Abstract 5512: Identification of circulating myeloid cells induced in advanced biliary cancer patients responding to anti-PD1 through combined single cell RNA sequencing and protein expression analysis
Authors: Keenan Bridget P. , McCarthy Elizabeth E. , Ilano Arielle , Lee David S. , Sun Yang , Sheldon Brenna , Cheung Alexander , Ye Chun Jimmie , Kelley Robin Kate , Fong Lawrence 
Published: September 2020

Abstract
Background: Patients with advanced biliary cancer (ABC) have low rates of response to immune checkpoint inhibition (CPI) monotherapy. We conducted a phase II clinical trial studying combination immunotherapy with GM-CSF and the anti-PD-1 antibody, pembrolizumab (NCT02703714), with preliminary objective response rate of 19%, higher than the reported response rate of 5.8% for pembrolizumab monotherapy in ABC. Mechanisms of response to CPI in ABC are unknown, and to our knowledge, there have not been studies of the immune response in this patient population at the single cell level.
Methods: Cryopreserved serial peripheral blood mononuclear cells (PBMC) samples from n=27 ABC patients treated with pembrolizumab plus GM-CSF were assessed with single cell RNA sequencing combined with CITE-Seq, the staining of cells with &gt;90 oligo-tagged antibodies. This allows for the simultaneous identification of protein and RNA expression on an individual cell basis. We used high-throughput multiplexing in which PBMC from different patients could be deconvolved from other mixed samples using individual specific SNPs (“demuxlet”). &gt;100 PBMC samples, including pre-treatment and 1 week, 3 weeks, and 6 weeks post-treatment, were studied. Nine of 27 patients were characterized as responders, while 18 were non-responders. We also performed CITE-Seq on PBMC from age/gender-matched healthy controls (n=9).
Results: Unbiased clustering revealed all major immune populations were present in PBMC from both ABC and controls, though the immune compositions differed, most strikingly within the myeloid compartment. A distinct population of CXCL5-expressing “cancer-associated myeloid cells”, distinct from classical and non-classical monocytes and dendritic cells, was found only within cancer patients. We also analyzed the effects of treatment on immune sub-populations within responders and non-responders and found that following anti-PD-1 monotherapy, there was an increased frequency and absolute number of non-classical monocytes in responders. Conversely, the addition of GM-CSF to anti-PD-1 therapy decreased the frequency of circulating non-classical monocytes, though there was a robust induction of pro-inflammatory genes following treatment with GM-CSF in responders. Regardless of responder status, GM-CSF also dramatically induced monocyte-derived dendritic cells within 1 week of administration.
Conclusions: The compositions of the myeloid compartment differed between ABC patients and healthy individuals, with a circulating cancer-associated myeloid population identified in ABC patients. Circulating non-classical monocytes increased following anti-PD-1 monotherapy in all patients, but declined after combination with GM-CSF in responders, suggesting that anti-PD-1-induced myeloid populations may be associated with response to anti-PD1. Pre-existing and induced myeloid cell states may represent a mechanism of response to anti-PD-1 and GM-CSF.
Citation Format: Bridget P. Keenan, Elizabeth E. McCarthy, Arielle Ilano, David S. Lee, Yang Sun, Brenna Sheldon, Alexander Cheung, Chun Jimmie Ye, Robin Kate Kelley, Lawrence Fong. Identification of circulating myeloid cells induced in advanced biliary cancer patients responding to anti-PD1 through combined single cell RNA sequencing and protein expression analysis [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 5512.