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Abstract

BACKGROUND:

While major progress has been made in diagnosis and treatment of gliomas based on molecules, molecular features of thalamic glioma have rarely been reported till now.

OBJECTIVE:

IDH1 mutation is important for prognosis of gliomas and represents a distinctive category of glioma. We intended to survey specific molecular abnormalities in high-grade thalamic gliomas (WHO III–IV).

METHODS:

We collected data of 50 and 93 newly diagnosed high-grade thalamic and superficial glioma patients respectively and conducted a comparative analysis of molecular characteristics between them. We analyzed expressions of molecules as follow: IDH1/2, P53, Ki-67, ATRX, PTEN, MMP9 and MGMT by Immunohistochemistry (IHC). Direct gene sequencing was performed to test the IDH1(R 132H) mutation.

RESULTS:

We found a significant difference of IDH1 mutation between those high-grade gliomas, with 92% (46/50) of the thalamic tumors and 71% (66/93) of the superficial gliomas showing IDH1 wild-type ( $p=$ 0.004). It also showed that IDH1 mutation in superficial glioblastomas 18.6% (13/70) occurred more than thalamic glioblastomas 2.6% (1/39) ( $p=$ 0.017). As to high-grade superficial gliomas, there were 26 patients with IDH1 mutation, which contained 7, 13, and 6 high, moderate and low Ki-67 expression gliomas, respectively. The IDH1 wild-type group (62 patients), was composed of 29, 30, and 3 high, moderate and low Ki-67 expression gliomas, respectively. There was a significant distinction between the IDH1 mutation and Ki-67 expressions ( $p=$ 0.024). We also noted that the occurrence of low Ki-67 expressions 23.1% (6/26) in IDH1 mutation group was outnumbered than IDH1 wild-type group 4.8% (3/62) ( $p=$ 0.018). In addition, we found PTEN negative correlated with MMP9 negative in thalamic high-grade gliomas, whereas no such difference was found in superficial gliomas ( $p=$ 0.016).

CONCLUSION:

The rare occurrence of IDH1 mutant high-grade thalamic gliomas strongly suggested that the high-grade thalamic glioma is another distinct tumor entity as compared to the high-grade superficial gliomas.

Keywords

High-grade Glioma thalamus superficial cerebrum IDH1 mutation

1. Introduction

Compared to non-thalamic gliomas, the morbidity of thalamic gliomas is significantly rare [1, 2, 3]. Thalamic gliomas have long been considered a difficult area for radical resection and diagnostic biopsy has been performed instead. Thus, there is no standardized criterion for treatment and there has not been any further research on the matter [4, 5, 6]. There are several reasons for the difficulty of treating thalamic gliomas: First, the thalamus is located in the deep area of the supratentorial which has complicated anatomical structures that make accessing and treating this lesion very difficult [4]. Second, the thalamic glioma has relatively high malignant properties and severe postoperative complications [7, 8]. Progress has been made in diagnosing and treating gliomas over the last two decades. It has already been proven that molecular diagnosis for gliomas is more valuable than merely relying on histological diagnosis in the effect of clarifying a diagnosis and predicting the outcome [9, 10]. IDH1 gene encodes isocitrate dehydrogenase 1, which has an oxidative carboxylation catalytic effect on isocitrate and induce the formation of a-ketoglutarate. Whereas IDH1 mutation promotes formation of high level of R-2-hydroxyglutarate (R-2-HG) which has been considered as a tumor metabolite contributing to the occurrence and development of gliomas [11, 16]. It has also been recognized as a great predictive biomarker for gliomas and represents a distinctive category of gliomas that drive its formation in a particular way [12, 13].

However, the characteristics of molecular abnormalities of thalamic gliomas are poorly understood in adults as compared to superficial cerebral gliomas. This study aimed to investigate the distinctions of molecular alteration between high-grade thalamic and superficial cerebral gliomas.

Our study found that IDH1 wild-type was mainly associated with high-grade thalamic gliomas, whereas no such features were found in high-grade superficial cerebral gliomas. We suggest that high frequency of IDH1 wild-type high-grade thalamic gliomas may be another evidence to turn out high-grade thalamic glioma as a different tumor entity as compared to other malignant gliomas with different locations.

Table 1
Clinical characteristics of thalamic and supratentorial superficial glioma patients

Variables	Thalamic	Superficial
	gliomas	gliomas
	( $n=$ 50)	( $n=$ 93)
Age at diagnosis, years
Mean	60	51.8
Median	55	45
Range	18–78	17–73
Gender
Male	33 (61.4%)	62 (66.7%)
Female	17 (38.6%)	31 (33.3%)
Pathological diagnosis
Anaplastic astrocytoma III	6 (10.5%)	12 (12.9%)
Anaplastic oligodendroglioma/	5 (8.8%)	11 (11.8%)
Oligoastrocytoma III
Glioblastoma multiforme IV	39 (68.4%)	70 (75.3%)

2. Materials and methods

We retrospectively selected a series of patients who initially underwent thalamic or superficial cerebral glioma surgical excision at West China Hospital (WCH) and collected their clinical materials from January 2013 to December 2016. We included patients based on two main criteria: First, they were diagnosed with gliomas as defined by the 2007 World Health Organization (WHO) classification of CNS tumors by at least two pathologists [14]. Second, preoperative magnetic resonance imaging (MRI) scans of all patients were reassessed to determine if the localizations of tumors were situated in the thalamus or superficial cerebral lobes respectively [15]. We defined “superficial cerebral glioma” as localized tumors completely within the cerebral lobes.

We excluded patients who weren’t diagnosed as glioma or whose localized tumor was situated neither in the thalamus nor in the superficial cerebral lobes. As there were only few cases of grade I and II thalamic gliomas, we precluded the low-grade (WHO I–II) thalamic and superficial gliomas. We also excluded patients whose preserved tumor tissue after surgical resection for pathological analysis was inadequate and patients who merely underwent biopsy instead of surgical excision for the relatively limited value of their tumor samples. Altogether, 50 patients with thalamic gliomas and 93 patients with high-grade superficial cerebral gliomas were identified and selected for this study. For thalamic gliomas, there were 24 tumors solely involving the thalamus. The remaining tumors not only had infiltrated the thalamus, but also infiltrated areas surrounding the thalamus which included the lateral ventricle (8 tumors), corpus callosum (7 tumors), basal ganglia (14 tumors), third ventricle (2 tumors), brain stem (2 tumors) and even cerebral lobes (14 tumors). The clinical characteristics of these patients are listed in Table 1. Immunohistochemistry (IHC) and direct gene sequencing were conducted by pathologists in WCH. The examining results were verified by at least two pathology experts individually. We firstly analyzed expressions of molecules as follow: IDH1/2, P53, Ki-67, ATRX, PTEN, MMP9 and MGMT by IHC. The primary antibodies for IHC included: IDH1 (clone H09; Dianova, Germany), p53 (MAB-0142, ZM-0408; Maixin), Ki-67 (790-4286; Roche), ATRX (HPA001906; Sigma), PTEN (ZA-0251; Zhongshan), MMP9 (MAB-0245, 56-2A4; Maixin), and MGMT (ZM-0461, UMAB56; Zhongshan). Methods of IHC and gene sequencing for further detecting the IDH1 (R132H) mutation have been described at length in prior studies [16, 17]. For the results of Ki-67 expressions through the method of IHC, we classified it into low ( $\leqslant$ 10% of cells positively stained), moderate (11–29% of cells positively stained) and high ( $\geqslant$ 30% of cells positively stained) Ki-67 expressions by evaluating the staining percentage of tumor cells.

Statistical analysis was conducted using SPSS version 20 for Windows (SPSS Inc). Both groups used the chi-square and fisher exact tests to examine the differences in categorical variables. A $p$ -value $<$ 0.05 was considered statistically significant.

Table 2
Immunohistochemistry differences of molecular abnormalities of high-grade gliomas between thalamus and superficial lobes

		Tumor localizations
		Thalamus	Superficial
Variables		( $n=$ 50)	lobes ( $n=$ 93)	$p$ -value
Molecular abnormalities
IDH1	Mutation	4	27	$p=$ 0.004
	Wild-type	46	66
P53	Positive	41	71	$p=$ 0.673
	Negative	6	13
MGMT	Positive	19	36	$p=$ 0.388
	Negative	23	31
Ki-67	Low	4	9	$p=$ 0.072
	Moderate	33	43
	High	11	36
PTEN	Positive	9	21	$p=$ 0.478
	Negative	20	33
ATRX	Expression	25	31	$p=$ 0.128
	Loss	1	7
MMP9	Positive	15	34	$p=$ 0.251
	Negative	13	17

Table 3

Correlations of molecular abnormalities and tumor grades in thalamic gliomas

		Thalamus ( $n=$ 50)
Variables		GBM-IV	Grade-III	$p$ -value
Molecular abnormalities
IDH1	Mutation	1	3	$p=$ 0.029
	Wild-type	38	8
Ki-67	Low	3	1	$p=$ 0.545
	Moderate	24	9
	High	10	1
ATRX	Positive	20	4	$p=$ 0.354
	Negative	1	1

Table 4

Differences of molecular abnormalities between thalamic and superficial cerebral gliomas according to different grades

		Group one (anaplastic glioma)			Group two (GBM)
		Tumor localizations		$p$ -value	Tumor localizations		$p$ -value
Variables		Thalamus	Cerebral		Thalamus	Cerebral
IDH1	Mutation	3	14	$p=$ 0.067	1	13	$p=$ 0.017
	Wild-type	8	9		38	57
Ki-67	Low	1	7	$p=$ 0.261	3	2	$p=$ 0.072
	Moderate	9	11		24	32
	High	1	5		10	31
MMP9	Positive	3	7	$p=$ 0.650	12	27	$p=$ 0.349
	Negative	4	5		9	12

3. Results

3.1 The expression status of molecular abnormalities in high-grade thalamic and superficial gliomas

Expression status of IDH1, P53, MGMT, Ki-67, ATRX, PTEN and MMP9 were examined by IHC in all 50 thalamic high-grade gliomas and 93 superficial high-grade gliomas. More than 50% of the cases presented mutations of P53, PTEN and MMP9 within both groups. Mutation rates of P53, PTEN and MMP9 across those two groups were 87.2% (41/47), 69% (20/ 29) and 53.6% (15/28) in the thalamic gliomas, 84.5% (71/84), 61.1% (33/54) and 66.7% (34/51) in the superficial gliomas respectively. The gene sequencing of IDH1 (R132H) showed that there was a full consistency of the results between IHC and gene sequencing in all samples. All 31 IDH1 (mutation) samples examined by gene sequencing were IHC positive and no samples showed IDH2 (R172) mutation. Only 8% (4/50) and 29% (27/93) of the thalamic and superficial high-grade gliomas showed IDH1 (R132H) mutation. The ATRX loss occurred only in 3.8% (1/26) and 18.4% (7/38) of the thalamic and superficial high-grade gliomas. Meanwhile, the low Ki-67 expression was only found in 8.5% (4/47) and 10.2% (9/88) of those two glioma entities respectively. There were no obvious differences of status of MGMT expression in both type of tumors. The specific data are listed in Table 2.

3.2 Less IDH1 mutation in thalamic GBM as compared to anaplastic thalamic gliomas

The clinical materials of all thalamic gliomas are displayed in Table 1. We conducted a comparison of correlations of molecular abnormalities and tumor grades in thalamic gliomas. As expected, we found that IDH1 mutations in the GBM group were less than anaplastic thalamic gliomas ( $p=$ 0.029). Whereas the expressions of Ki-67 had no significant difference between thalamic GBM and anaplastic gliomas ( $p=$ 0.545). Study conclusions are listed in Table 3.

3.3 Increased IDH1 wild-type was prevalent in the thalamic high-grade gliomas as compared to the superficial high-grade gliomas

Surprisingly, we still found a significant difference in IDH1 mutation between those two high-grade tumor entities with different localizations, with 92% of the thalamic tumors (46/50) and 71% of the superficial gliomas (66/93) showing IDH1 wild-type ( $p=$ 0.004). However, we didn’t found any difference of Ki-67 expressions between the thalamic gliomas and the superficial gliomas ( $p=$ 0.089). The immunohistochemistry conclusions of molecular abnormalities of high-grade gliomas between the thalamus and the superficial lobes are in Table 2. We also analyzed whether thalamic gliomas had any differences about molecular abnormality from superficial gliomas according to different grades. In our cohort, we specifically found that IDH1 mutation in superficial glioblastomas occurred more than thalamic glioblastomas ( $p=$ 0.017), whereas no significant distinction was found in anaplastic gliomas ( $p=$ 0.067). The details are listed in Table 4.

3.4 Low Ki-67 expression is particularly present in IDH mutant gliomas in superficial high-grade gliomas

We analyzed the relationships of molecular abnormalities in both the thalamic and superficial high-grade gliomas. It only showed that PTEN negative restricted with MMP9 negative in thalamic high-grade gliomas (Fig. 1A) ( $p=$ 0.016). As to superficial high-grade gliomas, there were 26 patients in the IDH1 mutation group, which contained 7, 13, and 6 high, moderate and low Ki-67 expression gliomas, respectively. The IDH1 wild-type group (62 patients), was composed of 29, 30, and 3 high, moderate and low Ki-67 expression gliomas, respectively. There was a significant distinction between the IDH1 mutation and Ki-67 expressions (Fig. 1B) ( $p=$ 0.024). Although no significant difference was detected about moderate and high Ki-67 expressions in superficial high-grade gliomas ( $p=$ 0.890 and $p=$ 0.084 respectively), we noted that the occurrence of low Ki-67 expressions 23.1% (6/26) in IDH1 mutation group was outnumbered than IDH1 wild-type group 4.8% (3/62) ( $P=$ 0.018). These results further demonstrate a correlation that low Ki-67 expression is particularly present in IDH mutant gliomas in superficial high-grade gliomas.

Figure 1.

(A) The relationship between MMP9 and PTEN in thalamic high-grade gliomas ( $P=$ 0.016). Regarding the MMP9 positive group, there were 8 PTEN positive and 7 PTEN negative respectively. In MMP9 negative group, there were only 1 sample showed PTEN positive, while samples of the PTEN negative were 12. (B) The relationship between IDH1 mutation and different levels of Ki-67 expressions in high-grade superficial gliomas ( $p=$ 0.024). Low Ki-67, Moderate Ki-67 and high Ki-67 mean $\leqslant$ 10% of cells positively stained, 11%-29% of cells positively stained and $\geqslant$ 30% of cells positively stained through the method of IHC, respectively.

4. Discussion

While the supra-tentorial superficial gliomas in adults have been considerably investigated, thalamic gliomas have not received the same attention. As known to all, there are many key molecules which have been identified as important for diagnosis and prognosis of gliomas, such as IDH1/2, P53, Ki-67 labeling index, ATRX loss, MGMT methylation, PTEN, MMP9 and so on [16, 18, 19, 20, 21, 22, 23]. In the present study, we focused on characteristics of molecular abnormalities in the thalamic high-grade gliomas. Thus we conducted a comparative analysis of high-grade thalamic and superficial cerebral gliomas. We were interested in correlations of different molecules in both groups. It showed that P53 positive occurred in the majority of both high-grade gliomas, whereas IDH1 mutation presented much less. This result was totally different from low-grade diffuse astrocytoma for the co-presenting of IDH1 mutation and P53 positive, which could explain the high malignancy and prove the prognostic importance of IDH1 mutation in high-grade gliomas [13].

It has been demonstrated that ATRX mutation represents good prognosis and to a great extent co-presents with IDH1 mutation in astrocytoma [18, 19]. ATRX gene mutation could trigger the development of abnormal telomeres in brain tumors and then induce the loss of ATRX expression [24]. There is a negative correlation between ATRX mutation and grades in gliomas [21]. In the present study, the ATRX mutation also presented only in a minority of thalamic and superficial high-grade gliomas, which means it may be a critical factor of high malignancy for thalamic high-grade gliomas. However, providing that the rare occurrence of ATRX mutation in high-grade gliomas, it could interpret the reason why we did not found a correlation between ATRX mutation and IDH1 mutation in our tumor cohorts [25].

The tumor suppressor gene PTEN is highly likely to mutate in high-grade gliomas with a percentage of 60%–70% [26]. This is in accordance with the results of thalamic and superficial high-grade gliomas in the present study. It also suggested that PTEN mutation could promote the highly malignant gliomas to come into being and predict a poor outcome [22, 27]. As regards MMP9, one of the matrix metalloproteinases, it plays an essential role in tumor invasiveness by degrading extracellular matrix and also is significantly associated with highly malignant gliomas [26, 28]. In this study, the expression rate of MMP9 in the thalamic gliomas was nearly the same as the superficial high-grade gliomas. Notably, we found PTEN negative correlated with MMP9 negative in thalamic high-grade gliomas, whereas no such difference was found in superficial gliomas. It has been reported that there was a negative correlation between PTEN and MMP9 mRNA expression in GBMs but was absent in anaplastic astrocytoma [23]. One of the reasons was that PTEN could inhibit glioma cell invasion by suppressing the proteolysis of extracellular matrix by MMP9 [29]. The reverse relationship between PTEN and MMP9 may due to a low expression of PTEN rather than an overexpression of MMP9, then proving the poor prognosis in thalamic gliomas from another point of view. More studies are needed to elucidate possible interactions of these genes in thalamic high-grade gliomas.

We initially found that the huge majority of the high-grade thalamic gliomas (92%) showed IDH1 wild-type and interestingly all thalamic gliomas with IDH1 mutation simultaneously infiltrated with superficial cerebrums. This was consistent with gliomas located in the deep brain or brain stem which were likely to be IDH1 wild-type [30, 31]. Our study also indicated that gliomas with IDH1 mutation mainly existed in superficial cerebral glioblastomas, as compared to thalamic GBM with rare occurrence [9]. Even though there was no significant difference of IDH1 mutation in anaplastic gliomas with different locations, it did show a high proportion of 60.9% of superficial gliomas with IDH1 mutation, comparing to only 27.3% of thalamic gliomas. One study demonstrated that gliomas localized in thalamic alone did not show any differences in survival rates from thalamic gliomas infiltrated with adjacent structures in pediatric high-grade gliomas [32]. Actually, in many cases with tumor infiltrated with thalamus and adjacent structures, it is difficult to judge its tumor cell origin or where the main tumor had arisen from. Then it do remind us of more tests aiming to investigate the molecular abnormalities and clinical courses between infiltrative and localized thalamic gliomas in adults may be warranted. IDH1 mutation has been recognized as a critical biomarker for diagnosing and predicting a favorable prognosis even in glioblastomas [9, 33]. Hartmann et al. reported that patients with IDH1 mutant GBM had favorable prognosis, whereas the result of patients with IDH1 wild-type anaplastic astrocytoma was the reverse [34]. All the above results strongly suggested that IDH1 mutation, an authoritative prognostic factor, associated with positive outcomes for patients with gliomas. Researches have suggested that the occurrence of IDH1 mutation is chiefly clustered in the frontal lobe [12, 35, 36]. Furthermore, most glioblastomas with IDH1 mutation were situated in the frontal lobe as well [12, 16, 17]. No wonder more and more researchers consider IDH1 mutant gliomas as a distinct entity of tumor and the specific mechanisms of the induction of IDH1 mutant gliomas is under development [13, 37].

To date, more and more researches support the idea that molecular information plays an important role in integrated diagnoses of tumors [38]. Therefore, it is suggested that biomarker-based stratification for each tumor entity should be conducted to provide better clinical implications and improve the diagnostic classification [39]. We owe our success of diagnosing and predicting outcomes of tumors accurately to a range of molecular abnormalities, such as IDH1 mutation, low ki-67 expression and ATRX loss, etc. [18, 40, 41]. In this study, we also found that IDH1 mutation in thalamic GBM was less than anaplastic thalamic gliomas. No significant difference was detected about moderate and high Ki-67 expressions between IDH1 mutation and wild-type groups in superficial high-grade gliomas, however the occurrence of low Ki-67 expressions in IDH1 mutation group was outnumbered than IDH1 wild-type group, which was in accordance with previous findings [16, 39]. As an indicator of the proliferation index, Ki-67 is one of the classical biomarkers of prognostic significance [16, 39, 42]. Research has demonstrated that a new model could effectively predict the prognosis of patients with astrocytic tumors by using IDH1, ATRX and Ki-67 expressions [19]. Other researches have also suggested that classification according to ki-67 and IDH1 could be more helpful for clinical judgement of predicting survival and adopting therapeutic regime [18]. To summarize, new classification based on molecular abnormalities for thalamic gliomas should be investigated in order to help predict overall survival.

In summary, molecular abnormalities of thalamic gliomas should be fully understood so as to guide treatment on account of its fatal outcome. Our results demonstrated that IDH1 wild-type mainly occurred in the thalamic high-grade gliomas as opposed to the superficial high-grade gliomas. The rare occurrence of IDH1 mutant high-grade thalamic gliomas strongly suggested that the high-grade thalamic glioma is another distinct tumor entity as compared to the high-grade superficial gliomas.

Conflict of interest

The authors report no potential conflict of interests.

Footnotes

Acknowledgments

The authors gratefully acknowledge the grant from Key Research and Development Project from the Department of Science and Technology of Sichuan Province, China (NO. 2017SZ0006).

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IDH1 status is significantly different between high-grade thalamic and superficial gliomas