Original article
Potential role of immune cell genetic variants associated with tumor microenvironment response in laryngeal squamous cell carcinoma (LSCC) in terms of clinicopathological features

https://doi.org/10.1016/j.prp.2021.153665Get rights and content

Abstract

Immunomodulatory signals regulate the self-tolerance, activation, priming and survival processes of T cells. Programmed cell death protein 1 (PD1), Programmed death-ligand 1 (PD-L1) inhibitory signals and CD27, CD28 costimulators have been detected for many solid organ cancers in tumor-infiltrating T cells. It was aimed to investigate the immune cell-based regulatory genetic variants in laryngeal squamous cell carcinoma (LSCC) in terms of clinicopathological features. Genotyping was performed by PCR-RFLP method for PD-1 rs2227981, PD-L1 rs2890658, CD28 rs3116496, CD27 rs2267966 genetic variants from genomic DNAs extracted from peripheral blood samples in One Hundred Thirty-Six individuals (Sixty-one LSCC and seventy-five controls). Analysis of SNPs was carried out according to multiple inheritance models (co-dominant, dominant, recessive, over-dominant and log-additive). There was no difference between LSCC and control groups in genotype/allele distribution for PD-1 and PD-L1 (p > 0.05). In the PD-1 overdominant model, the CT genotype was found to be high (p = 0.036) in those without a family history. The frequency of C allele (AC+CC) in the PD-L1 dominant model was higher in alcohol users and those with reflux (p = 0.024; p = 0.001 respectively). In the Dominant model for PD-L1, the AA genotype was lower in moderately and well-differentiated tumors than in poorly differentiated tumors (p = 0.02). CD27 AT and CD28 CT genotypes were found to be higher in LSCC patients compared to the control group (p = 0.009; p = 0.01 respectively), while linkage disequilibrium (LD) was detected between CD27 and CD28 (p = 0.02). In the CD28 dominant model, C allele (CT+CC) carriage was found to be high in those with family history and in those without reflux and perineural invasion (p = 0.01; p = 0.01; p = 0.03 respectively). In LSCC, PD-L1 rather than PD-1 has a prognostic effect in terms of clinicopathology, and the LD and clinicopathological relationships detected between CD28 and CD27 genotypes suggest that the hereditary immune checkpoint-dependent T cell traffic may be pathophysiologically important.

Introduction

Programmed cell death protein 1 (PD1) is a negative regulatory checkpoint inhibitor protein that is expressed more on tumor-infiltrating CD8(+) T cells than on B cells and monocytes. Although PD-1, which is expressed against most stimuli that under normal conditions, plays a critical role in the termination of the immune response, it causes an insufficient response of the T cell due to long-term exposure to antigen in chronic diseases such as cancer [1]. PD-1 expression in the activated T cell results from the induction of proinflammatory programmed death-ligand 1 (PD-L1) signals. The balance of this co-expression regulates T cell self-tolerance and the resolution process of inflammation [2]. PD-1 expression in the activated T cell results from the induction of proinflammatory PD-L1 signals. The balance of this co-expression regulates T cell self-tolerance and the resolution process of inflammation [2]. It supports the modulation process of effector T cells (Teff), although the function of the T cell is impaired by the PD-L1 signal. The PD-L1/PD-1 interaction reactivates antibody neutralization and cytotoxic T cell-mediated death of tumor cells. Possible imbalance in this co-expression is a critical mechanism for cancer cells to evade the immune response [3].

CD28 and CD27, which are positive inducers of the T-cell receptor (TCR) signaling on T cell, are expressed differently compared to tissue ligation. Thus, T cell preparation, activation and survival processes are modulated. Expressed in approximately 80% of CD4(+) T cells and 50% of CD8(+) T cells, the signal of CD28 can be directly suppressed by phosphorylation by PD-1 [4]. CD27 expressed from effector and naive T cells contributes to T cell differentiation processes through its ablation with CD70. While this signaling leads to optimal anti-tumor response, continuous CD27 signaling predisposes to tumor progression due to T cell depletion and Treg survival being affected [5]. Similarly, in chronic diseases such as cancer, the increase in PD-1 and CTLA-4 expression due to excessively repeated TCR signals causes downregulation of CD28, thus increasing tumor malignancy [6].

In the flow cytometry evaluation of cases with respiratory papilloma, high PD-1 expression was detected in CD4(+) T cells, while higher PD-L1 expression was detected in papilloma tissue compared to normal larynx tissue [7]. Similarly, in the immunohistochemical (IHC) evaluations of cases with nasopharyngeal tumors, high PD-1 expression was detected in CD8 (+) T cells, but no clinicopathological correlation was detected [8]. A positive correlation and dynamic tumor microenvironment were observed between high PD-1 level and tumor progression in laryngeal squamous cell carcinoma (LSCC) [9]. In the in situ hybridization evaluations of LSCC cases, HPV and PD-L1 positive cases were found to have better survival than the other two combinations [10]. While PD-L1 level was found to be higher than tumor-free surrounding tissue in IHC evaluations, it was also found to be associated with cancer stage, histological differentiation and lymph metastasis [11]. Higher PD-L1 mRNA level and correlation with tumor infiltrating lymphocyte density were found in LSCC tumors compared to tumor-free surrounding tissue [12].

PD-1 and PD-L1 gene polymorphisms have been associated with non-small cell lung, cervix, breast, colon and gastrointestinal cancers [13]. In the meta-analysis results of PD-1 rs2227981, which is the most frequently examined in the literature, its relation with general cancer risk was not found, but T allele and CT genotype carriage was found to be a risk factor for digestive system tumors[14]. Carriage of C allele was found to be associated with non-small cell lung cancer, and CT genotype carriage was associated with colon and gastric cancer [15], [16]. It has been observed that there is limited data in the literature reviews we have done in larynx or head and neck cancers (HNC) regarding the PD-1 rs2227981 and PD-L1 rs2890658 variants that we examined in our study. No significant difference was found between head and neck squamous cell carcinomas (HNSCC) group and the control group of PD-1 gene variants, including rs2227981. On the other hand, haplotypes created from these variants are significantly higher in the patient group than in the control group [17]. For PD-L1 rs2890658, the AC genotype was associated with breast cancer and esophageal squamous cell carcinoma, while the CC/AA genotypes were associated with higher survival in hepatocellular carcinoma and non-small cell lung cancer [18], [19], [20], [21].

Peripheral blood flow cytometry results in laryngeal carcinoma show that CD28 level is lower in the preoperative period compared to the postoperative period and control group [22]. It has been determined that CD8(+) CD28(+) T cells and CD4 (+) CD28(+) T cells are low in HNC [6], [23]. Correlation of CD28(+) T cell and Treg ratio with treatment response and high pd-1 expression were detected in patients with HNC [24]. In IHC evaluations, CD70 expression was correlated with tumor differentiation and tumor infiltrating lymphocyte density [25]. While CD27 expression was not detected in tumor tissue in 67 HNSCC cases, its expression was detected in the tumor microenvironment, and CD27 levels were significantly decreased after chemotherapy compared to before [26].

It is seen that CD27 gene polymorphisms have not been examined in the literature both in laryngeal or head and neck cancers, and in cancers in general, except for the study of Xu et al. In this study, the AT genotype of CD27 rs2267966 was found to be a risk-reducing factor in breast cancer, with a high frequency in the estrogen/progesterone negative and without lymphatic metastasis groups [27]. Although CD28 gene polymorphisms are widely studied in solid cancers, there are limited number of studies in laryngeal or head and neck cancers. The meta-analysis results of studies for rs3116496 on breast cancer, hepatocellular carcinoma, non-small cell lung cancer, and renal cell carcinoma generally showed that the frequency of T allele in cancers was lower than the frequency of C allele. It has been determined that this difference is not particularly important for the Asian population, but not for the European population [28]. Apart from the study in which combined genotypes including GT genotype for CD28 rs3116487 variant in oral cavity squamous cell carcinoma were associated with disease risk [29], no study was found in the literature for intronic variant CD28 rs3116496 and other CD28 variants in LSCC.

We aimed to examine the relationship between genotypes and haplotypes of PD-1, PD-L1, CD28, CD27 as T cell regulatory polymorphisms, disease risk and clinicopathological factors, since studies of immunotherapy and tumor microenvironment cancer biology in LSCC are limited in the literature.

Section snippets

Patients

The study design, protocols and informed consent was approved by both the Haydarpasa Numune Hospital Clinical Research Ethics Committee (Decree No. HNEAH-KAEK- 2017/KK/06). While the patient group was formed with LSCC patients from Haydarpaşa Numune Hospital Otorhinolaryngology/Head and Neck Surgery Clinic with HPV negative (HPV positives excluded) and pre-treatment period, the control group was selected from healthy individuals who did not have a three-generation cancer history. Smoking,

Results

While the mean age in LSCC was 62.22 ± 7.8 years, the mean age in the controls was 61.19 ± 7.6 years. The genotype distributions of PD-1, PDL-1, CD28 and CD27 according to the genetic models compared to the LSCC and control groups are shown in Table 2. No significant difference was found between LSCC and control groups for PD-1 and PDL-1 in all genetic models (p > 0.05). A significant difference was found between genotypes in the codominant model for CD28 (p = 0.013). In the recessive model for

Discussion

As a general approach, the state of immune mediators related to the tumor and tumor microenvironment in LSCC, as in almost all cancer types, contributes to supporting the antitumoral effect or tumor malignancy. After molecular characterization of PD-1 and PD-L1 in cancer, anti-PD-1/PD-L1 therapy started to be used in solid organ cancers due to the mechanics of blocking the immunomodulatory signal in lymphocytes or cancer cells. Although it has been determined that the combined use of

CRediT authorship contribution statement

Cem Horozoglu: Investigation, Formal analysis, Writing – review & editing. Dilara Sonmez: Investigation, Writing – review & editing. Seyda Demirkol: Investigation. Mehmet Tolgahan Hakan: Investigation. Islim Kaleler: Investigation. Ceylan Hepokur: Investigation. Aysegul Verim: Resources. Ilhan Yaylim: Conceptualization, Resources, Supervision, Formal analysis.

Acknowledgements

The present study was supported by a grant from the Scientific Research Projects Coordination Unit of Istanbul University (BYP-2019-32095). We would like to thank Biruni University Faculty of Medicine 3th term students Mrs. Hazal Karadag and Mrs. Asli Yildiz who assisted in tabulating the statistical data and drafting this manuscript.

Conflict of Interest

The authors declare that they have no competing interest.

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