Imatinib Mesylate as a Paradigm for the Development of Molecular Therapeutics

The case of imatinib (Gleevec; STI571) has shown how the use of a powerful, highly validated preclinical biomarker provided a convincing rationale for a novel targeted therapeutic; furthermore, its subsequent clinical development was also based around this key biomarker (Park et al., 2004). Imatinib was discovered in the late 1980s, and emerged as the lead compound from a series initially optimized against the platelet-derived growth factor (PDGFR) tyrosine kinase. Imatinib also selectively inhibits the ABL tyrosine kinases in vitro and blocks cellular proliferation and tumor growth of cells expressing BCR-ABL or v-ABL (Buchdunger et al., 2000).

Chronic myeloid leukemia (CML) is characterized by the BCR-ABL fusion protein that is formed by a reciprocal translocation between the long arms of chromosomes 9 and 22 (the Philadelphia chromosome). The BCR-ABL fusion protein functions as a constitutively activated tyrosine kinase. In the first phase I trials of imatinib in CML patients who were resistant to interferon-a, treatment with >300 mg/day had complete hematological responses (defined as a decrease in marrow blasts to 5% or less of total cellularity, a disappearance of blasts from the peripheral blood, an absolute neutrophil count of more than 1000 cm" , and a platelet count of more than 100,000 cm"3) in 98% of patients with chronic phase and 55% of those in blast phase (Druker et al., 2001). These striking results have been confirmed in further clinical testing, including a phase III trial showing statistically superior rates of cytogenetic (determined by percentage of Philadelphia chromosome-positive cells in metaphase in bone marrow) and hematological response with imatinib over standard therapy in patients with chronic-phase disease, as well as prolonging time to progression of accelerated-phase or blast-crisis disease (Kantarjian et al., 2002; O'Brien et al., 2003; Sawyers et al., 2002).

In the initial trials of imatinib, dose selection was guided by the rational selection of an appropriate PD endpoint: this involved assessment of BCR-ABL tyrosine kinase inhibition in circulating peripheral blood leukemic cells (Druker, 2002; Druker et al., 2001). Previous studies had demonstrated that in BCR-ABL expressing cells, the SH2, SH3 domain adaptor protein CRKL is the major tyrosine phosphorylated protein (Oda et al., 1994). The form of CRKL that is phosphorylated by BCR-ABL migrates more slowly on gel electrophoresis than the unphosphorylated form, allowing for the development of an assay that examined the relative proportion of phosphorylated CRKL before and during treatment (Senechal et al., 1998). Immunoblot assays demonstrating the degree of phosphorylation of CRKL showed that there is a plateau in inhibition of BCR-ABL above 250 mg. Together with the PK data, a dose of at least 400 mg was therefore recommended

(Druker et al., 2001). An MTD was not achieved with the agent, which has a high therapeutic index. In a subsequent phase III trial, quantitative realtime RT-PCR was used to measure levels of BCR-ABL transcripts in patients achieving complete cytogenetic response (Hughes et al., 2003). Risk of disease progression was found to inversely correlate with reduction of BCR-ABL mRNA compared with pretherapeutic levels. The proportion of patients with CML who had a reduction in BCR-ABL transcript levels of at least 3 log by 12 months of therapy had a negligible risk of disease progression during the subsequent 12 months (Deininger et al., 2005; Hughes et al., 2003). Use of RT-PCR has since been increasingly accepted for monitoring and assessment of response to imatinib, and the use of this molecular endpoint as a basis for clinical decision making has proved to be feasible and safe both in multicenter clinical trials (Hess et al., 2005; Martinelli et al., 2006).

Despite the stunning trial results for imatinib, which led to it being the first kinase inhibitor approved by the US FDA, the emergence of resistance to imatinib has shown that the new post-genomic designer drugs will not escape this age-old problem of cancer therapy (Workman, 2005c; Workman and Kaye, 2002). Resistance was first identified in patients who relapsed while receiving imatinib and was associated with point mutations that rendered the ABL-kinase resistant to the drug, or to a lesser extent with BCR-ABL gene amplification (Gorre et al., 2001; Krause and Van Etten, 2005). Crystallographic studies have revealed that imatinib binds to the ATP site of ABL only when the activation loop of the kinase is closed and thus stabilizes the protein in this inactive confirmation (Schindler et al., 2000). Mutations at 17 different amino acid positions within the BCR-ABL kinase domain have so far been identified (Branford et al., 2003; Shah et al., 2002). The majority of amino acid substitutions prevent BCR-ABL from achieving the inactive conformational state required for imatinib binding (Shah et al., 2002). Many of the mutations found in experimental systems were also found in treated patients. This has led to the development of several second-generation ABL kinase inhibitors with increased potency and activity against most imatinib-resistant mutants. Foremost among these is dasatinib (BMS-354825, Sprycel), a dual ABL/SRC kinase inhibitor (Shah et al., 2004) which has recently been approved by the FDA. This drug binds to ABL but in an open, active conformation, and has demonstrated clinical activity in patients with a wide range of imatinib-resistant BCR-ABL kinase domain mutations (Shah et al., 2004; Talpaz et al., 2006).

The ability of imatinib to inhibit the mutated c-KIT tyrosine kinase, which is associated with the rare malignancy GIST, has resulted in further success for imatinib. GISTs are highly refractory to standard chemotherapies, but response rates close to 60% in clinical trials were achieved with imatinib, transforming the management of this disease (Demetri et al., 2002;

van Oosterom et al., 2001). The highest activity appears to correlate with activating mutations at exon 11 of the KIT gene, with less activity in GISTs expressing exon 9 mutations or wild-type KIT (Heinrich et al., 2003a). Interestingly, 35% of GISTs with wild-type KIT have constitutively active PDGFRa, which is also inhibited by imatinib, most likely explaining the excellent clinical responses to the drug in this cohort (Arteaga and Baselga, 2004; Heinrich et al., 2003b). Tumor genotype is also of major prognostic significance for progression-free survival and overall survival in patients treated with imatinib for advanced GISTs. The presence of exon 9-activating mutations are the strongest adverse prognostic factor for response to imatinib, increasing the relative risk of death by 190% (P < 0.0001) when compared with KIT exon 11 mutants. Patients with exon 9 mutations hence benefit the most from the higher (800 mg daily) dose of the drug (Debiec-Rychter et al., 2006).

Confirmation of imatinib's activity in GISTs has been possible through the use of FDG-PET. Initial studies showed high FDG uptake in untreated patients on PET imaging (Antoch et al., 2004; van Oosterom et al., 2001). PET was shown to be superior to CT in detection of the earliest functional parameters indicative of tumor response induced by imatinib therapy (Stroobants et al., 2003; van Oosterom et al., 2001). Overall GIST disease status as evaluated by changes in size, density, and number of tumor nodules and vessels within the lesion correlated best with the reduction of maximum standardized uptake values (SUV) on FDG-PET (Choi et al., 2004). For responding patients, FDG uptake in the tumor decreases markedly from base line as early as 24 h after a single dose of imatinib (Demetri et al., 2002). Other c-KIT and PDGFR tyrosine kinase inhibitors are currently in clinical trials, and have shown activity in imatinib-refractory GIST, with FDG-PET used to confirm tumor response (Sarker et al., 2005, see Fig. 4).

B. EGFR Inhibitors

The family of ERBB family of membrane tyrosine kinase receptors comprises four members: EGFR/ERBB1, ERBB2, ERBB3, and ERBB4. EGFR and ERBB2 are involved in the development of numerous types of human cancer and have been pursued as novel anticancer agents (Hynes and Lane, 2005). In preclinical models, treatment of tumor cells with ERBBB inhibitors leads to down-regulation of AKT, ERK 1/2, SRC and STAT signaling, and inhibits cell proliferation. Small molecule inhibitors or antibodies have shown definite evidence of clinical activity, but their development has also been marked by some high-profile failures. In this section, we review the use of these inhibitors with regard to their clinical use and development of PD endpoints.

EGFR overexpression occurs in a number of malignancies, including non-small cell lung cancer (NSCLC), glioma, prostate, pancreatic, colorectal,

Pharmacodynamic Biomarkers for Molecular Cancer Therapeutics PET-CT after 7 days of CHIR258

Reduction of FDG uptake in liver metastases after 7 days dosing of CHIR258

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PET-CT after 7 days off CHIR258

Increase in FDG uptake in liver metastases after 7 days of no treatment with CHIR258

Fig. 4 PET-CT of a patient with gastrointestinal stromal (GIST) tumor patient showing a response to a novel c-KIT inhibitor CHIR258. This patient with imatinib-refractory GIST was treated on a phase I trial of a multitargeted tyrosinse kinase inhibitor, with activity against both c-KIT and PDGFR (Sarker et al., 2005). The drug was given on a 7 days on, 7 days off schedule, and PET-CT showed significant decrease in uptake in her liver metastases during treatment compared to 7 days off treatment. The patient experienced prolonged stable disease for 9 months on the drug. Figure courtesy of Dr. Gary Cook, Nuclear Medicine Department, Royal Marsden Hospital, Sutton, UK and Chiron Corporation, Emeryville, CA.

and head and neck tumors (Hynes and Lane, 2005). Indeed, EGFR was the first tyrosine kinase receptor to be linked to human cancer, and linkage of EGFR expression to outcome in NSCLC other tumor types led to the subsequent development of drugs against this target (Gschwind et al., 2004). Among the most prominent have been the small molecule inhibitors, gefitinib (Iressa) and erlotinib (Tarceva), and the monoclonal antibody cetuximab (Erbitux). The development of gefitinib and erlotinib will be discussed here.

Gefitinib and erlotinib are small molecule anilinoquinazolines that are selective, competitive inhibitors of ATP binding by EGFR; both drugs have been approved for use in advanced NSCLC. Gefitinib monotherapy showed partial response rates of 9-19% in phase II trials of patients with refractory advanced NSCLC (Kris et al., 2003). Erlotinib also improved disease-related symptoms, and disease-free and overall survival compared with placebo (Shepherd et al., 2005). However, the addition of either gefitinib or erlotinib to chemotherapy in the initial treatment of NSCLC showed no evidence of survival benefit (Giaccone et al., 2004; Herbst et al., 2004).

It was apparent from the very first studies that a subgroup of NSCLC patients with adenocarcinomas, and specifically those that were Asian, female, and never smoked, often had dramatic, durable responses to gefiti-nib. Subsequent sequencing of the EGFR gene in tumor tissues from these patients showed heterozygous somatic mutations within the tyrosine kinase domains of EGFR; these enhanced the responsiveness of the receptor to EGF ligand and increased its sensitivity to gefitinib and erlotinib, as well as preferentially activating the antiapoptotic AKT and STAT signaling pathways (Lynch et al., 2004; Pao et al., 2004; Sordella et al., 2004). However, in the National Cancer Institute of Canada (NCIC) randomized trial of erloti-nib in NSCLC, the accompanying molecular correlates study showed that expression of EGFR and amplification of the EGFR gene were significantly associated with response to erlotinib, but not increased survival; this suggests that erlotinib may depend for its activity on other signaling pathways that were not assessed in this study, such as AKT phosphorylation or ERBB3 status (Doroshow, 2005; Engelman et al., 2005; Tsao et al., 2005). The NCIC study contrasts with studies from Asia where EGFR mutant tumors are more common, and are associated with both significantly increased response rate and overall survival (Han et al., 2005; Mitsudomi et al., 2005). These studies exemplify the need for more attention to be placed on the appropriate selection of patients for molecular cancer therapeutics.

A series of highly elegant PD studies were performed in the original phase I trials of gefitinib. In these, skin was proposed as a surrogate tissue because of ease of access and high EGFR expression. In these studies by Baselga, Albanell, and colleagues, paired skin biopsies were taken pretherapy and on-therapy (day 28); immunohistochemical analysis of EGFR status, EGFR phosphorylation, ERK 1/2 phosphorylation, proliferation marker Ki67, p27KIP1, keratin 1, and phospho-STAT3 expression were performed in paraffin-embedded sections (Albanell et al., 2002; Baselga et al., 2002). Statistically significant inhibition of EGFR activation in the basal layer of interfollicular epidermis and in hair follicle keratinocytes was achieved in all paired skin samples during gefitinib treatment. ERK 1/2 phosphorylation was assayed as a downstream marker of EGFR signaling and showed a significant reduction in expression at day 28, providing proof of concept for target inhibition. In addition, cell proliferation index (using Ki67) was reduced, and there was increased expression of the CDK2 inhibitor p27KIP1 reflecting induction of G1 cell-cycle arrest, as previously demonstrated in preclinical models (Busse et al., 2000). Hematoxylin and eosin staining of skin during therapy showed that the stratum corneum of the epidermis was thinner and contained an increased number of apoptotic cells. All of these effects on the target and EGFR-dependent molecular endpoints were observed at every dose level, indicating a lack of dose-response effect. It is therefore likely that potentially biologically active concentrations were achieved at all dose levels (see Fig. 5).

Fig. 5 Immunohistochemical demonstration of pharmacodynamic changes in tumor and skin in response to treatment with EGFR inhibitors. Immunohistochemical sections of skin and tumor biopsies pretherapy versus on-therapy for (A) a patient with breast carcinoma treated with gefitinib and (B) a patient with NSCLC treated with erlotinib. The upper panels show sections of skin and the lower panels show sections of tumor. In each case the pretreatment samples are shown above the posttreatment samples. Both patients show inhibition of the phosphorylation of EGFR and MAPK (ERK 1/2) both in tumor and skin. In addition, a clear decrease of proliferation, as measured by the Ki67 marker, is observed. Figure courtesy of Dr. Federico Rojo and Dr. Jose Baselga, Val d'Hebron University Hospital, Barcelona, Spain.

Fig. 5 Immunohistochemical demonstration of pharmacodynamic changes in tumor and skin in response to treatment with EGFR inhibitors. Immunohistochemical sections of skin and tumor biopsies pretherapy versus on-therapy for (A) a patient with breast carcinoma treated with gefitinib and (B) a patient with NSCLC treated with erlotinib. The upper panels show sections of skin and the lower panels show sections of tumor. In each case the pretreatment samples are shown above the posttreatment samples. Both patients show inhibition of the phosphorylation of EGFR and MAPK (ERK 1/2) both in tumor and skin. In addition, a clear decrease of proliferation, as measured by the Ki67 marker, is observed. Figure courtesy of Dr. Federico Rojo and Dr. Jose Baselga, Val d'Hebron University Hospital, Barcelona, Spain.

Further studies are required with respect to the ongoing or completed phase II/III trials in an attempt to correlate the above biomarkers with response, survival, and EGFR mutational status. A number of limitations are apparent. There has been a lack of PD data from tumor tissue. PD effects measured in well-vascularized surrogate tissues such as skin may not predict for similar effects in poorly vascularized tumors. In addition, the activation of various signal transduction pathways in tumors might lead to different PD responses compared to healthy cells. Higher doses might be needed to inhibit EGFR activation in tumors than would be predicted by the effects measured in skin samples (Dancey and Freidlin, 2003).

In a recent phase II study of gefitinib at a dose of 500 mg/day in patients with advanced breast cancer, paired skin and tumor biopsies were obtained pretherapy and after 28 days of treatment, and PD assays performed as in the original phase I studies (Baselga et al., 2005). Gefitinib caused complete inhibition of EGFR phosphorylation in both skin and tumor (see Fig. 5). However, downstream consequences of receptor blockade were distinct in skin and tumor: ERK 1/2 phosphorylation was inhibited in both tissues, while gefitinib caused induction of p27KIP1 and a decrease in Ki67 in skin only. In addition, gefitinib did not result in a decrease in phosphorylated AKT in tumors (not done on skin). No complete or partial responses were observed in this study, indicating that in this setting inhibition of EGFR phosphorylation may be an indicator of target inhibition, but not of sensitivity to anti-EGFR agents. While inhibition of target may be required for antitumor effect, sensitivity to EGFR inhibitors is likely to be related to level of receptor dependence or "oncogene addiction" in the individual tumor.

A great deal has been learned from the assessment of biomarkers in the development of gefitinib. Unfortunately much of this was learned too late. The development of EGFR inhibitors illustrates the need for greater efforts to develop standardized assay procedures for assessing and predicting the effects of novel molecularly targeted agents, to standardize methods for the prospective collection of tumor samples, and to incorporate these assays into phase II/III clinical trials to maximize the likelihood of definitive clinical results (Doroshow, 2005).

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