Introduction

Ovarian cancer is the fourth most common cause of cancer death in women, with a worldwide incidence of 200,000 cases causing 115,000 deaths per year. As described elsewhere, the current standard of care for women with stage IC (and stage IB with adverse histological features) to stage III disease is surgery followed by adjuvant chemotherapy. Further lines of treatment can be given when patients relapse but cure is not possible. Ovarian cancer is considered to be a chemosensitive disease, with nearly 75% of patients initially responding to platinum-based treatment. The choice of agent upon relapse depends on the timing of relapse; if this is over 6 months from previous platinum administration, this can be repeated either as a single agent or as part of a combination regimen, as patients are still considered to be platinum sensitive. If relapse occurs within 6 months of previous platinum administration, non-platinum drugs such as pegylated liposomal doxorubicin are used. There is an increased understanding of the complicated biological pathways involved in the development of gynaecological cancers, their subsequent metastagenesis and the mechanisms involved in chemoresistance. Many clinical studies are underway investigating the use of novel agents in patients with gynaecological malignancies, either alone, concurrently with chemotherapy or sequentially, in an attempt to improve response rates/durations and thereby survival. This chapter will outline several of the new classes of novel agents which are at various stages of development, detail their putative mechanisms of action and their potential indications, and include brief summaries of their toxicity profiles from studies to date. Many of the phase II studies of new agents include patients in the platinum-refractory group, whose tumours are intrinsically resistant to chemotherapy and radiotherapy, and who therefore have low rates of response to conventional treatment.

Vascular Endothelial Growth Factor and Angiogenesis Inhibition

Angiogenesis or neovascularization is a normal physiological process involving the remodelling of vasculature and formation of new blood vessels. Angiogenesis plays a vital role in tumour formation and metastasis because both primary lesions and metastatic tumours must develop a new vascular supply in order to survive (Folkman 1971, 1990). Early initiation of angiogenesis is essential for cancer survival, and occurs when stimulatory factors overcome inhibitory factors, promoting the formation of new blood vessels (Bergers and Benjamin 2003).

Research investigating the molecular basis of angiogenesis has identified multiple pathways that contribute to tumour angiogenesis. These include vascular endothelial growth factor (VEGF), fibroblast growth factor and platelet-derived growth factor (PDGF). Based on the central role of VEGF in tumour angiogenesis and growth, this has emerged as the most promising therapeutic target for angiogenesis inhibition. The VEGF molecule interacts with cell surface VEGF receptors (VEGFRs) that initiate signalling through downstream pathways to promote growth and differentiation of vascular endothelial cells and formation of new blood vessels.

The biological roles of VEGF predict that its expression should be related to clinical outcome in cancer patients. Indeed, VEGF expression has been correlated with both disease-free survival and overall survival (OS) in a variety of gynaecological cancers. VEGF expression has been evaluated in women with ovarian carcinoma in several studies, which collectively provide consistent evidence that higher VEGF levels are associated with aggressive clinical behaviour in ovarian carcinoma. Kassim et al (2004) found some degree of VEGF expression in all ovarian cancer specimens examined, and the level of VEGF expression was significantly higher in tumour specimens compared with benign ovarian tissue. In addition, rising titres of VEGF from tumour specimens correlated with increasing stage and decreased survival. This relationship seems to be independent of important clinical and pathological prognostic factors. Furthermore, in women with ovarian cancer, high serum levels of VEGF are an independent risk factor for ascites, advanced-stage disease, undifferentiated histology, more metastases and decreased survival (Cooper et al 2002, Li et al 2004).

There are limited data regarding VEGF expression in endometrial cancer specimens. Holland et al (2003) documented VEGF expression in 100% of the endometrial cancer specimens examined. In addition, they found no expression of VEGF in benign endometrial tissue. The functional significance of the VEGF/VEGFR axis in endometrial cancer remains to be demonstrated.

In tissue specimens from patients with cervical cancer, VEGF expression has been noted immunohistochemically in 94% of samples (Loncaster et al 2000). Intratumour levels of VEGF are increased in patients with cervical cancer compared with levels in normal cervical tissue. For these women with cervical cancer, increasing intratumoral levels of VEGF also correlated with higher stage, increased risk of lymphovascular space invasion, greater likelihood of parametrial spread, and lymph node metastasis (Cheng et al 1999).

The biological and clinical significance of the VEGF pathway in tumour angiogenesis suggests its value as a therapeutic target in gynaecological malignancies. There are three main strategies for inhibition of the VEGF pathway for therapeutic applications. The first is to target VEGF itself, the second is to target VEGFR so as to prevent binding of VEGF, and the third is to inhibit tyrosine kinase activation and downstream signalling with small molecules that work at the intracellular level.

Bevacizumab (Avastin) is the first targeted agent to show significant single-agent activity in ovarian carcinoma. It is a recombinant humanized monoclonal antibody directed against VEGF. Bevacizumab has been tested in a number of phase II trials as monotherapy in epithelial ovarian cancer (EOC). Two such trials using single-agent bevacizumab in relapsed high-grade carcinoma of the ovary were presented at the American Society of Clinical Oncology (ASCO) meetings in 2005 and 2006 (Burger et al 2007, Cannistra et al 2007). They convincingly demonstrate bevacizumab’s significant single-agent activity in patients with relapsed EOC, with objective response rates (ORR) of 21% and 16%, respectively. Additionally, 52% and 64% of cases, respectively, achieved disease stabilization with median progression-free survival (PFS) of 4.4 and 4.5 months. These results are as good or better than typical rates from traditional second-line chemotherapeutic agents in this group of patients.

Cytotoxic and antiangiogenic agents can be used in combination for enhanced activity. It has been hypothesized that combining VEGF-targeted agents with frequently administered low-dose so-called ‘metronomic chemotherapy’ may have additive or synergistic antiangiogenic or antitumour effects. In 2005, Garcia et al presented preliminary data from a phase II trial of patients with recurrent ovarian cancer who were given bevacizumab biweekly with metronomic oral cyclophosphamide. The ORR was 28% and a further substantial proportion (57%) achieved disease stabilization for at least 6 months. The median PFS was 7.5 months and OS was 13 months. This protocol is now closed and the data are undergoing final analysis, but if consistent with preliminary results, these findings would provide the rationale for a phase II randomized trial of combination versus single-agent therapy. Additional reports of outcomes for patients with EOC and primary peritoneal cancer treated with bevacizumab include three historical case series of patients treated outside clinical trials with single-agent therapy or in combination with cytotoxic drugs, suggesting activity in more heavily pretreated patients with recurrent disease, and preliminary data in 2006 demonstrating the feasibility of the combination of traditional carboplatin/paclitaxel chemotherapy combined with bevacizumab in front-line therapy (Cohn et al 2006, Monk et al 2006, Penson et al 2006, Wright et al 2006a).

Based on the encouraging phase II trial data, bevacizumab has been rapidly incorporated into phase III trials, the major focus of which is on first-line treatment. Two similar randomized trials are now underway. The Gynaecologic Oncology Group 218 trial is designed to explore the role of bevacizumab in first-line treatment and in the maintenance setting, with a three-way randomization whereby all patients receive carboplatin/paclitaxel plus bevacizumab or placebo for six cycles, with those receiving bevacizumab further randomized to drug or placebo maintenance for 15 months, with the primary endpoint being OS. It is limited to patients with stage III or IV disease. In contrast, the Gynaecological Cancer Intergroup trial (ICON 7) is a non-placebo-controlled, smaller, two-arm trial in which patients receive either carboplatin/paclitaxel for six cycles or the same treatment plus bevacizumab every 3 weeks during chemotherapy and continuing for an additional 9 months, with the primary endpoint being PFS. The patient population for this trial includes all patients with at least high-risk, early-stage disease. Bevacizumab has also been tested in the phase II setting in combination with topotecan in women with platinum-refractory ovarian cancer (McGonigle et al 2008). To date, 22 patients have been enrolled, 18 of whom were evaluable; 22.2% had a partial response (PR) and 27.8% achieved disease stabilization. Toxicity was described as being acceptable.

Bevacizumab has also been given to women with cervical carcinoma. Wright et al (2006b) gave combination bevacizumab and 5-fluorouracil or capecitabine to women with recurrent cervical cancer. In this small retrospective group of six women, one patient achieved a complete response (CR), one achieved a PR and two achieved disease stabilization. Unfortunately, all four women eventually had disease progression at a median time of 4.3 months, and none had a progression-free interval of more than 6 months.

Unique toxicities have been ascribed to the administration of bevacizumab and other anti-VEGF molecules. Most of these toxicities (such as proteinurea, hypertension and bleeding) are generally mild and are either self-limiting or easily manageable. Other adverse effects, although uncommon, may be serious; these include arterial thromboembolism, wound-healing complications, and gastrointestinal perforation or fistulae.

Targeting the VEGF/VEGFR axis may affect the survival of both proliferating and quiescent endothelial cells, leading to the disruption of normal vasculature. The increase in arterial thromboembolic events, including cerebral infarction, transient ischaemic attacks, myocardial infarction and angina, may be related to this phenomenon. Hypertension is one of the most common side-effects of bevacizumab therapy, with an overall incidence of 22–32% (Gordon and Cunningham 2005). The mechanism responsible for bevacizumab-related hypertension is not fully understood, but it is thought to be related to decreased production of nitric oxide as a result of VEGF inhibition. Most patients with hypertension can be managed with oral antihypertensive agents.

VEGF is known to play a critical role in the physiological angiogenesis required for wound healing. This could be of particular importance when considering antiangiogenic therapy as front-line adjuvant treatment of ovarian cancer after cytoreductive surgery. Concerns about wound healing in postoperative patients have resulted in the decision to start bevacizumab/placebo therapy at cycle 2 within the Gynaecologic Oncology Group 218 trial. This would be more than 28 days after surgery.

The complication of bowel perforation is now well documented with bevacizumab and, although uncommon, is of concern. It has been suggested that the number of prior cytotoxic regimens and the presence of bowel obstruction might predispose to this complication, although our ability to identify high-risk patients requires further investigation.

Other agents targeting the VEGF pathway are also being developed rapidly. VEGF-Trap inactivates VEGF by acting as a decoy receptor for VEGF, preventing binding of the ligand to its natural receptors. Promising data have been reported regarding VEGF-Trap in preclinical and phase I studies in patients with advanced solid malignancies (Dupont et al 2004, Hu et al 2005). One patient in the phase I study had carboplatin-, taxane- and gemcitabine-resistant advanced ovarian cancer and achieved a response evaluation criteria in solid tumours (RECIST)-defined PR, a 67% reduction in CA125 levels, radiographic resolution of abdominal ascites and subjective improvement of performance status after four cycles of VEGF-Trap. It is currently being evaluated in the phase II setting in patients with recurrent ovarian carcinoma.

Cediranib (AZD2171) is a potent oral tyrosine kinase inhibitor (TKI) of VEGF1, 2, 3 and c-Kit. It has been tested in a phase II study reported at the ASCO meeting in 2008 as a single agent in recurrent ovarian, peritoneal or tubal cancer (Matulonis et al 2008). Preliminary results from 28 patients, 16 of whom had platinum-resistant disease, revealed an ORR of 18.5%. Common toxicities included hypertension (n = 13), fatigue (n = 5) and diarrhoea (n = 3). A further phase II study of this agent, reported in 2008, has shown similar promising efficacy data and toxicity profiles (Hirte et al 2008).

Sunitinib, an oral multitargeted TKI known to be effective against, amongst others, VEGFR and PDGF receptors, has recently been tested in the phase II setting as a single agent in patients with EOC, primary peritoneal and fallopian tube cancers (Biagi et al 2008). Preliminary results reported at the ASCO meeting in 2008 revealed that 12 out of 17 patients had a PR or disease stabilization. The tolerability profile of this drug is well known from its extensive use in other tumour types, and includes fatigue, mucositis, dysgeusia, hypertension, nausea and hand–foot reaction. Further studies involving larger patient groups are warranted in order to further assess the efficacy of sunitinib in gynaecological cancers.

Sorafenib, another multitargeted TKI (predominantly affecting VEGFR, PDGF receptors, Flt3 and c-Kit) has also been tested in the phase II setting in patients with recurrent EOC and primary peritoneal cancer, the results of which were presented at the recent ASCO meeting (Matel et al 2008). Seventy-three patients who had persistent or recurrent EOC who had been treated previously with one or two cytotoxic regimens, and who had progressed within 12 months of platinum-based therapy, were treated until disease progression or toxicity requiring cessation. The primary endpoint was PFS at 6 months. Response, OS and toxicity were also documented. Toxicities (grade three to four) included rash (n = 12), metabolic (n = 10), gastrointestinal (n = 3), cardiovascular (n = 2) and pulmonary (n = 2). Twelve out of 59 evaluable patients were progression free for at least 6 months. Two PRs were reported and 20 patients achieved disease stabilization. Mature data are awaited to further define the utility of this agent.

Sorafenib has also been studied in patients with uterine cancer in the phase II settting, and preliminary results have been reported (Nimeiri et al 2008). Patients with advanced or recurrent disease who had experienced one or fewer prior regimens were treated with sorafenib 400 mg bd. Fifty-five patients were enrolled. Grade three to four toxicities included hypertension (13%), hand–foot syndrome (13%) and anaemia (6%). The response rate in patients with advanced/recurrent disease was 5% (PR), and 50% of patients achieved disease stabilization. The median OS was 10.1 months. In patients with carcinosarcoma, 27% achieved disease stabilization. The completion of the trial is awaited.

The Epidermal Growth Factor Receptor Family

The human epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases has four identified members: EGFR or HER1, HER2, HER3 and HER4. This subgroup of receptors mediate cell growth, differentiation and survival, and are dysregulated in many types of cancer. Ligand binding to the extracellular domain of the receptors enables receptor homo- or heterodimerization, which initiates phosphorylation of the intracellular tyrosine kinase domain and activation of cell signalling to reduce apoptosis and increase tumour cell proliferation.

The HER pathway is involved in ovarian cancer pathogenesis (Berchuck et al 1990, Bartlett et al 1996, Campiglio et al 1999). The majority of ovarian cancer cell lines and tumours express high levels of a number of HER receptor ligands, as well as exhibiting amplification of EGFR and HER2, and expressing all HER receptors (Campiglio et al 1999). In EOC, attention has focused on EGFR (HER1) which is expressed in 19–92% of tumours, and expression of which is associated with a poor prognosis (Ford et al 1994, Lin et al 1994, Goff et al 1996, Ilekis et al 1997, Fischer-Colbrie et al 1997).

The goal of EGFR-targeted therapies is to disrupt phosphorylation, thereby increasing apoptosis and reducing cell proliferation signals. This disruption can be achieved in several ways: inhibition of dimerization, blocking of EGFR receptor ligand binding with humanized monoclonal antibodies, and inhibition of tyrosine kinase activity with small-molecule TKIs. In the majority of trials of anti-EGFR agents in patients with EOC, the agents are investigated as monotherapy in heavily pretreated women. Objective responses are relatively low but prolonged disease stabilization has been seen in a subset of women.

The small-molecule TKIs erlotinib (Tarceva) and gefitinib (Iressa) have been tested in phase II trials of patients with relapsed and pretreated EOC. A trial of erlotinib as monotherapy in women with advanced EGFR-positive cancers treated with a mean of three prior regimens was presented at the ASCO meeting in 2001 (Finkler et al 2001). The ORR was 8.8%, with a further 7% of patients achieving disease stabilization for more than 6 months. A similar trial of gefitinib in patients with recurrent or persistent ovarian carcinoma or primary peritoneal cancer, but in which EGFR expression was not required for inclusion, led to a response rate of only 3.7% and long-term disease stabilization for more than 6 months in a further 14.8% of patients (Schilder et al 2003). The median time to progression was approximately 2 months, but 10% of patients remained progression free at 8–12 months. Interestingly, all of these patients had EGFR-positive tumours. Unlike in lung cancer, where EGFR expression and the presence of a mutation in the tyrosine kinase domain of EGFR confer sensitivity to EGFR TKIs, the relationship between EGFR positivity, EGFR mutation and response to EGFR TKIs is not clearly defined in EOC. Better definition of this relationship may help to identify the subset of women with EOC that are likely to achieve clinically meaningful responses to TKIs, and therefore define the appropriate treatment population.

Monoclonal antibodies against EGFR have also been investigated. The most established anti-EGFR antibody is cetuximab (Erbitux). This has been evaluated in previously untreated women with EOC in combination with standard first-line carboplatin/paclitaxel chemotherapy and as maintenance therapy. An ORR of 87% was seen and toxicity was acceptable (Aghajanian et al 2005). Its use as monotherapy is currently under investigation. Another anti-EGFR monoclonal antibody, matuzumab, has been investigated in women with recurrent EGFR-positive EOC (Seiden et al 2005); although no objective responses were seen, 21% of women achieved disease stabilization for more than 6 months.

Monoclonal antibodies directed against HER2 have also been investigated in advanced EOC. Laboratory studies have suggested that 10–15% of women have tumours that overexpress HER2 graded as 2+ or 3+ positivity by immunohistochemistry (IHC). It is unclear whether this is predictive of clinical outcome in EOC. In breast cancer, where the role of HER2 receptor overexpression is well defined, it is only a predictive factor in those with IHC 3+ positivity and it is only this subset who derive clinical benefit from anti-HER2 therapies (Slamon et al 2001). In a phase II study of the anti-HER2 monoclonal antibody trastuzumab (Herceptin) in EOC, of the 837 patients who were registered and screened, only 95 (11%) had tumours with grade 2+ or 3+ positivity. Of these patients, only 41 were treated, with an ORR of only 7.3% (Bookman et al 2003). The study concluded that the clinical value of single-agent trastuzumab in recurrent ovarian cancer is limited by the low frequency of HER2 overexpression and low rate of objective response among patients with HER2 overexpression.

The monoclonal antibody pertuzumab was the first in a new class of targeted anticancer agents termed ‘HER dimerization inhibitors’. It binds to the dimerization site of HER2, inhibiting its interaction with other HER family members. Unlike HER1, -3 and -4, HER2 has no known ligand and assumes an open conformation, with its dimerization domain permanently exposed for interaction with other ligand-activated HER receptors. HER2 is the preferred partner for dimer formation, and subsequent pathway activation via receptor phosphorylation has been shown to drive ovarian tumour cell proliferation, even in the absence of HER2 overexpression. Pertuzumab was investigated as monotherapy in a phase II trial in patients with advanced, refractory or recurrent ovarian cancer (Gordon et al 2006). A PR was achieved in 4.3% of subjects, with 41% achieving disease stabilization. However, median PFS was 20.9 weeks for HER2-positive patients compared with 5.8 weeks for HER2-negative patients and 9.1 weeks for those of unknown HER2 status. It should be noted that these patients were heavily pretreated and the median number of prior therapies was five. The study concluded that given that pertuzumab blocks all HER2-mediated signalling, in contrast to targeted blockade of EGFR or HER2 overexpression, pertuzumab may result in higher rates of clinical activity in an appropriately selected population. A randomized phase II study evaluating the combination of carboplatin-based chemotherapy with pertuzumab in comparison with carboplatin-based therapy alone in patients with relapsed, platinum-sensitive ovarian cancer is currently underway. Preliminary data presented at the ASCO meeting in 2008 reveal that the addition of pertuzumab to carboplatin-based chemotherapy is well tolerated, but efficacy data are awaited.

The small-molecule TKI lapatinib is similar to gefitinib and erlotinib, except that it has similar specificity for EGFR and HER2. Data from initial clinical experience with lapatinib have only shown mild adverse events (rash, diarrhoea, nausea, vomiting) and no grade four events. In the original phase I trial, three subjects showed a PR and 12 patients achieved disease stabilization, including two with ovarian cancer (Spector et al 2005).

Poly(ADP-Ribose) Polymerase Inhibitors

BRCA1 and BRCA2 belong to a class of genes known as tumour suppressor genes. Their protein products are important for DNA double-strand break repair (Tutt and Ashworth 2002). Mutations in BRCA1 and BRCA2 predispose to a number of hereditary cancers, including breast and ovarian. Poly(ADP-ribose) polymerase (PARP) is a DNA-binding enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. Tumours in carriers of BRCA1 or BRCA2 mutations lack wild-type BRCA1 or BRCA2, but normal tissues retain a single wild-type copy of the relevant gene. This biochemical difference provides the rationale for an approach involving PARP inhibition to generate specific DNA lesions that require functional BRCA1 and BRCA2 for their repair. In a study by Farmer et al in 2005, BRCA1 and BRCA2 dysfunction was shown to profoundly sensitize cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. In other words, PARP inhibitors were found to be selectively cytotoxic to tumours lacking BRCA1 and BRCA2, where the specific mechanism that repairs damaged DNA is absent. In addition, PARP inhibitors have been shown to significantly enhance the effect of a number of widely used cytotoxics that damage DNA. Results from a phase I study of AZD2281, a novel, potent, orally active PARP inhibitor in patients with BRCA-deficient ovarian cancer, were presented at the ASCO meeting in 2008 (Fong et al 2008). The study demonstrated compelling activity in patients with BRCA-deficient ovarian cancer, with efficacy data revealing a PR rate of 44% and a further 25% of patients achieving disease stabilization. AZD2281 was well tolerated, toxicities being mainly gastrointestinal and mild, and little myelosuppression was seen. A randomized study of AZD2281 in BRCA-deficient ovarian cancer is planned.

mTOR

It is known that intracellular signal transduction pathways interact to regulate the cell cycle, apoptosis, cytoskeletal organization and senescence. An example of this is the mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that is a central regulator of cell growth, proliferation and survival (Beevers et al 2006). mTOR, in turn, is regulated by the phosphatidylinositol3-kinase (PI3K)/AKT pathway, a signalling cascade that is aberrant in over 70% of tumours (Meric-Bernstam and Mills 2004). Dysregulation of this pathway has been demonstrated in ovarian cancer.

The inhibition of mTOR results in a profound decrease in the transmission of proliferative signals through the PI3K/AKT pathway, leading to antiproliferative effects and angiogenesis inhibition. IHC analysis of ovarian cancer specimens has shown that both AKT and mTOR are frequently activated (phosphorylated) in these tumours (Altomare et al 2004). In the same study, the effects of AKT/mTOR activation on the therapeutic sensitivity of ovarian cancer cells to cytotoxic agents was tested. Augmentation of cisplatin-induced apoptosis was seen in cells exhibiting constitutive AKT activity when pretreated with the PI3K inhibitor LY294002. In contrast, ovarian cancer cell lines with low basal levels of AKT activity did not show increased cisplatin-induced apoptosis. In addition, inhibition of mTOR activity with rapamycin resulted in cell cycle arrest in those cells exhibiting constitutive AKT activity, but not in those cells with low levels of AKT activity. Collectively, these findings indicate that active AKT and downstream mTOR represent potentially important therapeutic targets in ovarian cancer.

Preclinical studies have demonstrated that rapamycin (a highly specific inhibitor of mTOR kinase) and its analogues inhibit proliferation not only in ovarian cancer cell lines but also in cervical and endometrial cancer cell lines, and in a broad range of murine syngeneic tumour models and human xenografts. Three rapamycin analogues are currently in clinical development as anticancer agents: AP23573, CCI-779 (temsirolimus) and RAD001 (everolimus).

AP23573 has been tested in a phase I trial in patients with advanced solid malignancies (Mita et al 2004). The dose-limiting toxicity was mucositis, and common treatment-related adverse events included mouth sores and rash. Four out of 32 (12.5%) patients experienced a confirmed PR and three (9%) additional patients had minor tumour regression. AP23573 has also been tested in a phase Ib combination trial with paclitaxel in patients with progressive solid tumours (Cresta et al 2007). The dose-limiting toxicities were mucositis, fatigue, myelotoxicity with prolonged moderate neutropenia, and skin rash. Pharmacodynamic analysis in peripheral blood mononuclear cells demonstrated no interference by paclitaxel on mTOR inhibition by AP23573. The trial concluded that combined therapy with AP23573 and paclitaxel is feasible and well tolerated, and recommended evaluation of the combination in phase II trials examining efficacy in specific tumours.

Preliminary results of a phase II trial of single-agent AP23573 in patients with advanced metastatic endometrial cancer were presented at the ASCO meeting in 2007 (Colombo et al 2007). The trial involved 45 patients, all of whom had progressive disease. The primary endpoint was a clinical benefit response, defined as a CR or PR or prolonged disease stabilization for at least four cycles. This was achieved, with 29% of patients having a clinical benefit response, including 10% with a PR. The most common toxicities were mucositis, fatigue, anaemia, diarrhoea and nausea/vomiting. This study reveals promising results of single-agent AP23573 in progressing endometrial cancer, and warrants further evaluation of the drug as monotherapy and in combination with other agents.

In preclinical models, CCI-779 or temsirolimus, an ester derivative of rapamycin, has shown antitumour effects across a wide variety of tumour types. An initial dose-escalation phase I trial administered temsirolimus weekly (Raymond et al 2004). Although thrombocytopenia was dose limiting and a reversible maculopapular rash and stomatitis were observed, the formal definition of a maximum tolerated dose was not met. In addition, objective partial and minor responses were observed at lower dose levels. Encouraging data from a phase II study of single-agent temsirolimus in chemotherapy-naïve patients with metastatic or recurrent endometrial cancer were presented at the ASCO meeting in 2006 (Oza et al 2006). The ORR was 21%, with 48% of patients achieving prolonged disease stabilization. These results are equivalent to those of currently used cytotoxic chemotherapy. A subsequent phase II study assessed the level of activity of temsirolimus in patients with metastatic and/or locally advanced recurrent endometrial cancer who had previously received chemotherapy (Oza et al 2008). The results of this study indicated that temsirolimus had modest activity, with only 7.4% of patients achieving a PR; however, 44% achieved disease stabilization with a mean duration of 3.5 months. A phase II trial of temsirolimus in the treatment of persistent or recurrent EOC or primary peritoneal cancer is currently underway.

RAD001 (everolimus) is an oral rapamycin analogue that selectively inhibits mTOR. It has shown encouraging single-agent clinical benefit in pretreated patients with recurrent endometrial carcinoma in a phase II study presented at the ASCO meeting in 2008 (Slomovitz et al 2008). Although none of the patients achieved a PR, 44% of patients achieved disease stabilization of more than 8 weeks. Interestingly, loss of PTEN expression, a tumour suppressor gene found in 40–60% of endometrial cancers, the loss of which leads to constitutive AKT activation and upregulation of mTOR activity, was found to be predictive of prolonged disease stabilization.

Protein kinase C

Protein kinase C (PKC) is a family of serine/threonine protein kinases which are centrally involved with the regulation of angiogenesis, cell growth and survival. Activated PKC-β promotes VEGF-mediated angiogenesis and has been implicated in the progression of malignancy, cell proliferation and inhibition of apoptosis. The activated form of PKC-β has been identified in a number of human tumours, including ovarian cancer.

Enzastaurin, an oral selective inhibitor of the PKC-β isozyme, has been shown to have antiangiogenic effects and to promote apoptosis by downregulating signalling through the PI3K pathway in several cancers (Graff et al 2005, Lee et al 2008). Preclinical studies have shown enzastaurin to have anticancer effects as a single agent and in combination with carboplatin and paclitaxel in breast and ovarian carcinomas (Teicher et al 2002, Keyes et al 2004). In a study of the effect of enzastaurin on ovarian cancer cell lines with selective resistance against various cytostatic drugs, taxane-resistant cells showed the most prominent response to low concentrations of enzastaurin (Meinhold-Heerlein et al 2006). In phase I trials, prolonged stabilization of disease was seen in several patients treated with enzastaurin, with acceptable toxicities (Carducci et al 2006). Synergistic activity with standard-of-care chemotherapies was seen, with no significant alterations in the pharmacokinetic variables of the cytotoxic drugs with which it was combined (Beerepoot et al 2006, Rademaker-Lakhai et al 2007). Enzastaurin is now being tested in ovarian cancer in the phase II setting by the Gynaecologic Oncology Group.

CA125

CA125 is a surface antigen that is expressed on the majority of EOCs and occurs at elevated levels in the serum of patients with ovarian cancer. Serum levels of CA125 are used to monitor responses to chemotherapy, relapse and disease progression in ovarian cancer patients. In 2001, the gene for the peptide moiety of CA125 was cloned and termed ‘MUC16’ because of its similarities to the mucin family of proteins (Yin and Lloyd 2001). The biological functions of CA125 are complex but, based on current knowledge, CA125 seems to enhance the malignant potential of ovarian cancer cells.

Monoclonal antibodies that target CA125 are now in development as a therapeutic strategy. One such drug, ovregovomab (Mab-813.13, Ovarex), behaves as an immunotherapeutic agent. Infusion of low-dose antibody results in the formation of circulating immune complexes which can trigger a cellular immune response targeting CA125 and the ovarian cancer. Reported data have shown an association between intratumoral T cells and improved clinical outcome, lending support for immune-mediated disease control (Zhang et al 2003). Ovregovomab has been compared with placebo in a randomized trial of women with ovarian cancer who had achieved complete remission after first-line chemotherapy. There was no OS advantage but subset analysis showed that half of the treated women generated an immune response to ovregovomab, and these women had superior survival to those without an immune response (Ehlen 2002, Berek et al 2004).

A phase III randomized trial was reported at the ASCO meeting in 2008 of maintenance oregovomab monoimmunotherapy versus placebo in patients with stage III/IV ovarian cancer (Berek et al 2008). Patients were treated until recurrence or for up to 5 years, and the primary endpoint was time to recurrence. Three hundred and seventy-one patients were randomized, but unfortunately there were no significant differences in the clinical outcomes between the two groups. Conclusions drawn were that although it was not effective as a single agent, its use in combination with other cytotoxic agents should be investigated further. Ovregovomab has been evaluated in combination with carboplatin/paclitaxel as front-line chemoimmunotherapy in patients with advanced ovarian cancer (Braly et al 2007). Chemotherapy-induced bone marrow suppression has been assumed to inhibit the generation of augmented immune responses; however, contrary to expectations, concurrent carboplatin/paclitaxel resulted in enhanced immune stimulation with ovregovomab.

A second drug, abagovomab (formerly ACA125), is an antibody which functionally mimics the CA125 antigen and induces humoral and cellular CA125-specific immunity. Abagovomab has been shown to generate immune responses in women with EOC (Wagner et al 2001). Indeed, those women who generated an immune response had a mean survival of 19.9 ± 3.1 months, compared with 5.3 ± 4.3 months in those women who did not develop a response. Efficacy testing is underway, although it is not yet clear whether this strategy will have clinical impact in EOC.

Src

Src, a non-receptor tyrosine kinase, is an attractive therapeutic target in ovarian cancer because it is expressed and activated in the majority of ovarian cancers and regulates a myriad of intracellular signal cascades responsible for critical tumour cell functions, including cell proliferation and differentiation, through extracellular stimulation by growth factors, growth hormones and integrins (Ishizawar and Parsons 2004). Moreover, the emerging role of Src in angiogenesis has recently been noted because of its upregulation of proangiogenic cytokines such as VEGF and interleukin-8 (Summy et al 2005). The ability of VEGF to disrupt endothelial barrier function, which has been correlated with tumour cell extravasation and metastasis, is mediated through Src.

Src has been found to be overexpressed in the majority of late-stage ovarian tumours, as well as a panel of ovarian cancer cell lines (Wiener et al 2003). A study by Han et al in 2006 showed that Src inhibition, through a novel small-molecule inhibitor AP23994, alone and in combination with cytotoxic chemotherapy, significantly reduced tumour growth in vivo in both chemotherapy-sensitive and chemotherapy-resistant preclinical ovarian cancer models. They concluded that Src inhibition might be an attractive therapeutic approach for patients with ovarian carcinoma.

Alpha Folate Receptor

Alpha folate receptor is a membrane-bound protein with high affinity for binding and transporting physiological levels of folate into cells. Folate is a basic component of cell metabolism and DNA synthesis and repair. Rapidly dividing cancer cells have an increased requirement for folate to maintain DNA synthesis; an observation supported by the widespread use of antifolates in cancer chemotherapy. Alpha folate receptor is overexpressed in the majority of EOCs but is largely absent from normal tissue. Overexpression is associated with increased tumour aggressiveness (Toffoli et al 1997). MORAb-003 is a humanized monoclonal antibody against alpha folate receptor. Binding of MORAb-003 to alpha folate receptor blocks phosphorylation, mediates the killing of alpha-folate-receptor-expressing tumour cells, and suppresses tumour growth in xenograft models. A phase I study of single-agent MORAb-003 showed no dose-limiting toxicities and no significant adverse events, and suggested efficacy in platinum-resistant EOC (Bell-McGuinn et al 2008).

A phase II trial of MORAb-003 as monotherapy or in combination with platinum and taxane in women with recurrent, platinum-sensitive EOC was presented at the ASCO meeting in 2008 (Armstrong et al 2008). In subjects receiving MORAb-003 with platinum and taxane, CA125 normalized in 58% of women after two cycles, 88% of women after four cycles and 100% of subjects after six cycles; RECIST scores of measurable disease improved in parallel. This is a significant increase in ORR compared with historical data for platinum/taxane alone. Based on these data, additional studies of MORAb-003 with chemotherapy in platinum-sensitive and platinum-resistant EOC are planned.

Novel Taxanes

Paclitaxel conjugates have an extended half-life compared with paclitaxel, leading to increased exposure of a tumour to the agent. One such agent that has shown activity in both platinum-sensitive and platinum-resistant ovarian cancer is CT2103. A phase II study of CT2103 in 99 heavily pretreated patients with recurrent ovarian, fallopian tube or peritoneal cancer has been carried out. Platinum-sensitive (n = 42) and platinum-refractory/-resistant (n = 57) patients were recruited, 61% of whom had received between three and 12 prior regimens (Sabbatini et al 2004). The response rate was 14% and 7% in platinum-sensitive patients and platinum-resistant patients, respectively, with a median time to progression of 2 months. Common toxicities reported were neuropathy (30% grade two or three) and hypersensitivity (8%).

Epothilones

Like the taxanes, epothilones are microtubule stabilizers and have activity against cancer cell lines which are resistant to paclitaxel and other cytotoxic drugs (Rowinsky 1997). They show activity in taxane-resistant tumours in vivo (Kolman 2005). This is partially due to the fact that they are not substrates for multidrug transporter proteins. Epothilone B (patupilone) has led to disease stabilization and responses in the phase I/II setting in various malignancies including cancer of the ovary, prostate, breast, colon, stomach and kidney (Mani et al 2004). Six epothilones are in early clinical trials for cancer treatment. Dilawari et al (2008) reported an expanded phase I study of ixabepilone 40 mg/m² in patients previously treated with taxanes (n = 44) who had ovarian, endometrial, fallopian tube, cervical, breast or another cancer. A range of one to 14 cycles was given, primarily to examine activity and neurotoxicity. Tumour marker response was seen in one-quarter of patients. Three patients had a PR and two had a minimal response. One-quarter of the patients had grade two to three neuropathy, which was preceded by vibration perception threshold changes. Other toxicities included hypersensitivity (n = 1) and neutropenia (grade three or more in 18 patients). There are also phase I data on the epothilone patupilone (EPO 906), both as a single agent and in combination with other chemotherapeutics, such as carboplatin (Gore et al 2005, Smit et al 2005, Baggstrom et al 2008, Fracasso et al 2008, Reid et al 2008).

Miscellaneous

Trabectedin (Yondelis, ecteinascidin-743) is a tetrahydroisoquinoline alkaloid derived from a Mediterranean marine organism which is able to bind to the minor groove in DNA. It thereby affects transcription factors involved in cell proliferation, blocks cell division in the G(2) phase and inhibits overexpression of the multidrug resistance-1 gene that leads to development of resistance to many anticancer drugs. It also interferes with nucleotide excision repair. Two phase II trials have reported single-agent activity in patients with platinum-sensitive and platinum-refractory recurrent ovarian cancer (Sessa et al 2005, Krasner et al 2007). In the platinum-sensitive group, 62 evaluable patients had an ORR of 29% [95% confidence interval (CI) 18.2–41.9] and median PFS was 5.1 months (95% CI 2.8–6.2). In the platinum-resistant group, out of 79 evaluable patients, the ORR was 6.3% (95% CI 2.1–14.2) and median PFS was 2 months (95% CI 1.7–3.5). The toxicity profile was manageable with 10% of patients having alanine aminotransferase elevation and 8% of patients having neutropenia (Krasner et al 2007). A second phase II study including patients with ovarian cancer resistant to platinum and taxanes (n = 30) or not (n = 29) has been reported (Sessa et al 2005). After two, three-weekly cycles, two PRs were seen and other responses were evident for up to 12.9 months (median 5 months). In the platinum-sensitive group, response rates were 43% (95% CI 23–65). The treatment was well tolerated.

The drug TLK-286 is thought to work by inhibiting glutathione-S-transferaseΠ, a detoxifying mechanism. A phase II study recruited 36 patients with platinum- and paclitaxel-refractory or -resistant ovarian carcinoma (Kavanagh et al 2005). Of the 94% of patients that were evaluable for response, 15% had objective tumour responses, one had a CR and four had a PR. Twelve patients achieved disease stabilization and this was associated with a decrease in CA125 and improvement in performance status. Median survival was 423 days. The compound was well tolerated and phase III studies are now underway.

A study investigating the additive benefit of proteosome inhibition in combination with liposomal doxorubicin in platinum-sensitive (n = 15) and platinum-resistant (n = 15) patients was reported at the ASCO meeting in 2008 (Scambia et al 2008). Preliminary results in the platinum-resistant cohort of patients showed prolonged disease stabilization, and four PRs were seen in the platinum-sensitive cohort. The main adverse events were gastrointestinal (77% grade two or less), neurotoxicity (25% grade two or less) and haematological toxicity (21% grade three or less). The study is still open and complete results are awaited with interest to further define the possible synergy with this combination.

Brostacillin is a drug that forms a covalent interaction with DNA when glutathione is present. Twenty-one platinum-resistant and platinum-refractory patients with EOC were treated with brostacillin in a phase II study (Lorusso et al 2008). Two patients had a PR which lasted over 6 months, one patient achieved disease stabilization for 24 weeks and the rest of the patients progressed. Reported toxicity included haematological toxicity (neutropenia in 71% of patients and thrombocytopenia in 43%) and weight decrease (9.5%). Further studies were felt to be warranted to further investigate the use of brostacillin in this patient group.

Integrins are molecules which are vital in forming cell-to-cell adhesions. Volociximab is an α5β1 integrin antibody that has been tested in patients with relapsed advanced EOC and primary peritoneal carcinoma in the phase I/II setting (Delmonte and Sessa 2008). Three patients were treated with 7 mg/kg qwk and six patients were treated with 15 mg/kg qwk in combination with liposomal doxorubicin until disease progression. Preliminary data suggest that this is a well-tolerated combination, with the main toxicities reported as asthenia (n = 6), nausea (n = 3), abdominal pain (n = 2), flu-like sumptoms (n = 2) and vomiting (n = 1). A phase II study is planned comparing liposomal doxorubicin alone with the combination of liposomal doxorubicin and volociximab.

Farnesyl transferase inhibitors block the activity of the RAS oncoprotein in tumours. BMS-214662 is a farnesyl transferase inhibitor that has been tested in the phase I setting in patients with advanced solid tumours in combination with paclitaxel and carboplatin (Dy et al 2005). Paclitaxel and carboplatin were given three weekly, and escalating doses of BMS-214662 were given on the first day of each cycle. Thirty patients were treated, and no pharmacokinetic interactions were seen between the three drugs. One patient with endometrial cancer and one patient with ovarian cancer had a PR. Disease stabilization was achieved in eight other patients. The combination was well tolerated with dose-limiting toxicities including neutropenia, thrombocytopenia, nausea and vomiting. A further phase I study tested BMS-214662 in combination with paclitaxel alone in patients with advanced solid tumours. Patients were treated (n = 26) with weekly paclitaxel and escalating doses of BMS-214662, and up to 94 courses of treatment (Bailey et al 2007). All the pretreated ovarian cancer patients had a PR, and pharmacokinetic studies showed no interaction with paclitaxel. Peripheral blood lymphocytes (PBLs) were taken to test for significant farnesyl transferase inhibition (>80%), which was seen at the end of the BMS-214662 infusion. Dose-limiting toxicities included grade four neutropenia, rises in serum transaminases and grade three diarrhoea.

Histone deacetylase (HDAC) inhibitors block the deacetylation of histones, a mechanism by which the regulation of gene expression is mediated. Two phase II studies were reported at the ASCO meeting in 2008 detailing results of HDAC inhibition in platinum-resistant epithelial ovarian tumours. The first study used belinostat (PXD101) in platinum-resistant patients (n = 18, group 1) or patients with micropapillary/borderline ovarian tumours (n = 12, group 2) (Mackay et al 2008). Tumour biopsies and PBLs were taken prior to treatment, and on the fourth day (out of 5 days) of treatment. Of the patients in group 1, nine patients achieved disease stabilization and six progressed. Of the patients in group 2, one patient had a PR, nine achieved disease stabilization and one patient had a CA125 response. Grade three adverse events included thrombosis (n = 3), dyspnoea (n = 2), fatigue (n = 2), elevated alkaline phosphatase (n = 2) and nausea (n = 2). The second study also tested belinostat but in combination with carboplatin and paclitaxel in platinum-resistant and platinum-sensitive patients. Eleven patients relapsed within 6 months of first platinum therapy. The ORR was 31%, which included one CR and 10 PRs. Sixteen patients achieved disease stabilization. Common grade three to four adverse events were neutropenia (n = 4), transaminitis (n = 4) and fatigue (n = 3). Recruitment to both studies is still ongoing.

Conclusion

The treatment of gynaecological malignancies in the future will no doubt make use of our increased understanding of the biology of these diseases. Whether the novel agents discussed above are used alone, in combination with each other or with other chemotherapeutics remains to be defined. The exact benefit of many of these agents in terms of response rates and disease-free survival is also largely unknown. In addition, it is uncertain whether they will simply be unaffordable in most health economies. However, this is certainly an exciting time in the field of gynaecological oncology, and it is hoped that the use of targeted agents will have a significant impact on disease progression and outcome in the near future.