Biological Profile of Primary and Secondary Lymphedema: Inflammatory, Endothelial, Metabolic, Lymphatic, and Fibrotic Markers

1. Background and Rationale Lymphedema is a chronic disorder characterized by impaired lymphatic transport and accumulation of interstitial fluid and macromolecules in affected tissues. It may be primary, resulting from congenital or genetically determined abnormalities of lymphatic development or function, or secondary, resulting from acquired damage to lymphatic vessels or lymph nodes. Secondary lymphedema may occur after cancer treatment, surgery, radiation therapy, trauma, infection, or other conditions that disrupt lymphatic drainage. Although impaired lymphatic drainage is central to the clinical phenotype, current evidence indicates that lymphedema is not solely a mechanical disorder. Persistent lymphatic dysfunction may induce a complex tissue response involving immune activation, endothelial dysfunction, metabolic alterations, adipose tissue accumulation, abnormal lymphangiogenic signaling, extracellular matrix remodeling, and progressive fibrosis. These processes may interact with each other and contribute to disease progression and clinical heterogeneity. The inflammatory response in lymphedema is characterized by infiltration and activation of immune cells and changes in cytokine signaling. In particular, type 2 immune responses and T helper 2 (Th2)-associated cytokines have been implicated in chronic inflammation and fibrosis. Endothelial activation may further contribute to altered vascular permeability and inflammatory cell recruitment. At the same time, metabolic and adipose tissue alterations may develop within chronically affected tissues and may also be reflected by systemic metabolic markers. Changes in lymphatic biology are another important component of disease pathogenesis. Lymphatic endothelial cells and their signaling pathways regulate lymphatic vessel formation, maintenance, and function. Vascular endothelial growth factors and their receptors, particularly the vascular endothelial growth factor C/vascular endothelial growth factor receptor 3 (VEGF-C/VEGFR-3) pathway, are important regulators of lymphangiogenesis. Abnormal regulation of these pathways may reflect an attempt to compensate for impaired lymphatic function but may not be sufficient to restore effective lymphatic transport. Progressive fibrosis represents an important late consequence of chronic lymphedema. Persistent inflammation and tissue remodeling may stimulate fibroblast activation and extracellular matrix deposition, resulting in increasing tissue stiffness and structural changes. Transforming growth factor beta and matrix metalloproteinases are among the pathways potentially involved in this process. Most available clinical studies have focused on secondary lymphedema, particularly after cancer treatment. The biological profile of primary lymphedema remains substantially less well characterized. Primary lymphedema is heterogeneous and may result from different genetic or developmental abnormalities, and it is not known whether its systemic and tissue-associated biological profile differs consistently from that of secondary lymphedema. A comparative assessment of primary and secondary lymphedema, together with healthy controls, may therefore provide additional insight into the biological processes associated with lymphatic dysfunction. Rather than focusing on a single biomarker, the present study will examine five interconnected biological domains: Th2-related inflammatory activity, endothelial activation, metabolic dysfunction, lymphatic biology, and tissue fibrosis. 2. Conceptual Framework The study is based on the hypothesis that the clinical manifestations of lymphedema arise from the interaction of multiple biological processes rather than from impaired lymphatic drainage alone. The five domains investigated in this study are therefore considered complementary components of a broader biological phenotype. For the primary analysis, one representative marker will be prespecified for each domain. These markers are intended to provide a pragmatic representation of the respective biological processes rather than to constitute validated diagnostic measures of the individual domains. Interleukin-13 (IL-13) will represent Th2-related inflammatory activity. Soluble vascular cell adhesion molecule-1 (sVCAM-1) will represent endothelial activation. Homeostatic model assessment of insulin resistance (HOMA-IR) will be used as a measure of systemic insulin resistance and metabolic dysfunction. Vascular endothelial growth factor C (VEGF-C) will represent lymphatic biological activity, particularly signaling associated with lymphangiogenesis. Transforming growth factor beta 1 (TGF-β1) will represent fibrotic tissue remodeling. Additional biomarkers will be evaluated in secondary and exploratory analyses to determine whether the findings obtained using the prespecified representative markers are supported by broader patterns within the same biological domain. 3. Study Population and Comparative Groups The study will compare three populations: individuals with primary lymphedema, individuals with secondary lymphedema, and healthy individuals without lymphedema. Participants with primary lymphedema will have clinically and, where appropriate, imaging-confirmed disease affecting an upper or lower extremity. Participants with secondary lymphedema will have a clearly established acquired cause of lymphatic impairment. Participants in the control group will have no clinical history or signs of lymphedema. The study population will be restricted to adults within the predefined age range to reduce the potential influence of substantial age-related differences in inflammatory, metabolic, endothelial, and lymphatic biomarkers. The groups will be comparable with respect to age and sex to the extent feasible. The study will be conducted at the Department of Dermatovenereology, University Medical Centre Ljubljana, with recruitment of eligible patients receiving clinical care at the institution and recruitment of healthy controls through appropriate public or direct invitations. 4. Clinical Characterization The study will include standardized clinical characterization of participants with lymphedema. Information will be collected regarding the onset, duration, presumed cause, affected limb and anatomical location, previous treatment, relevant medications, and history of cellulitis or erysipelas. The clinical examination will assess disease stage and manifestations relevant to lymphedema severity, including pitting edema, Stemmer sign, skin changes, and clinical evidence of tissue fibrosis. Objective assessment of limb volume will be performed using perometry, and tissue firmness will be assessed using a SkinFibrometer. Clinical characteristics will subsequently be evaluated in relation to biological markers to determine whether systemic and tissue-associated biological alterations correlate with the clinical expression of disease. 5. Biological Assessment Venous blood samples will be obtained during the study visit. Routine laboratory testing will be used to characterize the general metabolic, inflammatory, hematologic, hepatic, renal, thyroid, and cardiovascular status of participants and to identify potentially relevant confounding factors. The research biomarker panel will include markers representing inflammatory activity, endothelial activation, lymphatic biology, fibrosis and extracellular matrix remodeling, and metabolic and adipokine biology. The inflammatory panel will include tumor necrosis factor (TNF), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-13 (IL-13), interleukin-17 (IL-17), and interleukin-23 (IL-23). These markers will be used to explore inflammatory patterns, with particular attention to type 2 immune activity and broader inflammatory signaling. The endothelial panel will include soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), and E-selectin. These markers will be evaluated as indicators of endothelial activation and may provide information about systemic vascular-inflammatory interactions. The lymphatic biology panel will include vascular endothelial growth factor C (VEGF-C), vascular endothelial growth factor D (VEGF-D), vascular endothelial growth factor receptor 3 (VEGFR-3), and platelet factor 4 (PF4). These markers will be investigated to characterize pathways potentially involved in lymphatic vessel function, lymphangiogenesis, and lymphatic remodeling. Markers of fibrosis and extracellular matrix remodeling will include transforming growth factor beta 1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and matrix metalloproteinase-9 (MMP-9). Their concentrations will be assessed in relation to clinical evidence of tissue stiffness and fibrosis. Metabolic and adipocyte-associated markers will include leptin, resistin, and adiponectin. Standard metabolic parameters will also be used to calculate indices of insulin resistance and metabolic dysfunction. The laboratory methodology, analytical platforms, assay characteristics, sample handling, processing, storage conditions, and quality-control procedures will be specified in the study laboratory procedures before initiation of sample analysis. 6. Metabolic and Derived Variables Metabolic characterization will include fasting glucose, fasting insulin, hemoglobin A1c (HbA1c), lipid parameters, and other routinely measured biochemical variables. Homeostatic model assessment of insulin resistance (HOMA-IR) will be calculated using fasting glucose and fasting insulin according to the prespecified formula. The triglyceride-glucose (TyG) index will also be calculated as an additional exploratory marker of metabolic dysfunction. The visceral adiposity index (VAI) will be calculated using sex-specific formulas incorporating waist circumference, body mass index (BMI), triglycerides, and high-density lipoprotein (HDL) cholesterol. These derived measures will not replace direct clinical assessment but will provide additional information for investigating relationships between metabolic status and lymphedema-associated biological changes. 7. Skin Microbiome Analysis An exploratory component of the study will investigate the skin microbiome in participants with lymphedema. A standardized skin swab will be collected from a predefined site on affected skin and from a standardized comparison site. Potential factors that may influence microbiome composition will be recorded, including recent use of systemic or topical antibiotics, antiseptics, corticosteroids, emollients, and the interval since the last washing of the sampling site. Negative controls for sampling and laboratory processing will be incorporated to help identify environmental or procedural contamination. Microbiome samples will undergo sequencing and subsequent bioinformatic analysis using a prespecified laboratory and analytical workflow. The analysis will include assessment of microbial community composition and diversity. Alpha- and beta-diversity measures will be used to characterize within-sample diversity and differences in community composition between samples. The microbiome component is exploratory and is intended to generate hypotheses regarding potential relationships between the local skin environment, systemic biological markers, and clinical characteristics of lymphedema. 8. Exploratory Genetic Analysis A further exploratory component will be performed in participants with primary lymphedema who do not have a previously established genetic diagnosis. Selected gene symbols associated with primary lymphedema and lymphatic development will be investigated. The candidate gene panel is expected to include FLT4, FOXC2, GJC2, VEGFC, CCBE1, SOX18, and GATA2, although the final panel may be adjusted according to the capabilities and validated procedures of the performing laboratory. Genetic testing is intended to improve the biological characterization of primary lymphedema and to explore whether particular genetic variants are associated with differences in clinical or biomarker profiles. Participants undergoing genetic analysis will receive separate information regarding the nature and scope of genetic testing, potential uncertain or incidental findings, the handling and storage of genetic material, the reporting of clinically relevant findings, and the availability of genetic counseling where appropriate. Genetic samples will not be used for unrelated research purposes without appropriate additional consent. 9. Relationship Between Biological Domains and Clinical Phenotype A major objective of the study is to examine whether biological alterations are associated with the clinical severity and phenotype of lymphedema. The analysis will investigate associations between biomarkers and clinical measures including disease stage, limb volume, pitting edema, Stemmer sign, tissue firmness, and clinical evidence of fibrosis. The study will also assess relationships between the five biological domains. For example, inflammatory markers will be evaluated in relation to endothelial activation, metabolic indices, lymphatic markers, and markers of fibrosis. This approach is intended to determine whether the biological abnormalities associated with lymphedema occur as isolated changes or as coordinated patterns. The analysis will additionally consider potential effects of age, sex, body mass index, disease duration, anatomical location, and other predefined clinical characteristics. 10. Comparison of Primary and Secondary Lymphedema The direct comparison between primary and secondary lymphedema is a central component of the study. Primary and secondary lymphedema share the clinical consequence of impaired lymphatic transport but may differ in their underlying mechanisms. Primary disease may be associated with developmental or genetic abnormalities of the lymphatic system, whereas secondary disease results from acquired lymphatic injury. The study will therefore assess whether the five predefined biological domains and the broader biomarker profile differ between these two forms of lymphedema after accounting for relevant demographic and clinical characteristics. The results may help determine whether primary and secondary lymphedema share a common biological phenotype or whether distinct biological patterns can be identified. 11. Statistical Approach Statistical analyses will be performed according to a prespecified statistical analysis plan. Continuous variables will be assessed for distribution and summarized using appropriate descriptive measures. Categorical variables will be summarized using frequencies and percentages. The primary comparison will evaluate differences in the prespecified representative markers between the three study groups. Appropriate parametric or nonparametric methods will be selected according to the distribution and characteristics of the data. Pairwise comparisons will be performed where appropriate and corrected for multiple comparisons. Because five representative markers constitute the co-primary outcome measures, the statistical analysis will account for multiplicity using a prespecified adjustment procedure. The exact procedure and corresponding significance threshold will be defined before the primary analysis is performed. Associations between biomarkers and clinical measures will be evaluated using correlation analyses and multivariable regression models where appropriate. Multivariable models may include predefined covariates such as age, sex, body mass index, anatomical location, and duration of disease. Results will be reported using estimates of effect with 95% confidence intervals in addition to p-values. Missing data, outlying observations, and assumptions underlying statistical models will be assessed according to the prespecified analysis plan. Because the study includes a relatively broad biomarker panel and exploratory microbiome and genetic analyses, these analyses will be interpreted as exploratory and hypothesis-generating. Appropriate methods for controlling the false discovery rate or otherwise accounting for multiple testing will be applied where appropriate. 12. Sample Size and Exploratory Nature The planned sample consists of approximately 90 participants, with approximately 30 participants in each study group. The sample size reflects the feasibility of recruiting individuals with primary lymphedema, which is a relatively uncommon condition, while allowing comparative analysis across the three groups. The study is intended primarily to characterize biological patterns and generate hypotheses regarding mechanisms of disease. The sample size is therefore considered exploratory rather than definitive for establishing clinical efficacy or diagnostic thresholds. Where reliable prior estimates are available, a formal power calculation will be considered for the principal comparison. If adequate prior data are not available to support a reliable calculation, the study will be explicitly interpreted as a pilot or exploratory study, with emphasis on effect estimates and 95% confidence intervals rather than solely on statistical significance. Expected Contribution The study is designed to provide an integrated characterization of biological processes associated with primary and secondary lymphedema. By evaluating inflammatory, endothelial, metabolic, lymphatic, and fibrotic pathways within the same participants and relating these findings to standardized clinical measures, the study may provide a more comprehensive description of the biological phenotype of lymphedema. The comparison with healthy controls will help identify biological alterations associated with lymphedema in general, while the comparison between primary and secondary disease will explore whether different forms of lymphatic dysfunction are associated with distinct biological patterns. The exploratory microbiome and genetic analyses may provide additional hypotheses regarding local tissue factors and genetic determinants of disease heterogeneity. Ultimately, the findings may contribute to a broader understanding of lymphedema as a complex inflammatory, metabolic, lymphatic, and fibrotic disorder rather than solely a consequence of impaired lymphatic drainage. The results may provide a foundation for future prospective studies, validation of candidate biomarkers, identification of biological disease subtypes, and development of more mechanism-based diagnostic or therapeutic approaches. As this is an observational study, no experimental treatment will be administered and the study is not designed to evaluate treatment efficacy or establish causal relationships. Any associations identified will therefore require confirmation in appropriately designed longitudinal or interventional studies.