HGH1 and the immune landscape: a novel prognostic marker for immune-desert tumor microenvironment identification and immunotherapy outcome prediction in human cancers

ABSTRACT HGH1 homolog, a protein-coding gene, plays a crucial role in human growth and development. However, its role in human cancer remains unclear. For the first time, this study comprehensively evaluated the potential involvement of HGH1 in cancer prognosis and immunological function. To achieve this, data from various databases, including The Cancer Genome Atlas, Genotype Tissue Expression, Cancer Cell Lineage Encyclopedia, Human Protein Atlas, cBioPortal, Tumor Immune Estimation Resource and Immune Cell Abundance Identifier, were collated, as well as from one large clinical study, three immunotherapy cohorts and in vitro experiments. This study aims to elucidate the role of HGH1 expression in cancer prognosis and immune response. Our findings revealed a significant association between increased HGH1 expression and a worse prognosis across various cancer types. Predominantly, copy number variations were identified as the most common genetic mutations. Additionally, HGH1 was observed to not only regulate cell cycle-related functions to promote cell proliferation but also influence autoimmunity-related functions within both the innate and adaptive immune systems, along with other relevant immune-related signaling pathways. Gene set enrichment analysis and gene set variation analysis were used to substantiate these findings. HGH1 overexpression contributed to an immune-deficient (immune-desert) tumor microenvironment, which was characterized by a significant expression of immune-related features such as immune-related gene and pathway expression and the number of immune-infiltrating cells. Furthermore, the correlation between HGH1 expression and tumor mutational burden in four cancers and microsatellite instability in eight cancers was observed. This suggests that HGH1 has potential as an immunotherapeutic target. Immunotherapy data analysis supports this notion, demonstrating that patients with low HGH1 expression treated with immune checkpoint inhibitors exhibit improved survival rates and a higher likelihood of responding to immunotherapy than patients with high HGH1 expression. Collectively, these findings highlight the significant role of HGH1 in human cancers, illuminating its involvement in tumorigenesis and cancer immunity. Elevated HGH1 expression was identified to be indicative of an immune-desert tumor microenvironment. Consequently, the targeting of HGH1, particularly in combination with immune checkpoint inhibitor therapy, holds promise for enhancing therapeutic outcomes in patients with cancer.


Introduction
The incidence and mortality rates of malignant tumors have exhibited alarming increases over the past century, posing a severe threat to global public health [1].Despite notable breakthroughs in cancer treatment in recent years, the prognosis and survival rates for numerous cancer types remain poor [2,3].Recent studies have identified disorders within the tumor microenvironment (TME), particularly the tumor immune microenvironment (TIME), as a principal contributor to malignant tumor progression [4,5].However, in some cases, tumors can manifest as a "tumour immune desert", characterized by several comprised immune cell infiltration [6].This phenomenon fosters an inhospitable microenvironment for immunotherapy and allows tumor cells to evade immune surveillance, often resulting in poor treatment outcomes.Therefore, developing strategies to overcome the challenges posed by tumor immune deserts has emerged as a pivotal focus in cancer research.Investigating the underlying mechanisms of the TME in tumor progression is critical for the development of effective immunotherapeutic interventions to combat this phenomenon.
Human growth hormone (hGH) is a diverse endogenous protein encoded by the GH1 and GH2 genes located on chromosome 17 [7,8].While GH1 is primarily situated in the pituitary gland, GH2 is predominantly expressed in the placenta [9].GH1, a 191 amino acid single-chain protein with two disulfide bridges, accounts for approximately 80%-90% of the total circulating hGH [10].Previous studies have demonstrated HGH1's pivotal role in the regulation of cell growth, development and various metabolic pathways in vivo.For example, the levels of HIF-1 increase in association with the proximal HGH1 promoter region and mimic the reduction in hGH RNA induced by insulin [11].Moreover, it has received FDA approval for the treatment of various human dysfunctions [12,13].The tumor-promoting potential of hGH has been firmly established across multiple cancer types, encompassing breast cancer, colon cancer and prostate cancer.hGH accelerates tumorigenesis through both autocrine and paracrine effects on cancer cells and the TME [14][15][16].In vitro studies have demonstrated that autocrine hGH, identified in human melanoma, exerts direct control over epithelial-mesenchymal transition and invasion [17,18].hGH's mechanistic actions are mediated through its binding to the hGH receptor, thereby promoting the activation of signaling pathways known to drive tumoral progression, including the JAK/STAT pathway and its downstream effectors [19].Our study marks the first systematic exploration of HGH1's role in human cancer and tumor immunity.
In this study, we performed a comprehensive analysis of HGH1 involvement across various cancer types, encompassing HGH1 expression, its prognostic role and genetic alteration status, using integrated multi-omics patient data from multiple databases covering 33 cancer types.Moreover, Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA) revealed that HGH1 is not only involved in tumor progression but also exhibits an association with immune pathways across various tumor types.Finally, we delved into the interplay between HGH1, immune cell infiltration and immune-related gene expression.The presence of an immune-desert TME was indicated by the elevated expression of HGH1.Furthermore, immunotherapy data analysis confirmed that targeting HGH1 improves human cancer immunotherapy outcomes.Thus, this study aimed to investigate the role of HGH1 in human cancer development, progression and tumor immunity, with a view to offer novel insight into anti-tumor strategies.

Data source
Datasets comprising clinical information and gene expression profiles were obtained from The Cancer Genome Atlas (TCGA), Genotype Tissue Expression (GTEx) and Cancer Cell Line Encyclopedia databases.These datasets were downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/).

Collection and validation of clinical samples
Cervical squamous cell carcinoma and endocervical adenocarcinoma tissues were obtained from patients who underwent surgery at the Affiliated Hospital of Nantong University.Our cohort included six pairs of tissues collected between 2018 and 2020.The study received ethical approval from the Ethical Committee of the Affiliated Hospital of Nantong University (2021-K150-01).RNA was extracted from tumor tissues using TRIzol Reagent (Invitrogen), and cDNA was synthesized through reverse transcription using a PrimeScript™ RT Reagent Kit (TaKaRa, Shiga, Japan).Real-time quantitative polymerase chain reaction (qPCR) was performed in triplicate for each sample using a SYBR Premix Ex Taq II Reagent Kit (TaKaRa).The primer sequences for the target genes were as follows: HGH1 forward 5'-CCCGACAGGACATCACGAGT-3', reverse5'-GTCATACACGGCTCTCCTCTCT-3'; GAPDH forward 5'-TGACTTCAACAGCGACACCCA-3', reverse 5'-CACCCTGTTGCTGTAGCCAAA-3'.

Immunohistochemical staining
The Human Protein Atlas (HPA) (http://www.proteinatlas.org/) is a database containing immunohistochemical staining results from a wide range of tumors and normal tissues.We accessed data from HPA to explore the expression and the localization of HGH1 (Cat No. HPA042470; Atlas Antibodies, Sigma-Aldrich).Additionally, single-cell RNA sequencing (scRNAseq) analysis was conducted using publicly available genome-wide expression data encompassing all protein-coding genes across 536 individual cell type clusters corresponding to 15 different cell type groups.A specificity classification was performed to determine the expression of specific genes within these cell types [20].

Genetic alteration analysis
The cBioPortal database (http://www.cbioportal.org/) serves as a powerful tool for cancer genomics research, containing numerous cancer genomics datasets.We utilized this platform to analyze the frequency, mutation types and copy number alterations of HGH1 across various tumors.

Prognostic analysis
Kaplan-Meier analysis was performed to evaluate the overall survival (OS) of patients from TCGA cohort.Univariate Cox regression analyses were conducted to assess the significance of HGH1 in predicting OS, disease-specific survival (DSS), the disease-free interval (DFI), and the progressionfree interval (PFI) in human cancers.The Kaplan-Meier Plotter database, equipped to assess the impact of 54,675 genes on survival using data from 10,461 cancer samples, was employed to validate the correlation between HGH1 expression and survival in cervical squamous cell carcinoma.

GSEA and GSVA
GSEA, a robust analytical method, was employed for the interpretation of gene expression data, identifying statistically significant and consistent differences between different biological states in different groups [21].GSEA was performed using the R package "clusterprofiler", with an adjusted P-value <0.05 considered as statistically significant.Additionally, to assess the relative expression activity of each pathway and conduct a differential analysis of pathway expression activity between samples, we utilized the "ssGSEA" algorithm, based on gene expression data from single samples [22].Data encompassing hallmark pathway gene sets were sourced from the MSigDB database (https://www.gseamsigdb.org/gsea/msigdb/human/collections.jsp), and hallmark pathway scores were computed for various cancer types using the "GSVA" R package.

Tumor microenvironment or infiltration of immune cells
The estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using Expression Data (ESTIMATE) is a method to calculate stromal or immune scores, which represent the abundance of immune and stromal components within tumor tissues, respectively.Higher scores denote a greater proportion of the corresponding component in the TME.The ESTIMATE score and the sum of stromal and immune scores were calculated, which represents the integrated proportion of both components in the TME.Additionally, we assessed the correlation between HGH1 expression and ImmuneScore and StromalScore for each cancer using the' estimate' R package and Spearman's correlation analysis.Immune cell infiltration correlation analysis was performed using the TIMER2 database Patients in each TCGA tumor type were categorized into two groups (high and low HGH1 expression based on median HGH1 expression level) to compare the extent of immune cell infiltration.

Statistical analysis
All statistical analyses were performed using R version 4.1.1.Unless otherwise specified, Pearson's correlation coefficient was used for correlation analysis in this study.In the bioinformatics analysis section, the Wilcoxon test was used for comparison between two groups, while in the experimental section, the Student's t-test was used for difference analysis.Kaplan-Meier survival analysis and log-rank tests were used to compare the survival of different patient groups.A two-tailed p < 0.05 was considered statistically significant.

Expression of HGH1 in human cancers
First, HGH1 expression was evaluated using data from GTEx and TCGA.The analysis revealed that HGH1 was significantly overexpressed in 25 of the 33 cancers assessed, namely adrenocortical carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid neoplasm diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma, lower grade glioma, liver hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, pancreatic adenocarcinoma, prostate adenocarcinoma, rectum adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, testicular germ cell tumor, thymoma, uterine corpus endometrial carcinoma and uterine carcinosarcoma.Conversely, HGH1 was downregulated in three cancer types, namely acute myeloid leukemia, pheochromocytoma and paraganglioma and thyroid carcinoma (Figure 1a and Supplementary Table 1).Furthermore, we observed that HGH1 exhibited its highest expression in uveal melanoma tumor tissues, while its lowest expression was detected in kidney renal clear cell carcinoma tumor tissues (Figure 1b).Among normal tissues, HGH1 expression was highest in the testis tissues (Figure 1c).An overview of single cell type expression is provided in Figure 1d.Importantly, HGH1 displayed immune-related expression across various human tissues, encompassing adipose tissue, bone marrow, kidney, liver, skeletal muscle, breast, lymph nodes, ovary, spleen, stomach, endometrium and placenta.Finally, we demonstrated HGH1's intracellular localization in the endoplasmic reticulum, microtubules and nucleus of HEK293 and U-2 OS cell lines, with HGH1 expressed in all three sites (Figure 1e).To further validate the difference in HGH1 protein expression between normal and tumor tissues, we retrieved immunohistochemical data from patients with cancer in the HPA database.These data showed that HGH1 staining was weak in normal tissues; however, it was strong in the tumor tissues across various cancer types (Figure 2).These immunohistochemistry staining results were consistent with the HGH1 sequencing results at the transcriptome level.Collectively, these findings demonstrated that HGH1 is highly expressed in the majority of cancers.

Genetic alterations
To comprehensively evaluate HGH1 mutations in tumor tissues, we used the cBioPortal platform, analyzing data from 10,953 patients.HGH1 amplification was observed to be the predominant mutation type, with the highest amplification observed in ovarian serous cystadenocarcinoma (26.54%), followed by breast invasive carcinoma (10.42%) and esophageal adenocarcinoma (9.89%).Notably, diffuse large B-cell lymphoma exhibited a large number of deep deletions (Figure 3a), highlighting amplification as the prevailing type of HGH1 mutation.Figures 3b and c illustrate the threedimensional structure of HGH1 and the sites and types of HGH1 genetic alterations.Furthermore, our analysis of the relationship between HGH1 expression and copy number variation (CNV) revealed a significant positive correlation between HGH1 expression and CNV across all human cancer types assessed (Figure 3d).We further analyzed the CNV of HGH1. Figure 3e displays the proportions of various CNV types (including heterozygous amplification, heterozygous deletion, homozygous amplification and homozygous deletion) of HGH1 in the 33 types of cancers assessed.Thus, we identified the substantial presence of CNVs in HGH1.

Prognostic role of HGH1
We employed forest plots to determine the risk ratio statistics for overall survival (OS), diseasespecific survival (DSS), disease-free interval (DFI) and progression-free interval (PFI) for each cancer type included in this study, thereby elucidating the correlation between HGH1 expression and tumor prognosis.According to univariate Cox regression analysis, HGH1 emerged as a significant risk factor for OS in kidney renal clear cell carcinoma, sarcoma, uveal melanoma, breast cancer, skin cutaneous melanoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, mesothelioma, low-grade glioma, kidney renal papillary cell carcinoma, liver hepatocellular carcinoma and glioblastoma multiforme.Additionally, HGH1 was identified as a protective factor for OS in thymoma (Figure 4a, p < 0.05).Moreover, Cox regression analysis of DSS revealed that HGH1 was a risk factor for kidney renal clear cell carcinoma, mesothelioma, kidney renal papillary cell carcinoma, uveal melanoma, skin cutaneous melanoma, sarcoma, low-grade glioma, cervical squamous cell carcinoma and endocervical adenocarcinoma and glioblastoma multiforme (Figure 4b).Notably, high HGH1 expression predicted a shorter DFI in patients with mesothelioma (Figure 4c).Additionally, Cox regression analysis of the PFI revealed that HGH1 detrimentally affected patients with kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, uveal melanoma and mesothelioma (Figure 4d).Given our earlier observation that CNV was the predominant genetic alteration in HGH1-overexpressing cancers, we further examined the relationship between CNV and patient prognosis.Our analysis revealed that CNV significantly affected the OS, DSS, DFI and PFI of patients (Figure 4e).To validate our findings in cervical squamous cell carcinoma, a cancer type characterized by significantly higher HGH1 expression and poor prognosis, we measured HGH1 expression in six pairs of tissues using RT-qPCR.The analysis revealed that HGH1 expression was significantly higher in cervical squamous cell carcinoma surgical specimens compared to normal tissue (figure 4f).Subsequently, analyzing clinical data from 304 patients with cervical squamous cell carcinoma exhibited significantly worse survival outcomes compared to those in the low expression group, corroborating our previous results (Figure 4g).Thus, these results underscore the critical role of HGH1 in the prognosis of cancer.

Biological function
GSEA and GSVA were employed to explore the potential biological relevance of HGH1 in human cancers.Using the "clusterprofiler" package in the GSEA algorithm, we identified biological pathways potentially regulated by HGH1 across six cancers with comparable profiles (Figure 5).Our results indicated that HGH1 is closely associated with cell cycle-related pathways, such as "Cell Cycle", "Translation", "DNA Repair" and "RHO GTPase Effectors".Additionally, we also observed that HGH1 was widely involved in regulating immune-related pathways in human cancers, especially "Adaptive Immune System", "Innate Immune System", "Cytokine Signalling in Immune System", "Neutrophil Degranulation", "Antigen Processing: Ubiquitination & Proteasome Degradation" and "Class I MHC mediated Antigen Processing & Presentation" pathways.Furthermore, to identify signaling pathways potentially affected by HGH1 expression, we performed GSVA scoring based on 50 HALLMARK pathways.Figure 6 illustrates the relationships identified between HGH1 expression and signaling pathways.Thus, our analysis demonstrated that HGH1 significantly inhibited immune-related signaling pathways.
Additionally, an "ESTIMATE" algorithm was used to assess the association between HGH1 expression and stromal and immune scores (Figure 7a), revealing that HGH1 expression significantly influenced the immune score, stromal score and ESTIMATE score across most of the evaluated tumors.To validate these findings, we analyzed a TME-related pathway, which was involved in HGH1 expression, based on immunerelated data from TCGA and a large clinical study [23], including an immune-related pathway, chromophore-related pathway and DNA repair-related pathway.The findings revealed that HGH1 acts as a promoter of oncogenic pathways and inhibits immune-related pathways in human cancers (Figure 7b).Collectively, these results confirmed that HGH1 has a wide range of pro-oncogenic and immunosuppressive effects.

Tumor microenvironment analysis
We subsequently conducted an immune-related gene correlation analysis with HGH1 to further explore the relationship between HGH1 and the TIME.The analysis demonstrated that HGH1 was strongly associated with immunomodulatory genes, including immune activation genes (Figure 8a), chemokine genes (Figure 8b), chemokine receptor genes (Figure 8c) and MHC genes (Figure 8d).Importantly, the high expression of HGH1 was significantly associated with the low expression of most immune-related genes.These observations suggest that high HGH1 expression can often signify a relatively immunosuppressed TME in patients with cancer.
Dysfunctional immune cell infiltration can disrupt the immune system's ability to regulate tumor growth and may even promote tumor progression through immune escape.To verify that high HGH1 expression contributes to immunosuppression in the TME, we analyzed the relationship between HGH1 expression and immune cell infiltration using immune cell infiltration data from the TIMER2 database.Our analysis revealed that high HGH1 expression led to extensive reductions in immune cell infiltration within the TME (Figure 9).These results reaffirm the immunosuppressive role of HGH1 in human cancer and its propensity to create an immune-desert TME.

Immunotherapy response analysis
Microsatellite instability (MSI) and tumor mutation burden (TMB) serve as predictive factors for immunotherapy response in several tumor types.Elevated TMB or MSI tends to increase the likelihood of a positive response to immunotherapy.Our analysis revealed that the expression of HGH1 was associated with TMB in four cancer types and with MSI in eight cancer types (Figure 10a, b).These findings suggest that the expression of HGH1 may influence the response of patients to immunotherapy.
To validate this hypothesis, we collected HGH1 expression data from three groups of patients with various malignancies who had received immunotherapy.Kaplan-Meier analysis of these data revealed that patients with low HGH1 expression who received immune checkpoint inhibitors (ICIs) exhibited better OS than those with high OS who received ICI.Moreover, a higher proportion of patients with low HGH1 expression demonstrated favorable responses to immunotherapy compared to those with high HGH1 expression (Figure 10c- e).These findings underscore the significant impact of HGH1 expression on patients' responses to immunotherapy.

Discussion
Dynamic fluctuations in hGH secretion occur under different physiological and pathophysiological conditions [12,13].Recently, while hGH has been primarily studied in the context of endocrine disorders, its relevance in cancer has remained relatively unexplored [24].This comprehensive analysis aimed to elucidate the expression, prognostic value, biological function and immunological impact of HGH1 in human cancers.
This study initially focused on assessing HGH1 expression and its prognostic significance in various cancer types.HGH1 was found to be overexpressed in the majority of cancers analyzed.Immunohistochemical analysis further confirmed the high levels of HGH1 protein in most of the cancer types and revealed that HGH1 is mainly localized in the nucleus.Overall, these findings confirm the upregulation of HGH1 expression in various cancers, suggesting its potential as a diagnostic marker.Furthermore, HGH1 overexpression was also strongly associated with poor prognosis in a range of malignancies.HGH1 expression in cancer was found to be significantly influenced by CNV, impacting survival outcomes.These findings highlight HGH1 as a promising candidate for predicting cancer outcomes.
Another important finding of this study was the pivotal role played by HGH1 in cancer immunity.Increasingly studies have demonstrated that the evolution of malignant tumors is strongly influenced by the dysfunction of the TME, especially the TIME [25,26].The TIME of malignant tumors accelerates tumor growth, plays a crucial role in tumor progression and metastasis, predicts the clinical outcome of cancers and assesses the response of tumor cells to immunotherapy [27].Moreover, the immune status of a tumor has been closely associated with the composition and level of immune cell infiltration into its TME [28,29].The dysregulation of the cell cycle is also an important hallmark of cancer, leading to increased tumor cell division and proliferation [30].Our  study revealed that HGH1 not only promotes cell cycle-related pathways but also broadly inhibits immune-related pathways and other signaling pathways to promote tumorigenesis and tumor progression.Furthermore, HGH1 overexpression was observed to not only predict a reduced immune score, stromal score and ESTIMATE score in most tumors but also contribute to an immunodeficient TME through the inhibition of immune-related features, including immunerelated pathways, immune-infiltrating cells and immune-related gene expression.These findings suggest a potential association between recombinant hGH treatment and increased cancer incidence and mortality in patients with cancer history, as observed in a European cohort study [31].In contrast, this cohort study did not observe a similar trend in patients with growth failure without cancer history.These results reaffirm the immunosuppressive role of HGH1 in human cancers and indicate that HGH1 overexpression contributes to the development of an immunodeficient TME with an immune-desert phenotype.
High TMB and MSI scores are considered predictive factors for favorable immunotherapy outcomes [32].Current immunotherapy strategies include anti-tumor immune checkpoints such as PD-1 and CTLA-4 blocked by antibodies or small molecule inhibitors to "restart" the antitumor immune response.With the continuous advancements in ICI treatment, its application in tumor immunotherapy is increasing, significantly impacting clinical outcomes [33].Subsequently, we hypothesized that patients with low HGH1 expression could exhibit a positive response to immunotherapy.Therefore, we collected data from three immunotherapy cohorts receiving ICI to evaluate the impact of HGH1 in immunotherapy.Notably, patients with low HGH1 expression treated with ICI had better responses and longer survival rates compared to those with high HGH1 expression.These findings highlight the potential synergy between HGH1 targeting and ICI immunotherapy, emphasizing the potential benefits of combining HGH1-targeting strategies with immunotherapy.
Numerous strategies have been developed to target hGH action in cancer.In general, hGH-targeting strategies include inhibitors of hGH secretion, hGH receptor antagonists and inhibitors targeting hGH signal transduction pathways [34].Among these, peptide receptor antagonists have emerged as the most promising strategy, with pegvisomant exhibiting promise in clinical applications as an hGH receptor antagonist.As a standard treatment for acromegaly, pegvisomant has also demonstrated anti-tumor effects in breast and colorectal tumor xenografts, underscoring the rationale for investigating hGH receptor antagonists further in cancer treatment [35,36].Conversely, hGH has also been implicated in promoting resistance to radiotherapy, whereas pegvisomant treatment has been reported to inhibit endometrial tumor recurrence following radiotherapy [37,38].These findings emphasize the potential of combining hGH and and other relevant receptor-targeting treatment with radiotherapy to enhance the efficacy of cancer treatment.

Conclusion
This study sheds light on the multifaceted role of HGH1 in tumorigenesis and cancer immunity.Moreover, elevated HGH1 expression was identified as an immune-desert tumor microenvironment marker.Thus, targeting HGH1 in combination with ICI therapy holds promise for enhancing therapeutic outcomes in patients with cancer.This study offers valuable insights into the potential of HGH1 as a diagnostic marker and a therapeutic target, presenting novel perspectives for innovative anti-tumor strategies.

Figure 3 .
Figure 3. Genetic alterations of HGH1.HGH1 gene alterations in various cancers.(a) HGH1 gene mutation type analysis in various cancers by cBioPortal.(b) 3D protein structure of HGH1.Colored part means the binding region, while gray means the other part of HGH1.(c) The subtypes and distributions of HGH1 somatic mutations.(d) Correlation between HGH1 expression and CNV in indicated tumor types.(e) Pie plot summarizing the CNVs (including heterozygous amplification, heterozygous deletion, homozygous amplification, and homozygous deletion) of HGH1 in the indicated tumor types.

Figure 4 .
Figure 4. Prognostic Role of HGH1.(a-d) Univariate Cox regression analysis, for overall survival (OS), disease-specific survival (DSS), disease-free interval (DFI), and progression-free interval (PFI), of HGH1 in human cancers (red color represents significant results; P < 0.05).(e) Correlation between HGH1 CNV and OS.(f) The mRNA levels of HGH1 in 6 pairs of clinical samples were confirmed by RT-qPCR.(g) Results of prognostic analysis of HGH1 in the KM database.

Figure 5 .
Figure 5. GSEA of HGH1 in human cancers.The top-20 significant pathways identified through GSEA across the indicated tumor types (red color represents immune-related pathways).

Figure 6 .
Figure 6.GSVA of HGH1 in human cancers.GSVA results of 50 hallmark pathways from the MSigDB.

Figure 7 .
Figure 7.The relationship between HGH1 and regulation of the tumor microenvironment.(a) the heatmap represents the correlation between HGH1 expression and TME scores in human cancers.(b) Relationship between HGH1 and the tumor microenvironment (red represents positive correlation and blue represents negative correlation; darker the color, stronger is the correlation; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

Figure 10 .
Figure 10.Relationship of HGH1 with immune-related genes and immunotherapy.Radar plot of the correlation between HGH1 and (a) TMB or (b) MSI.(c-e) Kaplan-Meier OS analysis of HGH1 and the percentages of responsive and progressive patients in high-and low-HGH1 expression groups in the PMID32472114, GSE91061 Nivolumab Pre, and GSE91061 Nivolumab On cohorts (Abbreviations: CR, complete response; PR, partial response; PD, progressive disease; SD, stable diseae).