Detection of minimal residual leukemia by the polymerase chain reaction: potential implications for therapy

Clinica Chimica Acta, 217 (1993) 75-83 © 1993 Elsevier Science Publishers B.V. All rights reserved. 0009-8981193/$06.00

75

CCA05584

Detection of minimal residual leukemia by the polymerase chain reaction: potential implications for therapy Claus R. Bartram Section of Molecular Biology, Department of Pediatrics II, UniversiO,of UIm, UIm (Germany) (Received 6 May 1992; revision received 16 November 1992; accepted 17 November 1992)

Key words: Minimal residual disease (MRD); Polymerase chain reaction (PCR); Leukemia

Introduction

Over the past two decades impressive advances have been achieved in the treatment of human leukemia. However, disease relapse following successful remission induction remains a significant problem. Since most recurrences originate from malignant cells escaping therapeutic intervention, the development of adequately sensitive methods to detect residual disease is of major clinical importance. A related concern stem~ from the current view that a ,~till insufficiently characterized group of the eventually cured patients, in particuhr of the children, may in fact receive over= treatment and potentially face adverse long-term effects. Unfortunately, the quantity and dynamic behaviour of residual leukemia cells thus far remain largely enigmatic due to the inability of conventional methods including ¢ytomorphology, immunophenotyping, flow-cytometry, cytogen~tics, and Southern blot analysis to identify less than 1-5% malignant cells. The use of double-colour immunofluorescence has significantly improved the level of sensitivity with which neoplastic cells exhibiting phenotypic features that are absent on non-malignant hematopoietic counterparts can be identified [!,2]. More recently the application of polymerase chain reaction (PCR) strategies has set a new standard for the analysis of minimal residual disease (MRD) by permitting the identification of as few as one leukemia in 106 cells [3]. Several target sequences suitable for PCR analysis have been defined (Table 1). The enormous sensitivity raises expectations that PCR technology may offer unique tools Correspondence to: C.R. Bartram, Section of Molecular Biology, Department of PediatricsII, University of UIm, Prittwitzstrasse43, 7900 UIm, Germany.

76 TABLE I Detection of MRD in leukemia patients by PCR technique Marker locus

Leukemia

Target

I.

B-precursor ALL T-ALL, AML T-ALL

DNA

Functional region of rearranged Ig and TCR genes IgH (V-J), TCR-g (V-J), TCR6 (V-J, V-D, D-J) il. TAL-I-SlL recombination Site-specific deletion at 11>32 IlL Fusion region of chromosomal translocations BCR-ABL t(9:22) E2A-PBXI t(l'lP) PML-RARa t(i5;17) DEK-CAN t(6~9)

CML, B-precursor ALL B-precursor ALL AML-M3 AML

DNA

mRNA mRNA mRNA mRNA

to monitor individual responses to therapeutic efforts, to predict impending relapses prior to clinical manifestation and in turn to initiate alternative treatment with as small a leukemia burden as possible, to determine the quality of a bone marrow scheduled for autologous transplantation and to resolve the question of whether the complete eradication of leukemic cells is a conditio sine qua non to cure a patient or whether low frequencies of neoplastic cells can be effectively controlled by the immune system. Such information may eventually allow the design of patientadapted treatment protocols. In the following some aspects regarding possibilities and limitations of PCR methods for the diagnosis and monitoring of leukemia patients will be discussed. Clont~peclfle lg and TCR Probes Genes encoding immunoglobulin (lg) and T-cell receptor (TCR) chains are assembled from multiple segments which recombine during B- and T-cell differentia. tion [4,51. Accordingly, every lymphoid neoplasia is characterized by a unique junctional re$ion of rearranged Ig and/or TC~ loci generated by the recombination of different V, D and J elements and the random insertion or deletion of nucleotides at the joining site. Moreover, cross-lineage rearrangements of Ig or TCR genes are also observed in 10-20% of acute myeloid leukemias (AML). A variety of different PCR methods have been proposed for the generation of clone-specific junctional probes and their consecutive application in the evaluation of the remission status [6-8]. in principle, it is possible to design consensus primers which recognize the majority of V or J elenients of an lg or TCR locus or, alternatively, to synthesize specific amplimers that hybridize to individual V or J segments. The amplification product may then be isolated and directly used as a probe or a junction-specificoligomer can be prepared following sequence analysis. Other approaches comprise PCR using a single.sided oligonucleotide primer or PCR-mediated RNase protection analysis

!O, lOl.

77 TABLE II Detection levelof 71 clone-specificTCR6 probes ! ALL cell in

102-103

103-10's

104

ios

106

Cases

4

6

23

29

9

A strategy developed in our laboratory proceeds from the observation that the majority of ALL patients exhibit distinct patterns of TCR6 rearrangements that can readily be identified by immunogenotyping [6, ! 1,12]. Despite the limited repertoire of recombination, events realized in ALL, TCR6 loci show enormous junctional diversity due to imprecise joining and the insertion of N-region nucleotides. We have thus far amplified and isolated TCR6 junctional regions of 71 ALL patients (58 children, 13 adults) including 45 B-precursor ALL and 26 T-ALL, and used them directly as clone-specific probes. The detection limit of each probe varied due to differences in the site and composition of the junctional region, but allowed the identification of one neoplastic cell in 104-106 cells in the vast majority of cases (Table !I). In the few exceptions, sequence analyses of the junctional region and consecutive synthesis of oligonucleotide probes finally provided us with sufficiently sensitive tools. The detection sensitivity was determined semi-quantitatively by comparing the strength of the autoradiographic signals obtained after hybridization of the clonespecific probe to serial dilutions of the original leukemic material.

TABLE III Retroslmtive analysisof 71 ALL patients using clone-specificTCR6 probes (A) $$ Patients in continuous complete remission Therapeutic phase

Months post diagnosis

Number of st~mples

PCR Status

Consolidation Maintenance

I-6 7-24

20 35

16 12

Termination

> 24

~7

+

Ia

6 23

36

(B) 16 Patients with known relapse Persistence of leukemiacells 6-12 months prior to clinical relapse Detection of MRD 3-6 weeks before relapse Failure to detect relapsing leukemiacells due to secondary TCR rearrangement

12 patients 3 patients I patient

Evaluation of 92 (A) and 49 (B) bone marrow samplesobtained during completeclinical/haematological remission. sPatient relapsed 8 months after PCR analysis.

78

In a group of 55 ALL patients studied during continuous complete remission, bone m,~rro~ samples of most cases obtained during consolidation therap~ (i.e. a 6-month period of intensive treatment following remission induction) exhibited remaining leukemia cells at a level of one in 102-104 cells (Table IliA). A significant number of patients showed minimal residual ALL cells at frequencies of one in 103 to 106 cells even during the phase of maintenance therapy. Patients generally lacked evidence of residual disease after termination of treatment. However, much more important is the observation that longitudinal analyses disclosed marked individual differences in the intervals between achievement or clinical remission and eradication of residual leukemia below the detection level of PCR. It is interesting to note that these dynamic disparities in reduction of leukemia burden do not correlate with known risk factors and may thus define a novel component of the individual response to chemotherapy. A continuous, yet prolonged decline of neoplastic cells may be associated with a favourable course, while a steady increase of blasts apparently predicts clinical relapse. The latter view receives support from data obtained in a second group of 16 ALL patients, who relapsed during the course of the disease (Table IIIB). In most patients persistence and consecutive increase of leukemia cells could be identified several months prior to clinical manifestation. However, in three cases PCR analysis failed to detect neoplastic cells in multiple bone marrow samples obtained during maintenance therapy, yet revealed a PCR-positive result a few weeks before clinical relapse. A likely explanation is that this late detection v, as caused by a focal persistence of residual blasts [131. In another patient PCR analysis failed to detect leukemia relapse due to a secondary recombination at the TCR6 region that served as clone-specific marker. This technical pitfall highlights a limitation of any PCR strategy based on the immunogenotype of leukemia cells. Cional variations at rearranged TCR6 loci occur in about 15% of ALL patients who eventually relapse. However, the proportion of secondary Igll rearrangement o is significantly higher [2]. Moreover, up to 50% of B-precursor ALL exhibit multiple lgH gene rearrangements at initial presentation due to the presence of leukemia subelones [5,14]. These variations may often represent V-replacement events not affecting the D-N-J junctional region contained in a clone-s~cific probe [I 5l, however, this instability may make igH joining regions less suitable targets for PCR monitoring than TCR,y or TCR6 junctions. The data summarized above are in keeping with results obtained in other studies based on smaller numbers of patients [16-18]. TAL-I Gene Recombination Illegitimate V(D)J recombinase activity has been found to mediate a specific 90-kb deletion on chromosome Ip32, which results in fusion of the TAL-I and SIL genes [19-21]. Sequence analysis of TAL-! deletion regions revealed that the fusion-site differs in each leukemia due to random insertion and deletion of nucleotides com. parable with the generation of lg and TCR junctional regions. Since TAL-! deletions are observed on 10-30% of T-ALL patients, this genom;~c alteration represents an attractive target for i ~ R analysis aimed at the detectio~ of MRD [2,20,21]. However, results of resi~ctive studies have not been reported as yet,

79

Fusion Regions of Chromosomal Translocations A variety of leukemia-associated chromosomal abnormalities have recently become accessible to PCR analysis (Table I). The most important of these chromosomal defects is the Philadelphia (Ph) translocation, t(9;22). The Ph transiocation was originally discovered in chronic myelocytic leukemia (CML) but is also observed in acute leukemias. Cytogenetically the Ph chromosomes are indistinguishable between different leukemia entities. On the molecular level, however, two distinct subtypes have been defined [22]. The breakpoints of CML patients almost e~clusively map to the major breakpoint cluster region (M-bcr) on chromosome 22, while the majority of Ph-positive ALL patients show a translocation of ABL sequences into the minor breakpoint cluster region (m-her) of the BCR gene. In a retrospective study including 314 ALL cases a BCRoABL rearrangement was observed in 77/150 (51%) o~"adult B-precursor-ALL patients in contrast to only 6% of children with primary common ALL [23]. It is interesting to note that this remarkably high frequency of BCR-ABL-positive cases is maintained during the first 4 months of a prospective PCR analysis initiated in the German multicenter BMFT (Bundesministerium ffir Forschung und Techr~ologie) trial exhibiting 19 BCR-ABL-positive cases among 38 adult B-lineage ALL patients. In view of this high incidence of BCRABL-positive ALL in adults and the very poor prognosis associated with this leukemia subtype, the detection of chimeric BCR-ABL genes by PCR offers a helpful tool for a timely diagnosis as well as the monitoring of residual disease. Thus far we have analyzed 17 BM and 12 PB samples obtained from 12 Ph-positive ALL patients during complete clinical remission 3-14 months after initial diagnosis. Interestingly, 9 specimens, including BM samples of 3 patients, scored PCR negative. These preliminary data suggest that intensive chemotherapy can decrease the number of Phpositive cells below the detection level of PCR, at least in some patients. Similar results have recently been reported in Ph-positive ALL patients after bone marrow transplantation [24]. It is unlikely that data derived from MRD analysis in a particular leukemia entity can be generalized. This issue calls for independent evaluation ofeach hematopoietic neoplasia, taking into account biological discrepancies as well as different treatment modalities. A case in point is the detection of residual disease in CML patients after BMT. We have studied the remission status of 36 cases (Table IV). Patients who received a T-coil depleted marrow for prophylaxis of graft-versus-host disease (GVHD) exhibited residual disease years after transplantation and 4 relapsed cytogenetically or clinically during follow-up. This result suggests that T-cell depletion interferes with the complete eradication of CML cells due to a lack of graftversus-leukemia effect. This view is in line with the increase in clinical relapses after T-ceU depleted BMT [25]. In CML patients receiving an unmanipulated marrow the presence of GVHD may likewise influence the elimination of residual leukemia. All patients in complete remission for longer than 5 years showed no residual disease (Table IV) indicating that a complete eradication of the malignant cell clone is feasible and may be regarded as a prerequisite for curing CML [26]. The data summarized in Table IV in conjunction with cytogenetic, Southern blot and PCR analyses of other groups demonstrate that the persistence or fluctuation of Ph.positive cells may

80 TABLE IV MRD in 36 Ph-positive CML patients after BMT Number of patients I

7

!! a

29 6

b

23

Clinical fea:ures

PCR status ( > 6 months after BMT)

Follow-up (> 12 months after initial PCR)

T-cell depletion

7 positive cases (56-84 months after BMT)

2 cytogenetic 2 clinical relapses

6 tlegative cases

Complete remission

10 positive cases

2 PCR-ne~ative 2 cytogenetic relapses 3 clinical relapses

! 3 negative cases

1 PCR-positive I cytogenetic relapse

Unmanipulated marrow Long-time survivors (> 5 years) initial PCR analysis 7-35 months after BMT

IaitialPCR analysisof all patientswas performed during complete clinicaland cytogenetic remission.

no~ necessarily be associated with clinical relapse [27-29]. These observations emphasize the relevance of immunological mechanisms in the elimination of residual leuketnia and also pinpoint to substantial differences in the biology of MRD in this group of patients as compared with ALL patients receiving polychemotherapy. in addition to the BCR-ABL recombination, three other oncogene rearrangements corresponding to chromosomal translocations have been used as targets for the monitoring of residual leukemia by PCR (Table I). However, relatively few pa:icnts have thus far been studied with these molecl~lar markers [30-34]. Since the breakpoints of chromosomal translocations are often spread over large distances at the genomic level, hybrid mRI~A sequen~;es after reverse transcription into eDNA have to be used as amplification targets. Starting at the RNA level, however, requires more care in handling the cell samples as compared with DNA-based PCR strategies and poses problems in the quantification of residual disease, a difficulty which may eventually be overcome by the application of competitive PCR techniques [35]. A major drawback stems from the fact that similarly sized chimeric mRNA molecules are present in leukemia cells of different patients. In contrast to .junctional regions of lg and TCR genes, hybrid oncogene transcripts represent leukemia-specific rather than clone/patient-specific sequences. This makes it necessary to establish scrupulous precautions to minimize the danger of false= positive results caused by contamination. Necessary steps to identify or prevent cross-contaminations comwise the inclusion of multiple negative and positive con= trol samples, segregation of laboratory space for all procedures up to and including the setting up of an amplification reaction from those following amplification, prevention of aerosols during sample processing, handling of PCR products in ¢ hood, assignment of a set of positive-displacement micropipettes for preparing and pipet= ting components of the PCR.

81 TABLE V Pitfalls associated with the detection of MRD by PCR ~nalysi~ Cause

Consequence

Continuing ig or TCR rearrangements (in particular lgH locus) Limited sensitivity of junctional probe Degradation (in particular mRNA targets) Focal disease Contamination (in particular mRNA targets)

False-negative False-negative False-negative False-negative False-positive

Perspective Since all approaches mentioned throughout this article bear limitations (Table V) and specific advantages, the use of several methods will be necessary to analyse large numbers of patients and to confirm results derived from a single technique. It is also indicated to standardize methods and to control carefully for false-positive and falsenegative results. Along this line we have investigated 9 adult Ph-positive ALL patients by concurrent PCR analysis using TCR6 and UCB-ABL markers, respectively. Moreover, clone-specific TCR6 probes in conjun~-~t~ionwith the E2A-PBXI marker were used to study remission samples of 6 children with t(l;19) ALL. Mutual confirmation of results derived from both PCR strategies was obtained for all but one BM sample tested. In another cohort of patients a combinatio~ of multicolour immunophenotyping with the TCR6 PCR approach also revealed similar data [36]. At the present time the clinical meaning of detecting as few as one leukemia cell in 106 cells is far from being clear. However, tools to tackle this issue arc available and will be complemented in the nearer future by a broad spectrum of a~ditional PCR methods based on molecular markers characterizing neoplastic cells. Conclusive answers can only be expected from prospective trials enrolling large numbers of patients. Since the data derived from respective analyses may bear a profound impact on the therapy of hematopoietic malignancies including the development of individualized treatment protocols, it appears temr~ing to initiate these studies without further delay.

Acknowledgements I thank T.E. Hansen-Hagge, J.W.G. Janssen, M. Schmidtberger, C. Tell and S. Yokota for molecular analyses. I gratefully acknowledge the coop~;:~',on of, as well as many discussions with, A. Biondi, D. Campana and J.J.M. van Dongen. I thank the participants of the German Multicenter ALL Trials for Children ¢,BFM) and Adults (BMFT), namely D. Hoelzer, W.D. Luowig, J. Maurer, H. Riehm and T. Thiel for cooperation. The study of CML patients has been performed with R. Arnold. Finally, I thank A. Jacobs for editing the manuscript. Supported by the

82 Deutsche Forschungsgemeinschaf'~, Deutsche Krebshilfe and F6rderkreis f/Jr tumorund leuk/imiekranke K i n d e r Ulm.

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