No Association between Merkel Cell Polymavirus Infection and Keratoacanthoma in Korean Patients

Objectives: Keratoacanthoma (KA) is a relatively common benign tumor and resembles squamous cell carcinoma (SCC). The definitive cause of KA remains unclear, but trauma, ultraviolet light, chemical carcinogens, human papillomavirus, genetic factors, and immunocompromised status have been implicated as etiologic or triggering factors. Merkel cell polyomavirus (MCPyV) is suspected to cause the majority of cases of Merkel cell carcinoma (MCC). MCPyV-DNA was found significantly more frequently in MCC and only found in about one fourth of KAs. In a recent study, MCPyV was found in Korean patients with MCC. The aim of this study was to determine the presence of MCPyV in Korean patients with KA. Methods: Paraffin-embedded tissue samples were analyzed for the presence of MCPyV-DNA by polymerase chain reaction (PCR). A total of 105 KA samples were analyzed. Results: A study of MCPyV has not been reported about KA in Korean cases. In the present study the MCPyV was not detected with KA in the Korean patients. Conclusions: This supports that KA and MCPyV are not related to each other and MCVyP is not a major factor in the pathogenesis of KA.

Human polyomavirus DNA detection in keratoacanthoma and Spitz naevus: no evidence for a causal role.

Keratoacanthomas (KA) and Spitz naevus (SN) are both lesions with unknown aetiology; therefore, the possibility of a viral involvement, more specifically the involvement of human polyomaviruses (HPyV), was investigated. In total, 22 cases of KA and 25 cases of SN were tested for the presence of HPyVs. DNA was extracted and amplified by multiplex PCR and thereafter tested with a multiplex bead-based assay for HPyVs (BKPyV, JCPyV, KIPyV, WUPyV, MCPyV, TSPyV, HPyV6, 7 and 9) and two primate viruses (SV40 and LPyV). HPyV DNA was found in 20 of the 47 lesions. There was no significant difference in HPyV DNA detection frequency between patients diagnosed with KA and patients diagnosed with SN, nor any over-representation of a specific HPyV type in any of the two patient categories. In conclusion, evidence for a specific aetiological role of any of the above tested HPyVs in either KA or SN was not disclosed.

Frequent detection of human polyomavirus 6 in keratoacanthomas.

BACKGROUND: The recent discovery of the Merkel cell polyomavirus and its consistent association with Merkel cell carcinoma has drawn attention to the numerous recently discovered polyomaviruses and their possible involvement in the etiopathogenesis of non-melanoma skin cancer (NMSC). Data on the recently discovered human polyomavirus 6 (HPyV6) and its role in NMSC are sparse and in part controversial. METHODS: In the present study we tested a large number (n = 299) of NMSC specimens for the presence of human polyomavirus 6 (HPyV6) by DNA PCR and HPyV6 fluorescence in situ hybridization (FISH). In detail, 59 keratoacanthomas (KA), 109 basal cell carcinomas (BCC), 86 squamous cell carcinomas (SCC) and 45 trichoblastomas (TB) were tested for the presence of HPyV6. RESULTS: HPyV6 DNA PCR and subsequent sequence analysis revealed that 25 KAs (42.3 %), 23 BCCs (21.1 %), 8 SCCs (9.3 %) and 10 TBs (22.2 %) were HPyV6 positive. The presence of HPyV6 DNA was visualized and validated on the single cell level within the histomorphological context by HPyV6 fluorescence in situ hybridization. CONCLUSIONS: The high frequency of HPyV6 DNA in 42.3 % of KA possibly points to a role for HPyV6 in the etiopathogenesis of KAs. Although the detection rate of HPyV6 DNA in BCCs and TBs is within the previously reported detection range in normal skin, it does not exclude a possible role for HPyV6 in the carcinogenesis in a significant subset of these skin tumors.

Prevalence of human papillomavirus infection and RAS mutation in sporadic keratoacanthoma.

BACKGROUND: RAS gene activation and its association with human papillomavirus (HPV) infection have been extensively studied in various cancers. However, the correlation between RAS mutations and HPV in keratoacanthoma (KA) has not yet been investigated. METHODS: Detection of HPV DNA was performed by nested polymerase chain reaction in 28 KA specimens. Molecular analysis was also performed to identify oncogenic mutations (HRAS, KRAS, NRAS). Statistical analyses were performed using the Fisher's exact tests. RESULTS: HPV DNA was detected in eight (28.6%) of the 28 samples, and RAS mutations were detected in eight (28.6%). Six samples had an HRAS mutation, and two showed the NRAS mutation. The presence of an RAS mutation was significantly correlated with a history of chronic sun damage (P = 0.005). However, no significant correlation was observed between HPV infection and RAS mutation. CONCLUSIONS: Our findings suggest that mutational activation of the RAS gene is a common event in KA. However, RAS oncogene activation and HPV infection seem to represent two independent factors in the development of KA.

Human papillomavirus type 197 is commonly present in skin tumors.

Non-melanoma skin cancers commonly contain Human Papillomavirus (HPV), but the types found have varied depending on the polymerase chain reaction (PCR) primer systems used. Whole genome amplified DNA (not amplified by any specific PCR primers) from 91 skin lesions [41 squamous cell skin carcinomas (SCCs), 8 keratoacanthomas, 22 actinic keratoses, 3 basal cell carcinomas and 17 SCCs in situ] were sequenced. All samples were sequenced both at 160 Mb and 1.8 Gb sequencing depth per sample. The sequences from 10 different HPVs in 47/91 specimens were found. Sequences represented four established HPV types (HPV types 16, 22, 120, 124), two previously known putative types (present in GenBank) and four previously unknown HPV sequences (new putative types). The most commonly detected virus was cloned, sequenced and designated as HPV197. Type-specific real-time PCR detected HPV197 in 34/91 specimens. For comparison, a pool of the same samples after general primer PCR amplification was also sequenced. This revealed 40 different HPVs, but only two HPV types were detected both with sequencing without prior PCR and with sequencing PCR amplicons, suggesting that sequencing without prior PCR gives a more unbiased representation of the HPVs present. In summary, it was found that HPV can be sequenced from most skin disease specimens and HPV197 appeared to be the most commonly present virus.

Deep sequencing extends the diversity of human papillomaviruses in human skin.

Most viruses in human skin are known to be human papillomaviruses (HPVs). Previous sequencing of skin samples has identified 273 different cutaneous HPV types, including 47 previously unknown types. In the present study, we wished to extend prior studies using deeper sequencing. This deeper sequencing without prior PCR of a pool of 142 whole genome amplified skin lesions identified 23 known HPV types, 3 novel putative HPV types and 4 non-HPV viruses. The complete sequence was obtained for one of the known putative types and almost the complete sequence was obtained for one of the novel putative types. In addition, sequencing of amplimers from HPV consensus PCR of 326 skin lesions detected 385 different HPV types, including 226 previously unknown putative types. In conclusion, metagenomic deep sequencing of human skin samples identified no less than 396 different HPV types in human skin, out of which 229 putative HPV types were previously unknown.

Unbiased approach for virus detection in skin lesions.

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.

Prevalence of human polyomaviruses in common and rare types of non-Merkel cell carcinoma skin cancer.

BACKGROUND: Little is known about the association of human polyomaviruses (HPyVs) other than Merkel cell polyomavirus (MCPyV) with nonmelanoma skin cancer. OBJECTIVES: To evaluate the presence of HPyV6, HPyV7, trichodysplasia spinulosa-associated polyomavirus (TSV), also called HPyV8, and the recently discovered HPyV9 in basal cell carcinoma (BCC), actinic keratosis (AK), squamous cell carcinoma in situ (SCCis), squamous cell carcinoma (SCC), keratoacanthoma (KA), microcystic adnexal carcinoma (MAC) and atypical fibroxanthoma (AFX). METHODS: Archival paraffin-embedded samples (n = 193: 41 BCC, 31 AK, 8 SCCis, 52 SCC, 42 KA, 5 MAC and 14 AFX) were analysed for the presence of the respective HPyV by polymerase chain reaction (PCR). HPyV DNA loads (HPyV DNA copies per ß-globin gene copy) were determined in all HPyV-positive samples by quantitative real-time PCR. Immunohistochemical analysis of MCPyV large T-antigen (LTA) expression was performed using the monoclonal antibody CM2B4. RESULTS: MCPyV DNA was found in 29% of BCC, 19% of AK, 25% of SCCis, 27% of SCC, 29% of KA, 0% of MAC and 29% of AFX. MCPyV DNA loads never exceeded 0·3 MCPyV DNA copies per ß-globin gene copy (median 0·004). In the immunohistochemical analysis of MCPyV LTA expression, all evaluated samples (32 MCPyV DNA-positive samples) were LTA negative. HPyV6 DNA was found in 7% of BCC, 3% of AK, 12% of SCCis, 4% of SCC, 5% of KA, and 0% of MAC and AFX. HPyV6 DNA loads never exceeded 0·7 HPyV6 DNA copies per ß-globin gene copy (median 0·015). None of the 193 samples was positive for HPyV7, TSV or HPyV9 DNA. CONCLUSIONS: Our findings argue against a pathogenic role for MCPyV, HPyV6, HPyV7, TSV and HPyV9 in the analysed types of non-Merkel cell carcinoma skin cancer.

No evidence for a causal role of Merkel cell polyomavirus in keratoacanthoma.

BACKGROUND: Merkel cell polyomavirus (MCPyV) is a recently discovered virus that is monoclonally integrated into the genome of approximately 80% of all Merkel cell carcinomas (MCCs). While some evidence exists that MCPyV does not play a pathogenic role in other nonmelanoma skin cancers, such as basal cell carcinoma and squamous cell carcinoma (SCC), little is known about the presence of MCPyV in keratoacanthoma (KA). OBJECTIVES: To evaluate the prevalence, viral DNA-load, and large T(umor)-antigen expression of MCPyV in KA of immunocompetent patients and to compare the results with those found in SCC and MCC. METHODS: Paraffin-embedded tissue samples were analyzed for the presence of MCPyV-DNA by polymerase chain reaction (PCR). MCPyV-DNA load (MCPyV-DNA copies per beta-globin gene copy) was determined by using quantitative real-time PCR. Immunohistochemical analysis of the MCPyV large T-antigen was performed with the monoclonal antibody CM2B4. RESULTS: A total of 137 samples (42 KA, 52 SCC, and 43 MCC) were analyzed. MCPyV-DNA was found significantly more frequently in MCC (37/43, 86.0%) compared with KA (12/42, 28.6%) and SCC (14/52, 26.9%). Moreover, MCPyV-DNA loads were more than two orders of magnitude lower in KA and SCC compared with MCC (median/mean loads 0.005/0.015 [KA] vs 0.023/0.059 [SCC] vs 2.613/56.840 [MCC] MCPyV-DNA copies per beta-globin gene copy). All MCC analyzed (n = 3) expressed MCPyV large T-antigen, whereas 8 KA and 7 SCC were negative in immunohistochemistry. LIMITATIONS: The relatively small number of samples is a limitation. CONCLUSIONS: Our findings argue against a pathogenic role of MCPyV in KA and SCC.

High throughput sequencing reveals diversity of Human Papillomaviruses in cutaneous lesions.

There are at least 120 completely characterized human papillomavirus (HPV) types and putative new types are continuously found. Both squamous cell carcinoma of the skin (SCC) and other skin lesions commonly contain multiple cutaneous HPV types. The objective of this study was to achieve an improved resolution of the diversity of HPV types in lesions such as SCCs, actinic keratoses (AKs) and keratoacanthomas (KAs). Fresh frozen biopsies from 37 SCC lesions, 36 AK lesions and 92 KA lesions and swab samples from the top of the lesion from 86 SCCs and 92 AKs were amplified using the general HPV primers FAP and mixed to three pools followed by high throughput sequencing. We obtained 2196 reads with homology to HPV. In the pool of SCC/AK biopsies 48 different HPV types were found. Eighty-three types were found in the pool of SCC/AK swab samples and 64 types in the KA biopsies, respectively. For 9 novel putative HPV types most of the amplimer sequence was obtained, whereas for an additional 35 novel putative HPV types only partial amplimer sequences were obtained. Most of the novel putative types belonged to the genus Gamma. In conclusion, high throughput sequencing was an effective means to identify both known and previously unknown HPV types in putatively HPV-associated lesions and has revealed an extended diversity of HPV types.

The Bcl-xL inhibitor of apoptosis is preferentially expressed in cutaneous squamous cell carcinoma compared with that in keratoacanthoma.

Keratoacanthoma (KA) is difficult to histologically distinguish from squamous cell carcinoma (SCC). Therefore, although KA is a benign self-resolving skin lesion, KA is commonly treated as SCC. Biomarkers to distinguish KA and SCC would thus be desirable. In search for specific markers, paraffin-embedded tissue samples from 25 SCC and 64 KA were arranged in a tissue microarray (TMA) and stained for immunologic cell-markers CD3, CD20 and CD68 as well as for proteins considered of relevance in tumorgenesis, namely NF kappaB/p65, I kappaB-alpha, STAT3, p53, TRAP-1, pRB, phosphorylated pRb, Cyld, p21, p16(INK4), Survivin, Bcl-xL, Caspase 3, Bak, FLK-1/VEGF-r2 and Ki-67. In addition, the tumors were tested for presence of human papillomavirus by PCR. We detected that the two lesions differed significantly in expression of Bcl-xL which was present in 84% of the SCC compared with only 15% in the KA (p < 0.001). The lower expression of the antiapoptotic protein Bcl-xL in KA is consistent with a possible role of apoptosis in the regression of KA.

Characterisation of the first complete genome sequence of the roe deer (Capreolus capreolus) papillomavirus.

The complete genomic DNA of a novel roe deer (Capreolus capreolus) papillomavirus (CcPV1) was amplified and sequenced from fibropapillomatous skin lesions of a Hungarian roe deer. Viral DNA was detected by a pair of degenerate primers and the remaining genomic sequence was amplified by a long-template high-fidelity PCR and sequenced. The CcPV1 genome was 8032 bp long and contained open reading frames (ORFs) typical for Delta-papillomaviruses (E6, E7, E1, E2, E4, E5, E9, L2, and L1) and a 799 bp long untranslated regulatory region (URR). Phylogenetic analysis based on the 3861 bp long concatenated sequence of the E1-E2-L2-L1 ORFs and on separate alignments of all major ORFs using both neighbour-joining and maximum parsimony methods placed CcPV1 on a distinct branch between Ovine papillomavirus 1 and the other deer papillomaviruses within the Delta-papillomavirus genus, although pairwise nucleotide alignments of L1 ORF sequences determined highest identities with European Elk Papillomavirus (71.2%) and Reindeer Papillomavirus (70.3%).

HPV 6-positive giant keratoacanthoma in an immunocompetent patient.

Keratoacanthoma (KA) is a clinically distinct, rapidly growing lesion that generally presents as a solitary crateriform nodule in sun-exposed areas in elderly, fair-skinned individuals. A KA larger than 20-30 mm is referred to as giant keratoacanthoma, a relatively rare lesion especially in young patients. Such lesions grow rapidly with possible destruction of underlying tissues. In addition to ultraviolet exposure, KAs have also been associated with chemical carcinogens, chemical peels, genetic factors, chronic skin conditions that produce scarring, trauma and thermal burns. Immunosuppressed patients, especially after transplantation, also develop KAs. A viral etiology has been suggested but not confirmed. We encountered a case of giant keratoacanthoma (greater than 50 mm in diameter) with induration of underlying structures on the upper lip of a 39-year-old male sailor. The patient reported sudden appearance and rapid enlargement of the lesion in only three weeks. Biopsy of the cutaneous lesion and the characteristic clinical history suggested the diagnosis of keratoacanthoma. Total excision with primary closure of the defect by a nasolabial advancement flap was performed. Histological examination of the tumor mass confirmed the diagnosis of KA with infiltrative growth and perineural invasion. Immunosuppression was excluded by blood analyses, as were HIV, syphilis and hepatitis infections. Only low-risk genital HPV type 6 was detected in the lesion, suggesting a possible cocarcinogenic effect of HPV and UV light in a chronically sun-exposed patient.