Circulating Tumor Cells and Cell-Free DNA in Pancreatic Ductal Adenocarcinoma

Circulating Tumor Cells and Cell-Free DNA in Pancreatic Ductal Adenocarcinoma

The American Journal of Pathology, Vol. 189, No. 1, January 2019 ajp.amjpathol.org Pancreatic Cancer Theme Issue REVIEW Circulating Tumor Cells and...

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The American Journal of Pathology, Vol. 189, No. 1, January 2019

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Pancreatic Cancer Theme Issue

REVIEW Circulating Tumor Cells and Cell-Free DNA in Pancreatic Ductal Adenocarcinoma Tamara M.H. Gall, Samuel Belete, Esha Khanderia, Adam E. Frampton, and Long R. Jiao From the HepatoePancreato-Biliary Surgical Unit, Department of Surgery and Cancer, Imperial College, Hammersmith Hospital Campus, London, United Kingdom Accepted for publication March 26, 2018. Address correspondence to Tamara M. Gall, M.D., Department of Surgery and Cancer, Hammersmith Hospital, Du Cane Road, London, W12 0HS, United Kingdom. E-mail: [email protected]

Pancreatic cancer is detected late in the disease process and has an extremely poor prognosis. A bloodbased biomarker that can enable early detection of disease, monitor response to treatment, and potentially allow for personalized treatment would be of great benefit. This review analyzes the literature regarding two potential biomarkers, circulating tumor cells (CTCs) and cell-free DNA (cfDNA), with regard to pancreatic ductal adenocarcinoma. The origin of CTCs and the methods of detection are discussed and a decade of research examining CTCs in pancreatic cancer is summarized, including both levels of CTCs and analyzing their molecular characteristics and how they may affect survival in both advanced and early disease and allow for treatment monitoring. The origin of cfDNA is discussed, and the literature over the past 15 years is summarized. This includes analyzing cfDNA for genetic mutations and methylation abnormalities, which have the potential to be used for the detection and prognosis of pancreatic ductal adenocarcinoma. However, the research certainly remains in the experimental stage, warranting future large trials in these areas. (Am J Pathol 2019, 189: 71e81; https://doi.org/10.1016/ j.ajpath.2018.03.020)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a 5-year survival rate of only 2% to 9%. An estimated 279,000 individuals worldwide are diagnosed with PDAC each year, and it is the fifth most common cause of cancer death in the United Kingdom. The onset of symptoms and diagnosis is often late, and most patients have metastatic disease at diagnosis. However, genomic sequencing of PDAC suggests that it takes at least 15 years between the initiating mutations and metastatic potential.1 Those who are suitable for pancreatic resection have a much longer overall survival (OS), and in this group, smaller tumor size and lymph nodeenegative disease are associated with a further improvement in survival. The disease recurrence rate after surgical resection is high, with almost half developing disease within 18 months. These findings suggest that earlier identification of initial disease and disease recurrence would improve outcomes. Currently, the only noninvasive blood-based biomarker routinely used in clinical practice is CA 19-9. However, issues remain surrounding its sensitivity and specificity, and thus this has

led researchers to search for novel biomarkers. In this review, we have focused on two of these potential biomarkers: circulating tumor cells (CTCs) and cell-free DNA (cfDNA).

Circulating Tumor Cells Discovery In 1869, Thomas Ashworth reported that in a gentleman with multiple subcutaneous tumors of his thorax and abdomen, cells identical to those of the tumor were seen in the blood. This is the first written report suggesting that tumor cells in the blood may be responsible for metastases. Almost 90 years later in 1955, a more detailed study showed that cancer cells are present in the blood of patients with Disclosures: None declared. This article is a part of a review series on benign and neoplastic pancreatic lesions from their pathologic to molecular profiles and diagnoses.

Copyright ª 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.ajpath.2018.03.020

Gall et al colorectal cancer.2 Over the past decade, there has been renewed research interest in these CTCs, both as a liquid biopsy and as a prognostic marker for a variety of cancers. They are likely to play a key role in metastatic progression, a process known as the invasion-metastasis cascade comprising invasion, intravasation, migration, extravasation, and colonization.3

Origin CTCs are cells that circulate through the bloodstream and are thought to derive from the primary lesion. This blood-borne dissemination of cells from the primary tumor to distant organs may lead to metastatic disease. The origin of CTCs can be debated, and it is unclear whether they occur because of passive shedding of the tumor or active migration. Shedding could occur because of the detachment of clusters of connecting cells during tumor invasion into local vessels.4 Furthermore, tumor-induced angiogenesis leads to abundant blood vessels and an erosion-type mechanism with cells from the tumor separating from the mass lesion.5 Active vascular intravasation of cells may involve macrophages, with the resultant interaction inducing movement of tumor cells along collagen fibers toward blood vessels. Certainly, epidermal growth factor receptor and colony-stimulating factor 1, expressed by cancer cells, attract macrophages.6

Methods of Detection Levels of CTCs in the peripheral blood, however, are low, with approximately 1 cell per 105 to 107 mononuclear cells. Their detection is, therefore, extremely challenging. There are 40 to 50 different methods that have been used to detect circulating tumor cells in the literature.7,8 Essentially, CTCs can be positively or negatively enriched based on their biological properties or physical properties. Biological technologies depend on specific antibodies that bind to cell-surface markers on the CTCs.9 Methods for capturing CTCs based on their physical properties use enrichment methods based on the size, density, and electric charge of the CTCs.10 Filtrationbased systems and microfluidic cell sorting assume that tumor cells are larger than hematopoietic cells and trap these cells based on their size. Those using biological methods may have a high false-negative rate, missing tumor cells that do not have the specific cell-surface markers. However, those identifying CTCs by their physical properties are likely to have a higher false-positive rate, trapping some blood cells.8 Furthermore, researchers are looking at the individual properties of the CTCs, analyzing mutations within the DNA of the CTCs captured and enabling prediction of those that will respond to certain oncological therapies.11,12 The first US Food and Drug Administrationeapproved biological method was an automated detection system, CellSearch (Veridex, Huntingdon Valley, PA), validated in 2007.13 This kit is intended for the enumeration of CTCs of epithelial origin. Using ferrofluid nanoparticles with antibodies that target

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epithelial cell adhesion molecules, CTCs are separated from the bulk of other cells. The CTCs are then stained with antibodies to cytokeratins 8, 18þ, and/or 19þ, specific to epithelial cells. Leukocytes that may have contaminated the sample are stained with their specific antibody marker CD45. Finally, DAPI, a DNA stain, is used to highlight the nuclei of the cells. A fluorescent microscope is used to identify the CTCs, which are epithelial cell adhesion molecule, cytokeratin, and DAPI positive but negative for CD45. Clinical studies conducted with this system have demonstrated that CTCs are an independent predictor of progression-free survival (PFS) and OS in metastatic breast cancer,14,15 colorectal cancer,16 castrate-resistant prostate cancer,17 small-cell lung cancer,18 and nonesmall-cell lung cancer.19 Some CTCs, however, undergo epithelial-mesenchymal transition with down-regulated expression of cytokeratins and would not be detected by this system. Others have developed microfluidic systems using multiple antibody mixtures and alternate staining methods to try to capture more CTCs than antieepithelial cell adhesion molecule alone and to detect those that have undergone epithelial-mesenchymal transition and are cytokeratin negative.20e22 Some systems, such as the newer Screencell system (Caltag Medsytems Ltd, Buckingham, UK), rely on the larger size of the CTC rather than on the presence of surface antigens and have shown higher CTC detection rates.23,24 Certainly, in PDAC, Khoja et al25 captured CTCs in 40% (n Z 54) of patients using the CellSearch system compared with 93% using isolation by size of epithelial tumor cells. In essence, there is much heterogeneity in the methods used to detect CTCs, which leads to varying results in the literature. The concept of analyzing genetic and epigenetic mutations within the individual CTCs captured is novel and may enable us to predict those CTCs that are biologically active and more likely to cause metastatic disease.

CTC Detection in Pancreatic Cancer Despite an abundance of research into CTC numbers and their relationship with oncological outcomes in many other cancers, particularly breast cancer, there has been less in the literature regarding CTCs and PDAC. A literature search for relevant studies using the search terms CTC OR circulating tumor cells AND pancreatic adenocarcinoma OR PDAC was conducted on PubMed, Embase, Web of Science, and Cochrane Library databases. Case reports and research looking at non-blood CTCs (eg, peritoneal and bone marrow samples) were excluded. Twenty studies investigating CTCs in PDAC over the past 10 years were identified (Table 126e45). Kurihara et al45 found at least one CTC in the blood of 42% (n Z 26) of those with PDAC, whereas no patients with chronic pancreatitis (n Z 11) or healthy controls (n Z 10) had any CTCs detected. Interestingly, the median survival was 110.5 days for those who were CTC positive compared with 375.8 days for those who were CTC negative (P < 0.001). This is despite 87% of the

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CTCs and cfDNA in Pancreatic Cancer CTC-negative patients having stage IV disease. The survival difference persisted when analyzing only those with stage IV PDAC: 52.5 days for CTC-positive patients compared with 308.3 days for CTC-negative patients (P < 0.01). Similarly, de Albuquerque et al43 detected CTCs in 47.1% (n Z 34) of PDAC patients, compared with none in healthy controls (n Z 40). A shorter PFS (66 compared with 138 days; P Z 0.01) was observed for patients who had at least one CTC detected compared with those who were CTC negative. Earl et al35 also found poorer OS (88 compared with 393 days) in the 20% of PDAC patients who were CTC positive. A meta-analysis was conducted of nine cohort studies analyzing CTCs in PDAC patients.38 This showed that 43% (n Z 603) had positive CTCs from peripheral blood samples. These CTC-positive patients had significantly worse PFS than CTC-negative patients (hazard ratio, 1.89; 95% CI, 1.25e4.00; P < 0.001) and had worse OS (hazard ratio, 1.23; 95% CI, 0.88e2.08; P < 0.001). The suggestion that CTCs could be used as a prognostic marker for PDAC led Ren et al44 to examine CTC numbers after chemotherapy treatment in patients with PDAC. Two or more CTCs were detected in 80.5% (n Z 41) of patients, and again none was seen in healthy donors (n Z 20). This high percentage of patients with CTCs may be because all patients had advanced disease at the time of blood sampling. After one cycle of fluorouracil, only 29.3% had more than two CTCs identified, suggesting that CTCs could be used as an assessment of response to treatment, although this has not been reproduced.42 This limited evidence indicates that CTCs may have a future role in assessment of prognosis and treatment response in those with advanced PDAC. Whether CTCs could also be of value as a prognostic marker in those with earlier disease would be of interest. However, Bidard et al42 only identified CTCs in 5% (n Z 75) of patients with borderline resectable disease. Furthermore, in contrast to previous studies, more patients (9%; n Z 59) had CTCs detected after 2 months of chemotherapy, and there was no difference in PFS between those who were CTC positive or negative. Our own group examined CTCs in patients with resectable disease and identified CTCs in 50% of patients using the CellSearch system. A difference in survival was not observed (unpublished data, T.M.H.G., A.E.F., L.R.J.). CTCs were also examined in the portal circulation of those with resectable disease, and CTC positivity was found in 92% of patients.40 It is possible that many of these CTCs fail to enter the peripheral circulation. However, Bissolati et al34 reported a higher rate of liver metastases after 3 years of follow-up in those with CTCs identified in portal venous blood. Similarly, Tien et al32 found 85% of patients with portal venous CTCs developed liver metastases, compared with 13% with no portal venous CTCs. Compared with other epithelial cancers, a much smaller number of CTCs are seen in PDAC, and in fewer patients. For this reason, CTCs do not currently have a high enough

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sensitivity to be used for diagnosis, as a liquid biopsy. Certainly, only a sensitivity of 67% was found when distinguishing PDAC from other malignant pancreatic tumors26; and a sensitivity of 68% was found for distinguishing between PDAC and other pancreatic disease or healthy patients, including pancreatic pseudocysts, pancreatic serous cystadenomas, and solid pseudopapillary tumors.37 Indeed, CTCs have also been identified in 63% of those with benign disease,28 33% of patients with benign cystic lesions,41 64% with neuroendocrine tumors, 62% with intraductal papillary mucinous neoplasms, and 46% with chronic pancreatitis,36 implying a low specificity. Recently, an exciting addition to CTC research has been to analyze the specific molecular characteristics of the CTCs. This may lead to an understanding of the malignant potential of CTCs captured and add to the benefit of using CTCs as prognostic biomarkers. Dotan et al30 were able to measure MUC-1 from the CTCs captured by the CellSearch system. Increased MUC-1 tumor expression is associated with a poorer outcome in PDAC,46 and the presence of antieMUC-1 IgG antibodies correlates with improved survival.47 They were able to demonstrate that patients with MUC-1eexpressing CTCs (n Z 10) had a shorter median OS (2.7 months), compared with those with MUC1enegative CTCs (n Z 13; 9.6 months). Kulemann et al29 looked at KRAS mutation subtypes in CTCs from 58 PDAC patients. Those with a KRASG12V mutation (n Z 14) had a better OS (24.5 months) compared to those with other (10 months) or no detectable KRAS mutations (8 months; P Z 0.04). Poruk et al33 identified CTCs that expressed vimentin, a mesenchymal marker, as well as the epithelial cytokeratin markers. These CTCs have a strong association with metastatic potential in breast cancer, and evidence suggests that CTCs undergoing epithelial-mesenchymal transition may have more malignant potential.48 Of patients with resectable or borderline resectable PDAC, 78% (n Z 50) had CTCs expressing cytokeratins detected. Of these, 67% also expressed vimentin and had a shorter disease-free survival (9.5 compared with 13.5 months; P Z 0.02). Yu et al39 analyzed the gene expression profile of CTC RNA in 50 patients with advanced or locally advanced PDAC. They compared this with a model validated to predict chemotherapy sensitivity. Those predicted to be sensitive to chemotherapy had a longer disease-free survival (10.4 months) than those predicted to be resistant to chemotherapy (3.6 months; P Z 0.0001). Overall survival was also significantly different, 17.2 months in the sensitive group compared with 8.3 months in the resistant group (P < 0.0304).

Cell-Free DNA Discovery Mandel and Metais49 published an article in 1948 with the first description of circulating nucleic acids in human

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Gall et al Table 1

Studies Evaluating CTCs in PDAC Method

Main findings

2017 PDAC: 52 Other pancreatic malignancy: 10 2017 PDAC: 95 Controls: 48

Screencell

Sensitivity and specificity for PDAC: 67% and 80%, respectively

Immunostaining of CD45, DAPI, and CEP8-FISH

2017 PDAC: 8 NET: 9 Cholangiocarcinoma: 8 IPMN: 16 MCN: 1 Pancreatitis: 12 Controls: 9 2017 PDAC: 58 Controls: 10

Screencell

CTCs, more than two; sensitivity and specificity for PDAC: 75.8% and 68.7%, respectively CTC subtype-positive rates associated with tumor location Malignant: 51% CTC positive Benign: 63% CTC positive

Dotan et al30

2016 PDAC: 48

CellSearch

Ankeny et al31

2016 PDAC: 72 Controls: 39

Tien et al32

2016 PDAC/ampullary cancer: 60

Nanovelcro (California Nanosystems Institute, Los Angeles, CA) CMx platform (Taiwan Healthcare, Taiwan, Republic of China)

Poruk et al33

2016 PDAC: 50

ISET

Bissolati et al34

2015 PDAC: 20

CellSearch

Earl et al35

2015 PDAC: 45

CellSearch

Cauley et al36

Screencell

Han et al38

2015 PDAC: 105 Other pancreatic lesions: 74 Controls: 9 2015 PDAC: 22 Benign lesions: 6 Controls: 30 2014 PDAC: 603

Meta-analysis

Yu et al39

2014 PDAC: 50

Not stated

Gall et al40

2014 PDAC: 12

CellSearch

Rhim et al41

2014 PDAC: 11 Cystic lesions: 21 Controls: 19

GEDI

Reference Sefrioui et al

Year 26

Liu et al27

Rosenbaum et al28

Kulemann et al29

Zhang et al37

Sample size

Screencell

EpCAM-independent method

More than three CTCs, shorter overall survival CTC KRASG12V mutation trend to better overall survival No difference in overall survival between CTC-positive and CTC-negative patients Shorter overall survival in patients with CTC-expressing MUC-1 CTC sensitivity and specificity: 75.0% and 95.7%, respectively More CTCs detected from portal (58.3%) compared with peripheral (40%) venous blood 85% of patients with portal vein CTCs developed liver metastases compared with 13% of those with no portal vein CTCs 78% CTCs positive for CKs 67% of these also expressed vimentin Presence of CKs and vimentin associated with shorter overall survival 45% with CTCs Higher rate of liver metastases in patients with CTC positive from portal vein 20% with CTCs Shorter overall survival in CTC-positive patients (88 versus 393 days) 49% PDACs with CTCs 64% NETs with CTCs 62% IPMNs with CTCs 46% chronic pancreatitis with CTCs Sensitivity and specificity for PDAC: 68.18% and 94.87%, respectively Shorter disease-free survival and overall survival in CTC-positive patients Expression profiling of RNA from CTCs can predict chemotherapy response and disease-free and overall survival 92% with portal vein CTCs No difference in disease-free survival 73% PDACs with CTCs 33% cystic lesions with CTCs (table continues)

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CTCs and cfDNA in Pancreatic Cancer Table 1

(continued )

Reference

Year

Bidard et al42

2013 PDAC: 79

Sample size

Method

Main findings

CellSearch

5% PDACs with CTCs Shorter overall survival with CTC positive 47% PDACs with CTCs Shorter progression-free survival in CTC-positive patients More than two CTCs in 80.5% PDACs More than two CTCs in 29.3% after first cycle of 5-FU

de Albuquerque et al43 2012 PDAC: 34 Controls: 40 Ren et al44

Kurihara et al45

BM7 and VU1D9 (targeting mucin 1 and EpCAM, respectively) 2011 PDAC: 41 CA19-9eAlexa Fluor 488 Controls: 20 and CK8/18-Alexa Fluor 594 immunofluorescence (Thermo Fisher Scientific, Waltham, MA) 2008 PDAC: 26 CellSearch 42% PDACs with CTCs Chronic pancreatitis: 11 Shorter overall survival in CTC-positive patients Controls: 10

CK, cytokeratin; CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; FISH, fluorescence in situ hybridization; 5-FU, fluorouracil; IPMN, intraductal papillary mucinous neoplasm; ISET, isolation by size of epithelial tumor cell; MCN, mucinous cystic neoplasm; NET, neuroendocrine tumor; PDAC, pancreatic ductal adenocarcinoma.

plasma. After this, in 1977, it was observed that 173 patients with cancer had elevated levels of DNA in their serum compared with 55 healthy controls. This level was higher in those with metastatic disease and was found to be reduced after radiotherapy.50 In 1989, the DNA extracted from the plasma of cancer patients was seen to be identical to the corresponding cancer cells.51 Subsequently, several DNA mutations and microsatellite alterations associated with varying cancer subtypes have now been identified in plasma DNA. With huge improvements in DNA extraction and the advent of widespread PCR techniques, the oncological importance of cfDNA has been established. There is increasing evidence to suggest that specific genetic and epigenetic mutations in cfDNA may be diagnostic for certain tumors and may be useful to monitor treatment response. Furthermore, these specific mutations in cfDNA could have the potential to establish personalized oncological therapies all from a simple blood test.

Origin cfDNA is mostly a double-stranded molecule, consisting of small fragments (70 to 200 bp) and larger fragments with molecular weights of up to 21 kb, and occurs in both plasma and serum.52 Although cfDNA is actively released from cells as a part of normal metabolism, 4 to 40 times greater levels are seen in cancer patients.53 Indeed, in colorectal cancer, an estimated 3.3% of tumor DNA is released into the circulation daily.54 In healthy individuals, the cfDNA concentration ranges from 0 to 100 ng/mL of blood, which compares to concentrations of 0 to >1000 ng/mL of blood in cancer patients.55 There is debate as to the origin of cfDNA, but it is thought to be released from apoptosis, necrosis, and direct release from viable cells and from the lysis of CTCs. In cancer, there is a high cell turnover and hence increased programed apoptosis, which may explain higher plasma levels of cfDNA compared with other physiological states.52

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Further suggestion that apoptosis leads to the presence of plasma DNA is that a large proportion of cfDNA has a size of 180 to 720 bp, a character of cell death fragments.56 In addition, chloroquine, which induces apoptosis, also increases the concentration of cfDNA.57 Necrosis is commonly seen in cancer cells because of a higher growth rate of tumor tissue than angiogenesis, resulting in hypoxia. Phagocytosis of the necrotic cells by macrophages leads to release of cell components, including tumor DNA fragments.54 There may also be active release of DNA by tumor cells independent of apoptosis and necrosis. This has been previously demonstrated on lymphocytes.58 Finally, the lysis of CTCs may also contribute toward cfDNA.

cfDNA in Pancreatic Cancer As discussed above, healthy patients may have cfDNA detected in their circulation. Furthermore, as well as in neoplastic disease, increased levels may be seen in other physiological and pathologic conditions, including exercise, smoking, inflammatory disease, critical illness, sepsis, and trauma.59e61 Therefore, measuring total cfDNA is not clinically useful as a diagnostic marker for malignancy, because its sensitivity is generally low.53 However, there is a possibility of using cfDNA in cancer diagnosis and monitoring, by detecting tumor-specific genetic and epigenetic mutations. New developments in techniques for these analyses have enabled an accumulation of research in many malignancies over the past two decades. Specifically, cfDNA has been analyzed for gene mutations, loss of heterozygosity, methylation alterations, and microsatellite alterations. These changes may be some of the earliest events in malignant transformation and, therefore, their detection could lead to early clinical biomarkers. There is some limited research on cfDNA in PDAC. A literature search for relevant studies using the search terms cfDNA OR cell free DNA OR circulating DNA AND

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Gall et al Table 2

Studies Evaluating cfDNA in PDAC Year

Sample size

cfDNA analysis

Significant findings

2017

PDAC: 5

Cheng et al63

2017

PDAC: 188

56-Gene screening panel 60-Gene panel

Allenson et al64

2017

PDAC: 68 Controls: 54

KRAS mutation

Henriksen et al65

2017

Hadano et al66

2016

PDAC: 95 Chronic pancreatitis: 59 Controls: 27 PDAC: 105

Methylation, 10-gene panel screened KRAS mutation

Brychta et al67

2016

PDAC: 50 Controls: 20

KRAS mutation

Sausen et al68

2015

PDAC: 77

KRAS mutation

Kinugasa et al69

2015

PDAC: 75

KRAS mutation

Berger et al70

2016

KRAS and GNAS mutations

Earl et al35

2015

PDAC: 24 IPMN: 21 Controls: 38 PDAC: 31

Concordance of mutations with tumor sample in 28% New mutations with development of metastases KRAS G12V and ERBB2 exon 17 mutations were independently significantly associated with shorter survival Presence of KRAS G12 mutation associated with tumor responses observed on CT images in 76.9% KRAS mutations in 45.5% with localized disease and 57.9% with metastatic disease No correlation with survival KRAS mutations in 14.8% of controls More methylated genes in cancer Prediction model differentiated between PDAC and benign disease with a sensitivity of 76% and a specificity of 83% KRAS mutation in 31% Overall survival was 13.6 months in patients with mutant KRAS cfDNA and 27.6 months in those with wild-type KRAS KRAS mutations in 35% PDACs No KRAS mutations detected in controls No correlation seen between tumor stage, size, tumor content, and tumor cell load with the concentration of cfDNA in plasma KRAS mutations in 43% Disease progression using cfDNA was detected at an average of 3.1 months after surgery compared with 9.6 months using standard CT imaging KRAS mutation in 62.6% No correlation with survival Higher level of cfDNA in PDACs compared with IPMNs

Singh et al71

2015

PDAC: 127 Controls: 25

KRAS mutation

Sikora et al72

2015

Alu83 and Alu44 nucleotides

Takai et al73

2015

PDAC: 50 NET: 23 Chronic pancreatitis: 20 Controls: 23 PDAC: 259

Park et al74

2012

Methylation

Dabritz et al75

2012

PDAC: 16 Chronic pancreatitis: 13 Controls: 29 PDAC: 38

Pedersen et al76

2011

PDAC: 240 Controls: 240

Methylation

Reference Vietsch et al

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KRAS mutation

KRAS mutation

KRAS mutation

No significant difference in total cfDNA and survival Codon 12 KRAS-mutant cfDNA detected in 26% of patients cfDNA concentration increases with advanced disease stages; survival was 60 days in patients with the KRAS mutant cfDNA and 772 days in patients without the KRAS mutant cfDNA Higher levels of plasma cfDNA (>62 ng/mL) were associated with lower overall median survival time of 3 months compared with 11 months Presence of the KRAS gene was not found to be associated with any difference in survival Higher levels of cfDNA in PDACs

KRAS mutations in 47% inoperable PDACs and 8.3% resectable PDACs Altered methylation in PDACs compared with CPs and compared with controls KRAS mutations in 39% of PDAC patients Mutations associated with signs of progressive disease Prediction model of five CpG sites discriminated PDAC from controls (table continues)

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CTCs and cfDNA in Pancreatic Cancer Table 2

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Reference

Year

Sample size

cfDNA analysis

Significant findings

Liggett et al77

2010

Methylation

91.2% Sensitivity and 90.8% specificity for PDAC versus CP differentiation

Chen et al78

2010

PDAC: 30 Chronic pancreatitis: 30 Controls: 30 PDAC: 91

KRAS mutation

Dabritz et al79

2009

PDAC: 56 Chronic pancreatitis: 13

KRAS mutation

Melnikov et al80

2009

Methylation

Uemura et al81 Dai et al82

2004 2003

PDAC: 30 Controls: 30 PDAC: 28 PDAC: 15 Chronic pancreatitis: 10

Maire et al83

2002

PDAC: 47 Chronic pancreatitis: 31

KRAS mutation

KRAS mutations in 33% of PDAC patients Worse survival in those with mutations More KRAS mutations in PDAC versus chronic pancreatitis The addition of CA 19-9 gave 91% sensitivity for cancer diagnosis Prediction model of five promoters has 76% sensitivity and 59% specificity for PDAC detection KRAS mutations in 35% KRAS mutations in 73% of PDACs versus 20% of chronic pancreatitis KRAS þ CA 19-9 Z 66.67% sensitivity and 97% specificity for cancer detection KRAS mutations in 47% of PDACs versus 13% of chronic pancreatitis KRAS þ CA 19-9 Z 98% sensitivity and 77% specificity

KRAS mutation KRAS mutation

cfDNA, cell-free DNA; CT, computed tomography; IPMN, intraductal papillary mucinous neoplasm; NET, neuroendocrine tumor; PDAC, pancreatic ductal adenocarcinoma.

pancreatic adenocarcinoma OR PDAC was conducted on PubMed, Embase, Web of Science, and Cochrane Library databases. Twenty-three studies investigating CTCs in PDAC were identified over the past 15 years (Table 235,62e83). There are four main driver genes that are most frequently mutated in PDAC. These are KRAS, CDKN2A, SMAD4, and TP53, and their association with PDAC has been established for many years. Recently, exome sequencing of PDAC ascertained KRAS to be the most commonly mutated, seen in 90% to 95% of cases.84 Therefore, this gene has been the focus of much of the cfDNA research. Maire et al83 investigated for G12D mutations in codon 12 of KRAS, from the cfDNA of 47 patients with PDAC, most with stage IV disease, and found these in 47%. Interestingly, they also identified this mutation in 13% of those with chronic pancreatitis. Similarly, Dabritz et al79 used peptic nucleic acidemediated PCR clamping and real-time PCR with mutant-specific hybridization probes, to find codon 12 KRAS mutations in 36% of PDAC patients (n Z 56) and in 14% (n Z 13) of patients with chronic pancreatitis. Dai et al82 used PCR techniques to find KRAS mutations in 73% of PDAC patients (n Z 15), but again in 20% of chronic pancreatitis patients (n Z 10). Combining the detection of these mutations, with an increased CA 19-9, distinguishes PDAC from chronic pancreatitis with a sensitivity of 67% to 98% and a specificity of 77% to 97%.79,82,83 Levels of cfDNA have also been detected in patients with intraductal papillary mucinous neoplasms, although at a lower level than in PDAC.70 Furthermore, mutant KRAS has also been detected in 14.8% of healthy controls.64 This evidence suggests limited use of cfDNA KRAS mutations as a diagnostic biomarker.

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Interestingly, cfDNA KRAS mutations may be of more value as a prognostic biomarker. Chen et al78 analyzed 91 patients with inoperable PDAC. Those with detectable cfDNA codon 12 KRAS mutations had a significantly shorter survival (3.9 months) compared with those with wild-type KRAS (10.2 months; P < 0.001). In addition, Earl et al35 determined that 26% of patients (n Z 31) with PDAC had cfDNA codon 12 mutations of KRAS. These patients had a shorter survival (60 days) compared with those without the KRAS mutation (772 days; P Z 0.001). Hadano et al66 also demonstrated a shorter survival, 13.6 months, in 31% of patients (n Z 105) with mutant KRAS in cfDNA compared with 27.6 months in those with wild-type KRAS (P < 0.0001). In the largest study conducted73 with 259 PDAC patients, mutant KRAS was detected in cfDNA of 47% of patients with inoperable disease and 8.3% of those with resectable disease. The presence of the mutation was an independent prognostic factor for OS (hazard ratio, 3.04; P < 0.0001). In an analysis of cfDNA from 66 PDAC patients, a shorter survival was seen in those with mutant KRAS compared with wild-type KRAS. In contrast, the mutational status of the tumor DNA did not correlate with survival.69 A further link to cfDNA KRAS mutations and prognosis was established by Dabritz et al,75 who correlated the presence of KRAS mutations and computed tomographic (CT) findings. Of patients, 39% (n Z 38) had cfDNA KRAS mutations; however, on further analysis, only 9% of those with disease remission on CT had cfDNA KRAS mutations, compared with 75% of those with progressive disease on CT. KRAS mutations in cfDNA may also beneficial for predicting disease recurrence. PCR analyses performed at various time points after surgical

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Gall et al resection determined that patients with detectable mutations in their plasma were more likely to relapse than those with undetectable alterations (P Z 0.02). Furthermore, disease progression was detected 3.1 months after surgery, compared with 9.6 months using CT imaging (P Z 0.0004).68 However, others have failed to correlate the presence of KRAS mutations in cfDNA and survival. Uemura et al81 analyzed KRAS in plasma DNA in 28 patients with resectable PDAC and were able to identify mutations in 35%. No association was found between the presence of the cfDNA mutation and the size of the tumor or stage of the disease. Brychta et al67 found no correlation between mutant KRAS in cfDNA, which was detected in only 35% of PDAC patients (n Z 50), and the stage or grade of the disease. Allenson et al64 and Singh et al71 were also unable to determine a significant difference in survival when comparing patients with cfDNA mutant KRAS and those with cfDNA wild-type KRAS. Recently, the development of next-generation DNA sequencing analyzers has enabled rapid assessment of whole-exome DNA, using probes to >50 genes covering thousands of COSMIC (Catalogue of Somatic Mutations in Cancer) mutations. Cheng et al63 used next-generation sequencing from cfDNA in 188 patients with metastatic PDAC to identify at least one mutation in 83% of patients and a KRAS mutation in 72.3%. Specifically, only the KRAS G12V and ERBB2 exon 17 mutations were independently significantly associated with shorter survival. The presence of the KRAS G12 mutation was associated with tumor responses observed on CT images in 76.9% of patients and provided the earliest measurement of treatment in 60%. Vietsch et al62 analyzed the plasma of five PDAC patients at initial diagnosis with next-generation sequencing. An average of eight mutations was detected per sample, but concordance with the tumor sample was only 28%. Interestingly, after the development of disease metastases, 63% of mutations in cfDNA had not been detected initially, demonstrating the heterogeneity of the disease with progression. Our own group (unpublished data) used the ion torrent next-generation sequencer to analyze cfDNA in 16 patients with operable PDAC. A total of 256 singlenucleotide polymorphisms were identified, but only two gene mutations (APC and STK11) and no KRAS mutations were detected. DNA methylation is an important physiological process active in all cells. It occurs at CpG islands, found at the promoter region of genes. Abnormal methylation, such as the hypermethylation of a promoter region of a tumor suppressor gene, results in gene silencing.55 Hypomethylation leads to increased mutation rates and chromosome instability.85 Therefore, some research has examined methylation abnormalities in cfDNA. Altered methylation was detected in the cfDNA of 81.3% of PDAC patients, but also in 61.5% of those with chronic pancreatitis and in 3.5% of healthy controls.74 Others were able to use methylation abnormalities in 17 promoter regions of cfDNA to discriminate

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between PDAC and chronic pancreatitis, with a sensitivity of 91.2% and a specificity of 90.8%.77 Using a model of five promoter CpG sites, and a large cohort of 240 patients with PDAC, malignancy could be discriminated from healthy controls with a C-statistic of 0.76.76 Melnikov et al80 also developed a model of five promoters to discriminate between PDAC and healthy controls, with a sensitivity of 76% and a specificity of 59%. Henriksen et al65 analyzed the hypermethylation of 10 genes, and although cfDNA hypermethylation was detectable in both malignant disease and chronic pancreatitis, a much higher level was seen in PDAC. A diagnostic prediction model was able to differentiate between PDAC and benign disease, with a sensitivity of 76% and a specificity of 83%.65

Summary Pancreatic cancer has an aggressive and devastating biology. Insights into the molecular characteristics of this disease will provide valuable information, leading to earlier detection of the disease and the development of improved oncological therapies. The continuing analysis of circulating biomarkers is an exciting area of exploration, which may lead to personalized prognosis and treatment plans from a simple blood test. CTCs and cfDNA are two such biomarkers at the forefront of oncological research. There is promising published evidence that the presence of CTCs in advanced PDAC leads to shorter PFS and OS, and as levels are reduced after chemotherapy, they could be used to evaluate response to treatment. There is less evidence regarding the presence of CTCs in resectable PDAC and outcomes. However, in these patients, portal venous CTC detection could suggest a higher likelihood to develop liver metastases. As yet, CTCs may not distinguish between malignant and benign disease, reducing the potential benefit for disease diagnosis. However, recent advances in CTC research have evaluated the biological properties of captured CTCs, and this could be of future use in detection and prognostication. There are many markers in proteomic and genomic research that may increase the malignant potential of PDAC, yet, in terms of CTCs, the analyses are in their infancy, with only three investigated. Analyzing cfDNA for genetic abnormalities is also a promising area. Most research has examined KRAS mutations in plasma, and although detected in benign disease at lower levels, combination with CA 19-9 may increase diagnostic sensitivity and specificity. Most studies have also found shorter survival outcomes in those with mutant KRAS, and some evidence would suggest that the development of cfDNA mutant KRAS is an early marker of disease recurrence. With the advent of next-generation sequencing, an expansion of studies is anticipated in the coming years. Panels of genes with methylation abnormalities may also aid in the diagnosis of PDAC; however, there is not yet an established panel with potential clinical use. Published research is at the

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CTCs and cfDNA in Pancreatic Cancer experimental stage and currently involves small numbers of patients, using a variety of methods. Some of the encouraging results seen could partly be because of publication bias. We hope that the future will bring larger trials leading to the development of agreed protocols and the clinical use of both CTCs and cfDNA as markers of diagnosis, prognosis, and response to oncological treatments.

Supplemental Data Supplemental material for this article can be found at https://doi.org/10.1016/j.ajpath.2018.03.020.

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