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. 2025 May 1;31(9):1700-1710.
doi: 10.1158/1078-0432.CCR-24-3233.

Personalized Tumor-Specific Amplified DNA Junctions in Peripheral Blood of Patients with High-Grade Gliomas

Mohamed F Ali  1 , Cecile Riviere-Cazaux  2 , Sarah H Johnson  1 , Rebecca Salvatori  1   3 , Alan R Penheiter  1 , James B Smadbeck  1 , Stephen J Murphy  1 , Faye R Harris  1 , Lex F McCune  1 , Lucas P Carlstrom  2 , Michael T Barrett  4 , Farhad Kosari  1 , Leila A Jones  1 , Cristiane Ida  3 , Mitesh J Borad  5 , Bernard R Bendok  6   7   8   9   10 , Alfredo Quiñones-Hinojosa  11 , Alyx B Porter  12 , Maciej M Mrugala  12 , Kurt A Jaeckle  13 , Panos Z Anastasiadis  14 , Sani H Kizilbash  15 , John C Cheville  1   3 , David M Routman  16 , Terry C Burns  2 , George Vasmatzis  1
Affiliations

Personalized Tumor-Specific Amplified DNA Junctions in Peripheral Blood of Patients with High-Grade Gliomas

Mohamed F Ali et al. Clin Cancer Res. .

Abstract

Purpose: Monitoring disease progression in patients with high-grade gliomas (HGG) is challenging due to treatment-related changes in imaging and the requirement for neurosurgical intervention to obtain diagnostic tissue. DNA junctions in HGG often amplify oncogenes, making these DNA fragments potentially more abundant in blood than monoallelic mutations. In this study, we piloted a cell-free DNA approach for disease detection in the plasma of patients with HGG by leveraging patient-specific DNA junctions associated with oncogene amplifications.

Experimental design: Whole-genome sequencing of grade 3 or 4 isocitrate dehydrogenase-mutant or wild-type astrocytomas was utilized to identify amplified junctions. Individualized qPCR assays were developed using patient-specific primers designed for the amplified junction. ctDNA levels containing these junctions were measured in patient plasma samples.

Results: Unique amplified junctions were evaluated by individualized semi-qPCR assays in presurgical plasma of 18 patients, 15 with tumor-associated focal amplifications and three without tumor-associated focal amplifications. high copy-number junctions were robustly detected in the plasma of 14 of 15 (93.3%) patients with amplified junctions and none of the controls. Changes in junction abundance correlated with disease trajectory in serial plasma samples from five patients, including increased abundance of amplified junctions preceding radiographic disease progression.

Conclusions: In patients with grade 3 or 4 astrocytomas who had tumor-associated amplifications, patient-specific amplified junctions were successfully detected in assayed plasma from most patients. Longitudinal analysis of plasma samples correlated with disease trajectory, including cytoreduction and progression.

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Conflict of interest statement

M.F. Ali reports grants from NCI and the National Institute of Neurological Disorders and Stroke during the conduct of the study. J.B. Smadbeck reports other support from Veracyte outside the submitted work and employment with Veracyte, a company that is pursuing intellectual property in MRD Technologies that is not associated with this work. S.H. Kizilbash reports grants from FDA Office of Orphan Products Development during the conduct of the study as well as grants from LOXO Oncology, Orbus Therapeutics, CNS Pharmaceuticals, Wayshine Biopharma, Aminex Therapeutics, Apollomics, Nerviano Medical Sciences, Incyte, Celgene, and SonALAsense outside the submitted work. D.M. Routman reports other support from Adela outside the submitted work and has a patent for DNA methylation licensed to Exact Sciences and support from the NCI Paul Calabresi Program in Clinical/Translational Research at the Mayo Clinic Comprehensive Cancer Center (K12CA090628). T.C. Burns reports grants from NIH during the conduct of the study. G. Vasmatzis reports ownership of WholeGenome LLC. No disclosures were reported by the other authors.

Figures

Figure 1.
Figure 1.
Workflow and results of ctDNA detection of amplified DNA junctions in presurgical plasma. A, Workflow of the assay to detect amplified DNA junctions in the plasma of patients with glioma. DNA from the tumor is sequenced using a mate pair or WGS protocol. DNA junctions are detected and used to design individualized ctDNA qPCR. Primers and probes are designed for the selected junctions and undergo a pipeline to test for sufficient specificity and sensitivity to be used in the ctDNA assays to interrogate the blood for the presence of tDNA. B, ctDNA results in the presurgical plasma of 18 patients are graphed according to the sample number. One somatic, case- and tumor-specific DNA junction was assayed for each case. Copy-number junctions and chromosome locations are listed below each case. Low copy-number junctions were tested in three patients, and high copy-number junctions were tested in the remaining 15. All junctions were also tested in the same patient’s tDNA and tumor tissue DNA from other patients, and most were tested in PBMC except for PT118E, 385B, 078B, and 088B. Red lines below patient IDs indicate patients with longitudinal plasma samples available for testing. CN, copy number.
Figure 2.
Figure 2.
Longitudinal monitoring of ctDNA in the plasma of PT385B, a patient with an EGFR-amplified GBM. Plasma samples were obtained from the time of surgery through adjuvant TMZ. MRI images were obtained for the corresponding time points along the time course, as well as 11 weeks after the last plasma samples when radiographic recurrence was observed.
Figure 3.
Figure 3.
Longitudinal monitoring of ctDNA in plasma from PT088B, a patient with CDK4- and EGFR-amplified GBM. Plasma samples were obtained from the time of surgery through observation after completion of standard-of-care chemoradiation and adjuvant TMZ, including disease progression. Correlative imaging is shown.
Figure 4.
Figure 4.
Longitudinal monitoring of ctDNA in plasma from PT078B, a patient with a CDK4-amplified GBM. Plasma samples were obtained from the time of surgery, through a clinical trial of pembrolizumab with standard-of-care chemoradiation and adjuvant TMZ, and then throughout radiographic progression while on regorafenib, bevacizumab, and subsequently lomustine. Correlative imaging is shown.
Figure 5.
Figure 5.
Longitudinal monitoring of ctDNA in plasma from PT304E, a patient with an EGFR-amplified recurrent GBM. Plasma samples were obtained at the beginning, at the time of surgery for a recurrent GBM, through the patient’s participation in a dendritic cell vaccine clinical trial and on bevacizumab. Correlative imaging is shown at each time point.
Figure 6.
Figure 6.
Longitudinal monitoring of ctDNA in plasma from PT426E, a patient with an MAML2-amplified grade 4 IDH-mutant astrocytoma. Plasma samples were obtained at the time of re-resection for a recurrent grade 4 IDH-mutant astrocytoma and at the time of biopsy for dural lesions positive for leptomeningeal carcinomatosis. Correlative imaging is shown for both time points, including the thoracic spine imaging demonstrating increased contrast enhancement consistent with leptomeningeal disease (LMD).
See this image and copyright information in PMC

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