Validation of four candidate pancreatic cancer serological biomarkers that improve the performance of CA19.9

Pancreatic cancer is the tenth most common cancer type and the fourth leading cause of cancer-related deaths [1]. Diagnosis of small, early-stage tumors that can be surgically resected offers patients the best chances for survival and can increase five-year survival rates from ~5% to 20-30%, or higher at specialized treatment centers [1, 2]. Unfortunately, given the asymptomatic nature of its early stages, its aggressive disease course, and limitations of current detection technologies, fewer than 20% of patients are diagnosed with resectable disease.

Currently, detection of pancreatic cancer is based largely on various imaging modalities, such as computed tomography (CT), endoscopic ultrasound (EUS) and magnetic resonance imaging (MRI) [3, 4]. More definitive preoperative diagnoses of pancreatic cancer typically requires invasive means such as endoscopic retrograde cholangiopancreatography (ERCP) which enables tissue sampling or endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) [5, 6]. The major drawback of all of these methods for the optimal management of pancreatic cancer patients is that they are primarily utilized after the onset of symptoms (i.e. predominantly after the onset of late-stage disease). They are also associated with relatively high operating costs, and can be somewhat time consuming and/or invasive in nature. In this regard, implementation of highly sensitive and specific biomarkers or marker panels for pancreatic cancer can further enhance detection strategies by offsetting many of the limitations described above [5, 6].

The current clinically used marker for pancreatic cancer is carbohydrate antigen 19.9 (CA19.9). CA19.9 is a sialylated Lewis A-active pentasaccharide detected primarily on the surface of mucins in the serum of pancreatic cancer patients [7, 8]. Although elevated CA19.9 levels have been associated with advanced stages of the disease, they have also been associated with benign and inflammatory diseases [8‐10]. For early-stage pancreatic cancer detection, CA19.9 has a reported sensitivity of ~55% and is often undetectable in many asymptomatic individuals [7]. In addition, CA-19.9 is associated with Lewis antigen status and is absent in individuals with blood group Le(a^-b^-) (~10% of the general population) [7, 11]. Taken together, CA19.9 alone lacks the necessary sensitivity and specificity for pancreatic cancer detection and according to the American Society of Clinical Oncology Tumor Markers Expert Panel, CA19.9 is recommended only for monitoring response to treatment in patients who had elevated levels prior to treatment [12].

With the aim of identifying new biomarkers for pancreatic cancer, we previously performed proteomic analysis of conditioned media (CM) from six pancreatic cancer cell lines, one ‘normal’ pancreatic ductal epithelial cell line and six pancreatic juice samples using two dimensional LC-MS/MS [13]. A total of 3479 nonredundant proteins were identified with high confidence. Three strategies were then utilized to mine the list of identified proteins for putative candidate pancreatic cancer biomarkers: (1) differential protein expression analysis between the cancer and normal cell lines using label-free protein quantification, (2) an integrative analysis, concentrating on the proteins consistently identified in the multiple pancreatic cancer biological fluids subjected to proteomics analysis, and (3) analysis of tissue specificity through mining of publically available databases [13]. Of the candidates identified in our previous work, the current study details the validation of four candidates, REG1B, SYCN, AGR2 and LOXL2. These four candidates were selected based on commercially available ELISA kits for validation, as well as preliminary verification studies in smaller sample sets as described in our previous publication for AGR2 [13], and conducted in-house thereafter for the other three candidates (data not shown).

Due to the lack of a single highly sensitive and specific marker for many diseases, including for various measurable outcomes of pancreatic cancer, research has shifted to the development of panels of markers to achieve improved performance [16]. In the current study, four pancreatic cancer biomarker candidates (SYCN, REG1B, AGR2 and LOXL2) delineated through our previous integrated proteomics analysis of cell line conditioned media and pancreatic juice [13], were validated in two sample sets of serum/plasma containing a total of 432 samples. Individually, CA19.9 performed best when compared to the tested candidates; however in combination, three panels were found to significantly improve the performance of CA19.9 in discriminating healthy from PDAC in a training and validation set (Table 4, Figure 1). Additionally, several of the analyzed candidates, particularly SYCN and REG1B show the ability to capture PDAC cases missed by CA19.9 (Additional file 1: Table S6). Several panels were also identified which significantly improved the diagnostic accuracy of CA19.9 for discriminating early-stage from disease-free subjects (Additional file 1: Tables S7 and S8). In terms of distinguishing PDAC from benign disease and PDAC from other cancers, aside from CA19.9, AGR2 showed the best discrimination between these two groups (Additional file 1: Table S2).

Syncollin is a protein that is highly expressed in pancreatic acinar granules. Specifically, it is a zymogen granule protein found on the inner surface of the granule membrane with a role in the concentration and maturation of zymogens, as well as in the regulation of exocytosis [17, 18]. A few recent studies have described its presence and function in granules in other tissue types [19]. Syncollin has been identified in a qualitative proteomic analysis of pancreatic juice from patients with pancreatic cancer, and was elevated in serum from a murine model of pancreatic cancer [20, 21]; however to our knowledge, this is the first report of its study and extended validation through ELISAs in human serum. In the present study SYCN was significantly elevated in patients with pancreatic cancer when compared to healthy controls in both sample sets, as well as when compared to benign disease controls. SYCN also showed the ability to best capture samples which had CA19.9 within normal limits (Figure 2), and was significantly elevated in early PDAC when compared to healthy controls. Given that the majority of pancreatic cancers are believed to arise from ductal cells (or possibly acinar cells that undergo acinar-to-ductal metaplasia) [22, 23], elevation of SYCN in the circulation may be a secondary effect of the growing tumor through local tissue destruction. In pancreatic cancer, this has been recently studied for the protein transthyretin (TTR), an islet cell protein that is elevated in pancreatic juice from pancreatic cancer patients through destruction of islet cell architecture in the presence of invasive cancer [24]. A similar mechanism of local tissue destruction causing increased release of the protein may be occurring with SYCN. However, the unavailability of stage information for many of the PDAC cases prevented a comparison of early versus late stage SYCN levels in the present study and therefore, firm conclusions cannot be made regarding this at the present time. REG1B belongs to a family of proteins, encoded by the human REG genes, that is present in pancreatic acinar cells and promotes regeneration of pancreatic islets [25]. REG family members have been associated with pancreatic cancer or related diseases in the past. REG1A, a protein that is highly similar to REG1B has been implicated in pancreatitis, and other REG family members such as REGIV and hepatocarcinoma-intestine-pancreas/pancreatitis associated protein I (HIP/PAPI) have shown potential diagnostic utility for pancreatic cancer [26, 27]. REGIV and REG1A have also been shown to be elevated in gastric cancer and may serve as prognostic indicators [28‐30]. Both REG1A and REGIII were also found elevated in plasma from a mouse model of pancreatic cancer [21]. To the best of our knowledge, REG1B expression in serum has not been studied as a pancreatic cancer biomarker. In the present study, REG1B was significantly elevated in pancreatic cancer serum/plasma compared to healthy and benign disease controls, and it was a component of all three panels found to significantly improve the AUC of CA19.9 in our training and validation analyses (Tables 3 and 4).

Both REG1B and SYCN were identified in our previous proteomics discovery work as candidate pancreatic cancer biomarkers due to their presence in pancreatic juice from PDAC patients along with their identification as proteins highly tissue specific to the pancreas based on mining of several tissue specificity databases [13]. Interestingly, both proteins performed similarly in the validation study presented in this paper, being significantly elevated in comparisons between PDAC and healthy and PDAC and benign controls; however neither protein was able to significantly differentiate other cancers from PDAC in the studied samples. The ‘other cancer’ group in this study had a large fraction of serum samples from colon cancer patients. SYCN has not previously been studied in other cancers; however REG family gene expression, although not REG1B specifically, has been shown previously to be elevated in colon cancer and inflammatory bowel disease [31, 32].

AGR2 is a protein initially identified in Xenopus laevis that was shown to be crucial during ectoderm developmental stages of embryogenesis for formation of anterior structures [33]. AGR2 is a member of the protein disulfide isomerase (PDI) family, found localized to both the endoplasmic reticulum and cell surface [33]. Its role in normal human structures is still somewhat unclear; however it has been implicated in many cancer types, including pancreatic cancer [34‐36]. In pancreatic cancer cells, AGR2 has been shown to be involved in invasion and dissemination through posttranscriptional regulation of cathepsins D and B [34]. Recently, Chen et al. [37] found AGR2 to be overexpressed in pancreatic juice from patients with pancreatic intraepithelial neoplasia – III (PanIN3) and their ELISA results showed this protein to have potential diagnostic utility for pancreatic cancer in pancreatic juice; however these findings did not translate into their serum analysis. AGR2 was highly overexpressed in our previous integrated proteomic analysis of cell lines and pancreatic juice as well, and our preliminary verification studies showed it to be significantly elevated in plasma from pancreatic cancer patients [13]. In the present study, although significantly elevated in one sample set, AGR2 performed somewhat poorly in discriminating PDAC from healthy/non-cancer controls; however interestingly, it performed best, after CA19.9, in discriminating benign and other cancers from PDAC (Additional file 1: Table S2). AGR2 is a protein shown to promote tumor growth, cell transformation and migration [38]; however it is unclear as to why AGR2 was able to distinguish other cancers from PDAC in this study. As mentioned above, a large number of samples in the ‘other cancer’ group were colon cancer samples and it is possible that AGR2 serum levels are increased at higher levels in PDAC compared to colon cancer. A study from 2006 [39], also shows AGR2 mRNA levels to be downregulated in colon cancer; however firm conclusions cannot be made.

LOXL2 is an extracellular matrix protein involved in the epithelial to mesenchymal transition of cells and is highly expressed in desmoplastic/fibrotic stroma [40]. It has been shown to be upregulated in many cancer types and is believed to play a role in cancer metastasis. LOXL2 silencing in pancreatic cancer cells has shown improved sensitivity to gemcitabine therapy [41] and decreased tumor growth in gastric cancer [42]. In our serum/plasma analysis, the general ability of LOXL2 to distinguish PDAC from controls was not significant.

Validation of biomarkers and translation of markers from the bench to the clinic is a rigorous process [43]. The goal of this study was to preliminarily validate a set of the candidates identified in our previous proteomics work [13]. Of the candidates validated, several were able to significantly distinguish between case and control groups, with multiple panels demonstrating the ability to significantly improve the performance of CA19.9.

According to recent research, there is at least a ten-year window during early-stage pancreatic cancer development, followed by another seven years before it metastasizes [44]. The greatest applicability of markers for the early detection of pancreatic cancer would likely be as a pretest to an imaging modality in screening and surveillance programs for detecting developing pancreatic cancers in high-risk patient groups. One of the limitations of this study was the lack of staging information for all cases. In this regard, further validation of the candidates and panels presented in this study is warranted in larger sample sets of individuals with early-stage disease, as well as those with precursor lesions such as PanIN lesions. Additionally, consideration of the markers/panels presented in this study for other measurable outcomes of pancreatic cancer, such as monitoring response to treatment and assessing disease recurrence is also warranted.