The prognosis of gliomas with different molecular subtypes in the era of intensity-modulated radiation therapy (IMRT)

Purpose: This study aimed to evaluate the prognosis of glioma patients with different molecular subtypes of who treated with intensity-modulated radiation therapy (IMRT). Methods: We collected 45 glioma patients treated in our hospital between January 2017 and December 2020. All enrolled patients received postoperative IMRT and were divided into two groups based on the Isocitrate dehydrogenase (IDH status). Overall survival (OS) and progression-free survival (PFS) were estimated retrospectively. Results: The median follow-up was 22 months (range 2–108.5 months). The 1-year OS of IDH-mut group and ΙDH-wild group was similar (77.3% vs. 81.5%, p = 0.16). While the 1-year PFS of IDH-mut group was significantly higher than that in ΙDH-wild group (90.4% vs. 39.8%, p = 0.0051). Subgroup analysis revealed that the 1-year PFS of IDH-mut/1p/19q codeletion group and IDH-mut/1p/19q noncodeletion group was significantly higher than in IDH-wild type patients. For patients with IDH-mut/MGMT-methylation, the outcome was no significant difference in OS, but PFS was longer than other subtypes. Conclusion: This retrospective study showed that 1-year PFS of patients with IDH mutated was better than IDH-wild type patients. In subgroups analysis, the outcomes were shown that patients with IDH-mut/ 1p/19q codeletion and patients with IDH-mut/1p/19q noncodeletion had longer 1-year PFS than IDH-wild type patients, but the OS was similar between the subgroups. Patients with IDH-mut/MGMT-methylation had the best prognosis in the whole subgroups. However, these results still need further confirmation of large sample size, prospectively, randomized controlled trails.


INTRODUCTION
rate and prolong survival time. However, even if these patients received standard postoperative care, the 1-year overall survival (OS) still only 14.4 months [3]. Therefore, how to improve OS and reduce the local recurrence rate has become the research direction of many neuro-oncologists. Since on the 2016 World Health Organization (WHO) fourth revised edition proposed adding molecular typing as an important basis element for glioma diagnosis [4], the diagnosis, classification, prognosis and treatments of gliomas made great progress. The 5th edition of World Health Organization (WHO) classification of Central Nervous System Tumors released in 2021 [5] integrated the histological characteristics and molecular classification of glioma, and proposed a new tumor classification standard, which provides an important basis for the diagnosis, treatment and the classification of glioma. The new classification is no longer based on the cross solid tumors, but based on tumor phenotype, molecular subtype and biological similarity, which is more individualized than the previous classification based on the tumor gross type. Radiotherapy, as one of the most important postoperative treatment methods for highgrade gliomas, can bring significant survival benefits to patients. Previous studies [6,7] have shown that the survival time of high-grade gliomas is closely related to the start time of radiotherapy, and early postoperative treatment can effectively prolong the survival time of patients with glioma. Early in 1996, Karim AB et al. [8] compared the efficacy of low-dose and high dose radiotherapy for low-grade gliomas, the results showed that there was no significantly difference between the two groups. In 2002, Shaw E et al. [9] carried out a randomized phase III study about the efficacy of lowdose (50.4 Gy/28f) compared with high-dose (64.8 Gy/36f) radiotherapy for low-grade gliomas. The outcomes revealed that patients who received higher doses of radiation had lower overall survival than those who received lower doses. Moreover, the incidence of radiation brain necrosis in high-dose radiotherapy group was also higher than low-dose radiotherapy group. The standard treatment for glioblastoma is STUPP regimen [10]. Despite patients treated with the standard method of STUPP, the median overall survival still only 14 months [11]. Walker MD et al. [12] found the doseeffect relationship in the radiotherapy of malignant gliomas in 1979. Bleehen NM et al. [13] also compared the efficacy of different radiation doses of 45 Gy and 60 Gy on the grades 3 and 4 astrocytomas. The results also confirmed that patients treated with 60 Gy had better prognosis. Is boosting the dose of local radiotherapy beneficial to patients with high-grade gliomas? A study by RTOG /EORTG showed boosting radiation dose to 70 Gy also didn't benefit patients either [14]. Similarly, a randomized phase III study 93-05 by RTOG also revealed that boosting dose (15 to 24 Gy × 1f) on the basis of 60 Gy still didn't bring benefit to patients [15]. Piroth MD et al. [16] integrated boost IMRT with FET-PET to delineate the radiotherapy target volumes in glioblastoma patients, and boosted the radiation dose to 72 Gy, but the outcomes still didn't improve patients' survival rate. Isocitrate dehydrogenase (IDH), is a key rate-limiting enzyme in tricarboxylic acid cycle. Some studies had confirmed that high-grade glioma patients with IDH mutation had better prognosis [17][18][19][20], but the prognostic value of IDH for low-grade diffuse glioma is still unclear [21]. However, most of the previous researches on the prognosis of new molecular subtypes of gliomas are based on conventional radiation or 3-dimensional conformal radiation (3D-CRT), what about intensity modulated radiation therapy (IMRT)? what's the prognosis of patients with different molecular types in the era of IMRT? Do advanced radiation techniques benefit patients with different molecular classifications of glioma, or do patients with different molecular subtypes of glioma have inherently poor outcomes? There were rare studies about prognosis of patients with different molecular subtypes gliomas treated with IMRT. Therefore, our study retrospectively collected glioma patients based on the molecular subtypes who treated in our centre only received IMRT after surgery, and explored the prognosis of different molecular subtypes of gliomas.
Clinical data on patients' age, gender, pathological type, WHO grade, preoperative tumor size, surgical resection method, radiotherapy dose, recurrence site from the medical history system of The Second Affiliated Hospital of Guangxi Medical University. All the patients received radiotherapy 4-8 weeks after surgery. According to the status of IDH, we divided enrolled patients into two groups: IDH mutate group (IDH-mut group) and IDH wild group (IDH-wild group), regardless their histopathology status. Patient characteristics were summarized in Table 1, the flow chart was shown in Figure 1.

Molecular analysis
DNA was extracted from tumor tissues by standard methods, and all the samples were examined by experienced pathologists to ensure every sample had 80% or more tumor cell content. The techniques to determine MGMT promoter methylation by one-stage methylation-specific PCR [22] (methylation-specific primers generated a band on agarose gel in the lane containing PCR products was scored as positive). Frozen samples were used for IDH1 or IDH2 analysis. The genomic region spanning wild-type R132 of IDH1 was analyzed by direct sequencing using the following primers:5′TGTGTTGAGATGGACGCCTATTTG and 3′TGCCACCAACGACCAAGTC as the manufacture's protocol. The genomic region spanning wild-type R172 of IDH2 was analyzed by direct sequencing using the following primers: IDH2f 5-GCCCGGTC TGCCACAAAGTC and IDH2r 5-TTGGCAGACTC CAGAGCCCA as the manufacture's protocol.1p/19q codeletion was excluded by fluorescence in situ hybridization (FISH).

Radiotherapy
All the enrolled patients were received 6 MV-X ray liner accelerator of intensity modulated radiation therapy (IMRT). Patients were immobilized with an individual head-neck-shoulder thermoplastic mask in the supine position to ensure reproducibility of patient positioning during planning CT and the following radiation. The slice thickness was 3 mm of the CT scan.

Target volumes
The target volumes were administered according to the protocol of the EORTC and RTOG. Target delineation as follows:

Protocol of EORTC
The gross tumor volume (GTV) included the contrastenhancing regions which present on T1-weighted preoperative MRI scans and all the surgical areas. The clinical target volume (CTV) was determined by the GTV with a margin up to 2 cm. The planning target volume (PTV) was created by extending a 3 to 5 mm margin of the corresponding CTV.

Protocol of RTOG
The gross tumor volume-1 (GTV-1) as the EORTC described before, but GTV-1 included the perifocal edema regions which was visible on T2 or FLAIR sequence. The clinical target-1 (CTV-1) was based on the GTV with a margin of up to 2 cm (extending 2.5 cm margin if it didn't have perifocal edema). The planning target volume-1 (PTV-1) was the CTV1 extended 3 to 5 mm margin. The gross tumor volume-2 (GTV-2) included the surgical regions and all the contrast-enhancing regions detected on T1-weighted MRI. CTV-2 defined as the GTV-2 The prescription dose were as follows: the patients who diagnosed with low-grade gliomas treated with 54 Gy, but if patients had visible residual tumor on MRI, the dose boosted to 60 Gy, the PCTV dose was 45 to 54 Gy. For high-grade gliomas, the prescription dose was PGTV 60 Gy and PCTV 54 Gy.

Statistical analysis
The

Follow-up
In the first two years, follow-up and MRI were performed every 3 months after RT, and thereafter they were performed every 6 months until tumor progression or death.

Data availability statement
All data were presented in the manuscript and supplementary materials.
Multivariate analysis of 45 valid cases showed no significant prognostic factor in OS and PFS. Univariate analysis revealed that only IDH status was significant prognostic factor of PFS (Table 3).

DISCUSSION
The WHO classification of brain tumor in version 1,2016 [4] proposed molecular characteristics as one of the most important characteristics for gliomas, especially isocitrate dehydrogenase (IDH) 1/2, which is strongly associated with the prognosis and related to tumor grade. Molecular features become more important to gliomas. In the cIMPACT-NOW Consortiun for taxonomy of primary brain tumors suggested to reclassify those patients who had IDH wild-type diffuse gliomas as diffuse astrocytic gliomas, IDH wild-type with molecular features of glioblastoma, WHO 4 [24,25]. Some of the researches also confirmed that patients who had IDH wild-type display a poor survival as patients with IDH wild-type glioblastoma [26]. Most of the previous studies based on the conventional radiation technology or 3D-CRT, with the development of radiotherapy technology, are these advances bring some benefit for glioma patients based on the molecular characteristics? Our study retrospectively collected glioma patients regardless of their pathology, clinical features, surgical information and adjuvant treatments, reclassified the patients based on their molecular characteristics. In our study, patients with IDH mutated had better PFS than those in IDHwild group ((90.4% vs. 39.8%), but did not significantly differ in OS between the two groups. These results were similar to Michael Weller et al. 's founding [19]. In the study of Qi SongTao, he also found that IDH mutation, MGMT promoter methylation and 1p19q codeletion were related to prolong PFS [27]. However, there were       [30]. Although our study had some flaws, but the results demonstrated the prognosis of patients with different molecular types of gliomas under the same radiation technology. It more powerful to distinguish whether the progress of technology or molecular characteristics benefit for glioma patients.
In conclusion, this retrospective study showed that patients with IDH mutation had better PFS than those patients with IDH-wild type. In terms of IDH-AGING mut/1p/19q codeletion and IDH-mut/1p/19q noncodeletion of glioma patients, the outcomes were shown longer PFS than patients with IDH-wild type, but no difference in OS among the subgroups. Patients with IDH-mut/MGMT-methylation had the best prognosis in the subgroups. However, these results still needs further confirmation of large sample size, prospectively, randomized controlled trails.

AUTHOR CONTRIBUTIONS
All authors made a significant contribution to the research, whether that is in the study design, acquisition of data, statistical analysis and interpretation, as well as took part in drafting, revising, or reviewing the article.

ACKNOWLEDGMENTS
This work was finished by the team of Department of Radiation Oncology, The Second Affiliated Hospital of Guangxi Medical University, and thank you for all my colleagues.

CONFLICTS OF INTEREST
The authors declare no conflicts of interest related to this study.

ETHICAL STATEMENT AND CONSENT
This study was approved by the clinical research Ethics Committee of The Second Affiliated Hospital of Guangxi Medical University. The requirement for informed consent was waived because the medical records used in this study were obtained from previous clinical treatments and it will not adversely affect the rights and health of the subject. We declare that we would protect the confidentiality of personal information of research subjects.

FUNDING
This work was supported by the grants from the project of Middle-aged and Young Teachers' Basic Ability Promotion Project of Guangxi (CN) (No: 2020KY03023).