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There is increasing evidence that the spatial organization of cells within the tumor-immune microenvironment (TiME) of solid tumors influences survival and response to therapy in numerous cancer types. Here, we report results and demonstrate the applicability of quantitative single-cell spatial proteomics analyses in the TiME of primary and recurrent human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) tumors. Single-cell compositions of a nine patient, primary and recurrent (n = 18), HNSCC cohort is presented, followed by deeper investigation into the spatial architecture of the TiME and its relationship with clinical variables and progression free survival (PFS). Multiple spatial algorithms were used to quantify the spatial landscapes of immune cells within TiMEs and demonstrate that neoplastic tumor-immune cell spatial compartmentalization, rather than mixing, is associated with longer PFS. Mesenchymal (αSMA+) cellular neighborhoods describe distinct immune landscapes associated with neoplastic tumor-immune compartmentalization and improved patient outcomes. Results from this investigation are concordant with studies in other tumor types, suggesting that trends in TiME cellular heterogeneity and spatial organization may be shared across cancers and may provide prognostic value in multiple cancer types.

Image cytometry enables quantitative cell characterization with preserved tissue architecture; thus, it has been highlighted in the advancement of multiplex immunohistochemistry (IHC) and digital image analysis in the context of immune-based biomarker monitoring associated with cancer immunotherapy. However, one of the challenges in the current image cytometry methodology is a technical limitation in the segmentation of nuclei and cellular components particularly in heterogeneously stained cancer tissue images. To improve the detection and specificity of single-cell segmentation in hematoxylin-stained images (which can be utilized for recently reported 12-biomarker chromogenic sequential multiplex IHC), we adapted a segmentation algorithm previously developed for hematoxlin and eosin-stained images, where morphological features are extracted based on Gabor-filtering, followed by stacking of image pixels into n-dimensional feature space and unsupervised clustering of individual pixels. Our proposed method showed improved sensitivity and specificity in comparison with standard segmentation methods. Replacing previously proposed methods with our method in multiplex IHC/image cytometry analysis, we observed higher detection of cell lineages including relatively rare T H 17 cells, further enabling sub-population analysis into T H 1-like and T H 2-like phenotypes based on T-bet and GATA3 expression. Interestingly, predominance of T H 2-like T H 17 cells was associated with human papilloma virus (HPV)-negative status of oropharyngeal squamous cell carcinoma of head and neck, known as a poor-prognostic subtype in comparison with HPV-positive status. Furthermore, T H 2-like T H 17 cells in HPV-negative head and neck cancer tissues were spatiotemporally correlated with CD66b + granulocytes, presumably associated with an immunosuppressive microenvironment. Our cell segmentation method for multiplex IHC/image cytometry potentially contributes to in-depth immune profiling and spatial association, leading to further tissue-based biomarker exploration. © 2019 International Society for Advancement of CytometryKey terms cell segmentation; T H 17 cell phenotypes; tumor immune microenvironment AS emergence of immunotherapy has been revolutionizing cancer therapeutic strategies, the demands on biomarkers optimizing various treatment options have been constantly increasing (1-4). Profiling cellular complexities of tumor tissue provides a powerful platform to understand immune characteristics associated with clinical response to immunotherapy; thus, in situ tumor profiling and imaging have been increasingly important toward tissue-based biomarker development.Single-cell segmentation is a key technology in digital image analysis, as it enables the quantitative assessment of cell frequency, localization, and phenotypes (5-7). Previously, based on single-cell segmentation, we built an image quantitation platform with multiplex immunohistochemistry (IHC) and image cytometry platform to evaluate in situ immune characteristics where 12 immu...

hyroid cancer is the most common endocrine cancer in children, with accelerating incidence over the past 2 decades. 1,2 Similar to thyroid cancer in adults, approximately 90% of pediatric thyroid cancer is composed of papillary thyroid carcinoma (PTC), while follicular thyroid carcinoma and medullary thyroid carcinoma account for the remaining 10% of cases. 3 However, pediatric PTC in particular frequently presents with more advanced disease and higher rates of recurrence and persistent disease than in adults. 3,4 Rates of extracapsular extension have been reported in up to 50% of children vs 30% of adults, regional nodal involvement in up to 80% vs 50%, and distant metastasis in up to 30% vs 5%. 4,5 Multifocal disease has been found in up to 65% of pediatric patients with thyroid cancer compared with approximately 38% of adults with thyroid cancer. 4,6 Associations between multifocal disease and higher risk of recurrence and persistent disease in children have been suggested in some studies. 7,8 Current guidelines and consensus statements recommend total thyroidectomy in nearly all pediatric patients with PTC, largely driven by the increased prevalence of multifocality and associated risk of recurrence and persistent disease. 3,9 When compared with lobectomy, total thyroidectomy has been shown in some studies to lower the risk of recurrence and persistent disease from 35% to 6% in pediatric IMPORTANCE Current guidelines recommend total thyroidectomy for the majority of pediatric thyroid cancer owing to an increased prevalence of multifocality. However, there is a paucity of information on the exact prevalence and risk factors for multifocal disease-knowledge that is critical to improving pediatric thyroid cancer management and outcomes.OBJECTIVE To determine the prevalence and risk factors for multifocal disease in pediatric patients with papillary thyroid carcinoma (PTC). DESIGN, SETTING, AND PARTICIPANTSThis multicenter retrospective cohort study included patients 18 years or younger who underwent thyroidectomy for PTC from 2010 to 2020 at 3 tertiary pediatric hospitals and 2 tertiary adult and pediatric hospitals in the US.MAIN OUTCOMES AND MEASURES Demographic and clinical variables, including age, family history of thyroid cancer, autoimmune thyroiditis, prior radiation exposure, cancer predisposition syndrome, tumor size, tumor and nodal stage, PTC pathologic variant, and preoperative imaging, were assessed for association with presence of any multifocal, unilateral multifocal, and bilateral multifocal disease using multiple logistic regression analyses. Least absolute shrinkage and selection operator analysis was performed to develop a model of variables that may predict multifocal disease. RESULTSOf 212 patients, the mean age was 14.1 years, with 23 patients 10 years or younger; 173 (82%) patients were female. Any multifocal disease was present in 98 (46%) patients, with bilateral multifocal disease in 73 (34%). Bilateral multifocal disease was more accurately predicted on preoperative imaging than unila...