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© 2000 American Society for Clinical Oncology Fluorescence In Situ Hybridization on Peripheral-Blood Specimens Is a Reliable Method to Evaluate Cytogenetic Response in Chronic Myeloid LeukemiaFrom the Laboratory of Hematology and Clinical Hematology Department, University Hospital, Nantes, France. Address reprint requests to Hervé Avet-Loiseau, MD, Laboratoire d’Hématologie, Institut de Biologie, 44093 Nantes Cedex, France; email havetloiseau{at}chu-nantes.fr
PURPOSE: To evaluate the usefulness of fluorescence in situ hybridization (FISH) on peripheral-blood specimens to evaluate the cytogenetic response to treatment in patients with chronic myeloid leukemia (CML). PATIENTS AND METHODS: In a first attempt, we analyzed 62 bone marrow specimens using interphase FISH and compared the results with those of conventional cytogenetics. In a second step, we analyzed 60 paired sets of bone marrow and peripheral-blood specimens with interphase FISH. RESULTS: The results of interphase FISH agreed with conventional cytogenetics on bone marrow for most patients, and only minor differences were found (r = .98). The comparison of interphase FISH on bone marrow versus peripheral-blood specimens showed a strong correlation between these two specimen sources (r = .97). CONCLUSION: Our results confirmed that FISH is a sensitive technique for the evaluation of response to treatment in patients with CML. Moreover, our study suggests that follow-up of cytogenetic response to therapy can be evaluated on peripheral-blood specimens, thus enabling an easier and more frequent evaluation of patients. The next step will be to evaluate this technique in a large prospective trial to define the prognostic value of complete remissions evaluated by FISH.
TRANSLOCATION t(9;22)(q34;q11) is the hallmark of chronic myeloid leukemia (CML).1 Whereas the translocation is observed in up to 100% of metaphase cells at diagnosis, the percentage of Philadelphia (Ph)-positive cells decreases with therapy, especially with interferon.2-6 Thus, response to treatment can be evaluated by following the percentage of Ph-positive metaphase cells. Many clinical trials have demonstrated the usefulness of this therapy response monitoring, which shows that the best outcome is enjoyed by patients with the best response, ie, patients attaining the lowest percentage of Ph-positive cells.2-6 According to the percentage of residual Ph-positive cells, patients are classified into the following four response groups: (1) patients without cytogenetic response (> 90% Ph-positive cells); (2) patients with minor cytogenetic response (35% to 90% Ph-positive cells); (3) patients with major cytogenetic response (1% to 34% Ph-positive cells); and (4) patients with complete cytogenetic response (0% Ph-positive cells).5 In clinical practice, cytogenetic evaluation of response to interferon is performed every 3 to 6 months on bone marrow cells. Although cytogenetics is an effective technique to evaluate response to therapy, it displays several limitations. First of all, it is a time (and money) consuming technique. Secondly, because interferon induces marrow hypoplasia, the number of assessable metaphases might be extremely low, thus preventing the determination of an accurate percentage of residual Ph-positive cells. Therefore, evaluation of the cytogenetic response might be inadequate because of this low number of analyzable metaphases. Finally, cytogenetics requires bone marrow harvesting, which prevents repetitive analyses. Consequently, a technique enabling response evaluation on interphase blood cells would be highly desirable. Fluorescence in situ hybridization (FISH) enables a rapid detection of chromosomal rearrangements, even on interphase cells, and thus, avoids the requirement of metaphase obtention. Several studies using FISH for detection of the BCR-ABL fusion have been reported.7-10 However, these studies were limited by the sensitivity of the technique on interphase cells. Because probes used in these studies detected a single fusion on the Ph chromosome, false positivity might be as high as 10%, preventing the use of this technique in patients with a better response. Recent commercially available probes improved the sensitivity of the technique by including either a second fusion signal on the derivative chromosome 9 or a split of the ABL probe. Recent reports demonstrated that these new probes allow the detection of residual disease at the 1% level.11,12 To evaluate the usefulness and accuracy of FISH on peripheral-blood specimens for the evaluation of cytogenetic response in CML, we conducted the following three-step study using a commercially available probe: (1) we determined the false-positive rate of interphase FISH on marrow samples of patients with other myeloid malignancies and on peripheral-blood samples of healthy individuals; (2) we compared conventional cytogenetic evaluation with interphase FISH on bone marrow samples; and (3) we compared interphase FISH on simultaneously harvested paired sets of marrow and blood specimens from CML patients.
Patients To determine the FISH experiment positivity cutoff, we selected 14 patients with myeloid malignancies (nine patients with acute myeloid leukemia and five patients with myeloproliferative syndrome but lacking any BCR-ABL rearrangement). To obtain an unambiguous cutoff, control specimens were blindly analyzed by being mixed with specimens from CML patients in complete cytogenetic response. For the second part of the study (ie, comparison of cytogenetics and interphase FISH on bone marrow), we selected 79 samples obtained from 32 patients that were representative of the different cytogenetic response groups. Of these patients, 26 were receiving interferon-based front-line therapy, five patients were autografted for high-risk or accelerating CML, and one patient was in relapse after allogeneic transplant. Finally, the third part of the study (ie, comparison of interphase FISH on marrow and blood specimens) was performed in 60 specimens from 48 patients with CML at different stages of the disease. These 48 patients were either under front-line interferon therapy (31 patients), analyzed after unmanipulated autologous bone marrow transplantation for high-risk or accelerating CML (nine patients), or in relapse after allogeneic bone marrow transplantation (eight patients).
Cytogenetic and FISH Experiments
Sensitivity of FISH on Marrow and Blood Specimens To define the positivity cutoff level of the probe, we analyzed 14 bone marrow samples from patients with acute myelogenous leukemia (nine patients) or Ph-negative myeloproliferative syndrome (five patients). To have an objective definition of this cutoff, we blindly added specimens from patients with CML in complete cytogenetic response. Cells lacking BCR-ABL rearrangement displayed two red (R) signals, corresponding to the ABL probe, and two green (G) signals, corresponding to the BCR probe (thus, the normal GGRR configuration). Cells with a BCR-ABL rearrangement, ie, Ph-positive cells, displayed two red signals (ie, both normal and derivative chromosomes 9), one green signal (ie, normal chromosome 22), and one yellow (Y) signal, corresponding to the fusion of the ABL and BCR probes on the Ph chromosome. Therefore, Ph-positive configuration was RRGY. In each case, 300 nuclei were scored, and, consequently, positive cutoff values have been determined on a total of 4,200 cells. Whereas the mean percentage of Ph-positive cells in CML patients in complete cytogenetic response was 1% (SD = 0.4%), in controls, it was 0.17% (SD = 0.33%; range, 0% to 3% per 300 nuclei). Thus, the positivity cutoff for bone marrow was fixed at 1.2% (mean + 3 SD). Finally, to address the issue of inter- and intraobserver variability, we randomly selected 20 samples, which were blindly analyzed, first twice by one observer and then by two different observers. The most significant difference between the first observer and the second two was 3% in a patient with a high percentage of Ph-positive cells (76% and 79%, respectively). Thus, the method is highly reproducible. We found in a few CML patients an abnormally high percentage of cells with an RGY configuration. To analyze this configuration, we observed metaphase cells. Surprisingly, these patients presented a Ph chromosome bearing a fusion (Y signal) but lacked a residual signal on the derivative chromosome 9. We determined the positivity cutoff for this minor configuration (ie, RGY) on controls. This configuration was observed in 3.52% of cells (SD = 2.25%); thus, the cutoff was fixed at 10.5% (mean + 3 SD). Because of this low sensitivity level, patients with this configuration were excluded from the analysis. Finally, to define the cutoff for blood specimens, we analyzed 10 peripheral-blood samples from healthy volunteers, and 400 nuclei for each control were scored. These normal controls were also mixed with slides from patients in complete cytogenetic response. The mean percentage of Ph-positive nuclei was 0.3% (SD = 0.27%). Thus, the positivity cutoff on blood was fixed at 1.2%.
Comparison Between Cytogenetics and Interphase FISH on Bone Marrow Specimens
In 17 specimens (from four patients), we detected few cells with RRGY configuration but a high number of cells with an RGY configuration. This configuration corresponded to cells with a Ph chromosome but no residual signal on the derivative chromosome 9. In 12 cases, the percentage of positive cells (RGY) was below the cutoff value (10.5%) and, therefore, not assessable for comparison with cytogenetics. In the other five cases, the percentages of cells presenting the RGY configuration by FISH were 20%, 26%, 40%, 93%, and 94.5% versus 18%, 10%, 56%, 87%, and 94% by cytogenetics. The correlation in these cases was good. To evaluate the incidence of this configuration, we analyzed 61 samples corresponding to CML patients at diagnosis. We found such a configuration in eight (13%) of 61 patients, including three patients displaying both configurations, ie, RRGY and RGY. Of note, subsequent analyses revealed a progression of percentage of cells with the RGY configuration in two patients and stability in one.
Comparison Between Interphase FISH on Bone Marrow Cells and on Peripheral-Blood Cells
Several recent studies have demonstrated the accuracy of FISH in the analysis of BCR-ABL rearrangements in CML patients.11-15 These reports have demonstrated that this technique was able to detect all types of BCR-ABL fusions, even those present in patients with a so-called masked Ph chromosome. Moreover, it has been shown that analysis can be performed on interphase nuclei. Most of all, recent publications describe new probes using different strategies, enabling a sensitivity level below the 1% threshold.12 In this study, we performed the following three-step analysis: (1) determination of the sensitivity threshold of interphase FISH; (2) comparison of cytogenetics and interphase FISH on bone marrow specimens; and (3) comparison of interphase FISH on bone marrow and peripheral-blood specimens for measuring the residual disease. Using a commercially available probe, we have analyzed 62 bone marrow samples corresponding to different cytogenetic response stages. Despite few minor differences, we found a highly significant correlation between cytogenetic and FISH results. Interestingly, in six of 15 patients in complete cytogenetic response, we observed residual Ph-positive cells with FISH, whereas the opposite situation was found in only one of 10 patients. These seven patients with slightly discordant results were all patients with a small residual Ph-positive cell population. In six of these seven cases, the subsequent cytogenetic analysis confirmed the FISH results (five cases with cytogenetic relapse and one case with conversion to complete cytogenetic remission). However, despite these minor discrepancies, cytogenetics and interphase FISH on bone marrow specimens seemed in good agreement (r = .98). Moreover, we had the opportunity to analyze one patient with Ph-negative CML. Using FISH, we demonstrated that this patient had a masked Ph chromosome and that the monitoring of response to therapy was feasible by FISH. Finally, we showed that the technique is highly reproducible because intra- and interobserver variability was below 3% in patients with residual Ph-positive cells. In the first part of our analysis, we isolated 17 samples with an unusual RGY configuration. Because the percentage of cells with this configuration was clearly above the cutoff value, we decided to analyze a cohort of patients with CML at diagnosis. We had a frozen sample available for 61 patients at diagnosis. Among them, we observed this specific configuration in eight patients; in five patients, all the metaphase cells displayed this RGY disposition, whereas in three patients, we found RGY and RRGY cells. In all eight patients, this configuration corresponded to the absence of the residual signal on the derivative chromosome 9. Two hypotheses could be proposed, ie, either a 9q34 breakpoint was located upstream of the ABL gene (on the centromeric side), or there was a deletion of the 5' part of the ABL gene associated with the translocation. Studies to elucidate this question are currently in progress. However, because this specific configuration is associated with a lower sensitivity in the detection of Ph-positive interphase cells, we analyzed samples obtained at diagnosis for each patient to determine his own configuration. All the patients with this RGY configuration and analyzed under therapy displayed it at diagnosis. Thus, this specific abnormality does not seem to be acquired during evolution. However, because it is associated with a lower sensitivity, these patients have to be diagnosed at diagnosis to propose the appropriate method for evaluation of response. Because FISH results were highly correlated with cytogenetics on bone marrow, we moved on to the analysis of peripheral-blood specimens. We analyzed 60 paired sets of bone marrow and peripheral-blood specimens, collected at the same time for patients with CML and various cytogenetic responses. Comparable results were obtained, with an excellent correlation (r = .97). Thus, based on this large scale study, cytogenetic response might reliably be monitored by analyzing interphase peripheral-blood cells. This study confirms in a larger cohort three other reports that suggested the feasibility of interphase FISH on peripheral-blood specimens to monitor cytogenetic response.12,15,16 Thus, evaluation of response to therapy could be performed directly on peripheral-blood specimens by interphase FISH. This strategy presents two main advantages; it does not require bone marrow harvesting, and it is more sensitive because many more cells might be analyzed. Moreover, this strategy is cost-effective. Most (if not all) cytogenetic laboratories are equipped with fluorescence microscopes. Thus, the only extra cost is the probe (approximately $40 per patient). However, because this technique on blood does not require any culture, time and money are spared. Overall, the analysis time is much shorter with interphase FISH. Because the duration of analysis using conventional cytogenetics is highly dependent on the number of analyzable metaphases, it is extremely difficult to calculate a mean time (and cost). Nevertheless, even in specimens with a high number of metaphases, FISH analysis is quicker than cytogenetic examination. We propose the following strategy to monitor the cytogenetic response: interphase FISH evaluation on peripheral blood and classical cytogenetic examination if Ph-positive cell percentage increases. This strategy would permit a more careful follow-up of patients by repeating analyses. Finally, is this technique usable to assess minimal residual disease? In other words, what is the lowest level of Ph-positive cells detectable using FISH? Recent reports claimed that this threshold is far below the 1% level. To answer this important question, we performed a double study on both bone marrow and peripheral-blood specimens. To obtain an unambiguous threshold, we blindly analyzed marrow and blood specimens without BCR-ABL rearrangements, mixed with samples corresponding to patients in complete remission or with less than 5% Ph-positive cells. We think that this kind of analysis is the only way to unambiguously assess the positivity cutoff. If only normal samples are analyzed, observers might be influenced by the previous knowledge of the cell status and, thus, may underestimate the positivity threshold. With this strategy, we found similar thresholds with bone marrow and peripheral blood, ie, less than 1.5% (0.3% when negative samples were not blindly analyzed). This cutoff is similar to that of cytogenetics when analyzing 50 metaphase cells. Thus, FISH does not permit a quantitative evaluation of minimal residual disease in patients in complete cytogenetic response. For this category of patients (10% to 30% of patients2-5,17), more sensitive quantitative methods (ie, quantitative reverse transcriptase polymerase chain reaction) are necessary. Interphase FISH will not be adequate for a second category of patients, ie, those with the RGY configuration, who represent roughly 15% of patients. Actually, the positivity threshold for these patients is close to 10% and, thus, too high for a sensitive evaluation of response in patients who achieve a major cytogenetic response. Thus, interphase FISH will be an adequate method to quantify response to therapy for at least 85% of patients, with the classical RRGY configuration. This study clearly showed that interphase FISH on blood specimens is highly correlated with cytogenetics. One possible limitation would be to miss a clonal evolution, ie, apparition of secondary cytogenetic changes, usually associated with disease acceleration. However, in our experience, this situation is usually associated with an increase in the percentage of Ph-positive cells. Thus, any increase of this percentage should imply a classical cytogenetic evaluation of bone marrow cells. The next step would be to evaluate this technique in a large prospective trial to define the prognostic value of complete remissions evaluated by FISH.
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Copyright © 2000 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
