Purpose or Case Report: Currently, biopsy serves as the gold standard to accurately diagnose disease in pediatric sarcoma patients, but the risks of anesthesia and surgery, along with a failure to characterize the true heterogeneity of disease make this method less than ideal. Progression and response to therapy are monitored by radiologic exams which lack the sensitivity to detect early relapse. Cell free DNA (cfDNA) is released into the plasma as they undergo apoptosis and necrosis. ctDNA represents a small fraction of cfDNA in cancer patients and contains tumor specific alterations. It holds promise as a highly sensitive and specific biomarker. A limitation in applying liquid biopsy in clinical practice is the need to develop PCR or other DNA analysis methods to detect alterations specific to a single patient. We have developed a more widely applicable off the shelf test that does not involve a patient specific design.
Methods & Materials: Our CAPP Seq technique involves designing a selector comprised of custom designed oligonucleotide probes that tile across genomic regions of interest. These oligonucleotide probes are used to enrich for the relevant ctDNA via hybrid capture, followed by ultra deep sequencing to analyze alterations in the selected regions. Our selector was applied to pretreatment plasma samples from newly diagnosed or newly relapsed pediatric sarcoma patients. Plasma samples were analyzed at key timepoints over the course of treatment.
Results: Pediatric sarcoma patients had higher levels of cfDNA when compared to published levels in adult cancer patients. Canonical translocations were detected in the plasma of 13/14 (93%) pediatric sarcoma patients. This was confirmed by analysis of matched tumor samples, when available. Patients with metastatic disease had higher ctDNA levels compared to nonmetastatic patients. ctDNA levels correlated with clinical
course and, in some cases, rising ctDNA levels predicted relapse, earlier than was clincally apparent by imaging studies.
Conclusions: ctDNA analysis holds promise as an ultrasensitive and specific tool for monitoring tumor burden. Our assay was able to detect ctDNA in the plasma of metastatic and nonmetastatic pediatric sarcoma patients at diagnosis. Furthermore, we demonstrated that ctDNA levels correlated with clinical response to therapy. In some cases, ctDNA levels proved more sensitive than imaging, detecting minimal residual disease and
predicting relapse.