The impact of attaining a minimal disease state after high-dose melphalan and autologous transplantation for multiple myeloma

Authors


Professor Gareth J Morgan, University of Leeds, Algernon Firth Building, The General Infirmary at Leeds, Great George Street, Leeds LS1 3EX. E-mail: Garethm@pathology.leeds.ac.uk

Abstract

Initial studies with high-dose therapy (HDT) in myeloma suggest some beneficial effects of attaining a complete response (CR); however, the effect on survival is difficult to assess owing to inconsistencies in the definition of response between studies. We have analysed 96 newly diagnosed patients aged less than 65 years who received HDT and assessed the effect of response on survival using electrophoresis, immunofixation and fluorescent IgH polymerase chain reaction (PCR) to define CR. Patients received induction chemotherapy with C-VAMP (adriamycin, vincristine, methylprednisolone, cyclophosphamide) followed by melphalan 200 mg/m2 and reinfusion of peripheral blood stem cells. There was a high response to C-VAMP [CR = 24%, partial response (PR) = 64%], with all but one patient improving the depth of response after HDT (CR = 69%, PR = 31%). The progression-free survival (PFS) and overall survival (OS) were excellent at a median of 46·4 months and 72+ months. There was a trend towards an improved PFS in patients with an immunofixation-negative CR compared with patients with a PR (49·4 months, 41·14 months; P = 0·26). This was not evident when electrophoresis was used to define CR. The method used to define CR did not impact on the overall survival and fluorescent IgH PCR failed to add any additional prognostic information. This study supports the widespread use of the European Bone Marrow Transplantation group (EBMT) response criteria and suggests that immunofixation should be performed on all patients who become electrophoresis negative.

The conventional treatment of myeloma 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 (Blade et al, 1994). After the introduction of infusional chemotherapy, such as VAD (vincristine, adriamycin, dexamethasone), the number of patients responding to treatment and the level of response achieved has increased (Samson et al, 1989). 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 (McElwain & Powles, 1983). 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 beneficial effects of achieving a complete response (CR) and conceptually attaining a CR is seen as the first step to achieving a cure (Gore et al, 1989). Despite this, several studies have failed to show a plateau of survival, suggesting that all patients have residual disease, which eventually leads to relapse (Attal et al, 1996; Barlogie et al, 1997). It has been suggested that the level of response after high-dose chemotherapy may influence 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 important indicator of eventual outcome and is therefore an important therapeutic goal. Furthermore, additional treatment such as a second high-dose procedure might be of benefit to those patients who fail to achieve a CR following the initial HDT. The available data are difficult to interpret because the definition of a CR varies between studies. In order to improve the reporting of response rates, the European Bone Marrow Transplantation group/International Bone Marrow Transplant Registry (EBMT/IBMTR) have recently published guidelines in which stringent criteria define each response category (Blade et al, 1998).

We have evaluated response as a prognostic factor in a series of 96 cases of multiple myeloma treated with HDT. We have looked at the time to reach a maximum response and the level of response achieved using both the standard response criteria suggested by Gore et al (1989) and the new EBMT response criteria. Subsequently, these have been correlated with progression-free and overall survival.

Patients and methods

Patients and treatment A total of 96 newly diagnosed patients who received high-dose therapy during a 5-year period (October 1993 to November 1998) were included in the analysis. All patients fulfilled the criteria for diagnosis of myeloma adopted in the Medical Research Council (MRC) myeloma trials and were deemed to require treatment. Patients were aged less than 65 years (mean age 55·2 years, range 35·1–65·4 years), with no previous history of malignancy, severe pulmonary or cardiac disease, and were considered suitable to tolerate a high-dose procedure. The standard non-transplant option offered to patients was ABCM (adriamycin, carmustine, cyclophosphamide, melphalan). The isotype paraprotein distribution was IgG 62·5%, IgA 16·7%, IgD 3·1%, Bence-Jones protein (BJP) 16·7% and non-secretory 1%. The serum β2 microglobulin (β2m) was > 4 mg/l in 52·1%, the plasma creatinine > 130 μg/l in 20·8% and the haemoglobin level was < 10 g/dl in 44% of cases. (Table I). The median follow-up from diagnosis was 35·7 months (range 8·6–70·6 months).

Table I.  Characteristics of the 96 patients who received high-dose melphalan with peripheral blood stem cell transplant.
 Patient characteristics (n = 96)
Age (years) (Median)55·2 (35·1–65·4)
Sex (Male/Female)55·2%/44·8%
Isotype
 IgG62·5%
 IgA16·7%
 IgD3·1%
 Light chain (BJP)16·7%
 Non-secretory1·0%
β2m (mg/l)
 < 447·9%
 4–828·7%
 > 823·4%
Haemoglobin (g/dl)
 > 1235·6%
 8–1251·8%
 < 812·6%
Creatinine (μmol/l)
 < 13079·2%
 > 13020·8%

Initial treatment was with C-VAMP (d 1–4; adriamycin 9 mg/m2 i.v. and vincristine 0·4 mg i.v., d 1–5; methylprednisolone 1 g/m2 i.v./p.o. and d 1, 8 and 15; cyclophosphamide 500 mg i.v.) at 21-d intervals until maximum response. Peripheral blood stem cells were mobilized at this time (after a minimum of three cycles of C-VAMP) using cyclophosphamide (2–4 g/m2) and granulocyte colony-stimulating factor (G-CSF, 5 μg/kg s.c.). High-dose melphalan (HDM) was given at 200 mg/m2 followed by the reinfusion of at least 2 × 106 CD34+ cells/kg. All patients received interferon (Roferon-A 3 megaunits 3×/week) starting 3 months after the procedure and continued until relapse.

Response to treatment was monitored using serum electrophoresis and immunofixation at 3 weekly intervals until 6 weeks post HDM and then 3 monthly thereafter. Immunofixation of urine (concentrated 100-fold) was performed on the cases with Bence-Jones myeloma. Bone marrow aspirates and trephines were carried out before treatment, at the time of maximum response, and 3 monthly during the follow-up period. Response to treatment was assessed using two criteria, the traditional response criteria (Gore et al, 1989) and the EBMT/IBMTR/ABMTR criteria (Blade et al, 1998). The criteria suggested by the EBMT have a more stringent definition of CR. Both criteria require less than 5% plasma cells on bone marrow aspirate and biopsy, but the EBMT criteria require negative immunofixation compared with negative electrophoresis in the traditional criteria; an increase from immunofixation negative to positive is considered a relapse in the EBMT criteria compared with an increase in 25% of the paraprotein in the traditional criteria.

Polymerase chain reaction (PCR) Immunoglobulin heavy chain (IgH) PCR was performed using DNA extracted using standard phenol chloroform methods and amplified with a 5′-FITC (fluorescein isothiocyanate)-labelled primer to a consensus region of the JH gene and a primer to the consensus framework 3 region, as previously described (Owen et al, 1996). Electrophoresis and analysis were performed using an Applied Biosystems automated DNA sequencer (ABI model 373A). Electrophoretograms produced from the fluorescence intensity data, representing the size and relative amount of each PCR product as a peak on a histogram, were used to monitor the presence of clonal rearrangements. The criteria for a positive result have been described previously (Evans et al, 1998). Although this method is less sensitive than allele-specific PCR, it consistently retains a sensitivity of 1 in 104 (Owen et al, 1996).

Statistical methods Survival curves were estimated using the Kaplan–Meier method and the Cox regression model was used for multivariate analysis. Calculations were undertaken using the computer-based SPSS statistical package.

Results

In this series, the response to induction chemotherapy was excellent, with 88% of patients responding to C-VAMP using the traditional criteria [CR = 24%, partial response (PR) = 64%, minimal response (MR) = 9%, no response (NR) = 3%]. After high-dose melphalan, all but one patient improved the depth of their response, with the complete response rate rising to 69%. In the 11 patients who showed minimal or no response to C-VAMP but went on to receive HDM, three patients achieved a CR and seven patients a PR. One patient who achieved a minimal response to C-VAMP died from pneumocystis carinii on d 21 post HDM. (Fig 1). Using the more stringent EBMT criteria reduced the overall complete response rate after HDM from 69% to 53%. The reason for this discrepancy was that 16 patients who were electrophoresis negative remained immunofixation positive (Table II).

Figure 1.

A graphical representation of the response rates following CVAMP and high-dose melphalan showing a dramatic increase in the complete response rate as defined using the EBMT criteria.

Table II.  Response rates after CVAMP and HDM showing a dramatic increase in the number of complete responses after HDM.
 CVAMPHDM
TraditionalEBMTTraditionalEBMT
  1. The percentages are expressed using both the traditional and EBMT criteria. The main difference between the criteria is in the definition of complete response: the traditional criteria require a negative electrophoresis while the EBMT criteria are more stringent, requiring a negative immunofixation.

Complete response (%)24186953
Partial response (%)64703147
Minimal response (%)9900
No response (%)3300

The median progression-free survival of the entire group was 46·4 months (CI 37·8–54·9 months) with the median overall survival having not yet been reached (median 72+ months). (Fig 2). To date, 24 patients have died: one from pneumocystis carinii post transplant, 19 from myeloma, two from leukaemia and two from unrelated causes. Presentation β2m was a major prognostic factor for both progression-free and overall survival (P = 0·0007, P = 0·002 respectively), whereas presentation creatinine showed significance for overall survival only (P = 0·03). The initial response to C-VAMP chemotherapy was not a prognostic factor for either progression-free or overall survival (P = 0·5, P = 0·4 respectively). When maximum response post transplant was considered, the system used for the classification of complete response became important. Using the EBMT criteria that requires a negative immunofixation for a CR, there was a trend towards an improved progression-free survival in patients with a CR compared with patients with a PR (P = 0·26, median 49·4 months and 41·4 months respectively). (Fig 3A). This trend was not evident when using the traditional criteria (P = 0·9). There was no difference in the overall survival of patients who achieved a CR using either criteria compared with that of those patients who achieved a PR (Fig 3B). All other factors including age, sex, haemoglobin, paraprotein type and pretransplant creatinine were not significant for progression-free or overall survival.

Figure 2.

Kaplan–Meier curves showing the progression-free (·······) and overall survival (–––) for 96 newly diagnosed patients who received CVAMP and high-dose therapy.

Figure 3.

Kaplan–Meier curves showing the progression-free and overall survival for patients achieving a complete and partial response after high-dose therapy as defined by the EBMT criteria. There is a trend towards an improved progression-free survival in those patients who achieve a complete response (negative immunofixation) (–––) compared with those who achieve a partial response (·······). (A) Median CR 49·4 months, 47% at 5 years; median PR 41·1 months, 34% at 5 years. (B) Median CR not reached, 58% at 5 years; median PR not reached, 64% at 5 years.

We then considered the impact of the time taken to achieve a maximum response in the 50 patients who achieved a CR using the EBMT criteria. Fourteen patients (28%) had achieved their maximum response before HDM, 16 patients (32%) within 3 months post transplant, and the remaining 40% took up to 12 months to reach their maximum response (3–6 months, n = 11; 6–9 months, n = 4; and 9–12 months, n = 5). (Fig 4). Neither progression-free nor overall survival was affected by the time taken to achieve a CR (P = 0·79, P = 0·55 respectively).

Figure 4.

A graphical representation of the time taken to reach complete response (EBMT criteria) following CVAMP + HDM, highlighting the fact that only 60% of patients reach their complete response in 3 months.

In order to evaluate how immunofixation compared to PCR analysis, we examined a subset of patients who had an amplifiable IgH rearrangement at presentation and for whom a follow-up bone marrow was available. Of the 50 patients who achieved a CR defined using the EBMT criteria, 14 patients fulfilled these criteria. Using our technique, which is sensitive to 1 in 104 cells, all of these patients were PCR negative. To date, eight of these patients have relapsed and, at the time of relapse, all patients demonstrated the same IgH PCR rearrangement as at presentation.

During follow-up, seven patients (7%) developed an oligoclonal banding pattern on electrophoresis at a median of 3 months (range 2–5 months) post HDM. Six patients were in an immunofixation-negative CR and one in a PR with a minimal detectable IgGk paraprotein of 2 g. To determine whether this was a consequence of oligoclonal reconstitution or represented disease progression, PCR was performed on four of the cases that had an informative IgH rearrangement at presentation. At the time of testing when the oligoclonal banding pattern was present, the presentation IgH rearrangement was not demonstrable in any of the cases. This would suggest that the pattern seen was due to oligoclonal reconstitution with normal B cells. With a median follow-up of 38·9 months (range 9·0–45·7 months), all of the patients remain alive, although two patients have relapsed and are receiving second-line therapy. There was no difference in the survival of this group of patients compared with patients achieving a CR without oligoclonal banding.

Discussion

The complete response rate of 24% that we describe after induction therapy with C-VAMP compares favourably with other published studies of presentation cases using similar regimens (C-VAMP, VAD), in which 25–28% of patients achieved a CR after induction therapy using electrophoresis to detect paraprotein (Samson et al, 1989; Raje et al, 1997). In addition, our complete response rate is superior to less intensive combination regimens for which CR rates of 5% have been reported (MacLennan et al, 1992). The overall CR and PR rates of 69% and 31% after autologous transplant are superior to the majority of previous studies for which CR rates of 31–51%, measured using electrophoresis, and PR rates of 41–65% have been reported (Attal et al, 1992, 1996; Harousseau et al, 1995; Algere et al, 1998; Lenhoff et al, 2000). Another group that uses a similar induction regimen has reported equally high response rates (Cunningham et al, 1994; Powles et al, 1997), suggesting that the use of C-VAMP to maximum response followed by HDM is an effective therapeutic strategy for inducing a response. Alternatively, the greater response rates may reflect the use of melphalan 200 mg/m2 compared with 140 mg/m2 plus total body irradiation (TBI) (Attal et al, 1992, 1996). Our series also compares favourably with other more intensive sequential therapeutic regimens for which, after two transplants, CR and PR rates of 48% and 47% are seen (Barlogie et al, 1997, 1999).

The progression-free and overall survival of this group of patients is excellent (median PFS 46·4 months, median OS 72+ months) and compares favourably with other studies that have shown progression-free survivals of 32–43 months (Harousseau et al, 1995; Lenhoff et al, 2000). As in other studies, the level of serum β2m was the major prognostic factor. Previous studies have demonstrated that the presence of chemoresponsive disease during high-dose therapy regimens is of prognostic importance (Attal et al, 1992; Harousseau et al, 1995, Algere et al, 1998), although the effect of the depth of response to the treatment is uncertain. It would seem reasonable to postulate that patients who achieve a CR would have an improved outcome; however, the results of a number of studies have been inconsistent (Harousseau et al, 1995; Barlogie et al, 1997; Algere et al, 1998). In our study, there was a trend towards an improved progression-free survival in patients who attained a CR using the EBMT response criteria (i.e. negative immunofixation) compared with patients with a PR. This trend was not seen when CR was defined using electrophoresis alone. This finding 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 immunofixation performed. Previous studies have suggested that immunofixation will detect a paraprotein at a level of 0·05 g/l; however, how this compares with IgH PCR is unknown. We therefore examined the immunofixation negative cases further. All the 14 informative cases that were immunofixation negative were also IgH PCR negative, using a PCR with a sensitivity of 1 in 104. A more sensitive allele-specific oligonucleotide (ASO-PCR) that can detect one tumour cell in 106 has been used to detect residual disease in a number of other studies (Corradini et al, 1999, Martinelli et al, 1999). These studies demonstrated that about 80% of patients in an immunofixation-negative CR have detectable disease and, although the numbers in these studies were small, there was a suggestion that PCR-positive patients have an increased chance of relapse compared with those patients who become PCR negative. This data, in combination with our results, would suggest that there is little additional benefit in using fluorescent IgH PCR to monitor patients who become immunofixation negative. In addition, for PCR monitoring to be clinically relevant, the more sensitive ASO PCR approach should be used.

Interestingly, 40% of patients who achieved a CR failed to do so within 3 months of receiving HDM. Despite patients starting on interferon maintenance at 3 months, 25% of cases achieving a CR after high-dose therapy took longer than 6 months. The time to achieve a CR has been previously examined in 33 patients, of whom 24 achieved a CR after autologous transplantation (Singhal et al, 1995). In this study, 79% of patients cleared the paraprotein within 3 months and 96% in 6 months. Two other recent publications support these findings in which 90% of CRs occurred in 6–18 months (Barlogie et al, 1999; Lenhoff et al, 2000). The time taken to achieve a CR has a number of implications for both the reporting of data and the use of immunotherapy. In the sequential high-dose chemotherapy setting, some of the responses attributed to the final therapy may in fact be the result of previous therapies. We would therefore suggest that caution is required in the interpretation of response rates after each round. It has recently been suggested that immunotherapy should be offered only to patients achieving a CR (Kwak et al, 1997; Hart et al, 1999). Our data would suggest that up to 40% of patients might miss the opportunity of benefiting 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.

A number of patients develop oligoclonal banding after HDT, leading to difficulties in the interpretation of electrophoretic strips. The incidence of 7% in our series of patients is in keeping with another published series of 550 patients in which the incidence was 8·7% (Zent et al, 1998). The appearance of new serum M components of different isotypes may suggest that clonally related myeloma cells expressing variant isotypes are present in the patients after high-dose procedures. Using fluorescent IgH PCR, we were 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. Another group using a more sensitive ASO-PCR technique has also confirmed this finding (Guikema et al, 1998). Further evidence for the benign nature of this pattern is that there was no difference in the progression-free or overall survival for these patients compared with the complete response group as a whole. This has been noted previously (Zent et al, 1998) and supports the EBMT/IBMT/ABMTR recommendations, which suggest that oligoclonal banding seen on electrophoresis should not preclude the definition of a complete response (Blade et al, 1998).

This study confirms the efficacy of high-dose chemotherapy and PBSCT as induction chemotherapy yielding high response rates. However, our data failed to show any evidence of a subgroup of patients who achieve durable remission. Available data from randomized trials have not yet conclusively shown that high-dose chemotherapy improves survival: the important trial of the Intergroupe Francais du Myelome (IFM) shows no survival advantage when allowance is made for the prognostic factors of the patient populations (Attal et al, 1996). We believe that further randomized trials measuring survival and quality of life remain essential in this field.

Acknowledgments

G.J.M. is supported by the Leukaemia Research Fund and P.J.S. by the Imperial Cancer Research Fund.

Ancillary