Prevalence of the single-nucleotide polymorphism rs11554137 (IDH1105GGT) in brain tumors of a cohort of Italian patients

IDH mutational status is required for proper diagnosis according to the WHO criteria revised in 2016. The single nucleotide polymorphism (SNP) rs11554137 (IDH1105GGT) at codon 105 of IDH1 has been reported in patients with several tumor types, including those with glioma. The aim of this study is to investigate the prevalence of IDH1105GGT in a cohort of brain tumors, and its association with clinicopathologic features and IDH1 and IDH2 missense mutations. Exon 4 of IDH1 and IDH2 was analyzed in a series of brain tumors classified according to current WHO criteria. DNA from control individuals was analyzed to infer the prevalence of IDH1105GGT in the reference population. Analysis was performed using next generation sequencing. IDH1105GGT was three times more frequent in patients with tumors (44/293 cases, 15.0%) vs. population controls (6/109, 5.5%) (p = 0.0102). IDH1105GGT was more frequent in grade III tumors (26.1%) compared to grade II (10.9%, p = 0.038) and grade IV tumors (13.7%, p = 0.041). IDH1105GGT was more frequent in grade II and III tumors without an IDH tumor missense mutation (43.8%) than in those with (11.5%, p = 0.005). The IDH1105GGT SNP likely represents an important genetic marker, worthy of additional investigation to better understand the clinical and biological features of IDH-WT infiltrating gliomas.

IDH mutational status is required for proper diagnosis according to the WHO criteria revised in 2016. The single nucleotide polymorphism (SNP) rs11554137 (IDH1 105GGT ) at codon 105 of IDH1 has been reported in patients with several tumor types, including those with glioma. The aim of this study is to investigate the prevalence of IDH1 105GGT in a cohort of brain tumors, and its association with clinicopathologic features and IDH1 and IDH2 missense mutations. Exon 4 of IDH1 and IDH2 was analyzed in a series of brain tumors classified according to current WHO criteria. DNA from control individuals was analyzed to infer the prevalence of IDH1 105GGT in the reference population. Analysis was performed using next generation sequencing. IDH1 105GGT was three times more frequent in patients with tumors (44/293 cases, 15.0%) vs. population controls (6/109, 5.5%) (p = 0.0102). IDH1 105GGT was more frequent in grade III tumors (26.1%) compared to grade II (10.9%, p = 0.038) and grade IV tumors (13.7%, p = 0.041). IDH1 105GGT was more frequent in grade II and III tumors without an IDH tumor missense mutation (43.8%) than in those with (11.5%, p = 0.005). The IDH1 105GGT SNP likely represents an important genetic marker, worthy of additional investigation to better understand the clinical and biological features of IDH-WT infiltrating gliomas.
The isocitrate dehydrogenase (IDH) family includes three isozymes (IDH1, IDH2, IDH3) involved in the Krebs cycle as active participants in NADPH production. These proteins also play an important role in the cellular control of oxidative damage 1,2 . The IDH1 protein is localized to the cytoplasm and peroxisome, while IDH2 and IDH3 are located in mitochondria 3 . IDH1 mutations were first implicated in carcinogenesis by a high-throughput study of the mutational landscape of breast and colorectal cancers 4 . Since then, mutations in IDH1 or IDH2 genes have been detected in many different tumors, primarily gliomas (>80% of grade II and grade III gliomas) 5 , acute myeloid leukemia (AML, ~15% of cases) 6,7 and chondrosarcomas (~50% of cases) 8 . IDH mutations have been reported, albeit with a lower prevalence, in thyroid carcinoma (5-15% of cases) 9,10 , cholangiocarcinoma (15-20% of cases) 11 , and other solid neoplasms [12][13][14][15] . Among brain tumors IDH mutations are identified in over 80% of grade II and grade III gliomas (astrocytomas, oligodendrogliomas) 16,17 and in about 5% of glioblastomas (GBM) 16 . According to the 2016 World Health Organization (WHO) classification of Central Nervous System tumors, establishing whether a brain tumor is IDH mutated or wild-type (WT) is a crucial requisite for the classification of gliomas 18 .
Usually, synonymous single nucleotide polymorphisms (SNPs) do not change protein function, insofar as the amino acid sequence of the protein is not affected by the nucleotide change. Some silent SNPs, however, may lead to a protein defect, for example when they are localized in a splicing site 23,24 . In the case of the IDH1 gene, Wagner et al. (2010) found a silent SNP in a cohort of cytogenetically normal AML samples, that changes codon 105 of exon 4 from "GGC" (Gly) to "GGT" (Gly) 25 . The SNP (p.G105G, rs11554137:C > T -IDH1 105GGT , minor allele frequency 0.0569) has since been frequently reported in AML and is linked to an adverse prognosis [26][27][28] . It has also been reported in brain tumors in a study of patients with gliomas (grade II to IV) from France and Germany 29 and in a series of Bulgarian GBM patients 30 , as well as in thyroid tumors (both carcinomas and adenomas) 9,10 .
The role and biologic significance of the IDH1 105GGT SNP in tumorigenesis is poorly understood, but it appears to be associated with increased IDH1 mRNA levels leading to altered NADPH production 25,29 .
The aim of the present study was to assess the prevalence of the IDH1 105GGT SNP in a cohort of Italian patients with brain tumors classified according to 2016 WHO criteria, and investigate its association with clinicopathologic features and IDH tumor missense mutations.
We found a statistically significant difference in the prevalence of IDH1 105GGT between tumor grades, with the highest frequency among tumors belonging to grade III, compared to grades II and IV (p = 0.038 and p = 0.041 respectively, Chi-squared test, Fig. 1B). Also among gliomas, IDH1 105GGT is more frequent in grade III than in grade II or IV cases (p = 0.039 and p = 0.046, respectively). No statistically significant difference in prevalence was observed between grades II and IV, for both tumors and gliomas (p = 0.5765 and p = 0.6546, respectively, Chi-squared test). IDH1 105GGT and other IDH mutations. We observed an IDH1 or IDH2 mutation in 51 of 64 (79.7%) grade II tumors (all 51 cases were gliomas and 37 of these harbored the common p.R132H IDH1 mutation), in 36 of 46 (78.3%) grade III tumors (all 36 cases were gliomas and 31 harbored p.R132H), and in 4 of 183 (2.2%) grade IV tumors (all p.R132H).
Among 91 IDH-mutated gliomas, 11 (12.1%) also carried IDH1 105GGT . In 31 gliomas, IDH1 105GGT was detected in the absence of any IDH missense mutation. In accordance with data previously reported 29 , we found no correlation between the presence of IDH missense mutations in the tumor and the presence of IDH1 105GGT (p = 0.4749, Fisher's exact test) (Fig. 1C). However, IDH1 105GGT was more frequent in grade II and III gliomas without than with IDH missense tumor mutations (43.8% vs 11.5% respectively -p = 0.005, Fisher's exact test) (Fig. 1D). The SNP was also more frequent in grade II and III gliomas lacking IDH missense mutations than in GBM lacking IDH missense mutations (43.8% vs. 13.4%, p = 0.005, Fisher's exact test) (Fig. 1D). IDH1 105GGT and histological subtypes. In grade II and III tumors, IDH1 105GGT was more frequent in astrocytomas (13 of 63 cases −20.6%) than in oligodendrogliomas (4 of 40 cases −10%), but the difference did not reach statistical significance (p = 0.1837, Fisher's exact test). Even after the inclusion of GBMs in the astrocytoma group (38 of 246-15.4%), the prevalence of IDH1 105GGT was not statistically different between the oligodendroglial and astrocytic lineages (p = 0.4743, Fisher's exact test). IDH1 105GGT and age. The age of patients harboring IDH1 105GGT ranged from 26 to 74 years (mean 51.7ys).
These patients were slightly younger than those without the SNP (mean age 53.2ys; age range: 17-84ys), but the difference was not statistically significant (p = 0.4476, Mann Whitney test). Figure 2 summarizes the statistical relationship between age, IDH1 105GGT and IDH missense tumor mutations. There are significant differences among patient age and the distribution of IDH1 105GGT and that of IDH missense tumor mutations. In particular, patients bearing only the SNP (mean age 55.7 years) were older that patients bearing both the SNP and IDH missense tumor mutations (mean 45.6ys, p < 0.05, Tukey's multiple comparisons test), or those bearing only missense mutations (mean 42.3ys, p < 0.001, Tukey's multiple comparisons test).

Discussion
In our Italian cohort, the prevalence of the IDH1 105GGT SNP was considerably higher in patients with brain tumors compared to the control population (15.5% vs 5.5%, respectively).
Few studies have analyzed IDH1 105GGT in brain tumors, likely because routine molecular pathology methods do not always allow its identification. IDH1 105GGT status cannot be inferred by immunohistochemical methods, or by the mutation-specific PCR assays commonly used to diagnose p.R132H. Furthermore, sequencing requires the design of specific primers to include codon 105. Our NGS primers allowed us to reliably diagnose the SNP genotype in all samples.
Wang et al. in a cohort of French and German patients with gliomas, did not find a statistical correlation between IDH1 105GGT and tumor histological grade 29 . In our cohort of Italian patients, we found a statistical association of IDH1 105GGT with grade III gliomas, in particular with grade III astrocytomas. Importantly, among grade II and III gliomas, IDH1 105GGT was more frequent in those cases without IDH missense tumor mutations (Fig. 1D). Previous studies reported this polymorphism as an adverse prognostic factor in patients with acute myeloid leukemia 25 ; findings in the series of Wang et al. suggested a strong association with adverse outcome in patients with malignant glioma 29 . No association of the IDH1 105GGT SNP with survival was found in the GBM series of Stancheva et al. 30 .
Although the functional effects of this polymorphism are still unclear, prediction analysis has shown that nucleotide 315 of the IDH1 gene may be within a putative Exonic Splicing Silencer (ESS) motif (ESRsearch Tool, http://esrsearch.tau.ac.il/) 31 . A nucleotide substitution in this region could lead to a protein defect due to incorrect regulation of constitutive or alternative splicing. Moreover, IDH1 105GGT may be in linkage disequilibrium with other "tumor predisposing" variants.
Current opinion favors the existence of two major glioma groups: IDH-mutant gliomas, that are typically grade II and III tumors with a relatively favorable prognosis and IDH-WT tumors with a worse prognosis. As the large majority of IDH-WT tumors are grade IV, some authors have suggested that IDH-WT astrocytomas are in fact under-sampled IDH-WT GBMs and that they should be treated accordingly 32 . However, some subsets of IDH-WT low-grade gliomas do not have the molecular characteristics of GBM. These tumors likely represent other entities on a biological level. Some IDH-WT astrocytomas correspond to so called "pediatric type" tumors, sharing genetic and epigenetic features with pilocytic astrocytomas 33 .
In this context, the IDH1 105GGT SNP may represent an important marker to further dissect and understand the clinical and biological features of IDH-WT infiltrating gliomas. Additional studies are warranted to clearly define the genetic profile and clinical outcome of patients with the IDH1 105GGT SNP.

Methods
Case selection. A total of 293 consecutive cases of primitive brain tumors (64 grade II tumors, 46 grade III tumors, 183 grade IV tumors) were retrieved from the archives of Anatomic Pathology of Bellaria Hospital (Bologna, Italy). Samples were diagnosed and reclassified according to 2016 WHO criteria 18 . Patients were 181 males (61.8%) and 112 females (38.2%), aged from 17 to 84 years (mean age 52.9ys). Control DNA samples were analyzed from the peripheral blood of 109 individuals who underwent blood testing at the same institution to infer the prevalence of IDH1 105GGT in the reference population. None of the controls was affected by brain tumor or other neoplastic diseases. The study was approved by Ethic Committee of All information regarding the human material was managed using anonymous numerical codes and all samples were handled in compliance with the Helsinki Declaration (https://www.wma.net/policies-post/ wma-declaration-of-helsinki-ethical-principles-for-medical-research-involving-human-subjects/). IDH1 and IDH2 analysis. All analyses were performed on DNA from formalin fixed and paraffin embedded (FFPE) specimens, extracted with the QuickExtract FFPE DNA Extraction Kit (Epicentre, Madison, WI, U.S.A.). Control DNA from blood specimens were extracted using the MasterPure DNA Purification Kit (Epincentre, Madison WI, USA). IDH1 (exon 4, codons 96-138) and IDH2 (exon 4, codons 151-178) amplicons were generated using the following primers: IDH1 Fw 5′-GAAACAAATGTGGAAATCACCA-3′, IDH1 Rv 5′-TCACATTATTGCCAACATGACT-3′; IDH2 Fw 5′-AGCCCATCATCTGCAAAAA-3′, IDH2 Rv 5′-TGTGGCCTTGTACTGCAGA-3′. The IDH1 105GGT SNP (rs11554137) is 27 codons (81 nucleotides) upstream of the IDH1 hot spot codon (p.R132), well within the DNA region amplified by our set of primers.
Categorical variables were compared using the Chi-square test or Fisher's exact test. Continuous variables were compared using the Mann-Whitney test. Statistical comparison among IDH1 alterations and age was determined by the one-way analysis of variance (ANOVA) with Tukey's multiple comparison test. A p-value < 0.05 was considered as statistically significant. Statistical analyses were performed using GraphPad Prism 6.01 (GraphPad Software).
Data availability. The datasets analysed during the current study are available from the corresponding author on reasonable request.