Lung Cancer Study Develops New Test to Differentiate Multiple Primary Tumors from Metastasis
A recent study by Mayo Clinic researchers published in the Journal of Clinical Oncology addresses the question of whether multiple lesions in lung cancer patients represent true metastasis versus independent primary lesions, which is a difficult clinical problem with significant implications. Using next-generation DNA sequencing, researchers developed a chromosomal rearrangement–based test to differentiate multiple primary tumors from metastasis. This critical distinction typically represents the difference between aggressive local therapy for patients deemed to have independent primary tumors versus less aggressive, or even palliative systemic, therapy for patients thought to have metastatic disease—diagnosed when the tumor cells are interrelated, developed from a common lineage, and spread secondary malignant growths beyond the primary tumor.
Current methods for determining independent primary tumors from metastasis are largely based on provider-specific clinical and/or pathologic assumptions. There are presently no ancillary tests that allow this distinction to be made with any reasonable clinical accuracy. “Pathology review is the current gold standard,” says Stephen Murphy, Ph.D., a research scientist in the Mayo Clinic Center for Individualized Medicine, and co-first author on the paper. “When a patient presents with distal pulmonary nodules, biopsies from each nodule are reviewed by pathologists who assess relatedness through the presence or absence of common histological features. However, pathologists often disagree with contrasting calls in maybe twenty percent of cases.”
For this study, tumor specimens from patients with known independent primary tumors and metastatic lesions were used for lineage test development, which was then applied to multifocal lung tumors. Laser capture microdissection (LCM) was performed separately for each tumor. “However, in the final test version, most tumors will have high enough tumor volumes to perform the test directly on bulk extracted tissues without LCM.” says Dr. Murphy. Eliminating LCM from the final version will make the test less costly and more user-friendly for clinicians.
Genomic DNA was amplified directly from captured cells using methodology developed in the research group, and next-generation sequencing was performed using the Illumina Mate Pair protocol. A total of 41 tumor samples were sequenced, with a range of 3 to 276 breakpoints per tumor identified. “Genomic rearrangements are common in lung tumors, and breakpoints are the fusion junctions where chromosomes have shuffled, forming new chromosomal structures,” says Dr. Murphy.
Lung tumors predicted to be independent primary tumors based on different histologic subtypes did not share any somatic genomic rearrangements. In patients with paired primary lung tumors and distant metastases, shared rearrangements were identified in all tumor pairs, emphasizing the tumor specificity of identified breakpoints.
Cases 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.
Remarkably, the study found that samples, clearly classified as either independent primary tumors or metastases, based on clinical and histologic parameters, can be accurately separated into independent primary tumors and metastases based on genomic data. Most significantly, none of the detected breakpoints were shared between tumors from different patients. “These breakpoints really are very unique to each tumor,” says Dr. Murphy. “We’ve now sequenced more than 1,500 tumor samples, and we’ve never seen an identical breakpoint in non-related tumors.”
Dr. Murphy and the Mayo Biomarker Discovery research team already foresee wider applications for this test once it’s refined. “Squamous cell carcinomas frequently manifest concurrently in the head, neck, and lungs of the same patient and are generally indistinguishable using traditional histopathology,” he says. “Additionally, with non-synchronous tumors, this test will aid in determining whether a re-emerging tumor is a metastatic recurrence or a new independent primary. Basically, this test will aid clinicians in defining independence or metastatic disease with any tumor pair.”