Safety of Aflibercept in Metastatic Colorectal Cancer: A Literature Review and Expert Perspective on Clinical and Real-World Data.

Background: Metastatic colorectal cancer (mCRC) represents a substantial health burden globally and an increasing challenge in Asian countries. Treatment options include chemotherapy plus a vascular endothelial growth factor (VEGF) inhibitor (such as bevacizumab, aflibercept or ramucirumab), or anti-epidermal growth factor receptor (EGFR) therapies. Aflibercept, a recombinant fusion protein, has been approved for treatment of mCRC in combination with FOLFIRI for patients whose disease progresses during or after treatment with an oxaliplatin-containing regimen, based on its efficacy and tolerability profile in clinical trials. This report aims to provide an overview of both clinical and real-world evidence and experience on the use of aflibercept in routine clinical practice, with a focus on European, American and Asian populations. Methods: A literature search was conducted in PubMed (on 28th February 2019) using the search terms ("aflibercept") and ("Colorectal"OR"CRC") to identify publications containing information on aflibercept-containing regimens. Results: The adverse events (AE) profile was similar between geographical locations. Across trials, real-world and retrospective studies, grade ≥ 3 hypertension and proteinuria were amongst the most frequently reported AEs. Conclusions: The safety profile of aflibercept is generally manageable and comparable across various geographic locations.


Introduction
The global burden of colorectal cancer (CRC) is expected to increase by 60% to more than 2.2 million new cases and 1.1 million deaths by 2030. It is the second most frequently diagnosed cancer and the fourth leading cause of cancer-related death worldwide [1,2]). In Europe, 2018 estimates indicate that CRC is the second most frequently occurring cancer and cause of death from cancer [3]. Increases in incidence and mortality have been seen in Eastern Europe, where as other European countries have exhibited an increase in incidence but a decline in mortality [1]. In Asia, CRC rates are increasing rapidly, and it is now the third most frequently occurring malignancy in both men and women. Mortality from CRC has been increasing in Asian countries over the last decade, with the exception of Japan and Singapore [1,4]. According to the vital statistics of Japan in 2015, CRC was the most frequent cause of cancer deaths in women [5]. A multinational cohort study involving four Asian countries (Taiwan, Korea, Japan, and Hong Kong) demonstrated that the incidence of young-onset CRC has This paper aims to evaluate current safety data reported for aflibercept in mCRC using clinical and real-world/retrospective data. After reviewing these data, insights on patient management from different perspectives will be provided in order to help inform treatment decisions in this disease setting.

Methods
A literature search was conducted in PubMed using the search terms ("aflibercept") and ("colorectal"OR"CRC") to identify publications available cumulatively until March 13, 2020 containing information on aflibercept-containing regimens. Identified publications were reviewed and those including safety data were selected. Both clinical trials and real-world data (including observational and retrospective studies) were included. Review articles and reports with a focus on efficacy were excluded from the analysis. In order to obtain studies reporting safety data from a sufficient number of patients, individual case reports/case series with < 5 patients were also excluded. Google Scholar was used to access an article known to have only been presented at a congress ( Figure  1).

Aflibercept Mechanism of Action and Relevance To Safety Profile
Aflibercept is a recombinant fusion protein composed of the second immunoglobulin (Ig)-like domain of FLT1 (VEGFR1) joined to the third Ig-like domain of KDR (VEGFR2) fused to the Fc portion of human IgG1 (Figure 2). Aflibercept differs from other anti-VEGF agents by functioning as a decoy receptor with high-affinity for other angiogenic factors, VEGF-B and placental growth factor (PlGF). Additionally, PlGF has been proposed as a mechanism of bevacizumab resistance.

Aflibercept Mechanism of Action and Relevance To Safety Profile
Aflibercept is a recombinant fusion protein composed of the second immunoglobulin (Ig)-like domain of FLT1 (VEGFR1) joined to the third Ig-like domain of KDR (VEGFR2) fused to the Fc portion of human IgG1 (Figure 2). Aflibercept differs from other anti-VEGF agents by functioning as a decoy receptor with high-affinity for other angiogenic factors, VEGF-B and placental growth factor (PlGF). Additionally, PlGF has been proposed as a mechanism of bevacizumab resistance.
Consequently, aflibercept's binding affinity for PlGF has been hypothesized to contribute to its efficacy in the post-bevacizumab setting [7].
Cancers 2020, 12, x FOR PEER REVIEW Consequently, aflibercept's binding affinity for PlGF has been hypothesized to contribute to its efficacy in the post-bevacizumab setting [7].
Tumors exploit angiogenic pathways to facilitate rapid and uncontrolled proliferation. Using anti-angiogenics inhibits the formation of new vessels within the tumor and leads to hypoxia and tumor death. Although angiogenesis occurs at a lower rate in adults, these pathways are used for vascular homeostasis. For example, VEGF promotes the release of nitric oxide, a vasodilator, which may explain why anti-angiogenics are frequently associated with hypertension. Proteinuria, another frequent adverse event (AE) associated with anti-angiogenics, may be due to VEGF reductions leading to decreased nephrin expression, subsequently resulting in podocyte dysfunction. Antiangiogenics are also associated with non-cardiovascular AEs, due to the complexity of the VEGF signaling pathway [9,10].

Global Studies
The safety and efficacy of aflibercept has been evaluated in several global clinical trials ( Figure  3). VELOUR was an international randomized, placebo-controlled Phase III trial comparing the efficacy and safety of aflibercept plus FOLFIRI (n = 612) and placebo plus FOLFIRI (n = 614) in patients with mCRC who had previously received oxaliplatin based chemotherapy. Addition of aflibercept to FOLFIRI significantly improved efficacy outcomes compared with placebo plus FOLFIRI.  [8]). mAB, monoclonal antibody; MKI, multikinase inhibitor; PlGF, placental growth factor; VEGF, vascular endothelial growth factor; VEGF-R, vascular endothelial growth factor receptor. VEGFR-1 is transcribed from the Fms-related receptor tyrosine kinase (FLT1) gene, VEGFR-2 from the Kinase Insert Domain Receptor (KDR) and VEGFR-3 from the FLT3 gene.
Tumors exploit angiogenic pathways to facilitate rapid and uncontrolled proliferation. Using anti-angiogenics inhibits the formation of new vessels within the tumor and leads to hypoxia and tumor death. Although angiogenesis occurs at a lower rate in adults, these pathways are used for vascular homeostasis. For example, VEGF promotes the release of nitric oxide, a vasodilator, which may explain why anti-angiogenics are frequently associated with hypertension. Proteinuria, another frequent adverse event (AE) associated with anti-angiogenics, may be due to VEGF reductions leading to decreased nephrin expression, subsequently resulting in podocyte dysfunction. Anti-angiogenics are also associated with non-cardiovascular AEs, due to the complexity of the VEGF signaling pathway [9,10].

Global Studies
The safety and efficacy of aflibercept has been evaluated in several global clinical trials ( Figure 3). VELOUR was an international randomized, placebo-controlled Phase III trial comparing the efficacy and safety of aflibercept plus FOLFIRI (n = 612) and placebo plus FOLFIRI (n = 614) in patients with mCRC who had previously received oxaliplatin based chemotherapy. Addition of aflibercept to FOLFIRI significantly improved efficacy outcomes compared with placebo plus FOLFIRI. The experimental arm demonstrated a statistically significant benefit in overall survival (OS; median 13.50 months versus 12.06 months; p = 0.0032), progression-free survival (PFS; median 6.90 The experimental arm demonstrated a statistically significant benefit in overall survival (OS; median 13.50 months versus 12.06 months; p = 0.0032), progression-free survival (PFS; median 6.90 months versus 4.67 months; p < 0.0001), and response rates (19.8% versus 11.1%; p = 0.0001) compared with the control arm. The most frequently occurring Grade ≥ 3 AEs in patients who received aflibercept plus FOLFIRI were neutropenia (laboratory measurements: 36.7%, versus 29.5% for placebo plus FOLFIRI), hypertension (19.3%, versus 1.5% for placebo plus FOLFIRI), diarrhea (19.3%, versus 7.8% for placebo plus FOLFIRI), asthenia (16.8%, versus 10.6% for placebo plus FOLFIRI), stomatitis and ulceration (13.8%, versus 5.0% for placebo plus FOLFIRI) and infections and infestations (12.3%, versus 6.9% for placebo plus FOLFIRI). AEs observed at ≥ 5% in either arm are summarized in Table 1. Rates of any grade arterial thrombotic events were 1.5% for placebo plus FOLFIRI (0.5% grade 3) and 2.6% for aflibercept plus FOLFIRI (0.8% grade 3 and 1.0% grade 4) [11]. In a post hoc analysis, the incidence of grade 3/4 AEs was higher for patients ≥ 65 years of age than for those < 65 years of age in both the aflibercept (89.3% vs 80.5%) and placebo (67.4% vs 59.4%) arms. Interaction tests for grade 3/4 antiangiogenic agent-related AEs suggested no heterogeneity between the older and younger patient populations (p > 0.1) [12]. Similarly, another post hoc analysis did not identify any differences in all grade and grade 3/4 AEs between the subgroup of patients with better and poorer efficacy [13]. Importantly, the majority of grade 3/4 AEs occurred within 4 cycles of treatment, in a small percent of treatment cycles, and were mostly reversible [14].
The Aflibercept Safety and Quality-of-Life Program (ASQoP) was an open-label single-arm trial evaluating the effect of second-line aflibercept plus FOLFIRI on the quality of life and safety of patients with mCRC in a real-world setting (in a similar patient population as the VELOUR study). An interim analysis based on 779 patients demonstrated that aflibercept-related AEs were similar in ASQoP and VELOUR, though the frequency of some AEs appeared lower in ASQoP, which may reflect more flexible n FOLFIRI dosing, or more experience in AE management (Table 2; [15,16]).
The AFFIRM study compared aflibercept in combination with modified FOLFOX6 (mFOLFOX6; n = 119) with mFOLFOX6 alone (n = 117) for the first-line treatment of mCRC ( Figure 3). Rates of AEs were generally increased in the aflibercept arm compared with mFOLFOX6 alone, and the most frequently occurring grade ≥ 3 AEs were neutropenia (36.1% vs 29.3%), hypertension (35.3% vs 1.7%), peripheral sensory neuropathy (16.8% vs 17.2%), and diarrhea (13.4% vs 5.2%). One patient died as a result of an intracranial hemorrhage (unrelated to aflibercept) and four others from infections (also determined not related to study treatment) [17]. In a separate analysis of the AFFIRM study data, no association was found between certain biomarkers (somatic mutations in oncogenic drivers of mCRC, common single-nucleotide polymorphisms in VEGF pathway genes and plasma markers) and AEs [18]. Table 1. Summary of adverse events (any grade) ≥ 5% in the VELOUR trial of placebo plus FOLFIRI vs aflibercept plus FOLFIRI [11].
A Phase II study evaluated aflibercept in patients with mCRC, in Canada and the US, who had received prior bevacizumab (n = 51) or who were bevacizumab-naïve (n = 24) (Figure 3). The most frequently occurring grade 3/4 AEs were hypertension (13.5%) and proteinuria (10.8%). Ten patients discontinued aflibercept due to AEs, including five patients for severe hypertension and proteinuria [23].

Asian Studies
Several clinical trials evaluating second-line aflibercept have been undertaken across Asia. A Phase III study of aflibercept plus FOLFIRI in Asian patients with pre-treated mCRC included 111 evaluable patients ( Figure 3). The most frequently occurring grade ≥ 3 AEs were neutropenia (29%), hypertension (18%), diarrhea (17%) and stomatitis (11%). There were two treatment-related deaths in this patient group, four in the aflibercept plus FOLFIRI group and one in the placebo plus FOLFIRI only group [24].
Aflibercept had a consistent safety profile across clinical studies and regions. The most frequent AEs included neutropenia, hypertension and gastrointestinal issues.
An Italian study of 781 patients who received aflibercept plus FOLFIRI in the real-world setting reported a higher rate of AEs versus other studies (Figure 4). Hypertension (28.5%), neutropenia (27.5%; from laboratory data), asthenic conditions (20.0%), diarrhea (17.0%), and stomatitis (13.0%) were the most frequent grade 3/4 toxicities. One toxic death occurred during the study period due to sepsis, without neutropenic complications. There was no significant worsening of HRQL during treatment [39]. In a smaller Italian study of 74 patients with mCRC treated with either second-line FOLFIRI/bevacizumab (Arm A; n = 31) or FOLFIRI/aflibercept (Arm B; n = 43), the most frequently occurring grade 3 toxicity was neutropenia (16.3% in Arm B). Grade 3 cardiovascular events occurred in 7.0% of patients in Arm B. No grade 4 events were observed [40].
QoLiTrap was a multinational non-interventional study evaluating the quality of life of 1500 German patients with mCRC previously treated with FOLFIRI plus aflibercept (Figure 4). The most frequent AEs were diarrhea, oral mucositis, fatigue, nausea, and hypertension, predominantly grade ≤ 2 [41].
A retrospective study conducted in Taiwan evaluated aflibercept monotherapy in patients with mCRC (N = 15) who had refractory ascites failing to respond to standard chemotherapy. No drug-related AEs were reported; however only a small number of patients were evaluated [45].
Two studies assessing aflibercept plus FOLFIRI in 62 patients and 16 patients with mCRC, respectively, have reported a higher incidence of hematologic AEs compared with other larger studies [25,26].
Overall, safety profiles reported in registry and real-world studies were consistent with those reported in clinical trials. However, rates of grade ≥ 3 AEs occurred were lower compared with those observed in clinical trials.

Management of aflibercept-Associated Adverse Events in mCRC
AEs frequently associated with aflibercept therapy for CRC, irrespective of severity, include neutropenia, leukopenia, proteinuria, hypertension, diarrhea, stomatitis, fatigue, decreased weight, decreased appetite, elevated liver enzymes, abdominal pain, dysphonia, increased serum creatinine and headache. Severe AEs have included serious or fatal risk of bleeding, gastrointestinal perforation, and delayed wound healing, though some are related to chemotherapy (generally FOLFORI) [46][47][48].
The precise mechanisms responsible for the toxicities of aflibercept are not yet fully elucidated. However, hypertension may be influenced by VEGF interaction with the angiotensin system, and proteinuria may be related to VEGF effects on the renal glomerulus [47,48]. Thrombosis and hemorrhage could be related to the role of VEGF in vascular integrity [48]. Recommendations for managing the most clinically-relevant AEs associated with aflibercept therapy have been published and focus on hypertension, proteinuria, gastrointestinal perforation, thromboembolism and hemorrhage [48]. These are summarized in Table 3. Table 3. General guidelines for aflibercept dosing and schedule modification due to adverse events as categorized by CTCAE 4.0 * [48].

Event
Action to Be Taken   Hypertension  Grade 3  Grade 4 If not controlled with medication, discontinue aflibercept Discontinue aflibercept Proteinuria > 2 g protein/24 h Grade 4 proteinuria (nephrotic syndrome) Hold aflibercept until proteinuria improves to < 2 g of protein/24 h Discontinue aflibercept in patients with > 2 g proteinuria/24 h that does not resolve within 3 months after holding aflibercept. Work-up for proteinuria such as renal biopsy should be considered Discontinue aflibercept

Management of hypertension and proteinuria
Across trials, real-world and retrospective studies, grade ≥ 3 hypertension and proteinuria are among the most frequently reported AEs. Aflibercept prescribing information contains guidance for timely identification and management of hypertension. Patients receiving aflibercept should have their blood pressure monitored at least every 2 weeks or more regularly as clinically indicated throughout treatment and hypertension should be actively controlled with appropriate therapy. Aflibercept should be withheld in patients whose hypertension is not adequately controlled and only resumed at a reduced dose (2 mg/kg) for subsequent cycles once hypertension is controlled. Aflibercept should be permanently discontinued for development of hypertensive crisis or hypertensive encephalopathy (Aflibercept PI, 2016). Note that there is no clinical trial experience with aflibercept in patients with New York Heart Association (NYHA) class III/IV heart failure, as it was an exclusion criterion.
There is also specific guidance for management of proteinuria and patients should be monitored by urine dipstick analysis and/or urinary protein creatinine ratio (UPCR) during aflibercept therapy. In case of dipstick ≥ 2+ or UPCR > 1, 24-h urine collection is mandated. Aflibercept should be withheld for proteinuria ≥ 2 g/24 h and only resumed when resolved to < 2 g/24 h Aflibercept should be suspended for recurrent proteinuria and resumed at a reduced dose (2 mg/kg) on resolution. Aflibercept should be permanently discontinued for development of nephrotic syndrome or thrombotic microangiopathy [48].

Management of Aflibercept-Related Adverse Events in My mCRC Patients-A European Perspective
The European Society for Medical Oncology (ESMO) guidelines provide clinical recommendations for the management of mCRC and are implemented routinely in European clinical practice, though not for the management of treatment-related AEs [49].
In my experience, the most frequently occurring AEs in patients who receive aflibercept are hypertension and proteinuria. Management of hypertension is achieved through anti-hypertensive medication, effective patient education and active monitoring throughout treatment. If blood pressure is not controlled by anti-hypertensive medication, temporary discontinuation of aflibercept is considered. Importantly, patients with a persistent blood pressure of ≥ 150/90 are instructed to contact the emergency room for immediate treatment. Proteinuria is effectively managed by regular monitoring by dipstick test throughout treatment and temporary treatment discontinuation.
The frequency and intensity of AEs related to chemotherapy (FOLFIRI) can be potentiated by aflibercept e.g. mucositis and diarrhea. These are often managed by FOLFIRI dose reductions or a temporary discontinuation in aflibercept treatment.

Management of Aflibercept-Related Adverse Events in My mCRC Patients-An Asian Perspective
The ESMO guidelines have been adapted into pan-Asian consensus guidelines to take into account ethnic differences in toxicity and efficacy of certain systemic treatments in patients of Asian ethnicity [50]. Both guidelines are generally aligned with only a few revisions added in the pan-Asian adaptation. Japanese Society for Cancer of the Colon and Rectum (JSCCR) guidelines for the treatment of CRC [5] have been recently updated and now include aflibercept as one of the options in the category of VEGF-targeted agents [51]. However, as with the ESMO guidelines, there is no focus on management of AEs.
In my experience, the most common AEs observed with aflibercept plus FOLFIRI are consistent with those reported in clinical trials: neutropenia, leukopenia, hypertension, diarrhea, fatigue and decreased appetite. If AEs occur due to this combination treatment, the causative agent should be identified, if possible. These toxicities can be managed using symptomatic treatment for prevention, and in the majority of cases of AEs, aflibercept treatment is continued. In rare cases, treatment may need to be changed or discontinued but, according to UPCR guidelines, this is only with the occurrence of serious (grade ≥ 3) proteinuria with inadequate treatment response, according to UPCR guidelines. Dose reductions of 5-fluorouracil should be considered in cases of grade ≥ 3 neutropenia, grade ≥ 3 fatigue and diarrhea. Reduction of aflibercept should be considered in cases of for grade ≥ 3 proteinuria.
Rarely, granulocyte-colony stimulating factor (G-CSF) is used for prophylactic and therapeutic treatment of neutropenia. Low-risk patients without fever will not receive G-CSF even if they have grade 4 neutropenia when using FOLFIRI with aflibercept. However, the use of G-CSF varies across intuitions. Antimicrobial agents should be administered in cases of febrile neutropenia.
In the management of hypertension, patients are started with a calcium antagonist; there is a paucity of evidence for effective front-line treatment with angiotensin-receptor blockers (ARBs) in this setting. Calcium channel antagonists are also considered to be effective because of their relatively strong antihypertensive effect and the mechanism of action that reduces vascular smooth muscle cell contraction caused by impaired NO signaling induced by VEGF inhibitors. We consider the use of ARBs, angiotensin-converting-enzyme (ACE) inhibitors, beta-blockers and diuretics, dependent on the patient's condition, but most frequently use a combination of calcium antagonist plus ARB or ACE inhibitor. Hypertension could generally be improved by stopping or reducing the dose of VEGF inhibitors, since hypertension with the causative drug is reversible.
In cases of diarrhea, symptomatic treatment may be incorporated using loperamide or infusion treatment (for the worst cases of dehydration only). Prophylactic treatment with 5-hydroxytryptamine type 3 and steroids are used for the prevention of vomiting. Thus, we find that the safety profile of aflibercept is manageable; most AEs can be controlled without the need to discontinue treatment.

Discussion
Our review of the AEs reported among patients with mCRC receiving aflibercept in different populations and settings indicates that the range, incidence and severity of aflibercept-associated AEs is generally evenly distributed across geographies and in some instances appear to be less frequently reported in real-world/retrospective studies compared with clinical trials.
In the US real-world study by Ivanova and colleagues, although patients receiving aflibercept were more heavily pretreated and potentially less robust compared with the VELOUR trial, AE rates were similar to or lower than the VELOUR trial [42]. However, the lower incidence of reported AEs in the post-marketing setting may be due to underreporting as the reliance is on spontaneous reporting, which may fail to capture all occurring AEs [52].
Guidance for the management of clinically relevant AEs is available [48]. Of note, strict monitoring of blood pressure and immediate management of hypertension during therapy is mandatory [19]. In the large randomized trial of aflibercept plus FOLFIRI versus placebo plus FOLFIRI, although grade 3/4 AEs were more frequent in the aflibercept arm, they occurred in early treatment cycles and decreased sharply following initial presentation [14].

Conclusions
Aflibercept is an antiangiogenic agent that targets VEGF-A, VEGF-B and PlGF. It was approved for the treatment of patients with mCRC who had progressed on oxaliplatin following the pivotal VELOUR trial.
Data suggests that the AE profile of aflibercept combination therapy trends towards being more favorable in the real-world compared with clinical trial settings, with no new safety signals identified irrespective of geographical location. Real-world studies reported neutropenia, hypertension and gastrointestinal AEs as being frequently associated with aflibercept, similar to clinical trials. However, the rates at which these occurred was often lower in real-world studies compared with clinical trials. This may be a result of under-reporting or improved patient management. Furthermore, AEs are generally manageable and comparable across various geographic locations. As such, safety experience gained in clinical trials can translate into the clinical setting across various countries, for example, identifying when extra vigilance and close monitoring may be appropriate.