Transcriptional Expressions of SCNs Family Correlates With Overall Survival in Patients With Breast Cancer

doi:10.21203/rs.3.rs-944468/v1

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Transcriptional Expressions of SCNs Family Correlates With Overall Survival in Patients With Breast Cancer

https://doi.org/10.21203/rs.3.rs-944468/v1

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Background: Voltage-gated sodium (Nav) channels encoded by SCNs are heteromeric protein complexes containing pore-forming α subunits together with non-pore-forming β subunits. Ion channels play an important role in the regulation of many cellular processes during normal physiology, and increasingly recognized for their contribution to pathophysiology, including cancers. Numerous studies in the last years have reported the expression of SCNs in metastatic cells of many cancers and their upregulation have been evident in promoting migration, invasion and metastasis, whereas it remains unclear whether distinct SCNs family members play an important role in the development and progression of BC.

Results: The study investigated the roles of SCNs in the prognosis of BC using ONCOMINE, UALCAN, Kaplan-Meier Plotter, GEPIA, Metascape, LinkedOmics databases. The results showed that transcriptional and proteinic expressions of 9 SCNs were downregulated in patients with BC, including SCN1A~4A, 7A, 9A and SCN2B~4B. low expressions of 11 SCNs members were found to be significantly associated with poorer overall survival (OS) in BC patients (P＜0.01), including SCN2A, 3A, 5A, 7A, 9A~11A and SCN1B~4B. Moreover, prognostic value of mRNA expression of SCNs could only be seen in HER2-negative BC patients when we performed subgroup analysis.

Conclusions: These results indicated that SCNs could be prognostic biomarkers for survivals of BC patients. Some medicines that regulate SCNs might provide new targets for BC treatment.

Epigenetics & Genomics

Voltage-gated sodium channel

Breast cancer

Biomarker

SCNs

Prognosis

Breast cancer (BC) is the most commonly diagnosed cancer in women and the second leading cause of cancer-related death among females around the world. And BC has also been the second leading cause of death in America, and the sixth in China[1–3]. During the past few decades, the morbidity of BC is increasing in most markedly developing countries, due to a combination effect of social and economic factors, including the delayed childbirth, obesity and physical inactivity. In many developed countries, the incident rate of BC is increasing significantly. Therefore, BC brings a huge burden for both patients and society.

Prognosis of BC patients has been improved due to the increase in existing treatment regimens including surgery, chemotherapy, radiation therapy, endocrine therapy, and/or targeted therapy. However, unfortunately, many patients who are diagnosed and treated at early stages still suffer from local or distant tumor recurrence. It is necessary to detect biomarkers individually or in combination to assist early diagnosis, therapeutic strategy determination and prognosis prediction. To date, the lack of knowledge about precise molecular targets has limited the treatment of BC. Therefore, there is an urgent need for finding effective potential molecular biomarkers of BC progression and identifying potential therapeutic targets, to improve treatment strategies and prolong the survival time of BC patients.

Increasing relevance researches have reported that voltage-gated sodium ion (Nav) channels play a key role in the occurrence and development of tumors including BC[4–7]. Nav channels encoded by SCNs are heteromeric protein complexes composed of α subunit formed by one pore and two β subunits formed by non-pores. Nav channels are classically responsible for initiation and conduction of action potential in electrically excitable cells (including neurons and muscle cells). However, Nav channels are also expressed in many "non-excitable" cells, including fibroblasts, glial cells, immune cells and cancer cells, and their role remains to be studied. In BC cells, Nav channels regulate cellular morphology, migration, invasion and metastasis. And Nav was expected to be a novel strategy for BC therapy. Nav channels consist of the large α-subunit, without or with the much smaller β-subunits. Nine a-subunit isoforms (Nav1.1 to Nav1.9) of Nav channels are encoded in nine homologous members (SCN1A to SCN5A and SCN8A to SCN11A). Five β subunits, including β1 and its splice variants β1B and β2-β4 (encoded by the genes SCN1B to SCN4B)[8].

Increasing evidence showed that sodium ion channel was an important factor in the occurrence and development of cancer cells. Na+ could flow into the cell through sodium channel Nav1.5 (encoded by SCN5A) in BC cell line MDA-MB-231, which caused intracellular alkalinization and extracellular adjacent cell membrane acidification through sodium-hydrogen exchanger to increase degradation and invasion of pH-dependent extracellular matrix[9, 10]. However, some studies showed opposite results about other SCNs family[11–13]. Nevertheless, it remains unclear whether distinct SCNs family members play an important role in the development and progression of BC. In this study, we analyzed the expression of different SCNs family members, predicted pathways and the correlation with clinical prognosis in BC patients.

ONCOMINE database

ONCOMINE database (www.oncomine.org) is a cancer microarray database and web-based data mining platform, aims at facilitating stimulate new discovery from genome-wide expression analyses and compare the transcriptome data in most major types of cancer with respective normal tissues[14, 15]. The individual gene expression levels of 14 SCNs family members were analyzed by ONCOMINE database. We compared mRNA levels of BC vs. normal samples datasets. Gene rank was the median rank for one target gene over all analyses. The selection criteria was p value < 0.05 as a threshold, 2-fold change and gene rank in the top 10%. For each gene, comparison between BC patients and normal control samples analysis was performed.

UALCAN

UALCAN database (http://ualcan.path.uab.edu) is a comprehensive, user-friendly and interactive web resource for analyzing cancer transcriptome data from the Cancer Genome Atlas (TCGA)[16]. It was used to analyze relative expression of genes in cancer and normal samples, as well as in various tumor stage subgroups. In this study, it was utilized not only to compare the expression level of the genes in BC and normal samples, but also to estimate the effect of the expression level in various BC stage subgroups.

Kaplan-Meier plotter

The prognostic significance of the mRNA expression of SCNs family genes in BC was evaluated using the Kaplan-Meier plotter (www.kmplot.com), an online database which is available to assess the prognostic effect of genes expression on survival in designate cancers[17]. With the purpose to assess prognostic value of each SCNs gene, the patients were divided into high expression group and low expression group according to the median expression of the gene. Then we investigated the overall survival (OS) of BC patients by Kaplan-Meier survival plot, and calculated log rank p-value to evaluate significance. Moreover, subgroup analyses of OS were performed by dividing patients based on epidermal growth factor receptor 2 (HER2)-positive and HER2-negative subtypes.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

Gene Expression Profiling Interactive Analysis (GEPIA) is a newly developed interactive web server for estimating RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from TCGA and Genotype-tissue Expression dataset projects[18]. GEPIA provides customizable functions such as differential expression analysis, correlation analysis, profiling plotting, detection of similar genes, survival analysis, and dimensionality reduction analysis. We analyzed SCNs family genes and 10 similar expression neighbor genes of each SCN by GEPIA2.0 (http://gepia2.cancer-pku.cn/).

Metascape (http://metascape.org) is a web-based portal designed to provide a comprehensive gene list annotation and analysis resource for experimental biologists[19]. We conducted GO annotation and KEGG pathway enrichment analysis by Metascape. The GO terms categories are grouped by biological process (BP), cellular component (CC) and molecular function (MF). Only terms with P-value < 0.05 and minimum count of 3 were considered to be significant. Protein–protein interaction enrichment analysis was also performed by Metascape database.

Gene set enrichment analysis (GSEA)

LinkedOmics (http://www.linkedomics.orglogin.php) is a publicly available portal comprising multiomics data from 32 TCGA cancer types. It uses preprocessed and normalized data from the Broad TCGA Firehose and Clinical Proteomic Tumor Analysis (CPTAC) data portal to reduce redundant efforts and focus on the discovery and interpretation of attribute associations, and thus complements existing cancer data portals[20]. In our study, GSEA analysis was conducted by LinkedOmics Dataset, and FDR < 0.05 was set as the cut-off criteria.

Differential expression of different SCNs family members in BC patients

In order to explore the differential expression of individual SCNs members in BC patients, mRNA expression was analyzed by ONCOMINE database. As were shown in Figure 1, mRNA expressions of 14 SCNs family members in 20 types of cancers were compared with normal samples. Significantly downregulation of mRNA expressions in SCN1A~4A,7A,9A,2B~4B were found in BC patients comparing with normal samples in multiple datasets. Low expression of SCN1A~4A,7A,9A,2B~4B was shown in TCGA breast datasets (n=593), compared with normal tissues. Moreover, Radvanyi dataset also observed significant downregulation of SCN9A in BC tissue (fold change: -2.870, p=0.005)[21]. In Radvany dataset, significant low expression of SCN2B was found in BC samples compared to normal tissues (fold change: -2.892, p=0.002)[21]. Similarly, in other seven datasets, significant downregulation of SCN4B was found in BC tissues (all p<0.01)[21–26]. However, Finak dataset observed significant overexpression of SCN4A in BC tissues compared to normal tissues (fold change: 2.725, p=1.03E-14)[27]. And Turashvili dataset observed an increase of SCN1B mRNA expression in BC tissues (fold change: 2.937, p=8.56E-5)[28].

Then, the mRNA expressions of 14 SCNs members were further explored by UALCAN database, which was different from ONCOMINE database. As was shown in Figure 2, mRNA expressions of 11 SCNs members were significantly downregulated in primary BC tissues compared to normal samples (all p<0.001), including SCN1A~5A,7A,9A,2B~4B. These results are similar to those from ONCOMINE database.

Association of mRNA expressions of SCNs family members with different tumor stages of BC patients

After mRNA expressions were found to be downregulation in BC patients, we analyzed the correlation between mRNA expression of different SCNs family members with different tumor stages of BC by UALCAN.

As were shown in Figure 3, mRNA expressions of SCN2A~5A, 7A, 9A, 11A, 2B~4B in individual stages were significantly downregulated when comparing with normal samples (all P༜0.001). However, as the tumor grade increased, the mRNA expression of SCNs didn’t show a significant declining trend. The results may be due to the small sample size.

Prognostic value of mRNA expressions of SCNs family in BC patients

Kaplan-Meier plotter was applied to analyze the prognostic value of the mRNA expressions of SCNs family members in BC patients. The results showed that mRNA expressions of most of the SCNs family members were significantly associated with BC patients’ prognosis. As were shown in Figure 4, low expression of mRNA in SCN2A (HR=0.74, 95% CI: 0.64-0.86, p=9.9e-05), SCN3A (HR=0.85, 95% CI: 0.77-0.94, p=0.0013), SCN5A (HR=0.86, 95% CI: 0.78-0.95, p=0.0034), SCN7A (HR=0.63, 95% CI: 0.54-0.73, p=1.2e-09), SCN9A (HR=0.77, 95% CI: 0.66-0.9, p=0.00072), SCN9A (HR=0.79, 95% CI: 0.72-0.88, p=7.1e-06), SCN11A (HR=0.8, 95% CI: 0.72-0.89, and p=1.5e-05), SCN1B (HR=0.81, 95% CI: 0.74-0.9, p=6.3e-05), SCN2B (HR=0.61, 95% CI: 0.52-0.71, p=1.3e-10), SCN3B (HR=0.76, 95% CI: 0.69-0.84, p=1.4e-07), SCN4B (HR=0.64, 95% CI: 0.55-0.75, p=1e-08) were significantly associated with poorer OS of BC patients. However, SCN1A/4A/8A mRNA expression showed no significant correlation with prognosis in BC patients. These results indicated that mRNA expressions of SCN2A/3A/5A/7A~11A, 2B~4B were significantly associated with the prognosis in BC patients and they might be exploited as promising biomarkers for predicting the poor survival in BC.

Prognostic value of mRNA expression of SCNs in HER2-positive/HER2-negative BC patients

After mRNA expressions of SCN2A/3A/5A/7A~11A, 2B~4B were found to be significantly associated with BC patients’ prognosis, we then tried to assess the prognostic value of mRNA expression of SCNs in different subgroups. In HER2-negative BC patients, low mRNA expressions of most of the SCNs family members were significantly associated with poor prognosis. As were shown in Figure 5, lower mRNA expression of SCN2A (HR=0.69, 95% CI: 0.58-0.83, p=4.4e-05), SCN3A (HR=0.82, 95% CI: 0.73-0.92, p=0.00072, SCN5A (HR=0.85, 95% CI: 0.76-0.95, p=0.0057), SCN7A (HR=0.57, 95% CI: 0.47-0.68, p=2.1e-10), SCN9A (HR=0.8, 95% CI: 0.67-0.95, p=0.011), SCN10A (HR=0.8, 95% CI: 0.72-0.9, and p=0.00017), SCN11A (HR=0.86, 95% CI: 0.76-0.96, and p=0.0074), SCN1B (HR=0.82, 95% CI: 0.73-0.92, p=0.00068), SCN2B (HR=0.56, 95% CI: 0.47-0.67, p=1.6e-10), SCN3B (HR=0.76, 95% CI: 0.68-0.85, p=2.9e-06), SCN4B (HR=0.59, 95% CI: 0.5-0.71, p=5.7e-09) were significantly associated with shorter OS of BC patients. SCN1A/4A/8A mRNA expression showed no correlation with prognosis of BC patients. This result is similar to the whole patients. However, in HER2-positive BC subgroup (Figure 6), no significant correlation between mRNA expressions of most of the SCNs family members and prognosis except SCN9A (HR=0.69, 95% CI: 0.51-0.94, p=0.017). The results suggested that expressions of SCNs family members were correlated with clinical prognosis only in HER2-negative BC patients.

Predicted functions and pathways of SCNs and their similar expression neighbor genes in BC patients

We further analyzed 10 similar expression neighbor genes of each SCNs family by GEPIA2.0 and constructed an integrated network with SCNs and neighbor genes in BC patients by Metascape.

In Figure 7, genes including UNC79, SYNJ1 and DNM1 were significantly related to SCNs family. As was shown in Figure 8, GO enrichment analysis indicated that these genes were significantly involved in 3 terms in cellular components including GO:0001518 (voltage-gated sodium channel complex), GO:0030424 (axon), GO:0033268 (node of Ranvier), 8 terms in biological processes including GO:0051650 (establishment of vesicle localization), GO:0008585 (female gonad development), GO:0061025 (membrane fusion), GO:0042475 (odontogenesis of dentin-containing tooth), GO:0006575 (cellular modified amino acid metabolic process), GO:0030198 (extracellular matrix organization), GO:0050890 (cognition), GO:0010976 (positive regulation of neuron projection development), and 2 terms in molecular functions including GO:0099106 (ion channel regulator activity), GO:0008017 (microtubule binding).

To further explore the functions and pathways of SCNs genes, we performed GSEA enrichment analysis of each SCNs gene by LinkedOmics, respectively. 12 of SCNs family were enriched in “Proteasome”, 11 of SCNs family were enriched in “Base excision repair” and “DNA replication”, and 10 of them were enriched in “Mismatch repair” and “RNA polymerase”. As were shown in Figure9, most of SCNs genes were downregulated except for SCN1A which was upregulated in these pathways.

Ion channels are ubiquitously expressed throughout the body, being responsible for virtually basic cellular processes such as cell proliferation, solute transport, maintenance of membrane potential, nerve signaling and control of muscle contraction, invasion and many other activities[5]. The role of ionic channels, mainly potassium channels, has been extensively studied in cell proliferation. However, the possible involvement of sodium channels hasn’t been deeply described in tumors before[29]. Nav channels play a crucial role in development and propagation of action potentials in neurons and muscle cells. Nav activities control not only basic impulse generation and conduction but also directional and patterned growth, including target-specific axonal migration and regional synaptic connectivity[30–32]. Nav has also been related to several hereditary diseases of excitable tissues[30, 33], and more complicated pathological disorders, including chronic pain syndromes[34], epilepsy[35], ischaemic stroke[36], and Alzhei-mer’s disease[37].

Some studies have shown that the expression of α and β subunits of Nav was upregulated in various cancers including prostate, lung, and cervical cancer, which has been observed both in vitro and in vivo systems[29, 38–41]. It was found that they promoted the progress of metastasis through the Na+ currents carried by α subunits and adhesion interactions regulated by β subunits, such as invasion, migration, endocytosis and gene expression.

Results from our study showed that mRNA expressions of 11 SCNs family members were significantly downregulated in BC tissues compared to normal samples including SCN1A~5A, 7A, 9A, 11A and SCN2B~4B. And the results are similar to those from two different databases including ONCOMINE and UALCAN. The difference might come mainly different datasets, different tumors or specific SCN genes.

The roles of SCN1A/2A in cancer development are not well-reported, as well as its regulatory mechanism[42]. Wu et al revealed that SCN3A was downregulated in BC, SCN3A was mainly involved in driving membrane depolarization and action potential propagation. And membrane depolarization was associated with the pathogenesis of BC[43]. Besides, Nigam et al revealed that the drug Centchroman can induce G0/G1 arrest and caspase-dependent apoptosis by involving the mitochondrial membrane depolarization in human breast cancer cells[44]. Considering this, SCN3A might be a key regulator in the depolarization process of membrane in BC.

Nav1.5 is an α subunit of Nav, encoded by SCN5A. Studies have shown that Nav1.5 is usually highly expressed in metastatic BC cells in the form of neonatal splicing. The abnormal activation and expression of Nav1.5 triggers a variety of cellular mechanisms, including changes in H+ efflux, promotion of epithelial to mesenchymal transition (EMT) and the expression of cysteine cathepsin, to enhance the transfer and invasion of BC cells in vitro and in vivo[45]. Kamarulzaman et al. and Zhang et al. revealed the expression level of Nav1.5 in the strongly metastatic MDA-MB-231 BC cell line was significantly higher than in the weakly metastatic MCF-7 cells[46, 47]. Nelson et al. and Yamaci et al. also reported that SCN5A expression was significantly increased in BC tissues, which promoted growth and metastatic dissemination and was an independent predictor of poor prognosis compared with its expression in normal breast tissues[48–50]. In our study, the expression of SCN5A was downregulated in BC patients comparing with normal samples, and the overexpression of SCN5A was associated with poorer OS (P=0.0034). The regulatory mechanism of SCN5A in BC still needs to be further explored.

There were few studies on other SCNxA families in BC. Nav1.6 (encoded by SCN8A) and Nav 1.7(encoded by SCN9A), were reported that mRNA levels in human cervical cancer samples were ~40-fold and ~20-fold higher than in noncancerous cervical biopsies[11]. Study from Diaz et al showed that the expression and functional activity of Nav1.2, Nav1.4, Nav1.6, and Nav1.7 sodium channels were associated with human cervical cancer[29]. Diss et al. and Bennett et al. reported that Nav1.7 sodium channel was overexpressed in prostate metastatic cells in both animals and human prostate cancer[38, 51].

SCNxB have been demonstrated to interact with pore-forming Nav through covalent or non-covalent binding, to regulate their transport to the plasma membrane, with biophysical and pharmacological properties[52–56]. In addition, they also have other specific cellular functions, including neurite outgrowth, axonal fasciculation and interaction with glial cells[57]. The expression of SCNxB and their physiological role in non-excitable cells have not been characterized. There is not as much researches on their participation in oncogenic processes as that of Nav proteins[58].

In vitro study indicated that the expression of SCN1B in weakly invasive BC cells was higher than in strongly invasive BC cells. In weakly invasive MCF-7 cells, the downregulation of SCN1B reduced reduced adhesion and increased migration. Nelson et al. revealed that SCN1B gene was downregulated in BC compared with normal breast tissues[12]. In contrast, another study showed that SCN1B was overexpressed in BC patients, compared with non-cancer samples[59]. It was proposed that the proinvasive effect of SCN1B depended on both Nav-dependent and Nav-independent mechanisms, relying on the extracellular Ig domain of the protein[12].

Concerning the SCNxB, Chioni et al. found that weakly metastatic BC cells expressed considerably higher levels of SCN1B, SCN2B, and SCN4B mRNAs than strongly metastatic BC cells[59]. Another study identified that the SCN4B gene is expressed in normal epithelial cells and the decrease of SCN4B protein levels in breast cancer biopsies is associated with high-grade primary and metastatic tumors[13]. In cancer cells, reducing the expression of SCN4B can increase RhoA activity, promote cell migration and invasion, enhance primary tumor growth and metastatic spreading, by facilitating the acquisition of an amoeboid-mesenchymal hybrid phenotype, and SCN4B overexpression reduces BC cells invasiveness and tumor progression, which is similar to our result that the SCN4B gene is a tumor-suppressor gene[13].

In our results of GSEA enrichment analysis, Proteasome, Base excision repair, DNA replication, Mismatch repair and RNA polymerase were significantly enriched. And most of SCNs genes were low expression in these pathways except SCN1A. These results implied that SCNs might participate in stabilizing and repairing genomic DNA. We predicted that loss of SCNs could induce abnormal transcription to cause major dysfunction and even cause cancers.

In order to further explore the prognostic value and clinical significance of SCNs family, we divided into two subgroups including HER2-positive/HER2-negative BC patients. In the subgroup analysis, prognostic value of mRNA expression SCNs family only showed significance only in BC HER2-negative patients. Measurement of HER2 is routinely performed in all new, recurrent or metastatic invasive BC cases when feasible. Overexpression of HER2 drives tumor growth by constitutive activating MAPK and PI3K/AKT signaling pathways, thereby enhancing cell proliferation, invasion and metastasis. The development and progression of HER2-negative BC by other signaling pathways that contain SCNs genes. Moreover, only patients that are HER2-positve can receive targeted drugs (trastuzumab, lapatinib, and pertuzumab)[60]. The significance of HER2 gene has been demonstrated by correlation between overall survival and recurrence time with the HER2 gene amplification in BC[61]. There still are 80~85% of BC patients are HER2-negative, which usually lack validated targeted therapy, with conventional chemotherapy. The results of our study indicated that SCNs might be prognostic biomarkers for survivals of HER2-negative BC patients. Some medicines that regulate SCNs might provide new targets for HER2-negative BC treatment.

There were still some limitations in this study. (i) All the data analysis was retrieved from online databases; (ii) We did not assess the impact of clinical factors on prognostic value of SCNs in BC; (iii) We did not further explore the potential mechanisms of distinct SCNs in BC. There were still many unresolved questions that require further investigation: (i) Is it possible to make SCNs a biomarker of early diagnosis of BC; (ii) Can we develop additional agents to specifically activate specific SCN genes to improve efficacy and reduce side effects; and (iii) How can we accumulate medicine processes and promote the clinical application of relevant agents to improve the prognosis of BC patients. Further studies including larger sample sizes are required to verify our findings and to explore the clinical application of the SCNs members in the treatment of BC.

In conclusion, the results showed that transcriptional and proteinic expressions of 9 SCNs were downregulated in patients with BC, including SCN1A~4A, 7A, 9A and SCN2B~4B.

Besides, lower expressions of 11 SCNs members were found to be significantly associated with poorer overall survival (OS) in BC patients (P<0.01), including SCN2A, 3A, 5A, 7A, 9A~11A and SCN1B~4B. Moreover, prognostic value of mRNA expression of SCNs could only be seen in HER2-negative BC patients when we performed subgroup analysis. These results indicated that SCNs could be prognostic biomarkers for survivals of BC patients. Some medicines that regulate SCNs might provide new targets for BC treatment.

Acknowledgements

We would like to thank Yang Li for the advice and assistance with server administration.

Authors’ contributions

All authors critically reviewed the manuscript and contributed to conception and design of the protocol. PWX, ZY and SXJ had involved in drafting the manuscript or revising it critically for important intellectual content. LY reviewed and modified the manuscript.

Funding

This research was supported in part by Beijing Municipal Administration of Hospitals Clinical Medicine Development of Special Funding Support (code, ZYLX201805), National Major Scientific and Technological Special Project for “Significant New Drugs Development” during the Thirteenth Five-year Plan Period (code, 2017ZX09304017), and Beijing Municipal Science and Technology Project (code, Z191100006619094).

Availability of data and materials

The data and materials are available at ONCOMINE, UALCAN, Kaplan-Meier Plotter, GEPIA, Metascape, LinkedOmics databases during the current study.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Transcriptional Expressions of SCNs Family Correlates With Overall Survival in Patients With Breast Cancer

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Abstract

Figures

Background

Methods

ONCOMINE database

UALCAN

Kaplan-Meier plotter

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis

Gene set enrichment analysis (GSEA)

Results

Differential expression of different SCNs family members in BC patients

Association of mRNA expressions of SCNs family members with different tumor stages of BC patients

Prognostic value of mRNA expressions of SCNs family in BC patients

Prognostic value of mRNA expression of SCNs in HER2-positive/HER2-negative BC patients

Predicted functions and pathways of SCNs and their similar expression neighbor genes in BC patients

Discussions

Conclusion

Declarations

References

Additional Declarations

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