Johnen et al. BMC Cancer (2017) 17:386 DOI 10.1186/s12885-017-3375-5
RESEARCH ARTICLE
Open Access
Calretinin as a blood-based biomarker for mesothelioma Georg Johnen1*, Katarzyna Gawrych1, Irina Raiko1, Swaantje Casjens1, Beate Pesch1, Daniel G. Weber1, Dirk Taeger1, Martin Lehnert1, Jens Kollmeier2, Torsten Bauer2, Arthur W. Musk3,4,5, Bruce W. S. Robinson3,5, Thomas Brüning1 and Jenette Creaney3,5
Abstract Background: Malignant mesothelioma (MM) is a deadly cancer mainly caused by previous exposure to asbestos. With a latency period up to 50 years the incidence of MM is still increasing, even in countries that banned asbestos. Secondary prevention has been established to provide persons at risk regular health examinations. An earlier detection with tumor markers might improve therapeutic options. Previously, we have developed a new bloodbased assay for the protein marker calretinin. Aim of this study was the verification of the assay in an independent study population and comparison with the established marker mesothelin. Methods: For a case-control study in men, a total of 163 cases of pleural MM and 163 controls were available from Australia, another 36 cases and 72 controls were recruited in Germany. All controls had asbestosis and/or plaques. Calretinin and mesothelin were determined by ELISA (enzyme-linked immunosorbent assay) in serum or plasma collected prior to therapy. We estimated the performance of both markers and tested factors potentially influencing marker concentrations like age, sample storage time, and MM subtype. Results: Calretinin was able to detect all major subtypes except for sarcomatoid MM. Calretinin showed a similar performance in Australian and German men. At a pre-defined specificity of 95% the sensitivity of calretinin reached 71% and that of mesothelin 69%, when excluding sarcomatoid MM. At 97% specificity, the combination with calretinin increased the sensitivity of mesothelin from 66% to 75%. Sample storage time did not influence the results. In controls the concentrations of calretinin increased 1.87-fold (95% CI 1.10–3.20) per 10 years of age and slightly more for mesothelin (2.28, 95% CI 1.30–4.00). Conclusions: Calretinin could be verified as a blood-based marker for MM. The assay is robust and shows a performance that is comparable to that of mesothelin. Retrospective analyses would not be limited by storage time. The high specificity supports a combination of calretinin with other markers. Calretinin is specific for epithelioid and biphasic MM but not the rarer sarcomatoid form. Molecular markers like calretinin and mesothelin are promising tools to improve and supplement the diagnosis of MM and warrant further validation in a prospective study. Keywords: Mesothelioma, Sarcomatoid, Epithelioid, Biphasic, Asbestos, Biomarker panel, Early diagnosis, Calretinin, Mesothelin, Plasma, Serum
* Correspondence: [email protected] 1 Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Institute of the Ruhr University Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Johnen et al. BMC Cancer (2017) 17:386
Background Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes with an unfavorable prognosis. Clinical symptoms are often nonspecific and in most cases the tumor is detected at an advanced stage. Early detection, preferable with noninvasive or minimally-invasive methods, could improve therapeutic approaches and outcomes. MM is typically associated with a previous exposure to asbestos; with a latency period of 20 to 50 years. Asbestos has been classified as a human carcinogen by the International Agency for Research on Cancer (IARC) for nearly 30 years and subsequently, its production, processing, and use has been restricted or banned in many countries [1, 2]. However, a global asbestos ban as a measure of primary prevention does not yet exist and a number of nations still produce and/or use asbestos on a large scale. This continued use, coupled with the long latency between exposure and tumor incidence means that the number of new MM cases is still increasing. In Germany it is expected that the peak incidence of MM cases will occur around 2020 [3], and a similar trend is predicted for Australia [4]. In both Australia and Germany medical surveillance is offered to occupationally asbestos-exposed people for early detection of cancer (secondary prevention) and those that develop MM receive compensation. A surveillance program aimed at an early detection of MM could improve therapy options if, as in some other cancers, early diagnosis and therapy improves survival. Currently, diagnosis of MM requires tissue or cellular material that is examined by a specially trained pathologist, usually applying a panel of immunohistochemical markers [5]. Therefore, minimally-invasive procedures such as simple blood tests could greatly improve prognosis if early detection and treatment become possible [6]. A number of blood-based biomarkers for the detection of MM has been described, however, no single marker has sufficient sensitivity to detect all tumors, particularly the sarcomatoid subtype [7]. Thus, there is still a need for additional novel biomarkers, e.g., to offer more options for the assembly of marker panels with sufficient sensitivity and specificity [8]. Previously, we have developed an assay to detect calretinin in serum and plasma samples [9]. Calretinin is a 29 kDa calcium-binding protein originally found in neurons that is also expressed on the surface of mesothelial cells and overexpressed in MM [10–12]. Using primary cells from a mouse model Blum et al. demonstrated that mesothelial cell proliferation and migration was increased or decreased by overexpression or absence of calretinin, respectively, hinting at a possible target for a new therapeutic approach [13]. Calretinin is extensively used in antibody panels for the clinical diagnosis of MM by immunohistochemistry, including the sarcomatoid subtype [5, 11, 14, 15]. We found, in a
Page 2 of 12
small number of samples, that soluble calretinin was elevated in the blood of individuals with MM relative to healthy and asbestos-exposed controls [9]. A difference between plasma and serum samples was not evident and the antigen showed a high stability. We now present data on the verification of the calretinin assay in a larger and independent study population from Australia and Germany and compare its performance with that of the established marker mesothelin [7, 16–21]. We also addressed the specific question of the utility of calretinin for identifying MM cases of sarcomatoid histology in blood, which was not answered in the previous study.
Methods Study population and collection of samples
We used a case-control design to address specific questions. Firstly, to determine the performance of the calretinin assay for different MM histologies, we selected a series of male cases (n = 83) from Australia with similar numbers of samples with epithelioid (n = 27), biphasic (n = 28) or sarcomatoid (n = 28) histology. To enrich the number of cases of sarcomatoid histology it was necessary to use samples collected up to 15 years previously. A random selection of samples, stored for a similar length of time from age-matched individuals from Australia with benign asbestos-related disease (for reason of homogeneity, we selected pleural plaques only) was used as reference group (n = 88). These cases and controls are referred to as group 1 (Table 1). Secondly, to verify our original findings that calretinin is elevated in the blood of MM patients [9] we analysed two additional independent sample sets, from Australian (group 2) and German (group 3) collections of more recent origin that represented a more typical clinical setting. Both groups were similar in composition regarding subtypes and age at blood drawing, to allow comparison of a possible influence of the country of origin as surrogate for potential differences by type of control or sample handling. To adjust for subtype composition four cases of sarcomatoid MM from Germany, which originally had 40 cases in total, were excluded. In total, group 2 consisted of 80 male MM cases and 75 matched controls, and group 3 consisted of 36 male MM cases and 72 controls. Because a large proportion of asbestos exposures, particularly heavy exposures, occurred in occupational settings, a typical but also more challenging target population of a future application of the tumor markers would consist of persons with known asbestos exposure and benign asbestos-related diseases to whom regular health examinations by social security institutions and statutory accident insurances are offered. Therefore, the controls from Germany (group 3) were selected from a surveillance cohort of the statutory accident insurances for patients with asbestosis and/or plaques. All workers had previous asbestos exposure and a recognized occupational disease based on these pathologies. In group 2 (Australia),
Johnen et al. BMC Cancer (2017) 17:386
Page 3 of 12
Table 1 Characteristics of the study population (male cases and controls from Australia and Germany) Characteristics
Australia
Germany
Group 1 Total
Group 2
Group 3
Mesothelioma
Controls
Mesothelioma
Controls
Mesothelioma
Controls
83
88
80
75
36
72
Histological subtype Epithelioid
27 (32.5%)
48 (60.0%)
28 (77.8%)
Sarcomatoid
28 (33.7%)
0
0
Biphasic
28 (33.7%)
12 (15.0%)
4 (11.1%)
Not specified
0
20 (25.0%)
4 (11.1%)
Pathologic changes in controls Plaques
88 (100%)
55 (73.3%)
0
Asbestosis
0
0
44 (61.1%)
Asbestosis and plaques Year of blood drawing Median (range)
0
20 (26.7%)
28 (38.9%)
2005
2006
2012
2012
2011
2010
(1996–2011)
(2000–2010)
(2011–2013)
(2011–2013)
(2008–2014)
(2009–2014)
69.5 (52–84)
70 (41–89)
72 (53–90)
70 (34–85)
71 (43–83)
Age at blood drawing [years] Median (range)
70 (53–84)
Calretinin storage time [months] Median (IQR)
81.5 (42.9–111)
59.8 (32.8–75.7)
17.2 (7.4–23.2)
19.3 (8.8–28.3)
3.6 (1.9–7)
10.9 (3.7–19.5)
n.a.
n.a.
21.3 (11.5–27.4)
21.5 (11.1–30.4)
7.5 (4.8–15.1)
26.8 (19.1–31.9)
N < limit of detection
30 (36.1%)
61 (69.3%)
20 (25.0%)
73 (97.3%)
12 (33.3%)
62 (86.1%)
Median (IQR)
0.79 (
RESEARCH ARTICLE
Open Access
Calretinin as a blood-based biomarker for mesothelioma Georg Johnen1*, Katarzyna Gawrych1, Irina Raiko1, Swaantje Casjens1, Beate Pesch1, Daniel G. Weber1, Dirk Taeger1, Martin Lehnert1, Jens Kollmeier2, Torsten Bauer2, Arthur W. Musk3,4,5, Bruce W. S. Robinson3,5, Thomas Brüning1 and Jenette Creaney3,5
Abstract Background: Malignant mesothelioma (MM) is a deadly cancer mainly caused by previous exposure to asbestos. With a latency period up to 50 years the incidence of MM is still increasing, even in countries that banned asbestos. Secondary prevention has been established to provide persons at risk regular health examinations. An earlier detection with tumor markers might improve therapeutic options. Previously, we have developed a new bloodbased assay for the protein marker calretinin. Aim of this study was the verification of the assay in an independent study population and comparison with the established marker mesothelin. Methods: For a case-control study in men, a total of 163 cases of pleural MM and 163 controls were available from Australia, another 36 cases and 72 controls were recruited in Germany. All controls had asbestosis and/or plaques. Calretinin and mesothelin were determined by ELISA (enzyme-linked immunosorbent assay) in serum or plasma collected prior to therapy. We estimated the performance of both markers and tested factors potentially influencing marker concentrations like age, sample storage time, and MM subtype. Results: Calretinin was able to detect all major subtypes except for sarcomatoid MM. Calretinin showed a similar performance in Australian and German men. At a pre-defined specificity of 95% the sensitivity of calretinin reached 71% and that of mesothelin 69%, when excluding sarcomatoid MM. At 97% specificity, the combination with calretinin increased the sensitivity of mesothelin from 66% to 75%. Sample storage time did not influence the results. In controls the concentrations of calretinin increased 1.87-fold (95% CI 1.10–3.20) per 10 years of age and slightly more for mesothelin (2.28, 95% CI 1.30–4.00). Conclusions: Calretinin could be verified as a blood-based marker for MM. The assay is robust and shows a performance that is comparable to that of mesothelin. Retrospective analyses would not be limited by storage time. The high specificity supports a combination of calretinin with other markers. Calretinin is specific for epithelioid and biphasic MM but not the rarer sarcomatoid form. Molecular markers like calretinin and mesothelin are promising tools to improve and supplement the diagnosis of MM and warrant further validation in a prospective study. Keywords: Mesothelioma, Sarcomatoid, Epithelioid, Biphasic, Asbestos, Biomarker panel, Early diagnosis, Calretinin, Mesothelin, Plasma, Serum
* Correspondence: [email protected] 1 Institute for Prevention and Occupational Medicine of the German Social Accident Insurance (IPA), Institute of the Ruhr University Bochum, Bürkle-de-la-Camp-Platz 1, 44789 Bochum, Germany Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Johnen et al. BMC Cancer (2017) 17:386
Background Malignant mesothelioma (MM) is a highly aggressive tumor of the serous membranes with an unfavorable prognosis. Clinical symptoms are often nonspecific and in most cases the tumor is detected at an advanced stage. Early detection, preferable with noninvasive or minimally-invasive methods, could improve therapeutic approaches and outcomes. MM is typically associated with a previous exposure to asbestos; with a latency period of 20 to 50 years. Asbestos has been classified as a human carcinogen by the International Agency for Research on Cancer (IARC) for nearly 30 years and subsequently, its production, processing, and use has been restricted or banned in many countries [1, 2]. However, a global asbestos ban as a measure of primary prevention does not yet exist and a number of nations still produce and/or use asbestos on a large scale. This continued use, coupled with the long latency between exposure and tumor incidence means that the number of new MM cases is still increasing. In Germany it is expected that the peak incidence of MM cases will occur around 2020 [3], and a similar trend is predicted for Australia [4]. In both Australia and Germany medical surveillance is offered to occupationally asbestos-exposed people for early detection of cancer (secondary prevention) and those that develop MM receive compensation. A surveillance program aimed at an early detection of MM could improve therapy options if, as in some other cancers, early diagnosis and therapy improves survival. Currently, diagnosis of MM requires tissue or cellular material that is examined by a specially trained pathologist, usually applying a panel of immunohistochemical markers [5]. Therefore, minimally-invasive procedures such as simple blood tests could greatly improve prognosis if early detection and treatment become possible [6]. A number of blood-based biomarkers for the detection of MM has been described, however, no single marker has sufficient sensitivity to detect all tumors, particularly the sarcomatoid subtype [7]. Thus, there is still a need for additional novel biomarkers, e.g., to offer more options for the assembly of marker panels with sufficient sensitivity and specificity [8]. Previously, we have developed an assay to detect calretinin in serum and plasma samples [9]. Calretinin is a 29 kDa calcium-binding protein originally found in neurons that is also expressed on the surface of mesothelial cells and overexpressed in MM [10–12]. Using primary cells from a mouse model Blum et al. demonstrated that mesothelial cell proliferation and migration was increased or decreased by overexpression or absence of calretinin, respectively, hinting at a possible target for a new therapeutic approach [13]. Calretinin is extensively used in antibody panels for the clinical diagnosis of MM by immunohistochemistry, including the sarcomatoid subtype [5, 11, 14, 15]. We found, in a
Page 2 of 12
small number of samples, that soluble calretinin was elevated in the blood of individuals with MM relative to healthy and asbestos-exposed controls [9]. A difference between plasma and serum samples was not evident and the antigen showed a high stability. We now present data on the verification of the calretinin assay in a larger and independent study population from Australia and Germany and compare its performance with that of the established marker mesothelin [7, 16–21]. We also addressed the specific question of the utility of calretinin for identifying MM cases of sarcomatoid histology in blood, which was not answered in the previous study.
Methods Study population and collection of samples
We used a case-control design to address specific questions. Firstly, to determine the performance of the calretinin assay for different MM histologies, we selected a series of male cases (n = 83) from Australia with similar numbers of samples with epithelioid (n = 27), biphasic (n = 28) or sarcomatoid (n = 28) histology. To enrich the number of cases of sarcomatoid histology it was necessary to use samples collected up to 15 years previously. A random selection of samples, stored for a similar length of time from age-matched individuals from Australia with benign asbestos-related disease (for reason of homogeneity, we selected pleural plaques only) was used as reference group (n = 88). These cases and controls are referred to as group 1 (Table 1). Secondly, to verify our original findings that calretinin is elevated in the blood of MM patients [9] we analysed two additional independent sample sets, from Australian (group 2) and German (group 3) collections of more recent origin that represented a more typical clinical setting. Both groups were similar in composition regarding subtypes and age at blood drawing, to allow comparison of a possible influence of the country of origin as surrogate for potential differences by type of control or sample handling. To adjust for subtype composition four cases of sarcomatoid MM from Germany, which originally had 40 cases in total, were excluded. In total, group 2 consisted of 80 male MM cases and 75 matched controls, and group 3 consisted of 36 male MM cases and 72 controls. Because a large proportion of asbestos exposures, particularly heavy exposures, occurred in occupational settings, a typical but also more challenging target population of a future application of the tumor markers would consist of persons with known asbestos exposure and benign asbestos-related diseases to whom regular health examinations by social security institutions and statutory accident insurances are offered. Therefore, the controls from Germany (group 3) were selected from a surveillance cohort of the statutory accident insurances for patients with asbestosis and/or plaques. All workers had previous asbestos exposure and a recognized occupational disease based on these pathologies. In group 2 (Australia),
Johnen et al. BMC Cancer (2017) 17:386
Page 3 of 12
Table 1 Characteristics of the study population (male cases and controls from Australia and Germany) Characteristics
Australia
Germany
Group 1 Total
Group 2
Group 3
Mesothelioma
Controls
Mesothelioma
Controls
Mesothelioma
Controls
83
88
80
75
36
72
Histological subtype Epithelioid
27 (32.5%)
48 (60.0%)
28 (77.8%)
Sarcomatoid
28 (33.7%)
0
0
Biphasic
28 (33.7%)
12 (15.0%)
4 (11.1%)
Not specified
0
20 (25.0%)
4 (11.1%)
Pathologic changes in controls Plaques
88 (100%)
55 (73.3%)
0
Asbestosis
0
0
44 (61.1%)
Asbestosis and plaques Year of blood drawing Median (range)
0
20 (26.7%)
28 (38.9%)
2005
2006
2012
2012
2011
2010
(1996–2011)
(2000–2010)
(2011–2013)
(2011–2013)
(2008–2014)
(2009–2014)
69.5 (52–84)
70 (41–89)
72 (53–90)
70 (34–85)
71 (43–83)
Age at blood drawing [years] Median (range)
70 (53–84)
Calretinin storage time [months] Median (IQR)
81.5 (42.9–111)
59.8 (32.8–75.7)
17.2 (7.4–23.2)
19.3 (8.8–28.3)
3.6 (1.9–7)
10.9 (3.7–19.5)
n.a.
n.a.
21.3 (11.5–27.4)
21.5 (11.1–30.4)
7.5 (4.8–15.1)
26.8 (19.1–31.9)
N < limit of detection
30 (36.1%)
61 (69.3%)
20 (25.0%)
73 (97.3%)
12 (33.3%)
62 (86.1%)
Median (IQR)
0.79 (
