Coordinate Regulation of NF-κB Subunit Expression in EBV Negative, but Not EBV Positive, Pediatric Hodgkin’s Lymphoma
Authors: Horton Terzah M. , Sheehan Andrea N , Lopez-Terrada Dolores , Hutchison Robert Edward , Narendra Sonia , Higman Meghan A. , Wu Meng-Fen , Liu Hao 
Published: April 2020

Abstract
The nuclear factor-kB (NF-kB) pathway plays an integral role in Hodgkin’s lymphoma (HL), and may enhance survival of Hodgkin Reed Sternberg (H/RS) cells following chemotherapy by enhancing an anti-apoptotic phenotype. Previous studies have shown that both canonical (NF-kB/RelA or c-Rel) and non-canonical (NF-kB/RelB) NF-kB pathways are constitutively active in adults with HL. Epstein-Barr virus (EBV) infection has also been implicated in the constitutive activation of NF-kB in H/RS cells. Although LMP1 has been postulated to activate NF-kB through processing of the NF-kB subunit p100, it is unclear if this is the sole mechanism of EBV-induced activation of NF-kB and which specific NF-kB pathways (canonical versus non-canonical) are involved. There is also limited published information on EBV activation of NF-kB in pediatric HL.
We have examined specimens from 105 pediatric HL patients (61 male, 85 Caucasian, age range 1–21y [median age 15y], 87 nodular sclerosing, 8 mixed cellularity, 7 lymphocyte predominant, 3 NOS) and 10 lymphoid controls for NF-kB and IkB subunit expression by immunohistochemistry (IHC) using a HL tissue microarray obtained from the BioPathology Center (Columbus, OH) created from samples archived by the Children’s Oncology Group. All patients had intermediate-risk HL and were treated on the same protocol (AHOD0031). After CD30 staining evaluation, 14 (4%) of the 322 HL and control microarray spots (including 3 HL patients) were excluded from analysis for either weak CD30 staining or insufficient H/RS cells. The remaining 102 HL patients (282 spots on the microarray) were examined for both canonical and non-canonical NF-kB pathway subunit expression (RelA, RelB, c-Rel, IkB-a and IKK-a). Slides were also evaluated for EBV status using LMP-1 (IHC) and EBER (in situ hybridization) (concordance: 93%). Staining intensity (3-point scale) and localization (nuclear vs. cytoplasmic) of NF-kB, IkB, and IKK subunits was assessed and compared to controls, and EBER+ HL samples (n=19) were compared to EBER− HL samples (n=83). As previously demonstrated using standard IHC, both canonical (RelA p&lt;0.01, c-Rel, p&lt;0.05) and non-canonical (RelB, p&lt;0.01) NF-kB subunits, as well as IkB-a (p&lt;0.01) and IKK-a (p&lt;0.01) were elevated in the HL patients when compared to controls (Wilcoxon rank-sum test). EBV+ and EBV− tumors showed similar levels of NF-kB activation in both canonical and non-canonical pathways. Overall, as expected, there was a strong correlation between nuclear canonical pathway NF-kB c-Rel expression and IkB-a (Spearman correlation coefficient R=0.36, p&lt;0.01,), as well as a trend toward correlation between the non-canonical NFk-B/Rel B and IKK-a (R=0.25, p&lt;0.1) and canonical NF-kB-RelA and IkB-a (R=0.26, p&lt;0.1). Surprisingly, after correcting for multiple comparisons by Holm’s method, there was a very strong correlation between nuclear RelB and c-Rel expression (0.5, p&lt;0.01) as well as between cytoplasmic IkB-a and IKK-a (R=0.37, p&lt;0.01), suggesting coordinate upregulation of both the canonical and non-canonical NF-kB pathways. Although there were no statistically significant differences in NF-kB, IkB and IKK subunit expression in the EBV+ vs. EBV− HL tumors (Wilcoxon rank sum test), there were substantial differences in the coordinate regulation of NF-kB subunits in EBV+ vs. EBV− HL samples. EBV+ samples showed little evidence of NF-kB coordinate upregulation. EBV− samples, however, showed significant coordinate regulation of NF-kB pathway components, with strong correlations between canonical and non-canonical NF-kB pathway subunit expression (nuclear c-Rel- IkB-a [R=0.39, p&lt;0.01], cytoplasmic RelB-IKK-a [R=0.39, p&lt;0.01], nuclear c-Rel-RelB [R=0.56, p&lt;0.01] and cytoplasmic IkB-a-IKK-a [R=0.42, p&lt;0.01). In addition to being the first report to examine NF-kB pathway activation by tissue microarray, this is the first report examining NF-kB activation pathway in EBV+ vs. EBV− tumors in pediatric patients. These data suggest that pediatric HL has similar NF-kB pathway activation to adults, and that EBV+ HL are less likely to have canonical and non-canonical NF-kB coordinate regulation than EBV− HL.