**9. Potential predictive biomarkers for anti-angiogenic agents**

In view of the variable outcomes seen in the clinic, there is a need for the development of validated predictive biomarkers of response for anti-angiogenic therapy. In this way, patients who will derive benefit from such agents could be appropriately selected, whilst those that will not derive benefit (either at the outset or during therapy) could be selected for alternative, more effective therapy. Such a strategy would not only improve clinical outcomes but would also reduce the unnecessary burden of (a) toxicity to the patient, and (b) cost to the economy. Despite extensive international research in this field, there is currently no biomarker which predicts benefit or resistance to anti-angiogenic agents that is approved for routine clinical practice. The following are amongst several which have been investigated in the clinical setting.

#### **9.1 Circulating biomarkers**

Circulating biomarkers are an attractive tool for patients and clinicians as 'liquid biopsies' are relatively non-invasive and easy to perform, as compared with tissue biopsies of tumour with associated risks and potential technical difficulties depending on tumour site. VEGF levels have been studied as a potential biomarker with high levels associated with poorer outcomes [85]. Findings regarding its utility as a predictive biomarker have been more inconsistent [85]. An analysis of four randomised phase 3 trials investigated circulating VEGF level as a prognostic and predictive biomarker in mCRC, lung cancer and RCC which included bevacizumab in the treatment regimen. Tumour specimens were also tested for VEGF level. This

*Current Cancer Treatment*

during breaks in chemotherapy [76].

chemotherapy must be considered. Current alternative theories based mostly on preclinical data include: (1) direct synergy between the anti-angiogenic effects of bevacizumab and potential anti-angiogenic effects of chemotherapy [75], (2) targeting of VEGF signalling directly in cancer cells by bevacizumab [21], (3) chemotherapy may inhibit resistance to bevacizumab, because chemotherapy suppresses the tumour recruitment of myeloid cells that have been implicated in resistance to bevacizumab [76], (4) bevacizumab may prevent tumour rebound that may occur

It should be noted that vessel normalisation facilitated by anti-angiogenic agents may provide therapeutic benefit through other mechanisms, which are independent of chemotherapy delivery. For example, in glioblastoma patients, vessel normalisation induced by single agent VEGF-targeted therapy may prolong survival due to other effects, such as oedema control or improved tumour oxygenation [77]. There are two other curious observations that have yet to be properly explained. Firstly, the synergistic effect of adding bevacizumab to chemotherapy does not occur in all tumour types. For example, the addition of bevacizumab does not lead to improvements in outcome in advanced breast cancer [78]. Secondly, VEGFR TKIs show single

agent activity without the need for co-administration of chemotherapy [21].

done in order to understand the mechanisms involved.

Recent insight into these two curious observations has been reported. A study examining both clinical and mouse tumour tissue specimens demonstrated that tumour types utilising a vasculature surrounded by a well-developed stroma (e.g. mCRC, NSCLC) respond better to bevacizumab when it is added to chemotherapy as opposed to tumour types that utilise a vasculature without a well-developed intervening stromal component (e.g. mRCC, PNET) which respond better to VEGF TKIs alone [79]. This suggests that tumour cell interactions with different stromal components may influence response to different anti-angiogenic agents and how they synergise with concomitant drugs. However, there is still much work to be

**8. Synergy of anti-angiogenic agents with immunomodulatory therapy**

apy with other treatments including VEGF inhibitors are being explored.

studies have recently or are still in the process of investigating this.

A series of pre-clinical studies have shown that the use of anti-angiogenic agents along with immune checkpoint inhibitors (ICI) as a combination therapy has a synergistic and enhanced effect on the tumour when compared to either ICI therapy or anti-angiogenic therapy alone. Immunotherapy has emerged as a promising treatment option for many cancer types, offering hope for patients with the demonstration of improved outcomes including durable responses in some. Unfortunately, there are still many patients that either have short lived responses to such therapies or none at all. To overcome resistance mechanisms, combinations of immunother-

Since 2013, pre-clinical investigations in mice with various tumours have indicated that the combination of ICI and anti-angiogenic agents results in prolonged overall survival [80]. It has been observed that the VEGF can cause the upregulation of immune checkpoint molecules such as PD-1 and as a result, the use of anti-VEGF agents has been seen to reduce the expression of PD-1 on cytotoxic T lymphocytes [81]. Thus, the combination of using both anti-VEGF agents as well as anti-PD-1 agents could have a synergistic effect on inhibiting further tumour development [81]. Through the encouraging findings of pre-clinical investigations, many clinical

There are a multitude of clinical studies supporting the role of bevacizumab in the positive immune modulation of the tumour microenvironment and its beneficial effects when combined with the immune checkpoint PD1/PDL1 and

**48**

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, 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 had improved PFS [90].

#### **9.2 Levels of tumour VEGF isoforms**

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

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].

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

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

**51**

*DOI: http://dx.doi.org/10.5772/intechopen.89667*

(capecitabine and irinotecan) plus bevacizumab [99].

**9.4 VEGF polymorphisms**

**9.5 Radiological parameters**

*9.5.1 Baseline vascular perfusion on imaging*

*Angiogenesis and Its Role in the Tumour Microenvironment: A Target for Cancer Therapy*

interleukin 8, osteopontin, VEGF and TIMP1) was able to distinguish a sub-group of patients that derived a significantly greater overall survival benefit from this agent.

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

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].

Functional clinical imaging, taking into account tumour vasculature or metabolic activity by utilising CT, MRI or PET scanning, either prior to commencing treatment or following brief exposure of patients to therapy, may be a useful tool for predicting response or resistance to anti-angiogenic therapy [102]. For conventional cytotoxic chemotherapy, imaging has been employed to assess therapy response based on change in tumour size, as defined by RECIST (Response Evaluation Criteria In Solid Tumours). However, biological agents, such as bevacizumab and TKIs, may be cytostatic in terms of their mechanism of action, thus size may not be the only parameter that needs to be considered when assessing response and outcome. Examination of various parameters such as blood flow and tumour

morphology may provide additional important predictive information.

Several studies have examined pre-treatment levels of tumour perfusion and whether they can predict outcome. For example, enhanced levels of vessel perfusion interleukin 8, osteopontin, VEGF and TIMP1) was able to distinguish a sub-group of patients that derived a significantly greater overall survival benefit from this agent.
