Immunophenotypical detection of minimal residual disease in acute leukemia

Critical Reviews in Oncology/Hematology 32 (1999) 175 – 185 www.elsevier.com/locate/critrevonc

Immunophenotypical detection of minimal residual disease in acute leukemia J.F. San Miguel a,*, J. Ciudad b, M.B. Vidriales a, A. Orfao b, P. Lucio c, A. Porwit-MacDonald d, G. Gaipa e, E. van Wering f, J.J.M. van Dongen g a

Hematology Department, Hospital Uni6ersitario de Salamanca, Paseo de San Vicente 58 -182, 37007 Salamanca, Centro de In6estigacio´n del Cancer (CIC), Uni6ersity of Salamanca-CSIC, Spain b Flow Cytometry Department, Uni6ersidad de Salamanca, Spain c Hematology Department, Instituto Portugues de Oncologia, Lisbon, Portugal d Pathology Department, Karolinska Hospital, Stockholm, Sweden e Hematology Department, Fondazione Tettamanti, Monza, Italy f Dutch Leukemia Study Group, The Hague, The Netherlands g Immunology Department, Rotterdam Uni6ersity, Rotterdam, The Netherlands Accepted 1 June 1999

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

175

2. Strategies for the immunophenotypic detection of minimal residual disease in AL . . . . . . .

176

3. Clinical value of immunophenotypic detection of minimal residual disease . . . . . . . . . . . 3.1. Acute myeloblastic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Acute lymphoblastic leukemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

178 178 179

4. Future directions and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

182

5. Reviewer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

183

Biography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185

1. Introduction The ability to distinguish between normal and leukemic cells, particularly when the leukemic cells are present in low numbers, has been a challenging task for hematologists for many years, due to its important clinical implications. The limitations of morphology to make such a distinction has activated the search for other methods that allow a more precise identification of leukemic cells based on the presence of specific markers on the malignant clone. Such markers/techniques include, (i) a chromosomal abnormality detected by conventional cytogenetics or fluorescence in situ hybridisation (FISH); (ii) a leukemic in vitro growth * Corresponding author. Tel.: + 34-23-291384; fax: + 34-23294624. E-mail address: [email protected] (J.F. San Miguel)

pattern assessed by cell cultures; (iii) DNA content aneuploidy detected by flow cytometry; (iv) an abnormal antigenic marker profile detected by immunophenotyping flow cytometry; and (v) a molecular marker analysed by polymerase chain reaction (PCR). From these techniques, immunophenotyping and PCR are the most attractive options for distinguishing residual leukemic cells from normal cells. The final goal of detecting low numbers of residual leukemic cells (minimal residual disease (MRD)) is to obtain a more precise evaluation of the effectiveness of treatment in order to: (1) design patient-adapted post-remission therapies which would reduce the risk of both relapse and overtreatment; (2) to predict impending relapses prior to clinical manifestations; (3) make a better assessment of the quality of the stem cell harvested for autologous transplant and the efficacy of purging methods; and (4)

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to facilitate early therapeutic interventions (i.e. donor lymphocyte infusions) following transplantation. In this review, we will discuss the most relevant data published on immunophenotypical detection of MRD in acute leukemia (AL), together with our own experience and that accumulated through a European study devoted to the standardisation of techniques for the detection of MRD in AL (BIOMED-1 Concerted Action). The first part of the review will deal with the theoretical basis and current strategies for the immunophenotypical detection of MRD in AL (including both acute lymphoblastic leukemia (ALL) and acute myeloblastic leukemia (AML)), while the second part will focus on the clinical value of those studies for relapse prediction in AL.

2. Strategies for the immunophenotypic detection of minimal residual disease in AL The therapeutic strategies available used for the treatment of patients with acute leukemia result in a high complete remission (CR) rate. However, many of these patients will eventually relapse due to the persistence of low numbers of leukemic cells which are undetectable by conventional cytomorphological criteria whose detection limit is between 1 and 5 leukemic cells among 100 normal cells. The leukemic load under this level is called MRD. As previously mentioned, in order to identify MRD, more sensitive techniques are needed for detecting lower numbers of leukemic cells. At least, from the theoretical point of view, immunophenotyping is an optimum method for the investigation of MRD due to its speed and relative simplicity [1 – 12]. However, it has the disadvantage that neoplastic cells usually lack leukemic specific antigens, with the exception of some proteins resulting from fusion genes such as BCR/ABL and molecules such as NG2 (chondroitin sulfate proteoglican) which are present in blast cells from precursor B-ALL cases with t(4;11) and t(11;19) but not in normal BM cells [13,14]. In addition, phenotypic switches may occur leading to false negative results. Since a prerequisite of any MRD techniques must be the capacity to discriminate leukemic cells from normal cells even when they are present in low numbers in a sample, it is important to define first the phenotypic profiles of normal lymphoid and myeloid cells so that, the phenotypic differences in leukemic cells, if they exist, will become apparent. Conventionally, it has been considered that leukemic cells reflect the immunophenotypic characteristics of normal cells blocked at a certain differentiation stage, and therefore the presence of normal cells will hamper leukemic cell detection. However, several reports from the early reports showed that leukemic phenotypes frequently display deviations from the normal antigen expression during hemato-

poiesis development [1,15,16]. These observations were the basis for understanding abnormal antigen expression both in ALL [2,3] and AML [16,17] and layed the foundations for the identification of low numbers of abnormal cells in T-ALL using microscopic techniques [4]. In fact, they based their analysis in the concept of ‘ectopic’ antigen expression (cells positive for both TdT and T cell antigens outside of the Thymus) (see below) but without specifically mentioning this term. Moreover, using large panels of monoclonal antibodies (MoAb) in triple antigen stainings, analysed at flow cytometry, our group as well as several others [2,5,7,9,16,18–28] have confirmed that leukemic cells frequently display an uncommon or aberrant phenotype that allows their distinction from normal cells. Two major criteria may be used to recognise that a phenotype is leukemic-associated: the existence of ectopic phenotypes and the presence of antigenic aberrancies [9,11,12]. Ectopic antigen expression refers to the detection of cells with a particular phenotype, outside their normal homing sites (as an example, TdT+cells are never present in CNS in normal individuals and therefore their detection is specific of leukemic infiltration). Within the antigenic aberrancies we include three major categories: (i) cross-lineage antigen expression (the presence of either lymphoid-associated antigens on myeloid cells and vice-versa or T cell-associated antigens in Precursor-B-ALL and vice-versa); (ii) maturational related asynchronous antigen expression (presence of two antigens that are never co-expressed in normal hemopoiesis such as CD34 and CD11b) and (iii) antigen overexpression (reactivity for an antigen which is found at abnormally high levels compared to normal cells). It should be noted that in previous reports Terstappen et al. [16,17,21] described four categories, but we prefer to include the fourth one (‘absence of cell surface antigens’) under the heading of maturational asynchronous expression since in most cases the absence of an antigen results in an asynchronous phenotype. The incidence of phenotypic aberrancies has been extremely varied in the literature [5,25,27,29–39], mainly due to the heterogeneity of the panel of MoAb used and to the criteria employed for the definition of these aberrancies. In our experience, 85% of precursorB-ALL, 100% of T-ALL and 88% of AML patients display aberrant phenotypes at diagnosis (Table 1). Moreover this incidence could be even higher (up to 100%) upon expanding the panel of antigens explored (i.e. CD45 is not expressed in a significant number of ALL [40]). Therefore, according to these results immunophenotypical detection of MRD is feasible in most AL patients. Moreover a high proportion of cases showed more than one aberrant criteria. As shown in Table 2, in B- lineage-ALL we have found that the most common type of leukemia associated phenotype is

J.F. San Miguel et al. / Critical Re6iews in Oncology/Hematology 32 (1999) 175–185 Table 1 Incidence of leukemia-associated phenotypes in acute leukemiaa B-ALL (%) (n =100) Crosslineage Ag expression Asynchronous Ag expression Ag over-expression Ectopic phenotypes Total

T-ALL (%) (n= 50)

the presence of CD34 + CD11b+ cells. Reactivity for CD2 lymphoid associated antigen was the most frequent lineage infidelity in AML leukemic cells, while for antigen overexpression, the most common is that of CD34 (Table 4). Based on these data the lack of leukemic specific antigens should not be considered as a pitfall for MRD detection in AL, since most patients display phenotypic aberrancies that can be used as MRD targets to allow the distinction of leukemic from normal cells. The conventional strategy for the immunophenotypical detection of MRD has been generally based on the identification, during follow-up, of residual leukemic cells with the same phenotypic aberrancy as that detected at diagnosis. Consequently, a possible limitation of this approach is the existence of phenotypic changes during disease evolution. Several groups including our own [7,38,42–46], have observed that at relapse, changes in the expression of individual markers are relatively common both in AML and ALL (60 and 30% of cases, respectively, display changes at relapse, although the incidence is significantly lower if the data does not refer to cases but to all antigens explored). However, these changes do usually not affect those antigens involved in the definition of the leukemic associated phenotype; in fact in our experience this only occurs in 16% of AML, 18% of B-ALL and 20% of T-ALL cases. In addition, in a high proportion of AL patients, blast cells simultaneously display two or more phenotypic aberrations and at least one of them remains constant at relapse; these results are consistent with those reported by Campana et al. [7]. Therefore, the simultaneous investigation of all aberrancies detected at diagnosis will reduce the possibility of false negative results, due to the occurrence of phenotypic switches. Moreover, it should be emphasised that the high incidence of phenotypic changes previously re-

AML (%) (n =150)

75

60

27

39

38

80

35 5 85

0 90 100

9 0 88

a

More than one aberrant criterion was found in 60% of B-ALL, 82% of T-ALL, and 85% of AML.

the presence of cross-lineage antigen expression as defined by either the presence of myeloid-associated or T-cell related antigens. Regarding asynchronous antigen expression, the most common are the coexpression of CD34+ CD22+ + and CD10+ +CD20 + + . CD10 overexpression is also particularly frequent in Precursor-B-ALL [41]. In T-ALL the presence of ectopic phenotypes is the most important criteria as reflected by the existence of combinations that in normal individuals are restricted to the cortical thymus but are absent or extremely rare in normal BM. Nearly all T-ALL coexpress TdT and T cell antigens such as CD3 and CD5 or CD4/CD8, or have a weak expression of surface CD3. The incidence and distribution of both cross-lineage and asynchronous antigen expression are similar to those observed in B-ALL (Table 3). Finally, in AML the most common phenotypic aberration is the presence of asynchronous antigen expression, such as

Table 2 Leukemic phenotypes in B-ALL Total: 85% Cross-lineage Ag expression 75%

Asynchronous Ag expression

Ag Over-expression

Ectopic phenotypes and leukemic Ag*

39%

35%

10%

CD13 CD33 CD65 CD5 CD15 CD4 CD14 CD2

CD34+CD22++ CD10++CD20++ CD22++CD20− CD22+CD45− CD20+CD45− CD34+CD10− CD10-CD20d TDT+FMC7+ CD10++CD34−

59% 44% 25% 14% 25% 6% 8% 2%

* Not explored by our group.

7% 13% 4% 6% 9% 16% 5% 20% 9%

CD10 CD20 CD34

177

31% 1% 5%

NG2 (Chondroitin sulfate proteo.) in t(4;11) and t(11;19) KOR-SA 3544 in t(9;22)

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Table 3 Leukemic phenotypes in T-ALL Total: 100% Crosslineage Ag expression 60% CD13 CD33 CD19 CD117

Asynchronous Ag expression 38% 50% 33% 17% 33%

TCR+CD3dim CD34+CD3+ CD4+CD5-CD2− CD1a+CD2−

ported [7,42–46] may be related to two possible technical pitfalls: (a) some changes published in the literature were not based on stringent criteria, since either different MoAb clones or distinct fluorochrome conjugated were used at diagnosis and at relapse; and (b) until recently, the immunophenotypic detection of MRD was based on the antigenic characteristics of the predominant blast cell population at diagnosis. However, it is well known that several leukemic subpopulations with different phenotypic characteristics may be present at diagnosis [21,36], and a minor one might be the resistant clone responsible for relapse. Fig. 1 shows an example of promyelocytic leukemia that relapsed with an immature CD34+ phenotype (100% of cells) although at diagnosis most leukemic cells were CD34 − . However a careful analysis of the phenotype of blast cells at diagnosis shows that a small subpopulation (10% blast cells) of them were already CD34+. This small subpopulation would not be considered for the follow-up analysis of MRD using conventional criteria for the immunophenotypical analysis of patients in morphological CR, but eventually it was the cell clone responsible for the relapse. In our experience the presence of more than one blast cell subpopulation at diagnosis is particularly common in AML (74% of cases have two or more subpopulations) but less common in precursor-B-ALL (24%) and T-ALL (40%)[36,47]. Interestingly, these cell subsets frequently correspond to different stages of maturation of the neoplastic clone as assessed by their phenotypic profile. According to this observation, the investigation of MRD should be based on the phenotypic characteristics of each subpopulation, with special attention paid to the more immature ones. Based on this background, the strategy for MRD detection must include, as a first step, the identification of all phenotypic aberrancies and subpopulations of leukemic cells present at diagnosis. For that purpose, at the European BIOMED study, we have designed specific panels of MoAb in triple antigen combinations for both AML and ALL, specifically defined to identify lineage infidelities and asynchronous antigen expression. Once the aberrations are identified, we select the best combination of MoAb and fluorochromes for the

Ectopic phenotypes \90% 30% 11% 8% 8%

TdT+cCD3/CD5/CD4/CD8+ CD3dim+ CD4+CD8+CD3− CD1a+

95% 48% 19% 24%

identification of each of the phenotypic aberrancies detected in order to have an appropriate custom-built phenotypic probe. For MRD studies, all follow-up samples are analysed using a two-step procedure in which 106 cells, acquired by an appropriate live gate, are screened for the possible persistence of residual cells with the same phenotype as that of the leukemic cells identified at diagnosis. The sensitivity of this approach, based on dilutional experiments, ranges between 10 − 4 and 10 − 5 depending on the type of aberrancy and the normal background (Fig. 2).

3. Clinical value of immunophenotypic detection of minimal residual disease

3.1. Acute myeloblastic leukemia Table 5 summarises some of the most relevant series published concerning immunophenotypic detection of MRD in AML [4,7,23,25,28,48]. Initial MRD studies in AML [4,23] were based on double stainings, using the aberrant coexpression of TdT and myeloid markers, analysed by fluorescence microscopy, as the target for identification of residual disease during follow-up. These studies showed that the persistence or gradual increase of phenotypically aberrant cells was constantly associated with relapse. The immunological finding preceded by a period of 14–38 weeks the morphological relapse. However, some relapses also occurred in cases that were considered to be MRD negative. A very similar picture has been more recently reported by Campana and Pui [7], using multiparameter flow cytometry for the analysis of 13 children in CR after BMT: in four patients residual leukemic cells were observed and all relapsed within 2 months after immunophenotypical detection of MRD; from the remaining nine patients in which leukemic cells were not detected, seven remained in CR with a median followup of\ 1 year after treatment. Instead of focusing on sequential studies, Reading [25] and Wo¨rmann [48] have explored the significance of detecting phenotypically aberrant cells in the first BM aspirate obtained after achieving morphological

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CR. In Reading’s series the six patients who had \ 0.2% aberrant cells relapsed (between 1 and 7 months later), while only 1 of the 19 patients withB0.2% relapsed. In the Wo¨rmann series [48], the persistence of residual disease (defined as\ 0.5% cells with the leukemic phenotype) in the first remission BM was associated with a significantly lower disease free survival (DFS) (20 vs. 62% DFS at 3 years) and the same occurred with the persistence of leukemic cells after consolidation (6 vs. 60% DFS at 3 years). Sievers et al. [28] have reported on 39 AML patients (25 treated with chemotherapy and 10 with allogenic transplantation). In half of the patients leukemic blast cells were detected by multidimensional flow cytometry in the BM specimen obtained from the time that first morphological remission was achieved. The detection of leukemic cells in these patients was predictive of a more rapid relapse (median time of 153 days after diagnosis as compared to 413 days for MRD negative cases). We have explored the value of MRD investigation in a total 371 follow-up BM samples corresponding to 51 AML patients [49]. Our aim was to focus on the BM samples in morphological CR obtained after induction and intensification therapy, because these two moments may be particularly relevant for further therapy decisions. The median number of residual cells at the end of induction was 4 ×10 − 3 (range between 4 ×10 − 2 and 1× 10 − 4) while at the end of intensification it was four times less (1×10 − 3) (range between 2× 10 − 2 and 2× 10 − 5). Upon correlating the incidence of relapses with the number of residual blast cells identified as displaying a leukemic-associated immunophenotype, it was observed that, in the first remission BM obtained after induction therapy, using a threshold of 5×10 − 3 residual leukemic cells, we were able to differentiate two groups of patients with a significant different incidence of relapses (20% relapses in the group with low MRD

179

levels vs. 67% relapses in patients with high MRD levels, P= 0.002) These differences were maintained for the BM obtained after intensification therapy, using a threshold of 2× 10 − 3 for discrimination of the two risk groups. The prognostic impact of MRD detection at these two time points was confirmed in the analysis of relapse free survival (RFS). Using the same cut-off values two significantly different AML subsets could be discriminated with median RFS of 17 months versus not reached for patients with high and low MRD levels, respectively. Finally, we explored whether or not the number of residual leukemic cells was related to the functional expression of multidrug resistance at diagnosis as assessed by the rhodamine-123 efflux in a subgroup of 26 patients; our results showed that both after induction and after intensification therapy those patients with high residual disease levels displayed a significantly higher rhodamine-123 efflux at diagnosis [49].

3.2. Acute lymphoblastic leukemia Table 6 shows several series published in which the clinical value of immunophenotypical detection of MRD in ALL is analysed [4,5,22,50,51]. Although usually based on small numbers of patients initial studies [4,5,22] using double stainings (combining TdT with T cell markers or aberrant myeloid antigens expression) during follow-up, clearly showed that a gradual increase in the number of residual aberrant cells was generally associated with relapse. The immunological finding preceded by 4–25 weeks (mean 15 weeks) the morphological relapse. Griesinger et al. [50], using multiparametric flow cytometry, have shown that the presence of \1% cells with a leukemia-associated phenotype (LAIP) at two consecutive studies in remission BM is a powerful predictor of relapse (86% of LAIP+ cases relapse), while low numbers of LAIP+

Table 4 Leukemic phenotypes in AML cross-lineage Total: 88% Cross-lineage infidelity

Asynchronous Ag expression

Ag over-expression

27%

80%

9%

CD2 CD7 CD19 CD22 CD5

13% 11% 9% 4% 3%

CD117+33+DR− CD34+117+33+DR− CD34−14−15−33+ CD34+33−13+ CD34+15+117−

26% 20% 15% 12% 9%

CD34 HLA DR CD33 CD117

Abnormal light scatter patterns 28% 2% 2% 2% 1%

High FSC/SSC (myeloid) CD19 CD117 CD2 CD34

7% 4% 3% 2%

Low FCS/SSC (lymphoid) CD13 CD33 CD15 CD14

8% 4% 3% 2%

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Fig. 1. CD34 antigen expression in blast cells at diagnosis and at relapse in a patient with M3 leukemia.

cells during chemotherapy was compatible with continuous CR. In addition, Coustan-Smith et al. [51] have shown in a large series of 158 children with ALL, that the proportion of patients with detectable leukemic cells by flow cytometry (using triple-marker analysis) decreases along the treatment course of the disease. Thus, the proportion of patients with residual disease ( \ 0.1% leukemic cells) at the end of remission induction was 23%; 17% at 3 months; and 5 and 4% at months+ 7 and + 12, respectively, while none of the 65 samples examined at the end of therapy (2 years) had evidence of disease. The immunological detection of residual leukemic cells at any of these time-points during the course of treatment was significantly associated with a higher incidence of relapses and this parameter remained as a significant predictor of relapse after adjusting for well-known prognostic factors such as leukocyte counts, age and specific cytogenetic abnormalities. In addition, the presence of residual disease at the end of induction therapy correlated with adverse genetic abnormalities such as the Ph chromosome and MLL gene rearrangements [51], while MRD negativity correlated with hyperdiploidy and abnormalities of the TEL gene. In a series of 53 ALL cases (37 precursor-B-ALL, 16 T-ALL; 24 children and 29 adults) uniformly treated according to the chemotherapy protocols established by the Spanish Pethema Cooperative Group we have explored, whether or not an increase during follow up in

the number of residual aberrant cells could predict relapse. An increase in the level of MRD was considered to exist when the number of aberrant cells gradually increased at two consecutive studies or if in one study the number of leukemic cells was higher than\ 10 − 3 (this occurred in 23 out 53 (43%) of our patients). Relapse rate in patients with an increase in the level of MRD was 90% compared to only 22% in patients with stable MRD levels (P =0.0001). The same relationship was observed when children and adults as well as B and T-lineage ALL were separately analysed. The increase in the number of leukemic cells detected immunologically preceded by 1099 months the morphological and/or clinical relapse. The prognostic value of this criteria was confirmed in the analysis of RFS: patients with increased levels of MRD displayed a significantly shorter RFS (P= 0.0004). We have also explored the influence on disease outcome of the number of aberrant leukemic cells detected at different time-points during the course of treatment: after remission induction, at consolidation (month+ 4), during maintenance (month+12) and once the patients were out of treatment. It was observed that relapse rate was higher in patients with a number of residual blast cells\ 10 − 3 after induction (92 vs. 56%; P=0.02), after consolidation (93 vs. 61%; P= 0.04), during maintenance therapy (93 vs. 27%; P=0.0003) and once the patients were out of treatment (100 vs. 14%; P= 0.01) [52].

J.F. San Miguel et al. / Critical Re6iews in Oncology/Hematology 32 (1999) 175–185

181

Fig. 2. Dilutional experiment of a sample of T-ALL expressing CD7 and CD34 antigens diluted in normal bone marrow, showing a sensitivity level of 10 − 4.

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4. Future directions and conclusions Classically, investigation of MRD has been based on the identification at diagnosis of phenotypic aberrancies as a target for the detection of MRD. However, this strategy has several theoretical technical pitfalls: (i) it requires the existence of a phenotypic aberrancy as well as a diagnostic sample, and therefore cannot be systematically applied to all patients; (ii) it requires technical expertise; and (iii) it may be relatively expensive. For this reasons the search for alternative approaches for the detection of MRD may be of great value. For many years, hematologists have interpreted the existence of alterations in normal hemopoietic differentiation in patients with acute leukemia in CR, such as a decrease in platelet counts or hemoglobin levels, as a sign of a possible relapse prior to or concomitant with the appearance of leukemic cells in the BM. In line with this finding we have recently shown that in AML patients relapse is commonly preceded by the existence of abnormalities in the normal growth pattern of CFU-GM [53]. Based on this background, it can be hypothesised that the persistence of leukemic hematopoiesis would affect normal differentiation. Accordingly, subsequently the detection of immunophenotypical alterations in the normal differentiation patterns, such as the existence of an increase in the proportion of the more immature cell subsets or the presence of cells outside of the normal pathways — probably residual leukemic cells — could help to predict a possible relapse. Therefore, the strategy of this approach would be based on the advantage of flow cytometry for the construction of dot plot

displays for normal differentiation that would allow us to visually distinguish clusters of abnormal leukemic cells from normal cells as well as abnormalities in the normal differentiation pathway. Sievers et al. [28] have shown that a uniform three color panel of monoclonal antibodies was sufficient to detect low levels of AML cells post treatment. Preliminary data from our own institution shows that this may be a very useful and cost-effective approach, which is consistent with a similar observation recently reported by M. Borowitz [40]. Moreover, in line with these studies Vervoordeldonk et al. [54] have shown that during the first year after diagnosis the detection of increased numbers of CD34+ CD19+ or CD34+ CD22+ CD20− cells indicated the likelihood of relapse. The European Cooperative group involved in the BIOMED-1 study (BMH1-CT94 1675) is now exploring this possibility in B and T lineage ALL as well as in AML. In summary, two approaches could be considered for the immunophenotypical investigation of MRD: one is well established and based on the use of custom-built phenotypic probes for the immunophenotypical detection of aberrancies that allow the distinction between leukemic and normal cells; the second would be based on the analysis of changes in the normal differentiation pathway. Regarding the clinical value of these studies, the data which has been generated so far indicates that the level of MRD is a useful marker for monitoring treatment efficacy, and reflects in vivo sensibility to chemotherapy; this particularly applies to the evaluation of the quality of remission. Interestingly, the threshold used for discriminating two risk groups

Table 5 Clinical results in AML No. of cases Follow-up Campana*

MRD

Relapses/total

(%)

Reference

(7)

+ −

4/4 1/3

(100) (33)

[4]

Addriansen*

(15)

+ −

9/9 1/6

(100) (17)

[23]

Campana and Pui

(13) BMT

+ −

4/4 2/9

(100) (22)

[7]

6/6 1/10

(100) (10)

[25]

1st CR BM Reading

(16)

\0.2% B0.2%

Wo¨rmann**

(96)

\0.5% B0.5%

Sievers***

(39)

+ −

62% RFS 3y 20% RFS 3y 153 days 413 days

[48]

[28]

* Studies performed with fluorescence microscopy and dual stainings (TdT/CD13). ** RFS at 3 years after induction (after consolidation: DFS 6 vs. 60%). (%MRD+after induction and consolidation: 63 and 46%). *** Risk of early relapse is 2.8 times higher.

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183

Table 6 Clinical value of MRD in ALL No. of cases

MRD

Relapses/total

(%)

Reference

Campanaa

37

+ −

15/15 7/22

(100) (32)

[4]

Van Dongena

28

+ −

11/12 1/16

(92) (6)

[22]

8

+ −

1/2 0/6

(50) (0)

[5]

46

+ −

12/14 0/32

(86) (0)

[50]

128

+ −

(42)* (7)

[51]

Dracha

Griesingerb,c

Coustan-Smithc

a

Gradual increase during follow-up. Detection of\1% LAIP+cells at two consecutive studies. c Threshold for MRD+\1% and\0.1% in series 4 and 5, respectively. * Relapse rate at 3 years according to MRD evaluation at week 14 (PB0.0002). b

among those patients in morphological CR (around 10 − 3) is highly reproducible in independent series of patients. Regarding follow-up studies, in our opinion immunophenotypic detection of MRD should focus on specific treatment points which are relevant for further therapeutical decisions. Although we have not discussed the area of transplantation, immunophenotyping is a valuable tool to assess the quality of the stem cell harvested for transplantation. Finally, the time has probably now come to design prospective clinical trials based on MRD information.

5. Reviewer This paper was reviewed by George Janossy, MD, PhD, Department of Immunology, Royal Free Hospital, Pond Street, London NW3 2QG, UK.

[3]

[4]

[5]

[6]

[7]

[8]

[9]

Acknowledgements This work was partially supported by national grants from Spain (CYCIT SAF 94-038 and AECC-95). This study is integrated in the European BIOMED1 Concerted Action (BMH-CMT 94-1675).

[10]

[11]

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Biography Professor Jesu´s F. San Miguel is Professor of Hematology at the University of Salamanca and Head of the Hematology Department at the University Hospital of Salamanca. His main field of interest is leukemia and myeloma and during the last 4 years he has coordinated a European study (invoving 14 institutions) for standardization of minimal residual disease. He is a member of the advisory board of the International Myeloma Foundation and Multiple Research Foundation. He has published over 200 papers in international journals.