STAT3 Expression and Its Correlation with PD-L1 Expression in Non-Hodgkin's Lymphoma and Hodgkin's Lymphoma at Dr. Saiful Anwar Regional Public Hospital in Malang, Indonesian Population

Background Lymphomas are malignant lymphocyte neoplasms that globally account for 10% of cancers in individuals aged <20 years. Malignant lymphomas are divided into Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL). Despite the availability of many therapeutic modalities for lymphoma, such as Brentuximab vedotin, Nivolumab, and Pembrolizumab, it is still necessary to identify appropriate strategies with minimal side effects. Immunotherapy is a promising approach, exemplified by targeting JAK/STAT3 signaling, which can inhibit tumor growth and enhance antitumor immune responses. Hence, STAT3 (signal transducer and activator of transcription 3) is a promising therapeutic target. PD-L1 (programmed death-ligand 1), an immune checkpoint molecule, is used as a frontline treatment for various cancers. This study aims to determine STAT3 expression and its correlation with PD-L1 expression in NHL and HL to serve as a basis for further research on anti-STAT3 and its combination with other therapy targets. Methods Samples were obtained from paraffin blocks of patients with confirmed diagnoses of NHL and HL, and then immunohistochemical staining was carried out with PD-L1 and STAT3 antibodies. The collected data were then analyzed using SPSS. Results Among the 10 HL patients, no patients (0%) expressed STAT3, while nine patients (90%) expressed PD-L1. Among the 10 NHL patients, 1 patient (10%) expressed STAT3, while six patients (60%) expressed PD-L1. There were no significant differences in STAT3 expression and PD-L1 expression between HL patients and NHL patients. There was no correlation between STAT3 and PD-L1 expression in HL and NHL because almost all STAT3 expressions were negative. Conclusion Although this study revealed no differences between STAT3 and PD-L1 expression in HL and NHL and no significant correlation between STAT3 and PD-L1 expression in HL and NHL, this may serve as the basis for understanding the role of STAT3 and PD-L1 in the regulation of HL and NHL, which may be useful for further research targeting STAT3 and PD-L1 immunotherapy in HL and NHL.


Introduction
Lymphomas, a heterogeneous group of malignant lymphocyte neoplasms, account for 10% of cancers in individuals aged <20 years globally and have more than 90 subtypes.Malignant lymphomas are divided into Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL).In the USA, NHL ranks as the sixth most frequent cause of cancer-related death, while globally, in 2020, 0.2% of newly reported cancer deaths were due to HL.According to GLOBOCAN 2022, the incidence in Asia for HL is 37.3% and for NHL is 42.5%.In Indonesia, Dr. Saiful Anwar Regional Public Hospital in Malang reported 408 cases of lymphoma from 2018 to 2023, consisting of 56 cases of HL and 352 cases of NHL.Lymphatic tissue, bone marrow, or extranodal sites may be involved in lymphoma, with extranodal sites being more common among NHL patients.Examples of extranodal sites include Waldeyer's ring, salivary glands, orbit, paranasal sinuses, thyroid glands, larynx, and skin [1][2][3][4][5][6].
Tere are many available lymphoma therapeutic modalities, and immunotherapy is a promising approach.Te US Food and Drug Administration (FDA) have approved Brentuximab vedotin, Nivolumab, and Pembrolizumab as immunotherapy agents.Te selection of targeted agents and optimal dosage is important, even though the overall survival (OS) of lymphoma patients has been improved by new immunochemotherapeutic regimens.In a clinical trial by Carsulo et al., durvalumab (anti-PD-L1) demonstrated an 18% overall response rate (ORR) and 8% complete response rate (CRR) in difuse large B-cell lymphoma and a 59% ORR and 27% CRR in follicular lymphoma.However, 63% of patients experienced serious adverse events, mostly related to infection.Terefore, it is necessary to identify appropriate strategies with minimal side efects.Te challenge lies in the limited immune response to immunotherapy, prompting consideration of combination therapy to improve cancer therapy outcomes [7][8][9][10][11][12].
High NF-κB activity, resulting from genetic alterations in the toll-like receptor (TLR) and B-cell receptor (BCR) signaling pathways, induces the production of IL-6 and IL-10, leading to constitutive activation of JAK1 and STAT3 (signal transducer and activator of transcription 3).Tis activation promotes cell survival and proliferation, modulates the tumor microenvironment, promotes tumor immune evasion, and is associated with inferior clinical outcomes.Targeting JAK/STAT3 signaling can inhibit tumor growth and enhance antitumor immune responses, making STAT3 a promising therapeutic target in lymphoma.A clinical trial conducted by Li et al. regarding Napabuscin, a STAT3 inhibitor, showed potent cytotoxicity against NHL cells without signifcant toxicity [8,13].
PD-L1 (programmed death-ligand 1), an immune checkpoint molecule, binds the inhibitory PD-1 receptor on T-cells and suppresses T-cell activation.PD-1/PD-L1 inhibitors have been used as frontline treatments for various cancers, such as non-small cell lung cancer, metastatic melanoma, and renal cell carcinoma.So far, the anti-PD-L1 approved by the FDA is atezolizumab, avelumab, and durvalumab.However, a clinical trial conducted by Casulo found that durvalumab-anti-PD-L1-showed a limited therapeutic efect, with more than 50% of patients experiencing side efects such as transaminitis, increased bilirubin, diarrhea, thyroid disorders, pruritus, cerebral ischemia, and gastrointestinal perforation [9,10,14].
Tis study aims to determine the diferences between STAT3 and PD-L1 expression in NHL and HL, as well as the correlation between STAT3 expression and PD-L1 expression in NHL and HL.It also seeks to provide a basis for further research on anti-STAT3 and its combination with other therapy targets to improve patient outcomes.

Sample Collection.
Tis study was a cross-sectional study conducted in May 2023 at the Anatomical Pathology Laboratory, Dr. Saiful Anwar Regional Public Hospital, Malang.Data were collected from parafn blocks of patients who had a confrmed diagnosis of NHL and HL from January 2018 until May 2023.
Te sample size was calculated with the following formula [15]: Te sampling method used was simple random sampling from patient populations that met the inclusion and exclusion criteria.Te inclusion criteria included NHL and HL parafn blocks from biopsy samples confrmed histopathologically and/or immunohistochemically.Te exclusion criteria included lost or damaged parafn blocks or when the remaining tissue was insufcient for further examination in this study.

Immunohistochemistry Staining and Evaluation of PD-L1
Expression.PD-L1 IHC 22C3 pharmDx was used for immunohistochemical (IHC) staining, with tonsil tissue as the control tissue.Shortly, rehydrated deparafnized sections were subjected to the heat antigen retrieval technique.PD-L1 expression was evaluated using the Tumor Proportion Score (TPS).TPS represents the percentage of viable tumor cells showing partial or complete membrane staining (≥1+) relative to all viable tumor cells present in the sample (positive and negative).PD-L1 staining was considered negative if TPS <1%, positive if TPS ≥1%, and high PD-L1 expression if TPS ≥50%.Intensity of staining (+1 � weak, +2 � moderate, and +3 � strong staining) was also recorded [16,17].

Immunohistochemistry Staining and Evaluation of STAT3
Expression.STAT3 antibody (21E7) GeneTex was used for immunohistochemical (IHC) staining, with skin tissue as the control tissue.Shortly, rehydrated deparafnized sections were subjected to the heat antigen retrieval technique.STAT3 expression was evaluated from 5 highpower felds.STAT3 staining was considered negative if nuclear staining was <10% of tumor cells, low positive if nuclear staining was 10-50% of tumor cells, and high STAT3 expression if nuclear staining was ≥50% of tumor cells [18,19].
Advances in Hematology 2.4.Statistical Analysis.Te normality of the data was assessed using the Shapiro-Wilk test, and because the results were not normally distributed, nonparametric tests were used.Te comparison of PD-L1 and STAT3 immunoexpression between NHL and HL was assessed using the Mann-Whitney test.Te correlations between immunoexpression of PD-L1 and STAT3 were assessed using Spearman correlation.All analyses were conducted using Statistical Package for the Social Sciences (SPSS) software v20 [20].

Correlation between STAT3 Expression and PD-L1
Expression in HL and NHL.Te Spearman correlation test results between STAT3 expression and PD-L1 expression in HL patients could not be determined due to all STAT3 expressions being negative (Table 5).
Since all STAT3 expressions in HL patients were negative, a linear correlation between STAT3 and PD-L1 levels in HL patients could not be illustrated (Figure 2).
Based on the results of the Spearman correlation test between STAT3 expression and PD-L1 expression in NHL patients, a correlation coefcient value of 0.430 was obtained with a signifcance value of 0.214 (p > 0.05).Terefore, it can be concluded that there is no signifcant relationship between STAT3 expression and PD-L1 expression in NHL patients.In other words, high or low STAT3 expression in NHL patients is not associated with increased or decreased PD-L1 levels (Table 6).
Based on the linearity graph between STAT3 expression and PD-L1 expression in NHL patients, it appears that there is a positive linear relationship.However, due to the small sample size of NHL patients, the correlation test results did not yield signifcant fndings (Figure 3).

Discussion
In our study, almost all STAT3 expressions are negative, which is inconsistent with fndings from other studies.For instance, Sefens et al. reported that 38% of peripheral Tcell lymphoma cases showed positive STAT3 staining, while Skinnider et al. found STAT3 expression in 87% of classical HL, 46% of B-cell NHL, and 73% of T-cell NHL cases.STAT3 mediates the expression of various genes and plays a critical role in numerous cellular and biological processes, including cell proliferation, survival, diferentiation, migration, angiogenesis, and infammation.Consequently, abnormal STAT3 expression promotes malignant transformation and tumor progression through oncogenic gene expression that have been observed in numerous human cancers, and lymphoma is one of them.Moreover, STAT3 plays an important role in evading immune surveillance.Consequently, targeting JAK/STAT3 signaling can inhibit tumor growth and enhance antitumor immune responses, making STAT3 a promising therapeutic target in lymphoma [13,21,22].Te reason for the predominantly negative STAT3 expression in our research could be attributed to the small sample size and minimal variation among samples.Terefore, we hope that this study can serve as a foundation for further research using larger and more diverse samples to yield more signifcant results.

Advances in Hematology
Xie et al. reported that PD-L1 is expressed in 70-87% of HL cases, and compared to HL, some subtypes of NHL exhibit lower PD-L1 expression.A study by Uccela et al. also found that PD-L1 expression is positive in more than 80% of HL cases but less than 30% of NHL cases.In certain lymphomas, such as primary mediastinal large B-cell lymphoma (PMBL), plasmablastic lymphoma, nodular sclerosis, and mixed cellularity cHL, T-cell/histiocyte-rich B-cell lymphoma, as well as virus-associated malignancies (Epstein-Barr virus-associated DLBCL and Human Herpes Virus 8-associated Primary Efusion Lymphoma), PD-L1 expression has also been reported.Tis discrepancy occurs because alterations of the 9p24.1 chromosome, which afect PD-L1 activation, are more frequent (61%) in HL [23][24][25].Tis fnding aligns with our study, where PD-L1 expression was observed more frequently in HL (90%) than in NHL (60%).
In our study, due to the predominantly negative STAT3 expression observed, there is no signifcant relationship between STAT3 expression and PD-L1 expression in HL and NHL patients.Some studies have also reported that a poorer prognosis is associated with PD-L1 or STAT3 expression.Additionally, multiple oncogenic pathways leading to the expression of PD-L1 by upregulating STAT3 expression have been documented.Exposure to infammatory factors in the tumor microenvironment can induce PD-L1 expression,    Te limitation of this study includes the small sample size and the inherent variability associated with immunohistochemistry examination for PD-L1.Various factors may infuence the results, such as tumor type, tissue sampling and preparation methods, duration of parafn block storage, the sensitivity of the clone used, diferences among manufacturers, protocols employed, and scoring methods.Additionally, gene expression may not always correlate perfectly with protein expression [30][31][32][33].To address these limitations, future research should focus on specifc tumor subtypes, utilize the recommended PD-L1 clone 22C3, and include parafn blocks stored within a defned timeframe (2019-2023).Further confrmatory, larger-scale studies are needed to compare gene and protein expression of PD-L1.Additionally, studies regarding the combined use of anti-STAT3 with anti-PD-L1 therapies are warranted.

Conclusions
Although this study revealed no diferences in STAT3 and PD-L1 expression between HL and NHL and no signifcant correlation between STAT3 and PD-L1 expression in HL and NHL, this may serve as the basis for understanding the role of STAT3 and PD-L1 in the regulation of HL and NHL, which may be useful for further research targeting STAT3 and PD-L1 immunotherapy in HL and NHL.

Data Availability
Te underlying data for "STAT3 Expression and Its Correlation with PD-L1 Expression in non-Hodgkin's Lymphoma and Hodgkin's Lymphoma at Dr. Saiful Anwar Regional Public Hospital in Malang, Indonesian Population" can be accessed at https://doi.org/10.6084/m9.fgshare.25801537.v1.Te data are available under the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Ethical Approval
Tis study was approved by the Ethical Board for Research of Dr. Saiful Anwar Regional Public Hospital, Malang, on August 18, 2023, with protocol number 400/208/K.3/302/2023.Advances in Hematology

Figure 2 :
Figure2: Correlation between STAT3 expression and PD-L1 expression in HL patients using SPSS v20.Since all STAT3 expressions in HL patients are negative, a linear line cannot be drawn between STAT3 and PD-L1 levels in HL patients.

Figure 3 :
Figure3: Correlation between STAT3 expression and PD-L1 expression in NHL patients using SPSS v20.Although a positive linear relationship between STAT3 expression and PD-L1 expression is observed in NHL patients, the correlation is not signifcant due to the small sample size.

Table 1
and NHL (p > 0.05).However, there was a signifcant difference in the age of patients diagnosed with HL and NHL (p < 0.05), with HL cases tending to occur in younger patients aged 11-30 years and NHL cases tending to occur in older patients aged 41-60 years.

Table 1 :
Univariate analysis of patients' characteristics based on gender and age group.

Table 2 :
STAT3 expression in HL and NHL.

Table 3 :
PD-L1 expression in HL and NHL.

Table 4 :
PD-L1 staining intensity in HL and NHL.

Table 5 :
Correlation between STAT3 expression and PD-L1 expression in HL.

Table 6 :
Correlation between STAT3 expression and PD-L1 expression in NHL.