Novel biomarkers and endoscopic techniques for diagnosing pancreaticobiliary malignancy

The UK incidence of pancreatic ductal adenocarcinoma is 9 per 100,000 population, and biliary tract cancer occurs at a rate of 1–2 per 100,000. The incidence of both cancers is increasing annually and these tumours continue to be diagnosed late and at an advanced stage, limiting options for curative treatment. Population-based screening programmes do not exist for these cancers, and diagnosis currently is dependent on symptom recognition, but often symptoms are not present until the disease is advanced. Recently, a number of promising blood and urine biomarkers have been described for pancreaticobiliary malignancy and are summarised in this review. Novel endoscopic techniques such as single-operator cholangioscopy and confocal endomicroscopy have been used in some centres to enhance standard endoscopic diagnostic techniques and are also evaluated in this review.


Introduction
In the UK, pancreatic ductal adenocarcinoma (PDAC) is the 10 th commonest cancer and has an incidence of 9 per 100,000 population 1 , and biliary tract cancer (BTC) (including intra-and extra-hepatic cholangiocarcinoma and gallbladder cancer) has an incidence of 1-2 cases per 100,000 population 2 . Long-term survival is poor; 5-year survival is less than 4% for both tumours 3,4 . Often these tumours are diagnosed late, when patients have advanced disease and curative surgical resection is no longer possible.
Globally the highest incidence of PDAC is seen in Northern Europe and North America 5 , where the rates are 3 to 4 times higher than in tropical countries 6 . Overall incidence is increasing 5 , and as most tumours are sporadic, this rising incidence is attributed to differences in lifestyles and exposure to environmental risk factors 7 , such as smoking [8][9][10][11][12][13][14][15] , diabetes mellitus, chronic pancreatitis 1,15,16 and obesity 17 .
In BTC, the variations in incidence seen globally are even more pronounced; and the highest incidence is in northeastern Thailand (96 per 100,000 men) 18 , which has a population with high levels of chronic typhoid and infestation of liver fluke (Clonorchis sinensis and Opisthorchis viverinni) 18 . Other BTC risk factors seen in all populations include older age 18 , primary sclerosing cholangitis 19 , intraductal stones and rare biliary cystic diseases 20 . Inflammatory bowel disease, chronic viral hepatitis, cirrhosis, smoking, diabetes, obesity and excess alcohol consumption may also increase the risk of BTC 20-22 .
Despite improved diagnostic techniques, detecting pancreaticobiliary malignancy remains a significant clinical challenge. A common presentation of these tumours is a biliary stricture with or without a mass lesion. The differential of an indeterminate biliary stricture is broad, and often the associated symptoms and radiological findings overlap between benign and malignant conditions, often making differentiation-particularly between cancer, primary sclerosing cholangitis and IgG4-related disease-impossible without further investigations, typically by endoscopic retrograde cholangiopancreatography (ERCP) or endoscopic ultrasound (EUS) [23][24][25] . However, biliary brush cytology is also an imperfect test, although specificity is high (96-100%), sensitivity for malignancy remains low (9-57%) and in early disease when tumours are small, sensitivities are even lower 26,27 . Therefore, patients frequently require multiple procedures to obtain a final diagnosis [28][29][30] .
So there has been growing interest in the development of simple tests to streamline the diagnosis to pancreaticobiliary malignancy and guide appropriate and timely therapy for patients. Identifying better diagnostic tools for PDAC and BTC would also make screening and surveillance possible, particularly in high-risk populations 4,8,31 . This would enable the detection of tumours at an earlier stage when curative resection is possible, leading to substantial improvements in survival 32 . This review provides an overview of the latest innovations in diagnostic biomarkers and endoscopic techniques for pancreaticobiliary malignancy.

Methods
We performed a systematic review of the literature by using PubMed, EMBASE and the Cochrane Library. The search was limited to studies published in the English language between January 2013 and March 2017. Medical Subject Headings (MeSH) terms were decided by a consensus of the authors and included "pancreatic cancer" or "cholangiocarcinoma" and "biomarker". The search was restricted to title, abstract and keywords. Articles that described outcomes for fewer than five patients were excluded. Case reports, abstracts and reviews were excluded. All references were screened for potentially relevant studies not identified in the initial literature search.
The following variables were extracted for each report when available: number of malignant and benign cases, sensitivity, specificity and area under the curve (AUC). One hundred ten articles were included in the final review.

Serum biomarkers and blood tests
Carbohydrate antigen (CA) 19-9 is the most widely used tumour marker in pancreaticobiliary malignancy. Overall sensitivity (78-89%) and specificity (67-87%) are low, and in around 7% of the population who lack the Lewis (a) antigen, CA19-9 will remain negative 33 . In small tumours, sensitivity decreases further. The marker can also be elevated in a number of other malignant diseases (for example, gastric adenocarcinoma) and benign diseases, particularly those causing jaundice (for example, primary biliary cirrhosis, cholestasis and cholangitis), and in smokers 34 . In addition, variation has been reported among commercially available assays, which may impact on interpretation 35 . Therefore, to improve the sensitivity of the marker in current clinical practice, it is always interpreted in the context of cross-sectional imaging findings 33 .
Other commercially available tumour markers that have a role in diagnosing pancreaticobiliary cancer include carcinoembryonic antigen (CEA) and CA125. CEA is a glycosyl phosphatidyl inositol cell surface-anchored glycoprotein that is involved in cell adhesion. When elevated, it is highly suggestive of colorectal cancer, but it is also increased in about a third of patients with BTC 36-38 . CA125 is a protein encoded by the MUC16 gene and is a large membrane-associated glycoprotein with a single transmembrane domain. When elevated, it is suggestive of ovarian cancer, but it is also increased in about 40-50% of patients with pancreaticobiliary malignancy, particularly when there is peritoneal involvement 38 .
Owing to the limitations of existing biomarkers, over the last few years several studies have evaluated various combinations of biomarkers to supplement or ultimately replace existing biomarkers. Biomarker panels using combinations of markers, often including CA19-9, have been particularly successful in detecting small tumours and early disease. Validation studies have also shown that these markers can differentiate PDAC from relevant benign conditions and in some cases detect tumours up to 1 year prior to diagnosis with a specificity of 95% and a sensitivity of 68% 7 (Table 1 and Table 2). In pancreaticobiliary malignancy and PDAC in particular, metastatic disease occurs at a very early stage in tumour development. This is demonstrated by the fact that patients who underwent resection of small primary tumours (<2 cm) with no clinical evidence of metastatic disease had a 5-year survival after pancreatectomy of less than 18% owing to recurrent metastatic disease 39 . Tumour development is driven by a series of cumulative genetic abnormalities; therefore, genetic and epigenetic changes have been explored as diagnostic targets in circulating tumour cells, cell-free DNA (cfDNA) and non-coding RNA (Table 3-Table 5). Owing to the position and composition of pancreaticobiliary tumours, tissue samples are frequently acellular, making diagnostics challenging. Recently, the utility of next-generation sequencing was explored as a technique that allows the detection of low-abundance mutations and abnormalities in small amounts of material 40 . Changes in the metabalome are also being explored as a potential diagnostic tool in pancreaticobiliary malignancy 41 .

Bile and biliary brush biomarkers
Patients with an indeterminate stricture on cross-sectional imaging are typically referred for an ERCP and biliary brushing with or without endobilary biopsy to obtain tissue for diagnosis, with or without therapeutic stenting 28 . Although these techniques do not compromise resection margins in potentially resectable cases, sensitivity remains low (9-57%) and patients frequently have to undergo multiple procedures to obtain a diagnosis 28-30 . Bile can be easily obtained at the time of ERCP and, owing to its proximity to the tumour, is a potentially important source of diagnostic biomarkers in these cancers (Table 6). Unfortunately, owing to the invasiveness of ERCP, the role of these biomarkers is limited to diagnosis rather than screening or surveillance in these tumours.

Urinary biomarkers
Urine provides a very easy and acceptable source for biomarker analysis. In BTC, a 42-peptide panel (consisting mostly of fragments of interstitial collagens) correctly identified 35 of 42 BTC patients with a sensitivity of 83% and a specificity of 79% 42 . In PDAC, the three-biomarker panel (LYVE-1, REG1A and TFF1) has been validated in a multi-centre cohort of 371 samples. When comparing PDAC stage I-IIA (resectable disease) with healthy urines, the panel achieved AUCs of 0.97 (95% confidence interval of 0.93-1.00). The performance of the urine biomarker panel in discriminating PDAC stage I-IIA was superior to the performance of serum CA19-9 (P=0.006) 43 (Table 7).

Symptoms and cancer decision support tools
Recently, pre-diagnostic symptom profiles have been investigated as an alternative way of detecting hepato-pancreato-biliary (HPB) cancers at an early stage 8,9,16,44 . It is now recognised that the onset of PDAC and BTC is heralded by a collection of gastrointestinal and constitutional symptoms 45 . Although overlap occurs with other benign and malignant conditions, certain symptoms such as back pain, lethargy and new-onset diabetes have been identified as particularly suggestive of PDAC. Commonly performed blood tests such as liver function tests, glucose and haemoglobin also typically become abnormal in the months preceding diagnosis 46 . Therefore, cancer decision support tools     have been produced from combinations of symptoms and risk factors. In the UK, they have been introduced into general practices in 15 cancer networks to date 8 , and their utility is currently being audited 47 . Modification to existing tools to enhance their diagnostic accuracy can be expected in the future.

Endoscopic ultrasonography
If there is a mass lesion on cross-sectional imaging, endoscopic ultrasonography with fine-needle aspiration (EUS-FNA) provides an alternative method for visualising and sampling the extra-hepatic biliary tree, pancreas, gallbladder or peri-hilar lymph nodes. EUS-FNA has a diagnostic accuracy for PDAC of between 65% and 96% 48,49 . In BTC, a single-centre study reported a sensitivity of 73%, which was significantly better in distal compared with proximal tumours (81% versus 59% respectively, P=0.04) 50 . Recently, developed fine core biopsy needles appear to have improved diagnostic accuracy over traditional FNA needles, but randomised trials are awaited 49,51,52 . Rapid onsite examination by a cytopathologist is used in some centres, particularly in North America, and has been shown to improve the yield of EUS-FNA in individual centres 53,54 but this trend has not been borne out in recent randomised controlled trials 55 .
To improve the diagnostic accuracy of EUS, it can also be combined with novel adjuncts such as contrast agents (SonoVue ® ), transient elastography (TE) or confocal laser endomicroscopy (CLE). TE allows the measurement of the tissue firmness, which tends to be increased in malignant tissue. In a recent singlecentre study from the UK, quantitative strain measurements were found to have high sensitivity but low specificity for the detection of PDAC 56 . The technology to perform the techniques is available on most modern EUS machines and adds little time to the overall procedure time. The technique can be performed equally well by endosonographers with limited experience 57,58 and is particularly advantageous in cases where the diagnosis remains uncertain after standard EUS has been performed 59 . Contrast-enhanced EUS is performed with agents such as SonoVue ® and allows visualisation of the early arterial phase and late parenchymal phase enhancement of the pancreas. Pancreatic tumours are generally hypovascular compared with the surrounding parenchyma 60,61 . Dynamic contrast EUS is a relatively novel method that allows the non-invasive quantification of the tumour perfusion compared with the pancreatic parenchyma by using software that is now built into a number of EUS scanners. The use of this technology is evolving but is expected to be most applicable when predicting tumour response to chemotherapeutic agents, particularly new drugs against vascular angioneogenesis 62,63 .
Recently, a needle-based confocal endomicroscope has also been developed which can be passed through a 19G FNA needle to assess indeterminate masses, cysts or lymph nodes. Malignancy in the hepatobilary tract is identified by the presence of irregular vessels, vascular leakage and large dark clumps (Figure 1)  During ERCP, a "CholangioFlex" confocal probe (Mauna Kea Technologies, Paris, France) can be placed down the working channel of a cholangioscope or duodenoscope to obtain real-time CLE images, which are akin to standard histology (Figure 1). If the images obtained from a point on the biliary mucosa contain dark areas, this is highly suggestive of malignancy 79,80 . The diagnostic accuracy of probe-based CLE was recently validated in a prospective multi-centre international study with 112 patients (71 with malignant lesions). Tissue sampling alone had a sensitivity, specificity and diagnostic accuracy of 56%, 100% and 72%, respectively. In comparison, ERCP with probe-based CLE had a sensitivity, specificity and diagnostic accuracy of 89%, 71% and 82%, respectively. Diagnostic accuracy increased to 88% when probe-based CLE and tissue sampling results were combined 81 . CLE is also feasible in the pancreatic duct during pancreaticoscopy but, owing to concerns over pancreatitis, is rarely used. In a case report by Meining et al., the presence of a main ductintraductal papillary mucinous neoplasia was confirmed by clear views of typical finger-like projections 82 . Intraductal ultrasound in small studies has also been shown to have a diagnostic accuracy of up to 90% 83 .

Conclusions
Currently, the most widely used tumour marker in pancreaticobiliary malignancy is CA19-9. However, its use is limited by its elevation in a number of other benign and malignant conditions. Furthermore, it is not produced in approximately 7% of the population who are Lewis antigen-negative and is often undetectable when tumours are small. Over the last few years, a number of very promising biomarker panels have been identified which can detect tumours at an early stage when curative intervention could be possible. These markers are subject to ongoing validation studies but appear likely to be implemented into screening programmes, particularly for high-risk groups, in the near future.
Novel endoscopic techniques such as per-oral cholangioscopy and confocal endomicroscopy can enhance the diagnostic accuracy of standard techniques and are increasingly available in large-volume centres worldwide.

Competing interests
The authors declare that they have no competing interests.

Grant information
SPP is supported in part by National Institutes of Health grant P01CA8420. Part of the work was undertaken at University College London Hospitals/University College London, which received a portion of funding from the Department of Health's National Institute for Health Research Biomedical Research Centres funding scheme.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 62.