A A Ancillary

Testing, Committee IV, L. Layfield, Chair

b by Layfield Thu Aug 16, 2012 3:10 pm

Introduction:
Utilization of Ancillary Studies in Pancreatic/Biliary Cytology Members: Lester Layfield, MD Chair - Cytopathologist University of Utah Philippe Vielh, MD, PhD Cytopathologist - Institut Gustave Roussy, France Loren Joseph, MD AMP - Cytopathologist University of Chicago Armando Filie, MD Cytopathologist NCI, Bethesda, MD Ralph Hruban, MD Surgical Pathologist NCI, Bethesda, MD Nirag Jhala, MD Cytopathologist University of Pennsylvania Hormoz Ehya, MD Cytopathologist Fox Chase Cancer Center Martha Pitman, MD Cytopathologist MGH, Harvard University

Ancillary Testing for Bile Duct Brushings Cytologic evaluation of bile duct brushings is known to have a suboptimal sensitivity ranging from 4-61%.1-11 A number of approaches have been developed to augment the purely cytologic analysis of bile duct brushing specimens. These have included protocols to improve diagnostic sensitivity of brushing specimens designated as inconclusive, indeterminate or negative following on-site cytopathologic examination.12-15 Ancillary procedures potentially useful in interpretation of negative and indeterminate cytologic results include intraductal ultrasound examination, digital image analysis, immunolabeling, fluorescence in-situ hybridization, KRAS mutational analysis and sequential mutational analysis.1-14,16 One of the new imaging modalities include intraductal ultrasound (IDUS). IDUS has been shown to be a safe and readily performed procedure.17-20 IDUS probes operate at high frequency and produce detailed images of the bile duct wall and adjacent structures. Although some studies have shown good diagnostic sensitivity and specificity for IDUS, others have shown low sensitivity and specificity in patients with primary sclerosing cholangitis.12,18,19 Prior studies have demonstrated that endoscopic evaluation during ERC of bile duct brushings can improve the diagnostic accuracy of the cytologic examination and the combination of clinical (ERCP) findings with cytology is valuable in evaluation of biliary strictures.15 The use of intraductal ultrasound evaluation appears to further enhance the diagnostic accuracy of the combination of imaging and cytologic evaluation. Digital image analysis (DIA) has been used to identify abnormalities of

nuclear DNA content using spectrophotometric techniques.21-23 Digital image analysis utilizes Feulgen reactions which hydrolyze DNA into constituent nucleic acids that stoichiometrically bind to the Feulgen dye. Using this technique, DNA ploidy can be assessed by a variety of commercially available image analyzers.21,24 DNA ploidy status is assessed on the collected cells using a histogram generated by commercially available quantitative DNA analysis programs. Results are characterized as diploid, aneuploidy or tetraploid. Aneuploid and tetraploid results are most likely indicative of malignancy.25 The sensitivity of digital image analysis does not appear to improve diagnostic accuracy beyond that achievable with routine cytology for patients with primary sclerosing cholangitis. However, in patients without primary sclerosing cholangitis the technique does appear to improve diagnostic sensitivity. Digital image analysis appears to have excellent specificity for the diagnosis of carcinoma but only moderate sensitivity.22,23 Thus, digital image analysis appears to have diagnostic characteristics similar to routine cytology in that a positive test is highly accurate but a negative result is of little clinical value. KRAS gene mutation analysis has been used as an ancillary testing procedure for both analysis of pancreatic juice bile duct brushings and fine-needle aspirates of solid and cystic pancreatic masses.26-28 The majority of studies involving KRAS mutations have investigated their relationship with pancreatic adenocarcinoma29-32 and genetic progression in pancreatic duct lesions and intraductal papillary mucinous neoplasms.33-35 KRAS mutational analysis of pancreatic juice has even been used as a potential method for the early diagnosis of pancreatic carcinoma.36-38 Although KRAS mutations can be found in hyperplastic, dysplastic and malignant pancreatic duct epithelium,26-33 several studies have shown that KRAS mutation analysis is a sensitive test for pancreatic adenocarcinoma.39-41 Sturm et al27 studied 312 consecutive patients with extrahepatic biliary stenosis and found that conventional cytology combined with KRAS mutational analysis was more sensitive than conventional cytology alone. Kipp et al studied 35 cases of pancreatic adenocarcinoma brushing cytology samples collected during ERC and demonstrated a combined sensitivity of 86% for K-ras mutation and fluorescence in-situ hybridization analyses.42 Hruban et al34 have described a progression model for pancreatic carcinoma in which both KRAS and HER2/neu molecular abnormalities are seen in low grade dysplasias as well as malignancies. Other authors have described them in pancreatic duct hyperplasia and chronic pancreatitis.30,31 Reicher et al26 has demonstrated some utility for KRAS mutational analysis in the evaluation of EUS-FNA specimens. They described KRAS mutations in approximately 9% of benign specimens and 56% of malignant

specimens. Importantly, KRAS mutational analysis was helpful in dividing atypical specimens into benign and malignant categories. Of the two cases in Reichers study considered cytologically atypical but on follow-up demonstrated to be benign, none contained KRAS mutations.26 On the other hand, in cases considered atypical with malignant follow up, 20% of cases (2/10) demonstrated KRAS mutations. Additional studies will be necessary to demonstrate the value of KRAS mutational analysis in both bile duct stricture cytologies and FNAs of solid pancreatic masses. Multiple Mutational Analysis The potential application of analytic techniques for evaluating sequential mutation accumulation in bile duct brushing specimens has been studied. Lapkus et al have shown that there is considerable overlap in the spectrum of mutational markers in pancreatic duct and biliary brushings but the temporal profile of accumulation of these mutations differs significantly between pancreatic and biliary neoplasms.16 These authors studied the time course of mutation accumulation in pancreatic and biliary tract lesions by microdissecting cell clusters on the basis of cytomorphologic features (and analyzing the presence of loss of heterozygosity) with a panel of fifteen markers (1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, 22q) as well as point mutations in K-ras using PCR/capillary electrophoresis. Distinctive frequencies of mutational events in these lesions were found.16 While distinctive frequencies for sequential mutation accumulation were demonstrable, diagnostic utility of this approach is still to be determined. Fluorescence In-situ Hybridization Analysis Fluorescence in-situ hybridization (FISH) analysis of bile duct brushing specimens for polysomy using a commercially available DNA probe set (UroVysion; Abbot Molecular, USA) has been reported as a useful technique by a number of authors.11-13,25,36-38 This kit utilizes probes targeting the pericentromeric regions of chromosomes 3 (CEP3), 7 (CEP7), and 17 (CEP17) as well as chromosomal bands 9p21 (LSI 9p21). The method can be automated using the Bioview Imaging Duet system. Others25 have developed probe sets of their own based on known chromosomal alterations in genes associated with pancreatic carcinoma including: TP53, CDKN2A/p16, EGFR and others. In a series of 93 pancreaticobiliary brushings, Barr Fritcher et al37 demonstrated a specificity of 100% and a sensitivity of approximately 60% for the identification of carcinoma using FISH probes targeting centromeric regions of chromosomes 3, 7, 17 and the 9p21 band. They considered a specimen as positive for malignancy by FISH when five or more cells showed polysomy (>2 signals in at least 2 of the 4 probes). In that study, FISH analysis outperformed routine cytology and review consensus cytology of the brushing specimens. Boldorinni et al43 also

reported similar findings with FISH brushing specimens outperforming routine cytology. In that study, the sensitivity of FISH was 90% with 94% specificity. The positive predictive value was 98% and negative predictive value was 75% for FISH testing. Levy et al12 reported similar success with FISH. In that study, the authors considered trisomy for chromosome 7 to be benign. In a series of morphologically negative cytologies, FISH was able to suggest malignancy in 62% of cases.12 Barr Fritcher et al44 investigated the utility of ancillary cytologic studies including digital image analysis and FISH in a series of 498 consecutive patients with pancreaticobiliary strictures. They found that FISH had a sensitivity of 42.9% and was significantly better than the sensitivity of routine cytology (20.1%) when equivocal cytologies were considered negative. They concluded that FISH had a higher sensitivity without compromising specificity.13 Of all the ancillary techniques available for analysis of cytology specimens obtained by brushings from pancreaticobiliary strictures, FISH appears to improve diagnostic sensitivity the most over that achievable by routine cytology.12,13,15,26,43,44,45 While not directly addressing bile duct brushings, Kubiliun et al14 recommended the use of FISH for the diagnosis of pancreatic carcinoma in inconclusive cytologic evaluations. It appears that a similar approach is successful for the evaluation of negative and inconclusive pancreaticobiliary tract brushing specimens. Ancillary Testing for Pancreatic Cystic Lesions A number of ancillary tests have been proposed for aiding in the diagnosis of cystic lesions of the pancreas. These vary from measuring carcinoembryonic antigen (CEA) and amylase levels in cyst fluid, to histochemical stains (mucin) of cytologic smears to mutational analysis (HER2/neu, KRAS, GNAS, TP53) and measures of polysomy.35,46,47 Staining of smear preparations for mucin by a variety of techniques including the mucicarmine stain can also be performed. Staining for mucin can only be performed on direct smear preparations and is not diagnostically valid when performed on liquid-based preparations. Staining of smear preparations for mucins aids in the establishment of mucinous differentiation for the lining epithelium, however, it cannot separate benign from malignant lesions. Documentation of mucinous differentiation in cystic pancreatic lesions is clinically helpful in that it points to either a mucinous cystic neoplasm or an intraductal papillary mucinous neoplasm. Cyst fluid amylase is elevated in pseudocysts and in cysts that communicate with the pancreatic duct system but not in neoplastic cystic lesions. High amylase combined with low CEA suggests pseudocyst but high amylase and high CEA suggests an IPMN. Cyst

fluid CEA level is a reliable indicator of mucinous differentiation for a cyst, but unfortunately, it cannot reliably predict the presence of absence of malignancy.48 Originally, a CEA value of 192 ng/mL was used as a cut point bur more recent data suggests a cut point of 110 ng/mL. Some authors have demonstrated that CEA antigen level is helpful in separating malignant from pre-malignant cysts.46 Mean CEA levels of cyst fluid demonstrated a striking difference between benign and malignant cystic lesions. Benign cysts had a mean level of 5237 ng/mL and malignant cysts had a mean CEA level of 108,360 ng/mL. The difference was clinically significant with a p=.001 for separation of benign and dysplastic groups and a p<0.5 for the separation of dysplastic from malignant cysts.46 DNA analysis may also aid in the separation of benign from malignant cystic lesions. The success of DNA analyses depends on the amount of recoverable DNA.9 Measurements of cyst fluid DNA can be quantitated using a variety of commercially available techniques.46 The concentration of DNA is correlated with optical density (OD) as measured at a wavelength 260/280 and the mean concentration of DNA present within a fluid of pancreatic cystic lesions as documented by OD from a low of 6.5 in benign cysts to 16.5 in malignant cysts.46 Mutational analysis for KRAS mutations and LOH have been reported to be helpful in the separation of benign from malignant cysts.39,40 Khalid et al46,47 reported an absence of K-ras point mutations and LOH in benign cysts but an increasing frequency of mutations in premalignant cysts and malignant cysts. The number of mutations differed significantly between malignant and premalignant lesions (p=.003) and between malignant and benign cysts (p<.001).46 In addition, the sequence of mutation accumulation was also significantly different between pre-malignant and malignant lesions. Malignant cysts tended to acquire K-ras mutations first followed by allelic loss. The occurrence of a K-ras point mutation with or without subsequent allelic loss was significantly associated with malignancy (p<.001).46 The finding of LOH for a variety of tumor suppressor genes in addition to K-ras point mutations is strong evidence of malignancy.46,47 Others35 have had less success in using mutational analysis for the distinction of benign from malignant cystic lesions. Chadwick et al35 demonstrated that while K-ras point mutations were more common in malignant lesions than in benign lesions, but they also could be found in both benign and malignant intraductal papillary mucinous tumors.35 Others have had a similar experience with solid pancreatic neoplasms.33,43 Shen et al49 studied the utilization of a commercially available test that combines the detection of K-ras mutation, LOH and DNA quantity/quality in the diagnosis of pancreatic cystic lesions. The

concordance between the clinical consensus diagnosis and the commercial test was high with the commercial test showing a sensitivity of 83% and specificity of 100% for a malignant cyst and a sensitivity of 86% and specificity of 93% for a benign mucinous cyst. The authors concluded that the molecular analysis of pancreatic cyst fluids adds diagnostic value to the preoperative diagnosis.49 From the available data, it appears that the analysis of pancreatic cyst fluid for mucin by histochemical stains on direct smears and CEA level are helpful diagnostic adjuncts for the recognition of a cystic lesion showing mucinous differentiation. Cyst fluid amylase levels appear to be of great value for the recognition of pseudocysts. The utility of LOH (1p, 3p, 5q, 9p, 9q, 10q, 17p, 17q, 21q and 22q) analysis in the fluid of cystic lesions in the distinction of benign from malignant lesions may require additional larger studies. CA 125 in pancreatic cyst fluid: A few studies have investigated the cyst fluid CA 125 levels to assess the usefulness of this marker in determining the cyst type and discriminating between benign and malignant neoplasms.50-54 These studies have demonstrated that while the CA 125 levels are generally low in pseudocysts and high in cystadenomas, significant overlap exists between serous cystadenomas and mucinous cystic neoplasms. Furthermore, low levels of CA 125 have been reported in cystic islet cell tumors,55 and high levels in ciliated enteric duplication cyst56 and lymphoepithelial cyst.57 For these reasons, cyst fluid CA 125 assay does not seem to have an important role in the evaluation of pancreatic cysts. Ancillary Testing for Solid Pancreatic Neoplasms A number of ancillary procedures have been investigated for the detection of malignancy in solid pancreatic masses. K-ras point mutation analysis, FISH and DNA ploidy analysis have been used as potential methods for separating benign and malignant masses by FNA.13,25,48, 49,58-70 Pancreatic adenocarcinoma is believed to develop from either intraductal papillary mucinous neoplasms or more commonly from pancreatic intraepithelial neoplasia by the accumulation of a series of mutations.34 In this progression model for pancreatic carcinoma, activating point mutations in the K-ras oncogene and HER2/neu overexpression/amplification appear to be early events.58-65 K-ras mutations are found in nearly 45% of low grade pre-malignant lesions of the pancreas and their frequency increases along with increasing degrees of dysplasia.59 Additional mutations affecting other oncogenes occur later in the course of progression towards

adenocarcinoma with p16 showing increasing frequency of mutation in PanIN-II.34,65 TP53 mutations appear to be a relatively late event occurring in PanIN-III and invasive adenocarcinoma. While K-ras mutations are relatively common, p16 and p53 mutations are less frequent even in adenocarcinomas.35 A retrospective study by Khalid et al demonstrated the application of LOH and K-ras mutation analysis of EUS-FNA material in differentiating autoimmune pancreatitis from pancreatic cancer. K-ras mutation was detected in 10 of 11 (91%) of pancreatic cancer cases that yield DNA amplification and in none of the autoimmune pancreatitis cases suggesting that K-ras mutation in pancreatic mass FNA is associated with malignancy and may aid in the distinction from benign processes like autoimmune pancreatitis.40 Fluorescence in situ hybridization (FISH) has been shown to significantly improve diagnostic sensitivity without loss of specificity.14,38 The technique can be applied using commercially available probe sets. Disadvantages are that the method is time consuming and requires a fluorescence microscope. FISH analysis when combined with routine cytology has a sensitivity of approximately 85%.14 Because routine cytology has an excellent specificity, fluorescence in situ hybridization is most useful in improving diagnostic accuracy for cases judged as cytologically negative or inconclusive. Kubiliun et al14 have also recommended the use of FISH for the evaluation of cases judged on site as inconclusive or negative. In addition, Nodit et al67 explored the utility of microsatellite loss analysis in the diagnosis of pancreatic endocrine tumors. They concluded that microsatellite loss analysis of EUS-FNA material obtained from pancreatic endocrine tumors could be performed reliably and that losses of 3p, 6pq and 10pq along with gains of 5q, 12q, 18q and 20q were associated with a malignant behavior.67-70 Loss of heterozygosity studies appear to have some technical limitations. Because these techniques (when using cytologic material) require microdissection of small numbers of tumors followed by amplification of DNA, stochastic effects may alter the validity of test results. Additionally, the presence of polysomy or amplification in a cell population being analyzed by LOH may result in allelic imbalance due to other issues than loss of suppressor genes. Polysomy of chromosome 17 might result in one TP53 allele appearing more prominent than the other in some of these assays. Thus, techniques must be used that normalize overall signals to assure validity. A number of additional techniques have been investigated for their use in determining the presence of malignancy in pancreaticobiliary lesions. These include investigation of microRNAs, circulating tumor

cells and circulating nucleic acids. microRNA MicroRNAs are short (20-25 nucleotides) RNA molecules containing regions of complementarity to various mRNAs. Specific binding of a miRNA to a mRNA blocks translation. miRNA can be robustly quantified by real-time PCR and localized by ISH. Steele et al7l has reviewed profiling studies of pancreatic adenocarcinoma. Some miRNA , like miR-21 and miR-155, are highly expressed in early lesions.72-74 Most studies have analyzed resection specimens, one study analyzed fine needle aspirates75, none have assayed brushings. The clinical utility remains to be more firmly established, especially in comparison and conjunction with other techniques. Circulating Tumor Cells Circulating tumor cells (CTD),76 tumor DNA2,77 tumor RNA and now miRNA have been unequivocally demonstrated for most epithelial malignancies, including pancreatic cancer.78-80 Circulating tumor cells can be selected by size, immunocapture or flow cytometry and analyzed by IHC, DNA sequencing, RT-PCR, and FISH. Cytomorphology has received little attention.81 Some approaches allow imaging followed by molecular testing. The number of tumor cells per ml of blood varies from one cell to thousands for a given tumor type, determinants of number are unknown. A confounding factor is that the most common selection method, immunocapture targeting keratins or EpCAM, misses cells which have undergone EMT and which might account for the majority of CTC. The FDA has approved measurement of CTC in a limited setting for colon, breast and prostate cancer. Circulating nucleic acids On a molar basis, circulating tumor DNA, RNA and miRNA are much more abundant than CTC but can be more difficult to associate with a specific tumor. KRAS mutations in circulating DNA have been demonstrated in association with many malignancies.82,77 Small panels of miRNA show moderate to good ability to identify patients with specific tumors. Overexpression of miR-21, miR-155 and miR-210 have been shown to distinguish patients with pancreatic adenocarcinoma from healthy controls.83,84 The ability to follow such a result non-invasively and to correlate it with ambiguous cytology has obvious appeal but more extensive studies, in particular with a wider range of controls (such as pancreatitis) are needed. Similarly, the study of methylated DNA promoter sequences in circulating DNA has been shown to distinguish patients with cancer from healthy controls for numerous malignancies. Two studies for pancreatic cancer have identified several of the many potentially methylated promoters found in pancreatic carcinoma as useful for screening circulating methylated DNA.85,86 In particular hypermethylation of NPTX2 is demonstrated to

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