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).
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).
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).
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 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).
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 (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 (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).
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