BiliSeq Next-Generation Sequencing Testing Provides Molecular Approach to Improving Cancerous Bile Duct Stricture Diagnosis

Accurately differentiating cancerous from benign bile duct strictures has been a persistent diagnostic challenge. Endoscopic retrograde cholangiopancreatography (ERCP) can locate and characterize a stricture, but pathologic assessment of the tissue brushings and biopsies has recognized sensitivity limitations ranging from 8% to 67% in the literature. Cytologists apply deliberately rigid criteria for a positive diagnosis of cancer, meaning that a positive result is reliable, but many cancerous strictures produce specimens in which the malignant cells are scarce, damaged, or difficult to distinguish from other factors like inflammatory changes. The result is a high rate of false negatives, and many patients cycle through repeat procedures before reaching a definitive diagnosis. This leads to delays in treatment, among other consequences.

"The specificity of pathologic assessment has always been good. If it's positive, it's positive. But the sensitivity has been less than flipping a coin," says Adam Slivka, MD, PhD, professor of medicine and associate chief, Clinical Affairs, Division of Gastroenterology, Hepatology and Nutrition at UPMC and the University of Pittsburgh School of Medicine. "You get very few to no false positives, but you can see a lot of false negatives."

BiliSeq, a next-generation sequencing (NGS) assay developed at UPMC in a collaboration between Dr. Slivka’s group and the molecular pathologists at UPMC, is designed to be used alongside standard pathologic assessment to improve diagnostic accuracy. Applied to ERCP-obtained biliary specimens, BiliSeq analyzes cancer-associated gene mutations and fusion genes directly from the specimen's nucleic acid rather than relying only on cellular morphology. The BiliSeq test returns a binary result within seven to 10 days. When a test is positive for cancer, it can also identify if the patient carries a therapeutically actionable genomic alteration that may inform or otherwise change the treatment plan.

How BiliSeq Was Developed

The molecular diagnostic work that helped to create BiliSeq began in work with pancreatic cysts. Starting in 1994, Dr. Slivka and colleagues pursued molecular approaches to identifying cancerous and pre-cancerous pancreatic cysts, and over time, UPMC became a leading center for that work with the PancreaSeq platform. About 15 years ago, Dr. Slivka turned his attention to the bile duct and cholangiocarcinoma, a cancer that is difficult to diagnose by standard tissue sampling. He began taking extra brushings of bile duct strictures and sending them to Aatur Singhi, MD, PhD, associate professor, Department of Pathology, University of Pittsburgh School of Medicine, to run through the existing pancreatic sequencing assay. A meaningful number returned positive results for cancer-associated mutations, and those findings were affecting how patients were managed.

After obtaining an IRB and launching a formal study, Dr. Singhi then customized the gene panel for bile duct cancer biology, accounting for where the genomic profile of biliary cancers overlaps with pancreatic cancer and where it is different. The original version of BiliSeq started with a smaller number of genes, and now in its current iteration covers hundreds of cancer-associated genes and fusion genes. BiliSeq has been available for routine clinical use for close to 10 years.

How the Test Works

Outside providers can request a BiliSeq test directly from UPMC. Biliary specimens, including brushings and/or biopsies, are submitted at the time of ERCP in a nucleic acid stabilization buffer. The vial is shipped to UPMC's CLIA and CAP accredited molecular diagnostics laboratory within 24 to 48 hours of the procedure. DNA and RNA are extracted, sequenced against the BiliSeqV3 panel, and results are returned to referring providers within seven to 10 days.

Because BiliSeq detects genomic alterations rather than relying only on a pathologist’s interpretation of cellular morphology, the test results are a purely objective measure of the sample. This is a particular advantage when specimen cellularity is low or when inflammation obscures the picture, exactly the conditions under which conventional cytology most often fails.

"BiliSeq is less expensive and less labor intensive than other molecular based available tests, and the result is not open to interpretation," Dr. Slivka says. "It is a yes or no answer that gets patients on a straight line to a diagnosis without repeat testing or unnecessary procedures."

Why BiliSeq Performs Better Than Other Testing Platforms for Bile Duct Cancer

Fluorescence in situ hybridization (FISH), which evaluates chromosomal copy number abnormalities as a marker of malignancy, is commonly used as an adjunct to cytology in the workup of bile duct strictures. Originally developed for bladder cancer, FISH improves sensitivity somewhat over cytology alone but can still miss a significant proportion of biliary cancers and has a higher false positive rate than cytology It is also labor intensive, subject to interpretive variability between readers, and provides no information about specific gene mutations or therapeutically actionable molecular targets.

BiliSeq overcomes the sensitivity gap inherent in FISH and other testing modalities by looking directly at bile duct cancer-associated genetic mutations. When used in combination with standard pathologic assessment, BiliSeq substantially improves the likelihood of reaching a correct diagnosis. In clinical terms, if BiliSeq returns a positive result, the positive predictive value for cancer is more than 95%. If it returns a negative result, the negative predictive value is close to 90%.

BiliSeq has particular value in patients with primary sclerosing cholangitis (PSC), a chronic inflammatory liver disease in which approximately 15% of patients develop cholangiocarcinoma over their lifetime. The inflammatory background of PSC specimens can make cytologic interpretation alone suboptimal. A positive BiliSeq result can definitively establish a cancer diagnosis in cases where pathology returns only atypical or equivocal findings.

Identifying Actionable Genomic Targets to Improve Treatment Planning

Beyond just its ability to accurately detect the presence of bile duct cancer, BiliSeq has also been designed to look for mutations that can be targeted with existing therapeutics, thereby altering the clinical and therapeutic decision-making process, which is something cytology and FISH testing are not powered to do. Approximately 15% to 20% of BiliSeq tests have found a specific mutation for which a targeted therapy is currently available. KRAS is one of the more common genetic alterations seen in bile duct cancers and reported in BiliSeq. Recent development and U.S. Food and Drug Administration approval of KRAS inhibitors will dramatically increase the number of patients eligible for molecularly-targeted therapy.

The ability to find and act on these types of mutations can and has dramatically altered the treatment trajectory and outcomes for some patients.

“When these patients get standard chemotherapy treatments along with more targeted therapies designed specifically for the genetic mutations their cancers harbor, they can do remarkably better, " Dr. Slivka says. “We have seen this dynamic play out in some of our patients. Not only can we find their cancers faster and with greater accuracy, for some, BiliSeq findings change our treatment planning.”

The proportion of patients with actionable targets has grown as the BiliSeq panel has evolved over time. The current focus is on extending the panel beyond genomic analysis to include RNA expression data with the goal of identifying a broader range of molecular targets as drug development in biliary cancers advances.

New Data from a Six-Year Prospective Study of BiliSeq

Dr. Slivka and colleagues published a six-year, prospective, multi-institutional study of BiliSeqV2 and V3, on March 27 in Gastroenterology. The article, “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,” provides the most comprehensive assessment of the BiliSeq assay's real-world diagnostic performance to date.

The study examined data on 2,865 biliary specimens collected from more than 2,080 patients at 28 institutions. BiliSeqV2/V3 testing demonstrated 82% sensitivity and 98% specificity for neoplastic strictures, compared with 44% sensitivity and 99% specificity for standard pathologic assessment. Combined, the two modalities reached 88% sensitivity and 97% specificity.

"The sensitivity and specificity we are seeing with BiliSeq represent a significant improvement over what we have been accustomed to with standard cytologic assessment of biliary specimens," Dr. Slivka says.

For full details on the study design and findings, please read the press release, titled “Molecular Test Doubles Detection of Bile Duct Cancer.”

Submitting Samples to UPMC for Testing with BiliSeq

BiliSeq testing is not limited to UPMC patients. UPMC processes specimens for a growing number of institutions in the United States. Outside centers sign an agreement with UPMC and receive a collection vial and mailer for specimen submission. Specimens are processed in the UPMC Molecular & Genomic Pathology Laboratory and results are reported back to the referring team. Hospitals and health care systems that have their own NGS sequencing infrastructure can also obtain the published methodology developed by Dr. Slivka and colleagues and run the assay themselves on their own patient samples.

Inquiries from Providers and Hospitals

To learn more or get in touch about BiliSeq testing, referring physicians and institutions can contact the UPMC Molecular & Genomic Pathology Laboratory at 844-647-5227, or visit the website for more details on available tests, including BiliSeq, along with forms and specimen requirements.