Mitochondrial Deoxyribonucleic Acid Copy Number Elevation As a Predictor for Extended Survival and Favorable Outcomes in High-Grade Brain Tumor Patients: A Malaysian Study

Background: Investigating the role of mitochondrial DNA (mtDNA) alterations and their impact on brain tumor progression remains a significant focus in cancer research. The research aimed to explore the specific contributions of mtDNA copy number changes and their correlations with patient survival, large mtDNA deletions, and TFAM mutations in brain tumor patients. Methods: A total of 41 patients with confirmed brain tumors underwent DNA extraction from both tumor tissues and blood samples. The relative mtDNA copy number in comparison to the nuclear genome was quantified using quantitative real-time polymerase chain reaction (qRT-PCR). Long-range PCR assessed large-scale mtDNA deletions, and Sanger sequencing was applied to detect exon 4 TFAM mutations. Results: Analysis revealed significantly increased mtDNA copy numbers in brain tumor tissues (80.5%) compared to matched blood samples (P < .001). Median delta Ct (∆Ct) values were 7.35 in cancerous tissues and 11.81 in blood (P < .001), with median relative mtDNA content of 0.0123 and 0.0006, respectively (P < .001). Patients with higher mtDNA copy numbers experienced longer overall survival periods (P = .045) and notably favorable outcomes, particularly in high-grade tumor cases (P = .016). Furthermore, a single-nucleotide deletion was identified in exon 4 of TFAM in a patient with glioblastoma IV, while no large-scale mtDNA deletions were found in brain tumor patients. Conclusion: Our study strongly supports the role of increased mtDNA copy numbers as a reliable predictor for improved survival and positive outcomes in high-grade brain tumor patients.


Introduction
Brain tumors rank among the deadliest human malignancies, causing substantial morbidity and mortality worldwide.The Global Cancer Observatory report estimated a global incidence of 308 102 new cases and 251 239 deaths from brain tumors in 2020. 1 Various cellular and molecular research methods highlight the molecular diversity of brain tumors, arising from the progressive gathering of genetic mutations and epigenetic alterations. 2et, comprehending the origins of both malignant and non-malignant brain tumors, especially regarding the genetic aspects of the mitochondrial genome, remains a challenging endeavor.
Renowned for their vital involvement in cellular energy production and contribution to reactive oxygen species (ROS), mitochondria play a crucial role in maintaining metabolic homeostasis and regulating the process of cell death.Possessing their own genome, mitochondria are believed to have evolved independently from nuclear DNA.The suspicion of mitochondrial dysfunctions playing a role in boosting tumor growth and facilitating cancer progression has persisted for an extended period.
the mechanisms governing abnormal mtDNA content in cancer progression are currently limited and lack clarity.Alterations in mtDNA copy number are thought to play a role in fostering genomic instability, thereby enhancing the advancement of tumors.Up to this point, both an augmentation and reduction in mtDNA copy number have been identified as potential risk factors for cancer in human studies. 4gnificantly, the regulation of mtDNA replication influences the quantity of mtDNA copies, a process mediated by distinct enzymes and proteins, such as mitochondrial transcription factor A (TFAM).TFAM, a transcription factor encoded in the nucleus and functioning within mitochondria, holds a pivotal position in both mtDNA replication and the intricate process of mitochondrial biogenesis. 5Supported findings suggest that TFAM has a strong correlation with the abundance of mtDNA copies and plays a crucial role in regulating mtDNA expression. 5ells depleted of mtDNA exhibit diminished mitochondrial function, lowering TFAM protein expression in both the liver and skeletal muscle. 6Furthermore, cells with TFAM knockdown demonstrate a decrease in mtDNA copy numbers, as observed in esophageal squamous cell carcinoma. 7terations or deletions within mtDNA sequences have the potential to induce irregularities in mitochondrial copy numbers.In these scenarios, defects in mtDNA can lead to deletions by causing double-strand DNA breakage. 8Significantly, large-scale mtDNA deletions, often associated with mtDNA content levels, have been commonly observed in the aging process and mitochondrial myopathies and have recently been revealed in various human cancers. 9,10 the present study, we assessed the alterations that occur in the mtDNA genome and the role of mtDNA content in brain tumor patients.The implications of the aberrant mtDNA copy number in brain tumors demand novel observations, specifically in distinct tumor types and grades.Hence, this research aimed to investigate alterations in mtDNA and variations in copy numbers among Malaysian patients diagnosed with brain tumors, aiming to explore their connections with survival rates and other clinical characteristics.

Case Population
The study involved 41 brain tumor tissues obtained from the Department of Neurosciences, Universiti Sains Malaysia, between 2019 and 2023.To serve as a comparison, peripheral blood samples were collected from the same patients, serving as the control group.These samples specifically comprised histologically confirmed primary brain tumors, classified by neuropathologists following World Health Organization (WHO) guidelines.Additionally, individuals who had undergone prior neoadjuvant therapy and cases of tumor recurrence were excluded from this study.This study followed the principles outlined in the Declaration of Helsinki, and written informed consent was obtained from all participants.Ethical approval for the study was granted by the Research Ethics Committee of Universiti Sains Malaysia (approval no: USM/JEPem/17050269, Date: 26th July 2019).

DNA Extraction
DNA extraction was carried out using the Geneaid Isolation Kit (Geneaid Biotech Ltd., New Taipei City, Taiwan) following the manufacturer' s protocols.The concentration and quality of the isolated DNA were assessed using the NanoDrop ND1000 spectrophotometer and 1% gel agarose electrophoresis.All eligible DNA samples were preserved at −80°C until further analysis.

mtDNA Content Analysis
The relative mtDNA copy number was assessed via quantitative polymerase chain reaction (qPCR) by concurrently amplifying mtDNA (ND1 gene) and nuclear DNA (β-actin gene) using 2× Brilliant III SYBR Green Master Mix with Low Rox on the Mx3005P Real-time PCR System (Agilent Technologies, Inc., Santa Clara, Calif, USA) for all available samples.Primers for ND1 were 5'-TC TCACC ATCGC TCTTC TAC-3 ' as the forward primer and 5'-TT GGTCT CTGCT AGTGT GGA-3 ' as the reverse primer.For the β-actin gene, 5'-CA TGTGC AAGGC CGGCT TCG-3 ' was used as the forward primer and 5'-CT GGGTC ATCTT CTCGC GGT-3 ' as the reverse primer.The cycling conditions were 95°C for 3 minutes, followed by 40 cycles of 15 seconds at 95°C and 20 seconds at 60°C.The samples were run in triplicates, and the average threshold cycle number (Ct) values for both genes were used to determine the relative mtDNA content.The determination of mtDNA content was measured using the formula 2 × 2−∆ CT (CtmtDNA -CtnDNA) and 2−∆∆ CT method.

Long-Range Polymerase Chain Reaction for Large-Scale mtDNA Deletions Analysis
The process of detecting mtDNA large-scale deletions was conducted following similar protocols used previously, albeit with slight modifications. 10Two divided fragments of the entire mtDNA, which are 7.8 kb and 9.3 kb, were performed by long-range PCR.For the 7.8 kb amplification, 30 cycles were used with 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 3 minutes 30 seconds, and a final elongation at 72°C for 10 minutes.For the amplification of the 9.3 kb, the cycling conditions used were 30 cycles of 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 5 minutes, with a final elongation at 72°C for 10 minutes.The reaction mixture used for both amplifications was Phusion High Fidelity (ThermoFisher Scientific, Waltham, Mass, USA) on the SureCycler 8800 Thermal Cycler (Agilent Technologies, Inc., Santa Clara, Calif, USA).The products were then checked on a 1% agarose gel in TAE buffer for 55 minutes at 75 volts.

TFAM Mutation Analysis
The amplification of exon 4 of TFAM was performed as stated previously. 2 The PCR was conducted using the SureCycler 8800 Thermal Cycler (Agilent Technologies, Inc., Santa Clara, Calif, USA).A volume of 20 µL total reaction was performed using Phusion High Fidelity (ThermoFisher Scientific) for 30 cycles of 98°C for 10 seconds, 68°C for 30 seconds, and 72°C for 30 seconds, and a final elongation at 72°C for 7 minutes.The PCR products were then purified using QIAamp Purification Kit (QIAGEN, Hilden, Germany).

Sanger Sequencing
The PCR products were purified and subjected to Sanger sequencing using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, Calif, USA) on an ABI Prism 3700 DNA Analyzer automated sequencer (Applied Biosystems, Foster City, Calif, USA).The sequencing of the purified PCR products was executed with the same

Main Points
• Brain tumors are heterogeneous diseases, and the molecular landscape is complex and complicated.
• Diverse molecular alterations in mitochondrial DNA (mtDNA), including point mutations, deletions, insertions, microsatellite instability, polymorphisms, and changes in mtDNA copy number, have been identified and characterized in human brain tumors.
• Brain tumor screening in Malaysian patients reveals an increase in mtDNA copy number.
• The association of the increased mtDNA copy number and the clinicopathological features of brain tumor patients show no significant differences.
• The elevated mtDNA copy number shows higher overall survival in patients and a better prognosis in the high-grade tumor group.
primers used in the PCR amplification process.
Electropherogram results were aligned using BLAST software from the NCBI site, and the complementary DNA sequences for the TFAM gene (NC_000010.11)available in the database were used as references.

Statistical Analysis
The statistical analysis was conducted using Statistical Package for the Social Sciences (SPSS), version 28 (IBM SPSS Corp.; Armonk, NY, USA).
The mtDNA content values are presented as the median, the interquartile range (IQR), and the fold difference.The Mann-Whitney U-test was applied to analyze the relationship between mtDNA content and clinicopathological features among brain tumor patients, and the Pearson correlation coefficient was used to determine the relationship of the mtDNA content between cancerous tissues and blood controls.The chisquare difference test was used to compare the differences between the case groups.Kaplan-Meier survival analysis, using the log-rank test to determine significance, was performed to evaluate the survival of patients based on variations in mitochondrial DNA copy numbers.Survival duration was measured from the initial surgery day to the last clinical follow-up or relapse occurrence.The results were considered statistically significant at a P-value less than .05.

Clinicopathological Features
The clinicopathological characteristics of the patients are summarized in Table 1 In this study, we found no correlation between the average Ct values of MTND1 amplification with the ACTB amplification in cancerous tissues (r = 0.185; P = .248).Conversely, a significant correlation of both amplifications was observed in blood controls (r = 0.327; P = .039)(Figure 1A).

mtDNA Copy Number in Cancerous Tissues and Paired Blood Specimens
The present findings demonstrated that mtDNA content was significantly increased in cancerous brain tumor tissues (33/41) than those in the blood controls (chi-square difference test, P < .001)(Figure 1B).The ∆Ct values between cancerous tissues and blood controls showed a 4.5 cycle difference (∆∆CT) (Mann-Whitney U-test, P < .001),and the fold change of mtDNA content (2−∆∆ CT ) was 22.6 cycles.Additionally, there was a marked difference between relative mtDNA content in cancerous tissues and blood controls (2 × 2−∆ CT ) (Mann-Whitney U-test, P < .001)(Table 2).

Associations of Increased mtDNA Content and Clinicopathological Features in Brain Tumors
The associations between the relative content of mtDNA and other clinical parameters of 41 brain tumor cases, including gender, age at the time of diagnosis, tumor grades, and histological tumor types, were analyzed (Table 3).At the α = 0.05 level of significance, the outcomes show [median male (IQR) = 0.0063 (0.03), median female (IQR) = 0.0186 (0.04); Z = 240.5;P > .05]that there was no significant difference in the median score (P = .379)between males and females.Similarly, the results for the age group also showed no significant difference (P = .In this study, Kaplan-Meier survival analysis was used to compare the overall survival rates between 2 groups based on mtDNA copy number (Figure 1C).The results indicated that patients in the increased mtDNA copy number group showed an average survival of 48 months, whereas those in the decreased mtDNA copy number group had an average survival of approximately 11 months (P = .045).This finding suggests that individuals in the increased mtDNA copy number group experienced significantly longer survival durations in comparison to those in the decreased mtDNA copy number group.
Furthermore, an examination was conducted to analyze the survival outcome concerning the groups categorized by mtDNA copy number and clinical variables.The analysis revealed that high-grade tumor patients with increased mtDNA copy numbers exhibited better survival rates compared to those in the decreased mtDNA copy number group (P = .016)(Figure 1D).However, no significant impact on the survival times of brain tumor patients was found in relation to other clinical parameters such as age, gender, and histological tumor types.

Large-Scale mtDNA Deletion Analysis
Large-scale deletions of the mtDNA genome were considered in the brain tumor patients based on the absence of amplification PCR products at 7.8 kb and 9.3 kb (Figures 2A and  B).The presence of these 2 amplicons indicated no large-scale mtDNA deletions in the samples.
For 41 of the samples examined, the long-range PCR assay failed to detect samples that harbored large-scale mtDNA deletions.

TFAM Mutation
A fragment of exon 4 of TFAM was successfully amplified by specific primers, indicating a single PCR amplicon with the desired product at 200 bp in size.In the present study, TFAM mutation analysis uncovered that only 1 patient (1/41) exhibited nucleotide changes in patients who suffered from GBM IV.According to the electropherogram data, a single deletion of nucleotide A at position 3360 (Lys3360) caused no change in the amino acid sequence was observed (Figure 3).

Discussion
The prevailing consensus acknowledges that changes in mtDNA copy number and malfunctioning mitochondria play crucial roles in cancer advancement, extensively studied across various research. 5,11Despite adequate evidence connecting irregular mtDNA levels to tumor development, the origins and underlying understanding of this anomaly remain uncertain.Therefore, this study aimed to evaluate the functions of altered mtDNA copies, integrating mutation analyses, potentially influencing the invasiveness of brain tumors.
In this study, we assessed the mtDNA copy number in brain tumor tissues and blood samples.The findings revealed that the relative mtDNA content was significantly elevated in 33 out of 41 (80.5%) brain tumor tissues compared to their corresponding blood controls.As far as we know, our recent findings represent the initial observation of mtDNA content across various types and grades of brain tumors, encompassing gliomas, meningiomas, schwannomas, and medulloblastomas.3][14][15] The summary of previous reports of mtDNA content variations in most glioma cases has been documented in Table 4.
Additionally, our findings align with earlier research that noted a greater ratio of mtDNA content in tumor tissues compared to non-tumor tissues in GBM patients. 16Correspondingly, research conducted by Zhang et al. revealed heightened mtDNA copy numbers in glioma patients' tumor cases compared to controls, strongly linked to glioma susceptibility. 17In contrast, studies by Shen H et al 18 and Shen J et al 19 reported a decreased mtDNA copy number in glioma tissues compared to those in corresponding non-tumorous specimens.
The present study demonstrated elevated mtDNA copy numbers per cell ranging from 2 -2.99 to 2 -13.84 in tumorous tissues compared to those in peripheral blood controls, which ranged from 2 -6.48 to 2 -15.57 .Additionally, the average mtDNA content between brain tumorous tissues and blood controls appeared to have statistically significant differences.These outcomes indicate the enhanced sensitivity of the measurement of mtDNA alterations in cancerous tissues compared to blood specimens, providing a reassuring clinical appraisal of tumor progression.A comparable finding was reported when assessing mtDNA copy numbers in exosomes derived from plasma and brain tissue of glioblastoma (GBM) patients.The researchers observed reduced mtDNA content in both exosomes and brain tissues of tumor samples compared to the control group, suggesting that exosome analysis could serve as an alternative method for evaluating mtDNA copy numbers and highlighting the potential of mtDNA copy number as a biomarker for glioblastoma development. 20dditionally, the evaluation of mtDNA content in multiple cancers has shown vast fluctuation, suggesting that mitochondrial copies are not particularly in stringent regulation. 21,22Certainly, the amount of mtDNA copy number may vary depending on tissue types and is mostly present mostly in high-energy cells such as skeletal, cardiac muscles, and brain cells. 3 theory, cancer cells display degradation of mtDNA, characterized by a significant buildup of oxidative stress, suggesting heightened glycolytic activity and insufficient adenosine triphosphate (ATP) production. 23The irregular mtDNA genome may lead to inefficient oxidative phosphorylation, resulting in elevated ROS levels and reduced ATP synthesis rates.As a result, it is hypothesized that elevated mtDNA content represents a compensatory response to mitochondrial respiratory deficiencies and mtDNA damage. 24Earlier research indicated a notable decrease in ATP synthesis within oncocytic thyroid tumors exhibiting elevated mitochondrial numbers and mtDNA content. 25imilarly, a recent study unveiled reduced ATP levels in mice displaying depressive-like behavior, alongside heightened mtDNA copy numbers. 26 this present study, variations in relative mtDNA content between cancerous tissues and blood controls were prominent among female patients and younger patients of age <40.Moreover, increased mtDNA content was more evident in grade I and II and non-glial tissues of brain tumors.However, our results noted that there were no significant associations detected between increased mtDNA copy numbers and those clinicopathological characteristics in cancerous brain tumor tissues.A previously published study also uncovered that there were no statistical differences between increased mtDNA copy number with clinical parameters in patients with glioma cases. 17The inability to clarify these connections could be due to the study' s small sample size across various tumor types, despite the significant difference in mtDNA content between cancerous tissues and blood samples.Thus, the increased sample size is warranted in determining the association between mtDNA content and clinical features in brain tumors.
Considering the significant variations of mtDNA copy number in brain tumor patients, we further examined the associations between the variable mtDNA content and the survival of the patients.Our findings confirmed that higher mtDNA content was linked to longer overall survival compared to lower mtDNA content among the patients.This observation aligns with previous studies that highlighted the correlation between increased mtDNA copy numbers and improved overall survival in GBM patients. 14,15dditionally, our observations revealed that elevated mtDNA content displayed a favorable prognosis in high-grade tumor patients.A prior study similarly emphasized the significance of the increased mtDNA content in the survival of high-grade glioma patients. 13Likewise, an intriguing investigation conducted by Sravya et al 11 unveiled an inverse relationship between low mtDNA content and the survival rates of highgrade tumor cases.Consequently, it was hypothesized that an elevated mtDNA copy number could potentially contribute to the clinical outcomes observed in patients with brain tumors.
In the present finding, we detected a deletion of nucleotide A in TFAM sequences in a GBM patient (1 out of 41).The patient carried a single deletion with no amino acid changes and showed an increased mtDNA content level, indicating that there is no significant impact of those changes in the regulation of mtDNA content.However, a previous study revealed a high level of TFAM truncating mutation in 100% of cell lines and 74.4% of tissue samples of colorectal cancer, and the results also demonstrated mutated TFAM samples with decreased mtDNA content and mitochondrial instability. 5Another salient study found that heterozygous TFAM mutation reduces mtDNA copy number by up to 40% in vivo, while the homozygous mutation is embryonically lethal. 27Recently, reduced TFAM protein expression level was significantly associated with decreased mtDNA content and serves as a poor prognosis variable in non-small cell lung cancer. 28 is acknowledged that driver mutations contributing to tumor advancement are strongly linked to irregularities in mtDNA copy numbers.Previous studies highlighted that the expression of metabolic genes within mtDNA and the occurrence of somatic mutations expedite impaired mitochondrial functions by affecting mtDNA content. 5Nevertheless, it was stated that the alterations of several proteins might not necessarily influence the mtDNA replication process. 29An initial investigation revealed a slower rate of TFAM recovery compared to the mtDNA copy number in cells that had their mtDNA replenished with ethidium bromide, indicating that a higher level of mtDNA content might not rely heavily on increased TFAM levels. 30onetheless, TFAM comprises various functional domains that primarily control mtDNA content by engaging in specific and nonspecific sequence interactions within the mtDNA, ensuring the integrity of mitochondrial respiratory functions.Consequently, post-translational modifications influencing the turnover or stability of the TFAM protein can significantly impact the regulation of mtDNA content. 31 addition, we also examined large-scale deletions of mtDNA by the long-range PCR method that has been done in many functional studies. 10,32Our outcomes uncovered that none of the patients exhibit those deletions and were in line with a previous study by Danda et al 10 which showed no large deletions found in patients with fibromyalgia syndrome.Nonetheless, an earlier study discovered a 12.2% of 8.7 kb deletion and 2.2% of ~5 kb deletion occurred in blood specimens of colorectal cancer. 33A different prior report detected a high prevalence of large mtDNA deletions (3938 and 4388 bp) in cancerous tissues compared to the non-cancerous counterparts of breast cancer. 34ile large deletions in mtDNA have traditionally been traditionally viewed as infrequent occurrences (sporadic events) in comparison to point mutations, their substantial impact becomes more pronounced in aging, mitochondrial diseases, and cancers. 35This disparity likely arises from large deletions within the mtDNA, resulting in the loss or truncation of multiple structural genes responsible for encoding mitochondrial respiratory enzyme subunits. 33Moreover, the susceptibility of the mtDNA genome to damage and the presence of an impaired repair system might result in the prolonged persistence of mutated and deleted species within the cell, rather than undergoing proper repair.This scenario could potentially elevate oxidative stress, consequently fostering the development of cancer.
Limitations, drawbacks, or shortcomings: Considerations should be given to the limitations of our study.First, despite incorporating all available data, the sample size remains small.A smaller sample size can introduce biases and random errors, necessitating a larger study to thoroughly explore potential connections between mtDNA irregularities and clinical traits in these patients.Second, mtDNA copy numbers for the normal control were measured from whole blood DNA, chosen due to its easy accessibility.Non-tumor tissues near the brain tumor were not used as controls because highgrade tumors grow quickly, invade, and damage surrounding normal brain tissues, making them challenging to use as controls without harming nearby healthy tissues.Alternatively, the patient's own peripheral blood was used as a control.Third, variations in age, sex, and phenotypes among study groups might impact the diversity observed in these associations.For instance, the prevalence of mtDNA content in this study could be influenced by older age cases, as mtDNA copy number tends to be lower in aged populations.Conclusively, addressing these needs entails an expanded sample size and a meticulously planned research framework to comprehensively evaluate the connection between mtDNA content variations and clinical characteristics.
In conclusion, this study represents the first investigation in Malaysia exploring the correlation between variations in mtDNA copy numbers and the occurrence of brain tumors.The findings highlight elevated mtDNA copy counts in brain tumor tissues in contrast to controls, indicating possible significance within this cancer context and hinting at improved survival rates among brain tumor patients.Furthermore, future research should delve into the detailed mechanisms and potential roles underlying mitochondrial genome alterations and copy number variations in tumor progression.

Figure 1 .
Figure 1. A. mtDNA amplifications are correlated with nDNA amplifications in blood controls (P = .039,r = 0.327).B. Comparison of mtDNA content in cancerous brain tissues and blood controls.C. The overall survival plot of brain tumor patients with high and low mtDNA copy number.D. The survival plot of high-grade brain tumor patients with high and low mtDNA copy number.

Figure 2 .
Figure 2. A. The mtDNA genome of the samples was amplified in 9.3 kb, implying the absence of large-scale mtDNA deletions.B. The mtDNA genome of the samples was amplified in 7.8 kb, implying the absence of large-scale mtDNA deletions in brain tumors.Lane M, marker; lane 1-3, cancerous tissues; lane 4-6, blood controls; lane 7, no-template control to monitor contamination.

Figure 3 .
Figure 3. Detection of a single deletion of nucleotide A (Lys3360) in exon 4 of TFAM sequences by direct sequencing.
MTND1 gene, which constitutes total mtDNA, ranged from 22.05 to 32.38 in cancerous brain tissues and from 27.13 to 34.74 in peripheral blood, respectively.The average Ct values of the ACTB sequence, representing nDNA, ranged from 16.69 to 22.67 in cancerous tissues and from 17.86 to 23.22 in blood controls, respectively.The average Ct values of ACTB were less compared to the average Ct values of MTND1, indicating a greater level of nDNA than that of mtDNA level in all cases of brain tumors.
. A total of 41 patients with brain tumors were enrolled in this study, 23 (56.1%) were males, and 18 (43.9%)werefemales,with their age at diagnosis ranging from 5 to 73 years (mean: 41.9 ± 18.63).According to WHO classification, these brain tumors were categorized into 2 groups, glial (58.5%) and non-glial (41.5%) cases, and 2 distinct tumor grades, low grade (I and II; 61.0%) and high grade (III and IV; 39.0%) tumors.meningiomaIIconstitute 7.3% (3/41) of all cases.Determination of mtDNA Content in Co-extracted SamplesTotal DNA of mtDNA and nuclear DNA were co-extracted from paired cancerous tissues and blood samples.The results revealed that the average Ct values for the

Table 1 .
Clinicopathological Features of Brain Tumor Cases

Table 2 .
Correlation of mtDNA Content in Cancerous Tissues and Paired Controls a Mann-Whitney U-test.b Pearson correlation test.c Chi-square difference test.

Table 3 .
Data of Brain Tumor Patients and the Relationships Between Increased mtDNA Content in Brain Tumor Tissues and Clinicopathological Parameters a Median (IQR).b Mann-Whitney U-test.