Formalin-fixed Paraffin-embedded Breast Cancer Tissue Research Articles

Association between Cyclooxygenase-2 and Indoleamine 2,3-Dioxygenase Expression in Breast Cancer Patients from Pakistan.

Background:Tumors use several immunosuppressive mechanisms to evade immune destruction. Cyclooxygenase-2 (COX-2) expression may be a driver of immunosuppression in breast cancer, but the mechanisms involved remain elusive. COX-2 expression induces the expression of indoleamine 2,3 dioxygenase (IDO) in tumor cells. IDO is an immunosuppressive enzyme which is involved in tumor immune escape mechanisms in breast cancer. Our aim was to evaluate the association between COX-2 and IDO expression to find evidence of immunosuppression in Pakistani breast cancer patients. Methods:Immunohistochemical analysis was performed to evaluate the expression of COX-2, IDO, estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) on formalin-fixed paraffin-embedded breast cancer tissues of 100 patients. Univariable and multivariable logistic regression model was used to identify the independent risk factors of COX-2. Results:A total of 100 patients were included with a mean age and standard deviation of 48.28 ± 11.83. A significant association was observed among COX-2, IDO, ER, PR and tumor grade. In multivariable analysis, three variables were identified as significant independent risk factors for high COX-2: IDO expression high; [adjusted odds ratio (AOR) 6.51; 95% confidence interval (CI) (2.00-21.20), p=0.001], ER; [AOR 5.62; 95% CI (1.80-17.84), p=0.002] and age [AOR 1.04; 95% CI (1.00-1.10), p=0.05] respectively. Conclusion:Our data showed that high IDO expression is associated with high COX-2 expression in Pakistani breast cancer patients. The co-expression of both enzymes may suggest their role in disease pathogenesis. Hence the concurrent targeting of COX-2 and IDO may be a promising therapy for breast cancer.

Association of ESR1 Mutations and Visceral Metastasis in Patients with Estrogen Receptor-Positive Advanced Breast Cancer from Brazil.

Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy in estrogen receptor-positive advanced breast cancer and have been recognized as a prognostic and predictive biomarker as well as a potential therapeutic target. However, the prevalence of ESR1m in real-world patients has not been adequately described. Therefore, we sought to evaluate the prevalence of ESR1m in metastatic samples from Brazilian patients with estrogen receptor-positive (ER+) advanced breast cancer previously treated with endocrine therapy. The presence of ESR1m was evaluated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue using real-time quantitative polymerase chain reaction (RT-qPCR). Mutations in codons 380, 537, and 538 of the ESR1 gene were analyzed. Out of 77 breast cancer samples, 11 (14.3%) showed mutations in the ESR1 gene. ESR1m were detected in a variety of organs, and the D538G substitution was the most common mutation. In visceral metastasis, ESR1m were detected in 25% (8/32) of the samples, whereas in nonvisceral metastasis, ESR1m were detected in 6.7% (3/45) of the samples. The odds of a sample with visceral metastasis having an ESR1 mutation is 4.66 times the odds of a sample of nonvisceral metastasis having an ESR1 mutation (95% CI: 1.13–19.27; p value = 0.0333). Our study indicates that the prevalence of ESR1m in samples from Brazilian patients with metastatic ER+ breast cancer is similar to that described in patients included in clinical trials. We observed an association of ESR1m with visceral metastasis.

Amplified fluorescence imaging of HER2 dimerization on cancer cells by using a co-localization triggered DNA nanoassembly.

Convenient and sensitive detection of human epidermal growth factor receptor 2 (HER2) dimerization is highly desirable for molecule subtyping and guiding personalized HER2 targeted therapy of breast cancer. A colocalization-triggered DNA nanoassembly (CtDNA) strategy was developed for amplified imaging of HER2 dimerization. It exploits (a) the advantage of the specificity of aptamer proximity hybridization, and (b) the high sensitivity of hairpin-free nonlinear HCR. The mechanism of step-by-step hairpin-free nonlinear HCR for DNA dendritic nanoassembly was studied by native polyacrylamide gel electrophoresis, atomic force microscopy and fluorometry. The results revealed a high specificity, sensitivity, and excellent controllability of the DNA dendritic nanoassembly. The method was used to identify HER2 homodimers and HER2/HER3 heterodimers in various breast cancer cell lines using fluorescence microscopy. It was then extended to image and quantitatively evaluate HER2 homodimers in clinical formalin-fixed paraffin-embedded breast cancer tissue specimens. This revealed its remarkable accuracy and practicality for clinical diagnostics. Graphical abstract Schematic presentation of amplified imaging of human epidermal growth factor receptor 2 (HER2) dimerization on cancer cell surfaces by using a co-localization triggered DNA nanoassembly (CtDNA).

The composition of T cell infiltrates varies in primary invasive breast cancer of different molecular subtypes as well as according to tumor size and nodal status

T lymphocytes are the most numerous immune cells in tumor-associated infiltrates and include several subpopulations of either anticancer or pro-tumorigenic functions. However, the associations between levels of different T cell subsets and breast cancer molecular subtypes as well as other prognostic factors have not been fully established yet. We performed immunohistochemistry for CD8 (cytotoxic T cells (CTL)), FOXP3 (regulatory T cells (Tregs)), and GATA3 (Th2 cells) in 106 formalin-fixed paraffin-embedded invasive breast cancer tissue samples and analyzed both the numbers and percentages of investigated cells in tumor-associated infiltrates. We observed that triple-negative breast cancer (TNBC) and HER2+ non-luminal breast tumors were associated with more numerous CTLs and Tregs and a higher Treg/Th2 cell ratio as compared with luminal A subtype. A higher Treg percentage was related to a decreased hormone receptor expression, an increase in the Ki67 level, a greater tumor size of luminal tumors, and the presence of lymph node metastases. Moreover, differences in the composition of T cell infiltrates were associated with HER2 status and histologic grade and type, and a distinct immune pattern was observed in tumors of different phenotypes regarding pT stage and nodal status. The results of our work show the diversity of T cell infiltrates in primary invasive breast cancers of different phenotypes and suggest that progression of luminal or non-luminal tumors is related to distinct tumor-associated T cell composition.

Prognostic value of tumor cell DNA content determined by flow cytometry using formalin-fixed paraffin-embedded breast cancer tissues.

The use of formalin-fixed paraffin-embedded (FFPE) tumor tissues in flow cytometry (FCM)-based determination of tumor cell DNA content is more complicated than the use of fresh-frozen tissues. This study aimed to accurately measure tumor cell DNA content from FFPE tissues by separating tumor cells from stromal cells through FCM and investigating its prognostic impact. We separately measured the DNA contents of tumor cells and stromal cells by gating with pan-cytokeratin and vimentin (FCMC/V). We evaluated tumor cell DNA contents [DNA index (DI)] of 290 FFPE tumor tissues and classified them into low and high DI groups, using a cutoff DI value determined through an unbiased computational method. The distribution of DI was bimodal, and a cutoff value was determined at a DI of 1.26. The high-DI tumors were associated with aggressive phenotypes and had significantly worse distant recurrence-free intervals (DRFI) than low-DI tumors. Multivariate analysis revealed that lymph node metastasis, Ki67, and DI were independent factors affecting DRFI. Accordingly, patients with low-DI/low-Ki67 tumors had excellent outcomes compared with other tumor types. Multiploid tumors were associated with increased lymphocytic infiltration and aggressive phenotypes. The DI of FFPE tumors could be precisely determined through FCMC/V. A combination of DI and Ki67 analyses may be able to predict the prognoses of breast cancer patients with greater accuracy.

Indoleamine 2,3-dioxygenase expression and overall survival in patients diagnosed with breast cancer in Pakistan.

BackgroundImmune dysfunction in breast cancer patients is well established. Indoleamine 2,3-dioxygenase (IDO) is an immunosuppressive enzyme that is linked with progression of cancer. IDO is overexpressed in triple-negative breast cancer (TNBC) cases.Materials and methodsWe conducted the first study to analyze IDO expression and overall survival in breast cancer cases in Pakistan. Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), and human EGF receptor 2 (HER2) was evaluated by immunohistochemistry. Formalin-fixed paraffin-embedded breast cancer tissues of 100 (TNBC, n=49 and non-TNBC, n=51) patients were obtained from Shaukat Khanum Memorial Cancer Hospital and Research Centre. IDO expression was analyzed in association with clinicopathological features and overall survival. A total of 100 patients were classified based on the ordinal IDO score variables as low, medium, and high. In addition, overall mean age and SD of patients was 48.28±11.82.ResultsImmunohistochemical analysis showed that high IDO was observed in the TNBC patients (65.3%) compared to that in the non-TNBC patients (33.3%). Multivariable analyses showed that TNBC was an independent risk factor for high IDO expression. Overall survival was also significantly associated with IDO score.ConclusionOur study showed that IDO protein expression is higher in TNBC patients (P<0.01) and may suggest its role in disease pathogenesis. TNBC might be effectively treated with IDO inhibitors. Furthermore, high IDO expression is considerably associated with overall decreased patient survival. IDO might be utilized as a potential biomarker and immunotherapeutic target in breast cancer patients.

Stability of oestrogen and progesterone receptor antigenicity in formalin-fixed paraffin-embedded breast cancer tissue over time.

Use of archived formalin-fixed paraffin-embedded (FFPE) tissue is a standard method for evaluation of proposed prognostic and predictive tumour markers. However, little is known of the preservation of biomarker expression in old FFPE tumour blocks. We investigate the quality of immunohistochemical (IHC) oestrogen (ER) and progesterone receptor (PR) evaluation in FFPE tissue over time (1978-2000) using a large breast cancer tissue microarray (N=573) with access to receptor analyses in cytosol (CYT) at diagnosis, coexpression of other biomarkers and follow-up data. We found a good correlation between ER analysed with CYT at diagnosis and ER analysed with IHC in archived FFPE tissue from the same tumour. ER evaluation did not seem to be affected by tissue storage time. Nor was there any time-dependent difference in ERIHC correlation with other biomarkers (HER2, Ki67) or survival. Discordant cases were more often classified as ER-positive with IHC than with CYT. For PR, however, we found an increased correlation between methods in more recent time periods. This may possibly be explained by more reliable PRIHC results in newer samples, although other explanations may also contribute. Our results indicate stable ER expression in FFPE tissue archived for up to 40years.

Clinicopathological significance of cancer stem cell markers CD44 and ALDH1 expression in breast cancer.

CD44 and aldehyde dehydrogenase 1 (ALDH1) has been reputed to be cancer stem cell (CSC) markers in breast cancer. Yet, the clinicopathologic and prognostic significance of these markers remain unclear. In this study, we have investigated the expression of these markers and their relation with conventional clinicopathologic tumor characteristic including molecular subtype. CD44 and ALDH1 expression were investigated by immunohistochemistry in a series of 157 formalin-fixed paraffin-embedded breast cancer tissues. Overall, CD44 and ALDH1 are, respectively, detected in 33% (52 of 157) and 7% (10 of 157) of breast cancer cases. We also observed that CD44 expression was associated with histological grade (p = 0.005). For ALDH1, we found that its expression is more frequent with elderly women (> 50 years, p = 0.03). The investigation of relationship between the stem cell phenotype and breast cancer molecular subtype, revealed that CD44 and ALDH1 expression was more frequent in basal-like tumors (p = 0.005). Among the two cancer stem cell markers tested, ALDH1 showed a strong association with the basal marker EGFR (p = 0.05). These findings suggest that CD44 and ALDH1 play a role in the clinical behavior in breast cancer and might be interesting biomarkers and therapeutic targets.

A2A Receptors Are Highly Expressed in Young Patients with Luminal B and Triple Negative Breast Cancer Subtypes and Are Associated with a Higher Proliferate Index

Background and ObjectivesNumerous Molecules in the tumor microenvironment orchestrate the body's immune response against tumor cells thereby affecting tumor cell growth and proliferation. Adenosine is produced in the hypoxic tumor milieu and by stimulating its receptors (A2AR), plays an important immunosuppressive role that helps tumor cells evade the body s immune mechanisms. Furthermore, stimulation of these receptors also has a profound role in promoting the tumor vasculature and contributing to tumor aggressiveness. The present study aimed at investigating A2AR expression in breast cancer tissue and its association with different clinicopathological parameters of the tumor.MethodsThis study was conducted on 30 formalin‐fixed paraffin‐embedded (FFPE) human breast cancer tissues. Sections were immunohistochemically stained with of A2AR antibodies. Clinical and pathological data were retrospectively obtained from the patients' records in the archives of the pathology department at Alexandria University, Egypt.ResultsA2ARs were expressed not only on the tumor‐infiltrating immune cells (TILs) but were also found on the tumor cells themselves in 73.3% (n=22) of breast cancer tumors. Their expression on the tumor cells was significantly higher in younger age patients (&lt; 50 years) (88.2%) compared to older ones (54.5%) [p=0.044]. A2AR expression was also significantly associated with a Luminal B as well as triple‐negative tumors [p=0.028] compared to Luminal A and Her2‐enriched tumors. Patients expressing A2A receptors had a significantly higher expression of Progesterone receptors [P=0.031] compared to ER expression [P=0.098]. The majority of A2AR expressing tumors had a mitotic index score of 2 [p=0.013] as well as a significantly higher proliferative index (ki‐67 &gt;20%) [p=0.018]. No association was observed between A2AR expression and tumor size, type, grade, Lymph node status, the percentage of TILs or patient survival after 2 years of follow‐up.ConclusionOur study confirmed that A2AR were strongly expressed on breast cancer tumor cells in younger patients particularly those of luminal B and triple negative subtypes. Their expression was associated with a significantly higher proliferative index suggesting a possible role of these receptors in imparting a more aggressive phenotype and that targeting these receptors could have a potential role in improving the outcome of these patients.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quantifying Tertiary Lymphoid Structure-Associated Genes in Formalin-Fixed Paraffin-Embedded Breast Cancer Tissues.

Tertiary lymphoid structures (TLS) have been detected in several types of human solid tumors. These structures are thought to regulate local adaptive immune responses that can promote or antagonize tumor progression. Despite positive prognostic values associated with a TLS presence in several studies, discrepancies still exist. TLS are structurally organized entities composed of varying numbers of multiple cell types making their assessment in tumor tissues, particularly biopsies, challenging. Immunohistochemical staining of TLS-related cell populations is the most frequently used method for identifying and scoring them; however, TLS-related gene expression has also been explored. The protocols described are detailed to allow the user to quantify TLS-related gene expression on formalin-fixed paraffin-embedded human breast tumor tissues.

Abstract 2251: Tumor tissue gene expression in association with survival of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive type of cancer with limited treatment options. Previous studies have shown that gene expression profiles were associated with TNBC prognosis. Information on specific genes that are predictive of TNBC outcome is limited. Using data and samples from a cohort of 469 TNBC cases from Shanghai Breast Cancer Survival Study (SBCSS), we systematically evaluated expressions of 302 genes in tumor tissue with survival of TNBC. Genes included in the study are PAM50 genes, genes encoding drug metabolizing enzymes and, genes implicated in TNBC biology and progression based on literature reviews. Gene expression levels were measured in total RNA isolated from formalin-fixed paraffin-embedded breast cancer tissues using the NanoString nCounter assay. Cox regression was applied to evaluate disease-free survival (DFS) and overall survival (OS) in association with gene expression. Analysis was adjusted for known predictors of TNBC outcome, including age, disease stage, basal like subtype and DUSP4 gene expression. During a median follow-up of 5.3 years (range: 0.7-8.9 years), 100 deaths and 92 recurrences/breast cancer deaths were documented. Expression levels of 17 genes were significantly associated with OS, and 15 genes with DFS (P&amp;lt;0.05). The top 5 most significant genes are EOMES (Hazard Ratio, HR = 0.88; 95% Confidence Interval, 95% CI: 0.82-0.96), GZMK (HR = 0.91, 95% CI: 0.85-0.97), TUBB3 (HR = 1.12, 95% CI: 1.03-1.21), SIT1 (HR = 0.90, 95% CI: 0.83-0.98) and ZNF224 (HR = 0.89, 95% CI: 0.81-0.97) for OS, and TUBB3 (HR = 1.14, 95% CI: 1.05-1.24), KRT14 (HR = 1.08, 95% CI: 1.02-1.14), BAG1 (HR = 1.19, 95% CI: 1.05-1.35), CD44 (HR = 0.73, 95% CI: 0.58-0.92) and EOMES (HR = 0.89, 95% CI: 0.82-0.97) for DFS. Study is on-going to replicate the findings of this study. Citation Format: Shuyang Wang, Qiuyin Cai, Hui Cai, Pingping Bao, Jie Wu, Fei Ye, Wei Zheng, Ying Zheng, Xiao-Ou Shu. Tumor tissue gene expression in association with survival of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2251. doi:10.1158/1538-7445.AM2017-2251

Abstract LB-257: Estrogen signaling in mature luminal and luminal progenitor cells of BRCA2 carriers and non-carriers

Abstract Background: Carriers of mutations in the breast cancer susceptibility gene BRCA2 have a 50-80% lifetime risk of developing breast cancers, about 70% of which are positive for estrogen receptor (ER), a key oncogenic hormone receptor. Metastatic ER+ tumors also respond to PARP inhibitors associated with their DNA repair defect. There remains much to understand regarding estrogen signaling in BRCA2 carriers and preventing cancers in this high-risk population. Methods: We studied the transcriptomes and in-vitro growth of luminal progenitors and mature luminal cells derived from primary human normal breast tissue obtained from BRCA2 carriers and wild-type patients undergoing prophylactic mastectomy and reduction mammoplasty procedures, respectively. Subsequently, formalin-fixed paraffin-embedded breast cancer tissue samples from twelve case-control matched pairs of BRCA2 carriers and wild-type patients were investigated for ER genomic binding, H3K27Ac enhancer marks, and gene expression changes. Results: Luminal progenitors and mature luminal cells have different gene expression patterns. Unsupervised clustering of transcriptomes from normal tissue of three pairs of BRCA2-mutant and wildtype patients suggested that BRCA2 mutation status can segregate transcriptomes from luminal progenitors but not mature luminal cells. These differential transcriptomes in luminal progenitor cells from BRCA2 carriers were enriched for cell proliferation genes. In agreement, greater proliferative phenotype was observed in BRCA2-mutant luminal progenitor cells in comparison to the wild-type cohort. Interestingly, clustering analyses of twelve pairs of breast tumors indicated no clear differences between ER cistromes, enhancer marks and gene expression between BRCA2 carriers and wildtype patients. Because luminal progenitor cells are more likely to contribute to tumorigenesis, we hypothesize that the identified BRCA2-mutant signature in normal progenitor cells could be retained in tumors obtained from BRCA2 carriers. Conclusions: There are differential gene expression patterns between luminal progenitors and mature luminal cells obtained from normal human breast tissue. The transcriptomes in the luminal progenitor but not mature luminal cells segregate differently based on the BRCA2 mutation status. This identified BRCA2-mutant gene signature in luminal progenitor cells is enriched for pro-proliferation pathways, and BRCA2-mutant luminal progenitor cells exhibit higher cell proliferation, suggesting a potential cell-of-origin in BRCA2-associated breast cancers. Importantly, no significant differences in transcriptomes and ER cistromes are observed in breast tumors from BRCA2 carriers and non-carriers, indicating a need to identify BRCA2-mutant gene signature that may assist in segregating ER+ breast cancers in this high-risk populations. The knowledge from this study may assist in pioneering strategies for cancer prevention in high-risk BRCA2 mutation carriers. Citation Format: Hari Singhal, David Chi, Elgene Lim, Housheng He, Raga Vadhi, Prakash K. Rao, Henry W. Long, Andrea L. Richardson, Judy Garber, Myles Brown. Estrogen signaling in mature luminal and luminal progenitor cells of BRCA2 carriers and non-carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-257. doi:10.1158/1538-7445.AM2017-LB-257

Development and evaluation of a novel RT-qPCR based test for the quantification of HER2 gene expression in breast cancer

Accurate measurement of Human epidermal growth factor receptor (HER2) gene expression is central for breast or stomach cancer therapy orientation and prognosis. The current standards testing methods for HER2 expression are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH).In the current study, we explored the use of quantitative real time reverse transcription-PCR (RT-qPCR) as a potential method for the accurate relative quantification of the HER2 gene using formalin fixed paraffin embedded (FFPE) breast cancer biopsy samples. The main aim of the current study is to measure the level of concordance of RT-qPCR based quantification of HER2 overexpression with both IHC and FISH. Accordingly, an endogenous control gene (ECG) is required for this relative quantification and should ideally be expressed equivalently across tested samples. Stably expressed ECGs have been selected from a panel of seven genes using GenEx V6 software which is based on geNorm and NormFinder and statistical methods. Quantification of HER2 gene expression was performed by our RT-qPCR-based test and compared to the results obtained by both IHC and FISH methods.HER2 gene quantification using RT-qPCR test was normalized using the two ECGs (RPL30 and RPL37A) that were successfully identified and selected from a panel of seven genes as the most stable and reliable ECGs. We evaluated a total of 216 FFPE tissue samples from breast cancer patients. The results obtained with RT-qPCR in the current study were compared to both IHC and FISH data collected for the same patients. In addition to an internal evaluation, an external evaluation of this assay was also performed in a recognized pathology center in Europe (Clinic Barcelona Hospital Universitari, Spain) using 116 FFPE breast cancer tissue samples. The results demonstrated a high concordance between RT-qPCR and either IHC (98%) or FISH (72%) methods. Accordantly, the overall concordance was 85%.To our knowledge, this is the first study using the specific combination of RPL30 and RPL37 as reference genes for an accurate HER2 gene quantification in FFPE biopsy samples. Although further clinical validation regarding evolution and therapeutic response using RT-qPCR for the quantification of HER2 expression are still needed, the present study constitutes definitely a factual element that the RT-qPCR based assay may constitute a valid complementary test to accurately measure HER2 expression for a better treatment orientation.

Reliable gene expression profiling of formalin-fixed paraffin-embedded breast cancer tissue (FFPE) using cDNA-mediated annealing, extension, selection, and ligation whole-genome (DASL WG) assay.

BackgroundThe difficulties in using formalin-fixed and paraffin-embedded (FFPE) tumour specimens for molecular marker studies have hampered progress in translational cancer research. The cDNA-mediated, annealing, selection, extension, and ligation (DASL) assay is a platform for gene expression profiling from FFPE tissue and hence could allow analysis of large collections of tissue with associated clinical data from existing archives, therefore facilitating the development of novel biomarkers.MethodRNA isolated from matched fresh frozen (FF) and FFPE cancer specimens was profiled using both the DASL whole-genome (WG) platform, and Illumina BeadArray’s, and results were compared. Samples utilized were obtained from the breast cancer tumour bank held at the Cambridge University Hospitals NHS Foundation Trust.ResultsThe number of reliably detected probes was comparable between the DASL and BeadArray platforms, indicating that the source of RNA did not result in a significant difference in the detection rates (Mean probes- 17114 in FFPE & 17400 in FF). There was a significant degree of correlation between replicates within the FF and FFPE sample sets (r2 = 0.96–0.98) as well as between the two platforms (DASL vs. BeadArray r2 = range 0.83–0.89). Hierarchical clustering using the most informative probes showed that replicate and matched samples were grouped into the same sub-cluster, regardless of whether RNA was derived from FF or FFPE tissue.ConclusionBoth FF and FFPE material generated reproducible gene expression profiles, although there was more noise in profiles from FFPE specimens. We have shown that the DASL WG platform is suitable for profiling formalin-fixed paraffin-embedded samples, but robust bioinformatics analysis is required.

MicroRNA-711 is a prognostic factor for poor overall survival and has an oncogenic role in breast cancer.

MicroRNAs are important in cancer development and progression. In the present study, the clinical significance and function of microRNA-711 (miR-711) expression in breast cancer were investigated. The expression level of miR-711 was analyzed in breast cancer tissue samples using reverse transcription-quantitative polymerase chain reaction. Cell proliferation, colony formation, apoptosis and Transwell assays were performed in breast cancer cell lines transfected with miR-711 mimics or inhibitors, or control sequence. miR-711 was found to be upregulated in 30 formalin-fixed paraffin-embedded breast cancer tissue samples compared with paired non-cancerous breast tissues (P<0.05). Furthermore, a higher miR-711 expression was demonstrated to be associated with poor overall and disease-free survival times in 161 breast cancer patients, and miR-711 was identified as an independent prognostic factor using multivariate Cox regression analysis. In vitro, overexpression of miR-711 resulted in a significant increase in proliferation, colony formation, migration and invasion of breast cancer cells. By contrast, downregulating miR-711 inhibited cell proliferation, colony formation, migration and invasion and enhanced the rate of apoptosis of breast cancer cells. To the best of our knowledge, the present study is the first to demonstrate that miR-711 is an independent prognostic factor and serves an important oncogenic function in breast cancer, suggesting that miR-711 is a potential biomarker of prognosis and a molecular therapeutic target in breast cancer.

Clinical utility of reverse phase protein array for molecular classification of breast cancer.

Reverse Phase Protein Array (RPPA) represents a sensitive and high-throughput technique allowing simultaneous quantitation of protein expression levels in biological samples. This study aimed to confirm the ability of RPPA to classify archival formalin-fixed paraffin-embedded (FFPE) breast cancer tissues into molecular classes used in the Nottingham prognostic index plus (NPI+) determined by immunohistochemistry (IHC). Proteins were extracted from FFPE breast cancer tissues using three extraction protocols: the Q-proteome FFPE Tissue Kit (Qiagen, Hilden, Germany) and two in-house methods using Laemmli buffer with either incubation for 20min or 2h at 105°C. Two preparation methods, full-face sections and macrodissection, were used to assess the yield and quality of protein extracts. Ten biomarkers used for the NPI+ (ER, PgR, HER2, Cytokeratins 5/6 and 7/8, EGFR, HER3, HER4, p53 and Mucin 1) were quantified using RPPA and compared to results determined by IHC. The Q-proteome FFPE Tissue Kit produced significantly higher protein concentration and signal intensities. The intra- and inter-array reproducibility assessment indicated that RPPA using FFPE lysates was a highly reproducible and robust technique. Expression of the biomarkers individually and in combination using RPPA was highly consistent with IHC results. Macrodissection of the invasive tumour component gave more reliable results with the majority of biomarkers determined by IHC, (80% concordance) compared with full-face sections (60% concordance). Our results provide evidence for the technical feasibility of RPPA for high-throughput protein expression profiling of FFPE breast cancer tissues. The sensitivity of the technique is related to the quality of extracted protein and purity of tumour tissue. RPPA could provide a quantitative technique alternative to IHC for the biomarkers used in the NPI+.

97P Multiplex PCR of reference genes for accurate quantification of genes expression in formalin-fixed paraffin-embedded breast cancer tissues

97P Multiplex PCR of reference genes for accurate quantification of genes expression in formalin-fixed paraffin-embedded breast cancer tissues

Abstract A26: The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance

Abstract Background. The crosstalk between estrogen receptor alpha (ERα) and growth factor receptors is an essential part of tamoxifen resistance and the vascular endothelial growth factor/ tyrosine kinase domain receptor (VEGF/KDR) signaling pathway is clinically significant in the mechanisms of this resistance. The aim of the present study was to analyze the association of distribution pattern of ERα expression, VEGFR2 expression level, ESR1 and KDR single nucleotide polymorphisms (SNPs) with tamoxifen response in hormone receptor-positive primary breast cancer. Material and methods. Formalin-fixed paraffin-embedded and fresh frozen breast cancer tissue of the primary tumor was obtained from 97 hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen at the Tomsk Cancer Research Institute. The distribution patterns of ERα expression and Ki-67 protein expression were determined by immunohistochemistry. The quantitative expression levels of VEGFR2 (CD309) and p-Akt473 was measured using a flow cytometry. TaqMan SNP assays were used for genotyping ESR1+30T&amp;gt;C (rs2077647), ESR1 2014G&amp;gt;A (rs2228480), KDR-604T&amp;gt;C (rs2071559) and KDR 1192G&amp;gt;A (rs2305948) polymorphisms. Progression-free survival (PFS) was used as end-point for survival analyses. Results. Patients who did not benefit from tamoxifen treatment (tamoxifen resistance group -TR) showed significantly higher heterogeneous ERα expression than tamoxifen sensitive patients (TS; 81.1% and 58.3% respectively, P = 0.021). We found that ESR12014A mutant allele carriers were more prevalent in TR patients than in TS group (26.3% vs. 8.0%, respectively, P = 0.009). Similarly, the KDR -604T allele of rs2071559 was statistically significant related with tamoxifen resistance (P = 0.034). However, the KDR 1192G allele of rs2305948 were significantly less common in the tamoxifen progressive group compared to the tamoxifen sensitive patients (38.0% vs. 90.0%, respectively, P = 0.035). In addition, the variant homozygote KDR 1192GG (vs. AA) of rs2305948 was associated with VEGFR2 expression (r = -0.524, p = 0.012). VEGFR2 expression levels also showed borderline significant correlation with p-Akt473 expression (r = 0.368, p = 0.070). The Kaplan-Meier survival analysis demonstrated a significant relationship of heterogeneous distribution of ERα expression with poor survival of tamoxifen treated patients (log-rank P = 0.009). Moreover, the PFS for the patients with the KDR -604TT genotypes of rs2071559 were worse than for the patients with the CC genotype (log-rank P = 0.123). Conclusion. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation can serve as potential predictors for hormone receptor-positive breast cancer patients treated with adjuvant tamoxifen and thus provide more information for optimizing patient's selection to tamoxifen adjuvant therapy. Citation Format: Nataliya Babyshkina, Sergey Vtorushin, Stanislav Patalyak, Tatyana Dronova, Elena Slonimskaya, Nadejda Cherdyntseva. The distribution pattern of ERα expression, VEGFR2 expression level and its genetic variation as potential biomarkers for tamoxifen resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A26.

Quantification of HER2 by Targeted Mass Spectrometry in Formalin-Fixed Paraffin-Embedded (FFPE) Breast Cancer Tissues

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Abstract 2773: Association of ALDH1A1 gene expression with survival of triple-negative breast cancer

Abstract Aldehyde dehydrogenases (ALDH) are a family of enzymes that catalyze aldehyde into carboxylic acids via the NAD(P)+-dependent oxidation. Several ALDH family members have been identified in humans, including ALDH1A1, ALDH1A3, ALDH2, ALDH3A1, and ALDH4A1. ALDH1A1 is a crucial element in the retinoic acid signaling pathway regulating the self-renewal and differentiation of normal stem cells. It ALDH1A1 has been suggested as one of breast cancer stem cell markers and may play an important role in breast cancer prognosis. However, previous research on ALDH1A1 and breast cancer prognosis has yielded conflicting results. We analyzed the association between mRNA expression of ALDH1A1 in tumor tissues and survival of triple-negative breast cancer (TNBC: ER-/PR-/HER2-) using data and samples from three cohorts of breast cancer patients, including 469 cases from the Shanghai Breast Cancer Survival Study (SBCSS), 86 cases from the Nashville Breast Health Study (NBHS), and 47 cases from the Southern Community Cohort Study (SCCS). Gene expression levels were measured in total RNA isolated from microdissected archival formalin-fixed paraffin-embedded breast cancer tissues using Nanostring nCounter assays. The associations between ALDH1A1 mRNA expression levels and recurrence/breast cancer mortality and overall mortality were evaluated by multivariate survival analysis using the Cox regression model. Data from the SCCS and NBHS were combined in the analysis because both studies were conducted among US women and had a small sample size. The levels of ALDH1A1 mRNA expression were consistently and positively associated with disease free and overall survival independent of age at diagnosis and TNM stage in both Chinese and US women with TNBC. Compared to those with ALDH1A1 mRNA expression below the median, TNBC patients with ALDH1A1 mRNA expression above the median had a reduced risk of recurrence/breast cancer mortality (HR = 0.60, 95% CI: 0.39-0.92) and overall mortality (HR = 0.70, 95% CI: 0.47-1.05) in the SBCSS and reduced overall mortality in the SCCS/NBHS (HR = 0.27,95% CI: 0.10-0.74). Additional analyses of the SBCSS cohort stratified by basal-like breast cancer subtype showed that ALDH1A1 mRNA expression was similarly associated with reduced risk of recurrence/breast cancer mortality and total mortality in both basal-like and non-basal like TNBC patients, although the point estimates were not significant due to the smaller sample size. In addition, we found that patients with a higher tumor grade had a lower level of ALDH1A1 mRNA expression but did not observe an association with TNM stage. Our study suggests that the mRNA expression of ALDH1A1 in tumor tissue may be associated with favorable clinical outcomes in patients with TNBC. Further investigations are needed to understand the biological mechanism(s) underlying the observed association. Citation Format: Yan Liu, Qiuyin Cai, Ying Zheng, Michelle L. Baglia, Yinghao Su, Sarah Nechuta, Ping-Ping Bao, William Blot, Wei Zheng, Xiao-Ou Shu. Association of ALDH1A1 gene expression with survival of triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2773. doi:10.1158/1538-7445.AM2015-2773