Detection of IDH1 Mutation in cfDNA and Tissue of Adult Diffuse Glioma with Allele-Specific qPCR

Background: The World Health Organization (WHO) classification of central nervous system (CNS) tumors necessitates testing of isocitrate dehydrogenase (IDH) 1/2 gene mutation in patients with adult-type diffuse glioma (ADG) for better disease management. In clinical practice, the testing of IDH1 is primarily achieved using immunohistochemistry (IHC) specific to IDH1-R132, which carries a sensitivity of 80% and specificity of 100%. However, in some cases, non-specific background staining or regional heterogeneity in the protein expression of IDH1 may necessitate confirmatory genetic analysis. Robust and reliable assays are needed for IDH1/2 mutation testing. The aim of the current study was to detect IDH1 mutation in cfDNA and tissue of adult-type diffuse glioma with allele-specific qPCR. Materials and Methods: In the current study, IDH1-R132H mutation was analyzed in tumor tissue with paired cell-free DNA (cfDNA) in patients with ADG (n = 45) using IHC and competitive allele-specific Taqman PCR (CAST-PCR). Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissue and matched serum for cfDNA using commercially available kits. CAST-PCR with IHC for the detection of IDH1-R132H mutation was also compared. Results: The IDH1-R132H mutation was detected in 46.67% (21/45) cases and 57.78% (26/45) cases using IHC and allele-specific CAST-PCR. In cfDNA of matched IDH1-mutant FFPE tissue DNA, IDH1-R132H mutation was detected in 11.54% (3/26) using CAST-PCR. The concordance rate for IDH1-R132Hmutation between IHC and CAST-PCR was 80.77% (21/26). Conclusion: The CAST-PCR assay is more precise and sensitive for IDH1-R132Hdetection than traditional IHC, and IDH1-R132H mutation detection using cfDNA may add to the current methods of glioma genomic characterization.


Detection of IDH1 Mutation in cfDNA and Tissue of Adult
Diffuse Glioma with Allele-Specific qPCR and molecular diagnostic criterion for ADG in the 2016 WHO classification marked a significant departure from the previous morphology-alone classification (Louis et al., 2016;Parsons et al., 2008). IDH1/2 mutation is a feature of 'oligodendroglioma, IDH-mutant, and 1p19qcodeleted' and 'astrocytoma IDH-mutant' while IDHwildtype is a key diagnostic marker for 'Glioblastoma, IDH-wildtype' and 'Diffuse pediatric-type high-grade glioma, H3-wildtype, and IDH-wildtype' as recommended by the 2021 WHO classification of CNS (Brat et al., 2020;Louis et al., 2021).
Determining IDH1 mutation is crucial for diagnosis and selecting an appropriate treatment strategy. Typically, the first step in treating a glioma is to perform the safest radical resection to provide enough tumor tissue for a reliable diagnosis. Regardless of tumor grade, any glioma expressing IDH-wildtype should be regarded as glioblastoma, IDH-wildtype (Louis et al., 2021) and treated with aggressive chemoradiotherapy according to the Stupp protocol (Stupp et al., 2005. The Editorial Process: Submission:10/14/0000 Acceptance:03/10/2023 treatment of gliomas expressing mutated variations of IDH should be guided by the presentation of clinical and molecular features. For radically resected low-grade tumors exhibiting both the 1p/19q co-deletion and an IDH mutation, one might even consider omitting oncotherapy altogether and recommend watchful follow-up (Weller et al., 2017;Weller et al., 2021).
In clinical practice, the testing of IDH1/2 mutation is primarily based on immunohistochemistry (IHC) specific for IDH1/2 protein expression, which is limited in terms of sensitivity, and cross-reactivity. In some cases, non-specific background staining or regional heterogeneity in IDH1-R132H protein expression may necessitate confirmatory genetic analysis. Assays such as competitive allele-specific TaqMan Polymerase chain reactions (CAST-PCR) are characterized by their high sensitivity and specificity to detect minimal amounts of mutated DNA in a sample containing large amounts of normal wildtype DNA (Barbano et al., 2015;Bolton et al., 2015). It can robustly detect mutant alleles at values as low as 0.1% in a wildtype background and has >99% concordance with other technologies, including technology based on digital PCR and Sanger sequencing (Yang et al., 2018).
In recent years, various approaches have been developed, including "liquid biopsies" (nucleic acid extracted from biological fluids such as plasma, urine, and cerebrospinal fluid (CSF)) for detecting the IDH mutation (Satomi et al., 2022); D2HG detection in body fluids; and advanced MRI imaging with specific D2HG detection by magnetic resonance spectroscopy (MRS) (Fujita et al., 2022;Mithraprabhu et al., 2021;Tuna et al., 2022). However, none of them is currently used in clinical practice. A non-invasive, rapid, sensitive, and cost-effective method for IDH mutation analysis is needed to enhance diagnosis and predict survival. Analysis of cellfree nucleic acids has entered clinical practice in tumors like lung and colon cancer (Kolenčík et al., 2020;Kwapisz, 2017). The current study evaluated the IDH1-R132H mutation in cfDNA and respective tissue of adult-type diffuse glioma using a CAST-PCR assay. Further, the sensitivity and effectiveness of the CAST-PCR assay were compared with IHC for IDH1-R132H.

Study samples
Our study included histologically confirmed ADG patients (n=45) according to the WHO 2021 CNS classification. When the study commenced, cases were diagnosed based on the WHO 2016 classification of CNS tumors (Louis et al., 2016). They have been reclassified as per WHO 2021 CNS classification, and the staining of IDH1-IHC was used for IDH1 status in all cases (Louis et al., 2020;Louis et al., 2021). All IDH-mutated astrocytomas have been categorized as astrocytoma IDHmutant. IDH-wildtype cases were further classified on the histological features, including proliferation, necrosis, and mitosis. The Institutional ethics committee cleared the study (IEC No.26/18), and all study participants gave informed consent and have therefore been performed under the ethical standards of the Declaration of Helsinki (World Medical Association, 2013)

Sample collection
Formalin-fixed paraffin-embedded (FFPE) tumor tissue blocks were obtained from the departmental tumor archive after histopathological diagnosis, and peripheral blood (3.0ml) was collected in silica gel vials (BD Vacutainer, UK) from post-op patients who underwent biopsy or with remnant tumor. Samples were collected within 22-65 days (Q1-Q3) after the procedure. The serum was separated by centrifugation at 4,000 rpm for 10 minutes and stored at −80°C until further processing. All the samples were processed within 02 hours of collection.

Immunohistochemistry (IHC) of Isocitrate Dehydrogenase 1 (IDH1)
Immunohistochemical analysis of IDH1 was performed using Anti-IDH1 (R132H) (Dianova, USA Clone: H09unconj) in a dilution of 1:50. All Immunohistochemical assays were performed on a VENTANA BenchMark XT automated staining instrument according to the manufacturer's instructions with onboard deparaffinization, retrieval, and staining (Ventana Medical Systems, Inc., Tucson, USA). An expert pathologist (NH) independently analyzed all immunohistochemically stained sections.

Staining interpretation of IDH1
The staining interpretation of IDH was as follows: Mutant: intense cell cytoplasm and nucleus of tumor cells, wildtype: weak diffuse staining of cytoplasm and staining of macrophages. Normal/residual glial and vascular endothelial cells were internal negative controls.

DNA extraction from formalin-fixed paraffin-embedded (FFPE) tissue and serum
FFPE tumor tissue was used to extract genomic DNA. The tumor area was defined on a Hematoxylin and Eosin (HE) stained slide and a corresponding section marked for DNA isolation. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany, Cat No. #56404). Serum cell-free DNA (cfDNA) was extracted using the ChargeSwitch ® gDNA 1 mL Serum Kit (Invitrogen, USA) as per the manufacturer's instructions. The quality and quantity of the DNA were measured using Nanodrop (DeNoVix, USA, Model #DS-11).

IDH1 mutation detection by CAST-PCR
IDH1-R132H mutation was determined in 45 FFPE tissue DNA with paired cfDNA using CAST-PCR assay (TaqMan ® Mutation Detection Assay). Each case was amplified using a reference assay (#Hs00001019_rf) and a mutation assay (#Hs00000981_mu) in a 20µl reaction, using a 10µl of genotyping master mix (#4371353), 1µl of primer and probe for reference and mutation assay, 1-9 µl of template DNA (up to 50ng) and volume were brought to 20μl by nuclease-free water. Real-time amplification was performed using the AriaMx Real-Time PCR system (Agilent Technologies, USA) as per the manufacturer's instructions. In each batch, NTC was run to check for cross-contamination (Figure 1i). ΔCT values were IDH1 Mutation Detection with CAST-PCR (FFPE) tissue Out of 45 cases, IHC successfully detected IDH1-R132H mutation in 46.67% (21/45) of the cases. An IDH1-R132H mutation was determined in FFPE tissue DNA and cfDNA. The CAST-PCR assay successfully detected the IDH1-R132H mutation in 26/45 (57.78%) of FFPE DNA. Five cases showed mutations in the CAST-PCR test, but IHC showed they were all wildtype (Figure 1e-h). The rate of concordance for IDH1-R132H mutation between IHC and CAST-PCR was 80.77% (21/26) (p= 0.000; k= 0.780) ( Table 2 and Supplementary Table 1).

Statistical analysis
The IBM SPSS v22 Statistical Package for Social Sciences (SPSS, IBM, USA) was used for the statistical analysis. Cohen's kappa statistics tested the agreement between IHC and CAST-PCR in FFPE and cfDNA. Reverse Kaplan-Meier was used to compute the median follow-up time. Kaplan-Meier and log-rank analyses were performed to assess survival concerning each parameter; differences were considered significant when p ≤ 0.05.

Discussion
It has been found that IDH1 gene mutations are common in diffuse gliomas (Parsons et al., 2008). Based on the mutation profiles of glioma subtypes and primary and recurrent tumors, IDH1 mutations are considered one of the most significant genetic modifications in gliomagenesis (Johnson et al., 2014;Yan et al., 2009) that can aid in the diagnosis and prognosis. In addition, patients with known IDH1 mutations may benefit from new IDH1-targeted chemotherapeutic regimens under development (Golub et al., Mellinghoff et al., 2020).
A study by Matthias Preusser et al., (2011) showed the need for confirmatory genetic analysis in cases with non-specific background staining and/or regional heterogeneity of IDH1-R132H expression using the DIA H09 antibody (Preusser et al., 2011). Although Sanger sequencing is the "gold standard" for detecting mutations because of its low rate of false positives and high specificity, it has several drawbacks, including low sensitivity, long assay times, the necessity for high-quality tissue samples, and manual interpretation (Gao et al., 2016). Furthermore, next-generation sequencing (NGS), which is used to detect various mutations, has the drawback of taking too much time and being expensive to discover a single genetic variant. An alternative technique for mutation detection is a real-time CAST-PCR assay (Roma et al., 2013). Competitive allele-specific TaqMan PCR allows the selective amplification of minor alleles and blocks the amplification of non-mutant alleles.
In a significant subset of patients, immunohistochemical reactivity is not detected; it is likely but not certain that the tumor is IDH-wildtype. The study by Andrews and Prayson (2020) recommends PCR testing for all patients whose tumor is negative by IHC. However, this is not always performed, both because it is expensive and in some patient groups (e.g., elderly patients with tumors demonstrating necrosis), it is almost always negative (i.e., these are almost invariably glioblastoma). So, keeping this in mind, we decided to test the CAST PCR in IDH-negative tumors of all age groups.
In the current study, real-time PCR, combined with  were reported as mutated in CAST-PCR while were wild type in IHC. The reason leading to this inconsistency may be due to the sensitivity and specificity of antibodies (Preusser et al., 2011). Our results showed that the IDH1 mutation detection rate in gliomas was significantly different (p=0.000) between IHC and CAST-PCR (κ=0.780). A study by Agarwal et al., (2013) compared the performance of IHC and DNA sequencing for IDH1 mutation and found a concordance rate of 88% (44/50) (Agarwal et al., 2013). Similarly, in our study, the concordance rate between CAST-PCR and IHC in detecting IDH1 mutation was 80.77%. Moreover, the CAST-PCR assay was more sensitive than IHC in identifying the IDH1 mutation. Further, cfDNA and FFPE DNA show concordant results only in 11.54% of cases; this low concordance may be due to a lack of tumor-derived DNA or a low copy number of mutated DNA. All cases with cfDNA-positive for IDH1 on CAST-PCR were <55 years, including two cases of oligodendroglioma, IDH-mutant, 1p19q co-deleted, CNS WHO grade 2 and 3, respectively, and one case of Glioblastoma, IDH-wildtype CNS WHO grade 4 by IHC analysis.
We have included only the IDH1-R132H mutation in both IHC and CAST-PCR, and other mutations of IDH1 & IDH2 were not analyzed. However, these mutation types become secondary due to low frequency because their testing costs may burden the patients. Sanger sequencing is considered the gold standard for IDH1 & IDH2 mutation detection; however, we could not validate our results using the sequencing method.
Finally, the CAST-PCR technique for detecting glioma IDH1 gene mutations has high sensitivity, good reproducibility, ease of use, and accurate results. It offers a more precise method for detecting mutations in the IDH1 gene in formalin-fixed paraffin-embedded tissue samples but lacks sensitivity using an alternative source, cfDNA, in the case of tissue scarcity. Despite the small sample size, evidence suggests that CAST-PCR assays outperform IHC. More sensitive techniques may be required to detect IDH mutations in cfDNA.