Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western populations. It is characterized by the clonal proliferation and accumulation of small, mature-appearing B lymphocytes in the peripheral blood, bone marrow, and lymphoid tissues [1]. The clinical course of CLL is markedly heterogeneous, with some patients exhibiting indolent disease requiring no immediate therapy, while others experiencing rapid progression and resistance to conventional treatment modalities [2].

The CLL International Prognostic Index (CLL-IPI), incorporating five parameters (patient age, clinical stage, immunoglobulin heavy chain variable region (IGHV) mutation status, TP53 gene status, and serum β2-microglobulin (β2-MG) levels) was introduced to address this clinical variability [3]. Of these, β2-MG, a low-molecular-weight protein associated with tumor burden and immune activation, has been widely validated as a prognostic biomarker in CLL and other lymphoid malignancies [4]. Similarly, TP53 mutations and deletions are strongly correlated with poor treatment outcomes and reduced survival [5].

Beyond molecular determinants, systemic inflammation is increasingly recognized as a critical contributor to tumor biology in hematologic malignancies. Biomarkers such as C-reactive protein (CRP), albumin, ferritin, and composite indices like the CRP-to-albumin ratio (CAR) have demonstrated prognostic significance in various cancers, including CLL [3,6,7]. CAR, which reflects both systemic inflammation and nutritional status, has emerged as an independent predictor of overall survival in newly diagnosed CLL patients [3]. Albumin, a negative acute-phase reactant, typically declines in advanced disease and serves as a surrogate for nutritional depletion and chronic inflammation [3,6].

Ferritin, another acute-phase reactant, is elevated in a range of malignancies such as acute leukemias, multiple myeloma, and lymphomas [6–8]. Ferritin not only reflects iron metabolism but also modulates immune responses, potentially contributing to immune evasion and leukemogenesis [7]. High serum ferritin levels have been shown to correlate with tumor mass and disease activity [6]. Vitamin D, a steroid hormone with immunomodulatory properties, has also been implicated in CLL pathogenesis. Deficiency of 25-hydroxyvitamin D3 [25(OH)D] is common among CLL patients and may predispose to immune dysfunction and increased infection risk [1].

While individual markers such as β2-MG, ferritin, CRP, and vitamin D have demonstrated prognostic relevance, limited research has explored their combined utility in treatment-naïve CLL patients. Investigating the interaction between molecular abnormalities and inflammatory or nutritional biomarkers could provide a more holistic and accessible approach to risk stratification, particularly in resource-constrained settings.

The present study aims to evaluate the association between selected inflammatory and nutritional biomarkers, including CRP, albumin, CAR, ferritin, vitamin D3 with clinical stage in newly diagnosed, untreated CLL patients. By integrating conventional clinical staging with biomarker profiles, this study seeks to elucidate their prognostic value and potential role in guiding early risk stratification.

Continuous variables were summarized as means ± standard deviation or median with interquartile range depending on the distribution. One-way Analysis of Variance (ANOVA) was utilized to compare the means of normally distributed continuous variables across Rai stages, while the Kruskal-Wallis test was used for non-normally distributed variables. When the assumption of homogeneity of variances was violated, Welch's ANOVA was employed as an alternative statistical test. Post-hoc comparisons were done using the Dunn test for non-normally distributed variables. Continuous variables were visualized across Rai stages using box plots to explore distribution patterns. Multivariable linear regression models were performed to assess the association between each biomarker and Rai clinical stage, adjusting for age and sex. Stage 0 was used as the reference category. Categorical variables were compared across Rai stages using the Chi-square test. Data were analyzed using R software (version 4.5.0), and a p-value <0.05 was considered statistically significant.

The study included 150 newly diagnosed, treatment-naïve CLL patients, of whom 62.7% were male. The mean age of the patients was 64.08 ± 7.41 years. These patients were compared with 150 age- and sex-matched healthy controls. On comparing the Rai clinical stages, there was no statistically significant difference in mean ages (Welch’s ANOVA: p-value = 0.676), indicating demographic homogeneity across the clinical spectrum of patients.

Table 1 shows that serum biomarkers and hematological indices display significant differences in CLL patients compared to healthy controls. The serum ferritin, β2-MG, CRP, LDH, CAR, and ESR were significantly elevated in the patient group (all p-value <0.001), reflecting increased systemic inflammation due to tumour burden. On the other hand, albumin and vitamin D3 levels were markedly lower in CLL patients (p-value <0.001), suggesting chronic nutritional deficiency and potential immunomodulatory dysfunction. Hematological profiles revealed typical features of CLL, including anemia, thrombocytopenia, and pronounced leukocytosis, all consistent with underlying disease pathophysiology.

β2-MG: serum β2-microglobulin; LDH: Lactate dehydrogenase; ESR: Erythrocyte sedimentation rate; CRP: C-reactive protein; CAR: C-reactive protein-to-albumin ratio; Hb: Hemoglobin; WBC: White blood cell count

Progressive changes in biomarker levels were observed across Rai stages, highlighting their potential utility in clinical disease stratification (Table 2).

SD: Standard deviation; β2-MG: β2-Microglobulin; LDH: Lactate dehydrogenase; ESR: Erythrocyte sedimentation rate; CRP: C-reactive protein; CAR: C-reactive protein-to-albumin ratio; Hb: Hemoglobin; WBC: White blood cell count

In multivariable linear regression analyses, levels of ferritin, β2-MG, LDH, ESR, CRP, and the CRP/albumin ratio, as well as WBC and platelet count, were significantly associated with CLL stages after adjusting for age and sex. Ferritin, β2-MG, LDH, ESR and CRP levels, and WBC progressively increased with advancing stage, while albumin, Hb, vitamin D3, and platelet counts showed significant declines, particularly in stages III and IV (p-values <0.01 for all comparisons). Age and sex were not significant predictors in any of the models. These results underscore the independent prognostic value of these routinely accessible biomarkers in staging and monitoring CLL progression.

Ferritin (Figure 1), β2-MG, and LDH levels increased significantly from Rai Stage 0 through Stage III, with a slight decline or plateau observed at Stage IV (p-value <0.001). These patterns support their role as surrogate markers for tumour proliferation and metabolic activity.

Figure 1.

Serum biomarker trends across Rai stages in treatment-naïve chronic lymphocytic leukemia (CLL) patients. A) Mean Ferritin Levels (ng/mL) increase progressively with disease stage, peaking at Stage III and declining slightly at Stage IV, indicating its correlation with tumor burden. B) C-reactive protein–to-albumin ratio CAR demonstrates early-stage fluctuation followed by a sharp rise at Stage IV, underscoring systemic inflammation and nutritional compromise in advanced disease. C) Vitamin D3 Levels (ng/mL) decline steadily from Stage 0 to Stage IV, supporting its role in immunologic dysfunction and disease severity. D) C-reactive protein (CRP) levels (mg/L) show a progressive and significant increase across Rai stages, aligning with systemic inflammatory progression in CLL.

The CAR (Figure 1), a composite index of inflammation and nutritional status, was markedly elevated in Stages III and IV (p-value <0.001). Hematologic parameters worsened with advancing stage, characterized by increased severity of anemia and thrombocytopenia, as well as higher WBC counts (p < 0.001), reflecting disease progression.

Albumin levels declined steadily with disease progression, decreasing from 4.21 g/dL in Stage 0 to 2.66 g/dL in Stage IV (p-value <0.001), indicative of worsening nutritional status and increased systemic inflammation. Similarly, vitamin D3 levels showed a continuous downward trend from 30.85 ng/mL in Stage 0 to 12.72 ng/mL in Stage IV (p-value <0.001), reinforcing its relevance as a prognostic marker of immune dysregulation in CLL (Figure 1)

Markers of inflammation, including CRP (Figure 1) and ESR, increased substantially in later Rai stages (p-value <0.001), consistent with an intensifying inflammatory response in advanced disease.

All values are presented as means with 95% confidence intervals

This study aimed to determine whether commonly available inflammatory and nutritional biomarkers, including ferritin, CRP, albumin, the CAR, LDH, ESR, and vitamin D3, are associated with clinical stage and disease burden in newly diagnosed, treatment-naïve CLL patients. The findings of this research indicate that elevated levels of ferritin, CRP, ESR, LDH, and CAR were significantly associated with more advanced Rai stages, while serum albumin and vitamin D3 levels showed a progressive decline with increasing disease severity. Among these, ferritin and CAR emerged as particularly strong surrogate markers of tumor burden and systemic inflammation, consistent with prior research highlighting their prognostic relevance in hematologic malignancies [3]. These results reinforce the potential utility of these routine laboratory biomarkers in the initial risk stratification of CLL, particularly in resource-constrained settings where molecular testing may not be readily available.

In the current cohort of newly diagnosed, treatment-naïve CLL patients, elevated serum ferritin levels were significantly associated with more advanced clinical stages. This finding suggests that ferritin may serve as a surrogate marker of both tumor burden and disease activity. The results are consistent with prior research in hematologic malignancies such as acute myeloid leukemia, non-Hodgkin lymphoma, and multiple myeloma, in which high ferritin levels have been linked to more aggressive disease and poorer survival outcomes [8,11,12]. The mechanistic basis for this association is multifactorial: ferritin reflects not only systemic inflammation and iron overload, but also plays a role in tumor biology through oxidative stress modulation, impairment of lymphocyte differentiation, and immune evasion [7,13]. However, the prognostic role of ferritin in CLL remains somewhat controversial, with some studies failing to show a consistent correlation between ferritin and disease severity [14]. This variability may reflect differences in patient selection, presence of comorbidities such as autoimmune hemolytic anemia or liver dysfunction, and timing of biomarker measurement relative to disease progression. Furthermore, given the role of ferritin as an acute-phase reactant, elevated levels may sometimes reflect non-malignant inflammatory processes, potentially limiting its specificity in early-stage or indolent CLL. Nonetheless, the consistent elevation of ferritin in advanced stages across hematologic cancers supports its integration alongside other prognostic markers such as β2-MG, CAR, and TP53 mutation status in risk assessment models.

A growing body of evidence also points to the prognostic significance of vitamin D3 in CLL. In the present study, vitamin D3 levels declined progressively with increasing Rai stage, a finding that aligns with prior reports documenting widespread vitamin D deficiency among CLL patients. Shanafelt et al. were among the first to demonstrate that 25(OH)D levels below 25 ng/mL were associated with nearly twice the risk of disease progression and shorter time to first treatment [15]. Subsequent studies have validated these observations. For instance, Ito et al. found that low vitamin D3 levels correlated significantly with high β2-MG levels and advanced Rai/Binet stages [16]. The biological rationale is grounded in the immunoregulatory function of vitamin D: its active form, 1,25-dihydroxyvitamin D, modulates immune cell activity by enhancing cytotoxic T cells and natural killer cell function while suppressing pro-inflammatory cytokines [17]. These mechanisms are particularly relevant in CLL, which is characterized by chronic immune dysregulation and impaired immune surveillance. Despite compelling observational data, there is a lack of large-scale randomized controlled trials evaluating the impact of vitamin D supplementation on clinical outcomes in CLL. Whether deficiency is a causative factor or merely a marker of disease burden remains unclear, but vitamin D assessment may offer a valuable component of initial prognostic evaluation.

The findings of this study also confirmed that LDH levels were significantly elevated in patients with advanced CLL, supporting its role as a surrogate marker for tumor burden and cellular turnover, as highlighted in several previous studies [18–20]. A stage-wise increase in LDH from Rai 0 to IV was observed in the present cohort, consistent with the previous pattern [18]. Although LDH is not included in the CLL-IPI, these results suggest that it has clinical relevance as a reflection of metabolic activity and tumor dynamics. Additionally, inflammatory markers such as ESR and CRP showed progressive elevations with advancing disease stage, corroborating findings from earlier studies by Conroy et al. and Hallek et al., who identified these markers as reflective of systemic inflammatory load in lymphoproliferative disorders [1,21]. The data here also showed increased prevalence of hypoalbuminemia in higher Rai stages, reinforcing the value of albumin as a negative prognostic factor, particularly when interpreted in the context of CRP. The CAR, a novel index incorporating both inflammation and nutritional status, was significantly elevated in patients with progressive disease, echoing previous findings by Tang et al. [3]. These integrated trends suggest that a composite panel involving LDH, CRP, ESR, albumin, and CAR could provide a more comprehensive and accessible risk stratification model. Discrepancies with earlier studies may reflect population heterogeneity, comorbidity profiles, or stage distribution. Future prospective studies are warranted to validate the clinical utility of these biomarkers in broader, real-world settings.

Although both Rai and Binet staging systems are used interchangeably in CLL prognostication, the findings of this study showed a more consistent and progressive association between biomarker levels and Rai stage. This may be due to the greater staging resolution offered by the five-tier Rai system, which enables more precise stratification of laboratory and inflammatory markers, particularly in early-stage disease. In contrast, the broader grouping in the Binet system may obscure intermediate biological variations. These findings suggest that, for biomarker-based prognostic modeling, Rai staging may provide more granularity for research purposes, especially in early or indolent CLL.

Beyond classical tumor burden and inflammatory biomarkers, growing attention has been directed toward evaluating the role of BMI and geographic residence in the pathogenesis and progression of CLL. Several large, population-based cohort studies have identified a modest but significant association between elevated BMI and increased risk of developing CLL, particularly among males [22,23]. A meta-analysis further quantified this risk, revealing a 25% increase in CLL incidence for every 5 kg/m² rise in BMI [24]. The underlying mechanisms are hypothesized to involve obesity-induced immune dysregulation, chronic low-grade inflammation, and dysregulated cytokine signaling, all of which may contribute to lymphomagenesis [25,26].

Despite these associations, BMI has not been incorporated into major prognostic models such as the CLL-IPI, and most clinical investigations have failed to establish a robust link between BMI and disease stage, cytogenetic abnormalities, or treatment response [1,27]. While not a direct surrogate for disease severity, BMI may impact broader clinical variables including treatment tolerance, immune competence, and metabolic health, especially in older or comorbid populations [28].

The potential influence of place of residence, specifically urban versus rural environments, has also been explored in epidemiologic studies, though findings remain largely indirect. Geographic variation in CLL incidence has been observed in registry-based datasets, which may reflect differences in environmental exposures, healthcare access, and diagnostic intensity, rather than intrinsic biological differences [22,23]. Rural residence has been associated with increased exposure to pesticides, zoonotic agents, and radon, each of which may elevate CLL risk [24,26,27]. Moreover, individuals living in rural settings may face barriers to early diagnosis, molecular testing, and specialist access, which could influence disease progression and care quality [1,25,29]. Nonetheless, current evidence does not demonstrate a consistent correlation between residence type and CLL stage at diagnosis. These insights suggest that while BMI and geographic factors may not directly affect clinical staging, they may influence disease risk, access to care, and long-term outcomes, thereby warranting further investigation through large-scale, prospective studies.

Although patient characteristics such as BMI and residence (urban vs. rural) were recorded, analysis did not identify significant correlations between these factors and clinical stage. However, future research in larger cohorts may better elucidate the role of such demographic factors in disease progression.

While this study did not assess regional prevalence patterns of CLL, we acknowledge that this represents an important gap in the literature, particularly in underrepresented non-Western populations. Future investigations should aim to clarify how geographic variation may shape disease burden and biomarker expression.

This study highlights the clinical relevance of readily available inflammatory and nutritional biomarkers, such as ferritin, β2-MG, LDH, CRP, albumin, CAR, ESR, and vitamin D3, in relation to disease burden and staging in treatment-naïve CLL patients. The findings of the present study demonstrate that elevated levels of ferritin, β2-MG, LDH, CRP, ESR, and CAR are significantly associated with advanced Rai stages, while declining albumin and vitamin D3 levels reflect worsening nutritional and immunologic status.

Among these, ferritin and β2-MG emerged as particularly strong indicators of tumor burden, while CAR and vitamin D3 levels may reflect the broader interplay between systemic inflammation and immune dysregulation. These routinely measured, cost-effective biomarkers have the potential to enhance initial risk stratification and clinical decision-making, especially in settings where molecular diagnostics are limited. Their integration into clinical assessment frameworks may enhance early risk stratification and management in resource-constrained environments.

Future prospective, longitudinal studies incorporating survival outcomes and molecular profiles are warranted to validate the prognostic value of these biomarkers and assess their integration into existing clinical models for individualized patient management in CLL.