Agnese Vivaldi

Clinical Significance of Serum Mesothelin in Patients with Mesothelioma and Lung Cancer

Purpose: High levels of serum-soluble mesothelin family proteins (SMRP) have been found to be associated with malignant mesothelioma (MM), but not lung cancer (LC). To verify the clinical role of this marker for both these tumors, we tested serum SMRP in the largest population of thoracic cancers ever assembled. Experimental Design: SMRP blood concentrations were measured in 107 patients with MM, 215 patients with LC, 130 patients with benign respiratory diseases (BRD), and 262 controls. Statistical comparison between mean serum SMRP levels in all groups was done and receiver operating characteristic curves were constructed to evaluate the performance of this marker. Results: SMRP levels were significantly higher in patients with MM and LC than in patients with benign respiratory diseases and controls (P < 0.001). The area under the receiver operating characteristic curve for serum SMRP discriminating MM and controls was 0.77 (95% confidence interval, 0.71-0.83), with a best cutoff of 1.00 nmol/L (sensitivity, 68.2%; specificity, 80.5%). In both MM and LC, serum SMRP levels did not differ significantly between early and late stages. High SMRP levels proved to be an independent negative prognostic factor in patients with MM. Conclusions: Our data confirm that serum SMRP is a promising marker for the diagnosis, prognosis, and clinical monitoring of MM. We found that serum SMRP dosage may prove helpful in LC diagnosis as well. These data may also have positive repercussions on secondary preventive medical strategies for workers previously exposed to asbestos.

SV40 Enhances the Risk of Malignant Mesothelioma among People Exposed to Asbestos: A Molecular Epidemiologic Case-Control Study

We conducted a case-control study on asbestos exposure and presence of SV40 in tumor samples of malignant mesotheliomas (MMs) and bladder urotheliomas (BUs). PCR analysis revealed the presence of SV40 DNA (SV40+) in eight (42.1%) MMs and 6 (33.3%) BUs. The odds ratio for MM Asb- and SV40+ was 0.4 [95% confidence interval (95% CI), 0.03-4.0], for Asb+ and SV40- was 3.6 (95% CI, 0.6-21.0), and for Asb+ and SV40+ was 12.6 (95% CI, 1.2-133.9). Our results suggest that SV40 increases the risk of MM among individuals exposed to asbestos.

Simian virus 40-like DNA sequences in human papillary thyroid carcinomas

Sequences of the SV40 virus, a virus of Asian macaques, have been found in human tumors, such as pleural mesotheliomas, ependimomas and choroid plexus tumors. Transgenic mice carrying the SV40 large T gene under the transcriptional control of the thyroglobulin gene promoter, develop thyroid dedifferentiation and follicular thyroid cell proliferation, leading to thyroid hyperplasia and adenocarcinomas. On these bases we investigated the presence of SV40 DNA sequences in 69 samples of papillary thyroid carcinomas (PTC) and in other thyroid and non-thyroid carcinomas, as well as in benign thyroid diseases. By Southern blot and PCR amplification followed by sequence analysis, we found the presence of SV40-related sequences integrated in the tumoral DNA of three cases of PTC. At least the 203 bp fragment of the aminoterminus of large T antigen, the 294 bp fragment of the VP1 gene and the 483 bp entire regulatory region were present in the tumoral DNA of these patients. SV40 sequences were not found in tissues other than PTC. Our results demonstrate that, in addition to previous findings in mesotheliomas and brain tumors, SV40 is somehow linked to papillary thyroid carcinoma. Although our data do not demonstrate a causative role in the development of PTC, this possibility must be considered and requires further studies.

Expression analysis of facilitative glucose transporters (GLUTs) in human thyroid carcinoma cell lines and primary tumors

Fluorine-18-fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET) is based on cell capability to take-up glucose. While a significantly higher expression of the glucose transporter GLUT1 has been reported in thyroid tumors only few data are available on the expression of other GLUT isoforms. We studied several GLUT isoforms expression in thyroid tumor cell lines deriving from anaplastic (ARO, FRO), papillary (NPA), follicular (WRO) and medullary (TT) human thyroid carcinoma. GLUT1 and GLUT3 were also studied in 157 human thyroid malignant and benign tissues. Quantitative Real-time RT-PCR analysis revealed that GLUT1 mRNA levels were higher in less-differentiated cells (ARO, FRO) while GLUT3 mRNA levels were prevalent in well-differentiated cells (NPA, WRO). Accordingly, Western blot showed high expression and correct membrane targeting of GLUT1 protein in ARO and FRO and of GLUT3 protein in NPA and WRO. All cell lines were able to take-up different rates of (3)H-deoxy-glucose. The analysis of GLUT1 and GLUT3 mRNA expression in human thyroid tissues showed the prevalence of GLUT1, but not of GLUT3, in malignant with respect to normal tissues. Finally, both GLUT1 and GLUT3 showed a slightly higher expression in anaplastic than in well-differentiated tumors. In conclusion, we showed that GLUT1 and GLUT3 were the most important glucose transporters in the thyroid tumoral cells. In particular GLUT1 was the most prevalent in less-differentiated cells (ARO and FRO) while GLUT3 was the most prevalent in well-differentiated cells (NPA and WRO). A similar pattern of expression was found for GLUT1 but not for GLUT3 in human thyroid tumors.

In silico and in vitro analysis of rare germline allelic variants of RET oncogene associated with medullary thyroid cancer

Germline and somatic RET oncogene mutations are found in 98% hereditary and 40% sporadic medullary thyroid carcinomas. Our aim was to analyse by in silico and in vitro assays the transforming activity of six rare RET mutations (T338I, V648I, M918V, A883T, S904F and M848T). Six known RET mutations were used as controls. The in silico analysis showed the highest score value (i.e. 65) for S904F, M848T, M918T and C634R, whereas L790F, G691S, T338I and V648I had 0 score. Intermediate score values were obtained by A883T (score=55), M918V, V804M and Y791F (score=15). The in vitro focus formation assay showed that cells transfected with S904F, M918T, M848T or C634R generated the largest number of focus formation units (FFU). Intermediate numbers of FFU were observed in cells transfected with M918V, V804M, Y791F or A883T, while cells transfected with L790F, G691S, T338I or V648I showed a number of FFU similar to control cells. A positive correlation between the in silico score and in vitro FFU was found (P=0.0005). Only cells transfected with M918T or C634R grew faster and generated higher number of colonies in soft agar than control cells. However, the cells that were transfected with V804M produced an intermediate number of colonies. In conclusion, two of the six rare RET mutations, S904F and M848T possessed a relatively high transforming activity but a low aggressiveness; the other four mutations T338I, V648I, M918V and A883T were low or non-transforming, and their ability to induce tumoural transformation might be related to particular genetic conditions.

Re-differentiation of thyroid carcinoma cell lines treated with 5-Aza-2′-deoxycytidine and retinoic acid.

We studied cell growth rate, mechanisms of growth inhibition, phenotype re-differentiation, expression of RARalpha, beta, gamma and differentiation thyroid genes before and after combined treatment with 5-Aza-CdR and RA (5-Aza/RA) of human thyroid carcinoma cell lines (FRO, WRO, TT). Furthermore, the activity and localization of the re-expressed sodium-iodide-symporter (NIS) protein was analyzed. After 5-Aza/RA treatment, all cell lines showed a significant reduction in cell growth. This was associated with apoptosis in the TT, with inhibition of cell proliferation in the WRO, and with cell cycle impairment in FRO and WRO. FRO and WRO treated with 5-Aza/RA lost the ability to grow in soft agar. FRO re-expressed thyroid transcription factor-1 and thyroglobulin, TT and WRO re-expressed PAX-8 and FRO and TT re-expressed RARbeta and NIS mRNA. Despite this expression, they were unable to take up iodine: a cytoplasmic localization of NIS protein was demonstrated in FRO. In conclusion, besides a significant reduction in cell growth rate and in the ability to grow in soft agar, treatment with 5-Aza/RA partially re-differentiated FRO and induced expression of NIS mRNA and protein in FRO and TT, but this treatment was unable to restore the functional activity of NIS, likely because it was located into the cytoplasm without reaching the plasma membrane.

Combined Serum Mesothelin and Plasma Osteopontin Measurements in Malignant Pleural Mesothelioma

Introduction: Malignant pleural mesothelioma (MPM) is a lethal tumor related to asbestos exposure. At present, the only instruments for screening and diagnosis are based on radiological tests, posing evident economic and radio-protectionist problems. Some authors are evaluating biological indicators, such as plasma osteopontin (pOPN) and serum soluble mesothelin-related peptides (SMRP). This study aimed to evaluate whether a combination of these two markers could increase sensitivity and specificity in diagnosis of epithelioid MPM. Methods: We enrolled 93 healthy subjects, 111 individuals with benign respiratory disease (BRD), and 31 patients with MPM, histologically and/or cytologically confirmed. SMRP and pOPN levels were determined using commercially available enzyme-linked immunosorbent assay kits. Though a logistic regression analysis, SMRP and pOPN were combined and translated into a new index, called “combined risk index.” Results: Differences in both SMRP and pOPN mean values between epithelial MPM patients and healthy subjects or BRD patients were statistically significant (p < 0.0001), whereas there was no difference in SMRP and pOPN mean values between healthy subjects and BRD patients. The performance in MPM diagnosis resulted improved by the combination of the two markers. The results of our study should be confirmed by a larger scale and, possibly, a multicenter study, which could better take into consideration the influence of some possible confounding factors such as glomerular filtration rate and other blood parameters. Conclusions: We combined SMRP and pOPN dosages to increase diagnostic accuracy. This study showed for the first time that combined SMRP and pOPN measurements can increase both sensitivity and specificity in terms of combined risk index.

Comparison between plasma and serum osteopontin levels: usefulness in diagnosis of epithelial malignant pleural mesothelioma

Background A potential role of serum osteopontin (OPN) and serum mesothelin-related peptide (SMRP) in the diagnosis of malignant pleural mesothelioma (MPM) has been recently reported. Although the most important data regarding the role of OPN in MPMs derive from the markers measurement in serum samples, most commercial laboratory kits for OPN assay are suitable only for measuring plasma levels, as indicated by the manufacturers. Our study aimed to evaluate the influence of preanalytic variables on serum and plasma OPN, to compare serum and plasma OPN in the same population, and to assess whether OPN levels can aid in the diagnostic distinction of patients with MPM versus benign respiratory disease (BRD) and healthy subjects exposed to asbestos. Methods The influence of preanalytic variables such as the length of storage at different temperatures and the number of thawings of samples on serum and plasma OPN measurements were evaluated. We measured OPN in 239 plasma samples from 207 asbestos-exposed subjects including 94 healthy controls and 113 subjects with BRD, and 32 patients with epithelial MPM, employing a commercially available ELISA. Serum OPN was measured in 196 of the same 239 samples from 80 healthy subjects, 92 BRD patients and 24 MPM patients. Results We found that both serum and plasma OPN levels were influenced by storage at –80°C and by the number of thawings, while serum OPN was influenced also by storage at room temperature. Plasma and serum OPN levels were significantly higher (p<0.0001) in patients with epithelial MPM than in the healthy control group and the BRD group. The application of a ROC curve for plasma OPN resulted in an AUC value of 0.780 with a best cutoff of 878.65 ng/mL, with a sensitivity of 68.8% and a specificity of 84.5%. The AUC for sOPN was 0.725 with a best cutoff of 16.06 ng/mL, with a sensitivity of 62.5% and a specificity of 87.3%. Within the control group no significant correlation was observed between age, duration of asbestos exposure, pack-years in current smokers, lung function or imaging parameters and plasma or serum OPN. Conclusions These data suggest that plasm OPN and serum OPN are not influenced by confounding factors such as age, smoking habits and asbestos exposure. Plasma and serum OPN may be useful markers in the diagnosis of epithelial MPM in addition to traditional radiological exams. However, in our opinion plasma OPN is preferable to serum OPN because it is more stable and measurements of OPN in serum are less reliable.

Low prevalence of the somatic M918T RET mutation in micro-medullary thyroid cancer.

BACKGROUND The prevalence of RET somatic mutations in sporadic medullary thyroid cancer (MTCs) is ∼40%-50%, and the most frequent somatic mutation is M918T. RET-positive MTCs have been demonstrated to have a more advanced stage at diagnosis and a worse outcome. AIMS The aim of the present work was to compare the prevalence of RET somatic mutations in sporadic microMTCs (<1 cm) and in larger MTCs. PATIENTS We analyzed the M918T RET point mutation in 160 sporadic MTC cases. Tumors were classified according to their size: group A, <1 cm; group B, >1 and <2 cm; group C, >2 and <3 cm; and group D, >3 cm. RESULTS The overall prevalence of the somatic M918T RET mutation was 19.4% (31/160). RET mutations were distributed differently among the four groups. The prevalence was 11.3% (6/53) in group A, 11.8% (8/68) in group B, 31.8% (7/22) in group C, and 58.8% (10/17) in group D, exhibiting an increase with increasing size of the tumor. When comparing the prevalence of mutations in the four groups, we found a lower prevalence in microMTCs (p<0.0001). CONCLUSIONS The overall prevalence of RET somatic mutations was lower than expected, and the prevalence of the somatic M918T RET mutation was significantly lower in microMTCs than in larger tumors. To explain this finding, we can hypothesize either that other oncogene(s) might be responsible for the majority of microMTC, thus identifying a tumor subset, or that the RET mutation might, or might not, occur later during tumor progression.

Polymorphisms in the putative micro-RNA-binding sites of mesothelin gene are associated with serum levels of mesothelin-related protein

Background Serum mesothelin, also known as soluble mesothelin-related protein (SMRP), reportedly shows increased levels in epithelial-type malignant pleural mesothelioma, but sometimes also arrives at high values in healthy asbestos-exposed subjects. Objectives This study aimed to investigate whether single nucleotide polymorphisms in the 3′untranslated region (3′UTR) of the mesothelin-encoded gene (MSLN) are associated with the SMRP levels measured in serum. Methods The 3′UTR of the mesothelin gene was genotyped in 59 healthy asbestos-exposed subjects, selected on the basis of their SMRP levels. Direct sequencing did not show any new polymorphism, but enabled us to genotype two known SNPs (rs1057147, rs57272256). Differences in the mean values of SMRP in wild-type and variant heterozygote groups were calculated. Results High levels of SMRP in healthy asbestos-exposed subjects were significantly associated with polymorphism rs1057147 (G<A). Regarding rs57272256, there was no statistically significant difference between wild-type and heterozygote groups. Our study suggests that rs1057147 polymorphism can affect mesothelin expression. Although these data need to be confirmed with a larger number of cases, this study warrants further research in order to better understand the relationship between mesothelin polymorphisms and SMRP.