Deilson Elgui de Oliveira, MS, Maura M. Bacchi, MD, Eliane S. Abreu, MD, Ligia Niero-Melo, MD, and Carlos E. Bacchi, MD. Abstract

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1 Hematopathology / HODGKIN DISEASE IN BRAZIL Hodgkin Disease in Adult and Juvenile Groups From Two Different Geographic Regions in Brazil Characterization of Clinicopathologic Aspects and Relationship With Epstein-Barr Virus Infection Deilson Elgui de Oliveira, MS, Maura M. Bacchi, MD, Eliane S. Abreu, MD, Ligia Niero-Melo, MD, and Carlos E. Bacchi, MD Key Words: Hodgkin disease; Epstein-Barr virus; In situ hybridization; Brazil Abstract We analyzed clinicopathologic data, immunophenotype, and Epstein-Barr virus (EBV) status in 96 cases of Hodgkin disease (HD) in juveniles (younger than 20 years) and adults (20 years or older) from 2 distinctive states in Brazil. We studied 34 juvenile (group 1) and 16 adult (group 2) cases from Ceara and 31 juvenile (group 3) and 15 adult (group 4) cases from São Paulo. Ceara has a socioeconomic profile similar to a developing country; São Paulo is in better economic condition. Mixed cellularity (MC) was the major histologic subtype among groups 1 (22 [65%]), 3 (21 [68%]), and 4 (7 [47%]); nodular sclerosis (NS) was more frequent in group 2 (8 [50%]). EBV infection was observed in 61 cases (64%), including the following (among others): group 1, MC, 22 (65%) and NS, 4 (12%); group 2, NS, 3 (19%) and MC, 2 (12%); group 3, MC, 16 (52%) and NS, 1 (3%); and group 4, MC, 7 (47%). There was predominance of EBV+ HD cases in group 1 compared with group 3. HD in Brazilian patients is highly associated with EBV infection, but geographic differences reflect histologic subtypes and age distribution. Since Thomas Hodgkin first described Hodgkin disease (HD) in 1832, 1 much has been discussed about its cause and pathogenesis. The first clarification of its neoplastic origin was the description of Hodgkin and Reed-Sternberg (H-RS) cells, which are the morphologic hallmarks 2 of this kind of lymphoma. Nodular sclerosis (NS), mixed cellularity (MC), lymphocyte depletion (LD), and nodular lymphocyte predominance (LP) are the 4 histologic subtypes of HD, although some debate exists in the literature about LD being considered a distinct entity. 3 The Epstein-Barr virus (EBV) has been implicated in numerous human diseases, 4,5 including endemic Burkitt lymphoma, 6 undifferentiated nasopharyngeal carcinoma, 7,8 some non-hodgkin lymphomas 9,10 and HD. 11,12 EBV is the causative agent of infectious mononucleosis, and by adult life, virtually every person has serologic findings of a previous EBV infection. Through sensitive molecular biologic techniques, EBV can be found in malignant cells in up to 100% of cases of endemic Burkitt lymphoma and undifferentiated nasopharyngeal carcinoma. 7 Worldwide, EBV has been found in H-RS cells in frequencies that range from 32% (England) 13 to 94% (Peru). 14 This marked variation is observed mainly due to geographic factors, although methods also must be considered. In addition, the existence of different histologic patterns in association with specific epidemiologic aspects 15 suggests that HD has several distinct factors that can contribute to malignant transformation. There are rare and incomplete data about the frequency of HD subtypes in the Brazilian population, and the prevalence of EBV infection in patients with HD is unknown. To gain this information, we studied selected clinical and pathologic features and EBV status in juvenile and adult forms of American Society for Clinical Pathology Am J Clin Pathol 2002;118:

2 Elgui de Oliveira et al / HODGKIN DISEASE IN BRAZIL HD in Brazil. The patients were from 2 distinctive geographic areas: the state of São Paulo (São Paulo) and the state of Ceara (Ceara), in the southeast and northeast parts of the country, respectively. São Paulo is one of the most important industrial sites in Latin America. In 1999, its population was estimated at about 35 million, with 93% living in urban areas. On the other hand, the urban population in Ceara accounts for only 67% of its 7 million habitants. 16 These and other social and demographic indicators indicate a higher living standard in São Paulo, equivalent to that of developed countries, than in Ceara, where the socioeconomic indicators are more like those of developing countries. Herein we present and discuss the characteristics of HD in 2 Brazilian populations and compare them with data available in the literature. Materials and Methods Surgical biopsy specimens from a total of 96 cases of HD, both juvenile (younger than 20 years) and adult (20 years or older) forms, were obtained from medical institutions located in São Paulo (46 cases) and Ceara (50 cases), Brazil. Four groups were created as follows: group 1, 34 juvenile HD cases from Ceara; group 2, 16 adult HD cases from Ceara; group 3, 31 juvenile HD cases from São Paulo; and group 4, 15 adult HD cases from São Paulo. The 2 hospitals in Ceara that provided the cases are public institutions in which basically only patients in the low or very low socioeconomic class are treated. On the other hand, the vast majority of the cases from São Paulo came from private pathology laboratories, in which, in general, the patients belong to the middle or high socioeconomic class. All tissues were formalin-fixed and paraffin-embedded. H&E-stained sections were analyzed histologically, and the cases were classified according to the World Health Organization classification. 17 The cases in which the amount of tissue was enough only to diagnose HD without further classification were designated as HD not otherwise specified. Immunohistochemical analysis with monoclonal antibodies against CD30 (Ki-1 antigen; H-RS cells), CD15 (granulocyte-associated antigen; H-RS cells), CD20 (B cells), and CD45RO (T cell associated antigen) were performed to characterize the immunophenotype of the neoplastic cells. A monoclonal antibody against EBV latent membrane protein 1 (LMP-1) was used to assess expression of this oncogenic viral product. Briefly, deparaffinized sections were immunostained using the standard avidin-biotin-peroxidase technique with the aforementioned primary antibodies. Antigen retrieval was performed with microwave treatment 18 for all markers except LMP-1, for which trypsin was used as the pretreatment. We used 3,3'-diaminobenzidine as the chromogen and counterstained sections with methyl green or Harris hematoxylin. RNA in situ hybridization was performed to detect EBV in H-RS cells by using a biotinylated probe for Epstein-Barr encoded RNAs 1 (EBER-1), a viral transcript present in high levels in infected cells (about 10 7 copies per nucleus). The protocol was described elsewhere. 19 We used descriptive statistics and the chi-square test for results analysis. Results Table 1 and Table 2 summarize the available clinical data for HD cases. There was a male predominance in all groups studied, with the exception of group 1, in which the male/female ratio was 1. In the majority of cases (49/96 [51%]), the cervical lymph node was the anatomic location most frequently involved. Other common sites were supraclavicular (7/96 [7%]), axillary (8/96 [8%]), and inguinal lymph nodes (7/96 [7%]). There was no significant difference in the anatomic site of HD at diagnosis among most of the groups studied. However, patients in group 1 had a higher proportion of cervical lymph node involvement at diagnosis than did patients in group 3 (P <.01). The distribution of HD cases with regard to histologic subtype is given in Table 3. Mixed cellularity Image 1A was the major histologic subtype observed among groups 1, 3, and 4, whereas the NS type was more frequent in group 2. The LD subtype was seen in groups 1, 2, and 4, while the LP subtype was found only in group 3. Table 1 Sex and Age Distribution in 50 Cases of Hodgkin Disease From Ceara and 46 Cases From São Paulo, Brazil Group * M/F Ratio Mean ± SD (y) Coefficient of Variation (%) Median (y) Range (y) 1 (n = 34) 1:1 9.9 ± (n = 16) 1.3: ± (n = 31) 1.8: ± (n = 15) 1.5: ± * For a description of the groups, see the Materials and Methods section. Age 26 Am J Clin Pathol 2002;118:25-30 American Society for Clinical Pathology

3 Hematopathology / ORIGINAL ARTICLE Table 2 Anatomic Location of Hodgkin Disease at Diagnosis in 96 Cases From Brazil * Group Site 1 (n = 34) 2 (n = 16) 3 (n = 31) 4 (n = 15) Total (N = 96) Lymph node Cervical 24 (71) 9 (56) 12 (39) 4 (27) 49 (51) Supraclavicular 2 (6) 0 (0) 3 (10) 2 (13) 7 (7) Axillary 4 (12) 3 (19) 1 (3) 0 (0) 8 (8) Inguinal 1 (3) 1 (6) 3 (10) 2 (13) 7 (7) Other 3 (9) 3 (19) 12 (39) 7 (47) 25 (26) * Data are given as number (percentage). For a description of the groups, see the Materials and Methods section. Table 3 Distribution of Histologic Subtypes of Hodgkin Disease in 96 Cases From Brazil * Mixed Nodular Lymphocyte Lymphocyte Not Otherwise Group Cellularity Sclerosis Depletion Predominance Specified 1 (n = 34) 22 (65) 10 (29) 1 (3) 0 (0) 1 (3) 2 (n = 16) 6 (38) 8 (50) 2 (12) 0 (0) 0 (0) 3 (n = 31) 21 (68) 5 (16) 0 (0) 4 (13) 1 (3) 4 (n = 15) 7 (47) 4 (27) 1 (7) 0 (0) 3 (20) * Data are given as number (percentage). For a description of the groups, see the Materials and Methods section. In virtually all cases of classic HD (MC, NS, and LD subtypes), the immunophenotype of neoplastic cells was CD30+ Image 1B, CD15+ Image 1C, and negative for T- cell markers. A subset of cases showed CD20 expression. A complete panel of markers was not performed in all cases owing to scarcity of tissue in the paraffin blocks. In group 1, 3 cases of MC were CD20+. All LP cases in group 3 were CD20+ and CD30, CD15, and CD45RO, as previously described. 20 Table 4 summarizes these data. EBV infection was assessed by using 2 distinct methods: LMP-1 detection Image 1D by immunohistochemical analysis and RNA in situ hybridization with a biotinylated probe for EBER-1 Image 2. Although both showed similar results, EBV status was based on EBER-1 positivity, as RNA in situ hybridization is more sensitive than immunohistochemical analysis. EBV infection was observed only in H-RS cells in different frequencies for each HD subtype considered. Overall, EBV infection was associated with the MC subtype of HD in groups 1 (P <.0001), 3 (P <.05), and 4 (P <.005). In group 2, we did not observe a statistically significant predominance of EBV infection in any of the HD subtypes. In group 1, EBV+ cells were observed in all cases of the MC subtype and in fewer than half of the NS cases; in group 2, EBV was found in more than one third of the NS cases and in one third of the MC cases; in group 3, EBV was present in about three fourths of the MC cases and one fifth of the NS cases; and in group 4, EBV positivity was found in all cases of MC but no cases of the NS and LD subtypes Table 5. By using the chi-square test, we verified a significant predominance of EBV+ cases in group 1 compared with groups 2 (P <.001) and 3 (P <.05). On the other hand, EBV+ cases among adults were more frequent in group 4 than in group 2 (P <.05) Figure 1. A C Image 1 A, Mixed cellularity Hodgkin disease (H&E, 400). B-D, Immunohistochemical studies showing expression by Hodgkin and Reed-Sternberg cells of CD30 (B, avidin-biotin complex [ABC] method, 400), CD15 (C, ABC method, 400), and latent membrane protein 1 (D, ABC method, 400). B D American Society for Clinical Pathology Am J Clin Pathol 2002;118:

4 Elgui de Oliveira et al / HODGKIN DISEASE IN BRAZIL Table 4 Immunophenotype and Epstein-Barr Virus Status in 92 Cases of Classic Subtypes of Hodgkin Disease From Brazil * Group CD20 CD15 CD30 CD45RO LMP-1 EBER-1 1 (n = 34) 3/34 (9) 33/34 (97) 33/34 (97) 0/34 (0) 27/34 (79) 28/34 (82) 2 (n = 16) 3/16 (19) 6/7 (86) 7/7 (100) 0/7 (0) 2/7 (29) 5/16 (31) 3 (n = 27) 3/24 (12) 17/22 (77) 24/24 (100) 0/23 (0) 17/27 (63) 18/27 (67) 4 (n = 15) 3/10 (30) 11/12 (92) 11/12 (92) 0/11 (0) 8/13 (62) 10/15 (67) EBER-1, Epstein-Barr encoded RNAs 1; LMP-1, latent membrane protein 1. * Four cases of the lymphocyte predominance type were excluded. Data are given as number of cases positive/number of cases tested (percentage). Immunohistochemical analysis or RNA in situ hybridization was performed. For a description of the groups, see the Materials and Methods section. Image 2 Mixed cellularity Hodgkin disease. RNA in situ hybridization for Epstein-Barr virus EBER-1 transcript showing nuclear positivity in virtually all Hodgkin and Reed- Sternberg cells (methyl green counterstaining, 400). Discussion Epidemiologic data on HD have provided important clues regarding its etiopathogenesis. It was proposed that the bimodal age behavior could be best explained by the existence of dissimilar risk factors that also influence the morphologic features of HD In addition, increasing evidence implicates EBV infection in the pathogenesis of HD. 24 Despite the ubiquitous distribution of EBV in the population worldwide, the presence of the EBV genome and viral products restricted to H-RS cells points to some key role of this virus in the development of HD. 25 In HD cases, EBV is found with different frequencies among distinct age groups and geographic areas and even among different subtypes. Higher rates of EBV infection in HD were observed in developing countries such as Peru (94%), 14 Mexico (70%), 26 and Korea (69%), 27 where HD is one of the most common neoplasms in children. Conversely, proportionally low rates of infection were observed in England (32%), 13 the United States (43%), 28 and Germany (58%). 29 Recently, new and interesting data have been added regarding EBV infection and HD in different regions around the world. Kandil et al 30 studied 48 patients with HD from Saudi Arabia and found that half of their cases were EBV+. Furthermore, EBV infection was observed predominantly in patients younger than 14 years (58% vs 28% in patients older than 14 years; P <.08). 30 In 2001, Clarke et al 31 reported a low frequency of EBV infection in HD in women from the United States (53/311 cases; 17.0%). The EBV+ HD cases, which occurred mostly among patients of races other than white and older than 45 years (51%; P <.001), frequently manifested as the NS subtype (53% vs 34% MC) 31 and had a high disease stage (Ann Arbor stages III and IV). A low frequency of EBV infection and a strong association with the NS subtype also were observed in a series of 84 Table 5 Distribution of Epstein-Barr Virus (EBV) Infection and Histologic Subtypes of Hodgkin Disease in 96 Cases From Brazil * Lymphocyte Lymphocyte Not Otherwise Group Mixed Cellularity Nodular Sclerosis Depletion Predominance Specified 1 (n = 34) 22/22 (100) 4/10 (40) 1/1 (100) 0/0 (0) 1/1 (100) 2 (n = 16) 2/6 (33) 3/8 (38) 0/2 (0) 0/0 (0) 0/0 (0) 3 (n = 31) 16/21 (76) 1/5 (20) 0/0 (0) 0/4 (0) 1/1 (100) 4 (n = 15) 7/7 (100) 0/4 (0) 0/1 (0) 0/0 (0) 3/3 (100) * Data are given as number of EBV+ cases/number of cases of the histologic subtype (percentage). For a description of the groups, see the Materials and Methods section. 28 Am J Clin Pathol 2002;118:25-30 American Society for Clinical Pathology

5 Hematopathology / ORIGINAL ARTICLE HD cases from the United Kingdom (23% EBV+), 32 probably representing the profile expected for a population with a high living standard. To address whether the variation in frequency of EBV positivity in HD is due to socioeconomic factors, Takeuchi et al 33 studied HD cases diagnosed in Tokyo between 1955 and EBV was detected in 43 (40.6%) of 106 HD cases, with a decreasing rate of EBV positivity in the NS subtype during this period. 33 The authors claimed that this phenomenon was linked to the improvement of the living standard and changes in lifestyle in Japan during the second half of the 20th century. Largely based on MacMahon s 34 multiple etiology hypothesis for HD, 34 some authors have suggested updated models based on new molecular epidemiologic data. 12,15,24 It was proposed that HD could be divided into 3 entities on the basis of age and EBV status. The first comprises cases of childhood HD, which are invariably EBV+ and more often of the MC subtype. The same features could be verified in the second entity, which affects older adults and can be explained by immunosuppression and EBV reactivation later in life. The third entity affects young adults and has no link to EBV infection; those cases are frequently of the NS subtype and more often associated with a high living standard. Chances are that a distinct infectious agent would be implicated in these cases. 24 In our study, most of the Brazilian patients with HD were males (55/96 [57%]) and younger than 15 years (50/96 [52%]). Mixed cellularity was the predominant histologic subtype, and the EBV genome was detected in 61 (64%) of all cases studied. These data indicate that regarding HD, Brazil has a profile similar to that of some countries in Latin America and Asia. As Brazil is a large, continental, and heterogeneous country, we also split these data to consider 2 distinct geographic regions, São Paulo and Ceara. Data from the Secretary of Health of Brazil show that in 1998, infectious and parasitic diseases and perinatal illness were responsible for 16.3% of all deaths in Ceara, whereas in São Paulo, the same disorders represented 9.2% of the deaths. 35 This is some of the available information emphasizing the socioeconomic differences between these areas. For our cases, at the time of diagnosis patients with HD from Ceara were younger than those from São Paulo. However, in Ceara, EBV infection was more prevalent in juvenile cases of HD, while in São Paulo, it was found predominantly among adults. These data are in agreement with the hypothesis that EBV could have a more important role in the pathogenesis of HD in adults from developed areas and in children from developing regions. However, we did not observe a significant difference in the EBV status among all cases of HD from Ceara (33/50 [66%] EBV+) and all cases from São Paulo (28/46 [61%] EBV+). EBV-Positive Cases by RISH (%) * Juvenile Age Groups Adults Figure 1 Epstein-Barr virus (EBV) infection in 96 cases of Hodgkin disease from Ceara (black bars) and São Paulo (white bars), Brazil. * P <.05. RISH, RNA in situ hybridization. Number of Hodgkin Disease Cases Ceara São Paulo Ceara São Paulo Ceara São Paulo Age Ranges (y) Considering the morphologic features of HD, age, and EBV status for the cases in our study, it was not possible to match our cases with the 3 entities suggested by MacMahon. 34 The profile expected for a developing country (mostly EBV+ and the MC subtype) was present in juvenile cases of HD from both São Paulo and Ceara. Also, interestingly, half of the HD cases of young adults from São Paulo were EBV+ and of the MC subtype, and the matched group from Ceara was predominantly EBV and of the NS subtype Figure 2. It seems that factors other than socioeconomic profile and age distribution influence the relationship among HD development, its morphologic features, and EBV infection. HD continues to be a fascinating disease, and more studies are necessary to understand the peculiarities of its geographic and age distributions and the true influence of EBV infection on its pathogenesis. * MC EBV+ MC EBV NS EBV+ NS EBV Other EBV+ Other EBV Figure 2 Age distribution, Epstein-Barr virus (EBV) infection and histologic subtypes of 96 cases of Hodgkin disease from Ceara (n = 50) and São Paulo (n = 46), Brazil. MC, mixed cellularity; NS, nodular sclerosis. American Society for Clinical Pathology Am J Clin Pathol 2002;118:

6 Elgui de Oliveira et al / HODGKIN DISEASE IN BRAZIL From the Department of Pathology, Botucatu School of Medicine UNESP, Botucatu, Brazil. Address reprint requests to Dr Bacchi: Dept of Pathology, Botucatu School of Medicine UNESP, PO Box 564, Botucatu, Sao Paolo, Brazil Acknowledgments: We are indebted to Paulo Curi, PhD, who provided statistical analysis, and Celene Maria C. Gandin and Marcos Roberto Franchi for expertise in immunostaining. References 1. Hodgkin T. On some morbid appearances of the absorbent glands and spleen. Med Chir Trans. 1832;17: Küppers R, Rajewsky K. The origin of Hodgkin and Reed/Sternberg cells in Hodgkin s disease. Annu Rev Immunol. 1998;16: Mason DY, Banks PM, Chan J, et al. Nodular lymphocyte predominance Hodgkin s disease: a clinicopathological entity. Am J Surg Pathol. 1994;18: Schmidt CW, Misko I. The ecology and pathology of Epstein- Barr virus. Immunol Cell Biol. 1995;73: Griffin BE, Xue S. 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