Targeted next generation sequencing platform detected multiple mutations

Issue: October 2012

QUEBEC CITY — Using targeted next generation sequencing via the AmpliSeq cancer panel on the Ion Torrent platform, researchers at the University of Pittsburgh were able to test for 740 mutations across a broad spectrum of human cancer genes. According to researchers, this finding could improve sensitivity of the molecular diagnosis in thyroid surgical and fine needle aspiration samples.

Perspective from Nikolaos Stathatos, MD

“This study demonstrates that thyroid FNAs and tissue specimens can be successfully analyzed by targeted next generation sequencing (NGS) using Ion Torrent (Life Technologies) platform. Detection of hundreds of important cancer-specific mutations can be performed on thyroid FNA samples in a cost efficient way, with high accuracy and can be used clinically,” Marina Nikiforova, MD, FCAP, associate professor and director of the Molecular Anatomic Pathology laboratory at the University of Pittsburgh Medical Center, told Endocrine Today.

Marina Nikiforova

Nikiforova and colleagues evaluated the benefits of a novel next-generation sequencing platform known as Ion Torrent PGM for clinical testing of thyroid fine-needle aspiration (FNA) and surgical distribution.

The researchers looked at 60 thyroid samples, including 24 FNAs, 25 frozen, and 11 formalin-fixed, paraffin-embedded tissues that were previously tested using single-gene approach. They were analyzed for all 740 mutations in 46 genes available on the AmpliSeq cancer panel, according to researchers.

Data indicated that all of the FNA samples with indeterminate cytology diagnosis as well as resected specimen representing all major types of thyroid cancer were successfully analyzed. Particular advantage of using this novel technology was that as little as 10 ng of DNA was sufficient to adequately test all of the samples. Importantly, in addition to being able to detect all previously known mutations, the platform allowed the researchers to detect additional genetic alterations in thyroid FNAs and in resected specimen, according to researchers.

“In addition to common mutations in thyroid cancer (BRAF and RAS), NGS allowed to detect other rare mutation types in (KRAS codon 61, NRAS codon 12, PIK3CA, TP53, RET and other) at the same cost. Therefore, targeted next generation sequencing for a large mutational panel improves sensitivity of mutation detection in thyroid FNA and tissue specimens and helps to establish cancer diagnosis with high precision,” Nikiforova said.

Additional data found that germline missense variants were detected in the MET, ATM and KDR genes. Also, the researchers found RET M918T mutation in FNA sample previously diagnosed as suspicious for papillary carcinoma by cytology, allowing a diagnosis of medullar carcinoma before surgery.

The researchers said these findings could lead to efficient detection of multiple mutations in limited samples while remaining highly cost-effective.

“The commercial panel of mutations (AmpliSeq, Ion Torrent) that we used and reported in this study is a new advance in molecular diagnostics of thyroid nodules because it allows detecting multiple mutations at the same time and does not increase cost of testing,” Nikiforova said.

“However, even this novel approach can be improved further. For example, this panel of cancer genes is designed to broadly test for multiple mutations in different cancer types, some of which are not important in thyroid carcinogenesis. Therefore, we have designed and are currently validating a custom Thyroid Cancer Panel for targeted next generation sequencing, which will result in further reduction of the test cost. Another further direction will be in include testing for chromosomal rearrangements that are currently not analyzed in this panel.”

Nikiforova said she and colleagues hope that targeted next generation sequencing will offer a new molecular tool for establishing diagnosis of thyroid cancer preoperatively in FNA samples in a very cost efficient way.

For more information:

Nikiforova MN. O33. Presented at: the American Thyroid Association Annual Meeting; Sept. 19-23, 2012; Quebec City.

Disclosure: The researchers report no relevant financial disclosures.

Perspective

Nikolaos Stathatos, MD

This is an exciting time for thyroid oncology. Our ability to diagnose and assess thyroid cancer continues to improve daily with new techniques like this next generation sequencing approach. However, just like with any new method, the appropriate testing needs to be done and that takes time. As promising as it may be, extensive clinical testing will have to be done first. It is an expensive technique and unless we have more data about its clinical applicability, widespread use is probably not a good idea.

Dr. Nikiforova's group is extremely active and I have no doubt they will continue to update us on the utility of this new method in the daily clinical practice. However, I would suggest caution until such data becomes available. If there was one thing made clear at this year's ATA meeting, is that there is no uniform agreement about genetic testing for thyroid cancer, even for the commercially available methods.

Nikolaos Stathatos, MD

Massachusetts General Hospital

Boston, MA

Disclosures: Stathatos has no relevant financial disclosures.