Driver mutations in the CTNNB1 gene (encoding β-catenin) are a hallmark of sporadic hepatoblastoma (HBL). Our results show that CTNNB1 circulating tumour DNA (ctDNA) is readily detected in patients diagnosed with localised HBL, with serial sampling along the course of therapy and follow up providing a sensitive mechanism to monitor tumour dynamics and response to treatment. This exciting potential for CTNNB1 ctDNA to serve as a biomarker for treatment response in HBL holds clinical value, and requires assessment in a larger cohort of mixed tumour stages and recurrent disease.
Liquid biopsy is rapidly gaining traction for potentially revolutionizing cancer diagnosis and treatment through blood-based utilization of shed biomolecules. This approach can provide a global picture of the cancer in real time, at multiple time points, and with minimal invasiveness. In this review, we familiarize cancer biobanks with the principles used for liquid biopsy work and highlight unique aspects of applying liquid biopsy approaches to pediatric cancers to enable high-quality and efficient translational research.
Background: MYCN amplification (MNA), segmental chromosomal aberrations (SCA) and ALK activating mutations are biomarkers for risk-group stratification and for targeted therapeutics for neuroblastoma, both of which are currently assessed on tissue biopsy. Increase in demand for tumor genetic testing for neuroblastoma diagnosis is posing a challenge to current practice, as the small size of the core needle biopsies obtained are required for multiple molecular tests. We evaluated the utility of detecting these biomarkers in the circulation. Methods: Various pre-analytical conditions tested to optimize circulating-tumor DNA (ctDNA) copy number changes evaluations. Plasma samples from 10 patients diagnosed with neuroblastoma assessed for SCA and MNA using single nucleotide polymorphism (SNP) array approach currently used for neuroblastoma diagnosis, with MNA status assessed independently using digital-droplet PCR (ddPCR). Three patients (one in common with the previous 10) tested for ALK activating mutations p.F1174L and p.F1245I using ddPCR. Results: Copy number detection is highly affected by physical perturbations of the blood sample (mimicking suboptimal sample shipment), which could be overcome using specialized preservative collection tubes. Pre-analytical DNA repair procedures on ctDNA before SNP chromosome microarray processing improved the lower limit of detection for SCA and MNA, defined as 20% and 10%, respectively. We detected SCA in 10/10 (100%) patients using SNP array, 7 of which also presented MNA. Circulating-free DNA (cfDNA) and matched tumor DNA profiles were generally identical. MNA was detected using ddPCR in 7/7 (100%) of MNA and 0/12 (0%) non-MNA cases. MNA and ALK mutation dynamic change was assessed in longitudinal samples from 4 and 3 patients (one patient with both), respectively, accurately reflected response to treatment in 6/6 (100%) and disease recurrence in 5/6 (83%) of cases. Samples taken prior to targeted treatment with the ALK inhibitor Lorlatinib and 6–8 weeks on treatment showed reduction/increase in ALK variants according to response to treatment. Conclusions: These results demonstrate the feasibility of ctDNA profiling for molecular risk-stratification, and treatment monitoring in a clinically relevant time frame and the potential to reduce fresh tissue requirements currently embedded in the management of neuroblastoma.
Driver mutations in CTNNB1 are a hallmark of hepatoblastoma and offer a common biomarker for a liquid biopsy approach based on the presence of CTNNB1 circulating tumor DNA (ctDNA). We developed and investigated the utility of a quantitative universal next-generation sequencing (NGS) ctDNA assay for hepatoblastoma (QUENCH) to detect CTNNB1 ctDNA and assessed the links between ctDNA and current clinical indicators/biomarkers in hepatoblastoma. Applied to patients with hepatoblastoma, we demonstrate quantitation of various variants including single base substitutions and deletions down to 0.3% variant allele frequency, with 65% sensitivity and 100% specificity at the patient level, to allow biopsy-free tumor genotyping and sensitive ctDNA quantitation. CtDNA positivity correlates with tumor burden and ctDNA levels correlate with macroscopic residual disease and treatment response, thus providing promising evidence for the utility of quantitative ctDNA detection in hepatoblastoma.
Driver mutations in CTNNB1 are a hallmark of hepatoblastoma (HB) and offer a common biomarker for a liquid biopsy approach that is based on the presence of CTNNB1 circulating tumor DNA (ctDNA). Initial results 1 showed an association of CTNNB1 ctDNA variant allele frequency (VAF) identified through digital droplet PCR (ddPCR) and the levels of serum Alpha-fetoprotein (AFP, current HB biomarker) throughout the course of treatment of three patients with HB. However, applying custom probe-designed ddPCR assays may pose challenges for real-time data at diagnosis. Our primary objective is to investigate the utility of a universal next-generation sequencing (NGS) assay to detect CTNNB1 ctDNA in patients with HB. Our secondary objective is to compare the levels of: (1) NGS ctDNA, (2) ddPCR ctDNA, (3) AFP, and verify if they correlate with tumor burden and treatment response. We developed and tested a custom NGS assay covering exons 2 to 4 of CTNNB1 for detection of somatic mutations in plasma. We used the QIASeq platform which: (1) is based on a single primer extension and, thus, captures both single nucleotide variants (SNV) and structural variants (SV), and (2) integrates unique molecular indexing (UMI), thus allowing determination of ctDNA levels from VAF. Our cohort included 18 patients, 13 with Sanger sequencing confirmed CTNNB1 somatic mutations in the matched tumors: 6 missense SNVs and 7 deletions ranging from 96 to 348 base pairs. Our NGS assay was able to accurately detect mutations in 9/13 (69%, 7 SV and 2 SNV) of the Sanger confirmed cases, and SV and missense SNV in 2/5 (40%) cases pending orthogonal Sanger confirmation. Available ddPCR data for 3 patients (8 samples) 1 and corresponding NGS data showed similar ctDNA levels (deviation ranging between 1.2-8.5%). NGS CTNNB1 variants were detected in samples taken at initial diagnosis in 8/9 (89%), following neoadjuvant chemotherapy in 4/9 (44%), post-operatively for fully resected localized disease in 0/4 (0%), and metastatic recurrence in 1/5 (20%), suggesting a positive correlation between CTNNB1 variants and tumor burden. AFP levels were available for 24/27 samples, with abnormal levels detected in 23 samples. The ctDNA levels determined from NGS did not correlate with AFP levels; however, longitudinal plasma samples (n=9) showed similar dynamics of both ctDNA and AFP, reflecting the response to treatment. To conclude, we show that ctDNA detectable with our universal HB NGS assay is a good surrogate marker of tumor burden and shows correlation with treatment response. SV, prevalent in approximately 50% of cases of HB, may be inadequately captured and thereby misrepresented in probe-capture NGS platforms but were highly detectable in our assay. Further work is needed to understand the interplay of both AFP and ctDNA in HB monitoring. Reference: 1. Kahana-Edwin S, McCowage G, Cain L, et al. Exploration of CTNNB1 ctDNA as a putative biomarker for hepatoblastoma. Pediatr Blood Cancer. 2020. doi:10.1002/pbc.28594 Citation Format: Smadar Kahana-Edwin, Andre E. Minoche, Lucy E. Cain, Geoffrey McCowage, Sarah E. Woodfield, Sanjeev A. Vasudevan, Sarah Kummerfeld, Jonathan Karpelowsky. Utility of CTNNB1 ctDNA as a biomarker for hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 582.
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