Hypoxia-Inducible Factor-Alpha (HIF-α) Activation in Non-Hodgkin’s Lymphoma (NHL): Relationship to the Thioredoxin Family and Correlation with Survival by Tissue Microarray (TMA).
Authors: Evens Andrew M. , Schumacker Paul T. , Helenowski Irene B. , Singh Amar , Dokic Danijela , Keswani Anjeni , Kordeluk Elizabeth , Raji Adekunle , Winter Jane N. , Jovanovic Borko D. , Holmgren Arne , Nelson Beverly P. , Gordon Leo I. 
Published: April 2020

Abstract
HIFs are a family of heterodimeric transcription factors that activate multiple oncogenic signaling pathways. Thioredoxins, including thioredoxin-1 (Trx1) and thioredoxin reductases-1 and -2 (TrxR1 and TrxR2), regulate protein redox state and promote HIF stabilization. Elevated HIF and thioredoxin levels are associated with poor prognosis in solid tumors. We studied HIF-1α and HIF-2α in TMAs from diffuse large B-cell (DLBCL) and follicular (FL) lymphoma patient specimens, and thioredoxins and HIF in NHL cell lines by immunoblotting. Different levels of HIF expression were seen as 54% of DLBCL TMA cases had moderate-to-high HIF-1α expression compared with 20% of FL (p=0.001). 44% of DLBCL versus 11% of FL cases had moderate-to-high expression of both HIF-1α and HIF-2α (p=0.0017); while 56% of the DLBCL and 32% of FL showed at least low expression of both HIF-1α and 2α (p=0.042). On the other hand, 27% of FL and 25% of DLBCL samples had no detectable expression of either HIF-1α or HIF-2α.
TMA Analysis for HIF Follicular lymphoma (n=45) Diffuse large B-cell lymphoma (n=37) HIF-1α expression* p value** *Levels of HIF expression (%) were determined to be approximate medians of underlying empirical mass functions. 1 FL and 2 DLBCL specimens contained inadequate material for HIF-2α evaluation. **p values for comparison of FL with DLBCL using Fisher’s exact test. Absent (0%) 24/45 (53%) 13/37 (35%) 0.12 Low (1–10%) 12/45 (27%) 4/37 (11%) 0.10 Moderate (11–49%) 5/45 (11%) 10/37 (27%) 0.09 High (50–100%) 4/45 (9%) 10/37 (27%) 0.04 HIF-2α expression* Absent (0%) 19/44 (43%) 12/35 (34%) 0.49 Low (1–10%) 3/44 (7%) 5/35 (14%) 0.46 Moderate (11–49%) 5/44 (11%) 9/35 (26%) 0.14 High (50–100%) 17/44 (39%) 9/35 (26%) 0.24
With all newly-diagnosed FL and DLBCL cases combined (median follow-up 26 months), high expression of both HIF-1α and HIF-2α (n=11) was associated with 2-year event-free survival (EFS) of 62% and overall survival (OS) 80%, compared with 2-year EFS of 72% and OS 88% for patients with &lt;high HIF-1α and HIF-2α (n=48) expression (p=0.03 and p=0.16, respectively). High HIF-1α alone was associated with inferior 2-year EFS compared with &lt;high expression (31% vs 79%, p=0.005) with 2-year OS of 63% vs 89% (p=0.03). Analysis of FL and DLBCL subgroups showed similar inferior survival trends with high HIF-1α/HIF-2α expression. In addition, the NHL cell lines SUDHL4 and Raji demonstrated evidence of normoxic HIF-2α stabilization that was not seen in normal lymphocytes, while a small amount of normoxic HIF-1α stabilization was also seen. We found increased TrxR1 expression in Namalwa, HF1, SUDHL4, Raji, and Ramos cell lines, while only Namalwa, HF1 and SUDHL4 showed Trx1 and TrxR2 activation. These data demonstrate for the first time that HIF and the thioredoxins are activated in NHL and that HIF expression may influence prognosis.