Molecular target therapy for gastroenteropancreatic endocrine tumours: Biological rationale and clinical perspectives

Critical Reviews in Oncology/Hematology 72 (2009) 110–124

Molecular target therapy for gastroenteropancreatic endocrine tumours: Biological rationale and clinical perspectives Gabriele Capurso a,1 , Nicola Fazio b,1 , Stefano Festa a , Francesco Panzuto a , Filippo De Braud b , Gianfranco Delle Fave a,∗ a

Digestive and Liver Disease Unit, S. Andrea Hospital, II Medical School, University “La Sapienza”, Via Di Grottarossa 1035-1039, 00189, Rome, Italy b Unit of Clinical Pharmacology and New Drugs, European Institute of Oncology, Milan, Italy Accepted 28 January 2009

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Targeting angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Bevacizumab (BEV) (AvastinTM , Roche) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Sunitinib malate (SU11248, SutentTM , Pfizer) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sorafenib (BAY 43-9006, NexavarTM , Bayer Pharmaceuticals Corporation) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Vatalanib (PTK787/ZK222584; Schering AG, Novartis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Thalidomide (ThalidomideTM ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Endostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Blocking receptor tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. The EGF receptor family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Gefitinib (Iressa® , ZD1839, Astra Zeneca, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Other receptor tyrosine kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Imatinib (Gleevec® ; Novartis Pharma, Basel, Switzerland) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Blocking the mammalian target of rapamycin pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Temsirolimus (Torisel® , Wyeth) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Everolimus (RAD001, Novartis Pharma, Basel, Switzerland) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Other possible therapeutic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Gastroenteropancreatic endocrine tumours (GEP ETs) represent a relatively rare and heterogeneous group of neoplasms whose therapy can be challenging. The poorly differentiated, fast-growing cases are treated with chemotherapy. In the slow-growing ones, biotherapy is usually performed. Several categories of targeted therapies have been studied for their treatment in vitro and in vivo. A critical review of molecular alterations suggests a rationale for targeting angiogenesis, and the phosphatidylinositol 3 kinase (PI3 K)/AKT/mammalian target of rapamycin (mTOR) pathway. Accordingly, antiangiogenic agents and mTOR inhibitors are presently the most tested agents in phase II and III studies. Bevacizumab, some multitarget inhibitors, and mTOR inhibitors showed promising results in patients with advanced GEP ETs. A limited

∗ 1

Corresponding author. Tel.: +39 06 33775691; fax: +39 06 33775526. E-mail address: [email protected] (G. Delle Fave). The two authors contributed equally.

1040-8428/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.critrevonc.2009.01.008

G. Capurso et al. / Critical Reviews in Oncology/Hematology 72 (2009) 110–124

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activity has been reported for imatinib and epidermal growth factor receptor (EGFR) inhibitors. Combinations of molecular targeted therapies with different sites of action, and somatostatin analogues may be relevant to avoid molecular escape pathways. Future trials should include more homogeneous groups of patients and pay more attention to the subgroup with progressive disease. © 2009 Elsevier Ireland Ltd. All rights reserved. Keywords: Pancreatic endocrine tumours; Carcinoids; Targeted therapy; Angiogenesis; Tyrosine kinase; mTOR

1. Introduction Gastroenteropancreatic (GEP) endocrine tumours (ETs) represent a heterogeneous group of neoplasms deriving from the gastrointestinal (GI) tract and pancreas diffuse neuroendocrine system [1]. They can be further subdivided according to their primary site of development [pancreatic endocrine tumours (PETs) vs gastrointestinal tract carcinoids], clinical behaviour (stable or progressive disease – SD or PD), histological features (well or poorly differentiated), and presence of clinical syndromes due to hormones hypersecretion from cancer cells (“functioning” vs “non functioning” tumours). Even if considered fairly rare entities, their incidence seems to be increasing to up to five cases per 100,000 persons per year [2], with autopsy series reporting even higher incidences. In addition, due to their long-term survival, their prevalence is even higher than those of oesophageal, gastric, pancreatic and hepatobiliary cancers in the US [2]. Accordingly, a recent analysis of the American Surveillance, Epidemiology, and End Results (SEER) databases suggests that, while PETs represent only 1% of pancreatic neoplasms by incidence, their prevalence is close to 10% of all pancreatic cancers, reflecting their “indolent” nature [3]. Nevertheless, although GEP ETs are often well-differentiated and usually characterized by a relative slow growth-rate, a high percentage of stable disease, and a prolonged long-term survival irrespective of treatment [4], a subset of patients, especially those with PETs, display a rapid tumour growth leading to early death [4,5]. Many patients instead live for several years with metastatic disease, experiencing debilitating symptoms caused both by hypersecretion of various bioactive products and/or by tumour mass effect [6]. Given their heterogeneity and the increasing number of therapeutic options, the optimal management of patients with GEP ETs should presently involve different specialities in a multidisciplinary effort aimed to achieve a highly individualised therapeutic approach tailored on the patient’s specific characteristics. Surgery still represents the mainstay curative treatment in patients with limited disease. When cure is not possible because of advanced loco-regional disease or metastases, alternative medical treatments are available. In these cases it is particularly important to define whether the tumour is in progression before treatment to better evaluate drug efficacy, since many GEP ETs may remain stable for a long time even without treatment. Somatostatin analogues (SA) play a relevant role, being highly effective in controlling hormone hypersecretion and

symptoms in functioning tumours [7]. Their action on tumour growth in progressive disease is variable, with response rates being around 50%, and with a lower efficacy observed in PETs [8,9]. The effect of SA is however limited to disease stabilization, and partial response (PR) with tumour shrinkage is exceptional. Interferon (IFN)-alpha, alone or in combination with SA, is able to induce a good biochemical response, but rarely has a measurable objective response on tumour masses [10]. GEP ETs are usually not an ideal target for traditional, DNA-damaging cytotoxic agents, given their typical low proliferative indexes. Modest evidence of benefit has been reported, and indications are usually limited to poorly differentiated or highly aggressive tumours [11]. Metastatic disease can be treated with different locoregional approaches, such as hepatic artery embolization, chemoembolization or other ablative treatments, with good results in the control of symptoms and in the reduction of mass effects, obviously with palliative intents [12]. Peptide receptor radionuclide therapy (PRRT), taking advantage of the expression of somatostatin receptors on the surface of cancer cell, has yielded excellent results in the treatment of advanced GEP ETs [13], and should be considered as one of the main therapeutic options. However, some limits to its use are still represented by its relatively limited availability, possible lack of somatostatin receptors expression in some tumours, and systemic toxicity. In this scenario, new strategies are required to face the main therapeutic challenge, the treatment of progressive, advanced disease. Targeted therapy, interfering with key growth and angiogenic molecular pathways, represents a promising approach for GEP ETs treatment. A profound, specific knowledge of molecular biology and genetics of these tumours is necessary to implement its use. Aim of this paper is to provide a critical, updated review on the role of targeted drugs scrutinizing preclinical and clinical trials designed to evaluate their performances in the treatment of GEP ETs, and to discuss the biological rationale of these strategies. The review is based on a systematic search conducted in June 2008 and further updated in September 2008.

2. Targeting angiogenesis The role of angiogenesis in tumour growth and metastasis development is well recognized. The inhibition of neoangiogenesis is considered a valid treatment approach, achieving

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good results in a number of solid tumours [14]. Angiogenesis is a multistep process, including stimulation of endothelial proliferation by cytokines, degradation of extracellular matrix proteins and migration of endothelial cells mediated by adhesion molecules. GEP ETs seem an appropriate model for targeting of angiogenesis. Indeed, a transgenic mouse model (Rip1-Tag2) in which the animals develop PETs [15] has been studied in depth, showing that angiogenesis switch is a key mechanism in this process [16]. The same authors have also demonstrated that VEGF and its receptors are expressed in both normal, hyperplastic and neoplastic islet cells, and that matrix metalloproteinases (MMPs) play a pivotal role in stimulating angiogenesis [17]. Inhibition of angiogenesis is effective in reducing angiogenesis and tumour growth in the same model [18–20]. Data obtained from GEP ETs patients also suggested that these tumours are highly vascular, express proangiogenic molecules such as VEGF, and VEGF expression correlates with a more aggressive tumour behaviour [21–23]. On the other hand, other studies indicate that PETs present a wide range of microvascular density (MVD), with malignant tumours showing lower MVD and VEGF expression than benign ones [24]. Moreover, the VEGF gene was found to be down-regulated in both primary and metastatic lesions obtained from progressive, malignant PETs [25]. These contrasting observations may suggest that GEP ETs could differ (i.e. GI tract carcinoids from PETs, benign from malignant ones) in terms of neoangiogenic phenomena, and that different steps in tumours angiogenesis occur at different disease stages, with VEGF possibly becoming less relevant in more advanced stages or in less differentiated tumours. Agents affecting the VEGF pathway include drugs targeting VEGF itself (antibody or “trap”), antibodies or small molecules targeting intracellular domains of the VEGF receptor (VEGFR) and those of other tyrosine kinases, and drugs inhibiting the intranuclear production of VEGFR mRNA, such as angiozyme. A number of other compounds with antiangiogenic activity have been evaluated or are under evaluation for the treatment of EP ETs patients. 2.1. Bevacizumab (BEV) (AvastinTM , Roche) BEV is a humanized monoclonal antibody which blocks VEGF. It has been approved by FDA for clinical use in the following different malignancies and setting: as firstline treatment of advanced colorectal cancer, combined with irinotecan and/or fluorouracil and folates in February 2004; as second-line for advanced colorectal cancer combined with FOLFOX-4 in June 2006; as first-line for non-small cell lung cancer combined with carboplatin and taxol in October 2006; and as first-line for advanced breast cancer combined with taxol in February 2008. Treatment with BEV upregulates the expression of factor Sp1 and its downstream target genes transcription in human carcinoid cells. Combined treatment with BEV and

mithramycin A, a Sp1 inhibitor, produced synergistic tumour suppression, consistent with suppression of the expression of Sp1 and its downstream target genes. Considering that Sp1 is an important regulator of multiple angiogenic factors expression, BEV-initiated upregulation of Sp1 and subsequent overexpression of its downstream target genes may affect the potential angiogenic phenotype and the effectiveness of antiangiogenic strategies for the treatment of human carcinoid tumours [26]. In GEP ETs patients, BEV was investigated together with pegylated (PEG)-IFN alpha-2b in a randomized phase II study conducted in a mixed cohort of 44 patients with metastatic or unresectable carcinoid tumours from various sites (mostly midgut) on stable doses of octreotide. During an 18-week period after which both groups were switched to combined therapy with all three drugs, BEV seemed to be superior as compared to PEG-IFN, both in stabilizing disease and producing rapid decline of blood tumour supply (monitored by functional CT scan). Progression-free survival (PFS) at 18 weeks was 95% vs 68% (p = 0.02) [27]. The overall PFS, calculated from study entry for all 44 patients, was 63 weeks. Patients with PD at study entry had shorter PFS duration. However, a specific sub-analysis of patients with PD at study entry has not been reported. In the BEV arm, four patients (18%) obtained a partial response, as compared with no patients in the PEG-IFN arm. Seventy percent of BEV and 68% of PEG-IFN arm patients showed SD. Twenty-four patients underwent functional CT (12 per arm). In the BEV arm significant decreases in blood flow (BF), blood volume (BV), and permeability surface (PS) were described. No significant changes in tumour blood flow parameters were observed with PEG-IFN. Unfortunately, no definitive conclusions about the correlation between functional CT scan findings and clinical outcome can be drawn, due to the small number of patients studied. Particularly, only two of the responder patients had functional CT. Based on the results of this trial, a randomised phase III trial comparing octreotide plus INF alfa-2b or BEV in the treatment of patients with metastatic or locally advanced unresectablecarcinoid tumour was undertaken and is presently ongoing [127, see web references]. In another phase II clinical trial, the efficacy and safety of BEV in combination with temozolomide (an oral dacarbazine analogue) were explored in a mixed group of 34 patients with advanced GEP ETs, with a partial response in 14% (all PETs) and stable disease in 79% overall [28]. Due to the anticipated lymphopenia, patients received prophylaxis with trimethoprim/sulfamethoxazole and acyclovir, avoiding clinical complications. Patients on octreotide remained on octreotide at stable dose. However, in this study the baseline disease behaviour (SD or PD) has not been reported. At ASCO 2008 the preliminary results of a study using capecitabine, oxaliplatin, and BEV were presented [29]. Thirteen of a planned 37 patients had been enrolled. Ten of the 13 had PD at study entry. Among them, four PR and six SD were observed. Treatment was well tolerated, even though a

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transient increase in pain at the sites of disease was documented in six patients. Another study presented at the same meeting illustrated the results of a combination of FOLFOX6 and BEV in patients with progressive neuroendocrine carcinomas. Among 13 assessable patients, 1/5 carcinoids (20%), 2/6 PETs (33%) and 1/1 poorly differentiated PET had a PR. Nevertheless, one arterial thromboembolic event and one treatment-related death caused by sepsis occurred [30]. BEV is also under investigation in combination with Panzem® NCD (methoxyestradiol) in patients with locally advanced or metastatic carcinoid tumours [128]. Panzem has multiple mechanisms of action, including angiogenesis inhibition, disruption of microtubule formation and down-regulation of hypoxia inducible factor-one alpha (HIF1alpha). Other ongoing trials with BEV in neuroendocrine tumours are the NCT00609765 trial [129], which examines BEV, Fluorouracil, Doxorubicin and Streptozocin in locally advanced and metastatic PETs, and the NCT00607113 trial [130], employing BEV and RAD001 (Everolimus) in advanced low or intermediate grade neuroendocrine carcinoma. 2.2. Sunitinib malate (SU11248, SutentTM , Pfizer) Sunitinib (SUT) is a multitargeted tyrosine kinase inhibitor with antiproliferative and antiangiogenic effect, with activity against VEGFR, platelet derived growth factor receptor (PDGFR), c-KIT, Flt-3 and RET [31]. It has been approved by FDA for the second-line treatment of gastrointestinal stromal tumours (GIST) in January 2006, and as first-line treatment of advanced renal cell carcinoma in February 2007. In a phase I trial, 28 patients with different types of cancer received between 50 mg and 150 mg/day of SUT. Out of the four patients with a neuroendocrine carcinoma, one had a partial response and a second achieved a minor response [32]. The efficacy and safety of SUT have recently been assessed in a large phase II study of 109 patients with advanced, unresectable GEP ETs treated with repeated 6week cycles of SUT (50 mg/day, 4 weeks on and 2 weeks off). Radiological response was observed in 13.5% and 5.1% of PETs and carcinoid tumours, respectively, along with high percentages of SD and an acceptable safety profile [33]. Unfortunately, it has not been specified whether treatment showed efficacy in patients with PD at study entry. The median time to progression (TTP) was 10.2 months for patients with carcinoids and 7.7 for patients with PETs. Moreover, analyzing plasma levels of a panel of soluble proteins from patients enrolled in the same trial, the authors characterized potential biomarkers of biological response to SUT, concluding that sVEGFR-3 may be a novel biomarker of the biological activity of SUT, and that IL-8 may be of particular interest as a potential predictor of response in patients with GEP ETs [34].

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At present, a phase III double blind randomized trial sponsored by Pfizer, evaluating SUT vs placebo in patients with progressive, advanced/metastatic, well-differentiated EP ETs is ongoing [131]. Another Pfizer-sponsored phase II study, currently recruiting patients, will test sequence combination of selective hepatic artery embolization and SUT in patients with welldifferentiated, metastatic EP ETs with measurable liver metastases [132]. 2.3. Sorafenib (BAY 43-9006, NexavarTM , Bayer Pharmaceuticals Corporation) Sorafenib (SOR) is an oral multitarget inhibitor with high activity against Raf kinase (wild-type Raf-1 or the B-Raf V600E oncogene), which is overactivated in a variety of solid tumours. SOR also shares the molecular targets of SUT. It has also been approved by FDA for the second-line treatment of advanced renal cell carcinoma in December 2005, and for the treatment of advanced hepatocellular carcinoma in November 2007. Preclinical studies demonstrated the expression of Rap1 and B-Raf in GEP ETs, and proved a negative effect of SOR on tumour growth, with increased apoptosis in both BON and INS-1 cell lines. However, these findings were only detectable at high concentrations of SOR, therefore possibly through targets other than Raf [35]. Moreover, to possibly add confusion, it has previously been suggested that ZM336372, which on the opposite is a Raf-1 activator, is able to reduce hormone secretion and inhibit proliferation of BON cells [36]. The only clinical data on SOR in the treatment of GEP ETs were presented at ASCO 2007. Ninety-three patients (43 PETs and 50 carcinoids) with metastatic disease were treated with SOR, 400 mg po BID. Primary endpoint was tumour response according to RECIST. Only 10% of both PET and carcinoid subgroups had a PR, while grade 3-4 toxicities were observed in 43% of patients. This preliminary report suggested a modest efficacy and a relevant incidence of side effects [37]. An international multicenter phase II study co-ordinated by the Mayo Clinic and aimed to evaluate the efficacy of SOR in metastatic EP ETs has recently been suspended [133]. 2.4. Vatalanib (PTK787/ZK222584; Schering AG, Novartis) Vatalanib (VAL) is a potent, low molecular weight, oral, selective inhibitor of the VEGF receptor tyrosine kinases VEGFR-1 (Flt-1) and, in particular, VEGFR-2 (FLK-1/KDR). This small molecule is capable, at higher concentrations, to also block c-KIT, PDGFR-beta and cFMS TKs [38]. VAL has being tested as single drug and in combination with chemotherapeutic agents in patients with metastatic colorectal cancer, advanced pancreatic can-

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cer, primary or secondary myelodisplastic syndrome, ovarian cancer, advanced prostate and renal cell cancer and glyoblastoma multiforme. A trial designed by the European Neuroendocrine Tumour Society (ENETS) has been started since September 2005, but no preliminary results are available. At ASCO 2008 two abstracts on VAL in neuroendocrine tumours were presented. Pavel et al. [39] reported the results of 20 patients with advanced EP ETs, with PD according to RECIST criteria at study entry, and a Ki67 <15%. Three patients stopped treatment early and a total of seven had to quit treatment due to side effects. Stable disease was reported in 8/16 patients (50%), and it was maintained in 4/15 (27%) at 12 months. In another study [40], 17 patients were treated with VAL. Among the 10 assessable patients, 9 obtained a SD/minor response at CT scan or Somatostatin Receptor Scintigraphy (SRS).

also seems to be critical in affecting a number of downstream antiangiogenic signalling pathways. Phase I studies of recombinant human ENDO (rhENDO) in patients with advanced cancers evaluated the tolerability, pharmacokinetic and pharmacodynamic of the drug, but the lack of firm data regarding the optimal therapeutic doses constitute a limitation for all sort of clinical trial. A multicenter phase II study of rhENDO in 42 patients with metastatic neuroendocrine tumours did not show any significant tumour or biochemical regression, nor was associated with evidence of a cytostatic effect, even if the treatment was associated with minimal toxicity [44]. The majority (80%) of patients in this study experienced SD as the best response to treatment but, as already stressed, this observation might reflect the intrinsic indolent nature of these tumours, as part of the study group had stable disease before entry as well.

2.5. Thalidomide (ThalidomideTM ) 3. Blocking receptor tyrosine kinases Thalidomide (THL) is an orally available immunomodulatory drug, acting through the inhibition of TNF-alfa production, which also obtains a potent antiangiogenic effect in vivo [41]. As far as angiogenesis is concerned, it acts by interfering with the VEGF and bFGF pathways. A multi-institutional phase II trial assessed the efficacy of this orally bioavailable drug in combination with temozolomide (a cytotoxic alkylating agent) in a cohort of 29 patients with unresectable or metastatic neuroendocrine tumours, including 15 carcinoids and 11 PETs, whose baseline tumour behaviour (SD or PD) was not specified [42]. Treatment was associated with an overall radiological response rate of 25% and the best results were observed among the subgroup of patients with PETs, with a 45% partial response rate. Unfortunately, a high incidence of adverse events occurred. In particular, 60% G3-4 lymphopenia, with three cases of opportunistic infections were observed, and 55% of patients withdrew due to toxicity within a 8-month period. Currently, a phase II trial, sponsored by Memorial SloanKettering Cancer Centre and in collaboration with the National Cancer Institute, is evaluating the effectiveness and the safety of THL in patients with metastatic, low-grade neuroendocrine tumours [134]. 2.6. Endostatin Endostatin (ENDO) is a 20 kD peptide derived from the proteolysis of carboxy-terminal region of collagen XVIII, a structural constituent of human blood vessels, which has been shown to have an antiangiogenic activity through the inhibition of vascular endothelial cells migration and proliferation [43]. ENDO contains a heparin-binding motif, and may exert some of its antiangiogenic effects through the interactions with the heparan sulphate proteoglycans glypican-1 and -4. Binding of ENDO to alfa-5 beta-1 integrin on the cell surface

The surface of GEP ET cells presents several growth factor receptors, including receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR), the stem cell factor (SCF) receptor c-KIT and the platelet derived growth factor receptor [45–48]. At present, a number of different drugs specifically designed to block the activation of these targets and their downstream pathways are available. 3.1. The EGF receptor family EGFR (also called ErbB-1) is part of a receptor tyrosine kinase family also including HER-2/Neu (ErbB-2), HER-3 (ErbB-3) and HER-4 (ErbB-4) [49], whose activation after interaction with their ligands leads to a number of downstream cascade molecular events involving cell proliferation and transformation. Papouchado et al. explored the expression and activation of EGFR in a series of GEP ETs, confirming the expression of EGFR and its phosphorylation in carcinoids, and, to a lesser extent, in PETs too. Nevertheless, expression of phosphorylated-EGFR was found to be an unfavourable prognostic marker only in PETs [50]. Shah et al. confirmed the expression and the activation of EGFR in an ample series of GEP ETs, describing in addition the concomitant activation of downstream pathways, such as those of AKT and ERK [51]. However, EGFR mutations have not been found in GEP ETs [52]. The expression and amplification of HER-2/Neu were studied in patients with gastrinoma [53]. HER-2 was not expressed in most tumours, but its overexpression correlated with a more aggressive tumour behaviour. However, there are no clinical data on the use of HER-2 inhibitors such as trastuzumab (Herceptin® ; F. Hoffmann-La Roche Ltd., Basel, Switzerland), in GEP ETs.

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3.1.1. Gefitinib (Iressa® , ZD1839, Astra Zeneca, Sweden) Gefitinib (GEF) is a small molecule inhibitor of the EGFR, acting on its intracellular domain. In vitro preclinical studies demonstrated that blocking EGFR-TK with GEF results in growth inhibition, apoptosis and cell-cycle arrest in GEP ET cell lines [54]. Recently, GEF was tested in a phase II study of 96 patients with metastatic GEP ETs and progressive disease. At 6 months, 61% of carcinoid and 31% of PET patients had progression-free survival. One partial and one minor response were observed in the 40 patients with carcinoid, and two partial and one minor response in the 31 patients with PET. In addition, 32% of carcinoids and 14% of PETs had a SD for a period exceeding by at least 4 months the time to progression documented prior to study entry. GEF was overall well tolerated [55]. A similar Mayo Clinic trial is ongoing but no longer enrolling patients [135]. 3.2. Other receptor tyrosine kinases c-KIT (CD117) is a type III tyrosine kinase receptor which, once activated by its ligand, stem cell factor, induces the dimerization and autophosphorylation of the receptor at specific tyrosine regions, which act as docking sites for other intra-cytosolic proteins important for intracellular signal transduction. Abnormal expression of c-KIT and/or SCF has been described in a variety of solid tumours, and activating mutations of c-KIT are a typical feature of GIST [56–58]. Several studies have investigated the expression of cKIT, together with other receptor tyrosine kinases in GEP ETs, by immunohistochemistry [47,59–61]. The results are inconsistent and, as hypothesized for other cancer types, inter-studies disagreement may be explained by different antibodies employed or different immunohistochemistry protocols (see Table 1). Carcinoids also express PDGF and its receptors (PDGFRs). PDGF expression has been found in 70% of a series of 30 midgut carcinoid and PETs [62]. In the same study, PDGFR-alfa has been found on both tumour cells and the adjacent stroma, thus suggesting a possible autocrine loop supporting tumour proliferation. Moreover, PDGFRbeta has been described in the stroma of carcinoid tumours and PDGFR-alfa expression found to be more intense in metastatic than in primary tumours. In a previous study [63], PDGFR-beta expression has also been described as more intense in the stroma and small capillaries around cancer cell

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clusters, suggesting that carcinoid cells may simultaneously produce PDGF and up-regulate PDGFR-beta in a paracrine fashion. 3.2.1. Imatinib (Gleevec® ; Novartis Pharma, Basel, Switzerland) Imatinib (IMA) is a small molecule selectively targeting bcr-Abl, PDGFR-beta, c-KIT and ARG. After the demonstration of significant responses, it was the first selective tyrosine kinase inhibitor approved for cancer treatment, in chronic myeloid leukemia and GISTs [64]. In a preclinical study, IMA was shown to inhibit neuroendocrine tumour cell growth independently of c-KIT expression, possibly interfering with other tyrosine kinases or through tyrosine kinase-independent mechanisms [65]. In a phase II Novartis-sponsored trial, IMA at a starting dose of 800 mg daily, did not induce any objective radiological response in 15 patients with advanced carcinoid tumour [66]. In another trial designed by the same authors, of 27 assessable patients, only one showed partial response, 17 had stable disease, including 7 of the 14 with progressive disease at entry. Seven patients who achieved a biochemical response had a significantly superior PFS as compared to patients without biochemical response [67]. Another phase II study of IMA, at doses ranging from 400 to 800 mg/day, showed poor response and scarce safety in a heterogeneous small group of endocrine tumours [68]. Overall, evidence for the use of IMA in GEP ETs is poor, and a clear demonstration of over-activation or mutations of cKIT or PDGFR is needed to justify the use of similar receptor tyrosine kinase inhibitors in further clinical trials involving GEP ETs patients. 3.3. Blocking the mammalian target of rapamycin pathway The mammalian target of rapamycin (mTOR) is a serinethreonine kinase involved in the regulation of cell growth and death trough apoptosis. It plays a critical role in transducing a number of different signals mediated through the phosphatidylinositol 3 kinase (PI3 K)/protein kinase B (AKT) pathway, principally by activating downstream protein kinases required for both ribosomal biosynthesis and translation of key mRNAs of proteins necessary for cell-cycle progression [69]. The signalling pathways upstream mTOR include several tumour suppressors, such as PTEN, NF1 (neurofibromato-

Table 1 Rationale for evaluating novel therapies in clinical trials in patients with GEP ETs. Reference numbers in brackets. Target

Target expression and drug efficacy in animal model

Target expression and drug efficacy in cell lines

Target expression in human samples

Activation/mutations in human samples

Ongoing clinical trials

VEGF EGFR c-KIT mTOR

Yes [15–20] Not evaluated Not evaluated Yes [84]

Yes [26] Yes [53] Yes [64,66] Yes [80,81,84,87–89]

Yes [21–23], No [24,25] Yes [44–47] Yes [46,59], No [47,58,60,66] Yes [86,88]

Not evaluated Yes [49,50,52], No [51] Not evaluated Yes [86]

Yes No No Yes

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The interest in this pathway has also been implemented by results of a number of preclinical studies. Indeed, the antiproliferative effect of rapamycin (RAP) in GEP ETs cell lines has been described [81,82]. As somatostatin and octreotide themselves have been reported to decrease the activity of PI3 K/AKT/mTOR pathway [83,84] the possible role of a combination of SA and mTOR inhibitors also constitutes an intriguing issue. This aspect has been evaluated in depth by Moreno and colleagues [85], who aimed at determining the anti-tumour activity of RAP, octreotide and their combination both in vitro (BON and H727 cell lines), and in vivo (nude mice with subcutaneous injected BON cells) models. While the efficacy of RAP in suppressing tumour growth both in vivo and in vitro was confirmed, octreotide alone was not effective, nor its association led to any further improvement of RAP activity. Finally, while RAP inhibited mTOR activity, as suggested by the decline in its downstream effectors (phospo-S6 and phospo-4E-BP1), the addition of octreotide was not able to avoid the negative feedback leading to an increase in AKT phosphorylation. This last issue is critical, and fits with the decreased response to the drug and the shorter TTP reported in a recent clinical trial of glioblastoma patients treated with RAP [86].

Fig. 1. Schematic representation of the PI3 K/AKT/mTOR pathway. A green colour indicates overexpression or activation, red colour indicates reduced expression or deactivating mutations. Overall the balance of such events suggests an important role for this signalling pathway in GEP ETs.

sis), the kinase LKB1, and oncogenes such as Ras and Raf [70]. mTOR also mediates the effects of a number of growth factors such as IGF-1 and VEGF (see Fig. 1). These signalling pathways converge on the tuberous sclerosis complex (TSC1/TSC2), which inhibits the mTOR activator Rheb, a small GTPase. In turn, activation of the mTOR pathway enhances the activity of HIF-1alpha and of VEGF itself [71]. Rapamycin (sirolimus), a complex macrolide and highly potent fungicide and immunosuppressant is a highly specific inhibitor of mTOR. Moreover, this drug prevents cyclindependent kinase (cdk) activation, inhibits retinoblastoma protein phosphorylation, and accelerates the turnover of cyclin D1 with consequent inhibitory effects at the G1 /S phase transition [72]. Tumours exhibiting constitutively activated PI3 K/AKT/ mTOR signalling due to mutations or loss of the above mentioned tumour suppressor genes (PTEN or TSC), or overexpression of upstream genes, are potentially susceptible to mTOR inhibitors [73]. A remarkable rationale for targeting this pathways in PETs and carcinoids is represented by the recognised relation existing between TSC [74,75], PTEN [76,77], NF1 [78], the VHL [79,80] genes, and the development of GEP ETs.

3.3.1. Temsirolimus (Torisel® , Wyeth) Temsirolimus (TEM) is a derivative of sirolimus and has been approved by FDA for the treatment of advanced renal carcinoma in May 2007. It showed interesting results in other solid tumours. A multicenter phase II study aimed to evaluate the efficacy and safety of TEM in 37 patients with advanced, progressive neuroendocrine carcinoma (21 carcinoids and 15 PETs) has been published in 2006 [87]. A secondary aim of the study was to evaluate the pharmacodynamics of this drug and to look for possible markers associated with response to therapy in archival specimens of tumour biopsies obtained before and after treatment. Weekly doses of TEM, 25 mg, were administered intravenously. Radiological responses were similar between carcinoids and PETs, with PR rates of 4.8% and 6.7%, respectively. Twenty patients showed SD for at least 2 months. Therefore, some tumour control (PR plus SD) was achieved in 23 out of the 36 patients (63.9%). The median TTP was 6 months, and the 1-year progression-free and overall survival rates were 40.1% and 71.5%, respectively. The most frequent drug-related adverse events of all grades expressed as percentage of patients were: fatigue (78%), hyperglycaemia (69%) and skin rash/desquamation (64%). As for mechanisms, TEM appeared to down-regulate the phosphorylation of S6, a protein downstream of mTO. Higher baseline levels of phosphoryated-(p)mTOR were a good predictor of response. Increases in pAKT and decreases in p-mTOR after treatment were associated with an increased TTP. In conclusion, in this first study TEM as a single agent showed some activity in this population of GEP ETs, along with an acceptable toxicity profile. Unfortunately, to the best of our knowledge, there

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are currently no other open trials recruiting patients to evaluate the efficacy of TEM alone or in combination with other agents in GEP ETs. 3.3.2. Everolimus (RAD001, Novartis Pharma, Basel, Switzerland) Everolimus (EVE) is a potent, orally available mTOR inhibitor under investigation, as a single agent or in combination, for the treatment of both solid tumours and haematological malignancies. An initial preclinical study demonstrated a constitutive activation of the PI3 K/AKT/mTOR signalling pathway in BON cells, and showed that EVE was able to induce growth inhibition through G0/G1-phase arrest and apoptosis in this model [88]. Similar findings have been reported by others since then. Hörsch et al. reported growth arrest in both INS-1 and BON cells treated with EVE [89]. Interestingly, a pronounced synergism with octreotide has been reported. In the same preliminary report, expression and phosporylation of mTOR were detected by immunohistochemistry in an ample group of insulinomas and GI carcinoids. The efficacy of EVE was confirmed in a rat insulinoma cell line model as well [90]. In September 2008, the final results of the first phase II study designed to evaluate the efficacy and safety of EVE in patients with low to intermediate grade neuroendocrine carcinomas have been published [91]. In this study, EVE at daily doses of 5 or 10 mg orally in combination with depot octreotide (30 mg IM every 28 days) has been studied in 60 patients with advanced, low-grade neuroendocrine carcinomas (29 PETS and 31 carcinoids), 39 (65%) of whom had documented PD at study entry. According to RECIST criteria, in an intention-to-treat analysis, there were 13 patients (22%) with PR, and 42 (70%) with SD. Response rate was slightly higher in PETs than in carcinoids (PR 27% vs 17%), and higher with the 10 mg than with the 5 mg EVE dose (PR 30% vs 13%). The overall PFS was 60 weeks, higher in the 10 mg than in the 5 mg dose group (72 weeks vs 50 weeks), and in the carcinoid (63 weeks) group as compared to PETs (50 weeks). As expected, patients with PD at study entry had a shorter PFS (50 weeks) as compared to patients with SD at study entry (74 weeks). A questionable issue however, is represented by the lack of separate data for response analysis for PET and carcinoid patients with PD at entry. Treatment was well tolerated with aphtous ulcers constituting the most common, all-grade, toxicity and fatigue, diarrhoea, and hypophosphatemia as the most common grade 3/4 toxicity (around 10% of patients). A number of other clinical EVE trials sponsored by Novartis are in different stages of development. An open label phase II study with EVE, recruiting patients with PET after failure of chemotherapy has now been completed [136]. Its preliminary results were presented at the last European Society of Medical Oncology (ESMO) meeting in September 2008 by James Yao [92]. Patients were divided in two strata: 115 received EVE, 10 mg/day (stratum 1) and 45 EVE, 10 mg/day plus octreotide LAR 30 mg every 4 weeks (stratum 2). Response rate in stratum 1 was the main objective. Central

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reviewed PR was 7.8% in stratum 1 and 4.4% in stratum 2. A central reviewed tumour control (PR + SD) of 76.5% was observed in stratum 1 and 82.2% in stratum 2. Median exposure to EVE was 222 days in stratum 1 and 226 in stratum 2. A central reviewed median PFS was 9.3 months in stratum 1 and 12.9 months in stratum 2. EVE was well tolerated in both strata, with asthenia, fatigue, and thrombocytopenia being the most common grade 3 adverse events occurring in more than 5% of patients. The authors conclude that higher SD and PFS in stratum 2 may be due to added octreotide LAR effect. A randomized phase III, double blind, placebo-controlled study comparing octreotide depot plus placebo vs octreotide plus EVE in patients with functioning, advanced carcinoid tumours has recently closed the accrual [137]. Its results have not yet been presented. Finally, a multicenter European open label phase II study (RAMSETE) is incoming. It will evaluate the efficacy and safety of EVE as monotherapy for the first-line treatment of patients with non functioning GEP NETs [138]. Further studies are evaluating the combination of EVE and temozolomide in patients with advanced PETs [139] and of EVE and VAL in patients with advanced solid tumours, including GEP ETs [140]. Atiprimod is a novel proapoptotic and antiangiogenic compound acting through the deactivation of mTOR/AKT and STAT3 signalling pathways. Preliminary results of a study employing this drug at different dosages in patients with progressive carcinoid tumours are interesting, with an overall good safety profile and disease stabilization in around 90% of patients in a short term follow-up. Long-term results and more details on the study population are awaited [93].

4. Other possible therapeutic targets PGH synthase-2, better known as Ciclooxigenase-2 (COX-2), was found to be overexpressed in a Japanese immunohistochemical study of 38 gastrointestinal carcinoids, particularly in rectal carcinoids, and significantly at the tumours advancing edges, suggesting a pivotal role in tumour progression [94]. In addition, two studies demonstrated enhanced COX-2 expression in about 50% of PETs [95,96]. It is still debated whether COX-2 may play a role in carcinoid progression or if it represents only a molecular signature (marker) of tumour inflammation. An interesting issue is represented by the possible value of COX-2 as a prognostic biomarker, with a close correlation between its overexpression and tumour progression/proliferation, both in midgut carcinoid and malignant PETs [96,97]. Furthermore, in human neuroendocrine tumour cell lines, its inhibition through nonsteroidal anti-inflammatory drugs (with different profile of selectivity for COX isoforms), has been shown to block proliferation via G1 cell-cycle arrest [98]. However, the presently available data on the possible effects of COXibs or other atypical non-steroideal anti-

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inflammatory drugs in patients with GEP ETs, are poor and disappointing. As far as we know, only one clinical trial assessed the efficacy of a Cox-Inhibitor drug, with rofecoxib, given in combination with capecitabine, in patients with metastatic neuroendocrine tumours. This study was however stopped early, since in the first nine patients no response was observed, but some toxicity was invariably present [99]. Another molecular pathway deserving exploration as a possible target for GEP ETs therapy is that of the insulin-like growth factors (IGF) system. Several studies have described mRNA [100] and protein expression [101,81,102] of IGF and its receptors in human GEP ET cells. In addition, it has been demonstrated in vitro that IGF is a crucial autocrine regulator of cell proliferation in carcinoid cell lines, as suggested by the co-distribution of the two antigens [81]. The importance of the IGF system in tumourigenesis and progression of PETs has also been proved in animal models [103]. Since IGF-I receptor activation can lead to PI3K and AKT phosporylation, the concomitant use of EVE and IGF-IR inhibitors results in favourable, addictive effects on proliferation and apoptosis in transformed tumour cells [104]. Promising results from such a combination strategy [105] suggest a rational basis to design future clinical trials in GEP ETs. Presently, a phase II clinical trial sponsored by the Memorial Sloan-Kettering Cancer Centre is recruiting patients with metastatic neuroendocrine tumour to evaluate the efficacy of MK-0646, a humanized IgG1 monoclonal antibody able to bind the IGF-1R, and to reduce the downstream tyrosine kinase signalling [141]. Src is a family of proto-oncogenic, non-receptor tyrosine kinase including nine members. Src-like kinases act downstream of growth factor receptors and integrins transmitting messages that are crucial for several aspects of cell growth and metabolism, as for example cell-cycle regulation, cell adhesion and motility [106]. Overexpression of Lck, a member of Src family, has been recently demonstrated in metastatic progressive PETs [25]. The expression and activity of Src have been also described in PET cell lines and tissues. Inhibition of Src activity has been shown to interfere with adhesion, spreading and migration of cells [107]. Accordingly, a rationale for using Src inhibitors in PETs does exist. AZD0530 is a potent oral inhibitor of Src kinase that has been shown preclinically to block phosphorylation of paxillin (Pax), and focal adhesion kinase (FAK), downstream mediators of motility and invasion, with the potential to prevent metastases and invasion. In a phase I study of AZD0530, the inhibition of Src activity through the use of biomarkers has been demonstrated, for the first time in human cancers [108]. Histone deacetylases (HDAC) are another promising novel target for cancer therapy. Acetylation and deacetylation of histones are part of complex epigenetic processes covering a fundamental role in the modulation of gene transcription for eukaryotic cell biology. HDAC inhibition in vitro through trichostatin or sodium butyrate or MS-275, inhibits proliferation in a dose-dependent manner, also inducing apoptosis

and cell-cycle alterations of ETs cell lines [109]. A proteomic analysis of ETs cell lines investigated the effects of trichostatin before and after treatment, showing encouraging results in agreement with previous studies [110]. FR901228, a depsipeptide and histone deacetylase inhibitor, also called romidepsin, is presently under investigation in a phase II clinical trial sponsored by the Arthur G. James Cancer Hospital and Richard J. Solove Research Institute, with the collaboration of National Cancer Institute in patients with locally advanced or metastatic neuroendocrine tumours. At the moment, this study has stopped patient recruitment [142]. The 26S proteasome is a multicatalytic protease belonging to the ubiquitin-proteasome-system. It is a 2.5 Mda molecular machine which catalyzes the degradation of damaged, misfolded or unneeded proteins involved in cell-cycle regulation, apoptosis and angiogenesis. Although multiple mechanisms seem to be involved, proteasome inhibition may prevent degradation of proapoptotic proteins or enhance degradation of anti-apoptotic proteins, permitting the activation of programmed cell death in neoplastic cells. This seems an attractive novel target for the development of anti-cancer therapy [111]. Bortezomib [Velcade (Millennium Pharmaceuticals Inc., Cambridge, MA), formerly known as PS-341] is a specific, potent and reversible inhibitor of the 26S proteasome. The drug has received approval from the FDA and EMEA for the treatment of refractory myeloma, and is under investigation for a number of solid tumours. It has been evaluated in a phase II trial in 16 patients with metastatic GEP ETs. Stable disease was noted in 69% of cases, with no objective response and high percentage of grade 3 toxicity events, mainly peripheral neuropathy and diarrhoea [112]. Nelfinavir (Viracept® ) is an antiretroviral drug, belonging to the class of protease inhibitor, and used in the treatment of the human immunodeficiency virus infections. In 2007 the drug was recalled from the market because of fears that batches of the drug could have been contaminated with potentially cancerogenic substances. Ironically, Nelfinavir is currently under investigation for a possible “repositioning”, after the recent discoveries of its potential as anti-cancer drug. Preclinical studies [113] have extended the biological activity of this drug, showing its pleiotropic effects in vitro and in vivo. It can induce caspase-mediated apoptosis and caspase-independent cell death (characterized by induction of Endoplasmic Reticulum stress and autophagy), and can also inhibit AKT activation from different signalling pathways in tumour cells; moreover, it decreased the viability of a panel of drug-resistant breast cancer cell lines and inhibits the growth of NSCLC xenografts. Recently, a phase I trial has evaluated Nelfinavir for the first time as anti-cancer agent in combination with chemoradiotherapy, showing acceptable toxicity and promising activity in patients with locally advanced adenocarcinoma of pancreas [114]. A phase I trial is studying the side effects and the best dose of Nelfinavir for the treatment of patients with

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metastatic, refractory, or recurrent solid tumours, including GEP ETs [143].

5. Conclusions Although GEP ETs are relatively rare malignancies, their incidence is increasing, possibly due to the improvements in diagnostic techniques. In addition, given their indolent nature, their prevalence is also on the rise. The scarce sensitivity to conventional chemoterapeutics and the high rate of metastatic disease, render GEP ETs an ideal “model” to focus on in the search of new antiproliferative treatments. When evaluating clinical trials of molecularly targeted therapies in GEP ETs, the main concern is the high heterogeneity of the investigated patients, which make intra-population and inter-populations analysis difficult. Evaluation of tumour behaviour (stable vs progressive disease) before treatment is particularly important, as disease stabilization is an acceptable response in patients with welldocumented progressive disease. As far as therapeutic options are concerned, patients with well-differentiated, low-grade GEP ETs are commonly managed with SA, whereas patients with poorly differentiated, high-grade tumours are better approached with conventional chemotherapy. A third category of patients, those with welldifferentiated tumours, a clearly progressive or very advanced disease, and an intermediate proliferation rate, represent a true challenge. These are often treated with PRRT, when somatostatin receptors are expressed, and trials comparing such an approach with targeted therapy are advisable. Moreover, molecular targeted agents should be especially evaluated in the setting of patients with tumour progression without a response to biotherapy. In this review we first evaluated the data developed in preclinical studies, which described the expression and the role of the supposed main therapeutic targets of interest for the treatment of GEP ETs (see Table 1). Consistency of the data suggesting the actual expression and/or mutations/activation of each target protein is highly variable. In some cases, a clear efficacy of the single drugs has not been demonstrated with hard evidences, both in vitro and in vivo. However, a critical role for angiogenesis and for the mTOR pathway in GEP ETs emerges. We also extensively reviewed all the published literature evaluating the efficacy of targeted therapy in GEP ETs patients, including preliminary reports. As expected, the main limit for the critical interpretation of these results is represented by their heterogeneity and lack of detailed clinical information. Indeed, only a few studies employing targeted agents included patients with documented progressive disease before treatment and, unfortunately, data on differentiation, proliferation rates or the relationship between these parameters and the observed results have seldom been reported. With these limitations is difficult to define the real efficacy of treatment on spontaneous tumour growth. Fig. 2

Fig. 2. Schematic representation of the results of the clinical trials employing novel targeted agents in GEP ETs for which data on patients with progressive disease at study entry were available. The summarized results regard these patients with progressive disease only. Reference numbers in brackets. PET = pancreatic endocrine tumour and CTs = carcinoid tumours.

summarizes the results obtained in patients with progressive disease. Selected studies are those in which separated data for PETs and carcinoids were available. In these patients, both VEGF and mTOR inhibitors seem to offer promising results, while IMA and GEF appear to have a limited efficacy. Similar conclusions can be drawn examining the PFS rates listed in Table 2. In the three major studies using BEV, IMA and EVE, similar PFS have been reported for EVE and BEV, while the results with IMA are disappointing. When examining in more detail the possible indication for the use of antiangiogenic agents in patients with GEP ETs, several factors should be considered. A major issue is the apparent loss of expression of VEGF and the related reduction of MVD in more advanced PETs [24,25,115]. In these conditions cancer cells are exposed to hypoxia. As HIF-1 regulates negatively a number of other pathways including the mTOR one [116], and since it may increase chemoresistance [117], this aspect may be critical in choosing which drug to employ together with angiogenesis inhibitors. Overall, use of antiangiogenic agents such as BEV seems to represent a promising option for patients with progressive, well-differentiated endocrine carcinomas, possibly coupled with chemotherapy, even if in a study employing BEV and FOLFOX toxicity rates were high [30]. Future studies should clarify which subset of patient will benefit from this combination. A relevant role for the mTOR pathway in GEP ETs seems plausible for the quote of tumours with mutations or downregulation of the tumour suppressor genes upstream of mTOR [74–80] (Fig. 1). However, the single inhibition of mTOR alone is probably not enough, as a number of escape pathways seem to develop, both in vitro and in vivo, when mTOR is deactivated, including those following the phosporylation of AKT [86,118]. The role of SA in decreasing AKT phosphorylation seems to be limited [85]. A number of alternative rational targets to be tested together with mTOR inhibitors include inhibitors of PI3K itself [119], of the IGF axis [120] of Src [121] or MNK

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Table 2 Efficacy of the main employed drugs in trials in which data on progressive disease (PD) at entry and progression-free survival (PFS) were reported. Reference numbers in brackets. Treatment (reference)

Patientsa

Grade

Metastatic disease

PD at entry

Median PFS (weeks)

BEV + octreotide [27] RAD001 + octreotide [90] Imatinib [66]

44 CTs 31 CTs, 29 PETs 27 CTs

Well-differentiated Well-differentiated Not reported

100% Not reported 100%

23/44 (52%) 39/60 (60%) 14/27 (52%)

66 (all CTs) CTs 63; PETs 50 23.6 (all CTs)

a

CT: carcinoid tumour; PET: pancreatic endocrine tumour.

[122] many of which are in clinical development with preclinical results suggesting an interesting synergy with mTOR inhibitors. The best strategy, once again, has yet to be determined, and specific preclinical and clinical studies in GEP ETs are presently lacking. Finally, combining antiangiogenic drugs and mTOR inhibitors is an attractive possibility. The interaction between these pathways is relevant (Fig. 1), as the VEGF axis acts through the mTOR pathway, and the PI3K pathway is critical for endothelial cells [123,124]. Moreover, the inhibition of mTOR activity by hypoxia is reduced by TSC2 or PTEN deficiency [125]. Therefore, in more advanced tumours, where VEGF expression seems to be decreased and HIF-1 increased [24,115], coupling a block of the mTOR pathway with VEGF inhibitors may be an effective approach. Interesting data have been reported in an animal model of hepatocellular carcinoma with the combination of rapamycin and BEV showing potent additive effects [126]. In conclusion, even if the diffusion of these new agents in the clinical setting is fast-growing and the reported preliminary results exciting, more data from well-designed randomized trials are needed before these new approaches are included in standard therapeutic algorithms. In this view, design of future clinical trials must define and select homogeneous populations of tumour types, as preliminary evaluation of available data suggest a clear difference in response rates between PETs and carcinoids. A clear definition of the individual progression rate, before and after study entry, should also be provided (i.e. stable, slowly progressive, and rapidly progressive disease). Moreover, patients should be classified according to specific histological guidelines developed for GEP ETs (WHO classification, TNM stage), and response stratified according to these parameters and proliferation index, as evaluated by Ki67 immunostaining. Trials should ideally be designed by multidisciplinary teams of clinician scientists (gastroenterologist, surgeon, oncologist, pathologist, radiologists), keeping in mind the aforementioned issues.

Conflict of interest statement The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. We are grateful to Dr Massimo Marignani for his critical revision of scientific content and of English style and grammar.

Reviewers Robert T. Jensen, M.D. National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Digestive Diseases Branch, 10 Centre Drive, Bethesda, MD 20892-1804, United States. Kjell Öberg, M.D., Ph.D., Professor, University Hospital, Department of Endocrine Oncology, Uppsala, SE-751 85, Sweden.

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Biographies Gabriele Capurso, M.D., Ph.D. is a gastroenterologist at the Digestive & Liver Disease Unit of S. Andrea Hospital, University of Rome, “La Sapienza” with a contract professorship at the Medical School. He completed a Ph.D. in digestive oncology. He is particularly involved in the care of patients with digestive endocrine tumours and with pancreatic disorders, and in related research activity. He is an active member of the American Gastroenterology Association (AGA), American Pancreatic Association (APA), European Neuroendocrine Tumour Society (ENET), the Italian Society of Gastroenterology (SIGE) and the Italian Society of Pancreatology (AISP). He signed some 40 publications in referenced journals. Nicola Fazio, M.D. is an internist and medical oncologist. He is senior deputy director of the Division of Medical Oncology, at the European Institute of Oncology, of Milan. He is actively involved in clinical management and clinical research of patients with gastroenteropancreatic malignancies, in particular neuroendocrine tumors. He is active member of several scientific societies, including European Neuroendocrine Tumour Society (ENETS), American Society of Medical Oncology (ASCO), European Society of Medical oncology (ESMO), Associazione Italiana di Oncologia Medica (AIOM). He is co-author of more than 50 articles published in referenced journals. Stefano Festa, M.D. graduated in medicine in at the Second Medical School, “La Sapienza” University of Rome. He is involved in clinical and research activities at the Digestive & Liver Disease Unit of S. Andrea Hospital, Rome. Francesco Panzuto, M.D., Ph.D. is a gastroenterologist at the Digestive & Liver Disease Unit of S. Andrea Hospital, University of Rome, “La Sapienza” with a contract professorship at the Medical School. He completed a Ph.D. in digestive oncology. He is particularly involved in the care of patients with digestive endocrine tumours and with GIST, and in related research activity. He is an active member of the

American Gastroenterology Association (AGA), European Neuroendocrine Tumor Society (ENET), and the Italian Society of Pancreatology (AISP). He signed some 40 publications in referenced journals. Filippo De Braud, M.D. is an oncologist, since December 2000, he is director of the Clinical Pharmacology and New Drugs Development Unit at the European Institute of Oncology, Milano Italy and a member of Scientific and Technical Commission (CTS) of the Italian Agency for Medicine (AIFA) (The Commission for drugs approval of the Italian Ministry of Health) since February 2001. He is also active member of several professional societies: American Society for Clinical Oncology (ASCO); American Association for Cancer Research (AACR); European Society for Medical Oncology (ESMO); Società Italiana di Oncologia Medica (AIOM); Connective Tissue Oncology Society (CTOS); Società Italiana di Farmacologia (SIF). During his training and earlier work experiences has been involved in basic and clinical research at National Cancer Institute of Milan (Italy) Royal Free Hospital School of Medicine in London, Wayne State University of Detroit and Institute Gustave Roussy of Paris. He is co-author of some 130 articles published in referenced journals. Gianfranco Delle Fave, M.D. is a gastroenterologist. He is professor of medicine at the University of Rome “La Sapienza”, and head of the Digestive & Liver Disease Unit. He is involved in the prevention of digestive neoplasms, in the care of patients with such malignancies and with pancreatic disorders. He is particularly implicated in clinical and basic research in the field of digestive endocrine tumours. He is the past president of the Italian Society of Pancreatology (AISP), the vice-president of the Italian Federation of Digestive Diseases Societies (FISMAD), and an active member of the American Gastroenterology Association (AGA), American Pancreatic Association (APA), European Neuroendocrine Tumor Society (ENET) and of the Italian Society of Gastroenterology (SIGE). He signed some 200 publications in referenced journals.