Identification of Independent Primary Lung Tumors and Tumors with Metastases Using Next-Generation DNA Sequencing
By LabMedica International staff writers Posted on 25 Nov 2014 |
A team of oncologists and surgeons used a next-generation DNA sequencing (NGS) technique to determine whether non-small-cell lung cancer patients with multiple tumors carried independent primary tumors or a single primary tumor that had metastasized.
For patients with multiple tumors, distinguishing independent primary tumors from metastases could mean the difference between correct identification of early stage cancer (primary tumors) that may be cured by surgery and incurable late-stage disease (multiple metastases).
Investigators at the Mayo Clinic (Rochester, MN, USA) utilized an Illumina (San Diego, CA, USA) mate-pair library protocol to sequence the DNA from 41 tumor samples (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]) taken from patients with known independent primary lung tumors and metastatic lesions.
Mate pair library preparation generates long-insert paired-end DNA libraries useful for a number of sequencing applications. This technique requires that following DNA fragmentation, the DNA fragments are end-repaired with labeled deoxynucleotide bases. The DNA fragments are circularized, and non-circularized DNA is removed by digestion. Circular DNA is fragmented, and the labeled fragments (corresponding to the ends of the original DNA ligated together) are affinity purified. Purified fragments are end-repaired and ligated to Illumina paired-end sequencing adapters. Additional sequences complementary to the flow cell oligonucleotides are added to the adapter sequence with tailed PCR primers. The final prepared libraries consist of short fragments made up of two DNA segments that were originally separated by several kilobases. These libraries are ready for paired-end cluster generation, followed by sequencing utilizing an Illumina NGS platform.
Results from DNA sequencing revealed that in patients with primary lung tumors and paired distant metastases, shared chromosomal rearrangements occurred in all tumor pairs. In contrast, lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data was found for more than 82% of the samples.
“Clinicians often end up working with inexact, imperfect information to make potentially life-altering decisions for how we treat our patients,” said senior author Dr. Dennis Wigle, a thoracic surgeon at the Mayo Clinic. “Now we have a test that may be able to tell us whether we are dealing with independent primary tumors or metastatic disease. It is not definitive in terms of a test that we can roll out to clinical practice tomorrow, but it does show proof of principle. We need to expand this study to analyze more tumor samples and get more data on the sensitivity and specificity of this test. Then we will need to explore how we can make this test a part of standard diagnostic testing and finally assess how it will impact clinical care. Hopefully this test will provide an inroad for clinicians faced with scenarios where they do not know what course of action is best for their patients.”
The study was published in the November 10, 2014, online edition of the Journal of Clinical Oncology.
Related Links:
Mayo Clinic
Illumina
For patients with multiple tumors, distinguishing independent primary tumors from metastases could mean the difference between correct identification of early stage cancer (primary tumors) that may be cured by surgery and incurable late-stage disease (multiple metastases).
Investigators at the Mayo Clinic (Rochester, MN, USA) utilized an Illumina (San Diego, CA, USA) mate-pair library protocol to sequence the DNA from 41 tumor samples (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]) taken from patients with known independent primary lung tumors and metastatic lesions.
Mate pair library preparation generates long-insert paired-end DNA libraries useful for a number of sequencing applications. This technique requires that following DNA fragmentation, the DNA fragments are end-repaired with labeled deoxynucleotide bases. The DNA fragments are circularized, and non-circularized DNA is removed by digestion. Circular DNA is fragmented, and the labeled fragments (corresponding to the ends of the original DNA ligated together) are affinity purified. Purified fragments are end-repaired and ligated to Illumina paired-end sequencing adapters. Additional sequences complementary to the flow cell oligonucleotides are added to the adapter sequence with tailed PCR primers. The final prepared libraries consist of short fragments made up of two DNA segments that were originally separated by several kilobases. These libraries are ready for paired-end cluster generation, followed by sequencing utilizing an Illumina NGS platform.
Results from DNA sequencing revealed that in patients with primary lung tumors and paired distant metastases, shared chromosomal rearrangements occurred in all tumor pairs. In contrast, lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data was found for more than 82% of the samples.
“Clinicians often end up working with inexact, imperfect information to make potentially life-altering decisions for how we treat our patients,” said senior author Dr. Dennis Wigle, a thoracic surgeon at the Mayo Clinic. “Now we have a test that may be able to tell us whether we are dealing with independent primary tumors or metastatic disease. It is not definitive in terms of a test that we can roll out to clinical practice tomorrow, but it does show proof of principle. We need to expand this study to analyze more tumor samples and get more data on the sensitivity and specificity of this test. Then we will need to explore how we can make this test a part of standard diagnostic testing and finally assess how it will impact clinical care. Hopefully this test will provide an inroad for clinicians faced with scenarios where they do not know what course of action is best for their patients.”
The study was published in the November 10, 2014, online edition of the Journal of Clinical Oncology.
Related Links:
Mayo Clinic
Illumina
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