Bone Marrow Angiogenesis in Chronic Lymphocytic Leukemia: Relationship to Classical and Modern Prognostic Factors.
Authors: Smolej Lukas , Zak Pavel , Benesova Petra , Belada David , Hrudkova Monika , Maly Jaroslav 
Published: April 2020

Abstract
Background: Angiogenesis is considered a potential prognostic factor in chronic lymphocytic leukemia (CLL). Elevated levels of various angiogenic factors have been identified in plasma or serum of CLL patients. However, results of studies assessing bone marrow neovascularization in CLL are controversial, in part due to different antibodies used for immunohistochemical identification of endothelial cells and different methods of assessing microvessel density (MVD). Moreover, there are insufficient data regarding relationship of bone marrow angiogenesis to other prognostic markers in CLL such as clinical stage, pattern of marrow infiltration, genetic abnormalities detected by fluorescent in situ hybridization (FISH), or mutation status of immunoglobulin heavy-chain variable-region genes (IgVH).
Aims: To quantify MVD in bone marrow biopsies from CLL patients and control group and to assess its relationship to other prognostic factors.
Methods: We analyzed bone marrow biopsy specimens from 22 untreated patients with CLL (16 males, 6 females, median age 62 years, range, 31–74). Control group consisted of 17 biopsies from individuals without evidence of malignant disease in bone marrow. Neovascularization was assessed using immunohistochemical staining of endothelial cells in bone marrow trephine biopsies with anti-CD34 monoclonal antibody. Microvessel density was assessed under light microscope equipped with image analysis software and calculated using hot spot method, i.e. identification of three loci with highest accumulation of microvessels under low (100x) magnification and counting microvessels in three high-power fields (400x magnification) per hot spot. MVD was expressed as mean number of microvessels per mm2. CLL cohort was further divided into subgroups according to clinical course (stable, n=11 vs. progressive, n=11), Rai stage (0, n=9 vs. I-IV, n=13), pattern of marrow infiltration (non-diffuse, n=11 vs. diffuse, n=11), genetic abnormalities (favourable, i.e. no abnormality or del13q14 as a sole aberration, n=10 vs. unfavourable, i.e. all other abnormalities, n=11), and IgVH mutation status (mutated, n=7 vs. unmutated, n=14).
Results: MVD was significantly elevated in CLL group in comparison to controls (mean ± standard deviation [SD], 75.6 ± 50.6, 95% confidence interval [CI], 53.2–98.1/mm2 vs. 47.4 ± 21.8, 95% CI, 36.2–58.6/mm2, p=0.039). However, there were no significant MVD differences between CLL subgroups with regard to clinical course, pattern of marrow infiltration, Rai stage, FISH abnormalities or IgVH mutation status. Interestingly, when each subgroup was compared to controls, only patients with diffuse bone marrow infiltration (p=0.011), Rai stage I-IV (p=0.014) and mutated IgVH genes (p=0.019) had significantly increased MVD.
Conclusions: This study shows that microvessel density is significantly elevated in CLL. However, this difference is maintained in three subgroups only: diffuse pattern of infiltration, mutated IgVH genes and Rai stage I-IV. We did not observe significant MVD differences between CLL subgroups with regard to classical or modern prognostic factors. Large prospective studies are necessary to confirm our results and elucidate the real clinical relevance of bone marrow angiogenesis in CLL.