CD3+/CD4+ cells combined with myosteatosis predict the prognosis in patients who underwent gastric cancer surgery

Abstract Background This study aimed to investigate the predictive capacity of lymphocyte subpopulations, sarcopenia and myosteatosis for clinical outcomes in patients who underwent gastric cancer surgery. Additionally, the prognostic significance of CD3+/CD4+ cells in conjunction with myosteatosis was explored. Methods A cohort of 190 patients with gastric cancer who underwent surgery and received computed tomography scans between July 2016 and December 2017 at our institution was examined. Complete clinical information and peripheral lymphocyte subpopulations were available for all patients. A comprehensive array of statistical methodologies was employed to scrutinize variances in both clinical and pathological characteristics among patients, with the aim of identifying autonomous prognostic determinants requisite for the development of a nomogram. Subsequent assessment of the predictive efficacy of the nomogram was conducted via calibration curve analysis. Results The study comprised a cohort of 190 participants, encompassing 126 males (66.32%) and 64 females (33.68%), with a mean age of 58.47 (±11.37) years. Patients were stratified into three groups based on CD3+/CD4+ cells and myosteatosis, with 24 in Group 1, 87 in Group 2 and 79 in Group 3. Notably, patients in the third group exhibited significantly shorter progression‐free survival (PFS) (hazard ratio [HR] = 0.208, P < 0.001) and overall survival (OS) (HR = 0.193, P < 0.001). The subset of peripheral blood lymphocytes exhibited elevated levels of CD3+/CD4+ cells (HR = 2.485, P < 0.001) and heightened CD4+/CD8+ ratios (HR = 1.705, P = 0.038), whereas diminished CD19+ cell counts (HR = 0.210, P = 0.032) correlated with improved OS in patients. The individuals presenting with sarcopenia (HR = 4.089, P = 0.023) and myosteatosis (HR = 2.857, P < 0.001) displayed reduced OS. The multivariate Cox regression analysis showed that pathological tumour–node–metastasis stage, CD19+ cells, sarcopenia and CD3+/CD4+ cell–myosteatosis were identified as independent prognostic factors for PFS and OS in patients. The constructed nomograms for PFS and OS yielded C‐index values of 0.839 (95% confidence interval [CI]: 0.798–0.880) and 0.836 (95% CI: 0.792–0.879), respectively. The calibration analysis demonstrated that the nomograms accurately predicted the 3‐ and 5‐year survival rates of PFS and OS in patients. Conclusions Lymphocyte subsets, including CD3+/CD4+ cells, CD4+/CD8+ ratio and CD19+ cells, are indicative of clinical prognosis in gastric cancer surgery patients. Body composition parameters, such as sarcopenia and myosteatosis, are also associated with the patient's prognosis. The combination of CD3+/CD4+ cells with myosteatosis demonstrates enhanced prognostic value, enabling the identification of patients at high risk of post‐operative metastasis and recurrence.


Introduction
Gastric cancer stands as a formidable public health menace, ranking fifth among the most prevalent malignant tumours and occupying the fourth position in terms of cancer-related fatalities globally. 1Despite the array of treatment modalities available, surgical intervention remains the primary therapeutic avenue for individuals afflicted with gastric cancer.Nevertheless, even with radical resection, patients face a considerable risk of recurrence and metastasis. 2,3Consequently, an imperative exists to investigate novel prognostic indicators that can facilitate the precise selection of treatment strategies for patients.
The immune system constitutes a pivotal component in impeding tumorigenesis and resisting tumour progression. 4S1 Upon detecting aberrant cells, the immune system initiates an intricate cascade of cellular and molecular signals, activating immune cells to mount a response and ultimately eliminate these abnormal cells. 6,7ence, individuals with compromised immune function are predisposed to heightened risks of tumour recurrence and metastasis. 8S3 Numerous studies have also emphasized the utility of body composition as a prognostic predictor in individuals with tumours. 11While body mass index (BMI) stands as the most commonly employed metric, its consistency with clinical outcomes in tumour patients has been shown to be variable. 12ignificantly, BMI fails to consider the distribution of adipose tissue or the mass and quantity of skeletal muscle.Myosteatosis, characterized by abnormal adipose tissue distribution within and between muscle cells, results in excessive fat deposition within the muscle, which is a pathological state linked to diminished muscle mass, limb functionality and physical performance.S4 Severe myosteatosis has been associated with an unfavourable prognosis in gastric cancer 14 and other malignancies. 15he reciprocal interplay between the immune system and tissue wasting collectively contributes to tumour progression.Cancer-associated tissue wasting and weight loss primarily arise from factors such as anorexia, inflammation and the release of tumour-secreted molecules, which elevate energy expenditure and precipitate a state of metabolic disruption within the organism. 16,S7 This study endeavours to elucidate the correlation between lymphoid subpopulations, severe myosteatosis and the prognosis of gastric cancer patients who underwent surgery.Moreover, the study aims to amalgamate CD3+/CD4+ cells with severe myosteatosis to formulate a novel and more comprehensive prognostic index with the intent of accurately appraising the recurrence and progression risks in gastric cancer patients.

Patient cohort
This retrospective study was conducted with the approval of the Ethics Committee of Harbin Medical University Cancer Hospital, rendering the need for informed consent unnecessary.The investigation involved a cohort of 190 consecutive patients diagnosed with gastric cancer who underwent surgical procedures at Harbin Medical University Cancer Hospital between July 2016 and December 2017.These patients also underwent lymphatic subset testing.The analysis of data related to the 190 patients and their clinical profiles adhered to the principles outlined in the Helsinki Declaration and its subsequent amendments.The inclusion criteria stipulated that all patients must have undergone surgical intervention, undergone lymphatic subset testing and received abdominal CT scans at Harbin Medical University Cancer Hospital.All CT scans were performed 30 days before surgery.A standard radical gastrectomy consists of a gastrectomy with D2 lymphadenectomy and resection of N1 and N2 lymph nodes, as defined by the Japanese Classification of Gastric Carcinoma. 18onversely, exclusion criteria comprised patients with chronic ailments, those exhibiting an acute inflammatory response, individuals concurrently diagnosed with other primary malignancies in addition to gastric cancer and those with incomplete clinical data.The flow chart for clinical case selection is shown in Figure S1.Comprehensive clinical and pathological information was gathered and stored in the electronic medical records system.

Data acquisition
Patients were subjected to follow-up assessments through telephone consultations or outpatient visits.These followups occurred every 3-6 months during the initial 2 years post-surgery, followed by intervals of every 6-12 months for the third to fifth years and subsequently on an annual basis.Progression-free survival (PFS) was calculated from the date of surgery initiation to the occurrence of disease progression, mortality, cessation of follow-up or the final follow-up occasion.Disease progression was identified through chest and abdominal X-rays or CT.Overall survival (OS) was defined as the interval from the date of surgery initiation to mortality, cessation of follow-up or the last follow-up occasion.The electronic medical records system served as the repository for compiling patients' clinical and pathological data.

Evaluation of sarcopenia and myosteatosis
A single radiologist, who remained unaware of clinical information, conducted the interpretation of CT scans utilizing imaging software to assess skeletal muscle density (SMD) and additional body composition metrics.Following this, two separate researchers, operating independently, conducted an audit of the initial estimations by reassessing a randomly selected 30% subset of CT images.The outcomes of this auditing process consistently fell within a margin of ±5.0%.Subsequently, the CT data for each patient were imported into 3D Slicer (Version 4.10.2,www.slicer.org)for a comprehensive assessment of the cross-sectional area of skeletal muscles at the third lumbar vertebra (L3) and the mean SMD, measured in Hounsfield units (HU) across the entire muscle region.The HU thresholds for skeletal muscle were set within the range of À29 to 150.The skeletal muscle index (SMI) for L3 was computed as the ratio of skeletal muscle area (in square centimetres) to the square of the patient's height (in square metres).Concurrently, peripheral lymphatic subset levels were systematically quantified and analysed through a dedicated protein analyser (BD FACSCanto).
Optimal cut-off points for lymphatic subsets were determined via receiver operating characteristic (ROC) analysis, with OS serving as the predictive endpoint for patient mortality.The area under the ROC curve (AUC) was employed to assess the predictive capacity of lymphatic subsets.Sex-specific SMI cut-off values of 42.2 cm 2 /m 2 for men and 33.9 cm 2 /m 2 for women were used to define sarcopenia. 19Myosteatosis was defined using the mean SMD, with a cut-off value of <41 HU for patients with a BMI of <25 kg/m 2 and <33 HU for those with a BMI of 25 kg/m 2 or higher. 20Patients with CD3+/CD4+ cell ≥ 42.05% and myosteatosis comprised Group 1, while patients with CD3+/CD4+ cell < 42.05% and without myosteatosis were categorized under Group 3. The remaining cases were placed in Group 2.

Statistical analysis
Continuous variables were presented as means with standard deviations or as medians with interquartile ranges.Categorical variables were expressed as percentages.One-way analysis of variance (ANOVA), Kruskal-Wallis rank sum test and chi-square test or Fisher's exact test were utilized for comparisons between continuous and categorical variables, respec-tively.Kaplan-Meier survival curves and log-rank tests were employed to compute and compare survival rates and times.Utilizing the Cox proportional hazards model, univariate analysis was conducted, taking into account factors demonstrating significant prognostic relevance with a P value below 0.05.All identified prognostic factors underwent systematic optimization based on Akaike information criterion (AIC) values to prevent overfitting.Variables optimized through stepwise regression were subsequently integrated into the multifactor Cox proportional hazards model analysis to ascertain independent prognostic factors for both PFS and OS.Hazard ratios (HRs) and 95% confidence intervals (CIs) were used to assess relative risks.Nomograms were constructed to predict 1-, 3-and 5-year survival probabilities for PFS and OS.Calibration curve analysis was employed to assess the prognostic predictive ability of the nomogram.All statistical analyses were performed using R 4.1.3(Vienna, Austria), GraphPad Prism 8.0 (La Jolla, CA, USA) and IBM SPSS Statistics 25 (Chicago, IL, USA), with two-sided P values < 0.05 considered statistically significant.

Survival analysis for lymphocyte subsets
Utilizing Cox regression analysis, we identified significant associations between certain lymphatic subgroup indicators and the prognostic outcomes of patients with gastric cancer.The determination of optimal cut-off values for CD3+/CD4+ cells, CD4+/CD8+ ratio and CD19+ cells through ROC curve analysis yielded values of 42.05%, 1.95 and 14.85%, respectively.Subsequently, patients were stratified based on these cut-off values, revealing distinct survival rates and HRs for PFS and OS.For CD3+/CD4+ cells, 118 patients exhibited levels below 42.05%, with 1-, 3-and 5-year PFS rates of 91.5% (95% CI:  1A,D).

Construction of nomograms to predict progression-free survival and overall survival
This study determined that independent prognostic factors for PFS and OS were CD19+ cells, pTNM stage, sarcopenia and CD3+/CD4+ cell-myosteatosis.The C-index and 95% CI for predicting the survival probability of PFS and OS were 0.839 (0.798-0.880) and 0.836 (0.792-0.879), respectively (Figure 4A,B).The calibration analysis demonstrated that the nomograms accurately predicted the 3-and 5-year survival rates of PFS and OS in patients (Figure 5A,B).

Discussion
Gastric cancer stands as a prevalent digestive tract malignancy in China, ranking third in both incidence and mortality rates. 21espite advancements in the treatment modalities for gastric cancer patients, leading to improved survival rates, a substantial number still face unfavourable prognoses. 22Thus, there is an urgent imperative to identify novel indicators for accurate prognosis prediction among patients who underwent gastric cancer surgery.Inflammation, a well-established cancer risk factor, significantly contributes to malignant tumour development and progression. 23Infiltration of tumours by inflammatory cells has emerged as an independent prognostic factor influencing patient outcomes. 24While previous studies have concentrated on immune cell infiltration within the tumour microenvironment, 25,S8 the exploration of peripheral blood lymphocyte subsets in tumour patients remains relatively underexplored.The maintenance of a normal immune state crucially hinges on the coordination of diverse immune cells, particularly peripheral lymphocyte subpopulations.The constancy of lymphocyte subsets is perturbed in pathological states.Prior investigations have highlighted the favourable impact of higher peripheral blood levels of CD3+/CD8+ cells and lower levels of CD3À/CD56+ cells on OS in patients with nasopharyngeal carcinoma. 26A retrospective study involving 74 patients with advanced non-small cell lung cancer during immunotherapy demonstrated prolonged OS with elevated levels of CD3+/CD4+ and CD8+ T cells and reduced levels of NK cells. 27In a more extensive retrospective study comprising   482 patients with metastatic breast cancer, high levels of CD3 + T cells and CD3+/CD4+ T cells were associated with a poorer prognosis. 28Notably, patients with elevated circulating NK cell counts in progressive gastric cancer exhibited a more favourable prognosis. 29S9 In our current study, Cox regression analysis of peripheral lymphoid subpopulations revealed an unfavourable prognosis for patients with elevated CD3+/CD4+ cell levels, a high CD4 +/CD8+ ratio and diminished CD19+ cell counts.Examination of body composition unveiled that patients with sarcopenia and myosteatosis exhibited poorer prognoses.This study, for the first time, explored the association between CD3 +/CD4+ cell-myosteatosis and the prognosis of patients who underwent surgery for gastric cancer.The findings established CD3+/CD4+ cell-myosteatosis as an independent prognostic factor for both PFS and OS in patients who underwent radical gastric cancer surgery.Additionally, pTNM stage, sarcopenia and CD19+ cell counts were identified as independent prognostic factors for PFS and OS.
Potential mechanisms underlying the predictive capabilities of CD3+/CD4+ cell binding myosteatosis in gastric cancer surgery outcomes were considered.CD3+/CD4+ cells, pivotal players in the immune system, assume crucial roles in recruiting, activating and regulating various aspects of the adaptive immune response. 30While CD4+ T lymphocyte subpopulations may not share identical roles, they collectively contribute to tumour immunity. 31The distribution of T lymphocyte subpopulations varies significantly at different tumour stages, indicating their potential role as determinants of tumour progression. 32T helper (Th)1 cells mediate anti-tumour immune responses, with Th2 and regulatory T (Treg) cells exhibiting immunosuppressive properties.In early tumorigenesis, anti-tumour effects mediated by CTL and Th1 predominate.However, as tumours progress, the increasing number and proportion of tumours promote the rapid growth and dominance of T lymphocytes.S11 Elevated fat density may signify inflammatory changes, with serum inflammatory cell levels correlating with prognosis in various tumours.The muscle microenvironment, comprising neutrophils, monocytes and T lymphocytes, secretes mediators influencing repair and remodelling during skeletal muscle injury. 35Consequently, poor muscle mass, reflected by myosteatosis, may imply underlying impaired host immune defence, affecting the recurrence of metastasis in patients.
Furthermore, the immune system may engage in interactions with tissue wasting, consequently contributing to tumour recurrence and distant metastasis.S12 Within the tissues of individuals afflicted with malignant tumours, the predominant inflammatory infiltrating cells consist primarily of T lymphocytes. 37In instances where tumour patients exhibit cachexia, significant alterations occur in key signalling factors within the body, thereby further reducing the number of T lymphocytes, disrupting T cell metabolism and impeding differentiation. 38,S13,S14 Consequently, this disruption in immune system functionality leads to a decrease in immune surveillance of the tumour, ultimately resulting in tumour progression and recurrence. 39espite the significant findings, certain limitations must be acknowledged.This study constitutes a single-centre retrospective analysis with a relatively modest sample size.The exclusive focus on gastric cancer patients who underwent surgery might limit the generalizability of the findings.Additionally, the determination of optimal cut-off values for lymphatic subgroups heavily depends on ROC curves, with potential variations based on regional factors.To enhance the robustness and generalizability of our conclusions, multicentre prospective studies with larger sample sizes across diverse malignancies are warranted.

Conclusions
In summary, our study establishes associations between peripheral lymphoid subsets, body components and clinical outcomes in patients who underwent gastric cancer surgery.The novel combination of CD3+/CD4+ cells with myosteatosis emerged as a highly effective indicator, offering heightened predictive efficacy for identifying recurrence and metastasis in post-surgery gastric cancer patients.These findings provide valuable insights into the prognostic landscape of gastric cancer and lay the groundwork for future research avenues.