Spatially resolved immune microenvironmental profiling for follicular thyroid carcinoma with minimal capsular invasion.

Spatial profiles of the tumor-immune microenvironment are associated with disease progression and clinicopathological factors in various cancers. Follicular thyroid carcinoma (FTC) is the second most common thyroid cancer, where the presence of capsular invasion or angioinvasion determines the pathological diagnosis; however, little is known about the immune microenvironment profiles associated with the acquisition of invasive potential of FTC. In this study, we focused on FTC with minimal capsular invasion, and the spatially resolved immune microenvironment of FTC was studied in the discovery (n = 13) and validation cohorts (n = 40). CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, tumor-associated macrophages, CD66+ granulocytes, mature dendritic cells, and mast cells were quantitatively evaluated in single tissue sections, via a 12-marker multiplex immunohistochemistry and image cytometry. Cell densities and compositions of immune cells were spatially stratified by six tissue regions including tumor center, subcapsular region, capsular invasion, adjacent stroma of capsular invasion, peritumoral stroma, and adjacent normal. Lymphoid cell lineages in the tumor center and subcapsular regions were significantly lower than those in adjacent normal and peritumoral stroma, potentially related to the lymphoid lineage exclusion from the intratumoral regions of FTC. Interestingly, immune cell composition profiles in the capsular invasive front were distinct from those of intratumoral region. The ratios of T cells to CD66b+ granulocytes with capsular invasion were significantly higher than those without capsular invasion, suggesting the presence of a unique immune microenvironment at the invasive front between tumor foci and stroma. In addition, tumor cells at the capsular invasive front showed significantly higher expression of tumor programmed cell death ligand 1 (PD-L1) than those at the tumor center. This study revealed spatial immune profiles associated with capsular invasion of FTC, providing new insights into the mechanisms underlying its development and initial invasion.

Prognostic significance of spatial immune profiles in human solid cancers.

Immune-based tumor characteristics in the context of tumor heterogeneity are associated with suppression as well as promotion of cancer progression in various tumor types. As immunity typically functions based on intercellular contacts and short-distance cytokine communications, the location and spatial relationships of the tumor immune microenvironment can provide a framework to understand the biology and potential predictive biomarkers related to disease outcomes. Immune spatial analysis is a newly emerging form of cancer research based on recent methodological advances in in situ single-cell analysis, where cell-cell interaction and the tissue architecture can be analyzed in relation to phenotyping the tumor immune heterogeneity. Spatial characteristics of tumors can be stratified into the tissue architecture level and the single-cell level. At the tissue architecture level, the prognostic significance of the density of immune cell lineages, particularly T cells, is leveraged by understanding longitudinal changes in cell distribution in the tissue architecture such as intra-tumoral and peri-tumoral regions, and invasive margins. At the single-cell level, the proximity of the tumor to the immune cells correlates with disease aggressiveness and therapeutic resistance, providing evidence to understand biological interactions and characteristics of the tumor immune microenvironment. In this review, we summarize recent findings regarding spatial information of the tumor immune microenvironment and review advances and challenges in spatial single-cell analysis toward developing tissue-based biomarkers rooted in the immune spatial landscape.

Rapid multiplex immunohistochemistry for characterizing tumor-immune microenvironment.

Intratumoral immune profiles are related to prognosis and therapeutic efficacy, and could result in personalized treatments based on biomarkers. To develop a multiplex, quantitative, and rapid tissue evaluation method based on the clinically established standard immunohistochemistry (IHC), a 6-marker rapid multiplex IHC was developed based on our previously reported 14-marker multiplex IHC by reducing the number of labels and accelerating the staining procedure. First, fewer labels were required to identify the same immunological features linked to prognosis in 14-marker multiplex IHC analyses. The six selected markers showed a significant correlation with the 14 markers in the immune classification. Next, a rapid staining protocol was developed by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 h and 49 min, as opposed to the several days required for conventional multiplex IHC. Validation of benign tonsil and head and neck cancer tissues revealed a significant correlation between rapid and conventional 6-makrer multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cell ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable quantitative assessment of the tumor-immune microenvironment on a clinically feasible time scale, which promotes the development of tissue biomarker-guided therapeutic strategies.

Clinical, genomic and immune microenvironmental determinants of nivolumab response in head and neck squamous cell carcinoma.

Background: In view of improving biomarkers predicting the efficacy of immunotherapy for head and neck squamous cell carcinoma (R/M HNSCC), this multicenter retrospective study aimed to identify clinical, tumor microenvironmental, and genomic factors that are related to therapeutic response to the anti- Programmed cell death protein 1 (PD-1) antibody, nivolumab, in patients with R/M HNSCC. Methods: The study compared 53 responders and 47 non-responders, analyzing formalin-fixed paraffin-embedded samples using 14-marker multiplex immunohistochemistry and targeted gene sequencing. Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-1 ligand (PD-L1) expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. The frequency of natural killer cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Age-stratified analysis showed nivolumab response was linked to high CPS and lymphoid-inflamed profiles in patients aged ≥ 65. In contrast, lower NLR in peripheral blood counts was associated with response in patients aged < 65. Notably, TP53 mutation-positive group had lower CPS and T cell densities, suggesting an immune-excluded microenvironment. Patients with altered tumor suppressor gene pathways, including TP53, CDKN2A, and SMAD4 mutations, had lower CPS, higher smoking index, and were associated with poor responses. Conclusion: Nivolumab treatment efficacy in HNSCC is influenced by a combination of clinical factors, age, prior treatment, immune environmental characteristics, and gene mutation profiles.

Genomic and immune microenvironment profiling in a case of metastatic intrathyroid thymic carcinoma.

Metastatic intrathyroid thymic carcinoma (ITTC) is a rare cancer with no effective drugs for controlling. This case report has shown genomic and immune microenvironment profiles in metastatic ITTC and emphasized an immunosuppression via a PD-1/PD-L1 pathway, possibly strengthening the rationale for immune checkpoint blockade as a novel treatment.

A 14-Marker Multiplexed Imaging Panel for Prognostic Biomarkers and Tumor Heterogeneity in Head and Neck Squamous Cell Carcinoma.

Recent advances made in treatment for head and neck squamous cell carcinoma (HNSCC) highlight the need for new prediction tools to guide therapeutic strategies. In this study, we aimed to develop a HNSCC-targeting multiplex immunohistochemical (IHC) panel that can evaluate prognostic factors and the intratumor heterogeneity of HNSCC. To identify IHC-based tissue biomarkers that constitute new multiplex IHC panel, a systematic review and meta-analysis were performed to analyze reported IHC biomarkers in laryngeal and pharyngeal SCC in the period of 2008-2018. The Cancer Genome Atlas (TCGA) and Reactome pathway databases were used to validate the prognostic and functional significance of the identified biomarkers. A 14-marker chromogenic multiplex IHC panel including identified biomarkers was used to analyze untreated HNSCC tissue. Forty-five high-quality studies and thirty-one candidate tissue biomarkers were identified (N = 7062). Prognostic validation in TCGA laryngeal and pharyngeal SCC cohort (N = 205) showed that ß-catenin, DKK1, PINCH1, ADAM10, and TIMP1 were significantly associated with poor prognosis, which were related to functional categories such as immune system, cellular response, cell cycle, and developmental systems. Selected biomarkers were assembled to build a 14-marker panel, evaluating heterogeneity and polarized expression of tumor biomarkers in the tissue structures, which was particularly related to activation of Wnt/ß-catenin pathway. Integrated IHC analysis based on a systemic review and meta-analysis provides an in situ proteomics tool to assess the aggressiveness and intratumor heterogeneity of HNSCC.

Spatial Profiles of Intratumoral PD-1+ Helper T Cells Predict Prognosis in Head and Neck Squamous Cell Carcinoma.

Background: Functional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC). Methods: A total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma. Results: Tissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of TH1/TH2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b+ granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1)+ helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163+ tumor-associated macrophages (TAM) provided the strongest correlation with PD-1+ helper T cells, and cases with a high density of PD-1+ helper T cells and CD163+ TAM had a significantly shorter overall survival than other cases. Conclusion: This study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1+ helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC.

Radiation-induced angiosarcoma of the parotid gland after postoperative radiotherapy for hypopharyngeal carcinoma.

Secondary carcinogenesis within the irradiation range is one of the most severe problems in cancer survivors. A 60-year-old woman developed hypopharyngeal carcinoma, and she received radical surgery and postoperative radiotherapy. Eight years later, brown pigmentation and induration were observed in the left subaural region. Fine-needle aspiration biopsy revealed malignancy and the parotid tumor was diagnosed as recurrence of hypopharyngeal carcinoma. Neoadjuvant chemotherapy followed by radical parotidectomy was performed. The pathological diagnosis was angiosarcoma, which was most likely induced by past irradiation. About two months after surgery, lung metastases were detected. Docetaxel did not affect to lung metastases, but paclitaxel therapy was partially effective. The lung tumors increased in size, and brain metastases developed, resulting in death. Both neoadjuvant chemotherapy and radical surgery played important roles in the local disease control. Administration of newer agents as adjuvant chemotherapeutic agent should also be considered for improving the prognosis.