Biomarkers of Systemic Inflammation in Patients with Glioblastoma: An Analysis of Correlation with Tumour-Related Factors and Survival
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.323885
Source of Support: None, Conflict of Interest: None
Keywords: Cell counts, glioblastoma, LMR, NLR, PLR, progression-free survival, survival, systemic inflammationKey Message: A significant inflammatory response was found in patients with GBM and correlated with clinical features, the molecular profile of the tumour and poor survival. Several BMSIs that were found to influence PFS (ANC > 5230 or AMC > 810 or NLR > 9.49 or PLR > 381.36 or LMR < 1.87) were combined into an InfP. The InfP negative group had more than double the
Cancers have been shown to induce a state of chronic inflammation in the body. The magnitude of the systemic inflammatory response (SIR) can be estimated by measuring the levels of a heterogeneous group of entities, which can collectively be called the biomarkers of systemic inflammation (BMSIs). The BMSIs include proteins (albumin, C-reactive protein), the erythrocyte sedimentation rate, etc. The most commonly evaluated BMSIs (and the ones that most directly reflect the SIR) are the blood cell counts (CCs) and blood cell-count ratios (CCRs), such as the neutrophil-lymphocyte ratio-NLR, platelet-lymphocyte ratio-PLR, etc. The pre-treatment levels of these BMSIs have been shown to have prognostic significance for several solid-organ cancers such as those arising in the lung, stomach, pancreas, breast, bladder, colon, etc.,,,,,,,
The brain has traditionally been considered an immune-privileged site. However, evidence is now accumulating that pre-treatment levels of the BMSIs are deranged in patients with glioblastoma (GBM) as well.,,, Elevated levels of CCRs such as the NLR, PLR and combined platelet-neutrophil lymphocyte ratio (CoPNLR), and reduced levels of the lymphocyte-monocyte ratio (LMR), have all been associated with a shorter progression-free survival (PFS) in patients with GBM.,,, However, there has also been some conflicting evidence in the literature, with some studies reporting either no association of these markers with survival, or associating higher levels of NLR with longer overall survival.
This, therefore, is a three-part study. In the first part, we attempted to establish if there was any significant alteration in blood-cell counts (CCs) and blood cell count ratio-based BMSIs (CCRs) in patients with GBM when compared with a healthy control population. In the second part, we evaluated if there were any significant correlations between the CCs and CCRs and patient- and tumour-related factors. In the third part, we determined survival-based cut-points for the CCs and CCRs and examined if any of the determined thresholds affected survival.
This was a retrospective study approved by the institute ethics committees of the participating institutions (TMC/IEC III//900506 and JIP/IEC/SC/2014/10/706). Cases were identified by querying prospectively maintained databases in 2 of the participating institutions to identify all patients treated between 2007 and 2017, who had a histopathologic diagnosis of GBM (WHO grade IV). Patients with treatment-naïve GBM who had undergone the first surgery/biopsy at the participating institutions were included for analysis. Patients with recurrent lesions, those with documented infections, autoimmune diseases or other cancers, and those who had undergone biopsy/surgery of the brain tumour elsewhere, were all excluded from the analysis. The Control group was drawn from the set of (presumably) healthy persons who underwent pre-employment health tests at two of the participating institutions.
Absolute CCs from the haemogram were compared between cases and controls – absolute neutrophil count (ANC), absolute lymphocyte count (ALC), absolute monocyte count (AMC), absolute basophil count (ABC), and absolute eosinophil count (AEC). The following blood count-based CCRs were computed: NLR = (ANC/ALC), PLR= (platelet count/ALC), LMR= (ALC/AMC), granulocyte-agranulocyte ratio (GrAgr) = [(ANC + ABC + AEC)/(ALC + AMC)], ELR= (AEC)/(ALC) and dNLR= [(ANC)/(total leucocyte count - ANC)]. The systemic inflammation index (SII) was calculated as SII= [(platelet count x neutrophil count)/lymphocyte count]. The CoPNLR was scored as follows – score of 0 if platelet count <300000/cu mm and NLR <3, score of 2 if NLR > 3 and platelet count >300,000 and the score documented as 1 for all other combinations.,
Patient- and tumour-related factors and survival analyses
Patient-related factors such as the duration of symptoms, presence of neurologic deficits at presentation, etc., were obtained from the EMRs. Tumour volumes were calculated based on linear lesion dimensions measured on the pre-operative contrast and fluid-attenuated inversion recovery (FLAIR) magnetic resonance image (MRI) sequences using the simplified formula for the volume of an ellipse [v = (abc)/2]. The extent of resection (EoR) data was scored based on operative notes and postoperative MR images. All patients underwent (or were referred for) radiotherapy and chemotherapy after surgery. The PFS was defined as the time interval between surgery and documented radiologic recurrence/progression of disease.
Statistical analyses were performed on Stata v14.2 (StataCorp, College Station, Texas). Normally distributed continuous variables were compared using the Student's t-test, whereas non-normally distributed variables and categorical variables were compared using the Wilcoxon rank-sum or Kruskal-Wallis tests as appropriate. Bivariate analyses were carried out to correlate various tumour-related factors with the BMSIs. Outcome (PFS)-based threshold determination for the BMSIs was performed using the X-tile analysis software. The Cox proportional hazards model was used to test for the effect of the determined thresholds of the BMSIs on the PFS. The alpha level was set at 0.05 for significance in all analyses.
Data pertaining to 743 individuals were included in the analysis - 335 Controls and 408 Cases. The median age of the Control group was 25 years and that of the Cases group was 55 years (z = –17.7, P < 0.0001). The male: female ratio for the Control group was 5.8:1 whereas the gender ratio for the Case group was 2.2:1 (χ2 = 26.8, P < 0.0001).
Part 1: BMSIs––Cases vs. Controls
The CCs and CCRs were compared between cases and controls. All measured CCs were significantly different between cases and controls. The platelet count, total leucocyte count (TLC), ANC, AMC and ABC were significantly higher whereas the ALC and AEC were significantly lower in cases compared with controls [Table 1]. All the CCRs were significantly elevated in cases when compared with controls, except the ELR and LMR, which were significantly lower in the cases [Table 1].
Since the Case and Control groups were not ab initio matched for age and sex (vide supra), we repeated the analyses separately for age-and gender-matched groups. This matching resulted in 76 age-matched male Controls being compared with 38 male Cases and 42 age-matched female Controls compared with 21 female Cases (Case: Control = 1:2). Despite age- and sex-matching, the differences in the BMSIs between Cases and Controls persisted as for the unmatched groups [Table 2]. The TLC, ANC, AMC, and ABC were significantly higher whereas the ALC and the AEC were significantly lower in cases compared with controls. All CCRs were significantly higher in the cases than in the controls, except the ELR and LMR, which were higher in controls.
Part 2: BMSIs – correlations with patient- and tumour-related factors
The Case cohort comprised 408 patients treated for GBM. The mean duration of symptoms was 3.5 months (SD-10, range 1–156 months). The mean contrast-enhancing tumour size was 5.1 cms (SD-1.6, range 1–9 cms) and the mean contrast-enhancing tumour volume was 103.6 cc (SD-77.7, range 3–342 cc). The mean tumour size on FLAIR sequences was 7 cms (SD-3, range 3-14 cms) and the mean FLAIR tumour volume was 115.2 cc (SD-65.2, range 5.5–401 cc). Two hundred and forty patients (61%) had either gross-total (GTR) or near-total resection (NTR); the rest (n = 152, 39%) had subtotal resection (STR) or biopsy. The median Ki67 labelling index of the tumours (MIBLI) was 20 (range 3–90) [Supplementary Table 1].
The bivariate analysis matrix for the correlation of patient- and tumour-related factors with CCs and CCRs is displayed in [Supplementary Table 2]. Preoperative performance status (measured on the Karnofsky Performance Scale, KPS), altered sensorium at presentation, raised intracranial pressure (ICP) at presentation, ATRX status of the tumour and p53 status of the tumour correlated significantly with several BMSIs [Supplementary Table 2]. Patients who had a KPS ≥70 at presentation had a significantly higher AMC, AEC and LMR and a significantly lower NLR than those with KPS <70. Patients who had symptoms/signs of raised ICP at presentation had a significantly higher TLC, ANC, NLR, and dNLR and a significantly lower AEC than those without [Table 3]. Patients who were in altered sensorium at presentation had a significantly higher TLC, ANC, NLR, GrAgr, SII, dNLR and significantly lower AEC, ABC, LMR and ELR than those with preserved sensorium. Loss of ATRX correlated with higher ANC, NLR, GrAgr and dNLR and lower AEC and ABC (compared with lesions with retained ATRX). Lack of p53 overexpression was associated with lower AEC and significantly higher NLR, PLR, CoPNLR, GrAgr, SII, ELR and dNLR than those with p53 overexpression [Table 3].
Part 3: BMSIs––analysis of Progression-Free Survival
PFS data was available for 120 patients; the median PFS was 11.7 months (range 0.3-113 months). Twenty patients (17%) had not progressed at last follow-up, the median PFS for this group was 32 months. We generated thresholds for CCs that may have an impact on PFS using the X-tile software [Figure 1]. ANC >5230/cu mm and AMC >810/cu mm were identified as possible thresholds. We performed Cox regression using the proportional hazards model around these thresholds and generated Kaplan–Meir survival curves. ANC >5230/cu mm was associated with a shorter PFS (HR 1.95, 95% CI 1.17–3.23, P = 0.01, [Figure 2]a), as was AMC >810/cu mm (HR 1.83, 95% CI 1.17–2.87, P = 0.008, [Figure 2]b).
We then determined thresholds for the CCRs. X-tile analysis generated the following cut-point based thresholds – NLR – 9.49, LMR – 1.87 and PLR – 381.36. Besides the X-tile generated cut-points, we also dichotomized cases into groups around the following thresholds, previously reported in the literature as being of significance for survival – NLR – 4 and 5, PLR – 150 and 175, LMR – 3.8, CoPNLR – 1 and SII – 600000.,,, We then performed survival analysis around all of these thresholds using the Cox proportional hazards model. An NLR ≤9.49 (HR 1.68, 95% CI 1.01–2.78, P = 0.043), PLR ≤381.36 (HR 2.62, 95% CI 1.13–6.05, P = 0.024) and LMR >1.87 (HR 0.053, 95% CI 0.32–0.87, P = 0.013) were associated with longer PFS [Figure 2]c, [Figure 2]d, [Figure 2]e. Multivariate analysis including all these CCs and CCRs (ANC >5230, AMC > 810, NLR > 9.49, PLR > 381.36 and LMR < 1.87) showed that ANC > 5230 was able to independently influence survival (HR 1.77, 95% CI 1.04–3.01, P = 0.036).
We defined an inflammatory profile (InfP) using these parameters. The InfP was defined as being positive if any one of the following were to be true - ANC > 5230 or AMC > 810 or NLR > 9.49 or PLR > 381.36 or LMR < 1.87. Cases were then divided into two groups––group 0 with a positive InfP and group 1 with a negative InfP. Group 1 had a significantly longer median PFS (745 days) than group 2 (301 days, HR 0.4, 95% CI 0.22–0.69, P = 0.001, [Figure 2]f).
There are few studies in the literature that unequivocally report alterations in the levels of BMSIs in patients with brain tumours, when compared with healthy individuals. We have previously shown that the NLR was elevated in a cohort of patients with brain tumours (all WHO grades) when compared with a healthy control group. The present study included a homogenous group of patients with GBM, compared with a healthy control population. In this study, the pre-treatment levels of all CCRs and CCs were altered in Cases when compared with Controls. Some recent data demonstrates that the levels of BMSIs vary by age and gender.,, However, in the present study, the differences in the BMSIs between cases and controls persisted despite age- and sex-matching, and thus, this appears to be a robust finding described for the first time [Table 2].
The consistent pattern seen in patients with GBM was a higher TLC, ANC, AMC, ABC, NLR, PLR, CoPNLR, SII and dNLR, and lower ALC, AEC, LMR and ELR, when compared with controls. Elevation of the TLC, ANC, AMC, ABC, NLR, PLR, GrAgr, SII and dNLR all indicate the presence of an SIR. The AEC and ELR indicate an atopic process and depression of these indices indicates suppressed atopy. The AMC and LMR reflect cell-mediated immunity (as opposed to the non-specific SIR) and reduced AMC and LMR indicate suppression of the cell-mediated immune response. Depression of cell-mediated immunity, and specifically T-cell function, have been described in GBM., Thus, the pattern of alterations seen in patients with GBM is reflective of an ongoing nonspecific SIR, with depression of the atopic and cell-mediated immune responses.
Patient- and tumour-related factors
Patients presenting with symptoms or signs of raised ICP and those who presented with an altered level of consciousness displayed patterns of alterations of the CCs and CCRs that were indicative of the inflammatory phenotype [Table 3]. The pattern of alterations in the BMSIs in patients with poor KPS mirrored that for altered sensorium––higher ALC and NLR, and lower ALC and LMR. The patterns of alterations in the BMSIs for these three clinical features, although not exactly congruent, all unequivocally suggested the presence of an SIR. This appears to be a novel finding, since there are no prior reports in the literature regarding the association of pre-treatment altered level of consciousness, presence of raised ICP and poor KPS with the presence of an SIR, as reflected by altered BMSIs.
There is currently no data that reports any correlation of the molecular profile of GBM with the SIR. The only paper to date where the association of the BMSIs with the molecular profile of GBMs has been studied reported an absence of correlation between NLR, LMR, PLR and tumour IDH status. In the present study, however, patients who had tumours with loss of ATRX and lack of p53 overexpression were clearly associated with an inflammatory phenotype of changes in the CCs and CCRs [Table 3]. Elevated levels of CCRs as well as loss of ATRX both connote poor prognosis in GBM, and thus, this correlation appears logically consistent. However, it is difficult to explain why patients with p53 overexpression had a lower SIR, since p53 mutation has been thought to be associated with worse prognosis in GBM. However, some data show that p53 gain-of-expression mutations are not necessarily associated with a shorter PFS in patients with GBM. p53 has potent anti-inflammatory actions and this action may be the reason for the observed association of p53 with lower levels of the CCRs and by extension, a longer PFS. Thus, it appears clear that certain molecular changes such as loss of ATRX and lack of p53 overexpression are associated with an inflammatory phenotype; this is a novel finding.
BMSIs and Progression-free Survival
The association of NLR with survival in patients with GBM has been well established. The threshold for the pre-treatment NLR to impact survival as determined by this study was 9.49, for PLR - 381.36 and that of the LMR was 1.87. No cut-point that affected PFS could be determined for the other CCRs. The thresholds for the BMSIs that appear to impact PFS vary across several studies and much larger scale pooled analyses will be required to determine the cut-points that have true prognostic implications.,,, Besides the CCRs, several CCs were found to be altered in patients with GBM in this study. Of these, ANC > 5230 and AMC > 810 were found to be associated with shorter PFS. Not only has pre-treatment ANC been found to correlate with survival, it has also been shown to predict response to bevacizumab.
The BMSIs that were found to have thresholds that influenced PFS (ANC > 5230 or AMC > 810 or NLR > 9.49 or PLR > 381.36 or LMR < 1.87) were combined into an InfP. It was found that having a positive InfP strongly affected survival, with the InfP negative group having more than double the median PFS (745 days) as the InfP group (301 days, vide supra). Thus, having a derangement of any of the 4 identified parameters above the thresholds identified for the InfP is associated with poorer PFS.
Limitations and implications
This study was retrospective in nature and thus the degree to which variables affecting the BMSIs could be controlled was limited. However, all patients were of Indian ethnicity and the observed alterations in the BMSIs in patients with GBMs compared with controls persisted even after age- and sex-matching. Since the centres participating in this study are national referral hospitals, many patients were referred back to their hometowns for chemo-radiation after surgery and therefore, follow-up data was not available for a large proportion of the operated cohort.
The possible implications of this data are many. It appears clear now that the SIR connotes poor prognosis for patients with GBM. Although some studies have shown no benefit of pharmacologically suppressing the SIR, some other data do show some benefit from doing so.,,, This is an area for future research and clinical trials. No previous data shows the association of BMSIs with the molecular profile of GBM. However, this study established definite associations between ATRX and p53 and BMSIs. It is possible that a combination of BMSI levels/thresholds can, in the future, be developed as a surrogate marker for a particular tumour molecular profile.
Distinguishing between treatment change and tumour recurrence during the post-treatment follow-up of a patient with GBM remains difficult., It has recently been shown that the levels of NLR decrease following treatment with surgery, radiation and temozolamide. This decrease in the NLR during and after chemo-radiation was an independent prognostic factor for survival in GBM. This suggests the possibility of using levels of BMSIs as markers to distinguish radiation necrosis from recurrence, especially when serially monitored over time. This would require prospective longitudinal studies to establish.
Several BMSIs were deranged in patients with GBM when compared with healthy controls. Clinical features such as altered sensorium at presentation, raised ICP at presentation and poor pre-treatment KPS correlated with changes in the BMSIs suggestive of an SIR. Loss of ATRX and lack of overexpression of p53 also correlated with an inflammatory phenotype of changes in CCs and CCRs. Thresholds that affected PFS could be identified for the ANC, AMC, NLR, PLR and LMR. Derangement of any one of these parameters above the determined thresholds correlated with poorer survival.
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Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]