DNA/RNA-Based NGS Test Improves Diagnostic Accuracy in Bile Duct Strictures

The integration of expanded next-generation sequencing (NGS) panels into the evaluation of biliary specimens significantly improved the detection of neoplastic strictures compared with traditional pathologic assessment, according to findings from a 6-year, prospective, multi-institutional study published in Gastroenterology.¹ The study, which evaluated 2116 patients across 28 medical institutions, demonstrated that the BiliSeqV2/V3 testing platform achieved 82% sensitivity and 98% specificity for neoplastic structures, compared with the 44% sensitivity and 99% specificity of standard pathology.

By identifying actionable genomic alterations in approximately 23% of malignancies, the NGS-based approach also provides a direct pathway toward precision oncology in a disease space where therapeutic options have historically been limited.

“For decades, in bile duct cancer we’ve known that a negative biopsy doesn’t always rule out cancer,” stated Adam Slivka, MD, PhD, professor of medicine and associate chief of Clinical Affairs in the Division of Gastroenterology, Hepatology and Nutrition, at the University of Pittsburgh School of Medicine, and medical director of the GI Service Line for UPMC Health System, in a press release on the study.² “That uncertainty drives repeat testing and sometimes surgery without clear answers.”

Trial Overview and Diagnostic Performance

The prospective study analyzed 2908 biliary specimens, including brushings, biopsies, and bile samples, collected between 2018 and 2024. Investigators utilized 2 iterations of the platform: BiliSeqV2 (targeting 28 cancer-associated genes and 167 fusions) and the expanded BiliSeqV3 (161 genes and 763 fusions). Molecular results were compared with clinical, imaging, and pathologic parameters including diagnostic pathology and/or at least 1-year follow-up.

The combined BiliSeqV2/V3 testing achieved an overall sensitivity of 82%, which increased to 88% when combined with pathology; the specificity was 97%. In a subanalysis of 548 patients with indeterminate biliary strictures, BiliSeqV2/V3 achieved a 77% sensitivity and 98% specificity. Notably, the platform performed well in high-risk populations; among 327 patients with primary sclerosing cholangitis (PSC), NGS sensitivity alone was 85%, and NGS sensitivity plus pathology was 89%, whereas traditional pathology detected only 26% of neoplastic cases.

Clinical Context and Unmet Needs

Distinguishing between benign biliary disease and cholangiocarcinoma remains a diagnostic bottleneck.^3,4 While endoscopic retrograde cholangiopancreatography is a sensitive modality for locating strictures, the resulting cytology or brushings are notoriously insensitive, with reported sensitivities as low as 8% to 20% in some cohorts.⁴ Although fluorescence in situ hybridization (FISH) is often used as an adjunct to detect aneuploidy, it frequently yields false positives in patients with underlying inflammation, such as those with PSC.⁵

The BiliSeq platform utilizes a specialized nucleic acid lysis and stabilization buffer that allows for the collection of specimens directly in a vial rather than on smears or slides. This method preserves DNA and RNA quality, facilitating a 7-to-10-day turnaround time for results that can directly inform surgical or oncological management.

Genomic Alterations

Beyond its diagnostic utility, BiliSeqV3 identified targetable molecular alterations in 23% of the positive malignancies. The most frequent actionable findings involved the MAPK pathway (56%), including ERBB2, BRAF V600E, and KRAS G12C alterations. Other significant findings included DNA repair mutations (40%), including ATM, BRCA1/2, and MLH1, as well as IDH1/2 mutations.

In this cohort, NGS findings modified patient management in 30% of cases with targetable alterations. Among those who received therapy based on these results, 66% exhibited disease stability or a measurable radiographic response. In one notable instance, the detection of a TMPRSS2::ERG fusion in a biliary specimen—a marker highly specific for prostate cancer—led to the diagnosis of metastatic prostatic adenocarcinoma that had previously been misidentified as a primary biliary stricture.

Expert Framing and Limitations

The findings suggest that NGS-based testing should be considered a standard component of the diagnostic workup for biliary strictures, particularly for patients with PSC or those with negative initial cytology.

The authors noted several study limitations. While the specificity was high (98%), 16 false-positive cases were identified, which involved mutations in KRAS, TP53, and GNAS at low variant allele frequencies. These findings underscore the necessity of interpreting molecular results within the broader clinical and radiographic context. Additionally, while the study was multi-institutional, the testing was centralized at a CLIA-certified laboratory, which may limit immediate accessibility for some smaller community practices.

References

1. Das R, Kleinberger J, Sawas T, et al. DNA/RNA-based next-generation sequencing (NGS) improves the early diagnosis and management of neoplastic bile duct strictures: a six-year, prospective, multi-institutional, real-time study. Gastroenterology. 2026. doi:10.1053/j.gastro.2026.02.040
2. Molecular test doubles detection of bile duct cancer. News release. UPMC Hillman Cancer Center. March 27, 2026. Accessed April 1, 2026. https://tinyurl.com/3mf9jfes
3. Singhi AD, Nikiforova MN, Chennat J, et al. Integration of next-generation sequencing into the pathologic evaluation of biliary brushings and biopsies. Gut. 2020;69(1):52-61. doi:10.1136/gutjnl-2018-317817
4. Rizzo A, Brandi G. First-line Chemotherapy in Advanced Biliary Tract Cancer: Ten Years After the ABC-02 Trial. Cancer Treat Res Commun. 2021;27:100335. doi:10.1016/j.ctarc.2021.100335
5. Gonda TA, Viterbo D, Gausman V, et al. Mutation profiling and fluorescence in situ hybridization in the diagnosis of indeterminate biliary strictures. Clin Gastroenterol Hepatol. 2017;15(6):913-919.e1. doi:10.1016/j.cgh.2016.12.013