Pleural mesothelioma patients and ex-exposed asbestos workers are immunologically poor responders to oncogenic Merkel cell polyomavirus

Introduction

Pleural mesothelioma (PM) is an aggressive tumor of the serous cavities (1-3). The inhalation of asbestos, a fibrous silicate oncogenic and immunomodulatory mineral, is the main PM causative factor (4,5). Since PM arises and progresses mainly among workers ex-exposed to asbestos (WEA), this cancer is considered an occupational disease. PM predicting factors are almost completely unknown (6-8). Clinical signs in early disease stages are absent or non-specific, and PM is diagnosed in advanced stages (9-12). Genetic predispositions, such as mutations in the BRCA1-associated protein-1 (BAP1) gene, are known to contribute to PM onset (13). The potential involvement of oncogenic viruses, such as the polyomavirus simian virus 40 (SV40), in asbestos-related tumors has long been debated (14), but it highlights the possibility of viral involvement in PM.

Merkel cell polyomavirus (MCPyV) is the main causative agent of Merkel cell carcinoma (MCC) (15,16). The viral DNA integration into the host genome, the expression of two viral oncoproteins large T (LT) and small T (sT) antigens, and mutations resulting in LT truncation (tLT) (17), are considered key events in MCC onset. These oncoproteins, along with structural proteins viral protein (VP)1 and VP2, play significant roles in the virus lifecycle and are widely used in immunological studies (18). MCPyV is a ubiquitous virus in humans with a reported seroprevalence of 60–80% (19). Initial exposure to MCPyV typically occurs early in life, followed by the establishment of lifelong asymptomatic infection in immunocompetent individuals. However, in conditions of immune system impairment, MCPyV activity can increase, favoring MCC onset. Since immunosuppression plays an important role in increasing viral activity, patients with immunocompromised/oncological diseases might be at risk of developing MCPyV infection (20,21).

Although the immunological response against polyomaviruses such as SV40 has been evaluated in PM patients and asbestos-exposed individuals, the MCPyV serology in these classes of patients/individuals remains unknown. The previously reported findings on SV40 raised the hypothesis of whether MCPyV might be immunologically associated to asbestos-related tumors, such as PM. Given this context, this study aimed to investigate whether MCPyV might be immunologically associated with asbestos-related tumors such as PM. To this aim, sera from PMs and WEAs were analyzed for MCPyV serology using a recently developed indirect immunoassay based on specific MCPyV VP mimotopes (22). A group of healthy subjects (HS) was included as control. MCPyV seroprevalence/serological profiles were determined. To assess the humoral immune status of PMs and WEAs, the total fraction of serum immunoglobulin G (IgG) was determined. MCPyV serology was then longitudinally assessed in relation to PM patient’s overall survival (OS) and to WEA exposure factors. Moreover, the presence of MCPyV DNA and VP1 and LT messenger RNAs (mRNAs) was evaluated in set of formalin-fixed and paraffin-embedded (FFPE) PM specimens obtained from patients unrelated to PM serum donors. This study seeks to understand if MCPyV contributes to the pathogenesis of PM and whether this association could have implications for diagnosis and prognosis, for patients with asbestos-related tumors. We present this article in accordance with the STROBE reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-198/rc).

Methods

Sera

This is a cross-sectional study. PM (n=108), WEA (n=102), and HS (n=110) sera were collected and coded with indications of age, gender, and clinical and exposing factors information (Table 1). Mean age ± standard deviation (SD) of the PM, WEA, and HS groups were 71±11, 66±6, and 64±13 years (P>0.05), respectively. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. Written informed consent was obtained from all participants. The Ethical Committee of Ferrara Hospital, Italy, approved the study (No. 151078). Among the WEA group, 40 sera were collected at Villa Scassi Hospital, Genoa, Italy, from workers previously exposed to asbestos, either retired or still actively employed in the dock/shipyard as longshoremen involved in loading/unloading operations or in the iron- and steel-mill industries. These workers were identified from the historical database of the Italian Agency for Occupational Accident Insurance. The Ethical Committee of Liguria Region, Italy, approved the study (No. 54/2017). HS sera were obtained from discarded laboratory samples at the clinical laboratory analysis unit, University Hospital of Ferrara, Ferrara, Italy, after routine blood analyses and prior to their scheduled destruction by incineration (18,23). HS hospital records indicated that blood parameters were in the normal index range. PM, WEA, and HS sera were obtained under standardized conditions from overnight fasting subjects/patients. Sera were immediately isolated from collected blood, aliquoted, and stored at −80 ℃ until immunological testing. Eligibility criteria were: (I) WEA individuals aged >18 years old with a confirmed past of asbestos exposure; (II) PM patients with clinically diagnosed PM; and (III) HS individuals aged >18 years old with no history of asbestos exposure and clinically diagnosed PM or other tumors.

Cumulative asbestos exposure

Cumulative asbestos exposure was measured according to the quantitative approach developed by the German Confederation of Trade Associations adopted by the Italian Agency for Occupational Accident Insurance (24). To determine the average daily fiber concentration [C; measured in fiber-year load per cubic centimeter (cc)] to which a worker has been exposed over 1 year, calculations were based on the airborne fiber levels in their specific workplace considering an 8-hour workday and a 240-day work year, using the following equation: C = F × t × k, where F = concentration of airborne fibers in the specific workplace setting/occupational duty, t = duration of exposure in hours, and k=5.21×10⁻⁴, a constant representing the total number of annual working hours [1/(8 h/day × 240 days/year) =1/1,920=5.21×10⁻⁴]. By multiplying the fiber-year load by the number of years of exposure, we obtained the total cumulative dose expressed in fibers/cm³ of air (ff/cc) per total exposure years (24).

MCPyV-specific immunoassays

The MCPyV serology was investigated using an indirect immunoassay (22). Two linear peptides/mimotopes named MCPyV VP1 S and VP2 F were employed for detecting anti-MCPyV IgGs. Plates were coated with 5 µg of peptide for each well, diluted in 100 µL of coating buffer 1×, incubated at 4 ℃ for 16 h and then washed with washing buffer (WB). Plates were treated with 200 µL/well of blocking solution, incubated at 37 ℃ for 90 min, after which each well was filled up with 100 µL of 1:20 diluted sera in low cross-buffer. Solutions were purchased from Candor Bioscience, Wangen, Germany. Plates were then incubated at 37 ℃ for 90 min. Each well was rinsed with WB before adding the goat anti-human IgG heavy (H) and light (L) chain specific peroxidase-conjugate secondary antibody (Merck, Darmstadt, Germany) diluted 1:10,000. Plates were incubated at room temperature (RT) for 90 min. Wells were rinsed with WB and then added with 100 µL of ABTS (Sigma, Milan, Italy), incubated at RT for 45 min and read using a spectrophotometer (Thermo Electron, Vantaa, Finland) at a wavelength (λ) of 405 nm. Technical control sera were as reported (22). The cut-off value of each assay was determined as the mean of the optical density (OD) readings of 3 MCPyV-negative control sera plus 3 SDs of mean (mean +3 SDs) (25). Sera were positive when reacting to both S and F peptides. Serological profiles of anti-MCPyV IgGs were reported as median [interquartile range (IQR)] ODs for S and F peptides. Experiments were performed in triplicate.

Total IgG antibodies

Total serum IgG concentration was determined using the human IgG total enzyme-linked immunosorbent assay (ELISA) kit (Thermo, Milan, Italy). Diluted sera and unbound horseradish peroxidase (HRP)-conjugated anti-human total IgG antibody were incubated with an anti-human total IgG-coated antibody in pre-coated wells. Upon incubation, unbound HRP-conjugated anti-human total IgG antibody was removed during a washing step, and a substrate solution that is reactive with HRP was added. ODs were read at a λ of 450 nm. Total IgG concentration was measured in duplicates, interpolated from the total IgG standard curves, and corrected for sample dilution. The final total serum IgG concentration was reported as mg/mL, with a reference range in HS of 8–16 mg/mL (26).

MCPyV DNA detection by droplet-digital polymerase chain reaction (ddPCR)

Viral DNA was evaluated in randomly chosen FFPE PM specimens (n=50) and healthy pleura (HP) specimens (n=15). FFPEs were obtained from an additional group of PM patients unrelated to PM serum donors. Total DNA was extracted from FFPEs using the QIAmp DNA FFPE Tissue Extraction Kit (Qiagen, Milan, Italy). A total of 100 ng of input DNA was analyzed by ddPCR using a QX200 Droplet Digital system (Bio-Rad, Milan, Italy) for the detection and quantification of MCPyV DNA (15). A threshold line was employed to discriminate positive and negative droplets. EIF2C1 housekeeping gene was used to determine human cell equivalents. DNA isolation/H₂O controls were included. Viral DNA loads are reported as mean MCPyV DNA load ± standard error of mean (SEM) copy/cell.

MCPyV VP1 and LT mRNAs detection by quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from FFPEs using the RNeasy FFPE Kit (Qiagen). Total RNA (250 ng) was retro-transcribed using the Improm II (Promega, Fitchburg, WI, USA) reverse transcription system while complementary DNAs (cDNAs) were analyzed for MCPyV LT and VP1 mRNA expressions by qPCR using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) (27,28). Plates were run on CFX96 Touch Real-Time PCR (Bio-Rad). Three technical replicates per sample were used (29). qPCR data were analyzed using the ΔCt method, with normalization to the housekeeping gene GAPDH, which was included as an internal control in each reaction. MCPyV VP1 and LT mRNA levels are reported as mean 1/ΔCt ± SEM.

Statistical analysis

A two-sided Chi-squared test was applied for statistically analyze the MCPyV seroprevalence. Quantitative values were analyzed using the D’Agostino Pearson normality test, while parametric/non-parametric tests were applied according to normal/non-normal variables, respectively (30). MCPyV rates were assessed in relation to patient’s OS at 99 months using the Kaplan-Meier model (31); receiver operating characteristic (ROC) curves were used to calculate the area under the curve (AUC) for evaluating the reliability of MCPyV serology as PM and/or asbestos-exposure biomarker. Sensitivity, specificity, and Youden’s (J) index were computed. Spearman’s correlation (r) and linear regression (R²) coefficients were employed to assess the relationships between OD levels and asbestos exposure parameters in WEA samples, as well as between viral DNA copy number and mRNA levels. ddPCR data were analyzed using the QuantaSoft tool (Bio-Rad). Statistical analyses were done with GraphPad Prism version 8.0 (GraphPad, La Jolla, CA, USA). P<0.05 was considered statistically significant.

Results

Serum IgG antibodies to MCPyV in PM patients, WEA, and HS

Sera from PM, WEA, and HS were analyzed for their IgG reactivity to MCPyV VP1 S and VP2 F peptides (Table 2). S peptide-positive sera were also positive for F peptide and vice versa; a few sera were negative for S peptide while testing positive for F peptide, and vice versa. The combined overall rate of anti-MCPyV IgGs, for both peptides, was 26.9% (29/108), 27.5% (28/102), and 60.9% (67/110), in PM, WEA, and HS sera, respectively (Figure 1A, Table 2). The difference between PM and HS and between WEA and HS was statistically significant (P<0.001). No difference in MCPyV seroreactivity was found between PM and WEA (P>0.05) (Figure 1A, Table 2).

Figure 1 Seroprevalence and serological profiles of anti-MCPyV IgG antibodies and total IgG concentration in sera. (A) MCPyV seroprevalences were evaluated in PM patients (n=108), WEA (n=102), and HS (n=110). (B,C) Serological profiles of serum antibody reactivity to VP1 S (B), VP2 F (C) peptides evaluated in PM patients, WEA, and HS. Immunologic data are reported as OD value readings at λ 405 nm for sera analyzed in indirect immunoassay. In the scatter dot plot, each dot represents the dispersion of ODs of each sample. The median is indicated by the line inside the scatter plot with the IQR in PM patients, WEA, and HS. (D) Total IgG concentration values in serum samples from PM patients, WEA, and HS, reported as mg/mL. Data are presented as IgG mean values and their ranges marked by short horizontal lines. The dashed line indicates the cut-off value. ****, P<0.0001. HS, healthy subjects; IgG, immunoglobulin G; IQR, interquartile range; MCPyV, Merkel cell polyomavirus; OD, optical density; PM, pleural mesothelioma; VP, viral protein; WEA, workers ex-exposed to asbestos.

Serological profiles of circulating anti-MCPyV IgGs determined in PM (n=108), WEA (n=102), and HS (n=110), are detailed in Figure 1. ODs for S peptide were 0.08, 0.08, and 0.16 in PM, WEA, and HS, respectively. ODs for F peptide were 0.09, 0.09, and 0.33 in PM, WEA, and HS, respectively. Both S and F peptide ODs were lower in both PM, and WEA compared to HS (P<0.001).

No difference in MCPyV seroreactivity and ODs were found for both peptides, either considered alone or in combination, between HS males and females (P>0.05, data not shown).

Total serum IgG concentration

Total IgG levels were evaluated in PM (n=108), WEA (n=102), and HS (n=110) sera to evaluate their humoral immune status (32). The dispersion of total IgGs as mean (mg/mL) ± SD for each sample is shown in the scatter dot plot (Figure 1D). Total IgGs were detectable in all patients/individuals. The mean total IgG concentration (mg/mL) ± SD was 4.29±2.13, 5.72±2.2, and 4.55±2.07 in PM, WEA, and HS sera, respectively (P>0.05).

MCPyV serology as asbestos biomarker

ROC curves were generated to determine the reliability of our assay in discriminating PM and/or WEA from HS according to serum anti-MCPyV IgG positivity/ODs. For PM, the assay demonstrated 77.3% and 91.8% sensitivity, and 77.8% and 93.5% specificity, with J of 0.55 and 0.85, for S and F peptides, respectively. Areas under the curve (AUCs) for S and F peptides in PM and HS resulted as 0.85 and 0.98, respectively. The difference between the AUCs for S/F peptides were statistically significant compared to that of a worthless test (AUC =0.5, P<0.001, Figure 2A). For WEA, the assay demonstrated 79.1% sensitivity and 88.2% specificity, with a J of 0.67 for S peptide and 95.5% sensitivity and 93.1% specificity, with a J index of 0.89 for F peptide. AUCs resulted as 0.89 for S peptide and 0.98 for F peptide. The difference between AUCs for both peptides was statistically significant compared to that of a worthless test (P<0.001, Figure 2B).

Figure 2 MCPyV serology-based discrimination of PM patients and WEA from HS by ROC curve analysis. ROC curves were built in order to determine the reliability of VP1 S and VP2 F peptides in discriminating PM patients (n=108), WEA (n=102) from HS (n=110) according to anti-MCPyV IgG positivity/OD levels. (A) AUCs for VP1 S and VP2 F peptides in the comparison between PM and HS. (B) AUCs for VP1 S and VP2 F peptides in the comparison between WEA and HS. The diagonal line shows an AUC value of 0.5 which is representative of a worthless test. AUC, area under the curve; HS, healthy subjects; IgG, immunoglobulin G; MCPyV, Merkel cell polyomavirus; OD, optical density; PM, pleural mesothelioma; ROC, receiver operating characteristic; VP, viral protein; WEA, workers ex-exposed to asbestos.

Longitudinal analyses and PM subtypes

Kaplan-Meier model was used to longitudinally evaluate a possible association between MCPyV seropositivity and PM (n=108) patient’s OS during a 99 months follow-up. No significant differences were found (P>0.05, Figure 3A). Serum anti-MCPyV IgG rates were determined in PM stratified in epithelioid (n=40), sarcomatoid (n=25), and biphasic (n=43) histotypes. Sera from epithelioid, sarcomatoid and biphasic PMs react to combined S and F peptides with an overall rate of 40% (16/40), 28% (7/25), and 14% (6/43), respectively. A lower MCPyV-seroprevalence was found in biphasic PMs compared to epithelioids (P=0.01, Figure 3B). MCPyV serological profiles were evaluated in the three PM histotypes (Figure 3C,3D). Lower ODs were determined for S peptide in biphasics compared to epithelioids (P<0.001, Figure 3C). F peptide ODs resulted similar across epithelioid, sarcomatoid and biphasic PMs (P>0.05, Figure 3D).

Figure 3 OS analysis, seroprevalence and serological profiles of anti-MCPyV IgG antibodies evaluated in PM patients, and total IgG concentration in sera. (A) Kaplan-Meier model was used to longitudinally evaluate an association between MCPyV seropositivity and PM (n=108) patient’s OS during 99 months follow up. No significant differences were observed between MCPyV-seropositive and seronegative PMs (P>0.05). (B) Seroprevalences were compared in epithelioid (n=40), sarcomatoid (n=24), and biphasic (n=43) PM histotypes. (C,D) Serological profiles of serum antibody reactivity to VP1 S (C) and VP2 F (D) peptides were evaluated in the three PM histotypes. Immunologic data are reported as OD value readings at λ 405 nm for sera analyzed in immunoassay. In the scatter dot plot, each dot represents the dispersion of ODs of each sample. The median is indicated by the line inside the scatter plot with the IQR in PM epithelioid (n=40), sarcomatoid (n=24), and biphasic (n=43). (E) Total IgG concentration values in serum samples from CaP-PM (n=19), M-PM (n=11), P-PM (n=8), CiP-PM (n=5), and no-PM (n=17), reported as mg/mL. Data are presented as IgG mean values and their ranges marked by short horizontal lines. (F) Seroprevalences were compared in PM patients under CaP-PM (n=19), M-PM (n=11), P-PM (n=8), CiP-PM (n=5), and no-PM (n=17). (G,H) Serological profiles of serum antibody reactivity to VP1 S (G) and VP2 F (H) peptides were evaluated in PM patients undergoing different therapeutic regimens. (C,D,G,H) Immunologic data are reported as OD value readings at λ 405 nm for sera analyzed in immunoassay. In the scatter dot plot, each dot represents the dispersion of ODs of each sample. The dashed line indicates the cut-off value. *, P<0.05; ***, P<0.001. CaP-PM, PM patients under carboplatin + pemetrexed; CiP-PM, PM patients under cisplatin + pemetrexed; IgG, immunoglobulin G; IQR, interquartile range; M-PM, PM patients under mixed therapy; MCPyV, Merkel cell polyomavirus; no-PM, PM patients under no chemotherapy; OD, optical density; OS, overall survival; P-PM, PM patients under pemetrexed alone; PM, pleural mesothelioma; VP, viral protein.

Total serum IgG concentration, MCPyV serology, and PM treatment regimens

Treatment regimen data were collected from PM patients (n=60). Then, sera from PM patients under carboplatin + pemetrexed (CaP-PM, n=19), mixed therapy (M-PM, n=11), pemetrexed alone (P-PM, n=8), cisplatin + pemetrexed (CiP-PM, n=5), alongside a sub-cohort of PM patients under no chemotherapy (no-PM, n=17), were evaluated for total IgG concentration and for their IgG reactivity to MCPyV S and F peptides. The mean total IgG concentration (mg/mL) ± SD was 3.97±1.9, 3.04±1.6, 3.34±2.26, 3.59±1.7, and 4.25±1.37 in CaP-PM, M-PM, P-PM, CiP-PM, and no-PM, respectively (P>0.05, Figure 3E). Sera from CaP-PM, M-PM, P-PM, CiP-PM, and no-PM react to combined S and F peptides with an overall rate of 36.8% (7/19), 36.4% (4/11), 12.5% (1/8), 60% (3/5), and 29.4% (5/17), respectively (P>0.05, Figure 3F). MCPyV serological profiles were evaluated in PM patients stratified according to the therapeutic regimen. Both S and F peptide ODs were similar across CaP-PM, M-PM, P-PM, CiP-PM, and no-PM cohorts (P>0.05, Figure 3G,3H).

MCPyV serology and exposing factors

Correlations among serum anti-MCPyV IgG presence and WEA exposing factors were evaluated. WEA were stratified according to the level of cumulative asbestos exposure, i.e., 0–8 ff/cc (n=27), 8.1–55 (n=57), and >55 ff/cc (n=18). Sera from 0–8, 8.1–55, and >55 ff/cc reacted to combined S and F peptides with an overall rate of 44.4% (12/27), 26.3% (15/57), and 5.6% (1/18), respectively. A lower anti-MCPyV IgG rate was found in >55 ff/cc compared to 0–8 ff/cc (P<0.001, Figure 4A). No correlations were determined among serum anti-MCPyV IgG rates and the remaining WEA exposing factors (P>0.05, Figure 4B,4C).

Figure 4 Seroprevalence of anti-MCPyV IgG antibodies among WEA stratified for asbestos exposing factors. (A) Seroprevalences were compared in WEA (n=102) stratified into three subgroups according to the level of cumulative asbestos exposure, i.e., 0–8 (n=27), 8.1–55 (n=57), and >55 ff/cc (n=18). (B) Seroprevalences were compared in WEA (n=102) stratified into four subgroups according to the years since last previous asbestos exposure, as follows: 0–10 (n=20), 11–20 (n=46), 21–30 (n=28), and >30 years (n=8). (C) Seroprevalences were compared in WEA (n=102) stratified into three subgroups according to the years of asbestos exposure, as follows: 0–20 (n=30), 21–30 (n=40), and >30 years (n=32). **, P<0.01. ff/cc, fibers/cm³ of air; IgG, immunoglobulin G; MCPyV, Merkel cell polyomavirus; VP, viral protein; WEA, workers ex-exposed to asbestos.

Serological profiles of anti-MCPyV IgGs in terms of median (IQR) ODs were assessed in WEA stratified according to exposing factors (Table 2, Figure 5). Lower median ODs were found for S peptide in >55 ff/cc compared to both 0–8 and 8.1–55 ff/cc (P<0.001, Figure 5A). F peptide median ODs resulted lower in >55 ff/cc than in 0–8 ff/cc (P=0.01, Figure 5B). ODs were then compared among WEA stratified according to years of asbestos exposure i.e., 0–20 (n=30), 21–30 (n=40), and >30 years (n=32). Median ODs for both S and F peptides were similar across 0–10, 11–21, 21–30, and >30 years groups (P>0.05, Figure 5G,5H). Lower median ODs were detected for S peptide in >30 years compared to 0–20 years (P=0.01), while no differences were detected when were compared to 21–30 years (P>0.05). Similar median ODs for F peptide were detected in 0–20, 21–30, and >30 years (P>0.05, Figure 5G,5H). To better evaluate the correlation between OD levels and WEA exposing factors, r and R² coefficients were evaluated. An inverse correlation was found between cumulative asbestos exposure and OD levels, with r values of −0.328 (P<0.001) and −0.3862 (P<0.001) and R² values of 0.06202 (P=0.01) and 0.05861 (P=0.01) for S and F peptides, respectively (Figure 5C). Conversely, a direct correlation was detected between years since last asbestos exposure and OD levels for S peptide, with an r of 0.2267 (P=0.02) and an R² of 0.04187 (P=0.04, Figure 5F). Lastly, an inverse correlation was determined between years of asbestos exposure and OD levels for both peptides. Specifically, r values of −0.3015 (P=0.002) and −0.2397 (P=0.02), and R² values of 0.05753 (P=0.02) and 0.00149 (P>0.05) for S and F peptide, respectively (Figure 5I).

Figure 5 Serological profiles of serum antibody reactivity to MCPyV VP1 S, VP2 F peptides and correlation analysis between OD levels and asbestos exposure parameters in WEA. Immunologic data are from WEA (n=102) stratified according to: (A-C) the level of cumulative asbestos exposure, i.e., 0–8 (n=27), 8.1–55 (n=57), and >55 ff/cc (n=18); (D-F) years since last previous asbestos exposure, i.e., 0–10 (n=20), 11–20 (n=46), 21–30 (n=28) and >30 years (n=8); and (G-I) the years of asbestos exposure, i.e., 0–20 (n=30), 21–30 (n=40) and >30 years (n=32). Results are reported as OD value readings at λ 405 nm for sera analyzed in indirect immunoassay. (A,B,D,E,G,H) In the scatter dot plot, each dot represents the dispersion of ODs for each sample. The median is indicated by the line inside the scatter plot with the IQR in stratified WEAs. The dashed line indicates the cut-off value. (C,F,I) Linear regression and Spearman correlation analyses were conducted to explore the relationships between OD levels and asbestos exposure parameters. (C,I) An inverse correlation was found between OD levels and both (C) asbestos cumulative exposure and (I) years of asbestos exposure for both peptides. (F) A direct correlation was reported between OD levels and years since the last asbestos exposure for VP1 S peptide. *, P<0.05; ***, P<0.001. ff/cc, fibers/cm³ of air; IQR, interquartile range; MCPyV, Merkel cell polyomavirus; OD, optical density; VP, viral protein; WEA, workers ex-exposed to asbestos.

Detection of MCPyV DNA and VP1 and LT mRNAs in PM tissues

The presence of MCPyV DNA was evaluated by MCPyV-specific ddPCR in FFPE PM specimens (n=50) from an additional cohort of PM patients and in HP specimens (n=15) (33) (Figure 6A,6B). MCPyV DNA was revealed in 32% (16/50) of FFPEs from PM patients, while HP specimens were MCPyV-negative (P=0.01). The mean viral DNA load ± SEM detected in MCPyV-positive FFPEs from PM patients was 0.39±0.2. MCPyV LT and VP1 mRNAs were next investigated in MCPyV DNA-positive FFPEs (n=16). All FFPE samples were positive for VP1 mRNA, with a mean 1/ΔCt value ± SEM of 0.1±0.01, while 69% (11/16) of FFPE samples were positive for LT mRNA, with a mean 1/ΔCt ± SEM value of 0.07±0.004 (P=0.02, Figure 6C). Spearman’s correlation analysis between MCPyV DNA load and VP1 or LT mRNA levels revealed no significant associations (P>0.05, Figure 6D).

Figure 6 Detection of MCPyV DNA and VP1 and LT mRNAs in FFPE PM specimens and HP tissues. (A) The presence of MCPyV DNA was investigated in PM (n=50) and HP (n=15) FFPEs. (B) The mean MCPyV DNA load was quantified by ddPCR in MCPyV-DNA positive FFPEs (n=16) and reported as mean MCPyV DNA mean viral load copy/cell. (C) MCPyV VP1 and LT mRNA levels, reported as mean 1/ΔCt, were measured by qPCR. B and C panels: Error bars indicate the SEM. (D) No correlations were determined between MCPyV DNA load and VP1 and LT mRNA levels. *, P<0.05. ddPCR, droplet-digital polymerase chain reaction; FFPE, formalin-fixed and paraffin-embedded; HP, healthy pleural; LT, large T; MCPyV, Merkel cell polyomavirus; mRNA, messenger RNA; PM, pleural mesothelioma; SEM, standard error of mean; VP, viral protein; qPCR, quantitative polymerase chain reaction.

Discussion

In this study, sera from PM, WEA, and HS were studied for MCPyV serology. PM sera reacted to MCPyV antigens at a low rate and ODs compared to HS, indicating that although PM patients carry circulating anti-MCPyV IgGs, they might be prone to a reduction in their immunological response to MCPyV. The immune dysregulation in PM could be attributed to an impairment of immune function, typical of oncologic patients, which impairs specific anti-viral immune responses (34). MCPyV establishes a ubiquitous and asymptomatic infection in immunocompetent individuals. However, in conditions of immune impairment, an increase in MCPyV replication and oncogenic activity can occur (35). We therefore evaluated the presence of MCPyV DNA and mRNAs in PM specimens from an additional cohort of patients unrelated to PM serum donors. Since a reduced fraction of PM sera reacted for anti-MCPyV IgGs, we reasoned that randomly chosen tumor specimens, from unrelated PM patients, could be MCPyV DNA/mRNA-positive. Nearly one-third of tumors tested MCPyV DNA-positive, with the majority also expressing viral mRNAs, while normal pleural specimens were negative. Our findings contrast with those of an early study that performed qPCR to assess the presence of MCPyV genomic sequences in a cohort of fresh-frozen mesothelioma samples, reporting no positive detections (36). However, since we employed ddPCR, which is more sensitive compared to PCR (37), variations in assay sensitivity may account for these discrepancies. Our data suggest that PM patients might be facing a reduced ability to present MCPyV antigens, possibly leading to an increased MCPyV activity in tumors.

Intriguingly, WEA had a similar rate and serum anti-MCPyV IgG ODs compared to PMs, but lower than those in HS. Moreover, WEA with the highest asbestos cumulative exposure had the lowest MCPyV seroprevalence and ODs compared to WEA with the lower exposure. Spearman correlation analysis revealed an inverse correlation between MCPyV serology and years of asbestos exposure, while a direct correlation was found with years since last asbestos exposure. These data further support an inverse correlation between MCPyV serology and asbestos exposure. Asbestos may therefore depress the anti-MCPyV immune response in WEA (38). A similar mechanism might also be conceivable for patients affected by PM, which is an asbestos-driven tumor. Indeed, as an immunosuppressive mineral, asbestos can contribute to a decline in tumor immunity, thus favoring the PM onset. Also, the immunosuppressive effect of asbestos allows SV40 to escape the anti-viral immune system, preventing immune lysis of SV40-positive cells (39). Chronic inflammation (40), characterizing both PMs and WEA (41,42), might also play a role. Asbestos can cause dysfunctions in immunocompetent cells, resulting in tumor immunity decline (43), while chronic inflammation conditions are also known to impair the immune response to viral infections (44). Evaluating the MCPyV DNA load in white blood cells and/or studying the human leukocyte antigen system in PM and WEA should be further considered.

We subsequently evaluated any possible correlation between MCPyV serology and the WEA tobacco exposure, without finding any correlation. We can infer that tobacco smoke should not be considered as a risk factor for the reduced immunological response to MCPyV in WEA.

Notably, total IgG analysis, conducted to evaluate the humoral immune status of PMs and WEA, indicated similar levels among groups, in agreement with reference ranges (26). Conversely, MCPyV serology demonstrated high sensitivity and specificity in distinguishing PM and WEA from HS (45). Although the immune response against other viruses was not investigated, our findings suggest that PM and WEA are more likely experiencing a specific impairment of their immune surveillance to MCPyV. Previous data indicated that immunocompromised individuals/oncologic patients are poor responders to MCPyV (46). These patients/individuals might be at risk of developing MCC, as a result of the decline in their immune response to MCPyV (47). While a similar process cannot be excluded in PM and WEA, the nature of this immunological defect remains unknown.

The connecting link could be the susceptibility of respiratory tract cells to MCPyV infection, and possibly human mesothelial cells (HMCs). Since MCPyV DNA has been identified in various respiratory tract locations (48-50), a potential respiratory transmission route of this human tumor virus cannot be excluded. MCPyV might become latent in other sites through systemic spread, as theorized for other polyomaviruses (51). However, as the MCPyV reservoir cell type has not been determined yet, investigating whether this human polyomavirus can infect HMCs warrants further exploration.

Biphasic PM patients exhibited a lower MCPyV serology compared to epithelioid PM patients, while a non-significant decrease in anti-MCPyV IgGs was observed in sarcomatoid patients compared to epithelioid ones. As defined by the World Health Organization (WHO) classification (52), the biphasic histotype is characterized by the presence of both epithelioid and sarcomatoid cells within the same tumor. Among PM histotypes, epithelioid is the most common (53), while sarcomatoid is the most aggressive (54,55). Although our findings suggest a potential susceptibility of biphasic PM patients to MCPyV, variations in sample size across study cohorts might account for our immunological data. In this context, larger patient cohorts are needed in future research to comprehensively assess MCPyV serology across PM histotypes.

No significant differences were observed between PM MCPyV-positive and negative patients during the long-term follow-up of 99 months. Moreover, MCPyV serology showed no significant association with therapy regimen in PM patients. Based on our data, the evaluation of MCPyV serology as a prognostic indicator for PM is unlikely.

This study has limitations. First, the mere detection of MCPyV DNA and mRNAs in PM specimens does not provide definitive evidence that this virus is the causative agent in the onset of PM. Functional studies in PM and HMC cells should be conducted to validate this assumption. Second, while we examined the presence of serum IgGs against MCPyV VP antigens, investigating circulating IgGs against MCPyV oncoproteins may also be warranted.

Conclusions

In conclusion, we present the first evidence of MCPyV serology in PM and WEA. Our results collectively underscore the significance of the lower MCPyV serology observed in PM and WEA. It is likely that both groups are experiencing a specific impairment in their immune response to MCPyV. Elucidating whether MCPyV plays a role in PM is a future area of study.

Acknowledgments

We thank Professor Georgia Emma Gili for revising the English text of the manuscript.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-198/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-198/dss

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-198/prf

Funding: This work was supported, in part, by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milan, Italy (No. MFAG 21956 to J.C.R.; No. IG 21617 to M.T.; No. IG 21390 to G.M.); University of Ferrara, FAR grants 2021 (to F.M. and M.T.); Bando Giovani anno 2024 per Progetti di Ricerca Finanziati con il Contributo 5×1000 anno 2022 (to J.C.R.); and Bando Giovani anno 2022 per Progetti di Ricerca Finanziati con il Contributo 5×1000 anno 2020 (to C.M.).

Conflicts of Interest: All authors, except M.M., who has passed away, have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-198/coif). C.M. reports the funding from Bando Giovani anno 2022 per Progetti di Ricerca Finanziati con il Contributo 5×1000 anno 2020 and a AIRC fellowship for Italy (ID: 26829). E.C. reports a grant of Veronesi Post-Doctoral Fellowship from April 2023 to March 2024. G.M. reports the funding from AIRC (No. IG 21390). J.C.R. reports the funding from AIRC (No. MFAG 21956), Bando Giovani anno 2024 per Progetti di Ricerca Finanziati con il Contributo 5×1000 anno 2022, and a grant of Umberto Veronesi Foundation with a ‘Post-Doctoral Fellowship 2024’. M.T. reports the funding from AIRC (No. IG 21617). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethical Committee of Ferrara Hospital, Italy (No. 151078) and the Ethical Committee of Liguria Region, Italy (No. 54/2017). Written informed consent was obtained from all participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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