Faculty Bibliography

Early in solid tumor development, antigens are presented in tumor-draining lymph nodes (tdLNs), a process that is necessary to set up immune surveillance. Recent evidence indicates that tdLNs fuel systemic tumor-specific T cell responses which may halt cancer progression and facilitate future responses to immunotherapy. These protective responses, however, are subject to progressive dysfunction exacerbated by lymph node (LN) metastasis. We discuss emerging preclinical and clinical literature indicating that the tdLN is a crucial reservoir for systemic immunity that can potentiate immune surveillance. We also discuss the impact of LN metastasis and argue that a better understanding of the relationship between LN metastasis and systemic immunity will be necessary to direct regional disease management in the era of immunotherapy.

INTRODUCTION/UNASSIGNED:Quantitative, multiplexed imaging is revealing complex spatial relationships between phenotypically diverse tumor infiltrating leukocyte populations and their prognostic implications. The underlying mechanisms and tissue structures that determine leukocyte distribution within and around tumor nests, however, remain poorly understood. While presumed players in metastatic dissemination, new preclinical data demonstrates that blood and lymphatic vessels (lymphovasculature) also dictate leukocyte trafficking within tumor microenvironments and thereby impact anti-tumor immunity. Here we interrogate these relationships in primary human cutaneous melanoma. METHODS/UNASSIGNED:We established a quantitative, multiplexed imaging platform to simultaneously detect immune infiltrates and tumor-associated vessels in formalin-fixed paraffin embedded patient samples. We performed a discovery, retrospective analysis of 28 treatment-naïve, primary cutaneous melanomas. RESULTS/UNASSIGNED:Here we find that the lymphvasculature and immune infiltrate is heterogenous across patients in treatment naïve, primary melanoma. We categorized five lymphovascular subtypes that differ by functionality and morphology and mapped their localization in and around primary tumors. Interestingly, the localization of specific vessel subtypes, but not overall vessel density, significantly associated with the presence of lymphoid aggregates, regional progression, and intratumoral T cell infiltrates. DISCUSSION/UNASSIGNED:We describe a quantitative platform to enable simultaneous lymphovascular and immune infiltrate analysis and map their spatial relationships in primary melanoma. Our data indicate that tumor-associated vessels exist in different states and that their localization may determine potential for metastasis or immune infiltration. This platform will support future efforts to map tumor-associated lymphovascular evolution across stage, assess its prognostic value, and stratify patients for adjuvant therapy.

BACKGROUND:Lymphocyte activation-gene 3 (LAG-3) is an emerging next-generation immune checkpoint molecule. We aim to define the expression pattern of LAG-3 in cutaneous squamous cell carcinoma (cSCC) as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. OBJECTIVE:To define the expression pattern of LAG-3 in cSCC as a first step to understand the role of LAG-3 in cSCC prognosis and therapy. METHODS:To test whether LAG-3 is expressed on cSCC infiltrating lymphocytes, we isolated CD8 + T lymphocytes from three SCC tumors using flow cytometry and performed single-cell RNA sequencing for LAG-3 and programmed cell death protein -1 (PD-1). In addition, we evaluated LAG-3 mRNA expression in formalin-fixed, paraffin-embedded tissue using NanoString technology. RESULTS:Single-cell RNA sequencing showed that LAG-3 is expressed more than PD-1 in CD8 + tumor infiltrating lymphocytes (50.8% vs 35.2%, respectively). Quantifying LAG-3 mRNA expression showed that compared with normal skin, LAG-3 mRNA is approximately 8 fold higher in immunocompetent associated SCC tumors and approximately 2 fold higher in transplant associated SCC tumors ( p -values <.05). In addition, LAG-3 mRNA was expressed 7.2 fold higher in T2a SCC tumors compared with normal skin ( p -value <.05). CONCLUSION:Lymphocyte activation-gene 3 is expressed on SCC infiltrating T lymphocytes at a higher percentage than PD-1. In addition, LAG-3 mRNA expression is significantly higher in SCC tumors. Ongoing studies will be performed to define its role as an immune-related biomarker and as a therapeutic target.

BACKGROUND:Vulvar squamous cell carcinoma (vSCC) is a rare tumor with a good prognosis when treated at a localized stage. However, once regional/distant metastasis occurs, vSCC can be rapidly fatal. Thus, it is important to identify tumor prognostic features so that high-risk cases can be prioritized for further diagnostic workup and treatment. OBJECTIVE:To estimate the risk of regional/distant metastasis at presentation and sentinel lymph node status for vSCC based on histopathologic characteristics. METHODS:A retrospective cohort study of 15,188 adult vSCC cases from the National Cancer Database diagnosed from 2012 to 2019. RESULTS:We provide specific estimates of the risk of clinically positive nodes and metastatic disease at presentation and sentinel lymph node positivity according to tumor size, moderate/poor tumor differentiation, and lymph-vascular invasion. These histopathologic factors were all significantly associated with the tested clinical outcomes in a multivariable analysis. Moderate (hazard ratio, 1.190; P < .001) and poor differentiation (hazard ratio, 1.204; P < .001) and lymph-vascular invasion (hazard ratio, 1.465; P < .001) were also associated with significantly poorer overall survival. LIMITATIONS/CONCLUSIONS:Data on disease-specific survival not available in the data set. CONCLUSIONS:We demonstrate the association of the histopathologic characteristics of vSCC with clinically important outcomes. These data may provide individualized information when discussing diagnostic/treatment recommendations, particularly regarding sentinel lymph node biopsy. These data may also guide future staging and risk stratification efforts for vSCC.

IMPORTANCE:Merkel cell carcinoma (MCC) is a rare cutaneous malignant neoplasm with increasing incidence and high mortality. Although it is accepted that the optimal treatment for localized tumors is surgical, the data surrounding the optimal surgical approach are mixed, and current National Comprehensive Cancer Network guidelines state that Mohs micrographic surgery (MMS) and wide local excision (WLE) can both be used. The current National Comprehensive Cancer Network guidelines do not advocate a preference for MMS or WLE and suggest that they can be used interchangeably. OBJECTIVE:To evaluate the association of surgical approach with overall survival after excision of localized T1/T2 MCC. DESIGN, SETTING, AND PARTICIPANTS:This retrospective cohort study used the National Cancer Database to assess adults with T1/T2 MCC who were diagnosed between January 1, 2004, and December 31, 2018, with pathologically confirmed, negative regional lymph nodes and treated with surgery. The National Cancer Database includes all reportable cases from Commission on Cancer-accredited facilities. Data analysis was performed from October 2022 to May 2023. EXPOSURE:Surgical approach. MAIN OUTCOMES AND MEASURES:Overall survival. RESULTS:A total of 2313 patients (mean [SD] age, 71 [10.6] years; 1340 [57.9%] male) were included in the study. Excision with MMS had the best unadjusted survival, with mean (SE) survival rates of 87.4% (3.4%) at 3 years, 84.5% (3.9%) at 5 years, and 81.8% (4.6%) at 10 years vs 86.1% (0.9%) at 3 years, 76.9% (1.2%) at 5 years, and 60.9% (2.0%) at 10 years for patients treated with WLE. Patients treated with narrow-margin excision had similar survival as those treated with WLE, with mean (SE) survival rates of 84.8% (1.4%) at 3 years, 78.3% (1.7%) at 5 years, and 60.8% (3.6%) at 10 years. On multivariable survival analysis, excision with MMS was associated with significantly improved survival compared with WLE (hazard ratio, 0.59; 95% CI, 0.36-0.97; P = .04). High-volume MCC centers were significantly more likely to use MMS over WLE compared with other centers (odds ratio, 1.99; 95% CI, 1.63-2.44; P < .001). CONCLUSIONS AND RELEVANCE:In this cohort study, the use of MMS (compared with WLE) was associated with significantly improved survival for patients with localized MCC with pathologically confirmed negative lymph nodes treated with surgery. These data suggest that Mohs surgery may provide a more effective treatment for MCC primary tumors than conventional WLE, although the lack of randomization and potential for selection bias in this study highlight the need for future prospective work evaluating this issue.

Anemia commonly occurs in systemic lupus erythematosus, a disease characterized by innate immune activation by nucleic acids. Overactivation of cytoplasmic sensors by self-DNA or RNA can cause erythroid cell death, while sparing other hematopoietic cell lineages. Whereas chronic inflammation is involved in this mechanism, less is known about the impact of systemic lupus erythematosus on the BM erythropoietic niche. We discovered that expression of the endosomal ssRNA sensor human TLR8 induces fatal anemia in Sle1.Yaa lupus mice. We observed that anemia was associated with a decrease in erythromyeloblastic islands and a block in differentiation at the CFU-E to proerythroblast transition in the BM. Single-cell RNAseq analyses of isolated BM erythromyeloblastic islands from human TLR8-expressing mice revealed that genes associated with essential central macrophage functions including adhesion and provision of nutrients were down-regulated. Although compensatory stress erythropoiesis occurred in the spleen, red blood cell half-life decreased because of hemophagocytosis. These data implicate the endosomal RNA sensor TLR8 as an additional innate receptor whose overactivation causes acquired failure of erythropoiesis via myeloid cell dysregulation.