FAM83A: A Prospective Diagnostic Biomarker with Immunological Relevance in Lung Adenocarcinoma- Associated Malignant Pleural Effusion

FAM83A: A Prospective Diagnostic Biomarker with Immunological Relevance in Lung Adenocarcinoma- Associated Malignant Pleural Effusion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article FAM83A: A Prospective Diagnostic Biomarker with Immunological Relevance in Lung Adenocarcinoma- Associated Malignant Pleural Effusion Hangfeng Liu, Jia Yao, Yulan Liu, Liping Wu, Zhiwei Tan, Jie Hu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4453916/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Background : Malignant pleural effusion (MPE) is most frequently observed in patients with advanced lung adenocarcinoma (LUAD). Pleural fluid cytology is a less invasive procedure than pleural biopsy. Therefore, it is urgently required to discover new effective biomarkers for LUAD-associated pleural fluid cytology. Methods: mRNA sequencing and clinical data of LUAD were downloaded from TCGA and OncoSG databases. Differential gene expression analysis, survival analysis and immune cell infiltration analysis were carried out on the LUAD datasets. The expression levels of FAM83A, TFF-1, and NapsinA were assessed in 94 pairs of LUAD tumor and adjacent normal tissues, as well as in pleural effusion cell blocks obtained from 40 LUAD and 21 non-neoplastic patients, using immunohistochemistry. Results: Bioinformatics analysis demonstrated that FAM83A was screened out as a candidate biomarker for pleural fluid cytology due to its obvious difference in LUAD tissues and its influence on overall or disease-free survival, and histological grade. Immunohistochemistry showed that FAM83A expression was amplified in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs. Immune cell infiltration analysis revealed that FAM83A expression was significantly correlated with immune cell infiltration, such as a positive association with macrophage infiltration. Meanwhile, FAM83A staining was positive in 37 cases of LUAD pleural effusions, and was negative in 20 cases of non-neoplastic pleural effusions. Additionally, the expression pattern of FAM83A in LUAD pleural effusions was relatively consistent with that of TFF-1 and NapsinA, and was even a little stronger in some LUAD pleural effusions that were weakly positive or negative for TTF1 or NapsinA. Conclusions: Our work reveals FAM83A as a promising immune-related biomarker not only in LUAD biopsy specimens but also in LUAD pleural effusion. Our finding is of importance for providing a new and effective option for pleural fluid cytology. Malignant pleural effusion Lung adenocarcinoma FAM83A Immune infiltration Biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction Lung cancer remains the leading cause of cancer-related mortality worldwide. Lung cancer is classified into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), accounting for 85% and 15% respectively [ 1 ]. Lung adenocarcinoma (LUAD) is the major histological subtype of NSCLC [ 1 ]. Malignant pleural effusion (MPE) is a common clinical complication of malignancies, with a rate of 10% ~ 15% in advanced NSCLC in initial diagnosis and a higher rate in later treatment course [ 2 ]. Once the diagnosis of MPE in NSCLC is confirmed, patients will be classified into advanced TNM stages with a median overall survival of 5.5 months [ 3 ]. Thus, the distinction between MPE and benign pleural effusion is the first and critical step for early initiation of clinical intervention and adequate management. Unfortunately, some NSCLC patients with MPE have undefined diagnosis due to negative cytology. Therefore, it is essential to explore effective means to achieve global control of NSCLC patients with MPE. The diagnosis of malignant pleural effusion is mainly made by pleural fluid cytology and pleural biopsy. Pleural biopsy is an invasive procedure that may result in various complications including hemorrhage and infection. By contrast, pleural fluid cytology is a less-invasive tool. However, pleural fluid cytology still lacks enough sensitivity or specificity, which has not yet supplanted pleural biopsy. In recent years, some significant advances towards the use of biomarkers in pleural fluid cytology have been achieved. Some biomarkers like lactate dehydrogenase (LDH), carcinoembryonic antigen (CEA), carbohydrate antigen 153 (CA153) and adenosine deaminase (ADA) have been commonly used in pleural fluid [ 4 – 6 ]. In additional, numerous studies investigated the possibility of other novel biomarkers to improve the diagnostic ability of pleural fluid cytology, such as reactive oxygen species modulator 1 (ROMO1), hyaluronic acid (HA), chitinase-3-like protein 1 (YKL-40), thymidine kinase 1 (TK1), pro-cathepsin D, and programmed death-ligand 1 (PD-L1) [ 7 – 12 ]. To our knowledge, there is no consensus as to which biomarker is most effective to distinguish pleural effusion. Hence, it is urgently required to discover new effective biomarkers for differentiating malignant from benign pleural effusion. Using differentially expressed genes from mRNA sequencing data of LUAD tumors and para-cancerous tissues in TCGA database as an entry point, in this study, we screened new candidate biomarkers, which may have a favorable discrimination ability for the properties of pleural effusion. The diagnostic value was verified not only in tissue specimens but also in pleural effusion. With this work, family with sequence similarity 83 member A (FAM83A) stood out as a potential diagnostic biomarker in differentiating malignant from benign pleural effusion in LUAD patients. Moreover, FAM83A may have a novel immunomodulatory function during the development of LUAD. 2 Materials and methods 2.1 Bioinformatics analysis of FAM83A expression profile and clinical features in patients with LUAD RNA sequencing and clinical data of LUAD was downloaded from cBioPortal for Cancer Genomics ( http://www.cbioportal.org ). The data contains 4 datasets of LUAD-1 (OncoSG, Nat Genet 2020), LUAD-2 (TCGA, Firehose Legacy), LUAD-3 (TCGA, Nature 2014) and LUAD-4 (TCGA, PanCancer Atlas). LUAD-2, LUAD-3, and LUAD-4 datasets from TCGA were integrated, consisting of 517 LUAD tumors and 59 adjacent normal tissues. LUAD-1 dataset contains a list of 169 Chinese LUAD samples with clinical information. The DESeq2 R package ( https://bioconductor.org/packages/release/bioc/html/DESeq2.html ) was used to detect differentially expressed genes (DEGs) between 517 LUAD and 59 normal samples. The cut-off criterion of DEGs was adjusted p value < 0.01 and |log2 fold change| ≥1. The difference in overall survival or disease-free survival was assessed between LUAD patients with high FAM83A expression and those with low FAM83A expression by the log-rank test. The hazard ratios with 95% confidence intervals and log-rank P-values were calculated and displayed on the plot. The difference in FAM83A expression was compared between well, moderately, or poorly differentiated group by student's t test in 169 Chinese LUAD samples. 2.2 Correlation analysis of FAM83A expression and immune cell infiltration Based on the gene expression profile of 517 TCGA-LUAD samples, the immune cell infiltration of each sample was evaluated with the immunedeconv R package ( https://grst.github.io/immunedeconv ). immunedeconv was an R package that integrates six latest algorithms, including TIMER, CIBERSORT, EPIC, MCP-counter, quanTIseq, and xCell. Spearman's correlation coefficient analysis was used to investigate the correlations among different types of immune cells and to assess the association between FAM83A expression and the levels of infiltration of each immune cell type. Correlation analysis and visualization were conducted using the ggClusterNet R package ( https://github.com/taowenmicro/ggClusterNet/ ). p value < 0.05 considered statistically significant. 2.3 Patient information and clinical samples This study protocol related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital. Informed consent was obtained for experimentation with human subjects. The clinical samples were divided into two parts, which were collected at the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital. The first part was primary tumor specimens and paired normal lung tissues from 94 LUAD patients, who underwent surgical treatment. All patients in the first part met the following inclusion criteria: (a) Patients with a definite diagnosis of lung adenocarcinoma; (b) No other malignant tumors; (c) Patients at clinical stage I-IV classified by TNM classification of American Joint Committee on Cancer (AJCC); (d) Patients with complete clinical and pathological information including age, sex, TNM stage, and clinical stage. The second part consisted of pleural effusion from 40 LUAD patients and 21 non-neoplastic patients. Inclusion criteria for LUAD pleural effusion: (a) Pathological examination confirmed that the surgical tissue specimen was lung adenocarcinoma; (b) No other malignant tumors. Inclusion criteria for non-neoplastic pleural effusion: (a) Patients with no malignant tumors; (b) no cancer cells in pleural effusion. 2.4 Immunohistochemistry (IHC) Tissue specimens were fixed in 4% phosphate-buffered paraformaldehyde and subsequently embedded in paraffin. Fresh pleural effusion was placed for 20–30 mins, then the precipitate was collected and centrifuged at 2500 rpm for 5 mins. After discarding the supernatant, the precipitate was resuspended and centrifuged in 75% alcohol and 95% alcohol respectively. Subsequently, the precipitate was fixed in 10% formalin for 2–4 hours. Finally, well-formed cell pellet was transferred to a dehydrator for final processing with the same procedure used for the histology tissue. Immunohistochemistry was performed on three µm sections of paraffin-embedded tissues or pleural fluid cell pellet. Following antibodies were used in immunohistochemical staining: anti-FAM83A (Proteintech, Chicago, #20618-1-AP), anti-TTF1 (MXB®Biotechnologies, Fuzhou, China, #MAB-0677) and anti-Napsin A (MXB®Biotechnologies, Fuzhou, China, #MAB-0704). After using diaminobenzidine (DAB) substrate and counterstaining with hematoxylin, the target signals were observed using an Olympus BX53 microscope. Rabbit pre-immune serum was used as the primary antibody for the negative control. Staining intensity was scored as follows: 0 = negative staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. Patients with scores of 0 and 1 were classified in the low expression group, and patients with scores of 2 and 3 were classified in the high expression group. 2.5 Statistical analysis SPSS software (version 26.0; IBM, Chicago, IL) was used for statistical analysis. Chi-square test was used to analyze the correlation between clinicopathological characteristics and FAM83A expression. P value < 0.05 was statistically significant. 3 Results 3.1 FAM83A was a potential diagnostic and prognostic biomarker in LUAD To screen new diagnostic biomarkers for differentiating malignant from benign pleural effusion in lung adenocarcinoma (LUAD), we first aggregated RNA sequencing data of LUAD tumors and para-cancerous tissues from TCGA database, including 517 tumors and 59 normal tissues. By comparing gene expression between tumor and normal tissues, we observed a large amount of differentially expressed genes, in which FAM83A was conspicuous due to its obvious difference (log2 fold change was 6.79 and adjusted p value was 4.56E-177) (Fig. 1 A). As shown in Fig. 1 B, FAM83A was obviously up-regulated in LUAD tumors compared with normal tissues, indicating its favorable discrimination ability. For exploring the clinical significance of FAM83A in tumor progression, we further investigated the effect of FAM83A expression on overall survival and disease-free survival. Results showed that LUAD patients with high FAM83A expression had worse overall survival and disease-free survival than those with low FAM83A expression (Fig. 1 C,D). To further explore the influence of FAM83A on Chinese LUAD patients, we analyzed the relationship between FAM83A expression and overall survival or histological grade by mining transcriptomic dataset of 169 LUAD in individuals of Chinese ancestry. Significantly, Chinese LUAD patients with high FAM83A expression presented shorter overall survival than those with low FAM83A expression (Fig. 1 E). Moreover, FAM83A expression level in poorly differentiated LUAD was higher compared with that in well or moderately differentiated LUAD (Fig. 1 F). Collectively, these findings provided evidence to suggest that high expression of FAM83A was associated with poor prognosis in LUAD patients and FAM83A had the potential discriminability in lung tissues. 3.2 High FAM83A expression was associated with poor pathological features To determine the expression pattern of FAM83A in LUAD lung tissues, we quantified the protein level of FAM83A in 94 LUAD lung tissues and their paired normal counterparts by immunohistochemistry. Consistent with the mRNA expression pattern, elevated protein level of FAM83A was observed in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs, accounting for 94.7% of all specimens examined (Table. 1, Fig. 2 ). TFF-1 and NapsinA are common immunohistochemical markers for LUAD. To compare the diagnostic value of FAM83A, TFF-1 and NapsinA in LUAD, TFF-1 and NapsinA staining were also performed on LUAD lung tissues. Results showed that the expression pattern of FAM83A was relatively consistent with that of TFF-1 and NapsinA (Fig. 2 ). Meanwhile, the correlation between FAM83A expression and different clinicopathological characteristics of 94 LUAD patients was also analyzed. According to immunohistochemistry score, 94 LUAD patients were divided into high FAM83A group (n = 64) and low FAM83A group (n = 30). Correlation analysis showed that high FAM83A expression was associated with T classification ( p <0.001), lesion size ( p <0.001) and pleural invasion ( p <0.001) (Table 2 ). Other clinical parameters (age, gender, AJCC clinical stage, M classification and N classification) showed no statistical difference between the two groups (Table. 2). Therefore, these data further indicated that high FAM83A expression was related to poor pathological features and had the diagnostic value in LUAD lung tissues. Table 1 Protein expression of FAM83A in 94 LUAD tissues and paired normal tissues Group n Positive Negative Positivity rate χ 2 P Paired normal tissue 94 3 91 3.2% 157.433 < 0.001 LUAD tissue 94 89 5 94.7% Table 2 Correlation analysis between FAM83A and clinical characteristics of LUAD Clinical characteristics n FAM83A expression(%) χ 2 P Low expression High expression Gender Male 40 9(22.5%) 31(77.5%) 2.840 0. 092 Female 54 21(38.9%) 33(61.1%) Age(years) ≤50 17 7(41.2%) 10(58.8%) 0.819 0.365 >50 77 23(29.9%) 54(70.1%) Lesion size ≤10mm >10mm Pleural invasion no yes Stage 15 79 62 32 12(80.0%) 18(22.8%) 29(46.8%) 1(3.1%) 3(20.0%) 61(77.2%) 33(53.2%) 31(96.9%) 18.992 18.506 <0.001 <0.001 Ⅰ+Ⅱ 93 30(32.3%) 63(67.7%) 0.474 0.491 Ⅲ+Ⅳ 1 0(0.0%) 1(100.0%) T classification T1 + T2 73 30(41.1%) 43(58.9%) 12.676 <0.001 T3 + T4 21 0(0.0%) 21(100.0%) M classification M0 93 30(32.3%) 63(67.7%) 0.474 0.491 M1 1 0(0.0%) 1(100.0%) N classification N0 89 30(33.7%) 59(66.3%) 2.475 0.116 N1 5 0(0.0%) 5(100.0%) 3.3 Association between FAM83A expression levels and immune cell infiltration in LUAD Previous studies have confirmed that the immune microenvironment has an important effect on the prognosis of patients with LUAD. To better investigate the relationship between FAM83A expression levels and immune cell infiltration in LUAD, we calculated the infiltration expression of 22 immune cells in LUAD. The results revealed positive correlations between FAM83A expression and neutrophil, mast cell resting, myeloid dendritic cell activated, macrophage M2, macrophage M1, macrophage M0, NK cell activated, NK cell resting, T cell regulatory (Tregs), T cell follicular helper, T cell CD4 + memory activated, T cell CD4 + naïve, T cell CD8 + (Fig. 3 ). Conversely, FAM83A exhibited negative correlations with Eosinophil, Mast cell activated, Myeloid dendritic cell resting, Monocyte, T cell gamma delta, T cell CD4 + memory resting, B cell plasma, B cell memory and B cell naïve (Fig. 3 ). In summary, FAM83A potentially plays a novel immunomodulatory role in LUAD tumor immunity, suggesting its potential as a future target for lung cancer immunotherapy. 3.4 FAM83A has the potential to distinguish malignant from benign pleural effusion. Based on the above analysis of bioinformatics and immunohistochemistry in LUAD lung tissues, we speculated that FAM83A might be a novel diagnostic biomarker for pleural effusion. To verify our hypothesis, FAM83A、TFF-1 and NapsinA expression was performed on pleural effusion cell blocks from 40 LUAD patients and 21 non-neoplastic patients by immunohistochemistry. The results showed that 92.5% positive FAM83A expression was observed in LUAD pleural effusion, while 95.2% negative FAM83A expression was observed in non-neoplastic pleural effusion (Table. 3). And it was verified that FAM83A positive expression in pleural effusion cell mass was essentially the same as that of TTF-1 and NapsinA (Fig. 4 A ,B), and even FAM83A staining was more pronounced in some LUAD pleural effusions that were weakly positive for TTF-1 or NapsinA (Fig. 4 B,C). Thus, the above results suggested that FAM83A was a promising diagnostic biomarker for LUAD pleural effusion. Table 3 Protein expression of FAM83A in 21 non-neoplastic pleural effusion and 40 LUAD pleural effusion Group n Positive Negative Positivity rate χ 2 P Non-neoplastic pleural effusion 21 1 20 4.8% 45.131 < 0.001 LUAD pleural effusion 40 37 3 92.5% 4 Discussion Malignant pleural effusion (MPE) is most frequently observed in patients with lung adenocarcinoma (LUAD), indicating advanced or progressive stage. Pleural fluid cytology is a less-invasive tool for the diagnosis of MPE. The discovery of effective biomarkers is critical for pleural fluid cytology. In the present study, using bioinformatic analysis as a powerful tool, we screened out a favorable target gene, FAM83A . Further immunohistochemistry confirmed that FAM83A was a potential immune-related biomarker not only in LUAD tissue but also in pleural effusion. FAM83 family has eight members (FAM83A to FAM83H) that share a highly conserved N-terminal DUF1669 domain [ 13 ]. One study showed that DUF1669 domain is responsible for facilitating the interaction between FAM83 members and CK1 isoforms, which regulate cell division and apoptosis [ 14 ]. Multiple studies showed that FAM83 members are elevated in many tumors and are implicated in cancer growth, metastasis, and therapy resistance [ 13 , 15 ]. As the smallest member, FAM83A was frequently studied in various tumors. In breast cancer, FAM83A expression is upregulated and associated with poor prognosis [ 16 – 18 ], and contributed to EGFR-TKI resistance by interacting with and causing phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR [ 19 ]. Additionally, FAM83A is overexpressed in HER2-positive breast cancer cells and its knockdown severely inhibits proliferation and induces apoptosis [ 20 ], FAM83A expression is correlated with the chemoresistance and stemness of triple-negative breast cancer cells [ 21 ]. In ovarian cancer, FAM83A expression is upregulated and exerts pro-tumorigenic functions by affecting the Akt/Wnt/β-catenin pathway [ 22 ]. In pancreatic cancer, FAM83A is overexpressed and associated with poorer overall survival and disease-free survival [ 23 ], and promotes cancer stem cell-like traits and chemoresistance by WNT/β-catenin and TGF-β signaling pathways [ 24 , 25 ]. In pancreatic ductal adenocarcinomas, FAM83A expression is elevated and drives cell survival and tumorigenicity through a MEK/ERK-FAM83A feed-forward loop [ 26 ]. In cervical cancer, the role of FAM83A is controversial or contradictory, one study showed that FAM83A exerts a tumor‑suppressive role by regulating integrins [ 27 ], whereas two other studies have reported that FAM83A promotes cancer progression through the PI3K/AKT/mTOR pathway, as well as the epithelial mesenchymal transition and Wnt signaling pathways [ 28 , 29 ]. In head and neck squamous cell carcinoma, FAM83 induces EMT by activating the Wnt/β-catenin signaling pathway, which promotes tumor cell proliferation and metastasis [ 30 ]. In hepatocellular carcinoma, FAM83A is upregulated and correlated with poor progression-free survival time, and induces migration, invasion, and metastasis by activate EMT signaling and forming a FAM83A/PI3K/AKT/c-JUN positive-feedback loop [ 31 ]. Thus, abnormal expression of FAM38A is involved in the development of various cancers, including breast, pancreas, ovarian, pancreatic duct gland, cervix, head and neck squamous cell carcinoma and liver. Compared to other cancers, relevant studies about the oncogenicity of FAM83A were most in lung cancer. Liu et al. showed that FAM83A is overexpressed in lung cancer tissues and closely associated with poor lung cancer survival, and its knockdown significantly suppresses the proliferation, migration, invasion ability, and EGFR/MAPK/CHKA signaling of lung cancer cell lines [ 32 ]. Zheng et al. showed that high expression of FAM83A is correlated with advanced TNM stage and poor prognosis in lung cancer, and promotes cell proliferation and invasion by regulating Wnt and Hippo signaling pathways and EMT process [ 33 ]. Hu et al. presented that FAM83A expression is amplified and associated with poor prognosis in NSCLC, and promotes tumorigenicity at least partly via ERK and PI3K/Akt/mTOR pathways [ 34 ]. Zhou et al. reported that FAM83A expression was significantly increased and positively associated with tumor metastasis and poor survival of NSCLC patients, and promotes NSCLC cell migration and invasion by inducing EMT via PI3K/ATK/Snail signaling [ 35 ]. Richtmann et al. reported that FAM83A expression is elevated and associated with poor survival prognosis, and is involved in proliferation, anchorage-independent growth, migration, and EGFR pathway in NSCLC [ 36 ]. Five different cohort studies showed that LUAD tissue has higher FAM83A expression than adjacent lung tissue, and high FAM83A expression exhibits poor clinical outcomes such as advanced stage and shorter overall survival [ 37 – 41 ]. In addition, FAM83A drives PD-L1 expression via ERK signaling and FAM83A/PD-L1 co-expression correlates with poor prognosis in LUAD [ 40 ]. Based on the above researches, we can conclude that FAM83A is highly expressed in lung cancer tissues, and its high expression is associated with poor prognosis. Moreover, FAM83A promotes its aggressive oncogenic behavior in lung cancer through multiple pathways, including EGFR/MAPK/CHKA, PI3K/ATK/Snail, Wnt, Hippo, EMT, PI3K/Akt/mTOR, ERK and EGFR. Thus, FAM83A is emerging as an intriguing lung cancer biomarker that warrants further investigation, for example, the discovery and production of FAM83A inhibitors. Despite ample evidence that FAM83A is a promising biomarker for various tumors, to date, all efforts were concentrated on tissue samples, including LUAD. It is known that pleural effusion is a common symptom of advanced LUAD, and the distinction between MPE and benign pleural effusion is essential for its treatment. To address the diagnostic value of FAM83A in LUAD pleural effusion, our study firstly provided the evidence that FAM83A may be a favorable diagnostic biomarker for pleural fluid cytology in LUAD. In our work, 37 of 40 LUAD pleural effusion cases were positive for FAM83A staining, while 20 of 21 non-neoplastic pleural effusion cases were negative, reflecting its high sensitivity in pleural effusion. Moreover, our work also has observed the amplified expression of FAM83A in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs, which is consistent with other studies and collectively supports FAM83A as a credible biomarker of LUAD biopsy specimen. Although the new IASLC/ATS/ERS LUAD classification has recommended TTF-1 or Napsin A as an immunohistochemical marker for biopsy or cytology specimen [ 42 ], the variable sensitivity of TTF-1 and Napsin A may still lead to an inappropriate treatment [ 43 ]. Therefore, combination of FAM83A and TTF-1 or Napsin A would be a promising answer to this question. In conclusion, our work presented a comprehensive set of bioinformatic and experimental evidence establishing FAM83A as a potential immune-related biomarker not only in biopsy specimen but also in pleural effusion. More importantly, our findings provide a new and effective option for pleural fluid cytology. Declarations Acknowledgements Not applicable. Author Contributors Xiaolin Zhang and Shuanghua Cheng: concept and design. Yulan Liu, Liping Wu and Zhiwei Tan: immunohistochemistry. Jie Hu and Shigao Chen: collect specimens and corresponding clinical information. Hangfeng Liu and Jia Yao: clinical data analysis. Hangfeng Liu: draft the manuscript. All authors have read and agreed to the published version of the manuscript. Funding This work was supported by Medical Youth Innovation Project of Sichuan Province (Q22060)，and Research Project of China Baoyuan Investment Co., Ltd. (CBYI202102). Competing interests The authors declare that they have no competing interests. Patient consent for publication Informed consent was obtained for experimentation with human subjects. Ethics approval This study protocol related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. RNA sequencing and clinical data of LUAD was downloaded from cBioPortal for Cancer Genomics (http://www.cbioportal.org). The data contains 4 datasets of LUAD-1 (OncoSG, Nat Genet 2020), LUAD-2 (TCGA, Firehose Legacy), LUAD-3 (TCGA, Nature 2014) and LUAD-4 (TCGA, PanCancer Atlas). 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Elevated FAM83A expression predicts poorer clincal outcome in lung adenocarcinoma. Cancer Biomark 2019; 26:367-73. Zhou F, Wang X, Liu F, et al. FAM83A drives PD-L1 expression via ERK signaling and FAM83A/PD-L1 co-expression correlates with poor prognosis in lung adenocarcinoma. Int J Clin Oncol 2020; 25:1612-23. Liu X, Fu M, Xia D, et al. Overexpression of FAM83A Is Associated with Poor Prognosis of Lung Adenocarcinoma. Journal of oncology 2022; 2022:8767333. Travis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011; 6:244-85. Porcel JM, Palma R, Bielsa S, et al. TTF-1 and napsin A on cell blocks and supernatants of pleural fluids for labeling malignant effusions. Respirology 2015; 20:831-3. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Jun, 2024 Reviews received at journal 28 May, 2024 Reviews received at journal 26 May, 2024 Reviewers agreed at journal 24 May, 2024 Reviewers agreed at journal 22 May, 2024 Reviewers invited by journal 22 May, 2024 Editor assigned by journal 22 May, 2024 Submission checks completed at journal 22 May, 2024 First submitted to journal 21 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4453916","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307773324,"identity":"baa5c7e1-2048-4425-b56e-ba4918969f17","order_by":0,"name":"Hangfeng Liu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Hangfeng","middleName":"","lastName":"Liu","suffix":""},{"id":307773325,"identity":"d584f30c-45f0-4c8e-9759-263d8d56182c","order_by":1,"name":"Jia Yao","email":"","orcid":"","institution":"West China Biomedical Big Data Center, West China Hospital, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Yao","suffix":""},{"id":307773326,"identity":"33691e92-c002-4113-b2b9-0c8247ebcdf8","order_by":2,"name":"Yulan Liu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yulan","middleName":"","lastName":"Liu","suffix":""},{"id":307773327,"identity":"c3b5d9f0-5c76-495a-8077-9b23e3014c03","order_by":3,"name":"Liping Wu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Liping","middleName":"","lastName":"Wu","suffix":""},{"id":307773328,"identity":"0a4faf2c-c66b-4cbb-8514-e77c9e3df0ec","order_by":4,"name":"Zhiwei Tan","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Zhiwei","middleName":"","lastName":"Tan","suffix":""},{"id":307773329,"identity":"f089d664-7225-441d-956a-7c131079cbc5","order_by":5,"name":"Jie Hu","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Hu","suffix":""},{"id":307773330,"identity":"95009862-b2a6-46f0-94ea-d7ad85d19721","order_by":6,"name":"Shigao Chen","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shigao","middleName":"","lastName":"Chen","suffix":""},{"id":307773331,"identity":"b5f51a53-5d0f-4813-8c46-c21c0d8d1cb6","order_by":7,"name":"Xiaolin Zhang","email":"","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Zhang","suffix":""},{"id":307773332,"identity":"97276891-d6cf-432e-ae5f-b275e4840572","order_by":8,"name":"Shuanghua Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACCWYQaSDBzM/MfPgBCVoKLNgl29nSDIjTAiY/VPAbnOdRkCBKi2Q77+EXQIdJGx/mYTBgqLGJJqhFmpkvzQKoxdjsMO+BBwzH0nIbCGmRY+YxMwBqSTY7zJdgwNhwmHgt9ZubeQwkiNIizcxj/AAUyAbMxGqRbOYxY0gAapE4DAzkBGL8InH+jPGHD3/qmPn7Dx9+8KHGhrAWIGCTSIAxE3CrQgHMH4hUOApGwSgYBSMVAACFJTJ8/qZcaAAAAABJRU5ErkJggg==","orcid":"","institution":"China National Nuclear Corporation 416 Hospital","correspondingAuthor":true,"prefix":"","firstName":"Shuanghua","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2024-05-21 09:57:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4453916/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4453916/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":57947080,"identity":"10414db4-0fb5-4215-8e08-89287281684e","added_by":"auto","created_at":"2024-06-07 20:15:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2743417,"visible":true,"origin":"","legend":"\u003cp\u003eBioinformatics analysis revealed that FAM83A was overexpressed and associated with poor prognosis in LUAD. \u003cstrong\u003eA\u003c/strong\u003e The volcano plot of mRNA expression based on TCGA-LUAD dataset. \u003cstrong\u003eB\u003c/strong\u003e Relative expression of FAM83A in tumor and normal tissues using TCGA-LUAD datasets. \u003cstrong\u003eC,D\u003c/strong\u003e The influence of FAM83A expression on overall survival \u003cstrong\u003eC \u003c/strong\u003eand disease-free survival \u003cstrong\u003eD\u003c/strong\u003ebased on TCGA-LUAD dataset.\u003cstrong\u003e E\u003c/strong\u003e The influence of FAM83A expression on overall survival in Chinese LUAD patients using OncoSG-LUAD dataset. \u003cstrong\u003eF\u003c/strong\u003e The difference of FAM83A expression between well, moderately, or poorly differentiated group based on OncoSG-LUAD dataset.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4453916/v1/2258c3d02e9363161895a62a.png"},{"id":57947620,"identity":"88ffa53d-d75e-4887-a348-8813f7f6b9e2","added_by":"auto","created_at":"2024-06-07 20:23:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3017978,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative examples of FAM83A, TFF-1 and NapsinA immunohistochemical staining in LUAD tumor and paired normal tissues.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4453916/v1/db0b5797defe1dfe2b29cc44.png"},{"id":57947078,"identity":"8faf8ed1-5e88-4f41-951f-0d8bffe15e83","added_by":"auto","created_at":"2024-06-07 20:15:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":319755,"visible":true,"origin":"","legend":"\u003cp\u003eCorrelation between the FAM83A expression level and immune cell infiltration in TCGA-LUAD. The heatmap shows the associations among 22 immune cell populations in LUAD. The color blue or red represents positive or negative associations respectively, and the size of the circle of each grid represents the correlation coefficient. The line red or green represents the positive or negative correlation between FAM83A and each immune cell.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4453916/v1/fb796094f5922ce596960a56.png"},{"id":57947079,"identity":"2b0e2680-3373-442e-9fb8-bc8f37a97dd7","added_by":"auto","created_at":"2024-06-07 20:15:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3646499,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative examples of immunohistochemical staining of FAM83A, TTF-1 or Napsin A in LUAD and non-neoplastic pleural effusion. \u003cstrong\u003eA\u003c/strong\u003eStaining results of cell block in non-neoplastic pleural effusion. \u003cstrong\u003eB,C\u003c/strong\u003eStaining results of cell block in LUAD pleural effusion.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4453916/v1/4bca94a2d9b65fa3c98501ce.png"},{"id":57947622,"identity":"09a358ba-35ec-46a3-959c-4813a540d072","added_by":"auto","created_at":"2024-06-07 20:24:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10949957,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4453916/v1/d4ef942c-c73f-42ee-b05d-afc237fb8e78.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"FAM83A: A Prospective Diagnostic Biomarker with Immunological Relevance in Lung Adenocarcinoma- Associated Malignant Pleural Effusion","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eLung cancer remains the leading cause of cancer-related mortality worldwide. Lung cancer is classified into non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), accounting for 85% and 15% respectively [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Lung adenocarcinoma (LUAD) is the major histological subtype of NSCLC [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Malignant pleural effusion (MPE) is a common clinical complication of malignancies, with a rate of 10% ~ 15% in advanced NSCLC in initial diagnosis and a higher rate in later treatment course [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Once the diagnosis of MPE in NSCLC is confirmed, patients will be classified into advanced TNM stages with a median overall survival of 5.5 months [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thus, the distinction between MPE and benign pleural effusion is the first and critical step for early initiation of clinical intervention and adequate management. Unfortunately, some NSCLC patients with MPE have undefined diagnosis due to negative cytology. Therefore, it is essential to explore effective means to achieve global control of NSCLC patients with MPE.\u003c/p\u003e \u003cp\u003eThe diagnosis of malignant pleural effusion is mainly made by pleural fluid cytology and pleural biopsy. Pleural biopsy is an invasive procedure that may result in various complications including hemorrhage and infection. By contrast, pleural fluid cytology is a less-invasive tool. However, pleural fluid cytology still lacks enough sensitivity or specificity, which has not yet supplanted pleural biopsy. In recent years, some significant advances towards the use of biomarkers in pleural fluid cytology have been achieved. Some biomarkers like lactate dehydrogenase (LDH), carcinoembryonic antigen (CEA), carbohydrate antigen 153 (CA153) and adenosine deaminase (ADA) have been commonly used in pleural fluid [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In additional, numerous studies investigated the possibility of other novel biomarkers to improve the diagnostic ability of pleural fluid cytology, such as reactive oxygen species modulator 1 (ROMO1), hyaluronic acid (HA), chitinase-3-like protein 1 (YKL-40), thymidine kinase 1 (TK1), pro-cathepsin D, and programmed death-ligand 1 (PD-L1) [\u003cspan additionalcitationids=\"CR8 CR9 CR10 CR11\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. To our knowledge, there is no consensus as to which biomarker is most effective to distinguish pleural effusion. Hence, it is urgently required to discover new effective biomarkers for differentiating malignant from benign pleural effusion.\u003c/p\u003e \u003cp\u003eUsing differentially expressed genes from mRNA sequencing data of LUAD tumors and para-cancerous tissues in TCGA database as an entry point, in this study, we screened new candidate biomarkers, which may have a favorable discrimination ability for the properties of pleural effusion. The diagnostic value was verified not only in tissue specimens but also in pleural effusion. With this work, family with sequence similarity 83 member A (FAM83A) stood out as a potential diagnostic biomarker in differentiating malignant from benign pleural effusion in LUAD patients. Moreover, FAM83A may have a novel immunomodulatory function during the development of LUAD.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bioinformatics analysis of FAM83A expression profile and clinical features in patients with LUAD\u003c/h2\u003e \u003cp\u003eRNA sequencing and clinical data of LUAD was downloaded from cBioPortal for Cancer Genomics (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbioportal.org\u003c/span\u003e\u003cspan address=\"http://www.cbioportal.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The data contains 4 datasets of LUAD-1 (OncoSG, Nat Genet 2020), LUAD-2 (TCGA, Firehose Legacy), LUAD-3 (TCGA, Nature 2014) and LUAD-4 (TCGA, PanCancer Atlas). LUAD-2, LUAD-3, and LUAD-4 datasets from TCGA were integrated, consisting of 517 LUAD tumors and 59 adjacent normal tissues. LUAD-1 dataset contains a list of 169 Chinese LUAD samples with clinical information. The DESeq2 R package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioconductor.org/packages/release/bioc/html/DESeq2.html\u003c/span\u003e\u003cspan address=\"https://bioconductor.org/packages/release/bioc/html/DESeq2.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to detect differentially expressed genes (DEGs) between 517 LUAD and 59 normal samples. The cut-off criterion of DEGs was adjusted p value\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and |log2 fold change| \u0026ge;1. The difference in overall survival or disease-free survival was assessed between LUAD patients with high FAM83A expression and those with low FAM83A expression by the log-rank test. The hazard ratios with 95% confidence intervals and log-rank P-values were calculated and displayed on the plot. The difference in FAM83A expression was compared between well, moderately, or poorly differentiated group by student's t test in 169 Chinese LUAD samples.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Correlation analysis of FAM83A expression and immune cell infiltration\u003c/h2\u003e \u003cp\u003eBased on the gene expression profile of 517 TCGA-LUAD samples, the immune cell infiltration of each sample was evaluated with the immunedeconv R package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://grst.github.io/immunedeconv\u003c/span\u003e\u003cspan address=\"https://grst.github.io/immunedeconv\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). immunedeconv was an R package that integrates six latest algorithms, including TIMER, CIBERSORT, EPIC, MCP-counter, quanTIseq, and xCell. Spearman's correlation coefficient analysis was used to investigate the correlations among different types of immune cells and to assess the association between FAM83A expression and the levels of infiltration of each immune cell type. Correlation analysis and visualization were conducted using the ggClusterNet R package (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/taowenmicro/ggClusterNet/\u003c/span\u003e\u003cspan address=\"https://github.com/taowenmicro/ggClusterNet/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Patient information and clinical samples\u003c/h2\u003e \u003cp\u003eThis study protocol related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital. Informed consent was obtained for experimentation with human subjects. The clinical samples were divided into two parts, which were collected at the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital. The first part was primary tumor specimens and paired normal lung tissues from 94 LUAD patients, who underwent surgical treatment. All patients in the first part met the following inclusion criteria: (a) Patients with a definite diagnosis of lung adenocarcinoma; (b) No other malignant tumors; (c) Patients at clinical stage I-IV classified by TNM classification of American Joint Committee on Cancer (AJCC); (d) Patients with complete clinical and pathological information including age, sex, TNM stage, and clinical stage. The second part consisted of pleural effusion from 40 LUAD patients and 21 non-neoplastic patients. Inclusion criteria for LUAD pleural effusion: (a) Pathological examination confirmed that the surgical tissue specimen was lung adenocarcinoma; (b) No other malignant tumors. Inclusion criteria for non-neoplastic pleural effusion: (a) Patients with no malignant tumors; (b) no cancer cells in pleural effusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eTissue specimens were fixed in 4% phosphate-buffered paraformaldehyde and subsequently embedded in paraffin. Fresh pleural effusion was placed for 20\u0026ndash;30 mins, then the precipitate was collected and centrifuged at 2500 rpm for 5 mins. After discarding the supernatant, the precipitate was resuspended and centrifuged in 75% alcohol and 95% alcohol respectively. Subsequently, the precipitate was fixed in 10% formalin for 2\u0026ndash;4 hours. Finally, well-formed cell pellet was transferred to a dehydrator for final processing with the same procedure used for the histology tissue. Immunohistochemistry was performed on three \u0026micro;m sections of paraffin-embedded tissues or pleural fluid cell pellet. Following antibodies were used in immunohistochemical staining: anti-FAM83A (Proteintech, Chicago, #20618-1-AP), anti-TTF1 (MXB\u0026reg;Biotechnologies, Fuzhou, China, #MAB-0677) and anti-Napsin A (MXB\u0026reg;Biotechnologies, Fuzhou, China, #MAB-0704). After using diaminobenzidine (DAB) substrate and counterstaining with hematoxylin, the target signals were observed using an Olympus BX53 microscope. Rabbit pre-immune serum was used as the primary antibody for the negative control. Staining intensity was scored as follows: 0\u0026thinsp;=\u0026thinsp;negative staining; 1\u0026thinsp;=\u0026thinsp;weak staining; 2\u0026thinsp;=\u0026thinsp;moderate staining; and 3\u0026thinsp;=\u0026thinsp;strong staining. Patients with scores of 0 and 1 were classified in the low expression group, and patients with scores of 2 and 3 were classified in the high expression group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e \u003cp\u003eSPSS software (version 26.0; IBM, Chicago, IL) was used for statistical analysis. Chi-square test was used to analyze the correlation between clinicopathological characteristics and FAM83A expression. \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 FAM83A was a potential diagnostic and prognostic biomarker in LUAD\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eTo screen new diagnostic biomarkers for differentiating malignant from benign pleural effusion in lung adenocarcinoma (LUAD), we first aggregated RNA sequencing data of LUAD tumors and para-cancerous tissues from TCGA database, including 517 tumors and 59 normal tissues. By comparing gene expression between tumor and normal tissues, we observed a large amount of differentially expressed genes, in which FAM83A was conspicuous due to its obvious difference (log2 fold change was 6.79 and adjusted \u003cem\u003ep\u003c/em\u003e value was 4.56E-177) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, FAM83A was obviously up-regulated in LUAD tumors compared with normal tissues, indicating its favorable discrimination ability. For exploring the clinical significance of FAM83A in tumor progression, we further investigated the effect of FAM83A expression on overall survival and disease-free survival. Results showed that LUAD patients with high FAM83A expression had worse overall survival and disease-free survival than those with low FAM83A expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC,D). To further explore the influence of FAM83A on Chinese LUAD patients, we analyzed the relationship between FAM83A expression and overall survival or histological grade by mining transcriptomic dataset of 169 LUAD in individuals of Chinese ancestry. Significantly, Chinese LUAD patients with high FAM83A expression presented shorter overall survival than those with low FAM83A expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Moreover, FAM83A expression level in poorly differentiated LUAD was higher compared with that in well or moderately differentiated LUAD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Collectively, these findings provided evidence to suggest that high expression of FAM83A was associated with poor prognosis in LUAD patients and FAM83A had the potential discriminability in lung tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 High FAM83A expression was associated with poor pathological features\u003c/h2\u003e \u003cp\u003eTo determine the expression pattern of FAM83A in LUAD lung tissues, we quantified the protein level of FAM83A in 94 LUAD lung tissues and their paired normal counterparts by immunohistochemistry. Consistent with the mRNA expression pattern, elevated protein level of FAM83A was observed in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs, accounting for 94.7% of all specimens examined (Table. 1, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). TFF-1 and NapsinA are common immunohistochemical markers for LUAD. To compare the diagnostic value of FAM83A, TFF-1 and NapsinA in LUAD, TFF-1 and NapsinA staining were also performed on LUAD lung tissues. Results showed that the expression pattern of FAM83A was relatively consistent with that of TFF-1 and NapsinA (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Meanwhile, the correlation between FAM83A expression and different clinicopathological characteristics of 94 LUAD patients was also analyzed. According to immunohistochemistry score, 94 LUAD patients were divided into high FAM83A group (n\u0026thinsp;=\u0026thinsp;64) and low FAM83A group (n\u0026thinsp;=\u0026thinsp;30). Correlation analysis showed that high FAM83A expression was associated with T classification (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001), lesion size (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) and pleural invasion (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.001) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Other clinical parameters (age, gender, AJCC clinical stage, M classification and N classification) showed no statistical difference between the two groups (Table. 2). Therefore, these data further indicated that high FAM83A expression was related to poor pathological features and had the diagnostic value in LUAD lung tissues.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein expression of FAM83A in 94 LUAD tissues and paired normal tissues\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePositivity rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePaired normal tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e3.2%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e157.433\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLUAD tissue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e94.7%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCorrelation analysis between FAM83A and clinical characteristics of LUAD\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eClinical characteristics\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e\u003cem\u003eFAM83A\u003c/em\u003e expression(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLow expression\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHigh expression\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e9(22.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e31(77.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.840\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0. 092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e21(38.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e33(61.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge(years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e7(41.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10(58.8%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.819\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.365\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e23(29.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e54(70.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesion size\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026le;10mm\u003c/p\u003e \u003cp\u003e\u0026gt;10mm\u003c/p\u003e \u003cp\u003ePleural invasion\u003c/p\u003e \u003cp\u003eno\u003c/p\u003e \u003cp\u003eyes\u003c/p\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e79\u003c/p\u003e \u003cp\u003e62\u003c/p\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e12(80.0%)\u003c/p\u003e \u003cp\u003e18(22.8%)\u003c/p\u003e \u003cp\u003e29(46.8%)\u003c/p\u003e \u003cp\u003e1(3.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3(20.0%)\u003c/p\u003e \u003cp\u003e61(77.2%)\u003c/p\u003e \u003cp\u003e33(53.2%)\u003c/p\u003e \u003cp\u003e31(96.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.992\u003c/p\u003e \u003cp\u003e18.506\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅠ+Ⅱ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e30(32.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63(67.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.491\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eⅢ+Ⅳ\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT classification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1\u0026thinsp;+\u0026thinsp;T2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e30(41.1%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e43(58.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.676\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026lt;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT3\u0026thinsp;+\u0026thinsp;T4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM classification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e30(32.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e63(67.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.474\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.491\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN classification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e30(33.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59(66.3%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.475\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0(0.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5(100.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Association between FAM83A expression levels and immune cell infiltration in LUAD\u003c/h2\u003e \u003cp\u003ePrevious studies have confirmed that the immune microenvironment has an important effect on the prognosis of patients with LUAD. To better investigate the relationship between FAM83A expression levels and immune cell infiltration in LUAD, we calculated the infiltration expression of 22 immune cells in LUAD. The results revealed positive correlations between FAM83A expression and neutrophil, mast cell resting, myeloid dendritic cell activated, macrophage M2, macrophage M1, macrophage M0, NK cell activated, NK cell resting, T cell regulatory (Tregs), T cell follicular helper, T cell CD4\u003csup\u003e+\u003c/sup\u003e memory activated, T cell CD4\u003csup\u003e+\u003c/sup\u003e na\u0026iuml;ve, T cell CD8\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Conversely, FAM83A exhibited negative correlations with Eosinophil, Mast cell activated, Myeloid dendritic cell resting, Monocyte, T cell gamma delta, T cell CD4\u003csup\u003e+\u003c/sup\u003e memory resting, B cell plasma, B cell memory and B cell na\u0026iuml;ve (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In summary, FAM83A potentially plays a novel immunomodulatory role in LUAD tumor immunity, suggesting its potential as a future target for lung cancer immunotherapy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.4 FAM83A has the potential to distinguish malignant from benign pleural effusion.\u003c/h2\u003e \u003cp\u003eBased on the above analysis of bioinformatics and immunohistochemistry in LUAD lung tissues, we speculated that FAM83A might be a novel diagnostic biomarker for pleural effusion. To verify our hypothesis, FAM83A、TFF-1 and NapsinA expression was performed on pleural effusion cell blocks from 40 LUAD patients and 21 non-neoplastic patients by immunohistochemistry. The results showed that 92.5% positive FAM83A expression was observed in LUAD pleural effusion, while 95.2% negative FAM83A expression was observed in non-neoplastic pleural effusion (Table. 3). And it was verified that FAM83A positive expression in pleural effusion cell mass was essentially the same as that of TTF-1 and NapsinA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA ,B), and even FAM83A staining was more pronounced in some LUAD pleural effusions that were weakly positive for TTF-1 or NapsinA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB,C). Thus, the above results suggested that FAM83A was a promising diagnostic biomarker for LUAD pleural effusion.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein expression of FAM83A in 21 non-neoplastic pleural effusion and 40 LUAD pleural effusion\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePositive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePositivity rate\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eχ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNon-neoplastic pleural effusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.8%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e45.131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLUAD pleural effusion\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e92.5%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eMalignant pleural effusion (MPE) is most frequently observed in patients with lung adenocarcinoma (LUAD), indicating advanced or progressive stage. Pleural fluid cytology is a less-invasive tool for the diagnosis of MPE. The discovery of effective biomarkers is critical for pleural fluid cytology. In the present study, using bioinformatic analysis as a powerful tool, we screened out a favorable target gene, \u003cem\u003eFAM83A\u003c/em\u003e. Further immunohistochemistry confirmed that FAM83A was a potential immune-related biomarker not only in LUAD tissue but also in pleural effusion.\u003c/p\u003e \u003cp\u003eFAM83 family has eight members (FAM83A to FAM83H) that share a highly conserved N-terminal DUF1669 domain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. One study showed that DUF1669 domain is responsible for facilitating the interaction between FAM83 members and CK1 isoforms, which regulate cell division and apoptosis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Multiple studies showed that FAM83 members are elevated in many tumors and are implicated in cancer growth, metastasis, and therapy resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. As the smallest member, FAM83A was frequently studied in various tumors. In breast cancer, FAM83A expression is upregulated and associated with poor prognosis [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and contributed to EGFR-TKI resistance by interacting with and causing phosphorylation of c-RAF and PI3K p85, upstream of MAPK and downstream of EGFR [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, FAM83A is overexpressed in HER2-positive breast cancer cells and its knockdown severely inhibits proliferation and induces apoptosis [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], FAM83A expression is correlated with the chemoresistance and stemness of triple-negative breast cancer cells [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In ovarian cancer, FAM83A expression is upregulated and exerts pro-tumorigenic functions by affecting the Akt/Wnt/β-catenin pathway [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In pancreatic cancer, FAM83A is overexpressed and associated with poorer overall survival and disease-free survival [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and promotes cancer stem cell-like traits and chemoresistance by WNT/β-catenin and TGF-β signaling pathways [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In pancreatic ductal adenocarcinomas, FAM83A expression is elevated and drives cell survival and tumorigenicity through a MEK/ERK-FAM83A feed-forward loop [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In cervical cancer, the role of FAM83A is controversial or contradictory, one study showed that FAM83A exerts a tumor‑suppressive role by regulating integrins [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], whereas two other studies have reported that FAM83A promotes cancer progression through the PI3K/AKT/mTOR pathway, as well as the epithelial mesenchymal transition and Wnt signaling pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In head and neck squamous cell carcinoma, FAM83 induces EMT by activating the Wnt/β-catenin signaling pathway, which promotes tumor cell proliferation and metastasis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In hepatocellular carcinoma, FAM83A is upregulated and correlated with poor progression-free survival time, and induces migration, invasion, and metastasis by activate EMT signaling and forming a FAM83A/PI3K/AKT/c-JUN positive-feedback loop [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Thus, abnormal expression of FAM38A is involved in the development of various cancers, including breast, pancreas, ovarian, pancreatic duct gland, cervix, head and neck squamous cell carcinoma and liver.\u003c/p\u003e \u003cp\u003eCompared to other cancers, relevant studies about the oncogenicity of FAM83A were most in lung cancer. Liu et al. showed that FAM83A is overexpressed in lung cancer tissues and closely associated with poor lung cancer survival, and its knockdown significantly suppresses the proliferation, migration, invasion ability, and EGFR/MAPK/CHKA signaling of lung cancer cell lines [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Zheng et al. showed that high expression of FAM83A is correlated with advanced TNM stage and poor prognosis in lung cancer, and promotes cell proliferation and invasion by regulating Wnt and Hippo signaling pathways and EMT process [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Hu et al. presented that FAM83A expression is amplified and associated with poor prognosis in NSCLC, and promotes tumorigenicity at least partly via ERK and PI3K/Akt/mTOR pathways [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Zhou et al. reported that FAM83A expression was significantly increased and positively associated with tumor metastasis and poor survival of NSCLC patients, and promotes NSCLC cell migration and invasion by inducing EMT via PI3K/ATK/Snail signaling [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Richtmann et al. reported that FAM83A expression is elevated and associated with poor survival prognosis, and is involved in proliferation, anchorage-independent growth, migration, and EGFR pathway in NSCLC [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Five different cohort studies showed that LUAD tissue has higher FAM83A expression than adjacent lung tissue, and high FAM83A expression exhibits poor clinical outcomes such as advanced stage and shorter overall survival [\u003cspan additionalcitationids=\"CR38 CR39 CR40\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In addition, FAM83A drives PD-L1 expression via ERK signaling and FAM83A/PD-L1 co-expression correlates with poor prognosis in LUAD [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Based on the above researches, we can conclude that FAM83A is highly expressed in lung cancer tissues, and its high expression is associated with poor prognosis. Moreover, FAM83A promotes its aggressive oncogenic behavior in lung cancer through multiple pathways, including EGFR/MAPK/CHKA, PI3K/ATK/Snail, Wnt, Hippo, EMT, PI3K/Akt/mTOR, ERK and EGFR. Thus, FAM83A is emerging as an intriguing lung cancer biomarker that warrants further investigation, for example, the discovery and production of FAM83A inhibitors.\u003c/p\u003e \u003cp\u003eDespite ample evidence that FAM83A is a promising biomarker for various tumors, to date, all efforts were concentrated on tissue samples, including LUAD. It is known that pleural effusion is a common symptom of advanced LUAD, and the distinction between MPE and benign pleural effusion is essential for its treatment. To address the diagnostic value of FAM83A in LUAD pleural effusion, our study firstly provided the evidence that FAM83A may be a favorable diagnostic biomarker for pleural fluid cytology in LUAD. In our work, 37 of 40 LUAD pleural effusion cases were positive for FAM83A staining, while 20 of 21 non-neoplastic pleural effusion cases were negative, reflecting its high sensitivity in pleural effusion. Moreover, our work also has observed the amplified expression of FAM83A in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs, which is consistent with other studies and collectively supports FAM83A as a credible biomarker of LUAD biopsy specimen. Although the new IASLC/ATS/ERS LUAD classification has recommended TTF-1 or Napsin A as an immunohistochemical marker for biopsy or cytology specimen [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], the variable sensitivity of TTF-1 and Napsin A may still lead to an inappropriate treatment [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Therefore, combination of FAM83A and TTF-1 or Napsin A would be a promising answer to this question.\u003c/p\u003e \u003cp\u003eIn conclusion, our work presented a comprehensive set of bioinformatic and experimental evidence establishing FAM83A as a potential immune-related biomarker not only in biopsy specimen but also in pleural effusion. More importantly, our findings provide a new and effective option for pleural fluid cytology.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXiaolin Zhang and Shuanghua Cheng: concept and design. Yulan Liu,\u0026nbsp;Liping Wu\u0026nbsp;and\u0026nbsp;Zhiwei Tan: immunohistochemistry.\u0026nbsp;Jie Hu\u0026nbsp;and\u0026nbsp;Shigao Chen: collect specimens and corresponding clinical information. Hangfeng Liu and Jia Yao: clinical data analysis. Hangfeng Liu: draft the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Medical Youth Innovation Project of Sichuan Province (Q22060)，and Research Project of China Baoyuan Investment Co., Ltd. (CBYI202102).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInformed consent was obtained for experimentation with human subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study protocol related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Ethics Committee of the Second Affiliated Hospital of Chengdu Medical College, China National Nuclear Corporation 416 Hospital.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. RNA sequencing and clinical data of LUAD was downloaded from cBioPortal for Cancer Genomics (http://www.cbioportal.org). The data contains 4 datasets of LUAD-1 (OncoSG, Nat Genet 2020), LUAD-2 (TCGA, Firehose Legacy), LUAD-3 (TCGA, Nature 2014) and LUAD-4 (TCGA, PanCancer Atlas).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLi Y, Ge D, Gu J, et al. A large cohort study identifying a novel prognosis prediction model for lung adenocarcinoma through machine learning strategies. BMC Cancer 2019; 19:886.\u003c/li\u003e\n\u003cli\u003eTaghizadeh N, Fortin M, Tremblay A. US Hospitalizations for Malignant Pleural Effusions: Data From the 2012 National Inpatient Sample. Chest 2017; 151:845-54.\u003c/li\u003e\n\u003cli\u003eWilliam WN, Jr., Lin HY, Lee JJ, et al. 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Cancer Manag Res 2020; 12:12623-33.\u003c/li\u003e\n\u003cli\u003eZhao J, Zhao F, Yang T, et al. FAM83A has a pro-tumor function in ovarian cancer by affecting the Akt/Wnt/\u0026beta;-catenin pathway. Environmental toxicology 2022; 37:695-707.\u003c/li\u003e\n\u003cli\u003eZou W, Wang H, Wu D, et al. ncRNA-mediated upregulation of FAM83A is associated with poor prognosis and immune infiltration in pancreatic cancer. Frontiers in endocrinology 2023; 14:1093042.\u003c/li\u003e\n\u003cli\u003eChen S, Huang J, Liu Z, et al. FAM83A is amplified and promotes cancer stem cell-like traits and chemoresistance in pancreatic cancer. Oncogenesis 2017; 6:e300.\u003c/li\u003e\n\u003cli\u003eZhou C, Zhu X, Liu N, et al. B-lymphoid tyrosine kinase-mediated FAM83A phosphorylation elevates pancreatic tumorigenesis through interacting with \u0026beta;-catenin. Signal transduction and targeted therapy 2023; 8:66.\u003c/li\u003e\n\u003cli\u003eParameswaran N, Bartel CA, Hernandez-Sanchez W, et al. 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Journal of translational medicine 2021; 19:423.\u003c/li\u003e\n\u003cli\u003eLiu C, Peng X, Li Y, et al. Positive feedback loop of FAM83A/PI3K/AKT/c-Jun induces migration, invasion and metastasis in hepatocellular carcinoma. Biomed Pharmacother 2020; 123:109780.\u003c/li\u003e\n\u003cli\u003eLiu PJ, Chen YH, Tsai KW, et al. Involvement of MicroRNA-1-FAM83A Axis Dysfunction in the Growth and Motility of Lung Cancer Cells. Int J Mol Sci 2020; 21.\u003c/li\u003e\n\u003cli\u003eZheng YW, Li ZH, Lei L, et al. FAM83A Promotes Lung Cancer Progression by Regulating the Wnt and Hippo Signaling Pathways and Indicates Poor Prognosis. Front Oncol 2020; 10:180.\u003c/li\u003e\n\u003cli\u003eHu H, Wang F, Wang M, et al. FAM83A is amplified and promotes tumorigenicity in non-small cell lung cancer via ERK and PI3K/Akt/mTOR pathways. Int J Med Sci 2020; 17:807-14.\u003c/li\u003e\n\u003cli\u003eZhou F, Geng J, Xu S, et al. FAM83A signaling induces epithelial-mesenchymal transition by the PI3K/AKT/Snail pathway in NSCLC. Aging (Albany NY) 2019; 11:6069-88.\u003c/li\u003e\n\u003cli\u003eRichtmann S, Wilkens D, Warth A, et al. FAM83A and FAM83B as Prognostic Biomarkers and Potential New Therapeutic Targets in NSCLC. Cancers (Basel) 2019; 11.\u003c/li\u003e\n\u003cli\u003eYu J, Hou M, Pei T. FAM83A Is a Prognosis Signature and Potential Oncogene of Lung Adenocarcinoma. DNA Cell Biol 2020; 39:890-9.\u003c/li\u003e\n\u003cli\u003eZhang JT, Lin YC, Xiao BF, et al. Overexpression of Family with Sequence Similarity 83, Member A (FAM83A) Predicts Poor Clinical Outcomes in Lung Adenocarcinoma. Med Sci Monit 2019; 25:4264-72.\u003c/li\u003e\n\u003cli\u003eZhang J, Sun G, Mei X. Elevated FAM83A expression predicts poorer clincal outcome in lung adenocarcinoma. Cancer Biomark 2019; 26:367-73.\u003c/li\u003e\n\u003cli\u003eZhou F, Wang X, Liu F, et al. FAM83A drives PD-L1 expression via ERK signaling and FAM83A/PD-L1 co-expression correlates with poor prognosis in lung adenocarcinoma. Int J Clin Oncol 2020; 25:1612-23.\u003c/li\u003e\n\u003cli\u003eLiu X, Fu M, Xia D, et al. Overexpression of FAM83A Is Associated with Poor Prognosis of Lung Adenocarcinoma. Journal of oncology 2022; 2022:8767333.\u003c/li\u003e\n\u003cli\u003eTravis WD, Brambilla E, Noguchi M, et al. International association for the study of lung cancer/american thoracic society/european respiratory society international multidisciplinary classification of lung adenocarcinoma. J Thorac Oncol 2011; 6:244-85.\u003c/li\u003e\n\u003cli\u003ePorcel JM, Palma R, Bielsa S, et al. TTF-1 and napsin A on cell blocks and supernatants of pleural fluids for labeling malignant effusions. Respirology 2015; 20:831-3.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Malignant pleural effusion, Lung adenocarcinoma, FAM83A, Immune infiltration, Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-4453916/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4453916/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: Malignant pleural effusion (MPE) is most frequently observed in patients with advanced lung adenocarcinoma (LUAD). Pleural fluid cytology is a less invasive\u003c/p\u003e\n\u003cp\u003eprocedure than pleural biopsy. Therefore, it is urgently required to discover new effective biomarkers for LUAD-associated pleural fluid cytology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e mRNA sequencing and clinical data of LUAD were downloaded from TCGA and OncoSG databases. Differential gene expression analysis, survival analysis and immune cell infiltration analysis were carried out on the LUAD datasets. The expression levels of FAM83A, TFF-1, and NapsinA were assessed in 94 pairs of LUAD tumor and adjacent normal tissues, as well as in pleural effusion cell blocks obtained from 40 LUAD and 21 non-neoplastic patients, using immunohistochemistry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eBioinformatics analysis demonstrated that FAM83A was screened out as a candidate biomarker for pleural fluid cytology due to its obvious difference in LUAD tissues and its influence on overall or disease-free survival, and histological grade. Immunohistochemistry showed that FAM83A expression was amplified in LUAD tissues compared with paired normal tissues in 89 out of 94 pairs. Immune cell infiltration analysis revealed that FAM83A expression was significantly correlated with immune cell infiltration, such as a positive association with macrophage infiltration. Meanwhile, FAM83A staining was positive in 37 cases of LUAD pleural effusions, and was negative in 20 cases of non-neoplastic pleural effusions. Additionally, the expression pattern of FAM83A in LUAD pleural effusions was relatively consistent with that of TFF-1 and NapsinA, and was even a little stronger in some LUAD pleural effusions that were weakly positive or negative for TTF1 or NapsinA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur work reveals FAM83A as a promising immune-related biomarker not only in LUAD biopsy specimens but also in LUAD pleural effusion. Our finding is of importance for providing a new and effective option for pleural fluid cytology.\u003c/p\u003e","manuscriptTitle":"FAM83A: A Prospective Diagnostic Biomarker with Immunological Relevance in Lung Adenocarcinoma- Associated Malignant Pleural Effusion","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-07 20:15:50","doi":"10.21203/rs.3.rs-4453916/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-05T08:43:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-28T08:18:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-27T01:03:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239281690680932568866438567583989091264","date":"2024-05-24T11:47:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195307190520708349454676638619709296410","date":"2024-05-22T12:24:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-22T11:43:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-22T06:28:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-22T06:27:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Oncology","date":"2024-05-21T09:55:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"dion","sideBox":"Learn more about [Discover Oncology](https://www.springer.com/12672)","snPcode":"","submissionUrl":"","title":"Discover Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7d1c88cc-5301-4d2e-9c64-14036fbbe975","owner":[],"postedDate":"June 7th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-18T09:14:08+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-07 20:15:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4453916","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4453916","identity":"rs-4453916","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}