Just Accepted by Leukemia & Lymphoma
In Europe expression of EBNA2 is associated with poor survival in EBV-positive diffuse large B-cell lymphoma of the elderly Christiane Stuhlmann-Laeisz, Alisa Borchert, Leticia Quintanilla-Martinez, Sylvia Hoeller, Alexandar Tzankov, Ilske Oschlies, Markus Kreuz, Ralf Trappe, Wolfram Klapper DOI: 10.3109/10428194.2015.1040014 Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
Abstract Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly occurs by definition in patients above the age of 50 years without any known underlying immunodeficiency. We investigated the incidence and clinical relevance of this subtype in Europe with special attention to the EBV-latency type. Among the 598 DLBCL, 15 EBV-positive lymphomas fulfilling the criteria of EBV-positive DLBCL of the elderly were identified (2.5 %). Patients with EBV-positive DLBCL expressing EBNA2 showed a significantly poorer overall survival than patients with EBNA2-negative EBV-positive DLBCL (p=0.0156). The incidence of EBV-positive DLBCL of the elderly in Europe is much lower than in Asian countries (2.5 % of all cases of DLBCL). Interestingly, the likelihood of EBV positivity did not increase with age in patient above 50 years. Among EBV-positive DLBCL of the elderly a subgroup with EBV-latency type III expressing EBNA2 can be identified, which shows a poor outcome.
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In Europe expression of EBNA2 is associated with poor survival in EBV-positive diffuse large B-cell lymphoma of the elderly
Christiane Stuhlmann-Laeisz1, Alisa Borchert1, Leticia Quintanilla-Martinez2, Sylvia Hoeller3, Alexandar Tzankov3, Ilske Oschlies1, Markus Kreuz4, Ralf Trappe5, Wolfram Klapper1 1
Department of Pathology, Hematopathology Section and Lymph Node Registry,
University of Kiel, Germany, 2Department of Pathology, University of Tübingen,
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Germany, 3Department of Pathology, University of Basel, Switzerland, 4IMISE, Leipzig, Germany, 5Second Department of Medicine, University of Kiel, Germany, 5DIAKO Bremen, Bremen Germany, 1these authors contribute equally
Corresponding Author: Dr. med. C. Stuhlmann-Laeisz, University Hospital Schleswig-Holstein , Hematopathology Section and Lymph Node Registry Kiel , Arnold-Heller-Straße 3, Haus 14, 24105 Kiel, Tel: 0049-431-597-3394, Fax: 0049-431-597-4129
Short title: EBNA2 in EBV-positive DLBCL Abstract Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly occurs by definition in patients above the age of 50 years without any known underlying immunodeficiency. We investigated the incidence and clinical relevance of this subtype in Europe with special attention to the EBVlatency type. Among the 598 DLBCL, 15 EBV-positive lymphomas fulfilling the criteria of EBV-positive DLBCL of the elderly were identified (2.5 %). Patients with EBV-positive DLBCL expressing EBNA2 showed a significantly poorer overall survival than patients with EBNA2-negative EBV-positive DLBCL (p=0.0156). The incidence of EBV-positive DLBCL of the elderly in Europe is much lower than in Asian countries (2.5 % of all cases of DLBCL). Interestingly, the likelihood of EBV positivity did not increase with age in patient above 50 years. Among EBV-positive DLBCL of the elderly a subgroup with EBV-latency type III expressing EBNA2 can be identified, which shows a poor outcome.
Keywords: Ebstein-Barr virus, DLBCL, latency type, age
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INTRODUCTION DLBCL is the most frequent aggressive B-cell lymphoma in adults in Western Europe [1]. It represents a morphologically, molecularly and clinically very heterogeneous group of diseases [2]. The current WHO classification defines EBV-positive DLBCL of the elderly as a newly recognized subtype of DLBCL. EBV-positive DLBCL of the elderly occurs by definition in patients above 50 years, who are not immunocompromised [3,4]. Shimoyama et al. [5] showed that the incidence of EBV-positive DLBCL of the elderly increases with advanced age. We evaluated the incidence of EBV-positive DLBCL among DLBCL cases
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and the association with patient age for several reasons. First, in our personal diagnostic experience we have been facing DLBCL in very old patients above 80 years but rarely identified EBV. Second, the association of EBV in DLBCL with age was demonstrated primarily for Asian patients [6]. Third, European patient cohorts published so far are still too small to obtain reliable incidence data [7,8]. Fourth, we recently analyzed the association of molecular features of DLBCL with patient age extensively in a cohort containing too few EBV-positive lymphomas for a reliable analysis [9]. EBV-positive lymphoid malignancies are characterized by different prevailing latency types of the virus. These different latency types are defined as a specific expression pattern of EBV-associated antigens that are associated with certain stages of the viral life cycle [10,11]. Latency type III, defined by the expression of EBER, LMP1 and EBNA2, is supposed to represent an active phase of production of virus associated proteins [10] and occurs in lymphoproliferations with reduced immunity against EBV due to reduced antibody response to EBV-specific antigens [12]. It is thus predominantly found in the setting of immunosuppression [3]. The oncogenic role of EBV in lymphomagenesis has not yet been completely elucidated. It is known that the different entities of EBV-positive B-cell lymphomas are characterized by different expression patterns of EBV-associated proteins and RNA, reflecting different latency types. The overall survival and the progression free survival of EBV-positive DLBCL of the elderly seems to be poorer than in EBV-negative DLBCL in the same age group [13–16]. Recently published studies describe both latency type II and III occurring in EBV-positive DLBCL of the elderly [3,7,17,18]. Hoeller et al. [7] performed a survival analysis that indicated a trend towards poorer survival in EBV-positive DLBCL with latency type III than in lymphomas with latency type II. However the number
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of cases analyzed in this study was too small to draw reliable conclusions. In summary, we present a large cohort of DLBCL patients, which is representative for Europe and analyze the incidence of EBV-positive DLBCL of the elderly in the context of age association and patients` survival. MATERIALS AND METHODS Patients 436 Patients above 50 years at the time point of diagnose suffering from DLBCL diagnosed in the area of Schleswig-Holstein, Germany, were identified from the files of the Lymph
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Node Registry between 1995 and 2011. The median age was 71 years (range 50 – 98 years). 295 biopsy specimens were available for EBV encoded RNA (EBER) in situ hybridization. Evaluable results were obtained for 247 DLBCL. EBV-positive DLBCL of the elderly was diagnosed if more than 50% of the lymphoma cells were positive for EBV-associated RNA [4] and if no immunosuppression was recorded in the patient’s files. To obtain reliable data regarding the incidence of EBV-positive DLBCL, the cohort was combined with two previously published series of DLBCL from Basel, Switzerland [7] and Tübingen, Germany [8] yielding a total cohort of 674 DLBCL cases of patients from Europe. In Basel DLBCL cases diagnosed between 1988 and 2001 and reclassified according to the WHO-criteria were included. These cases represented primary diagnoses of patients from the local region of north-western Switzerland and the province of Tyrol Austria and did not include consultational cases. In Tübingen DLBCL in patients 50 years and older between 2000 and 2009 were selected. The final number of patients with evaluable data for EBER was 639. In this final cohort the median age was 70 (range 12-97 years); 598 patients were above 50 years; their median was 71 years (range 50-98 years). Analysis was based on tissue availability and quality. The level information regarding underlying immunodeficiency differed from patient to patient. No patient was reported to suffer from an immune dysregulatory disease or to have received immunosuppressive medication by the physician upon admission of the specimen. Additionally, treating physicians were contacted and specifically asked for underlying disease and medication. In order to evaluate the clinical effect of EBV-latency type on patient outcome and on the overall survival within the group of EBV-positive DLBCL, the 15 EBV-positive cases identified in the cohorts of Basel, Tübingen and Kiel were augmented by 15 additional EBV-
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positive cases fulfilling the criteria of EBV-positive DLBCL of the elderly identified among consultation cases in Kiel and received from outside of Schleswig-Holstein. Since this cohort of EBV-positive DLBCL might be enriched for EBV- positive DLBCL, it was used only for the survival analysis and not for the analysis of the incidence.
The study was performed according to the guidelines of the local ethics advisory board of each contributing center. Immunohistochemistry and EBER in situ Hybridization Formalin fixed, paraffin embedded (FFPE) biopsy specimens were cut and stained for
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hematoxylin and eosin, CD20, CD10, BCL2, KI67, BCL6, MUM1, LMP1, EBNA2 and ZEBRA as well as EBER in situ hybridization using Leica Bond-MAX staining systems and reagents (Leica, Hamburg, Germany). Supplementary table 2 lists the antibodies and staining procedures. The biopsy specimens were categorized as pleomorphic or monomorphic dependent o the histomorphological features of the cases as described by Oyama et al [19]. 18 cases were available for review and 5/18 (28%) presented as pleomorphic subtype and 13/18 (72%) cases were of the monomorphic subtype. 17 cases were available for review regarding specific histomorphological features found in EBV-positive DLBCL of the elderly such as necrosis and angiotropism as recently described [19]. 16/17 (94 %) cases presented with necrosis and/or ulceration. 8/16 (50 %) showed angiotropism of the neoplastic cells. 17 cases could be evaluated for classification as germinal center like (GCB) or non-germinal center like (non-GCB DLBCL) [20] using the expression of CD10, BCL6 and MUM1. 3/17 (17.6%) were of GCB subtype and 14/17 (82.4%) cases were of non-GCB subtype. Statistical analysis Overall survival (OS) was defined either as the time point from diagnosis until death of any cause or till the time point of last follow up. OS in EBV-positive DLBCL of the elderly as a function of EBNA2 expression was estimated according to Kaplan and Meier and KaplanMeier survival curves were performed with GraphPad Prism® version 5. Age association of EBV status in DLBCL was calculated by logistic regression analysis as previously described [9].
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RESULTS Incidence and age distribution of EBV-positive DLBCL in Europe 247 DLBCL specimens diagnosed in major oncologic centers in Schleswig-Holstein could be evaluated for EBV screening by EBER in situ hybridization. Among these cases 9 showed reactivity for EBER. Six of these cases were retrospectively excluded because an underlying immunodeficiency was identified in the patient's files. Thus, 3/247 lymphomas fulfilled the criteria of EBV-positive DLBCL of the elderly, reflecting an incidence of 1.21% among DLBCL in Schleswig-Holstein, northern Germany. The median age of the patients
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was 72 years (range 59 – 73). To analyze the incidence in Europe and the association of the EBV-status with age, we combined our series with previously published series from Basel, Switzerland [7], and Tübingen, Germany [8], yielding a final number of 639 evaluable cases of DLBCL. In this combined cohort 598 patients were above 50 years with a median age of 71 (50-98 years). Among these a total number of 15 (2.5%) DLBCL showed positivity for EBER and fulfilled the criteria of EBV-positive DLBCL of the elderly. The median age in the EBV-positive group was 72 years (range 57-88 years). Figure 1A shows the frequency and the age related distribution of EBV-association in DLBCL. EBV-association was detectable within the age span of 50-89 years. The highest incidence, 6/202 (2.97 %), was seen in patients between 70 and 79 years, the age in which most cases of DLBCL occurred (30.6 %, figure 1A). None of the lymphomas in patients above 90 years of age were EBVpositive. However, due to the low absolute number of DLBCL in patients above 90 years, the amount of analyzed specimens was rather low too. We therefore analyzed whether EBVpositivity in DLBCL is associated with patient age at diagnosis using logistic regression, which calculates the likelihood of EBV-association at any age independent of the number of analyzed samples in the respective age group. As illustrated in Figure 1B, there was no statistically significant association of EBV-positivity with patient age (p= 0.8522) in patients above 50 years. EBV-latency type and survival analysis Since we identified only a limited number of cases of EBV-positive DLBCL of the elderly in our population based series from Europe, we added 15 cases identified among consultation cases in Kiel, Germany, for a survival analysis. Since the consultation cases might be biased towards EBV-positive DLBCL, these cases were omitted from the analysis of the incidence.
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Supplementary table 1 summarizes the clinical characteristics of all 30 EBV-positive DLBCL of the elderly. The EBV-latency type was obtained by staining for LMP1 and EBNA2, as described [10]. 20/28 (71.4 %) biopsy specimens displayed latency type I and II with positivity for EBER and LMP1 but no expression of EBNA2 (Figure 2). 8/28 (28.6 %) cases showed latency type III with positivity for EBER, LMP1 and EBNA2 (Figure 3). 16/18 (89%) cases with detailed histomorphological review concerning polymorphic, monomorphic and Hodgkin-like subtype as described by Oyama et al. [19] were analyzed for the prevailing latency type. 4/18 (22%) of the EBNA2-positive cases were of monomorphic
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subtype and 1/18 (6%) displayed polymorphic subtype, whereas 7/18 (39%) of the EBNA2negative cases presented with monomorphic and 4/18 (22%) showed polymorphic subtype. None of the cases showed Hodgkin-like subtype. Clinical characteristics of the EBNA2 positive and negative EBV-positive DLBCL are outlined in supplementary table 3. Patients suffering from EBV-positive DLBCL with latency type III (EBNA2-positive) showed significantly shorter OS than patients with latency type I/II (no EBNA2 expression). The median survival of EBNA2-positive cases was 267 days and of EBNA2-negative cases 4383 d (p = 0.0156; Figure 4). DISCUSSION DLBCL is the most common aggressive B-cell lymphoma in Western countries. It is a very heterogeneous group of lymphomas with respect to clinical presentation, morphology, immunophenotype and molecular features [1,2]. The current WHO classification defines EBV-positive DLBCL of the elderly as a subgroup of DLBCL occurring in immunocompetent patients usually above 50 years. Age and the accompanying decrease in immunocompetence have been suspected to be involved in the potential oncogenic effect of EBV-reactivation in B-cells [3,4,21,22]. EBV-positive DLBCL of the elderly were initially described in Asian patient cohorts, in which they are supposed to have a rather high incidence of up to 14% among all DLBCL cases that increases with advanced age [4,13,23]. Analysis of smaller cohorts suggest the incidence of DLBCL in Western countries to be 1% and 14% [4,13,14,19,21,24]. To obtain a representative picture of the incidence of EBVpositive DLBCL of the elderly among DLBCL in Europe we analyzed a large cohort of DLBCL from major hematopathological centers. Since these diagnostic centers do not diagnose all DLBCL in a certain region, we cannot exclude a certain bias in the samples we
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analyzed. However, due to the large size of the cohort, the distribution over three centers and an unbiased selection from the archives, we consider our cohort fairly representative for Europe. The incidence (2.5%) of EBV-positive DLBCL of the elderly among all DLBCL in Europe is markedly lower than in Asian countries, where the incidence is described as being about 14% [4]. Most interestingly, EBV-positive DLBCL of the elderly was detected in those age groups in which the probability of suffering from DLBCL is highest, and the likelihood that a patient would suffer from an EBV-positive DLBCL did not increase with age in the age
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group of patients above 50 years. Our data suggests that EBV-association of DLBCL in immunocompetent patients in Europe may not be caused by an age-accompanying decrease in immune surveillance. EBV-associated lymphomas and lymphoproliferative diseases are much more common in Asian countries than in Europe, most likely due to differences in genetic predisposition. Differences in the incidence of EBV-association might also be explained by different cut-offs of EBER used. In our personal experience, it is rare that less than the majority of lymphoma cells stain positive for EBER. In the current complete series 27/30 (90%) of lymphomas showed more >50% positive lymphomas cells, a cut-off which has also been used in previous studies [4]. It is important to note, that the differential diagnosis between EBV-positive DLBCL and other EBV-positive lymphoproliferations might be challenging [25] since the polymorphic subtype of EBV-positive DLBCL might show morphologic and immunphenotypic overlap to lymphoproliferations of other type [26]. The observed difference in the incidence (lower in Europe) and age association (no increased likelihood with age) remains exclusive so far. Our data might influence screening strategies for EBV in DLBCL in Europe. We suggest that histological findings like necrosis (present in 94% of all cases in our cohort) and angiotropism rather than patient age and should prompt the pathologist to search for EBV. The current literature reports that both latency type II and III occur in EBV-positive DLBCL of the elderly [3,4,7,18]. Hoeller et al. [7] analyzed the impact of EBNA2 expression on the overall survival in 8 cases of EBV-positive DLBCL of the elderly. 2/8 (25%) cases were positive for EBNA2 and displayed poorer OS than EBNA2-negative cases; the analyzed groups were, however, too small for a statistical analysis. Using a large cohort of patients we showed that approximately one third of EBV-positive DLBCL of the elderly in Europe
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display latency type III (with expression of EBNA2) including the two EBNA2-positive cases detected in the cohort analyzed by Hoeller et al. [7]. The percentage of EBV-positive DLBCL expressing EBNA2 is comparable to Asian cohorts [4,6]. In our study this subgroup of lymphomas shows a significantly poorer OS compared to EBNA2-negative cases. The group of EBNA2-positive lymphomas showed a tendency towards a higher stage and unfavourable International Prognostic Index (IPI) group. However, the incomplete data and the cohort size do not allow any definite conclusion (supp. table 3). Ok et al. [27] recently analyzed EBV-association in a large cohort of patients of all age
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groups with diagnosed DLBCL. In contrast to our results the EBNA2-positive cases did not display a shorter OS. The difference in the prognostic role of EBNA2 expression between our and the published study might be explained by different patient selection criteria. In fact 7/28 EBV-positive DLBCL in the study by Ok and colleagues were younger than 50 years and over the half of the patients were from the USA, and thus might have had a completely different (immuno-) genetic background due to the different ethnic composition of the USA and European population with much higher Asiatic allelic promiscuity in the former. One might speculate that the EBV-positive DLBCL of the elderly consist of two subgroups, (a) lymphomas with EBNA2-positivity/latency type III with an age-associated decline in immunocompetence and a poor outcome and (b) lymphomas with latency type I or II, in which immunodeficiency might play a less important role in the pathogenesis of the lymphoma. The latter group might be similar to classical Hodgkin lymphoma, which is also frequently EBV-positive, associated with latency type II [25] and arises in immunocompetent patients. EBV-positive DLBCL of the elderly are a morphologically heterogeneous entity. The data presented in our study indicate further biological and clinical subgroups. We suggest that testing for the EBV-latency type is a useful tool for identifying a clinically more aggressive subgroup of lymphomas. Nevertheless, the prognostic role of the latency type has to be confirmed in future studies using unbiased large cohorts of EBVpositive DLBCL. In summary, we show that the incidence of EBV-positive DLBCL of the elderly is significantly lower in Europe compared to Asian countries. Furthermore, the probability to suffer from an EBV-positive DLBCL does not increase with increasing age in the European
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population. Finally, our data suggest, that the latency type in EBV-positive DLBCL of the elderly is associated with outcome of the patients. ACKKNOWLEDGEMENTS AND DECLARATION OF INTERESTS The authors would like to thank Charlotte Botz‐von Drathen and Olivera Batic for their excellent technical assistance. The work was supported by the Dr. Werner Jackstädt‐ Stiftung Junior Excellence Research Group “Mechanisms of B‐cell lymphoma development at old age as a basis for age adjusted therapeutic strategies“. We are indebted to the physicians who treated the patients and the pathologists who referred the cases to us for
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contributing specimens and clinical information and to Kay Dege for editing the manuscript. CSL, AB, LQM, SH, AT, IO and WK selected cases and analyzed slides. MK performed statistical analysis. RT provided clinical data. CSL and WK wrote the manuscript. All authors have approved the final version of the manuscript. The authors have no conflict of interest to report.
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References 1. Armitage JO, Weisenburger DD. New approach to classifying non-Hodgkin's lymphomas: clinical features of the major histologic subtypes. Non-Hodgkin's Lymphoma Classification Project. J. Clin. Oncol. 1998;16(8):2780-2795. 2. Swerdlow SH. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: International Agency for Research on Cancer; 2008. World Health Organization classification of tumours; 2. 3. Adam P, Bonzheim I, Fend F, Quintanilla-Martínez L. Epstein-Barr virus-positive
Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
diffuse large B-cell lymphomas of the elderly. Adv Anat Pathol. 2011;18(5):349-355. 4. Oyama T, Yamamoto K, Asano N, et al. Age-related EBV-associated B-cell lymphoproliferative disorders constitute a distinct clinicopathologic group: a study of 96 patients. Clin. Cancer Res. 2007;13(17):5124-5132. 5. Shimoyama Y, Asano N, Kojima M, et al. Age-related EBV-associated B-cell lymphoproliferative disorders: diagnostic approach to a newly recognized clinicopathological entity. Pathol. Int. 2009;59(12):835-843. 6. Shimoyama Y, Yamamoto K, Asano N, Oyama T, Kinoshita T, Nakamura S. Agerelated Epstein-Barr virus-associated B-cell lymphoproliferative disorders: special references to lymphomas surrounding this newly recognized clinicopathologic disease. Cancer Sci. 2008;99(6):1085-1091. 7. Hoeller S, Tzankov A, Pileri SA, Went P, Dirnhofer S. Epstein-Barr virus-positive diffuse large B-cell lymphoma in elderly patients is rare in Western populations. Hum. Pathol. 2010;41(3):352-357. 8. Hofscheier A, Ponciano A, Bonzheim I, et al. Geographic variation in the prevalence of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly: a comparative analysis of a Mexican and a German population. Mod. Pathol. 2011;24(8):1046-1054. 9. Klapper W, Kreuz M, Kohler CW, et al. Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma. Blood. 2012;119(8):18821887. 10. Klein E, Kis LL, Klein G. Epstein-Barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions. Oncogene. 2007;26(9):1297-1305.
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11. Thorley-Lawson DA, Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med. 2004;350(13):1328-1337. 12. Cen H, Williams PA, McWilliams HP, Breinig MC, Ho M, McKnight JL. Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders. Blood. 1993;81(5):1393-1403. 13. Park S, Lee J, Ko YH, et al. The impact of Epstein-Barr virus status on clinical outcome in diffuse large B-cell lymphoma. Blood. 2007;110(3):972-978.
Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
14. Chang S, Lu Y, Lu C, et al. Epstein-Barr virus is rarely associated with diffuse large B cell lymphoma in Taiwan and carries a trend for a shorter median survival time. J. Clin. Pathol. 2013. 15. Montes-Moreno S, Odqvist L, Diaz-Perez JA, et al. EBV-positive diffuse large B-cell lymphoma of the elderly is an aggressive post-germinal center B-cell neoplasm characterized by prominent nuclear factor-kB activation. Mod. Pathol. 2012;25(7):968982. 16. Ahn J, Yang D, Duk Choi Y, et al. Clinical outcome of elderly patients with EpsteinBarr virus positive diffuse large B-cell lymphoma treated with a combination of rituximab and CHOP chemotherapy. Am. J. Hematol. 2013;88(9):774-779. 17. Ok CY, Papathomas TG, Medeiros LJ, Young KH. EBV-positive diffuse large B-cell lymphoma of the elderly. Blood. 2013;122(3):328-340. 18. Liu F, Asano N, Tatematsu A, et al. Plasmablastic lymphoma of the elderly: a clinicopathological comparison with age-related Epstein-Barr virus-associated B cell lymphoproliferative disorder. Histopathology. 2012;61(6):1183-1197. 19. Oyama T, Ichimura K, Suzuki R, et al. Senile EBV+ B-cell lymphoproliferative disorders: a clinicopathologic study of 22 patients. Am. J. Surg. Pathol. 2003;27(1):1626. 20. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275-282.
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21. Franceschi C, Capri M, Monti D, et al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech Ageing Dev. 2007;128(1):92-105. 22. Roschewski M, Wilson WH. EBV-associated lymphomas in adults. Best Pract Res Clin Haematol. 2012;25(1):75-89. 23. Kuze T, Nakamura N, Hashimoto Y, Sasaki Y, Abe M. The characteristics of EpsteinBarr virus (EBV)-positive diffuse large B-cell lymphoma: comparison between EBV(+) and EBV(-) cases in Japanese population. Jpn J Cancer Res. 2000;91(12):1233-1240.
Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
24. Wada N, Ikeda J, Hori Y, et al. Epstein-barr virus in diffuse large B-Cell lymphoma in immunocompetent patients in Japan is as low as in Western Countries. J. Med. Virol. 2011;83(2):317-321. 25. Stuhlmann-Laeisz C, Oschlies I, Klapper W. Detection of EBV in reactive and neoplastic lymphoproliferations in adults-when and how? J Hematop. 2014;7(4):165170. 26. Dojcinov SD, Venkataraman G, Pittaluga S, et al. Age-related EBV-associated lymphoproliferative disorders in the Western population: a spectrum of reactive lymphoid hyperplasia and lymphoma. Blood. 2011;117(18):4726-4735. 27. Ok CY, Li L, Xu-Monette Z, et al. Prevalence and Clinical Implications of Epstein-Barr Virus Infection in de novo Diffuse Large B-Cell Lymphoma in Western Countries. Clin Cancer Res. 2014.
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Figure legends Figure 1: Age related incidence and distribution of EBV-negative DLBCL and EBVpositive DLBCL of the elderly in Europe (n=639) A: White bars represent EBV-negative DLBCL, black bars EBV-positive DLBCL of the elderly in percent of all analyzed cases. The numbers of analyzed cases are indicated in the lower panel. Age groups are outlined on the x-axis, percentage of positive lymphomas on the y-axis B: Logistic regression analysis of EBV-negative DLBCL and EBV-positive DLBCL of the elderly (p=0.8522). The x-axis represents the age. The y-axis illustrates EBV association.
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Green dots represent EBV-negative DLBCL, red dots EBV-positive DLBCL. The black horizontal broken line defines the mean value of EBV-positive DLBCL in the whole cohort. The slightly sloping blue line resembles the probability of suffering from an EBV-positive DLBCL in relation to increasing age.
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Figure 2: EBV-positive DLBCL of the elderly without EBNA2 expression. A: Diffuse infiltration of a lymph node by medium-sized lymphoid blasts with intermingled smaller lymphocytes (hematoxylin and eosin, original magnification 200 x, insert, original magnification 1000 x). B: The blasts show strong positivity for CD20 (CD20 original magnification 200 x). C: The lymphoma cells are positive for EBV-associated RNA in more than 50% of all tumor cells (EBER in situ hybridization, original magnification 200 x, insert, original magnification 1000 x). D: Expression of EBV-specific nuclear antigen 2 (EBNA2) is not detectable in the lymphoma cells (EBNA2, original magnification 200 x, insert
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original magnification 1000 x).
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Figure 3: EBV-positive DLBCL of the elderly with EBNA2 expression. A: Diffuse infiltration of a lymph node by pleomorphic blastic lymphoid cells accompanied by necrosis (hematoxylin and eosin, original magnification 200 x, insert upper right, original magnification 1000 x). Insert lower right: The blasts show an angiotropic growth pattern (hematoxylin and eosin, original magnification 1000 x). B: The blasts show heterogeneous positivity for CD20, with some blasts more strongly positive and others more weakly positive (CD20 original magnification 200 x). C: The lymphoma cells show nuclear positivity for EBV-associated RNA in more than 50% of all lymphoma cells in the diffuse
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lymphoma compartment and in the angiotropic lymphoma compartment (EBER in situ hybridization original magnification 200 x, C insert upper right and lower right, original magnification 1000 x). D: The lymphoma cells display nuclear positivity for EBNA2 (EBNA2 original magnification 200 x, D insert original magnification 1000 x).
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Figure 4: Overall survival of patients with EBNA2-positive and EBNA2-negative EBVpositive DLBCL of the elderly The Kaplan-Meier survival curve illustrates EBNA2-positive (continuous line, n=8) and EBNA2-negative (broken line, n=20) EBV-positive DLBCL of the elderly (p = 0.0156). The y-axis represents the percentage of patients alive. The x-axis shows the observation period in
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days.
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Table legends
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Supplementary table 1: Clinical characteristics of EBV-positive DLBCL of the elderly ca se no .
localisation
1
lymph node
57
2
thoracal bulky disease
3
age at ge diagno nd sis er
status after first therapy
stage
therapy
m
na
58
m
na
na 6x CHOP, radiothera py
PR
soft tissue
62
m
IIIA
CHOP
na
4
lymph node
63
m
IIA
R-CHOP
CR
5
64
m
na
na
na
6
lymph node gastrointestin al
65
m
II
na
na
7
lymph node
70
f
III
CHOP
na
8
left tonsil
71
m
na
na
na
9
cervix uteri
72
f
na
10
lymph node
77
m
IIIA
11
lymph node
77
f
IIA
na 2 x RBendamus tin 4 x CHOP Bendamus tin
na
sur viv al dea d dea d aliv e aliv e aliv e dea d aliv e
follow up in days 4383
831 1035 1065 5753 61 2253
na
na dea d
na 18
na
dea d
91
12
lymph node
79
m
na
na
na
dea d dea d
13
lymph node
81
f
na
na
na
na
na
14
86
m
na
na
na
88
f
IIBE
na
16
lymph node
62
f
IA
none watch and wait
17
83
f
IA
na
na
18
lymph node retropilonidal , lymph node
69
f
IIB
R-CHOP
na
19
lymph node
66
f
IIIB
PR
20
retroperitone al mass
62
m
IIIBE
na
dea d
21
lymph node
75
f
IIIB
R-CHOP pre-phase Chemoimmunoth erapy RBendamus tin
na dea d dea d dea d aliv e aliv e
na
15
lymph node intestine, lymph node
22
jejunum
58
m
na
CHOP
na
23
stomach
84
f
na
na
na
24
lymph node
59
m
IVB
R-CHOP
CR
25
lymph node
59
m
na
na
na
26
lymph node
80
m
IIIA
R-CHOP
CR
PR
na
na
dea d dea d dea d dea d aliv e dea d
335 274
na 630 135 1434 1162
260 76 28 100 721 1707 1092
18
27
soft tissue left leg
67
m
IBE
81
m
29
tonsil intraabdomin al mass
76
30
paraaortal
73
28
CR
na
R-CHOP tonsillar section
f
IIIB
R-CHOP
PR
m
IIA
R-CHOP
CR
CR
aliv e aliv e aliv e aliv e
988 258 968 952
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Clinical characteristics of all EBV-positive DLBCL (n=30). PR = partial remission, CR = complete remission, R = Rituximab, CHOP = Cyclophosphamide, Hydroxyrubicin, Vincristine, Predisolone; Benda = Bendamustin, na = not available, f = female, m = male
19
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Supplementary table 2: Antibodies for immunohistochemistry and ISH probes.
target
manufacturer
clone
Host
dilution
antigene retrieva
CD20
in-house antibody
JH1
mouse monoclonal
1:50
Ki67
Thermo Scientific
SP6
rabbit monoclonal
1:300
lmp1
DAKO
CS. 1-4
1:50
EBNA2
Fitzgerald
PE2
mouse monoclonal mouse monoclonal
Bond Epitope Retri Solution 1, 20 min 100 C Bond Epitope Retri Solution 2, 20 min 100 C no antigene Retriva
bcl2
DBS
100/D5
mouse monoclonal
1:25
bcl6
DCS
BL6.02
mouse monoclonal
1:50
mum1
DAKO
Mum1P
mouse monoclonal
1:50
CD10
Novocastra
56C6
mouse monoclonal
1:10
target
manufacturer
probe
1:50
Bond Epitope Retriv Solution 2, 30 min 100 C Bond Epitope Retriv Solution 1, 20 min 100 C Bond Epitope Retriv Solution 2, 30 min 100 C Bond Epitope Retri Solution 1, 20 min 100 C Bond Epitope Retri Solution 1, 30 min 100 C
antigene retrieva
EBV encoded Novocastra Bond ™EBER Probe (fluorescin-conjugated, 600ng/mL), RNA, EBER Bond ready to use reagent Antibodies for immunohistochemistry and ISH probes. The antibodies and probes used in this study are listed in the table and were used according to the manufacturer’s instructions. Immunostaining parameters concerning antibody dilution, antigen retrieval, developing reagents and performing systems are listed.
20
Bond Enzyme 1 , 15
Supplementary table 3: Clinical characteristics of the EBNA2-positive and negative EBV-positive DLBCL.
gender (male:female) age (in years) ann arbor stage III/IV IPI high/intermediate first therapy R-CHOP CR after first therapy
EBNA2-positive n=8 7:1 median 70 (range 59 – 79) 4/6 (67%) 1/1 (100%) 2/6 (33%) 3/3 (100%)
EBNA2-negative n=20 11:9 median 71 (range 57 – 88) 4/11 (36%) 1/3 (33%) 5/11 (45%) 6/6 (100%)
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Clinical characteristics of the EBNA2-positive and negative EBV-positive DLBCL for patients with assessable latency type (n=28). (IPI= international prognostic index, R-CHOP= Rituximab + Cyclophosphamid + Hydroxydaunorubicin (Doxorubicin) + Vincristin + Predniso(lo)n, CR= complete response)
21
In Europe expression of EBNA2 is associated with poor survival in EBV-positive diffuse large B-cell lymphoma of the elderly Christiane Stuhlmann-Laeisz, Alisa Borchert, Leticia Quintanilla-Martinez, Sylvia Hoeller, Alexandar Tzankov, Ilske Oschlies, Markus Kreuz, Ralf Trappe, Wolfram Klapper DOI: 10.3109/10428194.2015.1040014 Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
Abstract Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly occurs by definition in patients above the age of 50 years without any known underlying immunodeficiency. We investigated the incidence and clinical relevance of this subtype in Europe with special attention to the EBV-latency type. Among the 598 DLBCL, 15 EBV-positive lymphomas fulfilling the criteria of EBV-positive DLBCL of the elderly were identified (2.5 %). Patients with EBV-positive DLBCL expressing EBNA2 showed a significantly poorer overall survival than patients with EBNA2-negative EBV-positive DLBCL (p=0.0156). The incidence of EBV-positive DLBCL of the elderly in Europe is much lower than in Asian countries (2.5 % of all cases of DLBCL). Interestingly, the likelihood of EBV positivity did not increase with age in patient above 50 years. Among EBV-positive DLBCL of the elderly a subgroup with EBV-latency type III expressing EBNA2 can be identified, which shows a poor outcome.
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In Europe expression of EBNA2 is associated with poor survival in EBV-positive diffuse large B-cell lymphoma of the elderly
Christiane Stuhlmann-Laeisz1, Alisa Borchert1, Leticia Quintanilla-Martinez2, Sylvia Hoeller3, Alexandar Tzankov3, Ilske Oschlies1, Markus Kreuz4, Ralf Trappe5, Wolfram Klapper1 1
Department of Pathology, Hematopathology Section and Lymph Node Registry,
University of Kiel, Germany, 2Department of Pathology, University of Tübingen,
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Germany, 3Department of Pathology, University of Basel, Switzerland, 4IMISE, Leipzig, Germany, 5Second Department of Medicine, University of Kiel, Germany, 5DIAKO Bremen, Bremen Germany, 1these authors contribute equally
Corresponding Author: Dr. med. C. Stuhlmann-Laeisz, University Hospital Schleswig-Holstein , Hematopathology Section and Lymph Node Registry Kiel , Arnold-Heller-Straße 3, Haus 14, 24105 Kiel, Tel: 0049-431-597-3394, Fax: 0049-431-597-4129
Short title: EBNA2 in EBV-positive DLBCL Abstract Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly occurs by definition in patients above the age of 50 years without any known underlying immunodeficiency. We investigated the incidence and clinical relevance of this subtype in Europe with special attention to the EBVlatency type. Among the 598 DLBCL, 15 EBV-positive lymphomas fulfilling the criteria of EBV-positive DLBCL of the elderly were identified (2.5 %). Patients with EBV-positive DLBCL expressing EBNA2 showed a significantly poorer overall survival than patients with EBNA2-negative EBV-positive DLBCL (p=0.0156). The incidence of EBV-positive DLBCL of the elderly in Europe is much lower than in Asian countries (2.5 % of all cases of DLBCL). Interestingly, the likelihood of EBV positivity did not increase with age in patient above 50 years. Among EBV-positive DLBCL of the elderly a subgroup with EBV-latency type III expressing EBNA2 can be identified, which shows a poor outcome.
Keywords: Ebstein-Barr virus, DLBCL, latency type, age
1
INTRODUCTION DLBCL is the most frequent aggressive B-cell lymphoma in adults in Western Europe [1]. It represents a morphologically, molecularly and clinically very heterogeneous group of diseases [2]. The current WHO classification defines EBV-positive DLBCL of the elderly as a newly recognized subtype of DLBCL. EBV-positive DLBCL of the elderly occurs by definition in patients above 50 years, who are not immunocompromised [3,4]. Shimoyama et al. [5] showed that the incidence of EBV-positive DLBCL of the elderly increases with advanced age. We evaluated the incidence of EBV-positive DLBCL among DLBCL cases
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and the association with patient age for several reasons. First, in our personal diagnostic experience we have been facing DLBCL in very old patients above 80 years but rarely identified EBV. Second, the association of EBV in DLBCL with age was demonstrated primarily for Asian patients [6]. Third, European patient cohorts published so far are still too small to obtain reliable incidence data [7,8]. Fourth, we recently analyzed the association of molecular features of DLBCL with patient age extensively in a cohort containing too few EBV-positive lymphomas for a reliable analysis [9]. EBV-positive lymphoid malignancies are characterized by different prevailing latency types of the virus. These different latency types are defined as a specific expression pattern of EBV-associated antigens that are associated with certain stages of the viral life cycle [10,11]. Latency type III, defined by the expression of EBER, LMP1 and EBNA2, is supposed to represent an active phase of production of virus associated proteins [10] and occurs in lymphoproliferations with reduced immunity against EBV due to reduced antibody response to EBV-specific antigens [12]. It is thus predominantly found in the setting of immunosuppression [3]. The oncogenic role of EBV in lymphomagenesis has not yet been completely elucidated. It is known that the different entities of EBV-positive B-cell lymphomas are characterized by different expression patterns of EBV-associated proteins and RNA, reflecting different latency types. The overall survival and the progression free survival of EBV-positive DLBCL of the elderly seems to be poorer than in EBV-negative DLBCL in the same age group [13–16]. Recently published studies describe both latency type II and III occurring in EBV-positive DLBCL of the elderly [3,7,17,18]. Hoeller et al. [7] performed a survival analysis that indicated a trend towards poorer survival in EBV-positive DLBCL with latency type III than in lymphomas with latency type II. However the number
2
of cases analyzed in this study was too small to draw reliable conclusions. In summary, we present a large cohort of DLBCL patients, which is representative for Europe and analyze the incidence of EBV-positive DLBCL of the elderly in the context of age association and patients` survival. MATERIALS AND METHODS Patients 436 Patients above 50 years at the time point of diagnose suffering from DLBCL diagnosed in the area of Schleswig-Holstein, Germany, were identified from the files of the Lymph
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Node Registry between 1995 and 2011. The median age was 71 years (range 50 – 98 years). 295 biopsy specimens were available for EBV encoded RNA (EBER) in situ hybridization. Evaluable results were obtained for 247 DLBCL. EBV-positive DLBCL of the elderly was diagnosed if more than 50% of the lymphoma cells were positive for EBV-associated RNA [4] and if no immunosuppression was recorded in the patient’s files. To obtain reliable data regarding the incidence of EBV-positive DLBCL, the cohort was combined with two previously published series of DLBCL from Basel, Switzerland [7] and Tübingen, Germany [8] yielding a total cohort of 674 DLBCL cases of patients from Europe. In Basel DLBCL cases diagnosed between 1988 and 2001 and reclassified according to the WHO-criteria were included. These cases represented primary diagnoses of patients from the local region of north-western Switzerland and the province of Tyrol Austria and did not include consultational cases. In Tübingen DLBCL in patients 50 years and older between 2000 and 2009 were selected. The final number of patients with evaluable data for EBER was 639. In this final cohort the median age was 70 (range 12-97 years); 598 patients were above 50 years; their median was 71 years (range 50-98 years). Analysis was based on tissue availability and quality. The level information regarding underlying immunodeficiency differed from patient to patient. No patient was reported to suffer from an immune dysregulatory disease or to have received immunosuppressive medication by the physician upon admission of the specimen. Additionally, treating physicians were contacted and specifically asked for underlying disease and medication. In order to evaluate the clinical effect of EBV-latency type on patient outcome and on the overall survival within the group of EBV-positive DLBCL, the 15 EBV-positive cases identified in the cohorts of Basel, Tübingen and Kiel were augmented by 15 additional EBV-
3
positive cases fulfilling the criteria of EBV-positive DLBCL of the elderly identified among consultation cases in Kiel and received from outside of Schleswig-Holstein. Since this cohort of EBV-positive DLBCL might be enriched for EBV- positive DLBCL, it was used only for the survival analysis and not for the analysis of the incidence.
The study was performed according to the guidelines of the local ethics advisory board of each contributing center. Immunohistochemistry and EBER in situ Hybridization Formalin fixed, paraffin embedded (FFPE) biopsy specimens were cut and stained for
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hematoxylin and eosin, CD20, CD10, BCL2, KI67, BCL6, MUM1, LMP1, EBNA2 and ZEBRA as well as EBER in situ hybridization using Leica Bond-MAX staining systems and reagents (Leica, Hamburg, Germany). Supplementary table 2 lists the antibodies and staining procedures. The biopsy specimens were categorized as pleomorphic or monomorphic dependent o the histomorphological features of the cases as described by Oyama et al [19]. 18 cases were available for review and 5/18 (28%) presented as pleomorphic subtype and 13/18 (72%) cases were of the monomorphic subtype. 17 cases were available for review regarding specific histomorphological features found in EBV-positive DLBCL of the elderly such as necrosis and angiotropism as recently described [19]. 16/17 (94 %) cases presented with necrosis and/or ulceration. 8/16 (50 %) showed angiotropism of the neoplastic cells. 17 cases could be evaluated for classification as germinal center like (GCB) or non-germinal center like (non-GCB DLBCL) [20] using the expression of CD10, BCL6 and MUM1. 3/17 (17.6%) were of GCB subtype and 14/17 (82.4%) cases were of non-GCB subtype. Statistical analysis Overall survival (OS) was defined either as the time point from diagnosis until death of any cause or till the time point of last follow up. OS in EBV-positive DLBCL of the elderly as a function of EBNA2 expression was estimated according to Kaplan and Meier and KaplanMeier survival curves were performed with GraphPad Prism® version 5. Age association of EBV status in DLBCL was calculated by logistic regression analysis as previously described [9].
4
RESULTS Incidence and age distribution of EBV-positive DLBCL in Europe 247 DLBCL specimens diagnosed in major oncologic centers in Schleswig-Holstein could be evaluated for EBV screening by EBER in situ hybridization. Among these cases 9 showed reactivity for EBER. Six of these cases were retrospectively excluded because an underlying immunodeficiency was identified in the patient's files. Thus, 3/247 lymphomas fulfilled the criteria of EBV-positive DLBCL of the elderly, reflecting an incidence of 1.21% among DLBCL in Schleswig-Holstein, northern Germany. The median age of the patients
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was 72 years (range 59 – 73). To analyze the incidence in Europe and the association of the EBV-status with age, we combined our series with previously published series from Basel, Switzerland [7], and Tübingen, Germany [8], yielding a final number of 639 evaluable cases of DLBCL. In this combined cohort 598 patients were above 50 years with a median age of 71 (50-98 years). Among these a total number of 15 (2.5%) DLBCL showed positivity for EBER and fulfilled the criteria of EBV-positive DLBCL of the elderly. The median age in the EBV-positive group was 72 years (range 57-88 years). Figure 1A shows the frequency and the age related distribution of EBV-association in DLBCL. EBV-association was detectable within the age span of 50-89 years. The highest incidence, 6/202 (2.97 %), was seen in patients between 70 and 79 years, the age in which most cases of DLBCL occurred (30.6 %, figure 1A). None of the lymphomas in patients above 90 years of age were EBVpositive. However, due to the low absolute number of DLBCL in patients above 90 years, the amount of analyzed specimens was rather low too. We therefore analyzed whether EBVpositivity in DLBCL is associated with patient age at diagnosis using logistic regression, which calculates the likelihood of EBV-association at any age independent of the number of analyzed samples in the respective age group. As illustrated in Figure 1B, there was no statistically significant association of EBV-positivity with patient age (p= 0.8522) in patients above 50 years. EBV-latency type and survival analysis Since we identified only a limited number of cases of EBV-positive DLBCL of the elderly in our population based series from Europe, we added 15 cases identified among consultation cases in Kiel, Germany, for a survival analysis. Since the consultation cases might be biased towards EBV-positive DLBCL, these cases were omitted from the analysis of the incidence.
5
Supplementary table 1 summarizes the clinical characteristics of all 30 EBV-positive DLBCL of the elderly. The EBV-latency type was obtained by staining for LMP1 and EBNA2, as described [10]. 20/28 (71.4 %) biopsy specimens displayed latency type I and II with positivity for EBER and LMP1 but no expression of EBNA2 (Figure 2). 8/28 (28.6 %) cases showed latency type III with positivity for EBER, LMP1 and EBNA2 (Figure 3). 16/18 (89%) cases with detailed histomorphological review concerning polymorphic, monomorphic and Hodgkin-like subtype as described by Oyama et al. [19] were analyzed for the prevailing latency type. 4/18 (22%) of the EBNA2-positive cases were of monomorphic
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subtype and 1/18 (6%) displayed polymorphic subtype, whereas 7/18 (39%) of the EBNA2negative cases presented with monomorphic and 4/18 (22%) showed polymorphic subtype. None of the cases showed Hodgkin-like subtype. Clinical characteristics of the EBNA2 positive and negative EBV-positive DLBCL are outlined in supplementary table 3. Patients suffering from EBV-positive DLBCL with latency type III (EBNA2-positive) showed significantly shorter OS than patients with latency type I/II (no EBNA2 expression). The median survival of EBNA2-positive cases was 267 days and of EBNA2-negative cases 4383 d (p = 0.0156; Figure 4). DISCUSSION DLBCL is the most common aggressive B-cell lymphoma in Western countries. It is a very heterogeneous group of lymphomas with respect to clinical presentation, morphology, immunophenotype and molecular features [1,2]. The current WHO classification defines EBV-positive DLBCL of the elderly as a subgroup of DLBCL occurring in immunocompetent patients usually above 50 years. Age and the accompanying decrease in immunocompetence have been suspected to be involved in the potential oncogenic effect of EBV-reactivation in B-cells [3,4,21,22]. EBV-positive DLBCL of the elderly were initially described in Asian patient cohorts, in which they are supposed to have a rather high incidence of up to 14% among all DLBCL cases that increases with advanced age [4,13,23]. Analysis of smaller cohorts suggest the incidence of DLBCL in Western countries to be 1% and 14% [4,13,14,19,21,24]. To obtain a representative picture of the incidence of EBVpositive DLBCL of the elderly among DLBCL in Europe we analyzed a large cohort of DLBCL from major hematopathological centers. Since these diagnostic centers do not diagnose all DLBCL in a certain region, we cannot exclude a certain bias in the samples we
6
analyzed. However, due to the large size of the cohort, the distribution over three centers and an unbiased selection from the archives, we consider our cohort fairly representative for Europe. The incidence (2.5%) of EBV-positive DLBCL of the elderly among all DLBCL in Europe is markedly lower than in Asian countries, where the incidence is described as being about 14% [4]. Most interestingly, EBV-positive DLBCL of the elderly was detected in those age groups in which the probability of suffering from DLBCL is highest, and the likelihood that a patient would suffer from an EBV-positive DLBCL did not increase with age in the age
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group of patients above 50 years. Our data suggests that EBV-association of DLBCL in immunocompetent patients in Europe may not be caused by an age-accompanying decrease in immune surveillance. EBV-associated lymphomas and lymphoproliferative diseases are much more common in Asian countries than in Europe, most likely due to differences in genetic predisposition. Differences in the incidence of EBV-association might also be explained by different cut-offs of EBER used. In our personal experience, it is rare that less than the majority of lymphoma cells stain positive for EBER. In the current complete series 27/30 (90%) of lymphomas showed more >50% positive lymphomas cells, a cut-off which has also been used in previous studies [4]. It is important to note, that the differential diagnosis between EBV-positive DLBCL and other EBV-positive lymphoproliferations might be challenging [25] since the polymorphic subtype of EBV-positive DLBCL might show morphologic and immunphenotypic overlap to lymphoproliferations of other type [26]. The observed difference in the incidence (lower in Europe) and age association (no increased likelihood with age) remains exclusive so far. Our data might influence screening strategies for EBV in DLBCL in Europe. We suggest that histological findings like necrosis (present in 94% of all cases in our cohort) and angiotropism rather than patient age and should prompt the pathologist to search for EBV. The current literature reports that both latency type II and III occur in EBV-positive DLBCL of the elderly [3,4,7,18]. Hoeller et al. [7] analyzed the impact of EBNA2 expression on the overall survival in 8 cases of EBV-positive DLBCL of the elderly. 2/8 (25%) cases were positive for EBNA2 and displayed poorer OS than EBNA2-negative cases; the analyzed groups were, however, too small for a statistical analysis. Using a large cohort of patients we showed that approximately one third of EBV-positive DLBCL of the elderly in Europe
7
display latency type III (with expression of EBNA2) including the two EBNA2-positive cases detected in the cohort analyzed by Hoeller et al. [7]. The percentage of EBV-positive DLBCL expressing EBNA2 is comparable to Asian cohorts [4,6]. In our study this subgroup of lymphomas shows a significantly poorer OS compared to EBNA2-negative cases. The group of EBNA2-positive lymphomas showed a tendency towards a higher stage and unfavourable International Prognostic Index (IPI) group. However, the incomplete data and the cohort size do not allow any definite conclusion (supp. table 3). Ok et al. [27] recently analyzed EBV-association in a large cohort of patients of all age
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groups with diagnosed DLBCL. In contrast to our results the EBNA2-positive cases did not display a shorter OS. The difference in the prognostic role of EBNA2 expression between our and the published study might be explained by different patient selection criteria. In fact 7/28 EBV-positive DLBCL in the study by Ok and colleagues were younger than 50 years and over the half of the patients were from the USA, and thus might have had a completely different (immuno-) genetic background due to the different ethnic composition of the USA and European population with much higher Asiatic allelic promiscuity in the former. One might speculate that the EBV-positive DLBCL of the elderly consist of two subgroups, (a) lymphomas with EBNA2-positivity/latency type III with an age-associated decline in immunocompetence and a poor outcome and (b) lymphomas with latency type I or II, in which immunodeficiency might play a less important role in the pathogenesis of the lymphoma. The latter group might be similar to classical Hodgkin lymphoma, which is also frequently EBV-positive, associated with latency type II [25] and arises in immunocompetent patients. EBV-positive DLBCL of the elderly are a morphologically heterogeneous entity. The data presented in our study indicate further biological and clinical subgroups. We suggest that testing for the EBV-latency type is a useful tool for identifying a clinically more aggressive subgroup of lymphomas. Nevertheless, the prognostic role of the latency type has to be confirmed in future studies using unbiased large cohorts of EBVpositive DLBCL. In summary, we show that the incidence of EBV-positive DLBCL of the elderly is significantly lower in Europe compared to Asian countries. Furthermore, the probability to suffer from an EBV-positive DLBCL does not increase with increasing age in the European
8
population. Finally, our data suggest, that the latency type in EBV-positive DLBCL of the elderly is associated with outcome of the patients. ACKKNOWLEDGEMENTS AND DECLARATION OF INTERESTS The authors would like to thank Charlotte Botz‐von Drathen and Olivera Batic for their excellent technical assistance. The work was supported by the Dr. Werner Jackstädt‐ Stiftung Junior Excellence Research Group “Mechanisms of B‐cell lymphoma development at old age as a basis for age adjusted therapeutic strategies“. We are indebted to the physicians who treated the patients and the pathologists who referred the cases to us for
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contributing specimens and clinical information and to Kay Dege for editing the manuscript. CSL, AB, LQM, SH, AT, IO and WK selected cases and analyzed slides. MK performed statistical analysis. RT provided clinical data. CSL and WK wrote the manuscript. All authors have approved the final version of the manuscript. The authors have no conflict of interest to report.
9
References 1. Armitage JO, Weisenburger DD. New approach to classifying non-Hodgkin's lymphomas: clinical features of the major histologic subtypes. Non-Hodgkin's Lymphoma Classification Project. J. Clin. Oncol. 1998;16(8):2780-2795. 2. Swerdlow SH. WHO classification of tumours of haematopoietic and lymphoid tissues. Lyon: International Agency for Research on Cancer; 2008. World Health Organization classification of tumours; 2. 3. Adam P, Bonzheim I, Fend F, Quintanilla-Martínez L. Epstein-Barr virus-positive
Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
diffuse large B-cell lymphomas of the elderly. Adv Anat Pathol. 2011;18(5):349-355. 4. Oyama T, Yamamoto K, Asano N, et al. Age-related EBV-associated B-cell lymphoproliferative disorders constitute a distinct clinicopathologic group: a study of 96 patients. Clin. Cancer Res. 2007;13(17):5124-5132. 5. Shimoyama Y, Asano N, Kojima M, et al. Age-related EBV-associated B-cell lymphoproliferative disorders: diagnostic approach to a newly recognized clinicopathological entity. Pathol. Int. 2009;59(12):835-843. 6. Shimoyama Y, Yamamoto K, Asano N, Oyama T, Kinoshita T, Nakamura S. Agerelated Epstein-Barr virus-associated B-cell lymphoproliferative disorders: special references to lymphomas surrounding this newly recognized clinicopathologic disease. Cancer Sci. 2008;99(6):1085-1091. 7. Hoeller S, Tzankov A, Pileri SA, Went P, Dirnhofer S. Epstein-Barr virus-positive diffuse large B-cell lymphoma in elderly patients is rare in Western populations. Hum. Pathol. 2010;41(3):352-357. 8. Hofscheier A, Ponciano A, Bonzheim I, et al. Geographic variation in the prevalence of Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly: a comparative analysis of a Mexican and a German population. Mod. Pathol. 2011;24(8):1046-1054. 9. Klapper W, Kreuz M, Kohler CW, et al. Patient age at diagnosis is associated with the molecular characteristics of diffuse large B-cell lymphoma. Blood. 2012;119(8):18821887. 10. Klein E, Kis LL, Klein G. Epstein-Barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions. Oncogene. 2007;26(9):1297-1305.
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11. Thorley-Lawson DA, Gross A. Persistence of the Epstein-Barr virus and the origins of associated lymphomas. N Engl J Med. 2004;350(13):1328-1337. 12. Cen H, Williams PA, McWilliams HP, Breinig MC, Ho M, McKnight JL. Evidence for restricted Epstein-Barr virus latent gene expression and anti-EBNA antibody response in solid organ transplant recipients with posttransplant lymphoproliferative disorders. Blood. 1993;81(5):1393-1403. 13. Park S, Lee J, Ko YH, et al. The impact of Epstein-Barr virus status on clinical outcome in diffuse large B-cell lymphoma. Blood. 2007;110(3):972-978.
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14. Chang S, Lu Y, Lu C, et al. Epstein-Barr virus is rarely associated with diffuse large B cell lymphoma in Taiwan and carries a trend for a shorter median survival time. J. Clin. Pathol. 2013. 15. Montes-Moreno S, Odqvist L, Diaz-Perez JA, et al. EBV-positive diffuse large B-cell lymphoma of the elderly is an aggressive post-germinal center B-cell neoplasm characterized by prominent nuclear factor-kB activation. Mod. Pathol. 2012;25(7):968982. 16. Ahn J, Yang D, Duk Choi Y, et al. Clinical outcome of elderly patients with EpsteinBarr virus positive diffuse large B-cell lymphoma treated with a combination of rituximab and CHOP chemotherapy. Am. J. Hematol. 2013;88(9):774-779. 17. Ok CY, Papathomas TG, Medeiros LJ, Young KH. EBV-positive diffuse large B-cell lymphoma of the elderly. Blood. 2013;122(3):328-340. 18. Liu F, Asano N, Tatematsu A, et al. Plasmablastic lymphoma of the elderly: a clinicopathological comparison with age-related Epstein-Barr virus-associated B cell lymphoproliferative disorder. Histopathology. 2012;61(6):1183-1197. 19. Oyama T, Ichimura K, Suzuki R, et al. Senile EBV+ B-cell lymphoproliferative disorders: a clinicopathologic study of 22 patients. Am. J. Surg. Pathol. 2003;27(1):1626. 20. Hans CP, Weisenburger DD, Greiner TC, et al. Confirmation of the molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue microarray. Blood. 2004;103(1):275-282.
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21. Franceschi C, Capri M, Monti D, et al. Inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans. Mech Ageing Dev. 2007;128(1):92-105. 22. Roschewski M, Wilson WH. EBV-associated lymphomas in adults. Best Pract Res Clin Haematol. 2012;25(1):75-89. 23. Kuze T, Nakamura N, Hashimoto Y, Sasaki Y, Abe M. The characteristics of EpsteinBarr virus (EBV)-positive diffuse large B-cell lymphoma: comparison between EBV(+) and EBV(-) cases in Japanese population. Jpn J Cancer Res. 2000;91(12):1233-1240.
Leuk Lymphoma Downloaded from informahealthcare.com by Kainan University on 04/29/15 For personal use only.
24. Wada N, Ikeda J, Hori Y, et al. Epstein-barr virus in diffuse large B-Cell lymphoma in immunocompetent patients in Japan is as low as in Western Countries. J. Med. Virol. 2011;83(2):317-321. 25. Stuhlmann-Laeisz C, Oschlies I, Klapper W. Detection of EBV in reactive and neoplastic lymphoproliferations in adults-when and how? J Hematop. 2014;7(4):165170. 26. Dojcinov SD, Venkataraman G, Pittaluga S, et al. Age-related EBV-associated lymphoproliferative disorders in the Western population: a spectrum of reactive lymphoid hyperplasia and lymphoma. Blood. 2011;117(18):4726-4735. 27. Ok CY, Li L, Xu-Monette Z, et al. Prevalence and Clinical Implications of Epstein-Barr Virus Infection in de novo Diffuse Large B-Cell Lymphoma in Western Countries. Clin Cancer Res. 2014.
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Figure legends Figure 1: Age related incidence and distribution of EBV-negative DLBCL and EBVpositive DLBCL of the elderly in Europe (n=639) A: White bars represent EBV-negative DLBCL, black bars EBV-positive DLBCL of the elderly in percent of all analyzed cases. The numbers of analyzed cases are indicated in the lower panel. Age groups are outlined on the x-axis, percentage of positive lymphomas on the y-axis B: Logistic regression analysis of EBV-negative DLBCL and EBV-positive DLBCL of the elderly (p=0.8522). The x-axis represents the age. The y-axis illustrates EBV association.
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Green dots represent EBV-negative DLBCL, red dots EBV-positive DLBCL. The black horizontal broken line defines the mean value of EBV-positive DLBCL in the whole cohort. The slightly sloping blue line resembles the probability of suffering from an EBV-positive DLBCL in relation to increasing age.
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Figure 2: EBV-positive DLBCL of the elderly without EBNA2 expression. A: Diffuse infiltration of a lymph node by medium-sized lymphoid blasts with intermingled smaller lymphocytes (hematoxylin and eosin, original magnification 200 x, insert, original magnification 1000 x). B: The blasts show strong positivity for CD20 (CD20 original magnification 200 x). C: The lymphoma cells are positive for EBV-associated RNA in more than 50% of all tumor cells (EBER in situ hybridization, original magnification 200 x, insert, original magnification 1000 x). D: Expression of EBV-specific nuclear antigen 2 (EBNA2) is not detectable in the lymphoma cells (EBNA2, original magnification 200 x, insert
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original magnification 1000 x).
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Figure 3: EBV-positive DLBCL of the elderly with EBNA2 expression. A: Diffuse infiltration of a lymph node by pleomorphic blastic lymphoid cells accompanied by necrosis (hematoxylin and eosin, original magnification 200 x, insert upper right, original magnification 1000 x). Insert lower right: The blasts show an angiotropic growth pattern (hematoxylin and eosin, original magnification 1000 x). B: The blasts show heterogeneous positivity for CD20, with some blasts more strongly positive and others more weakly positive (CD20 original magnification 200 x). C: The lymphoma cells show nuclear positivity for EBV-associated RNA in more than 50% of all lymphoma cells in the diffuse
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lymphoma compartment and in the angiotropic lymphoma compartment (EBER in situ hybridization original magnification 200 x, C insert upper right and lower right, original magnification 1000 x). D: The lymphoma cells display nuclear positivity for EBNA2 (EBNA2 original magnification 200 x, D insert original magnification 1000 x).
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Figure 4: Overall survival of patients with EBNA2-positive and EBNA2-negative EBVpositive DLBCL of the elderly The Kaplan-Meier survival curve illustrates EBNA2-positive (continuous line, n=8) and EBNA2-negative (broken line, n=20) EBV-positive DLBCL of the elderly (p = 0.0156). The y-axis represents the percentage of patients alive. The x-axis shows the observation period in
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days.
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Table legends
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Supplementary table 1: Clinical characteristics of EBV-positive DLBCL of the elderly ca se no .
localisation
1
lymph node
57
2
thoracal bulky disease
3
age at ge diagno nd sis er
status after first therapy
stage
therapy
m
na
58
m
na
na 6x CHOP, radiothera py
PR
soft tissue
62
m
IIIA
CHOP
na
4
lymph node
63
m
IIA
R-CHOP
CR
5
64
m
na
na
na
6
lymph node gastrointestin al
65
m
II
na
na
7
lymph node
70
f
III
CHOP
na
8
left tonsil
71
m
na
na
na
9
cervix uteri
72
f
na
10
lymph node
77
m
IIIA
11
lymph node
77
f
IIA
na 2 x RBendamus tin 4 x CHOP Bendamus tin
na
sur viv al dea d dea d aliv e aliv e aliv e dea d aliv e
follow up in days 4383
831 1035 1065 5753 61 2253
na
na dea d
na 18
na
dea d
91
12
lymph node
79
m
na
na
na
dea d dea d
13
lymph node
81
f
na
na
na
na
na
14
86
m
na
na
na
88
f
IIBE
na
16
lymph node
62
f
IA
none watch and wait
17
83
f
IA
na
na
18
lymph node retropilonidal , lymph node
69
f
IIB
R-CHOP
na
19
lymph node
66
f
IIIB
PR
20
retroperitone al mass
62
m
IIIBE
na
dea d
21
lymph node
75
f
IIIB
R-CHOP pre-phase Chemoimmunoth erapy RBendamus tin
na dea d dea d dea d aliv e aliv e
na
15
lymph node intestine, lymph node
22
jejunum
58
m
na
CHOP
na
23
stomach
84
f
na
na
na
24
lymph node
59
m
IVB
R-CHOP
CR
25
lymph node
59
m
na
na
na
26
lymph node
80
m
IIIA
R-CHOP
CR
PR
na
na
dea d dea d dea d dea d aliv e dea d
335 274
na 630 135 1434 1162
260 76 28 100 721 1707 1092
18
27
soft tissue left leg
67
m
IBE
81
m
29
tonsil intraabdomin al mass
76
30
paraaortal
73
28
CR
na
R-CHOP tonsillar section
f
IIIB
R-CHOP
PR
m
IIA
R-CHOP
CR
CR
aliv e aliv e aliv e aliv e
988 258 968 952
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Clinical characteristics of all EBV-positive DLBCL (n=30). PR = partial remission, CR = complete remission, R = Rituximab, CHOP = Cyclophosphamide, Hydroxyrubicin, Vincristine, Predisolone; Benda = Bendamustin, na = not available, f = female, m = male
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Supplementary table 2: Antibodies for immunohistochemistry and ISH probes.
target
manufacturer
clone
Host
dilution
antigene retrieva
CD20
in-house antibody
JH1
mouse monoclonal
1:50
Ki67
Thermo Scientific
SP6
rabbit monoclonal
1:300
lmp1
DAKO
CS. 1-4
1:50
EBNA2
Fitzgerald
PE2
mouse monoclonal mouse monoclonal
Bond Epitope Retri Solution 1, 20 min 100 C Bond Epitope Retri Solution 2, 20 min 100 C no antigene Retriva
bcl2
DBS
100/D5
mouse monoclonal
1:25
bcl6
DCS
BL6.02
mouse monoclonal
1:50
mum1
DAKO
Mum1P
mouse monoclonal
1:50
CD10
Novocastra
56C6
mouse monoclonal
1:10
target
manufacturer
probe
1:50
Bond Epitope Retriv Solution 2, 30 min 100 C Bond Epitope Retriv Solution 1, 20 min 100 C Bond Epitope Retriv Solution 2, 30 min 100 C Bond Epitope Retri Solution 1, 20 min 100 C Bond Epitope Retri Solution 1, 30 min 100 C
antigene retrieva
EBV encoded Novocastra Bond ™EBER Probe (fluorescin-conjugated, 600ng/mL), RNA, EBER Bond ready to use reagent Antibodies for immunohistochemistry and ISH probes. The antibodies and probes used in this study are listed in the table and were used according to the manufacturer’s instructions. Immunostaining parameters concerning antibody dilution, antigen retrieval, developing reagents and performing systems are listed.
20
Bond Enzyme 1 , 15
Supplementary table 3: Clinical characteristics of the EBNA2-positive and negative EBV-positive DLBCL.
gender (male:female) age (in years) ann arbor stage III/IV IPI high/intermediate first therapy R-CHOP CR after first therapy
EBNA2-positive n=8 7:1 median 70 (range 59 – 79) 4/6 (67%) 1/1 (100%) 2/6 (33%) 3/3 (100%)
EBNA2-negative n=20 11:9 median 71 (range 57 – 88) 4/11 (36%) 1/3 (33%) 5/11 (45%) 6/6 (100%)
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Clinical characteristics of the EBNA2-positive and negative EBV-positive DLBCL for patients with assessable latency type (n=28). (IPI= international prognostic index, R-CHOP= Rituximab + Cyclophosphamid + Hydroxydaunorubicin (Doxorubicin) + Vincristin + Predniso(lo)n, CR= complete response)
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