Gene-expression analysis and the basal-like breast cancer subtype

Analysis of the patterns of gene expression in breast cancer suggests that it is not a single entity, but is comprised of several biologically distinct subtypes with characteristic molecular profiles. These molecular profiles confirm the clinical impression that estrogen receptor (ER)-negative differs from ER-positive, and expands our understanding by identifying breast cancer subtypes, including the basal-like and human epidermal growth factor receptor (HER)2/ER subtypes within the ER-negative subset, and the luminal A and B subtypes within ER-positive disease. The basal-like subtype is characterized by the low expression levels of the ER-related and the HER2-related group of genes, and therefore is often 'triple negative' on clinical assays for these proteins. This review discusses the molecular profiles of breast cancer with a focus on the clinical characteristics of, and treatment options for, the basal-like subtype.     

Clinical significance of basal-like breast cancer in Chinese women in Heilongjiang province

Background: Our objective was to clarify the clinical and biological characteristics of basal-like breastcancer (BLBC) and non-basal-like breast cancer (TN3BKE) in Heilongjiang. Methods: We examined, byimmunohistochemistry, expression of biological markers cytokeratin (CK) 5/6 and epidermal growth factorreceptor (EGFR) and B cell specific moloney murine leukemia virus integration site 1( Bmi-1) in triple-negativebreast cancer (TNBC). We studied the correlation between BLBC and several factors related to tumor progression,along with its prognostic value. Results: In the 229 cases of operable TNBC, BLBC was detected in 178 (77.7%)and TN3BKE- in 51 (22.2%). There was no significant difference in clinicopathological factors between them,However, BLBC was significantly associated with Bmi-1 expression (P=0.000) and shorter disease-free survival(DFS) (P = 0.045) and overall survival (OS) (P = 0.041). Conclusions: Compared with the non-basal group,patients with BLBC have a high expression of Bmi-1 and a poor prognosis.     

Calpain system protein expression in basal-like and triple-negative invasive breast cancer

BackgroundBasal-like and triple-negative breast tumours encompass an important clinical subgroup and biomarkers that can prognostically stratify these patients are required.Materials and methodsWe investigated two breast cancer tissue microarrays for the expression of calpain-1, calpain-2 and calpastatin using immunohistochemistry. The first microarray was comprised of invasive tumours from 1371 unselected patients, and the verification microarray was comprised of invasive tumours from 387 oestrogen receptor (ER)-negative patients.ResultsThe calpain system contains a number of proteases and an endogenous inhibitor, calpastatin. Calpain activity is implicated in important cellular processes including cytoskeletal remodelling, apoptosis and survival. Our results show that the expression of calpastatin and calpain-1 are significantly associated with various clinicopathological criteria including tumour grade and ER expression. High expression of calpain-2 in basal-like or triple-negative disease was associated with adverse breast cancer-specific survival (P = 0.003 and <0.001, respectively) and was verified in an independent cohort of patients. Interestingly, those patients with basal-like or triple-negative disease with a low level of calpain-2 expression had similar breast cancer-specific survival to non-basal- or receptor- (oestrogen, progesterone or human epidermal growth factor receptor 2 (HER2)) positive disease.ConclusionsExpression of the large catalytic subunit of m-calpain (calpain-2) is significantly associated with clinical outcome of patients with triple-negative and basal-like disease.     

FoxM1、c-myc蛋白在基底细胞样型乳腺癌中的表达及其临床意义

目的 探讨FoxM1和c-myc蛋白在基底细胞样型乳腺癌（BLBC）、非基底细胞样型乳腺癌（non-BLBC）及癌旁正常乳腺组织中的表达情况及二者之间的相互关系.方法 采用免疫组织化学法检测66例BLBC及其癌旁正常乳腺组织、70例non-BLBC组织FoxM1和c-myc蛋白的表达情况,采用Spearman相关分析法分析其与病理临床特征的关系.结果 BLBC、non-BLBC及癌旁正常乳腺组织中FoxM1蛋白阳性表达率分别为77.3％（51/66）、60.0％（42/70）和13.6％（9/66）,c-myc蛋白阳性表达率分别为71.2％（47/66）、54.3％（38/70）和22.7％（15/66）,FoxM1和c-myc蛋白在BLBC和non-BLBC组织中表达率均明显高于癌旁正常乳腺组织,且在BLBC组织中的表达明显高于non-BLBC组织,差异均有统计学意义（均P＜ 0.05）.在BLBC中FoxM1和c-myc蛋白表达呈正相关（r=0.294,P＜ 0.05）,且FoxM1和c-myc蛋白的表达与BLBC淋巴结转移及临床分期有关（P＜0.05）.结论 FoxM1和c-myc蛋白参与了BLBC的发生、发展,并且FoxM1和c-myc蛋白有一定的协同作用.     

Differences in autophagy-related activity by molecular subtype in triple-negative breast cancer

The aim of this study was to assess the expression of significant components of autophagy including beclin-1, light chain (LC) 3A, LC3B, and p62 in the molecular subtypes of triple-negative breast cancer (TNBC) and to evaluate the implications of the results. Tissues from 119 cases of TNBC were used for a tissue microarray. Expression of cytokeratin (CK) 5/6, epidermal growth factor receptor (EGFR), claudin 3, claudin 4, claudin7, E-cadherin, androgen receptor (AR), and gamma-glutamyltransferase 1 (GGT-1) was detected by immunohistochemical staining of the tissue microarrays. According to the results, the 119 cases of TNBC were subclassified into basal-like type (CK5/6-positive and/or EGFR-positive group), molecular apocrine type (AR-positive and/or GGT-1-positive group), claudin low type (claudin 3-, claudin 4-, or claudin 7-negative and/or E-cadherin-negative group), mixed type (having the features of more than two types), or null type (none of the above). Immunohistochemical staining for autophagy-related markers including beclin-1, LC3A, LC3B, and p62 was performed to evaluate the difference between clinicopathological parameters. TNBCs were categorized as basal-like type (36 patients, 30.3?%), molecular apocrine type (8 patients, 6.7?%), claudin low type (16 patients, 13.4?%), mixed type (37 patients, 31.1?%), and null type (22 patients, 18.5?%). Expression of nuclear p62 was higher in the molecular apocrine type and claudin low type than in other types of TNBC (p?=?0.008). Expression of beclin-1 was higher in molecular apocrine type than in other TNBC types (p?=?0.039). Expression of LC3A and LC3B showed no difference between the molecular subtypes. Multivariate Cox analysis revealed that the negative expression of p62 was associated with shorter disease-free survival [p?=?0.012; odds ratio, 3.192; 95?% confidence interval (CI), 1.293?C7.882] and shorter overall survival (p?=?0.009; odds ratio, 3.895; 95?% CI, 1.409?C10.771). Among the subtypes of TNBC, molecular apocrine breast cancer showed a higher expression of nuclear p62 and beclin-1 than others, which reflected higher autophagy activity.     

P-cadherin expression and basal-like subtype in breast cancers

Breast cancer is considered as one of the multifactorial diseases. The aim of the current study is to investigate the association between P-cadherin and molecular subtypes of breast cancer, especially the basal-like subtype. Two hundred and thirteen breast–invasive ductal carcinomas were involved in this study. The expressions of P-cadherin were detected via immunohistochemistry. The 213 cases were divided into luminal A, luminal B, HER2 overexpression subtype, and normal breast-like and basal-like subtypes according to the standard of molecular breast cancer subtypes. In addition, the expressions of CK5/6 and CK14 were detected to distinguish between the normal breast-like and the basal-like subtypes. P-cadherin expression was found in 91 cases of 213 breast–invasive ductal carcinomas, with a positive rate of 42.7 %. P-cadherin correlated negatively with estrogen receptor (ER) (p = 0.001) and progesterone receptor (p = 0.001), whereas it positively correlated with histologic grade (p = 0.003), NPI (p = 0.005), p53 (p = 0.038), and Ki67 (p = 0.022). P-cadherin expression showed a strong correlation with recurrence and distant metastasis (p = 0.009), and invasion of the vascular and soft tissues (p = 0.004). Moreover, P-cadherin expression existed in the basal-like and non-basal-like subtypes. During prognosis, P-cadherin expression was associated with decreased disease-free survival in patients (p = 0.009) and overall survival (OS) (p = 0.005). In addition, multivariate analysis showed that tumor grade (p = 0.021), ER (p = 0.015), clinical stage (p = 0.001), and P-cadherin (p = 0.033) were significant predictors of OS. The current data suggest that P-cadherin may be used to distinguish the basal-like subtype and to predict the outcome in view of the relationship with DFS and OS. Furthermore, P-cadherin expression may be useful in making treatment decisions.     

Hypomethylation of the MMP7 promoter and increased expression of MMP7 distinguishes the basal-like breast cancer subtype from other triple-negative tumors

Identification of novel targets for the treatment of basal-like breast cancer is essential for improved outcomes in patients with this disease. This study investigates the association of MMP7 expression and MMP7 promoter methylation with subtype and outcome in breast cancer patient cohorts. Immunohistochemical analysis was performed on a breast cancer tissue microarray and validated in independent histological samples. MMP7 expression significantly correlated with patient age, tumor size, triple-negative (TN) status, and recurrence. Analysis of publically available datasets confirmed MMP7 gene expression as a prognostic marker of breast cancer metastasis, particularly metastasis to the brain and lungs. Methylation of the MMP7 promoter was assessed by methylation-specific PCR in a panel of breast cancer cell lines and patient tumor samples. Hypomethylation of the MMP7 promoter significantly correlated with TN status in DNA from patient tumor samples, and this association was confirmed using The Cancer Genome Atlas (TCGA) dataset. Evaluation of a panel of breast cancer cell lines and data from the Curtis and TCGA breast carcinoma datasets revealed that elevated MMP7 expression and MMP7 promoter hypomethylation are specific biomarkers of the basal-like molecular subtype which shares considerable, but not complete, overlap with the clinical TN subtype. Importantly, MMP7 expression was identified as an independent predictor of pathological complete response in a large breast cancer patient cohort. Combined, these data suggest that MMP7 expression and MMP7 promoter methylation may be useful as prognostic biomarkers. Furthermore, MMP7 expression and promoter methylation analysis may be effective mechanisms to distinguish basal-like breast cancers from other triple-negative subtypes. Finally, these data implicate MMP7 as a potential therapeutic target for the treatment of basal-like breast cancers.     

Depletion of CCN1/CYR61 reduces triple-negative/basal-like breast cancer aggressiveness

Triple-negative/basal-like breast cancer (BC) is characterized by aggressive biological features, which allow relapse and metastatic spread to occur more frequently than in hormone receptor-positive (luminal) subtypes. The molecular complexity of triple-negative/basal-like BC poses major challenges for the implementation of targeted therapies, and chemotherapy remains the standard approach at all stages. The matricellular protein cysteine-rich angiogenic inducer 61 (CCN1/CYR61) is associated with aggressive metastatic phenotypes and poor prognosis in BC, but it is unclear whether anti-CCN1 approaches can be successfully applied in triple-negative/basal-like BC. Herein, we first characterized the prevalence of CNN1 expression in matched samples of primary tumors and metastatic relapse in a series of patients with BC. We then investigated the biological effect of CCN1 depletion on tumorigenic traits in vitro and in vivo using archetypal TNBC cell lines. Immunohistochemical analyses of tissue microarrays revealed a significant increase of the highest CCN1 score in recurrent tissues of triple-negative/basal-like BC tumors. Stable silencing of CCN1 in triple-negative/basal-like BC cells promoted a marked reduction in the expression of the CCN1 integrin receptor α_vβ₃, inhibited anchorage-dependent cell growth, reduced clonogenicity, and impaired migration capacity. In an orthotopic model of triple-negative/basal-like BC, silencing of CCN1 notably reduced tumor burden, which was accompanied by decreased microvessel density and concurrent induction of the luminal epithelial marker E-cadherin. Thus, CNN1/CYR61-targeting strategies might have therapeutic value in suppressing the biological aggressiveness of triple-negative/basal-like BC.     

Clinical trials of immunotherapy in triple-negative breast cancer

Breast Cancer Research and Treatment - This meta-analysis aimed to investigate whether receptor (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor receptor 2...     

Caveolin 1 and Caveolin 2 are associated with breast cancer basal-like and triple-negative immunophenotype

Caveolin-1 (CAV1) and caveolin 2 (CAV2) are the principal structural proteins of caveolae, sphingolipid and cholesterol-rich invaginations of the plasma membrane involved in vesicular trafficking and signal transduction. Over the recent years there has been controversy about their role in breast cancer and their suitability as markers of basal-like phenotype. Caveolin-1 and CAV2 protein expression was assessed on a tissue microarray containing 880 unselected invasive breast cancer cases, by means of immunohistochemistry. Caveolin-1 and CAV2 expression was observed in 13.4 and 5.9% of all breast cancer, respectively. Their expression was strongly associated with high histological grade, lack of steroid hormone receptor positivity (ER and PR), and expression of basal markers (basal cytokeratins, P63, P-cadherin). Furthermore, there was a significant association between CAV1 and CAV2 expression and basal-like phenotype. On univariate analysis only CAV2 had a prognostic impact on breast cancer-specific survival; however, this was not independent from other traditional markers on multivariate analysis. Our results demonstrate that both CAV1 and CAV2 are associated with basal-like phenotype. Further studies are warranted to determine whether they play an oncogenic role in basal-like/triple-negative breast cancer development or are just surrogate markers for this subgroup.     

Efficacy of everolimus, a novel mTOR inhibitor, against basal-like triple-negative breast cancer cells

Patients with triple‐negative breast cancers (TNBCs) typically have a poor prognosis because such cancers have no effective therapeutic targets, such as estrogen receptors for endocrine therapy or human epidermal growth factor receptor 2 (HER2) receptors for anti‐HER2 therapy. As the phosphatidylinositol 3′ kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) cascade is activated in TNBCs, mTOR is a potential molecular target for anticancer therapy. In this study, we investigated the antitumor activities of everolimus, an oral mTOR inhibitor, in nine TNBC cell lines. Everolimus effectively inhibited cell growth at concentrations under 100 nM (IC₅₀) in five cell lines and even in the 1‐nM range in three of the five cell lines. To identify specific characteristics that could be used as predictive markers of efficacy, we evaluated the expressions of proteins in the mTOR cascade, basal markers, and cancer stem cell markers using western blotting, fluorescent in situ hybridization (FISH), or immunohistochemistry. All five of the sensitive cell lines were categorized as a basal‐like subtype positive for either epidermal growth factor receptor (EGFR) or CK5/6, although resistant cell lines were not of this subtype and tended to exhibit the characteristics of cancer stem cells, with decreased E‐cadherin and the increased expression of Snail or Twist. In vivo assays demonstrated antitumor activity in a mouse xenograft model of basal‐like breast cancer, rather than non‐basal breast cancer. These results suggest that everolimus has favorable activity against basal‐like subtypes of TNBCs. Epidermal growth factor receptor and CK5/6 are positive predictive markers of the TNBC response to everolimus, while cancer stem cell markers are negative predictive markers.     

Low TLR9 expression defines an aggressive subtype of triple-negative breast cancer

Toll-like receptor-9 (TLR9) is a DNA receptor widely expressed in cancers. Although synthetic TLR9 ligands induce cancer cell invasion in vitro, the role of TLR9 in cancer pathophysiology is unclear. We discovered that low tumor TLR9 expression is associated with significantly shortened disease-specific survival in patients with triple negative but not with ER+ breast cancers. A likely mechanism of this clinical finding involves differential responses to hypoxia. Our pre-clinical studies indicate that while TLR9 expression is hypoxia-regulated, low TLR9 expression has different effects on triple negative and ER+ breast cancer invasion in hypoxia. Hypoxia-induced invasion is augmented by TLR9 siRNA in triple negative, but not in ER+ breast cancer cells. This is possibly due to differential TLR9-regulated TIMP-3 expression, which remains detectable in ER+ cells but disappears from triple-negative TLR9 siRNA cells in hypoxia. Our results demonstrate a novel role for this innate immunity receptor in cancer biology and suggest that TLR9 expression may be a novel marker for triple-negative breast cancer patients who are at a high risk of relapse. Furthermore, these results suggest that interventions or events, which induce hypoxia or down-regulate TLR9 expression in triple-negative breast cancer cells may actually induce their spread.     

Epidemiology of basal-like breast cancer

Risk factors for the newly identified “intrinsic” breast cancer subtypes (luminal A, luminal B, basal-like and human epidermal growth factor receptor 2-positive/estrogen receptor-negative) were determined in the Carolina Breast Cancer Study, a population-based, case–control study of African-American and white women. Immunohistochemical markers were used to subtype 1,424 cases of invasive and in situ breast cancer, and case subtypes were compared to 2,022 controls. Luminal A, the most common subtype, exhibited risk factors typically reported for breast cancer in previous studies, including inverse associations for increased parity and younger age at first full-term pregnancy. Basal-like cases exhibited several associations that were opposite to those observed for luminal A, including increased risk for parity and younger age at first term full-term pregnancy. Longer duration breastfeeding, increasing number of children breastfed, and increasing number of months breastfeeding per child were each associated with reduced risk of basal-like breast cancer, but not luminal A. Women with multiple live births who did not breastfeed and women who used medications to suppress lactation were at increased risk of basal-like, but not luminal A, breast cancer. Elevated waist-hip ratio was associated with increased risk of luminal A in postmenopausal women, and increased risk of basal-like breast cancer in pre- and postmenopausal women. The prevalence of basal-like breast cancer was highest among premenopausal African-American women, who also showed the highest prevalence of basal-like risk factors. Among younger African-American women, we estimate that up to 68% of basal-like breast cancer could be prevented by promoting breastfeeding and reducing abdominal adiposity. An erratum to this article can be found at     

Molecular features of the basal-like breast cancer subtype based on BRCA1 mutation status

BRCA1-mutated breast cancer is associated with basal-like disease; however, it is currently unclear if the presence of a BRCA1 mutation depicts a different entity within this subgroup. In this study, we compared the molecular features among basal-like tumors with and without BRCA1 mutations. Fourteen patients with BRCA1-mutated (nine germline and five somatic) tumors and basal-like disease, and 79 patients with BRCA1 non-mutated tumors and basal-like disease, were identified from the cancer genome atlas dataset. The following molecular data types were evaluated: global gene expression, selected protein and phospho-protein expression, global miRNA expression, global DNA methylation, total number of somatic mutations, TP53 and PIK3CA somatic mutations, and global DNA copy-number aberrations. For intrinsic subtype identification, we used the PAM50 subtype predictor. Within the basal-like disease, we observed minor molecular differences in terms of gene, protein, and miRNA expression, and DNA methylation variation, according to BRCA1 status (either germinal or somatic). However, there were significant differences according to average number of mutations and DNA copy-number aberrations, and four amplified regions (2q32.2, 3q29, 6p22.3, and 22q12.2), which are characteristic in high-grade serous ovarian carcinomas, were observed in both germline and somatic BRCA1-mutated breast tumors. These results suggest that minor, but potentially relevant, baseline molecular features exist among basal-like tumors according to BRCA1 status. Additional studies are needed to better clarify if BRCA1 genetic status is an independent prognostic feature, and more importantly, if BRCA1 mutation status is a predictive biomarker of benefit from DNA-damaging agents among basal-like disease.     

三阴乳腺癌的临床特征及预后的研究

目的探讨三阴乳腺癌（ER、PR、Her-2均为阴性）的临床特征及预后。方法随机抽取山东省立医院1995年10月-2000年10月间的手术切除石蜡包埋标本116例,采用免疫组化技术,检测053、Her-2、VEGF、EGFR蛋白表达情况,并做三阴乳腺癌与临床特征及预后相关性分析。结果116例乳腺癌患者中三阴乳腺癌22例,占19．0％。三阴乳腺癌与肿瘤大小、淋巴结转移数目、组织学分级、p53、EGFR蛋白均相关（P〈0．05）,与年龄、月经状况、VEGF蛋白无相关性。中位随访96（32-123）个月,22例三阴乳腺癌患者中,有12例（54．5％）发生复发转移,而94例非三阴乳腺癌患者中,20例（21．3％）发生复发转移（P〈0．05）。三阴乳腺癌表达不同的患者间总生存时间（OS）有统计学差异（P〈0．05）,而无病生存时间差异无统计学意义（P=0．2877）。Cox模型多因素回归分析显示,肿瘤大小、淋巴结转移数、组织学分级、是否为三阴乳腺癌是预测总生存率的独立预后因素。结论三阴乳腺癌通过免疫组化方法确定,有特定的临床特征,预后明显差于对照。其临床较为有效的治疗方法尚不明确,有待进一步深入研究。     

三阴性乳腺癌患者的临床特征与预后分析

Objective  

We investigated the clinical characteristics, and the prognostic factors of triple-negative breast cancer.     

侵袭性细胞基底样乳腺癌的生物学特征

最新的基因微阵列分析将乳腺癌分为5个亚型：LuminalA（ER＋）、LuminalB（ER＋）、类似正常组织的乳腺癌、Her-2／neu基因过度表达以及细胞基底样乳腺癌。研究证实,后两者无复发生存期短,预后较其他亚型差。不同于erbB-2／Her-2过表达亚型,细胞基底样乳腺癌亚型的高侵袭行为的生物学特征仍不清楚。研究发现,细胞基底样乳腺癌免疫组化特征为ER（-）、PR（-）以及Her-2（-）,而表达CK5／6、CK17、Her-1等,并且与侵袭性较强的遗传性乳腺癌BRCAl基因突变有关。在细胞基底样乳腺癌亚型中常表达一种小的热休克蛋白αB晶体蛋白,作为一种癌基因,其过度表达导致乳腺上皮细胞凋亡失控,从而抑制MAPK／ERK激酶通路。由此推测αB-晶体蛋白可能是细胞基底样乳腺癌亚型治疗的新靶点。     

The triple-negative subtype: new ideas for the poorest prognosis breast cancer

Triple-negative breast cancer accounts for about 15%-20% of all breast cancers. Patients with triple-negative subtype have a significantly increased risk of relapse and death. A panel of specific molecular alterations like high rate of p53 mutations, frequent loss of function of BRCA1, and several tyrosine kinase activations has been shown in this specific phenotype. An optimal chemotherapy regimen for these cancers remains to be determined, representing a major challenge for patient management. DNA alkylating agents, as cisplatin, were shown to be particularly effective in the neoadjuvant setting for patients with the disease. Targeted therapies are being successfully developed. Poly (ADP-ribose) polymerase-1 inhibitors induce tumor response as a single agent in BRCA1-mutated breast cancer and might sensitize cancer cells to cisplatin in the triple-negative subpopulation. Chemotherapy is a cornerstone of current clinical practice for this type of disease. Progress might derive from refined biology-driven phase II trials that will also integrate targeted agents with chemotherapy.