Pediatr Blood Cancer Low Expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 is Associated With Poor Prognosis in Pediatric Adrenocortical Tumors (ACT) Fabı ´ola A. Leite, MD, MSc, 1 Regia C. P. Lira, MSc, 1 Paola F. Fedatto, MSc, 1 Sonir R. R. Antonini, MD, PhD, 1 Carlos E. Martinelli Jr, MD, PhD, 1 Margaret de Castro, MD, PhD, 2 Luciano Neder, MD, PhD, 3 Leandra N. Z. Ramalho, MD, PhD, 3 Silvio Tucci Jr, MD, PhD, 4 Maria J. Mastelaro, MD, PhD, 5 Ana L. Seidinger, PhD, 5 Izilda A. Cardinalli, MD, 5 Jose ´ A. Yunes, PhD, 5 Silvia R. Brandalise, MD, PhD, 5 Luiz G. Tone, MD, PhD, 1 and Carlos A. Scrideli, MD, PhD 1* INTRODUCTION Adrenocortical tumors (ACT) are very rare in children and adolescents and represent about 0.2% of all pediatric cancers [1–4]. However, in Southern Brazil the incidence is 10–15 times more frequent than the worldwide occurrence and an inherited germline p.R337H mutation in the TP53 gene is considered to be responsible for this higher incidence and has been detected in over 80% of Brazilian patients with childhood ACT [5–7]. The differentiation of malignant and benign forms of childhood ACT is very difficult and, to date, there are no histological or molecular markers that can reliably distinguish them [2,3,8–10]. Also, in contrast to what is observed in adults, histological classification by the Weiss criteria does not accurately predict clinical outcome and is not entirely applicable to pediatric tumors [11,12]. Due to the rarity of these tumors, their molecular pathogenesis is poorly understood and several studies have been conducted to try to determine potential markers associated with biological behavior and prognosis [3,12–14]. In several human cancers, low expression of the human histocompatibility complex (HLA) genes, especially class II genes, has been associated with more aggressive disease [15–18], including adult adrenocortical tumors [19]. In pediatric ACT, only one study conducted on a small number of patients and using microarray technology described lower expression levels of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes in carcinomas compared to adenomas [20]. The aim of the present study was to evaluate the expression profile of these HLA class II genes and their association with biological and clinical features of pediatric ACT. MATERIALS AND METHODS Subjects and Tissue Samples The present study analyzed 58 of 60 consecutive pediatric patients with ACT diagnosed between October 1991 and July 2009 at two reference centers in Southeast Brazil. Two patients were excluded due to lack of good quality RNA for the study. Ten non- neoplastic adrenal pediatric samples collected during nephrectomy due to Wilms’ tumor and before chemotherapy were used as control. Disease stage was based on the modified Sandrini’s classification of Background. Low expression of HLA class II antigens has been associated with more aggressive disease in several human malignan- cies including adult adrenocortical tumors (ACT), but their clinical relevance in pediatric ACT needs to be investigated. Procedure. This study analyzed the expression profile of three class II histocompati- bility genes (HLA-DRA, HLA-DPA1, and HLA-DPB1) in 58 consecutive pediatric ACT (13 adenomas and 45 carcinomas) by quantitative real time PCR and their association with clinical and biological features. HLA-DPA1 protein level was determined by immunohistochemistry. Results. A significant association (P < 0.01) was observed between lower expression levels of the three genes analyzed and poor prognostic factors such as age 4 years, tumor size 200 cm 3 , tumor weight 100 g, and metastatic disease; the presence of an unfavorable event and death. Underexpression of the HLA-DRA, HLA-DPA1, and HLA-DPB1 genes were associated with lower 5-year event-free survival (EFS) (P ¼ 0.017, P < 0.001, and P ¼ 0.017, respectively). Cox multivariate analysis showed that HLA- DPA1 was an independent prognostic factor (P ¼ 0.029) when analyzed in association with stage IV, age and tumor size. Significantly lower EFS was also observed in patients with negative/weak immunostaining for HLA-DPA1 (P ¼ 0.002). Similar results were observed when only patients classified as having carcinomas were analyzed. Conclusion. Our results suggest that lower expression of HLA-DRA, HLA-DPA1, and HLA-DPB1 genes may contribute to more aggressive disease in pediatric ACT. HLA- DPA1 immunostaining may represent potential aggressiveness marker in this tumor. Pediatr Blood Cancer # 2014 Wiley Periodicals, Inc. Key words: adrenocortical tumors; children; HLA class II; HLA-DPA1; prognosis Additional Supporting Information may be found in the online version of this article at the publishers web-site. 1 Department of Pediatrics, Ribeira ˜o Preto Medicine School, University of Sa ˜o Paulo, Ribeira ˜o Preto, Brazil; 2 Department of Internal Medicine, Ribeira ˜o Preto Medicine School, University of Sa ˜o Paulo, Ribeira ˜o Preto, Brazil; 3 Department of Pathology, Ribeira ˜o Preto Medicine School, University of Sa ˜o Paulo, Ribeira ˜o Preto, Brazil; 4 Department of Surgery, Ribeira ˜o Preto Medicine School, University of Sa ˜o Paulo, Ribeira ˜o Preto, Brazil; 5 Centro Infantil Boldrini/State University of Campinas, Campinas, Brazil Grant sponsor: Fundac ¸a ˜o de Amparo a Pesquisa do Estado de Sa ˜o Paulo (FAPESP); Grant number: 2010/7020-9 Conflict of interest: Nothing to declare.  Correspondence to: Carlos A. Scrideli, Department of Pediatrics, Ribeira ˜o Preto Medicine School, University of Sa ˜o Paulo, Avenida Bandeirantes 3900, 14049-900 Ribeira ˜o Preto, SP, Brazil. E-mail: scrideli@fmrp.usp.br Received 6 March 2014; Accepted 5 May 2014  C 2014 Wiley Periodicals, Inc. DOI 10.1002/pbc.25118 Published online in Wiley Online Library (wileyonlinelibrary.com).