J Clin Oncol 23: by American Society of Clinical Oncology INTRODUCTION

Transcription

1 VOLUME 23 NUMBER 18 JUNE JOURNAL OF CLINICAL ONCOLOGY O R I G I N A L R E P O R T From the Departments of Pediatrics and Hematopathology, German Lymph Node Registry, University of Kiel, Kiel; Second Department of Pediatrics, Helios Klinikum Berlin, Berlin; and Department of Pediatrics, University of Münster, Münster, Germany. Submitted September 29, 2004; accepted March 7, Supported by the Schleswig-Holstein Cancer Society, Kiel, Germany. Authors disclosures of potential conflicts of interest are found at the end of this article. Address reprint requests to: Alexander Claviez, MD, Department of Pediatrics, University of Kiel, Schwanenweg 20, Kiel, Germany; a.claviez@pediatrics.uni-kiel.de by American Society of Clinical Oncology X/05/ /$20.00 DOI: /JCO Impact of Latent Epstein-Barr Virus Infection on Outcome in Children and Adolescents With Hodgkin s Lymphoma Alexander Claviez, Markus Tiemann, Heike Lüders, Matthias Krams, Reza Parwaresch, Günther Schellong, and Wolfgang Dörffel A B S T R A C T Purpose The prognostic significance of latent Epstein-Barr virus (EBV) infection in Hodgkin s lymphoma (HL) is debated controversially. Especially in the pediatric age group, no conclusive data are available. Patients and Methods Eight hundred forty-two children and adolescents (median age, 13.7 years) from pediatric multicenter treatment studies HD-90 and HD-95 were studied for latent EBV infection in Hodgkin s and Reed-Sternberg cells by immunostaining against latent membrane protein 1 (LMP-1). Results were compared with established risk factors. Results Two hundred sixty-three patients (31%) were LMP positive. EBV infection correlated with sex (39% male v 23% female; P.001), histologic subtype (69% mixed cellularity v 22% nodular sclerosis v 6% lymphocyte predominance; P.001) and young age. With a median follow-up of 4.9 years, 820 patients (97%) are alive. Probability of overall survival at 10 years ( standard deviation) for EBV-negative and -positive patients was 98.1% 0.6% and 95.1% 1.4%, respectively (P.017 by log-rank test). A negative effect of EBV infection became evident for patients with nodular sclerosis subtype Bennett II (P.02), and those treated for advanced stages (P.003). In multivariate analysis, LMP positivity was an independent factor for adverse outcome (RR 3.08). Probability of failure-free survival (FFS) in LMP positive and negative patients was 89.1% 2.3% and 84.1% 3.9%, respectively (P.86). Conclusion With effective combined treatment modalities in pediatric HL, latent EBV infection has no influence on FFS but is associated with an inferior overall survival in crucial subgroups. J Clin Oncol 23: by American Society of Clinical Oncology INTRODUCTION Insight for a causative role of Epstein-Barr virus (EBV) infection in the pathogenesis of Hodgkin s lymphoma (HL) has been accumulated from numerous clinicopathologic studies during recent years. 1-6 EBV infection in HL has been shown to be latent and monoclonal. In latent EBV infection, 11 genes are expressed, encoding two small nonpolyadenylated RNAs (EBER-1, EBER-2), six nuclear proteins (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-LP) and three integral latent membrane proteins (LMP-1, LMP-2A, LMP-2B). 7 In contrast to transplant-associated lymphomas where the whole spectrum of these genes is expressed (latency type III) and the restricted expression pattern observed in Burkitt s lymphoma (latency type I: EBNA-1, EBER-1, EBER-2), an intermediate pattern (latency type II) is seen in HL. Here, LMPs, EBERs and EBNA-1 are 4048

2 EBV Infection in Pediatric Hodgkin s Lymphoma detectable. 8 Although the oncogenic potential of EBV has been demonstrated in vitro, 9 the prognostic significance of latent EBV gene products found in tumor biopsies from patients with HL is debated controversially. In several recently published larger studies, latent EBV infection has been identified as a favorable prognostic parameter in HL, at least in a subset of patients The question, whether latent EBV infection is of prognostic clinical significance in children with HL has, however, been addressed in only small series to date. 15,16 To get more insight in the impact of EBV infection on clinical outcome in pediatric and adolescent patients with HL, a large cohort of patients enrolled into two consecutive pediatric multicenter trials was studied for the presence of latent EBV infection. Histopathologic findings were correlated with clinical data and outcome. PATIENTS AND METHODS Patients Eight hundred forty-two patients from the two most recent trials, the German-Austrian pediatric multicenter Deutsche Arbeitsgemeinschaft für Leukämieforschung und Behandlung im Kindesalter HD-90 (N 272; 32%) and the multinational HD-95 (N 570; 68%) of the Society of Pediatric Oncology and Hematology (GPOH), were included. Diagnosis of HL was made between October 1990 and August Four hundred sixty-five of the patients (55%) were male and 377 patients (45%) were female (male-to-female ratio, 1.2:1). Median age of the patients was 13.7 years (range, 2.2 to 20.2 years). Clinical stages were classified as follows: 88 patients (10%) had stage I, 470 patients (56%) had stage II, 172 patients (21%) had stage III, and 112 patients (14%) had stage IV. B symptoms were present in 274 patients (33%). Median follow-up of the patients was 4.9 years (range, 3.9 months to 12.1 years). Treatment Design of the two treatment studies has been outlined in detail elsewhere. 17,18 Briefly, patients received stratified, riskadapted treatment according to their allocated treatment groups (TGs) for early, intermediate and advanced stages, respectively. Patients in TG 1 (stages I, IIA), received two cycles, patients in TG 2(I E,II E A, IIB, IIIA) received four cycles, and patients in TG 3 (II E B, IIIB, III E, IV) received six cycles of four-drug polychemotherapy followed by involved field radiotherapy (IF-RT). The first two chemotherapy cycles in girls were vincristine, procarbazine, prednisone, and doxorubicin (OPPA); in boys, vincristine, procarbazine, prednisone, and doxorubicin (OEPA) were used. In the OEPA regimen, etoposide was administered instead of procarbazine because increased rates of testicular dysfunction were observed in a previous trial. In trial HD-95, male patients with stage IIIB or III E B initially received OPPA instead of OEPA. In TG 2 and TG 3, OPPA/OEPA was followed by two or four cycles of COPP (cyclophosphamide, vincristine, procarbazine, prednisone), respectively. Patients in trial HD-90 received 25 Gy, 25 Gy, or 20 Gy IF-RT, respectively, according to their TGs. In order to further reduce potential late effects from radiotherapy, in study HD-95, no IF-RT was given to those patients achieving a complete remission following chemotherapy. For the remaining patients, IF-RT dosage generally was reduced to 20 Gy. Patients with an insufficient tumor volume regression ( 75%) or significant residual tumor volume ( 50 ml) received boost doses up to 35 Gy. Histology Diagnosis of HL was established in all cases by morphologic criteria on the basis of the Rye classification and modified according to WHO classification when indicated. 19,20 Histologic subtypes were as follows: 90 patients (11%) had lymphocyte predominant HL (NLPHL) subtype, 549 patients (65%) had nodular sclerosis (NSHL) subtype (NSHL Bennett I, n 414; NSHL Bennett II, n 128; not specified, n 7), 190 patients (23%) had mixed cellularity (MCHL) subtype, five patients ( 1%) had lymphocyte-rich classical HL (LRCHL), six patients ( 1%) had lymphocyte depletion (LDHL) subtype and in two patients ( 1%) no subtype could be determined. Immunohistochemistry Latent EBV infection was determined with a monoclonal antibody directed against LMP-1 (clone CS1-4, Dako, Hamburg, Germany) as described previously. 15 To increase immunoreactivity, slides were subjected to microwave pretreatment as outlined elsewhere. 21 Biopsies were also immunostained for the presence of CD30 (Ber-H2) and CD20 (L26) antigens, respectively. 22,23 Visualization was performed either with streptavidin biotin complex (streptabc) or alkaline phosphatase-anti alkaline phosphatase (APAAP) technique. 24,25 Statistical Analysis Categoric data were compared by 2 test and continuous data were analyzed by the Mann-Whitney test. Tests were two-sided. Overall survival (OS) was calculated from the start of treatment to the date of death of any cause or last follow-up examination. Failurefree survival (FFS) was calculated from the start of therapy to the date of progression, relapse, death as first event and related to HL or last follow-up. Cases of death in remission or unrelated to HL were censored. Event-free survival (EFS) was defined from therapy start to first event (progression, relapse, second malignant neoplasia, death of any cause) or date of last contact. Probabilities of OS, EFS and FFS were estimated by Kaplan-Meier analysis and differences were compared by the log-rank test. Independent prognostic significance of factors examined was determined using multivariate regression analysis of survival data based on the Cox proportional hazard model. Calculations were done with SPSS for Windows (SPSS Inc., version , Chicago, IL) and SAS for Windows software (SAS Institute Inc., version 6.12, Cary, NC). RESULTS Immunohistochemistry In the present study, latent EBV infection of Hodgkin s and Reed-Sternberg (H&RS) cells, as determined by immunostaining against LMP-1 could be detected in 263 (31%) of 842 pediatric and adolescents patients with primary HL. This figure is well in line with results from other studies analyzing the prevalence of latent EBV infection in patients with HL from industrialized countries. 3 As expected, significant differences concerning LMP positivity were found among the histologic subtypes with predominance of

3 Claviez et al MCHL subtype (131 of 190 v 122 of 549 patients with NSHL subtype; P.001). Among patients with NSHL histology, EBV infection was present in 88 (21%) of 414 cases with subtype Bennett I and in 33 (26%) of 128 cases with subtype Bennett II (P.346). Only a minority of NLPHL cases (5 of 90; 6%) were positively stained for LMP-1 (Fig 1). EBV Infection, Sex, and Age Latent EBV infection was more often detectable in boys than in girls (180 of 465 v 83 of 377; P.001; Table 1). In boys as well as in girls, MCHL subtype was strongly associated with high rates of EBV positivity (75% of boys and 52% of girls were LMP positive). Moreover, EBV infection was significantly associated with younger age. While EBV positivity was found in 30 (73%) of 41 children younger than 5 years, only 44 (17%) of 266 adolescents older than 15 years were EBV infected (Fig 2). Sixty-seven percent of patients younger than 10 years were LMP-1 positive compared to 28% of those 10 years old or older (P.001). Clinical Characteristics As depicted in Table 1, no differences between patients with and without latent EBV infection were found with respect to stage, presence of B symptoms and allocation to the treatment group. There was, however, a significant difference of the proportion of EBV infected patients (HD-90: 41% LMP positive v HD-95: 27% LMP positive) within the two treatment studies (Table 1). This finding is most probably related to the different age distribution in the two trials, as the proportion of patients 15 years of age, in which NSHL is the predominant subtype is higher in the HD-95 treatment study than in trial HD-90 (202 [35%] of 570 v 64 [24%] of 272). Both pediatric trials, HD-90 and HD-95, included patients up to 18 years of age at diagnosis. In the HD-95 trial, substantially more adolescent patients have been referred who would have been previously treated primarily in protocols designed for adult patients. Table 1. Characteristics of Patients with Hodgkin s Lymphoma With Respect to Latent EBV Infection (N 842) Parameter EBV Positive (n 263) EBV Negative (n 579) No. % No. % Sex, male Age, years.001 Median Range Histologic subtype NLPHL NSHL MCHL LRCHL LDHL Not classified Clinical stage I II III IV B symptoms Treatment group Treatment study HD HD Abbreviations: EBV, Epstein-Barr virus; NLPHL, lymphocyte predominant Hodgkin s lymphoma; NSHL, nodular sclerosis; MCHL, mixed cellularity; LRCHL, lymphocyte-rich classical Hodgkin s lymphoma; LDHL, lymphocyte depletion. OS and FFS With a median follow-up of 4.9 years, 820 (97%) of 842 patients are alive. Probability of OS ( standard deviation [SD]) at 10 years was 97.2% 0.6% (Fig 3). Four patients died of causes not strictly related to HL (accident, 2; thrombosis, 1; encephalitis, 1). Two of them were EBV positive P Fig 1. Distribution of the three major histological subtypes, NLPHL (lymphocyte predominant Hodgkin s lymphoma), NSHL (nodular sclerosis), and MCHL (mixed cellularity) in relation to Epstein-Barr virus (EBV) status. Latent membrane protein (LMP) positivity is shown by dark bars, and LMP negativity by light bars. Fig 2. Age dependency of latent membrane protein (LMP) status in Hodgkin s lymphoma showing the highest frequency of Epstein-Barr virus (EBV) infection in the age group younger than 5 years JOURNAL OF CLINICAL ONCOLOGY

4 EBV Infection in Pediatric Hodgkin s Lymphoma Fig 3. Overall survival (OS), failure-free survival (FFS), and event-free survival (EFS) for the entire study population. and two were EBV negative. The remaining 18 patients died as a result of HL. Of those, 10 were EBV positive and eight were EBV negative. Three patients developed a second malignant neoplasia with no relation to latent EBV infection between 6.7 and 9.9 years following treatment. These tumors were a thyroid cancer in one patient, multiple basal cell carcinomas in another patient, and an astrocytoma in the third patient. The first two patients had EBV positive HL, whereas the latter was EBV negative. All three patients are alive to date. These events were censored for FFS. Probabilities of EFS and FFS in the whole cohort of patients at 10 years were 86% 2% and 87% 2%, respectively. Seventy-seven (9.2%) of 842 patients experienced a tumor progression or recurrence of their disease. Boys had a slightly inferior FFS compared with girls, which was of borderline significance (83.3% 3.2% v 91.7% 1.9%; P.059). Allocation to treatment intensity did not affect FFS significantly, but patients in TG 3 had an inferior OS compared with those in TG 1 or 2. The estimated OS rates were 93.6% 1.5% v 99.0% 0.5%; P.001. With respect to OS and FFS, there was no statistically significant difference between patients treated in trial HD-90 and HD-95. In addition, age was not of importance on outcome for either of the end points. Univariate Analysis In univariate analysis, LMP-positive patients had a slightly inferior OS compared with noninfected patients (Fig 4A). The probability rates for both groups at 10 years were 95.1% 1.4% and 98.1% 1.6%, respectively (P.017). In contrast, LMP positivity was not identified as a significant prognostic factor for FFS (Fig 4B). The estimated FFS at 10 years for the 263 EBV-positive patients was 89.1% 2.3% compared with 84.1% 3.9% for the 579 EBV negative patients (P.86). The influence of EBV infection on outcome was further assessed by analyzing allocation to treatment arms. Patients Fig 4. Outcome of the study population showing (A) an inferior probability of overall survival (pos) for Epstein-Barr virus infected patients but (B) no effect on probability of failure-free survival (pffs). LMP, latent membrane protein. with early stages had an excellent prognosis. One hundred twenty-five of 126 LMP positive patients and 232 of 233 LMP negative are alive to date (P.66). The corresponding figures for FFS were 120 of 126 and 227 of 233, respectively (P.34). In patients with intermediate stages (TG 2), there was also no significant difference in OS and FFS for patients with or without latent EBV infection. Probability of OS was 98.3% 1.7% v 98.2% 1.3% (P.89). The corresponding rates for FFS were 87.6% 4.4% v 89.9% 3.2% (P.5). In patients with advanced disease (TG 3), however, OS was inferior for EBV-positive patients (Fig 5A). Probability of OS was 86.4% 4.0% in LMP-positive patients in contrast to LMP negative patients, for whom probability was 96.4% 1.3% (P.003). Probability of FFS in LMPpositive patients was 85.7% 4.0% and for LMP negative patients it was 86.6% 2.6% (P.69). The EBV status generally had no significant influence on prognosis in patients according to their histologic subtype with one exception. Patients with nodular sclerosis and increased numbers of H&RS cells (NSHL subtype Bennett II) who were LMP positive had a poorer OS compared with LMP-negative patients (Fig 5B). Again, FFS in this subgroup was not statistically different with respect to EBV status (81.4% 6.9% v 83.8% 4.6%; P.52). With respect to patient age and EBV status, no difference in OS and FFS was observed

5 Claviez et al Fig 5. Overall survival OS with regard to latent membrane protein (LMP) status in (A) patients treated for advanced disease (n 280) and (B) those with nodular sclerosis Hodgkin s lymphoma (NSHL) subtype Bennett II (n 128). among young children, school-age children and adolescents (data not shown). Factors achieving a significant effect on OS and FFS in univariate analysis are presented in Table 2. NSHL subtype Bennett II, presence of B symptoms, and allocation to TG 3 have been shown previously to be of prognostic significance in the HD-95 trial. 26 Multivariate Analysis Factors that were significant in univariate analysis were included in the multivariate analysis. In a Cox regression model, LMP positivity emerged as an independent prognostic factor associated with an inferior OS. This effect was not observed for FFS. In addition, allocation to TG 3 also was associated with a poorer OS rate. For FFS, presence of B symptoms remained as the only statistically significant factor (Table 3). DISCUSSION In this study on 842 pediatric and adolescent patients with primary HL, latent EBV infection of H&RS cells was associated with an inferior OS, especially in patients with NSHL subtype Bennett II (sheets of H&RS cells make up more than 50% in at least 25% of the nodules) and those treated for advanced disease (TG 3). This observation was confirmed by multivariate analysis. LMP positivity, however, did not emerge as a significant prognostic parameter for FFS. The rates obtained for OS and FFS from patients included in the present study did not differ from those reported before for studies HD-90 and HD ,18 EBV infection in our cohort of patients was not associated with stage distribution, presence of B symptoms, and allocation to TG (Table 1), thus enabling us to study the effect of latent EBV infection on outcome. Studies investigating the impact of latent EBV infection on outcome of patients with HL have demonstrated conflicting results. The main findings of the available studies investigating this issue are summarized in Table 4. While EBV infection was reported to be a favorable prognostic parameter in nine studies, two studies observed the opposite effect, and in nine studies no statistically significant difference was observed ,27-39 Several reasons may be attributable for the differences on outcome, including the technique employed for the detection of EBV infection, patient selection, efficacy of primary treatment, and choice of the study end point. Demonstration of latent EBV infection in HL for routine diagnostic purposes can be reliably performed by immunohistochemistry with LMP-1 antibody, although in situ hybridization with EBER probes is considered as the more sensitive method. 40 Comparable results for LMP-1 and EBER-1 expression have been reported previously. 13 In contrast to EBER-1, however, LMP-1 is not expressed in nonmalignant bystander cells. 4 In our small previous study, we found corresponding results for LMP-1 and EBER-1 staining in 20 of 22 analyzed cases. 15 In the majority of the above mentioned studies, the expression of LMP was studied for the demonstration of latent EBV infection. In one of the cited studies, polymerase chain reaction was used for EBV detection, but no discrimination is possible between malignant and reactive cells unless single-cell approaches are selected. 29 For the evaluation of EBV infection as a new prognostic factor a sufficient sample size, patient homogeneity and standardized treatment are essential prerequisites to draw firm conclusions. Eight of the studies listed included fewer than 100 patients, and only three had more than 200 patients. A multivariate analysis was performed in 10 studies. In the study reported by Murray et al 13 with standardized therapy, response rates were superior for EBV positive patients (80% v 69%). The rates for OS and FFS at 2 and 5 years were higher in the EBV infected group compared to noninfected patients. A statistical significant difference, however, was observed for 2-year FFS only (P.02). The authors speculated that a different immune response exists between EBV positive and negative patients. Morente et al 10 identified clinical factors (advanced stage, older age, presence of B symptoms), a proliferation rate 20% (as determined by Ki-67 antigen expression) and expression of retinoblastoma protein less than 20% being associated with 4052 JOURNAL OF CLINICAL ONCOLOGY

6 EBV Infection in Pediatric Hodgkin s Lymphoma Variable Table 2. Risk Factors in Pediatric Hodgkin s Lymphoma on Prognosis by Univariate Analysis No. of Patients Overall Survival 5 Year 10 Year Failure-Free Survival 5 Year 10 Year % SD % SD P % SD % SD Sex Male Female LMP Positive Negative Treatment group and B symptoms Absence Presence Extranodal (stage I-III) With Without Stage I-III IV Histology NSHL type II Others Abbreviations: SD, standard deviation; LMP, latent membrane protein; NSHL, nodular sclerosis Hodgkin s lymphoma. P inferior survival. LMP expression was associated with improved survival in multivariate analysis, but had no impact on achieving a complete remission in a univariate logistic regression model. In the study by Naresh et al, 14 EBER expression was associated with superior overall and relapsefree survival at 10 years (85% v 64% and 60% v 44%, respectively), which was shown by multivariate analysis also. The authors, however, did not specify the treatment regimens administered to the patients. Subgroup analysis in several studies demonstrated that, at least in part, younger patients with HL and EBV infection had a superior outcome compared with older patients. 11,27,33 Theoretically, effects of latent EBV infection may differently influence the outcome of patients with HL. The oncogenic potential of LMP has been demonstrated in rodent cells by altering cell morphology, resistance of the growth-inhibiting effect of a medium with low serum content, loss of contact inhibition, and induction of tumor growth in nude mice. 9 LMP may upregulate the expression of the antiapoptotic bcl-2 gene, but no conclusive results have been reported about the prognostic significance of that finding. A large recent study points to an adverse prognosis in Bcl-2 protein positive patients compared to Bcl-2 negative patients treated with ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine) regimens. 41 LMP-1 is also capable of activating nuclear factor-kappa B (NF- B), thus mediating cytokine production, proliferation, and apoptosis. 42 Thus, one might expect that LMP expression exerts an adverse effect on patients with HL. Moreover, mutations in the LMP-1 gene resulting in loss of antigenicity of the protein have been associated with a more aggressive course of HL. 43 In those studies in which a favorable effect of EBV infection has been shown, the authors speculate on the immunogenic potential of cytotoxic T lymphocytes as a target for EBV-infected malignant cell population in HL. 44 In EBV-induced post-transplant lymphoproliferative disease following solid organ or hematopoietic stem cell transplantation, the efficacy of this approach has been demonstrated. 45,46 Recent work also suggests that this approach is feasible in selected patients with relapsed HL, but these procedures have not become routine practice yet. 47 Table 3. Influence of Latent EBV Infection in Pediatric Hodgkin s Lymphoma on Prognosis by Multivariate Analysis Using a Cox Regression Model Variable Parameter Estimate SE Risk Ratio Overall survival Latent membrane protein Treatment group Failure-free survival B symptoms Abbreviations: EBV, Epstein-Barr virus. P

7 Claviez et al Table 4. Overview on Published Studies With Respect to EBV Status on Prognosis in Hodgkin s Lymphoma Patient Characteristic Author No. of Patients Age (years) Range Median Male Sex (%) III/IV (%) B Symp toms (%)- EBV Positive EBV Genome/ Method Multivariate Analysis Main Findings of EBV Infection on Outcome Clarke NA LMP/EBER Yes OS worse ( 45 years) Herling NA LMP Yes No difference Flavell NA EBER Yes FFS better (males, 45 years) Murray NA LMP No FFS better Morente LMP Yes Longer OS Fellbaum NA NA NA NA NA PCR Yes No difference Krugmann NA LMP/EBER No FFS better Enblad LMP/EBER Yes No difference Naresh NA 78 LMP/EBER Yes OS and RFS better Montalban NA LMP Yes OS and FFS better Enblad NA LMP/EBER No No difference Glavina-Durdov LMP Yes DFS better (I/II, no B symptoms, 30 years) Axdorph LMP/EBER No No difference Oudejans NA NA NA NA NA NA No PFS worse Vassallo LMP/EBER Yes OS better (LMP 10%) Stark NA NA 34 LMP Yes OS and DFS worse Vestlev LMP Yes No difference Armstrong NA NA 36 LMP/EBER No No difference Engel LMP/EBER No OS better Claviez LMP/EBER No No difference Abbreviations: EBV, Epstein-Barr virus; NA., not available; LMP, latent membrane protein; EBER, EBV-encoded RNA; OS, overall survival; FFS, failure-free survival; RFS, relapse-free survival; DFS, disease-free survival; PFS, progression-free survival. Denotes mean. The majority of studies included some children, but only two small studies focused exclusively on pediatric patients. 15,16 The larger of these studies, on 47 children, suggested an improved outcome for patients with EBV infection. Data on outcome were available for 36 patients. Twenty-three (96%) of 24 EBV-infected children and 7 (58%) of 12 EBV-negative children were alive without disease (P.01). Median OS (63 v 47 months) but not relapse-free survival was longer for patients with latent EBV infection. 16 Due to heterogeneous patient cohorts from different geographic regions, the proportion of EBVpositive patients varied from 27% to 51% in Europe and North America, and the highest frequencies were found in studies from India and South Africa, both exceeding 70%. This finding corresponds to results of a large epidemiologic study, demonstrating an earlier infection in dependency of the socioeconomic status. 3 As critically discussed by Herling et al, 28 selection of the study end point may be an important factor. The authors state that the use of OS is problematic, because factors unrelated to the primary treatment may also affect the outcome. Natural limitation of life expectancy, which may be of major importance in the elderly population, however, is of minor impact in young patients. Concerning the efficacy of any first-line regimen, we agree with Herling et al that FFS is generally the more appropriate variable compared with OS. All studies evaluating the prognostic impact of EBV infection in HL should provide both end points. On the basis of our findings, we speculate that expression of LMP does not exhibit an adverse effect with respect to treatment response. Thus, EBV-infected H&RS cells do not exhibit a survival advantage. Especially in patients with localized and intermediate disease, current combined modality treatment is sufficient and abrogates the possible negative biologic effects of LMP. Patients from TG 3 and those with NSHL subtype Bennett II, however, showed a poorer OS rate. It can therefore be assumed that persistence of LMP appears as an adverse risk factor only in subgroups of patients with higher risk for treatment failure. This was also confirmed but not amplified when specific but smaller subgroups (stage II E B, IIIB, IV) were analyzed separately on outcome with respect to LMP status. Several findings from the present study confirm previously reported observations, such as correlation of EBV infection with MCHL subtype. 3 The predominance of EBV infection in male patients may be attributable in part to the distribution of histologic subtypes, because NSHL subtype was found more frequently in girls than in boys (79% v 54%, respectively). Analyzing MCHL subtype separately, boys were found to express LMP more often than girls (75% v 52%). The previously described correlation of young age with EBV infection in HL 48 was also supported 4054 JOURNAL OF CLINICAL ONCOLOGY

8 EBV Infection in Pediatric Hodgkin s Lymphoma by our findings. Median age of EBV-positive patients was 11.1 years in contrast to 14.4 years in those without infection (P.001). MCHL subtype was associated with younger age (median age, 11.2 years) compared with NSHL subtype (median age, 14.3 years), which was also statistically significant (P.001). So far, there is no conclusive proof to explain this fact. One may speculate that an immature immune system reacting to EBV infection is also more susceptible to develop MCHL. Speculations about a prognostic effect of EBV infection on the course in HD are tempting. To prove both a possible adverse effect by the oncogenic capacity of LMP-1 or a putative protective effect due to elucidation of a specific immune response of EBV infection, large series of patients with comparable clinical criteria and standardized therapeutic regimens are indispensable. In this large study on 842 patients receiving highly effective risk-adapted combined chemotherapy regimens with low-dose radiotherapy, EBV positivity did not emerge as an independent risk factor on FFS. LMP positivity, however, was associated with a poorer OS in a subgroup of patients with higher risks for treatment failure. Authors Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. REFERENCES 1. Weiss LM, Movahed LA, Warnke RA, et al: Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin s disease. N Engl J Med 320: , Pallesen G, Hamilton-Dutoit SJ, Rowe M, et al: Expression of Epstein-Barr virus latent gene products in tumour cells of Hodgkin s disease. Lancet 337: , Glaser SL, Lin RJ, Stewart SL, et al: Epstein-Barr virus-associated Hodgkin s disease: Epidemiologic characteristics in international data. Int J Cancer 70: , Jarrett RF, MacKenzie J: Epstein-Barr virus and other candidate viruses in the pathogenesis of Hodgkin s disease. Semin Hematol 36: , Hummel M, Anagnostopoulos I, Dallenbach F, et al: EBV infection patterns in Hodgkin s disease and normal lymphoid tissue: Expression and cellular localization of EBV gene products. Br J Haematol 82: , Wu TC, Mann RB, Charache P, et al: Detection of EBV gene expression in Reed- Sternberg cells of Hodgkin s disease. Int J Cancer 46: , Rickinson AB, Kieff E: Epstein-Barr virus, in Fields BN, Knipe DM, Howley PM, et al (eds): Virology, Vol 2 (ed 3). Philadelphia, PA, Lippincott-Raven, 1996, pp Grässer FA, Murray PG, Kremmer E, et al: Monoclonal antibodies directed against the Epstein-Barr virus-encoded nuclear antigen 1 (EBNA1): immunohistologic detection of EBNA1 in the malignant cells of Hodgkin s disease. Blood 84: , Wang D, Liebowitz D, Kieff E: An EBV membrane protein expressed in immortalized lymphocytes transforms established rodent cells. Cell 43: , Morente MM, Piris MA, Abraira V, et al: Adverse clinical outcome in Hodgkin s disease is associated with loss of retinoblastoma protein expression, high Ki67 proliferation index, and absence of Epstein-Barr virus-latent membrane protein 1 expression. Blood 90: , Flavell KJ, Billingham LJ, Biddulph JP, et al: The effect of Epstein-Barr virus status on outcome in age- and sex-defined subgroups of patients with advanced Hodgkin s disease. Ann Oncol 14: , Krugmann J, Tzankov A, Gschwendtner A, et al: Longer failure-free survival interval of Epstein-Barr virus-associated classical Hodgkin s lymphoma: A single-institution study. Mod Pathol 16: , Murray PG, Billingham LJ, Hassan HT, et al: Effect of Epstein-Barr virus infection on response to chemotherapy and survival in Hodgkin s disease. Blood 94: , Naresh KN, Johnson J, Srinivas V, et al: Epstein-Barr virus association in classical Hodgkin s disease provides survival advantage to patients and correlates with higher expression of proliferation markers in Reed-Sternberg cells. Ann Oncol 11:91-96, Claviez A, Tiemann M, Peters J, et al: The impact of EBV, proliferation rate, and Bcl-2 expression in Hodgkin s disease in childhood. Ann Hematol 68:61-66, Engel M, Essop MF, Close P, et al: Improved prognosis of Epstein-Barr virus associated childhood Hodgkin s lymphoma: Study of 47 South African cases. J Clin Pathol 53: , Schellong G, Pötter R, Brämswig J, et al: High cure rates and reduced long-term toxicity in pediatric Hodgkin s disease: The German- Austrian multicenter trial DAL-HD-90: The German-Austrian Pediatric Hodgkin s Disease Study Group. J Clin Oncol 17: , Dörffel W, Lüders H, Rühl U, et al: Preliminary results of the multicenter trial GPOH-HD 95 for the treatment of Hodgkin s disease in children and adolescents: Analysis and outlook. Klin Pädiatr 215: , Lukes RJ, Butler JJ: The pathology and nomenclature of Hodgkin s disease. Cancer Res 26: , Stein H, Delsol G, Pileri S, et al: Hodgkin lymphoma, in Jaffe ES, Harris NL, Stein H, et al (eds): World Health Organization classification of tumours. Pathology and genetics of tumours of hematopoietic and lymphoid tissues. Lyon, France, IARC Press, 2001, pp Cattoretti G, Pileri S, Parravicini C, et al: Antigen unmasking on formalin-fixed, paraffinembedded tissue sections. J Pathol 171:83-98, Schwarting R, Gerdes J, Dürkop H, et al: BER-H2: A new anti-ki-1 (CD30) monoclonal antibody directed at a formol-resistant epitope. Blood 74: , Ishii Y, Takami T, Yuasa H, et al: Two distinct antigen systems in human B lymphocytes: Identification of cell surface and intracellular antigens using monoclonal antibodies. Clin Exp Immunol 58: , Hsu SM, Raine L, Fanger H: Use of avidinbiotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29: , Cordell JL, Falini B, Erber WN, et al: Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32: , Schellenberg D, Lüders H, Rühl U, et al: Prognostic factors in pediatric Hodgkin s disease: Results of the German multicenter trial GPOH-HD 95. Leuk Lymphoma 42:80, 2001 (suppl 2; abstr) 27. Clarke CA, Glaser SL, Dorfman RF, et al: Epstein-Barr virus and survival after Hodgkin disease in a population-based series of women. Cancer 91: , Herling M, Rassidakis GZ, Medeiros LJ, et al: Expression of Epstein-Barr virus latent membrane protein-1 in Hodgkin and Reed-Sternberg cells of classical Hodgkin s lymphoma: Associations with presenting features, serum interleukin 10 levels, and clinical outcome. Clin Cancer Res 9: , Fellbaum C, Hansmann ML, Niedermeyer H, et al: Influence of Epstein-Barr virus genomes on patient survival in Hodgkin s disease. Am J Clin Pathol 98: , Enblad G, Sandvej K, Sundstrom C, et al: Epstein-Barr virus distribution in Hodgkin s disease in an unselected Swedish population. Acta Oncol 38: , Montalban C, Abraira V, Morente M, et al: Epstein-Barr virus-latent membrane protein 1 expression has a favorable influence in the outcome of patients with Hodgkin s disease treated with chemotherapy. Leuk Lymphoma 39: ,

9 Claviez et al 32. Enblad G, Sandvej K, Lennette E, et al: Lack of correlation between EBV serology and presence of EBV in the Hodgkin and Reed- Sternberg cells of patients with Hodgkin s disease. Int J Cancer 72: , Glavina-Durdov M, Jakic-Razumovic J, Capkun V, et al: Assessment of the prognostic impact of the Epstein-Barr virus-encoded latent membrane protein-1 expression in Hodgkin s disease. Br J Cancer 84: , Axdorph U, Porwit-MacDonald A, Sjoberg J, et al: Epstein-Barr virus expression in Hodgkin s disease in relation to patient characteristics, serum factors and blood lymphocyte function. Br J Cancer 81: , Oudejans JJ, Jiwa NM, Meijer CJ: Epstein-Barr virus in Hodgkin s disease: More than just an innocent bystander. J Pathol 181: , Vassallo J, Metze K, Traina F, et al: Expression of Epstein-Barr virus in classical Hodgkin s lymphomas in Brazilian adult patients. Haematologica 86: , Stark GL, Wood KM, Jack F, et al: Hodgkin s disease in the elderly: A populationbased study. Br J Haematol 119: , Vestlev PM, Pallesen G, Sandvej K, et al: Prognosis of Hodgkin s disease is not influenced by Epstein-Barr virus latent membrane protein. Int J Cancer 50: , Armstrong AA, Lennard A, Alexander FE, et al: Prognostic significance of Epstein-Barr virus association in Hodgkin s disease. Eur J Cancer 30A: , Herbst H, Steinbrecher E, Niedobitek G, et al: Distribution and phenotype of Epstein-Barr virus-harboring cells in Hodgkin s disease. Blood 80: , Rassidakis GZ, Medeiros LJ, Vassilakopoulos TP, et al: BCL-2 expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease predicts a poorer prognosis in patients treated with ABVD or equivalent regimens. Blood 100: , Bargou RC, Emmerich F, Krappmann D, et al: Constitutive nuclear factor-kappab-rela activation is required for proliferation and survival of Hodgkin s disease tumor cells. J Clin Invest 100: , Knecht H, Bachmann E, Brousset P, et al: Deletions within the LMP1 oncogene of Epstein-Barr virus are clustered in Hodgkin s disease and identical to those observed in nasopharyngeal carcinoma. Blood 82: , Sing AP, Ambinder RF, Hong DJ, et al: Isolation of Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes that lyse Reed-Sternberg cells: Implications for immune-mediated therapy of EBV Hodgkin s disease. Blood 89: , Rooney CM, Smith CA, Ng CY, et al: Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 92: , Khanna R, Bell S, Sherritt M, et al: Activation and adoptive transfer of Epstein-Barr virusspecific cytotoxic T cells in solid organ transplant patients with posttransplant lymphoproliferative disease. Proc Natl Acad Sci U S A 96: , Roskrow MA, Suzuki N, Gan Y, et al: Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for the treatment of patients with EBV-positive relapsed Hodgkin s disease. Blood 91: , Armstrong AA, Alexander FE, Paes RP, et al: Association of Epstein-Barr virus with pediatric Hodgkin s disease. Am J Pathol 142: , JOURNAL OF CLINICAL ONCOLOGY