Invasive cSCC Research Articles

MiR-181a Promotes Multiple Protumorigenic Functions by Targeting TGFβR3

Cutaneous squamous cell carcinoma (cSCC) comprises 15‒20% of all skin cancers and has a well-defined progression sequence from precancerous actinic keratosis to invasive cSCC. To identify targets for chemoprevention, we previously reported a cross-species analysis to identify the transcriptional drivers of cSCC development and identified miR-181a as a potential oncomiR. We show that the upregulation of miR-181a promotes multiple protumorigenic properties by targeting an understudied component of TGFβ signaling, TGFβR3. miR-181a and TGFβR3 are upregulated and downregulated, respectively, in cSCC. miR-181a overexpression (OE) and TGFβR3 knockdown (KD) significantly suppresses UV-induced apoptosis in HaCaT cells and in primary normal human epidermal keratinocytes. In addition, OE of miR-181a or KD of TGFβR3 by short hairpin RNA enhances anchorage-independent survival. miR-181a OE or TGFβR3 KD enhances cellular migration and invasion and upregulation of epithelial‒mesenchymal transition markers. Luciferase reporter assays demonstrate that miR-181a directly targets the 3'-untranslated region of TGFβR3. miR-181a upregulates phosphorylated SMAD3 levels after TGFβ2 administration and results in elevated SNAIL and SLUG expression. Finally, we confirm invivo that miR-181a inhibition compromises tumor growth. Importantly, these phenotypes can be reversed with TGFβR3 OE or KD in the context of miR-181a OE or KD, respectively, further highlighting the physiologic relevance of this regulation in cSCC.

Overview of guideline recommendations for the management of high‐risk and advanced cutaneous squamous cell carcinoma

AbstractCutaneous squamous cell carcinoma (cSCC) is the second most common form of skin cancer. National and international associations have issued evidence‐ and consensus‐based guidelines to offer clinicians a framework to optimally manage patients with invasive cSCC. Current updated guidelines regarding the recommendations on the management of patients with high‐risk and advanced cSCC include EDF/EADO (European) Guidelines 2020, US National Comprehensive Cancer Network guidelines 2021, American Academy of Dermatology guidelines 2018, British Association of Dermatology guidelines 2020 and German guidelines 2020. This review presents the guideline recommendations on the definition of high‐risk and advanced cSCC, surgical treatment and safety margins, definitive and adjuvant radiotherapy and systemic treatments. The recommendations across guidelines may converge, diverge or in some cases not be able to provide a recommendation, highlighting open questions to be answered by future studies.

The Role of p53 in Progression of Cutaneous Squamous Cell Carcinoma.

Simple SummarySkin cancers are the most common types of cancer worldwide, and their incidence is increasing. Epidermal keratinocyte-derived cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. The most important risk factor for cSCC is long-term exposure to solar ultraviolet radiation, which induces oncogenic mutations in epidermal keratinocytes. The most common mutations are inactivating mutations in tumor suppressor p53, which result in accumulation of additional mutations. Recently, the role of p53 in the progression and invasion of cSCC has also been elucidated. In this review we will discuss the role of p53 in development of cSCC and as a potential new therapeutic target in advanced cSCC.Skin cancers are the most common types of cancer worldwide, and their incidence is increasing. Melanoma, basal cell carcinoma (BCC), and cutaneous squamous cell carcinoma (cSCC) are the three major types of skin cancer. Melanoma originates from melanocytes, whereas BCC and cSCC originate from epidermal keratinocytes and are therefore called keratinocyte carcinomas. Chronic exposure to ultraviolet radiation (UVR) is a common risk factor for skin cancers, but they differ with respect to oncogenic mutational profiles and alterations in cellular signaling pathways. cSCC is the most common metastatic skin cancer, and it is associated with poor prognosis in the advanced stage. An important early event in cSCC development is mutation of the TP53 gene and inactivation of the tumor suppressor function of the tumor protein 53 gene (TP53) in epidermal keratinocytes, which then leads to accumulation of additional oncogenic mutations. Additional genomic and proteomic alterations are required for the progression of premalignant lesion, actinic keratosis, to invasive and metastatic cSCC. Recently, the role of p53 in the invasion of cSCC has also been elucidated. In this review, the role of p53 in the progression of cSCC and as potential new therapeutic target for cSCC will be discussed.

Response of advanced cutaneous squamous cell carcinoma to immunotherapy: case report

The most common cancer in the United States is non-melanoma skin cancer. Cutaneous squamous cell carcinoma (cSCC) is the second most common non-melanoma skin cancer after basal cell carcinoma. It develops in the middle and outer layers of the skin. Its precursor is actinic keratosis, which can progress to squamous cell carcinoma in situ, invasive cSCC, and finally metastatic cSCC. About 20% of non-melanoma skin cancers are squamous cell and the remaining 80% are basal cell. Unlike basal cell, squamous cell carcinoma has the propensity to metastasize. This commonly occurs with squamous cell carcinoma (SCC) thicker than 2 millimeters. The risk of metastasis and local recurrence increases with 6 mm thickness and desmoplasia. The risk factors are excessive sun or ultraviolet light (tanning beds) exposure, immunosuppression (either having a weakened immune system or taking immunosuppressive therapy) and fair skin. Therefore, it most commonly affects skin in the head and neck area such as scalp, ears, lips, face, neck or the back of the hands. The treatment for local cutaneous squamous cell cancer is mainly surgery; excisional surgery, Moh's surgery, cryosurgery, curettage and electrodessication, laser surgery or radiation therapy, photodynamic therapy or topical agents such as fluorouracil or imiquimod. However, cSCC that is locally advanced, such as involvement of regional lymph nodes, or has metastasized to distant organs or tissue, is not amenable to surgery or radiation alone. Immunotherapy with cemiplimab, a programmed cell death 1 (PD-1) inhibitor, is a US Food and Drug Administration (FDA) approved therapeutic option for locally advanced and metastatic cSCC for patients who are not candidates for or whose disease is not susceptible to curative surgery or radiation therapy. Cemiplimab is a humanized recombinant immunoglobulin monoclonal antibody that binds to and blocks PD-1 receptor found on T cells inhibiting T-cell proliferation and cytokine production. We present a case of locally advanced cSCC with regional lymph nodes metastases, which achieved clinical remission, utilizing a unique approach of therapy combining a checkpoint inhibitor, Cemiplimab and radiotherapy.

Mismatch Repair Protein Expression and Microsatellite Instability in Cutaneous Squamous Cell Carcinoma.

There exist relatively sparse and conflicting data on high-level microsatellite instability (MSI-H) and deficient mismatch repair (dMMR) in cutaneous malignancies. We aimed to determine the expression profiles of MMR proteins (MSH2, MSH6, MLH1, and PMS2) in different progression stages of cutaneous squamous cell carcinoma (cSCC, 102 patients in total) by immunohistochemistry, and search for MSI-H in patients with low-level MMR or dMMR using multiplex-PCR. Low-level MMR protein expression was observed in five patients: One patient with primary cSCC < 2 mm thickness and low-level MLH1, three patients with primary cSCC > 6 mm (including one with low-level MSH2, as well as MSH6 expression, and two with low-level PMS2), and one patient with a cSCC metastasis showing low-level MSH2 as well as MSH6. Intergroup protein expression analysis revealed that MLH1 and MSH2 expression in actinic keratosis was significantly decreased when compared to Bowen’s disease, cSCC < 2 mm, cSCC > 6 mm, and cSCC metastasis. In cases with low-level MMR, we performed MSI-H tests revealing three cases with MSI-H and one with low-level MSI-L. We found low-level MMR expression in a small subset of patients with invasive or metastatic cSCC. Hence, loss of MMR expression may be associated with tumour progression in a small subgroup of patients with non-melanoma skin cancer.

The Role of Circular RNAs in Keratinocyte Carcinomas.

Simple SummaryKeratinocyte carcinomas include BCC and cSCC and represent the most frequent cancer among fair-skinned people. Tumor development is associated with mutations and dysregulation of many genes involved in biological processes such as cell proliferation, differentiation and apoptosis. The expression of these genes is controlled in many ways, including transcriptional and post-transcriptional control by circular RNAs. In recent studies, a number of circular RNAs have been identified that are dysregulated in BCC and cSCC. Biological functions relevant to tumor development were shown for some of these circRNAs, which may represent biomarkers for disease progression and targets for novel treatment approaches.Keratinocyte carcinomas (KC) include basal cell carcinomas (BCC) and cutaneous squamous cell carcinomas (cSCC) and represents the most common cancer in Europe and North America. Both entities are characterized by a very high mutational burden, mainly UV signature mutations. Predominately mutated genes in BCC belong to the sonic hedgehog pathway, whereas, in cSCC, TP53, CDKN2A, NOTCH1/2 and others are most frequently mutated. In addition, the dysregulation of factors associated with epithelial to mesenchymal transition (EMT) was shown in invasive cSCC. The expression of factors associated with tumorigenesis can be controlled in several ways and include non-coding RNA molecules, such as micro RNAs (miRNA) long noncoding RNAs (lncRNA) and circular RNAs (circRNA). To update findings on circRNA in KC, we reviewed 13 papers published since 2016, identified in a PubMed search. In both BCC and cSCC, numerous circRNAs were identified that were differently expressed compared to healthy skin. Some of them were shown to target miRNAs that are also dysregulated in KC. Moreover, some studies confirmed the biological functions of individual circRNAs involved in cancer development. Thus, circRNAs may be used as biomarkers of disease and disease progression and represent potential targets of new therapeutic approaches for KC.

LB797 Immune nano-scintillator mediated novel triad photodynamic therapy reversing the tumor immunosuppressive microenvironment against invasive skin Squamous Cell Carcinoma

Therapy of cutaneous squamous cell carcinoma (cSCC) is a big challenge when it progresses into invasive cSCC which involves a deep range with a high degree of malignancy. Photodynamic therapy is a promising method utilized in tumor therapy while its applications are limited due to the low penetration depth of light in tissues and weak efficacy of each single treatment. Herein, a novel nanoplatform of X ray responsive, photosensitizer and immunologic adjuvant imiquimod loaded nano-scintillator (ScMMI) mediated triad photodynamic therapy (X-IPDT) was constructed to accomplish the inhibition of cSCC growth, benefiting from the deep penetration ability of X ray and simultaneous enhanced immunological effect.

Serum matrix metalloproteinase-13 as a diagnostic biomarker for cutaneous squamous cell carcinoma

BackgroundA significant proportion of newly diagnosed patients with cutaneous squamous cell carcinoma (cSCC) have metastasis and eventually die of the disease, necessitating the exploration of novel biomarkers for early detection of cSCC aggressiveness, risk assessment and monitoring. Matrix metalloproteinase-13 (MMP-13) has been implicated in cSCC pathogenesis. Serum MMP-13 levels have been shown to predict survival in patients with esophageal SCC, but their diagnostic value for cSCC has not been explored.MethodsWe conducted a case-control study to examine serum MMP-13 as a biomarker for cSCC. Patients with cSCC undergoing surgical resection and health controls undergoing plastic surgery were recruited. ELISA for measurement of serum MMP-13 and immunohistochemistry for detection of tissue MMP-13 were performed, and the results were compared between the case and the control group, and among different patient groups. ROC curve analysis was performed to determine the diagnostic value of serum MMP-13 levels.ResultsThe ratio of male to female, and the age between the case (n = 77) and the control group (n = 50) were not significantly different. Patients had significantly higher serum MMP-13 levels than healthy controls. Subjects with stage 3 cSCC had markedly higher serum MMP-13 levels than those with stage 1 and stage 2 cSCC. Patients with invasive cSCC had remarkably higher serum MMP-13 than those with cSCC in situ. Post-surgery serum MMP-13 measurement was done in 12 patients, and a significant MMP-13 decrease was observed after removal of cSCC. Tumor tissues had a remarkably higher level of MMP-13 than control tissues. Serum MMP-13 predicted the presence of invasive cSCC with an AUC of 0.87 (95% CI [0.78 to 0.95]) for sensitivity and specificity of 81.7 and 82.4%, respectively for a cut-off value of 290 pg/mL. Serum MMP-13 predicted lymph node involvement with an AUC of 0.94 (95% CI [0.88 to 0.99]) for sensitivity and specificity of 93.8 and 88.5%, respectively for a cut-off value of 430 pg/mL.ConclusionSerum MMP-13 might serve as a valuable biomarker for early detection of cSCC invasiveness and monitoring of cSCC progression.

Icelandic study of cutaneous squamous cell carcinoma (cSCC) epidemiology highlights the worldwide burden of disease and challenges of insitu and invasive cSCC registry data.

Icelandic study of cutaneous squamous cell carcinoma (cSCC) epidemiology highlights the worldwide burden of disease and challenges of insitu and invasive cSCC registry data.

Invasive and insitu squamous cell carcinoma of the skin: a nationwide study in Iceland.

The worldwide incidence of cutaneous squamous cell carcinoma (cSCC) is increasing. To evaluate the tumour burden of insitu and invasive cSCC in Iceland, where the population is exposed to limited ultraviolet radiation. This whole-population study used the Icelandic Cancer Registry, which contains records of all insitu and invasive cSCC cases from 1981 to 2017. Incidence of cSCC was evaluated according to age, anatomical location, residence and multiplicity, and trends were assessed using joinpoint analysis. Age-standardized rates (WSR) and age-specific incidence rates per 100000 person-years were calculated, along with cumulative and lifetime risks. Between 1981 and 2017, insitu cSCC WSR increased from 1·2 to 19·1 for men and from 2·0 to 22·3 for women. Invasive cSCC WSR rose from 4·6 to 14 for men and from 0·3 to 13·2 for women. The average number of insitu cSCC lesions was 1·71 per woman and 1·39 per man. Women developed more insitu cSCCs than invasive cSCCs in almost all anatomical locations, whereas men developed more invasive cSCCs, mostly on the head and neck. The rates of insitu cSCC were higher in Reykjavik compared with rural areas. Furthermore, women more commonly developed multiple insitu lesions. For lip cSCCs, invasive lesions occurred more frequently than insitu lesions among both sexes. Joinpoint analysis showed that insitu cSCC in women exhibited the most rapid incidence increase. cSCC has become an increasingly significant public health problem in Iceland. Tanning bed use and travelling abroad may contribute to skin cancer development. Public health efforts are needed to stem the behaviours leading to this rapid rise in cSCC.

Dermoscopy as a Tool in Differentiating Cutaneous Squamous Cell Carcinoma From Its Variants.

Dermoscopic features of cutaneous squamous cell carcinoma (cSCC) have been widely studied, but their accuracy should be further investigated. This study assessed the diagnostic accuracy of a set of predetermined dermoscopic structures for 3 variants of cSCC, namely Bowen disease, keratoacanthoma and invasive cSCC. Dermoscopic images of 56 histopathologically confirmed cSCC lesions (9 Bowen disease lesions, 7 keratoacanthomas, and 40 invasive cSCCs) were examined, and the diagnostic accuracy of dermoscopic structures was assessed. Discriminative ability of statistically significant positive predictors was determined using receiver operating characteristic (ROC) curves, and defined as an area under the ROC curve >0.700. Dermoscopic structures with statistical significance and discriminative ability were: for Bowen disease, clustered glomerular vessels and erosions; for keratoacanthoma, a central keratin plug; and for invasive cSCC, a mixed color of the background. Clustered and glomerular vessels had, for Bowen disease, perfect diagnostic accuracy, with: sensitivity of 88.9% for both features; specificity of 97.9% and 93.6%, respectively; positive predictive value (PPV) of 88.9% and 72.7%, respectively; and negative predictive value (NPV) of 97.8% for both. Erosions had, for BD, high specificity (87.2%) and NPV (91.1%), but low sensitivity (55.6%) and PPV (45.5%). A central keratin plug had, for keratoacanthoma, high specificity (87.8%) and NPV (93.5%), but low sensitivity (57.1%) and PPV (40%). A mixed background color had, for invasive cSCC, high specificity (81.3%) and PPV (89.7%), but low sensitivity (65%) and NPV (48.2%). Dermoscopy accurately differentiates BD, through clustered glomerular vessels, from keratoacanthoma and invasive cSCC. Dermoscopic structures of keratoacanthoma and invasive cSCC overlap, and only histopathologic analysis differentiates them precisely.

Cutaneous Squamous Cell Carcinoma: From Pathophysiology to Novel Therapeutic Approaches.

Cutaneous squamous cell carcinoma (cSCC), a non-melanoma skin cancer, is a keratinocyte carcinoma representing one of the most common cancers with an increasing incidence. cSCC could be in situ (e.g., Bowen’s disease) or an invasive form. A significant cSCC risk factor is advanced age, together with cumulative sun exposure, fair skin, prolonged immunosuppression, and previous skin cancer diagnoses. Although most cSCCs can be treated by surgery, a fraction of them recur and metastasize, leading to death. cSCC could arise de novo or be the result of a progression of the actinic keratosis, an in situ carcinoma. The multistage process of cSCC development and progression is characterized by mutations in the genes involved in epidermal homeostasis and by several alterations, such as epigenetic modifications, viral infections, or microenvironmental changes. Thus, cSCC development is a gradual process with several histological- and pathological-defined stages. Dermoscopy and reflectance confocal microscopy enhanced the diagnostic accuracy of cSCC. Surgical excision is the first-line treatment for invasive cSCC. Moreover, radiotherapy may be considered as a primary treatment in patients not candidates for surgery. Extensive studies of cSCC pathogenic mechanisms identified several pharmaceutical targets and allowed the development of new systemic therapies, including immunotherapy with immune checkpoint inhibitors, such as Cemiplimab, and epidermal growth factor receptor inhibitors for metastatic and locally advanced cSCC. Furthermore, the implementation of prevention measures has been useful in patient management.

The Genomic Landscape of Actinic Keratosis

Actinic keratoses (AKs) are lesions of epidermal keratinocyte dysplasia and are precursors for invasive cutaneous squamous cell carcinoma (cSCC). Identifying the specific genomic alterations driving the progression from normal skin to skin with AK to skin with invasive cSCC is challenging because of the massive UVR-induced mutational burden characteristic at all stages of this progression. In this study, we report the largest AK whole-exome sequencing study to date and perform a mutational signature and candidate driver gene analysis on these lesions. We demonstrate in 37 AKs from both immunosuppressed and immunocompetent patients that there are significant similarities between AKs and cSCC in terms of mutational burden, copy number alterations, mutational signatures, and patterns of driver gene mutations. We identify 44 significantly mutated AK driver genes and confirm that these genes are similarly altered in cSCC. We identify azathioprine mutational signature in all AKs from patients exposed to the drug, providing further evidence for its role in keratinocyte carcinogenesis. cSCCs differ from AKs in having higher levels of intrasample heterogeneity. Alterations in signaling pathways also differ, with immune-related signaling and TGFβ signaling significantly more mutated in cSCC. Integrating our findings with independent gene expression datasets confirms that dysregulated TGFβ signaling may represent an important event in AK‒cSCC progression.

Beyond the dermis—high-risk invasive squamous cell carcinoma: a retrospective review

Most cutaneous squamous cell carcinomas (cSCC) are low risk and can be treated with simple excision or ablation. High-risk cSCC require invasive treatment, including radical surgery. We present our experience in treating invasive cSCC of the pelvis and extremities. A retrospective review of the data of patients with invasive cSCC, indicated for surgery between 2014 and 2018, from a single institution was carried out. A total of 19 patients (nine men, 10 women) were included in the study. Mean age was 62 years; mean tumour size was 8.6cm). Of the 19 patients, five patients with paraplegia with cSCC arising from hard-to-heal ulcers died of infection or bleeding after surgery or systemic therapy. Also, nine patients with localised cSCC underwent margin-negative resection with or without radiation; one patient experienced disease relapse. Of the participants, two patients with previous transplants and multifocal aggressive cSCC underwent numerous resections but succumbed to disease, and two patients who presented with locally recurrent disease after previous positive margin resection and radiation underwent re-resection but developed recurrent disease. Prognosis for invasive cSCC largely depends on clinical setting. Tumours arising from ulcers in patients with paraplegia have a poor prognosis regardless of treatment. Invasive cSCC in transplant patients are often multifocal and often recur. Debulking procedures are associated with local recurrence despite radiation. Patients presenting with localised disease have a favourable prognosis with wide resection, flap coverage and adjuvant therapy.

Photodynamic therapy as an alternative treatment in patients with invasive cutaneous SCC where surgery is not feasible: Single center experience.

The aim of this study was to demonstrate the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) as an alternative treatment in cutaneous squamous cell carcinoma (cSCC) patients who are not fit for surgery. Thirty-three invasive cSCC patients who, for some reasons, cannot undergo surgery were enrolled in this study. All patients received plum blossom needle (PBN) pretreated ALA-PDT combined with topical application of 5% imiquimod cream. Two patients dropped the study because of severe pain and two patients discontinue treatment due to lack of response. Of 29 patients, who completed the treatment, 5 patients had complete response after 2-9 sessions of PDT and these patients had no recurrence till 18 months after treatment. Twenty-four patients achieved partial response and are satisfied with treatment outcome in terms of decreased symptoms and improved quality of life. PBN pretreated PDT in combination with topical imiquimod may be a viable treatment option for non resectable cSCC lesions.

Risk factors and timing of subsequent cutaneous squamous cell carcinoma in patients with cutaneous squamous cell carcinoma: A retrospective cohort study

Information about the frequency and timing of subsequent cutaneous squamous cell carcinoma (cSCC), along with associated risk factors, is limited. However, this information is crucial to guide follow-up care for these patients. To evaluate the risk and timing of subsequent cSCC in patients who presented with an initial diagnosis of cSCC. Retrospective review of an institutional review board-approved, single-institution registry of invasive cSCC. All patients had at least 2 primary cSCCs diagnosed on 2 separate dates 2months apart. A total of 299 primary cSCCs were included. At 6months from initial cSCC diagnosis, 18.06% (n=54) of patients developed subsequent cSCC; at 1year, 31.77% (n=94); at 3years, 67.56% (n=202); and at 5years, 87.96% (n=263) developed subsequent cSCC. Risk factors associated with subsequent cSCC include age at initial diagnosis (hazard ratio [HR], 1.02; 95% confidence interval, 1.004-1.027; P=.008), T2 stage (HR, 1.66; 95% CI, 1.07-2.57; P=.025), and poor tumor grade. Tumor grades well, moderate, and unknown have HRs of 0.21 (P<.001), 0.16 (P .001), and 0.25 (P=.001), respectively. Of patients who develop subsequent cSCC, 18.06% do so within 6months, and 31.77% do so within 1year of initial cSCC diagnosis. Patients with advanced age, poor histologic differentiation, and American Joint Committee on Cancer T2 stage are at highest risk. Close clinical follow-up after the initial diagnosis is recommended.

Comparison of the eighth edition of the TNM and Brigham Women's Hospital staging systems for cutaneous squamous cell carcinoma of the head and neck: a six-year review.

This study was undertaken with the aim to compare the T stages in a series of cutaneous squamous cell carcinoma (cSCC) patients using both the eighth edition of the Union for International Cancer Control (UICC) TNM Classification of Malignant Tumours (TNM8) and Brigham and Women's Hospital (BWH) staging systems. This would allow comparison of the two to determine suitability with regards to T stage and the effect on local recurrence and nodal disease. This was a six-year retrospective cohort study of patients with primary invasive cSCC of the head and neck who were diagnosed and treated at Western Sussex Hospitals Trust in the United Kingdom between 2007 and 2012. The TNM8 and BWH staging systems were applied to these primary cSCCs. A total of 695 invasive cSCCs treated in 604 patients over six years were identified. Most patients were male (76%), with a mean (range) age of 81 (50-103) years. The most common location for local recurrence was the scalp (n=26, 58%). Regional metastasis occurred most commonly in the parotid gland (n=20, 63%). All tumours were classified using both staging systems. Specifically, 432 tumours remained in the same T stage (61%), and 192 were downstaged (27%) and 71 upstaged using the BWH (10%). The median (SD) follow-up time was 23 (28) months (range 1-123). The BWH alternative staging system overlapped with the TNM8 in high-stage and low-stage tumour assignment. The highest percentage of local recurrence and regional metastasis occurred in T2b tumours.

Abstract 1540: Cutaneous squamous cell carcinoma: Evidence of epithelial to mesenchymal transition

Abstract Cutaneous Squamous Cell Carcinoma (cSCC) is the second most common form of skin cancer, frequently arising from sun-induced mutations. Most clinical cases of cSCC are non-invasive and achieve complete cure through tumor excision. However, advanced cSCC is associated with high mortality, thus research aimed at understanding factors influencing metastasis of invasive cSCC is needed. Cancer metastasis is a multistep process, including epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells undergo a dedifferentiation process whereby they transition to a highly mobile and invasive mesenchymal cell type with metastatic capacities. Changes in expression of several proteins, including TGF-β, vimentin, and E-cadherin have been identified as a hallmark of EMT, which may be promoting cancer progression. We hypothesize that during development of cSCC, cells within the lesions are undergoing EMT that facilitates invasion into the tissues. In situ and invasive cSCC tissues were obtained from excised tumors being prepped for histological sections from Mohs surgery; once clean margins were identified, adjacent normal tissues (ANT) were obtained during suturing. For all tissues, total protein was isolated; western blots were performed to determine expression of TGF-β1,2,3, Bcl-2, E-cadherin, phosphorylated E-cadherin, and vimentin. Analysis of MMP activity for each tissue type was performed using a FRET assay. For all proteins analyzed, cSCC tissues had higher levels of expression compared to ANT, with invasive cSCC tissues demonstrating increased protein expression over in situ samples (p&amp;lt;0.05 for all proteins). Compared to ANT, cSCC tissues also demonstrate significantly higher MMP activity both after 2 hours (p&amp;lt;0.01) and 4.5 hours (p&amp;lt;0.001). An oncology array performed using protein isolated from these cutaneous samples showed differences in the proteome profile between normal and cancer tissue, including some EMT-related markers. Immunohistochemistry (IHC) and immunofluorescent (IF) staining was performed using fixed cSCC tissue sections for the expression pattern of E-cadherin, cytokeratin, and vimentin. IHC staining reveals an inverse relationship between cytokeratin and vimentin expression, with poorly differentiated areas within lesions showing increased vimentin staining versus areas that are well-differentiated. Furthermore, IF staining reveals that poorly differentiated cells also have reduced E-cadherin staining at the junctions between cells. Our results show that cSCC is undergoing EMT at the projections of the lesions, indicating that this cancer has the potential to acquire a more aggressive phenotype. Tissues exhibiting higher rates of EMT may lead to a poorer prognosis if left untreated. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, Elizabeth E. Hull, Kathryn J. Leyva. Cutaneous squamous cell carcinoma: Evidence of epithelial to mesenchymal transition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1540.

Assessment of Cutaneous Squamous Cell Carcinoma (cSCC) In situ Incidence and the Risk of Developing Invasive cSCC in Patients With Prior cSCC In situ vs the General Population in the Netherlands, 1989-2017

The incidence rates of keratinocyte cancer are increasing globally; however, the incidence rates of cutaneous squamous cell carcinoma (cSCC) in situ and the risk of developing subsequent invasive cSCC remain unknown. To estimate annual population-based age-standardized incidence rates of histopathologically confirmed cSCC in situ stratified by sex, age, and body site and to assess the risk of developing invasive cSCC among patients with cSCC in situ compared with the general population. This nationwide epidemiological population-based cohort study used cancer registry data to identify all patients with a first incident of histopathologically confirmed cSCC in situ between January 1, 1989, and December 31, 2017. In addition, all patients with cSCC in situ who subsequently had a first incident of invasive cSCC were identified up to June 11, 2019. Data were analyzed between March 18 and November 12, 2019. Age-standardized incidence rates per year for cSCC in situ, standardized to the 2013 edition of the European Standard Population, were calculated by sex, age, and body site. Cumulative risks, standardized incidence ratios, and absolute excess risks were calculated to assess the risk of invasive cSCC in patients with cSCC in situ compared with the general population. In this population-based cohort study of 88 754 patients with a first incident of cSCC in situ between January 1, 1989, and December 31, 2017, 58.8% were women; the median age was 75 years (interquartile range [IQR], 67-82 years) for women and 73 years (IQR, 65-80 years) for men. Increasing incidence rates were observed, with the highest incidence rates in 2017 among women in general (71.7 cases per 100 000 person-years) and among men 80 years and older (540.9 cases per 100 000 person-years). The most common body site among women was the face (15.9 cases per 100 000 person-years) and among men was the scalp and/or neck (12.3 cases per 100 000 person-years). After 5 years of follow-up, among patients with cSCC in situ, the cumulative risk of developing an invasive cSCC at any anatomic location was 11.7% (95% CI, 11.6%-11.9%) in men and 6.9% (95% CI, 6.8%-7.0%) in women (P < .001). The standardized incidence ratio was highest in the first year of follow-up among both men (16.6; 95% CI, 15.7-17.5) and women (15.1; 95% CI, 14.2-16.1). This study reports the first nationwide incidence rates of cSCC in situ to date. The increasing incidence rates of cSCC in situ and the high risk of developing invasive cSCC among patients with cSCC in situ may increase the health care burden associated with precursors of keratinocyte cancer and highlight the need to include cutaneous skin cancer precursor lesions when exploring policies to address skin cancer care.

626 Solar simulated light induces cutaneous SCC in inbred mouse strains: Development of a clinically relevant mouse model

To address the need for clinically relevant cutaneous squamous cell carcinoma (cSCC) mouse models, we exposed SKH-1, FVB/N, Balb/c and C57BL/6 mice to solar simulated light (SSL), the environmental etiologic factor in human disease. Outbred SKH-1 mice are highly susceptible to SSL-induced tumor formation. However, an outbred strain limits the ability to evaluate MHC-restricted, antigen-specific T cell responses and perform studies with genetic mutants. The susceptibility of inbred strains FVB/N, Balb/c and C57BL/6 is unknown. UVA-340 bulbs were used to deliver SSL, because the ultraviolet spectrum closely resembles that from the sun. Mice were exposed to SSL 3 times per week starting with 1.2 kJ/m2 UVB and 13.9 kJ/m2 UVA. The dose was increased by 10% weekly to a maximum dose of 10 kJ/m2 UVB and 115.4 kJ/m2 UVA. SKH-1 mice received 22 weeks of SSL exposure with a cumulative dose of 257 kJ/m2 UVB and 2,967 kJ/m2 UVA. Other strains received 42 weeks of SSL exposure with a cumulative dose of 856 kJ/m2 UVB and 9,884 kJ/m2 UVA. SKH-1 mice were most susceptible to SSL-induced tumor formation with a median tumor onset of 19 weeks compared with 38 and 41 weeks for FVB/N and Balb/c mice, respectively (p < 0.0001). Tumors were histologically confirmed as papillomas (cSCC in situ) and invasive cSCC. At 44 weeks after the initiation of SSL exposure, no tumors had developed in C57BL/6 mice. A disadvantage of the SSL-induced tumor model is the long timeframe required to generate tumors. To address this disadvantage, we generated cell lines from the SSL-induced cSCC tumors in inbred mice. Thus, Balb/c and FVB/N mice are susceptible to SSL-induced cSCC formation. Syngeneic cSCC cell lines can be developed as transplantable cSCC models. These complementary approaches will facilitate future investigation.