Role of inflammatory markers in differentiation and prognostication of gliomas – A prospective cohort study

Abstract

Background:

The tumor microenvironment is an indispensable component of the tumorigenesis of gliomas. Retrospective studies reveal that certain inflammatory markers could represent the aggressive variants due to the systemic inflammation associated with them. The primary objective is to study the association between procalcitonin (PCT), C-reactive protein (CRP), and hematological inflammatory markers in gliomas. The secondary objective is to prognosticate gliomas using the Karnofsky performance score (KPS) at 6 months based on these markers.

Methods:

This prospective cohort study comprises 100 patients diagnosed with glioma radiologically. Multivariate logistic regression analysis was performed to assess the association of the biomarkers with KPS, and the Odds ratio (OR) was calculated.

Results:

92.8% of all glioma patients had PCT <0.05 ng/dL, and 85.4% had low CRP of <0.3 mg/L. High-grade gliomas (HGGs) had a low median basophil count compared to low-grade gliomas (P = 0.02). HGGs had increased values of hematological inflammatory markers compared to the low-grade group. The OR of low KPS (<70) is 7.4 times more in the high neutrophil–lymphocyte ratio group (P < 0.05).

Conclusion:

We found no significant association between PCT, CRP, and inflammatory biomarkers with grades of gliomas. However, there was a significant difference in the distribution of basophil count between the two groups. Further studies are required to validate their role in gliomas.

INTRODUCTION

The incidence of brain tumors in India was noted to be 5–10 per hundred thousand population, occupying about 2% of all malignancies.[5] The tumor microenvironment (TME) comprises a plethora of cellular and extracellular proliferative and inflammatory markers. Specifically, in gliomas, the TME is an indispensable component of tumorigenesis.[3] By virtue of the systemic inflammation associated with the gliomas, certain inflammatory markers can be detected in the serum that could represent the aggressive variants of gliomas. Some of the previously studied serum inflammatory markers include procalcitonin (PCT), C-reactive protein (CRP), and hematologic markers, including neutrophil-to-lymphocyte ratio (NLR), platelet-tolymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), and red blood cell distribution width (RDW). These inflammatory markers have been well-studied in other medical conditions, such as pneumonia and COVID-19. The utility of PCT includes tracking sepsis, antimicrobial stewardship, and as a prognostic indicator in patients with various infections. Studies have shown that PCT is elevated in glioma and can help in prognostication among its grades. The studies in the past, however, had the limitations of a smaller sample size, and the majority of them were of a retrospective study design. Since the sample size of the previous study was only 35, we studied the role of PCT with a larger sample in a prospective study design.[6]

The novelty of this study is that it is a prospective study that assesses the inflammatory markers and the prognosis, since most similar studies done in the past were of retrospective design.[4,10,12,19] The expected outcomes of the study are to find a positive correlation and increased levels of inflammatory markers such as PCT, CRP, NLR, PLR, LMR, and RDW.

MATERIALS AND METHODS

Study design and setting

This prospective cohort study was conducted from January 1^st, 2023, to June 15^th, 2024. The study was approved by the Institute of Ethics Committee (IEC) (JIP/IEC-OS/2022/291). The primary objective is to study the association between inflammatory markers such as PCT, CRP, NLR, PLR, LMR, and RDW with gliomas. Based on these markers, the secondary objective is to prognosticate gliomas using the Karnofsky performance status (KPS) scale at 6 months. The STROBE checklist was utilized for the reporting of this study. These hematological markers were specifically chosen as the previous retrospective studies have shown a significant correlation to prognosis. PCT was estimated from the patient’s serum using VIDAS B.R.A.H.M.S kit using the enzyme-linked fluorescent assay technique. CRP levels were estimated using standard CRP kits with the same blood sample, and standardized laboratory protocols were followed.

Patient selection

We employed a consecutive sampling method.

Inclusion criteria

The patients diagnosed with glioma radiologically at admission both elective and emergency cases were taken into the study after obtaining informed consent.

Exclusion criteria

Patients with tumor recurrence, histopathological reports other than glioma, history of autoimmune diseases such as rheumatoid arthritis, sarcoidosis, ankylosing spondylosis, psoriasis, and recent history of infection, renal failure, and heart disease were excluded from the study.

Study procedure

These patients underwent blood sampling for serum PCT, CRP, NLR, PLR, LMR, RDW, AEC, ABC, and RDW. Prognostication was done after 6 months of enrolment using the KPS scale.

Outcomes

The patients were divided based on the histopathology report into low-grade (WHO Grade 1 and 2) and high-grade groups (WHO Grade 3 and 4).

The NLR was calculated by dividing the absolute neutrophil count by the absolute lymphocyte count. PLR was obtained by dividing the platelet count by the absolute lymphocyte count. The LMR was obtained by dividing the absolute lymphocyte count by the absolute monocyte count.

Statistical analysis

We calculated the sample size to be 100 using OpenEpi software with a confidence interval of 95% and an alpha error of five. The association between serum PCT and CRP levels, NLR, PLR, LMR, and RDW was assessed using the Chi-square test and P-values were noted. Mann–Whitney U test was used to test the difference in the distribution of eosinophils and basophils between the two groups. Fisher’s exact test was used whenever any value in the contingency table was <5. The mean difference was estimated between PCT values in the two groups of low and high values, and Mann–Whitney U test was used as it followed a non-normal distribution. Multivariate logistic regression analysis was performed to assess the association of NLR, PLR, LMR, AEC, ABC, and RDW (independent variables) with KPS (dependent variable), and Odds ratio (OR) was calculated. Based on the results, the relative risk was calculated between the patients with high and low PCT levels.

RESULTS

The study included 47 males and 53 females, with a median age of 47 (33–57). The glioma grade was classified as low grade (Grades 1 and 2) and high grade (Grades 3 and 4). Among the glioma patients, 15 patients (16.9%) were of low grade, and 64 patients (74.9%) were of high grade, with Grade 4 patients occupying about 54% of all glioma cases [Figure 1]. The two groups were homogeneous in gender. (Chi-square - 0.9, P = 0.34). Out of all the patients that were included as gliomas based on pre-operative imaging, eight patients were not operated on for multiple reasons, two patients had descriptive biopsies, and 11 patients had diagnoses other than glioma.

Figure 1:

Distribution of the grades of glioma.

PCT

We noted that 92.8% (71) of all the patients diagnosed with glioma had PCT values <0.05 ng/dL. 5.2% (4) had a score between 0.5 and 2 ng/dL, and 2.1% (2) had more than 2 ng/dL values [Figure 2]. The mean difference between PCT values between the low-grade and high-grade gliomas (HGGs) calculated by Mann–Whitney test was 0.18, with a minimum of 0.05 and a maximum of 0.56 (P = 0.85). The relative risk between the low (<0.5) and high PCT (>0.5) is 1.11 times more in the HGG group (CI - 0.12–10.42).

Figure 2:

Distribution of procalcitonin among the two groups.

CRP

We also noted that 85.4% (65) of all the patients diagnosed with glioma had low CRP values of <0.3 mg/L. 13.5% (10) had a moderate score between 0.3 and 3 mg/L, and 1% (1) had high values of more than 3 mg/L.

The cutoffs for the hematological parameters were determined by performing a receiver operating characteristic (ROC) curve analysis. All the cutoffs were determined by selecting a value with an area under the curve of more than 0.5, with values of 0.63 for NLR, 0.61 for PLR, 0.54 for LMR, and 0.513 for RDW. HGGs were noted to have increased NLR, PLR, LMR, and RDW values compared to the low-grade group as shown in Figures 3 and 4.

Figure 3:

Distribution of neutrophil–lymphocyte ratio (NLR) in low and high-grade gliomas.

Figure 4:

Distribution of red cell distribution width (RDW) among the two groups.

Table 1 compares the frequencies of hematological parameters, PCT, and CRP with the grade of gliomas. The association between PCT, CRP, inflammatory biomarkers, and the grades of glioma was tested using the Chi-square test, and it was not found to be statistically significant.

Table 1:

Comparison of the association between hematological parameters and the grades of gliomas.

The median AEC count was 12.65 (0–54) in the low-grade group and 5.86 (0–827) in the high-grade group. There was no significant difference in the distribution of eosinophils between the low-grade and the high-grade groups.

We noted that the median ABC count was 19.6(8.6, 52.3) in the low-grade group and 9.5 (1.4, 25.5) in the high-grade group, as shown in Table 2. There was a significant difference in the median ABC values between the low-grade and the high-grade groups, such that HGGs had a lower median basophil count than low-grade gliomas (P = 0.02).

Table 2:

Comparison of absolute basophil count between the two groups.

The OR of low KPS (<70) is 7.38 times more in the high NLR group as compared to the low NLR group (C.I - 1.36–40.07, P < 0.05). As shown in Table 3, the other parameters did not significantly correlate with low KPS except for the NLR. At 6-month follow-up, 16 patients had died. Fourteen of them were diagnosed with HGG, and the other two patients had low-grade glioma.

Table 3:

Correlation of high values of hematological parameters with low Karnofsky performance status (<70).

The difference in KPS between the two groups was analyzed using the Wilcoxon signed-rank test. It was noted that there is no significant difference in the KPS score at baseline and after 6 months following surgical intervention in low-grade gliomas, whereas in HGGs, there was a significant reduction in KPS from a median of 80 (70–90) to 70 (0–100), P < 0.05.

DISCUSSION

Gliomas are a subset of brain tumors associated with local and systemic inflammation. Multiple studies have proven that the TME is the critical driver of mutagenesis in gliomas. We can utilize this attribute of gliomas to know the severity of the disease tumor burden and relate it to the patient’s prognosis. PCT and CRP are routinely utilized to identify autoimmune conditions, the source of sepsis, its progression, and response to antibiotics. Previous studies have shown the utility of hematological inflammatory markers such as NLR, principally along with PLR, LMR, and RDW, in predicting the severity of glioma and prognosis.[2,4,6] The study showed a slight predisposition of gliomas in females compared to Ravichandran, et al.: Role of inflammatory markers in gliomas males (53% vs. 47%). Of the patients that presented to our emergency and elective departments, 74.9% of them had symptomatic presentations and were noted to be HGGs. The relevance of PCT in oncology, specifically in gliomas, is an active research area. In a web-based and PubMed search, only one article was found to study the relationship between elevated PCT and HGGs.[6] This study indicates that PCT levels could aid in differentiating between gliomas and other types of brain lesions, such as abscesses, where PCT levels are typically higher.[6] Elevated PCT levels have been observed in various malignancies such as lung and ovarian tumors, suggesting a possible link between tumor burden, aggressiveness, and PCT expression. In our study, most glioma patients’ PCT values were predominantly negative. We observed that 92.8% of the glioma cases had PCT values of <0.05 ng/dL, and 5.2% had values between 0.5 and 2 ng/dL. About 2.1% of them had values of more than 2 ng/dL, which were HGGs. The relative risk between the low and high PCT groups is 1.11 times higher in the HGG group, but only two patients had high PCT levels. However, our study shows no significant association between PCT values and the grades of gliomas. CRP is another non-specific inflammation biomarker elevated in various conditions, including infections, autoimmune diseases, and malignancies. The combined use of PCT and CRP as inflammatory biomarkers in gliomas offers a multifaceted approach to understanding the TME. Several studies have demonstrated that glioma patients and meningioma patients with elevated pre-operative CRP levels have shorter overall survival (OS) and progression-free survival compared to those with normal CRP levels.[2,14] Bunevicius et al. (2018) also found that raised high-sensitivity CRP levels were associated with a greater mortality risk in meningioma cases with an OR of 3.65; P = 0.04.[2] We observed that 85.4% of all the patients diagnosed with glioma had low CRP values of <0.3 mg/L, 13.5% of them had a moderate score between 0.3 and 3 mg/L, and only 1% had a high CRP value of 4.8 mg/dL. The moderate and high values of CRP were observed exclusively in the group of HGGs, whereas the low-grade glioma patients had CRP values within the normal levels (0.3 mg/dL). However, this association is not statistically significant. This finding is consistent with the studies done previously. Cote et al. (2021), in their study of 417 gliomas, noted no significant association between CRP and white blood cells with glioma genesis.[4]

NLR, PLR, LMR, and RDW are markers of systemic inflammation and have been widely studied as prognostic indicators in gliomas. Elevated NLR has been associated with poor OS and progression-free survival in glioma patients.[1] It is also a simple and cost-effective biomarker easily obtained from routine blood tests. This study showed that among patients with high NLR, 84% belonged to the HGG group. The low-grade and high-grade groups obtained similar results with PLR, LMR, and RDW values. Although the frequencies of high levels of these inflammatory biomarkers were higher in the HGG group, suggesting weak associations, there were no significant associations between these biomarkers and the grade of gliomas. The study conducted by Serban et al. showed no significant association between the inflammatory markers and the prognosis and groups of glioma similar to our study.[12] Similar to NLR, PLR has been explored as a prognostic glioma marker. An elevated PLR indicates a pro-inflammatory and prothrombotic state conducive to tumor progression, as seen in a study on laryngeal cancer.[9] Higher PLR values correlate with poorer overall and progression-free survival in glioma patients, suggesting its potential as a prognostic marker.[16] Its utility may be enhanced by combining with other inflammatory markers, such as NLR.[1] LMR reflects the balance between the immune surveillance function of lymphocytes and the tumor-promoting activity of monocytes. A low LMR has been associated with poor prognosis in glioma patients, indicating a compromised immune response and increased tumor-promoting inflammation.[13,17] RDW measures the variation in the size of red blood cells and indicates underlying inflammation and nutritional deficiencies. Elevated RDW has been associated with poor prognosis in various cancers such as Hodgkin’s lymphoma, multiple myeloma, and diffuse large B-cell lymphoma.[8,18] Few studies showed that an increased RDW of >13.2 was associated with an increase in 1-year and overall mortality.[15] In our study, we noted no significant association between high and low RDW values and the grade of gliomas.

We also found that there was a significant difference in the distribution of basophils between the low-grade and the high-grade groups (P = 0.02). In previous retrospective studies, increased basophil counts were positively correlated with improved progression-free survival rates and decreased susceptibility to gliomas.[10,19] We noted a similar finding in our study. The median absolute basophil count was higher in the low-grade group, with a median of 19.6 (8.6, 52.3) compared to 9.5 (1.4, 25.5) in the high-grade group. However, the natural history of the disease and the development of secondary glioblastoma have yet to be discovered and require further prospective studies. We also noted that the median absolute eosinophil count was higher in the low-grade group (12.65 [0–54]) than in the high-grade group (5.86 [0–827]). However, this finding was not statistically significant. We noted that the odds of low KPS (<70) at 6 months are 7.38 times higher in patients with NLR of >3.5 compared to the low NLR group. This finding was statistically significant. We also noted no significant association between PLR, LMR, RDW, and KPS in multivariable logistic regression analysis. Our study analyzed the difference in the KPS at baseline and 6 months between the low and HGGs and observed a reduction in the median KPS by 10 points. This difference was noted to be statistically significant (P < 0.05). In another similar study, it was observed that elevated neutrophil percentage and increased NLR were associated with poor outcomes in patients with brain metastases.[11] They also noted that increased absolute lymphocyte count and percentage of lymphocytes had a positive correlation (P < 0.05). A systematic review of 137 publications revealed that seven studies showed poor OS in patients with gliomas with high NLR values. In addition, high NLR was associated with HGGs in six studies.[7]

Key results

We noted from the above findings that there is no statistically significant association between PCT and CRP levels, along with NLR, PLR, LMR, and RDW values. However, the group with high NLR seems to be associated with poor Karnofsky performance scores (P < 0.05). In addition, HGGs had a significant reduction of 10 points in the KPS score after 6 months of surgical intervention. The median absolute basophil count was higher in the low-grade group compared to the high-grade group (P < 0.05). However, there was no significant difference in the distribution of eosinophils between the two groups. These findings are partially similar to those of previous studies. From our knowledge and a Google/PubMed-based search, we found only one publication to test the association of PCT and the grades of gliomas. However, multiple authors suggested that PCT could be associated with glioma risk.[6] CRP has been studied in multiple articles, and the largest of them was done by Cote et al. [4] with a fairly large sample size, has shown no significant association similar to our study.

Clinical implications

From the findings of this study, we can infer that although gliomas are associated with an inflammatory milieu as part of their etiopathogenesis, serum CRP and PCT were of limited value in prognostication and differentiation of gliomas. However, as patients with high NLR have been associated with higher grades of gliomas and poorer KPS scores, this information could help in closely monitoring and frequently imaging high-grade glioma patients post-surgery. We could also consider performing extensive IHC markers on seemingly innocuous variants of gliomas in those with elevated NLR values. Since these markers are cost-effective and easily available, these markers could be used as surrogate markers for following up these patients.

Strengths and limitations

The strength of this study could be that it is a prospective cohort study. Many studies in the past had been performed retrospectively based on stored data of glioma patients. This study is also the second to evaluate the frequencies and association of PCT with low grade and HGGs. The possible weakness of this study could be its sample size. Secondarily, the sensitivities and specificities of the NLR, PLR, LMR, and RDW cutoff points were only marginally more than 50% as generated by ROC analysis. Another limitation of the study is the wide variability in the NLR values, which leads to wider confidence intervals. This could also be explained partly by the small sample size. Hence, this study could be extrapolated in the future with a larger sample size, including other possible variables that can be used to analyze their confounding effects. We could also estimate the PCT, CRP, and other inflammatory markers in the CSF and compare them with the serum values in future studies.

CONCLUSION

The role of inflammatory biomarkers such as PCT and CRP in gliomas is a promising area of research. We have noted that high NLR is a significant prognostic marker of poor KPS. While these biomarkers alone may not suffice for definitive diagnosis or prognosis, their integration into a broader diagnostic and prognostic framework could enhance the clinical management of glioma patients. Further research is needed to validate their prognostic utility and to explore their potential integration into clinical decision-making processes for glioma patients. In particular, quantification of inflammatory markers in the CSF, conducting multicenter studies, or using longitudinal biomarker tracking and inclusion of more specific inflammatory markers could give a more significant prognostication of gliomas.

Ethical approval:

The research/study was approved by the Institutional Review Board at JAWAHARLAL INSTITUTE OF POSTGRADUATE MEDICAL EDUCATION AND RESEARCH, number JIP/IEC-OS/2022/291, dated 18/11/2022.

Declaration of patient consent:

The authors certify that they have obtained all appropriate patient consent.

Financial support and sponsorship:

Nil.

Conflicts of interest:

There are no conflicts of interest.

Use of artificial intelligence (AI)-assisted technology for manuscript preparation:

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

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