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Alpha Internexin: A Surrogate Marker for 1p/19q Codeletion and Prognostic Marker in Anaplastic (WHO grade III) Gliomas
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.293453
Keywords: 1p/19q codeletion, anaplastic gliomas, ATRX, IDH, INA, surrogate marker
Diffuse gliomas are a common type of primary brain neoplasms in adults. In the WHO 2007 classification of tumors of the central nervous system (CNS), they were classified purely based on their morphological appearance and graded on a scale of II–IV of increasing malignancy, with histological examination of an adequately sampled tissue being the “gold standard” for diagnosis.[1],[2],[3] However, the recent WHO 2016 classification of CNS tumors defines gliomas by both histological and molecular parameters with the generation of an integrated diagnosis based on phenotypic and genotypic features of each tumor entity. According to this classification, isocitrate dehydrogenase (IDH) gene mutations (IDHmut) and 1p19q codeletion are tumor defining factors for diffuse oligodendrogliomas (ODG) and anaplastic oligodendrogliomas (AO). On the other hand, diffuse astrocytomas (DA) and anaplastic astrocytomas (AA) are defined as tumors that lack the 1p19q codeletion and demonstrate loss of expression of the Alpha thalassemia/mental retardation syndrome X-linked gene (ATRX loss) and express mutant p53 protein. Given the results of molecular testing, oligoastrocytomas remain as a “Not Otherwise Specified (NOS)” entity. At present, immunohistochemistry (IHC) to demonstrate IDH1(R132H) mutant protein, i.e., IDH1 positivity, is considered as a surrogate for the mutational status of IDH1(R132H), with DNA sequencing for rare IDH1&2 mutations being advocated for tumors which are negative for IDH1(R132H) on IHC, which in turn enables the identification of a small group of the IDH-wild type (IDHwt) diffuse gliomas.[4],[5] At present, for assessing 1p/19q chromosome copy number status, two major methodologies exist – polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) assays and fluorescence in-situ hybridization (FISH). LOH assays are based on comparing multiple polymorphic alleles in tumor DNA versus normal blood DNA. Because not all alleles are informative, it is necessary to amplify multiple loci for each chromosomal arm. In addition, the assay requires available normal DNA, possibly delaying test results. Both these drawbacks limit the applicability of LOH assays to study 1p19q status. FISH uses fluorescence-labelled probes to study chromosomes directly on tissue sections, which can be a time-consuming undertaking; it nevertheless preserves tissue architecture and is, therefore, a preferred procedure in diagnostic laboratories. Other techniques may also impact testing in the near future, for example, array comparative genomic hybridization.[6] The currently available methodologies for assessing 1p/19q codeletion status, namely PCR-based LOH assays and FISH technique, are both laborious, cumbersome, equipment, and manpower intensive; costly; difficult to standardize; and require technical expertise, and hence, are not widely available in the economically constraint environment of developing countries. On the other hand, IHC is a methodology which is well established, can be automated, and is relatively easy to standardize, cheaper, and is widely available in most of diagnostic/pathology laboratories worldwide. Recently, there are several novel markers whose role is being explored in gliomas, one of them being alpha internexin (INA), a class IV intermediate filament of approximately 66 kDa present in developing neuroblasts and in the central nervous system of adults.[7],[8],[9],[10],[11],[12],[13] The gene encoding INA maps to 10q24.33 and the INA protein, a major component of the intermediate filament network in small interneurons and cerebellar granule cells, is involved in neuronal development and has been suggested to play a role in axonal outgrowth. INA is expressed in normal neurons and axons but not in the glial cells, suggesting that INA expression in glial tumors should be considered an abnormal feature.[14],[15] In their study, Ho and Liem[16] found that the neurofilament protein expression pattern is a good marker for identifying tumor cell origin and differentiation status. Ducray et al.[13],[17] in their gene expression profiling studies found that 1p/19q codeleted gliomas displayed a proneural gene expression profile, with INA being one of the most overexpressed neuronal genes and its expression highly predicted the presence of 1p/19q codeletion and they proposed INA as a surrogate marker for 1p/19q codeletion status in oligodendrogliomas[8],[9],[12],[16],[17],[18] Although a few studies have given intermediate/contradictory results,[7],[11],[18] INA positivity in gliomas has been consistently reported to be a marker associated with good prognosis and long-term survival.[8],[9],[10],[17],[19],[20] In our previous study,[1] we explored the prognostic significance of the “integrated” histomolecular subgroups in a cohort of WHO grade III anaplastic gliomas (AG) using the molecular markers proposed by the ISN Haarlem guidelines.[4] Because FISH technique is an expensive tool for assessing the 1p19q status, in the present study, we studied the expression of the novel marker INA by IHC in our cohort of patients to explore its potential to serve as a surrogate marker for 1p/19q codeletion status in AOs and to assess its prognostic value in patients with AGs.
In this study, we performed IHC for INA expression on the retrospective cohort that is detailed in our previous study.[1] The study was carried out among 91 adult patients diagnosed with AG. Patient selection, pathology review, methodology for IHC for IDH1(R132H) and ATRX, sequencing for IDH status, FISH for 1p/19q chromosome copy number status, clinical data, patient follow-up, and survival have been published in our prior study.[1] IHC for INA expression was performed on 4-μm tissue microarray (TMA) sections using anti-INA antibody (1:500, Abcam, UK Cat No: ab7259). Briefly, following the initial processing steps, the slides were incubated overnight with the primary antibody. This was followed by incubation with the secondary antibody (MACH1, Biocare medical), and 3, 3'-diaminobenzidine (Sigma-Aldrich, St Louis, Missouri, USA) was used as chromogenic substrate. We used normal cerebellum as the positive control with each batch of staining and the entrapped neurons, and traversing axons in tumor tissue served as positive internal controls in INA negative tumors [Figure 1]. The cytoplasmic staining intensity was graded as +2 (strong positive staining), +1 (weak positive staining), and 0 (negative/no staining); the labelling index (LI) for INA was expressed as the percentage of cells that were positively stained (+2 staining intensity) among the total number of cells that were counted. In our study, only a staining intensity of +2, i.e., strong positive staining was considered to label the tumor for INA and the result was given as positive (LI >10%) or negative (LI ≤10%).[7],[8],[9],[10],[13],[17]
Statistical analysis Data were analyzed using the statistical package SPSS15 (Chicago, Illinois). Variables were tested for normal distribution and nonparametric tests were used where required. Chi-square test was used to assess the molecular parameters and pathological distribution. Student's t-test and analysis of variance (ANOVA) were used to study the relationship between quantitative and qualitative variables with two or more than two categories, respectively. Spearman rank correlation coefficient was used to assess significant associations between different molecular markers and AG subgroup alterations and intermarker correlation. RFS or OS were analyzed using Kaplan–Meier curves. Factors that were significant in univariate analysis (P < 0.05) were subjected to multivariate analysis (Cox regression models). Significant correlation between two parameters was taken at 95% confidence interval.
Descriptive statistics The study cohort comprised 91 patients of AGs with a male to female (gender) ratio of 2.6:1. The median and mean age at diagnosis was 36.0 and 37.6 ± 1.05 years, respectively (range: 19–64 years).[1] The histomolecular subgroups that were derived earlier following testing for IDH (R132H) and sequencing for other rare IDH1&2 mutations, ATRX and p53 expression, and 1p19q codeletion status, included 48 AO, 35 AA, and 8 AA-IDHwt, constituting 52.7%, 38.5%, and 9% of cases, respectively. Recurrence was seen in 55/91 cases (60.4%) during the study period (21 AO, 26 AA, and 8 AA-IDHwt). The median and mean overall survival (OS) for the cohort was 69.3 and 63.8 ± 2.8 months with a range of 24.4 to 92.4 months, whereas the median recurrence free survival (RFS) was 53.4 months with a mean of 53.1 ± 2.7 months with a range of 4.6 to 87.7 months.[1] Expression of alpha internexin in the histomolecular subgroups of anaplastic gliomas and intermarker correlation According to IHC results, INA expression was strong cytoplasmic with nil to weak nuclear staining of the tumor cells [Figure 1]. The frequency of INA expression across the diagnostic subgroups of AG is detailed in [Table 1].
Analysis for intermarker correlation [Table 2] showed that INA positivity not only correlated significantly with 1p/19q codeletion status (P < 0.001 and Rho = 0.804) but it also had a similar molecular association as 1p/19q codeletion, in that, its expression showed a positive correlation with IDHmut (P = 0.006 and Rho = 0.307) and a negative correlation with ATRX loss (P < 0.001 and Rho = −0.402). Because a few studies have suggested INA positivity on IHC as a surrogate marker for oligodendrogliomas and the 1p/19q codeleted status,[9],[13],[17],[21] we explored the role of INA as a surrogate marker for 1p/19q chromosome codeletion status in AG and found that INA positivity on IHC showed a significant positive correlation with 1p/19q codeletion with a sensitivity of 87.5%, specificity of 93.0%, a positive predictive value (precision) of 93.3%, and a diagnostic odds ratio of 93:1 in AGs [Figure 2], proving that INA positivity on IHC is a good surrogate marker for 1p/19q codeletion status in WHO grade III gliomas. In summary, INA expression showed a significant positive correlation with AO (P < 0.001 and Rho = 0.804) and negative correlation with AA (P < 0.001 and Rho = −0.646) and AA-IDHwt (P = 0.006 and Rho = −0.307) [Table 1].
Prognostic significance of alpha internexin expression in the integrated diagnostic subgroups of anaplastic gliomas On univariate survival analysis, in the entire group of AG, INA positivity was associated with significantly prolonged median OS compared to INA negative tumors (92.4 months v/s 45.1 months, respectively; P < 0.001). Similarly, median RFS was also significantly more in INA positive tumors when compared to INA negative tumors (67.5 months v/s 36.5 months; P < 0.001) [Figure 3]a, [Figure 3]b and [Table 3]. Furthermore, within AOs, INA positivity significantly improved RFS (P = 0.022), with the OS trending towards significance (P = 0.094) [Figure 3]c, d and [Table 3].
In our study cohort of 91 AG tumors, INA positivity on IHC was seen in 45 cases, whereas the remaining 46 were negative. The staining characteristic of INA on IHC was robust with strong cytoplasmic staining of the neurons and axons (positive internal control) and tumor cells, with nil to very weak staining of nuclei. Although INA is normally not expressed in glial tissue, its expression in glial tumors seems specific and has been proposed to be a good marker for identifying tumor cell origin and differentiation status.[7],[8],[9],[10],[11],[12],[13],[14],[15],[16] Previous studies in the era of WHO 2000 and 2007 classification of CNS tumors, which were based purely on histomorphological criteria had found INA expression to be significantly more frequent in oligodendrogliomas than in astrocytic tumors and associated with better progression free survival than the INA-negative oligodendrogliomas.[8],[9],[10],[17],[19],[20] Further, studies have shown INA to be overexpressed in 1p/19q codeleted gliomas,[13],[17] and even though a few other studies reported discordant results,[7],[11],[18] numerous studies have proposed INA as a surrogate marker for 1p/19q codeletion status in oligodendrogliomas[9],[10],[13],[17],[19],[20] and as a marker of good prognosis, as well as long-term survival in gliomas.[8],[9],[10],[17],[19],[20] However, the correlation of INA expression with the histomolecular subgroups of AG, as per the recent WHO 2016 classification, has not been described so far. In the present study, we classified AGs based on the current WHO 2016 classification of CNS tumors where IDH mutation and 1p/19q codeletion are markers defining AO and found that INA positivity showed a significant positive correlation with AO and negative correlation with AA (IDHmut and IDHwt). INA positivity not only correlated significantly with 1p/19q codeletion but also had a similar molecular profile as 1p/19q codeletion, correlating positively with IDHmut and negatively with ATRX loss.[4],[5],[22],[23],[24],[25],[26],[27],[28] On exploring the role of INA positivity on IHC as a surrogate marker for 1p/19q codeletion and by extension for the diagnosis of AOs, we found that INA positivity showed a positive predictive value (precision) of 93.3%, with a diagnostic odds ratio of 93:1, proving that INA positivity on IHC is a good surrogate marker for 1p/19q codeletion and by extension is a potential diagnostic marker for AO in WHO grade III AGs. These findings are consistent with the published literature,[8],[12] where the sensitivity was 96–100% and specificity was 70.8–86%, with a positive predictive value of 72.5–76%. In line with the previous studies that have proposed INA to be a good prognostic marker,[8],[9],[13],[17] in our study, on univariate survival analysis in AGs, INA positivity was associated with significantly prolonged survival with a 47.3 and 31 months increase in median OS and RFS, respectively, compared to INA-negative tumors. Furthermore, the current WHO 2016 classification is inherently prognostic due to the incorporation of the tumor genotype in defining diagnostic entities, where the “histomolecularly” defined AOs are known to have a better survival compared to the AAs (IDHmut and IDHwt). Interestingly, we found that within the group of AOs, INA positivity significantly improved RFS with OS trending towards significance, conferring a median survival advantage of 28.6 and 23.1 months, respectively, compared to INA-negative AOs, thus highlighting its prognostic value among AO tumors.
In conclusion, the current methodologies for assessing 1p/19q codeletion status, i.e., PCR-based LOH assays and FISH assay are both equipment and manpower intensive, costly, and are not widely available compared to IHC which is a well-established methodology, can be automated, is easier to standardize, cheaper, and is available in most diagnostic/pathology laboratories. We propose INA positivity on IHC as a surrogate marker for 1p/19q codeletion and by extension a diagnostic marker for AOs and a prognostic marker in AGs. Although this study has been carried out on WHO grade III gliomas, in our view, the results can be extrapolated to WHO grade II/diffuse gliomas. In a resource-constraint setting, we propose inclusion of INA staining along with the recommended panel for IDH mutations for the diagnosis and classification of gliomas as a practical adaptation of the recommendations of the WHO 2016 classification of CNS tumors. Further validations on other independent and larger cohorts of diffuse gliomas would further strengthen the findings of our study.[29],[30] Acknowledgements We kindly acknowledge Prof. GV Giri, Kidwai Hospital, Bangalore for his kind support, and thank Nagabhishek Natesh, Prem K, Chandrashekar MR, Manjunath R, and Raju B from NIMHANS, Bangalore for their help with collection of tumor samples, patient coordination, technical, and statistical assistance. We would also like to thank Medgenome Labs Pvt Ltd, Bangalore for their technical assistance in gene sequencing and analysis. Funding This study was funded by “0546-Neuro-Oncology Research Fund” NIMHANS, Bangalore, India. Financial support and sponsorship Nil. onflict of interest All the authors disclose that there is no conflict of interest relevant to the subject matter under consideration in this article. The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this paper.
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]
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