Molecular diagnosis in breast cancer

Breast cancer is a complex and heterogeneous disease, encompassing a plethora of entities with distinct biological features and clinical behaviour. The advent of high throughput molecular methods has allowed a systematic characterization of the genomic landscape of breast cancer, revealing a profound heterogeneity in this disease. These methods are having a profound effect on the understanding of breast cancer. Some have already been incorporated in clinical practice, such as the prognostic ‘gene signatures’ that allow the tailoring of therapy in the subgroup of patients with oestrogen receptor (ER)-positive and HER2-negative breast cancer. In this review, we discuss the contribution of the main molecular methods in breast cancer research and how this information is changing our approaches to the diagnosis and management of this disease. We also address novel developments in the diagnosis and management of HER2-positive breast carcinomas and familial breast cancer.     

HER2 status in molecular apocrine breast cancer: associations with clinical,pathological, and molecular features

Molecular apocrine breast cancer (MABC) is a distinct subtype of breast cancer. The purpose of this study was to investigate the relationship between HER2 status and clinicopathologic characteristics of MABCs from Chinese Han cohort. A cohort of 90 MABC patients were enrolled. Immunohistochemical method was performed to analyze the molecular expression, and the human epidermal growth factor receptor 2 (HER2) amplification was verified by fluorescence in situ hybridization (FISH). By studying these 90 MABC cases, the majority of studied patients were premenopausal young women (median age 48 yr) with high grade tumors. We also found that MABCs had high positive expression rates of HER2, CK8, CD44, CD166, p53 and BRCA1, the elevated Ki-67 labeling index, and favorable prognosis. There was a significantly higher incidence of lymph node metastasis and lower CD166 positive rate in HER2-negative patients compared to HER2-positive patients (54.5% vs. 37.0%, P = 0.044 and 72.7% vs. 91.3%, P = 0.021, respectively). The CK5/6 and EGFR expression rates were significant higher in HER2-negative cases than in HER2-positive cases, suggesting that there is overlap between MABC with HER2-negative phenotype and basal-like breast cancer. In addition, HER2 positive was found to be significantly associated a poor overall survival in MABCs. In conclusion, HER2 are highly expressed, and HER2 positivity could be considered as a significant biomarker of poor prognosis in MABC. The results also suggest that a subtype tumor with distinct patterns of molecule expression depending on HER2 status presented in MABC.     

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

     

Deletion of the inhibitor of growth 4 (ING4) tumor suppressor gene is prevalent in human epidermal growth factor 2 (HER2)-positive breast cancer

Inhibitor of growth 4 (ING4) is a candidate tumor suppressor gene that was shown to be deleted in 10% to 20% of breast cancers by array comparative genome hybridization analysis. We developed fluorescent in situ hybridization to detect the ING4 gene directly in the tissue samples on tumor tissue microarrays. We evaluated the ING4 gene status in 1033 breast cancer tissue samples and observed that ING4 was deleted in 16.5% (170/1033) of all breast cancers. ING4 deletion was significantly associated with Her2 overexpression: of the tumors with ING4 deletion, 23.8% (39/164) were human epidermal growth factor 2 (HER2) positive, as compared with 14.1% (115/814) of the tumors without ING4 deletion (P = .002). In addition, the tumors with ING4 deletion were more likely to belong to the HER2 molecular subtype (estrogen receptor negative/progesterone receptor negative/human epidermal growth factor positive) of breast cancer, compared with the other subtypes (28.4% HER2 versus 15.7% all, P = .002). ING4 deletion did not affect survival outcome of all patients with breast cancer (P = .797) or of the patients with HER2-positive tumors (P = .792). We conclude that ING4 deletion in breast cancer is relatively common, as 1 in 6 breast cancer harbors ING4 deletion. Furthermore, ING4 deletion is more prevalent in HER2-positive tumors, suggesting a functional antagonistic relationship between the ING4 tumor suppressor and the HER2 oncogene. These results sustain the view that ING4 is a tumor suppressor in breast cancer and suggest that ING4 deletion may contribute to the pathogenesis of HER2-positive breast cancer.     

Spotlight on Trastuzumab in the Management of HER2-Positive Metastatic and Early-Stage Breast Cancer

Trastuzumab (Herceptin) is a humanized monoclonal antibody used in the treatment of breast cancer that overexpresses human epidermal growth factor receptor 2 (HER2), which is associated with clinically aggressive disease and a poor prognosis.The addition of intravenous trastuzumab to first-line chemotherapy improved the time to disease progression, objective response rate, duration of response, and overall survival in randomized, multicenter trials in women with HER2-positive metastatic breast cancer. As such, trastuzumab has become the standard of care in this setting, despite its high acquisition cost and potential for cardiac events, and is licensed for use in combination with paclitaxel (Europe and the US) or docetaxel (Europe). In addition, trastuzumab monotherapy is approved for use in patients with HER2-positive metastatic breast cancer who have previously received chemotherapy for their metastatic disease. Recent data from large phase III trials with trastuzumab in the adjuvant setting revealed significant improvements in disease-free and overall survival. Thus, trastuzumab is also rapidly becoming a standard component of adjuvant therapy for patients with HER2-positive early-stage breast cancer.     

Immunohistochemical evaluation of human epidermal growth factor receptor 2 and estrogen and progesterone receptors in invasive breast cancer in women

Introduction

Estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) expression are crucial in the biology of breast carcinoma. HER-2/neu gene is amplified and overexpressed in 15-30% of invasive breast cancers. HER-2-positive breast cancers have worse prognosis than HER-2 negative tumors and possess distinctive clinical features. The aim of this study was to assess the expression of HER2 in cancer tissue of patients with invasive breast cancer in correlation with tumor type, histological grade, tumor size, lymph node status, and expression of estrogen receptor and progesterone receptor.

Material and methods

Formalin-fixed, paraffin-embedded tissues from 40 patients with invasive HER-2-positive breast cancer and from 191 patients with HER-2-negative breast cancer were used in this study. HER2 expression was determined using the test HerceptTest™ DAKO.

Results

Among 231 cases of breast cancer, 18 invasive lobular carcinomas and 213 invasive ductal carcinomas were diagnosed. Sixty percent of HER-2-positive breast cancers were ER-positive compared with 77% in the HER-2-negative group (p = 0.002). The expression of PR was observed in 43% of HER-2-positive breast cancers and in 72% of HER2-negative tumors (p = 0.003). Excessive expression of HER2 protein was detected in 60% of patients positive for estrogen receptors, which may worsen prognosis in these patients.

Conclusions

Determination of HER2 overexpression in breast cancer patients, allows for a determination of a group of patients with a worse prognosis.     

The assessment of HER2 status and its clinical implication in breast cancer

Amplification and overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is associated with an adverse prognosis. The introduction of trastuzumab and lapatinib has substantially improved the clinical outcomes of patients with HER2-positive breast cancer. The key element of the successful application of anti-HER2 therapies in real-world has been the selection of candidates for treatment based on the level of HER2 positivity of the tumor. HER2 status of breast cancer is clinically assessed by HER2 protein expression with immunohistochemistry (IHC) or HER2 gene amplification with in situ hybridization (ISH). The 2018 American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) HER2 guideline focused update revised the HER2 scoring criteria. Digital image analysis (DIA) has emerged as an objective and reproducible IHC scoring method and the ASCO/CAP HER2 guideline has acknowledged DIA as a diagnostic modality. In this article, we aim to review the assessment of HER2 status and its clinical application in breast cancer.     

Fluctuation of HER2 Expression in Breast Carcinomas during the Menstrual Cycle

The hormonal milieu at time of tumor surgery seems to have a significant impact on survival in premenopausal breast cancer patients. Indeed, surgery performed during the follicular phase of the menstrual cycle was suggested to correlate with a poor prognosis. To investigate the relationship between prognosis and menstrual cycle at time of surgery, we analyzed the expression of some markers associated with tumor aggressiveness, such as the hormone receptors, HER2, p53, Bcl2, and cathepsin D in breast carcinomas obtained from 198 premenopausal women who underwent surgery during different phases of the menstrual cycle. HER2 overexpression was found to fluctuate in hormone receptor-positive tumors. In actual fact, 20% of the tumors removed during the follicular phase scored HER2-positive, versus 8% of those removed during the luteal phase. Similarly, a number of hormone receptor-positive tumor specimens, obtained from the same patients during follicular and luteal phases, were scored HER2-positive when the sample was removed during the follicular phase and HER2-negative when removed in the luteal phase. Southern blot analysis of the HER2 gene indicated that, in hormone receptor-positive cases, the overexpression of HER2 is often not associated with gene amplification. The finding that overexpression of the HER2 gene, associated with tumor aggressiveness, can fluctuate according to the hormonal milieu may explain the increased survival of patients operated during the luteal phase. It is also relevant to the selection and treatment of patients most likely to benefit from anti-HER2 antibody therapy.     

XeNA: Capecitabine Plus Docetaxel, With or Without Trastuzumab, as Preoperative Therapy for Early Breast Cancer

Combinations of capecitabine and a taxane are highly active in metastatic breast cancer, and synergy between capecitabine and docetaxel has also been demonstrated. Such combinations potentially would provide a promising non–anthracycline-based alternative for patients with early breast cancer. Non-anthracycline preoperative regimens are a particularly interesting proposition in human epidermal growth factor receptor 2 (HER2)-positive breast cancer, as they offer less cardiotoxicity and thus can be used concomitantly with preoperative trastuzumab therapy. Capecitabine plus docetaxel (XT) and trastuzumab with XT (HXT) are promising non-anthracycline regimens for the preoperative treatment of women with HER2-negative and HER2-positive breast cancer, respectively. The Xeloda in Neoadjuvant (XeNA) trial, an open-label, multicenter, phase II study, independently assesses the efficacy of preoperative XT in HER2-negative and HXT in HER2-positive breast cancer. A particularly important feature of the XeNA study is the use of pathologic complete response (pCR) plus near pCR (npCR) as the primary endpoint. pCR is associated with long-term survival, and although it is valuable as a surrogate marker, pCR has some limitations. Measurement of residual breast cancer burden (RCB) has been proposed as a more practical alternative to predict survival after preoperative chemotherapy. The combination of RCB-0 and RCB-I (npCR) expands the subset of patients shown to benefit from preoperative chemotherapy, and achievement of pCR or npCR is associated with long disease-free survival. In XeNA, the sum of pCR and npCR will facilitate correlative studies designed to identify patients most likely to benefit from XT and HXT and may expedite the clinical evaluation of these novel preoperative regimens.     

Analysis of differentially expressed proteins between HER2 positive and triple negative breast cancer and their prognostic significance

Both triple negative breast cancer (TNBA) and HER2-positive breast cancer lack expression of estrogen receptor alpha (ER) and progesterone receptor (PR), while human epidermal growth factor receptor 2 (HER2) in TNBC is also negative. This study aimed to identify the differentially expressed proteins (DEPs) between TNBC and HER2-positive breast cancer and to improve understanding of their role in the prognosis of breast cancer. By analyzing the breast cancer data set in The Cancer Proteome Atlas (TCPA) database, 15 DEPs between TNBC and HER2-positive breast cancer were identified. GO and pathway enrichment analysis were performed on DEPs, and the protein–protein interaction (PPI) network was constructed. The overall survival (OS) analysis of the breast cancer protein dataset in the Kaplan-Meier plotter showed that low expression of ACC1 suggested a higher OS of HER2-positive breast cancer (HR = 5.34, P < 0.05) and TNBC (HR = 2.88, P < 0.05). And TNBC patients with high TBA1B (HR = 0.16, P < 0.01) or low INPP4B (HR = 3.47, P < 0.05) expression have a better prognosis. Our research provides new insights into the prognostic indicators of TNBC and HER2-positive breast cancer, which could be further studied.     

Prognostic significance of HER2 gene amplification according to stage of breast cancer

It is well known that the amplification of the HER2 gene is closely associated with poor prognosis of breast cancer. However, there is controversy about the clinical significance of HER2 according to lymph node status in breast cancer. The aim of this study was to identify the differences in the prognostic significance of HER2 gene amplification according to the stages of breast cancer. We prepared a tissue array for fluorescence in situ hybridization (FISH) with breast cancer specimens from the surgery in 1994 to 1999. Total 338 cases of breast cancer were enrolled and the median follow-up period was 6.3 yr. The detection rates of HER2 gene amplification were as follows: 10.3% in stage I, 22.3% in stage II, and 43.8% in stage III. On survival analyses HER2-positive groups showed worse prognosis in stage III of breast cancer, but not in stage I or II. Multivariate analyses with a Cox-regression model also revealed that HER2 amplification was an independent prognostic factor only in stage III breast cancer. Regarding HER2 gene amplification as a prognostic factor of breast cancer, the clinical significance of the gene was found to be confined to advanced breast cancer.     

Amplification and overexpression of the ABCC3 (MRP3) gene in primary breast cancer

The ATP-binding cassette (ABC) of active transporters comprises a group of proteins that which facilitate efflux of anticancer drugs from cancer cells. We focused on the gene amplification and protein expression of ABCC3 (also known as MRP3) in breast cancer cell lines and clinical tumor samples. Fluorescence and chromogenic in situ hybridization, using an ABCC3-specific probe, was used to analyze 11 breast cancer cell lines and 112 clinical tumor samples. The results of ABCC3 were correlated with the amplification status of HER2 and topoisomerase II alpha (TOP2A), which are located close to ABCC3 at 17q12-q21. Immunohistochemistry was used to assess ABCC3 protein overexpression. Of the cell lines studied 6 HER2-positive lines and 1 HER2-negative line exhibited amplification of ABCC3. In the HER-2-negative clinical tumor samples, only 4/55 (7.3%) exhibited ABCC3 amplification. In the HER2-positive tumors, ABCC3 was amplified in 16/57 tumors (28.1%, P=0.0059). TOP2A did not exhibit any consistent coamplification pattern. ABCC3 (MRP3) protein overexpression was more common in tumors with gene amplification (P=0.069). In silico analysis of 804 breast cancers with matched gene expression and copy number microarray data revealed significant differences ABCC3 across the molecular subtypes. Specifically, increased ABCC3 mRNA and gene copy numbers were most prominent in HER2 amplified and/or HER2-enriched classified tumors. Moreover, differential ABCC3 mRNA levels were found within the HER-2 amplified subset when stratified by the estrogen receptor status. We conclude that ABCC3 is frequently amplified and overexpressed in HER2-positive breast cancer, and something that warrants further studies correlating the results with therapeutic outcome.     

Evaluating TIMP-1, Ki67, and HER2 as markers for non-sentinel node metastases in breast cancer patients with micrometastases to the sentinel node

The aim was to investigate whether the biochemical prognostic markers TIMP-1, Ki67, and HER2 could predict metastatic spread to non-sentinel nodes (NSN) in breast cancer patients with micrometastases to sentinel node (SN). We included all breast cancer patients with micrometastases to SN operated between 2001 and 2007 at the Department of Breast Surgery, Herlev Hospital. The study was designed as a matched case-control study with 25 cases with micrometastases to SN and, in addition, metastatic spread to NSN and 50 matched controls with micrometastases to SN, but without NSN metastases. Patient and tumor characteristics were retrieved from the Danish Breast Cancer Cooperative Group database. Immunohistochemical analyses of TIMP-1 and Ki67 and measurements of HER2 on formalin-fixed paraffin-embedded tumor tissue were performed. No significant differences in the immunoreactivity of TIMP-1 and Ki67 were found between patients with and without NSN metastases. Six of seven HER2 positive patients did not have NSN metastases, but the results did not reach statistical significance. Despite being prognostic markers in breast cancer, TIMP-1 and Ki67 could not predict NSN metastases in women with micrometastatic disease to SN. Larger studies are needed to further validate HER2 as a marker for NSN metastases in these patients.     

HER2 testing in gastric cancer

Molecular therapies targeting HER2 are part of the established drug armamentarium in breast carcinoma. Now the ToGA trial, an international multicenter phase III clinical study, involving 24 countries globally, has shown that the anti-HER2 humanized monoclonal antibody Trastuzumab is effective in prolonging survival in HER2-positive carcinoma of the stomach and the gastroesophageal junction (GEJ). Similarly to breast carcinoma, >20% of gastric cancers show HER2 overexpression and/or amplification, and this percentage increases to 33% in GEJ tumors. Thus, as in breast carcinoma, pathologists are now asked to evaluate HER2 status in gastric carcinoma samples. As validated in the ToGA trial, the HER2 testing criteria that must be used in evaluating both gastric carcinoma biopsies and surgical specimens significantly differ from those routinely applied in breast carcinoma. The main variations with regard to the pattern of reactivity in HER2-expressing cells are as follows: the completeness of membrane staining is not a "conditio sine qua non" and the number of stained cells necessary to consider a case as positive is different. We must also take note of the much more frequent heterogeneity of HER2 positivity in gastric cancer compared with breast carcinoma and the less stringent correlation between HER2 amplification and protein overexpression that is observed in gastric carcinoma, where more than 20% of cases may carry HER2 amplification, although of low level, without HER2 expression. In these patients, in the ToGA trial, there was no apparent benefit from adding Trastuzumab to chemotherapy: for this reason the European Medicines Agency, while approving usage of Trastuzumab for metastatic adenocarcinoma treatment, indicated HER2 testing by immunohistochemistry as first evaluation assay, followed by fluorescence in situ hybridization in 2+ equivocal cases. HER2 testing in gastric carcinoma is a new field, opening several opportunities: for patients with gastric cancer, this is a new promising therapeutic option; for pathologists, strengthening our role in therapy selection and emphasizing our duty of providing accurate and reproducible HER2 testing results; for all interested in understanding the biology of gastric and GEJ cancer and in discovering new possible molecular therapy targets.     

Recurrence and survival rates among early breast cancer cases with triple negative immunophenotype

BACKGROUND: Axillary lymph node status, hormonal receptors (HR) and HER2 expression are significant prognostic factors for early breast cancer. Triple negative immunophenotype (HER2 and HR negative) is associated with a high frequency of recurrence and lower overall survival. The objective was assess clinical behavior, recurrence and survival of patients with triple negative early breast cancer and patients with other immunophenotypes. MATERIAL AND METHODS: We carried out a retrospective study among women with stages I-IIB over 18 years with determination of HR and HER2 expression by immunohistochemical assay. We identified 5 groups: triple negative, triple positive, HER2 negative & HR positive, HER2 positive & HR negative, HER2 negative & 1 HR positive. We recorded age, date of diagnosis, clinical stage, tumor size, axillary lymph node status, ER, PR, HER2, p53, angiogenesis, Ki67, type of surgery, adjuvant treatment, time to recurrence, number and recurrence site and overall survival. RESULTS: 17 patients (15.4%) had triple negative phenotype, 14 (12.7%) triple positive, 52 (47.3%) were localized in group 3, 11 (10%) in 4 and 16 (14.5%) in group 5. Triple negative phenotype was associated with increased cellular proliferation (p < 0.000); being young (median 43 years), large tumor size (median size 2.5 cm) lower proportion of patients in stage I and high frequency of p53 positive (78.5%). We observed a high frequency of recurrence and death among the triple negative group and among the HER2 positive and HR negative cases. CONCLUSIONS: Triple negative breast cancer is more common among young women and is associated with a high frequency of recurrence and mortality. Clinical behavior among triple negative breast cancer cases is aggressive and displays a similar clinical profile that observed among HER2 positive and HR negative patients.     

Sonographic detection of thyroid cancer in breast cancer patients

The purpose of our study was to analyze the incidence of incidental thyroid cancers which were detected by simultaneous sonographic examination of breast and thyroid glands. Between January 2001 and March 2004, 518 patients were diagnosed with breast cancer after modified radical mastectomy (n=369) or breast conserving surgery (n=149). We screened thyroid glands when we examined breast for diagnosis and follow-up after surgery. If we found the sonographic finding of suspicious for malignancy in thyroid, we immediately performed ultrasound-guided fine needle aspiration biopsy (FNAB). Forty-two cases showed suspicious sonographic findings and of those, 18 cases (42.9%) were determined to have suspicious malignant cytology by ultrasound guided FNAB. Among 518 breast cancers, total 13 cases (2.5%) were diagnosed with papillary carcinoma after thyroidectomy. The mean longest diameter of the thyroid masses was 9.9 mm (range 1-30 mm). Six cases (6/13, 46.2%) were diagnosed as simultaneous breast and thyroid cancers, and the rest of the thyroid cancers were detected after 6 to 33 months (mean 16.5 months) after surgery. In conclusion, the patients with breast cancer had a high incidence (2.5%) of thyroid cancer. Sonographic screening is useful for the early detection of thyroid cancer.     

Circular RNA hsa_circ_0001598 promotes programmed death-ligand-1-mediated immune escape and trastuzumab resistance via sponging miR-1184 in breast cancer cells

Approximately 25% of breast cancer (BC) patients are HER2-positive. Trastuzumab is used as a targeted therapy drug to treat HER2-positive BC patients; however, the drug resistance remains a big challenge. Circular RNAs (circRNAs) are reported to be involved in drug resistance, but the role of circ_0001598 has never been studied in BC. First, we identified the expression of circ_0001598 by RT-qPCR in BC. The gain-of-function and loss-of-function studies were applied to study the functional roles of circ_0001598 and its target gene. We observed upregulation of circ_0001598 in BC tissues, especially in trastuzumab-resistant BC samples. We further identified that miR-1184 is a functional target of circ_0001598. Moreover, it was found that programmed death-ligand 1 (PD-L1) was a direct target of miR-1184. The oncogenic effects of circ_0001598 in promoting BC cell growth, trastuzumab-resistance, PD-L1 expression, and escaping of CD8 T cell killing were abolished after the restoration of miR-1184. In conclusion, we demonstrate that circ_0001598/miR-1184/PD-L1 signaling plays a crucial role in the regulation of BC progression and trastuzumab-resistance phonotypes, which suggests that circ_0001598 may be a molecular target to treat HER2-positive BC patients.

     

HER2 testing in gastric cancer: a practical approach

Trastuzumab in combination with capecitabine or 5-fluorouracil and cisplatin is approved by the European Medicines Agency for the treatment of patients with human epidermal growth factor receptor 2 (HER2)-positive (immunohistochemistry 3+ or immunohistochemistry 2+/fluorescence in situ hybridization-positive or immunohistochemistry 2+/silver in situ hybridization-positive) metastatic adenocarcinoma of the stomach or gastro-esophageal junction. Approvals are underway in other countries, with recent approvals granted in the United States and Japan. Experience and data from trastuzumab use in breast cancer have highlighted the importance of quality HER2 testing and scoring to ensure accurate identification of patients eligible for treatment. HER2 testing in gastric cancer differs from testing in breast cancer due to inherent differences in tumor biology; gastric cancer more frequently shows HER2 heterogeneity (focal staining) and incomplete membrane staining. Consequently, gastric cancer-specific HER2 testing protocols have been developed and standardized and it is imperative that these recommendations be adhered to. Given the predictive value of HER2 protein levels with response in the trastuzumab for GAstric cancer study (ToGA), immunohistochemistry should be the initial testing methodology and fluorescence in situ hybridization or silver in situ hybridization should be used to retest immunohistochemistry 2+ samples. Wherever possible, bright-field methodologies should be used as these are considered to be superior to fluorescent methodologies at identifying heterogeneous staining. Specific training is required before embarking on HER2 testing in gastric cancer, irrespective of the experience of HER2 testing in breast cancer. This paper provides the most up-to-date practical guidance on HER2 testing and scoring in patients with gastric and gastro-esophageal junction cancer, as agreed by a panel of expert pathologists with extensive experience of HER2 testing particularly reflecting the European Medicines Agency-approved indication. It is anticipated that these recommendations should ensure accurate and consistent HER2 testing, which will allow appropriate selection of patients eligible for treatment with trastuzumab.     

Novel HER2 Aptamer Selectively Delivers Cytotoxic Drug to HER2-positive Breast Cancer Cells in Vitro

ABSTRACT: BACKGROUND: Aptamer-based tumor targeted drug delivery system is a promising approach that may increase the efficacy of chemotherapy and reduce the related toxicity. HER2 protein is an attractive target for tumor-specific drug delivery because of its overexpression in multiple malignancies, including breast, gastric, ovarian, and lung cancers. METHODS: In this paper, we developed a new HER2 aptamer (HB5) by using systematic evolution of ligands by exponential enrichment technology (SELEX) and exploited its role as a targeting ligand for delivering doxorubicin (Dox) to breast cancer cells in vitro. RESULTS: The selected aptamer was an 86-nucleotide DNA molecule that bound to an epitope peptide of HER2 with a Kd of 18.9 nM. The aptamer also bound to the extracellular domain (ECD) of HER2 protein with a Kd of 316 nM, and had minimal cross reactivity to albumin or trypsin. In addition, the aptamer was found to preferentially bind to HER2-positive but not HER2-negative breast cancer cells. An aptamer-doxorubicin complex (Apt-Dox) was formulated by intercalating Dox into the DNA structure of HB5. The Apt-Dox complex could selectively deliver Dox to HER2-positive breast cancer cells while reducing the drug intake by HER2-negative cells in vitro. Moreover, Apt-Dox retained the cytotoxicity of Dox against HER2-positive breast cancer cells, but reduced the cytotoxicity to HER2-negative cells. CONCLUSIONS: The results suggest that the selected HER2 aptamer may have application potentials in targeted therapy against HER2-positive breast cancer cells.     

Low expression of HER2 protein in breast cancer is biologically significant

HER2 status is routinely tested using immunohistochemistry or FISH following the licensing of a therapeutic agent targeting HER2. However, neither of these methods provides quantitative information relating to the 70-80% of patients with levels of expression lower than the assay detection thresholds. In this study, radioimmunohistochemistry was used to detect quantitative HER2 protein expression in 178 breast cancers. Survival analysis was performed, as were correlations with known prognostic variables and with overexpression of other HER family members. It is demonstrated that the populations expressing very high and very low levels of HER2 are each associated with increased risk of cancer-specific death on survival analysis (p = 0.0043). The group with low levels of HER2 was more likely to be of higher grade, EGFR-positive and ER/HER3/HER4-negative. HER2-positive cases were frequently ER-negative/HER3-positive, whilst cases with normal HER2 expression were often ER-positive/HER4-positive. The aggressive nature of the tumour group with low HER2 expression may be explained by actions of other HER family members, particularly EGFR, but whether these or other factors have a negative regulatory effect on HER2 expression remains to be determined.