In this study, we have shown that serum concentrations of Leptin and Osteopontin at the time of UM diagnosis can identify patients with poor metastasis-free survival. These two proteins were included in the serUM-Px prognostic test which assigns patients to a metastatic risk category. Herein, low serum Leptin concentrations and high serum Osteopontin concentrations correlate with the highest metastatic risk category at the time of UM diagnosis. Patients had gradually shorter metastasis-free survival and a greater incidence of UM-related mortality with each increasing metastatic risk category.
Previous studies have found a significant difference in plasma levels of Osteopontin between patients with and without detectable metastases.[38, 39] These studies showed that tumor marker levels increase before the existence of metastasis that can be identified by imaging. Osteopontin is a 314-amino acid phosphoglycoprotein that is a component of the noncollagenous bone matrix. This protein has been described in the role of diverse physiological roles such as chemotaxis, cell migration and adhesion, angiogenesis, apoptosis, cell-extracellular matrix interactions, and immune regulation. Osteopontin actively promotes the tumorigenic phenotype and contributes to metastatic spread. Elevated serum levels of Osteopontin have been described in patients with advanced or metastatic cancer. Recently, increased Osteopontin levels have been observed in patients with metastatic UM, which correlates with our results.[47–49]
The other variable in serUM-Px – Leptin – has been described in various types of tumor cells, including breast, prostate, colon and endometrium where Leptin has been implicated as a growth factor for these cancers.[50–54] Leptin does not only play a role in food intake and energy balance but also functions as a pro-inflammatory adipokine with a broad range of activities including cytokine production, cellular immunity, and inflammation.[55–57] Leptin may also promote tumor growth by signaling through normal endocrine pathways. Physiologic binding of leptin to its receptors on hypothalamic neurons leads to Thyrotropin-releasing hormone (TRH) production by these cells. Ellerhorst et al. have shown that melanoma cells express TRH and that TRH induces proliferation of these cells, which raises the possibility of Leptin as an inducer of melanoma TRH production and secretion, accounting in part for its growth-promoting effects.
The serUM-Px metastatic risk categories were not associated with primary tumor BAP-1 expression, which is a well-established strong prognostic marker.
Considering that this correlation was examined in a very limited sample of 15 tumors, we suspect that the non-significant correlation (p = 0.056) may represent a type II error, which should be investigated in a larger cohort.
Considering that most patients diagnosed with UM desire prognostic information and that most current testing alternatives entail an invasive procedure unless the tumor eye is enucleated, liquid biopsies based on peripheral blood samples are an attractive alternative. We suggest that the newly developed serUM-Px test may be seen as an alternative to FNABs and transvitreal biopsies. serUM-Px has the benefit of being a test that reflects the risk of lethal course, is relatively inexpensive, minimally invasive, and has a low risk profile regarding complications. Consequently, prognostic testing can be made available for all UM patients, regardless of treatment modality.
Strengths of this study include the complete control of patients’ follow-up. We had access to detailed data regarding the tumor and patient characteristics, as well as survival data from clinical records that were accessible regardless of where in the country the patient resides, which enabled robust correlation to the outcome where no patient was lost to follow-up. Further, our serUM-Px test predicted metastatic disease many years before macrometastases developed, whereas most other similar tests have relied on repeated sampling to reveal macrometastases at the time of or just before they become radiologically detectable. Another of the foremost strengths of this study is simultaneously one of its considerable limitations; the > 20-year storage of the serum samples at -80°C allowed for long follow-up. However, no fresh samples were included. Even though previous studies indicate that serum samples can be stored deep-frozen even for decades without protein degradation, the protein concentrations observed herein do not necessarily reflect concentrations in fresh samples.[60–62]
This study has several other limitations. The results were based on a relatively small cohort of patients with moderately few metastatic events. The latter is likely a result of inclusion of a cohort with quite small tumors. Tumor size is strongly associated with virtually all other prognostic factors in UM, including ciliary body involvement, BAP1 mutation, gene expression class 2, monosomy 3, tumor cell type, and patient age.[63–65] Several of these factors were not included in our data and we cannot assess their correlation with serUM-Px. Further, serum Leptin levels have a diurnal variation that follows the circadian rhythm with peak levels at night. Even though all of our blood samples were taken in the daytime, some patients may have been classified differently if their blood sample had been drawn at a different time of the day. Leptin levels also correlate to patient body mass and fat tissue volumes, with obese patients having higher serum concentrations. We did not collect data on patient weight or similar in this project, which would have helped us investigate if the patients with high Leptin levels and low metastatic risk were associated with obesity. This association merits further investigation in future studies.