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October 2002, Volume 16, Number 10, Pages 1959-1973
Table of contents    Previous  Article  Next   [PDF]
Spotlight on Acute Promyelocytic Leukemia
Acute promyelocytic leukemia: a model for the role of molecular diagnosis and residual disease monitoring in directing treatment approach in acute myeloid leukemia
D Grimwade1 and F Lo Coco2

1Division of Medical and Molecular Genetics, Guy's, King's and St Thomas' School of Medicine and Dept of Haematology, University College London Hospitals, London, UK

2Department of Cellular Biotechnologies and Hematology, Università la Sapienza, Rome, Italy

Correspondence to: D Grimwade, Division of Medical and Molecular Genetics, Guy's, King's and St Thomas' School of Medicine, Cancer Genetics Laboratory, 8th Floor, Guy's Tower, Guy's Hospital, London SE1 9RT, UK; Fax: 020 7 955 8762

Abstract

Acute promyelocytic leukemia (APL) is characterized by a number of features that underpin the need for rapid and accurate diagnosis and demand a highly specific treatment approach. These include the potentially devastating coagulopathy, sensitivity to anthracycline-based chemotherapy regimens, as well as unique responses to all-trans retinoic acid and arsenic trioxide that have revolutionized therapy over the last decade. The chromosomal translocation t(15;17) which generates the PML-RARalpha fusion gene has long been considered the diagnostic hallmark of APL; however, this abnormality is not detected in approximately 10% cases with successful karyotype analysis. In the majority of these cases, the PML-RARalpha fusion gene is still formed, resulting from insertion events or more complex rearrangements. These cases share the beneficial response to retinoids and favorable prognosis of those with documented t(15;17), underscoring the clinical relevance of molecular analyses in diagnostic refinement. In other cases of t(15;17) negative APL, various chromosomal rearrangements involving 17q21 have been documented leading to fusion of RARalpha to alternative partners, namely PLZF, NPM, NuMA and STAT5b. The nature of the fusion partner has a significant bearing upon disease characteristics, including sensitivity to retinoids and arsenic trioxide. APL has provided an exciting treatment model for other forms of AML whereby therapeutic approach is directed towards cytogenetically and molecularly defined subgroups and further modified according to response as determined by minimal residual disease (MRD) monitoring. Recent studies suggest that rigorous MRD monitoring, coupled with pre-emptive therapy at the point of molecular relapse improves survival in the relatively small subgroup of PML-RARalpha positive patients with 'poor risk' disease. Advent of 'real-time' quantitative RT-PCR technology seems set to yield further improvements in the predictive value of MRD assessment, achieve more rapid sample throughput and facilitate inter- and intra-laboratory standardization, thereby enabling more reliable comparison of data between international trial groups.

Leukemia (2002) 16, 1959-1973. doi:10.1038/sj.leu.2402721

Keywords

acute promyelocytic leukemia (APL); RT-PCR; minimal residual disease (MRD); real-time quantitative RT-PCR (RQ-PCR)

Biological and molecular characteristics of acute promyelocytic leukemia: implications for targeted therapeutic approaches

Clinical features

Acute promyelocytic leukemia (APL) is associated with a number of features that emphasize the need for rapid and accurate diagnosis and demand a highly specific treatment approach. Of critical importance is the potentially severe coagulopathy that often accompanies the disease, which can induce thrombotic and more often hemorrhagic complications, that are the most prevalent clinical features at presentation. The coagulopathy is frequently triggered or further exacerbated by initiation of chemotherapy, and led to the demise of up to a quarter of patients in early studies. However, increased awareness of this problem, prompt initiation of ATRA-based therapy, together with improvements in supportive care have seen a reduction in induction death rates to approximately 10% or less in more recent clinical studies.1,2,3,4

Establishing a diagnosis of APL is also important in view of its unique therapeutic profile. In particular, APL was the first disease for which differentiation therapy in the form of retinoids such as all-trans retinoic acid (ATRA) that directly target the underlying molecular lesion (see below) has been successfully used in clinical practice. ATRA has transformed the management of APL over the last decade, with recent studies revealing that it is most effectively deployed simultaneously with chemotherapy for remission induction (reviewed in Ref. 5). This approach has led to significant reductions in relapse rates and improved overall survival in comparison to chemotherapy alone.2,6,7,8 Further reductions in relapse risk may be achieved by use of ATRA as maintenance therapy either alone or in combination with 6-mercaptopurine and methotrexate.3,8 Distinct from other forms of acute myeloid leukemia (AML), APL is also characterized by striking sensitivity to anthracyclines accompanied by low levels of primary drug resistance and MDR expression,9,10,11 as well as by reduced dependence on cytarabine.4,12

Once complete remission (CR) has been achieved, APL is now considered one of the most favorable subsets of AML13 and bone marrow transplantation (BMT) is no longer routinely undertaken in first CR by a number of trial groups. Indeed this is supported by studies recently reported by the UK Medical Research Council (MRC), which demonstrated that any benefit for autologous or allogeneic BMT in terms of reduced relapse risk was more than offset by transplant-related morbidity and mortality.14,15 Therefore, attention has become focused upon identification of the relatively small group of patients at high risk of relapse following first-line therapy, who could benefit from additional treatment whilst in first morphological CR. Hence, as discussed below, monitoring for evidence of minimal residual disease (MRD) as a means of determining treatment approach is playing an increasingly important role in the management of APL patients. In addition to possible anticipation of impending relapse, thereby allowing early institution of salvage therapy, molecular monitoring provides the opportunity to rationalize therapy thereby sparing unnecessary toxicity in patients who maintain a status of molecular remission (see below for definitions). While the combination of ATRA and anthracycline-based chemotherapy is currently considered as the 'gold standard' first-line treatment for patients with APL, a variety of other therapeutic approaches with efficacy in the disease are currently under investigation. These include antibodies targeting CD33, which is expressed at the blast surface in virtually all cases of APL, and arsenic trioxide which has proved highly successful in the treatment of relapse, including patients resistant to other treatment modalities.16,17 More recently there has been interest in determining whether these agents could also have a role to play as potential first-line therapies.18,19

Molecular pathogenesis of APL

For many years, since its discovery 25 years ago, the t(15;17) has been considered the diagnostic hallmark of APL.20,21 Over a decade ago, the breakpoint regions were cloned and the significance of the response to retinoids noted in earlier clinical studies became apparent.22,23 The translocation was found to disrupt a previously uncharacterized gene, PML at 15q22, and the gene encoding the retinoic acid receptor alpha (RARalpha) located at 17q12-21.22,23,24,25,26,27 The resultant PML-RARalpha fusion protein has the potential to disrupt both PML- and RARalpha- dependent pathways (reviewed in Ref. 28). PML exhibits growth suppressor and pro-apoptotic activity and is predominantly localized within multi-protein nuclear body structures (PML nuclear bodies) that are disrupted in the presence of the PML-RARalpha fusion protein (reviewed in Ref. 29). RARalpha, which at physiological levels of its ligand (RA) typically functions as a transcriptional activator at retinoid response elements, is implicated in normal myeloid differentiation (reviewed in Ref. 30).

Whilst the t(15;17) was originally considered to be present in all cases of morphological APL,21 more recent studies have shown that almost 10% with successful cytogenetic analysis actually lack the classic t(15;17)2,31,32,33 (see Figure 1). In the majority of these cases, molecular analysis nevertheless reveals the presence of an underlying PML-RARalpha fusion gene formed as a result of insertion events, which may be cytogenetically cryptic, or more complex rearrangements.33,34,35,36,37 In other cases, rearrangements of 17q21 lead to fusion of RARalpha to alternative partner genes, ie PLZF (promyelocytic leukemia zinc finger), NPM (nucleophosmin), NuMA (nuclear mitotic apparatus) and STAT5b38 associated with t(11;17)(q23;q21), t(5;17)(q35;q12-21), t(11;17)(q13;q21) and der(17) respectively.38,39,40,41,42 It is now apparent that the nature of the fusion partner has a significant bearing upon disease characteristics, particularly the responsiveness to ATRA and arsenic trioxide.43,44

Over the last few years it has become clear that recruitment of nuclear corepressor complexes and histone deacetylase (HDAC) to retinoid response elements of downstream target genes, accompanied by hypermethylation, which are induced by APL-associated RARalpha fusion proteins at physiological levels of RA, play an important role in mediating the differentiation block that characterizes the disease.45,46 This contrasts with the situation at pharmacological levels of ligand (RA/ATRA), in which displacement of the corepressor complex from the fusion protein and alteration of the methylation profile of downstream target genes are correlated with transcriptional activation and induction of differentiation.45,46 Interestingly, the PLZF-RARalpha fusion protein has been shown to bind nuclear corepressor molecules to the amino-terminal portion of PLZF which are not displaced in the presence of retinoid and may account for the relative insensitivity of this subtype of APL to conventional doses of ATRA (reviewed in Ref. 47). Nevertheless, recent evidence suggests that PLZF-RARalpha associated APL may not be completely resistant to differentiating agents, responding to ATRA when combined with G-CSF48 or hydroxyurea,49 or to combinations of agonists of protein kinase A and retinoid-X-receptors.50 It is becoming apparent that occurrence of chromosomal rearrangements generating chimeric transcription factors which target corepressor/HDAC complexes to regulatory genes playing a key role in hemopoietic differentiation is a recurrent theme in hematological malignancy. This would suggest that HDAC inhibitors, possibly in combination with demethylating agents, may also have a therapeutic role (reviewed in Ref. 51). Preliminary evidence arising from studies of primary or secondary ATRA resistance in APL associated with PLZF-RARalpha49,52 or PML-RARalpha53 respectively suggests that this is indeed the case. Therefore, characterization of the underlying molecular lesion in newly diagnosed cases of APL is valuable to determine optimal treatment strategy in individual patients. Furthermore, this has the additional benefit of defining targets for minimal residual disease (MRD) monitoring, which as discussed below has been shown to be a key independent prognostic variable in this disease.

Establishing a diagnosis of APL: merits and potential limitations of current laboratory techniques

As mentioned above, rapid establishment of a diagnosis of APL is critical for optimal patient management. This relates particularly to the significant risk of hemorrhagic death if the bleeding diathesis is not addressed and the increased risk of relapse associated with poorer survival if patients with retinoid-responsive disease are not treated with ATRA in combination with chemotherapy. Indeed, ATRA carries an added advantage, in that it rapidly leads to improvement in the disease-associated coagulopathy. Diagnostic approaches are essentially focused on establishing the presence of an underlying PML-RARalpha fusion gene, which predicts a favorable response to ATRA and arsenic trioxide, and identifying cases with an underlying PLZF-RARalpha fusion which account for almost 1% APL and in which these drugs are ineffective when used as the sole therapeutic agents. In practice these biologically distinct subtypes of APL can usually be distinguished on the basis of characteristic morphological features.54 The most commonly used laboratory techniques include cytogenetic analysis, fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (RT-PCR) and PML immunostaining (see below). In the rare situation of morphologically confirmed, but PML-RARalpha negative APL, cytogenetic analysis may reveal a rearrangement of 17q12-21 indicating that RARalpha is fused to a known alternative fusion partner, the identity of which can subsequently be confirmed by RT-PCR. However, if conventional cytogenetic analysis proves unhelpful in PML-RARalpha negative APL, FISH using RARalpha probes or Southern analysis may establish whether RARalpha is rearranged and RT-PCR analysis for the presence of the various alternative RARalpha-fusion genes performed.

Cytogenetic analysis

Cytogenetic analysis now forms part of the mandatory routine 'work-up' for any newly diagnosed patient with AML. Karyotype has been shown to be a key prognostic determinant in this disease predicting likelihood of successful remission induction, relapse risk and overall survival and is being increasingly used to dictate treatment approach in large-scale clinical trials (reviewed in Ref. 55). In the majority of patients with morphological APL, presence of an underlying PML/RARalpha rearrangement is indicated by detection of the t(15;17) (reviewed in Ref. 56). However, analysis of multicenter clinical trials reveals that this abnormality is not identified in approximately 15% cases of suspected APL, the majority of which nevertheless still have underlying PML/RARalpha rearrangements.2 In some instances this reflects a failure of cytogenetic technique; for example, direct chromosomal examination can lead to reporting of a falsely normal karyotype reflecting residual normal marrow elements whose growth is favored relative to APL blasts.57 This shortcoming is usually overcome by more prolonged culture of leukemic blasts prior to cytogenetic assessment, which has since been adopted as standard practice for analysis of cases with suspected APL. Nevertheless despite this approach, in some cases of APL reported as having a normal karyotype, nested RT-PCR detects both PML-RARalpha and reciprocal RARalpha-PML fusion transcripts, implying that the t(15;17) was present, but had been missed by cytogenetic analysis.2 PML/RARalpha rearrangements may also result from insertion events, in which chromosomes 15 and 17 are typically of normal appearance, or from more complex aberrations involving multiple chromosomes.33 The occurrence of these phenomena clearly implies that additional molecular techniques such as RT-PCR, Southern, PML-immunofluorescence or FISH are required to complement conventional cytogenetics to confirm a diagnosis of APL. However, conventional cytogenetics should not be abandoned, since it is clearly fundamental to the identification of alternative translocations, the characterization of which has provided considerable insights into the pathogenesis of APL as a whole. Furthermore, cytogenetic analysis is of value in the identification of secondary chromosomal aberrations; the biological and clinical significance of which is currently uncertain. The majority of studies have suggested that the presence of additional cytogenetic abnormalities does not confer a deleterious effect on outcome (reviewed in Ref. 55). Indeed, preliminary evidence suggests that a favorable outcome is also observed in cases in which t(15;17) coexists with cytogenetic features that would otherwise predict an unfavorable prognosis such as 3q aberrations, -5/del(5q), -7 or occurs within a complex karyotype; however, this remains to be confirmed in much larger numbers of patients.55

Advantages and disadvantages of FISH

FISH can provide a valuable second-line approach to characterize cases in which conventional cytogenetics does not reveal the classic t(15;17). A potential advantage of the FISH technique is the wide variety of sample types that can be used for analysis. These include cells cultured for conventional karyotyping, which permit evaluation of chromosomes (metaphase FISH) and/or nuclei (interphase FISH). In addition, bone marrow and peripheral blood smears, or cytospins can be used for interphase FISH. This can be particularly useful in situations in which conventional cytogenetic analysis and/or RT-PCR are unsuccessful, especially when the sample contains very few cells.

Although chromosome paints may be of value in characterizing complex or variant translocations, they are not suitable for investigation of the majority of cases with PML-RARalpha fusions due to insertions, since the amount of rearranged chromosome 15- or 17-derived material is generally too small to be discerned. Therefore, characterization of cryptic PML-RARalpha fusions is dependent upon locus-specific probes that flank or span the chromosomal breakpoint. Probes specific to the PML and RARalpha loci are available commercially. However, it is important to be aware of the size and localization of these probes, since these factors need to be taken into account in assessing the results obtained, particularly in patients lacking the classic t(15;17). Ideally relatively small cosmid probes that are specific for the PML-RARalpha fusion should be used, since its generation by small insertion events can be potentially missed when much larger probes are used. Furthermore, probe sets designed to detect the reciprocal RARalpha-PML fusion gene will fail to yield fusion signals in situations in which the PML-RARalpha fusion results from insertions or simple variant translocations, since in such cases the reciprocal is not formed.33 A further caveat relates to the interpretation of interphase FISH, since normal chromosomes 15 and 17 can lie in close proximity in some nuclei, thereby giving rise to false positive signals. To address this issue, it is important to establish a threshold of positivity using several normal samples (mean percentage of positive cells +3 s.d.), which is about 10% with currently available probes.

Role of RT-PCR in molecular diagnosis of APL; implications for other forms of AML

Of the various molecular techniques available, RT-PCR affords a number of advantages provided precautions are taken to avoid false positives due to contamination and false negatives due to poor RNA quality, failures of RT or PCR steps, or use of inappropriate primers due to occurrence of unusual breakpoints (reviewed in Ref. 56). RT-PCR can provide relatively rapid confirmation of a clinical diagnosis of APL,58 and in this context peripheral blood, as well as bone marrow, is suitable for analysis. Furthermore, it can be used to rapidly (cf Southern analysis) determine PML breakpoint patterns, which have in some studies been correlated with a variety of disease characteristics and considered to provide prognostic information.2,59,60,61,62,63,64,65 A number of studies have suggested that presence of a bcr 3 (S-form) PML breakpoint may confer a poorer prognosis. However, this effect was more marked in studies in which ATRA was used as single agent induction therapy and may be related to decreased ATRA sensitivity observed in vitro in bcr3 cases.66 In more recent studies employing simultaneous ATRA and chemotherapy for induction, any such effect was not sufficiently strong to merit any modification in treatment approach.2,67,68 Bcr 2 (V-form) breakpoints have also been associated with a poorer outcome. However, since such breakpoints occur in only 5% patients and are heterogeneous at the molecular level,69 the numbers studied to date have been insufficient to exclude the possibility that any differences were related to chance rather than a genuine biological effect. Determination of PML breakpoint is also pertinent to the choice of primers for subsequent monitoring for MRD using conventional end-point or quantitative 'real-time' RT-PCR (RQ-PCR) approaches (reviewed in Ref. 70). Diagnostic RT-PCR analysis has the further advantage that it permits detection of reciprocal RARalpha-PML transcripts which provide an additional potential target for MRD monitoring which is available in approximately 75% of patients.2,71

The importance of molecular diagnosis for the optimal management of patients with suspected APL was initially suggested by Miller et al,72 and subsequently underlined by several prospective studies including the recent MRC ATRA trial. Patients with solely molecular evidence of the PML-RARalpha fusion were found to share the favorable prognosis of those with documented t(15;17); whilst cases lacking molecular and cytogenetic evidence for an underlying PML/RARalpha rearrangement were found to have a poorer outcome.2 This latter group had been entered into the trial on the basis of a clinical suspicion of APL. However, in each case subsequent central morphological review excluded this diagnosis. This study carries important implications for other forms of AML, since it would suggest that cases with cryptic rearrangement of core binding factor (CBF) genes, ie AML1-ETO or CBFbeta-MYH11 fusions are likely to be biologically similar to those with documented t(8;21) or inv(16)/t(16;16), respectively. As such they are likely to have a favorable prognosis to standard chemotherapy and can be spared routine use of BMT in first CR, which has been shown to confer no overall survival advantage in patients with documented t(8;21) or inv(16)/t(16;16).14,15 Overall, previous studies suggest that up to 15% of AML with evidence of an underlying AML1-ETO or CBFbeta-MYH11 gene fusion lack the typical respective cytogenetic abnormality (reviewed in Ref. 73), thereby providing an important rationale for molecular screening for such rearrangements. This could not only serve to increase the numbers of patients with a fusion gene target who could be monitored for MRD; but also identify those who would be suitable for tailored therapeutic approaches including molecularly targeted strategies, which are likely to play an increasing role in the management of leukemia patients in the future.

Role of immunofluorescence / immunostaining

Since APL constitutes a hematological emergency, rapid diagnosis is of paramount importance. Whilst conventional RT-PCR approaches,58 as well as more novel RQ-PCR techniques can provide a result within 1 day, there has been considerable interest in immunofluorescence using polyclonal or monoclonal PML antisera which can be performed within 4 h, whereby a diagnosis of PML-RARalpha positive APL is confirmed through detection of a characteristic microparticulate nuclear staining pattern.32,74,75 The technique is reliable provided: (1) cellular disruption is excluded (through use of phase contrast, or nuclear counterstaining); (2) cell spreads are sufficiently thin to prevent blasts from overlapping; and (3) the material examined contains sufficient APL blasts (ie peripheral blood derived from patients presenting with leukopenia will give false negative results if blasts are absent). The technique is also suitable for the analysis of unfixed, unstained crude buffy coat and bone marrow smears (either fresh or stored at -20°C), lending itself to the analysis of archival material. It is now clear that detection of a microparticulate nuclear staining pattern by PML-immunofluorescence in blasts is specific to cases of AML expressing the PML-RARalpha fusion protein, thereby identifying the group of patients likely to benefit from retinoids and arsenic trioxide. In cases in which RARalpha is fused to an alternative partner, eg PLZF, NuMA, NPM or in documented cases of APL lacking a rearrangement of RARalpha, a wild-type PML nuclear staining pattern has been reported.33,37,42,76 ATRA sensitivity varies amongst APL with alternative fusion partners; moreover, arsenic seems unhelpful in PLZF-RARalpha-associated disease44,52and untested in the remainder. For patients with morphological APL associated with a wild-type PML nuclear localization pattern, immunofluorescence is also of potential value in the rapid identification of those with underlying NPM/RARalpha rearrangements which are also retinoid responsive.33,76,77 Monoclonal nucleophosmin antibodies which were originally developed for diagnosis of NPM-ALK-associated non-Hodgkin's lymphoma,78 show a nucleolar pattern in APL cases with an underlying PML/RARalpha rearrangement and other subtypes of AML.33,76 However, in the very rare form of APL associated with an NPM-RARalpha fusion secondary to t(5;17), NPM is delocalized from the nucleoli and a microspeckled nuclear staining pattern is observed.33,76

PML immunofluorescence has proved to be very valuable in rapidly establishing a clinical diagnosis of APL and providing a confirmatory test in cases considered to have cryptic (15;17) rearrangements on the basis of FISH or RT-PCR analyses. The importance of distinguishing PML-RARalpha-associated APL is also important in view of the trend towards use of less intensive chemotherapy regimens for such patients, which are likely to be inadequate for other forms of AML. Although PML immunofluorescence staining is a very useful approach, it should not be considered as a replacement for RT-PCR, since it cannot distinguish between PML breakpoints and lacks the sensitivity to be of any value for minimal residual disease detection.

Should routine molecular screening for PML/RARalpha rearrangements be undertaken in all newly diagnosed AML?

Whilst the t(15;17) has traditionally been considered one of the defining features of APL, there have been a number of reports describing the occurrence of this chromosomal rearrangement with an associated PML/RARalpha fusion in other subtypes of AML.79,80,81,82 Nevertheless, these cases where tested were found to share the microparticulate PML nuclear staining pattern and ATRA sensitivity of patients with M3 morphology, further underlining the close relationship between the presence of the PML-RARalpha fusion protein, disruption of PML nuclear bodies and the ATRA response. The occurrence of such cases has given rise to the proposal that all newly diagnosed cases of AML should be routinely screened for evidence of PML/RARalpha rearrangements, with the rationale that additional patients would be identified for whom molecularly targeted therapies could be employed. However, a recent study in which screening for PML-RARalpha and RARalpha-PML fusion genes by nested RT-PCR was performed in 530 cases of AML lacking the t(15;17) and classified as having FAB types other than M3, revealed only one positive case.81 Central morphological review later revealed this to have been a missed case of M3v and indeed the t(15;17) was detected at the time of relapse. No cases were found to have an underlying PML-RARalpha fusion gene amongst 300 patients with non-M3 AML entered into the GIMEMA treatment protocols (Lo Coco et al, unpublished observations). Moreover, no cases with cryptic PML/RARalpha rearrangements were found when molecular screening was targeted to 54 cases of AML with trisomy 8,83 which is the most common secondary cytogenetic abnormality associated with the t(15;17).13 Therefore, presence of the PML-RARalpha fusion gene in AML subtypes other than M3 is an extremely rare phenomenon. This would suggest that molecular diagnostic techniques designed to detect the PML-RARalpha fusion should be restricted to morphological APL, and additionally targeted only to cases of AML associated with severe coagulopathy, an APL like immunophenotype (CD9+, CD13+, CD33+, sialylated CD15+, HLA-DR-, ± M3v-associated features of CD2, CD34 and/or CD19 positivity), or in which morphological examination reveals even small populations of cells suspicious of APL. Such proposals would appear at first sight to be at variance with results of other studies involving screening of AML for a range of chromosomal rearrangements using multiplex PCR84 or combinations of FISH and RT-PCR85 in which cases with PML-RARalpha fusions in the absence of the t(15;17) were reported. However, in neither of these studies were morphological features described and certainly all cases reported to have cryptic (15;17) rearrangements identified by the former study were actually found to have APL on morphological review (Niels Pallisgaard, personal communication). Therefore, this would again lend support to the view that generalized screening for the PML/RARalpha rearrangement amongst AML FAB types other than M3 using RT-PCR is of questionable value and emphasizes the importance of morphology in identifying this disease. On the other hand, a number of institutions take an alternative view, considering that the PML-RARalpha fusion is too important to miss and prefer to routinely screen all AML cases, which may be most cost effectively undertaken by PML immunostaining.

Minimal residual disease monitoring

Rationale for MRD detection in APL

In view of the relatively favorable prognosis of PML-RARalpha-positive APL following standard first-line therapy with ATRA and anthracycline-based chemotherapy regimens which achieve relapse rates of less than 30% (reviewed in Ref. 5), there is now a growing consensus that routine use of hemopoietic stem cell transplantation (HSCT) is no longer appropriate to consolidate CR1 in such patients. However, in order to achieve further improvements in cure rates, it has become increasingly important to identify the relatively small subgroup of patients with poor prognosis, in whom relapse could potentially be averted through treatment modification. This could involve more intensive consolidation, use of HSCT in first remission or alternative therapeutic approaches such as arsenic trioxide. A number of pre-treatment characteristics have been identified that predict an increased risk of relapse, including WBC and platelet counts2,68,86 (reviewed in Ref. 5), CD56 status,87,88 presence of Flt3 mutations89 and PML breakpoint pattern in some but not all studies90 (reviewed in Ref. 56), This has led to the development of a risk-adapted treatment approach by the GIMEMA and PETHEMA groups, whereby the intensity of consolidation therapy is modified according to presenting WBC and platelet counts. Although this is clearly a step in the right direction, pre-treatment characteristics currently lack the precision to ensure optimal treatment intensity according to the requirements of individual cases (reviewed in Ref. 70). This has engendered a great expectation that MRD monitoring, which has already been shown to provide independent prognostic information in APL, would fulfil its promise to permit more precise identification of patients otherwise destined to relapse, thereby ensuring targeting of additional therapy solely to those most likely to benefit.

Predictive value and limitations of conventional RT-PCR assays for MRD detection in APL patients treated with ATRA and chemotherapy

A number of studies employing conventional PML-RARalpha assays, which typically detect up to one APL cell/104 non-leukemic cells, have shown that extended courses of ATRA and combination chemotherapy ultimately induce PCR negativity in the vast majority of patients.2,65,67,91,92,93 Achievement of molecular remission is an important therapeutic goal in APL; since persistence of PML-RARalpha fusion transcripts until the end of chemotherapy consolidation, which occurs in 2-8% patients,2,4,67,94 has been found to be highly predictive of relapse,95 which can however be averted by additional therapy such as allogeneic BMT (Ref. 96 and Lo Coco et al, manuscript in preparation). However, rather frustratingly, the majority of patients who ultimately go on to relapse have been shown to test PCR negative in the marrow at the end of consolidation therapy.2,67,95,97,98 This indicates that PCR assessment at this single time-point cannot be relied upon to determine the optimum treatment approach in individual patients. Therefore, whilst it is clear that reduction of PML-RARalpha transcripts to below the threshold detectable by standard RT-PCR assays (one in 104) is a prerequisite for long-term survival and may currently represent our best therapeutic goal, achievement of PCR negativity cannot be equated with cure. The failure to detect residual disease in the marrow at the end of consolidation therapy in a significant proportion of patients who ultimately relapse has highlighted the relative insensitivity of such assays, which has been ascribed to inefficiency of the RT step, and also to relatively low expression of PML-RARalpha.99 Indeed, analyses based upon RNA derived from peripheral blood have been found to be even less sensitive,100 thereby accounting for the reliance upon bone marrow sampling for MRD detection in this disease to date. However, it also remains a possibility that in many cases, failure to detect residual disease is a reflection of sample quality giving rise to 'false-negative' results; whilst very occasionally MRD analyses performed on marrow will fail to predict isolated extramedullary relapses.95 In view of the relative insensitivity of the conventional nested PML-RARalpha RT-PCR assay, a number of different approaches have been taken to attempt to more accurately identify a subgroup of patients at particularly high risk of subsequent relapse, as discussed below.

Strategies to increase predictive value of MRD monitoring

Investigation of RT-PCR assays with enhanced sensitivity: A number of groups have evaluated nested RT-PCR assays for the detection of reciprocal RARalpha-PML fusion transcripts as a means of determining whether the inherent improvement in sensitivity (typically 1 log) over the standard PML-RARalpha assay confers any additional benefit in terms of predictive value.2,97,101,102 In an analysis of 105 cases entered into the MRC ATRA trial, performance of both assays in parallel was found to enhance MRD detection. Disappearance of detectable RARalpha-PML transcripts was frequently noted to lag behind PML-RARalpha following initiation of therapy,2 in accordance with the differential sensitivities of the respective assays established in dilution studies of the NB4 cell line.97 Indeed the RARalpha-PML assay led to the detection of residual disease in an additional 20% patients whilst in morphologic remission following induction or consolidation chemotherapy courses. However, the increase in sensitivity afforded by the RARalpha-PML assay did not improve the predictive value of MRD assessment performed at the end of consolidation. The majority of patients who ultimately relapsed had levels of disease at this time-point that still fell below the detection limit of the more sensitive assay; whereas detection of RARalpha-PML transcripts was not necessarily indicative of impending relapse.2 Nevertheless, monitoring RARalpha-PML transcripts in parallel with PML-RARalpha may still have some merit, since a recurrence of RARalpha-PML positivity could provide an earlier warning of impending relapse, than is currently provided by the less sensitive PML-RARalpha assay. Indeed use of both assays in parallel could therefore identify patients at higher or lower risk of relapse, which could be used to modify frequency of MRD assessment following the end of consolidation, accordingly.

A further strategy to try to improve the predictive value of MRD monitoring in APL, involved modifications to the nested RT-PCR technique as a means of increasing assay sensitivity.101,103 However, this was found to lead to the detection of PML-RARalpha and RARalpha-PML transcripts in patients in long-term remission.101,103 This is of interest since it suggests that patients with APL considered to be cured of their disease still have evidence of residual disease, in accordance with data previously acquired in patients with t(8;21) associated AML.104,105 It would appear that inter-study variation in the detection of disease-related transcripts in AML patients in long-term remission is more likely to reflect differences in the sensitivity limits of the respective assays rather than necessarily implying differences in tumor biology associated with remission. These findings suggest that residual disease is held in check by immunological mechanisms; and/or is consistent with the concept that PML-RARalpha and AML-ETO are in themselves insufficient to mediate their respective forms of AML, such that leukemic remission reflects eradication of clones of cells which acquired additional oncogenic events. Either way, the development of such sensitive RT-PCR reactions makes it difficult to distinguish between patients who are likely to be cured of their disease from those who are destined to relapse. This has prompted the development of a competitor-based quantitative nested RT-PCR assay which is more helpful in this respect,106 although such methods are too labor-intensive to be routinely applied to the analysis of large numbers of patient samples.

Evaluation of kinetics of molecular remission achievement on outcome: As discussed above, routine MRD assessment following completion of therapy fails to identify all patients who ultimately relapse. Therefore it was of interest to determine whether MRD monitoring performed earlier in the treatment course suggests that the rate of clearance of MRD is also an independent prognostic factor in APL, as had previously been demonstrated in ALL (reviewed in Ref. 107) and suggested by immunophenotypic MRD analysis in other forms of AML.108 In the study reported by the GIMEMA group, which employed the AIDA protocol, no correlation was observed between PCR status after induction and subsequent relapse risk.67 This is consistent with the concept that detection of PML-RARalpha transcripts at this stage could very well relate to differentiating leukemic cells subject to subsequent apoptosis, which cannot be distinguished by conventional RT-PCR protocols from residual APL blasts. However, the MRC study, in which PCR status using PML-RARalpha and RARalpha-PML assays was determined after each course of chemotherapy, suggested that the rate of clearance of disease-related transcripts is indeed an independent prognostic factor in APL.2 Detection of transcripts at any stage following induction or during consolidation therapy was associated with an increased risk of relapse. This trend was found to be most predictively useful following the third course of chemotherapy when most patients were evaluable, coinciding with the timing of bone marrow harvesting in the MRC AML 10 and 12 trials. Detection of disease-related transcripts at this stage predicted a significantly increased risk of relapse, associated with poorer overall survival (see Figure 2a, b). Furthermore, all the patients with evidence of residual disease at this time had low presenting leukocyte counts confirming delayed clearance of disease-related transcripts as an independent prognostic factor in APL. Whilst this result should be confirmed in a larger group of patients receiving uniform therapy including extended ATRA, it would suggest that molecular monitoring can identify subgroups of patients at high risk of relapse in the context of a relatively homogeneous disease. It is possible that this group of patients could benefit from additional consolidation therapy, including BMT in first remission. However, it may be more reasonable to use MRD profiles following induction and consolidation chemotherapy as a means of determining the most appropriate frequency for subsequent MRD assessment. Patients deemed to be at increased risk of relapse on the basis of pretreatment characteristics and MRD profile could be monitored more rigorously post-consolidation than those considered to be at much lower risk of relapse. This approach has the potential advantages that additional therapy is only targeted to the relatively small subgroup of patients destined to relapse and that such treatment can be delivered at the point of molecular relapse when it is likely to be more efficacious and associated with less morbidity (see below).

Predictive value of molecular surveillance post-consolidation: implications for treatment of molecular relapse: In a recent study the Italian GIMEMA group reported that recurrence of PCR positivity detected by three monthly surveillance marrows performed after completion of therapy was highly predictive of relapse.95 Using such a strategy approximately 70% relapses were successfully predicted, with the majority (81%) of patients who ultimately relapsed converting to PCR positivity within the first 6 months following completion of therapy. Median time from detection of molecular relapse to hematological relapse was 3 months, with a range of 1-14 months. Results from the MRC ATRA trial have highlighted the relatively poor prognosis of patients relapsing following first-line therapy with ATRA and chemotherapy,2 with only 64% achieving a second remission, associated with a 2-year survival of 42%, which are similar to data from other groups.109

Interestingly however, a subsequent study by the GIMEMA group has suggested that outcome may be improved if pre-emptive treatment is given at the time of molecular relapse, rather than awaiting frank hematological relapse.109 In this study, molecular relapse was defined as reappearance of the PML-RARalpha fusion in two successive marrow samples collected during post-consolidation monitoring. Confirmation of positive results by the second sample, avoided problems of 'false-positive' results due to PCR contamination, misinterpretation of non-specific PCR bands, or sample misidentification at any point between bone marrow aspiration and the issuing of results. Furthermore, the requirement for two PCR positive samples to define molecular relapse when using qualitative RT-PCR assays helps in excluding some patients with falling PML-RARalpha transcript levels who may not necessarily require additional more intensive therapy. In a pilot study considering 14 patients treated in molecular relapse with ATRA and chemotherapy, second molecular CR was obtained in 12, indeed in seven of 12 patients this was achieved with 30 days ATRA therapy alone. Eight patients entering second molecular CR subsequently proceeded to ABMT. Overall, the estimated 2-year survival of patients treated at the point of molecular relapse was 92%, which was significantly better than 44% for a previous series of 37 patients receiving the same treatment, but initiated at frank hematological relapse.109

By analyzing a larger patient series with a longer observation period, the GIMEMA group observed that a significant survival advantage was still detectable, at 5 years, comparing 32 patients who received treatment for molecular relapse (OS = 79%) with 94 patients who were given the same therapy at the time of hematologic disease recurrence (OS = 39%) (P < 0.01) (see Figure 3). Interestingly, dissection of the latter category (ie hematologic relapses) revealed that, amongst the 94 patients, 54 had inadequate PCR monitoring (marrow sample not collected or not sent at the appropriate time to the reference laboratory), whilst considering only the 40 patients who were sampled and analyzed correctly as requested in the study, 30 (75%) relapses were predicted by a positive test and 10 (25%) were not (ie samples tested negative in the last assessment prior to hematologic recurrence). In predicted cases, the median time interval between first conversion to PCR-positivity and relapse was 2-3 months (Lo Coco, unpublished observations).

Taking into account intervals between recurrence of PCR-positivity and onset of clinical relapse in both the above study and a recently published survey by the New York group,65 it may be recommended that post-consolidation marrow samples be collected for PCR analysis at 2-3 monthly intervals during the first year, and then at lower frequency (eg every 4-6 months) during the 2nd and 3rd years after completion of therapy. However, it is also evident that the frequency of PCR testing might be adapted depending on pre-therapy patient characteristics. It appears in fact that relapses are more commonly observed in patients with high initial leukocyte counts, as demonstrated in all large recently published APL trials.2,3,67,68,86 Because most events are observed within the first 6-8 months post-consolidation, even more stringent PCR-monitoring (eg every month) might be proposed for this patient category during this period. Similarly stringent monitoring would also be appropriate for patients with delayed clearance of disease related transcripts (ie PCR positive after the third course of chemotherapy), since this group has been shown to be at high risk of relapse (see Figure 2a).2 Conversely, a subgroup accounting for some 20% of newly diagnosed cases, and characterized by low WBC and high platelet (greater than 40 ´ 109/l) count at presentation, has been shown to be at negligible risk of relapse in a combined GIMEMA and PETHEMA study.68 Hence, it is advisable that the latter group be spared the risk of unnecessary treatment-related toxicity, as well as the inconvenience of frequent marrow sampling for molecular studies during follow-up.

Finally, the small but important group of patients who test positive at completion of front-line induction and consolidation, accounting for 2-8% of cases, can be defined as molecularly resistant. Such patients have to be considered at very high risk of disease recurrence associated with an extremely poor prognosis, and as such deserve immediate aggressive treatments including allogeneic BMT (see paragraph below).

Role of MRD monitoring in patients undergoing transplantation procedures

A further clinical context in which molecular monitoring appears extremely promising, is the prediction of outcome following transplantation procedures. However, in view of the overall favorable prognosis of APL, the body of evidence collected thus far is relatively small. In an Italian study comprising 15 patients undergoing ABMT in second CR, PCR status immediately prior to transplant was found to be a key indicator of subsequent relapse-free survival.110 Each of the seven patients with PML-RARalpha fusion transcripts detectable pre-transplant ultimately relapsed, whilst seven of eight patients testing negative at this stage maintained long-term remission. However, the role of ABMT was not entirely clear since only two patients had received combined ATRA and chemotherapy for initial induction. Therefore, it remains a possibility that the prolonged second CR obtained in some patients could have reflected a favorable response to salvage therapy with ATRA and chemotherapy rather than any additional benefit from ABMT consolidation per se. In this study, harvesting was performed in second morphologic remission, such that the PCR status of the graft material and marrow prior to transplant were invariably the same. Similar results were obtained in the MRC ATRA trial in which harvesting was undertaken in first CR. Each of the four cases testing PCR negative prior to ABMT undertaken in second CR, who received PCR negative marrow, remained disease-free at a median follow-up of 28 months.56 Only a very few studies have also considered molecular monitoring and outcome in situations in which the PCR status of the marrow prior to transplant and the graft material were not identical. Sanz et al reported prolonged remission following peripheral blood stem cell transplantation (PBSCT) performed in an APL patient in second molecular CR in which the graft had RT-PCR evidence of disease contamination. In this case, PML-RARalpha fusion transcripts were detectable in the marrow 1 month post PBSCT; all subsequent RT-PCR analyses were negative.111 Similarly, Thomas et al reported a favorable outcome following autologous PBSCT in second CR in a patient with evidence of residual disease within harvested stem cells, but testing PCR negative within the marrow immediately prior to transplant.112 In the MRC ATRA trial, four APL patients were identified with molecular evidence of residual disease in the bone marrow immediately prior to transplant. Three received ABMT using PCR negative marrow harvested in first CR, leading to long-term remissions in two patients, whilst the other case relapsed within 3 months of transplant. The remaining patient underwent allogeneic BMT with a successful outcome.56 Similarly, long-term remission has been reported in four of five further cases with evidence of residual disease in the marrow who underwent allogeneic BMT.96,113,114

Recent studies have also suggested a role for molecular monitoring after transplantation procedures. Marrow derived from patients in long-term remission following autologous and allogeneic transplants has been found to be PCR negative using conventional RT-PCR assays.56,112,114,115,116 Whilst there are relatively few data relating to the immediate post-transplant period, studies published to date would suggest that the presence of residual disease 3 months post-transplant is predictive of relapse.110,114 Interestingly, two cases of APL with evidence of significant residual disease at this stage post-ABMT, as detected by interphase FISH, have been successfully treated with ATRA. In both instances molecular remission was achieved and prolonged clinical remission has been maintained following discontinuation of retinoid therapy.117 The successful eradication of residual disease in these patients suggests that molecular monitoring should be routinely performed post-transplant, particularly in view of the relatively large number of treatment options available for treating molecular relapse in this setting.

MRD monitoring in APL patients treated with novel therapeutic agents

MRD monitoring has also been undertaken in APL patients treated using more experimental approaches, the precise role of which in the management of the disease remains to be determined. Most experience has been gained with arsenic trioxide, which when used as initial treatment for newly diagnosed APL has occasionally been found to induce molecular remissions.118 Relatively high rates of molecular remission have also been reported amongst patients treated with arsenic for frank hematologic relapse.18,118 However, preliminary evidence would suggest that molecular remissions attained with arsenic are generally not sustained, such that additional consolidation therapy is required (Lo Coco, unpublished observations).

A number of preliminary studies have evaluated the efficacy of antibodies targeting CD33 in APL. Both the mouse monoclonal antibody M195 conjugated to 131I and non-radioisotope-conjugated humanized M195 antibody (HuM195) were found to induce molecular remissions in a significant proportion of patients with residual disease following induction therapy, although HuM195 was less efficacious in the context of frank relapse.119,120 More recently, an alternative engineered human antibody targeting CD33 linked to the antitumor antibiotic calicheamicin (CMA-676/Mylotarg) has been evaluated (reviewed in Ref. 121). Preliminary evidence suggests that this could be of benefit to the treatment of APL, achieving high rates of molecular remission when used as front-line therapy in combination with ATRA.19 Furthermore, Mylotarg was also found to be effective in inducing long-term molecular remission in a patient in third relapse following previous autologous PBSCT, ABMT and arsenic trioxide122 and appears to be of value in the treatment of molecular relapse (Lo Coco et al, unpublished observations).

Other agents with efficacy in APL include liposomal ATRA, which attains much higher and more sustained serum concentrations than are achieved with conventional preparations of the drug.123 In a single-center study liposomal ATRA was found to lead to much higher rates of PCR negativity than are normally observed with conventional ATRA as single agent therapy.123 Clinical experience with HDAC inhibitors in APL is currently extremely limited. The exciting initial observation by Warrell et al,53 that the addition of the HDAC inhibitor sodium phenylbutyrate to ATRA led to clinical and molecular remission in a patient with multiply relapsed resistant PML-RARalpha positive APL, suggests that this approach has great promise that could be translated to other forms of leukemia. However, evaluation of HDAC inhibitors in subsequent patients has been somewhat disappointing and may be dependent upon the nature of mutations acquired within the region of the PML-RARalpha fusion gene encoding the ligand-binding domain.124,125,126,127 Nevertheless, it will be of interest to determine whether HDAC/ATRA combinations possibly in conjunction with demethylating agents could be of particular value in the context of molecular relapse at relatively early stages of the disease, potentially predating the acquisition of such mutations. Similarly, other novel therapeutic approaches, such as arsenic trioxide and antibodies targeting CD33, may prove to be most effective when deployed in the context of minimal residual disease rather than frank relapse.

Evaluation of quantitative RT-PCR approaches in PML-RARalpha-associated APL

Rationale for quantitative RT-PCR for MRD assessment in APL: Whilst conventional 'end-point' RT-PCR assays have clearly been shown to provide important prognostic information suitable for directing treatment approaches, studies to date have highlighted a number of limitations to such a strategy (see above). In particular, assays with a sensitivity limit of one in 104 fail to detect residual disease in the majority of patients tested at the end of intensive consolidation chemotherapy who ultimately go on to relapse.2,95 Although regular molecular monitoring post-consolidation improves prediction rates, about a quarter of relapses are still preceded by a PCR negative result.95 This could be a reflection of the relative insensitivity of the assay, patchy involvement of leukemia within the marrow, the kinetics of the relapse process, or 'false-negative' results due to insufficient or poor quality RNA. Attempts to increase assay sensitivity through modifications to RT and PCR steps led to the detection of fusion transcripts in patients in long-term remission, thereby precluding distinction of those cured of their disease from patients destined to relapse.103 This led to evaluation of a competitive RT-PCR approach whereby PML-RARalpha was quantified relative to ABL to control for RNA quality and quantity.100 This study suggested that ABL was a suitable control gene on the basis that its degradation rate was comparable to that of PML-RARalpha mRNA, which is important for the interpretation of MRD data derived from clinical trials in which transit time to the laboratory varies between samples. Furthermore, preliminary data suggested that patients maintaining CR can be distinguished from patients undergoing relapse on the basis of fusion gene transcript levels detected whilst in morphological remission and that relapses are preceded by a rise in detectable PML-RARalpha transcripts. Whilst these methods are too laborious to apply to patients entered into large-scale multi-center clinical trials, they nevertheless suggest that 'real-time' quantitative approaches (RQ-PCR) could be of value for the management of patients with APL.

Studies of 'real-time' quantitative RT-PCR (RQ-PCR) in APL

A number of groups have established RQ-PCR assays for the detection of PML-RARalpha fusion transcripts,128,129,130,131,132,133,134 with encouraging preliminary results. Assays based upon hybridization or hydrolysis probe technology have been shown to be highly reproducible and relatively sensitive, detecting less than 10 plasmid copies of the fusion gene and a single APL cell amongst 104 to 105 non-APL filler cells. The study by Cassinat et al provided preliminary evidence for differing kinetics of molecular remission achievement following ATRA and chemotherapy.130 Rigorous MRD monitoring in a series of 15 patients undertaken by Mitterbauer et al,132 successfully predicted relapses in five patients which were preceded by a rise in PML-RARalpha transcript levels. In an analysis of 47 patients derived from the MRC ATRA trial using PML-RARalpha primer sets developed by the Europe Against Cancer Group,128 RQ-PCR was found to increase detection rates of MRD in comparison to the conventional nested RT-PCR assay.133 Furthermore, this study highlighted the benefits of RQ-PCR as a means of identifying samples with inadequate RNA that could potentially give rise to 'false negative' PCR results. Such samples were found to have very low levels of expression of RARalpha which was evaluated as a control gene, and therefore could not enable MRD detection at anywhere approaching the maximal theoretical sensitivity achieved with good quality RNA. Since conventional 'end-point' assays invariably detected RARalpha transcripts in such suboptimal samples, previous studies based upon this methodology would have failed to exclude them. In addition to reducing the frequency of 'false negative' results, RQ-PCR was found to provide an independent prognostic factor in accordance with previous analyses in the same patient group using conventional nested RT-PCR.2 Detection of PML-RARalpha transcripts at the end of consolidation was predictive of subsequent relapse. Patients with detectable PML-RARalpha transcripts following the third course of chemotherapy had a significantly increased risk of relapse (60%) in comparison to those testing PCR negative at this stage (15% relapse risk).133 Therefore, whilst studies to date evaluating RQ-PCR in APL are relatively small, they nevertheless provide strong support for the adoption of this approach for prospective MRD monitoring in large-scale clinical trials. Furthermore, the ready standardization of RQ-PCR assays not only facilitates quality control, but also for the first time provides a really excellent opportunity for more reliable comparison of MRD data between national and international trial groups.

Concluding remarks and future prospects

Over the next few years it is likely that molecular analysis will assume an even greater role in determining and modulating treatment in individual patients with AML. This is particularly pertinent for patients with PML-RARalpha associated APL, due to the increasing range of therapeutic options available, which may prove to be particularly efficacious in the elimination of residual disease, revealed by RT-PCR assays, following first-line treatment with ATRA and anthracycline-based chemotherapy. MRD monitoring may be complemented by molecular screening for acquisition of mutations in the ligand-binding domain of PML-RARalpha within residual leukemic clones, which may confer retinoid resistance,124,125,126,127 serving as a guide as to whether ATRA or ATRA/HDAC inhibitor combinations are likely to be of value. Other treatment options for patients with evidence of residual disease post-consolidation include further conventional chemotherapy, arsenic trioxide, antibodies targeting CD33 and possibly for patients with a suitable donor, allogeneic HSCT. MRD monitoring has also been shown to be of value in determining whether patients are likely to benefit from autologous transplantation procedures undertaken in second morphological CR. In particular, patients with PML-RARalpha transcripts detected immediately prior to transplant receiving contaminated stem cells typically relapse, whilst patients with no evidence of MRD prior to transplant with a PCR negative graft generally have a favorable outcome.110 Additional studies are required to determine the likely outcome for patients in which PCR status of the graft and marrow immediately prior to autologous HSCT differ (reviewed in Ref. 70). Previous studies suggest that MRD monitoring is also of value as a means of determining need for additional therapy in the post-transplant setting for patients undergoing autologous or allogeneic procedures (reviewed in Ref. 70). In contrast to PML-RARalpha associated APL, in which transplantation procedures have little to offer except for high risk patients in first CR or as consolidation therapy for treatment of relapse, for those with the PLZF-RARalpha fusion, BMT in first CR should be given serious consideration if at all feasible, given the poorer prognosis and more limited treatment options available.33,43

Overall, MRD monitoring studies undertaken in APL patients have enabled greater rationalization in the use of HSCT in first CR or in more advanced disease. This enables targeting of dose intensification to the small subgroup of patients most likely to benefit, sparing unnecessary toxicity in the remaining patients who are likely to be cured of their disease. It is hoped that such an approach could be applied to the management of other subsets of AML, particularly those with AML1-ETO or CBFbeta-MYH11 in which routine use of BMT in first CR confers no overall survival advantage due to the relatively favorable prognosis with chemotherapy alone.14,15 Nevertheless, as indicated by experience gained in APL, such an approach could be particularly helpful in small subsets of 'high risk' patients identified by MRD monitoring. A potential stumbling block has been the controversy surrounding the role of conventional RT-PCR assays in predicting treatment outcome in CBF leukemias (reviewed in Ref. 135). However recent studies using quantitative RT-PCR suggest that patients at high risk of relapse can be distinguished from those maintaining long-term remission.106,136,137 If this is confirmed to be the case; patients with persistently high levels of residual disease may very well benefit from BMT in first morphological CR, whilst pre-emptive therapy may be of value to patients with documented molecular relapse (ie fusion gene transcript levels above the threshold value136,137).

With greater understanding of the molecular pathogenesis of other forms of acute leukemia, it seems highly likely that further molecularly targeted approaches will be developed. This is already exemplified by the evaluation of drugs targeting the RAS pathway which is activated by mutations in at least 10% AML,138 by specific Flt3 inhibitors139 and by the development of the tyrosine kinase inhibitor Glivec, which was designed to target BCR-ABL associated with Ph positive leukemias, but which could also possess activity in leukemias over-expressing c-kit (reviewed in Ref. 140). Agents targeting Flt3 could be of particular interest in APL, since approximately a third of cases harbor mutations in the gene encoding the receptor.89 A further important target for future treatment strategies could be provided by HDAC, since it is now clear that a number of chromosomal rearrangements associated with leukemia generate chimeric transcription factors that target corepressor/HDAC complexes to regulatory genes playing a key role in hemopoietic differentiation. In addition to RARalpha-fusion proteins that characterize APL, these include AML1-ETO generated by t(8;21) in AML and TEL-AML1 formed by t(12;21) in ALL (reviewed in Ref. 141). Clearly the success of such approaches is highly dependent upon the reliability of molecular diagnostic strategies to identify patients who would be suitable candidates.

In all these aspects APL, provides a valuable model for the merits of molecular diagnosis of leukemias, with modulation of therapy according to the underlying genetic aberration and treatment response as determined by MRD monitoring. While conventional qualitative nested RT-PCR assays have provided important prognostic information suitable for clinical decision-making in this disease; preliminary studies indicate that semi-automated quantitative RQ-PCR assays could yield further improvements in optimizing patient management. RQ-PCR has a number of potential advantages, enabling more precise investigation of kinetics of molecular remission achievement following initial therapy and its impact on subsequent relapse risk, evaluating responses to treatment for molecular or frank relapse and directing the need for additional therapy accordingly. Furthermore, through parallel quantification of housekeeping gene transcripts, RQ-PCR serves to identify poor quality samples that could otherwise give rise to 'false negative' PCR results. Overall, RQ-PCR provides an excellent opportunity for greater standardization and reliability of MRD detection within multi-center trials, thereby facilitating comparison of data between international trial groups. With increasing interest in the use of MRD monitoring for clinical decision-making, participation in national and international external quality control schemes that have become established over the last few years in order to optimize methodology, reproducibility and reliability of qualitative and quantitative assays,128,142,143,144 has become of paramount importance. It is hoped that RQ-PCR will enable even more precise tailoring of treatment according to molecular response, serving to identify subgroups of patients at high risk of relapse who could be targeted for additional therapy thereby improving their long-term outcome. Since such patients constitute a minority with APL and given the potential benefits of identifying those at high risk of relapse, whose outcome can be improved by pre-emptive therapy, APL provides an excellent model to stimulate cooperation at the multinational level. Moreover, it is hoped that molecular monitoring will fulfil the equally pressing requirement to reliably identify patients destined to remain in CR, who could potentially be spared excessive therapy with its inherent risk of treatment complications including secondary leukemia and myelodysplasia.

Acknowledgements

DG is supported by the Leukaemia Research Fund of Great Britain and FLC by AIRC, MIUR Cofin. 70% and Ministero della Salute. We are grateful to Marina Lafage-Pochitaloff for helpful discussions.

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Figures

Figure 1 Underlying molecular lesion detected in cases of morphological APL with successful karyotype analysis. Frequencies of cytogenetic and molecular subgroups are based on a study of 611 cases of morphological APL with successful karyotype analysis by the European Working Party.33 Overall to date, 15 cases with the t(11;17)(q23;q21)/PLZF-RARalpha fusion33,39,40,43,44,48,49,52 and four cases with the t(5;17)/NPM-RARalpha fusion33,41,76,145 have been reported, respectively. The NuMA-RARalpha and STAT5b-RARalpha fusions have been reported in only one case each.38,42

Figure 2 Relapse risk (a) and overall survival (b) according to PCR status following three courses of chemotherapy amongst patients entered into the UK Medical Research Council ATRA trial. P-R, PML-RARalpha; R-P, RARalpha-PML. Figure kindly provided by Georgina Harrison, Clinical Trials Service Unit, Oxford, reprinted from Ref. 70, by permission of Baillière Tindall.

Figure 3 Outcome of patients treated by the GIMEMA group receiving pre-emptive therapy at the point of molecular relapse in comparison to a historical control group treated for frank hematological relapse. Reproduced courtesy of WB Saunders146 and Baillière Tindall.70

Received 8 May 2002; accepted 21 June 2002
October 2002, Volume 16, Number 10, Pages 1959-1973
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