TLR9 expression is associated with PD-L1 expression and indicates a poor prognosis in patients with peripheral T-cell lymphomas

Journal Pre-proof TLR9 expression is associated with PD-L1 expression and indicates a poor prognosis in patients with peripheral T-cell lymphomas Jingrong Qian, Hongxue Meng, Bowen Lv, Jie Wang, Yingying Lu, Shu Zhao, Wenhui Li

PII:

S0344-0338(19)31602-4

DOI:

https://doi.org/10.1016/j.prp.2019.152703

Reference:

PRP 152703

To appear in:

Pathology - Research and Practice

Received Date:

1 August 2019

Revised Date:

6 October 2019

Accepted Date:

19 October 2019

Please cite this article as: Qian J, Meng H, Lv B, Wang J, Lu Y, Zhao S, Li W, TLR9 expression is associated with PD-L1 expression and indicates a poor prognosis in patients with peripheral T-cell lymphomas, Pathology - Research and Practice (2019), doi: https://doi.org/10.1016/j.prp.2019.152703

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

TLR9 expression is associated with PD-L1 expression and indicates a poor prognosis in patients with peripheral T-cell lymphomas

Jingrong Qiana,1, Hongxue Mengb,1, Bowen Lva,1, Jie Wanga, Yingying Lua, Shu Zhaoc,*, Wenhui Lia,*

Department of Clinical Laboratory, Harbin Medical University Cancer Hospital,

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a

Harbin, Heilongjiang 150081, P. R. China. b

Department of Pathology, Harbin Medical University Cancer Hospital, Harbin,

Department of Medical Oncology, Harbin Medical University Cancer Hospital,

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c

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Heilongjiang 150081, P. R. China.

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Harbin, Heilongjiang 150081, P. R. China.

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Jingrong Qian1, Hongxue Meng1 and Bowen Lv1 contributed equally to the work and

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should be considered co-first authors.

Wenhui Li*(email:[email protected]) and Shu Zhao* (email:zs_1881@163. com)

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are corresponding authors.

Correspondence: Wenhui Li Department of Clinical Laboratory, Harbin Medical University Cancer Hospital, No.150 Haping Road, Nangang District, Harbin, Heilongjiang Province, China.

Zip Code: 150081 Tel: 86-015344510273; Email: [email protected]

Correspondence: Shu Zhao Department of Medical Oncology, Harbin Medical University Cancer Hospital, No.150

Zip Code: 150081 Tel: 86-013946158616; Email: [email protected]

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Highlights

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Haping Road, Nangang District, Harbin, Heilongjiang Province, China.

Expression of TLR9 and PD-L1 in peripheral T-cell lymphomas (PTCL).

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Correlation between TLR9 and PD-L1 expression and clinicopathological

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features

TLR9 and PD-L1 expression on tumor cell indicates a poor prognosis in PTCL

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Univariate and multivariate analyses of prognostic factors in PTCL

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Abstract

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Toll-like receptor9 (TLR9), a member of pattern recognition receptors, play an important role in tumor immunologic surveillance. However, the clinical impact of TLR9 and programmed cell death-ligand 1 (PD-L1) in peripheral T-cell lymphomas (PTCL) remains unclear. In this study, we examined the expression of TLR9 and PDL1 by immunohistochemical staining in patients with PTCL, and evaluated the clinical

significance between expression and clinicopathological features. We found that the rates of high expression of TLR9 and PD-L1 on tumor cells were 65.3% and 45.8% in PTCL, respectively. TLR9 expression was associated with PD-L1 expression in PTCL. Moreover, TLR9 expression was associated with gender, ECOG score, Ki-67 expression, while PD-L1 expression was associated with the number of extranodal involvement and platelet count. High expression of either TLR9 or PD-L1 indicated a

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poor survival rate for patients with PTCL. Multivariate analysis confirmed that high expression of TLR9 and PD-L1 were unfavorable prognostic factors for patients with PTCL. Thus, TLR9 and PD-L1 expression might be important on the point of

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prognostic markers in PTCL.

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Keywords: TLR9, PD-L1, peripheral T-cell lymphomas, survival time.

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1. Introduction

Peripheral T-cell lymphomas (PTCL) is an uncommon and aggressive disease,

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which accounts for approximately 10%–15% of all non-Hodgkin lymphomas (NHL) [1]. According to the WHO classification, PTCL is subdivided into 28 histological

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subtypes, highlighting the cytological and phenotypic heterogeneity of this disease [2]. Compared with B cell lymphoma, symptoms

and

worse

patients with PTCL have aggressive clinical

survival

time

when

treated

with

traditional

immunochemotherapy [3]. Despite advances in the treatment of PTCL over the past few years, the prognosis of patients with PTCL has not improved significantly, and

many patients still have recurrence after first-line treatment. Thus, exploring potential prognostic indicators and therapeutic targets are still necessary for PTCL. Toll-like receptor (TLR9), a receptor for non-methylated CpG DNA, induces cytokines as well as many molecules to initialize the immune response [4]. Recent studies found that TLR9 is expressed not only in immune cells, but also in various types of tumor cells, such as lung, breast, glioma and pancreatic tumor cells. Some studies and the high expression

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suggested that TLR9 was involved in the tumorigenesis,

levels of TLR9 was correlated with tumor progression and poor prognosis [5-7], but others have failed to draw a similar conclusion [8, 9]. It is unclear whether the in patients with PTCL.

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expression levels of TLR9 has prognostic significance

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Programmed cell death ligand 1 (PD-L1) is involved in the inhibition of the adaptive immune system by binding to programmed cell death-1 (PD-1) receptor, which

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results in evade host immune surveillance [10]. Previous studies suggested that PD-L1

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expression on tumor cells be correlated with tumor progression and worse prognosis [11]. Moreover, blockade of the PD-1/PD-L1 pathway by anti-PD-1 or anti-PD-L1

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monoclonal antibodies may enhance anti-tumor effects in malignancy, such as diffuse large B-cell lymphoma, Hodgkin lymphoma melanoma and lung cancer [12-14].

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However, the clinical significance of PD-L1 expression in PTCL has not been reported. In this study, we retrospectively investigated the expression of TLR9 and PD-L1

in patients with PTCL, and then evaluated the clinical significance between expression and clinicopathological features. We further evaluated the relationship between TLR9 and PD-L1 expression in this study. This is the first report describing the

clinicopathological features and outcomes of TLR9 and PD-L1 expression in PTCL.

2. Materials and Methods

2.1 Patients From January 2006 to October 2018, a total of 144 patients were diagnosed with

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PTCL at Harbin Medical University Cancer Hospital. All cases were reviewed

according to present World Health Organization criteria (WHO 2016) [2]. A total of 144 patients had a complete clinicopathological and follow-up data. The research was

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approved by the ethic committee of Harbin Medical University Cancer Hospital.

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Informed consent was obtained from all patients and complied with the Declaration of

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Helsinki.

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2.2 Determination of TLR9 and PD-L1

Immunohistochemical staining was performed on each of paraffin-embedded

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biopsy specimens obtained from 144 patients with PTCL, a 4-mm-thick formalin-fixed paraffin-embedded section was submitted. Based on previous literature reports,

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immunohistochemical staining was performed [15]. The primary antibodies used for staining include: anti-CD3 (ab5690, 1: 50, Abcam, Cambridge, UK), anti-TLR9 (ab134368, 1:100; Abcam, Cambridge, UK), and anti-PD-L1 (ab205921, 1:100; Abcam, Cambridge, UK).

2.3 Evaluation of immunohistochemical staining In this study, we use serial sectioning during immunohistochemical staining, serial sections were used for T cell marker CD3 staining, tumor cell labeled leukocyte common antigen (LCA) staining and TLR9 / PD-L1 staining. Malignant T cells were visualized by morphology and CD3 staining, and multiple serial sections staining were used for interpretation to indicate TLR9/PD-L1 expression and localization. The

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method is consistent with previous literature reports [16, 17, 18, 19]. Additionally, the stained tissue sections were scored separately by two pathologists blinded to the

clinicopathological parameters. For PD-L1, based on previous studies, the samples with

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< 5% stained tumor cells were defined as negative. The PD-L1 expression was

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considered positive if membranous staining was observed in 5% or more of the lymphoma cells. According to staining intensity, the expression levels were further

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classified into three groups: weak staining (light brown, +), moderate staining (brown,

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++), and intense staining (dark brown, +++). The 5% threshold was based on a previous phase I trial of anti-PD-L1 agents and studies of malignancies involving the same clone

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antibody [20, 21, 22]. For TLR9, percentages of positively stained tumor cells were divided into 0: no positive tumor cells; 1: <10% positive tumor cells; 2: 10–50%

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positive tumor cells; and 3: >50% positive tumor cells. Staining intensity was classified as 0: no staining; 1: weak staining (light yellow); 2: moderate staining (yellowish brown); and 3: strong staining (brown). The staining index was staining intensity score × percentage score. Specimens for which the staining index score >4 were considered to have high TLR9 expression and those with staining index scores ≤4 were considered

to have low TLR9 expression [23].

2.4 Statistics Clinicopathological characteristics of the patients were compared using the chisquare test or Fisher’s two-sided exact test. The correlations between the groups were assessed Spearman’s correlation test. The Kaplan-Meier method was used to estimate

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overall survival (OS) and progression-free survival (PFS), and the log-rank test was performed to determine significant differences. The Cox-regression analyses, both univariate and multivariate, were used to identify the independency of these expression

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status. P-values < 0.05 were considered statistically significant. Data were analyzed

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using IBM SPSS software (version 22.0; IBM Corporation, New York, NY, USA).

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3. Results

3.1 Clinicopathological characteristics of PTCL patients

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A total of 144 patients with PTCL were included in the study (Table 1). Among these patients, 93 patients (64.6%) were male, and 51 patients (35.4%) were female.

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The median age was 58 years (range 7–82 years). According to the Ann Arbor classification system, 105 patients (72.9%) had advanced disease (Ann Arbor stage III/IV). 81 patients (56.2%) had systemic B symptom at the time of diagnosis, 93.8% of patients had elevated platelet (PLT) count, and 68.1% of patients had elevated serum levels of β2-microglobulin (β2-MG). An intermediate international prognostic score (IPI)

of low-intermediate risk or intermediate-high risk was seen in 64.6% of patients.

3.2 Expression of TLR9 and PD-L1 in PTCL To clarify the correlation between TLR9 and PD-L1 expression, we first evaluated TLR9 and PD-L1 expression in PTCL. TLR9 is normally expressed in immune cells such as dendritic cells and B cells, however, recent studies suggested that PD-L1 is

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inducible expressed in tumor cell and tumor-infiltrating macrophages, and TLR9 is also

expressed in tumor cells. The expression of TLR9/PDL1 on tumor cells was considered

in the present study. According to immunohistochemical staining (Fig.1), of 144 PTCL

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tissues, the high expression of TLR9 on tumor cells was noted in 65.3% of cases, and

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the low expression of TLR9 on tumor cells was found in 34.7% of patients. For PD-L1 expression, the high expression of PD-L1 on tumor cells was noted in 45.8% of cases,

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and the low expression of PD-L1 on tumor cells was found in 54.2% of patients,

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respectively.

We further evaluate the relationship between TLR9 and PD-L1 expression in

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patients with PTCL. Among patients with PD-L1 high expression, 68.2% of the patients (45/66) had TLR9 high expression, while 31.8% of the patients (21/66) had TLR9 low

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expression. Additionally, there was a significantly association between PD-L1 and TLR9 expression in patients with PTCL (Spearman’ r =0.509, P=0.037).

3.3 Correlation between TLR9 and PD-L1 expression and clinicopathological features

Based on the expression levels of TLR9, we dichotomized patients into two groups: the TLR9 high expression group and the TLR9 low expression group, and then compared the clinicopathological characteristics between the two groups (Table 1). Among patients with TLR9 high expression, 67 patients (71.3%) were male, 65 patients (69.1%) had advanced stage disease, and 68 patients (72.3%) had increased serum β2MG levels. TLR9 high expression was significantly associated with male (P=0.021),

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ECOG score (P=0.034), Ki-67 expression ≥ 45 (P=0.032). Compared to patients with PD-L1 low expression, PD-L1 expression was associated with extra nodal involvement

(P= 0.034) and increased PLT count (P=0.031). However, we found no significant

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correlations between PD-L1 expression and age, gender, histological type, tumor size,

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B symptom, ECOG score, IPI score, serum LDH levels or Ki-67 expression. According to 2016 World Health Organization (WHO) classification criteria,

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PTCL are composed of 28 different entities, highlighting the cytological and phenotypic

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heterogeneity of this diseases. We also explored the expression differences of TLR9 and PD-L1 in PTCL with different histological types. We found that the rates of high

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expression of TLR9 on tumor cells were 71.1% in peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS), 69.3% in angioimmunoblastic T-cell lymphoma

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(AITL), 55.0% in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALK+ ALCL), and 68.4% in anaplastic lymphoma kinase-negative anaplastic large cell lymphoma (ALK- ALCL). The rates of high expression of PD-L1 on tumor cells were 58.8% in PTCL-NOS, 50.0% in AITL, 30.0% in ALK+ ALCL, and 31.6% in ALK- ALCL, respectively. The rate of PD-L1 high expression was higher in

PTCL-NOS and AITL groups. However, there was no statistical difference between any two groups in PD-L1 expression (data not shown).

3.4 Survival of PTCL patients according to TLR9 and PD-L1 expression The 5-year overall survival (OS) rate was 41%, and the 5-year progression-free survival (PFS) rate was 22% for 144 PTCL patients. Patients with TLR9 high

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expression had lower 5-year PFS rate (18% vs. 28%, Fig. 2A; Table 2) and significantly lower 5-year OS rate (31% vs. 61%, P=0.004; Fig. 2B; Table 2) than patients with TLR9

low expression. Patients with PD-L1 high expression had significantly shorter 5-year

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PFS rate (13% vs. 31%, P=0.002; Fig. 2C; Table 2) and significantly shorter 5-year OS

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rate (34% vs. 79%, P<0.001; Fig. 2D; Table 2) than patients with PD-L1 low expression. Furthermore, PTCL patients with high expression of both TLR9 and PD-L1 had

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significantly shorter 5-year PFS rate (12% vs. 27%, P=0.022; Fig.2E; Table 2) and

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significantly shorter 5-year OS rate (38% vs. 55%, P<0.001; Fig.2F; Table 2) than

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patients with the single-high and double low expression.

3.5 Univariate and multivariate analysis of TLR9 and PD-L1 expression with

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clinicopathological variables Univariate analysis showed that PD-L1 high expression, Ann Arbor stage (III or

IV), B symptom, elevated leukocyte count, decreased lymphocyte count and elevated serum β2-MG levels were unfavorable prognostic predictors for PFS (P<0.05; Table 3), and PD-L1 high expression, TLR9 high expression, age ≥ 60, Ann Arbor stage (III or

IV), ECOG score ≥ 2, high IPI score, and elevated serum β2-MG levels were unfavorable prognostic predictors for OS (P<0.05; Table 4). Multivariate analysis with PD-L1 expression, Ann Arbor stage, B symptoms, and β2-MG level for OS showed that PD-L1 high expression remained as a significant poor prognostic factor [HR, 2.601;95%CI, 1.507-4.491; P=0.001] (Table 4, multivariate analysis 1). Similarly, multivariate analysis with TLR9 expression and other factors for

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OS indicated that TLR9 high expression were a significant unfavorable prognostic

factor for patients with PTCL [HR, 2.089; 95%CI, 1.136-3.841; P=0.018] (Table 4,

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multivariate analysis 2).

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4. Discussion

In the study, we analyzed the clinical significance of TLR9 and PD-L1 expression

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in patients with PTCL, and demonstrated that the high expression of TLR9 and PD-L1

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were unfavorable prognostic factors for patients with PTCL. Our study involved a relatively large number of patients with PTCL, which will disclose the clinical and

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biological significance of TLR9 and PD-L1 expression in PTCL. Infections and inflammation are important regulators of tumorigenesis and

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progression. TLR9 is an important member of the innate immune system, and ligand binding to TLR9 activates several signaling factors, such as nuclear factor-κB (NF-κB), characterized by increased production of inflammatory mediators [4, 24]. It has been reported that TLR9 is correlated with the invasion and metastasis of human pancreatic carcinoma, and Ntoufa S et al. reported that TLR9 induces cell proliferation and

chemoresistance through NF-κB activation in different B cell malignancies [25, 26]. However, TLR9 expression in patients with PTCL had not been studied. In the study, we found that TLR9 was expressed in tumor cells in patients with PTCL, and TLR9 high expression was associated with poor survival rate in enrolled patients with PTCL. Noteworthy, TLR9 high expression was associated with more risk factors with prognostic significance, including older age, high ECOG score and Ki-67 expression.

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Those may indicate that PTCL patients with high TLR9 expression may have an increased risk of disease progression.

PD-L1 may be inducible expressed in tumor cells and have been described as vital

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“co-inhibitors” of host T-cell immunity by binding to PD-1, which induce resistance to

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killing mediated by cytotoxic T lymphocytes and induction of T-cell unresponsiveness [27]. Previous reports showed that high PD-L1 expression in tumor cells relate to poor prognosis in malignancy, such as breast cancer, gastric

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clinical progression and

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cancer, lung cancer, diffuse large B cell lymphoma (DLBCL), natural killer/T-cell lymphoma (NK/TCL) [11]. In this study, we found that PD-L1 high expression in tumor

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cells was associated with poor survival rate in patients with PTCL. Wilcox et al reported that the expression of PD-L1 in tumor cells suppress T-cell proliferation and play a role

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in immune evasion in T cell–derived non-Hodgkin lymphoma, which is consistent with our results [19]. In addition, some in vitro studies reported that PD-L1 in tumor cells by binding to PD-1 on T-cells negatively regulates T cell synthesis of interleukin-2 (IL2) and interferon-γ (IFN-γ), consequently also leads to apoptosis of CD8+ cytotoxic Tlymphocytes [28]. Those suggested that the PD-1/PD-L1 expression may contribute to

a worse clinical outcome in patients with PTCL. In our study, we further found that TLR9 expression was associated with PD-L1 expression in patients with PTCL. An ‘r’ coefficient is 0.509 by spearman’s correlation test, indicating that the correlations between PD-L1 and TLR9 expression is medium strong positive correlation. Some studies in vitro demonstrated that TLR9 agonist increased immune checkpoint gene expression, such as PD-L1 expression [29]. Liu J et

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al. has been reported that PD-L1 expression was induced by IFN-γ and TLR ligands in isolated multiple myeloma cells plasma cells through MyD88- and MEK-dependent

pathway [30]. This may explain the possible relationship between TLR9 and PD-L1

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expression. In addition, it has been reported that PD-L1 may be inducible expressed in

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tumor cells, and some inflammatory factors, such as Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and granulocyte-macrophage colony stimulating factor

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(GM-CSF), are important factors to induce PD-L1 expression. We speculate that TLR9

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may induce tumor cells to release inflammatory factors and promote PD-L1 expression, which inhibit the function of immune effector cells. This may explain the worse

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prognosis in TLR9/PD-L1 double positive group in patients with PTCL. Future functional analyses are necessary to elucidate the correlation between TLR9 and PD-

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L1 expression.

PTCL are composed of 28 different entities according to 2016 WHO

classification criteria, suggesting the cytological and phenotypic heterogeneity of this group. In this study, we also analyzed the effects of TLR9 and PD-L1 expression on the prognosis of PTCL in different pathological types. Previous literature reports that

ALK+ ALCL express a high level of PD-L1 as a result of the constitutive activation of multiple oncogenic signaling pathways downstream of ALK activity [31, 32]. However, in this study, we found that the rates of high expression of PD-L1 in ALK+ ALCL was 30%, respectively. And Marzec M et al. reported overexpression of PD-L1 in ALK+ ALCL biopsy specimens with a positive ratio of 34–100% in tumor cells of the analyzed cases [33]. We will expand the sample size and further explore the mechanisms that

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influence PD-L1 expression in ALK+ ALCL. Additionally, despite their significant differences in pathologic appearance and clinical presentation, the most common

entities, PTCL-NOS, AITL, ALK+ ALCL, ALK- ALCL, which account for ∼60% of

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cases and tend to be treated similarly. And we found that TLR9 and PD-L1 high

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expression on tumor cells significantly was associated with poor survival rate in enrolled patients with PTCLs. We will further expand the number of cases to prove our

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finding.Moreover, PTCL have been associated with viral infection, particularly

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infection with Epstein-Barr virus (EBV), through whole-transcriptome RNA sequencing (RNA-seq) data and related clinical research [34, 35, 36, 37]. Perhaps

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because of the limitations of retrospective analysis, information on some potentially important clinical variables, e.g. Epstein-Barr virus positivity, are lacking, which would

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have affected PD-L1 positivity rates and outcome. The infection status and clinical value of EBV in various subtypes of PTCL is the direction we will further explore. Furthermore, PD-L1 expression was far more common on tumor-infiltrating monocytederived cells. It has been reported that PD-L1 was expressed on tumor-infiltrating histiocytic cells in 22% to 73% in T-cell lymphoproliferative disorders, and PD-L1,

expressed by immature dendritic cells (iDCs), inhibit T-cell activation and cytokine production [19, 38]. Soluble PD-L1 levels in patients with PTCL have been studied and higher levels is associated with inferior outcomes, in which soluble PD-L1 could be originating from the tumor microenvironment [39]. Therefore, the exploration of PDL1 expression in the tumor microenvironment in patients with PTCL would be a future project.

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In conclusion, we investigated the expression level of TLR9 and PD-L1 in a

relatively large population with PTCL, and suggested that TLR9 and PD-L1 expression

on tumor cells may be used as a biomarker for the prognosis in patients with PTCL. In

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any case, careful clinical studies are needed to determine whether blockade of the PD-

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Conflict of Interest

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to PD-L1 expression patterns.

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1/PD-L1 pathway can be an effective treatment strategy for patients of PTCL according

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The authors declare that they have no conflict of interest.

Compliance with ethical standards

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All procedures performed involving human participants in our study were in accordance with the ethical standards of the Institutional and/or research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. The research was approved by the ethic committee of Harbin Medical University

Cancer Hospital. Informed consent was obtained from all patients and complied with the Declaration of Helsinki.

Acknowledgments The authors thank Dr. Ken H. Young, MD. PhD, department of Hematopathology, the University of Texas MD Anderson Cancer Center, who provided medical guidance and

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input during the conduct of the study and critical review of the article.

The study was supported by University of Texas MD Anderson Cancer Center Sister Institution Network Fund, the Natural Science Foundation of Heilongjiang Province

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(grant number H2018045) and Heilongjiang Provincial Returned Scholars Science

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Fund (grant number LC2015035).

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Fig. 1. Representative immunohistochemical images of PD-L1 and TLR9 expression in PTCL tissues. (A) PD-L1 + tumor cells of PTCL. (B) PD-L1 ++ tumor cells of PTCL. (C) PD-L1 +++ tumor cells of PTCL. (D) + of TLR9 expression; (E) ++ of TLR9 expression; (F) +++ of TLR9 expression. Fig.2. Kaplan Meier analysis for OS and PFS according to TLR9 and PD-L1 expression in PTCL patients. (A, B) Kaplan-Meier analysis for PFS and OS according to TLR9 expression in patients

with PTCL; (C, D) Kaplan-Meier analysis for PFS and OS according to PD-L1 expression in patients with PTCL; (E, F) Kaplan-Meier analysis for PFS and OS according to the expression of both TLR9 and PD-L1 in patients with PTCL.

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Fig. 1. Representative immunohistochemical images of PD-L1 and TLR9 expression in PTCL tissues.

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(Fig.1 original figure)

(Fig.1 revised figure)

Fig 2. Kaplan Meier analysis for PFS and OS according to TLR9 and PD-L1 expression in PTCL patients.

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A

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B

D

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Table 1. Clinicopathologic characteristics of 144 patients with peripheral T-cell lymphomas. TLR9 N (%) (N=144)

59 44

0.051

47 31

38 28

0.744

93 (64.6) 51 (35.4)

26 24

67 27

0.021

55 23

38 28

0.106

17 (11.8) 88 (61.1) 20 (13.9) 19 (13.2)

5 27 9 6

12 61 11 13

0.743

7 44 14 13

10 44 6 6

0.148

116 (80.6) 28 (19.4)

39 11

77 17

0.572

133 (92.4) 11 (7.6)

46 4

87 7

0.905

15 (10.4) 129 (89.6)

5 45

10 84

0.905

39 (27.1) 105 (72.9)

10 40

29 65

63 (43.8) 81 (56.2)

24 26

39 55

127 (88.2) 17 (11.8)

48 2

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35 15

51 15

0.360

69 9

64 2

0.055

12 66

3 63

0.034

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65 13

23 55

16 50

0.480

0.453

29 49

34 32

0.084

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0.163

79 15

0.034

68 10

59 7

0.682

40 (27.8) 56 (38.9) 37 (25.7) 11 (7.6)

18 16 15 1

22 40 22 10

0.088

26 25 21 6

14 31 16 5

0.257

75 (52.1) 69 (47.9)

30 20

45 49

0.165

41 37

34 32

0.900

42 (29.2) 102 (70.8)

13 37

29 65

0.542

19 59

23 43

0.168

9 (6.2) 135 (93.8)

2 48

7 87

0.416

8 70

1 65

0.031

68 (47.2) 76 (52.7)

28 22

40 54

0.124

36 42

32 34

0.780

29 (20.1) 115 (79.9)

8 42

21 73

0.366

12 66

17 49

0.122

46 (31.9) 98 (68.1)

20 30

26 68

0.131

23 55

23 43

0.492

42 (29.2) 102 (70.8)

9 41

33 61

0.032

24 54

18 48

0.646

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P-value Low High (N=78) (N=66)

85 (59.0) 59 (41.0)

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Age (years) < 60 ≥ 60 Gender Male Female Histological type PTCL-NOS AITL ALK(+) ALCL ALK(-) ALCL Primary site Nodal Extra nodal Tumor size < 7.5cm ≥ 7.5cm Extranodal involvement (s) 0-1 >1 Ann Arbor stage I-II III-IV B symptoms Absent Present ECOG score 0-1 2-4 IPI score Low risk Low-intermediate risk Intermediate-high risk High risk LDH level (U/L) < 250 ≥ 250 HGB level (g/L) < 120 ≥ 120 Platelet count (10^9/L) < 100 ≥ 100 Leukocyte count (10^9/L) < 7.2 ≥ 7.2 Lymphocyte count (10^9/L) < 0.7 ≥ 0.7 β2-MG level (mg/L) < 3.0 ≥ 3.0 Ki-67 < 45 ≥ 45

P-value Low High (N=50) (N=94)

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Variable

PD-L1

Abbreviations: PTCL-NOS, peripheral T-cell lymphoma not otherwise specified; AITL, angioimmunoblastic T-cell lymphoma; ALK+ ALCL, anaplasticlymphoma kinas anaplastic large cell lymphoma; ECOG, Eastern Cooperative Oncology Group; IPI, international prognostic index;

LDH, lactate dehydrogenase; HGB, Hemoglobin count; β2-MG, β2-microglobulin.

Table 2. Survival of PTCL patients according to TLR9 and PD-L1 expression. PFS N (%)

OS

5‑year PFS rate

P-value

(95% CI)

5‑year OS rate

P-value

(95% CI) 0.429

TLR9

0.004

Low

50 (34.7)

28% (12.32-43.68)

61% (43.36-78.64)

High

94 (65.3)

18% (6.24-29.76)

31% (19.24-42.76) 0.002

PD-L1 Low

78 (54.2)

31% (13.36-48.64)

High

66 (45.8)

13% (5.16- 20.84)

<0.001 79% (53.52-1.0448) 34% (10.48-57.52) <0.001

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0.022

TLR9&PD-L1 Low *

99 (68.7)

27% (13.28-40.72)

55% (41.28-68.72)

High *

45 (31.3)

12% (2.20-21.80)

38% (24.28-51.72)

Abbreviations: OS, overall survival; PFS, progression-free survival

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*‘High group’ means those patients with both TLR9 high expression and PD-L1 high expression

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together; whereas all other patients with either TLR9 low expression or PD-L1 low expression were classified as ‘Low group’

Table 3. Univariate and multivariate analysis of prognostic factors for PFS in PTCL. Univariate analysis HR (95% CI) Age (years) ≥ 60 vs. < 60

PTCL-NOS vs. AITL +

ALK ALCL vs. AITL -

P-value

HR (95% CI)

P-value

1.467(0.938-2.293)

0.093

1.150(0.618-2.139)

0.660

0.370(0.168-0.812)

0.013

0.817(0.438-1.523)

0.525

0.864(0.503-1.485)

0.598

0.617(0.250-1.523)

0.295

1.209(0.584-2.505)

0.609

1.929(1.177-3.163)

0.009

1.429(0.818-2.499)

0.210

1.646(0.952-2.848)

0.075

1.630(1.059-2.511)

0.026

1.436(0.886-2.326)

0.142

1.284(0.800-2.061)

0.300

1.539(0.812-2.914)

0.186

1.536(0.989-2.385)

0.056

1.317(0.872-1.990)

0.190

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ALK ALCL vs. AITL

HR (95% CI)

0.055

na

Male vs. female

P-value

Multivariate analysis 2*

1.511(0.991-2.303)

Gender Histological type

Multivariate analysis 1*

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Variable

Primary site

Extranodal vs. Nodal Tumour size

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≥ 7.5 cm vs. < 7.5 cm

Extranodal involvement > 1 vs. 0-1

Ann Arbor stage III-IV vs. I-II

B symptoms Present vs. Absent ECOG score 2-4 vs. 0-1 IPI score Higher vs. Lower LDH levels (U/L) ≥ 250 vs. < 250

HGB levels (g/L) < 120 vs. ≥ 120

0.846(0.525-1.362)

0.491

1.458(0.587-3.623)

0.416

1.522(1.003-2.310)

0.049

1.763(1.046-2.970)

0.033

1.746(1.096-2.781)

0.019

0.633(0.401-1.002)

0.051

1.191(0.765-1.855)

0.439

1.918(1.260-2.921)

0.002

Platelet count (10^9/L) < 100 vs. ≥ 100 Leukocyte count (10^9/L) ≥ 7.20 vs. < 7.20 Lymphocyte count (10^9/L) < 0.7 vs. ≥ 0.7 β2-MG level (mg/L) ≥ 3.0 vs. < 3.0

1.743(1.086-2.799)

0.021

1.573(0.977-2.534)

0.062

1.168(0.744-1.833)

0.500

Ki-67 ≥ 45 vs. < 45 TLR9 High vs. Low PD-L1 2.048(1.335-3.142)

0.001

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High vs. Low

Abbreviations: PFS, progression-free survival; HR, hazard ratio; CI, confidence interval; PTCL-NOS, peripheral Tcell lymphoma not otherwise specified; AITL, angioimmunoblastic T-cell lymphoma; ALK+ ALCL, anaplasticlymphoma kinas anaplastic large cell lymphoma; ECOG, Eastern Cooperative Oncology Group; IPI, international prognostic index; LDH, lactate dehydrogenase; HGB, Hemoglobin count; β 2-MG, β2-microglobulin.

*The variables included in multivariate analysis 1 for PFS were PD-L1, Ann Arbor stage, B symptoms, and β2-MG

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level. *The variables included in multivariate analysis 2 for PFS were TLR9, Ann Arbor stage, B symptoms, and β2MG level.

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Table 4. Univariate and multivariate analysis of prognostic factors for OS in PTCL. Univariate analysis Variable

≥ 60 vs. < 60

1.912(1.174-3.112)

0.009

1.697(0.981-2.935)

0.058

Gender

na

Male vs. female Histological type

PTCL-NOS vs. AITL +

ALK ALCL vs. AITL -

ALK ALCL vs. AITL

0.843(0.413-1.722)

0.640

0.192(0.060-0.618)

0.006

0.438(0.198-0.972)

0.042

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Primary site

P-value

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HR (95% CI) Age (years)

Extranodal vs. Nodal

0.704(0.369-1.344)

0.287

0.728(0.264-2.009)

0.540

1.328(0.532-3.318)

0.544

2.111(1.182-3.770)

0.012

1.401(0.853-2.301)

0.183

2.531(1.233-5.197)

0.011

1.962(1.188-3.239)

0.008

1.076(0.662-1.748)

0.768

Multivariate analysis 1*

Multivariate analysis 2*

HR (95% CI)

HR (95% CI)

P-value

P-value

1.528(0.923-2.531)

0.099

1.454(0.867-2.438)

0.156

1.962(1.081-3.560)

0.027

2.194(1.202-4.004)

0.010

Tumour size

≥ 7.5 cm vs. < 7.5 cm

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Extranodal involvement > 1 vs. 0-1

Ann Arbor stage III-IV vs. I-II

B symptoms

Present vs. Absent ECOG score 2-4 vs. 0-1 IPI score Higher vs. Lower LDH levels (U/L) ≥ 250 vs. < 250

HGB levels (g/L) < 120 vs. ≥ 120

0.992(0.580-1.697)

0.978

1.416(0.441-4.547)

0.559

1.532(0.942-2.492)

0.086

1.761(0.986-3.143)

0.056

1.196(0.659-2.172)

0.556

1.416(0.782-2.565)

0.251

2.323(1.317-4.095)

0.004

2.194(1.222-3.939)

0.008

1.726(0.951-3.133)

0.073

1.532(0.942-2.492)

0.086

2.269(1,274-4.038)

0.005

2.089(1.136-3.841)

0.018

2.720(1.591-4.653)

0.001

Platelet count (10^9/L) < 100 vs. ≥ 100 Leukocyte count (10^9/L) ≥ 7.20 vs. < 7.20 Lymphocyte count (10^9/L) < 0.7 vs. ≥ 0.7 β2-MG level (mg/L) ≥ 3.0 vs. < 3.0 Ki-67 ≥ 45 vs. < 45 TLR9 High vs. Low PD-L1 2.601(1.507-4.491)

0.001

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High vs. Low

Abbreviations: OS, overall survival; HR, hazard ratio; CI, confidence interval; PTCL-NOS, peripheral T-cell lymphoma

not

otherwise

specified;

AITL,

angioimmunoblastic

T-cell

lymphoma;

ALK+

ALCL,

anaplasticlymphoma kinas anaplastic large cell lymphoma; ECOG, Eastern Cooperative Oncology Group; IPI, international prognostic index; LDH, lactate dehydrogenase; HGB, Hemoglobin count; β2-MG, β2-microglobulin.

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*The variables included in multivariate analysis 1 for OS were PD-L1, age, Ann Arbor stage, lymphocyte count and β2-MG level. *The variables included in multivariate analysis 2 for OS were TLR9, age, Ann Arbor stage,

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lymphocyte count and β2-MG level.