SAN FRANCISCO (GenomeWeb) – Researchers at the University of Pittsburgh Medical Center have developed a next-generation sequencing-based test that can help clinicians spot cancerous pancreatic cysts by identifying mutations found in DNA from the cyst fluid.
Approximately 15 percent of pancreatic cancers develop from common cysts, but there are currently no good methods to differentiate benign from cancerous cysts.
The researchers described the validation of the targeted NGS test in a study published in Gut last month, showing that it is more sensitive than other methods that are based primarily on morphology. They are now offering the test to all patients at the University of Pittsburgh Medical Center who have a pancreatic cyst, as well as to physicians outside of UPMC who want to order it.
Aatur Singhi, lead author of the study, said that the goal is to enable earlier diagnosis of pancreatic cancer and hopefully to improve survival rates. "We often find pancreatic cancer when it's too late," Singhi said. About 85 percent of pancreatic cancer patients are diagnosed after they already have advanced local or metastatic disease, he said, and the five-year survival rate is terrible, at just 9 percent.
If clinicians are able to more accurately determine whether a pancreatic cyst is benign or cancerous, that could help make diagnoses earlier and potentially improve survival rates.
Pancreatic cysts are typically found incidentally, often on an MRI that was performed for an unrelated purpose. If a physician finds a pancreatic cyst, there are several methods to determine whether it is cancerous, including an endoscopic ultrasound and biopsy, and other imaging-based tests, but they often have sensitivities of less than 50 percent. More recently, molecular testing has started to be adopted, but Singhi said that NGS-based molecular tests had the potential to be much more sensitive.
It's important to have a good way to distinguish cancerous cysts from benign ones because the cysts are common and surgical removal can cause serious problems. Singhi estimated that some 4 to 6 million Americans had pancreatic cysts and that most were benign. Surgical removal has a range of severe side effects: Up to 4 percent of patients die within 90 days after surgery and 30 to 40 percent have long-lasting effects, including early onset diabetes, pancreatitis, losing the ability to digest certain foods, and chronic pain, he said.
For the study, the researchers designed a targeted sequencing assay that focused on 11 genes known to be related to pancreatic cancer. For instance, cysts that have activating mutations in the KRAS or GNAS genes are associated with two types of precancerous cysts — intraductal papillary mucinous neoplasms and mucinous cystic neoplasms — while mutations in TP53, PIK3CA, and PTEN are associated with advanced neoplasia.
They analyzed pancreatic cyst fluid from nearly 600 patients, used Sanger sequencing to look for mutations in a subset of 159 of those patients, and also compared the molecular findings with ultrasound results. Follow-up data was available for 102 cases.
The researchers designed the assay on Thermo Fisher Scientific's Ion Torrent platform and were able to call mutations down to minor allele frequencies of 3 percent at a sequencing depth of 500x.
Mutations in KRAS or GNAS were identified in 308 cases, while mutations in TP53, PTEN, PIK3CA, or AKT1 were identified in 35 cases. Diagnostic data was available for 102 patients who underwent surgery, and the researchers were able to determine allele frequency thresholds for the genes to determine whether a cyst was cancerous to achieve an overall sensitivity of 89 percent and a specificity of 100 percent. By comparison, other non-molecular methods had sensitivities of 42 percent and lower and specificities between 74 and 98 percent.
Singhi said that the lab has been running the assay for the last three and a half years, initially as a research assay, but as a clinical test for the last two years. In addition, since completing the study, the researchers realized that of the 11 genes originally included, only seven were necessary, so they reduced the size of the assay. The test costs $750 and is reimbursed through the hospital's own health plan as well as some other insurance companies, Singhi said.
Aside from continuing to offer the NGS-based test from its clinical lab, Singhi said, the team is also looking to improve it. For instance, he said, the current version does not evaluate copy number alterations, and the team is now working on validating a method that analyzes several SNPs along a given gene to determine whether there is a deletion. It will also consider adding genes to the assay, depending on evidence in the literature.
Ultimately, he said, the researchers would like to further refine the test so that it can go beyond identifying pancreatic cysts that are already on their way toward becoming cancerous. "We'd like to expand the test into a risk stratification test," he said, identifying patients whose cysts are at risk for developing cancer in the future so they can be monitored.