Immunohistochemical study of uveal melanoma and its cellular microenvironment

Introduction. Uveal melanoma pathogenesis is determined by a number of factors, including the tumor molecular genetics, the organism’s immune response, and other ones. One of the approaches to studying the peculiarities of pathogenesis of this cancer is to determine the local subpopulations of lymphocytes and macrophages in combination with the study of the proliferative activity of tumor cells.

Objective – to study the immunohistochemical features of uveal melanoma and its cellular microenvironment.

Materials and methods. 24 enucleated eyes with uveal melanoma (144 histological and 216 immunohistochemicalpreparations) without previous treatment were analyzed. Cells of the immune microenvironment were analyzed: lymphocyte subpopulations and CD 68+ and CD 163+ antigens expressed by macrophages in the melanoma stroma and 2–3 mm from it. The tumor cell proliferation index Ki-67 was diagnosed.

Results. All tissue samples of uveal melanoma revealed the presence of lymphocytes in the microenvironment of tumor cells. A large proportion of the studied subpopulations of lymphocytes were T-cytotoxic CD28+ lymphocytes (absolute content: 607.3 ± 431.2, relative: 18.84 % ± 12.12 %) (p = 0.018). A smaller proportion, but in equal proportions, were  T-helpers CD4+, T-cytotoxic CD8+ and CD25+ lymphocytes (p = 0.6). The absolute number of natural killer cells subpopulation CD16+ was lower compared to CD56+ (p = 0.05). However, an almost equal relative content of the studied subpopulations was noted (p = 0.9). Histological examination revealed the presence of uveal melanoma macrophages in the microenvironment of the tissue. The immunohistochemical study of CD68+ and CD163+ antigens expressed by anti-inflammatory and pro-tumor macrophages showed that their absolute and relative content in the uveal melanoma tissue is almost the same with a slight predominance of CD163+ (p = 0.7). Immunohistochemical analysis showed that the nuclei of melanoma cells contain, on average, 575.2 ± 388.5 significant cells of the Ki-67 proliferation protein. This protein was found in 16.69 ± 10.88 % of tumor cells.

Conclusion. Immunohistochemical study allows to identify subpopulations of lymphocytes infiltrating the tumor, to determine the subtypes of macrophages and to estimate the Ki-67 index of tumor cell proliferation. The data obtained will make it possible to further evaluate the significance of individual immune cells (in particular, T-cytotoxic CD28+ lymphocytes) in the pathogenesis of uveal melanoma in order to develop targeted effects, substantiate new immunotherapeutic approaches to the treatment of primary tumors and reprogramming altered immune cells.

1. Brovkina A.F., Panova I.E., Saakyan S.V. Ophthalmic oncology: new over the past two decades. Vestnik oftal’mologii = Ophthalmology bulletin 2014;130(6): 13–9. (In Russ.).

2. Saakyan S.V., Balatskaya N.V., Katargina L.A. et al. Subpopulation composition of peripheral blood lymphocytes in uveal melanoma. Meditsinskaya immunologiya = Medical immunology 2019;21(4):765–72. (In Russ.). DOI: 10.15789/1563-0625-2019-4-765-772.

3. Saakyan S.V., Amiryan A.G., Tsygankov A.Yu. et al. Clinical, pathological and molecular genetic features of uveal melanoma with a high risk of metastasis. Rossiyskiy oftal’mologicheskiy zhurnal = Russian Ophthalmological Journal 2015;8(2):47–52. (In Russ.).

4. Fidler I.J., Kim S.J., Langley R.R. The role of the organ microenvironment in the biology and therapy of cancer metastasis. J. Cell Biochem 2007;101(4): 927–36. DOI: 10.1002/jcb.21148.

5. Saakyan S.V., Zakharova G.P., Myakoshina E.B. Mast cells in the microenvironment of uveal melanoma. Arkhiv patologii = Archive of pathology 2019;81(6):63–70. (In Russ.). DOI: 10.17116/patol20198106163.

6. Johansson J., Siarov J., Kiffin R. et al. Presence of tumor-infiltrating CD8+ T cells and macrophages correlates to longer overall survival in patients undergoing isolated hepatic perfusion for uveal melanoma liver metastasis. Oncoimmunology 2020;10;9(1):1854519. DOI: 10.1080/2162402X.2020.1854519.

7. Lee N., Zakka L.R., Mihm M.C. Jr. et al. Tumour-infiltrating lymphocytes in melanoma prognosis and cancerimmunotherapy. Pathology 2016;48(2):177–87. DOI: 10.1016/j.pathol.2015.12.006.

8. Bronkhorst I.H., Ly L.V., Jordanova E.S. et al. Detection of M2-macrophages in uveal melanoma and relation with survival. Invest Ophthalmol Vis Sci 2011;3;52(2):643–50. DOI: 10.1167/iovs.10-5979.

9. Toivonen P., Mäkitie T., Kujala E. et al. Microcirculation and tumor-infiltrating macrophages in choroidal and ciliary body melanoma and corresponding metastases. Invest Ophthalmol Vis Sci 2004;45(1):1–6. DOI: 10.1167/iovs.03-0622.

10. Mooy C.M., de Jong P.T., Van der Kwast T.H. et al. Ki-67 immunostaining in uveal melanoma. The effect of preenucleation radiotherapy. Ophthalmology 1990;97(10):1275–80. DOI: 10.1016/s0161-6420(90)32420-x.

11. Al-Jamal R.T., Kivelä T. KI-67 immunopositivity in choroidal and ciliary body melanoma with respect to nucleolar diameter and other prognostic factors. Curr Eye Res 2006;31(1):57–67. DOI: 10.1080/02713680500478535.

12. Tian T., Olson S., Whitacre J. et al. The origins of cancer robustness and evolvability. Integr Biol 2011;3(1):17–30. DOI: 10.1039/c0ib00046a.

13. Lane A.M., Kim I.K., Gragoudas E.S. Survival rates in patients after treatment for metastasis from uveal melanoma. JAMA Ophthalmol 2018;136(9):981–6. DOI: 10.1001/jamaophthalmol.2018.2466.

14. Rantala E.S., Hernberg M., Kivela T.T. Overall survival after treatment for metastatic uveal melanoma: a systematic review and meta-analysis. Melanoma Res 2019;29(6):561–8. DOI: 10.1097/CMR.0000000000000575.

15. Heppt M.V., Heinzerling L., Kähler K.C. et al. Prognostic factors and outcomes in metastatic uveal melanoma treated with programmed cell death-1 or combined PD-1/cytotoxic T-lymphocyte antigen-4 inhibition. Eur J Cancer 2017;82:56–65. DOI: 10.1016/j.ejca.2017.05.038.

16. Roma-Rodrigues C., Mendes R., Baptista P. V. et al. Targeting Tumor Microenvironment for Cancer Therapy. Int J Mol Sci 2019;20(4):840. DOI: 10.3390/ijms20040840.

17. Bronkhorst I.H.G., Jager M.J. Uveal melanoma: the Inflammatory microenvironment. J Innate Immun 2012;4:454–62. DOI: 10.1159/000334576.

18. Saakyan S.V., Zakharova G.P., Myakoshina E.B. Cellular microenvironment of uveal melanoma: clinical and morphological correlations and predictors of poor prognosis. Molekulyarnaya meditsina = Molecular Medicine 2020;18(3):27–33. (In Russ.). DOI: 10.29296/24999490-2020-03-04.

19. Frank G.A., Kuznetsova O.A., Zavalishina L.E. et al. Investigation of PD-L1-status of breast cancer using monoclonal antibody SP142 and prospects for determining therapeutic tactics. Arkhiv patologii = Archive of pathology 2019;81(5):5–10. (In Russ.). DOI: 10.17116/patol2019810515.

20. Oble D.A., Loewe R., Yu P. et al. Focus on TILs: prognostic significance of tumor infiltrating lymphocytes in human melanoma. Cancer Immun 2009;9(1):3.

21. McLean I.W., Saraiva V.S., Burnier M.N. Pathological and prognostic features of uveal melanomas. Can J Ophthalmol 2004;39:343–50. DOI: 10.1016/s0008-4182(04)80004-8.

22. Qin Y., Bollin К., Petaccia de Macedo M. et al. Immune profiling of uveal melanoma identifies a potential signature associated with response to immunotherapy. J Immunother Cancer 2020;8(2):e000960. DOI: 10.1136/jitc-2020-000960.

23. Krishna Y., McCarthy C., Kalirai Н. et al. Inflammatory cell infiltrates in advanced metastatic uveal melanoma. Human Pathology 2017;66:159–66. DOI: org/10.1016/j.humpath.2017.06.005.

24. Ly L.V., Bronkhorst I.H.G., van Beelen E. et al. Inflammatory cytokines in eyes with uveal melanoma and relation with macrophage infiltration. Invest Ophthalmol Vis Sci 2010;51(11):5445–51. DOI: 10.1167/iovs.10-5526.

25. Coussens L.M., Werb Z. Inflammation and cancer. Nature 2002;420(6917):860–7. DOI:10.1038/nature01322.

26. Tobal K., Deuble K., Mccartney A. et al. Characterization of cellular infiltration in choroidal melanoma. Melanoma Res 1993;3(1):63–5. DOI: 10.1097/00008390-199304000-00009.

27. Mantovani A. Tumor-associated macrophages in neoplastic progression: a paradigm for the in vivo function of chemokines. Lab Invest 1994;71(1):5–16.

28. Sun X., Kaufman P.D. Ki-67: more than a proliferation marker. Chromosoma 2018;127(2):175–86. DOI:10.1007/s00412-018-0659-8.

29. Novikova I.A., Maksimova N.A., Pozdnyakova V.V. et al. Some immunohistochemical features of skin melanocytic formations. Sovremennye problemy nauki i obrazovaniya = Modern problems of science and education 2016;5:1–8. (In Russ.).