Identification of α1,2-fucosylated signaling and adhesion molecules in head and neck squamous cell carcinoma.

Head and neck cancer is the seventh most common cancer in the world, and most cases manifest as head and neck squamous cell carcinoma. Despite the prominent role of fucosylated carbohydrate antigens in tumor cell adhesion and metastasis, little is known about the functional role of fucose-modified glycoproteins in head and neck cancer pathobiology. Inactivating polymorphisms of the fut2 gene, encoding for the α1,2-fucosyltransferase FUT2, are associated with an increased incidence of head and neck cancer among tobacco users. Moreover, the presence of the α1,2-fucosylated Lewis Y epitope, with both α1,2- and α1,3-linked fucose, has been observed in head and neck cancer tumors while invasive regions lose expression, suggesting a potential role for α1,2-fucosylation in the regulation of aggressive tumor cell characteristics. Here, we report an association between fut2 expression and head and neck cancer survival, document differential surface expression of α1,2-fucosylated epitopes in a panel of normal, dysplastic, and head and neck cancer cell lines, identify a set of potentially α1,2-fucosylated signaling and adhesion molecules including the epidermal growth factor receptor (EGFR), CD44 and integrins via tandem mass spectrometry, and finally, present evidence that EGFR is among the α1,2-fucosylated and LeY-displaying proteins in head and neck cancer. This knowledge will serve as the foundation for future studies to interrogate the role of LeY-modified and α1,2-fucosylated glycoproteins in head and neck cancer pathogenesis. Data are available via ProteomeXchange with identifier PXD029420.

Myeloid Cells Are Enriched in Tonsillar Crypts, Providing Insight into the Viral Tropism of Human Papillomavirus.

Viruses are the second leading cause of cancer worldwide, and human papillomavirus (HPV)-associated head and neck cancers are increasing in incidence in the United States. HPV preferentially infects the crypts of the tonsils rather than the surface epithelium. The present study sought to characterize the unique microenvironment within the crypts to better understand the viral tropism of HPV to a lymphoid-rich organ. Laser-capture microdissection of distinct anatomic areas (crypts, surface epithelium, and germinal centers) of the tonsil, coupled with transcriptional analysis and multiparameter immunofluorescence staining demonstrated that the tonsillar crypts are enriched with myeloid populations that co-express multiple canonical and noncanonical immune checkpoints, including PD-L1, CTLA-4, HAVCR2 (TIM-3), ADORA2A, IDO1, BTLA, LGALS3, CDH1, CEACAM1, PVR, and C10orf54 (VISTA). The resident monocytes may foster a permissive microenvironment that facilitates HPV infection and persistence. Furthermore, the myeloid populations within HPV-associated tonsil cancers co-express the same immune checkpoints, providing insight into potential novel immunotherapeutic targets for HPV-associated head and neck cancers.

Magnitude of benefit for adjuvant radiotherapy following minimally invasive surgery in intermediate to high risk HPV-positive oropharyngeal squamous cell carcinoma.

OBJECTIVE: To determine the outcomes and toxicities of minimally-invasive surgery with adjuvant intensity-modulated radiotherapy +/- chemotherapy (AT) compared to definitive surgical therapy (ST) in a contemporary cohort of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC). METHODS: From 2005 to 2013, a consecutive cohort of 190 HPV-positive OPSCC patients was retrospectively reviewed from multi-institutional databases maintained by the Departments of Otorhinolaryngology and Radiation Oncology. A total of 116 AT patients and 42 ST patients with intermediate or high risk pathologic features were included in the final analysis. All patients received minimally invasive surgery. Time to recurrence and time to death from the onset of surgery were evaluated. Toxicity data collected included dysphagia or xerostomia requiring feeding tube placement >6 months, or mandibular osteonecrosis requiring surgery or hyperbaric oxygen. RESULTS: All AT patients received IMRT to a median dose of 60 Gy. Chemotherapy delivered to 67.2% of AT patients. AT group included more high-risk patients given higher nodal classification (p = 0.005) and extracapsular extension (p = 0.0005). AT improved disease-free survival (HR 2.77, CI 1.22-6.28; p = 0.02) and local-regional control (HR 14.83, CI 3.240-67.839; p = 0.001). Disease-free survival with AT and tumor extracapsular extension was improved when compared to ST (HR of 4.34, CI 1.540-12.213; p = 0.006). Dysphagia or mandibular osteonecrosis toxicity after AT vs. ST of 19.0% vs. 2.4%. CONCLUSIONS: AT improved local-regional control and disease-free survival but was associated with greater toxicity. The recurrence benefit was most pronounced in tumors with extracapsular extension.

Preoperative Localization of Recurrence in the Thyroidectomy Bed Using a Radioactive Iodine125 Seed.

Intraoperative localization of nonpalpable recurrent thyroid cancer has been reported using needle localization, intraoperative ultrasound (US), dye injection, and radio-guided surgery. We describe the alternative technique of radioactive seed localization (RSL) in 3 patients with residual or recurrent papillary thyroid cancer. This technique has been used for many years in the setting of nonpalpable breast cancer, where it has been shown to be safe and has been associated with greater surgeon satisfaction as well as improved patient tolerability, cosmesis, and outcomes compared to needle localization. In addition, RSL allows complete decoupling of the radiology and surgery schedules. RSL was successful in our 3 patients with regard to safety, patient tolerability, and scheduling.

Differential Expression of Immune-Regulatory Genes Associated with PD-L1 Display in Melanoma: Implications for PD-1 Pathway Blockade.

PURPOSE: Blocking the immunosuppressive PD-1/PD-L1 pathway has antitumor activity in multiple cancer types, and PD-L1 expression on tumor cells and infiltrating myeloid cells correlates with the likelihood of response. We previously found that IFNG (interferon-gamma) was overexpressed by tumor-infiltrating lymphocytes in PD-L1(+) versus PD-L1(-) melanomas, creating adaptive immune resistance by promoting PD-L1 display. This study was undertaken to identify additional factors in the PD-L1(+) melanoma microenvironment coordinately contributing to immunosuppression. EXPERIMENTAL DESIGN: Archived, formalin-fixed paraffin-embedded melanoma specimens were assessed for PD-L1 protein expression at the tumor cell surface with IHC. Whole-genome expression analysis, quantitative (q)RT-PCR, IHC, and functional in vitro validation studies were used to assess factors differentially expressed in PD-L1(+) versus PD-L1(-) melanomas. RESULTS: Functional annotation clustering based on whole-genome expression profiling revealed pathways upregulated in PD-L1(+) melanomas, involving immune cell activation, inflammation, and antigen processing and presentation. Analysis by qRT-PCR demonstrated overexpression of functionally related genes in PD-L1(+) melanomas, involved in CD8(+) T-cell activation (CD8A, IFNG, PRF1, and CCL5), antigen presentation (CD163, TLR3, CXCL1, and LYZ), and immunosuppression [PDCD1 (PD-1), CD274 (PD-L1), and LAG3, IL10]. Functional studies demonstrated that some factors, including IL10 and IL32-gamma, induced PD-L1 expression on monocytes but not tumor cells. CONCLUSIONS: These studies elucidate the complexity of immune checkpoint regulation in the tumor microenvironment, identifying multiple factors likely contributing to coordinated immunosuppression. These factors may provide tumor escape mechanisms from anti-PD-1/PD-L1 therapy, and should be considered for cotargeting in combinatorial immunomodulation treatment strategies.

Evidence for a role of the PD-1:PD-L1 pathway in immune resistance of HPV-associated head and neck squamous cell carcinoma.

Human papillomavirus-associated head and neck squamous cell carcinomas (HPV-HNSCC) originate in the tonsils, the major lymphoid organ that orchestrates immunity to oral infections. Despite its location, the virus escapes immune elimination during malignant transformation and progression. Here, we provide evidence for the role of the PD-1:PD-L1 pathway in HPV-HNSCC immune resistance. We show membranous expression of PD-L1 in the tonsillar crypts, the site of initial HPV infection. In HPV-HNSCCs that are highly infiltrated with lymphocytes, PD-L1 expression on both tumor cells and CD68+ tumor-associated macrophages is geographically localized to sites of lymphocyte fronts, whereas the majority of CD8+ tumor-infiltrating lymphocytes express high levels of PD-1, the inhibitory PD-L1 receptor. Significant levels of mRNA for IFN-γ, a major cytokine inducer of PD-L1 expression, were found in HPV+ PD-L1(+) tumors. Our findings support the role of the PD-1:PD-L1 interaction in creating an "immune-privileged" site for initial viral infection and subsequent adaptive immune resistance once tumors are established and suggest a rationale for therapeutic blockade of this pathway in patients with HPV-HNSCC.

Colocalization of inflammatory response with B7-h1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape.

Although many human cancers such as melanoma express tumor antigens recognized by T cells, host immune responses often fail to control tumor growth for as yet unexplained reasons. Here, we found a strong association between melanocyte expression of B7-H1 (PD-L1), an immune-inhibitory molecule, and the presence of tumor-infiltrating lymphocytes (TILs) in human melanocytic lesions: 98% of B7-H1(+) tumors were associated with TILs compared with only 28% of B7-H1(-) tumors. Indeed, B7-H1(+) melanocytes were almost always localized immediately adjacent to TILs. B7-H1/TIL colocalization was identified not only in melanomas but also in inflamed benign nevi, indicating that B7-H1 expression may represent a host response to tissue inflammation. Interferon-γ, a primary inducer of B7-H1 expression, was detected at the interface of B7-H1(+) tumors and TILs, whereas none was found in B7-H1(-) tumors. Therefore, TILs may actually trigger their own inhibition by secreting cytokines that drive tumor B7-H1 expression. Consistent with this hypothesis, overall survival of patients with B7-H1(+) metastatic melanoma was significantly prolonged compared with that of patients with B7-H1(-) metastatic melanoma. Therefore, induction of the B7-H1/PD-1 pathway may represent an adaptive immune resistance mechanism exerted by tumor cells in response to endogenous antitumor activity and may explain how melanomas escape immune destruction despite endogenous antitumor immune responses. These observations suggest that therapies that block this pathway may benefit patients with B7-H1(+) tumors.

The tryptophan-rich motifs of the thrombospondin type 1 repeats bind VLAL motifs in the latent transforming growth factor-beta complex.

Transforming growth factor-beta (TGF-beta) is secreted as a latent complex of the latency-associated peptide (LAP) and the mature domain, which must be activated for TGF-beta to signal. We previously identified thrombospondin 1 (TSP1) as a physiologic activator of TGF-beta in vitro and in vivo. The WSXW sequences in the type 1 repeats of TSP1 interact with the mature domain of TGF-beta, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF-beta latent complex. However, the binding site for the WSXW sequence was not identified. In this report, we show that the WSXW sequences bind the (61)VLAL sequence in mature TGF-beta and also bind (77)VLAL in LAP. A glutathione S-transferase (GST) fusion protein of the second TSP1 type 1 repeat (GST-TSR2) binds immobilized VLAL peptide. VLAL peptides inhibit binding of LAP and mature TGF-beta to soluble GST-TSR2 and immobilized WSXW peptide. VLAL peptide inhibits TSP1-mediated activation of recombinant and endothelial cell-derived latent TGF-beta. Furthermore, TGF-beta or LAP deleted in the VLAL sequence fails to bind immobilized WSXW or soluble GST-TSR2, indicating that binding to both VLAL sequences is important for association of TSP1 and the latent complex. Additionally, TSP1 is unable to activate latent TGF-beta when VLAL is deleted from the mature domain. These data show that the WSXW motif binds VLAL on both LAP and mature TGF-beta, and these interactions are critical for TSP1-mediated activation of the TGF-beta latent complex.

Molecular interactions that confer latency to transforming growth factor-beta.

A major point of regulation of transforming growth factor-beta (TGF-beta) function is through control of activation of the latent TGF-beta complex, which consists of the latency associated peptide (LAP) secreted in non-covalent association with mature TGF-beta. Activation involves proteolysis, dissociation, or altered binding of LAP. However, the mechanism by which LAP confers latency to TGF-beta is poorly understood. Previously, we identified a conserved sequence near the N terminus of LAP as a site of thrombospondin-1 (TSP1) binding to the latent complex. Now we show that expression of the TGF-beta1-latent complex deleted in the TSP1 binding site ((54)LSKL) of LAP (DeltaLSKL LAP) results in secretion of LAP, but not of mature TGF-beta. DeltaLSKL LAP also fails to bind soluble or immobilized TGF-beta1. Consistent with an inability to bind the mature domain, DeltaLSKL LAP is unable to confer latency to TGF-beta, suggesting that the LSKL sequence is important, not only for TSP1 binding and activation, but also for binding to the mature domain. We identified the sequence (94)RKPK in the receptor-binding region of mature TGF-beta1 as the binding site for LAP. Peptides of the RKPK sequence bind LAP and inhibit LAP/TGF-beta association. RKPK peptides also activate latent TGF-beta, presumably by disrupting LAP-mature TGF-beta interactions. These studies provide a molecular basis for both latency and activation by TSP1 through the LSKL sequence of LAP binding to the RKPK sequence of mature TGF-beta.

A novel assay for the quantification of invasion from raft cultures of lung carcinomas.

Assays that conveniently quantify invasion of carcinoma cells in vitro have generally measured the passage of dissociated cells into a matrix. Although these assays have been helpful in identifying relative differences between different carcinoma cell lines or types, the requirement for dissociation overlooks the possible modulation of invasion by cell-cell interactions among carcinoma cells. Described here is a novel assay that quantifies invasion of a matrix placed above intact, multilayered raft cultures of lung carcinoma cell lines A549 and H520. The assay was performed by placing a porous membrane coated with matrix at the air interface of the raft cultures for varying lengths of time, after which the cells invading the matrix were enumerated. The numbers of cells invading increased in a relatively linear fashion from 24 to 72 h, and the absolute numbers within each cell line were reproducible with multiple sets of raft cultures prepared at different times. It was also found that this assay could quantify differences in invasion caused by changes in matrix composition. It is concluded that this assay can reproducibly quantify carcinoma cell invasion from three dimensional raft cultures.

Differential expression and biodistribution of cytokeratin 18 and desmoplakins in non-small cell lung carcinoma subtypes.

Adenocarcinoma (AC), squamous cell carcinoma (SCC) and adenosquamous carcinoma (ASC) of the lung are morphologically distinguished in part by cyto-architectural features. However, little is known about the relative expression and distribution of cyto-architectural proteins among AC, SCC and ASC. Initial microarray analysis revealed significant differences in expression of two cyto-architectural genes in AC, SCC and ASC. Desmoplakin (DP) 1 and 2, which link desmosomes to intermediate filaments, was strongly expressed in SCC relative to AC and ASC. Cytokeratin 18 (CK18), an intermediate filament that is commonly linked to desmoplakin, was strongly expressed in AC and ASC relative to SCC. Western blot analysis demonstrated that AC and ASC had abundant CK18 protein, whereas CK18 was weakly detected in SCC. DP 1 and 2 are strongly expressed in SCC and minimally expressed in AC and ASC. However, the ratio of one to the other is the same in SCC and AC, but DP2 is lost in ASC. Microscopic analysis with fluorescence-labeled antibodies for CK18 and DP 1 and 2 revealed abundant membrane localization of DP and minimal perinuclear localization of CK18 in SCC. In contrast, in both AC and ASC, the CK18 protein was diffusely distributed within the cytoplasm, and DP showed both membranous and cytoplasmic localization. In conclusion, the data here shows that AC, SCC and ASC each have specific patterns of DP 1 and 2 and CK18 gene expression, protein content and biodistribution.