In a recent study published in Journal of Thoracic Oncology, the Network Genomic Medicine Lung Cancer, which was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer, performing molecular analyses of patients, demonstrates the feasibility of using a highly multiplexed targeted resequencing assay for detecting somatic mutations in patients for lung cancer diagnosis.

These researchers decided that an Ion AmpliSeq Custom DNA Panel containing 102 amplicons of 12 different genes (Life Technologies) was ideal for their needs because of the flexibility of the CP design, and initial testing which indicated that previously determined variants could be detected in routine diagnostics.

However, the Network Genomic Medicine Lung Cancer also wanted to sequence the multiplex PCR product obtained from the Ion AmpliSeq panel on a MiSeq Benchtop sequencer. Using the MiSeq they multiplex 44 patients with a mean coverage of 4000×, a quality Q30 score of over 95% and a minimal coverage of 500 in 99% of the amplicons. They were able to do this by incubating the PCR products with NEXTflex DNA Adenylation Mix and then ligating NEXTflex DNA Barcode adapters to the fragments. 10 cycles of PCR were then run and the samples were sequenced on an Illumina MiSeq (Fig. 1). Using this protocol, sample preparation was reduced by 4 ½ hours and turnaround time was reduced from 12 days to 10 days when compared to conventional dideoxy-sequencing, while augmenting the number of targets.

DNA-Module-and-Barcode-Flow-Chart-for-Journal-of-Thoracic-Oncology

This comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, the Network Genomic Medicine Lung Cancer identified driver mutations at the expected prevalence. Furthermore they found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas.

Overall, these data demonstrate the utility of NGS analysis in a clinical routine setting, and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

König, K. et al. (2015) Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients. Journal of Thoracic Oncology. 10 :7, 1049–1057. doi: 10.1097/JTO.0000000000000570.