Abstract
Despite the establishment of the traditional prognostic factors for breast cancer, patients belonging to the same histological and molecular subgroup often present quite different outcomes. Recently, the introduction of gene expression profiling, assessed by RT-qPCR and microarray DNA analysis, offered a view of the whole cell gene activity and the ability to identify new transcripts that are associated with outcome. This review aimed to gather all recent trials about new breast cancer prognostic factors, focusing on the most promising one, the FGD3 gene, and to discuss the real feasibility of a molecular approach in everyday clinical practice. In conclusion, all literature concerning this subject indicated that expression of the FGD3 gene is a strong marker of good prognosis in breast cancer patients and that immunohistochemistry represents an efficient, inexpensive, reproducible evaluation method, affordable also by small Institutions.
Breast cancer is the most common cancer in women and one of the most accurately characterized in terms of biomolecular and pathological features. Traditional prognostic and predictive factors allow defining prognosis and response to treatment in breast cancer patients. Tumor size, lymph node involvement, lymph vascular invasion, tumor grading, histological type, multifocal disease, presence of ductal carcinoma in situ (DCIS) component, surgical margins status and biomolecular type (Luminal A, Luminal B, HER-2 positive, triple negative) are well established markers of disease-free survival (DFS) and overall survival (OS). However, within the same subgroup, patients often have different outcomes. The presence of other unknown markers of survival may account for such differences. Therefore, there is a continuous effort to identify new prognostic factors, that could help to better predict the individual outcome within the same histological and molecular subgroup of breast cancer patients (1).
The introduction of RT-qPCR and microarray DNA analysis made it possible to use a gene expression profiling (GEP) for the diagnosis of patients with certain breast cancer characteristics (2). Thanks to GEP, it is possible to analyze all the transcribed mRNAs of a single cell, so we can have a picture of a cell's gene activity and expressed proteins. Oncotype DX (3) and MammaPrint (4) are two examples of this new molecular approach: they analyze, respectively, 21 and 70 genes using RT-PCR and microarray DNA methods. ASCO 2016 Guidelines (5) and St Gallen Consensus Conference 2017 (6) recommend the use of GEP to assess the need for adjuvant chemotherapy in addition to hormonotherapy in ER/PgR-positive, HER-2-negative, lymphnode-negative patients. However, GEP is not commonly used in every institution around the world because of its high costs and the difficulty in interpretation.
The present study aims to review the most promising prognostic factors, recently established using the new techniques, underlining the advantages and discussing the feasibility of a molecular approach.
We performed a MEDLINE literature search, using PubMed, to find the most recent articles on new breast cancer prognostic factors. We focused our attention on the FGD3 gene, which seemed to be one of the most promising recently identified markers of outcome. We collected all the publications about the FGD3 gene, using keywords like breast cancer, outcome, prognostic factors and FGD3 gene. We identified 9 eligible research articles regarding FGD3 gene characteristics and functions, which we included in our review. Among them, five studies investigated the role of FGD3 as marker of outcome in breast cancer. The findings of these studies are summarized in Table I.
FGD3 Gene as a Promising Prognostic Factor in Breast Cancer
In 2013, Margolin et al. carried out a crowdsourced research study in order to identify genes associated with breast cancer prognosis: the Sage Bionetwork/DREAM Breast Cancer Prognosis Challenge (BCC), based on a dataset of 1981 breast cancer samples (METABRIC Data Set) (7). During the BCC, several genes came up as potential markers of prognosis. One of the most promising genes was FGD3 (Facio-Genital Dysplasia gene 3). This gene is localized on the long arm of chromosome 9 (Chr9q22.31) and encodes for FYVE, RhoGEF and PH-Domain containing protein 3. The FGD3 gene was isolated and mapped for the first time in mice in 2000 by Pasteris et al. (8). Their study aimed to identify the genes involved in Facio-Genital Dysplasia (Aarskog syndrome), an X-linked human developmental disease characterized by facial and genital dimorphisms, multiple skeletal alterations and low height, traditionally attributed to FGD1 gene mutations. The FGD1 gene is a guanine nucleotide exchange factor which activates the Rho GTPase Cdc42, resulting in cytoskeleton modifications and cellular growth and differentiation, and it represents the founding member of a family of disease involved genes, all sharing the same activity on Cdc42, one of them being FGD3.
Hayakawa et al. (9) further investigated the activity of FGD3 gene transcription products, showing that it had an inhibitory action on cell migration, differently from FGD1 gene whose transcription resulted in stimulation of cell migration.
Following the pathway of Margolin et al.'s Challenge (7), Cheng et al. (10) examined the correlation for each BCC gene with breast cancer patients' survival. They found that the FGD3 gene was the top-ranked protective gene for breast cancer, as high level of FGD3 expression correlated with a longer survival. They also showed that silencing FGD3 gene always resulted in silencing of the Sushi Domain Containing 3 gene (SUSD3), as they are genomically adjacent to each other on the long arm of chromosome 9. Therefore, the so-called FGD3-SUSD3 metagene represented the most protective gene in the METABRIC Data Set (SUSD3 was the second top-ranked protective gene, following FGD3). The lack of FGD3-SUSD3 expression was more strongly associated to poor outcome than the lack of ER expression. In addition, they found that ER-negative breast cancers tended to express low level of FGD3-SUSD3 metagene. In a further study by Moy et al. in 2015 (11), SUSD3 was found to be highly expressed in ER-positive breast cancers, as a promoter of estrogen-dependent cell proliferation and a regulator of cell-cell and cell-substrate adhesion. It represented a marker of good prognosis, as its high expression correlated with a stronger response to aromatase inhibitors therapy. Following Cheng et al.'s evidences, in 2014 Ou Yang et al. published a follow-up study with the aim of developing a new breast cancer prognostic test, called BCAM (Breast Cancer Attractor Metagenes) (12). The BCAM test was composed of the FGD3-SUSD3 metagene, other seven attractor metagenes (CIN, MES, LYM, END, CD68, DNAJB9 and CXCL12), tumor size and number of positive lymph nodes. Their most important finding was that traditional breast cancer molecular subtypes, ER/PgR status and HER-2 status did not provide any additional prognostic information when FGD3-SUSD3 and the other attractor metagenes were taken into account. They also compared the prognostic performance of the BCAM test with already established genomic assays, as OncotypeDX and MammaPrint, in several breast cancer datasets, demonstrating that the BCAM test outperformed them in all cases where significance was reached. In 2017, Willis et al. (13) confirmed the prognostic value of the FGD3 gene in a large cohort of breast cancer patients, compared to well established proliferation genes like MKI67, AURKA and PCNA, assessed by RT-qPCR. FGD3 outperformed the others (including the related SUSD3 gene) in predicting patients' outcome: higher expression levels of the gene were strongly associated with better prognosis. Moreover, they found that lower FGD3 expression correlated with higher degree of lymph node involvement. They also demonstrated its prognostic value in head and neck squamous cell carcinoma, lung adenocarcinoma, cervical squamous cell carcinoma, bladder urothelial carcinoma and sarcoma. FGD3 gene's involvement in other kinds of carcinoma has already been reported, yet its prognostic significance has not: in 2005 a work by Lacroix et al. underlined FGD3's role in the oncogenic process of follicular thyroid carcinoma with PAX8-PPARγ1 translocation, suggesting it might represent a molecular target of PPARγ (14). Recently, Chao et al. carried out a confirmation study on FGD3 as a marker of outcome in head and neck squamous cell carcinoma, remarking its correlation with favorable outcome (15). Finally, Willis et al. by RT-qPCR also noticed that estradiol stimulation increased FGD3 mRNA expression levels, due to the presence of an ESR1 binding site within the gene (13).
FGD3 Expression in Young Breast Cancer Patients
Persuaded by these interesting findings, we carried out a study in our Institution, focusing on the prognostic role of FGD3 in a cohort of 60 young (≤40 years) breast cancer patients (16). Our purpose was to identify a marker of survival, which could better define the outcome in a subgroup of patients with a well-known higher tumor aggressiveness and, in particular, predict the outcome of patients with diseases belonging to the same molecular subgroups yet presenting different prognosis. We used immunohistochemistry (IHC) to assess FDG3 expression in our breast cancer samples, as it is a simple and inexpensive method that can easily be used even in small Institutions. We compared FGD3 expression with traditional prognostic factors, showing that higher FGD3 expression was a significant independent prognostic factor in terms of OS and that lower expression was significantly associated with decreased OS and DFS. Even stratifying and adjusting by AJCC stage, FGD3 expression retained its significance in terms of OS, as patients with early AJCC stage and low-FGD3-expressing disease had a poorer outcome than those with advanced AJCC stage and high-FGD3-expressing disease. In addition, lower FGD3 expression was associated with a more widespread lymph node involvement and, as FGD3 IHC staining intensity decreased, the involved lymph nodes rate increased.
Conclusion
The need to introduce new prognostic factors for breast cancer striving hard for precision medicine, led to the development of new prognostic tests, based on genomic/molecular assays. The clinical implications of these promising findings are however still controversial because all studies were carried out in very large cohorts, using expensive and not easily reusable methods. Nevertheless, the FGD3 gene seems to be eligible to be investigated by easily affordable methods. Indeed, our recent work showed the applicability of FGD3 expression as a significant predictor of outcome in breast cancer patients belonging to the same subgroup but presenting different outcomes, even when assessed by an inexpensive, easily reproducible method, such as IHC.
The FGD3 gene implication on oncogenic process and its prognostic role in several cancers is nowadays well established. In particular in breast cancer, the available evidences suggest that FGD3 could help to better stratify patients, detecting those at higher risk. In addition, its demonstrated inhibitory role on cellular migration and lymph node involvement may be useful to identify patients with higher probability of lymph node spreading (even though not already detected by staging investigation) and influence individual treatment to improve the outcome.
Finally, the correlation between estradiol stimulation and the increase in FGD3 gene products could represent an interesting field of further studies. Despite the well-known stimulating effect of oestrogens on breast cancer cells, the minor aggressiveness of ER-positive diseases is established (17). Based on the above, FGD3 could play a key role in this pathogenic mechanism and be a potential target of new personalized treatments.
Further studies are required to better define FGD3's clinical role, however our findings, along with previous observations, strongly support the idea that FGD3 evaluation by IHC could bring a considerable advance in the prognostic assessment of breast cancer patients. The relatively simple and inexpensive evaluation of FGD3 expression by IHC may enhance the introduction of this test into every day clinical practice in many countries.
Footnotes
Authors' Contributions
TS: Conceptualization, data curation, investigation, critical review and editing; IR: Data curation, investigation, writing original draft and editing.
Conflicts of Interest
All Authors declare that there are no conflicts of interest regarding this study.
- Received May 11, 2020.
- Revision received May 25, 2020.
- Accepted May 27, 2020.
- Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved