A Novel Method Using Fine Needle Aspiration from Tumor-Draining Lymph Nodes Could Enable the Discovery of New Prognostic Markers in Patients with Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell cancer (cSCC) is the second most common form of skin cancer, characterized by abnormal, accelerated growth of squamous cells. When caught early, most cSCCs are curable. About 5 percent of the cSCC cases have advanced to such an extent, generally metastatic, that they are far more dangerous, with very poor prognosis and challenging to treat. All efforts to find biomarkers, in blood or in the tumor itself, for early identification of patients with a risk for metastasis have so far failed. The present study describes a novel method that enables the identification of lymphocyte markers in tumor-draining lymph nodes. Six patients with advanced cSCC were analyzed using a combination of a sentinel lymph node biopsy (SLNB) protocol, fine needle aspiration (FNA), and flow cytometry. Immunological results from the sentinel nodes were combined with corresponding data from peripheral blood and unfixed tumor tissues. The result demonstrates a striking difference between the subsets of T-cells from the three compartments. Our interpretation of this first pilot study is that the ability to follow specific immunological markers on lymphocytes in tumor-draining lymph nodes will enable the identification of novel prognostic biomarkers not detectable in material from blood and tumor tissues.

Regulatory B cells producing IL-10 are increased in human tumor draining lymph nodes.

The contribution of different immune cell subsets, especially T cells, in anti-tumor immune response is well established. In contrast to T cells, the anti-tumor contribution of B cells has been scarcely investigated. B-cells are often overlooked, even though they are important players in a fully integrated immune response and constitute a substantial fraction of tumor draining lymph nodes (TDLNs) known also as Sentinel Nodes. In this project, samples including TDLNs, non-TDLNs (nTDLNs) and metastatic lymph nodes from 21 patients with oral squamous cell carcinoma were analyzed by flow cytometry. TDLNs were characterized by a significantly higher proportion of B cells compared with nTDLNs (P = .0127). TDLNs-associated B cells contained high percentages of naïve B cells, in contrary to nTDLNs which contained significantly higher percentages of memory B cells. Patients having metastases in TDLNs showed a significantly higher presence of immunosuppressive B regulatory cells compared with metastasis-free patients (P = .0008). Elevated levels of regulatory B cells in TDLNs were associated with the advancement of the disease. B cells in TDLNs were characterized by significantly higher expression of an immunosuppressive cytokine-IL-10 compared with nTDLNs (P = .0077). Our data indicate that B cells in human TDLNs differ from B cells in nTDLNs and exhibit more naïve and immunosuppressive phenotypes. We identified a high accumulation of regulatory B cells within TDLNs which may be a potential obstacle in achieving response to novel cancer immunotherapies (ICIs) in head and neck cancer.

Radiotherapy-Dose Escalated for Large Volume Primary Tumors-And Cetuximab with or without Induction Chemotherapy for HPV Associated Squamous Cell Carcinoma of the Head and Neck-A Randomized Phase II Trial.

The leading cause of death for patients with HPV associated squamous cell carcinoma of the head and neck (SCCHN) after treatment with chemoradiotherapy (CRT) nowadays is peripheral metastasis. This study investigated whether induction chemotherapy (IC) could improve progression free survival (PFS) and impact on relapse pattern after CRT. METHODS: Eligible patients in this multicenter, randomized, controlled, phase 2 trial had p16-positive locoregionally advanced SCCHN. Patients were randomized in a 1:1 ratio to either RT with cetuximab (arm B) versus the same regimen preceded by two cycles of taxotere/cisplatin/5-FU (arm A). The RT dose was escalated to 74.8 Gy for large volume primary tumors. Eligibility criteria included patients of 18-75 years, an ECOG performance status 0-1, and adequate organ functions. RESULTS: From January 2011 to February 2016, 152 patients, all with oropharyngeal tumors were enrolled, 77 in arm A and 75 in arm B. Two patients, one in each group, withdrew their consent after randomization, leaving 150 patients for the ITT analysis. PFS at 2 years was 84.2% (95% CI 76.4-92.8) in arm A and 78.4% (95% CI 69.5-88.3) in arm B (HR 1.39, 95% CI 0.69-2.79, p = 0.40). At the time of analysis, there were 26 disease failures, 9 in arm A and 17 in arm B. In arm A, 3 patients had local, 2 regional, and 4 distant relapses as first sites of recurrence, and in arm B, 4, 4, and 9 relapses in corresponding sites. Eight out of 26 patients with disease progression had salvage therapy and 7 were alive NED (no evidence of disease), at 2 years. Locoregional control was 96% in arm A and 97.3% in arm B and OS 93% and 90.5%, respectively. Local failure as first site of recurrence was low, in 4.6% of patients and was similar for T1/T2 and T3/T4 tumors (n.s). Nevertheless, out of 7 patients with primary local failures, 4 were treated with the escalated RT dose. Toxicity was low and similar in the treatment arms. There was one fatal event in arm A where the combined effects of the drugs used in chemotherapy and cetuximab could not be ruled out. CONCLUSIONS: PFS, locoregional control and toxicity did not differ between the two arms, OS was high, and there were few local relapses. In arm B, more than twice as many patients had distant metastasis as the first site of relapse compared to arm A. The response to IC was found to define 29% of patients in arm A who did not have a tumor relapse during follow-up. An escalated dose of 74.8 Gy could mitigate the negative impact of large tumor volume but for some patients, even this intensified treatment was insufficient.

Sentinel node-assisted neck dissection in advanced oral squamous cell carcinoma-A new protocol for staging and treatment.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is used to improve the staging of and guide treatment in patients with early-stage T1-T2 N0 oral squamous cell carcinoma (OSCC). The role of sentinel nodes (SNs) and the use of SN-technique in advanced OSCC (T3-T4 and/or N+) remain to be evaluated. This study investigates the nodal drainage and the rate of positive SNs (SNs+) in all stages of OSCC. MATERIALS AND METHODS: In total, 85 patients with T1-T4 OSCC diagnosed 2019-2021 were included. We used a prolonged interval between peritumoral injection of radionuclide and SPECT-CT to include all SNs. RESULTS: Patients with advanced OSCC presented a higher proportion of contralateral lymphatic drainage and a higher rate of SN+ compared to patients with early-stage disease. T3-T4 and N+ tumors presented a tendency for a higher rate of contralateral lymphatic drainage compared to T1-T2 and N0 tumors (p = 0.1). The prevalence of positive nodes (SNs+) was higher among patients with advanced disease, T3-T4 versus T1-T2 (p = 0.0398). CONCLUSION: SN-assisted ND enables identification and removal of all SNs + and has the potential for more accurate staging and could possibly give prognostic advantages regarding regional recurrence for all OSCC patients, especially among those with advanced disease. The precise localization of the SNs + also suggests that a more individualized ND approach might be possible in the future even for patients with advanced OSCC.

Tumour-draining lymph nodes in head and neck cancer are characterized by accumulation of CTLA-4 and PD-1 expressing Treg cells.

INTRODUCTION: High Tregs infiltration within the tumour microenvironment (TME) of various cancers shows a positive correlation with poor prognosis. Despite the fact that tumour draining lymph nodes (TDLNs) are recognized as key organs playing a crucial role in response to immunotherapy and modulating anti-cancer immunity, the distribution of Tregs and their role in TDLNs remain uncertain thus far. The purpose of this project is to investigate the density of Tregs in TDLNs and non-TDLNs and their expression of immune checkpoint molecules - PD-1 and CTLA-4. METHODS: Samples including TDLNs, non-TDLNs and metastatic lymph nodes (LNs) from 23 patients with oral squamous cell carcinoma (OSCC) were analyzed by multicolour flow cytometry with a focus on Tregs population and expression of CTLA-4 and PD-1. RESULTS: TDLNs and metastatic LNs were characterized by a significantly higher infiltration of Tregs defined as CD4+FoxP3+CD25highCD127low cells and significantly higher expression of CTLA-4 and PD-1 on Tregs compared with non-TDLNs. Tregs in TDLNs and metastatic LNs co-expressed CTLA-4 and PD-1 abundantly. High expression of these immune check-point molecules correlated with positive N-stage but not with T-stage. CONCLUSION: TDLNs and metastatic LNs are characterized by a high accumulation of Tregs expressing high levels of CTLA-4 and PD-1. High infiltration of Tregs can be a potential driver of an immunosuppressive milieu in TDLNs that can, in turn, favour cancer progression. High accumulation of Tregs expressing CTLA-4 and PD-1 in TDLNs is associated with lymph node involvement, but not with the size of the primary tumour.

Navigation system for percutaneous tracheotomy.

BACKGROUND: Percutaneous dilatational tracheotomy (PDT) is a well-established method. The prerequisite is to identify anatomical landmarks of the neck. We introduce a three-dimensional navigation system - SafeTrach. AIMS/OBJECTIVES: We present an alternative technique using internal landmarks that can be used in patients with difficult anatomy. MATERIAL AND METHODS: The device is a forceps-like instrument with an outer and an inner shank. The later serves as a ventilation lumen and stabilizes the orotracheal tube in the midline of trachea. The outer shank acts as a three-dimensional guide for the puncturing needle. RESULTS: Out of 48 patients we have determined the level of puncture in 20 patients by using intraoperative measurements. The distance from the vocal cords to the puncture site was about 50 mm for men and 40 mm for women. In 13 of the patients who had had CT scans, we studied the distance between the vocal cords and the optimal puncture site and found the median distance for men 45 mm and for women 42 mm. CONCLUSIONS AND SIGNIFICANCE: With the studied navigation system one may use external or internal landmarks to indicate the puncture level in PDT. The device may minimize the risk of injuring the posterior tracheal wall.

CD4+ and CD8+ T cells in sentinel nodes exhibit distinct pattern of PD-1, CD69, and HLA-DR expression compared to tumor tissue in oral squamous cell carcinoma.

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.

Activation of T helper cells in sentinel node predicts poor prognosis in oral squamous cell carcinoma.

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.

A Novel Sentinel Lymph Node Approach in Oral Squamous Cell Carcinoma.

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.

Rapid nodal staging of head and neck cancer surgical specimens with flow cytometric analysis.

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 <1% malignant cells in four cases, not found in histopathology. Results from flow cytometry analysis can be obtained within 3 h of the time of biopsy. CONCLUSIONS: We show that flow cytometric analysis of nodal tissue is sensitive and reliable in identifying metastases of OSCC. Flow cytometry is inexpensive and fast, providing a possibility of perioperative diagnostics and immediate treatment planning.

A New Technique for Percutaneous Tracheotomy.

The objective of this study was to investigate a new technique for tracheal puncture during percutaneous dilatational tracheotomy (PDT). A new invention, known as SafeTrach, was used: this instrument allows exact localization of the puncture site with built-in protection of the posterior tracheal wall. Surgery was performed on 17 patients with this technique, and our experience is described in this report. The results showed that this new technique minimizes known risk factors compared with existing PDT techniques, including patients with disadvantageous anatomy.

A new safe and cost-effective percutaneous dilatational tracheotomy: SafeTrach.

Conclusion SafeTrach is a new simplified and safe technique to perform percutaneous dilatational tracheotomy (PDT) that eliminates known risk factors compared with existing percutaneous techniques. In the present clinical study, also patients with disadvantageous anatomy not suitable for conventional PDT (CPDT) were treated without complications using SafeTrach. PDT with SafeTrach (STPDT) offers an excellent solution for patients who need tracheotomy in connection with elective ear, nose, and throat (ENT) surgery. Objectives To assess a new technique for percutaneous tracheotomy. Methods Seventeen patients were tracheotomized with STPDT using SafeTrach for the initial penetration sequence and single step dilatational techniques for the dilatational sequence. The patients represented a variety of different neck anatomies. Fifteen patients were head- and neck cancer patients that were subjects of free flap transplants. Results This study showed that STPDT was safe and easy to perform and time-efficient. The median duration of the procedure was 11.5 min and the puncture was in all cases located in the midline of the trachea either between the 2nd and 3rd tracheal ring (n = 13) or between the 3rd and 4th ring (n = 4).