Metastatic progression defines the final stages of tumor evolution and underlies the majority of cancer-related deaths. The heterogeneity in disseminated tumor cell populations capable of seeding and growing in distant organ sites contributes to the development of treatment resistant disease. We recently reported the identification of a novel tumor-derived cell population, circulating hybrid cells (CHCs), harboring attributes from both macrophages and neoplastic cells, including functional characteristics important to metastatic spread. These disseminated hybrids outnumber conventionally defined circulating tumor cells (CTCs) in cancer patients. It is unknown if CHCs represent a generalized cancer mechanism for cell dissemination, or if this population is relevant to the metastatic cascade. Herein, we detect CHCs in the peripheral blood of patients with cancer in myriad disease sites encompassing epithelial and non-epithelial malignancies. Further, we demonstrate that in vivo-derived hybrid cells harbor tumor-initiating capacity in murine cancer models and that CHCs from human breast cancer patients express stem cell antigens, features consistent with the potential to seed and grow at metastatic sites. Finally, we reveal heterogeneity of CHC phenotypes reflect key tumor features, including oncogenic mutations and functional protein expression. Importantly, this novel population of disseminated neoplastic cells opens a new area in cancer biology and renewed opportunity for battling metastatic disease.

Background. Racial disparities among clinical trial participants present a challenge to assess whether trial results can be generalized into patients representing diverse races and ethnicities. The objective of this study is to evaluate the impact of race and ethnicity on treatment response in advanced non-small cell lung cancer (aNSCLC) patients treated with PD-1 or PD-L1 inhibitors through analysis of real-world data (RWD). Materials and Methods. A retrospective cohort study of 11,138 lung cancer patients treated at hospitals within the Mount Sinai Health System was performed. Patients with confirmed aNSCLC who received anti-PD1/PD-L1 treatment were analyzed for clinical outcomes. Our cohort included 249 aNSCLC patients who began nivolumab, pembrolizumab, or atezolizumab treatment between November 2014 and December 2018. Time-to-treatment discontinuation (TTD) and overall survival (OS) were the analyzed clinical endpoints. Results. After a median follow-up of 14.8 months, median TTD was 7.8 months (95% confidence interval 5.4not estimable [NE]) in 75 African American patients vs. 4.6 (2.4 -7.2) in 110 White patients (hazard ratio [HR] 0.63). Median OS was not reached (18.4 -NE) in African Americans vs. 11.6 months (9.7 -NE) in Whites (HR 0.58). Multivariable cox regression conducted with potential confounders confirmed longer TTD (adjusted HR 0.65) and OS (adjusted HR 0.60) in African American vs. White patients. Similar real-world response rate (42.6% vs. 43.5%) and disease control rate (59.6% vs. 56.5%) were observed in the African American and White patient populations. Further investigation revealed the African American group had lower incidence (14.7%) of putative hyperprogressive diseases (HPD) upon anti-PD1/PD-L1 treatment than the White patient group (24.5%). Conclusions. Analysis of RWD showed longer TTD and OS in African American aNSCLC patients treated with anti-PD1/PD-L1 inhibitors. Lower incidence of putative HPD is a possible reason for the favorable outcomes in this patient population. The Oncologist 2021;9999:• • Implications for Practice: There is a significant underrepresentation of minority patients in randomized clinical trials, and this study demonstrates we can use real world data to investigate the impact of race and ethnicity on treatment response. In retrospective analysis of advanced non-small cell lung cancer patients treated with PD-1 or PD-L1 inhibitors, African American patients had significantly longer time-to-treatment discontinuation and longer overall survival. Analysis of realworld data can yield clinical insights and establish a more complete picture of medical interventions in routine clinical practice.

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

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