Interphase fluorescence in situ hybridization (I-FISH) for the BCR-ABL translocation performed on peripheral blood (PB) white cells has been suggested as a surrogate for conventional bone marrow (BM) cytogenetics for monitoring patients with chronic myeloid leukemia (CML). I-FISH is faster, less costly, and does not require BM aspiration. For patients treated with interferon-alpha (IFN), a good correlation between the two methods has been demonstrated in several though not all studies. However, imatinib mesylate (STI571) has largely replaced IFN as the standard drug treatment for CML, raising the question if the results obtained in IFN-treated patients are applicable to patients on imatinib. We therefore compared the two methods in patients on imatinib and patients on other therapies, mainly IFN (collectively referred to as nonimatinib therapies). Our results demonstrate that the correlation between I-FISH and cytogenetics is much weaker in patients on imatinib than in patients on nonimatinib therapies. Correction of the I-FISH values for the proportion of lymphocytes barely improved the correlation, probably as a result of unpredictable proportions of Philadelphia-positive B cells. By contrast, I-FISH of PB neutrophils was much better correlated with BM cytogenetics. We conclude that I-FISH on unselected PB white cells is not suitable for monitoring patients on imatinib.
The molecular hallmark of chronic myeloid leukemia (CML) is the t(9;22)(q34;q11) (Philadelphia (Ph)) translocation, which is found in about 95% of all CML patients. As a result, a BCR-ABL fusion gene and protein are produced. The latter is a constitutively active tyrosine kinase that causes malignant transformation of a multipotent hemopoietic stem cell.1,2
Drug therapy of CML has traditionally relied on IFN as the only agent capable of producing long-term remissions, although only in a small subset of patients.3,4,5 The impressive rates of hematological and cytogenetic remissions seen with the Bcr-Abl-specific tyrosine kinase inhibitor imatinib have changed the therapeutic practice for patients who failed IFN.6,7 Based on vastly superior results in a randomized comparison with IFN/cytarabine, imatinib has now become the standard in newly diagnosed CML patients.8
The classical technique for diagnosis and follow-up of Ph-positive CML is G- or R-banding of BM metaphases. Disadvantages include high costs,9 the dependence on the growth of metaphases, and the fact that BM aspiration is an invasive and unpleasant procedure. Therefore, alternative methods like fluorescence in situ hybridization (FISH), reverse transcription-polymerase chain reaction (RT-PCR) or Southern blotting were evaluated as alternatives for monitoring.
Interphase FISH (I-FISH) to detect the BCR-ABL translocation in PB white cells has been compared to BM cytogenetics in a number of studies.10,11,12,13,14,15 Previous studies focused on patients treated with IFN-based drug therapy, allogeneic and autologous stem cell transplantation.10,13,16 While the correlation between I-FISH and BM metaphases is generally rather tight, there is considerable variation between studies, and there may be significant discrepancies between the two methods in individual patients. Most importantly, it is currently unknown if these results are applicable to patients on imatinib. In the present study, we compared I-FISH for BCR-ABL performed on PB leukocytes with BM cytogenetics in two cohorts of patients undergoing treatment with therapies other than imatinib and treatment with imatinib, respectively. Compared to patients on nonimatinib therapies, the correlation between PB FISH and BM cytogenetics was much weaker in patients on imatinib. Correction for lymphocytes (assessed by differential blood counts) did not significantly improve these results, while there was a much better correlation between BM cytogenetics and the Ph status of FACS-sorted PB neutrophils.
Materials and methods
Patients and samples (Table 1)
Paired BM and PB specimens were studied in 15 CML patients on various therapies other than imatinib (interferon-alpha (IFN) based, 10; hydroxyurea, 1; autologous stem cell transplantation followed by IFN, 2; allogeneic stem cell transplantation, 2) and 15 patients treated with imatinib. In the latter patients, PB leukocytes were also FACS sorted into neutrophils, monocytes, B- and T cells. Informed consent according to the Declaration of Helsinki was obtained in all cases.
Mononuclear cells were isolated by Ficoll–Hypaque density-gradient centrifugation. Neutrophils were separated based on size and granularity. Specific antibodies were used to separate monocytes (CD14), B- (CD19) and T cells (CD3). Phycoerythrin (PE)-conjugated anti-CD14 (MΦP9-PE) and anti-CD19 (J4.119-PE) antibodies were from Beckman-Coulter (Fullerton, CA, USA). Fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (SK7-FITC) was from Becton-Dickinson (Sunnyvale, CA, USA). Flow cytometry analysis and cell sorting were performed on a FACS Calibur (Becton Dickinson).
Preparation of cells for I-FISH
For prehybridization, PB specimens were incubated for 24 h in culture medium without cytokines (RPMI 1640, human AB serum, penicillin, streptomycin, L-glutamine). Next, colchicin was added, followed by another 3 h of incubation.
Preparation of cells for hypermetaphase FISH
BM aspirates were incubated for 24 h with colchicin in order to increase the number of metaphases for analysis.17
For detection of BCR-ABL, The LSI bcr-abl–DNA–ES probe (Vysis, Stuttgart, Germany) was used. Hybridization followed the manufacturer's protocol with slight modifications. FACS-sorted cells were immersed in 10 mM HCl/10% pepsin for 30 s at 37°C prior to hybridization. Signals were visualized under a Zeiss Axioskop microscope (Zeiss, Jena, Germany) using a FITC/Rhodamine dual band filter. A total of 25 metaphases or 500 interphase nuclei were analyzed in each sample if available. The cutoff for false-positive results is approximately 1% for unselected cells.18
All calculations were performed with the SPSS statistical package (SPSS GmbH, Munich, Germany). Categorical and noncategorical variables were compared by χ2 and Mann–Whitney U-test, respectively. Correlations were evaluated using Pearson's correlation coefficient.
The patient groups were comparable with respect to demographics and hematological parameters except that four patients in the imatinib group were in accelerated phase, whereas all other patients were in chronic phase (P<0. 05, Table 1). The median proportions of Ph-positive interphases in unselected PB white cells (43% with nonimatinib therapies, 40% with imatinib) and median Ph-positive BM metaphases (51 vs 52%) were also not different between the two groups.
Comparison between BM cytogenetics and I-FISH on unsorted PB white cells in patients treated with nonimatinib therapies
In all, 15 paired samples were available for analysis (Table 2). A median of 51.4% (range 0.6–100%) of BM metaphases and a median of 43.4% (range 0.5–82.7%) PB interphases were Ph positive. A strong correlation between the BM metaphases and PB interphases was observed (r=0.91, P<0.001, Figure 1a). However, there was a significant discordance in two patients (#3 and 9), where I-FISH detected a much lower proportion of BCR-ABL-positive cells.
Comparison between BM cytogenetics and PB I-FISH in patients on imatinib
In analogy to the patients on nonimatinib therapies, we examined BM and PB white cells in 15 patients on imatinib.
Unsorted white cells
In total, 13 paired samples were examined with a median of Ph-positive metaphases of 52% (range 8–100%) in BM and a median of Ph-positive interphases of 40% (range 2–80.8%) in PB. The correlation between BM cytogenetics and PB leukocytes was much weaker than in patients on nonimatinib therapies (r=0.6428, P=0.013, Figure 1b), with major discrepancies observed in a number of cases (Table 3). In CML, most B-lymphocytes and almost all T-lymphocytes are reportedly Ph negative.19 In order to correct for this, the results obtained from PB were corrected by subtracting the percentage of lymphocytes as given by the differential counts from the same samples. This approach has been used in several studies in patients on nonimatinib treatment.12,20,21,22 However, the correction led only to a very moderate improvement of the correlation coefficient to 0.6817 (P=0.005).
The weak correlation suggested that imatinib might preferentially eliminate certain subpopulations from the PB, while sparing others. We therefore separated the white blood cells from patients on imatinib according to their immunophenotype. False-positive cutoffs for FACS-sorted cells were established by analyzing 500 cells from each of five individuals with morphologically normal bone marrow. The false-positive cutoffs (mean+2 s.d.) were 0.2% for FACS-sorted neutrophils and T cells and 0.4% for monocytes and B cells. FACS-sorted cells from the 15 patients studied above were analyzed (Table 3).
The median Ph-positive BM metaphases were 40% (range 8–100%). In the PB, the median percentage of Ph-positive interphases was 35.2% (range 1.6–99.4%) in neutrophils, 24.8% (range 0–97.2%) in monocytes, 7.8% (range 0–38.2%) in B cells and 0.4% (range 0–7.4%) in T cells. As expected, there was no correlation between BM and PB for B cells (r=0.3772, P=0.15) and T cells (r=0.16, P=0.56). In contrast, there was a very good correlation between BM and PB neutrophils (r=0.89, P<0.001, Figure 2a), and reasonably good correlation between BM and PB monocytes (r=0.76, P=0.001, Figure 2b).
Several diagnostic methods are available to detect the BCR-ABL rearrangement at different biological levels. Conventional cytogenetics has been the gold standard for decades and is well validated in patients on IFN.3,4,23 Limitations of conventional karyotyping include its high costs, a considerable rate of failure due to lack of metaphases, and the need for bone marrow aspiration, an invasive and painful procedure. Detection of BCR-ABL in PB leukocytes by I-FISH has therefore been proposed as an alternative to conventional cytogenetics. In most direct comparisons, the two methods showed a good correlation, however, with considerable differences between individual studies.10,11,12,13,15,16 For example, Le Gouill et al observed an extremely good correlation (r=0.97) between I-FISH of PB and BM cytogenetics, while Lesser et al found a significant but considerably weaker correlation (r=0.78). In addition, individual cases may exhibit significant discrepancies.15,24 These discrepancies may be related not only to the analysis of interphase vs metaphase cells but also to the fact that in some patients, the proportion of clonogenic Ph-negative cells is higher in the PB than the BM.25 Our data show a good correlation between BM metaphases and PB interphases in unselected white blood cells, with a correlation coefficient of 0.91, exactly as recently reported by Schoch et al.13 Nonetheless, there were large discrepancies in some patients (#3 and 9, Table 2). Generally, PB IP-FISH results tended to be lower than BM metaphases, in accordance with other reports.13,15 This indicates that I-FISH may lead to a bias toward better responses.
In contrast, the correlation found in imatinib-treated patients, although significant, was much weaker (r=0.64, P=0.013), rendering I-FISH of total white blood cells of little use for the follow-up of individual patients. It has been proposed that the results of PB I-FISH should be corrected by subtraction of the lymphocytes, which are not usually part of the leukemic clone. However, correction for the lymphocytes (as assessed by standard differential blood count) only marginally improved the correlation coefficient. However, this shortcoming could be overcome by analyzing FACS-sorted PB neutrophils, for which again a good correlation with BM cytogenetics was found (r=0.89, P<0.001). Importantly, in our study, neutrophils were selected by flow cytometry based on their size and forward scatter distribution only, without the use of specific antibodies. Such separation is fast, inexpensive and thus useful for routine clinical applications in accordingly equipped laboratories. However, there were still significant discrepancies between the BM and PB neutrophils in several patients on imatinib (#7, 10, 13, Table 3). It has been suggested that BCR-ABL-negative neutrophils are preferentially released into the PB in some patients,24 but it remains unclear why this occurs in some patients while sparing others. Analysis of a larger cohort of patients will be necessary to determine the exact incidence of such large discrepancies.
A weaker correlation was also found for monocytes (r=0.76), making them a less suitable choice for clinical applications; moreover, their separation by FACS sorting with the use of antibodies is much more laborious and expensive than those of neutrophils. In keeping with published data,19,26,27 PB B cells were largely, and T cells almost exclusively Ph negative. However, there were large variations between individual patients with respect to the Ph status of B cells (Table 2). Thus, B cells may contribute to the Ph-positive cells in the PB in an unpredictable manner, which partially explains the large variations among individual patients. The apparent difference between patients on imatinib and patients on other therapies (mainly IFN) suggests that both agents may have a differential effect on the various populations of white cells. Unfortunately, it is no longer possible to test this, since most patients on IFN, unless in complete cytogenetic response, have been switched to imatinib.
Quantitative RT-PCR (Q-PCR) for the detection of BCR-ABL transcripts has emerged as yet another alternative to BM cytogenetics. Q-PCR is of established value in monitoring patients in complete cytogenetic response after allogeneic BMT28 or on IFN therapy,29 and is also widely used to follow patients on imatinib,30,31,32,33 where it may aid prognostication. A recent comparison found a tight correlation between Q-PCR, hypermetaphase FISH, I-FISH and conventional cytogenetics in the BM.13 However, the results await confirmation in controlled trials with survival or progression-free survival as end points.
Both Q-PCR and I-FISH measure static parameters, the percentage of cells with a BCR-ABL rearrangement and the average steady-state level of BCR-ABL mRNA, respectively. By contrast, metaphase karyotyping incorporates a functional test, namely the capacity of cells to proliferate in defined conditions, and this may aid its prognostic power. Achievement of a major cytogenetic response at 3 months predicts progression-free survival in patients treated with imatinib in chronic and accelerated phase.6,34 In addition, only conventional cytogenetics is capable of detecting additional chromosomal abnormalities in Ph-positive or Ph-negative cells. In the former cells, this appears to indicate disease progression;35 in the latter cells, it may be associated with a myelodysplastic syndrome.36 In both scenarios, conventional karyotyping provides vital information.
In summary, I-FISH of unselected white blood cells is not useful for monitoring of patients on imatinib. Analysis of neutrophils seems to improve the results and may be useful if a FACS sorter is available. It is likely that in the future, the various diagnostic tests will be used complementarily. Until validation in controlled trials with clinical end points is available, conventional karyotyping should remain the mainstay for monitoring CML patients.
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We thank Dr Haifa-Kathrin Al-Ali for dedicated patient care. We are grateful to Ms Christel Müller, Ms Christina Franke and Ms Daniela Klug for excellent technical assistance.
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Reinhold, U., Hennig, E., Leiblein, S. et al. FISH for BCR-ABL on interphases of peripheral blood neutrophils but not of unselected white cells correlates with bone marrow cytogenetics in CML patients treated with imatinib. Leukemia 17, 1925–1929 (2003). https://doi.org/10.1038/sj.leu.2403077
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