Invasive cSCC Research Articles

Fractal Dimension Analysis of the Tumor Microenvironment in Cutaneous Squamous Cell Carcinoma: Insights into Angiogenesis and Immune Cell Infiltration

The global incidence of cutaneous squamous cell carcinoma (cSCC), a prevalent and aggressive skin cancer, has risen significantly, posing a substantial public health challenge. This study investigates the tumor microenvironment (TME) of cSCC by focusing on the spatial distribution patterns of immune and vascular markers (CD31, CD20, CD4, and CD8) using fractal dimension (FD) analysis. Our analysis encompassed 141 cases, including 100 invasive cSCCs and 41 specimens with pre-invasive lesions exclusively, and the rest were peripheral pre-invasive lesions from the invasive cSCC class. The FD values for each marker were computed and compared between pre-invasive and invasive lesion classes. The results revealed significant differences in FD values between the two classes for CD20 and CD31 markers, suggesting distinct alterations in B cell distribution and angiogenic activity during cSCC progression. However, CD4 and CD8 markers did not exhibit significant changes individually. Still, the CD4/CD8 ratio showed a significant difference, suggesting a potential shift in the balance between T helper and cytotoxic T cell responses, impacting the immune landscape as lesions progressed from pre-invasive to invasive stages. These findings underscore the complexity and heterogeneity of the TME in cSCC and highlight the potential of FD analysis as a quantitative tool for characterizing tumor progression. Further research is needed to elucidate the implications of these differences in the clinical management of cSCC.

Epidemiologic trends in cutaneous squamous cell carcinoma from 2011 to 2021 at All Africa Leprosy, Tuberculosis, and Rehabilitation Training Center (ALERT) in Addis Ababa, Ethiopia

Cutaneous squamous cell carcinoma (cSCC) is the most common keratinocyte carcinoma in dark-skinned ethnic groups. Available studies are primarily focused on white populations, with fewer data available for black Africans, including Ethiopians. Therefore, less priority may be given to treatment and prevention in this population. To determine the prevalence and risk factors of cSCC among patients presenting to All Africa Leprosy, Tuberculosis, and Rehabilitation Training Center , Addis Ababa, Ethiopia. A literature review was performed to determine existing knowledge. A retrospective descriptive cross-sectional study was then conducted based on stored data of confirmed cases of cSCC from 2011 to 2021 at ALERT. Demographics, other parameters related to cSCC (size, anatomic location, degree of differentiation, metastasis, and recurrence), and potential risk factors including scars, burns, HIV status, xeroderma pigmentosum, and leprosy were collected and entered on a prepared data extraction sheet and analyzed using SPSS version 25 manufactured by IBM. Among 15,075 total pathologic samples reviewed, 3.8% (n=570) were reported histopathologically as invasive cSCC (n=437), keratoacanthoma, or (squamous cell carcinoma in-situ (Bowen disease). 50.3% (n=287) occurred in female, and the mean age of affected patients was 50.1years (SD 17.2). Nearly 70% were reported after >1year of symptoms, including morphologic change, ulceration, and nonhealing wound. HIV status was not universally screened but was positive in 9.8% (46/437). Cases were commonly found to occur on the lower extremities (X2=2.7196, Pr=0.099) and >4cm in size (46.3%). Among 274 cases with adequate histologic description, 82.5% (n=226) were well-differentiated. Loco-regional metastases comprised 6.0% (n=34) and 46.7% (n=266) had ulceration. The biologic significance of ulceration in this population is unclear. Ulceration may indicate chronic wounds or Marjolin's ulcers. High rates of metastasis call for a better understanding of risk factors, preventive measures, and early diagnosis. This model of research may serve as a foundation for future nationwide investigations through the Federal Ministry of Health of Ethiopia's Disease Prevention and Control Directorate.

Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. Cancer-associated fibroblasts (CAFs) promote progression of cancer, but their role in cSCC is largely unknown. We examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. We utilized multiplexed fluorescence immunohistochemistry for profiling CAF landscape in metastatic and non-metastatic primary human cSCCs, in metastases, and in premalignant epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and results were correlated with clinical and histopathological parameters including disease-specific mortality. Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression to invasive cSCC. Elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with metastasis risk of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Multimarker combination defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis, and independently predicted poor disease-specific survival. These results identify high PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ by CAFs as potential biomarkers for risk of metastasis and poor prognosis.

Epstein-Barr Virus DNA Is Associated With Conjunctival Squamous Cell Carcinomas: A Case-Control Study From Zimbabwe.

Incidence of conjunctival squamous cell carcinoma (cSCC) in Zimbabwe is >30-fold higher than the global average. cSCC risk is notably higher among people with human immunodeficiency virus, implicating impaired immune response and a yet unknown infectious etiology. Formalin-fixed, paraffin-embedded blocks from Zimbabwe, comprising conjunctival precancer (n = 78), invasive cSCC cases (n = 148) and nonmalignant eye lesions (n = 119), were tested for multiple DNA viruses using Luminex bead-based technology. Epstein-Barr virus (EBV) type 1 positivity was strongly associated with cSCC diagnosis (adjusted odds ratio [aOR], 5.6 [95% confidence interval {CI}, 3.0-10.4) and marginally associated with precancer (aOR, 2.1 [95% CI, 1.0-4.5]). On analyzing EBV transcriptional activity with any of LMP1, EBNA1, and BZLF1, RNA transcripts were detected in 5 of 112 controls, 3 of 67 precancers, and 10 of 139 cases and none were associated with conjunctival case status. Our EBV DNA data suggest that EBV may play a role in cSCC. However, the low detection rate of EBV RNA supports further investigation to infer causality.

Cutaneous Squamous Cell Carcinoma: Clinico-Dermoscopic and Histological Correlation: About 72 Cases.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer, accounting for 20% of malignant skin tumors. Dermoscopy is a very useful tool for diagnosing cSCC, and its findings are confirmed through histopathological studies. to describe the different dermoscopic structures of invasive cSCC and investigate their association with the clinical form and histopathological grade of differentiation. We conducted a cross-sectional study, collecting all patients diagnosed with squamous cell carcinoma over a period of 5 years. The study population was divided into two groups based on histological differentiation (well-differentiated and non-well-differentiated) and clinical form ( nodulo-ulcerative and nodular). Various dermoscopic parameters were compared between these groups. Out of 72 invasive cSCC, 81.9% were well-differentiated, while 18.1% were non-well-differentiated. The clinical form of cSCC was nodulo-ulcerative in 83.3% of cases and nodular in 16.7%. Well-differentiated tumors showed dotted, glomerular and hairpin vessels, along with a predominant white pattern characterized by centrally distributed keratin as well as white circles, and whitish perivascular halo (P < 0.05). The distribution of these white structures was radial in nodulo-ulcerarive lesions, whereas in nodular lesions, their distribution, as well as that of keratin, was more diffuse (P < 0.05). Non-well-differentiated tumors showed a combined white-red pattern with the predominance of arborizing vessels (P < 0.05). Our results show the reliability of dermoscopy as a tool for distinguishing between well- and poorly differentiated cSCC. This distinction is characterized by an increase in predominantly arborizing vessels and a corresponding decrease in white structures as the tumor progresses from a well-differentiated to a poorly differentiated state. Additionally, the nodulo-ulcerative form exhibits a central distribution of keratin, while the nodular form displays a diffuse distribution.

Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is the second most frequent of the keratinocyte-derived malignancies with actinic keratosis (AK) as a precancerous lesion. To comprehensively delineate the underlying mechanisms for the whole progression from normal skin to AK to invasive cSCC, we performed single-cell RNA sequencing (scRNA-seq) to acquire the transcriptomes of 138,982 cells from 13 samples of six patients including AK, squamous cell carcinoma in situ (SCCIS), cSCC, and their matched normal tissues, covering comprehensive clinical courses of cSCC. We identified diverse cell types, including important subtypes with different gene expression profiles and functions in major keratinocytes. In SCCIS, we discovered the malignant subtypes of basal cells with differential proliferative and migration potential. Differentially expressed genes (DEGs) analysis screened out multiple key driver genes including transcription factors along AK to cSCC progression. Immunohistochemistry (IHC)/immunofluorescence (IF) experiments and single-cell ATAC sequencing (scATAC-seq) data verified the expression changes of these genes. The functional experiments confirmed the important roles of these genes in regulating cell proliferation, apoptosis, migration, and invasion in cSCC tumor. Furthermore, we comprehensively described the tumor microenvironment (TME) landscape and potential keratinocyte-TME crosstalk in cSCC providing theoretical basis for immunotherapy. Together, our findings provide a valuable resource for deciphering the progression from AK to cSCC and identifying potential targets for anticancer treatment of cSCC.

Cutaneous squamous cell carcinoma of the eyelid in northern latitudes, a 25-year experience in Finland.

To evaluate the incidence, clinical features, diagnostic challenges, management and prognosis of cutaneous squamous cell carcinoma of the eyelid (ecSCC) in southern Finland, northern Europe, latitude 62° N. Patients were identified from the Finnish Cancer Registry and the Helsinki University Hospital databases during a 25-year period (1998-2022). Age, sex, location, clinical and histopathological diagnosis, treatment and outcome were retrieved. Cutaneous squamous cell carcinoma of the eyelid (ecSCC) was diagnosed in 58 patients. The mean age-standardized incidence was 1.03 per 100 000. Median age at the time of histopathological diagnosis was 79 (range 55-93) years; sex ratio was 0.52. Clinical diagnosis in the referral was ecSCC in only three patients. The most frequent misdiagnosis (38%) was basal cell carcinoma (BCC). One or more of the known risk factors (smoking, history of extensive sun exposure, systemic immunosuppression and previous insitu cSCC/cSCC) were documented in 71% of the patients. More than one third (38%) of the patients developed insitu SCC elsewhere on the skin; one third (31%) of the patients had invasive cSCC elsewhere. During the median follow-up time of 24 months, three patients experienced local recurrence, four patients developed metastatic disease (median 19 months) and two patients died of metastatic ecSCC. The estimated incidence of ecSCC in Finland (predominantly white Caucasian) was higher than in a previous study from Europe. Clinical diagnosis of ecSCC is difficult and often misdiagnosed as BCC. Immunosuppression as a risk factor should noticed. Recurrences of ecSCC, which may be lethal, were infrequent.

Topical Treatment of Cutaneous Squamous Cell Carcinoma in Situ and the Impact of Clinical Risk Factors and Positive Histologic Margins at the Time of Diagnosis.

Limited data exist for the efficacy of topical 5-fluorouracil (5-FU) and imiquimod for cutaneous squamous cell carcinoma (cSCC) in situ (cSCCis) with positive histologic margins at the time of diagnosis. Identify the efficacy of topical 5-FU and imiquimod in the treatment of cSCCis with positive histologic margins at the time of diagnosis in relation to clinical risk factors. Pathology records were screened at a single institution from 2014 to 2021 for cSCCis with positive histologic margins. Patients were included if they were treated with curative intent with topical 5-FU or imiquimod. Recurrences were evaluated in relation to multiple clinical risk factors. Of 215 patients treated with 5-FU or imiquimod after biopsy-proven cSCCis, 19 patients had recurrent cSCCis and 1 patient had upstaging to invasive cSCC. Recurrence was more likely in larger lesions at the time of initial biopsy ( p = .033) and in patients treated with topical imiquimod compared with topical 5-FU ( p < .01). Topical 5-FU is an appropriate therapy for cSCCis in the correct clinical scenario. Extra consideration should be taken for use of 5-FU in larger diameter cSCCis lesions. Although limited by sample size, our study does not support the use of imiquimod for cSCCis.

Abstract PO-074: The evolution of the tumor immune microenvironment in immunosuppressed patients with cutaneous squamous cell carcinoma

Abstract Immunosuppression is a major risk and prognostic factor for cutaneous squamous cell carcinoma (cSCC). Immune checkpoint inhibition is approved for the treatment of cSCC, yet most immunosuppressed patients are ineligible for immunotherapy. Better understanding on the tumor microenvironment of cSCC in the context of immunosuppression (i.e., organ transplant recipients, patients with hematological malignancies or autoimmune diseases or acquired immunodeficiencies such as HIV) is of utmost importance for proper implementation of immunotherapy as a therapeutic alternative for these patients. In this study, we characterized the immune cell phenotype of cSCC from immunocompetent (IC) and immunosuppressed (IS) patients treated at two Cancer Centers. We also compared the immune phenotype changes through the steps of skin carcinogenesis between IC and IS patients, and among individuals with distinct causes for immunosuppression. To evaluate the TME dynamics during tumorigenesis, we analyzed specimen of normal epithelium, keratinocytic intraepidermal neoplasia (including carcinoma in situ and actinic keratosis), or invasive cSCC. The analysis of cSCC tumor microenvironment was performed by multiplex immunofluorescence (Opal 12-Color), CyTOF and single-cell RNA sequencing. Our analysis revealed that densities of CD68+ and effector T-cells are diminished in IS patients compared with IC. An analysis of precursor lesions revealed that the density of tumoral CD8+LAG3+ T-cells in patients with hematological malignancies was significantly lower than in patients with other types of immunosuppression. Compared with patients with other types of immunosuppression, patients with hematological malignancies had a higher density of stromal CD4+ T cells in precursor lesions but a lower density of CD20+ B cells. Overall, IS patients had worse outcomes than IC patients, and the densities of CD68+ and CD8+LAG3+ cells were prognostic in this cohort. CD8+LAG3+ was significantly associated with DSS of IS patients indicating the impact of dysfunctional cytotoxic T cells on these patients’ survival. Our findings provide a detailed insight into the underlying immune alterations from tumor inception through growth and invasion, in patients with different types of immunosuppression. This dataset will serve as a benchmark data to guide clinical trials aiming to treat this patient population and provide a potential rationale for designing safer immunotherapy clinical trials for the population of IS patients with cSCC or other head and neck cancers. Citation Format: Mica D. E. Glaun, Frederico O. Gleber-Netto, Priyadharsini Nagarajan, Tongxin Xie, Shamima Akhter, Yu Han Chen, Erez Baruch N. Baruch, Michael K. Wong, Emily Y. Chu, Kenneth Y. Tsai, Elsa R. Flores, Deborah A. Silverman, Ryan P. Goepfert, Murat Cobanoglu, Jared Burks, Padmanee Sharma, James P. Allison, Jeffrey N. Myers, Neil D. Gross, Moran Amit. The evolution of the tumor immune microenvironment in immunosuppressed patients with cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-074.

Abstract 2101: Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma

Abstract Purpose: Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer and the metastatic disease is associated with poor prognosis. At present, no established biomarkers are available for predicting the metastasis risk and prognosis of patients with cSCC. Cancer-associated fibroblasts (CAFs) promote the progression of cancer, but their role in cSCC is largely unknown. Here, we have examined the potential of CAF markers in the assessment of metastasis risk and prognosis of primary cSCC. Experimental Design: We utilized multiplexed fluorescence immunohistochemistry for profiling of CAF landscape in metastatic and non-metastatic primary human cSCCs, the actual metastases, and in premalignant and benign epidermal lesions. Quantitative high-resolution image analysis was performed with two separate panels of antibodies for CAF markers and the results were correlated with clinical and histopathological parameters including disease-specific mortality. Results: Increased stromal expression of fibroblast activation protein (FAP), α-smooth muscle actin, and secreted protein acidic and rich in cysteine (SPARC) were associated with progression of premalignant epidermal lesions to invasive cSCC. Furthermore, elevation of FAP and platelet-derived growth factor receptor-β (PDGFRβ) expression was associated with the risk of metastasis of primary cSCCs. High expression of PDGFRβ and periostin correlated with poor prognosis. Furthermore, a multi-marker defined CAF subset, PDGFRα-/PDGFRβ+/FAP+, was associated with invasion and metastasis and independently predicted poor disease-specific survival. Conclusions: These results show, that fibroblast activation evolves during the progression of cSCC. High PDGFRβ expression alone and multimarker combination PDGFRα-/PDGFRβ+/FAP+ are potential biomarkers for the progression of primary cSCC to metastatic stage, and for poor prognosis. Citation Format: Jaakko S. Knuutila, Pilvi Riihilä, Liisa Nissinen, Roosa Kallionpää, Teijo Pellinen, Veli-Matti Kähäri. Cancer-associated fibroblast activation predicts progression, metastasis, and prognosis of cutaneous squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2101.

High-risk mucosal human papillomavirus-associated squamous cell carcinoma of the fingers presents distinctive clinicopathological characteristics and outcomes: a bicentric retrospective study.

High-risk mucosal human papillomavirus (HR-HPV) seems to play a role in cutaneous squamous cell carcinoma (cSCC), particularly in nail tumours, where genitodigital transmission has been suggested. The role of HR-HPV in nonungual cSCC of the finger needs to be clarified. To evaluate the prevalence, clinicopathological characteristics, surrogates and outcomes of HR-HPV in cSCC of the finger. This was an observational bicentric study including patients with an excised in situ or invasive cSCC located on the finger. Differences in HR-HPV and non-HR-HPV tumours were evaluated. Forty-five patients (45 tumours) were included. HR-HPV was detected in 33% of cases (22% HPV type 16). The mean age was lower in patients with HR-HPV than in those with non-HR-HPV (62·4 vs. 81·1 years, P = 0·001). HR-HPV tumours were smaller (10 mm vs. 15 mm, P = 0·07) and more frequently intraepidermal (60% vs. 20%, P = 0·004). The absence of elastosis (P = 0·030) and inflammation (P = 0·026) and the presence of basaloid morphology (P = 0·003) were surrogates of HR-HPV detection. Mean p16 positivity was 61% in HR-HPV and 36% in non-HR-HPV tumours (P = 0·061). Recurrence after surgery was more common in HR-HPV tumours (58% vs. 34%), although this was not statistically significant. HR-HPV was detected in 27% of the nonungual tumours. HR-HPV-associated cSCC of the finger appears in younger patients, is smaller and is less infiltrative than non-HR-HPV tumours. The presence of a basaloid morphology and the absence of elastosis and inflammation could be used as markers for HR-HPV detection. The high prevalence of HR-HPV in nonungual cSCC suggests its aetiopathogenic role in these tumours.

In transit metastases of cutaneous squamous cell carcinoma: A single institution case series

To the Editor: In transit metastasis of cutaneous squamous cell carcinoma (cSCC), defined as foci of cSCC originating within dermal or subcutaneous tissue, clinically distinct from the primary tumor, and occurring before the first echelon of regional lymph nodes, is a rare entity and manifestation of end-stage disease. Immunosuppressed patients, specifically solid organ transplant recipients, are at an increased risk of invasive and metastatic cSCC. Since 2004, there have been 3 published case series describing in transit metastasis of cSCC including a total of 124 patients. 1 Carucci J.A. Martinez J.C. Zeitouni N.C. et al. In-transit metastasis from primary cutaneous squamous cell carcinoma in organ transplant recipients and nonimmunosuppressed patients: clinical characteristics, management, and outcome in a series of 21 patients. Dermatol Surg. 2004; 30: 651-655 Crossref PubMed Google Scholar , 2 Ma J.H.Y. Wu A. Veness M. et al. In-transit metastasis from squamous cell carcinoma. Dermatol Surg. 2016; 42: 1285-1292 Crossref PubMed Scopus (14) Google Scholar , 3 Smile T.D. Ruiz E.S. Kus K.J.B. et al. Implications of satellitosis or in-transit metastasis in cutaneous squamous cell carcinoma: a prognostic omission in cancer staging systems. JAMA Dermatol. 2022; 158: 390-394 Crossref Scopus (0) Google Scholar Since the first 2 case series, therapy has shifted to multimodal treatment. 1 Carucci J.A. Martinez J.C. Zeitouni N.C. et al. In-transit metastasis from primary cutaneous squamous cell carcinoma in organ transplant recipients and nonimmunosuppressed patients: clinical characteristics, management, and outcome in a series of 21 patients. Dermatol Surg. 2004; 30: 651-655 Crossref PubMed Google Scholar ,2 Ma J.H.Y. Wu A. Veness M. et al. In-transit metastasis from squamous cell carcinoma. Dermatol Surg. 2016; 42: 1285-1292 Crossref PubMed Scopus (14) Google Scholar We performed a retrospective review of cSCC patients with in transit metastasis from 2015 to 2021 at a single academic institution.

Relationship between PD-L1 expression and prognostic factors in high-risk cutaneous squamous and basal cell carcinoma.

This study aimed to investigate the programmed cell death-ligand 1 (PD-L1) expression in cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) and its relationship with prognostic factors in tumors that are not in the head and neck region and are therefore relatively less exposed to the sun. This retrospective cross-sectional study included 25 invasive cSCC and 42 BCC cases with a diameter ≥ 2 cm located outside the head and neck region from 2010 to 2018. The biopsy samples were examined based on the membranous PD-L1 (22C3 clone) staining. Staining results were scored as follows: 0, no staining (negative); 1, < 10% PD-L1 positivity of tumor cells; and 2, ≥ 10% PD-L1 positivity of tumor cells. PD-L1 positivity was not seen in any BCC cases, whereas 11 (44%) of cSCC cases were PD-L1 positive. No significant relationship was observed between PD-L1 expression and prognostic parameters, including tumor diameter, tumor depth, and lymphovascular or perineural invasion in the cSCC group. PD-L1 expression was not associated with prognostic factors in the early stages of BCC and SCC located outside the head and neck region. Therefore, investigating the PD-L1 expression seems to be more relevant in patients with advanced-stage disease.

A Hybrid Epithelial to Mesenchymal Transition in Ex Vivo Cutaneous Squamous Cell Carcinoma Tissues.

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, ZEB1, SNAI1, MMPs, vimentin, and E-cadherin are hallmarks of an EMT process occurring within cancer cells, including cSCC cells, EMT within tissues is not an “all or none” process. Using patient-derived cSCC and adjacent normal tissues, we show that cells within individual cSCC tumors are undergoing a hybrid EMT process, where there is variation in expression of EMT markers by cells within a tumor mass that may be facilitating invasion. Interestingly, cells along the outer edges of a tumor mass exhibit a more mesenchymal phenotype, with reduced E-cadherin, β-catenin, and cytokeratin expression and increased vimentin expression. Conversely, cells in the center of a tumor mass retain a higher expression of the epithelial markers E-cadherin and cytokeratin and little to no expression of vimentin, a mesenchymal marker. We also detected inverse expression changes in the miR-200 family and the EMT-associated transcription factors ZEB1 and SNAI1, suggesting that cSCC EMT dynamics are regulated in a miRNA-dependent manner. These novel findings in cSCC tumors provide evidence of phenotypic plasticity of the EMT process occurring within patient tissues, and extend the characterization of a hybrid EMT program occurring within a tumor mass. 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.

Recent Advances in the Diagnosis and Management of High-Risk Cutaneous Squamous Cell Carcinoma.

Simple SummaryCutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer. Although most cSCCs are effectively treated with surgery, there are some tumors at higher risk for relapse, progression to nodal metastasis, or, rarely, death. An important issue concerns the early diagnosis and appropriate treatment of these few high-risk cSCCs, as they are associated with poorer prognosis and may more frequently progress to advanced cSCCs. This review discusses the characteristics of high-risk cSCC and how to identify and manage patients with these tumors.High-risk cSCC is defined as invasive cSCC staged as N0 (without detectable regional lymph nodes) and M0 (without distant metastasis), that has features associated with a higher risk of poorer prognosis. The focus of this review is on the recent advances in the diagnosis and management of high-risk cSCC. The interest in high-risk cSCC relies on its higher risk of progression to advanced cSCC, as it represents the main pool of cSCCs that give rise to advanced tumors. Assessment of the risk is thus particularly relevant for common cSCC to identify the few with a high-risk risk of local recurrence, metastasis, or disease-specific death among all other low-risk tumors. The timely diagnosis and effective treatment of high-risk cSCCs may halt their further progression and aim to prevent and lower the incidence of advanced cSCCs. Clearance of the tumor with negative surgical margins is the main goal of surgery, which is the primary treatment of cSCC. It seems that it is difficult to discern the group of high-risk cSCCs that may benefit from adjuvant RT, as a universal beneficial effect for a cSCC with any high-risk factor which was resected with clear surgical margins has not been established. In the case of a high-risk cSCC with positive margins after surgery, and re-excision not feasible, post-operative radiotherapy is performed when possible. Recommendations on further management are discussed. Regarding the follow-up of patients diagnosed with high-risk cSCC, factors to consider regarding the frequency and intensity of the follow-up schedule include the risk and possible time of occurrence of metastasis from 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.

A comparison of invasive squamous cell carcinoma greater than 1 year after treatment with 5-fluorouracil, imiquimod, or photodynamic therapy with aminolevulinic acid

Studies comparing 5-fluorouracil (5-FU), imiquimod, and photodynamic therapy with aminolevulinic acid (PDT-ALA) have evaluated the efficacy of destroying actinic keratosis (AK). However, this end point may not directly translate to cutaneous squamous cell carcinoma (cSCC) prevention. No study to date has evaluated these field therapies for cSCC prevention in the long term, defined as greater than 1-year posttreatment. Determine the time to surgically treat invasive cSCC development after treatment with 5-FU, imiquimod, or PDT-ALA beginning 1-year posttreatment. Retrospective cohort study using the Optum Clinformatics Data Mart database from 2012 to 2019 RESULTS: The rate of cSCC development in patients treated with 5-FU showed no significant difference compared with imiquimod (0.99; 95% CI, 0.90-1.08). PDT-ALA was worse than 5-FU (1.27; 95% CI, 1.19-1.36) and imiquimod (HR, 1.29; 95% CI, 1.17-1.43). Other known predictors of cSCC were consistent with previous literature. The location of field therapy could not be determined with a claims database. 5-FU is not superior to imiquimod beginning 1year posttreatment, despite previously demonstrated superior AK destruction efficacy, but was superior to PDT-ALA. Conflating AK destruction and cSCC prevention efficacy may not be appropriate. Future prospective studies should aim to use an end point of cSCC development.

Histopathologic features predictive of metastasis and survival in 230 patients with cutaneous squamous cell carcinoma of the head and neck and non-head and neck locations: a single-center retrospective study.

Staging systems for cutaneous squamous cell carcinoma (cSCC) produce inconsistent risk stratification. The aim of this study was to identify further prognostic parameters for better stratification. We retrospectively analysed the prognostic significance of clinicopathologic parameters of 230 patients who underwent primary excision of invasive cSCC of the head and neck (n = 115) and non-head and non-neck (n = 115) locations. In addition to known high-risk features, we analysed tumour nest shape, invasion pattern, lymphoid response pattern and tumour budding. On multivariable analysis, lymphovascular invasion (LVI) and high tumour budding predicted worse disease-specific survival, and ulceration, LVI and high tumour budding predicted worse overall survival. Only ulceration was independently associated with risk of nodal metastasis. High tumour budding, LVI and ulceration are independently associated with poor outcome in cSCC and may be used to refine cSCC prognostic stratification, which is crucial to optimize clinical decision and to identify patients who are more likely to benefit from more aggressive interventions or clinical trials.

Risk of Invasive Cutaneous Squamous Cell Carcinoma After Different Treatments for Actinic Keratosis

Treatment of actinic keratosis (AK) aims to prevent cutaneous squamous cell carcinoma (cSCC). However, whether AK can progress into invasive cSCC is a matter of debate, and little is known about the effect of treatment on preventing cSCC. To evaluate the risk of invasive cSCC and factors that may contribute to increased risk in patients with multiple AKs. In this secondary analysis of a multicenter randomized clinical trial, 624 patients with a minimum of 5 AKs within an area of 25 to 100 cm2 on the head were recruited from the Department of Dermatology of 4 hospitals in the Netherlands. Long-term follow-up was performed from July 1, 2019, to December 31, 2020. Patients were randomized to treatment with 5% fluorouracil, 5% imiquimod cream, methylaminolevulinate photodynamic therapy, or 0.015% ingenol mebutate gel. The primary outcome was the proportion of patients with invasive cSCC in the target area during follow-up. Secondary outcomes were the associations between risk of invasive cSCC and a priori defined potential prognostic factors, including type of treatment, severity of AK (Olsen grade), history of nonmelanoma skin cancer, and additional treatment. Of the 624 patients (558 [89.4%] male; median age, 73 years [range, 48-94 years]) in the study, 26 were diagnosed with a histologically proven invasive cSCC in the target area during follow-up. The total 4-year risk of developing cSCC in a previously treated area of AK was 3.7% (95% CI, 2.4%-5.7%), varying from 2.2% (95% CI, 0.7%-6.6%) in patients treated with fluorouracil to 5.8% (95% CI, 2.9%-11.3%) in patients treated with imiquimod. In patients with severe AK (Olsen grade III), the risk was 20.9% (95% CI, 10.8%-38.1%), and the risk was especially high (33.5%; 95% CI, 18.2%-56.3%) in patients with severe AK who needed additional treatment. In this secondary analysis of a randomized clinical trial, risk of invasive cSCC was highest in patients with Olsen grade III AK and was substantially increased in patients who received additional treatment. These patients should be closely followed up after treatment. ClinicalTrials.gov Identifier: NCT02281682.

PD-L1 blockade potentiates the antitumor effects of ALA-PDT and optimizes the tumor microenvironment in cutaneous squamous cell carcinoma

ABSTRACT Immune checkpoint blockade (ICB) is a powerful oncologic treatment modality for a wide variety of human malignancies, but the patient response rate to this treatment remains low, especially in patients with cutaneous squamous cell carcinoma (cSCC). 5-Aminoleuvulinic acid-photodynamic therapy (ALA-PDT) is widely used to treat cancerous and precancerous skin diseases, but the value of ALA-PDT in the treatment of invasive cSCC is debatable. Our previous studies have shown that ALA-PDT can induce antitumor immune responses by promoting the immunogenic death of tumor cells. However, it is unclear whether ALA-PDT exerts synergistic effects with ICB in cSCC. Here, we report that PD-L1 blockade potentiates the antitumor effects of ALA-PDT both on primary and distant tumors, and optimizes the tumor microenvironment in cSCC. In this study, we first detected PD-L1 expression in patients with different grades of cSCC. Then we found the combination of anti-PD-L1 monoclonal antibody (mAb) and ALA-PDT killed tumor cells by apoptosis- and/or ferroptosis-mediated immunogenic cell death (ICD) and stimulated systemic immune response, as well as building the immunological memory response to prevent tumor recurrence. Furthermore, we found that combination therapy can be used to recruit tertiary lymphoid structure (TLS)-like intratumoral lymphoid aggregates, which may promote tumor-infiltrating lymphocyte (TIL)-mediated antitumor immunity. In summary, our work demonstrates that ICB treatment with an anti-PD-L1 antibody is a promising strategy that may potentiate the antitumor effects of ALA-PDT in cSCC.