|Year : 2020 | Volume
| Issue : 1 | Page : 12-18
Mitochondrial DNA content in tumor tissue and blood of patients with glioblastoma – A reliable biomarker?
Palavalasa Sravya1, AS Uday Krishna2, Vani Santosh3, Arivazhagan Arimappamagan4
1 Department of Clinical Neurosciences, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
2 Department of Radiation Oncology, Kidwai Memorial Institute of Oncology, Bengaluru, Karnataka, India
3 Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
4 Neurosurgery, National Institute of Mental Health and Neurosciences, Bengaluru, Karnataka, India
|Date of Submission||14-May-2020|
|Date of Acceptance||04-Jun-2020|
|Date of Web Publication||2-Jul-2020|
Dr. Arivazhagan Arimappamagan
Department of Neurosurgery, National Institute of Mental Health and Neurosciences, Hosur Road, Bengaluru, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: Recent studies have shown that mitochondrial DNA (mtDNA) content in the tumor tissue is associated with patient survival in glioblastoma (GBM). However, mtDNA content in the blood of patients with GBM and its variation during treatment has not been documented. Hence, we assessed mtDNA content in blood and tumor tissue of GBM patients, its association with survival, and the variation of mtDNA content in blood with treatment.
Materials and Methods: GBM tissue obtained from 20 patients, 2 ml blood collected from the same patients at the time of presentation and following radiation therapy (RT), 2 ml blood obtained from 20 healthy volunteers were assessed for mtDNA content using quantitative real-time polymerase chain reaction (comparative ct method). The mtDNA content is expressed as the mtDNA/nuclear gene ratio.
Results: The mean mtDNA copy number in the GBM tissue was 492.3819, while in the blood of patients, it was 41.76 ± 19.45. In healthy volunteers' blood, it was 55.97 ± 5.78 (P = 0.04). The median overall survival was 13 months in patients with high tumor mtDNA content and 10 months in patients with low tumor mtDNA content (P = 0.04). Patients with low-mtDNA content in the blood had a relatively higher content in the tumor tissue (correlation coefficient − 0.26; P = 0.27). No significant difference was noted between mtDNA content in preoperative blood (41.76 ± 19.45) and post-RT blood sample (44.16 ± 12.83) (P = 0.6).
Conclusion: High GBM tumor mtDNA copy number is associated with better overall survival. mtDNA content in the blood is lower in GBM patients when compared with healthy volunteers. mtDNA content in the blood of the patients did not vary following surgery and RT.
Keywords: Blood biomarker, glioblastoma, mitochondrial DNA content, radiation therapy
|How to cite this article:|
Sravya P, Uday Krishna A S, Santosh V, Arimappamagan A. Mitochondrial DNA content in tumor tissue and blood of patients with glioblastoma – A reliable biomarker?. Int J Neurooncol 2020;3:12-8
| Introduction|| |
Glioblastoma (GBM) is an aggressive disease with a poor prognosis. The currently established prognostic biomarkers belong to the nuclear genome of the tumor tissue. The current standard of care in these patients is maximal safe resection of the tumor followed by adjuvant radiation and chemotherapy, as per Stupp's regime. The response to radiochemotherapy in GBM is assessed mainly through neuroimaging, which can be greatly influenced by factors such as steroid intake, radiotherapy, and chemotherapy itself and, more recently, by immunotherapy (treatment effect). In this scenario, a simultaneous search for a blood-based biomarker that can signify treatment response and evaluate nonresponders could be of use. A novel biomarker, i.e., the mitochondrial DNA (mtDNA) content has been gaining attention in GBM recently. While some studies have evaluated mtDNA in tumor tissue and correlated it with the prognosis,, simultaneous interest to assess mtDNA in blood also has been generated. It was found to be low in GBM tumor tissue, and higher mtDNA content is associated with better survival in these patients., Very few studies have evaluated the levels of mtDNA in patients with glioma. Prior studies have generated contradicting reports regarding the mtDNA content in the blood of glioma patients. Two studies reported that mtDNA content is high in glioma (all grades) when compared to healthy controls., However, they differed in grade-specific results, where one study showed that the mtDNA content in blood increased with grade of glioma, while the other study showed that GBM blood mtDNA content was lower than Grade II and Grade III gliomas together. Yet another study showed that mtDNA content was lower than the healthy calibrator DNA. This study further showed that high mtDNA content in blood is associated with poor prognosis in glioma. However, these studies have not compared mtDNA content in the blood of the patients with that in the tumor. Moreover, GBM constituted only approximately one-third of the patient cohort in these studies. Furthermore, the changes in mtDNA content in the blood of the patients during treatment have not been studied so far. The present study aims to add to the scarce knowledge regarding the mtDNA status in blood of patients with glioma and attempted to decipher whether the levels vary after therapy. Hence, we evaluated the survival significance of mtDNA content in GBM tumor tissue, whether mtDNA content in the blood of GBM patients recapitulates that in the tumor and whether intensity-modulated radiation therapy (IMRT) to the brain along with concomitant temozolomide (TMZ) affects the mtDNA content in the blood.
| Materials and Methods|| |
The prospective observational study involved 20 adult patients with lobar high-grade glioma who underwent surgery at our institute in the year 2017 and histologically diagnosed to have GBM. The study was approved by the Institute Ethics Committee. The patients were recruited for the study after obtaining written informed consent. Clinical details were collected. All patients underwent treatment for the tumor as per the standard of care. Tumor tissue resected during surgery was archived as formalin-fixed paraffin-embedded (FFPE) blocks. Patients were included in the study only if the histopathology was GBM. Two millilitre blood in ethylenediaminetetraacetic acid (EDTA) coated container was collected from these patients at two time points, i.e., preoperatively and postradiation therapy (RT) with concomitant TMZ. All patients underwent maximal safe resection followed by IMRT (60 Gy in 30 fractions or 59.4 Gy in 33 fractions) along with concomitant TMZ. The patients were followed up till November 2019 or until death.
Following written informed consent, 2 ml blood from 20 healthy volunteers was collected in EDTA-coated containers. The controls without a history of any form of cancer, epilepsy, genetic syndromes, diabetes mellitus, or other metabolic disease were recruited. Controls age was frequency matched with the age of the patients (<50 and 50).
Genomic DNA from tumor tissue was isolated from FFPE tissue using Qiagen QIAamp DNA FFPE tissue kit (cat no. 56404). For DNA isolation from blood, Macherey-Nagel NucleoSpin blood kit (cat no. 740951.10) was utilized. Briefly, the tissue was deparaffinized and subjected to lysis. The blood sample was directly subjected to lysis. The DNA from the lysate was then purified using the column method.
Mitochondrial DNA content assessment
Isolated DNA was utilized for mtDNA content assessment using quantitative real-time polymerase chain reaction. mtDNA content was assessed in GBM tumor tissue and blood of the corresponding patients. The procedure was performed using 2 Taqman probes, one specific for mitochondrial ND1 and the other specific for nuclear RNAseP gene used as the endogenous control. The mtDNA content was normalized to RNAse P (endogenous control). mtDNA content was calculated as the mtDNA/nuclear gene ratio (mtND1/RNAse P). [Table 1] shows the primer and probe sequences utilized in the study.
|Table 1: Primer and probe sequences for mitochondrial DNA copy number assay|
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The data were tested for normality, which demonstrated a nonnormal distribution. Kaplan–Meier survival analysis was used to study the survival significance of mtDNA content in blood and GBM tissue. Man–Whitney U-test was used to compare mtDNA content in the blood of GBM patients with that of healthy control blood. Wilcoxon signed-rank test was performed for paired sample analysis of mtDNA content in tumor tissue, preoperative blood and post-RT blood. Spearman correlation was performed to study the correlation between mtDNA content in blood versus tumor tissue.
| Results|| |
Clinical characteristics of the cohort
The clinical characteristics of the patients are summarized in [Table 2]. The mean age of the patients was 52.45 ± 8.6 years (range: 34–72 years). The cohort consisted of 11 male and 9 female patients. The control population comprised 13 males and 7 females. The mean age of controls was 49 ± 10.8 years (range: 25–60 years). Age did not correlate significantly with mtDNA content in the blood of the patients as well as controls. We noted no difference in mtDNA content between male and female patients. The median overall survival in the cohort was 13 months.
When stratified into high and low tumor mtDNA content (based on the median mtDNA/nuclear gene ratio of 451.195), the patients with high tumor mtDNA content had a higher median overall survival (20 months vs. 9 months; P = 0.04). When stratified into high and low preoperative blood mtDNA content (using median mtDNA/nuclear gene ratio of 47.10 as cut off), the lower blood mtDNA content group had a higher median overall, though not statistically significant (13 months vs. 10 months; P = 0.91). However, the small number of patients in this cohort limits the strength of these observations [Figure 1].
|Figure 1: Kaplan–Meier survival curves assessing prognostic significance of mitochondrial DNA content in tumor tissue and blood of patients with glioblastoma. (a) Overall survival in patients with low and high mitochondrial DNA content in tumor tissue. (b) Overall survival in patients with low and high mitochondrial DNA content in blood|
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Blood mitochondrial DNA content in glioblastoma patients versus healthy controls
We compared the mtDNA content in the blood of patients with that of the healthy volunteer blood. Interestingly, mtDNA content was found to be lower in the blood of patients when compared to healthy controls (P = 0.04) [Figure 2]. The mean mtDNA/nuclear gene ratio in the blood of patients was 41.76 ± 19.45. In healthy control blood, the ratio was 55.97 ± 5.78. We also noted that while there is a larger range of mtDNA content in blood of GBM patients, the mtDNA content in healthy controls had a narrow range, with most values around the median.
|Figure 2: Graphical representation of mitochondrial DNA content in blood of glioblastoma patients compared with that in healthy volunteers|
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Correlation between mitochondrial DNA content in blood and tumor tissue
Since blood and tumor tissue mtDNA content cannot be compared as a percentage of normal, the mtDNA/nuclear gene ratio is depicted for comparison. mtDNA/nuclear gene ratio in the blood of patients was 41.76 ± 19.45, while in the tumor tissue, it was 470 ± 206.8 (P< 0.001). The inter-patient variation of mtDNA content in tumor tissue was not reflected in the blood. The correlation coefficient was − 0.26 (P = 0.27) [Figure 3] showing that patients with higher mtDNA content in tumor tissue had lower mtDNA content in blood, though not statistically significant.
|Figure 3: Graphical representation of mitochondrial DNA content in blood and tumor tissue of glioblastoma patients. (a) The correlation between the mitochondrial DNA content between blood and tumor tissue of the patients. (b) Compares the median mitochondrial DNA copy number in blood and the median mitochondrial DNA content in the tumor tissues of the patients|
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Variation in mitochondrial DNA content in blood after radiation therapy with concomitant temozolomide therapy
We then evaluated the variation in mtDNA content values during therapy. Blood samples were collected preoperatively and after the completion of radiotherapy and concomitant chemotherapy with TMZ. We noted that the mean mtDNA/nuclear gene ratio in preoperative blood sample was 41.76 ± 19.45, and the post-RT mean ratio was 44.16 ± 12.83 (P = 0.6). Hence, no significant difference was observed in mtDNA content in blood at two-time points, namely preoperative and post-RT [Figure 4].
|Figure 4: Graphical representation of mitochondrial DNA content in blood of glioblastoma patients before and after surgery followed by radiation therapy|
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| Discussion|| |
mtDNA is a relatively unexplored part of the genome in cancer and more so in brain cancer. Studies in the last decade have attempted to study mtDNA, particularly the content of mtDNA, and elucidate its potential involvement in glioma pathogenesis. Reznik et al. have shown that low mtDNA content in glioma tissue is associated with poor survival. Correia et al. and Zhang et al. have shown that mtDNA content is lower than normal in gliomas as that it is lower in the higher grade gliomas., The study reiterates this finding in GBM tumor tissue. Dardaud et al. have recently shown that high mtDNA copy number is associated with better survival in young patients with GBM. Our study also indicated that a high mtDNA copy number in the tumor tissue is associated with better overall survival in GBM.
While more research in tumor tissue is warranted and rightfully pursued, more accessible and less invasive biological markers have always been sought after in the field of oncology. Considerable interest has resulted in blood/serum-based biomarkers in many cancers such as prostate cancer, and germ cell tumors. The components of liquid biopsy, namely, cell-free tumor DNA, cell-free RNA, tumor educated platelets, and proteins have been studied in several cancers, and several promising biomarkers were identified.,,, Therefore, it is pertinent to understand the status of expression of any potential biomarker in blood and how this information can be leveraged for advancing knowledge and in clinical practice. Therefore, while studies focus on mtDNA in tumor tissue, a simultaneous quest to evaluate its expression in blood can provide a multidimensional understanding of the scenario.
mtDNA content in blood has been assessed in very few cancers, and only three studies have evaluated the mtDNA in the blood of patients with glioma, with conflicting results. The studies differed in the fraction of blood studied for mtDNA content assessment. One study involved whole blood  while two others studied the leukocyte mtDNA content., In other cancers, high leukocyte mtDNA content was associated with an increased risk of thyroid cancer, melanoma, prostate cancer. However, the prognostic significance of mtDNA content in blood of these patients was not well studied. In GBM, there is very limited evidence pertaining to mtDNA content in blood, its correlation with tumor tissue mtDNA content, its survival significance and its variation with treatment.
Our study, for the first time, shows that mtDNA content in the blood of GBM patients is lower than that in their tumor tissue. However, the blood mtDNA content was not reflective of that in the tumor tissue in each patient. Our rationale behind assessing mtDNA content in blood was that since GBM tumor blood vessels are leaky, mtDNA might also escape into the bloodstream and be reflective of the mtDNA content in the tumor tissue. However, as some studies showed, anthropometric measures, factors like smoking and other lifestyle differences  might influence the mtDNA content. Moreover, we had assessed whole blood mtDNA content, which evaluates both cell-free mtDNA as well as leukocyte mtDNA together. Cell-free mtDNA and leucocyte mtDNA content may differ, as was described in major depressive disorder  and cell-free mtDNA content may be a more appropriate marker to study in future. Furthermore, the sample size assessed in this study is very small to arrive at a conclusion. Previous studies have either assessed the mtDNA content in the blood ,, or in the tumor tissue., These studies have revealed the prognostic significance of mtDNA content in blood or tumor tissue in different cohorts. However, these studies could not correlate both parameters in the same cohort. We studied the mtDNA content in both blood and tumor tissue in each patient. We noted that higher mtDNA content in tumor tissue is associated significantly with better overall survival (P = 0.04), which is in line with previous studies. However, contrary to the existing scarce literature, we found no significant association between mtDNA content in blood and patient survival.
Another significant finding of our study is that the mtDNA copy number in the blood of the patients with GBM is lower than in healthy subjects. The mtDNA content in the blood of healthy individuals is known to differ from that of several disease conditions. Even in normal physiological conditions, mtDNA copy number in blood is found to vary. Cell-free mtDNA has gained popularity in recent years, and it was found to vary with age, anthropometric measurements such as weight, body mass index, waist-to-hip ratio, psychiatric disorders such as depression, inflammation  and so on. One study showed that peripheral blood mtDNA content increases during severe intra-abdominal infection after trauma. Therefore, several factors apart from disease states influence the mtDNA content in blood. However, one recent study which evaluated a large cohort of healthy controls versus metastatic colon cancer patients inferred from the results that while there was variation in the healthy subjects, there was a significant difference in cell-free mtDNA content between cancer and healthy subjects where cancer patients showed much lower cell-free mtDNA content in the blood than the healthy subjects.
Another factor that can influence mtDNA content in the blood is treatment. Some studies evaluated the changes in blood mtDNA content after cardiac surgery , and orthopedic surgery. These studies have demonstrated a general increase in mtDNA content in the immediate postoperative period. One study further showed that the elevation of mtDNA content lasts for about 5 days and persistent elevation of mtDNA content suggests the inflammatory process. However, very little information exists regarding the mtDNA content during the course of disease. The possible effect of adjuvant therapy on mtDNA content in the blood of GBM patients was hitherto not studied. We evaluated if the value varies at two time points and show for the first time, that IMRT (60 Gy in 30 fractions or 59.4 Gy in 33 fractions) to the tumor bed along with concomitant TMZ, does not affect the mtDNA content in the blood. The rationale behind this aspect of the study was that TMZ is administered orally to the patients, and hence, we surmised that it might influence mtDNA in blood, which is prone to changes in response to several factors. However, this information needs to be understood with its limitations. The time interval required for a change of mtDNA content to occur in blood following an influence/stimulus is not clear. Furthermore, we did not measure the values in the postoperative period before initiation of RT for logistic reasons. An ideal blood-based biomarker should be able to accurately reflect the tumor tissue load in the body as well as response to therapy and development of recurrence. A more elaborate study of mtDNA content in different fractions of the blood at multiple time points and correlation with the radiological tumor can conclusively answer this question, though the present findings are not supportive of its possible candidacy for such approach.
The principal limitation of our study is that the sample size is small to evaluate the statistical significance of our findings. However, this study is a pilot study, and the results are encouraging and suggest that mtDNA content in tumor tissue can be a reliable prognostic biomarker. We have also shown that mtDNA content in blood needs to be studied at multiple time points during treatment in a large cohort.
| Conclusion|| |
Thus, our study reiterates the prognostic significance of mtDNA content in GBM tissue and adds to the scarce literature on mtDNA content in blood of GBM patients. We demonstrate that mtDNA content in blood is lower in GBM patients than in healthy controls. For the first time, we show that mean mtDNA content in blood is lower than the tumor tissue in GBM, and the blood mtDNA content does not vary after surgery and IMRT with concomitant TMZ therapy.
The authors would like to acknowledge the technicians at the neurooncology lab, NIMHANS, Mr. Chandrashekhar, and Mr. Suresh, for their participation in the collection of tissue and blood samples.
Financial support and sponsorship
The work presented in this paper was funded by TVS student bursary (by T S Srinivasan foundation, India) awarded to Dr. Palavalasa Sravya (TVSB/007/306/2017/01037).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]