Rodents as an animal model for studying tooth extraction-related medication-related osteonecrosis of the jaw: assessment of outcomes.

OBJECTIVE: To assess the outcomes of several rodent animal models for studying tooth extraction-related medication-related osteonecrosis of the jaw (MRONJ). DESIGN: After a search of the databases, 2004 articles were located, and 118 corroborated the inclusion factors (in vivo studies in rodents evaluating tooth extraction as a risk factor for the development of MRONJ). RESULTS: Numerous studies attempting to establish an optimal protocol to induce MRONJ were found. Zoledronic acid (ZA) was the most used drug, followed by alendronate (ALN). Even when ZA did not lead to the development of MRONJ, its effect compromised the homeostasis of the bone and soft tissue. The association of other risk factors (dexamethasone, diabetes, and tooth-related inflammatory dental disease) besides tooth extraction also played a role in the development of MRONJ. In addition, studies demonstrated a relationship between cumulative dose and MRONJ. CONCLUSIONS: Both ZA and ALN can lead to MRONJ in rodents when equivalent human doses (in osteoporosis or cancer treatment) are used. Local oral risk factors and tooth-related inflammatory dental disease increase the incidence of MRONJ in a tooth extraction-related rodent model.

Assessing PD-L1 Expression in Head and Neck Squamous Cell Carcinoma: Trials and Tribulations.

Immune checkpoint inhibitors have improved the outcome of patients diagnosed with inoperable recurrent or metastatic head and neck squamous cell carcinoma. However, as only a subset of head and neck cancer patients benefit from this treatment, biomarkers predicting treatment response help guide physicians in their clinical decision-making. PD-L1 expression assessed by immunohistochemistry is the single most clinically relevant biomarker predicting response to PD-1-blocking antibodies. Here, we discuss in which clinical context assessment of PD-L1 expression is instrumental for the choice of therapy, how pathologists score it, and how it affects the approval of anti-PD-1 antibodies. Furthermore, we discuss the heterogeneity of PD-L1 expression and review technical aspects of determining this prominent biomarker-knowledge that might influence clinical decision-making.

CXCL9:SPP1 macrophage polarity identifies a network of cellular programs that control human cancers.

Tumor microenvironments (TMEs) influence cancer progression but are complex and often differ between patients. Considering that microenvironment variations may reveal rules governing intratumoral cellular programs and disease outcome, we focused on tumor-to-tumor variation to examine 52 head and neck squamous cell carcinomas. We found that macrophage polarity-defined by CXCL9 and SPP1 (CS) expression but not by conventional M1 and M2 markers-had a noticeably strong prognostic association. CS macrophage polarity also identified a highly coordinated network of either pro- or antitumor variables, which involved each tumor-associated cell type and was spatially organized. We extended these findings to other cancer indications. Overall, these results suggest that, despite their complexity, TMEs coordinate coherent responses that control human cancers and for which CS macrophage polarity is a relevant yet simple variable.

Developing a definition of immune exclusion in cancer: results of a modified Delphi workshop.

Checkpoint inhibitors represent an effective treatment approach for a variety of cancers through their inhibition of immune regulatory pathways within the tumor microenvironment (TME). Unfortunately only a minority of patients with cancer achieve clinical benefit from immunotherapy, with the TME emerging as an important predictor of outcomes and sensitivity to therapy. The extent and pattern of T-cell infiltration can vary prominently within/across tumors and represents a biological continuum. Three immune profiles have been identified along this continuum: 'immune-desert' or 'T-cell cold' phenotype, 'immune-active', 'inflamed', or 'T-cell hot' phenotype, and 'immune excluded' phenotype. Of the three profiles, immune excluded remains the most ill-defined with no clear, universally accepted definition even though it is commonly associated with lack of response to immune checkpoint inhibitors and poor clinical outcomes. To address this, 16 multidisciplinary cancer experts from around the world were invited to participate in a symposium using a three-round modified Delphi approach. The first round was an open-ended questionnaire distributed via email and the second was an in-person discussion of the first round results that allowed for statements to be revised as necessary to achieve a maximum consensus (75% agreement) among the rating committee (RC). The final round questionnaire was distributed to the RC via email and had a 100% completion rate. The Delphi process resulted in moving us closer to a consensus definition for immune exclusion that is practical, clinically pertinent, and applicable across a wide range of cancer histologies. A general consensus of the role of immune exclusion in resistance to checkpoint therapy and five research priorities emerged from this process. Together, these tools could help efforts designed to address the underlying mechanisms of immune exclusion that span cancer types and, ultimately, aid in the development of treatments to target these mechanisms to improve patient outcomes.

Effects of periodontitis on cancer outcomes in the era of immunotherapy.

Periodontitis results from dysbiosis of the oral microbiome and affects up to 70% of US adults aged 65 years and older. More than 50 systemic inflammatory disorders and comorbidities are associated with periodontitis, many of which overlap with immunotherapy-associated toxicities. Despite the increasing use of immunotherapy for the treatment of cancer, uncertainty remains as to whether the microbial shift associated with periodontal disease can influence response rates and tolerance to cancer immunotherapy. We herein review the pathophysiology of periodontitis and the local and systemic inflammatory conditions related to oral dysbiosis, and discuss the overlapping adverse profiles of periodontitis and immunotherapy. The effects of the presence of Porphyromonas gingivalis, a key pathogen in periodontitis, highlight how the oral microbiome can affect the hosts' systemic immune responses, and further research into the local and systemic influence of other microorganisms causing periodontal disease is necessary. Addressing periodontitis in an ageing population of people with cancer could have potential implications for the clinical response to (and tolerability of) immunotherapy and warrants further investigation.

Targeting TBK1 to overcome resistance to cancer immunotherapy.

Despite the success of PD-1 blockade in melanoma and other cancers, effective treatment strategies to overcome resistance to cancer immunotherapy are lacking1,2. Here we identify the innate immune kinase TANK-binding kinase 1 (TBK1)3 as a candidate immune-evasion gene in a pooled genetic screen4. Using a suite of genetic and pharmacological tools across multiple experimental model systems, we confirm a role for TBK1 as an immune-evasion gene. Targeting TBK1 enhances responses to PD-1 blockade by decreasing the cytotoxicity threshold to effector cytokines (TNF and IFNγ). TBK1 inhibition in combination with PD-1 blockade also demonstrated efficacy using patient-derived tumour models, with concordant findings in matched patient-derived organotypic tumour spheroids and matched patient-derived organoids. Tumour cells lacking TBK1 are primed to undergo RIPK- and caspase-dependent cell death in response to TNF and IFNγ in a JAK-STAT-dependent manner. Taken together, our results demonstrate that targeting TBK1 is an effective strategy to overcome resistance to cancer immunotherapy.

Chemoradiation therapy alters the PD-L1 score in locoregional recurrent squamous cell carcinomas of the head and neck.

PD-L1 testing guides therapeutic decision-making for head and neck squamous cell carcinoma (HNSCC). We sought to understand whether chemoradiation therapy (CRT) influences the PD-L1 combined positive score (CPS) and other biomarkers of response to immunotherapy. PD-L1 expression was assessed using immunohistochemistry, and bulk RNA sequencing was performed on 146 HNSCC patients (65 primary sites, 50 paired local recurrences, and 31 paired regional recurrences). PD-L1 was scored using the CPS of ≥1, ≥20, and ≥50. Overall, 98 %, 54 %, and 17 % of HNSCCs had a CPS ≥1, ≥20, and ≥50, respectively. When using a cut-off of ≥1, CRT did not significantly change CPS at the locoregional recurrent site. However, there were significant changes when using CPS ≥20 or ≥50. The CPS changed for 32 % of patients when using a CPS ≥20 (p < 0.001). When using a CPS ≥50, there was a 20-23 % (p = 0.0058-0.00067) discordance rate at the site of locoregional recurrence. Oral cavity cancers had a significantly higher discordant rate than other primary sites for CPS ≥50, 44 % (8/18, p = 0.0058) and 58 % (7/12, p = 0.00067) discordance at the site of local and regional recurrence, respectively. When evaluating the 18 gene IFN-É£ signature predictive of response to anti-PD-1 blockade, there was a statistically significant increase in the IFN-É£ signature in recurrent larynx cancer (p = 0.02). Our study demonstrates that when using a higher cut-off of CPS ≥20 and ≥50, a repeat biopsy may be warranted after CRT for local and regional recurrent HNSCCs.

Human Papillomavirus-Directed Therapeutics for Human Papillomavirus-Associated Oropharyngeal Cancer.

ABSTRACT: Despite the availability of prophylactic human papillomavirus (HPV) vaccines, there is a growing incidence of HPV-associated head and neck squamous cell carcinomas (HPV-HNSCC) worldwide. The viral etiology of HPV-HNSCC provides an opportunity to develop personalized immune-based therapies, which target the unique viral- or tumor-specific proteins. Novel HPV-targeted immunotherapeutic approaches in clinical development are reviewed. Early results from these trials highlight new opportunities and potential challenges ahead. Immunotherapies for HPV-associated HNSCCs will require a tailored combinatorial approach based on preexisting mechanisms of host immune resistance. As the field continues to identify the relevant HPV types 16 and 18 immunogenic epitopes that are presented by diverse HLA class I alleles, improved HPV-targeted biologics and clinical monitoring tools can be developed and applied to a broader cancer patient population.

Pulmonary manifestations of chronic HPV infection in patients with recurrent respiratory papillomatosis.

Human papillomavirus (HPV) types 6 and 11 can infect the squamous epithelium of the respiratory tract. Up to 8·9% of patients with HPV-associated recurrent respiratory papillomatosis (RRP) have pulmonary involvement. Pulmonary manifestations of HPV infection are associated with considerable morbidity, in part because treatment options and management guidelines are lacking. Patients with pulmonary RRP have a 32-times increased lifetime risk of malignant transformation compared with the overall RRP population. We review the clinical and radiographic presentation, pathological features, and genetics of pulmonary RRP, and we provide management algorithms based on our clinical experience with this complex patient population. In patients with suspected pulmonary involvement, tissue-sparing procedures to address growing lesions might be warranted given the chronicity and multifocality of the disease over a patient's lifetime. However, malignant transformation of pulmonary lesion(s) warrants standard-of-care treatment for primary lung squamous cell carcinoma. Large cohort studies are needed to understand the clinical course of pulmonary RRP and to identify molecular markers of increased risk of malignant transformation in order to develop guidelines for optimal and standardised surveillance and treatment.

Multiplexed single-cell analysis of FNA allows accurate diagnosis of salivary gland tumors.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST). Single-cell homogenates and FNA samples were profiled by FAST cyclic imaging and computational analysis. A prediction model was generated using a training set of 151,926 cells from primary SGTs (N = 26) and validated on a separate cohort (N = 30). Companion biomarker testing, such as neurotrophic tyrosine receptor kinase (NTRK), was also assessed with the FAST technology. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell homogenate samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single marker increased the diagnostic accuracy (0.82 as compared to 0.65-0.74, respectively), independent of the cell number sampled. NTRK expression was also assessed by the FAST assay, highlighting the potential therapeutic application of this technology. Application of the novel multiplexed single-cell technology facilitates rapid biomarker testing from FNA samples at low cost. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision-making.

A rabbit model to investigate temporomandibular joint osteochondral regeneration.

OBJECTIVE: The objective of this study was 2-fold: (1) to describe the rabbit temporomandibular joint (TMJ) anatomy and (2) to provide a detailed, step-by-step description of a minimally invasive approach to perform a standard osteochondral TMJ defect that can be used to investigate the regenerative potential of biomaterials. STUDY DESIGN: This study was performed in 2 steps. In the first, a total of 8 rabbit carcasses (n = 16 joints) were used to study the normal TMJ anatomy and histology to develop a minimally invasive approach to access the articulating surface of the condyle to perform a standard osteochondral defect. In the second, the surgical procedure was performed in 10 live animals to evaluate the feasibility of the model and to evaluate the regenerative potential of a biodegradable light-cured hydrogel seeded with stem cells (results not shown). RESULTS: The cartilage of the mandibular condyle showed 4 layers: fibrous, proliferative, hypertrophic, and a zone of calcified cartilage. Positive safranin O staining was observed in the cartilage. The mean duration of the procedure (from incision to last stitch) was 35.5 (±9.21) minutes. All animals survived the procedures without any major complications. CONCLUSIONS: This animal model represents an easy and nonmorbid surgical approach to rabbit TMJ.

Diagnostic challenges and successful organ-preserving therapy in a case of secretory carcinoma of minor salivary glands.

BACKGROUND: Secretory carcinoma is a more recently described subtype of salivary gland carcinoma that may pose diagnostic challenges and frequently harbors NTRK fusions that may successfully be targeted by TRK inhibitors in advanced disease. CASE: We present the case of a female patient with secretory carcinoma arising in the base of tongue with persistent disease after debulking surgery and definitive chemoradiation. As an alternative to salvage surgery, which would have resulted in significant impairment of swallowing and speech function, a targeted therapy with the TRK-inhibitor larotrectinib against an identified ETV6-NTRK3 fusion product was initiated. Larotrectinib treatment has been well tolerated, resulted in durable complete response and the patient maintains good swallowing and speech function. CONCLUSION: The presented case underscores the importance of the accurate diagnosis of secretory carcinoma. It further highlights the impact of molecular testing as targeted therapies may play an important role in the management of advanced salivary gland cancers.

Rapid Serial Immunoprofiling of the Tumor Immune Microenvironment by Fine Needle Sampling.

PURPOSE: There is increasing effort to discover and integrate predictive and/or prognostic biomarkers into treatment algorithms. While tissue-based methods can reveal tumor-immune cell compositions at a single time point, we propose that single-cell sampling via fine needle aspiration (FNA) can facilitate serial assessment of the tumor immune microenvironment (TME) with a favorable risk-benefit profile. EXPERIMENTAL DESIGN: Primary antibodies directed against 20 murine and 25 human markers of interest were chemically modified via a custom linker-bio-orthogonal quencher (FAST) probe. A FAST-FNA cyclic imaging and analysis pipeline were developed to derive quantitative response scores. Single cells were harvested via FNA and characterized phenotypically and functionally both in preclinical and human samples using the newly developed FAST-FNA assay. RESULTS: FAST-FNA samples analyzed manually versus the newly developed deep learning-assisted pipeline gave highly concordant results. Subsequently, an agreement analysis showed that FAST and flow cytometry of surgically resected tumors were positively correlated with an R2 = 0.97 in preclinical samples and an R2 = 0.86 in human samples with the detection of the relevant tumor and immune biomarkers of interest. Finally, the feasibility of applying this minimally invasive approach to analyze the TME during immunotherapy was assessed in patients with cancer revealing local antitumor immune programs. CONCLUSIONS: The FAST-FNA is an innovative technology that combines bio-orthogonal chemistry coupled with a computational analysis pipeline for the comprehensive profiling of single cells obtained through FNA. This is the first demonstration that the complex and rapidly evolving TME during treatment can be accurately and serially measured by simple FNA.

Resident Kupffer cells and neutrophils drive liver toxicity in cancer immunotherapy.

Immunotherapy is revolutionizing cancer treatment but is often restricted by toxicities. What distinguishes adverse events from concomitant antitumor reactions is poorly understood. Here, using anti-CD40 treatment in mice as a model of TH1-promoting immunotherapy, we showed that liver macrophages promoted local immune-related adverse events. Mechanistically, tissue-resident Kupffer cells mediated liver toxicity by sensing lymphocyte-derived IFN-γ and subsequently producing IL-12. Conversely, dendritic cells were dispensable for toxicity but drove tumor control. IL-12 and IFN-γ were not toxic themselves but prompted a neutrophil response that determined the severity of tissue damage. We observed activation of similar inflammatory pathways after anti-PD-1 and anti-CTLA-4 immunotherapies in mice and humans. These findings implicated macrophages and neutrophils as mediators and effectors of aberrant inflammation in TH1-promoting immunotherapy, suggesting distinct mechanisms of toxicity and antitumor immunity.

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.

A Randomized Phase 2 Study of Pembrolizumab With or Without Radiation in Patients With Recurrent or Metastatic Adenoid Cystic Carcinoma.

PURPOSE: We evaluated the safety and efficacy of pembrolizumab (pembro) ± radiation therapy (RT) in a phase 2 study among patients with progressive, metastatic adenoid cystic carcinoma (ACC). METHODS AND MATERIALS: Eligible patients had metastatic ACC with progression within the last year and ≥1 measurable lesion. Patients were randomized to pembro alone or with RT to 30 Gy in 5 fractions (pembroRT). The primary endpoint was objective response rate outside the RT field. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and local RT responses. RESULTS: We randomized 20 patients (10 per arm) from 2017 to 2018. We did not observe objective response outside of the radiation treatment field; stable disease (SD) was the best response in 12 (60%) patients and was not different per arm (7 pembro, 5 pembroRT, P = .65). A tumor growth rate decrease (TGR) of >25% was noted among 7 of 12 patients and >75% in 4 patients. There were local responses in the irradiated field among all evaluable pembroRT patients. Median PFS and OS were 4.5/not reached for pembroRT and 6.6 / 27.2 months for pembro patients. One patient developed grade 3 liver enzyme elevation after 27 cycles of therapy. Correlative analyses confirm low levels of programmed death-ligand 1 expression (PD-L1), and CD8 infiltrating T-cells. We identified associations between local response and both MYB/NFIB translocation and PD-L1 expression and between changes in systemic immune populations and RT. CONCLUSIONS: Pembrolizumab and pembroRT were well tolerated. We observed no objective responses, but 60% of patients with PD before the study achieved SD, the majority with decreased TGR and half (n = 10) with clinical benefit (SD >6 months). We observed favorable local responses within the RT field. Additional strategies are needed to further delay progression and effect response.

Defining best practices for tissue procurement in immuno-oncology clinical trials: consensus statement from the Society for Immunotherapy of Cancer Surgery Committee.

Immunotherapy is now a cornerstone for cancer treatment, and much attention has been placed on the identification of prognostic and predictive biomarkers. The success of biomarker development is dependent on accurate and timely collection of biospecimens and high-quality processing, storage and shipping. Tumors are also increasingly used as source material for the generation of therapeutic T cells. There have been few guidelines or consensus statements on how to optimally collect and manage biospecimens and source material being used for immunotherapy and related research. The Society for Immunotherapy of Cancer Surgery Committee has brought together surgical experts from multiple subspecialty disciplines to identify best practices and to provide consensus on how best to access and manage specific tissues for immuno-oncology treatments and clinical investigation. In addition, the committee recommends early integration of surgeons and other interventional physicians with expertise in biospecimen collection, especially in clinical trials, to optimize the quality of tissue and the validity of correlative clinical studies in cancer immunotherapy.

New technology on the horizon: Fast analytical screening technique FNA (FAST-FNA) enables rapid, multiplex biomarker analysis in head and neck cancers.

PD-L1 profiling was recently approved by the US Food and Drug Administration as a companion diagnostic for anti-PD1 treatment in patients with head and neck cancer, ushering in a new era for precision medicine. However, the routine development and implementation of such testing is still limited by current clinical workflows and the lack of better and more comprehensive alternatives. In this review, the authors discuss the real-world challenges of clinically based biomarker testing and highlight the advantages of developing fine-needle aspiration (FNA)-based biomarker testing that would enable frequent and serial tumor sampling. A conceptual and technological innovation is introduced, fast analytical screening technique (FAST)-FNA (FAST chemistry-enabled FNA), which is being developed to inform immunotherapy treatment options in patients with head and neck cancer and to assist with the development of the next generation of predictive biomarkers.

High Expression of Programmed Death Ligand 1 and Programmed Death Ligand 2 in Ophthalmic Sebaceous Carcinoma: The Case for a Clinical Trial of Checkpoint Inhibitors.

PURPOSE: To evaluate the expression of programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) in ocular adnexal sebaceous carcinoma (OASC), and to appraise these findings within the context of recent comparable studies. DESIGNS: Retrospective case series. METHODS: Twenty cases of primary OASC were immunostained for PD-L1, PD-L2 and CD8. PD-L1 and PD-L2 expression were graded with both the combined positive score (CPS) and the tumor proportion score (TPS). Both raw CPS and TPS were reported, as well as positivity with TPS and CPS ≥1. CD8 expression was graded on a 0-3 scale. Charts were reviewed for clinical correlations. The results of the current study were compared with results of similar recent investigations. RESULTS: For the 20 cases, mean expression of PD-L1 with CPS was 29.7 (range 0-101.5) and with TPS was 12.2 (range 0-95.8); mean expression of PD-L2 with CPS was 7.9 (range 0-37.3) and with TPS was 1.9 (range 0-12.9). PD-L1 CPS ≥1 was detected in 95% of OASC, while PD-L1 TPS ≥1 was found in 75%. PD-L2 CPS ≥1 was present in 60%, while only 20% had PD-L2 TPS ≥1. Immune cells appeared to contribute to a substantial proportion of PD-L1 and PD-L2 positivity, and a conspicuous CD8-positive T-lymphocytic infiltrate was present in most tumors. Significant correlations were identified between tissue expression of PD-L1, PD-L2, and CD8. Tissues with greater levels of PD-L1 tended to express higher levels of PD-L2 and CD8. The degree of PD-L1 and PD-L2 expression was also associated with the area in millimeters squared of the immunostained tumor, suggesting that tumor sampling may influence interpretation of PD-L1 and PD-L2 expression in ocular adnexal tumors. CONCLUSIONS: The current and preceding studies confirm that PD-L1 and PD-L2 are expressed in a high percentage of OASCs. These results support the premise that checkpoint inhibitor drugs hold considerable therapeutic promise for patients with OASC and stimulate the institution of clinical trials.