Identification of independent primary tumors and intrapulmonary metastases using DNA rearrangements in non-small-cell lung cancer

Purpose: Distinguishing independent primary tumors from intrapulmonary metastases in non-small-cell carcinoma remains a clinical dilemma with significant clinical implications. Using next-generation DNA sequencing, we developed a chromosomal rearrangement-based approach to differentiate multiple primary tumors from metastasis. Methods: Tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal tumors. Laser capture microdissection was performed separately for each tumor. Genomic DNA was isolated using direct in situ whole-genome amplification methodology, and next-generation sequencing was performed using an Illumina mate-pair library protocol. Sequence reads were mapped to the human genome, and primers spanning the fusion junctions were used for validation polymerase chain reaction. Results: A total of 41 tumor samples were sequenced (33 adenocarcinomas [ADs] and eight squamous cell carcinomas [SQCCs]), with a range of three to 276 breakpoints per tumor identified. Lung tumors predicted to be independent primary tumors based on different histologic subtype did not share any genomic rearrangements. In patients with lung primary tumors and paired distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the patient specificity of identified breakpoints. Multifocal AD and SQCC samples were reviewed independently by two pulmonary pathologists. Concordance between histology and genomic data occurred in the majority of samples. Discrepant tumor samples were resolved by genome sequencing. Conclusion: A diagnostic lineage test based on genomic rearrangements from mate-pair sequencing demonstrates promise for distinguishing independent primary from metastatic disease in lung cancer.