Expression Level, Tissue Distribution Pattern and Prognostic Impact of Vascular Endothelial Growth Factor (VEGF) and Its Receptors Flt-1 and KDR in Different Subtypes of Non-Hodgkin Lymphoma (NHL).
Authors: Jørgensen Judit M. , Sørensen Flemming B. , Bendix Knud , Nielsen Johan L. , Funder Anette , Tainola Tapio , Sørensen Annette B. , d’Amore Francesco 
Published: April 2020

Abstract
Background: VEGF is the main regulator of tumour angiogenesis via its receptors, Flt-1 and KDR. It is expressed by tumour cells and normal stroma cells, and, beyond angiogenesis, it can stimulate tumour growth via autocrine and paracrine pathways.
Material and methods: Pre-therapeutic lymph-node biopsies from 152 NHL cases were investigated. These included 62 cases of follicular lymphoma (FL), 20 of which had histologically verified transformation to aggressive lymphoma, 46 cases of diffuse large B-cell lymphoma (DLBCL) and 44 cases of peripheral T-cell lymphoma (PTCL). VEGF, Flt-1 and KDR were investigated at protein level by immunohistochemistry, and at mRNA level by non-isotopic’ in situ’ hybridisation. For the latter, a fluorescein-conjugated probe was obtained from pGEM3Zf plasmid containing 1–581 bp of human VEGF cDNA. The staining intensity was scored semiquantitatively (0–3) and subdivided in ‘focal’or ‘diffuse’depending on morphological evaluation. Expression of VEGF and its receptors was correlated to the morphologically estimated microvascular density (Chalkley method).
Results: VEGF protein expression was detected in 88 % FL, 91% DLBCL and 95% PTCL. As expected, PTCL had a stronger and more diffuse staining pattern than FL and DLBCL. Flt-1 protein expression was detected in 95 % FL, 96% DLBCL and in virtually all cases of PTCL. Lymphoma cells expressing KDR were also seen in all biopsies. A diffuse staining pattern was more frequent in FL. VEGF mRNA was expressed in all subtypes at similar levels although DLBCL displayed a diffuse staining pattern more frequently than other histologies. Higher VEGF protein expression in lymphoma cells was correlated with higher microvascular density (p=0.005), while a negative correlation was observed between receptor expression (Flt-1 and KDR) and Chalkley count (p=0.01 and 0.003, respectively). Patients with FL, who had diffuse VEGF staining of lymphoma cells, had poorer overall survival than those with focal staining (p=0.04). Diffuse KDR staining was correlated with a higher risk of transformation (p=0.05). In fact, a remarkably high microvessel density was found in interfollicular as opposed intrafollicular areas and high interfollicular Chalkley count was correlated with histologic transformation to DLBCL (p=0.01). In ‘de novo’ DLBCL, high KDR expression was correlated with poor treatment response (p=0.03) and a grade 3 KDR intensity in lymphoma cells had a significant adverse impact on overall survival (p&lt;0.001). In PTCL, a diffuse tissue distribution of VEGF mRNA correlated to poorer overall survival (p=0.0015). Flt-1 expression had no influence on outcome in the investigated lymphoma subtypes
Conclusion: VEGF and its receptors are expressed by lymphoma cells in all investigated NHL subtypes. The staining pattern and intensity of VEGF and KDR differed in the three subtypes, while Flt-1 distribution was similar. High VEGF and KDR expression had adverse prognostic impact in DLBCL and PTCL.