DNA/RNA-based next-generation sequencing (NGS) allows earlier diagnosis of neoplastic bile duct strictures, a prospective multicenter real-time study found.

Reporting in Gastroenterology, on the 6-year, 28-institution study, Aatur D. Singhi, MD, PhD, an associate professor of pathology at the University of Pittsburgh Medical Center (UPMC) in Pittsburgh, and colleagues noted that distinguishing between benign and neoplastic bile duct stricture remains challenging. Pathologic assessment of conventionally obtained specimens has limited sensitivity, particularly among patients with primary sclerosing cholangitis (PSC), but NGS of specimens offers a promising diagnostic approach.

While endoscopic retrograde cholangiopancreatography (ERCP) and magnetic imaging cholangiopancreatography are sensitive enough to define the location and nature of a disease in the biliary tree, both techniques can be diagnostically inaccurate and require pathologic assessment of biliary specimens, explained Singhi, who is also director of the UPMC Developmental Laboratory.

“Patients can present with biliary strictures — and we are seeing a rise in these — due to a variety of underlying etiologies such as autoimmune conditions like PSC, but they can also be associated with malignancies, such as pancreatic cancer or cholangiocarcinoma,” Singhi told Medscape Medical News. Current modalities to determine stricture etiology are insufficient. “When we use imaging alone to determine if it’s malignant or benign, the results are more of a coin toss. Similarly, with pathology looking at cells that have been brushed or biopsied in this area where there’s so much background inflammation, this becomes very challenging.”

BiliSeqV2/V3 was developed to objectively determine the presence of a neoplasm, which could be associated with cholangiocarcinoma, pancreatic cancer, an ampullary neoplasm, or a neuroendocrine tumor, and sometimes even an occult metastatic process, Singhi explained. “We think this test could be prioritized in certain indications, but it won’t replace pathological evaluation,” Singhi said. “In theory, BiliSeq can replace FISH [fluorescence in situ hybridization] testing, a 20-year-old technology, with more modern-day sequencing modalities.”

Methodology

The investigators applied BiliSeqV2 testing (28 cancer-associated genes and 167 fusion genes) and BiliSeqV3 testing (161 cancer-associated genes and 763 fusion genes) to 2908 brushings, biopsies, and bile obtained from 2116 patients. They compared molecular results with clinical, imaging, and pathologic parameters, including diagnostic pathology, and/or at least 1-year follow-up.

BiliSeqV2/V3 testing was performed for 2865 (99%) specimens from 2080 (98%) patients. Based on follow-up from 1979 (95%) patients, BiliSeqV2/V3 demonstrated 82% sensitivity and 98% specificity for a neoplastic stricture. In comparison, pathologic assessment had a sensitivity of just 44% and a specificity of 99%, while combining BiliSeqV2/V3 with pathologic assessment improved sensitivity to 88% and maintained a high specificity of 97%.

High-risk populations, such as Hispanic individuals, germline carriers, and patients with PSC, also showed improvement in sensitivity with BiliSeqV2/V3 (74%-86%) vs pathologic assessment (26%-50%). Furthermore, actionable molecular alterations were identified in 20% of BiliSeqV3-positive neoplasms and were used to alter patient management in 30% of these cases.

In addition, BiliSeq can decrease the number of repeat ERCPs and the number of adverse events associated with these repeated procedures, Singhi said. “Moreover, it improves the ability to get patients to surgery, as opposed to the advancing of cancer while we’re still trying to make a diagnosis. Sequencing is also cheaper than a multiprobe FISH assay, so it has significant benefits for our healthcare system, for patients, and for the ability to do testing that’s more comprehensive.”

Offering an external perspective on the study, Matias A. Avila, PhD, a professor of biochemistry and molecular biology at Center for Applied Medical Research University of Navarra in Pamplona, Spain, said the technologies underlying BiliSeqV and a different assay his group has tested are highly comparable. Both rely on NGS panels to detect genetic alterations. “The findings reported in this study are consistent with our previous observations with Bilemut,” he told Medscape Medical News. The approach in the current paper, however, incorporates additional molecular features, including gene fusions, and uses an NGS panel with approximately three times more genes.

“When evaluating performance using bile as a liquid biopsy matrix, our assay demonstrated higher sensitivity, although with somewhat lower specificity,” Avila said. “As noted by the authors, this difference may be influenced by how limits of detection are defined for various specimen types.”

With these assays offering advantages over conventional diagnostic methods, Avila agreed they have the potential to replace certain tests such as FISH, while serving as complementary tools alongside existing approaches. “Importantly, NGS assays provide clinically actionable molecular insights that can inform patient management and therapeutic decision-making, information not typically obtained through current standard diagnostic workflows,” he said. “Implementing NGS-based analyses of biliary specimens could be transformative in the evaluation of suspected bile duct strictures.” Therefore, increasing awareness of this approach among the medical community is essential. “We really hope that these technologies will soon be incorporated into clinical practice guidelines issued by major medical and scientific societies.”

In summary, applying BiliSeqV2/V3 testing to ERCP-obtained specimens improved the diagnostic evaluation of bile duct strictures, achieving higher sensitivity, especially in PSC, and maintained high specificity compared with traditional methods. This study highlights the importance of NGS for precise diagnosis and therapeutic intervention.

This study was supported in part by the National Institutes of Health, the National Pancreas Foundation, the Sky Foundation, and the Pancreatic Cancer Action Network. 

Singhi reported receiving honoraria from Foundation Medicine, Inc., and Olympus Corporation of the Americas. No other authors had conflicts of interest to disclose.