AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma.

AHNAK/Desmoyokin is a giant protein which has been recently linked to reorganization of the actin cytoskeleton, cellular migration and invasion. Here, we investigated the role of AHNAK in the pathophysiology of larynx carcinoma-one of the major subtypes of head and neck cancer. To this end, we analysed AHNAK expression in tumor tissues from 83 larynx carcinoma patients in relation to overall survival. We found that tumoral AHNAK overexpression significantly associated with poor survival of these patients both in univariate and multivariate analysis. In further studies, we combined the prognostic value of AHNAK with selected markers of inflammation, such as macrophage migration inhibitory factor (MIF) and tumor-infiltrating neutrophils (CD66b-positive cells). Both MIF and neutrophils have been linked to enhanced tumoral migration and poor clinical outcome in patients with orohypopharynx carcinoma-another major subtype of head and neck cancer. Interestingly, we found that synchronous high levels of AHNAK and MIF or AHNAK and neutrophils, respectively, were stronger predictors of poor survival than AHNAK alone. Synchronous high levels of all three markers were the strongest predictors of poor survival in our patient cohort. Taken together, our findings propose novel strategies for an accurate prognosis in larynx carcinoma and suggest potential mechanisms of inflammation-mediated tumor progression

Neutrophils activate tumoral CORTACTIN to enhance progression of orohypopharynx carcinoma

CORTACTIN is an actin-binding protein critically involved in cellular migration and invasion. Here, we investigated the role of CORTACTIN in the pathophysiology of orohypopharynx carcinoma one of the major subtypes of head and neck cancer. To this end, we analyzed CORTACTIN expression in tumor tissues from 89 orohypopharynx carcinoma patients in relation to clinical parameters. We found that high tumoral CORTACTIN expression associated with poor survival, higher T-stage, and higher lymph node metastasis (N-stage) in these patients. Next, we combined the prognostic values of tumoral and stromal cell biological parameters in our patient cohort. We determined the potential interaction of tumoral CORTACTIN with tumor-infiltrating neutrophils, which have been previously linked to poor clinical outcome in orohypopharynx carcinoma patients with advanced disease. Interestingly, we found that patients with both high tumoral CORTACTIN expression and high neutrophilic infiltration had significantly worse clinical outcome than all other patients in our cohort. These findings suggest that tumoral CORTACTIN and tumor-infiltrating neutrophils might be functionally linked during progression of orohypopharynx carcinoma. In vitro, we showed that neutrophils released soluble factors which phosphorylated CORTACTIN in the tumor cells and promoted their migration. Furthermore, we demonstrated that strong CORTACTIN phosphorylation significantly correlated with strong neutrophilic infiltration in tumor tissues from orohypopharynx carcinoma patients. Taken together, our findings unravel a novel mechanism of tumor-stroma interaction, which might be relevant for a more accurate prognosis and improved therapeutic strategies in this tumor entity

Patterns of tumor infiltrating lymphocytes in adenoid cystic carcinoma of the head and neck

Adenoid cystic carcinoma (ACC) is a rare malignancy in the head and neck. The prognosis remains poor and late recurrences often occur after 5 years and later. To date, there are no reliable prognostic markers for ACC. In several solid tumors, tertiary lymphoid structures (TLS) are associated with improved survival. This study aims to investigate the role of distribution patterns of tumor infiltrating immune cells (TIL) in ACC. A cohort of 50 patients from three different cancer centers was available for analysis. Sections were stained for CD3, CD4, CD8 and CD20 and evaluated with regard to their distribution of TIL. Patterns were determined as infiltrated-excluded, infiltrated-inflamed and presence of tertiary lymphoid structures. About half of the cases showed an infiltrated-excluded TIL pattern and only a minority of six cases had TLS present within the tumor. Within the inflamed phenotype CD3+ cells were by far the most abundant lymphocyte subtype, and within this compartment, CD8+ T cells were predominant. There was no influence on overall or disease-free survival by any of the TIL patterns. This indicates that ACC is a tumor with very low immunogenicity and even abundance of lymphocytes does not seem to improve prognosis for this disease. Therefore, the observed lack of response towards immunotherapy is not surprising and other methods to induce recognition of ACC by the immune system must be found

During early stages of cancer, neutrophils initiate anti-tumor immune responses in tumor-draining lymph nodes

Tumor-draining lymph nodes (LNs) play a crucial role during cancer spread and in initiation of anti-cancer adaptive immunity. Neutrophils form a substantial population of cells in LNs with poorly understood functions. Here, we demonstrate that, during head and neck cancer (HNC) progression, tumor-associated neutrophils transmigrate to LNs and shape anti-tumor responses in a stage-dependent manner. In metastasis-free stages (N0), neutrophils develop an antigen-presenting phenotype (HLA-DR+CD80+CD86+ICAM1+PD-L1-) and stimulate T cells (CD27+Ki67highPD-1-). LN metastases release GM-CSF and via STAT3 trigger development of PD-L1+ immunosuppressive neutrophils, which repress T cell responses. The accumulation of neutrophils in T cell-rich zones of LNs in N0 constitutes a positive predictor for 5-year survival, while increased numbers of neutrophils in LNs of N1-3 stages predict poor prognosis in HNC. These results suggest a dual role of neutrophils as essential regulators of anti-cancer immunity in LNs and argue for approaches fostering immunostimulatory activity of these cells during cancer therapy

Human Endomucin : Distribution Pattern, Expression on High Endothelial Venules, and Decoration with the MECA-79 Epitope

Endomucin is a typical sialomucin that we recently identified on the surface of mouse endothelial cells and on putative hematopoetic clusters of the dorsal aorta in the embryo. We have generated a panel of monoclonal antibodies (mAbs) against the extracellular part of human endomucin and polyclonal antibodies against the cytoplasmic part. Using immunohistochemistry endomucin was specifically detected on endothelial cells of blood and lymphatic vessels of all analyzed human tissues. In addition, the polyclonal antibodies stained the epithelium of the epidermis as well as epithelial and myoepithelial cells of the eccrine and apocrine glands in the skin. This nonendothelial staining could only be seen with a subset of mAbs if the staining procedure was amplified. Although high endothelial venules (HEVs) were not significantly stained with mAbs against endomucin, the polyclonal antibodies clearly detected endomucin on HEVs in lymphatic organs of the mouse and human, suggesting HEV-specific glycosylation affecting recognition by the mAbs. Indeed, endomucin isolated from human and mouse lymphoid organs carried the MECA-79 epitope that defines a set of L-selectin ligands on HEVs called peripheral node addressins. We conclude that human and mouse endomucin are endothelial sialomucins with the potential to function as L-selectin ligands

Effect of Antigen Retrieval Methods on Nonspecific Binding of Antibody–Metal Nanoparticle Conjugates on Formalin-Fixed Paraffin-Embedded Tissue

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissues provides important diagnostic and prognostic information in pathology. Metal nanoparticles (NPs) and, in particular, surface-enhanced Raman scattering (SERS) nanotags as a new class of labeling reagents are promising to be used for multiplexed protein profiling on tissue sections. However, nonspecific binding of NPs onto the tissue specimens greatly hampers their clinical applications. In this study, we found that the antigen retrieval method strongly influences the extent of nonspecific binding of the antibody–SERS NP conjugates to the tissue. Our SERS labels comprised ca. 70 nm Au nanostars coated with ethylene glycol-modified Raman reporter molecules for hydrophilic stabilization and subsequent covalent bioconjugation to antibodies. We systematically investigated the influence of heat- and protease-induced epitope retrieval (HIER and PIER, respectively) on the immunostaining quality of prostate-specific antigen (PSA) on human prostate tissue sections. The best staining results were obtained with PIER. Pretreatment of the tissue sections by HIER led to selective but nonspecific adsorption of the antibody–Au nanostar conjugates onto epithelial cells, while enzymatic treatment within PIER did not. In addition to gold nanostars, also other types of metal NPs with different shapes and sizes (including ca. 20 nm quasi-spherical Au NPs and ca. 60 nm quasi-spherical Au/Ag nanoshells) as well as tissue sections from different organs (including prostate and breast) were tested; in each case the same tendency was observed, i.e., PIER yielded better results than HIER. Therefore, we recommend PIER for future NP-based tissue immunostaining such as immuno-SERS microscopy. Alternatively, for antigens that can only be unmasked by heating, PEGylation of the NPs is recommended to avoid nonspecific binding

Dissimilar patterns of tumor-infiltrating immune cells at the invasive tumor front and tumor center are associated with response to neoadjuvant chemotherapy in primary breast cancer

Abstract Background Tumor-infiltrating lymphocytes (TILs) are described as an important immune modulator in the tumor microenvironment and are associated with breast cancer (BC) outcome. The spatial analysis of TILs and TIL subtype distribution at the invasive tumor front (ITF) and the tumor center (TC) might provide further insights into tumor progression. Methods We analyzed core biopsies from 87 pre-therapeutic BC patients for total TILs and the following subtypes: CD3+, CD4+, CD8+, CD20+ and CD68+ cells in correlation to clinicopathological parameters and disseminated tumor cells (DTCs) in the bone marrow. Results TILs and TIL subtypes showed significantly different spatial distribution among both tumor areas. TILs, especially CD3+ T cells were associated with the tumor status and tumor grading. BC patients responding to neoadjuvant chemotherapy had significantly more TILs and CD3+ T cells at the TC. The presence of DTCs after NACT was related to CD4+ infiltration at the TC. Conclusion The dissimilar spatial association of TILs and TIL subtypes with clinicopathological parameters, NACT response and minimal residual disease underlines the necessity of detailed TIL analysis for a better understanding of immune modulatory processes