Results and Discussion

There were 84 suitable cases for analysis. The median follow up of the living patients was 14.3 years and 20 patients underwent transformation. The IPI was significant for both OS and risk of transformation (RT) in univariate analysis (P=0.0001 and P=0.01, respectively). Estimated 10-year OS and RT for all patients were 65% and 20%, respectively. TVD₉₀ ranged from 50.3 to 144 microns (median 78.4) and these data were divided into quartiles. In univariate analysis the quartile (21 cases) with low TVD₉₀ (i.e. high MVD) showed inferior OS (P=0.0001) and an increased RT (P=0.01) (Figure 2). These 21 cases are defined as Low TVD₉₀ while the three other quartiles (63 cases) are defined as High TVD₉₀. Clinical characteristics are summarized in Table 1. As most cases showed a distinct perifollicular pattern, we correlated TVD₉₀ with follicle size in each case. There was a significant association between low TVD₉₀ and small follicle size (χ², P=0.01). Low TVD₉₀ also correlated with younger age (χ², P=0.034), increased IPI (χ², P=0.01) and increased CD68⁺ macrophages (χ², P=0.025). In univariate analysis, TVD₉₀ and IPI are significant for both OS and RT, but not age or follicle size. In this cohort, CD68⁺ macrophages predicted OS (P=0.0004), but not RT (P=0.38). In a Cox’s model including TVD₉₀, CD68⁺ and IPI, all three markers were independent variables for OS, TVD₉₀ (RR=2.5, 95% CI=1.1–5.0, P=0.01), CD68⁺ (RR=2.2, 95% CI=1.0–5.0, P=0.05) and IPI (RR=2.9, 95% CI=1.5–5.5, P=0.001). Finally, in a Cox’s model for RT, only IPI significantly predicted RT (P=0.008), while TVD₉₀ was of borderline significance (P=0.08). Angiogenesis plays a crucial role in the growth and progression of human solid tumors.13 In most tumor types, increased MVD correlates with increased disease progression and decreased survival. Similar results have been reported in hematopoietic tumors, including multiple myeloma and lymphoma.3,4,14,15 In murine lymphoma models and in lymphoma patients, circulating endothelial cells and serum vascular endothelial growth factor (VEGF) levels appear to correlate with lymphoma volume and increased angiogenesis.16,17 In contrast, previous studies of MVD in FL have resulted in conflicting results. Koster et al. showed increased MVD to be associated with a more favorable OS in a series of 36 uniformly treated patients given CVP and interferon (IFN) α2b followed by IFN maintenance.6 In a later report, Jorgensen et al. analyzed 107 FL cases with heterogeneous treatments and found increased interfollicular MVD predicted inferior OS and increased transformation to DLBCL.7 In both studies, MVD was calculated using quantification of focal “vessel hot spots” within the tumor. In FL, however, the vessels show a heterogeneous distribution. Therefore, in order to avoid a scoring bias, we studied whole sections of FL biopsies and quantified automatically the average distance from CD34 stained vessel that included 90% of tumor cells: TVD₉₀. We believe this is a more accurate measurement of vessel density. Because of vessel predominance within the interfollicular and perifollicular areas, TVD₉₀ was correlated with the average size of the follicles in each case. Interestingly, cases with low TVD₉₀ correlated with small sized follicles although follicle size itself did not affect survival or risk of transformation.

In the current study, all 84 patients were treated uniformly with multi-agent chemotherapy and radiation, while in the series by Koster et al. all 36 patients were treated with CVP chemotherapy and IFN followed by IFN maintenance. In addition to the difference in cohort size, different therapeutic regimens may explain the contradictory results. IFN has both immunomodulatory and anti-angiogenic effects and thus may have been more effective in tumors with increased MVD.18 Radiation has been shown to induce tumor cells to secrete cytokines capable of inhibiting apoptosis in endothelial cells, thereby diminishing treatment response.19,20 It remains possible that this treatment modality influenced survival and transformation risk in our study.

It is well known that angiogenesis in cancer is critically influenced by the local tumor microenvironment.21 Using the same uniformly treated cohort, we previously showed that increased numbers of Lymphoma-Associated Macrophages (LAM) is associated with adverse outcome.9 Similar to solid tumor-associated macrophages, these LAM, possibly originating from bone marrow derived myeloid cells, may be attracted by hypoxia and tumor-derived chemotactic factors and show a distinct phenotype that promotes angiogenesis.22–24 Consistent with this hypothesis, we show a significant association between TVD₉₀ and LAM.

In summary, this study confirms the clinical relevance of increased angiogenesis affecting both FL survival and transformation risk in a series of advanced-stage FL patients uniformly treated with chemotherapy and radiotherapy. These findings suggest MVD as a useful biomarker in initial therapeutic decisions of patients with FL, and thus may provide a rationale for trials of anti-angiogenic therapy in FL patients with increased MVD.