Current perspectives on genotype classification and individualized drug targeting in triple-negative breast cancer

Triple negative breast cancer (TNBC), a special subset of breast cancer, refers to negative expressions of estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth receptor 2 (HER2). It is associated with extreme local recurrence and distant metastasis with highly invasive character. With advances in genomics, the bases of molecular classification of TNBC now include the heterogeneity of its expression at the molecular level and clinical pathology, apart from classical immunohistochemistry. Every subtype of TNBC has different individualized target drugs, which include epidermal growth factor receptor (EGFR) inhibitor, poly-AD-ribose polymerase (PARP) inhibitor, anthracycline or paclitaxel, immunotherapy and vascular endothelial growth factor receptor (VEGFR) inhibitor. Combinations of target drugs are also used. Thus, there are no widely recognized standards of genotype classification and individualized drug targeting in TNBC. In this review, relevant studies and latest developments on TNBC are presented.


INTRODUCTION
Evidence has shown that breast cancer is widely considered as a leading cause of death among women, and it has been associated with significantly increasing morbidity in recent years [1].In triple-negative breast cancer (TNBC), a subtype of breast cancer which is about 15 % of breast cancers, ERs, PR, and HER2 are not expressed [2,3].Due to its poor prognosis, high local recurrence rate and distant metastasis, TNBC does not respond well to targeted therapies [4].
Previous reports indicated that in TNBC, the different expressions of genic subtypes produce different hereditary events.For example, mutations in RAD51D, MRE11 and PALB2 may confer a higher risk of TNBC in the subtypes [9].Moreover, it has been reported that the high BRCA1 rs80350973 mutation associated with Ki-67 indices might be a predictor of prognosis of TNBC [10].Thus, these important genic hereditary events may provide critical guide for prediction of genic targeted therapies in TNBC.
The rapid developments in investigative technologies of tissue chip and tissue microarrays have facilitated studies on genotype classifications in TNBC, resulting in enhanced understanding and useful development of genic targeted therapies.

Genotype classification of triple-negative breast cancer
In 2000, the utilization of tissue chip and microarrays from the report of Perou et al [11] led to the discovery of much greater differences in gene expressions between various breast cancers.On the basis of that finding, five basic subtypes of breast cancer were proposed, which include luminal A, luminal B, ERBB-2 (+), basallike and normal-like subtypes.A part of these subtypes pertains to triple-negative phenotype which was reported in 587 cases with TNBC by Lehmann et al [12].Thus, the classification of TNBC was molecularly categorized as basal-like 1 (BL1) with the genic characteristics of cell cycle and DNA damage response, and basal-like 2 (BL2) subtypes with growth factor signaling and myo-epithelium marker; and immune-modulatory (IM) subtype.Others are mesenchymal (M) and mesenchymal stem-like (MSL) subtypes with expression of high differentiation and genes of growth factor pathways; and luminal androgen receptor (LAR) subtype activated by luminal signal [13].
Analysis of distinct molecular profiles by Burstein et al [14] identified the clinically-relevant TNBC subtypes as LAR, mesenchymal (MES), basallike immunosuppressed (BLIS), and basal-like immune-activated (BLIA), with each specific molecular target identified within the TNBC subtypes.Therefore, "triple-negative" is thought to refer to the common complexity of heterogeneous TNBC.Individualized drug targeting treatments related to heterogeneous TNBC and their clinical features are subjects of further studies.

Individualized drug targeting in TNBC from genotype classification
Due to lack of the expression of ER and HER2, TNBC is not sensitive to endocrine therapy and trastuzumab therapy.Thus, the patients have drug resistance, resulting in tumor recurrence, metastasis and poor prognosis.In addition, the signaling pathways, targets and molecular mechanisms of TNBC are too complex for easy development of individualized drug targeting.

ADP-ribose polymerase receptor inhibitor
The members of poly ADP-ribose polymerase (PARP) PARP-1, PARP-2 and PARP-3 participate in each step of DNA damage repair [15].When PARP inhibitor affected on cancer cells with BRCA1/2 mutations, the inhibition of PARP activity can prevent the formation, thus, the dependent PARP of DNA damage repair recombination were not resolved, at last, the DNA single-strand break (SSB) without repair led to the DNA stalled replication forks to convert the DNA double-strand breaks (DSBs), causing the apoptosis because of the homologous recombination (HR) lacked of BRCA1/2, which was called the "PARP-BRCA synthetic lethality" [16].Meanwhile, the theory uncovered the mechanisms of PARP inhibitors intermediates selectively killed the HR defective cells to provide the epidemiological evidences.Studies by Evans et al [17] found that patientderived xenografts (PDXs) harbored a heterogeneous set of genomic alterations consistent with the TNBC subtypes, which could capture the molecular and phenotypic heterogeneity of TNBC.Thus, beyond germline BRCA1/2, the PARP inhibitor could alter tumors and cause tumor regression in a variety of molecular subtypes in TNBC.Clinical trials by Lee et al [18] showed the phase I/Ib study of olaparib and carboplatin in 28 women with TNBC.With olaparib 400mg bid+carboplatin AUC4 as the maximum tolerated dose, the complete response (CR) was one patient (69+ months), while the partial response (PR) was 5 patients (median 4 months, range: 4 -7 months).Res, February 2018; 17(2): 361 The clinical trial revealed that the combination of olaparib and carboplatin had modest activity in sporadic TNBC patients.However, the study was at phase I/Ib, and the sample size was small.At present, there are a number of clinical experiments regarding combinations of PARP inhibitors with other chemotherapeutic drugs such as cisplatin, carboplatin and topotecans.The efficacy of combination treatment for BRCArelated breast cancer has attained approximately 70 %.However, the combination of olaparib and topotecan presented greater side effects in hematology [19][20][21].

Platinum drugs
Generally, platinum drugs can directly impact on DNA synthesis through the crosslinking of DNA which limits DNA unwinding and replication, resulting in anti-cancer effects.Studies by Bignon et al [22] showed pCR group and non-pCR patients with TNBC or deleterious BRCA1 or BRCA2 mutation who received anthracycline/taxane-based neo-adjuvant chemotherapy had pathologic complete response (pCR).The pCR of BRCA1 and BRCA2 mutation carriers were 38.3 % [95 % CI, 26 %-55 %] and 66 % respectively, and 42.6 % [95 % CI, 29.2 %-56.8 %].The disease-free survival (DFS) and overall survival (OS) in the pCR group were much better than those in the non-pCR group, indicating a high pCR rate after therapy in BRCAmutated TNBC which is of prognostic value.Failure of TNBC patients to reach effective pCR in neo-adjuvant chemotherapy means poor prognosis.This theory is of interest to researchers in the study of the benefits of platinum drugs in treating tumors.A clinical trial has studied the feasibility of using homologous recombination deficiency (HRD) score to predict response of TNBC patients to platinumcontaining neo-adjuvant chemotherapy drugs [23].Indeed, the efficacy of platinum drug therapy for TNBC is currently at the clinical trial phases, with emphasis on neo-adjuvant chemotherapies and chemotherapies in advanced TNBC patients [24,25].However, this does not involve the trial phases of assisted therapy for TNBC after operation, which requires a large number of clinical data for verification.

Epidermal or vascular endothelial growth factor receptor inhibitors
Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) are both classic structures of receptor tyrosine kinase (RTK); the former inevitably induces tumor cell proliferation, invasion, metastasis and angiogenesis by activating Ras-Raf-MAPK, JNK and PI3 pathways [26,27].Overexpression of VEGR also affects these processes.The major inhibitors of EGFR are RTK inhibitor and anti-EGFR monoclonal antibody (mAb).These inhibitors could be potential targets for TNBC therapy [28].
In trials conducted by Baselga et al [29], a group of 173 patients with TNBC were randomly given either cisplatin combined with cetuximab or cisplatin alone.Compared with cisplatin group, the overall response rate (ORR) of the cisplatin + cetuximab group increased from 10.3 % to 20 %.The median overall survival (OS) was in the range of 9.4 to 12.9 months, while the progression-free survival (PFS) increased from 1.5 months to 3.7 months.This showed that the addition of cetuximab to cisplatin could double ORR and prolong PFS and OS of TNBC patients.The novel targeted drug apatinib is an oral VEGFR which has been shown to exhibit certain effectiveness against TNBC [30].Multicenter phase II studies revealed that the recommended dose of 750 mg apatinib/day was better than 500 mg/day with respect to controlling the disease and adverse events.Research teams have examined VEGF-dependent biomarkers in patients using apatinib, to identify advanced breast cancer population sensitive to the drug [30,31].

Hepatocyte growth factor receptor inhibitor
MET protein encoded by MET oncogene is a membrane-bound tyrosine kinase implicated in the formation and/or progression of TNBC.After activation by hepatocyte growth factor receptor (HGF), MET signaling pathway can induce the transformation of epithelia-mesenchymal transition (EMT) to augment cancer cell migration and invasion.Studies have confirmed that overexpression of c-Met can independently predict poor outcomes in breast cancer [32].The progression of TNBC can be promoted by aberrant dysregulation of the receptor tyrosine kinase c-Met.A new report has shown that the common flavanol glycoside rutin is a potential lead molecule for preventing and controlling c-Met-dependent breast malignancies [33].Through bioassay-guided identification, rutin was found to inhibit breast cancer cell proliferation, migration and invasion.When rutin was injected into nude mouse at 30 mg/kg, 3 times/week, the growth of MDA-MB-231/GFP orthotopic xenograft was significantly reduced in TNBC [33].

PI3K/AKT/mTOR pathway inhibitor
The viability, proliferation, amplification, migration, invasion and angiogenesis in several physiological and pathological processes are induced and mediated by the PI3K/AKT/mTOR pathway.The PI3K/AKT/mTOR pathway is overly activated in TNBC [34].In a recent phase I trial of mTOR inhibition at the University Texas MD Anderson Cancer Center [35], advanced TNBC patients were treated with rapamycin combined with liposomal doxorubicin and bevacizumab.The results showed objective RR of 21 %, complete response (CR) of 8 %, partial response of 13 %, and the data showed disease stability for at least 6 months in 19 % patients, a 40 % clinical benefit rate, while 74 % of the patients had aberrant PI3K pathway.
A randomized trial tested liposomal doxorubicin, bevacizumab, and temsirolimus (DAT) or liposomal doxorubicin, bevacizumab, and everolimus (DAE) to identify aberrations in PI3K pathway, with a view to improving the possibility of targeting the pathway to enhance chemotherapy response [35].Rhizomaamorphophalli has also been shown to be a potential targeting therapeutic agent for TNBC [35].Studies by Wu et al [36] found, through flow cytometry analysis that MDA-MB-231 cells treated with RhA were arrested at the S phase, because RhA decreased the migration and invasion of MDA-MB-231 cells, so that PI3K/AKT/mTOR pathway was inhibited.This suggests that RhA is a potential therapeutic candidate in the treatment of TNBC through the inhibition of the PI3K/AKT/mTOR pathway.

Androgen receptor inhibitor
The androgen receptor (AR) accounts for 10 % of LAR subtypes of TNBC, which is driven by AR signal and is sensitive to AR retardants.In ER subtypes, ERα and ERβ are two particular receptors which are closely associated with aggressive phenotypes of breast cancer [37].This novel study revealed that the TNBC cell line can express endogenous ERβ2 only, and that its expression increased in TNBC cells and decreased cell proliferation and invasion.The expression of ERβ2also decreased the expression of prolyl hydroxylase 3 (PHD3) gene and is linked to increased hypoxia inducible factor 1α (HIF-1α) protein.These findings elucidated the importance of ERβ2 in enhancing TNBC cell proliferation and invasion, and also revealed that modulation of ERβ and ERβ1 signaling contributes to the invasive characteristics of TNBC [37].Enzalutamide is a new-style anti-androgen drug.A well-designed multi-center Simon's phase II clinical trial evaluated the efficacy of enzalutamide on ARpositive TNBC, and found that clinical benefit rate (CR or PR or stable disease) was 42 % during a 16-week follow-up.The data showed that non-luminal AR subtype of TNBC had low level of AR expression, indicating that it benefited from the AR-targeted therapy [38].
Steroid hormones in the supernatant of MDA-MB-453 and primary cancer-associated fibroblasts (CAFs) were used by Kikuchi et al to study the correlation between androgen synthetic enzymes and CAFs [39].The results revealed that the expressions of 17βHSD2, 17βHSD5, and 5α-reductase1 were increased by CAFs.In addition, 17βHSD2 and 17βHSD5 in IL-6 were partially dependent on phosphorylated STAT3, while the induction of HGF-mediated 5α-reductase1 was linked to phosphorylated ERK, suggesting that intra-tumoral androgen metabolism in ER-negative breast cancer are regulated IL-6 and HGF.These findings are helpful as novel references in individualized targeted therapy.

FINAL REMARKS
The genotype classification of TNBC is necessary for understanding the biological characteristics and clinical manifestation, and for developing individualized drug targeting for TNBC.Comprehensive clinical studies have shown that cytotoxic chemotherapy is the main therapeutic approach to TNBC.Indeed, PARP inhibitor and platinum drugs are very effective for treating TNBC with BRCA mutation.Researchers consider that these are not limited to this patient, gene homologous recombination is taken into consideration in the hope of development of clinical practices; It would be a significant hallmark to demonstrate effectiveness and tolerance of AR inhibitors, since AR is a molecular target with high positive expression in TNBC.In all, more clinical trials of subgroups in larger populations with heterogeneity of TNBC subtypes should be particularly focused on, so as to improve TNBC prognosis.Due to crosslinking of TNBC and signaling pathways, the application of different combinations of individualized drug targets offers great hopes for the treatment of TNBC.

Conflict of Interest
No conflict of interest associated with this work.

Contribution of Authors
We declare that this work was done by Xiao-tian Ma, Shou-hua Rong, Yu-chao Zhang, Li-ting Jia in this article and all liabilities pertaining to claims February 2018; 17(2): 363 relating to the content of this article will be borne by the authors.All authors read and approved the manuscript for publication.Xiao-tian Ma conceived and designed the study, Shou-hua Rong and Yu-chao Zhang collected and analyzed the data, Li-ting Jia wrote the manuscript.ABT-888, a PARP inhibitor, and carboplatin in vitro and in vivo using BRCA1 and BRCA2 isogenic models. Mol Cancer Ther 2012;11(9): 1948-1958. 22. Bignon L, Fricker JP, Nogues C, et