Chromogranin A is a reliable serum diagnostic biomarker for pancreatic neuroendocrine tumors but not for insulinomas

Pancreatic neuroendocrine tumors (PNETs) are a group of rare tumors. The prevalence and incidence have increased over the past 3 decades [1‐5]. The clinical presentations of PNETs are very complicated due to excess of gut peptides produced by functioning PNETs while symptoms of nonfunctioning PNETs (NF) are obscure [1, 3]. In addition, most of PNETs could be biologically aggressive [1, 3, 6]. Thus, the earlier and accurate diagnosis of PNET is important to facilitate surgical resection and/or to initiate appropriate medical management such as molecular targeted therapy, biotherapy and other intensive care.

Chromogranin A (CgA) is a 46-kDa glycoprotein, member of the granin family, exists within all type of neurons, normal neuroendocrine cells and is expressed in NET cells [7, 8]. Over the past 2 decades, many studies reported and confirmed that CgA was a reliable diagnostic biomarker for NETs including gastroentero-pancreatic NETs (GEP-NET) [1, 3, 5‐20] and also might be a prognostic biomarker for NETs [21]. Moreover, several studies showed that peripheral blood levels of CgA were increased in endocrine-associated tumors, for example, breast cancer [22] and prostate cancer [23, 24]. Recently, elevated serum/plasma levels of CgA were found in a number of non-endocrine solid tumors, such as hepatic carcinomas [25, 26] and pancreatic cancer [27]. Examination of CgA levels could be used not only for diagnosis but also for prognostic evaluation in these tumors [21, 23, 25, 27].

In recent ENETS and NANETS consensus guidelines, CgA was considered as the most practical and useful serum tumor marker in PNET patients [28, 29]. Some studies suggested that testing blood CgA should be mandatory for NET diagnosis [7]. However, few attention has been paid to the insulinoma which is the most common type of functioning PNETs [7, 8, 14, 15, 17, 19, 21, 30‐32]. In addition, peripheral blood levels of CgA are not routinely tested in Chinese patients with GEP-NET. Using CgA for clinical diagnosis has not been officially approved by Sino Food Drug Administration (SFDA) because little data have been reported.

Thus, the present study is to verify the utility of CgA in diagnosis of PNETs, focusing on its diagnostic value in insulinoma. We found that serum levels of CgA were not significantly elevated in patients with insulinomas, compared to the higher levels of CgA in other PNETs. This finding was rarely reported in previous studies.

Circulating CgA levels have been confirmed to be useful diagnostic marker for NETs, with a high specificity and sensitivity [1, 3, 5‐19, 28]. In the present study, we verified the diagnostic value of serum CgA in a series of patients with non-insulinoma PNETs, in agreement with previous studies. The sensitivity and specificity were 65.6% and 91.9%, respectively, similar to the rate of 67% and 96% when using CIS Bio kits, as Ardill and Erikkson described [12]. Similar to previous studies [9‐11, 14, 16, 41, 42], we found that CgA levels in patients with gastrinomas were much higher than those in patients without gastrin-secreting PNETs.

The interesting finding in the study was that serum CgA levels were not elevated in patients with insulinomas, including one patient with extensive liver metastases (48 ng/ml). Most of previous studies on PNETs did not clarify this unusual biochemical feature of insulinoma, the most common subtype of PNETs [7, 8, 13‐16, 19, 20, 31] but Wouter de Herder pointed out in a review that blood levels of CgA were rarely slightly elevated in subjects with insulinomas [43] and Portela-Gomes GM et al. mentioned in a review that well-differentiated NETs expressed CgA epitopes except insulinomas [44]. A recently published guideline which was revised by the UK and Ireland Neuroendocrine Tumor Society and the British Society of Gastroenterology addressed that CgA would not raised in benign insulinomas [45]. Moreover, Nobels et al. studied more than 200 NETs and found that serum CgA levels were rarely slightly elevated in patients with insulinomas (elevated in 2 of 21 patients, range 63–236 ng/ml, upper cut-off value was 220 ng/ml) [9]. Another study showed that CgA levels were not elevated in 5 cases of insulinomas [13]. In present study, we focused on insulinomas and our findings were very similar to their data which showed only a small part of patients with insulinomas (7/57, 12%) had a slightly increased level of CgA (>100 ng/ml, the highest level: 164 ng/ml). With a relatively low specificity (73%), serum CgA was not a reliable and practicable biomarker for diagnosis of insulinoma. This finding is important. Some studies suggested that testing CgA levels should be mandatory for PNETs diagnosis [7]. Furthermore, according to recent North American Neuroendocrine Tumor Society (NANETS) and European Neuroendocrine Tumor Society (ENETS) consensus guidelines [28, 29] as well as ESMO guidelines for NETs diagnosis [5], CgA was considered as a general biomarker for NETs, and CgA can be used as a marker in patients with both Functional PNET and NF-PNET [28, 29]. Insulinoma is the most common subtype of functioning PNETs [31, 35, 37, 46]. However, whether serum levels of CgA should be tested in patients with insulinomas has not been well clarified in those guidelines for NETs diagnosis. Our data and previous reports [9, 13] showed that insulinoma could be an exception for measuring serum levels of CgA for diagnostic purpose. It maybe not necessary to test CgA levels in patients with insulinomas although this issue needs to be further validated in more cases and in multiple clinical centers. In addition, using different commercial kits or assay could be useful to further validate our findings because the antibodies used in different assay were raised against the different domain or epitopes of the CgA molecular [12, 15, 17, 47].

The underlying mechanism of low CgA levels in patients with insulinomas is not clear. Nobels et al. speculated the serum levels of CgA were only slightly elevated in subjects with small NETs, such as insulinomas, pituitary adenomas [9]. However, there is disagreement in the literatures whether the serum CgA levels correlate with the extent of NETs or size of these tumors [7, 21, 32, 48]. In present study, we did not observe the correlation between the tumor size and CgA levels in PNETs (P = 0.545) and in insulinomas alone (P = 0.942). Some of non-insulinoma PNETs with relatively small size still had very high serum levels of CgA, for example, CgA level was 4572 ng/ml in a gastrinoma of 1.5 cm in size, and the highest CgA level in present study was more than 9000 ng/ml in a glucagonoma of 2.5 cm in size. Furthermore, one patient with a NF of 3.5 cm in size had a CgA level of 5772 ng/ml whereas another patient with a NF of 8 cm in size had a CgA level of 59 ng/ml.

Many previous studies [10, 13, 15, 41, 42, 49] and our present data showed that CgA levels in patients with NETs metastases were much higher than that in patients with localized NETs. Thus, it may hypothesized that few metastases in insulinomas would be the reason for low serum level of CgA in insulinomas (only one metastatic insulinoma in our serial). We found CgA levels in localized insulinomas were similar to that in localized non-insulinomas, P = 0.693. This might imply the above hypothesis could be partly true. In fact, most of insulinomas (>90%) are benign, absent metastasis in majority of insulinomas is one of the main characteristics of this unique tumor, and in our hospital, more than 95% of insulinomas are benign [37]. However, we noticed that the rate of elevated CgA levels in patients with localized non-insulinomas was significantly higher than that in patients with localized insulinomas, P = 0.015. In addition, one report showed that in 9 of the 10 patients with gastrinoma, CgA values were raised, even in the absence of metastasis [41]. These data suggested that metastasis could be one of determinant factors for high levels of CgA in PNETs, but not the only one. The tumor subtype could be another important determinant for CgA serum levels. Nevertheless, more patients with metastatic insulinomas were needed to validate the low levels of CgA in insulinomas although it might be quite difficult to do so due to the limited numbers of malignant insulinomas.

It was reported that pancreastatin, a CgA-derived peptide (CgA residues 250–301) with biological activity, inhibited the releasing of insulin by islet beta cells [7, 50] and insulinoma cell line [51]. Gayen et al. [52] observed an inverse relationship between pancreastatin and insulin. This CgA-derived peptide might antagonize the effect of insulin via the Akt/FOXO-1 and, administration of insulin could result in low plasma levels of pancreastatin in mice (the basal pancreastatin level dropped significantly following insulin injection). In majority of insulinomas, a great deal amount of insulin is secreted by tumor cells. We speculate that high levels of insulin in patients with insulinomas might inhibit the secretion of CgA in these tumor cells. A recent study demonstrated that insulin and proinsulin were released in patients with insulinomas in response to arterial calcium stimulation, whereas CgA was not released [53].

One research suggested that CgA targeted to secretory granules in association with protein secretogranin III, a member of granin family, in pituitary and pancreatic endocrine cells [54]. If other proteins such as secretogranin III were broken down, the secretion of CgA would be disrupted. The mechanisms of hormones and peptides secretion were very complicated, it maybe concerned with molecular cellular biology and the alterations of tumor cell functions.

Other than the low blood levels of CgA in insulinomas, the biomedical behaviors of insulinomas were quite different from other PNETs. For example, its low rate of malignancy (<10%), the relatively low rate of positive Octreotide scintigraphy can be identified in benign insulinomas comparing with non-insulinoma PNETs because many insulinomas do not express somatostatin receptor subtypes [35]. All of these unusual features of insulinomas indicated that unique molecular cellular aspects and/or functions existed in insulinoma cells.

It is hard to explain why the serum level of CgA was postoperatively elevated in one patient who had normal liver and kidney functions. This patient did not suffer with other disease or take PPI or H2 receptor blocker. The sample was detected repeatedly and the same results were obtained.

In conclusion, our findings suggested that CgA is not a reliable biomarker for insulinomas, hence, examination of blood CgA levels could not be recommended in patients with insulinoms according to Nobels’ [9] and our data. The mechanisms underlying low serum levels of CgA in insulinomas would appear to warrant further investigation.

The study revealed that the circulating CgA levels in patients with insulinomas were not obviously elevated, although we did validate the diagnostic value of serum CgA in a series of patients with non-insulinoma PNETs.