**9.4 VEGF polymorphisms**

*Current Cancer Treatment*

had improved PFS [90].

**9.2 Levels of tumour VEGF isoforms**

mCRC, but this failed to predict outcomes [92].

found that higher baseline circulating VEGF levels were associated with poorer clinical outcomes but levels did not predict response to bevacizumab [86]. There is early evidence from small and exploratory studies to suggest soluble VEGFR-1, with higher levels being associated with poorer outcomes with anti-angiogenic treatments, however larger studies are required to confirm these findings [87].

Other potential circulating biomarkers have also been investigated. In mCRC,

Levels of VEGF expression in a tumour could be a determinant of responsiveness to anti-VEGF therapy. Some small studies have demonstrated a relationship between baseline VEGF expression and response, however these findings have not been consistently replicated in large clinical trials and are often more informative as prognostic rather than predictive biomarkers [91]. Data from more recent prospective studies, however, have shown more consistency in the use of VEGF as a biomarker. A large randomised trial in patients with advanced breast cancer treated with bevacizumab demonstrated a significant association between high circulating levels of VEGF and survival benefit [78]. VEGF expression in tumours was investigated in the large phase III clinical trial of bevacizumab plus chemotherapy in

There are multiple reasons why using VEGF expression as a biomarker could be problematic: (1) advanced tumours express numerous pro-angiogenic factors in addition to VEGF which could confer resistance to bevacizumab irrespective of the amount of VEGF produced [93], (2) differences in the intensity of VEGF expression might be too small to be clinically relevant, (3) hypoxia, which is promoted by antiangiogenic therapy, is an important inducer of VEGF expression and might, therefore, lead to increased VEGF production in the presence of bevacizumab treatment; indeed, anti-angiogenic agents have been shown to induce expression of VEGF even in tumour naïve hosts [94], (4) variations in methodology across centres (including sample handling, the use of different scoring systems and non-validated antibodies) have a significant effect on biomarker trial results [95], (5) it is very challenging to standardise cut-offs for low and high VEGF levels, due to: (a) different methods used to measure VEGF at different centres and (b) differences in biology that occur

between racial groups, tumour types and different stages of disease [95].

Studies which have investigated other single circulating factors (such as FGF2, and r soluble VEGFR2) have also yielded contradictory and unsatisfactory conclusions [96]. Interestingly, however, recent clinical work in mRCC patients treated with anti-angiogenic TKIs suggests that profiling multiple circulating factors in the blood could have a more powerful prognostic and predictive role than assessing levels of single factors alone [97]. In this study, when patients with mRCC were treated with the TKI pazopanib, a biomarker signature of six factors (HGF, interleukin 6 and

**9.3 Levels of alternative pro-angiogenic growth factors**

elevated IL-8 levels at baseline were associated with a shorter PFS in patients treated with chemotherapy (FOLFIRI) and bevacizumab [88]. Elevated LDH and neutrophil levels have been found to independently predict poorer survival in patients treated with chemotherapy plus bevacizumab [89]. A promising predictive biomarker for response to bevacizumab based therapy in CRC appears to be circulating endothelial cells, with studies showing that patients with lower circulating endothelial cells at baseline undergoing treatment with bevacizumab based therapy

**50**

Polymorphisms in VEGF or VEGF receptors have been proposed to predict outcome from anti-angiogenic therapy. As these are generally binary in nature, they are attractive biomarkers since they may be easier to measure and apply prospectively. In metastatic breast cancer, polymorphisms in VEGF and VEGFR2 were analysed in several retrospective subset analyses in patients treated with chemotherapy, with or without bevacizumab. Two polymorphisms within the VEGF promoter/5′ untranslated region, VEGF alleles −2578AA and −1154AA, were significantly associated with improved OS in the bevacizumab plus paclitaxel group when compared to the −2578CA/−2578CC and −1154GA/−1154GG alleles. In contrast, they did not have prognostic power for OS in the chemotherapy-only arm [98]. The predictive power of the −2578AA and −1154AA VEGF alleles was also reported in a retrospective subset analysis of patients with metastatic colorectal cancer that received either FOLFIRI (leucovorin, fluorouracil, and irinotecan) plus bevacizumab or XELIRI (capecitabine and irinotecan) plus bevacizumab [99].

More recently, the role of VEGFR1 polymorphisms was studied in a large meta-analysis pooling DNA data from two phase III trials in patients with advanced pancreatic cancer treated with bevacizumab. VEGFR1 −1213AC/−1213CC alleles were significantly associated with poor outcome in patients receiving bevacizumab when compared to VEGFR1 −1213AA alleles [100]. To understand how this VEGFR1 polymorphism functionally affects VEGFR1 expression and how it might explain its correlation with poor outcome in patients receiving bevacizumab, Lambrechts and colleagues performed an *in vitro* study where the mutant codon of Tyr1213 was transiently overexpressed in HEK293T cells. Lysates from these cells demonstrated a significant increase in expression and signalling of VEGFR1 compared to HEK293T cells harbouring the wild type codon, thus providing a biological rationale for the role of this polymorphism as a negative predictive marker of response [100]. A significant correlation of the VEGFR −1213 with poor outcome was also corroborated by a subsequent study in patients with mRCC treated with sunitinib [101].
