Isocitrate dehydrogenase 2 mutation is a frequent event in osteosarcoma detected by a multi-specific monoclonal antibody MsMab-1

Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 occur in gliomas, acute myeloid leukemia, and cartilaginous tumors. Somatic mosaic IDH1/2 mutations are also reported in Ollier disease and Maffucci syndrome, which are characterized by multiple central cartilaginous tumors. Although IDH1/2 mutation analysis against osteosarcoma has been performed in several studies, no IDH1/2 mutation has been reported. Herein, we newly report the IDH2-R172S mutation in three of 12 (25%) osteosarcoma patients, which was detected by direct DNA sequencing. No monoclonal antibody (mAb) has been reported against IDH2-R172S mutation. However, we demonstrate that the IDH2-R172S peptide was recognized by our established multi-specific anti-mutated IDH1/2 mAb, MsMab-1, in enzyme-linked immunosorbent assay. Western blot analysis revealed that MsMab-1 reacts with PA tag combined recombinant proteins of IDH2-R172S. Furthermore, MsMab-1 stained IDH2-R172S-expressing osteosarcoma tissues in immunohistochemistry. The MsMab-1 stained nine of 32 (28.1%) osteosarcomas in a tissue microarray. This report is the first describing IDH2 mutations in osteosarcoma, which can be detected by MsMab-1 mAb. Taken together, these results show that MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing osteosarcoma.


Introduction
Although osteosarcoma is the most common primary malignant bone tumor in children and young adults, the advance of aggressive systemic chemotherapy has improved the survival rate for osteosarcomas. Nevertheless, the survival rate of osteosarcoma patients with primary lung metastases remains poor compared to that of patients with localized disease [1]. Moreover, multidrug combination chemotherapy for osteosarcoma entails ototoxicity, cardiac toxicity, and secondary malignancies [2]. To resolve these problems, it is expected to be necessary to develop molecular targeted agents with high tumor specificity.
Somatic mutations of isocitrate dehydrogenase (IDH) 1 and IDH2 were first found in gliomas [3] to convert a-ketoglutarate to oncometabolite R(-)-2-hydroxyglutarate (2-HG), although IDH1 and IDH2, respectively, catalyze the oxidative carboxylation of isocitrate to a-ketoglutarate in cytosol and mitochondria [4]. Results of 2-hydroxyglutarate dehydrogenase deficiency show that 2-HG accumulates in association with the inherited metabolic disorder 2-hydroxyglutaric aciduria because 2-hydroxyglutarate dehydrogenase changes 2-HG to a-ketoglutarate [5]. Patients with 2-hydroxyglutarate dehydrogenase deficiencies are known to face increased risk of brain tumors because they accumulate 2-HG in the brain and develop leukoencephalopathy. Furthermore, elevated 2-HG levels in the brain contribute to increased risk of cancer by increasing reactive oxygen species (ROS) concentrations [6]. IDH1/2 mutations were also reported in acute myeloid leukemias (AML) [7] and cartilaginous tumors [8][9][10]. Kerr et al. [11] also reported that IDH1/2 mutations were detected in chondrosarcoma, but not in chondroblastic osteosarcomas, suggesting that mutation analysis of IDH1/ 2 has yielded a promising biomarker for distinguishing chondrosarcoma from chondroblastic osteosarcoma.

Cell lines and tissues
Chinese hamster ovary (CHO)-K1 and U-2 OS osteosarcoma cell line were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured at 37°C in a humidified atmosphere of 5% CO 2 and 95% air in RPMI 1640 and Dulbecco's modified Eagle medium (DMEM), respectively, including 2 mmol/ L L-glutamine (Nacalai Tesque Inc., Kyoto, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies Inc., Carlsbad, CA). This study examined 12 osteosarcoma patients who underwent surgery at Yamagata University. The ethical committee of the Yamagata University Faculty of Medicine approved this study. Informed consent for obtaining samples and for subsequent data analyses was obtained from each patient or the patient's guardian. Tissue microarrays of osteosarcomas were purchased from Cybrdi Inc. (Frederick, MD). The pathological diagnosis of all specimens in this study was confirmed by a pathologist (Prof. Mitsunori Yamakawa, Yamagata University School of Medicine).

Results
Mutational analysis of IDH1/2 in osteosarcoma IDH1/2 mutation analysis against osteosarcoma has been performed in several studies [8,11], but no IDH1/2 mutation has been reported yet. For this study, we analyzed IDH1 and IDH2 mutations using 12 osteosarcoma specimens with direct DNA sequencing ( Table 1). All samples were primary tumors obtained by biopsy before chemotherapy or surgery. Of the 12 patients, 2 showed lung metastasis before chemotherapy. In accordance with previous reports, no IDH1 mutation was observed in 12 samples. In contrast, three of 12 (25%) osteosarcoma samples, two of which were osteoblastic osteosarcoma (OB) and one of which was high-grade surface osteosarcoma (HGS), possessed IDH2 mutations. Two OB patients with IDH2 mutation showed grade 3 histological necrosis after preoperative chemotherapy. Continuous disease-free survival was assessed for all three patients with IDH2 mutations (75-128 months). It is noteworthy that all three IDH2 mutations were of IDH2-R172S (AGG>AGT; Fig. 1A), which is also frequently observed in chondrosarcomas [8]. To confirm this result, we performed subcloning of these three PCR products. As presented in Figure 1B, all three cases included the IDH2-R172S mutant sequence (OS10, 14/16 (87.5%); OS11, 2/17 (11.8%); and OS12, 6/17 (35.3%)), demonstrating that all three osteosarcoma samples include IDH2-R172S mutations.

Discussion
This study demonstrated that osteosarcoma possesses IDH2 mutations, especially IDH2-R172S, which is also frequently observed with chondrosarcoma, which is another primary malignant bone tumor (Fig. 1). Amary et al. [8] reported that no IDH1/2 mutation was detected in non-cartilaginous neoplasms. In that report, osteosarcoma samples from 222 patients, 19 osteosarcoma cell lines, and one chordoma cell line were tested for IDH1 mutations using the high-throughput MassARRAY platform: 64.4% (143 samples) of these were also analyzed for IDH2 mutations, including all chondroblastic osteosarcomas. The discrepancy between our detection of IDH2 mutations in osteosarcomas and their analysis has not been resolved; it might be dependent on ethnic differences. The 12 patients for whom data are presented in Table 1 are all Japanese. The 32 patients in tissue microarray (Table 5) are all Chinese. In the near future, we expect to analyze IDH1/2 mutations using many more Japanese or Chinese osteosarcoma patients. Although we used subcloning method to confirm that OS12 possesses IDH2-R172S mutation (Fig. 1), MsMab-1 mAb did not stain OS12 in immunohistochemistry (Table 1), probably We previously established several anti-mutated IDH1/2 mAbs: HMab-1 against IDH1-R132H [14], SMab-1 against IDH1-R132S [16], GMab-r1 against IDH1-R132G [17], KMab-1 against IDH2-R172K [20], MMab-1 against IDH2-R172M [20], and WMab-1 against IDH2-R172W [21]. However, no mono-specific anti-IDH2-R172S mAb has been developed. Unexpectedly, we found that MsMab-1 detected IDH2-R172S, which was discovered in 25% of osteosarcoma patients in this study (Table 1). Moreover, MsMab-1 reacted with IDH2-R172G, which was reported in gliomas [3]. Because MsMab-1 did not react with IDH2-R172K or IDH2-R172W, and because it reacted weakly with IDH2-R172M, the combination of MsMab-1 with mono-specific mAbs such as KMab-1, MMab-1, and WMab-1 might still be necessary to detect all IDH1/2 mutations. For MsMab-1 reactivity, the discrepancy between ELISA (Tables 3 and 4) and Western blot analyses (Fig. 2) is expected to be resolved in the near future. MsMab-1 stained nine of 32 (28.1%) osteosarcomas (Fig. 4), indicating that MsMab-1 is extremely useful for the immunohistochemical detection of mutated IDH1/2 in osteosarcomas. The staining pattern of mutated IDH1/2 by MsMab-1 looks very heterogeneous (Fig. 4), which is different from that of glioma tissues [22]. Although we investigated 44 osteosarcoma patients in this study (Tables 1 and 5), we could not discuss the location of prepared tissues such as invasive or central portion, because we used biopsy samples from 12 patients (Table 1) and commercially available tissue microarray (Table 5). In a recent report, the staining pattern of IDH1-R132H by clone H09 mAb [19] in enchondroma of Ollier disease is heterogeneous, and the percentage of tumor cells positive for mutant IDH1 ranges from 50% to 90%, indicating intraneoplastic mosaicism [10]. Because intraneoplastic and somatic mosaicism has not been reported in osteosarcomas, the heterogeneity of MsMab-1 staining remains to be clarified.
Recently, two novel drugs were developed against mutated IDH1 and mutated IDH2. An inhibitor of mutant IDH1, AGI-5198, specifically suppressed the activity of mutant IDH1 and blocked the production of 2-HG [26]. Furthermore, AGI-5198 induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation under conditions of 2-HG inhibition. Blockade of mutant IDH1 impaired the growth of glioma cells possessing mutant IDH1, but not that of IDH1 WT glioma cells without appreciable changes in genome-wide DNA methylation. Another inhibitor of mutant IDH2, AGI-6780, selectively inhibits the tumorassociated mutant IDH2-R140Q [27]. AGI-6780 induced differentiation of TF-1 erythroleukemia and primary human AML cells in vitro. Although we found IDH2-R172S of osteosarcomas in this study, no inhibitor against IDH2-R172S has been discovered. New agents must be developed to improve the survival of patients diagnosed with osteosarcoma [28], although several clinical trials have been conducted using various agents activated in osteosarcoma: imatinib, a tyrosine kinase inhibitor targeting platelet-derived growth factor (PDGF)-A against refractory or relapsed solid tumors [29]; sorafenib, a multikinase inhibitor targeting mitogen-activated protein kinase (MAPK), vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), and KIT against relapsed unresectable osteosarcomas [30]; trastuzumab, a mAb to HER2 against metastatic osteosarcoma patients [31]. Nevertheless, no dramatic effect has been observed. To improve the treatment of osteosarcoma further, drugs targeted against not only a molecular overexpression but also gene mutations such as mutated IDH1/2 must be developed. Serum 2-HG is elevated in Ollier disease and Maffucci syndrome [9]. Dinardo et al. [32] recently reported that serum 2-HG levels predict IDH mutations and clinical outcome in AML. Their data confirm that serum measurement of an oncometabolite 2-HG provides useful diagnostic and prognostic information. The combination of immunohistochemistry using our developed anti-mutated IDH1/2 mAbs and serum measurement of an oncometabolite 2-HG is expected to improve osteosarcoma patient selection for IDH1/2-targeted therapy. Unfortunately, we could not check the 2-HG level before surgery, although we investigated 12 osteosarcoma patients. Serum measurement of 2-HG should be important in osteosarcomas; therefore, we will check the 2-HG before and after surgery of osteosarcoma patients in the next study.
It was reported that the potential for IDH mutations to produce 2-HG depends on allele specificity and subcellular compartmentalization [33]. The cellular 2-HG production from cytosolic IDH1 mutation is dependent on the activity of a retained WT IDH1 allele. The expression of mitochondrial IDH2 mutations led to robust 2-HG production in a manner that is independent of WT mitochondrial IDH function. Among the IDH2 mutations at R172 and R140, IDH2-R172 mutations consistently led to greater 2-HG accumulation than IDH2-R140 mutations did. From this perspective, it is important to elucidate the IDH mutations exactly in immunohistochemistry using mono-specific anti-mutated IDH1/2 mAbs such as HMab-1, SMab-1, GMab-r1, KMab-1, MMab-1, and WMab-1 as well as multi-specific mAbs MsMab-1 and MsMab-2. In the near future, IDH1/2 mutations will be acknowledged as useful diagnostic markers for osteosarcomas.