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The chromogranin-A (CgA) in prostate cancer
Archives of Gerontology and Geriatrics 43 (2006) 117–126 www.elsevier.com/locate/archger
The chromogranin-A (CgA) in prostate cancer Salvatore Ranno, Massimo Motta, Elvira Rampello, Corrado Risino, Ettore Bennati, Mariano Malaguarnera * Department of Senescence, Urological and Neurological Sciences, University of Catania, Cannizzaro Hospital, Via Messina 829, I-95126 Catania, Italy Received 12 April 2005; received in revised form 8 September 2005; accepted 14 September 2005 Available online 8 November 2005
Abstract Prostate cancer is one of the most frequent tumors in men. The neuroendocrine differentiation in prostate cancer has become more widely recognized and has attracted considerable attention as a potentially new finding with major diagnostic, prognostic and therapeutic implications. We investigated the role of the serum concentrations of CgA in a group of 57 patients with prostate cancer and in 61 elderly subjects with benign prostate hyperplasia (BPH). Neuron-specific enolase (NSE) is the most frequently employed marker to detect neuroendocrine features. Serum prostate-specific antigen (PSA), CgA and NSE levels were determined. Comparing prostate cancer group versus BPH group, the CgA level difference was 63.00 ng/ml ( p < 0.0001) and the PSA level difference was 50.86 mcg/ ml ( p < 0.0001). Between prostate cancer group and control group the CgA level difference was 94.3 ng/ml ( p < 0.0001), the PSA level difference was 52.91 mcg/ml ( p < 0.0001), and the NSE level difference was 1.34 mg/l ( p < 0.0001). Patients with higher CgA levels had poorer prognosis and survival, compared to those with lower CgA levels. These results support the concept that serum CgA level determination before treatment is a potential prognostic factor for prostate cancer. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chromogranin-A (CgA); Prostate cancer; Benign prostate hyperplasia (BPH); Prostate-specific antigen (PSA); Neuron-specific enolase (NSE)
1. Introduction Prostate cancer is one of the most frequent tumors in men, with an incidence of 55–65/ 100.000 inhabitants in European countries. It has been predicted that the current incidence * Corresponding author. Present address: Viale Andrea Doria 69, I-95126 Catania, Italy. Tel.: +39 095 726 2008; fax: +39 095 726 2011. E-mail address: [email protected] (M. Malaguarnera). 0167-4943/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2005.09.008
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of 79,543 new cases per year will rise up to 118,175 new cases by 2020 in Europe, and it will be the second leading cause of cancer-related deaths (Jensen et al., 1990; Jemal et al., 2002). Prostate tumors range from small, slowly growing lesions to aggressive tumors which metastasize rapidly, and there is a controversy about which screening-detected lesion will become clinically significant (Neal and Donovan, 2000). The normal prostate tissue consists of three cell types originating from the totipotent stem cells: (1) the basal cells; (2) the secretory, or exocrine cells, expressing the prostatespecific antigen (PSA); and (3) the neuroendocrine cells (NE) (Xue et al., 1998). The NE cells are present diffusely in the prostate of the newborn, but they quickly decrease in the peripheral zone immediately after the birth, and reappear after the puberty. During the puberty, the number of NE prostate cells increases, and reaches the optimal level between 25 and 54 years. In adult ages these cells are primarily located in periuretral prostate tubules (Daneshmand et al., 2005). The peptide most frequently produced by the NE prostate cells is chromogranin-A (CgA) and/or the neuron-specific enolase (NSE). CgA and NSE are the most frequently employed markers to detect neuroendocrine features, either at the tissue level or in general circulation. CgA is a member of the granin family and it is utilized as a cellular marker for endocrine and NE cells. CgA is a pro-hormone and its proteolysis constitutes a key element of its physiology. This process releases biologically active peptides having different paracrine and autoendocrine functions. The proteolysis is tissue-specific and the fragmentation of granin–proteins differs depending on its location. Although the function of CgA is not well known, it seems to be related to its calcium-binding property (Iacangelo et al., 1986). CgA appears to be stable in plasma at room temperature for extended periods (O’Connor et al., 1989) and can be measured by radioimmunoassay or enzyme-linked immunoadsorbent assay (Syversen et al., 1994). Thus, it is a promising candidate for simple and reliable assessment of prostate neuroendocrine activity. In addition, circulating CgA, due to different release characteristics and its longer half-life in plasma, may be less prone to rapid fluctuations in its plasma levels, than the catecholamines (Hsiao et al., 1990). Among the molecules studied, CgA seems to be most accurate and reliable marker for neuroendocrine tumors (NET). There are several reasons for this, as follows: (1) the specificity of CgA is very high regardless of the analytical method or the type of biological sample used; (2) CgA is highly expressed by cells of neuroendocrine origin, both normal and tumoral; (3) CgA identifies the NET and the metastases regardless of their localization; (4) CgA is expressed both by functioning and non-functioning NET; and (5) accurate and easy assays have been developed for plasma CgA determination (Fracalanza et al., 2005). NSE is the neuron-specific isomer of the glycolytic enzyme 2-phospho-D-glycerate hydrolase or enolase (Schmechel et al., 1987). It is a widely used immunohistochemical and serum marker for NE tissues and it is especially known as a marker for small-cell lung carcinoma (Akoun et al., 1985) and in the Merkel-cell tumors (Oishi and Sato, 1988; Cunningham et al., 1992). We investigated the role of the serum concentrations of CgA and NSE in a group of patients with prostate cancer and with benign prostate hyperplasia (BPH) and the relationship between the pre-treatment serum CgA and NSE levels, and clinical stages of the disease.
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2. Patients and methods A total of 57 patients with prostate adenocarcinoma (PAC) and 61 patients with BPH were enrolled between 1999 and 2003. In the same time, 44 elderly patients (control group) who were randomly selected from applicants for an annual health check-up were examined. They were judged as normal on the basis of the results of physical examination and laboratory findings. All patients and controls were screened for interfering factors on CgA assay as proton pump inhibitors, steroids, and renal failure. As a matter of fact, all these conditions can generate false positive results. Histological diagnosis of PAC was confirmed by needle biopsy, transurethral resection or sub-capsular prostatectomy. Only patients who have given written informed consent to use the frozen sera for analysis were included in the study. Blood samples were taken from the patients and sera were immediately frozen and stored at 20 8C until analysis. The analytical precision was evaluated by calculating within and between assay coefficient of variation (CV) on 10 replicates and 5 runs, respectively. The within and between assay CV was 4.0% and 6.1%, respectively. Serum PSA levels were determined at least once every 2 months using the tandem R-Kit (Hybritech, San Diego, CA, USA), i.e., the storage time of the samples was at worst cases 60 days. A commercial solid phase, two site immuno-radiometric assay was used to detect serum CgA (CgA-RIA CT, CIS Biointernational ORIS Group, Gif-sur-Yvette, France). Two monoclonal antibodies were prepared against sterically remote sites on the CgA molecule. The first was coated into the solid phase (coated tube), the second radiolabeled with iodine125 and used as a tracer. CgA present in the standards or the samples to be tested are sandwiched between the two antibodies. Following the formation of the coated antibody, antigen-iodinated antibody sandwich, the unbound tracer is easily removed by a washing step. The radioactivity bound to the tube is proportional to the concentration of CgA in the sample. Within assay CV ranged from 6.1% to 8.4%, and between assay from 5.2% to 6.7%, respectively. The normal range for serum levels of CgA in a control population is reported as 20–100 ng/ml (Wu et al., 1999), while the average of our 44 control subjects was 45.8 23.2 (see in Section 3). NSE was measured using an immuno-radiometric analysis (IRMA) based on two monoclonal antibodies, one of which was labeled with I125, while the other was combed to magnetizable mono-dispersed polymer particles. Bound radioactivity was counted in Wallac Wizard 1470 Automatic Gamma Counter. The within and between assay CV was 4.9% and 6.1%, respectively. A cutoff value of 10 mg/l NDE separated the normal from the elevated values (Paus and Nustad, 1989). Clinical chemistry tests were performed in the medical center laboratory using standard methods. Fasting blood samples were taken at enrolment from the participants. 2.1. Gleason’s grading The biopsies and surgical specimens were graded by the method of Gleason and Mellinger (1974), and patients were given the highest score, if there was more than one. Our referee pathologist reviewed all out-of-house biopsy results. The following categories
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Table 1 The TNM staging system in prostate cancer (Hoedemaeker et al., 2000) Stage
Description of properties
Primary tumor (T) Tx T0
Primary tumor cannot be assessed No evidence of primary tumor
T1 T1a T1b T1c
Clinically inapparent tumor, not palpable or visible by imaging Tumor incidental, found in 5% or less of resected tissues Tumor incidental, found in more than 5% of resected tissues Tumor identified by needle biopsy (performed because of high PSA)
T2 T2a T2b
Tumor confined within the prostate gland Tumor involves one lobe Tumor involves both lobes
T3 T3a T3b
Tumor extends through the prostatic capsule Extracapsular extensions (unilateral or bilateral) Tumor invades seminal vesicles
T4
Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, and/or pelvic wall
Regional lymph nodes (N) Nx N0 N1
Regional lymph nodes have not been observed No regional lymph node metastasis Regional lymph node metastasis is present
Distant metastases (M) Mx M0 M1 M1a M1b M1c
Distant metastasis has not been found No distant metastasis Distant metastasis observed In the non-regional lymph nodes In the bones At other sites
of Gleason’s grading have been applied: well-differentiated 2–4; intermediate-grade: 5–6; and high-grade: 7–10 (Gleason and Mellinger, 1974). 2.2. The clinical stages of the tumors The tumor–node–metastasis (TNM) system is the most widely used means for classifying the extent of cancer spread. TNM classification of malignant tumors provides the internationally accepted standards to describe and categorize cancer stages and progression (Chisholm et al., 1994). Definitions for classifying the primary tumor (T) are the same for clinical and for pathologic classification. There are four stages of local tumor growth, from T1 (incidental) to T4 (invasion of neighboring organs). T1 represents an ‘incidental’ state, where the tumor is detected by chance following transurethral resection or by biopsy following PSA testing. At this stage, the tumor remains undetectable by palpation (digital rectal examination; DRE) or ultrasonography. T4 represents advanced
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disease, where the tumor has invaded neighboring organs. The nodal stage (N0–N1) and the metastatic stage (M0–M1C) reflect the clinical spread of the disease to lymph nodes and distant sites (metastasis), respectively. A full description of the TNM staging system is shown in Table 1. In our studies we used only the part T of the TNM classification, in order to reveal the correlation between them and the measured laboratory parameters, as described later. 2.3. Statistical analysis All data are presented as mean standard deviation (S.D.). The following two-tailed tests at p < 0.05 level of significance were used to evaluate the results: the Mann–Whitney U-test was used in the case of two independent samples and the Spearman’s rank correlation coefficient test was used to test for univariate relationships between variables. Differences between groups were analyzed with chi-square tests for categorical variables. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc., Chicago, IL, USA).
3. Results Table 2 presents the mean basic parameters of the three groups studied. Only insignificant differences were observed in the age, systolic and diastolic blood pressure (SBP and DBP, respectively), the heart frequency, and body weight. However, the difference of mean CgA level of prostate cancer group was 94.3 ng/ml higher ( p < 0.0001), the PSA was 52.9 mcg/ml higher ( p < 0.0001) and the NSE was 1.38 mg/l higher ( p < 0.0001), than in the control group (Table 2). If comparing prostate cancer group to BPH group, the mean difference in CgA levels was 63.0 ng/ml ( p < 0.0001), the PSA difference was 50.9 mcg/ml ( p < 0.0001) and the NSE difference was 1.05 mg/l ( p = 0.004) (Table 2). Similar comparison of the BPH to control group resulted in mean differences of 31.2 ng/ml ( p < 0.0001), 2.1 mcg/ml ( p < 0.0001), and 2.4 mg/l ( p < 0.0001) for the CgA, PSA, and the NSE levels, respectively (Table 2). Table 2 The main characteristics of the groups of our study population Parameter
Prostate cancer
BPH
Controls
Number Age (years) SBP (mmHg) DBP (mmHg) Heart frequency (min 1) Weight (kg) CgA (ng/ml) PSA (mcg/ml) NSE (mg/l)
57 74 6.7 156 34 78 13 79 13 74 8 140.1 53.2 55.1 17.4 3.8 2.1
61 71 3.2 141 36 80 11 77 18 77 9 77 32 4.2 2.1 4.8 1.7
44 70 4.2 (60–81) 144 42 77 13 80 14 71 10 45.8 23.2 2.2 0.7 2.4 0.9
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Table 3 Distribution of the results in the prostate tumors according to their Gleason’s grades Grade-ranges
Number
CgA (ng/ml)
NSE (mg/l)
PSA (mcg/ml)
Low-grade (2–4) Intermediate-grade (5–6) High-grade (7–10)
23 15 19
111.0 54.0 140.7 34.5 174.63 42.9
3.4 2.2 4.0 2.0 4.1 2.2
44.1 16.0 55.9 14.0 67.9 12.1
The Gleason’s score comparisons are presented in Table 3. Low-grade cases showed significantly lower CgA levels ( 29.7 ng/ml in average, p < 0.001), than the intermediategrade group, while the differences in NSE and PSA levels were only 0.6 mg/l ( p = 0.408) and 11.8 mcg/l ( p < 0.01), respectively, i.e., remained insignificant (Table 3). If comparing low-grade versus high-grade groups, the CgA and PSA levels were significantly higher in the high-grade group: the mean difference was 63.7 ng/ml ( p < 0.0001), and 23.8 mcg/l ( p < 0.0001), respectively, but the NSE levels remained invariate, being different only by 0.7 mg/l ( p = 0.313), i.e., insignificant in statistical terms (Table 3). Similar comparisons between the intermediate-grade versus high-grade groups gave similar results. Namely, the CgA and PSA were higher in the latter group, by 34.0 ng/ml ( p < 0.0001) and 12.0 mcg/l ( p = 0.011) in average, respectively, while the NSE levels did not change significantly, since the mean difference was only 0.1 mg/l ( p = 0.880) (Table 3). The distribution of the mean values of the measured parameters was also calculated for the different clinical stages (Table 4). The comparison between T1 and T2 stages revealed higher values in T2 group, in CgA levels of 38.8 ng/ml ( p < 0.01), in the NSE levels 0.9 mg/l ( p = 0.032), and in the PSA levels 24.3 mcg/l ( p < 0.0001). Comparing the T2 and T3 stages, again significant increases were observed in CgA levels 48.6 ng/ml ( p = 0.003) and in NSE levels 1.6 mg/l ( p = 0.028), of the T3 group, while PSA levels remained invariate (1.3 mcg/l; p = 0.769). Table 4 also demonstrates that CgA and PSA levels were significantly higher in the T3 group as compared to Tx (by 42.9 ng/ml; p = 0.037 and by 13.0 mcg/l; p = 0.038), respectively, whereas the NSE values were not different from each other in these groups ( p = 0.511). If comparing the stages T1 and T3, the differences of all three parameters proved to be strongly significant: CgA, NSE, and PSA levels were higher in the T3 group ( p < 0.0001; p = 0.003; and p < 0.0001, respectively) (Table 4).
Table 4 The distribution of the results according to the clinical stages of the tumors Stages
Number
CgA (ng/ml)
NSE (mg/l)
PSA (mcg/ml)
T1 T2 T3 Tx
12 15 19 11
92.4 28.6 131.2 36.7 179.8 50.2 136.9 52.3
2.4 1.0 3.2 1.0 4.9 2.6 4.2 2.4
37.8 14.3 62.1 11.3 63.4 13.7 50.4 19.0
Note: The meaning of stages is shown in Table 1.
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T1 versus Tx stages differed significantly in cases of CgA ( p = 0.018) and NSE ( p = 0.021) levels, while the existing small difference in PSA mean values remained insignificant ( p = 0.084) (Table 4). In case of T2 stage, all three parameters displayed only insignificant differences to the means of Tx stage: for CgA, NSE, and PSA means we found p = 0.716; 0.163; and 0.061, respectively.
4. Discussion Prostate adenocarcinoma remains androgen dependent in its early stages, but after androgen withdrawal, relapses to an androgen insensitive disease (Gittes, 1991). The underlying mechanisms determining the progression to androgen insensitivity are poorly understood. Although both the management policy for early prostate cancer and the routine population screening for this disease remain matters of debate, increasing the rate of early diagnosis and treatment would seem to be a desiderable goal (Wilt, 2002). Prostate cancer has had a much lower profile and it seems unlikely that the population at risk is sufficiently aware of the disease and the possibilities for early treatment and cure. The results of the few published studies of public awareness of prostate cancer support this view (Price et al., 1993; Mainous and Hagen, 1994). However, these relatively small studies were conducted more than 10 years ago, and only one of the not too old studies assessed the knowledge of a variety of issues concerning prostate cancer (Egawa et al., 1998). After its discovery 20 years ago, the PSA has been considered to be the most valuable tool in the early detection, staging, and monitoring of prostate cancer (Polascik et al., 1999). However, PSA turned out to be not specific for prostate cancer, since elevated serum PSA levels were detected also in patients with BPH and prostatitis. One of the limitations of PSA as a tumor marker is that there is a substantial overlap in serum PSA values between men with BPH and prostate cancer. PSA testing resulted in findings of prostate cancer at earlier stages and in younger men, than before, however, the situation has been equilibrated during the recent years (Jemal et al., 2002). In addition, a recent analysis found that the pre-treatment PSA levels fell by a rate of 0.8% per year in average between 1988 and 1997 (Jani et al., 2001). The NE cells have been detected in benign prostate hypertrophy, prostate intraepithelial neoplasms (PIN), and in all the stages of prostate carcinoma. Recently, several serum and genetic markers have been associated with the pathogenesis of prostate cancer, suggesting their potential role as prognostic factors. The concept of neuroendocrine differentiation in prostate cancer has become more widely recognized and has attracted considerable attention as a potentially new finding with major diagnostic, prognostic, and therapeutic implications. The physiological role of prostate neuroendocrine cells is unknown, but they are supposed to be involved both in the regulation of prostate growth and differentiation (Abrahmoson, 1999). The neuroendocrine cells in both benign and malignant prostate tissues can be identified immunohistochemically with the use of antibodies against a number of products produced by these cells, including NSE, CgA, and members of the calcitonin gene family. Little is known about the specific role that the neuroendocrine cells
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have in the prostate or their relationship to the pathogenesis, growth, and prognosis of prostate diseases (Sciarra et al., 2003). Serum levels of the neuroendocrine marker CgA could reflect the NE activity of prostate carcinoma and could be used during the follow-up evaluation of advanced prostate carcinoma. In contrast to CgA, the utility of measuring NSE in patients with advanced prostate cancer is uncertain. The incidence of high NSE levels in patients with prostate cancer was lower than that of high chromogranin levels (Cussenot et al., 1998). NSE and CgA serum levels were very high in patients with hormone therapy-resistant prostate cancer than in patients with BPH and localized prostate cancer (Hvamstad et al., 2003). However, the determination of NE markers in serum may not be specific for prostate activity and only the analysis of a very large stratified population may determine whether there is a significant serum marker of NE activity in prostate cancer. It has been observed that serum CgA levels reflect the entity of NE differentiation of the prostate carcinoma in relationship to the stage of disease (Sciarra et al., 2004). Therefore, the measurement of CgA is useful, both from the diagnostic and prognostic points of view. Particularly elevated values of serum CgA levels are observed in a patient with hormonerefractory prostate carcinoma and they are associated to worse prognosis (Berruti et al., 2005). Low levels of CgA in the circulation are independent of age. The importance of increased CgA levels in serum was first shown in patients with pheochromocytoma and then demonstrated for other endocrine cancers (Ferrari et al., 1999; Cotesta et al., 2005). Elevated circulating levels have been seen in several neuroendocrine tumors, including pheochromocytomas (O’Connor and Frigon, 1984; O’Connor and Deftos, 1986), carcinoid tumors (Syversen et al., 1993), and neuroblastomas (Hsiao et al., 1990). The sensitivity and specificity of CgA in any NETs vary between 70% and 95% and between 70% and 80%, respectively. The highest accuracy has been observed in tumors characterized by an intense secretory activity, although the specificity and sensitivity remain very high also in non-functioning tumors. (Anderson and Lynch, 1993; Capella et al., 1995; Hauser et al., 1995; Neary et al., 1997). The clinical use of other neuro-hormonal markers has been limited by their in vitro instability, which requires special sampling, handling, and storage procedures. In contrast, CgA may be measured in serum or plasma collected with standard equipment, stored at room temperature for prolonged periods, and analyzed without extraction procedures. The measurement of neuroendocrine markers such as CgA in the blood of prostate cancer patients certainly constitutes a more objective assessment of the neuroendocrine differentiation of tumors, as it may correspond to the entire primary tumor cell population and its associated metastasis. The NE cells have been detected in benign prostate hypertrophy, PIN and in all the stages of prostate carcinoma. Patients with higher CgA level had poor survival and showed poor prognosis, compared to those with lower CgA level; these results support the notion that pretreatment serum CgA level determination is a potential prognostic factor for prostate cancer. The subjects with higher CgA levels had a higher grade of Gleason’s score. From these data we can realize that this method has not only a diagnostic use, since it allows us to evaluate the neuroendocrine differentiation of prostate cancer. The importance of CgA does not concern only the diagnostic and prognostic aspects, but also the therapeutic approach: in fact, CgA could permit a correct evaluation of therapy with somatostatin, which is the therapy of tumors with neuroendocrine differentiation (Berruti et al., 2001).
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Jensen, M., Steve, J., Moller, H., Remard, H., 1990. Cancer in the European Community and its member states. Eur. J. Cancer 26, 1167–1256. Mainous, A.G., Hagen, M.D., 1994. Public awareness of prostate cancer and the prostate-specific antigen test. Cancer Pract. 2, 217–221. Neal, D.E., Donovan, J.L., 2000. Prostate cancer: to screen or not to screen? Lancet Oncol. 1, 17–24. Neary, P.C., Redmond, P.H., Houghton, T., Watson, G.R.K., Bouchier-Hayes, D., 1997. Carcinoid disease. Review of the literature. Dis. Colon Rectum 40, 349–362. O’Connor, D.T., Deftos, L.J., 1986. Secretion of chromogranin A by peptide-producing endocrine neoplasms. N. Engl. J. Med. 314, 1145–1151. O’Connor, D.T., Frigon, R.P., 1984. Chromogranin A, the major catecholamine storage vesicle soluble protein. J. Biol. Chem. 259, 3237–3247. O’Connor, D.T., Pandian, M.R., Carlton, E., Cervenka, J.H., Hsiao, R.J., 1989. Rapid radioimmunoassay of circulating chromogranin A: in vitro stability, exploration of the neuroendocrine character of neoplasia, and assessment of the effect of organ failure. Clin. Chem. 35, 1631–1637. Oishi, S., Sato, T., 1988. Elevated serum neuron-specific enolase in patients with malignant pheochromocytoma. Cancer 61, 1167–1170. Paus, E., Nustad, K., 1989. Immunoradiometric assay for ag and gg-enolase (neuron-specific enolase) with use of monoclonal antibodies and magnetizable polymer particles. Clin. Chem. 35, 2034–2037. Polascik, T.J., Oesterling, J.E., Partin, A.W., 1999. Prostate-specific antigen: a decade of discovery. What we have learned and where we are going. J. Urol. 162, 293–306. Price, J.H., Colvin, T.L., Smith, D., 1993. Prostate cancer: perception of African–American males. J. Natl. Med. Assoc. 85, 941–947. Schmechel, D.E., Marangos, P.J., Martin, B.M., Winfield, S., Burkhart, D.S., Roses, A.D., Ginns, E.I., 1987. Localization of neuron-specific enolase (NSE) mRNA in human brain. Neurosci. Lett. 76, 233–238. Sciarra, A., Mariotti, G., Gentile, V., Voria, G., Pastore, A., Monti, S., Di Silverio, F., 2003. Neuroendocrine differentiation in human prostate tissue: is it detectable and treatable? Br. J. Urol. Int. 91, 438–445. Sciarra, A., Voria, G., Monti, S., Mazzone, L., Mariotti, G., Pozza, M., D’Eramo, G., Silverio, F.D., 2004. Clinical understaging in patients with prostate adenocarcinoma submitted to radical prostatectomy: predictive value of serum chromogranin A. Prostate 58, 421–428. Syversen, U., Mignon, M., Bonfils, S., Kristensen, A., Waldum, H.L., 1993. Chromogranin A and pancreastatinlike immunoreactivity in serum of gastrinoma patients. Acta Oncol. 32, 161–165. Syversen, U., Jacobsen, M.B., O’Connor, D.T., Ronning, K., Waldum, H.L., 1994. Immuno-assay for measurement of chromogranin A and pancrea-statin-like immunoreactivity in humans: correspondence in patients with neuroendocrine neoplasia. Neuropeptides 26, 201–206. Wilt, T.J., 2002. Clarifying uncertainty regarding detection and treatment of early-stage prostate cancer. Semin. Urol. Oncol. 20, 10–17. Wu, T.L., Chang, C.P., Tsao, K.C., Sun, C.F., Wu, J.T., 1999. Development of a microplate assay for serum chromogranin A (CgA): establishment of normal reference values and detection of elevated CgA in malignant diseases. J. Clin. Lab. Anal. 13, 312–319. Xue, Y., Smedts, F., Verhofstad, A., Debruyne, F., De la Rosette, J., Schalken, J., 1998. Cell kinetics of prostate exocrine and neuroendocrine epithelium and their differential interrelationship: new perspectives. Prostate Suppl. 8, 62–73.
The chromogranin-A (CgA) in prostate cancer Salvatore Ranno, Massimo Motta, Elvira Rampello, Corrado Risino, Ettore Bennati, Mariano Malaguarnera * Department of Senescence, Urological and Neurological Sciences, University of Catania, Cannizzaro Hospital, Via Messina 829, I-95126 Catania, Italy Received 12 April 2005; received in revised form 8 September 2005; accepted 14 September 2005 Available online 8 November 2005
Abstract Prostate cancer is one of the most frequent tumors in men. The neuroendocrine differentiation in prostate cancer has become more widely recognized and has attracted considerable attention as a potentially new finding with major diagnostic, prognostic and therapeutic implications. We investigated the role of the serum concentrations of CgA in a group of 57 patients with prostate cancer and in 61 elderly subjects with benign prostate hyperplasia (BPH). Neuron-specific enolase (NSE) is the most frequently employed marker to detect neuroendocrine features. Serum prostate-specific antigen (PSA), CgA and NSE levels were determined. Comparing prostate cancer group versus BPH group, the CgA level difference was 63.00 ng/ml ( p < 0.0001) and the PSA level difference was 50.86 mcg/ ml ( p < 0.0001). Between prostate cancer group and control group the CgA level difference was 94.3 ng/ml ( p < 0.0001), the PSA level difference was 52.91 mcg/ml ( p < 0.0001), and the NSE level difference was 1.34 mg/l ( p < 0.0001). Patients with higher CgA levels had poorer prognosis and survival, compared to those with lower CgA levels. These results support the concept that serum CgA level determination before treatment is a potential prognostic factor for prostate cancer. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Chromogranin-A (CgA); Prostate cancer; Benign prostate hyperplasia (BPH); Prostate-specific antigen (PSA); Neuron-specific enolase (NSE)
1. Introduction Prostate cancer is one of the most frequent tumors in men, with an incidence of 55–65/ 100.000 inhabitants in European countries. It has been predicted that the current incidence * Corresponding author. Present address: Viale Andrea Doria 69, I-95126 Catania, Italy. Tel.: +39 095 726 2008; fax: +39 095 726 2011. E-mail address: [email protected] (M. Malaguarnera). 0167-4943/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2005.09.008
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of 79,543 new cases per year will rise up to 118,175 new cases by 2020 in Europe, and it will be the second leading cause of cancer-related deaths (Jensen et al., 1990; Jemal et al., 2002). Prostate tumors range from small, slowly growing lesions to aggressive tumors which metastasize rapidly, and there is a controversy about which screening-detected lesion will become clinically significant (Neal and Donovan, 2000). The normal prostate tissue consists of three cell types originating from the totipotent stem cells: (1) the basal cells; (2) the secretory, or exocrine cells, expressing the prostatespecific antigen (PSA); and (3) the neuroendocrine cells (NE) (Xue et al., 1998). The NE cells are present diffusely in the prostate of the newborn, but they quickly decrease in the peripheral zone immediately after the birth, and reappear after the puberty. During the puberty, the number of NE prostate cells increases, and reaches the optimal level between 25 and 54 years. In adult ages these cells are primarily located in periuretral prostate tubules (Daneshmand et al., 2005). The peptide most frequently produced by the NE prostate cells is chromogranin-A (CgA) and/or the neuron-specific enolase (NSE). CgA and NSE are the most frequently employed markers to detect neuroendocrine features, either at the tissue level or in general circulation. CgA is a member of the granin family and it is utilized as a cellular marker for endocrine and NE cells. CgA is a pro-hormone and its proteolysis constitutes a key element of its physiology. This process releases biologically active peptides having different paracrine and autoendocrine functions. The proteolysis is tissue-specific and the fragmentation of granin–proteins differs depending on its location. Although the function of CgA is not well known, it seems to be related to its calcium-binding property (Iacangelo et al., 1986). CgA appears to be stable in plasma at room temperature for extended periods (O’Connor et al., 1989) and can be measured by radioimmunoassay or enzyme-linked immunoadsorbent assay (Syversen et al., 1994). Thus, it is a promising candidate for simple and reliable assessment of prostate neuroendocrine activity. In addition, circulating CgA, due to different release characteristics and its longer half-life in plasma, may be less prone to rapid fluctuations in its plasma levels, than the catecholamines (Hsiao et al., 1990). Among the molecules studied, CgA seems to be most accurate and reliable marker for neuroendocrine tumors (NET). There are several reasons for this, as follows: (1) the specificity of CgA is very high regardless of the analytical method or the type of biological sample used; (2) CgA is highly expressed by cells of neuroendocrine origin, both normal and tumoral; (3) CgA identifies the NET and the metastases regardless of their localization; (4) CgA is expressed both by functioning and non-functioning NET; and (5) accurate and easy assays have been developed for plasma CgA determination (Fracalanza et al., 2005). NSE is the neuron-specific isomer of the glycolytic enzyme 2-phospho-D-glycerate hydrolase or enolase (Schmechel et al., 1987). It is a widely used immunohistochemical and serum marker for NE tissues and it is especially known as a marker for small-cell lung carcinoma (Akoun et al., 1985) and in the Merkel-cell tumors (Oishi and Sato, 1988; Cunningham et al., 1992). We investigated the role of the serum concentrations of CgA and NSE in a group of patients with prostate cancer and with benign prostate hyperplasia (BPH) and the relationship between the pre-treatment serum CgA and NSE levels, and clinical stages of the disease.
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2. Patients and methods A total of 57 patients with prostate adenocarcinoma (PAC) and 61 patients with BPH were enrolled between 1999 and 2003. In the same time, 44 elderly patients (control group) who were randomly selected from applicants for an annual health check-up were examined. They were judged as normal on the basis of the results of physical examination and laboratory findings. All patients and controls were screened for interfering factors on CgA assay as proton pump inhibitors, steroids, and renal failure. As a matter of fact, all these conditions can generate false positive results. Histological diagnosis of PAC was confirmed by needle biopsy, transurethral resection or sub-capsular prostatectomy. Only patients who have given written informed consent to use the frozen sera for analysis were included in the study. Blood samples were taken from the patients and sera were immediately frozen and stored at 20 8C until analysis. The analytical precision was evaluated by calculating within and between assay coefficient of variation (CV) on 10 replicates and 5 runs, respectively. The within and between assay CV was 4.0% and 6.1%, respectively. Serum PSA levels were determined at least once every 2 months using the tandem R-Kit (Hybritech, San Diego, CA, USA), i.e., the storage time of the samples was at worst cases 60 days. A commercial solid phase, two site immuno-radiometric assay was used to detect serum CgA (CgA-RIA CT, CIS Biointernational ORIS Group, Gif-sur-Yvette, France). Two monoclonal antibodies were prepared against sterically remote sites on the CgA molecule. The first was coated into the solid phase (coated tube), the second radiolabeled with iodine125 and used as a tracer. CgA present in the standards or the samples to be tested are sandwiched between the two antibodies. Following the formation of the coated antibody, antigen-iodinated antibody sandwich, the unbound tracer is easily removed by a washing step. The radioactivity bound to the tube is proportional to the concentration of CgA in the sample. Within assay CV ranged from 6.1% to 8.4%, and between assay from 5.2% to 6.7%, respectively. The normal range for serum levels of CgA in a control population is reported as 20–100 ng/ml (Wu et al., 1999), while the average of our 44 control subjects was 45.8 23.2 (see in Section 3). NSE was measured using an immuno-radiometric analysis (IRMA) based on two monoclonal antibodies, one of which was labeled with I125, while the other was combed to magnetizable mono-dispersed polymer particles. Bound radioactivity was counted in Wallac Wizard 1470 Automatic Gamma Counter. The within and between assay CV was 4.9% and 6.1%, respectively. A cutoff value of 10 mg/l NDE separated the normal from the elevated values (Paus and Nustad, 1989). Clinical chemistry tests were performed in the medical center laboratory using standard methods. Fasting blood samples were taken at enrolment from the participants. 2.1. Gleason’s grading The biopsies and surgical specimens were graded by the method of Gleason and Mellinger (1974), and patients were given the highest score, if there was more than one. Our referee pathologist reviewed all out-of-house biopsy results. The following categories
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Table 1 The TNM staging system in prostate cancer (Hoedemaeker et al., 2000) Stage
Description of properties
Primary tumor (T) Tx T0
Primary tumor cannot be assessed No evidence of primary tumor
T1 T1a T1b T1c
Clinically inapparent tumor, not palpable or visible by imaging Tumor incidental, found in 5% or less of resected tissues Tumor incidental, found in more than 5% of resected tissues Tumor identified by needle biopsy (performed because of high PSA)
T2 T2a T2b
Tumor confined within the prostate gland Tumor involves one lobe Tumor involves both lobes
T3 T3a T3b
Tumor extends through the prostatic capsule Extracapsular extensions (unilateral or bilateral) Tumor invades seminal vesicles
T4
Tumor is fixed or invades adjacent structures other than seminal vesicles: bladder neck, external sphincter, rectum, levator muscles, and/or pelvic wall
Regional lymph nodes (N) Nx N0 N1
Regional lymph nodes have not been observed No regional lymph node metastasis Regional lymph node metastasis is present
Distant metastases (M) Mx M0 M1 M1a M1b M1c
Distant metastasis has not been found No distant metastasis Distant metastasis observed In the non-regional lymph nodes In the bones At other sites
of Gleason’s grading have been applied: well-differentiated 2–4; intermediate-grade: 5–6; and high-grade: 7–10 (Gleason and Mellinger, 1974). 2.2. The clinical stages of the tumors The tumor–node–metastasis (TNM) system is the most widely used means for classifying the extent of cancer spread. TNM classification of malignant tumors provides the internationally accepted standards to describe and categorize cancer stages and progression (Chisholm et al., 1994). Definitions for classifying the primary tumor (T) are the same for clinical and for pathologic classification. There are four stages of local tumor growth, from T1 (incidental) to T4 (invasion of neighboring organs). T1 represents an ‘incidental’ state, where the tumor is detected by chance following transurethral resection or by biopsy following PSA testing. At this stage, the tumor remains undetectable by palpation (digital rectal examination; DRE) or ultrasonography. T4 represents advanced
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disease, where the tumor has invaded neighboring organs. The nodal stage (N0–N1) and the metastatic stage (M0–M1C) reflect the clinical spread of the disease to lymph nodes and distant sites (metastasis), respectively. A full description of the TNM staging system is shown in Table 1. In our studies we used only the part T of the TNM classification, in order to reveal the correlation between them and the measured laboratory parameters, as described later. 2.3. Statistical analysis All data are presented as mean standard deviation (S.D.). The following two-tailed tests at p < 0.05 level of significance were used to evaluate the results: the Mann–Whitney U-test was used in the case of two independent samples and the Spearman’s rank correlation coefficient test was used to test for univariate relationships between variables. Differences between groups were analyzed with chi-square tests for categorical variables. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc., Chicago, IL, USA).
3. Results Table 2 presents the mean basic parameters of the three groups studied. Only insignificant differences were observed in the age, systolic and diastolic blood pressure (SBP and DBP, respectively), the heart frequency, and body weight. However, the difference of mean CgA level of prostate cancer group was 94.3 ng/ml higher ( p < 0.0001), the PSA was 52.9 mcg/ml higher ( p < 0.0001) and the NSE was 1.38 mg/l higher ( p < 0.0001), than in the control group (Table 2). If comparing prostate cancer group to BPH group, the mean difference in CgA levels was 63.0 ng/ml ( p < 0.0001), the PSA difference was 50.9 mcg/ml ( p < 0.0001) and the NSE difference was 1.05 mg/l ( p = 0.004) (Table 2). Similar comparison of the BPH to control group resulted in mean differences of 31.2 ng/ml ( p < 0.0001), 2.1 mcg/ml ( p < 0.0001), and 2.4 mg/l ( p < 0.0001) for the CgA, PSA, and the NSE levels, respectively (Table 2). Table 2 The main characteristics of the groups of our study population Parameter
Prostate cancer
BPH
Controls
Number Age (years) SBP (mmHg) DBP (mmHg) Heart frequency (min 1) Weight (kg) CgA (ng/ml) PSA (mcg/ml) NSE (mg/l)
57 74 6.7 156 34 78 13 79 13 74 8 140.1 53.2 55.1 17.4 3.8 2.1
61 71 3.2 141 36 80 11 77 18 77 9 77 32 4.2 2.1 4.8 1.7
44 70 4.2 (60–81) 144 42 77 13 80 14 71 10 45.8 23.2 2.2 0.7 2.4 0.9
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Table 3 Distribution of the results in the prostate tumors according to their Gleason’s grades Grade-ranges
Number
CgA (ng/ml)
NSE (mg/l)
PSA (mcg/ml)
Low-grade (2–4) Intermediate-grade (5–6) High-grade (7–10)
23 15 19
111.0 54.0 140.7 34.5 174.63 42.9
3.4 2.2 4.0 2.0 4.1 2.2
44.1 16.0 55.9 14.0 67.9 12.1
The Gleason’s score comparisons are presented in Table 3. Low-grade cases showed significantly lower CgA levels ( 29.7 ng/ml in average, p < 0.001), than the intermediategrade group, while the differences in NSE and PSA levels were only 0.6 mg/l ( p = 0.408) and 11.8 mcg/l ( p < 0.01), respectively, i.e., remained insignificant (Table 3). If comparing low-grade versus high-grade groups, the CgA and PSA levels were significantly higher in the high-grade group: the mean difference was 63.7 ng/ml ( p < 0.0001), and 23.8 mcg/l ( p < 0.0001), respectively, but the NSE levels remained invariate, being different only by 0.7 mg/l ( p = 0.313), i.e., insignificant in statistical terms (Table 3). Similar comparisons between the intermediate-grade versus high-grade groups gave similar results. Namely, the CgA and PSA were higher in the latter group, by 34.0 ng/ml ( p < 0.0001) and 12.0 mcg/l ( p = 0.011) in average, respectively, while the NSE levels did not change significantly, since the mean difference was only 0.1 mg/l ( p = 0.880) (Table 3). The distribution of the mean values of the measured parameters was also calculated for the different clinical stages (Table 4). The comparison between T1 and T2 stages revealed higher values in T2 group, in CgA levels of 38.8 ng/ml ( p < 0.01), in the NSE levels 0.9 mg/l ( p = 0.032), and in the PSA levels 24.3 mcg/l ( p < 0.0001). Comparing the T2 and T3 stages, again significant increases were observed in CgA levels 48.6 ng/ml ( p = 0.003) and in NSE levels 1.6 mg/l ( p = 0.028), of the T3 group, while PSA levels remained invariate (1.3 mcg/l; p = 0.769). Table 4 also demonstrates that CgA and PSA levels were significantly higher in the T3 group as compared to Tx (by 42.9 ng/ml; p = 0.037 and by 13.0 mcg/l; p = 0.038), respectively, whereas the NSE values were not different from each other in these groups ( p = 0.511). If comparing the stages T1 and T3, the differences of all three parameters proved to be strongly significant: CgA, NSE, and PSA levels were higher in the T3 group ( p < 0.0001; p = 0.003; and p < 0.0001, respectively) (Table 4).
Table 4 The distribution of the results according to the clinical stages of the tumors Stages
Number
CgA (ng/ml)
NSE (mg/l)
PSA (mcg/ml)
T1 T2 T3 Tx
12 15 19 11
92.4 28.6 131.2 36.7 179.8 50.2 136.9 52.3
2.4 1.0 3.2 1.0 4.9 2.6 4.2 2.4
37.8 14.3 62.1 11.3 63.4 13.7 50.4 19.0
Note: The meaning of stages is shown in Table 1.
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T1 versus Tx stages differed significantly in cases of CgA ( p = 0.018) and NSE ( p = 0.021) levels, while the existing small difference in PSA mean values remained insignificant ( p = 0.084) (Table 4). In case of T2 stage, all three parameters displayed only insignificant differences to the means of Tx stage: for CgA, NSE, and PSA means we found p = 0.716; 0.163; and 0.061, respectively.
4. Discussion Prostate adenocarcinoma remains androgen dependent in its early stages, but after androgen withdrawal, relapses to an androgen insensitive disease (Gittes, 1991). The underlying mechanisms determining the progression to androgen insensitivity are poorly understood. Although both the management policy for early prostate cancer and the routine population screening for this disease remain matters of debate, increasing the rate of early diagnosis and treatment would seem to be a desiderable goal (Wilt, 2002). Prostate cancer has had a much lower profile and it seems unlikely that the population at risk is sufficiently aware of the disease and the possibilities for early treatment and cure. The results of the few published studies of public awareness of prostate cancer support this view (Price et al., 1993; Mainous and Hagen, 1994). However, these relatively small studies were conducted more than 10 years ago, and only one of the not too old studies assessed the knowledge of a variety of issues concerning prostate cancer (Egawa et al., 1998). After its discovery 20 years ago, the PSA has been considered to be the most valuable tool in the early detection, staging, and monitoring of prostate cancer (Polascik et al., 1999). However, PSA turned out to be not specific for prostate cancer, since elevated serum PSA levels were detected also in patients with BPH and prostatitis. One of the limitations of PSA as a tumor marker is that there is a substantial overlap in serum PSA values between men with BPH and prostate cancer. PSA testing resulted in findings of prostate cancer at earlier stages and in younger men, than before, however, the situation has been equilibrated during the recent years (Jemal et al., 2002). In addition, a recent analysis found that the pre-treatment PSA levels fell by a rate of 0.8% per year in average between 1988 and 1997 (Jani et al., 2001). The NE cells have been detected in benign prostate hypertrophy, prostate intraepithelial neoplasms (PIN), and in all the stages of prostate carcinoma. Recently, several serum and genetic markers have been associated with the pathogenesis of prostate cancer, suggesting their potential role as prognostic factors. The concept of neuroendocrine differentiation in prostate cancer has become more widely recognized and has attracted considerable attention as a potentially new finding with major diagnostic, prognostic, and therapeutic implications. The physiological role of prostate neuroendocrine cells is unknown, but they are supposed to be involved both in the regulation of prostate growth and differentiation (Abrahmoson, 1999). The neuroendocrine cells in both benign and malignant prostate tissues can be identified immunohistochemically with the use of antibodies against a number of products produced by these cells, including NSE, CgA, and members of the calcitonin gene family. Little is known about the specific role that the neuroendocrine cells
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have in the prostate or their relationship to the pathogenesis, growth, and prognosis of prostate diseases (Sciarra et al., 2003). Serum levels of the neuroendocrine marker CgA could reflect the NE activity of prostate carcinoma and could be used during the follow-up evaluation of advanced prostate carcinoma. In contrast to CgA, the utility of measuring NSE in patients with advanced prostate cancer is uncertain. The incidence of high NSE levels in patients with prostate cancer was lower than that of high chromogranin levels (Cussenot et al., 1998). NSE and CgA serum levels were very high in patients with hormone therapy-resistant prostate cancer than in patients with BPH and localized prostate cancer (Hvamstad et al., 2003). However, the determination of NE markers in serum may not be specific for prostate activity and only the analysis of a very large stratified population may determine whether there is a significant serum marker of NE activity in prostate cancer. It has been observed that serum CgA levels reflect the entity of NE differentiation of the prostate carcinoma in relationship to the stage of disease (Sciarra et al., 2004). Therefore, the measurement of CgA is useful, both from the diagnostic and prognostic points of view. Particularly elevated values of serum CgA levels are observed in a patient with hormonerefractory prostate carcinoma and they are associated to worse prognosis (Berruti et al., 2005). Low levels of CgA in the circulation are independent of age. The importance of increased CgA levels in serum was first shown in patients with pheochromocytoma and then demonstrated for other endocrine cancers (Ferrari et al., 1999; Cotesta et al., 2005). Elevated circulating levels have been seen in several neuroendocrine tumors, including pheochromocytomas (O’Connor and Frigon, 1984; O’Connor and Deftos, 1986), carcinoid tumors (Syversen et al., 1993), and neuroblastomas (Hsiao et al., 1990). The sensitivity and specificity of CgA in any NETs vary between 70% and 95% and between 70% and 80%, respectively. The highest accuracy has been observed in tumors characterized by an intense secretory activity, although the specificity and sensitivity remain very high also in non-functioning tumors. (Anderson and Lynch, 1993; Capella et al., 1995; Hauser et al., 1995; Neary et al., 1997). The clinical use of other neuro-hormonal markers has been limited by their in vitro instability, which requires special sampling, handling, and storage procedures. In contrast, CgA may be measured in serum or plasma collected with standard equipment, stored at room temperature for prolonged periods, and analyzed without extraction procedures. The measurement of neuroendocrine markers such as CgA in the blood of prostate cancer patients certainly constitutes a more objective assessment of the neuroendocrine differentiation of tumors, as it may correspond to the entire primary tumor cell population and its associated metastasis. The NE cells have been detected in benign prostate hypertrophy, PIN and in all the stages of prostate carcinoma. Patients with higher CgA level had poor survival and showed poor prognosis, compared to those with lower CgA level; these results support the notion that pretreatment serum CgA level determination is a potential prognostic factor for prostate cancer. The subjects with higher CgA levels had a higher grade of Gleason’s score. From these data we can realize that this method has not only a diagnostic use, since it allows us to evaluate the neuroendocrine differentiation of prostate cancer. The importance of CgA does not concern only the diagnostic and prognostic aspects, but also the therapeutic approach: in fact, CgA could permit a correct evaluation of therapy with somatostatin, which is the therapy of tumors with neuroendocrine differentiation (Berruti et al., 2001).
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