An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

Background: Formalin fixed paraffin embedded (FFPE) tissues remains the standard method for fixation and storage of tissues for clinical pathology use. Protein extraction from these tissues remains challenging due to the reduced quality and amount of extracted proteins. Despite multiple successful attempts, isolation of proteins from FFPE tissue sections necessitates routine use of xylene, a highly toxic organic solvent. We previously showed that proteins can be efficiently extracted with a novel technique that utilizes hot distilled water as a substitute for xylene with a quality adequate for western blot analysis. However, its major drawback is the need to use an entire or major part of the paraffin-embedded tissue block. To address these issues we developed a new xylene-free method for protein extraction from FFPE tissue sections of around 8 μm thickness. Methods: A total of 44 different types of FFPE tissues sections of 8 μm thickness were obtained from various archived FFPE specimens which include: 17 colorectal cancer (5-6 years), 7 breast cancer (3 years), 3 thyroid cancer (2 years), 4 ovarian cancer (2 years), 8 uterine cancer (1 year), and 5 prostate cancer (1 year). Deparaffinization was conducted by gentle treatment of each section with hot distilled water (≈90°C) for less than 10 seconds. Deparaffinized tissues were then placed in a cell lysis buffer and Laemli buffer and incubated at 100°C for 5-10 minutes. The extracted proteins were quantified using NanoDrop Spectrophotometer and evaluated using western blot analysis for the presence of AKT and beta-actin. Results: Using this method, a significant amount of proteins was successfully isolated with an average amount of 2.670 μg/μl ± 0.623 or 1068 μg/8 μm tissue section. Compared to the standard protein extraction method using xylene the amount of proteins extracted in this experiment is at least four times greater. Protein extracts were of good quality and efficiently analyzed by western blot experiment. Moreover, the isolated proteins showed a similar migration pattern compared to the positive control protein on SDS-PAGE but with better bands’ intensity and clarity. Protein kinase B (PKB/AKT) was successfully identified in all specimens, and beta-actin protein was resolved with an efficiency higher than 80%. Hence, this technique has enabled us to efficiently extract and detect selected proteins from archived samples for up to 6 years. Conclusion: We developed an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissues sections and adequate for western blot analysis. Hence, utilizing this technique can further aid in the identification of tissue protein biomarkers of various diseases, monitor cancer progression, and improve the diagnosis. It remains to be determined if this method of protein extraction form FFPE is adequate for use in proteomic analyses. Citation Format: Anthony Mansour, Noha Bejjani, Carole Dagher, Rajaa Chatila, Wissam H. Faour. Optimized xylene-free protein extraction method from formalin-fixed paraffin-embedded tissue sections for western blot analysis. [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 LB-288. doi:10.1158/1538-7445.AM2015-LB-288

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10-20 tissue sections or whole tissue blocks, which prevents better resolved analyses. Nevertheless, it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissue of interest are limited. Here, we present F FPE tissue with a ntibody-guided c hromatin t agmentation with sequencing (FACT-seq), highly sensitive method to efficiently profile histone modifications in FFPE tissue by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7-pA-Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We showed a very small piece of FFPE tissue section containing ~4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. In archived FFPE human colorectal and human glioblastoma cancer tissue, H3K27ac FACT-seq revealed disease specific super enhancers. In summary, FACT-seq allows researchers to decode histone modifications like H3K27ac and H3K27me3 in archival FFPE tissues with high sensitivity, thus allowing us to understand epigenetic regulation. Graphical abstract: ( i ) FFPE tissue section; ( ii ) Isolated nuclei; ( iii ) Primary antibody, secondary antibody and T7-pA-Tn5 bind to targets; ( iv ) DNA purification; ( v ) In vitro transcription and sequencing library preparation; ( vi ) Sequencing.

Spatial genomics and digital pathology continue to advance at a rapid pace, generating large amounts of data. There is a critical unmet need for advanced tools to expedite spatial data generation, visualization, and analysis, to facilitate an improved understanding of health and disease, especially with the advancement of personalized cancer treatments. In this study we present a novel method for the extraction and analysis of genomic information from specific regions of interest (ROI) within a series of formalin-fixed paraffin embedded (FFPE) breast cancer adenocarcinoma tissue sections. An artificial intelligence-based tumor profiling algorithm first identified tumor, stroma, inflammatory, and necrotic regions within the tissue, and segmentation results were confirmed by a Board Certified Pathologist. Automated lysate extraction was then performed in the selected ROIs using ink-jet technology and novel ink chemistry. The process was performed directly on the tissue slide and the crude lysates generated were transferred to a standard collection tube for subsequent sequencing. DNA was extracted from the selected areas before being purified and analyzed using a multi-cancer panel. Library QC showed optimal coverage at an average of >95% uniformity. Sequencing results showed an average coverage of 95% at 250x across all amplicons of the panel. Variant calling was successfully performed. Sequencing data is presented to demonstrate the high coverage achieved using this workflow as well as the resulting tumor heterogeneity. This novel technology provides an efficient solution for the enrichment of tumor content with high sequencing coverage from FFPE tissue sections. Novel spatial data visualization and analysis techniques allowed a comprehensive assessment of tumor heterogeneity. Further studies on both DNA and RNA should be conducted to validate this approach for clinical molecular testing. Citation Format: Bidhan Chaudhuri, Katie Konigsfeld, Marisa Sanchez, Jason Smith, Casey Laris, Claire Weston, John Butler. A novel AI-driven spatial genomics platform enables tumor enrichment and analysis of heterogeneity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6575.

e21135 Background: To understand the dynamics of anti tumor immune responses (IR) in the setting of the ageing immune system, we examined immunologically relevant gene transcripts in formalin fixed paraffin embedded (FFPE) primary cutaneous melanoma (PCM) samples derived from five young (age <50 years) and five elderly (age >or=70 years) patients by qRT-PCR. Methods: A total of 10 FFPE tumor blocks were collected between 2004-2007 of patients with PCM at the University of Connecticut Health Center. Total RNA was extracted from FFPE tissue sections and random primed cDNA was generated. Pre amplification using pooled Taqman assays and Universal PCR Master Mix (Applied Biosystems, USA) was performed for 14 cycles to ensure an adequate amount of starting material for qPCR. Using 15 ng total RNA, the quantification of human IR genes (IL-2, INFg, TNFa, TGFb1, IL-10, Foxp3, and IDO-1) and transcripts of succinate dehydrogenase (SDHA) (as endogenous control) was accomplished by qRT-PCR amplification of a cDNA using TaqMan Universal PCR Master Mix and TaqMan gene expressions assay probes. All reaction assays including that of SDHA were performed in triplicate on an ABI 7500 Fast system (Applied Biosystems). Cycle threshold (Ct) values for each mRNA were normalized to SDHA (DCT) and represented as RQ=2-DCT. The average RQ was calculated for all ten samples in both cohorts and Student’s T-test was performed to evaluate the statistical significance of the IR gene expression. Results: While no significant difference of expression of Th1 cytokines (IL-2, IFNg, TNFa) or Th2/3 cytokines (IL-10, TGFb) could be detected within the two groups, FOXp3 expression was significantly decreased in PCM in the elderly. Conclusions: Our results suggest ageing may be associated with less regulatory responses in situ in PCM and that more expanded analyses of the nature of the IR in PCM are likely to provide information with implications on prognosis and immune based treatment options for elderly PCM subjects.

ABSTRACTA variety of techniques have been developed as diagnostic tools for the differential diagnosis of tumours produced by Marek’s disease virus from those induced by avian leukosis virus and reticuloendotheliosis virus. However, most current techniques are unreliable when used in formalin-fixed paraffin-embedded (FFPE) tissues, which often is the only sample type available for definitive diagnosis. A collection of tumours was generated by the inoculation of different strains of Marek’s disease virus, reticuloendotheliosis virus or avian leukosis virus singularly or in combination. FFPE tissue sections from tumour and non-tumour tissues were analysed by optimized immunohistochemistry (IHC) techniques and traditional as well as quantitative polymerase chain reaction (PCR) with newly designed primers ideal for DNA fragmented by fixation. IHC and PCR results were highly sensitive and specific in tissues from single-infected birds. Virus quantity was higher in tumours compared to non-tumour spleens from Marek’s disease (MD) virus-infected birds. Thus, using FFPE sections alone may be sufficient for the diagnosis of MD by demonstration of high quantities of viral antigens or genome in tumour cells, along with the absence of other tumour viruses by traditional PCR, and if standard criteria are met based on clinical history and histology. IHC furthermore allowed detection of the specific cells that were infected with different viruses in tumours from birds that had been inoculated simultaneously with multiple viruses. Following validation with field samples, these new protocols can be applied for both diagnostic and research purposes to help accurately identify avian tumour viruses in routine FFPE tissue sections.

Microplastic (MP) pollution is increasingly acknowledged as a critical environmental and public health issue. This study sought to establish a robust, clinically compatible method for detecting MP particles in deparaffinized formalin-fixed paraffin-embedded (FFPE) human colon tissue sections, using protocols compatible with routine clinical pathology. We employed mid-infrared photothermal (MIP) microscopy—also referred to as optical photothermal infrared (OPTIR) spectroscopy—as a non-destructive, high-resolution technique for chemical characterization and spatial mapping of polymer particles in intact FFPE samples. Following OPTIR analysis, identical sections underwent hematoxylin and eosin (H&E) staining to facilitate precise histopathological evaluation in defined regions of interest. Using this integrated workflow, we detected and localized polyethylene (PE), polystyrene (PS), and polyethylene terephthalate (PET) particles (21 PE particles, 1 PS particle, and 1 PET fiber) within distinct tissue areas. Subsequent histological assessment revealed characteristic inflammatory features near to these identified MP particles. To our knowledge, this represents the first demonstration of a diagnostic workflow that enables combined infrared spectroscopic and histopathological analysis of MPs in routinely processed human FFPE tissue. This approach offers a promising avenue to elucidate the role of microplastic accumulation in human disease and supports further investigation into potential mechanistic links between MP exposure and inflammatory processes in the colon.

Background: Black women with residual estrogen receptor (ER)-positive breast cancer after neoadjuvant chemotherapy (NAC) have worse distant recurrence free survival (DRFS) compared to White women. Distant metastases result from motile breast cancer stem cells (BCSCs) disseminating through intravasation portals on blood vessels called tumor microenvironment of metastasis (TMEM) doorways. Each TMEM doorway is composed of an actin-regulatory protein (Mena) expressing cancer cell, a macrophage expressing high levels of receptor tyrosine kinase TIE2, and an angiopoietin-expressing endothelial cell. High TMEM doorway density predicts metastatic potential in patients with ER+/HER2- breast cancer. The function of TMEM doorways can be pharmacologically blocked using TIE2 inhibitors. We hypothesized that racial disparity in DRFS in patients with residual ER-positive disease post-NAC is due to difference in density of TMEM doorways, BCSCs, and motile cells in the residual breast tumor microenvironment (TME). Methods: We performed a retrospective, multi-institutional study of the TME in the residual disease post-NAC from 200 patients. Triple immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tissue sections was used to visualize TMEM doorways. Immunofluorescent staining of FFPE tissue sections for SOX9 and MenaINV was used to visualize BCSCs and motile cells, respectively. The density of TMEM doorways, BCSC and motile cells was determined by automated image analysis. The relationship between the density of TMEM doorways, stem and motile cells, and DRFS was examined using Kaplan Meier log-rank test. Comparisons were made for the entire cohort, and separately for the two most prevalent molecular subtypes: ER+/HER2- and triple negative (TN). The analysis included 200 samples of residual breast cancer post-NAC: 100 from Black and 100 from White women. Results: Black compared to White women were more likely to develop distant recurrence (49% vs 34%, p=0.04), to receive mastectomy (70% vs 51%, p=0.009), and to have TN subtype (35% vs 21%, p<0.0001). Median time-to-follow-up was 70 months (range 2.8-160.7), and was similar between Black and White patients (67.7 vs 71.0 months, p=0.7). There was no racial disparity in DRFS in the overall cohort or TN subtype, but there was a trend towards worse survival in Black women in the ER+/HER2- subtype (Log-Rank p=0.09). TMEM doorway density was higher in Black women in the entire cohort (p=0.001) and in the ER+/HER2- subtype (p=0.01), while there was no difference in the TN subtype. TMEM doorway density was prognostic for DRFS in all patients (Log-Rank p=0.009) and the ER+/HER2- subtype (Log-Rank p=0.001), but not in the TN subtype. There was a trend towards higher BCSC density in the entire cohort (p=0.06) and the ER+/HER2- subtype (p=0.09), but there was no difference in the TN subtype. There was no racial disparity in motile cell density in the entire cohort as well as in the two subtypes. BCSC density and motile cell density were not prognostic for DRFS. Conclusion: Worse DRFS in Black, compared to White, women with ER-positive residual breast cancer post-NAC may be due to differences in TMEM doorway and BCSC density. Further studies are necessary to determine if the outcome of patients with residual disease and high TMEM doorway density post-NAC could be improved by blocking TMEM doorway function with TIE2 inhibitors. Citation Format: Gina Kim, Saeed Asiry, Isabelle Oktay, Yu Lin, Xianjun Ye, Esther Cheng, Nurfiza Ladak, John Condeelis, Esther Adler, Paula Ginter, Timothy D'Alfonso, David Entenberg, Maja Oktay. Racial disparity in post-neoadjuvant chemotherapy residual breast tumor microenvironment [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-36.

We explored three approaches to create tissue-specific knock-in mice by generating knock-in mice in which a substrate-docking site of the PDK1 protein kinase was ablated in Cre-expressing tissues in a way that prevented activation of one of its substrates, p70 ribosomal S6 kinase (S6K), but not another (protein kinase B (PKB)). Employing two of the approaches, termed the "heterozygous" and "minigene" methods, we generated mice in which Cre-expressing skeletal and cardiac muscle produced the mutant rather than wild type PDK1. Consistent with this, injection of these mice with insulin only induced activation of PKB but not S6K in muscle tissues. We have also demonstrated that insulin-stimulated glucose uptake proceeds normally in knock-in mice, consistent with the notion that PKB mediates this process. In contrast to conditional knock-out of PDK1 in muscle, the knock-in mice did not develop dilated cardiomyopathy, suggesting that PKB plays a key role in protecting mice from heart failure. The third knock-in strategy that was evaluated, termed the "inversion" method, did not proceed with high efficiency. We discuss the merits and disadvantages of each of the conditional knock-in approaches, along with the applications for which they may be most suited, and suggest how they could be further refined.

Next-generation sequencing (NGS) is routinely used for detection and determination of genetic mutations. While formalin fixed paraffin embedded (FFPE) samples have been a blessing for ensuring tumor tissue integrity, library construction for NGS using FFPE samples has been extremely challenging. Need for extraction of high quality and amplifiable DNA and RNA can make the workflow significantly more complex and challenging. The recent years have been witnessing a growing interest in precision medicine, and in monitoring biological systems via a multiomics approach. While such approaches can offer a holistic perspective of a system, they can be challenging owing to their inherent complexities, sample limitations, and lack of appropriate sample preparation methods. In addition to genomic data, proteomic data from FFPE samples are also gaining importance as more biomarkers are being discovered and optimized for detection and monitoring of diseases and their progression under specific therapy. Discovery and characterization to monitoring and quantification of proteins from FFPE samples can be complex – when it comes to ensuring extraction, purification, and digestion of proteins in high quality and quantity. Adaptive Focused Acoustics® (AFA®) Technology is an advanced acoustic technology that employs highly controlled bursts of focused high-frequency acoustic energy to efficiently and reproducibly proess samples in a temperature-controlled and non-contact environment This study presents an optimal use of AFA based workflows along with kits and methods for extraction, isolation, purification of TNA (DNA and RNA) from one 10 μm FFPE scroll. In addition, proteins can be extracted, purified (using a protein aggregation capture protocol), and digested in an expedited process in the same plate, also from one 10 μm FFPE scroll. The results not only show high yield, purity, and amplifiability for TNA but also reproducible and high yield of proteins and peptides – thereby offering every researcher achieve high quality, reliable, and reproducible data. Citation Format: Debadeep Bhattacharyya. Total Nucleic Acid to Proteins from FFPE: Confident Data with Comprehensive Sample Prep Workflows [abstract]. In: Proceedings of Frontiers in Cancer Science 2024; 2024 Nov 13-15; Singapore. Philadelphia (PA): AACR; Cancer Res 2025;85(15_Suppl):Abstract nr P65.

Innate Immune Microenvironment of Indolent B Cell Splenic Lymphomas: Role in Diagnosis and Treatment

Background: Breast and other solid tumors exhibit high and varying degrees of intra-tumor heterogeneity that make the management and treatment of these diseases rather difficult. In order to study clinically relevant tumor heterogeneity in detail we performed an in-depth analysis of the clonal architecture of primary chemoradiation-naive breast cancers and their lymph node metastases. We combined DNA content-based flow cytometry and ploidy analysis with high-definition array comparative genomic hybridization (aCGH) and next-generation sequencing technologies to interrogate the genomes and transcriptomes of multiple biopsies from the tumors and involved lymph nodes. Thus rather than inferring the presence of distinct tumor cell populations, our flow-sorting based approach of first identifying and defining the clonal populations and then interrogating their genomes provides a highly objective method of exploring the clinical significance of tumor heterogeneity in solid tumors. Material and methods: We used DAPI-based DNA content measurements and flow sorting to isolate nuclei from distinct clonal populations of diploid and aneuploid tumor cells in surgical tumor samples and distant lymph nodes from chemoradiation naive patients. In parallel, matching samples were processed for pathological, transcriptome, and IHC based assays. Each distinct sorted tumor cell population was then interrogated with aCGH and next generation sequencing. For each patient, the detailed profiles of ploidy, copy number aberrations and mutations were used to recreate and map the lineages present in the primary tumor and lymph. In one detailed case, we used 12 fresh frozen sections morphologically mapped from within a chemo-naïve primary invasive ductal carcinoma of histological grade 3 with lymph node involvement, 2-3 fresh frozen sections from 2 out of 5 lymph nodes with metastatic carcinoma, formalin-fixed paraffin-embedded (FFPE) tissue sections from 2 more of the involved lymph nodes and 5 morphologically mapped FFPE samples of normal tissue adjacent to the tumor. Results: We identified multiple coexisting aneuploid populations within biopsies from individual patients. Our clonal analysis determined the genomic aberrations arising in lineages that spread throughout the primary tumor and later bifurcated to distinct lymph nodes, revealing distinct therapeutic targets in the trunk and branches of the tumor. We identified driver events including high-level focal amplifications (e.g. SARC) and homozygous deletions (e.g. ROBO1 and ROBO2), that converge on distinct cellular signaling pathways. Conclusion: The approach highlighted here has broad implications in the study of tumor heterogeneity in breast and other solid tumors by providing a unique ultra-high resolution view of tumor heterogeneity that can advance more effective treatment and clinical management of patients with this disease. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD4-7.

We have recently developed a MALDI imaging mass spectrometry (MALDI-IMS) method to spatially profile N-linked glycans in frozen and formalin-fixed paraffin-embedded (FFPE) tissue sections and tissue microarrays (TMAs). Tissues are incubated with peptide N-glycosidase, and released N-glycans are detected directly using MALDI-FTICR, linked directly with tissue histopathology. Other methods to detect the localization of glycans in tissues rely on detection of broader glycan structural motifs (i.e., lectins or carbohydrate antigen antibodies), whereas our method is able to simultaneously identify and distinguish 40 or more components of the N-glycome on a single slide. To demonstrate the ability of MALDI-IMS to generate biomarker panels, pancreatic cancer tissue blocks and six TMAs containing matched tumor and non-tumor regions from over 70 patients were profiled. Aberrant glycosylation, such as elevated CA-19-9, is well documented in pancreatic cancer, making this an ideal sample set. To best analyze this complex data set, we developed an in-house analysis script that can extract spectra from tissue cores, and return non-biased statistics for observed glycan ions. Panels were generated using a training set of data and tested on external validation data set. The most accurate panel of 12 glycans achieved an overall sensitivity of 92.9% and specificity of 86.7%. Structural identification of N-glycans has been confirmed by techniques such as on-tissue CID, ethylation analysis, sequential glycosidase digestions, and comparison to glycan structural databases. Furthermore, imaging of entire FFPE sections matched with histological analysis revealed a vast diversity of N-glycan localizations, which could distinguish not only tumor from normal tissue, but also regions of pancreatitis and pancreatic intraepithelial neoplasia. The generated glycan tissue maps will also be used to determine specific glycoprotein carriers of biomarker candidate glycans in tissue and biofluid samples. Citation Format: Richard R. Drake, Thomas W. Powers, Benjamin A. Neely. A MALDI imaging mass spectrometry approach using tissue microarrays to identify an N-glycan biomarker panel for pancreatic cancers. [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 540. doi:10.1158/1538-7445.AM2015-540

Fluorescence in situ hybridisation (FISH) is a molecular cytogenetic technique, which is regularly applied to formalin-fixed paraffin-embedded (FFPE) tissue sections of a variety of cancers to assess chromosomal aberrations. However, high-quality FISH requires optimal enzymatic digestion, and insufficient digestion is not noted until the hybridisation signals are evaluated in the fluorescence microscope. As a consequence, FISH results may be unreliable, and the experiment might have to be repeated. To solve this problem, we developed a new method for real-time evaluation of enzymatic tissue digestion. Termination of enzyme activity at the proper time facilitates successful hybridisation, and experiments do not have to be repeated. We first performed FISH on 20 FFPE samples, which had been pepsin digested for different times, and this revealed distinct morphological changes within the nucleus and perinuclear space that were detectable by light microscopy. These observations suggested that the presence of intact and clear bare nuclei, surrounded by a translucent perinuclear space, might serve as an indicator of adequate digestion. We developed a protocol for assessment of this indicator, based on morphological features, and applied this to a collection of 400 tissue samples, partly of breast cancer and partly of different types of lymphoma, prior to FISH. The FISH success rate was 99.5% (398/400), which was significantly higher than that of the conventional method. In all successful cases, morphological signs of adequate digestion were paralleled by easily interpretable FISH signals. This new method for the real-time assessment of digestion quality improved the success rate of FISH and in addition was simple and rapid.

Background: Worldwide, biopsy is the gold standard for pathological assessment of cancer. Routinely, immunohistochemistry (IHC) can be performed to evaluate various tumor markers on serial sections of formalin-fixed paraffin-embedded (FFPE) tissue. Comprehensive molecular analysis of biopsy is limited by the microscopic size of samples and the scarcity of quantitative tools for FFPE tissue examination. Development of the new tools for molecular analysis of biopsy is needed to stratify, as early as possible, a patient’s individual risk for tumor aggressiveness and sensitivity to therapy to avoid potential over- and under treatment. Objective: To assess the feasibility of direct mRNA expression profiling of all three classic clinical markers of cancer progression - estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) in routinely collected FFPE core needle biopsy (CNB) of the breast. Methods: We obtained tissue from the Department of Pathology and Laboratory Medicine at the University of Pennsylvania, the University of Pennsylvania Tumor Tissue and Biospecimen Bank and the Cooperative Human Tissue Network. Steroid hormone receptor and HER2 status, determined by FDA-approved methods, was obtained from the surgical pathology reports. For direct (i.e. without amplification) mRNA profiling, we ran the Panomics/Affymetrix QuantiGene Plex 2.0 assay on Luminex according to manufacturer’s protocol. After the removal of low-abundance housekeeping genes expressing samples, the relative activity of target genes, ERS1, PGR, ERBB2 encoding ER alpha, PR and HER2 was detected. mRNA profiling was completed in 107 breast samples: 6 clinically normal (N) cases from reduction mammoplasty, 17 ductal carcinoma in situ ( DCIS) on CNB , 42 invasive ductal carcinoma (IDC) and 42 non-malignant matched tissue from surgical excisions. Groups were compared using one-way analysis of variance (ANOVA). Results: A pair-wise comparison among tissue groups revealed 3-fold higher expression levels of ERS1 in ER+ DCIS on CNB (P=. 055), and statistically significantly higher expression levels of ERS1 in the ER+/PR+ (P= .021) and ER+/PR- (P= .004) subgroups of IDC than in the N group. Remarkably, compared with N tissue, ERS1 levels were already decreased in non-malignant tissue adjacent to ER-/PR-/HER2- subgroup of IDC and were further significantly decreased in ER-/PR-/HER2- IDC itself (P= .023). Compared to the N group, significant decreases in PGR levels were found only in ER-/PR-/HER2- subgroup of IDC (P= .003), whereas significant increases in ERBB2 levels were found in both HER2+ DCIS on CNB (P= . 037) and HER2+IDC in excisions (P= .005). Conclusions: Our study, for the first time, proved the feasibility of using FFPE CNB as an RNA source for direct mRNA expression profiling. These findings support the notion that IHC evaluation of ER, PR, and HER2 status in the CNB DCIS may be complemented by concurrent detection of ESR1/PGR/ERBB2 mRNA levels. Citation Format: Indira Prabakaran, Paul J. Zhang, Marina Guvakova. The diagnostic power of direct mRNA profiling in breast cancer core biopsy. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr B24.

The majority of biopsies in both basic research and translational cancer studies are preserved in the format of archived formalin-fixed paraffin-embedded (FFPE) samples. Profiling histone modifications in archived FFPE tissues is critically important to understand gene regulation in human disease. The required input for current genome-wide histone modification profiling studies from FFPE samples is either 10–20 tissue sections or whole tissue blocks, which prevents better resolved analyses. But it is desirable to consume a minimal amount of FFPE tissue sections in the analysis as clinical tissues of interest are limited. Here, we present FFPE tissue with antibody-guided chromatin tagmentation with sequencing (FACT-seq), the first highly sensitive method to efficiently profile histone modifications in FFPE tissues by combining a novel fusion protein of hyperactive Tn5 transposase and protein A (T7−pA−Tn5) transposition and T7 in vitro transcription. FACT-seq generates high-quality chromatin profiles from different histone modifications with low number of FFPE nuclei. We proved a very small piece of FFPE tissue section containing ∼4000 nuclei is sufficient to decode H3K27ac modifications with FACT-seq. H3K27ac FACT-seq revealed disease-specific super enhancers in the archived FFPE human colorectal and human glioblastoma cancer tissue. In summary, FACT-seq allows decoding the histone modifications in archival FFPE tissues with high sensitivity and help researchers to better understand epigenetic regulation in cancer and human disease.