Divisione Universitaria di Ematologia, Azienda Ospedaliera S Giovanni Battista di Torino, Torino, Italy

Correspondence to: Dr M Boccadoro, Divisione Universitaria di Ematologia, Azienda Ospedaliera S Giovanni Battista di Torino, Via Genova 3, 10126 Torino, Italy

Multiple myeloma (MM) is characterized by the expansion of tumor plasma cells in bone marrow (BM), but neoplastic cells have been consistently detected in peripheral blood (PB). Peripheral blood progenitor cell (PBPC) collections have been widely used to support high-dose therapy for MM patients. A flow cytometric technique has been used to detect plasma cells in PB and PBPC harvests. High CD38 expression identified these cells, and their nature was confirmed by the coexpression of specific antigens, such as CD138 and cytoplasmic immunoglobulins. Malignant plasma cell reinfusion could negatively affect response rate and survival, as demonstrated in other hematological malignancies. To address this issue, the relationship between the number of reinfused plasma cells, response to chemotherapy and event-free survival (EFS) have been analyzed. Sixty-four MM patients were treated with intensified chemotherapy at diagnosis. They were mobilized with cyclophosphamide and G-CSF, and then treated with melphalan 100 mg/m² (MEL100) followed by PBPC support. A second course was given after 2 months, and a third to patients not in complete remission. There was no correlation between the number of reinfused plasma cells and response rate after this intensified chemotherapy: patients attaining complete remission received 3.6 ´ 10⁶/kg CD38⁺ cells, while those with a partial or no response received 5.6 and 2.9 ´ 10⁶/kg CD38⁺ cells. Similarly, there was no correlation between the number of reinfused plasma cells and EFS. Patients receiving less than 4.85 ´ 10⁶/kg CD38⁺ cells experienced a median EFS of 34.2 months as opposed to 36.4 months for those receiving more than 4.85 ´ 10⁶/kg CD38⁺ cells (P = 0.7). Recurrence of the disease is consistently observed in MM: our data suggest that in vivo residual tumor cells, rather than reinfused plasma cells are more likely to be responsible for relapse. Bone Marrow Transplantation (2000) 25, 25-29.

multiple myeloma; plasma cells; PBPC; purging

Multiple myeloma (MM) is a B cell lineage neoplastic disease characterized by uncontrolled proliferation of tumor plasma cells in bone marrow (BM), monoclonal immunoglobulin production and multiple osteolytic bone lesions. With conventional chemotherapy (melphalan and prednisone) complete remission (CR) is reached in less than 5% of patients and only 5% survive beyond 10 years.¹ Intensified chemotherapy regimens have therefore been proposed to improve the outcome of symptomatic MM patients, and their superiority has been demonstrated in several studies.^2,3,4,5,6 The feasibility of high-dose chemotherapy has been markedly improved by the reinfusion of peripheral blood progenitor cells (PBPC), which induce more rapid hemopoietic reconstitution than the BM-derived stem cells.⁷ With the introduction of PBPC and cytokines such as G- or GM-CSF as support for high-dose therapy, the mortality after autologous transplantation has dramatically reduced. MM usually affects elderly people with a median age of 60-70 at diagnosis. The low toxicity of this approach has extended its applicability to a large patient population, up to 70 years.⁸

Contamination of stem cell harvest products by monoclonal plasma cells in MM patients has been investigated by several authors. Neoplastic plasma cells have been identified in the peripheral blood (PB) during mobilization and in leukapheresis products.^9,10,11 Tumor cell contamination in PBPC harvest was significantly lower than in BM.^9,12 A significant reduction of neoplastic plasma cells in PBPC harvests is also obtained after repeated high-dose courses.¹³ However, either with in vivo purging - high-dose chemotherapy followed by autologous transplantation¹⁴ - or with in vitro purging techniques - positive selection of CD34⁺ cells from the apheresis products - residual tumor cells were always detectable by molecular biology analysis.¹⁵ This contamination is thought to influence disease recurrence in patients with hematological and extra-hematological malignancies.¹⁶ However, whether relapse is due to the reinfusion of malignant plasma cells after autologous transplantation or to the inability of submyeloablative regimens to eradicate them remains to be demonstrated.

In this study we have evaluated the role of plasma cell contamination in leukapheresis products on response rate and event-free survival (EFS) of 64 MM patients at diagnosis treated with an intensified regimen and PBPC support.

Patients and methods

Patient characteristics

Between March 1995 and November 1997, 64 MM patients were enrolled at diagnosis in a pilot study carried out at our institution. MM was diagnosed according to the Southwest Oncology Group (SWOG) criteria. Patient characteristics at diagnosis were: age 60.5 (range 42-73), 2-microglobulin 3 mg/l (range 0.4-22), labelling index 0.6% (range 0.1-4.7), BM plasmacytosis 30% (range 1-90), serum hemoglobin 11.5 mg/dl (range 5-15) and serum creatinine 1 mg/dl (range 0.7-3.8).

Treatment regimen

All patients received three courses of DAV (dexamethasone 40 mg p.o., on days 1-4; adriamycin, 50 mg/m² i.v., on day 1; vincristine 1 mg i.v., on day 1) as debulking therapy. Subsequently, they received cyclophosphamide 4 g/m² at day 0, followed by G-CSF 10 g/kg daily s.c. from day 3 to the last day of leukapheresis. Leukapheresis was initiated when circulating CD34⁺ cells exceeded 10/l. Three to four procedures were performed on each patient, with a Fresenius AS 104 Cell Separator (Schweinfurt, Germany). Leukapheresis products were cryopreserved. Patients received melphalan 100 mg/m² (MEL 100 protocol)¹⁷ on day 30. At day 31 PBPC were reinfused. Melphalan administration was repeated every 2 months for a total of two courses in patients reaching CR, and three in those reaching partial remission (PR). A minimum of 3 ´ 10⁶/kg CD34⁺ cells were reinfused after each course to ensure rapid engraftment.

Criteria for response

Clinical response was evaluated by serial assessment of myeloma M-component in serum and urine samples, analyzed by standard electrophoresis, and BM plasmacytosis. PR was defined as an M-component decrease >50%, a 90% decrease of Bence-Jones proteinuria and 50% reduction of BM infiltration, without any increase in size or number of lytic bone lesions. CR was defined as disappearance of serum and urine M-component by standard electrophoresis and BM plasmacytosis <5%. Relapse was defined as reappearance of serum or urine M-component, or an increase >50% from the lowest level of serum M-component, or an increase in size or number of lytic bone lesions. Progression was defined as any increase >25% of serum M-component or an increase in size or number of lytic bone lesions during the induction treatment.

PBPC and circulating plasma cells evaluation

The number of PBPC was determined by direct immunofluorescence on whole blood, using the anti-CD34 monoclonal antibody and flow cytometry analysis, as previously described.¹³ Analysis was performed with a FACScan analyser (Becton Dickinson, San Jose, CA, USA) equipped with a filter set for FITC-PE dual-color fluorescence. Data acquisition and analysis were performed with the FACScan Research Software and each measurement included at least 40 000 cells. The frequency of cells expressing CD34⁺ was calculated as the percentage of all analyzed cells. Dead cells were excluded on the basis of forward-and side-scatter analysis. The number of circulating CD34⁺ cells per l of blood was obtained by multiplying this percentage by the total number of leukocytes in 1 l of blood.

The amount of circulating plasma cells in PB and leukapheresis products was also determined daily during mobilization by direct immunofluorescence and cytofluorimetric analysis. Plasma cells were identified by the high expression of the CD38 antigen.¹⁸ One hundred microliters of heparinized whole blood or 2-3 ´ 10⁶ cells from the leukapheresis collections were incubated with 10 l of PE-conjugated anti-CD38 MoAb (Leu-17; Becton Dickinson) or 10 l of anti-CD38 and 10 l of fluorescein (FITC)- conjugated anti-CD138 (B-B4; IQP, Groningen, The Netherlands).¹⁹ RBC lysis, washes and cytofluorimetric analysis were performed as previously described.¹³ Only cells expressing CD38^bright were considered as plasma cells; their plasma cell nature was confirmed by CD38/CD138 coexpression and by the expression of cytoplasmic immunoglobulins (cylg). To evaluate the presence of cylg, the cell membrane was first labeled with anti-CD38 PE: cells were then treated with the FIX & PERM cell permeabilization kit (Caltag Laboratories, San Francisco, CA, USA) and labeled with the appropriate amount of FITC-conjugated goat anti-human lg (Caltag).

Statistical analysis

Values were compared by one-way analysis of variance (ANOVA). The actuarial duration of EFS was plotted as curves according to Kaplan and Meier.²⁰ Differences between the curves were appraised with the log-rank method. The EFS was evaluated by stratifying patients according to the median number of plasma cells reinfused and plotted from the beginning of the treatment. Differences were considered statistically significant when P < 0.05.

Results

CD38⁺ and CD34⁺ cells in PB and PBPC harvests

Plasma cells were consistently detected in PB and PBPC harvests. The number of circulating CD38⁺ and CD34⁺ cells was evaluated daily from day 10 to day 13 after cyclophosphamide. Absolute number of CD38⁺ cells was 41.1/l (range 0.5-162.8); percentage was 0.29 (range 0.1-2.9). Peripheral CD34⁺ cells were 85.1/l (range 5.7-430.5); percentage was 0.45 (range 0.05-3.8). The absolute number and percentage of reinfused CD38⁺ and CD34⁺ cells were determined after the first melphalan course and at the end of the treatment. Data are shown in Table 1.

Response rate and CD38+ reinfusion

Sixty-one out of 64 patients were evaluable for response at the end of the intensified regimen (three were non-secretory MM). Also patients unresponsive to DAV underwent the intensified regimen. Twenty-nine achieved CR (47%), 23 PR (38%), and nine (15%) did not respond (NR). At the end of treatment, those in CR had received 3.62 ´ 10⁶/kg (range 0.6-30.9) CD38⁺ cells, those in PR 5.65 ´ 10⁶/kg (range 0.6-19.8) CD38⁺ cells, and finally NR patients 2.9 ´ 10⁶/kg (range 0.78-11.2) CD38⁺ cells (Figure 1). Thus, the amount of reinfused plasma cells did not correlate with the response to chemotherapy (P = 0.44).

Event-free survival and CD38+ reinfusion

Patients were stratified into two groups according to the median number of reinfused plasma cells. Their clinical characteristics and prognostic factors were not statistically different (Table 2). Median EFS was 34.2 months for 32 patients who received less than 4.85 ´ 10⁶/kg CD38+ cells and 36.4 months for 32 patients who received more than 4.85 ´ 10⁶/kg CD38⁺ (Figure 2). No statistical difference in terms of EFS was observed between the two groups (P = 0.7). Median follow-up was 30.1 months. Median overall survival was not reached.

Discussion

Reinfusion of neoplastic cells during autotransplant may be related to disease recurrence, especially in hematological diseases such as various leukemias and MM, where both hematological progenitor and neoplastic cells infiltrate BM. Plasma cells and progenitor cells move into PB during mobilization procedures and are collected by leukapheresis. However, we found no correlation between the number of reinfused plasma cells, response to chemotherapy and patient outcome.

Different techniques have been applied to clear contaminating plasma cells from BM or PBPC harvests, including the use of monoclonal antibodies recognizing plasma cells, or CD34⁺ positive selection.^21,22 However, the few clinical data on plasma cell reinfusion after high-dose chemotherapy do not support use of these approaches. Barlogie's group evaluated the role of several parameters on outcome in patients treated with high-dose chemo-radiotherapy and rescued with BM progenitor cells. The percentage of BM plasma cells was not a prognostic factor.²³ Similar results have also been reported by the Intergroup Français du Myeloma (IFM).²⁴ They found that response rate and survival of three groups of patients supported, after melphalan 140 mg/m² plus TBI, with BM progenitor cells, PBPC, and purified CD34⁺ cells were not statistically different. Even highly purified hematopoietic progenitor cells did not result in a higher proportion of patients reaching CR.

Three years ago we started a pilot study to evaluate the toxicity and efficacy of an intensified approach in elderly MM patients. This included PBPC mobilization with cyclophosphamide and repeated courses of melphalan 100 mg/m² supported by PBPC reinfusion (MEL100 protocol). Seventy-one patients with a median age of 63 years entered this study, and the outcome was compared with a similar historical control group matched for age and ₂-microglobulin.¹⁷ Response to chemotherapy was strikingly increased in the intensified group (47% vs 5%), and survival was significantly prolonged. The procedure was well tolerated and suitable for the treatment of elderly patients. Sixty-four of these patients have now been analyzed for the role of reinfused plasma cells contaminating PBPC harvests.

Previous molecular analyses showed that neoplastic plasma cells are present in virtually all PBPC harvests.¹⁴ Molecular analysis is based on polymerase chain reaction (PCR) amplification of the complementary determining region III (CDRIII) of the immunoglobulin heavy chain gene and allows the detection of up to 10^-5-10^-6 tumor cells; however, CDRIII sequencing and specific primers and probes are required for each individual patient. Thus, PCR is a complex technique that recognizes very low numbers of neoplastic plasma cells, but does not allow their quantitation.

Cytofluorimetric analysis is a less sensitive technique, that detects up to 10^-3-10^-4 plasma cells, but does not prove that they belong to the tumor clone. When the cytoplasmic immunoglobulin kappa/lambda ratio is clearly unbalanced, the presence of monoclonal plasma cells is confirmed. However, molecular study consistently showed the presence of neoplastic cells even in patients with a normal cytofluorimetric kappa/lambda ratio (data not shown). Thus, both these techniques are unreliable in identifying the exact number of contaminating tumor cells. Cytofluorimetric analysis is preferred in large clinical studies for the easy quantification of contaminating plasma cells, containing various numbers of monoclonal elements.²⁵

The number of reinfused plasma cells in our protocol is surprisingly high. At the end of the MEL100 courses, patients had received up to 30.94 ´ 10⁶/kg plasma cells, although no relationship between the amount of reinfused plasma cells and outcome is apparent. Patients receiving a high number of plasma cells may reach CR, whereas others receiving a minimal amount may be resistant to the treatment or characterized by a shortened remission duration.

Plasma cells may be regarded as end stage cells, unable to reseed the BM. Myeloma stem cells may be located upstream along the B cell differentiation pathway. We have previously described the presence of immature B cells with heavy-chain immunoglobulins expressing the same CDRIII as neoplastic plasma cells.²⁶ These cells can be considered as myeloma precursors, and their proportion in the myeloma cell population varies from patient to patient. Thus, the number of myeloma precursors, rather than the number of plasma cells, could be related to outcome. However, positive selection of CD34⁺ cells could remove myeloma precursors, but their use after high-dose chemotherapy did not result in an obvious clinical advantage.²⁷ The chemoresistance of myeloma cells is a more likely explanation: their number still present in vivo is so high that the reinfused cells do not influence outcome. It has been calculated that at least 10⁹ myeloma cells are left after autotransplantation in remission patients. Thus, no effect can be expected on patient outcome by the reinfusion of a number of plasma cells 2 to 3 logs lower than the number of residual plasma cells.²⁷

Whatever the biological explanation of the lack of correlation between the number of reinfused plasma cells and patient outcome, our observation suggests that current in vitro purging techniques are unlikely to prevent myeloma recurrence and improve patient prognosis.²⁸ It appears, therefore, that the goal remains in vivo tumor cell eradication with the development of more effective treatments or alternative strategies, such as idiotype vaccination or dendritic cell infusions.²⁹

Acknowledgements

This work was supported in part by Associazione Italiana Ricerca Cancro (AIRC), Associazione Italiana Leucemie (AIL), and Ministero Universitá e Ricerca Scientifica e Tecnologica (MURST).

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Figure 1 Number of reinfused plasma cells (CD38⁺ ´ 10⁶/kg) and response to intensified chemotherapy with PBPC support. Of the 61 MM evaluable patients, nine did not respond (NR), 29 reached complete remission (CR) and 23 partial remission (PR) (see text for definitions). Each dot represents the amount of plasma cells reinfused in each patient according to response to chemotherapy. Horizontal lines represent median CD38⁺ ´ 10⁶/kg value.

Figure 2 Event-free survival (EFS) according to the number of plasma cells (CD38⁺ ´ 10⁶/kg) contaminating the PBPC reinfused after intensified chemotherapy. EFS was 34.2 months for patients receiving less than 4.85 ´ 10⁶/kg CD38⁺ cells; 36.4 months for those receiving more than 4.85 ´ 10⁶/kg (P = 0.7).

Table 1  Plasma cell contamination (CD38⁺) in PBPC (CD34⁺) harvests reinfused in MM patients after intensified chemotherapy

Table 2  Patient characteristics according to the number of reinfused plasma cells (CD38⁺) after intensified chemotherapy