Low expression of isocitrate dehydrogenase 1 (IDH1) R132H is associated with advanced pathological features in laryngeal squamous cell carcinoma

Recent developments in genomic sequencing have led to the identification of somatic mutations in isocitrate dehydrogenase 1 (IDH1) in various malignancies. IDH1 R132H is the most common mutation of IDH1, which affects codon 132 and results in the conversion of amino acid residue arginine (R) to histidine (H). This study is designed to evaluate the association between the expression of IDH1 R132H and clinicopathological characteristics in laryngeal squamous cell carcinoma (LSCC). The expression pattern and clinical significance of IDH1 R132H were investigated in tissue microarrays (TMAs) of 50 LSCC tumors as well as adjacent normal tissues using immunohistochemistry. Then the exons of the 12 tumor samples with negative/weak positive staining were sequenced by applying polymerase chain reaction (PCR). The results demonstrated that the cytoplasmic expression of IDH1 R132H was downregulated in tumor cells compared to adjacent normal tissues. A statistically significant association was found between a low level of cytoplasmic expression of IDH1 R132H protein and an increase in histological grade (p < 0.001), perineural invasion (p = 0.019), and lymph node involvement (p < 0.001). The exon4 sequencing results showed that only one sample was positive for IDH1 R132H mutation. IDH1 R132H expression was observed in 39 (78.0%) LSCC samples. These findings indicate that low cytoplasmic expression of IDH1 R132H may have clinical significance in LSCC patients and is associated with more aggressive tumor behavior and progression of the disease, which can help improve potential treatment in patients with LSCC. Further investigations are needed to understand the biological function of IDH1 R132H and larger sample size to confirm our findings.


Introduction
Laryngeal cancer is the most common type of head and neck cancer, accounting for approximately 185,000 new cases and 100,000 deaths worldwide in 2020, indicating a high mortality rate (Sung et al. 2021). Further, it has been estimated that newly diagnosed cases and mortality rates in the USA will be approximately 12,500 and 4000, respectively, during 2022 (Siegel et al. 2022). The most prevalent subtype of laryngeal cancer (about 95%) is laryngeal squamous cell carcinoma (LSCC; Johnson et al. 2020). Although the survival rate of LSCC changes from 80% in the early stage to less than 50% in the advanced stage, the overall 5-year survival rate is about 60% (Forastiere et al. 2018). Despite recent advances in the treatment of LSCC, which include surgery, radiotherapy, chemotherapy, and immunotherapy, it still has a worse outcome, making treatment challenging and the prognosis unsatisfactory (Carlisle et al. 2020;Megwalu and Sikora 2014). As a result, the identification of tumor markers provides a valuable and promising tool with greater efficacy that assists in the diagnosis, treatment, and improvement of prognosis.
Isocitrate dehydrogenase (IDH) is an enzyme of the Krebs cycle (TCA cycle), which catalyzes the oxidative decarboxylation of isocitrate and produces α ketoglutarate (α-KG), CO2, as well as NAD(P)H that protects the cells from reactive oxygen species (ROS; Al-Khallaf 2017; Stoddard et al. 1993). There are three different human IDH isoforms, comprising IDH1, IDH2, and IDH3. The location of IDH1 is cytoplasm and peroxisomes, while IDH2 and IDH3 are localized in mitochondria (Tommasini-Ghelfi et al. 2019). IDH1 and IDH2 are homodimeric and NADP+ dependent, reversible catalyzed reactions with no known allosteric modifiers, whereas IDH3 is heterotetrameric (2α, 1β, 1γ) and NAD + dependent, catalyzed irreversibly and regulated by different allosteric effectors (Reitman and Yan 2010). IDH1, the most commonly identified mutated gene, plays an important role in varying cellular functions, including lipid metabolism, glucose sensing, differentiation, DNA repair, and redox states (Ronnebaum et al. 2006;Molenaar et al. 2014). Mutation in IDH1 led to converting α-KG to D isomer of 2-hydroxyglutarate (D2HG). Accumulation of D2HG, an oncometabolite, leads to hypoxia-inducible factor 1α (HIF-1α) degradation and epigenetic alterations such as DNA demethylases, histone modification, and chromatin remodeling. These changes could affect cancer formation and progression (Molenaar et al. 2014;Dang et al. 2009;Robertson et al. 2019). The fourth exon of the IDH1 gene encodes three arginine residues, namely R100, R109, and R132, which are significant for the proteins' activity (Xu et al. 2004). IDH1 mutations are heterogenous missense mutations restricted to a single arginine residue, R132, in the active region of the enzyme (Cairns and Mak 2013). It has been discovered that there are five distinct R132 mutations that result in different amino acid exchanges, including histidine (R132H), cysteine (R132C), glycine (R132G), leucine (R132L), and serine (R132S), with R132H being the most prominent mutation (Paschka et al. 2010;Yan et al. 2009).
A growing body of evidence has indicated that IDH1 mutations are involved in various malignancies (Chotirat et al. 2012;Yang et al. 2012). For the first time, IDH1 mutation (IDH1 R132C) was recognized in breast and colorectal cancers in 2006 (Sjöblom et al. 2006). Following that, Parsons et al. found the IDH1 R132H mutation in glioma (Parsons et al. 2008). It was found that mutations in IDH1 occurred in grade 2 and 3 glioma patients and more frequently in secondary glioma than primary (Yan et al. 2009;Kloosterhof et al. 2011). Besides, IDH1 mutations were observed in patients with acute myeloid leukemia (AML; Mardis et al. 2009), nonepithelial melanoma (Shibata et al. 2011), chondrosarcoma (Amary et al. 2011), prostate cancer (Ghiam et al. 20122), intrahepatic cholangiocarcinoma (Borger et al. 2012), etc. It has been found that the rate of IDH1/2 mutations is low (11.8%) in laryngotracheal chondrosarcoma, suggesting a different mode of tumorigenesis needing further investigation (Tallegas et al. 2019).
Although the role of IDH1 mutation was examined in various cancers, its role in LSCC is undetermined. Therefore, the current study was designed for the first time to investigate the expression patterns of IDH1 R132H protein expression in a series of LSCC tumor samples, and its association with different clinicopathological parameters, including histological grade, TNM stage, lymphovascular invasion, perineural invasion, and lymph node involvement.

Investigation of IDH1 based on data mining
cBio Cancer Genomics Portal (cBioPortal; https:// www. cbiop ortal. org/) is an online visual exploration tool for multidimensional cancer genomics data such as The Cancer Genome Atlas (TCGA; Cerami et al. 2012). Therefore, the IDH1 gene was searched in the cBioPortal database to further evaluate most mutations of this gene and alterations in protein for head and neck cancer tissue samples. Moreover, to investigate the expression of IDH1 in mRNA levels in patients with head and neck cancer, the UCSC Xena Browser database (https:// xenab rowser. net/) was applied and a box plot mRNA expression analysis was conducted on normal, primary tumor, and metastatic tissues. UCSC

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Xena for tumor and normal samples obtained from public and private, multi-omic, and clinical/phenotype data such as TCGA and the Genotype-Tissue Expression (GTEx; Goldman et al. 2019). Finally, investigation of this biomarker as part of a literature review was done in neoplasm disease class of DisGeNET plug-in (Piñero et al. 2020) as well as protein-protein interaction (PPI) network using stringApp (confidence score ≥ 0.4; Doncheva et al. 2019) in Cytoscape software (Shannon et al. 2003).

Sample size calculation
The sample size was determined by using the following formula: The prevalence of the IDH1 expression (57.89%) was taken from research conducted by Chen et al. (2016). The alpha value, d, and power were considered as 0.05 and 0.14, and 90%, respectively. Z statistic for a confidence level was 1.96 for a 95% confidence level.
The calculated sample size was 47, and we used 50 tumor samples.

Patient's characteristics and tumor samples
A total of 50 formalin-fixed paraffin-embedded (FFPE) sample tissues from LSCC samples were collected from the pathology laboratory of Rasoul Akram hospital, Tehran, Iran, from 2018 to 2019. Patients diagnosed with primary LSCC who had undergone laryngectomy surgery but had not received chemotherapy or radiotherapy were considered eligible for participation in this study. In addition, the hematoxylin and eosin (H & E)-stained slides and medical archival documents of enrolled patients were retrieved to obtain clinicopathological and demographic data, including gender, age, tumor size, histological grade, TNM stage, lymphovascular invasion, perineural invasion, and lymph node involvement. Ethical approval was attained by the Ethics Committee of the Iran University of Medical Sciences (IR.IUMS.FMD. REC.1399.276) to use the patient's tissues, and assurance was given to keep the patient's information confidential.

Tissue microarray (TMA) construction
The H&E slides were examined by an expert pathologist (N.Sh) to identify and mark the three most representative areas of the tumor's various regions. Then the selected areas in each block were punched with a diameter of 0.6 mm using TMA equipment (Minicore; Alphelys, France) and precisely transferred into new recipient paraffin TMA blocks. Due to tumor heterogeneity as a major concern throughout the TMA procedure, three copies were constructed from each tumor sample's TMA block. TMA slides were obtained by cutting sections of TMA blocks to a thickness of about 4 μm, which were transferred to an adhesive-coated slide system. Lastly, the mean expression of three cores was calculated for each tissue sample to enhance the accuracy and validity of the data analysis.

Immunohistochemistry (IHC) staining
After TMA blocks were sectioned, all prepared slides were dewaxed at 60 °C for 1 h, deparaffinized, and rehydrated with xylene and graded ethyl alcohol. To suppress endogenous peroxides, 0.3% hydrogen peroxide (H 2 O 2 ) was added into the slides for 20 min at room temperature. After washing tissue sections in Tris-buffered saline (TBS), antigens were retrieved from tissue slides by autoclaving them for 10 min in citrate buffer (pH 6.0). Then, the slides were incubated with primary antibody, GenAb™ Monoclonal Anti-IDH1 R132H [Clone IHC132-1] dilution of 1/100, overnight at 4 °C. The next day, TMA sections following three times washing with TBS, were incubated with mouse anti-rabbit secondary antibody for 30 min at room temperature, then visualized by diaminobenzidine (DAB peroxidase substrate) and counterstained with hematoxylin. Eventually, the slides were dehydrated in graded ethyl alcohol, cleared in xylene, and mounted for evaluation. For the negative control, instead of using primary antibody, incubation with only TBS was carried out.

Evaluation of immunostaining
Immunostaining of IDH1 R132H on tissue microarray LSCC slides was evaluated by two independent pathologists (N.Sh., and N.E.), who were blinded to the clinicopathological and prognostic information, through a semiquantitative scoring system. A consensus was attained for all tissue samples. Three scoring systems were used to evaluate the level of IDH1 R132H expression: the intensity of staining, the percentage of positive tumor cells, and the H-score. The immunostaining intensity of IDH1 R132H was visually scored as 0, negative, or no staining; 1, weak staining; 2, moderate staining; and 3, severe staining. The percentage of positive tumor cells was scored ranging from 0 to 100% and categorized into four groups as follows: < 25, 25-50%, 51-75%, and > 75%. The total score was determined using the histochemical score (H-score), which was calculated by multiplying the intensity of the staining and the percentage of positive cells, resulting in a score between 0 and 300 for each core. In the current study, the median H-score was considered to categorize samples as high or low IDH1 R132H expression.

DNA extraction
Genomic DNA (gDNA) extraction from FFPE was carried out by applying the E.Z.N.A DNA extraction Kit (Omega Bio-Tek, Inc), according to the manufacturer's instructions. Mmutation hotspots within IDH1 exon 4 were analyzed. Proper positive and negative controls were included in each run. The programs were analyzed by trained personnel. To validate the results, gDNA underwent direct sequencing, namely Sanger sequencing.

Polymerase chain reaction (PCR)
Two primers were used in this protocol. The sense primer sequence was 5-CGG CTT GTG AGT GGA TGC -3 (position 691-710) and the antisense primer was 5-TGC TTA ATG GGT GTA GAT AC-3 (position 829-809). The forward and reverse primers amplified a product of approximately 300 bp.
The amplification conditions were: 95 °C for 8 min; 35 cycles at 94 °C for 30 s, 60 °C for 35 s, 72 °C for 45 min, and 72 °C for 5 min. The PCR reaction mixture for the set of primers contained: 12 μl Master AMPLIQON Taq 2 × Master Mix Red 1.5 mM Mgcl2, 1 μl of each primer, 6 μl H 2 O 2 , and 80 ng gDNA in a total volume of 2 μl. The PCR products were purified through ExoI and Fast-AP according to the manufacturer's instructions. The sequencing reaction was performed using BigDye™ Terminator v3.1 Cycle Sequencing Kit (based on manufacturer's instructions) and 2 μl primers. Each amplification contained positive (IDH1 gDNA) and negative (distilled water) controls to exclude crossover contamination.

Statistical analysis
All the obtained IHC data were analyzed using SPSS Statistics 25.0 software (IBM, NY, USA). Categorical data were reported as N (percent), whereas quantitative data were reported as mean (SD) and median (Q1-Q3). Pearson's 2 and Spearman's correlation tests were employed to determine the significance of the association and correlation between IDH1 R132H protein expression and clinicopathological variables. Moreover, to make pairwise comparisons between the groups, Kruskal-Wallis and Mann-Whitney U tests were performed. A p value of < 0.05 was regarded as statistically significant. The sequencing data of the PCR was analyzed through Sequencing Analysis software.

Bioinformatics approaches
The IDH1 mutations from 1438 samples in five head and neck cancer studies (cBioPortal) indicated 10 missense mutations which led to five changes in IDH1 protein (Fig. 1).  (cBioPortal). A This graphical view shows the protein domains and the positions of mutations. B The frequency and information for missense mutation type in the IDH1 gene that leads to five changes in its protein Also, the results of the TCGA database for 564 tissue samples via UCSC Xena exhibited mRNA expression level of IDH1 to a small extent, higher in tumor tissues with metastatic feature than primary tissues and normal tissues in patients with head and neck cancer, although it was not significant (one-way ANOVA, p = 0.08801 (f = 2.441), Fig. 2).
The PPI information from the STRING database indicated that IDH1 has a high confidence score with ACO1, ACO2, GOT2, IDH3B, and OGDH, the dysregulation of which have important roles in cancer progression and tumorigenesis (Smolková and Ježek 2012;Remacha et al. 2017;Fig. 3). Also, according to our findings in the literature review and DisGeNET data, the IDH1 gene was investigated in various cancers (Dang et al. 2016;Shait Mohammed et al. 2022), but there has been less research on the expression pattern for protein levels (Fig. 4).

Patients' characteristics
In this cross-sectional study, the sample population comprised a total of 50 LSCC patients with a mean age of 61 years (SD = 8.33; range 44-78 years). Of them, 49 cases were men (98.0%), and 1 was a woman (2.0%). Out of 50 patients, 29 (58.0%) were ≥ 61 years old and 21 (42.0%) were < 60 years old. The tumor size ranged from 1.5 to 7.5 cm, with a mean tumor size of 3.9 cm. The tumors size was classified into two groups based on the mean size. Overall, 23 (46.0%) cases showed tumor size of less than 3.9 cm, and 27 (54.0%) cases had tumor sizes of more than 3.9 cm.
The clinicopathological characteristics of our samples are shown in Table 1. PPI network for IDH1 protein was created by StringApp plug-in using Cytoscape software with a confidence score ≥ 0.4

Evaluation of IDH1 R132H expression in laryngeal squamous cell carcinoma and adjacent normal tissue samples using IHC
The IDH1 R132H expression level was assessed by utilizing the IHC technique on the TMA section with three varied scoring methods as follows: intensity of staining, percentage of positive tumor cells, and H-score. The IDH1 R132H expression pattern was observed only in the cytoplasm of both LSCC tissue samples and adjacent normal tissues. Based on the median IDH1 R132H H-score value (120) as the cutoff, patients were categorized into either low (≤ median of H-scores) or high (> median of H-scores) expression. A low expression of IDH1 R132H was observed in 29 (58.0%) LSCC samples, whereas higher IDH1 R132H expression was found in 21 (42.0%) samples. Furthermore, LSCC tissue samples showed IDH1 R132H expression with variable staining intensities, including 5 (10.0%) negative, 7 (14.0%) weak, 17 (34.0%) moderate, and 21 (42.0%) strong ( Table 2). The mean expression of IDH1 R132H in adjacent normal tissues was higher than that in tumor tissues samples (Fig. 5).

Cytoplasmic expression of IDH1 R132H and its association with clinicopathological features of laryngeal squamous cell carcinoma
To examine the association between IDH1 R132H expression and clinicopathological features, Pearson's χ 2 test was applied. Also, Spearman's correlation analysis was executed to show the correlation between the expression of IDH1 R132H and clinicopathological parameters. The results showed a statistically significant association between the cytoplasmic expression of IDH1 R132H Fig. 4 Obtained data about IDH1 in the DisGeNET library. Investigation of the IDH1 gene based on the DisGeNET database by Cytoscape shows that this gene is involved in various cancers and histological grade of the tumor in terms of intensity (p < 0.001) as well as perineural invasion in terms of H-score (p = 0.019). Moreover, our analysis showed a significant association between IDH1 R132H expression and lymph node involvement in both terms of intensity and H-score (All p < 0.001) in LSCC patients. However, the results of Spearman's correlation exhibited a significant inverse correlation between IDH1 R132H expression and histological grade (p = 0.002), lymph node involvement (p < 0.001), as well as perineural invasion (p = 0.019). In other words, higher expression of IDH1 R132H correlated with decreased histological grade, lymph node involvement, and perineural invasion. As exhibited in Table 3, there was no significant association between IDH1 R132H expression and other important clinicopathological characteristics. In addition, Kruskal-Wallis and Mann-Whitney U tests also showed significant differences between the median level of IDH1 R132H expression and perineural invasion (p = 0.035; Fig. 6).

Evaluation of IDH1 R132H mutation in negative/ weak positive staining intensities samples in laryngeal squamous cell carcinoma using PCR
To identify IDH1 R132H mutation, 12 LSCC samples with negative and weak positive staining intensities were assessed using gene sequencing with PCR technique. The IDH1 R132H exon4 sequencing results indicated that 11 of the 12 samples were negative for IDH1 R132H mutation and only one was positive (Fig. 7). Overall IDH1 R132H mutation were detected in 39 (78.0%) LSCC patients through using IHC and PCR methods. A flowchart of the process of samples selection is exhibited in Fig. 8.

Associations between status of IDH1 R132H mutation and clinicopathological features in laryngeal squamous cell carcinoma
All LSCC samples were divided into two groups, defined as positive IDH1 R132H or negative IDH1 R132H mutation. Pearson's χ 2 test observed a significant association between positive IDH1 mutation and histological grade (p < 0.001) as well as lymph node involvement (p = 0.014). We did not observe any association and correlation between the status of IDH1 R132H mutation and other clinicopathological characteristics. Furthermore, non-parametric Kruskal-Wallis and Mann-Whitney U tests were applied to evaluate differences between the tumor size among diverse IDH1 mutation groups. Our results did not display any differences between tumor size and various statuses of IDH1 R132H mutation (Table 4).    (

Discussion
LSCC is one of the most common cancers of the head and neck region, accounting for 90-95% of all laryngeal cancers; its incidence has significantly increased in the last decade (Siegel et al. 2022). Due to the lack of apparent symptoms in the early stage and the occurrence of lymph node metastases, the majority of patients present with advanced-stage cancer at diagnosis, which affects the prognosis of patients (Cooper et al. 2004). Given these facts and the limitations of currently available biomarkers, it appears crucial to explore more effective biomarkers that would help determine the appropriate treatment and monitoring of recurrence and metastasis precisely. Our in silico finding indicated IDH1 expression changes as well as mutations through bioinformatics tools in LSCC samples, which have been also observed in various types of cancer studies based on the DisGeNET database. Therefore, in the present study, one mutation, IDH1 (R132H), and its expression were investigated. The results of the experiments using the IHC method indicated that the cytoplasmic expression of IDH1 R132H was upregulated in adjacent normal tissues compared to LSCC samples. It has been shown that low cytoplasmic expression of IDH1 R132H was found to be associated with an increase in histological grade and high lymph node involvement. However, we need to use a larger sample size to confirm our findings due to a low number of patients with poorly differentiated and those with positive lymph node involvement. Further, our findings showed that there was an adverse association between the cytoplasmic expression level of IDH1 R132H and increasing perineural invasion, but no association between the cytoplasmic expression of IDH1 R132H and other clinicopathological features was found.
IDH1, an NADP + enzyme of the Krebs cycle, is localized in the cytoplasm and peroxisome, which converts isocitrate to α-KG (Xu et al. 2004). It participates in several cellular metabolic functions (lipid and glucose metabolism) and protects the cells from ROS and radiation (Lee et al. 2002). It has been discovered that IDH1 induces tumor suppressor genes, including ubiquinone oxidoreductase core subunit S1, guanine nucleotide binding protein gamma 4, and TNF alpha-induced protein 1, which have been shown to inhibit cancer progression via the effects on ROS, chemokine receptor biology, and nuclear factor κB (NFκB) signaling (Calvert et al. 2017). Additionally, according to the STRING-PPI network, IDH1 was correlated with proteins that have important roles in cancer, such as ACO1, ACO2, OGDH, IDH3B, and GOT2. It has been reported that in highly glycolytic cancer cells, to survive under conditions of limited glucose and hypoxia, IDH1 and aconitase (ACO) are required to generate lipogenic citrate from glutamine-derived α-KG (Smolková and Ježek 2012;Metallo et al. 2011). Moreover, it has been shown that IDH1 and oxoglutarate dehydrogenase (OGDH) are correlated with the development and progression of clear cell renal cell carcinoma (ccRCC) and serve as potential prognostic biomarkers (Zhang et al. 2021). Significantly, IDH1 mutations, along with germline mutations in IDH3B and glutamic-oxaloacetic transaminase 2 (GOT2), a TCA cycle enzyme, are involved in pheochromocytomas/paragangliomas predisposition and cancer metabolism through alterations in Krebs cycle metabolite ratios (Remacha et al. 2017;Lane et al. 2020). In addition, it has been found that IDH1 mutations are associated with the establishment and maintenance of cancer stem cells (CSCs). CSCs are a subpopulation of cancer cells with stem cell-like features that may differentiate into many cell types; they are the source of all cancer cells and cause therapeutic resistance and disease The results showed that there was a statistically significant association between the mean of IDH1 R132H expression and perineural invasion patients (p = 0.035) recurrence (Kahlert et al. 2009;Yan et al. 2013). IDH1 mutations and IDH-related pathways may be important molecular targets for affecting CSC components in human malignancies and improving the prognosis of the disease (Zhang et al. 2022). Mutation in IDH1 leads to less binding to isocitrate and reduced formation of α-KG; Subsequently, it contributes to the formation of D2HG (Dang et al. 2009;Zhao et al. 2009;Pietrak et al. 2011). The building up of D2HG is thought to function as an oncometabolite with a variety of potential tumorigenicities (DiNardo et al. 2013;Ward et al. 2012;Lu et al. 2012;Krell et al. 2013). It has been established that D2HG has an α-KG antagonist role, which competitively inhibits α-KG-dependent dioxygenases due to the significant structural similarity that hydroxylates key proteins such as HIF-1α (Zhao et al. 2009;Xu et al. 2011;Jiang et al. 2018). Hence, in IDH1 mutant tumor cells, HIF-1α hydroxylation, a degradation enzyme, is inhibited and causes its upregulation in cancer cells, which leads to tumor progression (Xu et al. 2011;Sasaki et al. 2012;Metellus et al. 2011). Also, the methylcytosine dioxygenase TET is inhibited by 2HG, leading to impaired tumor differentiation (Guo et al. 2011;Figueroa et al. 2010).
The most prevalent IDH1 mutation is a heterozygous point mutation in which guanine is altered to adenine at position 395 (C.395G>A), resulting in the substitution of arginine at position 132 with histidine (R132H) at the IDH1  (Parsons et al. 2008;Olar et al. 2012;Bleeker et al. 2009). There is accumulating evidence indicating that the IDH1 R132H (mIDH1) protein is expressed in cancers, including cholangiocarcinoma, AML, and glioblastoma (Parsons et al. 2008;Zhou et al. 2022;Marcucci et al. 2010). Besides, its mutations are associated with several cancerrelated processes (invasion, migration, and cell proliferation) and are involved in distinct regulatory signaling pathways Shen et al. 2020;Yan et al. 2018). It has been reported that the IDH1 R132H mutation suppressed PTEN expression through activating the Notch1/HES1 and PI3K/AKT pathways, thereby promoting malignant behavior in T-cell acute lymphoblastic leukemia cells (Liu et al. 2022). In addition, the IDH1 R132H mutation downregulated ICAM-1/CD54 in glioma cells, representing an immunomodulation role of mutant IDH1 (Ma et al. 2021). It also contributes to the progression of non-small cell lung cancer by promoting migration and proliferation (Yan et al. 2018).
Several studies demonstrated low expression of IDH1 at the protein level, such as breast cancer, gastric cancer, and colorectal cancer (Liu et al. 2018;Li et al. 2016), which are in agreement with our findings. There have been many reports concerning IDH1 expression and clinicopathological parameters in various tumor tissues. In accordance with our findings, previous studies revealed an inverse correlation between IDH1 expression and histological grade as well as lymph node involvement in colorectal cancer, gastric cancer, and ccRCC, suggesting high IDH1 expression can be an independent prognosis of favorable outcomes Laba et al. 2018). Besides, Liu, W.S. and colleagues discovered that low expression of IDH1 correlated with lymph node metastasis and high stage of breast cancer, leading to increased snail expression through activating HIF-1α and NFκB signaling. They identified that low expression of IDH1 could be an important marker for inhibiting cell migration and invasion of breast cancer cells (Liu et al. 2018).
It has been established that the IHC technique has 92.3% sensitivity, 97.9% specificity, and 96.7% accuracy, while PCR has 100% sensitivity, specificity, positive predictive values, negative predictive values, and accuracy to represent IDH1 mutation (Malueka et al. 2020). Therefore, to confirm the negative IHC results, DNA sequencing of 12 LSCC tumor samples with negative and weak positive staining intensities was performed. As Gondim, D. D. and colleagues reported, three false-negative results from IHC was found, while in contrast PCR results indicated one false-negative test (Gondim et al. 2019). Our findings indicated that the IDH1 R132H mutation was observed in 78% of LSCC tumor samples, which consisted of 38 positive IHC results and 1 positive PCR. In this regard, previous studies have demonstrated that this mutation is present in about 80% of grade II-III gliomas and secondary glioblastomas (Bleeker et al. 2009;Balss et al. 2008;Kang et al. 2009). As a result, IDH1 R132H immunostaining is considered a first-line diagnostic test, and PCR could be used to examine non-immunoreactive samples (Sporikova et al. 2022). We suggest using both IHC and PCR to detect IDH1 mutations. In general, our findings for the first time indicated that low expression of IDH1 R132H is associated with high pathological features consisting of histological grade, high lymph node involvement, as well as perineural invasion. Indeed, IDH1 R132H, which is expressed in the cytoplasm at the protein level and is recognized by IHC and PCR methods, could be a specific biomarker in LSCC. However, the limitations of this study are the small sample size and lack of survival data to investigate the prognostic role of IDH1 in LSCC. Extending the clinical follow-up time with a larger patient sample is recommended.

Conclusion
In summary the current study is the first to exhibit that decreased cytoplasmic expression of IDH1 may have a clinical significance in LSCC cases and is associated with increased invasiveness and poor outcomes. Further, it is found that 78% of the LSCC tumor samples has IDH1 R132H mutations. To sum up, investigation of the expression pattern of IDH1 in the cytoplasm as a predictor indicator of cancer progression in LSCC patients' tumor biopsies is useful. However, further research is required using a larger sample size to confirm our findings.