Assessment and Prognostic Significance of a Serum Cytokine Panel in Diffuse Large B-cell Lymphoma

<jats:title>Abstract</jats:title><jats:p><jats:bold>Objective</jats:bold>To assess the contents of circulating cytokines in patients with diffuse large B-cell lymphoma (DLBCL), and to examine their relationship with clinicopathological manifestations and prognosis.<jats:bold>Method</jats:bold>We recruited 72 DLBCL patients, 11 chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) patients, and 56 healthy controls from our hospital between the period of January 2017 and January 2020, and measured 7 serum cytokine contents using Beckman Navios flow cytometry. The cytokine level was compared between DLBCL patients and healthy controls using one way ANOVA. Two-sided Spearman test was employed for relationship evaluation between circulating cytokine levels and clinicopathological characteristics, IPI score, and short-term treatment response within DLBCL patients. The inter-group comparison of cytokine levels employed the Mann Whitney test. The support vector machine (SVM) was utilized for the cytokine evaluation-based prediction of DLBCL patient short-term treatment response. Lastly, survival curves were used to assess correlation between the aforementioned cytokines and overall survival.<jats:bold>Result</jats:bold>The IL-6, IL-10, and IFN-γcontents were markedly enhanced among DLBCL patients, as opposed to healthy controls (<jats:italic>P</jats:italic> &lt; 0.05). Patients with enhanced circulating LDH expressed elevated IL-10 level (P &lt; 0.05), and patients with augmented CRP expressed upregulated IL-6 and IL-10 levels, and the rise in IL-6 levels was positively associated with serum CRP (<jats:italic>P</jats:italic> = 0.00,<jats:italic>r</jats:italic> = 0.66). Additionally, the international prognostic index (IPI) risk stratification of DLBCL patients was strongly associated with the circulating IL-6 and IL-10 contents. Enhanced IL-6, IL-10, and TNF-α levels often produced worse short-term treatment efficacies (<jats:italic>P</jats:italic> &lt; 0.05). Moreover, the accuracy of short-term treatment response prediction model of DLBCL patients, obtained using SVM, was 81.63%. Using long-term follow-up, we further revealed that the DLBCL patients who expired within one year exhibited enhanced circulating IL-6 and IL-10 levels, compared to patients who survived, however, the IL-17 level was drastically reduced (<jats:italic>P</jats:italic> &lt; 0.05). Despite the aforementioned evidences, we observed no marked association between the specified cytokines and overall survival (OS).<jats:bold>Conclusion</jats:bold>The IL-6, IL-10, IL-17, TNF-α, and IFN-γ contents can potentially serve as biological indicators of DLBCL tumor immune status, and a combined application with the IPI score can be a robust indicator for DLBCL patient prognosis. Our findings provide novel ideas for the clinical treatment of DLBCL patients.</jats:p>

section_content: Diffuse large B-cell lymphoma (DLBCL) is a mature B-cell malignancy that is frequently found among adults with non-Hodgkin's lymphoma (NHL) [1]. In China, DLBCL accounts for 40. 1% of all NHL incidences, and it has garnered increasing attention due to its enhanced morbidity and lethality. At present, DLBCL pathogenesis is unclear, and based on a few reports, DLBCL is closely associated with in ammation-triggered immune dysfunction, a condition mediated by cytokines [2, 3]. R-CHOP (rituximab, cyclophosphamide, adriamycin, vincristine, and prednisone) is the current rst-line treatment for DLBCL, and it produces satisfactory remission rates in most patients. However, there is a considerable proportion of patients who are either insensitive to R-CHOP or relapse early [4, 5]. Owing to the high heterogeneity of DLBCL patient prognosis, it is extremely crucial to establish an appropriate patient prognostic strati cation method. The international prognostic index (IPI) score, a current gold standard for DLBCL patient prognostic assessment, does not accurately re ect the role of immune escape in disease progression. Moreover, the IPI accuracy is remarkably low with standard treatment, even in combination with rituximab (R) [6]. Thus, it is essential to identify novel biomarkers to enhance the IPI scoring system. Foreign investigations revealed that the expression pro les of certain cytokines are strongly correlated with the onset, severity, and prognosis of DLBCL [7] [8] [9]. At present, there are limited relevant studies in China. In view of the differences observed between domestic and foreign environments, patient pathogenic factors, and high DLBCL heterogeneity, herein, we examined the cytokine expression pro les of DLBCL patients in China using ow cytometry. Our ndings will provide new ideas for the prognostic analysis and clinical treatment of DLBCL worldwide.
section_name: Introduction

section_name: Materials and methods

section_content: In all, we recruited 111 patients with de nite B-NHL, con rmed via pathological biopsy, from our hospital between January 2017 and January 2020, and our diagnostic criteria were in accordance with the reported literature. Among the selected participants were 11 CLL/SLL patients (6 males and 5 females, age 55-85 years, median age 72 years); 72 primary DLBCL patients (46 males, 26 females, age 36-91 years, median age 65 years); and 21 DLBCL patients in remission after treatment ( age 36-74 years, median age 61 years). Among patients who were excluded from the study were those with autoimmune diseases, severe infections, secondary tumors, and other blood disorders. In addition, we employed 56 healthy volunteers as the control cohort (34 males and 22 females, age 24-78 years, median age 55 years). The gender and age of all four subject cohorts were comparable to one another (P = 0. 461, 0. 114). The nal follow-up was conducted in January 2021; the minimum follow-up period was 12 months; and the follow-up was terminated upon patient expiration during follow-up.
section_name: General information

section_content: We collected general clinical information from all DLBCL patients, namely, lactate dehydrogenase (LDH), C-reactive protein (CRP), ultrasonography, imaging, such as, radiographs, tomography (CT) or PET-CT, bone marrow aspiration, and biopsy. Patients also received an IPI score based on 5 factors including age, behavioral status, Ann Arbor stage, LDH expression level, and number of invaded sites of extra nodal lesions. 

Among the 72 DLBCL patients, three were automatically discharged without treatment, and 69 were treated with chemotherapy, according to the R-CHOP regimen over a 21-day treatment period. All patients were treated for 3 courses, prior to the e cacy evaluation, based on the NCCN Clinical Practice Guidelines in Oncology: B-Cell Lymphomas. The complete and partial remission patients made up the effectively treated cohort of 21 cases, and the disease progression and non-remission patients were classi ed under the ineffectively treated cohort of 28 cases. 

In all, 39 DLBC patients had available long-term follow-up data, and the remaining were lost to follow-up. In addition, 16 patients expired during a span of 1-year (follow-up period), and 23 patients survived during the period ≥ 1 year. These patients were categorized into the deceased and survived cohorts.
section_name: Sub-cohort of DLBCL patients

section_content: We employed a Beckman Navios ow cytometer and the human Beckman Th1/Th2/Th17 CBA cytokine kit (as per kit directions) to assess the serum concentrations of 7 speci ed cytokines among the untreated patients and healthy controls in each group prior to treatment. We collected 5 ml of fasting venous blood (arm) from all subjects, incubated at room temperature for 2 h, then separated the serum via a 5-min centrifugation at 1000g, prior to storage at 4℃. The interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ measurement was completed within 24 hours.
section_name: Cytokine assays

section_content: The SPSS 20. 0 statistical analysis software was employed for all data analyses. Multi-group counting data was assessed using one-way ANOVA. Comparisons in cytokine expressions were done with the Mann-Whitney test. The Kruskal Wallis test was employed for the comparison of cytokine pro le differences between the aforementioned two patient cohorts. Lastly, we performed correlation analysis based on cytokines and different CRP levels and IPI scores using the two-sided Spearman test. Signi cance was considered at P < 0. 05.
section_name: Statistical methods

section_content: Support vector machine (SVM) was utilized for the DLBCL patient prognosis prediction model, and it was generated via combination of the analytical results of cytokines, as determined by ow cytometry.
section_name: Generation of the prediction model

section_content: Expression pro les of 7 cytokines among the primary DLBCL patients, primary CLL patients, and normal control volunteers The IL-6, IL-10 (P < 0. 01), and IFN-γ (P < 0. 05) contents were markedly enhanced among primary DLBCL patients versus controls (Table 1 ). Meanwhile, the TNF-α levels exhibited a slight increase in primary DLBCL patients, but, it did not reach signi cance. We also observed no changes in the IL-2, IL-4, and IL-17 expressions between the two cohort (P > 0. 05, Table 1, Supplementary Fig. 1 ). Moreover, unlike comparison with the primary DLBCL patients, we observed no statistical differences in cytokine levels between the primary CLL patients and healthy controls (P > 0. 05, Table 1 ), suggesting that the serum cytokine levels are speci c to the early diagnosis of DLBCL patients.
section_name: Results

section_content: Apart from a rise in the serum IL-6 levels (P < 0. 05), we observed no signi cant alterations in the serum cytokine expressions of 21 DLBCL patients who achieved sustained remission following standard treatment and healthy controls during the sustained remission period (P > 0. 05, Table 2 ). This suggested an intimate relationship between cytokine expressions and disease progression among DLBCL patients.
section_name: Cytokine expression in DLBCL patients in sustained remission after treatment

section_content: Elevated LDH expression among DLBCL patients is closely associated with poor prognosis. Herein, we revealed that patients with enhanced serum LDH expressed considerably higher IL-6 and IL-10 contents than patients with normal LDH (Z = 2. 368, 3. 143, P = 0. 018, 0. 002), while the remaining cytokines were not signi cantly different (P > 0. 05, Fig. 1 ). Likewise, we revealed that patients with elevated CRP exhibited markedly higher serum IL-6 and IL-10 levels, compared to patients with normal CRP (Z = 3. 257, 2. 159, P = 0. 001, 0. 031, Fig. 2A-E ). Moreover, the magnitude of IL-6 elevation displayed a signi cant positive correlation with serum CRP (P = 0. 00, r = 0. 66, Fig. 2F ). Alternately, both TNF-α and IFN-γ exhibited a signi cant trend of elevation only in patients with CRP > 50 mg/L and > 100 mg/L, respectively (P < 0. 05, Supplementary Table 1 ). 2 ), and there was a positive correlation between the IPI score and serum IL-6 and IL-10 levels (IL-6: P = 0. 007, r = 0. 380; IL-10: P = 0. 002, r = 0. 438, Fig. 3I ). Moreover, even though the TNF-α difference between the two cohorts did not reach statistical (H = 3,474, P = 0. 324), there was a substantial elevation in the cohort with IPI of 2, relative to the cohort with IPI of ≥ 3 (Fig. 3E ).
section_name: Association between serum cytokine levels and clinicopathological features among DLBCL patients at rst presentation

section_content: The predictive model of short-term treatment response among DLBCL patients using the support vector machine (SVM) analysis of cytokines The 49 primary DLBCL patients included 21 effectively treated and 28 ineffectively treated patients. Based on our analysis, compared to the effectively treated patients, the ineffectively treated patients expressed higher serum IL-6 and IL-10 levels (P < 0. 01), as well as TNF-α levels (P < 0. 05, Table 3 ). Alternately, the IFN-γ content was not statistically different between the two cohorts, despite a strong rise among the ineffectively treated versus effectively treated patients (Fig. 4 ). 

The aforementioned cytokine data was separated into two categories, 80% for training and 20% for validation. Using the e1071 package and the random number set to 123, we performed optimization of the penalty coe cient C via tune. svm. The optimization range was between 0. 005-1, optimization step was 0. 005, and gamma was set to 1. 

Lastly, the optimal SVM model was derived as follows: the type was C-classi cation, the SVM kernel was radial, and the optimal C was 0. 895. Using this optimal SVM model, the prediction test group accuracy was 78. 57% (Supplementary Fig. 2 ), and the area under the ROC curve was 0. 73 (Fig. 5 ).
section_name: Associations between serum cytokine pro les and IPI scores among DLBCL patients at rst presentation

section_content: Among the 39 DLBCL patients with long-term follow-up, 16 expired (deceased cohort) and 23 survived (survived cohort) during the 12-month follow-up. The deceased patients expressed higher serum IL-6 and IL-10 levels, whereas, the serum IL-17 content was higher among the survived patients (P < 0. 05), and there were no signi cant changes in the remaining cytokines (P > 0. 05, Table 4 ). Interestingly, based on the survival plots, IL-6 alone did not signi cantly correlate with the survival prognosis of DLBCL patients (Fig. 6 ).
section_name: Relationship between clinical survival and serum cytokine levels among DLBCL patients

section_content: DLBCL is a highly aggressive form of NHL, and it is associated with an enhanced morbidity and mortality rate. Thus, it is imperative to identify novel observational indicators that can assist in the early diagnosis, clinical treatment, and assessment of DLBCL patient prognosis. More recently, the detection of cytokines and related receptors expression levels have become an essential component of basic and clinical immunology research, as the serum contents of cytokines gain increasing importance in the elds of early diagnosis of clinical diseases, prognosis, and even in assessing the e cacies of antitumor drugs and formulating individualized treatment plans [10]. Herein, we detected the cytokine expression levels of DLBCL patients using ow cytometry in order to provide novel ideas for the early diagnosis, clinical treatment and prognostic analysis of DLBCL. 

In recent decades, in the process of exploring new methods for the diagnosis and treatment of B-NHL, particularly DLBCL, research on IL-6, IL-10, TNF-α, and IFN-γ detection has gradually increased. IL-6 is a potent cytokine that accelerates the growth and differentiation of B lymphocytes, and it is an essential component of the lymphoma microenvironment. It also induces tumor blood vessel formation, destroys adhesion between tumor cells, and strongly prevents the anti-tumor effect of the body, thereby promoting growth, differentiation, and anti-apoptosis of tumor cells, which initiates a vicious cycle [11, 12]. Although IL-10 is known to possess antitumor effects mediated by CD8 + T cell responses, in the presence of CD19 + tumor cells, elevated IL-10 levels can potentially serve as a growth factor for tumorigenic B lymphocytes, and facilitate tumor cells to escape the immune system in an autocrine form [13, 14]. Moreover, it protects tumor cells from apoptosis via upregulation of the Bcl-2 expression, which provides an overall pro-tumor effect [15]. TNF-α is among the rst cytokines released during in ammation, and it is critical for the initiation of the cytokine cascade. Elevated serum TNF-α and associated receptors (sTNF-R1 and sTNF-R2) levels are strongly associated with shorter overall survival in numerous tumors [16, 17]. However, the role of TNF-α in DLBCL remains unclear. IFN-γ has classical antitumor effects, but it may be tolerated or even surpassed by its pro-tumor effects in the tumor microenvironment. Some studies demonstrated that IFN-γ greatly enhances oncogenic activity via the B invasive lymphoma protein 1/ADP ribose convertase 9 (BAL1/ARTD9) in high-risk DLBCL patients [18]. The role of IL-17, a characteristic Th17 cell-secreted cytokine in tumors, remains controversial. It was earlier demonstrated that in B-NHL, elevated transforming growth factor beta (TGF-β) levels inhibit Th17 cell differentiation while promoting differentiation of regulatory T cells, which produces a weakened anti-tumor mechanism, thereby enabling tumor immune escape [19]. 

Herein, we demonstrated signi cantly different expressions of the DLBCL and CLL cytokines, two typical representatives of aggressive and inert B-cell lymphomas. The expressions of IL-6, IL-10, and IFN-γ were signi cantly higher among DLBCL patients than in healthy controls. TNF-α was also strongly upregulated between the two cohorts, which con rmed that the correlation between aberrant cytokine expressions and DLBCL occurrence. No signi cant differences were observed in these cytokines between the CLL/SLL patients and healthy controls. 

Although some prior studies revealed that the serum IL-6 and IL-10 levels remain elevated in CLL/SLL patients, these are primarily concentrated within certain high-grade patients [20]. In this study, we demonstrated to a certain extent that serum cytokines have limited diagnostic signi cance for the inert B-cell lymphoma represented by CLL, however, it is speci c for DLBCL. Additionally, among DLBCL patients, the aforementioned cytokines, apart from IL-6, often returned to normal levels upon continued remission of the disease. This suggested that cytokines also have a strong relationship with disease progression, thus providing an idea for the treatment of DLBCL patients. 

Meanwhile, elevated LDH and CRP expressions were associated with poor prognosis in DLBCL patients, and we revealed via grouping that patients with enhanced serum CRP or LDH levels expressed higher IL-6 and IL-10 contents, and that the elevation of these factors was positively associated with serum CRP, which was consistent with the reports of Nacinovic et al [21]. Furthermore, there was a strong correlation between the IPI risk strati cation and circulating IL-6 and IL-10 levels in DLBCL patients. The IL-10 was signi cantly higher in low-risk IPI patients, relative to the high-risk IPI patients, and the difference was greater with elevated scores. Our e cacy observations revealed that elevated IL-6, IL-10, and TNF-α levels often predicted worse treatment effectiveness, which was consistent with the ndings of Dlouhy et al [7]. We further tested cytokines using the SVM technique, and the accuracy of our optimal SVM model to predict short-term treatment e cacies among DLBCL patients was 81. 63%. 

To a certain extent, this con rmed that cytokines are an important indicator of DLBCL treatment response. 

Using long-term follow-up, we demonstrated that DLBCL patients who expired within one clinical year expressed enhanced serum IL-6 and IL-10 levels, as well as strongly diminished IL-17 levels, compared to the patients who survived. In contrast, using survival curves, we did not observe a signi cant association between IL-6 levels and overall survival of DLBCL patients. This suggested, to some extent, that serum cytokines have some limitations in indicating long-term prognosis of DLBCL patients. 

In conclusion, the IL-6, IL-10, IL-17, TNF-α and IFN-γ contents can serve as prognostic indicators for the assessment of tumor immune status in DLBCL. Moreover, in combination with the IPI score, they can be important indicators of DLBCL prognosis, and may also provide a basis for the precise treatment and direction of novel and e cacious targeted therapies. Tables
section_name: Discussion

section_name: Acknowledgements

section_content: Not applicable.
section_name: Acknowledgements

section_name: Funding

section_content: This study was supported by the Foundation of Science Technology Department of Zhejiang Province (No. 

LGF22H080012), Zhejiang Provincial Medical Technology Plan Project (No. 2022KY505, No. 2020KY052 ).
section_name: Funding

section_name: Availability of data and materials

section_content: Not applicable.
section_name: Availability of data and materials

section_name: Declarations

section_content: Authors' contributions SX, LZ and LW collected and analyzed the data, drew fgures and tables, and contributed in writing the manuscript. SW, LZ and SX performed the statistical analysis; XT procured the funding for this study. XT, WN, SW participated in the design of the study, gave administrative or logistical support for this study, and reviewed drafts of the paper. All the authors agreed with the conclusions of this review and approved the nal manuscript.
section_name: Declarations

section_content: This study was authorized by the Ethics Committee of the Zhejiang Provincial People's Hospital (2021QT150).
section_name: Ethics approval and consent to participate

section_content: Not applicable.
section_name: Consent for publication

section_content: The authors declare that they have no competing interests.
section_name: Competing interests

section_content: The ROC curve of circulating cytokine SVM prediction model for the short-term treatment response of DLBCL patients. 

The survival curve analysis of the individual serum factor IL-6 in DLBCL patients.
section_name: Groups

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supplementary gureandtable. zip
section_name: Supplementary Files