Surgery 000 (2018) 1-6
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Expression of programmed death ligand 1 and 2 in adrenocortical cancer tissues: An exploratory study
John F. Tierney, MDª,*, Alyx Vogle, BSª, Jennifer Poirier, PhDª, Irene M. Min, PhDb, Brendan Finnerty, MDb, Rasa Zarnegar, MDb, Sam G. Pappas, MDª, Theresa Scognamiglio, MD, Ritu Ghai, MDd, Paolo Gattuso, MDd, Thomas J. Fahey III, MDb, Xavier M. Keutgen, MDª
a Rush University Medical Center, Department of Surgery, Division of Surgical Oncology, Chicago, Illinois
b NewYork-Presbyterian Hospital/Weill Cornell Medical Center, Department of Surgery, New York, New York
NewYork-Presbyterian Hospital/Weill Cornell Medical Center, Department of Pathology, New York, New York
d Rush University Medical Center, Department of Pathology, Chicago, Illinois
ARTICLE INFO
Article history: Accepted 25 April 2018 Available online xxx
ABSTRACT
Background: Inhibition of the interaction of programmed death 1 with programmed death ligand 1 and 2 has been used successfully for treatment of multiple advanced cancers, but expression has not been studied in adrenocortical carcinoma. In this study, we investigated programmed death ligand 1 and 2 expression in adrenocortical carcinoma to determine the potential usefulness of checkpoint inhibitors in these malignant neoplasms.
Methods: A total of 56 tissue samples from patients with adrenocortical carcinoma (34) and benign adrenal tissues (22) were identified. Immunohistochemistry was performed for programmed death lig- and 1, programmed death ligand 2, and CD8 and scored for membranous staining on adrenal and stromal tissue according to the immunoreactive score and absolute percentage, respectively. Descriptive statistics, a Mann-Whitney U test, and Fisher exact tests were calculated.
Results: In total, 15 adrenocortical carcinoma (44%) stained positive for programmed death ligand 2 and 1 adrenocortical carcinoma for programmed death ligand 1 (P =. 03). Adrenocortical carcinoma samples were more likely to express programmed death ligand 2 on tumor cells or in stromal tissues than be- nign samples (OR=2.3, P =. 03). There was no relationship between programmed death ligand 2 and CD8 expression (P =. 08). There were also no relationships between programmed death ligand 2 or CD8 ex- pression and tumor characteristics.
Conclusion: Programmed death ligand 2, but not programmed death ligand 1, is expressed commonly in adrenocortical carcinoma samples. The utility of certain checkpoint inhibitors should, therefore, be evaluated in further studies.
@ 2018 Elsevier Inc. All rights reserved.
Introduction
Adrenocortical carcinoma (ACC) is a rare but lethal malignancy with an estimated worldwide incidence of 2 per million.1 It is the second most aggressive endocrine malignancy behind anaplastic thyroid cancer, and its overall median survival of 32 months has not improved in the past 3 decades.1 Complete resection is con- sidered the most important component of treatment, but patients
frequently recur locally or distally, even after a curative resection. Systemic therapy with mitotane, etoposide, doxorubicin, and cis- platin has a low objective response rate and does not improve overall survival, even in an adjuvant setting.2 Phase II investiga- tions of newer therapies, such as sunitinib and vascular endothelial growth factor (VEGF)-inhibitors, also have not shown encouraging response rates.3,4 Therefore, novel, effective therapies are needed for this disease.
Programmed death 1 (PD-1) is a tyrosine-kinase receptor pro- tein expressed by B- and T-lymphocytes that is involved in regu- lation of the immune response.5,6 Specifically, PD-1 inhibits T-cell proliferation when activated by either of its ligands, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2).5 Blockade of the interaction between PD-1 and PD-L1 has been well
# Presented at the annual meeting of the American Association of Endocrine Sur- geons, Durham, North Carolina, May 6-8, 2018.
* Corresponding author: Rush University Medical Center, Surgery, 1750 West Har- rison Suite 785, Chicago, IL 60612.
E-mail address: john_f_tierney@rush.edu (J.F. Tierney).
https://doi.org/10.1016/j.surg.2018.04.086
0039-6060/ 2018 Elsevier Inc. All rights reserved.
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described in many cancers, including non-small cell lung cancer, esophageal cancer, triple-negative breast cancer, and melanoma.7,8 The only previous study to examine the role of PD-L1 in ACC, how- ever, found that its expression on tumor cells was limited to a low percentage of samples without correlation with clinical outcomes or pathologic features.9
Despite the more intense focus on PD-L1 in tumor immunother- apy, there is some evidence that PD-L2 is associated with de- creased survival and decreased presence of CD8+ tumor-infiltrating lymphocytes in patients with esophageal cancer and could be a viable treatment target for many cancers because it has a much greater affinity for the PD-1 receptor than PD-L1.6,10
We, therefore, sought to examine the expression levels of PD-L1 and PD-L2 in human ACC samples to determine whether
checkpoint inhibitors should be considered as a therapeutic option in these patients. Secondary objectives included correlating PD- L1 and PD-L2 expression with presence of tumor-infiltrating CD8+ lymphocytes and with pathologic and clinical features.
Methods
A total of 56 tissue samples from patients with ACC, indetermi- nate adrenocortical tumors (ACT), adrenal adenomas (AA), and nor- mal adrenal tissue (NA) were obtained from separate institutional biorepositories at Rush University Medical Center (RUMC) and NewYork-Presbyterian/Weill Cornell Medical Center (NYP-WCMC). The RUMC Institutional Review Board approved this study, the re- search offices of each institution approved material transfer, and all
J.F. Tierney et al./Surgery 000 (2018) 1-6
| Percentage of positive cells (A) | Intensity of staining (B) | IRS (A 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 |
data obtained from NYP-WCMC were de-identified before transfer. Consent and Health Insurance Portability and Accountability Act authorization were waived owing to the retrospective nature of the study.
Clinical and pathologic data, including patient sex, age at di- agnosis, presence of metastases at diagnosis, whether the tumor secreted hormones, and survival were collected from the medical records when available. Samples were classified according to their diagnosis on final clinical pathology performed at the time of tu- mor removal. Samples that did not meet Weiss criteria for ACC but had more than 5 mitoses per 50 high-power fields were consid- ered diagnosed as 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
Five-micron sections were made from formalin-fixed, paraffin- embedded tissue from 34 primary ACCs, 16 AAs, 3 NAs, and 3 ACTs. Samples from RUMC were mounted on individual glass slides, and samples from NYP-WCMC were obtained on a tissue microar- ray (TMA) because these samples had been cored and mounted previously.
PD-L1, PD-L2, and CD8 staining was performed using commer- cially available antibodies. Slides were first deparaffinized with xy- lene and rehydrated. A heat-mediated retrieval was performed us- ing citrate buffer (SignalStain® Citrate Unmasking Solution, Cell Signaling Technology, Danvers, MA). Endogenous peroxidases were blocked using hydrogen peroxide. Slides were incubated for 2 h with animal-free blocking solution (Cell Signaling Technology, Danvers, MA) for protein blocking. Slides were then incubated overnight with rabbit monoclonal anti-PD-L2 (Abcam, ab214221, 1:250, Cambridge, MA), mouse monoclonal anti-PD-L1 (Dako, 22c3 pharmDx, Santa Clara, CA), and rabbit polyclonal anti-CD8 (Ab- cam, ab4055, 1:200, Cambridge, MA) antibodies. Rabbit Signal- Stain® Boost Detection reagent secondary (Cell Signaling Technol- ogy, Danvers, MA) was then applied. Slides were developed using SignalStain® DAB Substrate Kit (Cell Signaling Technology, Dan- vers, MA) according to the manufacturer’s instructions and coun- terstained with hematoxylin (Harris Hematoxylin, BBC Biochemi- cal, Mount Vernon, WA). Finally, they were washed, dehydrated in alcohol and xylene, mounted, and coverslipped.
Immunohistochemical scoring
All samples were reviewed and scored blindly by 2, board- certified pathologists and scored for PD-L1 and PD-L2 expression on tumor cell membranes according to the percent of positive tu- mor cells and intensity. Intensity was scored as absent (0), low (1), moderate (2), or high (3). Each tumor sample on the TMA was rep- resented by 1 to 3 TMA cores; the percentages and intensities from each core were averaged.
For evaluation of tumor tissue, percentage scores were then converted to the following integer values on a 0-4 scale: a value of 0 was assigned to 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 calculated by multiplying the intensity score and the integer value for the percentage score, as described previ- ously (Table 1).11 Positive staining of adrenal samples in this study was defined as an IRS ≥6 (>50% of positive cells and ≥2+ staining intensity).
Positive staining of stromal tissue was determined by the per- cent of stromal cells that stained positive, with a cutoff of ≥10%, as described previously.10 CD8+ tumor infiltrating lymphocyte stain- ing was classified as absent, focal, or diffuse. When differences in IRS, percent of positive stromal cells, or CD8+ lymphocyte infiltra- tion existed between pathologists, the average score was used.
Statistical analysis
Descriptive statistics were calculated for the rate of PD-L1, PD- L2, and CD8+ lymphocyte positivity in tumor and stromal tissues according each histologic diagnosis. The proportion of ACC sam- ples expressing PD-L2 and the proportion expressing PD-L1, and the rates of PD-L2 positivity between ACC and benign samples in adrenal and stromal tissues, were both compared using Fisher ex- act tests. For ACC samples, relationships between PD-L2 positiv- ity and CD8+ lymphocyte infiltration, metastases at diagnosis, pa- tient 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 R 3.3.2.12
Results
Samples from 34 ACC, 3 ACT, 16 AA, and 3 NA were analyzed. A total of 19 samples mounted on individual glass slides were ob- tained from the RUMC institutional biorepository and 37 tissue cores mounted on a TMA were obtained from NYP-WCMC. Suffi- cient stromal tissue was present to analyze stromal protein expres- sion in 34 samples.
PD-L1 tumor staining was observed in only 1 ACC sample. No benign samples stained positive for PD-L1. Of note, the ACC sample that expressed PD-L1 did not also express PD-L2.
Strong PD-L2 tumor was found in 15 ACC and stromal stain- ing in 8 ACC samples. Although tumor or stromal PD-L2 expres- sion individually was not greater when comparing malignant to benign lesions (P =. 11 and P =. 16), ACC samples were more likely to express PD-L2 than benign samples when all samples with pos- itive tumor and stromal tissue were included (odds ratio [OR] 2.3, P =. 03; Table 2). ACC samples were also more likely to express PD- L2 than to express PD-L1 (P < . 001).
ACC samples on the TMA were more likely to be PD-L2 positive in the tumor and stroma than samples on individual glass slides (P =. 013 and < . 001).
A total of 65% (22 of 34) of ACC samples were either focally or diffusely positive for CD8+ tumor infiltrating lymphocytes. There was no relationship between PD-L2 expression in tumor or stroma and CD8+ lymphocyte infiltration (P =. 46, P =. 14). There was also no difference in the presence of CD8+ tumor infiltrating lympho-
J.F. Tierney et al./Surgery 000 (2018) 1-6
| ACC (%) | AA (%) | NA (%) | ACT (%) | All benign | P value* | |
|---|---|---|---|---|---|---|
| Number of samples | 34 | 16 | 3 | 3 | 22 | |
| RUMC | 13 (38) | 3 (19) | 0 (0) | 3 | 6 (27) | |
| NYP-WCMC | 21 (62) | 13 (81) | 3 (100) | 0 | 16 (73) | |
| PD-L1 positive (adrenal tissue) | 1 | 0 | 0 | 0 | 0 | 1 |
| PD-L2 positive (adrenal tissue) | 15 (44) | 4 (25) | 0 | 1 | 5 (23) | .16 |
| PD-L2 positive (stroma) | 8 (37) | 1 | 0 | 0 | 1 | .11 |
| PD-L2 positive (adrenal + stroma) | 18 (53) | 4 (25) | 0 | 1 | 5 (23) | .03 |
| CD8 absent | 12 (35) | |||||
| CD8 focal | 18 (53) | |||||
| CD8 diffuse | 4 (12) |
* P value refers to comparison between ACC and total benign groups
| Clinical characteristic | Tumor | Stroma | ||||
|---|---|---|---|---|---|---|
| PD-L2 positive (%) | Patient number | P value | PD-L2 positive (%) | Patient number | P value | |
| Metastases at diagnosis | 5 (42) | 20 | .64 | 3 (33) | 16 | .21 |
| Male sex | 5 (50) | 33 | .7 | 3 (50) | 22 | .62 |
| Hormone-secreting | 3 (38) | 14 | .62 | 3 (43) | 11 | .99 |
| Age > median (51.5 y) | 8 (62) | 29 | .14 | 3 (30) | 22 | .67 |
cyte or PD-L1 expression between the TMA and individual slide groups (P =. 13 and P =. 99).
Of the patients with ACC, 10 of 33 were male (30%), and the median age was 52 years. Of the 14 ACCs, 8 (57%) secreted hor- mones, most commonly cortisol. In total, 60% of patients (12 of 20) had metastases at diagnosis. There was no relationship between sex, age, metastases at diagnosis, or functional status, and PD-L2 positivity in the tumor or stroma (all P values >.1). There was no difference in sex, age, metastases at diagnosis, or functional status between the TMA and individual slide groups (all P values >.2 -. 7; Table 3).
Discussion
In this study, we sought to investigate the expression of PD- L1 and PD-L2 in ACC tumor samples. We demonstrated that al- though PD-L1 is rarely expressed in ACC, PD-L2 is strongly ex- pressed in nearly half of the ACCs, and that PD-L2 is expressed more frequently in the tumor or stromal tissues of ACCs than in benign samples, suggesting that it may be an interesting target for therapy.
Blockade of the interaction of PD-1 with its ligands, PD-L1 and PD-L2, has been shown to improve survival in a variety of cancers.5,7,8,13-15 PD-L1 is commonly tested in clinical pathology to determine if patients with non-small cell lung cancer and esophageal adenocarcinoma will respond to treatment with block- ade of the PD-1 pathway, but some patients who are PD-L1 neg- ative will also respond to this therapy.16 It has been hypothesized that PD-L2 expression could promote response to PD-1 inhibition in PD-L1 negative patients.13 Although PD-L1 is tested and ex- pressed more commonly in tumor tissue of all cancer types, PD-L2 has greater affinity for PD-1.6 It was initially posited that PD-L1 is constitutively expressed on all tissues, whereas PD-L2 is only ex- pressed by antigen-presenting cells, but this is now known to be untrue.6,10 PD-L2 expression has been shown to be upregulated in the presence of certain stimuli in the microenvironment, including Th2 cytokines such as IL-4, IFN-gamma, and toll-like receptor lig- ands, and has been demonstrated in cancer fibroblasts from multi- ple tumor types, although these mechanisms have not been stud- ied in ACC.6 PD-L2 expression in tumor tissue, therefore, is an at- tractive area for further investigation.
When a PD-1 receptor on the surface of a lymphocyte is acti- vated by its ligand, downstream effects eventually lead to impaired cytokine production and cytolysis, and eventually, to apoptosis of the lymphocyte.6 Physiologically, PD-1 activation dampens the im- mune response to protect against autoimmune and ineffective im- mune responses, but its mechanism also leads to immune dysfunc- tion in chronic viral infections and cancer.5 Tumor cell expression of a PD-1 ligand such as PD-L2 allows tumor cells to target host T-cells, and thereby evade the immune response.9 T-lymphocytes themselves upregulate PD-L1 expression on tumor cells by secret- ing IFN-gamma. 14 Although this pathway has been better described for PD-L1, PD-L2 expression is also IFN-gamma-dependent.
A subset of drugs targeting the PD-1 pathway, including ate- zolizumab, bind to PD-L1 and, therefore only block the interaction of PD-1 with PD-L1. Other drugs, however, including nivolumab and pembrolizumab, bind PD-1 directly and block its interaction with both PD-L1 and PD-L2.13 The use of this second class of drugs is more likely to be beneficial in ACC because PD-L1 expression ap- pears to be rare in this cancer.
Only 1 previous study by Fay et al reported PD-L1 expression in ACC by examining 28 samples. The authors found that PD-L1 was expressed in only 10% of ACCs but by tumor infiltrating mononu- clear cells in 68% of cases.9 The rate of PD-L1 expression on tumor tissue reported in our study (3%) was less than the rate reported previously, and no PD-L1 expression was seen in stromal tissue. Al- though the study by Fay et al determined that a sample was pos- itive for PD-L1 if 10% of tumor cells were positive, the ACC sam- ples in our study would still have been considered negative even at this lesser threshold. After reviewing both studies, we can con- clude that PD-L1 expression in ACC is uncommon. The previously reported high expression of PD-L1 on tumor-infiltrating mononu- clear cells combined with the expression of PD-L2 on tumor tissue and in stroma reported in this study suggests that the PD-1 path- way is still perhaps a viable target for immunotherapy for a select number of patients with ACCs.
This study has several limitations. First, the number of ACC samples is relatively small, which limits the power of any com- parisons with clinical and pathologic features. These tumors are rare and thus acquiring a large n value is difficult. Second, im- munohistochemistry has an inherent observational bias because it uses subjective scoring from a pathologist. We tried to correct
for this by having 2 pathologists assess these samples indepen- dently and blindly. Third, we were unable to analyze the effect of PD-L2 expression on patient survival because long-term follow-up and treatment information were not available and the number of patients was small. The available clinical information, such as the presence of metastases at diagnosis, does not necessarily corre- spond with tumor aggressiveness and was not available for all pa- tients. Inhibition of thePD-1 pathway has been an effective therapy even in cancers for which there is no apparent relationship be- tween increased PD-L1 expression and prognosis.17,18 Fourth, some samples were assessed using a TMA whereas others were mounted on individual glass slides. We initially hypothesized that the use of a TMA might lead to underestimation of PD-L1 and PD-L2 expres- sion if these proteins were not expressed in the area of the tumor used for the TMA core, but we found that TMA samples were ac- tually more likely to express PD-L2 than the samples on individual slides. The majority of samples on individual slides that did not express PD-L2 had no staining but expression was relatively uni- form in samples that did express the marker, so it is likely that the greater expression on the TMA accurately represents the tumor at large.
Despite these limitations, we have shown that PD-L2 is ex- pressed in nearly half of ACCs and could represent a viable target for further investigation. Future research should focus on testing of PD-1 inhibitors such as pembrolizumab in preclinical cell line and animal models as well as correlation of PD-L2 expression with pa- tient survival.
Conflicts of interest
The authors have indicated that they have no conflicts of inter- est regarding the content of this article.
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14. Webb JR, Milne K, Kroeger DR, Nelson BH. PD-L1 expression is associated with tumor-infiltrating T cells and favorable prognosis in high-grade serous ovarian cancer. Gynecol Oncol. 2016; 141:293-302.
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16. Arrieta O, Montes-Servín E, Hernandez-Martinez J-M, Cardona AF, Casas-Ruiz E, Crispín JC, et al. Expression of PD-1/PD-L1 and PD-L2 in peripheral T-cells from non-small cell lung cancer patients. Oncotarget. 2017;8:101994-102005.
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Discussion of Paper Number 22
Expression of programmed death ligand 1 and 2 in adrenocortical cancer tissues: An exploratory study
Dr Brian Untch (New York, NY): I think this has a lot of interest for us because we are in the midst of finishing a phase II study with pembrolizumab for advanced patients with ACC.
We have been picking patients mostly based on microsatellite instability and neoantigenic burden, and I am wondering if we should be using PD-L2 IHC instead. I would like your comments on that.
Secondly, I imagine some of these patients have undergone some genomic sequencing because it looked like it was a large group. So I am wondering if you have done any of that, and if you have been able to correlate genomic lesions with PD-L2 expression.
Dr John F. Tierney: I think working with PD-L2 IHC could po- tentially be usable to help select patients for that study. And I am curious if you have been looking at PD-L1 IHC for those patients, and if you found that PD-L1 was expressed on their tumors.
Then to your second question, many of these patients dated back a couple of decades, so I don’t know how many of them un- derwent genomic sequencing.
Dr John Phay (Columbus, OH): For specific checkpoint in- hibitors, there’s certain levels of what is considered positivity for the tumor cells. And for some, it’s as low as 25%. Did you see many more positive cells, if you lowered your criteria?
Dr John F. Tierney: We would not have seen that many more positive cells-especially for PD-L1. We would have seen a few more for PD-L2. But if we had lowered the threshold for PD-L1, we would not have seen any more positive cells.
Dr Janet Li (New York, NY): I am interested to know if you had any patients that only stained positive in the stroma and not in the tumor and if you anticipate that would affect tumor infiltration of the T-cells.
Dr John F. Tierney: We did have, I believe, only 3 patients who stained positive in the stroma and not in the tumor.
If the stroma is expressing the ligand, then that could still serve to bind PD-1 and therefore inhibit the T-cell efficacy, so that’s been
J.F. Tierney et al./Surgery 000 (2018) 1-6
seen in other cancers that stroma expression of PD-L1 can inhibit T-cell.
Dr Mark Cohen (Ann Arbor, MI): Did you happen to correlate PD-L2 expression levels with mitotic rate? It might be interesting to look at that.
Dr John F. Tierney: We did not.
Dr Edwin Kaplan (Chicago, IL): Very nice talk. Could you tell us the ones who are not that familiar with this, a drug like KEYTRUDA is a PD-L1 inhibitor I believe. What are the PD-L2 inhibitors?
Dr John F. Tierney: Actually, KEYTRUDA is a PD-1 inhibitor. PD- 1 is the receptor that’s on the T-cells. And then PD-L1 and PD-L2 bind PD-1.
But KEYTRUDA is a PD-1 inhibitor, and that will block both PD-L1 and PD-L2 from binding PD-1, so the PD-L1 in-
hibitors will only block PD-L1, but PD-1 inhibitors will block both.
There aren’t any specific PD-L2 inhibitors, but the PD-1 in- hibitors could potentially work for PD-L2 positive tumors.
Dr Oliver Gimm (Linkoping, Sweden): Very nice presentation. Thank you. As we know, these tumor cells are very heterogenous. I was wondering whether you were able to see a correlation be- tween those patients that were doing well and those that were doing less well.
Dr John F. Tierney: We didn’t have very long-term follow-up data for most of our patients, so we were not able to correlate that, unfortunately.