Analysis of angiogenic and stromal biomarkers in a large malignant mesothelioma cohort

Highlights

• Agents targeting angiogenic pathways such as bevacizumab have demonstrated efficacy in mesothelioma. An analysis of the combination of stromal and vascular biomarkers is relevant to the use of multi-angiokinase targets of PDGF, FGF and VEGF in malignant mesothelioma to help determine their prognostic implications.

• High CD31 was an independent poor prognostic factor and high PDGF-CC expression was associated with poor survival in mesothelioma.

• There was a high incidence of positive VEGF and FGFR IHC in the large mesothelioma cohort but no significant correlation between prognosis and immune expression was found.

Abstract

Background

Malignant mesothelioma (MM) is an aggressive malignancy of the pleura and other mesothelial membranes. Agents targeting vascular endothelial growth factor (VEGF) such as bevacizumab; and multi-kinase inhibitors such as nintedanib [angiokinase inhibitor of VEGF, platelet-derived growth factor (PDGF) receptor and fibroblast growth factor receptor (FGFR)] have recently demonstrated efficacy in MM.

Methods

Tissue microarrays (TMAs) were created from formalin-fixed, paraffin-embedded tissue samples obtained from 326 patients with MM who were treated surgically. PDGF-CC, FGFR-1, VEGF and CD31 expression were analysed by immunohistochemical (IHC) staining. The H-score method assigned a score of 0–300 to each sample, based on the percentage of cells stained at different intensities. CD31 was evaluated via Chalkley’s method to evaluate microvessel density. We evaluated the association between expression of the biomarkers, clinicopathological factors and outcomes, in patients with MM.

Results

CD31 high (≥5) was seen in only 31/302 (10.3%) irrespective of histology. PDGF-CC high (≥150) was seen in 203 /310 (65%) of all samples. VEGF high (≥80) was seen in 219/322 (68%) of all MM with 143/209 (68%) of epithelioid histology. FGFR-1 high (≥40) was seen in 127/310 (41%) of all MM. There was no association of VEGF and FGFR-1 IHC with survival nor differences between histological subtypes.

There was a non-significant trend towards poorer survival in epithelioid tumours with increased PDGF-CC expression (OS 18.5 vs 13.2 months; HR 0.7928; 95% CI 0.5958 to 1.055, P = 0.1110). High CD31 score was associated with significantly poorer survival (OS 12 vs 8.6 months; HR 0.48; 95% CI 0.2873 to 0.7941, P = 0.0044). Of the 31 patients with high CD31 scores; 23/31 (74%) were also high for PDGF-CC and 20/31 (64%) with high VEGF scores. CD31 was found to be an independent prognostic factor in multivariate analysis (HR 1.540; 95% CI 1.018 to 2.330; p = 0.041).

Conclusions

High CD31 was an independent poor prognostic factor and high PDGF-CC expression was associated with poor survival in MM. Abrogating these pathways may have prognostic implications.

Introduction

Angiogenesis is a complex process involving the interaction between tumour cells, growth factors and cells within the tumour microenvironment (TME). The purported primary stimulus for angiogenesis in the TME is the hypoxia-driven activation of hypoxia-inducible factor-1α, and the subsequent activation of VEGF, along with many other growth factors including platelet derived growth factors, endothelial growth factors and fibroblast growth factor.[1], [2], [3] The interaction between these cells within the TME and the supporting growth factors induce pro-inflammatory mediators which supports proliferation and survival of malignant cells, stromal remodelling and propagates angiogenesis-related factors.[4], [5], [6], [7]

VEGF is a growth factor that is linked to angiogenesis and stimulates neovascularization of tumours. In malignant mesothelioma (MM), elevated levels of VEGF and its receptor have been detected by immunohistochemistry^8,9 and correlated with the density of microvessels, increased tumour necrosis and poorer survival.¹⁰

A wide range of VEGFR tyrosine kinase inhibitors and monoclonal antibodies against VEGF (such as bevacizumab) have been trialled in mesothelioma. The Mesothelioma Avastin Cisplatin Pemetrexed Study (MAPS) study by the French Cooperative Thoracic Intergroup (IFCT) showed the addition of bevacizumab to pemetrexed plus cisplatin significantly improved overall survival (OS) from 16.1 months with doublet chemotherapy alone to 18.8 months with the combination of chemotherapy and bevacizumab.¹¹ The combination of chemotherapy with bevacizumab now exists as a new standard of care for the management of MM. Given that steadily rising health care costs are not sustainable, the use of bevacizumab in the management of malignant mesothelioma for a potential 2.7 months improvement in OS are often limited by the cost-benefit calculations.

The FGF pathway is involved in signal transduction pathways that regulate processes such as cell proliferation, differentiation and angiogenesis as well as playing a fundamental role in carcinogenesis.[12], [13], [14] FGF inhibitors are currently being investigated as a potential therapeutic agent for MM and include oral FGF inhibitors such as AZD4547 and BGJ398.^15,16 Schelch et al also investigated the role of FGFR1 inhibition using genetic constructs or tyrosine kinase inhibitors and demonstrated suppression of mesothelioma cell growth in vitro and in vivo; as well as increasing the sensitivity to cisplatin treatment and irradiation.¹⁷ FGF/FGFR-targeting strategies are currently explored in many clinical studies to evaluate its efficacy in the management of mesothelioma.

Platelet-derived growth factors (PDGF) are receptor tyrosine kinases that regulate diverse cellular functions, including cell proliferation, transformation, migration and embryonic development. The PDGF/PDGFR pathway has been implicated in mesothelioma carcinogenesis with both autocrine and paracrine mechanisms of proliferation.[18], [19], [20], [21], [22], [23], [24] PDGFs consists of four different polypeptide chains (PDGF-A, -B, -C and -D) which form disulphide-linked homodimers (PDGF-AA, -BB, -CC and -DD) or heterodimers (only one known heterodimer PDGF-AB).¹⁸ PDGF-CC has been reported to induce angiogenic activity indirectly, via upregulation of VEGF^19,20 and directly, via activation of PDGF-Rα and Rα − Rβ receptors.²¹ PDGF-CC has recently been shown to be a negative prognostic factor in triple negative breast cancer.²² Given PDGF-CC’s role in angiogenesis and paracrine functions, this could be an ideal biomarker to investigate in mesothelioma.

There are several small molecule tyrosine kinase inhibitors (TKIs) that target several receptor tyrosine kinases including VEGF that have been studied in MM and are summarized in our previous review article.²³ Positive data from the MAPS study has definitely lead to further interest in other anti-angiogenic agents being trialled in MM. In particular, orally available angiokinase inhibitors such as nintedanib were of significant interest over the last few years. Nintedanib is an orally available, triple angiokinase inhibitor that inhibits the VEGF, PDGF, and FGF receptors. The addition of nintedanib to pemetrexed plus cisplatin resulted in 4 months PFS improvement in the phase II LUME-Meso study.²⁴ Unfortunately, the encouraging efficacy signal observed for nintedanib in Phase II was not confirmed in a larger Phase III trial.²⁵ Previous studies of antiangiogenic treatment in MM with the pan-VEGFR inhibitor cediranib²⁶ and the multikinase inhibitor sorafenib²⁷ (inhibits VEGFRs and PDGFRs) also showed only limited activity.

While predictive biomarker studies are crucial to determine the subset of patients who may benefit from the addition of an anti-angiogenic agent, to date none have been forthcoming. CD31 (also known as platelet endothelial cell adhesion molecule-1) is an endothelial marker used to assess microvessel density with preclinical data showing significantly lower microvessel areas were present in tumors treated with nintedanib in xenograft models.²⁸ The complementary LUME-Meso biomarkers study had reviewed the plasma levels of 58 angiogenic factors (Human AngiogenesisMAP® panel, Myriad RBM) including single-nucleotide polymorphisms (SNPs) in genes for mesothelin (MSLN), VEGFR1 (FLT1) and VEGFR3 (FLT4) and assessed microvessel density via CD31 immunohistochemistry assessment of archival biopsy samples. Although there were potential signals for greater PFS and OS benefits in patients with low plasma endoglin and major homozygous VEGFR1/3 genotypes from the phase II study; no biomarkers showed any conclusive significant association with nintedanib efficacy.²⁹ A phase II trial evaluating vatalanib (a small molecule TKI that inhibits VEGFR1-3, with an inhibitory action also against c-KIT and PDGFRα) in previously untreated patients also demonstrated no correlation between baseline VEGF or PDGF levels and response, PFS or survival.³⁰

In the setting of these new anti-angiogenic therapeutic agents which are of interest in MM, we sought to evaluate VEGF, PDGF, FGFR, and CD31 by immunochemistry in MM to determine their frequency of expression and assess their associated prognostic implications.