NGS was performed on distinct small biopsy types, including EUS-FNAs or FNBs, brushings, and pancreatic juice, whereas several NGS panels were utilized. The initial material used for nucleic acid extraction was either fresh, directly collected for further NGS testing [25,55,61], frozen [23,57], also derived from formalin-fixed, paraffin-embedded tissue or cell blocks [70,71,85], residual liquid-based cytology (LBC) samples [58,62], or cytology slide scraping [59,60]. Mutations in the KRAS, TP53, CDKN2A, and SMAD4 genes were the most common ones detected in the PDAC patients tested [22,60,72,77]. Although KRAS mutations, as an early carcinogenic step, were also found in non-malignant cases, TP53 and SMAD4 alterations indicated HGD or carcinoma, triaging surgically fit patients for surgery [22,23,24,25,26,27].

A few of the published studies aimed to unravel the value of NGS in the preoperative evaluation of pancreatic cysts, in order to reduce unnecessary surgical procedures. This challenging task emerges more often in recent years, as more incidental cysts are detected, following the prevalent use of enhanced imaging technology [86]. To manage pancreatic cysts effectively, physicians should generally decipher if: (a) the cysts are mucinous or non-mucinous (the latter can be either non-neoplastic or benign with minimal malignant potential which can safely be managed conservatively), and (b) there is presence of at least HGD within the cysts; these would be classified as high-risk cysts, which are triaged for surgery [42]. In accordance with the literature, this review also found that the presence of KRAS mutations supported the diagnosis of a mucinous (IPMN or MCN) over a non-mucinous cyst (e.g., pseudocyst or SCA), whereas GNAS mutations favored IPMN over MCN [25,29,65,66,76,79]. NGS enhanced the diagnostic accuracy of EUS-FNA cytology to detect neoplastic mucinous cysts and differentiate them from the non-mucinous ones [49]. Notably, evidence indicated that NGS was more sensitive than the cytologic examination or elevated CEA cystic fluid levels (≥192 ng/mL), which are the two modalities traditionally used to evaluate pancreatic cysts [25,48,52,68]. For instance, Ren et al. showed the combination of cytologic examination, elevated CEA cystic fluid levels, and NGS reached a sensitivity of 94.1% and a specificity of 100% for the detection of neoplastic mucinous cysts [48]. Apart from discriminating between mucinous and non-mucinous pancreatic cysts, NGS was able to robustly identify high-risk cysts. A few studies indicated that specific mutations detected with NGS were associated with cystic neoplasms exhibiting HGD or invasion [25,76,87]. Rosenbaum et al. examined 113 pancreatic cystic fluid lesions from 105 patients and reported that SMAD4, TP53, CDKN2A, or NOTCH1 mutations indicated the presence of IPMN with high-grade dysplasia or cancer. Of interest, NGS combined with cytology improved the overall diagnostic accuracy to detect IPMNs and identify the high-risk IPMNs [25]. Similarly, Jones et al. also found that the presence of SMAD4, TP53, or CDKN2A alterations, discovered with NGS, indicate IPMNs with high-grade dysplasia or invasion [76].

Likewise, some teams utilized pancreatic juice-based NGS to recognize HGD or cancer while evaluating solid or cystic pancreatic lesions. For instance, TP53 or multiple KRAS mutations were associated with invasive IPMNs [24,67]. Furthermore, Suenaga et al. tested the pancreatic juice from a mixture of pancreatic cancer and precursors (with both LGD and HGD) under surveillance, in addition to normal controls. They found that patients with HGD or cancer exhibited higher numbers and concentration of mutations other than KRAS/GNAS (also a higher overall mutation concentration) in their pancreatic juice. Mutations in TP53 and/or SMAD4 or a high SMAD4/TP53 mutation score were associated with HGD or cancer, whereas none of them were detected in the controls. Thus, NGS facilitated the stratification of high-risk patients under pancreatic surveillance, by identifying the patients harboring at least HGD [23]. Yu et al. applied pancreatic juice-based NGS in a cohort of 115 pancreatic solid and cystic lesions (34 PDACs, 57 IPMNs, and 24 non-neoplastic controls). They reported that PDAC patients showed higher mutation concentrations than IPMNs or controls. Although TP53 and SMAD4 mutations were associated with PDACs, they were also detected in 15/57 and 1/47 of IPMNs, respectively, but in none of the controls. Notably, two high-risk patients of the cohort under surveillance showed TP53/SMAD4 mutations more than a year before their cancer diagnosis [26].

Apart from its use in diagnosis and preoperative risk stratification of pancreatic solid and cystic lesions, small biopsy-based NGS also showed potential in identifying potentially actionable alterations in PDAC patients. Takano et al. found such alterations in 22.4% of the cases tested [50], whereas Elhanafi et al. identified actionable mutations in the BRAF, MET, ERBB2, ARID1A, and BRCA1 genes in a few of the PDACs tested [70]. Lastly, Valero et al. reported at least one mutation in 17/19 of PDAC patients of their cohort, whereas KRAS, TP53, SMAD4, and ARID1A mutations were the ones most commonly detected. Notably, actionable alterations (e.g., in ATM or mTOR genes) were also found in some samples [46].

Whereas most studies focused on PDAC and its precursors, small biopsy-based NGS was also used to evaluate the molecular profile of other pancreatic neoplasms, pointing to a specific diagnosis or providing additional prognostic and therapeutic information. Gleeson et al. tested 90 primary and 32 metastatic PanNETs from the liver and reported that the former most often harbored MEN1, DAXX, ATRX, and TSC2 mutations. In addition, they found that alterations in TSC2, KRAS, and TP53 genes were associated with poor prognosis, whereas they also identified potentially actionable alterations in some members of the mTOR pathway (PTEN, TSC2, and PIK3CA) in 10% of primary and 12.5% metastatic NETs tested [74]. Whereas KRAS mutations were often in PDACs and IPMNs, they were not detected in the PanNET cases tested in two studies [58,83]. VHL mutations indicated a diagnosis of SCA in some studies. Of interest, Vestrup Rift et al. found that although mutations in KRAS and GNAS genes were the most common ones found in IPMNs, they were not detected in the three SCAs tested [66,68,76]. Furthermore, the presence of a CTNNB1 mutation indicated SPN; Kubota et al. found a CTNNB1 mutation in all seven SPNs, yet in just 1/11 NETs and in none of the PDACs, acinar cell carcinomas and pancreatitis cases of their cohort [81].

Evidence has shown that FNA-based NGS was highly concordant with its matched tissue-based molecular analysis, where it often revealed additional alterations, modifying the management plan of the patients [54,61,73,75,85]. In addition, it exhibited superior sensitivity than PCR or Sanger sequencing [83]. Rapid on-site evaluation (ROSE), besides improving the diagnostic accuracy of EUS-FNA, facilitated the acquisition of material for subsequent NGS testing, sparing the patients from additional invasive procedures [60]. Of interest, FNB was more likely to result in adequate material for subsequent NGS testing than FNA (OR: 4.95; 95% CI: 1.11–22.05; p = 0.04) [70], whereas larger gauge biopsy needles were more likely to result in successful NGS findings [64].