Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r - 0.8) for expression of PD-L1. We compared PD-L1 expression by flow cytometry using a 1 % cutoff for positivity in the tumor cell population and a 1 % cutoff of cells with at least 1+ intensity in immunohistochemically stained tissue sections as positive. Ten of 12 lung tumor samples were concordant while 2 were discordant. PD-L1 expression by flow cytometry varied widely (1.2-89.4 %) even in the positive concordant cases. In addition, PD-L1 expression in the aneuploid tumor population did not necessarily agree with the expression in the diploid tumor population. Fine, unequivocal quantification of PD-L1 on tumor and immune cells in NSCLC may allow for better prediction of response to therapies. The present study also offers a technology that can create a universal sample type from either FNA or fresh tissue.
Background: An integrative system capable of detecting proteomic, genomic and DNA content from cell isolates obtained by fine needle aspiration (FNA) biopsy may offer distinct advantages in diagnosing breast cancer and monitoring response to therapy. Cellular Multiplex™ is such a system. An initial pilot study evaluating this technology established a series of variables that could separate normal from cancerous elements using cells obtained from an FNA performed on excised tumors and reduction mammoplasty specimens. In order for the technology to be clinically relevant, it must perform robustly on intact tumors. The current study was therefore undertaken to validate Cellular Multiplex™ on cells obtained by FNA performed on intact tumor at the time of diagnosis. Methods: Patients undergoing lumpectomy requiring either needle or 125I seed localization were identified. FNA was performed on intact tumor (A samples) at the time of radiographic localization prior to lumpectomy and repeated on the excised tumor (B samples). Cells obtained by FNA were placed in a proprietary fixative then hybridized and stained to detect multiple mRNA and protein targets along with DNA content. Estrogen receptor, progesterone receptor and HER2 were included in the panel of targets and compared to the routine clinical pathology report. Cell morphology was assessed by mean corpuscular volume. Samples were analyzed using an EC800 (Sony Biotechnology, San Jose, CA) and the results from matched A and B samples were compared using the Mann-Whitney Wilcoxson Rank Sum test. The study is designed to enroll 50 patients. Here we report an analysis of the first 9 cases. Results: The cell number obtained from the excised tumors were 3-4 times greater (median to median) than obtained from the intact tumor. There was no statistical difference in the expression of the 2 mRNA targets, 8 protein targets, DNA content and cell morphology between the A and B samples. The parameters derived from Cellular Multiplex matched the standard pathologic features reported on the clinical pathology report in 8 of 9 cases. In the one discrepant case, Cellular Multiplexing detected ER positive cells in a case where standard pathologic evaluation with immunohistochemistry reported the tumor to be estrogen receptor negative. Conclusions: This interim analysis demonstrates that the Cellular Multiplex technology is working well using cells obtained by FNA performed on intact tumors with readouts matching those obtained from excised specimens. If confirmed in the remaining patients, these data suggest that this technology will be applicable for the evaluation of intact tumors thereby making it relevant for multiple clinical indications including diagnosis and monitoring response to neoadjuvant therapy. Citation Format: Mittendorf EA, Dogan B, Morgan R, Chargin A, Wu Y, Cornett-Risher S, Shults K. Integrative pathology: Analysis of cellular multiplex technology to detect proteomic, genomic and DNA data from fine needle aspiration biopsy specimens. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-17.
Background: Human papillomaviruses are a family of DNA viruses that infect the epithelium leading to the formation of lesions with the ability to progress into carcinoma in many forms. Research into causality of head and neck cancers has found a link to HPV infections affiliated with better prognosis. In addition, advances in immuno-oncology have brought immunotherapy to the forefront of cancer treatment across cancer types. Here we present data that demonstrate a combined diagnostic approach to quantify two markers important in the management of head and neck cancer. Methods: Swabs were collected from patients of Institut Gustave Roussy with lesions of the oral pharynx. Swabs were collected and placed into a collection vial with a proprietary fixation/permeabilization solution (IncellCollect, IncellDx, Inc.) and shipped overnight on cold packs for processing. Upon receipt, samples were passed through a 35 µM filter to remove aggregates. Cells were labeled with CD45 antibodies to separate epithelial from immune cells, RNA in situ hybridization with E6, E7 mRNA probes (HPV OncoTect) was performed, and these cells were labeled with PD-L1 Ab (clone 28-8) prior to analysis on the flow cytometer (CytoFlex, Beckman Coulter, Inc). Samples were also stained with DAPI to identify single nucleated cells, and to analyze cell cycle. Results: We analyzed samples from 8 patients with oral cancer with the combined E6, E7 mRNA/PD-L1 protein assay by flow cytometry. The percentage of tumor cells expressing PD-L1 averaged 9.1% in all of the samples. Of the HPV DNA positive tumor samples, the percentage of cells overexpressing HPV E6, E7 mRNA was 4.8%. Interestingly, the percentage of cells overexpressing E6, E7 mRNA that also expressed PD-L1 was statistically lower (1.4%) than the percentage of all tumor cells that expressed PD-L1 (P=0.01). Conclusions: We report a novel flow cytometric assay to quantify both HPV E6, E7 mRNA and PD-L1 simultaneously in single cells. Further, we found the expression of PD-L1 in tumor cells with HPV E6, E7 mRNA to be significantly lower than the expression of PD-L1 in tumor cells not expressing HPV E6, E7 mRNA. These findings may influence prognosis and treatment strategies. Note: This abstract was not presented at the conference. Citation Format: Rian J. Morgan, Amanda N. Chargin, Haitham Mirghani, Bruce K. Patterson. Simultaneous quantification of HPV oncogene (E6, E7 mRNA) and PD-L1 protein expression in oral cancer samples using flow cytometry [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 49.
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