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Citation: Kumar A, Bellayr IH, Singh HS, Puri RK (2021) IL-13Ra2 gene expression is a biomarker of adverse outcome in patients with adrenocortical carcinoma. PLoS ONE 16(2): e0246632. https:// doi.org/10.1371/journal.pone.0246632
Editor: Pankaj K. Singh, University of Nebraska Medical Center, UNITED STATES
Received: August 5, 2020
Accepted: January 24, 2021
Published: February 16, 2021
Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability Statement: All Adrenocortical Carcinoma files are available from the Genomic Commons database. https://portal.gdc.cancer.gov/ projects/TCGA-ACC.
Funding: This work was supported by the Intramural Research Program of the Center for Biologics Evaluation and Research (CBER), U.S. Food and Drug Administration.
Competing interests: The authors have declared that no competing interests exist.
RESEARCH ARTICLE
IL-13Ra2 gene expression is a biomarker of adverse outcome in patients with adrenocortical carcinoma
Abhinav Kumar 1, lan H. Bellayr1”, Hridaya S. Singh2, Raj K. Puri 1*
1 Division of Cellular and Gene Therapies, Tumor Vaccines and Biotechnology Branch, Center for Biologics and Evaluation Research, US Food and Drug Administration, Silver Spring, Maryland, United States of America, 2 Department of Zoology, Chaudhary Charan Singh University, Meerut, Uttar Pradesh, India
a Current address: National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, United States of America
Abstract
Adrenocortical carcinoma (ACC) is a rare but aggressive endocrine malignancy that usually results in a fatal outcome. To allow the better clinical management and reduce mortality, we searched for clinical and molecular markers that are reliable predictor of disease severity and clinical outcome in ACC patients. We determined a correlation between the overexpres- sion of IL-13Ra2 and the clinical outcome in ACC patients using comprehensive data avail- able in The Cancer Genome Atlas (TCGA) database. The dataset of 79 ACC subjects were divided into groups of low, medium, or high expression of IL-13Ra2 as determined by RNA- seq. These patients were also stratified by length of survival, overall survival, incidence of a new tumor event, incidence of metastasis, and production of excess hormones. We report a correlation between IL-13Ra2 expression and survival of subjects with ACC. High expres- sion of IL-13Ra2 in ACC tumors was significantly associated with a lower patient survival rate and period of survival compared to low expression (p = 0.0084). In addition, high IL- 13Ra2 expression was significantly associated with a higher incidence of new tumor events and excess hormone production compared to low or medium IL-13Ra2 expression. Within the cohort of patients that produced excess hormone, elevated IL-13Ra2 expression was significantly associated with a lower survival rate. Additionally, IL-13Ra1 had a potential relationship between transcript level and ACC survival. Our results and promising antitumor activity in preclinical models and trials indicate that IL-13Ra2 expression is an important prognostic biomarker of ACC disease outcome and a promising target for therapeutic treat- ment of ACC.
Introduction
Adrenocortical carcinoma (ACC) is a highly aggressive malignancy that originates in the outer layer of the adrenal gland. While most tumors of the adrenal gland are benign and common with a prevalence of 1-10%, ACCs are rare and occur with an annual incidence of 0.7 to 2.0
cases per million individuals. Clinically, ACC can be broadly divided into four stages, stage I and II tumors are restricted to organs where surgical removal is the common treatment, while advanced ACC stages defined as III and IV are highly fatal [1].
Approximately half of all ACC cases are discovered due to excess adrenal hormone pro- duced by the patient. In these cases, the risk of mortality is high as the tumor has grown signifi- cantly and metastasized. Therefore, early detection is needed to reduce the high mortality rate from ACC. Treatment often includes tumor resection, adrenolytic drug mitotane, and cyto- toxic therapy, but these options often have only moderate success [2]. Additional treatment options are needed to reduce the high mortality from this disease. Genes that are uniquely overexpressed in ACC may be promising targets for prognosis, early detection and therapeutic targets that could help manage this difficult disease.
We have previously reported that IL-13Rx2 is overexpressed in several types of cancer including renal cell carcinoma [3], glioblastoma multiforme [4], ovarian cancer [5], colorectal cancer [6] and pancreatic cancer [7]. We have also reported that IL-13Rx2 mRNA and protein is overexpressed in malignant ACC tumors compared to benign and normal samples [8]. IL- 13Ro.2 was found to influence cell division and invasion in ACC [8]. An earlier genome-wide gene expression profiling study of malignant and benign ACC tumors reported that IL-13Rx2 gene was transcriptionally upregulated by 24-fold in malignant compared to benign ACC tumors and had an excellent diagnostic accuracy for distinguishing malignant from benign adrenocortical tumors [9].
IL-13Ro2 is a component of the IL-13 receptor complex that consists of IL-13Rx1, and IL-4Rx chains [10-12]. IL-13 binds to IL-13Ral chain with low affinity and then recruits IL- 4Ra chain to form a high affinity receptor for signal transduction. On the other hand, IL-13 binds to IL-13Rx2 with high affinity and can mediate signal transduction through this chain in diseased fibroblasts and tumor cells [6, 11]. It has been reported that extracellular domain of IL-13Rx2 is cleaved and serves as a decoy receptor for IL-13. Because IL-13Ro2 binds IL- 13 with higher affinity than IL-13Rx1 [13, 14], it thereby allows sequestration of the ligand away from IL-13Ral for IL-13 signaling. It is proposed that this sequestration can be an apo- ptosis escape mechanism for tumor cells induced by IL-13 [15]. Inhibition of apoptosis of tumor cells that selectively express IL-13Rx2 suggests that IL-13Ro2 may act as an oncogene [16].
We have explored the therapeutic potential of IL-13Rx2 and reported that it can be targeted with an immunotoxin consisting of IL-13 and Pseudomonas exotoxin (IL-13-PE38QQR). We have tested this molecule in various Phase 1 and 2 clinical trials in patients with glioma and renal cell carcinoma [17]. Based on the overexpression of IL-13Rx2 in ACC tumors, we have performed a Phase 1 study in subjects with ACC [18]. Our results identified a maximum toler- ated dose (MTD) and recommended further testing of this molecule at MTD in additional patients with ACC [18].
In this study, using a sizable dataset, we have examined whether the IL-13Ra2 gene is asso- ciated with prognosis in ACC patients. We analyzed IL-13Ra2 gene expression in patients with different clinical parameters. We accessed the National Cancer Institute’s (NCI) database, The Cancer Genome Atlas (TCGA), and analyzed for the overexpression of IL-13Rx2 in ACC in RNAseq datasets. Related genes that are involved in the formation of IL-13R complex (IL- 13Ral and IL-4R) and program cell death ligand (PD-L1), as a control, were also evaluated with respect to patient age, hormone production, new tumor events, and tumor metastasis. Our analysis identified a link between IL-13Ro2 and the outcome of subjects with ACC. High expression of IL-13Ra2 in ACC tumor samples resulted in a low survival rate and associated with tumor reoccurrence and excess hormone production.
Materials and methods
Dataset
The National Cancer Institute’s (NCI) TCGA database has collected a comprehensive dataset on clinical and genomic characterization of ACC patients through a network of physicians, sci- entists and bioinformatics experts. The available information includes the age, sex, racial back- ground, clinical stage, and RNA sequence information from the tumor samples of individual ACC patients. We analyzed the RNAseq and corresponding clinical data files for 79 ACC patients which were downloaded in January 2017 from the TCGA Data portal. These datasets are now available from the Genomic Data Commons (GDC) Legacy archive. Protocols and procedural information for the data collection can be found at the GDC website. Additionally, GDC web applications were used in March 2017 to investigate mutations in ACC.
RNAseq data set was used to determine the IL-13Ra2 transcript expression level in the tumors from the ACC patients. From RNASeqV2 platforms expression data was produced. MapSplice was used to do the alignment and RSEM software to perform the quantitation tran- scription expression abundance. Data was normalized using RPKM (Reads Per Kilobase of transcript, per Million mapped reads) and reference genome hg37 producing Level 3 expres- sion data (https://www.ncbi.nlm.nih.gov/assembly/GCF 000001405.13/) [19].
For gene expression-based analyses, 79 ACC patients were stratified into three groups based on expression levels of the IL-13Rx2 transcript copy. The low expression group (n = 26) had an average IL-13Rx2 transcript copy of 4.19 ± 3.30 that ranged from 0.35-10.06 among the 26 patients. The medium expression group (n = 26) had an average IL-13Rx2 transcript copy of 47.78 ± 35.32 that ranged from 10.47-135.55 among the 26 patients. The high expression group (n =27) had an average IL-13Rx2 transcript copy of 1310.28 ± 1603.21 that ranged from 143.98- 7451.91 among the 27 patients. This strategy of dividing patients into three groups based on IL- 13Ro2 transcript level within the tumor was used as the basis to evaluate any associations between the IL-13Ro2 expression level and disease progression and outcomes in ACC patients.
The GDC database also provides the genetic mutations in individual tumor gene sequences that exhibited altered expression in global transcriptional analysis in ACC tumors. However, analysis of dataset did not reveal any genetic mutations in the IL-13Ra2 sequence in the 79 ACC tumors for which RNASeq data is available.
Statistical analysis and software
Fishers exact test was used to show statistical significance (p ≤ 0.05) for categorical variables, such as patient survival, excess hormone production, new tumor events, and tumor metastasis using the Graph Pad suit of online tools (www.graphpad.com/quickcalcs/catMenu). The final survival was calculated at the endpoint (7 years) provided in TCGA dataset. We divided num- ber of alive subjects by total number of subjects to determine the percentage of overall survival. Kaplan-Meier survival analysis was performed to compare patient survival between different IL-13Ra2 expression levels using the Graph Pad Prism software. Primary data transformation and analysis was done through the JMP Genomics software suit (JMP Genomics 6.1). The GDC data portal and exploration web tools were used to acquire publicly available gene expression and mutational data in ACC (https://portal.gdc.cancer.gov/projects/TCGA-ACC).
Results
Patient characteristics, clinical information and disease outcome
Demographics data is summarized in Table 1. Among the 79 ACC subjects, 60.8% were female and 39.2% were male, the average age at diagnosis was 46 (range 14-83 years old) and the five-
| Characteristics | N | |
| Total | 79 | |
| Sex | Male | 31 |
| Female | 48 | |
| Stage at Diagnosis | I | 9 |
| II | 37 | |
| III | 16 | |
| IV | 15 | |
| Metastatic Disease | Yes | 17 |
| No | 60 | |
| Sites of Metastasis | Liver | 5 |
| Lung | 5 | |
| Multiple Sites | 5 | |
| Brain | 1 | |
| Lymph Node | 1 | |
| Treatment Prior to Resection | Yes | 0 |
| No | 79 | |
| Adjuvant Treatment | Radiation | 14 |
| Mitotane | 43 | |
| Age at Diagnosis | Mean | 46 |
https://doi.org/10.1371/journal.pone.0246632.t001
year survival rate was 65%. Gender and age at diagnosis did not have a significant effect on sur- vival rate. Data pertaining to the clinical stage of ACC, incidence of a new tumor event, inci- dence of metastasis, and production of excess hormone were available for most of the subjects. Among the patient dataset, clinical stage classification was available for 77 of the 79 ACC sub- jects; 11.7% (9 subjects) had Stage I ACC and an 88% survival rate. 48.1% (37 subjects) had Stage II ACC and an 84% survival rate. 20.8% (16 subjects) had Stage III ACC and a 62.5% sur- vival rate. 19.5% (15 subjects) had Stage IV ACC and a 26% survival rate. Of the subjects with excess hormone production, as classified by the TCGA, 16 expressed excess cortisol, 16 expressed excess cortisol and androgen, 8 expressed excess androgen, 3 expressed excess Min- eralocorticoids, 2 expressed excess androgen and estrogen, 2 expressed excess estrogen, and 1 expressed excess cortisol and Mineralocorticoids.
Summary of demographic and clinical information on ACC subjects whose tumor samples were used to generate the transcriptional profiling data used in this study. ACC samples and clinical outcomes were collected from 79 subjects and deposited in the publicly available TCGA database. Metastatic and stage data for two subjects was unavailable.
Among the 79 ACC samples, clinical information regarding new tumor events was accessi- ble for 73. Among these 73 subjects, the incidence of a new tumor event, defined as reoccur- rence after initial treatment, was 47.9% (35 subjects). Subjects with a new tumor event had a 42% survival rate compared to 94% survival of subjects with no new tumor event. Among 77 ACC subjects, the incidence of metastasis was 22.1% (17 subjects). Subjects with metastatic tumors had a 29% survival rate (compared to 80% survival of subjects with non-metastatic tumors) and metastasis occurred in the lung (5 subjects), liver (5 subjects), brain (1 subject), lymph node (1 subject), or multiple sites (5 subjects).
Data available for 74 ACC subjects, 48 (64.9%) produced excess hormone. Subjects that produced excess hormone had a 56% survival rate compared to 88% survival of subjects that did not produce excess hormone. Hormones produced in excess included cortisol (16
subjects), cortisol/androgen (16 subjects), androgen (8 subjects), androgen/estrogen (2 sub- jects), estrogen (2 subjects), mineralocorticoids (3 subjects), and cortisol/mineralocorticoids (1 subject).
Comparison of IL-13Ra2 expression and ACC survival
The major objective of this study was to investigate the association of IL-13 receptor complex genes expression with disease outcome and hence could be used as potential biomarker and target for therapeutic treatment of ACC.
We analyzed the gene expression in RNASeq dataset for a possible relationship between the expression of IL-13Ro2 gene and survival from ACC during the 7-year period (the maximum period for which patient follow up data is available in the database). Using global transcrip- tional profiles of tumors at the TCGA database, we divided the 79 ACC patients into groups of low (n = 26), medium (n = 26), or high (n = 27) expression of IL-13Ra2. To calculate the over- all survival the number of alive subjects was divided by the total number of subjects. As shown in (Fig 1A), our analysis showed that the overexpression of IL-13x2 in ACC tumors is associ- ated with a lower patient survival time compared to low expression of IL-13Rx2 (p = 0.0112) (Fig 1B). The moderate expression of IL-13Ra2 showed no statistical significance when com- pared with low or high expression groups.
High expression of the IL-13Rx2 gene correlated with a lower rate of survival of ACC sub- jects. Subjects with low expression of IL-13Rx2 had an 85% survival rate. In contrast, subjects with medium and high IL-13Rx2 expression had a 62% and 48% survival rate, respectively (Fig 1C). Using the Fisher’s Exact Test, a statistically significant difference in the survival rate of subjects with low (n = 26) versus high (n = 27) expression of IL-13Rx2 was observed (p = 0.0084) (Fig 1D). When comparing low vs medium (n = 26) and medium versus high expressions no statistically significant difference was found among groups.
IL-13Rx2
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| b | Group | N | Overall Survival |
|---|---|---|---|
| IL-13Ra2 Low | N = 26 | 85% | |
| IL-13Rx2 High | N =27 | 48% |
| Group | Log Rank | Significance |
|---|---|---|
| Low vs High | p =. 0112 | Significant |
d
| Group | % Survival | n Survival |
|---|---|---|
| Low | 85% | 22 |
| Medium | 62% | 16 |
| High | 48% | 13 |
| Group | Symbol | Statistical Significance |
|---|---|---|
| Low vs High | - | p = . 0084 |
Fig 1. IL-13Ra2 expression and survival analysis of patients with ACC. 79 ACC patients were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-13Ra2 expression and Kaplan-Meier survival analysis were performed to determine overall survival time over 7-year period (Fig 1A and 1B) and survival rate (Fig 1C and 1D). Median survival and confidence intervals were not provided at the TCGA database. Due to medium expression of IL-13Ra2 showing no statistical significance when compared with low or high expression, moderate expression group was not included in the figure. Data were analyzed for different IL-13Ra2 expression levels and ACC survival using the Graph Pad Prism software. P values are shown comparing high vs. low expression.
https://doi.org/10.1371/journal.pone.0246632.9001
| Group | % New Tumor Event Occurrence |
|---|---|
| Low | 26% |
| Medium | 58% |
| High | 62% |
| Group | Symbol | Statistical Significance |
|---|---|---|
| Low vs Medium | * | p = . 048 |
| Low vs High | ** | p = . 0245 |
a
ACC Patients (n=78)
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New Tumor Event
| Group | New Tumor Event % Survival | No New Tumor Event % Survival |
|---|---|---|
| Low | 42% | 100% |
| Medium | 33% | 100% |
| High | 31% | 80% |
Fig 2. Comparison between IL-13Ra2 expression levels and new tumor occurrence. 79 ACC subjects were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-13Ra2 expression and the new tumor occurrence in these expression groups (Fig 2A and 2B) and their relationship with survival in subjects who developed new tumor events and those without new tumor events (Fig 2C and 2D) was assessed. P values are shown where significant.
https://doi.org/10.1371/journal.pone.0246632.g002
Comparison of IL-13Ra2 expression and new tumor events
The frequency of a new tumor event, defined as reoccurrence after initial treatment, was 48% (38 subjects) among 78 ACC subjects (data for one subject was not provided). Subjects with a new tumor event had a 29% survival rate compared to a 95% survival rate of subjects with no new tumor event. A new tumor event occurred significantly more frequently in ACC subjects with medium (58%) (p = 0.0202) or high (62%) (p = 0.0042) IL-13Ra2 expression compared to low (26%) IL-13Ra2 expression (Fig 2A and 2B). However, among the 35 ACC patients with a new tumor event, the level of IL-13Rx2 expression did not have a significant effect on the sur- vival rate (Fig 2C and 2D).
Comparison of IL-13Ra2 expression and metastasis
The incidence of metastasis was 22.1% (n = 17) among 77 ACC subjects and subjects with met- astatic tumors had a significantly lower survival rate compared to subjects with non-metastatic tumors (24% versus 76% survival rate) (p = 0.0001). For IL-13Rx2, there was no significant dif- ference in the incidence of tumor metastasis among ACC subjects with low, medium, and high expression. Instead, metastasis occurred at an even rate between all three expression levels; the metastatic tumor incidence was 23% in ACC subjects with low IL-13Rx2 expression, 20% in ACC subjects with medium IL-13Rx2 expression, and 23% in ACC subjects with high IL- 13Rx2 expression (Fig 3A and 3B). Low IL-13Rx2 expression (n = 6) was associated with a 33% survival rate of ACC subjects with tumor metastasis while ACC subjects without meta- static tumors (n = 20) showed a 100% survival rate (p = . 001). In contrast, medium (n = 5) IL- 13Ro2 expression was associated with a 20% survival rate of ACC subjects with tumor metasta- sis while ACC subjects with medium (n = 20) IL-13Rx2 expression and no metastasis showed a 70% survival rate (p = . 1206). Similarly, high (n = 6) IL-13Rx2 expression was associated with 16.7% survival rate of ACC subjects with tumor metastasis along with a survival rate of 60% for subjects without metastatic tumors (n = 20) (p =. 1602). Thus, subjects with low IL-
| Group | % Metastasis Occurrence |
|---|---|
| Low | 23% |
| Medium | 20% |
| High | 23% |
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| Group | Metastasis % Survival | No Metastasis % Survival |
|---|---|---|
| Low | 33% | 100% |
| Medium | 20% | 70% |
| High | 17% | 60% |
Fig 3. Comparison between IL-13Ra2 expression levels and metastasis occurrence. 77 ACC subjects were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-13Ra2 expression and the metastasis occurrence in these expression groups (Fig 3A and 3B) and their relationship with survival in subjects who developed metastasis and those without metastasis was assessed (Fig 3C and 3D).
https://doi.org/10.1371/journal.pone.0246632.g003
13Ro2 expression showed statistically significant correlation between tumor metastasis and sur- vival while this was not the case for medium and high expressors of IL-13Rx2 (Fig 3C and 3D)
Comparison of IL-13Ro2 expression and production of excess hormone
Among 79 ACC subjects, adrenal hormone excess data was available for 74 ACC subjects. In these subjects, 64.9% (48 subjects) produced excess hormone and subjects that produced excess hormone had a significantly lower survival rate compared to subjects that did not pro- duce excess hormone (52% versus 85% survival rate) (p = 0.006). Interestingly, we found that excess hormone production occurred significantly less frequently in ACC subjects with low (40%) compared to medium (75%) or high (80%) expression of IL-13Rx2 (p = 0.0209 for low versus medium and p = 0.0086 for low versus high) (Fig 4A and 4B). Although low IL-13Rx.2 was associated with an 80% survival rate whereas medium and high IL-13Rx2 was associated with a 55% and 35% survival rate, respectively, these results were not statistically significant (Fig 4C and 4D).
Analysis of IL-13Ra1, IL-4Ra and PD-L1 expression and survival of subjects with ACC
Since IL-13R and IL-4R constitute a part of IL-13 receptor complex, we also analyzed any pos- sible association between IL-13Ral and IL-4Ra expression and ACC survival. In addition, we analyzed a relationship between PD-L1 expression and ACC survival. PD-L1 is a checkpoint inhibitor and shown to play a major role in suppressing T cell immunity during cancer. We divided the 79 ACC patients into groups of low (n = 26), medium (n = 26), or high (n = 27) transcription level of IL-13Ra1. We observed a relationship between IL-13Ral expression and ACC survival. Though the difference in length of survival was not statistically significant (p>0.05), our analysis showed that higher expression of IL-13al in ACC tumors may be asso- ciated with a lower length of survival compared to low expression of IL-13Ral (Fig 5A and
a
ACC Patients (n=74)
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% Adrenal Hormone Excess
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Adrenal Hormone Excess
| Group | % Hormone Excess Occurrence |
|---|---|
| Low | 40% |
| Medium | 75% |
| High | 80% |
| Group | Symbol | Statistical Significance |
|---|---|---|
| Low vs Medium | * | p = . 209 |
| Low vs High | ** | p = . 0086 |
| Group | Hormone Excess % Survival | No Hormone Excess % Survival |
|---|---|---|
| Low | 80% | 87% |
| Medium | 55% | 66% |
| High | 35% | 100% |
Fig 4. Comparison between IL-13Ra2 expression and adrenal hormone excess. 74 ACC subjects were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-13Ra2 expression and their relationship with hormone excess occurrence was assessed (Fig 4A and 4B). P values are shown comparing low vs. medium and low vs. high IL- 13Ra2 expression. For comparison between IL-13Ra2 expression, adrenal hormone excess and survival, and their correlation with survival in subjects who developed excess hormone and those without excess hormone was assessed (Fig 4C and 4D).
https://doi.org/10.1371/journal.pone.0246632.g004
5B). In contrast with IL-13Ra2 gene expression, subjects with low expression of IL-13Rx.1 had a 42% survival rate whereas subjects with medium and high IL-13Ral expression had a 77% and 74% survival rate, respectively (Fig 5C and 5D). Using the Fisher’s Exact Test, there was a
IL-13Ra1
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| Group | N | Overall Survival |
|---|---|---|
| IL-13Ra1 Low | N = 26 | 42% |
| IL-13Rx1 High | N = 27 | 76% |
| Group | Log Rank | Significance |
|---|---|---|
| Low vs High | p > .05 | Not Significant |
d
| Group | % Survival | n Survival |
|---|---|---|
| Low | 42% | 11 |
| Medium | 77% | 20 |
| High | 74% | 20 |
| Group | Symbol | Statistical Significance |
|---|---|---|
| Low vs Medium | * | p = . 0227 |
| Low vs High | ** | p = . 0267 |
Fig 5. IL-13Ral expression and survival analysis of patients with ACC. 79 ACC patients were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-13Ra1 (Fig 5A and 5B) expression and Kaplan-Meier survival analysis was performed to determine survival time over 7-year period. In addition, IL-13Ral expression and its relationship with ACC overall survival was assessed (Fig 5C and 5D). Data was analyzed for different IL-13Ral expression levels and ACC survival using the Graph Pad Prism software. P values are shown comparing high vs. low expression.
https://doi.org/10.1371/journal.pone.0246632.g005
statistically significant difference in the overall survival rate of subjects with low (n = 26) versus medium (n = 26) (p = 0.0227) and low (n = 26) versus high (n = 27) (p = 0.0267) expression of IL-13Rx1. Consistent with the observed relationship between IL-13Ral expression and ACC survival, the age at death was also significantly higher in patients with medium (58.76 years) or high (62.33 year) versus low (34.76 years) IL-13Ral expression. No statistical significance was observed between IL-13Ral expression and the other clinical features measured from ACC patients. There was also no significant difference in the incidence of excess adrenal hormones among ACC subjects with low, medium, and high IL-13Ra1 expression. Similarly, among the 48 ACC patients with excess adrenal hormones, the level of IL-13Ra.1 expression did not influ- ence the survival rate. Additionally, there was no significant difference in the incidence of tumor metastasis among ACC subjects with low, medium, and high IL-13Ra1 expression. Also, among the 17 ACC patients with tumor metastasis, the level of IL-13Ra.1 expression did not influence the survival rate. However, new tumor events occurred significantly more fre- quently in ACC subjects with low (64%) versus medium (34.6%) IL-13Ral expression (p = 0.05). Importantly, among the 35 ACC patients with a new tumor event, the survival rate was significantly lower in patients with low (n = 16) versus elevated (medium and high) (n = 22) IL-13Ra.1 expression; patients with low IL-13Ral expression had a 12.5% survival rate whereas patients with elevated IL-13Ral expression had a 50% survival rate (p = 0.0356).
79 ACC patients were also divided into groups of low (n = 26), medium (n = 26), or high (n = 27) for the IL-4Ra and PD-L1 expression. However, there was no statistical significance observed between the expression of IL-4Ra and ACC survival (Fig 6). Similarly, PD-L1 expres- sion had no statistically significant relationship with survival in the 79 ACC subjects (Fig 7).
Discussion
By analyzing the National Cancer Institute’s TCGA database, we demonstrated that IL13Rx2 gene expression is related with the survival of patients with ACC where analysis indicated that
| Group | N | Overall Survival |
|---|---|---|
| IL-4Ra Low | N = 26 | 58% |
| IL-4Rx High | N = 27 | 77% |
| Group | Log Rank | Significance |
|---|---|---|
| Low vs High | p>.05 | Not Significant |
IL-4Ra
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| Group | % Survival | n Survival |
|---|---|---|
| Low | 57% | 15 |
| Medium | 57% | 15 |
| High | 78% | 21 |
Fig 6. IL-4Ra expression and survival analysis of patients with ACC. 79 ACC patients were divided between high (n = 27), medium (n = 26), and low (n = 26) IL-4Ra expression and Kaplan-Meier survival analysis was performed to determine survival time over 7-year period (Fig 6A and 6B). In addition, IL-4Ra expression and its relationship with ACC survival was assessed (Fig 6C and 6D). Data was analyzed for different IL-4Ra expression levels and ACC survival using the Graph Pad Prism software. P values are shown comparing high vs. low expression.
| b | Group | N | Overall Survival |
|---|---|---|---|
| PD-L1 Low | N = 26 | 46% | |
| PD-L1 High | N = 27 | 25% |
| Group | Log Rank | Significance |
|---|---|---|
| Low vs High | p > .05 | Not Significant |
PD-L1
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| Group | % Survival | n Survival |
|---|---|---|
| Low | 54% | 14 |
| Medium | 65% | 17 |
| High | 74% | 20 |
Fig 7. PD-L1 expression and survival analysis of patients with ACC. 79 ACC patients were divided between high (n = 27), medium (n = 26), and low (n = 26) PD-L1 expression and Kaplan-Meier survival analysis was performed to determine survival time over 7-year period (Fig 7A and 7B). In addition, PD-L1 expression and its relationship with ACC survival was assessed (Fig 7C and 7D). Data was analyzed for different PD-L1 expression levels and ACC survival using the Graph Pad Prism software. P values are shown comparing high vs. low expression.
https://doi.org/10.1371/journal.pone.0246632.g007
high IL-13Rx2 expression is associated with negative clinical outcomes as measured by four different metrics. First, subjects with high IL-13Rx2 expression had a lower survival rate and reduced length of survival than subjects with low IL-13Rx2 expression. Second, subjects with medium and high IL-13Rx2 expression had a higher incidence of a new tumor events than subjects with low IL-13Rx2 expression. Third, subjects with medium and high IL-13Rx2 expression exhibited a higher incidence of excess hormone production than subjects with low IL-13Rx2 expression. Fourth, among subjects with excess hormone production, patients with high IL-13Ro2 expression had a significantly lower survival rate compared to patients with low IL-13Rx2 expression.
Our results illustrate a relationship between high IL-13Rx2 expression and poor prognosis in patients diagnosed with ACC in agreement with our previous observations in patients with human glioblastoma multiforme (GBM) [4] which also utilized the NCI’s TCGA database. In previous studies, we analyzed IL-13Rx2 expression by immunohistochemistry and RT-PCR in tumors derived from patients with ovarian [20] and head and neck cancer [21]. In both reports, we observed that high IL-13Ra2 expression is associated with advanced stage disease. Collectively, these results indicate that IL-13Ro2 could be a good prognostic biomarker in patients with tumors that express high levels of IL-13Rx2.
The significance of IL-13Ro2 expression in ACC tumors in not clear. It is possible that this receptor is mutated in ACC. Therefore, we searched the TCGA database for possible muta- tions in the sequence of the IL-13Rx2 gene from ACC tumor samples. However, the TCGA database did not report any IL-13R genetic mutations in the IL-13Ro.2 gene suggesting that antigenic polymorphism did not contribute to the IL-13Ro.2 overexpression.
It is of interest to note that high expression of IL-13Ral in ACC was associated with higher % of survival. This relationship was found in male patients only. Consistent with this observa- tion, the age at death was also significantly higher in patients with high versus low IL-13Rx1 expression. Another interesting finding was that low IL-13Ral expression corresponds with
new tumor events in ACC patients. In addition, we noted that survival from a new tumor event was related with an elevated IL-13Ral expression.
Previous studies reported that IL-13Ral can restrict tumor progression; IL-13 binding to IL-13Ral activates Stat6 which promotes caspase-3 mediated apoptosis [15, 16, 22, 23]. Studies that have investigated the role of IL-13Ral in cancer are conflicted. For example, Kwon et al showed that high expression of IL-13Ral was associated with a lower risk of recurrence and cancer-induced mortality in patients with oral cavity squamous cell carcinoma [24]. In con- trast, high IL-13Ra.1 expression was significantly associated with clinicopathological parame- ters of aggressive phenotypes and with reduced survival in patients with invasive breast cancer [25].
Because of varying observations of higher survival with high IL-13Ra1 expression and con- tradictory relationships with other clinical parameters, we did not consider IL-13Ral a clear prognostic biomarker. On the other hand, IL-13Ra2 appears to be a reliable biomarker in ACC. The association between elevated IL-13Ro2 gene expression and adverse clinical out- come suggests that measurement of IL-13Ro2 in ACC patients could be used to differentially diagnose and identify patients at highest risk for a poor prognosis who could benefit from IL- 13Rx2 targeted therapy.
We also found that there was no significant correlation between transcriptional expression of PD-L1 and ACC survival. However, new tumor events occurred significantly more fre- quently in ACC subjects with low (68%) compared to high (29%) (p = 0.0101) or medium and high (38%) (p = 0.0156) expression of PD-L1. Similar to our results, Fay et al reported that there was no relationship between PD-L1 expression and ACC survival and clinic-pathologic parameters such as such as stage, grade, or excessive secretion of hormones [26].
Mitotane, which blocks hormone production, is administered in some ACC as treatment and shown to demonstrate overall 30% efficacy measured as stable disease or partial remission [2]. Interestingly, ribonucleotide reductase large subunit 1(RRM1) and cytochrome p450 2W1 (CYP2W1) expression levels has been associated with response to mitotane therapy and pro- longed tumor-free survival [27, 28]. We investigated whether IL-13Rx2 expression level can serve as a surrogate to monitor the efficacy of mitotane treatment in ACC patients. Analysis of the TCGA dataset revealed no statistical significance in survival between the low and high IL- 13Rx2 expressing ACC who had undergone mitotane treatment. However, these results should be interpreted with caution because of low sample size in the study sub-groups and the modest efficacy of mitotane against ACC.
Novel therapies are needed to increase the survival rate of ACC. Our results clearly indicate that patients with elevated levels of IL-13Rx2 are at significantly higher risk of an adverse out- come. Therefore, a therapeutic treatment that targets IL-13Ro.2 may improve the prognosis and clinical outcome of subjects expressing elevated levels of IL-13Rx2. Preclinical studies sug- gest that IL-13Rx2 may be a promising therapeutic target for ACC. Studies have shown that IL-13-Pseudomonas exotoxin (IL-13-PE38QQR) is highly cytotoxic in vitro and in vivo to sev- eral types of IL-13Ro2-positive cancer cells including ACC cells. Jain et al., demonstrated that the IL-13Ro:2-positive ACC cell line (NCI-H295R) is highly sensitive to IL-13-PE cytotoxin [8]. Furthermore, in this same study, it was shown that treatment of animals with IL-13-PE resulted in significant tumor regression and prolonged survival in a murine xenograft model of ACC. In addition, a Phase I clinical trial in patients with metastatic ACC demonstrated that IL-13-PE is safe and well tolerated and showed some activity in this disease. However, most patients developed neutralizing antibodies to immunotoxin which limited further administra- tion of IL-13-PE. Immunodepletion prior to treatment is being considered in future clinical trials to improve the effectiveness of IL-13-PE as a therapeutic treatment for ACC [18].
In summary, our results clearly establish that the levels of IL-13Ra2 gene expression play an important role in ACC pathogenesis and may serve as a prognostic biomarker of disease pro- gression and adverse outcome in these patients. Additionally, mining of the TCGA datasets may allow development of IL-13Ra2 gene detection-based test to guide decisions on case man- agement, treatment options and monitoring the progress of ACC patients.
Acknowledgments
The results shown here are in part based upon data generated by the TCGA Research Network. A special thanks to the Research Participation Program at CBER administered by the Oak Ridge Institute for Science and Education through the US Department of Education and the U.S. Food and Drug Administration agreement. We are very thankful to Dr. Jing Qin, NIAID for statistical review of our data and Drs. Marc Walderhaug and Shyh-Ching Lo of CBER, FDA for their thorough evaluation and helpful comments on our manuscript.
Author Contributions
Conceptualization: Abhinav Kumar, Ian H. Bellayr, Raj K. Puri.
Data curation: Abhinav Kumar.
Formal analysis: Abhinav Kumar.
Resources: Raj K. Puri.
Supervision: Ian H. Bellayr, Hridaya S. Singh, Raj K. Puri.
Writing - original draft: Abhinav Kumar.
Writing - review & editing: Abhinav Kumar, Ian H. Bellayr, Hridaya S. Singh, Raj K. Puri.
References
1. Fassnacht M, Allolio B. Clinical management of adrenocortical carcinoma. Best Pract Res Clin Endocri- nol Metab. 2009; 23(2):273-89. Epub 2009/06/09. https://doi.org/10.1016/j.beem.2008.10.008 PMID: 19500769.
2. Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, et al. Adrenocortical carcinoma. Endocr Rev. 2014; 35(2):282-326. Epub 2014/01/16. https://doi.org/10.1210/er.2013-1029 PMID: 24423978; PubMed Central PMCID: PMC3963263.
3. Obiri NI, Leland P, Murata T, Debinski W, Puri RK. The IL-13 receptor structure differs on various cell types and may share more than one component with IL-4 receptor. J Immunol. 1997; 158(2):756-64. Epub 1997/01/15. PMID: 8992992.
4. Han J, Puri RK. Analysis of the cancer genome atlas (TCGA) database identifies an inverse relationship between interleukin-13 receptor alpha1 and alpha2 gene expression and poor prognosis and drug resis- tance in subjects with glioblastoma multiforme. J Neurooncol. 2018; 136(3):463-74. Epub 2017/11/24. https://doi.org/10.1007/s11060-017-2680-9 PMID: 29168083; PubMed Central PMCID: PMC5805806.
5. Fujisawa T, Joshi BH, Puri RK. IL-13 regulates cancer invasion and metastasis through IL-13Ralpha2 via ERK/AP-1 pathway in mouse model of human ovarian cancer. Int J Cancer. 2012; 131(2):344-56. Epub 2011/08/23. https://doi.org/10.1002/ijc.26366 PMID: 21858811.
6. Barderas R, Bartolome RA, Fernandez-Acenero MJ, Torres S, Casal JI. High expression of IL-13 recep- tor alpha2 in colorectal cancer is associated with invasion, liver metastasis, and poor prognosis. Cancer Res. 2012; 72(11):2780-90. Epub 2012/04/17. https://doi.org/10.1158/0008-5472.CAN-11-4090 PMID: 22505647.
7. Shimamura T, Fujisawa T, Husain SR, Joshi B, Puri RK. Interleukin 13 mediates signal transduction through interleukin 13 receptor alpha2 in pancreatic ductal adenocarcinoma: role of IL-13 Pseudomo- nas exotoxin in pancreatic cancer therapy. Clin Cancer Res. 2010; 16(2):577-86. Epub 2010/01/14. https://doi.org/10.1158/1078-0432.CCR-09-2015 PMID: 20068108.
8. Jain M, Zhang L, He M, Patterson EE, Nilubol N, Fojo AT, et al. Interleukin-13 receptor alpha2 is a novel therapeutic target for human adrenocortical carcinoma. Cancer. 2012; 118(22):5698-708. Epub 2012/ 05/10. https://doi.org/10.1002/cncr.27629 PMID: 22570059; PubMed Central PMCID: PMC3416952.
9. Fernandez-Ranvier GG, Weng J, Yeh RF, Khanafshar E, Suh I, Barker C, et al. Identification of bio- markers of adrenocortical carcinoma using genomewide gene expression profiling. Arch Surg. 2008; 143(9):841-6; discussion 6. Epub 2008/09/17. https://doi.org/10.1001/archsurg.143.9.841 PMID: 18794420.
10. Zdanov A, Wlodawer A. A new look at cytokine signaling. Cell. 2008; 132(2):179-81. Epub 2008/02/05. https://doi.org/10.1016/j.cell.2008.01.006 PMID: 18243092.
11. Wills-Karp M, Finkelman FD. Untangling the complex web of IL-4- and IL-13-mediated signaling path- ways. Sci Signal. 2008; 1(51):pe55. Epub 2008/12/26. https://doi.org/10.1126/scisignal.1.51.pe55 PMID: 19109238; PubMed Central PMCID: PMC4446705.
12. Suzuki A, Leland P, Joshi BH, Puri RK. Targeting of IL-4 and IL-13 receptors for cancer therapy. Cyto- kine. 2015; 75(1):79-88. Epub 2015/06/20. https://doi.org/10.1016/j.cyto.2015.05.026 PMID: 26088753.
13. Fichtner-Feigl S, Strober W, Kawakami K, Puri RK, Kitani A. IL-13 signaling through the IL-13alpha2 receptor is involved in induction of TGF-beta1 production and fibrosis. Nat Med. 2006; 12(1):99-106. Epub 2005/12/06. https://doi.org/10.1038/nm1332 PMID: 16327802.
14. Arima K, Sato K, Tanaka G, Kanaji S, Terada T, Honjo E, et al. Characterization of the interaction between interleukin-13 and interleukin-13 receptors. J Biol Chem. 2005; 280(26):24915-22. Epub 2005/05/05. https://doi.org/10.1074/jbc.M502571200 PMID: 15870068.
15. Thaci B, Brown CE, Binello E, Werbaneth K, Sampath P, Sengupta S. Significance of interleukin-13 receptor alpha 2-targeted glioblastoma therapy. Neuro Oncol. 2014; 16(10):1304-12. Epub 2014/04/ 12. https://doi.org/10.1093/neuonc/nou045 PMID: 24723564; PubMed Central PMCID: PMC4165413.
16. Hsi LC, Kundu S, Palomo J, Xu B, Ficco R, Vogelbaum MA, et al. Silencing IL-13Ralpha2 promotes glio- blastoma cell death via endogenous signaling. Mol Cancer Ther. 2011; 10(7):1149-60. Epub 2011/05/ 21. https://doi.org/10.1158/1535-7163.MCT-10-1064 PMID: 21596889; PubMed Central PMCID: PMC3132296.
17. Joshi BH, Puri RK. IL-13 receptor-alpha2: a novel target for cancer therapy. Immunotherapy. 2009; 1 (3):321-7. Epub 2009/05/01. https://doi.org/10.2217/imt.09.8 PMID: 20635949.
18. Liu-Chittenden Y, Jain M, Kumar P, Patel D, Aufforth R, Neychev V, et al. Phase I trial of systemic intra- venous infusion of interleukin-13-Pseudomonas exotoxin in patients with metastatic adrenocortical car- cinoma. Cancer Med. 2015; 4(7):1060-8. Epub 2015/03/15. https://doi.org/10.1002/cam4.449 PMID: 25767039; PubMed Central PMCID: PMC4529344.
19. See L, Comber A, Salk C, Fritz S, van der Velde M, Perger C, et al. Comparing the quality of crowd- sourced data contributed by expert and non-experts. PLoS One. 2013; 8(7):e69958. Epub 2013/08/13. https://doi.org/10.1371/journal.pone.0069958 PMID: 23936126; PubMed Central PMCID: PMC3729953.
20. Kioi M, Kawakami M, Shimamura T, Husain SR, Puri RK. Interleukin-13 receptor alpha2 chain: a poten- tial biomarker and molecular target for ovarian cancer therapy. Cancer. 2006; 107(6):1407-18. Epub 2006/08/12. https://doi.org/10.1002/cncr.22134 PMID: 16902988.
21. Han J, Kioi M, Chu WS, Kasperbauer JL, Strome SE, Puri RK. Identification of potential therapeutic tar- gets in human head & neck squamous cell carcinoma. Head Neck Oncol. 2009; 1:27. Epub 2009/07/16. https://doi.org/10.1186/1758-3284-1-27 PMID: 19602232; PubMed Central PMCID: PMC2719634.
22. Rahaman SO, Sharma P, Harbor PC, Aman MJ, Vogelbaum MA, Haque SJ. IL-13R(alpha)2, a decoy receptor for IL-13 acts as an inhibitor of IL-4-dependent signal transduction in glioblastoma cells. Can- cer Res. 2002; 62(4):1103-9. Epub 2002/02/28. PMID: 11861389.
23. Viita H, Pacholska A, Ahmad F, Tietavainen J, Naarala J, Hyvarinen A, et al. 15-Lipoxygenase-1 induces lipid peroxidation and apoptosis, and improves survival in rat malignant glioma. In Vivo. 2012; 26(1):1-8. Epub 2012/01/03. PMID: 22210710.
24. Kwon M, Kim JW, Roh JL, Park Y, Cho KJ, Choi SH, et al. Recurrence and cancer-specific survival according to the expression of IL-4Ralpha and IL-13Ralpha1 in patients with oral cavity cancer. Eur J Cancer. 2015; 51(2):177-85. Epub 2014/12/09. https://doi.org/10.1016/j.ejca.2014.11.010 PMID: 25483786.
25. Park MH, Kwon HJ, Kim JR, Lee B, Lee SJ, Bae YK. Elevated Interleukin-13 Receptor Alpha 1 Expres- sion in Tumor Cells Is Associated with Poor Prognosis in Patients with Invasive Breast Cancer. Ann Surg Oncol. 2017; 24(12):3780-7. Epub 2017/06/22. https://doi.org/10.1245/s10434-017-5907-2 PMID: 28634667.
26. Fay AP, Signoretti S, Callea M, Telomicron GH, Mckay RR, Song J, et al. Programmed death ligand-1 expression in adrenocortical carcinoma: an exploratory biomarker study. J Immunother Cancer. 2015;
3:3. Epub 2015/03/15. https://doi.org/10.1186/s40425-015-0047-3 PMID: 25767716; PubMed Central PMCID: PMC4357210.
27. Volante M, Terzolo M, Fassnacht M, Rapa I, Germano A, Sbiera S, et al. Ribonucleotide reductase large subunit (RRM1) gene expression may predict efficacy of adjuvant mitotane in adrenocortical can- cer. Clin Cancer Res. 2012; 18(12):3452-61. Epub 2012/05/02. https://doi.org/10.1158/1078-0432. CCR-11-2692 PMID: 22547773.
28. Ronchi CL, Sbiera S, Volante M, Steinhauer S, Scott-Wild V, Altieri B, et al. CYP2W1 is highly expressed in adrenal glands and is positively associated with the response to mitotane in adrenocortical carcinoma. PLoS One. 2014; 9(8):e105855. Epub 2014/08/22. https://doi.org/10.1371/journal.pone. 0105855 PMID: 25144458; PubMed Central PMCID: PMC4140842.