number of CFU-GM cells was 1346.5/ml, which also occurred on median day 8 (5–10), with no significant difference between groups A and B.

    Table 2. Table II. Peak values of white blood cells (WBC), CD34+ cells and colony-forming unit-granulocyte macrophage (CFU-GM) following lenograstim administration.Thumbnail image of
  • a

    Values represent median (range). Group A: α-IFN discontinued the day before lenograstim administration; group B: α-IFN maintained during lenograstim administration.

  • Collection

    Leukaphereses were performed on days 5–12 and the median number of collections was three (two to six). The median number of days of G-CSF administration before the first LK was 6 (4–7) and did not differ significantly between centres. 22/23 patients had a successful collection, in whom the target number of 104 CFU-GM/kg collected was achieved with one (n = 20) or two LK (n = 2); one (n = 12), two (n = 6) or more than two (n = 4) LK were needed to collect ≥106 CD34+ cells/kg. Only one patient failed to achieve a successful collection; the lowest peak values of CD34+ cells (2/μl) and CFU-GM (42.4/ml) had been observed in this patient.

    Table 3. Table III. Median number of mononuclear cells (MNC), CD34+ cells and colony-forming unit-granulocyte macrophage (CFU-GM) collected in each patient (all leukaphereses): comparison between group A versus group B, and major cytogenetic responders (MCR) versus complete cytogenetic responders (CCR).Thumbnail image of
  • a

    Group A: α-IFN discontinued the day before lenograstim administration; group B: α-IFN maintained during lenograstim administration. Abbreviation: BW = body weight.

  • Cytogenetic analysis

    Of the 67 LK performed, cytogenetic analysis was possible on 47 (containing 10 or more evaluable metaphases) that were obtained from 20 patients; three patients were not evaluable for cytogenetic analysis. A total of 1107 metaphases (25.5 per LK) were analysed. The median number of Ph-positive metaphases per LK was 0 (0–66.6), with no significant differences between group A (median 3; 0–52) and group B (median 0; 0–66.6) or between those patients in CCR (median 0; 0–66.6) or MCR (median 3.5; 0–52) at study entry. No Ph-positive metaphases (CCR) were detected in 25 (53%) LK; in 18 LK the percentage of Ph-positive cells was < 35% (MCR) and only four LK had > 35% Ph-positive metaphases (36%, 50%, 52% and 67%, respectively). The percentage of CCR LK was higher in group B patients (20/33; 61%) than in group A patients (5/14; 36%), but this difference was not statistically significant ( 4 Table IV). Interestingly, 15/25 LK performed in CCR patients were Ph-negative, but 10 LK contained a substantial proportion of Ph-positive metaphases. In contrast, 10/22 LK performed in MCR patients were entirely Ph-negative (no significant difference between CCR and MCR patients).

    Table 4. Table IV. Karyotypic evaluation of 47 evaluable leukaphereses (LK): comparison between group A versus group B, and major cytogenetic reesponders (MCR) versus complete cytogenetic responders (CCR). Values represent absolute numbers (and percentages) of leukaphereses with 0, 1–35 and > 35% Ph-positive cells. Thumbnail image of
  • a

    Group A: α-IFN discontinued the day before lenograstim administration; group B: α-IFN maintained during lenograstim administration.

  • Residual leukaemic cells were analysed in some centres using a nested polymerase chain reaction (PCR) technique (with a sensitivity of 1:106 cells) in 22 Ph-negative LK (corresponding to 11 patients) ( Bilhou-Nabera et al, 1992 ). BCR-ABL rearrangements were detected in 11 LK, with no residual disease found in the 11 other LK. Two of these BCR-ABL-negative LK occurred in a patient who was not in CCR at study entry. No PCR analysis was performed on LK with Ph-positive metaphases.

    Final evaluation

    Of the 23 patients enrolled, one failed to achieve a successful collection (see above) and was considered a failure as defined by the study protocol (despite only five Ph-positive metaphases having been found among 92 metaphases originating from three LK). The remaining 22 patients were considered successful in terms of dose of cells collected; 12 of these patients were classed as ‘complete success’ (as previously defined) and four were classed as ‘partial success’, using cytogenetics. Three patients were not evaluable as they had no LK with > 10 available metaphases. Two patients had no leukaphereses showing < 35% Ph-positive metaphases; one of these had 14 Ph-positive cells among 30 evaluable metaphases (from two LK) and for the other only 10 metaphases were obtained, from one LK, of which five were Ph-positive. The final patient, who had a successful collection (1.2 × 106 CD34+ cells and 1.6 × 104 CFU GM/kg) after three LK had >10 metaphases analysed in two of the three LK; no Ph-positive cells were found among seven, 34 and 27 metaphases analysed. As these two evaluable LK had < 106 CD34+ cells and 104 CFU-GM/kg, this patient was classified as a failure according to the evaluation criteria defined in the study protocol (although this classification could be discussed). Thus, a total of four patients were considered to be failures ( 5 Table V). There was no statistically significant difference in the number of patients with complete success between either group A and group B or those patients who were either CCR and MCR at baseline ( 5 Table V). The baseline characteristics of the 12 patients who achieved complete success did not significantly differ from those of the other 11 patients ( 6 Table VI).

    Table 5. Table V. Summary of results for all 23 patients: group A versus group B, and complete cytogenetic responders (CCR) versus major cytogenetic responders (MCR).Thumbnail image of
  • a

    * Failure = either unsuccessful collection (n = 1) or > 35% Ph-positive cells in leukaphereses (n = 2). In addition, one patient had unsuccessful collection on LK evaluable for cytogenetics.† Fewer than 10 metaphases analysed.

  • Table 6. Table VI. Baseline characteristics of all patients according to response to lenograstim.Thumbnail image of
  • a

    Abbreviations: WBC, white blood cell; MCR, major cytogenetic response; CCR, complete cytogenetic response. Others = patients with partial response, failure and those not evaluable.* No statistically significant intergroup differences.

  • Safety

    Thirteen patients experienced adverse events including bone pain (n = 7) and headache (n = 6). 10 of these events were considered possibly or probably related to rhG-CSF administration and were easily reversible following discontinuation of rhG-CSF. Lenograstim was well tolerated at the site of injection (i.e. there was no injection site pain). Two to four months following lenograstim administration, no patient had developed resistance to α-IFN or transformation.

    DISCUSSION

    1. Top of page
    2. Abstract
    3. MATERIALS AND METHODS
    4. RESULTS
    5. DISCUSSION
    6. Acknowledgements
    7. References

    This study demonstrated that low doses of lenograstim (150 μg/m2/d, equivalent to 5 μg/kg/d), can mobilize haemopoietic progenitor cells into the blood of CML patients who have responded to IFN. Previous studies have indicated that G-CSF (lenograstim or filgrastim) can mobilize stem cells either in healthy volunteer donors ( Korbling et al, 1995 ) or in patients with haematological malignancies or solid tumours who are candidates for subsequent autologous transplantation ( Sheridan et al, 1992 ). In healthy donors the usual dose of G-CSF administered has ranged from 3 to 10 μg/kg/d for 4–8 d ( Korbling et al, 1995 ; Grigg et al, 1995 ). Peak CD34+ cells or CFU-GM usually occurs following 4–5 d administration of G-CSF, and in most cases, only one LK is needed to collect the target number of 3 × 106 CD34+ cells/kg or 5 × 104 CFU-GM/kg ( Korbling et al, 1995 ; Grigg et al, 1995 ). There is a trend for better collections with higher doses of G-CSF (≥10 μg/kg) ( Grigg et al, 1995 ). Not surprisingly, the results of blood cell mobilization and collection in healthy donors are more favourable than those observed in our study performed in α-IFN-treated patients with CML.

    In patients with solid tumours or haematological malignancies who receive G-CSF alone as a mobilizing treatment before autologous transplantation, CD34+ cell and CFU-GM mobilization usually occurs following approximately 5 d treatment with G-CSF (usual dose 5 μg/kg/d), but seems to be less pronounced than in healthy individuals ( Sheridan et al, 1992 ; Bensinger et al, 1993 ). In some such patients, particularly those who have previously received intensive or prolonged chemotherapy, the standard growth factor dose of 3–5 μg/kg is insufficient to mobilize blood stem cells ( Tricot et al, 1995 ). For these patients it may be useful to increase the dose of growth factor ( Bensinger et al, 1993 ) or to combine growth factor with other cytokines.

    Recombinant α-IFN has been shown have a mild inhibitory effect on haemopoiesis both in in vitro studies ( Neuman & Fauser, 1982) and in patients treated with α-IFN. The Italian Cooperative Study Group (1993) reported that, in patients with CML who had previously received α-IFN for several months, marrow stem cell collection was very difficult, even when α-IFN was discontinued several weeks before collection. Thus, when the present study commenced, it was not known whether G-CSF could mobilize progenitor cells into the blood of α-IFN-pretreated patients. Consequently, in order to facilitate blood stem cell mobilization, it was decided to discontinue α-IFN before administering lenograstim and to commence LK following 8–12 d of treatment with lenograstim. After the first patients had been treated, it was observed that WBC, CFU-GM and CD34+ cell mobilization was adequate to collect a sufficient number of progenitor cells. This favourable effect of lenograstim was subsequently confirmed in those patients who continued α-IFN treatment during lenograstim administration; in total, 22/23 patients enrolled in the study achieved successful mobilization. These results demonstrate that rhG-CSF is capable of reversing, at least partially, the haematological toxicity of long-term treatment with α-IFN. Whether this effect is dose-dependent remains unknown and could be addressed in a subsequent study, specifically in patients with CML who are at higher risk of poor mobilization, e.g. those patients who receive the highest cumulative doses of α-IFN.

    The second objective of this study was to assess the capacity of rhG-CSF to preferentially mobilize Ph-negative blood cells in CML patients with a good response to α-IFN. Although some cell lines transfected with the BCR-ABL gene have become growth-factor independent, few studies have shown a differential effect of growth factors on normal versus leukaemic (CML) progenitors. Such an effect has been suggested with interleukin-1, interleukin-4 or some other cytokines, e.g. TNF-α, leukaemia-inhibiting-factor or macrophage-inflammatory-protein-1-α, but, to date, this has not led to significant clinical applications ( Estrov et al, 1991 , 1993; Piacibello et al, 1990 ; Chasty et al, 1995 ). We have also reported that, using a liquid culture system and a combination of three cytokines (interleukin-1, interleukin-3 plus stem cell factor), Ph-negative BCR-ABL-negative progenitors could be grown more easily than their Ph-positive counterparts, suggesting that these three cytokines preferentially stimulate normal progenitors ( Jazwiec et al, 1995 ). Few studies have compared the effect of G-CSF on normal and CML progenitors, but it is generally acknowledged that G-CSF can promote CFU-GM growth from both normal and CML mononuclear cells; G-CSF is also produced by maturing neoplastic cells from patients with CML, and could subsequently contribute to the expansion of leukaemic mature cells ( Klein et al, 1990 ). Bedi et al (1994 ) have shown that growth factors (and G-CSF) can favour terminal differentiation of CML CD34+ cells more importantly than that of normal counterparts. Giralt et al (1993 ) have reported that in some patients with CML who relapse following AlloBMT, G-CSF re-induced complete remission and it was hypothesized that this effect resulted from preferential stimulation of Ph-negative cells. In the present study some results suggest that — at least in some patients — lenograstim preferentially mobilized Ph-negative BCR-ABL cells. Similar results were reported by Carreras et al (1997 ) who obtained Ph-negative LK in one patient who had minimal cytogenetic response during treatment with α-IFN. These experimental and clinical observations suggest that, at least in some patients with CML, normal (i.e. Ph-negative, BCR-ABL-negative) progenitors are more sensitive to stimulation by rhG-CSF than leukaemic progenitors. However, this needs to be confirmed in subsequent studies.

    The Genoa group, and others, have reported that in CML patients either at diagnosis or who did not respond to α-IFN, it was possible to collect Ph-negative progenitor cells after mobilization with both chemotherapy and G-CSF ( Carella et al, 1993 , 1998). In our study performed in CML patients with a good response to α-IFN, the proportion of patients who could achieve Ph-negative LK, after mobilization with G-CSF alone, was higher than that observed by Carella et al (1998 ) in patients treated with chemotherapy and G-CSF. Moreover, the procedure was very safe and could be performed on an out-patient basis; however, patients mobilized by chemotherapy and G-CSF have severe myelosuppression lasting 2–3 weeks and which requires hospitalization. Therefore, we think that for CML patients who have responded to α-IFN, a low-dose of G-CSF, without chemotherapy, is sufficient to mobilize normal progenitor cells. However, this observation needs to be confirmed in a larger group of patients. In addition, other points such as the optimal dose of G-CSF and, more importantly, the utility of these Ph-negative progenitors to reconstitute long-term Ph-negative haemopoiesis when transplanted to patients with CML, also remain to be investigated. This point could not be answered in the present study as the 23 patients included had a good response to α-IFN and thus no indications for ASCT. As no significant adverse effects were experienced by the patients in the present study, these remaining questions can be addressed safely in future studies.

    Acknowledgements

    1. Top of page
    2. Abstract
    3. MATERIALS AND METHODS
    4. RESULTS
    5. DISCUSSION
    6. Acknowledgements
    7. References

    This study was supported by Chugaï Rhone-Poulenc.

    References

    1. Top of page
    2. Abstract
    3. MATERIALS AND METHODS
    4. RESULTS
    5. DISCUSSION
    6. Acknowledgements
    7. References
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