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.

Non-Invasive Diagnosis of Sun Damaged Skin: Actinic Keratosis Vs Squamous Cell Carcinoma.

IMPORTANCE: Actinic keratosis (AK) is a premalignant lesion that has a1% to 10% potential of progression to squamous cell carcinoma (SCC), but it is not possible to determine which lesions are at higher risk. OBJECTIVE: This study examined the epidermal genetic profiles of actinic keratosis and SCC through non-invasive techniques seeking to develop a biopsy-free method for AK monitoring and aid in the early diagnosis of developing SCC. DESIGN: Ribonucleic acid (RNA) was collected from adhesive tape strips and gene expression levels were measured. A threshold fold change >2 and adjusted P-value <0.05 were used to determine differentially expressed genes. SETTING: Single center dermatology clinic. PARTICIPANTS: Patients who presented to the clinic with lesions suspicious of non-melanoma skin cancer that had never been previously biopsied. MAIN OUTCOME AND MEASURE: RNA was extracted via non-invasive biopsy and sequenced. Low quality samples were filtered out and the remaining samples underwent differential gene expression analysis by DESeq2 in R package. A threshold of fold change >2 and adjusted P-value <0.05 was used for determination of differentially expressed genes. The differentially expressed genes that overlapped between the corrected and uncorrected groups were the most significant for analysis. RESULTS: From 47 lesions, 6 significant differentially expressed genes were found between AK and SCC, and 25 significant differentially expressed genes between in-situ SCC and invasive SCC. Individual samples showed similarities based on diagnosis, suggesting mutations were specific to the disease and not the individual. CONCLUSIONS AND RELEVANCE: These findings highlight which genes may play a role in AK progression to SCC. The genomic differences between in-situ and invasive squamous cell carcinoma open an opportunity for early diagnosis of squamous cell carcinoma and risk prediction of actinic keratosis. J Drugs Dermatol. 2023;22(5): doi:10.36849/JDD.7097.

Transcriptomic Study on Human Skin Samples: Identification of Two Subclasses of Actinic Keratoses.

Actinic keratoses (AKs) are sun-damaged skin areas that affect 20% of the European adult population and more than 50% of people aged 70 years and over. There are currently no clinical or histological features allowing us to identify to which clinical class (i.e., regression or progression) an AK belongs. A transcriptomic approach seems to be a robust tool for AK characterization, but there is a need for additional studies, including more patients and elucidating the molecular signature of an AK. In this context, the present study, including the largest number of patients to date, is the first aiming at identifying biological features to objectively distinguish different AK signatures. We highlight two distinct molecular profiles: AKs featuring a molecular profile similar to squamous cell carcinomas (SCCs), which are called "lesional AKs" (AK_Ls), and AKs featuring a molecular profile similar to normal skin tissue, which are called "non-lesional AKs" (AK_NLs). The molecular profiles of both AK subclasses were studied, and 316 differentially expressed genes (DEGs) were identified between the two classes. The 103 upregulated genes in AK_L were related to the inflammatory response. Interestingly, downregulated genes were associated with keratinization. Finally, based on a connectivity map approach, our data highlight that the VEGF pathway could be a promising therapeutic target for high-risk lesions.

Significant Association of Poly-A and Fok1 Polymorphic Alleles of the Vitamin D Receptor with Vitamin D Serum Levels and Incidence of Squamous Cutaneous Neoplasia.

Vitamin D3, a prohormone, is converted to circulating calcidiol and then to calcitriol, the hormone that binds to the vitamin D receptor (VDR) (a nuclear transcription factor). Polymorphic genetic sequence variants of the VDR are associated with an increased risk of breast cancer and melanoma. However, the relationship between VDR allelic variants and the risk of squamous cell carcinoma and actinic keratosis remains unclear. We examined the associations between two VDR polymorphic sites, Fok1 and Poly-A, and serum calcidiol levels, actinic keratosis lesion incidence, and the history of cutaneous squamous cell carcinoma in 137 serially enrolled patients. By evaluating the Fok1 (F) and (f) alleles and the Poly-A long (L) and short (S) alleles together, a strong association between genotypes FFSS or FfSS and high calcidiol serum levels (50.0 ng/ml) was found; conversely, ffLL patients showed very low calcidiol levels (29.1 ng/ml). Interestingly, the FFSS and FfSS genotypes were also associated with reduced actinic keratosis incidence. For Poly-A, additive modeling showed that Poly-A (L) is a risk allele for squamous cell carcinoma, with an OR of 1.55 per copy of the L allele. We conclude that actinic keratosis and squamous cell carcinoma should be added to the list of squamous neoplasias that are differentially regulated by the VDR Poly-A allele.

Factors for risk stratification of patients with actinic keratosis using integrated analysis of clinicopathological features and gene expression patterns.

BACKGROUND: Actinic keratosis (AK) is considered as precursor lesion of invasive squamous cell carcinoma. Molecular studies on AK are limited because of too small size of the biopsy specimen to obtain enough DNA or RNA. METHODS: Twenty biopsy cases of AK, followed by second same-sited biopsies, were included. Ten cases were diagnosed with total regression (regression group), while the other 10 were diagnosed with invasive carcinoma (progression group) in the follow-up biopsies. Using digital spatial profiling (DSP) technology, whole-gene expression analysis defined by specific regions of interest was performed for all 20 cases. After the clinicopathological features were assessed, separate and integrated analyses of these features and gene expression patterns were performed using machine-learning technology. All analyses were performed on both lesion keratinocytes (KT) and infiltrated stromal lymphocytes (LC). RESULTS: Among the 18,667 genes assessed, 33 and 72 differentially expressed genes (DEGs) between the regression and progression groups were found in KT and LC respectively. The primary genes distinguishing the two groups were KRT10 for KT and CARD18 for LC. Clinicopathological features were weaker in risk stratification of AK progression than the gene expression patterns. Pathways associated with various cancers were upregulated in the progression group of KT, whereas the nucleotide-binding oligomerization domain (NOD)-like receptor signalling pathway was upregulated in the progression of LC. CONCLUSION: Gene expression patterns were effective for risk stratification of AK progression, and their distinguishing power was higher than that of clinicopathological features.

Targeted deep sequencing reveals genomic alterations of actinic keratosis/cutaneous squamous cell carcinoma in situ and cutaneous squamous cell carcinoma.

Actinic keratosis (AK) and cutaneous squamous cell carcinoma in situ (CIS) are two of the most common precursors of cutaneous squamous cell carcinoma (cSCC). However, the genomic landscape of AK/CIS and the drivers of cSCC progression remain to be elucidated. The aim of our study was to investigate the genomic alterations between AK/CIS and cSCC in terms of somatic mutations and copy number alterations (CNAs). We performed targeted deep sequencing of 160 cancer-related genes with a median coverage of 515× for AK (N = 9), CIS (N = 9), cSCC lesions (N = 13), and matched germline controls from 17 patients. cSCC harboured higher abundance of total mutations, driver mutations and CNAs than AK/CIS. Driver mutations were found in TP53 (81%), NOTCH1 (32%), RB1 (26%) and CDKN2A (19%). All AK/CIS and cSCC lesions (93.5%), except two, harboured TP53 or NOTCH1 mutations, some of which were known oncogenic mutations or reported mutations in normal skin. RB1 driver mutations were found in CIS/cSCC (36.4%) but not in AK. CDKN2A driver mutations were found more frequently in cSCC (30.8%) than in AK/CIS (11.1%). Among recurrent (≥3 samples) CNAs (gain in MYC and PIK3CA/SOX2/TP63; loss in CDKN2A and RB1), MYC (8q) gain and CDKN2A (9p) loss were more frequently detected in cSCC (30.8%) than in AK/CIS (11.1%). Ultraviolet was responsible for the majority of somatic mutations in both AK/CIS and cSCC. Our study revealed that AK/CIS lesions harbour prevalent TP53 or NOTCH1 mutations and that additional somatic mutations and CNAs may lead to cSCC progression in AK/CIS lesions.

Genome-wide association study of actinic keratosis identifies new susceptibility loci implicated in pigmentation and immune regulation pathways.

Actinic keratosis (AK) is a common precancerous cutaneous neoplasm that arises on chronically sun-exposed skin. AK susceptibility has a moderate genetic component, and although a few susceptibility loci have been identified, including IRF4, TYR, and MC1R, additional loci have yet to be discovered. We conducted a genome-wide association study of AK in non-Hispanic white participants of the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (n = 63,110, discovery cohort), with validation in the Mass-General Brigham (MGB) Biobank cohort (n = 29,130). We identified eleven loci (P < 5 × 10-8), including seven novel loci, of which four novel loci were validated. In a meta-analysis (GERA + MGB), one additional novel locus, TRPS1, was identified. Genes within the identified loci are implicated in pigmentation (SLC45A2, IRF4, BNC2, TYR, DEF8, RALY, HERC2, and TRPS1), immune regulation (FOXP1 and HLA-DQA1), and cell signaling and tissue remodeling (MMP24) pathways. Our findings provide novel insight into the genetics and pathogenesis of AK susceptibility.

Genomic Progression of Precancerous Actinic Keratosis to Squamous Cell Carcinoma.

The mechanism underlying the progression of actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) in situ (SCCIS) to SCC remains unclear. To investigate this, we performed regional microdissection and targeted deep sequencing in SCC (n = 10) and paired adjacent sun-damaged epidermis (SE)/AK/SCCIS (n = 13) samples to detect mutations and copy number alterations. Most (11/13) SE/AK/SCCIS tissues harbored ≥1 driver alterations, indicating their precancerous nature. All pairs except one showed genome architectures representing the genomic progression of SE/AK/SCCIS to SCC with common trunks and unique branches (seven parallel and five linear progression cases). SE/AK/SCCIS tissues tended to harbor lower mutation/copy number alteration burdens than SCC tissues, but most of them had driver mutations, including NOTCH1 and TP53 mutations. SCC-specific genomic alterations included TP53, PIK3CA, FBXW7, and CDKN2A mutations and an MYC copy number gain, but they were heterogeneous among cases, suggesting that a single gene or pathway does not explain the progression of AK to SCC. In multiregion analyses of AK lesions, only some AK samples were related to SCC. In conclusion, the SE/AK/SCCIS genomes may have previously acquired truncal driver alterations, such as NOTCH1 and TP53 mutations, which promote parallel or linear progression to SCC on an acquisition of additional genomic alterations.

Clarifying Progress on the Genomic Landscape of Actinic Keratosis.

Most cutaneous squamous cell carcinomas (cSCCs) arise from actinic keratoses (AKs), making these premalignant lesions attractive targets for therapeutic intervention before transformation. In a new article of the Journal of Investigative Dermatology, Thomson et al. (2021) characterize the genetic alterations in AKs and identify significantly mutated drivers associated with risk factors such as UVR or azathioprine along with signaling pathways that may regulate the progression from AK to cSCC.

Transcriptomic analysis identifies differences in gene expression in actinic keratoses after treatment with imiquimod and between responders and non responders.

The presence of actinic keratoses (AKs) increases a patient's risk of developing squamous cell carcinoma by greater than six-fold. We evaluated the effect of topical treatment with imiquimod on the tumor microenvironment by measuring transcriptomic differences in AKs before and after treatment with imiquimod 3.75%. Biopsies were collected prospectively from 21 patients and examined histologically. RNA was extracted and transcriptomic analyses of 788 genes were performed using the nanoString assay. Imiquimod decreased number of AKs by study endpoint at week 14 (p < 0.0001). Post-imiquimod therapy, levels of CDK1, CXCL13, IL1B, GADPH, TTK, ILF3, EWSR1, BIRC5, PLAUR, ISG20, and C1QBP were significantly lower (adjusted p < 0.05). Complete responders (CR) exhibited a distinct pattern of inflammatory gene expression pre-treatment relative to incomplete responders (IR), with alterations in 15 inflammatory pathways (p < 0.05) reflecting differential expression of 103 genes (p < 0.05). Presence of adverse effects was associated with improved treatment response. Differences in gene expression were found between pre-treatment samples in CR versus IR, suggesting that higher levels of inflammation pre-treament may play a part in regression of AKs. Further characterization of the immune micro-environment in AKs may help develop biomarkers predictive of response to topical immune modulators and may guide therapy.

Loss of retinoic acid receptor-related receptor alpha (Rorα) promotes the progression of UV-induced cSCC.

Cutaneous squamous cell carcinoma (cSCC) is prevalent in the world, accounting for a huge part of non-melanoma skin cancer. Most cSCCs are associated with a distinct pre-cancerous lesion, the actinic keratosis (AK). However, the progression trajectory from normal skin to AK and cSCC has not been fully demonstrated yet. To identify genes involved in this progression trajectory and possible therapeutic targets for cSCC, here we constructed a UV-induced cSCC mouse model covering the progression from normal skin to AK to cSCC, which mimicked the solar UV radiation perfectly using the solar-like ratio of UVA and UVB, firstly. Then, transcriptome analysis and a series of bioinformatics analyses and cell experiments proved that Rorα is a key transcript factor during cSCC progression. Rorα could downregulate the expressions of S100a9 and Sprr2f in cSCC cells, which can inhibit the proliferation and migration in cSCC cells, but not the normal keratinocyte. Finally, further animal experiments confirmed the inhibitory effect of cSCC growth by Rorα in vivo. Our findings showed that Rorα would serve as a potential novel target for cSCC, which will facilitate the treatment of cSCC in the future.

MiR-130a Acts as a Tumor Suppressor MicroRNA in Cutaneous Squamous Cell Carcinoma and Regulates the Activity of the BMP/SMAD Pathway by Suppressing ACVR1.

Cutaneous squamous cell carcinoma (cSCC) is a malignant neoplasm of the skin resulting from the accumulation of somatic mutations due to solar radiation. cSCC is one of the fastest increasing malignancies, and it represents a particular problem among immunosuppressed individuals. MicroRNAs are short noncoding RNAs that regulate the expression of protein-coding genes at the post-transcriptional level. In this study, we identify miR-130a to be downregulated in cSCC compared to healthy skin and precancerous lesions (actinic keratosis). Moreoever, we show that its expression is regulated at the transcriptional level by HRAS and MAPK signaling pathway. We demonstrate that overexpession of miR-130a suppresses long-term capacity of growth, cell motility and invasion ability of human cSCC cell lines. We report that miR-130a suppresses the growth of cSCC xenografts in mice. Mechanistically, miR-130a directly targets ACVR1 (ALK2), and changes in miR-130a levels result in the decreased activity of the BMP/SMAD pathway through ACVR1. These data reveal a link between activated MAPK signaling and decreased expression of miR-130a, which acts as a tumor-suppressor microRNA in cSCC and contribute to a better understanding of the molecular processes during malignant transformation of epidermal keratinocytes.

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 TGFß signaling significantly more mutated in cSCC. Integrating our findings with independent gene expression datasets confirms that dysregulated TGFß signaling may represent an important event in AK‒cSCC progression.

Actinic Keratoses: Reconciling the Biology of Field Cancerization with Treatment Paradigms.

This Perspective briefly reviews the relationship between UV-induced mutations in habitually sun-exposed human skin and subsequent development of actinic keratoses (AKs) and skin cancers. It argues that field therapy rather than AK-selective therapy is the more logical approach to cancer prevention and hypothesizes that treatment early in the process of field cancerization, even prior to the appearance of AKs, may be more effective in preventing cancer as well as more beneficial for and better tolerated by at-risk individuals. Finally, the Perspective encourages use of rapidly advancing DNA analysis techniques to quantify mutational burden in sun-damaged skin and its reduction by various therapies.

Identification of potential gene drivers of cutaneous squamous cell carcinoma: Analysis of microarray data.

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer with an increasing incidence. As a pre-cancerous condition, actinic keratosis (AK) has an up to 20% risk of progression to cSCC. This study aims to define the potential genes that associated with genesis and progression of cSCC, thereby further identify critical biomarkers for the prevention, early diagnosis, and effective treatment of cSCC.Two datasets GSE42677 and GSE45216 were downloaded from the GEO. Microarray data analysis was applied to explore the differentially expressed genes (DEGs) between cSCC samples and AK samples. Then functional enrichment analysis, protein-protein interaction (PPI) network, and drug-gene interaction analysis were performed to screen key genes.A total of 711 DEGs, including 238 upregulated genes and 473 downregulated genes, were screened out. DEGs mainly involved in pathways as extracellular matrix (ECM)-receptor interaction, hematopoietic cell lineage, phosphatidylinositol 3-kinase (PI3K-Akt) signaling pathway, and focal adhesion. Candidate genes, including upregulated genes as JUN, filamin A (FLNA), casein kinase 1 delta (CSNK1D), and histone cluster 1 H3 family member f (HIST1H3F), and downregulated genes as androgen receptor (AR), heat shock protein family H member 1 (HSPH1), tropomyosin 1 (TPM1), pyruvate kinase, muscle (PKM), LIM domain and actin binding 1 (LIMA1), and synaptopodin (SYNPO) were screened out. In drug-gene interaction analysis, 13 genes and 44 drugs were identified.This study demonstrates that genes JUN, FLNA, AR, HSPH1, and CSNK1D have the potential to function as targets for diagnosis and treatment of cSCC.

UV biomarker genes for classification and risk stratification of cutaneous actinic keratoses and squamous cell carcinoma subtypes.

Currently, there is no sensitive molecular test for identifying transformation-prone actinic keratoses (AKs) and aggressive squamous cell carcinoma (SCC) subtypes. Biomarker-based molecular testing represents a promising tool for risk stratifying these lesions. We evaluated the utility of a panel of ultraviolet (UV) radiation-biomarker genes in distinguishing between benign and transformation-prone AKs and SCCs. The expression of the UV-biomarker genes in 31 SCC and normal skin (NS) pairs and 10 AK/NS pairs was quantified using the NanoString nCounter system. Biomarker testing models were built using logistic regression models with leave-one-out cross validation in the training set. The best model to classify AKs versus SCCs (area under curve (AUC) 0.814, precision score 0.833, recall 0.714) was constructed using a top-ranked set of 13 UV-biomarker genes. Another model based on a 15-gene panel was developed to differentiate histologically concerning from less concerning SCCs (AUC 1, precision score 1, recall 0.714). Finally, 12 of the UV-biomarker genes were differentially expressed between AKs and SCCs, while 10 genes were uniquely expressed in the more concerning SCCs. UV-biomarker gene subsets demonstrate dynamic utility as molecular tools to classify and risk stratify AK and SCC lesions, which will complement histopathologic diagnosis to guide treatment of high-risk patients.

Glutathione S-Transferase Theta 1 / Mu 1 Null Genotype and Risk of Development of Actinic Keratosis in Pakistani Population.

OBJECTIVE: To assess the frequency and association of Glutathione S-Transferase Theta 1 and Glutathione S-Transferase Mu 1 null genotypes in development of actinic keratosis (AK) in a group of Pakistani population. STUDY DESIGN: Case-control analytical study. PLACE AND DURATION OF STUDY: Department of Biochemistry, Islamic International Medical College, Rawalpindi in collaboration with Department of Dermatology, Railway Hospital and Rural Health Center, District Health Office, Rawalpindi from September 2018 to September 2019.  Methodology: A total of 86 participants were included in this study with 27 biopsy proven cases of AK and 59 matched controls. Blood samples were collected after obtaining written informed consent; and DNA was extracted by Chelex™ method. Multiplex PCR (M-PCR) was done to find respective allelic frequencies of GSTM1 and GSTT1 genes in both cases and controls.  Results: Mean age of participants in cases and controls was 62.93 ±10.29 years and was 61.42 ±9.96 years, respectively. There were 18 males (66.7%) and 9 females (33.3%); and 43 males (72.9%) and 16 females (27.1%) in cases and controls, respectively. There was a significant association of GSTT1 null genotype with AK (OR: 2.72, 95% CI: 1.05-7.05, p = 0.037). There was a positive correlation between GSTT1 null genotype and AK (r = 0.225, p = 0.037). CONCLUSION: GSTT1 null genotype has a significant association for AK development in the studied Pakistani population. Key Words: Actinic keratosis, Glutathione S-Transferase Mu 1, Glutathione S-Transferase Theta 1, Polymerase chain reaction, Squamous cell carcinoma.

Identification of differentially expressed genes in actinic keratosis samples treated with ingenol mebutate gel.

Actinic keratosis is a common skin disease that may progress to invasive squamous cell carcinoma if left untreated. Ingenol mebutate has demonstrated efficacy in field treatment of actinic keratosis. However, molecular mechanisms on ingenol mebutate response are not yet fully understood. In this study, we evaluated the gene expression profiles of actinic keratosis lesions before and after treatment with ingenol mebutate using microarray technology. Actinic keratoses on face/scalp of 15 immunocompetent patients were identified and evaluated after treatment with topical ingenol mebutate gel 0.015%, applied once daily for 3 consecutive days. Diagnostic and clearance of lesions was determined by clinical, dermoscopic, and reflectance confocal microscopy criteria. Lesional and non-lesional skin biopsies were subjected to gene expression analysis profiled by Affymetrix microarray. Differentially expressed genes were identified, and enrichment analyses were performed using STRING database. At 8 weeks post-treatment, 60% of patients responded to ingenol mebutate therapy, achieving complete clearance in 40% of cases. A total of 128 differentially expressed genes were identified following treatment, and downregulated genes (114 of 128) revealed changes in pathways important to epidermal development, keratinocyte differentiation and cornification. In responder patients, 388 downregulated genes (of 450 differentially expressed genes) were also involved in development/differentiation of the epidermis, and immune system-related pathways, such as cytokine and interleukin signaling. Cluster analysis revealed two relevant clusters showing upregulated profile patterns in pre-treatment actinic keratoses of responders, as compared to non-responders. Again, differentially expressed genes were mainly associated with cornification, keratinization and keratinocyte differentiation. Overall, the present study provides insight into the gene expression profile of actinic keratoses after treatment with ingenol mebutate, as well as identification of genetic signatures that could predict treatment response.

Invasive squamous cell carcinomas and precursor lesions on UV-exposed epithelia demonstrate concordant genomic complexity in driver genes.

Although squamous cell carcinomas (SCC) are the most frequent human solid tumor at many anatomic sites, the driving molecular alterations underlying their progression from precursor lesions are poorly understood, especially in the context of photodamage. Therefore, we used high-depth, targeted next-generation sequencing (NGS) of RNA and DNA from routine tissue samples to characterize the progression of both well- (cutaneous) and poorly (ocular) studied SCCs. We assessed 56 formalin-fixed paraffin-embedded (FFPE) cutaneous lesions (n = 8 actinic keratosis, n = 30 carcinoma in situ [CIS], n = 18 invasive) and 43 FFPE ocular surface lesions (n = 2 conjunctival/corneal intraepithelial neoplasia, n = 20 CIS, n = 21 invasive), from institutions in the US and Brazil. An additional seven cases of advanced cutaneous SCC were profiled by hybrid capture-based NGS of >1500 genes. The cutaneous and ocular squamous neoplasms displayed a predominance of UV-signature mutations. Precursor lesions had highly similar somatic genomic landscapes to SCCs, including chromosomal gains of 3q involving SOX2, and highly recurrent mutations and/or loss of heterozygosity events affecting tumor suppressors TP53 and CDKN2A. Additionally, we identify a novel molecular subclass of CIS with RB1 mutations. Among TP53 wild-type tumors, human papillomavirus transcript was detected in one matched pair of cutaneous CIS and SCC. Amplicon-based whole-transcriptome sequencing of select 20 cutaneous lesions demonstrated significant upregulation of pro-invasion genes in cutaneous SCCs relative to precursors, including MMP1, MMP3, MMP9, LAMC2, LGALS1, and TNFRSF12A. Together, ocular and cutaneous squamous neoplasms demonstrate similar alterations, supporting a common model for neoplasia in UV-exposed epithelia. Treatment modalities useful for cutaneous SCC may also be effective in ocular SCC given the genetic similarity between these tumor types. Importantly, in both systems, precursor lesions possess the full complement of major genetic changes seen in SCC, supporting non-genetic drivers of invasiveness.