Abstract

1. Introduction

The transcriptional activity of the human genome (>90% of the genome) produces long non-coding RNAs (lncRNAs) whose functions are mostly unknown. The extremities of linear chromosomes, or telomeres, actively synthesize a heterogeneous population of lncRNAs named TERRA (telomeric repeat-containing RNA) with various sizes (from 100 bases to 10 Kb) and containing the canonical telomeric repeat sequence UUAGGG, as well as sequences unique to the subtelomeric region of each chromosome [1,2]. TERRA transcripts localize at telomeres and interact with the telomerase RNA subunit (TERC) and with the telomerase catalytic subunit (TERT) [3,4]. Several studies have established the role of TERRA in telomere homeostasis and its implication in cancer. In this regard, TERRA has been shown to sustain the activation of DNA damage response pathways at dysfunctional telomeres [5,6] and to facilitate completion of telomere DNA replication in cancer cells [7]. Importantly, TERRA depletion associates with an increase of telomerase activity, supporting the notion that TERRA can negatively regulate telomerase in mammalian cells [2,3,4]. Expression studies conducted by various technologies have revealed the dysregulation of TERRA in various solid tumors, a frequent downregulation in specific tumor types vs. their normal tissue counterparts [8], but also its overexpression in medulloblastoma [9]. Moreover TERRA expression is increased upon the treatment of human cancer cell lines with chemotherapeutic agents, suggesting that TERRA transcripts may participate in the cellular response and/or resistance to chemotherapy [10,11,12]. For hepatocellular carcinoma (HCC), it is known that telomerase activation is a hallmark of this malignant condition [13] and that TERRA may participate in telomerase regulation in this cancer type [8]. HCC is the most common type of primary liver cancer and worldwide is the fifth cancer and the third cause of cancer mortality; it is a highly heterogeneous disease and it occurs most often in people with chronic liver diseases (liver hepatitis, alcohol hepatitis, viral B or C hepatitis, steatohepatitis, cirrhosis). Also for this reason, the HCC clinical management remains difficult: very often the diagnosis reveals an advanced HCC that usually is not submitted to surgical resection and a resectable HCC frequently leads to recurrent tumors [14,15,16,17]. To date, the treatment options are limited and advancing in the molecular characterization of HCC is a real challenge. Also, early diagnosis represents a critical step for the management of HCC and the identification of novel biomarkers of HCC will be of foremost importance.

The aim of the current study was to characterize TERRA expression in HCC, determining by qPCR its expression levels in HCC tissues and in HCC cell lines as well as its circulating amount in the plasma of HCC patients and in the cell secretomes. Further, we assessed in the same biological specimens the expression of TERC and of TERT mRNA in relation to TERRA. Our results highlight that both cellular and extracellular expressions of TERRA and TERC are dysregulated in HCC, as well as the cellular expression of TERT mRNA.

2. Results

2.1. Dysregulated Expression of TERRA, TERC and TERT mRNA in HCC Tissues

To characterize the expression of TERRA in HCC patients, we first examined TERRA levels in paired HCC and peritumoral tissues by RT qPCR. TERRA expression from different telomeres, analyzed using specific primer pairs (as described in the Materials and Methods, TERRA1 = 1_2_10_13q; TERRA2 = 15q; TERRA3 = XpYp), resulted to be significantly downregulated (Figure S1). TERRA expression was obtained as a mean of the different TERRA relative quantifications and, as shown in Figure 1a by the qPCR data, TERRA was significantly downregulated in HCC tissues compared with their matched peritumoral tissues (RQ_HCC average vs. RQ_PT average, p-value = 0.02).

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Figure 1

Tissue expression of TERRA. (a) TERRA relative quantification mean comparison between PT and HCC. Wilcoxon test was used. (b) TERRA fold-change. Histograms represent R-values and bars upper and lower limits. Histograms are ordered by increasing R-values. The corresponding cases are listed on x axis. Upper and lower dot lines define the range of gene expression variation. (c) ROC curve analysis of TERRA in tissues. Sensitivity: percent of correctly classified HCC patients. Specificity: percent of correctly classified non-HCC. AUC: area under the ROC curve; CI: confidence interval. * p < 0.05.

The R values refer to the ratio RQ_HCC/RQ_PT and, among the HCC tested, 19/25 of cases (76%) showed downregulation of TERRA expression, whereas 6/25 cases (24%) showed its upregulation (Figure 1b). No correlation was observed between TERRA expression and the clinicopathological characteristics of HCC patients (Table S1). ROC curve analyses, generated for the different TERRA subpopulations considered, using the primer pairs mentioned above, revealed that each TERRA target had a significant capability in discriminating HCC from peritumoral tissues. Further logistic regression model of classifiers showed that the use of the combined TERRA molecules might represent a significant successful discriminating tool to distinguish HCC from peritumoral tissue (Figure 1c and Table 1) (AUC: 0.79; CI: 0.563–0.829; p-value = 0.0382).

Table 1

Diagnostic performance of TERRA in HCC tissues.

TARGET	Sensitivity	Specificity	AUC	SE	95% CI	p-Value
TERRA1	80%	64%	0.706	0.075	0.560–0.826	0.0064 **
TERRA2	84%	52%	0.698	0.074	0.551–0.819	0.008 **
TERRA3	84%	52%	0.712	0.072	0.567–0.831	0.0036 **
TERRA1+TERRA2	52%	80%	0.686	0.075	0.540–0.810	0.0166 *
TERRA1+TERRA3	52%	80%	0.71	0.072	0.565–0.830	0.0233 *
TERRA2+TERRA3	44%	80%	0.712	0.072	0.567–0.831	0.0149 *
TERRA1+TERRA2+TERRA3	44%	80%	0.709	0.073	0.563–0.829	0.0382 *

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* p < 0.05; ** p < 0.01.

Given the importance of telomerase in HCC, we also investigated the expression of TERC and TERT mRNA in the paired HCC and peritumoral tissues by qPCR. For TERC expression, the data obtained displayed a trend of upregulation in HCC tissues compared with the PT ones (Figure 2a, RQ_HCC average vs. RQ_PT average, p-value = ns) and receiver operating characteristic (ROC) curve analysis did not disclose a significant capability in distinguishing HCC from PT tissues (Figure 2c) (AUC:0.51; CI: 0.34–0.67; p-value = 0.89).

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Figure 2

Tissue expression of TERC. (a) TERC relative quantification mean comparison between PT and HCC. Wilcoxon test was used. (b) TERC fold-change. Histograms represent R-values and bars upper and lower limits. Histograms are ordered by increasing R-values. The corresponding cases are listed on x axis. Upper and lower dot lines define the range of gene expression variation. (c) ROC curve analysis of TERC to discriminate HCC from normal tissue. AUC: area under the ROC curve’ CI: confidence interval.

TERT mRNA expression (Figure 3a) was significantly upregulated (p-value = 0.019) at different orders of magnitude in HCC tissues compared to the PT ones (Figure 3b). ROC curve analysis of TERT mRNA expression revealed a successful significant ability in discriminating HCC from PT tissues (Figure 3c) (AUC: 0.716; CI: 0.57–0.86; p-value = 0.008). These findings suggest that quantification of TERRA and TERT mRNA may represent a discriminating tool to distinguish HCC from peritumoral tissue.

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Figure 3

Tissue expression of TERT mRNA. (a) TERT mRNA relative quantification mean comparison between PT and HCC. Wilcoxon test was used. (b) TERT mRNA fold-change. Histograms represent R-values and bars upper and lower limits. Histograms are ordered by increasing R-values. The corresponding cases are listed on x axis. Upper and lower dot lines define the range of gene expression variation. (c) ROC curve analysis to discriminate HCC from normal tissue. AUC: area under the ROC curve; CI: confidence interval. * p < 0.05; ** p < 0.01.

2.2. Determination of Circulating TERRA and TERC in the Plasma of HCC Patients

Since TERRA has been found in human blood plasma and in the culture medium of human cell lines [18,19], to characterize further TERRA expression in HCC, we measured its amount in the plasma of HCC patients and in healthy individuals; due to the lack of a known reference gene that would allow the normalization of circulating TERRA, we report qPCR data as the average raw cycle thresholds (Ct) in plasma samples [20,21]. As shown in Figure 4 and Table 2, circulating TERRA1 and TERRA3 amounts, corresponding to TERRA transcripts expressed from telomeres 1_2_10_13q (TERRA1) and telomeres XpYp (TERRA3), were significantly higher in the plasma of HCC patients compared to healthy subjects (Figure 4a,c, p-values = 0.0023 and 0.031, respectively) and TERRA2 amounts (corresponding to the 15q transcripts) revealed a trend of upregulation (Figure 4b, p-value = ns).

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Figure 4

Plasma levels of TERRA. (a–c) TERRA levels in terms of C_t in plasma of healthy (C) and HCC individuals. TERRA expression from different telomers was analyzed using specific primers (TERRA 1 = hTel 1_2_10_13q; TERRA 2 = hTel 15q; TERRA 3 = hTel XpYp). Parametric unpaired two-tailed t-test. (d) ROC curve analysis of TERRA to discriminate HCC from healthy individuals. * p < 0.05, ** p < 0.01, *** p < 0.001.

Table 2

Diagnostic performance of circulating TERRA in plasma.

TARGET	Sensitivity	Specificity	AUC	SE	95% CI	p-Value
TERRA1	60%	100%	0.747	0.079	0.604–0.859	0.001 **
TERRA2	52%	92%	0.622	0.084	0.474–0.756	0.146
TERRA3	56%	88%	0.686	0.08	0.540–0.810	0.02 *
TERRA1+TERRA2	64%	100%	0.742	0.081	0.599–0.856	0.002 **
TERRA1+TERRA3	64%	92%	0.763	0.074	0.622–0,872	0.0004 ***
TERRA2+TERRA3	52%	96%	0.648	0.083	0.500–0.778	0.07
TERRA1+TERRA2+TERRA3	60%	100%	0.765	0.074	0.624–0.873	0.0004 ***

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^* p < 0.05; ** p < 0.01; *** p < 0.001.

No correlation was observed between circulating levels of TERRA and the clinicopathological characteristics of HCC patients (Table S2). ROC curve analyses were generated for different TERRA subpopulations to inquire as to their capability to act as tools to successfully distinguish HCC from healthy subjects. In particular, the logistic regression model of TERRA classifiers evidenced the significant combined use of TERRA targets (Figure 4d and Table 2) (AUC: 0.765; CI: 0.624–0.873; p-value = 0.0004). To support these findings, we calculated the average Ct values of TERRA in the 2 groups of individuals and we obtained significantly higher levels in plasma from HCC patients in comparison to healthy control subjects (Figure S2, p-value = 0.038). Further, we evaluated the correlation by Spearman’s analysis of TERRA levels between the HCC tissues and the plasma samples from the same patients. The R-value was 0.3096 and the p-value was not significant (p value = 0.1410). Thus, in our cohort there was no correlation between TERRA levels in HCC solid biopsies and liquid biopsies (Figure S3). Further, as shown in Figure 5, the ddPCR determination of TERC in plasma revealed a significantly higher level in HCC patients vs. control subjects (2.118 vs. 0.826 copies/μL, p-value = 0.0019); and the ROC analysis gave an AUC value = 0.8120 with a p-value = 0.0002. On the contrary, TERT mRNA was not detectable in liquid biopsy specimens (data not shown). These findings suggest that the combined detection of circulating TERRA and TERC may represent a promising approach for the diagnosis of HCC.

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Figure 5

Plasma levels of TERC. (a) TERC levels in terms of copies/µL in plasma of control subjects (C) and HCC patients. Unpaired t-test was used. (b) ROC curve analysis of TERC to discriminate HCC from healthy individuals. ** p < 0.01; *** p < 0.001.

2.3. Characterization of Cellular and Extracellular TERRA in HCC Cell Lines

Considering the evidence provided by the present study on the altered expression of TERRA, TERC and TERT mRNA in HCC tissues and on the detection of TERRA and TERC in the plasma of HCC patients, we aimed to explore the cellular and extracellular expression of these transcripts in human HCC cell lines in order to gain mechanistic insights on the function of TERRA in HCC. Since TERRA transcripts may participate in the cellular response to anticancer compounds [10,11,12], first we assessed the transcript expression in HA22T/VGH and SKHep1C3 cells (Figure 6) and monitored the possible expression variation following an anticancer drug treatment, in particular the sorafenib treatment in sensitive and resistant cells (sf and SR cells, respectively) [22,23]; this is because sorafenib is one of the drugs used by clinicians for the treatment of advanced HCC [14].

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Figure 6

Intra- and extracellular expression of TERRA and telomerase transcripts in HCC cell lines. The expression levels of TERRA (a), TERC (b) and TERT mRNA (c) were measured in HA22T/VGH and SKHep1C3 untreated, resistant to sorafenib (SR) and treated with 15 µM sorafenib. The levels of TERRA (d) and TERC (e) were measured in EVs from HA22T/VGH and SKHep1C3 cells untreated, resistant to sorafenib (SR) and treated with 15 µM sorafenib. As described in Materials and Methods, gene expression level was normalized to the number of particles released per each biological sample, respectively. The histograms represent the mean values of 2 experiments, bars are ± SEM. Unpaired two tailed t-test; * p < 0.05, ** p < 0.01, *** p < 0.001.

As shown in Figure 6a, the SK-Hep1 C3 cells express a very high amount of TERRA compared to HA22T/VGH cells (RQ_SKHep1C3 = 67.63 vs. RQ_HA22T/VGH = 1.03; fold increase, FI = 65.9) and a lower amount of TERC and TERT mRNA compared to HA22T/VGH cells (Figure 6b,c). These cell lines are both undifferentiated HCC cells and can be considered as models of the heterogeneity of HCC; in particular, the HA22T/VGH cells might represent a model of the HCC set with TERRA down-modulation and the SKHep1C3 may be a model of HCC with TERRA upregulation. Both cell lines responded similarly to sf treatment, enhancing TERRA expression and with the FI higher in the HA22T/VGH cells which are characterized by a low basal level (FI_TERRA = 7.70 and FI_TERRA = 1.91 in HA22T/VGH and SK-Hep1C3 cells, respectively). The expression variation does not seem to depend on the sensitivity or resistance of the cells to sf, since this effect of the drug appears similar in both conditions. Both cell lines also responded similarly to sf treatment enhancing TERC and TERT mRNA expression, for TERC with a low FI (FI_TERC = 1.3 and FI_TERC = 1.14 in HA22T/VGH and SKHep1C3 cells, respectively) and for TERT mRNA with a higher FI in HA22T/VGH cells (FI_TERT = 5.01 in HA22T/VGH cells and a FI_TERT = 1.3 in the SKHep1C3 cells). We then isolated extracellular vesicles (EV) from the secretome of both cell lines to determine the amounts of extracellular transcripts detectable in the EV fraction. For TERRA, the results shown in Figure 6d revealed that: (i) TERRA was detectable in the EVs of both HCC cell lines, the amount of TERRA ascertained in the EVs of HA22T/VGH cells being very high, and (ii) both cell lines displayed a lower amount of TERRA in the EVs of sf-treated cells, thus showing an opposite trend of expression variation compared to the intracellular one. The TERRA size detected in the EVs was in a size range of less than 500 nt as revealed by Northern analysis (Figure S4). For TERC, it was detectable (Figure 6e) in the EVs derived from HA22T/VGH cells and, in this case, sf treatment led to expression variations similar to the cellular one, that is, an increase of the TERC amount. TERC was not detectable in the EVs derived from SK-Hep1C3 cells even when ddPCR was employed to determine its amount and the EVs of both HCC cell lines did not display TERT mRNA molecules. In particular, these findings reveal that the HCC cells expressing low TERRA levels (HA22T/VGH) show a very high TERRA amount into the EVs. Nevertheless, in both cell lines the sf treatment leads to a cellular TERRA increase and at the same time causes a TERRA decrease in the EVs.

3. Discussion

The expression variations of the lncRNA TERRA in cancer has generated a growing interest on the biology of these telomeric transcripts in various solid tumors (larynx and colon cancer, medulloblastoma, glioblastoma, head and neck cancer, cervical and endometrial carcinoma) [2,9,24,25,26,27,28], but its real functional role in a given cancer type remains to be elucidated. In the present work, for the first time, we determined TERRA expression in HCC by qPCR, in HCC tissues and in the plasma of HCC patients. We used a qPCR assay that did not detect all TERRA transcripts, but it detected the most abundant ones. In particular, we used three primer pairs to quantify TERRA expression levels transcribed from seven telomeres (TERRA1: 1_2_10_13q; TERRA2: 15q; TERRA3: XpYp) [29]. Furthermore, Diman et al. [30] demonstrated that the primer pair named TERRA1 (1_2_10_13q) detected the transcripts expressed from the telomeres: 1_2_4_10_13_22q. Thus, according to Diman et al., our qPCR assay should recognize TERRA transcripts expressed from nine telomeres. Furthermore, Montero et al. [31] demonstrated that TERRA is mainly transcribed from the 20q subtelomere in human cells and the primer pairs able to detect this transcript were not included in our qPCR assay. Moreover, considering the role of TERRA in the telomere function and the importance of the telomerase enzyme in the biology of HCC, in the present study, we also assessed the RNA expression of the main components of the telomerase complex, the RNA subunit TERC and the telomerase catalytic subunit TERT mRNA. We found a significant TERRA downregulation in HCC tissues vs. the corresponding PT ones and a significantly higher amount of TERRA in the plasma of HCC patients. In both cases, for the first time, ROC curves indicated significant capabilities in distinguishing HCC tissues from PT ones and HCC patients from control subjects. Further, the qPCR determinations of TERRA downregulation in HCC tissues, consistent with the in situ hybridization data reported by Cao et al. [8], could be utilized as specific characterization of the malignant tissue (of HCC or other cancer types), being the qPCR technique frequently used in research laboratories.

For TERRA transcripts in the plasma, an RNA-seq study [18] revealed the presence of TERRA molecules in the plasma of 2 normal subjects and of 19 patients with various cancer types (prostate, stomach, breast, colon, kidney, lung, duct, liver, melanoma, ovarian); however, to our knowledge, there are no reports on circulating TERRA in a small or large cohort of patients with the same type of cancer, or even reports on the PCR determinations of TERRA amounts in the plasma of cancer patients. Hence, the data reported in our study are the first concerning TERRA expression in the plasma of a cohort of patients with the same cancer type, HCC, in comparison to healthy individuals.

For the overexpressed amount of TERRA detected in the plasma of HCC patients, we cannot know the origin of the circulating forms of TERRA, if they derive from the malignant cells or not. Despite this, our data indicate that the plasma determination of TERRA amounts can be taken into account as a possible non-invasive molecular indicator of HCC diagnosis and, in order to ascertain this hypothesis, we are planning a multicenter study to examine a larger cohort of patients.

For TERC and TERT mRNA expression we found an upregulation in HCC tissues, which was significant for the TERT mRNA. For measuring the level of TERC and TERT mRNA in plasma, we used ddPCR, the best technology to assess absolute quantifications of transcripts, especially the circulating ones. The TERC amounts detected in the plasma of HCC patients were significantly higher, while TERT mRNA was not detectable, thus indicating that in HCC patients the cells do not secrete the detectable extracellular form of TERT mRNA. The ROC analysis of circulating TERC expression revealed a significant ability in discriminating HCC patients from controls. Concerning TERC in cancer, it is known from in situ and fluorescence hybridization data (ISH, FISH) that is overexpressed in lung, oral, prostate and cervix carcinoma [10]; further, only Novakovic et al., to our knowledge, reported nested-PCR data that revealed the presence of TERC in the plasma of healthy controls and of patients with breast cancer, melanoma and thyroid cancer, but did not allow the TERC amount to be considered as a tumor marker [29]. Hence, our findings would indicate the determination of circulating TERRA and TERC in HCC patients as specific non-invasive molecular indicators of this malignant condition.

Subsequently, in the present work, to address the problem of the dysregulation of TERRA, TERC and TERT mRNA in HCC from a biological point of view, we characterized two HCC cell lines for the expression of these transcripts and, for the first time, we report data on their expression both at the cellular and extracellular levels, indicating that the two HCC cell lines may represent the heterogeneity of HCC. In particular, the HA22T/VGH and SKHep1C3 cells, at low and very high TERRA expressions, respectively, may represent the HCC cases with TERRA downregulation and upregulation, respectively. HA22T/VGH cells producing low TERRA amounts expressed high levels of TERC and TERT mRNA, and this result is consistent with previous evidence indicating that TERRA depletion associates with increased levels of TERC [3]. Both HCC cell lines secrete TERRA molecules into the secretome-derived EVs. The HA22T/VGH cells secreted very high amounts of TERRA and low amounts of TERC. As expected, TERT mRNA was not detected as an extracellular form in any condition. Further, concerning the effect of an anticancer drug on TERRA expression, both cell lines similarly responded to sorafenib treatment enhancing the expression amount of TERRA, TERC and TERT mRNA at the cellular level. HA22T/VGH reacted to sf increasing the cellular expression of TERRA, TERC and TERT mRNA and decreasing the secretion of TERRA and TERC; SKHep1C3 cells reacted to sf increasing the cellular expression of TERRA, but much less TERC and TERT mRNA, and decreasing the secretion of TERRA and by not secreting both TERC and TERT transcripts. Sorafenib is a multikinase inhibitor exerting its antitumor activity mainly affecting the proliferation and apoptosis of cancer cells, known to influence gene expression at the level of mRNAs, ncRNAs and genome methylation with mechanisms that have not been elucidated [22,23,32]. Hence, further effort is needed to understand the intracellular events that led to an increase of TERRA in cells treated with sf. From a general point of view, it is known that TERRA is a heterogeneous group of ncRNAs generated from multiple different telomeres and with various size (from 100 to 10 Kb); it has been proposed that its upregulation can be regulated by stress inducing the processing of TERRA molecules into smaller fragments then secreted into the EVs. It seems that TERRA containing EVs are relatively stable and circulate in extracellular spaces, including plasma, but it is not known how the smaller extracellular forms are generated. As reported by Wang et al. [19], the extracellular molecules of TERRA can be breakdown products of full-length cellular TERRA or an alternative form that is encapsulated into the EVs and secreted in extracellular spaces. It is likely that TERRA containing EVs can transfer signaling information to surrounding cells impacting the extracellular microenvironment. The complex combination of factors that comprise EVs and the type of recipient cells sensing the EVs may determine the nature of the signaling and response.

Hence, this part of the present study is the first effort to characterize the cellular and extracellular expression of TERRA, TERC and TERT mRNA in HCC cell lines demonstrating the capability of these cells to secrete TERRA and TERC transcripts into the EVs and thus opening a new scenario in the biology of HCC cells. Novel issues will be addressed to understand the role of TERRA and TERC containing EVs in transferring signaling to surrounding cells in vitro and in an HCC tumor xenograft.

In conclusion, in the current work we have assessed the cellular and extracellular expression of TERRA, TERC and TERT mRNA studying HCC tissues, the plasma of HCC patients and the cells and secretomes of two HCC cell lines. Our novel findings obtained by qPCR show that HCC tissue is significantly characterized by TERRA downregulation and by TERT mRNA upregulation; the plasma of HCC patients revealed an overexpression of TERRA and TERC, thus indicating novel non-invasive indicators of HCC whose clinical diagnostic significance will be investigated further in a larger cohort of patients. It is clear that among HCC tissues there are two sets, one with TERRA down-modulation, the other with TERRA upregulation. Moreover, the characterization of two HCC cell lines, representative of this feature of HCC heterogeneity, has clearly shown the ability of HCC cells to produce and secrete TERRA and TERC into the EVs; the ability to produce TERT mRNA that is not secreted; and the ability to respond to sorafenib treatment increasing TERRA expression. From a general point of view, our findings contribute to the basic knowledge of the cellular and extracellular forms of TERRA and TERC produced by cultured cancer cell lines and those detectable in HCC tissues and in the plasma of HCC patients.

4. Materials and Methods

Supplementary Materials

The following supporting information can be downloaded at www.mdpi.com/article/10.3390/ijms23116183/s1.

Funding Statement

This research was supported by CIB (Biotechnology Interuniversity Consortium), the Lega Italiana Lotta ai Tumori (LILT), Fondazione EULO Brescia, Italy (to GDP); the Italian Ministry of University and Research (FFRB grant) (to AS); the University of Brescia (local grants) (to AS, GDP and NP); Fondazione Cassa di Risparmio Trento e Rovereto (to VGD); and Associazione Italiana Ricerca sul Cancro (AIRC), grant MFAG2019 ID 22840 (to EC). The funding bodies played no role in the design of the study; in the collection, analysis, and interpretation of data; or in writing the manuscript.

Author Contributions

Conceptualization, G.D.P. and A.S.; methodology, M.M., I.G., J.C. and K.J.; investigation, A.S., M.M., I.G., J.C., V.G.D., K.J. and E.C.; resources, N.P. and S.M.; data curation, G.D.P., A.S., V.G.D., E.C. and N.P.; writing—original draft preparation, G.D.P., A.S., M.M. and I.G.; writing—review and editing, G.D.P., A.S., V.G.D., E.C. and N.P.; supervision, G.D.P. and A.S.; funding acquisition, G.D.P., A.S., V.G.D., E.C. and N.P. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committees of ASST Civili Hospital of Brescia on 2 October 2012 (NP1230).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data supporting the findings of this study are contained within the contents of this article. The datasets generated during this study will be freely provided by the corresponding author upon request.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

References