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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.

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.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).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.This animal model represents an easy and nonmorbid surgical approach to rabbit TMJ.

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.

Purpose: Radiotherapy kills cancer cells by causing DNA damage, and stimulates a systemic antitumor immune response by releasing tumor antigen and endogenous adjuvant within the tumor microenvironment. However, radiotherapy also induces the recruitment of immunosuppressive myeloid cells, which can interfere with the antitumor immune responses elicited by apoptotic tumor cells. We hypothesized that local delivery of vaccine following radiotherapy will lead to the priming of antigen-specific CTL immune responses and render immunosuppressive myeloid cells susceptible to killing by the activated CTLs.Experimental Design: Using several antigenic systems, we tested whether intratumoral injection of antigenic peptide/protein in irradiated tumors would be able to prime CTLs as well as load myeloid cells with antigen, rendering them susceptible to antigen-specific CTL killing.Results: We show that by combining radiotherapy and targeted antigenic peptide delivery to the tumor, the adjuvant effect generated by radiotherapy itself was sufficient to elicit the priming and expansion of antigen-specific CTLs, through the type I IFN-dependent pathway, leading to synergistic therapeutic antitumor effects compared with either treatment alone. In addition, using two different types of transgenic mice, we demonstrated that CTL-mediated killing of stromal cells in tumors by our approach is important for tumor control. Finally, we confirmed the efficacy of this approach in our preclinical model using two clinically tested therapeutic human papilloma virus (HPV) vaccines.Conclusions: These data serve as an important foundation for the future clinical translation of radiotherapy combined with a clinically tested therapeutic HPV vaccine for the control of HPV-associated cancers. Clin Cancer Res; 20(3); 644–57. ©2013 AACR.

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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.

This file contains Supplementary Figure 1. Supplementary Figure 1 shows the staining pattern of tumoral PD-L1 staining.

This file contains Supplementary Figure 2. Supplementary Figure 2 shows tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs.

This file contains Supplementary Figures 3-9. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

PDF file - 941K, Characterization of E7-specific CD8+ T cell activation in tumor-bearing mice treated with radiation and intratumoral vaccination with E7 peptide.

This file contains Supplementary Table 1. Supplementary Table 1 shows the immunohistochemical staining for PD-L1, PD-1, CD4, and CD8 in the samples analyzed in the manuscript.

This file contains Supplementary Table 2. Supplementary Table 2 shows that the proliferative capacity of PD-1 expressing CD8, CD4, and FoxP3+ TILs are decreased compared to non-PD-1 expressing TILs in OTSCC.

PDF file - 809K, Evaluation of antitumor effect after radiation combined with intratumoral E7 peptide treatment in TLR9-/- and WT C57BL/6 mice.

This file contains the figure legends for supplementary figures 1-9. Supplementary Figure 1. Staining pattern of tumoral PD-L1 staining. Supplementary Figure 2. Tumoral PD-L1 staining in association with PD-1, CD8, and CD4 TILs. Supplementary Figure 3. Representative multi-color immunofluorescence staining shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 4. Representative multi-color immunofluorescence staining of a PD-L1 negative tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 5. CD4+ T cells show markedly more specific PD-L1 localization in OTSCC tumors compared to CD8+ T cells. Supplementary Figure 6. Representative multi-color immunofluorescence staining of an OTSCC tumor shows that PD-1+CD4+ T cells demonstrate greater PD-1 expression than PD1+CD8+ T cells. Supplementary Figure 7. Higher magnification multi-color immunofluorescence staining of a representative OTSCC tumor stained with PD-L1, PD-1, CD4, and CD8. Supplementary Figure 8. Diffuse tumoral PD-L1 staining shows low infiltration of CD4+ and CD8+ T cells. Supplementary Figure 9. Representative multi-color immunofluorescence staining of FoxP3, PD1, and Ki67 in OTSCC.

Oral tongue squamous cell carcinoma (OTSCC) is the most common oral cavity tumor. In this study, we examined the basis for the activity of programmed cell death protein (PD-1)-based immune checkpoint therapy that is being explored widely in head and neck cancers. Using multispectral imaging, we systematically investigated the OTSCC tumor microenvironment (TME) by evaluating the frequency of PD-1 expression in CD8+, CD4+, and FoxP3+ tumor-infiltrating lymphocytes (TIL). We also defined the cellular sources of PD-1 ligand (PD-L1) to evaluate the utility of PD-1:PD-L1 blocking antibody therapy in this patient population. PD-L1 was expressed in 79% of the OTSCC specimens examined within the TME. Expression of PD-L1 was associated with moderate to high levels of CD4+ and CD8+ TILs. We found that CD4+ TILs were present in equal or greater frequencies than CD8+ TILs in 94% of OTSCC and that CD4+FOXP3neg TILs were colocalized with PD-1/PD-L1/CD68 more frequently than CD8+ TILs. Both CD4+PD1+ and CD8+PD1+ TILs were anergic in the setting of PD-L1 expression. Overall, our results highlight the importance of CD4+ TILs as pivotal regulators of PD-L1 levels and in determining the responsiveness of OTSCC to PD1-based immune checkpoint therapy. Cancer Res; 77(22); 6365–74. ©2017 AACR.

Purpose: Advanced-stage cancers are extremely difficult to treat and rarely result in a cure. The application of oncolytic viruses is a potential strategy for controlling advanced-stage cancer because intratumoral (i.t.) injection of an oncolytic virus, such as vaccinia virus, results in tumor cell lysis and subsequent release of tumor antigens into the microenvironment. Furthermore, the viruses can serve as a vehicle for delivering genes of interest to cancer cells.Experimental Design: In the current study, we hypothesize that in tumor-bearing mice primed with DNA encoding an immunogenic foreign antigen, ovalbumin (OVA) followed by a boost with i.t. administration of vaccinia virus encoding the same foreign antigen, OVA, can generate enhanced antitumor effects through the combination of viral oncolysis and tumor-specific immunity.Results: We observed that tumor-bearing mice primed with OVA DNA and boosted with vaccinia encoding OVA (Vac-OVA) generated significant therapeutic antitumor effects as well as induced significant levels of OVA-specific CD8+ T cells in two different tumor models. Furthermore, treatment with Vac-OVA not only kills the tumor and stromal cells directly but also renders the tumor cells and surrounding stromal cells susceptible to OVA-specific CD8+ T-cell killing, resulting in enhanced antitumor therapeutic effects.Conclusions: Thus, the current study may provide a novel therapeutic strategy for the control of advanced-stage cancers.

PDF file - 1656K, Characterization of the distribution of CD11b+ myeloid cells and CD11b+ Gr-1+ MDSCs in tumor, spleen and DLN after radiation.

Supplementary Data from Combination of Viral Oncolysis and Tumor-Specific Immunity to Control Established Tumors

This file contains Supplementary Table 3. Supplementary Table 3 shows the clinicopathological characteristics of the patient cohort.

PDF file - 2157K, Characterization of different subsets of MDSCs in spleens, DLNs and tumors following the various treatments.

PDF file - 89K

PDF file - 723K, Immune mediated long-term tumor protection experiment.

Supplementary Table 1 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

PDF file - 28K, PD-1 expressing CD8+ T cells in non-cancerous tonsil tissue are able to produce IFN-g after stimulation

Immunotherapy and chemotherapy are generally effective against small tumors in animal models of cancer. However, these treatment regimens are generally ineffective against large, bulky tumors. We have found that a multimodality treatment regimen using DNA vaccination in combination with chemotherapeutic agent epigallocatechin-3-gallate (EGCG), a compound found in green tea, is effective in inhibiting large tumor growth. EGCG was found to induce tumor cellular apoptosis in a dose-dependent manner. The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, combined DNA vaccination and oral EGCG treatment provided long-term antitumor protection in cured mice. Cured animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7, but not a challenge of B16 7 weeks after the combined treatment, showing antigen-specific immune responses. These results suggest that multimodality treatment strategies, such as combining immunotherapy with a tumor-killing cancer drug, may be a more effective anticancer strategy than single-modality treatments. [Cancer Res 2007;67(2):802–11]

Supplementary Figure 3 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

XLSX file - 9K, IFN-g production from PD-1(+) and (-) TILs isolated from PD-L1 (+) and (-) HPV-HNSCCs; data corresponding to Figure 6B.

Supplementary Figure 1 from Epigallocatechin-3-Gallate Enhances CD8+ T Cell–Mediated Antitumor Immunity Induced by DNA Vaccination

Supplementary Figure 5 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

PDF file - 36K, Detection of PTEN by western blot and PD-L1 expression by flow cytometry in HPV(-) head and neck cancer lines

Supplementary Figure 4 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

Supplementary Table 3 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

Supplementary Figure 2 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

Supplementary Table 2 from Ectopic Expression of X-Linked Lymphocyte-Regulated Protein pM1 Renders Tumor Cells Resistant to Antitumor Immunity

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.

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-ɣ signature predictive of response to anti-PD-1 blockade, there was a statistically significant increase in the IFN-ɣ 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.

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.

Purpose To determine if there is a biologic rationale for using checkpoint inhibitor drugs targeting programmed cell death ligand 1 (PD-L1) and PD-L2 in the treatment of adenoid cystic carcinoma of the orbit. Methods Twenty-three cases of adenoid cystic carcinoma involving the orbit (13 primary lacrimal gland, 5 secondarily extending into the orbit, and 5 unspecified) were examined histopathologically. Immunohistochemistry for PD-L1, PD-L2, and CD8 was performed. Charts were reviewed for clinical correlations. Results Expression of PD-L1 and of PD-L2 was overall low in adenoid cystic carcinoma (mean expression 1.4 ± 0.9 of 5 for PD-L1, mean 0.83 ± 1.1 of 5 for PD-L2), and tumor-infiltrating CD8-positive T-lymphocytes were sparse (mean 1.1 ± 0.51 of 3). Only 13 of the 23 (57%) cases expressed PD-L1 as a combined positive score ≥1 of cells. No associations were found between expression levels of these markers and patient sex, tumor site of origin, Tumor, Node, Metastasis stage, or patient outcome. A significant association was observed between stromal PD-L1 expression and tumor histopathologic subtype (p = 0.05), and between tumor PD-L1 expression and prior exposure to radiation (p = 0.03). Conclusions Checkpoint inhibitor drugs may have limited impact in the treatment and clinical course of orbital adenoid cystic carcinoma based on the low frequency of CD8 infiltrate and low expression of PD-L1 and PD-L2. Pretreatment with radiation, however, may improve tumor response to checkpoint inhibitor drugs.

The immune landscape of cancer determines its responsiveness to immunotherapy. Tumors infiltrated with CD8+ T cells (immune-active tumors) are more likely to respond to immunomodulatory agents. However, immune activation often is counterbalanced by strong immunosuppressive mechanisms that are necessary to maintain homeostasis but consequentially can facilitate the survival of cancer cells in the immunocompetent host, a concept defined as compensatory immune suppression. TReg cells contribute to compensatory immune suppression, and therapies targeting the immunosuppressive TReg population are being actively explored. Wang et al. characterize a subset of peripheral circulating CD45−FOXP3hi TReg II cells that phenotypically and functionally parallel the activity of their intratumoral counterparts. The findings are paradigm shifting and may provide a potential liquid-based tool to evaluate the immunosuppressive activity of intratumoral TReg cells; they may also allow temporal assessment of whether the fine balance between immune rejection versus tolerance is achieved with various applied therapies.

Therapeutic cancer vaccines have met limited clinical success. In the setting of cancer, the immune system is either tolerized and/or has a limited tumor-specific T cell repertoire. In this study, we explore whether intratumoral (IT) vaccination with an HPV vaccine can elicit quantitative and qualitative differences in immune response as compared to intramuscular (IM) vaccination to overcome immune resistance in established tumors. We report that IT administration of an HPV-16 E7 peptide vaccine formulated with polyinosinic–polycytidylic acid (Poly (I:C)) generated an enhanced antitumor effect relative to IM delivery. The elicited anti-tumor effect with IT vaccination was consistent among the vaccinated groups and across various C57BL/6 substrains. IT vaccination resulted in an increased frequency of PD-1(hi) TILs, which represented both vaccine-targeted and non-vaccine-targeted tumor specific CD8(+) T cells. Overall, the CD8(+)/Treg ratio was increased within the tumor microenvironment using IT vaccination. We also assessed transcriptional changes in several immune-related genes in the tumor microenvironment of the various treated groups, and our data suggest that IT vaccination leads to upregulation of a broad complement of immunomodulatory genes, including upregulation of interferon gamma (IFNγ) and antigen presentation and processing machine (APM) components. IT vaccine delivery is superior to traditional IM vaccination routes with the potential to improve tumor immunogenicity, which has potential clinical application in the setting of accessible lesions such as head and neck squamous cell carcinomas (HNSCCs).

PURPOSE: Novel cancer immunotherapies, called immune checkpoint inhibitors, have demonstrated clinical efficacy in the treatment of squamous cell carcinomas of the head and neck. Tissue expression of programmed cell death 1 ligand 1 (PD-L1) and programmed cell death 1 ligand 2 (PD-L2) has been shown to predict tumor response to these drugs. We examine the expression of prognostic immune biomarkers, PD-L1 and PD-L2, in invasive ocular surface squamous neoplasia. DESIGN: Retrospective case series. METHODS: Eighteen cases of ocular surface or ocular adnexal invasive squamous cell carcinomas were identified in pathology case files of the Massachusetts General Hospital/Massachusetts Eye and Ear Infirmary and at the Wills Eye Hospital accessioned between January 1, 2014 and January 1, 2017. Immunohistochemical staining for PD-L1, PD-L2, CD8, and p16 was performed and graded in a standardized fashion. RESULTS: PD-L1 and PD-L2 were expressed on tumor cells to varying degrees, and also on some stromal cells and endothelial cells. Stromal and endothelial cell expression was also seen in control conjunctival specimens. Tumor expression of PD-L1 and PD-L2 was present on the cell membranes. All 18 (100%) of the tumors expressed PD-L1: 7 (39%) expressed a high level, 3 (17%) expressed a medium level, and 8 (44%) expressed a low level. Only 9 (50%) tumors expressed PD-L2 and it was at a low level. The expression of PD-L1 in tumor cells correlated with the presence of CD8-positive cytotoxic T lymphocytes among tumor cells (P < .01) and with the presence of CD8-positive cells in the surrounding stroma (P = .04). CONCLUSIONS: A subset of ocular invasive conjunctival squamous carcinomas express high levels of PD-L1 and CD8 and therefore may respond therapeutically to immune checkpoint inhibition.