In accordance with the Helsinki Declaration, all patients provided written informed consent for use of their samples, and the collection and use of the samples received Institutional Review Board (IRB) approval.
Archival tissue samples from 118 patients who had locally advanced or metastatic breast cancer and had failed treatment with anthracycline-, taxane-, and trastuzumab-containing regimens were studied. Specimens were obtained from patients who had histologically confirmed invasive breast cancer with Stage IIIB, Stage IIIC with T4 lesion, or Stage IV disease (GSK Study EGF100151), with documentation of ERBB2 overexpression (IHC 3+ or IHC 2+ with fluorescence in situ hybridization [FISH] confirmation) [28, 29]. Microarray analysis was performed at Response Genetics, Inc (Los Angeles, CA, USA).
Frozen tissue samples from 29 patients who were presurgical, treatment-naive, with Stage IA or Stage IB, resectable NSCLC were studied (GSK Study VEG105290). Microarray analysis was performed at Weill Medical College of Cornell University (New York, NY, USA).
Archival breast tumor specimens were formalin-fixed and embedded in paraffin. The quality of tumor tissue RNA extraction was assessed by laser capture microdissection. Reverse transcription-polymerase chain reaction (RT-PCR) for 300 base pair (bp) of the β-actin gene was used for quality control of these samples. Samples with a cycle time (CT) of < 32 at the 300 bp level were acceptable for microarray analysis. mRNA and cDNA were prepared from archival breast tumor tissues and frozen NSCLC samples using the Affymetrix HG-U133 Plus2 array (Affymetrix, Inc., Santa Clara, CA, USA). RNA microarray gene signal intensity was normalized using the robust microchip analysis (RMA) method described by Irizarry et al . Samples with RMA < 50 are below the level of detection by this method.
Samples of normal tissue cDNA and tumor tissue cDNA were obtained from Cytomyx (Lexington, MA, USA). Seven samples of normal breast tissue were studied, and 41 breast carcinoma samples, including tissue from cancers of Stage I, Stage II, and Stage IIIB were analyzed. Eight normal lung specimens and lung tissue samples from 40 tumors, ranging from Stages IA to IV were examined. Ovarian samples included 7 normal ovarian tissues and 41 ovarian tumors ranging from Stages I to IV.
MPL gene expression was measured using FAM-TAMRA-labeled primers and probes in a 7900HT thermal cycler using a standard 40-cycle profile with 9600 emulation. Following amplification, calculations were performed to determine the relative abundance of MPL normalized to 3 housekeeping genes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin (ACTB), and cyclophilin A (PPIA). Each result is presented as a normalized value of mRNA. EPOR, ERBB2, and IGF1R were also assessed to provide data for comparable receptors. Primers and probes were custom made by Integrated DNA Technologies (Coralville, IA, USA). Sequences for the primers and probes were previously described  and are listed in Additional file 1: Table S1.
qRT-PCR data analysis
CT values for genes of interest were normalized to the internal housekeeping genes run in the reaction using in-house software. Raw abundance value was calculated using the following equation: Abundance = 10e[(40-CT)/3.35]. Samples were scaled relative to each other using the geometric mean of the set of valid housekeeping gene data points for that sample. Each data point was then expressed as the ratio of the housekeeping gene abundance in the sample to the average of that housekeeping gene in all samples and marked invalid if it had statistically inconsistent behavior with the other housekeeping genes in those samples of similar tissue types. Samples with a relative abundance of < 50 are below the level of detection of this method.
IHC for TPO-R protein expression
Formalin-fixed, paraffin embedded (FFPE) controls and FFPE specimens of breast, lung, and ovarian cancer were procured by Mosaic Laboratories under an IRB-reviewed protocol that allows for use of remnant, de-identified, or anonymized human samples for in vitro analysis under the guidelines defining Exemption from Human Subject Research as defined by the Office of Human Research Protection. FFPE tissue samples of breast, lung, and ovarian cancer were also procured from OriGene (formerly Cytomyx) for analysis. A FFPE block of N2C-Tpo cells, a Tpo-dependent megakaryocytic leukemia cell line, and of normal bone marrow were used as positive controls for TPO-R expression.
Immunohistochemical analysis was performed in accordance with Mosaic Laboratories’ Standard Operating Procedures. Briefly, the procedure was performed manually using the Envision™ system and ancillary reagents (Dako, Carpinteria, CA). Specimens were sectioned at a thickness of 4 microns, mounted onto positive-charged glass slides, dried, deparaffinized, and rehydrated. Following rehydration, tissue sections were incubated in Envision peroxidase for 5 minutes to quench endogenous peroxidase. Heat-induced epitope retrieval was performed using Rip Tide buffer for 40 minutes at 95°C. Slides were incubated with anti-CD110 antibody (clone 1.78.1) (BD Biosciences) diluted in diluent (Dako) for 30 minutes. Slides were then rinsed twice in Splash-T buffer for 5 minutes each. Signal was visualized using Envision + Mouse HRP detection reagent (Dako) for 30 minutes, followed by 3,3 diamino benzidine according to manufacturer’s instructions. Slides were rinsed with water, counterstained with hematoxylin (Dako), dehydrated through graded alcohols, cleared in xylene, and coverslipped for microscopic evaluation.
Staining was evaluated by a pathologist; evaluation of reactivity involved a combination of the following: cellular localization of staining, staining intensity, subcellular localization, and percentage of cells staining in the primary component of the tissue type of interest. The CD110 IHC assay was scored on a semi-quantitative scale, and the percentage of cancer cells staining at each of the following 4 levels was recorded: 0 (unstained), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). An H-score was calculated based on the summation of the product of percent of cells stained at each intensity, using the following equation: (3 × % cells staining at 3+) + (2 × % cells staining at 2+) + (1 × % cells staining at 1+). H-score values range from 0–300. (Additional file 2: Table S2).
Cell line proliferation assays
Cell lines were obtained from the American Type Culture Collection (Walkersville, MD, USA). All cells were maintained in log-phase growth in their respective media. The breast cancer cell lines (MCF-7, BT474, and HCC1937), lung cancer cell lines (A549, NCI-H226, NCI-H460, and NCI-H510), and ovarian cell lines (OVCAR4 and SKOV-3) were grown in RPMI 1640 medium with 10% fetal calf serum (FCS). The ovarian carcinoma cell line OVCAR3 was grown in RPMI 1640 medium with 20% FCS, 1% sodium pyruvate, and 1% v/v glutamine.
All experiments were performed with milled, monoethanolamine salt form SB-497115-GR (eltrombopag) resuspended in water to 10 mg/mL, and diluted in Iscove’s Modified Dulbecco’s Medium (IMDM) with 1% FCS. Recombinant human TPO (rhTPO) was purchased from R&D Systems (Minneapolis, MN, USA) and diluted in IMDM.
Cells for the Cell Titer Glo® assay were plated at 1 × 103 cells/well in 96-well plates in medium containing 10% FCS and allowed to adhere for 24 hours. Cells were treated with eltrombopag at 0, 0.1, 0.4, 1, 4, 10, and 40 μg/mL. In breast and ovarian cancer cell lines, eltrombopag was also tested at 100 μg/mL. rhTPO at 100 ng/mL was also tested in these experiments. Cells were incubated for 72 hours at 37°C in 5% CO2 after the addition of eltrombopag or rhTPO. Active cell determinations were performed using the Cell Titer Glo® reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocol. Results were reported as relative luminescence units (RLU).
The calculated mean and standard deviations were produced using triplicate samples for each experiment. The IC50 was determined using XLfit version 4.2.1, utilizing the best fit model for each data set.
Western blotting for TPO-R protein expression
Lung cancer cell lines, A549, NCI-H226, NCI-H460, and NCI-H510, were grown as described above.
Western blots for TPO-R protein expression were performed on reduced cell lysates of log-phase growth lung cancer cell lines (50 μg/well) on a NuPage 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) with MOPS running buffer. Precision Protein Dual Color Standards (Bio-Rad, Hercules, CA, USA) were used. The gels were transferred to nitrocellulose and stained with a rabbit polyclonal anti-TPO-R primary antibody (Upstate Biotechnology Inc., Lake Placid, NY, USA; Cat# 06–944) and analyzed with an Odyssey® infrared imager (LI-COR Biosciences, Lincoln, NE, USA).