Received: 21 March 2019 Accepted: 25 March 2019 DOI: 10.1002/pbc.27757 Pediatric Blood & Cancer The American Society of Pediatric Hematology/Oncology LETTER TO THE EDITOR PD-L1 expression in pediatric Epstein-Barr virus positive classic Hodgkin lymphoma is not associated with 9p24.1 amplification To the Editor: Dilly-Feldis et al 1 have recently investigated expres- sion of programmed death 1/programmed cell death ligand 1 (PD1/PD- L1) in 42 French children with classic Hodgkin lymphoma (cHL). They reported that PD1/PD-L1 proteins were expressed at similar extent in 31 patients cured or in remission (mean age 15 years), and in 11 patients relapsed or refractory (mean age 14 years). Moreover, they found that PD-L1 protein was more intensely expressed in cells of 11 Epstein-Barr virus (EBV) positive cases. cHL is rarely observed in children <14 years of age in Western coun- tries. In developing countries of Asia, Africa, and South America, the disease is more common even in early infancy (<5 years of age) and is often associated with EBV infection. 2–4 It was speculated that early onset of cHL in those countries is probably related to early EBV infec- tion of children occurring in >90% of cases by the age of 6 years. 2 cHL has an inflammatory-like background. We speculated on the possibility that immaturity of the immune system during early infancy might cause dysregulation of PD1/PD-L1 expression. PD1/PD-L1 proteins were investigated in 53 cases of cHL occurring in children <14 years of age. Tissue sections were immunostained for PD1, PD-L1 (22C3), CD4, CD8, and CD68. Comparisons were made between children younger than 5 years (20 cases) or older (33 cases), between EBV+ (40 cases) and EBV– (13 cases) cHL, and between nodular sclerosis (NS, 30 cases) and mixed cellularity (MC, 23 cases) subtypes. It was found that the age of the children did not affect the expression of any of the investigated antigens. On the contrary, percentages of CD8+ T lymphocytes and PD-L1+ cells were significantly higher in EBV+ cases as compared with EBV– cases (Figure 1); a further difference was noted in the percentage of PD1+ cells in tumor areas that was significantly higher in NS subtype (P < .01; Student’s t test) as compared with MC, independently of EBV infection. Thus, our findings confirm previous reports 1,5–7 and indicate that EBV infection and histological subtypes are more relevant than age in determining cellular composition of cHL lesions even during early infancy. It was reported that PD-L1 expression is upregulated by EBV infection 8,9 or by chromosome 9p24.1 alterations, 7–9 and that the two mechanisms are mutually exclusive. 8,9 We have investigated by fluorescence in situ hybridization (FISH; Empire Genomics, NY) alterations of chromosome 9p24.1 in 12 cases. Results indicated that seven of seven pediatric EBV+ cases were negative for 9p24.1 amplification and that two of three EBV – cases (MC subtype) had evidence of amplification (probe ratio 4:1 and 5:1) (Figure 1D); two further EBV – cases were not evaluable. In conclusion, our findings are consistent with the view that EBV infection and 9p24.1 amplification are alternative mechanisms of PD-L1 upregulation in pediatric cHL, and that the microenvironment of EBV+ cases, perhaps through IFN- , is particularly effective in inducing PD-L1 expression. ACKNOWLEDGMENTS This study was supported by a grant from the Istituto Pasteur Italia Fondazione Cenci Bolognetti and by a grant “Programmi di Coop- erazione Internazionale Didattica e Scientifica con i paesi in via di sviluppo,” from Sapienza University Rome. The study is part of the collaborative scientific program “Baghdad Resolve” between Sapienza University and the Children’s Welfare Teaching Hospital, Baghdad Col- lege of Medicine, Iraq. CONFLICT OF INTEREST The authors declare that there is no conflict of interest. ORCID Stefania Uccini https://orcid.org/0000-0001-9318-7860 Stefania Uccini 1 Mazin F. Al-Jadiry 2 Giuseppina Pepe 3 Stefania Scarpino 3 Salma A. Al-Hadad 2 Luigi Ruco 3 1 Istituto Pasteur Italia, Rome, Italy 2 Children’s Welfare Teaching Hospital, Baghdad College of Medicine, Baghdad, Iraq 3 Department of Clinical and Molecular Medicine, Patology Unit, Sant’Andrea Hospital, Sapienza University, Rome, Italy Correspondence Luigi Ruco, Department of Clinical and Molecular Medicine, Pathology Unit, Sant’Andrea Hospital, Sapienza University, Via di Grottarossa 1035, 00189 Rome, Italy. Email: luigi.ruco@uniroma1.it Pediatr Blood Cancer. 2019;e27757. c  2019 Wiley Periodicals, Inc. 1 of 2 wileyonlinelibrary.com/journal/pbc https://doi.org/10.1002/pbc.27757