Clinical Liquid Biopsy Testing for Detection of Actionable Genomic Alterations in Children and Young Adults With Advanced Solid Tumors on the GAIN Study

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The most frequent diagnoses were osteosarcoma (n=9) and neuroblastoma (n=8), and 16 distinct tumor types were represented.

“The most common diagnoses were OS (n = 9) and NBL (n = 8), with a total of 16 distinct tumor types.”

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Detectable ctDNA tumor fraction in at least one sample was found in most patients with neuroblastoma (6/8, 75%), osteosarcoma (5/9, 56%), other sarcoma types (4/6, 67%), and other nonsarcoma tumors (4/7, 57%).

“Most patients with NBL (6 of 8, 75%), OS (5 of 9, 56%), other sarcoma types (4 of 6, 67%), and other nonsarcoma tumors (4 of 7, 57%) also had detectable ctDNA TF in at least one sample.”

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Among all 92 liquid biopsy samples, 51 (55%) had detectable ctDNA, with a median tumor fraction of 23% and a range of 0.4% to 96%.

“Among all 92 liquid biopsy samples, 51 (55%) had detectable ctDNA (median TF 23% [range, 0.4%–96%])”

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Detection of copy number alterations by ctDNA sequencing was lower overall, but was higher with tumor fraction at least 1% (40%, 35 of 87) than with tumor fraction below 1% (2%, 2 of 89).

“There is lower detection of CNAs via ctDNA sequencing, though still higher with TF ≥ 1% (40%, 35 of 87) compared with TF < 1% (2%, 2 of 89; Fig 3A).”

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Median time from sample collection to return of results was 9 days, with a range of 7 to 27 days.

“The median time for return of results from sample collection was 9 days (range, 7–27 days).”

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Five additional samples failed F1LCDx because of inadequate DNA (n=4) or quality control failure (n=1) and were excluded, giving a successful result return rate of 92 of 97 samples (95%).

“Five additional samples were unsuccessful for F1LCDx because of inadequate DNA (n = 4) or failure to meet quality control metrics (n = 1) and were excluded from analysis. Therefore, the rate of successful return of results was 92 of 97 samples (95%).”

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The most frequently altered genes across tissue and ctDNA were TP53 (12.6%, 37 of 293 variants), EWSR1 (6.5%, 19 of 293), and BRAF, MYCN, and RICTOR (each 3.4%, 10 of 293).

“The most frequently altered genes in tissue and ctDNA cumulatively were TP53 (12.6%, 37 TP53 variants/293 total variants), EWSR1 (6.5%, 19 of 293), BRAF, MYCN, and RICTOR (each 3.4%, 10 of 293).”

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Seventy-two percent of patients had metastatic disease at diagnosis, and all patients had active disease at the time of first liquid biopsy.

“Most patients (72%) had metastatic disease at diagnosis, and all patients had active disease at the time of first liquid biopsy.”

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Median age at diagnosis was 13.1 years, with a range of 0.7 to 23.3 years.

“The median age at diagnosis was 13.1 years (range, 0.7–23.3 years).”

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Among samples with tumor fraction at least 1%, detection of tissue short variants and translocations in ctDNA varied by tumor type: 100% (27/27) in Ewing sarcoma, 83% (5/6) in neuroblastoma, 43% (6/14) in osteosarcoma, 100% (7/7) in other sarcomas, and 91% (10/11) in other nonsarcoma tumors.

“In samples with TF ≥ 1%, we observed differences in the detection of short variants and translocations in ctDNA that were present in tissue across tumor types, with 100% (27 of 27) detected in EWS, 83% (5 of 6) in NBL, 43% (6 of 14) in OS, 100% (7 of 7) in other sarcomas, and 91% (10 of 11) in other nonsarcoma tumors (Fig 3B, Appendix Table A1).”

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Ninety-two F1LCDx liquid biopsy samples collected between February 2023 and November 2024 were analyzed, with a median of two samples per patient and a range of one to six.

“A total of 92 F1LCDx liquid biopsy samples collected between February 2023 and November 2024 were included in the analysis, with a median of two liquid biopsy samples per patient (range, 1–6).”

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ctDNA was detected in 42% (15 of 36) of patients' first liquid biopsy samples, with a median tumor fraction of 33% and a range of 0.55% to 96%.

“with detection in 42% (15 of 36) of patients’ first liquid biopsy samples (median TF 33% [range, 0.55%–96%]).”

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Seventy-two percent of patients (26 of 36) had at least one ctDNA sample with a clinically relevant genomic alteration, while 89% (32 of 36) had at least one clinically relevant alteration in tumor tissue.

“Among all patients in our cohort, 72% (26 of 36) had at least one ctDNA sample with a clinically relevant genomic alteration identified and 89% (32 of 36) had at least one clinically relevant alteration detected in tumor tissue.”

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ALK mutations were detected in neuroblastoma ctDNA in this cohort, including two cases with wild-type ALK in diagnostic tissue, and one patient had successful targeted treatment.

“We similarly detect ALK mutations in NBL ctDNA in our cohort, including two cases with wild-type ALK in diagnostic tissue, prompting successful targeted treatment in one patient.”

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At progression in Patient 30, a new HRAS p.Q61L mutation emerged and a pre-existing TP53 p.L194P variant expanded.

“At the time of progression, a new HRAS p.Q61L mutation (VAF 0.18%) emerged and a pre-existing TP53 p.L194P variant expanded, suggesting clonal tumor evolution and resistance to ALK inhibition.”

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Patient 9 with Ewing sarcoma had an mTOR p.I2500M mutation found in ctDNA and was treated with everolimus, but died shortly after starting therapy before further disease assessment.

“Patient 9 with EWS had an mTOR p.I2500M mutation (known functional, VAF 0.38%) discovered in ctDNA and was subsequently treated with mTOR inhibitor everolimus. However, the patient died shortly after initiation of therapy before further disease assessment.”

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Patients with progressive disease had a mean absolute change in ctDNA tumor fraction of +12.2% (SD 18.7%), compared with -0.1% (SD 14%) in stable disease and -23.4% (SD 30.6%) in partial response, with a statistically significant difference between partial response and progressive disease (P < .001).

“Patients with PD had a mean absolute change in ctDNA TF of +12.2% (standard deviation 18.7%), compared with −0.1% in patients with SD (standard deviation 14%) and −23.4% in patients with PR (standard deviation 30.6%), as demonstrated in Figure 4, with a statistically significant difference between PR and PD (P < .001).”

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Targetable mutations were identified across a range of pediatric solid tumors, but multiple rare histologies in the cohort had no ctDNA or targetable variants identified, requiring further investigation of ctDNA shedding.

“While we identified targetable mutations across a range of pediatric solid tumors, there were multiple rare histologies in our cohort in which ctDNA and targetable variants were not identified, requiring further investigation of ctDNA shedding.”

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Patient 29 with malignant peripheral nerve sheath tumor had an ROS1 translocation and EGFR p.R222C mutation in ctDNA, prompting ROS and EGFR inhibitor recommendations, although neither was pursued.

“Patient 29 with malignant peripheral nerve sheath tumor had an ROS1 translocation (likely functional) and EGFR p.R222C mutation (known functional, VAF 0.14%) in ctDNA, prompting ROS and EGFR inhibitor recommendations, although neither was pursued.”

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Patient 6 with neuroblastoma had an ALK p.F1174L mutation in a ctDNA sample with tumor fraction 64% that was not identified in archival tumor tissue, but this result did not change clinical care because of the patient's clinical instability.

“Separately, Patient 6 with NBL was also found to have an ALK p. F1174L mutation (known functional, VAF 2.2%) in a ctDNA sample with TF 64% that was not identified in archival tumor tissue. However, this result did not affect clinical care because of the patient’s clinical instability.”

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A growing body of evidence shows many clinical uses for ctDNA in adults with solid tumors, from prognostication and minimal residual disease evaluation to detection of targetable alterations.

“There is a growing body of data demonstrating numerous clinical applications for ctDNA in adults with solid tumors, ranging from prognostication and evaluation of minimal residual disease (MRD) to detection of targetable alterations.7–12”

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A more recent study found that serial clinical ctDNA sampling in high-risk neuroblastoma helped monitor disease status and clonal tumor evolution.

“A more recent study demonstrated that serial clinical ctDNA sampling in patients with high-risk neuroblastoma (NBL) was useful for monitoring disease status and clonal tumor evolution.19”

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Classified as probably not a substantial claim. May combine two claims.

Clinical impact of F1LCDx results was assessed through medical record review, and a result counted as impactful only when the treating physician explicitly documented that ctDNA contributed to management.

“We determined the clinical impact of results from F1LCDx reports via review of the EMR. Results were considered clinically impactful only if the treating physician explicitly documented that ctDNA results contributed to management.”

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ctDNA sequencing was done in a CLIA-certified and CAP-accredited laboratory using the validated, FDA-approved F1LCDx next-generation sequencing panel.

“ctDNA sequencing was performed in a CLIA-certified, College of American Pathologists–accredited laboratory (Foundation Medicine), using the validated, FDA-approved NGS panel assay F1LCDx.20”

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The study was approved by the Institutional Review Board at participating institutions.

“The study enrolled patients from clinical sites throughout the United States and was approved by the Institutional Review Board at participating institutions.”

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Children and young adults with advanced solid tumors continue to have poor outcomes.

“Outcomes remain poor for children and young adults with advanced solid tumors.1–5”

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• AssumptionRests on an assumption: A lack of non-invasive clinical biomarkers for care and novel clinical trials contributes to poor outcomes in children and young adults with advanced solid tumors.

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This assay was not assessed as a means of detecting minimal residual disease.

“However, we did not assess this assay as a means of detecting MRD.”

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Comparison of genomic findings between tissue and ctDNA was limited by differences in sequencing panels, curation pipelines, and sample timing, although genes absent from both panels were excluded from comparative analyses.

“The comparison of genomic findings in tissue and ctDNA was limited by differences in sequencing panels, curation pipelines, and sample timing although we attempted to correct for this by excluding genes from comparative analyses that were not present in both panels.”

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A single ctDNA assay was used, so generalizability to other assays is unknown.

“We used a single ctDNA assay, so generalizability to other assays is unknown.”

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The detection rate of actionable alterations in ctDNA with F1LCDx is higher in adult tumors than in this pediatric cohort, likely because of differences in tumor burden and the lack of truncal driver alterations in rare pediatric tumors.

“We acknowledge that the detection rate of actionable alterations in ctDNA using the F1LCDx assay is higher in adult tumors than our pediatric cohort, which is likely related to differences in tumor burden between the cohorts and the lack of truncal driver alterations in rare pediatric tumors.”

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Measuring ctDNA burden gives key information about the sensitivity of ctDNA for detecting actionable variants at a given time and may be useful even when disease burden is relatively low.

“Thus, measurement of ctDNA burden provides key information regarding the sensitivity of ctDNA to detect actionable variants at a specific time point and may be useful even when disease burden is relatively low.”

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Previous studies have found that children with solid tumors often have clinically relevant variants detected by ctDNA assays designed to capture common pediatric tumor copy number alterations and translocations.

“Previous studies have found that children with solid tumors have frequently detected variants of clinical relevance via ctDNA assays designed to capture common pediatric tumor CNAs and translocations.”

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ctDNA tumor fraction is not only an important biomarker for quantifying disease burden but also helps determine whether to reflex a negative liquid biopsy to a tissue test in pediatric solid tumors.

“We additionally demonstrated that ctDNA TF is not only an important biomarker in quantifying disease burden but also aids in determining whether to reflex a negative liquid biopsy to a tissue test in pediatric solid tumors.”

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A well-established relationship exists between ctDNA burden and disease status in adult solid tumors, as shown by ctDNA use for minimal residual disease detection and recurrence monitoring in colorectal cancers.

“A salient example of the well-established relationship between ctDNA burden and disease status in adult solid tumors is the use of ctDNA for MRD detection and monitoring for recurrence in colorectal cancers.10–12”

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Reliance on clinical documentation in the electronic medical record limited assessment of the clinical impact of ctDNA sequencing and identification of correlation between ctDNA changes and radiographic disease response.

“Reliance on clinical documentation in the EMR limited the assessment of clinical impact of ctDNA sequencing and identification of correlation between ctDNA changes and radiographic disease response.”

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Interpretation of very low or undetectable ctDNA tumor fraction is limited and is suspected to be multifactorial, including low disease burden, low ctDNA shedding, or assay inability to detect ctDNA because of tumor genomics not optimally matched to the assay.

“We acknowledge that this limits the interpretation of ctDNA TF that is very low or undetectable, which we suspect is multifactorial, including low disease burden, low ctDNA shed, or inability of the assay to detect ctDNA because of underlying tumor genomics that are not optimally matched to the assay.”

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The F1LCDx ctDNA assay was effective for detecting ctDNA in peripheral blood, noninvasively identifying key genomic variants particularly when ctDNA tumor fraction was at least 1%, and monitoring disease burden in children and young adults with advanced solid tumors.

“In conclusion, the F1LCDx ctDNA assay was effective in detecting ctDNA in peripheral blood, noninvasively identifying key genomic variants particularly with ctDNA TF ≥1%, and monitoring disease burden in children and young adults with advanced solid tumors.”

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The emergence of HRAS p.Q61L and expansion of TP53 p.L194P in Patient 30 suggest clonal tumor evolution and resistance to ALK inhibition.

“At the time of progression, a new HRAS p.Q61L mutation (VAF 0.18%) emerged and a pre-existing TP53 p.L194P variant expanded, suggesting clonal tumor evolution and resistance to ALK inhibition.”

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This study shows that ctDNA burden may be a useful marker of response to systemic therapy across multiple pediatric solid tumor types, with statistically significant changes in ctDNA tumor fraction between partial response and progressive disease.

“Our study shows that ctDNA burden may be a useful marker of response to systemic therapy across multiple pediatric solid tumor types, with statistically significant changes in ctDNA TF between PR and PD.”