Malignant pleural mesothelioma (MPM) is characterized by very long latency periods, sudden clinical onset, and extremely poor prognosis [1]. Identifying a panel of biomarkers to diagnose early grades is still the goal of many researchers [2,3]. While MPM is a rare cancer, its incidence is expected to increase dramatically due to the worldwide use of asbestos, the main etiological factor, over the past decades. Indeed, prolonged inhalation of asbestos fiber can form oxidized iron bodies, leading to the production of reactive oxygen species (ROS) and inducing reactive hyperplasia [4] that represents a primary step in mesothelioma development.

Gamma-Glutamyltransferase (GGT) is a type II membrane glycoprotein composed of a heavy and a light subunit linked by non-covalent bonds. GGT plays a crucial role in the translocation of amino acids across the plasma membrane. The activity of plasma GGT has been used for a long time as a liver function test and marker of alcohol abuse [5], but, more recently, increased GGT has been shown as a marker of oxidative stress [6], leading to cancer development and progression [7]. Accordingly, we showed that exposure to a subtoxic concentration of crocidolite asbestos triggered a GGT overexpression in THP-1 macrophagic cells, suggesting the possible involvement of GSH/GGT-dependent pro-oxidant reactions in the pathogenesis of MPM [8].

Malignant mesothelioma is an asbestos-related cancer of the serosal membranes that can affect pleura, peritoneum, pericardium, and the tunica vaginalis testis, characterized by chemo- and radio-resistance. The worldwide use of asbestos in the last century and its high bio-persistence account for a large proportion of people exposed to asbestos for occupational or environmental reasons. To date, however, no effective diagnostic tools exist for the surveillance of exposed individuals, and most MPMs are diagnosed at advanced stages. For this reason, many authors are evaluating the significance of biological indicators as biomarkers for the screening and early diagnosis of MPM.

Serum mesothelin-related protein (SMRP) is considered to be the most reliable marker [2,14].

SMRP by itself and other biomarkers currently under investigation do not possess an optimal combination of sensitivity and specificity. Nevertheless, some authors have demonstrated that the combination of different biomarkers can improve the diagnostic power [2,15,16].

Recently, numerous epidemiological studies have definitely ascertained that serum GGT elevation, even within the normal reference range, is associated with higher mortality of all causes, as well as with cardiovascular [17] and cancer-related mortality in the general population, independently from liver disease and alcohol abuse [13,18,19]. The association between serum GGT levels and the incidence of cancer (in general) and some site-specific cancer types was investigated in two large population-based cohort studies [18,19] and reviewed by Kunutsor et al. [13]. These studies reported a significant association between GGT levels and increased risk of developing digestive and respiratory/intra-thoracic malignancies in both genders. In all these previous studies, cancer risk correlated with total GGT. There is also consistent literature showing a correlation between total GGT amount and arterial blood hypertension, Diabetes Mellitus Type II (DMII) [20,21,22], cardiac [13,23], and hepatic diseases [5,24], but no data is yet available regarding any specific pattern of GGT fractional activity. In our study, a specific GGT fractional enzymatic activity has not been associated with any of the latter diseases. It must be outlined that all these subjects had total GGT values below two-fold the upper reference bound, limiting the possibility that our data are affected by the presence of subclinical hepatic alterations. Moreover, most people in the three groups had total GGT values below 55 U/l (290 subjects, 92.4%). However, we found that mesothelioma patients have a specific GGT fractional pattern, demonstrating that GGT-fractions may have a specific diagnostic meaning regardless of the total amount of GGT.

The most interesting result from the GGT fraction analysis is that the pattern associated with MPM differs not only from healthy subjects but also from those previously found in other diseases, such as muscular dystrophy [17], non-alcoholic fatty liver, and liver diseases in general [10,25]. These non-neoplastic patterns were characterized by an increase in total GGT and all single fractions, with s-GGT particularly specific for hepatic damage. MPMs showed a sensible increase in all GGT-fractions compared to the two combined control groups (ne-HS + pe-W), confirming previous observations. Additionally, MPMs were also characterized by a remarkable, though not statistically significant, decrease in the f-GGT fraction. Since the physio- and pathologic meaning of GGT-fractions is still far from being well known, there is no chance for a sensible, comprehensive interpretation of our data. Nevertheless, only the b- and m-GGT among all fractions were significantly different between pe-W and ne-HS, and the b-GGT fraction is also the only one to be spontaneously released in the culture medium by GGT-expressing tumor cell lines or by activated inflammatory cells, as demonstrated in previous in vitro experiments [9,26,27]. Furthermore, the b-GGT fraction increase had been previously attributed to the inflammatory component of a heterogeneous group of diseases [10,26], and total GGT levels have been associated with established markers of inflammation [28,29]. Our pe-W group included several lung parenchymal diseases and mono-bilateral pleural plaques mostly due to asbestos. On this basis, the difference in b-GGT activity between ne-HS and pe-W could be attributed to an inflammatory thoracic condition, either clinical (those having pleural or parenchymal pathologies) or sub-clinical phlogosis, due to the presence of asbestos fibers potentially responsible for continuous inflammatory triggering or possibly to the sum of the two circumstances. The observation that the pe-W group free from pleural and parenchymal disease still had a median value of b-fraction activity higher than ne-HS (statically significant though with lesser strength) supports this theoretical explanation. Most likely, the physio-pathological role of any single GGT fraction is different, and this might explain the different up- or down-regulation observed in their activity according to different health conditions.

Due to the lack of specific symptomatology, MPM is frequently diagnosed at late stages when the chances of effective treatments are very poor. For this reason, several potential biomarkers of early diagnosis have been investigated. By itself, none of these biomarkers reached the levels of sensitivity and specificity required for screening purposes. Therefore, some authors have proposed using combined risk indexes deriving from the simultaneous application of different biomarkers. This approach has proved attractive to allow for an increase in diagnostic accuracy. To date, the combination of SMRP and plasma osteopontin (pOPN) provided the best diagnostic performance, with an AUC value of 0.873 ± 0.05 [2]. Interestingly, the diagnostic accuracy of the combined risk index given by SMRP and b-/f-GGT ratio was slightly higher (0.875), with high sensitivity and specificity. While the accuracy of the combined SMRP-b/f GGT reported here is still too low to allow its employment in the clinical practice, these results suggest the eligibility of GGT fractions activity for a selected panel of early diagnosis biomarkers to be tested in future research. Furthermore, analyzing the GGT fractions on a larger sample could allow a better estimation of the real sensitivity and specificity of GGT as a biomarker for MPM.

In conclusion, this study demonstrated that the epithelioid subtype MPM is characterized by a specific pattern of GGT fractional activity, regardless of the total amount of GGT. The peculiarity of this enzymatic profile is given by a significant increase in the activity of the b-GGT and m-GGT fractions, along with a simultaneous decrease in the f-GGT fraction, which is constantly much more represented in healthy people. The b/f-GGT activity ratio resulted as the best performing parameter in ROC analysis among all fractions or rate combinations of them. The use of b/f-GGT in combination with SMRP further increases the diagnostic power. Non-exposed healthy subjects and exposed people, regardless of any benign respiratory disease, showed different b-GGT fraction activity. A new targeted research design is necessary to assess the role of b-GGT fractional activity as a biomarker of asbestos exposure. The complexity of the physio-pathological role of what we simplistically refer to as GGT activity, suggesting, however, that further research is needed to exploit its potential in both clinical and preventive applications.