Formalin-fixed Paraffin-embedded Breast Cancer Tissue Research Articles

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

Abstract 3331: Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines

Abstract Background: Breast radiotherapy is currently “one size fits all” regardless of breast cancer subtype (eg. luminal, basal). Emerging clinical data suggests that these distinct subtypes of breast cancer have unique patterns of response to radiation. There is a clinical need to identify radiation response biomarkers in order to provide individualized and optimal radiotherapy. Here we report on gene expression profiles associated with radiation response from: i) a rare clinico-genomic data in women treated with preoperative breast radiation and, ii) data from a pilot study of 16 biologically diverse breast tumor cell lines. Methods: Microarray analysis was done using the Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) and the Affymetrix HU133A2.0 arrays. RNA was harvested from paired pre- and post-radiation formalin-fixed paraffin-embedded breast cancer tissue in 26 patients treated with radiation prior to surgical resection. In addition, RNA was obtained from 16 biologically diverse breast cancer cell lines exposed to radiation treatment (5Gy and 0Gy). The genes with the highest coefficient of variation were selected and a paired sample t-test was used to evaluate the significance of these changes. We compared the top 100 significantly induced human genes with genes identified as differentially expressed in the 16 breast cancer cell lines. Findings of interest were validated using qPCR, IHC and wWestern blotting assays. Results: Fourteen genes had significant adjusted p-values for differential expression in the human data and were differentially expressed among the radiation responsive versus non-responsive cell lines. Differential expression of genes involved in: apoptosis, cell cycle and MAPK signaling pathways were observed in both the luminal human tumors and the largely luminal radiation responsive cell lines. Differential expression of FAS, a critical modulator of programmed cell death, was highly significant. Conclusion: The known function of FAS suggests a biologically plausible role in breast subtype specific radiation response. Induction of FAS is seen in human tissue, and preferentially in luminal breast cancer cell lines. Citation Format: Sharareh Siamakpour-Reihani, Wei Chen, Chen-Ting Lee, Yingchun Zhou, Kouros Owzar, Jen-Tsan Chi, Janet K. Horton. Gene expression profiling after radiation in human breast cancer specimens and breast cancer cell lines. [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 3331. doi:10.1158/1538-7445.AM2015-3331

HER-2 assessment in formalin-fixed paraffin-embedded breast cancer tissue by well-based reverse phase protein array.

BackgroundThe human epidermal growth factor receptor-2 (HER-2) expression level is a critical element for determining the prognosis and management of breast cancer. HER-2 targeted therapy in breast cancer depends on the reliable assessment of HER-2 expression status but current standard methods are lacking a rigorous quantitative assay. To address this challenge, we developed an assessment of HER-2 expression method by well-based reverse phase protein array (RPPA).ResultsWell-based RPPA is based on a robust protein isolation methodology paired with a novel electrochemiluminescence detection system. HER-2 value of well-based RPPA significantly correlated with dot blotting results (R2 = 0.939). By well-based RPPA, we successfully detected HER-2 expression in 76 human breast formalin-fixed paraffin-embedded tissue samples. We observed 93.4% (71/76) concordance between well-based RPPA and current HER-2 immunohistochemical assessment guideline. When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000). Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively.ConclusionsHER-2 value by well-based RPPA was correlated with the current HER-2 status guideline, suggesting that this normalized HER-2 assessment may offer advantages over unnormalized current immunohistochemical assessment methods.

Validation of DNA promoter hypermethylation biomarkers in breast cancer--a short report.

DNA promoter hypermethylation of tumor suppressor genes is known to occur early in cancer development, including breast cancer. To improve early breast cancer detection, we aimed to investigate whether the identification of DNA promoter hypermethylation might be of added value. The methylation status of a panel of 19 candidate genes (AKR1B1, ALX1, ARHGEF7, FZD10, GHSR, GPX7, GREM1, GSTP1, HOXD1, KL, LHX2, MAL, MGMT, NDRG2, RASGRF2, SFRP1, SFRP2, TM6SF1 and TMEFF2) was determined in formalin-fixed paraffin-embedded normal breast and breast cancer tissue samples using gel-based methylation-specific PCR (MSP). The promoters of the AKR1B1, ALX1, GHSR, GREM1, RASGRF2, SFRP2, TM6SF1 and TMEFF2 genes were found to be significantly differentially methylated in normal versus malignant breast tissues. Based on sensitivity, specificity and logistic regression analyses the best performing genes for detecting breast cancer were identified. Through multivariate analyses, we found that AKR1B1 and TM6SF1 could detect breast cancer with an area under the curve (AUC) of 0.986 in a receiver operating characteristic (ROC) assessment. Based on our data, we conclude that AKR1B1 and TM6SF1 may serve as candidate methylation biomarkers for early breast cancer detection. Further studies are underway to evaluate the methylation status of these genes in body fluids, including nipple aspirates and blood.

A Comparison of Different Methods for Rna and Dna Extraction From Formalin – Fixed Paraffin-Embedded Tissues From Different Cancer Samples

RNA and DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. we compared quantity of RNA extracted by two different protocols from 14 recently archived from patients suffered from different cancers distributed among formalin-fixed paraffin-embedded (FFPE) breast cancer tissues ,thyroid cancer tissues and Cervical uterus carcinoma tissues by using Guanidine isothiocyanate (GTC)with phenol-chloroform (protocol-1) and Silica Gel Column(SGC) dependent on spin column purification-based ( protocol- 2), to assess, which technique is the most efficient and reproducible in terms of total yield and purity. The results showed RNA isolated with SGC technique was characterized by higher mean concentration in a range (80-180) µg/ml ,but it give positive results to 12 sample with degradation in comparison with RNA isolated by the (GTC) technique (protocol-1), comparison with total RNA extraction from human blood ( two distinguished bands ) .In this study comparative methods have been performed to analyze the efficiency for extraction and purification of Genomic DNA from six selective FFPE Tissues samples, revealing that the extraction of DNA by using extraction modified method give good result with yield higher mean concentration of DNA in a range (160-260) µg/ml.The lysis of FFPE Tissues was enhanced by increased the concentration of proteinase K to30 mg\ml for 2 hour at 65C, which considered the best time for lysis tissues and heated comparable with results that obtained from lysis tissues by using 20mg\ml for (24 -48 hours) at 55C

Fusion transcript discovery in formalin-fixed paraffin-embedded human breast cancer tissues reveals a link to tumor progression.

The identification of gene fusions promises to play an important role in personalized cancer treatment decisions. Many rare gene fusion events have been identified in fresh frozen solid tumors from common cancers employing next-generation sequencing technology. However the ability to detect transcripts from gene fusions in RNA isolated from formalin-fixed paraffin-embedded (FFPE) tumor tissues, which exist in very large sample repositories for which disease outcome is known, is still limited due to the low complexity of FFPE libraries and the lack of appropriate bioinformatics methods. We sought to develop a bioinformatics method, named gFuse, to detect fusion transcripts in FFPE tumor tissues. An integrated, cohort based strategy has been used in gFuse to examine single-end 50 base pair (bp) reads generated from FFPE RNA-Sequencing (RNA-Seq) datasets employing two breast cancer cohorts of 136 and 76 patients. In total, 118 fusion events were detected transcriptome-wide at base-pair resolution across the 212 samples. We selected 77 candidate fusions based on their biological relevance to cancer and supported 61% of these using TaqMan assays. Direct sequencing of 19 of the fusion sequences identified by TaqMan confirmed them. Three unique fused gene pairs were recurrent across the 212 patients with 6, 3, 2 individuals harboring these fusions respectively. We show here that a high frequency of fusion transcripts detected at the whole transcriptome level correlates with poor outcome (P<0.0005) in human breast cancer patients. This study demonstrates the ability to detect fusion transcripts as biomarkers from archival FFPE tissues, and the potential prognostic value of the fusion transcripts detected.

Abstract P4-04-13: Could genes associated with response or resistance to radiotherapy be identified in breast cancer patients?

Abstract Background. An absolute 10-year reduction of locoregional/distant relapse and 15-year reduction of mortality from breast cancer (BC) after breast conserving surgery by radiotherapy (RT) are about 16% and 3.8%, respectively (EBCTCG, Lancet 2011;378:1707–16). Similarly, after radical mastectomy absolute 5-year reduction of local recurrence by RT is 17% and 15-year BC mortality was reduced by 5.4% (EBCTCG, Lancet 2005,366:2087–2106). Predictive factors of response to RT are less frequently investigated compared to predictive factors for systemic therapy. The ideal model for researching the predictive value of markers is a neo-adjuvant setting. We have analyzed genes associated with different response to RT using neo-adjuvant model in locally-advanced (LA) BC patients (pts). Patients and methods. This retrospective analysis included LA non-inflammatory BC pts treated with preoperative (P)RT alone with a total dose of 45 Gy in 15 fractions every second day to the breast and regional lymph nodes. Radical mastectomy was performed 6 weeks after PRT to all pts and adjuvant systemic therapy was administered as per protocol. Response to PRT was classified as pathological complete response (pCR) if there is no evidence of tumor in the breast after mastectomy, partial response (PR) if regression of tumor was &amp;gt;30%, and no response if regression was &amp;lt;30%. We have analyzed global gene expression profiles by microarray analysis in a set of 14 samples of formalin-fixed paraffin-embedded (FFPE) invasive breast cancer tissues. Results. We have identified a group of 134 LABC pts treated with PRT between 1997 and 2000, which was the routine practice at that time according to the protocol for diagnosis and treatment of BC. Fourteen FFPE tumor samples, 7 from responders to PRT and 7 from non-responders to PRT, were collected for gene expression profile analysis. Unsupervised clustering analysis (Pearson correlation, un-centered metrics) over top 20% most variable genes, revealed clear separation of non-responders and responders. Unsupervised clustering analysis of paired pre- and post-PRT samples indicating small differences in expression profile in response to PRT within non-responders that were clustering together, unlike responders that were separated in the unsupervised analysis showing grater differences in their gene expression. We have found 98 genes significantly differentially expressed [Fals Discovery Rate (FDR) &amp;lt; 0.05] between responders and non-responders to PRT. Molecular functions significantly associated with this gene list were determined using Ingenuity Pathway Analysis and included regulation of cellular growth and proliferation, cell cycle, cell death and survival as top scoring function. Interestingly, canonical pathways for G2/M DNA damage checkpoint regulation and GADD45 genotoxic stress signaling were significantly enriched (FDR&amp;lt;0.05) among differentially expressed gene list between RT resistant and RT sensitive tumors. Conclusion: The strength of these results is that, although obtained on a small number of pts, these tumours have been exposed only to RT without systemic therapy administered either previously or concomitantly with PRT. Further confirmatory research of potentially RT sensitive/resistant genes is warranted. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-04-13.

Abstract P4-19-03: Breast cancer oriented biobank and associated biobank management system: Preliminary analysis on 9043 samples from 274 individuals

Abstract Four years ago, after our Baylor College of Medicine (BCM) Breast and Cancer Centers experience, we decided to initiate a related translational research Project at the Breast Center Buenos Aires (BCBA) in Argentina. This included the development of a breast cancer-oriented Management System for clinical and lab data attached to a biorepository of tumor and normal breast tissue with matched blood specimens. We strictly followed ISBER Best Practices for Biorepositories, and shared the same protocols and strategies with BCM to facilitate international collaborative breast cancer research, being extremely aware of international standards. Objectives: Creation of a breast cancer-oriented blood, breast tissue and tumor biobank for translational research purposes. Creation of a Biobank Management and Tracking System, with a breast cancer database for associated epidemiological, pathological, clinical, and follow-up data of each patient. Methods: From April 2011 to June 2013 we processed blood and tissue samples from BCBA. Blood is collected at several time points during the breast cancer disease process: pre-surgical, pre-systemic treatment, and, if applicable, in the metastatic setting. Blood products are stored as whole blood, plasma, buffy coat, red blood cell pellet, serum and clot at -80°C or in GenPlates®. Fresh tissue and tumor samples were collected during surgical or core biopsy proceedings and stored fresh at -80°C in 1 ml cryovials and as FFPE (formalin-fixed paraffin embedded) tissue. Upon enrollment, participants completed an extensive epidemiological and risk factor questionnaire, which is supplemented by medical record abstraction for relevant pathological and clinical data, and re-contacted once a year for follow-up. Preparation and adaptation processes were compliant with the Population Sciences Biorepository and Smith Breast Center Tumor Bank at BCM: 1) IRB-approved informed consent documents 2) Epidemiological and risk factor questionnaires (Core/Breast module); 3) Blood and tissue collection and processing protocols. A web-based data management and tracking system was specifically designed for the BCBA biobank. Results: To date, we have collected 9043 samples, from 274 individuals, in 293 sample collections. Of 5449 frozen samples, 5054 (92.7%) are blood and 395 (7.3%) are tissue. We also collected 3264 blood samples in GenPlate® wells from 68 patients on 17 plates, and extracted DNA from them into Gentegra® tubes, all stored at room temperature. 230 FFPE breast cancer tissue biopsies from 20 surgical specimens, given by the pathologist after diagnosis, stored in cassettes at room temperature in our lab, were also included in the system starting from January 2013. All of them have been classified by the physician into three categories: Healthy Control 15.3% (n = 42), Benign 55.1% (n = 151) and Cancer 29.6% (n = 81). We collected epidemiological, and cancer data from all of them in our system. Conclusion: It took nearly 4 years from inception to realization for this biobank; however, the potential benefit to translation breast cancer research is large. The overall value of this biobank will depend on the number of individuals/samples accrued, the follow-up attained and data accuracy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-19-03.

Fusion transcript discovery in formalin-fixed paraffin-embedded human breast cancer tissues and its relation to tumor progression.

11018 Background: While several recently discovered gene fusions already play an important role in personalized cancer treatment, many cancer gene fusions remain to be discovered. Next generation sequencing has enabled identification of many rare gene fusion events in fresh or frozen solid tumors. There is a need to detect gene fusions in transcriptomes of formalin-fixed paraffin-embedded (FFPE) tumor tissue, for which there is long-term clinical outcome data. We therefore sought to develop bioinformatics methods to detect fusion transcripts in FFPE tissue and to characterize their association with clinical outcomes. Methods: RNA sequencing libraries were created and sequenced from tumor biopsy tissues (Plos One 2012 7(7): e40092) of two ER+ breast cancer cohorts consisting of 136 and 77 patients, for which clinical outcomes were available. The fusion junctions were nominated by the RNA-seq aligner GSNAP and further filtered to consider discontinuous expression patterns at exon/intron levels. Results: A total of 108 candidate fusion transcripts were detected and RT-PCR assays confirmed 89% of the top ranking fusion transcript candidates. The majority (82%) of identified fusion gene partners are listed in the COSMIC database of known cancer sequence variations. Of note, several patients expressed multiple fusion transcripts that are significantly associated with tumor progression (P&lt;0.001), including genes associated with cell proliferation and cellular metabolism. Furthermore, these patients also harbored inter-chromosomal gene fusions. It is noteworthy that several gene fusions were present in multiple patients. In one of these recurrent fusions the estrogen receptor gene acts as the fusion pair donor. Conclusions: Novel bioinformatics approaches developed here demonstrate the ability to detect fusion transcripts as biomarkers from archival FFPE tissues that associate with breast cancer progression. Some gene fusions were common in multiple patients and deserved further study.

Bond oracle HER2 IHC assay for identifying low to intermediate HER2-expressing breast cancer.

e11509 Background: HER2 targeted therapies are effective treatment for breast tumors that overexpress the HER2 protein to a high level (IHC score of 3+) or have amplification of the HER2 gene (HER2/CEP17 &gt;2). However, the majority of breast tumors exhibit low to intermediate expression of HER2 (IHC 1+ and 2+), for which there are limited treatment options. A new cancer vaccine candidate, E75 or nelipepimut-S, offers prevention of reoccurrence in these patients by generating a specific immune response to a HER2 peptide. To date, no companion diagnostic test is validated to differentiate HER2 at these levels of expression. The BOND Oracle HER2 IHC assay (Leica Biosystems) is being validated to reliably identify IHC HER2 1+ and 2+ cases by correlating to independent analytical measures of HER2 expression. Methods: Quantitative PCR was used to determine HER2 DNA copy number and mRNA expression in eight breast cancer cell lines spanning HER2 IHC scores of 0 to 3+ and 45 formalin fixed, paraffin embedded (FFPE) invasive breast tumor samples. HER2 receptor load was determined for the eight cell lines by flow cytometry. Data was correlated to HER2 expression levels as determined by manual interpretation using the BOND Oracle HER2 IHC assay. Results: Previous testing by Leica Biosystems (Table) demonstrates the relationship between HER2 receptor load, HER2 copy number, and HER2 IHC status on four assay control breast cancer cell lines. Preliminary results also show that HER2 mRNA expression correlates with IHC staining with the BOND Oracle HER2 IHC Assay in FFPE breast cancer tissue samples. Conclusions: Improving the discrimination of HER2 protein expression (IHC 1+, 2+) using the Bond Oracle HER2 IHC assay in breast cancer tumors will identify patients for new treatments in development such as E75. [Table: see text]

Abstract P1-07-19: Mass Spectrometry Based Quantitative Analysis of the HER Family receptors in FFPE Breast Cancer Tissue

Abstract The human EGF receptor family (HER's) consists of two clinically validated drug targets (EGFR and HER2), a third (HER3) currently under investigation for its possible role in the acquisition of multidrug resistance and a fourth (HER4), the role of which is still matter of debate. Drugs inhibiting EGFR or HER2 show significant antitumor activity in the clinic, however, acquisition of resistance is a hallmark of these and most targeted therapies. In the case of EGFR and HER2, one of the emerging resistance mechanisms is the co-expression of HER3. Indeed, recent reports show that inhibition of the PI3K pathway leads to upregulation of HER3, and subsequent resistance. Clinical analysis of protein levels in formalin fixed paraffin embedded (FFPE) tissues is limited to immunohistochemistry (IHC), which is semi-quantitative and requires significant amounts of tissue. Moreover, the vast majority of research groups consider specific HER3 staining by IHC particularly challenging. However, accurate measurement of these targets is critical both for properly defining treatment groups and predicting patterns of resistance. In order to address these issues, we used trypsin digestion mapping and stable isotope labeled peptides to develop a panel of quantitative mass spectrometric (MS) assays to measure the levels of EGFR, HER2, HER3 and other clinically relevant targets in FFPE breast cancer tissue. These quantitative MS assays were then multiplexed to analyze 1μg of tumor protein. In this study, we multiplexed HER family analysis on 31 HER2 positive breast cancers. Tumor tissue was microdissected from FFPE sections, and subjected to quantitative MS analysis of EGFR, HER2, HER3 as well as IGF1R and cMET. Quantitation of HER2 showed a broad range of HER2 expression in these tissues. The highest expresser measured 26 fmol/ug tumor tissue, representing amplification and massive protein over expression. In contrast, five tissues showed low levels of HER2 expression, below 1 fmol/ug, similar to HER2 non-amplified cell lines. This suggests that MS quantitation can identify patients with low expression of HER2 who are unlikely to respond to trastuzumab therapy. As a matter of fact, 3 of these 5 low expressing patients had outcome data and showed no response to trastuzumab treatment. In 28 of 31 patient tissue samples, HER3 showed low levels of expression (100–300 amol/ug tumor tissue) similar to HER3 expression in cell lines, and comparable to low expressing EGFR and HER2 cell lines. The remaining 3 patients had no detectable HER3. This study demonstrates the feasibility of measuring HER3 in multiplex in FFPE breast cancer tissue. Based on the low but widespread expression of HER3 in this cohort, it may be most useful to assess HER3 expression after initial treatment as a marker of potential resistance to targeted therapies. Taken together, these data demonstrate that a sensitive and quantitative assay to measure oncoproteins in FFPE clinical samples may help stratify patients with variable expression of these targets. Our quantitation of oncogene expression from clinical samples uses a small amount of tissue, is clinically applicable and alleviates the problem of scoring either positive or negative for the expression of a given protein. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-19.

Abstract P2-05-06: Quantitative measurement of HER2 expression in breast cancers: comparison with “real world” HER2 testing in a multi-center Collaborative Biomarker Study (CBS) and correlation with clinicopathological features

Abstract Background: Accurate determination of HER2 status is critical in determining appropriate therapy for breast cancer patients. The HERmark® assay is a novel method to quantitatively measure HER2 total protein expression (H2T) in breast cancer. In this study, we compared HERmark H2T with central laboratory HER2 retesting and local (site reported) HER2 testing of formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. The quantitative total HER2 measurements (H2T) by HERmark and results of local HER2 tests were correlated with tumor pathohistological characteristics and overall survival of breast cancer patients. Methods: 232 FFPE breast cancer tissues were provided by 11 CBS study sites for HER2 testing by the HERmark assay and central laboratory IHC re-testing performed in blinded fashion. Local HER2 immunohistochemistry and/or fluorescence in situ hybridization (FISH) results and valid HERmark H2T and central HER2 IHC results were obtained in 192 cases for analysis. Results: H2T showed a significant correlation with central HER2 IHC staining intensity (P &amp;lt; 0.0001). The concordance rates of positive and negative HERmark status (excluding equivocal) with those of local HER2 status determined by the CBS sites, and with those of central HER2 IHC status were 84% (Kappa = 0.68) and 96% (Kappa = 0.91), respectively. Higher H2T levels significantly correlated with higher tumor grade (p = 0.007) and negative ER/PR status (p = 0.002). Twenty-six (14%) cases showed discordant (conversion of negative and positive) results between local HER2 status and HERmark status. Of the discordant cases, HERmark significantly agreed with H-score of central HER2 IHC retesting (p = 0.014), as compared with local HER2 status. The concordant negative group (local HER2 negative/H2T low) demonstrated better overall survival (OS) (HR = 0.198, p = 0.0001), compared to that of concordant positive group (local HER2 positive/H2T high). The concordant negative group also showed better OS than that of discordant local HER2 negative/H2T high group (HR = 0.065, p = 0.0003), but showed no significant difference in OS as compared to that of discordant local HER2 positive/H2T low group (HR = 1.774, p = 0.499).). In 24 cases (13%) considered to be “triple negative” by local HER2, ER and PR testing, HERmark re-classified 4 cases (17%) as HER2 positive. Conclusions: H2T by HERmark yields a continuum of quantitative HER2 protein measurements that shows an excellent correlation with central HER2 IHC retesting and confirms the known correlations between HER2 expression with tumor grade and ER/PR status. OS results of concordant HER2 positive or negative groups (between local HER2 testing and HERmark H2T) confirmed that HER2 positive patients (excluding adjuvant trastuzumab therapy) have worse OS than patients with HER2 negative disease. However, in the HERmark and local HER2 discordant groups, OS appeared to track better with H2T by HERmark and not with the local HER2 status. Novel quantitative HER2 measurements may identify patients with false (+) and (−) HER2 status by local HER2 testing and may provide added clinical value to routine “real world” HER2 testing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-06.

Detection of Long Non-Coding RNA in Archival Tissue: Correlation with Polycomb Protein Expression in Primary and Metastatic Breast Carcinoma

A major function of long non-coding RNAs (lncRNAs) is regulating gene expression through changes in chromatin state. Experimental evidence suggests that in cancer, they can influence Polycomb Repressive Complexes (PRC) to retarget to an occupancy pattern resembling that of the embryonic state. We have previously demonstrated that the expression level of lncRNA in the HOX locus, including HOTAIR, is a predictor of breast cancer metastasis. In this current project, RNA in situ hybridization of probes to three different lncRNAs (HOTAIR, ncHoxA1, and ncHoxD4), as well a immunohistochemical staining of EZH2, is undertaken in formalin-fixed paraffin-embedded breast cancer tissues in a high throughput tissue microarray format to correlate expression with clinicopathologic features. Though overall EZH2 and HOTAIR expression levels were highly correlated, the subset of cases with strong HOTAIR expression correlated with ER and PR positivity, while the subset of cases with strong EZH2 expression correlated with an increased proliferation rate, ER and PR negativity, HER2 underexpression, and triple negativity. Co-expression of HOTAIR and EZH2 trended with a worse outcome. In matched primary and metastatic cancers, both HOTAIR and EZH2 had increased expression in the metastatic carcinomas. This is the first study to show that RNA in situ hybridization of formalin fixed paraffin-embedded clinical material can be used to measure levels of long non-coding RNAs. This approach offers a method to make observations on lncRNAs that may influence the cancer epigenome in a tissue-based technique.

Let-7 MicroRNAs Induce Tamoxifen Sensitivity by Downregulation of Estrogen Receptor α Signaling in Breast Cancer

MicroRNAs (miRNAs) play an important regulatory role in breast tumorigenesis. Previously, we found that let-7 miRNAs were downregulated significantly in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. In this study, we further found that endogenous levels of let-7b and let-7i miRNAs are inversely correlated with levels of estrogen receptor (ER)-a36, a new variant of ER-α66, in the FFPE tissue set. Bioinformatic analysis suggested that ER-α36 may be another target of let-7 miRNAs. To test this hypothesis, cotransfection of let-7 mimics or inhibitors together with full-length or a fragment of ER-α36 3'UTR luciferase construct was performed, and we found that let-7b and let-7i mimics suppressed the activity of reporter gene significantly, which was enhanced remarkably by let-7b and let-7i inhibitors. Both mRNA and protein expression of ER-α36 were inhibited by let-7 mimics and enhanced by let-7 inhibitors. Furthermore, ER-α36 mediated nongenomic MAPK and Akt pathways were weakened by let-7b and let-7i mimics in triple negative breast cancer cell line MDA-MB-231. The reverse correlation between let-7 miRNAs and ER-α36 also exists in Tamoxifen (Tam)-resistant MCF7 cell line. Transfection of let-7 mimics to Tam-resistant MCF7 cells downregulated ER-α36 expression and enhanced the sensitivity of MCF7 cells to Tam in estrogen-free medium, which could be restored by overexpression of ER-α36 constructs without 3'UTR. Our results suggested a novel regulatory mechanism of let-7 miRNAs on ER-α36 mediated nongenomic estrogen signal pathways and Tam resistance.

Abstract P3-09-05: Let-7 microRNAs Regulate a Novel Variant of Estrogen Receptor Alpha, ER-≥36, Nongenomic Estrogen Signal Pathway, and Tamoxifen Resistance in Breast Cancer

Abstract Background: MicroRNAs (miRNAs) have important regulatory functions in breast cancer tumorigenesis. We previously found that let-7 miRNAs were significantly downregulated in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. Material and Methods: QRT-PCR was used to check let-7 family miRNAs in FFPE tissues and breast cancer cell lines as well as expression of estrogen receptor (ER)-≥36, a variant of ER-≥66, after let-7 miRNA transfecton. Immunoblot analysis was employed to check protein expression in FFPE tissue and breast cancer cell lines. Luciferase reporter assay was used to detect direct regulation of let-7 miRNA on ER-α expression. MTT assay was applied for cell proliferation after transfection of let-7 miRNAs. Results: We found that there was an inverse correlation between the expression of ER-≥36 and several members of let-7 family miRNAs in the FFPE tissue set. Let-7 miRNA sequences match sequence in the 3’ untranslated region (3’ UTR) of ER-≥36, indicating ER-≥36 may be a target of let-7. Cotransfection of let-7 mimics with ER-≥36 3’ UTR luciferase construct decreased the activity of reporter gene. Conversely, let-7 inhibitors enhanced the reporter gene activity. Transfection of let-7 mimics inhibited both the mRNA and protein levels of ER-≥36 and further inhibited the non-genomic estrogen pathway mediated by ER-≥36 in MDA-MB-231 cells. On the contrary, transfection of let-7 inhibitors enhanced the ER-≥36 expression at both mRNA and protein levels in 184A1 cells. The high expression of ER-≥36 in tamoxifen resistant MCF7 cells can be inhibited by transfection of let-7 mimics and sensitivity toward tamoxifen is enhanced. Conclusion: Let-7 miRNAs regulate the expression of ER-≥36, which is involved in non-genomic estrogen pathway and tamoxifen resistance. Let-7 could be therapeutic target for breast cancer treatment. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-09-05.

Quantitation of p95HER2 in Paraffin Sections by Using a p95-Specific Antibody and Correlation with Outcome in a Cohort of Trastuzumab-Treated Breast Cancer Patients

p95HER2 is an NH(2)-terminally truncated form of HER2 that lacks the trastuzumab binding site and is therefore thought to confer resistance to trastuzumab treatment. In this report, we introduce a new antibody that has enabled the first direct quantitative measurement of p95HER2 in formalin-fixed paraffin-embedded (FFPE) breast cancer tissues. We sought to show that quantitative p95HER2 levels would correlate with outcome in trastuzumab-treated HER2-positive metastatic breast cancer. The novel p95HER2 antibody used here was characterized for sensitivity, specificity, and selectivity over full-length HER2. Quantitative p95HER2 levels were measured in 93 metastatic breast tumors using a VeraTag FFPE assay to determine the correlation of p95HER2 levels with outcomes. Within a cohort of trastuzumab-treated metastatic breast cancer patients, high levels of p95HER2 were found to correlate with shorter progression-free survival [hazard ratio (HR), 1.9; P = 0.017] and overall survival (HR, 2.2; P = 0.012) in patients with tumors selected to be HER2 positive by the VeraTag HER2 assay. For those with tumors found to be fluorescence in situ hybridization positive, elevated p95HER2 correlated similarly with shorter progression-free survival (HR, 1.8; P = 0.022) and overall survival (HR, 2.2; P = 0.009). We have successfully generated an antibody that can specifically detect p95HER2, and developed an assay to quantify expression in FFPE tumor specimens. Using this novel assay, we have identified a group of HER2-positive patients expressing p95HER2 that have a worse outcome while on trastuzumab. As p95HER2 retains sensitivity to kinase inhibitors, measurement of p95HER2 in breast tumor sections may be useful in guiding treatment for patients with HER2-positive breast cancer.

In Situ Identification of Putative Cancer Stem Cells by Multiplexing ALDH1, CD44, and Cytokeratin Identifies Breast Cancer Patients with Poor Prognosis

A subset of cells, tentatively called cancer stem cells (CSCs), in breast cancer have been associated with tumor initiation, drug resistance, and tumor persistence or aggressiveness. They are characterized by CD44 positivity, CD24 negativity, and/or ALDH1 positivity in flow cytometric studies. We hypothesized that the frequency or density of these cells may be associated with more aggressive tumor behavior. We borrowed these multiplexed, flow-based methods to develop an in situ method to define CSCs in formalin-fixed paraffin-embedded breast cancer tissue, with the goal of assessing the prognostic value of the presence of CSCs in breast cancer. Using a retrospective collection of 321 node-negative and 318 node-positive patients with a mean follow-up time of 12.6 years, we assessed TMAs using the AQUA method for quantitative immunofluorescence. Using a multiplexed assay for ALDH1, CD44, and cytokeratin to measure the coexpression of these proteins, putative CSCs appear in variable sized clusters and in 27 cases (of 490), which showed significantly worse outcome (log rank P = 0.0003). Multivariate analysis showed that this marker combination is independent of tumor size, histological grade, nodal status, ER-, PR,- and HER2-status. In this cohort, ALDH1 expression alone does not significantly predict outcome. We conclude that the multiplexed method of in situ identification of putative CSCs identifies high risk patients in breast cancer.

Abstract 2999: Assessment of microRNA expression in two-dimensional histopathological fields of breast cancer

Abstract Breast cancer is the second most common type of cancer among women with an expected ∼192,000 newly diagnosed cases in the United States this year alone. Early detection methods are becoming increasingly important for guiding treatment options and decisions. Currently, standard molecular diagnostic tests of breast cancer characterize patient cases by estrogen receptor (ER), progesterone receptor (PR) and Her2neu status. The recently discovered biomolecule, microRNA (miRNA), is being assessed as a novel diagnostic tool; however, since miRNA is a regulatory molecule it is likely that expression is cell-type and location specific, thus studies that don't specifically study malignant and normal cells may mask miRNA patterns within tissue. The goal of our study was to evaluate miRNA expression within sub-regions of formalin-fixed paraffin-embedded breast cancer tissue. Utilizing a combination of laser-based and manual microdissection techniques, we evaluated the expression of several breast cancer associated miRNA via TaqMan chemistry. We dissected selected areas of tumor and normal cells, as well as a grid-like pattern over entire tissue sections to separately evaluate each histopathological region. The data suggest there is heterogeneity of miRNA expression within the tumor and the surrounding normal tissue. In order study this phenomenon in more detail, we are now adapting a new two-dimensional amplification (2D-PCR) device to measure miRNA levels across tissue specimens with fine resolution. This approach to examining miRNA profiles in tissue sections may lead to novel findings with diagnostic or prognostic implications for breast cancer patients. 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 2999.

Toll‐like receptor 9 expression in breast and ovarian cancer is associated with poorly differentiated tumors

Toll-like receptor 9 (TLR9) activates the innate immune response when exposed to non-methylated CpG-DNA. TLR9 was recently shown to be expressed by cancer cells which have been previously characterized by global hypomethylation. We set out to examine the expression and molecular activity of TLR9 in breast and ovarian cancer cells. Firstly, we confirmed higher levels of hypomethylated DNA in the serum of patients with metastatic breast cancer (n = 18) versus age-matched tumor-free women (n = 18). In breast cancer cell lines and tissues, TLR9 mRNA expression was associated with estrogen-receptor (ER) status (n = 124, P = 0.005). Expression also correlated with increasing tumor grade in both breast (P = 0.03) and ovarian cancer specimens (n = 138, P = 0.04). Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) breast cancer tissues revealed higher TLR9 protein expression in hormone-receptor (HR)-negative specimens (n = 116, P < 0.001). Using an in vitro scratch assay, we observed that cell lines transfected to overexpress TLR9 demonstrated increased cellular migration when stimulated with CpG-DNA. When assessing the molecular activity of TLR9 in breast cancer, we found a strong positive correlation of nuclear factor-kappa B (NF-kappaB) activity with TLR9 mRNA expression (correlation coefficient r = 0.7, P < 0.001). Finally, immunofluorescence analysis of BT-20 and Hs578T breast cancer cell lines showed partial colocalizations of CpG-DNA with TLR9, which diminished when the cells were exposed to methylated CpG-DNA (mCpG-DNA) or control GpC-DNA. In summary we demonstrate that TLR9 expression is associated with poor differentiation in breast and ovarian cancer specimens, and that TLR9 overexpression and stimulation with hypomethylated DNA augments the migratory capacity of cancer cell lines.