Clinical Utility of Assessing CDKN2A Status in Recurrent Astrocytomas

doi:10.21203/rs.3.rs-4758261/v1

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Clinical Utility of Assessing CDKN2A Status in Recurrent Astrocytomas

https://doi.org/10.21203/rs.3.rs-4758261/v1

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Background: IDH-mutant astrocytomas exhibit a more indolent natural history and better prognosis compared to their IDH-wild type counterparts. The 2021 WHO classification integrated CDKN2A/Bhomozygous deletion as a crucial criterion for grading these tumors, emphasizing its prognostic implications. FISH assay is commonly used to assess CDKN2A status, but guidelines for interpreting FISH results for glioma prognostication are not well-defined in the literature.

Methods: We conducted an ambispective study involving 22 cases of recurrent IDH-mutant astrocytomas, including primary tumor samples. Histopathological assessments, including WHO grading and molecular profiling, were performed. Immunohistochemistry confirmed IDH mutation status, and FISH analysis evaluated CDKN2A homozygous deletion.

Results: Homozygous CDKN2A deletion was detected in only 1/22(4.8%) of primary tumors, which was grade 3 astrocytoma and 35.3% of recurrent grade 2/3 tumors. Patients harboring CDKN2A deletions exhibited significantly reduced overall survival compared to those without (67.7 vs 137.6 months). Progression-free survival did not significantly differ between groups.

Conclusion: Our findings highlight the clinical relevance of CDKN2A assessment in recurrent IDH-mutant astrocytomas and its utility as a prognostic marker. We propose a selective approach to FISH testing, focusing on primary grade 3 and all recurrent cases, to optimize diagnostic accuracy and inform personalized treatment strategies.

Astrocytoma

CDKN2A

IDH mutant astrocytoma

Recurrent astrocytoma

FISH

Gliomas are the most prevalent central nervous system (CNS) tumors, accounting 30% of the all primary CNS tumors [1]. The WHO CNS5 classification has unified all IDH-mutant diffuse astrocytic tumors into a single category: Astrocytoma, IDH-mutant. These tumors are then graded as CNS WHO grade 2, 3, or 4. Notably, the grading system no longer relies solely on histological criteria as the presence of cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) homozygous deletion warrants a higher tumor grade as WHO grade 4, irrespective of the absence of histological features including microvascular proliferation or tumor necrosis [2]. CDKN2A deletion is recognized as a marker of poor prognosis in gliomas and often correlates with more aggressive tumor behavior and poorer patient outcomes [3, 4]. The frequency of CDKN2A deletion might vary by region and population, but comprehensive global data is lacking.

The assessment of CDKN2A/B alterations in gliomas can be performed through various methodologies, each with distinct advantages. These methods include sequence analysis, fluorescence in situ hybridization (FISH) for detecting homozygous deletions, immunohistochemical (IHC) analysis of CDKN2A expression, and loss of heterozygosity (LOH) analysis using polymorphic microsatellites on chromosome 9p [5–7]. Despite these methodologies and the critical role of CDKN2A/B alterations in the prognosis and treatment of gliomas, there is no consensus or standardized protocol recommended by the WHO CNS tumor classification for assessing CDKN2A/B alterations in gliomas. Available data regarding the use of the fluorescence in situ hybridization (FISH) assay are highly variable, as various studies have used different cutoff values for reporting positive homozygous CDKN2A deletion [3, 4, 8–10]. This lack of standardization highlights the need for further research and collaboration to establish unified guidelines that can enhance the diagnostic and prognostic accuracy for glioma patients.

This study aims to ascertain the prevalence of homozygous CDKN2A deletion and its clinical significance in recurrent IDH-mutant diffuse astrocytomas. This current research aims to bridge this gap and facilitate more efficient and cost-effective utilization of this recently suggested molecular markers in clinical practices.

This study involved an ambispective examination conducted in the Neuropathology Laboratory at the Neurosciences Center, All India Institute of Medical Sciences, New Delhi. A total of 22 cases diagnosed with recurrent astrocytoma (aged > 18 years), and available primary tumor, were retrieved from the departmental archives (2015–2024). The study was approved by the institutional ethical committee. The study specifically focused on astrocytomas with IDH mutations diagnosed within this timeframe. Three independent neuropathologists (V.S., M.C.S., and H.J.) conducted a thorough blinded review of the histopathological characteristics. Grading was performed both according to the WHO CNS 4 or 5 classification criteria [2, 11] with or without access to the molecular profiling data for the cases.

Immunohistochemistry

Immunohistochemical staining was performed on 5-micrometer-thick sections of tumor samples fixed in formalin and embedded in paraffin (FFPE) using an automated immunostainer (Benchmark XT, Ventana, Tucson, AZ, USA) and standard protocols, including pretreatment using cell conditioning 1 buffer (Ventana) for 52 min and standard Ventana signal amplification.. Immunohistochemistry (IHC) was performed using the specific antibodies including IDH1-R132H (H09, Dianova, mouse monoclonal, 1:50), ATRX (Sigma-Aldrich, 1:400), p53 (Santa Cruz Biotechnology Inc., 1:200), and Ki-67/MIB-LI (DAKO, 1:200). The chromogen used in the experiment was diaminobenzidine.

The presence and absence of characteristics such as intensity, staining pattern, and distribution were meticulously observed. For the assessment of IDH, combined staining in the cytoplasm and nucleus was considered indicative of immunopositivity. For ATRX, the presence or absence of nuclear staining, along with the retention of expression in endothelial cells (which served as internal controls), was classified as retained or lost expression, respectively. Strong nuclear staining of p53 in more than 10% of tumor cells was classified as positive. The Ki-67 labeling index was calculated as the percentage of positively stained tumor cell nuclei.

Fluorescence in situ hybridization (FISH) assay for Homozygous CDKN2A Deletion

FISH assay was performed in all histologically grade 2 and 3 tumors. The study employed commercially available sets of fluorochrome-labeled probes manufactured by Vysis Inc., Abbott Laboratories SA. It included a locus-specific probe for CDKN2A (9p21) paired with a corresponding reference centromeric probe for chromosome 9.

In the analysis, signals were evaluated in a minimum of 200 non-overlapping, intact cell nuclei. To serve as controls, sections from non-neoplastic cortical tissue, obtained from epilepsy surgery specimens, were utilized for each probe pair. The identification of homozygous CDKN2A deletion was based on the simultaneous absence of both signals from the locus-targeted probe and the presence of a signal from the reference probe. In accordance with previous research, a cutoff of 30% was employed to define a significant CDKN2A homozygous deletion [4, 12].

Clinical details/Survival analysis

The demographic details, tumor location, size and surgical outcomes of the cases were recorded from the Medical records available in our institute. The study assessed survival outcomes, including overall survival (OS) and progression-free survival (PFS).

Statistical Analysis

Statistical analyses were conducted using RStudio, with additional packages including ggplot, survminer, and ggsurvplot. Associations between clinicopathological factors were assessed using the chi-square test for categorical variables and the nonparametric t-test, specifically the Mann-Whitney U-test, for continuous variables. Survival analysis was carried out employing the Kaplan-Meier method, and the log-rank test was utilized for comparison. All statistical tests were two-sided, and p-values less than 0.05 were considered indicative of statistical significance.

A total of 22 recurrent IDH mutated astrocytoma with available corresponding primary tumor were analysed. The mean patient age of cohort was 34.3 ± 8.64 years (range; 19–47 years) at the time of diagnosis of recurrent tumors. Male-to-female ratio was 6.3:1, including 19 male and 3 female patients. In the study, the primary 18 tumors were histologically grade 2 and four were grade 3 astrocytomas (Fig. 1). Among the recurrent cases, seven tumors were histologically grade 2, ten were grade 3, and five were grade 4 astrocytomas.

Homozygous CDKN2A Deletion analysis

Homozygous deletion of CDKN2A occurred in a single (1/22; 4.8%) primary tumor with histomorphological characteristics corresponding to grade 3, and 6 out of 17 (35.3%) recurrent tumors. Among these recurrent tumors, only one tumors was histologically classified as grade 2, and five tumors as grade 3. None of tumor classified as histologically grade 4 was assessed for the homozygous deletion of CDKN2A (Table).

Survival Analysis

The mean overall survival (OS) for patients with CDKNA2 deletion was significantly lower at 67.7 months compared to 137.6 months for patients without the deletion. In contrast, the mean progression-free survival (PFS) was comparable between the two groups, with patients exhibiting a PFS of 47.4 months for those with CDKNA2 deletion and 47.8 months for those without the deletion. Out of 22 patients, 15 were alive till the last date of follow up, 4 died of the disease and 3 lost to the follow up. Interestingly, 5/6 patients with CDKNA co-deletion are live and single cases lost to the follow up (Fig. 2).

Selective Approach for CDKN2A FISH Assay

On FISH analysis only single case of grade 3 tumors showed homozygous CDKN2A deletion with an overall frequency of 4.5%. However, in the recurrent cases, 6/17 (31.8%) histologically grade 2/3 tumors were positive for CDKN2A homozygous deletion. Therefore, we propose that the FISH assay may be limited only to all primary grade 3 and all recurrent cases irrespective of histological grade to analyze homozygous CDKN2A deletion status.

The progression of IDH-mutant astrocytomas from grade 2/3 to grade 4 is a complex process involving multiple genetic and epigenetic alterations. Recent studies have highlighted the importance of integrating CDKN2A deletion status into the grading system for IDH-mutant astrocytomas, as it is associated with poorer overall survival (OS) [13, 14]. CDKN2A/B homozygous deletion is a strong adverse prognostic factor in IDH-mutant astrocytoma, rendering a median overall survival of ~ 50–68% shorter compared to those without the deletion [15]. Based on this, the 2021 WHO classification has officially adopted CDKN2A/B homozygous deletion as a criterion to upgrade IDH-mutant astrocytoma to WHO grade 4, even in the absence of glioblastoma-specific histological features. However, the clinical benefits of analyzing the CDKN2A homozygous deletion in IDH-mutant astrocytoma remain poorly understood with limited literature [2–4, 14–17].

In the present study on analysis of 22 recurrent IDH mutant astrocytoms, FISH analysis revealed homozygous CDKN2A deletion in one grade 3 primary tumor (4.5%) and six recurrent grade 2/3 tumors (31.8%). Therefore, we strongly recommend that FISH analysis for homozygous CDKN2A deletion should be performed on all primary grade 3 and recurrent cases, irrespective of histological grade.

In a previous study from our center on IDH-mutant astrocytomas, homozygous CDKN2A deletion was conspicuously absent in grade 2 and rare in primary grade 3 IDH-mutant astrocytomas, occurring in only 10.9% of grade 3 cases. CDKN2A deletion was more frequent in recurrent (40%) than primary (2.76%) gliomas. Half of the CDKN2A-deleted cases had poor outcomes, with recurrence or death within 5–36 months. We proposed a selective approach to CDKN2A FISH testing in resource-limited settings, restricting it to high-risk cases with features of anaplasia, p16 loss, or tumor recurrence in order to optimize cost-effectiveness without compromising the identification of deleted cases [12].

A few other studies have investigated the frequency of CDKN2A deletions in grade 2 and 3 gliomas. Notably, Marker et al. reported the rarity of high-level CDKN2A homozygous deletion in primary tumors and its increased frequency in recurrent tumors. They defined a threshold of ≥ 30% of tumor cells with homozygous deletion to identify high-risk patients within grade 4 tumors. In contrast, grade 2 and 3 tumors rarely exceeded that cut-off and did not show worse survival, indicating the lack of prognostic significance of CDKN2A homozygous deletion by FISH in lower-grade IDH-mutant astrocytomas [17].

The study by Kocakavuk et al. examined the prognostic significance of hemizygous CDKN2A deletion in IDH-mutant gliomas without 1p/19q codeletion (IDHmut-noncodel). The authors analyzed copy number variation profiles of 1,256 initial and 494 recurrent IDHmut gliomas across four independent datasets [6–9]. They found that CDKN2A hemideletion was associated with significantly shorter OS in IDH mutant 1p19q non-codeleted gliomas. The frequency of CDKN2A hemideletion more than doubled in recurrent cases compared to initial diagnosis, suggesting it may become more prevalent over the course of the disease [18].

In summary, high levels of CDKN2A homozygous deletion are uncommon in primary IDH-mutant astrocytomas but become more frequent in recurrent tumors. The presence of CDKN2A deletion is associated with more aggressive histological features and poorer clinical outcomes, especially in the recurrent setting. CDKN2A deletion is rare in primary IDH-mutant grade 2/3 astrocytomas but more common in recurrent tumors, where it is associated with shorter overall and progression-free survival. Assessing CDKN2A status, especially in recurrent tumors, can help guide prognosis and management of astrocytoma patients.

ACKNOWLEDGMENTS

We would like to acknowledge the efforts of technical and application specialists at the Neuropathology Laboratory, All India Institute of Medical Sciences, New Delhi, India.

ETHICS APPROVAL

This study was approved by the Institute Ethics Committee.

CONSENT TO PARTICIPATE

Informed consent was obtained from all patients or their relatives if the patient was deceased.

FUNDING

None.

CONFLICTS OF INTEREST

The authors have no conflict of interest to declare that are relevant to the content of this article.

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Table: Frequency of homozygous CDKN2A deletion in different grades of primary and recurrent astrocytic tumours

Homozygous CDKNA2 deletion

Histologically

Grade 2

Histologically

Grade 3

Histologically Grade 4

Primary Tumors

0/17

N=1/4 (25%)

None

Recurrent Tumors

N=1/7 (14.3%)

N=5/10 (50%)

Not performed

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Reviewers agreed at journal
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20 Aug, 2024
Editor assigned by journal
29 Jul, 2024
First submitted to journal
28 Jul, 2024

You are reading this latest preprint version

Clinical Utility of Assessing CDKN2A Status in Recurrent Astrocytomas

Status:

Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Immunohistochemistry

Clinical details/Survival analysis

Statistical Analysis

RESULTS

Homozygous CDKN2A Deletion analysis

Survival Analysis

Selective Approach for CDKN2A FISH Assay

DISCUSSION

Declarations

References

Table

Status:

Version 1