These results demonstrate that massively parallel sequencing can be used to develop personalized biomarkers based on somatic rearrangements. We were able to identify tumor-specific markers in each of the six breast and colorectal cancer cases analyzed. Moreover, we demonstrated that the identified breakpoints can be used to detect tumor DNA in the presence of large quantities of normal DNA and in patient plasma. These results highlight the sensitivity and specificity of the approach and suggest broad clinical utility of the PARE method.
Virtually all tumors of clinical consequence are thought to have rearranged DNA sequences resulting from translocations and copy number alterations, and these sequences are not present in normal human plasma or nontumor tissues. A recent genome-wide analysis of 24 breast cancers showed that all analyzed samples contained at least one genomic rearrangement that could be detected by next generation sequencing (24
). From a technical perspective, PARE derived clinical assays should have no false positives: The PCR amplification of aberrant fusions of DNA sequences that are normally thousands of base pairs apart or on different chromosomes should not occur in nontumor DNA. In contrast, approaches that rely on monitoring of residual disease by analysis of somatic single-base alterations in specific genes are limited by polymerase error rates at the bases of interest (25
). The PCR process generates background single-base mutations that are identical to bona fide mutations but does not generate false-positive rearrangements with carefully chosen primers. Because of the higher signal-to-noise ratio thereby obtained, PARE theoretically permits more sensitive monitoring of tumor burden.
The PARE approach, however, is not without limitations. Although somatic alterations in oncogenes and tumor suppressor genes persist throughout the clonal evolution of a tumor, it is conceivable that some rearranged sequences could be lost during tumor progression. The identification of several PARE biomarkers, each specific for different chromosomal regions, would mitigate this concern, as it is unlikely that all such markers would be lost in any particular patient. Another limitation is the cost of identifying a patient-specific alteration. In this prototype study, we obtained an average of 194.7 million reads per patient, resulting in ~200 tags in each 3-kb bin. The current cost for such an assay is ~$5000, which is expensive for general clinical use. This cost is a consequence of the high physical coverage and the inefficiencies associated with stringent mapping of 25-bp sequence data to the human genome. As read quality and length continue to improve, less stringent mapping criteria and lower physical coverage will permit analyses similar to those in this study but with substantially less sequencing effort. Moreover, the cost of massively parallel sequencing, which has decreased substantially over the last 2 years, continues to spiral downwards. Finally, there are clinical settings where the fraction of any DNA from tumors, including rearranged sequences, in the patient plasma is exceedingly small and undetectable. To be detectable by PARE, there must be at least one rearrangement template molecule in the plasma sample analyzed. When disease burden is this light, PARE may yield false-negative results. Larger studies will be needed to determine the clinical utility of PARE and its prognostic capabilities.
Despite these caveats, there are numerous potential applications of PARE. These include the more accurate identification of surgical margins free of tumor and the analysis of regional lymph nodes as well as the measurement of circulating tumor DNA after surgery, radiation, or chemotherapy. Short-term monitoring of circulating tumor DNA may be particularly useful in the testing of new drugs, as it could provide an earlier indication of efficacy than is possible through conventional diagnostic methods such as computed tomography (CT) scanning. Given current enthusiasm for the personalized management of cancer patients, PARE affords a timely method for uniquely sensitive and specific tumor monitoring.