Novel investigational drugs for gastric cancer

1.Introduction
2.EGFR
3.HER2/neu
4.VEGF
5.Matrix metalloproteinase
6.Mammalian target of rapamycin (mTOR)
7.Conclusion
8.Expert opinion
Review

Novel investigational drugs for gastric cancer

Loredana Vecchione, Michele Orditura, Fortunato Ciardiello &
Ferdinando De Vita†
Second University of Naples School of Medicine, Division of Medical Oncology ‘F. Magrassi e A. Lanzara’, Department of Clinical and Experimental Medicine and Surgery, c/o II Policlinico Via S. Pansini 5, 80131 Naples, Italy

Background: Gastric cancer still represents a leading cause of death worldwide. Several cytotoxic agents have demonstrated activity and combination regimens improve progression-free survival, overall survival and quality of life. Nevertheless, now there is no standard therapy for advanced gastric cancer patients. Objective: To evaluate the role of new investigational agents. Methods: We analysed Phase I, II and III studies that evaluated tailored drugs directed against the epidermal growth factor receptor (EGFR), the c-erbB2, the vascular endothelial growth factor (VEGF), the vascular endothe- lial growth factor receptor (VEGFR), the matrix metalloproteinases (MMP) and the mammalian target of rapamycin (mTOR). Conclusion: Data from Phase II trials indicate the potential of improved efficacy of chemotherapy when administered in combination with bevacizumab and cetuximab. Trastuzumab results are ongoing, while marimastat has not obtained clinical developments even if it has demonstrated to be an active drug in this setting of patients.

Keywords: advanced gastric cancer, monoclonal antibodies, small molecule tyrosine kinase inhibitors, target therapy

Expert Opin. Investig. Drugs (2009) 18(7):945-955

1.Introduction

Despite a reduction in mortality, gastric cancer still represents the fourth most common cancer and the second leading cause of cancer deaths worldwide. In 2008, in the USA, there were an estimated 21,500 new cases and 10,800 deaths from gastric cancer [1]. In the last three decades, there has been a relative increase in the incidence of adenocarcinomas of the gastroesophageal junction (GEJ) and gastric cardia in respect of adenocarcinomas of the pylorus and the stomach body [2]. Even if adenocarcinomas of distal esophagus and cardia (GEJ) have different clinicopathological features compared with distal gastric cancers, the chemotherapy approaches remain similar for both entities. Gastric cancer is generally diagnosed at advanced stage because screening is not done in most western countries. By contrast, in eastern countries, such as Japan where esophagogastroduodenoscopy (EGDS) is done routinely, early detection is more common.
In case of early detection and for localized tumors (T1,2,3 N0 or T1,2,3 N 1,2), surgery remains, at present, the milestone, even if 5-year survival rates are still modest (35 – 45% in western countries). To reduce both loco regional and distant recurrences, combined approaches of chemo- and radiotherapy before and after surgery have been used. Even though they improve disease-free survival and overall survival, these combined schemes of chemo- and radiotherapy are still under investigation. This means that now there is no standard treatment in both the preoperative and the adjuvant setting for patients who receive a potentially curative resection [3-8].

At the time of diagnosis, most patients present an inoperable, locally advanced (T3 N3 or T4 any N) or metastatic (M+) gastric cancer (AGC). Prognosis for these patients is very poor, with a 5-year survival rate of approximately 10 – 15%, and a median overall survival of 3 – 4 months for people who receive best supportive care (BSC). By contrast, patients who receive palliative chemotherapy show an improvement in quality of life and median overall survival of 9 – 11 months versus 3 – 4 months for those who receive BSC [9-12].
Several cytotoxic agents have shown activity in gastric cancer, and a combination treatment has shown a significant survival benefit over single-agent therapy [13]. The most active combinations are the epirubicin-cisplatin-5 fluorouracil regimen (ECF) with a median overall survival of 8.2 months and the docetaxel-cisplatin-5 fluorouracil regimen (DCF) with a median overall survival of 9.2 months [14,15]. Recently, it has been shown that capecitabine can replace 5-fluorouracil and oxaliplatin can replace cisplatin in a triplet regimen [16]. At this time the best survival results are obtained with three-drug regimens containing fluorouracil, an anthracycline and a platin.
Several molecular targeting agents that have determined survival advantages in other cancer types are now under investigation in gastric cancer, and some early clinical studies have shown promising activity.
This review focuses on the clinical development of tailored therapies in advanced gastric cancer, with a particular interest on drugs directed against EGFR, the c-erbB2 (HER2), VEGF, matrix metalloproteinases (MMP) and mammalian target of rapamycin (mTOR).

2.EGFR

EGFR belongs to the erbB family, a family of four related cell membrane receptors. It is a transmembrane glycoprotein with an extracellular domain, a hydrophobic transmembrane domain and an intracellular domain with tyrosine kynase (TK) activity. Ligands for the extracellular domain are the EGF, the transforming growth factor-α (TGF-α), amphi- regulin, epiregulin, heregulin, neuregulin and betacellulin. The ligand–receptor interaction induces a receptor dimerization with another EGFR monomer (homodimerization) or with another member of the erbB family (heterodimerization). This induces the activation of the receptor TK intracellular domain and the EGFR autophosphorylation with a complex intracellular signal transduction cascade promoting cancer cell division, migration, angiogenesis and apoptosis inhibition. EGFR is overexpressed in several epithelial malignancies such as head and neck squamous cell carcinoma (HNSCC), colon-rectal cancer, esophageal cancer, pancreatic cancer and non-small-cell lung cancer (NSCLC).
It is also highly expressed in approximately one-third of advanced-stage gastric cancers [17]. Even though, until now, there have been no other similar studies, the experience of our institution shows how EGFR positivity could be useful

in predicting recurrence better than the extent of the primary tumor and node metastasis in radically resected gastric cancer patients [18]; the contemporary association of high serum con- centrations of VEGF correlate with worse outcome, providing evidence that the evaluation of VEGF and EGFR expression might select gastric cancer patients with poor prognosis who may benefit from more intensive treatments and target therapies [19].
Moreover, EGF is present in about 25 – 30% of gastric cancers and is correlated with the degree of gastric wall invasion and lymph nodes metastasis giving a 5-year survival rate worse than that of patients with EGF-negative tumors [20,21]. The worse prognosis in patients with EGF-positive and EGFR-overexpressed gastric cancer creates the rationale for the administration of anti-EGFR drugs.
EGFR can be inhibited by blocking the extracellular domain (with monoclonal antibodies (mAb) such as cetuximab (C225) and panitumumab) or by blocking the TK intracellular domain (with small-molecole tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib).
Cetuximab, a chimeric mAb directed against EGFR, has been approved alone or in combination with irinotecan for advanced colon rectal cancer refractory to irinotecan-based chemotherapy and in combination with radiotherapy for locally advanced HNSSC.
In a Phase II study (the FOLCETUX study), 38 patients with locally advanced or metastatic gastric or GEJ adenocar- cinoma were enrolled to receive first-line chemotherapy with cetuximab in combination with 5-fluorouracil, folinic acid, irinotecan (FOLFIRI: CPT 11 180 mg/m2 i.v. on day 1, LFA 100 mg/m2 i.v. followed by 5-FU 400 mg/m2 i.v. bolus, and 600 mg/m2 i.v. 22-h continuous infusion on days 1 and 2 every 2 weeks) for a maximum of 24 weeks. The overall response rate (ORR) was 44.1% with a disease control (complete response (CR) plus partial response (PR) plus stable disease (SD)) of 91.2%. The median time to response was 6 weeks. The median time to progression (TTP) was 8 months. At the time of the analysis the median overall survival (OS) was not reached. The major toxicity was hematological with 42.1% of grade 3 – 4 neutropenia. The authors also evaluated the correlation between the ORR, the TTP and the OS and pathological biomarkers as p53, Ki67, timidilate syntetase (TS) and EGFR. The finally analysis indicated that p53, Ki67, TS and EGFR expression did not correlate with ORR and that low TS expression was predictive of better TTP [22].
In combination with oxaliplatin and 5-fluorouracil (FUFOX scheme: oxaliplatin 50 mg/m2 plus 5FU 2000 mg/m2 plus DL- folinic acid 200 mg/m2 days 1,8,15,22 q.d. 36), cetuximab was administered in 52 metastatic gastric cancer patients [23]
giving an ORR of 65.2%, a TTP of 7.6 months and an OS of 9.5 months. In accordance with the EGFR status, the response rate was 76.5% for the undetectable tumors and 54.2% for the detectable ones.
As demonstrated in a Phase II study, the combination of oxaliplatin and irinotecan (oxaliplatin 85 mg/m2 biweekly

and irinotecan 125 mg/m2 biweekly) gives an ORR of 58%, a TTP of 5.3 months and an OS of 9.5 months when administered as first-line therapy in advanced gastric cancer patients [24]. To ameliorate these results, the same authors tried to evaluate this combination in association with cetuximab in another Phase II trial. This study, presented at the 2008 ASCO meeting, shows that the combination is feasible and that toxicity is not different from other polychemotherapy regimens. Main grade 1 – 2 toxicities were gastrointestinal and hematological, while few grade 3 – 4 hematological and gastrointestinal toxicities were recorded. Efficacy data have not been presented in the abstract [25].
In a multicenter nonrandomized Phase II study, cetuximab was given at standard dose (loading dose of 400 mg/m2 2-h infusion followed by 250 mg/m2 1-h infusion weekly) with irinotecan 80 mg/m2 + 24-h continuous infusion of sodium folinate (Na-FA) 200 mg/m2 and 5-FU 1500 mg/m2, on days 1, 8, 15, 22, 29 and 36 every 50 days in advanced untreated gastric cancer patients. A total of 49 patients were enrolled, and in 29 response-evaluable patients the ORR was 55% with a tumor control rate of 100%, an acceptable toxicity profile and a TTP of about 5 months [26].
Zhang et al. tried to evaluate the association of cetuximab at standard weekly dose with cisplatin 80 mg/m2 plus capecitabine 2000 mg/m2 on days 1 – 14 every 3 weeks as first-line treatment in AGC patients. Among 38 patients enrolled, 25 were evaluable for response, giving 40% ORR and 92% disease control. Secondary objectives (i.e., TTP and OS) were not achieved [27].
Until now, the Italian Phase II DOCETUX study has not still reached its end points, TTP and OS. Nevertheless, the ORR in 42 out of 48 AGC patients who underwent first-line treatment with standard-dose cetuximab in combi- nation with cisplatin 75 mg/m2 plus docetaxel 75 mg/m2 day 1, 3 times weekly was 40.5% with a disease control of 78.6% and with grade 3 – 4 neutropenia as the main toxicity [28].
In advanced colon rectal cancer, cetuximab has been shown to reverse irinotecan (CPT-11) resistance when given in combination with this drug in patients refractory to CPT-11. Preclinical data show synergy between cetuximab and docetaxel. To evaluate whether cetuximab in combination with docetaxel can reverse docetaxel resistance in AGC patients, Tebbutt et al. designed a Phase II study of second-line therapy. Patients with AGC pretreated with docetaxel were recruited to receive docetaxel 30 mg/ml on days 1 and 8, 3 times weekly, with cetuximab at standard dose. In 35 evaluable patients, ORR was modest with progression-free survival (PFS) of 2.1 months, OS of 5.3 months and low rates of toxicity. Never- theless the modest activity when administered as salvage treatment, cetuximab is able to maintain PFS and OS as other second-line regimens [29]. Phase II studies of cetuximab in combination with standard chemotherapy in patients with advanced gastric cancer indicate that it may provide additional clinical benefit.

On the back of these findings, Phase III clinical trials have been initiated. Patient enrolment is now underway in the open-label EXPAND trial. This 800-plus patient study will investigate the efficacy and safety of cisplatin and capecitabine ± cetuximab as first-line treatment for metastatic gastric adenocarcinoma including GEJ adenocarcinoma. PFS will be the primary end point.
Matuzumab is a humanized IgG1 monoclonal antibody targeting EGFR. In a Phase I study, matuzumab in association with epirubicin, cisplatin and capecitabine (ECX) has shown promising activity [30]. This study was followed by a Phase II trial comparing this combination with ECX alone; as yet there are no available results.
TKIs have offered disappointing results. Doi et al. analyzed, in a Phase II study, 75 AGC pretreated patients who received gefitinib at 250 or 500 mg daily. They obtained a modest clinical activity with only 1% of patients achieving PR and 16% presenting SD [31].
In a second study, gefitinib at a dose of 250 mg daily administered in 20 pretreated patients with esophageal and GEJ carcinoma achieved 15% PR and 15% SD [32].
Finally, Rojo et al. randomized 70 patients with previously treated advanced gastric and GEJ cancer to receive gefitinib at 250 or 500 mg daily. Tumor biopsies were obtained at screening and on day 28 of treatment to evaluate EGFR, phosphorylated EGFR (pEGFR) and phosphorylated mitogen- activated protein kinase (pMAPK). The results of the study indicated that, even though gefitinib showed a biologic activity inhibiting the EGFR activation with a reduction of EGFR phosphorylation, there was no clinical benefit. Further- more, gefitinib did not significantly inhibit the intratumoral phosphorylation of MAPK and Akt [33].
Previously mentioned trials with anti-EGFR inhibitors are listed in Table 1; ongoing Phase II and III trials of cetuximab are listed in Table 3.

3.HER2/neu

The HER2/neu protein is part of the EGFR family and, as the other members, it is a transmembrane TK receptor with an extracellular ligand-binding domain, a short transmembrane domain and an intracellular domain with TK activity. Until now, no ligands have been found for its extracellular domain, but it seems to be the preferred heterodimerization partner for other members of the HER family. As an oncogene, the protein overexpression in the cellular membrane secondary to the gene amplification is responsible for cellular abnormal proliferation. HER2 overexpression and/or amplification is present in about 10 – 34% of invasive breast cancers; it corre- lates with poor prognosis and represents a negative predictive factor of response to cytotoxic and endocrine therapy but a positive predictive factor of response to biological agents directed against it [34]. HER2 also appears overexpressed in colon, bladder, ovarian, endometrial, lung, uterine, cervix, head and neck, and esophageal carcinomas. In gastric cancer

Table 1. Trials with anti-EGFR inhibitors (first-line setting for cetuximab and advanced lines for gefitinib).

Regimens
No. of patients
G/GEJ ORR (%) TTP OS Grade 3/4
toxicity (%)
Author

FOLFIRI + cetuximab

38

34/4

44.2

8

16

Neutropenia (42.1) Diarrhea (7.9)

Pinto, 2007 [22]

FUFIRI + cetuximab 49 34/15 55 2 NA Diarrhea (16) Moehler, 2008 [26]

FUFOX + cetuximab
52
2/25
65.2
7.6
9.5 Neutropenia (6) Diarrhea (33)
Lordick, 2007 [23]

CDDP/CAPE + cetuximab
38
38/0
40
NA
NA Neutropenia (14.2) Diarrhea (6.3)
Zhang, 2008 [27]

CDDP/docetaxel + cetuximab 66 54/12 40 4 NA Neutropenia (39.4) Pinto, 2008 [28]
Gefitinib alone 75 NA 1 NA NA NA Doi, 2003 [31]
Gefitinib alone 20 NA 15 NA NA NA Adelstein, 2005 [32]

CDDP/CAPE: Cisplatin/capecitabine; FUFOX: 5FU/oxaliplatino/sodium folinate, FUFIRI: 5FU/irinotecan/ sodium folinate; G/GEJ: Gastric/gastroesophageal junction; NA: Not applicable; ORR: Overall response rate; OS: Overall survival; TTP: Time to progression.

it is positive in about 9 – 38% of cases with concordance between overexpression in immunohistochemistry (IHC) and amplification by FISH (fluorescence in situ hybridization) or CISH (cromogen in situ hybridization) [35,36]. Moreover, the HER2 expression varies depending on histology and on the primary tumor localization. In fact, it is highly overexpressed in intestinal tumor type rather than in diffuse ones and in GEJ tumors rather than in gastric tumors [36-39]. In particular, these differences are more evident in the TOGA trial, where tumor samples have been centralized and it has been found that HER2 is positive in 34% of intestinal type, 6% of diffuse type and 20% of mixed localizations. Furthermore, a recent update shows that it is present in 34% of GEJ tumors and 20% in gastric cancer samples [38]. The role of HER2 as prog- nostic factor in gastric cancer has been controversial because some of the initial studies failed to find an association with prognosis [40,41]. Nevertheless, recent studies have demonstrated a direct correlation between HER2 expression and poorer survival and have found HER2 to be an independent prognostic factor of survival in multivariate analysis [42-45].
Trastuzumab, a humanized mAb directed against the extra- cellular domain of HER2/neu receptor, enhances survival rates both in primary and in metastatic HER2-positive breast cancer patients. In preclinical studies, in vitro and in vivo, trastuzumab was shown to be able to inhibit the growth of HER2-positive but not of HER2-negative gastric cell lines [39,46].
Cellular growth inhibition is enhanced when it is given in combination with cytotoxic agents active against human gastric cancer cell lines such as paclitaxel, docetaxel, capecitabine, cisplatin or irinotecan [47,48]. In particular, when administered with cisplatin, it resulted in a synergistic inhibitory effect, whereas trastuzumab plus 5-FU or plus oxaliplatin obtained an additive effect.
In the clinical setting, there are at least three trials evaluating the role of trastuzumab in HER2-positive gastric or gastroe- sophageal adenocarcinoma patients.
In a Phase II study, 21 patients with HER2-positive AGC underwent first-line chemotherapy with trastuzumab (8 mg/kg day 1 – loading dose in first cycle – and 6 mg/kg for main- tenance doses) and cisplatin at 75 mg/m2 day 1 every 21 days until progression or unacceptable toxicity. Preliminary results on 17 assessable patients showed a 35% ORR and 52% disease control with an acceptable toxicity profile [49].
Nicholas et al. are evaluating, in a multicenter single-arm Phase II study, the activity of trastuzumab given with cisplatin and docetaxel in patients with HER2-positive metastatic gastric or GEJ cancer. Cytotoxic doses are 75 mg/m2 for cisplatin and docetaxel and 8 mg/kg (cycle 1) as loading dose and subsequent doses 6 mg/kg for trastuzumab all on day 1, every 21 days. In 2006, 9 (16%) of 55 screened patients resulted as HER2-positive. Best responses registered in five assessable patients were one CR, three PR and one SD with 100% disease control. Grade 3 – 4 peripheral neuropathy, abdominal cramping and neutropenia were probably due to chemotherapic agents rather than to the biological drug [50].
The ToGA is a multicenter, Phase III, open-label trial designed to evaluate if the combination of trastuzumab, cisplatin and 5-FU/capecitabine is able to improve the OS of HER2-positive AGC patients with respect to the cisplatin and 5-FU/capecitabine regimen alone. Secondary end points are PFS, TTP, ORR, disease control, duration of response and quality of life between the two treatment arms. So far, 400 out of 584 patients have been randomized and first efficacy data are expected in 2009 [38].
Until now, only one study has assessed the trastuzumab efficacy and safety when given alone at doses of 4 mg/kg (first administration) and of 2 mg/kg (subsequent adminis- trations) in HER2-positive AGC patients progressed to a platinum-based or a 5-fluoropyrimidine-based chemotherapy. In April 2006, only 3 out of 33 patients were IHC 3+ and thus eligible for trastuzumab therapy; so far, no updates are available [51].

Lapatinib, a small-molecule TKI with dual activity, is able to block the intracellular domain of both EGFR and HER2. In preclinical studies it inhibited the growth of two HER2- amplified gastric cancer cell lines and when combined with 5-fluorouracil, cisplatin, oxaliplatin or paclitaxel showed an additive or synergistic effect [52].
In a Phase II study, lapatinib was administered at 1500 mg orally daily in patients with HER2-positive AGC. The primary end point was ORR; secondary end points were time to treat- ment failure (TTF), OS, toxicities and the relationship between EGFR, ErbB2 and markers of angiogenesis with clinical outcome. Even though this study failed to reach the primary objective, because it obtained only a 12% ORR, the modest single-agent activity indicates that this targeted agent may warrant further investigation in combination regimens [53].
The ongoing LOGIC trial is a Phase III study that will evaluate in 410 patients whether the combination of lapatinib, oxaliplatin and capecitabine is able to improve the PFS of HER2 positive advanced gastric or gastroesophageal tumors with respect to the oxaliplatin and capecitabine combination.

4.VEGF

Since its inception more than three decades ago, the field of angiogenesis research has made significant progress [54]. During tumorigenesis, the process of angiogenesis, which is tightly and physiologically regulated, is often markedly disordered and requires the continued production of stimu- lators by tumor and stromal cells in excess of inhibitors. In addition, to provide nutrients and oxygen and remove catabo- lites, proliferating endothelial cells produce multiple growth factors that can promote tumor growth, invasion and survival. Therefore, angiogenesis provides both a perfusion effect and a paracrine effect to a growing tumor and tumor cells, while endothelial cells can drive each other to amplify the malignant phenotype. The VEGF is a potent mitogen for vascular endothelial cells both in physiological and in pathological processes. It consists of several proteins of 121,145,165,189 and 206 amino acids. The tissue predominant isoform is VEGF 165, whereas isoforms 121 and 165 are secreted and can be found in serum. VEGF plays an important role in cancer development because it promotes the endothelial cells’ migration and organization for the neovascularization of micrometastasis, metastasis and primary tumors. The multiple effects of VEGF are mediated through several different recep- tors including the TK receptors VEGFR1 (flt-1), VEGFR2 (KDR, flk-1) and VEGFR3 (flt4), with different binding specificities for each form of VEGF. The receptor activation results in slightly different effects, with VEGFR1 promoting differentiation and vascular maintenance, VEGFR2 inducing endothelial cells proliferation and vascular permeability and VEGFR3 stimulating lymphangiogenesis [55]. Although VEGFR is largely expressed on endothelial cells, VEGFRs have also been reported on inflammatory cells such as macrophages and tumor cells themselves [56]. High serum

concentrations of VEGF have been found in several solid tumors and correlate with poor prognosis.
In gastric cancer the VEGF expression increases with stage and tumor burden and represents a negative prognostic factor for survival [19,57,58]. Taken together, these data support the angiogenesis inhibition as a potential antitumor strategy: the most widely studied targets are VEGF and its receptors.
Bevacizumab is a recombinant humanized mAb. In contrast to the other immunological agents which target TK receptors on the cellular membrane, it interrupts the signal cascade by neutralizing the ligand, that is VEGF. Administered with different doses, with a minimum of 5 mg/kg every 2 weeks to a maximum of 15 mg/kg every 3 weeks, in combination with chemotherapy in the first-line setting, it has been shown to improve the OS of patients with advanced lung and breast cancer [59,60]. In metastatic colon rectal cancer it has obtained contrasting results because in combination with FOLFIRI regimens it has improved PFS and OS; in combination with FOLFOX and XELOX it has obtained an improvement only in PFS with no statistically significant improvement in OS [61,62].
In gastric cancer xenograft models, the use of bevacizumab resulted effective in inhibiting VEGF [63]. Shah et al., in a Phase II study, evaluated the combination of bevacizumab 15 mg/kg on day 1 with irinotecan 65 mg/m2 and cisplatin 30 mg/m2 on days 1 and 8, every 21 days in terms of TTP. A total of 47 patients with advanced gastric and GEJ unre- sected cancer were enrolled. With a median follow up of 12.2 months, the median TTP was improved by 75% over historical control with no difference for tumor localization (gastric vs GEJ). On the contrary, median TTP for patients with measurable disease was better than median TTP for patients with assessable and nonmeasurable disease (9.2 vs 6.4 months). The median OS was 12.3 months with advantage in measurable disease patients compared with nonmeasur- able disease patients (median OS of 15.4 vs 8.4 months). There was no increase in chemotherapy-related toxicity, and bevacizumab-specific toxicity was grade 3 hypertension recorded in 28% of patients. Thromboembolic events and gastrointestinal hemorrhages were similar to those reported in other studies [64], showing that the addition of bevacizumab to chemotherapy in untreated and unresected AGC patients is safe [65].
In combination with docetaxel 70 mg/m2 on day 1 and oxaliplatin 75 mg/m2 on day 1, bevacizumab at 7.5 mg/kg administered every 3 weeks gave, in a Phase II study, 50% ORR and 100% disease control. Main grade 3/4 toxicities were neutropenia, neuropathy and hypertension. At the time of presentation, at the 2008 ASCO meeting, the study was still recruiting and in a total of eight evaluable patients with unresected AGC there were two gastrointestinal perforations. The authors concluded the regimen results to be active even if bevacizumab should not be used outside clinical trials [66]. Other Phase II studies with preliminary results are listed in Table 2 while ongoing trials are listed in Table 3.

Table 2. Phase II studies of bevacizumab in combination with chemotherapy in the first-line setting.

Regimens
No. of patients
G/GEJ
ORR (%)
TTP
OS
Grade 3/4 toxicity (%)
Author

CDDP/IRI

47

24/23

65

8.3

12.3

Neutropenia (27) Hypertension (28) Thromboembolism (25) Diarrhea (15)
Gastric perforation (6) GI bleeding (4)

Shah, 2006 [65]

CDDP/IRI/docetaxel
32
12/7/12
63
NR
NR
Neutropenia (22) Diarrhea (28) Thromboembolism (9)
Enzinger, 2008 [79]

FOLFOX
16
7/9
63
7
8.9
No thromboembolic events nor GI bleeding
Cohenuram, 2008 [80]

OXA/docetaxel
8
4/4
50
NR
NR
Neutropenia (38) Hypertension (13) GI bleeding (25)
Hammad, 2008 [81]

mTCF
21
15/5/1
71
NR
NR
Neutropenia (50) Thromboembolism (29) GI bleeding (4.8)
Jhawer, 2008 [82]

CDDP/IRI: Cisplatin/irinotecan; FOLFOX: 5-Fluorouracil, oxaliplatin; G/GEJ: Gastric/gastroesophageal junction; mTCF: Docetaxel, cisplatin, 5-fluorouracil; OXA/docetaxel: Oxaliplain/docetaxel.

Table 3. Ongoing Phase II and III trials with biological agents in advanced gastric cancer.

Regimens
Trials
No. of patients
Line of treatment
Primary end point
Therapy

DOCETUX II 70 I RR CDDP/DOCE + cetuximab
B164 II 70 I RR CDDP/DOCE + cetuximab

CALGB 80403 ECOG-E-1206
II
270
I
RR
ECF + cetuximab CDDP/IRI + cetuximab FOLFOX + cetuximab

NCT-130689 II 36 II RR Cetuximab
MATRIX II 70 I RR ECX
ECX + matuzumab
REAL-3 III 730 I OS EOX
EOX + panitumumab
EXPAND III 800 I PFS CDDP/capecitabine ± cetuximab
ToGA III 584 I OS CDDP/5FU or capecitabine ±
trastuzumab
LOGIC III 410 I PFS Capecitabine/oxaliplatin ±
lapatinib
AVAGAST III 760 I OS CDDP/CAPE
CDDP/CAPE + bevacizumab MAGIC-B III 1100 Peri-op OS ECX
ECX + bevacizumab

CDDP/CAPE: Cisplatin/capecitabine; CDDP/DOCE: Cisplatin/docetaxel; CDDP/IRI: Cisplatin/irinotecan; ECF: Epirubicin, cisplatin, 5-fluorouracil; ECX: Epirubicin, cisplatin, capecitabine; FOLFOX: 5-Fluorouracil, oxaliplatin; OS: Overall survival; RR: Response rate.

The inhibition of the VEGF pathway comprises several small molecule TKIs that are directed to VEGF receptor (VEGFR). VEGFR TKIs have shown antiangiogenetic effects in different gastric cancer preclinical models. Until now, there are a few Phase II studies but no Phase III studies that are testing these molecules in clinical trials. Among them there are sunitinib and sorafenib.
Sorafenib is a potent inhibitor of raf tyrosine kinase, VEGFR-2, VEGFR-3, and platelets growth factor receptor- beta (PDGFR-β). Forty-four AGC patients received sorafenib 400 mg twice daily orally, docetaxel 75 mg/m2 and cisplatin 75 mg/m2 on day 1 every 21 days. Sun et al. reported in this study a median PFS of 5.8 months and a median OS of 14.9 months with an ORR of 38.6% in 17 assessable patients. Main grade 3 toxicity was neutropenia while anorexia, hand–foot reactions, nausea, diarrhea and dehydration were grade 1 [67].
Sunitinib malate is an oral multitarget inhibitor of VEGFRs, PDGFRs, KIT, RET, and FLT3. Approved for metastatic renal cell carcinoma and gastrointestinal stromal tumours (GIST) refractory to imatinib, it has been tested as monotherapy in platinum or 5-FU pretreated AGC patients [68]. Of 21 patients evaluable for efficacy, 1 PR and 8 SD were obtained. Grade 3/4 main toxicities included hand-and-foot syndrome (10.5%), fatigue (7.9%), anorexia (7.9%) stomatitis (5.3%), neutropenia (29%), thrombocytopenia (29%) and anemia (11%) with seven experiencing serious sunitinib-related adverse events.

5.Matrix metalloproteinase

Matrix metalloproteinases (MMPs) are zinc-dependant proteolytic enzymes that play an important role in normal processes of cellular growth, differentiation and repair by breaking connective tissue proteins. Experimental studies indicate that MMPs produced by cancer cells are of critical importance in tumor growth, invasion and metastasis. A positive correlation between tumor progression and the expression of multiple MMP family members (MMP-1, MMP-2, MMP-7, MMP-9, MMP-11, MT1-MMP) in tumor tissues has been demonstrated in numerous human and animal studies [69]. The ratio of activated total MMP levels, especially MMP-2, has also been correlated with tumor aggressiveness [69]. Further support for the role of MMPs in cancer dissemination came from the demonstration that tissue inhibitor of metalloproteinases (TIMPs) were able to interfere with experimental metastasis [70]. In gastric cancer, several MMPs are overexpressed (i.e., MMP-2, MMP-7, MMP-9, MMP14) and among them MMP-2 results in being strongly associated with tumor progression and lymph nodes metastasis [71]. Based on numerous preclinical studies, it was proposed that targeting MMP activity might provide a mech- anism to prevent cancer dissemination [72]. Marimastat is the only matrix metalloproteinase inhibitor (MMPI) that has been shown in preclinical and clinical trials to be active in gastric cancer cell lines and in AGC patients [73,74].

Tierney et al. recruited 35 patients with advanced, inoperable gastric or GEJ tumors to receive marimastat at a dose of 50 mg twice daily (6 patients), then reduced to 25 mg once daily (29 patients) because of toxicity, to evaluate the safety and tolerability of this MMPI. Although no clear responses were reported, some patients presented clinical benefit with clear signs of activity. The drug activity was demonstrated because, at repetitive tumor biopsies, 32% of patients showed an increased fibrotic cover of the tumor, 26% had decreased hemorrhagic appearance and at least two cases had evidence of increased stromal fibrotic tissue compared with the basal specimens [75].
On this basis, a total of 369 both chemo-naive and pretreated AGC patients were enrolled in a double blind, randomized Phase III study of comparison between mari- mastat at 10 mg orally twice daily versus placebo. The pri- mary end point was OS and secondary objectives were TTP and quality of life. At the time of analysis, the primary objective was not met, even though there was an improvement in median OS and in 2-year OS in patients who received marimastat versus patients who received placebo (5.2 vs 4.5 months and 9% vs 3%, respectively). Moreover, a statistical improvement of median OS and 2-year OS was observed in the previously treated patients who received marimastat (8.4 vs 5.8 months and 18% vs 5%, respectively) [76]. Although marimastat demonstrated therapeutic benefit in AGC patients, no further developments of this MMPI have been done.

6.Mammalian target of rapamycin (mTOR)

The mammalian target of rapamycin (mTOR) belongs to the phosphoinoside 3-kinase-related kinase (PIKK) family that regulates the protein synthesis and the cellular growth, metabolism, proliferation and angiogenesis. The activation of growth factor receptors by extracellular ligands – both growth factors (EGF, VEGF and IGF) and nutrients (amino- acids, glucose and oxygen) – determines the phosphorylation of several intracellular signaling pathways such as the phos- phoinoside 3-phosphate-kinase (PI3K). PI3K activates Akt serin-threonin-kinase that phosphorylates mTOR. The activation of mTOR produces the phosphorylation of p70S6K (S6K) and eukaryotic initiation factor 4E binding protein 1 (4E-BP1), which have a critical role in protein synthesis. Loss of the suppressive PTEN gene expression, PI3K gene mutations and amplification of Akt result in constitutive activation of the upstream PI3K/Akt pathway observed in some tumors. Such activation increases the tumors’ susceptibility to mTOR inhibitors and provided the rationale in developing rapamycin (mTOR inhibitor) analogs in various cancer types. Thus, the inhibition of mTOR by rapamycin or other agents such as everolimus (RAD001), CCI-779, AP23576, produces cellular death by a reduction of protein synthesis and an increase of apoptosis. Rapamycin and its analogs temsirolimus and everolimus are specific inhibitors of mTOR that exert suppressive effects on

proliferation, invasion and metastasis and induce apoptosis of tumor cells. Apart from the direct effects of mTOR inhibitors on tumor cells, rapamycin and its analogs have potent antiangiogenic properties related to the suppression of VEGF signal transduction.
The mTOR pathway plays an important role in several solid tumors being important for survival, proliferation, angiogenesis and resistance to chemotherapy: recent reports suggest that PI3K/AKT/mTOR pathways are often activated in gastric cancer, where it has been recently demonstrated, in a preclinical study, that they are involved in the development of peritoneal carcinomatosis [77].
To our knowledge, until now, there is only one Phase II study, conducted by Muro et al., that is testing the activity of RAD001, an oral inhibitor of mTOR, in advanced gastric cancer patients. This is a Phase II study with a Simon 2-stage design. Pretreated AGC patients received RAD001 daily at a dose of 10 mg per day. The primary end point

Erlotinib and gefitinib do not seem active in gastric cancer in contrast with the esophageal and GEJ results.
Trastuzumab showed important results in in vitro and in vivo studies, though we are waiting for results of Phase II and III studies.
Bevacizumab seems to be active and safe, but Phase III studies are warranted to understand better its efficacy and toxicity profile. In conclusion, some target therapies might have an important role in gastric cancer patients, but the results of Phase III trials are pending. Furthermore, before administering one of these tailored agents, it seems indispens- able to select patients who really may benefit from these treatments and it could be reasonable to design a Phase III trial with a standard unique regimen avoiding the use of different schemes.

8. Expert opinion

was disease control rate lasting at least 8 weeks and secondary objectives were PFS, ORR and safety. Preliminary results have been presented at the 2008 ASCO meeting with a total of 24 patients enrolled. Independent radiological review was done in 17 patients who had completed 8 weeks follow-up for efficacy, reporting SD in 10 patients and progression disease (PD) in seven patients. Grade 3 stomatitis, anorexia, hyponatraemia and diarrhea were recorded in one patient. These results support the continuation of the trial and the second stage is ongoing [78].
•
Results from recent Phase III trials with conventional chemotherapeutic agents have shown encouraging activity in advanced gastric cancer, providing patients with addi- tional therapeutic options; however, survival continues to be suboptimal. Targeted therapy seems to be promising and it is hoped that its incorporation into the next genera- tion of clinical trials will improve outcomes and help advance future treatments. EGFR, the VEGF and its receptors represent at present the most promising therapeutic targets in gastric cancer.

• Strategies of EGFR targeting have been numerous with mainly

7.Conclusion

Gastric cancer still represents a leading cause of death world- wide. First-line chemotherapy has demonstrated improvement in overall survival, progression free survival and quality of life when administered in locally advanced or metastatic gastric cancer patients; the most active combinations are able to obtain a maximum of 9 – 10 months of overall survival. That is why it seems necessary to investigate new drugs such as tailored agents. In this regard, EGFR and VEGF expression may be evaluated as a crucial step because these molecules can be targeted with different agents (such as monoclonal antibodies and small molecules); however, there are few Phase II studies that have demonstrated the activity of these biological drugs in gastric cancer.
Data from Phase II trials indicate the potential of cetuximab to improve the activity of different regimens such as FOLFIRI, FUFOX, and docetaxel-cisplatin. Biomolecular analysis of these studies indicates that there is no correlation between EGFR expression evaluated by ICH or FISH and response to treatment. Furthermore, it is difficult that the K-RAS and BRAF status might have an important role in terms of cetuximab

•

•
monoclonal antibodies specifically binding the extracellular domain of the EGFR and enzymatic inhibitors of the intra- cellular part of the receptors with tyrosine kinase activity. Among anti-EGFR drugs, erlotinib and gefitinib do not seem active in gastric cancer. On the other hand, cetuximab in combination with different chemotherapeutic regimens is able to increase ORR, median TTP and OS in Phase II studies in both chemonaive and pretreated patients.
Several approaches to block VEGF activity are under evaluation: at this time, bevacizumab, a humanized antibody against VEGF, seems to be active and safe in association with chemotherapy, demonstrating the potential for combining antiangiogenic agents with conventional therapeutics in advanced gastric cancer.
However, these novel investigational drugs seem to have a potential role in gastric cancer patients, but the results of Phase III trials are still pending. Furthermore, tailored therapy for each patient is an important goal for improving the outcome of patients with gastric cancer: therefore, reliable predictive tools are needed to apply these treatments for potentially sensitive tumors only.

sensitivity because in advanced gastric cancer the frequency of mutations in KRAS and BRAF is significantly lower with respect to colorectal cancer and do not seem to be correlated with the response to cetuximab combination therapy.
Declaration of interest

The authors state no conflict of interest and has received no payment in preparation of this manuscript.

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Affiliation Loredana Vecchione, Michele Orditura,
Fortunato Ciardiello &
Ferdinando De Vita† MD PhD †Author for correspondence Second University of Naples, School of Medicine,
Division of Medical Oncology “F. Magrassi & A. Lanzara”, Department of Clinical and
Experimental Medicine and Surgery, c/o II Policlinico Via S. Pansini 5, 80131 Naples, ItalyBB-2516
Tel: +39 081 566 6713; Fax: +39 081 566 6728; E-mail: [email protected]