Novel insight into histological and molecular astrocytoma, IDH‐mutant, Grade 4 by the updated WHO classification of central nervous system tumors

Abstract Background The latest fifth edition of the World Health Organization (WHO) classification of the central nervous system (CNS) tumors (WHO CNS 5 classification) released in 2021 defined astrocytoma, IDH‐mutant, Grade 4. However, the understanding of this subtype is still limited. We conducted this study to describe the features of astrocytoma, IDH‐mutant, Grade 4 and explored the similarities and differences between histological and molecular subtypes. Methods Patients who underwent surgery from January 2011 to January 2022, classified as astrocytoma, IDH‐mutant, Grade 4 were included in this study. Clinical, radiological, histopathological, molecular pathological, and survival data were collected for analysis. Results Altogether 33 patients with astrocytoma, IDH‐mutant, Grade 4 were selected, including 20 with histological and 13 with molecular WHO Grade 4 astrocytoma. Tumor enhancement, intratumoral‐necrosis like presentation, larger peritumoral edema, and more explicit tumor margins were frequently observed in histological WHO Grade 4 astrocytoma. Additionally, molecular WHO Grade 4 astrocytoma showed a tendency for relatively longer overall survival, while a statistical significance was not reached (47 vs. 25 months, p = 0.22). TP53, CDK6, and PIK3CA alteration was commonly observed, while PIK3R1 (p = 0.033), Notch1 (p = 0.027), and Mycn (p = 0.027) alterations may affect the overall survival of molecular WHO Grade 4 astrocytomas. Conclusions Our study scrutinized IDH‐mutant, Grade 4 astrocytoma. Therefore, further classification should be considered as the prognosis varied between histological and molecular WHO Grade 4 astrocytomas. Notably, therapies aiming at PIK3R1, Notch 1, and Mycn may be beneficial.


| INTRODUCTION
Glioma is the most common primary malignant tumor of the central nervous system (CNS), with an average annual incidence of 4.50 per 100,000. 1 Current clinical treatment options for glioma include surgery, radiotherapy, chemotherapy, immunotherapy, targeted therapy, and tumor-treating fields. 2,3However, the prognosis remains unfavorable. 1Considering the poor prognosis status and the vacancy of widely recognized therapeutic targets, a more accurate classification standard or guideline is significantly needed for individualized treatment and prognosis prediction.The World Health Organization (WHO) initially integrated the classification and grading of CNS tumors in 1979. 4After several revisions and updates, 5 the fourth edition of the WHO classification of CNS tumors (WHO CNS 4 classification) mainly focused on the histological characteristics of tumors. 6,7][10][11] However, the independent prognostic value of the molecular markers in all age groups remains to be determined. 12Based on these findings, the latest fifth edition of the WHO classification of CNS tumors (WHO CNS 5 classification) released in 2021 focused on advancing the role of molecular diagnosis in CNS tumor classification.Notably, it combines histopathology and molecular pathology for comprehensive diagnosis to further predict prognosis and conduct comprehensive treatment decisions. 13n the WHO CNS 5 classification criteria, the diagnosis of IDH-mutant glioblastoma no longer exists.][15] CDKN2A encodes p14ARF and INK4a and plays a role in regulating tumor suppression. 16,179][20][21] Additionally, CDKN2B encodes INK4b, which inactivates CDK4/CDK6 and controls cell cycle progression. 22,23It is suspected that homozygous deletion of CDKN2A/B, which breaks the "brake" of tumor progression brake, nullifies the tumor-suppressive environment created by IDH-mutant astrocytomas. 24,25However, on a biomolecular level, detailed interactive mechanisms between CDKN2A/B and IDH1/2 remain unknown.Meanwhile, other molecular biomarkers indicating worse outcome in IDH-mutant astrocytoma, including NOTCH1 mutations and incomplete resections in oligocytoma and PIK3R1 mutations, RB1 mutations and CDK4 mutations and amplifications in astrocytoma. 26,279][30][31] There are few clinical studies that specifically focus on this subtype, and our understanding remains limited. 31,32ncreasing attention to the novel species of astrocytoma, IDH-mutant, Grade 4 from the WHO CNS 5 classification has led to extensive research and exploration.In this study, we describe the clinical, radiological, and molecular pathological features of astrocytoma, IDH-mutant, Grade 4 based on the WHO CNS 5 classification.Furthermore, we explored the similarities and differences between the two subtypes of histological WHO Grade 4 astrocytoma and molecular WHO Grade 4 astrocytoma and searched for predictive molecular markers to benefit patient prognosis.To PIK3CA alteration was commonly observed, while PIK3R1 (p = 0.033), Notch1 (p = 0.027), and Mycn (p = 0.027) alterations may affect the overall survival of molecular WHO Grade 4 astrocytomas.

Conclusions:
Our study scrutinized IDH-mutant, Grade 4 astrocytoma.Therefore, further classification should be considered as the prognosis varied between histological and molecular WHO Grade 4 astrocytomas.Notably, therapies aiming at PIK3R1, Notch 1, and Mycn may be beneficial.

K E Y W O R D S
2021 WHO classification of central nervous system tumors, astrocytoma, glioma, Grade 4, IDH-mutant, molecular alteration, radiological characteristics do so, we analyzed our institution's comprehensive data on glioma patients.Notably, we aimed to provide a database for deepening the academic understanding of astrocytoma, IDH-mutant, Grade 4, and clinical treatment options.

| Study population
Patients who underwent surgery at the Department of Neurosurgery at Peking Union Medical College Hospital from January 2011 to January 2022, 33 classified as astrocytoma, IDH-mutant, Grade 4 according to WHO CNS 5 classification criteria, were screened for this study.Finally, patients with complete clinical and pathological data were included in our analysis.Written informed consent was obtained from all patients included for the use of clinical samples.The study was approved by the Institutional Ethics Review Board of PUMCH (S-424) and conformed to the requirements of the Declaration of Helsinki.

| Clinical and radiological data collection
Clinical and radiological information was collected retrospectively from patients' medical records and examinations.Patient clinical information utilized in our analyses included gender, age at diagnosis, body mass index (BMI), preoperative Karnofsky performance status (KPS) score, clinical symptoms, the extent of operation resection (EOR), and postoperative comprehensive treatment regimen.In addition, we collected patient survival data and adverse events through outpatient and telephone followups, with overall survival (OS) defined as the time from the surgery date to the patient's death or last-recorded follow-up (treated as censored values).The median OS was defined as the overall survival achieved by 50% of patients.
Radiological features of involved patients were collected from preoperative magnetic resonance imaging (MRI) to analyze the number of tumors, tumor location, involvement of functional areas, maximum tumor diameter, maximum edema diameter, intratumoral-necrosis like presentation, intratumoral hemorrhage, cystic lesions, calcification, tumor margin clarity, and tumor performance in T1 weighted image (T1WI), T2WI, and T1-based contrastenhanced images.In contrast-enhanced MRI, the boundary between the tumor parenchyma and the edema area near the tumor or adjacent brain tissue is clearly defined as a clear tumor boundary.If it was not discernible by the naked eye, the tumor boundary was defined as blurred. 34

| Tumor pathology data collection
Histopathological and molecular pathological data were collected.Histopathological data were obtained from the Department of Pathology of Peking Union Medical College Hospital report, mainly including the Ki-67 index and tumor histological grade.For molecular pathology, formalin-fixed paraffin-embedding (FFPE) tumor samples were collected from enrolled patients.DNA extraction was conducted with QIAGEN 56404 Kit, and nucleotide concentration and purity were determined by Qubit 4.0 Fluorometer (Thermo Fisher Scientific) using the dsDNA HS Assay Kit and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) respectively.We screened 60 molecular markers, including EGFR, TERT, CDKN2A/B, MYB, and MYBL1, by summarizing the recently published studies on the mechanism of glioma development and prognostic factors.DNA extraction was conducted with QIAGEN 56404 Kit, and nucleotide concentration and purity were determined by Qubit 4.0 Fluorometer (Thermo Fisher Scientific) using the dsDNA HS Assay Kit and Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific) respectively.We analyzed the molecular alterations of each enrolled patient using next-generation sequencing (NGS), and used polymerase chain reaction (PCR)-based assays and fluorescence in situ hybridization methods (FISH) for verification.CNVkit was applied to define the copy number variation from DNA sequencing results.The objective criteria for copy number loss were the ratio of copy number < = 1.5 while the number of bin > = 0.3, and the objective criteria for copy number gain were the ratio of copy number > = 2.5 while the number of bin > = 0.3. 35The complete list of molecular markers is shown in Table S1.

| Identification of enrolled patients and group analysis according to WHO CNS5
According to the WHO CNS 5 classification criteria, astrocytoma, IDH-mutant, Grade 4 includes histological WHO Grade 4 astrocytoma with necrosis or microvascular proliferation (histological astrocytoma as defined in this study) and WHO Grades 2, 3 astrocytomas included homozygous CDKN2A and/or CDKN2B deletions (molecular astrocytoma as defined in this study, further examinations were conducted to ensure the homozygous deletion status for CDKN2A/B).We further explored the differences and similarities between the two subtypes regarding clinical information, radiological features, prognostic status, and molecular alterations.

| Statistical analysis
Normally distributed measures were expressed as means ± standard deviations (SDs), and student's t-test determined differences between groups.Non-normally distributed measures were expressed as medians (first quartile, third quartile) (M [Q1, Q3]) and analyzed between groups by the Kruskal-Wallis H test. Additionally, comparisons of categorical variables were performed using the chi-squared test.Molecular alterations in histological and molecular WHO Grade 4 astrocytoma were presented using oncoplots.Survival analysis was performed using the Kaplan-Meier method and Log-Rank test, and Kaplan-Meier curves presented the results.p < 0.05 was considered to be statistically significant.All statistical analyses were performed with SPSS (version 26.0, IBM) statistical software, and graphs were created using R Studio (PBC & Certified B Corp.®) and GraphPad Prism (9, GraphPad Software) software.

| Baseline information of patients with astrocytoma, IDH-mutant, Grade 4
A total of 33 patients (27 males and 6 females) with astrocytoma, IDH-mutant, Grade 4 were involved in this study, including 20 with histological and 13 with molecular astrocytoma.The mean age was 44 years, and the mean preoperative KPS was 83.In terms of clinical symptoms, 19 (57.6%) emerged with neurological deficits, and 15 (45.5%) exhibited seizures.For EOR, 57.6% (19/33) of patients received gross total resection, 27.3% (10/33) underwent subtotal resection, and 15.1% (5/33) experienced biopsy.For combination therapy, 20 patients received chemotherapy, 19 completed radiotherapy, and 6 underwent targeted treatment.Notably, statistical significances were observed in gender, histological grading, and Ki-67 levels, while the difference in gender may due to the limitation of sample size.The specific information is shown in Table 1.

| Radiological features of patients with astrocytoma, IDH-mutant, Grade 4
The essential diagnostic basis for intracranial tumors in clinical diagnosis is neurological imaging.We summarized and compared the radiological characteristics of patients in the histological and molecular astrocytoma groups.Due to incomplete radiological data for some patients, 14 patients with histological WHO 4 grade astrocytoma and 12 patients with molecular WHO 4 grade astrocytoma were ultimately included, and typical radiological images were shown (Figure 1).
Regarding tumor distribution, solitary tumors occurred more frequently than multiple tumors in both groups, and there were no significant differences in tumor side, specific lobar localization, and functional area involvement.MRI in both groups demonstrated low or mixed density on T1W1 and high density on T2W1.At the same time, in the contrastenhanced image, enhancement was present in 92.9% (13/14) of patients in the histological astrocytoma group and only 33.3% (4/12) of patients in the molecular astrocytoma group.No differences were observed in the histological and molecular WHO Grade 4 astrocytomas in the rate of intratumoral hemorrhage, cystic degeneration, and calcification.In contrast, the histological astrocytoma group showed larger peritumoral edema, higher incidence of intratumoral-necrosis like presentation, and more explicit tumor margin.However, statistical significance was not reached (p values were 0.186, 0.086, and 0.052, respectively).Table 2 summarizes the radiological features of WHO Grade 4 astrocytomas.

| Survival of patients with astrocytoma, IDH-mutant, Grade 4
In both subgroups, we further explored the survival of patients with astrocytoma, IDH-mutant, Grade 4. The mOS of histological astrocytoma was 25 months.In comparison, the mOS of molecular astrocytoma was 47 months, suggesting that molecular astrocytoma was more likely to have a better prognosis and more prolonged survival than the histological subgroup.However, no statistical significance was observed in the analysis due to the limited sample size (p = 0.22).These results and specific survival information are presented in Figure 2.

| Molecular features in patients with astrocytoma, IDH-mutant, Grade 4
Different patterns of molecular marker alterations exist in different gliomas, including mutations, copy number gains, or copy number losses of chromosomes, and genes.Notably, we analyzed and demonstrated the molecular alterations of tumors in two subgroups.As some patients did not undergo molecular pathological examination due to incomplete specimens, we ultimately included two patients with histological WHO Grade 4 astrocytoma and 13 with molecular WHO Grade 4 astrocytoma.Figure 3 shows the changes in molecular markers in both groups of patients.In addition to alterations in IDH1 and CDKN2A/B, common genetic alterations in patients with WHO Grade 4 astrocytoma included TP53, CDK6, and PIK3CA.

| Implications of molecular alterations with survival in patients with astrocytoma, IDH-mutant, Grade 4
Currently, molecular markers for clinical classification, prognosis, and therapeutic response prediction of astrocytoma include MGMT promoter methylation and homozygous CDKN2A and/or CDKN2B deletions. 8,36,37his study focused on the molecular pathology of astrocytoma, IDH-mutant, Grade 4. We also explored other clinical and molecular marker alterations associated with patient prognosis, providing clues for prognostic prediction and clinical decision-making.The results of univariate regression analysis suggested that alterations in PIK3R1 (p = 0.033), Notch1 (p = 0.027), and Mycn (p = 0.027) may be associated with shorter OS in molecular WHO Grade 4 astrocytoma.Likewise, patients with double genetic alterations also showed worse OS in the subgroup analysis (Figure 4, p = 0.002).We further analyzed the effect of other clinical and molecular marker alterations on survival in patients with histological and molecular WHO Grade 4 astrocytoma.However, no correlation was seen between other clinical and molecular marker alterations and the survival of patients (Figure S1,S2).

| DISCUSSION
In light of the WHO CNS 5 classification criteria, new insights and more precise recognitions have been made regarding gliomas.This study focused on astrocytoma, IDH-mutant, Grade 4, whose definition was considerably changed based on the latest WHO classification.Thirtythree patients were included, and differential clinical features were observed in histological and molecular subgroups.Detailed surgical plans including parameters concerning the localization of tumors, the presence of deep lesions, and the presence of seizures at onset, should facilitate a thorough analysis of the glioma. 38 T A B L E 2 Radiological characters of 26 patients with histological or molecular astrocytoma, IDH-mutant, Grade 4. Seven patients were excluded from this analysis due to a lack of radiological information.11][39][40][41][42][43][44] As IDH1/2 mutation status predated the knowledge of astrocytic tumor prognosis, homozygous deletion of CDKN2A/B was further proposed to be related to a poorer prognosis in patients with astrocytoma, IDHmutant, Grade 4. 8,[45][46][47][48][49] Taking CDKN2A/B into the evaluation scheme of IDH-mutant astrocytoma specifies a more straightforward prognosis-directed treatment pathway for various patients. 48However, whether the new classification effectively subgroups patients with similar prognoses remains questionable.One study from Pittsburgh found that survival was only affected by homozygous CDKN2A deletion in histological Grade 4 astrocytomas but not in Grades 2, 3. 50 As suggested from our cohort analysis, patients with molecular WHO Grade 4 astrocytoma tend to have a longer survival expectancy compared to those with histological WHO Grade 4 astrocytoma.Restricted by the total number of involved cases, the difference was not statistically significant.Other confounding factors, such as follow-up treatment and start-up physical status, may also impact overall survival in both groups.Therefore, further investigation is required to determine the differential outcomes for histological and molecular WHO Grade 4 astrocytomas.PIK3R1 and Notch1 mutations have been previously identified in patients with glioblastoma and low-grade astrocytoma, correlating with shorter overall survival. 26,51In addition, somatic mutations of PIK3R1 are suggested to promote gliomagenesis. 52WHO Grade 4 astrocytoma patients with PIK3R1 mutations are expected to benefit from PI3K inhibitors. 53Depending on Mycn, tumor-related MiR-29b is demonstrated to inhibit the growth of glioma. 54On the other hand, targeting Mycn can sensitize gliomas to antitumor treatment. 55Therefore, glioma patients with Mycn mutations may respond to the combination of PARP and CHK1 inhibitors. 56The finding of PIK3R1, Notch1, and Mycn F I G U R E 3 Oncoplot of the molecular alterations of the 15 patients with histological or molecular astrocytoma, IDH-mutant, Grade 4 based on the WHO 2021 classification of CNS tumors.The copy number variation was defined with CNVkit, and copy number gain was defined as the ratio of copy number ≥ 2.5 while the number of bin ≥ 0.3, and copy number loss was defined as the ratio of copy number ≤ 1.5 while the number of bin ≥ 0.3.Each column represents an individual patient, and the cube's color indicates the alteration status of each molecular characteristic.Blue: Mutation; Green: Copy number loss; Red: Copy number gain; Gray: Histological astrocytoma, IDH-mutant, Grade 4; Brown: Molecular astrocytoma, IDHmutant, Grade 4. alterations as negative prognostic factors in molecular WHO Grade 4 astrocytomas may shed light on understanding the biobehavioral progression and also potential targeted therapies for this specific subset of astrocytomas.

All astrocytoma, IDH-
However, our study has certain noted limitations.First, systematic bias is inevitable, considering the small number of participants.Also, the bias induced by the small sample size affected the accuracy and reliability of the molecular subgroup analysis, therefore may influence the occurrence of possible statistical significance.Second, some histological specimens and radiological results were lost due to storage issues, further causing selective bias.Lastly, the molecular marker panel applied in this study was based on published papers containing 60 tumor-related molecular biomarkers.Also, three patients showed enhancement in MRI underwent subtotal resection.Therefore, the possibility of undersampling may affect the accuracy of radiologic features.In follow-up studies, more biomarkers that correlate to the prognosis of IDH-mutant, Grade 4 astrocytomas, including tumor group-specific genomic methylation profiles and copy number variations (CNV), should be considered and evaluated.
To conclude, our study scrutinized the new classification of IDH-mutant, Grade 4 astrocytoma, and provided preliminary data for stratifying the effect of the WHO CNS 5 classification.Therefore, further and more precise classification should be considered, given the differential prognosis presented by histological and molecular astrocytomas.Furthermore, personalized therapies consisting of targeted molecular inhibitors for PIK3R1, Notch 1, and Mycn may exhibit beneficial effects in the corresponding population.
Additionally, significantly different sex distribution was observed due to the limited cohort size.For the radiological manifestations, astrocytoma IDH-mutant, Grade 4 typically shows T1 hypodensity, T2 hyperintensity, and rim enhancement around central necrosis, while glioblastomas are usually irregularly shaped, with ring-enhancement, central necrosis, and surrounding oedema.In terms of subgroup analysis, tumor enhancement in the T1 enhancement phase was more frequently observed in the histological subgroup, corresponding to a higher histological grade by definition.Also, intratumoral-necrosis like presentation, larger peritumoral edema, and more explicit tumor margin were more frequently observed in the histological subgroup.Notably, molecular WHO Grade 4 astrocytoma showed a better prognosis, presented by a relatively long overall survival; however, no statistical significance was archieved (47 vs.25 months, p = 0.22).TP53, CDK6, and PIK3CA alterations were commonly observed in the molecular subgroup.PIK3R1, Notch1, and Mycn alterations might affect the overall survival of molecular WHO Grade 4 astrocytomas.Therefore, research on potential interactions between PIK3R1/Notch1/Mycn alteration and CDKN2A/B homozygous deletion may shed light on the mechanism and therapeutic target on molecular astrocytoma, IDH-mutant, Grade 4. The WHO classification system has evolved as our understanding of gliomas has grown.Due to the close relationship between tumorigenesis and F I G U R E 1 (A-C) A 31-year-old female with astrocytoma, IDH-mutation, Grade 4 without CDKN2A/B deletion, with rim enhancement; (D-F) A 46-year-old male with astrocytoma, IDH-mutation, Grade 4 with CDKN2A/B deletion, without enhancement.
T A B L E 1Note: Significant values are presented in bold.