Studies on Genomic Alterations in HER2-Positive Breast Cancer–Focus on Design, Synthesis & Evaluation of Anilinoquinazoline Analogues as Potential HER2 inhibitors

Abstract

Human epidermal growth factor receptor 2-positive (HER2-positive) breast cancer is an aggressive breast cancer subtype characterized by HER2 overexpression/amplification. Genomic alterations of HER2 and others have been reported to be associated with, HER2 overexpression and prediction of trastuzumab-response. The current study was carried out to identify genomic alterations associated with HER2-positive breast cancer and evaluate their association with clinical outcome in response to trastuzumab therapy given to HER2- positive breast cancer patients. Global Sequencing Array (GSA) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques were used to determine alterations in HER2 and other HER2-interacting as well as signaling-related genes implicated in the disease. In addition, 20 formalin fixed paraffin-embedded (FFPE) tissue samples were also evaluated by GSA for identifying significant variations associated with the disease as well as response to trastuzumab therapy. A germline variant in HER2 gene (I655V) was found to be significantly associated with the risk of the disease (p < 0.01). A nonsense mutation in PTPN11 (K99X), a pathogenic CCND1 splice site variant (P241P), a hotspot missense mutation in PIK3CA (E542K) and a hotspot missense mutation in TP53 (R249S); were observed in 25%, 75%, 30% and 40% of the HER2-positive breast cancer tissue samples, respectively. Mutant CCND1 (P241P) and PIK3CA (E542K) were found to be significantly associated with reduced disease-free survival (DFS) in patients treated with trastuzumab (p: 0.018 and 0.005, respectively). These results indicate that HER2, PTPN11, CCND1 and PIK3CA genes are important biomarkers in HER2-positive breast cancer. Moreover, the patients harboring mutant CCND1 and PIK3CA exhibit a poorer clinical outcome as compared to those carrying wild-type CCND1 and PIK3CA. Development of resistance and disease-relapse are the major problems associated with trastuzumab. Tyrosine-kinase inhibitors (TKIs) present better option to address the issues associated with trastuzumab. However, problems of resistance and ineffectiveness as monotherapy; persist with the currently available TKIs as well. We synthesized anilinoquinazoline-based compounds and evaluated them for anti-proliferative activity against HER2-positive breast cancer. Of the synthesized compounds (HS-2, HS-3, HS-5, HS-8 and HS-9), three (HS-3, HS-5 and HS-8) were evaluated for biological activity. HS-8 proved to be most-effective against SKBR3 (HER2-positive breast cancer cells) (IC50=2.8µM) iv with a lesser cytotoxicity towards the MDA-MB-231 (Triple-negative breast cancer cells) (IC50=3.2µM) and no toxicity towards FR-2 (normal breast epithelial cells).