Formalin-fixed Paraffin-embedded Breast Cancer Research Articles

MicroRNA signatures in hereditary breast cancer

This study aims to identify signatures of miR associated with hereditary, BRCA1 or BRCA2 mutation positive breast cancer (BC), and non-hereditary BC, either sporadic (SBC) or non-informative (BRCAX). Moreover, we search for signatures associated with tumor stage, immunohistochemistry and tumor molecular profile. Twenty formalin fixed paraffin embedded (FFPE) BCs, BRCA1, BRCA2, BRCAX and SBC, five per group were studied. Affymetrix platform miRNA v.3.0 was used to perform miR expression analysis. ER, PR, HER2 and Ki67 protein expression was analyzed by immunohistochemistry. BRCA1, BRCA2 and RASSF1 methylation analysis, AURKA copy number variations, and BRCA1 and BRCA2 deletions, were studied by MLPA. We validated eight of the miR selected by the arrays in 77 BCs by qRT-PCR. The miR profiles associated with tumor features were studied applying the Sparse Partial Least Squares Discriminant Analysis. MiR discrimination capability to distinguish hereditary and non-hereditary BC was analyzed by the discriminant function. With 15 out of 1,733 hsa-miRs, it was possible to differentiate the four groups. BRCA1, BRCA2 and SBC were associated with clusters of hyper-expressed miRs, and BRCAX with hypo-expressed miRs. Hsa-miR-4417 and hsa-miR-423-3p expressions (included among the eight validated miRs) differentiated 70.1 % of hereditary and non-hereditary BCs. We found miR profiles associated with tumor features like node involvement, histological grade, ER, PR and HER2 expression. Regarding molecular parameters, we only found a weak association of miRs in BC harboring losses in AURKA. We conclude that array miR expression profiles can differentiate the four study groups using FFPE BC. However, miRs expression estimated by qRT-PCR differentiates only hereditary and non-inherited BCs. The miR expression array is a simple and rapid approach that could be useful to facilitate the identification of those SBC carrying genetic or epigenetic changes in BRCA genes responsible of BRCA-like phenotype. These patients could benefit from the treatment with PARP inhibitors.

Methods for evaluating HER2 status in breast cancer: comparison of IHC, FISH, and real-time PCR analysis of formalin-fixed paraffin-embedded tissue

Methods for evaluating HER2 status in breast cancer: comparison of IHC, FISH, and real-time PCR analysis of formalin-fixed paraffin-embedded tissue Hans Olsson,1,2 Agneta Jansson,3 Birgitta Holmlund,3 Cecilia Gunnarsson2,4 1Molecular and Immunological Pathology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden; 2Department of Clinical Pathology and Clinical Genetics, Östergötland County Council, Linköping, Sweden; 3Division of Oncology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden; 4Division of Genetics, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden Abstract: The human epidermal growth factor receptor 2 gene (HER2) is amplified in approximately 15%–20% of all breast cancers. This results in overexpression of the HER2 protein, which is associated with worse clinical outcomes in breast cancer patients. Several studies have shown that trastuzumab, a monoclonal antibody that interferes with the HER2/neu receptor, can improve overall survival in patients with HER2-positive breast cancer. Immunohistochemistry (IHC), combined with different methods for in situ hybridization, is currently used for routine assessment of HER2 status. The aim of the present study was to determine whether real-time polymerase chain reaction (PCR) can serve as a supplementary method for evaluation of HER2 status in primary breast cancer. For this purpose, 145 formalin-fixed paraffin-embedded primary breast cancer samples were tested by real-time PCR amplification of HER2, using amyloid precursor protein as a reference. The results were compared with HER2 status determined by fluorescence in situ hybridization (FISH) and IHC. The specificity, sensitivity, and reproducibility of real-time PCR were evaluated, and a comparison of formalin-fixed and fresh-frozen samples was performed. This showed concordance of 93% between real-time PCR and FISH, and 86% between real-time PCR and IHC. Therefore, we suggest that real-time PCR can be a useful supplementary method for assessment of HER2 status. Keywords: 17q, breast cancer, HER2, real-time PCR

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<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 >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]

Application of Selected Reaction Monitoring for Multiplex Quantification of Clinically Validated Biomarkers in Formalin-Fixed, Paraffin-Embedded Tumor Tissue

One of the critical gaps in the clinical diagnostic space is the lack of quantitative proteomic methods for use on formalin-fixed, paraffin-embedded (FFPE) tissue. Herein, we describe the development of a quantitative, multiplexed, mass spectrometry-based selected reaction monitoring (SRM) assay for four therapeutically important targets: epidermal growth factor receptor, human EGF receptor (HER)-2, HER3, and insulin-like growth factor-1 receptor. These assays were developed using the Liquid Tissue-SRM technology platform, in which FFPE tumor tissues were microdissected, completely solubilized, and then subjected to multiplexed quantitation by SRM mass spectrometry. The assays were preclinically validated by comparing Liquid Tissue-SRM quantitation of FFPE cell lines with enzyme-linked immunosorbent assay/electrochemiluminescence quantitation of fresh cells (R(2) > 0.95). Clinical performance was assessed on two cohorts of breast cancer tissue: one cohort of 10 samples with a wide range of HER2 expression and a second cohort of 19 HER2 IHC 3+ tissues. These clinical data demonstrate the feasibility of quantitative, multiplexed clinical analysis of proteomic markers in FFPE tissue. Our findings represent a significant advancement in cancer tissue analysis because multiplexed, quantitative analysis of protein targets in FFPE tumor tissue can be tailored to specific oncological indications to provide the following: i) complementary support for anatomical pathological diagnoses, ii) patient stratification to optimize treatment outcomes and identify drug resistance, and iii) support for the clinical development of novel therapies.

Abstract 3462: High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors.

Abstract The class I phosphatidylinositol 3’ kinases (PI3K) play a major role in proliferation and survival in a wide variety of human cancers, and activation of the PI3K pathway is thought to be an important driver in estrogen receptor positive (ER+) breast cancer. A key factor in successful development of drugs targeting this pathway will be development in appropriate molecular subsets. Important questions relevant to PI3K inhibitor development in ER+ breast cancers are whether these inhibitors will work equally well in luminal A compared to luminal B tumors, and whether gene expression signatures of pathway activation may have additional utility in patient stratification beyond PIK3CA mutation status alone. The goal of this study was to develop a methodology for high throughput profiling of ER+ breast cancers, in order to enable molecular subtyping of patients enrolled in clinical studies. To accomplish this, we developed an analysis platform to measure the relative expression of 90 breast cancer and PI3K pathway specific genes in formalin-fixed paraffin-embedded (FFPE) tissue. The content for this panel consists of genes known to be important for epithelial-mesenchymal biology, proliferation rate, and transcriptional output of the PI3K pathway. The 96 assay panel (including 6 housekeeping genes) was developed on the Fluidigm Biomark microfluidics platform and was extensively validated using well-characterized breast cancer cell lines and FFPE breast cancer samples of known subtypes based on immunohistochemistry for HER2, ER, and PR. All assays showed high levels of inter-and intra-chip reproducibility and were sensitive on standard curves down to 3ng RNA input. Using this method we were able to separate breast cancers into distinct molecular subtypes, as well as identify more proliferative luminal B type tumors. In addition, PIK3CA mutation status, a potential biomarker, was determined using a highly specific and sensitive qRT-PCR mutation assay, in order to allow comparison with the PI3K pathway activation signature. We extended these analyses to a small cohort of patient samples consisting of matched primary and metastatic tumor tissues, and report here the correlation of primary and matched metastatic ER+ breast cancer FFPE tumor samples at both the gene expression and mutational levels. We found that the majority of matched pairs were concordant for both mutation status and gene expression, though a subset did show differences. Future studies will examine the prognostic significance and clinical relevance of this gene signature. Citation Format: Erica B. Schleifman, Rupal M. Desai, Jill Spoerke, Cheryl Victoria Wong, Ilma Abbas, Carol O'Brien, Garret Hampton, Timothy Wilson, Hartmut Koeppen, Rajesh Patel, Teiko Sumiyoshi, Ling Fu, Rachel Tam, Rajiv Raja, Mark Lackner. High-throughput qRT-PCR expression profiling of estrogen receptor positive breast tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3462. doi:10.1158/1538-7445.AM2013-3462

The Use of Sensitive Chemical Antibodies for Diagnosis: Detection of Low Levels of Epcam in Breast Cancer

EpCAM is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues. Here we report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded primary breast cancers. This new aptamer, together with our previously described aptamer, showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody (323/A3) showed limited or no reactivity. Our results demonstrated a more robust detection of EpCAM using RNA aptamers over antibodies in clinical samples with chromogenic staining. This shows the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.

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 < 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.

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin-fixed, paraffin-embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.

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.

204P - Evaluation of PTEN and PIK3CA Status in Breast Cancer for Patient Selection

ABSTRACT PTEN and PIK3CA status are potential predictors of response to PI3K-pathway inhibitors. Therefore, we searched the most reliable platform to assess both PTEN expression and PIK3CA mutations in primary and metastatic breast cancer (BC). We cross-validated the assays between different institutions. Studies were performed using four different sample sets of formalin-fixed paraffin-embedded (FFPE) breast cancers from VHUH/VHIO and from Columbia University. PTEN alterations were genotyped in 14 TNBCs by OncoscanTM platform (Affymetrix) and were correlated to immunohistochemistry (IHC). PTEN loss of heterozygosity (LOH, n = 4) with or without overlapping PTEN mutation (n = 5) was concordant to PTEN protein loss (H-Score Disclosure Y. Wang: Yuker Wang is employee of Affymetrix Inc. All other authors have declared no conflicts of interest.

P3.06 Comparison of High Resolution Melting and Restriction Fragment Length Polymorphism to Genotype Snp in Formalin Fixed Paraffin-Embedded Breast Cancer Tissue

ABSTRACT SNPs of NQO1, p53, and MDM2 have been correlated with pathological response or overall survival of breast cancer patients treated with anthracycline based chemotherapy. Therefore, the ability to detect these SNPs may be useful for treatment decision. In this study, we genotyped FFPE breast cancer samples to detect those SNP. Objective The aim of this study was to know the accuracy of HRM (High Resolution Melting) method to genotype SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G's in comparison to RFLP (Restriction Fragment Length Polymorphism) from archived formalin fixed paraffin embedded (FFPE) breast cancer blocks. Materials and Methods FFPE blocks were collected from anatomic pathology laboratory. DNA were isolated, and PCR amplified followed by either RFLP or HRM methods. Restriction enzymes HinfI, BstUI, and MspA1I were used in RFLP to genotype NQO1, p53, and MDM2 SNPs, respectively. The primer design which been used was the same between HRM and RFLP. The results of HRM were analyzed using melting, normalization, and differentiation plot curves. Sequencing technique has been used to resolve discrepancy between RFLP and HRM results. Results We screened SNPs on breast cancer FFPE samples using HRM and RFLP. The rates of genotyping agreement between both methods are 69, 23% (9/13 samples), 80% (8/10 samples), and 100% (7/7 samples) for NQO1, p53, and MDM2 SNPs, respectively. Conclusion HRM is a faster method than RFLP to genotype important breast cancer SNPs of NQO1 P187S, p53 P72R, and MDM2 T309G. Nevertheless, RFLP is more reliable method than HRM when the used primers sets are not optimized for HRM.

Abstract 683: Evaluation of PTEN and PIK3CA status in breast cancer for patient selection: Cross-validation between institutions

Abstract PTEN and PIK3CA status are potential predictors of response to PI3K-pathway inhibitors. Therefore, we searched the most reliable platform to assess both PTEN expression and PIK3CA mutations in primary and metastatic breast cancer (BC). Cross-validation across platforms at SU2C sites was performed using three different sample sets: 18 formalin-fixed paraffin-embedded (FFPE) primary triple negative breast cancer (pTNBC) samples from VHUH/VHIO, 12 FFPE pTNBC samples from Columbia University and 21 FFPE all-comers BC from VHUH/VHIO. Moreover, we evaluated the transitions occurring between early and disseminated disease in 14 paired FFPE samples from VHUH/VHIO. PTEN alterations were genotyped in 18 triple pTNBCs by OncoscanTM platform (Affymetrix) and were correlated to immunohistochemistry (IHC, 138G6 CST antibody). PTEN loss of heterozygosity (LOH, n=4) or frameshift mutation (n=1) was concordant to PTEN protein loss (H-Score<50) by IHC in 4 samples; with the discordant LOH having an H-Score of 60. In the other two non-concordant samples (total of 3/18, 17% discordance), presence of non-tumor DNA, as assessed by genomic heterogeneity, impeded an accurate DNA copy number variation determination by OncoscanTM. PTEN protein loss by IHC was also cross-validated between two institutions (Columbia and VHIO). In a cohort of 12 pTNBCs containing eight PTEN low samples, only two samples were discordant (2/12, 17% discordance). Paired primary versus metastatic tumors were identified to transit either way, in the PTEN assessment by IHC (2/8 paired samples, 25% transition). PTEN IHC failed in most (3/5) metastatic bone samples due to Bouin's fixative required for handling of this tissue. PIK3CA mutations by OncoscanTM were concordant to MassARRAY (Sequenom) in 4 out of 5 mutated samples within the panel of 18 pTNBCs. The discrepancy (1/18, 6%) was most likely due to differences in the sensitivity of the two assays. In another cohort of 21 BC samples, PIK3CA mutational status was cross-validated by MassARRAY at two institutions (MDACC and VHIO). Using identical, customized panels we found that only two samples were discordant because of mutant allele frequency close to the sensitivity of the assay (10%). Manual review of the individual assays increased the concordance to 100%. Among 14 paired primary vs metastatic breast cancers we detected two transitions from wild type (WT) to H1047R mutation and one transition from E545K to WT (3/14, 21% transition), underscoring the need to determine PIK3CA status in metastatic lesions. The divergence evaluating both PTEN and PIK3CA status between institutions was due to cut-off and sensitivity of the assays respectively. For patient pre-screening purposes, MassARRAY and IHC can be performed at each SU2C site both in primary and metastatic breast cancer lesions. OncoscanTM is a valid, centralized platform for evaluation of PTEN and PIK3CA genomic alterations in BC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 683. doi:1538-7445.AM2012-683

Clinical relevance of Ki67 gene expression analysis using formalin-fixed paraffin-embedded breast cancer specimens

Ki67 is a protein associated with cell cycle activity and shows a good correlation with the growth fraction, which has been proposed as a prognostic or predictive marker in breast cancer. In this study, we aimed to analyze the expression levels of Ki67 (MKI67) messenger RNA (mRNA) derived from formalin-fixed paraffin-embedded (FFPE) tissues for comparison with the immunohistochemical Ki67 labeling index, and investigate the correlation coefficients with clinical outcomes. We analyzed the data of Ki67 mRNA from FFPE and matched fresh-frozen (FF) tissues based on a real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assay system in 203 cases of primary invasive breast cancer. The correlation between Ki67 mRNA expression of either FFPE or FF specimens and Ki67 labeling index was positive, as was the correlation between the FFPE and FF results (P<0.0001). Ki67 mRNA expression of FFPE specimens was significantly associated with clinicopathological characteristics: tumor size, lymph node status, nuclear grade, hormone receptors, human epidermal growth factor receptor2 (Her2) status, and tumor subtype. In prognostic results, Ki67 gene expression in the FFPE specimens revealed almost similar patterns of significance in Kaplan-Meier curves and univariate and multivariate relapse-free survival results as the Ki67 labeling index. Gene expression analysis of Ki67 of FFPE specimens could be successfully performed using RT-qPCR, closely resembling the significant clinical characteristics of Ki67 labeling index. These results confirm that Ki67 gene expression of FFPE specimens has potential for evaluation of cell cycle activity of breast cancer specimens.

Sec62 Bridges the Gap from 3q Amplification to Molecular Cell Biology in Non–Small Cell Lung Cancer

The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

P1-07-12: Assessment of Real World HER2 Status by Immunohistochemistry (IHC) and Fluorescence In Situ Hybridization (FISH) in Breast Cancers: Comparison with HERmark®, a Validated Quantitative Measure of HER2 Protein Expression.

Abstract Background: Accurate assessment of the HER2 status is critical in determining appropriate therapy for patients with invasive breast cancer. ASCO/CAP HER2 testing guidelines caution that up to 20% of routine HER2 testing by IHC/FISH may be unreliable (Wolff et al. JCO 2007;25:118). The HERmark assay is a novel quantitative HER2 protein measurement for determining HER2 status in breast cancer. Central HER2 testing showed high concordance (96-98%) with HERmark for positive and negative categories when equivocal subsets were excluded (Huang et al. Am J Clin Pathol 2010;134:303; Joensuu et al, 2008 SABCS, abstract 2071). In this study, we examined concordance between HERmark and routine HER2 testing by IHC and FISH from “real world” formalin-fixed, paraffin-embedded (FFPE) breast cancers submitted commercially for HERmark testing. Methods: 717 HERmark results on FFPE breast cancers tested from 2008 to 2010 and corresponding HER2 IHC/FISH results were reviewed. The IHC and FISH results, per pathology reports submitted at the time of HERmark testing, were compared to HERmark categorical (negative, equivocal, positive) results. Results: 590 (419) samples had IHC (FISH) and HERmark results available. Of these cases, 92% (94%) were either negative or equivocal by IHC (FISH). The HERmark testing reported 33% HERmark negative, 33% HERmark equivocal and 34% HERmark positive. Comparisons of HER2 status by IHC and FISH vs. HERmark are detailed in Table 1. 19% of cases classified as IHC 0 or 1+ were positive by HERmark; 35% classified as FISH negative were HERmark positive. Of the 158 triple negative cases by IHC/FISH (ER, PR and HER2 negative, data not shown), 38 (24%) were reclassified as HER2 positive by HERmark. Conclusions: Higher than expected discordance between HER2 IHC/FISH and the HERmark assay was observed in this study compared with central HER2 testing of prior study cohorts. Selection bias may have impacted these results as ≥ 92% of cases submitted for HERmark testing were either equivocal or negative by routine HER2 IHC/FISH. Central HER2 FISH retesting of these samples will be performed and compared with HERmark results to further evaluate the discordance observed in this analysis. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-07-12.

In situ hybridization and polymerase chain reaction methods for the detection of Epstein-Barr virus RNA in breast cancer specimens

The role of Epstein-Barr virus (EBV) as a cofactor in breast cancer is controversial and its association with breast cancer varies. In this study, EBV was detected by using in situhybridization technique (ISH) to detect Epstein-barr virus encoded RNA1 (EBER1) transcripts. Archival formalin-fixed paraffin embedded breast cancer tissue samples (n = 139) and normal breast tissue (n = 20) obtained from Hospital Tuanku Ja’afar were sectioned, stained and examined microscopically for nuclear staining and by DNA amplification of the same gene. By ISH, 83/139 (59.7%) and 12/20 (60%) were EBV positives in the breast cancer tissues and normal tissues, respectively. On the other hand, confirmation by polymerase chain reaction (PCR) found that additional six samples (89/139 or 64%) breast cancer tissues were positive for EBER1 gene. To further confirm the identity of these amplified products, two samples (UiTM-53 and UiTM-73) were sequenced, BLAST, analyzed phylogenetically and was found to be 100% similar to the EBV EBER1 gene sequences already deposited in the GenBank (accession numbers AB065135, FN545286, EF187853 and DQ883818). These preliminary findings suggest that there is a correlation between EBV and breast cancer but need further testing with more samples to confirm the role of EBV. Key words: Breast cancer, Epstein-Barr virus, EBER1 transcripts, in situ hybridization, polymerase chain reaction (PCR), tumour grading.

Human papillomavirus infection and increased risk of breast cancer in north of Iran.

28 Background: Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer deaths among women worldwide. Human papillomaviruses have been related to the pathogenesis of breast cancer. In this study, we have analyzed the possible relevance of HPV infection for breast cancer risk among Iranian women from north part of Iran, Mazandaran province. Methods: Polymerase chain reaction method with three different primer sets was applied for detection of HPVs in formalin-fixed paraffin-embedded breast cancer and breast fibroadema as case and control tissues, respectively. HPV genotypes were determined by phylogenetic analysis of viral genome sequences. Results: Of the 58 women with breast cancer, 25.9% (15 isolates) had positive test results for HPV DNA in breast tumor samples in contrast to 2.4% of women (1 out of 41) with non cancer status (P + 0.002). The infection of HPV had an OR of 14.247 (95% CI 1.558-130.284; P + 0.019). The HPV genotypes in samples of breast cancer patients were 26.67% for HPV-16 (4 isolates) and HPV-18 (4 isolates), 13.3% for HPV-23 (2 isolates) and HPV-6 (2 isolates), 6.67% for HPV-11 (1 isolate), HPV-15 (1 isolate) and HPV-124 (1 isolate) and one isolate could not be genotyped compared to HPV reference sequences, while the sole detected HPV in control specimens was HPV-124. Conclusions: Our study reveals that HPV infection and age are the risk factors in breast cancer development in the north part of Iran, Mazandaran province. The association between risk of breast cancer development and viral infection is open and deserves further investigation.

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.