Evaluation of Immunohistochemical Expression of ALK-1 in Gliomas, WHO Grade 4 and Its Correlation with IDH1-R132H Mutation Status

Background: Glioblastoma (GB), a grade 4 glioma is the most common primary malignant brain tumor in adults. Recently, the mutation status of isocitrate dehydrogenase (IDH) has been crucial in the treatment of GB. IDH mutant cases display a more favorable prognosis than IDH-wild type ones. The anaplastic lymphoma kinase (ALK) is expressed as a receptor tyrosine kinase in both the developing central and peripheral nervous systems. Increasing lines of evidence suggest that ALK is over-expressed in GB and represents a potential therapeutic target. Objectives: The goal of the current study was to investigate ALK-1 immunohistochemical expression in gliomas, grade 4, besides its correlation with IDH1-R132H mutation status and the clinicopathological parameters of the tumors. Material and methods: Seventy cases of gliomas, grade 4 were tested for immunohistochemical expression of ALK-1 & IDH1-R132H in the tumor cells. Results: ALK-1 immunoexpression was detected in 22.9% of our cases and IDH1-R132H mutation was detected in 12.9% of them. ALK-1 expression (100%) was only detected in the more aggressive IDH R132H-negative GBs. ALK-1 expression was also noted in the larger-sized tumors, more in males and patients older than the mean age. Conclusion: Our results suggest that mutations in ALK-1 may predict a more dismal prognosis since ALK expression was only noted in IDH-R132H negative GBs known to have a considerably poorer outcome compared to IDH-R132H mutant cases. GBs with detectable ALK-protein expression could potentially experience substantial clinical advantages through the utilization of newly introduced ALK inhibitors allowing personalized treatment to a subset of patients. Hence, future studies targeting ALK in IDH wildtype Glioblastomas including clinical trials on larger scales are recommended.


Introduction
Glioblastoma (GB), classified by the World Health Organization (WHO) as a grade 4 glioma, is the most common and deadly primary malignant tumor affecting the central nervous system (CNS) in adults accounting for approximately 60% of all CNS tumors [1].It has a grim prognosis, with an average survival time of 15 months [2].
The development of glioblastoma involves a complex array of genetic and epigenetic alterations, which result in various subsets of mutations.Some of these mutations have been shown to impact survival rates and response
Targeted therapy approaches for IDH-mutant gliomas involve direct targeting of mutant IDH and epigenetic modulators.Additionally, targeting essential metabolic enzymes, DNA repair enzymes, redox homeostasis, and immunotherapies in order to improve treatment outcomes [7].The anaplastic lymphoma kinase (ALK) gene, located on chromosome 2p23 belongs to the insulin receptor superfamily of receptor tyrosine kinases [8].Full-length ALK is specifically expressed during embryogenesis in the developing central and peripheral nervous systems and seems to play a role in regulating cell proliferation and differentiation [9].
Studies have indicated that the presence of ALK expression alone can activate tumorigenic functions in GB.In pre-clinical models, depleting ALK through knockdown or inhibitory antibodies has resulted in reduced tumor  [10].
Developing effective ALK inhibitors that can penetrate the blood-brain barrier poses a significant challenge.If researchers can identify ALK overexpression as a predictive marker for sensitivity to new ALK inhibitors, it would have important implications for the treatment of GB.This additional information could help guide targeted therapies and improve patient outcomes in the future [11].
T h i s s t u d y a i m e d t o i n v e s t i g a t e A L K -1 immunohistochemical expression in gliomas, grade 4, besides its correlation with IDH1-R132H mutation status and the clinicopathological parameters of the tumors.

Retrieval of Cases and Material Collection
This cross-sectional analytical study included 70 formalin-fixed, paraffin-embedded full-face tumor tissue sections of retrospective cases that were diagnosed as glioma grade 4 (GB).The cases were collected from the Pathology Department at EL Kasr El Aini Hospital over a 19-month period between July 2021 and January 2023.
The clinical data including patient age, gender, tumor site and size, and history of recurrence were recorded from the Pathology requests and reports of the cases.For the sake of confidentiality, the names of the patients were replaced by an ID number and this number was used afterward on the glass slides and in the datasheet.The study was approved by the Kasr Alainy Research Ethics Committee (REC) with an approval number code: MD-218-2021.
Exclusion criteria were cases with missing data and biopsies with extensive crushing, cautery artifacts, or insufficient material for immunohistochemistry.

Histopathological Examination
Each paraffin block was cut by rotatory microtome at 4 μm thickness then mounted on glass slides, stained with hematoxylin and eosin (H&E) for routine histopathological examination, and on charged slides for immunostaining.Confirmation of the histologic diagnosis and grading and recording of the presence of any histologic variant were done according to the WHO classification of central nervous system tumors [12].

ALK-1 & IDH-1 Immunohistochemical Staining & Evaluation
Immunostaining was done using IDH-1 R132H ready-to-use monoclonal mouse antibody (#400100295, clone QM002, isotype IgG1, Dako, Denmark) and ALK-1 ready-to-use monoclonal mouse antibody (#05278783001, clone ALK01, Roche diagnostics, USA).Staining was carried out using an automated immunostainer from Dako.Negative and positive control sections were included for each assay.Sections obtained from oligodendroglioma were used as the positive control for IDH-1 and sections obtained from a known ALK protein-positive anaplastic lymphoma were used as the positive control for ALK-1 according to the manufacturer's recommendations.
For IDH-1 evaluation, the immuno-expression was determined by assessing the proportion of positive cytoplasmic staining in tumor cells; cases in which ≥10 % of cells were stained were defined as positive [13].
Cases showing IDH-1 immunoexpression were termed astrocytoma, IDH-mutant, WHO grade 4, instead of the formerly used term; IDH-mutant glioblastoma.However, the term glioblastoma IDH wild type can't be used in our study as it refers to a histologically WHO grade 4 astrocytic tumor which lacks mutations in IDH1/IDH2 and Histone 3 genes, while only IDH1-R132H mutation was examined IDH1 in our study.
As for ALK-1 evaluation, the granular cytoplasmic staining of any number of neoplastic cells was considered positive.Weak or equivocal staining was considered negative [14].

Statistical Analysis
The histopathological and immunohistochemical data were imported into the statistical analysis software SPSS, version 26 (Armonk, New York, United States), for further statistical analysis.Simple descriptive statistics such as the arithmetic mean and standard deviation were used to summarize the quantitative data, while frequencies were employed for qualitative data.For comparison, the Chi-square (χ2) test was performed.A significance level of p-value less than 0.05 was considered statistically significant.

Results
This study included 70 cases of gliomas, WHO grade 4. Their age ranged from 5 to 76 years with a mean of 48.04 years.Regarding the gender of the studied cases, females were 48% and males were 52 %.The clinicopathological characteristics of the studied cases are summarized in Table 1, Figure 1 illustrates the basic histological features and variants of Gliomas, WHO grade 4.
In our study, we observed cytoplasmic positivity of IDH-1 in 9 out of 70 cases.Hence 12.9% of our cases were classified as Astrocytoma, IDH mutant, WHO grade 4. We found a significant association between IDH-1 expression and age of patients (P value= 0.007) as well as with the mean larger size of the tumor (P value= 0.011).However, we did not report any significant correlation between IDH-1 expression and gender (0.815), tumor site (0.520), histological variants (0.879), or history of recurrence (0.167).
Regarding ALK-1 expression, we detected positive cytoplasmic expression in 16 out of 70 cases (22.9%).We did not report any significant correlation between ALK-1 expression and any of the studied parameters: age (0.533), gender (0.082), tumor site (0.096), tumor size (0.064), histological variants (0.354), or history of recurrence (0.188).However, it is worth noting that a majority of the ALK-1 positive cases were older than the mean age, and ALK-1 expression was more frequently observed in larger tumors.
A summary of the correlation between ALK-1 and IDH-1 expression and various pathological characteristics is presented in Table 2, Figure 2 displays the immunostaining of Gliomas, WHO grade 4 with IDH-1 and ALK-1.Among the cases studied, 45 showed negative staining for both ALK-1 and IDH-1.Interestingly, ALK-1 expression (100%) was exclusively observed in the more aggressive IDH-R132H negative GB cases, as shown in Table 3.

Discussion
In some studies, overexpression of ALK in GB has been associated with increased tumor cell proliferation, poorer overall survival, and progression-free survival, suggesting a potential prognostic role.However, the available data remains limited and conflicting [8].
Despite the positive outcomes observed in pre-clinical and in vivo studies conducted on GB when ALK inhibitors were applied, there have been no clinical trials demonstrating a significant impact on the survival of GB patients with the use of ALK inhibitors, likely due to their limited ability to penetrate the blood-brain barrier and the challenges in achieving therapeutic concentrations in the brain [8,11].However, the second and third-generation ALK inhibitors have exhibited notable effectiveness in treating CNS metastases in ALK-rearranged non-small cell lung cancers (NSCLC), indicating their ability to penetrate the brain effectively [10,15].
In the present study, ALK-1 expression was higher in cases older than the mean age compared to those younger than the mean age, disagreeing with Ferguson et al. 2016 [20], yet, consistent with Karagkounis et al. 2017 [17], Elsers et al. 2021 [18], Hakeem et al. 2021 [19] as they all noted more frequent ALK overexpression in older cases.This difference might be ascribed to variations in environmental factors and sample size.As for the correlation of ALK-1 immunoexpression with the gender of the studied cases, we reported higher expression in males (14/16); a finding consistent with Karagkounis et al. 2017 [17], Elsers et al. (021 [18], and Hakeem et al. 2021 [19].The established male predominance in GBs might clarify those concordant findings. Concerning the relationship between the site of the tumor and ALK-1 immunoexpression, the frontal lobe showed the most frequent expression (7/16), disagreeing with Elsers et al. 2021 [18] where the parieto-occipital region showed the highest ALK-1 expression.
In our study, higher ALK-1 immunoexpression was noted in the tumors larger than the mean size (13/16); a finding in harmony with Elsers et al. 2021 [18], and Hakeem et al. 2021 [19].No statistically significant correlation was be detected between ALK-1 expression and the histologic variants of the tumors in our study.Regarding the association between tumor recurrence and ALK-1 immunoexpression, we observed positivity in only one recurrent case (8.3%), and the association was not statistically significant, which aligns with the findings of Hakeem et al. 2021 [19].However, Azab 2023 [21] demonstrated a significant correlation between tumor recurrence and the ALK staining score of tumors.This difference may be attributed to the smaller number of recurrent cases in our study compared to non-recurrent cases.In view of IDH-1 immunoexpression in the present study, 12.9% of our cases were revealed to be Astrocytoma, IDH mutant, WHO grade 4, as cytoplasmic positivity of IDH-1 was detected in 9 cases (9/70; 12.9%), in consistency with Liu et al. 2016 [22], and Houssaini et al. 2022 [23] where IDH-1 was expressed in 12.8% & 11.8% of their GB cases respectively.
Regarding the correlation of IDH-1 expression with the mean age, we found a statistically significant association, and most IDH-1 negative cases were relatively older age, consistent with Liu et al. 2016 [22], WHO (2021) [12], Houssaini et al. 2022 [23] and Kiraz et al. 2022 [24].However, the latter's results didn't reach statistical significance.Regarding the relationship between the gender of the studied cases and IDH-1 immunoexpression, we found a statistically insignificant association, being compatible with Liu et al. 2016 [22], and Houssaini et al. 2022 [23].
As for the correlation between the site of the tumor and IDH-1 immunoexpression, the frontal lobe showed the most frequent expression (3/9), unlike Liu et al. 2016 [22] who noted the most frequent expression in the temporal lobe.Even though, these relations were statistically insignificant in both studies.Concerning the relationship between the mean size of the tumor (5 cm) and IDH-1 immunoexpression, we found a statistically significant association and positivity was noted only in tumors larger than the mean size and none of the smaller ones, being inconsistent with WHO CNS5 by WHO (2021) [12], and Kiraz et al. 2022 [24] who observed that most larger tumors are IDH-1 negative.This difference might be attributed to the difference in the sample size.
With respect to the relationship between the histological variants of the tumor and IDH-1 immunoexpression, IDH-1 expression was expressed in 20% of GB with oligodendroglial features (GB-Os) and in 12.7% of GB, classic cases.This finding was consistent with Hinrichs et al. 2016 [25], and Appin et al. 2013 [26] who noted that GB-Os show consistently increased frequencies of IDH-1 mutations compared to classic GBs.This has received particular attention given the association of IDH-1 mutations with secondary GBs & oligodendrogliomas with improved clinical outcomes according to Riemenschneider et al. 2010 [27], and Appin et al. 2013 [26] studies.With respect to the relationship between tumor recurrence and IDH-1 immunoexpression, the frequency of IDH-1 expression was higher in the recurrent cases (25% of recurrent cases while 10.3% of non-recurrent cases), this finding was compatible with Choi et al. 2018 [5], Han et al. 2020 [4] and, WHO (2021) [12] who stated that lower-grade glioma with IDH mutation often recur with having undergone malignant transformation to a higher grade.
In our study, we detected ALK expression (16/16; 100%) in IDH-R132H negative GB cases, which is known to have a significantly worse prognosis compared to IDHmutant grade 4 astrocytomas, as reported by Gritsch et al. 2022 [28].Our findings align with the studies conducted by Ferguson et al. 2016 [20], Karagkounis et al. 2017 [17], and Bu et al. 2021 [29], all of which observed higher ALK-1 expression in IDH1-R132H negative or wildtype GBs.The survival analysis performed by Bu et al. 2021 [29] confirmed that the presence of ALK mutation was associated with shorter overall survival in glioma patients, particularly in IDH-WT-GB patients.Therefore, it is crucial to identify ALK mutations in the care and management of glioma patients due to the potential for a worse prognosis and the possible eligibility for ALKtargeting therapies.However, it is worth noting that another study reported that despite observing high ALK expression in their GB cases, ALK overexpression did not correlate with prognosis in their study [17].
In conclusion, current evidence reveals that ALK mutations are absent in IDH mutant, astrocytoma grade 4 cases, exclusively positive in the more aggressive IDH-negative-GBs and likely play a role in glial tumor cell proliferation, as their expression was significantly higher in larger tumors, indicating a worse prognosis.Furthermore, new targeted ALK inhibitors could offer significant clinical benefits for GB cases with detectable ALK-protein expression, allowing personalized treatment for a subset of patients.Further studies, including clinical trials, are needed to elucidate the modality of selecting the candidates among GB patients that are most likely to benefit from ALK-1 targeted immunotherapy.This includes which antibody clone to be used and the threshold for positivity.As for the prognostic impact of ALK-1 expression, more studies addressing the effect of ALK-1 immunoexpression on GBs outcome are still required, to resolve this controversial issue in the literature.

Table 1 .
The Clinicopathological Characteristics of the Studied Cases

Table 2 .
Correlation of IDH-1 and ALK-1 Expression with Various Clinicopathological Characteristics among the Studied Gliomas, WHO Grade 4 Cases