Benjamin R. Kipp

A Comparison of Routine Cytology and Fluorescence in situ Hybridization for the Detection of Malignant Bile Duct Strictures

BACKGROUND  AND  AIM:The aim of this study was to assess the relative sensitivities and specificities of fluorescence in situ hybridization (FISH) and routine cytology for the detection of malignancy in biliary tract strictures.METHODS:Bile duct brushing and aspirate specimens were collected from 131 patients being evaluated for possible malignant bile duct strictures. Both specimen types were assessed by FISH but only brushing specimens were assessed by cytology. The FISH assay used a mixture of fluorescently-labeled probes to the centromeres of chromosomes 3, 7, and 17 and chromosomal band 9p21 (Vysis® UroVysion) to identify cells having chromosomal abnormalities. A case was considered positive for malignancy if five or more cells exhibited polysomy.RESULTS:Sixty-six of the 131 patients had surgical pathologic and/or clinical evidence of malignancy. Thirty-nine patients had cholangiocarcinoma, 19 had pancreatic carcinoma, and 8 had other types of malignancy. The sensitivity of cytology and FISH for the detection of malignancy in bile duct brushing specimens in these patients was 15% and 34% (p < 0.01), respectively. The sensitivity of FISH for the bile aspirate specimens was 23%, and the combined sensitivity of FISH for aspirate and brushing specimens was 35%. The specificity of FISH and cytology brushings were 91% and 98% (p = 0.06), respectively.CONCLUSIONS:FISH is significantly more sensitive than and nearly as specific as conventional cytology for the detection of malignant biliary strictures in biliary brushing specimens. FISH may improve the clinical management of patients who are being evaluated for malignancy in bile duct strictures.

A prospective comparison of digital image analysis and routine cytology for the identification of malignancy in biliary tract strictures

BACKGROUND & AIMS Digital image analysis (DIA) allows quantification of nuclear DNA content and may help distinguish benign and malignant strictures of the biliary tract. METHODS One hundred ten consecutive patients undergoing endoscopic retrograde cholangiography for suspicious biliary tract strictures were enrolled in a prospective study comparing the accuracy of DIA and routine cytology (RC). Standard brush cytology sampling was performed twice by using 2 cytology brushes per patient. Both brushes were fixed in a single-specimen vial. Each specimen was formed into 1 pellet, and the sample was equally divided for evaluation by DIA and RC. DNA histograms were generated for ploidy analysis. The DIA criterion for malignancy was demonstration of aneuploidy. RESULTS Two patients had inadequate samples obtained for DIA analysis, 7 benign patients were excluded because of inadequate follow-up of less than 75 days, and 1 patient was lost to follow-up to clarify malignant versus benign disease. Of the remaining 100 patients, 56 strictures were malignant and 44 were benign. The sensitivities of DIA and RC were 39.3% and 17.9%, respectively (P = 0.014). The specificities of DIA and RC were 77.3% and 97.7%, respectively (P = 0.003). The accuracy of DIA (56.0%) was equivalent to RC (53.0%). CONCLUSIONS DIA is a valuable adjunct to RC for detecting malignant strictures of the biliary tract.

The genomic landscape of small intestine neuroendocrine tumors

Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 106 nucleotides (range, 0-0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways.

Prospective Evaluation of Advanced Molecular Markers and Imaging Techniques in Patients With Indeterminate Bile Duct Strictures

BACKGROUND AND  AIMS:Standard techniques for evaluating bile duct strictures have poor sensitivity for detection of malignancy. Newer imaging modalities, such as intraductal ultrasound (IDUS), and advanced cytologic techniques, such as digital image analysis (DIA) and fluorescence in situ hybridization (FISH), identify chromosomal abnormalities, and may improve sensitivity while maintaining high specificity. Our aim was to prospectively evaluate the accuracy of these techniques in patients with indeterminate biliary strictures.METHODS:Cholangiography, routine cytology (RC), intraductal biopsy, DIA, FISH, and IDUS were performed in 86 patients with indeterminate biliary strictures. Patients were stratified based on the presence or absence of primary sclerosing cholangitis (PSC).RESULTS:RC provided low sensitivity (7–33%) but high specificity (95–100%) for PSC and non-PSC patients. The composite DIA/FISH results (when considering trisomy-7 [Tri-7] as a marker of benign disease) yielded a 100% specificity and increased sensitivity one- to fivefold in PSC patients versus RC, and two- to fivefold in patients without PSC, depending on how suspicious cytology results were interpreted. For the most difficult-to-manage patients with negative cytology and histology who were later proven to have malignancy (N = 21), DIA, FISH, composite DIA/FISH, and IDUS were able to predict malignant diagnoses in 14%, 62%, 67%, and 86%, respectively.CONCLUSIONS:DIA, FISH, and IDUS enhance the accuracy of standard techniques in evaluation of indeterminate bile duct strictures, allowing diagnosis of malignancy in a substantial number of patients with false-negative cytology and histology. These findings support the routine use of these newer diagnostic modalities in patients with indeterminate biliary strictures.

Integrated genomic characterization reveals novel, therapeutically relevant drug targets in FGFR and EGFR pathways in sporadic intrahepatic cholangiocarcinoma.

Advanced cholangiocarcinoma continues to harbor a difficult prognosis and therapeutic options have been limited. During the course of a clinical trial of whole genomic sequencing seeking druggable targets, we examined six patients with advanced cholangiocarcinoma. Integrated genome-wide and whole transcriptome sequence analyses were performed on tumors from six patients with advanced, sporadic intrahepatic cholangiocarcinoma (SIC) to identify potential therapeutically actionable events. Among the somatic events captured in our analysis, we uncovered two novel therapeutically relevant genomic contexts that when acted upon, resulted in preliminary evidence of anti-tumor activity. Genome-wide structural analysis of sequence data revealed recurrent translocation events involving the FGFR2 locus in three of six assessed patients. These observations and supporting evidence triggered the use of FGFR inhibitors in these patients. In one example, preliminary anti-tumor activity of pazopanib (in vitro FGFR2 IC50≈350 nM) was noted in a patient with an FGFR2-TACC3 fusion. After progression on pazopanib, the same patient also had stable disease on ponatinib, a pan-FGFR inhibitor (in vitro, FGFR2 IC50≈8 nM). In an independent non-FGFR2 translocation patient, exome and transcriptome analysis revealed an allele specific somatic nonsense mutation (E384X) in ERRFI1, a direct negative regulator of EGFR activation. Rapid and robust disease regression was noted in this ERRFI1 inactivated tumor when treated with erlotinib, an EGFR kinase inhibitor. FGFR2 fusions and ERRFI mutations may represent novel targets in sporadic intrahepatic cholangiocarcinoma and trials should be characterized in larger cohorts of patients with these aberrations.

A Multivariable Model Using Advanced Cytologic Methods for the Evaluation of Indeterminate Pancreatobiliary Strictures

BACKGROUND & AIMS Ancillary cytologic tests including digital image analysis (DIA) and fluorescence in situ hybridization (FISH) have been developed to improve the sensitivity of routine cytology (RC) for the diagnosis of malignancy in pancreatobiliary strictures. The goal of this study was to retrospectively compare the performance of RC, DIA, and FISH on clinical brushing specimens. METHODS Endoscopic retrograde cholangiopancreatography brushings were obtained from 498 consecutive patients with pancreatobiliary strictures and analyzed by RC, DIA, and FISH as per standard practice. RC diagnostic categories included negative, atypical, suspicious, or positive. Aneuploid/tetraploid histograms were considered positive for DIA. FISH was performed using UroVysion (Abbott Molecular, Inc, Des Plaines, IL) and classified as negative, trisomy, tetrasomy, or polysomy. RESULTS The sensitivity of polysomy FISH (42.9%) was significantly higher than RC (20.1%) when equivocal RC results were considered negative (P < .001) with identical specificity (99.6%). There was a significant difference in time for diagnosis of carcinoma between FISH diagnostic categories (P < .001) and between RC diagnostic categories (P < .001). Logistic regression analysis revealed that polysomy FISH, trisomy FISH, suspicious cytology, primary sclerosing cholangitis status, and age were associated with carcinoma (P < .05). CONCLUSIONS Polysomy FISH had high sensitivity without compromise to specificity. DIA was not a significant independent predictor of malignancy. Multivariable modeling using RC, FISH, age, and primary sclerosing cholangitis status can be used to estimate the probability of carcinoma for an individual patient. We recommend including FISH as a routine test where available, along with RC, in the evaluation of indeterminate pancreatobiliary strictures.

Isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma

Somatic mutations in isocitrate dehydrogenase 1 and 2 genes are common in gliomas and help stratify patients with brain cancer into histologic and molecular subtypes. However, these mutations are considered rare in other solid tumors. The aims of this study were to determine the frequency of isocitrate dehydrogenase 1 and 2 mutations in cholangiocarcinoma and to assess histopathologic differences between specimens with and without an isocitrate dehydrogenase mutation. We sequenced 94 formalin-fixed, paraffin-embedded cholangiocarcinoma (67 intrahepatic and 27 extrahepatic) assessing for isocitrate dehydrogenase 1 (codon 132) and isocitrate dehydrogenase 2 (codons 140 and 172) mutations. Multiple histopathologic characteristics were also evaluated and compared with isocitrate dehydrogenase 1/2 mutation status. Of the 94 evaluated specimens, 21 (22%) had a mutation including 14 isocitrate dehydrogenase 1 and 7 isocitrate dehydrogenase 2 mutations. Isocitrate dehydrogenase mutations were more frequently observed in intrahepatic cholangiocarcinoma than in extrahepatic cholangiocarcinoma (28% versus 7%, respectively; P = .030). The 14 isocitrate dehydrogenase 1 mutations were R132C (n = 9), R132S (n = 2), R132G (n = 2), and R132L (n = 1). The 7 isocitrate dehydrogenase 2 mutations were R172K (n = 5), R172M (n = 1), and R172G (n = 1). Isocitrate dehydrogenase mutations were more frequently observed in tumors with clear cell change (P < .001) and poorly differentiated histology (P = .012). The results of this study show for the first time that isocitrate dehydrogenase 1 and 2 genes are mutated in cholangiocarcinoma. The results of this study are encouraging because it identifies a new potential target for genotype-directed therapeutic trials and may represent a potential biomarker for earlier detection of cholangiocarcinoma in a subset of cases.

Molecular profiling of cholangiocarcinoma shows potential for targeted therapy treatment decisions

Cholangiocarcinoma is a highly lethal cancer of the biliary tract. The intrahepatic subtype of cholangiocarcinoma is increasing in incidence globally. Despite technologic advancements over the past decade, little is known about the somatic changes that occur in these tumors. The goal of this study was to determine the frequency of common oncogenes in resected cholangiocarcinoma specimens that could provide potential therapeutic targets for patients diagnosed with cholangiocarcinoma. Formalin-fixed, paraffin-embedded tissue blocks from 94 resected cholangiocarcinomas were used to extract DNA from areas comprising more than 20% tumor. Specimens were evaluated using the Sequenom MassARRAY OncoCarta Mutation Profiler Panel (San Diego, CA). This matrix-assisted laser desorption/ionization-time of flight mass spectrometry single genotyping panel evaluates 19 oncogenes for 238 somatic mutations. Twenty-five mutations were identified in 23 of the 94 cholangiocarcinomas within the following oncogenes: KRAS (n = 12), PIK3CA (n = 5), MET (n = 4), EGFR (n = 1), BRAF (n = 2), and NRAS (n = 1). Mutations were identified in 7 (26%) of 27 extrahepatic cholangiocarcinomas and 16 (24%) of 67 intrahepatic cholangiocarcinomas. When combined with IDH1/2 testing, 40 (43%) of the 94 cholangiocarcinomas had a detectable mutation. MassARRAY technology can be used to detect mutations in a wide variety of oncogenes using paraffin-embedded tissue. Clinical testing for somatic mutations may drive personalized therapy selection for cholangiocarcinomas in the future. The variety of mutations detected suggests that a multiplexed mutation detection approach may be necessary for managing patients with biliary tract malignancy.

Primary Sclerosing Cholangitis Patients With Serial Polysomy Fluorescence In Situ Hybridization Results Are at Increased Risk of Cholangiocarcinoma

OBJECTIVES:A polysomy fluorescence in situ hybridization (FISH) result in a pancreatobiliary brushing from a patient with primary sclerosing cholangitis (PSC) is very worrisome for carcinoma. However, treatment is not recommended unless verified by corroborative evidence of malignancy because of less than perfect test specificity in this population. The aim of this study was to evaluate the clinical outcome of PSC patients with serial polysomy FISH results.METHODS:Patients with PSC underwent endoscopic retrograde cholangiopancreatography with brushings when clinically indicated per standard practice. Brushings were evaluated by routine cytology and FISH. Retrospective review identified patients with a polysomy FISH result without definitive imaging or pathological evidence of malignancy at the time of the first polysomy, who underwent follow-up examinations including subsequent FISH testing (n=30). Patient records were reviewed to determine clinical outcome.RESULTS:In all, 9 of 13 patients (69%) with a subsequent polysomy result (i.e., serial polysomy) were diagnosed with cholangiocarcinoma (CCA) compared with 3 of 17 patients (18%) with subsequent non-polysomy results (P=0.008). There was a significant difference in time to a diagnosis of CCA between PSC patients with serial polysomy compared with those with subsequent non-polysomy (P=0.01). In four patients with serial polysomy results, imaging/pathological evidence of CCA was not found until 1–2.7 years after the initial polysomy FISH result.CONCLUSIONS:FISH may detect polysomic cells in pancreatobiliary brushings before other pathological or imaging techniques identify CCA. Patients with serial polysomy FISH results are at higher risk for having CCA than those with subsequent non-polysomy FISH results.

False positive endoscopic ultrasound fine needle aspiration cytology: incidence and risk factors

Objective It is broadly accepted that the false positive (FP) rate for endoscopic ultrasound fine needle aspiration (EUS FNA) is 0–1%. It was hypothesised that the FP and false suspicious (FS) rates for EUS FNA are greater than reported. A study was undertaken to establish the rate and root cause of discordant interpretation. Design Using a prospectively maintained endoscopic database, cytohistological discordant EUS FNA examinations from 30 July 1996 to 31 December 2008 were identified retrospectively. Setting Tertiary referral centre. Main outcome measures Discordant FNA was defined by positive or suspicious FNA cytology in the absence of malignancy or neoplasm in the subsequent surgical pathology specimen, specifically in the absence of neoadjuvant therapy. Three cytopathologists conducted a blinded review of randomised discordant and matched specimens. Results FNA was performed in 5667/18 066 (31.4%) patients undergoing EUS, of whom 2547 had cytology results interpreted as ‘positive’ or ‘suspicious’ or ‘atypical’ for malignancy or neoplasm. Subsequent surgical resection without prior neoadjuvant therapy was performed in 377 patients with positive or suspicious cytology. The FP rate was 20/377 (5.3%) and increased to 27/377 (7.2%) when FS cases were included. The incidence of discordance was consistent over time (1996–2002: 10/118 (8.6%) vs 2003–2008: 17/259 (6.6%); p=0.5) and was higher in non-pancreatic FNA (15%) than pancreatic FNA (2.2%; p=0.0001). Two-thirds of the non-pancreatic FP cases involved sampling of perioesophageal or perirectal nodes in patients with luminal neoplasms or Barretts oesophagus. Following pathological re-review, discordance was attributed to translocated cell contamination/sampling error (50%) or cytopathologist interpretive error (50%). Conclusions These findings refute the accepted paradigm that FP cytology rarely occurs with EUS FNA. Further investigation revealed that FP FNA developed secondary to endosonographer technique or initial cytological misinterpretation, and is particularly likely when perioesophageal or perirectal nodes are aspirated in the setting of a luminal neoplasm or Barretts oesophagus. Further study is needed to determine the significance of these findings and potential impact on the performance of FNA and patient outcomes.