Immune Characters and Plasticity of the Sentinel Lymph Node in Colorectal Cancer Patients.

PURPOSE: This study is aimed at immunologically characterizing sentinel lymph nodes (SNs) in colorectal cancer (CRC) patients and identifying changes in immunological phenotype and function of SNs isolated from the tumor immunosuppressive microenvironment. METHODS: A total of 53 pairs of matched SNs and non-SNs (NSNs) were collected by using a lymph node tracer dye. Flow cytometry was performed to detect the immunophenotype of T cells as well as the expression of activation and inhibitory markers. Differential expression and distribution of characteristic immune cell markers were analyzed by multiplex immunohistochemistry (mIHC). Transcriptomics analysis was conducted to compare the differences in the expression of immune-related genes among lymph nodes. The ex vivo culture of lymph nodes was carried out to examine changes in immunological phenotypes and functions. RESULTS: Compared with NSNs, SNs harbored a significantly higher percentage of regulatory T cells (Tregs) but a lower proportion of MoMDSCs. As indicated in the mIHC assays, Tregs, T follicular helper (Tfh) cells, and M2 macrophages were mainly distributed in cortical areas, germinal centers, and subcapsular sinus areas, respectively, while significantly higher numbers of Tregs and Tfh cells were detected in SNs as compared to NSNs. Moreover, GSEA revealed that T cell activation genes and CD8+ T cell exhaustion-related genes are enriched in SNs and NSNs, respectively. The ex vivo culture led to an increase in the proportion of CD4+ cells, while activating T cells in SNs. In addition, SNs displayed a higher increase in the expression of cytokines IFN-γ, TNF-α, and sFas than NSNs. CONCLUSION: SNs are shown to be in an immune active state in vivo, while highly expressing inhibitory cytokines and suppressive markers. The ex vivo culture enhanced antitumor immunological function of SN-T cells, providing a starting material for adoptive cell therapy for CRC.

Picomets: Assessing single and few cell metastases in melanoma sentinel lymph node biopsies.

BACKGROUND: Lymph node involvement is a significant prognostic factor for melanoma. Both number of positive nodes and disease burden within a lymph node affects survival. However, the significance of few tumor cells within a single node and subsequent optimal management remains without consensus. We investigated the implications of minimal nodal disease on clinical outcomes. METHODS: We reviewed 752 patients who underwent lymph node sampling at time of primary melanoma resection at our institution over 15 years. We deemed patients who had 1 node with 1 to 4 atypical cells staining positive for either Melan-A or Sox-10 as having "picomets." We examined the initial clinicopathological features, subsequent management, and outcomes. RESULTS: Thirty-three patients (4%) met criteria for having picomets. The most common number of positively staining atypical cells was 1 (n = 13). Nodal staging at initial pathology review varied, and overall stage ranged from IA to IIIC. Four patients underwent further therapy, none of whom had recurrent disease. Of the 29 patients undergoing observation/surveillance only, 5 had disease recurrence (17%). CONCLUSION: Although patients with picomets had better outcomes than historical stage matched cohorts, a small subset had recurrent disease. Staging patients with picomets as "N0" may not reflect the true negative prognostic significance of picomets. A larger population of patients meeting picomets criteria is needed to draw further conclusions.

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.

Influence of locoregional lymph node aspiration cytology vs sentinel lymph node mapping and biopsy on disease stage assignment in dogs with integumentary mast cell tumors.

OBJECTIVE: To compare the effect of sentinel lymph node (SLN) histology vs locoregional lymph node (LRLN) fine needle aspiration (FNA) cytology on assigned disease stage and adjunctive treatment recommendations and describe the incidence of anatomic disparity between the LRLN and SLN. STUDY DESIGN: A pre-post study refers to a study design type in which subjects are compared pre and post the intervention of interest. ANIMALS: Seventeen dogs undergoing primary excision of 20 cutaneous and subcutaneous mast cell tumors (MCT). METHODS: Client-owned dogs presenting to the Cornell University Hospital for Animals for surgical removal of a cytologically confirmed cutaneous or subcutaneous MCT >1 cm in diameter were enrolled. Cytological examination of FNA from the LRLN was compared with histology of the SLN. The SLN was identified by indirect computed tomographic lymphangiography (ICTL) after peritumoral injection of iopamidol and scanning at 1, 3, 5, 10, and 15 minutes. Histopathologic node score > 1 was considered metastatic. After case review by an oncologist, LRLN FNA cytology was compared with SLN histology for effect on changes in stage assignment and adjunctive treatment recommendations. RESULTS: Mast cell tumors were graded as 2 low (n = 11), 2 high (n = 2), and subcutaneous (n = 7). Optimal scan timing was 10 minutes after injection of iopamidol. Sentinel lymph node differed anatomically from LRLN in 5 of 18 scans. Metastases were detected by histology in 9 of 20 SLN compared with in 1 of 20 FNA of LRLN (P = .001), changing stage and adjunctive treatment recommendations 8 of 20 tumors. Only 6 of 19 LRLN FNA samples were diagnostic. CONCLUSION: Sentinel lymph nodes were consistently identified with ICTL and differed from LRLN in one-quarter of tumors. Histopathological examination of SLN altered recommendations in half of the dogs compared with the previous standard of care. CLINICAL SIGNIFICANCE: Indirect computed tomographic lymphangiography and SLN excision should be considered as a new standard for dogs with MCT.

Sentinel lymph node biopsy with intraoperative touch imprint cytology (TIC) in breast cancer: experience of a mild-volume center.

Although considered the gold standard in treatment of EBC, sentinel node biopsy still remains a debated issue. What to do in case of positive sentinel node and the need of intraoperative histological examination are the most topics under discussion. In this study we have retrospectively evaluate our case series of 359 sentinel node biopsy in the managing of breast cancer from January 2011 to December 2018, focusing on the TIC technique for performing intraoperative examination. It results in 12,8% "FALSE NEGATIVE" rate, in which only 4,2% in macrometastases, with an overall sensitivity of 68,4% (macrometastases: 86%; micrometastases: 11%), overall specificity of 98,7% and an overall accuracy of 89,7%. The intraoperative examination of SLN allows to reduce delayed surgery procedures and greater therapeutic safety in case of mastectomy. The TIC method can be considered valid, simple and rapid in identifying macrometastases, also allowing to avoid under-staging. The low sensitivity for micrometastases is not a limit, considering that recent evidence has drastically reduced the indications for ALND in these cases. Further ongoing trials and the possible validation of NOMOGRAMMS and SCORE are necessary to identify low risk cases in which to definitively omit the ALND and/or even the SLNB itself.

Survival analysis and sentinel lymph node status in thin cutaneous melanoma: A multicenter observational study.

Mitotic rate is no longer considered a staging criterion for thin melanoma in the 8th edition of the American Joint Committee on Cancer Staging Manual. The aim of this observational study was to identify prognostic factors for thin melanoma and predictors and prognostic significance of sentinel lymph node (SLN) involvement in a large multicenter cohort of patients with melanoma from nine tertiary care hospitals. A total of 4249 consecutive patients with thin melanoma diagnosed from January 1, 1998 to December 31, 2016 were included. The main outcomes were disease-free interval and melanoma-specific survival for the overall population and predictors of SLN metastasis (n = 1083). Associations between survival and SLN status and different clinical and pathologic variables (sex, age, tumor location, mitosis, ulceration, regression, lymphovascular invasion, histologic subtype, Clark level, and Breslow thickness) were analyzed by Cox proportional hazards regression and logistic regression. SLN status was the most important prognostic factor for melanoma-specific survival (hazard ratio, 13.8; 95% CI, 6.1-31.2; P < 0.001), followed by sex, ulceration, and Clark level for patients who underwent SLNB. A mitotic rate of >2 mitoses/mm2 was the only factor associated with a positive SLN biopsy (odds ratio, 2.9; 95% CI, 1.22-7; P = 0.01. SLN status is the most important prognostic factor in thin melanoma. A high mitotic rate is associated with metastatic SLN involvement. SLN biopsy should be discussed and recommended in patients with thin melanoma and a high mitotic rate.

Selectively hampered activation of lymph node-resident dendritic cells precedes profound T cell suppression and metastatic spread in the breast cancer sentinel lymph node.

BACKGROUND: Immune regulated pathways influence both breast cancer (BrC) development and response to (neo)adjuvant chemotherapy. The sentinel lymph node (SLN), as the first metastatic site, is also the first site where BrC-induced suppression of immune effector subsets occurs. Since intricate knowledge of the phenotypic and functional status of these immune effector subsets is lacking, we set out to map the immune landscape of BrC SLN. METHODS: Viable LN cells from BrC SLN (n = 58) were used for detailed flowcytometry-assisted mapping of the immune landscape of BrC SLN in a comparative analysis with healthy (i.e. prophylactic mastectomy-derived) axillary lymph nodes (HLN, n = 17). Findings were related to clinicopathological characteristics. RESULTS: Our data show that BrC-induced immune suppression in tumor-involved SLN, as evidenced by increased Treg and MDSC rates as well as by a generalized state of T cell anergy, coincides with hampered activation of LN-resident (LNR) dendritic cell (DC) subsets rather than of migratory DC subsets. Importantly, suppression of these LN-resident DC subsets preceded profoundly disabled T cell effector functions in tumor-involved SLN. Furthermore, we provide evidence that the suppressed state of LNR-cDC is not only related to nodal involvement but is also related to high-risk breast cancer subtypes that lack expression of hormone receptors and may be a negative predictor of disease-free survival. CONCLUSION: These data thus provide new insights in the mechanisms underlying loco-regional immune suppression induced by BrC and how these relate to clinical outcome. They identify the LNR-cDC subset as a pivotal regulatory node in cellular immune suppressive pathways and therefore as a promising therapeutic target to combat immune suppression and secure the induction of effective antitumor immunity, e.g. in combination with neo-adjuvant chemotherapy. .

Analysis of Postoperative Complications After 303 Sentinel Lymph Node Identification Procedures Using the SentiMag® Method in Breast Cancer Patients.

BACKGROUND The objective of this paper was to assess the complications following sentinel lymph node biopsy (SLNB) in breast cancer patients using the SentiMag® method. MATERIAL AND METHODS The study material consisted of 368 patients who had received the SLNB procedure in combination with wide local excision (WLE), simple mastectomy or who had an autonomous SLNB procedure in the period from January 2014 to September 2017. The final study group consisted of 303 patients who attended follow-up consultations. RESULTS Sensory disturbances in the arm occurred in 12 patients (9.9%), including 3 patients (1.5%) after WLE and 9 patients (8.4%) after simple mastectomy. Restricted mobility in the upper limb was experienced by 9 patients (7.1%), including 3 patients (1.5%) after WLE and 6 patients (5.6%) after simple mastectomy. Minimal-degree lymphedema developed in 9 patients (7.5%), including 2 patients (1%) after WLE and 7 patients (6.5%) after simple mastectomy. A significant correlation was demonstrated between the incidence of these complications and the number of lymph nodes dissected. A significantly higher incidence of paresthesia and lymphedema was revealed for simple mastectomy with SLNB when compared to WLE with SLNB. Discolorations upon tracer administration were observed in 47 patients (15.5%). CONCLUSIONS SentiMag® is a safe sentinel lymph node identification method used in breast cancer and has a low risk of complications. The rate of complications increases together with the number of dissected lymph nodes and the extent of the surgery. The possibility of temporary discolorations on the skin should be communicated to the patients explicitly prior to surgery.

Lymph Node Cellular Dynamics in Cancer and HIV: What Can We Learn for the Follicular CD4 (Tfh) Cells?

Lymph nodes (LNs) are central in the generation of adaptive immune responses. Follicular helper CD4 T (Tfh) cells, a highly differentiated CD4 population, provide critical help for the development of antigen-specific B cell responses within the germinal center. Throughout the past decade, numerous studies have revealed the important role of Tfh cells in Human Immunodeficiency Virus (HIV) pathogenesis as well as in the development of neutralizing antibodies post-infection and post-vaccination. It has also been established that tumors influence various immune cell subsets not only in their proximity, but also in draining lymph nodes. The role of local or tumor associated lymph node Tfh cells in disease progression is emerging. Comparative studies of Tfh cells in chronic infections and cancer could therefore provide novel information with regards to their differentiation plasticity and to the mechanisms regulating their development.