Formalin-fixed Paraffin-embedded Breast Cancer Samples Research Articles

Interaction between VEGF-A and immune checkpoint targets in triple-negative breast cancer and mechanisms of immune evasion and tumor progression.

1096 Background: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment triple negative breast cancer (TNBC). However, not all patients with TNBC benefit from the addition of ICIs due to factors such as intrinsic and acquired resistance to ICIs, and complex interplay between tumor and immune cells in the tumor microenvironment (TME). Vascular endothelial growth factor (VEGF) promotes angiogenesis and potentially modulates tumor immune evasion. A phase I clinical trial has shown that a bispecific antibody to VEGF-A and PD-L1 produces encouraging tumor responses in advanced TNBC. In this study, we investigated the interaction between VEGF and various immune gene expression signatures including PD-L1 in a real-world breast cancer patient population. Methods: Comprehensive immune profiling, including the RNA-seq based gene expression assessment of 395 immune-associated genes, was performed on 120 formalin-fixed paraffin-embedded breast cancer samples. PD-L1 expression (Combined Positive Score, CPS ≥ 10%) was measured by immunohistochemistry (IHC; 22C3). Clinicopathologic variables were collected. Statistical associations between quantitative biomarkers, including 28 immune checkpoint genes, were calculated using Pearson correlations (r), and comparisons of quantitative biomarkers between groups were performed using the proportions test. For statistical significance, p<0.05 was required. Results: The study cohort included 29 patients (24.2%) with TNBC and 91 (75.2%) with non-TNBC (including ER+/HER2- and HER2+). Most patients had metastatic tumors (n=92, 76.7%) and invasive ductal carcinoma (n=83, 69.2%). Among the TNBC group, 21 patients had PD-L1 positive tumors (72.4%), defined by CPS ≥ 10.In non-TNBC, VEGF has significant, though weak (r<0.35) correlations with the expression of three checkpoint genes: AKT1 (r=0.35, p=0.00063), ICOS (r=0.34, p=0.00099), and TIM3 (r=0.29, p=0.0061). In TNBC, VEGF has significant, strong (r>0.47) correlations with the expression of three other checkpoint genes: PD-1 (r=0.47, p=0.0094), PD-L1 (r=0.57, p=0.0012), and PIK3CA (r=0.6, p=0.00051) However, in TNBC, VEGF gene expression does not have a statistical association with PD-L1 IHC (CPS ≥/< 10 (p=0.9)). Conclusions: Our findings suggest that there is an interplay between angiogenesis and an immunosuppressive tumor immune microenvironment in TNBC to a greater degree than in non-TNBCs. This supports the notion that angiogenesis mediators such as VEGF-A may enhance the expression of immunosuppressive checkpoint molecules, leading to immune evasion and tumor progression. Combination treatments of ICIs, especially anti-PD-1 therapy, with VEGF antibodies may be promising approaches to enhance the immune responses in TNBC, especially in tumors with CPS<10, where the current frontline treatment is cytotoxic chemotherapy.

Loss of Human Epidermal Receptor 2 Expression in Formalin-Fixed Paraffin-Embedded Breast Cancer Samples and the Rescuing Effect of Enhanced Antigen Retrieval and Signal Amplification.

As an important therapeutic target in breast cancer, HER2 expression assessed by immunohistochemistry plays a critical role in breast cancer treatment. Recent advances in HER2 antibody-drug conjugate therapy have enabled patients with HER2-low expression breast cancer to benefit from the drugs. However, it is not known whether the HER2-low expression in breast cancer FFPE blocks would be lost as storage time increased. In this study, we aimed to assess the loss of HER2 antigenicity in stored FFPE blocks of breast cancer and the rescue effect of modifying the protocol of antigen staining. We selected archived HER2-low breast cancer FFPE blocks with stored time ranging from 1 year to over 15 years and re-detected the expression of HER2. Our study showed that HER2 antigenicity loss increased with storage time and could cause false negativity in HER2-low detection. Moreover, we showed that by either increasing the antigen retrieval time or applying the tyramide signal amplification (TSA) kit, the HER2 signal can be rescued and detected in about half of the cases with HER2-low loss without causing false positivity.

HER2 copy number determination in breast cancer using the highly sensitive droplet digital PCR method

Human epidermal growth factor receptor 2 (HER)-positive breast cancer (BC) is characterized by an aggressive clinical course. In the case of HER2 overexpression/amplification, patients benefit from HER2-targeting therapies. Standardized diagnostic HER2 assessment includes immunohistochemistry (IHC) and/or in situ hybridization (ISH). The aim of this study was to compare this “gold standard” with the Droplet Digital™ polymerase chain reaction (ddPCR), a method that allows sensitive and precise detection of copy number variations (CNV) in FFPE (formalin-fixed, paraffin-embedded) DNA samples. Partitioning of the PCR reaction into 20,000 droplets enables a precise quantitative “CN” discrimination also in heterogeneous samples. FFPE breast cancer samples (n = 170) with routinely assessed HER2 status by IHC/ISH were retrospectively analyzed using the ddPCR CNV ERBB2 assay. Comparison of HER2 status assessment by the two methods revealed concordant results in 92.9% (158/170) of the cases. Discrepant cases were verified and interpreted. For ddPCR, a cut off value of 3 HER2 copies was set to distinguish between HER2-negative and HER2-positive BC. Results obtained with the ddPCR CNV ERBB2 assay were consistent and reproducible, and serial dilutions demonstrated a high stability and sensitivity of the method. The ddPCR CNV ERBB2 assay may be a specific and convenient tool to quantify HER2 copy numbers in BC samples. In our study, this method showed high reproducibility in accuracy of HER2 assessment compared to IHC/ISH analysis.

Abstract 2907: A metaproteomic approach to study the host-microbiota interaction in cancer tissue

Abstract Background: The microbiota has the ability to modulate cancer susceptibility through a systemic interaction with the host that occurs also at tissue level. Unraveling the functional meaning of the microbiota in the tumoral microenvironment is crucial to understand its causal role. Metagenomic approaches identify the microbial composition but not its functional role. In this study, we exploited a metaproteomic approach to investigate the presence of microbiota-derived proteins on breast cancer (BC) tumoral and healthy tissues using formalin-fixed paraffin-embedded (FFPE) samples, and to assess the interaction with human proteome Material and methods: Three tumoral regions (peripheral tumor, tumor core, in situ carcinoma) and 2 healthy areas (intra-tumoral fibrosis, healthy tissue) were selected on 5 FFPE BC samples, 3 triple negative and 2 luminal, chosen based on the year of inclusion in FFPE (2017-2018). The proteomic analysis was performed on 10 µm tissue slides, by nanoLC-MS/MS analysis and data analysis by Perseus and Maxquant. Results: The proteomic analysis revealed the presence of 13 microbial proteins. Seven proteins are involved in energy production and metabolism, 4 are chaperonins. Most of the proteins were found in the intra tumoral fibrosis. Two Chaperone proteins DnaK (Thermosipho melanesiensis etc. and Rhodothermus marinus) and an S-adenosylmethionine synthase (MAT2A) (Homo sapiens; Helicobacter hepaticus etc.) were overexpressed in tumor tissue compared to the healthy. 1073 human proteins were identified and 78 were overexpressed. KEGG analysis revealed that the proteins overexpressed in healthy tissues are involved in the maintenance of cell and tissue structure and function, protein digestion and absorption, mediator of innate immunity and nonspecific defense mechanism against pathogens. The proteins overexpressed in tumor tissues belong to protein processing, antigen processing and presentation, carbon metabolism and phagosome or E.coli infection pathways Discussion: A proteomic approach on tumor tissue can give functional information about the host-microbiota interactions. Our preliminary data shows an overexpression in tumor samples, in particular in intra tumoral fibrosis, of DnaKs and MAT2A. The first are bacterial chaperone proteins able to interfere with important pathways related to DNA damage control/repair, cell-cycle/apoptosis and is a cancer promoter. The second protein is an enzyme essential for methylation that can confer growth and survival advantage to cancerous cells, enhancing tumor migration. Most of the bacterial proteins identified are involved in energy production or chaperonins. Taken together the bacterial and human proteome results, it seems that bacteria supply the tumor cells with important proteins that promote cancer growth and which fit perfectly into the pathways most represented in the tumor. More studies are needed to confirm the hypothesis. Citation Format: Francesca Pirini, Tania Rossi, Soulaimane Aboulouard, Giovanni Martinelli, Sara Bravaccini, Michel Salzet. A metaproteomic approach to study the host-microbiota interaction in cancer tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2907.

Abstract P6-11-20: Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda

Abstract Background: The global burden of cancer is rising exponentially in countries with the least resources to manage it. Breast cancer mortality is higher in African women than European women, but neither race nor access to care completely account for this discrepancy. Molecular profiling and methylation analysis could be informative but is expensive and the feasibility of using archived samples from low-resource settings is not known. We present a retrospective tissue collection study of breast cancer patients diagnosed at the University of Rwanda to investigate the quality of DNA from FFPE. Methods IRB approval and Material Transfer Agreement were obtained. Fifty tumor blocks and eight sets of FFPE slides from 2014-2018 were transported from Rwanda to USA in 2 batches. Pathology was reviewed for concordance. DNA was extracted using the QIAamp DNA FFPE Tissue kit (Qiagen). Batch 1: DNA quality and quantity was assessed using the Qubit 3.0 fluorometer (Life Technologies). Batch 2: DNA quality was assessed using Kapa hgDNA pPCR QC Assay. Bisulfite conversion and array hybridization protocols for the Illumina EPIC methylation platform to detect 2.5% methylated DNA target were used. DNA methylation data generated using the Infinum MethylationEPIC BeadChip (Illumina, Inc.) were pre-processed using minfi (v1.30.0), prior to quality control using Enmix (v1.20.2). A detection P-value threshold of 1E-06 was used to identify poorly detected probes, and a maximum threshold of 5% low quality data was used to identify poorly performing samples and CpG loci. Outliers based on beta-value distributions were also flagged as poor-quality samples. Mean bisulfite intensity values were calculated using Infinium type I and II control probes. DNA methylation beta-values were calculated to assess the proportion of methylated alleles at each individual CpG locus. Results: Histology was 82% concordant. All samples in Batch 1 had >5% low quality CpG’s. FFPE processing was reviewed and buffered formalin was not used. For Batch 2 10% neutral buffered formalin was transported and delivered to Central University Hospital of Kigali in March 2019. Eight additional patient samples were obtained and 6 out of 8 had <5% low quality CpG’s. Conclusion: Complex molecular studies are feasible with international collaboration. Optimal reagents and processing are crucial for successful analysis. Quality checkpoints must be done early in the process to maximize the potential for interpretable results. Capacity-building to improve DNA quality is the first step toward optimizing cancer treatment and minimizing disparities as molecular profiling increasingly directs cost-effective treatment.These results confirm methods appropriate for future molecular research on breast cancer in low resource settings. Citation Format: Mary Dickinson Chamberlin, Victoria Forbes, Germaine Dushimimana, Arminja Kettenbach, Kristen Muller, Emile Musoni, Eric Rutaganda, Owen M. Wilkins, Fred W. Kolling, Brock Christensen. Capacity-building for molecular analyses of formalin-fixed paraffin-embedded breast cancer samples from Rwanda [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-11-20.

Mutational analysis of PI3K/AKT signaling pathway in tamoxifen exemestane adjuvant multinational pathology study.

Deregulation of key PI3K/AKT pathway genes may contribute to endocrine resistance in breast cancer (BC). PIK3CA is the most frequently mutated gene in luminal BC (35%); however, the effect of mutations in helical versus kinase domains remains controversial. We hypothesize that improved outcomes occur in patients with estrogen receptor–positive (ER positive) BC receiving endocrine therapy and possessing PIK3CA mutations. DNA was extracted from 4,540 formalin-fixed paraffin-embedded BC samples from the Exemestane Versus Tamoxifen-Exemestane pathology study. Mutational analyses were performed for 25 mutations (PIK3CAx10, AKT1x1, KRASx5, HRASx3, NRASx2 and BRAFx4). PIK3CA mutations were frequent (39.8%), whereas RAS/RAF mutations were rare (1%). In univariable analyses PIK3CA mutations were associated with significantly improved 5-year distant relapse-free survival (DRFS; HR, 0.76; 95% CI, 0.63 to 0.91; P = .003). However, a multivariable analysis correcting for known clinical and biologic prognostic factors failed to demonstrate that PIK3CA mutation status is an independent prognostic marker for DRFS (HR, 0.92; 95% CI, 0.75 to 1.12; P = .4012). PIK3CA mutations were more frequent in low-risk luminal BCs (e.g., grade 1 nodev 3, node-negative v -positive), confounding the relationship between mutations and outcome. PIK3CA mutations are present in approximately 40% of luminal BCs but are not an independent predictor of outcome in the context of endocrine therapy, whereas RAS/RAF mutations are rare inluminal BC. A complex relationship between low-risk cancers and PIK3CA mutations was identified. Although the PI3K/AKT pathway remains a viable therapeutic target as the result of ahigh mutation frequency, PIK3CA mutations do not seem to affect residual risk following treatment with endocrine therapy.

Androgenic pathways in the progression of triple-negative breast carcinoma: a comparison between aggressive and non-aggressive subtypes

One of the active intracellular pathways/networks in triple-negative breast carcinoma (TNBC) is that of the androgen receptor (AR). In this study, we examined AR and androgen-metabolising enzyme immunoreactivity in subcategories of TNBC to further elucidate the roles of androgenic pathways in TNBC. We utilised formalin-fixed paraffin-embedded breast cancer samples from ductal carcinoma in situ (DCIS) and invasive ductal carcinoma patient cohorts. We then used immunohistochemistry to classify these samples into basal-like and non-basal samples and to assess interactions between AR, androgen-metabolising enzymes and proliferation. To further substantiate our hypothesis and provide mechanistic insights, we also looked at the expression and regulation of these factors in publically available microarray data and in a panel of TNBC AR-positive cell lines. DCIS was associated with higher levels of AR and enzymes (p<0.02), although a similar difference was not noticed in basal and non-basal samples. AR and enzymes were correlated in all states. In TNBC cell lines (MDA-MD-453, MFM-223 and SUM185-PE), we found that DHT treatment up-regulated 5αR1 and 17βHSD5 suggesting a mechanistic explanation for the correlations observed in the histological samples. Publicly available microarray data in TNBC cases suggested similar patterns to those observed in histological samples. In the majority of settings, including publically available microarray data, an inverse association between AR and proliferation was detected. These findings suggest that decreases in AR and androgen-metabolising enzymes may be involved in the increased biological aggressiveness in TNBC development.

Abstract P6-06-34: Comparing recurrence risk scores derived from 4 immunohistochemistry stains (IHC4) versus oncoType DX

Abstract Background: Prognostically relevant biomarkers in breast cancers can be assayed using nucleic acid (e.g. OncoType DX) or protein based (e.g. immunohistochemistry (IHC)) detection stratagies. Nucleic acid tests measure many more markers compared to immunohistochemistry, but are weighted heavily for ER, PR, and HER2 in their prognostic scoring algorithms. Recent data suggest that a panel of 4 IHC stains (IHC4) may have equivalent prognostic ability to multigene assays. The aim of this study was to retrospectively compare IHC4 scores to OncoType DX recurrence scores in a large cohort of patients in our statewide health system. Methods: Formalin-fixed paraffin-embedded breast cancer samples previously submitted for OncoType DX testing between 2008 and 2011 were obtained and sections stained for ER, PR, HER2, and Ki67 (the IHC4 staining panel). Stained slides were scanned digitally and scored using customized pathologist assisted image analysis algorithms (Leica Biosystems Aperio, Vista CA). The resulting data were used in the modified “Magee Equation 3” (Klein et al. Mod Pathol. 2013. PMID: 23503643) to calculate an IHC4 recurrence score on a numerically comparable scale to that used in the OncoType score. The IHC4 and Oncotype DX scores were directly compared for concordance and correlation. Results: 280 breast cancer samples comprised the study cohort. The mean (median) IHC4 score was 20.13 (19), range: 11-42. The mean (median) OncoType score was 18.48 (16), range: 0-75. Using cut-offs of 0-17 for low risk, 18-30 for intermediate risk, and &amp;gt;30 for high risk, the concordance data is shown in Table 1. Comparison of scores from IHC4 and OncoType by Risk Category OncoType RS LowIntermedHigh Low80201IHC4 RSIntermed816819 High029RS = Recurrence Score Pearson's correlation coefficient for the entire set was 0.6669. The overall concordance was 56.1%; one-step discordance 43.6%; and two-step discordance: 0.3%. Eliminating the intermediate scores, the concordance was 98.8%. Conclusions: Reasonable agreement was seen between risk scores derived by IHC4 and OncoType DX. Only one discrepancy among 280 scores would have resulted in a different treatment recommendation (low risk by IHC4 and high risk by OncoType). If the IHC4 score is clearly high or low, one would not expect a dramatically different result from Oncotype DX, and the Oncotype DX test may not be needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-34.

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

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.

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.

Abstract P4-08-12: Gene Expression Profiling of Formalin-Fixed, Paraffin-Embedded (FFPE) Breast Cancer Samples and Analysis of Intrinsic Subtypes

Abstract Background: The advent of microarray technology has enabled robust, high throughput analysis of breast cancer (BC) transcriptomes. Indeed, molecular classification of BC has been revolutionized by the advent of Gene Expression Profiling (GEP). FFPE tumor samples have presented a technical challenge for GEP studies due to degradation of extracted RNA. Newer technologies have overcome this challenge, and have lead to generation of quality GEP data and thus, new insights using archived tissues. Of particular interest to our group has been application of these techniques to the study of triple negative breast cancer (TNBC). TNBC is a BC sub-type characterized by a lack of erbB2 gene amplification and estrogen and progesterone receptor expression. This clinically-defined BC sub-type carries a poor prognosis, is insensitive to hormonal or HER-2 targeted therapies, and displays different incidences among ethnic groups. A better understanding of the genetic and molecular mechanisms underlying TNBC is critical to improving clinic outcomes and developing tailorized therapies. Study Objective: We demonstrate utility of FFPE BC samples in obtaining consistent, reproducible GEP data, and apply this technology to validate the ability to identify intrinsic BC subtypes in unselected specimens, as well to identify differentially expressed genes in TNBC. Methods: RNA isolation and labelled cDNA preparation were performed from freshly cut FFPE sections. Samples were hybridized to a breast cancer focused gene expression array (Breast Cancer DSA Research Tool, Almac Diagnostics Inc). DSA chip quality was assessed on parameters selected automatically from GCOS report files per chip using MATLAB script based web application developed by Almac. Data pre-processing used the Resolver Error Model. All parameters including Raw Q, Background, Scaling Factor and all controls met quality criteria set by Affymetrix and Almac Dx SOPs. Hybridization results were assessed with Principal Component Analysis and Cluster Analysis in a Rosetta Resolver Gene Expression Data Analysis System to identify potential outliers, contamination, or intra-tumor heterogeneity. In total, 47 FFPE breast cancer samples covering a range of hormonal receptor status and sub-types were profiled, as were 28 TNBC FFPE tumor samples. Results: Cluster analysis demonstrated that the Almac Breast Cancer DSA was able to clearly separate the 47 tumor samples of the mixed subtype group into the previously described intrinsic subgroups. Moreover, the DSA array contains 167 probesets which correspond to the 40 of the PAM-50 gene set used as a subtype predictor (Parker et al 2009). Analysis of the TNBC samples using the 167 probeset (based on mean intensity for probes representing each of the 40 genes) showed 100% consistency with published results demonstrating a basal-like gene expression signature. Summary: We have shown that our study methodology used can reliably measure gene expression in FFPE BC samples, and that the Breast Cancer DSA can be used to evaluate intrinsic subtypes of BC specimens. Analysis of the TNBC cases showed complete concordance between the PAM-50 gene set and the corresponding genes in the DSA. These study results are being validated in a larger data set. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-08-12.

Abstract 327: Genomic profiling of sentinel lymph node breast cancer metastasis

Abstract The sentinel lymph node (SLN) is the first node in the axilla to harbor malignant cells in breast tumors with metastasis, and its positivity is an indication for axillary lymph node dissection. Despite significant improvement in the methods for detection of tumor cells at this site, there are a still significant number of false negative cases and the inability to detect malignant cells in lower abundance, such as in the case of micrometastasis. We propose to use methods based on the genomic alterations, which present higher sensitivity and accuracy, and can be used to augment the current methods to identify the epithelial malignant cells at the SLN. In this study, we performed CGH and array-CGH analysis in microdissected tumors cells from the SLN metastatic lesions to identify DNA copy number changes. We compared the changes observed in the SLN lesion to the ones present in the corresponding primary tumors (PT) from the same patients. Here we present the results from the initial analysis of 10 FFPE (formalin-fixed paraffin-embedded) breast cancer samples (5 pairs of SLN and PT). DNA copy number changes were detected by both methods in all the cases analyzed. A remarkable similarity in the DNA profiles between the SLN metastatic lesions and the corresponding PT were observed, despite the high level of heterogeneity observed among the cases. CGH analysis revealed most frequently gains of chromosomal regions (at both lesions) involving 1p, 1q, 6q, 9q, 11p, 11q, 13q, 16p, 17q, 19p, 19q and Xq. Array-CGH analysis detected both amplifications and deletions, affecting mostly the chromosome regions 1p (genes affected KLHL17, NOC2L, PPIAL4), 9q (ANKRD20A, COL5A), 11p (PSMD13, SIRT3), 15q (MAN2C1), 19p (MUC16, AZU1) and 20q regions (PSMA7). In addition, several genes of the HIST, FLJ, SLC and TRIM family as well as the homeobox families HOX and NK were involved in the alterations. FISH analysis is being conducted to confirm the array-CGH findings as well as the analysis of additional matching samples. The assessment of copy number changes in the SLN metastasis (and in its correspondent PT) using genomic profiling is a very sensitive method and will lead to the identification of genomic alterations that can reliably characterize breast tumor cells at the SLN lesions. Once these alterations are validated in larger series of patients, they can be used as molecular markers that can improve the predictive value of the current established methods of SLN evaluation in the identification of patients at higher risk of developing axillary lymph node metastasis. 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 327.

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Student t-test was applied, showing DNA/RNA spectral region (1400–1000 cm−1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer.

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

This work presents a pilot study to illustrate the potential of Fourier transform infrared (FT-IR) imaging in breast cancer research. Using this technique, we have acquired infrared (IR) microspectroscopic images from healthy and cancerous breast tissue section from one patient. First of all, a Studentt-test was applied, showing DNA/RNA spectral region (1400–1000 cm−1) as the most discriminant for the differentiation between healthy and tumor samples. Afterwards, a supervised pattern recognition method, Partial Least Squares (PLS) was used to develop an automated classifier to discriminate the two classes of data. Infrared spectra of independent IR measurements were used to test the classifier. The class identity was correlated with information obtained by histopathologic gold standard. The results showed that more than 95% of the training and validation spectra were correctly identified. We demonstrate that combination between IR microspectroscopic imaging and multivariate data analysis can be used as a complement to present diagnostic tools for breast cancer.

Reliability and Reproducibility of a RNA Preamplification Method for Low-density Array Analysis From Formalin-fixed Paraffin-embedded Breast Cancer Samples

Molecular signatures of tumors with prognostic and predictive value are now available. However, several technical problems prevent the performing of gene expression profiles in routine diagnostics. The highly sensitive fluorescence-based real-time quantitative reverse transcriptase-polymerase chain reaction procedure is able to analyze mRNA levels from formalin-fixed paraffin-embedded (FFPE) tissues, the most commonly available source of tumor samples with well-documented information. However, the time-dependent fragmentation and small amount of RNA extracted from FFPE requires a preliminary mRNA amplification step to amplify small amounts of mRNA without significantly distorting relative mRNA levels. The purpose of this study was to determine the performance of a commercial cDNA preamplification kit (TaqMan PreAmp Master Mix Kit) on RNA samples obtained from FFPE tissues, prepared, and archived for routine diagnosis. We tested the reliability and reproducibility of this procedure for performing low-density gene expression assay from FFPE tissue samples. The whole procedure reported herein is a powerful and reproducible approach for routine clinical purposes that can be performed even using poorer RNA quality samples and that allows the analysis of gene expression for 96 genes in stored samples.

Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues

Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.

Automated array-CGH optimized for archival formalin-fixed, paraffin-embedded tumor material

BackgroundArray Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH.MethodsIn this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use.ResultsOur protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method.ConclusionThis report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility.

RNA extraction from ten year old formalin-fixed paraffin-embedded breast cancer samples: a comparison of column purification and magnetic bead-based technologies

BackgroundThe development of protocols for RNA extraction from paraffin-embedded samples facilitates gene expression studies on archival samples with known clinical outcome. Older samples are particularly valuable because they are associated with longer clinical follow up. RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue is problematic due to chemical modifications and continued degradation over time. We compared quantity and quality of RNA extracted by four different protocols from 14 ten year old and 14 recently archived (three to ten months old) FFPE breast cancer tissues. Using three spin column purification-based protocols and one magnetic bead-based protocol, total RNA was extracted in triplicate, generating 336 RNA extraction experiments. RNA fragment size was assayed by reverse transcription-polymerase chain reaction (RT-PCR) for the housekeeping gene glucose-6-phosphate dehydrogenase (G6PD), testing primer sets designed to target RNA fragment sizes of 67 bp, 151 bp, and 242 bp.ResultsBiologically useful RNA (minimum RNA integrity number, RIN, 1.4) was extracted in at least one of three attempts of each protocol in 86–100% of older and 100% of recently archived ("months old") samples. Short RNA fragments up to 151 bp were assayable by RT-PCR for G6PD in all ten year old and months old tissues tested, but none of the ten year old and only 43% of months old samples showed amplification if the targeted fragment was 242 bp.ConclusionAll protocols extracted RNA from ten year old FFPE samples with a minimum RIN of 1.4. Gene expression of G6PD could be measured in all samples, old and recent, using RT-PCR primers designed for RNA fragments up to 151 bp. RNA quality from ten year old FFPE samples was similar to that extracted from months old samples, but quantity and success rate were generally higher for the months old group. We preferred the magnetic bead-based protocol because of its speed and higher quantity of extracted RNA, although it produced similar quality RNA to other protocols. If a chosen protocol fails to extract biologically useful RNA from a given sample in a first attempt, another attempt and then another protocol should be tried before excluding the case from molecular analysis.

Caveolin-1 Mutations in Human Breast Cancer: Functional Association with Estrogen Receptor α-Positive Status

A Japanese study reported that up to 16% of breast cancer samples harbor a sporadic mutation within the human Cav-1 gene, namely P132L. To date, however, no studies have examined the United States' population. Here, we developed a novel allele-specific real-time PCR assay to detect the Cav-1 P132L mutation in mammary tumor cells isolated by laser capture microdissection from formalin-fixed paraffin-embedded breast cancer samples. We report that the Cav-1 P132L mutation is present in approximately 19% of estrogen receptor alpha (ERalpha)-positive breast cancers but not in ERalpha-negative breast cancers. This is the first demonstration that the P132L mutation is exclusively associated with ERalpha-positive mammary tumors. We also identified six novel Cav-1 mutations associated with ERalpha-positive breast cancers (W128Stop, Y118H, S136R, I141T, Y148H, and Y148S). Thus, the overall incidence of Cav-1 mutations in ERalpha-positive breast cancers approaches 35% (greater than one-third). To mechanistically dissect the functional relationship between Cav-1 gene inactivation and ERalpha expression, we isolated primary mammary epithelial cells from wild-type and Cav-1-/- mice and cultured them in a three-dimensional system, allowing them to form mammary acinar-like structures. Under conditions of growth factor deprivation, Cav-1-deficient mammary acini displayed increased ERalpha levels and enhanced sensitivity toward estrogen-stimulated growth, with specific up-regulation of cyclin D1. Finally, we discuss the possibility that sporadic Cav-1 mutations may act as an initiating event in human breast cancer pathogenesis.