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.
References
1. Bilimoria KY, Shen WT, Elaraj D, Bentrem DJ, Winchester DJ, Kebebew E, et al. Adrenocortical carcinoma in the United States: treatment utilization and prognostic factors. Cancer. 2008;113:3130-3136.
2. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, et al. Com- bination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med. 2012;366:2189-2197.
3. Kroiss M, Quinkler M, Johanssen S, van Erp NP, Lankheet N, Pöllinger A, et al. Sunitinib in refractory adrenocortical carcinoma: a phase II, single-arm, open-label trial. J Clin Endocrinol Metab. 2012;97:3495-3503.
4. O’Sullivan C, Edgerly M, Velarde M, Wilkerson J, Venkatesan AM, Pittaluga S, et al. The VEGF inhibitor axitinib has limited effectiveness as a therapy for adrenocortical cancer. J Clin Endocrinol Metab. 2014;99:1291-1297.
5. Riley JL. PD-1 signaling in primary T cells. Immunol Rev. 2009;229:114-125.
6. Rozali EN, Hato SV, Robinson BW, Lake RA, Lesterhuis WJ. Programmed death ligand 2 in cancer-induced immune suppression. Clin Dev Immunol. 2012;2012.
7. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012; 12:252-264.
8. Beckers RK, Selinger CI, Vilain R, Madore J, Wilmott JS, Harvey K, et al. Pro- grammed death ligand 1 expression in triple-negative breast cancer is associ- ated with tumour-infiltrating lymphocytes and improved outcome. Histopathol- ogy. 2016;69:25-34.
9. Fay AP, Signoretti S, Callea M, Teló GH, Mckay RR, Song J, et al. Pro- grammed death ligand-1 expression in adrenocortical carcinoma: an ex- ploratory biomarker study. J Immunother Cancer. 2015;3:3.
10. Ohigashi Y, Sho M, Yamada Y, Tsurui Y, Hamada K, Ikeda N, et al. Clinical sig- nificance of programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer. Clin Cancer Res Off J Am Assoc Cancer Res. 2005;11:2947-2953.
11. Stierer M, Rosen H, Weber R, Hanak H, Spona J, Tüchler H. Immunohistochem- ical and biochemical measurement of estrogen and progesterone receptors in primary breast cancer. Correlation of histopathology and prognostic factors. Ann Surg. 1993;218:13-21.
12. R Core Team. Vienna, Austria: R Foundation for Statistical Computing. 2016. Avail- able from :. https://www.R-project.org/.
13. Yearley JH, Gibson C, Yu N, Moon C, Murphy E, Juco J, et al. PD-L2 expression in human tumors: relevance to anti-PD-1 therapy in cancer. Clin Cancer Res Off J Am Assoc Cancer Res. 2017;23:3158-3167.
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.
15. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Com- bined nivolumab and ipilimumab or monotherapy in untreated melanoma. N Engl J Med. 2015;373:23-34.
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.
17. Schalper KA, Carvajal-Hausdorf D, Mclaughlin J, Altan M, Velcheti V, Gaule P, et al. Differential expression and significance of PD-L1, IDO-1, and B7-H4 in hu- man lung cancer. Clin Cancer Res Off J Am Assoc Cancer Res. 2017;23:370-378.
18. Lin G, Fan X, Zhu W, Huang C, Zhuang W, Xu H, et al. Prognostic significance of PD-L1 expression and tumor infiltrating lymphocyte in surgically resectable non-small cell lung cancer. Oncotarget. 2017;8(48):83986-83994.
Discussion
Check for updates
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 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.