148 research article Dynamic expression of 11 miRNAs in 83 consecutive primary and corresponding recurrent glioblastoma: correlation to treatment, time to recurrence, overall survival and MGMT methylation status Bostjan Matos*1, Emanuela Bostjancic*2, Alenka Matjasic2, Mara Popovic3, Damjan Glavac2 1 Department of Neurosurgery, University Clinical Center, Ljubljana, Slovenia 2 Department of Molecular Genetics, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia 3 Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia Radiol Oncol 2018; 52(4): 422-432. Received 6 August 2018 Accepted 3 September 2018 Correspondence to: Damjan Glavac, Ph.D., Department of Molecular Genetics, Institute of Pathology, Korytkova 2, Faculty of Medicine, SI-1000 Ljubljana, Slovenia. Phone: +386 1 543 7180, Fax: +386 1 543 7181; E-mail: damjan.glavac@mf.uni-lj.si Disclosure: No potential conflicts of interest were disclosed. *Authors contributed equally to this work. Background. Glioblastoma (GBM) is the most common and the most malignant glioma subtype. Among numerous genetic alterations, miRNAs contribute to pathogenesis of GBM and it is suggested that also to GBM recurrence and resistance to therapy. Based on publications, we have selected 11 miRNAs and analyzed their expression in GBM. We hypothesized that selected miRNAs are differentially expressed and involved in primary as well as in recurrent GBM, that show significant expressional differences when different treatment options are in question, and that are related to certain patients and tumor characteristics. Patients and methods. Paraffin embedded tissues, obtained from primary and corresponding recurrent tumor from 83 patients with primary GBM were used. Eleven miRNAs (miR-7, miR-9, miR-15b, miR-21, miR-26b, miR-124a, miR-199a, let-7a, let-7b, let-7d, and let-7f) were selected for qPCR expression analysis. For patients who received temozolamide (TMZ) as chemotherapeutic drug, O6-methylguanine-DNA methyltransferase (MGMT) methylation status was defined using the methyl-specific PCR. Results. There was a significant change in expression of miR-7, miR-9, miR-21, miR-26b, mirR-124a, miR-199a and let-7f in recurrent tumor compared to the primary. In recurrent tumor, miR-15b, let-7d and let-7f significantly changed comparing both treatment options. We also observed difference in progression free survival between patients that received radiotherapy and patients that received radiotherapy and chemotherapy, and longer survival for patients who received chemotherapy after second surgery compared to not treated patients. miR-26b showed correlation to progression free survival and let-7f to overall survival. We did not find any expression difference between the tumors with and without methylated MGMT. Conclusions. Our data suggest that analyzed miRNAs may not only contribute to pathogenesis of primary GBM, but also to tumor progression and its recurrence. Moreover, expression of certain miRNAs appears to be therapy-dependent and as such they might serve as additional biomarker for recurrence prediction and potentially predict a therapy-resistance. Key words: glioblastoma; recurrent; radiotherapy; chemotherapy; miRNA; expression Introduction Gliomas are the most common primary brain tumors and are classified on the basis of histopatho- logical characteristics. Glioblastoma (GBM) is the most common and malignant glioma subtype characterized by rapid growth and poor prognosis.1 Like cancer in general, GBM develop as a Radiol Oncol 2018; 52(4): 365-369. doi: 10.2478/raon-2018-0037 149 Matos B et al. / miRNAs and their expression in glioblastoma consequence of genetic alterations that accumulate with tumor progression. However, pathogenesis of GBM recurrence is still poorly understood. Certain molecular pathways and novel biomarkers have been established as diagnostic, prognostic and predictive markers, such as is epidermal growth factor receptor (EGFR) amplification, O6-methylguanine-DNA methyltransferase (MGMT) methylation and others.1-3 Among novel biomarkers are non-coding RNAs, of which the most studied are microRNAs (miRNAs). The miRNAs are small RNAs that act as endogenous regulators of gene expressions and function in different physiological processes, contributing to pathogenesis of different types of cancer, including gliomas. miRNA expression might be a potential biomarker for GBM used as diagnostic support or for prognostic and therapeutic application.4-6 However, differential expression of certain miRNAs might be also involved in patho-physiological mechanisms of GBM recurrence, but there are limited data regarding miRNA expression as GBM progresses (at its recurrence).78 Comprehensive knowledge and understanding of the molecular pathways underlying disease progression, tumor recurrence and response to therapy might be of great importance in future development of more efficient therapies in GBM. Local infiltration and progressive growth of GBM tumor cells into the adjacent brain tissue invalidate any possibility for a radical surgical tumor resection.9 Consequently, recurrence is observed in almost all tumors.7 Additionally, the difficulty in treatment of this lethal disease is in tumor acquisition of several mechanisms of radio and chemo- resistance. In addition to methylation of MGMT10, a change in miRNA's expression is also a cause for drug resistance in GBM.11 Several miRNAs have been related to radio- and chemo-sensitivity as well as radio- and chemo-resistance of glioma cells, and recognized as crucial regulators during glioma pathogenesis and progression.12 However, a certain degree of this aberrant regulation may also be the consequence of treatment. In aim to characterize at least some miRNAs that might be involved in GBM formation/progression and recurrence, and may also contribute to therapy resistance, we selected 83 consecutive patients with operable recurrent GBM, where samples from first and from second surgery were available. We selected ten miRNAs, which expression was up-regulated (miR-9, miR-15b, miR-21, miR-26b, let-7a, let-7b, let-7d and let-7f) or down-regulated (miR-7 and miR-124a) in previous studies of GBM, and were differentially expressed from other glioma subtypes.13 We additionally included miR-199a, which is involved in tumorigenesis of various types of cancer, including GBM.14 The majority of these miRNAs have already been shown to be involved in different tumorigenic processes in GBM or glioma cell lines (Table 1).415 We hypothesized that: (i) selected miRNAs are deregulated in primary as well as in recurrent GBM; (ii) some miRNAs are also differently expressed between primary and recurrent tumor; (iii) expression of selected miRNAs in recurrent GBM is therapy dependent; (iv) progression free survival and overall survival is different between distinct therapies; (v) expression of some miRNAs is related to progression free survival and overall survival; and TABLE 1. List of the analysed miRNAs with known functions in glioma pathogenesis MicroRNAs Invasion Proliferation Tu growth Migration Cell cycle Survival/ cell death Other functions miR-7 + + + nd + + Differentiation, Vasculogenic mimicry miR-9 nd + nd + nd nd Self-renewal, Vasculogenic mimicry miR-15b + + + nd + + Angiogenesis miR-21 + + + + + + / miR-26b + + + + nd + Vasculogenic mimicry miR-124 + + + + + + Differentiation, Angiogenesis miR-199a nd + nd nd nd nd / let-7a nd + + + nd nd / let-7b + nd nd + nd nd / let-7d nd nd nd nd nd nd / let-7f + + nd + nd nd / + = involved in tumorigenic process in glioma; nd = not determined yet as involved in tumorigenic process; Tu = tumor; reviewed from Karsy et al.4, Lages et al.13, Silber et al.5, Visani et al.40, Zhang et al.6 Radiol Oncol 2018; 52(4): 422-432. 150 Matos B et al. / miRNAs and their expression in glioblastoma TABLE 2. Demographic and clinicopathological characteristics of patients included in the study Histopathological diagnosis GBM, WHO grade IV Number of cases 83 Mean age (Year) 50 ± 12.8 (min 8, max 71) Male Gender Female 50 (60%) 33 (40%) Karnofsky performance status > 80 83 trieved from the archive of the Institute of Pathology, Faculty of Medicine, University of Ljubljana. Samples were classified in two groups, i.e. primary GBM (sample tissue collected at first surgery) and corresponding recurrent GBM (sample tissue collected at second surgery). As control sample RNA FirstChoice Human Brain Reference Total RNA (Cat. no. 6050, Ambion; Invitrogen, USA) was used. The patients' data, including age, gender and therapy are summarized in Table 2. radiotherapy after first surgery 83/83 (100%) Adjuvant treatment chemotherapy after first surgery radiotherapy after second surgery 54/83 (65%) 15/83 (18%) chemotherapy after second surgery 47/83 (56%) MGMT methylated (tested in a subset of patients treated with TMZ) Primary GBM 38/47 (81%) Recurrent GBM 46/47 (98%) Time to recurrence (months) 0.84-67.30 Survival after second surgery (months) 0.03-25.75 Overall survival (months) 0.84-70.48 MGMT = methyl guanine methyl transferase; TMZ = temozolamide (vi) methylation status of MGMT is in correlation to certain miRNAs. Patients and methods Patients and tissue samples Our retrospective study included eighty-three patients with recurrent GBM. The data were obtained from the patients charts from hospital registry of the Institute of Oncology and from the Cancer Registry of Slovenia. Each patient underwent the first surgery and the second one after the first recurrence of GBM between January 1997 and November 2011 at the University Clinical Centre Ljubljana, Slovenia. The selected patients were consecutive; of the 1117 that underwent the first surgery for GBM, 83 underwent also the second surgeryat tumor recurrence since these patients were capable of re-operation according to clinical standards (Karnofsky performance status over 80). Patients received radiotherapy (RT) after the surgery, and RT with concomitant and adjuvant chemotherapy (ChT) after 2005. Tissue samples were fixed in 10% buffered formalin and embedded in paraffin (FFPE) for routine diagnostics. The grading criteria based on WHO classification was used.14 Paired tissue samples were re- RNA isolation from FFPE tissue samples Tissue samples were cut at 10 |om from FFPE tissue blocks and for the isolation procedure six to eight 10 |jm sections were used. Total RNA isolation was performed using miRNeasy FFPE Kit (Qiagen, Germany) according to the manufacturer's protocol. The RNA was eluted in 30 |jl of nuclease-free water. The yield was measured spectropho-tometrically using the NanoDrop-1000 (Thermo Scientific, USA) and the quality was evaluated on the Bioanalyzer 2100 (Agilent Technologies, USA). Quantitative real-time PCR (qPCR) All the reagents were from Qiagen, except where otherwise indicated. Quantitative PCR (qPCR) was carried out using the Rotor Gene-Q Real-Time PCR System and all the qPCR reactions were performed in duplicates or triplicates. Prior to qPCR analysis, two pools of RNA samples were created from FFPE tissue samples (primary, recurrence) and, along with reference RNA, tested for qPCR efficiency. Reverse transcription using miScript reverse transcription kit was performed in a 10 |jl reaction master mix with 50 ng of total RNA according to manufacturer instruction. The resulting cDNA was diluted 100-fold and qPCR reaction was carried out in 10 |jl PCR master mix, according to manufacturer instruction. As the reference genes, RNU6B and SNORD25 were used based on efficiency results. Tested miRNAs were: miR-7, miR-9, miR-15b, miR-21, miR-26b, miR-124a, miR-199a, let-7a, let-7b, let-7d, and let-7f. DNA isolation In a subset of patients that were treated with temo-zolamide (TMZ) (n = 47), MGMT methylation status was determined. Tissue samples from the first surgery and the surgery at first recurrence were cut at 10 |jm from FFPE tissue blocks and for the isolation procedure, six to eight 10 |jm sections were Radiol Oncol 2018; 52(4): 422-432. 151 Matos B et al. / miRNAs and their expression in glioblastoma Tissue samples _I_ Newly diagnosed GBM Treatment with radiotherapy Recurrent GBM Treatment with radiotherapy t 3 3 Î Treatment with radio- and chemotherapy Treatment with radio- and chemotherapy FIGURE 1. Schematic representation of the workflow and statistical comparisons. GBM = glioblastoma; Reference RNA = Human Brain Reference RNA; 1 = Willcoxon Signed Rank Test between primary glioblastoma and corresponding recurrent glioblastoma using ACt; 2 = normalization to Human Brain reference RNA resulting in AACt; 3 and 4 = Mann-Whitney test between independent groups of samples using AACt (normalization of glioblastoma samples to Human Brain Reference RNA) lyzed the GBM samples was blinded to all clinical information. Statistical analysis To present a relative gene expression the 2-AACl method was used.17 For paired samples (primary, recurrent), the calculated ACt was tested for statistical significance using Wilcoxon Signed Rank test. For independent group of samples, AACt was calculated relatively to Human brain Reference RNA and used for Mann-Whitney test (treatment, MGMT status, etc.). Kaplan-Meier curve was used to analyze difference in progression free survival (time to recurrence) and overall survival (different treatment options, miRNA expression). Correlations between scale variables (miRNA expression, survival) were calculated using Pearson's and Spearman's correlation coefficient. For all statistical tests, the SPSS analytical software (ver.24 SPSS Inc. IL) was used (cut-off point at p < 0.05). The workflow of comparisons is summarized in Figure 1. 1 1 used. Total DNA isolation was performed using QIAamp DNA FFPE Tissue Kit (Qiagen) according to the manufacturer's protocol. The DNA was elut-ed in 60 |jl of nuclease-free water. The yield was measured fluorescently using the Quant-It (Life Technologies) according to manufacturer instruction and Rotor Gene Q (Qiagen). MGMT methylation status analysis For determining the MGMT methylation status, methyl-specific polymerase chain reaction (MSP) was used in a two-step approach with primers previously described.16 Briefly, prior to MSP, 500 ng of DNA was used for bisulfite conversion using innuCONVERT Bisulfite Basic Kit according to manufacturer instruction (Analytik Jena) and stored at -20°C for subsequent MSP. For MSP, 15 ng of bisulfite converted DNA was used with 0.2 |jM of each primer for methylated form and 0.3 |jM of each primer for unmethylated form, 2 mM of dNTP and 0.25 U of Hot Master Polymerase (5 Prime), all in 10 |jl reaction. Amplification was performed according to manufacturer instruction using 59°C for primer annealing. In each run, fully methylated (EpiTect Control DNA, methylated, Qiagen) as well as fully unmethylated controls (EpiTect Control DNA, unmethlyated, Qiagen) were used as assay controls. Results were analyzed using 2% agarose gel. The investigator who ana- Compliance with ethical standards Study has been approved by Slovene National Medical Ethics Committee (Number 113/05/13). Results Expression of miRNAs is changed in primary and recurrent GBM Comparing expression of analyzed miRNAs to Human Brain Reference RNA (AACt), we found altered expression for all 11 analyzed miRNAs both in primary as well as in recurrent GBM. Whereas miR-9 and miR-21 were mainly up-regulated and miR-15b was mainly down-regulated in primary and recurrent GBM, all other miRNAs showed more heterogeneous expression when compared to the Human Brain Reference RNA. Results are summarized in Figure 2. miRNAs in recurrent GBM are differentially expressed compared to primary GBM Using calculated ACt and Wilcoxon Signed Rank test, we have observed significant alteration in expression for 7 miRNAs between primary and recurrent GBM. Five miRNAs, miR-9 (p = 0.016), miR-21 (p < 0.001), miR-26b (p < 0.001), miR-124 (p Radiol Oncol 2018; 52(4): 422-432. 426 Matos B et al. / miRNAs and their expression in glioblastoma miR-9 expression miR-9 A miR-9 B miR-7 A miR-7 B 4 3 2 1 0 s -1 3 -2 -3 -4 -5 -6 miR-15b expression imiR-isb a miR-15b B let-7a expression ■ let-7a A ■ l t-7a B 10 9 8 7 6 5 3 4 3 2 1 0 miR-21 expression miR-21 A ■ miR-21 B let-7b expression ■ let-7b A ■ let-7b B miR-26b expression «miR-26b a miR-26b B let-7d expression ■ let-7d A ■ let-7d B 10 8 6 4 2 0 -2 -4 -6 miR-124a expression BmiR-i24a a ■ miR-124a B let-7f expression ■ let-7d A ■ let-7d B 10 8 6 4 2 0 -2 -4 miR-199a expression imiR-199a a miR-199aB Surgical resection + RT Recurrent Tu Surgical resection + RT + ChT Recurrent Tu FIGURE 2. Expression of miRNAs in primary and recurrent GBM (compared to the Human Brain Reference RNA, AACt) with Table presenting Average Fold Changes. ChT = chemotherapy; RT = radiotherapy; A = primary GBM; B = recurrent GBM Radiol Oncol 2018; 52(4): 422-432. -3 -5 153 Matos B et al. / miRNAs and their expression in glioblastoma = 0.029) and miR-199a (p < 0.001), were down-regulated, and miR-7 (p = 0.001) and let-7f (p < 0.001) were up-regulated in recurrent tumors compared to first time surgery. However, all of these miRNAs were differentially expressed also in a subgroup treated with RT and ChT, whereas only four of these showed statistically significant differential expression in a subgroup treated with RT alone. Results are summarized in Figure 3 and Figure 2 (Average Fold Change Table). Distinctive expression of miRNAs after different treatment options in recurrent GBM Using Mann-Whitney test for comparing subgroup of patients treated with RT alone to patients treated with RT and ChT (analyzed miRNAs normalized to Human Brain Reference RNA, AACt), we observed that expression of certain miRNAs was different before treatment (at first surgery) as well as after treatment (at second surgery). Therefore, only those miRNAs that showed significantly differential expression only after treatment (at second surgery) were considered truly as a consequence of treatment. Both miR-15b and miR-7d were down-regulated compared to Human Brain Reference RNA after treatment, and there was significant change in expression between patients that received RT alone and those that received RT and ChT (p = 0.013 for miR-15b and p = 0.008 for let-7d). In patients treated with RT alone, let-7f was up-regulated compared to Human Brain Reference RNA at second surgery, whereas it was unchanged in patients that were treated with RT and ChT. There was significant change in expression of let-7f between both groups (p = 0.007). Results are summarized in Figure 4. Correlation between progression free survival, survival after second surgery, overall survival, treatment and miRNA expression There was weak, but statistically significant change in progression free survival between patients who received RT alone and those who received both, RT and ChT after first surgery (7 months and 11 months, respectively; p = 0.045). Results are summarized in Figure 5A. We have also calculated the survival time from second surgery. The number of patients who received RT alone or RT and ChT was too small for reliable analysis of survival time. However, there 4 2 0