Molecular investigation of some DNA viruses in mucosal melanoma: Case–control study

The association between viral infections and both cutaneous and mucosal melanoma (MM) has not been fully investigated. Here, we assessed the prevalence of the DNA of a broad range of viruses in 31 MMs and 15 biopsies of healthy mucosa (HM) using molecular methods. The parvoviruses CuV and B19V, herpesviruses HSV1, HSV2, EBV, HHV6, and HHV8, polyomavirus MCPyV, and α‐HPVs were not detected, or rarely found, in MMs, and in HM, of the digestive, respiratory, and female genital tract. The overall prevalence of β‐HPV in MMs was not significantly higher compared to that in HM (70.9% and 53.3% respectively; p = 0.514). However, the number of MMs positive for β‐HPV types belonging to Species 3 and 5 and for some viral types belonging to Species 1, 2, 3, and 5 were significantly higher compared with HM (p < 0.05). Moreover, compared to HM, the MM samples contained a significantly higher number of β‐HPV types, mainly belonging to Species 1, 3, and 5 (p < 0.05). Our data, although suggesting a role for certain β‐HPV types in MM oncogenesis, require additional investigation in larger populations to support this hypothesis.


| INTRODUCTION
Mucosal melanoma (MM) is a rare tumor, accounting for only 0.03% of all cancers and for 1.3% of all melanomas. 1However, MM is clinically relevant because it is more aggressive and has a worse prognosis than the more common cutaneous melanoma (CM). 1 The main risk factors for CM include ultraviolet (UV) radiation, fair skin, a high number of acquired melanocytic nevi, and family history. 2Conversely, exposure to UV radiation does not appear to be a risk factor for MM.To date, the etiology of MMs is still unknown and genetic and/or environmental risk factors have not been clearly identified. 3Although a direct correlation between cigarette smoking and MM could not be demonstrated, it is known that 66% of patients with melanoma of the nasopharyngeal tract report a history of smoking. 4,5Moreover, vulvovaginal MM is associated with chronic inflammation, local trauma, use of irritant agents, and family history of CM, 3 while anorectal MM seems to be more common in patients with HIV infection.
It is known that some viral infections are associated with a variety of human cancers.However, the role of viruses, as a risk factor for melanoma has not been fully investigated.

| Viral DNA detection
To detect DNA of HSV1 and 2, EBV, HHV6 and 8, B19V, CuV, and MCPyV, PCR amplification assays were used as described in previous reports (Table 2).To detect HSV1 and EBV DNA two SybrGreen Real-time PCRs were carried out using reaction mix containing 400 nM of each primer.After the initial activation step, 40 cycles of amplification (95°C for 15 s, 60°C for 30 s, and 60°C for 10 s) were performed, with acquisition for 10 s at 60°C, after the extension step.
The specificity of Sybr-Green real-time PCRs was verified by melting curve analysis.For Melting analysis, ramp from 65°C to 95°C was used, rising by 0,1°C each step.
To detect HSV2, HHV6, HHV8, B19V, CuV, and MCPyV TaqMan Real-Time PCRs were done using master mix containing 400 nM of each primer and 200 nM of probe.After initial activation step, 40 cycles of amplification (95°C for 15 s and 60°C for 30 s) were performed with acquisition during the extension step.The amplification reactions were performed using Rotor Gene Q (Qiagen).
The analytical sensitivity of the PCRs for detection of viral DNA, in our experimental conditions, was determined by endpoint titration of viral target sequences.As reported in Table 2, the sensitivity of the reactions used in this study varied from 100 to 550 genome copies/ml.
HPV detection and genotyping were carried out using line probe assays, based on the PCR amplification and reverse hybridization principle.In particular, after amplification with biotinylated primers, the biotinylated amplicons were hybridized with a type-specific oligonucleotide probes immobilized as parallel lines on membrane strip and detected with streptavidin-conjugated alkaline phosphate and BCIP/NBT chromogen.The bands were then analyzed against a reference sheet.The mucosal α-HPVs were detected and typed using Ampliquality HPV-type Express v3.0 kit (AB Analitica), which amplify a sequence localized in L1 region of viral genome and can detect 40 genotypes (Table 3).The skin β-HPVs were detected and typed using RHA Kit Skin (beta) HPV (Diassay) which amplify 117 bp sequence localized in E1 region of viral genome and is able to identify 25 genotypes (Table 3).

| Statistical analysis
Continuous variables are reported with median and range (min-max values).Given the limited number of samples, we used the exact Fisher test for the categorical variables, while for the continuous variables a nonparametric approach was used: Mann-Whitney test or Wilcoxon test for comparisons between two groups.The Odds Ratio with the 95% confidence interval (CI95%) is reported.
The beta-HPV species (from 1 to 5) were analyzed both as categorical variable (presence/absence for each species) and as continuous variable.For the first analysis, a sample was deemed positive for a viral species when at least one virus belonging to that species was found in that specific sample.For the latter case, for each sample (MM patients or healthy donors), we report the number of identified genotypes among those tested for each single species and for all the species grouped together.
The statistical analysis was performed using the R software version 4.2.2-"Innocent and Trusting" (R Core Team (2022).R: A language and environment for statistical computing.R Foundation for Statistical Computing, Vienna, Austria.URL https://www.R-project.org/) with RStudio.
As shown in Figure 1A, the overall number of β-HPV types detected in MM samples was significantly higher in comparison with HM samples (p = 0.0268); that is, on average, the number of β-HPV types detected in MM samples (median 3 with a range between 0 and 18) was higher than that of the control samples (median 1 with a range between 0 and 3 types).
The β-HPV types more frequently detected in MM samples belonged to Species 1, 2, and 3.In fact, in the MM samples, the number of the viral types belonging to these species spread in a wide range (0-8, 0-6, and 0-3 types, respectively), while in the HM samples, viruses belonging to only two species (Species 1 and 2, range 0-3 types were detected).
Moreover, the comparison of the number of β-HPV genotypes in MM and HM revealed a statistical difference between the number of β-HPV belonging to Species 1 (p = 0.0434), 3 (p = 0.0154), and 5 types (p = 0.00474).
The β-HPV types belonging to Species 1, 2, and 3 were also the most frequent in the specimens obtained from females.However, in this case, the statistical comparison between MM and controls revealed a significance only for HPVs belonging to Species 5 (p = 0,007, Figure 1B).The Figure 1C shows, that in males the overall number of β-HPV types detected in MM samples (median 3.5 in a range between 0 and 16) was higher in comparison with HM samples    The boxplot graph shows the median (black line) and the number of genotypes identified in each patient (black circles), while the whiskers were calculated using the following formula: IQR (interquartile range) × 1.5.p < 0.05 was considered statistically significant.HM, healthy mucosa; HPV, human papillomavirus; MM, mucosal melanoma.
(median 0 with a range between 0 and 3) but no statistical significance was demonstrated.
Our results suggest that some, but not all, β-HPV types could play a role in the pathogenesis of MM.
In the digestive tract (Figure 2B), the number of HPV types was significantly higher (p = 0.01) in MM (median 4 with a range between 0 and 18) than in HM (median 0 with a range between 0 and 3).
Moreover, the comparison of the number of β-HPV genotypes in MM and HM revealed a statistical difference between the number of β-HPV belonging to Species 2 (p = 0.00876), 3 (p = 0.0319), and 5 (p = 0.00579).The number of β-HPV Species 1 was also higher in MM compared with HM, but the difference did not reach statistical significance (p = 0.0511).There was no difference in the number of viral types in MM of the respiratory tract compared to controls (p = 1) (Figure 2A).

| DISCUSSION
To the best of our knowledge, this is the largest study investigating the presence and distribution of a broad range of DNA viruses in MMs by molecular methods.Here, we found that all MM were negative for CuV, HSV1, HSV2, EBV, HHV6, and HHV8 DNA, thus, we could exclude a role of these viruses in the pathogenesis of MM.
Data in the literature are in line with our results.Indeed, the CuV genomic sequences, first detected in one sample of CM by viral enrichment and high throughput sequencing, 6 were subsequently detected in only 2 out of 185 melanoma biopsies using specific realtime PCR. 19Some human herpesviruses were also only rarely detected in MM 13,14 Moreover, in our study, there was no significant association between B19V infection and MM.B19V DNA was detected rarely, both in MM and HM samples, and the viral load was generally low, suggesting an asymptomatic persistence rather than a causative role of the virus in MM pathogenesis.Indeed, it is known that a low copy number of B19V DNA can persist in various organs and tissues without any apparent pathological consequences. 20 contrast to MCPyV-induced Merkel cell carcinoma, where viral DNA is detected in about 80% of cases 21 and the viral load is at least 1 genome per cell, 22 our results show no significant difference in the prevalence of MCPyV between MM and HM, with a very low viral load in both tumors and controls.In line with our results, a limited copy number of MCPyV genome has been demonstrated in various organs and tissues without any apparent pathological consequences. 23Moreover, MCPyV, as well as other human polyomaviruses (PyVs) DNA, was not detected in primary MMs using sensitive Luminex assay 9 or nested type-specific PCRs. 12Accordingly, human polyomaviruses are presumably not major factors for the development of extracutaneous melanomas.
Regarding HPV infection, it is well known that the persistence of high-risk HPV (HR-HPV) types belonging to the α-HPV genus is associated with carcinomas of the mucosal anogenital tract and head and neck carcinomas. 24In the present study, α-HPV DNA, namely HR-HPV16 DNA, was detected in only 1 MM sample from the respiratory tract (nasal cavity).Thus, such a low prevalence of α-HPV with previous studies where α-HPV DNA was not found 11 or was rarely detected in MMs. 10 However, Rohwedder and colleagues 25 describe two cases of vulvar melanomas with multiple α-HPV types, including HPV16 and some putative novel α-HPV types identified by degenerated nested PCR in both the malignant melanoma and surrounding skin.They can also be found in the mucosa, 29 but their role in the pathogenesis of tumors is still unclear.
Several β-HPV species and different viral types have been detected in different types of lesions 30 and seem to have varying oncogenic potential.
A group of specific β-HPV types, indicated as EV-HPVs, has been associated with cutaneous squamous cell carcinoma (SCC) in patients with Epidermodysplasia Verruciformis (EV) 29 and in organtransplanted patients. 31Moreover, β-HPV Species 1 types have been more frequently detected in benign skin lesions than in the matched perilesional skin, while there was a tendency for types belonging to Species 2 to be associated with SCC and actinic keratoses. 30Patel and colleagues found that β-HPV Species 1 types were detected more often in SCC than in basal cell carcinoma (BCC). 32In our previous investigation, we found that the genotypes belonging to Species 1 were statistically more common in nonmelanoma skin cancers (NMSCs) than in paired perilesional skin, and this was also the case with BCC patients alone. 33Using a broad spectrum of degenerate and type-specific primers, Rohwedder et al. 11 identified β-HPV DNA (both cutaneous and EV-HPV) in 67% of vulvovaginal melanomas.The authors proposed that their findings may represent a molecular record of HPV involvement in pathogenesis or progression of melanoma, which is consistent with the strong but poorly defined association of cutaneous HPV types with nonmelanoma skin cancers.
In the present study, a high prevalence of β-HPV in analyzed samples and the large number of viral types detected (especially in MM) prompted us to hypothesize a possible association between MM and β-HPV infection.Although the overall prevalence of β-HPV in MM was not significantly higher compared to controls, we found that the prevalence of β-HPV belonging to Species 3 and 5 and of some viral types belonging to Species 1, 2, 3, and 5 were significantly higher in tumors than in healthy mucosa.In addition, compared to HM, the MM samples contained a significantly higher number of

Formalin-fixed
paraffin-embedded (FFPE) samples of primary MM tissues (n = 39) and biopsies from HM (n = 15) were retrospectively selected from the Archive of the Section of Pathology, Department of Health Sciences, University of Florence, Florence, Italy.Ethical Committee approval for the study was obtained from the Ethics Committee of Area Vasta Centro (Azienda Ospedaliero Universitaria Careggi, Florence, Italy), nr.2015/00037100; amendment 2017-389.T A B L E 1 Characteristics of the tissue samples analyzed in the study.

T A B L E 6
Distribution of β-HPV types in MM and HM.

F I G U R E 1
Number of β-HPV genotypes detected in MM and HM in all patients (A), in females (B), and in males (C).The number of different β-HPV genotypes and of different β-HPV species (from 1 to 5) was determined and then statistically analyzed using Wilcoxon signed rank test.
Number of β-HPV genotypes detected in MM and HM in the respiratory tract (A) or the digestive tract (B).The number of different β-HPV genotypes and β-HPV species (from 1 to 5) was detected and analyzed using Wilcoxon signed rank test.The boxplot graph shows the median (black line), the number of genotypes identified in each patient (black circles), and the whiskers calculated with the following formula: IQR × 1.5.p < 0.05 was considered statistically significant.HM, healthy mucosa; HPV, human papillomavirus; IQR, interquartile range; MM, mucosal melanoma.infection does not support the hypothesis of a causal association between α-HPV infection and MM.Moreover, our results are in line β-HPV types belonging to Species 1, 3, and 5.The viral types more frequently detected in those belonging to Species 1, 2, and 3 in tumors and 1 and 2 in controls.Therefore, in line with the literature data, our results suggest that some, but not all, β-HPV types could play a role in the pathogenesis of MM.Our results, although suggesting a role for certain β-HPV types in MM oncogenesis, due to the limited sample size, require additional larger studies to support this hypothesis.Moreover, further studies are needed to investigate the biological activity of viral types detected in tumors and controls.Determination of viral load, viral gene expression, and interference of β-HPV infection with cellular gene expression in tumors and in healthy mucosa could help understand the role of these viruses in MM.Interestingly, Paolini and colleagues demonstrated that the upregulation of p16INK4a and AKT/P13K pathways in BCC is often associated with the presence of β-HPV belonging to Species 2, suggesting that in a subtype of BCC, these viruses may have a role in the carcinogenesis or in other, still undefined, biological properties of these tumors.34In conclusion, the investigation of a broad range of DNA viruses, including the detection and typing of 40 mucosal and 25 cutaneous HPVs, represents a major point of the strength of this study.A potential limitation is the relatively low number of tissue samples analyzed.Overall, our data do not support a primary role for viruses in MM pathogenesis.However, some of them (mainly some β-HPV types widely detected in melanoma samples) could act as cofactors in the development of MM.Furthermore, since the MM samples contained a high number of viral types, we cannot exclude the concomitant carcinogenic action of coexisting viruses.Another possible explanation is that the viruses could persist on the mucosa as harmless commensals.Further studies are needed to understand the real significance of β-HPV DNA persistence in MM.
-one out of 39 MM specimens and 15 out of 15 HM samples had amplifiable DNA, since they were positive for housekeeping AP3B1 (Adaptor Related Protein complex 3 subunit Beta) gene by PCR, and these samples were tested further for the presence of viral DNA.
T A B L E 1 (Continued) Thirty Amplification assays used to detect viral DNA.
Viral DNA detection in MM and HM specimens.out of 31 (25%) MM and 8 out of 15 (53.3%)HM tested positive for MCPyV DNA.The difference was not statistically significant, and the viral load was extremely low in both tumors and controls (from 2 × 10 2 to 9 × 10 3 copies/10 6 cells for MM and from 1.6 × 10 2 to 4.8 × 10 3 copies/10 6 cell for HM) suggesting, also in prevalence of viral DNA in MM and HM found in the present study is reported in Table4.All samples (both tumors and controls) were negative for CuV, HSV1, HSV2, EBV, HHV6, and HHV8 DNA indicating that these viruses are not associated with the pathogenesis of MM.B19V DNA was detected in 4/31(12.9%)MMand in 2/15 (13.3%)HM, and the viral load was generally low in both types of samples (from 5.4 × 10 3 to 6.9 × 10 4 copies/10 6 cells for MM and from 5.8 × 10 4 to 6.3 × 10 4 copies/10 6 cell for HM) suggesting an asymptomatic, persistent infection rather than a causative role of the virus in MM pathogenesis.T A B L E 3 HPV types detectable in the present study.Note: p-Value was calculated with Fisher's exact test.p< 0.05 was considered statistically significant.Abbreviations: B19V, human parvovirus B19; CI, confidence interval; CuV, cutavirus; HHV, human herpesvirus; HM, healthy mucosa; HPV, human papillomavirus; HSV, herpes simplex virus; MCPyV, Merkel cell polyomavirus; MM, mucosal melanoma; na, not applicable; OR, odds ratio.Eight this case, asymptomatic persistence of viral DNA, not the etiologic role of the virus in MM.Regarding α-HPV, HR-HPV16 DNA was detected in only 1/31 (3.2%) MM samples collected from the respiratory tract (nasal cavity).Thus, such low prevalence of α-HPV infection does not support the hypothesis of a causal association between α-HPV infection and MM.The overall prevalence of β-HPV was high in both MM and HM (22/31 [70.9%] vs. 8/15 [53.3%], respectively) and no statistical difference was found.However, MM samples containing β-HPV Species 3 and 5 were significantly more frequent compared to HM specimens (10/31 [32.3%] vs. 0/15, p = 0.019 and 18/31 [58.1%) vs.