This investigation was also supported by NIH Research Grant CA-16359 from the NCI
This investigation was also supported by NIH Research Grant CA-16359 from the NCI. List of abbreviations CScarcinosarcomaHER2/ErbB2epidermal growth factor 4-Aminobutyric acid type 2 receptorT-DM1Trastuzumab-emtansineTtrastuzumabFISHfluorescence in situ hybridizationIHCimmunohistochemistryqRT-PCRquantitative real time polymerase chain reactionADCCantibody-dependent-cell-mediated-cytotoxicityMMMTMalignant Mixed Mllerian TumorsDM1maytansinoid cytotoxinASCO/CAPAmerican Society of Clinical Oncology and the College of American PathologistsPBLperipheral blood lymphocytesIVintravenousIACUCInstitutional Animal Care and Use CommitteeMFImean fluorescence intensityADCantibody-drug conjugateNKnatural killer Footnotes Competing interests The authors declare that they have no competing interests.. the subset of HER2 positive CS patients with disease refractory to chemotherapy. followed by developing a supportive CS model. Methods Establishment of Carcinosarcoma cell lines Study approval was obtained from the Institutional Review Board, and all patients signed consent prior to tissue collection according to the institutional guidelines. A total of eight primary uterine and ovarian CS cell lines were established from fresh tumor biopsy samples, as described previously [27]. Tumors were staged according to the International Federation of Gynecology and Obstetrics staging system. Patient characteristics are noted 4-Aminobutyric acid in Table 1. Table 1 Patient Characteristics hybridization (FISH) analysis was performed using the PathVysion HER2 DNA-FISH-Kit (Abbott Molecular Inc., Abbott Park, IL, USA) according to the manufacturers instructions and as previously described [27]. Fluorescent signals in at least 30 non-overlapping interphase nuclei with intact morphology were scored using an Olympus BX61 microscope (Olympus America Inc.) with a 60-x planar objective, using the filter that permits simultaneous green and red colors. Tumor cells were scored for the number of orange (HER2/neu) and green (CEP17) signals. HER2/neu amplification was defined by a HER2/neu/CEP17 copy ratio 2.0 Quantitative real-time polymerase chain reaction (qRT-PCR) RNA extraction from all carcinosarcoma cell lines and from normal control tissue was performed using AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germantown, MD) according to the manufacturers instructions. Total RNA (5 g) was reverse-transcribed using Superscript III (Invitrogen, Carlsbad, CA). Quantitative PCR was carried out 4-Aminobutyric acid to evaluate the expression level of HER2 (ERBB2, Assay ID: Hs00170433_m1, Applied Biosystems) in all samples with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using the recommended protocol by the manufacturer. Each reaction was run in duplicate. The internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Assay ID: Hs99999905_ml, Applied Biosystems), was used to normalize variations in cDNA quantities from different samples. The comparative threshold cycle (Ct) method was used for the calculation of amplification fold as specified by the manufacturer and previously described [27]. Analyses were performed using SDS software 2.2.2 (Applied Biosystems/Life Technologies). Flow cytometry assay for HER2 staining Trastuzumab GRK4 (Herceptin, Genentech, South San Francisco, CA), a humanized monoclonal antibody (mAb) of the IgG1 isotype that binds with high affinity to the extracellular domain of the HER2 receptor was used to assess HER2 expression on the surface of tumor cells. All the primary carcinosarcoma cell lines were incubated with 2.5 g/ml of trastuzumab for 30 minutes on ice. Rituximab (Rituxan, Genentech, South San Francisco, CA), a chimeric anti-CD20 monoclonal-antibody (mAb) was used as a negative control (2.5 g/ml). A fluorescein isothiocyanate-conjugated Goat F(ab)2 Anti-Human immunoglobulin was used as a secondary reagent (Invitrogen, Life Technologies). Analysis was conducted with a FACScalibur, using Cell Quest software (BD Biosciences, San Diego, CA, USA). Test for antibody-dependant cell-mediated cytotoxicity (ADCC) Standard 4-hours chromium (51Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-Hypaque-separated peripheral blood lymphocytes (PBLs) from several healthy donors, in combination with T-DM1 or trastuzumab against the CS target cell lines at effector to target ratios (E:T) of 10:1 and 20:1. The release of 51Cr from target cells was measured as evidence of tumor cell lysis after exposure of the tumor cells to 2.5 g/ml of T-DM1 or 2.5 g/ml of T. Concentration-response experiments were performed in order to determine the optimal antibody dosing for ADCC experiments. The negative control condition was the incubation of target cells alone. As a positive control condition,.