clitaxel in patients with EGFR mutations. In contrast, biomarker analysis also revealed that in EGFR mutation-negative patients, progression free survival was significantly shorter with gefitinib than with carboplatin/paclitaxel. Results from these studies suggest that therapy for NSCLC could be tailored according to mutational status in order to improve patient outcome. Increased EGFR gene copy number may be associated with improved response rates with TKI therapy, and possible survival benefits [47–49]. Studies comparing the relationship between EGFR gene copy number and patient outcome following gefitinib therapy in patients with advanced NSCLC concluded that high EGFR gene copy number was associated with better survival, and may potentially be Ridaforolimus effective for predicting the efficacy of gefitinib therapy [47, 48]. A multivariate analysis by Tsao and colleagues revealed that expression of an increased EGFR copy number, but not mutations in EGFR, was associated with improved survival with second or third line erlotinib in the BR21 trial. However, this did not translate into a survival advantage in the treatment group [49]. In contrast to the first-line IPASS trial, mutation analysis was problematic in the BR21 trial because the tissue analyzed was not obtained contemporaneously with treatment. Retrospective analyses in NSCLC

patients treated with TKIs have investigated the potential for EGFR expression as a biological marker. Evidence for a possible link between EGFR overexpression and Ridaforolimus mTOR inhibitor treatment sensitivity is less clear as results appear to be conflicting [50]. Therefore, EGFR expression may not be the optimal method for patient selection according to a specific treatment. Molecular markers of resistance to EGFR inhibition In patients benefiting from EGFR inhibition, acquired resistance inevitably develops, even in patients with EGFR mutations. A number of molecular events, in particular EGFR mutations, are associated with the development of resistance to TKI therapy following initial response. The T790M EGFR mutation is the most common; approximately 40–50% of cases with acquired resistance to first generation EGFR inhibitors can be accounted for by the T790M mutation, in exon 20 of the EGFR kinase domain. The mutation results in the insertion of a bulky methionine residue, which interferes with TKI access to the active site (“gatekeeper” mutation) [51, 52]. A molecular analysis of circulating tumour cells from 27 TKI-na?ve patients with metastatic NSCLC found the T790M mutation in cancer clones from 38% of patients. The presence of T790M, even before patient exposure to TKI, was associated with a significantly shorter Ridaforolimus 572924-54-0 progression-free survival compared with patients who did not have detectable levels of T790M [53]. Other mutations may also lead to resistance [54, 55]. T854A is a novel mutation, which leads to substitution of alanine for threonine at position 854 in exon 21 of EGFR and subsequent resistance to first-generation

TKIs [54]. A molecular analysis of tumor cells obtained from patients with acquired resistance discovered a further novel secondary mutation of the EGFR kinase domain, D761Y [55]. Results suggest that the D761Y mutation, located in exon 19, decreases the sensitivity of mutant EGFR to TKIs. Alterations in parallel signalling pathways may overcome the effects of TKI therapy, such as MET amplification [56, 57]. The presence of mutations in other gene pathways may be associated with intrinsic resistance and the lack of sensitivity to TKI therapy. An activating KRAS mutation is present in 15–25% of adenocarcinomas and is associated with lack of sensitivity to TKIs Two approaches have been developed to overcome the limitations associated with first-generation TKIs: (1) activity against multiple receptor targets and (2) irreversible (covalent) binding. Multiple targets Cancer development and progression is driven by a variety of complex processes and interactions; molecular pathways in a tumor can therefore be adaptable and redundant [13]. The ErbB receptors have various interactions within the receptor family, forming different homo- and heterodimers with each other [7, 8]. This allows HER2, which has no identified ligand, and HER3, which has no kinase activity, to become actively involved in signalling. Therefore, therapy focusing on a single target might be unlikely to achieve adequate, long-term disease control for many patients.

A number of studies have provided increasing evidence supporting the dual inhibition of two or more receptors rather than single receptor targeting. Preclinical experiments have demonstrated that ErbB receptors act synergistically to cause malignant transformation in NIH3T3 cells and that either receptor alone is insufficient to induce this effect [60, 61]. Studies have also demonstrated that tumor cells can overcome the effect of an agent targeted to a particular ErbB receptor, by the presence of ligand for an alternative receptor [62]. In breast cancer cells