Prognostic Value of Immunohistochemical Expression of MTAP and AKIP1 in IDH1 Mutant Astrocytoma

Background: Definite treatment for glioma is not exist, and with increased drug resistance, more effort should be paid to identify new prognostic biomarkers and molecular targets for therapy for glioma patients. Aim: The current study aimed to evaluate the immunohistochemical (IHC) expression of MTAP and A-Kinase Interacting Protein 1 (AKIP1) in astrocytoma and to investigate their association with the clinicopathological characters of these cases. Methods: Totally 66 cases of astrocytoma patients involved in this study. Cases underwent tumor resection and tissue sections were stained with MTAP, AKIP1 and IDH1 by IHC and evaluated in different grades of astrocytoma and their association with survival and response to therapy was investigated. Results: High AKIP1 expression was positively correlated with treatment resistance and progressive disease. Positive IDH and retained MTAP expressions had shown better treatment response rather than negative IDH and lost MTAP. High AKIP, negative IDH and loss of MTAP expressions were significantly associated with poor survival outcome. Conclusion: Irrespective to grade and IDH status, the loss of MTAP immunoreactivity and high AKIP1 expression are predictive factors in astrocytoma, and they may be used as a biomarker for guiding astrocytoma management and prognosis surveillance.


Introduction
Glioma is the most common primary neoplasm of CNS worldwide.According to Yehia et al., in Egypt, gliomas account for 37.3% of primary CNS tumor.Among all its subtypes, astrocytoma is the most frequent glial tumors (Yehia et al., 2018).The new WHO classification of CNS tumor introduces major changes in form of integration of molecular characteristics in addition to histologic features in the classification and prediction of the outcome of astrocytoma patients (Louis et al., 2021).
Direct DNA sequencing is the major competitor to immunohistochemistry in diagnosis and risk stratification of tumors, but it is unavailable at most medical centers.Accurate molecular tests need sufficient tissues, that is unavailable in most cases as all the small biopsies placed immediately in formalin.Concordance between IHC and molecular testing of IDH is about 88% to 99% and some studies revealed that IHC detected mutations more than sequencing (Zouet al., 2015).That is why searching for immunohistochemical markers that replace molecular testes and can reflect the nature and prognosis of the disease will be valuable.According to the mutations in

Prognostic Value of Immunohistochemical Expression of MTAP and AKIP1 in IDH1 Mutant Astrocytoma
isocitrate dehydrogenase (IDH), astrocytoma is classified into 3 subtypes: IDH mutant, IDH wild type, and not otherwise specified.The IDH mutation subtype has the potential to progress to glioblastoma multiforme (GBM) (Nishikawa et al., 2022).
CDKN2A HD is one of the markers that is assessed using molecular procedures as genomic hybridization, or multiplex ligation-dependent probe amplification (Brat et al.,2020) and (Jeuken et al., 2006).The finding of Satomi et al., (2021), work supports that the loss of MTAP (5'-methylthioadenosine Phosphorylase) immunoreactivity represents an acceptable predictive surrogate for CDKN2A HD.Homozygous deletion of MTAP is frequently associated with many types of tumors due to loss of its tumor suppression function through its role in metabolism of polyamine and purines (Tang et al., 2018).A-kinase interacting protein 1 (AKIP1) contributes to the activation of p65 and NF-kappaB.It also has a role in the process of different diseases such as cancer and inflammatory diseases.AKIP1 may have a role in the development, progression, and drug resistance of astrocytoma (Shen and Yao 2021).
The aim of this work is to evaluate the IHC expression of MTAP and AKIP1 in astrocytoma and to investigate their association with the clinicopathological characters of these cases.

Materials and Methods
From January 2018 to January 2022, sixty-six formalinfixed, paraffin-embedded tissue blocks of astrocytoma were gathered from the pathology departments' archives at the Faculty of Medicine at Zagazig University.The following cases are among those involved: There are 12 cases of grade 2 diffuse astrocytoma, 28 cases of anaplastic astrocytoma (G3), 26 cases of glioblastoma (G4).clinicopathological information was extracted from the hospital's medical records.
Age above 18 and under 80 at the time of surgery, a histologic diagnosis of primary low-or high-grade astrocytoma, and the availability and suitability of tumor tissues removed after surgery for immunohistochemistry (IHC) detection were all inclusion criteria.Grade 1 astrocytoma and cases with insufficient biopsy were excluded.Grading of astrocytoma was done based on the 2016 WHO classification (Louis et al., 2016).The Ethics Committee of Faculty of Medicine Zagazig university had approved this study by number (#9447) in accordance with Declaration of Helsinki.
According to feasibility, patients at the neurosurgery department underwent gross total resection or subtotal maximum safe resection or stereotactic biopsy.The patients were treated at Zagazig University's clinical and medical oncology departments.The patients received their treatment at clinical Oncology Department at Zagazig University, Radiotherapy (Rth) was delivered by three dimensional conformal radiotherapy in incomplete resected or surgically inaccessible, symptomatic low grade glioma (G2), dose 50 -54 Gy/ 25-27 fractions.The patients with karnofsky scale (KPS) ≥ 60 diagnosed by WHO G3,4, standard radiotherapy was delivered 60 Gy/30 fractions with concurrent and adjuvant temozolomide (TMZ), while the patients with poor KPS (< 60), Rth was delivered in hypo fractionation schedule 40 Gy/15 fractions ± concurrent or adjuvant temozolomide.MRI brain with gadolinium-enhancement was requested from 2 -8 weeks after Rth then every 3 -6 months, other radiological evaluation performed to exclude the metastatic spread.Data was collected from patient's files.Hematoxylin and eosin-stained histological sections of tumors were reviewed by 3 expert pathologists.

Immunohistochemistry
Using the standard streptavidin-biotin labeling technique and the LSAB kit (Dako, Glostrup, Denmark) with appropriate positive and negative controls, immunohistochemistry was done.From the paraffinembedded tumor tissue specimens, sections of 4mm were cut, then deparaffination.Using xylene, graded ethanol and microwave heating, rehydration and antigen retrieval for the sections were performed.After blocking the peroxidase activity, the sections were incubated with primary antibody.
The sections in the next day were incubated with Goat anti-Rabbit IgG (H+L).Secondary Antibody, HRP (1:10,000, Invitrogen, Waltham, MA) at room temperature for 1 hour.Finally, the staining and counterstaining of the sections were carried out using diaminobenzidine (Sigma-Aldrich, Louis, MO, USA) and hematoxylin (Sigma-Aldrich, Louis, MO).
Any nuclear and cytoplasmic positivity for IDH1 was considered IHC-positive and for scoring cases divided into negative, no tumor cells stained; partly positive, presence of stained and not stained tumor cells; and completely positive, all identifiable tumor cells stained and for statistical evaluation positive cases were categorized as IDH mutant and negative cases as IDH wild type (Preusser et al., 2011).
MTAP immunohistochemistry identified as a cytoplasmic staining with or without nuclear staining in normal cells (as endothelial cells).The loss of immunoreactivity to MTAP is defined as absence of cytoplasmic staining with keeping the reactivity to the internal positive controls; tumors that showed cytoplasmic MTAP immunoreactivity were considered to retain MTAP staining (Chapel et al.,2020).
Cases that failed to stain positive for internal positive controls were considered uninterpretable.

Clinicopathological finding and immunohistochemistry
Resume of clinicopathological features of involved cases represented in Table 1.Studying IDH status shows that there was a statistically significant association between wild type IDH and higher grade (P< 0.02).IDH mutation was noticed among cases with retained MTAP (P=0.04) and lower AKIP1 expression (P=0.001).The IDH mutant astrocytoma has a better prognosis than wild type.There was a statistically significant increase in median of progression free survival (P=0.006) and overall survival (P=0.002)among mutant cases (Table 2, Figure 1).
According to MTAP expression, cases classified into retained 28 /66 (42.4%) and lost 38/66 (57.6%).There was a significant association between retained MTAP expression and high AKIP1 expression (P< 0.001).It was found that median of progression free survival and overall free survival decreased among lost MTAP cases (P< 0.001).Loss MTAP is a strong predictor of short progression free survival and overall free survival (Table 2, Figure 2).Table 3, Figure 3, shows a significant association between AKIP1 expression and histological grade of astrocytoma (p=0.01).Also, it was found that 2. Treatment outcome, survival, and progression analysis (Figure 4): High AKIP1 expression was positively correlated with the higher AKIP1 expression was significantly associated with the progression of disease (P=0.001).Cases with low and mild expression of AKIP1 had better overall survival.

Discussion
Till now a definite treatment for astrocytoma has not existed.The increased incidence of drug resistance and restricted therapeutic options for astrocytoma requires more effort to identify prognostic biomarkers and new molecular targets for therapy to improve the long-term prognosis in astrocytoma patients.
The histopathological assessment of lower-grade astrocytoma in patients with IDH-mutation is particularly challenging, despite the revolution caused by the discovery of the IDH mutation.Homozygous deletions of the CDKN2A/B have a great prognostic value in IDH-mutant astrocytoma and incorporated in its updated classification.The adjacent location of MTAP gene to CDKN2A exposes it to frequent co-deleted (Marjon et al., 2016), explaining the good concordance between CDKN2A and the lack of MTAP staining.According to Satomi et al., (2021), MTAP immunohistochemistry was used as a proxy for CDKN2A homozygous deletion.Furthermore, loss of MTAP may increase the stemness of GBM and make it more susceptible to purine deprivation (Hansen et al., 2019).That is why we investigated the expression of MTAP in the studied group and evaluated its prognostic value irrespective to IDH status.
A-kinase interacting protein 1 (AKIP1), is one of the molecules that is suspected in initiation and progression of astrocytoma.It has a regulatory role in protein kinase A catalytic subunit and P65 interaction with subsequent activation of NF-kappaB cascade inducing resistance to treatement and astrocytoma stem cell differentiation (Liu et al., 2019) and (Feng et al., 2019).Silencing of AKIP1 associated with suppression of the tumor cell progression, opening door for the trial of its implication in cancer treatment (Chen et al., 2022).In the current work, IDH mutation was detected in low grade astrocytoma more frequently than in high grade astrocytoma and represents a good prognostic factor independent on the tumor grade, similar finding was reported by previous studies of Hartmann et al., (2010).According to Le et al., (2020), IDH mutation was valuable molecular predictor of response to Temozolomide in GBM and, its mutation was positively correlated with the presence of O6-Methylguanine-DNA methyltransferase.
In agreement with previous reports of Von Deimling (Von Deimling et al., 2018), there is a poor PFS and OS in patients of wild IDH in comparison to mutated IDH patients.In this study, IDH mutation was significantly associated with MTAP retained expression (p =0.04), and wild IDH associated with AKIP1 high expression (p= 0.001).As far as we know no previous work investigated this relation.The current work reported that MTAP expression is retained frequently in lower grade astrocytoma.On the other hand, loss of its expression observed in higher grade astrocytoma.These findings agree with Li et al., (2021), finding that concluded that copy number loss of MTAP, was found to be significantly enriched in GBM.Also, according to Becker et al., (2015) MTAP expression was still present in 85% of low grade pilocytic astrocytoma.
In terms of the relationship between MTAP expression and survival, our research showed that maintaining MTAP expression is associated with a favorable prognosis regardless of tumor grade and IDH status whereas losing MTAP expression is associated with a poor prognosis in different grades of astrocytoma.Additionally, Satomi et al., (2021), showed that MTAP deficiency was linked to a trend toward shorter PFS and a significantly shorter OS.According to Singh et al., (2021) MTAP reduction promotes temozolomide resistance.In agreement with the finding of Shen and Yao (2021) the current work revealed that AKIP1 is positively associated with higher WHO grade in astrocytoma patients.
According to previous report (Shen and Yao, 2021) and in accordance with our observations, overall survival (OS) was lowest in patients with AKIP1 higher expression, and greatest in patients with AKIP1 low expression.Furthermore, high AKIP1 expression was positively connected with treatment resistance and a progressing condition, whereas low expression associated with full responses.In other words, high AKIP1 immunohistochemical expression is linked to decreased overall survival and its high level predicts poor survival regardless of the tumor's grade, with a statistically significant rise in median PFS and OS among cases with low and mild AKIP1 expression compared to cases with moderate and high expression in all grades.This association is reasonable as Liu et al., (2020), found that the cytotoxicity of the chemotherapeutic agents can be blocked by the action of AKIP1 that enhances the ability of astrocytoma stem renewal.Moreover, Zhang et al., (2018) found that knockdown of the gene expressing AKIP1 inhibits cell cycle propagation, and arrest tumor growth as well as angiogenesis through deactivating of chemokines CXC motif ligand.
While Feng et al., (2019), reported that AKIP1 high expression not only protects tumor cells against the cytotoxic effect of chemotherapy (temozolomide), but also enhances the self-renew capacity of astrocytoma cells.It also increases the tumor recurrence rate and the drug    Concerning its role in cancer progression, AKIP1 plays an important role in epithelial mesenchymal transformation and metastasis through the carcinogenic process.It stimulates the stemness and increases the invasive capacity of malignant cells by different pathways as β-catenin and HIF-1α pathways (Luo et al., 2022).
To conclude, independent on IDH1 status, MTAP and AKIP1 are prognostic markers in astrocytoma that is why we recommend their evaluation in all cases of astrocytoma even low-grade cases.

Figure 1 . 1
Figure 1. 1 IDH1 Immunohistochemistry: a-a case of anaplastic astrocytoma all cells are not stained (x400).b-diffuse astrocytoma IDH stains scattered cells and most cells of the tumor are not labeled (x400).C. diffuse astrocytoma showing IDH positive immunostaining in all tumor cells immunostaining in all tumor cells (x200).

Figure 2
Figure 2. a. MTAP immunohistochemistry demonstrating the loss of cytoplasmic MTAP staining while the internal control is positively labelled.b.A case of IDH-mutant astrocytoma retaining cytoplasmic MTAP staining (x400).

Figure 3
Figure 3. A-kinase Interacting Protein 1 (AKIP1) Expression in Glioma.The representative image of AKIP1 a. negative, b. mild expression, c. moderate and d. high expression in astrocytoma (x400).

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Figure 4. Kaplan Meier Plots, Left panel: progression free survival, right panel :overall survival stratified by MTAP and AKIP expression.

Table 1 .
Demographic and Clinical Data of the Studied Cases treatment resistance and a progressive disease, while low expressed patients had achieved a complete response, as well as patients with positive IDH and retained MTAP expressions had shown better treatment response rather than negative IDH, lost MTAP expressed patients.MTAP and AKIP expressions are significantly associated with both OS and PFS independent of IDH status (Table4).Relation between progression free survival and overall survival based on IDH, MTAP and AKIP1 expression in relation to WHO grading among the studied cases represented in Table5&6.

Table 3 .
Relation between Clinicopathological Data of the Studied Cases and AKIP1 Expression

Table 4 .
Relation between Survival and AKIP and MTAP1 Expression according to IDH expression:

Table 6 .
Relation between Progression Free Survival and Overall Survival of and AKIP1 Expression According to WHO Grading among the Studied Cases resistance in astrocytoma patients, resulting in patients with AKIP1 high expression having a worse prognosis compared to cases with low AKIP1 expression.