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Encyclopedia of Cancer, Springer-Verlag Berlin Heidelberg (2015)
Pancreatic cancer is the ninth leading cause of cancer diagnosis and fourth leading cause of cancer- associated death in the world. Pancreatic cancer biomarkers refer to cellular or biochemical mechanisms to aid in the detection, diagnosis, or prognostication of pancreatic cancer. Primarily, this term insinuates pancreatic exocrine tumors, speciﬁcally adenocarcinoma, but can be universally applied to all cancers arising within the pancreas. Additionally, the source and nature of pancreatic cancer biomarkers is not inherently deﬁned and thus can denote any method to aid in its clinical detection or projection including but not limited to analysis of the proper tumor, tumor-surrounding tissues, implicated body secretions, and distant body ﬂuids.
The term pancreatic cancer (PC) biomarkers can be contextually applied to many entities for multiple functions. In general terms, it refers to any biochemical detection mechanism that can be applied in PC to offer clinical improvement. Considering physiologic aspects and clinical points of failure in highly lethal PC, this general term can have three distinct applications: improvement of diagnostic ability, providing prognostic information including likely patient time of survival and presence of therapeutic resistance mechanisms, and following of disease progression and treatment response.
Clinically, each of these applications has meaningful clinical utility. Improved prognostic abilities, especially in the context of therapeutic efﬁcacy, promise to aid physicians and patients in treatment planning, potentially altering treatment course for optimal patient outcome. Following of disease progression through biomarkers allows for rapid indication of treatment response, a therapeutic switch should progression be found during a treatment course, and a transition to palliative care at the earliest indicated time point, thereby preserving patient quality of life to the fullest extent possible. Currently, biomarker monitoring of PC patient progression is the only biomarker utility currently available clinically. This monitoring involves the analysis of plasma-based CA19-9, speciﬁcally the sialylated Lewis (a) carbohydrate antigen present on various mucin proteins, though this current method is plagued with non-speciﬁcity along with the fact that up to 15 % of people lack to ability to produce sialylated Lewis (a) altogether.
While each of these biomarker applications are doubtlessly important, the discovery of novel biomarkers to aid in PC diagnosis, especially early diagnosis, has the potential for the greatest clinical impact on patient overall survival and thus has been the subject of the majority of PC biomarker research. To date, the only treatment modality with proven ability to signiﬁcantly and substantially alter PC patient outcome is resection, providing a 5-year survival rate of 24.6 % and a median survival of 19.3 months as compared to 2.9 % and 8.4 months in similar-stage non-resected patients. This point is further illustrated by studies indicating 5-year survival rates of 10–20 %, 30–60 %, and >75 % in patients with resected pancreatic tumors 3 cm, >2 cm, and >1 cm in diameter, respectively. Unfortunately, few PC patients are diagnosed with resectable disease. Of the estimated 43,920 patients who will be diagnosed with PC in the United States in 2012, 80–85 % will not be surgical candidates at the time of disease discovery. Of these, approximately 25–30 % are diagnosed with borderline resectable disease, for whom chemoradiotherapy is utilized in an attempt to downstage the tumor to resectability but is met with only a 8–30 % rate of success. Hence, for 70–75 % of PC patients, their diagnosis is an unequivocal death sentence owing primarily to the lateness of disease discovery, a statistic that has remained virtually unchanged for the past 25 years. The principal cause attributing to the late discovery of PC is a general lack of speciﬁc or clinically worrisome symptoms until late in the disease course, mostly due to the anatomic and physiologic nature of the disease. Further, when PC does elicit symptoms, they are often general and thus lead neither the patient nor physician to suspect PC at the top of the differential diagnosis. As such PC can, for the majority of patients, be considered a symptom-free disease, resulting in a situation in which early diagnosis must come in the form of ﬁnding something that neither patient nor clinician knows they should be looking for. In such situations, the only method for disease detection is via screening mechanisms, such as are employed for prostate cancer (PSA), breast cancer (mammogram), and colorectal cancer (colonoscopy), putting stringent stipulations upon what such a test can entail for it to be clinically acceptable as discussed below. Despite decades of research into this ﬁeld, however, no diagnostic marker has yet proven clinically viable for PC.
Additionally, due to the anatomic location and physiologic functions of the pancreas, biomarker source can also be highly variable. Such sources include tumor and peri-tumoral tissue, pancreatic juice, peripheral plasma/serum, circulating cells, and other less directly implicated body ﬂuids such as urine and bile. With each source comes a differing set of positive and negative properties and theoretical ties to the PC disease state, thus regulating speciﬁc targets and global approaches for study to discover utile PC biomarkers (Table 1).
Of the various areas from which to test for PC biomarkers, the most direct is in the tissue itself. Many studies have been conducted in the attempt to discern normal from neoplastic pancreata, being met with varying degrees of success. Two methods exist by which to obtain and analyze such tissue, each with its own advantages and disadvantages.
Core biopsy-based analysis
Analysis of core biopsy specimens is one of the oldest methods for cancer detection and conﬁrmation. Objectively, this method brings with it several distinct advantages not present with any other method, including obtainment of high-cellularity samples allowing for morphological and histological evaluation across multiple criteria, inclusion of both tumoral and peri-tumoral tissue as well as the junction between the two, and the presence of live cells in their respective surrounding microenvironment(s). Such properties offer an unparalleled look at the tissue of interest and provide the ability to directly link the presence of one or more cellular or morphotypic features to the existence of neoplastic disease and/or that disease’s therapeutic and prognostic phenotype.
This method of pancreatic evaluation, however, does not come without multiple detracting attributes as well. Primarily, due to its depth and location within the abdomen, obtainment of core biopsies from the pancreas is no small order. As such, many medical centers have opted to forgo prooperative biopsies for PC altogether in favor of aspiration-based evaluation methods. Further, the size of the pancreas and invasiveness of the procedure preclude performing core biopsies without the presence of a previously identiﬁed pancreatic mass/lesion, thus preventing its application as a screening mechanism. Consequently, core biopsy-based PC biomarkers are segregated solely to functions of disease conﬁrmation with further potential for prognostic utility.
Table 1. Theoretical properties of PC biomarker sources
|Biomarker source||Minimally invasive||High sensitivitya||High speciﬁcityb||Screening applicability||Prognostic applicability||Early diagnostic ability||Notes|
|Core biopsy||X X X X||X X X X||Only utile once a pancreatic lesion is discovered through other methods|
|Aspirate||X||X||X X X||X X X X||X||Risk of iatrogenic acute pancreatitis is low but not insigniﬁcant|
|Pancreatic juice||X X||X||X X||X X||X X X||X||Lack of ductal cannulation reduces both invasiveness and speciﬁcity|
|Serum/ plasma||X X X||X X X||X X X X||X X||X X||Speciﬁcity must come from choice of ideal marker as biomarker elevation can come from anywhere in the body|
|CTCs||X X X||X||X X X X||X X X X||X X X||Detection greatly increases the likelihood of distant disease spread|
|PBMCs||X X X||X X X||X X X||X X X X||X X X||X X X||Preliminary studies indicate early and speciﬁc changes in expression proﬁling|
|Bile||X||X||X||X||X||May be best for ruling out hepatic and gallbladder pathology from differential diagnosis|
|Urine||X X X X||X X||X||X||X X||Speciﬁcity must come from choice of ideal marker as biomarker elevation can come from anywhere in the body|
PC pancreatic cancer, CTCs circulating tumor cells, PBMCs peripheral blood mononuclear cells
aLikelihood of tumor detection in a scenario of pancreatic tumor presence
bLow risk of false-positive results in patients lacking a pancreatic tumor
Methodological development for evaluation of free cells within an aspirate sample has sought to overcome multiple downfalls of the core biopsy-based evaluation methods. Accomplished either through transcutaneous aspiration of the main pancreatic duct with endoscopic ultrasound guidance or via direct aspiration following cannulation of the papilla of Vater through endoscopic retrograde cholangiopancreatography (ERCP), aspirate specimens are less invasive to obtain than their core biopsy counterparts and thus lend themselves better to patient screening. Further, aspiration of the main pancreatic duct allows for a theoretical evaluation of the entire ductal network, with potential of collecting cells and debris from ductal lesions throughout the pancreas. Aspirate evaluation also lends itself well to the analysis of both cytology and protein secretions, broadening the range of potential evaluable biomarkers.
However, aspiration-based biomarkers carry with them many issues and potential complications. Presence of a cell or protein in an aspiration specimen is reliant on its being free within, or loosely adherent to the wall of a pancreatic duct or ductile, thus limiting the nature of evaluable entities. Obtainment of ample cellularity to allow for conclusive cytology or immunohistochemistry, especially if staining for multiple markers is desired, is also by no means guaranteed in pancreatic aspiration. Further, there is a small, though not insigniﬁcant, risk of pancreatic duct aspiration inducing acute pancreatitis, an acute inﬂammatory disease with signiﬁcant morbidity and the realistic potential for patient mortality. Additionally, aspiration disallows the evaluation of cells within their natural environment(s) as well as the evaluation of considerable peri-tumoral tissue. As such, though its less invasive and more comprehensive nature makes aspiration superior to core biopsy evaluation for diagnostic means, it is still not a diagnostically viable mechanism and thus continues to be relegated to use in patients with suspicious lesions or symptomatology for conﬁrmatory purposes.
A body ﬂuid that has received substantial recent attention for PC diagnostics is pancreatic juice; the results of some such studies are reviewed in Chakraborty et al. Pancreatic juice, consisting of the secreted liquids, enzymes, chemicals, and sloughed cells from the pancreatic ductal system as it travels toward the duodenum, is endoscopically collected as it exits through the sphincter of Oddi and can be performed with or without the preceding step of secretin stimulation. Importantly, like aspirate evaluation, these methods have the ability to assess ﬂuid, chemicals, and cells from throughout the entire acinar and ductal network of the pancreas, rendering a previously identiﬁed area of concern within the pancreas unnecessary. Further, pancreatic juice collection is less invasive than aspiration and does not pose the risk of iatrogenic pancreatitis.
A myriad of downsides exist to pancreatic juice evaluation as well, though. For example, only a small volume is able to be collected without secretin stimulation, thereby limiting the tests which can be performed, but secretin stimulation itself serves to increase the volume of the pancreatic excretion and thus risks diluting relevant biomarkers to non-detectability. Additionally, collection of the juice from the duodenum risks sample contamination from both the small intestines and emptying stomach, potentially sacriﬁcing both sensitivity (through dilution) and speciﬁcity of a given biomarker for PC. Lastly, the presence of a PC biomarker within pancreatic juice requires a communication of the tissue of origin for that biomarker, be it PC or peri-tumoral tissue, with the main pancreatic duct, a necessity which may not always be the case.
Due to ease of accessibility, serum/plasma-based biomarkers have undergone the most extensive of study as a source of surrogate PC diagnostic or prognostic markers, the most famous of which is the currently FDA-approved CA19-9. An important advantage of these circulating biomarkers is the potential for detection of those derived from both tumor and stromal cells. The mildly invasive nature of their collection, accomplished via simple venipuncture, is also of signiﬁcant beneﬁt to their potential as a diagnostic- screening mechanism.
Unfortunately, the majority of such studies have been hampered by difﬁculty in isolating, identifying, and consistently detecting these low-abundance proteins. Additionally, as the search for PC biomarkers becomes farther removed from the pancreas, the greater likelihood of non-speciﬁcity exists as biomarkers from the entire body would be thus detectable. This is of particular importance with regard to inﬂammatory or general neoplastic markers which may have utility if attributable to the pancreas speciﬁcally but otherwise can lead to clinical confusion and patient misdiagnosis. Consequently, choosing of proper biomarkers that are fully speciﬁc to pancreatic neoplasia in the quest for plasma/serum-based surrogate detection, prognostication, and/or following of progression of PC is of the utmost importance.
Another tissue with high diagnostic potential and becoming of increasing interest is circulating cells derived from venipuncture. Regarding current biomarker interest, these cells come in two ﬂavors: circulating tumor cells (CTCs) and peripheral blood mononuclear cells (PBMCs). CTCs are of interest due to the fact that their presence indicates not only the presence of cancer but also an increased likelihood of the presence of distant metastases, detectable or not, as well as the fact that their mutational proﬁle can offer indications as to the prognosis and therapeutic responsiveness of both primary and metastatic lesions.
Unfortunately, these beneﬁts come at a steep price as the likelihood of detectable CTCs being present in patients with truly localized disease is low, thus making these methods of little beneﬁt as an early diagnostic mechanism. Additionally, CTCs only make up a small portion of circulating cells, causing increased complications as these cells must be speciﬁcally isolated for analysis.
The theory that PBMCs, consisting chieﬂy of monocytes, T cells, and B cells, may offer an early diagnostic test for PC with high utility is founded on two physiologically competing principles: (1) cancer cells are inherently antigenic, leading to a tumor-directed immune response and (2) as cancer develops, it gains the ability to evade the immune system through direct and indirect mechanisms. The effect that each of these principles instills upon the immune system as a whole results in both predictable and unpredictable alterations in immune cell populations as well as their transcriptional patterns, with the potential for monitoring to allow for a surrogate window into active immune modulation and its potential cause(s). Further, different cancers and immunogenic disease states utilize different combinations of methods for immune activation and suppression, providing a conceivable speciﬁcity to PBMC expression proﬁles as a biomarker mechanism. However, speciﬁc knowledge regarding cancer immune detection as well as immune evasion is currently in its infancy, making the discovery of cancer-speciﬁc and prognostic proﬁling a result of non-directed global approaches with obvious need for optimization prior to full assessment of clinical potential.
Other body fluids
Multiple other sources of surrogate markers have also been examined for the presence of PC biomarkers. PC patient urine, which beneﬁts from a fully noninvasive nature, has been analyzed against that from healthy and CP controls by mass spectrometry, indicating a total of 60 proteins that were differentially expressed. Analysis of bile, obtainable through ERCP, has also determined that 127 protein fragments are elevated in PC patients. Such studies, however, suffer from a lack of intra-study validation and are currently singular in nature. Thus, while such ﬂuids may represent mechanisms for future PC biomarker development, much future exploration is needed.
Table 2. False-positive to true-positive (FP:TP) ratios obtained by two theoretical tests (A and B) as well as CA19-9 when applied as a PC screening mechanism to various patient populations
|Test A||Test B||CA19-9|
|100 %||99 %||90 %||90 %||70 %||80 %|
|Screened population||Pretest probability||FP:TP ratioa||FP:TP ratioa||FP:TP ratioa|
|General population (>50 years old)||1:2,000||20.0||222.2||571.4|
|Family history (1+ relatives)||1:222–1:1,136||2.2–11.4||24.7–126.2||63.4–324.6|
|Family history (2+ relatives)||1:313–1:469||3.1–4.7||34.8–52.1||89.4–134.0|
|Family history (3+ relatives)||1:63||0.6||6.9||17.9|
|Recent-onset type II diabetes||1:125–1:172||1.3–1.7||13.9–19.1||35.7–49.1|
aFP:TP Ratio indicates the number of PC-free patients who will test positive for every screened patient with PC correctly diagnosed per a test with the given diagnostic abilities
Importance of an optimal testing population
Another point of importance in the discussion of PC biomarkers, particularly for biomarkers that are intended for diagnostic screening, is the statistical properties that such a marker must possess for clinical applicability. Primarily, screening tests must be mildly invasive and must have adequate performance with regard to sensitivity and speciﬁcity to elicit results that are ultimately beneﬁcial to both clinician and patient. For the majority of screening tests (PSA, colonoscopy, mammogram, etc.), this stipulation skews diagnostic candidates toward increasing sensitivity, owing to the consideration that the purpose of the test is to add clinical suspicion to disease presence with the results of which being ultimately conﬁrmed through more accurate, though often more expensive and invasive methods. Such conﬁrmatory testing, however, does not exist in PC, especially in the case of small, localized tumors. Hence, a positive PC screening test has a high likelihood of resulting in invasive exploratory procedures such as EUS-FNAs and laparoscopies which also often fail to discern small tumors of the pancreas, thus raising the potential for pancreatectomies or pancreatoduodenectomies without concrete evidence of their necessity. These circumstances result in a situation in which PC diagnostic testing must be skewed toward increasing speciﬁcity under the consideration that it is better to give a portion of PC patients false-negative results than to lead a healthy patient to believe that they have this deadly disease, thus subjecting them to a barrage of tests and procedures causing signiﬁcant ﬁnancial, physical, and psychological burden that will, in many cases, generate only inconclusive results. Adding to this is the fact that successful screening for PC, due to its relative rarity, must, at its end, come down to the analysis and minimization of the false-positive (FP) to true-positive (TP) ratio (Table 2).
The current annual incidence of PC in the United States is approximately 43,920 patients, a number that has been slowly creeping upward with time. As per the 2010 census, an approximated total of 93,555,000 people over the age of 50 (below which a diagnosis of PC is unlikely) are currently living in the United States, putting the per capita incidence of PC at about 1:2,000. Based on this, even a diagnostic test with perfect 100 % sensitivity and a speciﬁcity of 99 % will falsely diagnose 20 healthy people for every PC patient it correctly detects. With the lack of highly accurate conﬁrmatory tests available for PC, especially in early disease, combined with their invasiveness and expense, even this optimal false-positive:true- positive (FP:TP) ratio cannot be considered acceptable. Hence, though improvement in the diagnostic performance of currently available surrogate diagnostic methods is obviously necessary for clinical applicability, it alone will fail to produce tests with true viability. Instead, equal focus must be placed on optimal population(s) in which to administer such a test. Such populations must be well-deﬁned as well as have a signiﬁcant and substantial increase in risk of PC diagnosis over baseline. Based on current knowledge, three populations meet these criteria and may prove to provide enhancement of PC diagnostic test viability to allow for clinical applicability: chronic pancreatitis (CP), patients with a family history of PC, and patients with recent-onset type II diabetes mellitus (DM2).
Both hereditary and nonhereditary CP is well established to be a signiﬁcant risk factor for future PC diagnosis, a fact thought to primarily arise from the proneoplastic chronic inﬂammatory nature of the disease. While it is unclear what, if any, subsets of CP patients have the greatest PC risk, it appears that risk increases with time following CP diagnosis. For CP patients in general, however, it is estimated that the annual incidence of PC is 1.1–1.8 % of the population. Such increase in risk would allow a biomarker with the ideal characteristics described above (100 % sensitivity and 99 % speciﬁcity) to provide one to two TPs for each FP encountered, a potential that, though still not ideal, is a stark improvement over that allowed for in the general population. Further, this risk, and hence diagnostic biomarker utility, may be further heightened in speciﬁc CP population subsets, a potential which must be further explored.
Patients with a family history signiﬁcant for PC also represent an attractive population for diagnostic screening. Overall, it has been shown that patients with a PC-positive family history in any ﬁrst-degree relative are at a 1.76–9.0-fold increased risk for PC diagnosis as compared to the general population. This risk further increases with the number of affected ﬁrst-degree relatives, with patients having two or more affected relatives being at a 4.26–6.4-fold increased risk and a 32-fold increased risk observed in patients with >3 ﬁrst-degree relatives with a PC diagnosis. As such, this population represents a realistic group in which to institute PC screening; however, it is noteworthy that only 5–10 % of all PC patients are considered familial, and thus the overall impact on the disease will be low.
Recent associations between new-onset DM2 and PC have indicated that this too may be a group in which diagnostic screening for PC may be viable. Newly diagnosed DM2 patients have a substantially increased risk of a PC diagnosis as compared to the general population, as it is reported that up to 0.8 % of patients who are >50 years of age and diagnosed with DM2 will receive a PC diagnosis within the ﬁrst 3 years, placing the overall risk at 1:125.
PC biomarkers represent a wide array of entities derived from various locations within the body and designed for multiple potential purposes. Currently, a single biomarker, CA19-9, is approved for use in PC with proven, though suboptimal, utility for following patient progression but lacks the required speciﬁcity for diagnostic use. Biomarkers derived from the pancreas or peripancreatic tissue, such as tumoral and peri-tumoral tissues, pancreatic duct aspirate, and pancreatic juice, have the potential for the greatest speciﬁcity but are limited by their invasive natures. Peripherally derived biomarkers such as those from plasma/serum, CTCs, PBMCs, and urine are less invasive and thus are more conducive to screening for early PC diagnosis, though they may coincide with a signiﬁcant loss of biomarker speciﬁcity or an increasingly complex testing design to reduce the potential for false positivity. Of these, CTCs and PBMCs have the greatest likelihood to offer reasonable speciﬁcity, though CTCs may have little ability in localized disease while PBMC proﬁling requires substantial optimization prior to realization of its full diagnostic utility. Additionally, in the study of PC biomarkers, consideration of the population in which to apply the test is of equal importance as optimization of the test itself and should be addressed as each series of studies move forward toward clinical testing.
Of further note, the greatest current hindrance to establishment of an early diagnostic test for PC is the current lack of ability to discover the disease in its early stages, thus precluding the obtainment of relevant samples for study. This point highlights the utility of transgenic murine models in PC biomarker discovery, allowing the correlation of biomarker alteration with disease stage from preneoplastic through metastatic disease, though ample correlation across species to determine the generalizability of mouse-based results to human patients has yet to be undertaken and likely will require assessment for each biomarker study either individually or together in a global manner.
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