Introduction

Glioblastoma multiforme (GBM) is an aggressive brain tumor known for its high resistance to treatment. Despite multiple attempts using various immunotherapeutic approaches and combinations [1], GBM remains incurable. Patients with glioblastoma have a poor prognosis, with a median survival of 14.6 months and a 2-year survival rate of less than 26.5% [2]. In a recent study conducted on the Taiwanese population, it was found that the 1-year survival rate of GBM was only 50.3%, which was significantly lower (24.0%) compared to the 2-years survival rate [3]. There are two major contributing factors to this outcome. Firstly, GBM frequently recurs and metastasizes due to the rapid proliferation of infiltrative residual tumor cells. Secondly, tumor cells that are resistant to current chemotherapy contribute to tumor regrowth and recurrence, which is often inevitable. Therefore, there is an urgent need to explore approaches that can improve the outcomes of GBM patients.

In 2009, the Food and Drug Administration (FDA) granted accelerated approval for bevacizumab (BEV), also known as Avastin, a humanized anti-VEGF monoclonal immunoglobulin 1 (IgG1) antibody. BEV inhibits angiogenesis by disrupting the VEGF/VEGF-receptor signaling pathway, thereby exerting indirect antitumor activity [4]. Currently, BEV is used for the treatment of recurrent glioblastoma multiforme (rGBM) [5]. Subsequent clinical studies also have demonstrated the effectiveness of bevacizumab in increasing the objective response rate and median progression-free survival in patients with rGBM [

Materials and methods

Subjects

The study protocols were approved by the Medical Ethics Committee of Taichung Veterans General Hospital (Approval number: CF17263B-4). This retrospective study included 139 GBM patients ranging in age from 20 to 92 years. GBM diagnosis was confirmed through pathological examinations. Samples were obtained from the Department of Minimally Invasive Skull Base Neurosurgery, Neurological Institute, Taichung Veterans General Hospital, from 2010 to 2022, comprising the primary study cohort. Informed consent was obtained from all subjects or their legal guardians prior to surgery, and the collected samples were promptly frozen. The informed consent process involved providing detailed information about the study's objectives, procedures, potential risks, and benefits to the participants. They were given ample opportunity to ask questions and clarify any concerns before voluntarily providing their consent to participate. The study protocol and informed consent procedure were reviewed and approved by the relevant institutional ethics committee to ensure compliance with ethical guidelines and standards. All GBM patients underwent surgical resection and received concurrent chemoradiotherapy with temozolomide (Temozolomide (TMZ): 75 mg/m2/d) (CCRT), followed by adjuvant TMZ (150–200 mg/m2/d). Bevacizumab (10 mg/kg intravenously every 2 weeks until disease progression) was administered only to patients with recurrent GBM. The validation cohort consisted of 139 cases selected from the primary cohort based on the following criteria: (1) availability of follow-up data and samples, and (2) a post-operative survival time of more than one month. The obtained samples were immediately frozen after surgery. Overall survival (OS) time was defined as the time from the operation to the date of death or censored at the date of the last follow-up examination. The study end date was 31 March 2023. Commencing on May, 2012, the National Health Insurance Bureau of Taiwan broadened its coverage within the framework of health insurance benefits, encompassing the targeted drug bevacizumab's application in the treatment of adult patients experiencing relapsed glioblastoma multiforme. The administration of bevacizumab was typically synchronized with the emergence of disease progression, reflecting a commitment to transparency. This clinical determination was rooted in a thorough assessment of each patient's unique circumstances, alongside pertinent clinical variables. This systematic approach facilitated the precise deployment of bevacizumab, aligning its usage with the unmistakable signs of disease advancement. As a result, its focused deployment effectively managed the recurrence of glioblastoma. Nevertheless, it is imperative to acknowledge that certain individuals, prior to 2012, might not have received optimal counsel and treatment due to factors such as financial limitations or individual considerations.

Endpoint

The primary endpoint of this study focused on overall survival (OS), which was defined as the period in months starting from the initiation of the first surgery and extending until the time of death. In cases where patients were still alive at the point of data censoring, the OS was calculated up to the date of the last follow-up. The secondary endpoints encompassed progression-free survival (PFS) and the assessment of adverse events. PFS, specifically, was defined as the duration in months commencing from the initiation of bevacizumab treatment and extending to the occurrence of disease progression or death. If patients were alive and had not encountered disease progression during data censoring, the calculated time interval was extended to the date of the last follow-up. These endpoint definitions were selected to comprehensively evaluate treatment outcomes and patient experiences.

Genoty** assay

We adhered to SNP names in accordance with the guidelines set forth by the Human Gene Nomenclature Committee (HGNC). Genomic DNA was extracted from frozen tumor tissues for the genoty** assay. The CDKN1A c.93C > A (codon 31) polymorphisms were analyzed using a polymerase chain reaction (PCR)—restriction fragment length polymorphism (RFLP) assay. PCR–RFLP analysis is a rapid and straightforward technique employed as an additional method to detect genetic polymorphisms in GBM. However, this method has certain limitations. The sequences of partial CDKN1A c.93C > A patients were determined using a DNA autosequencer (GeneAmp PCR System 2700 Thermal cycler; Applied Biosystems) (Fig. 1b). The primer sequence and PCR conditions for CDKN1A c.93C > A are described in Table 1 and Fig. 1a. For each sample, the amplified PCR product was digested with the Blp I restriction enzyme (New England Biolabs, Beverly, Massachusetts, USA). The digested reactions were incubated for 16 h at 37℃ [23]. Subsequently, the genoty** assay was conducted on a 2% agarose gel using molecular weight markers and visualized after staining with ethidium bromide (Fig. 2a). The Ser allele harbors a single Blp I restriction site (GCTNAGC), resulting in two fragments of 89 bp and 183 bp, while the Arg allele remains undigested, yielding a single band of 272 bp (Fig. 2b). Each genoty** assay included positive and negative controls, and 10% of the samples were randomly selected and run in duplicates, showing 100% concordance. The results were reproducible with no discrepancies in genoty**. The Supplementary Materials contain the provided DNA sequencing data. Moreover, the heterozygote C/A genoty** of CDKN1A c.93C > A exhibited two signal peaks in the DNA sequencing data (Fig. 1b), consistent with expectations. Additionally, we analyzed two other genotypes of CDKN1A polymorphisms (CDKN1A c.168 + 16G > C, rs3176352G > C, IV2 + 16; CDKN1A 3’UTR c.*70C > T, rs1059234C > T, C70T) and found them to be highly linked with S31R (CDKN1A c.93C > A; C98A, rs1801270) (Fig. 1c). Unprocessed images of the DNA electric gel are provided in the Supplementary Materials. Additionally, the Supplementary Materials provide detailed experimental procedures for Methylation-specific PCR and the identification of the IDH1 gene.

Fig. 1
figure 1

illustrates the schematic diagram of various CDKN1A polymorphisms. a Detailed sequences and location of CDKN1A c.93C > A (codon 31) in PCR production are presented. The NCBI association number is NC_000006.11. Red-colored words indicate the primers and green words represent the restriction enzyme Blp I cut site. The asterisk denotes the nucleotide of the CDKN1A c.93C > A polymorphism. b The PCR products of CDKN1A c.93C > A were analyzed by DNA sequencing, revealing three types of polymorphisms: Ser/Ser, Arg/Arg, and Ser/Arg, respectively. The asterisk represents the variant nucleotide of CDKN1A c.93C > A polymorphism. The heterozygote C/A genoty** of CDKN1A c.93C > A showed two signal peaks. c Schematic diagram of various CDKN1A polymorphisms, including S31R (rs 1801270C > A), IVS2 + 16 (rs 3176352G > C), and C70T (rs 1059234C > T). Red numbers indicate the number of nucleotides for each of these polymorphisms. After analyzing several samples using PCR–RFLP analysis, these three polymorphisms exhibit a high degree of linkage disequilibrium. For example, when S31R showed CC types (n = 15), IVS also exhibited CC types (93.3%, n = 14). Similarly, when S31R showed CC types (n = 26), C70T also displayed CC types (88.4%, n = 23). The pattern continues accordingly

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Table 1 Primers and Restriction Enzyme Used for Genoty** variants of p21 gene polymorphism
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Fig. 2
figure 2

illustrates the Blp I PCR–RFLP analysis schematic diagram for the CDKN1A c.93C > A polymorphism. a The PCR products (272 bps) representing three types of CDKN1A c.93C > A polymorphism variants are shown: CC (homozygote), GG (homozygote), and CG (heterozygote). After digestion with the restriction enzyme Blp I, the CC genotype (Ser/Ser) is divided into two fragments (89 and 183 bps). The GG genotype (Arg/Arg) remains a single fragment of 272 bps due to the ineffectiveness of Blp I digestion. The CG genotype (Ser/Arg) results in three fragments (89, 183, and 272 bps) after Blp I digestion. b The Blp I PCR–RFLP analysis for the CDKN1A c.93C > A polymorphism is presented. M represents the DNA ladder. Lanes 1, 2, 5, and 9 show the Arg/Arg homozygotes, which are not cleaved by Blp I and display a 272-bp band. Lanes 3, 6, 10, and 15 represent the Ser/Ser homozygotes, which are cleaved by Blp I resulting in 183- and 89-bp bands. Lanes 4, 7, 8, 11, 12, 13, and 14 display the Ser/Arg heterozygotes with all three bands (272, 183, and 89 bp) after restriction digestion

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Statistical analysis

Demographic data were presented as patient counts (percentages) for categorical variables and were compared using either the chi-squared test or Fisher's exact test. The overall survival (OS) outcomes were estimated using the Kaplan–Meier method, and differences in survival were assessed using the log-rank test. To investigate the association between CDKN1A and the 2-year overall survival (OS) of GBM patients, as well as the cumulative impact of CDKN1A SNP on the 2-year OS of GBM, adjusted hazard ratios (aHRs) with 95% confidence intervals (CIs) were employed. These aHRs were calculated using Cox proportional-hazards analyses and were adjusted for all the previously mentioned patient-level factors. Statistical analysis was conducted employing the Statistical Package for the Social Sciences (IBM SPSS version 22.0). All statistical analyses adhered to a two-sided approach, and significance levels of p < 0.05 and p < 0.01 were considered statistically significant.

Results

Demographic and clinicopathological characteristics of the participants

This retrospective study included 139 glioblastoma patients, consisting of 58 males and 81 females. The average age of the participants was 56 years (range: 20–92). There were no statistically significant differences in gender distribution among the different genotypes of CDKN1A c.93C > A (codon 31) (p = 0.888). At the end of the study, 80.6% of the patients had passed away, while 19.4% were still alive, with a median survival of 16.8 months. The clinical characteristics of the patients and the associations between the utilization of bevacizumab and various patient characteristics are outlined in Table 2. It is worth noting that patients over the age of 70 generally exhibited a poorer prognosis after surgery. The different variants of CDKN1A c.93C > A polymorphisms did not show any significant correlations with patient age, gender, tumor number, tumor size, tumor occurrence, or response to bevacizumab treatment.

Table 2 The Associations Between Bevacizumab Utilization and Diverse Patient Characteristics
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Genoty**

Association between CDKN1A c.93C > A Polymorphism and Glioblastoma Risk.

Tables 2 and 3 present the frequencies of genotypes and alleles within the CDKN1A gene. In our glioblastoma cases, we observed frequencies of 23.02% (32/139) for Ser/Ser, 27.34% (38/139) for Arg/Arg, and 49.64% (69/139) for Ser/Arg genotypes. Notably, all observed results were found to conform to the principles of the Hardy–Weinberg equilibrium. Furthermore, an assessment of the relationship between the CDKN1A c.93C > A genotype and demographic as well as clinicopathological characteristics of GBM patients was conducted, encompassing factors such as IDH1 gene status and MGMT promoter methylation status (as demonstrated in Table 3). Tables 2 and 3 provide additional insight by demonstrating that specific potential risk factors associated with GBM, including age, gender, tumor count, tumor resection size, IDH1 gene status, and MGMT gene methylation status, did not exhibit significant associations with the CDKN1A genotype. We investigated the link between individual CDKN1A c.93C > A genotypes and their correlation with the 2-year overall survival (OS) among GBM patients. This evaluation was conducted using Cox proportional-hazards models, which were adjusted for variables such as age, gender, stage, and the usage of bevacizumab, as illustrated in Table 4. However, in both univariate and multivariate analyses, none of the computed hazard ratios achieved statistical significance.

Table 3 Clinical characteristics of GBM patients and CDKN1A genotype (c.93C > A)
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Table 4 Relationship between CDKN1A codon 31 SNP and 2-year overall survival of GBM patients
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Genotype effects on overall survival after bevacizumab treatment

The study encompassed the enrollment of 139 GBM patients, with a median follow-up duration spanning 18.7 months (as visually represented in Fig. 3a). Our investigation ventured further into a meticulous assessment of the implications associated with the amalgamation of bevacizumab and chemotherapy concerning the outcomes of progression-free survival (PFS) and overall survival (OS) within the cohort of GBM patients. Within our study, we operationalized "PFS" as the timeframe commencing from the commencement of bevacizumab treatment and extending to the occurrence of disease progression or mortality. This particular definition was meticulously selected to exclusively probe the influence of bevacizumab treatment on the twin facets of disease progression and survival outcomes, with an explicit focus on the temporal period post-administration of the treatment. Although the median PFS duration within the subset of patients subjected to CCRT plus bevacizumab treatment did not manifest a statistically significant expansion (as visually indicated in Fig. 3b) (median of 14.5 months), our scrutiny of OS outcomes divulged a notably constructive impact. This was evident in the comparative evaluation between individuals who exclusively underwent chemotherapy (N = 69) and those who underwent a combined therapeutic regimen encompassing bevacizumab (N = 70), as vividly depicted in Fig. 3c.

Fig. 3
figure 3

Kaplan–Meier curves depicting overall survival (OS) and Progression-Free Survival (PFS) in patients receiving CCRT and CCRT plus bevacizumab. a Overall survival duration for the entire patient cohort. b PFS in patients with glioblastoma treated with bevacizumab. c Kaplan–Meier curves illustrating overall survival in patients receiving standard CCRT treatment and CCRT plus bevacizumab treatment. ** indicates p < 0.001

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Statistical analysis demonstrated a significant improvement (log-rank p < 0.001) in median OS from 9.7 to 28.2 months for the CCRT plus bevacizumab group compared to the CCRT group (Fig. 3c). These findings from the retrospective study strongly suggest that bevacizumab can extend the OS of patients with recurrent GBM [24].

To further explore the relationship between the CDKN1A c.93C > A polymorphism and GBM, we analyzed the overall survival data and genoty** information of all GBM patients. Using the log-rank test and Kaplan–Meier survival curve analysis, we found that the OS analysis comparing the CDKN1A c.93C > A variants did not yield significant results (Fig. 4a). However, a slightly better survival rate was observed in patients with the AA (Arg/Arg) variant.

Fig. 4
figure 4

Genotypes of CDKN1A c.93C > A variants and Kaplan–Meier plots of overall survival (OS) for three groups of GBM patients. a Survival curves for GBM patients stratified by CDKN1A c.93C > A genotypes. b Comparison of estimated OS in patients with CDKN1A c.93C > A Ser/Ser genotypes between those treated with CCRT and those treated with CCRT plus bevacizumab. c Estimated OS in patients with CDKN1A c.93C > A Arg/Arg genotypes for CCRT and CCRT plus bevacizumab treatment. d Estimated OS in patients with CDKN1A c.93C > A heterozygous Ser/Arg genotypes for CCRT and CCRT plus bevacizumab treatment. ** indicates p < 0.001

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We also investigated the impact of combining bevacizumab with chemotherapy on the overall survival (OS) of GBM patients with different CDKN1A genotypes. As shown in Fig. 4b, patients with the CC (Ser/Ser) genotype who received CCRT plus bevacizumab had a median survival of 17.3 months, similar to those with CCRT alone (p = 0.833). Interestingly, in contrast, patients with the AA (Arg/Arg) and CA (Ser/Arg) genotypes exhibited significantly longer median survival when treated with CCRT plus bevacizumab (34.3 and 24.9 months, respectively) compared to CCRT alone (13.1 and 8.5 months, respectively) (p = 0.001 and p < 0.001, respectively, Fig. 4c and d). Furthermore, patients with the CC genotype who received CCRT alone exhibited a higher median survival compared to the other two genotypes. Overall, GBM patients with the AA (Arg/Arg) and CA (Ser/Arg) genotypes demonstrated significantly prolonged survival in the CCRT plus bevacizumab treatment group compared to those with the CC (Ser/Ser) genotype (Fig. 4).

These findings suggest that GBM patients with the AA (Arg/Arg) and CA (Ser/Arg) genotypes of CDKN1A c.93C > A have significantly longer overall survival intervals when treated with CCRT plus bevacizumab compared to those with the CC (Ser/Ser) genotype in the same treatment group.

We broadened our inquiry to delve into the relationship between CDKN1A c.93C > A and the 2-year overall survival, stratifying the data according to the methylation status of the MGMT promoter and the IDH1 gene status (as illustrated in Table 5). Despite conducting both univariate and multivariate analyses, none of the calculated hazard ratios achieved statistical significance. Finally, we conducted a comprehensive risk assessment to determine the potential survival benefits associated with the utilization of BEV. Drawing from our research results, we employed the CDKN1A SNP, IDH1 gene status, and MGMT promoter methylation level to categorize risks. A summary of these findings is presented in Table 6. Among GBM patients with the CDKN1A c.93C > A genotype polymorphism, both univariate and multivariate analyses unveiled a substantial escalation in the risk of mortality for individuals with AA or CA genotypes who did not use BEV. In contrast, patients with the CC genotype exhibited no notable association with BEV usage. A comparable scenario is also evident within the MGMT methylation and IDH1 gene mutation groups: irrespective of MGMT methylation or IDH1 mutation status, individuals who refrain from BEV usage face a notably elevated risk of mortality compared to patients who undergo BEV treatment (Table 6). The aforementioned findings collectively suggest that the utilization of BEV appears to confer survival advantages to GBM patients, with the exception of those with the CDKN1A c.93C > A CC (Se/Ser) genotype.

Table 5 The stratified impact of MGMT gene promoter region methylation, IDH1 gene status, and CDKN1A c.93C > A genotype on the 2-year overall survival of GBM patients
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Table 6 The stratified influence of CDKN1A c.93C > A genotype, MGMT gene promoter region methylation, IDH1 gene status, and the utilization of Bevacizumab on the 2-year overall survival of GBM patients
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This study exclusively involved GBM patients in Taiwan, making it particularly relevant to individuals with Asian heritage and nationality. To comprehensively explore the diversity of CDKN1A c.93C > A polymorphisms across different ethnicities, we conducted an extensive review of pertinent literature within the Asian population (references [19, 25,26,24, 44, 45], suggesting our results are consistent with previous report data. However, it should be noted that the treatment effectiveness of bevacizumab may vary among individuals and can be influenced by various factors, including tumor characteristics, patient's physical condition, and combination with other treatment modalities. Therefore, before using bevacizumab or any other medication, patients should engage in detailed discussions and evaluations with their doctors to determine the optimal treatment plan.

The available data suggest that the S31R polymorphism in the p21 gene may serve as a predictive marker for improved overall survival in patients undergoing bevacizumab treatment. However, it is essential to acknowledge that this conclusion remains speculative and lacks direct data support. When considering potential molecular mechanisms for this observation, several points are worth considering. Firstly, the polymorphism could impact cell cycle regulation since p21 plays a vital negative regulatory role during the G1/S and G2/M transitions of the cell cycle. Any functional or stability changes in the p21 protein due to the polymorphism might disrupt cell cycle regulation, potentially influencing tumor cell growth and proliferation. Secondly, polymorphism may also influence DNA damage repair pathways, as p21 is involved in cellular responses and DNA damage repair. The altered functionality of p21 due to the polymorphism could lead to changes in reactions to therapy-induced DNA damage, potentially affecting treatment effectiveness and patient survival. Lastly, considering bevacizumab's mechanism of action as an anti-angiogenic drug that inhibits VEGF activity to obstruct tumor blood supply, the p21 protein could be associated with angiogenesis inhibition. The polymorphism might affect the regulation of the VEGF pathway, potentially impacting the efficacy of bevacizumab treatment. It is important to reiterate that these speculations are based on limited evidence, and further research and clinical validation are necessary to confirm the validity of this conclusion and unravel the underlying molecular mechanisms.

Conclusion

In conclusion, our data suggest that the CDKN1A c.93C > A, S31R polymorphism may serve as a predictive marker for improved overall survival in patients undergoing bevacizumab treatment. Although our sample size is relatively small, these findings indicate a potential association between the Arg/Arg and Ser/Arg genotypes of the CDKN1A c.93C > A polymorphism and the beneficial effect of bevacizumab in glioblastoma treatment. However, further confirmation of these findings is warranted through additional larger studies and tissue-specific biological characterization.

Availability of data and materials

The data that support the findings of this study are available from Taichung Veterans General Hospital but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of Taichung Veterans General Hospital.