Formalin-fixed Paraffin-embedded Breast Cancer Tissue Research Articles

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method

Developing immunoassay for absolute quantitation of protein biomarkers in Formalin Fixed Paraffin Embedded (FFPE) samples promises improved objectivity, consistency and accuracy in daily clinical practice. The feasibility of Quantitative Dot Blot (QDB) method for this purpose was explored in this study. We were able to measure HER2 protein levels using 0.5 µg/sample total protein lysate extracted from 2 × 5 µm FFPE slices absolutely and quantitatively using QDB method in 332 breast cancer FFPE samples. HER2 levels measured using two clinically validated antibodies for immunohistochemistry respectively were highly correlated (r = 0.963). We also achieved area under the curve (AUC) at 0.9998 ± 0.0002 (p < 0.0001, n = 224) with IHC analysis, and 0.9942 ± 0.0031 (p < 0.0001, n = 319) with combined results from IHC and Fluorescence in situ hybridization (FISH) analyses when analyzed with Receiver Operative Characteristics analysis (ROC) respectively. When the results were converted dichotomously with optimized cutoffs from ROC analyses, we achieved 99.5% concordance with IHC; and 96.9% with combined results from both IHC and FISH analyses. Therefore, we were able to demonstrate QDB method as the first immunoassay platform for absolute quantitation of protein biomarkers in FFPE samples to meet the need of daily clinical practice, especially for local laboratories or laboratories in developing countries.

Abstract P2-11-08: ESR1 mutations are not a mechanism of primary resistance to aromatase inhibitors in ER-positive breast cancer treated with neoadjuvant endocrine therapy

Abstract Introduction: Mutations in the ESR1 gene (ESR1m) are important mechanisms of resistance to endocrine therapy (ET) in estrogen receptor-positive (ER+) advanced breast cancer and have been recognized as a prognostic and predictive biomarker as well as a potential therapeutic target. ESR1m prevalence has been described as 9-45% in cohorts of ET-resistant metastatic tumors in a variety of publications. We recently reported a prevalence of 25% in a cohort of ER+ breast cancer patients with visceral metastasis. However, the role of ESR1m as a mechanism of resistance to ET used in early-stage disease is not well studied. Neoadjuvant endocrine therapy (NET) is being increasingly explored, not only to allow less extensive surgery but also as a scientific tool, exploring biomarkers to predict outcomes. The preoperative endocrine prognostic index (PEPI) combining Ki67 score, ER Allred score, tumor size, and nodal status after NET is a surrogate of endocrine sensitivity and can identify a subgroup of patients with primary resistance to ET. In this study, we evaluated the prevalence of ESR1m in tumor samples from patients with ER+ breast cancer that were primarily resistant to neoadjuvant aromatase inhibitor (AI) therapy. Methods: We followed a prospective cohort of postmenopausal patients with ER+ HER2- stages II-III breast cancer treated with neoadjuvant endocrine therapy (NET). Patients were treated with anastrozole for a recommended period of at least three months. Tumor samples from patients with a pattern of primary endocrine-resistant tumors (defined as a PEPI Score higher than 3) were selected and analyzed for the presence of ESR1m. ESR1m were 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. Results: 127 patients were included in the cohort, of which 100 (79%) had completed NET and had surgery. Among these patients, the PEPI Score ranged from 0 to 3 in 70% (70/100), and 30% (30/100) had a PEPI Score of 4 or more and were selected. 23 patients were included in the analysis (6 did not consent or were lost to follow-up, and one was HER2-positive). Patients characteristics are summarized in Table 1. The median duration of NET was 22 weeks. All samples of tumor tissue from the surgical specimens after NET were evaluable. Quantification of DNA extraction and reference gene cycle threshold values confirmed that the material was adequate for the analysis. We compared these findings with a study from our group using the same methodology in patients with advanced disease where ESR1 mutations were detected in 25% (n=32) of patients with visceral metastasis of ER+ breast cancer resistant to endocrine therapy. ESR1 mutations were not identified in any of the 23 patients with early-stage ER+ breast cancer resistant to NET (p 0.01, Fisher´s exact test) Discussion: Growing evidence supports the notion that there are different mechanisms of primary and secondary endocrine resistance. Our study suggests that ESR1 mutations do not evolve rapidly and do not represent a common mechanism of primary endocrine resistance in the neoadjuvant setting. Therefore, ESR1m should be considered a mechanism of acquired endocrine resistance in the context of advanced disease. Further research should be conducted to identify factors associated with intrinsic resistance to ET. Citation Format: Tomas Reinert, Susana Ramalho, Vivian CA Vasconcelos, Leonardo R Silva, Ana Elisa R Silva, Camila A Andrade, Maria Beatriz PL Kraft, Guilherme P Coelho, Jovana Mandelli, Monique Binotto, Cesar Cabello, Geisilene RP Silva, Jose Bines, Carlos H Barrios, Matthew J Ellis, Marcia S Graudenz. ESR1 mutations are not a mechanism of primary resistance to aromatase inhibitors in ER-positive breast cancer treated with neoadjuvant endocrine therapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-11-08.

Performance of Automated Dissection on Formalin-Fixed Paraffin-Embedded Tissue Sections for the 21-Gene Recurrence Score Assay

This study aimed to compare the performance of MilliSect dissection and manual dissection. Twenty-five formalin-fixed paraffin-embedded (FFPE) breast cancer tissue blocks were selected for comparison. Specific areas of interest (AOIs) in invasive carcinoma on tissue sections were transferred to dissection slides by manual macrodissection or the MilliSect instrument. The comparison criteria were 1) the time required for dissection; 2) RNA concentration and purity; 3) RNA quantity of 5 housekeeping genes (by RT-qPCR); and 4) ER, PR, HER2, Ki-67 and recurrence score (RS) values (by the 21-gene assay). Then, tumor-adjacent tissues, including fibrocollagenous and epithelial tissues, from the same selected tissue blocks of 8 of 25 patients were scraped using the mesodissection method, and their RS values were assessed to evaluate the influence of tumor-adjacent tissues on the target AOIs. Ultimately, 4 AOIs of invasive ductal carcinoma (IDC) from 1 tissue block of another 4 patients with lymph node (LN) metastases each, LN tissue and a mixture of IDC and LN tissue from the other tissue block of the same 4 patients were mesodissected to evaluate the influence of infiltrating lymphocyte levels on the RS values of AOIs. In our experience, the MilliSect instrument, which provides process management documentation, required more time than manual macrodissection (on average, approximately 9.1 min per sample versus 5.8 min per sample, respectively). The RNA yield and quality of the dissected tissues were comparable for the 2 methods. However, the tumor-adjacent tissues of the AOIs may influence the RS to some extent. Tumor-infiltrating lymphocytes (TILs) can dramatically increase RSs, far exceeding the influence of tumor-adjacent fibrocollagenous and epithelial tissues. In conclusion, MilliSect mesodissection is comparable to manual dissection. This mesodissection tool may facilitate AOI alignment and the dissection process for the 21-gene RS assay. Samples whose adjacent tissues are intermixed with TILs warrant special attention.

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+.