Gene Expression Profiling of Fibroepithelial Lesions of the Breast

doi:10.21203/rs.3.rs-1836561/v1

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Gene Expression Profiling of Fibroepithelial Lesions of the Breast

https://doi.org/10.21203/rs.3.rs-1836561/v1

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Fibroepithelial lesions of the breast (FELs) are a heterogeneous group of neoplasms exhibiting a histologic spectrum ranging from fibroadenomas (FAs) to malignant phyllodes tumors (PTs). Despite published histologic criteria for their classification, it is common for such lesions to exhibit overlapping features, leading to subjective interpretation and inter-observer disagreements in histologic diagnosis. Therefore, there is a need for a more objective diagnostic modality to aid in the accurate classification of these lesions and to guide appropriate clinical management. In this study, the expression of 750 tumor-related genes was measured in a cohort of 34 FELs (5 FAs, 9 cellular FAs, 9 benign PTs, 7 borderline PTs, and 4 malignant PTs). Differentially expressed gene analysis, gene set analysis, pathway analysis, and cell type analysis were performed. Genes involved in matrix remodeling and metastasis (e.g., MMP9, SPP1, COL11A1), angiogenesis (VEGFA, ITGAV, NFIL3, FDFR1, CCND2), hypoxia (ENO1, HK1, CYBB, HK2), metabolic stress (e.g., UBE2C, CDKN2A, FBP1), cell proliferation (e.g., CENPF, CCNB1), PI3K-Akt pathway (e.g., ITGB3, NRAS) were highly expressed in malignant PTs and less expressed in borderline PTs, benign PTs, cellular FAs, and FAs. The overall gene expression profiles of benign PTs, cellular FAs, and FAs were very similar. Although a slight difference was observed between borderline and benign PTs, a higher degree of difference was observed between borderline and malignant PTs. Additionally, the macrophage cell abundance scores and CCL5 were significantly higher in malignant PTs compared with all other groups. Our results suggest that the gene expression profiling-based approach could lead to further stratification of FELs and may provide clinically useful biological and pathophysiological information to improve the existing histologic diagnostic algorithm.

Breast fibroepithelial lesions (FELs) are a heterogeneous group of biphasic neoplasms composed of both stromal and epithelial elements that range from indolent fibroadenomas (FAs) to malignant phyllodes tumors (PTs). They account for about 0.5-1.0% of all breast tumors(1). Fibroepithelial lesions with cellular stroma are inconsistently classified as cellular FA or PT. Studies have shown high interobserver variability in the distinction between cellular fibroadenoma and benign phyllodes tumor, even among expert breast pathologists(2). Moreover, despite World Health Organization published histologic criteria to distinguish benign, borderline, and malignant phyllodes tumors(1), the overlapping morphology remains problematic(3, 4). The clinical behavior of FELs is highly variable, and the malignant potential of the various subtypes is difficult to predict based on histologic features alone. (5–8).

In recent years, a wide range of studies have been performed on PTs and FAs with respect to their biomarker expression, genetic alterations, and molecular mechanisms, as potential tools to more reproducibly classify PTs and FAs(9–11). Gene expression profiling is an additional approach which might help predict the biological behavior of FELs and provide insights for their clinical management, though studies using this technique remain limited. Moreover, since malignant PTs have the potential to metastasize (12), and no effective targeted therapy has yet been reported, the finding of targetable genes/pathways for malignant PTs might also be of clinical use.

A variety of biomarkers such as Ki-67 and p53 protein expression have been proposed to help with subclassification of phyllodes tumors(13, 14), and the expression of c-kit (CD117), phospho-histone3, MDM2, ERG, CD31, CD34 have been reported to be predictive of malignant behavior of PT (13, 15, 16). However, these ancillary studies have not been shown to be independent predictive and prognostic biomarkers(17). In recent years, genomic sequencing has identified common mutations in fibroadenomas and phyllodes tumors such as MED12, RARA, and MDM12 genes suggesting the same cellular origin (18–20). However, the molecular alterations driving the pathogenesis of FELs are still poorly defined (21, 22). Moreover, detection of differentially expressed genes within the different subgroups of fibroepithelial neoplasms (FAs, benign, borderline, and malignant PTs) is limited. Thus, currently proposed molecular alterations and immunohistochemistry are of limited use in routine practice. Further studies to characterize the gene expression profiles of FELs may more reliably classify and stratify risk for this group of lesions.

To this end, we performed gene expression profiling across the different categories of breast fibroepithelial lesions, analyzing the expression of 750 tumor-related genes. Our aim was to characterize the gene expression profiles of the different subcategories of breast fibroepithelial lesions, to determine whether gene expression profiling might aid in the distinction between these subcategories and provide information relevant to disease risk stratification in this category of breast lesions with overlapping histologic features.

Patient sample selection

This is a retrospective study. A total of 34 fibroepithelial lesions between 2017 to 2021 were selected from the pathology database at Cedars-Sinai Medical Center, including fibroadenomas (n = 5), cellular fibroadenomas (n = 9), benign phyllodes tumors (n = 9), borderline phyllodes tumors (n = 7) and malignant phyllodes tumors (n = 4). All H&E slides were blindly reviewed by two in-house senior breast pathologists who confirmed the diagnoses according to 2019 World Health Organization (WHO) criteria(1). Clinical reports and follow-up data were available for all patients.

RNA extraction and Gene expression analysis

All hematoxylin and eosin (H&E) stained slides were reviewed by two pathologists to select the most representative area for microdissection. Three 10µm FFPE (Formalin-Fixed, Paraffin-Embedded) slides were cut for each case. Normal breast tissue was avoided during microdissection to eliminate contamination and signal dilution. Total RNA was extracted using the RNeasy FFPE kit (Qiagen, Hilden, Germany) and quantified by Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). 100ng of total RNA was used to measure the expression of 750 tumor-related genes and 20 housekeeping/reference genes using the nCounter platform (Nanostring Technologies, Seattle, WA, USA) according to the manufacturer’s protocol. Expression counts were then normalized using the nSolver 4.0 software and custom scripts in R 3.3.2. False Discovery Rate [FDR], was used to identify differential gene expression across sample groups. Genes with at least 2-fold or higher expression were considered differentially expressed. Comparison of differential expression, gene set analysis, and cell type analysis were also performed using nSolver 4.0 software.

Immunohistochemistry

Immunostaining for CD163 was performed on selected sections from paraffin-embedded tissue blocks of each case and was produced in accordance with optimal staining standards. The slides were prepared at the immunohistochemistry lab at Cedars-Sinai Medical Center. 4-µm thick sections were incubated with monoclonal mouse antibody to CD163 (Leica clone 10D6). Staining was performed on the Roche Ventana Benchmark Ultra (Tucson, AZ) automated slide stainer using an onboard heat-induced epitope retrieval method in a high pH buffer. Staining for CD163 was visualized using the Ventana Ultra view DAB Detection System. All slides were subsequently counterstained with Mayer's hematoxylin.

Statistical analysis

Statistical differences for numerical values were calculated using the ANOVA test. The difference was considered statistically significant at a P-value of less than 0.05 (p < 0.05). All statistical analyses were performed using GraphPad Prism, v9. (GraphPad Software, Inc, San Diego, CA). Heatmaps were generated in R using the ComplexHeatmap package. Differentially expressed genes between samples were determined by Limma with a p-value of 0.05 using a Bonferonni correction. Clustering was conducted using genes that were differentially expressed among different subgroups.

Gene expression profiling among fibroadenomas, cellular fibroadenomas and benign phyllodes tumors

Fibroadenomas (FAs), cellular FAs, and benign PTs shared overlapping gene expression profiles, with very few differentially expressed (DE) genes identified. Volcano plots demonstrating the distribution of the fold changes in gene expression levels and p-values are shown in Fig. 1. A few genes (FSTL3, DUSP2 CDKN2A) were slightly upregulated in cellular FAs in comparison to FAs. Only 3 genes (SERPINA1, COMP, TWIST1) were slightly differentially expressed in benign PTs when compared with cellular FAs and FAs.

Gene expression profiling differentiates malignant phyllodes tumors from borderline phyllodes tumors, benign phyllodes tumors and fibroadenomas

To visualize the difference in expression levels of the 750 cancer-related gene across the spectrum of FELs, we performed a hierarchical clustering of differentially expressed (DE) gene among all groups. Malignant PTs clustered distantly apart from all the other FELs (Fig. 2A). A total of 131 DE genes (63 upregulated and 68 downregulated) were identified in malignant PTs, compared to the other FELs. Volcano plots showing pair-wise comparisons of differentially expressed genes between malignant PTs and each of the remaining FELs are shown in Fig. 2B. While there were some differences in the gene expression profile between borderline and benign PTs (10 upregulated and 10 downpregulated genes), more distinctive differential gene expression was observed between borderline and malignant PTs (30 upregulated and 26 downregulated genes).

Distinct biological processes involved in malignant PTs

To identify the biological processes that are altered in malignant PTs, gene set analysis and pathway score were performed (Fig. 3). Expression of genes such as ENO1, HK1, CYBB, HK2, VEGFA, and ERBB2, which are hypoxia-related genes was significantly altered in malignant PTs. Also, genes involved in various cancer-associated intracellular signaling cascades, including interferon signaling (EIF2AK2, ICAM1, STAT2, IRF3), NF-kappa B signaling (NFKB1, IKBKG, IKBKB), PI3K-Akt (PIK3R1, PETN, PRLR) and TGF-beta signaling (TGFB1, ID4). were also differentially expressed in malignant PTs. Genes regulating varying biological processes, such as apoptosis (CDH1, BIRC5, PSMB5, TNFSF10), epigenetic regulation (HDAC11, BNIP3, MAP2K12), matrix remodeling, and metastasis (MMP9, TGFB1, LOXL2, CDH1, PLOD2 COL11A1, COL5A, MMP7), also showed distinctive expression patterns.

Macrophage abundance score is increased in malignant and borderline PTs

Cell type profiling analysis measures genes that are widely accepted to be characteristic of various cell populations (dendritic cells, macrophages, neutrophils, T cells, NK cells, etc). In the 34 FEL cases analyzed, macrophage abundance scores measuring macrophage-specific surface marker genes (CD68, CD84) were highest in malignant PTs, followed by borderline PTs (Fig. 4A). CD163 immunohistochemistry also highlighted increased macrophage infiltrations in malignant PTs (Fig. 4B).

In this study, we measured and analyzed the expression of 750 tumor-related genes in 34 breast fibroepithelial lesions, as a potential aid in their classification and to explore their underlying biology. Although fibroadenomas and benign phyllodes tumors are recognized as distinct entities, their common overlapping histologic features (particularly between cellular FA and benign PT) makes the distinction challenging in daily practice. There are several comprehensive genetic and proteomic studies of FELs suggests that FAs and benign PTs share similar gene and protein expression profile(9, 10, 18, 23). Our study revealed that fibroadenomas (FAs), cellular fibroadenomas, and benign phyllodes tumors (PTs) manifest similar gene expression profiles. Genes that are involved in different cancer-related pathways like MAPK, epigenetic regulation, matrix remodeling and metastasis, angiogenesis, TGF-beta signaling, Hedgehog signaling, and PI3K-Akt showed no difference when performing gene set analysis between these three groups. Similar patterns were also recognized in these three groups of FELs using cell type analysis. While the current treatment protocol for asymptomatic fibroadenomas is conservative management (such as annual breast examination and ultrasound as clinically indicated(24)), benign phyllodes tumors are treated with surgical intervention in many medical centers(25, 26). The overlapping histologic and gene expression profiles of FAs, cellular FAs, and benign phyllodes tumors could imply a similarly indolent clinical behavior and may suggest that a conservative management strategy may be appropriate for each of these entities.

A recent study revealed that borderline phyllodes tumor could be separated into either benign or malignant PTs using molecular assays(27). Interestingly, in our study, the hierarchical clustering heatmap show several borderline phyllodes cases clustered with the malignant PTs group, and some clustered closely with the benign phyllodes tumor group (supplement Fig. 2). This suggests that a subset of borderline PTs exhibits a gene expression profile which overlaps with that of malignant PTs. Various gene alterations were found both in malignant PTs and borderline PTs (supplement Fig. 3), which could imply that some malignant PTs derive from a subset of borderline PTs through clonal evolution and/or acquisition of somatic mutations leading to malignant transformation Prospective studies utilizing gene expression analysis with larger sample size could aid in optimizing the diagnostic and prognostic stratification of borderline PTs. Additional studies are also required to determine whether the remaining subset of borderline PTs lacking a gene expression profile comparable to that of malignant PTs may also progress to malignancy via de novo pathways.

Extracellular matrix-related gene alterations are known to promote metastases in various cancers(28–30). The ECM molecules are major components in the tumor microenvironment integral for cellular adhesion and harboring growth-factors(31). Fibroblasts secrete collagen which is the main structural protein of the ECM responsible for intercellular adhesion, differentiation, and integrity(32). Previous studies report ECM gene dysregulation such as, but not limited to, COL1A1, FN1, TIMP1, and MMP9, to play an important role in tumorigenesis in various malignancies and are associated with poor prognosis(31, 33–35). In this study, we also found higher expressions of collagen type I alpha 1 chain (COL1A1) as well as matrix metallopeptidases 7 and 9 (MMP7, MMP9) genes in the borderline and malignant PTs groups when compared with benign PTs. Further studies are required to construct and validate a comprehensive molecular panel composed of ECM-related genes might aid with proper risk stratification of borderline PTs.

Our gene expression profiling results identified many important cancer-related biological processes such as angiogenesis, matrix remodeling & metastasis, PI3K-Akt pathways, TGF-beta signaling, MAPK pathways, and hypoxia which discriminate malignant PTs from other FELs. This is consistent with our current understanding of the pathogenesis and biologic behavior of malignant PTs, where stromal overgrowth, increased angiogenesis, and extracellular matrix factors are profoundly involved in tumor progression(9). Pathway score and gene set analysis revealed that angiogenesis-related genes including VEGFA, ITGAV, NFIL3, FDFR1, and CCND2 are highly upregulated in malignant PTs. Recent studies have demonstrated that VEGFRs are expressed in several cancer cell types and may dictate tumor invasion(36). This finding could potentially predict response to the targeted therapy (angiogenesis inhibitor). Overexpression of EGFR and PDGFA are also present in malignant phyllodes tumors, which suggests the potential use of dual VEGFR/EGFR inhibitors (Vandetanib), a combination of anti-VEGFR and anti-EGFR and multikinase inhibitor (Panzopnib).

The growth and survival of malignant cells are often driven by constitutive activation in the mitogen-activated protein kinase (MAPK) and phosphor-inositide 3-kinase (PI3K/AKT) signaling pathways(37–39). VEGFR-2 mediated activation of PI3K/Alk cascade is also important for tumor survival. It is well established that no pathways exist independently, the crosstalk between the pathways promotes tumor cell proliferation, survival, and invasion. Gene set analysis (GAS) revealed that 28 genes (PIK3R1, PRLR, EIF4EBP1, NRAS, HRAS, MAP3K12, KIT), involved in PI3K/AKT and MAPK pathways, were differentially expressed in malignant PTs group. Moreover, genes in NK-kappa B (IKBKB, KIBKG, NFKB1) and NOTCH (NOTCH1, HDAC11, APH1B) signaling pathways are also differentially expressed. PIK3CA/KRAS and HRAS alterations have been already reported in borderline and malignant phyllodes tumor(40). In contrast to the previous study, we identified high frequency and more gene alterations in comparison with the previous data which implies a potential therapeutic benefit from IKK inhibitors and mTOR inhibitors.

An increased macrophage abundance score in malignant phyllodes tumors was found in this cohort by cell type analysis. The score is calculated by measuring the expression level of four macrophage-related genes: CD163, CD68, CD84, and MS4A4A. We found CD68, CD84, and CD163 to be upregulated in malignant PTs. Many studies have found that breast cancer with high tumor-associated macrophage (TAM) infiltration was significantly correlated with aggressive biological behavior and could serve as an independent predictor of overall survival and recurrence-free survival(41, 42). Previous studies also proposed that TAMs could stimulate myofibroblast differentiation and promote proliferation as well as invasion of phyllodes tumor by CCL18 driven NF-kB/PTEN/AKT axis(43). The interactions of the tumor microenvironment and tumor cells have been shown to drive the progression of various cancers, and TAMs are the most abundant inflammatory cell type within the microenvironment of malignant PTs(44). Additional TAM-associated markers are also identified in our study by differential expression analysis including CCL18, CCL5, MMP-9, and SPP-1(45). Consistent with prior signaling pathway alteration results(46–48), our data also suggest TAMs play a crucial role in the tumorigenesis of malignant phyllodes tumors. However, the specific pathways of TAMs interacting with myofibroblasts and other inflammatory cells leading to malignant progression would require further investigation.

Our analysis was limited by a relatively small cohort size. Future studies with larger sample size are needed to validate our findings. In addition, correlating the molecular result with long term clinical follow-up data might provide relevant information to guide clinical management.

In conclusion, this study provided a comprehensive profile of gene alterations across various subtypes of breast fibroepithelial lesions and might bring new insights in their classification and improve understanding of their pathogenesis. Our results raise the possibility that fibroadenoma, cellular fibroadenoma and benign phyllodes tumor might be merged in the same subgroup for the purpose of clinical management. Further studies would be of particular interest to predict the biological behavior of borderline phyllodes tumors.

CONFLICT OF INTEREST

The authors declare they have no conflict of interest.

AUTHOR CONTRIBUTIONS

X.L. F.D. performed study concept and design; X.L., E.V., H.M., M.C., F.D. performed development of methodology and writing, review and revision of the paper; X.L., J.L., MT.C, E.V. provided acquisition, analysis and interpretation of data, and statistical analysis; MT.C. provided technical and material support. All authors read and approved the final paper.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

IRB approval from Cedars-Sinai Medical Center was obtained (IRB#1577) and the study was performed in accordance with the Declaration of Helsinki.

FUNDING

All sources of funds supporting the completion of this manuscript are under the auspices of Cedars-Sinai Medical Center. Funding for the project was provided by breast research grants from Dr. Armando Giuliano.

DATA AVAILABILITY STATEMENT

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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There is NO conflict of interest to disclose.

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Gene Expression Profiling of Fibroepithelial Lesions of the Breast

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Version 1

Abstract

Figures

Introduction

Material And Methods

Patient sample selection

RNA extraction and Gene expression analysis

Immunohistochemistry

Statistical analysis

Results

Gene expression profiling among fibroadenomas, cellular fibroadenomas and benign phyllodes tumors

Distinct biological processes involved in malignant PTs

Macrophage abundance score is increased in malignant and borderline PTs

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1