Cancer Biomarkers - Volume 26, issue 4

Authors: Zhou, Chunhuan | Zhao, Juanjuan | Liu, Juanjuan | Wei, Sixi | Xia, Ying | Xia, Wansong | Bi, Ying | Yan, Zhiqiang | Huang, Hai

Article Type: Research Article

Abstract: Recent studies have shown that long noncoding RNAs (lncRNAs) have profound impacts on cancer development. In our previous study, we have confirmed that lncRNA small nucleolar RNA host gene 16 (SNHG16) is associated with poor prognosis and malignant phenotype of gastric cancer (GC). However, the biological function of lncRNA SNHG16 is still unclear. Here, we aimed to investigate the mechanisms underlying the roles of SNHG16 in GC. In this work, SNHG16 knockdown could inhibit epithelial-mesenchymal transition (EMT) and invasion of GC cells. Moreover, our results revealed that SNHG16 could promote EMT via down-regulation of Dickkopf WNT signaling pathway inhibitor 3 …(DKK3) in GC cells. In addition, SNHG16 was found to be upregulated whereas DKK3 was downregulated in tumor tissues compared with adjacent normal tissues. It showed that the expressions of SNHG16 and DKK3 were negatively correlated in clinical GC tissues. All these results suggested that SNHG16 promotes GC progression via regulation of DKK3 directly or indirectly. SNHG16 might be used as a putative biomarker for metastatic prediction in GC patients. Show more

Keywords: lncRNA SNHG16, epithelial-mesenchymal transition, DKK3, GC

DOI: 10.3233/CBM-190497

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 393-401, 2019

Authors: Wang, Yezhao | Xu, Suyuan | Chen, Yudan | Zheng, Xingyue | Li, Tianwen | Guo, Junming

Article Type: Research Article

Abstract: Gastric cancer is one of the most common cancers in the world. However, current medical technologies have not identified a reliable method to cure advanced gastric cancer, and early gastric cancer is difficult to diagnose. Therefore, we focused on circular RNAs (circRNAs) that have been proven to be involved in the carcinogenesis of gastric cancer. We first used quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to evaluate the expression levels of hsa_circ_0005654 in 301 tissues, including 122 healthy gastric mucosa samples, 68 paired tissues from early gastric cancer and adjacent nontumor mucosae obtained by submucosal dissection, and 43 chronic gastritis tissues. …Then, we analyzed the relationship between the expression levels of hsa_circ_0005654 and the clinicopathological characteristics of patients with early gastric cancer. We ultimately confirmed the clinical diagnostic value of hsa_circ_0005654 through generating receiver operating characteristic (ROC) curves and comparing the areas under the ROC curves (AUCs). Our data revealed that hsa_circ_0005654 was significantly downregulated in early gastric cancer tissues compared with matched normal mucosae (P < 0.001). Meanwhile, the expression levels of hsa_circ_0005654 in early gastric cancer tissues were also obviously lower than those in chronic gastritis tissues (P < 0.001). The AUCs of early gastric cancer tissues vs. paired normal adjacent mucosae, and that of early gastric cancer vs. healthy controls, were 0.927 and 0.924, respectively. These results clearly demonstrated that hsa_circ_0005654 may serve as a new and promising diagnostic biomarker for screening early gastric cancer. The AUC, sensitivity and specificity of hsa_circ_0005654 are significantly higher than those of present gastric cancer associated-biomarkers. Show more

Keywords: Early gastric cancer, circular RNA, hsa_circ_0005654, biomarker, clinical significance

DOI: 10.3233/CBM-190561

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 403-410, 2019

Authors: Cheng, Lin | Shi, Liang | Dai, Hong

Article Type: Research Article

Abstract: BACKGROUND: Breast cancer is a worldwide leading cause of cancer mortality and it is associated with numerous tumor suppressor genes and oncogenes. Growing evidence exists that different KLFs play pivotal roles of in human malignancies. However, the function of KLFs in breast cancer development has remained uncovered. OBJECTIVE: To explore the potential prognostic biomarkers among KLFs in breast cancer. METHODS: In the present study, by using multiple large open databases, such as Oncomine database, Kaplan-Meier Plotter, and bc-GenExMiner online software, we deeply analyzed the expressions and clinical values about KLFs in patients with …breast cancer. RESULTS: KLF4/5/8/9/10/15 were significantly down-regulated in breast cancer samples. KLF11 exerts significantly negative effect on the prognosis of patients, whereas expressions of KLF4/15 were associated with better prognosis. Moreover, the vital genes KLF4/11/15 showed significant association with clinical parameters including age, estrogen receptor, progesterone receptor, epidermal growth factor receptor-2, Scarff-Bloom-Richardson grade, and Nottingham prognostic index. CONCLUSIONS: Bioinformatics analysis suggested that KLF4/11/15, compared to other KLFs, might be potential prognostic indicators and treatment targets for breast cancer patients. Show more

Keywords: KLFs, breast cancer, bioinformatics analysis, prognosis

DOI: 10.3233/CBM-190199

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 411-420, 2019

Authors: Li, Xian Min | Cao, Li Li

Article Type: Research Article

Abstract: BACKGROUND: Metastatic gastric carcinoma (GC) is a typically incurable disease. The progression of anti-metastatic treatment is hampered because the underlying mechanisms regulating the metastasis of GC cell are not well illuminated. OBJECTIVE: Therefore, further elucidation of the molecular mechanism behind the metastatic traits of GC cells is needed for optimizing GC treatment. METHODS: The levels of GOLM1 and MMP13 in GC cells and tissues were measured by using qPCR assay. The growth of GC cells in vitro was detected using MTS and colony formation assays. The migration and invasion of GC cells …was analyzed using wound healing test and Transwell invasion assay. The level of MMP13 in GC cell was measured using immunoblotting and the level of GOLM1 was measured using immunofluorescence staining. The role of GOLM1 on the distant metastasis of GC SGC7910 cell was analyzed using experimental metastasis assay. Transplanted tumor model was constructed to analyze the influence of GOLM1 on GC cell growth in vivo . RESULTS: Here, we report that GOLM1 is over-expressed in GC and knockdown GOLM1 impairs the aggressive phenotypes of GC cell in vitro . Furthermore, downregulation of GOLM1 restrains the tumor growth of GC cell in nude mice. Nevertheless, upregulation of GOLM1 distinctly elevated the growth, migration ability and invasiveness of GC SGC7910 cell. Finally, GOLM1 increases the metastatic phenotypes of GC cell in a MMP13-dependent manner. CONCLUSIONS: Altogether, this investigation demonstrates the crucial function of GOLM1 in the progression of GC, which indicating GOLM1 as a potential target for GC treatment. Show more

Keywords: GOLM1, gastric carcinoma, MMP13, metastasis

DOI: 10.3233/CBM-190301

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 421-430, 2019

Authors: Zhu, Zhenpeng | He, Anbang | Lv, Tongde | Xu, Chunru | Lin, Lanruo | Lin, Jian

Article Type: Research Article

Abstract: Prolyl 4-hydroxylase, beta polypeptide (P4HB) protein has been found to be associated with tumorigenesis in many types of tumor, However, the relationship between P4HB and clear cell renal cell carcinoma (ccRCC) has not been clarified. In this study, we focus on the correlation between P4HB expression and ccRCC. Through the Cancer Genome Atlas (TCGA) database, Gene Expression Omnibus (GEO) database, our database and immunohistochemical (IHC) staining. Compared with adjacent normal tissues, both the mRNA and protein levels of P4HB in ccRCC tissues were enhanced. The Kaplan-Meier survival analysis showed that high expression of P4HB is correlated with poor prognosis in …both TCGA database and our own database. Multivariate survival analysis and Univariate analysis showed that P4HB expression and age are significantly correlative with poor prognose. All the results indicated that P4HB is correlated with poor prognosis in human clear cell renal cell carcinoma. Show more

Keywords: P4HB, ccRCC, TCGA, GEO

DOI: 10.3233/CBM-190450

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 431-439, 2019

Authors: Zhang, Xinpei | Liu, Bo | Zhang, Jilei | Yang, Xinrui | Zhang, Gaoqi | Yang, Siyuan | Wang, Jing | Shi, Jinlong | Hu, Kai | Wang, Jijun | Jing, Hongmei | Ke, Xiaoyan | Fu, Lin

Article Type: Research Article

Abstract: BACKGROUND : ACOT plays an important role in lipid metabolism and recent studies found that ACOT participates in some kinds of tumorigenesis. However, both the role of ACOT and its significance have not been revealed in AML. Therefore, we conduct this study in order to investigate the association between AML and ACOT , and hopefully contributed to the management of AML. METHODS : One hundred and fifty-six AML patients were enrolled in our study whose data were derived from the Cancer Genome Atlas database. There were 85 patients who received only chemotherapy and other 71 …patients underwent allo-HSCT. RESULTS : Patients in high ACOT7 group had a significant lower EFS and OS, while patients in high versus low expression levels of other types of ACOT showed no significant difference on the outcome. High level of ACOT7 related with poor outcome in both chemotherapy-only group and HSCT group. CONCLUSIONS : High expression level of ACOT7 indicates unfavorable outcome in AML patients. Allo-HSCT could not overcome the unfavorable effect of ACOT7 in these patients. Show more

Keywords: ACOT7, acute myeloid leukemia, chemotherapy, allo-HCST, prognosis

DOI: 10.3233/CBM-182287

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 441-449, 2019

Authors: Su, Fei | Wang, Bo-Fang | Zhang, Tao | Hou, Xiao-Ming | Feng, Mao-Hui

Article Type: Research Article

Abstract: BACKGROUND: It has been documented that transient receptor potential melastatin 7 (TRPM7) plays a pivotal role in the development of multiple cancers. However, the role of TRPM7 in human colorectal cancer (CRC) is poorly understood. Therefore, the aim of this study was to investigate the expression and significance of TRPM7 in CRC. METHODS: In this study, TRPM7 expression was first investigated in Gene Expression Omnibus (GEO), and then validated it with the data from our medical center. CCK-8, colony survival, transwell, and flow cytometry assays were employed to evaluate the effects of TRPM7 knockdown on the …CRC cell proliferation, migration, and invasion, as well as cell cycle and apoptosis. RESULTS: We observed markedly increased TRPM7 expression in CRC tissues. CRC patients with high expression of TRPM7 suggested deeper tumor infiltration, positive lymph node metastasis, distant metastasis, and advanced clinical stage. In addition, TRPM7 was also overexpressed in CRC cell lines. Downregulated TRPM7 in vitro suppressed CRC cell proliferation, migration, and invasion, as well as triggered cell cycle arrest at the G0/G1 phase, reduced the S phase, and promoted apoptosis. Importantly, decreased TRPM7 in CRC cells reversed the epithelial-mesenchymal transition (EMT) status, accompanied by downregulation of N-cadherin and upregulation of E-cadherin. CONCLUSION: Our study indicated that the expression of TRPM7 was positively correlated with tumor infiltration, lymph node metastasis, distant metastasis and clinical stage of CRC. Besides, decreased TRPM7 in vitro inhibited CRC cell proliferation, migration and invasion by modulating EMT. Show more

Keywords: TRPM7, colorectal cancer, proliferation, migration, invasion, epithelial-mesenchymal transition

DOI: 10.3233/CBM-190666

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 451-460, 2019

Authors: Su, Xuan | Lin, Li-Wen | Weng, Jie-Ling | Chen, Shu-Wei | Yang, Xin-Hua | Zhou, Da-Lei | Long, Ya-Kang | Shao, Qiong | Ye, Zu-Lu | Peng, Jun-Ling | Deng, Ling | He, Cai-Yun | Yang, An-Kui

Article Type: Research Article

Abstract: This study aimed to evaluate the association of potential functional tagging single nucleotide polymorphisms (tagSNPs) in BRAF and TSHR with papillary thyroid cancer (PTC). Two tagSNPs (rs6464149 and rs7810757) in BRAF and six tagSNPs (rs17630128, rs2075179, rs7144481, rs2371462, rs2268477, and rs2288496) in TSHR were genotyped in 300 cases of PTC and 252 healthy controls. There was no difference in the genotype frequencies of BRAF and TSHR between PTC patients and control subjects, suggesting no contribution of BRAF or TSHR polymorphisms to the susceptibility to PTC. We observed that a tagSNP located in …the 3’ untranslated region of TSHR , rs2288496, could affect the incidence of lymph node metastasis (LNM). The variant TC and TC + CC genotypes conferred an increased risk of LNM (for TC vs . TT: odds ratio (OR) = 2.01, 95% confidence interval (CI): 1.07–3.77; P = 0.030; for TC + CC vs . TT: OR = 1.87, 95% CI: 1.04–3.39, P = 0.038). Moreover, subjects carrying variant genotypes had higher TSH levels and lower thyroxine (T4) and Anti-TG levels compared with those in subjects carrying common genotypes. Our findings showed that PTC patients carrying the TSHR rs2288496 TC and CC variants were associated with higher TSH level and lower T4 and Anti-TG levels and were prone to developing LNM. To confirm these results, additional studies and functional experiments, especially in other ethnic populations, are needed. Show more

Keywords: BRAF, TSHR, TSH, polymorphism, differentiated thyroid cancer

DOI: 10.3233/CBM-190630

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 461-470, 2019

Authors: Gutkin, Dmitriy W. | Shurin, Michael R. | El Azher, Mounia Alaoui | Shurin, Galina V. | Velikokhatnaya, Liudmila | Prosser, Denise | Shin, Namhee | Modugno, Francesmary | Stemmer, Paul | Elishaev, Esther | Lokshin, Anna

Article Type: Research Article

Abstract: Ovarian cancer is the leading cause of death among gynecologic diseases in the USA and Europe. High-grade serous carcinoma (HGSC) of the ovary, the most aggressive type of ovarian cancer, is typically diagnosed at advanced stages when the 5-year survival is dismal. Since the cure rate for stage I HGSC is high, early detection of localized initial disease may improve patient outcomes. Serous tubal intraepithelial carcinoma (STIC) is considered to be a precursor lesion of HGSC. Discovery of biomarkers associated with STIC could aid in the development of an HGSC screening algorithm. Using immunohistochemical staining, we have demonstrated overexpression of …UCHL1, ADAMTS13, and GAPDH in patients’ STIC lesions, but not in cancer-free fallopian tubes. We additionally demonstrated a marked increase of T cells in perineoplastic stroma surrounding STIC lesions (largely CD4 + cells), but not in normal fallopian tubes and HGSC. FOXP3 + T regulatory cells are absent in STIC lesions but are present in HGSC. These observations indicate the microenvironment surrounding a STIC lesion may be immune promoting in contrast to the immune suppressive microenvironment of invasive carcinoma. In summary, we have identified UCHL1, ADAMTS13, and GAPDH as novel potentially useful markers associated with early stages of HGSC tumorigenesis and possibly contribute to STIC immunogenicity. The lack of immune suppression in the STIC microenvironment indicates that the immune system can still recognize and keep STIC controlled at this stage of the tumor development. Show more

Keywords: Ovarian cancer, high-grade serous carcinoma (HGSC), serous tubal intraepithelial carcinoma (STIC), biomarkers, immune response

DOI: 10.3233/CBM-190528

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 471-479, 2019

Authors: Yao, Yaoshan | Liu, Longyang | He, Wenhua | Lin, Xian | Zhang, Xuan | Lin, Zhongqiu | Zeng, Zhaoyang | Guo, Suiqun

Article Type: Research Article

Abstract: The aim of the present study was to investigate kinesin family member 7 (KIF7) expression in epithelial ovarian cancer tissues (paraffin-embedded tissues and fresh) and to explore its expression, association with clinicopathological parameters and prognostic value in patients with epithelial ovarian cancer. A total of 113 paraffin-embedded tumor tissues of epithelial ovarian cancer patients diagnosed and operated at the memorial hospital of Sun Yat-sen University Between December 2009 and March 2017 and 41 paratumor tissues were collected for the present study and were assessed for KIF7 expression using immunohistochemistry. Furthermore, 22 fresh epithelial ovarian cancer tissues and their matched paratumor …tissues were collected from the Integrated Hospital of Traditional Chinese Medicine, Southern Medical University, between August 2013 and March 2019 and subjected to reverse-transcription quantitative PCR analysis to detect the mRNA expression of KIF7. The expression of KIF7 was lower in cancer tissues than in paratumor tissues, and KIF7 expression was associated with recurrence-free survival and overall survival in epithelial ovarian cancer patients. Furthermore, multivariate logistic regression analysis indicated that low KIF7 expression was an independent predictor of poor survival in patients with epithelial ovarian cancer. In conclusion, KIF7 has a tumor suppressor role in epithelial ovarian cancer and is a useful independent prognostic predictor. It may hold important value for the clinical diagnosis and treatment of epithelial ovarian cancer. Show more

Keywords: kinesin family member 7, epithelial ovarian cancer, prognosis, immunohistochemistry, reverse-transcription quantitative PCR

DOI: 10.3233/CBM-190328

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 481-489, 2019

Authors: Ma, Ge | Song, Guoxin | Zou, Xuan | Shan, Xia | Liu, Qingxie | Xia, Tiansong | Zhou, Xin | Zhu, Wei

Article Type: Research Article

Abstract: OBJECTIVE: To determine the utility of plasma microRNAs (miRNAs) as biomarkers in cervical cancer (CC). METHODS: Some studies were conducted about the specific expression of plasma miRNAs in the diagnosis of CC. Plasma samples of 97 CC patients and 87 normal controls (NCs) were used to identify dysregulation of miRNAs in the training, testing, and external validation phases. Receiver operating characteristic (ROC) curves and area under the ROC curve (AUC) were used to evaluate the sensitivity and specificity of identified individual miRNAs and miRNA panels for the diagnosis of CC. Expression levels of specific miRNAs were …also examined in plasma exosomes and tissue samples of CC patients. RESULTS: Four plasma miRNAs (miR-146a-5p, miR-151a-3p, miR-2110 and miR-21-5p) which showed up-regulation were identified and validated in CC patients. A panel of the four miRNAs were constructed as potential diagnostic markers for CC. The AUCs of the panel of these four-miRNAs for the training, testing, and external validation phases were 0.911, 0.774, and 0.786, respectively. miR-146a-5p and miR-21-5p levels were all up-regulated in CC tissue specimens, whereas miR-146a-5p, miR-151a-3p, and miR-2110 levels were up-regulated in plasma exosomes. CONCLUSION: The signature of the four-miRNAs identified in peripheral plasma is a promising novel biomarker for the diagnosis of CC. Show more

Keywords: Plasma microRNA, cervical cancer, diagnosis, exosome, qRT-PCR

DOI: 10.3233/CBM-190256

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 491-500, 2019

Authors: Li, Jian | Jin, Boxun | Wang, Tiezheng | Li, Wenlei | Wang, Zhenshun | Zhang, Haitao | Song, Yunjun | Li, Ning

Article Type: Research Article

Abstract: BACKGROUND: The identification of high-sensitivity biomarkers for detection of hepatocellular carcinoma (HCC) from high-risk individuals is essential. OBJECTIVE: The present study was undertaken to identify and validate serum microRNAs (miRNAs) as potential biomarkers for hepatitis C virus (HCV)-related HCC. METHODS: Illumina sequencing was employed to screen the expression profiles of miRNAs in serum samples of HCV-related HCC patients and liver cirrhosis (LC) patients. RT-qPCR was used to confirm the altered miRNAs between the two groups. Moreover, candidate miRNAs were examined in serum samples of 40 HCC patients, 54 LC patients, 55 patients with …chronic HCV hepatitis and 45 healthy controls. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic performance of the miRNAs for the detection of HCC. RESULTS: Four miRNAs (miR-122-5p, miR-331-3p, miR-494-3p, miR-224-5p) were significantly increased and two miRNAs (miR-185-5p, miR-23b-3p) were significantly decreased in HCC patients compared to LC patients. ROC curve analysis demonstrated that the six miRNAs could be used as potential biomarkers for HCC detection. Combination of the six miRNAs could efficiently detect HCC in LC patients with the area under the ROC curve (AUC) of 0.995 and combination of the six miRNAs also provided high diagnostic accuracy (AUC = 0.961) for detection of HCC in non-HCC subjects. CONCLUSIONS: The six serum miRNAs can be utilized as a surrogate and non-invasive biomarker for HCV-related HCC diagnosis. Show more

Keywords: Hepatocellular carcinoma, MiRNAs, serum, hepatitis c virus, liver cirrhosis

DOI: 10.3233/CBM-181970

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 501-512, 2019

Authors: Manai, Maroua | Abdeljaoued, Syrine | Goucha, Aïda | Adouni, Olfa | Bettaieb, Ilhem | Bouzaien, Hatem | Rahal, Khaled | Birnbaum, Daniel | Bertucci, François | Gamoudi, Amor

Article Type: Research Article

Abstract: BACKGROUND: Male breast cancer (MBC) is a rare and aggressive disease. Thus, identification of new therapeutic targets is crucial. OBJECTIVE: Our objective was to evaluate the protein expression of MARCKS (Myristoylated Alanine-Rich C-Kinase Substrate) in MBC and to investigate its prognostic value. MATERIALS AND METHODS: MARCKS protein expression in tumor and stromal cells was analyzed by immunohistochemistry (IHC) in a retrospective series of 96 pre-chemotherapy MBC samples and 80 normal breast samples, from Tunisian patients treated at Salah Azaiez Institute. Correlations were searched between MARCKS expression and clinicopathological features including overall survival (OS). …RESULTS: MARCKS was overexpressed in epithelial tumor cells in 66% of the MBC samples versus 26% of normal samples (p = 1.40 × 10 - 7 ). Such positive MARCKS expression in epithelial tumor cells was associated with positive HER2 status (p = 4.0 × 10 - 3 ). It was associated with shorter OS in uni-and multivariate analysis. By contrast, stromal IHC MARCKS expression was correlated only with tumor grade. CONCLUSION: MARCKS tumor cell overexpression might in part explain the aggressiveness and the poor prognosis of MBC. MARCKS can represent a potential therapeutic target for MBC. Show more

Keywords: MARCKS, expression, male breast cancer, immunohistochemistry, survival

DOI: 10.3233/CBM-190637

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 513-522, 2019

Authors: Lv, Min | Wang, Fen | Wang, Xiaoyan | Zhang, Cuilan

Article Type: Research Article

Abstract: OBJECTIVE: This study aimed to assess the diagnostic value of human epididymis protein 4 (HE4) in the pleural effusion of lung cancer patients. METHODS: HE4 protein in the pleural effusion of 60 lung cancer patients was measured by electrochemiluminescence, in parallel with those from 56 patients with benign lung disease, and the association with malignant pleural effusion was evaluated. RESULTS: The level of HE4 in samples from lung cancer patients was significantly higher than the level for those with benign lung lesions (P =  0.001) and patients with lung …adenocarcinoma showed significantly higher levels of HE4 than those with squamous cell carcinoma and small cell carcinoma (P =  0.002 and P =  0.034, respectively). Using an optimal threshold of 652.2 pmol/L, the HE4 level distinguished malignant lung cancer from benign lesions with a sensitivity of 78.3% and a specificity of 75.0%. Moreover, the HE4 level differentiated adenocarcinoma from benign lesions with a sensitivity of 75.9% and a specificity of 85.7% when a threshold of 744.05 pmol/L was used. However, there was no significant difference in the 2 year survival rates of lung cancer patients with high and low HE4 concentrations in pleural fluid (P =  0.882). In addition, there was no significant difference in HE4 levels between tuberculous and inflammatory pleural effusions (P =  0.309). CONCLUSION: HE4 in the pleural fluid of lung cancer patients can be valuable in the diagnosis of malignant pleural effusion; however, it does not correlate with the prognosis of patients. Show more

Keywords: Lung cancer, malignant pleural effusion, diagnosis, prognosis, HE4, electrochemiluminescence

DOI: 10.3233/CBM-190840

Citation: Cancer Biomarkers, vol. 26, no. 4, pp. 523-528, 2019