High Microvascular Density Correlates with Poor Outcome in Patients with Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Rituximab Plus Chemotherapy (R-CT).

Abstract 1948

Poster Board I-971

Survival after treatment of diffuse large B-cell lymphoma (DLBCL) is influenced by differences in the tumor microenvironment. Gene expression profiling (GEP) studies have shown that the angiogenesis-related signature (stromal-2 signature) is prognostically unfavorable. However, the clinical and biological significance of angiogenesis quantified in tumor tissue sections of DLBCL from patients treated with rituximab plus chemotherapy (R-CT) is not yet fully explored. CD31, the platelet adhesion molecule PECAM1, is one of the genes included in the “stromal-2 signature” reported in the GEP studies. The objective of this study was to determine whether the microvessel density (MVD) and microvessel number (MVN) in DLBCL patients treated with R-CT were associated with the clinicopathological features of the tumors and related to the outcome of the patients. The MVD and MVN were assessed in a series of 160 patients with DLBCL from the Leukemia Lymphoma Molecular Profiling Project consortium (LLMPP) 86M /74F; median age 64 yrs. The GEP was investigated in 116 of these including 50 germinal center B (GCB), 55 activated B-cell (ABC) and 11 unclassifiable cases. An independent series of 129 patients from the Catalan Lymphoma-Study Group (GELCAB) (67M/62F; median age 64 yrs) was used to validate the results. Front-line treatment was R-CT in all cases of both series. Tissue Microarrays (TMA) were constructed from pretreatment biopsy specimens of de novo DLBCL. High grade B cell lymphoma otherwise unclassifiable, primary mediastinal B cell lymphoma, T-cell-rich B cell lymphoma, and tumors associated with immunodeficiency were excluded. All cases were stained in an automated immunostainer with an antibody against CD31 (DAKO). The MVD and MVN were quantified using digitalized images of the tumor using Olympus Cell B Basic Imaging Software. Microvessel areas were defined as vascular areas delineated by CD31+ staining. The MVD was calculated as the sum of all microvessel areas divided by the total area analyzed. The MVN was the sum of all identified individual vessels, divided by the total area analyzed. TMAs were independently scored by two observers and discrepancies were resolved over a double-headed microscope. To determine whether the angiogenic values scored using the TMA were representative of the tumor sample, whole tissue sections and TMA cores from the same tumor were evaluated in 40 cases and compared by a linear regression analysis. MVD and MVN were grouped in quartiles when necessary and considered high or low when above or below the 50^th percentile, respectively. Linear correlation analysis between the CD31 (+) MVD results on TMA cores and on the corresponding whole tissue sections in 40 cases showed a good correlation (R²=0.81). In the LLMPP cohort, DLBCL with an ABC profile showed higher MVD than those with GCB profile (p=0.05). In addition, higher MVD was observed in patients with advanced stage (p<0.01), but there was no significant correlation with other clinical features. 5-yr overall survival (OS) according to CD31(+) MVD was 74% vs. 47% for patients with low and high MVD respectively (p=0.0015). Both the International prognostic index (IPI) (relative risk 3.3; p=0.001) and MVD (relative risk 2.2; p<0.001) showed independent prognostic value for OS in a Cox model. In addition, MVD and GEP type (GCB vs. ABC) were also independent predictors of OS. MVN showed no meaningful relation with initial features or with OS. In the validation cohort from the GELCAB, all the above mentioned results were confirmed, including the influence of MVD on OS (5-yr OS 78% vs. 50% for low and high MVD, respectively; p=0.02) that was also independent of IPI in a Cox model. In conclusion, increased MVD is able to discriminate poor-risk patients in DLBCL treated with R-CT independently of the IPI risk groups. This finding highlights the relevance of angiogenesis in the behavior of these tumors and suggests that it may be an important parameter when assessing the impact of new therapies, particularly anti-angiogenic drugs.