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Keywords:

  • acute myeloid leukaemia;
  • t(8;21);
  • MDR1 expression;
  • relapse;
  • prognostic model

Summary

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

Acute myeloid leukaemia (AML) carrying t(8;21) has an overall favourable prognosis. However, relapse occurs and the impact of multidrug resistance gene (MDR1) expression on recurring disease in this group of patients is not known. We determined quantifiable MDR1 expression in the bone marrow of 28 AML patients with t(8;21) by a validated real-time polymerase chain reaction assay. Using MDR1 expression, white blood cell count and CD56 expression at diagnosis we observed complete concordance of predicted and observed relapses. A calculated logit out of these three variables was a strong independent prognostic factor for overall (P = 0·007) and disease-free survival (P = 0·002).

Adult t(8;21) acute myeloid leukaemia (AML) has a favourable prognosis with high complete remission (CR) rates of 85–90% (Grimwade et al, 1998). However, relapse occurs in a significant number of patients. So far, apart from age, a few characteristics at presentation have been suggested to predict a worse outcome in adult t(8;21) AML patients; a high initial white blood cell count (WBC) (Billstrom et al, 1997), an increased WBC index (product of WBC count and percentage of bone marrow blasts) (Nguyen et al, 2002) and expression of the CD56 antigen (Raspadori et al, 2001) seem to be of particular clinical importance. Moreover, the relapse rate may depend on therapy intensity (Byrd et al, 1999).

Another prognostic factor for treatment outcome in AML is the expression of the multidrug resistance gene (MDR1). Our group reported a correlation between t(8;21) and MDR1 expression in adult AML (Schaich et al, 2002). We have now examined, for the first time, quantifiable MDR1 expression by real-time polymerase chain reaction (PCR) in adult AML patients carrying the t(8;21) to evaluate the prognostic impact of MDR1 expression in a multivariate setting including other possible prognostic factors.

Patients, therapy and control group

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

A total of 28 adult AML patients with t(8;21) treated within the Süddeutsche Hämoblastosegruppe (SHG) AML96 trial were included. Double induction therapy and postremission therapy were completed as recently described (Schaich et al, 2001). Bone marrow samples were taken from the patients at diagnosis. Eight healthy bone marrow donors served as a control group.

MDR1 real-time PCR

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

Sample handling, mRNA extraction and c-DNA synthesis of bone marrow samples of patients and of healthy bone marrow donors were performed as previously described. Real-time PCR conditions were essentially as described previously (Illmer et al, 2002). Prior to mRNA extraction all samples were depleted of CD3 with CD3-coated dynabeads (Dynal, Hamburg, Germany) according to the manufacturer's recommendations, to avoid false positive MDR1 expression.

Cytogenetics

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

Chromosome analyses were performed on metaphases from direct preparations, as well as from 24 and 48 h cultures of bone marrow and/or peripheral blood samples according to routine laboratory procedures.

Statistical analysis

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

Differences in quantifiable MDR1 gene expression or other quantifiable parameters between the analysed subgroups were evaluated by two-tailed Mann–Whitney U-test and differences in binary parameters were evaluated by two-tailed Fisher's exact test. Correlation between MDR1 expression and numeric clinical parameters was tested by the method of Pearson.

Multivariate analyses of the influence of experimental or clinical parameters on the probability of relapse or survival were carried out by stepwise forward logistic or Cox regression, respectively, using the SAS software package (SAS Institute, Cary, NC, USA). The concordance of predicted and observed relapses was confirmed by the Hosmer–Lemeshow test. P < 0·05 was considered significant.

Results and discussion

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

The clinical features of patients included in the study are listed in Table I.

Table I.  Patient characteristics.
  1. AML, acute myeloid leukaemia; WBC, white blood cell count; MDR, multidrug resistance.

No. of patients28
Sex
 Male12
 Female16
Median age, years (range)46 (22–71)
Disease status
 De novo AML27
 Secondary AML1
Extramedullary disease
 Skin involvement1
 Central nervous system involvement1
Median WBC, ×109/l (n = 28) (range)11·2 (1·2–71·0)
Median marrow blast percentage (n = 28) (range)53 (18–84)
Median lactate dehydrogenase, U/l (n = 27) (range)942 (12–3756)
Median CD34 expression, % (n = 25) (range)64 (7–89)
Median CD56 expression, % (n = 23) (range)26 (0–83)
Median MDR1 expression (n = 28) (range)3172 (69–31 780) copies
Cytogenetics (n = 28)
 Associated loss of sex chromosome10
 Associated del(9q)3
 Two or more associated aberrations  5

After double-induction therapy, 27 of 28 (96%) patients acheived CR. Relapse occurred in 10 of 27 (37%) patients with a median CR duration of 11·6 (3·5–32·8) months. The 5-year overall survival was 56% and the 5-year disease-free survival was 51%.

In a first validation step of our approach, it was found that T-lymphocytes, represented by the CD3 positive fraction, had significantly higher MDR1 expression values than the corresponding negative fractions containing either normal haematopoietic cells or blasts (P < 0·001; data not shown). CD3 negative fractions of the AML study population contained significantly more MDR1 transcripts than their counterparts from healthy bone marrow donors (median expression 3172 vs. 364 copies; P < 0·001).

Relapsed patients had significantly higher MDR1 expression levels (P = 0·01) and a trend towards higher WBC at diagnosis (P = 0·06) than non-relapsed patients.

For 23 patients, MDR1 expression, age, WBC, CD56 expression and additional cytogenetic aberrations other than loss of one sex chromosome were included as potential prognostic factors for relapse in a stepwise forward logistic regression analysis. MDR1 expression and WBC reached significance (both P = 0·02), and in the third step, CD56 expression was also significant (P = 0·03). Interestingly, with these three parameters the Hosmer–Lemeshow test revealed a complete concordance between predicted and observed relapses.

Furthermore, a Cox model was used to evaluate the prognostic value of a calculated logit that included WBC, CD56 and MDR1 expression. This logit was found to be a strong independent prognostic factor for overall and disease-free survival (see Table II). Thus, overall survival was 100% after 4 years for patients with a calculated “logit” lower than the median level compared with 23% for those with a calculated logit higher than the median level (P = 0·006). This difference was even more prominent for disease-free survival (100% vs. 18%; P = 0·0004).

Table II.  Multivariate analysis for overall (OS) and disease-free survival (DFS).
 OSDFS
  1. LOS, loss of sex chromosome.

  2. *The logit variable was calculated by the formula: logit = −1346·02 + 7·096 × WBC + 10·772 × CD56 + 0·128 × MDR1 expression.

  3. §Hazard ratio was calculated per 1000 units of logit.

AgeP = 0·07P = 0·13
Associated cytogenetic aberrations without LOSP = 0·86P = 0·51
Logit*P = 0·007P = 0·002
Hazard ratio (95% confidence interval)§2·21 (1·24–3·95)2·20 (1·34–3·64)

As therapy-refractory recurrent disease is still the major cause of death in AML patients with t(8;21), prediction of relapse is very important. In a previously conducted study we demonstrated an increased frequency of MDR1 positivity in adult t(8;21) patients compared with AML patients with normal karyotypes (Schaich et al, 2002). Such an increase in MDR1 expression had previously only been described in paediatric t(8;21) AML patients (Pearson et al, 1996), who generally have a worse prognosis than adult cases (Leblanc & Berger, 1997).

In the present study, prediction of relapse was possible in the multivariate analysis for AML patients with t(8;21) by a calculated ‘logit’. The biological reasons for the highly predictive value of the combination of WBC, CD56 and MDR1 expression in t(8;21) patients are not known but it may be speculated that enhanced genetic instability may have led to these deregulated properties of affected AML patients. In this context, our data may deliver the basis for more focused biological analyses in the future. Additionally, the published model has to be used and proven in a large, prospective study, which is already in preparation by our study group. Until then, our data propose a model for sensitive prediction of relapse in a well-defined group of AML patients with possible implications for differential treatment strategies.

Acknowledgments

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References

We thank A. Liebkopf, C. Dill and S. Gotthardt for expert technical assistance, S. Roettger for critical reading of the manuscript and the following members of the German SHG AML96 study group for entering their patients into the trial: W. E. Aulitzky, L. Leimer (Robert-Bosch-KH Stuttgart), H. Bodenstein, J. Tischler (Klinikum Minden), F. Fiedler, R. Herbst (Klinikum Chemnitz gGmbH), M. Gramatzki, G. Helm (Universitätsklinikum Erlangen), F. Hirsch (Kreiskrankenhaus Offenburg), A. D. Ho (Universitätsklinikum Heidelberg), D. Huhn, O. Knigge (Virchow-Klinikum Berlin), A. Neubauer (Klinikum d. Philipps-Universität Marburg), H. Pohlmann, N. Brack (Städtisches KH München-Harlaching), J. G. Saal (Malteser Krankenhaus Flensburg), U. Schäkel (Universitätsklinikum Dresden), N. Schmitz (Allg. KH St. Georg Hamburg), H. Wandt, K. Schäfer-Eckart, T. Denzel (Städtisches Klinikum Nürnberg). The study was partly supported by grants from the Deutsche Krebshilfe (70–2210-Eh5) and the ‘Kompetenznetzwerk Akute und Chronische Leukämien’ sponsored by the Bundesministerium für Bildung und Forschung (BMBF).

References

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Patients, therapy and control group
  5. MDR1 real-time PCR
  6. Flow cytometry
  7. Cytogenetics
  8. Statistical analysis
  9. Results and discussion
  10. Acknowledgments
  11. References
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