Deep sequencing of circulating tumor DNA uncovers tumor genomic mutations and heterogeneity in pediatric diffuse midline glioma: results from a pilot study

Published in Cancer
Deep sequencing of circulating tumor DNA uncovers tumor genomic mutations and heterogeneity in pediatric diffuse midline glioma: results from a pilot study
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Central nervous system (CNS) tumors are the leading cause of cancer-related death in children. The deadliest of these tumors is diffuse midline glioma (DMG), a devastating disease that most often arises in the pons of the brainstem and portends a survival time of less than one year from diagnosis. The delicate neuroanatomical location of these tumors precludes their surgical resection and poses a challenge to obtaining tumor tissue for molecular diagnosis and study. However, access to tumor genomic information is increasingly essential for CNS tumor diagnosis and subtyping, which are now defined by tumor molecular alterations.

What did we do?

In “Circulating tumor DNA sequencing provides comprehensive mutation profiling for pediatric central nervous system tumors”, we addressed the paucity of available tissue specimens from children diagnosed with DMG by exploiting patient liquid biopsies. We performed a pilot study to investigate and optimize a targeted deep sequencing platform, the TruSight Oncology 500 ctDNA (TSO500ctDNATM) panel, and adapted this assay for use with circulating tumor DNA (ctDNA). We sequenced paired cerebrospinal fluid (CSF) and plasma cell free DNA from 10 children diagnosed with DMG. CSF was obtained intra-operatively during surgery, via lumbar puncture, or directly from the lateral ventricles during autopsy. Paired tumor tissue genomic DNA was analyzed using whole exome sequencing to verify mutations that were detected in ctDNA.

What did we find?

The proof-of-concept study revealed remarkable assay sensitivity and specificity, particularly in CSF specimens. Superior assay performance was achieved in CSF when compared to plasma, including higher depth of target exon coverage and concordance between mutation calls detected in paired CSF and tumor tissue specimens. Tumor-specific and prognostic mutations, including histone H3K27M mutations (found in >80% of DMGs), major driver gene alterations (e.g., tumor suppressor genes TP53 and PTEN), and copy number variations (e.g., growth factor signaling pathway genes PDGFRA, KRAS), were detected even in pre-treatment CSF specimens with extremely low starting DNA inputs (<5ng). These findings highlighted the sensitivity of our deep sequencing approach for detecting “needle in a haystack” mutations present at very low abundance.

Capturing genomic heterogeneity in patient liquid biopsies

Strikingly, ctDNA sequencing achieved more complete characterization of tumor genomic heterogeneity when compared to analyses of tumor tissue alone. We identified tumor-specific mutations in ctDNA that were initially filtered out of tumor sequencing results, due to low allelic frequency.  Closer inspection of raw sequencing data from paired tissue specimens revealed that these mutations were indeed present in the corresponding tumors, but at very low allelic frequency, indicating sub-clonality. Our ctDNA data provided a deeper glimpse into the heterogeneous genomic landscape of DMG tumors.

What’s next?

As shown in our pilot study, ctDNA deep sequencing provides an opportunity to profile tumor sub-clonal heterogeneity, even in cases where the sensitive tumor location restricts tissue access. Longitudinal ctDNA sequencing may facilitate monitoring of tumor sub-clonal evolution, without requiring repeated, invasive surgical tissue biopsies. Further studies are aimed at sequencing longitudinal CSF specimens from children diagnosed with DMG and other CNS tumors, to detect changes in tumor mutation landscape and to contextualize these changes with respect to treatments received, tumor radiographic features, and clinical status (e.g., disease progression, metastatic spread). This approach will be critical for defining the underlying genomic drivers of tumor response or resistance to therapy, particularly in the case of CNS tumors such as DMGs for which repeated tissue biopsies are almost never performed.

Translational impact

The findings presented in this article highlight the potential of incorporating ctDNA deep sequencing into the diagnosis and clinical management of DMGs and other pediatric CNS tumors. This approach provides a less-invasive alternative to the standard surgical tissue biopsy for molecular-based CNS tumor diagnosis, monitoring of tumor genomic evolution, and unraveling mechanisms of tumor resistance to therapy.  


Poster image highlights the utility of liquid biopsy analysis for capturing both clonal and sub-clonal tumor mutations in circulating tumor DNA, in contrast to tissue biopsy which may detect clonal mutations yet provide an incomplete picture of sub-clonal mutations present in heterogeneous tumors. Longitudinal ctDNA sequencing also has the potential to uncover tumor genomic evolution during therapy, providing new insights into molecular mechanisms of tumor response/resistance to treatment. Typical timing of specimen collection is indicated for liquid biopsy (where samples may be collected at both upfront diagnosis and longitudinally during therapy) and surgical tissue biopsy (typically only performed at upfront diagnosis for most patients with DMG). Figure created with BioRender.com.

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Cancer Biology
Life Sciences > Biological Sciences > Cancer Biology

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