Development of novel agents for bladder cancer

u p d a t e o n c a n c e r t h e r a p e u t i c s 3 ( 2 0 0 9 ) 160–169

available at www.sciencedirect.com

journal homepage: www.updateoncancer.com

Development of novel agents for bladder cancer Noah M. Hahn a,∗ , Thomas Powles b , Christopher J. Sweeney a,c a

Department of Medicine, Indiana University School of Medicine, Indiana University Melvin and Bren Simon Cancer Center, Indianapolis, IN, United States b Saint Bartholomew’s Hospital, London, London, United Kingdom c University of Adelaide, Royal Adelaide Hospital, Adelaide, Australia

a r t i c l e

i n f o

a b s t r a c t

Keywords:

Traditional treatment options for bladder cancer include transurethral resection and intrav-

Bladder Cancer

esical Bacillus Calmette Guerin for early stage disease and cystectomy or radiation therapy

Novel Agents

(with or without chemotherapy) for muscle-invasive disease. Platinum-based chemother-

Chemotherapy

apy improves patient outcomes in both the perioperative and metastatic setting. Despite an

Angiogenesis

increase in new therapeutic options over the past decade for many cancer patients, simi-

Epigenetics

lar advances in bladder cancer are limited. In recent years, an improved understanding of

HER-2

the molecular forces driving bladder cancer development and progression has unfolded.

EGFR

These discoveries create a set of innovative therapeutic opportunities in bladder cancer.

NF␬B

This review examines novel anti-cancer agents currently in clinical trials with preclini-

PI3 kinase

cal rationale to support evaluation in bladder cancer. In addition, strategies to match a

Clinical Trial Design

patient’s tumor to the most appropriate agent are discussed. This may provide a more rational approach to evaluating the role of emerging anti-cancer agents in bladder cancer. © 2009 Elsevier Ltd. All rights reserved.

1.

Background

In the United States, the urinary bladder is the fifth most common site of new human cancer diagnoses. In 2008, over 68,000 individuals were diagnosed with bladder cancer (BC), and more than 14,000 patients died from their disease, with urothelial carcinoma (transitional cell carcinoma – TCC) as the dominant histological pattern in over 90% of cases [1]. This review will concentrate on novel therapeutics relevant to the treatment of urothelial carcinoma. Standard therapy options for early stage disease include transurethral resection of bladder tumor (TURBT) often in combination with intravesical administration of Bacillus Calmette Guerin (BCG) or chemotherapeutic agents such as mitomycin-C and gemcitabine. In high-risk patients, pro-

gression to muscle-invasive disease requiring cystectomy or radiation therapy (with or without chemotherapy) occurs in 40% of individuals. Despite treatment with platinum-based chemotherapy, death due to BC occurs in 20–30% of all individuals diagnosed with BC [2–5]. Consequently, an urgent need exists to develop improved therapeutic agents for BC patients. In the past decade, multiple novel targeted therapeutic agents have been developed to augment and/or replace traditional cytotoxic chemotherapy regimens for patients with many types of cancer. Examples of these new classes of drugs include: modern cytotoxic agents, anti-angiogenesis agents as well as inhibitors of the human epidermal growth factor receptor (HER) family, nuclear factor kappa B (NF␬B) and phosphatidylinositol-3 kinase (PI3K) pathways, and epigenetic modifiers.

∗ Corresponding author at: Indiana University Cancer Center, Indiana Cancer Pavilion Room RT445, 535 Barnhill Drive, Indianapolis, IN 46202, United States. Tel.: +1 317 278 6871; fax: +1 317 274 3684. E-mail address: [email protected] (N.M. Hahn). 1872-115X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.uct.2009.04.001

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2.

Modern cytotoxic agents

In the 1980s, cytotoxic cisplatin-based combination chemotherapy regimens demonstrated improved overall survival compared to non-platinum and single-agent regimens [6–8]. Of these regimens, the combination of methotrexate, vinblastine, adriamycin and cisplatin (MVAC) established itself as the standard of care. In the 1990s the combination of gemcitabine and cisplatin (GC) became the new standard by demonstrating similar overall survival with an improved toxicity profile when compared to MVAC [9,10]. Subsequent efforts to evaluate new cytotoxic agents have predominantly added an agent to the GC back-bone or trialed new agents in the second-line setting. In recent years, modern cytotoxic agents have shown only modest activity in the treatment of bladder cancer.

3. Recent trials of modern cytotoxic agents in bladder cancer Clinical trial results of some of the modern cytotoxic agents in the treatment of bladder cancer are summarized in Table 1. The data in this table and the following discussion gives an overview of the efficacy and toxicity profile of cytotoxic drug development in this setting and thus a framework to design future trials. Vinflunine is a fourth generation fluorinated vinca-alkaloid which exerts its anti-neoplastic effects through stabilization of the microtubule complex [11]. In preclinical murine bladder cancer models, vinflunine demonstrated superior clinical efficacy compared to vinorelbine [12]. In a phase II study in 51 metastatic bladder cancer patients previously treated with a platinum-containing regimen, vinflunine delivered at 320 mg/m2 intravenously (iv) day 1 on a once every three-week schedule produced partial tumor responses in 18% of patients and stable disease in 67% of patients [13]. Median progression-free and overall survivals were 3.0 and 6.6 months. Toxicity was primarily hematologic with 10% of patients experiencing neutropenic fever and two-treatment related deaths. In a larger phase II trial of 150 post-platinum metastatic bladder cancer patients, vinflunine produced similar efficacy, toxicity, and survival results [14]. In a recent phase III study of vinflunine versus best supportive care in 370 previously platinum-treated metastatic bladder cancer patients,

vinflunine failed to show significant improvement in overall survival by intention-to-treat analysis [15]. The median overall survival data of 6.9 months on the vinflunine arm provides a useful benchmark for future second-line trials. Pemetrexed is a multi-targeted anti-folate agent with demonstrated activity in non-small cell lung carcinoma and malignant mesothelioma [16,17]. Pemetrexed is transported into malignant cells through membrane associated carriers and ion channels. Once within the cell, it is polyglutaminated to its active form and exerts its anti-neoplastic effect through potent inhibition of dihydrofolate reductase (DHFR), thymidylate synthase (TS), and glycineamide ribonucleotide transformylase (GARFT) [18,19]. In single-agent phase II studies in bladder cancer patients with progressive disease after prior platinum-based chemotherapy, pemetrexed has demonstrated clinical activity. Sweeney et al. treated 47 patients with pemetrexed on a once every three-week schedule at a dose of 500 mg/m2 iv day 1 with appropriate vitamin supplementation [20]. In this pretreated population, a 27% response rate was observed (6% complete response (CR), 21% partial response (PR)) with a median overall survival of 9.6 months. Toxicity was minimal with less than 10% grade 3 or 4 hematologic toxicity and under 3% non-hematologic grade 3 or 4 toxicity. In contrast, Galsky et al. observed a response rate of 8% in 13 similar post-platinum bladder cancer patients treated with an identical dose and schedule of pemetrexed [21]. This study was halted at the interim analysis as it did not meet the prespecified requisite response rate to continue into the second stage. Therapy was well tolerated with rare non-hematologic toxicity. Pemetrexed has also been studied in the first-line treatment of metastatic bladder cancer patients in combination with gemcitabine therapy [22]. In a phase II study in 64 chemonaïve patients with metastatic transitional cell carcinoma of the bladder, pemetrexed delivered at 500 mg/m2 iv day 1 and gemcitabine at 1250 mg/m2 iv days 1 and 8 on a once every three-week schedule resulted in partial and complete response rates of 16% and 5%, respectively. Median overall survival was 10.3 months. Combination treatment was associated with increased toxicity compared to single-agent pemetrexed therapy with grade 3 or 4 febrile neutropenia, transaminase elevation, dyspnea, and stomatitis in 17%, 12%, 8% and 5% of patients, respectively. Similar results with this combination were reported in a phase II Eastern Cooperative Oncology Group trial [23]. This regimen has not been taken forward

Table 1 – Clinical trial results with modern cytotoxic agents. Agent

Ref

Pemetrexed Pemetrexed Pemetrexed + gemcitabine Pemetrexed + gemcitabine Ixabepilione Vinflunine Vinflunine Vinflunine

[20] [21] [22] [23] [27] [13] [14] [15]

Population 2nd line 2nd line 1st line 1st line 2nd line 2nd line 2nd line 2nd line

N 47 13 64 46 42 51 150 370

Hem toxicity (%) <10 20 38 41 36 10 58 50

Non-hem toxicity (%)

RR (%)

PFS

OS

3 23 12 11 36 8 17 28

27 8 28 28 12 18 15 9

2.9 NR 3.1 NR 2.7 3.0 2.8 3.0

9.6 NR 8.1 NR 8.0 6.6 7.9 6.9

Ref, references; N, number of patients; Hem, hematologic; Non-hem, non-hematologic; RR, response rate; PFS, median progression-free survival in months; OS, median overall survival in months.

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Table 2 – Clinical trial results of anti-angiogenic agents in bladder cancer. Agent

Ref

Sunitinib Sunitinib Sorafenib Sorafenib

[31] [32] [44] [43]

Population 1st line 2nd line 1st line 2nd line

N

Hem toxicity (%)

17 45 17 22

20 18 NR NR

Non-hem toxicity (%)

RR (%)

PFS

OS

14 7 0 0

5.9 2.4 1.8 2.2

NR 6.9 NR 6.8

6 7 NR 19

Ref, references; N, number of patients; Hem, hematologic; Non-hem, non-hematologic; RR, response rate; PFS, median progression-free survival in months; OS, median overall survival in months.

based on the impression it was neither less toxic or more active than cisplatin-based therapy. Ixabepilone is a semi-synthetic epothilone which exerts its anti-neoplastic effects through disruption of the microtubule assembly process [24]. Ixabepilone binds to tubulin in a distinctly separate manner from taxanes, thereby offering promise for reduced cross-resistance [25,26]. In a phase II study in metastatic bladder cancer patients who had received prior treatment with a platinum-containing chemotherapy regimen, ixabepilone was administered at 40 mg/m2 iv day 1 on a once every three-week schedule [27]. Of 42 evaluable patients, partial responses were seen in 5 patients (12%). Median progression-free survival and overall survival were 2.7 and 8 months, respectively. Treatment was associated with grade 4 or higher toxicity in 27% of patients with one treatment related death due to neutropenic sepsis. Due to its modest activity and considerable toxicity, ixabepilone has not been studied further in bladder cancer patients.

4. Anti-angiogenesis agents in bladder cancer As with most solid malignancies, angiogenesis is an attractive target in BC [28–30]. Microvessel density and high vascular endothelial growth factor (VEGF) levels are both associated with a poor prognosis in this disease. This has resulted in the investigation of both monoclonal antibodies and VEGF tyrosine kinase inhibitors (TKI) in this setting. Sunitinib is a multi-targeted VEGF TKI. It has been investigated in two-phase II studies in unselected patients with metastatic BC [31,32]. A Spanish group reported interim analysis results from 17 patients deemed unfit for platinum-based chemotherapy due to impaired renal function who received single-agent sunitinib as first-line therapy. A partial response rate of 14% was observed; no complete responses were seen. However, median progression-free survival was respectable at 5.9 months indicating that this may be a useful agent [31]. A second study administered single-agent sunitinib as second-line BC therapy. Response rates were low, with a

progression-free survival of 2.4 months and an overall survival of 6.9 months. These results are in line with the results observed with other second-line chemotherapy agents in this setting and should not discourage further investigation of this drug [15,20,21,33]. A number of studies investigating sunitinib, in combination with chemotherapy in the metastatic and neoadjuvant setting, are planned. One trial of particular note is a randomized phase II study from the University of Michigan, which investigates maintenance sunitinib therapy after the completion of chemotherapy in this disease. It is hoped that this will result in a delay in time to progression, which may be the optimal endpoint for these drugs. The results are awaited [34]. Sorafenib and bevacizumab also target angiogenesis and are effective agents in other tumor types [35–40]. Sorafenib is a multi-targeted VEGF TKI which also inhibits RAF signaling, while bevacizumab is a monoclonal antibody targeting VEGF [41,42]. As a single agent, sorafenib failed to demonstrate any significant clinical activity, however, both agents are being investigated in combination with chemotherapy [43,44]. Results of clinical experience with anti-angiogenic agents in BC are summarized in Table 2. Three ongoing studies of note in metastatic BC patients are highlighted in Table 3. These include: the European Organisation for Research and Treatment of Cancer (EORTC) randomized study investigating gemicitabine and cisplatin with or without sorafenib, the Hoosier Oncology Group (HOG) trial investigating the combination of cisplatin, gemcitabine, and bevacizumab, and the Cancer and Leukemia Group B (CALGB) study investigating the combination of gemicitabine, cisplatin, and bevacizumab. Studies with translational endpoints identifying markers associated with response to therapy are urgently required in this area.

5.

Epigenetic agents

Epigenetics refers to inheritable changes in gene function that cannot be explained by DNA sequence changes [45]. Two common epigenetic mechanisms which affect gene function

Table 3 – Important ongoing trials of anti-angiogenic agents in bladder cancer. Organization EORTC HOG CALGB

Agents

Population

Phase

Cisplatin + gemcitabine ± sorafenib Cisplatin + gemcitabine + bevacizumab Cisplatin + gemcitabine ± bevacizumab

1st line mTCC 1st line mTCC 1st line mTCC

II/III II III

mTCC, metastatic transitional cell carcinoma.

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Table 4 – Epigenetic agents in development relevant to bladder cancer. Agent SAHA MGCD0103 5-Azacitidine Decitabine

Mechanism of action Pan-HDACi Selective-HDACi Promoter hypomethylation Promoter hypomethylation

Route of delivery PO PO IV/SQ IV

Disease approved

Bladder development phase

CTCL None MDS MDS

Phase II Preclinical Preclinical Preclinical

SAHA, suberoylanilide hydroxamic acid; HDACi, histone deacetylase inhibitor; PO, per oral; IV, intravenous; SQ, subcutaneous; CTCL, cutaneous T-cell lymphoma; MDS, Myelodysplastic Syndrome).

include hypermethylation of cytosine–guanine (CpG)-island rich gene promoters and histone deacetylation. Multiple investigators have demonstrated the importance of these two epigenetic processes in BC cell cycle control, cellular adhesion, apoptosis, and tumor suppressor functions [30,46–48]. Therapeutic agents aimed at these epigenetic targets are now in clinical development for the treatment of bladder cancer. Suberoylanilide hydroxamic acid (SAHA, vorinostat) is an orally bioavailable pan-histone deacetylase inhibitor (HDACi) recently approved for the treatment of cutaneous T-cell lymphoma [49]. In preclinical studies utilizing the T24 bladder cancer cell line, SAHA demonstrated a 9-fold increase in WAF1 (p21) expression that was directly correlated with increased histone acetylation and decreased proliferation [50]. In a recent human phase II study in 14 bladder cancer patients with advanced disease progressing after platinumbased chemotherapy, continuous treatment with SAHA at 200 mg twice daily produced stabilization of disease in 3 patients (21%), with no complete or partial responses [51]. Treatment was associated with grade 4/5 myelosuppression in 36% of patients. Further study at this dose in an advanced bladder cancer population was not recommended. In contrast to the pan-HDACi SAHA, MGCD0103 is an oral HDACi selective for the class I HDAC isoforms 1, 2, 3, and 11. In a preclinical study of the T24 human bladder cancer cell line, MGCD0103 demonstrated significant dose-dependent induction of histone acetylation [52,53]. Moreover, MGCD0103 demonstrated a three-fold greater reduction in T24 bladder cancer cell line proliferation, as compared to SAHA, with respective IC50 values of 0.66 ␮M (MGCD0103) and 2.3 ␮M (for SAHA). Re-expression of WAF1 (p21) and induction of apoptosis also occurred in a MGCD0103-dose-dependent fashion [53]. In a phase I study in patients with advanced solid organ tumors resistant to standard therapies, MGCD0103 demonstrated stabilization of disease lasting up to 11 months in 5 of 38 patients with a tolerable safety profile [54]. While currently under development for hematologic malignancies, MGCD0103 has promising preclinical bladder cancer activity worthy of further human study. 5-Azacitidine (AzaC) is a non-methylatable cytidine analog that is incorporated into DNA during S-phase resulting in the loss of DNA methylation and the reactivation of previously silenced genes [55]. At high doses, AzaC demonstrates cytotoxicity, while low AzaC doses elicit DNA hypomethylation and cellular differentiation [56–58]. In preclinical cancer studies, multiple investigators have shown the ability of AzaC to significantly reduce methylation of CpG promoter regions and restore expression and activity of silenced tumor suppressor genes [56–58].

Decitabine (DAC) is a structurally similar cytidine analog with hypomethylating effects [59]. In T24 bladder cancer cell line in-vitro experiments, DAC demonstrated a reduction from 93% to 56% in CDKN2A (p16) promoter DNA hypermethylation resulting in a significant increase in CDKN2A (p16) expression. Furthermore, when DAC-treated T24 cells were implanted into nude mice, mean tumor size at four weeks was nearly 10fold smaller (0.14 mm3 ) than implanted tumors derived from untreated T24 cells (1.5 mm3 ) [60]. No human bladder cancer clinical trials have been completed with either AzaC or DAC, however, multiple phase I trials in combination with conventional chemotherapy and HDACi’s are underway. A summary of epigenetic agents potentially relevant to bladder cancer is presented in Table 4.

6.

HER signaling pathway

The HER family of receptors are involved in activation of signal transduction pathways which affect cell survival, metastasis, and angiogenesis [61]. There are four specific receptors (HER-1–4). HER-1 (epidermal growth factor receptor [EGFR]) and HER-2 have been most closely studied and have produced ground-breaking drug developments in other tumor types. Less is known about the role of HER-3 and HER-4. Effective drugs targeting these receptors are not yet widely available. Both EGFR and HER-2 are over-expressed in bladder cancer and are associated with a poor prognosis [62–64]. Therefore, agents targeting EGFR and HER-2 have been investigated in BC. Unfortunately, the results have been difficult to interpret due to heterogeneity between trials including variations in patient populations and trial designs. Moreover, these studies have relied on radiological response criteria (RECIST) which appears to be a poor surrogate endpoint for these agents in other tumors [65,66]. For example, in the erlotinib versus best supportive care second/third line trial in non-small cell lung carcinoma, an overall survival advantage was seen with this EGFR TKI with only an 8.9% response rate by standard RECIST criteria [67]. Additionally, EGFR targeted therapy has often been given in combination with chemotherapy in single arm studies, making it difficult to determine the exact efficacy of the additional investigational drug [68,69]. EGFR is over-expressed in almost 50% of transitional cell bladder tumors and is associated with a poor prognosis [63,64,66]. Unlike other tumors, mutations of the kinase domain of EGFR or gene amplifications of EGFR are rare [70]. Initial in-vitro work suggested the EGFR TKI gefitinib interacted synergistically with chemotherapy [71]. However, in a single arm phase II study in chemotherapy naive BC patients

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Table 5 – Clinical trial results of HER-1 (EGFR) and HER-2 targeted agents in bladder cancer. Agent

Ref

Population

N

Hem toxicity (%)

Cisplatin + gemcitabine + gefitinib Carboplatin + gemcitabine + paclitaxel + trastuzumab Lapatinib

[69] [68]

1st line 1st line

55 57

20 75

[66]

2nd line

59

0

Non-hem toxicity (%)

RR (%)

PFS

OS

25 23

51 70

8.0 9.3

14.4 14.1

7

2

2.0

4.1

Ref, references; N, number of patients; Hem, hematologic; Non-hem, non-hematologic; RR, response rate; PFS, median progression-free survival in months; OS, median overall survival in months.

treated with gefitinib, cisplatin, and gemcitabine, an observed response rate of 51%, progression-free survival of 8 months, and an overall survival of 14 months did not appear better than historical controls of the chemotherapy alone [69]. Cetuximab is an EGFR targeted monoclonal antibody which is also being investigated in combination with chemotherapy in two randomized phase II studies in metastatic BC patients. Results are awaited. HER-2 receptors are also over-expressed by immunohistochemistry in approximately 50% of patients with metastatic BC [62,66]. Its over-expression is also associated with a poor prognosis. As with EGFR, gene amplification of the HER-2 receptor is a rare event in bladder cancer [72]. Trastuzumab is a HER-2 targeted monoclonal antibody which has demonstrated marked improvement in overall survival in HER-2 expressing breast cancer patients in both the metastatic and adjuvant settings when combined with cytotoxic chemotherapy [73–75]. A recent trial examined the combination of paclitaxel, carboplatin, gemcitabine, and trastuzumab in untreated HER-2 positive metastatic BC patients [68]. The combination was tolerable; although 22% experienced grades 1–3 cardiac toxicity and three-treatment related deaths occurred. Seventy percent of patients responded to therapy. Median time to progression and overall survival were 9.3 and 14.1 months, respectively. These results do not appear superior to cisplatin and gemicitabine therapy but are difficult to interpret due to the lack of randomization and the high response rate expected from triplet cytotoxic chemotherapy. Lapatinib is a specific EGFR and HER-2 targeted TKI. Over two thirds of patients with metastatic BC over-express one or both of these receptors [66]. Lapatinib has been investigated as a single agent in unselected patients as second-line therapy in a single-arm study in metastatic BC [66]. The response rates were disappointing; however, subset analysis suggested a survival benefit for patients over-expressing EGFR or HER2. Therefore, lapatinib continues to be investigated in BC, and two studies are ongoing in the metastatic setting. These include a phase I/II EORTC study investigating the combina-

tion of gemicitabine, cisplatin, and lapatinib and a randomized phase III study from the United Kingdom comparing maintenance lapatinib versus placebo after first-line chemotherapy in patients with metastatic EGFR or HER-2 expressing cancers with stable disease or better after 4–6 cycles of chemotherapy. This sequential use of chemotherapy and lapatinib is supported by preclinical data in this area [76]. Clinical trial experiences with HER-targeted agents and key ongoing clinical trials are summarized in Tables 5 and 6, respectively.

7.

NF␬B and PI3K pathways

As our understanding of biological processes that drive carcinogenesis increases, so does our ability to develop new therapies to inhibit bladder cancer development and progression. Two very interesting biological pathways which are emerging as being the focus of drug development efforts are nuclear factor kappa B and PI3 kinase.

7.1.

Nuclear factor kappa B and bladder cancer

The NF␬B superfamily of eukaryotic transcription factors plays an important role in carcinogenesis. NF␬B and its regulators orchestrate a wide variety of signal transduction pathways that mediate invasion, angiogenesis, organ specific homing of metastatic cells, evasion of apoptosis and chemotherapy resistance [77,78]. NF␬B is a heterodimeric complex of Rel family proteins which remains bound to its inhibitor I␬B in an inactive form in the cytoplasm. More than 150 extra-cellular signals can lead to activation of NF␬B through activation of I␬B kinase [79]. Extra-cellular stimuli including tumor necrosis factor alpha (TNF␣), interleukin-1 (IL-1), viral infections, phorbol esters, oxidants, chemotherapy and radiation lead to I␬B degradation and subsequent nuclear translocation of NF␬B. In normal cells NF␬B is tightly regulated, whereas it is constitutively active in many malignancies including prostate cancer, leukemia, breast and pancreatic cancer [80–85]. When translo-

Table 6 – Important ongoing trials of HER-pathway targeted agents in bladder cancer. Organization UMCC FCCC EORTC UK

Agents

Population

Phase

Cisplatin + gemcitabine ± cetuximab Cetuximab ± paclitaxel Cisplatin + gemcitabine + lapatinib Cytotoxic chemotherapy ± maintenance lapatinib

1st line mTCC 2nd line mTCC 1st line mTCC 1st line mTCC

II II I/II III

UMCC, University of Michigan Cancer Center; FCCC, Fox Chase Cancer Center; UK, United Kingdom; mTCC, metastatic transitional cell carcinoma.

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cated to the nucleus, NF␬B binds to specific promoters and initiates transcription of several anti-apoptotic genes such as Bcl2, BclxL, Bfl1/A1, cFLIP, XIAP, TRAF-1, TRAF-2, cIAP-1, cIAP-2 and MnSOD [78]. NF␬B has also been shown to inhibit tumor suppressors such as, Inhibitor of Growth 4 (ING4) and p53. NF␬B also regulates several angiogenic factors including VEGF and interleukin-8 (IL-8) [86]. This is relevant to the cancer process as the metastatic potential of tumor cells correlates with expression of angiogenic genes such as VEGF, basic fibroblast growth factor (bFGF), IL-8 and type IV collagenases [87–90]. These factors are produced and secreted by tumor cells, and thus, play a significant role in tumorigenesis and metastasis. NF␬B inhibition is, therefore, a bone fide strategy for anti-cancer drug development. Moreover, the significance of NF␬B inhibition is underscored by the observation that insertion of mutated I␬B into cancer cell lines results in super-repression of NF␬B and consequent decreased VEGF and IL-8 expression as well as decreased in vivo growth and angiogenesis of both ovarian cancer and melanoma cell lines [91]. Given the involvement of NF␬B in so many cancer related pathways, efforts have been directed to developing inhibitors of I kappa B kinases (IKK) and IKK-related kinases [92]. The anti-cancer activity of bortezomib is thought to, in part, be mediated by inhibition of NF␬B [93]. However, the anti-cancer activity of bortezomib as a single agent is most profound in myeloma and it is thought single-agent activity may be limited to diseases that may be most dependent on NF␬B [93,94]. Preclinical work evaluating the role of NF␬B in bladder cancer has been done. For example, p65/RelA nuclear expression has been correlated with c-FLIP and bcl-2 expression, higher grade disease and poor survival in both superficial and muscle-invasive carcinomas [95]. Furthermore, a germline polymorphism (−1186 T → G) in the NF-␬B binding promoter region of COX-2 was found to be associated with an increased risk of bladder cancer in a hospital based controlled study of 217 Korean individuals [96]. Further evidence supporting the potential for targeting NF␬B in bladder cancer can be gleamed from in-vivo studies of anti-cancer agents which have activity in bladder cancer cell lines by inhibiting NF␬B [97–99].

7.2.

PI3 kinase and bladder cancer

The phosphatidylinositol-3 kinase pathway is centrally located in many signaling processes and has been shown to play a key role in the processes that are vital to cancer cell proliferation, survival, invasion and metastases. PI3K is an attractive cancer target for a variety of reasons including: (a) it is very frequently mutated and displays aberrant pathology in cancer; (b) inhibition of PI3K sensitizes cancer cells to chemotherapy, radiation, and other targeted therapies; and (c) resistance to many commonly used cytotoxic and cytostatic therapies is mediated via an aberrant PI3K pathway [100,101]. As detailed in many reviews including one by Powis and colleagues, PI3K is activated by a number of mechanisms, including growth factor receptors, integrins, and Ras. PI3K converts phosphatidylinositols to phosphatidylinositol3-phosphates, which in turn activates AKT (thymoma in AKR mouse/protein kinase B). Activation of AKT is blocked by the tumor suppressor phosphatase and tensin homologue (PTEN). AKT then activates a multitude of downstream tar-

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gets, including focal adhesion kinase (FAK), inducible nitric oxide synthase (iNOS), and I␬kinase (IKK) and thus NF␬B, Bcl-xl (B-cell lymphoma mutant, extra long), glycogen synthase kinase-3 (GSK-3) and others [102]. Excess activity of the PI3K/AKT pathway has been seen in many cancers by virtue of activating mutations of PI3K and AKT as well as by loss of the tumor suppressor/regulator PTEN. Inhibitors of both PI3K and AKT have entered the clinical trial setting and may have a role in bladder cancer. Preclinical work supporting the evaluation of targeting this pathway in bladder cancer has also been performed. As with breast, prostate and lung cancer, activation of the PI3K pathway in bladder cancer has been associated with poor clinical outcomes including survival [103]. Other investigators have observed in-vitro that PI3K activation in bladder cancer cells makes them resistant to EGFR inhibition while others have shown PI3K inhibition blocks the ability of bladder cancer cells to invade and that primary bladder cancer tumors from patients often have high AKT phosphorylation [104,105]. As with other cell lines, inhibition of PI3K in bladder cancer cell lines augments the activity of anti-cancer therapies such as radiation therapy [106].

8. Predicting bladder cancer clinical activity with correlative studies Due to the ever-increasing number of novel agents under clinical development for the treatment of bladder cancer, novel trial design considerations are needed to most efficiently and reliably bring these agents forward from preclinical speculative observations to ultimate proof of principle drug approval. Mechanisms to predict drug sensitivity and, thereby, select patient populations with high probabilities of clinical success are under development. With traditional cisplatin-based therapy, bladder tumor ERCC1 gene expression profiles have demonstrated the ability to segregate otherwise homogenous patient cohorts into good and poor-prognosis groups. In a prospective phase II study of 57 metastatic bladder cancer patients treated with cisplatin, gemcitabine, and/or paclitaxel, tumor messenger RNA (mRNA) expression levels of BRCA1, ERCC1, and RRM1 were evaluated for their associations with progression-free and overall survival [107]. Patients with low bladder tumor ERCC1 mRNA expression profiles demonstrated a marked improvement in overall survival (25.5 vs. 15.4 months, p = 0.030) compared to patients with high ERCC1 mRNA expression. As this is not data from a randomized trial, it is unknown at this stage if this is a predictive marker for cisplatin sensitivity. At the very least, the data supports tumor ERCC1 mRNA expression as prognostic. A recent powerful tool to predict bladder cancer drug sensitivity a priori rather than by retrospective biomarker analysis is the coexpression extrapolation (COXEN) algorithm [108]. Through sophisticated informatics and biostatistical methodologies, COXEN leverages the existing public National Cancer Institute (NCI) Developmental Therapeutics Program drug sensitivity NCI-60 cell line data with Affymetrix® gene expression profiles and clinicopathologic human data from patients with tumors not contained in the NCI-60 cell lines to produce a COXEN score predictive of response to existing drugs. The NCI-60 cell lines have been tested for sensitivity

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Fig. 1 – Novel future bladder cancer clinical trial design considerations.

to over 100,000 compounds. By eliminating the need to repeat time-consuming and costly preclinical efficacy experiments, COXEN presents an exciting tool for clinical investigators to rapidly screen and select drugs for bladder cancer clinical development. The power of this drug development algorithm is maximally leveraged in the neoadjuvant setting by providing for rapid correlation of biomarkers with pathologic and molecular responses. Together, tumor gene expression profiles such as ERCC1 and tumor COXEN scores present clinicians with novel strategies in which biology driven treatment selection may now be possible. A model for future metastatic bladder cancer trial design based on predictive correlative studies is highlighted in Fig. 1.

9.

[6]

[7]

[8]

Conclusion

The number of novel therapeutic compounds available for clinical development for the treatment of bladder cancer is at an unprecedented level. Translation of promising preclinical data into improved patient outcomes will require sophisticated clinical trial design with an emphasis on selection of patients with cancers dependent on pathways blocked by the drug of interest. It is our charge as clinicians to deploy these drugs and tools in the most expeditious manner possible into the clinical trials setting and, in so doing, bring advances to routine clinical care of patients with bladder cancer in the shortest time possible.

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