Stromal Incorporation of VEGFR-1+, CD68+ and α-SMA+ Hemangiogenic Cells Correlates with Histologic Subtype in Non-Hodgkin’s Lymphoma.
Authors: Ruan Jia , Hyjek Elizabeth , Hooper Andrea T. , Vincent Loic , Kermani Pouneh , Christos Paul J. , Ikalowych Sherry , Coleman Morton , Hempstead Barbara , Leonard John P. , Chadburn Amy , Rafii Shahin 
Published: October 2019

Abstract
BACKGROUND: Tumor stromal environment has been increasingly recognized to contribute to tumorigenesis. Vascular endothelial growth factor receptor-1+ (VEGFR-1+) hematopoietic cells and alpha-smooth muscle actin+ (α-SMA+) stromal cells both contribute to tumor neo-angiogenesis. However, their roles in promoting neo-angiogenesis specifically in human lymphomas remain unknown.
METHODS: We examined the spatial localization of vascular and stromal cells expressing CD34 (vasculature), α-SMA (stromal cells), VEGFR-1 (hematopoietic cells and neo-vessels) and CD68 (myelomonocytic hematopoietic cells) by immunohistochemistry in 42 cases of non-Hodgkin’s lymphoma (NHL) specimens, which include diffuse large B-cell lymphoma (DLBCL, n=28), Burkitt lymphoma (BL, n=2), follicular lymphoma (FL, n=7), and chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL/SLL, n=5).
RESULTS: There was a significant increase in CD68+ hematopoietic cells and a profound increase in the tissue hemangiogenic index, as defined by the degree of infiltration of both VEGFR-1+ neo-vessels and CD68+ cells, in aggressive lymphomas including DLBCL as compared to the indolent subtypes. Specifically, CD68 cell counts (mean±S.E. in 200X high power field (HPF)) for aggressive vs. indolent vs. benign hyperplasia were: 235.48±16.91 (n=30) vs. 35.98±4.48 (n=12) vs. 79.06±12.41 (n=5), p<0.0001 by ANOVA test; hemangiogenic index for DLBCL vs. CLL vs. FL: 18.14±1.27% (n=5) vs. 3.39±0.97% (n=4) vs. 6.08±1.26% (n=5), p< 0.0001. Transformed DLBCL (from indolent subtypes) had a similar increase in CD68+ cells compared to de novo DLBCL (193.97±58.10 (n=5) vs. 241.77±18.15 (n=23), p=0.81). In DLBCL, CD68+ cells were localized to the peri-endothelial region of the VEGFR-1+ neo-vessels and stromal compartment. Remarkably, although the expression of alpha-smooth muscle actin (α-SMA) was barely detectable in the aggressive subtypes, there was a profound diffuse increase of α-SMA throughout the stromal compartment of CLL/SLL. Surprisingly, there was no correlation between the CD34+ microvessel density (MVD) and the lymphoma subtypes (aggressive vs. indolent vs. benign hyperplasia: 39.29±3.42 vs. 41.88±5.38 vs. 47.92±5.84, p=0.61).
CONCLUSIONS: These data introduce the novel concept that the extent of vessel density has no correlation with the histologic grade of lymphomas. However, the stromal hemangiogenic index, as quantified by the incorporation of CD68+, VEGFR-1+, and α-SMA+ cells, correlates with NHL subtypes. Increased incorporation of pro-angiogenic CD68+ cells and diminished localization of α-SMA+ cells to the peri-vascular zone may contribute to enhanced neo-angiogenesis and tumor growth in DLBCL; while decrease in the CD68+ cells and increase in the α-SMA+ cells may promote neo-vessel stability in CLL/SLL. Thus stromal hemangiogenic components in lymphoma could potentially be targeted for therapeutic intervention.