In the present study, we identify several 3-biomarker panels offering high SN/SP and significant improvement over CA 19-9 alone for the discrimination of PDAC from healthy controls and benign subjects. To the best of our knowledge, these results represent the most advanced validated biomarker discovery effort aimed at the development of improved screening methodologies for the detection of pancreatic cancer. Since the incidence of pancreatic cancer in the general population (~1% lifetime risk) is too low to warrant screening, strategies are being investigated to define patient cohorts in which the positive predictive value for early-stage pancreatic cancers and advanced precursor lesions is high enough to justify more costly and invasive testing 24
. Therefore, our study intended to develop a panel of biomarkers that can be used to screen populations at an increased risk for the development of pancreatic cancer.
At present, high risk populations are not readily available for the purpose of pancreatic cancer screening. Pancreatic cancer-prone families are the most widely accepted population of this type to date, but only account for 5 to 10% of all pancreatic adenocarcinomas. Furthermore, the genetic factors underlying this predisposition are uncharacterized in the majority of these families. As it would take many years to follow a cohort of these patients to determine which unaffected individuals in these familial pancreatic cancer kindreds would develop PDAC, it is not practical to use this population for biomarker discovery. However, many centers are actively investigating risk stratification models that will take into account genetic factors along with epidemiological information, such as smoking and alcohol use, or clinical history including diabetic status or history of gastric ulcer. This should provide appropriate populations with a high enough incidence of pancreatic adenocarcinoma to warrant surveillance.
The multimarker panels identified in our analysis were highly discriminatory for PDAC vs. Healthy subjects demonstrating sensitivities ranging from 77–88% with 90% SP and a lower discriminatory power of 70–76% with 90% SP for PDAC vs. Benign. The optimal panel identified in the comparison of PDAC vs. Healthy comprised of CA 19-9, ICAM-1 and OPG demonstrated significant improvement over CA 19-9 by ROC analysis, whereas the best panel for discrimination of Benign from PDAC did not offer a substantial improvement in terms of SN over CA 19-9 at a level of SP above 80% (). The AUC values we observed for CA 19-9 were within the reported range (0.72–0.84) of several recent studies25–26
. The panel of CA 19-9, ICAM-1 and OPG also demonstrated a high level of cancer selectivity when applied to colon, lung, and breast cancers. The relative performance of these biomarker panels coupled with the small number of analytes required to achieve that performance make them attractive candidates in the development of early detection strategies.
In the course of our evaluation, we have identified number of proteins that differ significantly in the sera of patients diagnosed with PDAC and healthy controls, and many of these were also significant in comparison to the Benign group with considerable reproducibility in biomarker trends across the multiple sampling sites. To the best of our knowledge, 19 of the observed associations between serum biomarker levels and PDAC have not been described previously: GH, PRL, PTH, sFas, sFasL, MMP-2, TIMP 2-4, MPO, EGFR, ApoAI-II, ApoCIII, OC, GLP-1, HE4, and TGII. Although recent reports have described significant associations between serum levels of OPG 27
and pancreatic cancer, to the best of our knowledge, our report is the first to characterize its diagnostic capacity. The significant biomarkers include representatives from a diverse set of biological families, particularly proteins with functions in such critical aspects of tumor development as growth, angiogenesis, metastasis, inflammation, etc., and encompass an array of factors likely to originate from the developing tumor, the tumor microenvironment and components of the systemic host response to the malignancy.
In the current study, PDAC was associated with circulating alterations of a number of known mediators of inflammatory processes and acute phase reactants. The relationship between tumorigenesis and inflammation has become a central theme in anticancer research and is the focus of increasing interest within the setting of pancreatic cancer. Malignant transformation can be closely associated with chronic infection and inflammation, while elevated levels of pro-inflammatory proteins could play a role as tumor promoters 28
. It has also been clear for some time now that a number of pro-inflammatory gene products including acute-phase reactants can also be produced by components or tumor microenvironment and tumor cells themselves to further mediate tumor growth 29
. For example, elevated circulating levels of SAA could reflect not only hepatic synthesis as part of the acute-phase response, but also increased release of these proteins by cancer cells, and a possible role for SAA in tumor growth, metastasis, and neovascularization has been investigated 30–31
. Therefore, these pro-inflammatory proteins could potentially be utilized as cancer biomarkers. In fact, it has been demonstrated that serum SAA levels are elevated in a broad spectrum of neoplastic diseases 5, 32
suggesting that it could act at least as a non-specific tumor marker. The combination of CRP, another acute-phase reactant, and sIFNα/βR was demonstrated to diagnose gastrointestinal and hepatobiliary-pancreatic cancer with a SN/SP of 94.6/88 33
. The production of pro-inflammatory cytokines by tumor and stromal cells, regulated by NF-kB, has been observed to mediate tumorigenic effects in the pancreas as part of a characteristic desmosplastic reaction 34
. We observed altered levels of the NF-kB responsive cytokines TNFα and IL-8 which have been specifically shown to inhibit apoptosis and increase invasiveness of pancreatic cancer cells, respectively 35–36
. Also notably altered was the ROS-mediating enzyme, MPO. MPO has demonstrated involvement in the generation of DNA strand breaks, sister chromatid exchanges, mutations, and the formation of DNA adducts 37
, and is associated with smoking, a known risk factor of pancreatic cancer 38
Our findings also support several hypotheses regarding the role of specialized pathways related to obesity, bone homeostasis, and tissue remodeling, in pancreatic cancer. These findings include alterations in number of apolipoproteins and adipokines, modulators of lipid and insulin metabolism. ApoA1 may represent an emerging biomarker of malignancy as circulating levels of ApoA1 have recently been linked to several cancer types including pancreatic 39
. The adipokines, Adiponectin and Leptin are currently under intense scrutiny stemming from links to obesity, inflammation and malignancy 40
. We observed altered serum levels of OPN, a biomarker previously associated with pancreatic cancer 11
, and several other bone related factors, OC and OPG. OPG is a secreted member of the tumor necrosis factor receptor superfamily, and altered serum levels of OPG have been associated with several malignancies including colorectal cancer, pancreatic cancer, liver metastases, multiple myeloma, Hodgkin’s disease, and non-Hodgkin’s lymphoma 27
. We report dysregulation of several metastasis-related proteins in PDAC. The MMPs and their natural inhibitors, TIMPs, have well-described tumorigenic roles and our observations regarding MMP-2 and TIMPs 1-4 in pancreatic cancer patients are consistent with previous findings 41
. The cellular adhesion mediator ICAM-1 has been previously linked to the development of PDAC 42
and was utilized as a part of multimarker panel for the classification of PDAC from healthy controls 19
. The expression of ICAM-1 is associated with PDAC cell sensitivity to T-cell-based immunotherapy in vitro 43
, and prognostic significance of preoperative sICAM-1 levels in several cancers has been demonstrated 44–46
. Correlation of TGII with metastatic properties of several cancers has been suggested 47–48
, and its role as a tumor marker in the development and progression of pancreatic cancer has been well documented 49–50
. We also observed the dysregulation of a number of growth factors, receptors and mediators of angiogenesis including EGFR, ErbB2, Angiostatin, Endostatin, Thrombospondin, and IGFBP-1.
In summary, an extensive analysis of circulating biomarkers in patients diagnosed with PDAC and benign pancreatic conditions resulted in a robust profile of alterations in biomarkers in PDAC patients, offering improved insights into the network of factors involved in the process of pancreatic tumorigenesis. A number of the alterations we identify are novel with regard to associations with PDAC as is the expanded use of several previously characterized serum biomarkers for diagnostic purposes. Although the ideal biomarker test would recognize premalignant conditions, to the best of our knowledge, such requisite retrospective samples are presently not available for biomarker discovery. Therefore, the current study was guided by the assumption that a subset of those biomarkers demonstrating differential expression in developed cancer would also be altered in premalignant conditions. Thus, our analysis of biomarker alterations present in serum at the time of diagnosis should pave the way for the subsequent identification of biomarkers of preneoplastic disease. Additionally, biomarker panels discriminating PDAC from benign disease may offer a high level of clinical utility in combination with conventional imaging modalities to provide a diagnostic role as opposed to the aforementioned screening application. This study presents proof-of-principle of the utility of a multiplexed evaluation of circulating biomarkers for the identification and validation of multimarker panels with high classification power for PDAC. Further studies employing this type of approach may result in the identification of more robust panels for both screening and diagnosis of PDAC.
These findings represent an evaluation of biomarkers potentially related to pancreatic cancer and do not promote the use of any individual biomarker considered herein for diagnostic purposes. Moreover, while the nature of our investigation does not permit the identification of specific mechanistic links between any particular biomarker and the development of pancreatic cancer, our results do provide a sound basis for subsequent targeted analyses of pancreatic cancer biomarkers.
Statement of Translational Relevance
Specific challenges associated with pancreatic cancer including ubiquitous symptomatic presentation, deep anatomical location, and aggressive etiology, have greatly hindered efforts to combat the disease. Although the low incidence of the disease in the general population renders population-based screening impractical, screening could greatly improve survival in appropriately targeted high-risk populations. The current absence of reliable biomarker testing for pancreatic cancer mandates the development of novel strategies for identifying and characterizing additional biomarkers. The evaluation of serum biomarker levels in patients diagnosed with pancreatic cancer and a spectrum of benign pancreatic conditions presented here provides compelling evidence for the emerging role of blood-based screening in the clinical management of this disease. These findings not only include the identification of multimarker panels capable of discriminating pancreatic cancer from benign conditions and healthy controls with high sensitivity and specificity, but also offer improved insight into the complex network of factors involved in pancreatic tumorigenesis.