|Year : 2021 | Volume
| Issue : 2 | Page : 22-26
CpG2 hypermethylation in the CD95L promoter is associated with survival in patients with glioblastoma: An observational study
NU Farrukh Hameed1, Yuyao Zhou1, Lei Jin1, Ying Xu2, Jie Zhang1, Hong Chen3, James Xue2, Jinsong Wu1
1 Department of Neurosurgery, Shanghai Medical College, Huashan Hospital, Fudan University, Shanghai, China
2 CANbridge Life Sciences Ltd., Beijing, China
3 Department of Pathology, Huashan Hospital, Shanghai, China
|Date of Submission||31-May-2021|
|Date of Decision||21-Jun-2021|
|Date of Acceptance||05-Jul-2021|
|Date of Web Publication||29-Jul-2021|
Dr. Jie Zhang
Department of Neurosurgery, Shanghai Medical College, Huashan Hospital, Fudan University, Shanghai 200040, China
Source of Support: None, Conflict of Interest: None
Background and Aim: Blockade of CD95/CD95 ligand (CD95 L) signaling is a promising therapeutic approach for the treatment of glioblastoma (GBM), while methylation of a single cytosine-phosphate-guanine site (CpG2) upstream of the CD95 L promoter has been identified as a prognostic biomarker for GBM. Here, we conducted the first investigation of CD95 L expression and CpG2 methylation levels in the CD95 L promoter in GBM patients. Materials and Methods: In this observational study, tissue samples were collected from 69 patients with a primary diagnosis of World Health Organization Grade IV GBM treated at the Department of Glioma Surgery, Huashan Hospital, Shanghai Medical College, Fudan University and tested for CD95 L expression using immunohistochemistry (IHC). The CpG2 methylation status of the samples was also evaluated, and its impact on overall survival (OS) was assessed by univariate analysis. The study was approved by the Hospital Institutional Review Board (approval No. 220) on July 7, 2015. Results: The IHC results showed a CD95 L detection rate of at least 43.5% for tissue samples with IHC scores of 2+ or 3+ and 78.3% for those with IHC scores of 1+, 2+, or 3+. Patients with high CpG2 methylation levels (≥52% higher than the median value; n = 32) had significantly longer median survival compared with those with low CpG2 methylation levels (n = 29) (22.95 vs. 14.5 months; P = 0.0084). GBM patients who underwent gross total tumor resection (n = 57) showed similar results. Those in the high CpG2 methylation group had longer median OS compared with that of patients in the low CpG2 methylation group (23.5 vs. 18.0 months; P = 0.0141). Conclusions: Our results showed a significant prevalence of CD95 L expression in GBM patients, whereas CpG2 hypermethylation within the CD95 L promoter was positively associated with survival. These findings support that CD95/CD95 L signaling blockade has potential as a therapeutic strategy targeting treatment-resistant GBM.
Keywords: Biomarker, CD95, glioblastoma, targeted therapy
|How to cite this article:|
Farrukh Hameed N U, Zhou Y, Jin L, Xu Y, Zhang J, Chen H, Xue J, Wu J. CpG2 hypermethylation in the CD95L promoter is associated with survival in patients with glioblastoma: An observational study. Glioma 2021;4:22-6
|How to cite this URL:|
Farrukh Hameed N U, Zhou Y, Jin L, Xu Y, Zhang J, Chen H, Xue J, Wu J. CpG2 hypermethylation in the CD95L promoter is associated with survival in patients with glioblastoma: An observational study. Glioma [serial online] 2021 [cited 2022 Dec 8];4:22-6. Available from: http://www.jglioma.com/text.asp?2021/4/2/22/322654
| Introduction|| |
Glioblastoma (GBM) is one of the most aggressive tumors of the central nervous system, accounting for approximately 15% of all brain tumors and having a generally poor prognosis. In patients receiving current standard treatment, radiotherapy with temozolomide – the median overall survival (OS) is <15 months, whereas the 2- and 5-year survival rates are 26.5% and 9.8%, respectively.,, Compared with many cancers, the advancement of GBM treatment has been relatively slow, with bevacizumab being the only targeted therapy currently approved for recurrent GBM in several countries., This highlights the need to identify the key genetic alterations underlying GBM pathogenesis and develop effective targeted therapies for the treatment of this disease.
CD95 is a cell surface protein belonging to the tumor necrosis factor receptor family. When bound to CD95 ligand (CD95 L), its natural ligand, CD95 mediates processes such as apoptosis and cancer cell growth, invasiveness, and migration.,,,, Blocking CD95/CD95 L signaling can restore tumor immune responses and inhibit GBM invasiveness. The development of asunercept (CAN008, APG101), a human CD95 receptor/Fc-fusion protein that blocks CD95 signaling through specific binding to CD95 L, has demonstrated the potential clinical utility of employing CD95/CD95 L blockade as a therapeutic strategy to treat GBM. In a Phase II trial, asunercept has shown promising efficacy in patients with progressive GBM who received re-irradiation plus asunercept versus those receiving radiotherapy alone, with improved rates of 6-month progression-free survival (20.7% vs. 3.8%, respectively) being reported. Furthermore, asunercept was shown to reduce the invasiveness of GBM cells in vitro and may also improve the effectiveness of radiotherapy.
Interest in epigenetic processes underlying cancer pathogenesis has increased over recent years,,, and several genetic alterations have been implicated in different GBM tumor subtypes. An important facet of GBM molecular pathogenesis that has been overlooked is the role of aberrant DNA methylation, which in humans manifests as the methylation of the cytosine in the cytosine-phosphate-guanine (CpG) dinucleotide.,, A substantial number of genes contain CpG islands in their promoter regions, most of which are normally unmethylated, regardless of whether or not the genes are being expressed. The clustering of methylated genes has been observed in GBM,, ovarian cancer, and gastrointestinal, and hematologic malignancies., In addition, data from the Phase II trial of asunercept revealed that a lower level of methylation at a single CpG site (CpG2) upstream of the CD95 L promoter was associated with an improvement in median OS for patients with progressive GBM receiving re-irradiation plus weekly asunercept versus radiotherapy alone (16.1 vs. 6.5 months, respectively). These data suggest that the CpG2 methylation level in the CD95 L promoter may be a prognostic factor for asunercept treatment in GBM. However, the effect of asunercept in Asian patients remains unknown. Here, we assessed the levels and prevalence of CD95 L expression as well as the level of CpG2 methylation in the CD95 L promoter in tumor samples from GBM patients to determine the potential prognostic impact of these biomarkers in an Asian population.
| Materials and Methods|| |
Patient selection and study design
Tumor samples were obtained from 69 patients with histologically confirmed Grade IV GBM treated at the Department of Glioma Surgery, Huashan Hospital, Shanghai Medical College, Fudan University in this observational study. Tumor tissues were first examined by neuropathologists, and tissue quality was ensured before molecular profiling was undertaken. GBM diagnosis was made according to the updated World Health Organization classification. Section slides were prepared from each patient's tumor tissue. The study was approved by the Hospital Institutional Review Board (approval No. 220) on July 7, 2015 and was performed in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice. Written informed consent was obtained from all the participants before inclusion in the study.
Collected tumor tissue samples were stored in the Glioma Tissue Bank of our hospital. Residual tumor tissue and corresponding normal tissue samples were collected for immunohistochemistry (IHC) and DNA methylation analyses. Medical records and follow-up information were collected from the glioma patient management system. OS was defined as the interval between the day of surgery and the date of the last follow-up or death.
CD95 ligand immunohistochemistry
Tissue sections were processed and stained using a recombinant anti-CD95 L rabbit monoclonal antibody (APG1181, lot 5288, Apogenix, Germany) following the manufacturer's protocol.
The CD95 L positivity rate has been reported to range from 30% to 60% in GBM patients;, accordingly, a minimum positivity rate of 30% was set as the benchmark for GBM patients in this study. Precise two-sided binomial 95% confidence intervals (CIs) were computed for the CD95 L positivity rate to determine if it could be defined as being higher than 30%.
IHC assays were used to detect the expression level of CD95 L in tumor cells using ScanScope; original magnification: ×20 (Leica, Germany). The levels of expression were graded as 0: no staining, 1+: weakly positive, 2+: strongly positive, and 3+: very strongly positive, and these were determined by neuropathologists based on CD95 L staining in tumor cells. In general, staining intensity was scored in areas where staining was strongest within the whole tumor section. In this study, 1+, 2+, and 3+ were defined as a positive CD95 L result. CD95 L-positive tissues were grouped into (a) 2+ or 3+ and (b) 1+, 2+, or 3+.
Cytosine-phosphate-guanine site methylation analysis
For DNA methylation analysis, the tumor tissue samples were rechecked for consistency, and only those that met laboratory requirements were analyzed. The rest were discarded. Total DNA extraction was performed using the AmoyDx FFPE DNA Extraction Kit (Cat# ADx-FF01, Xiamen, China) according to the instruction manual. DNA methylation transformation was carried out using the Qiagen Bisulfite Kit (Cat# 59824, Maryland, USA) following the manufacturer's instructions.
DNA methylation analysis results were sorted into high- and low-level groups. Samples having ≥52% more methylation relative to the median value were categorized into the “high-level” group while the rest were classified into the “low-level” group.
The results obtained were analyzed by statisticians using SAS version 9.4 (SAS Institute, Cary, NC, USA). Quantitative variables were expressed as the mean and standard deviation or median and interquartile range, and categorical variables were expressed as frequencies (%). Univariate analysis was performed to assess the association between CpG2 methylation in the CD95 L promoter and OS.
| Results|| |
CD95 ligand expression
A total of 69 patients with GBM were included in this study. The median age of the patients was 62 years, 63.8% were male, and most (92.8%) had a Karnofsky performance status score of 90–100. Most patients (82.6%) received gross total resection [Table 1]. Representative images of CD95 L staining intensity are shown in [Figure 1]. Thirty samples showed 2+ or 3+ IHC scores and the rate of positivity was 43.5% (95% CI 31.6%–56.0%); 54 samples showed 1+, 2+, or 3+ IHC scores, and the CD95 L positivity rate was 78.3% (95% CI 66.7%–87.3%).
|Figure 1: Representative images of the immunohistochemistry results for CD95 ligand staining in the glioblastoma specimens tested. (A) Top left: CD95 ligand positive, 3+ immunohistochemistry score; (B) top right: CD95 ligand-positive, 2+ immunohistochemistry score; (C) bottom left: CD95 ligand-positive, 1+ immunohistochemistry score; (D) bottom right: CD95 ligand-negative, an immunohistochemistry score of 0|
Click here to view
Cytosine-phosphate-guanine site methylation
The samples from all 69 patients met the quality criteria for DNA methylation analysis. Among these patients, the median CpG2 methylation level was 4002.0 concentration copies/reaction [Table 1]. Among the 61 isocitrate dehydrogenase (IDH)-negative GBM patients, the median CpG2 methylation level was 3986.3 concentration copies/reaction; 32 patients had high CpG2 methylation levels and demonstrated a significantly longer median survival compared with that of patients with low CpG2 methylation levels (n = 29) (22.95 vs. 14.5 months; P = 0.0084). A similar observation was made in the 57 patients who underwent gross total tumor resection; patients with high CpG2 methylation levels had a longer median OS compared with that of patients with low levels of CpG2 methylation (23.5 vs. 18 months; P = 0.0141).
| Discussion|| |
The prognosis for patients with GBM remains dismal, and the development of new therapeutic strategies is an urgent unmet need. In this regard, promising results have been reported for CD95/CD95 L signaling blockade with asunercept in patients with recurrent GBM. In addition, through the development of asunercept, the methylation status of CpG2 in the CD95 L promoter has been identified as a potential prognostic biomarker for GBM. The present study represents the first investigation of the expression levels and prevalence of CD95 L and the association between CpG2 methylation status and survival in GBM patients. Our results showed a significant prevalence of CD95 L (>43%) in the GBM population and an association between OS and high levels of CpG2 methylation in the CD95 L promoter in patients with IDH-negative GBM.
Among the 69 patients with GBM included in this study, we found a CD95 L detection rate of 43.5% using 2+ or 3+ IHC scores and 78.3% using 1+, 2+, or 3+ IHC scores. This result suggests that targeting CD95/CD95 L signaling represents an effective treatment strategy for patients with GBM. Asunercept is a fusion protein, in which the extracellular domain of human CD95 is fused to the Fc part of human immunoglobulin G1 and was developed to target the CD95 signaling pathway in the treatment of GBM.,, Asunercept can specifically bind to CD95 L and prevent its binding to CD95, thereby inhibiting CD95 ligand-mediated tumor cell growth and migration. Moreover, asunercept can prevent T-cell death by blocking the interaction between tumor-derived CD95 L and CD95 present on the surface of T cells, which induces their apoptosis.,
In our study, we observed that high CpG2 methylation levels favored OS in IDH-wild type GBM patients. This result is interesting in the context of the Phase II trial of asunercept, in which compared with patients with high CpG2 methylation levels, those with low levels of CpG2 methylation achieved a greater benefit from the addition of asunercept to re-irradiation versus re-irradiation alone. One interpretation of these observations is that patients with a low-CpG2 methylation status have a worse prognosis than those with a high-methylation status; consequently, they achieve greater benefit from the addition of asunercept to re-irradiation. Studies have shown that cancer cells become either unmethylated or hypermethylated at CpG sites in promoter regions. Hypermethylation can silence numerous genes in budding cancer cells including tumor-suppressor genes, thereby favoring tumorigenesis., However, gene silencing can be favorable under certain circumstances such as with genomic imprinting, X-chromosome inactivation, and host silencing of viral DNA. It has been suggested that CpG methylation in promoter regions inappropriately silences tumor-suppressor genes, which can explain the higher prevalence of methylated CpG sites in cancer patients compared with that in healthy patients., Accordingly, a possible cause for the improved OS seen in GBM patients with wild-type IDH and who underwent gross total resection and had high levels of CpG2 methylation in the present study may be that the weakened expression or the silencing of oncogenes resulting from the hypermethylation of upstream CpG2 sites in their promoter regions decreased the aggressiveness of these tumors. Studies have also shown that decreased CpG methylation promotes genetic instability and carcinogenesis,, which could explain the contrasting reports of shorter survival times in GBM patients with hypomethylated CpG2. GBM tumor cells are already characterized by marked genetic heterogeneity, a key reason for their aggressiveness and resistance to therapies, and increased instability resulting from CpG hypomethylation may further aggravate prognosis.
The main limitation of this study was the small patient sample size, which only permitted univariate analysis. Further studies involving larger number of patients are required to support and build on our findings.
| Conclusions|| |
CD95 L was found to be expressed in >43% of GBM patients, confirming CD95/CD95 L signaling as an attractive therapeutic target for the treatment of this disease. Moreover, CpG2 hypermethylation appears to be positively associated with survival in GBM patients and merits further exploration in the development of novel therapies for treatment-resistant GBM.
CpG2 detection was conducted by Gemu Huang of Amoy Diagnostics Co., Ltd. IHC detection was conducted by Quintiles Central Laboratory.
Financial support and sponsorship
This research was funded by CANbridge Life Sciences Ltd., Beijing, China and Shanghai Shenkang Hospital Development Center, China (No. SHDC12018114). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Institutional review board statement
The study was approved by the Hospital Institutional Review Board (approval No. 220) on July 7, 2015 and performed in accordance with the principles of the Declaration of Helsinki.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patients have given their consent for their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity.
Conflicts of interest
YX and JX work for CANbridge Life Sciences Ltd. Other authors declare no conflicts of interest.
Editor note: JW is an Editorial Board member of Glioma. The article was subject to the journal's standard procedures, with peer review handled independently of this Editorial Board member and their research groups.
| References|| |
Young RM, Jamshidi A, Davis G, Sherman JH. Current trends in the surgical management and treatment of adult glioblastoma. Ann Transl Med 2015;3:121.
Hamisch C, Ruge M, Kellermann S, Kohl AC, Duval I, Goldbrunner R, et al
. Impact of treatment on survival of patients with secondary glioblastoma. J Neurooncol 2017;133:309-13.
Stupp R, Hegi ME, Mason WP, van den Bent MJ, Taphoorn MJ, Janzer RC, et al
. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised Phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009;10:459-66.
Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJ, et al
. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987-96.
Sahebjam S, Sharabi A, Lim M, Kesarwani P, Chinnaiyan P. Immunotherapy and radiation in glioblastoma. J Neurooncol 2017;134:531-9.
Diaz RJ, Ali S, Qadir MG, De La Fuente MI, Ivan ME, Komotar RJ. The role of bevacizumab in the treatment of glioblastoma. J Neurooncol 2017;133:455-67.
Kleber S, Sancho-Martinez I, Wiestler B, Beisel A, Gieffers C, Hill O, et al
. Yes and PI3K bind CD95 to signal invasion of glioblastoma. Cancer Cell 2008;13:235-48.
Roth W, Isenmann S, Nakamura M, Platten M, Wick W, Kleihues P, et al
. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 2001;61:2759-65.
Richards DM, Merz C, Gieffers C, Krendyukov A. CD95L and anti-tumor immune response: Current understanding and new evidence. Cancer Manag Res 2021;13:2477-82.
Fouqué A, Debure L, Legembre P. The CD95/CD95L signaling pathway: a role in carcinogenesis. Biochim Biophys Acta 2014; 1846:130-41.
Levoin N, Jean M, Legembre P. CD95 structure, aggregation and cell signaling. Front Cell Dev Biol 2020;8:314.
Motz GT, Santoro SP, Wang LP, Garrabrant T, Lastra RR, Hagemann IS, et al
. Tumor endothelium FasL establishes a selective immune barrier promoting tolerance in tumors. Nat Med 2014;20:607-15.
Krendyukov A, Gieffers C. Asunercept as an innovative therapeutic approach for recurrent glioblastoma and other malignancies. Cancer Manag Res 2019;11:8095-100.
Wick W, Fricke H, Junge K, Kobyakov G, Martens T, Heese O, et al
. A Phase II, randomized, study of weekly APG101+reirradiation versus reirradiation in progressive glioblastoma. Clin Cancer Res 2014;20:6304-13.
Blaes J, Thomé CM, Pfenning PN, Rübmann P, Sahm F, Wick A, et al
. Inhibition of CD95/CD95L (FAS/FASLG) signaling with APG101 prevents invasion and enhances radiation therapy for glioblastoma. Mol Cancer Res 2018;16:767-76.
Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 2003;349:2042-54.
Kumar S, Gonzalez EA, Rameshwar P, Etchegaray JP. Non-Coding RNAs as Mediators of Epigenetic Changes in Malignancies. Cancers (Basel) 2020; 12:3657.
Zhang J, Yang C, Wu C, Cui W, Wang L. DNA Methyltransferases in Cancer: Biology, Paradox, Aberrations, and Targeted Therapy. Cancers (Basel) 2020; 12:2123.
Jaenisch R, Bird A. Epigenetic regulation of gene expression: How the genome integrates intrinsic and environmental signals. Nat Genet 2003;33 Suppl:245-54.
J Dabrowski M, Wojtas B. Global DNA methylation patterns in human gliomas and their interplay with other epigenetic modifications. Int J Mol Sci 2019; 20:3478.
Cheray M, Etcheverry A, Jacques C, Pacaud R, Bougras-Cartron G, Aubry M, et al
. Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme. Mol Cancer 2020;19:36.
Vavouri T, Lehner B. Human genes with CpG island promoters have a distinct transcription-associated chromatin organization. Genome Biol 2012;13:R110.
Dong Z, Cui H. Epigenetic modulation of metabolism in glioblastoma. Semin Cancer Biol 2019;57:45-51.
Uddin MS, Mamun AA, Alghamdi BS, Tewari D, Jeandet P, Sarwar MS, et al
. Epigenetics of glioblastoma multiforme: From molecular mechanisms to therapeutic approaches. Semin Cancer Biol 2020. doi: 10.1016/j.semcancer.2020.12.015.
Natanzon Y, Goode EL, Cunningham JM. Epigenetics in ovarian cancer. Semin Cancer Biol 2018;51:160-9.
Kim H, Kim YH, Kim SE, Kim NG, Noh SH, Kim H. Concerted promoter hypermethylation of hMLH1, p16INK4A, and E-cadherin in gastric carcinomas with microsatellite instability. J Pathol 2003;200:23-31.
Nordlund J, Syvänen AC. Epigenetics in pediatric acute lymphoblastic leukemia. Semin Cancer Biol 2018;51:129-38.
Rahmani M, Talebi M, Hagh MF, Feizi AA, Solali S. Aberrant DNA methylation of key genes and acute lymphoblastic leukemia. Biomed Pharmacother 2018;97:1493-500.
Louis DN, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee WK, et al
. The 2016 World Health Organization classification of tumors of the central nervous system: A summary. Acta Neuropathol 2016;131:803-20.
Merz C, Strecker A, Sykora J, Hill O, Fricke H, Angel P, et al
. Neutralization of the CD95 ligand by APG101 inhibits invasion of glioma cells in vitro
. Anticancer Drugs 2015;26:716-27.
Hanke N, Kunz C, Thiemann M, Fricke H, Lehr T. Translational PBPK modeling of the protein therapeutic and CD95L inhibitor asunercept to develop dose recommendations for its first use in pediatric glioblastoma patients. Pharmaceutics 2019;11:152.
Peter ME, Krammer PH. The CD95(APO-1/Fas) DISC and beyond. Cell Death Differ 2003;10:26-35.
Schmidt C. Larger companies dominate cancer companion diagnostic approvals. Nat Biotechnol 2011;29:955-7.
Strathdee G, Appleton K, Illand M, Millan DW, Sargent J, Paul J, et al
. Primary ovarian carcinomas display multiple methylator phenotypes involving known tumor suppressor genes. Am J Pathol 2001;158:1121-7.
Wenger A, Ferreyra Vega S, Kling T, Bontell TO, Jakola AS, Carén H. Intratumor DNA methylation heterogeneity in glioblastoma: Implications for DNA methylation-based classification. Neuro Oncol 2019;21:616-27.
Garcia-Manero G, Daniel J, Smith TL, Kornblau SM, Lee MS, Kantarjian HM, et al
. DNA methylation of multiple promoter-associated CpG islands in adult acute lymphocytic leukemia. Clin Cancer Res 2002;8:2217-24.
Issa JP. Methylation and prognosis: Of molecular clocks and hypermethylator phenotypes. Clin Cancer Res 2003;9:2879-81.
Toyota M, Ahuja N, Suzuki H, Itoh F, Ohe-Toyota M, Imai K, et al
. Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res 1999;59:5438-42.