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Article

Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma

by
Antonio d’Amati
1,2,3,
Gabriella Serio
3,
Andrea Quaranta
3,
Luigi Vimercati
4,
Michelina De Giorgis
2,
Loredana Lorusso
2,
Mariella Errede
2,
Vito Longo
5,
Andrea Marzullo
3,
Domenico Ribatti
2 and
Tiziana Annese
1,2,*
1
Department of Medicine and Surgery, LUM University, Casamassima, 70010 Bari, Italy
2
Department of Translational Biomedicine and Neuroscience, University of Bari Medical School, 70124 Bari, Italy
3
Department of Precision and Regenerative Medicine and Ionian Area, Pathology Unit, University of Bari “Aldo Moro”, 70124 Bari, Italy
4
Department of Interdisciplinary Medicine, Occupational Health Unit, University of Bari “Aldo Moro”, 70124 Bari, Italy
5
Thoracic Oncology Unit, IRCCS Istituto Tumori “Giovanni Paolo II”, 70124 Bari, Italy
*
Author to whom correspondence should be addressed.
Cancers 2025, 17(2), 252; https://doi.org/10.3390/cancers17020252
Submission received: 10 December 2024 / Revised: 9 January 2025 / Accepted: 9 January 2025 / Published: 14 January 2025
(This article belongs to the Special Issue Mesothelioma Epidemiology, Pathogenesis, Emerging Treatments and Pre-Clinical Models)
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Simple Summary

Peritoneal mesothelioma (PeM) is a rare, aggressive cancer with globally rising mortality rates. Its diagnosis is challenging due to a lack of specific clinical signs, relying primarily on peritoneal biopsy and immunohistochemical staining. Patients face a 5-year survival rate of around 5%, highlighting the urgent need for early detection and treatment. Our study provides a detailed morphological and molecular perspective on the PeM heterogeneity. Results validate the prognostic significance of the mitotic index, BAP1 loss and p16/CDKN2A status, highlighting meaningful differences in survival outcomes and emphasizing the relevance of morphological and molecular alterations in clinical practice. Moreover, several statistical correlations between the TERT mRNA level and other investigated morphological and molecular features were observed.

Abstract

Background/Objectives: Telomerase reverse transcriptase (TERT) is the catalytic subunit of the telomerase enzyme responsible for telomere length maintenance and is an important cancer hallmark. Our study aimed to clarify the mRNA expression of TERT in peritoneal mesothelioma (PeM), and to explore the relationship between its expression and the clinicopathological parameters and prognosis of patients with PeM. Methods: In a cohort of 13 MpeM patients, we evaluated histotype, nuclear grade, mitotic count, necrosis, inflammation, Ki67, BAP1, MTAP and p16 expression by immunohistochemistry, p16/CDKN2A status by FISH and TERT mRNA expression by RNAscope. Results: Our results showed several statistical correlations between TERT mRNA-score and other investigated features: (i) a poor positive correlation with BAP1 score (r = 0.06340; p ≤ 0.0001); (ii) a moderate positive correlation with p16 FISH del homo (r = 0.6340; p ≤ 0.0001); (iii) a fair negative correlation with p16 FISH del hetero (r = −0.3965; p ≤ 0.0001); a negative poor correlation with MTAP (r = −0.2443; p ≤ 0.0001); and (iv) a negative fair correlation with inflammatory infiltrate (r = −0.5407; p = 0.0233). Moreover, patients survive for a significantly longer time if they have a low mitotic index adjusted (2–4 mitotic figures per 2 mm2) (p ≤ 0.0001), are male (p = 0.0152), lose BAP1 (p = 0.0152), are p16 positive and present no deletion or heterozygous for p16 (p ≤ 0.01). Conclusions: TERT is highly expressed in PeM, but it is not one of the crucial factors in evaluating the prognosis of patients. Nevertheless, the results validate the prognostic significance of the mitotic index, BAP1 loss and p16/CDKN2A status.

1. Introduction

Peritoneal mesothelioma (PeM) is an aggressive and uncommon cancer of the abdominal cavity, comprising up to 30% of all mesotheliomas [1]. PeM is divided into three histopathological subtypes: epithelioid (80% of PeM), sarcomatoid (rare) and biphasic/mixed (13% of PeM) [2]. Epithelioid is the least aggressive among the three types and is associated with the most favorable outcomes. Sarcomatoid and biphasic tumors are more infiltrative and related to poorer outcomes [3]. Sarcomatoid is defined by a dense growth of spindle cells arranged in fascicles and shows a higher degree of cellular abnormality [4]. The biphasic subtype comprises at least 10% of epithelioid and sarcomatoid cellular features. In the past, patients were treated with systemic therapy with limited success [5]. More recently, cytoreductive surgery [6] and hyperthermic intraperitoneal chemotherapy [7] have been employed with substantial enhancements in survival rates [8,9]. In the frontline for disease management, immunotherapy, either alone or in conjunction with chemotherapy, has gained much attention in treating inoperable PeM [10]. The diagnosis of PeM is obtained after a comprehensive evaluation that includes medical history, physical examination, laboratory tests, radiographic investigations, and diagnostic laparoscopy with histopathological diagnosis on several peritoneal biopsies [11,12].
Concerning PeM pathogenesis, the connection with asbestos exposure is not as strong as with pleural mesothelioma, but the suggested rationale for the development of the disease is the same. Exposure to the carcinogen is believed to lead to the accumulation of tiny fiber particles that cause harm. One of the proposed pathogenetic mechanisms is the emission of ROS and RNS (reactive oxygen and nitrogen species) by mesothelial and inflammatory cells that damage DNA, leading to genetic instability and triggering an inflammatory response by attracting various chemicals and proteins to promote cell growth and cancer development [13,14]. Recently, in patients without asbestos exposure, loss of chromosome regions and tumor suppressor genes (TSGs), as well as gain of function mutations in oncogenes, were also linked to the onset of PeM [15,16,17]. The most frequently altered genes in PeM were BAP1, NF2, CDKN2A, CDKN2B and PBRM1 [16,18,19].
Telomerase reverse transcriptase (TERT) is the catalytic subunit of the enzyme telomerase responsible for telomere length maintenance [20,21]. TERT is an important cancer hallmark [22], primarily known for maintaining telomere length by preventing cellular senescence and ensuring immortality [23], but it also has telomere-independent roles responsible for tumor progression. It rewires tumor metabolism [24,25,26,27], as in glioblastoma [24,28], and promotes tumor initiation or progression, or both [29,30,31]. In a panel of alternative lengthening of telomeres (ALT) cells with recombinant expression of TERT and its variants, it was demonstrated that TERT expression provides protection against double-strand DNA damage separately from its involvement in telomere synthesis, and that it enhances the proliferation of oncogene-transformed cells by mitigating the inhibitory effects of telomere attrition and genotoxic stress generated by chemicals or metabolism [32]. TERT also affects gene expression by interacting with chromatin remodeling factors, regulating DNA methylation and activating its RNA-dependent RNA Polymerase (RdRP) to produce siRNAs [33]. It also exerts its function on the ubiquitin-proteasomal system, upregulating 26S proteasome activity and assembly, providing cytoprotective effects against apoptosis induced by proteasome dysfunction [34,35]. Moreover, it is involved in tumor invasiveness, promoting the local invasion of primary tumor cells, intravasation, survival of circulating tumor cells, extravasation, micrometastasis formation and macrometastatic growth [36]. Among the non-canonical biological functions of TERT, it influences different components of the tumor microenvironment, including inflammatory factors and the immune response [37,38]. Several studies have demonstrated that TERT mRNA levels in tumors are potential diagnostic and prognostic markers [39,40,41,42]. In colorectal cancer [43,44] and metastatic melanoma [45], tissue and plasma TERT mRNA levels were found to be independent markers of therapeutical response and, therefore, were prognostic of disease progression.
The present study aimed to evaluate TERT mRNA expression in PeM by RNAscope and correlate its levels with morphological and molecular factors that are currently used for prognosis and patient survival. We evaluated histotype, nuclear grade, mitotic count, necrosis, inflammation, Ki67, BAP1 and MTAP protein expression by immunohistochemistry, p16 protein expression by immunohistochemistry and its deletion by FISH.

2. Materials and Methods

2.1. Patients and Tissue Collection

This study was performed following the Declaration of Helsinki on archival material belonging to the project, which was reviewed and approved by the Ethics Committee of the University of Bari Aldo Moro (Accession number 5062, 22 June 2016).
Our study population consisted of archival tissue samples from 13 patients with peritoneal mesothelioma collected in 2003–2017. Information on patients’ sex, age, survival time (as months between the histological diagnosis and the date of death), asbestos exposure, and year of exposure were gathered via questionnaires and categorized based on the National Mesothelioma Register criteria (COR Apulia) (Table 1).
The biopsy specimens—formalin-fixed and paraffin-embedded—were cut into a series of sections (3 to 5 microns) used for all the morphological and molecular analyses. Histological sections were stained with hematoxylin-eosin and evaluated for the following: histotype (epithelioid, biphasic and sarcomatoid); nuclear atypia (from 1 (mild) to 3 (severe)); inflammatory infiltrate (scored as 1, low; 2, moderate; or 3, extensive) (Table 2); the presence of necrosis; mitotic count (score of 1, 2 or 3 based on mitoses in 1, 2–4 and ≥5, expressed on 2 mm2 and indicated as per the guidelines for grading in epithelioid pleural mesothelioma) [46]; or score of 1, 2 or 3 based on mitoses in 2–4, 5–9 and >10 expressed on 2 mm2 [47]); nuclear grade (score of 1–3 was assigned: 1, round uniform nuclei; 2, irregular nuclear contours, small nucleoli; and 3, pleomorphism and markedly irregular nuclear contours and prominent nucleoli [47]); immunostaining for Ki-67, BAP1, p16 and MTAP; p16 deletion by FISH; TERT mRNA expression by RNAscope.

2.2. Tert RNAscope

Sections of 4-μm from all FFPE tissue samples were cut on a microtome and mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA). The slides were baked in the HybEZTM Oven (Advanced Cell Diagnostics (ACD), Hayward, CA, USA) for 60 min at 60 °C, deparaffinized via incubating in xylene followed by 100% ethanol, and then pretreated according to the manufacturer’s instructions for manual assay using the RNAscope® 2.5 HD Reagent Kit (ref. 322360, Advanced Cell Diagnostics (ACD), Hayward, CA, USA). In brief, the pretreatment consisted of submerging the slides into pretreat-1 solution for 10 min, pretreat-2 for 15 min, and pretreat-3 for 30 min (ref. Pretreatment kit 322330, Advanced Cell Diagnostics (ACD), Hayward, CA, USA). The slides were then covered with the RNAscope® Probe–Hs-TERT-O1–Homo sapiens telomerase reverse transcriptase (TERT) transcript variant 1 mRNA (ref. 481961, Advanced Cell Diagnostics (ACD), Hayward, CA, USA), Hs-PPIB positive control probe (ref. 313901, Advanced Cell Diagnostics (ACD), Hayward, CA, USA) or negative control DapB probe (ref. 310043, Advanced Cell Diagnostics (ACD), Hayward, CA, USA) at 40 °C for 2 h. The Hs-PPIB probe was used as a control to ensure RNA quality. After the probes’ hybridizations, sections were subjected to signal amplification and detection using a Fast-RED solution.
Images of the whole slides were captured using the scanning platform Aperio ScanScope CS (Leica Biosystems, Nussloch, Germany) at a magnification of ×40. TERT mRNA semiquantitative analysis was performed on 6 randomly selected 0.12 mm2 layers per slide by an expert pathologist using the Aperio RNA ISH algorithm embedded in the ImageScope v.11.2.0.780 (Leica Biosystems, Nussloch, Germany). The obtained results were divided into five customized ranges/scores: 0, which includes cells that do not contain dots per cell; 1+, which includes cells containing 1–3 dots per cell; 2+, which includes cells containing 4–9 dots per cell; 3+, which includes cells containing 10–15 dots per cell; and 4+ which includes cells containing ≥16 dots per cell. Results were reported as mean ± sd. Statistical analysis was performed using two-way ANOVA analysis and Tukey’s multiple comparisons test with GraphPad Prism 7.04. p ≤ 0.05 was considered the limit for statistical significance.

2.3. BAP1, P16, MTAP, Ki-67 Immunohistochemistry

A broad panel of antibodies for immunohistochemistry was used, following WHO recommendations [48].
BRCA1-associated protein 1 (BAP1) (clone C4, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was classified as negative only if a complete absence of nuclear staining was observed in the presence of nuclear-positive lymphocytes.
P16 was analyzed using a monoclonal antibody (clone G175-405, BD Pharmingen; 1:50 dilution) and was assessed for the number of positive cells and the strength of cytoplasmic and/or nuclear staining (score 0: absent, score 1: focal or diffuse low-intensity staining, score 2: diffuse and moderate intensity staining, score 3: diffuse and high-intensity staining). P16 was deemed positive only for scores ≥ 2 [49].
MTAP protein expression was analyzed using a monoclonal antibody (clone 2G4 from Abnova Corp., Taipei City, Taiwan; 1:200 dilution). In accordance with previous similar studies, such as by Chapel et al. [50] and Hida et al. [51], a value of 1 was assigned to tumors that showed a complete loss of MTAP expression or a “faint” cytoplasmic positivity, and a value of 2 to tumors that showed “strong” cytoplasmic positivity in more than 1% of the neoplastic cells. Immunoreactivity was considered “strong” when tumor cells stained more intensely than the internal control (e.g., lymphocytes, endothelial cells, fibroblasts) and “faint” when they stained less intensely than the internal control. Cases without sufficient tumor tissue for evaluation or with a failed positive internal control were excluded from further analysis.
Ki67/MIB1 was evaluated (clone K5001, DAKO, Denemark, dilution 1:200) and was expressed as the percentage of positive cells in a total of at least 1000 cells. Positivity was also classified as either low (≤20%) or high (>20%) based on the median value.

2.4. Fluorescence In Situ Hybridization (FISH)

P16/cyclin-dependent kinase inhibitor 2A (CDKN2A) deletion was investigated using a dual color FISH locus-specific CDKN2A (9p21) commercially available probe (Abbott, Abbott Park, IL, USA) to detect chromosome 9 deletion [52]. A FISH locus-specific CDKN2A (9p21) probe (Abbott, Abbott Park, IL, USA) was used to detect chromosome 9 deletion. Vysis LSI CDKN2A/CEP 9 probes are provided in one vial as a mixture of the LSI CDKN2A (p16) probe, labeled with Spectrum Orange, and the CEP 9 probe, labeled with Spectrum Green. The LSI CDKN2A probe spans approximately 222 kb and contains several genetic loci, including D9S1749, DS1747, p16 (INK4B), p14 (ARF), D9S1748, p15 (INK4B) and D9S1752. The CEP 9 Spectrum Green probe hybridizes to alpha satellite sequences specific to chromosome 9, CE Marked. A minimum of 100 non-overlapped intact interphase nuclei of consecutive cells were scored for each case. A cut-off of 20% was used for homozygous deletion.
The heterozygous deletion was defined as occurring when >20% of the cells showed only one signal or a lower signal number than CEP9.

2.5. Statistical Analyses

Statistical analyses were performed in GraphPad Prism version 7.04 for Windows (GraphPad Software, La Jolla, CA, USA). The distribution of datasets was assessed using D’Agostino and Pearson Omnibus and Shapiro–Wilk normality tests.
Two-way ANOVA with post-hoc Tukey’s multiple comparisons test was used to calculate the significance level of the difference in TERT mRNA expression between the samples. Spearman nonparametric correlation analysis was used to assess the association between TERT mRNA expression and the other morphological and cytogenetic parameters. Survival curves were calculated using the Kaplan–Meier method and significance was assessed using the log-rank (Mantel–Cox) test. p values less than 0.05 in the two-tailed analyses were considered significant and are summarized in Figure panels as * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

3. Results

Thirteen patients, 11 male (84.6%) and 2 female (15.4%), with a mean age of 66.5 ± 8.6 years, were included in the study. Asbestos exposure (occupational and/or environmental) was known in 9 of the 13 patients (69.2%). Clinical data were collected for all patients (Table 1).

3.1. Histologic Evaluation

Tumor histotypes included seven (53.9%) epithelioid and six (46.1%) biphasic mesotheliomas (Table 1). Representative photomicrographs of epithelioid and biphasic mesotheliomas are shown in Figure 1.
Nuclear grade 1 was reported in one case (7.7%), grade 2 in five cases (38.5%), and grade 3 in seven cases (53.8%) (Table 3). Mitotic count was >10/2 mm2 in one case (14.3%) of epithelioid and four cases (66.6%) of biphasic mesothelioma, respectively (Table 3). Necrosis was observed in seven cases (53.8%): two (28.57%) epithelioid and five (71.43%) biphasic mesotheliomas. Inflammatory infiltrate was present in eight cases (61.5%): five (71.43%) epithelioid and three (50%) biphasic mesotheliomas. Desmoplastic stromal reaction was observable in six cases (46.2%): one (17.77%) epithelioid and five (83.33%) biphasic mesotheliomas, respectively. Ki67 value was >20% in two cases (28.6%) of epithelioid and four cases (66.6%) of biphasic histotypes (Table 3).

3.2. BAP1 Expression

In accordance with previous observations in a large cohort of mesotheliomas [53] (frequent loss of BAP1 expression, particularly in the epithelioid subtype), our immunohistochemical evaluation for BAP1 showed negativity in all seven (100%) cases of epithelioid and in two cases (33.3%) of biphasic mesotheliomas (Table 4).

3.3. P16 Expression and CDKN2A Status

Immunohistochemical evaluation for the p16 protein showed positivity in five cases (71.4%) and two cases (33.3%) of epithelioid and biphasic mesotheliomas, respectively (Table 4). FISH analysis for p16/CDKN2A revealed the absence of deletion in one case (14.3%) of epithelioid and one case (16.7%) of biphasic mesotheliomas, heterozygous deletion in three cases (42.8%) of epithelioid and one case (16.7%) of biphasic mesotheliomas, and homozygous deletion in three cases (42.8%) and four cases (66.7%) of epithelioid and biphasic mesotheliomas, respectively.

3.4. MTAP Expression

Immunohistochemical evaluation for the MTAP protein showed complete negativity in two cases of epithelioid (28.6%) and two cases of biphasic (33.3%) mesotheliomas (Table 4). Two cases (28.6%) of epithelioid and three cases (50%) of biphasic mesotheliomas, respectively, showed “faint” positivity (i.e., lower than the internal control) and were considered negative (i.e., MTAP loss of expression). On the other hand, three cases (42.8%) and one case (16.7%) of epithelioid and biphasic mesotheliomas, respectively, showed “strong” cytoplasmic positivity; hence, MTAP expression was considered retained.

3.5. TERT

The TERT mRNA expression quantification, represented as red dots (Figure 2), in malignant peritoneal mesothelioma showed a different expression panel among the different histotypes (Figure 3, Table 5).
In general, all cases presented an overexpression of TERT, but for some, there was an overexpression with cells showing more than 16 molecules/dots per cell. There was a mean of 23.9 ± 9 and 6.6 ± 6.4 percent of cells containing 1–3 dots per cell in epithelioid and biphasic mesotheliomas, respectively. Four cases (57.1%) of epithelioid and three cases (50%) of biphasic mesotheliomas showed cells containing 4–9 dots per cell, and only one case (14.3%) of epithelioid mesothelioma showed cells with 10–15 dots per cell and cells with ≥16 dots per cell.
No statistically significant difference was detected as regards survival in correlation with TERT mRNA expression (Figure 4), although the slope for the range 4+, which includes cells containing ≥16 dots per cell, was steeper, indicating a higher event rate (death rate) and, therefore, a possible worse survival prognosis. On the contrary, a flatter slope for 1+, which includes cells containing 1–3 dots per cell, was highlighted, indicating a lower event rate and, therefore, a better survival prognosis.
Our results (Figure 5) showed several statistical correlations among TERT mRNA-scores and other investigated features: (i) a poor positive correlation with BAP1 score (r = 0.06340; p ≤ 0.0001); (ii) a moderate positive correlation with p16 FISH del homo score (r = 0.6340; p ≤ 0.0001); (iii) a fair negative correlation with p16 FISH del hetero (r = −0.3965; p ≤ 0.0001); a negative poor correlation with MTAP score (r = −0.07435; p = 0.0420); and (iv) negative fair correlation with inflammatory infiltrate (r = −0.5407; p = 0.0233).
No correlations were found with the type of exposure, the years of exposure, the sex, the age score and survival, or with necrosis, mitotic index, nuclear grade and ki-67 (Figure 5).

3.6. Analysis of Survival

Taking all the cases of PeM together, patients’ survival ranged between 1 and 59 months (Table 1). For the epithelioid type, mean survival was 30.9 ± 20.2 months (range 4–59 months; CI: 12.2–49.6; median survival time 28 months); for the biphasic type, it was 9.8 ± 17.9 months (range 1–46; CI: −8.9–28.58; median survival time 2 months). Patients with epithelioid mesothelioma survived longer than those with a biphasic histotype (Figure 4).
Cases with lower adjusted mitotic index (mitoses in 2–4 cells) (p ≤ 0.0001), males (p = 0.0152), those with the loss of BAP1 (p = 0.0152), positivity for p16 in immunohistochemistry, p16 heterozygous deletion or no p16 deletion (depending on the employed score) (p ≤ 0.0001) showed better survival than their counterparts (Figure 4; Table 6).

4. Discussion

In the present single-center retrospective study on 13 patients affected by PeM, several morphological and molecular parameters were analyzed and correlated with TERT mRNA expression and survival in order to better understand the role of TERT and its relationships with the clinicopathological features of our cohort.
In tumors, the common result of the diverse mechanisms leading to the activation of TERT is an increase in TERT mRNA expression. The quantification of TERT mRNA may serve as a prognostic indicator in many different tumors, such as thyroid carcinoma [54,55], Wilm’s tumor [56], non-small cell lung cancers [57] and neuroblastoma [58].
Here, TERT mRNA expression was evaluated by RNAscope. Among the quantitative (real-time PCR) and qualitative (in situ hybridization) techniques for transcript analyses, RNAscope is a valid high-throughput method [59]. RNAscope assay enables the association between histomorphology and gene expression, accurately distinguishing between neoplastic and non-neoplastic tissues, making it a helpful diagnostic tool [60,61,62].
The use of the RNAscope assay to assess TERT status in FFPE tissue has already been evaluated and validated in tumor samples [63]. However, in our study, we used a specific alternative algorithm to quantify the mRNA signals (Aperio RNA ISH algorithm embedded in the ImageScope), and no data on PeM have previously been reported in the literature.
Although not significant, we found an increased TERT mRNA expression in epithelioid PeM cases compared to biphasic ones. Since our results reported that in the epithelioid histotype, the mitotic index is generally lower than in the biphasic one, while the presence of inflammatory infiltrate is more evident, the expression of TERT in epithelioid PeM could be related to the activation of non-canonical mechanisms, such as the recruitment and maintenance of inflammation. It has been reported that TERT has effects on inflammation and the immune response. For instance, the production of inflammatory cytokines IL-6 and TNF-α, which play a crucial role in cancer-related inflammation, depends also on TERT binding to NF-κB [64,65]. The expression of inflammatory markers INFγ, TNF, IL10 and PD-1 in a non-small-cell lung cancer mouse model was influenced by TERT [66]. Moreover, TERT is recognized by T cells and can induce an adaptive immune response [67]. For instance, TERT is required for melanoma progression, avoiding immune rejection and regression. [68]. In CD4+ T cell activation, TERT is transported from the cytoplasm to the nucleus via kinase Akt [69].
In contrast to a study by Xian et al. [70], we found a negative fair correlation between TERT expression and inflammatory infiltrate. A plausible explanation lies in the diversity between mesothelioma and head and neck tumors and their microenvironments. We believe that further studies will clarify the role of TERT in the PeM inflammatory microenvironment.
Moreover, we have evaluated the correlation between TERT expression and several features: histotype, nuclear grade, mitotic count, necrosis, inflammation, Ki67, BAP1 and MTAP protein expression by immunohistochemistry, p16 protein expression by immunohistochemistry and its deletion (CDKN2A gene) by FISH. Our results showed several statistical correlations among TERT mRNA-scores and other investigated features: (i) a poor positive correlation with BAP1 score (r = 0.06340; p ≤ 0.0001); (ii) a moderate positive correlation with p16 FISH del homo (r = 0.6340; p ≤ 0.0001); (iii) a fair negative correlation with p16 FISH del hetero (r = −0.3965; p ≤ 0.0001); a negative poor correlation with MTAP score (r = −0.2443; p ≤ 0.0001); and (iv) a negative fair correlation with inflammatory infiltrate (r = −0.5407; p = 0.0233).
A lower mitotic index (2–4 mitotic figures per 2 mm2) (p ≤ 0.0001), male sex (p = 0.0152), BAP1 loss (p = 0.0152), positivity for p16 by immunohistochemistry, CDKN2A heterozygous deletion or no CDKN2A deletion (depending on the employed score) (p ≤ 0.0001) predict for better overall survival independently of other parameters.
Previous studies have examined the prognostic significance of the mitotic index in peritoneal mesothelioma; univariate analysis showed that more prolonged overall survival was significantly associated with a low mitotic index [71,72].
In the United States and Europe, PeM incidence is higher in males vs. females [73,74]; in our cohort, 85% of enrolled patients were males. However, our data demonstrate an inverse trend because we found better overall survival in males vs. females [71,75], probably due to our small cohort size and because men often entered follow-up earlier than females due to anamnestic data of occupational asbestos exposure.
In accordance with the existing literature, we found a loss of BAP1 nuclear protein expression in 85% of our cases [18], and our data confirmed a better overall survival rate for patients with BAP1 protein loss independently of other clinicopathological features [72,76,77,78,79]. BAP1, as a tumor suppressor, is involved in chromatin remodeling and gene expression regulation. Emerging evidence (particularly for hepatocellular carcinoma) suggests that TERT could interact with BAP1 via the WNT/β-catenin signaling pathway, which is often activated in tumors with TERT overexpression. This interaction might disrupt BAP1’s ability to maintain genomic stability, facilitating tumor progression. Furthermore, TERT’s telomerase-independent functions, such as its role in modulating transcriptional networks, could influence BAP1 activity by altering the epigenetic landscape [80,81].
The results of immunohistochemistry for p16 expression and FISH analysis p16/CDKN2A status showed that 54% of our cases were positive for p16 protein expression and that 15% did not present CDKN2A deletions, while its homozygous deletion was found in 54% of cases and heterozygous deletion in 31%. As previously demonstrated by our group, confirmed by the results of this study and consistent with the literature, no deletion/heterozygous deletion of p16/CDKN2A resulted in the most prolonged survival [72,78]. Moreover, our data showed better overall survival in patients with p16 positivity by immunohistochemistry, in accordance with Borczuk [82], which demonstrated that p16 loss was independently linked to poorer survival outcomes. TERT expression has been linked to cell cycle regulation, where it may downregulate tumor suppressors like p16 through the activation of the PI3K/AKT/mTOR pathway. This pathway is known to promote cellular proliferation and bypass senescence mechanisms driven by p16 loss. Additionally, TERT’s role in telomere maintenance can indirectly influence p16 levels by mitigating telomere dysfunction, which otherwise triggers a p16-mediated senescence response [83].

5. Conclusions

This study enhances the understanding of PeM by exploring its morphological and molecular heterogeneity, emphasizing the role of specific markers—the mitotic index, BAP1 loss, and p16/CDKN2A status—in prognostic accuracy and clinical decision-making. The interplay between TERT, p16 and BAP1 likely creates a permissive environment for tumor development by promoting immortalization (via telomere elongation), evasion of growth suppression (via p16 inhibition) and genomic instability (via BAP1 inactivation). Although these mechanisms are supported by the current literature, further experimental studies, such as pathway-specific inhibition or knockdown models, would be required to definitively elucidate the molecular crosstalk. Despite achieving statistical significance, several limitations must be acknowledged: the cohort size was limited, the study lacked samples from the sarcomatoid subgroup and the retrospective nature of our analysis restricts the strength of our conclusions. Larger, prospective studies are needed to confirm these results and evaluate additional markers like TERT, which may improve clinical outcome stratification beyond histological features alone.

Author Contributions

Conceptualization, T.A. and A.d.; methodology, T.A.; software, M.E.; validation, A.d., V.L., L.V. and G.S.; formal analysis, V.L.; investigation, M.D.G. and L.L.; resources, G.S., A.M., A.Q., L.V. and D.R.; data curation, T.A.; writing—original draft preparation, T.A.; writing—review and editing, T.A., A.d., G.S. and A.Q.; visualization, T.A.; supervision, G.S., A.M. and L.V.; project administration, T.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was approved by the local Ethics Committee of Bari Policlinico University Hospital (Accession number 5062, 22 June 2016).

Informed Consent Statement

Not applicable. Patients and the public were not involved in the design, conduct, reporting or dissemination plans of this research.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Hematoxylin and eosin staining of peritoneal mesothelioma. In (A), a representative case of peritoneal mesothelioma, epithelioid histotype is observed. In (B), a representative case of peritoneal mesothelioma, biphasic histotype is observed. Scale bar: (A,B) 112 μm.
Figure 1. Hematoxylin and eosin staining of peritoneal mesothelioma. In (A), a representative case of peritoneal mesothelioma, epithelioid histotype is observed. In (B), a representative case of peritoneal mesothelioma, biphasic histotype is observed. Scale bar: (A,B) 112 μm.
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Figure 2. RNAscope of TERT on peritoneal mesothelioma. In (A), a representative case of peritoneal mesothelioma, epithelioid histotype, shows moderate TERT expression of up to 9 dots per cell. In (B), a representative case of peritoneal mesothelioma, biphasic histotype, shows low TERT expression of up to 3 dots per cell. In (C), a representative case of peritoneal mesothelioma, epithelioid histotype, shows high TERT expression of more than 16 dots per cell. In (D), a representative case of peritoneal mesothelioma, biphasic histotype, shows high TERT expression of up to 9 dots per cell. In all cases, TERT is found as usual in the nuclei but also in the cytoplasm. Scale bar: (A–D) 56 μm.
Figure 2. RNAscope of TERT on peritoneal mesothelioma. In (A), a representative case of peritoneal mesothelioma, epithelioid histotype, shows moderate TERT expression of up to 9 dots per cell. In (B), a representative case of peritoneal mesothelioma, biphasic histotype, shows low TERT expression of up to 3 dots per cell. In (C), a representative case of peritoneal mesothelioma, epithelioid histotype, shows high TERT expression of more than 16 dots per cell. In (D), a representative case of peritoneal mesothelioma, biphasic histotype, shows high TERT expression of up to 9 dots per cell. In all cases, TERT is found as usual in the nuclei but also in the cytoplasm. Scale bar: (A–D) 56 μm.
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Figure 3. TERT mRNA expression by RNAscope. Interleaved bar shows TERT mRNA expression in all the samples divided into five customized ranges: 0, which includes cells that do not contain dots per cell (not represented); 1+, which includes cells containing 1–3 dots per cell; 2+, which includes cells containing 4–9 dots per cell; 3+, which includes cells containing 10–15 dots per cell; and 4+, which includes cells containing ≥16 dots per cell.
Figure 3. TERT mRNA expression by RNAscope. Interleaved bar shows TERT mRNA expression in all the samples divided into five customized ranges: 0, which includes cells that do not contain dots per cell (not represented); 1+, which includes cells containing 1–3 dots per cell; 2+, which includes cells containing 4–9 dots per cell; 3+, which includes cells containing 10–15 dots per cell; and 4+, which includes cells containing ≥16 dots per cell.
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Figure 4. Kaplan–Meier survival of patients with peritoneal mesothelioma. In (A), survival data is shown based on TERT score: patients with high TERT mRNA expression showed shorter survival times than their counterparts (violet lines); not statistically significant. In (B), survival data is shown based on histological type: epithelioid type (line blue), biphasic type (line red); differences are not significant. In (C), survival data based on mitotic index adjusted is shown: 2–4 (line red), 5–9 (line blue) or >10 (line green) mitosis expressed on 2 mm2; p ≤ 0.0001. In (D), survival data based on sex is shown: male (line blue) and female (line red); p = 0.0152. In (E), survival data based on BAP1 is shown: positive (line blue) and negative (line orange); p = 0.0152. The dashed lines indicate SE.
Figure 4. Kaplan–Meier survival of patients with peritoneal mesothelioma. In (A), survival data is shown based on TERT score: patients with high TERT mRNA expression showed shorter survival times than their counterparts (violet lines); not statistically significant. In (B), survival data is shown based on histological type: epithelioid type (line blue), biphasic type (line red); differences are not significant. In (C), survival data based on mitotic index adjusted is shown: 2–4 (line red), 5–9 (line blue) or >10 (line green) mitosis expressed on 2 mm2; p ≤ 0.0001. In (D), survival data based on sex is shown: male (line blue) and female (line red); p = 0.0152. In (E), survival data based on BAP1 is shown: positive (line blue) and negative (line orange); p = 0.0152. The dashed lines indicate SE.
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Figure 5. Spearman’s correlation graphs between the customized score relative to the percentage of cells containing TERT mRNA (1+–4+ on the x-axis) and the score for a series of parameters obtained from morphological and cytogenetic analyses (y-axis). In (A), TERT mRNA correlation with BAP1, p16 and MTAP protein expression, and with p16 cytogenetic alterations. In (B), TERT mRNA correlation with histotype, atypia and inflammatory infiltrate. In (C), TERT mRNA correlation with asbestous exposure type and time, sex, age and survival month. In (D), TERT mRNA correlation with necrosis, mitotic indexes, nuclear grade and ki-67. Statistical significance was determined by linear regression analysis. Only the significant r and p-values are reported.
Figure 5. Spearman’s correlation graphs between the customized score relative to the percentage of cells containing TERT mRNA (1+–4+ on the x-axis) and the score for a series of parameters obtained from morphological and cytogenetic analyses (y-axis). In (A), TERT mRNA correlation with BAP1, p16 and MTAP protein expression, and with p16 cytogenetic alterations. In (B), TERT mRNA correlation with histotype, atypia and inflammatory infiltrate. In (C), TERT mRNA correlation with asbestous exposure type and time, sex, age and survival month. In (D), TERT mRNA correlation with necrosis, mitotic indexes, nuclear grade and ki-67. Statistical significance was determined by linear regression analysis. Only the significant r and p-values are reported.
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Table 1. Demographic characteristics of mesothelioma patients.
Table 1. Demographic characteristics of mesothelioma patients.
PatientsAsbestos
Exposure 		Year of
Exposure		SexAgeSurvival Time (Months)
Case 114M6447
Case 214M582
Case 3114M5911
Case 4126M647
Case 52Not countableM684
Case 6212F721
Case 73-F802
Case 82Not countableM5028
Case 9215M7246
Case 10110M591
Case 111Not countableM7259
Case 12127M6822
Case 1312M7845
For asbestos exposure score means: 1 = professional; 2 = environmental; 3 = no data.
Table 2. Morphological characteristics of mesothelioma patients’ biopsies relative to cell type.
Table 2. Morphological characteristics of mesothelioma patients’ biopsies relative to cell type.
PatientsHistotypeAtypieInflammatory
Infiltrate
Case 1111
Case 2232
Case 3121
Case 4231
Case 5122
Case 6231
Case 7232
Case 8121
Case 9231
Case 10232
Case 11121
Case 12121
Case 13122
Histotype score definitions: 1 = epithelioid; 2 = biphasic; 3 = sarcomatoid. Atypie score means: 1 = milde; 2 = moderate; 3 = severe. Inflammatory infiltrate score definitions: 1 = positive; 2 = negative.
Table 3. Morphological characteristics of mesothelioma patients’ biopsies relative to proliferation and necrosis.
Table 3. Morphological characteristics of mesothelioma patients’ biopsies relative to proliferation and necrosis.
PatientsNecrosisMitotic
Index-Pleura		Mitotic
Index Adjusted		Nuclear GradeKi-67
Case 123322
Case 213332
Case 322121
Case 413332
Case 513222
Case 613331
Case 713332
Case 822111
Case 912131
Case 1023232
Case 1122121
Case 1222121
Case 1313231
Necrosis score meanings: 1 = yes; 2 = no. Mitotic index-pleura score meanings: 1 = 1; 2 = 2–4; 3 = 5. Mitotic index adjusted meanings: 1 = 2–4; 2 = 5–9; 3 = >10. Nuclear grade score meanings: 1 = round uniform nuclei; 2 = irregular nuclear contours, small nucleoli; 3 = pleomorphism, markedly irregular nuclear contours and prominent nucleoli. Ki-67 score meanings: 1 = <20; 2 = >20.
Table 4. Histological characteristics of mesothelioma patients’ biopsies relative to BAP1, p16 and MTAP.
Table 4. Histological characteristics of mesothelioma patients’ biopsies relative to BAP1, p16 and MTAP.
PatientsBAP1IHC
p16		FISH p16aFISH p16b FISH
Del Homo		FISH
Del Hetero		IHC
MTAP
Case 12121222
Case 22111112
Case 32230211
Case 42230212
Case 52230211
Case 62230211
Case 71230212
Case 82111112
Case 92121221
Case 101230212
Case 112121222
Case 122130212
Case 132121222
BAP1 score meanings: 1 = positive; 2 = negative. IHC p16 score meanings: 1 = positive; 2 = negative. FISH p16a score meanings: 1 = no del; 2 = del hetero; 3 = del homo. FISH p16b score meanings: 0 = del omo; 1 = no del. FISH del homo score meanings: 1 = <20; 2 = >20. FISH del hetero meanings: 1 = <50; 2 = >50. IHC MTAP score meanings: 1 = negative; 2 = positive.
Table 5. TERT mRNA expression in mesothelioma patients’ biopsies.
Table 5. TERT mRNA expression in mesothelioma patients’ biopsies.
PatientsPercentage of Cells in ‘1+’Percentage of Cells in ‘2+’Percentage of Cells in ‘3+’Percentage of Cells in ‘4+’Score
Case 140.553.470.000.002
Case 20.910.000.000.001
Case 353.9024.761.660.174
Case 49.930.160.000.002
Case 54.800.000.000.001
Case 615.540.360.000.002
Case 76.340.040.000.002
Case 89.030.060.000.002
Case 96.520.000.000.001
Case 100.530.000.000.001
Case 116.310.000.000.001
Case 1245.065.370.000.002
Case 137.750.000.000.001
The “Percentage of Cells in” indicates the percentage of cells that contained 1–3 dots per cell (1+), 4–9 dots per cell (2+), 10–15 dots per cell (3+) or ≥16 dots per cell (4+). The colors of the lines are unique for each case and correspond to the colors used for the graph in Figure 3.
Table 6. Pathological factors predictive of peritoneal mesothelioma patient survival. Univariate analysis using log-rank test.
Table 6. Pathological factors predictive of peritoneal mesothelioma patient survival. Univariate analysis using log-rank test.
Variables
Months (95% CI)		No. of Cases
Univariate		Mean Survival (Min–Max)p-Value
Histology
Epithelioid730.86 (4–59)0.0526
Biphasic69.83 (1–46)
Sarcomatoid00
Mitotic count adjusted
2–4 = 1533.20 (11–59)≤0.0001
5–9 = 2316.67 (1–45)
>10 = 3511.80 (1–47)
Ki67
<20730.29 (1–59)0.0666
>20610.50 (1–47)
Sex
M1124.73 (1–59)0.0152
F21.50 (1–2)
BAP1
pos21.50 (1–2)0.0152
neg1124.73 (1–59)
IHC p16
pos735.57 (2–59)0.0022
neg64.33 (1–11)
FISH p16
no del215 (2–28)0.0049
del homo449.25 (45–59)
del hetero76.86 (1–22)
FISH p16
del homo637.83 (2–59)0.0044
no del76.86 (1–22)
FISH p16 del hetero
<5098.67 (1–28)0.0019
>50449.25 (45–59)
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MDPI and ACS Style

d’Amati, A.; Serio, G.; Quaranta, A.; Vimercati, L.; De Giorgis, M.; Lorusso, L.; Errede, M.; Longo, V.; Marzullo, A.; Ribatti, D.; et al. Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma. Cancers 2025, 17, 252. https://doi.org/10.3390/cancers17020252

AMA Style

d’Amati A, Serio G, Quaranta A, Vimercati L, De Giorgis M, Lorusso L, Errede M, Longo V, Marzullo A, Ribatti D, et al. Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma. Cancers. 2025; 17(2):252. https://doi.org/10.3390/cancers17020252

Chicago/Turabian Style

d’Amati, Antonio, Gabriella Serio, Andrea Quaranta, Luigi Vimercati, Michelina De Giorgis, Loredana Lorusso, Mariella Errede, Vito Longo, Andrea Marzullo, Domenico Ribatti, and et al. 2025. "Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma" Cancers 17, no. 2: 252. https://doi.org/10.3390/cancers17020252

APA Style

d’Amati, A., Serio, G., Quaranta, A., Vimercati, L., De Giorgis, M., Lorusso, L., Errede, M., Longo, V., Marzullo, A., Ribatti, D., & Annese, T. (2025). Analysis of TERT mRNA Levels and Clinicopathological Features in Patients with Peritoneal Mesothelioma. Cancers, 17(2), 252. https://doi.org/10.3390/cancers17020252

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