DNA mismatch repair pathway defects in the pathogenesis and evolution of myeloma
Mark R. Velangi3,
Elizabeth C. Matheson,
Gareth J. Morgan1,
Graham H. Jackson2,
Penelope R. Taylor2,
Andrew G. Hall and
Julie A.E. Irving
Northern Institute for Cancer Research, University of Newcastle upon Tyne, UK, 1 Department of Haematology, Leeds General Infirmary, Leeds, UK and 2 Department of Haematology, Royal Victoria Infirmary, Newcastle upon Tyne, UK
3 To whom correspondence should be addressed Email: ms.velangi{at}btinternet.com
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Abstract
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Genetic instability is a prominent feature in multiple myeloma and progression of this disease from monoclonal gammopathy of uncertain significance (MGUS) and smouldering myeloma (SMM) is associated with increasing molecular and chromosomal abnormalities. The DNA mismatch repair (MMR) pathway is a post-replicational DNA repair system that maintains genetic stability by repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication. Deficiencies in proteins pivotal to this pathway result in a higher mutation rate, particularly at regions of microsatellite DNA. We have investigated the proficiency of the MMR pathway in clinical samples and myeloma cell lines. Microsatellite analysis showed instability at one or more of nine loci examined in 15 from 92 patients: 7.7% of MGUS/SMM, 20.7% of MM/plasma cell leukaemia (PCL) and 12.5% of relapsed MM/PCL. An in vitro heteroduplex G/T repair assay found reduced repair in two cell lines, JIM1 and JIM3, and in two of four PCL cases and was associated with aberrant expression of at least one mismatch repair protein. Thus we show that MMR defects are found in plasma cell dyscrasias and the increased frequency during more active stages of the disease suggests a contributory role in disease progression.
Abbreviations: LOH, loss of heterozygosity; MGUS, monoclonal gammopathy of uncertain significance; MM, multiple myeloma; MMR, DNA mismatch repair; MSI, microsatellite instability; PCL, plasma cell leukaemia
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Introduction
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A multistep natural history of the aetiology and progression of multiple myeloma (MM) can be developed based on a number of clinical and laboratory observations. Occasionally MM is preceded by a stable asymptomatic phase known as monoclonal gammopathy of uncertain significance (MGUS). MGUS is found in
5% of the population over 60. Transformation to MM occurs at a rate of
1% per year (1). During the later stages of the natural history of patients with MM a proportion enter a leukaemic phase with the appearance of circulating plasma cells associated with alterations in adhesion molecule profiles. The evolution of MM is associated with a number of defined genetic alterations. An early event implicated in the pathogenesis of MM is a translocation between the IgH locus on 14q32 and a variety of oncogenes, including FGFR3 (4p16.3), MUM1/IRF4 (6p25), cyclin D3 (6p21), C-MAF (16q23) and C-MYC (8q24) (2). The structure of the breakpoints of these translocations suggests that the process of physiological switch recombination or aberrant recombination during the late stages of ontogeny is important in this process. As the disease progresses mutations in a range of oncogenes may be detected; these include FGFR3, P53 and, most frequently, N- or K-RAS. This increased rate of mutations suggests that there may be an underlying defect in the ability of transformed plasma cells to detect and eliminate alterations as they arise.
The DNA mismatch repair (MMR) pathway is the most important post-replicative repair process involved in the maintenance of genomic stability. Originally described in Escherichia coli, homologues of the bacterial proteins MutS and MutL, which play key roles in mismatch recognition and initiation of repair, have been identified in higher organisms, including man. The MMR pathway is responsible for correcting insertion/deletion loops, including those that occur in microsatellite DNA, and single basebase mispairs that occur after misincorporation during normal DNA replication (3). Inactivation of genes encoding proteins involved in this system result in a mutator phenotype that is associated with a predisposition to tumour development, both in mice and humans (4,5). A consequence of the mutator phenotype is the production of multiple replication errors in simple repetitive DNA sequences, resulting in a phenomenon known as microsatellite instability (MSI). MSI was first observed in subjects with colon cancer (57) and further studies demonstrated that MSI results from an underlying defect of the MMR genes, in particular hMLH1 and hMSH2 (7,8).
The finding of MMR defects in colon cancer led to studies of MSI in a variety of other malignancies. Strong evidence implicates defects in MMR genes with the development of defined subgroups of haematological disorders, in particular those of lymphoid origin. Studies in PMS2-, MLH1-, MSH2- and MSH6-deficient mice clearly establish a link between MMR gene defects and the development of lymphoid tumours (4,911). In humans, coding region mutations in both hMSH2 and hMLH1 have been identified in primary tumour samples from patients with lymphoblastic lymphoma and lymphoid cell lines (12) and MSI in lymphomas from human immunodeficiency virus positive individuals (13), adult T cell leukaemia (12) and in lymphoid cell lines (14). In addition, insertion/deletion mutations within the BAX G8 mononucleotide run have been reported in 4 of 29 (14%) human lymphoid tumour cell lines not selected for MMR deficiency (15). Although the overall findings of several studies that have investigated MSI occurrence in malignancies of the myeloid lineage indicate that MSI is uncommon in de novo acute myeloid leukaemia (16), it is more common in therapy-related myeloid leukaemia (17). Kindreds from four families with biallelic inactivation of either hMSH2 or hMLH1 developed a range of haematological malignancies, including chronic myeloid leukaemia (1821). To date, a detailed study of MMR proficiency in plasma cell dyscrasias has not been reported. In this study we report that MMR defects are frequent in cases which present as the more aggressive forms of MM, including plasma cell leukaemia (PCL), suggesting that loss of MMR proficiency may be responsible, at least in part, for the progression of these disorders.
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Materials and methods
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Patients and cell lines
Study samples were collected from patients attending hospitals within the former Northern Health Region of England. Local ethical approval was granted for this study and informed consent was obtained from all patients. Bone marrow samples were collected from 102 patients diagnosed with plasma cell dyscrasias and processed within 18 h of collection. Analysis of the age range, gender and paraprotein type of this cohort (Table I) suggest that it is representative of the general clinical spectrum of these disorders. Ten cases were excluded from the study due to either insufficient material or the presence of >10% non-malignant cells after sample purification. In the 92 cases where suitable material was available for further analysis 84 were obtained at presentation (15 MGUS, 11 SMM, 54 MM and 4 PCL) and 8 at relapse after treatment (7 MM and 1 PCL). In the case of PCL (case 51), a sample was alsoavailable at relapse (rel51). A diagnosis of PCL was made if >2 x 109 plasma cells were detected in the peripheral blood. Whole blood was collected at diagnosis or at the time of remission from all patients as a source of constitutive DNA. Confirmation of the presence of <1% plasma cells in these blood samples was made by cytological examination. A panel of human myeloma cell lines and control cell lines were examined. These are listed in Tables II and III.
CD138 purification
Bone marrow aspirate samples from all patients were initially either enriched for mononuclear cells by standard density gradient centrifugation using Lymphoprep (Nycomed UK Ltd, Birmingham, UK) or subjected to a standard red cell lysis procedure. Purification of plasma cells was achieved using B-B4 monoclonal antibodies as described previously (34) and CD138 Microbeads and MidiMacs columns (Miltenyi Biotec, Bisley, UK), used according to the manufacturer's instructions. Purity was assessed by cytological examination after May Grunwald-Giemsa staining. Samples containing <90% myeloma cells were excluded from further study, as described above.
Cytosol preparation
Cytosolic extracts were prepared from either cell lines or purified plasma cells. As the activity assay requires a minimum of 5 x 107 cells, assessments could only be performed on cell lines or cases of PCL. Cells were washed in ice-cold isotonic buffer [20 mM HEPES, pH 7.9, containing 5 mM KCl, 1.5 mM MgCl2, 0.2 mM phenylmethylsulphonyl fluoride, 1 mM dithiothreitol, 250 mM sucrose and EDTA-free protease inhibitor (one tablet/50 ml buffer; Boehringer Mannheim)] followed by ice-cold hypotonic buffer (as above but minus sucrose), before resuspension in twice the pellet volume of ice-cold hypotonic buffer. Cells were lysed mechanically using a glass homogenizer (Fisher Scientific), and progression of the lysis monitored microscopically by trypan blue exclusion. When
85% of the cells were lysed, nuclei were pelleted and the supernatant cleared by centrifugation at 12 000 g for 7 min. Aliquots of the lysate were stored at 80°C prior to analysis. Cytosolic protein concentration was measured using the bicinchinonic acid protein assay kit (Pierce & Warriner, Chester, UK).
Western blot
Cell lysates were prepared as described above, diluted in Laemmli sample buffer to a final protein concentration of 0.5 mg/ml and heated at 100°C for 5 min. Aliquots of 10 µg protein were loaded onto a 420% precast Trisglycine polyacrylamide gel (Invitrogen, Paisley, UK) and electrophoresed. Proteins were transferred onto Immuno-Blot PVDF membrane (Bio-Rad, Hemel Hempstead, UK) by electroblotting at 30 V overnight in CAPS buffer (10 mM CAPS, pH 11, with 10% methanol). After blocking in 5% skimmed milk in Tris-buffered saline containing Tween 20 (10 mM Tris, 100 mM NaCl, pH 7.5, plus 0.05% Tween 20) for 1 h, membranes were probed with either anti-MLH1, anti-PMS2 or anti-MSH2 mouse monoclonal antibodies at 2 µg/ml (BD Pharmingen, Oxford, UK) or with anti-MSH6 rabbit polyclonal antibody at 1:3000 (generously provided by Prof. Jiricny, Zurich). Primary antibodies were made up in 5% skimmed milk powder and incubated with the blots for 1 h at room temperature. Each blot was also probed for actin as a protein loading control, using mouse monoclonal anti-actin at 1:10 000 (Oncogene, CN Biosciences, Nottingham, UK). The secondary antibodies used were horseradish peroxidase-conjugated anti-mouse Ig (1:1000; BD Pharmingen) and horseradish peroxidase-conjugated anti-rabbit Ig (1:2000; Amersham Pharmacia Biotech, Little Chalfont, UK). Immune complexes were visualized using an enhanced chemiluminesence reagent (ECL-Plus; Amersham Pharmacia Biotech).
Functional assay/complementation
The ability of cytosolic extracts to repair DNA mismatches was assessed as described previously (35). Briefly, wild-type and mutant M13mp2 phage derivatives were used to prepare double-stranded DNA substrates that contain a nick in the ()-strand and mismatched or unpaired bases in the lacZ
-complementation gene (ß-galactosidase gene). Five nanograms of an M13mp2 phage heteroduplex substrate, prepared containing a G:T mispair and a nick in the strand containing the T, was incubated with 50 µg cell lysate and the repaired/unrepaired heteroduplex substrate, purified and electroporated into MMR-deficient E.coli NR 9162. Transformed bacteria were plated out with the
-complementation E.coli strain CSH50 onto minimal agar plates supplemented with isopropyl-ß-D-thiogalactopyranoside and X-Gal (Sigma, Dorset, UK). The resulting plaques were scored as pure blue, mixed burst or clear. A reduction in the percentage of mixed plaques and an increase in pure blue or clear plaques is indicative of heteroduplex repair. Repair efficiency was calculated as 1 (% mixed plaques in test sample ÷ % mixed plaques in a water-only control). Complementation assays were performed using human recombinant MutL
(190 ng) protein or MutS
(136 ng), kindly provided by Dr Giancarlo Marra (Institute for Medical Radiobiology, Zurich, Switzerland). The M13mp2 phage derivatives and E.coli strains were kindly provided by Dr Thomas Kunkel (National Institute of Environmental Health Sciences, Research Triangle Park, NC).
Microsatellite analysis
DNA was extracted from purified plasma cells and peripheral blood using the QIamp DNA Minikit (Qiagen Ltd, Crawley, Sussex). Yields ranged from 2 to 300 ng/µl. Nine microsatellite repeat markers mapping to a variety of chromosomes were studied using the matched tumour and normal DNA for MSI (ref). Included were repeats of mononucleotides (3), dinucleotides (5) and tetranucleotides (1). Primer sets were obtained from Research Genetics Inc. (Huntsville, AL) and the forward primer of each pair was fluorescently labelled using Beckman WellRed dye D3. PCR reactions were performed in a final volume of 25 µl containing the DNA template, 3 mmol/l MgCl2, 1x PCR buffer, 125 µmol/l dNTPs, 0.625 U AmpliTaq Gold DNA polymerase (PE Applied Biosystems), 200 nmol/l upstream and downstream PCR primers and 510 ng genomic DNA. Amplification was performed in a Perkin-Elmer 9700 GeneAmp PCR system (Applied Biosystems, Branchburg, NJ) after an initial denaturation at 95°C for 10 min, followed by 30 cycles of denaturation at 95°C for 45 s, primer annealing at 57°C for 45 s, primer extension at 72°C for 1 min and a final extension of 72°C for 7 min. One microlitre of each PCR reaction was combined with 40 µl of deionized formamide and 0.5 µl size standard-400 (Beckman Coulter UK, High Wycombe, UK) labelled with Beckman WellRed dye D1 and analysis was performed using CEQ2000XL (Beckman Coulter UK) for automatic sizing of fragments using a fluorescent detection method (36). MSI was defined as the addition of a new allele in the tumour DNA sample. Loss of an allele was regarded as complete loss of heterozygosity (LOH). All samples showing evidences of LOH or microsatellite instability were confirmed in an independent PCR.
Methylation analysis
Methylation profiles in the hMLH1 promoter were determined by bisulphite treatment of DNA and a methylation-specific PCR as previously described (37).
Mutation analysis
Mutational analysis of the coding region of the hMLH1 gene was carried out by denaturing HPLC on a Transgenomic WAVE machine using described methodologies (38). Mutational screening of exons 210 of the MSH6 gene, including intronic flanking regions, was performed using a direct sequencing approach (primer sequences available on request). PCR products were purified using a PCR Clean up kit (Qiagen) and sequenced using BIGDye terminator chemistry on an ABI automated sequencer.
Statistical analysis
The
2 test for trend was used to analyse statistical significance of observed differences in the frequency of microsatellite alterations in subgroups of plasma cell dyscrasias. An unpaired t-test was used in the analysis of differences in the functional assay. A P value of <0.05 was considered to be statistically significant.
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Results
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Microsatellite instability
As no constitutional DNA sample was available for the cell lines, we analysed MSI by examining three loci, BAT25, BAT26 and BAT40, which have mononucleotide repeats and are regarded as quasimonomorphic, i.e. predominantly monomorphic in Caucasian populations. Instability of the JIM3 cell line was found at the BAT40 locus only. Interestingly, no alteration was seen in the JIM1 line, derived from the same patient. A larger panel of nine microsatellite loci was tested in the patient group as constitutional DNA was available for direct comparison. Evidence of alteration of at least one microsatellite locus was found in 2 of 26 in the MGUS/SMM group and 12 of 58 in the newly diagnosed MM/PCL samples (see Figure 1). Of these 12 MM/PCL patients, 5 of 12 displayed alterations in at least two loci and 3 of 12 samples at three or more. One of eight MM/PCL relapse cases displayed an alteration of one or more microsatellite loci. These results are summarized in Tables IV and V. In the one PCL patient where matched samples were available (case 51), an increase in the degree of MSI was found at relapse (rel 51) when compared with the diagnosis sample, with the emergence of an alteration at the D18S69 locus on chromosome 18q21. Of interest we also demonstrated LOH at 1p32 in two cases and 17q in four cases. One sample (case 37) demonstrated LOH at both of these loci. As complete LOH and MSI can be indistinguishable by standard microsatellite analyses, the incidence of MSI may be under-reported.

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Fig. 1. Representative results of microsatellite analyses comparing normal (N) and plasma cell tumour (T) DNA at the D18S69 locus in case 19 (A) and the mycL locus in case 27 (B). In case 19 there was evidence of MSI and in case 27 there was complete LOH. The block arrow denotes the 180 bp marker.
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In vitro functional G:T repair assay
We have previously reported validation of the functional MMR assay repair of a G/T substrate. Assays were carried out on a range of cell lines with documented MMR defects (SW48, LOVO, HEC1A and HCT15). The MMR-proficient positive control TK6 gave a repair efficiency of 90% for a G/T substrate. The cell lines defective in MLH1 (SW48), MSH2 (Lovo), PMS2 (Hec-1-A) and MSH6 (HCT15) all showed little or no repair. Complementation experiments were performed in which addition of either recombinant MutL
or MutS
protein to these cell lines, dependent on their defect, restored repair activity (35). Results obtained from a panel of eight human myeloma cell lines are shown in Figure 2A. Of the eight tested, six demonstrated proficient repair activity. The repair proficiency of the JIM1 and JIM3 lines was
25%, significantly less than that obtained in the proficient lines (P = 0.002 and P < 0.0001, respectively) but greater than that in lines with documented MMR gene defects (35). Complementation with MutL
heterodimer restored activity to levels just lower than those obtained in the proficient TK6 control (Figure 2B), whilst complementation with MutS
had no effect on mismatch repair activity (data not shown). The requirement for large cell numbers (>5 x 107) in this assay limited us to the examination of three PCL cases only (patients 51, 61 and 87). In one of these (patient 51), samples were available both at diagnosis and at relapse, in the other two a sample was only available at diagnosis. Although slightly reduced, repair activity was not significantly different from the proficient control cell line in case 87 (P = 0.06). In case 61 the activity was similar to that in the JIM cell lines. Case 51 was proficient at diagnosis but at relapse showed a marked and significant fall in activity (P = 0.001). There was insufficient cytosol to perform complementation with MutL
in this case.

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Fig. 2. (A) Functional repair activity for a G/T mismatch in a panel of human myeloma cell lines and PCLs. The cell lines JIM1 and JIM3 demonstrate reduced repair activity compared with the TK6-proficient control (P = 0.002 and P < 0.0001, respectively). In addition, reduced activity was found in one PCL case (61) and one case of PCL at relapse (rel51) with a normal presentation sample (51). (B) Complementation of JIM1 and JIM3 with recombinant MutL demonstrates partial restoration of function.
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Western blotting
Expression of hMSH2, hMSH6, hPMS2 and hMLH1 was detected in all of the myeloma cell lines tested, including JIM1 and JIM3 (Figure 3). The results did not correlate with the functional assay. For example, expression of hMLH1 was relatively low in JIM1 and JIM3, but was not markedly less than in U266, which was found to be fully MMR proficient in G/T repair. Similarly, analysis of protein expression by western blotting in the cases of PCL analysed failed to show the complete loss seen in cell lines with mutated MMR proteins, although expression was markedly reduced in case 61. Case 51 at diagnosis, which had normal functional activity, had a truncated form of hMSH6. This truncated form was also visible, along with a normal form, in the JIM1 and JIM3 cell lines and at relapse in case 51.
Methylation-specific PCR
The methylation status of the hMLH1 promoter region was investigated in all patients in whom MSI was discovered and in all myeloma cell lines. No evidence of hMLH1 promoter methylation was found in any of the cell lines, including JIM1 and JIM3, or patient samples with microsatellite alterations (see Figure 4).

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Fig. 4. Methylation-specific PCR demonstrating absence of methylation in JIM1, JIM3 and patient samples 51 and rel51. For each sample paired PCR reactions were performed with unmethylated (UM) and methylated (Me) primers (M-marker). TK6 and SW48 served as controls. SW48 has a non-functional MLH1 gene due to methylation of the promoter region.
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hMLH1 and hMSH6 mutation analysis
Denaturing HPLC was used to screen for mutations in all 19 exons and variable amounts of flanking introns of the hMLH1 gene in the JIM1 and JIM3 cell lines. Chromatograms showed homoduplex patterns in all PCR products apart from that including exon 15, which gave a heteroduplex pattern for both cell lines (data not shown). Direct sequencing of these PCR products identified an intronic A
G transition at position 19 from the 5'-end of exon 15, a previously characterized single nucleotide polymorphism (39). The presence of a heteroduplex pattern excludes LOH in these cell lines.
Similarly, screening of the exonic regions and splice site of the MSH6 gene in patient 51 and the JIM1 and JIM3 cell line mutations did not reveal mutations that may account for a truncated hMSH6 protein.
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Discussion
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In this study we have investigated the role of MMR defects in the pathogenesis and progression of MM. Initial findings in a panel of human myeloma cell lines demonstrate reduced G:T repair activity by an in vitro heteroduplex repair assay in two of the eight cell lines studied. The two repair-deficient lines, JIM1 and JIM3, were derived from the same patient and demonstrated a similar reduction in MMR proficiency to
30% of the TK6 positive control. Complementation experiments with recombinant MutL
suggested that the defect was in activity or expression of hMLH1 or hPMS2. However, this reduced repair was not found to be associated with absence of expression of any of the four main MMR proteins, in contrast to results obtained in colorectal cell lines and other cell lines of lymphoid origin studied in our laboratory with genetic defects in one or more of the MMR genes (35). The observation of MSI at the BAT40 locus in JIM3 but not JIM1 cells is also in line with a partial MMR defect. Mutation analysis of the entire coding region revealed no abnormalities in hMLH1 in either JIM1 or JIM3 and the presence of a heteroduplex pattern in the PCR product for exon 15 excluded LOH at this locus. Methylation analysis of the hMLH1 promoter revealed no abnormalities, in contrast to previous reports of MMR defects in the absence of MMR gene mutations. Investigations are currently underway to exclude mutations in the hPMS2 gene as a cause of repair deficiency in the JIM cell lines.
The demonstration of reduced repair activity in the JIM1 and JIM3 cell lines led us to investigate whether similar findings could be demonstrated in primary patient samples. MSI analysis was conducted in 92 cases by comparing constitutive and plasma cell DNA at nine loci. Alterations in at least one locus were seen in 15 of 92 cases. Abnormalities in two or more loci were only seen in 7 cases. Analysis of MSI in HNPCC has established that abnormalities at three or more loci in a well-defined panel are normally present in cases with a defined genetic mutation in either the hMSH2 or hMLH1 gene. However, as emphasized by the international group who established these criteria, the same may not apply in sporadic tumours (40). One recent study examining the role of MSI in gastric lymphoma has demonstrated that microsatellite loci other than those typically mutated in HNPCC were more commonly found in this group of patients and a lymphoma panel was introduced. The RER phenotype (as defined by the NCI Workshop for HNPCC) of these individual lymphoma cases was found to differ depending on whether the cases were analysed by the HNPCC or the lymphoma panel (41). As yet, clear criteria for the diagnosis of MSI in non-HNPCC malignancies have not been established, so we have elected to describe numbers of alterations of microsatellite loci rather than to label a population as MSI-positive. In cases of MMR defects arising due to either mutations in other members of the MMR pathway, for example hMSH6, MSI has been noted to be less prominent. Finally, it has been suggested that attenuated MMR capability associated with reduced rather than absent expression of the MMR proteins may be associated with a reduced incidence of MSI (42). These observations suggest that low level MSI may indicate a significant decrease in MMR capability which falls short of complete absence. In order to test this hypothesis we performed an in vitro functional assay in three cases of PCL who presented with adequate material. One of these, patient 87, had no microsatellite alterations and full repair capability. Another (patient 61) had alterations at one locus and markedly reduced in vitro repair activity. In the final case, patient 51, samples were available before and after treatment with melphalan and prednisolone. Interestingly, analysis of these samples showed a marked reduction in repair activity after therapy, associated with the emergence of one altered locus. As MMR deficiency has been associated with the development of a range of chemotherapeutic agents, including the anthracyclines, it is possible that a MMR-defective drug-resistant subclone has emerged during chemotherapy. Unfortunately, insufficient cytosol was available to perform complementation analysis in any of the primary samples.
Analysis by western blotting revealed a marked decrease in expression of all the MMR proteins in case 61. In case 51 a lower molecular weight MSH6 band was detected both at presentation and at relapse (case rel51) and was also evident in the JIM1 and JIM3 cell lines. This truncated form was not attributable to truncating or splice site mutations and may be due to alternative splicing or protein degradation, although protease inhibitors were included in the sample preparation buffer and this form was not evident in any of the other samples analysed. Complete absence of expression was not noted in any case.
As transcriptional silencing of hMLH1 has been found in many solid tumours with MMR defects, promoter methylation analysis was performed in all the cases found to have MSI at one locus at least. This was found to be negative, suggesting that other causes of decreased expression may be responsible, including those acting to reduce transcription or decrease protein stability. Recently it has been shown that U937 myeloid leukaemia cells have low levels of MMR protein expression which may be stimulated by increasing protein kinase C activity (42). Increased MMR protein levels were associated with enhanced MMR function. These results suggest that some cell types have attenuated rather than absent MMR function. Studies are underway to establish if MMR protein expression in the JIM1 and JIM3 cell lines may be enhanced in a similar way.
A widely accepted model for the multistep pathogenesis of plasma cell dyscrasias (43) separates MM development into three phases, inactive, active and fulminant. The inactive phase comprises MGUS and SMM, the active phase MM and the fulminant phase relapsed MM and PCL. Genetic instability, such as RAS and P53 point mutations, is predominantly found in these latter two stages. Mutations of N- and K-RAS are rarely detected in MGUS and SMM (12.5%), but are more frequent in MM (54.5%) and PCL patients (50%) and in the majority at relapse (81%) (44). P53 mutations are relatively infrequent in MM at diagnosis, occurring in 03% of patients with inactive MM (4547), but become more frequent as the disease progresses, occurring in up to 20% of patients with advanced disease (48).
The observation of an increased frequency of mutations in advanced stages of MM led us to investigate whether defects of the DNA MMR pathway might be implicated in MM progression. We have demonstrated that MMR defects are uncommon in MGUS and SMM but are detected in the more active stages of the disease, suggesting that the presence of MMR defects may be a contributory factor in disease progression in some cases. Unfortunately, serial samples from individual patients were not available and since only 1% of MGUS cases per year progress, such an analysis would require a large, long-term prospective study. Defects in MMR proteins result in a mutator phenotype with a 100- to 700-fold increase in repetitive tract instability (49), often associated with frameshift mutations in important growth regulatory genes that possess mononucleotide repeats within their coding regions. Mutations in genes such as BAX and TGFßRII are well described in gastrointestinal and endometrial tumours and invariably lead to truncated proteins (50). Such mutations have been found in MSI-positive haematopoietic cell lines (51,52) but not, as yet, in clinical samples (53,54) and it may be that the pattern of genes targeted by MMR defects may vary in different tumour types (55). However, studies of the HPRT locus in MMR-defective cell lines show a generalized increase in the background point mutation rate in coding regions of many genes (56), potentially in the RAS family and/or P53, leading to the evolution of tumour cells with a selective growth advantage and a more aggressive tumour phenotype.
The identification of MMR defects in patients with plasma cell dyscrasias has therapeutic implications. As well as recognizing replicational errors, the MMR pathway also recognizes DNA adducts resulting from chemotherapeutic agents, but initiates apoptosis rather than repair. In MMR-deficient cells, chemotherapy-induced adducts are tolerated, resulting in selection of MMR-deficient, drug-resistant cancer cells. This tolerance phenomenon may be exploited to allow specific radiosensitization of MMR-deficient cells after treatment with radioactive halogenated thymidine analogues. These become incorporated into DNA and form reactive uracil residues and strand breaks after exposure to ionizing radiation (57). Thus, MMR-deficient plasma cell dyscrasias may define a new subgroup of patients who might benefit from specific targeted therapy with these novel agents.
In summary, we have provided evidence that MMR defects, although uncommon in MGUS and SMM, become more frequent as the disease progresses and may contribute to the development of plasma cell leukaemia through an increased rate of mutation of tumour suppressor genes such as P53 and/or oncogenes such as RAS.
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Acknowledgments
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We are grateful for the cooperation of members of The Northern Regional Haematologists Group in providing samples and clinical data for this study. The work was supported by grants from the Leukaemia Research Fund.
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Received July 11, 2003;
revised April 6, 2004;
accepted May 9, 2004.