BioDrugs (2013) 27:213–224 DOI 10.1007/s40259-013-0014-9

REVIEW ARTICLE

Regorafenib for Gastrointestinal Malignancies From Preclinical Data to Clinical Results of a Novel Multi-Target Inhibitor Giuseppe Aprile • Marianna Macerelli Francesco Giuliani

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Published online: 23 February 2013 Ó Springer International Publishing Switzerland 2013

Abstract Intracellular signals for cancer cell growth, proliferation, migration, and survival are frequently triggered by protein tyrosine kinases (TKs). The possibility of disrupting core disease pathways has led to development and widespread clinical use of specific TK inhibitors that in the past decade have markedly changed treatment strategies and impacted on overall outcomes. However, intrinsic resistance may limit the benefit of these drugs, and multiple escape routes compensate for the inhibited signaling. The disruption of several points of the same pathway and the simultaneous interference with different intracellular oncogenic processes have both been recognized as valuable strategies to maximize the therapeutic potential of this class of agents. In this scenario, regorafenib has emerged as a novel, orally active, multitarget compound with potent activity against a number of angiogenic and stromal TKs, including vascular endothelial growth factor receptor 2 (VEGFR-2), tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE-2), fibroblast growth factor receptor 1 (FGFR-1), and platelet-derived growth factor receptor (PDGFR). Moreover, the drug has the capability of blocking KIT, RET and V600 mutant BRAF. Starting from interesting preclinical results, this review describes the clinical development of regorafenib in gastrointestinal malignancies, focusing on data derived from cutting edge clinical trials that have provided evidence of efficacy in

G. Aprile (&)
M. Macerelli Department of Medical Oncology, University and General Hospital, Piazzale S Maria Misericordia, 1, 33100 Udine, Italy e-mail: [email protected] F. Giuliani Department of Medical Oncology, National Cancer Institute ‘‘G. Paolo II’’, Bari, Italy

pretreated patients with advanced colorectal cancer or gastrointestinal stromal tumors.

1 Introduction Preclinical evidence of the importance of phosphorylation by protein tyrosine kinases (TKs) in cancer cells [1–3] triggered a widespread hope that anticancer compounds targeting TKs would be useful agents in solid malignancies [4]. Interesting preclinical suggestions were soon followed by convincing clinical results [5] that showed how the use of the TK inhibitor (TKI) imatinib mesilate could change the natural history of gastrointestinal stromal tumors (GISTs) that were characterized by constitutive activation of the oncogenic kinase KIT. At the same time, the study of the human kinome helped to unfold the intricate network of the phosphorylation-based intracellular signaling [6]. Not surprisingly, in the following years a notable pipeline of oral TKIs was developed, with the aim of reshaping the treatment horizon of several solid tumors [7]. Despite the great interest surrounding all these novel targets and the initial success of specific inhibitors, clinical progress has been uneven and the need for further fine tuning has become progressively clear [8]. Indeed, while TKIs were revolutionary in the treatment of tumors driven by a single oncogenic kinase [9], the average survival benefit provided to patients with more complex diseases, though noteworthy, has been limited [10–14] or restricted to molecularly selected subpopulations [15]. Moreover, the issue of primary and secondary resistance has emerged. While redundant feedback loops and crosstalk between different signaling pathways create multiple salvage conduits and may compensate early on for the inhibited signaling, acquired mutations in the

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downstream effectors may cause secondary resistance within the course of therapy. Two main strategies have been pursued to delay or overcome resistance: (i) the parallel block of multiple points of the same pathway and (ii) the simultaneous inhibition of different oncogenic pathways, with the latter strategy being the more plausible [16]. Whether a combination of highly selective agents or a single multitarget drug should be used upfront to simultaneously inhibit different pathways is unclear [17, 18]. Combining selective agents may produce additive or synergistic effects and, although it is a potential source of unforeseen drug interactions, this strategy may allow high target selectivity with limited systemic toxicities [19]. On the contrary, the use of a single multitarget agent offers an advantage with its unique property of optimal target promiscuity. The flipside of having a wide target scope is the potential disadvantage of a narrow therapeutic window, because of likely increased toxicity from crossreactivity with normal tissues [20]. Nevertheless, clinically approved TKIs have, in general, shown favorable safety profiles, with low frequencies of serious adverse events (AEs) reported in phase III clinical trials [9, 11, 13, 14]. A potential limitation of the use of single multitarget inhibitors is inadequate activity against multiple intracellular targets, rather than an increase of toxicities; differing affinities for the receptors may result in diverse target inhibition. In the past decade, the development of novel therapies that target critical biologic pathways has greatly expanded treatment options for patients with advanced GIST or metastatic colorectal cancer (CRC). However, new drugs are needed to further extend patients’ overall survival. In this moving landscape, regorafenib (BAY 73-4506) was developed, an orally active multikinase inhibitor that is being developed and commercialized by a joint venture of Bayer and Onyx Pharmaceuticals [21]. Notably, regorafenib has shown promising results in patients with TKIresistant GIST or heavily pretreated advanced CRC, for whom there is currently no other therapy approved by any regulatory authority. This review aims at describing its mechanisms of action as well as preclinical and clinical development, specifically focusing on gastrointestinal malignancies.

2 Regorafenib: Mechanisms of Action and Targeted Pathways Regorafenib (BAY 73-4506) is a novel oral diphenylureabased multikinase inhibitor that simultaneously targets angiogenic and stromal TKs, including human vascular endothelial growth factor receptor 2 (VEGFR-2), tyrosine kinase with immunoglobulin-like and EGF-like domains 2

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(TIE-2), fibroblast growth factor receptor 1 (FGFR-1), PDGFR, and oncogenic kinases such as KIT and RET, along with p38 mitogen-activated protein kinase (MAPK), v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and its V600 mutant [22]. Its complex chemical structure (4-[4-({[4-choro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]-n-methylpyridine-2-carboxamide) is very similar to that of sorafenib (BAY 43-9006), except for the substitution of a hydrogen atom with a fourth fluorine atom in the central aromatic ring that is responsible for a broader spectrum of action. Angiogenesis is a key hallmark of cancer that contributes to tumor growth and metastases [23]. Amongst the many proangiogenic molecules that have been intensively studied, the different isoforms of VEGF, PDGF, and their receptors are the mainstay actors of tumor neovascularization [24]. Once activated, they trigger a number of proangiogenic pathways that increase vascular permeability, mediate degradation of the extracellular matrix, provide endothelial cells with mitogenic and survival signals, and eventually facilitate blood vessel growth and remodeling [25]. This complex mechanism is targeted by the TKIs, thus contributing to interrupt the downstream signaling [26]. Regorafenib, among its specific antiangiogenic properties, inhibits TIE-2. Primary activating mutations of KIT and PDGFRa genes encoding structurally aberrant TK receptors serve as primary drivers for the development of around 90 % of GIST, while a minority of patients has no mutations in these kinases (wild-type GIST). Despite the fact that patients may benefit from prolonged disease control with imatinib [9] and may further extend the advantage with sunitinib [27], there is the urgent need for other active TKIs to overcome acquired resistance to those compounds, which usually appears within 2 years for imatinib and 6 months for sunitinib after imatinib failure [28]. Multitarget inhibitors may fulfill this gap, and two independent phase II trials have shown a median progression-free survival (PFS) of approximately 5 months when resistant GIST patients are exposed to sorafenib [29, 30]. Along with sorafenib [31] and vandetanib [32], regorafenib may also inhibit the RET pathway. RET is a transmembrane receptor tyrosine kinase with an extracellular cadherin domain that binds calcium, cysteine-rich region, transmembrane domain, and intracellular kinase domain [33]. Mutations in the extracellular coding region (exons 8, 10, and 11) or in the intracellular kinase domain (exons 13 to 16) activate multiple transduction pathways, including RAS/ERK, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), v-src sarcoma viral oncogene homolog (SRC), PLCc, JNK, and STAT3 [34]. At the same time, regorafenib inhibits the ERKMAPK activated pathway, which is activated during the

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differentiation of intestinal epithelial cells; also, there is evidence that this pathway results in the activation of the pathogenesis and progression of CRC [35]. MAPK is a major signaling pathway in cell proliferation and integrates signals that affect differentiation, survival, and migration. Its signaling cascade is involved in different steps of both the proinflammatory response and the metastatic process [36], and, frequently, p38 MAPK is found to be aberrant in human cancers. Since the activation of the RAS/RAF/MEK/ERK cascade induces expression of VEGF [37], there is a link between angiogenesis and the MAPK pathway in CRC. Finally, regorafenib also acts against BRAF, a well known target for cancer therapy [38]. Somatic point mutations in exon 11 or exon 15 of BRAF occur in approximately 8 % of human tumors, most frequently in melanomas, colorectal adenocarcinomas, and thyroid cancers [39]. A single point mutation, V600E, accounts for approximately 90 % of cases, determines the lock of the kinase in the active conformation, and confers CRC cancer patients a dismal prognosis [40]. Currently, specific inhibitors of BRAF V600E have produced excellent results in melanomas [41, 42], but their usefulness for CRC patients is still unproven [43].

3 Preclinical Development of Regorafenib In vitro biochemical and cellular assays have shown that regorafenib may inhibit a number of important kinases within the nanomolar range [35]. Additional inhibited kinases include DDR2, EphA2, PTK5, p38a and b, while a few others (EGFR, PKC, MET, MEK, ERK1/2 and AKT) were unrepressed, even at high drug concentrations. In the same assays, regorafenib was shown to be a potent inhibitor of mutant receptor kinase KIT K642E and RET C634W, continually activated in GIST-882 and TT-thyroid cell lines, respectively. In addition, MAPK pathway inhibition has been evaluated and was interrupted via KRAS inhibition. Actually, regorafenib potently inhibits the serine/threonine kinase BRAF, a downstream target of the RAS signaling pathway, and its oncogenic mutant BRAF V600E. However, the inhibition of this pathway has not been reported in vivo. Notably, regorafenib showed antimetastatic activity in 19 out of 25 different human CRC cell lines [44]. In murine xenograft models, regorafenib has shown potent broadspectrum antitumor efficacy [35]. Tumor-bearing mice treated with a daily regorafenib dose of 10 or 30 mg/kg had significant tumor shrinkage. Remarkably, no animal lethality was noted, suggesting a high apparent therapeutic index with doses in the range of clinical efficacy. In vivo potent antiangiogenic activity was linked to simultaneous

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inhibition of VEGFR-2, TIE-2 and PDGFRb, that was shown to be more profound than inhibition of VEGF signaling alone. The pharmacodynamic effect of the drug on tumor vasculature has been assessed by dynamic contrastenhanced magnetic resonance imaging that showed significant decrease in tumor perfusion and extravasation of the tracer in tumor-bearing rats. Interestingly, no tumor regrowth was observed for 4 days after the last regorafenib dose. In preclinical tumor CRC cell line xenograft models in athymic mice (Colo-205, BRAF mut and KRAS wt; HT-29, BRAF mut and KRAS wt; HCT 15, BRAF wt and KRAS mut), as well as in MDA-MB-231 (breast cancer cell line, KRAS G13D or BRAF G464V mut), or 786-O (renal cancer cell lines, VHL gene deleted) murine models, regorafenib inhibits growth in a dose-dependent manner. A slow regrowth was observed at all doses within 9 days from treatment termination. With these compelling preclinical results shaping a strong rationale, regorafenib has been further studied in several early clinical trials conducted in different solid tumors.

4 Clinical Development of Regorafenib In the first-in-man phase I dose-escalation trial [45], 53 heavily pretreated patients with a median of three previous treatment lines were enrolled to evaluate the safety, pharmacokinetic, pharmacodynamic and efficacy profile of regorafenib. In the study, CRC, ovarian cancer, and melanoma were the most represented tumor types. Regorafenib was administrated as an oral solution for dose levels between 10 and 120 mg or as coprecipitate tablets (of 20 mg or 100 mg) for dose levels of 120, 160 or 220 mg, because of comparable bioavailability of the two oral formulations at higher doses. Eight dose cohorts were evaluated, with 220 mg once daily being the highest dose tested. In cohort 1, patients received regorafenib at a dose of 10 mg on day 1 and days 8–14 every 28 days; in the following cohorts, patients received increasing doses of regorafenib in a 21-days-on, 7-days-off schedule. Overall, the median treatment duration was 78 days (range 3–1,239) and across all dose levels, 50 patients (94 %) received 50 % or more of the planned dose, with 38 patients (72 %) receiving 70 % or more. Forty-four patients (83 %) experienced at least one treatment-related AE. The most frequently reported AEs were hoarseness (55 %), hand-foot skin reaction (HFSR) (40 %), mucositis (36 %), diarrhea (32 %), and hypertension (30 %). The most common grade 3–4 treatment-related AEs were HFSR (19 %), hypertension (11 %), diarrhea (8 %), and rash/desquamation (6 %). No toxic deaths were reported. As expected, the frequency of treatment-related AEs increased with dose levels. Patients enrolled in dose level cohorts of 10–60 mg

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tolerated the drug very well, without reporting dose-limiting toxicities (DLTs), dose reductions, temporary interruptions, or permanent discontinuation. After the evaluation of dose-limiting toxicities occurred in cohort 7 (160 mg) and cohort 8 (220 mg), the daily dose of 160 mg was established as the maximum tolerated dose (MTD) for regorafenib given in the 21-days-on, 7-daysoff schedule. Pharmacokinetic data have been also provided [45]. Among regorafenib-derived active metabolites, M-2 (N-oxyde metabolite; BAY 75-7495) and M-5 (N-oxyde/ N-desmethyl metabolite; BAY 81-8752) have multiple peaks of plasma concentration at steady-state, the first occurring after 1–4 hours and the others at 8 and 24 hours from administration. Preclinical studies have demonstrated pharmacological activity of M-2 and M-5 with efficacy similar to the parental compound. M2 and regorafenib itself may be further modified by glucuronidation. A dosedependent increase of plasma concentration of regorafenib was demonstrated up to 60 mg, but the same correlation was lacking for dose escalation up to 120 mg. Regorafenib, M-2, and M-5 present a long half-life (about 20–40 hours) that explained the accumulation of the drug and its metabolites in plasma after multiple doses. Regorafenib has time-linear pharmacokinetics and its accumulation is predictable. On the contrary, M-2 and M-5 concentrations vary with time. Elimination of M-2 is similar to that of the parent drug, while data suggested that M-5 may have a slower elimination with a prolonged half-life. In the above-quoted phase I study [45], antiangiogenic activity of the experimental drug was assessed by measuring changes in tumor perfusion with dynamic contrastenhanced magnetic resonance imaging (DCE-MRI). A significant decrease of 40 % was reported after 21 days with the dose levels of 120 mg (solution), 160 mg (tablet), and 220 mg (tablet). A decrease in plasma VEGFR-2 concentrations during cycles 1 and 3 was reported to be dose-dependent. Accordingly, plasma VEGF concentration increased during the 21 days of exposure to regorafenib and returned to baseline levels during the following 7 days off, suggesting that the intermittent schedule might cause angiogenic flares during the break periods. In the study, tumor response was assessed by RECIST (Response Evaluation Criteria In Solid Tumors) in 47 patients (88 %). Three patients had partial response (PR) and 32 had stable disease (SD) at 2 months on study treatment. Responding patients had renal cell carcinoma (60 mg oral solution, time to progression [TTP] 20.6 months), CRC (220 mg tablet, treatment discontinued for AE) and osteosarcoma (120 mg solution, TTP 8.3 months). The study recommended a 160 mg oral daily dose to be used in phase II trials testing a 21-days-on, 7-days-off schedule of regorafenib.

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Since activity of the drug was noted in patients with advanced CRC, the study was expanded to further evaluate the drug in this population [46]. Overall, 38 heavily pretreated Caucasian patients (median of four prior lines of therapy) were included in the analysis (15 in the doseescalation cohort and 23 in the extension cohort); 26 of them received regorafenib at 160 mg daily. Median age was 64 years (range 36–85), and the performance status (PS) was generally good (0–1 95 %). Previous systemic treatments included oxaliplatin (84 %), irinotecan (84 %), bevacizumab (53 %) and anti-EGFR antibodies (53 %). For the 26 patients included in the 160 mg dose level cohort, the median treatment duration was 49 days. Although six patients permanently discontinued regorafenib because of treatment-related AE, toxicities were easily manageable in the outpatient setting with few treatment reductions or interruptions. Twenty-seven (71 %) patients were evaluable for response. Disease control rate lasting at least 2 months was 74 %, with a median PFS of 107 days (95 % CI 66–161). The role of KRAS status in predicting regorafenib activity was also tested. An exploratory survival analysis showed slightly longer median PFS for mutant KRAS tumors compared with wild-type (84 vs 161 days), although the difference was not statistically significant. Nevertheless, the small sample size and the nature of the analysis prevented drawing any definitive conclusion about the impact of KRAS status on response to regorafenib therapy. The second phase I dose-escalation trial reported in the literature [47] assessed safety, pharmacokinetics and efficacy of continuous regorafenib in 38 heavily pre-treated advanced cancer patients (including CRC 16 %, thyroid 13 %, and head and neck cancer 13 %). AE frequencies were similar to those previously reported. DLTs in cycles 1–2 occurred in 2 out of 11 patients at a daily dose of 100 mg (HFSR, anemia/thrombocytopenia), in 3 out of 6 (HFSR n = 2; thrombocytopenia n = 1) at 120 mg, and in 4 out of 10 (HFSR n = 2; diarrhea n = 1; hyperbilirubinemia/AST increase n = 1) exposed to a dose of 140 mg. Consequently, the continuous daily dose of 100 mg was defined as MTD with clinical activity. Interestingly, disease control lasting at least 6 weeks was reported for 61 % of included patients. The same Investigators’ group presented a parallel phase I dose-escalation study conducted in advanced refractory non-small cell lung cancer (NSCLC) patients [48]. Twenty-three patients were treated with two different doses of regorafenib of 100 or 120 mg administered orally once daily. Median treatment duration was 84 days (range 12–281). AEs noted were similar to those already reported, except for mild-to-moderate hypothyroidism in 26 % of treated patients. Pharmacokinetic data confirmed plasma increase of the drug proportionally to dose exposure. Among 17 evaluable patients, 13 reached

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SD at 6 weeks after the start of treatment and 4 patients after 12 weeks. One patient with SD had a PFS of 279 days. 4.1 Phase II Trials Three phase II trials on regorafenib have been reported so far (49–51). The first is an open-label, phase II study that enrolled 49 untreated patients with renal cell carcinoma, with PS 0–1 and low or intermediate risk according to the Motzer criteria score [49]. Patients received regorafenib at a dose of 160 mg on a 21-days-on, 7-days-off schedule. The primary endpoint of the study was response rate. Renal failure occurred in 8 % of patients, most likely due to continued drug intake despite having dehydration. Of 33 evaluable patients, 27 % reported PR and 42 % SD. At the time of interim analysis, 35 patients were still on study. The second is a phase II, uncontrolled, open-label, international safety study in pretreated hepatocellular carcinoma (HCC) patients [50]. The primary aims of the trial were safety and tolerability; secondary endpoints were TTP, overall survival (OS), response rate, and disease control rate. A pharmacokinetic study of regorafenib and its M2 and M5 metabolites was also included. Thirty-six Child-Pugh A patients (median age 61 years) previously exposed to sorafenib were recruited and treated with an oral daily dose of 160 mg in a 3-weeks-on, 1-week-off schedule until disease progression (DP), patients’ refusal, or unacceptable toxicity. Median treatment duration was 15.5 weeks (range 2–36), with 15 patients still on treatment at the time of analysis. Regorafenib was discontinued because of DP in six patients, AE in 12 patients, consent withdrawn in two patients and death in one case. Overall, grade 3–4 AEs were limited, with fatigue (17 %), HFSR (14 %), and diarrhea (6 %) being the more frequently observed. Median TTP was 4.1 months. The disease control rate of 72 % (one PR and 25 SD) and the very interesting 6-month OS rate of 80 % suggested a promising activity of regorafenib in this population. The mechanism by which regorafenib may overcome resistance to sorafenib remains to be investigated in future studies. Finally, based on its ability to inhibit c-KIT and PDGFR in GIST cell lines [35], regorafenib has been investigated in patients with advanced or metastatic GIST who have progressed after treatment with imatinib and sunitinib, the only approved drugs to treat this disease. The primary objective of a recent multicenter, phase II trial was to assess clinical benefit, as defined by the composite of complete response, PR, and SD lasting at least 16 weeks, in 34 TKI-resistant GIST patients [51]. Secondary goals were PFS, safety, and tolerability of the drug. Any number of previous therapies for GIST was permitted, but previous

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exposure to sorafenib was among the exclusion criteria. Regorafenib was administrated at 160 mg/day with the usual 3-weeks-on, 1-week-off schedule until RECIST 1.1 DP, unacceptable toxicity, or patients’ withdrawal. Additionally, tumor genotyping (KIT, PDGFRa, and BRAF) was performed, while a separate consent was asked for optional tumor biopsies to be performed before the first dose of regorafenib and between day 10 and 21 of the first cycle. Median age of enrolled patients was 56 years (range 25–76), median number of prior regimens was 2 (range 2–10). Disease progression was the main reason for stopping both imatinib and sunitinib, and median times on the drugs were 21 and 13 months, respectively. Primary kinase mutation was available for 30 patients, and resulted in KIT exon 11 (19 patients), KIT exon 9 (3 patients), BRAF exon 15, or wild-type status for KIT and PDGFRa (8 patients). The median number of cycles administered per patient was 8 (range 2–17). At the final analysis, clinical benefit was documented in 25 patients (4 PR, 22 SD); two patients progressed early, and one withdrew consent. Median PFS for the whole cohort was 10 months, while median OS was not reached after a median follow-up of 11 months. The scope of toxicities was not different from expected, with grade 3 hypertension, HFSR and hypophosphatemia reported in 36 %, 24 %, and 15 % of patients, respectively. Three life-threatening AEs were reported (two cases of hyperuricemia and one thrombotic event). Although the small sample size precludes the drawing of strong conclusions, there was no statistically significant difference in the rate of clinical benefit among genotype groups. Immunoblotting analysis of biopsies repeated before treatment start and at day 15 demonstrated *50 % inhibition of KIT and AKT phosphorylation in 75 % of patients, all with SD lasting at least four cycles. Clinical activity of regorafenib was also studied, utilizing FDGPET/CT, a pharmacodynamic biomarker with imatinib and sunitinib in patients with GIST, documenting metabolic responses even when radiological assessment according to RECIST criteria confirmed the disease as stable [52]. 4.2 Phase III Trials Two phase III trials have been published: the first investigating regorafenib treatment in CRC, the second in GISTs. Following the rationale for regorafenib use in CRC and preliminary positive results in phase I trials, the multicenter, randomized, double-blind, placebo-controlled, phase III CORRECT (Patients with metastatic COloRectal cancer treated with REgorafenib or plaCebo after failure of standard Therapy, BAY 73-4506/14387) trial was conducted in 16 countries with 114 active centers to evaluate efficacy and safety of regorafenib in patients with advanced disease who

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had progressed during or within 3 months following last administration of approved standard therapy [53]. The primary efficacy endpoint of the study was OS, the secondary endpoints were PFS, objective response rate (ORR) and disease-control rate (DCR). Other endpoints included duration of response/SD, quality of life, pharmacokinetics, and evaluation of plasma biomarkers. Among others, key inclusion criteria to be fully satisfied at time of screening included age C18 years, pathological evidence of advanced colorectal adenocarcinoma, DP during or within 3 months after last therapy, life expectancy of at least 3 months, good Eastern Cooperative Oncology Group (ECOG) PS (0 or 1), and adequate bone marrow, liver, and renal function. Previous therapies included fluoropyrimidine, oxaliplatin, irinotecan, bevacizumab, and cetuximab or panitumumab (if KRAS wild-type). As expected, baseline characteristics were well balanced in the two study groups, including KRAS mutational status and number of prior anticancer therapies (approximately 60 % of patients had received C4 regimens of chemotherapy). To demonstrate a 33 % improvement in median OS (from 4.5 to 6 months), 760 patients were randomized 2:1 to regorafenib (160 mg daily in a 3-weeks-on, 1-week-off schedule) plus best supportive care (BSC) or placebo plus BSC. Stratification factors included prior treatment with VEGF-targeting drugs (yes versus no), time from diagnosis of metastatic disease (C18 months versus \18 months), and geographical origin. Before the final analysis, scheduled soon after approximately 582 death events were observed (1-sided overall a of 0.025), two interim analyses were preplanned and conducted by an Independent Data Monitor Committee. At the time of the second interim analysis (75 % of events required for final analysis) data showed an estimated hazard ratio (HR) for OS of 0.77 (95 % CI: 0.63–0.94; 1-sided p = 0.0051) with a median OS of 6.4 months for regorafenib versus 5.0 months for placebo. The estimated HR for PFS was 0.49 (95 % CI: 0.42–0.58; 1-sided p \ 0.000001) with a median PFS of 1.9 months (95 % CI: 1.88–2.17) for regorafenib and 1.7 months for placebo (95 % CI: 1.68–1.74). Notably, ORR was only 1.6 % for regorafenib versus 0.4 % for placebo while the DCR was 44.8 % for regorafenib (PR 1 %, SD 43.8 %) and 15.3 % (DP 0.4 %, SD 14.9 %) for placebo (p \ 0.000001), indicating that the strength of the drug is more in delaying progression than inducing tumor shrinkage. Thus, regorafenib is the first small molecule multitarget TKI with demonstrated efficacy in advanced CRC. Frequencies of AEs were as expected: fatigue 47.4 % (grade 3–4, 9.6 %); HFSR 46.6 % (grade 3, 16.6 %); diarrhea 33.8 % (grade 3–4, 7.2 %); anorexia 30.4 % (grade 3, 30.2 %); voice changes 29.4 % (grade 3, 0.2 %); hypertension 27.8 % (grade 3, 7.2 %); oral mucositis 27.2 % (grade 3, 3 %); and rash/desquamation 26 % (grade 3, 5.8 %). Mutated KRAS status was reported for 54.1 % of

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patients exposed to regorafenib and for 61.6 % of those that received placebo. Interestingly, no apparent effect of KRAS status on the primary efficacy outcome (PFS) was observed, even if a subgroup analysis showed that regorafenib produced significant OS advantage for patients with KRAS wt tumors (HR 0.65, 95 % CI 0.48–0.91) but not for those with mutated tumors (HR 0.87, 95 % CI 0.67–1.12) [54]. Waiting for marketing authorization, an ‘expanded access’, phase IIIb, prospective, interventional, open-label, singlearm, multicenter study of regorafenib started, providing the drug to CRC patients who have failed after all available standard therapies [55]. Based on preliminary results, the randomized, doubleblind, placebo-controlled, phase III GRID (GIST, Regorafenib In Progressive Disease) trial, a collaborative worldwide effort among academic and industrial research teams, was funded by Bayer HealthCare Pharmaceuticals and conducted in 17 countries across Europe, North America and Asia-Pacific [56]. The aim of the study was to evaluate efficacy and safety of regorafenib in patients with metastatic and/or unresectable GIST who have already failed on at least imatinib and sunitinib. Six months were sufficient to screen 234 patients. Of those, 199 were randomized 2:1 to receive regorafenib at 160 mg once daily on a 3-weeks-on, 1-week-off schedule plus BSC or placebo (same schedule) plus BSC, with the ambitious goal of obtaining a 100 % increase in PFS (HR 0.5). Patients who had been treated with any VEGFR inhibitors other than sunitinib were excluded. The same was true for patients with cardiovascular dysfunctions, including congestive heart failure, myocardial infarction within 6 months before study entry, cardiac arrhythmias requiring medical treatment, uncontrolled hypertension, or unstable angina. The primary endpoint was PFS judged as per independent blinded central review. Co-secondary endpoints were OS, TTP, ORR, DCR and duration of response. In addition, exploratory analyses were planned in order to verify the impact of tumor genotype on outcomes, screen for comprehensive kinase mutations in the plasma, and examine health-related quality of life. As expected, baseline characteristics such as median age, sex, race, number of prior lines of therapy, and ECOG PS were all well balanced between the two groups. Notably, heavily pretreated patients exceeded 40 % in both treatment arms (44.4 % in the regorafenib arm versus 40.9 % in the placebo arm), with nilotinib being the most frequent third-line treatment (21.8 % and 30.3 %, respectively). The primary endpoint was clearly met, since regorafenib significantly improved median PFS compared with placebo (4.8 versus 0.9 months, HR 0.27, 95 % CI; 0.19–0.39; 1-sided p \ 0.0001), and the PFS benefit was confirmed in all prespecified subgroups. At time of DP, patients were eligible for unblinding and crossover to open-label regorafenib if initially assigned to

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placebo. In all, 85 % were able to receive regorafenib after progression and, even among those patients, a 5-month PFS was reported. The small number of deaths (29 in the regorafenib arm, 17 in the placebo arm), makes data currently immature to evaluate overall survival, even if this parameter will most probably be uninformative, suffering the crossover effect because of this trial design. Median OS was not reached in either group, although a non-significant trend was noted in favor of patients who started regorafenib earlier in the course of care with an estimated HR of 0.77 (95 % CI 0.42–1.41). Interestingly, patients exposed to regorafenib had higher response rates (4.5 versus 1.5 %) and disease control rates (52.6 versus 9.1 %) compared with those who received placebo. The safety profile of regorafenib was commensurated with previous studies. The most common severe drug-related AEs were HFSR (19.7 %), hypertension (22.7 %), and diarrhea (5.3 %). Baseline GIST genotype was available for approximately half of the included patients. Exon 11 KIT mutation was found in 53.1 % of patients, exon 9 KIT mutation in 15.6 %. In the exploratory analysis, the advantage in PFS among those patients treated with regorafenib was similar to that reported for the entire population, regardless of mutational status. Results of regorafenib in progressive GIST after failure of imatinib and sunitinib satisfy an unmet clinical need, and this multitarget inhibitor may be proposed as a potential new standard of care for this patient population [56].

5 Ongoing Trials, Future Research, and Open Questions The ultimate goal to introduce new drugs in the advanced setting is to offer more opportunities to cancer patients who have failed standard therapies and are running out of valuable treatment options. Along this line, the benefit provided by a new drug to heavily pretreated patients with GIST, colorectal cancer or HCC may at least cover an area of high unmet need. Undoubtedly, regorafenib is a novel, orally active multikinase inhibitor with a strong preclinical rationale and promising clinical results. Still, a number of puzzling questions regarding regorafenib remains to be answered, and future clinical studies are still being designed to investigate on these issues. 5.1 Regorafenib as Single-Agent or in Combination Therapy? First of all, it is unclear whether it is better to employ regorafenib as a single agent or in combination with other drugs and if it should be used as last salvage treatment or moved to front-line therapy. Regarding its use as a single

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agent or in combination, a phase I study [57] is ongoing to evaluate the safety profile, MTD and pharmacokinetic interactions of first-line regorafenib in combination with pemetrexed and cisplatin in lung cancer. Moreover, a number of studies are ongoing in patients with advanced CRC to evaluate the safety and efficacy of the combination of regorafenib in first- and second-line treatment with common backbone regimens such as FOLFIRI or FOLFOX [58–60] [Table 1]. Hopefully, regorafenib will succeed where other TKI (e.g., vatalanib PTK787/ZK222584 in the CONFIRM (Colorectal Oral Novel Therapy for the Inhibition of Angiogenesis and Retarding of Metastases) trials, cediranib AZ2171 in the HORIZON studies) have failed. 5.2 Which is the Optimal Dose and Schedule? The intermittent 160 mg/day schedule (3 weeks on, 1 week off) has been tested in two randomized, phase III trials conducted in CRC and GIST, and may therefore be considered as the standard. However, a continuous daily dose of 100 mg was also proven to be feasible and have the potential advantage to avoid angiogenic flares during the rest period. Likewise, different doses of sunitinib (50 mg/ day 4 weeks on, 2 weeks off or 37.5 mg/day continuously) are equally effective and safe [61]. In the Strumberg phase I trial [46], regorafenib was reduced or interrupted in two out of three patients, and 25 % of those treated at the 160 mg dose level permanently discontinued the drug because of AEs. Moreover, in the CORRECT trial [54], the proportion of regorafenib-treated patients experiencing AEs leading to treatment discontinuation was seven times higher than that of patients who received placebo, while in GIST patients [51], approximately one third of patients could tolerate a maximum dose of 80 mg per day. Theoretically, pharmacokinetic studies may help clinicians in defining the ideal treatment dose and schedule. In a cohort of 79 GIST patients exposed to a daily imatinib dose of 400 mg (n = 36) or 600 mg (n = 37) [62], a lower trough level measured at steady-state seemed to be associated with outcome. Specifically, patients with minimum concentration lower than 1,110 ng/mL at day 29 had significantly shorter median TTP compared with those with a higher steady-state concentration of imatinib (11.3 months vs [30 months, p \ 0.0029). Although no survival differences were reported for patients with different trough levels, this preliminary report suggested that a low steady-state plasma level may contribute to imatinib failure. Plasma concentration-time profile at steady state of regorafenib and its major metabolites have also been reported [46]. Further studies, however, are needed to verify the relationships between drug plasma levels and clinical outcomes of imatinib and other TKIs.

Study title (acronym)

An Open-label Phase IIIb Study of Regorafenib in Patients With Metastatic Colorectal Cancer Who Have Progressed After Standard Therapy

Phase I, Open-label, Non-placebo Controlled Study to Determine the Safety, Pharmacokinetics, and Pharmacodynamics of BAY73-4506 in Combination With mFOLFOX6 or FOLFIRI as First or Second Line Therapy in Patients With Metastatic Colorectal

A Randomized, Double-blind, Placebo-controlled Phase III Study of Regorafenib Plus Best Supportive Care Versus Placebo Plus BSC in Asian Subjects With Metastatic Colorectal Cancer Who Have Progressed After Standard Therapy (CONCUR)

Multi-Center, Randomized, Placebo-Controlled Phase II Study of Regorafenib in Combination With FOLFIRI Versus Placebo With FOLFIRI as Second-Line Therapy in Patients With Metastatic Colorectal Cancer

An Uncontrolled, Open-label, Phase II Study in Subjects With Metastatic Adenocarcinoma of the Colon or Rectum Who Are Receiving First Line Chemotherapy With mFOLFOX6 (Oxaliplatin/ Folinic Acid/5-fluorouracil [5-FU]) in Combination With Regorafenib (CORDIAL)

Trial identifier

NCT01538680

NCT00934882

NCT01584830

NCT01298570

NCT01289821 II

II randomized 2:1

III

I

IIIb expanded access

Phase

Regorafenib ? mFOLFOX6

Regorafenib/ placebo ? FOLFIRI

Regorafenib

Regorafenib ? mFOLFOX6 or FOLFIRI

Regorafenib

Treatment

Regorafenib 160 mg po on days 4–10 and 18–24. In case of administration as a single agent during the study, 160 mg po daily for 3 weeks on, 1 week off

160 mg, po, daily, per 7-day cycle

160 mg po daily for 3 weeks of every 4-week cycle

160 mg po once daily from days 4–10 and from days 18–24 in combination with mFOLFOX6 or FOLFIRI

160 mg po daily for 3 weeks of every 4-week cycle

Schedule

First-line therapy in patients with metastatic CRC

Second-line therapy in pretreated patients with FOLFOX, KRAS or BRAF mutant with metastatic CRC

Pretreated Asian CRC patients who have failed all available standard therapies

CRC patients pretreated with no more than one previous chemotherapy for advanced disease

Pretreated CRC patients who have failed all available standard therapies

Included population

Table 1 Major ongoing trials with regorafenib as monotherapy or in combination with chemotherapy in gastrointestinal malignancies

Objective response rate

PFS

OS

Adverse event collection; effect of regorafenib on the PK of mFOLFOX6 and FOLFIRI

Safety

Primary endpoints

October 2013

Winter 2019

Spring 2014

Spring 2012

Spring 2014

Estimated study completion date

220 G. Aprile et al.

221

Autumn 2012

5.3 Will Mutational Analyses Ever Help in Selecting Patients with a Higher Chance to Benefit?

CRC colorectal cancer, HCC hepatocellular carcinoma, OS overall survival, PFS progression-free survival, PK pharmacokinetics, po orally

Child A HCC patients who cannot benefit from treatments of established efficacy and have failed sorafenib Regorafenib II An Uncontrolled Open Label Multicenter Phase II Safety Study of BAY73-4506 in Patients With Hepatocellular Carcinoma NCT01003015

160 mg po daily for 3 weeks of every 4-week cycle

Safety

Not specified Biomarker data. Evaluation of potential relationships between biomarker data and clinical activity Biopsy amenable, pretreated Asian CRC patients 160 mg po daily for 3 weeks of every 4-week cycle Regorafenib IIa A Phase IIA Proof of Concept Study of Regorafenib (Bayer 73-4506) in Biopsy-amenable Asian Colorectal Cancer Patients NCT01189903

Included population Schedule Treatment Phase Study title (acronym) Trial identifier

Table 1 continued

Primary endpoints

Estimated study completion date

Regorafenib for Gastrointestinal Malignancies

Available data does not seem to substantiate this hypothesis. In the exploratory analysis of a phase I trial [46], no clear difference in PFS between KRAS mutated and wildtype group were noted, even though median PFS of the wild-type group was 90 % longer (161 vs 84 days). Moreover, in the phase III trial enrolling CRC patients, a very low overall response rate was reported (1 %), and the likelihood to obtain prolonged SD seemed to be independent from KRAS or BRAF status [54]. Finally, despite the significantly longer PFS of patients with GIST tumors carrying the primary exon 11 KIT mutations compared with the PFS of patients with tumors with primary exon 9 mutations (p = 0.01), there was no statistically significant difference in the rate of clinical benefit among different genotype groups [51, 56]. 5.4 Do Patients Previously Exposed to VEGF Inhibitors Still Respond to Regorafenib? The answer seems to be yes. In the CORRECT trial, all the patients were previously exposed to (and failed) bevacizumab. Moreover, to carry on with antiangiogenic treatment beyond bevacizumab failure is a reasonable strategy, confirmed by the post-progression use of bevacizumab itself combined with a different backbone chemotherapy as in the ML18147 trial [63] or aflibercept combined with second-line FOLFIRI in the VELOUR (Aflibercept Versus Placebo in Combination With Irinotecan and 5-FU in the Treatment of Patients With Metastatic Colorectal Cancer After Failure of an OxaliplatinBased Regimen) study [64]. Accordingly, 100 % of GIST patients exposed to regorafenib in the US study [51] and in the GRID trial [56] have previously failed sunitinib. 5.5 How May Regorafenib Benefit Patients with GIST? Preclinical and clinical data have shown that secondary mutations in KIT account for the vast majority of TKI resistance. Based on the hypothesis that individual KIT mutant oncoproteins may affect drug sensitivity [65], Antonescu and colleagues investigated the efficacy of sorafenib, nilotinib, and dasatinib on a set of Ba/F3 cells expressing various imatinib-resistant KIT mutants. The exposure to sorafenib, the parental compound of regorafenib, resulted in inhibition of all the double KIT mutants tested, included those resistant to other TKIs. These mutations occur in exons 13 and 14, which encode the ATP binding pocket, or in exons 17 and 18, which encode the kinase activation loop. Since both imatinib and sunitinib are virtually useless against mutations affecting the

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activation loop of KIT or PDGFRa, it has been hypothesized that this could be the target of regorafenib. Alternatively, the new TKIs may inhibit other salvage signaling pathways. Indeed, the main hurdle in overcoming secondary resistance in GIST patients is due to the fact that multiple secondary mutations can be synchronously present at multiple metastatic locations in the same patient, hindering the efficacy of most TKIs, including regorafenib.

6 Conclusions In summary, regorafenib is a potent, orally active, multitarget inhibitor, exhibiting robust efficacy data in patients with heavily pretreated metastatic colorectal cancer or advanced TKI-refractory GIST. Interestingly, it appears to inhibit the cancer-promoting signals in a very unique way, retaining its broad activity even in patients whose cancers have developed resistance to all other standard treatments. Ongoing and future trials will shed light on a number of unanswered questions and help oncologists to optimize the use of the drug. A decade after the introduction of targeted agents in the clinical practice, there is need for new drugs that may extend survival and provide new hope to patients with life-threatening gastrointestinal malignancies. Regorafenib, indeed, is part of this story. Acknowledgements The authors want to thank Dr Jessica Menis, Clinical Fellow, EORTC, Brussels, Belgium and Dr Masoud Saman, Department of Otolaryngology – Head and Neck Surgery, New York Eye and Ear Infirmary, NY, USA for their valuable comments and friendly contribution in reviewing the manuscript. Competing interest statement The authors declare they have no competing financial interests. Funding source No sponsors were involved in the writing of the manuscript or in the decision to submit the manuscript for publication.

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