A substantial percentage of patients with primary lung cancer either present with, or subsequently develop, a second cancerous lesion in the lung. However, differentiating between a second, independent primary cancer and intrapulmonary metastasis of the primary lesion is often challenging.

Determination of tumor histology on pathologic examination of surgical specimens remains the standard for distinguishing these 2 types of tumors, and histologic determinations can be facilitated by tumor genomic assessments identifying common, early, driver mutations (eg, EGFR and KRAS). Nevertheless, limitations of these methods remain.

In this study, the method of mate-pair sequencing was used to more accurately evaluate the DNA “breakpoints” or “junctions” involved in complex chromosomal rearrangements leading to gene fusions.

The distinctive signatures found at somatic junctions “present ideal markers to define a common origin or lineage in the multiple tumor setting,” the study authors noted.

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Mate-pair sequencing is a process that facilitates DNA sequencing of longer DNA fragments. Most standard next-generation DNA sequencing processes are limited in that they typically work by piecing together DNA fragments that are in the range of 200 to 800 base pairs (bps). However, this limitation can interfere with the precise characterization of certain molecular alterations, such as gene-fusion junctions.2

In the mate-pair sequencing process, the ends of longer DNA fragments (2-5 kilobases) are biotinylated; this results in circularization of these fragments. The circular DNA is then cut into smaller length fragments, and the smaller fragments containing biotin are isolated and sequenced from both ends, facilitating maximal coverage of DNA sequences corresponding to junctional regions of gene rearrangements.2

A total of 76 distinct non-small cell lung cancer tumors from 37 patients were analyzed in the study. Data related to clinical tumor designation (based on histological, radiological data) as either a second primary or a metastatic lesion were abstracted from patient charts.  In addition, 2 pulmonary pathologists made independent histologic assessments of the tumor specimens (both pathologists had no prior knowledge of previous data).

Somatic DNA was extracted from tumor specimens, and sequenced using a standard clinical next-generation sequencing lung cancer assay designed to detect alterations in EGFR, BRAF, KRAS, HRAS, NRAS, ALK, ERBB2, and MET. In addition,tumorRNA was sequenced for ALK, ROS1, RET, and NTRK1 gene fusions.  Finally, somatic DNA extracted from tumor specimens was interrogated using mate-pair sequencing.

Of the 41 matched tumor pairs investigated, tumor lineage was possible to determine in 100% of specimen pairs using mate-pair sequencing to facilitate elucidation of shared and unique junctional regions of gene rearrangements. In contrast, 59%, 24%, and 17% were designated as independent primaries, related metastases, and indeterminate, respectively, on independent pathologic histological review.

When prior clinical assessment was compared with these 2 other methods (ie, histological review and mate-pair sequenced junctions), concordance was observed in only 63% of cases. Using the limited next-generation sequencing panel, 53% of the screened tumors had no gene mutations, and no gene fusions were detected in these specimens using the RNA sequencing assay.

“[In] future scenarios, lineage calling from mate-pair sequencing data could be auxiliary to therapeutic patient management testing, similar to the current lung actionable gene panels.” the study authors concluded.

References

  1. Murphy SJ, Harris FR, Kosari F, et al. Using genomics to differentiate multiple primaries from metastatic lung cancer [published online May 16, 2019]. J Thorac Oncol. doi: 10.1016/j.jtho.2019.05.008
  2. ECSeq Bioinformatics. What is mate pair sequencing for? https://www.ecseq.com/support/ngs/what-is-mate-pair-sequencing-useful-for. Updated March 20, 2017. Accessed May 29, 2019.