Archival Breast Cancer Tissue Research Articles

Systematic review and feasibility study on pre-analytical factors and genomic analyses on archival formalin-fixed paraffin-embedded breast cancer tissue

Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Abstract PO5-14-03: Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer

Abstract Discussion: Breast cancer is the most common malignancy diagnosed amongst women, with more the two million new cases identified globally each year. Thus, considerable resources have been devoted to a better understanding of the disease and how to optimize outcomes for patients. Genetics and genomics have become part of routine care with the latter used to inform treatment planning for patients with ER+ breast cancer (ER+ BC). A multitude of assays may be used to determine the need for adjuvant chemotherapy but none of the current tests on the market are guideline-recommended for making decisions related to neoadjuvant chemotherapy, surgery, or radiation. The 24-gene StemPrintER assay was developed to measure the “stemness” of tumors, or how much a tumor behaves like stem cells, which could have important and novel implications for breast cancer treatment planning. The assay has been validated for predicting prognosis using formalin-fixed paraffin-embedded (FFPE) tissue blocks from several clinical cohorts, including TransATAC. The goal of current and future research will be to determine whether the assay can determine the ideal surgical intervention for ER+BC patients. Early in silico work demonstrated a trend toward this predictive capacity for the assay and suggests the assay may be able to identify the benefit of mastectomy versus quadrantectomy. Here, we report on the ability to set up and run the assay in our lab using synthetic oligonucleotides and archival tissue blocks. Methods: Synthetic oligonucleotides and archival FFPE breast cancer tissue were used to set up and analytically validate the assay’s performance in two phases. The initial phase was intended to confirm the individual assay design performance by assessing linearity and precision and the ability to amplify RNA from FFPE, which confirmed compatibility with standard extraction methods. The second phase was intended to determine whether these methods could be implemented to reliably extract and amplify RNA from FFPE >10 years old (range: 10-14 years) in order to generate a StemPrintER score. The assay was run according to standard operating procedures for StemPrintER. Analyses were conducted Python 3.9.12. Results: Two assay runs with duplicate samples for each run using a 7-point dilution series demonstrated linearity (coefficient of correlation, r2) results ranging from 0.978 to 0.999 for each of the 24 genes in the assay. Amplification efficiency was between 100-101% for each gene. Using 14 FFPE samples from patients with triple-negative breast cancer, we demonstrated a consistent extraction yield of RNA from tumor blocks which produced amplification of each of the targets. Analysis of intra- and inter-run precision resulted in coefficients of variation (CV) ranging from 0.2-1.07% and inter-run CV’s ranging from 0.66%-5.56%. Utilizing FFPE >10 years old from ER+BC patients demonstrated that the lab could extract RNA from 100% of samples and amplify the material to obtain binary StemPrintER results. Conclusion: These data demonstrate that StemPrintER can be run reliably on FFPE tissue up to 14 years old. In addition, our laboratory has demonstrated that assay results are highly linear and reproducible. Additional studies are planned to build on preliminary surgical prediction data and determine whether the assay can meet the unmet need of identifying the ideal surgical intervention in patients with early-stage ER+/HER2- breast cancer. Citation Format: Catie Cronister, Brock Schweitzer, Hannah Gilmore, Tyler Nielsen. Analytical Validation of the StemPrintER Assay in Patients with Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-03.

Quality Assessment of Proteins and RNA Following Storage in Archival Formalin-Fixed Paraffin-Embedded Human Breast Cancer Tissue Microarray Sections.

Although the immunogenicity of formalin-fixed paraffin-embedded tissue sections can decrease during storage and transport, the exact mechanism of antigenic loss and how to prevent it are not clear. Herein, we investigated changes in the expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), E-cadherin, and Ki-67 in human breast tissue microarray (TMA) tissue sections stored for up to 3 months in dry and wet conditions. The positive rates of ER and PR expression were minimally changed after 3 months of storage, but the Allred scores of ER and PR stored in humid conditions decreased remarkably in comparison to fresh-cut tissue. The HER-2 antigenicity and RNA integrity of breast TMA sections stored in dry conditions diminished gradually with storage time, whereas the immunoreactivity and RNA quality of HER-2 in humid conditions decreased sharply as storage length increased. The area and intensity of E-cadherin staining in tissue sections stored in dry conditions did not change significantly and were minimally changed after 3 months, respectively. In contrast, the area and intensity of E-cadherin staining in tissue sections stored in humid conditions decreased significantly as storage length increased. Finally, the Ki-67 labeling index of tissue sections stored for 3 months in dry (9% decrease) and wet (31.9% decrease) conditions was decreased in comparison to fresh sections. In conclusion, these results indicate that water is a crucial factor for protein and RNA degradation in stored tissue sections, and detailed guidelines are required in the clinic.

Efficacy and Safety of Weekly Paclitaxel With or Without Oral Alisertib in Patients With Metastatic Breast Cancer

Elevated expression of AURKA adversely affects prognosis in estrogen receptor (ER)-positive and ERBB2 (formerly HER2)-negative and triple-negative breast cancer and is associated with resistance to taxanes. To compare paclitaxel alone vs paclitaxel plus alisertib in patients with ER-positive and ERBB2-negative or triple-negative metastatic breast cancer (MBC). In this randomized clinical trial conducted with the US Oncology Network, participants were randomized to intravenous (IV) paclitaxel 90 mg/m2 on days 1, 8, and 15 on a 28-day cycle or IV paclitaxel 60 mg/m2 on days 1, 8, and 15 plus oral alisertib 40 mg twice daily on days 1 to 3, 8 to 10, and 15 to 17 on a 28-day cycle. Stratification was by prior neo or adjuvant taxane and by line of metastatic therapy. Eligible patients were those who had undergone endocrine therapy, 0 or 1 prior chemotherapy regimens for MBC, more than 12 months treatment-free interval from neo or adjuvant taxane therapy, and with measurable or evaluable lytic bone-disease. Data were analyzed from March 2019 through May 2019. The main outcome was progression-free survival (PFS) with secondary end points of overall survival (OS), overall response rate, clinical benefit rate, safety, and analysis of archival breast cancer tissues for molecular markers associated with benefit from alisertib. A total of 174 patients were randomized, including with 86 randomized to paclitaxel and 88 patients randomized to paclitaxel plus alisertib, and 169 patients received study treatment. The final cohort included 139 patients with a median (interquartile range [IQR]) age of 62 (27-84) years with ER-positive and ERBB2-negative MBC, with 70 randomized to paclitaxel and 69 randomized to paclitaxel plus alisertib. The TNBC cohort closed with only 35 patients enrolled due to slow accrual and were not included in efficacy analyses. The median (IQR) follow-up was 22 (10.6-25.1) months, and median (IQR) PFS was 10.2 (3.8-15.7) months with paclitaxel plus alisertib vs 7.1 (3.8-10.6) months with paclitaxel alone (HR, 0.56; 95% CI, 0.37-0.84; P = .005). Median (IQR) OS was 26.3 (12.4-37.2) months for patients who received paclitaxel plus alisertib vs 25.1 (11.0-31.4) months for paclitaxel alone (HR, 0.89; 95% CI, 0.58-1.38; P = .61). Grade 3 or 4 adverse events occurred in 56 patients (84.8%) receiving paclitaxel plus alisertib vs 34 patients (48.6%) receiving paclitaxel alone. The main grade 3 or 4 adverse events with paclitaxel plus alisertib vs paclitaxel alone were neutropenia (50 patients [59.5%] vs 14 patients [16.4%]), anemia (8 patients [9.5%] vs 1 patient [1.2%]), diarrhea (9 patients [10.7%] vs 0 patients), and stomatitis or oral mucositis (13 patients [15.5%] vs 0 patients). One patient receiving paclitaxel plus alisertib died of sepsis. This randomized clinical trial found that the addition of oral alisertib to a reduced dose of weekly paclitaxel significantly improved PFS compared with paclitaxel alone, and toxic effects with paclitaxel plus alisertib were manageable with alisertib dose reduction. These data support further evaluation of alisertib in patients with ER-positive, ERBB2-negative MBC. ClinicalTrials.gov Identifier: NCT02187991.

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

Abstract P6-09-01: Central Ki67 analysis as a predictor for adjuvant capecitabine efficacy in early breast cancer (EBC) subtypes in US oncology trial 01062

Abstract Background: USON 01062 (O’Shaughnessy J, et al. Proc SABCS, 2010, abst S4-2) showed no improvement in the primary endpoint of disease-free survival (DFS) (median FU 5 yrs: HR 0.84, 95% CI: 0.67-1.05; p = 0.125) with the addition of capecitabine (X) to standard adjuvant chemotherapy, but showed improvement in OS (HR 0.68, 95% CI: 0.51-0.92; p = 0.011). Exploratory analysis of local pathology-assessed Ki67 suggested benefit from adjuvant X in pts with more highly proliferative cancers with Ki67 ≥ 10% (Pippen J et al. Proc ASCO, 2011, abst 500). The objective of this study is to determine whether centrally-performed Ki67 IHC results corroborate or refute this finding. Methods: 2610 pts with resected high risk EBC were randomized to receive 4 cycles of AC (doxorubicin 60mg/m2 and cyclophosphamide 600mg/m2) IV every 3 wks for 4 cycles followed by either docetaxel 100mg/m2 IV or docetaxel 75mg/m2 IV plus X 825mg/m2 PO bid for 14 days every 3 wks for 4 cycles. Archival primary breast cancer tissue was collected on 2000 pts for predictive biomarker analyses. Central Ki67 IHC was performed using the anti-Ki67 monoclonal antibody SP6 and was read by one pathologist (HK) according to published recommendations (Dowsett M, et al. JNCI 103:1-9, 2011). Results: Central Ki67 IHC has been performed on 1440 pts who had centrally-validated informed consents. The distribution of% Ki67-positive cells by locally-assessed ER/HER2 subtype is shown below. 45% of HR+ HER2- BCs had a Ki67 ≤ 10%, while 24% had a Ki67 11% to 20%, and 31% had a Ki67 > 20%. The concordance between the local vs central Ki67 results was low at 46% for Ki67 <10%, 49% for Ki67 10%-20%, and 76% for Ki67 > 20%. The central Ki67 results tended to be higher than the local testing results. Central mRNA classifiers were developed for ER, PR, HER2 and Ki67 using Fluidigm Microfluidics Dynamic Arrays and correlate highly with central IHC assessment of these markers. Conclusions: HR+ HER2- EBC is enriched for cancers with a low proliferative rate, a group of pts unlikely to benefit from the cell cycle-specific cytotoxic agent, capecitabine. Analyses of the impact of adjuvant X added to AC/T in EBC pts according to ER status, and according to Ki67 (analyzed as a binary and continuous variable) will be performed prior to SABCS, 2013. Number of Patients% Ki67 Pos CellsTotal *HR+TNHER2+/HR+HER2+/ HR-0-104163622222711-151391066151016-20126871615821-3018411539201031-1005751403423555Total144081042510790*Totals do not equal sum of subtype categories due to missing HER2 information Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-09-01.

Interobserver concordance of Ki67 labeling index in breast cancer: Japan Breast Cancer Research Group Ki67 Ring Study

The standardized assessment of Ki67 labeling index (LI) is of clinical importance to identify patients with primary breast cancer who could benefit from chemotherapy. In this study, we evaluated the interobserver concordance of Ki67 LI assessment. Six surgical pathologists participated and all the slides were prepared from archival breast cancer tissues fixed in 10% buffered formalin for 24 h and stained with MIB-1. Three independent studies were conducted. In the first study, 30 stained slides were assessed using two different methods: the scoring system, with a positive rate scored from 1 (0-9%) to 10 (90-100%) by visual estimate; and the counting method, with approximately 1000 cells counted in hot spots. In the second study, 20 tumors with Ki67 LI 5-25% were assessed, and in the third study, 15 printed photographs of stained slides were assessed to avoid variations by selecting different fields. In study 1, the counting system (intraclass correlation coefficient [ICC], 0.66 [95% confidence interval 0.52-0.78]) demonstrated a better correlation than the scoring system (ICC, 0.57 [0.42-0.72]). In study 2, the assessment for Ki67 LI of 5-25% demonstrated a correlation (ICC, 0.68 [0.50-0.81]) similar to that of study 1 (unrestricted range of Ki67 LI). In study 3, the assessment of Ki67 LI by counting yielded a good concordance (ICC, 0.94 [0.88-0.97]). In conclusion, there was better concordance with the counting system, and concordance was high when the assessed field was predetermined, indicating that the selection of the evaluation area is critical for obtaining reproducible Ki67 LI in breast cancer.

Analysis of indoleamine 2-3 dioxygenase (IDO1) expression in breast cancer tissue by immunohistochemistry

The immunosuppressive enzyme, indoleamine 2,3 dioxygenase (IDO), is overexpressed in many different tumor types including breast cancer. IDO inhibitors synergize with chemotherapy in breast cancer murine models. Characterizing IDO expression in breast cancer could define which patients receive IDO inhibitors. This study analyzed IDO protein expression in 203 breast cancer cases. The relationship between IDO, overall survival (OS), disease-specific survival (DSS), clinicopathologic, molecular, and immune tumor infiltrate factors was evaluated. Expression of IDO, estrogen receptor (ER), progesterone receptor (PR), human epithelial receptor 2, cytokeratin 5/6, epithelial growth factor receptor, phosphorylated AKT, neoangiogenesis, nitrogen oxide synthetase 2 (NOS2), cyclooxygenase 2 (COX2), FoxP3, CD8, and CD11b on archival breast cancer tissue sections was evaluated by immunohistochemistry. Associations between IDO and these markers were explored by a univariate and multivariate analysis. Survival was analyzed using Kaplan-Meier (OS) and Wilcoxon two-sample (DSS) tests. IDO expression was higher in ER+ tumors compared to ER- tumors. IDO was lower in those with higher neoangiogenesis. OS was better in ER+ patients with high IDO expression. DSS was better in node-positive patients with high IDO expression. IDO activity positively correlates with NOS2. COX2 as positively correlated with IDO on univariate but not multivariate analysis. There was a trend toward greater numbers of CD11b+ cells in IDO-low tumors. IDO protein expression is lower in ER- breast tumors with greater neoangiogenesis. Future clinical trials evaluating the synergy between IDO inhibitors and chemotherapy should take this finding into account and stratify for ER status in the trial design.

Multiplex layered immunohistochemistry to predict response of HER2-positive breast cancer patients to trastuzumab therapy.

136 Background: Since only 40% of HER2-positive breast cancer patients respond to trastuzumab therapy, there is a great clinical need for a test that will better predict which patients would be responders to trastuzumab therapy. Since no single biomarker will likely improve identification of responders, this study will determine the efficacy of multiple biomarkers to predict response of patients to trastuzumab therapy. Our company has developed a protein analysis platform, layered immunohistochemistry (L-IHC), for the analysis of multiple protein biomarkers from a single FFPE tissue section while retaining the histologic orientation. L-IHC is being used to develop a companion diagnostic test in which multiple protein biomarkers are analyzed in HER2-positive breast cancer specimens to predict which patients will respond to trastuzumab therapy. Methods: In this ongoing clinical exploratory study, analysis of pretreatment archival HER2-positive FFPE breast cancer tissue sections from patients whose subsequent therapy included trastuzumab and whose response to therapy (responders and non-responders) is known was performed using L-IHC. Tissue sections were probed using a panel of 14 biomarkers along the HER2 and mTOR pathways were analyzed. In a blinded fashion to the response data, the intensity of specific biomarker signals corresponding to cancer regions on an H&E was visually scored (0,1,2,3, & 4+). The pattern of biomarker expression was correlated with the responder status of the patient. Results: A total of 24 responders and 9 non-responders were analyzed. The sum of four of the 14 biomarkers including p-mTOR, p-4EBP1, pERK, and HIF1-alpha discriminated the responders and non-responders. Using 7 as a cutoff, this test correctly identified 21/24 responders and 7/9 non-responders for a sensitivity of 87%, specificity of 78%, and an accuracy of 82%. Conclusions: Results of this study strongly suggests that analysis of p-mTOR, p-4EBP1, pERK, and HIF1-alpha using L-IHC improves twofold the prediction of patient response to trastuzumab compared to Her2 alone.

Detection of mRNA for Nuclear Receptor Co-Activators 1 and 3 in Archival Breast Cancer Tissue and Surrounding Stroma: A Tissue Expression Study~!2009-01-14~!2010-02-10~!2010-07-20~!

Before the age of 75 years, approximately 10% of women will be diagnosed with breast cancer, one of the most common malignancies and a leading cause of death among women. The objective of this study was to determine if expression of the nuclear receptor coactivators 1 and 3 (NCoA1 and NCoA3) varied in breast cancer grades. RNA was extracted from 25 breast tumours and transcribed into cDNA which underwent semi-quantitative polymerase chain reaction, normalised using 18S. Analysis indicated that an expression change for NCoA1 in cancer grades and estrogen receptor alpha negative tissue (P= 0.028 and 0.001 respectively). NCoA1 expression increased in grade 3 and estrogen receptor alpha negative tumours, compared to controls. NCoA3 showed a similar, but not significant, trend in grade and a non-significant decrease in estrogen receptor alpha negative tissues. Expression of NCoA1 in late stage and estrogen receptor alpha negative breast tumours may have implications to breast cancer treatment, particularly in the area of manipulation of hormone signalling systems in advanced tumours.

Abstract 3742: FOXC1 is a potential prognostic marker with functional significance in basal-like breast cancer

Abstract Although basal-like breast cancer (BLBC) is a distinct molecular subtype, it has no defining gene or protein marker that can be used as the basis for therapy. Estrogen receptor (ER) and HER2 receptor guide treatment of luminal and HER2 breast cancer subtypes, respectively, but chemotherapy is still the only systemic treatment available for BLBC. In view of its poor clinical outcomes, high proliferation rates and preferential metastasis to the brain, there is an urgent need for effective targeted therapy of BLBC. In a previous study using cDNA microarray analyses and immunohistochemistry of archival breast cancer tissue specimens, we showed consistent and exclusive overexpression of FOXC1 in BLBC versus other molecular subtypes. FOXC1 overexpression was significantly associated with poor overall survival and brain metastasis. In the present study, we show that overexpression of FOXC1 in breast cancer cells with low endogenous FOXC1 levels increased cellular proliferation, migration, and invasion, and induced epithelial-mesenchymal transition. Knockdown of FOXC1 by shRNA in breast cancer cells with high endogenous FOXC1 expression had the opposite effect. Cell signaling studies showed that FOXC1 activated the NF-κB pathway by upregulating the peptidyl-prolyl isomerase Pin1 and downregulating the ubiquitin ligase SOCS-1. Pin1 binds to p65, inhibits the p65 interaction with IκBα, and thus enhances p65 nuclear localization and protein stability. SOCS-1 directly interacts with p65 and promotes its degradation. FOXC1 knockdown reduced p65 protein levels and NF-κB activity. The importance of the NF-κB pathway for FOXC1 function was confirmed by the increased sensitivity to NF-κB inhibitors in cells that overexpressed FOXC1. Because BLBC under-expresses ER, FOXC1 was inversely associated with ER, as expected. Interestingly, FOXC1 repressed ER transcription and activity via NF-κB signaling, and FOXC1 overexpression switched MCF-7 breast cancer cell growth from estrogen-dependent to estrogen-independent. This may explain why ER is not normally detected in BLBC. These results suggest that FOXC1 may be a specific diagnostic and prognostic biomarker for BLBC and may play an important role in regulating aggressive traits associated with BLBC. It might also serve as a potential molecular therapeutic target for BLBC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3742.

Identification of new genes associated with breast cancer progression by gene expression analysis of predefined sets of neoplastic tissues

Gene expression profiles were studied by microarray analysis in 2 sets of archival breast cancer tissues from patients with distinct clinical outcome. Seventy-seven differentially expressed genes were identified when comparing 30 cases with relapse and 30 cases without relapse within 72 months from surgery. These genes had a specific ontological distribution and some of them have been linked to breast cancer in previous studies: AIB1, the two keratin genes KRT5 and KRT15, RAF1, WIF1 and MSH6. Seven out of 77 differentially expressed genes were selected and analyzed by qRT-PCR in 127 cases of breast cancer. The expression levels of 6 upregulated genes (CKMT1B, DDX21, PRKDC, PTPN1, SLPI, YWHAE) showed a significant association to both disease-free and overall survival. Multivariate analysis using the significant factors (i.e., estrogen receptor and lymph node status) as covariates confirmed the association with survival. There was no correlation between the expression level of these genes and other clinical parameters. In contrast, SERPINA3, the only downregulated gene examined, was not associated with survival, but correlated with steroid receptor status. An indirect validation of our genes was provided by calculating their association with survival in 3 publicly available microarray datasets. CKMT1B expression was an independent prognostic marker in all 3 datasets, whereas other genes confirmed their association with disease-free survival in at least 1 dataset. This work provides a novel set of genes that could be used as independent prognostic markers and potential drug targets for breast cancer.

Expression analysis of carbohydrate antigens in ductal carcinoma in situof the breast by lectin histochemistry

BackgroundThe number of breast cancer patients diagnosed with ductal carcinoma in situ (DCIS) continues to grow. Laboratory and clinical data indicate that DCIS can progress to invasive disease. Carbohydrate-mediated cell-cell adhesion and tumor-stroma interaction play crucial roles in tumorigenesis and tumor aggressive behavior. Breast carcinogenesis may reflect quantitative as well as qualitative changes in oligosaccharide expression, which may provide a useful tool for early detection of breast cancer. Because tumor-associated carbohydrate antigens (TACA) are implicated in tumor invasion and metastasis, the purpose of this study was to assess the expression of selected TACA by lectin histochemistry on DCIS specimens from the archival breast cancer tissue array bank of the University of Arkansas for Medical Sciences.MethodsFor detection of TACA expression, specimens were stained with Griffonia simplicifolia lectin-I (GS-I) and Vicia vilosa agglutinin (VVA). We studied associations of lectin reactivity with established prognostic factors, such as tumor size, tumor nuclear grade, and expression of Her-2/neu, p53 mutant and estrogen and progesterone receptors.ResultsWe observed that both lectins showed significant associations with nuclear grade of DCIS. DCIS specimens with nuclear grades II and III showed significantly more intense reactivity than DCIS cases with nuclear grade I to GS-1 (Mean-score chi-square = 17.60, DF = 2; P = 0.0002) and VVA (Mean-score chi-square = 15.72, DF = 2; P = 0.0004).ConclusionThe results suggest that the expression of VVA- and GS-I-reactive carbohydrate antigens may contribute to forming higher grade DCIS and increase the recurrence risk.

HER-2 gene amplification by chromogenic in situ hybridisation (CISH) compared with fluorescence in situ hybridisation (FISH) in breast cancer—A study of two hundred cases

The purpose of the study was to compare two methods used to analyse HER-2 gene amplification (fluorescence in situ hybridisation (FISH) and chromogenic in situ hybridisation (CISH)), and determine the accuracy of the antibodies CB11 and HercepTest for immunohistochemical detection of HER-2 overexpression from archival breast cancer tissue. Additionally, interobserver variability in the interpretation of CISH and immunohistochemical tests was measured. Two hundred cases of invasive breast carcinoma diagnosed between 2000 and 2003 were selected. Immunohistochemistry (IHC) was performed with HercepTest and CB11, and gene amplification was determined by FISH (PathVision, Vysis) and CISH (Zymed) using tissue macroarrays. An excellent concordance (94.8%) was found between CISH and FISH. Considering FISH as gold standard, sensitivity of CISH was 97.5% and specificity 94%. Overall interobserver agreement of CISH was 97.5% and of IHC 84%. Both antibodies showed a sensitivity of 95.2% and a specificity of 70.7% (CB11) and 81.2% (HercepTest). Our results show that CISH is a highly accurate, reproducible and practical technique to determine HER-2 gene amplification. CB11 and HercepTest are good screening methods with a high sensitivity. The performance of tissue macroarrays to test HER-2 status by IHC, FISH and CISH has demonstrated to be an available and effective method to study large series of tumours.

Detection of mRNA levels for the estrogen alpha, estrogen beta and androgen nuclear receptor genes in archival breast cancer tissue

Previous studies in our laboratory have shown association of nuclear receptor expression and histological breast cancer grade. To further investigate these findings, it was the objective of this study to determine if expression levels of the estrogen alpha, estrogen beta and androgen nuclear receptor genes varied in different breast cancer grades. RNA extracted from paraffin embedded archival breast tumour tissue was converted into cDNA and cDNA underwent PCR to enable quantitation of mRNA expression. Expression data was normalised against the 18S ribosomal gene multiplex and analysed using ANOVA. Analysis indicated a significant alteration of expression for the androgen receptor in different cancer grades ( P=0.014), as well as in tissues that no longer possess estrogen receptor alpha proteins ( P=0.025). However, expression of estrogen receptors alpha and beta did not vary significantly with cancer grade ( P=0.057 and 0.622, respectively). Also, the expression of estrogen receptor alpha or beta did not change, regardless of the presence of estrogen receptor alpha protein in the tissue ( P=0.794 and 0.716, respectively). Post-hoc tests indicate that the expression of the androgen receptor is increased in estrogen receptor negative tissue as well as in grade 2 and grade 3 tumours, compared to control tissue. This increased expression in late stage breast tumours may have implications to the treatment of breast tumours, particularly those lacking expression of other nuclear receptor genes.

BRCA1 Gene Expression in Breast Cancer: A Correlative Study between Real-time RT-PCR and Immunohistochemistry

Breast cancer is a major cause of cancer-related mortality in women. There are major discrepancies concerning the usefulness of various antibodies in detecting breast cancer susceptibility gene 1 (BRCA1) protein and its subcellular localization. The aim of the present study was to determine the specificity and sensitivity of immunohistochemistry (IHC) as a screening method for demonstrating BRCA1 expression. BRCA1 gene expression in archival paraffin-embedded breast cancer tissues was studied simultaneously at the protein and mRNA levels, and the two findings were compared. Forty-eight archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression at protein level by IHC using four different antibodies against different BRCA1 epitopes and at mRNA level using real-time RT-PCR. BRCA1 mRNA expression was reduced or absent in 79% of the samples, and this finding correlated significantly with loss of BRCA1 protein expression in 83% of breast cancer tissues using one BRCA1 antibody studied (AB-1, against N-terminus epitope). The specificity of this antibody was 91.3%, and its sensitivity was 66.6%. There was no significant correlation between BRCA1 mRNA and protein expression as demonstrated by the remaining three antibodies. Antibody 8F7 had the highest sensitivity of 100%, but its specificity was 30.4% if mRNA levels were considered as the reference standard.

BRCA1 Gene Expression in Breast Cancer in Kuwait: Correlation with Prognostic Parameters

Objectives: This study assessed the BRCA1 gene expression in breast cancer in Kuwait, and compared it with other known prognostic factors for the disease. Materials and Methods: Forty-eight random samples of archival paraffin-embedded breast cancer tissues were studied for BRCA1 gene expression. Immunohistochemical method utilizing antibodies against different epitopes on the BRCA1 protein was used to study BRCA1 protein expression. In addition, for 29 patients, reverse transcription-polymerase chain reaction was used to detect BRCA1 mRNA expression. BRCA1 expression was correlated with age, histological type and grade of breast cancer, estrogen and progesterone receptor status, and C-erbB-2 expression. Results: No demonstrable BRCA1 mRNA and protein expression was found in 79 and 83% of the breast cancer tissues, respectively. A positive relationship was demonstrated between lack of BRCA1 (mRNA and protein) expression and high histological grade, negative estrogen and progesterone receptor status, and overexpression of C-erbB-2 in the breast cancer tissues. Conclusions: The study demonstrated lack of BRCA1 gene expression (mRNA and protein) in the majority of breast cancers in Kuwait and confirmed the inverse relationship between BRCA1 expression and parameters that determine poor prognosis in breast cancer.

Chromogenic in situ hybridization (CISH): a novel alternative in screening archival breast cancer tissue samples for HER-2/neu status.

BackgroundChromogenic in situ hybridization (CISH) is emerging as a practical, cost-effective, and valid alternative to fluorescent in situ hybridization in testing for gene alteration, especially in centers primarily working with immunohistochemistry (IHC).MethodsWe assessed Her-2/neu alteration using CISH on formalin-fixed paraffin-embedded primary invasive ductal carcinoma tumors in which IHC (CB11 antibody) had previously been performed, and we compared the results with IHC. The 160 selected cases were equally stratified randomly into the four IHC categories (scores of 0, 1+, 2+, and 3+). We also compared age at diagnosis and tumor histologic grade with IHC and CISH Her-2/neu.ResultsWe were able to perform and evaluate CISH successfully on all cases. The agreement between 3+ IHC and CISH-amplified cases as well as between all IHC and CISH Her-2/neu negative cases was 100%, and the concordance on all positive cases was 72.50%, with an overall agreement of 86.25%. All the discordant cases had 2+ IHC scores. Although we noted Her-2/neu positivity more in premenopausal women, the age at diagnosis was not significantly associated with IHC or CISH results. Similarly, although the small group of well-differentiated tumors was apparently Her-2/neu negative in both tests, no significant association was noted between any tumor histologic grade and either IHC or CISH results.ConclusionsCISH is easily integrated into routine testing in our laboratory. It is a necessary adjunct in determining the subset of non-amplified IHC-positive invasive tumors that will not benefit from trastuzumab therapy. Those cases with 2+ IHC results will be triaged and subjected to CISH. Her-2/neu testing should be done on all breast cancer cases regardless of age at presentation and tumor histologic grade.

The Characterization of CpG Methylation ofERα andERβ Gene in the Breast Cancer

Purpose: Aberrant methylation of promoter cytosine guanine dinucleotide (CpG) islands is known to be responsible for the alteration and silencing of cancer genes. The data presented here show that most methylations of Estrogen Receptorα (ERα) and ERβ are found at or near the transcriptional factor binding sites in the breast cancer tissues. Methods: Fifty archival breast cancer tissues and twentyfive normal tissues were selected and the status of the methylation and the transcription were investigated by bisulfite genomic sequencing and reverse transcription (RT) PCR. Results: Consequently, the hypermethylation of ERα and ERβ genes was found in 66.0% and 50.0% of 50 breast cancers, respectively. In particular, the methylation sites were frequently located near the CCAAT box (-363 and -375) for the ERα gene, and at or adjacent to binding sites of GATA (-217, -302) and Sp1 (+224, +227, +160) for the ERβ gene. The methylations at or near the binding sites were observed in most of the methylated cancers (ERα 87.9%, and ERβ 84.0%). The methylated cases were negatively correlated with the expression of ERα and ERβ RNA (P<0.01). In particular, tumors with CpG methylation of ERα and ERβ at or near the binding sites did not express mRNA, whereas those CpG methylation outside the sites showed moderate expression. Four tumors with methylated ERα genes at sites unrelated to the binding sites showed higher levels of protein expression than those with methylation at or near the sites (P=0.01). Conclusion: Although the number of samples was relatively small, our results suggest that DNA methylation in ERα and ERβ appears to take significant effect on transcriptional silencing and is most often present in the CpG sites at or near the putative transcriptional factor binding sites. We believe this finding offers a clue to the initiation or spread pattern of CpG methylation in human breast cancer. (Journal of Korean Breast Cancer Society 2004;7:8-16)

Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization.

We evaluated HER2/neu gene amplification by fluorescence in situ hybridization (FISH) in archival paraffin-embedded breast cancer tissues. Tumors from 63 human invasive breast cancers were categorized into two groups depending on whether the paraffin-embedded tissue blocks had been stored for more or less than 12 months duration. These were subjected to routine and modified FISH protocols. As microwave oven formalin fixation of tissues was carried out in the majority of the older archived specimens, the effect of this fixation method was also analyzed. FISH signals were obtained in all 13 archival specimens stored for less than 12 months. However, in 50 specimens stored for more than 12 months duration, the procedure was successful in only 10 specimens (20%), for which the pretreatment procedure had to be individually optimized for each specimen. There was no significant difference in the detection of FISH signals between microwave oven and routinely fixed specimens.