Abstract 4969: Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR
Authors: Nell Rogier J. , Versluis Mieke , Zoutman Willem H. , Van der Velden Pieter A. 
Published: September 2020

Abstract
The absolute quantification of adaptive immune cells in cancer is of clinical importance for diagnosis, prognosis and treatment. First, the cancer itself may be of lymphoproliferative origin, in which an accurate cell count facilitates the diagnosis. Alternatively, healthy T- and B-lymphocytes may be infiltrating a tumor, affecting tumor progression or representing a potential therapeutic target.
However, sample quantity and quality do not always allow to perform cell- or tissue-based quantitative analyses. As an alternative, we identified generic DNA markers of a variety of adaptive immune cell types. These markers were translated into a digital PCR experimental setup, enabling the absolute quantification of these cell types in bulk DNA.
The sensitivity and dynamic range of this approach were technically validated in control samples and calibration curves. Moreover, a biological validation showed a precision of our measurements comparable to flow cytometry (R2 &gt; 0.9). Importantly, as our sample requirements are much lower (5-20 ng of DNA instead of intact cells), this approach provides a novel, sensitive way for the accurate quantification of adaptive immune cells independent of the cellular context.
We evaluated our approach in the context of two clinical applications: diagnosis of vitreoretinal lymphoma by a liquid biopsy, and absolute deconvolution of the immune infiltrate in melanoma.
Vitreoretinal lymphoma is a clinically challenging masquerade syndrome, frequently mimicking a benign uveitis. To confirm the diagnosis, a vitreous biopsy may be obtained and analyzed for malignant cells. However, analysis may be impeded by low cellularity or loss of cellular integrity. With our approach, we could acquire absolute immune cell quantifications from only 10 uL vitreous fluid, independent of cellular context.
To analyze the immune infiltrate from bulk DNA or RNA samples in solid tumors, numerous deconvolution approaches are available. However, these methods typically rely on relative differences in gene expression, consequently lacking the precision to be compared with absolute measurements. Our DNA-based method allows to obtain absolute lymphocyte quantifications, comparable to (single) cell-based technologies. In both cutaneous and uveal melanoma, this facilitated an enhanced deconvolution of the bulk expression analysis.
In conclusion, we developed and validated a digital PCR-based approach to quantify immune cells in bulk DNA samples. A similar precision could be obtained compared to flow cytometry, but with much lower requirements concerning sample quality and quantity. Therefore, this method can be broadly applied in a wide range of sample types, supporting cancer diagnosis, prognosis and treatment.
Citation Format: Rogier J. Nell, Mieke Versluis, Willem H. Zoutman, Pieter A. Van der Velden. Absolute quantification of immune cells in bulk DNA samples and liquid biopsies using digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4969.