ENOX2-based early detection (ONCOblot) of asbestos-induced malignant mesothelioma 4–10 years in advance of clinical symptoms

Malignant mesothelioma is an aggressive, almost uniformly fatal, asbestos-induced cancer [1, 2]. It is a tumor of the serosal cavities, predominantly of the pleura and is generally widespread throughout the cavity at the time of presentation. Patients who are treated with supportive care have a median survival of only 9 months [3]. Those treated with the best available chemotherapy (pemetrexed and cisplatinum) have an average increased survival of only 10 weeks [4], with a median survival between 9 and 14 months [5]. However, in select patients with early-stage epithelial disease who undergo extra pleural pneumonectomy, followed by adjunct chemotherapy and radiotherapy, 5-year survival rates of 46 % have been reported [6]. Therefore, like most cancers, early detection of malignant mesothelioma has the potential to improve patient outcomes [7].

ENOX2 transcript variants of specific molecular weights and isoelectric points (pIs) are produced uniquely by patients with cancer [10]. Identification of cancer presence by detecting ENOX2 transcript variants produces a low incidence of both false positives and false negatives (>1 % for subjects with clinically confirmed cancers), as they are molecular signature molecules produced specifically by cancer cells and are absent from non-cancer cells [10]. The predictive correlation between ONCOblot findings and the onset of cancer is based on findings that support ENOX2 as a marker of cancer presence [11].

Considerable interest exists in the development of early screening tests for malignant mesothelioma in asbestos-exposed populations. Therefore, the present study was undertaken to determine if cancer-specific ENOX2 transcript variants might serve as biomarkers to detect the presence of malignant mesothelioma in advance of clinical symptoms. To this end, serum samples, collected prior to diagnosis as part of a prospective cohort study [12, 13], were examined by using the ONCOblot test. This examination of serum samples from asbestos-exposed subjects revealed that patients with a clinically confirmed diagnosis of malignant mesothelioma produced a consistent pattern of two ENOX2 transcript variants. These two transcript variants were detected well in advance of clinical symptoms. These findings indicate that the ONCOblot Tissue of Origin Cancer Detection Test might, if validated in other studies, provide a useful addition to the diagnostic repertoire of tests for the early detection of malignant mesothelioma [14, 15].

The identification of specific ENOX2 isoforms in sera can be indicative of the presence of cancer and also indicative of the cancer site. Malignant mesothelioma is characterized by the presence of two ENOX2 protein species of molecular weight 64 and 41 kDa, and pI 3.9 and 4.3, respectively.

All 17 patients who were diagnosed with malignant mesothelioma displayed both mesothelioma-specific protein ENOX2 isoforms. Importantly, both ENOX2 isoforms were required for a correct identification of malignant mesothelioma by using the ONCOblot test (Table 1).

Of the cancer types examined to date, the ONCOblot pattern for malignant mesothelioma most closely resembles that of bladder cancer (Table 1). However, the pI of the larger molecular weight transcript variant is sufficiently different to avoid mischaracterization (Table 1). In any case, differentiating these two cancers rarely presents a diagnostic dilemma clinically. For malignant mesothelioma two ENOX2 protein transcript variants are evident; indeed multiple ENOX2 isoforms are seen in approximately half of the different types of cancer (Table 1).

The two ENOX2 transcripts were apparent in the seven mesothelioma patients examined, 4–11 years before the clinical onset of disease (Fig. 2). This is an exciting finding and implies that production of ENOX2 proteins are an early event in carcinogenesis. To our knowledge, this is the earliest prediagnostic indicator of cancer thus far reported. The use of serum biomarkers for the early detection of cancer has been the goal of many individual researchers and research consortia, such as the Early Detection Research Network [16, 17]. While some serum biomarkers have been described, few are used in routine clinical practice [18], and most give a lead time of less than a year [19]. Biomarker utility is hampered by low levels of specificity combined with a propensity to yield false positives [20]. The only useful biomarker for mesothelioma is mesothelin which is elevated in between 15 and 40 % of individuals exposed to asbestos before diagnosis of mesothelioma [7]. Asbestos-exposed individuals represent an ideal cohort to evaluate prospective serum biomarkers for cancer detection, due to both their quantifiable exposure to a carcinogen and the well-established link to a specific cancer type, malignant mesothelioma.

The distribution of histological asbestos-related lung cancer is similar to that of lung cancers of other etiologies [21, 22]. The test has been evaluated previously for both non-small cell and small cell lung cancers (Table 1). Those cancers exhibit a single ENOX2 protein unique to lung cancer but can be distinguished by their pIs [8].

Of the subjects with benign disease, 60 % lacked ENOX2 proteins in their serum (Table 3). Both protein transcript variants were found in the serum of only one subject currently diagnosed with benign disease (subject ID 1268). For the remaining five subjects diagnosed with benign disease, only one protein transcript variant was detected. It is possible that the presence of one of the two mesothelioma-specific isoforms is an indicator of early pathological changes that predate the development of mesothelioma, as the transition from benign disease to malignant mesothelioma may be required for both transcript variants to be present. This issue would require longer follow-up to elucidate. Within the ONCOblot test, the presence of only the high molecular weight mesothelioma-specific ENOX2 transcript variant would be identified as ‘not in the database’ as no malignancy characterized to date produces a single ENOX2 transcript variant with a similar molecular weight and pI as this ENOX2 transcript variant. In contrast, the presence of only the low molecular weight transcript variant would be misidentified as a cancer of blood cell origin (Table 1).

Of note, for the three patients who were diagnosed with benign disease and examined in a time series, the detected ENOX2 spot size either remained constant or declined during the observational period. Two of these subjects (subject ID 1542 and 4288) produced only one ENOX2 transcript variant (Table 3). Although the remaining patient (subject ID 1268) produced both mesothelioma-specific transcript variants (Table 3), a steady decline of Protein 1–40 % of the initial amount detected was observed over a 9 year period. The largest spot diameter encountered in patients clinically diagnosed with mesothelioma was 6.6 mm representing a nearly tenfold increase in ENOX2 proteins in the serum compared to levels giving rise to a 2 mm diameter spot at early detection. It is possible, though not proven, that as the controls in this study have all been exposed to asbestos that these false positives may represent a pre-malignant stage of mesothelioma that has yet to become clinically meaningful. Furthermore, mesothelioma is recognized for the long latency period between asbestos exposure and malignancy, so it is possible that during this phase equilibrium is maintained between the host and the cancer. The presence of one of the mesothelioma-associated ENOX2 transcript variants may reflect this interaction. The immune system is capable of influencing the outcome of mesothelioma patients, as evidenced by the occasional finding of spontaneous mesothelioma regression accompanied by strong lymphocyte infiltration [23] and by spontaneous humoral responses [24]. This notion will be investigated further.

A test that can detect mesothelioma at an early stage might offer the prospect of early intervention as an approach to improve patient outcomes. The data from this study demonstrate that serum ENOX2 proteins characteristic of malignant mesothelioma can be detected in subjects 4–11 years before diagnosis based on clinical symptoms, and raises the possibility that the benefits of early intervention could be studied in such individuals.

In a population of asbestos-exposed subjects who eventually developed malignant mesothelioma, ENOX2 transcript variants characteristic of malignant mesothelioma were present in serum 4–10 years in advance of clinical symptoms. As with all biomarker studies, these observations require validation in a larger, independent cohort of patients and should include prospective as well as retrospective sampling.