Monitoring of minimal residual disease in chronic myeloid leukemia

Hematol Oncol Clin N Am 18 (2004) 657 – 670

Monitoring of minimal residual disease in chronic myeloid leukemia Stefan Faderl, MDa,*, Andreas Hochhaus MDb, Timothy Hughes, MDc a

Department of Leukemia, The University of Texas M.D. Anderson Cancer Center, Box 428, 1515 Holcombe Bouelvard, Houston, TX 77030, USA b III. Medizinische Klinik, Fakulta¨t fu¨r Klinische Medizin Mannheim der Universita¨t Heidelberg, Wiesbadener Strasse 7-11, 68305 Mannheim, Germany c Division of Haematology, Institute of Medical and Veterinary Science, Frome Road, Adelaide 5000, Australia

Chronic myeloid leukemia (CML) has become a paradigm in cancer medicine for how advances in understanding of the biology of the disease can be translated into the development of targets for therapy and how these same targets can be used effectively as molecular markers in the diagnosis and monitoring of the response of patients on therapy. Traditional response criteria (based on morphologic assessment of marrow and blood slides) and metaphase cytogenetics have proved useful in the initial prognostication of patients. Even patients in complete cytogenetic remission, however, still may harbor disease at levels undetectable with these methods yet sufficient to cause disease relapse. The degree to which the levels of residual disease can be reduced during therapy, therefore, has become an increasingly recognized objective of clinical trials in CML, assuming that reduction of this tumor load is of prognostic relevance. Two developments in CML are now converging to address this objective: (1) a shift from using qualitative polymerase chain reaction (PCR) assays toward real-time quantitative (RQ) techniques that allow monitoring of the kinetics of residual disease over time; and (2) three therapeutic scenarios (stem cell transplantation [SCT], interferon-a [IFN-a], and imatinib mesylate) in which the clinical value of these assays is currently being tested. As important as PCR testing of minimal residual disease has become, any appreciation of the significance of residual disease also has to be viewed in the context of the biology of the disease and its particular therapeutic approach. The ultimate goals remain uncontested: (1) to identify at

* Corresponding author. E-mail address: [email protected] (S. Faderl). 0889-8588/04/$ – see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.hoc.2004.03.010

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Table 1 Assays in the diagnosis and monitoring of patients who have chronic myeloid leukemia Parameter

Cytogenetics

I-FISH

H-FISH

D-FISH

Q-PCR

Number of cells analyzed Sensitivity Time of analysis Detection of clonal evolution False-positive results (%) Sample source

15 – 25

100 – 200

100 – 1000

100 – 200

10,000 – 1,000,000

10 1 – 10 12 h Yes

2

10 1 – 10 1h No

2

10 2 – 10 12 h No

4

10 1 – 10 1h No

3

10 4 – 10 6–8 h No

NA

5 – 10

NA

1–5

NA

BM

PB/BM

BM

PB/BM

PB/BM

6

Abbreviations: BM, bone marrow; D-FISH, double-probe FISH; FISH, fluorescence in situ hybridization; H-FISH, hypermetaphase FISH; I-FISH, interphase FISH; NA, not applicable; PB, peripheral blood; Q-PCR, quantitative PCR.

the earliest possible time point the patients likely to relapse, (2) to optimize patient therapy in a risk-adapted manner, and (3) to improve survival. This article primarily focuses on the use of PCR to monitor residual disease and the significance of PCR for patients treated with SCT, IFN-a, and imatinib.

Methods of detection of BCR-ABL –positive cells The Philadelphia chromosome (Ph) is a shortened chromosome 22 that results from a reciprocal translocation of the long arms of chromosomes 9 and 22, a translocation referred to as t(9;22)(q34;q11). Through this process, a segment from the ABL gene on 9q34 is transposed into the BCR gene on 22q11. The resulting BCR-ABL gene is then transcribed into a chimeric mRNA and eventually translated into fusion proteins of varying size (p190BCR-ABL, p210BCR-ABL, p230BCR-ABL) according to the exact breakpoint location of the genes involved [1]. Hence, it is possible to detect evidence of the BCR-ABL translocation at different levels of this process with varying sensitivities, depending on the technique that is applied (Table 1). Conventional cytogenetic analysis Cytogenetic analysis remains the ‘‘gold standard’’ for the diagnosis of CML. Its major advantage lies in its strength as a screening procedure for additional chromosomal abnormalities that allow early detection of clonal evolution as a marker of disease acceleration. On the other hand, cytogenetic analysis is labor intensive and time-consuming. It is further limited by the need to culture mitotic cells for analysis and the small number of metaphases (20 – 25) examined. Whereas the Ph is detectable in more than 90% of patients with CML at diagnosis, among the remaining 10%, about half show a normal chromosome 22 but will have molecular evidence of the BCR-ABL translocation. Although

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cytogenetic analysis is of limited value in the monitoring of residual disease because its sensitivity of 1% to 5% is too low, it is still superior to morphologic assessment of marrow smears.

Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) is performed by cohybridization of a BCR and ABL probe to denatured metaphase chromosomes or interphase FISH (I-FISH) nuclei. In normal cells, two red and green signals indicate the location of the normal ABL and BCR genes, respectively. In BCR-ABL –positive cells, a single red and green signal point toward the normal ABL and BCR gene, whereas the fusion of the red and green signal, frequently detected as yellow fluorescence, indicates the BCR-ABL fusion product. FISH analysis is rapid and allows evaluation of 100 to 2000 cells (in the case of hypermetaphase FISH [H-FISH]) [2– 4]. At that level of analysis, the sensitivity is sufficiently high (up to 10 3) for quantification of residual disease even in patients who have major and complete cytogenetic responses. Limitations of FISH lie in its potential for falsenegative and false-positive results, depending on the specific probes, culture conditions, and detection systems used. I-FISH allows direct visualization of the Ph translocation and the cryptic BCR-ABL rearrangements in interphase nuclei and metaphase cells; however, its high false-positive rate (up to 10%, especially at low Ph-positive percentage values) and its insufficient sensitivity make it unsuitable for analysis of minimal residual disease. Although double-color FISH has reduced the false-positive rate associated with I-FISH and, like I-FISH, can be used to evaluate blood and marrow samples with good correlation between the sample sources [5], its sensitivity is still suboptimal and the same limitations with regard to residual disease detection prevail. Furthermore, double-color FISH is not applicable to patients with 9q+ deletions. H-FISH combines a modified preparation of metaphases with FISH. Its advantages include higher sensitivity due to the higher number of metaphases analyzed, the lack of false-positive results, and more reliable quantification. Therefore, only H-FISH is of any benefit for analysis of minimal residual disease [6]. Reverse transcriptase polymerase chain reaction A patient who meets all of the criteria for a clinical and hematologic complete remission still may harbor up to 109 to 1010 residual leukemia cells that remain below the threshold of detection of morphologic assessment, conventional cytogenetic analysis, or FISH. The criteria for clinical remission, therefore, are somewhat arbitrary and, in reality, do not reflect a leukemia-free disease status. Rather, this residual pool of leukemic cells may result in disease recurrence. Nucleic acid amplification by PCR undoubtedly is the most sensitive assay to detect and monitor residual disease. PCR can detect 1 BCR-ABL – positive

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cell in a background of 104 to 106 normal cells and, thus, is at least up to four orders of magnitude more sensitive than any of the other assays. As with other chromosomal translocations, the breakpoints in CML are distant from each other at the DNA level but in much closer proximity after the BCR-ABL gene has been transcribed into RNA. Therefore, amplification of the RNA product through reverse transcription (RT)-PCR has become the procedure of choice [7]. The initial results of PCR testing for residual disease in patients with CML following SCT, albeit interesting, were inconsistent and conflicting, pointing to drawbacks of the PCR technique in generating false-positive or false-negative results [8 – 11]. The last decade, however, has witnessed substantial improvements, facilitating the use of PCR in clinical practice. Using housekeeping genes as controls (ABL, BCR, G6PD, and others) to monitor quality and quantity of mRNA and cDNA integrity, standardizing the methodology, and employing strict precautions to avoid contamination have helped to enhance the specificity of PCR [12]. Nested RT-PCR has increased the sensitivity of the assay by 2 to 3 logs by performing two consecutive PCR steps using a new set of primers internal to those used in the first round of amplification [13]. Qualitative PCR (ie, the assessment of the mere presence or absence of the BCR-ABL product by PCR) has been of very limited value for clinical use and is poorly suited to establish the kinetics of minimal residual disease over time [14]. One of the most significant steps in the development of PCR over the last few years, therefore, has been the application of quantitative assays. During competitive PCR, a synthetic competitor gene that serves as an internal control is coamplified at serial dilutions with the target gene [15,16]. The amount of BCRABL is determined by defining the concentration of the competitor gene at which equivalence to the concentration of the target gene is achieved. Although competitive PCR methods have generated useful results, the technique is complex and not always accurate and the results are not easily reproducible among different centers. RQ-PCR (Taqman and LightCycler systems) not only has simplified quantification of the residual disease markers but also has contributed to increased precision and accuracy of the PCR measurements, making RQ-PCR the current standard for PCR quantification [17 – 19]. During RQ-PCR, the accumulation of amplicons is detected by laser excitation of fluorochromes that are labeled to sequence-specific probes in addition to the PCR primers. During successive amplification cycles, the polymerase advances along the DNA strand. A laser that assays the PCR reaction detects the liberated probe, and the accumulation of the fluorescence intensity is recorded in ‘‘real time’’ for each PCR cycle. As for the LightCycler, the juxtaposition of two hybridization probes leads to the emission of fluorescence. RQ-PCR is rapid, time efficient, and cost effective. Furthermore, it has made PCR quantification accessible to more centers and, at the same time, facilitated interlaboratory standardization of methodology. It is hoped that RQ-PCR will make PCR results among different centers more reproducible and, thus, make comparisons between multicenter studies possible.

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Monitoring of minimal residual disease Allogeneic stem cell transplantation Monitoring residual disease following SCT, irrespective of the type of assay used (ie, qualitative versus quantitative PCR), has brought to light some general principles that favor the use of PCR techniques in predicting relapse. Whereas minimal residual disease can be detected in about 20% to 30% of patients following allogeneic SCT, the association between the presence of residual disease and relapse is highly dependent on two factors: time from transplant and type of transplant [20]. In a study of 364 patients using qualitative nested PCR, detection of the BCR-ABL transcript at 3 to 6 months following SCT had no impact on probability of relapse and prognosis [21]. During the window of 6 to 12 months following therapy, however, persistent PCR positivity for residual disease was associated with a higher risk of relapse (42% versus 3% in PCR-negative patients, P < 0.0001) and inferior survival (74% versus 83%, P = 0.002). The relative risk of relapse for PCR-positive patients at 6 to 12 months after SCT was approximately 26%. These results have been confirmed by other transplant groups. Detection of residual disease up to 6 months after transplantation is common, but about two-thirds of these patients will have no detectable residual leukemia with longer observation [16,22,23]. The relative insignificance of residual disease at the earlier stages of therapy highlights other important aspects of residual disease. First, a substantial contribution to the success of transplantation stems from the graft-versus-leukemia effect [24,25]. Assuming that the persistent BCR-ABL –positive cells reflect truly clonogenic cells that have survived the assault of the conditioning regimen, these cells may not yet have been eliminated by the ongoing effects of graft versus leukemia. Many, if not most, of these early surviving cells, therefore, still may be eliminated as a consequence of the stimulation of the host’s immune system by the donor-derived cells. On the other hand, cells that survive beyond that point may constitute a pool of residual cells from which relapse can occur. Two observations underscore the significance of the graft-versus-leukemia effect in this context. Although the frequency of the rate of detection of residual disease after SCT (25%) is equal, the rate of relapse in patients with residual disease is much higher when the donor is related than when the donor is unrelated (60% versus 10%) [21]. Likewise, a high risk of relapse is associated with the detection of BCR-ABL transcripts in recipients of T-cell –depleted transplants, which may be detected in up to 80% of these patients [24,26]. Persistent PCR negativity beyond 1 year after SCT has been associated with a good prognosis [27]. Residual disease kinetics and the significance of detection at different time points differ substantially. As much as qualitative PCR measurements have generated insights into the importance of residual disease following SCT, qualitative PCR results, especially from single time points, are not reliable in predicting the risk of relapse of individual patients over time and certainly are

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not capable of capturing the kinetics of residual disease throughout the posttransplant course of observation. Quantitative PCR is able to establish a pattern of rising, falling, or stable levels of residual disease. Olavarria et al [28] used quantitative RT-PCR to correlate presence or absence of residual disease early after SCT (3 –5 months) with outcome. Among 138 CML patients, PCR results were classified as negative, low positive (BCR-ABL transcripts <100/mg RNA or a BCR-ABL/ABL ratio <0.02%, see later discussion), or high positive (exceeding the previously mentioned thresholds). The cumulative incidences of relapse at 3 years were 16.7%, 42.9%, and 86.4%, respectively (P = 0.0001), thus establishing a striking correlation between BCR-ABL levels and risk of early relapse following SCT. The obvious corollary of trying to identify transplant patients who are at high risk of relapse is to intervene early with alternative therapies to prevent disease recurrence before it happens; therefore, a valid definition of molecular relapse is essential. The group at Hammersmith Hospital in the United Kingdom established a set of criteria for molecular relapse [7,28,29]. It should be emphasized that these criteria are empiric, based on experience mostly with RT-PCR and will be heavily influenced by increasing use of RQ-PCR. Although in the study by Olavarria et al [28], a threshold level of 0.02% seemed a strong predictor for relapse, especially in patients with previously negative or lower levels of BCRABL, further requirements for defining molecular relapse have been suggested: (1) repetition of the analysis twice within 2 to 3 months or (2) in case of higher BCR-ABL levels that exceed a BCR-ABL/ABL ratio of 0.05%, one more independent confirmatory analyses would be considered sufficient to establish molecular relapse. Interferon-a Until the approval of imatinib for patients who have early chronic-phase CML, IFN-a was the predominant therapy for patients who could not undergo SCT. About 10% to 25% of patients on IFN-a achieve complete cytogenetic responses, and long-term responders have been described [30]. The essential difference to transplanted patients, however, is that virtually all patients on IFN-a remain PCR positive [31]. This observation is of interest because it raises several issues that pertain to the usefulness of PCR techniques in this patient population and to the general considerations regarding the biologic uniqueness of responses that are achieved with IFN-a compared with SCT and imatinib. In quantifying BCR-ABL transcripts, Hochhaus et al [32] compared the cytogenetic response of patients on INF-a to the molecular response based on BCR-ABL/ABL ratios. They established cut-off points for cytogenetic complete responders (ratio of up to 2%), partial responders (2% –14%), and minor or nonresponders (> 14%). Because the predictive value of molecular responders should matter most in complete cytogenetic responders, the following question arises: can threshold values of BCR-ABL/ABL ratios be defined within this group of complete responders that would distinguish patients who will relapse

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from those who will maintain their response? In a study of 54 CML patients treated with IFN-a who achieved a complete cytogenetic response, all remained PCR positive [31]. Despite their failure to achieve complete molecular responses, however, some patients maintained a continuous complete remission of up to 5 years. At the time of maximal cytogenetic response, the median BCR-ABL/ ABL ratio was 0.045%, which was used as a threshold value in this study. Almost 50% of the complete cytogenetic responders who maintained residual disease above these levels relapsed versus <5% who remained below this cut-off point. The dynamics of response and relapse in patients treated with IFN-a not only are biologically intriguing (see later discussion) but also have practical relevance with respect to changing to alternative therapies or choosing the appropriate timing to safely discontinue therapy with IFN-a. Even after a decade’s use of IFN-a therapy in CML and though no universally agreed-on guidelines yet exist, it appears reasonable to aim for as low levels of residual disease as possible. Although a study by Kurzrock et al [33] suggested that some complete cytogenetic responders eventually become PCR negative if followed long enough, the value of PCR negativity in the setting of therapy with IFN-a still should be embraced very cautiously. Talpaz and colleagues [34] and others [35,36] observed that myeloid and erythroid colonies from blood and marrow samples still expressed BCR-ABL transcripts in patients who attained complete cytogenetic responses after IFN-a treatment and who also were PCR negative for BCR-ABL. Imatinib Imatinib has changed the therapeutic landscape substantially for CML patients. It now is considered first-line therapy in early chronic-phase and the most active single agent for patients in accelerated and blast phases. Highly illustrative in terms of gauging clinical and molecular response rates of imatinib in CML is the International Randomized Study of Interferon and STI571 (IRIS) [37,38]. In this study, 1106 newly diagnosed CML patients in early chronic phase were randomized to receive imatinib (553 patients) or IFN-a/cytarabine (553 patients). Crossover to the alternative group was allowed in case of treatment failure and intolerance. At 18 months of follow-up, the major and complete cytogenetic response rates of imatinib versus IFN-a/cytarabine were 87.1% versus 34.7% (P < 0.001) and 76.2% versus 14.5% (P < 0.001), respectively. The trial thus established high major and complete cytogenetic response rates and the superiority of imatinib over IFN-a– based therapy. Hughes et al [38] measured BCR-ABL levels in 313 patients who achieved a complete cytogenetic response with imatinib or the IFN-a combination during participation in the IRIS trial. RQ-PCR was used and the percentage of BCRABL/BCR was calculated. Three aspects of this study are of particular interest: the speed of reduction of BCR-ABL transcripts, the amount of suppression of minimal residual disease levels, and impact on progression-free survival. A 3-log reduction in BCR-ABL levels was achieved in 39% of all imatinib-treated

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patients but in only 2% of the IFN-a –treated patients. Overall, 13% of patients who received imatinib achieved a >4-log reduction; 3% had reductions of > 4.5 log, which approaches the current sensitivity of RQ-PCR assays. Two interesting observations have emerged from this and a smaller, subsequent subgroup analysis [39]. The median levels for imatinib-treated patients continue to decrease and have not reached a plateau as of 24 months. Furthermore, the IRIS study demonstrated that the achievement of at least a 3-log reduction (which has been termed a major molecular response) by 1 year of imatinib therapy is predictive of progression-free survival at 2 years. On the other hand, patients who did not achieve a >2-log reduction in the amount of BCR-ABL transcripts at 6 months had the highest risk of disease progression, rarely achieved a complete cytogenetic response, and never attained a major molecular response. Given that most patients achieve a complete cytogenetic response with imatinib relatively quickly, additional information from use of RQ-PCR assays and quantification of BCR-ABL transcripts permits stratification of patients into those who will remain in remission and those who are likely to progress. These results were confirmed in another subset analysis by Hochhaus and colleagues [40]. Among 139 early chronic-phase CML patients, 69 were treated with imatinib as first-line therapy. This group had a rapid decrease in BCR-ABL transcript levels that was superior in degree and speed compared with the IFN-a group. Residual disease was rarely eliminated; however, with molecular responses ongoing up to 24 months following treatment start, it is conceivable that some of these patients eventually will have undetectable BCR-ABL levels. A good correlation has been established between measurements of BCR-ABL transcripts in the marrow and peripheral blood, making blood samples an adequate source for molecular follow-up after a complete cytogenetic response has been established, especially in chronic phase [19,41]. Nevertheless, cytogenetic analysis should be repeated at regular intervals because Ph-negative clonal evolution has been observed in patients on imatinib that would not be detected by serial RQ-PCR measurements alone [42 –45].

Limitations and controversies PCR has become an indispensable tool for the detection and follow-up of minimal residual disease in CML. RQ-PCR, in particular, has emerged in recent years as a new technology on its way to becoming the standard assay for minimal residual disease monitoring. Although good correlations have been established between PCR quantification of the BCR-ABL transcripts and relapse in the setting of SCT, any laboratory test results that impact on clinical decision-making will have to be appraised critically. Although PCR techniques have been continuously refined over the last few years, the possibility of technical pitfalls leading to false-positive or falsenegative results should not be discarded. Sample contamination, inadequate sample volume, inappropriate sample source, loss of sensitivity of junctional

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probes, degradation of the target molecule, and poor quality RNA integrity can lead to inaccurate results [43,46,47]. Interlaboratory standardization of test procedures has become more important as results from different centers need to be comparable to arrive at useful clinical conclusions in the context of multicenter trials, as is exemplified in the IRIS trial of imatinib versus IFN-a/ cytarabine in newly diagnosed chronic-phase CML patients. In addition to purely technical aspects, the biologic diversity of the target cell has to be considered. Shifts in disease markers as reflected in oligoclonality, subclone formation, clonal evolution, and incomplete target sequence rearrangements in the CML cells can occur, leading to negative test results that falsely suggest absence of disease. Attention also should be paid to less frequent molecular fusion products (e6a2, e19a2, fusions lacking exon a2) [48] that highlight the need to identify correctly the transcript type at diagnosis as the basis for subsequent residual disease analysis. Multiplex PCR has been suggested as a suitable technique to perform this analysis [49]. An interesting observation is the fact that some patients maintain a continuous clinical remission even in the presence of minimal residual disease. Persistent molecular disease, therefore, does not always correlate with impending clinical relapse and does not necessarily establish an insurmountable obstacle to cure. That the presence of residual disease is compatible with ongoing clinical remissions has been described in other leukemias (ie, acute myeloid leukemia with translocation t[8;21] or inv[16]) in which serial PCR testing in remission has not been found useful for predicting relapse [50,51]. These observations draw attention to two interesting aspects of minimal residual disease: (1) the residual cells detected in CML patients are BCR-ABL positive but may lack the functional attributes of a leukemic cell and (2) tumor dormancy. Using a highly sensitive RT-PCR assay (1:108), several investigators were able to detect BCR-ABL transcripts in healthy individuals in an age-dependent manner [52,53]. These findings indicate the presence of leukemia-specific fusion genes in some hematopoietic cells without the accompanying clinical syndrome of leukemia. Therefore, gene fusion products by themselves are not always able to generate the leukemic phenotype but may require other, yet unidentified, leukemogenic ‘‘hits.’’ Alternatively, leukemic fusion genes may be expressed in hematopoietic cells that already have entered an apoptotic pathway before acquiring the characteristic leukemic attributes and that already have lost their clinical relevance. Closely associated with this phenomenon is tumor dormancy [54]. A tumor may be considered dormant when residual malignant cells are kept under growth control by certain host mechanisms but otherwise retain their neoplastic potential. Host immune surveillance, therefore, may play a major role in the containment of residual leukemic cells. In a study of 379 patients who survived relapse-free at least 18 months following SCT, BCR-ABL could be detected by PCR in about 25% [27]. The absolute risk of relapse in those patients was modest, however, with a 19% 3-year cumulative incidence following the first positive PCR assay. These constellations suggest that cure in a clinical sense does not always require elimination of all levels of disease and consequently, that not

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Table 2 Guidelines for follow-up for diagnosis and monitoring of patients who have chronic myeloid leukemia Assay

Therapy

Recommendation

Cytogenetic analysis

Any

‘‘Gold standard’’ for diagnosis Repeat at least yearly (identification of clonal evolution of Ph-positive or Ph-negative hematopoiesis)

FISH I-FISH

Any

H-FISH PCR

Any

SCT

IFN-a Imatinib

Pretreatment for baseline Every 3 months until Ph<10% As for I-FISH. No false-positive results. Can be used when Ph<10% Monitoring of minimal residual disease for patients who have achieved a complete cytogenetic response Testing at 4-wk intervals (peripheral blood) If PCR negative, test at 3- to 6-mo intervals If PCR levels increase, testing more frequently is recommended Quantitative PCR at 3-mo intervals Quantitative PCR at 3-mo intervals

all patients who have persistently detectable leukemia at the molecular level will need therapy.

Recommendations for monitoring during therapy Although certain key elements are emerging from currently published data, no universally accepted guidelines for monitoring patients who have CML and minimal residual disease have been established (Table 2). Routine cytogenetic testing remains the ‘‘gold standard’’ at diagnosis and should be repeated at intervals of 6 to 12 months to identify clonal evolution. After the number of Ph-positive metaphases has fallen below 10%, H-FISH and quantitative PCR should be applied for further monitoring of residual disease. Following SCT, it is recommended that quantitative PCR be performed every 4 weeks for as long as BCR-ABL transcripts continue to be identified. If the levels become undetectable, then testing intervals can be increased to every 3 months or every 6 months if PCR testing remains negative. In the case of increasing levels, PCR testing should be performed more frequently [55,56]. The question of what constitutes a molecular relapse has been dealt with earlier. After molecular relapse has been established, salvage therapy (eg, donor lymphocyte infusions after allogeneic SCT) should be instituted. It should be emphasized, however, that these guidelines have not been validated for patients who remain residual-disease positive after therapy with IFN-a or imatinib, and longer follow-up and experience is

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necessary, especially in the latter group, to fully appreciate the impact of PCR testing.

Summary Detection and monitoring of minimal residual disease has become one of the most prevalent topics in CML therapy. The goal of early detection of residual disease is to allow timely therapeutic intervention before overt relapse of disease occurs that has become resistant to therapy. The most powerful tool to serve this purpose is PCR. Major improvements in assay techniques have advanced PCR from a purely qualitative test with considerable variability of test results to an RQ assay with far more reproducible results than were possible before. At the same time, treatment of CML has changed dramatically since the introduction of imatinib. Integration of therapy and molecular assays such as PCR, in addition to a better understanding of the pathophysiology of CML, has assumed even more importance. Quantitative PCR testing has become the standard monitoring strategy for patients undergoing SCT. Although correlations exist between positive test results and probability of relapse, no absolute guidelines for monitoring have been established, especially for patients treated with imatinib. The real impact of the use of RQ-PCR on the validity of predicting relapse according to treatment (SCT, IFN-a, imatinib) will become apparent over time. It is unlikely, however, that any of the results can be fully appreciated without thorough integration of the molecular biology of CML and, as an extension to that, the influence that different therapeutic modalities exert on it.

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[9]

[10]

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[12] [13]

[14] [15] [16]

[17] [18]

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