Biochimica et Biophysica Acta 1815 (2011) 44–64

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Review

Current status of molecular markers for early detection of sporadic pancreatic cancer

Subhankar Chakraborty a, Michael J. Baine b, Aaron R. Sasson b,d, Surinder K. Batra a,b,c,⁎a Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USAb Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE, USAc Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USAd Department of Surgery, University of Nebraska Medical Center, Omaha, NE, USA

⁎ Corresponding author. Department of Biochemistry985870 Nebraska Medical Center, Omaha, NE 68198-58

E-mail address: sbatra@unmc.edu (S.K. Batra).

0304-419X/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.bbcan.2010.09.002

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 August 2010Received in revised form 23 September 2010Accepted 24 September 2010Available online 1 October 2010

Keywords:DiagnosisPanINBiomarkers

Pancreatic cancer (PC) is a highly lethal malignancy with near 100% mortality. This is in part due to the factthat most patients present with metastatic or locally advanced disease at the time of diagnosis. Significantly,in nearly 95% of PC patients there is neither an associated family history of PC nor of diseases known to beassociated with an increased risk of PC. These groups of patients who comprise the bulk of PC cases are termedas “sporadic PC” in contrast to the familial PC cases that comprise only about 5% of all PCs. Given the insidiousonset of the malignancy and its extreme resistance to chemo and radiotherapy, an abundance of research inrecent years has focused on identifying biomarkers for the early detection of PC, specifically aiming at thesporadic PC cohort. However, while several studies have established that asymptomatic individuals with apositive family history of PC and those with certain heritable syndromes are candidates for PC screening, therole of screening in identifying sporadic PC is still an unsettled question. The present review attempts to assessthis critical question by investigating the recent advancesmade inmolecular markers with potential use in theearly diagnosis of sporadic PC — the largest cohort of PC cases worldwide. It also outlines a novel yet simplerisk factor based stratification system that could be potentially employed by clinicians to identify thoseindividuals who are at an elevated risk for the development of sporadic PC and therefore candidates forscreening.

and Molecular Biology, Eppley Institute for Research in C70, USA. Tel.: +1 402 559 5455; fax: +1 402 559 6650.

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

Contents

1. Prologue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452. Pancreatic adenocarcinoma: evolution and progression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453. Limitations of clinical symptoms in the early diagnosis of pancreatic cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464. Risk factors and their role in pancreatic cancer screening and surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

4.1. Modifiable or lifestyle associated risk factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.2. Non-modifiable or genetic risk factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474.3. Chronic pancreatitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484.4. Type II diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494.5. Cystic neoplasms of the pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

5. Role of imaging in the early diagnosis of pancreatic cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.1. Endoscopic-ultrasound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505.2. Recent advances in pancreatic imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

6. PanIN lesions as a source of potential biomarkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517. Biomarkers in body fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

7.1. Serum and plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537.2. Pancreatic juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557.3. Other body fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

8. Advances in molecular diagnosis of pancreatic cancer: role of microRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569. Screening as a tool for early detection of pancreatic cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

ancer and Allied Diseases, University of Nebraska Medical Center,

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10. Management of the high-risk patient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5811. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

1. Prologue

It seems that pancreatic cancer (PC) has recently crept further andfurther into the consciousness of the general public. The news thatLuciano Pavarotti, the renowned opera singer and one of the legendary“three tenors”hadbeendiagnosedwithPC cameasa shock tomillionsofhis followers the world over. A year-and-a-half later, the famous actorand heartthrob, Patrick Swayze, also received the diagnosis of PC andsuccumbed to his disease in just 19 months. Former United Statespresident Jimmy Carter has been intimately affected by PC, having losthis father, mother, brother and two sisters to this malignancy. Perhapsthe most well known recent case of PC in the public eye was the case ofDr. Randy Pausch of Carnegie Mellon University who was diagnosedwith metastatic pancreatic adenocarcinoma and died from themalignancy in 2008. While his words, “If I don't seem as depressed ormorose as I should be, sorry to disappoint you,” from his “Last lecture”showed thewell-grounded couragewithwhich he accepted his disease,his well-publicized plight brought to the attention of the worldcommunity the insidious and relentless nature of this malignancy andthe need to diagnose PC at an early stage. The objective of this review isto revisit certain aspects of PC screening, highlight the latest advances inthe early diagnosis of this cancer, and suggest possible strategies thatcould help identify high-risk cases among those with no familial orknown genetic predisposition to PC.

2. Pancreatic adenocarcinoma: evolution and progression

The pancreas (derived from the Greek words, “pan” meaning “all”and “creas” meaning “flesh”) is a key organ in the field of medicinedue to its association with two diseases, diabetes mellitus and PC [1].The former accounts for an estimated 24 million cases in the UnitedStates alone (around 7.8% of the population) [2], while the latter is thefourth leading cause of cancer related deaths in the United Statesamong both men and women [3]. Although the ductal systemcomprises a small portion of the exocrine pancreas, 90% of pancreaticneoplasms are ductal in origin, of which more than 80% are invasiveadenocarcinomas. It is of interest to note that the term “pancreascancer” when used in common diction refers to conventional ductaladenocarcinoma (commonly referred to simply as simply “ductaladenocarcinoma”) arising from the gland. However, in the patholog-ical lexicon, this term also encompasses other less common variants ofductal adenocarcinoma (including the foamy gland, large duct andvacuolated patterns) as well as other carcinomas of ductal origin suchas colloid, medullary, squamous, adenosquamous, and undifferenti-ated carcinomas. The origin of the neoplastic cells in ductaladenocarcinoma is still unclear, although several sources have beensuggested including the non-neoplastic ductal epithelium, islet cells,and more recently, pancreatic stem cells [4,5]. The progression fromnon-neoplastic cells to invasive adenocarcinoma has been suggestedto occur though a series of pre-malignant lesions characterized byprogressively increasing dysplasia. These precursors, termed aspancreatic intraepithelial neoplasia (or PanINs), have well-definedmorphological characteristics — PanIN-1a (flat), PanIN-1b (papillarywithout dysplasia), PanIN-2 (papillary with dysplastic changes) andPanIN-3 (carcinoma-in situ). Two updates on PanIN lesions werepublished recently [6,7].

Adenocarcinoma of the pancreas can sometimes be associatedwith two other types of pancreatic lesions — intraductal papillary

mucinous neoplasm (IPMN) and mucinous cystadenomas of thepancreas. IPMNs are a unique category of pancreatic neoplasmswhichmanifest as a massive dilation of the intra-pancreatic ducts eitherinvolving the entire duct system or restricted to the main pancreaticduct (main duct IPMN) or its branches (branch duct-type IPMN) [8].IPMNs are broadly classified into invasive and non-invasive variants.The epithelium lining the non-invasive IPMNs shows a variety ofdifferentiation routes including gastric, intestinal, pancreatobiliaryand oncocytic patterns, the latter two being rare subtypes. Intestinaltype IPMNs, which are generally found in the main pancreatic ducthave a propensity to develop into mucinous non-cystic (colloid)carcinoma, while those in the branch ducts are usually of the gastrictype and are commonly benign. Pancreatobiliary IPMNs are generallyconsidered to be at a greater risk of progressing to PC [9]. From theclinical standpoint, IPMNs are associated with a significant risk ofmalignancy and hence demand surgical resection [10] (IPMNs havebeen recently reviewed in [11]). Mucinous cystadenomas representanother important group of pancreatic neoplasms which are distin-guished from IPMNs by the absence of a communication between thecyst and the adjacent pancreatic duct. All mucinous cystic neoplasmsare generally considered potentially malignant owing to the risk of anassociated cystadenocarcinoma. Serous cystic tumors, on the otherhand, are completely benign with no reported recurrence followingcomplete surgical resection [12,13].

Pancreatic cancer is one of the few malignancies with nearly 100%mortality once diagnosed. The median survival from the time ofdiagnosis to death ranges from 3 to 6 months. A major cause for thepoor prognosis is the resistance of PC cells to conventionalchemotherapy and radiotherapy [14]. Gemcitabine, a pyrimidineanalogue that targets cells in the S (synthesis)-phase of the cell cycle,is currently the mainstay of chemotherapy for PC. Recent reportsindicate that it is more efficacious when used in combination withCapecitabine (a pro-drug that is converted to 5-fluorouracil insidetumor cells) and platinum-based agents rather than as amonotherapy[15]. Surgery is recommended only for those patients withoutevidence of metastatic disease and without significant vascularinvolvement. Generally, malignant tumors of the head and periam-pullary region (when resectable) are managed by a pancreaticoduo-denectomy (with or without preservation of the pylorus), while thoseinvolving the body and tail are dealt with by a distal pancreatectomy.Segmental resection of the pancreas is reserved only for benigntumors or pre-malignant lesions in the body of the pancreas. Thesurgical management of PC has been reviewed by Michalski in arecent article [16].

There have been several recent articles that have discussed variousaspects of PC, including the common genetic alterations [17], currentmanagement strategies [18] as well as the efforts toward under-standing the molecular aspects of this malignancy [19]. Nearly all arein agreement on the point that there is currently no definitivetreatment for PC. Hence, there has been an emphasis on identifyingone or more markers that can detect cancer of the pancreas at asurgically resectable stage or even earlier (in the stage of dysplasia).Most such studies have been based on the principle that one or moregenes (and by extension, proteins) whose expression is upregulated(or appears de novo) or diminished (decreased or lost) in localizedcancer or the dysplastic lesions (compared to non-neoplastic ductalcells) holds promise as potential biomarkers for early diagnosis. Thecatch, however, is how to identify patients with PanIN lesions, who

Table 1Risk factor based stratification to identify patients at risk of developing sporadicpancreatic cancer.

Risk group Risk factor

High-risk (O.R.a ≥2) (i) Obesity (BMI ≥30)(ii) Current smokers(iii) Former or current smokers with an Arg188Hispolymorphism(in XRCC2) or a combination of an Asp312 (on exon 10)and Lys751haplotype in the XPD gene(iv) Type II diabetes mellitus of recent onset (b3 years)(v) History of pancreatitisb ≥1 year back(vi) Chronic pancreatitis(vii) History of symptoms suggestive of pancreaticcancerc for ≥3 years(viii) Endoscopic ultrasound findings of high-grade(moderate to severe) chronic pancreatitisd

Intermediate risk(O.R. ≥1.5 but b2)

(i) Blood group AB and B(ii) Overweight (BMI ≥25 but b30)(iii) Calories adjusted intake of saturated fat N25 g/daye

(iv) Race: African AmericansLow risk (O.R. b1.5) (i) Blood group A

(ii) History of gallstones(iii) Childhood exposure to environmental smoke

No significant risk (i) Former smokers who quit ≥15 years ago, irrespective ofthe number of pack years smoked(ii) Alcohol (independent of type and duration)f

(iii) Non-alcoholic beverages: tea, coffee, juices(iv) Red meat(v) Education status or income

Decreased risk ofpancreatic cancer

(i) Age at menarche ≥15 years(ii) ≥4 pregnancies(iii) Allergies (hay fever, seasonal allergies and allergy toanimalsg)

Possible risk factorsh (i) Patients with an attack of acute pancreatitis in thepresence of a pre-existing KrasG12D mutation(ii) Marital status (widowed or never married vs. currentlymarried)i

a Odds ratio.b No distinction made between acute or chronic pancreatitis.c Includes abdominal pain, unusual bloating, belching or heartburn, altered bowel

habits, symptoms of biliary obstruction, and general constitutional symptoms (fatigue,inability to sleep, anorexia and weight loss).

d According to the Cambridge criteria.e Only in males.f Could contribute indirectly through alcohol-induced chronic pancreatitis.g Irrespective of the type of animal.h These are the factors reported in single studies to be associated with an increased

risk of PC and need further confirmation.i The former group were shown to be at an elevated risk of pancreatic cancer in one

study.

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are almost always asymptomatic. If there could be a method toidentify these individuals, they could be valuable subjects inprospective studies for the detection of early molecular markers ofPC. An alternative approach has been to prospectively examinesamples from patients with clinical conditions known to increase therisk of PC (like chronic pancreatitis, type II diabetes mellitus, chronicsmokers, those with an occupational history of working in the rubberindustry, or those with a history of tropical or idiopathic pancreatitisor cystic fibrosis [20], to name a few) or healthy subjects with a familyhistory of PC or PC related syndromes. However, the number of suchstudies is too few to draw any significant conclusions.

3. Limitations of clinical symptoms in the early diagnosis ofpancreatic cancer

It is generally recognized that PC is an insidious disease with nospecific early clinical symptoms, except where the primary tumor islocated in the head of the pancreas. Then, the patient may presentearly with signs of biliary obstruction (obstructive jaundice). It isworthwhile to quote two recent studies that examined the utility ofclinical symptoms in the early diagnosis of PC. The first studycompared the frequency of symptoms reported by 120 patientswith PC with that in 180 matched control subjects. An importantobservation was that most patients with PC reported experiencingsymptoms within three years prior to the diagnosis of the malignancy[21]. The most commonly reported symptoms included abdominalpain, unusual bloating, belching or heartburn, altered bowel habits(either constipation or diarrhea), symptoms of biliary obstruction(jaundice, pale stools and pruritus) as well as general constitutionalsymptoms (fatigue, inability to sleep and weight loss). While the oddsratio for many of these symptoms was quite high (for instance N30 forabdominal pain and pale stools), suggesting that these are reportedmore commonly by patients with PC than the general population,their specificity remains a major issue as the same symptoms are alsoreported in many benign conditions. A second study [22] conductedamong Brazilian patients found that most patients diagnosed with PCexperienced asthenia, weight loss and anorexia which was unrelatedto the stage of the cancer. Notably, a longer interval between the onsetof symptoms and the initial diagnosis of pancreatic cancer wasassociated with the disease being first identified at a more advancedstage. Further, none of the patients with a tumor in the body or tail ofthe pancreas was diagnosed with Stage I disease (nearly 80% were instage IV at presentation). These studies suggest that while clinicalsymptoms lack specificity, clinicians should nonetheless maintain ahigh index of suspicion for an occult malignancy in patients whomeetthe criteria of the “high-risk group” (Table 1). Obstructive jaundiceappears to be the only condition with some specificity for PC andhence must be carefully investigated by the attending physician torule out a possible malignancy.

4. Risk factors and their role in pancreatic cancer screeningand surveillance

4.1. Modifiable or lifestyle associated risk factors

Several risk factors have been found over the years that areassociated with an increased risk of PC. A meta-analysis [23]examining 14 studies (6 case–control and 8 cohort studies) onEuropean or North American individuals found that the risk of PC wasnot significantly affected by the BMI (relative risk — 1.02 per unitincrease in BMI, 95% C.I.: 1.01–1.03). However, the relative riskincreased from 1.02 to 1.03 when corrected for smoking (p=0.04).Further, a correction for the presence of diabetes did not alter the risksignificantly nor was there a difference in the risk between males(1.03) and females (1.02). However, obese individuals (defined asthose with a BMI ≥30) did have a slightly higher risk (relative risk:

1.19) of developing PC compared to normal-weight individuals (BMIb25) (similar findings reported in [24,25]). Men and women withcentral adiposity were reported to be at a greater risk of PC comparedto those who report a peripheral weight gain (relative risk: 1.45; 95%C.I.: 1.02–2.07) [24]. Significantly, there was no relationship betweenthe extent of recreational physical activity and the risk of PC, evenwhen analyzed for the subset of individuals who were aged 40 yearsand above [24].

Several studies have now shown that smoking is a strong riskfactor for PC [26–30]. It has been suggested that smoking contributesto nearly one quarter of all cases of PC, making it the single mostprevalent risk factor for this disease [31]. A study of familial PC (FPC)kindreds found that smoking was an independent risk factor for PC(odds ratio (O.R.): 3.7) and smokers developed cancer of the pancreasnearly one decade earlier than non-smokers (59 years vs. 69 years)[32]. One population based case–control study [28] among Canadiansreported that smokers were at an elevated risk factor of developing PCcompared to never smokers. This risk was observed in both the sexes.Men with a smoking history of ≥35 pack years and women whosmoked 23 or more pack years were found to be at the highest risk ofdeveloping PC (O.R.: 1.46 and 1.84 respectively). However, neither the

47S. Chakraborty et al. / Biochimica et Biophysica Acta 1815 (2011) 44–64

amount of total alcohol consumed (even up to one drink every day)nor the type of alcoholic beverage (wine, beer or liquor) had any effecton their risk of developing ductal adenocarcinoma. Interestingly, thisstudy also found that neither drinking coffee (≥4 cups/day) nor theincome or the number of years of education had any impact on therisk of PC. A later study by the same group [27] revealed that anincreased weekly caloric intake and participation in ≥8.2 h ofstrenuous physical activity was associated with a higher and lowerrisk of PC, respectively (O.R.: 1.68 and 0.59, respectively).

Among women, four or more pregnancies and an older age at thetime of menarche (15 years or older) were associated with areduction in the risk of PC. Additionally, caloric intake and physicalactivity did not appear to alter the risk of PC among women. A historyof exposure to environmental tobacco smoke from childhood andonto adulthood, however, translated into a modest elevation in therisk for subsequent development of an adenocarcinoma of thepancreas, even among never smokers [26]. Further, this risk wasamplified in active smokers compared to those with a past history ofcigarette use (≥25 pack years).

A hospital based case–control study in northern Italy, whileconfirming the findings of the Canadian group, also found that the riskof PC among smokers was independent of the number of cigarettessmoked per day (even up to 20 or more per diem). Of interest was theobservation that among former smokers, the risk of PC fell to levelscomparable to never smokers after only 15 years or more postquitting smoking. Notably, this was independent of the number ofcigarettes smoked or the duration of the smoking habit [30].

In one case–control study [33] “heavy smokers” (defined as thosewith a history of N40 pack years) were at an increased risk of PC ifthey were carriers of at least one minor allele for the DNA repair geneXPD/ERCC2 at D312N (O.R.: 2.78. 95% C.I.: 1.3–6) or D711D (O.R.: 2.2,95% C.I.: 1–4.7). Thus, the risk of PC associated with smoking appearsto be modified by the presence of co-existing genetic alterations ingenes that regulate cellular response to DNA damage.

In a large prospective cohort study of over 1 million Americansover a 14-year period [34], black race (N1.5-fold greater risk in bothmen and women) and history of gallstones (1.3-fold higher risk onlyamongmen)were predictive of a risk of PC. The risk was also two-foldor higher in current male smokers who smoked 20 or more cigarettesper day (for women, a similar degree of risk was observed with 10 ormore cigarettes per day), or smoked for N25 years compared to neversmokers. Notably, the risk associated with cigarette smoking was notmodified by diet or other lifestyle factors examined. The studies alsoobserved no relationship between the levels of education, theconsumption of red meat, citrus fruits, juices, coffee or spirits andthe risk of PC. From the aforementioned studies, it appears thatspecific lifestyle traits of an individual have a considerable impact ontheir risk of developing PC.

A recent nested case–control study among 1,141 PC patients and7,954 controls British patients found that the use of non-steroidalanti-inflammatory drugs for about 2 years prior to the diagnosis of PCwas associated with a significantly decreased risk of PC (O.R. 0.75, 95%C.I.: 0.62–0.97) [35].

4.2. Non-modifiable or genetic risk factors

Although the role of genetic predisposition in sporadic PCs is stillunclear, a familial clustering of PC cases has been reported innumerous studies (summarized recently by Landi [36]). Based onthese reports, strategies have been recommended to screen asymp-tomatic individuals belonging to familial PC kindreds [37,38]. Severalgenetic alterations have been shown to be associated with ahereditary predisposition to PC. Some mutations predispose thecarriers to malignancies in other organs as well. Mutations in theBRCA1 gene for instance, are associated with an increased risk ofbreast, ovarian, uterine and fallopian tube malignancy in women and

breast and prostate cancer in men. In addition, carriers for a mutationin this gene are also at an increased risk for pancreatic, colon, gastric,lung cancer and melanoma [39]. While genetic changes are morefrequent in cases with an associated family history of PC (FPCkindreds), inactivating mutations in p16INK4a (also called cyclindependent kinase A or CDKN2A) are frequently observed in sporadicPC [39]. However, mutations in p16INK4a are not specific for PC (inwhich the common mutation is V126D), being also observed in othermalignancies such as familial cases of malignant melanoma (alsoV126D) and breast cancer (associated with an 113insArg mutation).The latter patients are also at an increased risk for PC [40].

In one study involving 18 German families with familial PC [41],each of whom had at least two first degree relatives with PC, and fivewho had at least one relative with both PC andmelanoma, none of thefamilies without an associated melanoma had a mutation in thep16INK4a gene, while two of the five families with both PC andmelanoma had germline truncating mutations (Q50X and E119X) inthe gene. A prospective study among patients enrolled in the NationalFamilial Pancreas Tumor Registry (NFPTR) found that the increase inrisk of PC among those with a family history (of PC) was generallyconfined to patients who were 60 years or older [42].

The PALB2 (also known as partner and localizer for BRCA2 orFANCN) gene located on chromosome 16 encodes for a protein thatstabilizes the BRCA2 oncoprotein in the nucleus [43] and has recentlybeen linked to a possible association with the risk of familial PC.Biallelic inactivating mutations in PALB2 have been previously linkedto Fanconi's anemia, specifically to subtype N which resembles theFanconi's anemia phenotype caused by a biallelic mutation in theBRCA2 gene [44]. PALB2 has also been shown (in several populationbased studies) to increase the susceptibility of patients to thedevelopment of breast cancer (the estimated risk ranging from 2.3-fold to 6-fold for carriers of mutations in the gene) [45]. A recent studycomparing tumor DNA from a patient with familial PCwith the humanreference genome revealed a germline deletion of four base pairs inthe PALB2 gene that translated into a frame shift mutation at codon 58of the gene [46]. Further sequencing of this gene in 96 patients with afamily history of PC revealed truncatingmutations in the gene in threepatients. Each of the three mutations identified (IVS5-1 GNT, 3116 delA and 3256 CNT) produced a different stop codon. Importantly, notruncating mutations were reported in a cohort of 1084 healthyindividuals [47], while only two healthy controls out of 1079 had amutation (1592delT) in the same gene in another study [45]. Agermline missense mutation (P239S) in Palladin, a gene located onthe chromosome locus 4q32–34 has also been suggested to be linkedto a familial clustering of PC [46]. However, subsequent studies havefailed to detect the presence of this mutation in FPC clusters [48–52].

While most PCs with a family history have been described inassociation with some form of a genetic alteration, an interesting caseof a family with six cases of pancreatitis and three of PC in the secondgeneration was recently described wherein the affected members hadnone of the known genetic alterations [53]. Further, themalignancy inthis cohort uniquely spared the head of the pancreas. Instead, acharacteristic fatty infiltration involving only the body and the tailwas observed. Clearly, the relationship between genetic factors andthe environment that modulates the risk of an individual developingPC is highly complex and needs to be elucidated further.

A prospective cohort study of 107,503 U.S. health professionalsfound that the ABO blood group of an individual could contributesignificantly to the risk factor for PC development [54]. Individuals withnon-O blood groups were found to be at a greater risk for thedevelopment of PC, with the risk being lower among group A thanamong group B individuals. Notably, the association between the ABOblood group and the risk of subsequent PC (as measured by the hazardratio) was not significantly modifiedwhen other purported risk factors,like advanced age (≥62.5 years), body mass index (≥25.7), physicalactivity (≥13.5metabolic equivalent task hours perweek) and smoking

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(nevervs. past/current smokers)were considered. Further, theRhgroupdid not appear to have an influence on the risk of PC.

Allergies, especially a history of hives, have been linked to the risk ofdeveloping cancer, although thedirection of the riskvaries fromstudy tostudy. While the presence of an allergy has been shown to increasethe risk of hematopoietic malignancies (leukemia, lymphoma andmyeloma) [55], it appears to reduce the risk of cancer at other sites [56].Several epidemiological studies have also examined the associationbetween a history of allergies and PC. One prospective study of over1 million Americans from 1982 to 2000 found that a history of hay feverwas associated with a significantly lower mortality among thosediagnosed with PC (compared to those without such a history) [57].Another hospital based case–control study by Olson et al. found that ahistory of allergies (regardless of the type of allergen) was associatedwitha significantly lower risk ofdevelopingPC [57], a viewsupportedbya more recent study in Canadians [58]. Interestingly, the presence of aG3017T variation in the IL-4 gene (an inducer of IgE synthesis togetherwith IL-13)was actually associatedwith a slightly increased risk of PC inallergy-prone individuals (O.R.: 1.47, 95% C.I.: 0.8–2.69), while the riskwas significantly reduced in thosewhodidnot report any allergies (O.R.:0.44, 95% C.I.: 0.2-0.99) [57]. The results of this pilot study seem tosuggest that genetic variations in genes encoding for cytokines(including SNPs and mutations) and other mediators of inflammationcould also affect the risk of PC, and possibly permit risk stratification forpatients.

Polymorphisms in the cytochrome P450 enzyme (CYP2A6),capable of activating several procarcinogens (including the highlycarcinogenic nitrosamines) has been linked to an increased risk ofsporadic PC (independent of smoking) [58], although further studiesare needed to confirm these observations. Single nucleotide poly-morphisms (SNPs), which are variations in the DNA sequence (amongdifferent individuals) involving a single nucleotide, have beenextensively studied for a possible direct association with the risk ofdeveloping PC, or alternatively altering the risk of the malignancyamong individuals with certain predisposing factors. However, moststudies to date have not revealed any significant relationship betweena SNP and the risk of PC in the general population. One study evenexamined the entire mitochondrial DNA for SNPs associated with therisk of PC [59], while another examined for possible correlation withsurvival [60], but none was observed. A recent study employing 178PC and 182 healthy controls identified a SNP in the gamma glutamyltransferase (GGT) gene that was significantly (pb0.05) associatedwith a risk of PC in a separate validation set [61]. A genomewideassociation analysis employing 3,851 PC and 3,934 healthy controlsrecently identified eight novel SNPs on chromosome loci 1q32.1 (5SNPs), 5p15.33 (1 SNP) and 13q22.1 (2 SNPs) that were significantlyassociated with a risk of developing PC [62]. The SNPs on 1q32.1 mapto the NR5A2 gene that encodes a nuclear receptor normallyexpressed (among other organs) in the exocrine pancreatic glands,while that on 5p15.3 maps to the CLPTM1L-TERT locus encoding twogenes that have both been associated with carcinogenesis. The SNP on1q32.1 however, mapped to a region devoid of any known genes.While these studies provide useful genetic markers to determine therisk of PC, whether (and how) the SNPs results in functionalalterations in the protein function and whether they act indepen-dently or in association with other risk factors remains to beinvestigated.

There appear to be an association between SNPs in DNA repairgenes and the risk of PC, specifically among smokers. An example isthe significantly increased risk of PC, among heavy smokers (definedas those with a history of≥22 pack years) who harbored an Arg188Hispolymorphism in the XRCC2 gene [63], while an aspartate (Asp) toasparagnine (Asn) polymorphism (genotype Asn312Asn) in exon 10 ofthe XPD gene significantly reduced the risk of PC ( O.R.:0.42 and 0.46compared to an Asp312Asp and Asp312Asn genotype respectively) onlyamong smokers [64]. Compared to PC-free individuals with the

312Asn-751Lys haplotype, patients with PC were also reported tohave a greater likelihood of an 312Asp-715 Gln haplotype at the XPDgene (O.R. 3.0, 95% C.I.: 1.3-6.9) in the same study.

As more and more genetic alterations are described, one hope isthat, in the future, a chip-based assay (similar to microarrays) can bedeveloped to screen patients with family histories of cancer ingeneral, and PC in particular, to identify mutations that could helpidentify asymptomatic individuals at high-risk for developing apancreatic malignancy.

4.3. Chronic pancreatitis

Although the true prevalence of chronic pancreatitis (CP) is notknown, it is estimated to range between 0.04% and 5% [65] (diagnosis,classification and genetics of CP excellently reviewed in [66]). There isa proven association between carcinoma of the pancreas and both thesporadic (chiefly tropical calcifying pancreatitis [67]) and hereditaryforms of CP (chiefly due to mutations in the PRSS1 [68] and cationictrypsinogen gene [69]), the standardized incidence ratio for develop-ment of PC in CP cases being 14–18, which is further increased bycigarette smoking [70]. Results from a prospective case–control study[71] seem to suggest that CPmay be closely related to PC, although theanswer to the question of a cause-and-effect relationship is not clear(the link between CP and PC was discussed in a recent article [72]). Arecent meta-analysis has suggested that the interval between CP andPC is nearly 20 years, with pancreatitis occurring within 1–2 yearsprior to the diagnosis of PC usually resulting from tumor relatedobstruction of the pancreatic duct [73]. Transgenic mice wherein thehuman interleukin-1β gene is expressed under the control of a ratelastase promoter develop features similar to those of severe CP [74].However, although the older mice developed features of tubularcomplexes, there were no PanIN lesions or tumors. This model couldbe useful to examine the relationship between CP and PC in moredetail. The possible role of pancreatic stellate cells, fibroblast-like cellsthat are normally quiescent but get activated during inflammation, inCP-associated carcinogenesis has also been suggested (reviewed in[75]). In support of a possible link between the two pathologies wasthe observation that abnormalities suggestive of CP were far morecommon (N70%) in patients who were classified as being at “high-risk” for PC compared to the prevalence of these changes in controlsubjects (b20%). Further, the study also found that “high-risk”patients, i.e. those with a family history of PC (defined as the presenceof PC in at least two first degree relatives or presence of an inheritedmutation known to predispose to PC) were more likely to haveabnormalities suggestive of CP on endoscopic-ultrasound (EUS) thanthose without such a history (odds ratio: 17.4). Significantly, theincidence of high-grade CP findings upon EUS examination (gradedaccording to the Cambridge classification [76]) was significantlygreater in the “high-risk” patients (53% showed moderate grade and5% severe grade chronic pancreatitis) compared to the controls (4%moderate grade and none with severe grade chronic pancreatitis). Afollow-up of 223 patients with non-hereditary CP (N70% of whichwere attributed to alcohol) revealed that the incidence of PC amongthese patients was nearly 6% during a 14-year period [77]. Notably,these patients were also at an increased risk for gastric andesophageal carcinoma, although the risk was less than that formalignancy of the pancreas.

Kras mutations have been demonstrated to occur in only a smallpercentage (4.4% of 429 micro dissected lesions) of non-hereditary/sporadic CP (in the absence of any mutated p53) in a study of 30resected specimens. Acute inflammation of the pancreas (acutepancreatitis) in the setting of a pre-existing activating Kras(G12D)mutation, however, resulted in rapid progression of PanIN lesions andaccelerated development of adenocarcinoma in a mouse model of PC[78]. This suggests that there is interplay between Kras activation andthe inflammatory cascade (possibly through cytokines) that enhances

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the risk of malignant transformation in the pancreas. Activatingmutations in Kras have been identified in the pancreatic juice andtissues from CP cases [79]. It would be of interest to examine whetherpatients with CP who have mutations in Kras and develop recurrentattacks of pancreatitis are at an accelerated risk of developing invasiveadenocarcinoma. This information would be crucial to make adecision on surgical removal of the diseased pancreas in theseindividuals.

Several studies have found that CP and PC are very similar in theprofile of genes expressed in them [80,81]. One study [82] found that aset of seven genes could discriminate between the two pathologieswith an accuracy of 92% in a randomly assigned training set. Alphaintegrin (α6β4 integrin) was shown to be highly expressed in PC andPanIN lesions but weakly in the normal pancreatic ducts and CPtissues, suggesting its potential use as a tissue based marker todistinguish the two closely related pathologies. Proteomics basedapproaches have revealed that Maspin [83], MUC4-p53 combination[84] Annexin-2 and Insulin-like growth factor binding protein 2(IGFBP-2) [85] are differentially overexpressed in PC compared to CP.A Western blot array (Powerblot, BD Biosciences) analysis of pooledprotein samples from normal, CP and PC derived pancreatic tissuesidentified more than 50 proteins that were upregulated in PCcompared to CP, while an almost equal number were downregulated[86]. Fig. 1 lists genes that were differentially expressed between PCand CP by a magnitude of at least five-fold.

However, several of these studies suffer from limitations, mostlyrelated to the study design (small sample size, no distinction betweenearly and late or resectable vs. unresectable PC) and lack of validation

Fig. 1. Recent update on proteins differentially expressed between chronic pancreatitis anddifficulty in distinguishing foci of adenocarcinoma in the setting of an underlying chronic pexpression. The figure shows the genes that are differentially upregulated specifically in chrnames of the respective proteins (http://www.genenames.org/). Only proteins that were fouby quantitative real-time RT-PCR. ¥Proteins that were validated by immunohistochemistry

of potential markers. In most cases, only a small percentage of themarkers identified were validated in a test set (which sometimes wastoo small in size). Thus, it is important that the results of these studiesbe first validated in blinded samples at multiple centers to identify themost promising molecules.

4.4. Type II diabetes mellitus

The relationship between adult onset diabetes mellitus, especiallywithin the first three years after the initial diagnosis, and thedevelopment of PC has been quite murky. The central questionremains: which came first, the diabetes or the cancer? While there isno denying the fact that the two do seem related, there does not seemto be a simple relationship between them. A study by Egawa et al.comparing patients with a family history of diabetes with thosewithout showed that pre-existing diabetes (≤3 years duration), wasmore likely to be associated with or lead to PC. Further, PC patientswith a family history of diabetes in a first degree relative were onaverage at least 5 years younger at diagnosis (61±9 vs. 65±11 years), had masses predominantly involving the body and tail ofthe pancreas (consistent with the higher concentration of beta cells inthese regions), and had cancer of a non-tubular type (includingintraductal papillary mucinous cancer, adenosquamous carcinomaand mixed ductal-endocrine cancer) [87]. In a nested case–controlstudy in a population of residents of Rochester (Minnesota) aged50 years and above [88], Chari and co-workers identified that amongnewly diagnosed type II diabetics, the risk of developing PC within thefirst three years was almost eight-fold higher than that in the general

pancreatic cancer. A major problem in the early diagnosis of pancreatic cancer is theancreatitis. Further, the two processes share many genes that show a similar pattern ofonic pancreatitis (left) and pancreatic cancer (right). The symbols represent the HUGOnd to be up to five-fold or more are included. *Proteins whose expression was validated(adapted from [198]).

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population. Perhaps, the most significant finding from the viewpointof surveillance was that 10 of the 18 patients diagnosed with PC in thestudy were diagnosed within six months of meeting the criteria fordiabetes. Further, only seven of the 18 diabetics who developed PChad a family history of diabetes. Notably, nine of the 18 patients hadsymptoms suggestive of malignancy at the time when they first metthe criteria for diabetes, though only three of the 18 cases wereresectable at the time of presentation (with PC). The proportion ofever smokers was also considerably higher among those diabeticswho developed PC (95% vs. 69% in those without PC) in this study. Arelative lack of a link, however, between adult onset diabetes and therisk of PC among FPC kindreds was also reported elsewhere [32]. Thecase–control study in Italy alluded to earlier [30] also examined therisk of PC among diabetics, specifically those who were on treatment.Diabetic patients who were on treatment but did not develop PC weretaken as controls in this study. The presence of diabetes emerged as asignificant risk factor for subsequent diagnosis of PC (relative risk, R.R.: 2.89 (95% C.I. :1.71–4.86)). A key observation was that while themean age of onset of diabetes mellitus (55.7 years vs. 55.4 years) andthe duration of the disease (6.6 years vs. 9.2 years) were comparablebetween cases and controls, PC cases were more likely to havereceived treatment for diabetes within 2 years before the diagnosis ofPC (R.R.: 4.61, (95% C.I. 1.99–11.53)). Further, the risk of PC decreasedprogressively with the time elapsed between the diagnosis (andtreatment) of DM and the diagnosis of PC (R.R.: 2.41 for casesdiagnosed 3–5 years before diagnosis of PC vs. 2.06 for casesdiagnosed more than 5 years before the diagnosis of PC). Notably,the type of treatment also appeared to influence the risk of PC withpatients on insulin at a significantly higher risk than those treatedwith oral hypoglycemics (R.R.: 7.68, 95% C.I.: 1.27–17.22). Mostsignificantly, diabetic patients treated with insulin for more than fiveyears were at a significant risk for developing PC (R.R.: 6.21, 95% C.I.:1.61–23.96). However, patients treated with oral hypoglycemics didnot show any increase in risk, even after five years of treatment.

4.5. Cystic neoplasms of the pancreas

Cystic tumors of the pancreas can be divided into two principaltypes— serous and mucinous cystic neoplasms. Serous neoplasms aregenerally benign. Mucinous cystic tumors, on the other hand, arepotentially malignant. Invasive cancer arising in a mucinous cyst(mucinous cystadenocarcinoma) is a lethal disease with a prognosissimilar to that of pancreatic adenocarcinoma [89]. In one study, allpatients identified with unresectable adenocarcinoma in cystic mucinproducing tumors of the pancreas were dead within 2–20 months ofdetection. Conversely, those with benign tumors (hyperplasia oradenoma) were alive without recurrence after surgery. In anotherretrospective analysis of 84 cases of cystic neoplasms at a singlecenter, none of the 77 patients without invasive carcinoma developedrecurrence following surgical resection of the lesion [89]. In sharpcontrast, five of the six who survived surgical resection forcystadenocarcinoma died of recurrence within five years of theirsurgery. All the five patients had 5–10 cm sized tumors. The solesurvivor had a singly microscopic focus of invasive carcinoma. Inanother single center study, 20% of patients with symptomatic cystswere found to have an associated adenocarcinoma on histopathologicexamination of the resected specimen in comparison to only 5% forincidental cysts [89]. Further, in this study, EUS was able to identifycysts in eight cases that were missed by both computerizedtomography (CT) and magnetic resonance imaging (MRI). Thus, theincidence of neoplastic changes is nearly four times greater insymptomatic cysts than silent ones, suggesting that patients in thisgroup are good candidates for close long-term follow-up.

Consequently, early detection of cystic tumors of the pancreaswarrants continuous follow-up to identify nascent changes associatedwith malignancy. However, hyperplastic and adenomatous tumors

are unremarkable in their features, being almost always less than 3 cmin size, associated with a main pancreatic duct (MPD) diameter lessthan 3 mm, have a characteristic absence of mural nodules and lack ofmucin extrusion from the ampulla of Vater. Cytology is also non-informative and serum CA 19-9 as well as CEA are usually withinnormal limits. However, in intraductal tumors (IDT), pancreatoscopyduring endoscopic retrograde cholangiopancreatography (ERCP) canvisualize the MPD and also measure its diameter.

5. Role of imaging in the early diagnosis of pancreatic cancer

5.1. Endoscopic-ultrasound

CP is considered a risk factor for PC. However, most cases of PCprobably arise sporadically in a pancreas afflicted by changes of CP[90,91]. PC in the setting of CP presents a diagnostic challenge owingto several reasons. First, CP often mimics changes seen in ductalcancer and the converse is also true. Further, the chronic inflamma-tion accompanying CP can often obscure a small malignant focus.These reduce the sensitivity and specificity of even endoscopicultrasound (EUS) guided fine needle aspiration (FNA), necessitatingadditional criteria or markers to distinguish foci of malignant changein the background of CP. One clue comes from the observation that CPseldom presents with obstructive jaundice [92]. So, the presence ofconcomitant jaundice in a patient with risk factors for CP (e.g. male,age b50 years, African-American descent with/without a history ofalcohol intake) should alert the radiologist to search carefully forminute foci of malignant change. Measures that increase the chancesof detecting isolated dysplastic/neoplastic foci include increasing thenumber of passes of the needle during EUS-guided FNA and surgicalexploration in FNA-negative cases with a high index of suspicion.However, EUS-FNA should not be performed in those patients with CPwithout an apparent mass, due to the risk of procedure relatedpancreatitis.

A prospective study by Le Blanc et al. [93] showed that theminimum number of passes of the needle during an EUS guided FNAto get an optimal diagnosis of a suspected pancreatic tumor is seven,while it is five in case of lymph nodes. By using this approach, theyachieved a sensitivity of 83.3%, with specificity, positive (PPV), andnegative predictive value (NPV) of 100%, 100% and 50% respectively.In the case of lymph nodes, the sensitivity, specificity, PPV, and NPV offive or more passes of the needle were 77%, 100%, 100% and 81%,respectively. This study also suggested that the lack of a cytopathol-ogist at the bedside was one of the key reasons for an inadequatesmear. Surgical histopathology and a one year clinical follow-up wereused as reference standards for this study [93].

Despite advances in other imaging techniques, EUS-FNA hasincreasingly become the gold standard for confirming the diagnosisof PC without subjecting the patient to surgery. A recent study [94]examined the performance of contrast-enhanced power Doppler(CEPD) and real-time sonoelastography (RTSE) in distinguishingbetween CP and PC. While a combination of the two techniquesdiagnosed PC with a sensitivity, specificity, accuracy, PPV and NPV of76%, 95%, 96%, 71% and 83% respectively, EUS-FNA still performedbetter (comparative performance 88%, 100%, 100%, 84% and 93%respectively).

Early detection of pancreatic neoplasms would logically requirethe detection of small lesions. In a retrospective study of over 1000FNAs conducted at a single institution, it was reported that EUS-guided FNA was more sensitive (86.5% vs. 80%), had comparablespecificity (97.3% vs. 100%), and PPV (97% vs. 100%), higher NPV(87.8% vs. 50%), fewer false negatives (6.8% vs. 16.7%) and a higheraccuracy (91.9% vs. 83.3%) than abdominal ultrasound-guided FNA indetecting intra-pancreatic lesions less than 3 cm in diameter.Moreover, the rate of inadequate specimens was significantly reducedby EUS-guided FNA (2%) as compared to percutaneous US or CT

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guided biopsy (18.5%). Several factors, including the presence of anon-site cytopathologist as well as the experience of the endoscopistand radiologist, can influence the accuracy of EUS-FNA. Given theconsiderations of time and cost, it is recommended that for patients atrisk of PC (from history and presence of risk factors as outlined inTable 1), EUS-FNA is the screening test of choice to detect lesionssmaller than 3 cm, even if abdominal US or the CT scan is negative. Forlarger lesions, there is no obvious difference in the three techniques,so a conventional US or CT guided percutaneous FNA to obtain tissuefrom suspicious masses is advisable in the interests of cost, time, andavailability of trained personnel. It is further recommended that allcases of atypical/inconclusive and suspicious FNA should be closelyfollowed by repeat biopsy or surgery if necessary, given that asignificant number of them progress to ductal cancer (82.2% and 58.6%of the atypical and suspicious cases, respectively, in one study) [95].

EUS has emerged as the frontrunner among various imagingmodalities being tested for screening patients to detect indolentpancreatic neoplasms. In a prospective study on high-risk patientsaimed at detecting asymptomatic PCs [71], EUS was more sensitivethan CT scan and endoscopic retrograde cholangiopancreatography(ERCP) in identifying silent pancreatic neoplasms. This study alsoconfirmed the safe nature of EUS, with mild post-procedural pain asthe only major complication. It is interesting to note that branch-typeintraductal papillary mucinous neoplasm (IPMN) was the mostcommon pancreatic neoplasm identified by EUS. Further, the IPMNadenomas identified were most frequently associated with PanINlesions of varying grades andwith CP (10 out of 12 patients with IPMNin another study had symptoms of CP [96]). In one high-risk patient,the IPMN lesion (obtained after surgical resection) was associatedwith carcinoma-in situ. It has previously been reported [97] thatseveral genes that are highly upregulated in PC are also similarlyderegulated in IPMNs (including lipocalin-2, galectin-3, claudin-4 andcathepsin E). These studies strengthen the premise that IPMN is aprecursor of invasive adenocarcinoma. A second high-risk patient inthis study [71] had a branch-type IPMN lesion with multiple foci ofPanIN-3 (high-grade dysplasia) and what appeared like adenocarci-noma. In another case report, Nüssler and co-workers reported that a54-year old womanwithmargin-negative, non-invasive IPMN treatedby segmental resection subsequently developed an adenocarcinomain the tail of the pancreas three years later [98]. A prospective study of12 patients with IPMN (referred to earlier [96]) observed extensivemultifocal intraductal changes involving most or all of the pancreas insix of the ten patients who underwent surgery for removal of thepancreas. The study concluded that while IPMNs in general have afavorable prognosis, a diffuse dilation of the main pancreatic ductusually indicates widespread involvement of the duct by the tumorand warrants total pancreatectomy.

A study focusing on cystic neoplasms of the pancreas reported thatIPMNs were the most common diagnoses among patients undergoingsurgery for a cystic lesion (either due to symptoms or other factorsincluding increasing size, patient anxiety, or doubtful findings uponimaging), being present in nearly 50% of the resected specimens [99].Notably, 38% of the main duct-IPMNs had an associated invasivecomponent.

5.2. Recent advances in pancreatic imaging

Many modalities are available to image the pancreas includingnon-invasive techniques like ultrasound, contrast-enhanced multi-detector computed tomography (CECT), magnetic resonance imaging(MRI), integrated positron emission tomography/computed tomog-raphy, and invasive techniques, like endoscopic retrograde cholan-giopancreatography (ERCP) and endoscopic-ultrasound (EUS)(reviewed in [100] and [101]). Advances in cytology including digitalimage processing and fluorescence in situ hybridization (FISH) areemerging as useful adjuncts to conventional cytology in the early

detection of PC. In a study of 446 patients, 80% of thosewith polysomicsignal on FISH (performed on ERCP brushings) were found to have PCby cytology, and nearly all were diagnosed with PC within 2 years ofidentification of a polysomy on FISH [102].Semiconductor nanocrys-tals or quantum dots (QDs) have emerged as novel agents for bio-imaging owing to several advantages over traditional organic dyebased imaging techniques including a higher quantum yield, greaterstability and a wider range of excitation wavelengths (ranging fromthe visible to near-infrared) that can be easily modulated by changingphysical characteristics (size, shape and composition of the nanopar-ticle). A major development has been the recent development of non-cadmium based QDs which were non-toxic to PC and immortalizedhuman cells over a wide concentration range [103]. These indiumphosphide-zinc sulphide QDs can be conjugated to biomolecules likeantibodies and have been shown in vitro to target specifically to thecells expressing the specific antigen. Plectin-1, recently identified as amarker for PC, was recently shown to be a specific marker todistinguish PC from CP immunohistochemically, while plectin-1-targeted peptides conjugated to magnetofluorescent nanoparticles(PTP-NP) allowed the detection of small PC by ex vivo MRI ingenetically engineered mouse models [104]. Manganese-(Mn) dopedquantum dots (MnQDs), which emit light in the near-infrared region(700 nm–1 μm) and can be solubilized stably in aqueous media (bysurface functionalization with lysine) and also conjugated tobiomolecules, have also been recently developed [105]. TheseMnQDs conjugated with anti-claudin 4, anti-mesothelin or anti-PSCA (prostate stem cell antigen) monoclonal antibodies wereinternalized into the PC cells Panc-1 and MiaPaca without significanttoxicity to the cells. When injected into mice, the MnQDs produced apeak at 828 nm, which was distinct from the autofluorescenceproduced by the mouse tissues. Importantly, a dose nearly fivetimes the routine dose for QDs used in imaging breast cancer cells insmall animals did not produce any toxicity or behavioral change inmice even 12 weeks after a single injection. These studies, althoughpreliminary, point toward exciting new developments that are poisedto provide better resolution imaging (employing QDs conjugated toproteins that are expressed by high-grade dysplasia, the stage ofprecursor lesion most definitely linked to risk of infiltratingadenocarcinoma). Low-coherence enhanced backscattering (LEBS)and elastic light scattering fingerprinting (ELF) based opticalmeasurements of the apparently healthy periampullary duodenaltissue have shown that high-risk cases (including those with a familyhistory) generate signals that lie in between that obtained fromhealthy individuals and those with PC [106], suggesting the utility offield-effects as useful tools to identify early malignancy.

6. PanIN lesions as a source of potential biomarkers

Analysis of differentially expressed genes in various PanIN lesionsisolated from patient tissue samples by micro dissection has revealedthat PanIN-2, and not PanIN-1 is the first stage in the development ofPC that is associated with significant genetic changes (PanIN-1b, 2, 3and ductal cancer showed 47, 438, 578 and 610 differentiallyexpressed genes respectively) [107]. Three main groups of differen-tially expressed genes were recognized in this study: those whoseexpressionwas lost from normal ducts very early during pathogenesis[including the genes for putative cytokine high in normal 1(SCGB3A1), amiloride sensitive cation channel 2 (ACCN2), and thetransmembrane mucin MUC13], genes whose expression was elevat-ed beginning from the stage of PanIN-1b and maintained until PC[including S100 calcium-binding protein (S100P), the trefoil factors 1and 2, and matrix metalloproteinase 1 (MMP 1)], and those for whichan increased expression was first detected only in the late stages ofdysplasia (i.e. PanIN-2, 3 or PC), which included the serine threoninekinase 11, fibronectin 1 and plastin 3 genes. Further, two additionalpatterns of gene expressionwere identified by the authors. A transient

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change in the expression of some genes was detected in the earlyPanIN stages that returned to levels comparable to normal tissue inthe later stage of pathogenesis. A more persistent pattern ofdifferential gene expression was also noted, particularly for genesimportant inmaintaining the cellular structure and those important indevelopment/differentiation. The transient change in gene expressioncan be explained by an activation of counter-regulatory genes that areturned on in response to early mutations that impair normal cellularfunctions. However, in the later stages of cancer progression, many ofthe normal control mechanisms in the cells are inactivated,presumably accounting for the reversal of the genetic changes seenin the earlier PanIN stages. The more persistent changes in expressionusually effect genes that are important in the remodeling of theextracellular matrix, an important change during tumorigenesis(these include upregulation of MMP 7, fibronectin 1, and type 3collagen as well as downregulation of MMP17). Based on the premisethat PanIN-2 is the earliest precursor of invasive ductal adenocarci-noma, the authors selected 22 genes that were significantlyupregulated in PanIN-2 compared to the normal ducts as potentialbiomarkers for early diagnosis. However, a major drawback of thisstudy was the lack of any chronic pancreatitis tissues in the analysis.As discussed earlier, CP shares several markers in common with PC(and PanINs) and thus one of the goals of biomarker discovery for theearly diagnosis of PC is to exclude proteins that are common to CP.One of the earliest altered oncoproteins is the mucin MUC1.Immunostaining with the PAM-4 monoclonal antibody againstMUC1 specifically labeled the dysplastic ducts but not the normalones, with a progressive increase from low to high-grade PanINs inone study [108].

An ideal biomarker, from the standpoint of both the patient andthe physician, is one that can also predict the prognosis following anearly diagnosis. One such possible marker is the epithelium specificmarker cytokeratin-20 (CK-20). Belonging to the category of acidicintermediate filaments, CK-20 is not expressed in normal fetal andadult pancreas, but is aberrantly expressed in 30–60% of pancreaticductal adenocarcinomas [109]. Further, it is also expressed in PanINlesions, but only when the associated cancer is also positive for CK-20.It is more commonly expressed in higher grade PanIN's (88% in PanIN-2 and 3 combined vs. 60% in PanIN-1). This suggests that in areasonable fraction of ductal cancers, the aberrant expression of CK-20 precedes the development of invasive cancer. Screening high-riskpatients for CK-20 expression in the pancreatic juice or in FNAspecimens could identify a subgroup of individuals with asymptom-atic changes of PanIN in their pancreas. CK-20 also has a prognosticrole, as CK-20 expressing cancers appear to have a significantly poorprognosis. In one study, none of the patients who expressed CK-20 intheir cancerswas alive beyond 26 months [110]. CEACAM-6 is anotherprotein which is strongly expressed in the PanIN-3 lesions, and likeCK-20, an inverse relationship is observed between CEACAM-6expression and survival of PC patients, with non-expression (in theprimary tumor) translating into the absence of nodal metastasis and alonger post-operative survival [111]. Class-III β tubulin (TUBB3) hasalso been shown to be absent in non-neoplastic ducts, but increasinglyexpressed with advancing grades of PanIN, reaching the highestexpression in adenocarcinoma [112].

A recent proteomic analysis [113] using micro dissected cells fromPanIN lesions of nine PC patients identified 31 proteins that weredifferentially expressed at one or more stages of intraepithelialneoplasia (compared to the non-neoplastic ducts). Immunohistochem-ical validation of five of these proteins (major vault protein (MVP),anterior gradient homolog 2 (AGR2), 14-3-3-sigma, annexin-4 andS100A10) was also done suggesting that these markers in combinationcould be used to correctly identify PanIN lesions, most pertinently inFNAs and cytopathological specimens. A similar approach (but usinggross rather than micro dissected specimens of normal pancreas,chronic pancreatitis, PanIN-3 and PC followed by mass spectrometry)

was adoptedby Pan et al. [114]. Using a cut-off of a≥1.75-fold change inPanIN-3 compared to the normal pancreas, 70proteinswere found tobeupregulated and 133 downregulated in PanIN-3 (compared to thenormal ducts). Interestingly,most of the enriched proteins dysregulatedin PanIN-3 lesions were related to cellular motility and remodeling ofthe cytoskeleton. As the actin cytoskeleton plays a central role in cellularinvasion,motility andmetastases, the findings of this study suggest thatdysregulation of proteins associated with cellular invasion (a changetypical of cancer) begins early on, i.e. in the high-grade dysplasticlesions. Particularly interestingwas thedysregulationof 18proteins thatare known to directly interact with c-MYC, a well known oncogenewhich is involved in nearly one-fifth of all human cancers. One of theproteins, β-tubulin, which was shown to be upregulated both in PanINand chronic pancreatitis tissues, was also shown to be differentiallyexpressed during the progression of PC in anearlier study [115]. Someofthe proteins (identified in [114]) were also found to be dysregulated insimilar direction in a study by Sitek and co-workers [113] (e.g. TPM2,EEF1A1). However, while Sitek et al. had observed a downregulation ofAnnexin A-IV (ANXA4), Pan and colleagues [114] found an upregulation(≈2.5-fold) in the gene in PanIN lesions. The study [114] also validatedthe overexpression of Laminin-β1, actinin-4 and galectin-1 by immu-nohistochemistrywhich revealed that while all threewere expressed inthe stroma (actinin-4 only in PC and galectin-1 in both PanIN and PCstroma), only actinin-4 was also overexpressed by the ductal epithe-lium. Fig. 2 depicts the proteins whose expression is altered during theprogression of PC from normal ducts to high grade dysplasia (PanIN-3).One of the promising markers for early stage pancreatic dysplasia isclaudin-18, which belongs to the family of tight junctional proteins.Claudin-18 is not expressed by the non-neoplastic ductal cells, butexpressed in low grade PanINs (PanIN-1)with a progressive increase inexpressionuntil infiltratingadenocarcinoma [116]. Further, a stronganddiffuse expression of claudin-18 by the PCs is also associated with abetter survival in PC patients suggesting its additional utility as aprognostic marker in ductal adenocarcinoma.

While the detection of biomarkers for PanIN lesions is the correctapproach toward realizing the dream of one or more sensitive andspecific biomarkers for pancreatic dysplasia, it is important to realizetwo things. First, PanIN lesions are quite frequent among patientswith chronic pancreatitis (and in one report, in serous cystadenoma[117], a benign cystic tumor of the pancreas). Second, the time fromthe onset of PanIN lesions to the development of invasive adenocar-cinoma and the absolute risk of PC with a given grade of PanIN is stillunknown. In one study [118], only one out of 9 patients with PanIN-3lesions in the setting of sporadic CP developed invasive adenocarci-noma after nearly 10 years. In the same study, none of the patientswith PanIN-2 (n=11) or PanIN-1 (n=31) developed ductaladenocarcinoma during follow-up. Further, the mean duration of CPin this cohort of patients was 8 years. This suggests that it takes nearlytwo decades from the time of onset of CP to develop invasiveadenocarcinoma and that only a few patients (about 10%) with high-grade dysplasia go on to develop invasive malignancy. More studiesare needed to clarify the association between CP, incidental pancreaticdysplasia and the risk of developing invasive adenocarcinoma.

Hisa et al. [119] in a study on small (≤2 cm diameter) ductalcarcinoma of the pancreas found that PanIN-3 lesions were commonlyspread outwithin 2.5 cm from the edge of themainmass (less than 25%were beyond 1 cm from the edge of the tumor). On the other hand,PanIN-2 lesions, although found adjacent to the mass (only 50% weremore than 1 cm beyond the mass edge), were discontinuous with themass and/or the PanIN-3 lesions. PanIN-1 lesions were found to bedistributed in a sporadic manner throughout the pancreas (nearly 90%of PanIN-1a and 95% of PanIN-1bweremore than 1 cm from the edge ofthe cancer). This study suggested that given their close proximity toinvasive adenocarcinoma, PanIN-3 lesions represent an intraductalextension of the cancer. Hence, surgical resection of, small (≤2 cm) PCsshould include, a margin of at least 11 mm (from the mass edge).

Fig. 2. Recent update on genes with a differential expression in the pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions. Pancreatic cancer develops from a series ofpre-malignant lesions termed as PanINs. There are four grades of PanIN— PanIN-1a, 1b, 2 and 3. Genes that are differentially expressed in PanIN lesions hold significant potential inthe early detection of adenocarcinoma of the pancreas. The figure shows genes that show a significant up- or downregulation in these precursor lesions compared to the non-neoplastic ducts of the pancreas. The gene name in the figure refers to its Entrez Gene ID (http://www.ncbi.nlm.nih.gov/gene). ¥Based on reactivity to the PAM-4 monoclonalantibody to MUC1. £Examination of S100P mRNA in micro dissected PanIN and PDAC tissues did not show any difference between PanIN and PDAC (adapted from [6,181,190–197]).

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A second model proposed for the development of PC is thetransformation of acinar cells into malignant ductal cells [120].According to this model, pancreatic acini transform gradually intoductules, a process accompanied by a loss of their enzyme-producingcell characteristics (including loss of prominent endoplasmic reticululmand zymogen granules, and loss of chymotrypsin reactivity), a reductionin cell height (manifesting as an enlargement of the lumen) andaccompanied by a fibro inflammatory reaction in the surroundingstroma. However, tubular complexes (as these precursor lesions arecalled) are also observed in CP and serous cystadenoma. Interestingly,while PanIN-1 and 2 lesions were also observed in the same sections,PanIN-3 lesionswere found only in associationwith PC and not in any ofthe 42 CP or 18 serous cystadenoma sections examined in the samestudy [120]. The acinar model of PC origin received further credencewhen a recent study [121] showed that knockout of an acinus restrictedtranscription factor Mist1 in KrasG12D expressing mice led to anaccelerated development of mPanIN (mouse PanIN) lesions in thepancreas and transformation of acinar cells into a ductal cell phenotype(in vitro) associated with an activation of epidermal growth factorreceptor (EGFR) andNotch signalingpathways in the transformingcells.

7. Biomarkers in body fluids

7.1. Serum and plasma

Serum and plasma (serum without fibrinogen and other clottingfactors) remain the most easily accessible tissues for diagnostic testingand, hence are an attractive medium for biomarker testing to screen for

early stage disease. An advantage of using serum is that it represents theproteins released from both the tumor cells and stroma, therebyincreasing the number of potential markers. However, studies toidentify potential markers of disease have been hampered by thedifficulty in isolating and identifying the low-abundance proteins.Immunodepletion of the 12 most abundant proteins, followed byfluorometric 2-DIGE (2-dimensional gel electrophoresis) and massspectrometry on plasma samples collected from patients with anestablished diagnosis of PC (twowith stage I, sevenwith stage II and onestage III disease) at three time points — just before surgery, 10 weekspost-operative and just before commencement of chemotherapy,identified two sets of proteins with potential prognostic significance[122]. The first set was comprised of 14 proteins that correlatedpositively with the tumor burden (these decreased after removal of thetumor), while the second set included eight proteins that wereselectively elevated in patients who had progression of the disease(defined as either recurrence of the tumor or death) compared to thosewithout any detectable tumor one year after surgery (summarized inFig. 3). Notably, while both the stage I patients were alive at one year,four of the stage II patients were dead from recurrence, reinforcing theurgency of an early diagnosis of PC. Another study identified mannose-binding lectin-2 (MBL2) andmyosin light chain kinase (MLCK) as beingsignificantly upregulated proteins in the serum of PC patients byfluorometric 2-DIGE followed by tandemMS and validation byWesternblotting [123]. The major drawback of this study however was that itonly included a single stage 1 patient who showed a significantelevation of MLCK but not MBL2 in his serum. An analysis of serumsamples from patients with PC (stages I–IV), benign pancreatic diseases

Fig. 3. Recent update on proteins whose expression correlates with tumor burden and clinical outcome in patients with pancreatic cancer. The prediction of tumor burden and earlyidentification of recurrence remain two key challenges in patients with established pancreatic cancer. Proteins that can predict these events are immensely useful in devisingprognostic algorithms, tailoring existing treatment strategies and devising new strategies for the treatment of pancreatic adenocarcinoma. The figure depicts proteins that have beenidentified as indicators of tumor burden (left) and recurrence or progression of disease (right) in pancreatic cancer. The unique NCBI identifiers for the proteins are as follows: C3(protein accession number: NP_000055.2), C4A (GenBank: AAA51855.1), CFH (Swiss-Prot: P08603.4), A1BG (PIR: 69990), GC (NP_000574), APOA4 (GenBank: AAA51748.1),SERPINF1 (GenBank: AAA60058.1), HPX (GenBank: AAH05395.1), β-2 microglobulin (GenBank: CAA23830.1), α-2 macroglobulin (PRF: 224053), α-2 microglobulin (GenBank:CAI15899.1), plasminogen (GenBank: AAH60513.1), α-2 HS glycoprotein (GenBank: BAA22651.1), serum albumin precursor (GenBank: AAF01333.1), and C1q B-chain precursor(GenBank: CAA26880.1).

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(including pancreatitis, serous cystadenoma, pancreatic pseudocyst,ampullary adenoma and diverticulitis), and healthy controls (high-riskindividuals from FPC kindreds undergoing surveillance) by antibodymicroarrays (using a two-color rolling circle amplification) revealedthat a set of proteins present in the serum could distinguish samples ofPC cases from thosewith benign diseases (anti-protein induced VitaminK antagonist-II (PIVKA-II) and CA15-3) and from healthy individuals(C-reactive protein, PIVKA-II, α1 antitrypsin, IgA, cathepsin D andalkaline phosphatase) with more than 90% sensitivity and specificity[124].

Another recent study identified elevated phospho-ERK1/2 levels inthe serum of PC patients as a potential adjunct to CA19-9 in thediagnosis of PC. However, there was a considerable overlap in theserum levels of ERK-1/2 and other phosphoproteins between patientswith PC and those with pancreatitis, which together with the lack of aspecific cut-off for levels of these phosphoproteins and the smallsample size of patients with resectable PC suggests the need forvalidation of the role of phosphoproteins as early markers of PC [125].These studies, though preliminary have established that antibodymicroarrays, like gene microarrays, are a valuable tool to identifydifferentially expressed proteins with diagnostic potential in theserum and plasma.

An observation often reported in literature is the discrepancybetween the level of expression of a protein and that of its transcriptfor a given type of cell. A good example is a study to identify thesecretomeof PC cells (Panc-1 chosen as prototype) by comparing itwiththat of normal pancreatic ductal cells (HPDE cells) [126],wherein nearly50% of the proteins did not show a correlation between their mRNA andprotein levels (overall correlation between RNA and protein expressionin this study was 0.28). Certain proteins may also show a reciprocalvariation in theirmRNAandprotein levels. For instance, the CD9 antigenwhich was upregulated nearly eight-fold in the Panc-1 secretome wasdownregulated two-fold at the mRNA level. One explanation for thisdiscrepancy between transcript and protein level could be the relativepreponderance of a post-transcriptional regulation in the case of certain

proteins (e.g. cytokines), which might explain the relative difference inthe abundance of the two forms. From the diagnostic standpoint, thisobservation underscores the importance of performing an analysis ofboth the transcriptome and the secretome, each of which has a uniqueset of differentially expressed genes (or proteins respectively).

Using multivariate analysis procedures including classification,regression tree and logistic regression, it was demonstrated [127] thatsurface-enhanced laser detection/ionization time of flight massspectrometry (SELDI TOF-MS) could be used to generate fingerprintsof cancer cells from serum samples. In this study, a theoretical modelcould correctly classify all of the PC serum samples in a randomlyassigned “test” set (i.e. 100% sensitive), while the specificity wasabout 94%. Notably, the individual discriminatory proteins were notidentified. Fingerprinting readily accessible body fluids from cancerpatients and comparing it with a database of similar data from a largebase of normal samples could ultimately provide the solution to earlyidentification of small neoplasms that would otherwise be missed onimaging.

The presence of a biliary obstruction can alter the proteomicexpression in the serum/plasma of patients with benign pancreaticdiseases. This is perhaps best exemplified by the false elevation ofCA19-9 in the serum of patients with obstructive jaundice in theabsence of any malignancy. However, most studies on biomarkers inPC do not distinguish between non-PC patients with and withoutconcomitant obstruction of the biliary tract. One study by Bloomstonet al. [128] identified fibrinogen γ as being highly expressed in sera ofPC patients (mean concentration: 51 mg/dl in 32 PC cases vs. nodetectable levels in healthy controls). However, no correction wasmade for serum bilirubin levels nor did the authors discuss the stageof malignancy in their study population. Recent studies suggest thatSELDI-MS is significantly better than the traditional technique ofMALDI-TOF MS in identifying biomarkers from body fluids. A studyamong Chinese patients [129] concluded that the traditional proteo-mic technique of 2-DE followed by matrix assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry was rather

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inefficient at identifying low-abundance proteins in the plasma. Mostof the differentially expressed proteins in their study were acutephase reactants and high abundance proteins (haptoglobin, Immu-noglobulin-J, hemoglobin and α1-antitrypsin). SELDI-MS is designedto overcome these problems. In this technique, the proteins (as littleas 20 μl of serum/plasma) are captured, concentrated and purified onthe small chemical surface of a SELDI chip, followed by themeasurement of the molecular weight (obtained from the mass/charge ratio) and the relative intensity of each protein captured on thechip by sensitive time of flight (TOF)-MS. Using this technique and ahigh resolution QqTOF instrument, four proteins, producing masspeaks of 8766 m/z, 17,272 m/z, 28,080 m/z and 14,779 m/z werefound to discriminate PC patients from healthy controls with highsensitivity (97%) and specificity (95%) [130]. Upon validation in atraining cohort of PC (including 1 stage I and 4 stage II patients ofwhom four, including the stage I were correctly identified) andhealthy controls at two institutions, this set of proteins had asensitivity and specificity of 91%. When combined with measurementof CA19-9, all the PC samples in the validation set were correctlyidentified, while six of the 39 controls were also positive (falsepositive rate of 15%). The identity of the four peaks remainsunresolved, chiefly owing to the difficulty in purifying the proteinsfrom these low intensity peaks without contamination from thesurrounding proteins. The limitations of this study included a lack ofsamples from patients with either pancreatic inflammation (acute orchronic) or benign pancreatic diseases. Analyzing serum samplesfrom 126 PC, 61 CP, 24 type 2 diabetics and 12 healthy controls bySELDI-TOF/MS Navaglia and co-workers identified 219 peakscorresponding to low molecular weight proteins (m/z range 1007–9255) [131]. A decision tree employing the binary recursivepartitioning method identified three peaks with a m/z ratio of 1526,1211 and 3519 that could correctly classify 100% of healthy controls(from CP, PC and type 2 DMs), 73% of type 2 diabetics (from CP andPC) and 70% of CPs (from PC patients). However, a receiver operatingcharacteristic (ROC) curve analysis revealed that this combination ofSELDI-TOF/MS peaks was not better than CA19-9 either alone or incombination in discriminating PC patients from the other groups(AUC for CA19-9 being 0.89, for SELDI-TOF/MS peaks alone 0.81 andfor the combination 0.86). When patients with type 2 DM alone wereconsidered, a combination of CA19-9 and the three SELDI-TOF/MSpeaks could correctly identify 100% of patients (n-24) who had type 2DM in the absence of CP or PC. However, while 97% of PC cases werecorrectly classified, only 77% of CP cases were accurately discrimi-nated by the combination. It remains to be seen whether the proteinsidentified by these studies can be detected by other less technicallydemanding techniques or serum/plasma based assays for possibleapplication as screening biomarkers.

A serum based assay to identify the circulating mucin MUC1reactive to the monoclonal antibody PAM4 revealed that the assaywas able to discriminate between patients with PC and pancreatitis/healthy controls with high specificity [132]. However, the study hadseveral limitations. It was not clear as to whether the PC patientstested had early or advanced disease. There was also no mention ofwhether the specificity of the assay for patients with PC vs. those withbenign pancreatic disease with obstructive jaundice was examined.Moreover, while the assay was highly specific, its sensitivity was stilllow, making it a better confirmatory than a screening serological test.

Serum levels of tumor specific growth factor (TSGF) above a cut-offof N71 U/ml was found to be 92% sensitive (83% specific) for PC, whichwas higher than that of CA242 and CA19-9 [133]. Significantly, whenthe performance of the three markers was compared across differentstages of PC, a general trend emerged wherein the markers were leastsensitive in detecting stage I and most sensitive in detecting stage IVcases. Several factors could explain this observation. Most studies onbiomarker discovery employ stage II-IV patients, with a pooling of thesamples, hence selecting for markers that are elevated in advanced

cancer. Further, the larger tumor burden with increasing stage couldtranslate into greater production of the tumor marker and hence itsappearance in the serum. TSGF was still the most sensitive indetecting stage I patients (sensitivity of 60% compared to 30% forCA242 and 40% for CA19-9), suggesting that it could be potentiallyuseful in identifying early stage disease. Interestingly, the level of twoserum markers (TSGF and CA242) was higher in patients with cancerof the head of the pancreas compared to those involving the body, tailor whole gland. PCs in the head are known to present earlier owing toobstruction of biliary outflow, while those in the body and tail areusually silent until the advanced stages.When the threemarkerswerecombined together (TSGF N71 U/ml, CA242 N20 U/ml and CA19-9N37 U/ml) they were 100% specific for the diagnosis of PC, while nomarker alone could reach perfect specificity. The onlymajor limitationof this study was not investigating the specificity of these markers inthe presence of benign conditions causing obstructive jaundice.

Macrophage inhibitory cytokine (MIC-1), a member of the TGF-βfamily is elevated in the serum of patients with PC. However, it is notvery useful to distinguish PC (specifically resectable cases) from CP(specificity of 44% compared to 86% for CA19-9 in one study [134]).The presence of diabetes or jaundice could not explain its lowspecificity in the cases examined. However, it was highly sensitive inidentifying PC cases (90% each in distinguishing PC from normal andchronic pancreatitis, respectively) which was better than that ofCA19-9 (62% sensitivity). Hence, MIC-1 could hold potential as aninitial screening test in high-risk patients. The authors of this studyalso pointed out a key issue: the need for a consistent assay for a givenmarker to avoid the issues of variability from one study to another.

7.2. Pancreatic juice

The pancreatic juice has recently received a lot of interest as apotential source of biomarkers of early stage neoplasia owing to itsdirect relationship to the ductal system of the pancreas [135,136]. Onestudy [137] found that the levels of certain heavy metals (present mostcommonly in cigarette and environmental smoke) were significantlyelevated in pancreatic juice from patients with PC. Of significance wasthe observation that an increase in levels of chromium (mean (±SE)level in healthy subjects: 9±14 μg/ml) by one standarddeviation abovethat seen in healthy controls was associated with a nearly three-foldincreased risk of PC (95% C.I.: 1.2–7.8). Further, if the mean levels ofchromium and selenium (mean (±SE) level in healthy subjects: 43±13 μg/ml) were added, an increase of 20 μg/ml in this sum wasassociated with the greatest risk of PC (O.R.: 5.8, 95% C.I.: 1.5–22).

The pancreatic juice contains a variety of cells. These includeerythrocytes, neutrophils, lymphocytes and ductal cells. It has beensuggested that an analysis of whole tissue for gene expression studieslike microarray might yield erroneous results owing to the overrepresentation of a particular cell type(s) in the specimen [138]. Toovercome this, one study [138] employed immunomagnetic separa-tion of ductal cells (both normal andmalignant) by selecting cells thatexpress the mucin MUC1 (a known epithelial marker) on theirsurface. Microarray analysis performed using these fractionated cellsyielded a set of genes that distinguished healthy subjects from thosewith pancreatic adenocarcinoma with the greatest accuracy. Whatwas apparent from this study was the fact that a single marker cannotdistinguish between the two conditions owing to heterogeneity ofexpression between normal and diseased cells. Hence, a combinationof markers, possibly arrayed onto a chip, can be used to screenfractionated epithelial cells (instead of the whole specimen) and acriterion established (based on analysis of large number of samples)that distinguishes between the various disease states with thegreatest accuracy.

A comparison of the proteome of pancreatic juice from patientswith PC with those diagnosed with other diseases (including IPMN,islet cell tumor, CP and serous cystadenoma of the pancreas) yielded a

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16.57 kDa protein, HIP/PAP-1 (hepatocarcinoma–intestine–pancreas/pancreatitis associated protein-1) [139]. ELISA performed on pancre-atic juice validated that this protein, which is normally released fromthe acini during pancreatitis, is elevated in the pancreatic juice ofpatients with PC but not in those with non-malignant pancreaticdiseases. HIP/PAP-1 expression in the acini (but not the ducts) wasalso confirmed by immunohistochemistry. However, the maindrawback of the study was that of the 28 patients with PC examined,only one was of stage I, and two were of stage 2. While the stage 1patient had high levels of HIP/PAP-1 in the pancreatic juice, both stage2 patients had HIP/PAP-2 levels that overlapped with that of the IPMNcases. Another recent study identified 20 proteins that weresignificantly elevated (pb0.05) at least 2-fold or greater in pancreaticjuice collected from three patients with PanIN-3 lesions compared to acontrol sample comprising of pooled pancreatic juice from fivepatients with benign pancreatic diseases (sphincter of Oddi dysfunc-tion and CP) [140]. Some of these proteins had previously been shownto be differentially expressed in PanIN lesions including anteriorgradient-2, MUC5AC, cytoplasmic actin and Annexin A4 (Fig. 2). Animportant observation in this study was the lack of correlationbetween elevation of secreted protein (specifically AGR-2) levels inthe pancreatic juice and serum from the same patient. A possiblereason is that circulating levels of these proteins were too low to bedetected by ELISA.

A global mRNA expression analysis on the pancreatic juice (on twodifferent types of chips-U133A and X3P) revealed that intact RNA couldbe isolated from thepancreatic juice and that between40 and 130 geneswere increased three-fold or more in PC samples compared to non-cancer controls (healthy individuals and CP), depending on the platformused [141].

Human telomerase reverse transcriptase (hTERT) is a majorcatalytic subunit of human telomerase and its mRNA levels correlateclosely with the activation of telomerase. It has been suggested thathTERT could have a possible role in the diagnosis of PC. Anexamination of hTERT expression in pancreatic juice samples(obtained after secretin stimulation) from patients with PC andIPMN revealed that while cytology was 47% sensitive and 57%accurate in distinguishing malignant cells from benign cells, a positivestaining for hTERT was nearly 85% sensitive and over 80% accurate inachieving this distinction [142]. Significantly, among the PC cases,hTERT staining correctly identified 17 out of 21 cases that werenegative for malignant cells by cytology. Among patients withinvasive IPMN, hTERT positivity correctly identified eight out ofeight cases negative by cytology, while among IPMN cases withcarcinoma-in situ, 14 of the 17 cases negative by cytology werepositive for hTERT staining in the cells. There appeared to be nopreference for hTERT expression among different grades of PC orbetween themain vs. branch duct IPMN. Importantly, the pathologistsreviewing the slides did so without knowledge of the diagnosis andnone of the cells obtained from patients with benign pancreaticdisease (IPMN with adenoma and CP) was positive for hTERT. Thediagnostic performance of applying hTERT immunostaining tocytology negative cases as a method to identify early malignancyand pre-malignant lesions (especially in high-risk individuals)appears to show great promise and needs to be examined in largerstudies.

Using a combination of 2-DIGE and tandem MS, a comparison ofthe proteome of pooled pancreatic juice from nine PC and nine controlsamples (included CP, gallstone induced pancreatitis, benign cysticneoplasm and cystic fibrosis), identified three proteins, namelymatrixmetalloproteinase-9, oncogene DJI and α-1B-glycoprotein precursorthat were differentially upregulated in PC patients [143]. While thestudy validated the expression of these three proteins in thepancreatic juice by Western blotting, validation of their expressionin tissues did not include any of the control conditions that were usedfor the initial discovery. Instead, normal pancreas was used as the

control. Analysis of MMP-9 levels in serum revealed that the levelswere significantly higher in PC patients than controls (CP and healthyindividuals). However, four of the five PC patients in this study hadmetastatic disease, while the stage of the disease in the remaining fivepatients was not discussed. It is possible that the high levels of MMP-9are representative of metastatic PC and hence need to be confirmed inearly stage patients.

The composition of pancreatic juice can be influenced considerablyby the presence of co-existing biliary obstruction. One study [144]found that by 2-DE, there were only seven spots that were moreprominent consistently in pancreatic juice from patients with cancerof the head of the pancreas compared to those with non-malignantdisease of the pancreas but with co-existent obstruction of the biliarytract. In another study [145], apolipoprotein A1, transthyretin andapolipoprotein E were significantly altered in PC patients (comparedto controls-CP and benign biliary disease) without considering theeffect of biliary obstruction (measured as the level of total serumbilirubin). However, after accounting for elevation of bilirubin(N17 μmol/l), only transthyretin levels (which is decreased in PCpatients) remained associated with the risk of PC.

7.3. Other body fluids

Urine has been examined as another non-invasive biological sourceof potential biomarkers. One study that employed 2-DIGE separationfollowed by mass spectrometry revealed that nearly 60 proteins weredifferentially expressed in PC compared to controls (CP and healthycontrols) [146]. However, the results could not be validated byWesternblotting. One of the reasons suggested has been that proteins in urinemight undergo extensive post-translational modification (chieflyglycosylation) and fragmentation which could account for the failureof antibodies to identify them. However, this study did not distinguishPC by stage, necessitating further studies to examine the utility ofurinary biomarkers in the diagnosis of PC.

Bile collected by ERCP from patients who presented with biliarystenosis of various etiologieswhenexaminedbymass spectrometrywasfound to contain 127protein fragments [147]. Of these, several includingmucin-1 (MUC1), matrix metalloproteinase 7 (MMP7) and neutrophilgelatinase associated lipocalin/lipocalin-2 (NGAL) [148] had beenpreviously reported to have a role in the pathogenesis of PC. Twoproteins, Carcinoembryonic antigen-related cell adhesion molecule 6(CEACAM-6) and MUC1 (identified using a monoclonal antibody thatdetects the CA19-9 antigen) were subsequently validated by Westernblot. CEACAM-6 is a membrane receptor linked to glycosylphosphati-dylinositol (GPI), which has been shown to be elevated in severalcancers. MUC1, on the other hand, is a membrane bound mucinexpressed by normal and tumor cells of the digestive tract which carriesthe cancer associated CA19-9 epitope (2,6 sialosyl-fucosyl lactote-traose). Both these proteins could be detected by Western blot in thesupernatant (obtained after centrifugation of the bile) rather than in thecell pellet, suggesting that these are secreted into the bile. Anobservation worthy of note is that most of the cholangiocarcinomaspecimens included in this study were also positive for CEACAM-6 andCA19-9 (3/3 for CEACAM-6 and 2/3 for CA19-9), suggesting that the twomalignancies share common dysregulated proteins. Given the dismalprognosis for patients with cholangiocarcinoma [149], it would beuseful to include samples from patients with this malignancy insubsequent studies together with those from PC when examining forearly diagnostic markers (role of bile as a source of biomarkers forhepatobiliary malignancies has been recently reviewed [150]).

8. Advances in molecular diagnosis of pancreatic cancer:role of microRNAs

In the recent years, an ever strengthening link has beendemonstrated between the altered expression, mutations, andmature

Table 2Mature microRNA signatures expressed differentially in the normal pancreas, chronicpancreatitis and pancreatic cancer tissues (modified from [160] and [162]).

Expression in the normal pancreas (compared to PDAC)

miRNA ID[160] Fold change miRNA ID [162] Fold change

miR_217 269.8 miR_148a 5.5miR_216 188.8 miR_148b 3.2miR_375 14.7 miR_375 2.2miR_494 8.1 miR_221 −3.4miR_29c 7.1 miR_181a −3.0miR_96 5.8 miR_21 −3.0miR_30a_3p 5.2 miR_155 −2.1miR_223 −10 miR_210 −3.0miR_31 −10 miR_181b −2.9

Expression in chronic pancreatitis (compared to PDAC)

miRNA ID Fold change miRNA ID Fold change

miR_217 77.4 miR_148a 4.5miR_216 52.9 miR_148b 3.0miR_375 6.2 miR_375 2.2miR_196b −5 miR_203 −4.1miR_196a −5 miR_221 −2.5miR_210 −8 miR_181d −2.2

Expression in normal pancreas (compared to chronic pancreatitis)

miRNA ID Fold change miRNA ID Fold change

miR_150 5.6 miR_497 2.0miR_96 −6.5 miR_494 −4.7miR_148b −8.5 miR_100_1/2 −3.3

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microRNA processing, and the susceptibility to, or progression of,cancers. MiRNA-21 has been demonstrated to be significantly over-expressed in both PC cell lines and tissues relative to normalpancreatic tissue [151]. Further, an A to G germline mutation wasidentified in the genomic DNA 59 bp upstream of the region encodingfor the pre-miRNA in one PC patient. However, luciferase based assaysrevealed no change in the transcription of the mature miRNA-21 as aresult of this mutation. Given that a germline mutation in the primaryprecursor ofmiR-16-1–miR-15a has been linked to a familial variant ofchronic lymphocytic leukemia (CLL) [152], it is possible thatmutations in certain human miRNAs could be linked to an increasedrisk of developing PC. Further, the same could also be linked to familialclustering of PC cases and to the survival and response of pancreatictumors to therapy.

MiR-155 and miR-21 transcripts were shown to be significantlyupregulated (mean: 12-fold) in areas of IPMN without invasion (bylocked nucleic acid in situ hybridization), while miR-155 transcriptswere detected in the pancreatic juice of six out of 10 IPMN cases but innone of the controls (patients with non-neoplastic pancreatobiliarydisorders) [153]. Small non-coding RNAs have recently been inves-tigated for the diagnostic role in PC. One small study [154] comprisedof 15 adenocarcinoma, eight CP and nine normal adjacent tissuesamples identified a signature of 24 non-coding RNAs that discrim-inated PC from the remaining two groups. The implication of thesefindings will need to be assessed in larger studies.

Using primary malignant tumor specimens from six different sites(breast, colon, pancreas, prostate, stomach and lungs) and amicroarray platform, a set of microRNAs has been identified that areshared by more than one type of solid tumor as well as those that arespecifically upregulated in PC tissues [155]. Lee et al. [156] usedmicroarray to profile the microRNA precursors overexpressed in PCtissues compared to pancreatitis, benign adjacent pancreas, andnormal pancreatic tissue. They also identified a set of microRNAs thatcould classify correctly 28 out of 28 PDACs, six out of six normalpancreatic tissue and 11 out of 15 benign pancreatic disease tissues ofa “test set.” Interestingly, exocrine pancreatic neoplasms appear tohave a miRNA expression profile distinct from pancreatic endocrinetumors (PETs) [157]. The mature form of miR-155, which isupregulated in PC, was found to be downregulated in PETs(comparing with normal pancreas). Further, upregulation of miR-204was found to be specific for insulinomas (no significant change inPC). Additionally, miR-21 was found to correlate with metastases ofPETs to the liver and also distinguished acinar cell carcinomas fromnon-tumor pancreatic tissue. Given mir-21's overexpression inseveral cancers, including pancreatic [158] and breast [159], and itscorrelation with advanced stage, metastases [159] and chemoresis-tance [158], it appears that it could emerge as a general indicator ofpoor prognosis in most patients with malignant tumors.

A study by Szafranska et al. [160] identified a different set of maturemiRNAs that were differentially expressed in chronic pancreatitis andPC (seven stage 2 and three stage 3) and compared to normal pancreatictissue (summarized in Table 2). Further, the difference between raw Ct(threshold cycle in real-time PCR) values of only twomiRNAs (miR-196and miR-217) provided a simple index that could distinguish diseasedpancreatic tissues (CP or PC) from normal pancreas independent of thetotal quantity of RNA sample. A subsequent analysis [161] confirmedthatmiRNA signatures can be used to discriminate between normal, CP,and PC in frozenfineneedle aspirate specimens (miR-217, 148, 130b and375 were downregulated, while miR-196a and miR-210 were upregu-lated in PC relative to CP). Further, an index similar to the one used fortissues earlier (Ct miR-196–Ct miR-217) was able to correctly classifypancreatic tissues into normal, CP and PC.MiR-196a expressionwas alsodemonstrated to be specific for malignant ductal cells, being expressedinnoneof thenon-pathologic ductal andacinar cells, butwith increasingexpression seen in PanIN lesions, culminating in 100% positivity in themalignant ductal cells. This study suggested for the first time that

miRNA-196a expression is turned on during the progression ofpancreatic adenocarcinoma and thereby could be useful in the earlydiagnosis of PC. Expression by the primary tumor of certain miRNAsalso has a bearing on patient survival, with some (miRNA-452, 105, 127,518-2, 187 andmiR-30a-3p) being associatedwith longer survival, whileothers (miR-196-a andmiR-219) were associated with reduced survivalin a cohort of PCpatientswithmetastasis to lymphnodes [162]. A strongexpression of miR-21 was also associated with shorter survival (15 vs.27 months in patients with strong vs. weak or focal miR-21 expressionrespectively (p=0.037) [163]), although no association with tumorstage or gradewas observed. Significantly, the expression ofmiRNAs (in[162]) did not correlate with any of the other genetic abnormalitiescommonly observed in PC (inactivation of TP53, CDKN2, SMAD4 oractivating mutations of KRAS), suggesting a unique role for miRNAs inthe pathogenesis of PC.

9. Screening as a tool for early detection of pancreatic cancer

Screening for cancer requires markers with high sensitivity [164].Implementation of screening programs appears to be currently theonly way to reduce themortality associatedwith PC, given the paucityof options for treatment beyond surgical resection. However, it is notcurrently in place for the general population owing to the relativelylow incidence of this malignancy and the lack of accurate, inexpensiveand non-invasive diagnostic tests for early disease [71]. For thepurpose of screening, two groups (kindreds) of at-risk individuals areconsidered: those with at least one first degree relative diagnosedwith PC, termed as familial PC kindreds (FPC) and those without anyrelatives affected with PC, termed sporadic PC kindreds (SPC). Thelatter group comprises the majority of PC diagnosed annually in theUnited States. Several studies have shown that the risk of PC amongthe FPC group increases with the number of affected first degreerelatives with PC (five-fold, six-fold and 32-fold increases in risk with1, 2 and 3 affected first degree relatives, respectively, in one study[165]).

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A consensus guideline on themanagement of patients at high-riskfor the development of PC recommended secondary screening on aresearch basis for patients with hereditary pancreatitis, families withmultiple cases of PC, individuals with even one documented case of amutation known to predispose to PC in the family, and individualsfrom kindreds of Peutz–Jeghers syndrome [166]. Knowledge of riskfactors that predispose to the development of PC, would help usstratify patients more effectively. This in turn, could help tailorscreening and surveillance strategies to better target those at high-risk and away from those at low risk. These low-risk individualscould be examinedwith less invasive techniques or not at all. Geneticfactors influence the risk of developing PC considerably, as evidencedby the racial difference in susceptibility to PC. Further, about 10% ofPC cases have a family history of the disease, suggesting that analtered expression or activity of one or more genes determines therisk of developing PC in a subset of PC cases [167,168]. However,most syndromes associated with PC are rare and usually associatedwith an increased prevalence of other cancers. Some of theseconditions include FAP (familial adenomatous polyposis, mutationof adenomatous polyposis coli tumor suppressor gene (TSG); 4.5-fold greater risk) [169], FAMMM (familial atypical multiple molemelanoma; mutations in the TSG CDKN2A/p16 or in a minority ofcases in CDK4), Li-Fraumeni syndrome (recessive mutations in p53TSG or CHK2), Peutz–Jeghers syndrome (autosomal dominantmutations in the STK11 gene) and the Lynch syndrome-II (mutationsin mismatch repair genes) [170]. Some individuals with mutations inthe BRCA2 TSG also have an increased risk of PC [171]. It is interestingto quote the results of a prospective study which included two high-risk patients [71], one with and the other without a 6174 delT BRCA2mutation in the germline. The first patient, with an Ashkenazi Jewishancestry and a history of ovarian and breast cancer, was normalduring baseline evaluation but found to have a 6-mm cyst in theuncinate process communicating with the pancreatic duct one yearafter the initial evaluation. Within the next six months, multiplelesions appeared in the liver, and a diagnosis of metastasis from aprimary pancreatic adenocarcinoma was made. In the secondpatient, there was a family history of BRCA2 mutation (althoughshe herself was negative). In her, an IPMN adenoma together withmultiple high-grade PanIN lesions was found. This study whilestrengthening the association of genetic mutations with screeningfor PC highlighted the complementary roles of EUS and CT scan inscreening patients suspicious of harboring an occult neoplasm of thepancreas. In another interesting report [172], PC surveillance by EUSand MRI resulted in the detection of asymptomatic PC in twomembers (a mother and daughter) of a Dutch family with p16-Leiden mutation (19 bp deletion in exon 2 of the CDKN2A gene) andwith an atypical presentation of the FAMMM syndrome (presence ofPC and melanoma in two members of the same family). Two otherfamily members were discovered to be carriers for the p16-Leiden, ofwhom one was treated for melanoma and carcinoma of the cheekwhile the other was healthy. Three other family members who werescreened turned out negative for the mutation. In this study, theindex case, a 76-year old woman, was found to have atrophy of thepancreatic tail and dilation of the pancreatic duct tapering to an ill-defined mass on EUS. A CT scan revealed a hypodense lesion in thetail of the pancreas. Histologic examination of the resected pancreasrevealed a poorly differentiated adenocarcinoma of the tail withperineural and nodal invasion (4/8 adjacent nodes) but with nodistant metastases. Upon follow-up, the patient was doing wellfollowing chemotherapy. Her 51-year old daughter had a small(1 cm) hypoechoic mass on EUS examination in the body of thepancreas at the level of the splenic artery suspicious for malignancy.During surgery, the regional lymph nodes were found to be free ofcancer and histology revealed a moderately differentiated adeno-carcinoma. It is notable that MRI and CT scans performed in the samepatient did not pick up any abnormality in the pancreas. One of the

carriers of themutation was also screened by EUS andMRI and foundto have side branch IPMN.

It has been posed that EUS is a cost-effective method of screeningasymptomatic patients, at least those from familial PC kindreds. Amodel-based analysis employing a hypothetical cohort of 100 patientsfrom a FPC kindred found that if every person who was found to havepancreatic dysplasia on screening agreed to undergo total pancrea-tectomy, a pretest probability of at least 16% (for occurrence ofpancreatic dysplasia) was required for a significant survival benefitresulting from endoscopic screening (by EUS and ERCP) [173].Survival benefit was measured as years of survival followingpancreatectomy and was found to be dependent on the sensitivityof the test being employed. Thus, a sensitivity of less than 84% for EUSand 68% for ERCP was predicted to actually result in fewer life-yearsfor the patients if screeningwas undertaken. This study also estimatedthat a screening procedure was not likely to produce an improvementin survival if the life-expectancy of the patient was less than 27 yearsat the time of screening. A temporal relationship was also evidentbetween the time of onset of pancreatic dysplasia and death from PC.It was estimated that for a givenmalignancy, screening high-risk caseswould not be appropriate if this period was more than 12 years (forPC, it has been estimated that the time from the onset of dysplasia toonset of invasive carcinoma is 10 years [174], while the mediansurvival after diagnosis has been estimated to be 0.8 years [175]).Inflation and costs of pancreatic surgery did not appear to affect theoverall cost-effectiveness of screening patients by endoscopy (EUSfollowed by ERCP). Rather, the two key factors that determined thecost-effectiveness of a screening test were the pretest probability ofpancreatic dysplasia and the sensitivity of the test. Importantly, as thepretest probability of dysplasia increased, the screening test could beless sensitive for the screening strategy to still remain effective (thestudy assumed a 20% prevalence of pancreatic dysplasia in theirhypothetical cohort and 90% sensitivity of EUS and ERCP in identifyingdysplastic lesions in the pancreas).

Germline mutations occur in only a small percentage of sporadicPC cases (7% of apparently sporadic cases had a germline mutation inBRCA2 in one hospital based study) [176]. This argues againstimplementing screening for genetic mutations in patients without asuggestive family history or known inherited syndrome who areconsidered at a high-risk for PC (e.g. CP and those diagnosedwith typeII diabetes within the last three years). Further, germline deletions canbe missed, even with standard sequencing techniques, potentiallyleading to false-negative results [177]. However, among asymptom-atic patients with a family history of PC or an inherited mutation, thestory is different. Up to 12% of patients with at least one first degreerelative with PC [178], and nearly 18% patients with three or morerelatives with PC have a germline mutation in BRCA2 [177]. Thus,employing a screening test for genetic mutations would be suitable asa first-line screening test in these patients.

10. Management of the high-risk patient

Cystic neoplasms of the pancreas, when diagnosed (usually byimaging), should be completely resected (except when a cleardiagnosis of serous cystic neoplasm is made, when a wait and watchpolicy is recommended [179]) and a thorough histopathologicexamination should be performed. If no evidence of tissue invasionis detected, the chances of recurrence are negligible and prolongedfollow-up is not recommended. However, if invasion is identified(invasive cystadenocarcinoma), the prognosis is generally poor [12].One of the largest prospective studies undertaken to examine thefactors that influenced mortality from PC [35] reported an inverseassociation between consumption of vegetables (carrots, tomatoes,squash/corn, green leafy vegetables, raw vegetables, and cabbage,broccoli, and Brussels sprouts) and the risk of PC. However, thisreduction in risk was observable only among men. Another case–

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control study reported a reduction in risk with intake of fruits [180].This finding could be useful as lifestyle modification advice to patientswho are determined to be at an increased risk of PC owing to theirlifestyle and dietary habits.

One study [181] aimed at examining the role of EUS and associatedguided FNA in the detection of pancreatic neoplasms represents apotentially useful strategy that can be applied for screening. In thisstudy, 110 patients who were suspicious for possible PC based on anenlargement of the head of the pancreas (HOP) or a dilation of thepancreatic duct (by CT or MRI), but who did not have any detectablemass (by any of these two modalities) were screened by EUS. Caseswhere a focal lesion was identified by EUS were subjected to FNA anda final diagnosis was made based on the cytopathological examina-tion. Out of 43 patients with a dilated pancreatic duct (irrespective ofpresence or absence of a dilated common bile duct), a focal pancreaticlesion was identified in 18, all of whom underwent FNA. Four weresubsequently diagnosed with adenocarcinoma. A dilation of thepancreatic duct with an abrupt proximal cut-off was more commonlyassociated with an adenocarcinoma of the pancreas (four out of 15cases) compared to a diffuse dilation of the pancreatic duct (no PCsamong 28 patients). Among patients with an enlarged HOP (n=67), afocal pancreatic lesion was identified in 14 of which two were foundto be primary pancreatic adenocarcinoma, one a metastatic tumor tothe pancreas, while 10 were benign cases (by FNA). 17 patients alsohad features of CP, of which 12 had no focal lesion while five had aconcomitant focal lesion. In this study, nearly 17% of all patients whounderwent CT or MRI for suspected PC had either an enlarged HOP(6%) or a dilated pancreatic duct (11%). The diagnostic accuracy of aEUS with or without FNA was 99% (95% C.I.: 97%–100%). Due to thefact that obstructive jaundice can also cause dilation of the pancreaticduct in the absence of PC [182], the authors of this study excluded allpatients with serum total bilirubin ≥1 mg/dl at the time of initialpresentation. In this study, based at the Saint Louis UniversityHospital, all patients who underwent EUS were rigorously followedthrough telephone calls and correspondence with referring andprimary care physicians. Those without any evidence of cancerunderwent repeat imaging by EUS/CT/MRI three, six or twelve -months after the initial procedure. Interestingly, there were nospecially trained radiologists employed to interpret the CT/MRIfindings in this study. The NPV of EUS in this study was 99% (95% C.I.: 0.97–1.0) strengthening its role as the most useful imagingmodality for screening suspicious patients.

11. Conclusions and perspectives

There is little doubt that a general screening for PC in the populaceis not practical, chiefly owing to its extremely low incidence (9 per100,000 per year) [183]. About 5–10% of PCs report a family history ina close family member and this constitutes an attractive cohort forscreening purposes (reviewed in [184]). However, the majority of PCsare sporadic. There is a need to identify certain “indicators”which canalert the attending physician to the possibility of an occult pancreaticneoplasm. The suspicious cases can then be screened by a CT scan orEUS (the latter being preferable given its high sensitivity andspecificity). Those patients in whom a suggestive lesion is discoveredcan then be investigated further by EUS-guided FNA to identify thenature of the lesion. Applying EUS to screen asymptomatic individualsof FPC kindred found that most individuals of this kindred hadfindings that were strongly suggestive of CP (most commonly thepresence of multiple hypoechoic foci, generally in the head or the tailregion). As these findings are also observed in patients who have CPsecondary to prolonged alcohol consumption, it has been recom-mended that the presence of EUS features suggestive of CP in thesetting of a positive history of chronic alcoholism entails a repeat ofthe same procedure after at least sixmonths of complete abstinence. If

the findings are still suspicious, an ERCP (pancreatogram) isrecommended.

“A pertinent question that often comes to mind is: to what extentis early diagnosis by imaging affected by observer-to-observervariation in interpretation of the results?” One study [185] tried toexamine this question by assessing the inter-observer agreement ininterpretation of EUS findings among 17 experienced endosonogra-phers before and after a workshop to draft consensus EUS findings forthe early diagnosis of PC in high-risk persons. The results revealed thatexcept for cysts, inter-observer agreement for all other conditions(including masses, CP lesions and even normal pancreas) was ratherpoor. Several factors, including poor video quality and experience ofthe endosonographer in identifying the subtle changes often observedin high-risk patients, were suggested as possible causes for the lack ofagreement. Thus, better resolution instruments and training ofsonographers in identifying the changes associated with high-riskcases appears to be a pressing requirement needed to improvedetection rates for early PC lesions.

A key point that comes to light from a review of the literature is theimportance of a careful history, specifically relating to the enumer-ation of risk factors for PC. Table 1 lists some of the points from thehistory which can be helpful pointers for identifying patients at ahigh-risk for PC.

Another question often asked is whether it is economically viableto perform an invasive procedure like EUS or an expensive one like CTon an asymptomatic individual. A suggestion has been that to keep thescreening procedure viable, asymptomatic at-risk cases should bescreened by a highly sensitive and relatively inexpensive test(preferably serological). Positive cases should then be screened bymore specific tests (again preferably serological) before proceeding tomore expensive imaging techniques like EUS [186]. Although theexact frequency of EUS based screening has not yet been established,it has been suggested (in [187]) that patients from FPC kindredsundergo EUS based screening annually, beginning 10 years earlierthan the age of onset of PC in the youngest case. Those patients (fromFPC kindreds) with normal EUS findings are recommended to befollowed-up at 2–3 year intervals. This interval is likely to be longerfor patients without a family history of PC or other contributinggenetic conditions. Patients who are assessed to be at high-riskfollowing screening have been offered the option of pancreatectomy(either total or distal) [187]. In theWashington experience [187], totalpancreatectomy was offered to family members of the high-risk FPCkindred, while distal pancreatectomy was offered to all otherasymptomatic patients with pancreatic dysplasia (non-FPC). Totalpancreatectomy in the latter cohort was limited only to those patientswith documented high-grade dysplasia in the pancreas.

Counseling is an important aspect of management of patients(both FPC and SPC types) in order to emphasize that currently there isno definitive test to detect early stage PC or pre-cancer. It is alsoimportant to understand the factors that may influence the desire ofasymptomatic individuals to seek advice for cancer screening. Theexperience from a study in Korea to understand the factors associatedwith screening seeking behavior in gastric cancer [188] found threemain factors that determined this behavior: 1. anxiety aboutdeveloping cancer, 2. having a family member who was havingregular check-ups and 3. being educated about the importance ofcancer screening tests (this last factor was the strongest factor linkedto the desire to seek screening).

Alexander Graham Bell, the great inventor, once remarked, “Greatdiscoveries and improvements invariably involve the cooperation ofmany minds. I may be given credit for having blazed the trail, butwhen I look at the subsequent developments I feel the credit is due toothers rather than me.” In this age of ever growing information, theemphasis has been on sharing information and resources. Inter-disciplinary and inter-institutional collaboration holds the key tosuccess in our endeavor to conquer this seemingly incurable

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malignancy. An example in this directionwas the recent creation of anopen access database (http://www.pancreasexpression.org/) [189]which contains gene expressionmeasurements from various PC types,PanIN lesions and CP. These types of public databases could serve asrepositories of information to enable a systematic unraveling of thepathways underlying PC initiation and progression.

As pointed out earlier, a large percentage of the studies aimed atthe discovery of biomarkers for PC either did not separate the patientsaccording to stage, history of chemotherapy, family history, andpresence or absence of important risk factors (e.g. smoking anddiabetes) or employed too small a sample size [135]. In some cases,there was a discrepancy in the results of two similar studies [160,162].Thus, another important need is to pay careful attention to the designof studies. Ideal groups of PC patients would be those in stage I or II(which are considered to be resectable stages) [190] who have not yetreceived any form of therapy (as this might modify expression ofgenes/proteins) and who have no family history of PC (SPC).Validation of the potential markers identified from a “training” setof carefully chosen subjects should then be validated in a “test” setthat should be as similar to the “training” set as possible. It is notsurprising that markers identified by global analysis often failvalidation due to inappropriate selection of samples for biomarkerdiscovery and validation (e.g. although cyclin-1 was one of the mostupregulated proteins in sera of PC patients by mass spectroscopy,several benign samples expressed the protein on subsequentvalidation by Western blot on a separate set of samples [191]).Where reports are conflicting (as in the case of the C677Tpolymorphism in the methylene tetrahydrofolate reductase genewhich has been linked to an increased risk of PC in the setting of folatedeficiency [192]), careful studies are needed to clear the controversy.

Thus, in conclusion, it would be fair to say that we have a large anddiverse armamentarium of biomarkers ranging from the shortmiRNAs to the huge mucin glycoproteins which, combined withexisting and emerging imaging techniques, has the potential toidentify this lethal malignancy at an early, potentially resectable stage.Exciting new developments continue to be made including thediscovery of new tumor suppressor genes like WWOX (WW-domain-containing oxidoreductase) [193] and the development ofmouse models that are bringing us ever closer to understanding thecellular origin of PC itself and to the dream of identifying occult PC inevery pre-symptomatic individual.

Acknowledgements

The authors on this work are supported by grants from theNational Institutes of Health (RO1 CA78590, UO1 CA111294, RO1CA133774, RO1 CA131944, and P50 CA127297) and the Departmentof Defense (PC074289 and BC074639). We thank Ms. Kristi L. Bergerfor editing the manuscript.

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