Colorectal cancer (CRC) is the third most prevalent cancer worldwide, with more than 800,000 new cases identified each year.¹

Although early diagnosis has resulted in a minor decline in CRC mortality over the past 10 years, 20% of patients still develop metastatic disease.² The prognosis of this disease is dismal once it becomes inoperable. A new age of molecular targeted therapy emerged as a result of rapidly expanding knowledges in the field of tumor biology. Specifically a monoclonal antibody, bevacizumab, targets vascular endothelial growth factor (VEGF).

It is possible to slow tumor angiogenesis by blocking VEGF from interacting with its receptors (VEGFRs).

The first phase III study evaluating the activity of an anti-VEGF agent in metastatic CRC (mCRC), compared irinotecan, bolus fluorouracil, and leucovorin (IFL) alone vs. IFL in combination with bevacizumab and it found that the overall survival (OS) increased from 15.6 months to 20.3 months (P = 0.001).³ These encouraging results led to a growing interest in this group of drugs, and many studies have been conducted better understand the mechanisms underlying tumor angiogenesis,^4,5 leading to the development of more than 50 new medications with anti-angiogenic properties.⁶

In a phase III trial (VELOUR),⁷ which combined aflibercept with irinotecan/5-fluorouracil (5-FU) as second-line treatment, aflibercept (VEGF-Trap), a fusion protein with high VEGF affinity, increased progression-free survival (PFS) and OS of patients with metastatic colorectal cancer.

We carried out a careful search of full-text papers in PubMed (www.ncbi.nlm.nih.gov/pubmed/; accessed on 30 June 2022) starting from 2017, using “colorectal,” “cancer,” “chemotherapy,” “VEGF,” and “epidermal growth factor receptor” as keywords. The full articles found were reviewed in detail. In addition, all abstracts from international congresses from 2020 to June 2022 were also reviewed.

Anti–VEGF agents in mCRC

Neoangiogenesis is a crucial step in the development of solid tumors which have difficulty expanding over 2 mm in diameter without the oxygen and nutrients provided by the circulatory system.^8,9

The basic endothelial growth process, vessel co-option, intussusceptive microvascular growth, glomeruloid angiogenesis, endothelial progenitor cell mobilization, and vasculogenic mimicry are among the developments that contribute to blood vessel creation in malignancies.⁵ Angiogenesis refers to endothelial formation from pre-existing capillaries, creates new vascular blood flows. The balance between pro- and anti-angiogenic factor controls neovascularization.¹⁰ The VEGF family is believed to contain the pro-angiogenic agents and the angiogenic switch is sought to be primarily controlled by VEGF-A.^11,12 VEGF stimulates angiogenesis by increasing endothelial cell migration and proliferation, changing blood vessel permeability, and regulating morphological and functional aspects.

Additionally, non-growing vascularization process can involve VEGF.^5,11

VEGF can draw circulating endothelial cell progenitors (CEPs) from the marrow to develop vascular structures. VEGF-induced vasculature in tumors exhibits immature structural development and aberrant function, as shown by irregularly dilated lumina, tortuous form, pericyte shortage, and high permeability.¹⁰ This aberrant vasculature causes an increase in interstitial fluid pressure (IFP), as well as a reduction in the transport of nutrients and oxygen, which in turn lead to further production of VEGF.¹³

IFP can further hamper the delivery of oxygen, nutrients, and cytotoxic chemotherapy.¹⁴

Studies have shown that many different tumor, including CRC, exhibit increased VEGF expression.^15,16

Additionally the hyper expression of VEGF has been proven to be connected to the development, invasion, and metastasis of CRC.^15,17

Trials of bevacizumab, a humanized monoclonal antibody against VEGF-A, demonstrated that VEGF is a crucial target for the treatment of solid cancers. Anti-angiogenic compounds (including anti-VEGF drugs) have been demonstrated to be effective through research in a variety of animal models. One such mechanism involves boosting the delivery of cytotoxic medications through vascular normalization.¹⁸ Anti-angiogenic drugs may also be able to prevent the repopulation of tumor cells during the period between chemotherapy treatments. Inhibiting the mobility of circulating endothelial cells (CECs) or their progenitors (CEPs) is thought to be a key mechanism by which anti-angiogenic drugs decrease tumor growth and increase chemotherapy sensitivity.^19,20

Bevacizumab in clinical trials

Bevacizumab is the first anti-VEGF agent to be approved for both the first and second-line therapy of mCRC. The effectiveness of anti-VEGF agents in the treatment of CRC was demonstrated by the experiences with bevacizumab. The combination of bevacizumab with chemotherapy, yielded excellent responses in terms of PFS and OS. It was found to be effective when used alone or in combination with anti-EGFR treatment.²¹

Bevacizumab has been widely included in some chemotherapy regimens to treat mCRC since a key trial demonstrated that it can increase OS. Bevacizumab has been shown to be effective when combined with the first-line chemotherapy regimens for CRC that are most frequently used in clinical practice, namely FOLFOX (leucovoin [LV], 5-FU, and oxaliplatin), FOLFIRI (LV, 5-FU, and irinotecan), and XELOX (capecitabine and oxaliplatin).²²

Bevacizumab and irinotecan

The first study that demonstrated the superiority of bevacizumab in combination with irinotecan was a phase III trial the conducted by Hurwitz et al³ that resulted in the approval of bevacizumab I for the first-line treatment of mCRC. Subsequently, continuous infusion of 5-FU replaced the 5-FU bolus, after a phase III study (BICC-C), demonstrated that the continuous infusion administration had lower toxicity and improved efficacy.²³ The median (PFS) of FOLFIRI compared to a modified IFL (mIFL) regimen was longer (7.6 vs. 5.9 months). Additionally, the FOLFIRI with bevacizumab arm had a considerably longer median (OS) than the mIFL with bevacizumab arm (28.0 vs. 19.2 months; P = 0.037).²⁴

Bevacizumab and oxaliplatin

Saltz et al²⁵ conducted a phase III trial where bevacizumab in combination with either FOLFOX or XELOX as the first-line treatment for mCRC, did not improve the overall response rate (38% in both arms) and OS (19.9 months vs. 21.3 months, P = 0.077) compared with either combination alone. However, there was a considerable PFS benefit (9.4 months) in the bevacizumab group and 8.0 months in the placebo arm (hazard ratio [HR], 0.83; 97.5% confidence interval [CI], 0.72 to 0.95; P = 0.0023); These findings were thought to be related to frequent interruption. Giantonio et al²⁶ conducted a phase III study of the second-line treatment of mCRC, the Eastern Cooperative Oncology Group study (ECOG 3200) and found that the combination of bevacizumab (10 mg/kg) with FOLFOX led to significantly better PFS (7.3 months vs. 4.7 months) and median survival (12.9 months vs. 10.8 months) than FOLFOX alone.

Bevacizumab and 5-fluorouracil

Some patients may be treated with a single agent such as 5-FU because they are unable to receive multi-drug chemotherapy due to issues with tolerance or other factors. A combined analysis of the findings from three distinct studies revealed that bevacizumab in combination with 5-FU is preferable to 5-FU alone.^27,28

Kabbinavar et al²⁷ conducted a phase II study comparing bevacizumab with FU/LV vs. FU/LV alone in patients with age > 65 years, ECOG status 1 or 2, serum albumin < 3.5 g/dL, and or prior abdominal/pelvic radiotherapy.

The median survival was 16.6 months for the FU/LV/bevacizumab group and 12.9 months for the FU/LV/placebo group (HR, 0.79; P = 0.16). The median PFS was 9.2 months (in the FU/LV/bevacizumab group) and 5.5 months (in the FU/LV/placebo group) (HR, 0.50; P = 0.0002). The response rates were 26.0% in the FU/LV/bevacizumab group and 15.2% FU/LV/placebo group (P = 0.055); the duration of response was 9.2 months (FU/LV/bevacizumab group) and 6.8 months (FU/LV/placebo group) (HR, 0.42; P = 0.088). Grade 3 hypertension was more common with bevacizumab (16% vs. 3%) but was controlled with oral medication and did not cause study drug discontinuation.

Bevacizumab beyond progression

The initial assessment of bevacizumab effectiveness in the post-progression therapy of mCRC was evaluated in the BRiTE nonrandomized prospective observational study. The results demonstrated good safety and significant efficacy. The median OS from the time of progression in the group that received post-progression therapy with the regimen containing bevacizumab was 19.2 months, compared to 9.5 months for the control arm.^29,30 Bennouna et al,³¹ conducted a phase III randomized open-label trial (ML18147) at 220 centers. They enrolled 820 patients and randomized them at 1:1 ratio to second-line chemotherapy with or without bevacizumab. The patients had experienced progression for less than 3 months after completing first-line chemotherapy. The primary end point OS was met demonstrating that bevacizumab after progression improved the median (OS) in the experimental arm compared to the control arm (11.2 months vs. 9.8 months; P = 0.0062).³¹

Bevacizumab in combination with EGFR inhibitors

Anti-VEGF and anti-epidermal growth factor receptor (anti-EGFR) agents are two different targeted therapies that have proven effective when used in combination with chemotherapy in mCRC. Studies have shown that EGFR has a strong impact on tumor-associated angiogenesis and that combining EGFR and VEGF signaling inhibitors for treatment has at least additive anticancer effectiveness.^32,33

Two-phase II studies, PACCE and CAIRO 2, have assessed the use of dual biologic treatments in conjunction with cytotoxic chemotherapy. In both studies, the addition of anti- EGFR resulted in a reduction of the primary end point with an increase in toxicity.^34,35

These results are further confirmed by a recently published phase III trial that compared FOLFOX6 combined with CB (5-FU, LV, bevacizumab, and cetuximab) to mFOLFOX6-B (modified FOLFOX6 + bevacizumab) for mCRC patients. The outcomes showed that the dual biologic FOLF-CB group efficacy was not greater than the mFOLFOX6-B group. The control group (mFOLFOX6-B) had higher patient satisfatction.³⁶

The median OS was 21 months in combination arm, and 19.5 months in the control arm, while the 12-month PFS rates were 45% and 32% respectively.

Bevacizumab in the adjuvant setting

According to experimental data, the early stages of tumorigenesis and development occur simultaneously to VEGF-induced angiogenesis. Therefore, in the early phase of disease development, when tumors are small, anti-VEGF treatment should be very effective. However, the poor findings of two-phase III trials in adjuvant setting (NSABP C-08 and AVANT)^37,38 did not allow the continuation of studies in this disease setting.

In the NSABP C-08 trial, 2,672 patients with stage II and III resected colorectal cancer, were enrolled in two arms. The experimental arm received FOLFOX6 combined with bevacizumab, while the control arm FOLFOX6 alone. The primary end point, disease-free survival (DFS) after 35,6 months of follow-up, was not met (HR, 0.89; 95% CI, 0.76 to 1.04; P = 0.15). In the AVANT phase III trial, 2,867 patients were enrolled in three arms.

FOLFOX4 with bevacizumab, FOLFOX4 alone and XELOX. The primary end point was DFS. After 46 months of follow-up, the HR for DFS was 1.17 (95% CI, 0.98–1.39; P = 0.07) for bevacizumab—FOLFOX4 vs. FOLFOX4, while it was 1.07 (95% CI, 0.90–1.28; P = 0.44) for bevacizumab with XELOX vs. FOLFOX4. Thus, the researchers concluded that, and for bevacizumab—XELOX vs. FOLFOX4 was 1.07 (95% CI, 0.90–1.28; P = 0.44), concluding that bevacizumab does not prolong DFSl when added to adjuvant chemotherapy in resected stage III colon cancer.

Aflibercept

The second extracellular domain of VEGFR1 and the third extracellular domain of VEGFR2 are fused to the Fc portion of immunoglobulin (Ig) G1 to create the completely human recombinant protein known as aflibercept.³⁹

Aflibercept, according to research in mouse models, binds to VEGF-B and placental growth factor in addition to binding all isomers of VEGF-A.^40,41 Aflibercept can bind to several isoforms of VEGF with a very high affinity (1 pM), which is superior to existing anti-VEGF drug.⁴²

Studies using multiple animal models have shown that aflibercept acts on endothelial cells, pericytes, and even the vascular basal membrane or VEGF receptor. These actions prevent the development and remodeling of new blood vessel, both of which were already seen with bevacizumab.^42,43

It is thought that the remodeling effects of anti-VEGF agents which normalize tumor vascularization, may explain the increased efficacy of chemotherapy when used in combination.⁴⁴

As previously discussed, aflibercept can reduce vascular density and growth, suppressing tumor growth, and improving radiation therapy efficacy in the treatment of neuroblastoma xenografts. It can also reduce the tumor burden and inhibit the metastasis of ovarian cancer when combined with paclitaxel.⁴⁵ Aflibercept is well tolerated and according to phase I trials, and the recommended dose is 4 mg/kg.^43,46

In phase I/II studies, the most frequently reported adverse events were fatigue, proteinuria, hypertension, nausea, and lymphopenia, with rare reports of grades 3–4 toxicity.^47-49

Thromboembolic events were less common in patients who received aflibercept treatment than in those treated with bevacizumab because the entire human protein sequences and VEGF have a 1:1 binding ratio.⁵⁰

Anti-VEGF treatment is frequently combined with other drugs since anti-VEGF monotherapy performed poorly in pivotal trials. Most phase II and phase III trials examined the effectiveness of aflibercept when combined with chemotherapy. Aflibercept has been used to treat a variety of tumor in clinical studies, including prostate cancer, non-small cell lung adenocarcinoma, ovarian cancer, and mCRC.^51,52

Aflibercept was approved after the results of a phase III trial (VELOUR) in combination with FOLFIRI as a second-line treatment for mCRC. The primary end point was OS. After one oxaliplatin-based therapy failed, 1,226 patients with mCRC received FOLFIRI and aflibercept (4 mg/kg) or FOLFIRI with placebo, every 2 weeks. PFS was 6.90 vs. 4.67 months (HR, 0.758; P = 0.00007), and objective response rate was 19.8% vs. 11.1% (P = 0.0001). In the aflibercept group, the median OS was 13.50 months, compared to 12.06 months in the placebo arm (HR, 0.817; P = 0.0032). In the aflibercept arm and the placebo arm, respectively, 26.6% and 12.1% of patients discontinued treatment due to side events.

The findings of the AFFIRM phase II trial, in which patients with mCRC received mFOLFOX6 with aflibercept or FOLFOX6 alone as first-line therapy, demonstrated that the PFS rate at 1 year for patients receiving mFOLFOX6 and aflibercept was comparable to observed in the control arm receiving mFOLFOX6 alone. The toxicity profile was consistent with other anti-VEGF drugs and similar to that observed in early phase I/II trials.

Ramucirumab

VEGFR-2 is the main mediator of the pro-angiogenic effects of VEGF-A and other growth factors. According to experimental data, the interaction between VEGF-A and VEGFR-2 is crucial for the development of blood vessels in tumors. This relationship is blocked by VEGFR-2 inhibition. A completely human IgG-1 monoclonal antibody called ramucirumab, binds to the VEGFR-2 extracellular domain with high affinity (kilodaltons 50 pmol/L), blocking all VEGF ligands. DC101, a rat antibody that likewise binds to the VEGFR-2 extracellular domain and prevents ligand binding, has been linked to significantly decreased tumor growth in preclinical investigations using human colon cancer xenograft mouse models.

Tabernero et al⁵³ conducted a phase III double-blind placebo-controlled trial that compared FOLFIRI combined with ramucirumab vs. FOLFIRI alone in second-line mCRC. The median OS was 13.3 months (95% CI, 12.4–14.5) in the ramucirumab arm vs. 11.7 months (95% CI, 10.8–12.7) in the placebo group (HR, 0.844; 95% CI, 0.730–0.976; P = 0.0219).

The addition of new therapies in the treatment of mCRC has in prolonged patient’s lives.

Nonetheless, an important issue is that is in the early stage of the disease (i.e., in adjuvant setting), where preclinical data indicate that neo angiogenesis plays a crucial role in tumorigenesis, but phase III randomized clinical trials have shown the opposite.

Even the timing of administration of anti-VEGF agents has been called into question by phase III randomized clinical trials. These agents should be administered intermittently before chemotherapy in order to normalize the vascular bed and allow the chemotherapy to permeate better, however, it has been observed that they can also be used after disease progression.^54,55

This has changed the idea that the prolonged use of anti-VEGF agent can activate alternative pro-angiogenic signals capable of creating resistance.⁵⁶

Data in the literature have shown that both anti-VEGF and anti-EGFR agents are effective in colorectal cancer. Preclinical data have also supported the hypothesis of possible synergistic activity between these two drug categories, which has induced investigators to combine them in clinical practice. However, the PACCE study and CAIRO 2 have rejected this hypothesis, advising against the combined use of these agents in current clinical practice.

Promising data have been published in the literature regarding the addition of metronomic chemotherapy to anti-VEGF drugs, including the use of vascular disrupting agents, which are capable of interacting synergistically with anti-VEGF agents in the process of normalizing the tumor vascular bed with an effect on CEPs.^57–59

There are still no validated markers predictive of response to anti-VEGF agents.⁶⁰ Many trials studied the trend of VEGF, placenta growth factor (PIGF), soluble VEGFR-2, and basic fibroblast growth factor (bFGF), during therapy, noting a change in the values compared to baseline, but without correlations showing that they could serve as predictive factors of the response to anti-VEGF therapy.⁶¹

Viable CECs and CEPs, which are crucial for tumor angiogenesis, have been extensively studied in recent years.

Previous research has shown that anti-angiogenic drugs may limit the recruitment or proliferation of endothelial progenitor cells (CEPs) in tumor tissue, and that CEC and CEP counts may serve as substitute biomarkers with predictive power.⁶²

Another field of study is the evaluation of circulating levels of CECs and CEPs. In recent trials it has been observed that anti-VEGF drugs can reduce the circulating levels of CECs and CEPs even in mild tumor tissues. This finding implies that these agents could be used as response drivers.⁶²

Finally, the last topic is to identify the best chemotherapy backbone for combining with anti-VEGF drugs to best exploit their effectiveness.

Bevacizumab, aflibercept and ramucirumab are very effective drugs when combined with first- and second-line chemotherapy for mCRC. Considerable work remains to be done to better integrate these drugs into the complex therapeutic strategy in mCRC in order to be able to fully exploit them, resolving persistent uncertainties. This implies the need for basic, translational and clinical investigative research. In the future, new opportunities will arise from the combination of anti-angiogenic drugs and immunotherapy.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

We thank all the authors for their contribution.

None.

No potential conflict of interest relevant to this article was reported.