Molecular monitoring of response to imatinib (Glivec®) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission

Abstract

A significant proportion of chronic myeloid leukemia (CML) patients achieve a major cytogenetic remission (MCR) to imatinib therapy after failing interferon (IFN) α-based protocols. We sought to determine levels of residual disease in patients with MCR using various molecular methods and to establish a relation between residual BCR-ABL transcript levels and rate of relapse in complete cytogenetic remission (CCR). Response was measured by conventional cytogenetic analysis, hypermetaphase and interphase fluorescence in situ hybridization (HM-FISH, IP-FISH) of bone marrow (BM) cells, qualitative nested and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for BCR-ABL transcripts. We investigated 323 peripheral blood (PB) and BM samples from 48 CML patients who achieved a complete (Ph+ 0%; n=41) or partial (Ph+ 1–34%; n=7) cytogenetic remission after 3–20 months of imatinib therapy. Prior to imatinib, 35 patients were in chronic phase (CP), eight in accelerated phase (AP), four in myeloid and one in lymphoid blast crisis. HM-FISH results correlated with ratios BCR-ABL/ABL in PB and BM. In patients with CCR, residual disease was detectable by HM-FISH (31%), IP-FISH (18%), and RT-PCR (100%). During follow-up, BCR-ABL became undetectable in two patients (one CP, one AP) by both nested and quantitative RT-PCR. CCR is ongoing in 30 evaluable patients, 11 patients have relapsed. At the time of best response, median ratios BCR-ABL/ABL were 2.1% (range 0.82–7.8) in patients with subsequent relapse and 0.075% (range 0–3.9) in patients with ongoing remission (P=0.0011). All 16 CP patients, who achieved ratios BCR-ABL/ABL <0.1% as best molecular response are in continuous remission, while 6/13 patients (46%) with ratios 0.1% have relapsed (P=0.0036). We conclude that: (i) in patients with CCR to imatinib, HM-FISH and RT-PCR usually reveal residual BCR-ABL+ cells; (ii) RT-PCR results derived from PB and BM are comparable in CP CML; and (iii) low levels of residual disease with ratios BCR-ABL/ABL <0.1% are associated with continuous remission.

Introduction

Chronic myeloid leukemia (CML) constitutes a clinical model for molecular-cytogenetic detection and therapy surveillance, since the disease is associated with a specific chromosomal aberration, the Philadelphia (Ph) translocation, t(9;22)(q34;q11). The molecular consequence is the BCR-ABL fusion gene, which is expressed as BCR-ABL mRNA and BCR-ABL fusion protein with constitutive tyrosine kinase activity.1 Imatinib (formerly STI571, Glivec®) is a selective inhibitor of BCR-ABL and specifically inhibits the growth of BCR-ABL-positive cell lines in vitro.2,3 In phase II studies, complete cytogenetic remission (CCR) was observed in 41% of chronic phase (CP) patients after failure of interferon α (IFN) therapy,4 17% of accelerated phase (AP) patients5 and 7% of patients in myeloid blast crisis (BC).6

The degree of tumor load reduction is an important prognostic factor for patients with CML on therapy.7,8 The standard method to monitor the response to therapy is cytogenetic analysis of bone marrow (BM) aspirates, which allows the comprehensive assessment of chromosomal aberrations. However, BM metaphases are required and aspiration and cultivation of proliferating cells are not always sufficient. The technique is relatively insensitive since usually only 20–50 metaphases are analyzed. After therapy with IFN, the stability of CCR depends on the risk profile of the patients9 and the level of residual BCR-ABL mRNA transcripts measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR).10 After imatinib treatment, the assessment of the response quality in patients with CCR is even more important, since the proportion of patients in CCR is significantly higher and achievement of CCR more rapid as compared to IFN.4 We sought to assess the impact of molecular methods for the detection and quantification of residual disease in patients with a major cytogenetic response (MCR, ie <35% Ph+ metaphases) to imatinib therapy and to establish a relation between levels of residual disease and stability of CCR. CP patients after IFN failure and patients in advanced disease were monitored by (i) qualitative and quantitative RT-PCR, (ii) hypermetaphase and (iii) interphase fluorescence in situ hybridization (HM-FISH, IP-FISH).

Patients and methods

Patients and samples

In total, 323 peripheral blood (PB) and BM samples from a total of 48 Ph and BCR-ABL positive CML patients (27 male, 21 female) who achieved a MCR (Ph+ <35%) by therapy with imatinib were analyzed. Median age at the start of therapy was 59.8 years (range 24.3–76.7). Patients were treated with imatinib within three multicenter phase II trials and an expanded access program and received 400–600 mg imatinib according to the trial protocols.4,5,6 By multiplex PCR,11 b3a2 transcripts were detected in 29, b2a2 in 14, and both transcript types in five patients. Prior to imatinib therapy, 35 patients were in chronic phase (CP), eight in AP, and five in BC four myeloid, one lymphoid). CP patients were pretreated with IFN for a median of 14 months (range, 1–135). Response to therapy was determined by conventional cytogenetical analysis of BM metaphases, molecular cytogenetics including HM-FISH and IP-FISH of BM cells, and real-time quantitative RT-PCR (Q-PCR) for BCR-ABL transcripts. After first CCR, patients were followed for a median period of 13 (range 0–35) months.

Cytogenetic analysis

Cytogenetic analyses were performed on BM aspirates within 24 h after aspiration. Metaphases from direct or short-term (24- or 48-h) cultures with or without colcemid exposure and addition of cytokines (erythropoietin, G-CSF, GM-CSF, SCF and IL3) were examined after Giemsa-banding.12 Between two and 70 metaphases were evaluated (median, 25). Cytogenetic response was evaluated according to standard criteria: complete response, 0% Ph+ metaphases; partial response, 1–34% Ph+ metaphases.13 Only results derived from at least 10 metaphases were considered for comparative analysis between the different methods. Cytogenetic, FISH and PCR analyses were performed on PB and BM samples taken simultaneously.

Fluorescence in situ hybridization

For hypermetaphase preparations, colcemid was added to the BM cultures for an additional 24 h after these had been incubated for 24 h in medium containing the above-mentioned cytokines incubation. IP-FISH was performed on slides from BM cells prepared for cytogenetic analysis. FISH analyses were performed by cohybridization of commercially available dual color translocation probes (BCR and ABL; Vysis, Downers Grove, IL, USA). CML cells were recognized by the juxtaposition of the BCR and ABL signals. The lab-specific cutoff level of false-positive results was 5% for IP-FISH and 0% for HM-FISH.12

RNA extraction and cDNA synthesis

Samples were processed on the same day after collection. Total leukocyte RNA was extracted from at least 20 ml of PB and 4 ml of BM after red cell lysis. RNA extraction was performed by CsCl gradient centrifugation14 or by commercially available extraction kits (RNeasy, Qiagen, Hilden, Germany). RNA was reverse transcribed using random hexamer priming and MMLV reverse transcriptase as described.14 cDNA samples were stored at −20°C.

Q-PCR and qualitative ‘nested’ PCR

Q-PCR for BCR-ABL, total ABL and glucose-6-phosphate dehydrogenase (G6PD) transcripts was performed using the LightCycler technology (Roche Diagnostics, Penzberg, Germany).15 The number of BCR-ABL transcripts per 2 μl cDNA was estimated by comparison to serial dilution of plasmid molecules (101–106 BCR-ABL plasmid molecules/2 μl). Normalized levels of residual disease were calculated as the ratio between BCR-ABL and total ABL and BCR-ABL and G6PD, respectively. If quantification revealed less than 10 BCR-ABL transcripts/2 μl or undetectable BCR-ABL by Q-PCR, qualitative nested PCR was performed16 using 5 μl cDNA. Positive results of these samples were expressed as the ratio of <10 BCR-ABL/number of control gene transcripts. Nested PCR was considered negative in case of at least 5 × 104 ABL transcripts/5 μl cDNA. All experiments included negative controls from all stages of the reactions and strict precautions were taken to prevent contamination.

Definition of relapse

Relapse after CCR was defined as the appearance of at least one Ph+ metaphase on two consecutive cytogenetic analyses or as loss of hematological remission with leukocytosis >10 × 109/l and/or immature precursors in PB.

Statistical analysis

The correlation of residual disease data was tested with the Spearman's rank coefficient. Q-PCR results from contemporaneous PB and BM samples were compared using the paired Mann–Whitney test. Follow-up data after CCR were analyzed with the Kruskal–Wallis test. The comparison of the BCR-ABL/ABL ratio in PB between patients who continued CCR vs relapsing patients was performed using the Mann–Whitney test. The influence of the level of residual disease on risk of relapse was determined by Fisher's exact test.

Results

Cytogenetic analysis

Prior to imatinib therapy, cytogenetic analysis revealed 76–100% Ph+ metaphases (median 100%). After a median duration of therapy of 5.5 months (range 2.6–20), CCR was observed in 41, PR in seven patients (Table 1). Follow-up of CCR patients revealed relapse in 11 patients (diagnosed in 10 patients by cytogenetics, in one patient by hematological criteria); of these, four patients (three in CP, one in AP prior to imatinib) remained in PR and seven patients (CP, n=3; AP n=1; myeloid BC, n=2; lymphoid BC, n=1) lost hematologic and cytogenetic remission. Median time to relapse after achieving CCR was 5 months (range 1.5–17).

Table 1 Stage of the disease and best cytogenetic response of patients investigated, n=48

FISH

HM-FISH was performed in 104 BM samples on a median of 106 (range 8–525) metaphases. IP-FISH was done in 116 cases on a median of 120 nuclei (range 55–459). In patients with CCR, HM-FISH revealed BCR-ABL positivity in 0–17% of metaphases (median 0), IP-FISH suggested BCR-ABL positivity in 0–16% of the evaluated nuclei (median 3.3). Considering the cutoff level of 0% for HM-FISH and 5% for IP-FISH, samples derived from patients who were considered in CCR by conventional cytogenetics demonstrated residual disease in 21/68 (31%) and 14/77 (18%) cases by HM- and IP-FISH, respectively (Table 2).

Table 2 Results during major (complete and partial) cytogenetic response to imatinib therapy

Q-PCR

A total of 237 Q-PCR results (PB and BM) were available for the comparison with FISH at partial and complete cytogenetic response to therapy. All patients were persistently positive for the BCR-ABL transcripts in PB samples. In two cases, BCR-ABL transcripts were undetectable by Q-PCR from PB and BM and nested PCR from BM, but nested PCR revealed BCR-ABL transcripts in PB. During CCR, the median ratio BCR-ABL/ABL was 0.60% (range 0.017–21%) in PB and 0.52% (range 0–34%) in BM, respectively; the median ratio BCR-ABL/G6PD was 0.022% (range 0.00060–0.66%) in PB and 0.026% (range 0–0.62%) in BM, respectively (Table 2).

Comparison of FISH and Q-PCR data

There was a good correlation between HM-FISH and Q-PCR results from PB and BM. The correlation was less pronounced between HM- and IP-FISH and between IP-FISH and Q-PCR results from PB and BM (Figures 1 and 2). When HM-FISH became negative during CCR, Q-PCR detected BCR-ABL mRNA in 80% (35/44) of cases in PB and in 83% (29/35) of cases in BM, respectively. Four of six BM samples, which were BCR-ABL negative by Q-PCR, remained positive in the qualitative-nested PCR.

Figure 1
figure1

Comparison of HM-FISH analysis and Q-PCR results in PB (panel a) and BM (panel b), expressed as the ratio BCR-ABL/ABL, in patients who achieved a major cytogenetic remission to imatinib.

Figure 2
figure2

Correlation of FISH and Q-PCR data in BM and PB in patients with major cytogenetic remission to imatinib. Correlation was good between HM-FISH and the ratios BCR-ABL/ABL in BM and PB, but not between IP-FISH and all other methods.

Comparison of BCR-ABL transcript levels in PB and in BM

In all, 102 sample pairs derived from PB and BM obtained simultaneously were available for comparison. The ratio BCR-ABL/ABL correlated well between PB and BM samples (r=0.77; P<0.0001; Figures 2 and 3). The median level of BCR-ABL transcripts, expressed as the ratio BCR-ABL/ABL, was 1.2 times higher in BM as compared to PB, but this difference was not significant.

Figure 3
figure3

Comparison of Q-PCR results from PB and BM (n=102 sample pairs), expressed as the ratio BCR-ABL/ABL, in patients with major cytogenetic remission to imatinib; dark symbols represent samples from patients with AP or BP prior to imatinib.

Quantification of internal controls

In 234 samples (PB and BM), G6PD transcripts were quantified as alternative internal standard to ABL. The ratios BCR-ABL/ABL and BCR-ABL/G6PD correlated with r=0.88 (P<0.0001; Figure 4). In cDNAs derived from PB, the median number of ABL transcripts was 17 530/2 μl cDNA (range, 659–188 000) and of G6PD transcripts 445 700/2 μl (range, 7200–3 800 000), respectively. In cDNAs from BM, the median number of ABL transcripts was 29 900/2 μl (range, 550–262 800) and of G6PD transcripts 588 000/2 μl (range, 4800–10 580 000), respectively.

Figure 4
figure4

Comparison of ABL and G6PD genes as internal standards in patients with major cytogenetic remission to imatinib.

Kinetics of the ratio BCR-ABL/ABL (PB) after CCR and association with subsequent relapse

At the time of first CCR, the median ratio BCR-ABL/ABL in PB was 1.4% (range 0.016–27%) in 40 samples available. Levels of residual disease in patients who subsequently lost their response were higher as compared to patients with continuous remission, but this difference was not significant (Table 4, Figure 5). Follow-up was performed for a median of 13 months (range 0–35) after first CCR. The best individual molecular response for all patients (n=41) was 0.086% (median; range 0–7.8); for CP patients (n=29) 0.089% (median; range 0–7.8), in AP (n=8) 0.20% (median; range 0–6.2) and in BC (n=4) 0.037% (median; range 0.013–0.71). Best responses in patients who subsequently relapsed were significantly higher as compared to patients with continuous CCR (P=0.0017; Table 4, Figure 6). In two patients, residual disease was undetectable: in a CP patient, BCR-ABL became intermittently undetectable 6 months after achieving CCR, in an AP patient BCR-ABL transcripts were undetectable on two consecutive PCR analyses 20 and 26 months after first detection of CCR. Levels of residual disease in patients with continuous response to the therapy gradually decreased after achieving CCR (P<0.0001; Table 3; Figure 5). Of 18 patients who achieved BCR-ABL/ABL ratios of 0.1%, nine relapsed (50%); in contrast, of the 23 patients who achieved BCR-ABL/ABL ratios of <0.1%, only two relapsed (8.7%, P=0.003).

Table 4 Residual disease in patients achieving a CCR by imatinib. Comparison of patients with loss of response vs patients with continuous response to imatinib at the time point of the first achieved CCR and at the time point of best molecular response (till the time point of relapse)
Figure 5
figure5

Kinetics of residual disease, expressed as the ratio BCR-ABL/ABL in PB, in patients with CR on imatinib therapy; dark symbols represent samples negative by Q-PCR, but positive by qualitative nested RT-PCR.

Figure 6
figure6

Best molecular response in PB during follow-up for 41 patients who achieved CCR. For the 11 patients who subsequently relapsed, the minimum ratio BCR-ABL/ABL in PB was significantly higher (P=0.0017) than in patients with ongoing response to therapy. The difference is even more pronounced in CP patients (open circles; P=0.0011), since two patients in advanced disease relapsed despite having achieved ratios BCR-ABL/ABL <0.1%.

Table 3 Residual disease measured as ratio BCR-ABL/ABL in PB according to time after achieving a complete cytogenetic remission (CCR) in 30 patients with continuous response to imatinib

Of 29 CP patients who achieved CCR, complete remission has been maintained in 23 cases, six patients have relapsed. The ratios BCR-ABL/ABL of patients who subsequently relapsed were not significantly different at the time of the first CCR, but significantly higher at the time of maximal response as compared to patients with continuous remission (P=0.0011; Table 4; Figure 6). All 16 patients with CP who achieved BCR-ABL/ABL ratios <0.1% are in continuous remission; in contrast, 6/13 patients (46%) with ratios BCR-ABL/ABL 0.1% have relapsed (P=0.0036). In CP patients, the only detectable difference between patients in continuous remission vs relapsing patients was the level of best response, while pretherapeutic prognostic parameters were not different (Table 5).

Table 5 Pretherapeutic parameters (median, range) of CP patients who subsequently achieved CCR on imatinib

Discussion

The aim of residual disease analysis is to enable a better assessment of the response of individual patients to treatment or to evaluate the efficacy of a particular treatment protocol on a group of patients.17,18,19 Cytogenetic banding analysis is still regarded the gold standard for defining the response of patients to treatment, but this technique has a routine sensitivity of about 1–5%, which is a particular drawback in patients with good response to the therapy.

Considering that patients with leukemia at presentation have a total burden of more than 1012 leukemic cells,20 patients without evidence of the Ph chromosome in the cytogenetical analysis still may harbor up to 1010 malignant cells. G-banding cytogenetical analysis provides information on only a relatively small number of cells making the detection of a low frequency of Ph+ cells problematic.21 The major advantage of conventional cytogenetics is the detection of other chromosomal aberrations, which may indicate an acceleration of the disease22 or clonal proliferation of Ph negative hematopoiesis.23,24,25

HM-FISH, based on improved culture techniques and computer-aided analysis of large numbers of metaphases markedly improves detection of residual disease, allows quantitative assessment of the proportion of Ph+ cells26 and has a higher sensitivity than chromosomal banding analysis.12 In our series, in patients with CCR on imatinib therapy, BCR-ABL-positive metaphases were detected by HM-FISH in 31% of the cases. A limitation of HM-FISH is, however, the inability of monitoring the nondividing Ph+ cells.26

IP-FISH for BCR and ABL does not depend on the cycling status of cells, but substantial levels of false-positive results make quantitative assessment of IP-FISH results in the setting of minimal residual disease analysis difficult.27,28 Owing to the high variation of BCR-ABL-negative lymphoid cells in the samples from imatinib-treated patients, IP-FISH results show a high degree of variation and do not provide any additional information for the management of patients with CCR achieved by imatinib. Correction of IP-FISH results for nonlymphoid cells only may improve the results,29 but is rather cumbersome and unprecise.

RT-PCR is by far the most sensitive method to detect residual disease in CML and able to detect a single leukemia cell in a background of 105–106 normal cells. In general, we observed a good correlation of HM-FISH results with Q-PCR results obtained from PB and BM. However, in 69% of CCR cases, HM-FISH was also negative and the only method capable to reveal and quantify residual disease was RT-PCR.

In an effort to improve the clinical relevance of RT-PCR techniques, several methods including the competitive RT-PCR and recently real-time RT-PCR procedures have been developed to quantify the level of residual disease in patients with CML.16,30,31,32,33,34,35,36,37 In this study, we used the LightCycler technology for detection and quantification of BCR-ABL transcripts using a previously described protocol, which enables to amplify less than 10 molecules per reaction and to detect one CML cell in 105 cells from healthy donors.15 A good correlation of cytogenetic response status and Q-PCR results after imatinib therapy has been demonstrated.38,39

In CCR patients, Q-PCR revealed BCR-ABL mRNA in 80% of cases in PB and 83% in BM even when HM-FISH was negative. For the majority of patients in CCR after imatinib therapy, qualitative-nested RT-PCR is of limited value in determining patients' response, since most patients remain positive with Q-PCR. However, nested RT-PCR is still of relevance in Q-PCR negative cases to reveal residual BCR-ABL transcripts below the detection level of Q-PCR in the individual sample.37 We observed concordant Q-PCR results in samples obtained from PB and BM. Thus, patients with good response to imatinib can be monitored using PB samples rather than BM, permitting more frequent and less invasive analysis.

Total ABL and G6PD mRNA transcripts were used as internal standards in order to standardize BCR-ABL mRNA levels for variability in RNA and cDNA quality. The good correlation of the individual ratios BCR-ABL/ABL and BCR-ABL/G6PD suggests that either gene may be used as internal standard. The advantage of using ABL is that the same pair of hybridization probes used for quantification of BCR-ABL can be applied. In case of RT-PCR negativity, the number of ABL or G6PD transcripts/volume cDNA reflects the quality of the sample and is therefore a measure for the power of the statement ‘undetectable BCR-ABL’. In our series, negative RT-PCR results were considered in the presence of >5 × 104 ABL transcripts/5 μl cDNA.

Undetectable BCR-ABL was rare after therapy with imatinib with current follow-up. Thus, median levels of residual disease were significantly higher than in CCR patients after allogeneic stem cell transplantation. Within 6 months after transplantation, about two-thirds of the patients become BCR-ABL negative.16,40 In contrast, levels of residual disease and proportion of patients with undetectable BCR-ABL in CCR to imatinib after IFN failure are similar to those in CCR to IFN10 considering the shorter treatment interval of imatinib (1.5 years) as compared to IFN (4 years).

Analyzing all samples investigated in nonrelapsing patients continuing imatinib therapy after CCR revealed that BCR-ABL levels decline over time. This suggests an ongoing process of quantitative disease depletion by imatinib. In some patients, however, cytogenetic or hematologic relapse occurs after CCR. The risk of relapse is significantly higher in advanced disease5,6,41 than in CP,4 but even in CP patients, resistance has been observed and may be correlated to the level of residual disease and duration of therapy.42 We 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. Relapse was rare in patients with ratios BCR-ABL/ABL of <0.1%.

None of the patients investigated stopped treatment after achievement of CCR. Thus, there is no information available whether remission could be maintained after withdrawal of imatinib in patients with low levels of residual disease.

We conclude, that residual disease can be detected by qualitative and quantitative RT-PCR in most patients treated with imatinib. Real-time Q-PCR is a reliable and sensitive method to monitor these patients during therapy. Our results demonstrate a greater sensitivity of nested RT-PCR than of Q-PCR. In CP patients, low levels of residual disease with ratios BCR-ABL/ABL <0.1% are associated with continuous remission, independent from pretherapeutic clinical parameters. Longer follow-up is required to determine the ability of imatinib to induce and maintain molecular remissions. Quantitative assessment of residual disease will certainly help to modify and individualize treatment over time. However, to reach this goal, standardized parameters to measure and express residual disease levels should be used within clinical trials. Therefore, an International Study on Standardization of BCR-ABL quantification has been launched recently, which will contribute to a harmonization in the calculation and expression of minimal residual disease parameters.19

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Acknowledgements

We are grateful to all colleagues and nursing staff from the referring centers for participating in this study. This work was supported by the Deutsche José-Carreras-Stiftung eV, the Forschungsfonds der Fakultät für Klinische Medizin, Mannheim and the competence network ‘Acute and chronic leukemias’, sponsored by the German Bundesministerium für Bildung und Forschung (Projektträger Gesundheitsforschung; DLR e.V.).

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Correspondence to A Hochhaus.

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Paschka, P., Müller, M., Merx, K. et al. Molecular monitoring of response to imatinib (Glivec®) in CML patients pretreated with interferon alpha. Low levels of residual disease are associated with continuous remission. Leukemia 17, 1687–1694 (2003). https://doi.org/10.1038/sj.leu.2403033

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Keywords

  • quantitative RT-PCR
  • chronic myelogenous leukemia
  • imatinib
  • minimal residual disease
  • FISH
  • molecular cytogenetics

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