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Fluorescence in situ hybridization compared with conventional cytology for the diagnosis of malignant biliary tract strictures in Asian patients
ORIGINAL ARTICLE: Clinical Endoscopy
Fluorescence in situ hybridization compared with conventional cytology for the diagnosis of malignant biliary tract strictures in Asian patients Roongruedee Chaiteerakij, MD, PhD,1,3 Emily G. Barr Fritcher, CT (ASCP),2 Phonthep Angsuwatcharakon, MD,3 Wiriyaporn Ridtitid, MD,3 Supakarn Chaithongrat, BSc,3 Apinya Leerapun, MD,4 Todd H. Baron, MD,1,5 Benjamin R. Kipp, PhD,2 Michael R. Henry, MD,2 Kevin C. Halling, MD, PhD,2 Rungsun Rerknimitr, MD,3,* Lewis R. Roberts, MBChB, PhD1,* Rochester, Minnesota, USA; Bangkok, Chiang Mai, Thailand; Chapel Hill, North Carolina, USA
Background and Aims: Fluorescence in situ hybridization (FISH) has improved the diagnostic performance of cytology for the evaluation of malignant biliary strictures in the United States and Europe. The utility of FISH for the diagnosis of biliary strictures in Asia is currently unknown. We aimed to compare the sensitivity of FISH and conventional cytology for the diagnosis of malignant biliary strictures in Thai patients. Methods: A prospective study was performed at 2 university hospitals between 2010 and 2013. Patients being evaluated for malignant-appearing biliary strictures were included (N Z 99). Bile duct brushings were collected and assessed by cytology and FISH. Sensitivities with 95% confidence intervals of cytology and FISH were the main outcome measures. Results: The overall sensitivities of cytology and FISH were 38% and 55%, respectively (P Z .001). For those with a diagnosis of cancer based on clinical evidence without biopsy confirmation (n Z 44), the sensitivities of cytology and FISH were 43% and 57%, respectively (P Z .06). For the 49 patients for whom a cancer diagnosis was confirmed by pathology, FISH had a significantly higher sensitivity than cytology, with a sensitivity of 53% versus 33%, respectively (P Z .008). Conclusions: FISH improves the diagnostic performance of cytology and can be used as a complementary tool to bile duct brushing and biopsy for the evaluation of malignancy in biliary strictures in Asian populations. (Gastrointest Endosc 2016;83:1228-35.)
Biliary tract strictures can be due to benign or malignant diseases. Differentiation of malignant from benign biliary strictures is crucial for appropriate management. However,
this is not always easily achieved because the presentations may be similar. Histopathology is the gold standard for the diagnosis of biliary malignancy, and tissue sampling by
Abbreviations: CCA, cholangiocarcinoma; CEA, carcinoembryonic antigen; FISH, fluorescence in situ hybridization; PSC, primary sclerosing cholangitis.
Current affiliations: Division of Gastroenterology and Hepatology (1), Department of Laboratory Medicine and Pathology (2), Mayo Clinic College of Medicine, and Mayo Clinic Cancer Center, Rochester, Minnesota, USA, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok (3), Thailand, Department of Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai (4), Thailand, Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, North Carolina, USA (5).
DISCLOSURE: Dr Halling and Mayo Clinic receive royalties from the sale of the UroVysion FISH probe set. All other authors disclosed no financial relationships relevant this publication. This work was supported by grant CA165076 from the National Institutes of Health; the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567); the Mayo Clinic Cancer Center (CA15083), and the Mayo Foundation (to L.R.R.); and the Mayo Clinic Center for Clinical and Translational Science (NCATS UL1 TR000135). See CME section; p. 1258. *Authors Rerknimitr and Roberts contributed equally to the article. Copyright ª 2016 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 http://dx.doi.org/10.1016/j.gie.2015.11.037
Reprint requests: Rungsun Rerknimitr, MD, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand or Lewis R. Roberts, MBChB, PhD, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. If you would like to chat with an author of this article, you may contact Dr Rerknimitr at [email protected] or Dr Roberts at [email protected].
Received March 23, 2015. Accepted November 25, 2015.
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endobiliary forceps biopsy and/or bile-duct brush cytology performed during ERCP are commonly used.1 Although brush cytology yields a high specificity of 90% to 100%, its sensitivity remains unsatistifactory2-7 and varies substantially, depending on biliary tract location (proximal vs distal biliary stricture), patient population (eg, primary sclerosing cholangitis [PSC] vs non-PSC), and criteria used for positivity.2-7 The sensitivity of brush cytology was reported as 15% to 20% in 6 studies comparing the performance of FISH with that of routine cytology.2-7 By comparison, in a systematic review of 16 studies of the utility of brush cytology, the pooled sensitivity of brush cytology for the detection of malignant biliary strictures was reported as 40%.8 The low sensitivity of brush cytology stems from inadequate tumor cellularity and sampling difficulties due to the desmoplastic nature of pancreatobiliary malignancies as well as the technical challenge of obtaining samples from difficult-to-access stricture locations. Additionally, the interpretation of histologic findings is sometimes difficult because of inflammation, fibrosis and necrosis, small tissue fragments, and the challenge of distinguishing cytologic atypia from invasive malignancy.9,10 Advanced cytologic methods have been developed to enhance the limited sensitivity of forceps biopsy and brush cytology. Among these methods, the fluorescence in situ hybridization (FISH) assay (Multiprobe UroVysion; Abbott Molecular, Inc, Des Plaines, Ill) has improved the performance of brush cytology.2-4,6 Briefly, the FISH assay uses 4 fluorescent-labeled probes that hybridize directly to the pericentromeric regions of chromosomes 3, 7, and 17 and the chromosome 9p21 (CDKN2A) locus. Application of the assay to cytology brushings allows determination of the number of copies of chromosomes 3, 7, and 17 and of the 9p21 locus that are present in individual cells by using different fluorescence colors for each probe. It has been shown that the presence of 5 or more cells with polysomy (ie, >2 copies) of 2 or more chromosomes or loci is diagnostic of malignancy.2-4 The UroVysion FISH assay has been shown to substantially improve the performance of brush cytology for diagnosis of malignant biliary strictures.2-4,7,11 The diagnostic sensitivity of FISH has been reported as 34% to 58%, with 91% to 100% specificity, compared with a sensitivity of 15% to 21% for conventional cytology in the same patient cohorts.2,4,6 Accordingly, UroVysion FISH is now widely used in the United States as an ancillary diagnostic tool for evaluating biliary strictures. FISH was recently shown to increase the sensitivity of routine brush cytology from 35% to 52% for the diagnosis of malignancy in pancreatobiliary strictures in European populations.7 Whether FISH, which is clinically useful for the assessment of biliary strictures in Western populations, is applicable to Asian populations is currently unknown. There are substantial differences in the etiology of malignant biliary tract diseases, particularly cholangiocarcinoma (CCA), between Western and Asian populations. Liver fluke infestation is the main cause of CCA in Asians, whereas PSC, noninfectious biliary tract www.giejournal.org
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disease, and other unknown risk factors are the main causes of CCA in Western populations. It has been shown that the spectrum of genetic alterations in liver fluke– associated CCA is different from the alterations found in non–liver fluke–associated CCA.12 TP53, SMAD4, MLL3, and GNAS were more frequently mutated in liver fluke– associated CCA, whereas BAP1 and IDH1 or IDH2 were more frequently mutated in non–liver fluke–associated CCA.12 It is therefore important to determine whether the FISH assay, which was originally developed by using samples from Western populations, can be applied to Asians. The primary goal of this study was to determine the utility of FISH for the detection of malignancy in biliary tract strictures in an Asian population. We conducted a prospective study to evaluate whether FISH improves the performance of conventional cytology for the diagnosis of malignancy in biliary strictures at 2 academic centers in Thailand.
METHODS Patient population This study was approved by the Institutional Review Boards at the Mayo Clinic (Rochester, Minn), Chulalongkorn University (Bangkok, Thailand), and Chiang Mai University (Chiang Mai, Thailand). Consecutive patients evaluated for malignant-appearing biliary tract strictures who underwent ERCP between March 2010 and December 2013 at Chulalongkorn University and Chiang Mai University were prospectively enrolled in the study. Patients for whom alternate diagnostic tests including EUS-FNA were performed were also included in this study. Patients were followed postprocedurally by outpatient visits or by telephone contact to ascertain the final diagnosis, vital status, and cause of death. The diagnosis of malignancy was confirmed by histopathologic examination of surgically resected specimens or biopsy specimens (either open or laparoscopic surgical, percutaneous needle, or endoscopic forceps biopsy); or by clinical criteria, a combination of symptoms (jaundice and/or weight loss [n Z 32]), evidence of a stricture and/or mass on radiologic imaging in the absence of acute cholangitis (n Z 44), serum CA19-9 level higher than 100 U/mL (n Z 33), or clinical or radiologic progression (40 patients had clinical progression, 2 had radiologic progression, and 22 had both clinical and radiologic progression) during a mean ( SD) followup time of 6.4 (7.2) months. The presence of a malignantappearing biliary tract stricture was defined as long (>2 cm), asymmetrical, irregular, or abrupt narrowing of the biliary tract.5,13,14 Telephone contact was made 1 year after ERCP in all patients to confirm the progression of malignancy or death from malignancy. Physicians who determined the final diagnosis were blinded to the results of FISH.
Specimen collection and processing Cytologic specimens were obtained from biliary stricture sites during ERCP by using a standard cytology brush Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1229
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(Cook Medical, Winston-Salem, NC). The brush was passed through the stricture site for at least 5 to-and-fro motions and then stirred in PreservCyt preservative solution (Hologic, Inc, Marlborough, Mass) to release the cells. The specimens were kept at room temperature until shipment to the Cytopathology Department at the Mayo Clinic, where FISH and cytology examinations were performed. Once the specimens arrived, each was divided equally for FISH analysis and cytology examination. PreservCyt solution has a shelf life of 2 years at room temperature, and all specimens were shipped within 1 to 2 months after collection.
FISH assay Half of each specimen was processed on a FISH slide by using a standard protocol, as previously described.4,14 FISH was performed by using the fluorescently labeled Vysis UroVysion probe set (Abbott Molecular). An experienced cytotechnologist who was blinded to the clinical information and corresponding cytology results evaluated signal patterns of the FISH slides by fluorescence microscopy. Specimens were considered positive if polysomy was identified in 5 or more cells. Polysomy was defined as gain of 2 or more copies for 2 or more probes within a cell.2 Images of FISH results are shown in Figure 1.
Conventional cytology The other half of each specimen was processed on ThinPrep slides (Hologic, Inc) and Pap stained. All slides were screened by 2 cytotechnologists and reviewed by an experienced cytopathologist who was blinded to the clinical information and the FISH results. The cytology results were classified by the cytopathologist as positive, suspicious, atypical, or negative for malignancy by using previously published standard criteria.15,16 Only the positive classification was called as positive for cytology.
Other data collection Patient medical records were reviewed to obtain baseline patient characteristics, clinical information at the time of diagnosis and at return visits, and the results of radiologic imaging at the time of the ERCP procedure and during follow-up. Levels of CA19-9 and carcinoembryonic antigen (CEA) measured within 7 days of the date of ERCP procedure were abstracted.
Statistical analysis Of 101 patients enrolled in this study, FISH diagnosis and/or conventional cytology diagnosis was not feasible due to inadequate cellularity in brushing samples for 7 patients (4 for FISH and 3 for cytology). For the remaining 94 patients, both FISH analysis and cytology examination were performed. Two patients lost to follow-up were excluded from analysis due to inadequate information for making a definite diagnosis. A total of 92 patients who had both FISH and cytology results and an adequate follow-up period were included in the initial analysis. 1230 GASTROINTESTINAL ENDOSCOPY Volume 83, No. 6 : 2016
Figure 1. Malignant cell showing polysomy (>2 copies of at least 2 chromosomal loci) by using fluorescence in situ hybridization probes to CEP 3 (red), CEP 7 (green), CEP 17 (aqua), and the 9p21 locus of chromosome 9 (gold). CEP, chromosome enumeration probe.
Because patients with inadequate cellularity in brushing samples may represent important cases in which too few cells are obtained for diagnosis and because this is one of the greatest challenges in this disease, we included the 7 patients who had inadequate cellularity in their brush samples in the final analysis. The samples with inadequate cellularity were coded as negative for FISH or cytology in the final analysis so that the estimated sensitivity and specificity were more reflective of the sensitivity and specificity that would be obtained when the test is implemented in real-life practice. The final analysis therefore comprised 99 patients (93 with malignancy and 6 with benign biliary strictures). Sensitivity and specificity along with the corresponding 95% confidence interval of FISH and cytology were estimated and compared by using the McNemar test or the c2 test as appropriate. Levels of CA19-9 and CEA in patients with malignancy versus benign biliary strictures were compared by using the Mann-Whitney U test. All analyses were performed by using JMP version 10.0 (SAS Institute Inc, Cary, NC). A P value <.05 was considered significant.
RESULTS Baseline characteristics Of the 99 patients, 46 (46%) were male with a mean age ( SD) of 64.9 13.1 years. None of the patients had underlying PSC or primary stone-induced stenoses. Two patients had a history of recurrent pyogenic cholangitis; 1 had a final diagnosis of CCA and the other had a final diagnosis of benign biliary stricture due to surgery. Ninety-three of the 99 patients (94%) were diagnosed with malignancy and 74 of 93 (80%) had an associated www.giejournal.org
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TABLE 1. Baseline characteristics of study patients
Characteristics
All subjects, no. (%)
Cancers diagnosed by clinical criteria, no. (%)
Cancers diagnosed by pathology, no. (%)
99
44
49
Total number Male Age, y, mean SD
46 (46)
25
21
64.9 13.1
66.3 13.9
63.6 12.3
Diagnosis Cancer
93/99 (94)
44/93 (47)
49/93 (53)
Cholangiocarcinoma
58 (62)
34 (77)
24 (49)
Pancreatic cancer
15 (16)
4 (9)
11 (22)
Ampullary cancer
7 (8)
1 (2)
6 (13)
Gallbladder cancer
6 (7)
1 (2)
5 (10)
Hepatocellular carcinoma
2 (2)
2 (5)
0 (0)
Mixed hepatocellular cholangiocarcinoma
2 (2)
1 (2)
1 (2)
Cancer of unknown origin
2 (2)
1 (2)
1 (2)
Lung cancer
1 (1)
0 (0)
1 (2)
Benign biliary stricture
6/99 (6)
mass. The most common cancers were CCA (n Z 58, 62%), followed by pancreatic cancer (n Z 15, 16%), and ampullary cancer (n Z 7, 8%) (Table 1). The final diagnosis of malignancy was confirmed histopathologically in 49 patients; 44 patients had a diagnosis of malignancy by using clinical criteria. The remaining 6 patients who had clinical presentation and radiologic findings suspicious for malignancy at the time of ERCP but did not have evidence of malignancy during 1 year of follow-up received a diagnosis of benign biliary strictures (1 postsurgical biliary stricture, 1 chronic pancreatitis, 1 common bile duct stone, 1 choledochal cyst, and 2 of undetermined cause). The median (interquartile range) levels of serum CA19-9 and CEA for patients with malignancy versus those without malignancy were 487.9 (135.8-1000) versus 32.0 (17.1-45.0) U/mL for CA19-9 (P Z .006) and 6.7 (3.4-25.9) versus 2.4 (2.1-3.0) ng/mL for CEA (P Z .03). The indications for ECRP were obstructive jaundice (94/99, 95%) and cholangitis (5/99, 5%). Ninety-three of the 99 patients (94%) had primary ERCP, and 6 (6%) were previously stented. EUS-FNA was performed in 16 patients (17%) who had a diagnosis of malignancy.
Sensitivity and specificity of FISH versus conventional cytology for diagnosis of malignant biliary strictures The diagnostic performance of FISH and conventional cytology are shown in Table 2. Polysomy was detected by FISH in 51 of 93 patients (55%) with malignancy, whereas positive cytology was observed in only 35 of the 93 patients (38%). Therefore, FISH had significantly better sensitivity than cytology for the diagnosis of malignancy by using biliary brushing specimens (55% vs www.giejournal.org
38%, P Z .001). The specificity was 100% for both FISH polysomy and positive cytology. When FISH and cytology were considered together (ie, either test was positive), the sensitivity for diagnosis of malignancy compared with FISH alone increased slightly to 59%. Of 58 patients with a diagnosis of malignancy who had negative (n Z 20, 34.5%), atypical (n Z 20, 34.5%), or suspicious (n Z 18, 31.0%) cytology results, polysomy was observed with the FISH assay in 20 patients, yielding a sensitivity of 34.5%. Endoscopic biopsies were performed on 13 of the 51 patients (25%) with positive FISH test results. Of the 13 patients undergoing endoscopic biopsy, specimens positive for malignancy were obtained in 6 patients, 1 specimen was suspicious for malignancy, specimens from 3 patients showed high-grade dysplasia, 2 patients had inadequate specimens, and 1 patient had a negative biopsy result. Thus, the sensitivity and specificity of endoscopic biopsy in those with available results were 46% and 100%, respectively. For patients with a clinical diagnosis of malignancy, 25 of 44 had positive results on FISH, whereas 19 of 44 had positive cytology results, resulting in a higher sensitivity with FISH than with cytology (56.8% vs 43.2%, P Z .06). Of the 49 patients who had pathological confirmation of a cancer diagnosis, 16 were positive by cytology, 26 were positive by FISH, and 28 were positive by cytology and/or FISH. The associated sensitivities were 32.7% (16/49) for cytology alone, 53.1% (26/49) for FISH alone, and 57.1% (28/49) for the combination of cytology and FISH, respectively. When classified by location of biliary strictures, the sensitivity of FISH in samples obtained from proximal biliary strictures was not different from that in samples Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1231
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TABLE 2. Performance of FISH and conventional cytology for detection of malignant biliary stricture Proportion of positive tests in patients with a diagnosis of cancer
Sensitivity, % (95% CI)
P value
A. Cytology negative FISH polysomy
2/20
10.0 (1.8-33.1)
FISH polysomy or trisomy with 9p21 loss
2/20
10.0 (1.8-33.1)
B. Cytology negative or atypical FISH polysomy
8/40
20.0 (9.6-36.1)
FISH polysomy or trisomy with 9p21 loss
8/40
20.0 (9.6-36.1)
FISH polysomy
20/58
34.5 (22.8-48.2)
FISH polysomy or trisomy with 9p21 loss
20/58
34.5 (22.8-48.2)
C. Cytology negative, atypical or suspicious
D. Entire cohort Cytology positive
35/93
37.6 (28.0-48.3)
Cytology and/or endoscopic biopsy positive
38/93
40.9 (30.9-51.6)
Reference .08
FISH polysomy
51/93
54.8 (44.2-65.1)
.001
FISH polysomy or trisomy with 9p21 loss
53/93
57.0 (46.3-67.1)
.0001
Combined FISH and/or positive cytology
55/93
59.1 (48.4-69.1)
<.0001
Combined FISH and/or positive cytology and/or endoscopic biopsy
57/93
61.3 (50.6-71.0)
<.0001
Cytology positive
19/44
43.2 (28.7-58.9)
Reference
Cytology and/or endoscopic biopsy positive
19/44
43.2 (28.7-58.9)
1.0000
FISH polysomy
25/44
56.8 (41.1-71.3)
.06
FISH polysomy or trisomy with 9p21 loss
26/44
59.1 (43.3-73.3)
.02
Combined FISH and/or positive cytology
27/44
61.4 (45.5-75.3)
.005
Combined FISH and/or positive cytology and/or endoscopic biopsy
27/44
61.4 (45.5-75.3)
.005
Cytology positive
16/49
32.7 (20.4-47.7)
Reference
Cytology and/or endoscopic biopsy positive
19/49
38.8 (25.5-53.8)
.08
FISH polysomy
26/49
53.1 (38.4-67.2)
.008
FISH polysomy or trisomy with 9p21 loss
27/49
55.1 (40.3-69.1)
.002
Combined FISH and/or positive cytology
28/49
57.1 (42.3-70.9)
.0005
Combined FISH and/or positive cytology and/or endoscopic biopsy
30/49
61.2 (46.2-74.5)
.0002
E. Clinical diagnosis
F. Pathological diagnosis
CI, Confidence interval; FISH, fluorescence in situ hybridization.
obtained from distal biliary strictures, ie, 57% versus 52% for proximal versus distal strictures, respectively (P Z .61) (Fig. 2A).
Performance of FISH and cytology for the diagnosis of different types of cancers Patients with a diagnosis of malignancy were classified into 3 groups, depending on cancer type: CCA (n Z 58), pancreatic cancer (n Z 15), or other malignancies (n Z 20). For the diagnosis of CCA, 30 CCA patients (52%) had positive FISH results and 23 (40%) had positive cytology results. Thus, FISH had a better sensitivity than cytology; however, the statistical difference was borderline (52% vs 40%, P Z .05) (Fig. 2B). For the diagnosis of pancreatic cancer, 8 patients were positive for FISH and 1232 GASTROINTESTINAL ENDOSCOPY Volume 83, No. 6 : 2016
5 were positive for cytology. Consequently, FISH had a substantially higher sensitivity than cytology for the diagnosis of pancreatic cancer, but the difference was not statistically significant due to the small number of patients (53% vs 33%, P Z .18). For the diagnosis of other malignancies, FISH results were positive in 13 patients, but only 7 had positive cytology results. FISH, therefore, had a significantly better sensitivity than cytology for the diagnosis of nonpancreatobiliary malignancies (65% vs 35%, P Z .01).
Signal patterns of the FISH assay The total number of brushings in this study with aneuploidy (ie, changes in number of chromosome copies in a cell) detected by FISH was 53. Of the 58 CCAs, www.giejournal.org
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Sensitivity (%) 100
80
60
40
57% 60%
Cytology
64% 64% 52% 54% 54%
Cytology/Biopsy
59%
FISH FISH/Trisomy or 9p21 loss FISH/Cytology
45% 45% 37% 30%
FISH/Cytology/Biopsy
20
0 Proximal (n = 47)
A
Distal (n = 46)
Sensitivity (%) 100
80
P = .01
P = .05 P = .02
60
P = .05 52% 53%
40
P = .01 57% 59%
P = .18
70% 65%65% 65%
60% 60%60%
53% 45%
40% 41%
35%
33%33%
20
0
B
Cholangiocarcinoma (n = 58)
Pancreatic cancer (n = 15)
Other cancers (n = 20)
Figure 2. Sensitivity of fluorescence in situ hybridization (FISH), cytology, and biopsy for the diagnosis of malignant biliary stricture by location of stricture and types of cancers.
31 (53%) had aneuploidy and 27 (47%) did not have aneuploidy. Of the 15 pancreatic cancers, 9 (60%) had aneuploidy and 6 (40%) did not have aneuploidy, and of the 7 ampullary cancers, 5 (71%) had aneuploidy and 2 (29%) did not have aneuploidy. All 6 gallbladder cancers and 2 hepatocellular carcinomas had aneuploidy. The rest of the cancers, including 2 mixed hepatocellular carcinoma-CCAs, 2 cancers of unknown primary origin, and 1 lung cancer, did not have aneuploidy. The vast majority of brushings with aneuploidy were polysomic (51/53, 96%). In addition to polysomic cells, 2 of these samples also contained a clone of cells showing www.giejournal.org
trisomy 7 but were classified as polysomy for statistical analysis because polysomy is the more advanced alteration. There were 2 specimens with abnormal FISH signal patterns other than multiple gains (ie, polysomy), which were classified as negative for statistical analysis. One specimen from a patient with pancreatic cancer contained cells at a frequency of 5% that displayed both trisomy 3 and 9p21 loss. Another brushing from a patient with CCA contained cells that displayed both trisomy 17 and 9p21 loss at a frequency of 40%. Interestingly, these abnormal FISH signal patterns are not commonly seen in patients evaluated at Mayo Clinic. There are no validated cutoff values Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1233
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in clinical practice for these abnormalities, but if trisomy with 9p21 loss was considered a positive result, then the sensitivity of FISH increased slightly to 57%. The most common nonpolysomic FISH abnormalities seen in the Mayo cohort are trisomy and tetrasomy. There were no Thai brushings classified as trisomy or tetrasomy, likely due to patient population dynamics (ie, high cancer prevalence with few inflammatory strictures in the Thai cohort).
Chaiteerakij et al
Making a definitive diagnosis of malignancy in patients with biliary strictures remains challenging. Despite advances in endoscopic techniques for obtaining samples from the biliary tract, the sensitivity of conventional cytology has not improved significantly. The application of ancillary diagnostic methods that can enhance the performance of cytology is therefore critically important. In this prospective multicenter study we found that FISH performed significantly better than conventional brush cytology for the diagnosis of malignant biliary strictures in biliary brushings obtained from Thai patients. These results suggest that FISH can be used in conjunction with forceps biopsy and brush cytology for the evaluation of biliary strictures in Asian patients. We found that FISH was significantly more sensitive than brush cytology (55% vs 38%). The specificity was 100% for both tests, albeit in the context of a very small number of true negative samples, making the specificity estimate less reliable. Interestingly, the overall sensitivity of polysomy FISH in this study was better than that previously reported from Western populations.2-5,7,17 Studies conducted in the United States observed an overall diagnostic sensitivity of polysomy FISH of 43% with a specificity of 99.6%.3 In 1 U.S. study, the sensitivity in non-PSC patients ranged between 31% and 48% with a specificity of 100%.2 A study of 81 patients conducted in Poland, among whom 2 patients had underlying PSC, reported that polysomy of any chromosome or trisomy of chromosome 3 or 7 had a sensitivity of 43% for the diagnosis of malignant pancreatobiliary stricture from bile duct brushing samples.7 Very recently, a study from Switzerland showed that FISH had a diagnostic sensitivity of 61% in patients with inconclusive cytology.17 Notably, the criteria for FISH polysomy in the last study were slightly different from those applied in other studies, ie, FISH was considered as positive if polysomy of chromosome 3, 7, or 17 was present in at least 4 cells or if deletion of 9p21 was present in at least 12 cells. The higher sensitivity reported by the last study may therefore be due to the different criteria for positive FISH. The main strengths of this study are as follows: (1) it is the first study evaluating the diagnostic performance of
FISH in the detection of malignant biliary strictures in brushing samples obtained from Asians and (2) the study was conducted in a prospective fashion at 2 hospitals. As a result, for patients with a clinical context suggestive of CCA, eg, older patients with a malignant-appearing biliary stricture and elevated CA19-9, but who are negative for malignancy by histology of endoscopic biopsy or conventional cytology, we now consider FISH polysomy as sufficient evidence of malignancy to justify treatment with chemotherapy or radiation therapy at our institutions. The main limitation of this study was the lack of ability to address specificity and negative predictive values due to the small number of patients with a definitive diagnosis of benign biliary stricture (n Z 6). The main purpose of this study was to determine the performance of FISH for the diagnosis of biliary tract malignancy. Patient enrollment was restricted to patients with a suspicion of malignancy. Patients with known benign strictures without any clinical suspicion of malignancy were not included in the study. Thus, the number of patients with benign biliary strictures in our cohort was small. Additionally, PSC, which induces benign strictures with malignant potential and is a common indication for ERCP in Western countries, is a very rare disease in the Thai population. In our patient cohort, none had PSC. Nonetheless, we succeeded in our aim to determine whether FISH performs better than routine cytology for the diagnosis of malignant biliary stricture in Asia. The assessment of the sensitivity of the test is more important than establishing its specificity, which has been rigorously determined in multiple previous studies.2-5,7,17 It is important to note that the reported specificity of FISH polysomy varies from 89% to 100%, depending on the study cohorts and institutions where FISH testing was performed.2-7 The specificity of FISH for the diagnosis of malignancy in biliary strictures was first investigated in 2004 and initially reported as 91%.4 With increasing experience in performing FISH over time, the specificity of FISH has been consistently reported as 98% to 100% since 2006.2,3,5,6 Interestingly, a recent study investigating the performance of the FISH test by using pancreatic duct brushing specimens showed a specificity of 89%.7 In this study, most patients with malignancy had pancreatic cancer, accounting for 57% of the patients with malignancy, whereas in the previous studies in which FISH achieved a specificity of 98% to 100%, the majority of patients were diagnosed with CCA, and patients with pancreatic cancer accounted for only 16% to 32% of the cohorts. The relatively low specificity in this last study could therefore be partially explained by the difference in study population. It is also worth mentioning that the sensitivity of FISH in the Thai cohort, in which the incidence of CCA is very high, was greater than that in the U.S. cohort, ie, 38% versus 15% to 20%.2-5 The higher sensitivity observed in the Thai samples is likely due to differences in the patient populations and variation in the pretest probability of malignancy.
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DISCUSSION
Chaiteerakij et al
Patients in this study typically presented at a more advanced stage than patients in the United States or Europe, resulting in larger tumor size for greater sample success and a higher likelihood of accumulated genetic alterations required to achieve a positive FISH test result. PSC is a known risk factor for CCA in patients of European heritage,18 and although the benefit of surveillance on survival has not yet been proved,19 such patients are often recommended to undergo surveillance for CCA.19 Therefore, PSC patients with CCA are more likely to have earlier stage tumors, which are more difficult to sample and diagnose, compared with non-PSC patients. A recent Italian study evaluated a very similar patient population of non-PSC patients with an 83% cancer prevalence and reported test performance attributes similar to those in the current study. The sensitivity of positive routine cytology and positive UroVysion FISH were 42% and 70%, respectively.20 Risk factors for the development of CCA and genetic signatures of tumor cells at the nucleotide level differ among ethnic and geographic patient cohorts. Recently, unique mutational patterns were identified in liver fluke–related CCA.12 Regardless of the differences in frequencies of mutated genes, chromosomal instability and aneuploidy remain characteristics of malignant cells. Therefore, it is conceivable that FISH would be diagnostically useful in an Asian population, and indeed our results support its clinical utility in Thailand. Although UroVysion FISH has consistently been shown to have a higher sensitivity than conventional cytology, a higher detection rate remains desirable. The UroVysion probe set was originally developed for the diagnosis of urothelial cancer. It is possible that a probe set designed and optimized specifically for pancreatobiliary malignancies could achieve a higher sensitivity, and such an effort is currently under way.21 Because there are differences in the genetic profiles of PSC-related CCA and liver fluke–associated CCA, another important future project is the development of a novel FISH test based on samples from Thailand. In conclusion, our study confirmed the value of FISH in diagnosing malignancy in patients presenting with biliary tract strictures in Thailand. These findings suggest that FISH can be used as a supplementary tool to bile duct biopsy and brushing in actual practice in Thailand for the evaluation of biliary strictures. Further study is required in Asian patients with benign biliary strictures to confirm the high specificity of a positive FISH result, as reported in other patient populations.
ACKNOWLEDGMENT The authors would like to thank Charlie DeReuil and Cook Endoscopy for supplying the cytology brushes used in this study.
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REFERENCES 1. Yoon WJ, Brugge WR. Endoscopic evaluation of bile duct strictures. Gastrointest Endosc Clin N Am 2013;23:277-93. 2. Moreno Luna LE, Kipp B, Halling KC, et al. Advanced cytologic techniques for the detection of malignant pancreatobiliary strictures. Gastroenterology 2006;131:1064-72. 3. Fritcher EG, Kipp BR, Halling KC, et al. A multivariable model using advanced cytologic methods for the evaluation of indeterminate pancreatobiliary strictures. Gastroenterology 2009;136:2180-6. 4. Kipp BR, Stadheim LM, Halling SA, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am J Gastroenterol 2004;99: 1675-81. 5. Barr Fritcher EG, Kipp BR, Slezak JM, et al. Correlating routine cytology, quantitative nuclear morphometry by digital image analysis, and genetic alterations by fluorescence in situ hybridization to assess the sensitivity of cytology for detecting pancreatobiliary tract malignancy. Am J Clin Pathol 2007;128:272-9. 6. Gonda TA, Glick MP, Sethi A, et al. Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures. Gastrointest Endosc 2012;75:74-9. 7. Smoczynski M, Jablonska A, Matyskiel A, et al. Routine brush cytology and fluorescence in situ hybridization for assessment of pancreatobiliary strictures. Gastrointest Endosc 2012;75:65-73. 8. Burnett AS, Calvert TJ, Chokshi RJ. Sensitivity of endoscopic retrograde cholangiopancreatography standard cytology: 10-y review of the literature. J Surg Res 2013;184:304-11. 9. Harewood GC, Baron TH, Stadheim LM, et al. Prospective, blinded assessment of factors influencing the accuracy of biliary cytology interpretation. Am J Gastroenterol 2004;99:1464-9. 10. Kocjan G, Smith AN. Bile duct brushings cytology: potential pitfalls in diagnosis. Diagn Cytopathol 1997;16:358-63. 11. Sokolova IA, Halling KC, Jenkins RB, et al. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine. J Mol Diagn 2000;2: 116-23. 12. Chan-On W, Nairismagi ML, Ong CK, et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infectionrelated bile duct cancers. Nat Genet 2013;45:1474-8. 13. Heimbach JK, Gores GJ, Haddock MG, et al. Predictors of disease recurrence following neoadjuvant chemoradiotherapy and liver transplantation for unresectable perihilar cholangiocarcinoma. Transplantation 2006;82:1703-7. 14. Gores GJ, Darwish Murad S, Heimbach JK, et al. Liver transplantation for perihilar cholangiocarcinoma. Dig Dis 2013;31:126-9. 15. Renshaw AA, Madge R, Jiroutek M, et al. Bile duct brushing cytology: statistical analysis of proposed diagnostic criteria. Am J Clin Pathol 1998;110:635-40. 16. Scudera PL, Koizumi J, Jacobson IM. Brush cytology evaluation of lesions encountered during ERCP. Gastrointest Endosc 1990;36:281-4. 17. Vlajnic T, Somaini G, Savic S, et al. Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology. Cancer Cytopathol 2014;122:627-34. 18. Tyson GL, El-Serag HB. Risk factors for cholangiocarcinoma. Hepatology 2011;54:173-84. 19. Rizvi S, Eaton JE, Gores GJ. Primary sclerosing cholangitis as a premalignant biliary tract disease: surveillance and management. Clin Gastroenterol Hepatol 2015;13:2152-65. 20. Boldorini R, Paganotti A, Andorno S, et al. A multistep cytological approach for patients with jaundice and biliary strictures of indeterminate origin. J Clin Pathol 2015;68:283-7. 21. Barr Fritcher EG, Voss JS, Brankley SM, et al. An optimized set of fluorescence in situ hybridization probes for detection of pancreatobiliary tract cancer in cytology brush samples. Gastroenterol 2015; 149:1813-24.
Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1235
Fluorescence in situ hybridization compared with conventional cytology for the diagnosis of malignant biliary tract strictures in Asian patients Roongruedee Chaiteerakij, MD, PhD,1,3 Emily G. Barr Fritcher, CT (ASCP),2 Phonthep Angsuwatcharakon, MD,3 Wiriyaporn Ridtitid, MD,3 Supakarn Chaithongrat, BSc,3 Apinya Leerapun, MD,4 Todd H. Baron, MD,1,5 Benjamin R. Kipp, PhD,2 Michael R. Henry, MD,2 Kevin C. Halling, MD, PhD,2 Rungsun Rerknimitr, MD,3,* Lewis R. Roberts, MBChB, PhD1,* Rochester, Minnesota, USA; Bangkok, Chiang Mai, Thailand; Chapel Hill, North Carolina, USA
Background and Aims: Fluorescence in situ hybridization (FISH) has improved the diagnostic performance of cytology for the evaluation of malignant biliary strictures in the United States and Europe. The utility of FISH for the diagnosis of biliary strictures in Asia is currently unknown. We aimed to compare the sensitivity of FISH and conventional cytology for the diagnosis of malignant biliary strictures in Thai patients. Methods: A prospective study was performed at 2 university hospitals between 2010 and 2013. Patients being evaluated for malignant-appearing biliary strictures were included (N Z 99). Bile duct brushings were collected and assessed by cytology and FISH. Sensitivities with 95% confidence intervals of cytology and FISH were the main outcome measures. Results: The overall sensitivities of cytology and FISH were 38% and 55%, respectively (P Z .001). For those with a diagnosis of cancer based on clinical evidence without biopsy confirmation (n Z 44), the sensitivities of cytology and FISH were 43% and 57%, respectively (P Z .06). For the 49 patients for whom a cancer diagnosis was confirmed by pathology, FISH had a significantly higher sensitivity than cytology, with a sensitivity of 53% versus 33%, respectively (P Z .008). Conclusions: FISH improves the diagnostic performance of cytology and can be used as a complementary tool to bile duct brushing and biopsy for the evaluation of malignancy in biliary strictures in Asian populations. (Gastrointest Endosc 2016;83:1228-35.)
Biliary tract strictures can be due to benign or malignant diseases. Differentiation of malignant from benign biliary strictures is crucial for appropriate management. However,
this is not always easily achieved because the presentations may be similar. Histopathology is the gold standard for the diagnosis of biliary malignancy, and tissue sampling by
Abbreviations: CCA, cholangiocarcinoma; CEA, carcinoembryonic antigen; FISH, fluorescence in situ hybridization; PSC, primary sclerosing cholangitis.
Current affiliations: Division of Gastroenterology and Hepatology (1), Department of Laboratory Medicine and Pathology (2), Mayo Clinic College of Medicine, and Mayo Clinic Cancer Center, Rochester, Minnesota, USA, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok (3), Thailand, Department of Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai (4), Thailand, Division of Gastroenterology and Hepatology, University of North Carolina, Chapel Hill, North Carolina, USA (5).
DISCLOSURE: Dr Halling and Mayo Clinic receive royalties from the sale of the UroVysion FISH probe set. All other authors disclosed no financial relationships relevant this publication. This work was supported by grant CA165076 from the National Institutes of Health; the Mayo Clinic Center for Cell Signaling in Gastroenterology (NIDDK P30DK084567); the Mayo Clinic Cancer Center (CA15083), and the Mayo Foundation (to L.R.R.); and the Mayo Clinic Center for Clinical and Translational Science (NCATS UL1 TR000135). See CME section; p. 1258. *Authors Rerknimitr and Roberts contributed equally to the article. Copyright ª 2016 by the American Society for Gastrointestinal Endoscopy 0016-5107/$36.00 http://dx.doi.org/10.1016/j.gie.2015.11.037
Reprint requests: Rungsun Rerknimitr, MD, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok, Thailand or Lewis R. Roberts, MBChB, PhD, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. If you would like to chat with an author of this article, you may contact Dr Rerknimitr at [email protected] or Dr Roberts at [email protected].
Received March 23, 2015. Accepted November 25, 2015.
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endobiliary forceps biopsy and/or bile-duct brush cytology performed during ERCP are commonly used.1 Although brush cytology yields a high specificity of 90% to 100%, its sensitivity remains unsatistifactory2-7 and varies substantially, depending on biliary tract location (proximal vs distal biliary stricture), patient population (eg, primary sclerosing cholangitis [PSC] vs non-PSC), and criteria used for positivity.2-7 The sensitivity of brush cytology was reported as 15% to 20% in 6 studies comparing the performance of FISH with that of routine cytology.2-7 By comparison, in a systematic review of 16 studies of the utility of brush cytology, the pooled sensitivity of brush cytology for the detection of malignant biliary strictures was reported as 40%.8 The low sensitivity of brush cytology stems from inadequate tumor cellularity and sampling difficulties due to the desmoplastic nature of pancreatobiliary malignancies as well as the technical challenge of obtaining samples from difficult-to-access stricture locations. Additionally, the interpretation of histologic findings is sometimes difficult because of inflammation, fibrosis and necrosis, small tissue fragments, and the challenge of distinguishing cytologic atypia from invasive malignancy.9,10 Advanced cytologic methods have been developed to enhance the limited sensitivity of forceps biopsy and brush cytology. Among these methods, the fluorescence in situ hybridization (FISH) assay (Multiprobe UroVysion; Abbott Molecular, Inc, Des Plaines, Ill) has improved the performance of brush cytology.2-4,6 Briefly, the FISH assay uses 4 fluorescent-labeled probes that hybridize directly to the pericentromeric regions of chromosomes 3, 7, and 17 and the chromosome 9p21 (CDKN2A) locus. Application of the assay to cytology brushings allows determination of the number of copies of chromosomes 3, 7, and 17 and of the 9p21 locus that are present in individual cells by using different fluorescence colors for each probe. It has been shown that the presence of 5 or more cells with polysomy (ie, >2 copies) of 2 or more chromosomes or loci is diagnostic of malignancy.2-4 The UroVysion FISH assay has been shown to substantially improve the performance of brush cytology for diagnosis of malignant biliary strictures.2-4,7,11 The diagnostic sensitivity of FISH has been reported as 34% to 58%, with 91% to 100% specificity, compared with a sensitivity of 15% to 21% for conventional cytology in the same patient cohorts.2,4,6 Accordingly, UroVysion FISH is now widely used in the United States as an ancillary diagnostic tool for evaluating biliary strictures. FISH was recently shown to increase the sensitivity of routine brush cytology from 35% to 52% for the diagnosis of malignancy in pancreatobiliary strictures in European populations.7 Whether FISH, which is clinically useful for the assessment of biliary strictures in Western populations, is applicable to Asian populations is currently unknown. There are substantial differences in the etiology of malignant biliary tract diseases, particularly cholangiocarcinoma (CCA), between Western and Asian populations. Liver fluke infestation is the main cause of CCA in Asians, whereas PSC, noninfectious biliary tract www.giejournal.org
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disease, and other unknown risk factors are the main causes of CCA in Western populations. It has been shown that the spectrum of genetic alterations in liver fluke– associated CCA is different from the alterations found in non–liver fluke–associated CCA.12 TP53, SMAD4, MLL3, and GNAS were more frequently mutated in liver fluke– associated CCA, whereas BAP1 and IDH1 or IDH2 were more frequently mutated in non–liver fluke–associated CCA.12 It is therefore important to determine whether the FISH assay, which was originally developed by using samples from Western populations, can be applied to Asians. The primary goal of this study was to determine the utility of FISH for the detection of malignancy in biliary tract strictures in an Asian population. We conducted a prospective study to evaluate whether FISH improves the performance of conventional cytology for the diagnosis of malignancy in biliary strictures at 2 academic centers in Thailand.
METHODS Patient population This study was approved by the Institutional Review Boards at the Mayo Clinic (Rochester, Minn), Chulalongkorn University (Bangkok, Thailand), and Chiang Mai University (Chiang Mai, Thailand). Consecutive patients evaluated for malignant-appearing biliary tract strictures who underwent ERCP between March 2010 and December 2013 at Chulalongkorn University and Chiang Mai University were prospectively enrolled in the study. Patients for whom alternate diagnostic tests including EUS-FNA were performed were also included in this study. Patients were followed postprocedurally by outpatient visits or by telephone contact to ascertain the final diagnosis, vital status, and cause of death. The diagnosis of malignancy was confirmed by histopathologic examination of surgically resected specimens or biopsy specimens (either open or laparoscopic surgical, percutaneous needle, or endoscopic forceps biopsy); or by clinical criteria, a combination of symptoms (jaundice and/or weight loss [n Z 32]), evidence of a stricture and/or mass on radiologic imaging in the absence of acute cholangitis (n Z 44), serum CA19-9 level higher than 100 U/mL (n Z 33), or clinical or radiologic progression (40 patients had clinical progression, 2 had radiologic progression, and 22 had both clinical and radiologic progression) during a mean ( SD) followup time of 6.4 (7.2) months. The presence of a malignantappearing biliary tract stricture was defined as long (>2 cm), asymmetrical, irregular, or abrupt narrowing of the biliary tract.5,13,14 Telephone contact was made 1 year after ERCP in all patients to confirm the progression of malignancy or death from malignancy. Physicians who determined the final diagnosis were blinded to the results of FISH.
Specimen collection and processing Cytologic specimens were obtained from biliary stricture sites during ERCP by using a standard cytology brush Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1229
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(Cook Medical, Winston-Salem, NC). The brush was passed through the stricture site for at least 5 to-and-fro motions and then stirred in PreservCyt preservative solution (Hologic, Inc, Marlborough, Mass) to release the cells. The specimens were kept at room temperature until shipment to the Cytopathology Department at the Mayo Clinic, where FISH and cytology examinations were performed. Once the specimens arrived, each was divided equally for FISH analysis and cytology examination. PreservCyt solution has a shelf life of 2 years at room temperature, and all specimens were shipped within 1 to 2 months after collection.
FISH assay Half of each specimen was processed on a FISH slide by using a standard protocol, as previously described.4,14 FISH was performed by using the fluorescently labeled Vysis UroVysion probe set (Abbott Molecular). An experienced cytotechnologist who was blinded to the clinical information and corresponding cytology results evaluated signal patterns of the FISH slides by fluorescence microscopy. Specimens were considered positive if polysomy was identified in 5 or more cells. Polysomy was defined as gain of 2 or more copies for 2 or more probes within a cell.2 Images of FISH results are shown in Figure 1.
Conventional cytology The other half of each specimen was processed on ThinPrep slides (Hologic, Inc) and Pap stained. All slides were screened by 2 cytotechnologists and reviewed by an experienced cytopathologist who was blinded to the clinical information and the FISH results. The cytology results were classified by the cytopathologist as positive, suspicious, atypical, or negative for malignancy by using previously published standard criteria.15,16 Only the positive classification was called as positive for cytology.
Other data collection Patient medical records were reviewed to obtain baseline patient characteristics, clinical information at the time of diagnosis and at return visits, and the results of radiologic imaging at the time of the ERCP procedure and during follow-up. Levels of CA19-9 and carcinoembryonic antigen (CEA) measured within 7 days of the date of ERCP procedure were abstracted.
Statistical analysis Of 101 patients enrolled in this study, FISH diagnosis and/or conventional cytology diagnosis was not feasible due to inadequate cellularity in brushing samples for 7 patients (4 for FISH and 3 for cytology). For the remaining 94 patients, both FISH analysis and cytology examination were performed. Two patients lost to follow-up were excluded from analysis due to inadequate information for making a definite diagnosis. A total of 92 patients who had both FISH and cytology results and an adequate follow-up period were included in the initial analysis. 1230 GASTROINTESTINAL ENDOSCOPY Volume 83, No. 6 : 2016
Figure 1. Malignant cell showing polysomy (>2 copies of at least 2 chromosomal loci) by using fluorescence in situ hybridization probes to CEP 3 (red), CEP 7 (green), CEP 17 (aqua), and the 9p21 locus of chromosome 9 (gold). CEP, chromosome enumeration probe.
Because patients with inadequate cellularity in brushing samples may represent important cases in which too few cells are obtained for diagnosis and because this is one of the greatest challenges in this disease, we included the 7 patients who had inadequate cellularity in their brush samples in the final analysis. The samples with inadequate cellularity were coded as negative for FISH or cytology in the final analysis so that the estimated sensitivity and specificity were more reflective of the sensitivity and specificity that would be obtained when the test is implemented in real-life practice. The final analysis therefore comprised 99 patients (93 with malignancy and 6 with benign biliary strictures). Sensitivity and specificity along with the corresponding 95% confidence interval of FISH and cytology were estimated and compared by using the McNemar test or the c2 test as appropriate. Levels of CA19-9 and CEA in patients with malignancy versus benign biliary strictures were compared by using the Mann-Whitney U test. All analyses were performed by using JMP version 10.0 (SAS Institute Inc, Cary, NC). A P value <.05 was considered significant.
RESULTS Baseline characteristics Of the 99 patients, 46 (46%) were male with a mean age ( SD) of 64.9 13.1 years. None of the patients had underlying PSC or primary stone-induced stenoses. Two patients had a history of recurrent pyogenic cholangitis; 1 had a final diagnosis of CCA and the other had a final diagnosis of benign biliary stricture due to surgery. Ninety-three of the 99 patients (94%) were diagnosed with malignancy and 74 of 93 (80%) had an associated www.giejournal.org
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TABLE 1. Baseline characteristics of study patients
Characteristics
All subjects, no. (%)
Cancers diagnosed by clinical criteria, no. (%)
Cancers diagnosed by pathology, no. (%)
99
44
49
Total number Male Age, y, mean SD
46 (46)
25
21
64.9 13.1
66.3 13.9
63.6 12.3
Diagnosis Cancer
93/99 (94)
44/93 (47)
49/93 (53)
Cholangiocarcinoma
58 (62)
34 (77)
24 (49)
Pancreatic cancer
15 (16)
4 (9)
11 (22)
Ampullary cancer
7 (8)
1 (2)
6 (13)
Gallbladder cancer
6 (7)
1 (2)
5 (10)
Hepatocellular carcinoma
2 (2)
2 (5)
0 (0)
Mixed hepatocellular cholangiocarcinoma
2 (2)
1 (2)
1 (2)
Cancer of unknown origin
2 (2)
1 (2)
1 (2)
Lung cancer
1 (1)
0 (0)
1 (2)
Benign biliary stricture
6/99 (6)
mass. The most common cancers were CCA (n Z 58, 62%), followed by pancreatic cancer (n Z 15, 16%), and ampullary cancer (n Z 7, 8%) (Table 1). The final diagnosis of malignancy was confirmed histopathologically in 49 patients; 44 patients had a diagnosis of malignancy by using clinical criteria. The remaining 6 patients who had clinical presentation and radiologic findings suspicious for malignancy at the time of ERCP but did not have evidence of malignancy during 1 year of follow-up received a diagnosis of benign biliary strictures (1 postsurgical biliary stricture, 1 chronic pancreatitis, 1 common bile duct stone, 1 choledochal cyst, and 2 of undetermined cause). The median (interquartile range) levels of serum CA19-9 and CEA for patients with malignancy versus those without malignancy were 487.9 (135.8-1000) versus 32.0 (17.1-45.0) U/mL for CA19-9 (P Z .006) and 6.7 (3.4-25.9) versus 2.4 (2.1-3.0) ng/mL for CEA (P Z .03). The indications for ECRP were obstructive jaundice (94/99, 95%) and cholangitis (5/99, 5%). Ninety-three of the 99 patients (94%) had primary ERCP, and 6 (6%) were previously stented. EUS-FNA was performed in 16 patients (17%) who had a diagnosis of malignancy.
Sensitivity and specificity of FISH versus conventional cytology for diagnosis of malignant biliary strictures The diagnostic performance of FISH and conventional cytology are shown in Table 2. Polysomy was detected by FISH in 51 of 93 patients (55%) with malignancy, whereas positive cytology was observed in only 35 of the 93 patients (38%). Therefore, FISH had significantly better sensitivity than cytology for the diagnosis of malignancy by using biliary brushing specimens (55% vs www.giejournal.org
38%, P Z .001). The specificity was 100% for both FISH polysomy and positive cytology. When FISH and cytology were considered together (ie, either test was positive), the sensitivity for diagnosis of malignancy compared with FISH alone increased slightly to 59%. Of 58 patients with a diagnosis of malignancy who had negative (n Z 20, 34.5%), atypical (n Z 20, 34.5%), or suspicious (n Z 18, 31.0%) cytology results, polysomy was observed with the FISH assay in 20 patients, yielding a sensitivity of 34.5%. Endoscopic biopsies were performed on 13 of the 51 patients (25%) with positive FISH test results. Of the 13 patients undergoing endoscopic biopsy, specimens positive for malignancy were obtained in 6 patients, 1 specimen was suspicious for malignancy, specimens from 3 patients showed high-grade dysplasia, 2 patients had inadequate specimens, and 1 patient had a negative biopsy result. Thus, the sensitivity and specificity of endoscopic biopsy in those with available results were 46% and 100%, respectively. For patients with a clinical diagnosis of malignancy, 25 of 44 had positive results on FISH, whereas 19 of 44 had positive cytology results, resulting in a higher sensitivity with FISH than with cytology (56.8% vs 43.2%, P Z .06). Of the 49 patients who had pathological confirmation of a cancer diagnosis, 16 were positive by cytology, 26 were positive by FISH, and 28 were positive by cytology and/or FISH. The associated sensitivities were 32.7% (16/49) for cytology alone, 53.1% (26/49) for FISH alone, and 57.1% (28/49) for the combination of cytology and FISH, respectively. When classified by location of biliary strictures, the sensitivity of FISH in samples obtained from proximal biliary strictures was not different from that in samples Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1231
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TABLE 2. Performance of FISH and conventional cytology for detection of malignant biliary stricture Proportion of positive tests in patients with a diagnosis of cancer
Sensitivity, % (95% CI)
P value
A. Cytology negative FISH polysomy
2/20
10.0 (1.8-33.1)
FISH polysomy or trisomy with 9p21 loss
2/20
10.0 (1.8-33.1)
B. Cytology negative or atypical FISH polysomy
8/40
20.0 (9.6-36.1)
FISH polysomy or trisomy with 9p21 loss
8/40
20.0 (9.6-36.1)
FISH polysomy
20/58
34.5 (22.8-48.2)
FISH polysomy or trisomy with 9p21 loss
20/58
34.5 (22.8-48.2)
C. Cytology negative, atypical or suspicious
D. Entire cohort Cytology positive
35/93
37.6 (28.0-48.3)
Cytology and/or endoscopic biopsy positive
38/93
40.9 (30.9-51.6)
Reference .08
FISH polysomy
51/93
54.8 (44.2-65.1)
.001
FISH polysomy or trisomy with 9p21 loss
53/93
57.0 (46.3-67.1)
.0001
Combined FISH and/or positive cytology
55/93
59.1 (48.4-69.1)
<.0001
Combined FISH and/or positive cytology and/or endoscopic biopsy
57/93
61.3 (50.6-71.0)
<.0001
Cytology positive
19/44
43.2 (28.7-58.9)
Reference
Cytology and/or endoscopic biopsy positive
19/44
43.2 (28.7-58.9)
1.0000
FISH polysomy
25/44
56.8 (41.1-71.3)
.06
FISH polysomy or trisomy with 9p21 loss
26/44
59.1 (43.3-73.3)
.02
Combined FISH and/or positive cytology
27/44
61.4 (45.5-75.3)
.005
Combined FISH and/or positive cytology and/or endoscopic biopsy
27/44
61.4 (45.5-75.3)
.005
Cytology positive
16/49
32.7 (20.4-47.7)
Reference
Cytology and/or endoscopic biopsy positive
19/49
38.8 (25.5-53.8)
.08
FISH polysomy
26/49
53.1 (38.4-67.2)
.008
FISH polysomy or trisomy with 9p21 loss
27/49
55.1 (40.3-69.1)
.002
Combined FISH and/or positive cytology
28/49
57.1 (42.3-70.9)
.0005
Combined FISH and/or positive cytology and/or endoscopic biopsy
30/49
61.2 (46.2-74.5)
.0002
E. Clinical diagnosis
F. Pathological diagnosis
CI, Confidence interval; FISH, fluorescence in situ hybridization.
obtained from distal biliary strictures, ie, 57% versus 52% for proximal versus distal strictures, respectively (P Z .61) (Fig. 2A).
Performance of FISH and cytology for the diagnosis of different types of cancers Patients with a diagnosis of malignancy were classified into 3 groups, depending on cancer type: CCA (n Z 58), pancreatic cancer (n Z 15), or other malignancies (n Z 20). For the diagnosis of CCA, 30 CCA patients (52%) had positive FISH results and 23 (40%) had positive cytology results. Thus, FISH had a better sensitivity than cytology; however, the statistical difference was borderline (52% vs 40%, P Z .05) (Fig. 2B). For the diagnosis of pancreatic cancer, 8 patients were positive for FISH and 1232 GASTROINTESTINAL ENDOSCOPY Volume 83, No. 6 : 2016
5 were positive for cytology. Consequently, FISH had a substantially higher sensitivity than cytology for the diagnosis of pancreatic cancer, but the difference was not statistically significant due to the small number of patients (53% vs 33%, P Z .18). For the diagnosis of other malignancies, FISH results were positive in 13 patients, but only 7 had positive cytology results. FISH, therefore, had a significantly better sensitivity than cytology for the diagnosis of nonpancreatobiliary malignancies (65% vs 35%, P Z .01).
Signal patterns of the FISH assay The total number of brushings in this study with aneuploidy (ie, changes in number of chromosome copies in a cell) detected by FISH was 53. Of the 58 CCAs, www.giejournal.org
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Sensitivity (%) 100
80
60
40
57% 60%
Cytology
64% 64% 52% 54% 54%
Cytology/Biopsy
59%
FISH FISH/Trisomy or 9p21 loss FISH/Cytology
45% 45% 37% 30%
FISH/Cytology/Biopsy
20
0 Proximal (n = 47)
A
Distal (n = 46)
Sensitivity (%) 100
80
P = .01
P = .05 P = .02
60
P = .05 52% 53%
40
P = .01 57% 59%
P = .18
70% 65%65% 65%
60% 60%60%
53% 45%
40% 41%
35%
33%33%
20
0
B
Cholangiocarcinoma (n = 58)
Pancreatic cancer (n = 15)
Other cancers (n = 20)
Figure 2. Sensitivity of fluorescence in situ hybridization (FISH), cytology, and biopsy for the diagnosis of malignant biliary stricture by location of stricture and types of cancers.
31 (53%) had aneuploidy and 27 (47%) did not have aneuploidy. Of the 15 pancreatic cancers, 9 (60%) had aneuploidy and 6 (40%) did not have aneuploidy, and of the 7 ampullary cancers, 5 (71%) had aneuploidy and 2 (29%) did not have aneuploidy. All 6 gallbladder cancers and 2 hepatocellular carcinomas had aneuploidy. The rest of the cancers, including 2 mixed hepatocellular carcinoma-CCAs, 2 cancers of unknown primary origin, and 1 lung cancer, did not have aneuploidy. The vast majority of brushings with aneuploidy were polysomic (51/53, 96%). In addition to polysomic cells, 2 of these samples also contained a clone of cells showing www.giejournal.org
trisomy 7 but were classified as polysomy for statistical analysis because polysomy is the more advanced alteration. There were 2 specimens with abnormal FISH signal patterns other than multiple gains (ie, polysomy), which were classified as negative for statistical analysis. One specimen from a patient with pancreatic cancer contained cells at a frequency of 5% that displayed both trisomy 3 and 9p21 loss. Another brushing from a patient with CCA contained cells that displayed both trisomy 17 and 9p21 loss at a frequency of 40%. Interestingly, these abnormal FISH signal patterns are not commonly seen in patients evaluated at Mayo Clinic. There are no validated cutoff values Volume 83, No. 6 : 2016 GASTROINTESTINAL ENDOSCOPY 1233
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in clinical practice for these abnormalities, but if trisomy with 9p21 loss was considered a positive result, then the sensitivity of FISH increased slightly to 57%. The most common nonpolysomic FISH abnormalities seen in the Mayo cohort are trisomy and tetrasomy. There were no Thai brushings classified as trisomy or tetrasomy, likely due to patient population dynamics (ie, high cancer prevalence with few inflammatory strictures in the Thai cohort).
Chaiteerakij et al
Making a definitive diagnosis of malignancy in patients with biliary strictures remains challenging. Despite advances in endoscopic techniques for obtaining samples from the biliary tract, the sensitivity of conventional cytology has not improved significantly. The application of ancillary diagnostic methods that can enhance the performance of cytology is therefore critically important. In this prospective multicenter study we found that FISH performed significantly better than conventional brush cytology for the diagnosis of malignant biliary strictures in biliary brushings obtained from Thai patients. These results suggest that FISH can be used in conjunction with forceps biopsy and brush cytology for the evaluation of biliary strictures in Asian patients. We found that FISH was significantly more sensitive than brush cytology (55% vs 38%). The specificity was 100% for both tests, albeit in the context of a very small number of true negative samples, making the specificity estimate less reliable. Interestingly, the overall sensitivity of polysomy FISH in this study was better than that previously reported from Western populations.2-5,7,17 Studies conducted in the United States observed an overall diagnostic sensitivity of polysomy FISH of 43% with a specificity of 99.6%.3 In 1 U.S. study, the sensitivity in non-PSC patients ranged between 31% and 48% with a specificity of 100%.2 A study of 81 patients conducted in Poland, among whom 2 patients had underlying PSC, reported that polysomy of any chromosome or trisomy of chromosome 3 or 7 had a sensitivity of 43% for the diagnosis of malignant pancreatobiliary stricture from bile duct brushing samples.7 Very recently, a study from Switzerland showed that FISH had a diagnostic sensitivity of 61% in patients with inconclusive cytology.17 Notably, the criteria for FISH polysomy in the last study were slightly different from those applied in other studies, ie, FISH was considered as positive if polysomy of chromosome 3, 7, or 17 was present in at least 4 cells or if deletion of 9p21 was present in at least 12 cells. The higher sensitivity reported by the last study may therefore be due to the different criteria for positive FISH. The main strengths of this study are as follows: (1) it is the first study evaluating the diagnostic performance of
FISH in the detection of malignant biliary strictures in brushing samples obtained from Asians and (2) the study was conducted in a prospective fashion at 2 hospitals. As a result, for patients with a clinical context suggestive of CCA, eg, older patients with a malignant-appearing biliary stricture and elevated CA19-9, but who are negative for malignancy by histology of endoscopic biopsy or conventional cytology, we now consider FISH polysomy as sufficient evidence of malignancy to justify treatment with chemotherapy or radiation therapy at our institutions. The main limitation of this study was the lack of ability to address specificity and negative predictive values due to the small number of patients with a definitive diagnosis of benign biliary stricture (n Z 6). The main purpose of this study was to determine the performance of FISH for the diagnosis of biliary tract malignancy. Patient enrollment was restricted to patients with a suspicion of malignancy. Patients with known benign strictures without any clinical suspicion of malignancy were not included in the study. Thus, the number of patients with benign biliary strictures in our cohort was small. Additionally, PSC, which induces benign strictures with malignant potential and is a common indication for ERCP in Western countries, is a very rare disease in the Thai population. In our patient cohort, none had PSC. Nonetheless, we succeeded in our aim to determine whether FISH performs better than routine cytology for the diagnosis of malignant biliary stricture in Asia. The assessment of the sensitivity of the test is more important than establishing its specificity, which has been rigorously determined in multiple previous studies.2-5,7,17 It is important to note that the reported specificity of FISH polysomy varies from 89% to 100%, depending on the study cohorts and institutions where FISH testing was performed.2-7 The specificity of FISH for the diagnosis of malignancy in biliary strictures was first investigated in 2004 and initially reported as 91%.4 With increasing experience in performing FISH over time, the specificity of FISH has been consistently reported as 98% to 100% since 2006.2,3,5,6 Interestingly, a recent study investigating the performance of the FISH test by using pancreatic duct brushing specimens showed a specificity of 89%.7 In this study, most patients with malignancy had pancreatic cancer, accounting for 57% of the patients with malignancy, whereas in the previous studies in which FISH achieved a specificity of 98% to 100%, the majority of patients were diagnosed with CCA, and patients with pancreatic cancer accounted for only 16% to 32% of the cohorts. The relatively low specificity in this last study could therefore be partially explained by the difference in study population. It is also worth mentioning that the sensitivity of FISH in the Thai cohort, in which the incidence of CCA is very high, was greater than that in the U.S. cohort, ie, 38% versus 15% to 20%.2-5 The higher sensitivity observed in the Thai samples is likely due to differences in the patient populations and variation in the pretest probability of malignancy.
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DISCUSSION
Chaiteerakij et al
Patients in this study typically presented at a more advanced stage than patients in the United States or Europe, resulting in larger tumor size for greater sample success and a higher likelihood of accumulated genetic alterations required to achieve a positive FISH test result. PSC is a known risk factor for CCA in patients of European heritage,18 and although the benefit of surveillance on survival has not yet been proved,19 such patients are often recommended to undergo surveillance for CCA.19 Therefore, PSC patients with CCA are more likely to have earlier stage tumors, which are more difficult to sample and diagnose, compared with non-PSC patients. A recent Italian study evaluated a very similar patient population of non-PSC patients with an 83% cancer prevalence and reported test performance attributes similar to those in the current study. The sensitivity of positive routine cytology and positive UroVysion FISH were 42% and 70%, respectively.20 Risk factors for the development of CCA and genetic signatures of tumor cells at the nucleotide level differ among ethnic and geographic patient cohorts. Recently, unique mutational patterns were identified in liver fluke–related CCA.12 Regardless of the differences in frequencies of mutated genes, chromosomal instability and aneuploidy remain characteristics of malignant cells. Therefore, it is conceivable that FISH would be diagnostically useful in an Asian population, and indeed our results support its clinical utility in Thailand. Although UroVysion FISH has consistently been shown to have a higher sensitivity than conventional cytology, a higher detection rate remains desirable. The UroVysion probe set was originally developed for the diagnosis of urothelial cancer. It is possible that a probe set designed and optimized specifically for pancreatobiliary malignancies could achieve a higher sensitivity, and such an effort is currently under way.21 Because there are differences in the genetic profiles of PSC-related CCA and liver fluke–associated CCA, another important future project is the development of a novel FISH test based on samples from Thailand. In conclusion, our study confirmed the value of FISH in diagnosing malignancy in patients presenting with biliary tract strictures in Thailand. These findings suggest that FISH can be used as a supplementary tool to bile duct biopsy and brushing in actual practice in Thailand for the evaluation of biliary strictures. Further study is required in Asian patients with benign biliary strictures to confirm the high specificity of a positive FISH result, as reported in other patient populations.
ACKNOWLEDGMENT The authors would like to thank Charlie DeReuil and Cook Endoscopy for supplying the cytology brushes used in this study.
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FISH/cytology for pancreatobiliary malignancy
REFERENCES 1. Yoon WJ, Brugge WR. Endoscopic evaluation of bile duct strictures. Gastrointest Endosc Clin N Am 2013;23:277-93. 2. Moreno Luna LE, Kipp B, Halling KC, et al. Advanced cytologic techniques for the detection of malignant pancreatobiliary strictures. Gastroenterology 2006;131:1064-72. 3. Fritcher EG, Kipp BR, Halling KC, et al. A multivariable model using advanced cytologic methods for the evaluation of indeterminate pancreatobiliary strictures. Gastroenterology 2009;136:2180-6. 4. Kipp BR, Stadheim LM, Halling SA, et al. A comparison of routine cytology and fluorescence in situ hybridization for the detection of malignant bile duct strictures. Am J Gastroenterol 2004;99: 1675-81. 5. Barr Fritcher EG, Kipp BR, Slezak JM, et al. Correlating routine cytology, quantitative nuclear morphometry by digital image analysis, and genetic alterations by fluorescence in situ hybridization to assess the sensitivity of cytology for detecting pancreatobiliary tract malignancy. Am J Clin Pathol 2007;128:272-9. 6. Gonda TA, Glick MP, Sethi A, et al. Polysomy and p16 deletion by fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures. Gastrointest Endosc 2012;75:74-9. 7. Smoczynski M, Jablonska A, Matyskiel A, et al. Routine brush cytology and fluorescence in situ hybridization for assessment of pancreatobiliary strictures. Gastrointest Endosc 2012;75:65-73. 8. Burnett AS, Calvert TJ, Chokshi RJ. Sensitivity of endoscopic retrograde cholangiopancreatography standard cytology: 10-y review of the literature. J Surg Res 2013;184:304-11. 9. Harewood GC, Baron TH, Stadheim LM, et al. Prospective, blinded assessment of factors influencing the accuracy of biliary cytology interpretation. Am J Gastroenterol 2004;99:1464-9. 10. Kocjan G, Smith AN. Bile duct brushings cytology: potential pitfalls in diagnosis. Diagn Cytopathol 1997;16:358-63. 11. Sokolova IA, Halling KC, Jenkins RB, et al. The development of a multitarget, multicolor fluorescence in situ hybridization assay for the detection of urothelial carcinoma in urine. J Mol Diagn 2000;2: 116-23. 12. Chan-On W, Nairismagi ML, Ong CK, et al. Exome sequencing identifies distinct mutational patterns in liver fluke-related and non-infectionrelated bile duct cancers. Nat Genet 2013;45:1474-8. 13. Heimbach JK, Gores GJ, Haddock MG, et al. Predictors of disease recurrence following neoadjuvant chemoradiotherapy and liver transplantation for unresectable perihilar cholangiocarcinoma. Transplantation 2006;82:1703-7. 14. Gores GJ, Darwish Murad S, Heimbach JK, et al. Liver transplantation for perihilar cholangiocarcinoma. Dig Dis 2013;31:126-9. 15. Renshaw AA, Madge R, Jiroutek M, et al. Bile duct brushing cytology: statistical analysis of proposed diagnostic criteria. Am J Clin Pathol 1998;110:635-40. 16. Scudera PL, Koizumi J, Jacobson IM. Brush cytology evaluation of lesions encountered during ERCP. Gastrointest Endosc 1990;36:281-4. 17. Vlajnic T, Somaini G, Savic S, et al. Targeted multiprobe fluorescence in situ hybridization analysis for elucidation of inconclusive pancreatobiliary cytology. Cancer Cytopathol 2014;122:627-34. 18. Tyson GL, El-Serag HB. Risk factors for cholangiocarcinoma. Hepatology 2011;54:173-84. 19. Rizvi S, Eaton JE, Gores GJ. Primary sclerosing cholangitis as a premalignant biliary tract disease: surveillance and management. Clin Gastroenterol Hepatol 2015;13:2152-65. 20. Boldorini R, Paganotti A, Andorno S, et al. A multistep cytological approach for patients with jaundice and biliary strictures of indeterminate origin. J Clin Pathol 2015;68:283-7. 21. Barr Fritcher EG, Voss JS, Brankley SM, et al. An optimized set of fluorescence in situ hybridization probes for detection of pancreatobiliary tract cancer in cytology brush samples. Gastroenterol 2015; 149:1813-24.
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