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Immunohistochemical expression of histone modification pattern in adult glioblastoma

1 Department of Pathology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India
2 Department of Neurosurgery, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, Tamil Nadu, India

Date of Submission30-Jan-2022
Date of Decision29-Aug-2022
Date of Acceptance29-Aug-2022
Date of Web Publication18-Nov-2022

Correspondence Address:
Lawrence D'Cruze,
Department of Pathology, Sri Ramachandra Medical College, Porur, Chennai - 600 116, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.jcrt_257_22

 > Abstract 

Background: Despite the growing advances in molecular research and therapeutics, glioblastomas are still considered highly invasive aggressive tumors with a median survival of 15 months. Genetic alterations have been studied in detail; however, additionally, there is now growing evidence on the role of epigenetic alterations in glioblastoma. Recently, histone modification patterns have been found to have a significant part in gene expression and prognosis. However, further research in this field is warranted to establish its role for the betterment of these patients with the deadly disease.
Aims: To determine the immunohistochemical expression of histone modifications like histone-3-lysine-18 acetylation (H3K18Ac) and histone-4-lysine 20 trimethylation (H4K20triMe) in glioblastoma patients
Materials and Methods: This is a retrospective study of 48 glioblastoma patients who underwent surgery. Immunohistochemistry (IHC) for tri-methyl-histone-H4 (Lys20) (H4K20triMe) and acetyl-histone-H3 (Lys18) (H3K18Ac) was performed in paraffin-embedded tissues manually, and the expression was noted. Data on the mitotic index and overall survival was collected and statistically analyzed.
Results: The mean age was 50 years with a M: F ratio of 1.6:1. Out of 48 cases, 60% (28 cases) demonstrated positivity for H3K18Ac and 98% (46 cases) for H4K20triMe. The pattern of expression was nuclear with increased expression adjacent to necrosis and at the invasive front. The overall median Q score for H3K18Ac was 1/12 and for H4K20triMe was 6/12. No significant statistical significance was observed between histone expression, Ki67%, and overall survival.
Conclusion: Histone modification patterns are being explored in detail in an array of tumors. They also have a potential role in glioblastoma for risk stratification and instituting appropriate treatment based on the prognosis. Epigenetic changes like histone modification patterns, in addition to genetics, can pave the way for a better molecular understanding of glioblastomas and provide hope in the future to improve the survival of these patients with deadly diseases.

Keywords: Glioblastoma, histone, immunohistochemistry, prognosis, survival

How to cite this URL:
Archana B, D'Cruze L, Sundaram S, Ramanathan K, Ganesh K. Immunohistochemical expression of histone modification pattern in adult glioblastoma. J Can Res Ther [Epub ahead of print] [cited 2022 Dec 9]. Available from: https://www.cancerjournal.net/preprintarticle.asp?id=361528

 > Introduction Top

Gliomas account for the majority (>70%) of brain tumors, of which glioblastomas are the most frequent, malignant, and aggressive tumors.[1] Despite recent advances in treatment, the survival of glioblastoma patients remains poor with a median survival time of 15 months that has not changed significantly in the past two decades.[2] Intensive molecular studies in the field of glioblastomas have revealed a variety of dysregulated genes. Recently, it has become clearly evident that epigenetic alterations, in addition to genetic changes, may be significant in the genesis and progression of glioblastomas.[3] Broad differences in treatment response and survival within glioblastoma patients also suggest that added factors like epigenetic changes could influence overall survival in these patients. In addition to DNA methylation which is the best-studied epigenetic change, histone modifications are known to play an important role in glioma genesis.[4] Modifications are known to be reversible changes and have a significant role in gene expression. They are an emerging field important from the “theranostic” point of view which includes therapeutics and diagnosis. Histone modification patterns include changes such as acetylation, methylation, phosphorylation, ubiquitination, etc. Histone modifications such as H3K18Ac and H4K20triMe are commonly identified that may have an impact on the overall survival of glioblastoma patients.[4] Intriguingly, many drugs like histone deacetylase inhibitors have been recently developed and clinically validated for use in cancers.[5] For cases with poor prognosis as indicated by the corresponding epigenetic profiles, more aggressive therapies could be considered to prolong their life.

 > Objectives Top

To determine the immunohistochemical expression of histone modifications like histone-3-lysine-18 acetylation (H3K18Ac) and histone-4-lysine-20 trimethylation (H4K20triMe) in glioblastoma patients

 > Methodology Top

Study design

This was a retrospective study.

Tissue samples from archival tissue blocks of 48 glioblastoma patients received in the Department of Pathology from Jan 2014 to 2018 were included in the present study.

Inclusion criteria: All glioblastoma patients

Exclusion criteria: Other grades of gliomas (grade 1 to 3)

Parameters such as demographic details and the mitotic index were collected and tabulated [Table 1].
Table 1: Clinical, demographic, and immunohistochemical characteristics of study sample

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Ethical approval

Institutional Ethics committee approval was obtained to use the archival material for research purposes and to review the medical records (IEC-N1/19/FEB/68/06). All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional or regional) and with the Helsinki Declaration of 1975.

Immunohistochemistry (IHC)

We used specific rabbit antibodies to detect two histone modifications, tri-methyl-histone-H4 (Lys20) (H4K20triMe) and acetyl-histone H3 (Lys18) (H3K18Ac), both from Cell Signaling Technology, and secondary antibody from GeNei (goat antirabbit IgG HRP).

Sections were deparaffinized in xylene and rehydrated in alcohol. Antigen retrieval was then performed. The endogenous peroxidase was quenched at room temperature with a solution of 0.3% hydrogen peroxide in methanol, and the sections were kept in a pressure cooker for about 10 minutes or 2 whistles having 0.01M sodium citrate buffer (pH 6.0–6.2). Immunohistochemical staining with primary antibodies for H4K20triMe and H3K18Ac was applied at a 1:200 dilution, overnight at room temperature, and then incubation was performed with the secondary antibody for 2 hours at room temperature. Chromogen detection was accomplished by tagging with diaminobenzidine. The sections were then counterstained with hematoxylin, dehydrated, and mounted with DPX. A semiquantitative assessment of the intensity and pattern of staining on tissue sections was performed independently by two observers.

The intensity of staining-I (score 1 to 3) and the percentage positivity of cells (nuclear staining) P (0–25%: score 1, 26–50%: score 2, 51–75%: score 3, and >76%: score 4) were calculated, and a total Q score was obtained by multiplying the intensity with the percentage positivity of cells for both the antibodies separately.

Q = I × P. The maximum Q score was 12

Survival analysis

Follow-up data was updated in August 2019. The overall survival (OS) time was taken from the date of surgery to the date of death as a result of any cause.

Statistical analysis

Correlation between the histone modification patterns assessed by IHC and variables like age and Ki67% was analyzed using RVersion 3.6 software. The probability of survival was estimated using the Kaplan–Meier curve.

 > Results Top

There were 48 glioblastoma patients who were included in this study. The median age of the study population was 50 years. The male (n = 30): female (n = 18) ratio was 1.6:1. Clinical, demographic, and immunohistochemical characteristics of these patients are summarized in [Table 1]. Out of 48 cases, 60% (28/48 cases) showed positivity for H3K18Ac and 98% (46/48 cases) showed positivity for H4K20triMe [Figure 1] and [Figure 2]. IHC expression of H3K18Ac and H4K20triMe was nuclear with the majority of cases showing a low level of heterogeneity. Few cases showed an increased perivascular pattern of staining. It was noted that there was increased staining adjacent to the areas of necrosis and at the invasive front [Figure 3]a, [Figure 3]b, [Figure 3]c, [Figure 3]d, [Figure 3]e, [Figure 3]f. Endothelial cells within the tumor and normal neural tissue adjacent to the tumor also showed positive IHC expression in a few cases.
Figure 1: Frequency of positive IHC staining for H3K18Ac

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Figure 2: Frequency of positive IHC staining for H4K20triMe

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Figure 3: Representative examples of immunohistochemical analysis of glioblastoma tissues, (a) 100×, maximum staining for H4K20triMe-Q score 12, (b) 100×, H4K20triMe-Q score 6, (c) 100×, maximum staining for H3K18Ac-Q score 12, (d) 100×, H3K18Ac-Q score 1, (e) 40×, perivascular staining of H4K20triMe, and (f) 100×, H4K20triMe interface between unstained necrotic fragment with adjacent positively stained tumor cells

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Q scores for both H3K18Ac and H4K20triMe ranged from a minimum score of 0/12 to a maximum score of 12/12. The overall median Q score for H3K18Ac was low (score of 1/12) and that for H4K20triMe was higher (6/12). The mean score for H3K18Ac was 3/12 and that for H4K20triMe was 7/12. The mean Ki67 labeling index was 34%. No significant relationship could be observed between IHC expression with patient age and Ki67%. We further divided all patients into four groups based on Q scores (<4 or ≥4) of the corresponding histone modification pattern (H3K18Ac and H4K20triMe) for prognostication [Figure 4].
Figure 4: Subgrouping according to the IHC expression level (Q score)

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All patients (100%) were dead at the time of this analysis. We managed to collect OS for 20 cases. The median follow-up for 20 cases (41%) period was 20 months, and the median OS was 6 months. 70% of these cases survived for less than a year. Only two cases survived for 21 months [Table 2]. The highest median OS of 8 months was noted in group 3 (H4K20triMe, Q score <4). Groups 1 to 4 were not associated with significant OS. Kaplan–Meier curves were plotted, and we found varying patterns of survival at every stage of the disease which was statistically insignificant due to lesser follow-up data [Table 2].
Table 2: Median OS of the different groups of glioblastoma patients

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 > Discussion Top

Glioblastomas are highly aggressive rapidly growing tumors that have a dismal survival despite advances in treatment modalities. Extensive efforts have been made to understand the genetic alterations, and a variety of dysregulated genes have been identified in cell cycles, DNA repair, apoptosis, cell migration, invasion, and angiogenesis.[2] The discovery of isocitrate dehydrogenase (IDH) especially has shed new light on the molecular profile in glioblastoma.[6] During the past two decades, increasing experimental data support the idea that epigenetic alterations have a crucial role in the development and progression of human cancers like prostate cancer, breast cancer, colon cancer, gastric cancer, lung cancer, and leukemias.[7],[8] The elucidation of epigenetic changes in glioblastomas such as aberrant DNA methylation, histone modification, and micro-RNA expression has changed our point of view on these heterogeneous tumors.[9] The prognostic significance of one such epigenetic change, DNA methylation by O6-Methylguanine-DNA Methyltransferase (MGMT), has been widely studied and established.[10],[11],[12]

Compared to other epigenetic changes, histone modification patterns have been less characterized. However, there is significant evidence to demonstrate the role of specific histone modification patterns with the overall prognosis in lung, breast, gastric, prostate, kidney, and colon cancers.[5],[13],[14],[15]

Evidence from a literature search of recent data shows that histone modifications may play a role in the genesis and progression of gliomas.[4] In our study, we assessed the expression of the two most important histone modification patterns, H3K18Ac and H4K20triMe, in glioblastomas. We divided our cases into four groups based on Q scores like that done by Liu et al.[4] for prognosis. However, there was no significant correlation between the groups with the OS in our study. Liu et al. demonstrated that H3K18 acetylation significantly influenced OS, demonstrating a greater survival for patients whose tumors expressed low levels of H3K18Ac similar to that reported by Tzao et al.[16] that results in esophageal carcinoma. However, previous reports have found that the levels of H3K18Ac were lower in cancers of the prostate, lung, kidney, and breast which predicted a poorer survival, which appears contrary to the above study.[13],[15],[17] Also, H4K20 trimethylation was found to influence survival with longer OS for patients whose tumors expressed higher levels.[4] Similarly in other studies also, the same histone modification profiles predicted opposite prognosis in different cancers that indicated that histone modification patterns may possess time and tissue-specific features of heterogeneity. Further studies are needed to characterize in more detail global histone modification patterns in glioblastoma. For those with worse prognosis as indicated by the specific epigenetic profiles, more aggressive treatment could be provided.

Histone deacetylase inhibitors are a promising new class of drugs useful in hematological and solid tumors. The use of these drugs is being studied and is in the clinical trial stage.[18] It has been proposed that these drugs could interact with histone modifications and improve the survival outcome and prevent metastasis. In addition, the prognostic value of these modification patterns will aid in the identification of glioma patients with different prognoses to modify chemotherapy and appropriately select suitable patients for optimal adjuvant treatments. In contrast with genetic mutations, epigenetic mutations are considered to be reversible through drug treatments.[19] Hence, this is an area of active research currently.

In a developing country like ours, due to improper follow-up of patients, we encounter a major challenge to collect accurately sufficient data on OS and disease-free intervals. To determine the accurate overall incidence and characteristic patterns of these alterations, there is a necessity for further study and analysis. Newer models such as glioblastoma patient-derived tumor stem cells grown in neurosphere culture will be added to epigenetic research. Furthermore, a combined approach of determining genetic, environmental, and epigenetic factors together has not been studied so far and the detection of histone modifications has never been tested in body fluids yet. All these could pave way for further mysteries on glioblastoma to unfold and develop potential optimum targets for therapy.

 > Conclusion Top

Very limited data have been published worldwide to validate the pattern of prognostic influence of histone modifications in glioblastoma. Further studies need to be carried out for a larger sample. In addition, we only detected the expression of histone modifications at the protein level by IHC, not at the mRNA level. We wish to expand our study by including all grades of diffuse gliomas and correlate it with the IDH mutation status, which is an established therapeutic genetic signature according to the WHO classification system of gliomas. Ultimately, combined genomic and epigenomic data should provide new predictive biomarkers of response to develop effective therapies and save patients suffering from this devastating disease.

Financial support and sponsorship

Sriher GATE project research grant, 2018.

Conflicts of interest

There are no conflicts of interest.

 > References Top

Müller Bark J, Kulasinghe A, Chua B, Day BW, Punyadeera C. Circulating biomarkers in patients with glioblastoma. Br J Cancer 2020;122:295-305.  Back to cited text no. 1
Martinez R, Schackert G. Epigenetic aberrations in malignant gliomas: An open door leading to better understanding and treatment. Epigenetics 2007;2:147-50.  Back to cited text no. 2
Nagarajan RP, Costello JF. Epigenetic mechanisms in glioblastoma multiforme. Seminars Cancer Biol 2009;19:188-97.  Back to cited text no. 3
Liu B-L, Cheng J-X, Zhang X, Wang R, Zhang W, Lin H, et al. Global histone modification patterns as prognostic markers to classify glioma patients. Cancer Epidemiol Biomarkers Prev 2010;19:2888-96.  Back to cited text no. 4
Zhou M, Yuan M, Zhang M, Lei C, Aras O, Zhang X, et al. Combining histone deacetylase inhibitors (HDACis) with other therapies for cancer therapy. Eur J Med Chem 2021;226:113825. doi: 10.1016/j.ejmech. 2021.113825.  Back to cited text no. 5
Liu A, Hou C, Chen H, Zong X, Zong P. Genetics and epigenetics of glioblastoma: Applications and overall incidence of IDH1 mutation. Front Oncol 2016;6:16. doi: 10.3389/fonc. 2016.00016.  Back to cited text no. 6
Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007:8:286-98.  Back to cited text no. 7
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Kreth S, Thon N, Kreth FW. Epigenetics in human gliomas. Cancer Lett 2014;342:185-92.  Back to cited text no. 9
Hegi ME, Liu L, Herman JG, Stupp R, Wick W, Weller M, et al. Correlation of O 6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. J Clin Oncol 2008;26:4189-99.  Back to cited text no. 10
Wick W, Weller M, Van Den Bent M, Sanson M, Weiler M, Von Deimling A, et al. MGMT testing-The challenges for biomarker-based glioma treatment. Nat Rev Neurol 2014;10:372-85.  Back to cited text no. 11
Reifenberger G, Hentschel B, Felsberg J, Schackert G, Simon M, Schnell O, et al. Predictive impact of MGMT promoter methylation in glioblastoma of the elderly. Int J Cancer 2012;131:1342-50.  Back to cited text no. 12
Elsheikh SE, Green AR, Rakha EA, Powe DG, Ahmed RA, Collins HM, et al. Global histone modifications in breast cancer correlate with tumor phenotypes, prognostic factors, and patient outcome. Cancer Res 2009;69:3802-9.  Back to cited text no. 13
Park YS, Jin MY, Kim YJ, Yook JH, Kim BS, Jang SJ. The global histone modification pattern correlates with cancer recurrence and overall survival in gastric adenocarcinoma. Ann Surg Oncol 2008;15:1968-76.  Back to cited text no. 14
Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, et al. Global levels of histone modifications predict prognosis in different cancers. Am J Pathol 2009;174:1619-28.  Back to cited text no. 15
Tzao C, Tung HJ, Jin JS, Sun GH, Hsu HS, Chen BH, et al. Prognostic significance of global histone modifications in resected squamous cell carcinoma of the esophagus. Mod Pathol 2009;22:252-60.  Back to cited text no. 16
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Cappellacci L, Perinelli DR, Maggi F, Grifantini M, Petrelli R. Recent progress in histone deacetylase inhibitors as anticancer agents. Curr Med Chem 2020;27:2449-93.  Back to cited text no. 18
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]

  [Table 1], [Table 2]


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