Acta Neurochir (2013) 155:1437–1442 DOI 10.1007/s00701-013-1802-1

CLINICAL ARTICLE - BRAIN TUMORS

Histone H1.0—a potential molecular marker with prognostic value for patients with malignant gliomas Nikolay Gabrovsky & Milena Georgieva & Maria Laleva & Konstantin Uzunov & George Miloshev

Received: 3 November 2012 / Accepted: 14 June 2013 / Published online: 29 June 2013 # Springer-Verlag Wien 2013

Abstract Background Histones are proteins closely associated with the DNA molecules and serve as a structural scaffold for the organization of chromatin. They play an important role in the regulation of gene expression by changing the level of DNA compaction. The special subtype of the linker histone family—H1 zero (H1.0) is generally expressed in non-dividing, terminally differentiated cells. The aim of our study is to investigate the correlation between the quantities of histone H1.0 in human gliomas, the histopathological grade and the overall survival. Material and method Twenty-nine (N=29) patients with intraaxial lesions underwent a microsurgical tumor resection. Tumor samples were snap-frozen in liquid nitrogen immediately after resection. Following a specific protocol, linker histones were extracted from the tumor specimens and the quantities of histone H1.0 were assessed. All patients were followed up prospectively. Results Of the 29 patients in our study (M:F=17:12), five had a grade II astrocytoma, seven had a grade III, and 17 had a grade IV, according to the World Health Organization (WHO) classification. At the end of the study, three patients were still alive. The mean quantities of H1.0 were: 23.3 for grade II tumors, 13.9 for grade III and 11.3 for grade IV tumors. The statistical analysis demonstrated that the histological grade, age and Karnofsky performance status (KPS) remain

N. Gabrovsky (*) : M. Laleva : K. Uzunov Department of Neurosurgery, University Hospital “Pirogov”, 21, Totleben blvd, 1606 Sofia, Bulgaria e-mail: [email protected] M. Georgieva : G. Miloshev Laboratory of Molecular Genetics, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria

among the most reliable predictive factors for the survival of patients with gliomas. Grade III–IV gliomas had significantly less histone H1.0 than grade II gliomas. Conformably, in a multivariate Cox regression analysis, H1.0 made a small but significant contribution (p<0.05) to survival rates. Conclusion Our study confirmed that histone H1.0 is a potential biological marker with prognostic value for the survival of patients with gliomas. The quantities of histone H1.0 are correlated to the histopathological grade of the tumor. The more aggressive and malignant gliomas tend to have lower quantities of histone H1.0. Keywords Glioma . Histone . Epigenetics . Prognostic factors

Introduction Malignant gliomas (MG) are the most frequent primary central nervous system (CNS) tumors. The prognosis of this illness remains dismal, with an inevitable recurrence and overall survival varying generally from 9 to 18 months. Currently, MG are incurable and the main hope remains in the further understanding of the processes of carcinogenesis and in the search for targets for new drug development. The relatively new discipline of epigenetics refers to inheritance that is not coded in the DNA sequence, but is due to modifications in DNA and chromatin. The research in epigenetics is one of the most rapidly expanding fields in cancer biology, because all malignancies are recognized not only as genetic, but also as epigenetic diseases. The realization of epigenetic program includes at least four biological phenomena—DNA methylation, posttranslational modifications of histones, dynamic shuffling of histone variants and not yet well understood functions of small noncoding RNAs. All these epigenetic mechanisms are thought to be crucial for the regulation of gene expression [1]. DNA in the eukaryotic

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because, for example, the quantity of this histone does not change in neuroendocrine tumors [8]. This controversy can be explained with the differences in origins and developmental stages of the tumor type. Therefore, to understand the relationship between H1.0 and malignancies, it is quite important to first assess the amount of this histone in any given cancer type, and then to deeply dissect the underlying mechanisms. Here, we present our results aiming to investigate a correlation between the quantities of the linker histone H1.0 in human gliomas, their histopathological World Health Organization (WHO) grade, and the time of overall survival. This kind of correlation is made for the first time, and we explore the potential of histone H1.0 to become a biological marker with prognostic value in patients with gliomas.

nucleus is compacted in a nucleoprotein complex called chromatin [2]. Histones are proteins closely associated with DNA molecules. In humans, five families of histones have been identified—H1, H2A, H2B, H3 and H4. The four core histones H2A, H2B, H3, and H4 (two molecules of each) serve as structural scaffold for the organization of the chromatin in a repetitive octamer structure called a nucleosome [3] (Fig. 1). In the recent decade, it became evident that histones have not only a structure-maintaining role, but also may influence gene expression by changing the level of DNA compaction [4]. The H1 histone family, also called the family of “linker histones”, binds to the linker DNA entering and exiting the nucleosome. In humans this family includes eleven different subtypes of lysine-rich proteins divided according to their temporal and spatial expression. Linker histones have an important function in the maintenance of genomic integrity and play a role in the transcriptional regulation of a subset of genes involved in aging, DNA repair, DNA methylation, imprinting and apoptosis [5, 6]. It is now becoming clear that every subtype of linker histones plays a different but specific role in genome organization and expression [7]. The special subtype of the linker histone family H1 zero is generally expressed in non-dividing, terminally differentiated mammalian cells [8]. Strong evidence has been accumulated that H1 zero (denoted H1.0 in humans) is connected with processes of cellular differentiation [9]. More importantly, the quantity of this linker histone subtype could be inversely correlated with the malignization of the cells. It has been shown that histone H1.0 is missing in certain cancer cell lines, including HeLa [10], the hepatoma cell line HepG2 and some carcinoma-derived cell lines. It has also been shown that H1.0 is in a lower amount or is absent in some tumors, such as hepatocarcinoma [11], Lewis lung carcinoma [12] and breast cancer [13]. However, a strict correlation between cancer cells and lowering of the amount of H1.0 could not be directly drawn

For the period 2004–2008, 29 patients with intraaxial lesions were included in our study. All patients were surgically operated and a craniotomy with microsurgical tumor resection was performed. In all cases, immediately after resection a piece of the tumor with a volume of 1.5–2 cm3 was frozen in liquid nitrogen and stored at −80 °C. All specimens underwent histological examination and tumor grading according to WHO. A specific extraction of linker histones from the frozen tumor specimens was performed according to the protocol published by Smith and Johns, 1980 [14] with some modifications [15]. The consecutive steps of specific linker histone isolation are as follow: All steps of linker histone isolation were performed on ice in the presence of cocktail of protease inhibitors (Protease inhibitor panel, Sigma-Aldirch, Cat. N: INHIB-1). Specimens from (approx. 1 cm3) tumor tissues were left to thaw on ice, cut to

Fig. 1 Artistic representation of the organization of chromatin in the nucleus. a The DNA double helix. b The nucleosome—core histones (red sphere) serve as structural scaffold for the organization of the

chromatin. Histone H1 binds to the linker DNA entering and exiting the nucleosome (yellow ellipsoide). c Further compaction of the DNA. d The chromosome

Material and methods

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small pieces by a scissor and homogenized by pestle in a homogenizer. During the homogenization, several milliliters of 1×PBS (0.14 M NaCl, 2.7 mM KCl, 1.47 mM KH2PO4, 8.1 mM Na2HPO4, pH 7) were added. The cells in the homogenate were lysed by 0.25 % Nonidet P40 for 10 min. After the lysis the freed nuclei were collected by centrifugation at 2,500×g for 15 min., the nuclear pellet was washed by 0.35 M NaCl, and the linker histones were extracted from it. The linker histones were obtained by specific perchroric acid extraction . Five volumes of 5 % perchloric acid was added to the nuclear pellet and the extraction continued for 1 h. After the extraction the nuclear suspension was centrifuged for 20 min at 5,000×g and the histones from supernatant were precipitated with 1/4 of the volume 100 % trichloroacetic acid. The precipitated linker histones were collected by centrifugation at 6,000×g for 20 min, washed by ethanol and ethanol-ether (1:1, v/v), air-dried and kept frozen until the time of analysis. The extracted proteins were separated on SDS-containing polyacrylamide gel electrophoresis (SDS—PAGE). After the electrophoresis, the proteins in the gels were stained overnight with 0.35 % solution of Coomassie Brilliant Blue G250 (Sigma-Aldrich). The quantity of the histone H1.0 was assessed by a specialized video-capturing system and a gel analysis system, GDS 7600 of UVP. The quantification of histone H1.0 was done by the specialized software GelPro 3 of Molecular Cybernetics Co. Under the conditions of 20 % polyacrylamide SDS gels, human linker histones generally separate to three distinctive fractions. All of the linker histone subtypes with the exception of H1.0 mixed together into two closely migrating bands from the first two more slowly migrating bands. H1.0 migrates faster than the others and thus forms the third, fastest migrating band [16]. For the purpose of our research, all linker histones on the gel were accepted as 100 %, and the percentage of histone H1.0 was calculated. In order to unambiguously confirm the H1.0 subfraction, Western blot analyses were additionally performed, using anti-H1.0 polyclonal serums [17]. All patients were followed up prospectively and detailed clinical data concerning the evolution of the disease were recorded. Main follow-up protocols were: physical examination, neurological examination, detailed symptoms description (including duration, intensity, frequency, progression and treatment applied), image study description (localization, volume/size, contrast enhancement of the lesion, peritumoral edema, midline shift), quality of life assessment—Karnofsky performance status (KPS), operative protocol details (intraoperative finding, macroscopic description of the tumor, extent of resection) and protocol for operative complications. The most important demographic and clinical variables with potential prognostic value for the survival, such as age, sex, KPS, histological grading, tumor location, adjuvant treatment and number of operations, were analyzed for all 29 patients using Statistica software (Table 1).

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Analyses of variance were performed on the amount of histone H1.0 (Table 2) and the quality of life measurements (Table 3) in order to study their potential connection with histological grades. A multivariate Cox regression determined the independent importance of different variables for the continuance of patients’ survival in number of weeks (see Table 4). Kaplan-Meier survival analysis was used to assess survival probability of the factors. Age was converted into a noncontinuous variable by dividing it into two groups: 1) age less than 55 years and 2) age above 55 years. Log-rank test was applied for evaluation of the statistical difference between the groups in each survival analysis. To determine the effect of continuous variables, such as the amount of histone H1.0 and the quality of life, a multivariate Cox regression analysis was performed. Age entered the multivariate analysis as a continuous variable. Non-continuous factors (sex, therapy, histology, tumor localization in a hemisphere and in the four lobes) were also included into the multivariate analysis.

Results From the 29 patients enrolled in our study (M:F=17:12), five were diagnosed to have a grade II astrocytoma, seven to have a grade III, and 17 to have a grade IV, according to the WHO classification. At the end of 2010, 26 of the patients exhibited tumor progression and passed away, while three of the patients were alive (all of them with grade II tumors). These particular patients were censored in some of the analyses. The demographic and clinical data, treatment type, tumor localization and the descriptive statistics concerning the survival times are presented in Table 1. Details about the age, sex, histopathological grade, mean time of survival, localization and adjuvant therapy are also included in Table 1. The measured quantities of H1.0 as a percentage of the total quantity of all linker histones were as follow: 23.3 % for grade II, 13.9 % for grade III and 11.3 % for grade IV tumors (Table 2). A one-way ANOVA was run on quality of life data with regard to histological grades (II, III, and IV) as an independent variable. The analysis showed a significant main effect of histology levels [F(2, 26)=6.07, p<0.01; ηp2=0.32]. A Bonferroni test suggested significant differences between grade II and grade IV (p<0.05). Grade III did not differ significantly from the two other grades (for means and other descriptive statistics, see Table 3). To assess the possible interconnection between the quantity of H1.0 and the histology level, a univariate analysis of variance (ANOVA) was run on H1.0 and

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Table 1 Characteristics of the patients and clinical data [means (M) are given along with standard deviations (SD) in parenthesis]. Mean, confidence interval (± 95 %; CI), median and range of survival time in weeks are given for each characteristic Characteristics

Number of patients

Number censored (ALIVE)

Mean survival time (CI) in weeks

median survival time (range)

Overall (N) Age (years) ≤ 55 Age (years) > 55 Sex Male Sex Female Radiotherapy Yes Radiotherapy No Chemotherapy Yes Chemotherapy No

29 17 12 17 12 13 16 5 24

3 3 0 1 2 2 1 1 2

54.4 (44.1–75.1) 73.5 (46.9–95.8) 27.3 (19.2–45.9) 47.2 (35.3–72.1) 64.0 (47.0–112.6) 66.4 (45.8–105.5) 44.6 (35.1–73.6) 67.0 (36.4–174.8) 51.8 (43.1–77.7)

27.0 (1–224) 67.0 (1–224) 22.0 (1–96) 26.0 (1–143) 52.5 (1–224) 59.0 (1–224) 24.5 (1–143) 61.0 (2–140) 26.5 (1–224)

Radiotherapy and Chemotherapy Yes Radiotherapy and Chemotherapy No Histology WHO grade II Histology WHO grade III Histology WHO grade IV Tumor location (overlapping) Frontal Temporal Parietal Occipital

3 26 5 7 17

1 2 3 0 0

60.0 (29.9–361.4) 53.7 (44.3–77.9) 17.8 (16.9–81.3) 111 (42.7–146.0) 41.8 (29.4–60.2)

61.0 (2–117) 26.5 (1–224) 2.0 (1–67) 99.0 (11–224) 26.0 (1–140)

4 19 13 4

0 3 1 0

43.3 (34.6–227.9) 52.6 (44.7–87.4) 64.0 (48.4–111.3) 56.3 (33.6–221.4)

19.0 (1–134) 27.0 (1–224) 35.0 (2–224)

Hemisphere Left Hemisphere Right

12 17

0 3

71.0 (46.9–112.3) 42.6 (33.6–68.7)

Histology H1.0 as an independent variable with three histological levels (II vs. III vs. IV). A main effect was obtained [F(2, 26)=7.56, p<0.01; ηp2 =0.37], which suggested that levels III (Mean H1.0=13.9) and IV (M=11.3) do not differ between each other in terms of mean H1.0. However, Bonferroni test showed that both levels are significantly different (p<0.05 and p<0.01) from level II (M=23.3). See Table 4 for more descriptive statistics. As far as Kaplan-Meier analysis is concerned, only III and IV histological grades were chosen to enter the

35.5 (11–143) 55.5 (1–224) 23.0 (1–143)

analysis, since the lack of sufficient data for the level II (five patients, three of which were censored, Table 1). It is clearly seen that patients with III level had better survival time than patients with IV level (Table 1 for descriptive statistics). To test the prognostic significance power of therapy effect and to avoid small number of cases whenever possible, the data were collapsed (radiotherapy or chemotherapy vs. no therapy). Neither therapeutic factors, nor demographic factors, nor tumor location factors appeared to be close to significance (Table 4).

Table 2 Quantities of H1.0 in regard to histological grade and age. Mean (M), standard deviation (SD), median, confidence interval (CI) and range of H1.0 quantity are presented Characteristics

M (SD)

median

CI

range

Histone H1.0, total Histone H1.0, II grade Histone H1.0, III grade Histone H1.0, IV grade Histone H1.0 by age, in years (≤ 55) Histone H1.0 by age, in years (> 55)

14.0 (7.4) 23.3 (4.4) 13.9 (8.7) 11.3 (5.2) 15.0 (7.6) 12.7 (7.0)

12.3 23.6 8.8 11.5 12.5 11.6

11.2–16.8 17.9–28.7 5.9–21.9 8.6–14.0 11.0–18.9 8.2–17.2

3.1–27.2 16.1–27.2 6.4–26.1 3.1–20.7 5.0–27.2 3.1–26.1

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Table 3 Quantities of quality of life (QoL) in regard to histological grade and age. Mean (M), standard deviation (SD), median, confidence interval (CI) and range of H1.0 quantity are presented Characteristics

M (SD)

median

CI

range

QoL, total QoL, II grade QoL, III grade QoL, IV grade

73.1 (13.7) 86 (5.5) 78.6 (10.7) 67.1 (13.1)

80 90 80 60

67.9–78.3 79.2–92.8 68.7–88.5 60.3–73.8

40–90 80–90 60–90 40–90

79 (12.2) 65 (11.7)

80 60

72.6–85.1 57.6–72.4

50–90 40–80

QoL at admission by age, in years (≤ 55) QoL at admission by age, in years (> 55)

Discussion In our study, we have analyzed the role of several factors with proven prognostic value for the survival of patients with malignant gliomas, including the histopathological grade, age and the KPS score. Among the many different variables that were assessed for their importance to the patients’ overall survival, just a few came out to have a significant weight in our data. For instance, patients’ age showed to be an important factor in both Kaplan-Meier and regression analyses, a result that is supported by numerous data that suggest that the older the patient, the worse the survival. Histology levels have also been shown to be important for the prognosis. Higher tumor grades proved to lead to worse surviving, but only in the Long-rank analysis. Our data may not be demonstrative, because two patients with grade II tumors died shortly after the operation for reasons unrelated to the brain pathology. On the other hand, all of the survivals in our study were with grade II tumors. Table 4 Univariate (Kaplan-Meier) and multivariate analyses. In a multivariate Cox regression analysis, age appeared to be the best predictor of survival (p<0.01). Histone H1.0 made a small but significant contribution (p<0.05) into survival times. None of other variables approached significance level Variable

Long-rank test, p =

Multivariate, Wald

Multivariate, p =

Sex Quality of life Age diagnosed Collapsed therapy Number of operations H1.0 Histology H1.0 Hemisphere Frontal Temporal Parietal Occipital

0.273 – 0.005 0.172 – – 0.008 0.426 0.415 0.919 0.343 0.850

0.383 1.577 8.631 1.083 1.902 5.565 1.868 1.906 2.180 0.230 2.366 0.588

0.536 0.209 0.003 0.298 0.168 0.018 0.172 0.167 0.140 0.631 0.124 0.443

The results of our study also demonstrated that a connection between quality of life according to the KPS score and the histological grades of the tumor does exist. Patients with higher histological grade have significantly lower level of life quality: 90 for grade II, 80 for grade III and 60 for grade IV tumors. These results confirm that the histological grade and KPS remain among the most reliable predictive factors for survival of patients with gliomas. Regarding the location of the lesion—left, right or in the different lobes—we did not find significant correlation to survival. However, we have to mention that all of the survivals in our series were with tumors located in the temporal lobe of the right hemisphere. A relation between a more extensive resection in the right temporal lobe and the longer survival could be made, but we don’t have computed tomography (CT)/magnetic resonance imaging (MRI) data to confirm this hypothesis. The other variable in our studies, the special H1 subtype histone H1.0, demonstrated interesting correlation. ANOVA results showed that histone H1.0 is intrinsically linked with the histology—one of the most reliable survival predictors. Grade III and grade IV gliomas had significantly less H1.0 than grade II gliomas. Conformably, in a multivariate Cox regression analysis, histone H1.0 made a small but significant contribution (p<0.05) to survival times. All these data confirm our hypothesis that there is an inverse correlation between the amount of H1.0 and the aggressiveness of the astrocytic tumor, and that the lower quantities of histone H1.0 in the tumor are related to a higher aggressiveness. Thus, for patients with less histone H1.0 in the tumors, a shorter survival time has to be expected. This correlation highlights the importance of this linker histone in tumorigenesis, and moreover, in the development and the outcome of malignant gliomas. Such a statement is quite logical, having in mind that H1.0 functions are linked to proliferation and differentiation of the cell. Interestingly, in unison with our data, it has been shown by other authors that the amount of H1.0 in different tissues correlates inversely with their proliferative activity [2, 18]. Moreover, heterologous overexpression of H1.0 in a cell line has a well-defined effect on the chromatin structure, resulting in a more compacted chromatin conformation compared to the

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overexpression of the other linker subtypes [19], and thus leading to significantly higher inhibition of transcription [14]. Several other studies have shown that histone H1.0 is missing in some cancer cell lines, such as the HeLa cell line [10], the hepatoma cell line HepG2, and carcinoma-derived cell lines [20]. It was also shown that H1.0 is in a lower amount or absent in some tumors, such as hepatocarcinoma [11] and Lewis lung carcinoma [12], which also correlates with our results. Interestingly, in thyroid neoplastic tissues [10] and in acute lymphoblastic leukemia [21], histone H1.0 is present in normal quantities. These data suggest a kind of cancer-specific change in H1.0 amounts. On the basis of our data, we believe that the lower amounts of H1.0 in high-grade gliomas correspond to dismal prognosis and higher tumour growth. These results highlight histone H1.0 as a potential molecular marker with predictive value, especially for malignant gliomas.

Conclusion Our study confirmed that histone H1.0 is a potential molecular marker with prognostic value for the extent of survival in patients with gliomas. The quantities of histone H1.0 are in correlation with the histopathological grade of the tumor, and the more aggressive and malignant gliomas tend to have lower quantities of histone H1.0. Further investigations are needed to assess the roles of linker histones, and especially H1.0, in glioma tumorigenesis. Acknowledgements N.G. is supported by the Bulgarian Science Fund, Grant number ТК02-468/2009. M.G. is supported by the Bulgarian Science Fund, Grant number DMU 02/8 and from the World Federation of Scientists, National Scholarship Programme. G. M. is supported by the Bulgarian Science Fund, Grant number DID 02/35. Conflicts of interest None.

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