Estimating copy number using next-generation sequencing to determine ERBB2 amplification status.

Assessing Erb-b2 receptor tyrosine kinase 2 (ERBB2) amplification status in breast and gastric cancer is necessary for deciding the best therapeutic strategy. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are currently used for assessing protein levels and gene copy number (CN), respectively. The use of next-generation sequencing (NGS) to measure ERBB2 CN in breast cancer is approved by the United States Federal Drug Administration as a companion diagnostic. However, a CN of less than 8 is evaluated as "equivocal", which means that some ERBB2 amplification cases diagnosed as "HER2 negative" might be false-negative cases. We reviewed the results of gene profiling targeting 160 cancer-related genes in breast (N = 90) and non-breast (N = 19) cancer tissue, and compared the ERBB2 CN results with the IHC/FISH scores. We obtained an estimated CN from the measured CN by factoring in the histological proportion of tumor cells and found that an ERBB2-estimated CN of 3.2 or higher was concordant with the combined IHC/FISH outcome in 98.4% (88/90) of breast cancer cases, while this was not always evident among non-breast cancer cases. Therefore, NGS-estimated ERBB2 CN could be considered a diagnostic test for anti-HER2 therapy after the completion of adequate prospective clinical trials.

Antitumor effect of the tyrosine kinase inhibitor nilotinib on gastrointestinal stromal tumor (GIST) and imatinib-resistant GIST cells.

Despite the benefits of imatinib for treating gastrointestinal stromal tumors (GIST), the prognosis for high risk GIST and imatinib-resistant (IR) GIST remains poor. The mechanisms of imatinib resistance have not yet been fully clarified. The aim of the study was to establish imatinib-resistant cell lines and investigate nilotinib, a second generation tyrosine kinase inhibitor (TKI), in preclinical models of GIST and imatinib-resistant GIST. For a model of imatinib-resistant GIST, we generated resistant cells from GK1C and GK3C cell lines by exposing them to imatinib for 6 months. The parent cell lines GK1C and GK3C showed imatinib sensitivity with IC50 of 4.59±0.97 µM and 11.15±1.48 µM, respectively. The imatinib-resistant cell lines GK1C-IR and GK3C-IR showed imatinib resistance with IC50 values of 11.74±0.17 µM (P<0.001) and 41.37±1.07 µM (P<0.001), respectively. The phosphorylation status of key cell signaling pathways, receptor tyrosine kinase KIT (CD117), platelet-derived growth factor receptor alpha (PDGFRA) and downstream signaling kinases: serine-threonine kinase Akt (AKT) and extracellular signal-regulated kinase 1/2 (ERK1/2) or the non-receptor tyrosine kinase: proto-oncogene tyrosine-protein kinase Src (SRC), was analyzed in established cell lines and ERK1/2 phosphorylation was found to be increased compared to the parental cells. Nilotinib demonstrated significant antitumor efficacy against GIST xenograft lines and imatinib-resistant GIST cell lines. Thus, nilotinib may have clinical potential for patients with GIST or imatinib-resistant GIST.

Establishment and characterization of novel cell lines and xenografts from patients with gastrointestinal stromal tumors.

At present, no suitable GIST model exists for the analysis of drug resistance or metastasis using established human gastrointestinal stromal tumor (GIST) cell lines or xenografts even though the molecular mechanisms of drug resistance, progression and metastasis require clarification. The aim of this study was to establish and characterize human GIST cell lines and xenografts that can be used for evaluating drug resistance or various new molecularly targeted therapies. GIST tissues from patients were cultured and implanted under the skin of NOG (NOD/Shi-scid, IL-2Rrnu) mice. Two new cell lines (GK1C and GK3C) and three xenografts (GK1X, GK2X and GK3X) were generated from these clinical samples. The established GIST cell lines and xenografts were investigated for tumorigenesis and imatinib sensitivity. These cell lines and xenografts showed characteristic GIST morphology and exhibited KIT expression profiles similar to those of the patient samples. In addition, these GIST cell lines and xenografts were sensitive to imatinib. In conclusion, new human GIST cell lines and xenografts were established and maintained through repeated passages. These models will enable further study of combination therapies and the mechanisms of resistance, and allow testing of novel targeted monotherapies and combination therapies.

Antitumor effect of cetuximab in combination with S-1 in EGFR-amplified gastric cancer cells.

Overexpression of human epidermal growth factor receptor (EGFR) has been detected in gastric cancer (GC) and is associated with poor outcomes. Combination treatment regimens with EGFR-targeting agents and cytotoxic agents are considered to be a potential therapeutic option for EGFR-overexpressing GC. Herein, we have investigated the effects of combination treatment with the oral fluoropyrimidine S-1 and the EGFR-targeting agent cetuximab in GC cells with or without EGFR overexpression. EGFR expression was determined by FACS and quantitative PCR in GC cells. Experimental 5-fluorouracil (5FU) was used instead of S-1 for in vitro experiments. The efficacy of 5FU or cetuximab monotherapy or combination 5FU/cetuximab therapy was examined in vitro and in vivo. Clinical specimens were examined for EGFR by immunohistochemistry (IHC). EGFR expression score was defined as strong membrane and cytoplasmic staining in at least 50-75% of cells. The combination of 5FU and cetuximab synergistically inhibited cell proliferation and exhibited an enhanced proapoptotic effect in GC cells with EGFR overexpression. Cetuximab also induced down-regulation of phosphorylation of EGFR and AKT, leading to diminished signaling. The antitumor effect of the combination of S-1 and cetuximab in vivo was also greater than that of either drug alone. Our preclinical findings thus indicate that the combination of S-1 and EGFR-targeting therapy is a promising treatment option for GC with EGFR overexpression.