Factori de prognostic în cancerul de sân triplu negativ

 Prognostic factors in early stages triple negative breast cancer

First published: 24 martie 2017

Editorial Group: MEDICHUB MEDIA


Background. Triple Negative Breast Cancer (TNBC) is an aggressive disease even in early stages. There is a 30% five-year recurrence rate in stage I-III primary operable population. By gene expression analysis, an intrinsic heterogeneity was identified corresponding to different chemo sensitivity. A tendency towards aggressive adjuvant treatment is observable even though there are good prognostic histologies. Methods. We conducted a systematic review in order to identify the prognostic factors studied in early stages of TNBC. Results. Young age, metaplastic histology, peritumoral vascular invasion, high tumoral grade and high expression of Ki-67 were consistently prognostic factors for early recurrence. From biomolecular point of view, we found that bazal like, and the more recently described claudin low subtype, along with BRCA mutational status are clearly associated with poor 5-year disease free survival (DFS) and overall survival (OS). Significantly associated with negative clinical outcome are also, EGFR overexpression, p53 deleterious mutation, Bcl2 negativity and androgen receptor down regulation. Despite having a questionable feasibility, the prognostic signatures, which in case of hormone negative tumors are based on immune modulatory genes, could lead to a new therapeutic approach and spare many newly diagnosed breast cancer patients from an aggressive therapeutic approach. Conclusion. Assessment of several biomarkers might indicate the high risk TNBC population is able to benefit from adjuvant chemotherapy.

triple negative breast cancer, early stage, early recurrence, prognostic factors


Cancerul de sân triplu negativ (CSTN) este o boală agresivă chiar și în stadiile inițiale. Există o rată de recidivă de 30% în primii 5 ani în stadiile I-III operate per primam. Prin analiza expresiei genelor, s-a identificat o heterogenitate intrinsecă ce se asociază cu sensibilitate diferită la tratamentul citostatic. O tendință către un tratament adjuvant agresiv este observată în practica clinică, în pofida factorilor histologici cu prognostic bun. Metode. Am întreprins o recenzie sistematică pentru a identifica factorii de prognostic studiați în stadiile inițiale ale CSTN. Rezultate. Vârsta tânără, histologia metaplastică, invazia vasculară peritumorală, gradul mare de diferențiere tumorală și expresia înaltă de Ki-67 sunt factori prognostici sugestivi pentru recurența precoce. Din punct de vedere biomolecular, am descoperit că subtipurile „bazal like” și recentul descris „claudin low”, alături de statusul BRCA mutant, sunt în mod clar asociate cu supraviețuire fără semne de boală și supraviețuire globală scăzute la 5 ani.
Supraexpresia EGFR, deleția p53, absența Bcl2 și scăderea expresiei receptorului de androgen sunt semnificativ statistic asociate cu evoluție clinică infaustă. În pofida unei fezabilități discutabile, semnăturile de prognostic, care în cazul tumorilor fără receptori hormonali sunt bazate pe gene imunomodulatoare, ar putea conduce la o nouă abordare terapeutică și ar evita un tratament agresiv la mulți pacienți cu neoplasm mamar nou diagnosticat. Concluzie. Cuantificarea unui număr suficient de markeri biologici ar putea identifica populația cu CSTN cu risc înalt, care ar beneficia de chimioterapie adjuvantă. 


Triple negative breast cancer (TNBC) accounts for 15% to 20% of all breast cancers, with almost 200.000 new cases each year(1). Triple negative immuno phenotype is an independent predictor factor of distant metastasis and low specific survival(2,3).
The lack of targeted adjuvant therapies determines a tendency towards aggressive chemotherapy even in very early stages. 
The European and North American guideline’s recommendations are for adjuvant chemotherapy starting from 0.6 cm pathological diameter(4,5).
Despite this approach, the 5-year relapse rate in stage I-III with adjuvant chemotherapy is about 30%, the median time to metastatic recurrence is less than 3 years and the 5-year survival rate after the metastatic event is less than 30%(1).
Patients with triple negative phenotype are three times likely to develop visceral metastases than other types of breast cancer, frequently in lung and brain(6). In very early stages, T1N0-1, 5-year disease free survival (DFS) and cancer specific survival are approximately 85%(7). Nevertheless, two thirds of early-stage patients treated conservatively by surgery remain disease free at five years without adjuvant chemotherapy(3).
From histologic and prognostic point of view, triple negative breast cancer goes from apocrine subtype, very low aggressive, to metaplastic highly aggressive and, from biomolecular stand point, almost 30% is represented in varied proportions by luminal and HER2 like clusters and more than 70% by basal like, demonstrating its heterogeneity(8,9).
In order to identify the clinical, histological and biomolecular factors that might subclassify TNBC into different prognostic groups to select patients who are, or not, candidates for aggressive adjuvant chemotherapy, we conducted a systematic review.


A general review was conducted based on PubMed and Medline databases using the searching terms: triple negative breast cancer, early stage, early recurrence, prognostic factors. Additional sources were identified from references cited in the articles identified by electronic searching.
The selection criteria for the retained articles were: period of publication between January 2005 and December 2015, early stages triple negative breast cancer with surgery like primary treatment treated or not treated by adjuvant chemotherapy population and investigating for the prognostic factors for relapsed at 3-5 years from diagnosis.


Usually in breast cancer population, the young age correlates with histologically higher-grade tumors, highly aggressive and for that reason we presume a poorer outcome in this age category(10). As compared to other subtypes, TNBC is already prevalent at younger ages, as older individuals make up nearly 20% of cases(11,12).
In a retrospective study of 653 early stage TNBC patients, 84% having basal like phenotype, for a mean follow-up of 88 months, younger age defined as below 53 years old, the mean age of the population, was statistically significant for both DFS and overall survival (OS): HR 1.630 (P = 0.033), respectively 1.8 (P = 0.001)(13). In a study extended over 37 years, a cohort made of women older than 70 years having early operable triple negative breast cancer, a better survival trend was noted, with 5-year breast cancer specific survival of 79% versus 73% in younger patients (p= 0.39). Without receiving any adjuvant chemotherapy, the five year regional relapse rate was 9% versus 14% and the metastases relapse rate 27% versus 30% in the elderly patients compared to their younger counterpart. It is worth mentioning that patients older than 70 years had not received any adjuvant chemotherapy(14).
Unlike the mentioned studies, the large recent Zhu W. et al. analysis made by searching the Surveillance, Epidemiology, and End Results (SEER) database and enrolling primary non-metastatic TNBC cases showed that patients over 70 years had a distinctly worse overall survival (HR: 3.042, p<0.001) and cancer-specific survival (HR: 2.125, p<0.001) than the younger patients. Despite the tumoral phenotype characteristics benign-like, a lower probability of lymph node metastasis (N1-3, 30.5% vs. 36.2%; p<0.001), early TNM stages (stage I, 42.5% vs. 35.2%; p<0.001) and a low differentiation grade (grade I/II, 28.4% vs. 17%; p<0.001), the under treatment seems to exert an important impact on outcome results in older-aged TNBC women. Only 92.8% of the older women underwent surgery and less than a half was given radiotherapy: significantly less than young patients (94.6%, p = 0.002 respectively 50.8%, p<0.001)(15).
Epidemiological studies show the highest prevalence of TNBC among younger premenopausal African-American women. The majority of studies have shown a tendency to worse survival associated with African race, in patients with triple negative tumors(16,17). 
In a 71% Afro-Americans population from 124 TN patients treated by chemotherapy and surgery, after a median follow-up of 23 months, 28% had an event versus 19% in Caucasian patients (p = 0.37). Three-year event free survival (EFS) and Breast Cancer Specific Survival (BCSS) trended towards the white race, 77% versus 64% (p = 0.20) and 92% versus 76% (p= 0.13) respectively, with similar results on multiple variable modeling (EFS: HR 0.62, p= 0.29; BCSS: HR 0.36, p = 0.18)(18). The same trend was observed in a larger retrospective study on 493 TNBC patients, the hazard ratio for the African-American women being 1.19 (p = 0.39) and 0.91 (p = 0.64) for OS and DFS, respectively, so without reaching the statistical difference(19).
Obesity is a modifiable lifestyle risk factor and it has been shown to be associated with increased risk of developing breast cancer, including triple negative phenotype(20). A substantial body of evidence exists linking Body Mass Index (BMI) to prognostic outcome among women with breast cancer but without statistical significance in triple negative population(21,22). In a study presented at the San Antonio Breast Cancer Symposium in 2011 by Sparano and colleagues, on the prognostic outcome of BMI in early stage breast cancer patients enrolled in the Eastern Cooperative Oncology Group adjuvant trials, it was found no significant association among women with triple negative phenotype(23). This data has been confirmed by other studies, Ademuyiwa et al. finding also no significant association between BMI and prognostic outcome among women with early stage TNBC, for a median follow-up of 37.2 months(24). The same question was addressed for 2311 stage I-III TNBC patients. By dividing the cohort into 3 groups, according to BMI, it was confirmed the absence of a significant increase in risk of distant metastases in overweight or obese patients, HR being 1.04 in BMI <25 vs. BMI 25-29.9 (p = 0.66), respectively 0.99 in BMI <25 vs. BMI ≥ 30 (p = 0.89)(25).
Histological subtype 
Invasive ductal carcinoma represents the majority of histopathological subtypes that can be seen in triple negative phenotype breast cancer. Besides ductal and lobular, less common carcinomas associated to better survival outcomes live under the name of special histologies(26).
A large analysis conducted by Montagna and colleagues, in triple negative breast cancer population primary treated by surgery, brought up a detailed presentation of survival indicators according to histological subtype. Thus, five-year DFS and OS were 77% and 84% for patients with ductal carcinoma, 56% and 89% for those with metaplastic carcinoma, and both 5-year DFS and OS were 100% for patients with adenoid cystic and medullary carcinomas, respectively. For patients with lobular histology the 5-year DFS was worse than for those with ductal subtype, 64.3%, but similarly in term of OS, 81.9%(27).
Regarding special histologies, there are few studies addressing the question of prognostic significance in a specific population. Adenoid cystic and secretory carcinoma with an incidence of less than 0.1% are associated to indolent clinical course and better prognosis, even without adjuvant chemotherapy(28). In a SEER Program study, Ghabach B et al. reported very interesting 5-year, 10-year and 15-year survival rates of 98%, 95% and 91% respectively in breast cancer population(29). Similar results have been reported by Veeratterapillay R et al., with a 100% rate of survival, without recurrence at 5 years(30).
Medullary carcinoma accounts for 3% to 5% of invasive breast carcinomas, and despite highly cellular proliferation and poor differentiation, positive outcomes have been reported(31). In a study on 41 patients study with medullary carcinoma of the breast, treated with primary radiotherapy with or without adjuvant chemotherapy, the 6-year local recurrence-free survival rate was 86%, while survival rates were 83%(32).
In contrast, metaplastic histology, which represents almost 1% of all breast carcinomas, poorly differentiated and heterogeneous, purely metaplastic or admixed with other types of invasive carcinoma(33,34) are principally basal like and associated with poor prognosis resistance to systemic cytotoxic therapy(35). In metaplastic breast cancer patients with lymph node metastasis having undergone adjuvant chemotherapy, a 3-year disease free survival rate of 44.4% versus 72.5% in other type triple negative invasive ductal carcinomas (p=0,025) has been reported(36). Rayson et al. analysis reported a 53% risk of 2-year locoregional recurrence, consistent with previous documented 5-year risk of 35% to 62% in node negative metaplastic breast cancer, significantly higher than 20%, the risk reported for other invasive breast cancers(37).
Tumor size and nodal involvement 
It is currently accepted that triple-negative phenotype is associated with larger size, grade 3 tumors, pushing margin, high risk of recurrence and distant metastasis. The relation between tumor size TNBC and lymph node status and survival is less clear, as over the years studies found discordant relations(38-40). Large cohort studies found mostly an inverse correlation, that could be explained by a lack of relationship between increasing tumor size and lymph node involvement in these type of breast cancer, indicating a preferentially hematogenously spread(41).
In a SEER study, assessing the correlation between breast cancer subtype and lymph node involvement, the triple-negative breast cancer subtype (n=361) had a significantly lower risk of lymph node positivity than the HR+/HER2- subtype (Odds ratio 0.686, p= 0.004)(42).
In one analysis designed to explore the prognostic role of Her2-neu score and Ki-67 in TNBC, the nodal status (N0 versus N1-3) was found to significantly correlate with DFS (p<0.0001) and OS (p=0.0005)(43). In a specifically designed study, the five-year metastasis-free survival was significantly higher for patients without lymph node involvement, 86%, compared to patients having one to three involved nodes, 76%, and 50% for those having more than three nodes (p = 0.038)(44).
Histological grade 
The histologic grade is a component of the Nottingham Prognostic Index (NPI) which consists of tumor size in centimeters, tumor grade (1-3) and a lymph node score (1-3) and is largely used to predict the survival in patients having operable breast cancer. In 227 cases of primary operable invasive triple negative breast carcinoma, the poor NPI score group (lymph node-positive, grade 3 tumors, and sized more than 1.5 cm) had a shorter OS (log-rank = 6.9, p = 0.008) and DFS (log-rank = 5.99, P = 0.014) compared to the subgroup of patients who did not receive chemotherapy(45). In another analysis made in a 164 TNBC cohort, NPI higher than 5.4 clearly separates the worse from moderate outcome, as nearly 72% of TNBC was grade III tumors, clearly indicating a significant correlation with survival. Tumor size appears to be also significant for survival, showing that patients with larger tumors carry about 3.2 fold increased risk of breast cancer related death, compared to patients with tumors smaller than 5 cm (p <0.001)(46).
Lymph vascular invasion 
The presence of lymph vascular invasion or peritumoral vascular invasion is a well-known indicator for a higher metastatic risk and overall poorer survival in breast tumors(47). In a specific study, peritumoral vascular invasion (PVI) was found as an independent predictor of overall mortality in triple negative invasive ductal carcinomas (HR 1.98, p<0.001)(48). By a more sensitive analysis (hematoxylin-eosin staining and classic immunohistochemistry combined) PVI was found to be prognostic even in node negative tumors, HR 2.41 (p=0.028), with a significant difference in term of five year metastasis free survival:  50.8% versus 87.5% in PVI negative population (p = 0.003)(49).
Tumor infiltration lymphocyte 
Recently, due to immunotherapy development, there is a revival of interest for the Tumor Infiltration Lymphocyte (TIL) phenomena that has been described decades ago.
TIL could be considered as an indicator of immune response degree and is presented more and more as a factor of favorable prognosis. High lymphocyte infiltration is recognized as significantly associated to the less aggressive medullary triple negative breast cancer subtype(50). 
Kreike and colleagues proposed a prognostic score associating TIL and central fibrosis. As so, four prognostic groups were identified: very good with moderate-extensive lymphocytic infiltrate and no central fibrotic zone, remaining free of metastases, little to no lymphocytic infiltrate in combination with no central fibrotic zone, having a 94% 5-year metastasis free survival, a group having moderate-extensive lymphocytic infiltrate in combination with a central fibrotic zone having 78% 5-year metastasis-free survival and the worst prognostic group with  lymphocytic infiltrate in combination with a central fibrotic zone with 5-year metastasis-free survival of 39% (P = 0.0008)(44).
A recent 8 studies meta-analyses confirmed that rich intra tumoral or stromal lymphocyte infiltration in triple negative phenotype is associated to a 30% reduction in the hazard of disease recurrence (HR = 0.70; p = 0.001), a 22% reduction in the risk of distant relapse (HR = 0.77; p = 0.0008) and a 34% reduction in the risk of death (HR = 0.66; p = 0.0003)(51).
The expression of the nuclear antigen human Ki-67 protein is associated with cell proliferation during all phases of the cell cycle excepting G0. It was shown that TNBC tumors present a higher expression of the protein than other phenotypes and correlate with nodal metastasis(52,53).
Assessment of tumoral cells proliferation by the mean of nuclear antigen Ki-67 is already used to classify breast tumors into different prognosis subgroups, its value being confirmed in several meta-analyses. The Ki-67 rate is usually measured on histological sections and is defined as the percentage of stained invasive carcinoma cells(54). 
In a study by Keam B and colleagues, in locally advanced TNBC, high Ki-67 expression, defined for a cut-off of 10%, has been shown to be a significant prognostic factor, being associated to a poor 3-year RFS (HR = 7.82, p = 0.002)(55).
The outcomes have also been confirmed in early stages for a much higher defined cut off (61%), by the association with shorter DFS (HR 3.813, p = 0.008) and OS (HR 2.351, p = 0.05) for a mean follow-up of 43 months(56).
In a differential analysis of basal like versus non basal like subtype, the correlation with Ki-67 (P >0.05) was found nonsignificant (p = 0.05), and that could be explained by their high proliferation rate. This could pose a limit to the ability of a proliferation marker to identify clinically distinct subgroups inside triple negative immune phenotype(57).
Others biomarkers  
Among the other biomarkers that could be noted in terms of prognostic significance in triple negative phenotype are mucins. These are normally present on the apical surface of gland-forming epithelial cells and, under malignant transformation they become easy to detect by immunohistochemistry laboratory methods(58).
By laboratory studies a small tissue-specific epithelial mucin (SBEM) was described, that is expressed only in mammary and salivary glands and that became used for the differential diagnosis of the primary origin of an unknown metastasis, especially in high grade and hormonal negative tumors. SBEM seems to be a potential specific marker for predicting hematogenous micro metastasis(59).
In 87 TNBC patients initially treated by surgery, in the group SBEM 3+ the median DFS and OS were found only 12 and 25 months versus 28 and 39 months in the SBEM <3+ group. High protein expression significantly correlated with DFS and OS, associating an HR of 3.370 (p = 0.008) and respectively of 4.185 (p = 0.004)(60).
Another marker which could have a therapeutical significance is Aurora A. The Aurora kinases, serine-threonine proteins that serve as regulators of mammalian cell division, are involved in chromosome segregation and cytokinesis. Aurora A and B have been implicated in tumor formation and progression and are overexpressed in a variety of cell lines(61,62). Overexpression of Aurora A (Aur-A), also known as STK15, BTAK or Aurora 2, in mouse mammary epithelium, resulted in genetic instability, subsequent tumor formation, enhanced cell migration and promoted breast cancer metastasis(63-65).
Analysis for Aur-A immunoreactivity in a cohort of 122 TNBC patients showed that high expression correlates with a significantly high recurrence rate, 47.6% vs. 20.3% (p = 0.002), within the first 3 years of follow-up and with a significantly inferior median OS than those with Aur-A low expression (67.5 months vs. 110 months, HR 3.631, P = 0.001). Along with high Ki-67, it predicted an inferior OS (P = 0.001) and PFS (P = 0.001) compared with low expression of both biomarkers group(66).
Basal like genotype
The triple negative tumors can be subdivided based on gene expression analysis in basal-like, normal like and the more recently described claudin-low molecular subtype which is uncommon and intriguing. The BRCA1-deficient breast category has also been individualized(67).
It’s worth noting that the triple negative breast cancer is an immunohistochemistry surrogate for the basal-like subtype, defined by cDNA microarray analysis(8).
Basal-like breast cancer represents about 75% of TNBC and 15% of all breast cancer, and has proved itself to hold prognostic significance. The neoplastic cells of this tumor type consistently express genes usually found in myoepithelial cells of the breast, including basal cytokeratin (CK) 5/6, 14 and 17 which are normally found in the basal layer of stratified epithelium(68-71).
From scientific perspective, microarray-based expression profiling analysis remains the gold standard for the identification of basal-like breast cancers but, due to the difficulty of applying genomic analysis in the everyday practice, researches have been made in order to simplify the method of identification. The correspondent in immunohistochemistry was obtained by the work of Nielsen and colleagues, who described a panel of four markers: ER-, HER2-, CK 5/6+ and HER1+, matching 100% specificity and 76% sensitivity. Cytokeratin expression was associated with low survival: median specific DFS was 8.8 vs. 13.2 years in negative group (p = 0.015)(72). The basal like population has an absolute 10% lower 10-year breast cancer survival than the negative phenotype, with a 1.62 times greater risk for breast cancer specific death and OS hazard ratio of 4.26 (p = 0.000741)(73). Besides, advances in high-throughput technology permitted the evidence of other less common phenotypes having distinct clinical outcomes such as medullary associated with BRCA pathway dysfunction, molecular apocrine, claudin-low, HER2-enriched without HER2 gene amplification(74).
Lehmann subtypes 
Lehmann and colleagues, through gene expression profiling from 21 breast cancer data sets and 587 cases of triple negative breast cancer, identified six triple negative molecular entities displaying significant differences in terms of incidence, risk factors, prognosis and response to treatment. There are two clusters of basal like, two of mesenchymal like, one immune related and one of androgen receptor expression. Basal like (BL) subtypes were found to express genes regulating cell proliferation pathway and in mesenchymal subtypes, genes for angiogenesis and associated with stem cells in stem like variant (MSL). In the immunomodulator (IM) subtype there are clusters for immune pathways and cytokine signaling activation and, in luminal androgen receptor (LAR), the hormonal pathways regulated by the androgen receptor are heavily enriched. The outcomes differed according to molecular subtype, RFS being significantly lower in the LAR subtype comparing to the BL1 (HR = 2.9), IM (HR = 3.2), and MSL (HR = 10.5) (P <0.05), also decreased in the M subtype compared with BL1 (HR = 2.6) and IM (HR= 2.9). Distant-metastasis free survival (DMFS) did not vary between subtypes (log-rank test, P = 0.2176), but M subtype had a significantly higher rate of metastases compared to the BL1 (HR = 2.4, P <0.05) and IM (HR = 1.9, P <0.06). The increased RFS in the absence of increased DMFS for patients in the LAR subtype suggested that recurrence could be due to local relapse(75).
In one original analysis, patients with TNBC and a Her2-neu negative score of 0 had a significantly worse DFS (p = 0.0021) and OS (p = 0.0105) compared to patients with a Her2-neu positive immunohistochemistry score of 1 or 2, negative in fluorescence in situ hybridization (FISH) test(76).
Claudin low
Recently described by microarray gene analysis, the claudin-low represents 5% of breast tumors and more than 25% of triple negative phenotype. It is characterized by mesenchymal features, low expression of cell-cell junction proteins (E-cadherin, occludin, and claudins 3, 4, and 7) and intense immune infiltrate(77). This signature gene expression derived from human tumor initiating cells (TICs) and mammary stem cells is the least differentiated phenotype along the mammary epithelial differentiation hierarchy(78).There are very few studies focused on the phenotypic and molecular characterization of claudin low (CL) subtype: in one large analysis of 673 samples, HR in triple negative versus other phenotypes was 1.7, with significant p value of 0.0012(79).
BRCA1 mutational status and BRCA ness 
Depending on the ethnic background and age of the investigated cohort, up to 20% of women with TNBC diagnosed at a young age or with a family history of breast cancer and about 14% of those with unselected family history carry a BRCA mutation. 60% to 80% of carriers of the BRCA1 germinal mutation display a TNBC phenotype. As regards to BRCA2, deleterious mutations were identified in 2-3% of TNBC and between 16% and 23% of BRCA2 carriers are TNBC(80). The clinical outcomes for women with sporadic breast cancer compared with those with BRCA-related cancers have been reported to be similar(81).
One of the first studies reporting the clinical outcome of 183 stage I-III women with triple negative breast cancer, according to BRCA1 status, treated by adjuvant alkylant agents, found no statistical difference in terms of freedom from distant metastasis (FFDM), HR 0.90 (p = 0.79), and breast cancer specific survival (BCSS) HR 0.73 (p = 0.48). The five-year OS was 82% for carriers and 74% for non-carriers, none of them with statistical significance. A propensity for brain metastasis was revealed in BRCA1 mutated population, with overall incidence of 58% versus 24% (p = 0.06)(82).
Sporadic TNBC can display clinical and molecular similarities to BRCA1 associated breast cancers. In those cases, the BRCA1 pathway may be dysfunctional despite the absence of somatic mutations, possibly due to epigenetic alterations, a concept termed “BRCAness”(83).
One of the mechanisms is the methylation of the BRCA1 promoter (BRCA1PM). In 39 TN primary tumors, this modification was detected in 30% of cases. Five-year RFS was 27% for patients having the methylation versus 62% for patients without BRCA1PM (p = 0.041) and five-year OS rate was 36% versus 77% (p = 0.004). The majority of the patients had received anthracycline (90%) and taxanes (69%) chemotherapy(84).
In a Xu et al. study evaluating the impact of BRCA1PM in Chinese breast cancer patients, a slightly better outcome was observed in subgroup BRCA1PM triple negative versus non triple negative. It’s worth mentioning that the majority of patients were treated in 1990s and received an adjuvant regimen of chemotherapy not including either anthracyclines or taxanes(85).
 It has previously been shown that the presence of a BRCA1 germline mutation and BRCA1PM typically do not coexist in the same tumor(86).
Cells without functional BRCA1 are deficient in homologous recombination repair. As a consequence, alternate repair mechanisms activate which results in genomic instability(83,87). The characteristics, gains and losses of genomic DNA have been used to identify tumors with the same pattern; some sporadic, non-BRCA1-mutant tumors that have the same genomic pattern of as BRCA1-mutated and can be classified as a BRCA1-like category(88-90).
It was found that there are 21 known DNA recombina­tion and repair genes, up-regulated in BRCA1 dysfunctional tumors that represent the BRCA1-like status. In a 112 TN breast cancer samples study assessing for mutational status in BRCA1, in 21 here mentioned genes and PIK3CA gene, it was showed that BRCA1 germline mutation and BRCA1 promoter methylation overlap with BRCA1-like status in 70% and 79%, respectively (p =0.02).  BRCA1-like tumors had a significantly worse prognosis than patients with a non-BRCA1-like tumor, HR 3.32 (P = 0.01). TP53 was found to be frequently mutated in BRCA1-like (P <0.05), while PIK3CA was frequently mutated in non-BRCA1-like tumors (P <0.05)(91).
The EGFR1, also known as ErbB1 and HER1, is a transmembrane glycoprotein receptor containing an extracellular ligand binding domain and an intracellular receptor tyrosine kinase domain(92).
EGFR overexpression in breast cancer is associated with large tumor size, poor differentiation, and poor clinical outcomes; at an intra-cellular level, it is associated with Ras-Raf-MEK-ERK signaling pathway dysregulation. Frequently, the overexpression reflects amplification and mutation of the EGFR gene and in TNBC patients it reached the rate of 50%, higher than that seen in other breast cancer subtypes. It approaches 90% in basal like, as it is, along with cytokeratin 5/6, a surrogate marker for this subtype(93-96). 
EGF-EGFR axis stimulation seems to have an important role in mechanism of metastasis in hormonal negative breast tumors(97).
In a study made in pT1-3, pN1-3 triple negative disease, EGFR overexpression, defined as more than 50% positive cells, showed to be a negative prognostic factor. Four-year DFS in the less expressed receptor group was 79%, respectively 52% (p = 0.0001), and 4-year OS was 87%, respectively 81% (P = 0.0004) with HR 2.39 (P = 0.004) for DFS and 2.34 (P = 0.01) for OS(98).
In basal like TNBC subtype, with nodal and distant metastases, a significantly higher intratumoral expression of EGFR and CK5/6 was shown as compared to those without metastases(52).
P53 is an intracellular tumor suppressor protein that induces senescence, cell-cycle arrest, or apoptosis when cells are exposed to various forms of stress, including DNA damage. TP53 gene mutation is associated with poor prognosis in many human tumors, including breast cancer(99).
Triple negative breast tumors are associated with a significantly higher expression of p53. TP53 mutations are seen in more than 80% of basal like subtype, furthermore associating PTEN loss in 35% of cases(100). The most common are the missense mutations, where one amino acid is substituted for another resulting in aberrant p53 protein(101).
In breast cancer cells, p53 positive immunostaining represents a surrogate for TP53 mutation, according to the observation that abnormal manufactured protein is retained in the cytoplasm(52).
In an analysis of two independent primary breast tumor case series, in triple negative patients, the HR for OS and EFS in p53 negative versus p53 positive population, for a median follow-up at 150 months, was 1.57 (p = 0.46), respectively 1.93 (p = 0.28), such without reaching the statistical significance(102). 
In a small cohort specific study, p53 positive 20 TNBC patients had a worse outcome, having shorter RFS than patients carrying a p53 negative tumor (p = 0.0206)(103).
Addressing the question of p53 prognostic value in the outcome of adjuvant anthracycline treated breast cancer patients, in triple-negative subgroup, Chae et al. found a significant difference for relapse-free survival (p = 0.005), but without significance in overall survival(104).
Interesting, in mouse models it was shown an endosomal recycling upregulation of epidermal growth factor receptor (EGFR) in P53 mutated triple negative breast cancer cells, increasing its oncogenic potency(105).
B-cell lymphoma 2 (Bcl2) is a protein that acts as an anti-apoptotic factor and prolongs G0 cell cycle. The tumorigenic potential of inappropriate Bcl2 protein expression was first described as a result of the chromosomal translocation t (14, 18) seen in subsets of non-Hodgkin’s lymphoma, in which it is associated with adverse outcome. Since this discovery, overexpression of Bcl2 protein has been identified in a variety of solid organ malignancies, including breast cancer(106).
Bcl2 is normally expressed in breast glandular epithelium and is known to be upregulated by estrogen receptor. In contrast to non-Hodgkin’s lymphoma, high Bcl2 level is associated to improved outcome in hormone receptor-positive node negative breast cancer(107). Bcl2 is one of the 21 genes validated in the prognostic signature, Oncotype DX(108).
In a large cohort analysis of 11.212 women with early-stage breast cancer, Bcl2 positive expression was found as a powerful positive prognostic marker in ER negative subgroup, independent of received adjuvant therapy (HR 0.63, P = 0.001)(109).
In a specific study, negative Bcl2 (Bcl2-) was observed in 70% of early primary TNBC and was significantly associated with high mitotic index. It has been shown to be an independent prognostic marker, nearly doubling the 10-year risk of death, HR 1.69 (P = 0.004) and recurrence, HR 1.64 (P = 0.0006). In Bcl2-, anthracycline exposed patients, a better breast cancer specific survival was found: 75% versus 68% in non-treated patients (P = 0.002)(110).
Androgen receptors
Androgen receptors (AR) occur in up to 90% of all breast cancer and are expressed in approximately 30% of TNBC. The cut-off generally selected for positivity is more than 10% nuclear-staining. It has been suggested that AR positivity should be associated with favorable outcomes in hormonal positive tumors, probably due to in vitro findings on the suppressive effect of ERα-positive pathway with antiproliferative effect(111,112). The majority of studies found in AR expressed triple negative population an improvement of OS and DFS, while others, mostly unspecific, showed no significant effects(113-115). In a specific study of a triple negative population, the AR negative immunostaining has been shown to be an independent prognostic factor of DFS and OS compared to AR positive patients (p = 0.032)(116).
The absence of AR expression correlates with other tumor features of aggressiveness. The Ki-67 values over 61% were described in more than 30% AR negative tumors versus 7.2% in AR positive triple negative tumors (p = 0.014)(117). In another analysis, the absence of expression was found associated with higher histologic grade (p = 0.001), development of recurrences (p = 0.038) and distant metastasis (p=0.04)(118).
Decreased AR intratumoral expression and increased EGFR and CK5/6 intratumoral expression may play a role in the development of metastases and may predict metastatic disease(52).
Cyclooxigenase 2
Cyclooxigenase 2 (Cox-2) enzyme was described as involved in cancer cell proliferation, resistance to apoptosis, mutagenesis, tumor angiogenesis and invasion. There has been no signaling of differences in Cox 2 expression between TNBC versus non TNBC, being around 30%; instead, it was shown to correlate with poor prognosis. In a recent study on 31 TN tumors, Cox-2 expression was significantly associated to overall survival (P = 0.005) for a median follow-up period of 5.5 years(120).
High VEGF levels within the primary breast tumor are correlated with shorter survival. TNBC has significant high levels of VEGF, and these have been described to associate with poor outcome regardless of tumor stage, with impaired breast cancer corrected survival BCCS, HR = 5.1 (P = 0.029), without reaching statistical significance for relapse free survival (HR = 2.1; P = 0.066)(121).
Biomarkers that remain to be explored
There are several genomic modifications that are translating in specific changes in biomarkers structure, among them being Fibroblast growth factor receptor 2(FGFR2), widely studied in recent times.
An analysis made in several datasets for FGFR2 gene amplification or overexpression found 4% in TNBC comparing to receptor positive breast cancers (p=0.0065)(122). FGFR2 amplification recruits macrophages to the tumor microenvironment and there is a strong correlation between macrophage density and poor prognosis, their recruitment leading to the promotion of cell invasion, angiogenesis, and immune suppression(122).
Prognostic signatures 
Multigene prognostic gene signatures provide standardized, complementary information to routine pathological variables and are now endorsed by ASCO, St. Gallen and NCCN guidelines as information that could assist therapeutic decision-making in ER-positive cancers(4,5,123).
These prognostic signatures fail to predict patient outcome in hormone negative breast cancer especially in the context of basal like breast cancer, because they are based on gene modules known to regulate or execute cell proliferation and the predictors described to date for hormone receptors negative breast cancer are linked mostly to expression of immune, inflammatory and chemokine networks genes.
One of the first signatures obtained by integrative analysis of three major microarray expression datasets was a seven immune response genes that were found downregulated in the estrogen receptor negative group while associating a great risk for distant metastasis (HR 2.02 p = 0.009)(124).
A forty five gene signature, applied for 48 stage I-III TNBC patients, proved a 98% predictive accuracy in distant recurrence and HR was 2.29 (p = 0.04) for a median follow-up period of 4.4 years. Functional network analysis of the 45 predictor genes revealed that deregulated TGF-β immune/inflammatory signaling may profoundly participate in metastatic invasion of triple-negative breast cancer(125).
In a larger population, 154 triple negative adjuvant treatment naïve breast cancer patients, a fourteen genes prognostic signature from which 8 genes being functionally linked to immune/inflammatory chemokine regulation, associates a HR 4.18 (p = 0.000097) in high versus low index groups(126).
A seven-gene Nano String signature feasible for paraffin-embedded studied in 203 stage I-III TNBC patients found 62% and 85% rates of distant recurrence in the low- and high-risk groups (p=0.001), respectively a HR of 2.967 (p= 0.000598) for distant metastasis free survival(127).
By combining two microarray-derived HRneg/Tneg prognostic signatures IR-7 and Buck-4, five genes signature Integrated Cytokine Score (ICS) applicable by RT-PCR assay platform was developed, based on its functional pathway linkage through interferon-γ and IL-10. In multivariate Cox proportional regression, HR was found 3.8 (P = 0.0003) and the dichotomization node-negative/ICS-low identified a subset of low-grade HR negative tumors with <10% 5-year distant recurrence risk(128).
One of the most recent signatures is that developed by a Dutch team from laboratory of proteomics from Rotterdam. It is an 89.5% sensitivity and 70.5% specificity protein signature which predicts 5-year metastasis-free survival of lymph node negative patients who did not receive systemic adjuvant therapy. The objective was the selection of TNBC patients that can be spared of the toxicity of adjuvant chemotherapy. It contains three proteins (FTH1, GANAB and STX12) that are associated with immunomodulation and cell death-associated pathways and FTH1 an iron storage protein that indirectly regulates the ratio of CD4+ T cells and CD8+ T cells by altering iron distribution. The predicted poor-prognosis patients had a HR of 12.45 (p = 0.001) to develop distant metastasis(129).


Factors that were consistently prognostics for early recurrence are young age, metaplastic histology, peritumoral vascular invasion, high tumoral grade and high expression of Ki-67.
From the biomolecular point of view, we found that basal like and more recently described claudin low subtype BRCA mutational status are clearly associated with worse 5-year DFS and OS. Significantly associated with negative clinical outcome is also EGFR overexpression, p53 deleterious mutation, Bcl2 negativity and androgen receptor downregulation.
The feasibility of prognostic signatures that in case of hormone negative are based on immune modulatory genes to lead to a new therapeutic approach and spare many newly diagnosed breast cancer patients for aggressive adjuvant chemotherapy remains to be proved.
A better assessment in every day practice of phenotypic heterogeneity of TNBC by the mean of tumoral biomarkers may allow improvement in planning and designing novel,  and individualized treatments for this disease.   n


1. Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res. 2007 Aug 1; 13(15):4429-34.
2. Swain S. Triple-Negative Breast Cancer: Metastatic Risk and Role of Platinum Agents. ASCO Clinical Science Symposium; 2008 June 3.
3. Haffty BG, Yang Q, Reiss M et al. Locoregional Relapse and Distant Metastasis in Conservatively Managed Triple  Negative Early-Stage Breast. Cancer J Clin Oncol. 2006 Dec 20; 24(36):5652-7.
4. Senkus E, Kyriakides S, Penault-Llorca F, et al. ESMO guidelines working group. Primary breast cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up. Ann Oncol 2013; 24(Suppl.  6):vi7e23.
5. NCCN guidelines version 3.2015
6. Cortazar P, Zhang L, Untch M et al. Pathological complete response and long-term clinical benefit in breast cancer: the CTNeoBC pooled analysis. Lancet. 2014 Jul 12; 384(9938):164-72.
7. Lai HW, Kuo SJ, Chen LS et al. Prognostic significance of triple negative breast cancer at tumor size 1 cm and smaller. Eur J Surg Oncol. 2011 Jan; 37(1):18-24.
8. Perou CM, Sørlie T, Eisen MB et al. Molecular portraits of human breast tumours.  Nature. 2000 Aug 17; 406(6797):747-52.
9. Prat A, Adamo B, Cheang MC et al. Molecular characterization of basal-like and non-basal-like triple-negative breast cancer. Oncologist. 2013; 18(2):123-33.
10. Azim HA Jr, Michiels S, Bedard PL et al. Elucidating prognosis and biology of breast cancer arising in young women using gene expression profiling. Clin Cancer Res. 2012 Mar 1; 18(5):1341-51.
11. Cancello G, Maisonneuve P, Rotmensz N et al. Prognosis and adjuvant treatment effects in selected breast cancer subtypes of very young women (<35 years) with operable breast cancer. Ann Oncol. 2010 Oct; 21(10):1974-81.
12. Billar JA, Dueck AC, Stucky CC et al. Triple-negative breast cancers: unique clinical presentations and outcomes. Ann Surg Oncol. 2010 Oct; 17 Suppl 3:384-90.
13. Thike AA, Iqbal J, Cheok PY et al. Triple negative breast cancer: outcome correlation with immunohistochemical detection of basal markers. Am J Surg Pathol. 2010 Jul; 34(7):956-64.
14. Syed BM, Green AR, Nolan CC et al. Biological characteristics and clinical outcome of triple negative primary breast cancer in older women - comparison with their younger counterparts. PLoS One. 2014 Jul 7; 9(7):e100573.
15. Zhu W, Perez EA, Hong R, Li Q, Xu B. Age-Related Disparity in Immediate Prognosis of Patients with Triple-Negative Breast Cancer: A Population-Based Study from SEER Cancer Registries PLoS One. 2015 May 28; 10(5):e0128345.
16. Lund MJ, Trivers KF, Porter PL et al. Race and triple negative threats to breast cancer survival: a population-based study in Atlanta, GA. Breast Cancer Res Treat. 2009 Jan; 113(2):357-70.
17. Dawood S, Broglio K, Kau SW et al. Triple receptor-negative breast cancer: the effect of race on response to primary systemic treatment and survival outcomes. J Clin Oncol. 2009 Jan 10; 27(2):220-6.
18. Sachdev JC1, Ahmed S, Mirza MM et al. Does race affect outcomes in triple negative breast cancer? Breast Cancer (Auckl). 2010 May 7; 4:23-33.
19. Pacheco JM, Gao F, Bumb C, Ellis MJ, Ma CX. Racial differences in outcomes of triple-negative breast cancer. Breast Cancer Res Treat. 2013 Feb; 138(1):281-9.
20. Phipps AI, Chlebowski RT, Prentice R et al. Body size, physical activity, and risk of triple-negative and estrogen receptor-positive breast cancer. Cancer Epidemiol Biomarkers Prev. 2011 Mar; 20(3):454-63.
21. Ewertz M, Jensen MB, Gunnarsdóttir KÁ et al. Effect of obesity on prognosis after early-stage breast cancer. J Clin Oncol. 2011 Jan 1; 29(1):25-31.
22. Phipps AI, Chlebowski RT, Prentice R et al. Body size, physical activity, and risk of triple-negative and estrogen receptor-positive breast cancer. Cancer Epidemiol Biomarkers Prev. 2011 Mar; 20(3):454-63.
23. Sparano JA, Wang M, Zhao F et al. Obesity at diagnosis is associated with inferior outcomes in hormone receptor-positive operable breast cancer. Cancer. 2012 Dec 1; 118(23):5937-46.
24. Ademuyiwa FO, Groman A, O’Connor T et al. Impact of body mass index on clinical outcomes in triple-negative breast cancer. Cancer. 2011 Sep 15; 117(18):4132-40.
25. Dawood S, Lei X, Litton JK et al. Impact of body mass index on survival outcome among women with early stage triple-negative breast cancer. Clin Breast Cancer. 2012 Oct; 12(5):364-72.
26.  Page DL. Special types of invasive breast cancer, with clinical implications. Am J Surg Pathol 2003; 27:832-5.
27. Montagna E, Maisonneuve P, Rotmensz N et al. Heterogeneity of triple-negative breast cancer: histologic subtyping to inform the outcome. Clin Breast Cancer. 2013 Feb; 13(1):31-9.
28. Azoulay S, Laé M, Fréneaux P et al. KIT is highly expressed in adenoid cystic carcinoma of the breast, a basal-like carcinoma associated with a favorable outcome. Mod Pathol. 2005 Dec; 18(12):1623-31
29. Ghabach B, Anderson WF, Curtis RE et al. Adenoid cystic carcinoma of the breast in the United States (1977 to 2006): a population-based cohort study. Breast Cancer Res. 2010; 12(4):R54.
30. Veeratterapillay R, Veeratterapillay S, Ward E et al. Adenoid cystic carcinoma of the breast: case report and review of literature. Ann R Coll Surg Engl. 2012 May; 94(4):e137-8.
31. Martinez SR, Beal SH, Canter RJ et al. Medullary carcinoma of the breast: a population-based perspective. Med Oncol. 2011 Sep; 28(3):738-44.
32. Fourquet A, Vilcoq JR, Zafrani B et al. Medullary breast carcinoma: the role of radiotherapy as primary treatment. Radiother Oncol. 1987 Sep; 10(1):1-6.
33. Reis-Filho JS, Milanezi F, Carvalho S et al. Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysis. Breast Cancer Res. 2005; 7(6):R1028-35.
34. Tavassoli FA, Devilee P: Tumors of the breast and female genital organs. Pathology and genetics of tumors of the digestive system. World Health Organization Classification of Tumors. Lyon, France: IARC Press; 2003:37–41.
35. Hennessy BT, Giordano S, Broglio K et al. Biphasic metaplastic sarcomatoid carcinoma of the breast. Ann Oncol. 2006 Apr; 17(4):605-13.
36. Bae SY, Lee SK, Koo MY et al. The prognoses of metaplastic breast cancer patients compared to those of triple-negative breast cancer patients. Breast Cancer Res Treat. 2011 Apr; 126(2):471-8.
37. Rayson D, Adjei AA, Suman VJ, Wold LE, Ingle JN.  Metaplastic breast cancer: Prognosis and response to systemic therapy. Ann Oncol 1999, 10(4):413–419.
38. Rakha EA, Reis-Filho JS, Ellis IO. Basal-like breast cancer: a critical review. J Clin Oncol. 2008 May 20; 26(15):2568-81.
39. Crabb SJ, Cheang MC, Leung S et al. Basal breast cancer molecular subtype predicts for lower incidence of axillary lymph node metastases in primary breast cancer. Clin Breast Cancer. 2008 Jun; 8(3):249-56.
40. Tan DS, Marchió C, Jones RL et al. Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients. Breast Cancer Res Treat. 2008 Sep; 111(1):27-44.
41. Dowsett M, Cuzick J, Wale C et al. Retrospective analysis of time to recurrence in the ATAC trial according to hormone receptor status: an hypothesis-generating study. J Clin Oncol. 2005 Oct 20; 23(30):7512-7.
42. Mattes MD, Bhatia JK, Metzger D et al. Breast Cancer Subtype as a Predictor of Lymph Node Metastasis according to the SEER Registry. J Breast Cancer. 2015 Jun; 18(2):143-8.
43. Schmidt G, Meyberg-Solomayer G, Gerlinger C et al. Identification of prognostic different subgroups in triple negative breast cancer by Her2-neu protein expression. Arch Gynecol Obstet. 2014 Dec; 290(6):1221-9.
44. Kreike B, van Kouwenhove M, Horlings H et al. Gene expression profiling and histopathological characterization of triple-negative/basal-like breast carcinomas. Breast Cancer Res. 2007; 9(5):R65.
45. Rakha EA, El-Sayed ME, Green AR et al. Prognostic markers in triple-negative breast cancer. Cancer. 2007 Jan 1; 109(1):25-32.
46. Albergaria A, Ricardo S, Milanezi F et al. Nottingham Prognostic Index in triple-negative breast cancer: a reliable prognostic tool?. BMC Cancer. 2011 Jul 15; 11:299.
47. Lee AH, Pinder SE, Macmillan RD et al. Prognostic value of lymphovascular invasion in women with lymph node negative invasive breast carcinoma. Eur J Cancer. 2006 Feb; 42(3):357-62.
48. Mohammed RA, Ellis IO, Mahmmod AM et al. Lymphatic and blood vessels in basal and triple-negative breast cancers: characteristics and prognostic significance. Mod Pathol. 2011 Jun; 24(6):774-85.
49. Sabatier R, Jacquemier J, Bertucci F et al. Peritumoural vascular invasion: a major determinant of triple-negative breast cancer outcome. Eur J Cancer. 2011 Jul; 47(10):1537-45.
50. Tamiolakis D, Simopoulos C, Cheva A et al. Immunophenotypic profile of tumor infiltrating lymphocytes in medullary carcinoma of the breast.  Eur J Gynaecol Oncol. 2002; 23(5):433-6.
51.  Ibrahim EM, Al-Foheidi ME, Al-Mansour MM, Kazkaz GA. The prognostic value of tumor-infiltrating lymphocytes in triple-negative breast cancer: a meta-analysis. Breast Cancer Res Treat. 2014 Dec; 148(3):467-76.
52. Peng Y. Potential prognostic tumor biomarkers in triple-negative breast carcinoma. Beijing Da Xue Xue Bao. 2012 Oct 18; 44(5):666-72.
53. Rhee J, Han SW, Oh DY et al. The clinicopathologic characteristics and prognostic significance of triple-negativity in node-negative breast cancer. BMC Cancer. 2008 Oct 23; 8:307.
54. Urruticoechea A, Smith IE, Dowsett M: Proliferation marker Ki-67 in early breast cancer. J Clin Oncol 2005, 23:7212-7220.
55. Keam B, Im SA, Lee KH et al. Ki-67 can be used for further classification of triple negative breast cancer into two subtypes with different response and prognosis. Breast Cancer Res. 2011 Mar 2; 13(2):R22.
56. Mrklić I, Ćapkun V, Pogorelić Z, Tomić S. Prognostic value of Ki-67 proliferating index in triple negative breast carcinomas. Pathol Res Pract. 2013 May; 209(5):296-301.
57. Ferguson NL, Bell J, Heidel R et al. Prognostic value of breast cancer subtypes, Ki-67 proliferation index, age, and pathologic tumor characteristics on breast cancer survival in Caucasian women. Breast J. 2013 Jan-Feb; 19(1):22-30.
58. Gendler S, Taylor-Papadimitriou J, Duhig T, Rothbard J, Burchell J. A highly immunogenic region of a human polymorphic epithelial mucin expressed by carcinomas is made up of tandem repeats. J Biol Chem. 1988 Sep 15; 263(26):12820-3.
59. Graham RA, Burchell JM, Taylor-Papadimitriou J. The polymorphic epithelial mucin: potential as an immunogen for a cancer vaccine. Cancer Immunol Immunother. 1996 Feb; 42(2):71-80.
60. Liu L, Liu Z, Qu S, Zheng Z, Liu Y, Xie X. Small breast epithelial mucin tumor tissue expression is associated with increased risk of recurrence and death in triple-negative breast cancer patients. Diagn Pathol. 2013 May 1; 8:71.
61. Keen N, Taylor S. Aurora-kinase inhibitors as anticancer agents. Nat Rev Cancer. 2004 Dec; 4(12):927-36.
62. Fu J, Bian M, Jiang Q, Zhang C. Roles of Aurora kinases in mitosis and tumori­gene­sis. Mol Cancer Res. 2007 Jan; 5(1):1-10.
63. Marumoto T, Zhang D, Saya H et al. Aurora-A - a guardian of poles. Nat Rev Cancer. 2005 Jan; 5(1):42-50.
64. Liu Q, Ruderman JV et al. Aurora A, mitotic entry, and spindle bipolarity. Proc Natl Acad Sci U S A. 2006 Apr 11; 103(15):5811-6.
65. Wang X, Zhou Y-X, Qiao W et al. Overexpression of aurora kinase A in mouse mammary epithelium induces genetic instability preceding mammary tumor formation. Oncogene. 2006 Apr; 25:7148–58.
66. Xu J, Wu X, Zhou WH et al. Aurora-A identifies early recurrence and poor prognosis and promises a potential therapeutic target in triple negative breast cancer. PLoS One. 2013; 8(2):e56919.
67. Krishnamurthy S, Poornima R, Challa VR, Goud YG. Triple negative breast cancer - our experience and review. Indian J Surg Oncol. 2012 Mar; 3(1):12-6.
68. Prat A, Perou CM. Deconstructing the molecular portraits of breast cancer. Mol Oncol. 2011 Feb; 5(1):5-23.
69. Bramwell VH, Pritchard KI, Tu D et al. A randomized placebo-controlled study of tamoxifen after adjuvant chemotherapy in premenopausal women with early breast cancer (National Cancer Institute of Canada-Clinical Trials Group Trial, MA.12). Ann Oncol. 2010 Feb; 21(2):283-90.
70. Cheang MC, Martin M, Nielsen TO et al. Defining breast cancer intrinsic subtypes by quantitative receptor expression. Oncologist. 2015 May; 20(5):474-82.
71. Yehiely F, Moyano JV, Evans JR, Nielsen TO, Cryns VL. Deconstructing the molecular portrait of basal-like breast cancer. Trends Mol Med. 2006 Nov; 12(11):537-44.
72. Nielsen TO, Hsu FD, Jensen K et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma. Clin Cancer Res. 2004 Aug 15; 10(16):5367-74.
73. Cheang MC, Voduc D, Bajdik C et al. Basal-like breast cancer defined by five biomarkers has superior prognostic value than triple-negative phenotype. Clin Cancer Res. 2008 Mar 1; 14(5):1368-76.
74. Ma CX, Luo J, Ellis MJ. Molecular profiling of triple negative breast cancer. Breast Dis. 2010; 32(1-2):73-84.
75. Lehmann BD, Bauer JA, Chen X et al. Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies. J Clin Invest. 2011 Jul; 121(7):2750-67.     
76. Schmidt G, Meyberg-Solomayer G, Gerlinger C et al. Identification of prognostic different subgroups in triple negative breast cancer by Her2-neu protein expression. Arch Gynecol Obstet. 2014 Dec; 290(6):1221-9.
77. Prat A, Parker JS, Karginova O et al. Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010; 12(5):R68.
78. Herschkowitz JI, Simin K, Weigman VJ et al. Identification of conserved gene expression features between murine mammary carcinoma models and human breast tumors. Genome Biol. 2007; 8(5):R76.
79. Sabatier R, Finetti P, Guille A et al. Claudin-low breast cancers: clinical, pathological, molecular and prognostic characterization. Mol Cancer. 2014 Oct 2; 13:228.
80. Couch FJ, Hart SN2, Sharma P et al. Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer. J Clin Oncol. 2015 Feb 1; 33(4):304-11.
81. Kriege M1, Seynaeve C, Meijers-Heijboer H et al. Distant disease-free interval, site of first relapse and post-relapse survival in BRCA1- and BRCA2-associated compared to sporadic breast cancer patients. Breast Cancer Res Treat. 2008 Sep; 111(2):303-11.
82. Lee LJ, Alexander B, Schnitt SJ et al. Clinical outcome of triple negative breast cancer in BRCA1 mutation carriers and noncarriers. Cancer. 2011 Jul 15; 117(14):3093-100.
83. Turner N, Tutt A, Ashworth A. Hallmarks of ‘BRCAness’ in sporadic cancers. Nat Rev Cancer. 2004 Oct; 4(10):814-9.
84. Sharma P, Stecklein SR, Kimler BF et al.The prognostic value of BRCA1 promoter methylation in early stage triple negative breast cancer. J Cancer Ther Res. 2014 Mar 19; 3(2):1-11.
85. Xu Y, Diao L, Chen Y et al. Promoter methylation of BRCA1 in triple-negative breast cancer predicts sensitivity to adjuvant chemotherapy. Ann Oncol. 2013 Jun; 24(6):1498-505.
86. Wei M, Grushko TA, Dignam J et al. BRCA1 promoter methylation in sporadic breast cancer is associated with reduced BRCA1 copy number and chromosome 17 aneusomy. Cancer Res. 2005 Dec 1;65(23):10692-9.
87. Wang H, Zeng ZC, Bui TA et al. Nonhomologous end-joining of ionizing radiation-induced DNA double-stranded breaks in human tumor cells deficient in BRCA1 or BRCA2. Cancer Res. 2001 Jan 1; 61(1):270-7.
88. Lips EH, Laddach N, Savola SP et al. Quantitative copy number analysis by Multiplex Ligation-dependent Probe Amplification (MLPA) of BRCA1-associated breast cancer regions identifies BRCAness. Breast Cancer Res. 2011 Oct 27; 13(5):R107.
89. Vollebergh MA, Lips EH, Nederlof PM et al. An aCGH classifier derived from BRCA1-mutated breast cancer and benefit of high-dose platinum-based chemotherapy in HER2-negative breast cancer patients. Ann Oncol. 2011 Jul; 22(7):1561-70.
90. Schouten PC, van Dyk E, Braaf LM et al. Platform comparisons for identification of breast cancers with a BRCA-like copy number profile. Breast Cancer Res Treat. 2013 Jun; 139(2):317-27.
91. Severson TM, Peeters J, Majewski I et al. BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential. Mol Oncol. 2015 Oct; 9(8):1528-38.
92. Downward J, Yarden Y, Mayes E et al. Close similarity of epidermal growth factor receptor and v-erb-B oncogene protein sequences. Nature. 1984 Feb 9-15; 307(5951):521-7.
93. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol. 1995 Jul; 19(3):183-232.
94.  Schulze WX, Deng L, Mann M. Phosphotyrosine interactome of the ErbB-receptor kinase family. Mol Syst Biol. 2005; 1:2005.0008. Epub 2005 May 25.
95. Burness ML, Grushko TA, Olopade OI. Epidermal growth factor receptor in triple-negative and basal-like breast cancer: promising clinical target or only a marker? Cancer J. 2010 Jan-Feb; 16(1):23-32.
96. Bhargava R1, Gerald WL, Li AR et al. EGFR gene amplification in breast cancer: correlation with epidermal growth factor receptor mRNA and protein expression and HER-2 status and absence of EGFR-activating mutations.  Mod Pathol. 2005 Aug; 18(8):1027-33.
97. Waters KM, Liu T, Quesenberry RD et al. Network analysis of epidermal growth factor signaling using integrated genomic, proteomic and phosphorylation data.  PLoS One. 2012; 7(3):e34515.
98. Viale G, Rotmensz N, Maisonneuve P et al. Invasive ductal carcinoma of the breast with the «triple-negative» phenotype: prognostic implications of EGFR immunoreactivity. Breast Cancer Res Treat. 2009 Jul; 116(2):317-28.
99. Brosh R, Rotter V. When mutants gain new powers: news from the mutant p53 field. Nat Rev Cancer. 2009 Oct; 9(10):701-13.
100. Koboldt DC, Fulton RS, McLellan MD et al. Comprehensive molecular portraits of human breast tumours. Nature. 2012 Oct 4; 490(7418):61-70.
101. Olivier M, Langerød A, Carrieri P et al. The clinical value of somatic TP53 gene mutations in 1,794 patients with breast cancer. Clin Cancer Res. 2006 Feb 15; 12(4):1157-67.
102. Biganzoli E, Coradini D, Ambrogi F et al. p53 status identifies two subgroups of triple-negative breast cancers with distinct biological features. Jpn J Clin Oncol. 2011 Feb; 41(2):172-9.
103. Coradini D, Biganzoli E, Ardoino I et al. p53 status identifies triple-negative breast cancer patients who do not respond to adjuvant chemotherapy. Breast. 2015 Jun; 24(3):294-7.
104. Chae BJ, Bae JS, Lee A et al. p53 as a specific prognostic factor in triple-negative breast cancer. Jpn J Clin Oncol. 2009 Apr; 39(4):217-24.
105. Shapira I, Lee A, Vora R, Budman DR. P53 mutations in triple negative breast cancer upregulate endosomal recycling of epidermal growth factor receptor (EGFR) increasing its oncogenic potency. Crit Rev Oncol Hematol. 2013 Nov; 88(2):284-92.
106. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM. Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science. 1984 Nov 30; 226(4678):1097-9.
107. Leung LK, Wang TT. Paradoxical regulation of Bcl-2 family proteins by 17beta-oestradiol in human breast cancer cells MCF-7. Br J Cancer. 1999 Oct; 81(3):387-92.
108. Paik S, Tang G, Shak S et al. Gene expression and benefit of chemotherapy in women with node-negative, estrogen receptor-positive breast cancer. J Clin Oncol. 2006 Aug 10; 24(23):3726-34.
109. Dawson SJ, Makretsov N, Blows FM et al. BCL2 in breast cancer: a favourable prognostic marker across molecular subtypes and independent of adjuvant therapy received. Br J Cancer. 2010 Aug 24; 103(5):668-75.
110. Abdel-Fatah TM, Perry C, Dickinson P et al. Bcl2 is an independent prognostic marker of triple negative breast cancer (TNBC) and predicts response to anthracycline combination (ATC) chemotherapy (CT) in adjuvant and neoadjuvant settings. Ann Oncol. 2013 Nov; 24(11):2801-7.
111. McNamara KM, Yoda T, Takagi K et al. Androgen receptor in triple negative breast cancer. J Steroid Biochem Mol Biol. 2013 Jan; 133:66-76. 
112. Castellano I, Allia E, Accortanzo V et al. Androgen receptor expression is a significant prognostic factor in estrogen receptor positive breast cancers. Breast Cancer Res Treat. 2010 Dec; 124(3):607-17.
113. Pistelli M, Caramanti M, Biscotti T et al. Androgen receptor expression in early triple-negative breast cancer: clinical significance and prognostic associations. Cancers (Basel). 2014 Jun 27; 6(3):1351-62.
114. He J, Peng R, Yuan Z et al. Prognostic value of androgen receptor expression in operable triple-negative breast cancer: a retrospective analysis based on a tissue microarray. Med Oncol. 2012 Jun; 29(2):406-10.
115. McGhan LJ, McCullough AE, Protheroe CA et al. Androgen receptor-positive triple negative breast cancer: a unique breast cancer subtype.  Ann Surg Oncol. 2014 Feb; 21(2):361-7.
116. Tang D, Xu S, Zhang Q, Zhao W. The expression and clinical significance of the androgen receptor and E-cadherin in triple-negative breast cancer. Med Oncol. 2012 Jun; 29(2):526-33.
117. Hu R, Dawood S, Holmes MD et al. Androgen receptor expression and breast cancer survival in postmenopausal women. Clin Cancer Res. 2011 Apr 1; 17(7):1867-74.
118. Park S, Koo J, Park HS et al. Expression of androgen receptors in primary breast cancer. Ann Oncol. 2010 Mar; 21(3):488-92.
119. Zhou L1, Li K, Luo Y et al. Novel prognostic markers for patients with triple-negative breast cancer.  Hum Pathol. 2013 Oct; 44(10):2180-7.
120. Linderholm BK, Hellborg H, Johansson U et al. Significantly higher levels of vascular endothelial growth factor (VEGF) and shorter survival times for patients with primary operable triple-negative breast cancer. Ann Oncol. 2009 Oct; 20(10):1639-46.
121. Turner N, Lambros MB, Horlings HM et al. Integrative molecular profiling of triple negative breast cancers identifies amplicon drivers and potential therapeutic targets. Oncogene. 2010 Apr 8; 29(14):2013-23.
122. Qian BZ, Pollard JW. Macrophage diversity enhances tumor progression and metastasis. Cell. 2010 Apr 2; 141(1):39-51.
123. Coates AS, Winer EP, Goldhirsch A et al. Tailoring therapies - improving the management of early breast cancer: St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2015. Ann Oncol. 2015 Aug; 26(8):1533-46.
124. Teschendorff AE, Miremadi A, Pinder SE, Ellis IO, Caldas C. An immune response gene expression module identifies a good prognosis subtype in estrogen receptor negative breast cancer. Genome Biol. 2007; 8(8):R157.
125. Kuo WH, Chang YY, Lai LC et al. Molecular characteristics and metastasis predictor genes of triple-negative breast cancer: a clinical study of triple-negative breast carcinomas. PLoS One. 2012; 7(9):e45831.
126. Yau C, Esserman L, Moore DH et al. A multigene predictor of metastatic outcome in early stage hormone receptor-negative and triple-negative breast cancer.  Breast Cancer Res. 2010; 12(5):R85.
127. Park YH, Jung HH, Do IG et al. A seven-gene signature can predict distant recurrence in patients with triple-negative breast cancers who receive adjuvant chemotherapy following surgery. Int J Cancer. 2015 Apr 15; 136(8):1976-84.
128. Yau C, Sninsky J, Kwok S et al.  An optimized five-gene multi-platform predictor of hormone receptor negative and triple negative breast cancer metastatic risk. Breast Cancer Res. 2013; 15(5):R103.
129. Liu NQ, Stingl C, Look MP et al. Comparative proteome analysis revealing an 11-protein signature for aggressive triple-negative breast cancer. J Natl Cancer Inst. 2014 Feb; 106(2):djt376.