miR-200 family as new potential prognostic factor of overall survival of patients with WHO G2 and WHO G3 brain gliomas

doi:10.21203/rs.3.rs-4888929/v1

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miR-200 family as new potential prognostic factor of overall survival of patients with WHO G2 and WHO G3 brain gliomas

https://doi.org/10.21203/rs.3.rs-4888929/v1

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Purpose

Gliomas are the predominant cause of cancer-related deaths among the young population. Even after incorporation of IDH1/2 mutations and 1p19q codeletion there are doubts regarding adjuvant treatment in WHO G2/G3 gliomas. miRNA molecules control about 30% of all genes, also many oncogenes, tumor suppressor genes and genes responsible for the response to ionizing radiation and systemic treatment. Patients with brain gliomas exhibit miRNA disorders. We aimed to evaluate the expression of miR-200 family members in relation to selected clinico- pathological factors and their prognostic value.

Material/Methods

We enrolled 53 patients diagnosed with WHO G2/G3 brain gliomas treated between 2012–2016. RT-qPCR based expression of miR-200 family was assessed in tumor and surrounding non-cancerous tissue. An analysis of selected clinico- pathological features was carried out. A logistic regression model was prepared for the miRNA signature. The predictive potential of the signature was assessed using the ROC curve. A stepwise backward regression model was used to select variables with a significant predictive potential related to OS.

Results

It was shown that miR-200a-3p, miR-200a-5p, miR-200c-5p, miR-141-3p and miR-429 can be independent predictors of survival. Better 2- and 5-year OS was associated with higher expression of miR-200a-3p, miR141-3p and lower expression of miR-200a-5p, miR-200c-5p, miR-429. The strongest predictors of survival were miR-200a-5p, miR-200b-3p, miR-200c-5p, miR-141-3p, miR-429, tumor volume and CTV.

Conclusion

Members of the miR-200 family exhibit prognostic value for 2- and 5-year OS. Presented predictive models of survival may be clinically useful for treatment optimization.

Glioma

miRNA

survival

brain tumor

prognostic factor

Patients with brain gliomas exhibit miRNA disorders. Members of the miR-200 family exhibit prognostic value for 2- and 5-year OS. Presented predictive models of survival may be clinically useful for adjuvant treatment optimization.

miRNA molecules control about 30% of all genes. Patients with brain gliomas exhibit miRNA disorders. The expression of miR-200 family in relation to selected clinico- pathological factors and their prognostic value was evaluated. 53 patients with WHO G2/G3 brain gliomas were enrolled. RT-qPCR based expression of miR-200 family was performed. A logistic regression model was prepared for miRNA signature. The predictive potential of the signature was assessed. We selected variables with a significant predictive potential related to OS. miR-200 members can be independent predictors of survival. Better OS was associated with higher expression of miR-200a-3p, miR141-3p and lower expression of miR-200a-5p, miR-200c-5p, miR-429. The strongest predictors were miR-200a-5p, miR-200b-3p, miR-200c-5p, miR-141-3p, miR-429, tumor volume and CTV. To best of our knowledge no such comprehensive involving all members of miR-200 family exist. Presented predictive models of survival may be clinically useful for treatment optimization.

Gliomas, tumors of glial origin, constitute approximately 80% of primary malignant brain tumors(1). The two most recent central nervous system WHO classifications tumors from 2016 and the subsequent modification in 2021 highlight the role of molecular diagnostics with isocitrate dehydrogenase(IDH) mutation and 1p19q codeletion as prominent prognostic and predictive factors(2, 3). Although these classifications have significantly improved prognostic and predictive efficiency, progress in treatment is insufficient, and new prognostic and predictive factors are needed. The microRNA (miR) are short (18–25 nucleotides) non-coding RNA fragments which regulate gene expression after the transcription process(4, 5). They control about 30% of mammalian genes, including oncogenes, tumor suppressor genes, and those involved in responses to ionizing radiation and systemic treatments; each miR can control hundreds of genes, impacting virtually all signal transduction pathways, making them excellent potential biomarkers for early disease diagnosis(6, 7). They act by interacting with the 3' non-translational regions (3'UTR) of target mRs, but also with other gene fragments such as 5'UTR, coding regions or promoters(4). Numerous scientific reports indicate that patients with gliomas exhibit significant miR dysregulation (8–12).

In several recent publications, the prognostic and predictive potential of the miR-200 family is considered in patients with various neoplasms(5). It consists of 5 particles: miR-200a, miR-200b, miR-200c, miR-141 and miR-429(13). They are located on two chromosomes. miR-200a, miR-200b, miR-429 on 1p36 and the remaining two on 12p13(13). Interestingly, different expression levels of miR-200 have been observed depending on the grade of gliomas(14). Preliminary studies indicate the role of miR-200a, miR-200b and miR-200c as single agents in the processes of growth, migration, and invasion of gliomas with various mechanisms involved in those processes(15–17). Additionally, the miR-200 family may be involved in the response to glioma treatment. miR-200a negatively correlated with the expression of the DNA repair enzyme O6-methylguanine methyltransferase (MGMT), which is crucial in the response to temozolomide used in glioma treatment (14). The mechanisms of action of miR-200 family described in the above studies may influence the response to chemotherapy. Certain data also indicate that microRNA-200c increases the radiosensitivity of human cancer cells, which should be investigated in the context of the widespread use of radiotherapy in the treatment of gliomas(18).

Increasing evidence indicates that members of the microRNA-200 family are closely associated with glioma initiation, progression, metastasis and treatment response of gliomas (13). The single miRNA strand evaluation may not be sufficient to obtain a clear answer about their impact on the prognosis (19). However, to our knowledge, there is a lack of comprehensive analyses that examine all members of this family in the context of gliomas.

The aim of this study was the assessment of the relative expression of all components of the miR-200 family in cancerous and adjacent non-cancerous tissue of patients with WHO G2 and WHO G3 brain gliomas, their relation to clinicopathological factors, the prognostic value of miR-200 family molecules based on the analysis of overall survival (OS).

We conducted an observational cohort study. Inclusion criteria were age between 20–80 years, diagnosis of WHO G2 or G3 brain glioma done 2012–2016, previous surgery (complete or partial macroscopic resection) or biopsy, adjuvant radical radiation therapy. Patients with glioma recurrence were excluded. A retrospective analysis of selected clinico- pathological features (histopathological diagnosis, Grade, tumor volume (TV), extent of resection (GTR, STR, biopsy), anatomical area occupied by the tumor, clinical target volume (CTV), the age and sex of patients) was carried out based on the medical records. In 2012–2015, histopathological diagnoses were made in accordance with the 4th edition of the WHO classification of the central nervous system tumors, and from 2016 in accordance with revision of the 4th edition. Tumor volume was assessed by a radiation oncologist (RO) in collaboration with a radiologist based on three tumor dimensions using preoperative phase T1 and phase T2 MRI. CTV was defined based on data downloaded from treatment planning systems. In the years in which the study was conducted, these volumes were determined uniformly, i.e. a 1.5 cm margin was added to the area of the bed/tumor and the enhancement in the T1 phase and the T2/FLAIR signal. The study was performed in accordance with the Declaration of Helsinki and was approved by the local Ethics Committee (KE-0254/349/2018). Due to the retrospective nature of the study, Medical University of Lublin Ethics Committee waived the need of obtaining informed consent.

Determination of the presence of 1p19q codeletion using the FISH method

To identify 1p/19q codeletion the FISH method was used. 4 µm sections of the unstained formalin-fixed, paraffin-embedded tissues were prepared using Vysis IntelliFISH Universal FFPE Tissue Pretreatment and Wash Reagent Kit (Abbott, USA), according to manufacturer’s protocol. Dual-color Vysis LSI 1p36/1q25 and LSI 19q13/19p13 FISH Probe Kit (Abbott, USA) was used. All probe pairs were co-denatured with the tissue sections and hybridized overnight at 37°C in separate slides. On hundred nuclei were assessed per each slide. The proportion of nuclei containing only one signal of 1p or 19q was calculated by evaluating nuclei possessing two control signals. Deletion was defined as a signal ratio ≤ 0.8 for the region of interest compared to the control probe. The % of nuclei containing deleted signals was also calculated.

DNA extraction, IDH1 (codon 132) and IDH2 (codon 172) sequencing

DNA extraction was performed with the QIA Amp DNA FFPE Tissue Kit (Qiagen, Germany) from three 5 µm sections per sample of FFPE tissue and measured on a NanoDrop ND-1000 spectrophotometer (ThermoScientific, USA). IDH1/2 mutation determinations were performed according to the methodology described by Deng (20). The obtained sequences were compared to the reference sequence of the genes: NM_005896.4 (IDH1) and NM_002168.4 (IDH2).

RNA extraction, RT PCR and quantitative PCR

miRNA extraction was performed with the miRNeasy FFPE Kit (Qiagen, Germany) from three 5 µm sections per sample of FFPE tissue. Each section was placed on a microscope slide to separate cancerous tissue from non-cancerous tissue. These two types of tissues were collected. The RNA yield was determined using a NanoDrop ND-1000 spectrophotometer (ThermoScientific, USA) and presence of small RNA was assessed by Agilent 2100 Bioanalyzer electrophoresis (Agilent Technologies, USA) using the RNA 6000 Pico Kit (Agilent Technologies, USA). Reverse transcription was performed using the TaqMan Advanced miRNA cDNA Synthesis Kit (ThermoFisher Scientific, USA) in a Tprofessional thermoblock (Biometra, Germany) according to the manufacturer's protocol. Expression levels were determined using a Real-Time PCR 7500 instrument (Applied Biosystems, USA). TaqMan Advanced miRNA Assays (ThermoFisher Scientific, USA) were used along with probes and primers commercially available for the studied miRNA family: hsa-miR-429, hsa-miR-141-3p, hsa-miR-200a-3p, hsa-miR-200a-5p, hsa-miR-200c-3p, hsa-miR-200c-5p, hsa-miR-200b-3p, hsa-miR-200b-5p. Relative expression was normalized to internal control with probe hsa-miR-26a-5p. All reactions were carried out according to manufacturer's protocols. Mean Ct values were calculated for all miRNAs and relative quantitative value was determined by using the 2 −∆∆CT method.

Statistical Analysis

The mean, standard deviation (SD), median and range (minimum, maximum) were used in the analysis of the collected data. The distribution of variables was examined based on the Shapiro-Wilk test. To test the significance of the difference in the relative expression level of a given miRNA between groups, the parametric Student's t-test for two independent variables and the ANOVA test for three or more independent variables were used. The parametric Student's t-test was also used as a post-hoc test for statistically significant results of the ANOVA test.

The analysis of Overall Survival (OS) from day of surgery was performed for the entire study group and with the determination of a cut-off point for a continuous variable (relative miRNA expression level), differentiating the probability of OS based on the Kaplan-Meier estimator (21). Moreover, a logistic regression model was prepared for the miRNA signature, i.e. the set of all tested miRNAs. Based on the predictions from the model, an analogous analysis of OS was performed, and the predictive potential of the signature was assessed using the Receiver Operating Characteristic (ROC) curve. A backward stepwise regression model was used to select clinical features and variables with significant predictive potential related to patient overall survival, and variables were sequentially eliminated based on the Akaike Information Criterion (AIC). The alpha level of statistical significance for all analyzes was set at 0.05. All analyzes were performed in the R v4.0.2 environment.

We enrolled 53 patients (29 (54.7%) men and 24 (45,3%) women) diagnosed with WHO G2 and WHO G3 brain gliomas (astrocytoma, oligoastrocytoma and oligodendroglioma) treated between 2012–2016. Clinical characteristics of the cohort are shown in Table 1. Main mass of the tumors was localized in the frontal lobe (22 cases; 41.5%), temporal lobe (16 cases; 30.2%) and in the parietal lobe (15 cases; 28.3%).

Table 1. Clinical characteristics of included patients (n=53).

Variable	n
Sex (male)	29 (54.7%)
Age (median; years)	41 (23-67)
TV (median; cm³)	49 (3.375-512)
CTV (median; cm3)	318 (226-420)
Histopatological subtype Astrocytoma Oligodendroglioma Oligoastrocytoma	30 (56.6%) 8 (15.1%) 15 (28.3%)



Grade WHO G2 WHO G3	27 (50.9%) 26 (49.1%)


IDH mutation status IDH1 + IDH2 +	33 (62.3%) 3 (5.7%)


1p19q codeletion WHO G2 WHO G3	12 (22.6%) 9 (75%) 3 (25%)


Surgery type GTR STR/biopsy	39 (73.6%) 14 (26.4%)

IDH1/IDH2 mutation and 1p19q codeletion

A mutation in codon 132 of the IDH1 and in codon 172 of the IDH2 gene was identified among 33 (62.3%) and 3 (5.7%) patients, respectively.

1p19q co-deletion, according to the EORTC interpretation criteria, was detected in 12 (22.6%) patients, 9 cases in the WHO G2 gliomas (Table 1).

Evaluation of miR-200 expression

A lower relative expression of miR-200b-5p (p = 0.0038), miR-200c-3p (p = 0.077), miR-141-3p (p = 0.011) was detected in tumor tissue. A higher relative expression of miR-200a-5p (p = 0.005) was reported in gliomas without 1p19q codeletion. WHO G2 gliomas presented higher relative expression of the miR-200c-3p (p = 0.025) compared to WHO G3 gliomas.

There were no differences in the relative expression of miR-200 family according to gender, IDH1/IDH2 mutations, the extent of resection, or TV. There was a difference in relative expression of miR-200a-5p (p = 0.0067), miR-200b-3p (p = 0.074) and miR-141-3p (p = 0.048) between histological types. The highest expression of miR-200a-5p was observed in oligodendroglioma. Higher miR-141-3p expression was associated with astrocytoma (Fig. 1).

Prognostic value of single miR-200 family molecules based on OS.

During a follow-up period of 203 months for an OS of a median of 94 months (range: 10–203), 30 (56.6%) patients died. Median survival time was 105 months (95% CI: 88-NA). The probability of 2- and 5-year OS was 94% (95% CI: 88–100%) and 74% (95% CI: 63–88%). Longer 2- and 5-year OS was associated with higher miR-200a-3p, miR-141-3p and lower miR-200a-5p, miR-200c-5p, miR-429 relative expression in the whole cohort. The analysis showed that the expression of miR-200a-3p, miR-200a-5p, miR-200c-5p, miR-141-3p and miR-429 correlates with OS (Fig. 2).

Higher miR-200a-3p expression leads to better prognosis.

Patients with high (i.e. above the cut-off) versus low expression of miR-200a-3p had better median OS which was 120 months (95% CI: 97-NA) and 71.5 months (95% CI: 43-NA), respectively (HR = 0.452, 95% CI: 0.214–0.953, p = 0.0327). The 2- and 5-year OS rates in high versus low expression groups were 95% (95% CI: 88–100%) and 78% (95% CI: 66–93%) versus 94% (95% CI: 83–100%) and 62% (95% CI: 43–91%), respectively.

Higher miR-141-3p expression leads to better prognosis.

Patients with high versus low expression of miR-141-3p expression group had better median OS was 123 months (95% CI: 123-NA) and 97 months (95% CI: 64-NA), respectively (HR = 0.443, 95% CI: 0.202–0.973, p = 0.0372). The 2- and 5-year OS rates in high versus low expression groups were 96% (95% CI: 88–100%) and 83% (95% CI: 68–100%) versus 93% (95% CI: 85–100%) and 67% (95% CI: 52–86%), respectively.

Lower miR-200a-5p expression leads to better prognosis.

Patients with low versus high expression of miR-200a-5p expression had better median OS which was 123 months (95% CI: 101-NA) and 86 months (95% CI: 56-NA), respectively (HR = 2.03, 95% CI: 0.969–4.23, p = 0.0555). The 2- and 5-year OS rates in low versus high expression groups were 94% (95% CI: 87–100%) and 80% (95% CI: 68–94%) versus 94% (95% CI: 84–100%) and 61% (95% CI: 42–88%), respectively.

Lower miR-200c-5p expression leads to better prognosis.

Patients with low versus high expression of miR-200c-5p expression had better median OS which was 105 months (95% CI: 101-NA) and 72.5 months (95% CI: 56-NA), respectively (HR = 2.18, 95% CI: 1.02–4.64, p = 0.0382). The 2- and 5-year OS rates in low versus high expression groups were 95% (95% CI: 88–100%) and 79% (95% CI: 68–93%) versus 93% (95% CI: 80–100%) and 57% (95% CI: 36–90%), respectively.

Lower miR-429 expression leads to better prognosis.

Patients with low versus high expression of miR-429 expression had better median OS which was 120 months (95% CI: 101-NA) and 88 months (95% CI: 56-NA), respectively (HR = 1.91, 95% CI: 0.907–4.01, p = 0.0835). The 2- and 5-year OS rates in low versus high expression groups were 97% (95% CI: 87–100%) and 79% (95% CI: 68–94%) versus 90% (95% CI: 84–100%) and 65% (95% CI: 42–88%), respectively.

A predictive survival model based on the entire miR-200 family

A logistic regression model was developed through estimating the probability of OS based on the net effect of changes in the relative expression level of all tested miRNA molecules, hereinafter referred to as the miRNA signature. Then, the predictive potential of the above signature was estimated using the ROC curve, along with determining the cut-off point according to which the prognosis of OS was predicted (Fig. 3).

The model was characterized by 63% sensitivity and 78% specificity. The positive predictive value was PPV = 79% and the negative predictive value was NPV = 62%. The number of false positives was FP = 5 and false negatives FN = 11. In the group classified below the model cut-off, the median OS was undetectable, and above the model cut-off it was 97 months (95% CI: 64–123). The 2- and 5-year OS rates in low versus high expression groups were 97% (95% CI: 90–100%) and 91% (95% CI:80–100%) compared to 73% (95% CI: 59–91%) and 74% (95% CI: 58–94%), respectively. A lower model value correlated with a better prognosis (HR = 1.9, 95% CI: 0.907–3.99, p = 0.0836) (Fig. 4).

A predictive survival model based on miR-200 family and clinico-pathological features.

Backward stepwise regression was performed to select clinico- pathological variables with significant predictive potential related to OS. Initially, the model included variables such as Grade, type of surgery, IDH1/IDH2 mutation, age, location, TV, CTV and the relative expression of miRNA molecules in the tumor tissue as potential predictors; the dependent variable was the patient's death from the disease. As a result of eliminating individual features, significant predictors of patient survival turned out to be TV (V cm3), CTV, relative expression of miR-200a-5p, miR-200b-3p, miR-200c-5p, miR-141-3p and miR − 429. The stages of backward elimination of variables from the model based on the Akaike Information Criterion (AIC), which is a qualitative measure of the model are shown in Fig. 5. In the final model the strongest predictors of OS were miR-200a-5p, miR-200b-3p, miR-200c-5p, miR-141-3p, miR-429, TV and CTV.

In our cohort the dominant histological subtype was astrocytoma (56.6%). Oligoastrocytoma and oligodendroglioma accounted for 28.3% and 15.1%, respectively. It should be taken into account that in the WHO 2021 classification, oligoastrocytoma do not constitute a separate subtype according to molecular criteria and would be classified as oligodendrogliomas or astrocytomas(22). Our findings align with existing epidemiological data, showing a 2–3:1 ratio of astrocytoma to oligodendroglioma occurrence, as reflected in our analysis (23, 24)

A well-known molecular prognostic factors for brain gliomas are IDH 1/2 mutations and 1p19q codeletion. Literature indicates that IDH1 mutations occur in 60–80% of WHO G2/G3 gliomas, while IDH2 mutations are much rarer, with a 1–6% incidence and both decrease with grade(25, 26, 27, 28). This corresponds to the results obtained in our analysis. The presence of 1p19q codeletion was found in 52.2% of gliomas with an oligodendroglial component. It was higher in WHO G2 compared to the WHO G3 gliomas − 75% and 27.3%, respectively, which as well stays in line with literature data (29, 30).

miR-200 family in glioma tissue and accompanying tissue.

Available literature, in many cases, analyzes only one strand of each miR-200 molecule. It should be emphasized that current knowledge about miRNAs is constantly expanding and indicates that both the 5' and 3' strands may be functional and perform compatible as well as distinct regulatory functions (31, 32). Analysis of only single strands of a given miRNA leaves many questions and constitutes an incomplete set of information in relation to many cancer diseases, including gliomas. Conducted own research, in accordance with the latest trends in miRNA characterization, analyzes the expression level of both miRNA strands, which contain a complete set of information.

First, a comparative analysis of each component and its impact on patient prognosis was performed in connection with well-known prognostic factors in the form of clinical and molecular features.

Expression of miR-200 family depending on Grade.

In our own analysis, a significantly lower level of miR-200c-3p expression was observed in WHO G3 gliomas. Wang et al. showed a reduced miR-200a expression in WHO G3 and WHO G4 gliomas. Patients with WHO G2 tumors were not included (33). Similarly, Liu et al., showed a significant reduction in miR-200a-3p expression in glioma tissues with higher Grade (34). Chen et al. showed a decrease in the expression of miR-200a with higher Grade, but most of the patients were with WHO G4 gliomas (35). Regarding miR-200b expression, a study which included patients with G1-G4 gliomas showed its significant decrease with increasing Grade (p = 0.002) (36). Similar results were obtained by Men et al. however, the differences between patients with WHO G2 and WHO G3 gliomas were less noticeable (37). The meta-analysis confirmed the results. There was a decrease in its expression between WHO G1/G2 and WHO G3/4 groups − 5.87 ± 1.77 vs. 3.13 ± 0.89, p < 0.01, respectively (38). Qin et al., compared WHO G2 and WHO G3/4 gliomas and showed a decrease in miR-200c expression with increasing Grade in tissues, as well as in glioma cell lines (p = 0.0064)(39). These results are consistent with our own analysis. In regard to miR-141 expression, negative correlation with higher Grade was found in three studies, two of them included additionally WHO G1 and G4 gliomas (40, 41, 42). One study showed positive correlation between the expression of miR-141-3p and Grade in WHO G1/G2 and WHO G3/G4 gliomas (p < 0.001) (43). Only one report about miR-429 expression was found. Thirty-seven patients with WHO G2 and 24 patients with WHO G3 gliomas were included. Positive correlation of its expression with increasing Grade was found (p < 0.05) (44).

Taking into account presented results, the differences shown may result from the small number of patients included. Additionally, it should be borne in mind that the discrepancies could also be caused by different cut-off points in relation to the assessment of the expression, considered as low or high level. Undoubtedly, the expression of individual components of the miR-200 family, changing with Grade, may be related to carcinogenesis and progression of gliomas, which requires further, broader analysis in the future.

Expression of miR-200 family depending on IDH 1/2 mutation and 1p19q codeletion.

There was no relationship between the expression of miR-200 family and the IDH1/2 mutation. However, a significant increase in the expression of the miR-200a-5p was noted in gliomas without 1p19q codeletion. So far, no combined analysis of miR-200 and IDH 1/2 mutations and/or 1p19q codeletion have been performed, as was done in our study. Undoubtedly the weak point of miR-200 expression studies in gliomas is the lack of reference of the miR-200 expression level to the new molecular classification which was also emphasized in meta-analysis (37).

Prognostic value of miR-200 family based on OS.

In our study, 2- and 5-year OS for WHO G2/G3 gliomas was 94% and 74%, respectively. These data are consistent with the systematic reviews and big cohort studies(45, 46, 47).

With regard to miR-200 family, our analysis showed a significant relationship between the expression of its components and OS. We have shown that WHO G2/G3 gliomas with higher miR-200a-3p and lower miR-200a-5p expression had better prognosis. Unfortunately, there are no publications directly comparing the OS with miR-200a expression. It has been found that the miR-200-3p inhibits the ability of glioma cells to survive, invade and proliferate (35). Similarly, it was shown that its reduced expression in glioma cells was associated with a worse response to chemotherapy and glioma neogenesis and progression (33). Therefore, it seems logical to assume that a reduced level of the miR-200a is associated with the development of clones resistant to systemic treatment, which results in worse OS. Unfortunately, it was not specified which strand the obtained results concerned.

Our analysis showed no relationship between the expression of both miR-200b and OS. Similar results were presented by Wang et al., in patients with WHO G1/G2 gliomas but higher expression of miR-200b led to better OS in WHO G3/G4 gliomas (p = 0.028) (36). It should be also noted that this is the only study that reported a significantly higher expression of miR-200b in glioma tissues compared to non-cancerous tissue, unlike the rest of the available literature data regarding this component (36). Men et al. showed that low miR-200b expression led to shorter 5-year OS and 5-year PFS in WHO G3/G4 gliomas. There were no differences in WHO G1/G2 group. These differences for PFS and OS in patients with it’s low expression compared to high expression group were 9.61 months (95% CI, 6.9–12.8) and 13.89 months (95% CI,11.06–17.83) and 14.66 months (95% CI, 9.82–20.92) and 20.19 months (95% CI, 13.68–26.29) (p = 0.023 and 0.012), respectively (37). However, it should be considered that histological types among WHO G2/G3 gliomas included only astrocytomas and anaplastic astrocytomas, without information regarding IDH mutation and 1p19q codeletion (37).

Our findings showed that low miR-200c-5p expression correlated with better prognosis. This is the first report of this type. Its important role in gliomagenesis and prognostication was previously highlighted (39). However, also patients with WHO G4 gliomas, ependymocytomas and anaplastic ependymocytomas were included (39).

We have also noted that higher miR-141-3p expression correlates with better OS. There is no other literature involving its impact on OS. However, there is research that may indirectly explain obtained results. It was previously shown that high miR-141 expression can strongly inhibit the proliferative and invasive potential of gliomas diminishing their ability to progress (40, 48).

High levels of miR-141-3p inhibits glioma tumorigenesis, which has been linked with miR-141-3p/YAP 1 regulation (42). It has been also shown, in vitro and in vivo, that miR-141-3p can inhibit formation of new vessels, cell division cycle and induce apoptosis of glioma cells. Mutual inverse correlation between miR141-3p and the EphA2 gene was detected (48).

Our study reported better 2- and 5-year OS in patients with lower miR-429 expression. Sun et al. showed a significantly higher probability of 5-year OS was noted in patients with low miR-429 expression (p < 0.001). The multivariate analysis confirmed correlation of miR-429 expression with OS (RR: 3.674, 95% CI: 1.990–6.784, p = 0.001) (44). A relationship between miR-429 and BMK-1 kinase expression was previously described by other authors. Material derived from cell lines and paraffin blocks of WHO G1-G4 gliomas were used. Also in this study a higher miR-429 expression led to better OS (p = 0.000) (49). Described results indicate an important role of miR-429 as a prognostic factor for OS of WHO G2/G3 gliomas.

Expression of one type of miRNA and its interactions with another may lead to contrasting prognostic value in different cancers and when compared to the determination of just single miRNAs. Therefore, studies that integrate a larger number of miRNA and clinicopathological features provide the highest prognostic and predictive value. This allows for the creation of reliable models that more accurately determine the prognosis of patients with a given group of variables, as indicated by meta-analyses (50, 51, 52). In our study, for the first time, the entire panel of the miR-200 family, including both strands, was used to create a logistic regression model estimating the probability of OS. The model based on all components had a sensitivity of 63% and a specificity of 78%. Moreover, the predictive potential of the signature reached AUC = 0.703. This indicates that the selected miRNA signature has a relatively high potential to distinguish patients according to prognosis. Patients with a lower model value had a significantly better prognosis. The difference in 2- and 5-year OS in the group with a low compared to high model value was 24% and 17%, respectively.

This indicates the potential clinical usefulness of the presented model when considering treatment strategies. Taking into account the multitude of gliomas clinical features and the range of therapeutic effects there is a strong need to search for prognostic models helping to decide whether adjuvant radiotherapy/ chemotherapy should be used and what types, doses and duration should be chosen. Perhaps the intensification of treatment in patients with a higher model value would lead to better results. However, this hypothesis requires testing in prospective clinical trials.

In the final step, clinicopathological features were integrated with miR-200 expression allowing more precise OS prediction. The 9 stages, backward stepwise elimination model, confirmed the strong predictive value of TV and CTV.

The impact of TV on OS of glioma patients have been previously described in the literature. Flores et al. showed that worse OS occurred in patients with TV ≥ 60 cm³ and/or ≥ 2000 mm² in the T2-Flair sequence before surgery (HR 3.72 and 3.93) (53). Another study demonstrated the worst OS when tumors were larger than 5 cm (HR = 1.56, 95% CI 1.12–2.19, p = 0.0089) (54). However, it should be borne in mind that there are also reports showing that there is no relationship between TV, extent of resection and OS in patients with anaplastic astrocytomas (55).

Chapman et al. showed a relationship between PTV and OS in HGG. For non-stereotactic techniques, PTV above 131cc significantly correlated with worse OS (56). Guram et al. showed no significant difference in OS depending on PTV created by addition of 0.4 cm, 1 cm or 2–3 cm margin in HGG (57). In our work, CTV was chosen, which corresponds better with the OS than the PTV. The relationship of CTV with OS and PFS, was analyzed in the work by Liu et al. Two groups, CTV defined according to the EORTC guidelines as a bed with edema and a 2 cm margin, and the other similar but in which edema was not included, were selected. During the median follow-up of 26.4 months, no differences in OS and PFS were shown (p = 0.418, p = 0.388) (58).

The presented reports, which are ambiguous, encourage clinical trials involving evaluation of CTV and the results of therapy. Perhaps smaller volumes may be used in patients treated for certain types of gliomas, depending on the planned adjuvant therapy regimen.

In our final ANOVA model, apart from TV and CTV, miR-200a-5p, miR-200b-3p, miR-141-3p and miR-429 were predictors of OS (p = 0.054). This model is the first tool of its kind that can be helpful in prediction of OS. Its use may help clinicians in difficult decisions related to the treatement intensification or de-intensification.

A limitation of our study is the relatively small sample size and the lack of analysis based on different therapeutic regimens. However, it remains the most comprehensive analysis of the miR-200 family available. It should be borne in mind that in WHO 2021 classification, some of included patients would be switched to other glioma types. Future trials should be conducted prospectively, on a larger cohort, taking into account all of the molecular features implemented in WHO 2021 classification and detailed data on adjuvant treatment.

In patients with WHO G2/G3 brain gliomas, the relative expression of miR-200 family shows differences between cancerous and non-cancerous tissue. Grade, histopathological subtype and the presence of 1p19q codeletion correlate with the expression of selected miR-200 molecules. miR-200 family has a prognostic value in terms of 2-year and 5-year OS. It is advisable to evaluate both strands of each miR-200 family member. Both prediction models, based on the entire miR-200 family signature and miR-200 family, TV and CTV demonstrate potential clinical utility but require confirmation in future studies.

Acknowledgements

The study was co-financed by Jakub hr. Potocki’s Foundation funds.

Declaration of disclosure

All authors declare that they have no conflicts of interest to disclose.

Declaration of author contributions

Mateusz Bilski contributed to conceptualization, methodology, writing - original draft, formal analysis and data curation, resources acquisition,

Marzanna Ciesielka contributed to writing - original draft and formal analysis,

Magdalena Orzechowska contributed to formal analysis, visualization,

Bożena Jarosz contributed to writing - review & editing,

Paulina Całka contributed to data curation, writing - review & editing,

Sylwia Bilska contributed to writing - review & editing,

Agata Banach contributed to data curation,

Aleksandra Grzywacz contributed to data curation,

Gabriela Czaja contributed to writing - review & editing,

Jacek Fijuth contributed to writing - review & editing and supervision,

Łukasz Kuncman contributed to formal analysis, validation, writing – original draft and review & editing and supervision.

Data availability statement

All data generated or analysed during this study are included in this published article [and its supplementary information files (qPCR raw data)]

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No competing interests reported.

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miR-200 family as new potential prognostic factor of overall survival of patients with WHO G2 and WHO G3 brain gliomas

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