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Ancillary techniques that help in refining the cytological diagnosis are invaluable. A good cell block containing ample material is required. Ancillary tests include biochemical and molecular tests and immunohistochemical and special stains. These tests have diagnostic and prognostic implications. The PSC made recommendations on the utilization of ancillary studies [29]. These are further expanded upon in subsequent chapters of this monograph. Indications for ancillary testing in the pancreas include the following scenarios: differentiation of benign from malignant ductal lesions, diagnosis of primary and metastatic neoplasms, work-up of suspected hematopoietic lesions, and preoperative work-up of pancreatic cysts.

Several studies have found KRAS mutations in the premalignant dysplastic lesions and invasive carcinomas of the pancreas [48–53]. Although KRAS mutation analysis is a sensitive test for pancreatic adenocarcinoma, it can also test positive in benign cases such as chronic pancreatitis [50, 51]. Current data do not support KRAS testing in solid pancreatic masses. The PSC recommends the use of commercially available UroVysion FISH testing (Abbott Molecular, Abbott Park, IL, USA) on brushings from pancreatobiliary strictures with cytological diagnosis of indeterminate atypia. This test analyzes individual cells for DNA abnormality to determine if the sample is positive or negative. FISH analysis outperforms routine cytology with very high specificity and much higher sensitivity for the identification of carcinoma [54–56].

For the assessment of pancreatic cystic lesions, ancillary tests that aid in the diagnosis are CA19-9, CEA, and amylase levels in the cyst fluid, and some special stains. Special stains such as periodic acid-Schiff (PAS) and PAS with diastase (dPAS) detect the presence of glycogen. Glycogen is present in the clear cytoplasm of the cuboidal cells of serous cystadenoma and in the zymogen granules of the acinar cell lesions. PAS, mucicarmine (for neutral mucin), and Alcian blue (acid mucin) are used to detect the presence of intracellular and extracellular mucin. The cyst fluid CEA level is a very good test to diagnose if the cyst is mucinous, in which case the level usually exceeds 192 ng/mL [57]. Cyst fluid amylase levels help to diagnose pseudocyst where the levels are typically elevated into thousands. This test, however, does not distinguish IPMN from MCN as both can show slightly elevated levels.

Immunostaining for Smad4 is a good surrogate for SMAD4 genetic alterations and is useful in establishing a diagnosis of adenocarcinoma as it is retained in benign pancreatic ducts and is lost in more than half of PDACs [58]. Overexpression of mesothelin favors a diagnosis of adenocarcinoma. Trypsin and chymotrypsin positivity helps to diagnose ACC.

For PanNETs, immunostains synaptophysin, chromogranin, and the proliferation marker Ki-67 can be extremely helpful. Ki-67 is of importance in the histologic grading of PanNETs, but its utility in cytological preparations remains to be determined [59]. Strong nuclear staining for beta-catenin is diagnostic for SPNs in the pancreas.

Fig. 2. Flowchart depicting an algorithmic approach to pancreatic lesions for diagnostic purposes.

Cystic lesions of the pancreas exhibit a specific distinctive mutational profile for each subtype. SCNs have VHL mutations, CTNNB1 mutation is diagnostic of SPNs, MCNs have mutations involving the RNF43, KRAS, TP53, and SMAD4 genes, and IPMNs have mutations involving the genes KRAS, RNF43, GNAS, P53, and SMAD4 [60, 61].