Minimal residual disease monitoring in multiple myeloma

Best Practice & Research Clinical Haematology Vol. 15, No. 1, pp. 197±222, 2002

doi:10.1053/beha.2002.0192, available online at http://www.idealibrary.com on

12 Minimal residual disease monitoring in multiple myeloma Faith E. Davies

MRCP, MD

Department of Health Clinical Scientist, Academic Unit of Haematology & Oncology, University of Leeds, Algernon Firth Building

Andrew C. Rawstron

PhD

Clinical Scientist, Haematological Diagnostic Service

Roger G. Owen

MRCP, MD MRCPath

Consultant Haematologist, Haematological Diagnostic Service

Gareth J. Morgan*

PhD, FRCP, FRCPath

Professor of Academic Haematology, Academic Unit of Haematology & Oncology, University of Leeds, Algernon Firth Building Leeds General In®rmary, Great George Street, Leeds LS1 3EX, UK

Traditionally, response to treatment in multiple myeloma has been measured by the serum or urinary paraprotein and the percentage of plasma cells in the bone marrow. The use of allogeneic and autologous transplantation has increased the complete response rate and overall survival in patients with myeloma, and in order to assess the e€ects of such treatments accurately more sensitive methods for assessing residual disease have been introduced. The aim of this chapter, therefore, is to describe the available techniques to assess response, monitor residual disease and predict relapse in myeloma. The traditional techniques of paraprotein measurement using electrophoresis and immuno®xation are compared with more sensitive approaches involving the polymerase chain reaction for detecting rearrangements of the immunoglobulin heavy-chain region and ¯ow cytometry for detecting malignant plasma cells. Emphasis is placed on the advantages and disadvantages of each method and its utility in the clinical setting. Key words: multiple myeloma; complete response; electrophoresis; immuno®xation; PCR; ¯ow cytometry.

Multiple myeloma represents about 1% of all haematological malignancies and has an incidence of about 2/100 000 population in the United Kingdom. The clinical picture involves a combination of bone destruction, immune de®ciency, bone marrow failure *Correspondence to: Professor Gareth J. Morgan, Academic Unit of Haematology & Oncology, University of Leeds, Algernon Firth Building, The General In®rmary at Leeds, Great George Street, Leeds LS1 3EX, UK. 1521±6926/02/010197‡26 $35.00/00

c 2002 Harcourt Publishers Ltd. *

198 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

and renal failure, and the diagnosis requires the presence of at least two of three characteristic features: a paraprotein or monoclonal immunoglobulin in the blood and/ or the urine, bone marrow in®ltration by malignant plasma cells and the presence of osteolytic bone lesions. The outlook for patients is poor, with a median survival of approximately 3.5 years, although with the recent introduction of high-dose therapy and a number of novel biologically based therapies there is a suggestion of improved survival. The conventional treatment of myeloma using melphalan, with or without steroids, frequently results in the achievement of a stable `plateau' phase during which patients may have minimal or no symptoms related to their disease. During this phase, however, patients still have a considerable tumour burden and survival has not been shown to correlate with the level of residual tumour or the extent of response to conventional therapy.1 Following the introduction of infusional chemotherapy, using combinations of vincristine, adriamycin, cyclophosphamide and steroids (e.g. VAD and cVAMP), the number of patients responding to treatment and the level of response achieved has increased.2 These responses are often short lived, and it was with the purpose of improving the duration of response that high-dose therapy (HDT) was introduced.3 Since this approach has been combined with peripheral blood stem cell rescue, its safety has improved and it is now widely used for the treatment of myeloma. Initial studies using HDT suggested some bene®cial e€ects of achieving a complete response (CR), and conceptually attaining a CR is seen as the ®rst step to achieving a cure.4 Despite this, several studies have failed to show a plateau of survival, suggesting that all patients have residual disease which eventually leads to relapse.5,6 It has been suggested that the level of response after high-dose chemotherapy may in¯uence outcome, and that patients who achieve a CR may have an improved survival. If this is true, it implies that the achievement of a CR is an indicator of outcome and is therefore an important therapeutic goal. It may also be possible to use the achievement of complete response as a way of comparing the e€ectiveness of di€erent treatment strategies. If response is to be used in this fashion, it is important to use uniform de®nitions and this is re¯ected in recent guidelines published by the European group for blood and bone marrow transplantation/International bone marrow transplant registry/ Autologous blood and marrow transplant registry (EBMT/IBMTR/ABMTR), in which stringent criteria are set to de®ne each response category7 (Table 1). In these guidelines a complete response is de®ned using serum or urine electrophoresis with immuno®xation and bone marrow examination. They have a more stringent de®nition of CR compared to the more traditional response criteria of Gore et al (1989)4; both approaches require less than 5% plasma cells on bone marrow aspirate and biopsy but the EBMT criteria require negative immuno®xation compared to negative electrophoresis in the traditional criteria; an increase from immuno®xation negative to positive is considered a relapse in the EBMT criteria compared to an increase in 25% of the paraprotein in the traditional criteria. However, a number of studies have shown that these methods are relatively insensitive and have demonstrated, using PCR and ¯ow cytometry, that residual disease is still present. Currently, the EBMT/IBMTR criteria do not include an assessment of minimal residual disease, as the criteria were designed for worldwide use in the transplant registries and some of the methods used are not available to all transplant centres and have not been standardized. The aim of this chapter therefore is to describe the available techniques for the detection of minimal residual disease (MRD) in myeloma, emphasizing the advantages/disadvantages of each method and its utility in the clinical setting.

MRD monitoring in multiple myeloma 199 Table 1. EBMT, IBMTR, ABMTR response criteriaa. Complete response (CR) 1. Absence of the original monoclonal paraprotein in serum/urine by routine electrophoresis and immuno®xation maintained for a minimum of 6 weeks. The presence of oligoclonal bands consistent with oligoclonal immune reconstitution does not exclude CR. 2. 55% plasma cells in a bone marrow aspirate and on trephine biopsy. 3. No increase in size or number of lytic bone lesions on radiological investigations, if performed (development of a compression fracture does not exclude response). 4. Disappearance of soft-tissue plasmacytomas. Patients in whom some, but not all, of the criteria for CR are ful®lled are classi®ed as PR. This includes patients in whom electrophoresis is negative but in whom immuno®xation has not been performed. Partial response (PR) 1. 450% reduction in the serum monoclonal paraprotein level, maintained for a minimum of 6 weeks. 2. Reduction in 24-hour urinary light-chain excretion either by 490% or to 5200 mg/24 hours, maintained for a minimum of 6 weeks. 3. For patients with non-secretory myeloma only, 50% reduction in plasma cells in a bone marrow aspirate and on trephine biopsy, maintained for a minimum of 6 weeks. 4. 450% reduction in the size of soft-tissue plasmacytoma. 5. No increase in size or number of lytic bone lesions on radiological investigations, if performed. Patients in whom some, but not all of the criteria for PR are ful®lled are classi®ed as MR. Minimal response (MR) 1. 25±49% reduction in the serum monoclonal paraprotein level maintained for a minimum of 6 weeks. 2. 50±89% reduction in 24-hour urinary light-chain excretion, which still exceeds 200 mg/24 hours, maintained for a minimum of 6 weeks. 3. For patients with non-secretory myeloma only, 24±49% reduction in plasma cells in a bone marrow aspirate and on trephine biopsy, maintained for a minimum of 6 weeks. 4. 25±49% reduction in the size of soft-tissue plasmacytomas. 5. No increase in size or number of lytic bone lesions on radiological investigations, if performed. MR also includes patients in whom some, but not all, of the criteria for PR are ful®lled. No change (NC) Not meeting the criteria of either minimal response or progressive disease. Plateau Stable values (within 25% above or below value at time response is assessed) maintained for at least 3 months. Relapse from CR 1. Re-appearance of serum or urinary paraprotein on routine electrophoresis or on immuno®xation con®rmed by at least one further investigation and excluding oligoclonal immune reconstitution. 2. 45% plasma cells in a bone marrow aspirate or trephine biopsy. 3. Development of new lytic bone lesions or soft-tissue plasmacytomas or de®nite increase in the size of residual bone lesions. Development of a compression fracture does not exclude continued response. 4. Development of hypercalcaemia (corrected 42.8 mmol/l) not attributable to any other cause. Progressive disease 1. 425% increase in the serum monoclonal paraprotein level which must also be an absolute increase of at least 5 g/l and con®rmed by at least one repeated investigation. 2. 425% increase in 24-hour urinary light-chain excretion, which must also be an absolute increase of at least 200 mg/24 hours and con®rmed by at least one repeated investigation. 3. 425% plasma cells in a bone marrow aspirate or trephine biopsy, which must also be an absolute increase of at least 10%. 4. De®nite increase in the size of existing lytic bone lesions or soft-tissue plasmacytomas. 5. Development of new lytic bone lesions or soft-tissue plasmacytomas. Development of a compression fracture does not exclude continued response. 6. Development of hypercalcaemia (corrected 42.8 mmol/l) not attributable to any other cause. aReproduced

from Blade et al (1998, British Journal of Haematology 102: 1115±1123) with permission.

200 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

METHODS OF ASSESSING MINIMAL RESIDUAL DISEASE IN MYELOMA Traditional methods for assessing response to treatment The most frequently occurring types of paraprotein characterizing myeloma are IgG (52%), IgA (22%), light-chain-only myeloma (16%), non-secretory myeloma (6.5%) followed by IgM and D myeloma which are extremely rare. There are a number of ways in which serum paraproteins can be measured. Traditionally proteins in the serum and urine have been separated according to their charge electrophoretically using either a cellulose acetate membrane or an agarose gel as the support medium with the resulting bands being visualized using a protein stain such as Ponceau S or Coomassie blue. These bands may also be quanti®ed using a densitometer to produce a tracing in which each band is translated into an inscribed peak whose height and width re¯ect the intensity of the band, and thus the paraprotein level. If a paraprotein is present the classical pattern of a1, a2, b and g bands is disrupted by an extra band, in which the position of the band within the classical pattern depends on the type of protein present. In order to identify the isotype of the paraprotein further, immuno®xation is required. In this technique several aliquots from the same sample are electrophoresed and incubated with a di€ering anti-heavy-chain or anti-light-chain antisera. The paraprotein is precipitated only by its own appropriate antiserum and can consequently be identi®ed. The level at which serum paraproteins can be detected varies with the separation and visualization technique used. Electrophoresis on paper and other carriers can detect 2±5 g/l, and high-voltage-zone (high-resolution) electrophoresis on agar or cellulose acetate membrane detects 1 g/l; however, the sensitivity is much worse if the band is hidden in the beta region. Although more technically demanding and labour intensive, immuno®xation results in the accurate quanti®cation of paraprotein and is sensitive at detecting a small monoclonal immunoglobulin in the presence of a normal or increased polyclonal background (0.05 g/l).8 The quantitative assessment of urinary light chain uses the same approach as outlined above but can be very dicult, and thus has some implications for residual disease detection. Twenty-four-hour urinary collection allows for the determination of levels over a period of time which helps to rule out variation due to a dilute sample; however, they are often dicult for patients to collect and are consequently incomplete. Other approaches utilize the urinary creatinine to normalize the result, enabling just one aliquot to be tested. These diculties in urinary assessment are re¯ected in the current de®nitions of response. Urinary assessments are now being superseded by the measurement of free light chain in the serum9, which is reliable and o€ers a degree of sensitivity which was not possible with previous methods. Another area of diculty is the non-secretary myeloma where there is no serum or urinary paraprotein to follow. Morphological assessment of the bone marrow o€ers one approach for assessing response that can be supplemented by clonality testing. However, these cases are often the result of illegitimate class switch recombination or deletion of the functional immunoglobulin allele, making the use of clonality markers dicult.10 Finding markers of clonality for such patients requires a thorough characterization of the presenting clone using an extensive panel of primers covering all possible immunoglobulin rearrangements. Polymerase-chain-reaction-based approaches for the assessment of MRD Southern blotting is considered to be the `gold standard' approach for the demonstration of clonality at a molecular level.11 Despite this, it has a number of major

MRD monitoring in multiple myeloma 201

drawbacks when applied to the study of myeloma. The most obvious of these is the lack of sensitivity, which is of particular relevance in a disease where, in most cases at presentation, the average plasma cell percentage is only of the order of 30%. It is for this reason that methods based on the polymerase chain reaction (PCR) for the detection of clonality have been developed for use in myeloma. There are a number of important factors that apply to all PCR methods when they are used to monitor residual disease. For e€ective monitoring using a single bone marrow aspirate, the target cell must be distributed evenly throughout the bone marrow. Myeloma is a patchy disease12, and in order to avoid false-negative results it is important to consider the need for multiple biopsies from di€erent sites. A further consideration is the level of sensitivity of the test and the ease with which the test can be performed. This is exempli®ed by the use of ¯uorescent IgH PCR which is readily applied to clinical material, compared to the more labour-intensive nature of allele-speci®c approaches. In these circumstances the clinical value of the extra log of sensitivity in a disease with a patchy marrow distribution needs to be carefully considered. An issue which becomes more important as the sensitivity of the test increases is the possibility of false-positive results. In myeloma there are a number of potential targets for the detection of clonality by PCR. The most commonly used is the immunoglobulin heavy-chain gene rearrangement. The structure of the immunoglobulin heavy-chain gene The immunoglobulin antibody molecule is made of two identical heavy and light chains held together by inter- and intra-chain disulphide bonds. Each immunoglobulin heavy and light chain complex contains three hypervariable complementary-determining regions (CDRs) ensuring tremendous diversity in combining speci®city for antigen and four conserved framework areas (FR). The CDRs are the most diverse region of the antibody and are unique to each immunoglobulin rearrangement and thus identify individual B cells or clonal B cell expansions. All six CDRs associate to form the antigenbinding site, whose conformation is maintained by the framework sequences. The CDR3 is in direct contact with antigen and is the most variable part of the molecule. Its extraordinary diversity of up to 1014 di€erent peptides results from several mechanisms. The complementary-determining regions of the immunoglobulin molecule are encoded by a number of genes located on chromosome 14 at position 14q32. There are about 150 variable genes (V), 30 diversity genes (D) and six joining genes (J) located at this site which recombine so that one variable gene is juxtaposed to one diversity gene and one joining gene (VDJ) (Figure 1). This VDJ gene combination is initially juxtaposed to the m constant gene of the heavy chain constant region; however, during a process called switch recombination, the constant gene, and thus the antibody class, is changed. The CDR3 encompasses the 30 end of variable gene, all of the diversity gene and the 50 end of the joining gene. To increase the diversity further, the VDJ region often contains N nucleotides, which are randomly inserted at both the V-D and D-J junctions by the enzyme terminal deoxynucleotidyl transferase (TdT). Random deletions also occur at the terminal nucleotides of rearranging V, D and J genes. Moreover, the D gene segment may be rearranged in reverse orientation (30 to 50 ), fused to another D gene (D-D) and transcribed in any of three potential reading frames depending on the V-D junctional sequence. The ®nal process in the formation of a speci®c CDR3 region is called somatic hypermutation, during which random mutations are introduced into the V region. In the germinal centre reaction this leads to an increase in the binding anity and speci®city of the antibody for its cognate antigen. All of these mechanisms contribute to the `CDR3

202 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan a. Germline genes. Variable genes V1

V2

V3

Diversity genes

Vn

D1

D2

D3

Joining genes

Dn

J1

J2

Constant genes δ

µ

Jn

J3

α

ε

b. Initially a D segment is joined to J segment. A N region is inserted under the influence of TdT.

deleted sequence

V1

V2

V3

D3

Vn

Dn

D1

J1

J2

N

D2

J3

γ

δ

µ

Jn

ε

c. A functional VDJ is generated by combining one of many V segments to the DJ region deleted sequence

V2

V1

V3

Vn

D1

N

D2

N

J3

Jn

µ

δ

γ

ε

d. During a primary immune response IgM is generated using a constant µ sequence.

V

D

J

Cµ

e. Following maturation of the immune response or during a secondary immune response IgG is generated by class switching to a contant γ region utilizing the switch region sequences.

V

D

J

Cγ

Figure 1. Schematic representation of VDJ recombination.

antibody ®ngerprint' which is unique to each B cell or B cell clone. The presence of such a unique region has been exploited in the investigation of B cell malignancies by using PCR to amplify the CDR3 sequence. An important consideration for the use of these techniques in the clinical setting is the ability to amplify the fragments of interest. In tumours such as CLL and ALL which have not encountered a germinal centre reaction, it is possible to amplify a fragment in nearly 100% of cases. In other tumours such as follicular lymphoma and myeloma, which have encountered the somatic hypermutation process, mutations have been introduced which can a€ect the binding sites of the primers used, reducing the number of cases in which a marker is available.

MRD monitoring in multiple myeloma 203

Consensus PCR-based approaches The principle of this approach relies upon ampli®cation of the rearranged V region using a consensus JH region primer and one of a number of potential V region primers. In the presence of a polyclonal population either a smear or a ®ngerprint is seen after electrophoresis. In the presence of a clonal population a single band of a speci®c size is seen which is unique to the clone (Figure 2). The ability to demonstrate clonal IgH rearrangements depends on the PCR strategy used. A number of di€erent primers spanning the IgH locus can be used to demonstrate clonal rearrangements.13 (Figure 3). These are either based on consensus sequences between the di€ering V family members or rely upon family-speci®c primers based on the framework 1 leader sequences. Pick-up rates for these primer sets are given in Table 2.14±17 Using the framework 3 (Fr3), PCR clonal rearrangements are detectable in up to 56% of cases. Single rearrangements predominate, with bi-allelic arrangements seen only rarely (55%). The size range for these products is 87±173 bp, with the majority (96%) lying within the normal ®ngerprint. The framework 1 family (Fr1f) primer sets utilize a single primer for each of the Fr1 families combined with the same consensus JH primer. These primers give similar pick-up rates for clonal rearrangements and bi-allelic rearrangements as the Fr3 primer sets, but give larger products (294±368 bp). We have assessed other primers, including Fr1 consensus and Fr2 primers, which had inferior pick-up rates to these standard approaches and did not detect additional rearrangements. We therefore recommend the routine use of Fr3 primers, and if a rearrangement is not detected we suggest the use of Fr1f primers. Using multiple JH primers can also improve pick-up compared to the use of a single JH consensus primer alone. In our experience a multiplex containing the JH consensus primer combined with JH3 and JH6 family primers can maximize the yield. The VH family gene usage in myeloma, detected by these methods, corresponds to what is expected in the normal situation, with VH3 rearrangements predominating while VH5 and VH6 rearrangements are rare (Table 3). In follow-up studies from presentation and relapse we found the size, sequence and type of rearrangement to be extremely consistent, making these rearrangements good markers to follow residual disease. This is in contrast to ALL where the size of the clone at presentation can di€er from that at relapse, making interpretation very dicult. Regardless of the primer combination used, it is not possible to detect clonal rearrangements in 20% of myeloma cases. This is thought to be because of a loss of VH primer binding sites secondary to somatic hypermutation. The extent of mutation seems high in myeloma, with a median of 8% nucleotides mutated compared with 2% in CLL and 4% in follicular lymphoma. Extensive mutation has also been demonstrated in V kappa genes; again, the extent of this seems to be greater in myeloma than in other lymphoproliferative disorders. In an attempt to yield more informative markers in myeloma other clonality targets have been investigated. Chief among these has been light-chain rearrangement18 and kappa-deleting-element rearrangements.19,20 Using these extra PCR approaches the yield of markers can reach as high as 93%. This may potentially be increased further by the use of partial rearrangements at these loci as targets. An important consideration in these approaches is the method used to detect the clonal rearrangement. Most approaches use acrylamide gel electrophoresis because the resolution provided by agarose gel electrophoresis is inadequate. A number of detection methods have been used, including ethidium bromide, radioactive and ¯uorescent labelling. During the initial development of these approaches radioactively labelled products were routinely used which improved sensitivity compared to ethidium visualization alone by up to one log. However, with the development of

204 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

Figure 2. An example of ¯uorescent framework 3 IgH PCR. (a) Fluorescent dyes speci®cally label standards and PCR products within the acrylamide gel, allowing accurate sizing of the products to within a single base. (b and c) Electrophoretograms produced from the ¯uorescence intensity data, indicate the size and relative amount of each PCR product as a peak on a histogram. (b) Clonal rearrangements appear as distinct peaks, and the computer analysis software is able to size rearrangements within a single base pair. (c) A polyclonal B cell pattern produces a ®ngerprint electrophoretogram, i.e. peaks in a normal distribution separated by three base pairs.

CDR1

FR2

N

D

87-173bp

192-285bp

294-368bp

FR3

FR3 consensus primer

CDR2

FR2 Primer

DH

N

J

JH Consensus primer

JH

C

CH

Figure 3. A schematic diagram of the immunoglobulin heavy chain gene locus showing the position of the primers used for framework 1 and 3 PCR. Only cells which have undergone recombination of the IgH gene will be ampli®ed. Variability in the size of the product is due to the highly diverse CDR3 region, which is created by the recombination of the V, D and J regions and with the random deletion and addition of nucleotides.

FR1

FRI consunsus primer

FRI family specific primers

VH

MRD monitoring in multiple myeloma 205

206 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan Table 2. Percentage pick-up rate of clonal IgH rearrangement with di€ering primer combinations. V region primers Fr3 Fr1f Fr3 ‡ Fr1f Fr1 con Fr2 Fr1con ‡ Fr2 Total

JH consensus (%)

JH mix (%)

59 59 77 48 48 63 80

61 63 82 50 52 68 84

Fr ˆ framework; f ˆ family; con ˆ consensus; mix ˆ JHcon, JH3 and JH6.

Table 3. VH family gene usage in multiple myeloma (MM). VH family VH1 VH2 VH3 VH4 VH5 VH6

MM (%)

Normal (%)

19 11 49 16 5 0

19 6 46 21 4 2

¯uorescent PCR, which has many advantages, it is no longer used. In the ¯uorescent PCR analysis system, ¯uorescent dyes speci®cally label standards and PCR products, allowing accurate sizing of the products to within a single base. Electrophoretograms produced from the ¯uorescence intensity data indicate the size and relative amount of each PCR product as a peak on a histogram, and are used to monitor the presence of clonal rearrangements. A polyclonal B cell pattern produces a ®ngerprint electrophoretogram, i.e. peaks in a normal distribution separated by three base pairs, whereas clonal rearrangements appear as distinct peaks. The computer analysis software is able to size rearrangements consistently to within a single base pair and is also able to size each peak within a ®ngerprint; this greatly facilities the identi®cation of clonal rearrangements of known size when they are present within a polyclonal background (Figure 2). This method consistently retains a sensitivity of 1 in 104, but is dependent on the position of the clonal peak within the ®ngerprint and the number of normal ampli®able B cells within the sample.21,22 Simple agarose gel electrophoresis does not o€er a reasonable alternative to these ¯uorescent approaches, lacking both sensitivity and speci®city. Heteroduplex analysis can, however, o€er a useful complementary test. This approach uses a non-denaturing acrylamide gel system and ethidium bromide visualization, following the formation of heteroduplexes by denaturing and rapidly cooling the ampli®ed PCR material. In this way, unique clone-speci®c heteroduplexes are formed which have their own unique mobility. The sensitivity is less than that of ¯uorescent PCR, but occasionally it can help to distinguish clonal rearrangement within a background of polyclonal B cells.19,23 Although clonal rearrangements of the TCR genes can be detected in myeloma they are unlikely to have occurred within the myeloma cells and therefore cannot be used as clonal markers.24,25

MRD monitoring in multiple myeloma 207

An important consideration when monitoring B cell disorders following high-dose therapy (HDT) is the e€ect of chemotherapy on normal B cells. In myeloma a number of patients develop oligoclonal banding on serum electrophoresis following HDT, leading to diculties in the interpretation of electrophoretic strips. The incidence is between 7 and 10%.26,27 The appearance of new serum M components of di€erent isotypes may suggest that clonally related myeloma cells expressing variant isotypes are present in the patients after high-dose procedures. Using ¯uorescent IgH PCR and allele-speci®c PCR (ASO-PCR), a number of groups have been unable to demonstrate any clonally related cells at the time of the appearance of oligoclonal banding, suggesting that these cells merely represent an imbalance in the regenerating B cells.27,28 Further evidence for the benign nature of this pattern is that there is no di€erence in the progression-free or overall survival for these patients.26,27 The EBMT/IBMT/ABMTR recommendations therefore suggest that oligoclonal banding seen on electrophoresis should not preclude the de®nition of a complete response.7 Allele-speci®c PCR approaches Allele-speci®c (ASO) PCR is undoubtedly the most sensitive approach for the detection of residual disease, with the majority of studies reporting sensitivities of one tumour cell in 105±6 normal cells. Its use is described widely elsewhere in this issue but, in short, PCR products are generated as outlined above and are sequenced. This sequence is used to design either clono-speci®c primers or probes for use in hybridization experiments. The increased sensitivity is the consequence of the detection of only tumour-speci®c immunoglobulin sequences and the lack of contaminating signal from the polyclonal background of normal B cells. However, designing optimum oligonucleotides for this purpose can be dicult, and normal B cell sequences are often detected leading to quite wide variations in the sensitivity of this approach. Consequently, it is important to validate the sensitivity of each probe/primer synthesized. Interestingly, we compared ¯uorescent PCR with clono-speci®c approaches in ALL and found that, although the clono-speci®c approach was more sensitive at detecting residual disease, there was considerable overlap between the two techniques.21 The complexity of the technique means that it is labour-intensive and slow, making clinical application dicult and suggesting that its role may be only practical in scienti®c studies. Adapting the PCR to give quantitative information One of the major requirements for a test aimed at detecting residual disease is an ability to quantify the level of disease. Monitoring of the serum with immuno®xation is straightforward, o€ers moderate sensitivity and is quantitative. Although ¯uorescent PCR may o€er slightly more sensitivity it is not quantitative and will, at maximum, detect 1:104 normal cells. Clono-speci®c approaches are generally more sensitive and will detect residual disease at a level of 1:105 normal cells. Used sequentially in this fashion, some idea of the level of residual disease can be obtained. More sophisticated approaches have been developed which can be of use clinically. Clono-speci®c PCR can be modi®ed to be semi-quantitative, and this has been improved by the application of limiting dilution approaches.29 In this technique multiple dilutions in normal DNA are made and the level of disease is calculated by a determination of the number of positive results in the dilutions. This approach is, however, complicated in myeloma because of the lack of 100% tumour DNA at presentation. Although of interest historically, the development of real-time PCR

208 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

technology has superseded this approach.12,30 Detection of target DNA is based on the cleavage of ¯uorescently labelled probes by the 50 -to-30 exonuclease activity of Taq DNA polymerase. These approaches have two main advantages. Fluorescence data are collected without post-PCR manipulation of samples, and quantitative data are obtained by using the number of cycles at which ¯uorescence crosses the baseline, the CT value. The most widely used approach utilizes Taqman probes31 (see Chapter 3 for details of the Taqman technique). However, this still requires the generation of clonospeci®c oligonucleotides, which is time-consuming and expensive. The optimum approach to minimize costs would be to generate a universal Taqman probe. To date this has not been possible; however, it is possible to generate clono-speci®c primers and to combine these with a limited set of probes dependent upon the JH primers used (Kneba and Evans, personal communication). Other targets for PCR Illegitimate class switch recombination, with the formation of chromosomal translocations involving chromosome 14q32, is a common feature of multiple myeloma, being detected in up to 60% of clinical samples using FISH-based approaches. The fact that these are relatively early events in the pathogenesis of multiple myeloma makes them ideal targets for detecting residual disease. A common recurring translocation in clinical material is the t(4;14)32,33 which is detectable in 10% of myeloma cases. As a consequence of the translocation, sequences from chromosome 4 are fused to the immunoglobulin switch regions on chromosomes 14. Following the translocation, the FGFR3 gene on chromosome 4 is overexpressed, and in clinical samples there is a one-to-one relationship between FGFR3 overexpression, the presence of the translocation making it suitable as a screening method. The other pertinent feature of the translocation is the generation of Ig/MMSET fusion transcript.32 Using RT-PCR it is possible to generate a sensitive speci®c PCR which will detect the majority of cases carrying a t(4;14) (Figure 4). Based on historical experience this is likely to be the most sensitive approach for monitoring residual disease in myeloma. A small number of translocations may be missed by these approaches but because the breakpoints at the DNA level are relatively conserved it is possible to use long-distance PCR approaches to detect the translocation at the DNA level.33 Flow cytometry methods for the assessment of MRD Plasma cells are terminally di€erentiated B cells and hence express a number of B cell antigens as well as myeloma-speci®c antigens. This speci®c cell-surface antigen pattern may be exploited by ¯ow cytometry, o€ering a quick and ecient method not only for quantifying low levels of neoplastic plasma cells but also for assessing normal immune reconstitution. Plasma cells may be identi®ed using four-colour ¯ow cytometry by high CD38, high CD138 and low CD45 expression. A sequential gating strategy is particularly important for assessing minimal disease states as it is necessary to eliminate contaminating events such as apoptotic cells and cellular debris. The expression of CD19 and CD56 is then used to distinguish between normal and neoplastic plasma cells. The former are consistently CD19‡ 56 , whereas the latter are CD19 or CD19‡ 56‡ . Up to 200 000 events are acquired, allowing a maximum sensitivity of detection of 0.01% in all cases34±36 (Figure 5).

2

3

4

5

6

7

Iµ

Chromosome 4 breakpoint in intron 3 of MMSET with splice va

1

Iµ

Iµ

Iµ

Chromosome 4 breakpoint in intron 3 of MMSET with splice variant

Chromosome 4 breakpoint in intron 2 of MMSET

PCR Primers

Ex 3

Exon 3

Exon 4

Exon 4

Exon 5

Ex 5

Exon 5

ms6r

Exon 6

Exon 6

Exon 6

Chromosome 4 MMSET

Lane 1: patient 1 intron 2 breakpoint Lane 2: patient 2 intron2 breakpoint Lane 3: patient 3 intron 2 breakpoint with splice variant Lane 4: patient 4 intron 3 breakpoint Lane 5: patient 5 intron 3 breakpoint Lane 6: cell line H929 (positive control) intron 3 breakpoint Lane 7: 100bp ladder

Chromosome 14 IgH locus

Figure 4. (a) A diagrammatic illustration of the di€erent t(4;14) IgHMMSET fusion products and the position of RT-PCR primers. (b) Ethidium bromide-stained agarose gel of IgHMMSET fusion products in ®ve patients.

180bp

438bp

1064bp

b.

a.

MRD monitoring in multiple myeloma 209

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Figure 5. Flow cytometric detection of neoplastic plasma cells. (a) The gating strategy used to detect plasma cells is designed to exclude the majority of contaminating events. This is particularly important for assessing minimal disease states, as contaminating events (apoptotic cells and cellular debris) are frequently higher in number than plasma cells in post-treatment samples. Analysis of CD38 versus CD138 expression (i) provides the best separation of plasma cells from other leukocytes, but is also subject to contamination with cells binding antibodies non-speci®cally. This can be detected on the CD38 versus CD45 plot (iii) to the right of the plasma cell population. As such, an initial region (R1) is set around cells expressing a high level of CD38 and CD138 (i), and a second region (R2) set on the light scatter of gated CD38‡ 138‡ cells (ii). A third region (R3) was set around the cells satisfying both R1 and R2 for CD38 and CD45 expression (iii). Regions R2 and R3 are then optimized until events falling within both of these regions are all CD138‡ and CD3 . (b) Representative plots from three individuals at 3 months post-transplantation. Horizontal quadrant markers are set according to the CD3 control for analysis of CD138 and CD19 expression. CD56 expression is weak on normal plasma cells, and the marker is set higher than control (at 100 as standard) as this provides a better discrimination between normal and neoplastic cells. The expression of CD19 PE and CD56 PE was then used to distinguish between normal and neoplastic plasma cells. The former are consistently CD19‡ 56dim (top row of plots) whereas the latter are CD19 or CD19‡ 56‡ (middle row of plots). The level of CD19 expression is broad on normal plasma cells, and up to 10% may be CD19 in comparison to control. Therefore, samples were classi®ed as containing neoplastic plasma cells only if more than 10% had an abnormal phenotype. The lower row shows a patient whose bone marrow contains mostly normal plasma cells, but there is a neoplastic population detectable that represents 15% of total plasma cells.

MRD monitoring in multiple myeloma 211

Proportion of patients Progression free

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Complete reponse (IF-), only normal plasma cells present post-transplant Complete reponse (IF-), neoplastic plasma cells present post-transplant Figure 6. Flow cytometric enumeration of neoplastic plasma cells provides additional prognostic information over immuno®xation. Kaplan±Meier analysis of progression-free survival for patients who achieve a complete (i.e. immuno®xation-negative) remission post-transplantation. Patients who have residual neoplastic plasma cells were compared against those who have only normal plasma cells at 3 months post-transplantation. Survival is shown from time of transplantation.

This ¯ow assay has several advantages over current methods for the assessment of residual disease in myeloma patients. The assay utilizes the same markers for each patient, is applicable to over 98% of patients and does not require knowledge of presentation characteristics to allow detection of residual disease. The sensitivity is approximately one log greater than that of consensus-primer ¯uorescent IgH-PCR. Although the sensitivity is lower than that of patient-speci®c PCR approaches, the assay can be performed rapidly, which means that it can be used routinely to aid treatment decisions. In contrast to ASO-PCR ± which measures all B-lineage cells with the same CDR3 sequence as the neoplastic plasma cells, including clonally related B lymphocytes ± this ¯ow method measures neoplastic plasma cells directly.34,35 The technique can also be used to give semi-quantitative measurement of residual malignant plasma cells. The actual number of plasma cells can be a€ected by the extent of contamination of the marrow aspirate with peripheral blood. To overcome this issue malignant plasma cell values are expressed as a percentage of normal white cells, and while this is not perfect, it at least provides a semi-quantitative method. The application of ¯ow cytometric monitoring also demonstrates the presence of normal plasma cells in the majority of patients, independently of the presence or absence of residual neoplastic plasma cells. Monitoring of patients after high-dose therapy using this approach has shown that residual plasma cells are detectable in approximately 30% of patients who have no evidence of disease with immuno®xation. Normal plasma cells return in 75% of cases, but are sustained in only 25% of cases and correlate with the recovery of normal immunoglobulin levels. There is a suggestion that those patients who have no detectable neoplastic plasma cells, and who also show sustained normal plasma cell recovery with normal immunoglobulin levels, have a prolonged remission post-high-dose melphalan. In comparison, those patients with either neoplastic plasma cells or who fail to regenerate sustained levels of normal plasma cells post-transplant with normal immunoglobulin levels have a poor prognosis as they are at high risk of early relapse37 (Figures 6 and 7).

212 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

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Normal plasma cells only post-transplant Neoplastic plasma cells present post-transplant Figure 7. Kaplan±Meier analysis of progression-free and overall survival, comparing patients with detectable neoplastic plasma cells at 3 months post-transplantation against those with only normal plasma cells present. The presence of neoplastic plasma cells at 3 months post-transplantation predicts early relapse. Survival is shown from time of transplantation.

THE CLINICAL ROLE OF RESIDUAL DISEASE MONITORING IN MYELOMA Monitoring response Complete response rates to conventional oral treatment ± as assessed by electrophoresis ± are in the order of 5±10%.38 Interestingly, in the MRC study the level of response did not correlate with survival but the attainment of plateau did. Plateau was de®ned as a stable state where patients have no symptoms attributable to active disease, the paraprotein level is stable and blood transfusion is not required. A further ®nding of this study was that the rate of response correlated inversely with outcome, rapid response being associated with poor outcome. The complete response rate with intravenous induction chemotherapy with VAMP, C-VAMP or VAD in presentation cases is higher, with 24±28% of patients achieving a CR post-induction therapy using electrophoresis to detect paraprotein.2,27,39 These rates can be further increased by using high-dose melphalan with peripheral blood stem cell or bone marrow support to consolidate induction chemotherapy. The overall CR and PR rates of 69% and 31% post-autologous transplant in our series27 are superior to the rates in the majority of previous studies where CR rates measured by electrophoresis of 31±51% and a PR rate of 41±65% have been reported.5,40±43 Another

MRD monitoring in multiple myeloma 213

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group which uses a similar induction regimen has reported equally high response rates44,45 suggesting that the use of C-VAMP to maximum response followed by HDM is an e€ective therapeutic strategy for inducing response. Alternatively, the greater response rates may re¯ect the use of melphalan 200 mg/m2 compared to 140 mg/m2 plus total body irradiation (TBI).5,41 Our series also compares favourably to other more intensive sequential therapeutic regimens where, following `double autografts', CR and PR rates of 48% and 47% are seen.6,46 The impact of depth of response on survival Although the e€ect of the depth of response on survival is uncertain, it would seem reasonable to postulate that patients who achieve a CR have an improved outcome. The results of a number of studies using serum electrophoresis have, however, been inconsistent.6,27,40,43,47 In our experience there was a trend toward an improved progression-free survival in patients who attain a CR with negative immuno®xation compared to patients with a PR (Figure 8). This trend was not seen when CR was de®ned using electrophoresis alone. This ®nding supports the more widespread use of the EBMT guidelines, and as a consequence all patients who become negative using electrophoresis should go on to have immuno®xation performed. An important question which needs to be fully addressed is whether the application of PCR-based technology can provide additional useful information compared to simple monitoring of serum or urinary paraprotein levels.27,48 A recent report has demonstrated that cases that were immuno®xation-negative were also IgH PCR-negative, using

214 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

a ¯uorescent PCR with a sensitivity of 1:104.27 A more sensitive ASO-PCR able to detect one tumour cell in 106 has been used to detect residual disease in a number of other studies.49±51 These studies demonstrate that about 50% of patients in an immuno®xationnegative CR have detectable disease with clono-speci®c PCR techniques. Although the number of cases in these studies is small, there is a suggestion that PCR-positive patients have a shorter progression-free survival compared to those patients who become PCRnegative. These data would therefore suggest that there is little additional bene®t for using ¯uorescent IgH PCR to monitor patients who become immuno®xation-negative, and that if PCR monitoring is to be clinically relevant the more sensitive ASO-PCR approach should be used. However, in the clinical trial setting, interpretation of data may be dicult. This is based on the argument that a marker is available for only 80% of cases, of which only 50% of cases will attain a complete response after intensive chemotherapy, and only 50% of these cases will become PCR-negative. This group would represent 20% of the total cases, and such a small group would lack the power to demonstrate a di€erence in outcome unless the initial study was very large indeed. Time to maximum response Whether electrophoresis, immuno®xation or PCR are used to de®ne a complete response, it is interesting to note that 20±40% of patients who achieve a CR post-highdose procedure fail to do so within 3 months of receiving HDM27,52, and even at 6±18 months only 90% of CRs will have occurred.43,46 The time taken to achieve a complete response has a number of implications for both the reporting of data and for the use of second transplant procedures and immunotherapy. In the sequential high-dose chemotherapy setting some of the responses attributed to the ®nal therapy may in fact be due to previous therapies, and we would suggest that caution is required in the interpretation of response rates following each round. It has also recently been suggested that immunotherapy should be o€ered only to patients achieving a CR53,54, suggesting that up to 40% of patients might miss the opportunity of bene®ting from such an approach if assessment is made at 3 months post-transplantation. Alternatively, if treatment is given at 3 months to patients not in a CR, apparent improvement in response may be due to a delayed response to the high-dose therapy. Response rates with allogeneic and autologous transplantation A very important consideration is whether there is a di€erence in either depth or numbers of responses between patients receiving an allograft rather than an autograft. There are few available data with which to address this question. Limited data generated on our own patients suggest that the time to maximum response using ¯uorescent IgH PCR is delayed in patients undergoing allogeneic transplantation conditioned with Cy/TBI compared to autologous transplantation with Mel 200 mg/m2 (Haynes and Russell, personal communication) (Figures 9 and 10). An Italian group have carried out a comparison of single and double autografts with allogeneic transplantation. The conditioning treatments used were 200 mg/m2 HDM or 200 mg/m2 HDM followed by 120 mg/m2 melphalan with 12 mg/m2 busulphan or CyTBI. Following treatment, relatively low numbers of complete responses were achieved: 38% for the allogeneic transplants and only 18% and 27% for the single and double autograft procedures.51,55 Analysis of the autografted patients showed that 16% achieved a molecular CR compared to 50% in the allogeneic group. Overall, those patients achieving a molecular CR were less likely to relapse, an observation which was

MRD monitoring in multiple myeloma 215 0

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Figure 9. Schematic diagram of the detection of minimal residual disease using IgH PCR after (a) autografting and (b) allografting w ˆ PCR-negative; W ˆ PCR-positive; T ˆ serological relapse; and Q ˆ clinical relapse. Note in the allografting group that PCR negativity can occur late after the transplantation procedure, suggesting a graft-versus-myeloma e€ect.

stronger in the autograft group. There were also similar di€erences to those observed by us in the sequential PCR results between the allogeneic and autograft groups. PCR negativity was achieved by 3 months in the autograft group whereas it was possible to become negative after this time in the allograft group, in some cases after 1 year. This

216 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan a.

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di€erence presumably re¯ects the graft-versus-myeloma e€ect present within the allografted group and is compatible with the observations we have made. Sequential monitoring to predict relapse It is dicult to make meaningful statements about sequential monitoring in order to predict relapse. There are few available data, and there is a reluctance to perform regular PCR analyses to generate these data due to the need for repeated bone marrows and costs. Analysis of the data, such as they are, suggests that if the approach

MRD monitoring in multiple myeloma 217

were to be successful it would have to be done regularly at 3 monthly intervals48,55,56 (Figure 9). It is likely to be of use only in those patients who become immuno®xationnegative. Overall, until treatments improve, clono-speci®c PCR approaches are not likely to be useful outside the clinical study setting, and ¯ow cytometry o€ers a more realistic prospect of obtaining useful clinical information. The bone marrow is the optimum source of material for analysis as it is possible to be PCR-positive in the bone marrow but negative in the peripheral blood (Figure 10). These approaches are likely to become more important clinically as approaches to treatment change. One of the main aims of treatment in myeloma must be to induce and maintain minimal-disease status. When e€ective maintenance strategies are available, it will be important to monitor disease responses at low levels. A good example of this is the use of donor lymphocyte infusions following T-cell-depleted mini-allogeneic transplantation, where the aim is to induce a graft-versus-myeloma e€ect. Monitoring the manipulation of stem cell harvests One of the major concerns regarding the re-infusion of autologous progenitor cells following a high-dose procedure is contamination of the harvest with myeloma cells and whether these cells have the ability to re-populate the marrow and contribute to the relapse of disease.57 In the majority of myeloma cases (70%), the contamination as measured by ¯ow cytometry and PCR is less than one tumour cell in 103±4 normal cells.29 The cases with high tumour contamination tend to be those with persistent disease within the bone marrow at the time of mobilization.14,56,58 Correlation with outcome is dicult to assess, although one group has suggested that individuals with no contamination have a greater chance of a CR after autografting58; however, the biological signi®cance of this is dicult to understand. Using a more sensitive oligospeci®c PCR, which is able to detect one tumour cell in 106 normal cells, there is evidence of contamination in almost 100% of cases.49 Whether these cells are clonogenic is a dicult question to address, but sensitive immunophenotypic tests suggest that the cells within apheresis products have a phenotype similar to that of myelomatous plasma cells from the bone marrow but express lower levels of syndecan1.34 There is no de®nitive evidence from mouse studies regarding this matter but, clearly, if these cells are re-infused they may contribute to a relapse of disease. A number of groups have tried to reduce/eliminate the tumour contamination of harvests by either selecting for stem cells expressing the CD34 antigen alone or combining this with a negative selection for myeloma cells. A tumour depletion of 3±5 logs can be achieved, and the resulting product can be infused without a€ecting engraftment29,30,48,56 (Figure 10). There are, however, a number of theoretical considerations in determining whether harvest manipulation is clinically relevant. For purging to be e€ective, the major source of contamination must be considered to be from the graft, with the patient being tumour-free, and previous trials of induction chemotherapy suggest that this is unlikely. Experience from syngeneic transplants supports this because, despite a perfect match between host and donor and the graft lacking contamination with tumour cells, there is no plateau on the survival curve and all patients eventually relapse.59 This implies that the tumour burden within the patient is probably more clinically relevant than the tumour contamination of the graft. It is therefore clear that if purging works, it will be e€ective in only a minority of cases; to show that purging is e€ective in improving survival will require huge studies. Recently reported have been the results of a large phase III randomized study, involving 190 patients, which assessed the clinical bene®ts of CD34 selection in

218 F. E. Davies, A. C. Rawstron, R. G. Owen and G. J. Morgan

myeloma. These data suggest that purging of the harvest material results in no di€erence in progression-free or overall survival.60,61 SUMMARY In conclusion, there is clearly a role for the detection of residual disease in myeloma, and this will become greater with the widespread application of high-dose procedures and other immune-based treatments that increase the number of complete responses. Practice points Reasons for monitoring residual disease in myeloma . quantify the level of response and correlate with outcome . quantify contaminating tumour cells in harvest material . monitor success of purging strategies . monitor e€ects of therapy at low levels of disease . regular monitoring to detect early relapse Measuring residual disease in myeloma . bone marrow morphology and immuno¯uorescent techniques . electrophoresis 2±5 g/l . immuno®xation 0.05 g/l . ¯uorescent IgH PCR 1:103±4 Increasing sensitivity . ¯ow cytometry 1:104 . clono-speci®c PCR 1:105±6 Potential PCR markers for monitoring residual disease in myeloma . immunoglobulin heavy-chain gene rearrangements . light-chain rearrangements . kappa-deleting element rearrangements . partial rearrangements . chromosome 14 translocations, e.g. t(4;14) The t(4;14) in myeloma . it occurs in 10% of cases of myeloma and Monoclonal Gammopathy of Uncertain Signi®cance (MGUS) . fuses the strong enhancer of the immunoglobulin gene with genes on chromosome 4 . Ig/MMSET fusion gene transcripts are detected in most cases: they can be used as targets following RT-PCR . the FGFR3 gene on chromosome 4 is over-expressed; this can be detected by RTPCR . at the DNA level the breaks are conserved and can be detected using longdistance PCR methodology Role of ¯ow cytometry in residual disease monitoring . as sensitive as ¯uorescent IgH PCR . quantify level of circulating tumour cells . quantify level of disease in bone marrow . detect malignant phenotype so that presenting material is not necessary . quantify normal plasma cells . monitor disease sequentially and predict outcome

#

MRD monitoring in multiple myeloma 219

Research agenda . the development of novel biologically based treatment approaches aimed at increasing complete response rates and maintaining responses . implementation of routine free light-chain assessment . the routine implementation of ¯ow cytometry to clinical trials in order to assess fully the response rates and the time to maximum response . the development of novel primer sets to maximize the detection of clonal rearrangements by PCR . the sequential application of consensus and clono-speci®c PCR methodologies to compare autologous and allogeneic transplant approaches and to assess fully the graft-versus-myeloma e€ect . full evaluation of quantitative approaches for monitoring residual disease ± in particular, a comparison of ASO-PCR with ¯ow cytometry

Currently, using the achievement of complete responses as an indicator of outcome o€ers a way of comparing di€erent treatment regimes, and monitoring the level of myeloma below clinically detectable thresholds o€ers the option of manipulating disease using novel non-cytotoxic approaches. Whereas, with conventional treatment, response did not correlate with outcome, current treatment strategies aim to maximize complete responses to minimize the residual burden of myeloma, and preliminary data suggest that such approaches may improve outcome. There is some evidence supporting the idea that the achievement of a molecular CR can improve outcome. However, escalating the level of conditioning therapy does not seem likely to improve the numbers of CRs beyond those already obtained. This particular aim is more likely to be obtained using non-cytotoxic approaches utilizing agents such as the thalidomide derivatives, proteosome inhibitors, antibody-directed therapy and immune-based approaches. Currently the graft-versus-myeloma e€ect, which is apparently seen in allogenic transplantation, seems able to exert a continuous e€ect on the myeloma clone and this can only be demonstrated using sensitive PCR-based approaches. Clearly, convenient peripheral-blood-based approaches are required and would be the optimum method for MRD detection because of patient discomfort from bone marrow procedures and the patchy nature of disease. However, at present, the best methods for assessment of MRD require bone marrow examination. Both PCR and ¯ow cytometry are more sensitive than paraprotein electrophoresis, and immuno®xation, and have complementary roles in the assessment of minimal residual disease. Acknowledgements The Leukaemia Research Fund and the Yorkshire Cancer Research support these authors.

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