Molecular analysis of circulating and disseminated tumor cells (CTCs/DTCs) has great potential as a means for continuous evaluation of prognosis and treatment efficacy in near-real time through minimally invasive liquid biopsies. To realize this potential, however, methods for molecular analysis of these rare cells must be developed and validated. Here, we describe the integration of imaging mass cytometry (IMC) using metal-labeled antibodies as implemented on the Fluidigm Hyperion Imaging System into the workflow of the previously established High Definition Single Cell Analysis (HD-SCA) assay for liquid biopsies, along with methods for image analysis and signal normalization. Using liquid biopsies from a metastatic prostate cancer case, we demonstrate that IMC can extend the reach of CTC characterization to include dozens of protein biomarkers, with the potential to understand a range of biological properties that could affect therapeutic response, metastasis and immune surveillance when coupled with simultaneous phenotyping of thousands of leukocytes.

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Multiplexed immune cell profiling of the tumor microenvironment (TME) in cancer has improved our understanding of cancer immunology, but complex spatial analyses of tumor-immune interactions in lymphoma are lacking. Here, we used imaging mass cytometry (IMC) on 33 cases of diffuse large B-cell lymphoma (DLBCL) to characterize tumor and immune cell architecture and correlate it to clinicopathological features such as cell of origin, gene mutations, and responsiveness to chemotherapy. To understand the poor response of DLBCL to immune checkpoint inhibitors (ICI), we compared our results to IMC data from Hodgkin lymphoma, a cancer highly responsive to ICI, and observed differences in the expression of PD-L1, PD-1, and TIM-3. We created a spatial classification of tumor cells and identified tumor-centric subregions of immune activation, immune suppression, and immune exclusion within the topology of DLBCL. Finally, the spatial analysis allowed us to identify markers such as CXCR3, which are associated with penetration of immune cells into immune desert regions, with important implications for engineered cellular therapies. This is the first study to integrate tumor mutational profiling, cell of origin classification, and multiplexed immuno-phenotyping of the TME into a spatial analysis of DLBCL at the single-cell level. We demonstrate that, far from being histopathologically monotonous, DLBCL has a complex tumor architecture, and that changes in tumor topology can be correlated with clinically relevant features. This analysis identifies candidate biomarkers and therapeutic targets such as TIM-3, CCR4, and CXCR3 that are relevant for combination treatment strategies in immuno-oncology and cellular therapies.

Diffuse large B cell lymphoma (DLBCL) is an aggressive and heterogenous entity characterized by its variable clinical and biological behaviour, and approximately 30% of patients experience relapsed or refractory disease after first-line therapy. We hypothesize that a better characterization of the tumor microenvironment (TME) might help identify patients who may benefit from individualized immunotherapies. Similar studies in this area have been limited by technical challenges - conventional highly multiplexed techniques require tissue disruptions that lose spatial information, while those that retain tissue architecture can only examine 6-8 markers simultaneously. We and others have previously reported that PD-L1 expression is correlated with decreased survival in a cohort of 85 DLBCL patients. In the present study, we characterized TME components, including their types, frequency and spatial interaction, in DLBCL using imaging mass cytometry (IMC), which allows high-dimensional, single-cell and spatial analysis of FFPE tissues at sub-cellular resolution. Using a panel of 32 antibodies, IMC was performed on a subset of our previously studied cohort. We examined 41 cores from 33 DLBCL cases, 17 GCB and 16 non-GCB, by Hans criteria. Clinical outcome data were available for 29 patients, 22 of whom had complete response (CR) to R-CHOP therapy while 7 had primary refractory disease. Using both supervised gating and unsupervised clustering algorithm, IMC data were analyzed for relevant immunophenotypes and compared across clinical outcome groups. The TME was mainly composed of 13.1% ± 1.9% (mean ± SE) CD4+ T-helper cells, 10.8% ± 1.1% CD8+ cytotoxic T cells, 6.3% ± 0.9% CD68+ macrophages, 2.7% ± 0.5% FoxP3+ regulatory T cells, and 58.1% ± 3.4% tumor cells. In non-GCB group, higher ratio of regulatory T cells was associated with refractory disease. In contrast, activated granzyme-B+/CD8+ cytotoxic T cells were more frequent in CR group, while markers of exhaustion (Tim3, Lag3) were found in patients with refractory disease. To gain functional insight into the various immune subsets, we performed spatial analysis of the immune cells and their relation to blood vessels and tumor cells. Nearest distance analysis showed that CD4+ cells were most tightly clustered around blood vessels in patients with CR, while in those with refractory diseases, CD4+ cells were further away from the vessels (p=0.03). On the contrary, distances between cytotoxic T cells and regulatory T cells showed no difference between CR and refractory patients. Together, these results show variable composition of the different immune cells and their spatial heterogeneity to be associated with the clinical outcome of DLBCL patients and that spatial analysis of immune cells should be explored as a potential biomarker for patients treated with immunotherapies. Citation Format: Monirath Hav, Erik Gerdtsson, Mohan Singh, Anthony Colombo, James Hicks, Peter Kuhn, Imran Siddiqi, Akil Merchant. Highly multiplexed imaging mass cytometry reveals immune cell composition and spatial heterogeneity in diffuse large B cell lymphoma associated with treatment outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2789.