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Anticancer immunotherapies have revolutionized cancer management, yet the effect of systemic anti‐programmed cell death protein 1 (PD‐1) treatment is predominantly studied in tumor‐infiltrating lymphocytes (TILs). Its impact on PD‐1 expressing cells in tumor‐draining lymph nodes (TDLNs) is not well understood and yet to be explored. Thus, further research aiming for better understanding of the PD‐1 pathway not only in tumor tissue but also in TDLNs is warranted. In this study, we investigated the expression of PD‐1, CD69, and HLA‐DR on CD4+ and CD8+ T cells by flow cytometry analysis of peripheral blood mononuclear cells (PBMCs), TDLNs, and tumor samples from patients with oral squamous cell carcinoma (OSCC). Our data showed that both helper and cytotoxic T lymphocytes in OSCC tissue were highly activated and expressed high level of PD‐1 (over 70% positivity). Lymphocytes in TDLNs and peripheral blood expressed significantly lower levels of PD‐1 and other activation markers compared to TILs. Moreover, we demonstrated that a significant fraction of PD‐1 negative TILs expressed high levels of human leukocyte antigen – DR isotype and CD69. In contrast, PD‐1 negative cells in TDLNs and PBMCs scarcely expressed the aforementioned activation markers. Furthermore, we proved that patients with a high percentage of CD3+ PD‐1+ cells in tumor‐draining lymph nodes had significantly lower disease‐free and overall survival rates (log‐rank test P = .0272 and P = .0276, respectively). Taken together, we proved that flow cytometry of lymph nodes in OSCC is feasible and may be used to investigate whether PD‐1 levels in TDLNs correspond with survival and potentially with response to anti‐PD‐1 therapy. Such knowledge may ultimately help guide anti‐PD‐1 treatment., Flow cytometry is a powerful tool that can be used in head and neck cancers to investigate immunological milieu of tumor‐draining lymph nodes. In this project, we provide a characterization of T cells within head and neck cancer tumors, tumor‐draining lymph nodes, and blood.

Background: Occult metastases are common in patients with oral squamous cell carcinoma (OSCC) which is why elective neck dissection, adjuvant radiotherapy or watchful waiting have been treatment options after surgical removal of the primary tumour. Sentinel lymph node biopsy (SLNB) has lately emerged as a novel possibility in treatment planning. Objectives: To establish a reliable and clinically useful protocol for SLNB in staging/elective neck dissection in oral cancer. Methods: Fourteen consecutive patients with T1-T2 N0 oral cancer were enrolled when scheduled for elective neck dissection. Results: This study outlines various techniques for improving SLNB in head and neck cancer. After evaluation, a combination of techniques was found to constitute a reliable, clinically adaptable work concept. The suggested procedure starts with the pre-surgical injection of radioactive technetium 99Tcm carried on tilmanocept (Lymphoseek ®) at the tumour site. The radioactivity in the lymph node is then visualized preoperatively with Single Photon Emission Computed Tomography (SPECT/CT). Intraoperatively, indocyanine green (ICG) is injected and a sentinel node is visualized with near-infrared light. To support the sentinel node detection, the surgeon uses a hand-held gamma detection probe. This approach results in a reproducible and reliable detection of sentinel nodes. Conclusion: This paper presents a novel protocol for the identification of the sentinel node in the head and neck region. The protocol additionally enables the use of flow cytometry analysis of resected lymph nodes.

Recurrence in oral squamous cell carcinoma (OSCC) significantly reduces overall survival. Improved understanding of the host’s immune status in head and neck cancer may facilitate identification of patients at higher risk of recurrence and improve patients’ selection for ongoing clinical trials assessing the effectiveness of immune checkpoint inhibitors (CPI). We aimed to investigate Sentinel Node-derived T-cells and their impact on survival. We enrolled prospectively 28 OSCC patients treated at Karolinska University Hospital, Stockholm, Sweden with primary tumour excision and elective neck dissection. On top of the standard treatment, the enrolled patients underwent sentinel node procedure. T cells derived from Sentinel nodes, non-sentinel nodes, primary tumour and PBMC were analyzed in flow cytometry. Patients who developed recurrence proved to have significantly lower level of CD4+ CD69+ in their sentinel node (31.38 ± 6.019% vs. 43.44 ± 15.33%, p = 0.0103) and significantly higher level of CD8+ CD HLA-DR+ (38.95 ± 9.479% vs. 24.58 ± 11.36%, p = 0.0116) compared to disease-free individuals. Survival analysis of studied population revealed that patients with low proportion of CD4+ CD69+ had significantly decreased disease-free survival (DFS) of 19.7 months (95% CI 12.6–26.9) compared with 42.6 months (95% CI 40.1–45.1) in those with high CD4+ CD69+ proportion in their Sentinel Nodes (log-rank test, p = 0.033). Our findings demonstrate that characterization of T-cell activation in Sentinel Node serves as a complementary prognostic marker. Flow cytometry of Sentinel Node may be useful in both patients’ surveillance and selection for ongoing CPI clinical trials in head and neck cancer.

Background: Detection of metastatic spread of head and neck cancer to cervical lymph nodes is essential for optimal design of therapy. Undetected metastases lead to mortality, which can be prevented by better detection methods. Methods: We analysed 41 lymph nodes from 19 patients with oral squamous cell carcinoma (OSCC). Each lymph node was divided in two, one half processed for histopathology and the other half dissociated into single-cell suspension, stained for the carcinoma cell markers cytokeratin 5/8 (CK5/8), epithelial cell adhesion molecule (EpCAM) and epithelial mucin (MUC-1), and analysed with flow cytometry. Flow cytometry data were compared with histopathology performed on serial sections and immunohistochemistry. Six cervical lymph nodes from cancer-free patients were used to establish baseline levels in flow cytometry. Results: Flow cytometry analysis (fluorescence-activated cell sorting; FACS) detected all six metastases confirmed by histopathology as well as the histologically negative nodes. Importantly, among nine sentinel lymph nodes, FACS analysis detected

Background Human papillomavirus (HPV) is a causative agent for tonsillar and base of tongue squamous cell carcinoma (TSCC/BOTSCC), as well as for cervical cancer. Premalignant stages in cervical cancer have been studied extensively, while little is known about premalignant stages in TSCC/BOTSCC and the role of HPV. Here we analyzed differences in gene and protein expression between high‐grade dysplasia and invasive cancer in both HPV‐positive (HPV+) and HPV‐negative (HPV−) TSCC/BOTSCC. Methods High‐grade dysplasia and invasive carcinoma were laser microdissected from HPV+ and HPV− TSCC/BOTSCC tumor sections. Differential gene expression was studied utilizing nanoString RNA‐panels and genes of interest were validated on the protein level by immunohistochemistry. Results Forty genes in the HPV+ tumors showed significantly different expression between high‐grade dysplasia and invasive cancer and 33 genes in the HPV− tumors. Five out of the nine most significant pathways showed similar increased activity in invasive cancer as compared to high‐grade dysplasia in both HPV+ and HPV− tumors. Lastly, significant differences in protein expression was confirmed for SPARC, psoriasin, type I collagen and galectin‐1 in both HPV+ and HPV− tumors. Conclusions This is to our knowledge the first study disclosing differences and similarities in gene expression between dysplastic and invasive HPV+ and HPV− TSCC/BOTSCC., It is debated if high‐grade dysplasia exists in HPV+ TSCC/BOTSCC, there is also a need for pathological invasion markers in HNSCC. Here we present similarities between HPV+ and HPV‐ high‐grade dysplasia, suggesting HPV+ high‐grade dysplasia is an entity. We also identify possible invasion markers in HNSCC.

Human epidermal growth factor receptor 2 (HER2) immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) are widely used semiquantitative assays for selecting breast cancer patients for HER2 antibody therapy. However, both techniques have been shown to have disadvantages. Our aim was to test a recent automated technique of combined IHC and brightfield dual in situ hybridization-gene protein detection platform (GPDP)-in breast cancer HER2 protein, gene, and chromosome 17 centromere status evaluations, comparing the results in accordance to the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from both 2007 and 2013. The GPDP technique performance was evaluated on 52 consecutive whole slide invasive breast cancer cases with HER2 IHC 2/3+ scoring results. Applying in turns the American Society of Clinical Oncology/College of American Pathologists recommendations for HER2 testing in breast cancer from 2007 and 2013 to both FISH and GPDP DISH assays, the HER2 gene amplification results showed 100% concordance among amplified/nonamplified cases, but there was a shift in 4 cases toward positive from equivocal results and toward equivocal from negative results. This might be related to the emphasis on the average HER2 copy number in the 2013 criteria. HER2 expression by IVD market IHC kit (Pathway®) has a strong correlation with GPDP HER2 protein, including a full concordance for all cases scored as 3+ and a reduction from 2+ to 1+ in 7 cases corresponding to nonamplified cases. Gene protein detection platform HER2 protein "solo" could have spared the need for 7 FISH studies. In addition, the platform offered advantages on interpretation reassurance including selecting areas for counting gene signals paralleled with protein IHC expression, on heterogeneity detection, interpretation time, technical time, and tissue expense.

We present a florid case of remitting seronegative symmetrical synovitis with pitting edema that was actually a paraneoplastic syndrome of an asymptomatic undiagnosed adenosquamous lung cell carcinoma. The arthritis led to a screening for lung cancer and an early enough diagnosis for a curative intervention.