Neratinib shows efficacy in the treatment of HER2 amplified carcinosarcoma in vitro and in vivo

Abstract

Objective Carcinosarcoma is a deadly gynecologic malignancy with few effective treatment options. The study of new therapies is difficult because of its rarity. The objective of this study was to determine the efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma. Methods The efficacy of neratinib in the treatment of HER2 amplified carcinosarcoma was determined in vitro using seven primary carcinosarcoma cell lines with differential expression of HER2/neu. Data regarding IC50, cell cycle distribution, and cell signaling changes were assessed by flow cytometry. The efficacy of neratinib was determined in treating mice harboring HER2 amplified carcinosarcoma xenografts. Results Two of seven (28.5%) carcinosarcoma cell lines were HER2/neu amplified. HER2/neu amplified cell lines SARARK6 and SARARK9 were significantly more sensitive to neratinib than the five non-HER2/neu amplified carcinosarcoma cell lines (mean±SEM IC50: 0.014μM±0.004 vs. 0.164μM±0.019 p=0.0003). Neratinib treatment caused a significant build up in G0/G1 phase of the cell cycle, arrest auto phosphorylation of HER2/neu and activation of S6. Neratinib inhibited tumor growth (p=0.012) and prolonged survival in mice harboring HER2 amplified carcinosarcoma xenografts (p=0.0039). Conclusions Neratinib inhibits HER2 amplified carcinosarcoma proliferation, signaling, cell cycle progression and tumor growth in vitro. Neratinib inhibits HER2/neu amplified xenograft growth and improves overall survival. Clinical trials are warranted.

Efficacy of neratinib in the treatment of HER2/neu-amplified epithelial ovarian carcinoma in vitro and in vivo

“…At the time of frozen section, small portions of viable tumor were collected for processing and establishment of primary cell lines. Tumor samples were processed and de-identified as previously described [14, 15]. Briefly, tumors were processed using a scalpel for mechanical disruption in an enzymatic solution of 0.14% collagenase type 1 (Sigma) and 0.01% DNase (Sigma, 2000 KU/mg) in RPMI 1640.…”

“…Neratinib’s efficacy was tested using tumor cell cultures established as cell lines using previously published protocols [15]. FISH and IHC testing were utilized to determine HER2/neu expression of each primary tumor [13].…”

“…FISH and IHC testing were utilized to determine HER2/neu expression of each primary tumor [13]. Briefly, cell lines that showed weak staining in<10% of cells were considered 0, those with staining in 10–30% of cells were classified as 2+, while cell lines with uniform staining in >30% of cells were classified as 3+ [15]. HER2/neu expression was determined by FISH assays in primary cell lines [16].…”

“…Solutions of different concentrations were then created by diluting the stock solution: 0.2, 0.02, 0.01, 0.002 and 0.0004 μM. Sterile water with 0.5% methylcellulose (Sigma Life Sciences, St. Louis, MO) and 0.4% polysorbate 80 were used to dissolve neratinib for in vivo experiments [15]. …”

“…Flow cytometry was utilized to read cell cycle, while FlowJo was used for the final analysis (FlowJo, Ashland, OR). The data from the cell cycle analysis experiments are expressed as mean ± standard deviation (mean ± SD) [15]. …”