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Elevated chromogranin A (CgA) serum levels in the patients with advanced pancreatic cancer
Archives of Gerontology and Geriatrics 48 (2009) 213–217
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Elevated chromogranin A (CgA) serum levels in the patients with advanced pancreatic cancer Michele Malaguarnera a, Erika Cristaldi b, Lisa Cammalleri b, Valentina Colonna b, Helga Lipari b, Alessandra Capici b, Andrea Cavallaro c, Massimiliano Beretta b, Innocenza Alessandria b, Salvatore Luca b, Massimo Motta b,* a
Department of Aging Sciences, Research Center on the Extreme Senescence, University of Catania, Via Messina 829, I-95126 Catania, Italy Department of Senescence, Urological and Neurological Sciences, Cannizzaro Hospital, University of Catania, Via Messina 829, I-95126 Catania, Italy c Department of Surgery of ‘‘Policlinico Gaspare Rodolico’’, University of Catania, Via Santa Sofia 86, I-95123 Catania, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2007 Received in revised form 10 January 2008 Accepted 14 January 2008 Available online 7 March 2008
The neuroendocrine differentiation in PC could potentially represent a new finding with diagnostic, prognostic and therapeutic implications. This study aimed at evaluating the clinical usefulness of CgA as a neuroendocrine (NE) serum-marker. We investigated the role of the serum concentration of CgA in a study group of patients with PC. CgA was significantly higher in the patients affected by PC as compared with the group of healthy subjects (HS) and those with chronic pancreatitis (CHP) (p < 0.001). Also the HS group differed significantly from the CHP control group in the serum CgA levels (p < 0.001). The serum carbohydrate antigen (CA19-9) level displayed a significant difference (p < 0.001) between the PC and the HS group. The PC and CHP groups, as well as the HS and CHP groups showed also significant differences in the CA19-9 levels (p < 0.001). One can conclude that the patients with higher CgA levels had poorer prognosis and survival, as compared to those with lower CgA levels. These results support the notion that the determination of serum CgA level before treatment may be a potential prognostic factor for PC. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pancreatic cancer Neuroendocrine components Chromogranin A Carbohydrate antigen (CA19-9)
1. Introduction PC ranks fifth behind carcinomas of the lung, colorectum, breast and prostate as a cause of cancer deaths (Ahlgren, 1996). The risk of developing PC increases with age; it has been estimated that this risk increases two- to threefold for each decade of life after 40 years. Worldwide incidence rates are highest in industrialized countries and lowest in African and Asian countries, suggesting that environmental factors linked to a ‘‘Western lifestyle’’ substantially increase the risk of pancreatic cancer. Pancreatic adenocarcinomas tend to be aggressive tumors that disseminate early and tend to follow similar patterns of metastatic spread in most patients. Patients with PC are vaguely unwell for a number of months before the development of overt symptoms that lead to the diagnosis of their illness. The conventional treatment approaches such as surgery, radiation, chemotherapy, or combinations of these, have had little impact on the course of this
* Corresponding author. Tel.: +39 095 726 2008; fax: +39 095 726 2011. E-mail address: [email protected] (M. Motta). 0167-4943/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2008.01.014
aggressive neoplasm. Therefore, we will be in a position to effectively diagnose, prevent and treat this disease, only if developing a detailed understanding of the biology of PC. A number of tumor-associated antigens, detectable in the serum of patients with PC have been described, the most useful one being the CA19-9 (Safi et al., 1990). CA19-9 is a mucin-associated CA produced by a normal pancreas, as well as by PC cells. Serum CA19-9, the sialylated Lewis blood group antigen determined by the monoclonal antibody 1116-NS19-9 (Herlyn et al., 1979) has been proven as the most sensitive and specific serum-marker for PC (Pleskow et al., 1989; Rollhauser and Fleischer, 1998). Persistently increased CA19-9 concentrations after resection of PC have been reported to be associated with poor survival, as compared with patients whose CA19-9 had been normalized (Glenn et al., 1988; Tian et al., 1992; Safi et al., 1997). Both exocrine PC and several exocrine tumors have been considered. Various studies have reported the existence of numerous exocrine PCs, containing argyrophil and hormone-immunoreactive cells (Compagno and Oertel, 1978; Suda and Hashimoto, 1979; Chen et al., 1985, 1988). The presence of argyrophil and hormone-immunoreactive cells in pancreatic ducts and carcinomas may be indicative
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of the close developmental relationship between endocrine and exocrine parts of the pancreas (Chen et al., 1988). CgA is an acidic glycoprotein, present in NE tissues, and its cosecretion with peptide hormones and neuropeptides makes it a suitable tissue- and serum-marker of neoplasms of NE origin (Deftos, 1991). CgA is a member of the granin family and is costored with catecholamines in adrenal medullary and sympathetic neuronal vesicles, is co-released with catecholamines during sympatho-adrenal activations in humans (O’Connor and Frigon, 1984; Takiyyddin et al., 1990). CgA is co-stored with catecholamines in adrenal medullary and sympathetic neuronal vesicles. During sympatho-adrenal activation, they are co-released. Recently, it has been reported that CgA is a strong and independent prognostic indicator in the patients with complicated myocardial infarction (Estensen et al., 2006). Higher plasma CgA levels are associated with increased long-term mortality after myocardial infarction (Omland et al., 2003). Moreover, higher levels of CgA in the patients with chronic heart failure are associated with elevated severity of symptoms and prognosis (Ceconi et al., 2002). It seems that the blockade of sympathetic nervous system with somatostatin is more effective than use of ganglion-blockers (Takiyyuddin et al., 1991). CgA acts as a pre-hormone with multiple proteolytic sites allowing production of multiple peptides with various physiological functions (Metz-Boutigue et al., 1993). It is also present in the widely distributed NE system of the bronchial and gastrointestinal tracts and of the skin (Nobles et al., 1998). 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 pancreatic NE 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). We investigated the role of the serum concentrations of CgA and the CA19-9 in a group of patients with PC and with pancreatitis, as well as the relationship between the serum CgA and CA19-9 levels found before the treatment, and the clinical stages of the disease. 2. Patients and methods
The patients were divided into four groups according to the stage of their disease: stage I (11 patients); stage II (12 patients); stage III (26 patients), and stage IV (26 patients). Written informed consent was obtained from each patient. The study had been approved by the Local Ethics Committee. 2.2. Serum collection and storage Venous blood samples were collected before treatment from each patient before surgery, centrifuged to obtain serum samples and stored at 80 8C until assayed. Clinical chemistry tests were performed in the medical center laboratory using standard methods. Fasting blood samples were taken at enrolment from the participants. 2.3. Assaying methods A commercial solid-phase two-site immunoradiometric assay was used to measure 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 iodine 125 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. The normal range for serum levels of CgA in a control population is reported as 20–100 ng/ml. The coefficients of variation within and between assay were 4.1 and 6.4%, respectively. CA19-9 was measured by microparticle enzyme immunoassay (MEIA) kits (Abbot, Chicago, IL, USA). The normal range of CA19-9 was between 0 and 50. The coefficients of variation within and between assay were 4.4 and 6.6%, respectively. The pathological staging of PC has recently been revised and it is based on the extent of tumor involvement of local and distant structures. Stage I tumors are limited to the pancreas. Stage II tumors are regionally invasive without involvement of the celiac or superior mesenteric arteries, but may involve regional lymph nodes. Stage III lesions are defined by direct involvement of the celiac or superior mesenteric arteries, and stage IV lesions are defined by the presence of the distant metastases.
2.1. Patients 2.4. Statistical analyses The presence of CgA in serum was evaluated in 75 consecutive patients (44 males and 31 females) with PC, and in two control groups. One of the control groups consisted of 30 patients (14 males and 16 females) affected by CHP, and the second one constituted by 20 HS (10 males and 10 females). Patients of the three groups were aged 65–80 years. All patients and control subjects were screened for interfering factors on CgA assay, such as proton pump inhibitors, steroids and renal failure. Patients with these conditions were excluded because could generate false positive results. None of the patients with PC had received chemoor radio-therapy before the blood sample collection. The diagnosis of PC was based on the clinical symptoms, on markedly elevated serum CA19-9 and typical findings on dynamic computed tomography. The diagnosis of PC was confirmed on pathohistological findings. Angiography and magnetic resonance imaging (MRI) were performed when considered necessary, to plan the surgical strategy. The new tumor, node, metastasis system (TNM) classification in gastroenterology was used to classify the tumors (Rosch, 1998).
All data are presented as mean SD. Discrete and continuous variables were compared using either Student’s ‘‘t’’ test or the Wilcoxon Mann–Whitney non-parametric test for unpaired data. Categorical variables were compared with either the x2-test or the Fisher exact test when requested. The Spearman’s rank correlation coefficient test was used to test for univariate relationships between variables. The following tests at p < 0.05 level significance were used to evaluate the results and was considered statistically significant. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc. Chicago, IL, USA). 3. Results 3.1. Comparison of the groups The baseline characteristics of HS, PC and CHP groups did not show any statistically meaningful differences in the general parameters, like age, systolic and diastolic blood pressure (SBP
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Table 1 The main parameters and results of the groups of our study population (mean SD, unless otherwise indicated) Parameters
HS group
PC group
CHP group
Male/female Age (years) SBP (kPa) DBP (kPa) Heart rate (b/m) Smokers (%) CgA (ng/ml) CA19-9 (U/l)
10/10 65.4 4.2 17.29 4.25 10.24 1.59 80 14 53% 36.0 22.0b,c 3.7 0.6b,c
47/28 65.8 5.7 18.08 3.19 10.37 1.33 84 12 50% 192.9 66.5a,b 171.69 91.69a,b
14/16 65.7 3.8 17.95 3.72 10.64 1.19 87 10 56% 96.0 31.0a,c 28.0 12.0a,c
Statistical comparisons: aPC vs. CHP: p < 0.001; bHS vs. PC: p < 0.001; cHS vs. CHP: p < 0.001.
and DBP, respectively), heart rate and smoking habits (Table 1). However, the circulating levels of CgA were elevated (>100 ng/ml) in 71% of patients with PC, and CgA values were not correlated with the calendar age in any of the groups. No significant differences in CgA values were found between males and females in any of the groups. The patients with CHP and PC had higher serum CgA levels than the HS group (Table 1). The mean difference in CgA between PC and CHP groups was 96.91 ng/ml (p < 0.001, 95% CI = 71.75–122.07), and in CA19-9 was 143.69 U/l (p < 0.001, 95% CI = 110.28–177.10). Comparing the HS and PC group, the mean difference in CgA was 156.91 ng/ml (p < 0.001, 95% CI = 126.85–186.97), and in CA19-9 it amounted to 167.99 U/l (p < 0.001, 95% CI = 127.12–208.86). The mean differences were between the HS and the CHP groups in CgA were 60 ng/ ml (p < 0.001, 95% CI = 43.87–76.73) and CA19-9 was 24.30 U/l (p < 0.001, 95% CI = 18.88–29.72). All these differences proved to be highly significant (Table 1). 3.2. Comparison of the four stages in the PC group The results distributed in the various stages of the PC group are shown in Table 2. Stage I compared to stage III was lower in CgA, the mean difference being 34.69 ng/ml (p < 0.05, 95% CI = 67.09 to 2.29); but this difference was 128.85 ng/ml (p < 0.001, 95% CI = 154.22 to 103.48) against the stage IV (Table 2). The mean difference in CA19-9 was low, but significant between the stage I and stage II: 16.38 U/l (p < 0.01, 95% CI = 28.60 to 4.16); the same difference increased against the stage III to 92.63 U/l (p < 0.001, 95% CI = 114.52 to 70.74); and grew even further against the stage IV: 212.05 U/l (p < 0.001, 95% CI = 246.02 to 178.08). If comparing the stage II versus stage IV, the mean difference in CgA was 121.27 ng/ml (p < 0.001, 95% CI = 146.65 to 95.89) (Table 2). The mean difference in CA19-9 between stage II and stage III was 76.25 U/l (p < 0.001, 95% CI = 96.60 to 5.90), but
Fig. 1. The mean concentrations of CgA (ng/ml) and CA19-9 (U/l) in the four stages of PC.
against stage IV the difference increased to 195.67 U/l (p < 0.001, 95% CI = 227.28 to 163.56) (Table 2, Fig. 1). If we compared the stage III and stage IV results, the mean difference in CgA was 94.16 ng/ml (p < 0.001, 95% CI = 118.5 to 69.82) and in CA19-9 was 119.42 U/l (p < 0.001, 95% CI = 144.5 to 94.34), respectively (Fig. 1). 3.3. Correlation between CA19-9 and CgA In the PC group, CgA and CA19-9 were strongly and significantly correlated: r = 0.85; p < 0.001 (Fig. 2). No correlation was observed in the HS and CHP groups. 4. Discussion This study demonstrated that CgA is altered in PC. Although the biological functions of CgA are not established with certainty, several clinical applications as marker for NE tumors are already in use or are being developed. Serum CgA has been recognized as a valuable marker for the detection of neuroblastoma, pheochromocytoma, small cell lung carcinoma and carcinoid tumors (Kimura et al., 1997; Stridsberg and Husebye, 1997). Recently, cellular clones with morphological and functional NE features have been demonstrated in non-endocrine tumors and elevated levels of serum CgA have been described in patients with carcinoma of the prostate, breast, liver and colon (Wu et al., 2000; Spadaro et al., 2005; Ranno et al., 2006). Measurement of NE markers such as CgA in the blood of PC patients certainly constitutes a more objective assessment of the NE differentiation of tumors, as it may correspond to the entire primary tumor cell population and its associated metastasis. Malignant pancreatic tumors are usually classified into those of the exocrine pancreas or those of endocrine pancreas and are generally categorized into three types (ductal, acinar or endocrine) (Kloppel et al., 1996). The exocrine pancreas includes two types of ductal cells and acinar cells, while the endocrine pancreas consists
Table 2 The distribution of the results according to the clinical stages of the tumors (mean SD, unless otherwise indicated) Stages
CgA (ng/ml)
Ca 19-9 (U/l)
Stage I (11) Median (range) Stage II (12) Median (range) Stage III (26) Median (range) Stage IV (26) Median (range)
135.00 28.03a,b 132 (82–181) 142.58 32.85 e 147.5 (97–192) 169.0 49.41a,g 149 (99–285) 263.85 37.10b,e,g 271 (176–322)
63.45 17.09b,c,d 62 (40–94) 79.83 10.61c,e,f 80.5 (67–96) 156.08 33.78d,f,g 167 (96–212) 275.50 53.98e,f,g 278 (187–361)
Statistical comparisons: astage I vs. III: p < 0.05; bstage I vs. IV: p < 0.001; cstage I vs. stage II: p < 0.01; dstage I vs. stage III: p < 0.001; estage II vs. stage IV: p < 0.001; f stage II vs. stage III: p < 0.001; gstage III vs. stage IV: p < 0.001.
Fig. 2. The correlation of CA19-9 (U/l) and CgA (ng/ml) concentrations in all patients with PC.
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of islet cells. Pancreatic tumors originate from one of these cell types, most often from ductal cells. In recent years, the coexistence of exocrine and endocrine elements in PC can be attributed to their common embryologic origin. In fact the theory that all endocrine cells derived from the neural crest has been abandoned in favor of a common precursor stem cell of endodermic origin from which both endocrine and exocrine cells (Pour and Schmied, 1999). Peters et al. (2000) in a review article on the embryologic development and growth of the pancreas noted that ductal cells could be stimulated to differentiate into islet cells. Depending on the point at which neoplastic transformation occurs along the pathway of proliferation and differentiation of these cells, a pancreatic neoplasm might exhibit ductal, acinar, or endocrine features. CgA has been used to indicate NE differentiation in tumors of epithelial origin. NE differentiation has several potential implications in the natural history and prognosis of PC (MetzBoutigue et al., 1998). Differentiation may occur in several epithelial tumors (gut, colon, pancreas). The de-differentiation of the epithelial cells, in fact, leads to a NE differentiation. NE tumors, on the other hand, are generally very well differentiated tumors and completely different from epithelial tumors. Between NE tumors and NE-differentiated epithelial tumors there are important differences. NE-differentiated epithelial tumors are not biologically active and do not respond better or different to any kind of treatment. So an early recognition of NE differentiation is very important, since NE tumors sometimes have a favorable prognosis. Furthermore, early identification of a NE neoplasm could allow better control of the syndrome that may be associated with a biologically active tumor. Finally, the use of a suitable prognostic indicator such as CgA represents a key step in management of pancreatic cancer. Other regulatory mechanisms are likely to be involved in this process. In adenocarcinoma the proliferation compartment is composed by exocrine cell types, while full differentiated NE tumor cells remain in a quiescent state (G0 phase of the cell cycle) (Anderson and Lynch, 1993; Capella et al., 1995; Hauser et al., 1995; Neary et al., 1997). Somatostatin-producing cells find in central and peripheral nervous system, in endocrine pancreas and in gut. Gastrointestinal tract and pancreas contain the largest amounts of somatostatin. Moreover, various studies have shoved the presence of somatostatin receptors in many tissues, including gut, brain, endocrine and exocrine pancreas, pituitary adrenals, thyroid, kidney and immune cells (Reichlin, 1983; Reisine, 1995; Reubi, 2004). The presence of somatostatin receptors (SST2) has been reported in a large number of human primary non-NE tumors, such as breast, colon and prostate cancer (Reubi et al., 1992, 1996; Schaer et al., 1997). The density of SST2 in several tumors is widely heterogeneous. The receptors are preferentially expressed in differentiated, rather than the anaplastic tumors. SST2 is selectively lost in 90% of the human pancreatic adenocarcinomas and derived pancreatic cell lines. The expression of SST2 suggests the possibility that its presence may represent a marker of cell differentiation in some tumors. Furthermore, the loss of expression of functional SST2 may be functionally involved in neoplastic progression (Pollak and Schally, 1998). It is possible that mutations in SST2 could cause a loss of check on proliferation in cancer cells (Zhang et al., 1995). Expression of SST2 in human PC cells also inhibits both in vitro and in vivo tumorigenic cells, and promotes a local and distant antitumor bystander effect with an increase in apoptosis and a decrease in Ki-67 proliferative index (Delesque et al., 1997; Rochaix et al., 1999; Reubi et al., 2001; Guillermet et al., 2003). The bystander effect results at least in part from increased serum somatostatin-like immunoreactivity levels and increased expres-
sion of functional SST2 receptors. The SST2 acts as a tumor suppressor gene for human pancreatic cancer. Somatostatin analogs can control hypersecretion in the patients with NE tumors that express somatostatin receptors. In SST2 tumors, clinical symptoms related to hypersecretion can be controlled by longterm administration of one of the somatostatin analogs (Lamberts et al., 1996; Patel, 1999; Bruns et al., 2002). In addition, these agents may also exert some anti-proliferative actions (Shojamanesh et al., 2002). Native somatostatin is an effective inhibitor of pancreatic exocrine secretion. Data suggest that its possible activity in preventing complications of pancreatic surgery. To inhibiting pancreatic exocrine secretion during surgery and in early postoperative period might empty pancreas from enzymes, thus preventing damages to pancreatic and peripancreatic tissues (Li-Ling and Irving, 2001; Poon et al., 2002). In our patients we observed an increase in serum CgA levels in relationship to stage of disease. The circulating levels of CgA were elevated (>100 ng/ml) in 71% patients with PC and in 23% patients with CHP. In none of the HSs were values overcoming the 100 ng/ ml. These data seem to confirm the clinical usefulness of CgA as serum-marker in the patients with pancreatic adenocarcinoma. Patients with higher CgA level present an advanced stage of disease if compared to those with lower CgA level (Fig. 1); these results support the notion that pretreatment serum CgA level determination is a potential prognostic factor for pancreatic cancer. From our data we can realize that the serum levels of CgA have not only a diagnostic value, since it allows evaluating the NE differentiation of PC. Besides the importance of CgA does not concern only the diagnostic and prognostic aspects, but also the therapeutic approach. In the presence of PC, somatostatin may affect cell growth both by indirect and direct effects, including inhibiting secretion of growth-promoting factors such as epidermal growth factor, insulin-like growth factor-1, and gastrointestinal hormones and direct binding to somatostatin receptors to trigger growth inhibition. These negative regulations are thought to be mediated via two separate pathways: one is by inhibiting mitogenic signaling of growth factor receptors resulting in the arrest of the cell cycle, and the other is by promoting apoptosis leading to cell death (Li et al., 2005). 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. Knowledge of this factor will likely contribute to determining the development of new diagnostic and therapeutic agents. At present, all cancer cells within tumor are treated like if they have the ability to drive tumor growth, invasion and metastasis. The ability to identify these crucial cells will allow efforts to develop new diagnostic marker and therapies to be focused on the cells that are responsible for the maintenance of the malignancy. References Ahlgren, J.D., 1996. Epidemiology and risk factors in pancreatic cancer. Semin. Oncol. 23, 241–250. Anderson, R.J., Lynch, H.T., 1993. Familial risk for neuroendocrine tumours. Curr. Opin. Oncol. 5, 75–84. Bruns, C., Lewis, I., Briner, U., Meno-tetang, G., Weckbecker, G., 2002. SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile. Eur. J. Endocrinol. 146, 707–716. Capella, C., Heitz, P.U., Ho¨fler, H., Solcia, E., Klo¨ppel, G., 1995. Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Arch. 425, 547–560. Ceconi, C., Ferrari, R., Bachetti, T., Opasich, C., Volterrani, M., Colombo, B., Parrinello, G., Corti, A., 2002. Chromogranin A in heart failure. A novel neurohumoral factor and a predictor for mortality. Eur. Heart J. 23, 967–974. Chen, J., Baithun, S.I., Ramsay, M.A., 1985. Histogenesis of pancreatic carcinomas: a study based on 248 cases. J. Pathol. 146, 65–76.
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Elevated chromogranin A (CgA) serum levels in the patients with advanced pancreatic cancer Michele Malaguarnera a, Erika Cristaldi b, Lisa Cammalleri b, Valentina Colonna b, Helga Lipari b, Alessandra Capici b, Andrea Cavallaro c, Massimiliano Beretta b, Innocenza Alessandria b, Salvatore Luca b, Massimo Motta b,* a
Department of Aging Sciences, Research Center on the Extreme Senescence, University of Catania, Via Messina 829, I-95126 Catania, Italy Department of Senescence, Urological and Neurological Sciences, Cannizzaro Hospital, University of Catania, Via Messina 829, I-95126 Catania, Italy c Department of Surgery of ‘‘Policlinico Gaspare Rodolico’’, University of Catania, Via Santa Sofia 86, I-95123 Catania, Italy b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 15 October 2007 Received in revised form 10 January 2008 Accepted 14 January 2008 Available online 7 March 2008
The neuroendocrine differentiation in PC could potentially represent a new finding with diagnostic, prognostic and therapeutic implications. This study aimed at evaluating the clinical usefulness of CgA as a neuroendocrine (NE) serum-marker. We investigated the role of the serum concentration of CgA in a study group of patients with PC. CgA was significantly higher in the patients affected by PC as compared with the group of healthy subjects (HS) and those with chronic pancreatitis (CHP) (p < 0.001). Also the HS group differed significantly from the CHP control group in the serum CgA levels (p < 0.001). The serum carbohydrate antigen (CA19-9) level displayed a significant difference (p < 0.001) between the PC and the HS group. The PC and CHP groups, as well as the HS and CHP groups showed also significant differences in the CA19-9 levels (p < 0.001). One can conclude that the patients with higher CgA levels had poorer prognosis and survival, as compared to those with lower CgA levels. These results support the notion that the determination of serum CgA level before treatment may be a potential prognostic factor for PC. ß 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Pancreatic cancer Neuroendocrine components Chromogranin A Carbohydrate antigen (CA19-9)
1. Introduction PC ranks fifth behind carcinomas of the lung, colorectum, breast and prostate as a cause of cancer deaths (Ahlgren, 1996). The risk of developing PC increases with age; it has been estimated that this risk increases two- to threefold for each decade of life after 40 years. Worldwide incidence rates are highest in industrialized countries and lowest in African and Asian countries, suggesting that environmental factors linked to a ‘‘Western lifestyle’’ substantially increase the risk of pancreatic cancer. Pancreatic adenocarcinomas tend to be aggressive tumors that disseminate early and tend to follow similar patterns of metastatic spread in most patients. Patients with PC are vaguely unwell for a number of months before the development of overt symptoms that lead to the diagnosis of their illness. The conventional treatment approaches such as surgery, radiation, chemotherapy, or combinations of these, have had little impact on the course of this
* Corresponding author. Tel.: +39 095 726 2008; fax: +39 095 726 2011. E-mail address: [email protected] (M. Motta). 0167-4943/$ – see front matter ß 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.archger.2008.01.014
aggressive neoplasm. Therefore, we will be in a position to effectively diagnose, prevent and treat this disease, only if developing a detailed understanding of the biology of PC. A number of tumor-associated antigens, detectable in the serum of patients with PC have been described, the most useful one being the CA19-9 (Safi et al., 1990). CA19-9 is a mucin-associated CA produced by a normal pancreas, as well as by PC cells. Serum CA19-9, the sialylated Lewis blood group antigen determined by the monoclonal antibody 1116-NS19-9 (Herlyn et al., 1979) has been proven as the most sensitive and specific serum-marker for PC (Pleskow et al., 1989; Rollhauser and Fleischer, 1998). Persistently increased CA19-9 concentrations after resection of PC have been reported to be associated with poor survival, as compared with patients whose CA19-9 had been normalized (Glenn et al., 1988; Tian et al., 1992; Safi et al., 1997). Both exocrine PC and several exocrine tumors have been considered. Various studies have reported the existence of numerous exocrine PCs, containing argyrophil and hormone-immunoreactive cells (Compagno and Oertel, 1978; Suda and Hashimoto, 1979; Chen et al., 1985, 1988). The presence of argyrophil and hormone-immunoreactive cells in pancreatic ducts and carcinomas may be indicative
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of the close developmental relationship between endocrine and exocrine parts of the pancreas (Chen et al., 1988). CgA is an acidic glycoprotein, present in NE tissues, and its cosecretion with peptide hormones and neuropeptides makes it a suitable tissue- and serum-marker of neoplasms of NE origin (Deftos, 1991). CgA is a member of the granin family and is costored with catecholamines in adrenal medullary and sympathetic neuronal vesicles, is co-released with catecholamines during sympatho-adrenal activations in humans (O’Connor and Frigon, 1984; Takiyyddin et al., 1990). CgA is co-stored with catecholamines in adrenal medullary and sympathetic neuronal vesicles. During sympatho-adrenal activation, they are co-released. Recently, it has been reported that CgA is a strong and independent prognostic indicator in the patients with complicated myocardial infarction (Estensen et al., 2006). Higher plasma CgA levels are associated with increased long-term mortality after myocardial infarction (Omland et al., 2003). Moreover, higher levels of CgA in the patients with chronic heart failure are associated with elevated severity of symptoms and prognosis (Ceconi et al., 2002). It seems that the blockade of sympathetic nervous system with somatostatin is more effective than use of ganglion-blockers (Takiyyuddin et al., 1991). CgA acts as a pre-hormone with multiple proteolytic sites allowing production of multiple peptides with various physiological functions (Metz-Boutigue et al., 1993). It is also present in the widely distributed NE system of the bronchial and gastrointestinal tracts and of the skin (Nobles et al., 1998). 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 pancreatic NE 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). We investigated the role of the serum concentrations of CgA and the CA19-9 in a group of patients with PC and with pancreatitis, as well as the relationship between the serum CgA and CA19-9 levels found before the treatment, and the clinical stages of the disease. 2. Patients and methods
The patients were divided into four groups according to the stage of their disease: stage I (11 patients); stage II (12 patients); stage III (26 patients), and stage IV (26 patients). Written informed consent was obtained from each patient. The study had been approved by the Local Ethics Committee. 2.2. Serum collection and storage Venous blood samples were collected before treatment from each patient before surgery, centrifuged to obtain serum samples and stored at 80 8C until assayed. Clinical chemistry tests were performed in the medical center laboratory using standard methods. Fasting blood samples were taken at enrolment from the participants. 2.3. Assaying methods A commercial solid-phase two-site immunoradiometric assay was used to measure 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 iodine 125 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. The normal range for serum levels of CgA in a control population is reported as 20–100 ng/ml. The coefficients of variation within and between assay were 4.1 and 6.4%, respectively. CA19-9 was measured by microparticle enzyme immunoassay (MEIA) kits (Abbot, Chicago, IL, USA). The normal range of CA19-9 was between 0 and 50. The coefficients of variation within and between assay were 4.4 and 6.6%, respectively. The pathological staging of PC has recently been revised and it is based on the extent of tumor involvement of local and distant structures. Stage I tumors are limited to the pancreas. Stage II tumors are regionally invasive without involvement of the celiac or superior mesenteric arteries, but may involve regional lymph nodes. Stage III lesions are defined by direct involvement of the celiac or superior mesenteric arteries, and stage IV lesions are defined by the presence of the distant metastases.
2.1. Patients 2.4. Statistical analyses The presence of CgA in serum was evaluated in 75 consecutive patients (44 males and 31 females) with PC, and in two control groups. One of the control groups consisted of 30 patients (14 males and 16 females) affected by CHP, and the second one constituted by 20 HS (10 males and 10 females). Patients of the three groups were aged 65–80 years. All patients and control subjects were screened for interfering factors on CgA assay, such as proton pump inhibitors, steroids and renal failure. Patients with these conditions were excluded because could generate false positive results. None of the patients with PC had received chemoor radio-therapy before the blood sample collection. The diagnosis of PC was based on the clinical symptoms, on markedly elevated serum CA19-9 and typical findings on dynamic computed tomography. The diagnosis of PC was confirmed on pathohistological findings. Angiography and magnetic resonance imaging (MRI) were performed when considered necessary, to plan the surgical strategy. The new tumor, node, metastasis system (TNM) classification in gastroenterology was used to classify the tumors (Rosch, 1998).
All data are presented as mean SD. Discrete and continuous variables were compared using either Student’s ‘‘t’’ test or the Wilcoxon Mann–Whitney non-parametric test for unpaired data. Categorical variables were compared with either the x2-test or the Fisher exact test when requested. The Spearman’s rank correlation coefficient test was used to test for univariate relationships between variables. The following tests at p < 0.05 level significance were used to evaluate the results and was considered statistically significant. Data were analyzed using the statistical package SPSS for Windows 7.5 (SPSS Inc. Chicago, IL, USA). 3. Results 3.1. Comparison of the groups The baseline characteristics of HS, PC and CHP groups did not show any statistically meaningful differences in the general parameters, like age, systolic and diastolic blood pressure (SBP
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Table 1 The main parameters and results of the groups of our study population (mean SD, unless otherwise indicated) Parameters
HS group
PC group
CHP group
Male/female Age (years) SBP (kPa) DBP (kPa) Heart rate (b/m) Smokers (%) CgA (ng/ml) CA19-9 (U/l)
10/10 65.4 4.2 17.29 4.25 10.24 1.59 80 14 53% 36.0 22.0b,c 3.7 0.6b,c
47/28 65.8 5.7 18.08 3.19 10.37 1.33 84 12 50% 192.9 66.5a,b 171.69 91.69a,b
14/16 65.7 3.8 17.95 3.72 10.64 1.19 87 10 56% 96.0 31.0a,c 28.0 12.0a,c
Statistical comparisons: aPC vs. CHP: p < 0.001; bHS vs. PC: p < 0.001; cHS vs. CHP: p < 0.001.
and DBP, respectively), heart rate and smoking habits (Table 1). However, the circulating levels of CgA were elevated (>100 ng/ml) in 71% of patients with PC, and CgA values were not correlated with the calendar age in any of the groups. No significant differences in CgA values were found between males and females in any of the groups. The patients with CHP and PC had higher serum CgA levels than the HS group (Table 1). The mean difference in CgA between PC and CHP groups was 96.91 ng/ml (p < 0.001, 95% CI = 71.75–122.07), and in CA19-9 was 143.69 U/l (p < 0.001, 95% CI = 110.28–177.10). Comparing the HS and PC group, the mean difference in CgA was 156.91 ng/ml (p < 0.001, 95% CI = 126.85–186.97), and in CA19-9 it amounted to 167.99 U/l (p < 0.001, 95% CI = 127.12–208.86). The mean differences were between the HS and the CHP groups in CgA were 60 ng/ ml (p < 0.001, 95% CI = 43.87–76.73) and CA19-9 was 24.30 U/l (p < 0.001, 95% CI = 18.88–29.72). All these differences proved to be highly significant (Table 1). 3.2. Comparison of the four stages in the PC group The results distributed in the various stages of the PC group are shown in Table 2. Stage I compared to stage III was lower in CgA, the mean difference being 34.69 ng/ml (p < 0.05, 95% CI = 67.09 to 2.29); but this difference was 128.85 ng/ml (p < 0.001, 95% CI = 154.22 to 103.48) against the stage IV (Table 2). The mean difference in CA19-9 was low, but significant between the stage I and stage II: 16.38 U/l (p < 0.01, 95% CI = 28.60 to 4.16); the same difference increased against the stage III to 92.63 U/l (p < 0.001, 95% CI = 114.52 to 70.74); and grew even further against the stage IV: 212.05 U/l (p < 0.001, 95% CI = 246.02 to 178.08). If comparing the stage II versus stage IV, the mean difference in CgA was 121.27 ng/ml (p < 0.001, 95% CI = 146.65 to 95.89) (Table 2). The mean difference in CA19-9 between stage II and stage III was 76.25 U/l (p < 0.001, 95% CI = 96.60 to 5.90), but
Fig. 1. The mean concentrations of CgA (ng/ml) and CA19-9 (U/l) in the four stages of PC.
against stage IV the difference increased to 195.67 U/l (p < 0.001, 95% CI = 227.28 to 163.56) (Table 2, Fig. 1). If we compared the stage III and stage IV results, the mean difference in CgA was 94.16 ng/ml (p < 0.001, 95% CI = 118.5 to 69.82) and in CA19-9 was 119.42 U/l (p < 0.001, 95% CI = 144.5 to 94.34), respectively (Fig. 1). 3.3. Correlation between CA19-9 and CgA In the PC group, CgA and CA19-9 were strongly and significantly correlated: r = 0.85; p < 0.001 (Fig. 2). No correlation was observed in the HS and CHP groups. 4. Discussion This study demonstrated that CgA is altered in PC. Although the biological functions of CgA are not established with certainty, several clinical applications as marker for NE tumors are already in use or are being developed. Serum CgA has been recognized as a valuable marker for the detection of neuroblastoma, pheochromocytoma, small cell lung carcinoma and carcinoid tumors (Kimura et al., 1997; Stridsberg and Husebye, 1997). Recently, cellular clones with morphological and functional NE features have been demonstrated in non-endocrine tumors and elevated levels of serum CgA have been described in patients with carcinoma of the prostate, breast, liver and colon (Wu et al., 2000; Spadaro et al., 2005; Ranno et al., 2006). Measurement of NE markers such as CgA in the blood of PC patients certainly constitutes a more objective assessment of the NE differentiation of tumors, as it may correspond to the entire primary tumor cell population and its associated metastasis. Malignant pancreatic tumors are usually classified into those of the exocrine pancreas or those of endocrine pancreas and are generally categorized into three types (ductal, acinar or endocrine) (Kloppel et al., 1996). The exocrine pancreas includes two types of ductal cells and acinar cells, while the endocrine pancreas consists
Table 2 The distribution of the results according to the clinical stages of the tumors (mean SD, unless otherwise indicated) Stages
CgA (ng/ml)
Ca 19-9 (U/l)
Stage I (11) Median (range) Stage II (12) Median (range) Stage III (26) Median (range) Stage IV (26) Median (range)
135.00 28.03a,b 132 (82–181) 142.58 32.85 e 147.5 (97–192) 169.0 49.41a,g 149 (99–285) 263.85 37.10b,e,g 271 (176–322)
63.45 17.09b,c,d 62 (40–94) 79.83 10.61c,e,f 80.5 (67–96) 156.08 33.78d,f,g 167 (96–212) 275.50 53.98e,f,g 278 (187–361)
Statistical comparisons: astage I vs. III: p < 0.05; bstage I vs. IV: p < 0.001; cstage I vs. stage II: p < 0.01; dstage I vs. stage III: p < 0.001; estage II vs. stage IV: p < 0.001; f stage II vs. stage III: p < 0.001; gstage III vs. stage IV: p < 0.001.
Fig. 2. The correlation of CA19-9 (U/l) and CgA (ng/ml) concentrations in all patients with PC.
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of islet cells. Pancreatic tumors originate from one of these cell types, most often from ductal cells. In recent years, the coexistence of exocrine and endocrine elements in PC can be attributed to their common embryologic origin. In fact the theory that all endocrine cells derived from the neural crest has been abandoned in favor of a common precursor stem cell of endodermic origin from which both endocrine and exocrine cells (Pour and Schmied, 1999). Peters et al. (2000) in a review article on the embryologic development and growth of the pancreas noted that ductal cells could be stimulated to differentiate into islet cells. Depending on the point at which neoplastic transformation occurs along the pathway of proliferation and differentiation of these cells, a pancreatic neoplasm might exhibit ductal, acinar, or endocrine features. CgA has been used to indicate NE differentiation in tumors of epithelial origin. NE differentiation has several potential implications in the natural history and prognosis of PC (MetzBoutigue et al., 1998). Differentiation may occur in several epithelial tumors (gut, colon, pancreas). The de-differentiation of the epithelial cells, in fact, leads to a NE differentiation. NE tumors, on the other hand, are generally very well differentiated tumors and completely different from epithelial tumors. Between NE tumors and NE-differentiated epithelial tumors there are important differences. NE-differentiated epithelial tumors are not biologically active and do not respond better or different to any kind of treatment. So an early recognition of NE differentiation is very important, since NE tumors sometimes have a favorable prognosis. Furthermore, early identification of a NE neoplasm could allow better control of the syndrome that may be associated with a biologically active tumor. Finally, the use of a suitable prognostic indicator such as CgA represents a key step in management of pancreatic cancer. Other regulatory mechanisms are likely to be involved in this process. In adenocarcinoma the proliferation compartment is composed by exocrine cell types, while full differentiated NE tumor cells remain in a quiescent state (G0 phase of the cell cycle) (Anderson and Lynch, 1993; Capella et al., 1995; Hauser et al., 1995; Neary et al., 1997). Somatostatin-producing cells find in central and peripheral nervous system, in endocrine pancreas and in gut. Gastrointestinal tract and pancreas contain the largest amounts of somatostatin. Moreover, various studies have shoved the presence of somatostatin receptors in many tissues, including gut, brain, endocrine and exocrine pancreas, pituitary adrenals, thyroid, kidney and immune cells (Reichlin, 1983; Reisine, 1995; Reubi, 2004). The presence of somatostatin receptors (SST2) has been reported in a large number of human primary non-NE tumors, such as breast, colon and prostate cancer (Reubi et al., 1992, 1996; Schaer et al., 1997). The density of SST2 in several tumors is widely heterogeneous. The receptors are preferentially expressed in differentiated, rather than the anaplastic tumors. SST2 is selectively lost in 90% of the human pancreatic adenocarcinomas and derived pancreatic cell lines. The expression of SST2 suggests the possibility that its presence may represent a marker of cell differentiation in some tumors. Furthermore, the loss of expression of functional SST2 may be functionally involved in neoplastic progression (Pollak and Schally, 1998). It is possible that mutations in SST2 could cause a loss of check on proliferation in cancer cells (Zhang et al., 1995). Expression of SST2 in human PC cells also inhibits both in vitro and in vivo tumorigenic cells, and promotes a local and distant antitumor bystander effect with an increase in apoptosis and a decrease in Ki-67 proliferative index (Delesque et al., 1997; Rochaix et al., 1999; Reubi et al., 2001; Guillermet et al., 2003). The bystander effect results at least in part from increased serum somatostatin-like immunoreactivity levels and increased expres-
sion of functional SST2 receptors. The SST2 acts as a tumor suppressor gene for human pancreatic cancer. Somatostatin analogs can control hypersecretion in the patients with NE tumors that express somatostatin receptors. In SST2 tumors, clinical symptoms related to hypersecretion can be controlled by longterm administration of one of the somatostatin analogs (Lamberts et al., 1996; Patel, 1999; Bruns et al., 2002). In addition, these agents may also exert some anti-proliferative actions (Shojamanesh et al., 2002). Native somatostatin is an effective inhibitor of pancreatic exocrine secretion. Data suggest that its possible activity in preventing complications of pancreatic surgery. To inhibiting pancreatic exocrine secretion during surgery and in early postoperative period might empty pancreas from enzymes, thus preventing damages to pancreatic and peripancreatic tissues (Li-Ling and Irving, 2001; Poon et al., 2002). In our patients we observed an increase in serum CgA levels in relationship to stage of disease. The circulating levels of CgA were elevated (>100 ng/ml) in 71% patients with PC and in 23% patients with CHP. In none of the HSs were values overcoming the 100 ng/ ml. These data seem to confirm the clinical usefulness of CgA as serum-marker in the patients with pancreatic adenocarcinoma. Patients with higher CgA level present an advanced stage of disease if compared to those with lower CgA level (Fig. 1); these results support the notion that pretreatment serum CgA level determination is a potential prognostic factor for pancreatic cancer. From our data we can realize that the serum levels of CgA have not only a diagnostic value, since it allows evaluating the NE differentiation of PC. Besides the importance of CgA does not concern only the diagnostic and prognostic aspects, but also the therapeutic approach. In the presence of PC, somatostatin may affect cell growth both by indirect and direct effects, including inhibiting secretion of growth-promoting factors such as epidermal growth factor, insulin-like growth factor-1, and gastrointestinal hormones and direct binding to somatostatin receptors to trigger growth inhibition. These negative regulations are thought to be mediated via two separate pathways: one is by inhibiting mitogenic signaling of growth factor receptors resulting in the arrest of the cell cycle, and the other is by promoting apoptosis leading to cell death (Li et al., 2005). 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. Knowledge of this factor will likely contribute to determining the development of new diagnostic and therapeutic agents. At present, all cancer cells within tumor are treated like if they have the ability to drive tumor growth, invasion and metastasis. The ability to identify these crucial cells will allow efforts to develop new diagnostic marker and therapies to be focused on the cells that are responsible for the maintenance of the malignancy. References Ahlgren, J.D., 1996. Epidemiology and risk factors in pancreatic cancer. Semin. Oncol. 23, 241–250. Anderson, R.J., Lynch, H.T., 1993. Familial risk for neuroendocrine tumours. Curr. Opin. Oncol. 5, 75–84. Bruns, C., Lewis, I., Briner, U., Meno-tetang, G., Weckbecker, G., 2002. SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile. Eur. J. Endocrinol. 146, 707–716. Capella, C., Heitz, P.U., Ho¨fler, H., Solcia, E., Klo¨ppel, G., 1995. Revised classification of neuroendocrine tumours of the lung, pancreas and gut. Virchows Arch. 425, 547–560. Ceconi, C., Ferrari, R., Bachetti, T., Opasich, C., Volterrani, M., Colombo, B., Parrinello, G., Corti, A., 2002. Chromogranin A in heart failure. A novel neurohumoral factor and a predictor for mortality. Eur. Heart J. 23, 967–974. Chen, J., Baithun, S.I., Ramsay, M.A., 1985. Histogenesis of pancreatic carcinomas: a study based on 248 cases. J. Pathol. 146, 65–76.
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