Langenbeck's Archives of Surgery

, Volume 403, Issue 2, pp 151–194 | Cite as

Can we better predict the biologic behavior of incidental IPMN? A comprehensive analysis of molecular diagnostics and biomarkers in intraductal papillary mucinous neoplasms of the pancreas

REVIEW ARTICLE

Abstract

Purpose

Predicting the biologic behavior of intraductal papillary mucinous neoplasm (IPMN) remains challenging. Current guidelines utilize patient symptoms and imaging characteristics to determine appropriate surgical candidates. However, the majority of resected cysts remain low-risk lesions, many of which may be feasible to have under surveillance. We herein characterize the most promising and up-to-date molecular diagnostics in order to identify optimal components of a molecular signature to distinguish levels of IPMN dysplasia.

Methods

A comprehensive systematic review of pertinent literature, including our own experience, was conducted based on the PRISMA guidelines.

Results

Molecular diagnostics in IPMN patient tissue, duodenal secretions, cyst fluid, saliva, and serum were evaluated and organized into the following categories: oncogenes, tumor suppressor genes, glycoproteins, markers of the immune response, proteomics, DNA/RNA mutations, and next-generation sequencing/microRNA. Specific targets in each of these categories, and in aggregate, were identified by their ability to both characterize a cyst as an IPMN and determine the level of cyst dysplasia.

Conclusions

Combining molecular signatures with clinical and imaging features in this era of next-generation sequencing and advanced computational analysis will enable enhanced sensitivity and specificity of current models to predict the biologic behavior of IPMN.

Keywords

Intraductal papillary mucinous neoplasm (IPMN) Pancreatic cyst Molecular diagnosis Biomarkers 

Abbreviations

BD

Branch duct

CA19-9

Carbohydrate antigen 19-9

CEA

Carcinoembryonic antigen

CT

Computed tomography

EUS

Endoscopic ultrasound

IPMN

Intraductal papillary mucinous neoplasm

LOH

Loss of heterozygosity

MCN

Mucinous cystic neoplasm

miRNA

MicroRNA

MPD

Main pancreatic duct

NGS

Next-generation sequencing

NPV

Negative predictive value

PDAC

Pancreatic ductal adenocarcinoma

PPV

Positive predictive value

SCA

Serous cystadenoma

SPN

Solid pseudopapillary neoplasm

TIL

Tumor-infiltrating lymphocyte

Introduction

Intraductal papillary mucinous neoplasms (IPMNs) are tumors with adenomatous proliferation of ductal epithelium. Mucin production causes dilatation of the ductal pancreatic system that can affect the main pancreatic duct (MPD), branch ducts (BDs), or both (mixed). IPMN of the pancreas was first reported in 1982 by Ohashi et al. as a category of pancreatic tumor distinct from known tumors of the exocrine pancreas [1]. However, that definition and classification of IPMN was not formally adopted by the World Health Organization until 1996, by which time additional studies identified the premalignant potential of IPMN histopathologic subtypes [2, 3, 4]. The language and the descriptions of the lesions have changed over time, being ever refined to improve discourse and research on IPMN (Fig. 1). It has been reported that 24% of patients at autopsy have a pancreatic cyst, of which 20% will contain atypia or high-grade dysplasia [5]. Thus, this is a prevalent disease with an incidence that has increased by 2–13% secondary to ubiquitous cross-sectional imaging and advancements in multidetector computed tomography (CT) imaging quality [6].
Fig. 1

IPMN nomenclature. The literature includes various definitions of the IPMN level of dysplasia over time. The current terms used in this study to discuss IPMN have been categorized, clarified, and standardized

As a result, IPMN has become the most common cystic precursor lesion of pancreatic ductal adenocarcinoma (PDAC), representing up to 25% of resected pancreatic neoplasms [7]. In the early era of IPMN diagnosis, research focused on the discovery of aberrant markers found in pancreatic cystic lesions; however, multiple groups have since focused their studies on the clinical signs and imaging characteristics of the disease, and these have evolved to form the basis of multiple consensus guidelines. Though the sensitivity of the current clinical treatment guidelines is satisfactory, the specificity remains poor [8]. As a result, surgical management is pursued in the setting of worrisome or high-risk radiographic features in order not to miss occult high-grade dysplasia or invasive cancer, though the majority of resected lesions in the country are low risk (low- or moderate-grade dysplasia) on final pathology. Surgical intervention often involves major pancreatic resection which carries a significant risk of mortality and morbidity [9]. Though the risk of invasive carcinoma in MD or mixed IPMNs approximates 50%, the risk is far less in BD-IPMN and may be minimal in small BD-IPMN with no high-risk features [10, 11] (Fig. 2). Thus, particularly for small BD-IPMN, enhanced guidelines with improved negative predictive value are desired.
Fig. 2

Levels of dysplasia and histopathology of IPMN phenotypes

General characteristics of IPMNs, radiographic diagnosis, cyst fluid composition, and their delineation from other pancreatic tumors have been well established, but knowledge regarding their growth and progression into malignancy remains poorly described. Current clinical guidelines will need to evolve to improve accuracy in determining the level of cyst dysplasia [12, 13, 14, 15], and additional molecular diagnostic data to distinguish high- from low-risk cysts is desperately needed. We have previously shown that EUS-FNA cytology has limited utility in surgical decision making for IPMN [16], and have focused our current research on identifying prognostic molecular biomarkers within the cyst fluid [17, 18, 19].

The aim of this review is to organize the current landscape of molecular markers in tissue, duodenal secretions, cyst fluid, saliva, and serum in order to identify the potential of specific oncogenes, tumor suppressor genes, glycoproteins, markers of the immune response, proteins, DNA/RNA mutations, and microRNA (miRNA) to elucidate the grades of cyst dysplasia and risk of malignant transformation.

Methods

A systematic review of the literature following the PRISMA guidelines was conducted (Fig. 3). A search of PubMed, MEDLINE, and the Cochrane Database of Collected Reviews was performed, surveying articles from January 2007 through September 2017. Keyword combinations of index terms including “Molecular OR Biomarkers” that were identified after a critical review of the current literature AND “Diagnosis” AND “IPMN” were combined (Supplementary table). Only original articles with data regarding molecular markers specifically from IPMNs were included. Unpublished manuscripts, abstracts, and case reports were excluded. Review articles and references lists, in select circumstances, directed searches of the embedded references and were manually reviewed for primary data. A total of 355 studies were identified. Titles and abstracts of all articles were reviewed, and of these, 158 studies were found to be of relevance for full review. After elimination of reviews and articles without a focus on biomarkers in IPMN, data were extracted and synthesized from 100 articles.
Fig. 3

PRISMA flow diagram. Evaluation and inclusions of articles relevant to the biological molecules used to identify IPMNs were evaualted with a particular focus on the levels of dysplasia

Results and discussion

Oncogenes

Mutations in genes involved in cell growth and proliferation or inhibition of apoptosis can result in unregulated cellular growth. Mutations in these proto-oncogenes cause aberrant expression of oncogenes that bypass the regular cell machinery leading to carcinogenesis. Kirsten RAS (KRAS), GNAS, phosphatidylinositol-3 kinases (PI3Ks), BRAF (MAPK signaling), telomerase reverse transcriptase expression, and hedgehog pathway oncogenes have been shown to be involved in IPMN progression.

KRAS

KRAS is located on chromosome arm 12p and encodes a membrane-bound guanosine triphosphate (GTP)-binding protein. It is implicated in signal transduction, cell proliferation, cell survival, and motility. KRAS mutations or oncogenic KRAS has common point mutations with a frequency ranging from 38.2 to 100% in IPMNs evaluated from pathologic specimens and cyst fluid [20, 21]. Many recent studies have shown that mutations in KRAS (50–83% of malignant IPMNs) or GNAS (25–83% of malignant IPMNs) or simultaneous mutations are present in up to 90% of IPMNs [22, 23, 24, 25]. These mutations are also present in IPMN with low-grade dysplasia from 40 to 90% (either KRAS or GNAS or both) of cases [3]. Several studies that have evaluated the diagnostic utility of mutant KRAS have found it to be a useful marker for characterizing focal pancreatic lesions but without sufficient specificity to differentiate high-risk lesions [26, 27]. When evaluating for the mutational abundance of KRAS across various grades of dysplasia, there was no significant difference found (87% in low-grade, 90.2% in intermediate-grade, and 70.7% in high-grade dysplasia) [26], supporting the idea that KRAS mutations are considered to be an early event in the neoplastic transformation of IPMNs [28].

Using resected pathologic specimens as the gold standard for identifying mutational expression, KRAS mutations are also identified in the pancreatic juices (duodenal fluid) of patients with varying abilities to identify IPMN (Table 1). KRAS mutations were detected in the duodenal fluid from a large percent of subjects pathologically confirmed to have pancreatic cancer (73, p = 0.0005); however, only 50% of patients with a clinical diagnosis of IPMN fluid were noted to have KRAS mutations in the fluid [29]. Further, 19% of patients that had no clinical evidence of a pancreatic cyst were also found to have KRAS mutations in the duodenal fluid, which the authors attributed to the potential of these patients to have benign microscopic pancreatic intraepithelial neoplasia (PanIN). Thus, KRAS mutational detection alone may not be as applicable to diagnosing IPMN as it is to PDAC, particularly in duodenal juice [29].
Table 1

Molecular diagnostics of IPMN. IPMN biomarker data reviewed from 2007 to 2017 garnered from pancreatic tissue, cyst fluid, pancreatic juices, serum/plasma, and saliva arranged by category of biomarker and in reverse chronology

1st author

Title

Year

Biomarkers

Sample collection

Lab technique

Key/important results

Oncogene, tumor suppressor genes, NGS, gene methylation

 Oncogenes

  Kadayifci

Value of adding GNAS testing to pancreatic cyst fluid KRAS and carcinoembryonic antigen analysis for the diagnosis of intraductal papillary mucinous neoplasms

2017

KRAS, GNAS, CEA

197 patients with cyst fluid

- IPMN, 108 patients

- Non-IPMN, 89 patients

Pathologic review, cytology, DNA analysis, and CEA

- GNAS+ in 51 patients (47.2%) with IPMN; 42 of these patients (82.3%) also had a KRAS mutation

- Adding GNAS to KRAS increased the diagnostic accuracy from 76.6 to 79.1% (p > 0.05)

- Adding GNAS to CEA increased the diagnostic accuracy from 66.4 to 80.7% (p < 0.05) but did not achieve a diagnostic superiority to KRAS testing alone (80.7 vs 76.6%, p > 0.05)

- Accuracy of CEA, KRAS, and GNAS combination was significantly better than that of all single tests (p < 0.05)

  Yang

Detection of mutant KRAS and TP53 DNA in circulating exosomes from healthy individuals and patients with pancreatic cancer

2017

KRAS, TP53

Exosomes from the serum of 171 healthy subjects and patients (PDAC, IPMN, CP, or others) who underwent pancreatic resection

Exosome isolation, DNA extraction, digital PCR

- In 48 serum samples from PDAC patients, 39.6% had KRASG12D mutations, and 4.2% had TP53R273H mutations

- KRASG12D and TP53R273H mutations were detected in exosomal DNA from 2 of 7 IPMN patients with KRASG12D, 1 of which also copresented with TP53R273H mutation

- Circulating exosomal DNA in 5 of 9 CP patients enabled the detection of KRASG12D mutation, and in 114 healthy subjects whom circulating exosomal DNA was derived, 2.6% presented with KRASG12D

  Berger

Detection of hot-spot mutations in circulating cell-free DNA from patients with intraductal papillary mucinous neoplasms of the pancreas

2016

KRAS, GNAS

Serums cf DNA of 21 IPMN patients, 38 healthy controls, 24 metastatic PDAC patients, 26 SCA patients, and 16 IGD IPMN patients (blood/tissue; IPMN resected)

Droplet digital polymerase chain reaction (ddPCR)

- Total amount of cfDNA can discriminate controls and patients with IPMN or PDAC

- GNAS mutations were found in the cfDNA from patients with IPMN, but not in patients with SCA or controls

- KRAS mutations were only seen in PDAC, and no IPMNs were identified consistently with this technique

  Bournet

Endoscopic ultrasound-guided fine-needle aspiration plus KRAS and GNAS mutation in malignant intraductal papillary mucinous neoplasm of the pancreas

2016

KRAS, GNAS

37 cyst fluid samples from IPMN patients with 18 pathologic tissue samples and 19 surgical biopsies/FNA analysis or follow-up (10 benign IPMNs, 27 malignant IPMNs)

KRAS and GNAS mutational assays

- Sensitivity, specificity, PPV and NPV, and accuracy of cytopathology alone to diagnose IPMN malignancy were 55, 100, 100, 45, and 66%, respectively

- When KRAS mutational analysis was combined with cytopathology, these values were 92, 50, 83, 71, and 81%, respectively

- GNAS assays did not improve diagnosis

  Ideno

Clinical significance of GNAS mutation in intraductal papillary mucinous neoplasm of the pancreas with concomitant pancreatic ductal adenocarcinoma

2016

GNAS, KRAS

- 110 FFPE tissue samples from IPMN patient with 16 PDAC patients

- GNAS status in IPMN versus 23 pancreatic fluid specimens

DNA extraction, mutational analysis

- GNAS mutation rate in IPMN with PDAC was significantly lower than that in IPMN without PDAC [4/16, (25%) vs 61/94 (65%); p = 0.0047]

- In 45 GNAS wild-type IPMNs, 10 (43%) of 23 gastric-type IPMNs had distinct PDAC, whereas only 2 (9%) of 22 nongastric-type IPMNs had distinct PDAC (p = 0.017)

- GNAS status in pancreatic fluid was consistent with that in tissue in 21 (91%) of 23 patients

  Sugiyama

Gli2 protein expression level is a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms

2016

SHH, Gli2

- 9 FFPE tissue samples from patients with benign/noninvasive IPMN

- 17 tissue samples from patients with invasive PDAC

IHC, tissue microarray

- GLI2 protein is expressed in both PanIN/IPMN and PDAC

- Cytoplasmic GLI2 level in PDAC was modest in comparison to that in PanIN/IPMN

- Hedgehog interacting protein was strongly expressed in the precursors, whereas the level in PDAC was significantly attenuated

- A strong correlation between sonic hedgehog and GLI2 staining was found in both human and murine pancreatic tumors

- GLI2 protein level could serve as a feasible marker of ligand-dependent hedgehog activation in pancreatic neoplasms

  Eshleman

KRAS and GNAS mutations in pancreatic juice collected from the duodenum of patients at high risk for neoplasia undergoing endoscopic ultrasound

2015

KRAS, GNAS

272 secretin-stimulated pancreatic juice samples (194 screened family history, 30 pancreatic cancer, 48 pancreatic cysts, pancreatitis, or normal)

Digital high-resolution melt-curve analysis, pyrosequencing, and mutational score

- KRAS mutations were detected in pancreatic juice from PDAC (73%) versus cancer screening (50%) versus controls (19%) (p = 0.0005)

- KRAS mutation detected 3 or more times (47%) in PDAC patients than screened subjects (21%) versus controls (6%, p = 0.002)

- Mutations in KRAS (but not GNAS) were found in similar percentages of patients with or without pancreatic cysts

  Kuboki

Molecular biomarkers for progression of intraductal papillary mucinous neoplasm of the pancreas

2015

KRAS, GNAS, EGFR, PIK3CA GNAO1, GNAQ, or GNAI2, MARK, pMARK, AKT, pAKT, nuclear accumulation of β-catenin, SMAD4 loss, and TP53 overexpression

172 tissue samples from IPMN patients

Direct sequencing, IHC, tissue microarray

- GNAS and KRAS mutations were detected in 48 and 56% of IPMNs, respectively

- Significant associations were observed between IPMN morphological types and GNAS mutations, KRAS mutations, the expression of phosphorylated MARK, AKT, phosphorylated AKT, nuclear accumulation of β-catenin, SMAD4 loss, and TP53 overexpression

- Histological grades associated with the expression of EGFR, pMAPK, AKT, and pAKT, nuclear β-catenin, SMAD4 loss, and TP53 overexpression - Invasive phenotypes associated with KRAS mutations, the nuclear β-catenin, and SMAD4 loss; - Worse prognosis associated with SMAD4 loss and TP53 overexpression

- Impacts of multiple molecular features revealed that TP53 overexpression was an independent prognostic factor (p = 0.030)

  Tan

GNAS and KRAS mutations define separate progression pathways in intraductal papillary mucinous neoplasm-associated carcinoma

2015

275 genes (including KRAS, GNAS, and RNF43)

38 tissue samples from patients with IPMN-associated PDAC (38 IC, 34 HGD, 19 LGD, 38 normal tissue)

Microdissection, DNA extraction and analysis, massive parallel sequencing

- GNAS mutations were more prevalent in colloid-type IC than tubular-type (89 vs 32%, respectively; p = 0.0001)

- KRAS mutations were more prevalent in tubular-type than colloid-type IC (89 vs 52%, respectively; p = 0.01)

- GNAS mutations were more prevalent in I-type (74%) compared with PB-type (31%) and G-type (50%; p = 0.02)

- The presence of these mutations did not vary according to the degree of dysplasia

  Al-Haddad

Performance characteristics of molecular (DNA) analysis for the diagnosis of mucinous pancreatic cysts

2014

KRAS, cyst fluid CEA, cytology

48 tissue samples from 286 patients with cyst fluid analysis

DNA molecular analysis, pathologic review

- Molecular analysis sensitivity of 50% and specificity of 80% in identifying mucin pancreatic cysts (accuracy of 56.3%)

- The combination of KRAS molecular analysis with cyst fluid CEA and cytology resulted in higher mucin pancreatic cyst diagnosis (sensitivity, specificity, and accuracy of 73.7, 70, and 72.9%, respectively)

  Hosada

GNAS mutation is a frequent event in pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas

2014

GNAS and KRAS

290 tissue samples from 284 patients with tumors (97 PDAC, 30 IPMN-IC, 61 IPMNs, 10 MCN, 52 PanNET, 16 acinar cell neoplasms, 10 SCA, and 14 SPN, 35 of PanIN-1, 25 of PanIN-2, and 17 of PanIN-3)

Microdissection, IHC, tissue microarray, mutational analysis

- 64% (39/61) of IPMNs and 37% (11/30) of IPMN-IC have a GNAS mutation

- GNAS mutations were rarely observed in PDAC (1%, 1/88) and PanINs (3%, 2/77), and not at all in MCN (0/10), PanNET (0/52), acinar cell neoplasms (0/16), SCA (0/10), and SPN (0/14)

  Singhi

Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts

2014

GNAS, KRAS

91 cyst fluid samples (41 IPMNs, 9 IPMN-IC, 16 MCNs, 10 PanNET, 9 SCA, 3 retention cysts, 2 pseudocysts, 1 lymphoepithelial cyst)

Mutational analysis, DNA molecular analysis

- GNAS mutations in 16 (39%) IPMNs and 2 (22%) IPMN-IC

- KRAS mutations in 28 (68%) IPMNs, 7 (78%) IPMN-IC, and 1 (6%) MCN

- Mutations in either gene were present in 34 (83%) IPMNs, 8 (89%) IPMN-IC, and 1 (6%) MCN

- GNAS and KRAS mutations had 100% specificity but 65% sensitivity for mucinous differentiation

- In IPMNs, mutations in either gene had 98% specificity and 84% sensitivity

  Takano

Deep sequencing of cancer-related genes revealed GNAS mutations to be associated with intraductal papillary mucinous neoplasms and its main pancreatic duct dilation

2014

KRAS, GNAS, and 46 cancer-related genes containing 739 mutation hotspots

152 pancreatic juices

- 9 normal pancreas

- 22 CP

- 39 PDAC

- 82, IPMN

48 FFPE tissue samples

- 6 IPMNS

- 42 PDAC

DNA extraction, multiplex PCR targeted

- KRAS and GNAS mutations were most frequently detected in both PDAC and IPMN cases

- Pancreatic juice

• GNAS mutations were detected in 7.7% of PDAC cases and 41.5% of IPMN cases (p = 0.001 vs others). All PDAC cases with GNAS mutations (n = 3) were accompanied by IPMN

• GNAS mutations in IPMN cases were associated with dilated MPD (p = 0.016)

- FFPE

• GNAS mutations were detected in 50% of PDAC cases concomitant with IPMN, 33.3% of PDAC cases derived from IPMN, and 66.7% of IPMN cases, while no GNAS mutations were detected in cases of PDAC without IPMN

  Dal Molin

Clinicopathological correlates of activating GNAS mutations in intraductal papillary mucinous neoplasm (IPMN) of the pancreas

2013

GNAS

54 tissue samples from patients with GNAS codon 201 mutational status and a separate cohort of 32 patients

Microdissection, DNA extraction, pyrosequencing

- GNAS-activating mutations were found in 64% of validation group versus previously reported prevalence of 57%

- Overall, 52 of 86 (61%) of IPMNs demonstrated GNAS mutations

- 100% of I-type IPMNs demonstrated GNAS mutations compared to 51% of G-type, 71% of PB-type, and 0% of O-type

  Kanda

Mutant GNAS detected in duodenal collections of secretin stimulated pancreatic juice indicates the presence or emergence of pancreatic cysts

2013

GNAS

291 pancreatic fluid versus intraoperative frozen tissue samples

Microdissection, pyrosequencing, high-resolution digital melt-curve analysis

- GNAS mutations were detected in pancreatic juice samples of 50/78 familial and sporadic cases with IPMNs (64.1%) and 15/33 (45.5%) with only small cysts (< 5 mm), but none of 57 disease controls

- In 97 subjects who had serial pancreatic evaluations, GNAS mutations predicted subsequent progression of pancreatic cysts

  Kato

aPKCλ/i is a beneficial prognostic marker for pancreatic neoplasms

2013

aPKCλ/i (atypical protein kinase-proto-oncogene and RAS-driven tumorigenesis)

115 PDCA and 46 IPMN tissue samples and 11 paired normal versus cancer tissue samples

RNA and DNA PCR, IHC, tissue microarray

- High expression levels of aPKCl/i were significantly correlated with a worse histological grade (p = 0.010) and advanced stage of the tumor (p = 0.0050) in IPMN patients

- aPKCl/i expression involved in malignant transformation of IPMNs

  Nikiforova

Integration of KRAS testing in the diagnosis of pancreatic cystic lesions: a clinical experience of 618 pancreatic cysts

2013

KRAS

618 cyst fluid samples, tissue pathology available for 142 patients

DNA molecular analysis

- 603 (98%) cyst fluid samples were satisfactory for molecular analysis

- Mutations in KRAS were detected in 232/603 (38%) aspirates with 320/603 (53%) specimens that were either less than optimal (38%) or unsatisfactory (15%) for cytopathologic diagnosis

- 142 patients’ tissue specimens (26%) consisted of 53 KRAS-mutated and 89 KRAS-wild-type cysts

- KRAS mutations had a specificity of 100%, but a sensitivity of 54% for mucinous differentiation

- KRAS had a sensitivity of 67 and 14% for IPMNs and MCNs, respectively

  Nissim

Genetic markers of malignant transformation in intraductal papillary mucinous neoplasm of the pancreas: a meta-analysis

2012

MUC1, MUC2, MUC5AC, KRAS, p53, human telomerase reverse transcriptase (hTERT), cyclooxygenase-2, and SHH

1235 IPMN tissue samples

Pathologic review

- hTERT and SHH showed the strongest association with malignant IPMN, whereas MUC5AC and KRAS showed weak association with IPMN histologic progression

  Talar-Wojnarowska

A comparative analysis of K-ras mutation and carcinoembryonic antigen in pancreatic cyst fluid

2012

KRAS, CEA

56 cyst fluid samples (39 patients with benign lesions, 17 premalignant/malignant lesions)

Mutational analysis, DNA molecular analysis

- CEA levels were higher in patients with malignant cysts (mean levels 238 ± 12.5 ng/ml) compared to benign lesions (mean levels 34.5 ± 3.7 ng/ml; p < 0.001)

- KRAS mutation correctly classified 11 of 17 patients with premalignant/malignant lesions (64.7% sensitivity and 97.4% specificity; p < 0.01)

- The presence of CEA and KRAS mutation correctly identified 16 of 17 premalignant/malignant cysts (94.1%)

  Wu

Recurrent GNAS mutations define an unexpected pathway for pancreatic cyst development

2011

169 genes (33 oncogenes, 136 tumor suppressor genes)

19 cyst fluid samples from patients IPMN were first analyzed, then additional 113 IPMNs versus 95 tissue samples of PDAC

Massive parallel sequencing and DNA molecular analysis

- GNAS mutations were present in 66% of IPMNs, and either KRAS or GNAS mutations could be identified in 96%

- In 7/8 cases of IPMN-associated IC, the GNAS mutations present in the IPMNs were also found in the invasive lesion

  Khalid

Pancreatic cyst fluid DNA analysis in evaluating pancreatic cysts: a report of the PANDA study

2009

Cyst fluid KRAS, CEA

113 patients with 40 malignant, 48 premalignant, and 25 benign cyst fluid analysis and tissue samples collected

DNA molecular analysis, pathologic review

- Cyst fluid KRAS mutation was helpful in the diagnosis of mucinous cysts (specificity 96%)

- Components of DNA analysis (ie. high KRAS DNA amount, allelic loss amplitude >82%) detected malignant cysts

- All malignant cysts with negative cytological evaluation (10/40) could be diagnosed as malignant by using DNA analysis

  Jang

Increased K-ras mutation and expression of S100A4 and MUC2 protein in the malignant intraductal papillary mucinous tumor of the pancreas

2009

KRAS, S1004A, MUC2

41 tissue samples of FFPE IPMNs (27 with benign IPMNs and 14 with HGD/IC)

IHC, tissue microarray, DNA extraction, and mutational analysis of KRAS

- KRAS mutations at codons 12 and 13 were detected in 13/37 (38.2%) of the IPMNs: in 5/24 (20.8%) of benign IPMNs and in 8/13 (61.5%) of malignant IPMNs (p = 0.028)

- The expression of S100A4 and MUC2 was increased in malignant IPMNs

  Hashimoto

Telomere shortening and telomerase expression during multistage carcinogenesis of intraductal papillary mucinous neoplasms of the pancreas

2008

Telomerase expression

68 frozen tissue samples

- 17 IPMN patients with 37 individual loci

- 15 PDACs

- 10 CP

- 83 adjacent noncancerous pancreas tissue

Telomere length by quantitative fluorescence in situ hybridization

- Telomeres were significantly shortened in 36 (97.3%) of 37 IPMN loci, with average telomere length decreasing with IPMN progression

- Marked telomere shortening was observed from the HGD (p < 0.001) versus IGD IPMNs, although shortened to a critical length by IGD

- Upregulated human telomerase reverse transcriptase expression was detectable and increased gradually with cancer development

- Telomerase activation and progression observed early in IPMN carcinogenesis from IGD to HGD IPMNs

  Satoh

Expression of sonic hedgehog signaling pathway correlates with the tumorigenesis of intraductal papillary mucinous neoplasm of the pancreas

2008

SHH and Gli1

41 patients with IPMN

- 19 frozen tissue samples from 15 IPMN patients

- 75 FFPE tissue samples from 33 IPMN patients

Microdissection IHC, tissue microarray, qRT-PCR

- SHH and Gli1 mRNAs were detected in all the examined lesions and 8 out of 19 lesions in IPMNs, respectively

- SHH and Gli1 mRNAs were likely to be upregulated from the LGD to IGD to IC cells

- IHC of SHH and Gli1 expression was correlated with the grade of cell atypia

  Schonleben

PIK3CA, KRAS, and BRAF mutations in intraductal papillary mucinous neoplasm/carcinoma (IPMN/C) of the pancreas

2008

PIK3CA, KRAS, BRAF

38 FFPE tissue samples (3 LGD, 4 IGD, 5 HGD, 24 PDAC, 2 MCN)

Direct genomic DNA sequencing

- 4 somatic missense mutations of PIK3CA within the 36 IPMN specimens (11%)

- 17 (47%) KRAS mutations

- 1 (2.7%) BRAF mutation

  Schonleben

Mutational analyses of multiple oncogenic pathways in intraductal papillary mucinous neoplasms of the pancreas

2008

EGFR and HER2, KRAS, BRAF, and PIK3CA

36 FFPE tissue samples of IPMN

DNA mutational analysis

- Identified 1 silent mutation of HER2, 17 (43%) KRAS mutations, 1 (2.7%) BRAF mutation, and 4 (11%) mutations of PIK3CA in the IPMN/IPMC samples

  Jang

Immunohistochemical expression of sonic hedgehog in intraductal papillary mucinous tumor of the pancreas

2007

SHH

55 FFPE tissue samples from IPMN patients

IHC, tissue microarray

- IHC SHH expression was noted in 6 (46.2%) of 13 LGD, 5 (35.7%) of 14 IGD, 12 (80%) of 15 HGD, and 11 (84.6%) of 13 IC

- SHH expression was significantly increased in malignant IPMN (HGD/IC) compared with nonmalignant IPMN (LGD/IGD) (82.1 vs 40.7%, p = 0.0005)

- All 3 cases of node metastasis showed IHC SHH expression in tumor cells of metastatic lymph nodes → critical role in the late stage of carcinogenesis of IPMN

 Tumor suppressor genes

  Sakamoto

Clinicopathological significance of somatic RNF43 mutation and aberrant expression of ring finger protein 43 in intraductal papillary mucinous neoplasms of the pancreas

2015

RNF43, GNAS, KRAS, SMAD4 loss, and p53 overexpression

176 resected FFPE IPMN tissue samples, of which 57 had frozen tissue samples available

NGS, IHC, tissue microarray, mutational analysis

- Somatic RNF43 mutations were found in 8 (14%) of the 57 examined cases

- Ring finger protein 43 expression is downregulated in 52 (29.5%) of the 176 examined cases

- RNF43 mutations were significantly associated with the downregulated expression of ring finger protein 43 (p = 0.011), GNAS mutation (p = 0.020), and mural nodule detection (p = 0.038)

  Meng

Expression of SOX9 in IPMN of pancreas

2014

SOX9 versus MUC1, MUC2, MUC5AC

27 pathologic tissue samples (19 IPMN cases with 78 lesions identified)

IHC, tissue microarray

- SOX9 was expressed in the entire epithelium once the neoplasms advanced to HGD and IC

  Garcia-Carrecedo

Loss of PTEN expression is associated with poor prognosis in patients with IPMN of the pancreas

2013

PIK3CA, AKT1, PDPK1/PDK1, PTEN, and Ki-67

36 FFPE tissue samples of IPMNs

Mutational analysis

- 3 IPMN cases had E17K mutation in the AKT1 gene, and 1 case harboring the H1047R mutation in the PIK3CA gene was detected among the 36 cases

- PDK1 was significantly overexpressed in the HGD versus LGD (p = 0.034) and in PB and I-type IPMN versus G-type IPMN (p = 0.020)

- Loss of PTEN expression was strongly associated with the presence of IC and poor survival in these IPMN patients (p = 0.014)

  Kanda

Mutant TP53 in duodenal samples of pancreatic juice from patients with pancreatic cancer or high-grade dysplasia

2013

TP53

180 duodenal samples of pancreatic juice versus intraoperative frozen tissue samples (28 patients)

Mutational analysis

- TP53 mutations were identified in 9.1% of IGD IPMNs (2/22), 17.8% of PanIN-2 (8/45), 38.1% of HGD IPMNs (8/21), 47.6% of PanIN-3(10/21), and 75% of PDAC (15/20); no TP53 mutations were found in PanIN-1 lesions or LGD IPMNs

- TP53 mutations were detected in the duodenal samples of pancreatic juice from 29/43 patients with PDAC (67.4% sensitivity) and 4/8 patients with high-grade lesions (PanIN-3 and HGD IPMN)

  Ozaki

Serine protease inhibitor Kazal type 1 and epidermal growth factor receptor are express in PDAC, IPMN, PanIN

2013

SPINK1, EFGR

63 surgical FFPE tissue samples

- 23 PDAC

- 21 IPMN

- 65 PanIN-1A, 32 PanIN-1B, 17 PanIN-2, 6 PanIN3

- 8 other pancreatic neoplasm

- 11 CP

IHC, tissue microarray

- Both SPINK1 and EGFR were expressed in almost all PanIN lesions

- Ductal origin—PDAC, IPMN, and MCN samples expressed SPINK1

- EGFR was expressed in 87% of PDAC and 48% of IPMN lesions

- IPMN lesions and malignant lesions expressed EGFR more often than LGD/IGD

  Raty

Cyst fluid SPINK1 may help to differentiate benign and potentially malignant cystic pancreatic lesions

2013

Serine protease inhibitor Kazal type 1 (SPINK1) and enzymatic marker (tumor-associated trypsin inhibitor)

61 cyst fluid samples collected confirmed by FFPE tissue samples

- 4 acute pancreatitis

- 17 pseudocyst

- 7 SCA

- 21 MCN

- 12 IPMN

Immunofluorometric analysis

- Acute pancreatitis patients had high SPINK1 levels

- Among chronic cysts, SPINK1 levels were significantly higher in patients with potentially malignant cysts (MPD/mixed duct IPMN and MCN) than with benign cysts (BD-IPMN and SCA, 480 vs 18 mg/l; p < 0.0001)

  Shroff

SOX9: a useful marker for pancreatic ductal lineage of pancreatic neoplasms

2013

SOX9 (SRY gene)

146 benign pancreas tissue samples (109 normal pancreas and 37 CP) and 136 paired tissue samples from PDAC patients (47 PanIN, 21 IPMN, 14 MCN, 10 SCA, 39 PanNET, 9 ACC, and 23 SPN)

IHC, tissue microarray

- Nuclear expression of SOX9 was detected in the centroacinar cells and ductal cells, but not in acinar or endocrine cells in 100% benign tissue

- Compared to benign tissue, IPMN had lower SOX9 expression (p < 0.05)

  Dal Molina

Loss of expression of the SWI/SNF chromatin remodeling subunit BRG1/SMARCA4 is frequently observed in intraductal papillary mucinous neoplasms (IPMNs) of the pancreas

2012

BRG1

66 tissue samples of FFPE IPMNs

IHC, tissue microarray

- Normal pancreatic epithelium strongly immunolabeled for BRG1

- Reduced BRG1 expression was observed in 32 (53.3%) of the 60 evaluable IPMN lesions and occurred more frequently in HGD (13 of 17 showed loss; 76%) compared to IGD (15 of 29 showed loss; 52%) and LGD (4 of 14 showed loss; 28%) (p = 0.03)

  Jury

Gene expression changes associated with the progression of intraductal papillary mucinous neoplasms

2012

62 genes

14 patients with 28 FFPE tissue samples

- 10 LGD, 5 IGD

- 6 HGD, 7 IC

Microdissection, RNA isolation, transcriptome amplification microarray, qRT-PCR

- 62 genes were identified as showing significant changes in expression (p < 0.05 and a 2-fold cutoff)

- Growth factor AY-related pathways were found in the progression of IPMN to malignancy

- Most highly upregulated genes included regenerating islet-derived 3 (REG3A), FN1, and COL1A1, whereas gastrokines 1 and 2 (GKN1 and GKN2) were the mostly highly downregulated

- Three of the top subnetworks altered between HGD and LGD IPMNs were built around the seeds of transforming growth factor A receptor 1 (TGF-AR1), TGF-A1, and the general TGF family

  Hayashi

PDCD4 expression in intraductal papillary mucinous neoplasm of the pancreas: its association with tumor progression and proliferation

2010

PDCD4 (programmed cell death tumor suppressor)

108 tissue samples of IPMN

IHC, tissue microarray

- PDCD4 expression was 79, 43, and 10% in LGD, IGD, and HGD, respectively

- PDCD4 expression had a strong relationship with p21 expression (p = 0.0001) and an inverse correlation with Ki-67 labeling index (r = − 0.6255, p = 0.0001)

  Lubezky

High-throughput mutation profiling in intraductal papillary mucinous neoplasm (IPMN)

2010

323 oncogenic mutations in 22 tumor-related genes (KRAS, p53, and PIK3CA)

27 FFPE tissue samples (14 LGD, 6 IGD, 7 HGD)

Chip-based mass spectrometer, microdissection, DNA extraction and analysis

- 9 KRAS mutations (LGD, 2/14; IGD, 1/6; HGD, 6/7), 3 p53 mutations (LGD, 1/14; HGD, 2/7), and 2 PIK3CA mutations (LGD, 1/14; HGD, 1/7)

- KRAS, p53, and PIK3CA mutations present in the invasive cancer were absent in the adjacent precursor cells in 50% of the cases

  Abe

Different patterns of p16INK4A and p53 protein expressions in intraductal papillary-mucinous neoplasms and pancreatic intraepithelial neoplasia

2007

p16INK4A expression, overexpression of p53 and Ki-67

47 tissue samples of FFPE IPMN (from 26 patients) and 42 PanIN lesions (from 16 patients)

IHC, tissue microarray

- Either the loss of p16INK4A expression or the overexpression of p53 was much more frequently observed among PanIN-3 than among HGDs in IPMN (p = 0.046 and 0.008, respectively)

- Ki-67 labeling index was correlated with the histological grades of both PanINs and IPMNs (p = 0.0001 and p = 0.0001, respectively)

  Miyasaka

The role of the DNA damage checkpoint pathway in intraductal papillary mucinous neoplasms of the pancreas

2007

p21WAF1, accumulation of p53

128 tissue samples of FFPE IPMN (LGD, IGD, HGD, IC)

IHC, tissue microarray, and expression of ATM, Chk2, and p21WAF1 and accumulation of p53

- Chk2 phosphorylation and expression of p21WAF1 was shown in LGD and showed a significant decreasing trend with the progression of atypia (p < 0.0001)

- p53 accumulation was mostly detected in malignant IPMNs

- DNA damage checkpoint provides a selective pressure for p53 mutation

 NGS

  Yu

Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms

2017

KRAS, GNAS, TP53, SMAD4, CDKN2A, RNF43, TGFBR2, BRAF, PIK3CA

Pancreatic juice from 115 subjects with 53 discovery and 62 validation cohorts:

- 38 PDAC

- 57 IPMN (surgical pathology or imaging findings)

- 24 controls

96 NGS reactions sequencing 9 genes

- PDAC and IPMN were more likely to have mutant DNA detected in pancreatic juice than controls (both p < 0.0001)

- TP53 and/or SMAD4 mutations were commonly detected in juice samples from patients with PDAC and were not detected in controls (p < 0.0001); mutant TP53/SMAD4 concentrations could distinguish PDAC from IPMN cases with 32.4% sensitivity and 100% specificity and from controls

- 2/4 patients who developed PDAC despite close surveillance had SMAD4/TP53 mutations detected in juice samples collected 1 year prior to their PDAC diagnosis

  Jones

Impact of next-generation sequencing on the clinical diagnosis of pancreatic cysts

2016

AKT1, ALK, APC, BRAF, CDH1, CDKN2A, CTNNB1, DDR2, EGFR, ERBB2, ESR1, FBXW7, FGFR1, FGFR2, FGFR3, FOXL2, GNA11, GNAQ, GNAS, HRAS, IDH1, IDH2, KIT, KRAS, MAP2K1, MET, NOTCH, NRAS, PDGFRA PIK3CA, PIK3R1, PTEN, RET, ROS1, SMAD4, SMO, STK11, TP53, and VHL

99 pancreatic cyst fluid samples (EUS-FNA) from 86 patients were analyzed by cytology, CEA, and targeted NGS

39 cancer genes targeted with NGS

- NGS impacted the clinical diagnosis by defining a cyst as mucinous in 48% of cysts without elevated CEA levels

- 20% of cysts that were nonmucinous by imaging were mucinous by NGS; in 14 nonspecific cysts, CEA levels were not elevated in 12 (86%), and NGS established a mucinous etiology in 3 (25%)

- KRAS or GNAS mutation supported an IPMN with nonmucinous CEA in 71%

- 7 cyst fluids (8%) had either a TP53 mutation, loss of CDKN2A or SMAD4 in addition to KRAS and/or GNAS mutations; 5/7 (71%) were clinically malignant with, high-grade cytology

- CEA was more specific for a mucinous etiology (100%), but NGS was more sensitive (86 vs 57%)

  Springer

A combination of molecular markers and clinical features improve the classification of pancreatic cysts

2015

BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL

130 tissue samples from patients with cystic neoplasm (12 SCA, 10 SPN, 12 MCN, 96 IPMN) and 99 cyst fluid samples collected

Mutational analysis, massively parallel sequencing

- Molecular markers and clinical features classified cyst type with 90–100% sensitivity and 92–98% specificity

- Molecular marker panel correctly identified 67/74 patients who did not require surgery; the number of unnecessary operations was reduced by 91%

  Amato

Targeted next-generation sequencing of cancer genes dissects the molecular profiles of intraductal papillary neoplasms of the pancreas

2014

ABL1, AKT1, ALK, APC, ATM, BRAF, CDH1, CDKN2A, CSF1R, CTNNB1, EGFR, ERBB2, ERBB4, EZH2, FBXW7, FGFR1, FGFR2, FGFR3, FLT3, GNA11, GNAS, GNAQ, HNF1A, HRAS, IDH1, JAK2, JAK3, IDH2, KDR/VEGFR2, KIT, KRAS, MET, MLH1, MPL, NOTCH1, NPM1, NRAS, p16, PDGFRA, PIK3CA, PTEN, PTPN11, RB1, RET, SMAD4, SMARCB1, SMO, SRC, STK11, TP53, VHL, RNF43

52 tissue samples of IPMN from 51 surgically treated patients (40 fresh-frozen and 12 FFPE)

NGS of 51 cancer genes and IHC, tissue microarray of p16 and SMAD4

- At least 1 somatic mutation was observed in 46/48 (96%) IPMNs; 29 (60%) had multiple gene alterations

- GNAS and/or KRAS mutations were found in 44/48 (92%) of IPMNs

- GNAS was mutated in 38/48 (79%) IPMNs and KRAS in 24/48 (50%), and these mutations coexisted in 18/48 (37.5%) of IPMNs

- RNF43 was the third most commonly mutated gene and was always associated with GNAS and/or KRAS mutations

- TP53 and BRAF gene mutations (10 and 6%) were only observed in HGD IPMNs

- P16 was lost in 7/34 IPMNs and 9/17 IPMN-IC

- SMAD4 was lost in 1/34 IPMNs and 5/17 IPMN-IC

- DNA from 7 cyst fluid aspirates identified 10/13 mutations detected in their associated IPMN

 Methylation

  Hong

Genome-wide CpG island profiling of intraductal papillary mucinous neoplasms of the pancreas

2012

CpG island methylation profiles

6 tissue samples of IPMNs (1 LGD, 3 IGD, 2 HGD) and matched pancreatic duct samples

Methylation CpG island amplification (MCAM), Agilent methylated CpG island amplification and microarray analysis, methylation-specific PCR (MSP)

- 11/13 genes evaluated by MSP were more commonly methylated in 61 IPMN versus 43 normal pancreas samples

- Several genes (BNIP3, PTCHD2, SOX17, NXPH1, EBF3) were significantly more likely to be methylated in IPMNs with HGD than with LGD

- SOX17, demonstrated a loss of protein expression by IHC in 22% (19/88) of IPMNs

- The most specific marker was BNIP3

  Nakayama

Hypermethylation-mediated reduction of WWOX expression in intraductal papillary mucinous neoplasms of the pancreas

2009

WW domain-containing oxidoreductase (WWOX tumor suppressor), SMAD4

41 tissue samples of FFPE IPMNs (20 LGD, 21 HGD/IC)

IHC, tissue microarray, methylation-specific PCR

- Loss or reduced WWOX immunoreactivity was detected in 3 (15%) of 20 LGD and 17 (81%) of 21 HGD/IC

- Hypermethylation of the WWOX regulatory site was detected in 1 (33%) of 3 WWOX LGD and 9 (53%) of 17 WWOX HGD/IC

- Reduction of WWOX expression was significantly correlated with a higher Ki-67

- Decreased WWOX expression was significantly correlated with a loss of SMAD4 expression in these IPMNs

  Hong

Multiple genes are hypermethylated in intraductal papillary mucinous neoplasms of the pancreas

2008

Isolated DNA for 7 genes (SPARC, SARP2, TSLC1, RELN, TFPI2, CLDN5, UCHL1) commonly aberrantly methylated in PDAC

50 tissue samples from IPMN patients and adjacent PDAC and control pancreas from 27 IPMN/PDAC patients and 10 PanNET

Microdissection, IHC, tissue microarray, methylation-specific PCR

- The mean percentage of genes methylated in PDAC with IPMN was significantly higher than that in noninvasive IPMN or peritumoral normal epithelial cells

- IC had significantly more methylated genes than LGD/IGD IPMN (44 ± 26%, p < 0.0001)

- The mean percentage of genes methylated in histologically normal pancreatic ductal cells from patients with PDAC (22 ± 17%) was significantly higher than that in normal ductal cells from patients with PanNET (4 ± 7%, p = 0.002)

- Aberrant DANN methylation increases with histologic grades of IPMN

 Miscellaneous

  Morimatsu

Insulin-like growth factor II messenger RNA binding protein-3 is a valuable diagnostic and prognostic marker of intraductal papillary mucinous neoplasm

2013

Insulin-like growth factor II messenger RNA-binding protein 3

190 tissue samples and 15 biopsy samples of IPMNs

IHC, tissue microarray

- IMP3 expression was recognized in 71.8% (28/39) of HGD IPMN and in 81.3% (26/32) of IPMN-IC, but it was not found in any IPMNs with LGD or IGD

- IMP3 expression sensitivity of 76.1% and a specificity of 100% (p = 0.001)

- IMP3 expression was increased in cancerous lesions compared to noncancerous lesions in biopsy specimens (p = 0.027)

- Disease-specific survival IPMN-IC was significantly shorter in the high-expression group (> 50% tumor staining) than in the low-expression group (≤ 50% tumor staining; p = 0.0069)

  Oda

Differential ezrin and phosphorylated ezrin expression profiles between pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasm, and invasive ductal carcinoma of the pancreas

2013

Ezrin and p-ezrin

131 IPMN, 47 PanIN, 59 PDAC tissue samples

IHC, tissue microarray

- Ezrin and p-ezrin (tyr354) expressions were significantly higher in IPMN with an associated IC compared with those in IPMN with HGD (p = 0.03 and p = 0.0007, respectively)

- In all grades of PanINs, ezrin and p-ezrin (tyr353) were highly expressed

- The negative p-ezrin (tyr353) expression group of PDAC showed a significantly worse prognosis than did the positive p-ezrin (tyr353) expression group by survival analysis (p = 0.04) and adverse prognostic factor by both univariate and multivariate analyses (p = 0.048 and p = 0.015)

  Yonaiyama

Epithelial cell adhesion molecules (EpCAM) overexpression is correlated with malignant potential of IPMNs of the pancreas

2013

EpCAM

51 tissue samples of FFPE IPMN (32 LGD, 6 IGD, 8 HGD, 5 IC)

IHC, tissue microarray

- EpCAM overexpression was found in 16 (31.4%) of tumor samples

- 5 predictors of malignancy in univariate analysis: serums CA19-9, MPD diameter, mural nodule, phenotype, and EpCAM overexpression

- In multivariate analysis, only EpCAM overexpression was identified and independent

  Bausch

Plectin-1 is a biomarker of malignant pancreatic intraductal papillary mucinous neoplasms

2009

Plectin-1

49 tissue samples FFPE IPMNs (6 LGD/IGD, 31 HGD/IC, 12 PDAC with metastasis from IPMN) and cyst fluid from 4 HGD/IC and 3 LGD/IGD

IHC, tissue microarray, and plectin expression assays

- 26/31 malignant IPMN and all 12 lymph node metastases were Plec-1 positive compared to 6 benign IPMN expressed Plec-1

- Specificity of Plec-1 in distinguishing malignant IPMN from benign IPMN was 83%, and its sensitivity was 84%

- All cyst fluid samples from malignant IPMN, but none of the three benign IPMN, were Plec-1 positive

Glycoproteins

 CEA, CA19-9

  You

Emerging role of tumor markers and biochemistry in the preoperative invasive assessment of intraductal papillary mucinous neoplasm of the pancreas

2016

Serum CA19-9, CA24-2, CEA, hsCRP

87 patients with surgical resection (serum samples) [4 LGD, 34 IGD, 16 HGD, and 33 IC]

Serum analysis and pathology review

- Elevated serum concentrations of CA19-9, CA24-2, CEA, and hsCRP were significantly associated with IC

- Multivariate analysis: increased CA19-9- and CEA-independent predictors of invasiveness

- Combination of CA19-9, CA24-2, and CEA improved the accuracy: sensitivity and specificity of 71.0 and 87.7%, respectively

  Kim

Clinical implication of serum carcinoembryonic antigen and carbohydrate antigen 19-9 for the prediction of malignancy in intraductal papillary mucinous neoplasm of pancreas

2015

Serums CEA and CA19-9

367 (250 LGD/IGD, 117 HGD/PDAC) patients with surgical biopsy-proven IPMN with preoperative serum tumor markers

Serum analysis and pathology review

- HGD and PDAC diagnosed in 117 (31.9%) patients

- Elevated serum CA19-9 was more frequent in patients with malignant [34.2%; invasive IPMN (47.9%) vs HGD (11.4%), p < 0.001] IPMN

- Multivariate analysis showed that MPD > 5 mm, mural nodules, and elevated serum CA19-9 (p < 0.001) were independent predictors of malignancy

- The sensitivity, specificity, and accuracy were 34.2, 92.4, and 73.8%, respectively, for elevated serum CA19-9

  Ngamruengphong

Cyst carcinoembryonic antigen in differentiating pancreatic cysts: a meta-analysis

2013

Cyst CEA

504 patients (8 studies) with cyst fluid samples

Cyst fluid analysis and pathology review

- Cyst CEA for malignant cyst ranged from 109.9 to 6000 ng/ml

- Pooled sensitivity of 63%, specificity of 63%, and diagnostic OR of 3.84

- Mucinous cysts (MCN and IPMN, 227 patients), with a pooled sensitivity and specificity of 65 and 66%, respectively; OR = 4.74

  Kucera

Cyst fluid carcinoembryonic antigen level is not predictive of invasive cancer in patients with intraductal papillary mucinous neoplasm of the pancreas

2012

Cyst CEA

47 EUS-FNA cyst fluid and surgical resection (11 MPD-IPMN, 24 BD-IPMN, 12 mixed)

Cyst fluid analysis and pathology review

- Cyst fluid CEA concentration increased as the pathology progressed from LGD (1261 ± 1679 ng/ml) to IGD (7171 ± 22,210 ng/ml) to HGD (10,807 ± 36,203 ng/ml)

- The mean CEA level decreased once invasive cancer developed (p = 0.869)

- The sensitivity, specificity, PPV, NPV, and accuracy of a cyst fluid CEA concentration greater than 200 ng/ml for the diagnosis of malignant IPMN (HGD and IC) were 52.4, 42.3, 42.3, 52.4, and 46.8%, respectively

  Othman

Carcino embryonic antigen and long-term follow-up of mucinous pancreatic cysts including intraductal papillary mucinous neoplasm

2012

CEA

143 patient initial EUS-FNA cyst fluid samples (58 patient surgical resection pancreatic cysts, while 85 patient surveillance)

Cyst fluid analysis and pathology review

- In the surgical group, the median CEA value for benign and malignant mucinous neoplasms was 796 and 438 ng/ml, respectively

- No correlation between CEA level and progression in cyst size

  Fritz

Role of serum carbohydrate antigen 19-9 and carcinoembryonic antigen in distinguishing between benign and invasive intraductal papillary mucinous neoplasm of the pancreas

2011

CEA and CA19-9

142 patients with IPMN pathology (37 LGD, 38 IGD, 17 HGD, 50 PDAC) with preoperative serum CEA and CA19-9

Serum analysis and pathology review

- 142 patients, with raised CEA and CA19-9 serum levels, were significantly associated with IPMN-IC

- 74% IPMN-IC had raised levels of CA19-9

- With a cutoff level of 37 units/ml, CA19-9 had a specificity of 85.9%, a NPV of 85.9%, a PPV of 74.0%, and accuracy of 81.7%

- 80% of patients with an IPMN-IC had raised serum levels of CA19-9 and/or CEA compared with only 18% of those with a noninvasive tumor (p < 0.001)

  Nagula

Evaluation of cyst fluid CEA analysis in the diagnosis of mucinous cysts of the pancreas

2010

Cyst CEA

In 267 patient EUS-FNA cyst fluid samples, 97 had pathologic specimens (42 LGD IPMN, 10 HGD IPMN, 12 LGD MCN, 2 HGD MCN)

Cyst fluid analysis and pathology review

- Mucinous cysts were identified in 66 of 97 (68%)

- CEA > 192 ng/ml had a sensitivity and specificity of 73 and 65%, respectively, for identifying mucinous cysts; cyst fluid CEA was not associated with malignancy (p = 0.85)

- 178 patients under surveillance by 8 (4%) developed radiographic changes necessitating surgery; pathology demonstrated 7 benign mucinous cysts and 1 retention cyst. CEA was not associated with radiographic progression (p = 0.37)

 Mucin

  Sinha

A gastric glycoform of MUC5AC is a biomarker of mucinous cysts of the pancreas

2016

MUC5AC, endorepellin

Cyst fluid samples from three independent cohorts of 49, 32, and 66 patients

Antibody-lectin sandwich assays, monoclonal antibodies to test for terminal, alpha-linked GlcNAc

- Biomarker panel comprising the previously identified glycoforms of MUC5AC and endorepellin gave 96, 96, and 87% accuracy for identifying mucinous cysts in the 3 cohort, and average sensitivity and specificity of 92 and 94%, respectively

- Alpha-linked glycoform of MUC5AC was unique to IPMN, whereas terminal beta-linked GlcNAc was increased in both IPMNs and MCN

- The enzyme that synthesizes the alpha-GlcNAc, A4GNT, was expressed in the epithelia of mucinous cysts especially in HGD regions

  Akimoto

Serum N-glycan profiles in patients with intraductal papillary mucinous neoplasms of the pancreas

2015

CEA, CA 19-9, Span-1, and duke pancreatic monoclonal antigen type 2 (DUPAN-2), serum N-glycan profiles

79 tissue samples from IPMN patients, including 13 invasive IPMNs

Glycome analysis assessed the relationship between N-glycan changes

- 70 glycans were identified, and their expression profiles were significantly different depending on the cyst size, the presence of an enhancing solid component, and the histological grade of the IPMN

- 9 glycans were highly expressed in patients with invasive IPMNs

- Glycan m/z 3195 had the highest diagnostic value for distinguishing invasive from noninvasive IPMNs

- High levels of m/z 3195 (OR = 20.5) and the presence of enhancing solid components (OR = 35.8) were significant risk factors for invasive IPMNs

  Yokoyama

Diagnosis of pancreatic neoplasms using a novel method of DNA methylation analysis of mucin expression in pancreatic juice

2014

DNA methylation electrophoresis of mucin genes

45 pancreatic juice samples with various lesions with paired pancreatic tissue from 17 fresh surgical specimens of PDAC

IHC, tissue microarray, and DNA methylation status of pancreatic fluid

- DNA methylation status of MUC1, MUC2, and MUC4 in pancreatic juice matched with the mucin expression in tissue

- Analyses of the DNA methylation status of MUC1, MUC2, and MUC4 were useful for differential diagnosis of PDAC with specificity and sensitivity of 87 and 80%, 100 and 88% for I-type IPMN, and 88 and 77% for G-type IPMN, respectively

  Hisaka

Potential usefulness of mucin immunohistochemical staining of preoperative pancreatic biopsy or juice cytology specimens in the determination of treatment strategies for intraductal papillary mucinous neoplasm

2013

MUC1, MUC2, MUC5AC

60 tissue samples of IPMNs, 4 preoperative tumor biopsies, and 10 preoperative pancreatic juice cytology

IHC, tissue microarray

- Mucin IHC of preoperative biopsy of surgically resected specimens and pancreatic juice cytology (of 10 specimens) were in agreement

  Masuda

MUC2 expression and prevalence of high-grade dysplasia and invasive carcinoma in mixed-type intraductal papillary mucinous neoplasm of the pancreas

2013

MUC2 expression and the presence of HGD and IC, in mixed IPMN

101 consecutive patients with tissue samples of IPMNs morphologically classified (I/G/PB/O) with MPD-IPMN 16 (12/4/0/0), mixed type 45 (16/28/1/0), and BD-IPMN 40 (0/38/1/1)

IHC, tissue microarray

- MUC2 expression in MPD was 75%, mixed-type 36%, and BD-IPMNs 0%

- In mixed-type IPMN, the prevalence of HGD and/or IC in MUC2-positive IPMN was significantly higher than that of MUC2-negative IPMN (HGD + IC, 88 vs 38%, p = 0.0017; IC, 50 vs 21%, p = 0.042)

- MUC2 expression is an independent predictive factor of HGD and IC in mixed IPMN

  Sai

Pancreatic duct lavage cytology with the cell block method for discriminating benign and malignant branch-duct type intraductal papillary mucinous neoplasms

2013

Pancreatic duct lavage cytology: MUC1, MUC2, MUC5AC, MUC6

44 patients with pancreatic duct lavage and cell block samples (BD-IPMN)

IHC, tissue microarray

- Cell block diagnosis was cancer positive in 11 patients and negative in 33

- The sensitivity, specificity, PPV, and NPV of this method were 92, 100, 100, and 97%, respectively

- The cytological and histological results of MUC1, MUC2, MUC5AC, and MUC6 agreed in 88% (15/17), 94% (16/17), 88% (15/17), and 100% (17/17), respectively

  Carrara

Mucin expression pattern in pancreatic diseases: findings from EUS-guided fine-needle aspiration biopsies

2011

MUC1, MUC2, MUC3, MUC4, MUC5A, MUC5B, MUC6, and MUC7 genes

90 patients with solid or cystic pancreatic lesion cyst fluid EUS-FNA

Cytological analysis, RNA extraction, and RT-PCR

- Prevalence of MUC1, MUC2, MUC4, and MUC7 in PDAC was 57.7, 51.4, 18.9, and 73.0%, respectively

- 50% of benign lesions and PanNET and 63% of IPMNs were positive for MUC1

- 25% of benign lesions, 86% of PanNETs, and 47% of IPMNs were positive for MUC2

- None of the benign lesions or PanNETs expressed MUC4

- MUC7 expression was highly significant for PDAC (p = 0.007) and HGD IPMN (p = 0.05)

  Maker

Pancreatic cyst fluid and serum mucin levels predict dysplasia in intraductal papillary mucinous neoplasms of the pancreas

2011

MUC1, MUC2, MUC4, and MUC5AC

40 of 147 cyst fluid samples (EUS-FNA) (21 LGD/IGD, 19 HGD/IC) as well as serum and FFPE

Enzyme-linked immunosorbent assays

- MUC2 and MUC4 cyst fluid concentrations were elevated in high-risk versus low-risk patients

- Corresponding serum samples revealed higher levels of MUC5AC in high-risk compared with low-risk patients (19.9 ± 9.3 vs 2.2 ± 1.1 ng/ml, p = 0.04)

- Histopathologic subtype was significantly associated with grade of dysplasia, and the intestinal subtype displayed increased MUC2

  Nakata

S100P is a novel marker to identify intraductal papillary mucinous neoplasms

2010

S100P vs MUC1, MUC2, MUC5AC

105 FFPE IPMN tissue samples

IHC, tissue microarray

- S100P expression was not detected in normal pancreatic ductal epithelium but was detected in all IPMNs (100%) with diffuse nuclear or nuclear/cytoplasmic staining

- MUC5AC was also expressed in most IPMNs (102/105; 97%)

- S100P was clearly expressed in the invasive component of IPMNs (32/32; 100% vs MUC5AC was expressed in only 23 cases of 32 invasive components (p = 0.01)

  Shimamoto

MUC1 is a useful molecular marker for malignant intraductal papillary mucinous neoplasms in pancreatic juice obtained from endoscopic retrograde pancreatography

2010

CEA, MUC1, and human telomerase reverse transcriptase

34 pancreatic juice samples and serums (13 PDAC, 7 IC/HGD, 2 IGD, 7 LGD, 5 CP)

Fluid, RNA extraction, qRT-PCR

- MUC1 expression in IC was significantly higher in LGD while IGD and HGD had significantly higher MUC1 expression than LGD (p = 0.0152)

- The sensitivity, specificity, PPV, NPV, and accuracy of MUC1 mRNA were 88.9, 71.4, 80.0, 83.3, and 81.3%, respectively

  Pizzi

Glucose transpoter-1 expression and prognostic significance in pancreatic carcinogenesis

2009

Glut-1, MIB-1. MUC1, MUC2

60 patients who had surgical resection for PDAC, 9 surgical tissue samples from IPMN patients

IHC, tissue microarray

- Glut-1 expression was not found in IPMN with LGD/IGD but in 60% of IPMNs with HGD

  Yonezawa

Expression profiles of MUC1, MUC2, and MUC4 mucins in human neoplasms and their relationship with biological behavior

2008

MUC1, MUC2, MUC4

34 FFPE IPMN tissue samples (G-type), 23 IPMN (I-type), 50 PDAC

IHC, tissue microarray, in situ hybridization compared vs clinical factors

- MUC1 or MUC4 expression is related with the aggressive behavior of human neoplasms and a poor outcome of the patients

- MUC2 expression tends to be related with the indolent behavior of human neoplasms and a favorable outcome of the patients, although indolent PB-IPMN neoplasms sometimes show invasive growth with MUC1 expression in the invasive areas

- MUC2 mucin in indolent PB-IPMN coincided with expression of MUC2 mRNA

- DNA methylation and histone modification at the MUC2 promoter may play an important role

 Miscellaneous

  Ohtsuki

Usefulness of KL-6 in the subtyping of intraductal papillary mucinous neoplasia of the pancreas, including carcinoma, dysplasia, and hyperplasia

2014

KL-6 (mucinous high-molecular-weight glycoprotein) and MUC1, MUC2, MUC5AC, MUC6, and MIB-1 ab

12 FFPE tissue samples of HGD and IC IPMN with associated dysplasia and hyperplasia and 6 FFPE tissue samples of LGD IPMN with dysplasia and hyperplasia

IHC, tissue microarray

- KL-6 antibody is a good IHC biomarker of the PB subtype of IPMN, but not the I - subtype

- Identifying IPMN subtypes based on KL-6 sustainability would be useful

  Shindo

Podoplanin expression in the cyst wall correlates with the progression of intraductal papillary mucinous neoplasm

2014

Podoplanin expression (alpha-smooth muscle actin) in cancer-associated fibroblast

130 FFPE tissue samples of IPMNs (35 LGD, 23 IGD, 33 HGD, 39 IC)

IHC, tissue microarray of alpha-smooth muscle actin

- PDPN+ layer increased with progression from IPMN with LGD to IPMN-IC

- Thickerpodopanin expression in IPMN with MPD involvement, non-G-type IPMN, and IPMN with mural nodules

- A high ratio (> 50%) of PDPN+ stromal fibroblasts was a predictor of poor outcome

  Takeshita

Clinicopathologic study of the MIB-1 labeling index (Ki67) and postoperative prognosis for intraductal papillary mucinous neoplasms and ordinary ductal adenocarcinoma

2012

MIB-1 (ki67)

60 tissue samples of FFPE IPMN (30 LGD, 8 HGD, 13 IC, 9 PDAC)

IHC, tissue microarray, and MIB-1 labeling index

- MIB-1 labeling index for IPMN with LGD (1.8%) was significantly lower than that for IPMN with HGD (14.2%)

- 5-year survival rates after resection were 100% for IPMN LGD, 83.3% for HGD, 53.8% for IPMN with an IC, and 10.3% for PDAC

Cytokines and immunology

 Gemenetzis

Neutrophil-to-lymphocyte ratio is a predictive marker for invasive malignancy in intraductal papillary mucinous neoplasms of the pancreas

2017

NLR, PLR

272 patients who underwent tissue resection for IPMN with serum collected

Pathologic review, serum analysis

- NLR and PLR were significantly elevated in patients with IPMN-IC (p < 0.001)

- Multivariate analysis: NLR > 4, IPMN cyst of size > 3 cm, presence of enhanced solid component, MPD dilatation > 5 mm, and jaundice were statistically significant variables

 Yip-Schneider

Prostaglandin E2: a pancreatic fluid biomarker of intraductal papillary mucinous neoplasm dysplasia

2017

PGE2

Cyst fluid 100 patients with IPMN and tissue pathology (47 LGD/IGD, 34 HGD, 20 IC IPMN)

PGE2 ELISA, pathology review

- Mean pancreatic cyst fluid PGE2 levels in HGD and IC IPMN were significantly higher than LGD/IGD IPMN

- Pre-op pancreatic cyst fluid CEA > 192 ng/ml and PGE2 at a threshold of 0.5 pg/l demonstrated sensitivity, specificity, and accuracy of 78, 100, and 86%, respectively, for detection of HGD/IC IPMN

 Beatty

Immunobiology and immunosurveillance in patients with IPMNs, premalignant precursors of PDAC

2016

Anti-MUC1, surface markers

88 patients with IPMN, 75 healthy controls, and 105 PDAC patients with FFPE tissue samples and serum

IHC, tissue microarray, anti-MUC1 ELISA, flow cytometry

- IPMN patients make MUC1-specific IgG

- Evidence of CD4 and CD8 T cell infiltration into IPMN areas of high dysplasia, suggesting an ongoing immune response within the lesions

- Increased levels of circulating myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) in some IPMN = T cell exhaustion

 Arima

The neutrophil-to-lymphocyte ratio predicts malignant potential in intraductal papillary mucinous neoplasms

2015

NLR, CA19-9

76 patients with tissue resection for IPMN and pre-op serum

Pathologic review, serum analysis

- Preoperative NLR was significantly higher in patients with IPMN-IC (2.51 ± 0.84) than in patients with LGD (2.01 ± 0.71, p = 0.0079) and healthy volunteers (1.37 ± 0.33, p < 0.0001)

- NLR was significantly reduced after curative tumor resection

- MD (p = 0.0231) and NLR > 2.074 (p = 0.0329) were independent predictors of IPMN-IC

- With combined criteria including guidelines, CA19-9 > 37 IU/ml and NLR > 2.074 show a high PPV of 78% and specificity of 96%

 Ishikawa

Effect of Twist and Bmi1 on intraductal papillary mucinous neoplasm of the pancreas

2014

EMT markers for Notch signaling (Twist, Bmi1, Jagged1, E-cadherin)

35 FFPE IPMN tissue samples

IHC, tissue microarray

- Positive expression of Twist and Bmi1 was observed in 40.0 and 42.9% of IPMNs

- Twist and Bmi1 expression was significantly higher in HDG and IC (p < 0.05) than in LGD

- High expression of Twist was correlated with Jagged1 expression and inversely correlated with expression of E-cadherin (p = 0.06 and p < 0.05)

- Recurrence rate was significantly higher in the group that showed simultaneously high expression of Twist and Bmi1 (p < 0.05)

 Kuroki

EZH2 is associated with malignant behavior in pancreatic IPMN via p27Kip1 downregulation

2014

EMT (EZH2, Ki-67, p27Kip1)

- 45 IPMN tissue samples, with a total of 181 lesions (48 normal ducts, 50 LGD, 53 IGD, 19 HGD, 11 IC)

- 20 FFPE IPMN lesions (9 IGD, 6 HGD, 5 IC) and 7 normal ducts

IHC, tissue microarray, RNA analysis, and RT-PCR

- Ki-67-positive nuclei were increased during IPMN progression

- EZH2 expression displayed a similar pattern (normal duct, LGD, IGD, HGD, IC) with cell proliferative activity; EZH2 expression in malignant IPMNs was significantly higher

- Positive correlation between EZH2 expression and Ki-67-positive nuclear ratio (p = 0.0001)

 Lahat

Epithelial-to-mesenchymal transition (EMT) in intraductal papillary mucinous neoplasm (IPMN) is associated with high tumor grade and adverse outcomes

2014

EMT (E-cadherin, vimentin ZEB-1), microRNA

58 IPMN tissue pathology (25 LGD, 18 HGD, 15 IC) and 5 normal pancreas FFPE tissue samples

IHC, tissue microarray, western blot, microRNA microarray

- E-cadherin expression was significantly lower in malignant versus LGD IPMN (p < 0.05)

- Vimentin expression was increased in HGD (p < 0.05)

- EMT was associated with increased lymph node metastasis and decreased survival of HGD/IC IPMN patients (p < 0.05

- ZEB-1 was exclusively expressed by malignant IPMN tumors

- 24 differentially expressed miRNAs (14 upregulated, 10 downregulated)

- miR-200c and miR-141 were downregulated in malignant versus LGD IPMNs(p < 0.05)

 Ikemoto

Indoleamine 2,3-dioxygenase affects the aggressiveness of intraductal papillary mucinous neoplasms through Foxp3+ CD4+ CD25+ T cells in peripheral blood

2013

IDO (kynurenine pathway) and FoxP3

12 tissue samples from IPMN patients, serum

Serum analysis, IHC, tissue microarray

- Pathologically aggressive IPMNs was significantly associated with the number of peripheral Foxp3+ Tregs (p = 0.05) and IDO-positive cells per high-power field (HPF) (p = 0.01)

- Patients with 7 or more IDO-positive cells/HPF had a significantly higher recurrence rate than those with less than 7 IDO-positive cells/HPF (p = 0.01, log-rank test)

 Lee

Inflammatory protein profiling of pancreatic cyst fluid using EUS-FNA in tandem with cytokine microarray differentiates between BD IPMN and inflammatory cysts

2012

Inflammatory mediator proteins (IMPs)

83 of 89 IMPs detected in 10 patient EUS-FNA cyst fluid samples

Multiplexed IMP-targeted microarray, IMP profiles

- 7 IMPs were detected in BD-IPMN, but not inflammatory cysts, while 11 IMPs were identified in inflammatory cysts, but not BD-IPMN

- GM-CSF expression was present in all inflammatory cyst samples

- HGF was in higher concentrations in inflammatory cysts compared to BD-IPMN

 Maker

Cyst fluid interleukin-1b (IL-1β) levels predict the risk of carcinoma in intraductal papillary mucinous neoplasms of the pancreas

2011

Interleukin-1 beta (IL-1β)

34 cyst fluid aspirates (6 LGD, 15 IGD, 13 HGD, 6 IC)

Multiplex sandwich immunoassay

- IL-5 and IL-8 concentrations were higher in the cyst fluid from patients in the high-risk group - IL-1β concentrations were also higher in the cyst fluid from patients with HGD/IC (n = 19) compared to those with LGD/IGD (n = 21, 539 ± 255 vs 0.2 ± 0.1 pg/ml, p < 0.0001)

- IL-1β remained a significant predictor of high-risk cysts after multivariate analysis

 Schmidt

PGE (2) in pancreatic cyst fluid helps differentiate IPMN from MCN and predict IPMN dysplasia

2008

PGE2

65 patient cyst fluid and tissue specimens

- 29 IPMNs (2 LGD, 12 IGD, 8 HGD, 7 IC)

- 12 PDAC, 11 MCN, 5 SCA, 1 PSN

PGE2 ELISA, pathology review

- Mean PGE2 levels (pg/μl) in IPMNs (2.2 ± 0.6) were greater than in MCNs (0.2 ± 0.1) (p < 0.05)

- The mean PGE2 level of IPMN by dysplastic stage was 0.1 ± 0.01 (LGD), 1.2 ± 0.6 (IGD), 4.4 ± 0.9 (HGD), and 5.0 ± 2.3 (IC)

- PGE2 level dropped in advanced cases with pancreatic ductal obstruction (0.3 ± 0) versus nonobstructing lesionsi (8.6 ± 2.9)

Proteomics

 Al Efishat

Multi-institutional validation study of pancreatic cyst fluid protein analysis for prediction of high-risk IPMN of the pancreas

2017

Cyst fluid proteomic analysis

Cyst fluid of 149 patients (89 low risk, 60 high risk)

- Training set, n = 104

- Validation set, n = 45

Multianalyte bead array analysis (Luminex), pathologic review

- All 4 cyst fluid markers (MMP9, CA72-4, sFASL, and IL-4) were overexpressed in patients with high-risk IPMN (p < 0.05)

- 2 predictive models based on preselected combinations of CF markers had concordance indices of 0.76 (model 1) and 0.80 (model 2)

 Kim

Biomarker development for intraductal papillary mucinous neoplasms using multiple reaction monitoring mass spectrometry

2015

Plasma proteins

184 plasma samples (11 plasma proteins identified)

Mass spectrometry, high-throughput multiple reaction monitoring

- 11 proteins were identified to differentiate healthy samples from IPMN (lactate dehydrogenase B, thiorexodine thrombospondin I, IGF BP2, IGF BP3, leucine-rich alpha-2 glycoprotein 1, β-thromboglobulin, kallikrein B, complement component 5, angiotensin I, carboxypeptidase N polypeptide 2)

- 11 proteins were selected as IPMN marker candidates with high confidence in 184 plasma samples, and a six-protein panel constructed by combining marker candidates had high discriminatory power in distinguishing between IPMN and controls

 Rebours

In situ proteomic analysis by MALDI imaging identifies ubiquitin and thymosin-b4 as markers of malignant intraductal pancreatic mucinous neoplasms

2014

Cyst fluid proteomic analysis

- BD-IPMN frozen section (LGD 10, HGD 10) for MALDI (45 tissue specimens and 25 cyst fluid samples)

MALDI analysis, IHC, tissue microarray, and fluid DNA analysis

- MALDI differential spectra of proteins were found in the two groups with significantly different intensities: HGD IPMNs characterized as high monomeric ubiquitin and an acetylated fragment of thymosin-b4

- Validation on tissue microarray and EUS - FNA samples confirmed that ubiquitin was overexpressed in HGD (p = 0.04 and p = 0.0004)

 Corcos

Proteomic assessment of markers for malignancy in the mucus of intraductal papillary mucinous neoplasms of the pancreas

2012

Cyst fluid proteomic analysis

43 cyst fluid/mucus samples obtained from surgically resected IPMN patients (21 LGD/IGD, 22 HGD/IC/PDAC)

Mass spectrometry (candidate protein expression profiles)

- 5 candidate proteins of interest [mass-to-charge ratio (m/z) 5217, 6326, 6719, 10,453, and 10,849 days) were selected by their high diagnostic accuracy and ability to distinguish between malignant and benign tumors

MicroRNA

 Wang

MicroRNA expression levels as diagnostic biomarkers for IPMNs

2017

Pancreatic microRNA

78 tissue samples of FFPE IPMN and control patients

Microdissection and qRT-PCR

- miR-210, miR-223, miR-221, miR-155, and miR-187 were differentially expressed in normal pancreas and IPMNs

- miR-21, miR-155, miR-187, miR-210, and miR-223 are significantly higher in IPMN patient samples compared to the CP

- miR-16, miR-17, miR-181a, and miR-187 that are differentially expressed between the IPMN and PDAC

 Vila-Navarro

MicroRNAs for detection of pancreatic neoplasia: biomarker discovery by next-generation sequencing and validation in 2 independent cohorts

2017

Pancreatic and cyst fluid microRNA

70 tissue samples and 95 cyst fluid samples

- Discovery set—18 surgical samples (11 PDAC, 4 IPMN, 3 controls)

- Validation

• Set 1—52 surgical samples (24 PDAC, 7 IPMN, 6 CP, 15 controls)

• Set 2—95 EUS-FNA samples (60 PDAC, 9 IPMN, 26 controls)

Microdissection and qRT-PCR, NGS

- 607 and 396 miRNAs were significantly deregulated in PDAC versus IPMN versus controls, and 40 miRNAs were commonly overexpressed in both PDAC and IPMN

- The first validation set showed upregulation of 31 miRNAs in PDAC samples and 24 miRNAs in IPMNs

- In the second validation set, 30 miRNAs were upregulated with 13 miRNAs (miR-93, miR-16, miR-548d-3p, miR-320a, miR-4468, miR-3120-3p, miR-4713-5p, miR-103a, miR-155, miR-4770, miR-181a, miR-221, and miR-151b) showing good discriminatory power of PDAC versus control and 30 miRNAs upregulated in IPMNs

 Abue

Circulating miR-483-3p and miR-21 is highly expressed in plasma of pancreatic cancer

2015

Plasma and pancreatic microRNA (miR-483-3p and miR-21)

10 PDAC and 13 IPMN tissue samples; plasma samples of 32 PDAC patients, 12 IPMN, and 30 controls

Serum analysis, RNA extraction, microdissection, and qRT-PCR

- Expression levels of miR-483-3p and miR-21 were detected in all examined plasma samples and were significantly higher in PDAC compared to controls (p < 0.01)

- Plasma miR-483-3p expression was significantly higher in PDAC patients than IPMN patients (p < 0.05)

- Plasma miR-21 level was associated with advanced stage (p < 0.05), metastasis to lymph node and liver (p < 0.01), and shorter survival (p < 0.01) of the PDAC patients

 Humeau

Salivary microRNA in pancreatic cancer patients

2015

Salivary microRNA

Saliva samples from EGD from 7 PDAC, 4 pancreatitis patients, 4 controls, and 2 IPMN

Saliva analysis, RNA extraction, and qRT-PCR

- Identified miR-21, miR-23a, miR-23b, and miR-29c as being significantly upregulated in saliva of PDAC patients versus control showing sensitivities of 71.4, 85.7, 85.7, and 57%, respectively, and specificity of 100%

- miR-23a and miR23b are overexpressed in the saliva of patients with IPMN

- miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis versus controls

- miR-216 was upregulated in PDAC versus pancreatitis patients with sensitivity of 50% and specificity of 100%

 Permuth-Wey

A genome-wide investigation of microRNA expression identifies biologically-meaningful microRNAs that distinguish between high-risk and low-risk IPMN of the pancreas

2015

Pancreatic microRNA

49 tissue samples of FFPE IPMN

- Discovery phase, 28 (19 HGD, 9 LGD)

- Validation phase, 21 (4 LGD, 4 IGD, 11 HGD, 2 IC)

Microdissection and qRT-PCR, microarray gene analysis, bioinformatics analysis

- 6 miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were downregulated in high-risk versus low-risk IPMNs and significantly distinguished between groups

- Low miR-99b expression was associated with MPD involvement (p = 0.021)

- Serum albumin levels were correlated with miR-99a (p = 0.004) and miR-100 expression (p = 0.008)

- Oncogenic targets of miR-130a (ATG2B, MEOX2), miR-342-3p (DNMT1), and miR-126 (IRS-1) were upregulated in high- versus low-risk IPMNs (p < 0.10)

 Permuth-Wey

Plasma microRNAs as novel biomarkers for patients with IPMN of the pancreas

2015

Plasma microRNA

44 IPMN and 25 controls patients (2 from IPMN patients, 1 from controls) rendered 66 plasma samples

Serum analysis, RNA extraction, and qRT-PCR

- 30-miRNA signature distinguished 42 IPMN cases from 24 controls (p = 0.002)

- 5-miRNA (miR-200a-3p, miR-1185-5p, miR-33a-5p, miR-574-3p, and miR-663b) discriminated between 21 malignant (HGD/IC) and 21 benign (LGD/IGD) IPMNs (p = 0.005)

- Paired plasma and tissue samples from patients with IPMNs can have distinct miRNA expression profiles of miR-484, miR-330, and miR-574-3p

 Wang

Next-generation sequencing of pancreatic cyst fluid microRNAs from low-grade benign and high-grade invasive lesions

2015

Cyst fluid microRNA

17 cyst fluid samples from patients with IPMN, MCN, and PDAC

- 6 low-risk (LGD/IGD)

- 8 high-risk (HGD)

- 3 PDAC

RNA extraction, qRT-PCR, NGS, pathway analysis

- 13 miRNAs (miR-138, miR-195, miR-204, miR-216a, miR-217, miR-218, miR-802, miR-155, miR-214, miR-26a, miR-30b, miR-31, and miR-125) were elevated, and 2 miRNAs (miR-451a and miR-4284) were depleted in the cyst fluids derived from IC

- Tumor suppressors miR-216a and miR-217 varied significantly in these cyst fluids

 Caponi

The good, the bad and the ugly: a tale of miR-101, miR-21 and miR-155 in pancreatic IPMNs.

2013

Pancreatic microRNA

86 tissue samples

- 65 invasive IPMN

- 16 noninvasive IPMN

- 5 normal pancreas tissues

Microdissection and qRT-PCR

- miR-21 and miR-155 were upregulated in invasive IPMNs versus noninvasive IPMNs versus normal tissues

- miR-101 levels were significantly higher in noninvasive IPMNs and normal tissues compared with invasive IPMNs

- High levels of miR-21 were associated with worse overall survival and also had a shorter median disease-free survival (10.9 vs 29.9 months, p = 0.01)

- miR-21 is an independent prognostic indicator for mortality, disease progression, and positive lymph node status

 Lubezky

MicroRNA expression signatures in IPMN of the pancreas

2013

Pancreatic microRNA

55 FFPE tissue samples (10 LGD, 5 IGD, 5 HGD, 10 IPMN-IC, 5 PDAC, 10 normal pancreas)

Microdissection, microarray, and qRT-PCR

- 14 miRNAs were differentially expressed in all IPMN tissues compared with normal pancreatic tissue

- Expression of 15 miRNAs was significantly different between LGD/IGD versus HGD/IPMN-IC (miR-217, miR-216a, miR-216b, miR-148a, miR-375, miR-130b, miR-21-star, miR-146a, miR-150, miR-214-star, miR-503, miR-32, miR-424-star, miR-708, and miR-155)

 Matthaei

miRNA biomarkers in cyst fluid augment the diagnosis and management of pancreatic cysts

2012

Pancreatic and cyst fluid microRNA

- 55 FFPE tissue samples and 15 cyst fluid samples

- Validation with 33 FFPE tissue samples and 50 cyst fluid samples

RNA extraction, microdissection, and qRT-PCR

- 26 and 37 candidate miRNAs that distinguish LGD from HGD IPMNs were found using FFPE and cyst fluid specimens, respectively

- 18 miRNAs, selected from FFPE and cyst fluid data, separated HGD from LGD IPMNs, SCA, and uncommon cysts, such as SPN and PanNET

- Differentiation between groups was found with a sensitivity of 89% and specificity of 100%

 Ryu

Elevated microRNA miR-21 levels in pancreatic cyst fluid are predictive of mucinous precursor lesions of ductal adenocarcinoma

2011

Cyst fluid microRNA (miR-21, miR-155, miR-221, miR-17-3p, miR-191)

40 cyst fluid samples

- 24 cystic precursor lesions included 14 IPMNs (11 noninvasive, 3 IC) and 10 MCNs

- 16 nonmucinous cysts included 11 SCA and 5 benign cysts

RNA extraction, microdissection, and qRT-PCR

- Significantly higher expression of miR-21, miR-221, and miR-17-3p was observed in the mucinous versus nonmucinous cysts (p < 0.01)

- The highest median area under the curve for miR-21, with a median specificity of 76% and sensitivity of 80%

 Habbe

MicroRNA miR-155 is a biomarker of early pancreatic neoplasia

2009

Pancreatic and cyst fluid microRNA (a panel of 12 miRNAs)

- 15 noninvasive frozen IPMN tissue samples

- 64 tissue samples (13 LGD, 31 IGDs, and 20 HGD)

- Pancreas fluid from 15 patients (10 IPMN, 5 CP)

In situ hybridization, microdissection, and qRT-PCR

- Significant overexpression by qRT-PCR of 10 of 12 miRNAs was observed in the 15 IPMNs versus matched controls (p < 0.05), with miR-155 (mean 11.6-fold) and miR-21 (mean 12.1-fold) demonstrating the highest relative fold changes in the precursor lesions

- Upregulation of miR-155 transcripts was observed in 6 of 10 (60%) IPMN-associated pancreatic juice samples compared to 0 of 5 (0%) disease controls

FFPE formaldehyde-fixed paraffin-embedded, PanNET cystic pancreatic neuroendocrine tumors, LGD low-grade dysplasia, IGD intermediate-grade dysplasia, HGD high-grade dysplasia, IC invasive carcinoma, CP chronic pancreatitis, SCA serous cystadenoma, VHL Von Hippel-Lindau, LOH loss of heterozygosity, qRT-PCR quantitative reverse transcription polymerase chain reaction, ACC acinar cell carcinoma, SPN solid pseudopapillary neoplasm, BP benign pancreas, I-type intestinal, G-type gastric, PB-type pancreatobiliary, O-type oncocytic, GM-CSF granulocyte-macrophage colony-stimulating factor, HGF hepatocyte growth factor, EUS-FNA endoscopic ultrasound-guided fine needle aspiration, MCN mucinous cystic neoplasm, hsCRP high-sensitivity C-reactive protein, mRNA messenger RNA

Combined cytopathology and mutation analysis of KRAS and GNAS together, however, enhances their individual contribution, and the sensitivity and specificity of an IPMN diagnosis increase to 92 and 50%, respectively, with a positive predictive value (PPV) and negative predictive value (NPV) of 71 and 81%. Further, this analysis correctly predicted malignancy in 80% of IPMNs [30]. When exploring the use of serum and circulating free DNA (cfDNA), KRAS mutations were only seen in PDAC and no IPMNs were identified consistently [31]. The same group performed a recent study of circulating exomes of DNA, in which a small amount of serum was purified to identify cancer-specific mutations. In 48 serum samples from PDAC patients, the KRAS mutation (G12D) was present in 39.6% of the samples, correlating with the prevalence in matched tumor tissue of 40–50% [32]. Thus, it is feasible to evaluate KRAS mutational analysis in tissue, cyst fluid, duodenal fluid, and plasma, and its presence is associated with PDAC; however, as a single marker, it is not ideal to identify the diagnosis of IPMN or the level of cyst dysplasia consistently as it is essentially an early indicator of pancreatic cell stress.

GNAS

GNAS is an oncogene that encodes the stimulatory G protein α-subunit (Gsα) of heterotrimeric G proteins. When activated by the exchange of GDP for GTP and the dissociation from G protein β- and γ-subunits, Gsα stimulates the cyclic AMP-generating enzyme adenylyl cyclase and other effectors. Most GNAS mutations are found with the highest incidence in IPMNs and IPMNs associated with PDAC rather than PDAC alone [24]. Mutations are most often at codon 201 or 227, which are known to induce gain of function of the encoded Gsα protein. Through multiple studies, it has been demonstrated that GNAS mutations in pancreatic fluid correlate with tissue mutations and are found in IPMN more so than other pancreatic cysts [33, 34]. A recent publication identified recurrent mutations in the GNAS gene in invasive adenocarcinomas associated with IPMN, possibly defining a new pathway for pancreatic tumorigenesis specifically in IPMN [26].

When IPMN surgical specimens are evaluated for KRAS and GNAS mutations, they do not vary according to the degree of dysplasia, suggesting that mutations in these genes occur early in IPMN carcinogenesis. GNAS mutations were more prevalent in invasive IPMN colloid carcinoma rather than tubular-type invasive IPMN (89 vs 32%; p = 0.0003). Conversely, KRAS mutations were more prevalent in tubular-type (89 vs 52%; p = 0.01) [23]. At the same time, adding GNAS testing to pancreatic cyst fluid KRAS mutational analysis increases the diagnostic accuracy in identifying IPMNs to 66–80.7% [33].

Further, similar to KRAS, GNAS can be sequenced from cfDNA isolated from IPMN patient’s plasma. Differences in GNAS codon mutation rates between IPMN and control patients were significant with a sensitivity and specificity for diagnosing IPMN of 81 and 84.2%, respectively. Additional studies evaluating GNAS mutational rates in cyst fluid [3], pancreatic fluid [35], and tissue [26] demonstrated compatible rates of codon 201 mutations; thus, similar to studies evaluating KRAS, GNAS mutations can be identified across study samples and are a biomarker of IPMN [31].

PI3K

PI3Ks are a complex family of lipid kinases involved in cell proliferation, differentiation, chemotaxis, survival, trafficking, and glucose homeostasis [36]. Phosphoinositide-3-kinase-catalytic-alfa gene (PI3KCA) mutations frequently elevate the lipid kinase activity of PIK3CA and trigger the AKT signaling pathway. Of interest in a recent univariate analysis regarding surgically resected IPMN specimens, higher histologic grade and higher pathologic stage were associated with significant elevations of phosphorylated AKT [37]. The frequency of PIK3CA somatic mutations was low in IPMN but, when present, was associated with late-stage malignant transformation [37, 38, 39].

BRAF/MAPK pathway

B-Raf is a serine/threonine kinase, encoded by BRAF on chromosome 7, and is part of the RAS/MAPK pathway. The RAS/MAPK pathway regulates proliferation, differential migration, and apoptosis of cells. Although only a minority of IPMN have a somatic mutation in this gene, by altering the Ras-Raf-MEK-ERK-MAPK pathway, it may accelerate neoplastic progression [38, 39]. More recent studies utilizing next-generation sequencing (NGS) have shown that the presence of phosphorylated MAPK is associated with low-grade and gastric-subtype IPMN, and not high-grade disease [37]. Clearly, BRAF mutations and alterations in signaling axis are important in the pathogenesis of IPMN and likely correlate with the level of dysplasia, though additional studies will be necessary to elucidate clinical utility as a biomarker.

Telomerase reverse transcriptase expression

Telomerase reverse transcriptase is an RNA-dependent DNA polymerase coded on chromosome 5 (human telomerase reverse transcriptase (hTERT) gene). Normally, apoptotic cell death is induced when telomeres become critically short; however, the telomerase can extend telomeres to extend cell life. With increasing IPMN dysplasia, telomere shortening is reduced with telomerase activity significantly enhanced in invasive carcinoma. hTERT expression is 87% in invasive carcinoma, 85% in high-grade dysplasia, 36% in moderate-grade dysplasia, and 16% in low-grade IPMN [40]. Thus, hTERT expression may be a useful marker of IPMN progression towards malignancy.

Hedgehog signaling pathway

Hedgehog (Hh) protein is a family of secreted signaling factors that regulates organogenesis. Sonic hedgehog (SHH) is highly expressed in tumor cells and is the most studied ligand in the pathway. In IPMN, SHH expression is detected in 46.2% of low-grade, 35.7% of intermediate-grade, 80% of high-grade, and 84.6% of invasive carcinomas [41]. Furthermore, SHH expression is detectable in pancreatic juice from IPMN, but it is absent in pancreatitis [42]. These data suggest that the activation of the SHH signaling pathway is associated with transformation from benign to malignant dysplasia in IPMN [42, 43]. It has also been shown to be involved in the progression of IPMN carcinoma to lymph node metastasis [41]. Recently, a ligand to SHH (GL1) was found to have higher expression in the cytosol of IPMN cells as compared to PDAC cells. Simultaneously, hedgehog-interacting protein (HIP), which binds to SHH directly and attenuates SHH signaling, was found to be increased in precursor lesions as compared to PDAC lesions [44].

Summary: oncogenes

IPMNs are more likely to have mutated GNAS expression than PDACs, while KRAS is the most abundant mutation found across pancreatic cysts including IPMN. These oncogenes have been evaluated in surgical tissue, cyst fluid, pancreatic juice, and serum. The level of dysplasia in IPMN may be differentiated based on the level of expression of BRAF, phosphorylated MAPK, hTERT, and SHH.

Tumor suppressor genes

As opposed to oncogenes, tumor suppressor genes protect cells from uncontrolled cell growth. However, mutations in these genes cause a loss or reduction in function that often leads to carcinogenesis. The most extensively studied tumor suppressor genes in IPMN development and progression include tumor suppressor gene p53 (TP53), CDKN2A (p16), DPC4 (SMAD4), Brahma-related gene 1 (BRG1/SMARCA4), and ring finger protein 43 (RNF43/ β-catenin).

TP53

The TP53 is located on the short arm of chromosome 17p. It protects against mutation accumulation by DNA damage through induction of apoptosis [45]. p53 is inactivated by missense mutations in sequences coding for the DNA-binding domain. In IPMN tissue, p53 overexpression correlates with the grade of dysplasia with no mutations in low-grade, 33.3% in intermediate-grade, 38.5% in high-grade [46], and near 100% in invasive carcinomas [46, 47, 48]. Similar to tissue samples, pancreatic juice TP53 mutations are undetectable in low-grade IPMN and low-grade PanIN and range from 38 to 50% in high-grade dysplasia and IPMN carcinoma [49]. Levels of mutations in TP53/SMAD4 in pancreatic secretions were significantly higher in patients with PDAC rather than in patients with IPMN pathology [50]. Overexpression of TP53 correlated with increased progression to invasive IPMN, worse prognosis, and increased recurrence rates [37]. Thus, TP53 mutation is typically observed in malignant IPMNs and invasive carcinoma and is detectable in tissue and pancreatic juice, and these mutations are considered a late event in IPMN lesions [29].

CDKN2A/p16

CDKN2A, or cyclin-dependent kinase inhibitor 2A, is located on chromosome 9p21. The gene codes for two proteins—INK4 family members p16 (p16INK4a) and p14arf. Both are tumor suppressor proteins that regulate the cell cycle. P16INK4a inhibits progression of the cell cycle by binding to CDKs (CKD4 and CKD6) and forcing gene silencing through retinoblastoma protein phosphorylation. While the loss of p16 protein is predominantly described in low/intermediate-grade IPMN, the gene is silenced in invasive carcinoma. The loss of the tumor suppressor effect of CDKN2A occurs in 10–60% of benign cysts and in 90–100% of malignant cysts with associated carcinomas, making it an early event in IPMN formation [51]. The loss of heterozygosity of the p16 gene increased from low-grade dysplasia (12.5%), to intermediate-grade dysplasia (20%), to high-grade dysplasia (33%), and to invasive carcinoma (100%) [48]. The loss of p16INK4A expression is higher in PanIN-3 lesions than high-grade IPMN lesions, suggesting a deviating tumorigenic pathway for both lesions [46]. CDKN2A mutations are also found in IPMN cyst fluid in association with KRAS/GNAS mutations. Together, the data support the concept that mutations in this gene occur early, but not consistently, in the development of IPMN carcinogenesis [52].

SMAD4/DPC4

SMAD4 is a nuclear transcription factor that activates transcription of cell cycle inhibitors via the transforming growth factor β (TGF-β) growth inhibition pathway. It is encoded by the deleted pancreatic cancer locus 4 (DPC4) tumor suppressor gene on the 18q21 locus. Allelic deletion or mutation of SMAD4 upregulates the retinoblastoma pathway, enabling cell progression from the G1 to S phase without regulation. SMAD4 expression is detected in all low-, intermediate-, and high-grade dysplasias in IPMNs, while it is lost in 75% of invasive IPMNs (carcinomas) [53]. SMAD4 mutations have also recently been associated with poor prognosis and recurrence of IPMN, with likely invasive components [35]. Digital NGS scores are able to detect mutant TP53 and/or SMAD4 in pancreatic juice to distinguish PDAC (54% NGS scores of ≥ 5) from high-grade dysplasia in IPMN (0% NGS scores of ≥ 5) with 32.4% sensitivity and 100% specificity and to distinguish IPMN from control pancreas with 64.7% sensitivity and 100% specificity (p < 0.01) [50]. Furthermore, the loss of SMAD4 expression was found to be significantly associated with IPMN that progresses to carcinoma, poorer prognosis, and greater chance of recurrence when compared to other tumor suppressor genes and oncogenes in pathologic specimens [37]. These features make the loss of SMAD4 expression a late aggressive marker of IPMN progression to carcinoma.

BRG1/SMARCA4

Transcriptional activator BRG1, also known as the ATP-dependent helicase SWItch/sucrose nonfermentable (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), is known to regulate gene transcription by altering the chromatin structure. The loss of BRG1 expression in IPMN tissue is associated with increasing degrees of dysplasia from low grade (28%) to intermediate grade (52%) and to high grade (76%) [54]. A small number of pancreatic adenocarcinomas are also known for biallelic inactivation of the gene, thereby downregulating chromatin remodeling and cell cycle regulation [55]. The exact role of BRG1 silencing in IPMN remains to be elucidated.

RNF43/Wnt pathway/β-catenin

Ligand-induced wingless-type (Wnt)/β-catenin signaling has been shown to be aberrant in IPMN lesions, some originating from mutations in RNF43. RNF43 is a ubiquitin E3 ligase that targets the frizzled receptor via interactions with the R-spondin protein as a form of negative feedback. RNF43 has been shown to inhibit Wnt/β-catenin signaling by reducing the membrane-level fizzled receptor conformation to bind Wnt ligand. In pancreatic cancer cells, RNF43 loss of function conferred uninhibited Wnt signaling and β-catenin nuclear translocation [56]. RNF43 mutations are found in IPMN tissue and were significantly associated with GNAS mutations (p = 0.020) and mural nodule detection (p = 0.038) [57].

β-Catenin (encoded by the CTNNB1 gene) is a protein involved in the regulation and coordination of cell–cell adhesion and gene transcription. β-Catenin nuclear accumulation is associated with worsening dysplasia of IPMN [57], with nuclear β-catenin identified in high-grade and invasive IPMN tissues [37]. These studies support that unregulated Wnt/β-catenin signaling is present to a greater degree in malignant IPMN.

Epigenetics and DNA methylation of tumor suppressor genes

Epigenetic gene silencing has been associated with tumor progression in gastric [58], breast [59], hepatocellular [60], and nonsmall cell lung carcinomas [61]. The target enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 (PRC2), mediates histone methyltransferase activity and functions as a transcriptional repressor involved in gene silencing in invasive IPMN. Kuroki et al. evaluated EZH2’s role in IPMN malignant transformation by silencing tumor suppressor proteins Ki-67, and p27Kip1. It was identified that EZH2 and Ki-67 were predominantly found in malignant pancreatic tissue (p = 0.0001) whereas p27Kip1 was found to a greater degree in noninvasive IPMN tissue (malignant vs benign: p = 0.05). EZH2 was associated with accelerated cell proliferation and malignant progression in IPMN via the downregulation of p27Kip1 [62].

In regard to DNA methylation, Hong et al. performed CpG island amplification followed by microarray analysis to compare the methylation status of IPMN with normal pancreatic ductal epithelium in resected tissue specimens [63]. They found differential methylation in 2259 genetic regions and identified a series of genes including BNIP3, PTCHD2, SOX17, NXPH1, and EBF3 that were characteristically hypermethylated in IPMN with high-grade compared to low-grade dysplasia.

Summary: tumor suppressor genes

Most tumor suppressor gene analyses have been performed on pathologic samples of IPMN except for pancreatic juice assessment of p53 overexpression and loss of function of SMAD4. Overexpression of TP53; loss of heterozygosity or reduced expression of CDKN2A (p16), DPC4 (SMAD4), BRG1 (SMARCA4), and RNF43; and nuclear location of β-catenin are all associated with worrisome phenotypes of IPMN.

Glycoproteins

Glycan-based serological assays are useful to detect serum biomarkers for cancer. Serum and cyst fluid analyses have focused on carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), and mucin expression levels in different pancreatic cyst types. Their role in diagnosing IPMN and level of dysplasia is herein evaluated.

CEA and CA19-9

CA19-9 and CEA have historically been used as serum tumor markers for pancreatic cancer. CEA is a 180-kDa cell surface glycoprotein which is elevated in the serum of over half of all patients with PDAC [64]. Cyst fluid CEA was evaluated in a prospective cooperative study where a level > 192 ng/ml was found to be superior to EUS, cytology, and other tumor markers in identifying a mucinous, as opposed to serous, cyst [65, 66]. CA19-9, the Lewis antigen, is a tumor-associated glycoprotein that is also elevated in the serum from the majority of patients diagnosed with PDAC [67]. When evaluating the utility of CA19-9 in diagnosing IPMN, a study compared CA19-9 and the diagnostic characteristics in imaging put forth by the international consensus guidelines to evaluate grades of dysplasia. When percentages with elevated (> 37 units/ml) serum CA19-9 were significantly higher, the more invasive the IPMN [67]. The diagnostic accuracy of serum CA19-9 in predicting malignant IPMN (73.8%) was comparable to imaging findings including mural nodularity, MPD dilatation greater than 10 mm, and thickened cyst walls [68]. Furthermore, it was found that CA19-9 was an independent predictor of malignancy. Serum concentrations of CEA and CA19-9 are higher in patients with malignant rather than benign IPMN and are especially high in patients with invasive IPMN [69]. Cyst fluid CEA levels increasing with malignant potential of IPMN have not been supported [70], and the ability of cyst fluid CEA to differentiate malignant from benign pancreatic cysts was not supported in multiple studies [18, 71, 72]. Hence, elevated serum levels of these markers are reflective of invasive cancer that is usually already radiographically evident, and their role in cyst fluid is limited to CEA as a marker of mucinous histology.

Mucin gene expression

Mucins (MUCs) are heavily glycosylated high-molecular-weight glycoproteins whose polypeptide chains have domains rich in threonine and serine [73]. In the pancreas, these glycoproteins play a role in protecting the ductal lining and allowing for duct renewal, epithelial differentiation, modulation of cell adhesion, and cell signaling. Alterations in MUC glycosylation have been observed in tumor tissues, and it has been hypothesized that they are important in tumorigenicity [74, 75, 76].

MUC1 and MUC3 are membrane-associated glycoproteins, whereas MUC2, MUC5A, MUC5B, and MUC6 are gel-forming mucins and MUC7 is a soluble mucin. Localization of MUC1 changes with dysplasia as it mainly exists on the luminal surface of normal pancreatic tissue rather than in the main pancreatic duct, acini, or islets. Other than the membrane-associated glycoproteins, the other classes of mucins are not expressed in normal pancreas, making their expression notably tumor-associated [73, 77].

Varying levels of MUCs corresponding with a level of dysplasia have been controversial but can be corroborated to some degree with the histopathologic subtype. Some studies revealed that IPMNs usually express MUC2, MUC5AC, and mostly MUC4, whereas they do not express MUC1 [78, 79]. The gastric subtype of IPMN is usually low-grade and typically expresses MUC5AC but not MUC1 or MUC2. Further evaluation of MUC5AC recently identified an alpha-linked glycoform of that can differentiate between IPMN and mucinous cystic neoplasms. This glycoform is not only specific for IPMN, but the enzyme that it has produced, A4GNT, is highly expressed in regions with high-grade dysplasia [80]. The intestinal subtype of IPMN typically expresses MUC2 while the more aggressive pancreatobiliary subtype typically expresses MUC1 [81].

This is in contrast to invasive ductal adenocarcinomas, which are characterized by the overexpression of MUC1 and the absence of MUC2. In a study of cyst fluid from patients with IPMN that underwent pancreatectomy, it was determined that MUC1 expression was very low in the cyst fluid in all IPMN groups with no correlation with the degree of cyst dysplasia. MUC5AC expression was much higher than MUC1 across all IPMN groups, but it too had no association between expression and the level of dysplasia. On the other hand, MUC2 and MUC4 levels tended to follow the degree of dysplasia with higher levels of expression in highly dysplastic and invasive cystic lesions [17].

A hallmark of pancreatic ductal adenocarcinoma is overexpression of MUC1 and MUC4 with alternative isoforms without tandem repeat array [82, 83]. These isoforms are associated with metastasis likely due to their anti-adhesive properties that hinder intercell and cell-to-matrix interactions [84]. Carrara et al. determined that 63% of IPMN cyst fluids were positive for MUC1 and 47% were positive for MUC2 expression [85], while MUC7 expression was associated with adenocarcinoma (p = 0.007) and IPMN (p = 0.05). Taken together, MUC expression in cyst fluid and resected tissues can help differentiate the biology of disease based on the IPMN histopathologic subtype. In the preoperative setting, cyst fluid analysis of MUC2 and MUC4 may identify high-risk cysts a priori.

Aberrant glycosylation of serum proteins

Aberrant glycosylation of serum proteins has been investigated in patients with pancreatic cancer where increased fucosylation is a common modification [86]. Analysis of serum N-glycan profiles in patients with various stages of IPMN showed that glycan m/z 3195, a tri-antennary glycan with fucose residues, exhibited the highest diagnostic value in distinguishing invasive from noninvasive IPMN (p = 0.0006) with 92.3% sensitivity and 66.7% specificity [87]. Still more work is needed to see how applicable this is in distinguishing IPMN levels of dysplasia.

Summary: glycoproteins

Elevated serum levels of CEA and CA19-9 are markers of invasive cancer, and cyst fluid is limited to CEA as a marker of mucinous histology. MUC5AC expression is elevated more consistently in IPMN lesions while levels of MUC2 and MUC4 expression in fluid tend to be greatest in high-risk lesions. Although aberrant protein glycosylation is found in cyst fluid and serum, currently, their quantification has more utility in diagnosing carcinoma than in determining an IPMN level of dysplasia.

Cytokines and immune response

Inflammation and neoplasia have a complex interplay in pancreatic dysplasia and can be a biologic indicator for IPMN. The difficulty is distinguishing whether the inflammatory response is a response to cyst dysplasia or conversely, if inflammatory cytokines cause a change in the tissue microenvironment, causing initiation of the dysplasia [88]. In both instances, quantification of the immune response in IPMN cyst fluid may serve as a biomarker of dysplasia [89].

Epithelial-to-mesenchymal transition (EMT), an early embryonic process associated with inflammation and pancreatic carcinogenesis, may have a role in IPMN development [90, 91]. In one study, E-cadherin loss, a hallmark of EMT, was not observed in the majority of low-grade IPMNs (n = 20; 82%) in comparison to carcinoma, and the data demonstrated a correlation between overexpression of EMT markers and a higher IPMN tumor grade. The Kaplan–Meier survival analysis demonstrated that low expression of E-cadherin was associated with decreased IPMN patient survival (p = 0.007) [92].

Maker et al. analyzed cytokine expression to evaluate both humoral and cell-mediated markers of the immune response in 40 IPMN cyst fluid samples from resected tumors. In a univariate analysis, IL-1β, IL-5, and IL-8 had higher levels of expression in the presence of high-grade dysplasia or invasive carcinoma. Multivariate analysis revealed that IL-1β levels alone predicted high- from low-risk cysts and remained a significant predictor when corrected for IPMN type and cyst size. IL-1β levels had a high sensitivity and specificity to correctly identify high- from low-risk cysts [18].

Prostaglandins represent another molecular marker of inflammation that may be evaluated in IPMN cyst fluid. In a small number of patients with BD and MPD-IPMN, prostaglandin E2 (PGE2) levels in the cyst fluid were measured and found to differentiate low- from high-risk lesions [93]. PGE2 is a product of cyclooxygenase-2 (COX-2), a key enzyme in the inflammatory pathway found to be overexpressed in malignancy. In a validation study, mean pancreatic cyst fluid PGE2 levels in low-grade, high-grade, and invasive IPMNs increased in stepwise fashion (p = 0.002) [94].

Identification of antigens expressed on premalignant IPMN would be ideal targets for immunotherapy. The tumor antigen MUC1 is a potential target given that elevated MUC1 IgG expression is associated with improved survival [95]. Furthermore, areas of high-grade dysplasia in IPMN demonstrated increased CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs) indicative of an innate anti-tumor immune response. In this study, increased levels of circulating myeloid-derived suppressor cells and regulatory T cells and evidence of T cell exhaustion were also identified in high-risk lesions [96]. Thus, the study of the IPMN tumor microenvironment supports the presence of inherent tumor antigen recognition tempered by an immunoregulatory milieu.

Similarly, serum neutrophil-to-lymphocyte ratio (NLR) and platelet-to-lymphocyte ratio (PLR) reflect the immune response in IPMN with increased ratios being associated with malignancy. NLR and PLR were significantly elevated in patients with IPMN-associated invasive carcinoma (p < 0.001) [97]. A separate study also found that a NLR > 2.074 had a high positive predictive value of 78% and specificity of 96% in diagnosing invasive IPMN [98].

Summary: cytokines and immune response

The immune response to IPMN is currently being evaluated in tissue, cyst fluid, and serum. Increasing tumor grade is associated with EMTs, the presence of CD4 and CD8+ TIL, and elevated levels of cyst fluid IL-1β and PGE2, while high ratios of NLR and PLR are associated with invasive disease.

Proteomics

Cyst fluid analysis is unique in that it provides the opportunity to study IPMN-secreted proteins. From large-scale multiplex analysis of proteins in IPMN cyst fluid, MMP9, CA72-4, sFASL, and IL-4 have been identified as a proteomic signature with the ability to discriminate high- from low-risk cysts with a concordance index of up to 0.8. In a multi-institutional validation study, the concordance index could be further increased when a clinical nomogram was included with the molecular data [99].

DNA and RNA mutational analysis

Recent advances in genomic technology have enhanced the ability to determine aberrant gene expression in small sample volumes of serum, cyst fluid, and pancreatic juice. Low yields of genetic material can be amplified, and their expression profiles can be compared between levels of dysplasia in IPMN.

Next-generation sequencing

NGS has enhanced the use of small volume samples with limited DNA/RNA to detect low-abundance somatic mutations. To avoid calling NGS-related error mutations, studies are required to have a standard of reproducibility and PCR confirmation of mutations [52]. NGS of KRAS, GNAS, loss of CDKN2A, loss of SMAD4, or overexpression of TP53 was found to change image-based cyst-type diagnosis in 12% of cases. These changes affected patient management (benign to malignant diagnosis, and vice versa). Of 53 cysts classified as mucinous by imaging, 36 (68%) did not have an elevated cyst fluid CEA level but NGS confirmed a mucinous etiology in 26 of the 36 cysts (72%) [52]. Similarly, NGS has been used to distinguish between PDAC and IPMN from endoscopically collected pancreatic juice samples. In this study, TP53/SMAD4 concentrations were found to differentiate PDAC from IPMN with 32.4% sensitivity and 100% specificity [50].

In a multicenter retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cyst adenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 IPMNs), cyst fluid was analyzed to identify mutated genes and loss of heterozygosity in BRAF, CDKN2A, CTNNB1/β-catenin gene, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL. Results were included in an algorithm to identify the cyst type and level of dysplasia. A combination of molecular signatures and clinical features revealed a sensitivity of 90–100% and specificity of 92–98% to appropriately identify 67/74 patients with low- or no-risk lesions. The authors estimated that surgical management could have been reduced by 91% with this tool. Specifically, the molecular features used to identify IPMN were the presence of GNAS, RNF43, loss of heterozygosity of chromosome 9, chromosome 1q aneuploidy, and chromosome 8 aneuploidy with clinical features including age ≥ 85 years, MPD dilation, cyst communication with MPD, and abdominal pain. This combination resulted in a specificity of 84% and sensitivity of 94% to correctly identify a pancreatic cyst that required surgical resection, including malignant IPMN [3]. These findings are similar to a previous study by the same group where whole exome sequencing of multiple types of cysts identified composite molecular and clinical features with an accuracy of 82% using multivariate organization of combinatorial alterations analytics [100].

Variations in microRNA expression

Tissue miRNAs

One in three protein-coding genes is small (19–25-nucleotide) noncoding RNAs known as miRNAs. Since miRNAs are stable in tissue and biofluids while also having tissue-specific expression patterns, they are a favorable biomarker for study in IPMN [101]. Dysregulation has been recognized in numerous human malignancies, including pancreatic ductal adenocarcinoma and precursor lesions [102, 103, 104, 105]. Elevated levels of miR-21 and miR-155 were identified in surgically resected noninvasive IPMN but were more frequently detected in IPMN with carcinoma in situ [106]. When comparing aspirated cyst fluid to resected tissue, significantly elevated concentrations of miR-21, miR-217, and miR-17-3p were found in mucinous cysts compared to nonmucinous cysts [107].

Global miRNA expression analysis of varying grades of IPMN exposed a hierarchical clustering of 15 miRNAs that were different between low- and intermediate-grade IPMNs compared to high-grade and invasive IPMNs [108]. Specifically, three miRNAs (miR-21, miR-708, miR-155) were elevated, and miR-217 was decreased in high-grade and invasive IPMNs compared to low- and moderate-grade IPMNs.

Since more than 1000 miRNAs exist, several recent studies have focused investigations on genome-wide miRNA expression in IPMN tissue. Matthaei et al. utilized high-throughput miRNA expression profiling in low-grade (n = 10) and high-grade (n = 12) IPMN tissues as well as cyst fluid from three low-grade and four high-grade IPMNs [109]. Logistic regression analysis led to the final identification of a nine-miRNA model (miR-24, miR-18a, miR-30a-3p, miR-92a, miR-106b, miR-342-3p, miR-99b, miR-142-3p, miR-532-3p), which allowed separation of all but one high-grade IPMN from low-grade IPMNs/SCAs with a sensitivity and specificity of 89 and 100%, respectively. Of note, miR-21 was upregulated in all grades of IPMN but did not predict the risk of malignant transformation. Similarly, Permuth-Wey et al. evaluated miRNA expression in IPMN tissue with the goal of discovering miRNAs that may differentiate high-risk IPMN from low-risk lesions. Six miRNAs (miR-100, miR-99b, miR-99a, miR-342-3p, miR-126, miR-130a) were downregulated in high-risk versus low-risk IPMN. The authors further evaluated the oncogenic targets of these miRNAs and found that ATG2B, MEOX2 (miR-130a), DNMT1 (miR-342-3p), and IRS-1 (miR-126) were upregulated in high- versus low-risk IPMNs (p < 0.10) [110]. Additional studies by Wang et al. identified yet additional miRNA that distinguished PDAC from IPMN with miR-16, miR-17, miR-181a, and miR-187 [111].

Cyst fluid miRNAs

Further evaluation of cyst fluid revealed combinations of miRNA associated with IPMN and levels of dysplasia. Tissue samples and cyst fluid were analyzed with NGS to compare miRNA patterns between PDAC, IPMN, and control specimens. Thirteen miRNAs (miR-93, miR-16, miR-548d-3p, miR-320a, miR-4468, miR-3120-3p, miR-4713-5p, miR-103a, miR-155, miR-4770, miR-181a, miR-221, and miR-151b) had high accuracy in discriminating PDAC from control specimens. Similarly, five miRNAs (miR-103a, miR-155, miR-181a, miR-181b, and miR-93) could discriminate IPMN from control specimens [112].

In a concurrent study, 13 miRNAs (miR-138, miR-195, miR-204, miR-216a, miR-217, miR-218, miR-802, miR-155, miR-214, miR-26a, miR-30b, miR-31, and miR-125) were found to be elevated in cyst fluid samples from pancreatic tumors and two miRNAs (miR-451a and miR-4284) were decreased as the level of dysplasia increased. Further, miR-216a and miR-217 significantly discriminated between low- and high-grade dysplasia/PDAC [113].

Circulating miRNAs

Plasma analysis of patients with PDAC revealed elevated circulating miR483-3p and miR-21 compared to both healthy controls and IPMN patients [114]. A 30-miRNA signature was identified that distinguished IPMN from healthy controls. These miRNAs included miR-378e, miR-579, miR-30e-5p, and miR-570-3p that together demonstrated a 2.09–4.32-fold higher expression in IPMN. As a single marker, miR-145-5p was most significantly associated with an IPMN diagnosis. Of the 30 miRNAs, a five-miRNA (miR-200a-3p, miR-1185-5p, miR-33a-5p, miR-574-3p, and miR-663b) signature discriminated between 21 high-risk and 21 low-risk (low- and moderate-grade dysplasias) IPMNs (p = 0.005). This five-miRNA IPMN signature detected malignant IPMN with 80.9% sensitivity, 52.5% specificity, 63.0% PPV, and 73.3% NPV. Pathway enrichment analysis of this group of miRNAs was associated with cell cycle and Wnt signaling, while the broader 30-miRNA signature was also associated with pathways in cancer, p53 signaling, and PI3K-AKT signaling, consistent with the data discussed in prior sections of this review [115].

Salivary miRNA

Salivary miRNA has been evaluated as a marker in various cancers. Humeau et al. demonstrated that four salivary miRNAs (miR-21, miR-23a, miR-23b, and miR-29c) could identify PDAC patients from cancer-free patients, while miR-210 and let-7c identified pancreatitis patients from healthy controls, and miR-216 discriminated pancreatitis from cancer. miR-23a and miR-23b were found in the saliva of patients diagnosed with IPMN; however these miRNAs were also present in the saliva of patients with pancreatitis, showing that these salivary miRNAs may be expressed in multiple nonspecific pancreatic pathologies [116]. Furthermore, salivary miR-23a overexpression has recently been associated with KRAS [117] and linked to EMT [118], which were discussed in prior sections of this review.

Summary: NGS and microRNA

NGS has enhanced the use of limited DNA/RNA to detect low-abundance somatic mutations. DNA mutational analysis in combination with clinical features may increase the strength of an IPMN diagnosis and potentially can predict the level of dysplasia. miRNA may also serve as a diagnostic marker of IPMN and may differentiate levels of cyst dysplasia. However, multiple different miRNAs have been identified in various studies with limited overlap. Furthermore, miRNA expression patterns appear to differ depending on the body fluid sampled. Pathway enrichment analysis of miRNA expression has enhanced our understanding of IPMN carcinogenesis, yet much work and validation is required to elucidate specific therapeutic or diagnostic targets.

Conclusions

Our understanding of the natural history of IPMN has greatly evolved in the last few decades, and the majority of research has concentrated on clinical and radiographic features associated with confirming the diagnosis and predicting malignant potential. Indeed, the European, AGA, and Fukuoka/modified Sendai criteria that currently guide our surgical management of these patients are based on these studies. In order to enhance the sensitivity of our guidelines and improve specific patient selection for resection, molecular diagnostics will need to be validated and incorporated into these algorithms. Much work has been done in the last 10 years to identify potential biomarkers from tissue, pancreatic juice, cyst fluid, plasma, and even saliva to include in these algorithms, as we have endeavored to review in this rigorous systematic review. Potential biomarkers with discriminatory ability can be found in various tissues, and sources including proteins, glycoproteins, RNA, DNA, and cytokines. Given the ability for cyst fluid to be obtained preoperatively and to reflect secreted glycoproteins, proteins, genetic material, and immunologic markers as representative of the whole cyst, we anticipate that cyst fluid, as well as endoscopically acquired pancreatic fluid in combination with high-throughput sequencing, will be a most likely source for future molecular diagnostics in this disease. The optimal signature has yet to be determined; however, it is clear that a combination of molecular markers and clinical signs and symptoms will improve our selection of patients for both surveillance and curative intent resection in the very near future.

Notes

Acknowledgements

The authors give thanks to Ms. Helena Vonville for the spreadsheets to guide, organize, and execute the systematic review.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

423_2017_1644_MOESM1_ESM.docx (33 kb)
ESM 1 (DOCX 32 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Surgery, Division of Surgical OncologyUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of Microbiology and ImmunologyUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Creticos Cancer Center at AIMMCChicagoUSA

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