IBC-Related Genetic Biomarkers
Due to the aggressive behavior of IBC and poor prognosis despite complex cancer treatment, recent researches have focused on investigating the molecular genetics of IBC to use targeted therapies in the treatment. Gene expression profiling attempts to characterize IBC, however, none of them have so far been able to identify a unique mutational or phenotypic profile specific to IBC. Several studies were performed that did not reveal a significant difference in gene expression between IBC and non-IBC.7 Ross et al reported the most frequently altered genes in 53 patients with IBC, which were not unique to IBC. Amplification of MYC (32%), PIK3CA (28%), HER2 (26%) and FGFR1 (17%) and mutation of p53 (62%), BRCA2 (15%) and PTEN (15%) were detected. Forty-two percent of patients with triple-negative IBC had MYC amplification, therefore, further studies of this finding are needed to improve the survival of this group of patients with a poor prognosis.21
In another study, somatic mutations in 24 patients with metastatic IBC were tested. Three major mutations were p53 (75%), PIK3CA (41,7%) and ERBB2 (16,7%). This was the first report of higher frequency of ERBB2 mutation in IBC, especially in patients with hormone-receptor positivity, which could be a potential target in treatment for HR+ IBC.22
Rana et al evaluated the results of genetic tests of 368 women with IBC. The germline mutations were found in 14.4% of patients. 7.3% had BRCA1 or BRCA2 mutations, 6.3% had other breast cancer gene mutation (PALB2, CHEK2, ATM, BARD1), and 1.6% had a non-breast cancer-associated mutation. The highest prevalence of germline mutations was among patients with triple-negative IBC (24%). The diversity of detected germline mutations suggests the need for further studies to assess the role of them in IBC.23
The role of p53 mutation in patients with IBC was evaluated in several studies. A study of 27 patients with IBC reported two mechanisms, which can subvert the normal function of p53 – cytoplasmic sequestration of the wild-type protein and direct nuclear mutations.24 A subsequent study evaluated the prognostic significance of p53. Riou et al reported an 8.6-fold higher risk of death in patients with a p53 mutation and nuclear overexpression of p53 protein compared to patients without these findings. There were also observed prognostic interactions with HR expression. ER-negative women with p53 nuclear overexpression had a 17.9-fold higher risk of death versus 2.8-fold in patients with p53 overexpression without ER-negativity.25
Small, non-coding RNAs – microRNAs (miRNAs) have also been actively investigated as molecular biomarkers for the diagnosis and prognosis of IBC tumors. MiRNAs influence tumor´s intrinsic and extrinsic components and also modulation of TME by regulation of the expression of genes by targeting mRNAs. Qi et al described 5 potential miRNAs as diagnostic molecular biomarkers in IBC (miR-301b, miR-451, miR-15a, miR-342-3p and miR-342-5p), some miRNAs associated with a better (miR-19a, miR-7, miR-324-5p) and with poorer prognosis (miR-21, miR-205).26 Other studies described the lower expression of miR-26b in IBC than in normal breast tissue and lower expression of miR-205 in IBC compared with non-IBC. Lower expression of both, miR-26b and miR-205, was associated with shorter distant metastasis-free survival and overall survival.27,28 The potential application of IBC associated miRNAs for diagnosis and prognosis of IBC requires further investigation.
The major goal of recently published studies was to identify differences in the gene expression of IBC from non-IBC breast cancers to find out targetable genomic drivers.
A novel gene called WNT1 inducible-signaling pathway protein 3 (WISP3) appears to operate as a tumor suppressor gene in IBC. About 80% of IBC is characterized by the loss of WISP3 (versus 21% in non-IBC). WISP3 is a protein able to inhibit the invasive potential of malignant cells and tumor cell growth.29
Another of the most highly overexpressed genes in IBC is the putative oncogene RhoC GTPase (90% in IBC versus 38% in non-IBC). RhoC GTPase is a member of Ras superfamily proteins and it is involved in cytoskeletal reorganization. Upregulation of the Rhoc GTPase gene leads to the release of angiogenic cytokines, enhanced invasiveness potential, and high motility of the tumors cells. RhoC GTPase is also associated with the upregulation of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-6 (IL-6), and interleukin-8 (IL-8), what is responsible for angiogenic stroma formation in IBC.30–32 RhoC GTPase protein, as mentioned above, plays a role in focal adhesion and invasion ability of IBC cells and in increasing metastatic capacity. RhoC activation is caused by caveolin 1, a cell membrane protein, which is upregulated in IBC and thus increases the invasiveness of cancer cells.33,34
Two studies reported high upregulation of the gene that encodes myristoylated alanine-rich C-kinase substrate (MARCKS) and the association between MARCKS overexpression and shorter metastasis-free survival (MFS) in patients with IBC. MARCKS is a substrate for protein kinase C and plays an important role in cell motility, phagocytosis, and regulation of the cell cycle. Concerning the association of MARCKS overexpression in IBC and association with poor MFS, MARCKS might represent a new potential therapeutic target in IBC.35,36
Boersma et al identified multiple pathways related to the endoplasmic reticulum stress response, that were differentially expressed in the tumor stroma of IBC versus non-IBC. Their findings suggest that the genomics of the stromal cells may play an important role in understanding the IBC phenotype.37
IBC tends to be a highly vascular tumor because of its high angiogenic and angioinvasive potential.38 Patients with IBC have greater lymphatic vessel density and higher levels of the vascular endothelial growth factor D (VEGF-D) compared to patients with non-IBC.39 VEGF-D plays an important role in angiogenesis and lymphangiogenesis of IBC. The VEGF receptor-3 is expressed in lymphatic endothelium and is activated by VEGF-C and VEGF-D ligands. The expression of VEGF-D was detected only in IBC cell lines.40–42 Thus, the efficacy of angiogenesis inhibitors in the treatment of IBC was examined. Neoadjuvant bevacizumab combined with trastuzumab and chemotherapy in HER2-positive women with IBC was efficacious and well tolerated in previously untreated IBC in Phase 2 trial (BEVERLY-2). The complete remission rate in the bevacizumab arm was markedly higher in this study than in previous works.43 In preclinical studies are evaluated platelet-derived growth factor-α (PDGFRα) inhibitors (BLU-285) and monoclonal antibodies (olaratumab). PDGFRα upregulation in tumor emboli also contributes to high angiogenesis in IBC.7
IBC cancer cells form structures that mimic the normal mammary gland, which can be the reason for high chemotherapy resistance, another intrinsic characteristic of IBC. The structures activate several signaling pathways, one of them is the epidermal growth factor receptor (EGFR) mediated pathway.7 EGFR is overexpressed in chemotherapy-resistant structures of IBC. In the study of 44 cases of IBC 30% was EGFR positive with significantly worse overall survival (OS) compared to EGFR-negative disease (P=0.01). The expression of growth factor receptors in IBC is associated with high recurrence rates and a higher risk of death; therefore, it may represent the new therapeutic targets.44
Alexander et al identified the new biomarker for therapy of IBC – Cyclin E, which plays an important role in tumors cells invasion and chemoresistance due to the regulation of many pathways important in the biology of IBC. They described a high expression of cyclin E in patients with IBC, but not correlation with cyclin E phenotype and poorer outcome as we can see in non-IBC tumors. Early targeting of this pathway may be beneficial in patients with chemotherapy resistance. We already have clinically available agents that target the cyclin E/CDK2 complex. One of these, dinaciclib, has had high toxicity in early trials,45–47 but in combination with other agents or treatment modalities and in metronomic dosing it could be a new option in therapy of IBC. CDK inhibitors could have a function in sensitization in post-mastectomy radiation, particularly in women with residual tumors, and so reduce the risk of recurrence.48
Enhancer of zeste homolog 2 (EZH2) was examined as a potential biomarker to identify patients with IBC treated with radiation with a high risk of locoregional recurrence, who may benefit from radiosensitizers. EZH2 status was tested in 62 patients with IBC who received pre- or post-operative radiation. Locoregional recurrence occurred in 16 women (25,8%), 15 of them had EZH2 expression. In addition, EZH2 positive IBC was associated significantly with negative ER status and TNR status.49
In ER-negative/HER2 positive IBC tumors, activation of the NF-κB pathway is often observed. This pathway is one of the main inflammation-mediated pathways and its activation can lead to the upregulation of antiapoptotic factors with subsequent resistance to chemotherapy. The activation was more frequently observed in IBC tumors (43% versus 4% in nonIBC).50–52
Preclinical data have demonstrated activation of the PI3K/mTOR and JAK/STAT pathways in IBC, along with the expression of inflammatory cytokines and TAMs.53 JAK/STAT pathway by promoting communication between extracellular peptide signals and cancer cell´s gene promoters supports the survival of cancer cells.54 In IBC, JAK/STAT dysregulation and subsequent isoforms pJAK2 and pSTAT3 activation and overexpression of IL-6 are observed. Jhaveri et al retrospectively analysed biomarkers expression of pJAK2, pSTAT3, IL-6, and others in IBC tumors and surrounding non-tumor tissue. Ninety-five percent of samples were pJAK2 positive, suggesting a mechanism of resistance after neoadjuvant chemotherapy. The activation of biomarkers was also demonstrated in surrounding non-tumor tissue.53,55 The combination of chemotherapy and JAK1 and JAK2 inhibitors in IBC is now being investigated in Phase I/II trial.56
Several studies have confirmed higher secretion of IL-6 in IBC tumors and significantly higher levels of serum IL-6 in IBC compared to nonIBC patients.57 The IL-6 inflammatory pathway activation is increasingly noted mainly during the lymphatic invasion of cancer cells.58 Further studies to investigate the IL-6 pathway as a therapeutic target in IBC are needed.
PIK3CA kinase, which plays an important role in the proliferation of cancer cells, is frequently mutated in breast cancer. IBC tumors are characterized by frequent genomic alterations in the HER/PI3K/mTOR pathway. PI3K may promote oncogenic signaling through mTOR activation. Marker of this activation is ribosomal S6 protein (pS6) which was positive in 100% IBC in Jhaveri´s study mentioned above.53,59 Thus, IBCs may benefit from an mTOR targeted therapy, which was demonstrated in the BOLERO-3 trial. There was the clinical benefit by mTOR inhibitor everolimus using in trastuzumab-resistant, HER2-positive patients.60
Finally, the inflammatory cyclooxygenase 2 (COX2) pathway is also well studied in IBC. COX2 is overexpressed in IBC compared to non-IBC and patients with COX2 overexpression have poorer overall survival compared to those with low COX2.61 Upregulation of COX2 and subsequently prostaglandin E2 (PGE2) overexpression is a result of chronic inflammation in TME.62 Table 1 reviews molecular biomarkers in IBC.
In conclusion, while there are several signalling pathways that show different activations in IBC compared to non-IBC setting, currently, no genetic aberration and/or activation of specific signalling pathways was identified in IBC patients, that is lacking in non-IBC setting.
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