1. 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 [1–6].

There are many available lymphoma therapeutic modalities, and immunotherapy is a promising approach. The US Food and Drug Administration (FDA) have approved Brentuximab vedotin, Nivolumab, and Pembrolizumab as immunotherapy agents. The 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 diffuse 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. Therefore, it is necessary to identify appropriate strategies with minimal side effects. The challenge lies in the limited immune response to immunotherapy, prompting consideration of combination therapy to improve cancer therapy outcomes [7–12].

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). This 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 significant 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 effect, with more than 50% of patients experiencing side effects such as transaminitis, increased bilirubin, diarrhea, thyroid disorders, pruritus, cerebral ischemia, and gastrointestinal perforation [9, 10, 14].

This study aims to determine the differences 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.

2. Methods

3. Results

4. STAT3 Expression in HL and NHL

Table 2 summarizes STAT3 expression in twenty patients with Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. Among the 10 Hodgkin’s lymphoma patients, none (0%) showed expression of STAT3, while all 10 (100%) exhibited no expression of STAT3. Among the 10 non-Hodgkin’s lymphoma patients, none (0%) had high expression of STAT3, 1 patient (10%) had low expression of STAT3, and 9 patients (90%) showed no expression of STAT3. The Mann–Whitney test revealed no significant difference in STAT3 expression between HL and NHL (p > 0.05).

5. PD-L1 Expression in HL and NHL

Table 2 summarizes PD-L1 expression in twenty patients with Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. Among the 10 Hodgkin’s lymphoma patients, 7 patients (70%) exhibited high expression of PD-L1, 2 patients (20%) showed low expression of PD-L1, and 1 patient (10%) had no expression of PD-L1. Among the 10 non-Hodgkin’s lymphoma patients, 3 patients (30%) had high expression of PD-L1, 3 patients (30%) showed low expression of PD-L1, and 4 patients (40%) exhibited no expression of PD-L1. The statistical analysis using the Mann–Whitney test revealed no significant difference in PD-L1 expression between HL and NHL (p > 0.05).

Tables 3 and 4 summarize PD-L1 staining intensity in twenty patients with Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. Among the 9 Hodgkin’s lymphoma patients with PD-L1 positivity, 1 patient (11.1%) exhibited +3 staining intensity, 7 patients (77.8%) showed +2 staining intensity, and 1 patient (11.1%) had +1 staining intensity. Among the 6 non-Hodgkin’s lymphoma patients with PD-L1 positivity, 4 patients (66.7%) had +2 staining intensity, and 2 patients (33.3%) exhibited +1 staining intensity (Figure 1).

5.1. Correlation between STAT3 Expression and PD-L1 Expression in HL and NHL

The 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).

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

			PD-L1 level in HL	STAT3 level in HL
Spearman’s rho	PD-L1 level in HL	Correlation coefficient	1,000
		Sig. (2-tailed)
		N	10	10
	STAT3 level in HL	Correlation coefficient
		Sig. (2-tailed)
		N	10	10

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 coefficient value of 0.430 was obtained with a significance value of 0.214 (p > 0.05). Therefore, it can be concluded that there is no significant 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).

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

			PD-L1 level in NHL	STAT3 level in NHL
Spearman’s rho	PD-L1 level in NHL	Correlation coefficient	1,000	0.430
		Sig. (2-tailed)		0.214
		N	10	10
	STAT3 level in NHL	Correlation coefficient	0.430	1,000
		Sig. (2-tailed)	0.214
		N	10	10

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 significant findings (Figure 3).

6. Discussion

In our study, almost all STAT3 expressions are negative, which is inconsistent with findings from other studies. For instance, Seffens et al. reported that 38% of peripheral T-cell 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, differentiation, migration, angiogenesis, and inflammation. 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]. The reason for the predominantly negative STAT3 expression in our research could be attributed to the small sample size and minimal variation among samples. Therefore, we hope that this study can serve as a foundation for further research using larger and more diverse samples to yield more significant results.

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 Effusion Lymphoma), PD-L1 expression has also been reported. This discrepancy occurs because alterations of the 9p24.1 chromosome, which affect PD-L1 activation, are more frequent (61%) in HL [23–25]. This finding 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 significant 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 inflammatory factors in the tumor microenvironment can induce PD-L1 expression, thereby facilitating immune evasion. PD-L1 protein expression in tumor cells is upregulated by interleukin (IL)-1, IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Through a STAT3-dependent mechanism, IL-6 plays an important role in the induction of PD-L1. Studies by Cheng et al. have shown that in hepatocellular cancer-associated fibroblasts, PD-L1 expression can be induced through the IL-6-STAT3 pathway, resulting in impaired T-cell function and immune suppression through the PD1/PD-L1 signaling pathway [26–29].

The limitation of this study includes the small sample size and the inherent variability associated with immunohistochemistry examination for PD-L1. Various factors may influence the results, such as tumor type, tissue sampling and preparation methods, duration of paraffin block storage, the sensitivity of the clone used, differences among manufacturers, protocols employed, and scoring methods. Additionally, gene expression may not always correlate perfectly with protein expression [30–33]. To address these limitations, future research should focus on specific tumor subtypes, utilize the recommended PD-L1 clone 22C3, and include paraffin blocks stored within a defined timeframe (2019–2023). Further confirmatory, 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.

7. Conclusions

Although this study revealed no differences in STAT3 and PD-L1 expression between 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.

Ethical Approval

This 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.

Disclosure

Reporting Guidelines. Repository name: The ARRIVE checklist 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 DOI: https://doi.org/10.6084/m9.figshare.24574273.v1.

Conflicts of Interest

The authors declare that they have no conflicts of interest.