PD-L1 Expression and Tumor Infiltrating Lymphocytes in Neurofibromatosis Type 1-Related Benign Tumors and Malignant Peripheral Nerve Sheath Tumors:An Implication for Immune Checkpoint Inhibition Therapy

Jin LIU ,Haibo LI ,Chengjiang WEI ,Qingfeng LI ,Zhichao WANG

1 Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China

2 Xiangya Medical College of Central South University,Changsha,Hunan 410013,China

3 Department of Plastic and Reconstructive Surgery,Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China

# These authors contributed equally to this manuscript.

ABSTRACT Background Neurofibromatosis type 1 (NF1) is an autosomal dominant inherited disorder.It can affect multiple systems of the body and cause severe disfigurement and discomfort in these patients.There are two types of neurofibromas,named cutaneous and plexiform neurofibromas.The latter type may transform into malignant peripheral nerve sheath tumors (MPNSTs).Surgical resection is difficult to perform owing to the complex tissue structure of neurofibromas;therefore,it is necessary to develop novel and effective therapies for the treatment of these tumors.Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1)-related immune checkpoint inhibitors have been proven effective for various cancers,and the positive expression of PD-L1 and tumor-infiltrating lymphocytes (TILs) has been recognized as a biomarker for the response to immune checkpoint therapy.Methods We conducted immunohistochemistry (IHC) staining to detect PD-L1 expression in plexiform neurofibroma and MPNST tissue samples.Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were performed to detect PD-L1 and PD-1 expression in MPNST cell lines.IHC staining was used to show immune cell infiltration in NF1 and MPNST tissues.Results IHC staining showed PD-L1 positive expression in neurofibromas and MPNST tumor tissues.In addition,qPCR and western blotting showed high expression of PD-L1 in MPNST tumor cells.IHC staining revealed that aberrant T lymphocytes infiltrated the plexiform neurofibroma and MPNST tumor tissues.Conclusion These results indicate that immune checkpoint mechanisms may play a pivotal role in the development of NF1-related tumors,and immune checkpoint inhibitors may be effective for managing neurofibromas and MPNSTs.Combined therapy with other molecular agents may be explored in the future.

KEY WORDS Neurofibromatosis type 1;Malignant peripheral nerve sheath tumor;PD-L1;Tumor-infiltrating lymphocytes;Immune checkpoint inhibition

INTRODUCTION

Neurofibromatosis type 1 (NF1) is an autosomal dominant inherited disorder affecting one in 2 000 to one in 4 000 people worldwide[1].It mainly affects the nervous system,skin,eyes,and bone and can cause severe disfigurement and discomfort in patients.NF1 patients are often diagnosed in early childhood through the presence of café-au-lait spots (observed in 99% of NF1 patients) and may develop neurofibromas in adulthood[2].Neurofibromas has two types:cutaneous neurofibromas and plexiform neurofibromas[3].Cutaneous neurofibromas are mostly present on or beneath the skin,whereas plexiform neurofibromas are characteristically hyper-proliferative with enlarged tumors that can affect entire body sections and encroach on vital organs and structures[2].In addition,plexiform neurofibromas may transform into highly invasive peripheral nerve sarcomas called malignant peripheral nerve sheath tumors (MPNSTs),with a possibility of approximately 10%[2].As an autosomal dominant inherited disorder,NF1 is characterized by NF1 gene mutation,which results in the loss of neurofibromin.The key role of neurofibromin,which is classically known as a GTPase activating protein (GAP) in the RAS,has been widely accepted in the pathogenesis of NF1.Loss of neurofibromin could lead to increased cell growth and survival through hyperactivation of RAS[4],which then transmits its growth-promoting signal through the V-Akt murine thymoma viral oncogene homolog (AKT)-mechanistic target of rapamycin (mTOR) and mitogen-activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) effector pathways[5].These patients may suffer through their lives;therefore,several measures have been attempted to manage this syndrome.Currently,the common methods for treating NF1 include surgical removal,laser ablation (for small lesions),electrodessication,and emollients (moisturizers).However,the tumor tissue of NF1 can be rich in blood vessels and nerves,especially in the case of plexiform neurofibromas,making the removal surgery complex and dangerous.In recent years,researchers have focused on developing targeted therapies for NF1.Several biologically targeted therapies,including imatinib,selective MEK inhibitors,and mTOR inhibitors,have been evaluated in clinical trials[6-8].

Programmed cell death-ligand 1 (PD-L1),also known as B7 homolog 1 or CD274,is a member of the immunoregulatory ligand B7 family[9].PD-L1 is known to induce inhibitory signals through interaction with programmed cell death protein 1 (PD-1) expressed on the cell surface of T cells,which results in the suppression of tumor-specific T cell responses[10].This mechanism plays a vital role in the process of tumor immune tolerance and immune escape.Currently,immune checkpoint inhibitors targeting the PD-1/PD-L1 interaction are effective against different types of malignant tumors,including melanoma,bladder cancer,non-small cell lung carcinoma,lymphoma,and breast cancer[9,11-12].In addition,it is recognized that high expression of PD-L1 and tumor-infiltrating lymphocytes (TILs) are predictive biomarkers for cancer response to immune checkpoint therapy[13-14].Previous reports have indicated that the abnormal immune system may play an important role in the pathogenesis of NF1,particularly T cells and mast cells[15];therefore,whether immune checkpoint mechanism influences the onset and development of NF1 and MPNST remains unknown.In the present study,we aimed to determine the expression of PD-1/PD-L1 in NF1,MPNST,and tumor cells.In addition,we detected immune cell infiltration in NF1-related tumor tissues,suggesting abnormal inflammation in tumor tissues.These results suggest the possibility of immune checkpoint therapy for NF1-related tumors.

MATERIALS AND METHODS

Cell Lines

One human Schwann cell line was purchased from the American Type Culture Collection (ATCC).Five MPNST cell lines (ST88-14,STS26T,T265,S462,and S462TY) were given by Prof.Vincent Keng[16]and Prof.Jilong Yang[17].All MPNST cell lines were derived from NF1 patients,except for STS26T.Cell lines were cultured in DMEM,10% FBS,and penicillin/streptomycin (Gibco,United States) and were tested mycoplasma negative every 3 months.The verification of cell lines was confirmed using short tandem repeat DNA profiling (Applied Biological Materials Inc.,Canada).

Patients and Specimens

Fresh human plexiform NF1 and MPNST tissues were obtained from surgical resection specimens of patients at the Shanghai Ninth People’s Hospital (Shanghai,China).The study was approved by the Ethics Committee of Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine (project ID:SH9H-2019-T163-2),and informed consent was obtained from all patients under institutional review board protocols.

Immunohistochemistry Staining

Tissue sections from NF1 and MPNST patients were dewaxed in xylene and rehydrated with decreasing concentrations of ethanol.After a quick wash,antigens were unmasked and retrieved by microwaves in citrate buffer.The primary antibody was incubated overnight at 4 °C.After incubation with the appropriate biotin-conjugated secondary antibody for 2 h at a routine time,the signal was detected with diaminobenzidine substrate (Vector Laboratories).The sections were counterstained with hematoxylin.The antibodies used were PD-L1 (Abcam,ab205921),CD3 (Abcam,ab16669),CD4 (Abcam,ab183685),CD8 (Abcam,ab217344),and CD20 (Abcam,ab64088).

Quantitative PCR

Total tissue and cell RNA were extracted following the procedure of the RNeasy kit (Qiagen,Canada).Complementary deoxyribonucleic acid (cDNA) was transformed using the PrimeScript RT Master Mix Kit (Takara,Japan).Quantitative PCR was performed on cDNA using SYBR Green System (Applied Biosystems),and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control.Relative expression was calculated using the ΔΔCT method (n=4).

Western Blot

Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime,China).Protein concentrations were determined using bicinchoninic acid protein assay reagent (Beyotime,China).The antibodies used were PD-L1 (Abcam,205921) and GAPDH (CST,2118).Amersham Imager 600 (General Electric Company,Boston,MA,USA) was used to detect the band signals.

RESULTS

Expression of PD-L1 in NF1-Related Benign Tumors and MPNSTs

To investigate the expression of PD-L1 in NF1-related tumor tissues,IHC staining was performed for PD-L1 in plexiform neurofibroma and MPNST tissue samples.Representative PD-L1 IHC staining results are shown in Fig. 1A and 1B.High expression of PD-L1 was detected in NF1 and MPNST tissues.Thereafter,RT-PCR and western blotting were performed to detect PD-L1 and PD-1 expression in MPNST cells.Five MPNST cell lines were detected,and one normal Schwann cell line was used as a control (Fig.1C-1D).Consistent with the IHC results,the expression of PD-L1 was significantly higher in the five MPNST cell lines than in normal Schwann cells.The PD-L1 protein levels in 26T,T264,S462,and S462TY cell lines,as shown by western blot,were higher than those of the control,which implied that MPNST tumor cells overexpress PD-L1 gene and immune checkpoint inhibition may be effective for this kind of tumor.In addition,the expression level of the PD-1 gene in MPNST cells showed no specific trend.

Fig.1 Expression of PD-L1 in NF1-related tumor tissues and tumor cells.(A) Representative immunostaining images of PD-L1 in neurofibroma tissue.(B) Representative immunostaining images of PD-L1 in MPNST tissue.(C) Relative PD-L1 and PD-1 mRNA levels in five MPNST cell lines and normal Schwann cell line;**P＜0.05.(D) Relative PD-L1 protein level in five MPNST cell lines and normal Schwann cell line.GAPDH was used as a loading control.

Infiltration of Immune Cells in NF1 and MPNST Tissues

It is widely accepted that PD-L1 induces immune inhibition signaling through its interaction with PD-1 on the surface of T cells,and PD-L1 positive tumors are often infiltrated by abnormal immune cells,which has previously been implicated as another biomarker for the response to immune checkpoint therapy;therefore,we performed IHC staining for CD3,CD8,CD4,and CD20 positive lymphocytes in NF1 and MPNST tumor tissues.Representative PD-L1 IHC staining results are shown in Fig.2.T cells (CD3,CD4,and CD8 positive) were infiltrated in NF1 and MPNST tumor tissues,whereas CD20 positive B cells were only scattered in tumor tissues,implicating a T cell-dominant immune microenvironment in NF1-related tumors.This infiltration of T cells may play a pivotal role in developing NF1-related tumors and suggest that PD-1/PD-L1-based immune checkpoint therapy may be a promising strategy for NF1 and MPNST.

Fig.2 Infiltration of immune cells in NF1 and MPNST tissues. Representative immunostaining images of CD3,CD4,CD8,and CD20 positive lymphocytes in neurofibroma and MPNST tissue. MPNST,malignant peripheral nerve sheath tumor.

DISCUSSION

NF1 is an autosomal dominant inherited disorder characterized by mutations in the NF1 gene[18].Clinical manifestations include neurofibroma,café-au-lait spots,and skeletal and neurological disorders,and the possibility of developing MPNSTs[4].Current management for NF1 and MPNST is surgical resection,which faces many obstacles;therefore,there is an urgent need to develop new therapeutic targets.In recent years,several methods of targeted therapy have been confirmed to be effective and are now under clinical trials,including imatinib,sirolimus,and selumetinib.In the present study,we confirmed PD-L1 expression in NF,MPNST tissues,and MPNST tumor cells.Moreover,IHC staining showed lymphocyte infiltration in NF1-related tumor tissues,indicating an abnormal inflammatory microenvironment in these tumor tissues.Targeting the immune system may be a promising strategy for treating NF1 and MPNST.

PD-L1 is a member of the B7 family of immunoregulatory ligands and is expressed in various types of malignant tumors.It induces inhibitory signals through interaction with PD-1 on the surface of T cells,which is an important mechanism for tumor immune escape.In recent years,PD-1/PD-L1 based immune checkpoint inhibition therapy has been proven effective for several malignant tumors[19];however,no biological agents have been applied in NF1 and MPNST treatment.Based on previous reports,the expression of PD-L1 on tumor cells and TILs is well accepted as a predictive biomarker for the response to immune checkpoint inhibitors[14];therefore,positive expression of PD-L1 in NF1,MPNST,and the infiltration of T lymphocytes indicate a possibility for immune therapy.Previous studies have implicated the key role of T lymphocytes in the development of NF1-related tumors[15,20].An in vitro study showed that the loss of the NF1 gene in T cells resulted in enhanced RAS activation and an increase in the number of both mature and immature T cells;however,the loss of NF1 also resulted in a reduction in proliferation with the T cell receptor and interleukin-2R stimulation[21].In addition,evidence shows that T lymphocytes participate in the immune escape of NF1-related tumors.In MPNST,transporter-activator protein 1 (TAP1),which loads peptide antigens onto major histocompatibility complex (MHC) classⅠmolecules and CD74,which function in the processing and transportation of MHC classⅡmolecules,are both significantly downregulated in Schwann cells,leading to resistance to T cell attack[22].A more recent study focused on all NF1-related tumors and observed a decrease in human leukocyte antigen-A/B/C gene levels by using gene analysis,which also attenuated the function of T cells[23].This study showed PD-L1 expression and T lymphocyte infiltration in plexiform neurofibroma and MPNST,particularly the infiltration of CD8+cytotoxic T cells,which may be another mechanism for T cell-related immune escape.In summary,these results suggest that targeting T lymphocytes and tumor immune checkpoint molecules may be an effective therapeutic strategy for NF1-related tumors.However,this study only showed the positive expression of PD-L1 in NF1-related tumors,and further functional experiments and clinical trials will be needed to explore the exact response of neurofibromas and MPNSTs to immune checkpoint inhibitors.In addition,PD-L1 expression can be regulated by multiple signaling molecules,including MYC,MEK/ERK,phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR),Kirsten rat sarcoma viral oncogene homolog.It has been suggested that combination with relevant molecular targeted therapy may augment the efficiency of checkpoint inhibitors[19].Due to the role of some oncogenic signaling molecules,such as MEK/ ERK and PI3K/AKT/mTOR,which have been implicated in the pathogenesis of NF1-related tumors[2],a combination of immune checkpoint therapy and targeted molecular therapy indicate a bright future for the management of NF1 and MPNST;however,further studies should be conducted.

FUNDING

This work was supported by the grants from the Youth Doctor Collaborative Innovation Team Project (QC201803) of Shanghai Ninth People’s Hospital of Shanghai Jiao Tong University School of Medicine,Shanghai Youth Top-Notch Talent Program (201809004),“Chenguang Program”supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (19CG18) and Science and Technology Commission of Shanghai Municipality (19JC1413),Shanghai Rising Star Program (20QA1405600),Innovative research team of high-level local universities in Shanghai (SSMUZDCX20180700),and Shanghai Municipal Key Clinical Specialty (shslczdzk00901).

ETHICS DECLARATIONS

Ethics Approval and Consent to Participate

This study was approved by the Ethics Committee of the Shanghai Ninth People’s Hospital,Shanghai Jiao Tong University School of Medicine (ID:SH9H-2019-T163-2).All participants provided written informed consent before study enrolment.

Consent for Publication

All the authors have consented to the publication of this article.

Competing Interests

The authors declare that they have no competing interests.The authors state that the views expressed in the article are their own and not the official position of the institution or funder.