Altered extracellular matrix correlates with an immunosuppressive tumor microenvironment and disease progression in younger adults with oral cavity squamous cell carcinoma.

Introduction: Oral cavity squamous cell carcinoma (OSCC) occurs most frequently in patients >60 years old with a history of tobacco and alcohol use. Epidemiological studies describe increased incidence of OSCC in younger adults (<45 years). Despite its poor prognosis, knowledge of OSCC tumor microenvironment (TME) characteristics in younger adults is scarce and could help inform possible resistance to emerging treatment options. Methods: Patients with OSCC were evaluated using TCGA-HNSC (n=121) and a stage and subsite-matched institutional cohort (n=8) to identify differential gene expression focusing on the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) processes in younger (≤45 years) vs. older adults (≥60 years). NanoString nCounter analysis was performed using isolated total RNA from formalin-fixed paraffin-embedded (FFPE) tumor samples. Stained tumor slides from young and old OSCC patients were evaluated for CD8+ T-cell counts using immunohistochemistry. Results: Younger OSCC patients demonstrated significantly increased expression of ECM remodeling and EMT process genes, as well as TME immunosuppression. Gene set enrichment analyses demonstrated increased ECM pathways and concurrent decreased immune pathways in young relative to old patients. Transcripts per million of genetic markers involved in ECM remodeling including LAMB3, VCAN, S100A9, COL5A1, and ITGB2 were significantly increased in tumors of younger vs. older patients (adjusted p-value < 0.10). Young patient TMEs demonstrated a 2.5-fold reduction in CD8+ T-cells as compared to older patients (p < 0.05). Conclusion: Differential gene expression impacting ECM remodeling and TME immunosuppression may contribute to disease progression in younger adult OSCC and has implications on response to evolving treatment modalities, such as immune checkpoint inhibitor therapy.

Predictive capacity of immune-related adverse events and cytokine profiling in neoadjuvant immune checkpoint inhibitor trials for head and neck squamous cell carcinoma.

OBJECTIVES: Certain low-level immune-related adverse events (irAEs) have been associated with survival benefits in patients with various solid tumors on immune checkpoint inhibitors (ICIs). We aimed to investigate the association between irAEs and response to neoadjuvant ICIs in patients with head and neck squamous cell carcinoma (HNSCC) and to identify differences in circulating cytokine levels based on irAE status. METHODS: This was a retrospective cohort study including three neoadjuvant clinical trials from July 2017 to January 2022: NCT03238365 (nivolumab ± tadalafil), NCT03854032 (nivolumab ± BMS986205), NCT03618654 (durvalumab ± metformin). The presence and type of irAEs, pathologic treatment response, and survival were compared. Canonical linear discriminant analysis (LDA) was performed to identify combinations of circulating cytokines predictive of irAEs using plasma sample multiplex assay. RESULTS: Of 113 participants meeting inclusion criteria, 32 (28.3%) developed irAEs during treatment or follow-up. Positive p16 status was associated with irAEs (odds ratio [OR] 2.489; 95% CI 1.069-6.119; p = 0.043). irAEs were associated with pathologic treatment response (OR 3.73; 95% CI 1.34-10.35; p = 0.011) and with higher OS in the combined cohort (HR 0.319; 95% CI 0.113-0.906; p = 0.032). Patients with irAEs within the nivolumab cohort had significant elevations of select cytokines pre-treatment. Canonical LDA identified key drivers of irAEs among all trials, which were highly predictive of future irAE status. CONCLUSIONS: irAEs are associated with response to neoadjuvant ICI therapy in HNSCC and can serve as clinical indicators for improved clinical outcomes. irAEs can be predicted by concentrations of several circulating cytokines prior to treatment.

Nivolumab and ipilimumab in combination with radiotherapy in patients with high-risk locally advanced squamous cell carcinoma of the head and neck.

BACKGROUND: The combination of nivolumab and ipilimumab has been approved for the treatment of multiple solid tumors. This was a phase I study investigating definitive radioimmunotherapy (RIT) with nivolumab and ipilimumab for the treatment of locally advanced (LA) squamous cell carcinoma of the head and neck (SCCHN). METHODS: Patients with newly diagnosed, stage IVA-IVB SCCHN eligible for cisplatin-based chemotherapy received nivolumab (3 mg/kg every 2 weeks for a total of 17 doses) and ipilimumab (1 mg/kg every 6 weeks for a total of 6 doses) starting 2 weeks prior to radiotherapy. The primary endpoint was safety of definitive RIT. Secondary endpoints included progression-free survival (PFS) and overall survival (OS). Exploratory endpoints included the association of baseline programmed death-ligand 1 (PD-L1) expression as well as on-treatment changes in immune bias with treatment outcomes. RESULTS: Twenty-four patients were enrolled. With a median follow-up of 36.1 months, grade 3 or higher treatment-related adverse events were reported in 21 individuals (88%); 5 individuals developed in-field soft tissue ulceration during consolidation immunotherapy, resulting in one fatality. The 3-year PFS and OS rates were 74% (95% CI 58% to 94%) and 96% (95% CI 88% to 100%), respectively. PD-L1 combined positive score (CPS) did not correlate with death or disease progression. Decreases in extracellular vesicle PD-L1 within the concurrent RIT phase were associated with prolonged PFS (p=0.006). Also, interval decreases in circulating interleukin (IL)4, IL9, IL12, and IL17a during concurrent RIT were associated with subsequent ulceration. CONCLUSIONS: Definitive RIT with nivolumab and ipilimumab has sufficient clinical activity to support further development. Early changes in circulating biomarkers appear able to predict treatment outcomes as well as ensuing in-field soft tissue ulceration. TRIAL REGISTRATION NUMBER: NCT03162731.

IL-8 correlates with nonresponse to neoadjuvant nivolumab in HPV positive HNSCC via a potential extracellular vesicle miR-146a mediated mechanism.

Therapy using anti-PD-1 immune checkpoint inhibitors (ICI) has revolutionized the treatment of many cancers including head and neck squamous cell carcinomas (HNSCC), but only a fraction of patients respond. To better understand the molecular mechanisms driving resistance, we performed extensive analysis of plasma and tumor tissues before and after a 4-week neoadjuvant trial in which HNSCC patients were treated with the anti-PD-1 inhibitor, nivolumab. Luminex cytokine analysis of patient plasma demonstrated that HPVpos nonresponders displayed high levels of the proinflammatory chemokine, interleukin-8 (IL-8), which decreased after ICI treatment, but remained higher than responders. miRNAseq analysis of tetraspanin-enriched small extracellular vesicles (sEV) purified from plasma of HPVpos nonresponders demonstrated significantly lower levels of seven miRNAs that target IL-8 including miR-146a. Levels of the pro-survival oncoprotein Dsg2, which has been to down-regulate miR-146a, are elevated with HPVpos tumors displaying higher levels than HPVneg tumors. Dsg2 levels decrease significantly following ICI in responders but not in nonresponders. In cultured HPVpos cells, restoration of miR-146a by forced expression or treatment with miR-146a-loaded sEV, reduced IL-8 level, blocked cell cycle progression, and promoted cell death. These findings identify Dsg2, miR-146a, and IL-8 as potential biomarkers for ICI response and suggest that the Dsg2/miR-146a/IL-8 signaling axis negatively impacts ICI treatment outcomes and could be targeted to improve ICI responsiveness in HPVpos HNSCC patients.

CD8+ and FoxP3+ T-Cell Cellular Density and Spatial Distribution After Programmed Death-Ligand 1 Check Point Inhibition.

OBJECTIVES: To analyze CD8+ and FoxP3+ T-cell cellular density (CD) and intercellular distances (ID) in head and neck squamous cell carcinoma (HNSCC) samples from a neoadjuvant trial of durvalumab +/- metformin. METHODS: Paired pre- and post-treatment primary HNSCC tumor samples were stained for CD8+ and FoxP3+. Digital image analysis was used to determine estimated mean CD8+ and FoxP3+ CDs and CD8+-FoxP3+ IDs in the leading tumor edge (LTE) and tumor adjacent stroma (TAS) stratified by treatment arm, human papillomavirus (HPV) status, and pathologic treatment response. A subset of samples was characterized for T-cell related signatures using digital spatial genomic profiling. RESULTS: Post-treatment analysis revealed a significant decrease in FoxP3+ CD and an increase in CD8+ CDs in the TAS between patients receiving durvalumab and metformin versus durvlaumab alone. Both treatment arms demonstrated significant post-treatment increases in ID. Although HPV+ and HPV- had similar immune cell CDs in the tumor microenvironment, HPV+ pre-treatment samples had 1.60 times greater ID compared with HPV- samples, trending toward significance (p = 0.05). At baseline, pathologic responders demonstrated a 1.16-fold greater CD8+ CDs in the LTE (p = 0.045) and 2.28-fold greater ID (p = 0.001) than non-responders. Digital spatial profiling revealed upregulation of FoxP3+ and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) in the TAS (p = 0.006, p = 0.026) in samples from pathologic responders. CONCLUSIONS: Analysis of CD8+ and FoxP3+ detected population differences according to HPV status, pathologic response, and treatment. Greater CD8+-FoxP3+ ID was associated with pathologic response. CD8+ and FoxP3+ T-cell distributions may be predictive of response to immune checkpoint inhibition. CLINICALTRIALS: gov (Identifier NCT03618654). LEVEL OF EVIDENCE: 3 Laryngoscope, 133:1875-1884, 2023.

Intracellular monitoring by dendritic cells - a new way to stay informed - from a simple scavenger to an active gatherer.

Dendritic cells (DCs) are required for the initiation of the adaptive immune response. Their ability to acquire antigens in the periphery is a critical step in this process. DCs express a wide variety of adhesion molecules and possess an extremely fluid plasma membrane that facilitates scavenging the extracellular environment and capturing material like exosomes, apoptotic bodies, and pathogens. Besides these standard routes, the acquisition of antigens by DCs can be further facilitated by tunneling nanotubes, trogocytosis, and gap junctions. However, in this article, we will argue that this is an incomplete picture, as certain observations in the literature cannot be explained if we assume DCs acquire antigens only through these means. Instead, it is more likely that DCs preferentially use adhesion molecules to form long-lasting cell-cell interactions to actively siphon material from cells they are in direct contact with. It is highly likely that DCs use this mechanism to continually capture membrane and cytosolic material directly from surrounding cells, which they scan to assess the health of the donor cell. Doing so would provide an array of advantages for the host immune system, as it would not be reliant on compromised cells to release antigens into the extracellular milieu. Therefore, we propose updating our view of DC antigen acquisition to include a process of active, contact-dependent capture of material directly from neighboring cell cytosol (cytocytosis), which we would term intracellular monitoring.

Tadalafil Enhances Immune Signatures in Response to Neoadjuvant Nivolumab in Resectable Head and Neck Squamous Cell Carcinoma.

PURPOSE: We hypothesize that the addition of the phosphodiesterase-5 inhibitor tadalafil to the PD-1 inhibitor nivolumab, is safe and will augment immune-mediated antitumor responses in previously untreated squamous cell carcinoma of the head and neck (HNSCC). PATIENTS AND METHODS: We conducted a two-arm multi-institutional neoadjuvant randomized trial in any-stage resectable HNSCC (NCT03238365). Patients were stratified at randomization by human papillomavirus (HPV) status. Patients in both arms received nivolumab 240 mg intravenously on days 1 and 15 followed by surgery on day 28. Those in the combination therapy arm also received tadalafil 10 mg orally once daily for 4 weeks. Imaging, blood, and tumor were obtained pretreatment and posttreatment for correlative analysis. RESULTS: Neoadjuvant therapy was well-tolerated with no grade 3 to 5 adverse events and no surgical delays. Twenty-five of 46 (54%) evaluable patients had a pathologic treatment response of ≥20%, including three (7%) patients with a complete pathologic response. Regardless of HPV status, tumor proliferation rate was a negative predictor of response. A strong pretreatment T-cell signature in the HPV-negative cohort was a predictor of response. Tadalafil altered the immune microenvironment, as evidenced by transcriptome data identifying enriched B- and natural killer cell gene sets in the tumor and augmented effector T cells in the periphery. CONCLUSIONS: Preoperative nivolumab ± tadalafil is safe in HNSCC and results in more than 50% of the patients having a pathologic treatment response of at least 20% after 4 weeks of treatment. Pretreatment specimens identified HPV status-dependent signatures that predicted response to immunotherapy while posttreatment specimens showed augmentation of the immune microenvironment with the addition of tadalafil.

Cytokine Profiling in Low- and High-Density Small Extracellular Vesicles from Epidermoid Carcinoma Cells.

Exosomes or small extracellular vesicles (sEVs) are membrane-bound nanoparticles that carry various macromolecules and act as autocrine and paracrine signaling messengers. In this study, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. In this study, sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation. They were then subjected to multiplex profiling of cytokines associated with the surface of intact sEVs. The results revealed a previously undescribed active sorting of cytokines onto the surface of low-density and high-density sEV subpopulations. Specifically, an altered surface presentation of desmoglein 2 decreased FGF-2 and VEGF in low-density sEVs. In addition, in response to desmoglein 2, IL-8 and RANTES were increased in low-density sEVs but only slightly decreased in high-density sEVs. Finally, IL-6 and G-CSF were increased dramatically in high-density sEVs. This comprehensive analysis of the cytokine production profile by squamous cell carcinoma‒derived sEVs highlights their contribution to immune evasion, pro-oncogenic and proangiogenic activity, and the potential to identify diagnostic disease biomarkers.

Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

Phase Ib Clinical Trial of IGV-001 for Patients with Newly Diagnosed Glioblastoma.

PURPOSE: Despite standard of care (SOC) established by Stupp, glioblastoma remains a uniformly poor prognosis. We evaluated IGV-001, which combines autologous glioblastoma tumor cells and an antisense oligonucleotide against IGF type 1 receptor (IMV-001), in newly diagnosed glioblastoma. PATIENTS AND METHODS: This open-label protocol was approved by the Institutional Review Board at Thomas Jefferson University. Tumor cells collected during resection were treated ex vivo with IMV-001, encapsulated in biodiffusion chambers with additional IMV-001, irradiated, then implanted in abdominal acceptor sites. Patients were randomized to four exposure levels, and SOC was initiated 4-6 weeks later. On the basis of clinical improvements, randomization was halted after patient 23, and subsequent patients received only the highest exposure. Safety and tumor progression were primary and secondary objectives, respectively. Time-to-event outcomes were compared with the SOC arms of published studies. RESULTS: Thirty-three patients were enrolled, and median follow-up was 3.1 years. Six patients had adverse events (grade ≤3) possibly related to IGV-001. Median progression-free survival (PFS) was 9.8 months in the intent-to-treat population (vs. SOC, 6.5 months; P = 0.0003). In IGV-001-treated patients who met Stupp-eligible criteria, PFS was 11.6 months overall (n = 22; P = 0.001) and 17.1 months at the highest exposure (n = 10; P = 0.0025). The greatest overall survival was observed in Stupp-eligible patients receiving the highest exposure (median, 38.2 months; P = 0.044). Stupp-eligible patients with methylated O6-methylguanine-DNA methyltransferase promoter (n = 10) demonstrated median PFS of 38.4 months (P = 0.0008). Evidence of immune activation was noted. CONCLUSIONS: IGV-001 was well tolerated, PFS compared favorably with SOC, and evidence suggested an immune-mediated mechanism (ClinicalTrials.gov: NCT02507583).

Discordant Responses Between Primary Head and Neck Tumors and Nodal Metastases Treated With Neoadjuvant Nivolumab: Correlation of Radiographic and Pathologic Treatment Effect.

PD-1 blockade represents a promising treatment in patients with head and neck squamous cell carcinoma (HNSCC). We analyzed results of a neoadjuvant randomized window-of-opportunity trial of nivolumab plus/minus tadalafil to investigate whether immunotherapy-mediated treatment effects vary by site of involvement (primary tumor, lymph nodes) and determine how radiographic tumor shrinkage correlates with pathologic treatment effect. PATIENTS AND METHODS: Forty-four patients enrolled in trial NCT03238365 were treated with nivolumab 240 mg intravenously on days 1 and 15 with or without oral tadalafil, as determined by random assignment, followed by surgery on day 31. Radiographic volumetric response (RVR) was defined as percent change in tumor volume from pretreatment to posttreatment CT scan. Responders were defined as those with a 10% reduction in the volume of the primary tumor or lymph nodes (LN). Pathologic treatment effect (PTE) was defined as the area showing fibrosis or lymphohistiocytic inflammation divided by total tumor area. RESULTS: Sixteen of 32 patients (50%) with pathologic evidence of LN involvement exhibited discordant PTE between primary sites and LN. In four patients with widely discordant adjacent LN, increased PTE was associated with increased infiltration of tumor CD8+ T cells and CD163+ macrophages, whereas stromal regulatory T cells were associated with low nodal PTE. RVR correlated with PTE at both primary tumor (slope = 0.55, p < 0.001) and in LN (slope = 0.62, p < 0.05). 89% (16/18) of radiographic non-responders with T1-T3 primary sites had no (n = 7) or minimal PTE (n = 9), whereas 15/17 (88%) of radiographic responders had moderate (n = 12) or complete (n = 3) PTE. CONCLUSION: Nivolumab often induces discordant treatment effects between primary tumor sites and metastatic lymph nodes within subjects. This treatment discordance was also demonstrated in adjacent lymph nodes, which may correlate with local immune cell makeup. Finally, although these data were generated by a relatively small population size, our data support the use of early radiographic response to assess immunotherapy treatment effect in HNSCC.

Cancer-Associated Fibroblast Density, Prognostic Characteristics, and Recurrence in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis.

INTRODUCTION: The progression and clinical course of head and neck squamous cell carcinoma (HNSCC) relies on complex interactions between cancer and stromal cells in the tumor microenvironment (TME). Among the most abundant of these stromal cells are cancer-associated fibroblasts (CAFs). While their contribution to tumor progression is widely acknowledged, and various CAF-targeted treatments are under development, the relationship between CAF density and the clinicopathologic course of HNSCC has not been clearly defined. Here we examine the published evidence investigating the relationship of cancer-associated fibroblasts to local recurrence and indicators of prognostic significance in HNSCC. METHODS: We conducted a meta-analysis of existing publications that compare the relationship between CAF density, local recurrence, and clinically significant pathologic criteria of disease development (T stage, nodal positivity, clinical stage, vascular invasion, perineural invasion, Ki67 expression, and differentiation). Thirteen studies met the selection criteria, providing a total study population of 926 patients. Forest plots and risk ratios were generated to illustrate overall relationships. RESULTS: Higher CAF density within the tumor microenvironment is associated with advanced T stage, nodal infiltration, clinical stage, vascular invasion, perineural invasion, Ki67 expression, and differentiation (p <0.05). High CAF density is also associated with increased rates of local recurrence (p <0.001). CONCLUSIONS: Across multiple studies, increased CAF density is correlated with histopathological criteria of poor prognosis in HNSCC. These findings highlight that CAFs may play a pivotal role in HNSCC development and progression. Staining for CAFs may represent a valuable addition to current pathologic analysis and help to guide prognosis and treatment. Understanding the mechanisms by which CAFs reciprocally interact with cancer cells will be crucial for optimization of TME-focused treatment of HNSCC.

miRNA- and cytokine-associated extracellular vesicles mediate squamous cell carcinomas.

Exosomes, or small extracellular vesicles (sEVs), serve as intercellular messengers with key roles in normal and pathological processes. Our previous work had demonstrated that Dsg2 expression in squamous cell carcinoma (SCC) cells enhanced both sEV secretion and loading of pro-mitogenic cargo. In this study, using wild-type Dsg2 and a mutant form that is unable to be palmitoylated (Dsg2cacs), we investigated the mechanism by which Dsg2 modulates SCC tumour development and progression through sEVs. We demonstrate that palmitoylation was required for Dsg2 to regulate sub-cellular localisation of lipid raft and endosomal proteins necessary for sEV biogenesis. Pharmacological inhibition of the endosomal pathway abrogated Dsg2-mediated sEV release. In murine xenograft models, Dsg2-expressing cells generated larger xenograft tumours as compared to cells expressing GFP or Dsg2cacs. Co-treatment with sEVs derived from Dsg2-over-expressing cells increased xenograft size. Cytokine profiling revealed, Dsg2 enhanced both soluble and sEV-associated IL-8 and miRNA profiling revealed, Dsg2 down-regulated both cellular and sEV-loaded miR-146a. miR-146a targets IRAK1, a serine-threonine kinase involved in IL-8 signalling. Treatment with a miR-146a inhibitor up-regulated both IRAK1 and IL-8 expression. RNAseq analysis of HNSCC tumours revealed a correlation between Dsg2 and IL-8. Finally, elevated IL-8 plasma levels were detected in a subset of HNSCC patients who did not respond to immune checkpoint therapy, suggesting that these patients may benefit from prior anti-IL-8 treatment. In summary, these results suggest that intercellular communication through cell-cell adhesion, cytokine release and secretion of EVs are coordinated, and critical for tumour growth and development, and may serve as potential prognostic markers to inform treatment options. ABBREVIATIONS: Basal cell carcinomas, BCC; Betacellulin, BTC; 2-bromopalmitate, 2-Bromo; Cluster of differentiation, CD; Cytochrome c oxidase IV, COX IV; Desmoglein 2, Dsg2; Early endosome antigen 1, EEA1; Epidermal growth factor receptor substrate 15, EPS15; Extracellular vesicle, EV; Flotillin 1, Flot1; Glyceraldehyde-3-phosphate dehydrogenase, GAPH; Green fluorescent protein, GFP; Head and neck squamous cell carcinoma, HNSCC; Interleukin-1 receptor-associated kinase 1, IRAK1; Interleukin 8, IL-8; Large EV, lEV; MicroRNA, miR; Palmitoylacyltransferase, PAT; Ras-related protein 7 Rab7; Small EV, sEV; Squamous cell carcinoma, SCC; Tissue inhibitor of metalloproteinases, TIMP; Tumour microenvironment, TME.

A Pilot Study of Radiation Therapy in Combination With Pembrolizumab in Patients With Metastatic Renal Cell Cancer.

OBJECTIVES: There is no study published regarding the benefit of radiation therapy (RT) in combination with immune checkpoint inhibitors (ICIs) for the treatment of metastatic renal cell cancer (mRCC). This report is part of an exploratory study aiming to determine the immunomodulatory activity of RT alone or in combination with pembrolizumab in solid tumors. MATERIALS AND METHODS: mRCC patients were treated with a combination of RT (8 Gy×1 or 4 Gy×5) followed by pembrolizumab with or without lead-in dose of pembrolizumab. Treatment response was measured based on the modified Response Evaluation Criteria in Solid Tumors criteria. Adverse events were monitored and graded. Pre-RT and post-RT tumor biopsies were obtained to evaluate programmed death-ligand 1 expression. Immune markers from peripheral blood before, during, and after treatment were analyzed using flow cytometry. RESULTS: Twelve mRCC patients who progressed on prior antiangiogenic therapy were enrolled. Half had 2 lines of prior therapy. Two patients (16.7%) had partial responses and were on study for 12.4 and 14.5 months. Three patients had stable disease for a period ranging from 4.2 to 10.4 months, whereas 7 patients had progressive disease. Median progression-free survival was 8.6 months and median overall survival was 32.3 months. Three patients had grade ≥3 events (hyperglycemia, thrombocytopenia, transaminitis). Biopsied tissue programmed death-ligand 1 expression and tumor-infiltrating lymphocytes were numerically higher in responders comparing to nonresponders (Modified Proportion Score 45% vs. 30.45%; tumor-infiltrating lymphocytes odds ratio 4.92). CONCLUSION: Combining RT with pembrolizumab in pretreated mRCC is well-tolerated and appears to have comparable efficacy with single-agent nivolumab.

Prognostic Significance of Tumor-Associated Macrophage Content in Head and Neck Squamous Cell Carcinoma: A Meta-Analysis.

Background: Head and neck squamous cell carcinoma (HNSCC) exists within a microenvironment rich in immune cells. Macrophages are particularly abundant in and around tumor tissue, and have been implicated in the growth, malignancy, and persistence of HNSCC (1). However, current literature reports variable degrees of association between the density of tumor-associated macrophages (TAMs) and clinicopathologic markers of disease (2, 3). These inconsistent findings may be a result of differences in approach to TAM detection. Authors have measured total TAMs in tumor tissue, while others have stained tumor samples for individual subtypes of TAMs, which include pro-inflammatory (M1-like) and immunosuppressive (M2-like). Our aim is to more clearly define the prognostic significance of the phenotypes of tumor-associated macrophages in HNSCC. Methods: We conducted a meta-analysis of the existing publications investigating the relationship between TAMs (total and M2-like subtype) and T stage, nodal involvement, vascular invasion, lymphatic invasion, and tumor differentiation (Figure 1). A total of 12 studies were included. Forest plots and risk ratios were generated to report overall effect. Results: Higher density of both total and M2-like subtype of TAMs in the tumor microenvironment is associated with advanced T stage, increased rates of nodal positivity, presence of vascular invasion, and presence of lymphatic invasion (p < 0.0001; Figures 2-9). There is no significant association between TAM density, either total or M2-like subtype, and tumor differentiation (Figures 10, 11). Conclusions: Increased density of TAMs, including those of the M2-like phenotype, correlate with poor clinicopathologic markers in HNSCC. Our findings warrant additional investigation into the subpopulations of TAMs, the mechanisms behind their recruitment and differentiation, and the associated influence of each phenotype on tumor growth and invasion. A greater understanding of TAM dynamics in HNSCC is critical for directing further research and employing TAM-targeted adjunct therapies.

Loss of CD169+ Subcapsular Macrophages during Metastatic Spread of Head and Neck Squamous Cell Carcinoma.

OBJECTIVE: The purpose of this study is to assess CD169 expression in metastatic and nearby tumor-free lymph nodes of patients with head and neck squamous cell carcinoma (SCC). STUDY DESIGN: Retrospective analysis based on immunohistochemistry. SETTING: Tertiary care center. SUBJECTS AND METHODS: The abundance of CD169+ cells in the subcapsular sinuses (SCSs) of lymph nodes was assessed immunohistochemically in paraffin-embedded tissue samples derived from 22 patients with oral cavity and oropharyngeal SCC. RESULTS: SCSs of lymph nodes harboring metastatic SCC contained significantly fewer CD169+ macrophages (106.5 ± 113.6 cells/mm2) compared to nearby tumor-free lymph nodes (321.3 ± 173.4 cells/mm2, P < .001). This observation extended to 21 of the 22 cases investigated. In addition, 6 patients who later developed recurrent disease contained lower numbers of CD169+ cells (268.6 ± 169.5 cells/mm2) in nearby tumor-free lymph nodes compared to 341.0 ± 176.1 cells/mm2 in those who remained disease free (P = .399). Human papillomavirus (HPV)-positive patients (n = 4) had a 6-fold lower number of CD169+ cells in metastatic nodes (61.2 ± 85.5 cells/mm2) compared to nearby tumor-free lymph nodes (369.5 ± 175.5 cells/mm2, P = .028). In comparison, HPV-negative patients had only a 3-fold reduction (116.6 ± 118.5 cells/mm2 vs 310.6 ± 176.2 cells/mm2, P < .001). CONCLUSION: Metastatic spread of SCC to regional lymph nodes is associated with lower abundance of CD169+ macrophages in the SCSs of draining lymph nodes. These results set the stage for an in-depth investigation into the mechanism(s) by which metastatic SCC controls CD169+ macrophage abundance and its significance as it relates to prognosis and treatment response.

Potential role of CSF cytokine profiles in discriminating infectious from non-infectious CNS disorders.

Current laboratory testing of cerebrospinal fluid (CSF) does not consistently discriminate between different central nervous system (CNS) disease states. Rapidly distinguishing CNS infections from other brain and spinal cord disorders that share a similar clinical presentation is critical. New approaches focusing on aspects of disease biology, such as immune response profiles that can have stimulus-specific attributes, may be helpful. We undertook this preliminary proof-of-concept study using multiplex ELISA to measure CSF cytokine levels in various CNS disorders (infections, autoimmune/demyelinating diseases, lymphomas, and gliomas) to determine the potential utility of cytokine patterns in differentiating CNS infections from other CNS diseases. Both agglomerative hierarchical clustering and mixture discriminant analyses revealed grouping of CNS disease types based on cytokine expression. To further investigate the ability of CSF cytokine levels to distinguish various CNS disease states, non-parametric statistical analysis was performed. Mann-Whitney test analysis demonstrated that CNS infections are characterized by significantly higher CSF lP-10/CXCL10 levels than the pooled non-infectious CNS disorders (p = 0.0001). Within the infection group, elevated levels of MDC/CCL22 distinguished non-viral from viral infections (p = 0.0048). Each disease group of the non-infectious CNS disorders independently showed IP-10/CXCL10 levels that are significantly lower than the infection group [(autoimmune /demyelinating disorders (p = 0.0005), lymphomas (p = 0.0487), gliomas (p = 0.0294), and controls (p = 0.0001)]. Additionally, of the non-infectious diseases, gliomas can be distinguished from lymphomas by higher levels of GRO/CXCL1 (p = 0.0476), IL-7 (p = 0.0119), and IL-8 (p = 0.0460). Gliomas can also be distinguished from autoimmune/demyelinating disorders by higher levels of GRO/CXCL1 (p = 0.0044), IL-7 (p = 0.0035) and IL-8 (p = 0.0176). Elevated CSF levels of PDGF-AA distinguish lymphomas from autoimmune/demyelinating cases (p = 0.0130). Interrogation of the above comparisons using receiver operator characteristic analysis demonstrated area under the curve (AUC) values (ranging from 0.8636-1.0) that signify good to excellent utility as potential diagnostic discriminators. In conclusion, our work indicates that upon formal validation, measurement of CSF cytokine levels may have clinical utility in both identifying a CNS disorder as infectious in etiology and, furthermore, in distinguishing viral from non-viral CNS infections.

Metformin Clinical Trial in HPV+ and HPV- Head and Neck Squamous Cell Carcinoma: Impact on Cancer Cell Apoptosis and Immune Infiltrate.

Background: Metformin, an oral anti-hyperglycemic drug which inhibits mitochondrial complex I and oxidative phosphorylation has been reported to correlate with improved outcomes in head and neck squamous cell carcinoma (HNSCC) and other cancers. This effect is postulated to occur through disruption of tumor-driven metabolic and immune dysregulation in the tumor microenvironment (TME). We report new findings on the impact of metformin on the tumor and immune elements of the TME from a clinical trial of metformin in HNSCC. Methods: Human papilloma virus-(HPV-) tobacco+ mucosal HNSCC samples (n = 12) were compared to HPV+ oropharyngeal squamous cell carcinoma (OPSCC) samples (n = 17) from patients enrolled in a clinical trial. Apoptosis in tumor samples pre- and post-treatment with metformin was compared by deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Metastatic lymph nodes with extra-capsular extension (ECE) in metformin-treated patients (n = 7) were compared to archival lymph node samples with ECE (n = 11) for differences in immune markers quantified by digital image analysis using co-localization and nuclear algorithms (PD-L1, FoxP3, CD163, CD8). Results: HPV-, tobacco + HNSCC (mean Δ 13.7/high power field) specimens had a significantly higher increase in apoptosis compared to HPV+ OPSCC specimens (mean Δ 5.7/high power field) (p < 0.001). Analysis of the stroma at the invasive front in ECE nodal specimens from both HPV-HNSCC and HPV+ OPSCC metformin treated specimens showed increased CD8+ effector T cell infiltrate (mean 22.8%) compared to archival specimens (mean 10.7%) (p = 0.006). Similarly, metformin treated specimens showed an increased FoxP3+ regulatory T cell infiltrate (mean 9%) compared to non-treated archival specimens (mean 5%) (p = 0.019). Conclusions: This study presents novel data demonstrating that metformin differentially impacts HNSCC subtypes with greater apoptosis in HPV-HNSCC compared to HPV+ OPSCC. Moreover, we present the first in vivo human evidence that metformin may also trigger increased CD8+ Teff and FoxP3+ Tregs in the TME, suggesting an immunomodulatory effect in HNSCC. Further research is necessary to assess the effect of metformin on the TME of HNSCC.

Exosomal αvß6 integrin is required for monocyte M2 polarization in prostate cancer.

Therapeutic approaches aimed at curing prostate cancer are only partially successful given the occurrence of highly metastatic resistant phenotypes that frequently develop in response to therapies. Recently, we have described αvß6, a surface receptor of the integrin family as a novel therapeutic target for prostate cancer; this epithelial-specific molecule is an ideal target since, unlike other integrins, it is found in different types of cancer but not in normal tissues. We describe a novel αvß6-mediated signaling pathway that has profound effects on the microenvironment. We show that αvß6 is transferred from cancer cells to monocytes, including ß6-null monocytes, by exosomes and that monocytes from prostate cancer patients, but not from healthy volunteers, express αvß6. Cancer cell exosomes, purified via density gradients, promote M2 polarization, whereas αvß6 down-regulation in exosomes inhibits M2 polarization in recipient monocytes. Also, as evaluated by our proteomic analysis, αvß6 down-regulation causes a significant increase in donor cancer cells, and their exosomes, of two molecules that have a tumor suppressive role, STAT1 and MX1/2. Finally, using the Ptenpc-/- prostate cancer mouse model, which carries a prostate epithelial-specific Pten deletion, we demonstrate that αvß6 inhibition in vivo causes up-regulation of STAT1 in cancer cells. Our results provide evidence of a novel mechanism that regulates M2 polarization and prostate cancer progression through transfer of αvß6 from cancer cells to monocytes through exosomes.

Macrophage type 2 differentiation in a patient with laryngeal squamous cell carcinoma and metastatic prostate adenocarcinoma to the cervical lymph nodes.

BACKGROUND: The tumor microenvironment often polarizes infiltrating macrophages towards a type 2, or M2 phenotype, that is characterized by expression of various cysteine-rich, scavenger receptors, including CD163. The primary function of M2 macrophages is to facilitate wound healing. As such, they are capable of providing metabolic support to a growing tumor, neovascularization, as well as protection from cytotoxic T cells. The tumor microenvironment contains a milieu of secreted factors and vesicles, which in certain circumstances can gain access to lymphatic vessels that drain to local lymph nodes. CASE PRESENTATION: We report a 59-year-old male with recurrent T4 squamous cell carcinoma (SCC) of the larynx with synchronous prostate adenocarcinoma confined to the prostate and regional pelvic lymph nodes, without metastatic disease. The patient underwent salvage total laryngectomy and bilateral neck dissection with final pathology revealing a recurrent moderately differentiated SCC involving the larynx as well as prostate cancer in draining level 4 cervical lymph nodes bilaterally. CD163 staining was performed on the primary tumor, a negative draining lymph node, and a level four lymph node with a focus of metastatic prostate cancer and compared to benign controls. The negative draining lymph node demonstrated a large CD163 population of cells as did the interface of the focus of prostate cancer and surrounding lymph node. CD163 levels were markedly increased in this patient compared to benign lymph node controls. The macrophage differentiation at the primary tumor in the larynx was strongly CD163 positive supporting an immune permissive environment for tumor growth and metastasis. CONCLUSION: We describe a unique case of solitary metastatic prostate cancer to cervical lymph nodes in the setting of a laryngeal cancer. These observations suggest that SCC-derived factors drive a tumor-supportive environment in draining lymph nodes dominated by an overwhelming number of CD163+, M2 macrophages. Lymph nodes that are 'primed' by SCC differentiation to M2 phenotype may be at higher risk of harboring metastases.