6 Molecular biology and leukaemia diagnosis

6 Molecular biology and leukaemia diagnosis MARK W O R W O O D M I C H A E L WAGSTAFF

INTRODUCTION The techniques of molecular biology involve the analysis, or manipulation of DNA, RNA and protein at the molecular level. For the diagnosis of leukaemia there are a number of possibilities, some of which are now becoming widely exploited: 1. 2. 3.

Detection of specific nucleotide sequence changes which represent part of the mechanism of malignant change. These may include point mutations, deletions and rearrangements. Detection of 'clonality' (a population of cells derived from a single cell). Analysis of gene rearrangements which can indicate the level of maturation of leukaemic cells.

Examples of all three approaches are described below, but first it is worth examining the techniques currently in use before considering their application. So far, most applications to leukaemia diagnosis have involved examination of DNA and we shall concentrate on D N A , referring briefly to use of RNA or protein sequences when relevant. TECHNIQUES FOR ANALYSIS OF DNA Methods for preparing DNA and RNA from blood cells and tissues are now well established and full details are available in the laboratory manual by Sambrook et al (1989). Special techniques for small samples, etc., are referred to later in this chapter.

Southern blotting In this technique (introduced by Southern, 1975) DNA is extracted from cells or tissues and is cut at specific sites by incubationwith one or more of the many specific restriction endonucleases available. The DNA fragments are then separated by electrophoresis, usually in agarose 'submarine' gels, and the fragments transferred to a nitrocellulose or nylon membrane by 'blotting'. After transfer, the DNA is fixed to the membrane and specific Baillidre's ClinicalHaematology---

Vol. 3, No. 4, October1990 ISBN0-7020--1475-3

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fragments may be detected using a labelled probe (a DNA sequence corresponding to the gene of interest). The technique is simple but timeconsuming. Usually preparation of DNA will require up to 48 h, incubation with restriction enzymes 18h, electrophoresis 24--48h, blotting 18h, hybridization and washing 24 h, and autoradiography anything from 3 h to 10 days, depending on the nature of the gene being studied and the labelling of the probe. Thus analysis of samples will take at least 7 days. Once a technique is in routine use and is well understood then simplification and acceleration may be possible; for example, the use of saturated NaC1 in DNA preparation, instead of the conventional extractions with phenol--chloroform (Miller et al, 1986) is a considerable simplification-although in some samples further purification using phenol-chloroform is necessary to reduce protein contamination. Incubation with restriction endonucleases can be reduced to a few hours after establishing the minimum time required for complete digestion. Thinner and smaller gels for electrophoresis are adequate in some cases and require running times of only a few hours. The classic Southern blotting can be replaced by vacuum blotting, which requires only an hour or so. With optimum conditions, hybridization and washing times can be reduced, and with efficient labelling only a few hours are needed for autoradiography. There are, however, a number of pitfalls. It is essential to check that digestion by the restriction endonuclease is complete--this is readily achieved by rapid electrophoresis of a small portion of the digest in a 'minigel' apparatus. It is often difficult to determine accurately the amount of D N A in a sample, particularly if there is contamination with R N A or protein. While such contamination may not prevent successful analysis of D N A fragments by Southern blotting, it will give an overestimate of the DNA concentration from the absorbance at 260 nm. Again, running the digested DNA in a 'minigel' and comparing the fluorescence seen in the presence of ethidium bromide with a standard D N A preparation will allow a more accurate estimate. Transfer of D N A fragments from the gel to the membrane by blotting is not quantitative, and larger fragments are transferred less efficiently than small fragments. Although depurination with HCI, before denaturation of D N A in the gel with NaOH, is supposed to improve transfer we have not found this additional step to be worthwhile. It is necessary to establish the optimum conditions for hybridization with the labelled probe--these will include the concentration of probe in the hybridization mixture, the stringency of the washing solutions and the temperature of washing. Finally, it should be remembered that if the membrane is exposed to the X-ray film in a cassette containing an intensifying screen there will not necessarily be a linear relationship between radioactivity and the degree of blackening of the film (see later). Despite these qualifications, Southern blotting is a technique that can be carefully monitored and validated. It is usually possible to hybridize the membrane to an alternative probe to show that D N A is present at the expected concentration in the right region of the membrane and so demonstrate that a missing band, or a particularly intense band, on the X-ray film is not an artefact of transfer or hybridization. It is easy to provide 'control'

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samples on either side of the sample of interest to demonstrate positive or negative results and also to estimate the size of fragments. It must be remembered that the mobility of D N A fragments during electrophoresis is dependent on D N A concentration, and it is therefore important to standardize the amount of DNA loaded onto the gel. This is especially important when using 'fingerprinting' techniques (see later). Evaluation of gene dosage is possible provided that precautions are taken to ensure a linear relationship between the amount of DNA and the signal intensity on the X-ray film. Polymerase chain reaction This technique was originally described by Saiki et al (1985), and it is no exaggeration to state that it has caused a revolution in genetic analysis. Using the polymerase chain reaction (PCR) a specific, predetermined DNA sequence may be amplified over a million times, even from trace amounts of starting material (< 10 ng). As a result of amplification, specific sequences can be rapidly analysed for point mutations, polymorphisms, etc. Often direct analysis of the amplified product may be possible (e.g. by gel electrophoresis without the use of probes to identify the fragments). In the PCR, two specific oligonucleotide primers (generally of 20-25 bases) are added to the sample DNA. They flank the sequence of interest-one primer being a copy of a section of the coding strand and the other of the non-coding strand (Figure 1). Binding of the primers to the DNA is achieved by denaturing the DNA at 90-95°C for about lmin and then allowing binding, or annealing, at 45-55°C. Addition of a DNA polymerase that acts on single-stranded DNA initiates synthesis of complementary DNA strands from the attached primers along both the coding and non-coding strands. If the process of denaturation, annealing and primer extension is then repeated, the newly synthesized strand as well as the original strand will also be duplicated. The reaction is carried out in the presence of the necessary deoxynucleotide triphosphates as well as excess primer. In the original technique primer extension was carried out using the Klenow fragment of Escherichia coil DNA polymerase I at 37°C. As the enzyme was denatured at high temperature, fresh enzyme was added during every cycle. A thermostable D N A polymerase from Thermus aquaticus (Taq polymerase) is now available. This can survive incubation at 90°C or more and is most active at 70°C. With a temperature of 50-60°C in the synthetic stage the primers hybridize specifically to their complementary sequences, so reducing non-specific amplification. Because of the stability of the enzyme it is not necessary to add new enzyme during each cycle of heating and cooling. Generally about 30 cycles are used for PCR, with incubation times of about 2 minutes for each stage of the reaction, but after 30 cycles the amount of Taq D N A polymerase becomes limiting. Amplification of approximately 106 times can be achieved under such conditions. Further amplification requires dilution of the product and initiation of a fresh amplification reaction, when factors of 109 may be achieved (Saiki et al, 1988). The process is usually automated with microprocessor-controlled heating blocks

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providing rapid changes of temperature. The exact reaction conditions vary with, for example, the length of sequence to be amplified. The maximum fragment length that can be amplified in this way is in the region of 3 kb; the longer the sequence the longer the extension time required. Although PCR was originally applied to purified DNA it is possible to achieve successful results with DNA extracted from cells but only partially purified. The PCR has been successfully applied to DNA from single hair roots (Higuchi et al, 1988), buccal epithelial cells from a mouthwash (Lench et al, 1988) and stored, paraffin-embedded biopsy specimens (Lai-Goldman et al, 1988).

Detection of amplified products Electrophoresis of the PCR product is usually carried out in polyacrylamide gels, but agarose is used for larger fragments. Staining with ethidium bromide allows determination of the size of the fragments in order to check the specificity of the amplification. Digestion of the PCR product with a restriction endonuclease before electrophoresis can also provide additional information on specificity, and it may facilitate the detection of a point mutation within the fragment. Apart from size differences generated by point mutations, it is also possible to detect point mutations in PCR products by probing with allele-specific labelled oligonucleotides (see'later). Using 'dot' or 'slot' blotting procedures, samples can be hybridized to oligonucleotide probes specific for wild-type or for mutant sequences. By careful adjustment of the hybridization and washing procedures, single nucleotide changes can be detected. Further details of the technique and its applications may be found in Erlich (1989).

Pitfalls encountered in PCR Two drawbacks are now becoming widely recognized. Both are counterparts of one of the two major advantages of the technique: its sensitivity and specificity. The first is contamination: only a trace of contaminating DNA from a 'positive' sample is needed to produce a 'positive' result from a negative sample. Macintyre (1989) has described the precautions necessary to avoid contamination. Using Southern blotting detection of a gene deletion would not be compromised even if 2% of the DNA was from a patient without the deletion, but PCR may yield a result indistinguishable from normal DNA. The second problem is the possibility of genetic variation affecting the primer sequences. An example of this has been given by Fujimura et al (1990). They estimated that at the D758 locus on chromosome 7, genetic variation might lead to a genotyping error once in every 200-300 independent chromosomes analysed.

Availability of DNA probes The choice of a suitable probe requires careful examination of the original description of the probe as well as its subsequent use, and, usually, discussion

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with the authors who have made it available. Generally, investigators have been generous in making probes available for scientific and medical research, but there have been instances where probes are the subjects of patents and where licences have been issued for commercial exploitation. Examples of this are the original mini-satellite probes for paternity testing in commercial laboratories. However, even in these cases, probes have been made available to the scientific community. There have also been instances where probes have been described but have not been released and are only available from a commercial supplier. It is becoming a general policy that journals do not encourage publication of work if the materials are not to be made available, A number of commercial suppliers can now provide probes for the BCR gene, the IgG genes and the T cell receptor (TCR) genes. However, the introduction of PCR technology circumvents the necessity to obtain a probe. Once the sequence is known, oligonucleotides can be synthesized relatively cheaply either in the laboratory or by firms specializing in DNA synthesis. Labelling of probes for Southern blotting or PCR Despite much effort to develop other labels, 32p remains the most widely used label for DNA or oligonucleotide probes. A number of methods for labelling nucleotide probes to high specific activities have become widely used: 1. 2. 3. 4.

'End-labelling' in which the ~/-phosphate of ATP is transferred to the Y-hydroxyl terminus in DNA or RNA by means of bacteriophage T4 polynucleotide kinase. 'Nick translation', in which E. coli DNA polymerase I replaces unlabelled nucteotides in double-stranded DNA with [ctYP]deoxynucleotides. 'Random primer' techniques in which oligonucleotide primers are used to produce radiolabelled probes by primer extension using the Klenow fragment of E. coli polymerase I. 'SP6' labelling of cloned DNA to produce highly radioactive singlestranded RNA.

All these techniques are described in detail in Sambrook et al (1989). The first method is generally used to label oligonucleotide probes or to label size markers for electrophoresis. The 'random primer' methods have to a large extent replaced the nick translation techniques for labelling of plasmid DNA or inserts from plasmids. A number of manufacturers provide kits for both nick-translation and random primer labelling. Single-stranded probes have a number of advantages which are sometimes of critical importance: unwanted D N A : D N A hybrids (probe-to-probe) are not formed and it is possible to prepare probes of known polarity, which make it possible to determine which strand of a DNA segment is transcribed into mRNA. In addition, 32p has the intrinsic advantage of being incorporated into the molecule without disturbing the structure or affecting ability to hybridize. So far, attempts to use non-radioactive labels have not met with general success.

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One interesting development that may permit automation of the PCR has recently been described by Chehab and Kan (1990). They made two primers, one for the normal [3-globin sequence and one specific for the sickle mutation: one was labelled with fluorescein and the other with rhodamine. In addition, an unlabelled oligonucleotide primer for the opposite strand of DNA was synthesized. By initiating the PCR with both of the labelled primers they were able to detect AA, AS and SS genotypes by fluorescence analysis of the amplified DNA. HAEMATOLOGICAL APPLICATIONS The techniques briefly described above originated as tools for purely scientific research. They clearly contribute to an understanding of those haematological diseases that have a genetic basis and they already play a part in diagnostic investigation of some of these conditions. In this section, we describe present applications and indicate their impact on haematological practice. It should be borne in mind that this is an area of rapid change. Chronic myeloid leukaemia In 1960 Nowell and Hungerford described the Philadelphia (Ph) chromosome. In 1973 Rowley suggested that this resulted from a reciprocal translocation between chromosomes 9 and 22 and this has since been more precisely described (see Chapter 5: t(9;22)(q34.1;q11.21). The Ph chromosome is present in haemopoietic cells in 95% of patients with chronic myeloid leukaemia (CML), in about 5% of patients with acute myeloid leukaemia (AML), 10% of children with acute lymphoblastic leukaemia (ALL) and up to 25% of adults with ALL. It is also found very occasionally in patients with polycythaemia rubra vera, myelofibrosis and refractory myelodysplasia; in most of these the Ph chromosome appears to be identical to that described in CML. (For a review of the molecular biology of CML see Dreazen et al, 1988b.) Two oncogenes map to relevant parts of the chromosomes involved in the Ph translocation, A B L to chromosome 9 (q34.1) and SIS to chromosome 22(q12.3-13.1) (Human Gene Mapping, 1990). A B L is the normal cellular homologue of the Abelson murine leukaemic virus transforming gene which causes pre-B cell leukaemias in mice. SIS is the normal homologue of the Simian sarcoma virus transforming gene and is also closely related to the gene coding for the [3-chain of platelet-derived growth factor. It is now known that the translocation involves both A B L (from 9 to 22) and SIS (22 to 9). The A B L gene is at the point at which breaks in chromosome 9 occur, but the SIS oncogene is a considerable distance from the breakpoint on chromosome 22 and there is no evidence of alteration of SIS expression in CML. The A B L gene contains approximately 230 kbp and has two alternative 5' exons (Ia and Ib) along with ten 3' exons (II-XI). Exons Ia and Ib are separated by a long intron. In the formation of the Ph

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Figure 2. Schematic representation of the normal BCR and A B L genes (not to scale) and the ABL-related transcripts present in normal and leukemic cells. (a) Exons of the A B L gene are depicted as open boxes above the line; the alternative first exons Ib and Ia and t h e A B L second exon are indicated by Roman numerals. Exons of the BCR gene are depicted as black boxes above the line. The 'major' breakpoint region is 1-5. The Roman numeral I denotes the first exon of the BCR gene. (b) ABL-related transcripts present in cells containing the Ph chromosome, mRNAs are depicted schematically, and their approximate sizes are given. For each transcript, the coding sequences derived from the BCR gene are shown as solid boxes, using the same numbering system as in (a). From Hooberman et al (1989) with permission,

Chromosome, breaks in chromosome 9 occur at variable sites, usually 5' to exon II of the ABL gene and typically in the Ia-Ib intron but only rarely 5' of exon Ib (Figure 2). Thus exons II-XI of A B L are translocated from chromosome 9 to chromosome 22, and sometimes exon Ia is also taken. However, there is much less variation in the position of the breakpoint on chromosome 22 (Figure 2). Here breaks are found in a section of D N A only a few kilobase-pairs long, which has been called the breakpoint cluster region or bcr (Groffen et al, 1984). This bcr region is part of the more than 70-kbp tong gene that has been called BCR. It includes 20 exons encoding a protein of 127 amino acids (Heisterkamp et al, 1985). In the human genome there are three related genes containing only the 3' region (including the last seven exons). These genes are not located on chromosome 22 and do not appear to be transcribed (Lifshitz et al, 1988). In Ph-positive CML, breaks in the BCR gene occur as shown in Figure 2 in the so-called major bcr region. In Ph-positive ALL, breaks occur in the 'minor bcr' region and the fused gene does not include exons from the major

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region. In normal cells the BCR gene is transcribed into mRNAs of 4.5 and 7kbp. The 7kb transcript includes the sequences found in the 4.5kb sequence. A B L is transcribed into mRNAs of 6 and 7 kb, which are initiated by different promotors. In cells containing the Ph chromosome there is a novel of 8.5 kbp ABL-related transcript which contains 3.3 kb of BCR 5' sequences as well as A B L sequences (Shtivelman et al, 1985). In normal cells, A B L mRNA transcripts are translated into 145 kDa proteins (called P145) which differ at the N-terminus according to the presence of exons Ia or Ib in the gene. In CML the chimeric 8.5 kbp BCR-ABL mRNA is translated into a protein of molecular mass 210 kDa. This includes 1104 amino acids from A B L and either 902 or 927 amino acids from the BCR protein according to the absence or presence of exon 3 in the mRNA. This fused protein (P210), unlike the normal P145, has significant tyrosine kinase activity in vitro (Konopka et al, 1984).

Prognostic and diagnostic application of molecular study of the BCR region Ph-negative CML. Although demonstration of a BCR rearrangement in a patient with a cytogenetically Ph-positive CML provides no additional diagnostic information, the technique may be of value where karyotyping is difficult or when only frozen or dead cells are available. Chan et al (1987) have described the use of a 5.8 kb BCR probe which appears to be able to detect rearrangements in all such patients studied. One obvious application is in confirming the diagnosis of CML in a suspected case which is Ph-negative on cytogenetic examination. A proportion of patients with 'Ph-negative CML' will be found to have a rearrangement in the bcr region and a diagnosis of CML can be established. The largest study reported is that of Wiedemann et al (1988) who analysed 28 cases of Ph-negative CML and found that eight showed rearrangements of BCR. Five of these cases were indistinguishable from Ph-positive CML on the basis of morphology, of blood and bone marrow, and in clinical course. Seven which did not show BCR rearrangement were reclassified as atypical CML and, in one case, chronic myelomonocyticleukaemia (CMML). Kantarjian et al (1988) investigated 23 patients with Ph-negative CML and 17 patients with CMML. Rearrangement of BCR was found in 11 of the CML patients (48 %) and in none of the patients with CMML. Among patients classified as Ph-negative, response to c~-interferon therapy was also examined. BCRrearrangement positive CML patients had similar characteristics to those with Ph-positive disease (a good response), but BCR-negative patients had a poor response. Kantarjian et al concluded that these molecular studies not only helped in the classification of CML but also allowed the identification of a subgroup of patients with clinical and prognostic characteristics similar to those of patients with Ph-positive CML. Recently Cogswell et al (1989) have examined 70 cases of CML. In patients with the Ph chromosome or with BCR rearrangement, aberrant RAS genes were not detected, but in seven of 13 patients without BCR rearrangement, mutant RAS genes were found. These findings suggest that

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Ph-negative CML might be better classified as a variant of chronic myelomonocytic leukaemia rather than as CML. Ph-positive CML. There has been considerable interest in the possibility that the site of the breakpoint may indicate the prognosis. Schaefer-Rego et al (1987) examined breakpoints in 26 patients with Ph-positive CML. In eight of the nine patients in blast crisis the breakpoints were in the 3' portion of the BCR gene, whereas in 17 patients in chronic phase the breakpoints were in the 5' region, suggesting a strong correlation between the 3' BCR breakpoint and blast crisis. Similar findings were reported by Eisenberg et al (1988). Dreazen et al (1988a) examined six patients with CML in whom the chronic phase had persisted for between 7 and 26 years and compared them with 20 patients in whom the chronic phase lasted less than 7 years. All patients had a rearrangement within the bcr region. In both groups there was a similar distribution of breakpoints within the major bcr (3' to 5'). Mills et al (1988) also found no correlation between the site of the breakpoint and the clinical phase of the disease, but they did report a striking correlation between site and time from presentation to blast crisis. Patients with a 5' breakpoint had a median time of 203 weeks, whereas for those with a 3' breakpoint the median was 52 weeks. Ogawa et al (1989) could find no correlation between site of the breakpoint in the BCR gene and clinical phase of the disease in a study of 15 Ph-positive patients, and Dyck and Bosco (1989) came to the same conclusion after a study of 46 patients. Shtalrid et al (1988) studied 108 patients with Ph-positive CML. All but one had a rearrangement within the major bcr. There was no correlation between the location of the breakpoint within the bcr and laboratory" or clinical features of the disease, but the duration of the chronic phase was found to be significantly longer in patients with a breakpoint in fragment 2. At present therefore, the value, to prognosis, of precise determination of the breakpoint within the major bcr is uncertain. Deletions in the BCR gene Shtalrid et al (1988), in their survey of 108 patients with Ph-positive CML, found large 3' BCR deletions in nine patients but did not observe any relationship to clinical outcome. Hirosawa et al (1988) concluded that deletions might be the reason that B C R - A B L rearrangements could not be detected in some CML patients. However use of the 5.8 kb probe described by Chan et al (1987a) may allow detection. A patient described by Reeve et al (1988) had both 3' and 5' rearrangements in the BCR region at diagnosis, but during the development of terminal acute leukaemia there was a progressive loss of germ-line BCR DNA, along with an increase in rearranged DNA consistent with acquired homozygosity. The deletion included a significant part of chromosome 22. PCR and detection of the B C R - A B L fusion genes Amplification of mRNA for the fused genes by PCR techniques has been

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described by Kawasaki et al (1988), Lee et al (1988), Dobrovic et al (1988), Morgan et al (1989), Lange et al (1989), Hooberman (1989), Lee et al (1989) and Roth et al (1989). The technique is very sensitive and relatively fast; it enables detection of cells with the rearrangement in numbers as low as 1 in 1000 (Dobrovic et al, 1988). It is potentially of considerable value in detecting residual disease after treatment, for instance after bone marrow transplantation for CIVIL.

Acute lymphoblastic leukaemia (ALL) The most frequent chromosome abnormality found in (ALL) is the Ph chromosome. Although apparently identical to that found in CML, it is present in only about 10% of patients (Third International Workshop on Chromosomes in Leukaemia, 1988). Molecular analysis has revealed differences between the breakpoints in these patients and those with CML. In Ph-positive ALL the BCR/ABL fusion protein may be either the 210 kDa (P210) product described in CML or a 185 kDa protein (Clark et al, 1987; Chan et al, 1987; Kurzrock et al, 1987). The mRNA coding for these proteins differs only in the presence or absence of certain exons in the middle of the BCR gene (Figure 2). Less of the BCR protein is present in the P185 than P210. In several cases of Ph-positive ALL it has been shown that the breakpoint occurs in the first intron of the BCR gene (Fainstein et al, 1987; Hermans et al, 1987; Rubin et al, 1988). Recently Denny et al (1989) have isolated the entire 130 kb BCR gene from a human cosmid library and have isolated a series of five single copy probes from the first intron (70 kb) of BCR. Using these they have located the breakpoints in eight out of 10 cases of Ph-positive ALL. All were located at the 3' end of intron I and were associated with an unusual restriction fragment length polymorphism caused by deletion of a 1 kb fragment containing Alu repeat sequences. The breakpoints were found within a 20 kb region of the intron. In two samples, breakpoints were found neither within intron I nor in the CML breakpoint cluster region. Denny et al suggested that if a deletion had occurred, which removed the probe sequences, then the rearrangement would not be detected. Large deletions have been described in the 3' region of the BCR gene in some patients with Ph-positive CML (Shtalrid et al, 1988). Rearrangements involving related BCR genes that are also located on chromosome 22 could also be possible (Croce et al, 1987) and might not be detected using single-copy probes. Heisterkamp et al (1989) have provided a survey of breakpoints in patients with ALL who were Ph-positive. Of 23 adult patients, 12 were found to have a breakpoint within the major bcr and 11 did not have major bcr breakpoint and did not produce the P210 fusion protein. Of these 11 patients, three either had a breakpoint in BCR intron I or produced P190. However, in nine children (14 years or less), for whom an adequate analysis was available, the breakpoint was in the first intron of the BCR gene and not in the major BCR. These findings have been confirmed in nine other adults by Schaefer-Rego et al (1988).

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Diagnostic strategy for BCR-ABL rearrangement It is clear that the ability to detect rearrangement involving the BCR gene can be of value in the diagnosis of CML and ALL. More detailed analysis of the site of the breakpoint within the 'major' or 'minor' regions is not yet of diagnostic or prognostic value. Detection by Southern blotting requires the two probes if both types of rearrangement are to be detected, and even after application of several probes it is still possible that a deletion may prevent detection of a rearrangement. However, we have been able to detect the BCR rearrangement in 29 out of 30 cases of CML with the Ph chromosome (Figure 3); the exception was one case in which only a few metaphases showed the translocation. Another valuable method might be detection of the fusion protein with monoclonal antibody (Walker et al, 1987). These techniques will distinguish between P210 and P185 but are relatively cumbersome and are not in routine use. The most promising method is, therefore, amplification of the B C R - A B L mRNA using PCR techniques, followed by electrophoretic separation of the amplified product to identify the type of fused message. CHROMOSOME TRANSLOCATIONS IN LYMPHOPROLIFERATIVE DISORDERS The Philadelphia chromosome is the most studied example of a translocation that is both characteristic of a type of haematological malignancy and has been analysed at the molecular level. There are a number of translocations specific for lymphoproliferative disorders (see Chapter 5). The best-known is Burkitt's lymphoma, where a marker 14q + chromosome was first described by Manolov and Manolova (1972). It is now known that three types of reciprocal translocation are found in Burkitt's lymphomas, all of which involve the M Y C gene (chromosome 8q24). The other chromo-

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somes taking part are 2, 14 and 22, and the breakpoints are at the loci for IgK IgH and Ig)t, respectively. The t(8;14)(q24;q32) is the most common--being present in about 75% of Burkitt's lymphomas (Croce, 1986). The MYC gene is known to be important in cell proliferation and in transformation (Cole, 1986). However it is not expressed in its germ-line configuration in normal B cells. In Burkitt's lymphoma the gene is expressed at levels similar to those found in proliferating normal cells, but it is only the translocated MYC that is expressed. This inappropriate expression is due to enhancers within the Ig loci. Molecular analysis of the translocations found in Burkitt's lymphoma indicate that there are two mechanisms (Haluska et al, 1990). In the endemic lymphoma occurring in equatorial Africa, associated with Epstein-Barr virus infection, there is no rearrangement of the MYC gene. The intact gene is present and the breakpoint on chromosome 14 involves the DH or JH segments of the IgH gene. The evidence suggests that the endemic Burkitt's lymphomas develop from a pre-B cell which is in the process of rearranging its Ig genes. Sporadic Burkitt's lymphoma occurs worldwide but is not closely associated with Epstein-Barr virus infection. The phenotype is of a more mature B-ceU than that of the endemic lymphoma. Here the translocation occurs immediately 3', or within, the MYC and in the switch region on chromosome 14. This suggests that the translocation arises at a later stage of differentiation, when switch recombinases are active (Hatuska et al, 1990). In the more common follicular lymphomas the t(14;18)(q32; 21) translocation is commonly found. This involves one of the JH segments of the IgH gene on chromosome 14 and a gene which has been called BCL-2 on chromosome 18. This gene encodes a protein that appears to have potential oncogenic activity (Reed et al, 1988; Vaux et al, 1988). The consequence of the translocation in B cells appears to be an increase in mRNA levels rather than an alteration in protein sequence (Tsujimoto et al, 1987). Two breakpoint regions have been identified on chromosome t8, with the twothirds of the breakpoints clustered within 2.8kb (the 'major breakpoint region') and the remainder in a second or minor breakpoint region (Cleary et al, 1986). DNA probes that can detect rearrangement of these two regions have been described by Cleary and Sklar (1985), and Cleary et al (1986) and Weiss et al (1987) have detected translocations in over 80% of cases of follicular lymphoma using these probes. However, other studies indicate a much lower incidence of translocations in the USA (Aisenberg et al, 1988; Amakawa et al, 1989) and in Europe (Pezzella et al, 1990). Pezzella et al have described the use of a PCR technique which makes it possible to detect translocations with D N A from paraffin-embedded sections. Thus detection at the molecular level can be a highly specific and sensitive aid to diagnosis. The t(11;14),translocation is observed in many cases of chronic lymphocytic leukaemia and also in some diffuse, small and large cell lymphomas. The translocation involves the IgH locus on chromosome 14 and a gene named BCL-1 (Erikson et al, 1984) on chromosome 11q13. A number of translocations involving TCR genes have been described for T cell leukaemias or lymphomas. The mechanisms appear to be analogous to

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M. WORWOODAND M. WAGSTAFF

translocations involving the Ig locus, but generally the other gene involved has not been described (Griesser et al, 1989).

Gene rearrangements in lymphoid malignancies Immunoglobulin and TCR studies Lymphoid leukaemias and lymphomas have traditionally been classified morphologically and more recently by specific monoclonal antibodies against cell-surface antigens (Chapter 4). The most recent additions to the diagnostic armoury are probes for the human immunoglobulin genes and the T-cell receptor genes (for review see Griesser et al, 1989). These probes allow a distinction between B and T cell malignancies to be made in many cases and provide means of detecting a clonal population, of indicating the position within the T or B cell lineage of malignant cells and of detecting residual disease after treatment.

Gene structure and localization The immunoglobulin molecules consist of two identical heavy chains (IgH) and two identical light chains (IgL). The structure is illustrated in Figure 4, together with germ-line gene organization and chromosome localization. There are two types of T-cell receptor. One contains a and [3 chains and the other ~/ and ~ chains. Again germ-line genomic organization and chromosome location are shown in the figure. Both Ig and TCR genes contain segments of D N A (exons) called variable (V), diversity (D, not Igkh or TCR~), joining (J) and constant (C). B Cells Kappa

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Figure4. Germ-linegenomicorganizationsof the humanimmunoglobulingenesIgH, IgK,Ig)t, and the T-cell receptor genes a,/3, Yand 6. V, variable; D, diversity;J, Joiningsegments;C, constant region genes. The chromosomallocationsare as follows:IgK,2pl 1; IgA, 22ql 1; IgH, 14q32; T-cell a, 6, 14qll; T-cell/3, 7q34; T-cell y, 7p15. From Griesser et al (1989) with permission.

MOLECULAR BIOLOGY AND LEUKAEMIA DIAGNOSIS

963

During B- or T-cell lineage commitment these genes undergo rearrangement, and this sequence of events may ultimately lead to the transcription of a functional Ig or TCR mRNA. First there is the combination of a D gene segment with a J segment. This DJ segment then combines with a V gene to form a variable region assembly which, with a C gene, gives the fully rearranged lg or TCR gene. During normal differentiation of B lymphocytes the IgH gene is the first to be rearranged. This may result in synthesis of cytoplasmic heavy chain. Next, rearrangement of K light chain genes occurs and, if no K chain is produced, IgA rearrangement takes place. A productive rearrangement leads to synthesis of p.K or txh polypeptides. If the rearrangement is not successful, the cell does not proceed beyond the pre-B stage. For the TCR it has been demonstrated that the genes are rearranged in the order TCRT, TCRfl and TCRa. TCR6 activation may occur at about the same time as TCR3, rearrangement. Each B or T cell produces an antigen receptor which is a molecule of unique specificity. Daughter cells will have receptors identical to those of the parent cell, unless a mutation occurs. A mature B cell and its progeny will have the same variable region sequences at the gene and p01ypeptide levels and will have either K or h light chains, but not both. Southern blotting with suitable probes makes it possible to detect clonality, to assign cell lineage, to detect cells of the same clone in different tissues and to detect the presence of residual, malignant cells after therapy. A minor leukaemic clone, under 1% of total cells, can be detected in bone marrow samples from patients who, morphologically, appear to be in complete remission (Zehnbaner et al, 1986). In terms of detection of residual disease, PCR techniques are even more powerful, with detection of a specific clone in 1 in 105 cells (see, for example, Lee et al, 1987). Clonality determination using PCR and primers for the V and J regions of the Ig genes has been demonstrated (Trainor et al, 1990) and the simplicity, speed and versatility of this approach will give it a very high value in the diagnosis of lymphoid disorders.

Clonal gene rearrangements in leukaemia During differentiation of a stem cell into a mature lymphocyte, Ig and TCR genes are rearranged and restriction-endonuclease sites are altered. Polyclonal cell populations do not show distinct bands on Southern blotting because many different rearrangements are present, but in a tumour most cells have the same rearrangement and so specific bands can be seen. In almost all tumours derived from mature B cells (B-ALL, B-CLL, hairy cell leukaemia, myeloma, non-Hodgkin's lymphoma) a monoclonal rearrangement of Ig genes is observed. This indicates that these tumours are derived from a single malignant B cell after rearrangements of its Ig genes (reviewed in Reis et al, 1988). In C-ALL there is usually rearrangement of at least one Ig heavy chain gene and, sometimes, rearrangement of light chain genes, indicating that these are also B cell tumours (reviewed in Reis et al, 1988). Tumours arising from mature T cells (T cell lymphomas, T-CLL) show clonal

964

M. W O R W O O D A N D M. W A G S T A F F

rearrangements of TCRfl and TCRy genes. Most malignancies described as immature T cell tumours, such as T-ALL, also show TCRfl rearrangements. However, in some lymphoid malignancies, both TCR and Ig genes are rearranged in the same cells or tissues. In general, rearrangements of both receptors are more commonly found in B-cell rather than T-cell disorders. The rearrangements differ from those described in T-cell malignancies. For example, only one Ig heavy chain allele may be rearranged, with the Ig light chains remaining in the germ-line state. There may only be rearrangement of one TCR~ or TCRy gene allele, whereas both alleles are usually rearranged in T cell malignancies. One explanation for such findings is in terms of 'lineage promiscuity' (Greaves et al, 1986). Yancopoulos et al (1986) suggested both the TCR and Ig genes are susceptible to the 'recombinase' enzyme that facilitates recombination of discontinuous segments of these genes. These inappropriate rearrangements do not proceed to completion. It is also possible that cells which fail to differentiate lack the signals present in more mature cells which terminate gene rearrangements (Davey et al, 1986). The TCRa gene has been particularly difficult to study because of its large size. The TCRa locus is about 1000 kb long (Griesser et al, 1988). The TCR6 genes are located within the TCRa gene between the V and J regions (Figure 3). TCRa rearrangement therefore leads to deletion of the corresponding TCRy locus. Surface expression of oL[3and y8 B are mutually exclusive in T cells. Analysis of TCRfl gene rearrangement has been widely applied in determining the clonality of lymphoid cell populations but the TCRy and 8 genes cannot be exploited in the same way. Normal polyclonal T cells show a number of discrete bands on Southern blotting, reflecting the restricted number of V regions (Macintyre and Sigaux, 1989). It is therefore difficult to detect a minority clone in the presence of polyclonal cells as this will merely cause a probably undetectable change in the intensity of one of the bands. Clonai analysis---use of X-chromosome gene inactivation

The techniques previously described can all be applied to the detection of clonal populations of cells but they depend on the presence of specific markers of leukaemic change (the Ph chromosome for example) or the presence of gene rearrangements that are only found in certain cells (Ig gene rearrangements in B-lymphoid cells). There is another technique which has a much more general application in terms of cell type, although so far it has been limited to a small minority of the population. This is the technique based on X-chromosome inactivation in females, the principles of which were first stated by Lyon (1972). Inactivation of one X-chromosome occurs at an early stage of embryogenesis, and the particular X chromosome inactivated in each progenitor cell remains the same in all its progeny. Thus, if two polymorphic forms of a protein [e.g. the enzyme glucose-6-phosphate dehydrogenase (G6PD) ] are coded for by genes on the X chromosome, only one will be expressed in the daughters of a particular progenitor cell. Normally a tissue contains a mixture of cells with respect to which X

965

MOLECULAR BIOLOGY AND LEUKAEMIA DIAGNOSIS

c h r o m o s o m e is i n a c t i v a t e d . If all the cells in a t u m o u r o r tissue e x p r e s s t h e s a m e i s o e n z y m e t h e n t h e y a r e all d e r i v e d f r o m o n e cell a n d t h a t t u m o u r is said to b e ' m o n o c l o n a l ' . T h e s a m e p r i n c i p l e s can be a p p l i e d at the g e n e level. R e s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m s ( R F L P ) can b e e x p l o i t e d to distinguish b e t w e e n t h e m a t e r n a l a n d p a t e r n a l c o p i e s of t h e X - c h r o m o s o m e genes. I n a d d i t i o 0 , c h a n g e s in t h e activity of m a n y g e n e s , including t h o s e o n t h e X c h r o m o s o m e , a r e a c c o m p a n i e d b y c h a n g e s in t h e m e t h y l a t i o n o f c y t o s i n e r e s i d u e s ( R a z i n a n d R i g g s , 1980; E h r l i c h a n d W a n g e , 1981). O f t e n , b u t n o t always, m e t h y l a t i o n is a s s o c i a t e d with an inactive gene. T h e s e c h a n g e s can b e detected by the action of restriction endonucleases that distinguish between methylated and unmethylated cytosine residues.

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pPB1.7 used as hybridizationprobe is indicated by the bar. The 5' end of the messenger RNA is located 250bp to the left of B1, and transcription continues through B3. (B) Diagrammatic representation of results that would be expected with monoclonal and polydonal tumours (see text). Lane 1, Barn HI digest; lanes 2 to 4, Barn HI plus Hha I digests; Lane 2, monoclonal tumour, 12 kb allele, active; lane 3, monoclonal tumour, 24 kb allele active; lane 4, polyclonal tumour. The pattern of the low molecular weight bands that appears after Hha I digestion varies between individual DNA samples, depending on the methylation status of sites H1 to H6 in the inactive chromosome. At least one of sites H2 to H6 is unmethylated in the inactive chromosome. From Vogelstein et al (1985) with permission. Abbreviations: Ba to B3, Barn HI restriction sites; Ha to H6, Hha I sites; 1 to 9, Hpa II sites.

966

M. W O R W O O D AND M. WAGSTAFF

This technique was first described using the enzyme hypoxanthine phospho-ribosyltransferase (HPRT, E.C.2.4.28; chromosome location Xq26). A map of part of the gene and the location of the probe applied by Vogelstein et al (1985) is shown in Figure 5. The Bam HI restriction sites B1 and B3 are present in all X chromosomes but B2 is a polymorphic site. About 27% of females are heterozygous at this locus. There are at least six Hha I sites between sites B1 and B2. Hha I cleaves at the sequence GCGC but does not cleave if either C nucleotide is methylated. Site I is unmethylated in active chromosomes. H2 to H6 are methylated in almost all active X chromosomes, but at least one of these sites is unmethylated in inactive X chromosomes. The results of Southern blotting after digestion with Barn HI and Hha I are shown diagramatically in Figure 5B. Digestion with Bam HI alone gives two restriction fragments of approx 24 and 12 kb in a woman who is heterozygous for this polymorphism. If the DNA is from normal tissue, the intensity of each band will decrease by about 50% after digestion with Hha I, as both chromosomes are inactivated to the same extent. If the DNA is from a single clone of cells then only one fragment will remain after digestion with Hha I. This is because one of the X chromosomes will be inactive in all cells and one active. The fragment corresponding to the active chromosome will show a very small change in mobility after digestion with Hha I because Hha I site I is unmethylated and the enzyme will cut the sequence here. However, there will be a small reduction in intensity after binding of the probe as some of sites 2 to 6 may also be unmethylated on the active chromosome. On the inactive chromosome at least one of sites H2 to H6 will be unmethylated. This means that the other Bam HI fragment will disappear and be replaced by the very small fragments that result from cutting the Barn HI fragments at sites Hz-H6. It is important to study normal cells from the same patients because X-inactivation shows a normal distribution between paternal and maternal chromosomes; the mean is about 50:50, but the ratio may range from less than 20 : 80 to more than 80 : 20 (Gartner and Riggs, 1983). In order to exploit the technique fully it is necessary to quantitate the changes in intensity that result from digestion with Hha I. To do this the same amount of DNA must be loaded in each gel track. (It should be remembered that using intensifying screens to enhance the signal from a 32p-labelled probe on a filter does not produce a linear response to radioactivity.) It may be necessary to use 'pre-flashed' films or autoradiography (i.e. without intensifying screens) with sensitive X-ray films. In any case, mixtures of allele fragments should be prepared by mixing DNA from subjects homozygous for either allele and demonstrating a linear relationship between the expected ratio of alleles and the ratios of intensity on X-ray film. The same principles apply to the use of the enzyme phosphoglycerate kinase (PGK, E.C.2.7.2.3; chromosome location Xq13) as a probe. Using both HPRT and PGK it should be possible to carry out clonal analysis in up to 50% of women, as about 30% are heterozygous for a BglI polymorphism in the PGK gene (Vogelstein et al, 1987); an example is given in Figure 6.

144 217 34 19

No, of patients studied 42 (HPRT) 62 (PGK) 12 9 7?

No. of patients heterozygous for HPRT or PGK*

* Hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase. Patients in remission.

Myelodysplastic syndromes AML

Normal

Diagnosis 1/42 0/40 7/8 9/9 2/7

No, monoclonal/ no, studied

Table 1. Clonal analysis using X-chromosome gene probes in female subjects. Reference Vogelstein et al (1989) Vogelstein et al (1989) Janssen et al (1989) Bartram et al (1989)

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968

M. WORWOOD AND M. WAGSTAFF

a

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e

Figure 6. Clonality studies using the PGK probe, a, homozygote; b,c, heterozygote, polyclonal; d,e, heterozygote monoclonal, a,b,d, after digestion with Eco RI, Bgl I and Bgl I; c,e, after further digestion with the methylation sensitive restriction enzymes Hpa II. Note the disappearance of one of the bands in lane e.

Table 1 summarizes the results of clonality studies in several groups of patients. We have found a slightly lower incidence of heterozygotes than by Vogelstein et al; this may be due to population variation. In our experience, confirmed by others, the PGK assay is technically more reliable than the H P R T system. This is possibly because the fragment sizes are smaller and because there are fewer problems caused by degraded D N A samples and poor transfer to the filter during blotting. The presence of repetitive sequences in both HPRT and PGK probes also causes difficulties and the necessity for multiple enzyme digestions (and hence precipitations) can lead to loss of DNA before electrophoresis. The value of the X-chromosome gene inactivation technique may be greatly enhanced if the highly informative X-chromosome probe, M27B, fulfils its promise (Abrahamson et al, 1989). This has a heterozygosity rate of over 90% and can also be used to detect X-inactivation. GENETIC FINGERPRINTING The studies of the BCR gene and immunoglobulin gen e rearrangements are examples of techniques that can detect clonality in certain types of leukaemia. Studies of X-gene inactivation extend detection of clonality to female patients. Are there techniques of more general application, male as well as female, and for other types of malignancy? Analysis of restriction fragment length polymorphisms (RFLP) of single copy genes might provide a sort of 'microcytogenetics'. In theory, one might

969

MOLECULAR BIOLOGY AND LEUKAEMIA DIAGNOSIS

screen samples of tumour DNA with a battery of probes and detect deletions or amplification of loci or, possibly, mutations affecting a restriction enzyme site. Usually, however, only a small proportion of subjects are heterozygous for a particular polymorphism, and many probes would be needed even for one chromosome. The 'mini-satellite' DNA probes introduced by Jeffreys et al (1985a, 1985b) appear to overcome both of these difficulties. They enable simultaneous detection of many loci, each of the loci being highly polymorphic. Hypervariable regions of DNA have been found within a number of human genes (for example Harvey RAS, Capon et al, 1983; globin, Higgs et al, 1981; insulin, Bell et al, 1982). The variation is in length of sequence and is due to the presence of a variable number of repeats of a short, sequence ('tandem repeat sequences'); it can be detected by the use of a restriction

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Figure 7. DNA Fingerprinting. DNA was prepared from granulocytes (G) and mononuclear cells (L) from four normal subjects. After digestion with Alu I, electrophoresis in 0.8% agarose and Southern blotting the membrane was hybridized to probe 33.6 (Jeffreys et al, 1985a). Note the extreme polymorphisrn and the consistent patterns from duplicate samples. Photograph kindly provided by S. R. Ridge, Cardiff.

970

M. WORWOOD AND M. WAGSTAFF

enzyme that does not cut within the region of repeated sequences. In fact, Jeffreys et al (1985a) exploited a considerable extension of this principle by introducing probes which detect multiallelic variation at a wide variety of sites. Weller et al (1984) screened a human genomic library using a probe from the hypervariable region of the human myoglobin gene and found that the sequences isolated contained variable numbers of repeats of a 10 bp 'core sequence' homologous to the chi recombination sequences (Smith et al, 1981) found in E. coli. These E. coli sequences are thought to be 'hot spots' for recombination, and it has been proposed that the variable number of repeats at loci detected by mini-satellite probes may have been generated by unequal chromatid exchange promoted by the presence of these sequences. This theory is supported by the location of mini-satellites on human chromosomes. Royle et al (1988) have shown by in situ hybridization that, unlike the Alu repeat sequences (>500000 copies in the human genome; Hardman, 1986), mini-satellite sequences detected by the 33.15 probe (Jeffreys et al, 1985a) are clustered around the chiasmata. Such a location obviously reduces the general applicability of these sequences in detecting genetic change. Use of the probes 33.6 and 33.15 has become widely known to the scientific community and the general public as 'DNA fingerprinting'. The technique has revolutionized forensic medicine, particularly in paternity testing, clarification of family relationship in immigration disputes and in the identification of specimens of body fluids or even hairs in criminal cases. The polymorphic nature of the sequences detected with the 33.6 and 33.15 probes is such that up to 30 bands in the 4-20 kb size range may be resolved after Southern blotting. The probability that two unrelated individuals will possess the same fragments after sequential hybridization to both probes is 5 X 10 -19 (Jeffrey et al, 1985b). For related individuals the probability is approximately 10-8 in sibs or between parents and children, and in the case of monozygotic twins the patterns will be identical. The low rate of mutation (0.5 X 10 4 tO 1.5 × 10 4 per locus per gamete) for the longest fragments (Jeffreys et al, 1985a) is also important in justifying the technique's use in forensic medicine. The ubiquitous nature of repeat sequences in itself creates technical difficulties in the handling of mini-satellite probes. Heterologous DNA cannot be used to prevent non-specific binding of the probes to the filter. Consequently it is necessary to employ a lengthy post-hybridization regime in order to create a balance between a strong signal and removal of nonspecifically bound probe. The technique is also of value in the detection of recipient cells after aUogeneic bone marrow transplantation (Weitzel et al, 1988). Depending on the differences between donor and recipient DNA, it is possible to detect from i to 10% of a population of recipient cells in the presence of donor cells (Min et al, 1988). Furthermore, the analysis does not depend on sex differences, differences in blood group or the presence of a specific disease marker. Another potential use is the detection of clonal markers in malignant cells. Thein et al (1987) examined the DNA fingerprint patterns obtained

MOLECULAR BIOLOGY AND LEUKAEMIA DIAGNOSIS

971

from normal and tumour tissues of patients with gastrointestinal tumours. Changes in the patterns were found in three out of five patients with colo-rectal carcinoma. The changes can be of several types--appearance of a new band, loss of a band, change in intensity of bands or combinations of these--and may provide clonal markers. Loss of a mini-satellite fragment may indicate a micro-deletion. Changes in DNA methylation (Goelz et al, 1985) may also affect DNA fingerprints. These can be excluded by the use of restriction enzymes such as Alu I and Hae III, which are not specific for sites that may be blocked by CpG methylation. The appearance of new bands in tumour DNA (Thein et al, 1987) presumably indicates changes in the length of existing minisatellites, perhaps by unequal sister-chromatid exchange. In any case, such differences in fingerprint pattern may provide markers for the study of clonality and progression of a tumour through the sequence of malignant changes. However, because the number of loci detected by the probes is relatively small (estimated at 30--40) it would be expected that mini-satellites would be affected only in a small proportion of patients, and this has been found to be the case. Locus specific mini-satellite probes may also have value in the detection of particular deletions. Thein et al (1988) used the polymorphic probe pg3 (sequence located at 7q31.3-qter) to detect loss of chromosome 7 in the DNA from leukocytes of 10 out of 118 patients with myelodysplasia (MDS); DNA from hair roots was used as the control. Monosomy 7, or del7 could only be found in only five of these patients by eytogenetic analysis. USE OF Y CHROMOSOME PROBES IN BONE MARROW TRANSPLANTATION The application of in situ hybridization with a Y chromosome probe in sex-mismatched transplants has been demonstrated; this is a valuable way of distinguishing between donor and recipient cells (Hutchinson, 1989). A PCR technique using Y chromosome sequences has also been described (Kogan et al, 1987), but it is difficult to see how such a method can be of diagnostic use at present. Two examples illustrate why this is the case. In a female patient transplanted with male, donor marrow a positive result does not distinguish between complete engraftment and chimaerism. In the reverse situation we have demonstrated the presence of Y chromosome sequences in a male patient 14 days after transplantation, whereas at 3 months they were not detectable. This was probably due to residual radiation-damaged cells but may have been interpreted as a failure to engraft. Thus the great sensitivity of PCR is a disadvantage in such cases. Its routine application in this context should await the development of reliable, quantitative methodology. SUMMARY

The diagnosis and classification of leukaemia started with simple morphological

972

M. WORWOODAND M. WAGSTAFF

examination and now embraces use of special stains, cytochemistry and immunophenotyping. Genetic studies have progressed from karyotyping to detection of genetic changes within genes. The methods described in this chapter are still at an early stage of development and, so far, have provided relatively little in the way of an extension of available diagnostic information. Sometimes the methods provide extensions to existing techniques, for example by the detection of bcr rearrangements in patients who have C M L or A L L but do not have a detectable Philadelphia chromosome. A n o t h e r example is retrospective diagnosis of gene rearrangements using D N A from slide preparations. However, it should be noted that it has only very recently been shown that there is likely to be a causal relationship between the Ph chromosome and leukaemia. Daley et al (1990) induced C M L in mice by bone marrow transplantation of cells infected with a retrovirus encoding P210 bCr/abl and Heisterkamp et al (1990) produced mice transgenic for a B C R / A B L P190 D N A construct and showed that the progeny died of acute leukaemia (mostly A L L ) . We have not summarized studies of the incidence of activated oncogenes such as R A S in leukaemia and myelodysplasia. Such oncogenes appear to be involved in many tumours and may well indicate either a predisposition to cancer or a particular stage of malignancy, but their analysis does not at present help in making a diagnosis. It is likely that, as we understand more about the nature of the malignant process, we shall be able to use genetic techniques to enhance considerably both diagnostic and prognostic precision.

REFERENCES Abrahamson G, Fraser NJ, Boyd Y, Craig I & Wainscoat JS (1989) A highly informative X-chromosome probe, M27B, can be used for the determination of tumour clonality. British Journal of Haematology 74: 371-377. Aisenberg AC, Wilkes BM & JacobsonJO (1988) The bcl-2 gene is rearranged in many diffuse B-cell lymphomas. Blood 71: 969-972. Amakawa R, Fukhuara S, Ohno H et al (1989) Involvementof bcl-2 gene in Japanese follicular lymphoma. Blood 73: 78%791. Bell GI, SelbyMJ & Rutter WJ (1982) The highlypolymorphicregion near the human insulin gene is composedof simple tandemly repeating sequences. Nature 295: 31-35. Capon DJ, Chen EY, LevinsonAD, SeeburgPH & Goeddel DV (1983) Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature 302: 33-37. Chart LC, Chen, P-M, PowelIset al (I987a) Molecular lesion in chronicgranulocyticleukemia is highly conserved despite ethnic and geographicalvariation. Leukemia I: 486-490. Chart LC, Karhi KK, Rayter SI et al (1987b) A novel abl protein expressed in Philadelphia chromosome positive acute lymphoblasticleukaemia. Nature 325: 635-637, Chehab FF & Kan YW (1990) Detection of sickle cell anaemia mutation by colour DNA amplification. Lancet i: i5-17, Clark SS, McLaughlinJ & Crist WM (1987) Unique formsof the abl tyrosinekinase distinguish Ph1positive CML from Ph~positive AML. Science 235: 85-88, Cleary ML, Galili N & SklarJ (1986)Detection of a secondt(14;18) breakpoint cluster regionin human follicular lymphomas. Journal of Experimental Medicine 164: 315-320. Cleary ML & Sklar J (1985) Nucleotide sequence of a t(14;18) chromosomalbreakpoint in

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