Originally published as JCO Early Release 10.1200/JCO.2007.12.8298 on December 17 2007

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High Numbers of Tumor-Associated Macrophages Have an Adverse Prognostic Value That Can Be Circumvented by Rituximab in Patients With Follicular Lymphoma Enrolled Onto the GELA-GOELAMS FL-2000 Trial

Danielle Canioni, Gilles Salles, Nicolas Mounier, Nicole Brousse, Marie Keuppens, Frank Morchhauser, Thierry Lamy, Anne Sonet, Marie-Christine Rousselet, Charles Foussard, Luc Xerri

From the Department of Pathology, Assistance Publique-Hopitaux de Paris, Hôpital Necker-Enfants Malades; Université Paris-Descartes, Paris; Department of Hematology, Hospices Civils de Lyon; Université Lyon 1, Lyon; Department of Hematology, Centre Hospitalo-Universitaire, Nice; Department of Hematology, Centre Hospitalier, Lille; Department of Hematology, Centre Hospitalier, Rennes; Departments of Pathology and Hematology Hôpital d'Angers, Angers; Department of Bio-Pathology, Institut Paoli-Calmettes; Université de la Méditerranée, Marseille, France; and Department of Hematology, Hopital Universitaire de Mont-Godinne, Yvoir, Belgium

Corresponding author: Danielle Canioni, MD, Department of Pathology, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, 75015, Paris, France; e-mail: danielle.canioni{at}nck.ap-hop-paris.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
Purpose High amounts of intratumoral macrophages have been shown to correlate with poor prognosis in patients with follicular lymphoma (FL) treated with chemotherapy without rituximab. We tried to establish whether intratumoral macrophage count (MC) definitely is able to predict the outcome of FL patients in the rituximab era.

Patients and Methods We analyzed immunohistochemical CD68 expression in 194 FL patients from the FL-2000 trial, randomly assigned to receive cyclophosphamide, doxorubicin, etoposide, prednisolone, and interferon (CHVP-I) or rituximab plus CHVP-I. Immunohistochemistry was performed on paraffin sections using anti-CD68 KP1 antibody, and stained macrophages were scored on high-power field (hpf) in either intrafollicular (IF) or extrafollicular (EF) areas.

Results For IF MC, the best cutoff point was estimated at 10 macrophages/hpf. Low IF MC was significantly associated with a better event-free survival (EFS; P = .011). However, this effect was observed only in the CHVP-I arm (P = .012) and not in the rituximab plus CHVP-I arm. Using a cutoff of 15 IF MC, we found no significant association with EFS. For EF MC, fewer than 22 macrophages/hpf were associated with better EFS in the CHVP-I arm (P = .02) but not in the rituximab plus CHVP-I arm.

Conclusion These results show that MC can predict outcome of FL patients and that rituximab is able to circumvent the unfavorable outcome associated with high MC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
Follicular lymphoma (FL) is usually considered as an indolent non-Hodgkin's lymphoma, with a median survival of 8 to 10 years.¹ Various prognosis predictors of debated value are used to predict the outcome of FL patients. One of the best parameters is the Follicular Lymphoma International Prognostic Index (FLIPI), including age, stage, number of nodal sites, hemoglobin level, and lactate dehydrogenase (LDH) as predictors of survival.² However, identification of patients at increased risk of treatment failure remains a crucial challenge.

Several retrospective studies have tried to define tumor-related biologic parameters that influence survival.^3-8 This effort recently culminated in gene profiling analyses, which suggested that survival of patients with FL is correlated with molecular features of nonmalignant immune cells present in the tumor.^8-10

Because of the complexity of molecular tests in routine practice, a possible solution would be to focus on easily reproducible immunohistochemical (IHC) markers. In this respect, high numbers of FOXP3-positive infiltrating T cells were reported to be associated with improved survival,¹¹ but these results remain controversial.¹²

Another candidate biomarker is the number of tumor-associated macrophages (TAMs). In fact, IHC quantification of TAMs using an anti-CD68 KP1 antibody was reported to be a surrogate for microarray gene signatures.¹³ However, this pioneering study was not fully confirmed in other series of patients.^12,14-16

In the recent FL-2000 study focusing on FL patients with high tumor burden, the Groupe d'Etude des Lymphomes de l'Adulte (GELA) demonstrated a significant survival advantage for a therapy associating rituximab with cyclophosphamide, doxorubicin, etoposide, prednisolone, and interferon (CHVP-I), when compared with CHVP-I alone.¹⁷ In the present study, we used anti-CD68 KP1 immunostaining to evaluate the value of TAM as a prognostic marker in patients treated prospectively in the FL-2000 study.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
The patients studied for CD68 expression at the protein level were a subset of the 358 patients entered onto the FL-2000 trial, a prospective multicenter study conducted by the GELA between May 2000 and May 2002.¹⁷

FL-2000 Protocol
Patient selection. Patients were included onto this trial on the basis of a histologically proven diagnosis of FL grade 1, 2, or 3A. Patients with histologic grade 3B FL were excluded. Histologic slides were reviewed by three pathologists. Patients were classified according to the WHO classification using histologic analysis of samples stained with hematoxylin and eosin, associated with usual IHC staining (CD20, CD5, CD10, and bcl2 antibodies).

Patients were eligible if they were between 18 and 75 years of age and had untreated FL with high tumor burden. They were required to have at least one of the following criteria: presence of "B" symptoms; Eastern Cooperative Oncology Group performance status 1; LDH more than the normal value; β₂-microglobulin 3 mg/L; largest tumor 7 cm, three distinct nodes 3 cm; or serous effusion, compression, or symptomatic splenomegaly. Patients were not eligible if they had CNS involvement, active cancer, or any serious active concomitant disease, or if their general status did not permit the administration of 12 courses of CHVP (a combination of cyclophosphamide 600 mg/m² on day 1, doxorubicin 25 mg/m² on day 1, etoposide 100 mg/m² on day 1 and prednisolone 40 mg/m² per day for 5 days). Patients were also excluded if they had a cardiac contraindication to doxorubicin therapy, positive serology for the HIV, or unresolved hepatitis B virus infection.

Eligible patients were randomly assigned to treatment by the study coordinating center. Treatment consisted of CHVP (one course a month for six courses + once course every 2 months for six additional courses) associated with interferon alfa-2a (4.5 MU three times a week) for 18 months or six courses (one per month for 6 months) of CHVP associated with 18 months of interferon alfa-2a combined with six infusions of 375 mg/m² of rituximab (days 1 and 8 of courses 3 and 4; day 1 of courses 5 and 6). The trial was approved by our Institutional Review Board and informed consent was obtained from all participants in accordance with the declaration of Helsinki.

Staging and Follow-Up
The stage of the disease was evaluated by physical examination, computed tomography scan of the chest and abdomen, bone marrow biopsy, and other investigations depending on the clinical symptoms. Patients were staged according to the Ann Arbor classification. Eastern Cooperative Oncology Group performance status was assessed and LDH was expressed as the maximum/normal value ratio. Patient response was assessed after the end of induction chemotherapy and was classified according to the International Workshop criteria.¹⁸

Follow-up procedures included physical examination every 3 months for the first 2 years, then every 6 months for 3 years, and then annually. Thoracic and abdominal computed tomography scans were performed every 6 months during the first 2 years and then annually.

IHC and Macrophage Count
Slides of formalin-fixed, paraffin-embedded tissues were stained centrally in the GELA pathology institute with an anti-CD68 antibody (clone KP1, dilution 1:50; DakoCytomation, Glostrup, Denmark) using a standard avidin biotin peroxidase technique and the chromogen 3-amino-9-ethylcarbazole. The number of CD68⁺ macrophages was initially determined using a low-power magnification (x100 magnification). Given that the number of macrophages inside and between the tumoral follicles varied, we decided to score CD68⁺ macrophages in these two distinct areas to obtain both intrafollicular macrophage count (IF MC) and extrafollicular macrophage count (EF MC). Both IF MC and EF MC were estimated separately on high power (x400) magnification from five different representative fields per case (where the staining was the strongest). Each x400 magnification field had 0.19 mm² in surface area. Only CD68⁺ cells displaying macrophagic/histiocytic morphology were counted. To make the determination of MC more objective, the scoring was performed independently by two different pathologists (D.C., M.K.). The interobserver reproducibility was assessed using the difference between the counts of each observer. The mean of the counts from each reader was then calculated and used for the statistical analysis.

Statistical Analysis
All analyses were performed on an intention-to-treat basis. To allow direct comparison with the data given in our previous report,¹⁷ the stopping date of the present analysis was also set at June 1, 2005.

Patient characteristics and response rates were compared by the ² and Fisher's exact tests. Event-free survival (EFS) was measured from the date of random assignment to that of disease progression, relapse, or death as a result of any cause or to the stopping date. Overall survival (OS) was measured from the date of random assignment to either death as a result of any cause or the stopping date. When the stopping date was not reached, the data were censored at the date of the last follow-up evaluation. Survival functions were estimated by the Kaplan-Meier method and compared by log-rank test. Differences between the results of comparative tests were considered significant if the two-sided P value was less than .05.

Because the FL-2000 trial was not stratified on CD68 expression, we controlled for the effects of prognostic factors on outcome due to sampling fluctuation in the treatment groups using multivariate analysis of survival. The potential prognostic factors for survival or relapse were the risk factors in the FLIPI index. A Cox model regression was fitted including FLIPI, MC, and treatment arm as explanatory variables. The interactions between risk factors and treatment were also included in the model. Given that MC was a continuous biologic variable, it was dichotomized by applying the standard split-sample approach. The resulting thresholds were checked by including cubic smoothing splines in the risk function of the Cox model.

Differences between the results of comparative tests were considered significant if the two-sided P value was less than .05. All statistical analyses were performed using SAS 9.13 (SAS Institute, Cary, NC) and Splus 2000 (MathSoft, Cambridge, MA) software.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
CD68 Expression and Baseline Characteristics
The histologic material of 345 of 358 patients enrolled onto the FL-2000 trial was reviewed centrally. A sufficient amount of biopsy specimen was available for 194 patients, which was investigated for CD68 IHC detection. All of these 194 biopsy specimens were obtained at the time of diagnosis, before any treatment. The characteristics of these 194 patients did not differ significantly from those of the overall population on the FL-2000 study in terms of demographic data, baseline characteristics, or clinical factors affecting outcome, including parameters of the FLIPI and treatment (Fig 1; Table 1).

View larger version (129K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Patterns of CD68 immunostaining in follicular lymphoma (FL) samples. (A) The most frequent pattern was as many CD68+ cells in intrafollicular areas than in extrafollicular areas. High-power view (x400) allows the identification of macrophages for counting (A, insert). (B) In other less frequent FL samples, only rare macrophages could be detected, whatever the area.

CD68 Expression and Clinical Outcome
Univariate analysis. The median follow-up was 42.9 months (range, 32.7 to 61.8 months). Three-year survival estimates for all patients were 61% ± 7% for EFS and 91% ± 4% for OS. Response rates are listed in Table 2. There was a significant impact of the addition of rituximab to CHPV-I in terms of complete response (42% for CHVP v 71% for CHVP plus rituximab; P < .001).

View larger version (14K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Relative risk (RR) of event according to (A) intrafollicular (IF), and (B) extrafollicular (EF) macrophage counting (MC). Spline curves show how the RR of event changes as the CD68 count varies. (···) indicates the 95% CI. The RR increased as the CD68 count increased and the best cutoff point was identified at the intersection with the line zero.

View larger version (13K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 3. Event-free survival (EFS) according to intrafollicular (IF) macrophage counting (MC). (A) A high IF MC had a significant adverse effect on EFS for all patients (3-year EFS, 75% ± 10% v 56% ± 8%; P = .01). The prognostic value on EFS of low IF MC was observed mainly in (B) follicular lymphoma (FL) patients who received cyclophosphamide, doxorubicin, etoposide, prednisolone, and interferon (CHVP-I; relative risk [RR] = 1.7; P = .01), (C) but not in those who received CHVP-I plus rituximab (RR = 1.4; P = .15).

Multivariate Analysis
The FL-2000 trial was not stratified on the basis of CD68 staining. Therefore, the apparent effect of CD68⁺ MC on EFS may have been due to sampling fluctuations with a limited number of patients in treatment arms. To counteract this drawback, the impact of CD68⁺ MC on patient EFS was evaluated here by multivariate analysis controlling for the effects of treatment and FLIPI. After adjustment for FLIPI (score 0 to 2 v score 3-5, RR for EFS by FLIPI = 1.9; P = .005), interactions between MC and treatment were significant (P < .05), indicating that treatment efficacy varied according to the pattern of risk factors. Introducing the individual FLIPI criteria with either the number of IF MC or EF MC in the Cox model indicated that the number of nodal sites and the MC remained, together with the treatment arm, the only independent factors retained in the model (not shown).

The prognostic value of EFS was then examined in each treatment arm. A low IF MC (< 10 macrophages/hpf) was observed mainly in FL patients who received CHVP-I (RR = 1.7; P = .01; Fig 3B), but not in those receiving CHVP-I plus rituximab (RR = 1.4; P = .15; Fig 3C). The prognostic value of low EF MC (< 22 macrophages/hpf) on EFS was significant in patients treated with CHVP-I (RR = 1.6; P = .02; Fig 4A) but not for patients treated with CHVP-I plus rituximab (RR = 1.1; P = .31; Fig 4B).

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 4. Event-free survival (EFS) according to extrafollicular (EF) macrophage counting (MC). The prognostic value on EFS of low MC was significant in (A) patients treated with cyclophosphamide, doxorubicin, etoposide, prednisolone, and interferon (CHVP-I; relative risk [RR] = 1.6; P = .02), (B) but not for patients treated with CHVP-I plus rituximab (RR = 1.1; P = .31). hpf, high-power field.

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

The anti-CD68 KP1 clone is an ideal candidate for routine IHC prognostic use, given that the corresponding epitope is highly resistant to usual fixatives. We decided to count TAM using the x400 magnification dry lens, which is recommended for the histologic grading of FL according to the WHO classification.³ In fact, the x1,000 oil lens used in a previous report¹³ requires an inconvenient manipulation in routine practice.

Another major difference between the previous report from Farinha et al¹³ and the present study is that we chose to count TAM on whole tissue sections, rather than on tissue microarray (TMA) sections. Counting on whole tissue sections can be easily done at the time of the initial histologic diagnosis. In addition, it is required for a reliable determination of the respective influence of IF MC versus EF MC. The interval between neoplastic follicles is variable from sample to sample, and thus the relatively small size of TMA cores may hamper the reliable evaluation of five representative IF or EF fields per sample.

These methodologic differences could probably account for the discrepancy between our cutoff values and the previously reported cutoff of 15 macrophages/hpf.¹³ The latter result represented the global count of macrophages, whatever their localization, performed with an oil lens on TMA cores.¹³ In contrast, when we used a cutoff of 15 IF MC in our series, we failed to find a significant association between IF MC and patients' EFS, which indicates that the use of an appropriate cutoff is an important requirement for prognostic purposes and may vary with the procedure of CD68 IHC interpretation and counting.

The variability of cutoff selection, as well as the heterogeneity of the patient characteristics and of the counting procedure, may explain the discrepancy between our findings and previous series of FL samples in which CD68 immunostaining alone had no prognostic value.^12,14-16 In this respect, it must be stressed out that the present report provides data from a prospective cohort of randomly included patients treated with uniform therapy.

Our results are in accordance with previous gene expression profiling studies showing that mRNA downregulation of genes related to macrophage/follicular dendritic cells function predicts a favorable outcome.¹⁰ Our data also agree with observations suggesting that high numbers of macrophages are correlated with inferior survival in epithelial cancers.¹⁹ To the extent that they have been investigated, TAMs have a phenotype and function similar to M2 macrophages, including poor cytotoxicity for tumor cells and promotion of tumor-cell proliferation induced by Th2 cytokines such as interleukin (IL) -4, IL-13, and IL-10.²⁰

In this study, patients with a high MC had significantly lower EFS. Nonetheless, it must be noted that the unfavorable impact of high MC was observed predominantly in patients treated with CHVP-I but not in those treated with CHVP-I plus rituximab. Given that the clinical data regarding presentation and outcomes indicate that the 194 patients analyzed for TAM content were representative of the whole population entered onto the FL-2000 trial, it is unlikely that these results could be related to any selection bias. Instead, they suggest that the beneficial effect of rituximab is optimal in patients with high MC, which suggests that the poor prognosis indicated by high MC in FL patients can be circumvented by rituximab. Alternatively, the reduction in the number of CHVP courses in the rituximab arm (six instead of 12) may also account for the different prognostic value of MC between the two arms.

Our findings thus favor the hypothesis that macrophages may act synergistically with anti-CD20–targeted therapy. Rituximab has been shown to induce apoptosis and to inhibit cell proliferation, but also to exert complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity.²¹ One can speculate that this latter mechanism could be facilitated by a high number of TAMs, and thus explain the diminished beneficial effect of rituximab in patients with low MC compared with those with high MC. This hypothesis is supported by recent studies showing that macrophages not only cooperate with complement and CCL3 in the eradication by rituximab of an established lymphoma xenograft,²² but are also able to kill chronic lymphocytic leukemia cells in the presence of rituximab via antibody-dependent cellular cytotoxicity.²³ Alternatively, it cannot be ruled out that the direct apoptotic action of rituximab against lymphoma cells could be efficient enough to overcome the promotion of B-cell growth by TAMs.

In conclusion, this study confirms the interest in CD68 immunostaining as a routine tool to predict outcome of FL patients with high tumor burden receiving chemotherapy, and highlights the influence of treatment strategies on its reliability. Although these results still have to be translated to a larger group of patients with FL presenting unselected characteristics, they could lead to improvements in risk stratification of FL patients, and thus offer the possibility of better therapeutic management and improved future trial design.

	Authors' Disclosures of Potential Conflicts of Interest

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
Conception and design: Danielle Canioni, Gilles Salles, Nicole Brousse, Luc Xerri

Provision of study materials or patients: Gilles Salles, Nicole Brousse, Frank Morchhauser, Thierry Lamy, Anne Sonet, Marie-Christine Rousselet, Charles Foussard, Luc Xerri

Collection and assembly of data: Danielle Canioni, Gilles Salles, Nicolas Mounier, Marie Keuppens, Luc Xerri

Data analysis and interpretation: Danielle Canioni, Gilles Salles, Nicolas Mounier, Marie Keuppens, Luc Xerri

Manuscript writing: Danielle Canioni, Gilles Salles, Nicolas Mounier, Luc Xerri

Final approval of manuscript: Danielle Canioni, Gilles Salles, Nicolas Mounier, Nicole Brousse, Frank Morchhauser, Thierry Lamy, Luc Xerri

	Appendix

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Appendix REFERENCES

 
The following centers and investigators participated actively in the FL-2000 trial: Salles G. (Hospices Civiles, Lyon, France), Haioun C. (Hopital Henri-Mondor, Créteil, France), Brice P. (Hopital Saint-Louis, Paris, France), Foussard C. (Centre Hospitalier d'Angers, France), Fermé C. (Institut Gustave Roussy, Villejuif, France), Lamy T. (Centre Hospitalier de Rennes, France), Bouabdallah R. (Institut Paoli Calmette, Marseille, France), Lederlin P. (Centre Hospitalo-Universitaire Brabois, Vandoeuvre les Nancy, France), Morschhauser F. (Centre Hospitalier de Lille, France), Rossi J.-F. (Centre Hospitalo-Universitaire Lapeyronie, Montpellier, France), Sebban C. (Centre Leon Berard, Lyon, France), Van Hoof A. (Centre Hospitalier, Bruges, Belgique), Colombat P. (Centre Hospitalier Bretonneau, Tours, France), Deconinck E. (Hopital Jean Minjoz, Besancon, France), Delwail V. (Hopital Jean Bernard, Poitiers, France), Audhui B. (Hopital Pasteur- Hopitaux Civils de Colmar, Colmar, France), Bosly A. (Université Catholique de Louvain, Mont-Godinne, Yvoir, Belgique), Gressin R. (Centre Hospitalo-Universitaire de Grenoble, Grenoble, France), Pignon B. (Centre Hospitalier de Dunkerque, Dunkerque, France), Tilly H. (Centre Henri Becquerel, Rouen, France), Casanovas O. (Hopital Saint Jean, Perpignan, France) Gabarre J (Hopital La Pitié Salpétrière, Paris, France), Maisonneuve H. (Centre Hospitalier Départemental, La Roche sur Yon, France), Blanc M. (Centre Hospitalier de Chambery, Chambery, France), Fruchart C. (Centre François Baclesse, Caen, France), Varet B. (Hopital Necker, Paris, France), Eisenmann J.-C. (Centre Hospitalo-Universitaire de Mulhouse, Mulhouse, France), Morel P. (CHU de Lens, Lens, France), Collignon J (Centre Hospitalo- Universitaire Hutois, Huy, Belgique), Fitoussi O. (Polyclinique Bordeaux Nord Aquitaine, Bordeaux, France), Flesch M. (Fondation Drevon, Dijon, France), Janvier M. (Centre René Huguenin, Saint Cloud, France), Lefort S. (Centre Hospitalier de Brive, Brive la Gaillarde, France), Peaud P.-Y. (Hopital de Valence, Valence, France), Van den Bossche M (Hopital Ste Elizabeth, Namur, Belgique), André M. (Centre Hospitalier Notre Dame, Charleroi, Belgique), Azagury M. (Centre Hospitalier de St Germain, St Germain en Laye, France); Bordessoule D. (Centre Hospitalo-Universitaire Dupuytren, Limoges, France), Bouabdallah K. (Centre François Magendie, Pessac, France), Cailleres S. (Centre Hospitalier du Pays d'Aix, Aix en Provence, France), De Prijck B. (Centre Hospitalier de la Citadelle, Liège, Belgique), Delmer A. (Centre Hospitalo-Universitaire Robert Debré, Reims, France), Dor J.-F. (Hopital La Fontonne, Antibes, France), Lepeu G. (Hopital Henri Duffaut, Avignon, France), Marit G. (Centre François Magendie, Pessac, France), Mineur P., (Hopital St Joseph, Gilly, Belgique), Nedellec G. (Hopital d'Instruction des Armées Percy, Clamart, France), Salles B. (Hopital de Chalon, Chalon sur Saone, France), Solal-Celigny P. (Centre Jean-Bernard, Le Mans, France), Soussain C. (Centre René Huguenin, Saint Cloud, France), Thyss A. (Centre Antoine Lacassagne, Nice), Valenza M. (Centre Hospitalier de Draguignan, Draguignan, France), Wetterwald M. (Centre Hospitalier de Dunkerque, Dunkerque, France).

	ACKNOWLEDGMENTS

 
We thank all of the pathologists who contributed actively to this study by providing biopsy specimens for the included patients; Denitza Muller and Roselyne Delepine for patient data monitoring; and Chafika Coppeaux and Karine Sardin for technical assistance.

	NOTES

 
published online ahead of print at www.jco.org on December 17, 2007.

Supported by the Programme Hospitalier de Recherche Clinique (Hospices Civils de Lyon, PHRC 2000-081) and the Ligue Nationale Contre le Cancer.

Presented in part at the Annual Meeting of the American Society of Hematology, December 9-12, 2006, Orlando, FL.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

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Submitted June 6, 2007; accepted October 11, 2007.

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