Can DNA cytometry be used for evaluation of malignancy and premalignancy in bile duct strictures in primary sclerosing cholangitis?

doi:10.1016/S0168-8278(00)80117-8

Journal of Hepatology

Volume 33, Issue 6, December 2000, Pages 873-877

https://doi.org/10.1016/S0168-8278(00)80117-8 Get rights and content

Abstract

Background/Aims: The significance of DNA ploidy determinations for diagnosing cholangiocarcinoma (CC) in primary sclerosing cholangitis (PSC) has not previously been evaluated. Knowledge of tumour cell ploidy by DNA cytometry may facilitate the evaluation of malignant and premalignant lesions in PSC.

Methods: Twenty-eight patients with CC were studied; 10 of the patients had PSC. Seventeen samples from 15 patients with PSC but without CC were used as controls for benign strictures. Gallbladder tissue from 100 patients with chronic cholecystitis was also analysed. DNA was measured using flow cytometry on cells from paraffin-embedded tissues.

Results: Tumours from patients with PSC displayed non-tetraploid DNA aneuploidy significantly more often (80%) than tumours from patients without PSC (39%) (p<0.05). CC from patients with PSC significantly more often displayed DNA aneuploidy: 80% (8/10) compared with 12% (2/17) in bile ducts in PSC without CC (p=0.0007). The frequency of DNA aneuploidy in gallbladder tissue from patients with chronic cholecystasis was 1% (1/100).

Conclusion: The high prevalence of DNA aneuploidy in PSC-related CC and the low prevalence in benign PSC strictures point to DNA cytometry as a possible future method for detecting malignant and premalignant changes in bile duct strictures in patients with PSC. This method may be useful in selecting PSC patients for liver transplantation.

Section snippets

Patients

Forty-four CCs were identified in a register including all cancer cases diagnosed at Huddinge University Hospital between 1989 and 1997. In four patients, no paraffin-embedded material was available and 12 samples were not evaluable by flow cytometry, due to bad quality of the samples or too much background disturbance. The remaining 28 tumours were included in the present study: 19 were diagnosed at biopsy and 9 at autopsy. Ten of the patients had PSC. The diagnosis of PSC was based on typical

Results

The median age at CC diagnosis was 62 years for tumour and PSC patients (range 27–86). The cancer patients without PSC were significantly older than those with PSC, as demonstrated in Table 1. Of the patients 57% (16/28) were women, and there was no difference in sex distribution among patients with and without PSC (Table 1). The diagnosis of PSC was made before the cancer diagnosis in all PSC patients. Of the PSC patients with CC, 6/10 had cirrhosis. Eighty percent (20/25) of all PSC patients

Discussion

In this study, 54% of CCs showed non-tetraploid DNA aneuploidy. Aneuploid and tetraploid tumours together represented 64%. In patients with PSC, 80% of the tumours showed DNA aneuploidy, a figure significantly higher than in patients without PSC (39%). CC in PSC develops at least two decades earlier than CC in patients without PSC and may therefore be a separate entity different from CC without concomitant PSC. The higher rate of aneuploidy among patients with CC and PSC appears to support this

Acknowledgements

This study received grant support from Ruth and Richard Juhlin's Fund, Glaxo Wellcome AB and from the Karolinska Institute.

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Performance of fluorescence in situ hybridization in biliary brushings with equivocal cytology: an institutional experience
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Biliary brushing (BB) cytology has a sensitivity of 15%-65% and specificity approaching 100% for detecting malignancy. Fluorescence in-situ hybridization (FISH) using the UroVysion probe set has been advocated to enhance the detection of malignancies with reported sensitivity of 43%-84%. We sought to evaluate the performance of FISH in BB with equivocal cytology at our institution.
Patients with atypical and suspicious BB with concurrent diagnostic FISH performed at our institution from 2014 to 2021 were identified through a query of our pathology database. FISH (using UroVysion probe set containing centromere enumeration probes to chromosomes 3, 7, and 17) was positive if at least 5 cells demonstrated polysomy. Electronic medical records were reviewed for pathology results and outcomes. Patients were classified malignant if they had positive pathology or documented clinical impression of malignancy and benign if they had negative pathology and/or documented benign clinical course for at least 12 months.
We identified 254 equivocal BB (238 atypical/16 suspicious) with concurrent FISH results from 191 patients (105 benign, 86 malignant). 12% (22/191) of patients were FISH positive. Twenty-four percent (21/86) of patients with malignancy had positive FISH but were nonspecific for pancreaticobiliary/ampullary adenocarcinomas. Almost all positive FISH were associated with malignancy (21/22; 95%). There was 1 positive FISH in a patient with primary sclerosing cholangitis who had a benign outcome.
The small number of positive FISH results in BB with equivocal cytology raises the question of the optimal criteria for malignancy. Using only polysomy could result in lower sensitivity.
The Cytomorphologic and Molecular Assessment of Bile Duct Brushing Specimens
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Citation Excerpt :
Polysomy reflects gains of two or more of the four probes in at least five cells and signifies chromosomal instability (Fig. 8), which, in the setting of a new stricture, is considered a positive test result with a 95% specificity for malignancy.42,43 Given that only approximately 80% of all pancreaticobiliary malignancies demonstrate chromosomal instability resulting in aneuploidy, FISH analysis has inherently limited sensitivity.44 A FISH test depends the cells analyzed that requires highly skilled technicians with excellent morphologic skills.
DNA flow cytometric analysis of paraffin-embedded tissue for the diagnosis of malignancy in bile duct biopsies
2020, Human Pathology
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This is crucial because well differentiated pancreaticobiliary tumors can mimic benign epithelium and glands, especially in the superficial samples obtained by brushing. To improve the diagnostic yield of different sampling techniques, several ancillary tools have been developed and include DNA flow cytometry [5–7], digital image analysis [8–12], KRAS mutational testing [13,14], fluorescence in situ hybridization (FISH) [15–21], and more recently, next-generation sequencing [22–24]. Although these tests have been shown to improve the diagnostic sensitivity in detecting malignancy over cytology alone, they are usually performed on brushing samples and often require a separate specimen for analysis.
Differentiation of reactive versus neoplastic epithelial changes can be challenging in bile duct biopsies. The samples are often scant, distorted, and mixed with significant inflammation, ulceration, and/or debris. Histological confirmation of malignancy is often required before the initiation of surgical therapy, and an erroneous diagnosis of malignancy can lead to unnecessary clinical management. Aneuploidy assessment by DNA flow cytometry was performed on formalin-fixed paraffin-embedded (FFPE) tissue from 63 bile duct biopsies: 10 with a malignant diagnosis (7 with adenocarcinoma and 3 with at least high-grade dysplasia [HGD]); 3 with an atypical diagnosis showing rare atypical glands/cells, concerning but not definite for malignancy; 28 likely reactive biopsies with acute/chronic inflammation, ulceration, and/or mild nuclear atypia; and 22 additional benign biopsies without significant inflammation, ulceration, or nuclear atypia. Aneuploidy was detected in 7 (70%) of the 10 biopsies with definite neoplasia (5 of 7 adenocarcinoma cases and 2 of 3 at least HGD cases), all 3 (100%) atypical biopsies, and none of the 50 benign biopsies. All 3 atypical cases with aneuploidy were subsequently found to have adenocarcinoma (n = 2) or HGD (n = 1). Among the 2 cases of at least HGD with aneuploidy, 1 case developed adenocarcinoma, but no follow-up information was available in the other case. The remaining 1 case of at least HGD, despite having normal DNA content, was found to have adenocarcinoma on follow-up. None of the 50 benign cases (further supported by normal DNA content) developed adenocarcinoma within a mean follow-up time of 37 months (range: 0–282 months). The estimated sensitivity of aneuploidy as a diagnostic marker of malignancy (adenocarcinoma and HGD) was 70%, with the specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%. In conclusion, DNA flow cytometry using FFPE tissue from bile duct biopsies demonstrates a high rate of aneuploidy (70%) in malignant cases and normal DNA content in all benign biopsies. Although the sample size is small, the results indicate that this assay can be potentially useful in challenging atypical cases, where morphological evaluation is limited by scarcity of atypical glands/cells, inflammation, and/or ulceration.
ERCP tissue sampling
2016, Gastrointestinal Endoscopy
Citation Excerpt :
Such molecular abnormalities have been identified in approximately 80% of biliary and pancreatic cancers.68 Aneuploidy has been reported in 80% of bile duct specimens from patients with PSC and cholangiocarcinoma.69 In 2004, Kipp et al70 published the first study comparing FISH with routine brush cytology.
An Optimized Set of Fluorescence in Situ Hybridization Probes for Detection of Pancreatobiliary Tract Cancer in Cytology Brush Samples
2015, Gastroenterology
Pancreatobiliary cancer is detected by fluorescence in situ hybridization (FISH) of pancreatobiliary brush samples with UroVysion probes, originally designed to detect bladder cancer. We designed a set of new probes to detect pancreatobiliary cancer and compared its performance with that of UroVysion and routine cytology analysis.
We tested a set of FISH probes on tumor tissues (cholangiocarcinoma or pancreatic carcinoma) and non-tumor tissues from 29 patients. We identified 4 probes that had high specificity for tumor vs non-tumor tissues; we called this set of probes pancreatobiliary FISH. We performed a retrospective analysis of brush samples from 272 patients who underwent endoscopic retrograde cholangiopancreatography for evaluation of malignancy at the Mayo Clinic; results were available from routine cytology and FISH with UroVysion probes. Archived residual specimens were retrieved and used to evaluate the pancreatobiliary FISH probes. Cutoff values for FISH with the pancreatobiliary probes were determined using 89 samples and validated in the remaining 183 samples. Clinical and pathologic evidence of malignancy in the pancreatobiliary tract within 2 years of brush sample collection was used as the standard; samples from patients without malignancies were used as negative controls. The validation cohort included 85 patients with malignancies (46.4%) and 114 patients with primary sclerosing cholangitis (62.3%). Samples containing cells above the cutoff for polysomy (copy number gain of ≥2 probes) were classified as positive in FISH with the UroVysion and pancreatobiliary probes. Multivariable logistic regression was used to estimate associations between clinical and pathology findings and results from FISH.
The combination of FISH probes 1q21, 7p12, 8q24, and 9p21 identified cancer cells with 93% sensitivity and 100% specificity in pancreatobiliary tissue samples and were therefore included in the pancreatobiliary probe set. In the validation cohort of brush samples, pancreatobiliary FISH identified samples from patients with malignancy with a significantly higher level of sensitivity (64.7%) than the UroVysion probes (45.9%) (P < .001) or routine cytology analysis (18.8%) (P < .001), but similar specificity (92.9%, 90.8%, and 100.0% respectively). Factors significantly associated with detection of carcinoma, in adjusted analyses, included detection of polysomy by pancreatobiliary FISH (P < .001), a mass by cross-sectional imaging (P < .001), cancer cells by routine cytology (overall P = .003), as well as absence of primary sclerosing cholangitis (P = .011).
We identified a set of FISH probes that detects cancer cells in pancreatobiliary brush samples from patients with and without primary sclerosing cholangitis with higher levels of sensitivity than UroVysion probes. Cytologic brushing test results and clinical features were independently associated with detection of cancer and might be used to identify patients with pancreatobiliary cancers.
An extended fluorescence in situ hybridization approach for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing cytology preparations
2013, Human Pathology
Citation Excerpt :
This indicates that, in CC, hyperdiploidy is a manifestation of a continuous clonal evolution, which remains active at the time of diagnosis because of the underlying genetic instability, as in other tumors [7]. Consistent with this view is that PSC-related CC cases, which are thought to follow a longer process of malignant transformation, in general present with higher ploidy values than sporadic cases of the disease [8-10]. In our patients, chromosome 17 was involved in all 31 hyperdiploid cases, followed by chromosome 7, in 29.
The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease.

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Can DNA cytometry be used for evaluation of malignancy and premalignancy in bile duct strictures in primary sclerosing cholangitis?

Abstract

Section snippets

Patients

Results

Discussion

Acknowledgements

Gastroenterology

Gastroenterology

Gastroenterology

Gastroenterology

Hepatology

Hepatology

Mayo Clin Proc

Gastroenterology

Gastroenterology

Significance of aneuploidy

Br J Surg

Prognostic significance of DNA ploidy in patients with stage II and stage III colon carcinoma

Cancer

Nuclear DNA content and pathology in radically treated pancreatic carcinoma

Cancer

Ploidy and proliferation patterns in colorectal adenocarcinomas related to Dukes' classification and histopathological differentiation

Acta Path Microbiol Immunol Scand

DNA aneuploidy and histologic dysplasia in long-standing ulcerative colitis

Dis Colon Rectum

Early detection of malignancy in ulcerative colitis. A flowcytometric DNA study

Cancer

Ulcerative colitis and cancer - with special reference to the increased colorectal cancer risk

Thesis Karolinska Institute, Stockholm

Prognostic value of Ki-67 expression, ploidy and S-phase fraction in patients with pancreatic cancer

Anticancer Res