Next Generation Sequencing (NGS) of Biliary Specimens Improves Preoperative Detection and Management of Malignant Bile Duct Strictures

December 19, 2019

The poor performance of current preoperative methods for differentiating between malignant and benign biliary strictures results in some patients undergoing surgical resection despite having benign strictures. Alternatively, this can result in a delay in the diagnosis of biliary cancer. Seeking a solution, Aatur Singhi, MD, PhD, and Adam Slivka, MD, PhD, developed a targeted NGS assay called BiliSeq. Testing endoscopic retrograde cholangiopancreatography (ERCP)-obtained biliary specimens against this assay offers clinicians a more reliable diagnosis compared to standard methods.

Benign biliary strictures are typically due to conditions such as sclerosing cholangiopathies, iatrogenic injury, and infection, while malignant strictures are related to carcinomas arising from the pancreatobiliary duct cell, ampulla of Vater, and liver cancers. The preferred methods for pathologic confirmation during endoscopic retrograde cholangiopancreatography (ERCP) are bile duct brushings and forceps biopsies, but the sensitivity of these approaches to detect malignancy can vary from eight to 67 percent. Several ancillary detection techniques have been developed, including digital image analysis, KRAS mutation testing, and multicolor fluorescence in situ hybridization (FISH), but these methods also fail to produce reliable diagnoses due to the wide variance in sensitivity and, in the case of the latter method, a propensity for subjective interpretation errors, which can only be avoided by an experienced pathologist performing labor intensive work. 

Distinguishing between the two is difficult, especially because certain conditions, like primary sclerosing cholangitis (PSC), are associated with benign strictures but are also associated with an increased risk of bile duct cancer and may harbor pre-cancerous changes.

The failure of current diagnostic methods for malignant biliary strictures often leads to repeated ERCP procedures and delayed clinical decisions that risk disease progression, and even more alarmingly, can lead to a high percentage of patients undergoing an unnecessary surgical resection. A more reliable method is required for preoperative diagnosis of malignant biliary duct strictures, and this serves as the motivation for this study of a promising new methodology involving NGS. 

NGS and Its Use in Previous Studies of Biliary Strictures

Sequencing of the human genome was once an expensive, time-consuming process, but the advent of NGS and the introduction of novel molecular diagnostics have created unparalleled opportunities for low-cost use of DNA sequencing in clinical and research environments. Consequently, our understanding of the genomic landscape of neoplasms arising in, or secondarily involving, the bile duct system has improved at an incredible pace in recent years.

These innovations have played a pivotal role in recent research studying the efficacy to distinguish between malignant and benign strictures. Bankov et al. published the results of an NGS assay that was used to test a retrospective cohort of patients and found a sensitivity of 81 percent. However, this study tested only 16 patients, did not evaluate biliary brushing specimens, and used DNA extracted from Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks for sequencing, resulting in such a low-quality of DNA that it had to be enriched before analysis. A more comprehensive assessment of NGS testing on bile duct specimens was reported by Dudley et al, which studied a cohort of 73 bile duct and eight main pancreatic duct brushing specimens, finding a sensitivity of 68 percent and a specificity of 97 percent. But even this larger study did not collect dedicated brushings for DNA isolation and extracted DNA from CytoLyt-preserved specimens. Unsurprisingly, 11 percent of their specimens had degraded to the point of failing NGS testing.

This previous literature has shown that NGS testing of bile duct strictures may have promise, but a more comprehensive study was required for definitive evaluation of the impact on patient management when genomic alterations are detected in bile duct specimens. 

Methodological Improvements and Results 

In our study, we developed a highly sensitive, targeted NGS assay called BiliSeq in a laboratory with Clinical Laboratory Improvement Amendments certification and accreditation by the College of American Pathologists. Investigation centered on 28 genes that are commonly mutated, amplified, and/or deleted in malignant neoplasms involving the bile duct system. 

During an initial training cohort followed by a validation cohort, a total of 163 biliary brushings and 172 biliary biopsies were collected for pathological evaluation. Rather than relying on DNA-extraction methods from processed pathology specimens that might have reduced the yield and quality of the genome, we performed a dedicated bile duct brushing and/or biopsy for each patient and submitted the resultant 160 brushings and 135 biopsies for BiliSeq testing. 

NGS was performed prospectively as part of clinical care with a 10-day turnaround in the UPMC Molecular & Genomic Pathology (MGP) Laboratory. We then compared the results of NGS testing with the results of diagnostic pathology of 145 surgically resected specimens or biopsy specimens and with the results of further clinical evaluations on 75 of the patients. BiliSeq was found to have a sensitivity of 73 percent and a specificity of 100 percent, an improvement over pathological evaluation alone for detecting at least high-grade biliary dysplasia involving the bile duct. The combination of both pathological and BiliSeq testing brought sensitivity up to an incredible 83 percent while maintaining a specificity of 99 percent.

A preliminary observation from this study is the striking improvement in sensitivity that BiliSeq allowed for patients with PSC. These patients’ strictures are typically caused by inflammation, so it is often challenging to procure a sample, and pathologic evaluation usually produces unreliable diagnoses. Routine cytologic evaluation tested at an eight percent sensitivity for PSC patients, but BiliSeq achieved a sensitivity of 83 percent for at least high-grade biliary dysplasia. This is compelling evidence to further explore the potential of BiliSeq to improve clinical outcomes for this high-risk patient population.

Clinical Implications of Results

These results highlight the diagnostic applicability of NGS-based assays to ERCP-obtained biliary specimens in the early detection and management of patients with indeterminate bile duct strictures. For clinicians, this testing can grant the ability to avoid unnecessary resections and to make evidence-based clinical decisions before the disease progresses.

Our results also have implications for the growing body of literature around precision medicine. BiliSeq testing identified two patients with ERBB2 amplifications, a genomic alteration known to respond well to trastuzumab in conjunction with standard first-line chemotherapy. Both patients exhibited measurable radiographic responses and normalization of serum CA19-9, and both are currently alive and well thanks to the ability of NGS to provide a personalized, targeted treatment option. To learn more about these patients, visit inside.upmc.com/deadly-diagnosis-precision-medicine.

References

Singhi AD, Nikiforova MN, Chennat J, Papachristou GI, Khalid A, Rabinovitz M, Das R, Sarkaria S, Ayasso MS, Wald AI, Monaco SE, Nalesnik M, Ohori NP, Geller D, Tsung A, Zureikat AH, Zeh H, Marsh JW, Hogg M, Lee K, Bartlett DL, Pingpank JF, Humar A, Bahary N, Dasyam AK, Brand R, Fasanella KE, McGrath K, Slivka A. Integrating Next Generation Sequencing to Endoscopic Retrograde Cholangiopancreatography (ERCP)-Obtained Biliary Specimens Improved the Detection and Management of Patients With Malignant Bile Duct Strictures. Gut. 2019. Epub ahead of print.
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