Interleukin-10: A Potential Prognostic Marker in Patients with Newly Diagnosed Multiple Myeloma

Background: Identification of high-risk patients with multiple myeloma (MM) is important for improving treatment outcomes. Efforts to identify significant prognostic markers are unremitting. Angiogenesis plays an important mechanism in the progression of MM. One of the mediators of this angiogenic process is interleukin-10 (IL-10). Aim: To assess the role of IL-10 as a prognostic marker in MM. Methods: This was a cross-sectional study that included 39 newly diagnosed patients with MM. Serum IL-10 level was measured using Magnetic Luminex® Assay multiplex. The relationship between IL-10 and tumor stage and other known prognostic markers in MM was studied. Results: The median (interquartile range) value of IL-10 was 3 (2.9 – 3.2) pg/mL. Interleukin-10 level differed significantly according to the revised International Staging System stage of MM, being higher in higher stages. The median (interquartile range) IL-10 level was 2.89 (2.79 – 2.95) pg/mL in stage I, 3 (2.9 – 3.1) in stage II and 3.2 (3.1 – 3.66) in stage III (p = 0.0009). In addition, higher IL-10 significantly correlated with lower hemoglobin (p = 0.002), lower albumin (p = 0.045), higher creatinine (p = 0.009), higher β2-microglobulin (p = 0.002), higher lactate dehydrogenase (p = 0.0007) and higher bone marrow plasma cell percentage (p = 0.015). Conclusion: The results support a prognostic role of IL-10 in MM and its pathogenesis.


Introduction
Multiple myeloma (MM) is a neoplasm of clonal plasma cells that originate from the post-germinal lymphoid B-cell lineage 1 . It is the second most frequent hematological malignancy after lymphoma and remains largely incurable disease.
Multiple bone marrow microenvironmental changes are involved in the development and progression of MM, as the support provided by the immune microenvironment changes to myeloma cells is crucial for their survival [2][3][4] .
Interleukin-10 (IL-10) is an immune-regulatory cytokine produced by many cells including macrophages, T-lymphocytes, and natural killer cells. Its role in cancer development is still controversial. It may promote tumor cell proliferation and metastasis via its immunosuppression functions 5 . Its anti-tumour effect may be due to the stimulation of natural killer and cytotoxic T-lymphocytes mediated killing of cancer cells 6 . Research is still required to evaluate the role of IL-10 in the tumorigenesis of MM. In addition, the prognostic role of IL-10 in MM has been suggested. A high level of IL-10 was found to correlate significantly with higher MM stage and β2microglobulin level 7 .
Our aim was to study the relationship between IL-10 level and other prognostic markers in MM.

Methods
This study was an observational cross-sectional study. It included 39 patients with newly diagnosed MM who presented to the Medical Oncology Department -South Egypt Cancer Institute and the Clinical Hematology Unit -Internal Medicine Department, Assiut University from July 2017 to July 2020.
All patients were proven to have MM according to the International Myeloma Working Group Criteria for the diagnosis of MM and Related Plasma Cell Disorders 8 .
All patients were diagnosed and evaluated at the outpatient clinic of the concerned department. They were subjected to full history taking, clinical examination and laboratory investigations. The laboratory investigations included complete blood picture (using Cell-Dyn Ruby, Abbott Diagnostics, USA), serum chemistry (creatinine, calcium, albumin, lactate dehydrogenase and β2microglobulin, using COBAS Integra 400 Plus, Roche, Switzerland), serum protein electrophoresis and immunofixation (using Pretty Interlab, Interlab, Italy) as well as bone marrow aspiration and trephine biopsies. Interphase fluorescence in situ hybridization (FISH) was performed to stratify the risk of the disease. The disease was staged according to the revised International Staging System (R-ISS).
Serum samples were collected from patients before starting anti-myeloma therapy using a serum separator tube and stored at -20 °C for further evaluation. Serum concentration of IL-10 was evaluated according to the manufacturer's instructions using Magnetic Luminex® Assay multiplex kits (Human Premixed Multi-Analyte Kit, R&D Systems, Bio-Techne, Minneapolis, USA).

Statistical analysis
Continuous data were presented as mean and standard deviation (SD) or median and range, while categorical data as frequency and percentage. For abnormally-distributed quantitative data, comparison between two groups was done using Mann-Whitney and between more than two groups by Kruskall Wallis test. Correlations were assessed using the Spearman's rank correlation coefficient.
Data analysis was done using the IBM Statistical Package for Social Science (SPSS) for Windows, Version 21.0. (Armonk, NY: IBM Corp).

Ethical considerations
The study was approved by the Institutional Review Board of the South Egypt Cancer Institute, Assiut University (approval No: 380).
An informed written consent was taken from all patients and controls.

Results
The demographic and clinical characteristics of patients are summarized in Table 1. Two-thirds were males and their mean age was 58 (± 8.8) years.  The correlation between IL-10 level and the studied variables is detailed in Table 2. The following variables significantly correlated with higher IL-10 level: higher R-ISS stage, lower hemoglobin level, lower albumin level, higher creatinine level, higher β2-microglobulin level, higher lactate dehydrogenase level, and higher percentage of plasma cells in the bone marrow. Although IL-10 level was higher in patients with osteolytic lesions and a calcium level above 12 mg/dL, this was not statistically significant.

Discussion
The interaction of plasma cells with bone marrow microenvironment mediates myeloma disease progression 9 . Multiple myeloma remains largely an incurable disease despite the development of many therapeutic agents, such as bortezomib and lenalidomide. Failure to cure is multifactorial and can be attributed to the underlying genetic aberrations and to the surrounding microenvironment 10 .
Accurate identification of high-risk patients and risk stratification are very important in improving outcomes of MM patients. A large number of prognostic markers have been described, but none of them completely explains the heterogeneity seen in this tumor 11 .
Interleukin-10 is an anti-inflammatory cytokine that plays a crucial role in the immune suppressive microenvironment in MM 12 . It is involved in the terminal differentiation of B-cells into plasma cells and enhances its proliferation. Furthermore, it seems to enhance the proliferation of MM cells 13 .
Previous studies showed that IL-10 level is higher in patients with MM when compared with controls without MM 12,14,15 . Wang et al 12 found that IL-10 serum concentration in their studied MM patients was much higher than in normal controls. Also, Alexandrakis et al 14 reported that serum level of IL-10 was significantly higher in newly diagnosed MM patients compared to healthy controls and its level was significantly related to high ISS stage. A similar finding was reported by Aref et al 15 .
Our study showed a significantly higher serum levels of IL-10 in MM patients with advanced ISS stages and this falls in line with previous studies 14 .
In addition to the strong association between IL-10 level and stage found in this study, IL-10 level positively correlated with other individual prognostic markers of MM, such as serum β2microglobulin and bone marrow plasma cells percentage. This is in agreement with Shekarriz et al 11 who demonstrated that there was a significant correlation between elevated IL-10 levels and serum β2-microglobulin concentration and a trend for a positive correlation between the IL-10 level and bone marrow plasma cells percentage. Wang et al 12 also found that the serum level of IL-10 was significantly higher in patients with higher bone marrow plasma cells percentage, as well as those with elevated serum β2-microglobulin and lactate dehydrogenase levels. Similarly, Gu et al 16 reported that serum IL-10 level showed significant positive correlations with lactate dehydrogenase and serum β2-microglobulin concentrations. This further confirms the possible prognostic role of IL-10 in MM.
In addition to IL-10, the prognostic role of other cytokines, like IL-6 and IL-17A, in MM has been recently suggested 16 . More research is needed to confirm the prognostic value of IL-10 and other cytokines and to study the value of incorporating them into the current staging systems.

Conclusion
The results of this study confirm the association between higher IL-10 level and advanced stage and other prognostic markers in patients with MM. The incorporation of IL-10 in risk stratification of MM may be considered. Further studies with larger sample and outcome evaluation are needed to validate its prognostic role.