A blood test that measures the amount of cell-free DNA (cfDNA) in the bloodstream – called a liquid biopsy – correlates with how patients will progress after they are diagnosed with glioblastoma (GBM), the deadliest and most common primary brain tumor in adults.
In a new study, researchers from the Abramson Cancer Center of the University of Pennsylvania are the first to show that patients with a higher concentration of cfDNA – circulating DNA that cancer and other cells shed into the blood – have a shorter progression-free survival than patients with less cfDNA, and that cfDNA spikes in patients either at the time of or just before their disease progresses.
The team also compared genetic sequencing of solid tissue biopsies in GBM side-by-side with the liquid biopsies and found that while both biopsies detected genetic mutations in more than half of patients, none of those mutations overlapped, meaning liquid biopsy may provide complementary information about the molecular or genetic makeup of each tumor.
Clinical Cancer Research, a journal of the American Association for Cancer Research, published the findings today.
“Doctors have begun using liquid biopsies more frequently to monitor certain cancers – particularly lung cancer – in recent years as research has shown their effectiveness in other disease sites.
But until now, there has been little focus on the clinical utility of liquid biopsy in brain tumors,” said the study’s senior author Erica L. Carpenter, MBA, Ph.D., director of the Liquid Biopsy Laboratory and a research assistant professor of Medicine at Penn.
The findings may eventually prove impactful for GBM patients. The disease is particularly aggressive, and while most estimates show there are around 11,000 new cases each year, the five-year survival rate is between five and 10 percent.
One of the challenges in treating GBM is that monitoring imaging can be an inaccurate way to detect disease progression. Moreover, the tumors themselves are usually heterogeneous – meaning that different parts of the tumor contain different genetic mutations – which means treatments focused on only one target are ineffective or only partially effective.
Another problem is that trying to track these mutations over time can be difficult, since getting a new tissue sample requires a repeat brain surgery.
While most patients do have their tumors surgically removed after initial diagnosis, additional procedures to monitor their disease over the course of treatment can be difficult and are invasive for patients.
These issues are especially difficult in patients who experience a recurrence, since when GBM comes back, it often returns with a vastly different genetic makeup.
“If our findings are validated by further studies, it would mean that these patients may be able to get a simple blood test that would give us a more accurate assessment than imaging of whether their disease has progressed or not, as well as more data on the mutations in their tumors,” said the study’s lead author Stephen J. Bagley, MD, MSCE, an assistant professor of Hematology-Oncology in Penn’s Perelman School of Medicine.
This study included 42 patients with newly diagnosed GBM.
They had blood tests at diagnosis, before surgery, and at regular intervals throughout their standard of care chemotherapy and radiation.
The 28 patients with a lower concentration of cfDNA pre-surgery – defined as cfDNA that was below the average of the total group – had almost double the progression free survival (median 9.5 months) compared with the 14 patients with higher concentrations (median 4.9 months).
In a sub-analysis of 20 patients, liquid biopsy detected at least one mutation in 11 patients, and all of those mutations were different than what was detected in analysis of each patient’s solid tumor biopsy.
The authors say the detection of additional mutations is especially exciting, since effective therapy for GBM will ultimately require combination therapies due to the heterogeneity of the tumor.
“If liquid biopsy can give us a more comprehensive view of the molecular profile of the tumor, we can potentially pick more effective combinations for each patient,” Bagley said.
The authors say this work is more hypothesis-generating than practice-changing at this point since the cohort of patients is relatively small.
However, they’ve continued to enroll patients and plan to perform a larger analysis in the future. They also plan to perform tumor DNA sequencing on multiple different samples of the resected tumors of each patient to learn about the full molecular profile of each.
Glioblastoma multiforme (GBM), a type of glioma, is the most aggressive type of primary brain tumor (PBT), with limited therapy options and a median survival of 12–15 months [1].
Comprehensive molecular profiling of PBTs can inform more detailed biological classification beyond traditional histopathology [2,3].
Development of therapies directed at molecular targets in gliomas and other PBTs is underway and holds promise as an improvement over current standard therapies [3–5].
However, trials of genomically matched therapies for brain tumors require next-generation sequencing (NGS) of a recent tissue sample, thus limiting progress; tissue requirements also limit the ability to identify and track mutation clonality and clonal evolution of tumors [6–8] and may miss important heterogeneous genomic events [9].
Additionally, recurrent glioblastomas are rapidly growing tumors, and obtaining a biopsy in order to complete molecular profiling is a time-consuming step.
Genomic profiling utilizing tissue samples obtained from invasive biopsy may not always be clinically feasible and is not without risk of morbidity or mortality [10,11].
Additionally, tissue biopsies may be found to have insufficient quantity or quality of material for NGS profiling.
Even when tissue sampling is feasible and sufficient for genomic analysis, tissue-based NGS may fail to capture a complete picture of the cancer’s genetic profile due to intra- and inter-tumor heterogeneity [8,12–15].
Recently, assays analyzing cell-free DNA (cfDNA) have become commercially available. These tests present an opportunity to genomically profiled patients’ tumors through a plasma sample without the need for an invasive tissue biopsy.
cfDNA contains fragments of circulating tumor DNA (ctDNA) released into circulation through apoptosis and/or active DNA release [16,17].
Given the short 2-h half-life of plasma cfDNA fragments in circulation and the ability to capture heterogeneity across multiple areas of a tumor, this technology provides an opportunity to assess cancer genomic signatures in real-time [18–20].
A prior study of plasma ctDNA yield across a variety of solid tumor types identified ctDNA alterations in less than 10% of patients with glioma [21].
The authors hypothesized that the blood–brain barrier is a physical obstacle preventing ctDNA from reaching peripheral circulation, suggesting limited clinical utility of such technology in this cancer type.
A recent study utilizing a comprehensive ctDNA analysis yielded a 51% cfDNA detection rate in patients with advanced primary glioblastoma [22] suggesting that ctDNA detection rate in primary brain tumors may vary by assay performance and/or histopathology and grade. We sought to evaluate the ability of a highly sensitive and specific cfDNA NGS assay to identify genomic alterations in patients with GBM and other PBTs, to further characterize ctDNA yield by histopathologic features, and to begin to explore the spectrum of genomic alterations identified in cfDNA in this clinically tested patient population.
Discussion
Contrary to other cfDNA studies which postulated that ctDNA would not cross the blood–brain barrier to reach systemic circulation, we found that half of the patients with primary brain tumors had detectable cfDNA alterations with 48.9% of these having a potentially genomically targetable alteration identified.
Among patients with GBM, who comprised just over half of this cohort, ctDNA alterations were detected 55% of the time.
This suggests that cfDNA analysis for GBM genomic profiling may be appropriate to consider prior to an invasive biopsy (performed solely to obtain tissue for genomic testing) and in patients for whom an invasive biopsy is not feasible or who decline.
Alterations were detected even more frequently in patients with meningioma, which is consistent with the absence of the blood–brain barrier present in other subtypes of primary brain cancer [28].
With an average VAF of 0.33% and a minimum VAF of 0.05% in this cohort, this study underscores the importance of utilizing a cfDNA assay with high sensitivity for detection of low-level alterations.
As seen in Table 2, the number of alterations and cfDNA VAF were both lower in this primary brain tumor cohort compared with a cohort of all solid tumors undergoing this cfDNA assay.
The mechanisms that influence the release of tumor DNA into the bloodstream are not entirely understood, and it is possible that the blood–brain barrier may limit the amount of ctDNA able to enter peripheral circulation from a primary brain tumor. The low VAFs observed in this study suggest that technical assay performance is of particular importance when selecting a commercial cfDNA platform for clinical use in this patient population in order to increase the likelihood of identifying these low-level alterations.
This study demonstrates a higher ctDNA alteration yield in patients with primary brain tumors than previously reported. Additionally, one quarter of samples had a ctDNA alteration detected that suggested eligibility for an off-label targeted therapy regimen.
Almost half of patients had a ctDNA alteration detected that suggested eligiblity for a targeted therapy clinical trial. This study suggests that the identification of genomic alterations in the cfDNA of patients with primary brain tumors is feasible.
This is promising for the continued development and execution of clinical trials of targeted therapies in this patient population, as the ease, convenience and safety of plasma cfDNA sampling has the potential to make genomic profiling a possibility when tissue is unavailable or unobtainable in the setting of advanced PBT.
Some of the alterations identified in this patient cohort do show potential for molecular targeted therapeutics, including BRAF/IDH1/IDH2 mutations, ERBB2/MET/EGFR/PDGFRA amplifications and mutations in DNA damage repair genes. For example, at the time of submission, trials using targeted therapies related to genes and pathways described in detail above (e.g., inhibition of RAF/MEK, EGFR and PARP, among others) were available in PBTs.
The option to detect these and other genomic alterations through cfDNA analysis may improve access to clinical trials investigating the use of these agents in the setting of primary brain tumors.
As described above, the exploratory analysis presented here utilizes data from an assay commercially available across solid tumor types.
Therefore, it is promising that the yield of clinically relevant genomic alterations using a liquid biopsy approach could be even higher from an assay specifically designed with PBTs in mind. However, this may introduce practical challenges, for example, the difficulty of implementing parallel epigenomic and RNA-based methodologies to assess methylation and splice variants, respectively.
Additionally, the evolution of personalized medicine has seen multiple pancancer approval for drugs targeting specific biomarkers (e.g., pembrolizumab for MSI-high tumors, larotrectinib for tumors with NTRK fusions) and continued success applying targeted therapies from one cancer type to another (e.g., anti-HER2 therapy common in breast cancer showing efficacy in colorectal cancer, BRAF/MEK inhibition common in melanoma showing efficacy in lung adenocarcinoma).
Trends such as these may support a broader, less PBT-specific approach to include identification of potential basket or umbrella drug trial targets. There has also been promising work done assessing cfDNA from cerebrospinal fluid [29,30], though this sample collection is still more invasive compared with peripheral blood draw. Future studies investigating ideal liquid biopsy assay composition and sample type may be warranted to further explore these questions [20].
It is important to note an underlying limitation of this study. As the cohort was based on samples submitted to a commercial laboratory, clinical information (including pathologic confirmation of diagnosis, or timing of cfDNA collection in relation to therapy regimen) was not available for all patients.
Sample collection may have occurred at various clinical time points (e.g., baseline vs stable disease vs progression) which may have affected ctDNA alteration detection rates and VAF. The likelihood of identifying genomic alterations shed by the tumor in plasma cfDNA is highest prior to treatment and at times of progressive disease, rather than when patients are clinically stable or in active treatment when ctDNA release into the blood is suppressed. However, these clinical details are not available for this cohort from a commercial laboratory, as this information is not required for clinical testing.
This preliminary analysis was intended to focus on overall detection rate of ctDNA in patients with PBTs using an available retrospective dataset, and a breakdown by specific molecular alterations would result in too small of numbers to draw meaningful formal correlative conclusions in this preliminary descriptive analysis.
An in-depth exploration of the specific alteration landscape would be best conducted in a cohort with samples collected at consistent and clinically appropriate timepoints (baseline active disease and/or progression) to maximize the likelihood of capturing the tumors’ genomic signatures through cfDNA. However, the preliminary spectrum of mutated genes in this cfDNA cohort is similar to that of published data from The Cancer Genome Atlas (TCGA) genomic analysis of tissue, including TP53, NF1, IDH1 and EGFR [31,32].
As this data is from clinical cfDNA analysis performed by a commercial laboratory that does not require detailed clinical data to order testing, genomic profiling results of corresponding tumor tissue for patients who may have had this analysis were not available for comparison in this patient cohort.
Any potential discordance may be due to the disease stage, treatment history and clinical status of the patients in the current cfDNA cohort. TCGA recruited patients without any prior therapies, while the current cohort enrolled patients who may have been treatment-naive or previously treated.
It is known that the spectrum of mutations observed in treatment-naive versus previously treated tumors differs due to tumor evolution following treatment. Other discrepancies in the results of the two cohorts may be related to sequencing coverage of the cfDNA assay (Supplementary Figures 1–4).
For example, the cfDNA assay cannot assess for large deletions, including EGFR vIII, and the detection of amplifications in cfDNA analysis is dependent on the level of ctDNA shed being high enough to distinguish CNAs from the vast quantities of germline cfDNA with normal copy number. Additionally, due to the ability of the cfDNA test to capture genomic heterogeneity across disease burden discordance may be due to detection of alterations that were not observed in tumor tissue testing from a single site biopsy.
A future study of tissue plasma alteration concordance in which paired samples are collected contemporaneously at clinically relevant timepoints per published concordance study criteria [33] would be valuable, though perhaps would be limited by the clinical feasibility of collecting tumor tissue at the time of advanced stage disease when plasma cfDNA analysis is clinically indicated.
The cfDNA assay utilized in this study attempts to report only alterations of somatic origin. However, discrimination between alterations of germline versus somatic origin becomes challenging in cases with high tumor burden and/or chromosomal instability [34].
It is also not possible to rule out hematopoietic origin of alterations through sequencing of cfDNA alone [35], and some alterations, like JAK2 V617F, occur more frequently in myeloproliferative neoplasms than in solid tumors. Therefore, similar to tissue-only testing [36,37], tumor-derived origin of alterations identified by NGS of cfDNA cannot be confirmed with certainty.
More information: Stephen Bagley et al. Clinical utility of plasma cell-free DNA in adult patients with newly diagnosed glioblastoma – a pilot prospective study, Clinical Cancer Research (2019). DOI: 10.1158/1078-0432.CCR-19-2533
Journal information: Clinical Cancer Research
Provided by Perelman School of Medicine at the University of Pennsylvania
