Amanda Chargin1, Rian Morgan1, Uma Sundram2, Keith Shults1, Ellen L Tsay1, Navneet Ratti3, Bruce K Patterson4. 1. IncellDx, Inc., 1700 El Camino Real, Menlo Park, CA, 94027, USA. 2. Department of Anatomic Pathology, William Beaumont Hospital, Royal Oak, MI, 48073, USA. 3. Tissue Diagnostics Inc., 2829 Depot Road, Suite 4B, Hayward, CA, 94545, USA. 4. IncellDx, Inc., 1700 El Camino Real, Menlo Park, CA, 94027, USA. brucep@incelldx.com.
Abstract
OBJECTIVE: We report a truly quantitative technology for PD-L1 expression in non-small cell lung cancer (NSCLC). In addition, we present a non-enzymatic technology that creates a cell suspension from fresh tumor tissue so that either fine-needle aspiration (FNA) or fresh tissue can be used in this assay. METHODS: Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, California) was performed on 4-mm punch biopsies. An FNA was taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, and PD-L1 and then stained with DAPI to identify intact, single cells, and to analyze cell cycle. RESULTS: Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r 2 - 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.
OBJECTIVE: We report a truly quantitative technology for PD-L1 expression in non-small cell lung cancer (NSCLC). In addition, we present a non-enzymatic technology that creates a cell suspension from fresh tumor tissue so that either fine-needle aspiration (FNA) or fresh tissue can be used in this assay. METHODS: Non-enzymatic tissue homogenization (IncellPREP; IncellDx, Menlo Park, California) was performed on 4-mm punch biopsies. An FNA was taken from the same tumor to create matched sample sets. Cells were labeled with antibodies directed against CD45, PD-1, and PD-L1 and then stained with DAPI to identify intact, single cells, and to analyze cell cycle. RESULTS: Comparing the IncellPREP homogenization and FNA demonstrated a strong correlation (r 2 - 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.