Despite advancements in cancer-related supportive care and the expanding range of treatment options, high relapse rates and poor prognoses in advanced-stage malignancies remain critical challenges in oncology [1-4]. Traditionally, oncologists have focused on intensifying treatment to reduce cancer relapse. Although intensified chemotherapy has proven effective in managing relapses across various cancers, its associated toxicity significantly increases the risk of treatment-related morbidity and mortality [5-7].
In recent years, immunotherapy has emerged as a promising approach in cancer treatment. Key modalities within this field include cell therapy, cytokine therapy, immune checkpoint inhibitors, and cancer vaccines [8-12]. The interaction between circulating immune effector cells and cancer cells plays a pivotal role in determining the success of immunotherapy [13-15].
This study aims to explore the relationship between lymphocyte subsets and treatment outcomes in patients with advanced-stage ovarian cancer who received concurrent conventional chemotherapy and cytokine-induced killer (CIK) cell therapy, using Picibanil and Aldesleukin (recombinant IL-2).

Case Enrollment
A retrospective chart review was conducted to analyze the prognosis of patients with advanced-stage ovarian cancer. Patients diagnosed with FIGO stage III or IV ovarian cancer at Chang Gung Memorial Hospital between October 2008 and April 2018 were included. The study was approved by the Chang Gung Memorial Hospital Research Ethics Committee (Project No. CMRPG3H1771). Patients with incomplete medical records or uncertain disease staging were excluded from the analysis.
Treatment Schema
Management of advanced-stage ovarian tumors followed a standard protocol of debulking surgery, followed by conventional adjuvant chemotherapy. Concurrently, cytokine-induced killer (CIK) cell therapy was administered as an adjunct immunotherapy, utilizing Picibanil and Aldesleukin (recombinant IL-2) to enhance the anti-tumor activity of immune effector cells.
Evaluation of Treatment Response
Treatment response was evaluated through radiological imaging techniques, including computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) scans. Follow-up intervals were determined based on clinical judgment. All imaging studies were independently reviewed by radiology experts, and any inconclusive findings were resolved by consensus among the reviewers.
Statistical Analysis
One month after the initiation of CIK add-on immunotherapy, complete blood counts (CBC), including differential counts and lymphocyte subset measurements, were analyzed to assess their prognostic impact. Continuous variables were compared using Mann-Whitney U tests, while categorical variables were analyzed with Fisher’s exact test. All statistical analyses were conducted using GraphPad Prism version 9.0 software.

A total of 25 patients diagnosed with advanced-stage ovarian cancer were included in this study. The baseline characteristics of the cohort are summarized in Table 1. The median follow-up duration was 3.9 years (IQR 2.4–9.0). Among the cases, 19 patients were categorized as FIGO stage III, and 9 were FIGO stage IV. Disease relapse occurred in 16 patients (65%), with 14 patients (56%) ultimately succumbing to the disease.
For prognostic analysis, the cohort was stratified into relapse and non-relapse subgroups (Table 2). The age at diagnosis and FIGO stage distribution were comparable between the two subgroups. However, a significantly lower mortality rate was observed in the non-relapse group (11.1% vs. 81.3%, P = 0.0021). Patients in the non-relapse group also had significantly longer follow-up periods (10.3 vs. 2.5 years, P < 0.0001).
Regarding complete blood count (CBC) analysis, the non-relapse subgroup demonstrated a significantly higher absolute lymphocyte count (ALC) compared to the relapse subgroup (1,485 vs. 951 cells/μL, P = 0.0014) and a significantly lower neutrophil-to-lymphocyte ratio (NLR) (1.5 vs. 2.8, P = 0.0171).
Analysis of lymphocyte subsets (Table 3) revealed that the non-relapse subgroup had a significantly higher cytotoxic T lymphocyte (CTL) percentage (13.5% vs. 9.9%, P = 0.0226). Survival analysis, shown in Figure 1, indicated a significantly higher 5-year overall survival (OS) rate in the non-relapse group (88.9% vs. 18.8%, P < 0.001).

Where ????????(????) is Physicians have long grappled with disease relapse in advanced-stage malignancies, despite advances in multi-agent and intensified chemotherapy regimens [1-3]. In this study, we explored the use of cytokine-induced killer (CIK) immunotherapy as an adjunct to conventional treatment in patients with advanced-stage ovarian cancer, aiming to improve survival outcomes. The observed relapse and mortality rates of 64% and 56%, respectively, align with survival data reported from other developed countries.

The CIK immunotherapy employed in this study utilized Picibanil and interleukin-2 (IL-2). Picibanil acts as an immune stimulant, enhancing natural killer (NK) cell and T-cell activity, while IL-2 modifies the immune microenvironment to augment the function of immune effector cells [16-19]. These effector cells, following antigen presentation, recognize and target malignant cells for elimination. The non-relapse subgroup in our study exhibited elevated levels of NK cells and cytotoxic T lymphocytes (CTLs), highlighting the potential connection between increased levels of these immune cells and improved survival outcomes.
The prognostic significance of complete blood counts (CBC) in cancer patients has been widely studied, particularly the role of the neutrophil-to-lymphocyte ratio (NLR) in predicting outcomes [20-25]. Neutrophils may promote inflammatory processes that enhance tumor proliferation, while lymphocytes, including T and B cells, are essential in antigen presentation and the activation of immune effector mechanisms to combat tumor cells. Imbalances in these immune cell populations can significantly affect prognosis [20, 21, 23]. Consistent with previous findings, our study demonstrates that higher absolute lymphocyte counts (ALC) and lower NLR are strongly associated with improved overall survival (OS). Although we also observed trends suggesting that a higher lymphocyte-to-monocyte ratio (LMR) and lower platelet-to-lymphocyte ratio(PLR) may confer survival advantages, these findings did not reach statistical significance.
Several limitations should be considered when interpreting these results. First, the retrospective design and small sample size may introduce bias, affecting the generalizability of the conclusions. Second, variations in follow-up intervals between clinicians could limit the precision of survival analyses. Finally, as this is a single-institution study, further caution is warranted in extrapolating the results to broader populations. Larger, prospective studies are necessary to confirm these findings.
Despite these limitations, our study successfully demonstrates that higher ALC and lower NLR are associated with enhanced survival outcomes in advanced-stage ovarian cancer patients. The balance of lymphocyte subsets plays a crucial role in sustaining immune effector cell function. Specifically, a higher CTL count, without disruption in the CD4-to-CD8 ratio, is linked to improved OS.
Increased absolute lymphocyte counts (ALC) and decreased neutrophil-to-lymphocyte ratios (NLR) are associated with improved survival outcomes in patients with advanced-stage ovarian cancer. Maintaining a balanced distribution of lymphocyte subsets is essential for the optimal function of immune effector cells. Our findings suggest that elevated cytotoxic T lymphocyte (CTL) levels, in conjunction with a stable CD4-to-CD8 ratio, are key factors contributing to improved overall survival.
Disclosures
The authors declare no conflicts of interest.
Acknowledgements
The study was supported by grant CMRPG3H1771, which was provided by Chang Gung University Hospital (Taoyuan, Taiwan). 

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