Overexpression of the ATP-dependent drug efflux pump ABCG2 is a major molecular mechanism of multidrug resistance in cancer and might be a predictive biomarker for drug response. Contradictory results have been reported for immunohistochemical studies of ABCG2 protein expression in colorectal cancer (CRC), probably because of the use of different antibodies and scoring approaches. In this study, we systematically studied six commercially available anti-ABCG2 antibodies, using cell lines with up-regulation of ABCG2, and selected one antibody for validation in CRC tissue. Furthermore, we established scoring guidelines for ABCG2 expression based on the clinically used guidelines for HER2 immunohistochemistry assessment in gastric cancer. The guidelines provide a semi-quantitative measure of the basolateral membrane staining of ABCG2 and disregard the apical membrane staining and the cytoplasmic signal. Intra-tumor heterogeneity in ABCG2 immunoreactivity was observed; however, statistical analyses of tissue microarrays (TMAs) and the corresponding whole sections from primary tumors of 57 metastatic CRC patients revealed a strong positive correlation between maximum TMA scores and whole sections, especially when more than one core was used. In conclusion, here, we provide validated results to guide future studies on the associations between ABCG2 immunoreactivity in tumor cells and the benefits of chemotherapeutic treatment in patients with CRC.

In this study we investigated the use of cancer cell protein expression of ABCG2 to predict efficacy of systemic first-line irinotecan containing therapy in patients with metastatic colorectal cancer (mCRC). From a Danish national cohort, we identified 119 mCRC patients treated with irinotecan containing therapy in first-line setting. Among these, 108 were eligible for analyses. Immunohistochemistry (IHC) analyses were performed on the primary tumor tissue in order to classify samples as high or low presence of ABCG2 protein. Data were then associated with patient outcome (objective response (OR), progression free survival (PFS) and overall survival (OS)). ABCG2 protein expression in the basolateral membrane was high (score 3+) in 33% of the patients. Exploratory analyses revealed a significant interaction between ABCG2 score, adjuvant treatment and OR (p = 0.041) in the 101 patients with evaluable disease. Patients with low ABCG2 (score 0–2) and no prior adjuvant therapy had a significantly higher odds ratio of 5.6 (Confidence Interval (CI) 1.68–18.7; p = 0.005) for obtaining OR. In contrast, no significant associations between ABCG2 expression and PFS or OS were found. These results suggest that measurement of the ABCG2 drug efflux pump might be used to select patients with mCRC for irinotecan treatment. However, additional studies are warranted before conclusions regarding a clinical use can be made. Moreover, patients with high ABCG2 immunoreactivity could be candidates for specific ABCG2 inhibition treatment in combination with irinotecan.

Today, chemotherapy and molecularly targeted therapies are the main therapeutic options in cancer treatment, but their effectiveness is limited by drug resistance, a major problem facing current cancer research. Since its establishment in the late 1980s, the NCI60 cell line panel has generated tremendous value, economic as well as societal, by contributing to the identification of mechanisms of drug action. This information, in turn, has been used to create new anti-cancer drugs for the benefit of patients. However, the cell lines comprised in the NCI60 panel are mostly derived from treatment of chemo-naïve patients (no medical treatment). Since early clinical trials of anti-cancer drugs most often recruit patients who have failed several different prior treatments, there is a need to develop a platform that builds on the concept underlying the NCI60 cell line panel and extend it to allow testing of potential new drugs in the setting of drug resistance. Moreover, it is also well-known that molecular drug resistance mechanisms might first appear following drug exposure and access to panels of matched drug-sensitive and drug-resistant isogenic cell lines, is thus mandatory for studying the underlying mechanisms of acquired drug resistance. We have initiated the establishment of DEN-50R, a novel and unique cell line-based drug screening platform within cancer treatment. The platform will, when fully developed, consist of human cancer cell lines representing the five most common cancer forms (colorectal, prostate, lung, breast cancer and malignant melanoma) and matched drug-resistant sublines for each of the five cancer forms. In a second phase additional cancer forms will be included. Resistance is developed against drugs and drug combinations currently used in the clinic, and with the introduction of novel drugs and regimens, new corresponding resistant sublines will be generated. All pairs of sensitive and resistant cell lines are thoroughly characterized with regard to cross-resistance to other drugs as well as to genomics, epigenomics, transcriptomics, and proteomics profiling. All data generated with the DEN-50R platform will be placed in the new National Danish Life Science Supercomputer with secure, regulated access providing a unique and innovative integrative analysis resource for academic, clinical and industrial researchers involved in anti-cancer drug development and cancer diagnostics at both pre-clinical and clinical levels. We envision that the DEN-50R be used to: • Test novel chemical entities for anti-cancer effects, including potential lack of cross-resistance thereby facilitating selection of analogs for further preclinical and clinical testing. • Test already known drugs for potential effects in drug resistant cell lines facilitating “repurposing” of drugs. • Reveal novel molecular drug resistant mechanisms allowing for targeted development of new drug entities that can circumvent drug resistance • identify novel predictive biomarkers for the most common drug entities further facilitating a personalized treatment approach. Citation Format: Jan Stenvang, Jose Moreira, Niels Frank Jensen, Signe Lykke Nielsen, Torben Orntoft, Ulrik Lassen, Cathrine Nordgaard, Stine Ninel Hansen, Haatisha Jandu, Anna Vind, Maria Andreasen, Camilla Cederbye, Julie Noer, Thiago Lima, Birgitte Martine Viuff, Soeren Brunak, Thomas Skoet Jensen, Peter Buhl Jensen, Joergen Drejer, Henrik Ditzel, Nils Brünner. DEN-50R - establishment of a novel and unique cell line based drug screening platform for cancer treatment. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B71.