IDH1 mutation is detectable in plasma cell-free DNA and is associated with survival outcome in glioma patients

Liquid biopsy recently emerged as a new approach to investigate the molecular profile of solid tumors by detecting gene alterations and obtaining potential prognostic and predictive biomarkers across different cancers [1‐3]. Liquid biopsy has gained interest due to its advantages being a minimally invasive, sensitive, repeatable, and feasible alternative to tissue biopsy, having the ability to capture heterogeneity across multiple areas of tumors [2, 4‐6]. The term “liquid biopsy” is mainly referred to the analysis of circulating free DNA (cfDNA) extracted from plasma, which contains a small amount of tumor DNA (ctDNA); however, this concept is also applied to different biological fluids such as blood, cerebrospinal fluid (CSF), urine, saliva, and to several analytes such as cell-free RNA, circulating tumor cells, extracellular vesicles (EVs), RNA and non-coding miRNA [7‐13]. Nowadays, several national and international recommendations are available, regarding the use of liquid biopsy in clinical practice, which mainly refer to the use of plasma cfDNA, and suggest the use of alternative sources only in specific clinical trials and research studies [14, 15]. However, when using liquid biopsy, one of the major challenge is represented by the false negative results, mainly related to anatomic barriers and disease biological characteristics [16, 17]. While cfDNA has been detected in several types of cancers including breast, lung, pancreatic, melanoma and colorectal cancer [18‐27], few studies have been able to identify cfDNA in peripheral blood in patients with glioma, due to cfDNA difficulties of crossing the blood–brain barrier (BBB), and since its release can change depending on histopathology, localization, and tumor grade [17, 28‐32].

Ideally, the use of the liquid source close to the disease site would be the optimal solution; therefore, the CSF-derived cfDNA would be the best source of biological material to study the molecular profile of brain tumors through liquid biopsy [33‐38]. Published studies using sensitive methods, such as digital PCR, demonstrated that higher concentrations of cfDNA may be found in CSF compared to plasma, suggesting that cfDNA in CSF could be used as a molecular marker to identify mutations and longitudinally monitor changes in brain tumors [39‐42]. However, CSF is an invasive, complex, and uncomfortable approach, requiring specific expertise and associated with additional risks following its repeated use [34].

Among brain tumors, high-grade gliomas are considered the most common malignancies in adults and their classification has traditionally been based on histopathological findings supplemented by tissue-based tests, grade, and genomic alterations [43‐46]. In particular, the presence of the IDH1 p.R132H amino acid substitution in tissue has been found to be the most common subtype with a 90% of prevalence among IDH1-mutant tumors [47‐50]. Different studies suggested that IDH-mutant gliomas have a significantly improved prognosis, independently of age and grade, as compared to IDH-wild-type tumors [51‐54]. Accordingly, the World Health Organization (WHO) classification of diffuse gliomas under the 2021 update has been dependent largely on IDH1 mutation status together with 1p/19q-codeletion [55‐57]. In particular, the new classification includes astrocytoma, IDH-mutant (Grade 2, 3, 4), oligodendroglioma, IDH-mutant, and 1p/19q-codeleted (Grade 2, 3) and GBM, IDH-wildtype (Grade 4) [57].

Therefore, the use of IDH1 mutation status as a prognostic biomarker of tumor molecular evolution has allowed easier and more accurate risk stratification of glioma patients and could be a key tool to improve their personalization of treatments. Moreover, IDH-1 and -2 recently became a predictive biomarker of response to specific IDH inhibitors, such as vorasidenib for low grade glioma [58].

In the present study, we assessed the feasibility of detecting plasma-cfDNA IDH1 mutation in gliomas and the results were correlated with survival and clinical characteristics of patients affected by gliomas.

Our study examined the feasibility of using liquid biopsy for patients affected by a brain tumor, and the association between the mutational status of IDH1 in plasma, survival outcomes and clinical characteristics of a cohort of glioma patients. In our study, a statistically significant concordance between IDH1 mutation detection in tissue and cfDNA was found (p = 0.0024), suggesting that liquid biopsy is a technique capable of integrating tissue biopsy, especially in elderly patients or patients with tumors close to critical areas. In particular, IDH1 mutation was detected in the cfDNA of 10 out of 21 patients harboring an IDH1 mutant tumor, and in 5 patients with IDH1 wild type tumor tissue (n = 41), suggesting that liquid biopsy may support tissue biopsy in the detection of IDH1 mutation. Similarly, previous studies were able to identify the IDH1 mutation using a digital PCR technology in the plasma of 50–60% of patients with IDH-mutant gliomas [29, 30]. Interestingly, all patients with IDH1 wild type tumor but with detectable IDH1 mutation in cfDNA had a subtotal resection, and a residual tumor burden was still present in the brain. Therefore, a shedding of cfDNA in the circulation could be expected as a consequence of this type of surgery [60, 61]. For this reason, our analysis could more accurately represent the entire heterogeneity of the tumor compared to tissue biopsy, explaining the detection of the IDH1 mutation in the cfDNA of a subgroup of patients negative on tissue. On the other hand, in our cohort eleven patients presented IDH1 mutated tumor tissue and no IDH1 mutation in plasma, suggesting that the blood-brain barrier could limit the release of cfDNA and consequently, the detection of IDH1 mutation. Recently, a meta-analysis highlighted the complexity of cfDNA analysis to detect the mutational status in glioma patients [28]. cfDNA analysis resulted to have high specificity (0.98; 95% CI 0.96–0.99) but a relatively moderate sensitivity (0.69; 95% CI 0.66–0.73) and a high grade of heterogeneity (I2 = 73.1%, p < 0.05) of sample source and assay methods [28]. These latter could affect the sensitivity of the cfDNA and the accuracy of the findings, suggesting that cfDNA could be used only as an auxiliary tool for molecular assessment of glioma [62].

In our study, patients were divided into different groups depending on the brain tumor’s histopathological subtypes and tumor grade, finding a higher rate of IDH1 mutation (AF%) in the diffuse astrocytoma, glioblastoma and oligodendroglioma. However, the association between tumor grade and the presence of IDH1 mutation (p = 0.10) was not statistically significant. Specifically, the majority of patients with a low-grade brain tumor correlated with the presence of the p.R132H mutation, while the high-grade group, which counted about 82% of patients, did not show IDH1 mutation. This is in accordance with previous studies, which reported that IDH1 mutation occurs in the majority of low-grade gliomas and less frequently in high-grade gliomas [50, 63, 64]. Accordingly, Yan et al. confirmed that IDH1 mutations are more frequent in G II–III astrocytomas and oligodendrogliomas and less frequently in GBM, underlining the correlation between the presence of IDH1 mutation and tumor grade [63].

To date, few data have reported the association between IDH1 mutations with tumor localization [65]. In the present study, we found that the majority of gliomas (57.2%) with IDH1 mutations were located in the frontal lobe, followed by gliomas affecting multiple sites (33.3%) and temporal or parietal tumors (9.5%), suggesting a potential relationship between IDH1 mutations and tumor localization.

Interestingly, our study evaluated the role of the IDH1 mutation detected in tissue and plasma and the survival outcome. Patients carrying IDH1 mutation p.R132H have a higher survival rate than patients not carrying the mutation. Accordingly, several studies highlighted the positive correlation between IDH1 p.R132H mutation and survival of patients with gliomas [66‐68]. Sanson et al. highlighted that the IDH1 codon 132 mutation is associated with the genomic profile of the tumor and constitutes an independent prognostic marker in G II to IV gliomas (p = 0.00021) [66]. Similarly, Polivka et al. affirmed that patients with IDH1 p.R132H mutation had a significantly longer OS than patients with wild-type IDH1 (270 versus 130 days; p < 0.024) [67].

A survival rate of 65.8 months was found in patients with a wild type IDH1 status in tissue but a mutant IDH1 status in plasma (T−/P+), which is approximately twice as long than the median OS of tissue (24.4 months) or plasma (35.8 months) IDH1 wild type patients of our cohort, assuming the real presence of the IDH1 mutation. However, due to the small sample size, appropriate statistical analyses could not be performed.

In our study, the evaluation of the O-6-methylguanine-DNA methyltransferase (MGMT) status was not available for all patients. In this context, different studies suggested the importance of combined IDH1 and MGMT analysis to predict survival in patients with different types of gliomas [65, 69]. Patients harboring IDH1 mutation and MGMT methylation had the more favorable outcomes, followed by patients with IDH1 mutation and unmethylated MGMT promoter, and patients without IDH1 mutation and unmethylated MGMT promoter [69]. There is compelling evidence that liquid biopsy, especially using cfDNA, offers an accurate and accessible approach to capture the landscape of brain tumor-related molecular alterations, allowing diagnosis and characterization of glioma patients [35, 38]. Liquid biopsy is already used in several tumors, especially for treatment monitoring, and recent several studies reported its usefulness in the management of glioma patients [38, 70‐73]. Moreover, liquid biopsy has found applicability as potential minimally invasive alternative to traditional tissue surgery, especially in difficult scenarios (i.e unavailable tissue, poor clinical conditions of patients), bypassing its spatial and temporal biases, and for the research of diagnostic and prognostic biomarkers, enabling clinicians to improve the neuro-oncology traditional monitoring of glioma patients [32, 33, 35‐37, 74‐76]. Moreover, the assessment of the IDH-1 and -2 status will become crucial since vorasidenib, a new oral brain-penetrant IDH-1/2 inhibitor inhibitor, showed linical activity in IDH-mutant gliomas [58]; therefore, its assessment will be relevant in order to treat patients with appropriate targeted treatments.

Our study confirms that liquid biopsy and cfDNA could be a complementary methods to tissue biopsy in the detection of IDH1 mutation, which can be used as a strong prognostic and predictive biomarker for a favorable clinical outcome in patients with glioma. The potential use of liquid biopsy opens an innovative field of circulating biomarkers research with a relevant impact on the characterization, prognosis, and clinical management of brain cancer.