Stem Cell Procurement

Hematopoietic stem cell mobilization with intravenous melphalan and G-CSF in patients with chemoresponsive multiple myeloma: report of a phase II trial

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Summary:

Multiple myeloma (MM) is an incurable hematologic malignancy for which autologous hematopoietic stem cell transplantation (HCT) is a standard therapy. The optimal method of stem cell mobilization is not defined. We evaluated intravenous melphalan (60 mg/m2), the most effective agent for MM, and G-CSF (10 μg/kg/day) for mobilization. End points were safety, adequacy of CD34+ collections, MM response, and contamination of stem cell components (SCC). In total, 32 patients were mobilized. There were no deaths or significant bleeding episodes; 14 patients (44%) required hospitalization for neutropenic fever. Median days of grade 3 or 4 neutropenia or thrombocytopenia were 7 (2–20) and 8 (3–17). Median mobilization days, CD34+ cells/kg and total leukaphereses were 16 (12–30), 12.1 million (2.6–52.8), and 2 (1–5) respectively. Four patients (12.5 %) failed to achieve the target of 4 million CD34+ cells/kg in five leukaphereses. Reduction in myeloma was seen in 11 patients (34%) with 3 (9%) achieving complete response; 15 (47%) maintained prior responses. Estimated MM contamination per SCC (N=48) was 0.0009% (range 0–0.1) and 0.21 × 10 4 cells per kg (range 0–41.2). Increased contamination was associated with increased patient age. This strategy for mobilization is feasible, frequently requires hospitalization and transfusion, and controls disease in most patients.

Main

Multiple myeloma (MM) is diagnosed in approximately 14 000 patients annually in the USA accounting for 10–15% of all hematologic malignancies. Although new therapies are in use, MM remains incurable with a median survival of about 4 years.1,2 Autologous hematopoietic cell transplantation (HCT) is standard therapy especially in younger patients.3,4,5 A recent phase III trial showed a survival advantage of 10 months in patients who underwent HCT, with a trend toward improved survival in patients with high-risk disease (β2 microglobulin >8.0 mg/l).6 All patients eventually relapse and sources of relapse include minimal residual disease post-HCT and possibly infusion of contaminating myeloma cells at the time of stem cell infusion.

The optimal method for stem cell mobilization has not been established for myeloma. Ideally, it should combine the collection of 4–10 million CD34+ cells per kg, minimal clonotypic contamination, and tumor reduction with limited toxicity. This target range for CD34+ cell collection is based on the current practice of collecting sufficient CD34+ cells to perform two or three HCT, including the option of tandem HCT as well as salvage therapy after relapse from tandem HCT. Factors that influence the collection of blood stem cells in myeloma patients include the amount of marrow infiltration by disease, amount of prior radiotherapy and number of previous chemotherapy regimens.7

Clonotypic cells circulate in the blood of myeloma patients and are routinely found in blood stem cell components (SCC) if sensitive reverse transcriptase (RT)-PCR is used;8,9 however, whether these clonotypic cells have proliferative potential remains unclear.10,11,12 A phase III trial testing CD34-selected autografts in myeloma patients failed to show a survival advantage compared to unselected autografts; however, the optimal conditioning regimen (tandem transplant with high-dose melphalan) was not employed in that trial.13,14,15 Furthermore, ample evidence exists to support the view that patients receiving autografts with increasing amounts of tumor cell contamination relapse frequently and have shorter remission durations, although this could reflect tumor burden at the time of HCT.16,17,18,19,20,21,22,23

Melphalan is the most effective single agent in the treatment of myeloma; its chronic use can be toxic to stem cells and can cause treatment-related leukemia.24,25 Perhaps, that is why it has not commonly been used for stem cell mobilization. We report the results of a clinical trial employing melphalan as both a stem cell mobilizing and a therapeutic agent. We show that intravenous melphalan (60 mg/m2) and G-CSF can be used for successful stem cell mobilization in patients with chemoresponsive myeloma; however, the dose of melphalan we used (60 mg/m2) is associated with frequent hospitalization and requires transfusion support. We also show that melphalan for mobilization also is able to reduce myeloma burden; that, using a sensitive and novel approach for assessing clonotypic contamination,18 SCC mobilized with melphalan and G-CSF contain minimal clonotypic contamination and that increased clonotypic contamination is associated with increased patient age.

Patients, materials and methods

Clinical trial

Patients 18 years of age or older en route to HCT and not previously mobilized for stem cell collection were eligible for this phase II Institutional Review Board (IRB)-approved trial, provided that they completed no more than 3 months prior to enrollment. Performance status 3 (ECOG) was acceptable provided that the poor performance status was due to bone disease or neuropathy related to myeloma. Criteria for exclusion included >3000 cGy prior total radiotherapy, >200 mg prior oral melphalan, previous stem cell mobilization or transplant, or significant cardiac or comorbid disease that would preculde an HCT. All patients provided written and informed consent.

Patients received intravenous melphalan at 60 mg/m2 as a single dose and began G-CSF 10 μg/kg/day subcutaneously on the day after chemotherapy. They received prophylaxis with ciprofloxacin 500 mg b.i.d. and fluconazole 100 mg qd, and were transfused for hemoglobin <8.0 g/dl or platelets <20 000/μl. Stem cell leukopheresis began when the leukocyte count on recovery from nadir exceeded 5000/μl. Leukapheresis continued until the target dose of 4 × 106 CD34+ cells per kg was achieved or to a maximum of five leukaphereses. Myeloma was evaluated in each patient before and after stem cell mobilization by serial M protein studies and bone marrow aspirates and biopsies.

The primary endpoints of the trial were safety and achieving a target collection of 4 × 106 CD34+ cells per kg in five leukaphereses. Secondary end points included the response of the myeloma to the melphalan and assessment of clonotypic contamination of SCC. Toxicities were scored using National Cancer Institute clinical toxicity criteria.26,27

Response of the myeloma to melphalan was described as maintained if disease status did not change. A reduction of >50% in the M protein was described as a response to melphalan mobilization, while a complete response required that serum and urine immunofixation be negative and the bone marrow biopsy be normal (ie no evidence of clonal plasma cells as assessed by staining for CD138, kappa and lambda). An increase of >25% in the M protein was considered progressive disease, as was occurrence of new lytic lesions or soft tissue plasmacytomas heralded by new symptoms.

Assessing clonotypic contamination

Bone marrow and SCC specimens were obtained for molecular studies as previously described.18 Clonal immunoglobulin (Ig) VL and VH genes were identified as previously, using PCR primers for consensus VH and Cα or Cγ regions and for Vκ and Vλ subgroups.18,28,29,30 Representative samples were obtained from each SCC without manipulation. To estimate the percent of clonotypic cells, limiting-dilution PCR with patient-specific CDR1/CDR3 primers was performed18,28,29,30,31,32,33,34 with DNA from 2 × 105 cells per SCC serially log-diluted (ie 10-fold) with buffy-coat DNA (limiting-dilution PCR). Specimens giving all negative results were assessed by RT–PCR, whereas specimens giving positive results underwent limiting-dilution PCR. PCR conditions were as previously described; annealing temperatures ranged from 50 to 64°C, depending on primer pair. The percent of clonotypic cells per SCC was calculated as previously described.18 The amount of contamination was estimated by multiplying the percent of clonotypic cells (using the higher of the VH or the VL estimate) by the total number of mononuclear cells collected per kg.

Statistics

Univariate analysis was conducted in order to find the association between achieving or failing to achieve the targeted dose of 4 × 106 CD34+ cells per kg in a maximum of five collections and other clinical or treatment variables, including gender, stage of disease, ECOG performance status, previous melphalan and radiation exposures, hemoglobin, β2 microglobulin, LDH, and CRP.35 In the univariate analysis, the association was assessed by Fisher's exact test, the Wilcoxon test, and Spearman's correlation coefficient.

Correlations between the two approaches to estimating SCC contamination were evaluated by paired t-test. Correlations between levels of contamination and clinical or treatment variables were evaluated by the Wilcoxon or Mann–Whitney tests, and Spearman's correlation coefficient.

Results

Patients

In total, 32 patients were enrolled from September 2000 to January 2003; clinical characteristics are shown in Table 1. The median age was 57 years (range 33–73) and 18 (56%) were men. In total, 24 (75%) had normal cytogenetics, two (6%) had chromosome 13 deletions, three (10%) had trisomy or tetrasomy of chromosome 11, and one had both 11 and 13 abnormalities. Initial induction chemotherapy included VAD (vincristine, doxorubicin and dexamethasone), pulse dexamethasone, TAD (thalidomide, doxorubicin and dexamethasone), C-VAMP (cyclophosphamide, vincristine, doxorubicin and methylprednisolone), DVD (Doxil, vincristine and dexamethasone), and melphalan and prednisone.

Table 1 Baseline characteristics

Stem cell collections

Four patients (12.5%) failed to reach the target dose of 4 × 106 CD34+ cells/kg in 5 days of leukapheresis. Of these, one had plasma cell leukemia, two had received prior thalidomide up to the day of i.v. melphalan, and one was severely ill with parenchymal brain lesions, likely infectious in origin. Of these, two had sufficient cells collected with a sixth leukapheresis and two were collected again with G-CSF mobilization (all four subsequently underwent SCT). The median number of days until initiation of leukapheresis was 16 (range 12–30). The median number of CD34+ cells/kg collected was 12.1 million × 106 (2.6–52.8). The median number of leukaphereses required to reach the target of 4 × 106 CD34+ cells/kg was 2 (1–6). Using univariate analysis the success or failure to collect stem cells, the CRP level was the only variable significantly different (higher) in patients who failed mobilization vs those who succeeded (P=0.02).

Responses

In total, 15 patients (47%) had maintained responses while eight responded with >50% M protein reductions and three had complete responses. Five (16%) had progressive disease and one had an inadequate bone marrow for assessment. Of the 32 patients, 30 subsequently underwent HCT. Two patients did not, a 36-year-old woman who died secondary to infection, respiratory failure and progression of disease 4.5 months after melphalan mobilization, and a 72 year-old woman who was hospitalized with headaches while neutropenic. She was found to have parenchymal brain lesions that were culture-negative and responded to treatment for both toxoplasmosis and cryptococcus. She never underwent a transplant.

Toxicities

All patients experienced grade 3 or 4 neutropenia. The median number of days of grade 4 neutropenia was 7 (0–19). In total, 16 patients (50%) experienced grade 3 or 4 anemia requiring transfusion. The median number of red blood cell transfusions required was 1 (range, 1–12). All patients experienced grade 3 or 4 thrombocytopenia and 28 (88%) required platelet transfusions; the median number of platelet transfusions was 2 (0–18). In total, 14 patients were hospitalized, 12 for neutropenic fever, one for congestive heart failure, and one for fever without neutropenia. Of those admitted with fever, bacteremia was documented in five (coagulase-negative Staphylococcus (n=3), Acinetobacter (n=1) and a gram negative bacillus (n=1)), and respiratory viruses in four, including respiratory syncytial virus and parainfluenza. The median number of days of hospitalization was 9 (3–50). One patient was in ICU for 12 days.

Clonotypic contamination

We evaluated 48 SCC from 28 patients, including 28 and 19 from the first and second leukapheresis, respectively, and one from a third leukapheresis. In total, 22 of the patients had heavy and light chain components to their monoclonal proteins (14 IgG, seven IgA, one IgM), and in 18 of 22 cases we were able to identify both heavy and light chain clonal genes. In four cases (three VL and the single IgM) we were unable to do so. Six patients had light chain myeloma (5κ, 1λ) and in all cases clonal genes were identified. All genes identified showed evidence of somatic hypermutation.

For 18 patients both VL and VH primers were designed; however, two of the VH primer sets continued to amplify bands similar in size to the target amplicon, despite using buffy-coat DNA from three normal donors as substrate and repeated redesign of primers (Most target amplicons were 220 bp, ranging from 159 to 255 bp). Therefore, those VH primer sets were deemed insufficiently specific for further use. For 10 patients, only VH (n=3) or VL (n=7) primers were designed; all passed specificity testing. In sum, 44 of 46 primer sets passed specificity testing (25 VL and 19 VH), and all of these 44 were found to have sensitivities <0.01%.

Results are available for all 48 SCC. In total, 16 VH primer sets passed testing for the 21 patients with IgG or IgA paraproteins, and all had companion VL primer sets, allowing the evaluation of 29 SCC for contamination by both approaches. Results for these 29 SCC were highly correlated (Figure 1; r2=0.91, P0.01). Of these, SCC successfully analyzed by both VL and VH PCR (n=29), 31% (9/29) had identical estimates by both methods; in six of the concordant cases the estimate was zero or none detected. The more sensitive RT–PCR assays in these six cases were positive by both methods (n=2), negative by both methods (n=3), and positive by VL and negative by VH PCR (n=1). Of the 48 SCC, eight were negative by DNA-based PCR.

Figure 1
figure1

In total, 29 SCC from 16 patients were analyzed by both VL and VH PCR. In (a), the values are shown as percentages of clonal MM cells based on limiting-dilution PCR results and were highly correlated (r2=0.91, P0.01, paired t-test, two-tailed). Means were 0.006% (horizontal lines). In (b), log10 values of the number of MM cells per kg are shown, and the correlation between the methods is apparent in the distribution of points.

VL and VH estimates differed for 69% of the SCC (20/29); in these instances, the higher value was employed as the estimate of contamination. VH PCR was significantly higher for 11 (2.3 vs 0.31 × 104 cells per kg, P<0.01 by one-tailed paired t-test), whereas VL PCR was higher for nine SCC (5.0 vs 4.2 × 104 cells per kg, P=0.07). Of the remaining 19 SCC, 14 could be evaluated with VL, and five with VH, primer sets only. For the 48 SCC assessed, median estimated clonotypic contamination was 0.0009% (0–0.1) or 0.21 × 104 cells per kg (0–41.2).

By univariate analysis, the presence or absence of contamination (dichotomous variable) did not correlate with stage of disease, previous thalidomide therapy, CRP, β2 microglobulin, or LDH. The presence of clonotypic contamination correlated with increasing age; those whose SCC contained clonotypic contamination were significantly older (P=0.02) (Figure 2).

Figure 2
figure2

The median age of patients with detectable contamination by DNA-based PCR (PCR-pos, n=22, filled circles) was 58 while that of patients with no detectable contamination (PCR-neg, n=6, open circles) was 48.5 years. The difference was significant (P=0.02, Mann–Whitney, one-tailed).

Discussion

Melphalan is the most effective single agent for the treatment of myeloma and demonstrates a dose-response.36,37 Several investigators have proposed using melphalan for stem cell mobilization in the past, and several studies have examined its use for mobilization in myeloma patients.38,39,40,41 In one such study, 17 of 21 patients were able to have more than 2.5 × 106 CD34+ blood stem cells collected per kg.38 In the other, 54 of 60 minimally pretreated patients had sufficient numbers of stem cells collected successfully (defined as more than 2.5 × 106 CD34+ blood stem cells per kg) after i.v. melphalan.39 All 60 patients had received two cycles of VAD, followed 1½ to 2 months later by 70 mg/m2 of intravenous melphalan with G-CSF at 5 μg/kg/day starting 4 days later. The median CD34+ cell yield was 4.2 × 106/kg (range, 2.5–11.5 × 106/kg) in two leukaphereses (range, 1–5).

In this study, although we attempted to improve stem cell collection by using G-CSF at 10 μg/kg, we did not begin collections until the total leukocyte count exceeded 5000/μl (had we used circulating CD34+ cells to guide collection, we may have been able to improve leukapheresis even further). We were nevertheless able to define the efficacy and toxicities of i.v. melphalan as both a mobilizing and therapeutic agent. Melphalan and G-CSF in this study provided effective mobilization; sufficient CD34+ cells were collected in the majority of patients to allow three HCT. Although the sample size is small, the correlation of elevated premobilization C-reactive protein levels with failure to achieve the target CD34+ cell dose suggests that baseline inflammation or increased IL-6 affected melphalan mobilization.42 Since all four patients in this category were subsequently collected and transplanted, the relevance of the association remains unclear.

Melphalan and G-CSF also provided additional therapeutic benefit. A third of patients had further disease reduction, including 10% who achieved complete response, as the result of the therapeutic effect of melphalan mobilization. In comparison to cyclophosphamide for mobilization, i.v. melphalan appears at least as effective and perhaps more so. In one of the few studies that examined myeloma response in mobilization, a median 21% reduction in paraprotein (and no complete responses) was observed in 105 chemoresponsive myeloma patients receiving 7 g/m2 of cyclophosphamide with 300 μg/day G-CSF for mobilization.9 With respect to the toxicities of i.v. melphalan and cyclophosphamide for mobilization, we compared this series with 15 consecutive chemoresponsive myeloma patients mobilized with cyclophosphamide (3000 mg/m2) and G-CSF at our center. Although not randomized, this comparison demonstrated that patients mobilized with cyclophosphamide had significantly fewer days of mobilization (10.0 vs 16.5, P0.01) and fewer collections (1 vs 2, P=0.02) than those receiving i.v. melphalan. Total CD34+ cells per kg collected were not significantly different.

Toxicities were notable. Although there were no treatment-related deaths, 44% of patients required hospital admission, usually for neutropenic fever, and all patients required transfusion support. The duration of the mobilization period (median 16 days) is a significant consideration with respect to patient tolerance and safety, particularly in comparison to the 10 days typical of mobilization with cyclophosphamide and G-CSF, or the 6–7 days typical of mobilization with G-CSF alone. It is clearly possible that melphalan for mobilization at the dose used (60 mg/m2) could result in toxicities that might markedly delay HCT.

In addition, in this study, we also asked what impact melphalan mobilization had on clonotypic contamination in the SCC collected and what variables influenced myeloma cell contamination of SCC. It is reasonable to assess SCC contamination as part of the development and use of novel mobilization strategies in patients with hematologic malignancies headed for HCT. We have previously reported data indicating that melphalan-mobilized SCC contain minimal clonotypic contamination and that the assessment of contamination is enhanced by the method we employed, using Ig VL and VH patient-specific PCR.18 We have also previously suggested that melphalan-mobilized SCC contain fewer clonotypic cells than SCC mobilized with cyclophosphamide or ifosfamide and G-CSF.18

In this report we further substantiate the claim that clonotypic contamination is minimal and, furthermore, that both Ig VL and VH patient-specific PCR approaches are useful. Indeed, as newer agents with activity in myeloma are incorporated into induction therapy, the clinical complete response (CCR) rate will increase. And, as the use of tandem high-dose melphalan HCT and novel maintenance and immunotherapy strategies push the CCR rate to 50% and higher, the challenge of following patients whose incurable disease has become undetectable by conventional testing will become more salient.43,44 PCR-based techniques such as the one we employ may then become more relevant and useful in the assessment of minimal residual disease.

Of particular interest is the association between clonotypic contamination and patient age. Such an association has not been previously described and merits further investigation. Less toxic induction therapy and mobilization regimens (such as G-CSF alone) are usually used in patients older than 70. Contamination of SCC has not been systematically studied in those patients. The impact of patient age on CD34+ cell mobilization in myeloma has previously been evaluated; low yields and delayed platelet recovery both appear to be associated with increased age.45 Our data suggest that concerns regarding SCC contamination in elderly patients may be warranted as well.

Finally, autologous HCT does not yet cure myeloma, although outcomes are improving. For this reason, it may be advantageous to provide flexibility of timing of autologous HCT in a way that more appropriately reflects the underlying biology of the disease in selected patients.46 Intravenous melphalan for mobilization may afford patients who wish to delay HCT an advantage since it also treats the disease; over three quarters of patients on this trial had stable disease or a further response to melphalan mobilization. Nevertheless, although we have shown that i.v. melphalan can be used effectively to mobilize adequate numbers of CD34+ cells with minimal clonotypic contamination, nearly half of the patients required hospitalization and all required transfusion during the 2–3-week period of mobilization. Despite these toxicities, selected patients seeking to defer HCT may benefit from melphalan mobilization, provided the risks are fully appreciated.

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Acknowledgements

We thank the MSKCC Cytotherapy Laboratory for assistance with stem cell samples and the Memorial Hospital nurses in the Adult Day Hospital and in-patient services who cared for our patients. This work was supported by NIH Grant CA05826, FDA Grant R01-002174-02 (RLC), the Graziano Fund, the Multiple Myeloma Research Foundation, the Mel Stottlemyre Myeloma Research Fund, the Donald Stein Myeloma Research Fund, the Werner and Elaine Dannheiser Fund for Research on the Biology of Aging of the Lymphoma Foundation, and Amgen.

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