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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 1 (January 1), 2000:
pp. 62-66
CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Molecular heterogeneity in complete cytogenetic responders after
interferon- therapy for chronic myelogenous leukemia: low levels of
minimal residual disease are associated with continuing remission
Andreas Hochhaus,
Andreas Reiter,
Susanne Saußele, Anja Reichert,
Michael Emig,
Jaspal Kaeda,
Beate Schultheis,
Ute Berger,
Patricia
C. A. Shepherd,
Norman C. Allan,
Rüdiger Hehlmann,
John M. Goldman, and
Nicholas C. P. Cross for the German CML Study Group and
the UK MRC CML Study Group
From the III. Medizinische Universitätsklinik, Fakultät
für Klinische Medizin, Mannheim der Universität Heidelberg,
Mannheim, Germany; Western General Hospital, Edinburgh, UK; Imperial
College School of Medicine, Hammersmith Hospital, Department of
Hematology, London, UK.
 |
Abstract |
A substantial minority of patients with chronic myelogenous
leukemia (CML) achieve a complete response (CR) to treatment with interferon- (IFN), defined as the disappearance of Philadelphia chromosome-positive metaphases. Currently it is unclear how long IFN
treatment should be continued for such patients. We used a competitive
reverse transcriptase-polymerase chain reaction (RT-PCR) to quantify
levels of BCR-ABL transcripts in 297 peripheral blood specimens
collected from 54 patients who had achieved CR with IFN. The median
duration of observation was 1.9 years (range, 0.3-11.0 years). Total
ABL transcripts were quantified as internal control and results were
expressed as the ratio BCR-ABL/ABL. All 54 patients had molecular
evidence of residual disease, although 3 patients were intermittently
PCR negative. The median BCR-ABL/ABL ratio at the time of maximal
response for each patient was 0.045% (range, 0%-3.6%). During the
period of observation 14 patients relapsed, 11 cytogenetically to
chronic phase disease and 3 directly to blastic phase. The median ratio
of BCR-ABL/ABL at maximal response was significantly higher in patients
who relapsed than in those who remained in CR (0.49% versus 0.021%,
P < 0.0001). Our findings show that the level of residual
disease falls with time in complete responders to IFN, but molecular
evidence of disease is rarely if ever eliminated. The actual level of
minimal residual disease correlates with the probability of relapse. We
suggest that for patients who reach CR, IFN should be continued at
least until relatively low levels of residual leukemia are achieved.
(Blood. 2000;95:62-66)
© 2000 by The American Society of Hematology.
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Introduction |
Since the initial description of the polymerase chain
reaction (PCR) in 1985,1 numerous applications for
amplification of specific DNA sequences have been developed. Because of
its remarkable sensitivity, PCR has become a standard technique for the
detection of minimal residual disease (MRD) after therapy of various
hematologic and infectious diseases.
Chronic myelogenous leukemia (CML) constitutes a clinical
model for molecular detection and therapy surveillance because this entity was the first leukemia known to be associated with a specific chromosomal rearrangement, the Philadelphia (Ph) translocation t(9;22)(q34;q11), and the presence of a novel chimeric gene, BCR-ABL. In 1989, the first encouraging results concerning detection of MRD by
PCR in patients with CML after allogeneic bone marrow
transplantation were reported.2 However, conflicting data
from a comparative multicenter study revealed serious problems for the
method with a high rate of false-positive results and provoked an open
discussion.3,4 Over the past 10 years, PCR has been
optimized and developed. Specificity has been considerably increased by
the partial standardization of methodology and the introduction
of rigorous precautions to avoid contamination.5
Sensitivity has been improved by using nested primer pairs and
performing 2 consecutive PCR steps. In view of the limited value of
qualitative PCR for monitoring CML patients after therapy,
quantitative BCR-ABL PCR assays were developed to monitor patients
after bone marrow transplantation6,7 or treatment with
interferon- (IFN)8,9 and are now in routine clinical use.
Interferon- may induce complete hematologic remission in about 70%
to 80% of patients with CML in early chronic phase.10 Complete response (CR) has been observed by cytogenetic or Southern blot analysis in 6% to 38% of patients.11-16 It is
generally agreed that the variability of response rates may be
explained by differences in the risk composition of patient groups.
Complete responders survive significantly longer than those without
cytogenetic remission,15 but some of these patients
will ultimately relapse with reappearance of the Ph-positive
clone.17 Using the reverse transcriptase-polymerase chain
reaction (RT-PCR) assay, persistent disease in CR has been documented in the great majority of cases.9,17-19 The aim
of our study was to assess the prognostic significance of sequential quantitative analysis of residual disease in CML patients in CR on IFN.
| |
Patients and methods |
Patients
Fifty-four patients who had achieved CR to treatment with IFN were
studied prospectively (36 male, 18 female; median age at diagnosis 48 years, range 5-76 years) with informed consent as a cooperative project
of the German and UK MRC CML study groups. Thirty-two patients were
treated with recombinant IFN-2a, 5 with recombinant IFN-2b, 1 with recombinant IFN-2c, 14 with lymphoblastoid IFN-1n, 1 with
IFN-2a and 2b, and 1 with IFN-2a and IFN-n1 consecutively. The
therapeutic goal in all patients was to treat them with the maximum
tolerable doses of IFN, starting with 3 million IU/d with increasing
dosages to 6, 9, or 12 million IU/d in the UK study11 or
starting with 5 million IU/m2/d in the German
study.12 The doses were adjusted according to
leukocyte and platelet counts to reach a level of 2000 to 4000 white
blood cells/µL and > 100 000/µL platelets. The median interval from diagnosis until start of IFN therapy was 0.2 year (range, 0-2.1 years). At diagnosis, 52 patients were Ph positive, 2 patients were Ph
negative/BCR-ABL positive. CR was defined as the disappearance of Ph
chromosome-positive metaphases or, for Ph-negative/BCR-ABL-positive patients, the disappearance of the rearranged BCR allele as assayed by
Southern blot analysis. The median time from start of IFN therapy to
first detection of CR was 1.6 years (range, 0.4-6.4 years). The median
follow-up period in CR was 1.9 years (range, 0.3-11.0 years). In 44 patients, CR lasted at least 1 year with 10 patients experiencing
long-lasting complete remissions of more than 5 years. In 6 patients
IFN has been withdrawn after 0.3 to 5.2 years (median, 4.4 years) from
CR because of stable CR (n = 3) or because of adverse side effects
(n = 3). Additional therapies administered prior to CR were
hydroxyurea (n = 30), busulfan (n = 2), hydroxyurea and busulfan
(n = 1), and cytosine arabinoside (n = 1). Prognostic risk
distribution according to Sokal20 was calculated for 51 patients: low risk, n = 27 (53%); intermediate risk, n = 17
(33%); high risk, n = 7 (14%). For 49 patients a new score
established for IFN-treated patients21 was assessed: low
risk, n = 27 (55%); intermediate risk, n = 19 (39%); high risk,
n = 3 (6%).
Cytogenetics and Southern blot analysis
Cytogenetic and DNA analyses were performed according to standard
protocols. For chromosome analysis bone marrow specimens were examined
on direct or short-term (24-hour) cultures. Southern blotting for M-bcr
rearrangements was performed as reported.22 A minimum
number of 20 metaphases was required to assess CR. If fewer than 20 metaphases were available for analysis, CR was only diagnosed if no
M-bcr rearrangement was detectable by Southern blot analysis. Relapse
was defined as the reappearance of at least 1 Ph-positive metaphase or,
alternatively, the reappearance of the rearranged M-bcr band in
Southern blot analysis.
Qualitative and quantitative RT-PCR analysis
RNA was extracted from leukocytes obtained from 20 mL peripheral
blood. Samples were received either locally or by mail and spent
between 1 to 3 days in transit. We have previously shown essentially
identical levels of residual disease in peripheral blood and bone
marrow.9 Conditions for RNA extraction, reverse transcription, and 2-step (nested) PCR for BCR-ABL have been
described.23 An estimate of the number of BCR-ABL
transcripts was made applying a competitive PCR titration
assay6,9 using a semilogarithmic dilution series of
101 to 106 molecules added to the same volume
of patient cDNA. The equivalence point between different competitor
concentrations was determined by densitometric analysis. ABL mRNA was
quantified in all samples as an internal standard using the same
competitor construct but different oligonucleotide
primers.9,24 The result was expressed as the percentage
ratio between BCR-ABL and ABL. BCR-ABL-negative results obtained from
10 samples with ABL equivalence points < 10.4-5
competitor molecules per reaction were regarded as of insufficient quality for sensitive detection of MRD and were therefore discarded. Strict precautions were taken to prevent contamination. All
experiments included negative controls from all stages of the reactions.
A total of 297 samples (1-13 per patient, median 5) were analyzed.
Seventy samples have been investigated before CR or after relapse, 227 samples were obtained at various time points during CR (median 1.7;
range, 0-11.0 years after first CR). Of these 227 samples, 196 were
studied during ongoing IFN- therapy in complete remission, 31 samples of 6 patients after withdrawal of IFN.
Statistical methods
Relapse-free survival analysis was based on Kaplan-Meier estimation
and groups were compared by log-rank test. Relapse-free survival was
dated from the first PCR analysis in complete remission to avoid
selection bias for the time interval between first detection of CR and
first PCR analysis. The impact of the minimum individual MRD level on
the stability of CR was proven by the Mann-Whitney test. The influence
of the level of residual disease and the relationship to prognostic
risk groups at diagnosis on the risk of relapse were determined by
Fisher's exact test.
| |
Results |
All 54 patients had evidence of residual BCR-ABL transcripts in
complete remission. Twenty-six patients (48%) expressed b3a2, 18 (33%) b2a2, 9 (17%) b2a2 and b3a2, and 1 (2%) b3a3 BCR-ABL transcripts. For 3 patients, nested PCR was intermittently negative (1 of 4, 2 of 7, and 1 of 11 samples, respectively). The median BCR-ABL/ABL ratio of all 227 samples investigated during complete remission was 0.07% (range, 0%-7.0%) and the median ratio of the 196 samples investigated during ongoing IFN therapy was 0.1% (range, 0%-7.0%). The lowest level of residual disease on sequential analysis (maximal response) for individual patients in CR ranged between 0% and
3.6% (median, 0.045%). Levels of residual disease in 153 samples of
40 non-relapsing patients declined over time after first CR
(Table), indicating an ongoing effect of
IFN in reducing the levels of MRD. Five of the 6 patients who had
ceased IFN therapy were in stable remission at the date of analysis,
2.2 to 9.5 years (median, 5.6 years) after IFN withdrawal. Levels of
MRD in these 5 patients ranged from 0% to 3.0% (median, 0.07%)
BCR-ABL/ABL. The sixth patient relapsed 2.5 years after IFN withdrawal
and had rising levels of MRD on sequential analysis.
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Table. Residual disease measured as ratio BCR-ABL/ABL transcripts
according to time in complete remission (CR) in 40 nonrelapsing
patients continuing IFN therapy
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Fourteen patients (26%) relapsed at 0.3 to 5.1 years (median, 1.3 years) after first diagnosis of CR and 0.2 to 3.9 years (median, 1.1 years) after first molecular analysis. Eleven patients relapsed into
chronic phase CML with the reappearance of Ph-positive metaphases or
M-bcr rearrangement. Three patients relapsed directly into blast crisis
(1 myeloid, 2 lymphoid). Four patients died, 3 of blast crisis and 1 of
liver failure in chronic phase CML (Figure
1). The relapsing patients have been
treated with IFN-2a (n = 9), IFN-2b (n = 1), IFN-n1
(n = 3), and IFN-2c (n = 1). Additional therapies in relapsing
patients were hydroxyurea (n = 10), busulfan (n = 1), and none
(n = 3). The relapsing patients were recruited from the German
studies (n = 7), from the UK studies (n = 6), or neither trial
(n = 1). There was no specific pattern of the type of IFN, additional
therapies, or allocation to a specific study in relapsing patients.
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| Fig 1.
Maximum response on sequential analysis for the 54 CML
patients in CR on IFN.
For the 14 patients who relapsed, the minimum BCR-ABL/ABL ratio
achieved was significantly higher (P < 0.0001, 2-sided
Mann-Whitney test) than that seen in patients who remained in CR.
 = relapse in chronic phase CML, n = 11;  = relapse in
blast crisis, n = 3;  = continuing complete remission,
n = 40.
|
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Of the 14 patients who relapsed, 13 had persistently high levels of MRD
(> 0.045% BCR-ABL/ABL, the median lowest level of MRD for patients
in CR) on sequential analysis while still in CR. Of the 27 patients who
failed to achieve BCR-ABL/ABL ratios of < 0.045%, 13 (48.1%) subsequently relapsed. In contrast, of the 27 patients who
did achieve BCR-ABL/ABL ratios of < 0.045%, only 1 patient
subsequently relapsed (3.7%; P =.0003). This patient, referred to above, ceased IFN therapy 2.5 years before relapse.
Relapse-free survival was significantly different between patients with
residual BCR-ABL/ABL ratios  0.045% as compared to patients with
ratios > 0.045% (P < .0001 2-sided log-rank test, Figure
2). We found no association between the
rate of relapse and the prognostic risk of individual patients at
diagnosis: for the Sokal score, 5 of 27 low-risk versus 8 of 24 intermediate/high-risk patients relapsed (P = .34; not
significant, Fisher's exact test); for the IFN-score, 6 of 27 low-risk versus 7 of 22 intermediate/high-risk patients relapsed
(P = .52; not significant, Fisher's exact test).
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| Fig 2.
Relapse-free survival from first PCR analysis of patients
in CR compared to maximum response to IFN therapy.
One of 27 patients with low levels of residual disease ( 0.045% BCR-ABL/ABL) relapsed compared to13 of 27 patients with
relatively high levels (> 0.045%, P < .0001, 2-sided
logrank test).
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Discussion |
Cytogenetic analysis is the standard method to ascertain the quality
of remission in patients with CML treated with IFN, but this technique
has a maximum sensitivity of only 10 2. Because
patients with leukemia at presentation usually have a total burden of
more than 1012 malignant cells,25 individuals in CR may harbor as few as zero or as many as 1010 leukemia cells.26 Conventional RT-PCR for BCR-ABL is routinely able
to detect residual disease down to a level of a single cell in a background of 105 to106 normal cells and
therefore is up to 4 orders of magnitude more sensitive than
cytogenetic analysis. Although rare BCR-ABL-positive cells
(1/107-108) have been detected in a significant
proportion of normal healthy adults,27,28 the finding of
BCR-ABL-positive myeloid progenitors in CML patients in CR strongly
supports the conclusion that the routine assay is detecting cells
derived from the leukemic clone.29
The finding of long-lasting cytogenetic remission in some CML patients
treated with IFN raises the question of whether this agent actually
cures the disease. Using standardized methods for blood processing, RNA
extraction, cDNA synthesis, and nested RT-PCR, we found evidence of
residual disease in all of 54 patients. Discordant findings in the
literature concerning the ability of qualitative PCR to detect
residual disease in CML patients in CR on IFN are probably attributable
to the sensitivity with which BCR-ABL transcripts have been detected in
different studies and samples. Quantification of BCR-ABL transcripts by
competitive PCR revealed levels of residual disease in CR that spanned
more than 4 orders of magnitude. Analyzing all samples
investigated in non-relapsing patients continuing IFN therapy after CR
revealed that BCR-ABL levels declined over time, consistent with the
findings of Kurzrock et al.18 This suggests an ongoing
process of quantitative disease depletion by IFN treatment. However, in
our study, all 10 patients with follow-up of 5 years or more in CR were
still BCR-ABL positive. It is possible therefore that the level of
residual disease declines slowly but eventually reaches a plateau. This
is consistent with the hypothesis that in some cases residual leukemia
cells have no capacity to regenerate clinically significant leukemia, a
situation tantamount to "operational" cure.
In some patients, however, cytogenetic or hematologic relapse occurs
after CR. We have found a significant difference in the risk of relapse
in patients with relatively high BCR-ABL transcript levels as compared
to patients with low levels. In particular, relapse was rare in
patients with BCR-ABL/ABL ratios of 0.045%. As compared to the
total population in randomized studies,21 complete
responders represent a relatively high proportion of low-risk patients
according to the Sokal score20 (53% versus 35%) or the
interferon score21 (55% versus 41%). However, we have
found that the risk of relapse after reaching CR does not depend on the
prognostic risk at diagnosis, but on the residual BCR-ABL levels in CR.
Furthermore, we found that only a minority of patients (1 of 6) who
have achieved low levels of MRD subsequently relapsed following
withdrawal of treatment (patient B, Figure 3). These findings suggest that it is
advisable to continue IFN treatment until at least relatively low
levels of BCR-ABL are achieved. If, for whatever reason, IFN is
discontinued, rising BCR-ABL levels in CR could be used as an indicator
to reinitiate treatment.
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| Fig 3.
Molecular monitoring in 3 representative patients in CR
on IFN therapy.
In patient A, a transient complete cytogenetic response was followed by
a hematologic, cytogenetic, and molecular relapse. In patient B,
relapse occurred after withdrawal of IFN because of adverse effects.
Patient C is in stable complete cytogenetic remission with very low
ratios BCR-ABL/ABL 10.4 years after initiation of IFN therapy. The
upper dotted line indicates the levels of 2% BCR-ABL/ABL at which
cytogenetic relapse is expected,9 and the lower dotted line
(0.1% BCR-ABL/ABL) the median ratio of all samples of patients
investigated in CR.
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| |
Acknowledgments |
We wish to thank all hematologists and cytogeneticists in Germany,
Great Britain, Switzerland, and Austria who provided samples and data.
Cooperating institutions include the following:
Germany: Hämatologische Gemeinschaftspraxis Aachen, U. Essers; Hämatologische Praxis Aachen, L. Habets;
Gemeinschaftspraxis Bad Oeynhausen, P. Harms;
Humboldt-Universität-Charité, Berlin, K. Possinger;
Zentralkrankenhaus St. Jürgen-Strasse, Bremen, C. R. Meier;
Medizinische Klinik und Poliklinik, Universität Düsseldorf,
C. Aul; St. Johannes-Hospital, Duisburg, M. Westerhausen; Nordwest-Krankenhaus Frankfurt, A. Knuth; Allg. Krankenhaus St. Georg,
Hamburg, R. Kuse; Zytogenetisches Labor, Med. Klinik, Universität Hamburg, D. K. Hossfeld; Medizinische Klinik und Poliklinik V, Universität Heidelberg, M. Bentz; Kreiskrankenhaus Herford, U. Schmitz-Huebner; Städt. Klinik Kassel, W. D. Hirschmann;
Caritas-Krankenhaus Lebach, D. Hufnagl; III. Medizinische Klinik
Mannheim, Universität Heidelberg, R. Hehlmann;
Gemeinschaftspraxis München, M. Fromm; Städt. Klinikum
Nürnberg, C. Falge, W. Brockhaus; Diakonie-Krankenhaus, Schwäbisch-Hall, H. H. Heißmeyer; Praxis Tübingen, M. Haen; Bundeswehrkrankenhaus Ulm, A. P. Schoengen; Zytogenetisches
Labor, Universität Ulm, B. Heinze; Kreiskrankenhaus
Waldbröl, L. Labedzki; Med. Poliklinik, Universität
Würzburg, M. Wilhelm.
United Kingdom: Aberdeen Royal Infirmary, A. A. Dawson; Gwynedd
Hospitals, Bangor, R. Williams; St. Helier Hospital, Carshalton, K. Wilson; Western General Hospital, Edinburgh, P. C. A. Shepherd, N. C. Allan; Epsom General Hospital, L. Jones;
Farnborough Hospital, A. K. Lakhani; Frimley Park Hospital, J. van de
Pette; St. Luke's Hospital, Guildford, G. Robbins; Huddersfield Royal
Infirmary, C. Carter; Walton Hospital, Liverpool, P. A. Stevenson; St.
John's Hospital Howden, Livingston, M. K. Cook; Hammersmith Hospital, London, J. M. Goldman, A. Chase, S. Coulthard; Royal Marsden Hospital, London, D. Catovsky; University College London, A. H. Goldstone; St.
Bartholomews Hospital, London, J. S. Lilleyman; Norfolk & Norwich
Hospital, G. E. Turner; Queen's Medical Centre, Nottingham, J. Davies;
Poole General Hospital, A. Worsley; Northwick Park Hospital, Watford,
C. D. L. Reid.
Switzerland: Kantonsspital Basel, A. Tichelli; Inselspital,
Bern, A. Tobler; Hämatologische Praxis, Breitenbach, J. Haberthür.
Austria: Universität Graz, Abt. Hämatologie, H. Sill.
We are grateful to Prof Dr J. Hasford and Dipl-Stat M
Pfirrmann, Institut für Medizinische
Informationsverarbeitung, Biometrie und Epidemiologie der
Universität München, Germany, for their valuable
statistical advice.
| |
Footnotes |
Submitted April 5, 1999; accepted August 24, 1999.
Supported by the Leukaemia Research Fund of Great Britain, the Dr.
Mildred Scheel Stiftung, the Deutsche
José-Carreras-Leukämiestiftung e.V., and the
Forschungsfonds der Fakultät für Klinische Medizin Mannheim
der Universität Heidelberg, Germany.
Reprints: Andreas Hochhaus, III. Medizinische
Universitätsklinik, Fakultät für Klinische
Medizin, Mannheim der Universität Heidelberg, Wiesbadener
Strasse 7-11, 68305 Mannheim, Germany; e-mail:
hochhaus{at}rumms.unimannheim.de.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Top
Abstract
Introduction