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Researchers Develop qPCR Assay to Detect Telomere Fusion in Pancreatic Cancer

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NEW YORK (GenomeWeb) – A team led by researchers at Johns Hopkins University of Medicine has developed a real-time PCR-based assay that they said could indicate the likelihood of mutating pancreatic lesions in cancer cells.

In a study published inThe Journal of Molecular Diagnostics last week, the researchers investigated the relationship between telomere length, telomerase activity,  and the presence of telomeric fusions in pancreatic cell specimens. They developed a nested qPCR-based assay to detect telomere fusions in pancreatic ductal adenocarcinomas, intraductal papillary mucinous neoplasms (IPMNs), and IPMN cyst fluids. The assay includes two rounds of PCR, with the second round applying a telomere repeat probe to detect the fusions.

In an email, Michael Goggins, the corresponding author for the study and a professor of pathology at JHU, said that he and his colleagues were interested in telomere-based biomarkers "because telomere shortening and telomerase activity is an important biological feature of the progression of precancerous lesions to invasive cancer."

In the study, the researchers examined 91 pancreatic cancer cell lines and xenograft samples, 93 IPMNs, 93 surgical cyst fluid samples, and normal pancreases. IPMN tissues and cyst fluid were obtained from patients undergoing partial pancreatic resections at the Johns Hopkins Hospital from 2004 to 2015.

To build the telomerase fusion assay, the researchers incubated an oligonucleotide mimicking a telomere end with a protein extract; nucleoside triphosphates containing deoxyribose as the sugar (dNTPs); and buffer, which allowed the telomerase to extend the oligo by adding TTAGGG repeats. They reported that the assay identified telomere fusions in 62 percent of pancreatic cancer cell lines and xenografts, and in 48 percent of IPMS with an associated invasive adenocarcinoma.

Further, they noted that "[b]ecause the assay does not require laborious experimental procedures, [the assay] can be readily applied to diagnostic laboratories to detect telomere fusions in clinical samples."

Using the assay, Goggins and his team identified telomere fusions in 22 of 31 of the pancreatic cancer cell lines, with all but one found using the telomere repeat probe. An additional telomere fusion was also detected in the human pancreatic duct epithelial cell line known to have chromosomal abnormalities, but not in human pancreatic nestin-expressing lines or in normal pancreatic tissues.

Among the 23 fusion-positive cell lines , 20 had a telomere-subtelomere fusion, where a short telomeric repeat from one chromosomal end fuses to a subtelomere region without repeats from the other chromosomal end. Three cell lines had fusions that contained small insertions between the telomeric repeats of one chromosome and the subtelomere of the other, the authors said in the study.

Telomere fusion sequences had a variable number of telomeric repeats, including the presence of telomeric sequences. Most fusions had variant repeat sequences, which are believed to contribute to telomere dysfunction and fusion formation.

Applying the fusion assay on 60 pancreatic cancer xenograft samples, the team found that 34 of the samples were fusion positive. They also detected telomere fusions in tumors with high-grade dysplasia .

In addition, the team noted that fusion-positive pancreatic cell lines had significantly shorter telomeres and higher telomerase activity than fusion-negative lines. Similarly, they found that telomeres are shorter in IPMN cells than in adjacent normal cells, but longer than in pancreatic cancer cell lines.  

While the team performed the fusion assay on DNA isolated from IPMN, normal pancreata, and normal duodenum, they failed to find telomere fusions in 39 normal pancreas and duodenal tissue samples.

The team found similar results when they performed the assay on cyst fluid samples derived from resected IPMNs. While no fusions were found in samples with low-grade dysplasia, the prevalence of fusions increased with histologic grade. Cyst fluids that contained telomere fusions were also more likely to have elevated levels of telomerase.

The authors argued that telomere fusions are nonclonal events and are only detected as rare events. With the assay, the team detected fusions using 1000 or more genome equivalents of DNA.

"Although telomere fusions that arise from critically short telomeres can cause chromosomal breakage-fusion-breakage cycles, most telomere fusions are not tolerated by the cell," the authors stated. "They represent transient events that arise in neoplastic cells before they die," and therefore "it is likely that telomere fusions were not found to be prognostic."

While the team succeeded in demonstrating the presence of shortened telomeres, sporadic telomeric fusions, and increased telomerase  in a modest proportion of pancreatic lesions, Loren Joseph, assistant professor of pathology at Harvard Medical School, stated in an editorial that "each biomarker shows only weak correlations with the presence of advanced lesions." Joseph noted that the clinical use of the assay may be limited to patients who go through pancreatic resection, and said that the weak correlations may not pertain to patients who undergo endoscopic ultrasound-guided fine needle aspiration biopsy for more ambiguous indications.

Based on the results, the team believes that telomere fusions can be detected in later stages of IPMN progression and can serve as biomarkers for predicting the presence of high-grade dysplasia and invasive cancer.

"Many patients will develop multiple fine needle aspirates of a mass lesion in the pancreas, where pathologists are not sure if it's cancer or a reactive change," Aatur Singhi, assistant professor at the University of Pittsburgh Medical Center Health Plan, said in an interview. "It's possible that the telomere fusion assay may be highly sensitive and specific for malignancies in the solid neoplasms, not just cystic pancreas."

Goggins and his team has submitted an application seeking a patent that describes  the telomere fusions and their detection in pancreatic cyst fluids.

"Accurate molecular analysis of pancreatic cyst fluid using telomere fusion testing, along with complementary molecular tests to detect mutations and other molecular changes, can improve clinical decision-making about the need for resection of pancreatic cysts," Goggins added.