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STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA
ADVANCES IN THE TREATMENT OF ADULT ACUTE LYMPHOCYTIC LEUKEMIA, PART I
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STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA Wendy Stock, MD, and Zeev Estrov, MD
During the past 2 decades, there has been considerable progress in the treatment of both childhood and adult acute lymphocytic leukemia (ALL). Currently, 70% to 90% of adults achieve a complete remission (CR) and 25% to 50% of these patients may experience prolonged disease-free survival (DFS) and may be cured of their disease. Unfortunately, most adults with ALL will ultimately experience a recurrence and die of their leukemia. Although most children with ALL may now be cured with current therapeutic regimens, the ability to distinguish good-risk patients from those who are likely to relapse has important clinical implications. Relapse, in most pediatric and adult cases, is thought to result from residual leukemia cells that remain following achievement of "complete" remission but are below the limits of detection using conventional morphologic assessment of the bone marrow. Sensitive techniques are now available to detect subclinical levels of residual leukemia, termed minimal residual disease (MRD). Study of MRD may have great usefulness for assessing the clinical efficacy of current and future strategies aimed at improving the cure rate in ALL and other malignancies; however, the clinical significance of MRD detection and monitoring remains to be determined. Several important questions must be answered by carefully controlled, prospective studies before the therapeutic interventions based on MRD monitoring strategies are implemented. First, does early detection of leukemia
From the Leukemia Program, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois (WS); and Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas (ZE)
HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA
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by sensitive techniques predict relapse? Second, if early detection is predictive of relapse, can early detection at a molecular level guide therapy (i.e., should the intensity of treatment be increased or decreased based on MRD results)? Also, does the treatment of a molecular relapse actually improve survival? Third, is it essential for the leukemic clone detected by sensitive MRD assays to be completely eliminated to achieve a cure? Studies of MRD may provide insights into the pathogenesis and biologic behavior of leukemia. Will sensitive molecular monitoring facilitate the identification of homeostatic mechanisms that modulate growth of the leukemic clone? This article focuses on current strategies for monitoring MRD in ALL, discusses the recent studies of MRD monitoring in ALL, and reviews the possible clinical relevance of MRD studies. METHODS FOR DETECTING MINIMAL RESIDUAL DISEASE A patient in clinical remission from leukemia can still harbor up to 1O1O leukemia cells that remain below the threshold of detection by morphologic methods.n During the last decade, a variety of techniques have been studied for the detection of residual disease. These methods include cytogenetics, cell culture systems, fluorescence in situ hybridization (FISH), Southern blotting, immunophenotyping, and polymerase chain reaction (PCR) techniques (Table l).5,6, s, 14, 22, 55, @,69, 70, 73, 74, 78, 79 The applicability of the techniques for MRD detection depends on three parameters: specificity (ability to discriminate between malignant and normal cells without false-positive results); sensitivity (detection limit of at least lop3, or detection of 1 malignant cell in a background of at least 1000 normal cells); and reproducibility and applicability (easy standardization and rapid collection of results for clinical application).” The detection limit of most of these is not lower than 1%to 5% malignant cells; however, depending on the phenotype and genotype of the leukemia, immunophenotyping and PCR techniques are able to detect lower frequencies of leukemia cells and fulfill the criteria for MRD detection. Immunophenotyping techniques using multicolor-gated flow cytometry are based on aberrant expression of antigens by the leukemia cell popul a t i ~ n .70,~ ,72 For example, in B-cell-lineage ALL, leukemic blasts may coexpress cross-lineage T-cell antigens (e.g., CD5 and CD7) or myeloid antigens (e.g., CD13 and CD33), have asynchronous expression of antigens, or drop or overexpress a specific antigen (particularly CD10).20,38 Recently by using two four-color combinations of antibodies (CD19, CD45, CD20, CDlO and CD19, CD45, CD9, CD34), aberrant leukemic blasts were identified in 81 of 82 pediatric B-lineage ALL cases.78In Tlineage ALL, leukemic blasts nearly always coexpress terminal deoxynucleotidyl transferase (TdT), CD2, cytoplasmic CD3, CD5, and CD7 in addition to other T-cell antigens. Cells with this immunophenotypic pattern are absent or occur very rarely in normal bone marrow or
Colony growth
Molecular markers
Karyotype analysis
Immunophenotyping
Marker
Flow cytometry (multiparameter) Fluorescence microscopy Cytogenetic studies Fluorescence in situ hybridization (FISH) Southern blotting Polymerase chain reaction Clone-specific Leukemia-specific (fusion gene) DNA RNA (reverse transcription-PCR) Clonogenic assay
Technique
Table 1. METHODS FOR DETECTION OF MINIMAL RESIDUAL DISEASE
510-4
10-1-10-2 10-4-10-2
10-2-10-4 10-2-10-4 10-'-10-2 10-2-10-4
Detection Limit
90
70-90 20-50
60-90
23
8, 24 (for review) 49, 53, 54 (techniques)
8, 47, 64 8, 22, 49, 55, 64, 66
5-40
References
5, 6, 8, 13, 14, 70, 73, 74, 78, 79
("/I
90-100
Applicability
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Thus, it is theoretically possible to detect MRD in most patients with ALL using immunophenotypic analysis, and this technique allows accurate quantification of the leukemic population. A recent study of 128 children with ALL showed sequential MRD monitoring using multiparameter flow to be a valuable predictor of re1ap~e.l~ Low-frequency normal hematopoietic progenitor cells, similar to leukemic blasts, may express the same cytoplasmic or surface-bound marker profile, however, rendering distinction between normal and malignant cells difficult in the MRD setting. There is also concern about the potential for change in immunophenotypic expression during disease progression resulting in a false-negative result.4O Because most recent studies of MRD in adult ALL have used PCR techniques, the remainder of this article focuses on these studies. Detection of Minimal Residual Disease by Polymerase Chain Reaction Using PCR for specific amplification of a DNA sequence or cDNA unique to the leukemia clone can permit identification of 1 malignant cell among lo4 to lo6 normal cells. Two types of PCR targets can be used to detect MRD in ALL patients: junctional regions of leukemia clone-specific rearranged immunoglobulin and T-cell receptor (TCR) genes, or leukemia-specific breakpoint fusion regions of chromosome rearrangements (translocations, deletions, or inversions) (Table 2). The deletion and random insertion of nucleotides during immunoglobulin and TCR gene rearrangement generates unique junctional sequences that can serve as clone-specific markers of the leukemia. The precise nucleotide sequence of the junctional region of the immunoglobulin or TCR genes in the leukemia cells can be identified at the time of diagnosis allowing the design of oligonucleotides specific to the junctional region. These oligonucleotides can then be used as patient-specific probes (or primers) for detecting MRD during and following treatment of the le~kemia.5~ Oligoclonality of immunoglobulin and TCR gene rearrangements at diagnosis is a relatively frequent phenomenon.', 68 Continuing rearrangements and secondary rearrangements that occur during the disease course might result in the loss of the specific junctional regions initially identified at diagnosis. Therefore, it seems to be important to monitor ALL patients with two or more independent PCR targets to prevent false-negative results during f o l l ~ w - u p . ~ * ~ ~ Leukemia-specific chromosome rearrangements are also useful PCR targets for detecting MRD. Oligonucleotide primers are designed at opposite ends of the breakpoint fusion region so that the PCR product contains the tumor-specific fusion sequences. Because the PCR products should not exceed about 2 kilobases (kb) in routine MRD studies, PCR amplification can only be used for chromosome aberrations in which the breakpoints cluster in a small area (preferably <2 kb). In ALL, DNAbased PCR for MRD can be performed for T-lineage ALL with TALI gene
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Table 2. FREQUENCY OF PCR TARGETS AVAILABLE FOR MRD DETECTION IN ALL Frequency (%) ALL Lineage
Chromosomal Abnormality
Molecular Target
Adults
Children
-45 -60 -80 -50 25-30 5-8 <5 2-3 4-5 <1 <1 <1 <1
-55 -60 -90 -35
B-lineage
-
TCRG (DNA) TCRy (DNA) IgH (DNA) IgK-Kde* (DNA) BCR-ABL (RNA) MLL-AF4 (RNA) TEL-AML1 (RNA) E2A-PBX1 (RNA) MYC-IgH (DNA) IL3-IgH1 (DNA) MLL-ENL (RNA) E2A-HLF (RNA) MLL-AF9 (RNA) TCRG (DNA) TCRG (DNA) TCRy (DNA) IgH (DNA) IgK-Kde (DNA) TALl deletion (DNA) RHOM2-TCRy TALl -TCRa H O X l l -TCRa
-70 -70 -85 -5-10 0 10-20 5-10 <1 1-3
4-6
3-5 25-30 5-6 1-2 <1 <1 <1 <1 -50 -50 -90 -10-20 0
‘The t(12;21) is a cryptic translocation only rarely identified using standard cytogenetics. PCR = polymerase chain reaction; MRD = minimal residual disease; ALL = acute lymphocytic leukemia; TCR = T-cell receptor gene rearrangements; IgH = immunoglobulin heavy chain rearrangements; IgK-Kde = Ig kappa gene deletional rearrangement joined to kappa-deleting element.
deletions (SIL-TALZ), the t(ll;l4)(pl3;qll) involving RHOMZ-TCRD, the t(1;14)(p32;qll) involving TALI-TCRD, and the t(10;14)(q24;qll) involving HOXZZ-TCRD.” In most of the chromosome translocations common to adult ALL, however, the breakpoints are spread over areas much larger than 2 kb; therefore, MRD detection and monitoring depends on identifying the resultant leukemia-specific fusion mRNA. This fusion mRNA can be used as a target for MRD analysis using PCR after the fusion mRNA (consisting of transcribed coding exons) is converted to cDNA using the enzyme reverse transcriptase (RT). This technique is known as reverse transcriptase PCR (RT-PCR). In ALL, RT-PCR is used to detect the following transcripts: BCX-ABL resulting from the t(9;22); E2A-PBXZ in cases of pre-B ALL with a t(1;19); MLL-AF4 in t(4;ll) leukemia; and the TEL-AMLZ resulting from the t(12;21) in precursor-B ALL (Table 2 ) . In monitoring MRD, PCR has the advantages of extraordinary sensitivity, speed, and minimal tissue requirements. The disadvantages of
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PCR for monitoring MRD encompass a variety of technical and biologic issues, includingz5 contamination of the reaction product (which can be avoided with strict quality control) the lack of reproducibility of results when small numbers of transcripts are present the risk of RNA degradation and inefficiency during conversion of mRNA to cDNA (which may reduce the sensitivity of RT-PCR monitoring) evolution of the leukemic clone subclone formation the presence of oligoclonal populations that can cause both falsenegative and false-positive results. Precise quantification of copy number using PCR has also been problematic in the past. Endpoint measurements, that is, analysis of the reaction product after amplification is completed, commonly have been used for quantification. These measurements depend on multiple dilutions or coamplification of standards (internal or external to the reaction system) and are cumbersome, error prone, and technically demandir~g.3~ New automated PCR techniques, however, will probably allow more precise and consistent quantitation of residual leukemic clones.15,35 One such technique, known as red time PCR, uses a fluorogenic probe, an oligonucleotide with both a reporter fluorescent dye and a quencher dye atta~hed.3~. 50 This probe is designed to anneal specifically between the forward and reverse primers used to amplify a specific target sequence. During PCR, the probe is cleaved by the 5’ nuclease activity of Taq DNA polymerase during primer extension. Cleavage of the probe separates the reporter dye from the quencher dye, generating a fluorescent reporter dye signal. With each cycle of amplification, additional reporter molecules are cleaved from their probes, increasing the fluorescence intensity in proportion to the amount of transcript produced during the logarithmic phase of PCR amplification. Recent studies of MRD monitoring using real time technology, some of which are discussed later, are beginning to provide new insights into the potential clinical significance of MRD detection. CLINICAL STUDIES MEASURING RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA BCR-ABL in Philadelphia ChromosomePositive Acute Lymphoblastic Leukemia
Reverse transcriptase polymerase chain reaction has been used to detect and monitor the BCR-ABL fusion gene associated with the t(9;22) that occurs in as many as 30% of adults with ALL.31,58 In adult patients expressing B-lineage surface antigens, approximately 50% of cases are
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BCR-ABL-positive ( + ). Most of the patients (approximately 70%-75%) have the p190 subtype; the p210 subtype of BCR-ABL that is present in almost all patients with CML occurs in approximately 25% to 30% of patients with ALL.8o The poor prognosis of adults with BCR-ABL+ ALL treated with combination chemotherapy justifies attempts to intensify treatment in first remission using allogeneic transplantation, when available for this group of patients (see article by Radich elsewhere in this issue). Allogeneic transplantation improves DFS for some of these patients; however, even with transplantation, the relapse rate for BCR-ABL+ ALL is quite high, ranging from 40% to 28, 65 Several studies of MRD status following allogeneic transplantation provide intriguing information about the risk of relapse in BCR-ABL+ ALL and may help identify patients who are likely to relapse. In the studies listed in Table 3, RTPCR techniques with a reported sensitivity of 1 leukemia cell in a background of lo5 normal cells were used to study MRD following 48, 56, 58 All these studies found allogeneic or autologous that patients who are consistently BCR-ABL negative following transplantation are unlikely to relapse and may become long-term survivors. Conversely, patients who are BCR-ABL following transplantation seem to be at high risk for subsequent relapse. In the largest published series (28 patients), Radich et a1 found that the relative risk (RR) for relapse was significantly higher for patients with a detectable BCR-ABL transcript following transplantation than for those without detectable BCR-ABL (RR = 5.7; P = 0.025)?8 The prognostic significance of the PCR assay remained after controlling for other clinical variables that could influence relapse risk. The risk of relapse was greater for patients with a p190 fusion transcript than for those with a p210 BCR-ABL. The median time from detection of a positive PCR result to relapse was 94 days. Additional insights into the kinetics of disappearance of BCR-ABL during
+
Table 3. CLINICAL RELEVANCE OF DETECTION OF BCWABL TRANSCRIPTS AFTER ALLOGENEIC OR AUTOLOGOUS TRANSPLANTATION FOR Ph ALL'
+
PCR = Positive Study
No. of Patients
p190 BCFUABL Gehly3' Miyamuraa Pre~dhomrne~~
Radich6' p210 BCWABL
Radich6'
PCR = Negative
No. of Patients
No. of Relapses (%)
No. of Patients
No. of Relapses (%)
4 15 9
1 7 5
28
15
1 (100) 7 (100) 5 (100) 9 (60)
3 8 4 13
0 1 (13) 0 2 (15)
8
8
1 (13)
0
0
*Results were obtained using qualitative, not quantitative PCR, usually within the first 100 days after transplantation. Ph + = Philadelphia chromosome-positive;ALL = acute lymphocytic leukemia; PCR = polymerase chain reaction.
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treatment will be obtained from current studies using real-time PCR techniques to quantify transcript number repr~ducibly.~~ Prospective PCR validation with quantitative techniques in a larger series of BCRA B L + patients is clearly required; however, with rigorous monitoring it may be possible to introduce novel therapies to eliminate residual disease and avoid the dire consequences of a clinical relapse in these patients.
Studies of Other Fusion Transcripts in Minimal Residual Disease in Acute Lymphocytic Leukemia: MLL-AF4 and E2A-PBX1 The M L L gene, located on chromosome band 11q23, is known to be involved in more than 30 translocations in acute leukemias (ALL and acute myeloid leukemia [AML]) and lymphomas. In ALL, the most common M L L gene abnormality is the MLL-AF4 (also known as ALLZAF4) fusion gene that results from the t(4;11)(q21;q23). It is found in approximately 3% to 6% of adults and children with ALL and is the most common cytogenetic abnormality in infants with ALL.3,46 The t(4;ll) has been associated with a very poor prognosis when standard chemotherapy regimens are employed, but improved survival has been noted in patients undergoing allogeneic transplantation in first remission or when intensive postremission treatment with high-dose cytarabine is A recently published prospective analysis of MRD monitoring in 25 patients with the MLL-AF4 transcript is the largest study of residual disease in these patients.'* In this study, qualitative RT-PCR analysis with a sensitivity of 1 in lo4 showed that in patients who were in a clinical remission at the end of high-dose induction and consolidation therapy (without bone marrow transplantation) continued PCR positivity (in 13 of 25 patients), or reconversion from negative to positive (in 5 of 25 patients) was predictive of relapse. Patients who remained PCR-negative during the first 3 to 6 months following diagnosis seemed to enjoy prolonged disease-free survival. These results are concordant with several other smaller MRD series that suggest that MLL-AF4 patients who remain persistently PCR-positive at early time points following chemotherapy or transplantation have a high likelihood of relapse and that PCR negativity following transplantation is highly predictive for relapse-free survival.'*,36, 41 Polymerase chain reaction monitoring studies have also been performed in ALL patients with the E2A-PBXZ fusion gene that results from the t(1;19)(q23;p13).Unlike the studies described previously, in one study of 41 children with a t(1;19)(q23;p13), qualitative RT-PCR detection of the E2A-PBXZ at the end of consolidation therapy was not predictive of treatment These results are similar to those noted in several smaller studies of MRD with the E2A-PBXZ fusion transcript.39,58 Discordance between matched blood and bone marrow samples was noted
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in a number of these emphasizing the need for careful comparison of these tissues in future MRD studies. Clone-Specific Polymerase Chain Reaction using Immunoglobulin Heavy Chain and T-cell Receptor Gene Rearrangements
Clone-specific PCR studies of MRD in ALL have been performed mostly in children; few studies have involved adults. The results from these trials have not always been consistent, and this variability may be ascribed to a number of factors: (1)PCR testing has been performed at different times during the treatment cycle; (2) some of the studies used qualitative PCR assays, whereas others involved a number of different methods for quantitation of the PCR product; (3) sample sources have been different (fresh bone marrow versus archival material scraped from bone marrow slides); and (4) the treatment regimens have varied considerably. Nevertheless, several conclusions can be drawn from these studies that are summarized in Table 4. Serial clone-specific PCR analyses of remission samples demonstrate that during the immediate postinduction period, from 40% to 70% of patients are still positive for MRD32,45 and that these levels often continue to decline during the first,6 months after initiation of thera~y.2~ Several of these studies suggested that detection of residual disease by PCR following induction (1to 2 months after starting therapy) has prognostic value.5,29, 34, 77 Patients with positive PCR results following induction therapy and those who did not show a consistent decrease in residual disease levels during the first 6 months of treatment had a significantly higher risk of relapse than did patients with low-level residual disease or negative PCR results. A number of recent large, prospective MRD-monitoring studies in pediatric ALL confirm these findings and suggest that quantitation of MRD may be critical because the probability of long-term relapse-free survival seems to be directly related to the level of residual disease, both early in the course of treatmen@9, 75 and at later time point^.^ In their semiquantitative MRD monitoring study of 246 children with ALL, Cave et a1 demonstrated in a multivariate analysis that PCR detection of more than lo-* residual blasts following intensive induction chemotherapy was the most powerful independent predictor of early r e l a ~ s e When .~ later time points during treatment were analyzed, a MRD burden greater than was associated with significantly higher relapse rates. In another large study of MRD involving 240 children with ALL, van Dongen et a1 found that combining semiquantitative MRD information from several time points during treatment identified three risk groups to be distinguished. Forty-three percent of patients were in a low-risk group with a 3-year relapse rate of only 2%; 43% were in an intermediate-risk group with a relapse rate of 23%; and 15% were in a high-risk group with a 75% relapse rate.75Other studies confirm the significance
+/-
Van D ~ n g e n ~ ~ 240 C
30 A
For~ni~~
+/-
+/-
+
After induction After consolidation 6 months after consolidation After 6 months After end of therapy After induction After 6 months After consolidation After induction After 4 months Before maintenance End of therapy Up to 35 months after end of therapy After induction After induction After 3 months After 6 months After induction After 3 months After 6 months After 12 months
Time of Testing
MRD = minimal residual disease; PCR = polymerase chain reaction; C = children; A = adults; = no quantitation.
24 C 50 C 27 A 10 A
Roberts62 Steenberged7 Brisco5 NizeP'
+ +/+
+/-
66 C
Go~lden~~
23 C
+
246 C*
Number
Cave9
Reference
Quant,itative Assay
Table 4. SELECTED STUDIES OF MRD USING CLONE-SPECIFIC PCR
+/ -
25 15 21 23 18 40 32 5 8 8 7 5 10 11 3 3 2 5 4 1
Relapse
= quantitative PCR assay;
20 12 18 9 9 5 8 5 2
-
63 32 38 33 28 98 47 6 16 16 8
Positive
PCR Results
=
7 8 25 5 6 2 18 0 0 0 0 0 0 6 3 0 0 2 1 1 3 -
Relapse
semiquantitative PCR assay;
88 95 215 29 30 71 166 148 1 1 8 7 2 22 9 1 1 5 5 10 9
Negative
PCR Results
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of serial, quantitative measurements of residual disease and indicate that the timing of disappearance and reappearance of marrow blasts is crucial in predicting those who are destined to relapse and those who will 51 For example, the presence of residual disease at remain in remis~ion.~, the time of maintenance therapy has recently been shown to be an accurate predictor of outcome in children with T-lineage ALL.17 Studies of MRD have also used clone-specific techniques in the setting of allogeneic stem cell transplantation for ALL. Sixty-four children and adolescents with ALL who were in morphologic remission were studied for the presence of MRD immediately before allogeneic transplantation. The presence of any MRD before transplantation was associated with a 2 year event-free survival (EFS)of only 17%, in contrast to an EFS of 73% for those who had no detectable MRD.43Detection of MRD following allogeneic transplantation has also correlated with higher relapse rates. The same authors studied 71 children and found that patients who exhibited MRD during the first 3 months following transplantation had a ninefold higher relapse rate than MRD-negative patients. The detection of MRD was quite unusual in those patients who remained in remission. Only one of 35 patients in CR had a positive PCR result for MRD when the study was performed more than 6 months following transplantation.44The presence of MRD using clone-specific PCR detection following allogeneic transplantation was also predictive of relapse in another study composed primarily of adults with ALL.60 Despite the finding from many of these studies that the presence of residual disease portends relapse following completion of combination chemotherapy or allogeneic transplant, other studies suggest that not all patients with MRD experience relapse. In the Cav6 study mentioned previously, about 20% of the patients with an MRD level greater than did not relapse. Molecular evidence of a residual leukemic clone has been detected in bone marrow for as long as 9 years following In a recent completion of therapy for ALL without a clinical relap~e.5~ study using a highly sensitive quantitative PCR assay combined with clonogenic assays to assess residual disease in children with ALL, MRD was demonstrated in 15 of 17 patients who nevertheless remained in remission from 2 to 35 months after completion of all treatment.62Longterm persistence of MRD in patients who remain in clinical remission has also been described in patients with AML with a t(8;21) and in patients who have undergone allogeneic stem cell transplantation for chronic myelogenous leukemia (CML).l0,43, 52, 61 Similar results have been described previously in patients with p210 BCR-ABL+ ALL and in children with t(1;19) ALL. Another recent study suggests that prolonged dormancy of the leukemic clone can be established. In this study of 1134 children who were long-term survivors of ALL, Vora et a1 studied 12 children who eventually relapsed more than 10 years after diagnosis.76 In 8 of these relapsed cases, the investigators were able to study DNA from the original diagnosis and from the relapsed bone marrow. In each relapsed sample they found the identical immunoglobulin heavy-chain (IgH) or TCR gene rearrangement that was present in the leukemic clone
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from the diagnostic bone marrow specimen. This finding suggests that the leukemic cells or clonal precursors survived for more than a decade. CLINICAL SIGNIFICANCE OF THE DETECTION OF MINIMAL RESIDUAL DISEASE IN LEUKEMIA The studies of MRD in ALL and other leukemias have assumed great importance because long-term DFS seems to depend on the ability to control residual disease. The initial generation of studies of MRD in ALL provides tantalizing preliminary data suggesting that, in certain subsets of disease, the absence of a leukemia-specific marker, such as p190 BCR-ABL, is predictive of long-term remission, whereas its persistence may portend relapse. The persistence of MRD is less clear when other molecular markers are used. It is clear, however, that the potential clinical usefulness of each type of MRD assay needs to be determined rigorously before results are used to modify treatment of individual patients or groups of patients. Before PCR is used for clinical interventions, it must be determined that such a test reveals identical results in different laboratories and that it has acceptable sensitivity, specificity, and positive and negative predictive values. Future studies of MRD in adult ALL will require prospective, sequential analyses on larger numbers of patients, as has been performed in the European pediatric ALL studies of MRD. The use of automated, quantitative real time PCR is becoming more widespread and may be essential for technique stanThe dardization and for the validation of the earlier ~ t u d i e s .69' ~ ~ ~current ~~ generation of MRD studies is likely to determine more clearly whether a threshold level may be defined above which patients are likely to relapse and whether serial monitoring will provide data that will be useful for future clinical interventions designed to minimize toxicity or prevent overt relapse. The European pediatric cooperative group, IBFM-SG, has just begun a prospective trial in which the results of a semiquantitative MRD assay are being used to stratify postremission treatment in children with ALL (JVD Van Dongen, personal communication, 2000). These studies also provide fascinating clues to the pathogenesis of these diseases and the biologic properties of residual leukemia cells. It seems that residual disease is a dynamic process, with the numbers of 62 Although shortly after residual leukemia cells fluctuating over therapy the levels of residual disease may fall below the detection limit of even highly sensitive molecular assays, the leukemia cells do not necessarily disappear in clinical remission. They may increase until clinically overt disease recurs, or they may fall below the threshold of detection again. A positive-negative-positive pattern of PCR results is common, and molecular relapse does not always predict clinical relapse. When the disease remains dormant for years, as described previously, the leukemic clone may have come under the control of an immunologic or other homeostatic mechanism. Alternatively, the detected cells containing a specific fusion gene transcript such as p210 BCR-ABL, or E2A-PBXZ,
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although members of the leukemic clone, may behave as preleukemic cells that require additional genetic mutations (perhaps acquired by a subclone of descendant cells at the time of diagnosis). These additional mutations 30, 33 Also, falsemay be essential for full leukemic transformati~n.~~? negative PCR results can occur with clone-specific PCR as a result of clonal evolution in IgH or TCR genes of the leukemia cells and therefore limit the predictive power of a negative result!, 51, 55 Furthermore, because leukemic blasts are considered malignant counterparts of normal, immature hematopoietic progenitor cells, they express closely related differentiation and lineage markers.62It is, therefore, possible that seemingly residual leukemic cells may in fact be normal hematopoietic cells. This observation is highlighted by the fact that disease-specific fusion gene products, including BCR-ABL, BCL2-IgH, MLL-AF4, and the partial tandem duplication of MLL have been detected with highly sensitive PCR techniques in healthy individuals.z, 63, 71 Molecular assay techniques such as PCR provide unparallel sensitivity for the detection of residual disease. These assays have contributed significantly to the understanding of residual disease in ALL and other hematologic malignancies. The usefulness of these powerful molecular techniques extends beyond detection of residual disease and is redefining the concept of that has been based on criteria formulated many years before the introduction of molecular biologic assessment. Eventually, the knowledge gained from MRD studies, including quantification of residual disease, understanding of the fluctuations in residual disease levels over time, identification of threshold levels of MRD that are predictive of relapse, and insights into the host mechanisms that control MRD over many years, may allow improved therapies to be developed for ALL and for other leukemias.
References 1. Beishuizen A, Hahlen K, Hagememeijer A, et al: Multiple rearranged immunoglobulin genes in childhood acute lymphoblastic leukemia of precursor B-cell origin. Leukemia 9:316, 1991 2. Biernaux C, Loos M, Sels A, et al: Detection of major bcv-abl gene expression at a very low level in blood cells of some healthy individuals. Blood 86:3118, 1995 3. Biondi A, Rambaldi A, Rossi V, et al: Detection of ALLl/AF4 fusion transcript by reverse transcription-polymerase chain reaction for diagnosis and monitoring of acute leukemias with the t(4;ll) translocation. Blood 82:2943, 1993 4. Biondi A, Yokota S, Hansen-Hagge TE, et al: Minimal residual disease in childhood acute lymphoblastic leukemia: Analysis of patients in continuous complete remission or with consecutive relapse. Leukemia 6:282, 1992 5. Brisco MJ, Condon J, Hughes E, et al: Outcome prediction in childhood acute lymphoblastic leukaemia by molecular quantification of residual disease at the end of induction. Lancet 343396, 1994 6. Campana D, Coustan-Smith E: The use of flow cytometry to detect minimal residual disease in acute leukemia. Eur J Histochem 40 (suppl 1):39, 1996 7. Campana D, Coustan-Smith E, Janoosy G: The immunological detection of minimal residual disease in acute leukemia. Blood 76:163, 1990
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8. Campana D, Pui C-H: Detection of minimal residual disease in acute leukemia: Meth-
odologic advances and clinical significance. Blood 85:1416, 1995 9. Cave H, van der Werff ten Bosch J, Suciu S, et al: Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia. N Engl J Med 339:591, 1998 10. Chang K-S, Fan Y-H, Stass SA, et al: Expression of AML1-ETO fusion transcripts and detection of minimal resiudal disease in t(8;21)-positive acute myeloid leukemia. Oncogene 8:983, 1993 11. Cimino G, Elia L, Rapanotti MC, et al: A prospective study of residual-disease monitoring of the ALLUAF4 transcript in patients with t(4;11) acute lymphoblastic leukemia. Blood 95:96, 2000 12. Cimino G, Elia L, Rivolta A, et al: Clinical relevance of residual disease monitoring by polymerase chain reaction in patients with ALL-I / AF-4 positive acute lymphoblastic leukemia. Br J Haematol 92659464, 1996 13. Ciudad J, San Miguel JF, Lopez-Berges MC, et al: Prognostic value of immunophenotypic detection of minimal residual disease in acute lymphoblastic leukemia. J Clin Oncol 16:4774, 1998 14. Coustan-Smith E, Behm FG, Sanchez J, et al: Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 351:550, 1998 15. Cross NCP: Qunatitative PCR techniques and applications. Br J Haematol 89:693, 1995 16. Dewald GW, Wyatt WA, Juneau AL, et al: Highly sensitive fluorescence in situ hybridization method to detect double BCR/ ABL fusion and monitor response to therapy in chronic myeloid leukemia. Blood 91:3357, 1998 17. Dibenedetto SP, Lo Nigro L, Pine Mayer S, et al: Detectable molecular residual disease at the beginning of maintenance therapy indicates poor outcome in children with Tcell acute lymphoblastic leukemia. Blood 90:1226, 1997 18. Dolken G, Illerhaus G, Hirt C, et al: BCL-2/ JH rearrangements in circulating B cells of healthy blood donors and patients with nonmalignant diseases. J Clin Oncol 141333, 1996 19. Donovan JW, Ladetto M, Zou G, et al: Immunoglobulin heavy-chain consensus probes for real-time PCR quantification of residual disease in acute lymphoblastic leukemia. Blood 95:2651, 2000 20. Drach J, Drach D, Glass1 H, et al: Flow cytometric deterination of atypical antigen expression in acute leukemia for the study of minimal residual disease. Cytometry 13393, 1992 21. Eber M, Battmer K, Kafert S, et al: Monitoring of BCR-ABL expression using ”realtime” RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation. Leukemia 133383, 1999 22. Engel H, Drach J, Keyhani A, et al: Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progenitor cells. Leukemia 13:568, 1999 23. Estrov Z, Grunberger T, Dube ID, et al: Detection of residual acute lymphoblastic leukemia cells in cultures of bone marrow obtained during remission. N Engl J Med 315:538, 1986 24. Estrov Z, Freedman MH: Detection of residual disease in acute lymphoblastic leukemia of childhood. Leuk Lymphoma 33:47, 1999 25. Fader1 S, Estrov Z: The clinical significance of detection of residual disease in childhood ALL. Crit Rev Oncol Hematol 473, 1998 26. Finiewicz KJ, Larson RA: Dose-intensive therapy for adult acute lymphoblastic leukemia. Semin Oncol 265-20, 1999 27. Ford AM, Bennett CA, Price CM, et al: Fetal origins of the TEL-AMLI fusion gene in identical twins with leukemia. I’roc Natl Acad Sci U S A 95:4584, 1998 28. Forman SJ, O’Donnell MR, Nademanee AP, et al: Bone marrow transplantation for patients with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 70:587, 1987 29. Foroni L, Coyle LA, Papaioannou M, et al: Molecular detection of minimal residual disease in adult and childhood acute lymphoblastic leukaemia reveals differences in treatment response. Leukemia 11:1732, 1997
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30. Gale KB, Ford AM, Repp R, et al: Backtracking leukemia to birth Identification of clonotypic gene fusion sequences in neonatal blood spots. Proc Natl Acad Sci U S A 94:13950, 1997 31. Gehly GB, Bryant AM, Lee AM, et al: Chimeric BCR-ABL messenger RNA as a marker for minimal residual disease in patients transplanted for Philadelphia chromosomepositive acute lymphoblastic leukemia. Blood 78:458, 1991 32. Goulden NJ, Knechtli CJC, Garland RJ, et al: Minimal residual disease analysis for the prediction of relapse in children with standard-risk acute lymphoblastic leukaemia. Br . Haematol 100:2?35, 1998 33. Greaves M: Silence of the leukemic clone. N E n d T Med 336:317, 1997 34. Gruhn 8, Hongen S, Yi H, et al: Minimal residual disease after intensive induction therapy in childhood acute lymphoblastic leukemia predicts outcome. Leukemia ~12:67i,*1998 35. Heid CA, Steven I, Livak KT, et al: Real time auantitative PCR. Genome Res 6:986, 1996 36. Hilden JM, Frestedt JL, Moore RO, et al: M o l e h a r analysis of infant acute lymphoblastic leukemia: MLL gene rearrangement and reverse transcriptase-polymerase chain reaction for t(4;11)(qZl;q23).Blood 86:3876, 1995 37. Hunger SP, Majilinde Z, Camitta BM, et al: E2A-PBXl chimeric transcript status at end of consolidation is not predictive of treatment outcome in childhood acute lymphoblastic leukemias with a t(1;19)(q23;p13): A Pediatric Oncology Group Study. Blood 91:1021, 1998 38. Hurwitz CA, Gore SD, Stone KD, et al: Flow cytometric detection of rare normal human marrow cells with immunophenotypes characteristic of acute lymphoblastic leukemia cells. Leukemia 6933, 1992 39. Izraeli S, Janssen WSG, Haas OA, et al: Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: A comparison between cytogenetic and polymerase chain reaction analysis. Leukemia 7671, 1993 40. Janossy G, Bollum FJ, Bradstock KF, et al: Cellular phenotypes of normal and leukemic hemopoietic cells determined by analysis with selected antiblody combinations. Blood 56:430, 1980 41. Janssen JWG, Ludwig W, Borkhardt A, et al: Pre-pre-B acute lymphoblastic leukemia: High frequency of alternatively spliced ALL1-AF4 transcripts and absence of minimal residual disease during complete remission. Blood 84:3835-3842, 1994 42. Jurlander J, Caligiuri M, Tuutu T, et al: Persistence of the AMLl/ETO fusion transcript in patients treatmed with allgeneic bone marrow transplantation for t(8;21) leukemia. Blood 88:2183, 1996 43. Knechtli CJC, Goulden NJ, Hancock JE et al: Minimal residual disease status before allogeneic bone marrow transplantation is an important determinant of successful outcome for children and adolescents with acute lymphoblastic leukemia. Blood 924072, 1998 44. Knechtli CTC. Goulden NT. Hancock IP, et al: Minimal residual disease status as a predictor df relaspe after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia. Br J Haematol 102:860, 1998 45. Kuang S-Q Gu L-J, Dong S, et al: Long-term follow-up of minimal residual disease in childhood acute lymphoblastic leukemia patients by polymerase chain reaction analysis of multiple clone-specific or malignancy-specific gene markers. Cancer Genet Cytogenet 88:110, 1996 46. Ludwig W-D, Rieder H, Bartram CR, et al: Immunophenotype and genotypic features, clinical characteristics, and treatment outcome of adult pro-B acute lymphoblastic leukemia: Results of the German multicenter trials GMALL 03/87 and 04/89. Blood 92:189%1909, 1998 47. Mancini M, Nanni M, Cedrone M, et al: Combined cytogenetic FISH and molecular analysis in acute promyelocytic leukaemia at diagnosis and in complete remission. Br J Haematol 91:878, 1995 48. Miyamura K, Tanimoto M, Morishima Y, et al: Detection of Philadelphia chromosomepositive acute lymphoblastic leukemia by polymerase chain reaction: Possible eradication of minimal residual disease by marrow transplantation. Blood 79:1366, 1992 Y
-
~-
~~
~~
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49. Morgan GJ, Pratt G: Modem molecular diagnostics and the management of hematological malignancies. Clin Lab Haematol 20:135, 1998 50. Nelson NC, Hammond PW, Matsuda E, et al: Real time quantitative PCR. Genome Res 6986, 1996 51. Nizet Y, van Daele S, Lewalle P, et al: Long-term follow-up of residual disease in acute lymphoblastic leukemia patients in complete remission using clonogenic IgH probes and the polymerase chain reaction. Blood 83:412, 1993 52. Nucifora G, Larson RA, Rowley JD: Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M 2 in long-term remission. Blood 82712, 1993 53. Pongers-WillemseMJ, Verhagen OJ, Tibbe GJ, et al: Real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia using junctional region specific TaqMan probes. Leukemia 12:2006, 1998 54. Pongers-Willemse MJ, Seriu T, Stolz F, et al: Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TALl deletions as PCR targets. Leukemia 13:110, 1999 55. Potter MN, Stewart CG, Oakhill A The significance of detection of minimal residual disease in childhood acute lymphoblastic leukemia. Br J Haematol 83:412, 1993 56. Preudhomme C, Henic N, Cazin B, et al: Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL).Leukemia 11:294, 1997 57. Privitera E, Rivolta A, Ronchetti D, et al: Reverse transcriptase/polymerase chain reaction follow-up and minimal residual disease detection in t(l;l9)-positive acute lymphoblastic leukemia. Br J Haematol92:653, 1996 58. Radich J, Gehly G, Lee A, et al: Detection of bcr-abl transcripts in Philadelphia chromosome-positive acute lymphoblastic leukemia after marrow transplantation. Blood 89:2602, 1997 59. Radich JP, Kopecky KJ, Boldt DH, et al: Detection of BCR-ABL fusion genes in adult acute lymphoblastic leukemia by the polymerase chain reaction. Leukemia 9:1668,1994 60. Radich J, Ladne P, Gooley T PCR-based detection of unique immunoglobulin V-D-J gene rearrangements in acute lymphoblastic leukemia predicts relapse after allogeneic bone marrow transplantation. Biol Blood Marrow Transplant 1:24, 1995 61. Radich JP, Gehly G, Gooley T, et al: Polymerase chain reaction detection of BCRABL fusion transcript after allogeneic marrow transplantation for CML: Results and implications in 346 patients. Blood 85:2632, 1995 62. Roberts WM, Estrov Z, Ouspenskaia MV, et al: Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N Engl J Med 336317, 1997 63. Schnittger S, Wormann 8, Hiddemann W, et al: Partial tandem duplications of the MLL gene are detectable in peripheral blood and bone marrow of nearly all healthy donors. Blood 921728, 1998 64. Secker-Walker LM, Prentice HG, Durrant J, et al: Cytogenetics adds independent prognostic information in adults with acute lymphoblastic leukaemia on MRC trial UKALL XA. Br J Haematol 91:878, 1995 65. Sierra J, Radich J, Hansen JA, et al: Marrow transplants from unrelated donors for treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia. Blood 90:1410, 1997 66. Seong D, Giralt S, Fischer H, et al: Usefulness of detection of minimal residual disease by "hypermetaphase" fluorescent in situ hybridization after allogeneic BMT for chronic myelogenous leukemia. Bone Marrow Transplant 19:565, 1997 67. Steenbergen EJ, Verhagen OJHM, van Leeuwen EF, et al: Prolonged persistence of PCR-detectable minimal residual disease after diagnosis or firs; relapse predicts poor outcome in childhood 8-precursor acute lymphoblastic leukemia. Leukemia 91726, 1995 68. Steenbergen EJ, Verhagen OJ, Van den Berg H, et al: Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to DJ joining) in childhood B precursor acute lymphoblastic leukemia. Leukemia 11:1258, 1997
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69. Stock W, Sher D, Dodge R, et al: Quantitative molecular monitoring of BCRIABL transcripts in adult acute lymphoblastic leukemia (ALL) using a real-time PCR assay: Pilot study from the Cancer and Leukemia Group B (CALGB 8762). Blood 94(suppl 1):287a, 1999 70. Terstappen LWMM, Loken MR Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials. Cytometry 9:548, 1988 71. Uckun FM, Hermann-Hatten K, Crotty M-L, et al: Clinical significance of MLL-AF4 fusion transcript expression in the absence of a cytogenetically detectable t(4;11)(q21;q23) chromosomal translocation. Blood 92810, 1998 72. Van Bekkum DW: Reflection on the detection and treatment of leukemia. In Lowenberg B, Hagenbeck A, (eds): Minimal Residual Disease in Acute Leukemia. Boston, Martinus Nijoff, 1984, pp 385-390 73. van Dongen JJM, Szczepanski, de Bruijn MAC, et al: Detection of minimal residual disease in acute leukemia patients. Cytokines and Molecular Theory 2121, 1996 74. van Dongen JJM, Breit TM, Adriaansen HJ, et al: Detection of minimal residual disease in acute leukemia by immunological marker analysis and polymerase chain reaction. Leukemia 6S1:47, 1992 75. van Dongen JJM, Seriu T, Panzer-Gmmayer ER, et al: Prognostic value of minimal residual disease in acute lymphoblastic leukaemia in childhood. Lancet 3521731, 1998 76. Vora A, Frost L, Goodeve A, et al: Late relapsing childhood lymphoblastic leukemia. Blood 922334, 1998 77. Wasserman R, Galili N, Ito Y, et al: Residual disease at the end of induction therapy as a predictor of relapse during therapy in childhood B-lineage acute lymphoblastic leukemia. J Clin Oncol 10:1879, 1992 78. Weir G, Cowan K, Le Beau P, et al: A limited antibody panel can distinguish Bprecursor acute lymphoblastic leukemia from normal B precursors with four color flow cytometry: Implications for residual disease detection. Leukemia 13:558, 1999 79. Wells DA, Sale GE, Shulman HM, et al: Multidimensional flow cytometry of marrow can differentiate leukemic from normal lymphoblasts and myeloblasts after chemotherapy and bone marrow transplantation. Am J Clin Pathol 110:84, 1998 80. Westbrook CA, Hooberman AL, Spin0 C, et al: Clinical significance of the BCR-ABL fusion gene in adult acute lymphoblastic leukemia: A Cancer and Leukemia Group B Study (8762). Blood 802983, 1992
Address reprint requests to: Wendy Stock, MD Leukemia Program University of Chicago 5841 S. Maryland M / C 2115 I208 Chicago, IL 60637
0889-8588/00 $15.00
+ .OO
STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA Wendy Stock, MD, and Zeev Estrov, MD
During the past 2 decades, there has been considerable progress in the treatment of both childhood and adult acute lymphocytic leukemia (ALL). Currently, 70% to 90% of adults achieve a complete remission (CR) and 25% to 50% of these patients may experience prolonged disease-free survival (DFS) and may be cured of their disease. Unfortunately, most adults with ALL will ultimately experience a recurrence and die of their leukemia. Although most children with ALL may now be cured with current therapeutic regimens, the ability to distinguish good-risk patients from those who are likely to relapse has important clinical implications. Relapse, in most pediatric and adult cases, is thought to result from residual leukemia cells that remain following achievement of "complete" remission but are below the limits of detection using conventional morphologic assessment of the bone marrow. Sensitive techniques are now available to detect subclinical levels of residual leukemia, termed minimal residual disease (MRD). Study of MRD may have great usefulness for assessing the clinical efficacy of current and future strategies aimed at improving the cure rate in ALL and other malignancies; however, the clinical significance of MRD detection and monitoring remains to be determined. Several important questions must be answered by carefully controlled, prospective studies before the therapeutic interventions based on MRD monitoring strategies are implemented. First, does early detection of leukemia
From the Leukemia Program, Section of Hematology/Oncology, University of Chicago, Chicago, Illinois (WS); and Department of Bioimmunotherapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas (ZE)
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by sensitive techniques predict relapse? Second, if early detection is predictive of relapse, can early detection at a molecular level guide therapy (i.e., should the intensity of treatment be increased or decreased based on MRD results)? Also, does the treatment of a molecular relapse actually improve survival? Third, is it essential for the leukemic clone detected by sensitive MRD assays to be completely eliminated to achieve a cure? Studies of MRD may provide insights into the pathogenesis and biologic behavior of leukemia. Will sensitive molecular monitoring facilitate the identification of homeostatic mechanisms that modulate growth of the leukemic clone? This article focuses on current strategies for monitoring MRD in ALL, discusses the recent studies of MRD monitoring in ALL, and reviews the possible clinical relevance of MRD studies. METHODS FOR DETECTING MINIMAL RESIDUAL DISEASE A patient in clinical remission from leukemia can still harbor up to 1O1O leukemia cells that remain below the threshold of detection by morphologic methods.n During the last decade, a variety of techniques have been studied for the detection of residual disease. These methods include cytogenetics, cell culture systems, fluorescence in situ hybridization (FISH), Southern blotting, immunophenotyping, and polymerase chain reaction (PCR) techniques (Table l).5,6, s, 14, 22, 55, @,69, 70, 73, 74, 78, 79 The applicability of the techniques for MRD detection depends on three parameters: specificity (ability to discriminate between malignant and normal cells without false-positive results); sensitivity (detection limit of at least lop3, or detection of 1 malignant cell in a background of at least 1000 normal cells); and reproducibility and applicability (easy standardization and rapid collection of results for clinical application).” The detection limit of most of these is not lower than 1%to 5% malignant cells; however, depending on the phenotype and genotype of the leukemia, immunophenotyping and PCR techniques are able to detect lower frequencies of leukemia cells and fulfill the criteria for MRD detection. Immunophenotyping techniques using multicolor-gated flow cytometry are based on aberrant expression of antigens by the leukemia cell popul a t i ~ n .70,~ ,72 For example, in B-cell-lineage ALL, leukemic blasts may coexpress cross-lineage T-cell antigens (e.g., CD5 and CD7) or myeloid antigens (e.g., CD13 and CD33), have asynchronous expression of antigens, or drop or overexpress a specific antigen (particularly CD10).20,38 Recently by using two four-color combinations of antibodies (CD19, CD45, CD20, CDlO and CD19, CD45, CD9, CD34), aberrant leukemic blasts were identified in 81 of 82 pediatric B-lineage ALL cases.78In Tlineage ALL, leukemic blasts nearly always coexpress terminal deoxynucleotidyl transferase (TdT), CD2, cytoplasmic CD3, CD5, and CD7 in addition to other T-cell antigens. Cells with this immunophenotypic pattern are absent or occur very rarely in normal bone marrow or
Colony growth
Molecular markers
Karyotype analysis
Immunophenotyping
Marker
Flow cytometry (multiparameter) Fluorescence microscopy Cytogenetic studies Fluorescence in situ hybridization (FISH) Southern blotting Polymerase chain reaction Clone-specific Leukemia-specific (fusion gene) DNA RNA (reverse transcription-PCR) Clonogenic assay
Technique
Table 1. METHODS FOR DETECTION OF MINIMAL RESIDUAL DISEASE
510-4
10-1-10-2 10-4-10-2
10-2-10-4 10-2-10-4 10-'-10-2 10-2-10-4
Detection Limit
90
70-90 20-50
60-90
23
8, 24 (for review) 49, 53, 54 (techniques)
8, 47, 64 8, 22, 49, 55, 64, 66
5-40
References
5, 6, 8, 13, 14, 70, 73, 74, 78, 79
("/I
90-100
Applicability
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Thus, it is theoretically possible to detect MRD in most patients with ALL using immunophenotypic analysis, and this technique allows accurate quantification of the leukemic population. A recent study of 128 children with ALL showed sequential MRD monitoring using multiparameter flow to be a valuable predictor of re1ap~e.l~ Low-frequency normal hematopoietic progenitor cells, similar to leukemic blasts, may express the same cytoplasmic or surface-bound marker profile, however, rendering distinction between normal and malignant cells difficult in the MRD setting. There is also concern about the potential for change in immunophenotypic expression during disease progression resulting in a false-negative result.4O Because most recent studies of MRD in adult ALL have used PCR techniques, the remainder of this article focuses on these studies. Detection of Minimal Residual Disease by Polymerase Chain Reaction Using PCR for specific amplification of a DNA sequence or cDNA unique to the leukemia clone can permit identification of 1 malignant cell among lo4 to lo6 normal cells. Two types of PCR targets can be used to detect MRD in ALL patients: junctional regions of leukemia clone-specific rearranged immunoglobulin and T-cell receptor (TCR) genes, or leukemia-specific breakpoint fusion regions of chromosome rearrangements (translocations, deletions, or inversions) (Table 2). The deletion and random insertion of nucleotides during immunoglobulin and TCR gene rearrangement generates unique junctional sequences that can serve as clone-specific markers of the leukemia. The precise nucleotide sequence of the junctional region of the immunoglobulin or TCR genes in the leukemia cells can be identified at the time of diagnosis allowing the design of oligonucleotides specific to the junctional region. These oligonucleotides can then be used as patient-specific probes (or primers) for detecting MRD during and following treatment of the le~kemia.5~ Oligoclonality of immunoglobulin and TCR gene rearrangements at diagnosis is a relatively frequent phenomenon.', 68 Continuing rearrangements and secondary rearrangements that occur during the disease course might result in the loss of the specific junctional regions initially identified at diagnosis. Therefore, it seems to be important to monitor ALL patients with two or more independent PCR targets to prevent false-negative results during f o l l ~ w - u p . ~ * ~ ~ Leukemia-specific chromosome rearrangements are also useful PCR targets for detecting MRD. Oligonucleotide primers are designed at opposite ends of the breakpoint fusion region so that the PCR product contains the tumor-specific fusion sequences. Because the PCR products should not exceed about 2 kilobases (kb) in routine MRD studies, PCR amplification can only be used for chromosome aberrations in which the breakpoints cluster in a small area (preferably <2 kb). In ALL, DNAbased PCR for MRD can be performed for T-lineage ALL with TALI gene
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Table 2. FREQUENCY OF PCR TARGETS AVAILABLE FOR MRD DETECTION IN ALL Frequency (%) ALL Lineage
Chromosomal Abnormality
Molecular Target
Adults
Children
-45 -60 -80 -50 25-30 5-8 <5 2-3 4-5 <1 <1 <1 <1
-55 -60 -90 -35
B-lineage
-
TCRG (DNA) TCRy (DNA) IgH (DNA) IgK-Kde* (DNA) BCR-ABL (RNA) MLL-AF4 (RNA) TEL-AML1 (RNA) E2A-PBX1 (RNA) MYC-IgH (DNA) IL3-IgH1 (DNA) MLL-ENL (RNA) E2A-HLF (RNA) MLL-AF9 (RNA) TCRG (DNA) TCRG (DNA) TCRy (DNA) IgH (DNA) IgK-Kde (DNA) TALl deletion (DNA) RHOM2-TCRy TALl -TCRa H O X l l -TCRa
-70 -70 -85 -5-10 0 10-20 5-10 <1 1-3
4-6
3-5 25-30 5-6 1-2 <1 <1 <1 <1 -50 -50 -90 -10-20 0
‘The t(12;21) is a cryptic translocation only rarely identified using standard cytogenetics. PCR = polymerase chain reaction; MRD = minimal residual disease; ALL = acute lymphocytic leukemia; TCR = T-cell receptor gene rearrangements; IgH = immunoglobulin heavy chain rearrangements; IgK-Kde = Ig kappa gene deletional rearrangement joined to kappa-deleting element.
deletions (SIL-TALZ), the t(ll;l4)(pl3;qll) involving RHOMZ-TCRD, the t(1;14)(p32;qll) involving TALI-TCRD, and the t(10;14)(q24;qll) involving HOXZZ-TCRD.” In most of the chromosome translocations common to adult ALL, however, the breakpoints are spread over areas much larger than 2 kb; therefore, MRD detection and monitoring depends on identifying the resultant leukemia-specific fusion mRNA. This fusion mRNA can be used as a target for MRD analysis using PCR after the fusion mRNA (consisting of transcribed coding exons) is converted to cDNA using the enzyme reverse transcriptase (RT). This technique is known as reverse transcriptase PCR (RT-PCR). In ALL, RT-PCR is used to detect the following transcripts: BCX-ABL resulting from the t(9;22); E2A-PBXZ in cases of pre-B ALL with a t(1;19); MLL-AF4 in t(4;ll) leukemia; and the TEL-AMLZ resulting from the t(12;21) in precursor-B ALL (Table 2 ) . In monitoring MRD, PCR has the advantages of extraordinary sensitivity, speed, and minimal tissue requirements. The disadvantages of
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PCR for monitoring MRD encompass a variety of technical and biologic issues, includingz5 contamination of the reaction product (which can be avoided with strict quality control) the lack of reproducibility of results when small numbers of transcripts are present the risk of RNA degradation and inefficiency during conversion of mRNA to cDNA (which may reduce the sensitivity of RT-PCR monitoring) evolution of the leukemic clone subclone formation the presence of oligoclonal populations that can cause both falsenegative and false-positive results. Precise quantification of copy number using PCR has also been problematic in the past. Endpoint measurements, that is, analysis of the reaction product after amplification is completed, commonly have been used for quantification. These measurements depend on multiple dilutions or coamplification of standards (internal or external to the reaction system) and are cumbersome, error prone, and technically demandir~g.3~ New automated PCR techniques, however, will probably allow more precise and consistent quantitation of residual leukemic clones.15,35 One such technique, known as red time PCR, uses a fluorogenic probe, an oligonucleotide with both a reporter fluorescent dye and a quencher dye atta~hed.3~. 50 This probe is designed to anneal specifically between the forward and reverse primers used to amplify a specific target sequence. During PCR, the probe is cleaved by the 5’ nuclease activity of Taq DNA polymerase during primer extension. Cleavage of the probe separates the reporter dye from the quencher dye, generating a fluorescent reporter dye signal. With each cycle of amplification, additional reporter molecules are cleaved from their probes, increasing the fluorescence intensity in proportion to the amount of transcript produced during the logarithmic phase of PCR amplification. Recent studies of MRD monitoring using real time technology, some of which are discussed later, are beginning to provide new insights into the potential clinical significance of MRD detection. CLINICAL STUDIES MEASURING RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA BCR-ABL in Philadelphia ChromosomePositive Acute Lymphoblastic Leukemia
Reverse transcriptase polymerase chain reaction has been used to detect and monitor the BCR-ABL fusion gene associated with the t(9;22) that occurs in as many as 30% of adults with ALL.31,58 In adult patients expressing B-lineage surface antigens, approximately 50% of cases are
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BCR-ABL-positive ( + ). Most of the patients (approximately 70%-75%) have the p190 subtype; the p210 subtype of BCR-ABL that is present in almost all patients with CML occurs in approximately 25% to 30% of patients with ALL.8o The poor prognosis of adults with BCR-ABL+ ALL treated with combination chemotherapy justifies attempts to intensify treatment in first remission using allogeneic transplantation, when available for this group of patients (see article by Radich elsewhere in this issue). Allogeneic transplantation improves DFS for some of these patients; however, even with transplantation, the relapse rate for BCR-ABL+ ALL is quite high, ranging from 40% to 28, 65 Several studies of MRD status following allogeneic transplantation provide intriguing information about the risk of relapse in BCR-ABL+ ALL and may help identify patients who are likely to relapse. In the studies listed in Table 3, RTPCR techniques with a reported sensitivity of 1 leukemia cell in a background of lo5 normal cells were used to study MRD following 48, 56, 58 All these studies found allogeneic or autologous that patients who are consistently BCR-ABL negative following transplantation are unlikely to relapse and may become long-term survivors. Conversely, patients who are BCR-ABL following transplantation seem to be at high risk for subsequent relapse. In the largest published series (28 patients), Radich et a1 found that the relative risk (RR) for relapse was significantly higher for patients with a detectable BCR-ABL transcript following transplantation than for those without detectable BCR-ABL (RR = 5.7; P = 0.025)?8 The prognostic significance of the PCR assay remained after controlling for other clinical variables that could influence relapse risk. The risk of relapse was greater for patients with a p190 fusion transcript than for those with a p210 BCR-ABL. The median time from detection of a positive PCR result to relapse was 94 days. Additional insights into the kinetics of disappearance of BCR-ABL during
+
Table 3. CLINICAL RELEVANCE OF DETECTION OF BCWABL TRANSCRIPTS AFTER ALLOGENEIC OR AUTOLOGOUS TRANSPLANTATION FOR Ph ALL'
+
PCR = Positive Study
No. of Patients
p190 BCFUABL Gehly3' Miyamuraa Pre~dhomrne~~
Radich6' p210 BCWABL
Radich6'
PCR = Negative
No. of Patients
No. of Relapses (%)
No. of Patients
No. of Relapses (%)
4 15 9
1 7 5
28
15
1 (100) 7 (100) 5 (100) 9 (60)
3 8 4 13
0 1 (13) 0 2 (15)
8
8
1 (13)
0
0
*Results were obtained using qualitative, not quantitative PCR, usually within the first 100 days after transplantation. Ph + = Philadelphia chromosome-positive;ALL = acute lymphocytic leukemia; PCR = polymerase chain reaction.
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treatment will be obtained from current studies using real-time PCR techniques to quantify transcript number repr~ducibly.~~ Prospective PCR validation with quantitative techniques in a larger series of BCRA B L + patients is clearly required; however, with rigorous monitoring it may be possible to introduce novel therapies to eliminate residual disease and avoid the dire consequences of a clinical relapse in these patients.
Studies of Other Fusion Transcripts in Minimal Residual Disease in Acute Lymphocytic Leukemia: MLL-AF4 and E2A-PBX1 The M L L gene, located on chromosome band 11q23, is known to be involved in more than 30 translocations in acute leukemias (ALL and acute myeloid leukemia [AML]) and lymphomas. In ALL, the most common M L L gene abnormality is the MLL-AF4 (also known as ALLZAF4) fusion gene that results from the t(4;11)(q21;q23). It is found in approximately 3% to 6% of adults and children with ALL and is the most common cytogenetic abnormality in infants with ALL.3,46 The t(4;ll) has been associated with a very poor prognosis when standard chemotherapy regimens are employed, but improved survival has been noted in patients undergoing allogeneic transplantation in first remission or when intensive postremission treatment with high-dose cytarabine is A recently published prospective analysis of MRD monitoring in 25 patients with the MLL-AF4 transcript is the largest study of residual disease in these patients.'* In this study, qualitative RT-PCR analysis with a sensitivity of 1 in lo4 showed that in patients who were in a clinical remission at the end of high-dose induction and consolidation therapy (without bone marrow transplantation) continued PCR positivity (in 13 of 25 patients), or reconversion from negative to positive (in 5 of 25 patients) was predictive of relapse. Patients who remained PCR-negative during the first 3 to 6 months following diagnosis seemed to enjoy prolonged disease-free survival. These results are concordant with several other smaller MRD series that suggest that MLL-AF4 patients who remain persistently PCR-positive at early time points following chemotherapy or transplantation have a high likelihood of relapse and that PCR negativity following transplantation is highly predictive for relapse-free survival.'*,36, 41 Polymerase chain reaction monitoring studies have also been performed in ALL patients with the E2A-PBXZ fusion gene that results from the t(1;19)(q23;p13).Unlike the studies described previously, in one study of 41 children with a t(1;19)(q23;p13), qualitative RT-PCR detection of the E2A-PBXZ at the end of consolidation therapy was not predictive of treatment These results are similar to those noted in several smaller studies of MRD with the E2A-PBXZ fusion transcript.39,58 Discordance between matched blood and bone marrow samples was noted
STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA
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in a number of these emphasizing the need for careful comparison of these tissues in future MRD studies. Clone-Specific Polymerase Chain Reaction using Immunoglobulin Heavy Chain and T-cell Receptor Gene Rearrangements
Clone-specific PCR studies of MRD in ALL have been performed mostly in children; few studies have involved adults. The results from these trials have not always been consistent, and this variability may be ascribed to a number of factors: (1)PCR testing has been performed at different times during the treatment cycle; (2) some of the studies used qualitative PCR assays, whereas others involved a number of different methods for quantitation of the PCR product; (3) sample sources have been different (fresh bone marrow versus archival material scraped from bone marrow slides); and (4) the treatment regimens have varied considerably. Nevertheless, several conclusions can be drawn from these studies that are summarized in Table 4. Serial clone-specific PCR analyses of remission samples demonstrate that during the immediate postinduction period, from 40% to 70% of patients are still positive for MRD32,45 and that these levels often continue to decline during the first,6 months after initiation of thera~y.2~ Several of these studies suggested that detection of residual disease by PCR following induction (1to 2 months after starting therapy) has prognostic value.5,29, 34, 77 Patients with positive PCR results following induction therapy and those who did not show a consistent decrease in residual disease levels during the first 6 months of treatment had a significantly higher risk of relapse than did patients with low-level residual disease or negative PCR results. A number of recent large, prospective MRD-monitoring studies in pediatric ALL confirm these findings and suggest that quantitation of MRD may be critical because the probability of long-term relapse-free survival seems to be directly related to the level of residual disease, both early in the course of treatmen@9, 75 and at later time point^.^ In their semiquantitative MRD monitoring study of 246 children with ALL, Cave et a1 demonstrated in a multivariate analysis that PCR detection of more than lo-* residual blasts following intensive induction chemotherapy was the most powerful independent predictor of early r e l a ~ s e When .~ later time points during treatment were analyzed, a MRD burden greater than was associated with significantly higher relapse rates. In another large study of MRD involving 240 children with ALL, van Dongen et a1 found that combining semiquantitative MRD information from several time points during treatment identified three risk groups to be distinguished. Forty-three percent of patients were in a low-risk group with a 3-year relapse rate of only 2%; 43% were in an intermediate-risk group with a relapse rate of 23%; and 15% were in a high-risk group with a 75% relapse rate.75Other studies confirm the significance
+/-
Van D ~ n g e n ~ ~ 240 C
30 A
For~ni~~
+/-
+/-
+
After induction After consolidation 6 months after consolidation After 6 months After end of therapy After induction After 6 months After consolidation After induction After 4 months Before maintenance End of therapy Up to 35 months after end of therapy After induction After induction After 3 months After 6 months After induction After 3 months After 6 months After 12 months
Time of Testing
MRD = minimal residual disease; PCR = polymerase chain reaction; C = children; A = adults; = no quantitation.
24 C 50 C 27 A 10 A
Roberts62 Steenberged7 Brisco5 NizeP'
+ +/+
+/-
66 C
Go~lden~~
23 C
+
246 C*
Number
Cave9
Reference
Quant,itative Assay
Table 4. SELECTED STUDIES OF MRD USING CLONE-SPECIFIC PCR
+/ -
25 15 21 23 18 40 32 5 8 8 7 5 10 11 3 3 2 5 4 1
Relapse
= quantitative PCR assay;
20 12 18 9 9 5 8 5 2
-
63 32 38 33 28 98 47 6 16 16 8
Positive
PCR Results
=
7 8 25 5 6 2 18 0 0 0 0 0 0 6 3 0 0 2 1 1 3 -
Relapse
semiquantitative PCR assay;
88 95 215 29 30 71 166 148 1 1 8 7 2 22 9 1 1 5 5 10 9
Negative
PCR Results
STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA
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of serial, quantitative measurements of residual disease and indicate that the timing of disappearance and reappearance of marrow blasts is crucial in predicting those who are destined to relapse and those who will 51 For example, the presence of residual disease at remain in remis~ion.~, the time of maintenance therapy has recently been shown to be an accurate predictor of outcome in children with T-lineage ALL.17 Studies of MRD have also used clone-specific techniques in the setting of allogeneic stem cell transplantation for ALL. Sixty-four children and adolescents with ALL who were in morphologic remission were studied for the presence of MRD immediately before allogeneic transplantation. The presence of any MRD before transplantation was associated with a 2 year event-free survival (EFS)of only 17%, in contrast to an EFS of 73% for those who had no detectable MRD.43Detection of MRD following allogeneic transplantation has also correlated with higher relapse rates. The same authors studied 71 children and found that patients who exhibited MRD during the first 3 months following transplantation had a ninefold higher relapse rate than MRD-negative patients. The detection of MRD was quite unusual in those patients who remained in remission. Only one of 35 patients in CR had a positive PCR result for MRD when the study was performed more than 6 months following transplantation.44The presence of MRD using clone-specific PCR detection following allogeneic transplantation was also predictive of relapse in another study composed primarily of adults with ALL.60 Despite the finding from many of these studies that the presence of residual disease portends relapse following completion of combination chemotherapy or allogeneic transplant, other studies suggest that not all patients with MRD experience relapse. In the Cav6 study mentioned previously, about 20% of the patients with an MRD level greater than did not relapse. Molecular evidence of a residual leukemic clone has been detected in bone marrow for as long as 9 years following In a recent completion of therapy for ALL without a clinical relap~e.5~ study using a highly sensitive quantitative PCR assay combined with clonogenic assays to assess residual disease in children with ALL, MRD was demonstrated in 15 of 17 patients who nevertheless remained in remission from 2 to 35 months after completion of all treatment.62Longterm persistence of MRD in patients who remain in clinical remission has also been described in patients with AML with a t(8;21) and in patients who have undergone allogeneic stem cell transplantation for chronic myelogenous leukemia (CML).l0,43, 52, 61 Similar results have been described previously in patients with p210 BCR-ABL+ ALL and in children with t(1;19) ALL. Another recent study suggests that prolonged dormancy of the leukemic clone can be established. In this study of 1134 children who were long-term survivors of ALL, Vora et a1 studied 12 children who eventually relapsed more than 10 years after diagnosis.76 In 8 of these relapsed cases, the investigators were able to study DNA from the original diagnosis and from the relapsed bone marrow. In each relapsed sample they found the identical immunoglobulin heavy-chain (IgH) or TCR gene rearrangement that was present in the leukemic clone
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from the diagnostic bone marrow specimen. This finding suggests that the leukemic cells or clonal precursors survived for more than a decade. CLINICAL SIGNIFICANCE OF THE DETECTION OF MINIMAL RESIDUAL DISEASE IN LEUKEMIA The studies of MRD in ALL and other leukemias have assumed great importance because long-term DFS seems to depend on the ability to control residual disease. The initial generation of studies of MRD in ALL provides tantalizing preliminary data suggesting that, in certain subsets of disease, the absence of a leukemia-specific marker, such as p190 BCR-ABL, is predictive of long-term remission, whereas its persistence may portend relapse. The persistence of MRD is less clear when other molecular markers are used. It is clear, however, that the potential clinical usefulness of each type of MRD assay needs to be determined rigorously before results are used to modify treatment of individual patients or groups of patients. Before PCR is used for clinical interventions, it must be determined that such a test reveals identical results in different laboratories and that it has acceptable sensitivity, specificity, and positive and negative predictive values. Future studies of MRD in adult ALL will require prospective, sequential analyses on larger numbers of patients, as has been performed in the European pediatric ALL studies of MRD. The use of automated, quantitative real time PCR is becoming more widespread and may be essential for technique stanThe dardization and for the validation of the earlier ~ t u d i e s .69' ~ ~ ~current ~~ generation of MRD studies is likely to determine more clearly whether a threshold level may be defined above which patients are likely to relapse and whether serial monitoring will provide data that will be useful for future clinical interventions designed to minimize toxicity or prevent overt relapse. The European pediatric cooperative group, IBFM-SG, has just begun a prospective trial in which the results of a semiquantitative MRD assay are being used to stratify postremission treatment in children with ALL (JVD Van Dongen, personal communication, 2000). These studies also provide fascinating clues to the pathogenesis of these diseases and the biologic properties of residual leukemia cells. It seems that residual disease is a dynamic process, with the numbers of 62 Although shortly after residual leukemia cells fluctuating over therapy the levels of residual disease may fall below the detection limit of even highly sensitive molecular assays, the leukemia cells do not necessarily disappear in clinical remission. They may increase until clinically overt disease recurs, or they may fall below the threshold of detection again. A positive-negative-positive pattern of PCR results is common, and molecular relapse does not always predict clinical relapse. When the disease remains dormant for years, as described previously, the leukemic clone may have come under the control of an immunologic or other homeostatic mechanism. Alternatively, the detected cells containing a specific fusion gene transcript such as p210 BCR-ABL, or E2A-PBXZ,
STUDIES OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOCYTIC LEUKEMIA
1301
although members of the leukemic clone, may behave as preleukemic cells that require additional genetic mutations (perhaps acquired by a subclone of descendant cells at the time of diagnosis). These additional mutations 30, 33 Also, falsemay be essential for full leukemic transformati~n.~~? negative PCR results can occur with clone-specific PCR as a result of clonal evolution in IgH or TCR genes of the leukemia cells and therefore limit the predictive power of a negative result!, 51, 55 Furthermore, because leukemic blasts are considered malignant counterparts of normal, immature hematopoietic progenitor cells, they express closely related differentiation and lineage markers.62It is, therefore, possible that seemingly residual leukemic cells may in fact be normal hematopoietic cells. This observation is highlighted by the fact that disease-specific fusion gene products, including BCR-ABL, BCL2-IgH, MLL-AF4, and the partial tandem duplication of MLL have been detected with highly sensitive PCR techniques in healthy individuals.z, 63, 71 Molecular assay techniques such as PCR provide unparallel sensitivity for the detection of residual disease. These assays have contributed significantly to the understanding of residual disease in ALL and other hematologic malignancies. The usefulness of these powerful molecular techniques extends beyond detection of residual disease and is redefining the concept of that has been based on criteria formulated many years before the introduction of molecular biologic assessment. Eventually, the knowledge gained from MRD studies, including quantification of residual disease, understanding of the fluctuations in residual disease levels over time, identification of threshold levels of MRD that are predictive of relapse, and insights into the host mechanisms that control MRD over many years, may allow improved therapies to be developed for ALL and for other leukemias.
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-
~-
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~~
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Address reprint requests to: Wendy Stock, MD Leukemia Program University of Chicago 5841 S. Maryland M / C 2115 I208 Chicago, IL 60637