Strong association of TLR2 and TLR3 polymorphisms with keratoacanthoma and common warts: a case-control study.

AIM: To determine variations in allele and genotype frequencies between keratoacanthoma (KA) and common warts (CW), compared with the control group, in three single nucleotide polymorphisms (SNPs) within the TLR2, TLR3, and TLR9 genes. METHODS: This case-control study involved samples from 161 patients with KA, 152 patients with CW, and 469 controls. DNA was isolated from formalin-fixed paraffin-embedded tissue sections. Three SNPs - rs4696480 in TLR2, rs7657186 in TLR9, and rs35213 in TLR3 - were genotyped with TaqMan Genotyping Assays on the 7500 Real-Time PCR System. RESULTS: TLR2 rs4696480 and TLR3 rs7657186 were significantly overrepresented in KA and CW compared with controls (P<0.001). The association was stronger for CW than for KA, as evidenced by higher frequencies of the A allele and AA genotype for rs4696480. Both KA and CW patients had higher frequencies of the G allele and GG genotype for rs7657186 than controls. rs7657186 was moderately associated with KA and CW, with the G allele and GG genotype being more prevalent in CW cases, where no AA homozygotes were found. CONCLUSION: Genetic variants in TLR2 (rs4696480) and TLR3 (rs7657186) genes may affect KA and CW development, influencing immune responses and susceptibility to these skin lesions. Further research is required to elucidate TLR expression patterns and their role in KA development.

Mosaic Muir Torre Syndrome: Keratoacanthoma as a Piece of the Puzzle.

ABSTRACT: Lynch syndrome is an inherited condition, which increases the risk of numerous visceral malignancies and cutaneous tumors such as keratoacanthomas and sebaceous tumors. It is typically identified by immunohistochemistry of tissue taken from tumors or through genetic testing with next-generation sequencing. Diagnosing Lynch syndrome becomes more complex when the individual is mosaic for the relevant pathogenic variant. There are very few cases of this reported in the medical literature. It is even more unusual for the diagnosis to be made based on testing of a keratoacanthoma lesion. We report a case where immunohistochemistry of a keratoacanthoma helped make a diagnosis of mosaic Lynch syndrome. We will explore how mosaicism should be considered when a phenotype is strong, even if next-generation sequencing reports no pathogenic or likely pathogenic variant and how lesions such as keratoacanthomas can have a role in the early detection and treatment of future malignancies.

Distinguishing Keratoacanthoma from Well-Differentiated Cutaneous Squamous Cell Carcinoma Using Single-Cell Spatial Pathology.

Keratoacanthoma (KA) is a common keratinocyte neoplasm that is regularly classified as a type of cutaneous squamous cell carcinoma (cSCC) despite demonstrating benign behavior. Differentiating KA from well-differentiated cSCC is difficult in many cases due to the substantial overlap of clinical and histological features. Currently, no reliable discriminating markers have been defined, and consequently, KAs are often treated similarly to cSCC, creating unnecessary surgical morbidity and healthcare costs. In this study, we used RNA sequencing to identify key differences in transcriptomes between KA and cSCC, which suggested divergent keratinocyte populations between each tumor. Imaging mass cytometry was then used to identify single-cell tissue characteristics, including cellular phenotype, frequency, topography, functional status, and interactions between KA and well-differentiated cSCC. We found that cSCC had significantly increased proportions of Ki67+ keratinocytes among tumor keratinocytes, which were dispersed significantly throughout non-basal keratinocyte communities. In cSCC, regulatory T-cells were more prevalent and held greater suppressive capacity. Furthermore, cSCC regulatory T-cells, tumor-associated macrophages, and fibroblasts had significant associations with Ki67+ keratinocytes as opposed to avoidances with KA, indicating a more immunosuppressive environment. Our data suggest that multicellular spatial features can serve as a foundation to enhance the histological discrimination of ambiguous KA and cSCC lesions.

Keratoacanthoma or cutaneous squamous cell carcinoma revealing a DNA mismatch repair default (Muir-Torre Syndrome).

Keratoacanthoma (KA) and well-differentiated cutaneous squamous cell carcinoma (cSCC) are hardly distinguishable clinically and histologically. They both can be seen in patients with hereditary non-polyposis colorectal cancer (HNPCC) or Lynch Syndrome, corresponding to DNA microsatellite instability. In our case, a young man had the excision of two rapidly growing skin tumours for which distinction between KA and cSCC was initially clinically and pathologically challenging. The diagnosis of well-differentiated cSCCs was made and the patient was treated with surgery. Ten years after the first cSCC, he was diagnosed with Muir-Torre syndrome, a variant of Lynch syndrome, with an heterozygote mutation of the MSH2 gene. This later diagnosis allowed to screen his family members for the same mutation and to adopt an appropriate follow-up regarding the risk of digestive tumours for him and his family. Furthermore, it is important to know that, in case of non-resectable cSCC occurring in this patient, immunotherapy using anti-PD1 antibody would probably be effective due to the known increased immunogenicity of MMR deficient tumours.

Differences in the mitochondrial microsatellite instability of Keratoacanthoma and cutaneous squamous cell carcinoma.

Keratoacanthoma (KA) is a common cutaneous neoplasm which often resembles typical squamous cell carcinoma (SCC) in both its clinical and historical presentation. Several studies have attempted to identify methods for distinguishing between KA and SCC, however, none of these have proven to play any obvious roles in these tumors. Given this we went on to evaluate mitochondrial microsatellite instability (mtMSI) in KA and SCC in an effort to understand these tumors better. DNA was isolated from paired normal and tumoral tissues donated by 57 KA patients and 43 SCC patients. MtMSI was then analyzed using eight microsatellite markers and was observed in 2 (3.5%) of the 57 KA patients and 8 (18.6%) of the 43 SCC patients, respectively. MtMSI was also shown to affect different locations depending on tumor type. In KA patients, mtMSI was detected at mitochondrial D514 D-loop and presented with (CA) n repeats, in contrast, all of the SCC patient experienced mtMSI at the D310 with (C)n repeats of the D-loop region. These differences in location were found to be significant, which may support the hypothesis that KA and SCC have different pathogenetic pathways. Our results also suggest that mtMSI may be a candidate for developing novel differential diagnostic methods for KA and SCC.

A case of Muir-Torre syndrome with a keratoacanthoma and sebaceous neoplasms: Clinicopathological features and a speculation on the pathogenesis of cutaneous tumor type.

Muir-Torre syndrome is a hereditary condition characterized by occurrence of sebaceous neoplasms or keratoacanthomas and visceral tumors. The most common mechanism for this syndrome is a constitutional defect in the mismatch repair genes. We report the case of a 67-year-old woman with a mutator L homologue 1 (MLH1) mutation. She had a history of endometrial and colorectal cancers. The patient presented with a typical keratoacanthoma on the right cheek and numerous sebaceous neoplasms on the face and trunk. Seven sebaceous adenomas and a low-grade sebaceous carcinoma were excised. Most sebaceous adenomas showed dermoscopic features such as some yellow comedo-like globules and curved vessels in creamy-white areas. Moreover, they revealed pathological features such as keratoacanthoma-like architecture and peritumoral or intratumoral lymphocytes. One of these sebaceous adenomas indicated histopathologically spontaneous regression and another was continuous with the hair follicle. Immunohistochemical staining for mismatch repair proteins revealed loss of expression for MLH1 and postmeiotic segregation increased 2 (PMS2) proteins in tumor cells nuclei in both keratoacanthoma and sebaceous adenoma. Nuclei in overhanging epithelial lips of the keratoacanthoma were also negative. These findings suggest that the type of Muir-Torre syndrome-related cutaneous tumor may have been affected by mismatch repair protein deficient sites in the pilosebaceous unit.

Keratoacanthoma: Update on the Debate.

ABSTRACT: Keratoacanthoma (KA) is a cutaneous tumor with a biphasic pattern of growth. A rapidly growing phase is usually followed by involution. KA occurs on sun-damaged skin. There are many listed causative associations, which include some therapeutic agents. Debate continues as to whether KA is a variant of squamous carcinoma (SCC) or a separate entity. Reporting of KA versus SCC is markedly inconsistent. Reasons for inconsistency include overlapping microscopic criteria, variants of KA with more aggressive features, and possibly medicolegal concerns. Genetic studies have shown some differences between the 2 entities. Activation of apoptotic pathways has been demonstrated in KA. Genetic studies have shown a possible role of human polyomavirus 6 in the pathogenesis of at least some KAs. Given that some cases of KA have components that behave as conventional SCCs, KA can be considered as a low-grade variant of SCC with some genetic differences.

Onycholemmal variant of keratoacanthoma centrifugum marginatum as an expression of mutated committed stem cells in a conceptual pathway.

We describe a 43-year-old woman with a 10-year history of grossly hyperkeratotic nodules which progressively extended over the right ring finger. These involuted leaving pale, atrophic skin in their wake. At presentation, the advancing border had an arciform series of nodules in the pattern of keratoacanthoma centrifugum marginatum. The presence of filiform keratinisation that encased the nail plate, gross onychogryphotic masses of keratin on the ventral finger surface and a flat nail-like plate of keratin on the dorsal finger surface were distinctive features. Skin biopsy showed epidermal acanthosis, gross papillomatous cutaneous horn formation that had onycholemmal features. The pathology differed from keratoacanthoma and was not crateriform or infundibulocystic. Although HPV was not detected on immunohistochemistry, pathogenesis may still represent an HPV-related transfection of onycholemmal keratin committed stem cells producing an onycholemmal variant of keratoacanthoma centrifugum marginatum. A conceptual model linked to advances in follicular stem cell biology is formulated to explore this case.

The role of p53, Ki-67 and laminin expression in the differential diagnosis of keratoacanthoma and well-differentiated SCC.

INTRODUCTION: We have aimed to evaluate the difference between the expression of p53, Ki-67, and laminin in keratoacanthoma and well-differentiated SCC (SCC) and to determine its importance in differential diagnosis. METHODS: This study totally included 46 cases consisting of 23 cases with keratoacanthoma and 23 with SCC. As well as age, gender, localization, and diameter of the lesion, the expression of p53, Ki-67 and laminin was evaluated. RESULTS: No statistically significant difference was found between KA and well-differentiated SCC in terms of diameter, age, and localization. There was a statistically significant difference between KA and well-differentiated SCC in terms of p53 and Ki-67 staining (P < 0.001). Increased expression of p53 and Ki-67 was found in well-differentiated SCC. A statistically significant correlation was present between the expression of p53 and Ki-67 in KA. A statistically significant difference was detected between KA and well-differentiated SCC in terms of laminin staining (P = 0.018). Increased laminin expression was determined in well-differentiated SCC. CONCLUSION: We have determined in this study that p53, Ki-67 and laminin may be used as adjuvant immunohistochemical markers in differential diagnosis of KA and well-differentiated SCC.

Features of multiple self-healing squamous epithelioma and Loeys-Dietz syndrome in a patient with a novel TGFBR1 variant.

Multiple self-healing squamous epithelioma (MSSE, Ferguson-Smith disease) and Loeys-Dietz syndrome (LDS) are allelic conditions associated with pathogenic variants in the transforming growth factor beta receptor 1 gene (TGFBR1). We describe a patient with a novel missense variant in this gene: c.664G > A, p.[Gly222Arg], who clinically presents with both syndromes. The patient also has a history of gastric antral vascular ectasia, which has not been reported previously in LDS.

Distinct variants affecting differential splicing of TGFBR1 exon 5 cause either Loeys-Dietz syndrome or multiple self-healing squamous epithelioma.

Variants in TGFBR1 have been reported to induce two completely distinct diseases, namely Loeys-Dietz syndrome (LDS) and multiple self-healing squamous epithelioma (MSSE). However, detailed mechanisms underlying this effect remain unknown. We report a Japanese familial case of LDS with a novel splice donor site variant in TGFBR1 gene (c.973 + 1 G > A; NG_007461.1). The intronic variant was predicted to mediate in-frame exon 5 skipping within the serine/threonine kinase (STK) domain, which may also be mediated by a similar TGFBR1 variant of a splice acceptor site in intron 4 (c.806-2 A > C), identified in a British familial case of MSSE. Therefore, ex vivo splicing and functional assays were performed in mammalian cells to evaluate the effect of these sequence variants. The MSSE variant activated a cryptic acceptor site at 76 bp downstream of the 3' natural splice acceptor site, which produced an out-of-frame transcript (r.807_882del, p.Asn270Thrfs*8). In contrast, the LDS variant generated two types of in-frame transcription products, r.[806_973del, 965_973 del], and produced two functionally inactivated proteins, p.[Asp269_Gln324del, Thr323_Gly325del], as a result of exon 5 skipping and the activation of a cryptic donor splice site at 9 bp upstream of the 5' natural splice donor site, respectively. Our results support the previously proposed but not yet approved mechanism that dominant-negative and truncating variants in STK domain induce LDS and MSSE, respectively.

Prevalence and molecular characteristics of DNA mismatch repair protein-deficient sebaceous neoplasms and keratoacanthomas in a Japanese hospital-based population.

BACKGROUND: Muir-Torre syndrome (MTS) is currently considered as a clinical variant of Lynch syndrome (LS). The clinical significance of the screening of patients with MTS-associated cutaneous tumors for the identification of LS has not yet been established. In addition, the prevalence and molecular characteristics of mismatch repair (MMR) protein deficiency in such tumors has scarcely been investigated in the Japanese population. METHODS: Immunohistochemistry (IHC) for MMR proteins (MLH1, MSH2, MSH6 and PMS2) was performed in formalin-fixed paraffin-embedded sections prepared from 16 sebaceous neoplasms (SNs) resected from 13 patients and 32 keratoacanthomas (KAs) resected from 31 patients at our institution between January 2005 and March 2014. Tumors showing MMR protein loss were further subjected to genetic analysis for detecting the presence of germline and/or somatic alterations of the MMR genes to identify the precise molecular mechanisms underlying the protein loss. RESULTS: Among the 16 SNs resected from 13 patients, eight SNs resected from five patients (38.5%) showed loss of expression of MMR proteins (MLH1/PMS2 loss, one patient; MSH2/MSH6 loss, four patients). Genetic analyses showed a pathogenic germline MSH2 mutation in one patient, somatic hypermethylation of the MLH1 promoter region in one patient, and somatic alterations of MSH2 without detectable germline mutations of MSH2 in three patients. None of the KAs examined in the study showed any loss of MMR protein expression. CONCLUSIONS: The efficacy of routine screening of cutaneous neoplasms known to be associated with MTS by IHC for MMR proteins to identify LS may be fairly limited. MMR protein loss as determined by IHC in SNs is not always diagnostic of LS, and appears, in most cases, to be a result of somatic inactivation of the MMR genes.

Squamous proliferations on the legs of women: Qualitative examination of histopathology, TP53 sequencing, and implications for diagnosis in a series of 30 cases.

BACKGROUND: Women with multiple squamous cell carcinomas (SCCs) of the legs have a striking clinical phenotype. Numerous tumors can develop in a short period of time. OBJECTIVE: Because histopathologic findings can vary in women with multiple SCC lesions, from keratoacanthoma-like to well-differentiated SCC, we hypothesized that TP53 variants might shed light on the appropriate classification. METHODS: We sequenced TP53 in 30 SCCs from 6 women who had multiple SCCs on their legs during a 21-month time frame. RESULTS: Histopathologic analysis showed 16 of the 30 lesions did not have prominent cytologic atypia and were distinguished by having expanded follicle-like structures composed of large, glassy, eosinophilic keratinocytes; these lesions resembled keratoacanthoma and were categorized as keratoacanthoma-like squamous proliferations (KASPs). The 14 remaining tumors had more prominent cytologic atypia and remained classified as SCC. Twenty of 30 tumors (including the KASPs) from the 6 different patients lacked detectable TP53 mutations. Ten of the 14 tumors that remained classified as SCC had detectable TP53 mutations. LIMITATIONS: This is a small series. CONCLUSION: These findings suggest that some cutaneous squamous proliferations on the legs of women with multiple lesions lack prominent cytologic atypia as well as TP53 mutations and might be more akin to keratoacanthoma than SCC or might represent a reactive phenomenon.

Expression of PD-L1 in keratoacanthoma and different stages of progression in cutaneous squamous cell carcinoma.

BACKGROUND: Programmed cell death 1 (PD-1) and its ligands (PD-L1) play a major role in the immune responses of a variety of cancers. OBJECTIVES: To investigate the expression of PD-L1 in different progression forms of cutaneous squamous cell carcinoma (cSCC) and keratoacanthoma (KA). METHODS: We performed immunohistochemical staining of 21 KA, 26 actinic keratoses (AK), 20 Bowen´s diseases (BD), and 26 high-risk cSCC. The staining patterns were assessed using the tumour proportion score and staining intensity evaluation. Immunohistology scores were statistically analysed. RESULTS: PD-L1 expression of tumour cells as well as tumour-infiltrating cells (TILs) was significantly higher in KA and cSCC when compared to AK and BD (P = 0.00028 and P = 0.00033, respectively). We observed a very strong positive correlation between the PD-L1 protein expression of tumour cells of KA and the PD-L1 protein expression of TILs (r = 0.97; P < 0.0001). A similar correlation was also found for cSCC (r = 0.86; P < 0.0001). The percentage of PD-L1 + tumours was 33.3% for KA and 26.9% for cSCC. Similarly, the percentage of PD-L1 + TILs in KA and cSCC was 33.3 and 34.6%, respectively. CONCLUSIONS: PD-L1 is differently expressed in cSCC and closely related non-melanoma skin cancer. cSCC exhibit PD-L1 expression in a fourth of cases, indicating that PD1/PD-L1 inhibitors might be beneficial in a proportion of patients with an inoperable or metastatic cSCC. Unlike AK and BD, TILs and tumour cells of KA and cSCC present very similar PD-L1 expression profiles indicating a common immune escape mechanism.

Possible Involvement of F1F0-ATP synthase and Intracellular ATP in Keratinocyte Differentiation in normal skin and skin lesions.

The F1F0-ATP synthase, an enzyme complex, is mainly located on the mitochondrial inner membrane or sometimes cytomembrane to generate or hydrolyze ATP, play a role in cell proliferation. This study focused on the role of F1F0-ATP synthase in keratinocyte differentiation, and its relationship with intracellular and extracellular ATP (InATP and ExATP). The F1F0-ATP synthase ß subunit (ATP5B) expression in various skin tissues and confluence-dependent HaCaT differentiation models was detected. ATP5B expression increased with keratinocyte and HaCaT cell differentiation in normal skin, some epidermis hyper-proliferative diseases, squamous cell carcinoma, and the HaCaT cell differentiation model. The impact of InATP and ExATP content on HaCaT differentiation was reflected by the expression of the differentiation marker involucrin. Inhibition of F1F0-ATP synthase blocked HaCaT cell differentiation, which was associated with a decrease of InATP content, but not with changes of ExATP. Our results revealed that F1F0-ATP synthase expression is associated with the process of keratinocyte differentiation which may possibly be related to InATP synthesis.

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice.

Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies.

Aberrant DNA Methylation in Keratoacanthoma.

BACKGROUND: Keratoacanthoma (KA) is a self-limiting epidermal tumor for which histopathological examination sometimes suggests malignancy. Based on inconsistent clinical views, KA can be regarded as both a benign tumor and a variant of squamous cell carcinoma (SCC). Aberrant DNA methylation frequently occurs in malignant tumors but it scarcely occurs in benign tumors. Whether aberrant methylation occurs in KA has not been previously examined. OBJECTIVE: The aim is to elucidate whether aberrant methylation of CpG islands (CGI) containing a high density of cytosine-guanine dinucleotide (CpG) sites occurs in KA. METHODS: Five SCC cell lines, two cultured samples of normal human epidermal keratinocytes (NHEKs), 18 clinical SCC samples, and 21 clinical KA samples were analyzed with Infinium HumanMethylation450 BeadChips, quantitative real-time methylation-specific PCR (RT-MSP) and/or bisulfite sequencing. RESULTS: Genome-wide analyses of NHEK, KA, and SCC indicated that there was a greater number of aberrantly hypermethylated CGIs in SCC than in KA and there were aberrantly hypermethylated CGIs which are common in both. Among the common hypermethylated CGIs, RT-MSP and bisulfite sequencing targeting CGIs located on CCDC17, PVR, and MAP3K11 gene bodies also showed that methylation levels were significantly higher in KA than in normal epidermis. Statistical analyses suggested that the methylation level of CGI located on PVR in SCC might be correlated to lymph node metastasis (P = 0.013, Mann-Whitney U test) and that the methylation level of CGI in MAP3K11 in KA might be correlated to age (P = 0.031, linear regression analysis). CONCLUSION: Aberrant DNA methylation occurs in KA.

Tp53/p53 status in keratoacanthomas.

BACKGROUND: Keratoacanthoma (KA) is a common keratinocytic skin neoplasm that typically develops rapidly and undergoes complete spontaneous regression. As the pro-apoptotic p53 protein may be involved in the lifecycle of KA, we studied the p53 status throughout the main stages of KA that include proliferation, maturation and regression in a large series of lesions. METHODS: One-hundred and twenty-four KAs were characterized with respect to age of the lesions both clinically and histopathologically, in addition to phenotypic characteristics such as cellular atypia, infiltration, inflammation and fibrosis. Tp53 mutations were detected by capillary electrophoresis, and p53 protein levels were assessed by immunohistochemistry. RESULTS: Tp53 mutations were detected in 49 cases (39.5%) and were associated with high p53 protein levels (p = 0.007) and histopathologic age of the lesions (p = 0.044). Significant association was also seen between high p53 protein levels and atypia (p = 0.036), whereas the association with infiltration showed borderline significance (p = 0.057). High p53 protein levels were significantly associated with gene mutations in transplanted, but not in non-transplanted patients. CONCLUSION: We show a high frequency of Tp53 mutations in KAs that is associated with increased p53 levels. The results indicate a role for the p53 protein in KA development.