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Indoleamine 2,3-Dioxygenase-1 Expression in Adrenocortical Carcinoma
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John F. Tierney, MD,ª Alyx Vogle, BS,ª Brendan Finnerty, MD,b Rasa Zarnegar, MD,’ Ritu Ghai, MD,” Paolo Gattuso, MD,’ Thomas J. Fahey III, MD,b and Xavier M. Keutgen, MDd,*
a Rush University Medical Center, Division of Surgical Oncology, Department of Surgery, Chicago, Illinois
b New York Presbyterian Hospital-Weill Cornell Medical Center, Department of Surgery, New York, New York
” Rush University Medical Center, Department of Pathology, Chicago, Illinois
d The University of Chicago Medical Center, Division of Endocrine Surgery, Department of Surgery, Chicago, Illinois
ARTICLE INFO
Article history: Received 23 July 2019 Received in revised form 30 May 2020
Accepted 17 June 2020 Available online xxx
Keywords: Adrenocortical carcinoma Endocrine surgery Surgical oncology
ABSTRACT
Background: Indoleamine 2,3-dioxygenase 1 (IDO-1) is overexpressed in many human car- cinomas and a successful target for therapy in mouse models. Prognosis of patients with advanced adrenocortical carcinoma (ACC) is poor due to the lack of effective treatments, and new therapies are therefore needed. Herein, we investigate whether IDO-1 is expressed in human ACC tissues.
Methods: 53 tissue samples from patients with ACC, adrenal adenoma (AA), adrenocortical tumors (ACTs), and normal adrenal were identified. Immunohistochemistry was per- formed on formalin-fixed, paraffin-embedded slides for IDO-1. Samples were scored for cytoplasmic staining as per intensity and the percent of positive cells and for stromal staining by percent of positive cells. Tumor characteristics, PD-L1, PDL-2, and CD-8+ T- lymphocyte expression were also determined.
Results: Samples from 32 ACC, 3 ACT, 15 AA, and 3 normal adrenal were analyzed. IDO-1 was expressed in tumor tissue in 22 of 32 ACC samples, compared with 8 of 15 AA sam- ple (P = 0.344). IDO-1 expression was significantly increased in stromal tissue of ACC samples (16 of 33), compared with AA samples (0 of 15) (P = 0.001). IDO-1 expression in ACC and AA samples was associated with PD-L2 expression (P = 0.034). IDO-1 expression in ACC stromal tissue was associated with CD8+ T-lymphocyte infiltration (P = 0.028).
Conclusions: IDO-1 is expressed in a majority of ACC samples. Its expression in tumor tissue is associated with PD-L2 expression, and expression in stroma is associated with CD8+ cell infiltration. IDO-1 inhibition, alone or in combination with PD-1 inhibition, could therefore be an interesting target in treatment of ACC.
@ 2020 Elsevier Inc. All rights reserved.
E-mail address: xkeutgen@surgery.bsd.uchicago.edu (X.M. Keutgen).
Introduction
Adrenocortical carcinoma (ACC) is a rare but lethal tumor, with an overall median survival after resection of only 32 mo.1,2 Stage IV disease conveys an even worse prognosis and puts patients at a three-fold increased risk of death compared with patients with stage I tumors.1 Furthermore, recurrence is common and often occurs soon after resection.3 Mitotane in combination with etoposide, doxorubicin, and cisplatin is considered the first-line treatment for advanced ACC, but less than a quarter of patients respond to this regimen, and it has not been shown to prolong overall sur- vival.4 Improved medical therapies are therefore needed, both in the adjuvant setting and for unresectable disease.
Indoleamine 2,3-dioxygenase-1 (IDO-1) is a heme-based enzyme that serves as the rate-determining step for trypto- phan catabolismin the kynurenine pathway.5,6 It was initially isolated from placenta, where it is thought to mediate maternal tolerance to the fetus but more recently has been implicated in a variety of human cancers.5-7 Multiple IDO-1 inhibitors are currently in preclinical development, and it is hypothesized that IDO-1 inhibition could work synergistically with programmed cell death protein-1 (PD-1) inhibition or that IDO-1 expression could predict the effectiveness of anti-PD-1 therapy.8-11 IDO-1 blockade is also thought to be partly responsible for the efficacy of imatinib against gastrointes- tinal stromal tumors.8
Our group has previously demonstrated that programmed death ligand-2 (PD-L2), but not programmed death ligand-1 (PD-L1), is overexpressed in the adrenal and stromal tissue of certain ACC samples, suggesting that PD-1 inhibitors are perhaps a potentially effective therapy in a subgroup patients with ACC.12 The role of IDO-1 in ACC, however, has not pre- viously been studied and remains unclear. We therefore sought to determine IDO-1 expression in ACC tumor and stromal tissue, compare expression levels to benign adrenal lesions, and determine whether IDO-1 expression is associ- ated with PD-L2 expression, CD8+ T-lymphocyte infiltration, or other clinical or pathological characteristics.
Methods
Fifty-three tissue samples from patients with ACC, adrenal adenoma (AA), indeterminate adrenocortical tumors (ACTs), and normal adrenal (NA) were obtained from separate insti- tutional biorepositories at Rush University Medical Center (RUMC) and New York Presbyterian-Weill Cornell Medical Center. The RUMC Institutional Review Board approved this study; the research offices of each institution approved ma- terial transfer, and all data obtained from NYP-WCMC were deidentified before transfer. Consent and Health Insurance Portability and Accountability Act authorization were waived due to the study’s retrospective nature.
Clinical and pathological data, including patient sex, age at diagnosis, presence of metastases at diagnosis, whether the tumor secreted hormones, and survival were collected from the medical records when available. Samples were classified in accordance with their diagnosis on final clinical pathology
performed at the time of tumor removal. The samples that did not meet Weiss criteria for ACC, a pathologic grading system used for ACC diagnosis, but had more than five mitoses per 50 high power fields were considered diagnosed as indetermi- nate ACTs by the clinical pathologist who reviewed the initial pathology. These tumors were treated as benign lesions in this study because the clinical course of these patients was not consistent with ACC.
Immunohistochemical analysis
5-micron sections were made from formalin-fixed paraffin- embedded tissue from 32 primary ACC tumors, 15 AAs, 3 NAs, and 3 ACTs and mounted on either individual slides or cores on a tissue microarray (TMA). IDO-1 staining was then per- formed using a method described previously.12 After slides were deparaffinized with xylene and rehydrated, citrate buffer (SignalStain Citrate Unmasking Solution, Cell Signaling Technology) was used for antigen retrieval. Endogenous per- oxidases were blocked using hydrogen peroxide. The slides were incubated for 2 h for with Animal-Free Blocking Solution (Cell Signaling Technology), and then incubated overnight with mouse monoclonal anti-Indoleamine 2, 3-dioxygenase antibody (Abcam, ab55305) at a concentration of 3ug/mL. Mouse SignalStain Boost Detection reagent secondary (Cell Signaling Technology) was then applied. SignalStain DAB Substrate Kit (Cell Signaling Technology) was used for devel- oping, and the slides were counterstained with hematoxylin (Harris Hematoxylin, BBC Biochemical). Finally, they were washed, dehydrated, mounted, and coverslipped. Information on PD-L1 and PD-L2 expression in tumor and stroma, and CD-8 T-lymphocyte infiltration, which were reported previously, was obtained from laboratory records.12
Immunohistochemical scoring
All samples were reviewed blindly by two board-certified pa- thologists at RUMC. The slides were scored for IDO-1 expres- sion in tumor cell cytoplasm as per the percent of positive cells and intensity. Intensity was scored as absent (0), low,1 moderate,2 or high.3 IDO-1 expression in the stromal tissue was scored as per the percent of positive cells. Each tumor sample on the TMA was represented by between one and three TMA cores; the percentages and intensities from each core were averaged.
For tumor tissue, percentage scores were converted to the following integer values on a 0-4 scale: a value of 0 was assigned to the slides that stained 0% of cells; 1 to <10%; 2 to 10%-50%; 3 to 51%-80%; and 4 to 81%-100%. An immunoreac- tive score (IRS) was then calculated by multiplying the in- tensity score and the integer value for the percentage score, as described previously (Table 1).12,13 Positive staining of adrenal samples in this study was defined as an IRS >6 (>50% of positive cells and >2+ staining intensity). Stromal tissue was considered positive if ≥ 10% of cells stained positive for IDO-1 as described previously.14
When differences in IRS, percent of positive stromal cells, or CD8+ lymphocyte infiltration existed between pathologists, the average score was used.
| Table 1 - Immunoreactive score (IRS). | ||
| Percentage of positive cells (A) | Intensity of staining (B) | IRS (An x B) |
| 0 = no positive cells | 0 = no reaction | 0-1 = negative |
| 1 = < 10% positive cells | 1 = mild reaction | 2-5 = weak expression |
| 2 = 10%-50% positive cells | 2 = moderate reaction | 6-12 = strong expression |
| 3 = 51%-80% positive cells | 3 = intense reaction | |
| 4 = 81%-100% positive cells | ||
| An IRS score > 6 was considered positive staining for this study. | ||
Statistical analysis
Descriptive statistics were calculated for the rate of IDO-1 expression in accordance with each histological diagnosis. IDO-1 expression in tumor and stromal tissue was compared between ACC and AA samples and with PD-L2 tumor and stromal expression among ACC and AA samples. For ACC samples, relationships between PD-L2 positivity and CD8+ lymphocyte infiltration, metastases at diagnosis, patient sex, and whether the tumor secreted hormones were also compared with Fisher exact tests. The relationship between PD-L2 positivity and patient age was compared using a Mann- Whitney U test. All analyses were conducted in SPSS v. 22.15 p- values ≤ 0.05 were considered significant.
Results
Samples from 32 ACC, 3 ACT, 15 AA, and 3 NA were analyzed, including 35 samples that were cored and mounted on a TMA. IDO-1 was expressed in tumor tissue with an IRS >6 in 22 of 32 ACC samples (69%), compared with 8 of 15 AA samples (53%) (P = 0.344). IDO-1 expression was significantly increased in stromal tissue of ACC samples (16 of 33, 47%) when compared with AA samples (0 of 15, 0%) (P = 0.001) (Table 2) (Figure).
15 of 30 ACC and AA samples (50%) that expressed IDO-1 in the adrenal tissue were also positive for PD-L2 in adrenal tis- sue, compared with only 3 of 17 samples (18%) that did not express IDO-1 (P = 0.034) (Table 3).
Among ACC samples, IDO-1 expression in the stroma was significantly associated with CD8+ T-lymphocyte infiltration. Of the 15 ACC samples that expressed IDO-1 in the stroma, 13 samples (87%) also stained positive for CD8+ T lymphocytes (P = 0.028). There was no association between IDO-1 expres- sion and PD-L1 expression (P = 0.29). The only ACC sample that stained positive for PD-L1 in the previous study did not express IDO-1 (Table 3).
There was no significant relationship between the pres- ence of metastases at diagnosis, hormone secretion, patient sex, or patient age, and IDO-1 expression in tumor or stromal tissue (P = 0.38-1). There was also no significant relationship between the use of the TMA and IDO-1 expression in tumor or stromal tissue (P = 0.08-0.14) (Table 3).
Discussion
This study demonstrates that IDO-1 is expressed in the tumor and stromal cells of ACC samples and significantly more frequently in stromal tissue of ACC, compared with AAs. IDO- 1 expression in stromal tissue of ACC samples is also associ- ated with CD8+ T-lymphocyte infiltration, and expression in adrenal tissue was associated with PD-L2 expression.
Physiologically, IDO-1 is involved in facilitating the pla- centa’s role in preventing T-cell-mediated rejection of the fetus by the mother.16 It has been implicated in numerous cancers, including renal cell carcinoma, breast cancer, and non-small-cell lung cancer.5,7,16-18 It is thought to participate in the tumor’s evasion of the immune system by depleting tryptophan from the tumor microenvironment, which then induces an immunosuppressive response by promoting the development of regulatory T-cells and inhibiting the function of cytotoxic T-cells.16,17,19 These deranged cytotoxic T-cells secrete interferon gamma, which then further increases IDO-1 expression, which perpetuates this immunosuppressive cycle.17
Previous studies demonstrated that tumor IDO-1 expres- sion in non-small-cell lung cancer and stromal IDO-1 expression in renal cell carcinoma are associated with an increased number of tumor infiltrating lymphocytes, similar to our finding that stromal IDO-1 expression was associated with CD8+ T-cell infiltration in ACC.17,18 Although IDO-1 was described primarily in tumor tissue of lung cancer, it was only expressed in stromal tissue in renal cell carcinoma.17 If IDO-1 indeed exerts its immunosuppressive effects by depleting
| Table 2 - IDO-1 immunohistochemical expression in adrenal samples as per IRS > 6. | |||||
| Ido-1 expression | ACC | AA | ACT | NA | P-value* |
| Adrenal tissue | 22 (69%) | 8 (53%) | 1 (33%) | 2 (67%) | 0.344 |
| Stromal tissue | 16 (47%) | 0 (0%) | 2 (67%) | 1 (33%) | 0.001 |
P-value refers to the comparison between ACC and AA.
A
B
C
D
1
E
F
tryptophan from the microenvironment, it should have similar effects regardless of whether it is expressed by tumor or stroma.
There was no association between IDO-1 and PD-L1 expression in lung cancer, which led the authors of a previ- ous study to hypothesize that lung cancer only evades the immune system through one mechanism.18 In this study, we described a relationship between IDO-1 expression in adrenal tissue and PD-L2 expression, which suggests that ACC
perhaps evades the immune response in multiple ways, although an interaction between IDO-1 and PD-L2 has not been studied to date.
Stromal IDO-1 expression has been shown to predict response to nivolumab, a PD-1 inhibitor, in renal cell carci- noma.17 High stromal expression of IDO-1 in ACC, therefore, perhaps should be considered as a marker to initiate anti-PD-1 therapy, particularly because PD-L1 expression in ACC is low, as our group has demonstrated previously.12 The patients who
| Table 3 - Patient, tumor, and pathological characteristics associated with IDO-1 expression in ACC tumor and stroma. | ||||
|---|---|---|---|---|
| Clinical characteristic | Tumor | Stroma | ||
| IDO-1 positive (%) | P-value | IDO-1 positive (%) | P-value | |
| Metastases at diagnosis | 7 (70%) | 1 | 8 (73%) | 0.38 |
| Male sex | 7 (87%) | 0.222 | 6 (67%) | 0.43 |
| Hormone secreting | 6 (75%) | 0.544 | 4 (50%) | 0.47 |
| PD-L1 positive | ||||
| PD-L2 positive (tumor) | 15 (83%) | 0.034 | 5 (28%) | 0.75 |
| PD-L2 positive (stroma) | 8 (89%) | 0.039 | 4 (44%) | 1 |
| CD8+ T-lymphocyte infiltration | 14 (67%) | 1 | 13 (62%) | 0.028 |
| Tissue microarray | 26 (74%) | 0.012 | 9 (35%) | 0.07 |
co-express IDO-1 and PD-L2 in tumor tissue might be partic- ularly good candidates for anti-PD-1 therapy, as PD-L2 is also thought to mediate the response to these drugs.20
Preclinical studies have demonstrated efficacy of IDO-1 inhibition in cell culture and mouse models of a variety of cancers.8,21,22 IDO-1 inhibition has also shown efficacy in combination therapies for stage IV prostate cancer and mel- anoma in phase II trials.8,23 More recently, however, a phase III trial of the IDO-1 inhibitor epacadostat in combination with pembrolizumab for stage IV or unresectable melanoma was ended early because the combination did not improve progression-free survival compared with pembrolizumab monotherapy alone, although the observed safety profile was consistent with previous phase II studies.24 Despite this finding, other phase II studies of IDO-1 inhibitors continue to recruit based on preclinical efficacy. In addition, the finding that IDO-1 expression in renal cell carcinoma is associated with response to anti-PD-1 therapy suggests that this tumor marker could perhaps affect patient management by being used to select patients for PD-1 inhibition, even if future trials of IDO-1 inhibitors fail.17
The role of immunotherapy in the treatment of ACC re- mains uncertain after the Avelumab in Metastatic or Locally Advanced Solid Tumors (JAVELIN Solid Tumor) trial investi- gating the PD-L1 inhibitor avelumab in ACC demonstrated an overall response rate of merely 6%.25 Based on our group’s previous research, however, it is not surprising that a PD-L1 inhibitor is not effective, as we demonstrated that PD-L2 but not PD-L1 was overexpressed in ACC.12 A trial using a PD-1 inhibitor might therefore lead to more promising results.
Cosentini et al. theorized that the avelumab was not effective because ACC secretes excess cortisol, leading to the known decrease in CD8+ lymphocyte infiltration observed in ACC and a localized immunosuppression not seen in other tumors.26 The authors suggested that pretreatment with drugs that target the WNT-beta-catenin or p53 pathways could improve immune responsiveness by boosting tumor- infiltrating lymphocytes.26 In the data presented here, we found that IDO-1 expression is associated with CD8+ lymphocyte infiltration. If Cosentini is correct that immuno- therapy is less effective in ACC because of the low number of tumor-infiltrating lymphocytes, it is possible that IDO-1 expression could be used as a marker to identify tumors that
are more likely to be immunoresponsive. It is premature, therefore, to discount the future role of immunotherapy in ACC, despite the disappointing results of the JAVELIN trial.
This study has several limitations. First, the sample size is necessarily small, as ACC is a rare disease. Second, we are unable to comment on IDO-1 expression’s impact on survival, as we do not have information on long-term follow-up for most patients in this cohort. Third, the significance of the IDO-1 expression seen in NA and ACT lesions is unclear, as there were only three samples in each of these groups. Finally, the future role of IDO-1 inhibition remains unclear because of the recent failure of the epacadostat phase III trial in melanoma. Nevertheless, IDO-1 stromal expression has been shown to be predictive of a response to PD-1 inhibition in renal cell carci- noma, which suggests a role for evaluation of IDO-1 expression, regardless of future IDO-1 inhibitor trial results. As systemic therapies have not held promise to improve progression-free and overall survival in patients with advanced metastatic ACC, we believe that exploring and understanding the immune microenvironment of these rare neoplasms may eventually allow for selection of a subgroup of patients who benefit from checkpoint or other immune therapies.
In summary, we found that IDO-1 is expressed in tumor and stromal samples of patients with ACC and that IDO-1 expression is also associated with PD-L2 expression and CD8+ T-cell infiltration. Further research should focus on characterizing the immune microenvironment of ACC and defining the role of IDO-1 expression in predicting response to anti PD-1 therapy in these tumors.
Acknowledgment
Author contributions: Tierney - conception and design, data collection, data analysis, drafting manuscript, critical revi- sion, final approval. Vogle: data collection, data analysis, final approval. Finnerty: critical revision, final approval. Zarnegar: critical revision, final approval. Ghai: data analysis, final approval. Gattuso: data analysis, final approval. Fahey: critical revision, final approval. Keutgen: conception and design, critical revision, final approval.
Funding: This work had no specific funding.
Disclosures
None of the authors have disclosures or competing interests to report. All authors have approved the final manuscript.
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