BiliSeq: A Molecular Test that Improves Bile Duct Cancer Detection

Evaluating bile duct obstructions remains difficult, even with advanced imaging techniques, as clinicians often cannot determine whether the underlying cause is benign or malignant. Although bile duct brushings and biopsies are routinely used for pathological assessment, their sensitivities range widely—from eight to 67 percent. As a result, these methods don’t always give clinicians a definitive diagnosis.

To address these limitations, researchers at the University of Pittsburgh School of Medicine and the University of Pittsburgh Medical Center Hillman Cancer Center developed BiliSeq, a 28-gene DNA-based next-generation sequencing (NGS) panel that detects genetic mutations associated with cancer in bile duct tissue.¹

In their new paper, published in Gastroenterology, the researchers tested two new versions of BiliSeq, V2 and V3, which included expanded DNA- and RNA-based NGS panels, on bile duct samples across a six-year, prospective, real-time study.² BiliSeq detected approximately 82 percent of bile duct cancers compared to 44 percent with pathology alone; however, when combined, BiliSeq increased cancer detection to nearly 90 percent. These findings underscore the benefit of this molecular tool in helping clinicians with cancer diagnosis and treatment decisions.

The researchers collected nearly 3,000 bile duct specimens—including brushings, biopsies, and bile—from more than 2,000 patients with bile duct strictures over a six-year period. They first conducted molecular testing on 217 patients using BiliSeqV2, which combined BiliSeqV1 with RNA testing of 167 fusion genes. Meanwhile, they analyzed the remaining 1,863 patients with BiliSeqV3, which included an expanded DNA/RNA-based panel of 161 genes and 763 fusion genes.

Both versions identified genomic alterations, and the team evaluated these findings against standard diagnostic approaches, including pathologic assessment and procedures, such as a serum blood test that measures the cancer biomarker carbohydrate antigen (CA) 19-9.

Notably, the researchers found that BiliSeqV2/V3 achieved a sensitivity of 82 percent and a specificity of 98 percent compared to tests measuring elevated CA19-9 and pathologic assessment, which demonstrated sensitivities of 69 percent and 44 percent respectively. However, combining BiliSeqV2/V3 with the pathologic assessment of biliary specimens improved the sensitivity to 88 percent and maintained a high specificity of 97 percent.

The researchers also observed important differences across patient subgroups. Hispanic and Asian patients may face a higher risk of bile duct cancer due to a greater likelihood of carrying germline pathogenic variants in cancer predisposition genes. In addition, BiliSeqV3 analysis revealed a higher prevalence of tumorigenic gene alterations in patients with primary sclerosing cholangitis, a liver condition characterized by bile duct inflammation and scarring that can further complicate diagnosis, particularly when relying on traditional pathology.

Using the identified mutations as a guide, the patients’ clinicians altered care management, resulting in disease stability noted upon a follow-up appointment. Based on these findings, the improved sensitivity of BiliSeqV2/V3 makes it a promising tool alongside traditional assessments to aid clinicians in early diagnosis and subsequent management of bile duct cancer, a boon for improving patient outcomes.