The Detection of Minimal Residual Disease in Acute Lymphoblastic Leukaemia
M. N. Potter S UMMA R Y. The detection of minimal residual disease in acute lymphoblastic leukaemia (ALL) can be achieved by assessing leukaemia-specific features at a cellular, chromosomal or molecular level. The application of the polymerase chain reaction to the amplification of leukaemia-specific chromosomal translocations and clone-specific immunoglobulin and T-cell receptor gene rearrangements allows assessment of the majority of cases of ALL. The sensitivity of detection of this technique is around one leukaemia cell in 10s normal marrow cells. A comparative review of the advantages and pitfalls of the different methods of detecting minimal disease is presented. The clinical relevance of such detection is discussed, with early results suggesting that this may have predictive value for future disease relapse.
Definition of Minimal Residual Disease At the time of diagnosis of acute lymphoblastic leukaemia (ALL), the total blast cell load in an adult is estimated to be at least 10’2.1~2 After 7 days of induction therapy blasts have usually disappeared from the peripheral blood. By day 28 there are no bone marrow leukaemia cells detectable by conventional means in over 95% of children treated on modern protocols. However, cessation of therapy soon after remission induction leads to prompt relapse in all cases,3 and even with present-day chemotherapy relapse occurs in one third of all cases of childhood ALL. Therefore, haematological or clinical remission does not correlate with true biological remission and improved methods of assessing remission states are required. The term minimal residual disease describes leukaemia cells present in the marrow at a level below that detectable by conventional methodology. For example, the standard haematological definition of remission is the presence of 5% blasts on light microscopy though
M. N. Potter, Department of Haematology-Oncology, Royal Hospital for Sick Children, St Michael’s Hill, Bristol, BS2 SBJ, UK. Blood Reviews (1992) 6, 68-82 0 1992 Longman Group UK Ltd
this may still correlate with minimal residual disease equivalent to a total of 10” body leukaemia cells.2 The purpose of this review is to discuss methods used in the detection of minimal residual disease and the potential clinical significance of such detection.
Methods Used in the Detection of Minimal Residual Disease (Fig. 1). Detection at a Cellular Level Morphological
Assessment. The simplest approach to the detection of residual leukaemia involves morphological assessment of bone marrow or other relevant tissue samples such as cerebra-spinal fluid or testicular biopsies. The presence of normal, nonmalignant blasts in bone marrow, limits the sensitivity of such detection to 5% unless the leukaemic cells have a highly distinctive cellular morphology eg the L3 form of B-ALL. The hypothesis that earlier detection of relapse could lead to an improved outcome led to a vogue for routine surveillance marrow examination throughout the chemotherapy programme. This hypothesis was not proven4*’ and in one study 72% of the relapses occurred within 1 month of a
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Morphology Immunology
Cell cultw.
Pranuture chromoaomrl condensxtlon fluoreocmce
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Southern blot PCR
Fig. 1 Methods of detection of minimal residual disease at a cellular, chromosomal and molecular level.
normal marrow aspirate.4 As well as having a high false negative rate for the prediction of future relapse, false positive results can occur in children due to the occurrence of a rebound lymphocytosis, often with immature forms, at the end of chemotherapy.‘j As well as routine marrow surveillance, studies of serial morphological assessment of extramedullary sites, notably the central nervous system (CNS) and testis, have been performed. In examination of the cerebra-spinal fluid for leukaemic blasts, false positive results can occur due to contamination with peripheral blood or altered cell morphology resulting from cytocentrifugation, viral infection or chemotherapy effects7 False negative assessment can also occur.’ Testicular relapse, with a subsequent bone marrow relapse in the majority of children, was a cause of treatment failure in up to 20% of boys in early chemotherapy trials. As a result of this, randomised trials of testicular radiotherapy were introduced such as the MRC UKALL VI and VII trials.’ However, there was no overall improvement in outcome and this led to the introduction of bilateral testicular biopsies in late treatment or at the end of therapy in order to predict which boys would relapse. Unfortunately, the false negative rate of testicular biopsy in the detection of residual leukaemia is high, due to sampling error and difficulties in distinguishing between testicular stromal cells, lymphocytes and lymphoblasts.9*‘0 The false positive rate is unknown because those patients interpreted as positive were re-treated. As well as these criticisms, with modern intensive chemotherapy, the rate of testicular relapse is falling (remarkably, less than 1% in the St Jude Study XI). l1 For these reasons routine testicular biopsies have now largely been abandoned and local radiotherapy (plus systemic chemotherapy) is reserved for boys with a biopsy-proven clinical testicular relapse. Immunological Assessment. (Reviewed by Campana et al.“) In general these techniques have been evalu-
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ated using fluorochrome-conjugated antibodies and visualisation by fluorescence microscopy or by analysis on a flow cytometer. Unfortunately, no single nuclear or surface antigen that can currently be recognised is truly leukaemia-specific. This has led to the development of double and even triple immunofluorescent analysis to look for antigen expression patterns which characterise leukaemia cells but are very rare or absent on normal marrow cells. Over 90% of cases of T-ALL express nuclear TdT in association with either CDl, CD3 or CD5,“,i3 This combination of markers is not found on normal T-cells outside the thymus gland. Therefore, immunological assessment is applicable to the majority of cases of T-ALL, and the sensitivity of detection can reach one leukaemic cell in 10’ normal marrow or blood cells (10P5) in experienced hands.“‘i3 The immunophenotype of common ALL (CALL), nuclear TdT, CD10 and CD19 expression, is also found in children in normal bone marrow and the postchemotherapy rebound phase,i4 post marrow transplantation, l5 and in other non-malignant conditions, e.g. transient erythroblastopaenia of childhood.i6 However, there is a subgroup of cases of B-lineage ALL which have asynchronous antigen expression causing marker patterns which occur rarely on normal marrow cells. These include cases expressing nuclear TdT in association with CD1 3, CD33, CD2, CDw65, cytoplasmic u or surface immunoglobulin. 12,i3 This may allow analysis of around one-third of cases of B-lineage ALL, but unfortunately the combination occurring most frequently in childhood ALL (TdT/ cytoplasmic u) is present on cells of normal infant and regenerating marrows, and the sensitivity of detection using this marker combination is only 10m2 to lo-3.12,‘3 To be useful in tracking minimal residual disease, the immunophenotypic patterns described above should be stable and not show changes between presentation and relapse. Campana et all3 described important phenotypic changes in 2 cases of ALL. One of 34 cases of TdT positive ALL showed loss of TdT expression at relapse, and 1 of 4 cases of pre B-ALL expressing TdT/cytoplasmic u showed loss of cytoplasmic u at relapse. The occurrence of phenotypic switches such as these could cause false negative results for the detection of minimal residual disease. The potential of immunological assessment in the tracking of minimal disease has been demonstrated by Campana et al in a study of 44 patients with acute leukaemia.13 In 19 patients [12 T-ALL, 3 B-lineage ALL, 4 acute non-lymphocytic leukaemia (ANLL)], there was evidence of minimal disease in samples considered to be in morphological remission-the level ranged from 0.02% to 5% of the mononuclear cell fraction. All 19 patients relapsed after 4-25 weeks (median 14.5 weeks) from the first abnormal immunological assessment. In the other 25 patients, no evidence of minimal residual disease was found despite repeat experiments. With a follow-up of 17- 114 weeks
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THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC
LEUKAEMIA
(median 28 weeks), 17 of the 25 were well and in complete morphological remission, but 8 have relapsed (6 with a marrow relapse and 2 with an isolated CNS relapse). The interval between the last (negative) immunological test and marrow relapse in these 6 patients ranged from 6-51 weeks (median 21.5 weeks). Therefore, although there were no false positive results associated with this technique, the false negative rate was disappointing, and could rise as follow up was still relatively short. In assessing extramedullary sites of leukaemia, the detection of TdT positive cells in cerebra-spinal fluid has, in certain cases of TdT positive leukaemia, been found to be a more accurate predictor of future CNS relapse than cytomorphology alone.17 However, TdT detection has not been found to be an accurate predictor of testicular relapse. This is due to the presence of TdT positive cells in the normal testis leading to potential false positivity.”
capacity, growth requirements, and drug sensitivity of occult leukaemias. However, the detection of minimal residual disease is limited by a proportion of leukaemias that will not grow in vitro (approximately 20%) and the lack of reproducibility of growth by different laboratories using the same conditionsparticularly for ALL.21 Future improvements may centre on an improved understanding of the growth factor requirements for acute leukaemias. Also, care is required when extrapolating from an in vitro model of leukaemia to the in vivo clinical situation. More elaborate culture systems with stromal elements, and transplantation into severe combined immunodeficiency (s.c.i.d) mice equipped with a human lymphopoietic system offer potentially more meaningful models for studying all aspects of normal stem cell and leukaemia cell biology, including the characterisation of minimal residual disease.
In vitro Culture of Leukaemia Cells.
Detection at a Chromosomal Level
In vitro assays for clonogenic leukaemia (leukaemia colony forming units or CFU-L) have been used to detect occult ALL.1g*20 The marrow is usually T-cell depleted before culture in a semi-solid medium in the presence of feeder cells. CFU-L can then be studied by colony and cell morphology, immunophenotyping, and by cytogenetic and molecular analysis. Estrov et al studied marrow cultures from 13 patients with ALL in morphological remission.‘g 6 of these (5 immediately post remission induction and 1 post bone marrow transplantation) showed evidence of CFU-L with the same immunological and karyotypic markers as the original leukaemic marrow. 4 of these patients relapsed (2, 5, 18 and 30 months after testing), but 2 remain in remission at 20 and 30 months post testing. Cultures from the other 7 patients did not yield CFU-L and this group continued in complete clinical remission with a followup of between 16 and 30 months post presentation. Miller et al studied 58 patients with ALL and ANLL in complete remission at the time of bone marrow harvest prior to autologous bone marrow transplantation.20 CFU-L were cultured from 35 of 43 patients with ANLL, and 10 of 15 patients with ALL. Southern blot analysis of immunoglobulin and T-cell receptor gene rearrangements from the CFUL DNA showed an identical clonal pattern to the original diagnostic marrows in four of these cases of ALL. In this report, in vitro growth did not predict the clinical outcome. However, in a second part to the study, the in vitro sensitivity of the occult CFUL to 4-hydroperoxycyclophosphamide was tested. In these patients, who had been conditioned with cyclophosphamide and had received marrow purged with 4-hydroperoxycyclophosphamide, lack of in vitro sensitivity was found to be the only factor that predicted relapse post marrow transplantation. In summary, the advantages of in vitro marrow culture include the ability to study the proliferative
Conventional cytogenetic analysis with banding techniques is important in the classification of ALL and most cases show either abnormalities of ploidy or structural rearrangements such as translocations. However, the tracking of minimal residual disease is limited by the laborious nature of the analysis and low sensitivity-which is similar to morphological assessment (5%). Other limitations of this technique include the need for in vitro culture, the inability to analyse non-dividing cells and to correlate results with cell phenotype, and the requirement for large chromosomal aberrations (covering 5- 10 Mb of DNA) for visibility. New methods for assessing cytogenetic abnormalities offer potential benefits including a role in the detection of minimal residual disease. Premature
Chromosomal Condensation. This technique involves the fusion of interphase blood or bone marrow cells with mitotic inducer cells and allows the karyotypic analysis of non-dividing cells, which can then be phenotypically recognised by combination with a cytochemical technique.22 The conformation of the condensed chromosomes also indicates the cell cycle status of the interphase cells at the time of fusion. Early studies indicated that bone marrow cells from patients with acute leukaemia tend to arrest in late Gl phase whereas normal marrow cells arrest in early Gl phase. Furthermore, a rising level of late Gl phase cells following a period of remission with low levels, was found to precede morphological relapse by about three months.22 Later work has suggested that this increase is due to a rise in the proportion of mature cells, often of more than one lineage, in late Gl rather than, or in addition to, an actual rise in leukaemic blast cells.22 Fluorescence
in situ Hybridisation. This technique involves denaturation of DNA and hybridisation to biotin labelled single stranded DNA probes. Bound probe is then visualised with fluorescent-labelled avi-
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din.23*24 Three types of probe are available. Firstly, probes have now been identified for nearly all the human chromosomes. They allow the analysis of chromosome copy number in interphase nuclei and metaphase spreads. Sensitivity of detection is limited by naturally occurring low level aneuploidy and hybridisation artefacts, but is currently less than 1%. There has been one recent report of the detection of minimal residual disease by this technique in a patient with ALL characterised by trisomy X.25 Secondly, whole-chromosome probes are composite probes designed to specifically hybridise to each chromosome along its whole length. They allow detection of structural rearrangements in metaphase spreads to a sensitivity level of 10e3. Thirdly, locus-spec$c probes allow recognition of specific chromosomal loci. For example, two colour fluorescent in situ hybridisation with probes for the abl oncogene on chromosome 9q34 and the bcr region on chromosome 22ql1, allows the detection of the Philadelphia chromosomal translocation.23~24 Instead of a random distribution, the presence of juxtaposed bcr and abl signals (within 1 urn in interphase nuclei) is indicative of the Philadelphia chromosome (Phr). This technique does not require prior mitotic stimulation and allows phenotype/genotype correlation on an individual cell basis. It is potentially of great value in the detection of minimal residual disease in cases of ALL with chromosomal translocations and deletions. Two further techniques have the advantage of automation by analysis with a flow cytometer. chromosome-specl&
Flow Karyotyping. This technique
is based on the analysis of chromosome suspensions obtained from IO3 to IO6 mitotic cells after in vitro culture and colcemid treatment.23*24 The chromosomes are stained with two fluorescent dyes, one binding specifically to GC-rich DNA and the other to ATrich DNA, and analysed by dual beam flow cytometry. Individual chromosomes can then be recognised on the resulting two-dimensional flow karyotype. This technique is most suited for recognising structural rearrangements of chromosomes. Limitations include the need for in vitro culture, the inability to separate normal chromosomes 9912 on the flow karyotype, heteromorphic variability amongst the chromosomes of normal individuals, and background interference resulting from nuclear and chromosomal fragments produced during chromosomal isolation.23,24 Measurement of the DNA Index. Difference in ploidy of leukaemia cells can be measured by flow cytometry after staining nuclei with a DNA-specific dye such as propidium iodide. 26 The DNA measurement is expressed as a DNA index, which is the ratio of the modal value of the cellular content of Go/Gl-phase leukaemic cells to normal diploid cells. A high initial DNA index at presentation is a good prognostic factor in childhood ALL.26 Furthermore, this tech-
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nique has identified the presence of minimal residual disease in a case of hypodiploid ALL,27 though the sensitivity of detection is relatively limited. Finally, karyotypic changes from presentation to relapse occurring in around 10% of cases of childhood ALL*’ could cause false negative results for the tracking of minimal residual disease by certain cytogenetic techniques.
Detection at the Molecular Level
This has largely involved two basic techniquesSouthern blotting and the polymerase chain reaction (PCR). In the process of Southern blotting,*’ DNA from the tissue of interest is digested with site-specific restriction enzymes. These recognise specific, recurring, palindromic sequences (usually 4-6 base pairs in length) and cleave double stranded DNA at these sites. The resulting fragments of DNA are separated by agarose gel electrophoresis and then transferred to nylon or nitrocellulose membranes. A radiolabelled complementary DNA probe is then used to identify the gene of interest. Rearrangement of a gene alters the position of the restriction sites relative to one another and this is detected as a shift in the band size after autoradiography. Germline positions for each enzyme/probe combination are documented. Absence of a signal may indicate gene deletion. The PCR (described in detail by Macintyre in Blood Reviews 1989)30 is a very powerful technique which can be used to amplify short segments (less than 10 kb) of double stranded DNA. Amplification is directed by two oligonucleotide primers, which are designed to be complementary to sequences flanking the segment of interest. The procedure involves repeated cycles of heat denaturation to render the DNA single stranded, primer annealing and, with the thermostable enzyme Taq polymerase, extension to form a new strand of complementary DNA. After 30 cycles there is a IO6 to 107-fold increase in the target DNA sequence. The sensitivity and specificity of this technique may be increased by performing two rounds of amplification; the second round using two primers internal to the initial pair (nested PCR). The amplification products are separated by gel electrophoresis, and may be visualised directly under ultraviolet light after ethidium bromide staining, or by using a radiolabelled internal probe after Southern transfer to a filter. These techniques have been used in the detection of minimal residual disease either by application to leukaemia-specific chromosomal translocations or to rearrangements of the immunoglobulin and T-cell receptor genes. Chromosomal Translocations and Minimal Residual Disease: The Philadelphia Chromosome, t (9;22) (q34;qll).
On cytogenetic analysis, this translocation is observed in over 90% of cases of chronic myeloid leukaemia
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THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC
(CML), 15520% of cases of adult ALL, and 4% of cases of childhood ALL. It results from a reciprocal exchange of genetic material between the long arms of chromosomes 9 and 22 with breakpoints in the abl protooncogene and the breakpoint cluster region (bcr) respectively. 31*32Although only one form of Ph’ chromosome is recognised by cytogenetic analysis, there are two distinct translocation sites detected at a molecular level. In CML and approximately half the cases of adult ALL, the usual breakpoint is between exons 2 and 3 or 3 and 4 of the major breakpoint cluster region (M-bcr). By contrast in most cases of childhood ALL, and some cases of adult ALL, the breakpoint is in the first intron of bcr, a region referred to as the minor breakpoint cluster region (m-bcr). In both rearrangements the bcr sequences join to the abl exon 2.33,34Two messenger RNA (mRNA) transcripts of differing length result from the fused bcr-abl genes, one of 8 kb from M-bcr breakpoints and one of 7 kb from m-bcr breakpoints. 35*36Whilst there is a large variation in the site of the breakpoints in rearranged DNA (over 200 kb DNA for the abl oncogene and 5.8 kb DNA for the bcr region), splicing out of sequences during the process of transcription results in these two more constant bcrfabl chimeric mRNA gene segments. These are amenable to amplification by the PCR following an initial reverse transcription step to form complementary DNA. The position of the primers used in this reaction are shown in Figure 2. Maurer et al studied a large series of cases of adult ALL (179 cases) and childhood ALL (87 cases at initial presentation) for the frequency of the bcr-abl rearrangement as assessed by the PCR.37 Significantly, 50% of cases of adult B-lineage ALL were positive by this technique: 32% of which had M-bcr breakpoints and 68% had m-bcr breakpoints. In contrast only 6% of children were positive when tested at disease presentation.37 All cases of T-lineage ALL tested negative. These results may have important biological and prognostic value-patients who tested PCR positive had a tendency towards a higher presenting white cell count, were older and had a significantly poorer prognosis than those who were negative. This may partly explain the difference in BCR
r-iGr-]y-----------J
a exon 2 exon 3
abl
M-bcdexon
G= abl
M-bcr(exonPVabl
abl
m-bcdexon
3Vabl
d
,exonl,~
BCR ______..... exon 2
............
BCR exon 1
lhbl
e
Fig. 2 Possible bcr/abl fusion mRNA transcripts in patients with ALL. Amplification across the different translocation points can be achieved with the primers shown after an initial reverse transcription step. In each type of rearrangement a product of differing size will result.
LEUKAEMIA
overall survival between children and adults with ALL. Furthermore, if these results are confirmed, PCR amplification of bcr-abl may be applicable to around half the cases of adult B-lineage ALL in the tracking of minimal residual disease. CML is the first malignancy to have been studied by the PCR for the assessment of minimal residual disease. Attention has been mainly focussed on patients following allogeneic bone marrow transplantation. Several studies with clinical follow-up have been reported38-50 and provide lessons which may be applicable to other diseases. For example, a number of problems are apparent when an attempt is made to correlate results from different studies: (1) The patient characteristics may be different, including age, duration of chronic phase, conditioning regime, T-cell status of donor marrow and post transplant immunosuppression; (2) The techniques used to detect the minimal disease have been different. For example many of the earlier studies used only a single round of PCR, and these results may not be comparable to the more sensitive nested PCR used in later studies (reported sensitivities are 1O-3 for single round PCR and 10e5 to lO-‘j for nested PCR41*44,50); (3) False positive results caused by contamination of the reaction mix with previously amplified material may have been a particular problem in some of the earlier reports (discussed by Hughes et a15i). Consequently, much more stringent precautions have been taken in recent studies47,50, and (4) The tissue analysed has varied between different studies (i.e. blood or bone marrow). Nevertheless, a number of general conclusions have been made. At a recent workshop results on 222 samples from 157 patients with CML from five centres were reported.48 Many early results (3-12 months post transplant) were positive although the majority later became negative in recipients of non-T-cell depleted marrow grafts. In contrast, in recipients of T-cell depleted grafts, and in those patients transplanted in accelerated or blast phase CML, the rate of positivity was higher. These results are in concordance with the higher rate of clinical relapse seen in these latter groups of patients. However, it is not clear to date whether PCR can accurately and reliably predict relapse in individual patients. For example, some patients have shown persistent positivity by PCR (and occasionally by cytogenetic analysis) but continue in clinical and haematological remission.50 Part of the problem with interpreting minimal disease results in CML may relate to the stem cell nature of this disease. A positive result does not necessarily equate to minimal residual leukaemia, but may reflect persistence of long-living memory lymphocytes,52 which may survive conditioning treatment.53.54 There has been one report of the use of the PCR to assess minimal residual disease in Ph’-positive ALL.55 8 patients (age not presented) in clinical
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remission, 3 of whom showed no evidence of the Ph’ chromosome on cytogenetic analysis, were positive by PCR. 4 patients were followed with serial samples after marrow transplantation; 1 patient became PCR positive after an initial negative phase and subsequently had a clinical relapse, 2 patients were PCR negative and remained in complete remission 9 and 12 months post transplant, and the fourth died of transplant-related complications. Clearly, further studies are required. Unlike CML, (a stem cell disorder), there is evidence that the Ph’ translocation is restricted to the leukaemic cells in Ph’-positive ALL56,57 which may make minimal residual disease results more meaningful in the context of predicting future relapse. t(8;14) (q24;q32). This translocation, involving the c-myc oncogene on chromosome 8 and the immunoglobulin heavy chain gene on chromosome 14, is difficult to amplify by the PCR because of variability in the site of the breakpoints. However, some success has recently been achieved by an ingenious method involving multiple primers. Complementary primers were designed to multiple repeat sequences within the switch J.I region (Sp) of the immunoglobulin heavy chain gene and to three areas of the c-myc gene (within the first intron and to 3’ and 5’ flanking sequences of the first exon of c-myc). Amplification was performed after selection of the appropriate primer sets from Southern blot assessment of the breakpoint regions, giving a sensitivity of detection of lO-‘j in a number of patient samples and cell lines.58 Therefore, this technique may be applicable to around one third of cases of sporadic Burkitt’s lymphoma or B-ALL which show breakpoints within c-myc and !$.I.~~ t(1;19) (q23;pl3). This translocation involving the fusion of the E2A gene from chromosome 19 with the PBXl homeobox gene from chromosome 1 occurs in around 25% of children with pre-B (cytoplasmic p positive) ALL. It results in a chimeric transcript with an identical sequence across the translocation point in each case examined. It is therefore analagous to the hcr/ahl chimeric gene in CML and likewise can be amplified by the PCR following an initial reverse transcription step.(j’ This has allowed the detection of E2A-PBXI transcripts in a patient in whom the translocation was not detected cytogenetitally, and in one patient has suggested the presence of minimal disease occurring before a clinical relapse.hO t(l;l4) (p34;qll). This translocation involves cleavage of the tal-1 gene on chromosome 1 and transposition of the 3’ end of this gene into the T-cell receptor cl/S gene locus on chromosome 14. Even though cytogenetic evidence of a t( 1;14) is present in only a small number of cases of T-ALL, a site-specific deletion, not visible on cytogenetic analysis, affects the tal-1 gene in around one quarter of cases of
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T-ALL.61 These rearrangements (deletions and translocations) vary in site by only a few bases from patient to patient. Amplification by the PCR has been reported with a sensitivity of detection of 10-5.62 As yet, tal-1 rearrangements have not been found in normal tissue, and this target gene offers the possibiIity of studying minimal disease in a significant proportion of cases of T-ALL. There is also evidence indicating a degree of clustering of breakpoints in two further chromosomal translocations: the t(ll;14)(p13;qll) which is found in a minority of cases of T-ALL, and the t(4;l l)(q21;q23) which is commonly found in cases of infant ALL.63,64 These translocations may also prove amenable to amplification by the PCR. In summary, the presence (if confirmed) of the bcr/ abl translocation in around one half of all cases of B-lineage ALL in adults makes this chimeric gene an attractive target for the study of minimal residual disease by PCR. In childhood ALL, the majority of cases do not have recurring chromosomal translocations (Table 1). Therefore, unless new clonal gene rearrangements are found (in a manner similar to the tal-I gene rearrangements in cytogenetically normal cases of T-ALL), a more generally applicable method of studying minimal disease by the PCR is required.
Point Mutations
and Minimal
Residual Disease
Point mutations of the ras proto-oncogene occur in approximately 20% of cases of adult ALL and 6% of cases of childhood ALL.65.66 These have been found predominantly in codons 12 and 13 of N-ras. Whilst it may be possible to detect low levels of cells with ras mutations against a normal cell population by the PCR and sequence specific oligonucleotide probes,66,67 this is technically difficult as there is only a one base mismatch from normal. Also, ras mutations may be altered or absent at relapse compared to presentation.68q69 Recent work has also identified subgroups of ALL with point mutations in the ~53 tumour suppressor gene7’ and deletions of interferon genes71,72-possibly associated with loss of an adjacent (as yet unidentified) tumour suppressor gene. These may provide further markers for the molecular detection of minimal residual disease, but further work is Table 1 Potential applicability of the PCR in the study of minimal residual disease in childhood ALL by amplification across chromosomal translocations
Chromosomal
translocation
t(9; 22) t(8; 14) t(l; 19) t(l; 14) and tal-I rearrangement t(l1; 14)(p13: qll) t(4; 11) Total (refs. 26, 37, 55, 58-64)
% of cases applicable study by PCR 6
M. N. Potter S UMMA R Y. The detection of minimal residual disease in acute lymphoblastic leukaemia (ALL) can be achieved by assessing leukaemia-specific features at a cellular, chromosomal or molecular level. The application of the polymerase chain reaction to the amplification of leukaemia-specific chromosomal translocations and clone-specific immunoglobulin and T-cell receptor gene rearrangements allows assessment of the majority of cases of ALL. The sensitivity of detection of this technique is around one leukaemia cell in 10s normal marrow cells. A comparative review of the advantages and pitfalls of the different methods of detecting minimal disease is presented. The clinical relevance of such detection is discussed, with early results suggesting that this may have predictive value for future disease relapse.
Definition of Minimal Residual Disease At the time of diagnosis of acute lymphoblastic leukaemia (ALL), the total blast cell load in an adult is estimated to be at least 10’2.1~2 After 7 days of induction therapy blasts have usually disappeared from the peripheral blood. By day 28 there are no bone marrow leukaemia cells detectable by conventional means in over 95% of children treated on modern protocols. However, cessation of therapy soon after remission induction leads to prompt relapse in all cases,3 and even with present-day chemotherapy relapse occurs in one third of all cases of childhood ALL. Therefore, haematological or clinical remission does not correlate with true biological remission and improved methods of assessing remission states are required. The term minimal residual disease describes leukaemia cells present in the marrow at a level below that detectable by conventional methodology. For example, the standard haematological definition of remission is the presence of 5% blasts on light microscopy though
M. N. Potter, Department of Haematology-Oncology, Royal Hospital for Sick Children, St Michael’s Hill, Bristol, BS2 SBJ, UK. Blood Reviews (1992) 6, 68-82 0 1992 Longman Group UK Ltd
this may still correlate with minimal residual disease equivalent to a total of 10” body leukaemia cells.2 The purpose of this review is to discuss methods used in the detection of minimal residual disease and the potential clinical significance of such detection.
Methods Used in the Detection of Minimal Residual Disease (Fig. 1). Detection at a Cellular Level Morphological
Assessment. The simplest approach to the detection of residual leukaemia involves morphological assessment of bone marrow or other relevant tissue samples such as cerebra-spinal fluid or testicular biopsies. The presence of normal, nonmalignant blasts in bone marrow, limits the sensitivity of such detection to 5% unless the leukaemic cells have a highly distinctive cellular morphology eg the L3 form of B-ALL. The hypothesis that earlier detection of relapse could lead to an improved outcome led to a vogue for routine surveillance marrow examination throughout the chemotherapy programme. This hypothesis was not proven4*’ and in one study 72% of the relapses occurred within 1 month of a
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Southern blot PCR
Fig. 1 Methods of detection of minimal residual disease at a cellular, chromosomal and molecular level.
normal marrow aspirate.4 As well as having a high false negative rate for the prediction of future relapse, false positive results can occur in children due to the occurrence of a rebound lymphocytosis, often with immature forms, at the end of chemotherapy.‘j As well as routine marrow surveillance, studies of serial morphological assessment of extramedullary sites, notably the central nervous system (CNS) and testis, have been performed. In examination of the cerebra-spinal fluid for leukaemic blasts, false positive results can occur due to contamination with peripheral blood or altered cell morphology resulting from cytocentrifugation, viral infection or chemotherapy effects7 False negative assessment can also occur.’ Testicular relapse, with a subsequent bone marrow relapse in the majority of children, was a cause of treatment failure in up to 20% of boys in early chemotherapy trials. As a result of this, randomised trials of testicular radiotherapy were introduced such as the MRC UKALL VI and VII trials.’ However, there was no overall improvement in outcome and this led to the introduction of bilateral testicular biopsies in late treatment or at the end of therapy in order to predict which boys would relapse. Unfortunately, the false negative rate of testicular biopsy in the detection of residual leukaemia is high, due to sampling error and difficulties in distinguishing between testicular stromal cells, lymphocytes and lymphoblasts.9*‘0 The false positive rate is unknown because those patients interpreted as positive were re-treated. As well as these criticisms, with modern intensive chemotherapy, the rate of testicular relapse is falling (remarkably, less than 1% in the St Jude Study XI). l1 For these reasons routine testicular biopsies have now largely been abandoned and local radiotherapy (plus systemic chemotherapy) is reserved for boys with a biopsy-proven clinical testicular relapse. Immunological Assessment. (Reviewed by Campana et al.“) In general these techniques have been evalu-
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ated using fluorochrome-conjugated antibodies and visualisation by fluorescence microscopy or by analysis on a flow cytometer. Unfortunately, no single nuclear or surface antigen that can currently be recognised is truly leukaemia-specific. This has led to the development of double and even triple immunofluorescent analysis to look for antigen expression patterns which characterise leukaemia cells but are very rare or absent on normal marrow cells. Over 90% of cases of T-ALL express nuclear TdT in association with either CDl, CD3 or CD5,“,i3 This combination of markers is not found on normal T-cells outside the thymus gland. Therefore, immunological assessment is applicable to the majority of cases of T-ALL, and the sensitivity of detection can reach one leukaemic cell in 10’ normal marrow or blood cells (10P5) in experienced hands.“‘i3 The immunophenotype of common ALL (CALL), nuclear TdT, CD10 and CD19 expression, is also found in children in normal bone marrow and the postchemotherapy rebound phase,i4 post marrow transplantation, l5 and in other non-malignant conditions, e.g. transient erythroblastopaenia of childhood.i6 However, there is a subgroup of cases of B-lineage ALL which have asynchronous antigen expression causing marker patterns which occur rarely on normal marrow cells. These include cases expressing nuclear TdT in association with CD1 3, CD33, CD2, CDw65, cytoplasmic u or surface immunoglobulin. 12,i3 This may allow analysis of around one-third of cases of B-lineage ALL, but unfortunately the combination occurring most frequently in childhood ALL (TdT/ cytoplasmic u) is present on cells of normal infant and regenerating marrows, and the sensitivity of detection using this marker combination is only 10m2 to lo-3.12,‘3 To be useful in tracking minimal residual disease, the immunophenotypic patterns described above should be stable and not show changes between presentation and relapse. Campana et all3 described important phenotypic changes in 2 cases of ALL. One of 34 cases of TdT positive ALL showed loss of TdT expression at relapse, and 1 of 4 cases of pre B-ALL expressing TdT/cytoplasmic u showed loss of cytoplasmic u at relapse. The occurrence of phenotypic switches such as these could cause false negative results for the detection of minimal residual disease. The potential of immunological assessment in the tracking of minimal disease has been demonstrated by Campana et al in a study of 44 patients with acute leukaemia.13 In 19 patients [12 T-ALL, 3 B-lineage ALL, 4 acute non-lymphocytic leukaemia (ANLL)], there was evidence of minimal disease in samples considered to be in morphological remission-the level ranged from 0.02% to 5% of the mononuclear cell fraction. All 19 patients relapsed after 4-25 weeks (median 14.5 weeks) from the first abnormal immunological assessment. In the other 25 patients, no evidence of minimal residual disease was found despite repeat experiments. With a follow-up of 17- 114 weeks
70
THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC
LEUKAEMIA
(median 28 weeks), 17 of the 25 were well and in complete morphological remission, but 8 have relapsed (6 with a marrow relapse and 2 with an isolated CNS relapse). The interval between the last (negative) immunological test and marrow relapse in these 6 patients ranged from 6-51 weeks (median 21.5 weeks). Therefore, although there were no false positive results associated with this technique, the false negative rate was disappointing, and could rise as follow up was still relatively short. In assessing extramedullary sites of leukaemia, the detection of TdT positive cells in cerebra-spinal fluid has, in certain cases of TdT positive leukaemia, been found to be a more accurate predictor of future CNS relapse than cytomorphology alone.17 However, TdT detection has not been found to be an accurate predictor of testicular relapse. This is due to the presence of TdT positive cells in the normal testis leading to potential false positivity.”
capacity, growth requirements, and drug sensitivity of occult leukaemias. However, the detection of minimal residual disease is limited by a proportion of leukaemias that will not grow in vitro (approximately 20%) and the lack of reproducibility of growth by different laboratories using the same conditionsparticularly for ALL.21 Future improvements may centre on an improved understanding of the growth factor requirements for acute leukaemias. Also, care is required when extrapolating from an in vitro model of leukaemia to the in vivo clinical situation. More elaborate culture systems with stromal elements, and transplantation into severe combined immunodeficiency (s.c.i.d) mice equipped with a human lymphopoietic system offer potentially more meaningful models for studying all aspects of normal stem cell and leukaemia cell biology, including the characterisation of minimal residual disease.
In vitro Culture of Leukaemia Cells.
Detection at a Chromosomal Level
In vitro assays for clonogenic leukaemia (leukaemia colony forming units or CFU-L) have been used to detect occult ALL.1g*20 The marrow is usually T-cell depleted before culture in a semi-solid medium in the presence of feeder cells. CFU-L can then be studied by colony and cell morphology, immunophenotyping, and by cytogenetic and molecular analysis. Estrov et al studied marrow cultures from 13 patients with ALL in morphological remission.‘g 6 of these (5 immediately post remission induction and 1 post bone marrow transplantation) showed evidence of CFU-L with the same immunological and karyotypic markers as the original leukaemic marrow. 4 of these patients relapsed (2, 5, 18 and 30 months after testing), but 2 remain in remission at 20 and 30 months post testing. Cultures from the other 7 patients did not yield CFU-L and this group continued in complete clinical remission with a followup of between 16 and 30 months post presentation. Miller et al studied 58 patients with ALL and ANLL in complete remission at the time of bone marrow harvest prior to autologous bone marrow transplantation.20 CFU-L were cultured from 35 of 43 patients with ANLL, and 10 of 15 patients with ALL. Southern blot analysis of immunoglobulin and T-cell receptor gene rearrangements from the CFUL DNA showed an identical clonal pattern to the original diagnostic marrows in four of these cases of ALL. In this report, in vitro growth did not predict the clinical outcome. However, in a second part to the study, the in vitro sensitivity of the occult CFUL to 4-hydroperoxycyclophosphamide was tested. In these patients, who had been conditioned with cyclophosphamide and had received marrow purged with 4-hydroperoxycyclophosphamide, lack of in vitro sensitivity was found to be the only factor that predicted relapse post marrow transplantation. In summary, the advantages of in vitro marrow culture include the ability to study the proliferative
Conventional cytogenetic analysis with banding techniques is important in the classification of ALL and most cases show either abnormalities of ploidy or structural rearrangements such as translocations. However, the tracking of minimal residual disease is limited by the laborious nature of the analysis and low sensitivity-which is similar to morphological assessment (5%). Other limitations of this technique include the need for in vitro culture, the inability to analyse non-dividing cells and to correlate results with cell phenotype, and the requirement for large chromosomal aberrations (covering 5- 10 Mb of DNA) for visibility. New methods for assessing cytogenetic abnormalities offer potential benefits including a role in the detection of minimal residual disease. Premature
Chromosomal Condensation. This technique involves the fusion of interphase blood or bone marrow cells with mitotic inducer cells and allows the karyotypic analysis of non-dividing cells, which can then be phenotypically recognised by combination with a cytochemical technique.22 The conformation of the condensed chromosomes also indicates the cell cycle status of the interphase cells at the time of fusion. Early studies indicated that bone marrow cells from patients with acute leukaemia tend to arrest in late Gl phase whereas normal marrow cells arrest in early Gl phase. Furthermore, a rising level of late Gl phase cells following a period of remission with low levels, was found to precede morphological relapse by about three months.22 Later work has suggested that this increase is due to a rise in the proportion of mature cells, often of more than one lineage, in late Gl rather than, or in addition to, an actual rise in leukaemic blast cells.22 Fluorescence
in situ Hybridisation. This technique involves denaturation of DNA and hybridisation to biotin labelled single stranded DNA probes. Bound probe is then visualised with fluorescent-labelled avi-
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din.23*24 Three types of probe are available. Firstly, probes have now been identified for nearly all the human chromosomes. They allow the analysis of chromosome copy number in interphase nuclei and metaphase spreads. Sensitivity of detection is limited by naturally occurring low level aneuploidy and hybridisation artefacts, but is currently less than 1%. There has been one recent report of the detection of minimal residual disease by this technique in a patient with ALL characterised by trisomy X.25 Secondly, whole-chromosome probes are composite probes designed to specifically hybridise to each chromosome along its whole length. They allow detection of structural rearrangements in metaphase spreads to a sensitivity level of 10e3. Thirdly, locus-spec$c probes allow recognition of specific chromosomal loci. For example, two colour fluorescent in situ hybridisation with probes for the abl oncogene on chromosome 9q34 and the bcr region on chromosome 22ql1, allows the detection of the Philadelphia chromosomal translocation.23~24 Instead of a random distribution, the presence of juxtaposed bcr and abl signals (within 1 urn in interphase nuclei) is indicative of the Philadelphia chromosome (Phr). This technique does not require prior mitotic stimulation and allows phenotype/genotype correlation on an individual cell basis. It is potentially of great value in the detection of minimal residual disease in cases of ALL with chromosomal translocations and deletions. Two further techniques have the advantage of automation by analysis with a flow cytometer. chromosome-specl&
Flow Karyotyping. This technique
is based on the analysis of chromosome suspensions obtained from IO3 to IO6 mitotic cells after in vitro culture and colcemid treatment.23*24 The chromosomes are stained with two fluorescent dyes, one binding specifically to GC-rich DNA and the other to ATrich DNA, and analysed by dual beam flow cytometry. Individual chromosomes can then be recognised on the resulting two-dimensional flow karyotype. This technique is most suited for recognising structural rearrangements of chromosomes. Limitations include the need for in vitro culture, the inability to separate normal chromosomes 9912 on the flow karyotype, heteromorphic variability amongst the chromosomes of normal individuals, and background interference resulting from nuclear and chromosomal fragments produced during chromosomal isolation.23,24 Measurement of the DNA Index. Difference in ploidy of leukaemia cells can be measured by flow cytometry after staining nuclei with a DNA-specific dye such as propidium iodide. 26 The DNA measurement is expressed as a DNA index, which is the ratio of the modal value of the cellular content of Go/Gl-phase leukaemic cells to normal diploid cells. A high initial DNA index at presentation is a good prognostic factor in childhood ALL.26 Furthermore, this tech-
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nique has identified the presence of minimal residual disease in a case of hypodiploid ALL,27 though the sensitivity of detection is relatively limited. Finally, karyotypic changes from presentation to relapse occurring in around 10% of cases of childhood ALL*’ could cause false negative results for the tracking of minimal residual disease by certain cytogenetic techniques.
Detection at the Molecular Level
This has largely involved two basic techniquesSouthern blotting and the polymerase chain reaction (PCR). In the process of Southern blotting,*’ DNA from the tissue of interest is digested with site-specific restriction enzymes. These recognise specific, recurring, palindromic sequences (usually 4-6 base pairs in length) and cleave double stranded DNA at these sites. The resulting fragments of DNA are separated by agarose gel electrophoresis and then transferred to nylon or nitrocellulose membranes. A radiolabelled complementary DNA probe is then used to identify the gene of interest. Rearrangement of a gene alters the position of the restriction sites relative to one another and this is detected as a shift in the band size after autoradiography. Germline positions for each enzyme/probe combination are documented. Absence of a signal may indicate gene deletion. The PCR (described in detail by Macintyre in Blood Reviews 1989)30 is a very powerful technique which can be used to amplify short segments (less than 10 kb) of double stranded DNA. Amplification is directed by two oligonucleotide primers, which are designed to be complementary to sequences flanking the segment of interest. The procedure involves repeated cycles of heat denaturation to render the DNA single stranded, primer annealing and, with the thermostable enzyme Taq polymerase, extension to form a new strand of complementary DNA. After 30 cycles there is a IO6 to 107-fold increase in the target DNA sequence. The sensitivity and specificity of this technique may be increased by performing two rounds of amplification; the second round using two primers internal to the initial pair (nested PCR). The amplification products are separated by gel electrophoresis, and may be visualised directly under ultraviolet light after ethidium bromide staining, or by using a radiolabelled internal probe after Southern transfer to a filter. These techniques have been used in the detection of minimal residual disease either by application to leukaemia-specific chromosomal translocations or to rearrangements of the immunoglobulin and T-cell receptor genes. Chromosomal Translocations and Minimal Residual Disease: The Philadelphia Chromosome, t (9;22) (q34;qll).
On cytogenetic analysis, this translocation is observed in over 90% of cases of chronic myeloid leukaemia
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THE DETECTION OF MINIMAL RESIDUAL DISEASE IN ACUTE LYMPHOBLASTIC
(CML), 15520% of cases of adult ALL, and 4% of cases of childhood ALL. It results from a reciprocal exchange of genetic material between the long arms of chromosomes 9 and 22 with breakpoints in the abl protooncogene and the breakpoint cluster region (bcr) respectively. 31*32Although only one form of Ph’ chromosome is recognised by cytogenetic analysis, there are two distinct translocation sites detected at a molecular level. In CML and approximately half the cases of adult ALL, the usual breakpoint is between exons 2 and 3 or 3 and 4 of the major breakpoint cluster region (M-bcr). By contrast in most cases of childhood ALL, and some cases of adult ALL, the breakpoint is in the first intron of bcr, a region referred to as the minor breakpoint cluster region (m-bcr). In both rearrangements the bcr sequences join to the abl exon 2.33,34Two messenger RNA (mRNA) transcripts of differing length result from the fused bcr-abl genes, one of 8 kb from M-bcr breakpoints and one of 7 kb from m-bcr breakpoints. 35*36Whilst there is a large variation in the site of the breakpoints in rearranged DNA (over 200 kb DNA for the abl oncogene and 5.8 kb DNA for the bcr region), splicing out of sequences during the process of transcription results in these two more constant bcrfabl chimeric mRNA gene segments. These are amenable to amplification by the PCR following an initial reverse transcription step to form complementary DNA. The position of the primers used in this reaction are shown in Figure 2. Maurer et al studied a large series of cases of adult ALL (179 cases) and childhood ALL (87 cases at initial presentation) for the frequency of the bcr-abl rearrangement as assessed by the PCR.37 Significantly, 50% of cases of adult B-lineage ALL were positive by this technique: 32% of which had M-bcr breakpoints and 68% had m-bcr breakpoints. In contrast only 6% of children were positive when tested at disease presentation.37 All cases of T-lineage ALL tested negative. These results may have important biological and prognostic value-patients who tested PCR positive had a tendency towards a higher presenting white cell count, were older and had a significantly poorer prognosis than those who were negative. This may partly explain the difference in BCR
r-iGr-]y-----------J
a exon 2 exon 3
abl
M-bcdexon
G= abl
M-bcr(exonPVabl
abl
m-bcdexon
3Vabl
d
,exonl,~
BCR ______..... exon 2
............
BCR exon 1
lhbl
e
Fig. 2 Possible bcr/abl fusion mRNA transcripts in patients with ALL. Amplification across the different translocation points can be achieved with the primers shown after an initial reverse transcription step. In each type of rearrangement a product of differing size will result.
LEUKAEMIA
overall survival between children and adults with ALL. Furthermore, if these results are confirmed, PCR amplification of bcr-abl may be applicable to around half the cases of adult B-lineage ALL in the tracking of minimal residual disease. CML is the first malignancy to have been studied by the PCR for the assessment of minimal residual disease. Attention has been mainly focussed on patients following allogeneic bone marrow transplantation. Several studies with clinical follow-up have been reported38-50 and provide lessons which may be applicable to other diseases. For example, a number of problems are apparent when an attempt is made to correlate results from different studies: (1) The patient characteristics may be different, including age, duration of chronic phase, conditioning regime, T-cell status of donor marrow and post transplant immunosuppression; (2) The techniques used to detect the minimal disease have been different. For example many of the earlier studies used only a single round of PCR, and these results may not be comparable to the more sensitive nested PCR used in later studies (reported sensitivities are 1O-3 for single round PCR and 10e5 to lO-‘j for nested PCR41*44,50); (3) False positive results caused by contamination of the reaction mix with previously amplified material may have been a particular problem in some of the earlier reports (discussed by Hughes et a15i). Consequently, much more stringent precautions have been taken in recent studies47,50, and (4) The tissue analysed has varied between different studies (i.e. blood or bone marrow). Nevertheless, a number of general conclusions have been made. At a recent workshop results on 222 samples from 157 patients with CML from five centres were reported.48 Many early results (3-12 months post transplant) were positive although the majority later became negative in recipients of non-T-cell depleted marrow grafts. In contrast, in recipients of T-cell depleted grafts, and in those patients transplanted in accelerated or blast phase CML, the rate of positivity was higher. These results are in concordance with the higher rate of clinical relapse seen in these latter groups of patients. However, it is not clear to date whether PCR can accurately and reliably predict relapse in individual patients. For example, some patients have shown persistent positivity by PCR (and occasionally by cytogenetic analysis) but continue in clinical and haematological remission.50 Part of the problem with interpreting minimal disease results in CML may relate to the stem cell nature of this disease. A positive result does not necessarily equate to minimal residual leukaemia, but may reflect persistence of long-living memory lymphocytes,52 which may survive conditioning treatment.53.54 There has been one report of the use of the PCR to assess minimal residual disease in Ph’-positive ALL.55 8 patients (age not presented) in clinical
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remission, 3 of whom showed no evidence of the Ph’ chromosome on cytogenetic analysis, were positive by PCR. 4 patients were followed with serial samples after marrow transplantation; 1 patient became PCR positive after an initial negative phase and subsequently had a clinical relapse, 2 patients were PCR negative and remained in complete remission 9 and 12 months post transplant, and the fourth died of transplant-related complications. Clearly, further studies are required. Unlike CML, (a stem cell disorder), there is evidence that the Ph’ translocation is restricted to the leukaemic cells in Ph’-positive ALL56,57 which may make minimal residual disease results more meaningful in the context of predicting future relapse. t(8;14) (q24;q32). This translocation, involving the c-myc oncogene on chromosome 8 and the immunoglobulin heavy chain gene on chromosome 14, is difficult to amplify by the PCR because of variability in the site of the breakpoints. However, some success has recently been achieved by an ingenious method involving multiple primers. Complementary primers were designed to multiple repeat sequences within the switch J.I region (Sp) of the immunoglobulin heavy chain gene and to three areas of the c-myc gene (within the first intron and to 3’ and 5’ flanking sequences of the first exon of c-myc). Amplification was performed after selection of the appropriate primer sets from Southern blot assessment of the breakpoint regions, giving a sensitivity of detection of lO-‘j in a number of patient samples and cell lines.58 Therefore, this technique may be applicable to around one third of cases of sporadic Burkitt’s lymphoma or B-ALL which show breakpoints within c-myc and !$.I.~~ t(1;19) (q23;pl3). This translocation involving the fusion of the E2A gene from chromosome 19 with the PBXl homeobox gene from chromosome 1 occurs in around 25% of children with pre-B (cytoplasmic p positive) ALL. It results in a chimeric transcript with an identical sequence across the translocation point in each case examined. It is therefore analagous to the hcr/ahl chimeric gene in CML and likewise can be amplified by the PCR following an initial reverse transcription step.(j’ This has allowed the detection of E2A-PBXI transcripts in a patient in whom the translocation was not detected cytogenetitally, and in one patient has suggested the presence of minimal disease occurring before a clinical relapse.hO t(l;l4) (p34;qll). This translocation involves cleavage of the tal-1 gene on chromosome 1 and transposition of the 3’ end of this gene into the T-cell receptor cl/S gene locus on chromosome 14. Even though cytogenetic evidence of a t( 1;14) is present in only a small number of cases of T-ALL, a site-specific deletion, not visible on cytogenetic analysis, affects the tal-1 gene in around one quarter of cases of
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T-ALL.61 These rearrangements (deletions and translocations) vary in site by only a few bases from patient to patient. Amplification by the PCR has been reported with a sensitivity of detection of 10-5.62 As yet, tal-1 rearrangements have not been found in normal tissue, and this target gene offers the possibiIity of studying minimal disease in a significant proportion of cases of T-ALL. There is also evidence indicating a degree of clustering of breakpoints in two further chromosomal translocations: the t(ll;14)(p13;qll) which is found in a minority of cases of T-ALL, and the t(4;l l)(q21;q23) which is commonly found in cases of infant ALL.63,64 These translocations may also prove amenable to amplification by the PCR. In summary, the presence (if confirmed) of the bcr/ abl translocation in around one half of all cases of B-lineage ALL in adults makes this chimeric gene an attractive target for the study of minimal residual disease by PCR. In childhood ALL, the majority of cases do not have recurring chromosomal translocations (Table 1). Therefore, unless new clonal gene rearrangements are found (in a manner similar to the tal-I gene rearrangements in cytogenetically normal cases of T-ALL), a more generally applicable method of studying minimal disease by the PCR is required.
Point Mutations
and Minimal
Residual Disease
Point mutations of the ras proto-oncogene occur in approximately 20% of cases of adult ALL and 6% of cases of childhood ALL.65.66 These have been found predominantly in codons 12 and 13 of N-ras. Whilst it may be possible to detect low levels of cells with ras mutations against a normal cell population by the PCR and sequence specific oligonucleotide probes,66,67 this is technically difficult as there is only a one base mismatch from normal. Also, ras mutations may be altered or absent at relapse compared to presentation.68q69 Recent work has also identified subgroups of ALL with point mutations in the ~53 tumour suppressor gene7’ and deletions of interferon genes71,72-possibly associated with loss of an adjacent (as yet unidentified) tumour suppressor gene. These may provide further markers for the molecular detection of minimal residual disease, but further work is Table 1 Potential applicability of the PCR in the study of minimal residual disease in childhood ALL by amplification across chromosomal translocations
Chromosomal
translocation
t(9; 22) t(8; 14) t(l; 19) t(l; 14) and tal-I rearrangement t(l1; 14)(p13: qll) t(4; 11) Total (refs. 26, 37, 55, 58-64)
% of cases applicable study by PCR 6
