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The frequency of PTEN germline mutations in Chinese breast cancer patients: The PTEN gene may not be closely associated with breast cancer in the Chinese population
Gene 744 (2020) 144630
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
The frequency of PTEN germline mutations in Chinese breast cancer patients: The PTEN gene may not be closely associated with breast cancer in the Chinese population
T
Yu Wua,c,1, Dabing Huangb,1, Huanhuan Zhangd, Xiaoling Wengd,e, Honglian Wangd, ⁎ ⁎ Qinghua Zhouf, Ying Wuf, Yi Shenf, Baining Sund, , Zhen Hue, a
State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Department, School of Life Science, Fudan University, Shanghai 200436, PR China Department of Oncology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui Province, 230001, PR China c Department of Biostatistics and Computational Biology, School of Life Sciences, Fudan University, Shanghai 200436, PR China d AITA Medical Research Institute, Shanghai 200000, PR China e Department of Breast Surgery, Fudan University Shanghai Cancer Center, 270 Dongan Road, Xuhui, Shanghai 200032, PR China f Department of Surgery, Luwan Branch of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Breast neoplasms PTEN Biological markers
Background: PTEN is a tumour suppressor gene that has been proven to be related to breast cancer incidence and tumour progression. The aim of this study was to investigate the frequency of PTEN mutations in breast carcinomas in China and the relationships of PTEN mutations with clinicopathological parameters and clinical outcomes. Material and methods: Trimmomatic, Burrows-Wheeler Aligner (BWA), ANNOVAR, SAMtools, and Sanger sequencing were used to analyse PTEN mutations and identify variants in Chinese breast cancer. The frequency of PTEN mutations and the relationships of PTEN mutations with clinicopathological parameters and clinical outcomes were evaluated in breast carcinomas in China. Results: The rate of PTEN germline mutation was 0.23% (n = 9) among 3955 unselected primary breast cancer patients. Of these 9 patients, 2 carried pathogenic mutations, and both were identified as having infiltrative carcinoma. One patient had a family history. The other 7 patients carried only PTEN germline variants that were not identified as pathogenic mutations. Conclusions: We studied the frequency of PTEN germline mutations in a sequential cohort of Chinese breast carcinoma patients. Based on these data, we hypothesize that the germline mutation of the PTEN gene is not closely related to the occurrence of breast cancer in the Chinese population. In the clinic, the PTEN germline mutation cannot be used as the basis for the detection of breast cancer.
1. Background Breast cancer is currently the most common cancer in women, with more than 1.3 million new cases occurring every year around the world. In the past few years, the outcomes of breast cancer have improved substantially as a result of recent advancements in the understanding of breast cancer biology and the development of new protocols for individual treatments (Torre et al., 2015). However, breast cancer
remains the leading cause of death in women, and approximately 450,000 persons have died from breast cancer (Li et al., 2017). It is important to identify potential biomarkers that could be used to screen high-risk patients and predict breast cancer prognosis. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), also known as mutated in multiple advanced cancers 1 (MMAC1), was identified on chromosome 10q23 as a tumour suppressor gene (Garcia et al., 1999). PTEN has recently been found to be
Abbreviations: ACMG, American College of Medical Genetics; AMP, Association for Molecular Pathology; IDC, Invasive Ductal Carcinoma; TNBC, Triple-Negative Breast Cancer ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Sun), [email protected] (Z. Hu). 1 Co-first authors. https://doi.org/10.1016/j.gene.2020.144630 Received 4 November 2019; Received in revised form 17 March 2020; Accepted 26 March 2020 Available online 28 March 2020 0378-1119/ © 2020 Published by Elsevier B.V.
Gene 744 (2020) 144630
Y. Wu, et al.
frequently mutated in human cancers, such as prostate cancer (Pesche et al., 1998), ovarian cancer (Han et al., 2018), breast cancer (Rhei et al., 1997), endometrial cancer (Risinger et al., 1997) and melanoma (Guldberg et al., 1997). A report showed that partial loss of normal PTEN tumour suppressor function could be compounded by additional disruption caused by the expression of inactive mutant PTEN protein (Papa et al., 2014). Many mechanisms can cause this loss of function of the PTEN gene, including missense and insertion mutations in the PTEN gene, deletion of the PTEN gene, reduced expression of PTEN because of promoter methylation, and suppression of PTEN enzyme activity (Leslie and den Hertog, 2014). Germline mutations in PTEN are also observed in Cowden syndrome (CS), which is an inherited cancer syndrome associated with a high risk of breast cancer (Liaw et al., 1997). In sporadic breast cancer, the frequency and relevance of PTEN alterations have not been elucidated completely. Therefore, we decided that a comprehensive investigation would be useful to clarify the mutation status and prognostic significance of PTEN. In this study, the frequency of PTEN mutations in breast carcinomas in China and the relationship between PTEN mutations, clinicopathological parameters and clinical outcomes were evaluated according to all of the currently available evidence.
Table 1 Clinical characteristics of the study participants.
2. Material and methods 2.1. Ethics statement Study subjects were recruited at 19 clinical centers in 11 Chinese provinces between 2012 and 2018. Before beginning the work, we orally explained our study objectives and procedures to all participants and obtained their permission to include their blood specimens in our study. Moreover, written informed consent was provided by each participant. The present study was reviewed and approved by the ethics committees of all the hospitals involved. 2.2. Sample acquisition A total of 3955 Chinese patients who were diagnosed with breast cancer were included in the present study. Clinical and pathological information, including age, ethnicity, menopausal status, type of tumour, disease stage, lymph node status and tumour size, were collected. Family history was defined as breast cancer patients having one or more cancer patients (any kind of cancer) as first-, second-, or third-degree relatives. Informed written consent was obtained from all participants. We declare that the experiments described in this study were performed in compliance with the current laws of the People’s Republic of China.
Variables
N
Percent (%)
Age at diagnosis ≤50 > 50 na
1779 737 1211
47.73% 18.63% 30.62%
Histology infiltrative carcinoma non-infiltrative carcinoma others na
2268 179 276 1232
57.35% 4.53% 6.98% 31.15%
Tumor size ≤1.5 cm > 1.5 cm na
841 2223 891
21.26% 56.21% 22.53%
ER status Positive Negative na
2351 1140 464
59.44% 28.82% 11.73%
PR status Positive Negative na
2263 1231 461
57.22% 31.13% 11.66%
HER2 status Positive Negative na
720 2392 843
18.20% 60.48% 21.31%
Molecular typing Luminal-A Luminal-B TNBC HER2 overexpression na
139 943 869 268 1736
3.51% 23.84% 21.97% 6.78% 43.89%
Family history Yes No na Total
902 2506 547 3955
22.81% 63.36% 13.83% 100.00%
3. Results 3.1. Patient characteristics We have summarized the clinicopathological characteristics of the patients in Table 1. There were 3955 unselected breast cancer patients included in this study, and 9 patients were tested for PTEN mutations. The mean age of these breast cancer patients was 58.83 years. A total of 2268 (57.35%) patients were diagnosed with infiltrative carcinoma, and 179 (4.53%) patients were diagnosed with non-infiltrative carcinoma. A total of 2351 (59.44%) patients had ER-positive status, and 2263 (57.22%) patients had PR-positive status. A total of 720 (18.20%) patients presented with a HER2-positive status. A total of 869 (21.97%) patients were classified with the triple-negative breast cancer (TNBC) molecular type, and 268 (6.78%) were classified with the HER2 overexpression molecular type. A total of 2506 (63.36%) patients had no family history.
2.3. Mutation analysis and validation First, Trimmomatic (Bolger et al., 2014) was applied to trim and crop the read pairs (FASTQ data) from the sequencing system. Then, the Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) was run to map the resulting reads to the hg19 reference genome. SAMtools (Li et al., 2009) was used to sort and index the mapping result, and then, the mpileup command was used to identify the variants. The variants were annotated with ANNOVAR (Wang et al., 2010), and the mutations were classified into 5 different categories according to the classification protocol of the professional practice guidelines of the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP). Multiple databases were used to check these classified alterations, such as ClinVar (public archive of interpretations of clinically relevant variants), Human Genome Mutation Database (HGMD), and dbSNP (a single-nucleotide polymorphism database). We validated the mutations classified as pathogenic mutations or probable pathogenic mutations by Sanger sequencing and achieved 100% concordance.
3.2. Prevalence of germline mutations in PTEN Germline mutations of PTEN were tested in a large cohort of 3955 unselected breast cancer patients using a gene panel. We validated the mutations classified as pathogenic mutations or probable pathogenic mutations. In the results, a total of 8 mutations were identified in 9 of 3955 (0.23%) unselected breast cancer patients (Table 2). Of these 8 mutations, 4 were SNPs, and 4 were the first germline mutations. Seven variants were in exon 9 of the PTEN mutation carriers, followed by one 2
Gene 744 (2020) 144630
Y. Wu, et al.
Table 2 Mutations and SNPs Identified in the Study Cohort. HGMD＿prot NP_000305.3:p.Q245* NP_000305.3:p.Y176C
Sig
Geno
Annotation
Gene＿id
hgvs＿c
3-RareModerate 5-ClinVarDamaging 3-RareModerate VUS VUS
0*1 0*1 0*1 0*1 0*1
ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862
3-RareModerate 3-RareModerate 5-ClinVarDamaging 3-RareModerate
0*1 0*1 0*1 0*1
missense_variant stop_gained missense_variant missense_variant splice_donor_variant&splice_region_variant& intron_variant missense_variant missense_variant frameshift_variant missense_variant
ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862
hgvs＿p
Rank
Exon_number
c.949G > A p.Val317Ile c.733C > T p.Gln245* c.527A > G p.Tyr176Cys c.949G > A p.Val317Ile c.253+4G > A
8 7 6 8 4
9 9 9 9 8
c.134 T > C c.644 T > C c.107delG c.923G > A
2 7 2 8
9 9 9 9
p.Val45Ala p.Phe215Ser p.Gly36fs p.Arg308His
(p. Gln245*), a stop-gained variant, and the other variant was chr10:89653806c.107delG (p. Gly36fs), a frameshift variant. The first pathogenic PTEN mutation was revealed in a 41-year-old woman presenting ER- and PR-positive status as well as HER2-negative status. She was diagnosed with left, stage I, non-specific infiltrating carcinoma. The second mutation identified in a 50-year-old woman who presented with HER2-positive status and ER/PR-negative status. She was identified with right infiltrating ductal carcinoma, and she also had a family history (her brother died of gastric carcinoma). In addition, among the 9 individuals who carried PTEN mutations, one patient was diagnosed with Paget disease, 2 had IDC, 4 had ER- and PR-positive status, and 2 presented with HER2-negative status. One patient was identified with TNBC molecular type, HER2 overexpression molecular type was detected in 2 individuals, luminal-A molecular type was also detected in 2 patients, and one patient presented luminal-B molecular type. Owing to the partial loss of clinical data, we failed to determine the other patients’ genotypes. In conclusion, the frequency determined in our study was similar to that reported by Li et al. (2018) but significantly differed from the result of Wu et al. (2016). The study by Wu Y et al. focused on the Chinese human population, while most patients in our cohort lived in Shanghai. Therefore, we considered that the regional disparity might account for the different frequencies of PTEN germline mutations in Chinese breast cancer patients. In addition, the rate of PTEN mutation in foreign breast cancer patients was also higher than that in our results in the Chinese population, which might be caused by the heterogeneity of these populations. According to our data, the rate (0.23%) of PTEN germline mutations in Chinese breast cancer patients was too small, and we hypothesized that the germline mutation of the PTEN gene was not directly related to the occurrence of breast cancer. Clinically, the PTEN gene might not be a biomarker in Chinese breast cancer patients, and germline mutation of the PTEN gene can still not be used as the basis for the detection of breast cancer.
mutation in exon 8. In the 9 PTEN mutation carriers, one mutation was a frameshift variant, one was a stop-gained variant, one was identified as a splice donor variant, splice region variant and intron variant, and others were missense variants. The frameshift variant and the stopgained variant were identified as pathogenic mutations. The frameshift variant was on chr10:89653, the stop-gained variant was on chr10:89717, and the splice donor variant was on chr10:89690. None of these variants has been included in the dbSNP database. 3.3. Clinical features and mutations Two patients who carried pathogenic PTEN mutations were both diagnosed with infiltrative carcinoma, but one of them had ER- and PRpositive status with HER2-negative status, while the other presented with HER2-positive status and ER- and PR-negative status. One patient was diagnosed with Paget disease, and one had invasive ductal carcinoma (IDC). The tumour sizes of 5 PTEN mutation carriers were larger than 1.5 cm. Four patients had ER- and PR-positive status, and 2 patients presented with HER2-negative status. One patient was classified as TNBC molecular type, 2 were HER2 overexpression molecular type, 2 were luminal-A molecular type, and one was luminal-B molecular type. One PTEN mutation carrier had a family history involving a brother who died of gastric carcinoma. 4. Discussion PTEN is the first identified tumour suppressor with lipid phosphatase activity. A report has demonstrated that PTEN is mutated or inactivated in a number of malignant tumours, including neuroglioma and endometrial, prostate, breast, liver and thyroid cancers (Lu et al., 1999). Somatic mutations in the PTEN gene were found at high frequency in breast cancer, with 3.8% in the Catalogue of Somatic Mutations in Cancer (COSMIC) database (Forbes et al., 2017). According to TCGA (The Cancer Genome Atlas) data, the percentage of PTEN was 3.2% (16/507). However, the rate of germline mutation of the PTEN gene was not that high. Recently, a study reported that the somatic mutation frequency of PTEN in a cohort of Chinese breast cancer patients was 0.32% (1/313) (Li et al., 2018), while another report showed that the percentage of PTEN mutations was 0.06% (5/8085) (Sun et al., 2017) in the Chinese population. In addition, Wu Y et al. (Wu et al., 2016) showed that the mutation frequency of the PTEN gene was 1.15% (2/131) among breast cancer patients in the China Hunan population. In addition, Esther Rhei et al. (Garcia et al., 1999) showed that one germline mutation of the PTEN gene was identified in 1 of 54 (1.85%) American primary breast cancers, and a retrospective study (Fulket al., 2019) showed a PTEN mutation rate of 0.42% (n = 7) in 1650 breast cancer and uterine cancer (BUC) patients. In our study, we determined that 9 patients carried germline mutations in 3955 unselected breast cancer patients (0.23%), as shown in Table 2, and 2 (0.05%, 2/3955) mutations were identified as pathogenic mutations in the PTEN gene; one was chr10:89717708c.733C > T
CRediT authorship contribution statement Yu Wu: Data curation, Formal analysis. Dabing Huang: Conceptualization, Investigation, Methodology, Funding acquisition. Huanhuan Zhang: Investigation, Methodology. Xiaoling Weng: Data curation, Formal analysis, Writing - original draft. Honglian Wang: Data curation, Formal analysis. Qinghua Zhou: Investigation, Methodology. Ying Wu: Investigation, Methodology. Yi Shen: Investigation, Methodology. Baining Sun: Data curation, Formal analysis, Writing - original draft. Zhen Hu: Conceptualization, Writing review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 3
Gene 744 (2020) 144630
Y. Wu, et al.
Acknowledgements
Leslie, N.R., den Hertog, J., 2014. Mutant PTEN in cancer: worse than nothing. Cell 157 (3), 527–529. Li, H., Durbin, R., 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25 (14), 1754–1760. Li, G., Guo, X., Chen, M., Tang, L., Jiang, H., Day, J.X., et al., 2018. Prevalence and spectrum of AKT1, PIK3CA, PTEN and TP53 somatic mutations in Chinese breast cancer patients. PLoS ONE 13 (9), e0203495. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., et al., 2009. Genome project data processing S: the sequence alignment/map format and SAMtools. Bioinformatics 25 (16), 2078–2079. Li, S., Shen, Y., Wang, M., Yang, J., Lv, M., Li, P., et al., 2017. Loss of PTEN expression in breast cancer: association with clinicopathological characteristics and prognosis. Oncotarget. 8 (19), 32043–32054. Liaw, D., Marsh, D.J., Li, J., Dahia, P.L., Wang, S.I., Zheng, Z., 1997. Germline mutations of the PTEN gene in Cowden disease, and inherited breast and thyroid cancer syndrome. Nat. Genet. 16 (1), 64–67. Lu, Y., Lin, Y.Z., LaPushin, R., Cuevas, B., Fang, X., Yu, S.X., et al., 1999. The PTEN/ MMAC1/TEP tumor suppressor gene decreases cell growth and induces apoptosis and anoikis in breast cancer cells. Oncogene 18 (50), 7034–7045. Papa, A., Wan, L., Bonora, M., Salmena, L., Song, M.S., Hobbs, R.M., et al., 2014. Cancerassociated PTEN mutants act in a Dominant-negative Manner to suppress PTEN protein function. Cell 157 (3), 595–610. Pesche, S., Latil, A., Muzeau, F., Cussenot, O., Fournier, G., Longy, M., et al., 1998. PTEN/ MMAC1/TEP1 involvement in primary prostate cancers. Oncogene 16 (22), 2879–2883. Rhei, E., Kang, L., Bogomolniy, F., Federici, M.G., Borgen, P.I., Boyd, J., 1997. Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in primary breast carcinomas. Cancer Res. 57 (17), 3657–3659. Risinger, J.I., Hayes, A.K., Berchuck, A., Barrett, J.C., 1997. PTEN/MMAC1 mutations in endometrial cancers. Cancer Res. 57 (21), 4736–4738. Sun, J., Meng, H., Yao, L., Lv, M., Bai, J., Zhang, J., et al., 2017. Germline mutations in cancer susceptibility genes in a large series of unselected breast cancer patients. Clin. Cancer Res. 23 (20), 6113–6119. Torre, L.A., Bray, F., Siegel, R.L., Ferlay, J., Lortet-Tieulent, J., Jemal, A., 2015. Global cancer statistics. CA Cancer J. Clin. 65 (2), 87–108. Wang, K., Li, M., Hakonarson, H., 2010. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38 (16), e164. Wu, Y., Jiang, B., Dai, X., Hu, X., Wang, S., Jiang, P., et al., 2016. PTEN and NBS1 gene mutations in familial breast cancer and early-onset breast cancer from Hunan Province in China. Zhong Nan Da Xue Xue Bao Yi Xue Ban 41 (2), 121–126.
This work was supported by the AITA Medical Research Institute. We thank Professor Liu Yun for his assistance with English language editing. We are grateful to all the medical institutions in China and the medical association for providing us with cancer incidence data. Funding This project was supported by the Fundamental Research Funds for the Central Universities (WK9110000086), the postdoctoral research funding of Anhui Province in 2019 (2019B371) and the Performance Project of Anhui Provincial Key Laboratory of Tumour Immunotherapy and Nutrition Therapy (1606c08236). References Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30 (15), 2114–2120. Forbes, S.A., Beare, D., Boutselakis, H., Bamford, S., Bindal, N., Tate, J., et al., 2017. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res. 45 (D1), D777–D783. Fulk, K., Milam, M.R., Li, S., Yussuf, A., Black, M.H., Chao, E.C., et al., 2019. Women with breast and uterine cancer are more likely to harbor germline mutations than women with breast or uterine cancer alone: a case for expanded gene testing. Gynecol Oncol pii: S0090-8258(18)31535-X. Garcia, J.M., Silva, J.M., Dominguez, G., Gonzalez, R., Navarro, A., Carretero, L., et al., 1999. Allelic loss of the PTEN region (10q23) in breast carcinomas of poor pathophenotype. Breast Cancer Res. Treat. 57 (3), 237–243. Guldberg, P., Thor Straten, P., Birck, A., Ahrenkiel, V., Kirkin, A.F., Zeuthen, J., 1997. Disruption of the MMAC1/PTEN gene by deletion or mutation is a frequent event in malignant melanoma. Cancer Res. 57 (17), 3660–3663. Han, C.Y., Patten, D.A., Richardson, R.B., Harper, M.E., Tsang, B.K., 2018. Tumor metabolism regulating chemosensitivity in ovarian cancer. Genes Cancer 9 (5–6), 155–175. https://doi.org/10.18632/genesandcancer.176.
4
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short communication
The frequency of PTEN germline mutations in Chinese breast cancer patients: The PTEN gene may not be closely associated with breast cancer in the Chinese population
T
Yu Wua,c,1, Dabing Huangb,1, Huanhuan Zhangd, Xiaoling Wengd,e, Honglian Wangd, ⁎ ⁎ Qinghua Zhouf, Ying Wuf, Yi Shenf, Baining Sund, , Zhen Hue, a
State Key Laboratory of Genetic Engineering and Collaborative Innovation Center for Genetics and Department, School of Life Science, Fudan University, Shanghai 200436, PR China Department of Oncology, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui Province, 230001, PR China c Department of Biostatistics and Computational Biology, School of Life Sciences, Fudan University, Shanghai 200436, PR China d AITA Medical Research Institute, Shanghai 200000, PR China e Department of Breast Surgery, Fudan University Shanghai Cancer Center, 270 Dongan Road, Xuhui, Shanghai 200032, PR China f Department of Surgery, Luwan Branch of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, PR China b
ARTICLE INFO
ABSTRACT
Keywords: Breast neoplasms PTEN Biological markers
Background: PTEN is a tumour suppressor gene that has been proven to be related to breast cancer incidence and tumour progression. The aim of this study was to investigate the frequency of PTEN mutations in breast carcinomas in China and the relationships of PTEN mutations with clinicopathological parameters and clinical outcomes. Material and methods: Trimmomatic, Burrows-Wheeler Aligner (BWA), ANNOVAR, SAMtools, and Sanger sequencing were used to analyse PTEN mutations and identify variants in Chinese breast cancer. The frequency of PTEN mutations and the relationships of PTEN mutations with clinicopathological parameters and clinical outcomes were evaluated in breast carcinomas in China. Results: The rate of PTEN germline mutation was 0.23% (n = 9) among 3955 unselected primary breast cancer patients. Of these 9 patients, 2 carried pathogenic mutations, and both were identified as having infiltrative carcinoma. One patient had a family history. The other 7 patients carried only PTEN germline variants that were not identified as pathogenic mutations. Conclusions: We studied the frequency of PTEN germline mutations in a sequential cohort of Chinese breast carcinoma patients. Based on these data, we hypothesize that the germline mutation of the PTEN gene is not closely related to the occurrence of breast cancer in the Chinese population. In the clinic, the PTEN germline mutation cannot be used as the basis for the detection of breast cancer.
1. Background Breast cancer is currently the most common cancer in women, with more than 1.3 million new cases occurring every year around the world. In the past few years, the outcomes of breast cancer have improved substantially as a result of recent advancements in the understanding of breast cancer biology and the development of new protocols for individual treatments (Torre et al., 2015). However, breast cancer
remains the leading cause of death in women, and approximately 450,000 persons have died from breast cancer (Li et al., 2017). It is important to identify potential biomarkers that could be used to screen high-risk patients and predict breast cancer prognosis. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN), also known as mutated in multiple advanced cancers 1 (MMAC1), was identified on chromosome 10q23 as a tumour suppressor gene (Garcia et al., 1999). PTEN has recently been found to be
Abbreviations: ACMG, American College of Medical Genetics; AMP, Association for Molecular Pathology; IDC, Invasive Ductal Carcinoma; TNBC, Triple-Negative Breast Cancer ⁎ Corresponding authors. E-mail addresses: [email protected] (B. Sun), [email protected] (Z. Hu). 1 Co-first authors. https://doi.org/10.1016/j.gene.2020.144630 Received 4 November 2019; Received in revised form 17 March 2020; Accepted 26 March 2020 Available online 28 March 2020 0378-1119/ © 2020 Published by Elsevier B.V.
Gene 744 (2020) 144630
Y. Wu, et al.
frequently mutated in human cancers, such as prostate cancer (Pesche et al., 1998), ovarian cancer (Han et al., 2018), breast cancer (Rhei et al., 1997), endometrial cancer (Risinger et al., 1997) and melanoma (Guldberg et al., 1997). A report showed that partial loss of normal PTEN tumour suppressor function could be compounded by additional disruption caused by the expression of inactive mutant PTEN protein (Papa et al., 2014). Many mechanisms can cause this loss of function of the PTEN gene, including missense and insertion mutations in the PTEN gene, deletion of the PTEN gene, reduced expression of PTEN because of promoter methylation, and suppression of PTEN enzyme activity (Leslie and den Hertog, 2014). Germline mutations in PTEN are also observed in Cowden syndrome (CS), which is an inherited cancer syndrome associated with a high risk of breast cancer (Liaw et al., 1997). In sporadic breast cancer, the frequency and relevance of PTEN alterations have not been elucidated completely. Therefore, we decided that a comprehensive investigation would be useful to clarify the mutation status and prognostic significance of PTEN. In this study, the frequency of PTEN mutations in breast carcinomas in China and the relationship between PTEN mutations, clinicopathological parameters and clinical outcomes were evaluated according to all of the currently available evidence.
Table 1 Clinical characteristics of the study participants.
2. Material and methods 2.1. Ethics statement Study subjects were recruited at 19 clinical centers in 11 Chinese provinces between 2012 and 2018. Before beginning the work, we orally explained our study objectives and procedures to all participants and obtained their permission to include their blood specimens in our study. Moreover, written informed consent was provided by each participant. The present study was reviewed and approved by the ethics committees of all the hospitals involved. 2.2. Sample acquisition A total of 3955 Chinese patients who were diagnosed with breast cancer were included in the present study. Clinical and pathological information, including age, ethnicity, menopausal status, type of tumour, disease stage, lymph node status and tumour size, were collected. Family history was defined as breast cancer patients having one or more cancer patients (any kind of cancer) as first-, second-, or third-degree relatives. Informed written consent was obtained from all participants. We declare that the experiments described in this study were performed in compliance with the current laws of the People’s Republic of China.
Variables
N
Percent (%)
Age at diagnosis ≤50 > 50 na
1779 737 1211
47.73% 18.63% 30.62%
Histology infiltrative carcinoma non-infiltrative carcinoma others na
2268 179 276 1232
57.35% 4.53% 6.98% 31.15%
Tumor size ≤1.5 cm > 1.5 cm na
841 2223 891
21.26% 56.21% 22.53%
ER status Positive Negative na
2351 1140 464
59.44% 28.82% 11.73%
PR status Positive Negative na
2263 1231 461
57.22% 31.13% 11.66%
HER2 status Positive Negative na
720 2392 843
18.20% 60.48% 21.31%
Molecular typing Luminal-A Luminal-B TNBC HER2 overexpression na
139 943 869 268 1736
3.51% 23.84% 21.97% 6.78% 43.89%
Family history Yes No na Total
902 2506 547 3955
22.81% 63.36% 13.83% 100.00%
3. Results 3.1. Patient characteristics We have summarized the clinicopathological characteristics of the patients in Table 1. There were 3955 unselected breast cancer patients included in this study, and 9 patients were tested for PTEN mutations. The mean age of these breast cancer patients was 58.83 years. A total of 2268 (57.35%) patients were diagnosed with infiltrative carcinoma, and 179 (4.53%) patients were diagnosed with non-infiltrative carcinoma. A total of 2351 (59.44%) patients had ER-positive status, and 2263 (57.22%) patients had PR-positive status. A total of 720 (18.20%) patients presented with a HER2-positive status. A total of 869 (21.97%) patients were classified with the triple-negative breast cancer (TNBC) molecular type, and 268 (6.78%) were classified with the HER2 overexpression molecular type. A total of 2506 (63.36%) patients had no family history.
2.3. Mutation analysis and validation First, Trimmomatic (Bolger et al., 2014) was applied to trim and crop the read pairs (FASTQ data) from the sequencing system. Then, the Burrows-Wheeler Aligner (BWA) (Li and Durbin, 2009) was run to map the resulting reads to the hg19 reference genome. SAMtools (Li et al., 2009) was used to sort and index the mapping result, and then, the mpileup command was used to identify the variants. The variants were annotated with ANNOVAR (Wang et al., 2010), and the mutations were classified into 5 different categories according to the classification protocol of the professional practice guidelines of the American College of Medical Genetics (ACMG)/Association for Molecular Pathology (AMP). Multiple databases were used to check these classified alterations, such as ClinVar (public archive of interpretations of clinically relevant variants), Human Genome Mutation Database (HGMD), and dbSNP (a single-nucleotide polymorphism database). We validated the mutations classified as pathogenic mutations or probable pathogenic mutations by Sanger sequencing and achieved 100% concordance.
3.2. Prevalence of germline mutations in PTEN Germline mutations of PTEN were tested in a large cohort of 3955 unselected breast cancer patients using a gene panel. We validated the mutations classified as pathogenic mutations or probable pathogenic mutations. In the results, a total of 8 mutations were identified in 9 of 3955 (0.23%) unselected breast cancer patients (Table 2). Of these 8 mutations, 4 were SNPs, and 4 were the first germline mutations. Seven variants were in exon 9 of the PTEN mutation carriers, followed by one 2
Gene 744 (2020) 144630
Y. Wu, et al.
Table 2 Mutations and SNPs Identified in the Study Cohort. HGMD＿prot NP_000305.3:p.Q245* NP_000305.3:p.Y176C
Sig
Geno
Annotation
Gene＿id
hgvs＿c
3-RareModerate 5-ClinVarDamaging 3-RareModerate VUS VUS
0*1 0*1 0*1 0*1 0*1
ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862
3-RareModerate 3-RareModerate 5-ClinVarDamaging 3-RareModerate
0*1 0*1 0*1 0*1
missense_variant stop_gained missense_variant missense_variant splice_donor_variant&splice_region_variant& intron_variant missense_variant missense_variant frameshift_variant missense_variant
ENSG00000171862 ENSG00000171862 ENSG00000171862 ENSG00000171862
hgvs＿p
Rank
Exon_number
c.949G > A p.Val317Ile c.733C > T p.Gln245* c.527A > G p.Tyr176Cys c.949G > A p.Val317Ile c.253+4G > A
8 7 6 8 4
9 9 9 9 8
c.134 T > C c.644 T > C c.107delG c.923G > A
2 7 2 8
9 9 9 9
p.Val45Ala p.Phe215Ser p.Gly36fs p.Arg308His
(p. Gln245*), a stop-gained variant, and the other variant was chr10:89653806c.107delG (p. Gly36fs), a frameshift variant. The first pathogenic PTEN mutation was revealed in a 41-year-old woman presenting ER- and PR-positive status as well as HER2-negative status. She was diagnosed with left, stage I, non-specific infiltrating carcinoma. The second mutation identified in a 50-year-old woman who presented with HER2-positive status and ER/PR-negative status. She was identified with right infiltrating ductal carcinoma, and she also had a family history (her brother died of gastric carcinoma). In addition, among the 9 individuals who carried PTEN mutations, one patient was diagnosed with Paget disease, 2 had IDC, 4 had ER- and PR-positive status, and 2 presented with HER2-negative status. One patient was identified with TNBC molecular type, HER2 overexpression molecular type was detected in 2 individuals, luminal-A molecular type was also detected in 2 patients, and one patient presented luminal-B molecular type. Owing to the partial loss of clinical data, we failed to determine the other patients’ genotypes. In conclusion, the frequency determined in our study was similar to that reported by Li et al. (2018) but significantly differed from the result of Wu et al. (2016). The study by Wu Y et al. focused on the Chinese human population, while most patients in our cohort lived in Shanghai. Therefore, we considered that the regional disparity might account for the different frequencies of PTEN germline mutations in Chinese breast cancer patients. In addition, the rate of PTEN mutation in foreign breast cancer patients was also higher than that in our results in the Chinese population, which might be caused by the heterogeneity of these populations. According to our data, the rate (0.23%) of PTEN germline mutations in Chinese breast cancer patients was too small, and we hypothesized that the germline mutation of the PTEN gene was not directly related to the occurrence of breast cancer. Clinically, the PTEN gene might not be a biomarker in Chinese breast cancer patients, and germline mutation of the PTEN gene can still not be used as the basis for the detection of breast cancer.
mutation in exon 8. In the 9 PTEN mutation carriers, one mutation was a frameshift variant, one was a stop-gained variant, one was identified as a splice donor variant, splice region variant and intron variant, and others were missense variants. The frameshift variant and the stopgained variant were identified as pathogenic mutations. The frameshift variant was on chr10:89653, the stop-gained variant was on chr10:89717, and the splice donor variant was on chr10:89690. None of these variants has been included in the dbSNP database. 3.3. Clinical features and mutations Two patients who carried pathogenic PTEN mutations were both diagnosed with infiltrative carcinoma, but one of them had ER- and PRpositive status with HER2-negative status, while the other presented with HER2-positive status and ER- and PR-negative status. One patient was diagnosed with Paget disease, and one had invasive ductal carcinoma (IDC). The tumour sizes of 5 PTEN mutation carriers were larger than 1.5 cm. Four patients had ER- and PR-positive status, and 2 patients presented with HER2-negative status. One patient was classified as TNBC molecular type, 2 were HER2 overexpression molecular type, 2 were luminal-A molecular type, and one was luminal-B molecular type. One PTEN mutation carrier had a family history involving a brother who died of gastric carcinoma. 4. Discussion PTEN is the first identified tumour suppressor with lipid phosphatase activity. A report has demonstrated that PTEN is mutated or inactivated in a number of malignant tumours, including neuroglioma and endometrial, prostate, breast, liver and thyroid cancers (Lu et al., 1999). Somatic mutations in the PTEN gene were found at high frequency in breast cancer, with 3.8% in the Catalogue of Somatic Mutations in Cancer (COSMIC) database (Forbes et al., 2017). According to TCGA (The Cancer Genome Atlas) data, the percentage of PTEN was 3.2% (16/507). However, the rate of germline mutation of the PTEN gene was not that high. Recently, a study reported that the somatic mutation frequency of PTEN in a cohort of Chinese breast cancer patients was 0.32% (1/313) (Li et al., 2018), while another report showed that the percentage of PTEN mutations was 0.06% (5/8085) (Sun et al., 2017) in the Chinese population. In addition, Wu Y et al. (Wu et al., 2016) showed that the mutation frequency of the PTEN gene was 1.15% (2/131) among breast cancer patients in the China Hunan population. In addition, Esther Rhei et al. (Garcia et al., 1999) showed that one germline mutation of the PTEN gene was identified in 1 of 54 (1.85%) American primary breast cancers, and a retrospective study (Fulket al., 2019) showed a PTEN mutation rate of 0.42% (n = 7) in 1650 breast cancer and uterine cancer (BUC) patients. In our study, we determined that 9 patients carried germline mutations in 3955 unselected breast cancer patients (0.23%), as shown in Table 2, and 2 (0.05%, 2/3955) mutations were identified as pathogenic mutations in the PTEN gene; one was chr10:89717708c.733C > T
CRediT authorship contribution statement Yu Wu: Data curation, Formal analysis. Dabing Huang: Conceptualization, Investigation, Methodology, Funding acquisition. Huanhuan Zhang: Investigation, Methodology. Xiaoling Weng: Data curation, Formal analysis, Writing - original draft. Honglian Wang: Data curation, Formal analysis. Qinghua Zhou: Investigation, Methodology. Ying Wu: Investigation, Methodology. Yi Shen: Investigation, Methodology. Baining Sun: Data curation, Formal analysis, Writing - original draft. Zhen Hu: Conceptualization, Writing review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 3
Gene 744 (2020) 144630
Y. Wu, et al.
Acknowledgements
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