Evaluation of Immune-Biomarker Expression in High Grade Soft Tissue Sarcoma : Emphasis of importance of HLA-DQA1 expression as a prognostic marker

Background: High-grade soft tissue sarcoma (STS) is a highly malignant neoplasm having an overall poor prognosis. Numerous prognostic factors determine tumor progression and patient outcome. Many immune-related cells identified in the tumor microenvironment play important roles in various tumor types. This study was undertaken to evaluate the expression of immune-related genes and to elucidate the correlation between these genes and prognosis in high grade STS. Methods: Twelve cases of formalin-fixed paraffin embedded tissue samples of high grade STS were included for gene expression analysis using the NanoString nCounter® System and 35 cases were used for immunohistochemistry. For comparison analysis, the patients were divided into two groups, depending on the overall survival (OS). Expressions of 770 Genes were analyzed using the nCounter® PanCancer Immune Profiling Panel of the NanoString nCounter® Analysis System. Immunohistochemistry for the most significantly altered genes was additionally performed. The correlation between gene expression and prognosis of high grade STS was then evaluated. Results: Of the 770 immune-related genes analyzed by NanoString nCounter® Analysis, several genes were identified as differentially expressed between the two groups. Based on the gene expression level and fold change, representative 13 genes were identified: 7 of the 13 candidate genes (C3, CD36, DOCK9, FCER2, FOS, HLA-DRB4, and NCAM1) were found to be significantly overexpressed in the poor prognostic group. In contrast, expressions of the other 6 immune-related genes (BIRC5, DUSP4, FOXP3, HLA-DQA1, HLA-DQB1, and LAG3) were increased in the good prognostic group. Positive expressions of HLA-DQA1, HLA-DQB1, and HLA-DRB4 were obtained in 74.3%, 34.3% and 48.6% tumors, respectively. The positive expression of HLA-DQA1 was associated with a significantly longer OS (p=0.028). . Conclusions: Analysis of gene expression determined that expressions of 13 immune-related genes were significantly different between two groups. HLA-DQA1 and HLA-DQB1 from the Nanostring nCounter® system. Some immune-related genes were identified, and immunohistochemistry was carried out to validate the prognostic significance of HLA-DQA1, HLA-DQB1, and HLA-DRB4. Our results indicate that the expression of HLA-DQA1 is significantly related to long-term survival, suggesting the potential to be used as an immune biomarker of good prognosis in high grade STS.


Background
Soft tissue sarcomas (STS) are rare malignant tumors that account for 1% of adult cancers. They are a heterogeneous group, comprising more than 50 different types [1]. The diagnosis of STS is based on the morphologic pattern and immunohistochemistry (IHC), in addition to the traditional hematoxylineosin (H-E) staining. An increasing number of specific genetic aberrations, including chromosomal translocations, gene amplification, and mutation have been identified in various types of sarcoma.
These findings led to the recommendation of conducting molecular tests such as reverse transcription polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and next generation sequencing (NGS) [2,3]. Histologic diagnosis is important in predicting the STS outcome. Sarcoma grading is also widely used as a predictor for outcome of the majority STS and several different grading systems have been developed [4,5]. Irrespective of the system used, high grade STS has a higher rate of metastasis and lower survival rate, and their identification may influence clinical decisions made during primary tumor treatment [6]. Although surgical resection is the primary treatment method, conventional chemotherapy is generally used in patients with high grade and/or advanced stage STS. In high-risk patients, the tumor is not completely resected and the prognosis is poor, despite additional treatment. Patients are also reported to suffer from treatment toxicity [7].
These data emphasize the urgent need for new therapies [8].
Many cells related to the immune system detected in the tumor microenvironment show genetic mutations that are essential for prognosis or treatment of the different tumor types [9]. Recent studies show that immunotherapy is widely used to effectively treat solid tumors. The FDA has also recently approved pazopanib, a multi-tyrosine kinase receptor inhibitor, after a study demonstrated that the treated group for STS showed an increased progression-free survival compared to the placebo group [8]. Immune checkpoint inhibitors targeting the PD-1/PD-L1 signaling axis are being explored as a new treatment modality in STS, and the evaluation of PD1-and PD-L1-positivity in STS could be possible criteria for selecting suitability of patients for PD1-based immunotherapy [10,11].
Analyzing immune-related genes of STS in the tumor microenvironment could consequently be helpful in identifying useful validated markers for predicting the prognosis and management of STS. The present study evaluated the expressions of immune-related genes in high grade STS using the NanoString nCounter® PanCancer Immune Profiling Panel, that is a novel multiplex gene expression   panel designed to quantitate 770 genes from different immune cell types and populations covering   both adaptive and innate immune responses, common checkpoint inhibitors, tumor-specific antigens, and housekeeping genes and applied to identify tumor-specific immune targets [12]. The selected genes were subsequently evaluated for clinicopathological significance through additional immunohistochemistry using paraffin blocks of the high grade STS.

Materials And Methods Patients and tissue samples
High grade STS tissue samples were obtained by surgical resection between January 1998 and December 2013, performed at the Pusan National University Hospital, Busan, Korea. Twelve formalinfixed paraffin embedded blocks were selected for gene expression analysis using the NanoString nCounter® System and 35 cases were subjected to immunohistochemistry. Diagnoses were confirmed by pathological analysis using the diagnostic criteria defined in the World Health Organization (WHO) classification and histologic grade was evaluated according to the French The mRNA data anaylsis was performed using the freely available nSolver™ analysis software (NanoString Technology), with the mRNA profiling data normalized using housekeeping genes. R software was used for the analysis. Changes greater than 2-fold upregulation or downregulation were considered significant. The genes greater than 2 fold change and p-value < 0.05 between the two groups were selected. The heat map of gene expression for differently expressed genes between the good and poor prognostic group was then plotted.

Immunohistochemical Staining And Analysis
Sections were transferred to poly-L-lysine-coated glass slides, dewaxed in xylene, and rehydrated in ethanol. Staining was performed using the BondMax autostainer and reagents (Vision Biosystems). HLA-DQB1, and HLA-DRB4 if CPS was 1 or more, and was considered as negatively expressed when was less than 1 [13].
Statistical analysis was conducted using the SPSS 22.0 software (SPSS, Chicago, IL, USA). The associations between overall survival and expressions of HLA-DQA1, HLA-DQB1, and HLA-DRB4 were assessed using Pearson's χ2 test, and calculated using the KaplanMeier logrank test. P < 0.05 was considered to indicate a statistically significant difference.

Results
NanoString nCounter® PanCancer Immune Profiling The 12 patients selected for gene expression analysis were aged 50 to 86 years (average 56 years).
Patient follow-up was done from the date of surgery until death or last visit to the hospital. The followup period ranged from 4 to 122 months (mean 68.3 months). Totally, 7 patients survived during the follow-up period and were classified as the good prognostic group, while 5 died of disease and were classified as the poor prognosis group.
Nanostring nCounter® Analysis was performed for all samples, and gene expressions were compared between the two groups. In the 770 immune-related genes panel of the nCounter® Analysis, a number of genes were found differentially expressed in the two groups, and 13 representative genes were selected based on gene expression levels and fold changes (Fig. 1). Of the 13 candidate genes, 7 genes (HLA-DRB4, NCAM1, CD36, CD3, FOS, FCER2, DOCK9) showed a significant increase in the poor prognostic group (Table 1). In contrast, the expressions of 6 immune-related genes (HLA-DQA1, HLA-DQB1, LAG3, FOXP3, BIRC5, DUSP4) were increased in the good prognostic group ( Table 2). The    Table 3.

Discussion
STSs have a mortality rate ranging from 40-60% for high-grade lesions. The predictors of survival time in patients with high grade STS include tumor size, histology, grading, margin status at resection, and the presence of pre-surgical metastasis [4]. Standard treatment of high grade STS includes surgical resection, with radiation therapy as a supplementary treatment and chemotherapy, if required. Based on the detected genetic mutations, target therapy may also be performed [14,15].
A recent study reports that co-administration of the monoclonal antibody Olaratumab, which acts against the platelet-derived growth factor receptor alpha (PDGFRα), and the traditional chemotherapy agent doxorubicin, increases the overall long-term survival rate [16]. A better understanding of prognostic factors is required to apprise patients for prognosis and treatment regimens.
A few studies have reported immune-response and action of immune checkpoint inhibitors in STS [8][9][10][11]. Recent years has seen the emergence of programmed death-1 (PD-1) and programmed death- The current study investigated expressions of diverse immune-related genes in high grade STS according to the prognosis, and further examined the correlation between immune-related genes and survival using formalin-fixed, paraffin embedded material. This was achieved on the fully automated and highly precise Nanostring nCounter® system used for gene expression analysis, which accurately detects and counts 770 transcripts from 24 different immune cells, including adaptive immune response, innate immune response, common checkpoint inhibitors, tumor-specific antigens, and housekeeping genes, using a small amount of mRNA based on the digital color-coded barcode technology [12]. The Nanostring nCounter® Sarcoma Fusion CodeSet has been recently introduced in the STS research area to assess fusion transcripts using formalin-fixed, paraffin-embedded material [17]. The custom-designed NanoString nCounter® based sarcoma assay is highly sensitive and specific in a clinical setting for sarcoma molecular diagnosis.
Our results reveal significant genetic variations between the survival group (good prognosis group) and DOD group (poor prognosis group). The criteria were applied to select an important gene, having a p value < 0.05 and a 2-or more fold change between the two groups. In all, 13 genes were selected; 7 immune-related genes (C3, CD36, DOCK9, FCER2, FOS, HLA-DRB4, and NCAM1) were significantly increased in the poor prognosis group. In contrast, the expression of 6 immune-related genes (BIRC5, DUSP4, FOXP3, HLA-DQA1, HLA-DQB1, and LAG3) were increased in the good prognosis group. NCAM1 (neural cell adhesion molecule 1) encodes a cell adhesion protein which is a member of the immunoglobulin superfamily. The encoded protein is reported to be involved in the expansion of T cells and dendritic cells, which play an important role in immune surveillance. Some reports that NCAM1 is related to therapeutic resistance in cancer [18,19]. CD36 plays a role in immune signaling as well as a scavenger receptor for fatty acid uptake that modulates cell-to-extracellular matrix attachment and TGF activation. CD36 has increasingly emerged as a prognostic marker associated with the metastatic process [20]. Complement C3 is useful in the development of novel strategies to improve the effectiveness of cancer immunotherapy [21]. FCER2 (Fc fragment of IgE receptor II) encoded by this gene is a B-cell specific antigen, and has essential roles in B cell growth and differentiation, and the regulation of IgE production, although it remains unknown how this gene plays a role in the development or treatment of cancer [22]. DOCK (decicator of cytokines) are a family of guanine-nucleotide exchange factors (GEF) for Rho GTP ases and FOS is a subunit of AP-1 transcription factor. Their function are not well understood in the function of immune system and cancer.
BIRC5, also called survivin, is a well-known cancer therapeutic target. BIRC5 covers a numerous mechanisms of action in immune responses as well as in molecular cancer diagnostics and therapeutics [23]. Survivin peptide immunogen-reactive antibodies are considered to exert an additional advantage for survivin immunotherapy. Survivin-specific T-cell reactivity strongly correlates with tumor response and patient survival [24]. This study revealed reduced levels of survivin in the poor prognosis group, hence, the possibility as a therapeutic target need to be considered in cases of high grade STS. FOXP3 (forkhead box P3) is a member of the forkhead/winged-helix family of transcriptional regulators and is associated with T cell function. Regulatory T cells expressing the transcription factor FOXP3 have a critical role in the maintenance of immune homeostasis and prevention of autoimmunity and cancer pathogenesis [25]. Due to their ability to suppress selfantigen responses, regulatory T cells with FOXP3 may have anti-tumor immune function, whereas increased FOXP3 expression in tumor tissue is reported to be associated with a better prognosis in some cancers. This discrepancy was identified particularly in colorectal cancers [26,27]. In the current study, FOXP3 expression was increased in the good prognosis group and decreased in the poor prognosis of high grade STS. These findings underline the need for accurate assessment of

Conclusion
We believe that this study is meaningful in research areas of immune-biomarker expressions of high grade STS. In spite of using formalin-fixed, paraffin embedded materials, good results were obtained from the Nanostring nCounter® system. Some immune-related genes were identified, and immunohistochemistry was carried out to validate the prognostic significance of HLA-DQA1, HLA-DQB1, and HLA-DRB4. Our results indicate that the expression of HLA-DQA1 is significantly related to long-term survival, suggesting the potential to be used as an immune biomarker of good prognosis in high grade STS.