Pancreatic Cystic Lesions: A Focused Review on Cyst Clinicopathological Features and Advanced Diagnostics

Macroscopic, endomicroscopic, and histologic findings and correlation are an integral part of the diagnostic evaluation of pancreatic cystic lesions (PCLs), as complementing morphologic features seen by different specialties are combined to contribute to a final diagnosis. However, malignancy risk stratification of PCLs with worrisome features can still be challenging even after endoscopic ultrasound guided-fine needle aspiration (EUS-FNA) with cytological evaluation. This review aims to summarize cyst clinicopathological features from the pathologists’ perspective, coupled with knowledge from advanced diagnostics–confocal laser endomicroscopy and cyst fluid molecular analysis, to demonstrate the state-of-art risk stratification of PCLs. This review includes illustrative photos of surgical specimens, endomicroscopic and histologic images, and a summary of cyst fluid molecular markers.


Introduction
Pancreatic cystic lesions (PCLs) are being detected more frequently due to the increasing use of cross-sectional imaging [1]. Our knowledge of different types of PCLs has grown dramatically in recent years. A multidisciplinary approach using a combination of radiological imaging, endoscopic ultrasound (EUS), cytology, cyst fluid analysis, and molecular profiling is most helpful in diagnosing and risk stratifying PCLs [2][3][4][5]. Despite these advancements, challenges still exist in the appropriate classification of PCLs as benign versus precancerous and accurate risk-stratification of intraductal papillary mucinous neoplasm (IPMNs). Unwarranted resection of benign PCLs (up to 15% of pancreatectomies) causes unnecessary mortality and morbidity [6], and there is continued surgical overtreatment of branch duct-IPMNs where up to 50% of lesions only reveal low-grade dysplasia [7].
Various guidelines and recommendations exist for the management of PCLs [8]. The authors' institution follows the 2017 international consensus guidelines for intraductal papillary mucinous neoplasm (IPMN) of the pancreas [1]. While the differentiation of most PCLs can be achieved through a combination of the clinical history, gender, imaging characteristics, cytology and cyst fluid analyses, malignancy risk stratification of PCLs with worrisome features can still be challenging even after EUS-FNA with cytological evaluation. This review aims to summarize cyst clinicopathological features from the pathologists' perspective, coupled with knowledge from advanced diagnostics-confocal laser endomicroscopy and cyst fluid molecular analysis-to demonstrate the state-of-art risk stratification of PCLs.
EUS-guided needle-based confocal laser endomicroscopy (nCLE) generates real-time endomicroscopic images of the PCLs. Coupled with intravascularly injected fluorescein dye, nCLE accentuates the vascular pattern of the lesion revealing distinct epithelial features.
Endomicroscopy: EUS-nCLE of IPMN shows finger-like papillary projections composed of an outer epithelium and inner vascular core [23]. Figure 2 shows the typical appearance of IPMNs as seen during real-time in vivo EUS-nCLE procedure. Figure 2A shows a BD-IPMN with low-grade dysplasia. The epithelium is thinner and more translucent. Comparatively, Figure 2B demonstrates BD-IPMN with high-grade dysplasia with thicker and darker epithelium indicative of cellular and nuclear stratification, respectively. Both computer-aided artificial intelligence algorithms and human-interobserver studies have revealed a high diagnostic accuracy in nCLE-guided differentiation of IP-MNs [24,25].
The three histologic subtypes of IPMNs are gastric type (~70%), intestinal type (~20%), and pancreatobiliary type (~10%) (Figure 3). Gastric type ( Figure 3A) resembles gastric foveolar epithelium, composed of tall columnar cells with basally oriented small nuclei and abundant cytoplasmic mucin. Scattered goblet cells may be seen. Gastric type is often associated with low-grade dysplasia and usually occurs in branch ducts [30]. Intestinal type ( Figure 3B) is the second most common histologic type, which most often resides in the main duct and is associated with high-grade dysplasia. It features hyperchromatic villous papillae, containing elongated and crowded nuclei, basophilic cytoplasm and variable amount of goblet cells [31][32][33][34]. Pancreaticobiliary type ( Figure 3C) contains amphophilic to eosinophilic cytoplasm, enlarged nuclei with nucleoli, and moderate cytoplasmic mucin. It typically involves the main duct and often shows high-grade cytoarchitectural atypia. Some consider the pancreatobiliary type a variant of high-grade gastric type [31,35].
The three histologic subtypes of IPMNs are gastric type (~70%), intestinal type (~20%), and pancreatobiliary type (~10%) (Figure 3). Gastric type ( Figure 3A) resembles gastric foveolar epithelium, composed of tall columnar cells with basally oriented small nuclei and abundant cytoplasmic mucin. Scattered goblet cells may be seen. Gastric type is often associated with low-grade dysplasia and usually occurs in branch ducts [30]. Intestinal type ( Figure 3B) is the second most common histologic type, which most often resides in the main duct and is associated with high-grade dysplasia. It features hyperchromatic villous papillae, containing elongated and crowded nuclei, basophilic cytoplasm and variable amount of goblet cells [31][32][33][34]. Pancreaticobiliary type ( Figure 3C) contains amphophilic to eosinophilic cytoplasm, enlarged nuclei with nucleoli, and moderate cytoplasmic mucin. It typically involves the main duct and often shows high-grade cytoarchitectural atypia. Some consider the pancreatobiliary type a variant of high-grade gastric type [31,35].
Grading of dysplasia: IPMNs is currently classified as low grade and high grade, based on the highest degree of cytoarchitectural atypia in the epithelium [14,36]. Lowgrade IPMNs are characterized by mucinous epithelium with mild to moderate atypia. The epithelium may be flat ( Figure 3A), show small tufting, or arrange into tall papillae but with bland cytology ( Figure 4A). High-grade IPMNs feature severe atypia, characterized by papillae with irregular branching and budding, nuclear stratification with loss of polarity, cellular pleomorphism, and increased mitoses ( Figure 4B-D). Notably, with increasing dysplasia, the papillae may lose the central fibrovascular core. In Figure 4B, while the bottom villous structure still has a fibrovascular core, the villi in the center of the image become slender without stromal core and the nuclei are rounded up, showing high-grade features including pleomorphism and loss of polarity.

Grading of dysplasia:
IPMNs is currently classified as low grade and high grade, based on the highest degree of cytoarchitectural atypia in the epithelium [14,36]. Lowgrade IPMNs are characterized by mucinous epithelium with mild to moderate atypia. The epithelium may be flat ( Figure 3A), show small tufting, or arrange into tall papillae but with bland cytology ( Figure 4A). High-grade IPMNs feature severe atypia, characterized by papillae with irregular branching and budding, nuclear stratification with loss of polarity, cellular pleomorphism, and increased mitoses ( Figure 4B, C, D). Notably, with increasing dysplasia, the papillae may lose the central fibrovascular core. In Figure 4B, while the bottom villous structure still has a fibrovascular core, the villi in the center of the image become slender without stromal core and the nuclei are rounded up, showing high-grade features including pleomorphism and loss of polarity.

Molecular alterations:
Somatic mutations in KRAS and GNAS are the two most common genetic alterations seen in IPMN, together seen in >95% of all IPMNs [37,38]. KRAS mutation is an early event and nearly a prerequisite in the pathogenesis of ductal neoplasms, including pancreatic ductal adenocarcinomas (PDAC). Activating GNAS mutations are enriched in the intestinal subtype [39][40][41]. RNF43 somatic mutations are seen in about 50% of IPMNs [37,42]. Changes in tumor suppressor genes, such as TP53, CDKN2A and SMAD4, as well as mutations of mTOR genes (PTEN, PIK3CA, AKT1) are associated with advanced neoplasia (high-grade dysplasia and invasion) [43][44][45][46][47][48][49]. Alterations of other genes in the mitogen-activated protein kinase (MAPK) pathway have also been detected in mucinous cysts, including BRAF, ERBB2, HRAS, and MAPK1. GNAS and BRAF mutations in mucinous cysts are fairly specific for IPMN [9,46]. IPMN-associated invasive carcinoma: Overall, about a third of resected IPMN are associated with invasive carcinoma. Main-duct IPMN (>10 mm) has higher rate of associated malignancy, up to 60%, whereas branch-duct IPMN (>3 cm) has a lower rate (15-20%) [50][51][52][53][54]. The two most common IPMN-associated invasive carcinomas are tubular (ductal) adenocarcinoma and colloid adenocarcinoma. Colloid adenocarcinomas almost always arise in a background of intestinal-type IPMN, typically harbor GNAS mutations, and carry a better prognosis. Tubular adenocarcinoma is morphologically similar to conventional pancreatic ductal adenocarcinoma (PDAC) and is associated with pancreaticobiliary/gastric-type IPMN, and KRAS mutation [40,55,56]. The invasive component can be very focal, therefore thorough (if not complete) sampling of the resected pancreas is warranted. but with bland cytology ( Figure 4A). High-grade IPMNs feature severe atypia, characterized by papillae with irregular branching and budding, nuclear stratification with loss of polarity, cellular pleomorphism, and increased mitoses ( Figure 4B, C, D). Notably, with increasing dysplasia, the papillae may lose the central fibrovascular core. In Figure 4B, while the bottom villous structure still has a fibrovascular core, the villi in the center of the image become slender without stromal core and the nuclei are rounded up, showing high-grade features including pleomorphism and loss of polarity.

Mucinous Cystic Neoplasm
Definition: Mucinous cystic neoplasm (MCN) of the pancreas is a mucin-producing cystic epithelial neoplasm that is associated with characteristic ovarian-type subepithelial stroma.
Epidemiology: The mean patient age is 48 years. MCN predominantly (>98%) occurs in women [14]. Increasing age correlates with a higher risk of invasive carcinoma, suggesting that progression occurs over a period of years.
Macroscopic appearance: The mean size of MCN is 6 cm (ranging 2 to 35 cm). MCNs with invasive carcinoma are larger (>5 cm, mean: 9 cm) [57]. MCNs can be unilocular or multilocular with a few septa. They do not communicate with the pancreatic ductal system and typically have a thick fibrous wall (≥3 mm, Figure 5A top arrow) [61]. The pathogenesis is unclear and thought to be related to ectopic embryonic remnants, which explains some "ancient" changes such as a thick fibrous wall and mural calcifications. High-grade MCNs often have mural nodules or papillary projections. Endomicroscopy: EUS-nCLE of MCNs shows horizon-type epithelial bands ( Figure  6A) of variable thickness without papillary conformation [23]. The epithelial bands are single or in multiple layers and are best observed when viewed tangentially to the nCLE probe. Further, MCNs can reveal clusters of inflammatory cells with areas of a dark background and fluorescent macrophages. Since MCNs may contain foci of atrophy and chronic inflammation, nCLE characterization of epithelial bands can sometimes be challenging during in vivo EUS. Inflammatory clusters of cells or debris can also be observed in pseudocysts, but such patients frequently have a history of pancreatitis and nCLE does not reveal any epithelial bands ( Figure 6B,C). Endomicroscopy: EUS-nCLE of MCNs shows horizon-type epithelial bands ( Figure 6A) of variable thickness without papillary conformation [23]. The epithelial bands are single or in multiple layers and are best observed when viewed tangentially to the nCLE probe. Further, MCNs can reveal clusters of inflammatory cells with areas of a dark background and fluorescent macrophages. Since MCNs may contain foci of atrophy and chronic inflammation, nCLE characterization of epithelial bands can sometimes be challenging during in vivo EUS. Inflammatory clusters of cells or debris can also be observed in pseudocysts, but such patients frequently have a history of pancreatitis and nCLE does not reveal any epithelial bands ( Figure 6B,C). Histopathology: The mucinous epithelium of MCNs is morphologically similar to that of IPMNs, and the dysplasia is also graded based on a two-tiered system [36]. It is not uncommon for the epithelium of MCN to be denuded in areas, sometimes extensively. Looking for the subepithelial ovarian-type stroma is helpful in such cases ( Figure 5B arrows), and the presence of this distinctive stroma is required for the diagnosis of MCN [14]. However, the ovarian-type stroma may be attenuated (hypocellular/hyalinized) in large MCNs, postmenopausal patients, or around areas with advanced neoplasia [57]. Positive immunohistochemical staining for progesterone receptor (60-90%) ( Figure 5C) and estrogen receptor (30%) may aid in the detection of the ovarian-type stroma [60,62].

Molecular alterations:
Activating mutations in codon 12 of KRAS is seen in 50-66% of MCNs as well as loss of function in RNF43 [9,41,67]. Unlike IPMN, GNAS mutations are rarely seen in MCN [43]. The gene mutations associated with advanced neoplasia in IPMN may be observed in MCN, including TP53, CDKN2A, SMAD4, and/or mTOR genes (PTEN, PIK3CA, AKT1) [9]. Histopathology: The mucinous epithelium of MCNs is morphologically similar to that of IPMNs, and the dysplasia is also graded based on a two-tiered system [36]. It is not uncommon for the epithelium of MCN to be denuded in areas, sometimes extensively. Looking for the subepithelial ovarian-type stroma is helpful in such cases ( Figure 5B arrows), and the presence of this distinctive stroma is required for the diagnosis of MCN [14]. However, the ovarian-type stroma may be attenuated (hypocellular/hyalinized) in large MCNs, postmenopausal patients, or around areas with advanced neoplasia [57]. Positive immunohistochemical staining for progesterone receptor (60-90%) ( Figure 5C) and estrogen receptor (30%) may aid in the detection of the ovarian-type stroma [60,62].

Serous Cystadenoma
Definition: Serous cystadenoma (SCA) is a benign epithelial neoplasm composed of uniform cuboidal, glycogen-rich clear cells that often form cysts containing serous fluid.
Epidemiology: The mean age at presentation is 58 years, with a female predominance (female: male = 3:1).
Location: SCA can occur anywhere in the pancreas; mostly (50-75%) in the body or tail. Macroscopic appearance: SCAs are well-circumscribed, multilocular cystic mass, without communication with the pancreatic ductal system. The mean size is about 4 cm with a wide range (1 to 25 cm in diameter) [68][69][70][71]. The cyst wall is typically thin (<3 mm) [61,72]. The microcystic variant shows characteristic honeycomb or sponge pattern ( Figure 5D,E). A central scar with a sunburst calcification pattern is present in 30% of cases. There are also rare macrocystic (oligocystic) variant and solid variant.
Endomicroscopy: EUS-nCLE of SCA shows an intricate fern pattern of vascularity ( Figure 6D,E) or also called superficial vascular network. This interstitial vascular pattern reveals parallel or inter-connected network of capillaries underneath the epithelium [23]. Trafficking red blood cells are frequently observed in the fine vascular meshwork during the in vivo EUS-nCLE procedure. There is variation in contrast (fluorescein) penetration within the capillaries; often, the capillaries are densely packed with red blood cells.
Histopathology: The lining epithelium of SCA consists of a single layer of low cuboidal epithelial cells with clear cytoplasm, due to abundant intracytoplasmic glycogen. Variably prominent capillary network is seen underneath the epithelium ( Figure 5F). Nuclear atypia and mitoses are typically absent. The neoplastic epithelium is immunoreactive for inhibin, which can be helpful in small biopsy or cytology specimens. Microforceps biopsy may show very scant cuboidal epithelial cells in a background of blood ( Figure 5G,H); positive inhibin staining helps to confirm the diagnosis ( Figure 5I) in the appropriate clinical setting. Molecular alternations: Germline or somatic alterations of the tumor suppressor gene VHL is present in SCAs [9,41,67,73]. In the setting of germline VHL mutation, there could be multifocal SCAs in the pancreas. Alterations in genes associated with IPMN, MCN, and PDAC, such as KRAS, GNAS, CDKN2A, and SMAD4, have not been reported in SCAs [41]. TP53 or TERT promoter mutations may be prognostically important, as they are associated with interval growth of cyst size [9].

Cystic Neuroendocrine Tumor
Pancreatic neuroendocrine tumors are typically well-circumscribed, solid tumors. However, hemorrhage and secondary degeneration may occur in the center of the tumor ( Figure 7A,B), resulting in a grossly cystic neuroendocrine tumor (cNET) with only viable tumor cells in the cyst wall. EUS-nCLE of cNET shows dark clusters (trabeculae) of cells separated by stroma [23]. The clustering of cells can conform to various shapes, commonly in cords or groups with occasional geometric formations ( Figure 6F). Histologically, cNET is composed of nests, trabeculae, or ribbons of neoplastic cells, separated by thin vascular fibrous septa ( Figure 7C). MEN1 alterations are highly specific for cNETs, but the sensitivity is low (27%) [9]. Loss of ATRX/DAXX and the presence of alternative lengthening of telomeres (ALT) are associated with poor prognosis [9]. cNETs with loss of heterozygosity (LOH) of ≥3 genes tend to have distant metastasis [9].

Solid Pseudopapillary Neoplasm
Definition: Solid pseudopapillary neoplasm (SPN) is a low-grade malignant tumor that lacks a specific line of pancreatic epithelial differentiation. It most likely arises from genital ridge cells that were translocated to the pancreas during embryogenesis [74].
Epidemiology: SPNs occur predominantly (90%) in young women. The mean age at presentation is 28 years.
Location: SPNs have a slight preference for the pancreatic tail [75,76].
Macroscopic appearance: SPNs are well-demarcated mass lesions, with variable solid and cystic components, and occasional calcifications. SPNs are typically large tumors (average size: 8 cm; range: 0.5-25.0 cm) [14]. The cystic component reflects degenerative changes secondary to hemorrhagic necrosis ( Figure 7D). Not surprisingly, small tumors tend to be more solid.
Endomicroscopy: EUS-nCLE of SPN is indistinguishable from cNET revealing groups or clusters of cells separated by interstitial spaces [23].

Solid Pseudopapillary Neoplasm
Definition: Solid pseudopapillary neoplasm (SPN) is a low-grade malignant tumor that lacks a specific line of pancreatic epithelial differentiation. It most likely arises from genital ridge cells that were translocated to the pancreas during embryogenesis [74].
Epidemiology: SPNs occur predominantly (90%) in young women. The mean age at presentation is 28 years.
Location: SPNs have a slight preference for the pancreatic tail [75,76]. Macroscopic appearance: SPNs are well-demarcated mass lesions, with variable solid and cystic components, and occasional calcifications. SPNs are typically large tumors (average size: 8 cm; range: 0.5-25.0 cm) [14]. The cystic component reflects degenerative changes secondary to hemorrhagic necrosis ( Figure 7D). Not surprisingly, small tumors tend to be more solid. [23].

Endomicroscopy: EUS-nCLE of SPN is indistinguishable from cNET revealing groups or clusters of cells separated by interstitial spaces
Histopathology: SPN is composed of epithelioid cells, forming pseudopapillary structures due to perivascular growth ( Figure 7E) and also crowded nests imparting a solid appearance in areas ( Figure 7F). SPNs may mimic a pancreatic NET, macroscopically, endomicroscopically, and histologically. Diffuse nuclear staining of β-catenin, as well as the expression of SOX11 and TFE3 by immunohistochemistry, are helpful to confirm the diagnosis of SPN [77][78][79][80]. Of note, SPNs may be positive for synaptophysin by immunohistochemistry, but chromogranin should be negative.

Pseudocyst
A pseudocyst is a collection of fluid contents walled off by fibrous tissue after episode(s) of pancreatitis ( Figure 7G). The location is usually outside the pancreas. EUS-nCLE of pseudocyst ( Figure 6B,C) reveals a dark background due to the absence of vascular interstitium and a true epithelium-lined cyst wall. Some auto-fluorescent inflammatory cells are usually present in pseudocysts [23]. Histologically, a cyst lining is absent, and the cyst wall is composed of fibroinflammatory tissue, often with evidence of old hemorrhage and necrosis (cholesterol clefts and pigments, Figure 7H).
Epidemiology: LECs are the most prevalent among squamous-lined epithelial cysts and account for approximately 0.5% of all PCLs. The mean patient age at presentation is 56 years, with a male predominance (80%) [84]. Dermoid cysts of the pancreas occur in a younger age group with a mean age of 23 (range 2-53 years) with no gender predominance [83]. The mean patient age for ECIPAS is 38 years [83] with a slight female predominance [90].
Location: LEC can occur anywhere in the pancreas (head, body, or tail) or can be found in an extra-pancreatic location [83,84,91,92]. Dermoid cysts can occur anywhere along the pathway of ectodermal cell migration and can been found in anywhere in the pancreas [93,94]. ECIPAS are primarily located in the tail of the pancreas [83,90].
Macroscopic Appearance: The mean size of LECs is approximately 5 cm (range 1.2-17 cm). They are often round and well-demarcated from the surrounding pancreas. They can be multilocular (60%) or unilocular (40%) in appearance. The contents of the cysts appear "cheesy" or "caseous" (signifying keratinaceous debris) or may less often be clear and serous [84]. Dermoid cysts of the pancreas contain a combination of both cystic and solid components. Ectodermal differentiation (skin, hair follicles, sweat glands, sebaceous material) is the most common, but structures from other germ layers (cartilage, bone, thyroid tissue, etc.) may be present [88]. The mean size of ECIPAS is 4.5 cm (range 2.3-6.5 cm). ECIPAS can be unilocular or multilocular. It may contain serous fluid but will lack presence of hair or skin appendages [83,95,96].
Endomicroscopy: LECs can reveal clusters of bright particles suggestive of keratinous debris otherwise can be heterogenous in appearance with a bland background (squamous epithelium) and lattice-type blood vessels [23,97]. ECIPAS can reveal cords of cells suggestive of ectopic splenic tissue [97].

Simple Mucinous Cyst
In the absence of a visible obstructive process, mucinous cysts that are >1 cm, without any features of IPMN or MCN, are classified as simple mucinous cysts [36,[102][103][104][105]. Cyst fluid analysis may show elevated CEA levels. Histologically, the mucinous epithelium is flat with rare tufting, but well-formed papillae are absent. The most common mutations include KMT2C (62%), KRAS and TP53 (15%) [52,106]. However, in 31% cases, no mutations are detected. A simple mucinous cyst is essentially a diagnosis of exclusion, and the main differential considerations include IPMN, MCN, and a retention cyst involved by pancreatic intraepithelial neoplasia.

Advanced and Emerging Diagnostic Tools for Pancreatic Cystic Lesions
Despite our ever-increasing knowledge of PCLs, there remains continued resection of benign cysts at high rates and resection of IPMNs with low-grade dysplasia. In this review, we have described features observed in specific PCLs using two advanced diagnostics (nCLE and cyst fluid NGS). Both techniques can enhance the diagnostic accuracy of cyst type and advanced neoplasia significantly [108], and they may be considered when radiomics and EUS cyst morphology are indicative of a high-risk PCL [2]. However, these advanced diagnostics are only available in limited centers currently, and large prospective studies are needed before incorporation into guidelines. Other emerging diagnostic tools for PCLs include Mass Spectrometry and Optical Coherence Tomography (OCT).
Needle-based confocal laser endomicroscopy (nCLE) provides real time, en-face (perpendicular to histologic sectioning planes) visualization of cyst lining at the microscopic level. Through a combined assessment of interstitial vascular pattern, cyst content fluorescence characteristics, and the "negative" image/architecture of the cyst epithelium, the accuracy of nCLE diagnosis of PCLs reaches over 90% [109,110]. In addition, nCLE evaluation of the "thickness" and "darkness" of the cyst epithelia allows grading of dysplasia and thus risk stratification of IPMNs [24]. However, the lack of adequate exposure/training for the real-time interpretation of images impede the widespread use of nCLE. Artificial intelligence (AI)-aided interpretation of nCLE images may help overcome some of the challenges [25].
Next generation sequencing of the nucleic acid present in the cyst fluid, offers molecular insight into the genetic makeup of the cells shed from the neoplastic cyst epithelium. Certain genetic alternations indicate specific pancreatic cyst types, and molecular evolution with additional gene mutations is correlated with neoplastic progression and advanced neoplasia [9]. Alternations involving KRAS, GNAS, and/or BRAF indicate mucinous cyst; GNAS and BRAF mutations in mucinous cysts are fairly specific for IPMN (rare in MCN). Advanced neoplasia in mucinous cysts is associated with alterations in TP53, SMAD4, CTNNB1, CDKN2A, and/or the mTOR genes (PTEN, PIK3CA, AKT1). Cystic pancreatic NETs are associated with MEN1 mutations and LOH for multiple genes. Loss of ATRX/DAXX and the presence of alternative lengthening of telomeres (ALT) are associated with poor prognosis in pancreatic NETs. SCAs often harbor VHL mutations; TP53 and TERT promoter mutations are seen in large-sized SCAs.
EUS through-the-needle biopsy (TTNB) represents a "bite" biopsy of the cyst wall by a microforceps introduced through a standard EUS 19-gauge FNA needle. TTNB generates larger tissue fragments and boasts good diagnostic yield (69.5%) and high histological accuracy (86.7%) in a meta-analysis of 454 patients [111]. Another meta-analysis demonstrates that TTNB has a higher sensitivity and specificity than cytology [112]. However, a relatively high rate of complications has been reported with mucinous cysts or cysts with connection to pancreatic duct. In a prospective study of 101 patients who underwent TTNB, adverse event rate was 9.9% (10 patients, including 9 acute pancreatitis and 1 fatality) [113]. Therefore, TTNB should be considered for patients where the necessity of an accurate diagnosis outweighs the risks [114]. We exclusively reserve TTNB for SCAs. Only when nCLE shows SCA (or non-mucinous) pattern, we will consider TTNB.
Mass spectrometry is a useful tool that measures the mass-to-charge ratio of ions and allows for the identification of the molecular weight, structure, and chemical formula of pure substances [115,116]. Additionally, mass spectrometry has been a helpful noninvasive tool that can assist in the early diagnosis of cancer by identifying both the presence of cancer and its progression over time [117,118]. Early mass spectrometry studies analyzing protein profiles obtained from pancreatic cyst fluid found unique peptide patterns for various benign PCLs [119,120], detected pancreatic tumor markers (such as Mucin family members, S100 proteins, CEA-related proteins) [121], and were able to distinguish between benign and malignant pancreatic lesions [120]. Notably, one study combined a novel biomarker panel of four proteins with CA19-9 and was able to diagnose pancreatic carcinoma with a sensitivity of 95% and specificity of 94.1% [122]. In a more recent 2018 diagnostic study, targeted mass spectrometry taken from pancreatic cyst fluid using protein biomarkers from mucin-5AC and prostate stem-cell antigen was able to identify advanced neoplasia with an accuracy of 96%. Targeted mass spectrometry can detect 95% of malignant/severely dysplastic lesions, compared with 35% and 50% for CEA and cytology respectively. Additionally, a panel of peptides taken from mucin-5AC and mucin-2 was able to discriminate between premalignant/malignant and benign lesions with an accuracy of 97%, outperforming cyst fluid CEA (61%) and cytology (84%) [123].
Optical Coherence Tomography is an imaging modality that features a spatial resolution at the µm level. OCT imaging has been applied to the examination of human eyes [124], skin [125], and cardiovascular tissue [126]. Recently, a prototype endoscopic OCT has been reported for early diagnosis and Endo-OCT image-guided brachytherapy of pancreatic cancer and precursor lesions [127].

Summary
Unlike pancreatic cancers that have known risk factors such as smoking, chronic pancreatitis, diet, and diabetes, most neoplastic pancreatic cysts do not have a clear etiology. Perhaps, they may be considered genetic diseases, caused by inherited (germline) and somatic mutations. Patients with certain hereditary cancer syndromes are known to be predisposed to developing pancreatic cysts. IPMNs have been reported in patients with McCune-Albright syndrome, Peutz-Jeghers syndrome, Lynch syndrome, familial adenomatous polyposis, hereditary breast and ovarian cancer syndrome, familial atypical multiple mole melanoma, and Carney complex [128]. SCNs develop in 90% of patients with von Hippel-Lindau syndrome (VHL). Overall, 10-20% of PanNETs are associated with hereditary syndromes including multiple endocrine neoplasia type 1, VHL, neurofibromatosis type 1, tuberous sclerosis, and others [14]. It is unclear why MCNs and SPNs have a female predominance. They could be related to persistent fetal periductal mesenchyme or translocated embryonic tissue from genital ridges that respond and proliferate in response to hormonal stimulation [14].