cSCC Tissue Research Articles

Oncolytic activity of a coxsackievirus B3 strain in patient-derived cervical squamous cell carcinoma organoids and synergistic effect with paclitaxel

BackgroundCervical squamous cell carcinoma (CSCC) is a prevalent gynecological malignancy worldwide. Current treatments for CSCC can impact fertility and cause long-term complications, underscoring the need for new therapeutic strategies. Oncolytic virotherapy has emerged as a promising option for cancer treatment. Previous research has demonstrated the oncolytic activity of the coxsackievirus B3 strain 2035 A (CVB3/2035A) against various tumor types. This study aims to evaluate the clinical viability of CVB3/2035A for CSCC treatment, focusing on its oncolytic effect in patient-derived CSCC organoids.MethodsThe oncolytic effects of CVB3/2035A were investigated using human CSCC cell lines in vitro and mouse xenograft models in vivo. Preliminary tests for tumor-selectivity were conducted on patient-derived CSCC tissue samples and compared to normal cervical tissues ex vivo. Three patient-derived CSCC organoid lines were developed and treated with CVB3/2035A alone and in combination with paclitaxel. Both cytotoxicity and virus replication were evaluated in vitro.ResultsCVB3/2035A exhibited significant cytotoxic effects in human CSCC cell lines and xenograft mouse models. The virus selectively induced oncolysis in patient-derived CSCC tissue samples while sparing normal cervical tissues ex vivo. In patient-derived CSCC organoids, which retained the immunohistological characteristics of the original tumors, CVB3/2035A also demonstrated significant cytotoxic effects and efficient replication, as evidenced by increased viral titers and presence of viral nucleic acids and proteins. Notably, the combination of CVB3/2035A and paclitaxel resulted in enhanced cytotoxicity and viral replication.ConclusionsCVB3/2035A showed oncolytic activity in CSCC cell lines, xenografts, and patient-derived tissue cultures and organoids. Furthermore, the virus exhibited synergistic anti-tumor effects with paclitaxel against CSCC. These results suggest CVB3/2035A could serve as an alternative or adjunct to current CSCC chemotherapy regimens.

Single cell transcriptomics reveals dysregulated immnue homeostasis in different stages in HPV-induced cutaneous squamous cell carcinoma.

In order to explore the huge impact of impaired immnue homeostasis on the occurrence and development of cutaneous squamous cell carcinoma (cSCC), and investigate characterization of the cellular components and their changes which is crucial to understanding the pathologic process of HPV-induced cSCC, we diagnosed and followed up on a very rare HPV-induced cSCC patient who progressed at a very fast rate and transferred to death quickly. We performed single-cell RNA sequencing (scRNA-seq) of 11 379 cells from the skin tissues of this patient with four different skin statuses after HPV infection. Immunofluorescence experiments were used for validation. scRNA-seq identified that CD52+ HLA-DOA- macrophages only existed in paracancerous cutaneous squamous cell carcinoma (pc-cSCC) and cSCC tissue. Besides, immune cells including CD8+ exhausted T cells and CD4+ regulatory T cells as well as matrix cells like MMP1+, and MMP11+ fibroblasts were gradually increased. Meanwhile, COMP+ ASPN+ fibroblasts gradually decreased. Cell interaction analysis revealed enhancement in interactions between monocytes/macrophages, fibroblasts and tumour-specific keratinocytes. scRNA-seq was performed in HPV-induced cSCC for the first time, to explore the correlation between infection and tumour. It is the first time to study the development of tumours from different stages of infection in HPV-induced cSCC. In this study, the tumour itself and the tumour microenvironment were both analysed and explored. And it was validated in clinical samples from different patients. Our findings reveal the dynamic immnue homeostasis from normal skinto cSCC tissue, this alteration might drive HPV-induced cSCC.

MiR-34a-5p suppresses cutaneous squamous cell carcinoma progression by targeting SIRT6

Cutaneous squamous cell carcinoma (cSCC) is a malignant tumor originating from epidermal or appendageal keratinocytes, with a rising incidence in recent years. Understanding the molecular mechanism driving its development is crucial. This study aims to investigate whether miR-34a-5p is involved in the pathogenesis of cSCC by targeting Sirtuin 6 (SIRT6).The expression levels of miR-34a-5p and SIRT6 were determined in 15 cSCC tissue specimens, 15 normal tissue specimens and cultured cells via real-time polymerase chain reaction (RT-qPCR). Pearson’s correlation analysis was conducted to evaluate the relationship between miR-34a-5p and SIRT6 expression levels in cSCC tissues. A431 and SCL-1 cells were transfected with miR-34a-5p mimic, negative control or miR-34a-5p mimic together with recombinant plasmids containing SIRT6 gene. Cell counting kit-8, clone formation assay, wound healing assay, and flow cytometry were employed to assess the effects of these transfections on proliferation, migration, and apoptosis, respectively. The interaction between miR-34a-5p and SIRT6 was characterized using a dual-luciferase reporter assay.MiR-34a-5p expression was down-regulated in cSCC tissues significantly, while the SIRT6 expression was the opposite. A negative correlation was observed between the expression of miR-34a-5p and SIRT6 in cSCC tissues. Furthermore, overexpression of miR-34a-5p led to a significant reduction in the proliferation and migration abilities of A431 and SCL-1 cells, accompanied by an increase in apoptosis levels and a decrease in SIRT6 expression levels. MiR-34a-5p was identified as a direct target of SIRT6. Importantly, overexpression of SIRT6 effectively counteracted the inhibitory effect mediated by miR-34a-5p in cSCC cells.Our findings suggest that miR-34a-5p functions as a tumor suppressor in cSCC cells by targeting SIRT6.

Risk-Stratification using the 40-Gene Expression Profile (40-GEP) Test Identifies Patients with Node Negative Cutaneous Squamous Cell Carcinoma (cSCC) at Higher Risk of Metastasis Who May Benefit from Adjuvant Radiation Therapy (ART)

ART is a standard treatment used to reduce the risk of metastasis and recurrence in moderate‒to‒high-risk cSCC patients. Indications for ART have been largely based on pathologic risk factors and informed by staging systems, and while radiation oncologists generally designate a >10% risk threshold for usage of ART, there is no consensus on which groups of tumors may benefit from ART. The 40-GEP test has been independently validated to predict a cSCC patient's risk for regional/distant metastasis in patients with one or more high-risk clinicopathologic factors and reports three biologic risk groups: Class 1 (low, ∼7%), Class 2A (moderate, 20-25%), and Class 2B (high risk, >50%) for metastasis. This study aims to evaluate whether a biomarker informed risk stratification approach using a 40-GEP result could refine the ability to select patients with node negative cSCC at higher risk of metastasis who are most likely to benefit from ART. In this retrospective study, all patients had primary cSCC tissue with verified clinicopathologic information of tumors with one or more high-risk factors, met clinical testing criteria, were comprehensively staged, and had outcomes data (n = 954). Patients with node positive disease, or those with nodal failure within 3 months of diagnosis were excluded (n = 19). From the n = 935, an intermediate risk population wherein ART is often considered was defined as Brigham and Women's Hospital (BWH) ≥T2a (n = 489). Kaplan-Meier survival analysis and log-rank test were used to assess metastasis free survival (MFS). Univariate Cox regression compared metastasis rates between 40-GEP results. The 3-year MFS rate for this eligible for ART cohort was 82.4% The 40-GEP demonstrated statistically significant risk stratification with MFS rates of 92.4%, 76.1% and 59.4% for Class 1, Class 2A and Class 2B, respectively (p<0.0001). Cox regression was significant for Class 2A and 2B compared to Class 1, with a 3.2-fold and 6.4-fold increase in metastasis, respectively (p<0.0001). 64% (59/92) of all metastases received a Class 2A result, and 44% (14/32) of Class 2B patients metastasized. 46% (223/489) of the cohort received a Class 1 result. Of patients staged BWH T1 (n = 446), those with a Class 2A and 2B had an 88.7% and 66.7% MFS rate, respectively. Within this eligible for ART population, patients with Class 2A or 2B 40-GEP results have inferior rates of MFS, while Class 1 patients have <10% risk of metastasis. Nearly half of this population received a 40-GEP Class 1 result and could be considered for treatment de-intensification trials. Conversely, patients with low-risk BWH T1 stage, who are traditionally not considered for ART, that received a Class 2A or 2B (>10% risk of metastasis) could be considered for adjuvant therapy.

Stathmin 1 and p53 Expression in Cutaneous Squamous Cell Carcinoma and Precursor Lesions.

Studies on the relationship between stathmin 1 (STMN1) and cutaneous squamous cell carcinoma (cSCC) are limited. We aimed to evaluate the relationship between clinicopathological factors and STMN1 and p53 expressions in cSCC and compare them with those in the precursor lesions of cSCC and normal tissue. A total of 195 patients, followed between January 2014 and December 2021, with diagnoses of primary cSCC (n = 129), in situ cSCC (n = 20), or actinic keratosis (n = 46), as well as 29 histopathologically normal tissue samples, were included in the study. Immunohistochemical staining for STMN1 and p53 was performed. In the cSCC group, STMN1 scores were higher in poorly differentiated ( P = 0.001) and ulcerated ( P < 0.001) tumors. A linear relationship between STMN1 score and tumor area, tumor thickness, and mitosis was found ( P = 0.001, P = 0.003, and P < 0.001, respectively). There was no statistically significant correlation between STMN1 and p53 scores. Our results support the previous view that STMN1 may be associated with some adverse clinicopathological and high-risk features of cSCC. To the best of our knowledge, this is the first and largest study to investigate STMN1 expression in cSCC, precancerous lesions of cSCC, and normal tissues.

CD8/PD-L1 immunohistochemical reactivity and gene alterations in cutaneous squamous cell carcinoma.

In recent years, several immune checkpoint inhibitors targeting programmed death-ligand 1 (PD-L1) or PD-1 have been developed for cancer therapy. The genetic background of tumors and factors that influence PD-L1 expression in tumor tissues are not yet elucidated in cutaneous squamous cell carcinoma (cSCC). CD8-positive tumor-infiltrating lymphocytes (TILs) are known to be related to tumor immunity. Here, we aimed to study the relationship between CD8/PD-L1 immunohistochemical reactivity and gene alterations in cSCC. Tumorigenic genes were examined to identify gene alterations using next-generation sequencing (NGS). We collected 27 cSCC tissue samples (from 13 metastatic and 14 non-metastatic patients at primary diagnosis). We performed immunohistochemical staining for CD8 and PD-L1, and NGS using a commercially available sequencing panel (Illumina Cancer Hotspot Panel V2) that targets 50 cancer-associated genes. Immunohistochemically, CD8-positive TILs showed a high positive score in cSCC without metastasis; in these cases, cSCC occurred predominantly in sun-exposed areas, the tumor size was smaller, and the total gene variation numbers were notably low. The tumor depth, PD-L1 positivity, and gene variation number with or without tumor metastasis were not related, but the gene variation number tended to be higher in cSCCs arising in non-sun-exposed areas. Tumor metastasis was more common in cSCC arising in non-sun-exposed areas, which decreased the number of TILs or CD8-positive cells. From a genetic perspective, the total gene alterations were higher in cSCC with metastasis. Among them, ERBB4 and NPM1 are presumably involved in cSCC tumorigenesis; in addition, GNAQ, GNAS, JAK2, NRAS, IDH2, and CTNNB1 may be related to tumor metastasis. These results provide information on potential genes that can be targeted for cSCC therapy and on immune checkpoint inhibitors that may be used for cSCC therapy.

Thioredoxin domain-containing protein 9 protects cells against UV-B-provoked apoptosis via NF-κB/p65 activation in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC), a type of non-melanoma skin cancer (NMSC), is the most common malignancy worldwide. Thioredoxin (TXN) domain-containing protein 9 (TXNDC9) is a member of the TXN family that is important in cell differentiation. However, the biological function of this protein in cancer, particularly cSCC, is still unknown. In the present study, our experiments revealed the protective effects of TXNDC9 on UV-B-irritated cSCC cells. The initial findings showed that TXNDC9 is significantly upregulated in cSCC tissue and cells compared to normal skin tissue and keratinocytes. UV-B radiation robustly induces the expression of TXNDC9, and UV-B-induced cSCC cell death is boosted by TXNDC9 deficiency. Moreover, cSCC cells lacking TXNDC9 displayed attenuated activation of the NF-κB pathway. Additional studies by inhibiting TXNDC9 confirmed this finding, as TXNDC9 deficiency attenuated UV-B radiation-induced translocation of NF-κB p65 from the cytoplasm to the nucleus of cSCC. In conclusion, our work demonstrates the biological roles of TXNDC9 in cSCC progression and may provide a novel therapeutic target to treat cSCC in the future.

Proteasome Subunit Alpha Type-7 Expression Suppresses Cutaneous Squamous Cell Carcinoma Progression by Inhibiting Cancer-associated Cytokines.

Cutaneous squamous cell carcinoma (cSCC) is a common non-melanoma skin cancer, and its incidence is increasing. Proteasome subunit alpha type-7 (PSMA7) has been found to be aberrantly expressed in several cancers. However, whether it functions as a tumor suppressor or oncogene in the pathogenesis of cancers, particularly cSCC, remains controversial. Here, we aimed to investigate the functions of PSMA7 in cSCC pathogenesis. Clinicopathological characteristics were evaluated in 131 patients with cSCC using tissue sections. The expression of PSMA7, nucleotide-binding oligomerization domain-containing protein 1 (NOD1), and mitochondrial antiviral signaling protein (MAVS) was determined in cSCC tissue sections using immunohistochemical staining. The effect of PSMA7 expression on the biological behavior of cSCC cells was investigated in vitro. High immunoreactivity of PSMA7 (high-PSMA7) was detected in 53 (40.5%) patients with cSCC and was significantly associated with histologic grade (p=0.008) and favorable recurrence-free survival (p=0.018). The expression of PSMA7 and NOD1 (p=0.026) and MAVS (p=0.032) was negatively correlated in cSCC tissues. Contrary to the results of the cohort study, cell viability and invasiveness significantly decreased after PSMA7 down-regulation in cSCC cells in vitro. mRNA expression of tumor necrosis factor-alpha, interleukin-1 alpha (IL-1α), IL-6, and IL-8 were significantly increased after PSMA7 down-regulation in cSCC cells (all p=0.002). PSMA7-mediated degradation of NOD1 and MAVS as well as the subsequent reduction of the cancer-associated cytokine network may be a crucial mechanism of the antitumoral function of PSMA7 in patients with cSCC.

LB888 Role of MFN2 gene as prognostic biomarker for cutaneous squamous cell carcinoma recurrence

Although vast amount of research has been done on genes related to cancers, it remains unclear whether Mitofusin2 (MFN2) functions as a tumor suppressor or oncoprotein. The impact of MFN2 on carcinogenesis is likely to be more complicated than expected, so further research is needed. We aimed to investigate the effect of MFN2 on the pathogenesis of cutaneous squamous cell carcinoma (cSCC) and analyzed the expression of MFN2 in cSCC tissue and investigated the influence of MFN2 expression on the biological behavior of cSCC.

Abstract LB022: A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues

Abstract While most cases of cutaneous squamous cell carcinoma (cSCC) are benign, invasive cSCC is associated with higher mortality and is often more difficult to treat. As such, understanding the factors that influence the progression of cSCC are important. Aggressive cancers metastasize through a series of evolutionary changes, collectively called the epithelial-to-mesenchymal transition (EMT). During EMT, epithelial cells transition to a highly mobile mesenchymal cell type with metastatic capacities. While changes in expression of TGF-β, vimentin, and E-cadherin are hallmarks, EMT is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues (ANT), we observed a significant increase in mRNA expression of TGF-β (p&amp;lt;0.05), TGF-βR2 (p&amp;lt;0.001), and E-cadherin (p&amp;lt;0.005) in cSCC tissues by qPCR. Immunoblots performed on homogenized tissues yielded significant and parallel increases in protein expression of TGF-β (p&amp;lt;0.05) and E-cadherin (p&amp;lt;0.05). We also observed increased expression of the EMT-associated transcription factors Zeb1 and Snai1 in cSCC tissue sections using immunohistochemistry. Interestingly, we observed a significant increase in expression of vimentin (p&amp;lt;0.01) by immunoblot and differential expression of four oncogenic and EMT-associated miRNAs; miR-31 expression was significantly higher in cSCC tissues (p&amp;lt;0.001), while we observed a significant decrease in expression of miR-34a (p&amp;lt;0.05), miR-200b, and miR-200c in cSCC tissues. Examination of ANT and cSCC tissue sections using immunofluorescence and immunohistochemistry showed differential, and reverse, expression of E-cadherin and vimentin among cells within an individual tumor. Collectively, our data support a hypothesis that cSCC tumors are undergoing a hybrid EMT program, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. This hybrid EMT program may be promoting both survival and invasiveness of the tumors. A better understanding of this hybrid EMT process may influence therapeutic strategies in more invasive disease. Citation Format: Christopher S. Pulford, Chandana K. Uppalapati, McKale R. Montgomery, Elizabeth E. Hull, Kathryn J. Leyva. A hybrid epithelial to mesenchymal transition in ex-vivo cutaneous squamous cell carcinoma tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB022.

TOPK Affects Autophagy of Skin Squamous Cell Carcinoma by Regulating NF-KB Pathway through HDAC1.

Objective To explore the effect and potential mechanism of T-LAK cell-originated protein kinase (TOPK) on autophagy in cutaneous squamous cell carcinoma (cSCC). Methods Human cSCC cancer tissue and paracancerous tissue samples were collected clinically; immunohistochemistry was used to detect the expression of TOPK, nuclear factor κB p65 (NF-κB p65), phosphorylated nuclear factor κB inhibitor α (p-IκBα), Beclin-1, and microtubule-associated protein 1 light chain 3 (LC3) in cSCC tissue; and immunofluorescence was adopted to detect the coexpression of NF-κB p65, p-IκBα, and TOPK in cSCC tissue. After TOPK silencing or overexpression, SCL-1 and A431 cells were treated with PDTC and 3-MA, respectively. RT-qPCR and Western Blot were used to detect the mRNA and protein expressions of histone deacetylase 1 (HDAC1) in TOPK-silenced/overexpressing cells. Western Blot was performed to detect the protein expressions of NF-κB p65, p-p65, IκBα, p-IκBα, Beclin-1, and LC3 in each group. Transwell and scratch healing experiments were used to detect the ability of cells to invade and migrate. The formation of autophagosomes in each group was observed by TEM. Results Compared with adjacent tissues, TOPK, NF-κB p65, p-IκBα, Beclin-1, and LC3 were highly expressed in cSCC cancer tissues; TOPK and NF-κB p65 were coexpressed; and TOPK and p-IκBα were expressed in cSCC cancer tissues both increased. The mRNA and protein levels of TOPK in human cSCC cells were significantly higher than those in human normal skin HaCaT cells. After TOPK knockout, the expression of HDAC1, p-IκBα/IκBα, NF-κB p65, p-p65, Beclin-1, LC3II/I proteins, cell invasion, and migration abilities were significantly reduced, and fewer autophagosomes were observed. Treatment with PDTC and 3-MA significantly downregulated NF-κB pathway protein activity and autophagy level and reduced cell migration and invasion ability. Conclusion TOPK promotes the malignant progression of cSCC by upregulating HDAC1 to activate the NF-κB pathway and promote autophagy.

M2 macrophage facilitated angiogenesis in cutaneous squamous cell carcinoma via circ_TNFRSF21/miR-3619-5p/ROCK axis.

In recent years, the role of circular RNA in cancer cells has been studied broadly; however, the functional significance of circular RNA in the regulation of the tumor microenvironment (TME) is not fully understood. In this study, we aimed to reveal the role of circ_TNFRSF21 in M2 macrophage-induced cutaneous squamous cell carcinoma (cSCC) angiogenesis. Quantitative polymerase chain reaction and Western blotting were performed to determine the levels of the indicated genes. Direct binding between circ_TNFRSF21 and miR-3619-5p, miR-3619-5p, and ROCK2 was verified by dual-luciferase activity. The migration and invasion of human umbilical vein endothelial cells were evaluated by wound healing and transwell assays. Tube formation was performed to detect in vitro angiogenesis. Circ_TNFRSF21 and ROCK2 were upregulated in cSCC tissue, while miR-3619-5p was downregulated. Circ_TNFRSF21 negatively regulated the expression of miR-3619-5p, while miR-3619-5p negatively regulated the expression of ROCK2. miR-3619-5p suppressed tube formation by inhibiting ROCK signaling. M2 macrophages facilitated tube formation via the circ_TNFRSF21/miR-3619-5p/ROCK2 axis. Our present study revealed that circ_TNFRSF21 was elevated in M2 macrophages and mediated M2 macrophage-induced tube formation in vitro.

Sirolimus diminishes the expression of GRO-α (CXCL-1) /CXCR2 axis in human keratinocytes and cutaneous squamous cell carcinoma cells

BackgroundOrgan transplant recipients show a high incidence for the formation of cutaneous squamous cell carcinoma (cSCC), while sirolimus appears to reduce the risk. GRO-α is a chemokine, which is overexpressed in many tumor entities and associated with malignant transformation. However, little is known about the expression and function of GRO-α in human cSCC. ObjectiveOur aim was to investigate the relevance of the GRO-α (CXCL-1)/ CXCR2 axis in human cSCC and the potential impact of sirolimus. MethodsWe analyzed the GRO-α expression in human keratinocytes, different cSCC cell lines as well as cSCC tissue and investigated its effect on cell proliferation and migration. Additionally, we incubated cells with sirolimus and measured the expression of GRO-α and its receptor CXCR2. ResultsWe showed that both constitutive as well as induced GRO-α expression is higher in in cSCC cell lines compared to keratinocytes and that GRO-α protein is detectable in human cSCC tissue. By GRO-α exposure and shRNA knock down, we identified GRO-α as a driving factor in proliferation and migration. Moreover, in a dermis equivalent GRO-α knocked down cSCC cell lines displayed a reduced capacity in tumor nest formation. Incubation with sirolimus significantly inhibited GRO-α expression in keratinocytes as well as tumor cell lines. Moreover, sirolimus decreased the expression of the corresponding receptor CXCR2. ConclusionTaken together, our results suggest that the GRO-α/CXCR2 axis plays a role in human keratinocyte carcinogenesis and might represent a molecular mechanism for the preventive effect of mTOR inhibitors in cSCC development.

LncRNA EZR‑AS1 knockdown represses proliferation, migration and invasion of cSCC via the PI3K/AKT signaling pathway.

Although long non-coding RNAs (lncRNAs) have been implicated in various human cancer types, the role of lncRNA ezrin antisense RNA 1 (EZR-AS1) in cutaneous squamous cell carcinoma (cSCC) remains unclear. The present study aimed to investigate the effect of lncRNAEZR-AS1 on cSCC and identify the underlying molecular mechanisms. EZR-AS1 expression was measured in cSCC tissue and cells detected using reverse transcription-quantitative PCR. Gain-of-function assays were performed in A431 cells, which have a relatively low expression of EZR-AS1, while loss-of-function assays were performed in SCC13 and SCL-1 colon cancer cells, which have a relatively high expression of EZR-AS1. Cell viability, proliferation, migration, invasion and apoptosis were assessed using MTT, plate cloning, wound healing, Transwell and flow cytometry assays, respectively. EZR-AS1 mRNA expression levels were significantly upregulated in cSCC tissues and cells compared with adjacent healthy tissues and HaCaT cells, respectively. Compared with the small interfering RNA (si)-negative control (NC) group, si-EZR-AS1 significantly inhibited SCC13 and SCL-1 cell proliferation, migration and invasion, but promoted cell apoptosis. By contrast, compared with the pc-NC group, EZR-AS1 overexpression significantly enhanced A431 cell proliferation, migration and invasion, but inhibited cell apoptosis. Moreover, focal adhesion kinase (FAK) was identified as a target of EZR-AS1, and EZR-AS1 knockdown significantly decreased FAK expression compared with the si-NC group. Moreover, EZR-AS1 knockdown significantly downregulated the protein expression levels of phosphorylated (p)-PI3K/PI3K and p-AKT/AKT in cSCC cells compared with the si-NC group. The PI3K agonist 740Y-P significantly reversed si-EZR-AS1-mediated effects on SCC13 and SCL-1 cell proliferation, migration, invasion and apoptosis. In conclusion, the present study demonstrated that si-EZR-AS1 inhibited cSCC cell proliferation, migration and invasion, and promoted cell apoptosis, potentially via regulating the PI3K/AKT signaling pathway. Therefore, the present study provided novel insights into the diagnosis and treatment of cSCC.

Dysregulation of tissue and serum microRNAs in organ transplant recipients with cutaneous squamous cell carcinomas.

Micro RNAs (miRNAs) are considered as promising biomarkers for skin cancer. By next generation sequencing (NGS), we measured and compared miRNA expression profiles of tumor, peri‐lesional, and control tissue of immunosuppressed organ transplant recipients (OTR), suffering from localized cutaneous squamous cell carcinoma (cSCC). Out of 490 miRNAs detected in cSCC tissue, 23 significantly regulated miRNAs were selected for quantitative targeted analysis in serum samples of our patients and control sera from OTR without cSCC. Two onco‐miRNAs (mir‐1290, mir‐1246), previously identified as crucial drivers for tumor initiation and cancer progression, were significantly and exclusively upregulated in both tumor tissue and serum. This finding suggests that miRNA dysregulation in cSCC tissue is reflected in serum even in patients without advanced disease and highly differentiated cSCCs.

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.

Abstract 4898: The contribution of human papilloma virus infection to cutaneous squamous cell carcinoma in patients with chronic lymphocytic leukemia

Abstract Background: Human papilloma virus (HPV) is the most common sexually transmitted disease worldwide. α-genus HPV have oncogenic potential in anogenital skin and mucosa. Transplant patients have an increased incidence of β-genus HPV-associated cutaneous squamous cell carcinoma (cSCC). The host immune system is particularly important in the pathogenicity of HPV infection. The antibody-mediated, humoral immune response clears virus particles from body fluids and prevents re-infection. Cell-mediated immunity clears the virus from infected cells, generating immune memory. Impaired cell-mediated immunity is considered a primary risk factor in HPV-induced cSCC in the immunosuppressed population. Patients with chronic lymphocytic leukemia (CLL), a B-cell malignancy characterized by impaired humoral immunity, are at increased risk of developing cSCC. The purpose of this study is to explore the potential role of HPV in malignancies with impaired humoral response. Methods Primary cSCC tissue DNA was isolated from patients diagnosed with CLL. The tissue was analyzed for the presence of HPV. Diagnostic specimens underwent consensus dermatopathology review. Peripheral blood analytes and clinical parameters for disease course were statistically analyzed in conjunction with histopathology. Results Eleven patients with CLL contributed 37 cSCCs. In 49% of the lesions, HPV was detected. Among the cSCC lesions, 27% of the lesions contained β-genus HPV and 30% contained α-genus HPV. The most frequently reported virus was HPV-27 (α-genus), with 16% of lesions containing this variant which is frequently associated with cutaneous warts rather than cSCC. A majority of the HPV-containing lesions displayed moderate dermal lymphocytic inflammation. The lesions were often documented in sun exposed areas such as the scalp and face. Interestingly, HPV-16 and HPV-18, often found in anogenital epithelia, were detected in sun exposed skin in 14% of the lesions examined. In those patients who had documented immunoglobulins, all had hypogammaglobulinemia. Conclusions We examined clinical, histopathologic, and viral genotypes of the cSCCs in patients with CLL. It is widely accepted that CLL patients have a higher incidence and recurrence of cSCC; however, the characteristics of the lesions and the potential association to HPV has not been reported. In our cohort, the lesions were as likely to contain HPV from the α- or β- genus. HPV-27, normally associated with warts, displayed an atypical pathology. The presence of HPV-16 and -18 in sun exposed areas, suggests a novel pathology in compromised humoral immunity. Although our analysis derived some interesting associations, the role for HPV in cSCC in patients with impaired humoral immunity requires further characterization. This is particularly important as we explore the role of vaccinations in reducing HPV-associated malignancies. Citation Format: Preeya Bhavsar, Mugahed Hamza, Sepideh Mehravaran, Qin He, Steven Tyring, Peter Rady, Bhuvaneswari Krishnan, Gustavo Rivero, Daniel N. Cohen, Iberia Romina Sosa. The contribution of human papilloma virus infection to cutaneous squamous cell carcinoma in patients with chronic lymphocytic leukemia [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 4898.

141 All-trans Retinoic Acid Inhibits Cell Proliferation through Upregulation of TET2 in Squamous Cell Cancer

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent non-melanoma skin cancer. All-trans retinoic acid (ATRA) has been previously proposed anti-tumor potential via inhibiting cancer cell growth by binding to the intracellular retinoic acid receptors (RAR). The ten-eleven translocation 2 (TET2) proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and loss-functions of TET2 leading to hypermethylation have been established in various cancers. However, its role in cSCC remains unknown. In the present study, we detected the expression of 5hmC, TET2 and retinoic acid receptor B (RARb) in normal skin, actinic keratosis (AK), Bowen disease and cSCC clinical samples by immunohistochemical staining. It’s significant downregulation of 5hmC and TET2 were observed in the AK, Bowen disease and cSCC tissues compared with normal skin, while expression of RARb was reduced. Human primary keratinocytes and cSCC cell lines (Scl-1 and HSC-1) were cultured in vitro, receiving stimulation of retinoic acid. The results showed that treatment with ATRA inhibited the proliferation of cSCC cell lines and increased the expressions of TET2 and RARb in cSCC cell lines. Moreover, ectopic overexpression of TET2 in cSCC cell lines induce upregulation of RARB while no obvious expression changes of TET2 were observed in RARB overexpressed cSCC cell lines. Our results suggest that TET2 and 5hmC were decreased in cSCC tissue and cSCC cell lines compared to normal tissues and human primary keratinocytes. In addition, ATRA promotes the expression of TET2 in cSCC cell lines, which is closely related to RARb. This study provides evidence that 5hmC and TET2 have the potentials to serve as biomarkers that identified presence and progression of cSCC. It identified a novel function of ATRA in promoting a TET-mediated epigenetic regulation suggesting that the availability of ATRA in cancer cells will have various effects on different epigenetic targets.

Validation of a 40-gene expression profile test to predict metastatic risk in localized high-risk cutaneous squamous cell carcinoma

Currentstaging systems for cutaneous squamous cell carcinoma (cSCC) have limited positive predictive value for identifying patients who will experience metastasis. To develop and validate a gene expression profile (GEP) test for predicting risk for metastasis in localized, high-risk cSCC with the goal of improving risk-directed patient management. Archival formalin-fixed paraffin-embedded primary cSCC tissue and clinicopathologic data (n=586) were collected from 23 independent centers in a prospectively designed study. A GEP signature was developed using a discovery cohort (n=202) and validated in a separate, nonoverlapping, independent cohort (n=324). A prognostic 40-GEP test was developed and validated, stratifying patients with high-risk cSCC into classes based on metastasis risk: class 1 (low risk), class 2A (high risk), and class 2B (highest risk). For the validation cohort, 3-year metastasis-free survival rates were 91.4%, 80.6%, and 44.0%, respectively. A positive predictive value of 60% was achieved for the highest-risk group (class 2B), an improvement over staging systems, and negative predictive value, sensitivity, and specificity were comparable to staging systems. Potential understaging of cases could affect metastasis rate accuracy. The 40-GEP test is an independent predictor of metastatic risk that can complement current staging systems for patients with high-risk cSCC.

AXIN2 and SNAIL expression predict the risk of recurrence in cutaneous squamous cell carcinoma after Mohs micrographic surgery.

Recurrence is a common complication observed during cutaneous squamous cell carcinoma (cSCC) treatment; however, biomarkers for predicting recurrence in cSCC remain unknown. The present study aimed to investigate the predictive value of axis inhibition protein 2 (AXIN2) and SNAIL expression in cSCC recurrence. AXIN2 and SNAIL expression was evaluated using immunohistochemistry in 111 cSCC tissue samples obtained from 18 patients who presented recurrence (recurrence interval, 1–91 months) and 93 patients who did not experience recurrence following Mohs micrographic surgery (MMS) during the follow-up period (156 months). Nomogram construction was performed using patients' clinicopathological characteristics and AXIN2 and SNAIL protein expression. The results demonstrated that high AXIN2 (histoscore >100) and SNAIL (histoscore >100) expression was detected in 35 and 44 cSCC tissues, respectively. Furthermore, the expression levels of AXIN2 and SNAIL were significantly associated in patients with cSCC (P=0.001). AXIN2 and SNAIL expression levels were significantly associated with tumor size (P=0.021 and P=0.044, respectively) and recurrence of cSCC (P=0.017 and P=0.042, respectively). In addition, the results of the Kaplan-Meier curve analysis revealed that recurrence-free survival was significantly associated with tumor size (P=0.025), differentiation status (P<0.001), AXIN2 expression (P=0.001) and SNAIL expression (P=0.001). Furthermore, the results of the multivariate analysis demonstrated that age (P=0.043), AXIN2 expression (P=0.001) and SNAIL expression (P=0.045) were independent risk factors for cSCC recurrence in the present cohort. A nomogram for predicting the 1-, 2-, 3-, and 5-year recurrence-free survival was developed for patients with cSCC by including independent risk factors with a concordance index of 0.75. The results suggested that high AXIN2 and SNAIL expression may be considered as potential risk factors for cSCC recurrence. This nomogram may therefore be useful to assess the probability of recurrence in patients with cSCC following MMS.