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Biomarkers for early diagnosis of pancreatic cancer Expert Rev. Gastroenterol. Hepatol. Early online, 1–11 (2014)
Claire Jenkinson1, Julie Earl2, Paula Ghaneh1, Christopher Halloran1, Alfredo Carrato2, William Greenhalf1, John Neoptolemos1 and Eithne Costello*1 1 Department of Molecular and Clinical Cancer Medicine, National Institute for Health Research Liverpool Pancreas Biomedical Research Unit, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK 2 Department of Medical Oncology, Ramo´n y Cajal University Hospital, Carretera Colmenar Km 9,100, 28034 Madrid, Spain *Author for correspondence: Tel.: +44 151 706 4178 Fax: +44 151 706 5826 [email protected]
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Pancreatic ductal adenocarcinoma is an aggressive malignancy with a 5-year survival rate of approximately 5%.The lack of established strategies for early detection contributes to this poor prognosis. Although several novel candidate biomarkers have been proposed for earlier diagnosis, none have been adopted into routine clinical use. In this review, the authors examine the challenges associated with finding new pancreatic cancer diagnostic biomarkers and explore why translation of biomarker research for patient benefit has thus far failed. The authors also review recent progress and highlight advances in the understanding of the biology of pancreatic cancer that may lead to improvements in biomarker detection and implementation. KEYWORDS: biomarker • diagnosis • jaundice • pancreatic cancer
Discussion
With an estimated 250,000 new cases diagnosed every year, pancreatic ductal adenocarcinoma (PDAC) remains the fifth leading cause of cancer death worldwide [1]. Unfortunately, unlike many other cancer types, there has been little improvement in survival rates for pancreatic cancer over the past four decades [2] and it has been projected to become the second leading cause of cancer-related death by 2030 [3]. Failure to detect the disease before it is advanced is a major problem and is mainly due to the fact that early warning signs are vague, while symptoms such as obstructive jaundice, weight loss or pain tend to become more apparent at later stages of the disease. Consequently, around 80% of patients are diagnosed with locally advanced or metastatic disease [1] ruling out potentially curative resection. Survival times for patients with advanced disease are generally low [4,5]. However, for patients who have undergone surgery to remove their tumour followed by chemotherapy, the 5-year survival is significantly better, at approximately 25% [6–8]. Currently, the only biomarker used in the routine management of pancreatic cancer is CA19-9, an epitope of sialylated Lewis blood group antigen [9,10]. A recent meta-analysis [11],
10.1586/17474124.2015.965145
which pooled summary estimates of 11 studies, found that CA19-9 had both a sensitivity and specificity of 80% for pancreatic cancer diagnosis. CA19-9 is upregulated in pancreatic inflammation and benign pancreatic disease [9] which hampers its specificity for pancreatic cancer. Moreover, approximately 5% of the population are Lewis ab negative and therefore do not secrete CA19-9 [12]. Thus, while CA19-9 is successfully used for predicting clinical course, improvements in sensitivity and specificity for reliable early PDAC detection are desirable [13]. Much effort has focused therefore on enhancing the performance of CA19-9 by including it within larger panels of markers [14–17]. How early should pancreatic cancer be detected to allow significant improvement to outcome?
If patients with pancreatic cancer are divided into those with resectable, locally advanced and metastatic disease, early detection should, at the very least, enhance the detection of patients in the resectable category. Biomarkers that would increase the proportion of patients who are eligible for potentially curative surgery should lead to an improvement in prognosis. Nonetheless, median survival after surgery for patients treated with chemotherapy such as
2014 Informa UK Ltd
ISSN 1747-4124
1
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Review
Jenkinson, Earl, Ghaneh et al.
fluorouracil plus folinic acid or single-agent gemcitabine remains just below 2 years [8], indicating that possibly even earlier intervention is required to radically alter survival times. There is evidence that pancreatic cancer is a systemic disease from its early stages. A study using genetically engineered mouse models of pancreatic cancer showed that pancreatic epithelial cells from lesions considered to be preneoplastic had spread into the stroma of the pancreas and had invaded the bloodstream [18]. The suggestion that this disease spreads from a very early stage has implications for early detection (see below) and for subsequent early intervention, which may need to concentrate on more rapid application of systemic treatments [19,20]. Scope of this review
Technological advances have opened up opportunities for diagnostic biomarker discovery in a number of areas. Advances in proteomic technologies, in both the range of mass spectrometry-based approaches and its increased sensitivity for detection of low abundance proteins, have made mass spectrometry a powerful tool for biomarker protein discovery. Advances in imaging and cell tracking are also gaining pace, while miRNAs, circulating cancer cells and circulating tumor DNA are areas with vast potential for facilitating cancer detection. For the purpose of this review we have largely subgrouped biomarkers based on their properties, rather than the sample type from which they originate. Biomarkers specifically relating to pancreatic cystic neoplasms, including intraductal papillary mucinous neoplasms (IPMNs), are not reviewed here. Protein markers Serum protein biomarkers
As a source of biomarkers, serum is minimally invasive to obtain and is a sample type that clinical laboratories are used to performing blood tests on. However, the large dynamic range of proteins in serum complicates biomarker discovery protocols [21]. Nonetheless, much effort has focused on developing blood-borne biomarkers for pancreatic cancer diagnosis. Given the immense genetic, environmental and cancerassociated variation across individuals, single biomarkers are unlikely to offer accurate diagnosis for everyone. The emphasis has therefore shifted toward developing panels of biomarkers to complement or surpass CA19-9 alone [15–17,22]. Using Luminex bead-based technology, Brand et al. [15] performed one of the most comprehensive pancreatic cancer biomarker studies to date, analyzing 83 proteins in 333 PDAC patients, 144 patients with benign pancreatic conditions and 227 healthy individuals. Samples were split into training and validation sets. A panel of CA19-9, ICAM-1 and OPG discriminated PDAC patients from healthy individuals with a sensitivity/specificity of 88/90% in a training set and 78/94% in an independent validation set. A different panel comprising CA19-9, CEA and TIMP-1 discriminated PDAC patients from benign subjects with a sensitivity/specificity of 76/90% in a training set and 71/89% in validation. doi: 10.1586/17474124.2015.965145
Alternative panels have also been proposed that can identify pancreatic cancer. Park et al. [22] measured serum levels of cathepsin D along with a number of tissue inhibitors of matrix metalloproteinases and matrix metalloproteinases in relatively large training and validation sets of pancreatic cancer patients (248 total) and control subjects (114 healthy controls, 102 patients with chronic pancreatitis). A panel comprising CA19-9, cathepsin D and MMP-7 improved the discrimination of cancer from controls over that of CA19-9 alone. A study by Faca et al. [17] highlighted the potential of including relevant mouse models to assist biomarker discovery. Analysis of the plasma proteome of genetically engineered mice with early versus advanced pancreatic cancer led to the identification of a panel of seven proteins (ALCAM, ICAM-1, LCN2, TIMP-1, REG1A, REG3 and IGFBP4) that differentiated newly diagnosed PDAC cases from chronic pancreatitis patients with an area under the receiver operating characteristic (ROC) curve (AUC), compared to CA19-9 alone which gave an AUC of 0.79. This study was also one of the first to use prediagnosis samples as part of the validation protocol for markers. Five of the proteins identified (LCN2, REG1A, REG3, TIMP-1 and IGFBP4) when combined with CA19-9 discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 months prior to the clinical diagnosis of pancreatic cancer with an AUC of 0.911. Detection of stage I disease with high sensitivity was recently reported using a monoclonal antibody called PAM4 which binds to the mucin MUC5AC [23], and has specificity for PDAC and its precursor lesions but not for normal or benign tissues [24]. Gold et al. [14] evaluated a PAM4-based serum immunoassay for pancreatic cancer detection. Using sera from 298 PDAC patients, 99 patients with other cancers, 120 patients with benign disease of the pancreas and 79 healthy adults, PAM4 demonstrated a sensitivity of 64% for PDAC patients with stage I disease. The sensitivity was higher (85%) for PDAC patients with advanced cancer, and combining PAM4 with CA19-9 demonstrated an improved overall sensitivity for PDAC of 84% compared to 77% for CA19-9 alone. Interestingly, PAM4 offered good discrimination between PDAC and chronic pancreatitis, a characteristic that is lacking from many reported biomarkers of PDAC. Makawita and colleagues [25] also demonstrated a panel of biomarkers to be superior to CA19-9 for the detection of stage I–II pancreatic cancer. This study evaluated four biomarkers, namely, regenerating islet-derived 1b (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein and lysyl oxidase-like 2 in two sample sets. A panel consisting of SYCN + REG1B + CA19-9 performed better than CA19-9 for the detection of early-stage (stage I–II) PDAC versus diseasefree controls. Protein biomarkers in tissue & subcellular fractions
Due to the invasiveness of procedures to obtain cancer tissue, this material is not commonly used as a screening tool. Blood is a more accessible and therefore optimal sample for biomarker Expert Rev. Gastroenterol. Hepatol.
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Biomarkers for early diagnosis of pancreatic cancer
testing. Nonetheless, pancreatic cancer tissue contains the highest concentrations of PDAC-specific markers, and as such, is the most direct source of proteomic cancer biomarkers [26]. The potential to identify cancer-specific biomarkers from the tumor make it an invaluable resource. Crnogorac-Jurcevic et al. [27] performed a large-scale immunoblotting analysis with more than 900 primary antibodies on cancer tissue, chronic pancreatitis and normal pancreas. Several proteins, such as UHRF1, ATP7A and AOX1, differed in their expression between chronic pancreatitis and pancreatic cancer, suggesting potentially important roles in pancreatic carcinogenesis. Chen et al. [28] measured the levels of complement C3, complement C4b1 and apolipoprotein E in tissue from pancreatic cancer patients of varying stages by immunohistochemistry, reverse transcription PCR (RT-PCR) and western blotting. The levels of all three were higher in the pancreatic cancer tissue compared to the normal tissue. They showed that complement C3 correlated with earlystage disease, while they suggested that complement C4b1 and apolipoprotein E could potentially be used as diagnostic markers of advanced PDAC due to their marked correlation with later stages and lymph node metastasis. Advances in proteomic techniques have enabled the combination of subcellular fractionation and mass spectrometry for the identification of potential biomarkers associated with pancreatic cancer within tissues. McKinney et al. [29] utilized matched tumor and nontumor sample pairs from patients undergoing tumor resection and then separated the tissues to obtain cellular protein fractions corresponding to cytosol, membrane, nucleus and cytoskeleton. With this approach, 1771 proteins in normal and 1985 proteins in pancreatic tissue with cancer were identified, and 104 proteins were found to be significantly changed in pancreatic cancer. Four secreted proteins, namely, biglycan, pigment epithelium-derived factor, thrombospondin-2 and TGF-binduced protein Ig-h3 precursor were validated as expressed in pancreatic cancer tissue. This study demonstrated the potential of subcellular fractionation as a powerful strategy for identification of differentially expressed proteins in pancreatic cancer. Protein markers in pancreatic juice
The advantage of body fluids such as urine is that they can be obtained without the requirement of an invasive procedure; however, urine is separated from the tumor by the circulation and kidneys. Pancreatic juice, by contrast, is in direct contact with the cancer and is excreted out of the body. Traditional serum biomarkers such as CEA and CA19-9 can be found in pancreatic juice [30] but with little or no diagnostic advantage over serum for pancreatic cancer patients, especially given the increased costs and risks of obtaining juice samples. Empirical approaches to proteomic analysis have identified more promising proteins [31], but as yet this promise has not borne fruit and none of these juice biomarkers have found their way to the clinic. DNA markers in pancreatic juice
Detection of specific cancer-related genetic or epigenetic changes in pancreatic juice samples has proved more productive. Notably informahealthcare.com
Review
detection of K-ras mutations in pancreatic juice has proven to be reasonably sensitive for the detection of pancreatic cancer [32], but it was found to give very poor specificity, especially when comparing chronic pancreatitis and pancreatic cancer. Other markers, notably mutations in p53, gave considerably better specificity [33], but with a reduction in sensitivity. Assay of methylation in various promoters has been found to be an effective alternative to mutation detection, the most effective being detection of methylation of the CDKN2a (p16) promoter [34]. The specificity and sensitivity can be improved further by combining mutation and methylation analysis [35], but this remains far from perfect and pancreatic juice sampling using Endoscopic Retrograde Cholangiopancreatography can result in induction of pancreatitis; Endoscopic Retrograde Cholangiopancreatography-induced pancreatitis can prove fatal. This risk can be reduced by prophylaxis using stents and nonsteroidal anti-inflammatory agents such as diclofenac [36]. The risk may be further reduced or eliminated by obtaining juice from the duodenum without cannulating the pancreatic duct. Promising results have been obtained from molecular analysis of samples obtained in this way [37,38]. miRNA markers
miRNAs are small RNAs (22–25 nt) that negatively regulate gene expression by binding to complementary mRNA resulting in gene silencing, translational repression or target degradation. The deregulation of some miRNAs has been identified as a mechanism responsible for cell transformation including pancreatic cancer development [39] miRNAs with diagnostic potential for pancreatic cancer are summarized in TABLE 1. Overexpression of miR-21 has been reported in PDAC and precursor pancreatic intraepithelial neoplasia (PanIN) lesions, and has been correlated with poor overall survival and chemoresistance to gemcitabine [40–42]. Moreover, miR-21 is known to be upregulated in several tumor types as it negatively regulates the tumor suppressor genes PTEN and PDCD4 [43]. miRNAs can be secreted or interchanged among cells in the form of microparticles [44] and thus can be detected in bodily fluids including serum. It has been estimated that healthy individuals have between 5 and 50 mg/ml of microparticles that increase under different disease conditions [45]. Thus, they are valuable minimally invasive diagnostic markers and have been exploited as cancer serum/plasma biomarkers in many tumor types [43]. miR-18a, miR-200, miR-210 and miR-375 have been reported as potential minimally invasive blood-based biomarkers for pancreatic cancer [46–49] although on an individual level they do not outperform CA19-9 in terms of sensitivity and specificity for the detection of PDAC. Li et al. [48] quantified serum levels of miR-200a/200b in the serum of 45 patients with PDAC, 11 with CP and 32 healthy controls. Elevated levels of miR-200a provided a sensitivity and specificity of 84.4 and 87.5%, respectively, for PDAC, while elevated levels of miR-200b delivered a sensitivity and specificity of 71.1 and 96.9%, respectively. Neither marker differentiated PDAC from CP. Despite this shortcoming, further studies are merited to verify the performance of miR-200 as a biomarker in larger doi: 10.1586/17474124.2015.965145
Review
Jenkinson, Earl, Ghaneh et al.
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Table 1. miRNAs reported as having diagnostic potential in pancreatic cancer. miRNA
Tissue
miR-21
PDAC Gemcitabine resistance Precursor lesions – PanINs
[40,41]
PanINs IPMN
[42,39]
miR-155
Ref. [40] [42,39]
[50] [42,52,53] [51]
miR-196
PDAC IPMN
miR-375
Plasma IPMN
[46]
miR-18a
Plasma
[49]
miR-210
PDAC IPMN Plasma
[108] [51]
PanINs IPMN Serum
[42] [51]
miR-200
[51]
[47]
[48]
miRNAs reported as having diagnostic potential in pancreatic cancer. PanIN: Pancreatic intraepithelial neoplasia; PDAC: Pancreatic ductal adenocarcinoma.
patient populations. Morimura et al. [49] found that miR-18a was raised in plasma of PDAC patients. Although this miRNA did not outperform CA19-9, levels of both miR-18a and CA19-9 dropped after tumor resection, suggesting that monitoring the miR-18a levels in the serum of patients in remission could support the detection of cancer recurrence. One problem with miRNA profiling in tissue or bodily fluids is that many miRNAs which have been found to be selectively expressed in advanced cancer have not been validated in earlier cancers or preneoplastic lesions. By contrast, miR-155, miR-196 and miR-210 have been reported to be associated with PDAC and PanIN lesions as well as IPMN [39,42,47,50–53]. Detecting changes in PanIN lesions is of great interest, since aberrant expression of miRNAs in these lesions may represent an early modification in pancreatic tumorigenesis. Ryu et al. [39] used quantitative RT-PCR to determine the levels of miR-21, miR-155 and miR-221 in 31 microdissected PanINs. They observed that miR-155 was significantly overexpressed in PanIN-2 and PanIN-3 lesions (2.6-times; p = 0.02 and 7.4-fold; p = 0.049, respectively). miR-21 was overexpressed only in PanIN-3 (2.5-fold; p = 0.02), while no change in expression was found for miR-221 in PanIN lesions compared with normal duct epithelium. du Rieu et al. [54] also explored the expression of miRNAs in PanIN lesions. They reported that miRNAs miR-21, miR-205 and miR-200 were elevated in PanIN lesions in a conditional KRAS (G12D) mouse model, while miR-222 production decreased. The levels of three miRNAs, miR-29c, miR-221 and let-7a, remained unchanged between normal ducts and PanIN lesions. doi: 10.1586/17474124.2015.965145
In a search for diagnostic miRNA candidates, Baraniskin et al. discovered that the discriminatory power of one promising candidate, miR-1246, actually came from U2 small nuclear RNA fragments. The group went on to show that U2 small nuclear RNA fragments are highly stable in serum and plasma and hold the potential as a novel diagnostic biomarker for PDAC.
[55]
Cytokines & chemokines
Panels of cytokines, chemokines and growth factors are also potential important diagnostic markers of pancreatic cancer. They are released by various components of the immune system and contribute to neoplastic transformation [56]. The expression of VEGF and bFGF was elevated, alongside an increase in CA19-9 levels, in the serum of patients with pancreatic cancer (stage I–IV; p < 0.0001) compared to healthy subjects [57]. Using LabMAPTM technology with classification trees, Zeh et al. [58] identified a multicytokine panel which, when used in combination with CA19-9, differentiated PDAC patients from control groups. Shaw et al. [59] measured serum levels of 27 cytokines in 241 subjects, including 127 with PDAC, 49 with chronic pancreatitis, 20 with benign biliary obstruction and 45 healthy controls. Having split samples into independent training and test sets, cytokine biomarker panels were identified using best fit logistic regression models. A panel of IP-10, IL-6, PDGF plus CA19-9 offered improved diagnostic performance over CA19-9 for distinguishing PDAC patients from patients with benign disease. These findings support a potential role for cytokine panels in the discrimination of PDAC from patients with benign pancreatic diseases, with validation in larger sample sets merited. Autoantibodies
Recently, several studies have shown that cancer patients produce detectable autoantibodies, that is, antibodies generated against certain tumor-associated antigens, which may be useful as bloodborne biomarkers for early detection of cancer [56]. In one of the largest studies into autoantibody detection in PDAC [60], autoantibodies to CTDSP1 (carboxy-terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1), MAPK9 (mitogenactivated protein kinase 9) and NR2E3 (nuclear receptor subfamily 2, group E, member 3) were analyzed in 300 pancreatic cancer patients and 300 controls. A significantly elevated antibody response was observed in disease cases for CTDSP1 (p = 0.004), MAPK9 (p = 0.0002) and NR2E3 (p = 0.0001) compared with the controls; however, discrimination between cancer cases and controls were relatively poor for each marker with AUC values of 0.62, 0.59 and 0.56 for NR2E3, MAPK9 and CTDPS1, respectively [60]. Novelli and colleagues identified autoantibodies in the sera of pancreatic cancer patients, with reactivity to two acidic isoforms of the glycolytic enzyme enolase (1/2), which had the power to discriminate PDAC patients from controls with 62% sensitivity and 97% specificity [61]. More recently, this group reported a high frequency of autoantibodies to Ezrin in a Expert Rev. Gastroenterol. Hepatol.
Biomarkers for early diagnosis of pancreatic cancer
genetically engineered mouse model of spontaneous PDAC [62]. The finding translated to PDAC patients, with 56% of PDAC patient sera (n = 120) displaying reactivity against Ezrin compared to only 12% of non-PDAC sera (n = 50) and 2% of sera from patients with chronic pancreatitis (n = 46).
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Circulating tumor cells
The detection of tumor cells in peripheral blood, the so-called circulating tumor cells (CTC), has offered the promise of using them as biomarkers for disease detection and management. Their presence in peripheral blood has been associated with lower progression-free and overall survival in several cancer types, and they have been proposed as early indicators of tumor spread as invasive, but localized tumors may shed CTC into the bloodstream before a metastasis is established [63]. CTC can enter the blood circulation by tumor cell shedding from the primary tumor, a process that may occur in the early stages of tumor formation [64,65]. Alternatively, tumor cells may enter the circulation through a process of epithelial- to-mesenchymal transition whereby they acquire mesenchymal features including motility and invasiveness [66]. Pancreatic epithelial cells were detected in peripheral blood of a PDAC mouse model that harbored early lesions, but prior to tumor formation [18], implying ample opportunity for early detection of CTC. Of note, the cells detected in this mouse model had undergone epithelial-mesenchymal transition. This fact has significant ramifications for strategies designed to monitor CTC for diagnosis or treatment, since CTC with a mesenchymal phenotype may evade detection that relies on the expression of epithelial markers such as EpCAM. Rhim et al. recently confirmed the detection of pancreas epithelial cells (CECs) in patient blood samples. Using microfluidic geometrically enhanced immunocapture, CECs were detected in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer, 8 of 11 (73%) with PDAC and in 0 of 19 cyst-free or cancer-free controls [67]. The rareness of CTC makes their detection and isolation challenging, and numerous strategies have been proposed for their identification. Positive selection is based on the expression of cell-surface markers, for example, the US FDA approved CellSearch system and negative selection technologies isolate CTC based on cell physicochemical properties such as size, density or surface charges such as the isolation by size of epithelial tumour cells (ISET) system. One reported study detected CTC in 11/24 patients with stage IV disease using the CellSearch system [68] and a comparative study of these two techniques in metastatic or inoperable pancreatic cancer patients detected CTC in 40% of patients using the CellSearch system as compared to 93% by ISET. On the whole, more CTC were detected by ISET than by CellSearch, mean 26 versus 2 CTC/7.5 ml of blood (range 0–240 vs 0–15) [69]. The low detection rate of CTC by the CellSearch system has been corroborated by an additional study of patients with 12 different types of metastatic cancers where pancreatic cancer patients had one of the lowest CTC levels among all the included cancer types (4/6 patients had
Review
Biomarkers for early diagnosis of pancreatic cancer Expert Rev. Gastroenterol. Hepatol. Early online, 1–11 (2014)
Claire Jenkinson1, Julie Earl2, Paula Ghaneh1, Christopher Halloran1, Alfredo Carrato2, William Greenhalf1, John Neoptolemos1 and Eithne Costello*1 1 Department of Molecular and Clinical Cancer Medicine, National Institute for Health Research Liverpool Pancreas Biomedical Research Unit, University of Liverpool, Daulby Street, Liverpool L69 3GA, UK 2 Department of Medical Oncology, Ramo´n y Cajal University Hospital, Carretera Colmenar Km 9,100, 28034 Madrid, Spain *Author for correspondence: Tel.: +44 151 706 4178 Fax: +44 151 706 5826 [email protected]
informahealthcare.com
Pancreatic ductal adenocarcinoma is an aggressive malignancy with a 5-year survival rate of approximately 5%.The lack of established strategies for early detection contributes to this poor prognosis. Although several novel candidate biomarkers have been proposed for earlier diagnosis, none have been adopted into routine clinical use. In this review, the authors examine the challenges associated with finding new pancreatic cancer diagnostic biomarkers and explore why translation of biomarker research for patient benefit has thus far failed. The authors also review recent progress and highlight advances in the understanding of the biology of pancreatic cancer that may lead to improvements in biomarker detection and implementation. KEYWORDS: biomarker • diagnosis • jaundice • pancreatic cancer
Discussion
With an estimated 250,000 new cases diagnosed every year, pancreatic ductal adenocarcinoma (PDAC) remains the fifth leading cause of cancer death worldwide [1]. Unfortunately, unlike many other cancer types, there has been little improvement in survival rates for pancreatic cancer over the past four decades [2] and it has been projected to become the second leading cause of cancer-related death by 2030 [3]. Failure to detect the disease before it is advanced is a major problem and is mainly due to the fact that early warning signs are vague, while symptoms such as obstructive jaundice, weight loss or pain tend to become more apparent at later stages of the disease. Consequently, around 80% of patients are diagnosed with locally advanced or metastatic disease [1] ruling out potentially curative resection. Survival times for patients with advanced disease are generally low [4,5]. However, for patients who have undergone surgery to remove their tumour followed by chemotherapy, the 5-year survival is significantly better, at approximately 25% [6–8]. Currently, the only biomarker used in the routine management of pancreatic cancer is CA19-9, an epitope of sialylated Lewis blood group antigen [9,10]. A recent meta-analysis [11],
10.1586/17474124.2015.965145
which pooled summary estimates of 11 studies, found that CA19-9 had both a sensitivity and specificity of 80% for pancreatic cancer diagnosis. CA19-9 is upregulated in pancreatic inflammation and benign pancreatic disease [9] which hampers its specificity for pancreatic cancer. Moreover, approximately 5% of the population are Lewis ab negative and therefore do not secrete CA19-9 [12]. Thus, while CA19-9 is successfully used for predicting clinical course, improvements in sensitivity and specificity for reliable early PDAC detection are desirable [13]. Much effort has focused therefore on enhancing the performance of CA19-9 by including it within larger panels of markers [14–17]. How early should pancreatic cancer be detected to allow significant improvement to outcome?
If patients with pancreatic cancer are divided into those with resectable, locally advanced and metastatic disease, early detection should, at the very least, enhance the detection of patients in the resectable category. Biomarkers that would increase the proportion of patients who are eligible for potentially curative surgery should lead to an improvement in prognosis. Nonetheless, median survival after surgery for patients treated with chemotherapy such as
2014 Informa UK Ltd
ISSN 1747-4124
1
Expert Review of Gastroenterology & Hepatology Downloaded from informahealthcare.com by Washington University Library on 01/15/15 For personal use only.
Review
Jenkinson, Earl, Ghaneh et al.
fluorouracil plus folinic acid or single-agent gemcitabine remains just below 2 years [8], indicating that possibly even earlier intervention is required to radically alter survival times. There is evidence that pancreatic cancer is a systemic disease from its early stages. A study using genetically engineered mouse models of pancreatic cancer showed that pancreatic epithelial cells from lesions considered to be preneoplastic had spread into the stroma of the pancreas and had invaded the bloodstream [18]. The suggestion that this disease spreads from a very early stage has implications for early detection (see below) and for subsequent early intervention, which may need to concentrate on more rapid application of systemic treatments [19,20]. Scope of this review
Technological advances have opened up opportunities for diagnostic biomarker discovery in a number of areas. Advances in proteomic technologies, in both the range of mass spectrometry-based approaches and its increased sensitivity for detection of low abundance proteins, have made mass spectrometry a powerful tool for biomarker protein discovery. Advances in imaging and cell tracking are also gaining pace, while miRNAs, circulating cancer cells and circulating tumor DNA are areas with vast potential for facilitating cancer detection. For the purpose of this review we have largely subgrouped biomarkers based on their properties, rather than the sample type from which they originate. Biomarkers specifically relating to pancreatic cystic neoplasms, including intraductal papillary mucinous neoplasms (IPMNs), are not reviewed here. Protein markers Serum protein biomarkers
As a source of biomarkers, serum is minimally invasive to obtain and is a sample type that clinical laboratories are used to performing blood tests on. However, the large dynamic range of proteins in serum complicates biomarker discovery protocols [21]. Nonetheless, much effort has focused on developing blood-borne biomarkers for pancreatic cancer diagnosis. Given the immense genetic, environmental and cancerassociated variation across individuals, single biomarkers are unlikely to offer accurate diagnosis for everyone. The emphasis has therefore shifted toward developing panels of biomarkers to complement or surpass CA19-9 alone [15–17,22]. Using Luminex bead-based technology, Brand et al. [15] performed one of the most comprehensive pancreatic cancer biomarker studies to date, analyzing 83 proteins in 333 PDAC patients, 144 patients with benign pancreatic conditions and 227 healthy individuals. Samples were split into training and validation sets. A panel of CA19-9, ICAM-1 and OPG discriminated PDAC patients from healthy individuals with a sensitivity/specificity of 88/90% in a training set and 78/94% in an independent validation set. A different panel comprising CA19-9, CEA and TIMP-1 discriminated PDAC patients from benign subjects with a sensitivity/specificity of 76/90% in a training set and 71/89% in validation. doi: 10.1586/17474124.2015.965145
Alternative panels have also been proposed that can identify pancreatic cancer. Park et al. [22] measured serum levels of cathepsin D along with a number of tissue inhibitors of matrix metalloproteinases and matrix metalloproteinases in relatively large training and validation sets of pancreatic cancer patients (248 total) and control subjects (114 healthy controls, 102 patients with chronic pancreatitis). A panel comprising CA19-9, cathepsin D and MMP-7 improved the discrimination of cancer from controls over that of CA19-9 alone. A study by Faca et al. [17] highlighted the potential of including relevant mouse models to assist biomarker discovery. Analysis of the plasma proteome of genetically engineered mice with early versus advanced pancreatic cancer led to the identification of a panel of seven proteins (ALCAM, ICAM-1, LCN2, TIMP-1, REG1A, REG3 and IGFBP4) that differentiated newly diagnosed PDAC cases from chronic pancreatitis patients with an area under the receiver operating characteristic (ROC) curve (AUC), compared to CA19-9 alone which gave an AUC of 0.79. This study was also one of the first to use prediagnosis samples as part of the validation protocol for markers. Five of the proteins identified (LCN2, REG1A, REG3, TIMP-1 and IGFBP4) when combined with CA19-9 discriminated pancreatic cancer cases from matched controls in blood specimens obtained between 7 and 13 months prior to the clinical diagnosis of pancreatic cancer with an AUC of 0.911. Detection of stage I disease with high sensitivity was recently reported using a monoclonal antibody called PAM4 which binds to the mucin MUC5AC [23], and has specificity for PDAC and its precursor lesions but not for normal or benign tissues [24]. Gold et al. [14] evaluated a PAM4-based serum immunoassay for pancreatic cancer detection. Using sera from 298 PDAC patients, 99 patients with other cancers, 120 patients with benign disease of the pancreas and 79 healthy adults, PAM4 demonstrated a sensitivity of 64% for PDAC patients with stage I disease. The sensitivity was higher (85%) for PDAC patients with advanced cancer, and combining PAM4 with CA19-9 demonstrated an improved overall sensitivity for PDAC of 84% compared to 77% for CA19-9 alone. Interestingly, PAM4 offered good discrimination between PDAC and chronic pancreatitis, a characteristic that is lacking from many reported biomarkers of PDAC. Makawita and colleagues [25] also demonstrated a panel of biomarkers to be superior to CA19-9 for the detection of stage I–II pancreatic cancer. This study evaluated four biomarkers, namely, regenerating islet-derived 1b (REG1B), syncollin (SYCN), anterior gradient homolog 2 protein and lysyl oxidase-like 2 in two sample sets. A panel consisting of SYCN + REG1B + CA19-9 performed better than CA19-9 for the detection of early-stage (stage I–II) PDAC versus diseasefree controls. Protein biomarkers in tissue & subcellular fractions
Due to the invasiveness of procedures to obtain cancer tissue, this material is not commonly used as a screening tool. Blood is a more accessible and therefore optimal sample for biomarker Expert Rev. Gastroenterol. Hepatol.
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Biomarkers for early diagnosis of pancreatic cancer
testing. Nonetheless, pancreatic cancer tissue contains the highest concentrations of PDAC-specific markers, and as such, is the most direct source of proteomic cancer biomarkers [26]. The potential to identify cancer-specific biomarkers from the tumor make it an invaluable resource. Crnogorac-Jurcevic et al. [27] performed a large-scale immunoblotting analysis with more than 900 primary antibodies on cancer tissue, chronic pancreatitis and normal pancreas. Several proteins, such as UHRF1, ATP7A and AOX1, differed in their expression between chronic pancreatitis and pancreatic cancer, suggesting potentially important roles in pancreatic carcinogenesis. Chen et al. [28] measured the levels of complement C3, complement C4b1 and apolipoprotein E in tissue from pancreatic cancer patients of varying stages by immunohistochemistry, reverse transcription PCR (RT-PCR) and western blotting. The levels of all three were higher in the pancreatic cancer tissue compared to the normal tissue. They showed that complement C3 correlated with earlystage disease, while they suggested that complement C4b1 and apolipoprotein E could potentially be used as diagnostic markers of advanced PDAC due to their marked correlation with later stages and lymph node metastasis. Advances in proteomic techniques have enabled the combination of subcellular fractionation and mass spectrometry for the identification of potential biomarkers associated with pancreatic cancer within tissues. McKinney et al. [29] utilized matched tumor and nontumor sample pairs from patients undergoing tumor resection and then separated the tissues to obtain cellular protein fractions corresponding to cytosol, membrane, nucleus and cytoskeleton. With this approach, 1771 proteins in normal and 1985 proteins in pancreatic tissue with cancer were identified, and 104 proteins were found to be significantly changed in pancreatic cancer. Four secreted proteins, namely, biglycan, pigment epithelium-derived factor, thrombospondin-2 and TGF-binduced protein Ig-h3 precursor were validated as expressed in pancreatic cancer tissue. This study demonstrated the potential of subcellular fractionation as a powerful strategy for identification of differentially expressed proteins in pancreatic cancer. Protein markers in pancreatic juice
The advantage of body fluids such as urine is that they can be obtained without the requirement of an invasive procedure; however, urine is separated from the tumor by the circulation and kidneys. Pancreatic juice, by contrast, is in direct contact with the cancer and is excreted out of the body. Traditional serum biomarkers such as CEA and CA19-9 can be found in pancreatic juice [30] but with little or no diagnostic advantage over serum for pancreatic cancer patients, especially given the increased costs and risks of obtaining juice samples. Empirical approaches to proteomic analysis have identified more promising proteins [31], but as yet this promise has not borne fruit and none of these juice biomarkers have found their way to the clinic. DNA markers in pancreatic juice
Detection of specific cancer-related genetic or epigenetic changes in pancreatic juice samples has proved more productive. Notably informahealthcare.com
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detection of K-ras mutations in pancreatic juice has proven to be reasonably sensitive for the detection of pancreatic cancer [32], but it was found to give very poor specificity, especially when comparing chronic pancreatitis and pancreatic cancer. Other markers, notably mutations in p53, gave considerably better specificity [33], but with a reduction in sensitivity. Assay of methylation in various promoters has been found to be an effective alternative to mutation detection, the most effective being detection of methylation of the CDKN2a (p16) promoter [34]. The specificity and sensitivity can be improved further by combining mutation and methylation analysis [35], but this remains far from perfect and pancreatic juice sampling using Endoscopic Retrograde Cholangiopancreatography can result in induction of pancreatitis; Endoscopic Retrograde Cholangiopancreatography-induced pancreatitis can prove fatal. This risk can be reduced by prophylaxis using stents and nonsteroidal anti-inflammatory agents such as diclofenac [36]. The risk may be further reduced or eliminated by obtaining juice from the duodenum without cannulating the pancreatic duct. Promising results have been obtained from molecular analysis of samples obtained in this way [37,38]. miRNA markers
miRNAs are small RNAs (22–25 nt) that negatively regulate gene expression by binding to complementary mRNA resulting in gene silencing, translational repression or target degradation. The deregulation of some miRNAs has been identified as a mechanism responsible for cell transformation including pancreatic cancer development [39] miRNAs with diagnostic potential for pancreatic cancer are summarized in TABLE 1. Overexpression of miR-21 has been reported in PDAC and precursor pancreatic intraepithelial neoplasia (PanIN) lesions, and has been correlated with poor overall survival and chemoresistance to gemcitabine [40–42]. Moreover, miR-21 is known to be upregulated in several tumor types as it negatively regulates the tumor suppressor genes PTEN and PDCD4 [43]. miRNAs can be secreted or interchanged among cells in the form of microparticles [44] and thus can be detected in bodily fluids including serum. It has been estimated that healthy individuals have between 5 and 50 mg/ml of microparticles that increase under different disease conditions [45]. Thus, they are valuable minimally invasive diagnostic markers and have been exploited as cancer serum/plasma biomarkers in many tumor types [43]. miR-18a, miR-200, miR-210 and miR-375 have been reported as potential minimally invasive blood-based biomarkers for pancreatic cancer [46–49] although on an individual level they do not outperform CA19-9 in terms of sensitivity and specificity for the detection of PDAC. Li et al. [48] quantified serum levels of miR-200a/200b in the serum of 45 patients with PDAC, 11 with CP and 32 healthy controls. Elevated levels of miR-200a provided a sensitivity and specificity of 84.4 and 87.5%, respectively, for PDAC, while elevated levels of miR-200b delivered a sensitivity and specificity of 71.1 and 96.9%, respectively. Neither marker differentiated PDAC from CP. Despite this shortcoming, further studies are merited to verify the performance of miR-200 as a biomarker in larger doi: 10.1586/17474124.2015.965145
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Jenkinson, Earl, Ghaneh et al.
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Table 1. miRNAs reported as having diagnostic potential in pancreatic cancer. miRNA
Tissue
miR-21
PDAC Gemcitabine resistance Precursor lesions – PanINs
[40,41]
PanINs IPMN
[42,39]
miR-155
Ref. [40] [42,39]
[50] [42,52,53] [51]
miR-196
PDAC IPMN
miR-375
Plasma IPMN
[46]
miR-18a
Plasma
[49]
miR-210
PDAC IPMN Plasma
[108] [51]
PanINs IPMN Serum
[42] [51]
miR-200
[51]
[47]
[48]
miRNAs reported as having diagnostic potential in pancreatic cancer. PanIN: Pancreatic intraepithelial neoplasia; PDAC: Pancreatic ductal adenocarcinoma.
patient populations. Morimura et al. [49] found that miR-18a was raised in plasma of PDAC patients. Although this miRNA did not outperform CA19-9, levels of both miR-18a and CA19-9 dropped after tumor resection, suggesting that monitoring the miR-18a levels in the serum of patients in remission could support the detection of cancer recurrence. One problem with miRNA profiling in tissue or bodily fluids is that many miRNAs which have been found to be selectively expressed in advanced cancer have not been validated in earlier cancers or preneoplastic lesions. By contrast, miR-155, miR-196 and miR-210 have been reported to be associated with PDAC and PanIN lesions as well as IPMN [39,42,47,50–53]. Detecting changes in PanIN lesions is of great interest, since aberrant expression of miRNAs in these lesions may represent an early modification in pancreatic tumorigenesis. Ryu et al. [39] used quantitative RT-PCR to determine the levels of miR-21, miR-155 and miR-221 in 31 microdissected PanINs. They observed that miR-155 was significantly overexpressed in PanIN-2 and PanIN-3 lesions (2.6-times; p = 0.02 and 7.4-fold; p = 0.049, respectively). miR-21 was overexpressed only in PanIN-3 (2.5-fold; p = 0.02), while no change in expression was found for miR-221 in PanIN lesions compared with normal duct epithelium. du Rieu et al. [54] also explored the expression of miRNAs in PanIN lesions. They reported that miRNAs miR-21, miR-205 and miR-200 were elevated in PanIN lesions in a conditional KRAS (G12D) mouse model, while miR-222 production decreased. The levels of three miRNAs, miR-29c, miR-221 and let-7a, remained unchanged between normal ducts and PanIN lesions. doi: 10.1586/17474124.2015.965145
In a search for diagnostic miRNA candidates, Baraniskin et al. discovered that the discriminatory power of one promising candidate, miR-1246, actually came from U2 small nuclear RNA fragments. The group went on to show that U2 small nuclear RNA fragments are highly stable in serum and plasma and hold the potential as a novel diagnostic biomarker for PDAC.
[55]
Cytokines & chemokines
Panels of cytokines, chemokines and growth factors are also potential important diagnostic markers of pancreatic cancer. They are released by various components of the immune system and contribute to neoplastic transformation [56]. The expression of VEGF and bFGF was elevated, alongside an increase in CA19-9 levels, in the serum of patients with pancreatic cancer (stage I–IV; p < 0.0001) compared to healthy subjects [57]. Using LabMAPTM technology with classification trees, Zeh et al. [58] identified a multicytokine panel which, when used in combination with CA19-9, differentiated PDAC patients from control groups. Shaw et al. [59] measured serum levels of 27 cytokines in 241 subjects, including 127 with PDAC, 49 with chronic pancreatitis, 20 with benign biliary obstruction and 45 healthy controls. Having split samples into independent training and test sets, cytokine biomarker panels were identified using best fit logistic regression models. A panel of IP-10, IL-6, PDGF plus CA19-9 offered improved diagnostic performance over CA19-9 for distinguishing PDAC patients from patients with benign disease. These findings support a potential role for cytokine panels in the discrimination of PDAC from patients with benign pancreatic diseases, with validation in larger sample sets merited. Autoantibodies
Recently, several studies have shown that cancer patients produce detectable autoantibodies, that is, antibodies generated against certain tumor-associated antigens, which may be useful as bloodborne biomarkers for early detection of cancer [56]. In one of the largest studies into autoantibody detection in PDAC [60], autoantibodies to CTDSP1 (carboxy-terminal domain, RNA polymerase II, polypeptide A, small phosphatase 1), MAPK9 (mitogenactivated protein kinase 9) and NR2E3 (nuclear receptor subfamily 2, group E, member 3) were analyzed in 300 pancreatic cancer patients and 300 controls. A significantly elevated antibody response was observed in disease cases for CTDSP1 (p = 0.004), MAPK9 (p = 0.0002) and NR2E3 (p = 0.0001) compared with the controls; however, discrimination between cancer cases and controls were relatively poor for each marker with AUC values of 0.62, 0.59 and 0.56 for NR2E3, MAPK9 and CTDPS1, respectively [60]. Novelli and colleagues identified autoantibodies in the sera of pancreatic cancer patients, with reactivity to two acidic isoforms of the glycolytic enzyme enolase (1/2), which had the power to discriminate PDAC patients from controls with 62% sensitivity and 97% specificity [61]. More recently, this group reported a high frequency of autoantibodies to Ezrin in a Expert Rev. Gastroenterol. Hepatol.
Biomarkers for early diagnosis of pancreatic cancer
genetically engineered mouse model of spontaneous PDAC [62]. The finding translated to PDAC patients, with 56% of PDAC patient sera (n = 120) displaying reactivity against Ezrin compared to only 12% of non-PDAC sera (n = 50) and 2% of sera from patients with chronic pancreatitis (n = 46).
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Circulating tumor cells
The detection of tumor cells in peripheral blood, the so-called circulating tumor cells (CTC), has offered the promise of using them as biomarkers for disease detection and management. Their presence in peripheral blood has been associated with lower progression-free and overall survival in several cancer types, and they have been proposed as early indicators of tumor spread as invasive, but localized tumors may shed CTC into the bloodstream before a metastasis is established [63]. CTC can enter the blood circulation by tumor cell shedding from the primary tumor, a process that may occur in the early stages of tumor formation [64,65]. Alternatively, tumor cells may enter the circulation through a process of epithelial- to-mesenchymal transition whereby they acquire mesenchymal features including motility and invasiveness [66]. Pancreatic epithelial cells were detected in peripheral blood of a PDAC mouse model that harbored early lesions, but prior to tumor formation [18], implying ample opportunity for early detection of CTC. Of note, the cells detected in this mouse model had undergone epithelial-mesenchymal transition. This fact has significant ramifications for strategies designed to monitor CTC for diagnosis or treatment, since CTC with a mesenchymal phenotype may evade detection that relies on the expression of epithelial markers such as EpCAM. Rhim et al. recently confirmed the detection of pancreas epithelial cells (CECs) in patient blood samples. Using microfluidic geometrically enhanced immunocapture, CECs were detected in 7 of 21 (33%) patients with cystic lesions and no clinical diagnosis of cancer, 8 of 11 (73%) with PDAC and in 0 of 19 cyst-free or cancer-free controls [67]. The rareness of CTC makes their detection and isolation challenging, and numerous strategies have been proposed for their identification. Positive selection is based on the expression of cell-surface markers, for example, the US FDA approved CellSearch system and negative selection technologies isolate CTC based on cell physicochemical properties such as size, density or surface charges such as the isolation by size of epithelial tumour cells (ISET) system. One reported study detected CTC in 11/24 patients with stage IV disease using the CellSearch system [68] and a comparative study of these two techniques in metastatic or inoperable pancreatic cancer patients detected CTC in 40% of patients using the CellSearch system as compared to 93% by ISET. On the whole, more CTC were detected by ISET than by CellSearch, mean 26 versus 2 CTC/7.5 ml of blood (range 0–240 vs 0–15) [69]. The low detection rate of CTC by the CellSearch system has been corroborated by an additional study of patients with 12 different types of metastatic cancers where pancreatic cancer patients had one of the lowest CTC levels among all the included cancer types (4/6 patients had
