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Malignant Lymphomas |
From the Departments of Clinical Hematology (JD, TEG, IG, CH), Pathology and INSERM U617 (PG, CC-B), Biostatistics (FH), Nuclear Medicine (EI, MM), and Radiology (AR) - H. Mondor Hospital, Paris XII University, Assistance Publique-Hôpitaux de Paris (AP-HP), Créteil, France; Departments of Clinical Hematology (CG) and Pathology (JB), St-Louis Hospital, Paris VII University, AP-HP, Paris, France
Correspondence: Corinne Haioun, Service d’Hématologie Clinique, Hôpital H. Mondor, 51, Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil, France. E-mail: corinne.haioun{at}hmn.aphp.fr
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ABSTRACT |
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Design and Methods: We investigated the expression of CD10, Bcl-6, and MUM1 in 81 patients with DLBCL evaluated early with 18FDG-PET. The tumors were classified as GC or nGC using the algorithm of Hans et al. The results of both methods were correlated with the patients’ characteristics and survival.
Results: CD10 was positive in 27/76 (36%), Bcl-6 in 43/74 (58%), and MUM1 in 33/73 (45%) interpretable cases. Thirty-eight (51%) were in the GC group, and 36 (49%) in the nGC group. With a median follow-up of 33 months, estimated 3-year event-free survival (EFS) of the whole population was 67%. There was no influence of GC/nGC phenotype on survival. Three-year EFS was 46% in the early PET-positive group versus 80% in the PET-negative group (p=0.0003).
Interpretation and Conclusions: The prognostic value of GC/nGC phenotype is not confirmed in this heterogeneous series, whereas early PET findings are confirmed to be a powerful predictor of outcome. The impact of treatment decisions based on early PET results should be evaluated.
Key words: diffuse large B-cell lymphoma, germinal center, immunohistochemistry, PET scan, prognosis.
Diffuse large B-cell lymphoma (DLBCL), despite being considered a single entity within the WHO classification of lymphoid tumors,1 is a heterogeneous disease in terms of clinical presentation, histopathology, and outcome. As new treatments appear, there is increasing interest in identifying the patients in whom conventional approach is likely to fail. Risk stratification currently relies mainly on the International Prognostic Index (IPI).2 This approach has proven useful in identifying high-risk patients who could benefit from consolidative high-dose therapy (HDT) after having reached a first remission.3,4 Among other parameters, expression of Bcl-2 can also predict outcome in DLBCL.5,6
Gene-expression profiling in DLBCL has brought an insight into the biological heterogeneity of the disease. Major subgroups were identified: germinal center B cell-like (GC), activated B cell-like or non-GC (nGC), the former showing a better prognosis.7,8 Immunohistochemistry has been evaluated as a surrogate for this molecular classification.9–14 The phenotypic GC and nGC groups are defined by the expression of GC or post-GC stage markers. While using variable definitions, some 9,11,12, but not all studies10,13,14 found a better prognosis for phenotypically-defined GC cases. 18Fluorodeoxyglucose positron emission tomography (18FDG-PET) scanning performed after a few cycles of chemotherapy has been shown to predict treatment outcome.15–20 In our recently published series of 90 patients,15 event-free and overall survival differed significantly between patients with a negative versus positive 18FDG-PET after two cycles of chemotherapy, independently from the IPI score.
Both methods appear promising in order to establish optimal risk-based treatment strategies, but, to the best of our knowledge, they have never been compared within the same patient population. Our objective was to conduct such a comparison in order to determine which method performs better in the clinical setting. We retrospectively studied the expression of Bcl-2, CD10, Bcl-6 and MUM1 on biopsies from 81 consecutive patients with DLBCL who had been prospectively investigated with early 18FDG-PET. We aimed at evaluating the prognostic impact of immunophenotype (phenotypic classification into GC and nGC groups and Bcl-2 expression), along with that of early 18FDG-PET imaging.
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Design and Methods |
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Inclusion criteria were age under 80 years, a centrally reviewed diagnosis of DLBCL, measurable disease, ECOG performance status of 0 to 2, and availability of paraffin-embedded tissue for immunohistochemical analysis. Patients with central nervous system involvement, positive human immunodeficiency serology, concomitant or previous cancer (except carcinoma in situ of the cervix), or any serious concomitant disease contraindicating chemotherapy were not included.
According to the declaration of Helsinski, the protocol was approved by our Institutional Review Board and all patients gave written informed consent. The study was sponsored by the Délégation à la Recherche Clinique of the Assistance Publique – Hôpitaux de Paris.
Pretreatment evaluation and follow-up
Before treatment, all patients were evaluated by physical examination, complete blood counts, routine chemistry including measurement of lactate dehydrogenase (LDH) levels, computed tomographic scan of the thorax, abdomen and pelvis, and bone marrow biopsy. Restaging was performed after the first two and four cycles of induction, at the end of treatment, then every 6 months for 2 years, and then yearly. Responses were classified according to the International Workshop criteria.21 Additionally, all patients underwent whole-body 18FDG-PET examination before starting treatment and after the first two chemotherapy cycles (see section 18FDG-PET).
Treatment
Forty-four patients (54%) were treated within randomized clinical trials conducted by the Groupe d’Etude des Lymphomes de l’Adulte (GELA). Induction treatment always included an anthracycline-based regimen, which was either CHOP (doxorubicin 50 mg/m2 day 1, cyclophosphamide 750 mg/m2 day 1, vincristine 1.4 mg/m2 day 1 and prednisone 40 mg/m2 days 1–5, repeated every 21 days for 4 courses, n=23) or one of the dose-intensified ACBVP (doxorubicin 75 mg/m2 day 1, cyclophosphamide 1,200 mg/m2 day 1, vindesine 2 mg/m2 days 1 and 5, bleomycin 10 mg days 1 and 5 and prednisone 60 mg/m2 days 1–5, every 15 days for four courses, n=48) or AC/ACE (doxorubicin 75 mg/m2 day 1, cyclophosphamide 1,000 mg/m2 day 1, and prednisone 60 mg/m2 days 1–5, for one course followed by three courses repeated every 15 days of the same drugs plus etoposide 150 mg/m2, n=10) regimens. Thirty-three patients (41%) received consolidative HDT after having reached CR, the others received CHOP-based or ACBVP-type3 sequential consolidation. Consolidative HDT was given to younger patients with two or three age-adjusted IPI factors at diagnosis,3,4 within or outside protocols (n=28), and to patients with one age-adjusted IPI factor and high Bcl-2 expression within the LNH98-2 GELA protocol.22 Thirty-seven patients (46%) received rituximab as a part of their treatment. Clinicians taking care of patients were blinded to the results of early 18FDG-PET and treatment decisions were taken only on the basis of conventionnal staging methods.
18FDG-PET
Modalities of 18FDG-PET image acquisition were as previously described.15 Images were interpreted by a consensus of two experienced observers blinded to clinical and radiological data. All foci of abnormal FDG uptake were scored for their extent and intensity using a three-point scale (1=low, 2=moderate, 3=high) within each lymphatic area, organ, and skeletal region. Then, each post-chemotherapy scan was scored as negative or positive. Negative was defined as having no residual abnormal uptake or as having a unique residual site (with an extent score of 1 associated with an intensity score of 1), while all the other previously hyper-metabolic sites were extinguished. This approach was successfully used by Mikhaeel and co-workers in a previous study.23 Positive was defined as having at least one residual site (with an extent score of 1) associated with an intensity score of 2 or 3, or as having two or more residual sites with any score of extent and intensity.
Immunohistochemical studies
All immunohistochemical studies were performed in the same laboratory under standardized conditions. Deparaffinized tissue sections were immunostained with antibodies including CD10 (56C6, Novocastra, Newcastle, UK), Bcl-2 (clone 124), Bcl-6 (P1F6) and MUM1/IRF4 (MUM1p) (DakoCytomation, Glostrup, Denmark) using an indirect immunoperoxidase method with a manual technique (Bcl-2, CD10) or an automated immunostainer (Ventana medical systems, Tucson, AZ, USA) (Bcl-6, MUM1). Antigen retrieval involved microwave heating pretreatment with three cycles of 5 minutes in 0.01M citrate buffer, pH 7.6 for Bcl-2, CD10 and Bcl-6 or in EDTA buffer pH 9 for MUM1. Positivity was rated independently by two observers (PG, JD) and discordant cases were resolved by review on a multiheaded microscope. We used the thresholds of 50% positive cells for Bcl-2 and 30% for CD10, Bcl-6 and MUM1.12, 24 Cases without any internal positive control were scored as not informative for the corresponding antibody. Patients were classified as having GC or nGC disease following the algorithm of Hans et al. (Figure 1A).12
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2 test. Overall survival (OS) was calculated from the date of enrolment to death from any cause or last follow-up. Event-free survival (EFS) was calculated from the date of enrollment to disease progression, relapse, death from any cause or last follow-up.21 Survival curves were estimated using the method of Kaplan-Meier and compared using the log-rank test.25 Multivariate analysis was performed with a Cox proportional-hazards regression model with EFS as the dependent variable.26 Differences between the results of comparative tests were considered statistically significant at a two-sided p<0.05. All statistical analyses were performed using Statistical Application System software (SAS, version 9, SAS Institute, Cary, NC, USA). |
Results |
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Three patients were not evaluated after the first four courses of chemotherapy (induction treatment) because of early death or disease progression. According to conventional staging methods, 62/78 (79%) patients were in complete remission (CR) or CR-unconfirmed (CRu), 9/78 (11%) were in partial remission (PR), and 7/78 (9%) had stable or progressive disease following induction. Treatment had to be interrupted following induction because of death or toxicity in four cases. At the end of the complete treatment procedure, 61/74 (82%) patients were in CR or CRu (including four patients who had converted from PR to CR or CRu), one patient was in persistent PR, and five patients had progressive disease after having shown an initial response.
With a median follow-up of 33 months, estimated 3-year OS and EFS rates of the entire population were 75% and 67%, respectively. Patients with a high-intermediate/high IPI score (3–5 factors) had a 3-year EFS of 52%, as opposed to 72% among the patients with a low-intermediate/low score (0–2 factors) (p=0.09). Three-year EFS was 61% and 73% in the Bcl-2-positive and negative groups, respectively (p=0.08). Survival did not differ according to the results of CD10, Bcl-6 or MUM1 immunostaining, and, most importantly, we did not observe any prognostic influence of the GC versus nGC profile (Figure 1B): 3-year EFS was 72% in the GC group and 64% in the nGC one (p=0.65). With a longer median follow-up (33 months versus 24 months), we confirmed our findings regarding the prognostic value of early 18FDG-PET scan: the 3-year EFS was 46% in the PET-positive group and 80% in the PET-negative group (p=0.0003). Three-year OS was 90% in the PET-negative group versus 52% in the PET-positive group (p<0.0001).
In a subgroup analysis, we evaluated the prognostic influence of early 18FDG-PET findings and GC versus nGC profile in the following populations: higher and lower-risk patients according to the IPI, patients who had or had not received rituximab, patients who had or had not received HDT. The predictive value of early 18FDG-PET results was observed in every analyzed subgroup, as was the absence of predictive value of the immunophenotypic GC versus nGC profile (data not shown). When excluding the group of patients in whom the diagnosis had been made on a mediastinal biopsy (which might not be only comprised of cases of primary mediastinal DLBCL), Bcl-2 positivity and GC status did not significantly predict 3-year EFS: 54% versus 74% (p=0.08) for Bcl2 and 72% versus 58% (p=0.39) for GC status. Early PET negativity was still highly predictive of outcome: 3 year EFS was 81% versus 36% in positive cases (p<0.0001).
The IPI risk groups, the results of Bcl-2 immunostaining and the results of early-PET were entered in a Cox proportional hazards model. A positive early PET was the only factor significantly associated with EFS, with a relative risk of 3.55 (p=0.0014).
Although the early PET results performed well as a prognostic predictor, 20% of the early PET-negative patients still relapse. It would be interresting to identify potential prognostic parameters within this subgroup. In this regard, Bcl-2 status and GC/nGC phenotype did not show predictive power in our series: among early PET-negative patients with interpretable Bcl-2 status (n=47), Bcl-2 positive patients (n=23) had a 77% 3-year EFS versus 87% in negative cases (n=24) (p=0.12). In this same group, GC patients had a 3-year EFS of 86% versus 78% in nGC cases (p=0.63). Likewise, no differences were observed according to Bcl-2 or GC/nGC status in the early PET positive group.
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Discussion |
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TOPABSTRACTDesign and MethodsResultsDiscussionReferences |
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We applied two innovative risk-stratification approaches to a series of DLBCL patients with heterogeneous presentations (nodal, extranodal or primary mediastinal), and treatments, fairly closely reflecting patients seeen in our everyday practice. The series was mainly composed of younger patients with high-risk disease (as determined by the IPI), who are the best candidates for innovative first-line treatment approaches, and thus for optimal risk stratification. Both methods have been recently introduced, and are thus currently undergoing active evaluation. Each method would potentially affect therapeutic decisions at different time points, as one delivers information at diagnosis, and the other only after a few cycles of therapy.
The large majority (91%) of patients could be classified as having GC or nGC disease using the algorithm of Hans, and 51% were in the GC group. We found that multiple extranodal disease sites and bone marrow involvement were more frequent in nGC cases. To the best of our knowledge, such differences in disease presentation according to phenotypic (or genotypic) profile have not been reported previously. In this series, the phenotypic profile had no impact on prognosis, although the 8% difference in EFS observed between the GC and nGC groups may suggest a trend favoring the GC subgroup. This is concordant with results observed by other investigators in the setting of first-line treatment,10,13 as well as in relapsed/refractory disease,14 but appears to contradict the results of others.9,11,12 The reasons for these discrepancies remain to be understood. We did not confirm the findings of Fields et al.20 who observed an impressive difference in EFS between Bcl2-positive and negative cases within their small population of interim PET-negative patients. This might be due to differences between the two studies, in particular regarding treatment. Another explanation, among several, is that interpretation of immunostaining patterns is prone to interobserver and inter-institutional variations, as has recently been shown for MUM1.27 In addition, our study was based on consecutively recruited patients with heterogeneous clinical presentations, and included patients with mediastinal lymphoma who likely belong to molecularly distinct entity.28,29
Almost half of the patients in this series received rituximab as part of their treatment. The addition of rituximab to multiagent chemotherapy has been shown to erase the predictive value of known prognostic markers, namely Bcl-2 and Bcl-6 expression.24, 30 This difference from other series homogeneously treated with chemotherapy only should be taken into account. Importantly, the original observation that GC and nGC cases (as determined by gene expression profiling) had different outcomes originates from series of patients treated without rituximab.7,8 The use of first-line HDT could have further modified the predictive value of individual prognostic markers. Interestingly, in our series, early 18FDG-PET scanning predicted survival in both the groups treated with and without rituximab or HDT. Some patients (n=14) received their treatment within the GELA 98-B2 protocol, in which patients with one adverse factor of the age-adjusted IPI with Bcl-2 expression received consolidative HDT in order to try to overcome the poor prognosis associated with Bcl-2 expression. Such an approach might have contributed to the lack of predictive value of Bcl-2 expression in the present series, but the low number of patients (n=5) potentially represents only a minimal bias.
With a longer follow-up of our previously published series,15 we confirmed the prognostic value of early 18FDG-PET scan results: 3-year EFS was 46% in the PET-positive group and 80% in the PET-negative group (p=0.0003).
In view of the conflicting results presented here and elsewhere,9–14 we believe that GC/nGC phenotype should be further evaluated in large and homogeneously treated series of patients before it can be widely applied for stratification of patients. Definitive knowledge on its potential value will need ongoing efforts in the field of technique standardization.27,31 The strong prognostic impact of early PET, as shown again here warrants prospective trials evaluating the impact of stratification through early 18FDG-PET used to propose a risk-adapted treatment approach in DLBCL.
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Acknowledgments |
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Footnotes |
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JD, PG, FR and CH designed research; EI, AR and MM performed and interpreted imaging studies; FH and JD analyzed data; JD, CG, TEG, IG and CH provided patient care and clinical data; PG, CC-B and JB performed the histopathological review; JD, PG, FR and CH wrote the paper; and all authors checked the final version of the manuscript.
The authors reported no potential conflicts of interest.
Received for publication October 9, 2006. Accepted for publication May 3, 2007.
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