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British Medical Bulletin 64:227-254 (2002)
© 2002 The British Council
Therapeutics targeting signal transduction for patients with colorectal carcinoma
Institute for Drug Development, Cancer Therapy and Research Center, and University of Texas Health Science Center at San Antonio, Texas, USA
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Abstract |
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Abstract
IntroductionThe cytotoxics developed for the treatment of patients with advanced colorectal cancer have yielded diminishing returns. Agents aimed at novel molecular targets are required to improve the prognosis of this disease. This review describes the most recent advances in the clinical development of therapies designed to block the function of several important signalling cellular proteins. Therapies discussed include agents targeting: (i) the epidermal growth factor receptor (EGFR) family; (ii) Ras via the inhibition of farnesyltransferase; (iii) Raf kinase; (iv) the mitogen-activated protein kinase pathway (MAPK, MEK, Erk); (v) Akt; and (vi) the apoptosis signalling pathways including NF-
B, Bcl-2 and the TRAIL receptor. The results of clinical trials of the first generation of such therapeutics to enter clinical evaluation in malignant diseases are presented. Potential advantages and disadvantages of these different therapeutic modalities are discussed and future challenges for the evaluation of these targeted agents in the clinic is presented. |
Introduction |
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While there have been improvements in the outcome of patients with colorectal cancer due to the use of ‘non-selective’ cytotoxic chemotherapeutics such as the fluoropyrimidines and the topoisomerase I inhibitors, the overall clinical impact of these therapeutics has been modest. The discovery of a plethora of subcellular targets and the rational generation of selective targeting agents has opened an era of new opportunities and extraordinary challenges. The specificity of these agents renders them capable of specifically targeting the inherent abnormalities of cancer cells, potentially resulting in less toxicity than traditional non-selective cytotoxics. Among the many new types of rationally designed agents are therapeutics targeting various strategic facets of growth signal transduction, malignant angiogenesis, survival, metastasis, and cell-cycle regulation. The evaluation of these agents is likely to require a radical departure from the traditional drug development paradigms in order to realize their full potential. It remains difficult, however, to predict which targets and associated therapeutic candidates will yield the most promising results in the treatment of colorectal cancer. This chapter will initially focus on novel therapeutics that disrupt the transduction of growth stimulatory signals. Growth factor signalling plays important paracrine and autocrine roles in modulating tumour cell proliferation and apoptosis. The epidermal growth factor (EGF) family of receptors (ErbB receptors) has been implicated in the pathogenesis and prognosis of colorectal carcinoma. Agents targeting these receptors, and their downstream signalling pathways which include Ras, Raf kinase, and the mitogen activated protein kinases (MEKK, MEK and Erk), are now being evaluated in the clinic (Fig. 1). Agents targeting these serial components of the growth factor receptor-Ras-Raf-MAPK (mitogen activated protein kinase) signalling pathway will be discussed in turn (Fig. 2). Furthermore, agents that promote the activation of the apoptosis pathway will also be discussed, including agents targeting bcl-2, Akt, NF-
B and the TRAIL receptor.
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Therapeutics targeting the epidermal growth factor receptor (EGFR) |
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EGFRs are integral components of the principal signalling cascade involved in regulating solid tumour growth. EGFR blockade is, therefore, a rational therapeutic approach to treating colorectal and other epidermoid malignancies, thereby inhibiting cancer cell proliferation and tumour progression1.
Anti-EGFR antibodies
Monoclonal antibodies to EGFR were initially generated by immunizing mice to raise monoclonal antibodies to the human receptor2–4. Murine antibodies, such as Mab 225 (IgG2a), could block ligand binding to the EGFR and inhibit EGF-stimulated EGFR tyrosine kinase activity, binding to the receptor with affinity comparable to its natural ligand TGF-
(Kd = 2 x 10-9 M). These antibodies prevented functional ligand binding and caused receptor dimerization and internalization, inhibiting tyrosine kinase-dependent phosphorylation, down-regulating EGFR expression, and blocking the EGFR signalling cascade. These antibodies inhibited EGF and TGF--induced anchorage dependent and independent growth of EGFR expressing cells in vitro. Anti-EGFR monoclonal antibodies have, however, often been demonstrated to be more effective in vivo than in vitro, possibly due to anti-angiogenic activity and/or enhancement of immune effector activity. in vivo, these antibodies delay tumour growth, induce tumour regression, enhance the activity of cytotoxic chemotherapy, and reduce metastases in animal models.
Chimeric monoclonal antibodies
To overcome the development of a human anti-mouse antibody (HAMA) immune response against murine monoclonal antibodies in treated patients human/mouse chimeric (or partially ‘humanized’) antibodies have been produced. These retain the small portion of murine protein sequences responsible for antigen binding, with the remainder of the molecule being composed of human immunoglobulin.
Cetuximab (C225) is a chimeric monoclonal antibody directed against the extracellular domain of the EGFR receptor in which the constant regions of the mouse monoclonal antibody were replaced by the constant regions of the human light chain 
1 heavy chain2-4. Cetuximab has a binding affinity of 1–2 x 10-10 M, which is approximately 10-fold greater than that of the natural EGFR ligands EGF and TGF- (1–2 x 10-9 M) and the parent mouse monoclonal antibody M225. Cetuximab induces EGFR dimerization, and internalization, inhibiting its signalling. Cetuximab arrests cells in the G1 phase of the cell cycle, inducing p27Kip1 expression, inhibiting the growth of EGFR-expressing tumour cells in vitro and in vivo. Cetuximab can inhibit tumour growth, reduce tumour volume, and increase survival in xenograft models of many EGFR expressing tumours2-4. Such effects are associated with apoptosis in some studies. Enhanced activity, including additive and synergistic interactions, have been noted with combinations of cetuximab and radiation, monoclonal antibodies against HER2, and a variety of cytotoxic agents.
The results of phase I studies of cetuximab4-6 indicate that cetuximab pharmacokinetics are non-linear and that saturation of drug elimination pathways occur at doses of 200 and 400 mg/m2. The higher dose was associated with zero-order clearance with an estimated half-life of 7 days. While 3% of patients developed detectable human anti-chimeric antibodies (HACA), which were occasionally neutralizing, there was little evidence for clinically significant production of HACAs in response to cetuximab following repeated weekly infusions.
Cetuximab is being principally developed on a once-a-week, intravenous infusion schedule as a component of multitherapeutic regimens. The principal reason for this strategy is preclinical data suggesting that the dominant antitumour effect of therapeutics targeting EGFR alone is likely to be delayed tumour growth. This may not impact patients with advanced malignancies as significantly as tumour regression plus delayed tumour growth. Achievement of early cytoreduction using therapeutics targeting EGFR in combination with radiation and/or chemotherapy in patients with advanced malignancies represents a logical developmental strategy. The prominent beneficial interactions between anti-EGFR therapeutics and other therapeutic modalities also support this strategy. However, the use of other potentially active therapeutic modalities in combination with cetuximab confounds the evaluation of the inherent activity and relative contribution of each modality in a non-randomized setting. Table 1 lists the relevant characteristics of several early clinical trials (phases I and II) of cetuximab administered as a single agent and in combination with other therapeutic modalities. To date, the most clinically relevant adverse events include an acneiform rash or folliculitis, which has occurred in approximately 80% of patients5-7. Severe (grade 3–4) allergic reactions have occurred in approximately 4% of patients, with 2% experiencing severe reactions within minutes of the first infusion. The skin reactions, which usually involve the face, upper chest, and back, are likely to be related to the role of EGFR in epidermal tissues. The severity of the skin reactions has been dose-related but not dose-limiting. Discontinuing treatment has resulted in complete resolution of symptoms without scarring, and treatment delay may be an effective means of controlling the severity of this toxicity. Other adverse effects that have been relatively uncommon include asthenia, fever, and elevations in hepatic transaminase levels.
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Antitumour activity has been noted in phase I and II trials of cetuximab-based single-agent and combination regimens (Table 1). Single-agent cetuximab has demonstrated activity in 5-fluorouracil (5-FU) and irinotecan (CPT-11)-refractory colorectal cancer (CRC) that expresses EGFR. A total of 57 patients were treated in a phase II study utilizing a 20 mg test dose, then a 400 mg/m2 loading dose over 2 h, followed by 250 mg/m2 over 1 h weekly. Six patients (11%; 95% CI, 4–22%) achieved a partial response8. Thirteen additional patients had stable disease or minor responses, in keeping with the primarily cytostatic effects of this agent.
A phase II trial of cetuximab plus irinotecan in patients with advanced colorectal carcinoma that had not responded to treatment with irinotecan (refractory or stable disease) has been reported9. Partial responses in 27 (22.5%) of 120 EGFR expressing patients (72% of patients screened expressed EGFR), with stable disease as the best response in a further nine patients (7.5%), was reported. The median duration of the partial responses was 84 days. Patients were treated with cetuximab 400 mg/m2 i.v. as a loading dose followed by 250 mg/m2 i.v. weekly plus irinotecan on an identical dose schedule to that administered prior to study. The most common adverse events in the study were diarrhoea, neutropenia, nausea, fatigue and acneiform rashes. Of the 89% of patients who reported a skin rash, 29% experienced a partial response, suggesting that rash serve as a surrogate of antitumour activity. With further evaluation, the rash may help identify patients who are likely to benefit and/or those receiving an adequate dose of this combination therapy.
A phase II trial of IMC-C225 plus weekly CPT-11, 5-FU and folinic acid in patients with previously untreated metastatic colorectal cancer whose tumours expressed EGFR has also been reported10. IMC-C225 was administered using a 400 mg/m2 loading dose, then 250 mg/m2 weekly without planned interruption. CPT-11 was administered at a dose of 125 mg/m2, followed by leucovorin at 20 mg/m2 and 5-FU at 500 mg/m2. CPT-11, 5-FU and folinic acid were given weekly for 4 weeks, repeated every 6 weeks. Eleven patients (44%; 95% CI, 24–68%) achieved a partial response. An additional five patients achieved a minor response of greater than 40% reduction in tumour size. The most commonly encountered adverse events were diarrhoea (grade 3/4, 33%) and neutropenia (grade 3/4, 33%), with one patient suffering a non-fatal myocardial infarction in the setting of diarrhoea and dehydration. An acneiform skin rash, predominantly on the face and upper torso, was seen in 26 of 27 patients, with 19% of patients having a National Cancer Institute Common Toxicity Criteria (NCI-CTC) grade 3 rash. Rashes improved with continued treatment, and no patient discontinued treatment due to skin toxicity. Nonetheless, dose modifications of CPT-11 and 5-FU were frequent, and 89% of patients required a decrease in CPT-11 and 5-FU doses within the first two cycles (12 weeks) of therapy. Dose reductions of IMC-C225 were not required. A starting dose of 100 mg/m2 of CPT-11, 20 mg/m2 folinic acid, and 400 mg/m2 of 5-FU with standard dose IMC-C225 has been recommended for phase III development.
Cetuximab-based combination regimens are now being evaluated in various phase III settings. Randomized trials in patients with advanced colorectal cancer include a study of cetuximab in combination with CPT-11, 5-FU, and folinic acid versus the chemotherapy regimen alone as first-line therapy, and a study of cetuximab plus irinotecan versus irinotecan alone.
Fully humanized monoclonal antibodies
A fully humanized IgG2 monoclonal antibody specific to EGFR, ABX-EGF (Abgenix, Inc.), has also been generated using XenoMouse® technology11. The XenoMouseTM transgenic strain was engineered to express human immunoglobulin genes and lacks functional mouse immunoglobulin genes. Xenomouse monoclonal antibodies do not contain mouse protein sequences and should not be immunogenic. Therefore, they have a slower clearance rate than mouse or mouse-derived monoclonal antibodies, facilitating repeated administration.
ABX-EGF binds EGFR with high affinity (5 x 10-11 M), blocking the binding of both EGF and TGF-, and inhibiting EGFR tyrosine phosphorylation and cell proliferation in EGFR-expressing human carcinoma cell lines. ABX-EGF has been demonstrated to result in complete eradication of established human tumour xenografts. No tumour recurrence was observed for more than 8 months following the last antibody injection, further indicating complete tumour cell elimination by the antibody. Inhibition of human pancreatic, renal, breast and prostate tumour xenografts expressing different levels of EGFR, was also achieved by ABX-EGF. Tumours expressing more than 17,000 EGFR molecules per cell showed significant growth inhibition when treated with ABX-EGF, but the monoclonal antibody had no effect on tumours that did not express EGFR11. Clinical development of ABX-EGF has been initiated. Preliminary results of a phase I study of ABX-EGF, administered weekly for 4 weeks in patients with various malignancies likely to express EGFR, indicate that the ABX-EGF is well-tolerated at doses (
1.0 mg/kg) predicted to induce antitumour activity. Drug-induced transient acneiform rash has been reported and human anti-human antibodies (HAHA) have not been detected in any patient to date. Disease-directed evaluations have recently begun in this and other centres. Studies in patients with colorectal cancer are on-going, including a single agent ABX-EGF phase II study and a randomized study of ABX-EGF in combination with 5-FU and CPT-11 in patients with untreated metastatic disease.
Receptor tyrosine kinase inhibitors Another approach to blocking these receptors involves the use of specific pharmacological agents that inhibit their tyrosine kinase activity, preventing receptor autophosphorylation and the phosphorylation of downstream signalling proteins12. Compound screening efforts resulted in many chemical structures that inhibited purified EGFR. Early inhibitors suffered from a lack of potency and specificity. Greater potency, specificity, and antitumour activity have been observed with newer generation inhibitors that include the quinazolines12,13. Members of this class of compounds demonstrate inhibitory potencies as low as 6 pM against the EGFR tyrosine kinase, with almost total specificity relative to other tyrosine kinases, and potent inhibition of all EGF-mediated processes in viable cells. A binding model has been proposed based on a three-dimensional model of EGFR tyrosine kinase and structural activity studies. The model shows that the chromophore binds to a narrow hydrophobic pocket that corresponds to the binding site for the adenosine base of ATP. Table 2 lists several small-molecule EGFR tyrosine kinase inhibitors in clinical development.
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ZD 1839 (Iressa®)
ZD 1839 (Fig. 1) inhibits EGFR tyrosine kinase with an IC50 value of 20 nM14. It exhibits minimal activity against other tyrosine kinases. ZD1839 induces increased levels of the cyclin-dependent-kinase inhibitor p27Kip1. This plays an essential role in ZD1839-induced cell cycle perturbation by decreasing CDK2 activity, resulting in cell cycle arrest in the G1 phase. ZD1839 inhibits EGF-stimulated growth of KB oral carcinoma cells (IC50, 80 nM) and the growth of ovarian, colon, and breast cancer cells in soft agar (IC50, 0.2–0.4 µM). Inhibition of growth is principally noted, but apoptosis is also observed at higher doses. Co-administration of ZD1839 increases the pro-apoptotic effects of all cytotoxics evaluated in EGFR overexpressing cells in a dose-dependent, supra-additive manner.
ZD1839 has excellent oral bioavailability, with little toxicity in mice. ZD1839 produced impressive activity against human tumour xenografts. Tumour growth is suppressed as long as treatment continues; however, re-growth usually occurs when ZD1839 treatment is discontinued. It is active against several human xenograft models including LOVO and GEO colon carcinomas. Studies performed in the APCMin mouse model of familial adenomatous polyposis also suggest that EGFR-blockade may have a role in cancer prevention, with inhibition of EGFR signalling resulting in a 90% reduction in the formation of adenomatous polyps15.
The preliminary results of four phase I studies of ZD1839 in patients with advanced solid malignancies are summarized in Table 316-19. Intermittent and continuous administration schedules were initially evaluated with the continuous schedule being selected for future development. The most common adverse effect of ZD1839 was an acneiform rash. Nausea, vomiting, and diarrhoea were also common, but severe (grade 3–4) adverse events were rare. Grade 3–4 diarrhoea was consistently observed in patients treated with ZD1839 at 700 mg/day in the intermittent (14 days-on treatment followed by 14 days-off treatment) study. However, ZD1839 was generally well tolerated at the lower dose levels. In the two trials evaluating continuous uninterrupted ZD1839 administration, severe toxicity, consisting of skin rash and diarrhoea, were repeatedly observed at doses exceeding 600 mg/day. Pharmacokinetic studies confirmed that ZD1839 is suitable for once-daily dosing and that biologically relevant serum concentrations (e.g. > IC90 for inhibition of EGFR tyrosine kinase in vitro) are achievable at doses exceeding 100 mg/day. Pharmacokinetics were dose proportional, with steady state being achieved by 7 days and the terminal half-life ranging from 27–48 h.
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Disease-specific studies with ZD1839 have focused on the treatment of patients with non-small-cell lung carcinoma until very recently. A single agent National Cancer Institute of Canada Clinical Trials Group (NCIC-CTG) study in EGFR unselected patients with colorectal carcinoma has also recently been reported20. While no objective responses were observed, several patients had tumour marker decrements suggesting antitumour activity. Moreover, successive biopsies performed pre- and post-therapy indicated decreased tumour cell proliferation rates and increased apoptotic rates in several patients. A further phase II trial of ZD1839 in patients with colorectal carcinoma is also on-going with progression-free survival as its primary end-point. A phase I/II trial of ZD1839 with 5-FU (370 mg/m2 daily for 5 days) and folinic acid utilizing the Mayo regimen (20 mg/m2 daily for 5 days) has been performed21. This has shown that this combination can be safely administered with promising antitumour efficacy. Pharmacokinetic studies indicated no significant change in mean exposure to either ZD1839 or 5-FU/LV when these are given in combination. Further combination studies are now in different phases of development.
OSI-774 (Tarceva®)
OSI-774 (Fig. 1) is a highly specific and reversible, ATP-competitive, quinazoline inhibitor of the EGFR tyrosine kinase22. In HN5 head and neck and DiFi colorectal carcinoma tumour cell lines, the IC50 for inhibition of EGF-mediated receptor autophosphorylation was 20 nM, with EGF-dependent mitogenesis and proliferation being reduced by 50%. OSI-774 showed significant in vivo activity in tumour xenograft studies. The feasibility of administering OSI-774 by intermittent, and uninterrupted daily oral dosing, schedules have been evaluated in early clinical, phase I, trials23,24. The most common toxicities of OSI-774 on both schedules included mild-to-moderate acneiform skin rashes, diarrhoea, nausea, and headache. The maximum tolerated dose was not reached on the intermittent schedule, although severe, manageable diarrhoea was observed in one subject. On the uninterrupted, oral, daily dosing schedule, diarrhoea was the principal dose-limiting toxicity which precluded escalation of OSI-774 doses above 150 mg/day. The majority of patients experienced an acneiform rash. The severity of the rash typically peaked by weeks 3–4, but treatment interruptions and dose reductions were not usually required for this. Pharmacokinetic studies revealed dose independent behaviour, with no evidence of drug accumulation with repetitive daily treatment. At the maximum tolerated dose (150 mg/day), minimum steady-state plasma concentrations (Css,min) averaged 1.2 ± 0.62 µg/ml. This exceeds concentrations (0.5 µg/ml) consistently capable of inhibiting the growth of EGFR-expressing tumours in preclinical models. One patient with metastatic colorectal carcinoma experienced a minor reduction in the size of metastases. The reports of phase II studies evaluating the activity of OSI-774 150 mg/day orally in previously treated patients with carcinomas of the lung, ovary, and head and neck have demonstrated single-agent activity. Randomized clinical trials have being designed in a variety of tumour types and treatment settings. Concurrent with disease-directed studies, the feasibility of administering OSI-774 in combination with other anticancer modalities is also being evaluated.
PKI 166
PKI 166 (Novartis) is a pyrrolopyrimidine with high selectivity for EGFR. The IC50 for EGF-stimulated growth in BALB mouse keratinocytes is 1.38 µM. It also suppresses the growth of A431 xenografts, as well as xenografts derived from human prostate cancer, non-small cell lung cancer (NSCLC), and breast cancer. PKI 166 is currently undergoing clinical evaluation in patients with solid malignancies25.
GWS572016
The ability of inhibitory compounds to blockade not only EGFR, but also HER2 may prove to be advantageous since signalling via HER2 may potentially lead to drug resistance. GWS572016 (also known as GW2016) is a 6-thiazolyquinazoline that has been selected for development because it reversibly inhibits the activation (phosphorylation) of both EGFR and HER2 in a dose-dependent manner. It has demonstrated potent tumour growth inhibitory activity in vitro (IC50 values below 0.15 µM) and in vivo. This agent was selected for further development. It is currently undergoing clinical evaluation26.
Reversible or irreversible inhibitors? The rationale for developing small molecules that irreversibly inhibit EGFR tyrosine kinase was based on the results of studies indicating that maximal antitumour activity occurs with prolonged suppression27. The optimal use of reversible inhibitors may require continued relevant plasma concentrations to keep the target suppressed. Irreversible inhibitors may be advantageous by eliminating existing kinase activity that can regenerate only when new receptors are synthesized. This may, however, be limited by the rate of receptor regeneration which appears to be relatively rapid (12–48 h). Irreversible compounds may, nonetheless, require that relevant plasma concentrations be attained for much briefer durations, minimizing the requirement for continuous drug administration. This may eliminate the requirement for therapeutics with long plasma half-lives without compromising efficacy and reducing toxicity due to non-specific interactions that may occur at high or prolonged plasma levels. For these reasons, irreversible inhibitors, like CI-1033 and EKB-569, are being developed (see Table 2).
CI-1033
CI-1033 is the dichloride salt of PD0183805, a 4-anilinoquinazoline, that acts as an ATP-binding site-directed inhibitor of activation of EGFR, HER2, erbB3, and erbB428. CI-1033 binds irreversibly with high affinity to all the four erbB family members, particularly EGFR and HER2. It inhibits isolated EGFR tyrosine kinase activity with an IC50 value of 1.5 nM and heregulin-mediated tyrosine phosphorylation in MDA-MB-453 human breast carcinoma, which expresses HER2, erbB3, and erbB4, with an IC50 value of 9 nM. The agent is also capable of inducing regressions of well-established A431 xenografts. The results of studies of long-term drug administration indicate that CI-1033 maintains tumour growth suppression for extended time-periods without the emergence of drug resistance. The activity of the compound appears to be independent of dose fractionation, with significant activity being obtained on dosing regimens ranging from once daily to once weekly. CI-1033 has been demonstrated to enhance the cytotoxic effects of other therapeutic modalities. For example, the agent enhances the cytotoxic effects of the topoisomerase inhibitor SN-38 in vitro.
In phase I studies29 of CI-1033 administered as a single oral dose once weekly for 3 weeks, every 4 weeks, and daily for 7 days every 3 weeks, the most common toxicities have been mild-to-moderate vomiting, diarrhoea, and acneiform rash. Thrombocytopenia and reversible dose-limiting hypersensitivity reactions, that appear to be prevented by antihistamine premedication, have also been observed in both studies. The elimination half-life averaged 5–6 h. CI-1033 has also been studied in 14 days on/7 days off, 21 days on/7 days off and 28 days on/7 days off schedules. Sixty-eight patients were treated at doses ranging from 2–220 mg/day. Using these schedules the dose-limiting toxicities were grade 3 stomatitis and rash (acneiform and maculopapular). The elimination half-life using this schedule was 4 h with no drug accumulation observed. Further clinical development utilizing protracted schedules is on-going.
EKB-569
EKB-569 (Genetics Institute) is a potent, specific, low-molecular weight oral compound that binds irreversibly to EGFR. The agent inhibits EGFR tyrosine kinase activity in the low nanomolar range and is efficient at blocking autophosphorylation of HER2 tyrosine kinase, albeit at much higher concentrations than EGFR. The agent inhibits growth of EGFR and HER2 overexpressing cancer cells and xenografts in preclinical studies. Preclinical studies in the APCmin/+ mouse, a murine model of familial adenomatous polyposis (FAP) with EKB-569 combined with sulindac, a prototypical non-steroidal anti-inflammatory drug, have demonstrated that this combination can prevent polyp generation. This indicates that this agent may be a powerful prophylactic agent in patients with FAP30. EKB-569 is currently undergoing clinical development on a daily, oral, uninterrupted schedule31. Using this regimen, this compound is well tolerated, with diarrhoea and acneiform rash being the commonest noted adverse event. NCI-CTC grade 3 diarrhoea was dose-limiting at a dose of 100 mg/day, with 75 mg/day being the recommended dose for future efficacy studies.
Other therapeutic modalities In addition to monoclonal antibodies and RTK inhibitors, other approaches used to target the EGFR include antisense therapies and immunoconjugates linking toxins to anti-EGFR antibodies or EGFR ligands.
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Targeting ras signal transduction |
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The ras signal transduction proteins are a family of guanosine triphosphatases (GTPases) that function as chemical switches, cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states32-35. Ras proteins transduce signals from receptor tyrosine kinases to a downstream cascade of protein kinases that regulate the growth and regulatory processes that are aberrant in malignant cells (Fig. 2). Activating mutations of ras occur in a significant proportion of colorectal (
50%) cancers. The central role in regulating these processes played by Ras and its downstream kinases, which include Raf kinase and the mitogen-activated kinases (see below), make this pathway an important target for novel anti-cancer therapeutics (see Fig. 2). Ras is synthesized as an inactive cytosolic peptide. It undergoes a series of post-translational modifications at the carboxy-terminus, which increase its hydrophobicity and facilitate its association with the cell membrane. This process is dependent on the enzyme farnesyltransferase (FTase), which adds a 15-carbon farnesyl isoprenoid group to the carboxy-terminal CAAX peptide sequence motif. This process allows its localization to the inner surface of the plasma membrane. FTase inhibition can block the function of Ras, turning off its signal transduction. This can also alter the activity of other farnesylated cellular proteins including RhoB, and the kinesins CENP-E and CENP-F.
Preclinical studies with FTase inhibitors suggest that their antitumour activity does not solely depend on the status of ras signalling. These compounds appear to be most active against tumours expressing H-ras > N-ras >> K-ras36. This may be due to alternative prenylation of K-Ras by GGTase-I when farnesylation is blocked. Several FTase inhibitors have been demonstrated to have in vivo activity in human xenograft models, with growth inhibition being observed in xenografts with and without activated ras oncogenes.
Several FTase inhibitors have undergone clinical evaluation33-35. The lead compounds currently in clinical development are R115777 (Janssen) and SCH66336 (Schering-Plough). R115777, the orally bioavailable methyl-quinolone non-peptidomimetic inhibitor (see Fig. 1) sharing structural similarities to the CAAX motif of Ras, was the first FTase inhibitor to enter phase I clinical evaluations. R115777 was initially administered twice daily for 5 days every 2 weeks at escalating doses (25–1300 mg twice orally). An unacceptably high rate of dose-limiting toxicity, consisting of neuropathy, fatigue with decreased performance status, and gastrointestinal complaints, were observed at the 1300 mg twice daily dose level, with lower dosing being well-tolerated. A patient with metastatic colorectal cancer who was treated with R115777 at a dose of 500 mg twice a day had a 46% decrease in carcino-embryonic antigen (CEA), with improved symptoms and stable disease for 5 months. When, however, R115777 was administered twice daily for 3 weeks every 4 weeks, neutropenia and thrombocytopenia were the principal dose-limiting side-effects with the maximum tolerated dose being 240 mg/m2 twice daily. When given continuously, the maximum tolerated dose was 300 mg bid, with neutropenia, thrombocytopenia and peripheral neuropathy being dose-limiting. A further colorectal cancer patient on this study had a minor response with a more than 50% decrement in serum tumour marker (CEA) levels.
Based on the observed preclinical activity in colorectal cancer models, and hints of anticancer activity in early clinical trials, a randomized double-blind placebo-controlled study of R115777 has been performed in patients with advanced refractory colorectal cancer37. This phase III trial compared R115777 (300 mg bid for 21 days every 28 days) to placebo in unselected patients who had failed two or more prior chemotherapy regimens. The primary endpoint of this study was overall survival, with the study being designed to detect a 50% increase in overall survival with a power of 85%. A total of 368 patients were randomized (2:1 ratio), with 235 patients receiving R115777. There was no significant difference in overall survival between the two arms (P = 0.396) with the median overall survival for patients on R115777 being 5.7 months (95% CI, 5.2–6.5) versus 6.1 months (95% CI, 5.2–7.8 months) for patients on placebo. This negative randomized study in unselected colorectal cancer patients suggests that treatment with the farnesyltransferase inhibitor R115777 may not result in any clinical benefit. However, this result may also mask a true benefit from farnesyltransferase blockade, in a subgroup of patients sensitive to FTase, such as the Ki Ras negative, or erbB receptor-positive subgroups.
FTase inhibitors have demonstrated cytotoxic and radiation-sensitizing properties in tumours in vitro and in vivo. Combination therapy with FTase inhibitors and cytotoxic agents are, therefore, being evaluated in early clinical trials. R115777 has been combined with 5-FU and leucovorin, with bimonthly fixed dose 5-FU/leucovorin using the de Gramont schedule (leucovorin 200 mg/m2/2 h, 5-FU 400 mg/m2 intravenous bolus, 5-FU 600 mg/m2 over 22 h on days 1 and 2)38. Patients received R115777 at doses ranging from 200–500 mg twice daily, with severe myelosuppression being dose-limiting at 500 mg twice daily. Further study of FTase inhibitors in combination with cytotoxics and radiation therapy are warranted for the treatment of colorectal malignancies.
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Targeting Raf-1 signalling |
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The proto-oncogene Raf-1 is a serine-threonine protein kinase identified as a downstream effector of Ras in the growth factor receptor signalling cascade (Fig. 2)39. The most extensively studied member of the Raf family is Raf-1, which was initially identified by homology to the v-Raf oncogene contained in oncogenic retroviruses. A 74 kDa protein, it comprises three main functional domains, including the amino-terminal domain that is involved in Ras binding, the kinase domain at the carboxyl terminal end and a negative regulatory domain that controls Raf-1 kinase activity. Constitutively activated mutated Raf-1 is known to possess transforming activity in a range of human cancers and mutated forms of the Raf oncogene have been observed in many tumour types. Raf-1 is also activated in cancer cells through activated growth factor signalling pathways upstream, in tumours cells with increased growth factor or Ras signalling. It has been described to play roles in the regulation of proliferation, differentiation and apoptosis, with recent studies utilizing knockout and knockin mice suggesting that its main role is in protecting cells from apoptosis. These and other studies indicate that there is significant crosstalk between the Raf-MEK-Erk pathway and the PI3 kinase-Akt pathway and indicate that Raf may have roles other than MEK activation.
Raf-1 is an important target for the development of anticancer drugs. Several approaches have been pursued to block Raf kinase activity include antisense molecules; inhibitors of Raf protein folding that target the heat shock protein (Hsp-90) molecular chaperones; and small pharmacological molecules. Development of the antisense 20-base phosphorothioate oligonucleotide ISIS 5132 has been discontinued. ISIS 5132 and the Hsp-90 inhibitors have been previously reviewed40,41.
A search for specific pharmacological inhibitors of Raf kinase has resulted in the identification of the bis-aryl ureas as lead active compounds, inhibiting a biochemical acellular assay at concentrations lower than 500 nM42. The activity of these lead compounds was then studied in a mechanistic cellular assay, where the ability of the compounds to inhibit oestrogen-induced MEK phosphorylation was evaluated in a murine 3T3 cell line transfected with an oestrogen receptor-Raf kinase fusion construct. Active compounds in this and other cellular screens led to the selection of the bis-aryl urea BAY 43-9006 as a candidate for clinical development. BAY 43-9006 (Fig. 1) was extensively studied in the K-ras dependent human colorectal cancer cell line HCT116, and was active when administered at oral doses ranging from 10–100 mg/kg per day for 14 days. Combination xenograft studies in the DLD-1 colon model revealed that CPT-11 and Bay 43-9006 were synergistic indicating that this compound may be best used in combination.
The first compound of its class to enter clinical trials, BAY 43-9006 has been evaluated in two phase I studies43,44. In the first study, patients were initially treated with weekly doses, and then by continuous oral daily treatment at 100, 200, 400 and 800 mg twice daily. Forty-six patients were treated with NCI-CTC grade 3 diarrhoea being dose-limiting in 2 of 6 patients at 800 mg bid per day continuously. Other observed toxicities included rash (grade 1 or 2; n = 9), pancreatitis (grade 3, n = 1 AT 100 mg) and fatigue (grade 2 or 3, n = 2 at 800 mg bid daily. One patient with hepatocellular carcinoma had a partial response at 400 mg bid per day. Biomarker studies performed in peripheral blood lymphocytes from treated patients revealed a dose-related inhibition of stimulated Erk phosphorylation, with stable suppression of Erk phosphorylation at the recommended phase II dose of 400 mg bid per day. A further phase I study utilizing a 28 days on and 7 days off schedule in a 35-day cycle has also been described. Twenty-five patients were reported to have received BAY 43-9006 utilizing initially a once weekly, then an every other day, and finally a 28 of 35 day schedule. At doses up to 200 mg bid, the MTD had still not been reached with grade 1 or 2 skin rash and diarrhoea being observed. A randomized discontinuation phase II efficacy study is planned for colorectal cancer patients with this compound.
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Targeting the mitogen activated protein kinase pathways |
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The mitogen activated protein kinases (MAPKs) are involved in several signal transduction cascades, and are critical components of the growth factor signalling pathways (Fig. 2)45. They are evolutionarily conserved three-kinase cascades comprising from upstream to downstream the MEK kinases (MEKKs), the MAPK/ERK kinases (MEKs), and the ERKs. The ERKs are activated directly by MEKs, which are dual-specificity protein kinases that generally recognize only certain ERKs as substrates, phosphorylating them on both a tyrosine and a threonine or serine residue. MEKs are activated by MEKKs, the most upstream kinases in this cascade, a structural diverse family of kinases. A number of pharmacological inhibitors that block the signalling of several members of the mitogen activated kinase cascade have been identified46,47. These include inhibitors of p44 ERK1 and p42 ERK2, as well as inhibitors of the p38 isoforms and inhibitors of MEK1 and MEK2.
The first agent targeting the MAPK signalling pathway to be evaluated in the clinic in the treatment of cancer patients is CI-1040 (Pfizer, Ann Arbor, MI, USA), formerly known as PD184352. CI-1040 is an oral, small molecule inhibitor of the dual specificity kinases MEK1 and MEK2 (Fig. 1). The blockade of MEK1/2 by CI-1040 specifically inhibits ERK1/2 phosphorylation with IC50 values for the enzymes below 20 nM. CI-1040 inhibits ERK1 and ERK2 activity in colon 26 carcinoma cells with an IC50 of 120 nM, also inhibiting the growth of tumour xenografts derived from this cell line. CI-1040 has been administered48,49 to patients with advanced carcinoma using a 21-days on, 7-days off, treatment schedule. At reporting, 220 courses had been administered to 78 patients. Studies on peripheral blood mononuclear cells and on tumour biopsies from treated patients revealed consistent decreases in phospho-ERK activity after treatment with CI-1040. Toxicities included fatigue, diarrhoea and rash, with CI-1040 generally well tolerated. An 800 mg bid dose has been recommended for future phase II studies. During this study, one patient with pancreatic cancer achieved a confirmed partial response lasting more than 6 months. Stable disease for 12 or more weeks has been observed in 30% of patients on this study. These encouraging results indicate that efficacy studies with CI-1040 will soon be underway.
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Apoptosis signalling pathways |
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A number of strategies have been investigated to enhance tumour cell apoptosis. These include treatment with the antisense molecules to the anti-apoptotic regulators of the bcl-2 family of proteins to enhance the apoptosis inducing effects of cytotoxic chemotherapies, small molecule inhibitors of the serine-threonine kinase Akt (also known as protein kinase B), agonistic antibodies to the TRAIL (TNF-related-apoptosis inducing ligand)-R1 receptor and inhibitors of NF-
B. Bcl-2 antisense (G3139) Many of the molecular genetic changes that occur during the transition from normal colonic epithelium to colorectal cancer have been identified40. Several of these represent potential therapeutic targets. One molecular pathway that is altered commonly in colorectal cancer and other malignancies is the bcl-2 apoptotic pathway, with either overexpression of the bcl-2 gene, or inactivating mutations of the bax gene. An excess of bcl-2 protein expression relative to that of bax inhibits apoptosis due to many types of pro-apoptotic signals including those generated by cytotoxics and radiation. Bcl-2 overexpression has been reported in 30–94% of pathological specimens of human colorectal cancer, whereas bcl-2 is only weakly expressed in the basal proliferative layer of normal colonic epithelium. Furthermore, bcl-2 overexpression in primary colon cancer specimens has been reported to be a negative prognostic factor with respect to recurrence and survival. The equilibrium between pro-apoptotic and anti-apoptotic signals in colon cancer may be critical in determining a tumour's responsiveness to chemotherapy-induced cell damage, with resistance being conferred by either overexpression of bcl-2 or a diminution of bax gene expression, resulting in a higher bcl-2:bax ratio which inhibits activation of apoptotic pathways. These molecular changes may have profound effects on the sensitivity of colon cancer to cytotoxic agents, including irinotecan. Therefore, strategies directed at down-regulation of bcl-2 may modulate resistance to chemotherapy. A specific strategy uses a bcl-2 antisense oligonucleotides to hybridize to bcl-2 mRNA. G3139 is an 18-mer antisense molecule, targeting the first six codons of the bcl-2 mRNA open reading frame sequence 5'-TCTCCCAGCGTGCGCCAT. Following hybridization, the mRNA undergoes degradative cleavage mediated through RNAase H, which leads to a decrease in bcl-2 protein expression and significant enhancement of chemosensitivity in vitro and in vivo. This effect is sequence-specific and does not occur with either 2-base mismatched or reverse-polarity antisense controls.
The feasibility of administering G3139 and irinotecan has been evaluated in phase I and II studies in patients with advanced colorectal malignancies. In these studies, G3139 was administered as continuous intravenous infusion for 7 days with irinotecan administered on day 6. Significant down-regulation of bcl-2 protein in peripheral blood mononuclear cells has been observed following G3139 therapy. At reporting50, 17 patients (8 previously exposed to CPT-11) had been treated with 60 courses of G3139/CPT-11 at G3139 doses ranging from 3 to 7 mg/kg/day and CPT-11 from 280 to 350 mg/m2. Grade 3 and 4 diarrhoea unresponsive to loperamide, grade 3 nausea and vomiting, grade 4 neutropenia 5 days and febrile neutropenia were dose limiting at G3139 5 mg/kg/day and CPT-11 350 mg/m2. The G3139 5 mg/kg/day and CPT-11 280 mg/m2 dose levels proved safe. Persistently stable disease over 12 courses was observed in a patient previously treated with CPT-11. In 3 of 9 CPT-11 naïve patients, there was 1 patient with an objective partial response, and 2 patients with stable disease after 2, 6 and 8 cycles, respectively. Further efficacy studies with bcl-2 antisense in colorectal cancer administered in combination with oxaliplatin and 5-FU are on-going.
Targeting the serine-threonine kinase Akt The proto-oncogene AKT is one of the most attractive kinase targets relevant to apoptotic pathways in cancer cells51. Also known as protein kinase B, AKT is negatively regulated by PTEN (or MMAC) which is frequently mutated in cancer cells. Studies in patients with advanced colorectal cancer have shown that about 17% of colon cancers have PTEN mutations, with a higher frequency being observed in microsatellite unstable tumours. Moreover, immunohistochemical studies indicate that AKT is highly overexpressed in approximately 57% of sporadic colorectal carcinomas, with overexpression also being noted in adenomas but not hyperplastic or normal colonic epithelium. Furthermore, phospho-specific antibodies have revealed that phospho-AKT is detectable in malignant, but not normal, epithelium suggesting that Akt signalling is an important target in colorectal cancer. Small molecule inhibitors of Akt are currently undergoing preclinical testing and may soon be evaluated in clinical trials.
Inhibition of NF-B
The nuclear factor (NF)-B family of transcription factors plays an important role in the regulation of a variety of biological responses including apoptosis, cell-cycle progression and differentiation52. Highly conserved in structure and function evolutionarily, these proteins are ubiquitously expressed and have been implicated in carcinogenesis. There are at least five members of the NF-B family in mammals: p50/p105, p65/RelA, c-Rel, RelB and p52/p100. Cloning of these subunits has revealed a conserved central region known as the Rel homology domain, which is involved in DNA binding, interactions with an inhibitory protein called IB and dimerization. While many forms of NF-B have been described the classic form is the heterodimer of the p65/RelA and p50 subunits.
NF-B is activated by a range of stimuli including growth factors and cytokines. This activation involves the regulation of the IB family of inhibitory proteins. Growth factor signalling induces IB phosphorylation by IB kinase (IKK), which targets IB for proteasomal degradation. Decreased IB activity activates NF-B, resulting in its translocation to the nucleus where it modulates the expression of a variety of genes including several apoptosis regulators, growth factors, cell adhesion molecules, and other transcription factors. A number of studies have demonstrated that NF-B signalling protects tumour cells from the induction of apoptosis following exposure to cytotoxic therapy, and that blockade of NF-B can potentiate the antitumour activity of cytotoxics. The inhibition of NF-B through the adenoviral delivery of a modified form of IB has been shown to sensitize chemoresistant tumours to the apoptotic potential of TNF- and CPT-11. This strategy bears further evaluation in patients with colorectal cancer in the clinic.
An inhibitor of NF-B, triptolide, which is also known as PG490, has been extracted from the Chinese herb Tripterygium wilfordii. PG490 is a diterpene triepoxide and can block NF-B activation, inhibiting tumour growth as a single agent and sensitizing tumour necrosis factor (TNF)-resistant tumour cell lines to TNF-. PG490 also sensitizes tumour cells to cytotoxic induced apoptosis53. PG490 is currently being evaluated in early clinical trials.
An alternative route to inhibiting NF-B is the non-specific blockade of the ubiquitin-proteasome pathway54. This pathway is the principal pathway for intracellular protein degradation, involving the marking of proteins labelled for degradation by poly-ubiquitin chains, followed by their degradation to peptides and free ubiquitin by a large multimeric protease, the proteasome. The proteasome exists in all eukaryotic cells and is involved in the regulation of IB degradation. Inhibitors of proteasomal enzymatic activity have been developed. Proteasome inhibition can inhibit NF-B function by blocking IB degradation, and the anticancer activity of these agents is thought to be in part related to the effect on this target. However, the ubiquitin-proteasome pathway regulates the degradation and function of a plethora of proteins that may play critical roles in oncogenesis including p53, p27, p21, the cyclins, ß-catenin, bcl-2 and bax. The precise mechanism of action of this family of compounds is, therefore, probably multifaceted.
The boronate compound PS-341 is the first proteasome inhibitor to be studied in the clinic, with several phase I and phase II clinical trials being reported54. Optimal drug dosing has been determined in these early clinical studies using a bioassay that analyzed residual proteasome enzymatic activity in peripheral blood mononuclear cells. PS-341 has been generally well tolerated with low-grade fever and/or fatigue, thrombocytopenia and diarrhoea the commonest documented toxicities. Skin rash and peripheral neuropathy has also been reported. Antitumour activity has been observed in several tumour types including multiple myeloma, androgen-resistant prostate cancer and non-small-cell lung carcinoma.
Targeting TRAIL (TNF related apoptosis inducing ligand) receptors The death receptor ligands tumour necrosis factor (TNF), Fas ligand (FasL) and TRAIL are all able to induce apoptosis by binding to their cell membrane receptors55. Recombinant forms of these ligands can potentiate the antitumour effects of cytotoxics agents in both in vitro and in vivo models. Such ligands have also been shown to substantially potentiate the antitumour activity of antibodies targeting ErbB receptors such as the EGFR targeting antibody C225, or cetuximab, in CPT-11-refractory colon cancer models. Studies with such agents have until now been limited by the toxicity of these potential therapeutics, with severe liver toxicity and sepsis-like toxicities. Native recombinant human TRAIL has been less toxic, although hepatocellular toxicity remains a concern. TRAIL mediates apoptosis through two death receptors, TRAIL-R1 and TRAIL-R2. These receptors are capable of initiating a cascade of signalling events that result in caspase activation and ultimately cell death. A fully human recombinant agonistic antibody to TRAIL-R1, TRM-1, is now undergoing phase I clinical evaluation. TRM-1 induces apoptosis in vitro and in vivo in colon cancer models including the human colon carcinoma cell line SW480, binding TRAIL-R1 with a calculated IC50 of 0.1–0.14 nM. It potentiates the antitumour activity of topoisomerase I inhibitors including CPT-11. Since TRAIL-R1 expression has been noted in colon cancer pathological specimens, if this antibody can be safely administered in active doses in patients with colorectal cancer it has significant promise in the treatment of this disease.
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Other important signalling targets |
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There are over two hundred new agents being tested in anti-cancer drug clinical trials. An increasingly large proportion of these drugs are being directed at inhibiting some form of intracellular signalling. An even larger number of compounds are being evaluated preclinically, and it is impossible for this review to fully cover this rapidly enlarging subject. These include drugs targeting the epithelial cell adhesion molecule EpCAM (17-1A antigen), the mucin MUC1, the insulin growth factor receptor, ß-catenin, the STAT (signal transducers and activators of transcription) and smad signalling proteins, and the cyclins and cyclin-dependent kinases. Therapeutics targeting these and many other targets are in various stages of preclinical or clinical evaluation.
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Future challenges |
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There are many challenges to the optimal evaluation of molecular targeted therapeutics. These include determining optimal dosage and administration schedule. Optimal dosing may be best defined as the dose resulting in the maximal achievable antitumour effect with minimal or tolerable toxicity. This may not always be the dose achieving the maximal biological effect as seen with the proteasome inhibitor PS-341. Neither is this the maximum tolerated dose. The latter is the dose utilized in the clinical development of most anticancer cytotoxic drugs. This change in the anticancer drug development paradigm will require the development of laboratory assays that can accurately measure the drug effect on the target in the clinic. For therapeutics blockading EGFR and its downstream signalling, techniques that permit the assessment of the phosphorylation-state of EGFR and its downstream signalling proteins, in tumour and surrogate tissues, may be helpful in selecting optimal clinical dosing.
Moreover, preclinical data and early clinical results indicate that major tumour regression is not likely to be the predominant effect of many molecular-targeted therapeutics. However, clinical evaluation of these agents is often performed in patients with advanced disease who require cytoreduction for clinical benefit. Maximal clinical benefit is likely to be obtained when these agents are utilized in combination with other therapeutic modalities (e.g. cetuximab and CPT-11). The prominent additive or synergistic interactions between these drugs and other therapeutic modalities support the development of these agents in combination regimens. Combination efficacy testing, however, confounds the evaluation of the relative contribution of molecular targeting in non-randomized studies. Nonetheless, future development of these agents is likely to involve their use in combination with other therapeutic modalities. Novel clinical trial designs such as the randomized discontinuation method, and the study of combinations of a cytotoxic and a targeted drug in the treatment of disease that has progressed on that cytotoxic are, therefore, being pursued.
Selecting the most appropriate study patients for disease-directed randomized clinical studies also presents a formidable challenge. Molecular tumoural heterogeneity, if unrecognized or unaccounted for, may confer different risks to different patients resulting in underpowered clinical trials56. These trials may then fail to detect a truly effective new therapy. Careful patient selection based on the tumoural expression of the drug target may be important in the design of these trials. This, however, may lead to missed opportunities if the mechanism of action of the drug in question is not fully understood. Moreover, target expression may be insufficient to predict antitumour activity. The anticancer activity of a specific signalling inhibitor may also largely depend on alterations in signalling both upstream and downstream of its target protein.
Also, while results with trastuzumab indicate that patients with the highest levels of cell-membrane HER2 expression attain maximal clinical benefit, this may not be applicable to EGFR. Unlike HER2, EGFR is very rapidly recycled, with a very short transit time at the cell surface, and preclinical data suggest that EGFR signalling may continue after receptor internalization. High levels of cell membrane EGFR expression may not, therefore, be necessary for clinical benefit with EGFR blockade. Clearly, optimal molecular determinants of activity for EGFR directed therapeutics and other molecular-targeted agents need to be evaluated to assist patient selection for these clinical trials.
Selecting patients for farnesyltransferase inhibitor therapy raises similar challenges. Initial efficacy studies of targeted treatment with these compounds has been largely to patients with a high incidence of K-Ras mutations. However, these agents are least active in K-Ras expressing preclinical models. This may have masked the presence of clinical benefit since the completed randomized study of R115777 in patients with metastatic colorectal cancer did not select patients based on Ras mutation status. Several objective responses have been noted with this agent in this tumour type suggesting that this agent may have clinically significant antitumour activity in a selected group of patients with colorectal cancer. Careful patient selection based on tumour molecular phenotyping is clearly likely to become a major issue in the development of targeted anticancer therapeutics.
Another significant challenge to developing targeted therapeutics arises from the difficulties relating to the selection of appropriate endpoints of clinical efficacy. Tumour regression has traditionally been utilized in phase II trials when screening for anticancer activity, but this may not be a useful surrogate for clinical benefit with targeted therapies whose primary effect is tumour growth delay. Alternative clinical trial designs which quantitate the ability of a drug to slow tumour growth, such as randomized discontinuation designs and multinomial designs, are being pursued as we search for alternative paradigms to guide the optimal development of these compounds57,58. Only adequately designed clinical trials will ensure that the usefulness of molecularly-targeted therapies is correctly assessed, and that potentially useful agents are not rejected on the basis of poor performance in an inadequately designed trial with inappropriate clinical and biological endpoints59.
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Acknowledgements |
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The authors are indebted to their patients, and all current and past members of the clinical trials and translational research teams at the Institute for Drug Development, Cancer Therapy and Research Center, as well as at the University of Texas Health Science Center, both at San Antonio. Dr de Bono is supported by grants from the American Society of Clinical Oncology, the Breast Cancer Research Foundation, as well as a Doris Duke Charitable Foundation Career Development Clinician Scientist Award and an NIH R21 award. The Institute for Drug Development is supported by an NCI Phase I trials grant.
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Footnotes |
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Correspondence to: Johann de Bono MBChB MRCP MSc PhD, Institute for Drug Development, Cancer Therapy and Research Center, 7979 Wurzbach Road, 4th Floor Zeller Building, San Antonio, TX 78229, USA
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