Use of immunohistochemical markers can refine prognosis in triple negative breast cancer

Background  

Basal-like breast cancer has been extensively characterized on the basis of gene expression profiles, but it is becoming increasingly common for these tumors to be defined on the basis of immunohistochemical (IHC) staining patterns, particularly in retrospective studies where material for expression profiling may not be available. The IHC pattern that best defines basal-like tumors is under investigation and various combinations of ER, PR, HER2-, CK5/6+ and EGFR+ have been tested.     

Estrogen receptor (ER) mRNA expression and molecular subtype distribution in ER-negative/progesterone receptor-positive breast cancers

We examined estrogen receptor (ER) mRNA expression and molecular subtypes in stage I–III breast cancers that are progesterone receptor (PR) positive but ER and HER2 negative by immunohistochemistry (IHC) or fluorescent in situ hybridization. The ER, PR, and HER2 status was determined by IHC as part of routine clinical assessment (N = 501). Gene expression profiling was done with the Affymetrix U133A gene chip. We compared expressions of ESR1 and MKI67 mRNA, distribution of molecular subtypes by the PAM50 classifier, the sensitivity to endocrine therapy index, and the DLDA30 chemotherapy response predictor signature among ER/PR-positive (n = 223), ER-positive/PR-negative (n = 73), ER-negative/PR-positive (n = 20), and triple-negative (n = 185) cancers. All patients received neoadjuvant chemotherapy with an anthracycline and taxane and had adjuvant endocrine therapy only if ER or PR > 10 % positive. ESR1 expression was high in 25 % of ER-negative/PR-positive, in 79 % of ER-positive/PR-negative, in 96 % of ER/PR-positive, and in 12 % of triple-negative cancers by IHC. The average MKI67 expression was significantly higher in the ER-negative/PR-positive and triple-negative cohorts. Among the ER-negative/PR-positive patients, 15 % were luminal A, 5 % were Luminal B, and 65 % were basal like. The relapse-free survival rate of ER-negative/PR-positive patients was equivalent to ER-positive cancers and better than the triple-negative cohort. Only 20–25 % of the ER-negative/PR-positive tumors show molecular features of ER-positive cancers. In this rare subset of patients (i) a second RNA-based assessment may help identifying the minority of ESR1 mRNA-positive, luminal-type cancers and (ii) the safest clinical approach may be to consider both adjuvant endocrine and chemotherapy.     

Among triple-negative breast cancers, HER2(0) breast cancer shows a strong tendency to be basal-like compared with HER2(1+) breast cancer: preliminary results

Background

Diagnosis of triple-negative breast cancer (ER-negative, PgR-negative, HER2-negative; TNBC) is performed by means of immunohistological staining. HER2-negative includes HER2(0) and HER2(1+), based on differences in the staining intensity, but there have been no reports on comparison of these two types in TNBC. Accordingly, this study was designed to investigate the possible differences in the biological characteristics of HER2(0) breast cancer and HER2(1+) breast cancer in TNBC.

Methods

Tissue specimens from 89 TNBC patients were immunohistochemically stained for CK5/6, EGFR, p53, Ki67, E-cadherin, TOP2A and Bcl-2. The expressions of these markers and the clinicopathological findings were compared between the HER2(0) patient group and the HER2(1+) patient group. When either CK5/6 or EGFR was positive, the specimen was judged to be the basal-like phenotype of breast cancer.

Results

The percentages of CK5/6- and/or EGFR-positive specimens in the HER2(0) and HER2(1+) groups were 44.9 and 16.8%, respectively, showing that there was a significantly greater number of basal-like phenotype patients in the HER2(0) group (p?<?0.01). The percentage of E-cadherin-positive specimens in the HER2(0) group was 66.6%, which was significantly greater than the 40.0% recorded in the HER2(1+) group (p?<?0.05). The respective percentages of TOP2A-positive specimens in the HER2(0) and HER2(1+) groups were 55.0 and 30.0%, and the difference was statistically significant (p?<?0.05).

Conclusion

In TNBC, HER2(0) breast cancer showed a strong tendency to include more of the basal-like phenotype compared with HER2(1+) breast cancer. The staining results indicated the possibility that HER2(0) breast cancer and HER2(1+) breast cancer have different characteristics.     

Basal markers and prognosis in luminal breast cancer

Purpose

Basal marker expression in triple-negative breast cancers identifies basal-like tumours, and thus separates the TN group into two prognostic groups. However, the expression and prognostic significance of basal markers in luminal breast cancers are poorly described. The aim of this study was to investigate the expression and prognostic value of basal markers (CK5, CK14 and EGFR) in luminal breast cancer.

Methods

A total of 1423 formalin-fixed, paraffin-embedded breast cancer tumours from a well-characterized cohort of Norwegian women, previously reclassified into molecular subtypes using IHC and ISH, were included in the study. For the present study, tumours expressing at least one of the basal markers CK5, CK14 or EGFR were defined as basal marker positive. Cumulative incidence of death from breast cancer and hazard ratio analyses were used to assess prognosis according to basal marker expression.

Results and conclusion

In total, 470 cases (33.0%) were basal marker positive. A higher proportion of the basal marker-positive tumours were of histopathological grade 3 compared to basal marker negative. For hormone receptor-positive, HER2-negative cases, we found better prognosis for basal marker-positive breast cancer compared to basal marker negative. For all subtypes combined, poorer prognosis for basal marker-negative cases was found in histopathological grade 2 tumours but not among grade 1 and 3.

     

Evaluation of biological pathways involved in chemotherapy response in breast cancer

Introduction

Our goal was to examine the association between biological pathways and response to chemotherapy in estrogen receptor-positive (ER⁺) and ER-negative (ER^-) breast tumors separately.

Methods

Gene set enrichment analysis including 852 predefined gene sets was applied to gene expression data from 51 ER^- and 82 ER⁺ breast tumors that were all treated with a preoperative paclitaxel, 5-fluoruracil, doxorubicin, and cyclophosphamide chemotherapy.

Results

Twenty-seven (53%) ER^- and 7 (9%) ER⁺ patients had pathologic complete response (pCR) to therapy. Among the ER^- tumors, a proliferation gene signature (false discovery rate [FDR] q = 0.1), the genomic grade index (FDR q = 0.044), and the E2F3 pathway signature (FDR q = 0.22, P = 0.07) were enriched in the pCR group. Among the ER⁺ tumors, the proliferation signature (FDR q = 0.001) and the genomic grade index (FDR q = 0.015) were also significantly enriched in cases with pCR. Ki67 expression, as single gene marker of proliferation, did not provide the same information as the entire proliferation signature. An ER-associated gene set (FDR q = 0.03) and a mutant p53 gene signature (FDR q = 0.0019) were enriched in ER⁺ tumors with residual cancer.

Conclusion

Proliferation- and genomic grade-related gene signatures are associated with chemotherapy sensitivity in both ER^- and ER⁺ breast tumors. Genes involved in the E2F3 pathway are associated with chemotherapy sensitivity among ER^- tumors. The mutant p53 signature and expression of ER-related genes were associated with lower sensitivity to chemotherapy in ER⁺ breast tumors only.     

Identification of typical medullary breast carcinoma as a genomic sub-group of basal-like carcinomas, a heterogeneous new molecular entity

Introduction

Typical medullary breast carcinoma (MBC) has recently been recognized to be part of the basal-like carcinoma spectrum, a feature in agreement with the high rate of TP53 mutations previously reported in MBCs. The present study was therefore designed to identify phenotypic and genetic alterations that distinguish MBCs from basal-like carcinomas (BLC).

Methods

Expression levels of estrogen receptor (ER), progesterone receptor (PR), ERBB2, TP53, cytokeratins (KRTs) 5/6, 14, 8/18, epidermal growth factor receptor and KIT, as well as TP53 gene sequence and high-density array comparative genomic hybridization (CGH) profiles, were assessed and compared in a series of 33 MBCs and 26 BLCs.

Results

All tumors were negative for ER, PR and ERBB2. KRTs 5/6 were more frequently expressed in MBCs (94%) than in BLCs (56%) (p = 0.0004). TP53 mutations were disclosed in 20/26 MBCs (77%) and 20/24 BLCs (83%). Array CGH analysis showed that a higher number of gains (95 regions) and losses (34 regions) was observed in MBCs than in BLCs (36 regions of gain; 13 regions of losses). In addition, gains of 1q and 8q, and losses of X were found to be common to the two groups, whereas gains of 10p (53% of the cases), 9p (30.8% of the cases) and 16q (25.8% of the cases), and losses of 4p (34.8% of the cases), and amplicons of 1q, 8p, 10p and 12p were the genetic alterations found to characterize MBC.

Conclusion

Our study has revealed that MBCs are part of the basal-like group and share common genomic alterations with BLCs, the most frequent being 1q and 8q gains and X losses; however, MBCs are a distinct entity within the basal-like spectrum, characterized by a higher rate of KRT 5/6 expression, a higher rate of gains and losses than BLCs, recurrent 10p, 9p and 16q gains, 4p losses, and 1q, 8p, 10p and 12p amplicons. Our results thus contribute to a better understanding of the heterogeneity in basal-like breast tumors and provide potential diagnostic tools.     

Circulating biomarkers in hepatocellular carcinoma

Purpose

Our aims are to determine levels of circulating cellular and protein biomarkers in hepatocellular carcinoma (HCC) patients and to analyse any relationships with clinical parameters.

Methods

Fifty-four consenting patients were recruited. Circulating tumour cells (CTCs) were enumerated (by CellSearch) and characterised via filtration [by isolation by size of epithelial tumour cells (ISET)] with downstream immunohistochemistry (IHC). Glypican-3 (GPC3) expression in tumour biopsies and CTCs (by IHC) was compared, and levels of circulating caspase-cleaved and full-length cytokeratin 18 (CK18, measured using M30 and M65 ELISAs) were examined as a putative prognostic factor and marker of tumour burden.

Results

CTCs were identified in 14 out of 50 (28 %) patients by CellSearch and in 19 out of 19 (100 %) patients by ISET. The presence of GPC3-positive CTCs by ISET was 100 % concordant with the presence of GPC3-positive cells in the original tumour (n = 5). No statistically significant correlations were observed between CTC number and clinical characteristics, although trends were noted between CTC subtypes, Child–Pugh score and tumour node metastasis stage. Serum M30 and M65 levels (as continuous variables) significantly correlated with overall survival (OS) in a univariate analysis (p = 0.003 and p < 0.001, respectively); M65 levels remained statistically significant in a multivariate analysis (p = 0.029).

Conclusions

This is the first study to detect GPC3-positive CTCs in HCC, important for drug development with this target. The significant association of circulating CK18 with OS in HCC further exemplifies the utility of circulating biomarkers in cancer.     

Effectiveness of neo-adjuvant systemic therapy with trastuzumab for basal HER2 type breast cancer: results from retrospective cohort study of Japan Breast Cancer Research Group (JBCRG)-C03

Purpose

While human epidermal growth factor receptor 2 (HER2) target therapies have significantly improved the prognosis of patients with HER2-enriched breast cancer, differing clinical benefits and gene expression analyses suggest a divergent HER2 subgroup. We aimed to investigate whether the basal HER2 subtype of breast cancer has distinguished characteristics.

Methods

We performed a substudy by using data from a retrospective multi-institutional cohort of JBCRG-C03. Between 2001 and 2011, we identified 184 eligible patients who received concurrent neo-adjuvant chemotherapy (NAC) with trastuzumab for hormone receptor-negative and HER2-positive breast cancer. We defined basal HER2 subtype breast cancer as HER2-positive, ER/PgR-negative, and basal markers (EGFR, CK14 or CK5/6) positive by immunohistochemistrical evaluation. The pathologic complete response (pCR) and disease-free survival (DFS) rates were compared between the two subtypes.

Results

A total of 127 (69.0%) patients achieved pCR after NAC and 29 (15.8%) patients experienced events of DFS within a 42 month median follow-up period (interquartile range 26–58 months). Although the basal HER2 subtype was related with poor DFS (3 year DFS: non-basal HER2, 95.0%; basal HER2, 86.9%; adjusted HR 3.4; 95% CI 1.2–14.5), neither the subtype (pCR: non-basal HER2, 75%; basal HER2, 66.7%; adjusted OR 0.60; 95% CI 0.27–1.28) nor the degree of expression of basal markers was significantly related with the pCR rate.

Conclusion

Basal HER2 phenotype showed poor DFS, but equivalent pCR rate after concurrent neo-adjuvant chemotherapy with trastuzumab. A different treatment approach to basal-HER2 type is needed even for cases that achieved adequate clinical response after NAC.

     

CDC73 mutations and parafibromin immunohistochemistry in parathyroid tumors: clinical correlations in a single-centre patient cohort

Objective

To determine if molecular and immunohistochemical (IHC) features of the HRPT2/CDC73 gene and its product, parafibromin, predict the natural history of parathyroid malignancy, particularly atypical adenoma, as seen in a single-centre patient cohort.

Methods

Matched tumor and non-tumor tissues were obtained from 46 patients with parathyroid carcinoma (CA) (n?=?15), atypical adenoma (AA) (n?=?14) and typical adenoma (TA) (n?=?17), as defined by standardized histopathological criteria. Exons and exon-intron boundaries of the CDC73 gene were sequenced to identify germline or somatic mutations. IHC staining for parafibromin was performed and scored as positive if nuclear staining was at least partially IHC-positive.

Results

Mutations of CDC73 were observed in 9/15 (60?%) CA, 2/14 (14?%) AA, and 1/17 (6?%) TA tumors. A recurrent two basepair mutation in exon 7 -- c.679_680delAG -- accounted for half of all identified mutations. Absence of parafibromin nuclear staining was noted in 8/12 (67?%) CA, 2/13 (15?%) AA, and 3/17 (18?%) TA tumors. Median follow up times were 88?months for CA, 76?months for AA, and 104?months for TA patients. One patient, a member of a previously reported multiplex family with a germline CDC73 mutation was found to have a second adenoma after removal of an atypical adenoma.

Conclusions

Molecular screening and IHC are both useful tools in the differential diagnosis of parathyroid tumors, but both have limited sensitivity and specificity. CDC73 mutations and negative immunostaining were common in atypical adenomas, but no local recurrence was observed in any case with successful surgical removal after follow-up periods of 27 to 210?months.     

Helix-loop-helix protein inhibitor of differentiation 4 (ID4) expression is an indicator of poor survival in Nigerian breast cancer women

Aim

This study investigates the prognostic features of helix-loop-helix protein inhibitor of differentiation 4 (ID4), a negative regulator of breast cancer-associated gene 1 (BRCA1) in breast cancer from Nigerian women, where the mode of treatments remains unclear.

Materials and methods

In this study, the protein expression of ID4 was investigated in a series of 235 Nigerian women, prepared as a tissue microarray (TMA), using immunohistochemistry.

Results

There was a positive correlation between ID4 expression and variables associated with poor prognosis and basalphenotype including high histological tumour grade, expression of epidermal growth factor receptor (EGFR), cytokeratins (CK5/6, CK14), placental cadherin (P-cadherin), p53 and Phosphoinositide-3-kinases (PI3KCA). A greater proportion of ID4 positive tumours lacked expression of BRCA1, oestrogen receptor (ER) and progesterone receptor (PR). Univariate and multivariate survival analysis showed that ID4 remained a prognostic factor for breast cancer-specific survival (BCSS) independent of tumour grade, size, lymph node involvement and basal-like phenotype.

Conclusion

ID4 may play a role in the development and progression of breast cancer in Nigerian women. ID4 could also potentially be used as a target for drug therapy in this poor prognostic ethnic group.     

Role of HGF in epithelial–stromal cell interactions during progression from benign breast disease to ductal carcinoma in situ

Introduction

Basal-like and luminal breast cancers have distinct stromal–epithelial interactions, which play a role in progression to invasive cancer. However, little is known about how stromal–epithelial interactions evolve in benign and pre-invasive lesions.

Methods

To study epithelial–stromal interactions in basal-like breast cancer progression, we cocultured reduction mammoplasty fibroblasts with the isogenic MCF10 series of cell lines (representing benign/normal, atypical hyperplasia, and ductal carcinoma in situ). We used gene expression microarrays to identify pathways induced by coculture in premalignant cells (MCF10DCIS) compared with normal and benign cells (MCF10A and MCF10AT1). Relevant pathways were then evaluated in vivo for associations with basal-like subtype and were targeted in vitro to evaluate effects on morphogenesis.

Results

Our results show that premalignant MCF10DCIS cells express characteristic gene expression patterns of invasive basal-like microenvironments. Furthermore, while hepatocyte growth factor (HGF) secretion is upregulated (relative to normal, MCF10A levels) when fibroblasts are cocultured with either atypical (MCF10AT1) or premalignant (MCF10DCIS) cells, only MCF10DCIS cells upregulated the HGF receptor MET. In three-dimensional cultures, upregulation of HGF/MET in MCF10DCIS cells induced morphological changes suggestive of invasive potential, and these changes were reversed by antibody-based blocking of HGF signaling. These results are relevant to in vivo progression because high expression of a novel MCF10DCIS-derived HGF signature was correlated with the basal-like subtype, with approximately 86% of basal-like cancers highly expressing the HGF signature, and because high expression of HGF signature was associated with poor survival.

Conclusions

Coordinated and complementary changes in HGF/MET expression occur in epithelium and stroma during progression of pre-invasive basal-like lesions. These results suggest that targeting stroma-derived HGF signaling in early carcinogenesis may block progression of basal-like precursor lesions.     

Crk adaptor proteins act as key signaling integrators for breast tumorigenesis

Introduction

CT10 regulator of kinase (Crk) adaptor proteins (CrkI, CrkII and CrkL) play a role in integrating signals for migration and invasion of highly malignant breast cancer cell lines. This has important implications, as elevated CrkI/II protein levels were observed in a small cohort of breast cancer patients, which identified a potential role for Crk proteins in breast cancer progression. Numerous in vitro studies identified a role for Crk proteins in cell motility, but little is known about how Crk proteins contribute to breast cancer progression in vivo.

Methods

The clinical significance of Crk proteins in human breast cancer was assessed by analyzing published breast cancer datasets using a gene expression signature that was generated following CrkII over-expression and by examining Crk protein expression in tissue microarrays of breast tumors (n = 254). Stable knockdown of Crk (CrkI/CrkII/CrkL) proteins was accomplished using a short hairpin RNA (shRNA)-mediated approach in two basal breast cancer cell lines, MDA-231 1833TR and SUM1315, where the former have a high affinity to form bone metastases. Both in vitro assays (cell migration, invasion, soft agar growth) and in vivo experiments (intra-cardiac, tibial and mammary fat pad injections) were performed to assess the functional significance of Crk proteins in breast cancer.

Results

A gene signature derived following CrkII over-expression correlated significantly with basal breast cancers and with high grade and poor outcome in general. Moreover, elevated Crk immunostaining on tissue microarrays revealed a significant association with highly proliferative tumors within the basal subtype. RNAi-mediated knockdown of all three Crk proteins in metastatic basal breast cancer cells established a continued requirement for Crk in cell migration and invasion in vitro and metastatic growth in vivo. Furthermore, Crk ablation suppressed anchorage independent growth and in vivo orthotopic tumor growth. This was associated with diminished cell proliferation and was rescued by expression of non-shRNA targeted CrkI/II. Perturbations in tumor progression correlated with altered integrin signaling, including decreased cell spreading, diminished p130Cas phosphorylation, and Cdc42 activation.

Conclusions

These data highlight the physiological importance of Crk proteins in regulating growth of aggressive basal breast cancer cells and identify Crk-dependent signaling networks as promising therapeutic targets.     

Estrogen receptor and HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors

Introduction

Estrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors, whereas human epidermal growth factor receptor (HER)2/neu-positive breast cancers are associated with worse prognosis. The objective of the present study was to determine whether ER-positive and ER-negative status relates to epigenetic changes in breast cancer-related genes. To evaluate epigenetic differences in tumor-related genes relating to ER and HER2/neu status of primary tumors, we examined the promoter methylation status of the promoter region CpG islands of eight major breast tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARβ2, and CDH1).

Methods

Paired ER-positive (n = 65) and ER-negative (n = 65) primary breast tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted from paraffin-embedded tumor tissue after microdissection, and methylation-specific PCR and capillary-array electrophoresis analysis were performed.

Results

In early stages of tumor progression (T1 and N0), RASSF1A and CCND2 were significantly (P < 0.05) more methylated in ER-positive than in ER-negative tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis positive group than in the negative group. Double negative (ER-negative, HER2/neu-negative) breast cancers had significantly lesser frequencies of RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035, respectively). Both ER and HER2/neu status correlated independently with these epigenetic alterations.

Conclusion

We demonstrated significant differences in tumor-related gene methylation patterns relevant to ER and HER2/neu status of breast tumors. This may be of significance in the assessment of targeted therapy resistance related to ER and HER2/neu status in breast cancer patients.     

不同分子分型的HR阴性乳腺癌临床病理特征及预后的研究

目的： 探讨激素受体 (hormone receptor,HR)阴性浸润性乳腺癌的分子分型情况,以及不同分子分型的临床病理特征和预后?方法：应用组织芯片技术及免疫组化将所选200例HR阴性[包括雌激素受体 (estrogen receptor,ER)和孕激素受体 (progesterone receptor ,PR)均为阴性]浸润性乳腺癌,依据HER2及基底细胞标记[包括细胞角蛋白5/6 (cytokeratin 5/6,CK5/6),细胞角蛋白14 (cytokeratin14,CK14)及上皮生长因子受体 (epidermal growth factor receptor,EGFR)]表达情况,分为以下类型：HER2过表达型、基底细胞样型及裸型;HER2过表达型又分两类亚型：单纯性HER2过表达型、基底细胞样-HER2过表达型;比较不同分子分型乳腺癌的病理特征及预后。结果：HR阴性乳腺癌具有病理分级高,临床分期晚,发病年龄近绝经期等特点,各分型间差异无统计学意义;基底细胞样-HER2过表达型远处转移率高于基底细胞样型 (P<0.05),其5年无病生存率及5年总生存率低于各分型 (P<0.05)。 结论：基底细胞样型并非影响乳腺癌预后的独立因素,基底细胞样-HER2过表达型乳腺癌较基底细胞样型具有更差的预后情况。     

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer

Introduction

Metastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.

Methods

We used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.

Results

Most brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.

Conclusions

Deregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.     

Gene expression signatures of morphologically normal breast tissue identify basal-like tumors

Introduction

The role of the cellular microenvironment in breast tumorigenesis has become an important research area. However, little is known about gene expression in histologically normal tissue adjacent to breast tumor, if this is influenced by the tumor, and how this compares with non-tumor-bearing breast tissue.

Methods

To address this, we have generated gene expression profiles of morphologically normal epithelial and stromal tissue, isolated using laser capture microdissection, from patients with breast cancer or undergoing breast reduction mammoplasty (n = 44).

Results

Based on this data, we determined that morphologically normal epithelium and stroma exhibited distinct expression profiles, but molecular signatures that distinguished breast reduction tissue from tumor-adjacent normal tissue were absent. Stroma isolated from morphologically normal ducts adjacent to tumor tissue contained two distinct expression profiles that correlated with stromal cellularity, and shared similarities with soft tissue tumors with favorable outcome. Adjacent normal epithelium and stroma from breast cancer patients showed no significant association between expression profiles and standard clinical characteristics, but did cluster ER/PR/HER2-negative breast cancers with basal-like subtype expression profiles with poor prognosis.

Conclusion

Our data reveal that morphologically normal tissue adjacent to breast carcinomas has not undergone significant gene expression changes when compared to breast reduction tissue, and provide an important gene expression dataset for comparative studies of tumor expression profiles.     

BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A

Introduction

Treatment of breast cancer is becoming more individualized with the recognition of tumor subgroups that respond differently to available therapies. Breast cancer 1 gene (BRCA1)-deficient tumors are usually of the basal subtype and associated with poor survival rates, highlighting the need for more effective therapy.

Methods

We investigated a mouse model that closely mimics breast cancer arising in BRCA1-mutation carriers to better understand the molecular mechanism of tumor progression and tested whether targeting of the Polycomb-group protein EZH2 would be a putative therapy for BRCA1-deficient tumors.

Results

Gene expression analysis demonstrated that EZH2 is overexpressed in BRCA1-deficient mouse mammary tumors. By immunohistochemistry we show that an increase in EZH2 protein levels is also evident in tumors from BRCA1-mutation carriers. EZH2 is responsible for repression of genes driving differentiation and could thus be involved in the undifferentiated phenotype of these tumors. Importantly, we show that BRCA1-deficient cancer cells are selectively dependent on their elevated EZH2 levels. In addition, a chemical inhibitor of EZH2, 3-deazaneplanocin A (DZNep), is about 20-fold more effective in killing BRCA1-deficient cells compared to BRCA1-proficient mammary tumor cells.

Conclusions

We demonstrate by specific knock-down experiments that EZH2 overexpression is functionally relevant in BRCA1-deficient breast cancer cells. The effectiveness of a small molecule inhibitor indicates that EZH2 is a druggable target. The overexpression of EZH2 in all basal-like breast cancers warrants further investigation of the potential for targeting the genetic make-up of this particular breast cancer type.