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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 3  |  Page : 598-603
ATRX protein is a potential prognostic marker in clear cell renal cell carcinoma


1 Department of Pathology, Yeungnam University College of Medicine, Daegu, South Korea
2 Department of Pathology, Yeungnam University Yeongcheon Hospital, Yeongcheon, South Korea

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Date of Submission27-Dec-2021
Date of Decision20-Jan-2022
Date of Acceptance23-Jan-2022
Date of Web Publication26-May-2022
 

   Abstract 


Objective: Cancer cells activate either telomerase or alternative lengthening of telomeres (ALT) to maintain telomere length and achieve immortalization. Alpha thalassemia/mental retardation X-linked (ATRX) is involved in chromatin remodeling. Mutations in ATRX genes are associated with the loss of nuclear expression and correlated with the ALT phenotype. ATRX expression has been evaluated in various cancers, especially sarcoma and neuroendocrine tumors, and its clinical significance has been shown to be diverse, depending on the tumor types. The role and prognostic value of ATRX expression in clear cell renal cell carcinoma (CCRCC) have not been elucidated. Methods: We investigated the messenger RNA (mRNA) expression levels of ATRX using the gene expression profiling interactive analysis (GEPIA) database and evaluated the expression of ATRX using immunohistochemical (IHC) staining in 302 CCRCC cases. Results: Loss of ATRX expression was significantly associated with larger tumor size, higher nuclear grade (NG), lymphovascular invasion (LVI), pathologic T (pT) stage, recurrence/metastasis, and stage. Although ATRX was not an independent prognostic factor, patients with loss of ATRX expression showed poor survival. Conclusion: Our findings suggest that loss of ATRX expression could be a potential biomarker for predicting aggressive tumor behavior and poor clinical outcomes in CCRCC.

Keywords: ATRX, biomarker, immunohistochemistry, prognosis, renal cell carcinoma

How to cite this article:
Baek J, Jang NR, Shim YR, Gu MJ. ATRX protein is a potential prognostic marker in clear cell renal cell carcinoma. Indian J Pathol Microbiol 2022;65:598-603

How to cite this URL:
Baek J, Jang NR, Shim YR, Gu MJ. ATRX protein is a potential prognostic marker in clear cell renal cell carcinoma. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Aug 15];65:598-603. Available from: https://www.ijpmonline.org/text.asp?2022/65/3/598/345855





   Introduction Top


Clear cell renal cell carcinoma (CCRCC) is the most common renal cell carcinoma (RCC) (approximately 75% of all RCCs) and has a more unfavorable prognosis than other RCC subtypes.[1],[2] Radical nephrectomy or partial nephrectomy is the standard treatment for localized RCC. However, 20%–40% of patients experience recurrence with a metastatic rate of 5%–15% after surgical resection.[3] Metastatic or inoperable CCRCCs are refractory to conventional chemotherapy and radiotherapy, with a 5-year survival rate of less than 10%.[4] Thus, a large series of studies have been performed over the past decades to identify potential biomarkers to predict disease progression and clinical outcomes. However, sensitive and specific biomarkers for the diagnosis and monitoring of CCRCC remain crucial. The treatments targeting the vascular endothelial growth factor using tyrosine kinase inhibitors, mammalian target of rapamycin (mTOR) inhibitors, and immune checkpoint inhibitors have improved the clinical outcome of CCRCC.[5],[6] However, there is still therapeutic resistance in metastatic CCRCCs. Furthermore, no reliable biomarker is known to assess the progression and act as a guideline to select the best treatment options.

Neoplastic cells activate the telomere maintenance mechanism (TMM) to achieve replicative immortality and unlimited proliferative capacity of cancer cells.[7] The alpha thalassemia/mental retardation X-linked (ATRX) gene encodes the ATRX protein, which is a chromosomal remodeling protein belonging to the SWItch/sucrose non-fermentable (SWI/SNF) family of DNA helicases and plays a crucial role in chromatin regulation and maintenance of telomerases.[8],[9] ATRX mutation leads to the activation of the alternative lengthening of telomeres (ALT) pathway and eventually results in chromosomal instability, tumorigenesis, and metastases.[8],[9]

This study aimed to determine the expression of ATRX genes by immunohistochemistry (IHC) in a large cohort of patients with CCRCC and evaluate its potential clinicopathological and prognostic implications. To the best of our knowledge, there have been no previous studies on the expression and prognostic significance of ATRX in CCRCC.


   Materials and Methods Top


Case selection

After excluding the patients who received preoperative therapies, had insufficient clinicopathologic data, or those with tissue unavailability for immunohistochemical (IHC) staining, we included a total of 302 patients with CCRCC who had undergone nephrectomy between May 2000 and October 2020 at Yeungnam University Medical Center and obtained clinical information from the patients' medical records. The duration of follow-up was evaluated from the date of surgery to the date of death or recent follow-up. The clinicopathologic features included patients' sex, age, tumor size, tumor necrosis, lymphovascular invasion (LVI), tumor, node, metastases (TNM) stage, recurrence/metastasis, and nuclear grade (NG, based on the 2016 WHO Classification of Tumours of the Urinary System and Male Genital Organs).[10] Tumor grading and staging were classified according to the eighth American Joint Committee on Cancer TNM staging system.[11]

Tissue microarrays (TMAs) were constructed from paraffin-embedded blocks of CCRCC tissues. Two to four 1.5 mm tissue cores that represented tumor areas corresponding to each patient were included in the TMAs. Non-neoplastic kidney and liver, gastric mucosa, lymph node, breast cancer, and uterine leiomyoma tissues were included as controls in each TMA section. One section from each TMA was stained with hematoxylin and eosin and further reviewed to confirm the histologic findings of the representative CCRCC.

IHC

IHC staining was performed on 4-μm-thick TMA sections using an automated slide stainer (Ventana BenchMark XT system Ventana Medical Systems, Tucson, AZ, USA) with cell conditioning 1 (CC1) solutions for the following antibody: ATRX (1:100; clone: HPA001906; Atlas Antibodies; Sweden). The primary antibodies were incubated for 40 min at 37°C, followed by amplification using an OptiView Detection Kit (Ventana Medical Systems) with diaminobenzidine as the chromogen and hematoxylin counterstaining, according to the manufacturer's protocol. IHC staining was analyzed without prior knowledge of the clinicopathological data. Consistent with the procedure followed in the previous studies, IHC interpretation was based on nuclear staining regardless of cytoplasmic staining; complete loss of nuclear staining was determined as loss of expression.[9] Normal kidney tubules, glomerulus, inflammatory cells, stromal cells, and endothelial cells served as an internal control for positive expression.

Statistical analysis

Statistical analyses were performed using the IBM Statistical Package for the Social Sciences (SPSS) version 25.0 (IBM Corp., Armonk, NY, USA). Correlations between categorical variables were analyzed using the Chi-square test or Fisher's exact test. Overall survival (OS) and disease-free survival (DFS) curves were estimated using the Kaplan–Meier method, and survival differences were compared using the log-rank test. Multivariate Cox regression analyses were performed using pathological variables that were recognized as significant in the univariate survival analyses. Statistical significance was defined as P < 0.05.


   Results Top


Case review

The cohort of 302 patients had a mean age of 58.4 years (range, 26–93 years). Of these, 222 (73.5%) were male. Tumor size ranged from 0.9 cm to 14.5 cm (mean, 4.47 cm). Follow-up data were available for 283 cases with a mean follow-up time of 85.6 months (range, 0–247 months; median, 75 months). During this period, 29 patients (9.6%) experienced recurrence/metastasis with a median time of 35.1 months (range, 0–191 months), while 38 patients (12.6%) showed CCRCC-specific mortality.

Messenger RNA (mRNA) expression of ATRX genes

We evaluated the gene expression profiling interactive analysis 2 (GEPIA2; http://gepia.cancer-pku.cn) database to identify the RNA-seq mRNA expression of ATRX in RCC from the Cancer Genome Atlas and Genotype-Tissue Expression (GTEx) projects (523 RCC and 100 normal samples). The ATRX mRNA levels were downregulated in RCC samples compared to those in normal kidney samples [Figure 1]. The ATRX gene expression gradually decreased as the cancer stage increased (P = 0.00447) [Figure 2]a. Patients with low ATRX gene expression tended to have shorter OS and DFS than those with high expression (P = 0.0028 and P = 0.0051, respectively) [Figure 2]b.{Figure 1}{Figure 2}

Loss of ATRX expression is related to tumor progression

Normal renal tubular epithelium and glomerular endothelial cells showed strong nuclear expression on IHC staining for ATRX. The ATRX IHC on whole tissue sections was conducted to identify the heterogeneity in 15 cases and showed a homogeneous staining pattern. Loss of ATRX expression was observed in 42.1% (127/302) of CCRCC specimens, respectively [Figure 3]. Loss of ATRX expression was significantly associated with larger tumor size (P < 0.001), higher NG (P < 0.001), higher pathologic T (pT) stage (P = 0.007), LVI (P = 0.004), recurrence/metastasis (P = 0.024), and higher stage (P < 0.001). The relationship between ATRX expression and clinicopathological features is summarized in [Table 1].{Figure 3}{Table 1}

Association of loss of ATRX expression and survival

Patients with loss of ATRX expression were significantly associated with shorter OS (203.051 vs. 218.267 months, P < 0.001) [Figure 4]. The Kaplan–Meier survival curve demonstrated that OS was significantly different according to the tumor size (215.780 vs. 209.353 vs. 134.478 vs. 70.938 months, P < 0.001), status of LVI (89.333 vs. 231.503 months, P < 0.001), NG (P < 0.001), status of recurrence/metastasis (81.702 vs. 237.703 months, P < 0.001), pT stage (242.187 vs. 110.014 vs. 81.930 months, P < 0.001), and stage (245.000 vs. 183.167 vs. 110.959 vs. 66.145 months, P < 0.001). DFS was shorter in patients with CCRCC with loss of ATRX expression (203.074 vs. 218.229, P < 0.001). The CCRCCs with less than 7.0 cm size did not show any significant DFS difference (215.780 vs. 212.074 months), but the average DFS was significantly decreased to 132.577 months in CCRCCs with 7–10 cm size and 67.031 months in those >10 cm (P < 0.001). The CCRCCs with LVI (92.724 vs. 232.368 months, P < 0.001), higher NG (P < 0.001), recurrence/metastasis (60.820 vs. 237.703 months, P < 0.001), higher pT (242.192 vs. 108.822 vs. 76.659 months, P < 0.001), and higher stage (245.000 vs. 183.167 vs. 110.803 vs. 37.655 months, P < 0.001) revealed significantly shorter DFS. In univariate analysis, CCRCCs with more than 4.0 cm size, LVI, recurrence/metastasis, pT2 and pT3 stages, stage III and IV, and loss of ATRX were associated with worse OS and DFS. Among them, LVI, and stage III and IV were the independent prognostic factors [Table 2] and [Table 3].{Figure 4}{Table 2}{Table 3}


   Discussion Top


Cancer cells activate either telomerase or ALT to maintain telomere length and achieve immortalization.[8] ALT occurs in approximately 10%–20% of all tumors and is associated with certain tumor types, with varying impacts on patient prognosis.[7],[10],[11] ALT-associated TMM is commonly detected in tumors of mesenchymal and neuroepithelial origin and is relatively rare in tumors of epithelial origin.[12],[13],[14] The ATRX genes are intimately linked to the ALT phenotype.[7],[15] ATRX loss has been strongly linked to DNA damage, replicative stress, and ALT[16]; however, the molecular mechanisms controlling the activation of ALT remain unclear.[15],[17]

The correlation between ATRX expression status and its clinical significance has been shown to be diverse, depending on the tumor type. Among ALT-associated cancers, pancreatic neuroendocrine tumors, liposarcoma, leiomyosarcoma, angiosarcoma, and human epidermal growth factor receptor 2 (HER2+) breast cancers are associated with poor prognosis. In contrast, glioma, neuroblastoma, and osteosarcoma have a better prognosis. Loss of ATRX expression in angiosarcoma and leiomyosarcoma is associated with a shorter DFS.[18],[19] Koschmann et al.[20] suggested that ATRX could be a malignancy marker because the loss of ATRX expression has been observed more frequently in primary glioblastomas than in grade II gliomas. In addition, ATRX-deficient gliomas demonstrated shorter median survival and increased genomic instability.[21] Loss of ATRX expression is associated with reduced survival in pancreatic neuroendocrine tumors and pulmonary carcinoid tumors.[22],[23] On the other hand, Je et al.[24] reported that ATRX may not contribute to gastric cancer, colorectal cancer, and prostate cancer development because positive ATRX immunostaining was observed in more than 90% of tumor cells, and there was no difference in the immunoactivity between normal cells and cancer cells.

In GEPIA results, downregulation of the ATRX gene was related to the stage and worse outcomes, demonstrated using gene-expression data. In this study, loss of ATRX expression was associated with more aggressive clinicopathologic features, such as larger tumor size, LVI, larger NG, recurrence/metastasis, higher pT stage, higher stage, and poor survival.

Ataxia telangiectasia and Rad3-related protein (ATR) inhibitors have been developed as novel drugs for ATRX-deficient tumors and have demonstrated high selectivity for alkaline phosphatase-positive cancer.[25] However, telomerase inhibitory therapies have recently been tested in clinical practice, and they are not predicted to be effective in alanine transferase-positive cancers.[26]

A limitation of this study is that it is a retrospective design and we did not evaluate the ATRX mutation. However, the IHC of ATRX is a surrogate marker for mutation.


   Conclusion Top


We assessed the mRNA expression levels of ATRX genes using the GEPIA database and evaluated the loss of ATRX expression by IHC in CCRCC. Loss of ATRX expression was significantly associated with aggressive clinicopathologic features and worse survival, which suggests that ATRX plays an important role in CCRCC progression and can be a potential biomarker to predict tumor aggressiveness and poor clinical outcome.

Ethical statements

This study was approved by the Institutional Review Board (IRB) of Yeungnam University Hospital (IRB No: 2021-04-032) and written informed consent was waived by IRB because of the retrospective nature of the study.

Conflict of interests

The authors declare no conflict of interests.

Financial support and sponsorship

Nil.



 
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Correspondence Address:
Mi-Jin Gu
Department of Pathology, Yeungnam University College of Medicine, 170 Hyeonchung-ro, Nam-gu, Daegu - 42415
South Korea
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_1256_21

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