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Keywords:

  • acute leukaemia;
  • minimal residual disease;
  • flow cytometry;
  • PCR;
  • quantitative PCR

In patients with acute leukaemia, studies of minimal (i.e. submicroscopic) residual disease (MRD) should improve measurements of treatment response and enable estimates of the residual leukaemic cell burden during clinical remission, thereby improving the selection of therapeutic strategies and, possibly, long-term clinical outcome. The most useful methods for MRD monitoring currently available are polymerase chain reaction (PCR) amplification of fusion transcripts and rearranged antigen-receptor genes, and flow cytometric detection of aberrant immunophenotypes. Several studies in patients with acute lymphoblastic leukaemia and acute myeloid leukaemia have demonstrated that MRD is a powerful and independent prognostic indicator. The strong association between MRD and risk of relapse was observed in children and adult patients irrespective of the methodology used to detect residual disease. This article discusses the relative advantages and disadvantages of each MRD assay, and reviews the reported correlations between MRD, clinical and biological features of the disease, and outcome.

Rationale for studies of mrd

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

A multitude of factors influence treatment response in patients with acute leukaemia (Lowenberg et al, 1999; Pui et al, 2001). Cell lineage, stage of maturation, karyotype and molecular abnormalities regulate expression of genes that control drug metabolism and apoptosis, and determine the leukaemic cells' capacity to grow and their sensitivity to chemotherapy. Other important factors include size of the tumour burden, dosage of drugs and their interaction, pharmacokinetic and pharmacogenetic variables, and compliance with treatment schedule. These parameters are associated with a varying risk of relapse but their predictive power is far from absolute and their use to make treatment decisions in individual patients is inherently limited.

In vivo measurements of leukaemia cytoreduction should reflect the combined effect of clinical and cellular variables; rather than predicting outcome, these measurements provide direct information on the effectiveness of treatment in each patient. However, estimates by conventional morphological techniques have limited sensitivity and accuracy: in most cases, leukaemic cells can be detected in bone marrow with certainty only when they constitute 5% or more of the total cell population. Such a high detection threshold results in the inability to measure fluctuations in the leukaemia tumour mass with accuracy, and resurgent disease can be diagnosed only at its most advanced stages. Methods for detecting MRD (i.e. submicroscopic) are at least 100-times more sensitive than conventional morphological techniques and allow a more stringent definition of ‘remission’ in patients with acute leukaemia, which is rapidly becoming the standard at many cancer centres (Fig 1).

image

Figure 1. Factors that influence response to treatment in patients with acute leukaemia.

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An essential premise of MRD studies is that MRD levels provide reliable estimates of the residual leukaemia mass. This assumes that leukaemic cells are homogeneously distributed throughout the bone marrow so that measurements of MRD performed on a small aliquot of bone marrow (perhaps, equivalent to one thousandth of the total active marrow) are representative of the total leukaemia burden. However, observations in patients (Mathe et al, 1966) and in animal models of leukaemia (Martens et al, 1987) indicate that there could be considerable heterogeneity in the distribution of leukaemic cells after treatment and that individual samples may not be informative. Another caveat is that MRD signals may not correspond to viable leukaemic cells with a capacity for cell renewal. Cell viability can only be determined with methods that use intact cells (e.g. flow cytometry) but it is impossible to determine with MRD methods that examine nucleic acid material (e.g. polymerase chain reaction). Moreover, even when viable, leukaemic cells may lack sustained self-renewal capacity, a feature that is not possible to assess on a routine basis. As discussed in this article, most of these reservations have been dispelled by the results of several correlative studies of MRD and treatment outcome. It is now clear that detectable MRD implies the presence of leukaemic stem cells capable of leading to disease recurrence.

Methodological options for mrd studies

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

The first MRD studies in patients with leukaemia were made soon after antibodies for leucocyte differentiation antigens became available. Strong expression of the common acute lymphoblastic leukaemia (ALL) antigen (CD10) and terminal deoxynucleotidyl transferase (TdT) in ALL cells, and apparent absence of these markers on normal peripheral blood cells, suggested the use of these molecules as markers of leukaemia. However, it was soon clear that a proportion of cells in the bone marrow (i.e. B-cell progenitors) expressed both CD10 and TdT (Janossy et al, 1980). The distinction between leukaemic lymphoblasts and normal lymphoid progenitors remains crucial for detecting MRD in patients with B-lineage ALL. In early studies, it was also noted that T-lineage ALL cells (and normal thymocytes) expressed TdT and T-cell markers whereas lymphoid cells in bone marrow and peripheral blood did not (Bradstock et al, 1981). This finding enabled the first productive MRD studies in patients with acute leukaemia, and remains the basis of MRD studies by immunological techniques in T-lineage ALL.

Over the following two decades, many methods to study MRD have been tested (Campana & Pui, 1995; Szczepanski et al, 2001). In ALL, the most reliable methods include flow cytometric profiling of aberrant immunophenotypes, polymerase chain reaction (PCR) amplification of fusion transcripts and chromosomal breakpoints, and PCR amplification of antigen-receptor genes. In acute myeloid leukaemia (AML), only the first two of these methods can be applied, as most patients lack antigen-receptor gene rearrangements. Owing to limited sensitivity (approximately 1–5%), conventional karyotyping and fluorescence in situ hybridization (FISH) are occasionally useful for clarifying the nature of morphologically suspicious blast cells, but cannot reliably detect submicroscopic leukaemia (Mancini et al, 2000). However, improvements in image analysis technology, allowing simultaneous visualization of morphological, immunophenotypic and FISH features, may enhance the usefulness of this approach (Bielorai et al, 2002). The success of methods based on differential properties of normal and leukaemic cells in culture has been limited to a few laboratories (Estrov et al, 1986; Uckun et al, 1993).

Many comprehensive reviews have addressed in detail the methodological aspects of MRD detection (Campana & Pui, 1995; van Dongen et al, 1999; Foroni et al, 1999; Lo Coco et al, 1999a; Szczepanski et al, 2001; Campana & Coustan-Smith, 2002; Liu & Grimwade, 2002). Table I and the following sections summarize the applicability and the main specific advantages and disadvantages of each technique.

Table I.  Applicability, and relative advantages and disadvantages of methods for MRD studies in patients with acute leukaemia.
MethodApplicabilityAdvantagesDisadvantages
ALLAML
PCR amplification of fusion transcripts40–50%20–40%Potential for very high sensitivity. Same set of reagents can be applied to multiple patients. Relatively rapid.RNA degradation (false-negative results). Unknown number of transcripts per cell (difficult quantification of MRD). Cross-contamination of RT-PCR products (false-positive results).
PCR amplification of IG and TCR genes90%10%Potential for very high sensitivity. Fixed number of targets per cell (relatively accurate quantification).Oligoclonality and clonal evolution (false-negative results). Tailor-made reagents for each patient (laborious and costly).
Flow cytometric detection of aberrant immunophenotypes95%75–85%Rapid. Accurate quantification. Can distinguish viable from apoptotic cells. Provides overview of haematopoietic status.Immunophenotypic shifts (false-negative results). Complex analysis.

Leukaemia-specific targets for pcr

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

Breakpoint fusion regions of chromosomal aberrations, and rearranged immunoglobulin (IG) and T-cell receptor (TCR) genes are leukaemia-specific sequences that have been used with consistent success in molecular studies of MRD. The value of another target, WT-1, is supported by some studies (Cilloni et al, 2002; Ogawa et al, 2003). However, WT-1 is also expressed by normal haematopoietic progenitors (Hosen et al, 2002) and has not been found to be reliable by some investigators (Elmaagacli et al, 2000). Internal tandem duplications (ITD) of the FLT3 gene occur in approximately 20–30% of AML patients and, in principle, could be used as targets for PCR-based MRD studies (Stirewalt et al, 2001). However, FLT3/ITD detected at diagnosis appear to be unstable, often becoming undetectable at relapse (Kottaridis et al, 2002; Shih et al, 2002).

With the exception of the TAL1 gene abnormalities, the genomic breakpoints of the most common known leukaemia fusion genes are spread over large distances within each gene locus. PCR analysis starting from DNA would require determination of the exact breakpoint in each patient, which is not practical (van Dongen et al, 1999). As the resulting mRNA is similar in many patients, RNA is the typical starting material for PCR-mediated amplification of breakpoint fusion sequences. After reverse transcription (RT) into cDNA, PCR amplification is applied using primers at opposite sites of the breakpoint fusion region (van Dongen et al, 1999). An advantage of using the breakpoint-fusion regions for MRD studies is their stability during the disease course. However, only 50% or less of ALL and AML cases in children and adults have specific chromosomal aberrations with well-defined breakpoint fusion regions (Look, 1997; Liu & Grimwade, 2002). Therefore, the applicability of this approach is restricted to certain subgroups of leukaemias.

The uniqueness of each immunoglobulin (Ig) and TCR molecule depends on the rearrangement and joining of the multiple variable (V), diversity (D) and joining (J) gene segments of the IG and TCR gene loci, on the deletion of nucleotides from the germline sequences of the rearranging gene segments, and on the random insertion of nucleotides at the junctional sites (Foroni et al, 1999; Pongers-Willemse et al, 1999). Thus, the sequences of the junctional regions of rearranged IG and TCR genes are signatures of each lymphoid cell clone, normal or malignant. For MRD studies, the various IG and TCR gene rearrangements must be identified in each patient at diagnosis. The sequence information enables the design of junctional region-specific oligonucleotides, which can be used as primers in the PCR procedure to specifically amplify the rearrangements of the malignant clone (Pongers-Willemse et al, 1999) or as probes to distinguish PCR products derived from leukaemic cells among those that are derived from normal lymphoid cells (Yokota et al, 1991).

Virtually all B-lineage ALL patients have rearranged IGH genes (van Dongen & Wolvers-Tettero, 1991). In addition, rearrangements of the IGK deleting element (Kde) occur at a relatively high frequency (approximately 60%) (Beishuizen et al, 1997). Most T-ALL patients have rearranged TCRB, TCRG and/or TCRD genes (van Dongen & Wolvers-Tettero, 1991), and cross-lineage TCR rearrangements are found in many patients with B-lineage ALL (Szczepanski et al, 1999). In the majority (approximately 90%) of B-lineage ALL patients, MRD can be revealed by junctional regions of IGH, IGK-Kde, TCRG and/or TCRD gene rearrangements (Pongers-Willemse et al, 1999), and in most (> 95%) T-ALL patients by TCRB, TCRG and/or TCRD (Kneba et al, 1995; Pongers-Willemse et al, 1999).

The need to develop individual probes or primers is one of the main limiting factors in the widespread application of MRD studies by PCR amplification of IG and TCR genes. Some investigators have attempted to bypass this need by identifying the leukaemic DNA on the basis of size and signal intensity after separation by high-resolution gel electrophoresis (Deane & Norton, 1990; Sykes et al, 1997). Polyclonal background levels vary but usually limit the sensitivity of this approach to the detection of one leukaemic cell among 103 normal cells.

IG and TCR gene rearrangements in B- and T-lineage ALL are prone to subclone formation and multiple IGH gene rearrangements are already found at diagnosis in 30% to 40% of B-lineage ALL patients (Beishuizen et al, 1994). This creates uncertainty about the prioritization of the clones that should be monitored in some patients. In addition, the emergence of subclones that are not apparent at diagnosis may occur, carrying the risk of false-negative results during MRD monitoring (van Dongen & Wolvers-Tettero, 1991; Beishuizen et al, 1994; Pongers-Willemse et al, 1999). In a recent analysis of 94 patients with B-lineage ALL, studied at diagnosis and relapse, 71% of the potential Ig and TCR targets for MRD analysis identified at diagnosis were preserved at relapse (Szczepanski et al, 2002). The most stable were IGK-Kde rearrangements and the least stable were incomplete TCRD rearrangements. Monoclonal rearrangements were significantly more stable than oligoclonal rearrangement. For these reasons, it has been recommended that at least two PCR targets should be used. In B-lineage ALL, these are available in approximately 70% of children and 50% of adults; in T-lineage ALL, two PCR targets (including TAL1 deletions) can be identified in approximately 90% of children and 85% of adults (Pongers-Willemse et al, 1999). Notably, clonotypic rearrangements of IG and TCR genes are found in only 50% of infants with t(4;11) ALL (Peham et al, 2002).

Quantification of mrd by pcr

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

Depending on the uniqueness of the sequence targeted and the quality of the material, PCR can detect one leukaemic cell in 103−106 normal cells. High sensitivity may paradoxically become a problem as it generates a propensity to false-positive results due to contamination, particularly when the same set of primers is applied to different patients. However, investigators are well aware of this potential problem and most take measures to minimize it.

A complication of MRD studies by PCR is related to the limited quantitative power of the technique. Nevertheless, under typical conditions and using appropriate techniques, the quantification of leukaemic cells by PCR amplification of single-copy genes (e.g. IGH and TCR genes) can be adequate (Sykes et al, 1992; Cave et al, 1994; Ouspenskaia et al, 1995; Pongers-Willemse et al, 1998; Neale et al, 1999). When RNA is the target molecule, additional potential pitfalls may render the correlation more imprecise. RNA is prone to degradation, and the efficiency of its initial conversion to cDNA by reverse transcriptase may vary. For example, it was reported that, using standard techniques, less than 1000 PML-RARA molecules could be obtained from 1 µg of diagnostic bone marrow RNA derived from approximately 106 acute promyelocytic leukaemia (APL) cells (Seale et al, 1996). Poor yield of PML-RARA cDNA would then lead to low sensitivity of the RT-PCR. Moreover, the number of transcripts per cell is unlikely to be homogeneous in all the leukaemic cell population and is also unlikely to remain stable in cells exposed to chemotherapy. Finally, levels of transcript expression in patients with the same disease may differ considerably (Krauter et al, 1999; Buonamici et al, 2002), which may affect the consistency of MRD measurements in a patient population.

MRD is traditionally quantified by comparing the PCR product obtained in the test sample with that of the patient's leukaemic cell DNA or RNA serially diluted into DNA or RNA from normal cells. Efforts to enhance the precision of the assay by competitive PCR or limiting dilution analysis have been effective (Sykes et al, 1992; Cave et al, 1994; Ouspenskaia et al, 1995) but the increased complexity of these approaches may hinder their routine application. Real-time quantitative PCR (RQ-PCR) appears to have solved some of the complications associated with PCR quantification. A fluorescent reporter is used in the PCR, and accumulation of fluorescence during the reaction (‘real-time’) is measured: the increase in fluorescence is proportional to the amount of target amplicon synthesized. Results are compared with those of serial dilutions of diagnostic material. This methodology can be applied to both breakpoint fusion region (Pallisgaard et al, 1999; Chen et al, 2001; de Haas et al, 2002) and antigen-receptor genes (Pongers-Willemse et al, 1998). In the latter case, to reduce the costs associated with designing fluorescent probes that match patient-specific sequences, probes matching germ line segments, such as V (Donovan et al, 2000; Verhagen et al, 2000), J (Bruggemann et al, 2000) and Kde regions (van der Velden et al, 2002a), and applicable to multiple patients can be used. Alternatively, some investigators have bypassed the requirement for fluoresceinated probes by using the DNA intercalating dye SYBR green I as a fluorescent reporter (Nakao et al, 2000; Li et al, 2002).

Immunophenotypes for mrd studies by flow cytometry

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

In patients with T-lineage ALL, MRD can be monitored by searching for cells expressing TdT and CD3 or other cell markers in bone marrow or peripheral blood (Campana & Coustan-Smith, 2002) (Fig 2). In B-lineage ALL and AML, one needs to identify aberrant phenotypes that are not expressed by normal bone marrow or peripheral blood cells (Fig 2). Therefore, MRD studies in these leukaemias are complicated by variations in the cellular composition and immunophenotype of normal bone marrow that occur with age and exposure to various agents. For example, proportions of early lymphoid progenitors (or ‘haematogones’) are low in the bone marrow of healthy adults and especially low in patients receiving corticosteroids or chemotherapy (Paolucci et al, 1979). In contrast, these cells are found in high proportions in young children and in patients after transplantation or chemotherapy (Lucio et al, 1999; van Lochem et al, 2000; Van Wering et al, 2000; McKenna et al, 2001). These conditions may uncover normal cells expressing phenotypes that are undetectable in studies of healthy individuals. Nevertheless, immunophenotypes that clearly distinguish B-lineage ALL cells from normal lymphoid progenitors and haematogones have been identified (Campana & Coustan-Smith, 2002).

image

Figure 2. Immunophenotypic differences between leukaemia cells and normal bone marrow cells. Flow cytometric dot plots shows expression of markers typically used for detecting MRD in T-lineage ALL, B-lineage ALL and AML (top row), and the expression of the same markers in bone marrow cells from healthy individuals (middle row) and from patients recovering after chemotherapy (bottom row). Dashed circles enclose areas of the dot plot corresponding to leukaemic cells in each case. Immunophenotypic analysis of T-lineage ALL cells was done on CD3+ cells, analysis of B-lineage ALL cells on CD19+ cells and analysis of AML cells on CD33+ and/or CD34+ cells (markers that were expressed in virtually all leukaemic cells at diagnosis). The analysis of the corresponding normal control subjects was done on the same cell subsets.

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Detection of MRD by flow cytometry in AML presents some specific difficulties. Owing to their immunophenotypic heterogeneity, AML cells usually spread across many areas of the dot plot instead of forming the tight cluster typical of ALL cells (Fig 2). Therefore, with any given marker combination, only a fraction of cells may be phenotypically abnormal. In addition, AML cells often have light scattering properties similar to those of normal cells with high autofluorescence. These features introduce complexity in the analysis, and may reduce the sensitivity of the assay. Nevertheless, sensitive MRD detection in AML is feasible. In a recent study using four-colour flow cytometry, 26 of 54 (48%) children with AML had leukaemia cells expressing immunophenotypes that allowed measurement of MRD with a sensitivity of one leukaemic cell among 104 or more normal cells; another 20 patients (37%) had immunophenotypes that enabled the detection of one leukaemic cell among 103 cells (unpublished observations). In adult AML, the proportion of patients that can be studied with a high degree of sensitivity may be larger. In one study, 46 of 53 patients had phenotypes that were found at frequencies of less than one in 104 cells in normal bone marrow, while seven had phenotypes found in normal bone marrow but at frequencies of less than one in 103 (San Miguel et al, 1997). In another study, 65 of 93 patients had a phenotype suitable for detection of one leukaemic cell in 104 normal cells (Venditti et al, 2000).

The advent of microarrays that enable the rapid analysis of global gene expression has opened new possibilities to identify markers of leukaemia. The results of one of our earlier studies (Chen J.S. et al, 2001) suggest that a comparison of the gene profiles of normal and leukaemic cells will identify new, widely applicable markers for MRD studies in both ALL and AML, and should ultimately allow the design of simple antibody panels for practical, reliable and universal monitoring of MRD.

One of the main causes of false-positive MRD results by flow cytometry is the use of inappropriate markers to distinguish leukaemic cells from normal cells. The range of normality needs to be established by extensive studies of bone marrow and peripheral blood cells collected not only from healthy individuals but also from patients at various stages of treatment (Campana & Coustan-Smith, 2002). The use of immunophenotypic combinations that ‘work’ only in samples collected at certain time points during treatment carries a high risk of error because the influence of individual variabilities in pharmacodynamics, pharmacogenetics and treatment compliance on normal lympho-haematopoiesis is unpredictable. A cause of false-negative MRD results is the occurrence of immunophenotypic shifts (Coustan-Smith et al, 1998; Baer et al, 2001). In our experience, however, the impact of this phenomenon on MRD results can be drastically reduced by using multiple sets of markers in each patient.

Quantification of mrd by flow cytometry

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

Two main variables influence MRD detection by flow cytometry: (a) the degree of morphological and phenotypic difference between target cells and the remaining cells, and (b) the number of cells that can be analysed. As discussed above, immunophenotypes that do not overlap with the normal counterparts of leukaemic cells must be carefully selected. The number of cells that can be analysed for each set of markers in clinical samples is usually less than 1 × 106. Because a distinct cluster of at least 10–20 dots is necessary to interpret suspect flow cytometric events, the maximum sensitivity of the assay under these circumstances would be 0·001% (Campana & Coustan-Smith, 2002). Therefore, a sensitivity of 0·01% (or one leukaemic cell in 104 normal cells) should be consistently achievable during routine MRD testing, even allowing for varying numbers of available cells and immunophenotypes that are not expressed in 100% of leukaemic cells.

There are various aspects of the laboratory procedure that need attention and have been previously discussed in detail (Campana & Coustan-Smith, 2002). Suffice it to say that spurious signals, which may have minimal impact during routine immunophenotyping of leukaemia, may be a source of major errors when studying MRD. False signals can derive from imperfect conditions of the buffers used, sample carry-over and non-specific binding of antibodies or fluorochromes to cells (Campana & Coustan-Smith, 2002).

Comparison of methodologies

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

To compare flow cytometry and PCR amplification of IGH genes, we studied serial dilutions of normal and leukaemic cells and found the two methods to be concordant (r2 = 0·962) (Neale et al, 1999). We then examined 62 bone marrow samples collected from children with ALL in clinical remission (Neale et al, 1999). In 12 samples, both techniques detected MRD levels ≥ 1 in 104. The percentages of leukaemic cells measured by the two methods correlated well (r2 = 0·978). Of the remaining 50 samples, 48 had MRD levels < 1 in 104. Results were discordant in only two samples: PCR detected two in 104 and five in 104 leukaemic cells, whereas the flow cytometric assay was negative; both patients were subsequently MRD negative by both assays and remain in clinical remission after completion of therapy. More recently, we compared the results obtained by using a panel of antibodies containing the new MRD marker CD58 with those of PCR amplification of IGH genes in 55 samples obtained from 29 patients at various time points during remission (Chen J.S. et al, 2001) (unpublished results). In 46 samples, MRD was < 0·01% by PCR analysis and negative by CD58 flow cytometric studies. By contrast, ≥ 0·01% leukaemic cells were detected by both methods in six samples, and the MRD estimates by the two methods were highly correlated. The two methods yielded discrepant results in only three of the 55 samples: one was shown by PCR to have 0·02% leukaemic cells, but no MRD was detected by flow cytometric analysis of CD58 expression; the two other samples were shown by CD58 analysis to have 0·01% leukaemic cells but by PCR to have less than 0·01% (0·0014% and 0·0022%) (unpublished observations). A study comparing the results of flow cytometry and PCR amplification of TCRG and TCRD genes to detect MRD in 89 bone marrow samples from 23 patients with ALL found concordant results in 70 samples. Discrepancies were more evident during the early phases of therapy, and were attributed to low cellularity and presence of putative PCR inhibitors (Malec et al, 2001).

A study comparing flow cytometry to RT-PCR detection of BCR-ABL transcripts in 23 bone marrow samples from patients with Philadelphia-chromosome ALL in remission observed concordant results in 18 samples (Munoz et al, 2000). In two samples the RT-PCR assay was positive while no cells (< 0·1%) were detectable by flow cytometry; the remaining three samples had 0·1–0·2% leukaemic cells by flow cytometry but no positive signal by RT-PCR. We also compared the results of flow cytometry with the results of RT-PCR amplification of fusion transcripts (BCR-ABL and MLL-AF4; unpublished observations). The methods gave concordant results in 25 of 27 remission bone marrow samples of children with B-lineage ALL in clinical remission (10 had MRD ≥ 0·01% positive, and 15 were MRD negative). Of the two remaining samples, one was negative by flow cytometry but positive (0·001%) by PCR; the other was positive by flow cytometry but negative by PCR (this patient had MRD that was detectable by both methods in prior and subsequent samplings). A similar comparison was performed with 40 samples obtained from 20 children with AML during treatment (unpublished observations). The molecular abnormalities studied were AML1-ETO, CBFB-MYH11, MLL-AF9 and MLL-AF10. In three samples, RT-PCR indicated a level of residual disease ≥ 0·1%. Two of these samples also had residual cells detectable by flow cytometry, whereas one sample, with 0·1% to 1% levels of residual disease by RT-PCR, did not; leukaemic metaphases were not detected by cytogenetic analysis. The remaining 37 samples had either undetectable leukaemic transcripts (n = 25) or weak signals corresponding to estimated levels of residual disease lower than 0·1% (n = 12), but seven of these (four with undetectable leukaemic transcripts and three with < 0·1% residual disease by RT-PCR) had = 0·1% residual disease by flow cytometry. The variable sensitivities of MRD detection by flow cytometry (0·01% in ALL; 0·1–0·01% in AML) and by RT-PCR (0·1% to 0·001%), and the limited quantitative capacity of the conventional (pre-RQ-PCR) RT-PCR technique used can explain the observed discrepancies. A correlation between flow cytometric results and real-time PCR detection of WT-1 was illustrated in a recent report (Trka et al, 2002).

Prognostic value of mrd in all

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

Several prospective studies in childhood ALL have defined the prevalence of MRD at different time points during treatment (Fig 3). Although there are variations, probably due to differences in chemotherapy schedule, these studies have conclusively demonstrated the clinical significance of MRD. In one multicentric study, MRD was measured in 178 patients by a competitive PCR assay targeting junctional sequences of IGH and TCR (Cave et al, 1998). The absence or presence and level of residual leukaemia during the first 6 months of therapy were significantly correlated with the risk of early relapse at each of the times studied. Patients with ≥ 1% leukaemic cells after the completion of induction therapy or those with ≥ 0·1% at later time points had a particularly high risk of relapse. Another multicentric study monitored MRD in 240 children with ALL treated according to national protocols of the International Berlin–Frankfurt–Munster (BFM) Study Group (van Dongen et al, 1998). This study also used PCR analysis of IGH and TCR genes as well as TAL1 deletions. MRD-positive patients had 5–10-fold higher relapse rates (39–86% at 3 years) than those who were MRD negative at the various follow-up times (3–15% at 3 years). MRD levels ≥ 1% at the end of induction treatment and before consolidation treatment were associated with a threefold higher relapse rate when compared with patients with a low degree of MRD, and with a 10- to 20-fold higher relapse rate when compared with MRD-negative patients. Combined MRD information from the first two follow-up time points was particularly informative, allowing the identification of three different risk groups: a low-risk group, comprising 43% of patients with a 3-year relapse rate of 2%[95% confidence interval (CI) 0·05–12%]; a high-risk group, comprising 15% of patients with a relapse rate of 75% (55–95%); and an intermediate group (43%), with a 3-year relapse rate of 23% (13–36%).

image

Figure 3. Prevalence of MRD during treatment in children with ALL. Bars represent the percentage of MRD-positive patients, according to the studies indicated. Black areas correspond to patients with ≥ 0·1% MRD in each study. Data were obtained from Coustan-Smith et al (1998) and unpublished results for the St Jude studies, from Cave et al (1998) for the European Organization for Research and Treatment of Cancer (EORTC) study, and from van Dongen et al (1998) for the I-BFM Study Group (I-BFM-SG) study.

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We used flow cytometry to prospectively study MRD in 195 children with newly diagnosed ALL enrolled in a single-institution chemotherapy programme (TOTAL XIII) (Coustan-Smith et al, 1998, 2000, 2002a). We found that detectable MRD (i.e. ≥ 0·01% leukaemic mononuclear cells) at each time point (d 19 of remission induction therapy, end of remission induction, and weeks 14, 32 and 56 of continuation) was significantly associated with a higher relapse rate (Fig 4). Patients with high levels of MRD at the end of the induction phase (≥ 1%) or at week 14 of continuation therapy (≥ 0·1%) had a particularly poor outcome. The incidence of relapse among patients with MRD at the end of the induction phase was 68% ± 16% (SE) if they remained MRD+ at week 14 of continuation therapy, compared with 7% ± 7% if MRD became undetectable. The persistence of MRD until week 32 was highly predictive of relapse: all four MRD+ patients relapsed vs two of the eight who converted to undetectable MRD status.

image

Figure 4. Cumulative incidence of relapse according to MRD in childhood ALL. MRD was measured by flow cytometry at d 19 of remission induction therapy, at the end of remission induction therapy (d 46), and at weeks 14 and 32 of continuation therapy (Coustan-Smith et al, 2000, 2002a). Solid lines indicate patients with residual disease detectable by flow cytometry; broken lines indicate residual-disease-negative patients.

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Of the 112 patients studied at d 19 of remission induction therapy, 53 had achieved a profound cytoreduction (MRD < 0·01%) despite the short chemotherapy period (Coustan-Smith et al, 2002a). The treatment outcome for this group of patients was outstanding: 3-year cumulative incidence of relapse was 1·9% ± 1·9%, as compared with 28·4% ± 6·4% for MRD+ patients (Fig 4). These results are in line with those of another study in which bone marrow samples collected at d 15 of therapy from 68 children with ALL were assayed by PCR amplification of antigen-receptor genes (Panzer-Grumayer et al, 2000). However, the proportion of children who achieved the lowest levels of MRD in the two studies was different: 21% in this series, 46% in ours.

In the studies outlined above, the prognostic value of MRD was independent of that of other known clinical and biological prognosticators of outcome. In our study, MRD remained a significant predictor in analyses that excluded patients at very high or very low risk of relapse by St Jude criteria, or that focused on patients with high risk of relapse by the criteria defined by the National Cancer Institute (Coustan-Smith et al, 2000; Pui et al, 2001). In the International BFM (I-BFM) study, MRD was also a strong predictor of outcome in children with medium-risk features (Biondi et al, 2000). In contrast to the I-BFM study (Willemse et al, 2002), we did not find higher levels of MRD in children with T-lineage versus those with B-lineage ALL (Coustan-Smith et al, 2000). The reason for this difference is not clear. Contributing factors could be differences in treatment protocol and/or patient composition (infants and patients aged 15–18 years old were included in the St Jude study).

Other investigators have used PCR (Gruhn et al, 1998; Gameiro et al, 2002; Nyvold et al, 2002) or flow cytometry (Ciudad et al, 1998; Farahat et al, 1998) to study MRD in patients with ALL and found a good correlation with outcome. The correlation between MRD detected by flow cytometry during treatment, and outcome in 108 children enrolled in the BFM 95 protocol in Austria was recently reported (Dworzak et al, 2002). These investigators converted percentages of MRD into number of blasts per volume of bone marrow. Patients with persistent disease (≥ 1 blast/µl) at d 33 and week 12 of treatment had a 100% probability of relapse, compared with 6% in all others. It was found, however, that the sensitivity of the markers used was limited in bone marrow samples from patients who were recovering post chemotherapy. A recent study showed that the predictive value of MRD monitoring extends to children receiving treatment for relapsed ALL (Eckert et al, 2001). Children with MRD levels < 0·1% at d 36 of therapy post relapse had a probability of event-free survival of 0·86, compared with zero for children with higher levels of MRD.

Fewer studies of the significance of MRD in adult ALL have been reported. In 85 adult patients with Philadelphia-chromosome-negative B-lineage ALL, in whom MRD was monitored by PCR amplification of IGH genes during the first 24 months of treatment, presence or absence of MRD correlated with clinical outcome and was most significant at 3–5 months after induction therapy and beyond (Mortuza et al, 2002).

In general, the collective evidence for a correlation between presence of MRD and increased risk of relapse in ALL is strong. Such correlation, however, was not observed in one study in which MRD was monitored by RT-PCR amplification of E2A-PBX1 fusion transcripts at the end of consolidation treatment in children with t(1;19)-positive ALL (Hunger et al, 1998). Whether a more precise quantification of MRD at this or at other time points during treatment would have identified patients with higher relapse rates, or whether MRD is not informative in this subset of ALL remains unclear. Another study based on RT-PCR amplification of MLL-AF4 transcripts found a good correlation between PCR findings and treatment outcome in 22 adults and three infants with t(4;11) ALL (Cimino et al, 2000).

Mrd in patients with all undergoing haematopoietic cell transplantation

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

MRD detection by PCR amplification of antigen-receptor genes was predictive of subsequent relapse in children with ALL who underwent allogeneic bone marrow transplantation (Knechtli et al, 1998a). The 2-year event-free survival estimate was 0% for the 12 patients who had high levels of MRD (estimated to be 0·1% to 1%) before transplantation, 36% for the 11 who had low levels of MRD (0·001% to 0·1%) and 73% for the 33 who had undetectable MRD. Not unexpectedly, the same group also found that the detection of MRD after transplantation is predictive of an unfavourable outcome (Knechtli et al, 1998b). Another study looked at MRD in remission bone marrow samples taken prior to conditioning therapy in 41 children with ALL (Bader et al, 2002). Patients were classified as having high levels of MRD if a clonal band was already evident after electrophoresis (n = 17), low levels of MRD if it was detected only after probing with a patient-specific probe (n = 10), and no detectable MRD (n = 14). The 5-year event-free survival for these groups was 23%, 48% and 78% respectively.

MRD monitoring of BCR-ABL fusion transcripts predicted the outcome of allogeneic or autologous bone marrow transplantation in adult patients with Philadelphia-chromosome-positive ALL (Radich et al, 1997). MRD status after allogeneic bone marrow transplantation, rather than before, was also found to be an important predictor of outcome in adults with Philadelphia-chromosome-negative ALL, whereas in the patients receiving an autologous transplant, MRD status before transplant was a more significant predictor (Mortuza et al, 2002). MRD detected by flow cytometry in bone marrow samples taken prospectively from 24 patients with ALL before starting the conditioning regimen was a significant predictor of outcome (Sanchez et al, 2002).

Detection of mrd in peripheral blood

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

Early studies found that in most patients with B-lineage ALL the level of MRD in the peripheral blood was at most one tenth that in the bone marrow (van Rhee et al, 1995; Brisco et al, 1997). We recently compared MRD measurements in 747 pairs of bone marrow and peripheral blood samples collected from 231 children during treatment for newly diagnosed ALL (Coustan-Smith et al, 2002b) (unpublished observations). MRD was detected in both marrow and blood in 78 pairs, and in the marrow but not in blood in 67 pairs; it was undetectable in the remaining 602 pairs. Remarkably, the marrow and blood findings were completely concordant in the 179 paired samples from patients with T-lineage ALL: for each of the 41 positive marrow samples, the corresponding blood sample was positive (Fig 5). In B-lineage ALL, however, only 37 of the 104 positive marrow samples had a corresponding positive blood sample. The results of a recently reported study using immunofluorescence analysis and RQ-PCR are in agreement with these data (van der Velden et al, 2002b). We also observed that peripheral-blood MRD in B-lineage ALL patients was associated with a very high risk of disease recurrence (Coustan-Smith et al, 2002b). The 4-year cumulative incidence of relapse in patients with B-lineage ALL was 80·0% ± 24·9% for those who had peripheral-blood MRD at the end of remission induction therapy but only 13·3% ± 9·1% for those with MRD confined to the marrow. Therefore, peripheral blood could be used to monitor MRD in patients with T-lineage ALL. In B-lineage ALL, the presence of MRD in peripheral blood appears to identify patients who are at a very high risk of relapse.

image

Figure 5. Distribution of MRD in bone marrow and peripheral blood of patients with T-lineage ALL. Values represent the percentage of residual disease determined by flow cytometry in paired bone marrow and peripheral blood samples at the indicated treatment intervals (Coustan-Smith et al, 2002b) (unpublished results).

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Mrd studies in genetic subgroups of aml

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

The lack of widely expressed molecular markers in AML cells precludes the systematic study of MRD by PCR (Liu & Grimwade, 2002). A recent study found that antigen-receptor genes were clonally rearranged in 9·5% of AML patients and that non-random genetic abnormalities with fusion transcripts were suitable for molecular studies of MRD in 16% (Boeckx et al, 2002). Therefore, correlative studies between MRD and treatment outcome have been performed only in selected groups of patients, almost exclusively adults (Table II). In APL, PCR detection of PML-RARA and RARA-PML transcripts during remission generally predicts relapse (Grimwade, 1999; Lo Coco et al, 1999a). In the UK Medical Research Council (MRC) all trans retinoic acid (ATRA) study, delayed clearance of PML-RARA transcripts during consolidation was associated with an increased risk of relapse (Burnett et al, 1999). In the study by the Gruppo Italiano Malattie Ematologiche Maligne dell′ Adulto (GIMEMA), recurrence of detectable transcripts in patients who were MRD negative after consolidation was also associated with a high relapse risk, a finding that suggests a potential for treatment at the time of molecular relapse (Diverio et al, 1998). The significance of detecting AML1-ETO transcripts in patients with AML t(8;21) during remission is less clear. Early studies showed that these transcripts persist for more than 5 years of remission after the completion of treatment (Nucifora et al, 1993), a finding that could be explained by the expression of AML1-ETO transcripts in normal monocytes, B cells and haematopoietic colony forming cells (Miyamoto et al, 2000). Despite the lack of a strict association between AML1-ETO transcripts and AML cells, careful quantification of these transcripts can help to monitor MRD (Morschhauser et al, 2000; Tobal et al, 2000). MRD studies have also been done in patients with AML and other cytogenetic abnormalities, such as inv(16). Patients that during remission had expression of CBFB-MYH11 fusion transcripts (normalized against expression of 18S RNA) in > 10 copies by RQ-PCR had a significantly shorter remission duration and higher risk of relapse than patients with a CBFB-MYH11 fusion transcript copy number < 10 (Marcucci et al, 2001). Predictive thresholds have also been identified by other investigators (Laczika et al, 2001; Buonamici et al, 2002; Guerrasio et al, 2002).

Table II.  Examples of correlative studies of MRD and treatment outcome in patients with AML.
AML SubtypeMRD methodLeukaemia markerNumber of patients studiedMost informative timepointMost informative MRD levelReference
  • *

    MRD negative at end of consolidation.

  • For MRD measured in bone marrow; 100 molecules/µg of RNA for measurements in peripheral blood.

  • Normalized against expression of 18S RNA.

APL, t(15;17)RT-PCRRARA-PML105After 3 courses of chemotherapy0·01%Burnett et al (1999)
PML-RARA    
APL, t(15;17)*RT-PCRPML-RARA163Post-consolidation0·01%Diverio et al (1998)
t(8;21)RT-PCRAML1-MTG8 (ETO)  25Post-consolidation/intensification1000 molecules/µg of RNATobal et al (2000)
t(8;21)RT-PCRAML1-MTG8 (ETO)  51Post-first consolidation0·01%Morschhauser et al (2000)
inv(16)RQ-PCRCBFB-MYH11  16Post-completion of chemotherapy10 CBFB/MYH11 transcript copiesMarcucci et al (2001)
M0–M2, M4–M6Flow cytometryAberrant immunophenotypes  56Post-consolidation0·035%Venditti et al (2000)
M0–M6Flow cytometryAberrant immunophenotypes126Post-induction0·1%San Miguel et al (2001)

Mrd studies in aml by flow cytometry

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

The presence of immunophenotypic abnormalities in bone marrow correlated with earlier occurrence of relapse in 19 of 35 children with AML in first morphological remission (Sievers et al, 1996). In a recent update, the same investigators found these abnormalities in 41 of 252 who responded to initial therapy, a finding that was associated with a poorer outcome (Sievers et al, 2003). One unique feature of the studies by Sievers and colleagues is that the immunophenotype of leukaemic cells in each patient was unknown at the time of the residual disease test. Therefore, the sensitivity of MRD detection afforded by this approach cannot be determined, and it is possible that some patients who appeared to be free of leukaemia according to this assay had leukaemic cells whose immunophenotype could not be distinguished with the markers used. In sequential studies performed in 53 patients with AML, the level of MRD at the end of the induction and intensification phases of therapy was correlated with the probability of subsequent relapse (San Miguel et al, 1997). Patients who had MRD > 0·5% during first remission or > 0·2% at the end of intensification therapy had a significantly higher rate of relapse than those patients who had a lower level of MRD. In a more recent report from the same group, the first bone marrow sample obtained after induction treatment that showed morphological remission was found to be very informative (San Miguel et al, 2001). Based on the MRD level, 126 patients could be divided into four groups. Eight patients had fewer than 0·01% leukaemic cells and none had relapsed at the time of the report; 37 patients had 0·01–0·1% leukaemic cells and a 3-year cumulative relapse rate of 14%; 64 patients had 0·1–1% leukaemic cells and a relapse rate of 50%; 17 patients had more than 1% residual cells and a relapse rate of 84%. In another study of 51 patients in whom MRD was examined after consolidation, the most predictive MRD cut-off value determined retrospectively was 0·035%: 17 of 22 patients with that or higher levels of MRD relapsed compared with five of 29 patients with lower MRD levels (Venditti et al, 2000). In patients with AML receiving autologous bone marrow transplantation, levels of cells expressing an aberrant immunophenotype in the autograft correlated with disease recurrence (Reichle et al, 1999).

Outstanding questions and future perpective

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

As discussed in this article, each of the three main methodologies for studying MRD in patients with acute leukaemia has relative advantages and disadvantages (Table I). The use of multiple approaches is desirable because it can increase the number of patients that can be studied and offset the limitations of individual methods as well as the accuracy of the individual measurements (Pui & Campana, 2000). However, such a combined approach is difficult to implement in most institutions.

The sensitivity that is required for productive MRD studies depends on the clinical question. Clearly, a high sensitivity is likely to be necessary when assessing MRD in stem cell autografts. For monitoring MRD in vivo, the results of correlative studies with outcome indicate that a MRD lower than 0·01% during early treatment identifies patients with a particularly good treatment outcome (Cave et al, 1998; Coustan-Smith et al, 1998, 2000; Diverio et al, 1998; van Dongen et al, 1998; Burnett et al, 1999; San Miguel et al, 2001). In ALL, a 0·01% level of sensitivity can be routinely achieved by flow cytometry and by PCR amplification of antigen-receptor genes. In AML, such a high level of sensitivity may be difficult to achieve consistently by either flow cytometry or RT-PCR. The simplification of MRD methods, for example by reducing the number of markers used by flow cytometry or by avoiding the use of patient-specific oligonucleotide in PCR reactions, invariably decreases the sensitivity of the assay. However, on the basis of the results of correlative studies, it can also be argued that a 0·1% level of sensitivity may be sufficient to identify patients at a high risk of relapse (Cave et al, 1998; Coustan-Smith et al, 1998, 2000; van Dongen et al, 1998; San Miguel et al, 2001).

Because of their complexity, methods for MRD detection can currently be performed in a few specialized laboratories. Multicentre studies incorporating MRD assays require shipment of specimens. While this is generally not a problem for PCR amplification of DNA sequences, which are stable over time and may be detectable even after cells have undergone apoptosis, it may seriously affect RNA integrity and the precision of RT-PCR-based MRD assays (Muller et al, 2002). As the process of apoptosis in most leukaemic cases begins once the cells are removed from the in vivo microenvironment and this process may affect protein expression, flow cytometric studies may also yield imprecise results in samples that are not studied immediately after collection. In this regard, methods to stabilize samples during shipment would be of great benefit. Because of the wide availability of flow cytometry, however, detection of MRD by this technique is also amenable to decentralization. In this regard, methods for rapid exchange of flow cytometric files between centres may be invaluable in ensuring homogeneity in intercentre MRD measurements (Lorenzana et al, 1999).

Methods for MRD have many potential applications in the clinical management of patients with acute leukaemia. Treatment intensification for patients with slow clearance of leukaemia and persistent or resurgent MRD has a strong theoretical basis, as it is well established that the size of tumour burden and the curability of cancer are related, and it is well supported by the results of correlative studies. A study indicated the effectiveness of early administration of salvage therapy to patients with APL after the reappearance of positive RT-PCR results (Lo Coco et al, 1999b). At St Jude Children's Research Hospital, MRD has been used as a criteria for treatment intensification in children with newly diagnosed ALL since 1998, and it has also been incorporated in the recently initiated AML2002 protocol for children with AML. Conversely, patients with profound cytoreductions at the early stages of therapy are clear candidates for less intensive (hence less toxic) treatment regimens. However, MRD negativity does not mean disease eradication and the possibility exists that reduction in treatment intensity will result in an increased rate of relapse, even in patients who readily achieve MRD-negative status. MRD-based treatment intervention studies that address the benefit of using MRD for both treatment intensification and reduction in childhood ALL have been initiated within the BFM group.

MRD studies are becoming an integral part of the modern management of patients with leukaemia. The selection of the methods to be used in each centre depends on the expertise and facilities available, and on collaborative links that can be established with laboratories that are proficient in MRD detection. The main challenge for expert MRD investigators is to simplify methods while maintaining or increasing their reliability, thus disseminating the potential benefits of MRD monitoring to all patients (Eden, 2002).

Acknowledgments

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References

I thank Elaine Coustan-Smith for providing data and critical review of the article. I also thank Geoffrey Neale and Ching-Hon Pui for helpful discussions, and members of the St Jude Hematologic Malignancies program for their support of MRD studies. This work was supported by grants CA60419 and CA21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC).

References

  1. Top of page
  2. Rationale for studies of mrd
  3. Methodological options for mrd studies
  4. Leukaemia-specific targets for pcr
  5. Quantification of mrd by pcr
  6. Immunophenotypes for mrd studies by flow cytometry
  7. Quantification of mrd by flow cytometry
  8. Comparison of methodologies
  9. Prognostic value of mrd in all
  10. Mrd in patients with all undergoing haematopoietic cell transplantation
  11. Detection of mrd in peripheral blood
  12. Mrd studies in genetic subgroups of aml
  13. Mrd studies in aml by flow cytometry
  14. Outstanding questions and future perpective
  15. Acknowledgments
  16. References
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