Analysis Of Formalin-fixed Paraffin-embedded Tissue Research Articles

Biopsy Proteome Scoring to Determine Mucosal Remodeling in Celiac Disease

Histologic evaluation of gut biopsies is a cornerstone for diagnosis and management of celiac disease (CeD). Despite its wide use, the method depends on proper biopsy orientation, and it suffers from interobserver variability. Biopsy proteome measurement reporting on the tissue state can be obtained by mass spectrometry analysis of formalin-fixed paraffin-embedded tissue. Here we aimed to transform biopsy proteome data into numerical scores that give observer-independent measures of mucosal remodeling in CeD. A pipeline using glass-mounted formalin-fixed paraffin-embedded sections for mass spectrometry-based proteome analysis was established. Proteome data were converted to numerical scores using 2 complementary approaches: a rank-based enrichment score and a score based on machine learning using logistic regression. The 2 scoring approaches were compared with each other and with histology analyzing 18 patients with CeD with biopsies collected before and after treatment with a gluten-free diet as well as biopsies from patients with CeD with varying degree of remission (n= 22). Biopsies from individuals without CeD (n= 32) were also analyzed. The method yielded reliable proteome scoring of both unstained and H&E-stained glass-mounted sections. The scores of the 2 approaches were highly correlated, reflecting that both approaches pick up proteome changes in the same biological pathways. The proteome scores correlated with villus height-to-crypt depth ratio. Thus, the method is able to score biopsies with poor orientation. Biopsy proteome scores give reliable observer and orientation-independent measures of mucosal remodeling in CeD. The proteomic method can readily be implemented by nonexpert laboratories in parallel to histology assessment and easily scaled for clinical trial settings.

Proteomics analysis of an individual formalin-fixed paraffin-embedded tissue section using isobaric-tag amplification.

The comprehensive analysis of formalin-fixed paraffin-embedded (FFPE) tissues is essential for retrospective clinical studies. However, detecting low-abundance proteins and obtaining proteome-scale data from FFPE samples pose analytical challenges in mass spectrometry-based proteomics. To overcome this challenge, our study focuses on implementing an isobaric labeling approach to improve the detection of low-abundance target proteins in FFPE tissues, thereby enhancing the qualitative and quantitative analysis. We employed an isobaric labeling approach utilizing synthetic peptides or proteins to enable the qualitative and quantitative measurement of target proteins in FFPE tissue samples. To achieve this, we incorporated tandem mass tag (TMT)-labeled recombinant proteins or synthetic peptides into TMT-labeled metastatic breast cancer FFPE tissues. Through this strategy, we successfully detect coexisting CD276 (B7-H3) and CD147 proteins while identifying over 6000 proteins using targeted analysis of individual FFPE tissue sections. Our findings provide compelling evidence that the incorporation of isobaric labeling, along with the inclusion of TMT-labeled peptides or proteins, greatly enhances the detection of target proteins in FFPE tissue samples. By employing this approach, we were able to obtain robust qualitative measurements of CD276 and CD147 proteins, showcasing its effectiveness in identifying more than 6000 proteins in FFPE samples. The integration of an isobaric labeling approach, in conjunction with synthetic peptides or proteins, presents a valuable strategy for enhancing the detection and validation of target proteins in FFPE tissue analysis. This technique holds immense potential in retrospective clinical studies, as it enables comprehensive analysis of low-abundance proteins and facilitating proteome-scale investigations in FFPE samples. By leveraging this methodology, researchers can unlock new insights into disease mechanisms and advance our understanding of complex biological processes.

RNA Extraction Method Impacts Quality Metrics and Sequencing Results in Formalin-Fixed, Paraffin-Embedded Tissue Samples

Archived formalin-fixed, paraffin-embedded (FFPE) tissue samples are being increasingly used in molecular cancer research. Compared with fresh-frozen tissue, the nucleic acid analysis of FFPE tissue is technically more challenging. This study aimed to compare the impact of 3 different RNA extraction methods on yield, quality, and sequencing-based gene expression results in FFPE samples. RNA extraction was performed in 16 FFPE tumor specimens from patients with diffuse large B-cell lymphoma and in reference FFPE material from microsatellite-stable and microsatellite-instable cell lines (3 replicates each) using 2 silica-based procedures (A, miRNeasy FFPE; C, iCatcher FFPE Tissue RNA) and 1 isotachophoresis-based procedure (B, Ionic FFPE to Pure RNA). The RNA yield; RNA integrity, as reflected by the distribution value 200; and RNA purity, as reflected by the 260/280 and the 260/230 nm absorbance ratios, were determined. The RNA was sequenced on the NovaSeq 6000 instrument using the TruSeq RNA Exome and SMARTer Stranded Total RNA-Seq Pico v3 library preparations kits. Our results highlight the impact of RNA extraction methodology on both preanalytical and sequencing-based gene expression results. Overall, methods B and C outperformed method A because these showed significantly higher fractions of uniquely mapped reads, an increased number of detectable genes, a lower fraction of duplicated reads, and better representation of the B-cell receptor repertoire. Differences among the extraction methods were generally more explicit for the total RNA sequencing method than for the exome-capture sequencing method. Importantly, the predicative value of quality metrics varies among extraction kits, and caution should be applied when comparing and interpreting results obtained using different methods.

The addition of FAIMS increases targeted proteomics sensitivity from FFPE tumor biopsies

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM. Limits of quantitation were determined by spiking synthetic peptides into a human spleen matrix. In addition, 20 clinical samples were analyzed using FAIMS-PRM and the quantitation of HER2 was compared with that obtained with the Ventana immunohistochemistry assay. FAIMS-PRM improved the overall signal-to-noise ratio over that from PRM and increased assay sensitivity in FFPE tissue analysis for four (HER2, EGFR, cMET, and KRAS) of five proteins of clinical interest. FAIMS-PRM enabled sensitive quantitation of basal HER2 expression in breast cancer samples classified as HER2 negative by immunohistochemistry. Furthermore, we determined the degree of FAIMS-dependent background reduction and showed that this correlated with an improved lower limit of quantitation with FAIMS. FAIMS-PRM is anticipated to benefit clinical trials in which multiple biomarker questions must be addressed and the availability of tumor biopsy samples is limited.

Atmospheric-Pressure Infrared Laser-Ablation Plasma-Postionization Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh-Frozen Tissue Sections with No Sample Preparation.

Mass spectrometry imaging (MSI) encompasses a powerful suit of techniques which provide spatially resolved atomic and molecular information from almost any sample type. MSI is now widely used in preclinical research to provide insight into metabolic phenotypes of disease. Typically, fresh-frozen tissue preparations are considered optimal for biological MSI and other traditional preservation methods such as formalin fixation, alone or with paraffin embedding (FFPE), are considered less optimal or even incompatible. Due to the prevalence of FFPE tissue storage, particularly for rare and therefore high-value tissue samples, there is substantial motivation for optimizing MSI methods for analysis of FFPE tissue. Here, we present a novel modality, atmospheric-pressure infrared laser-ablation plasma postionization (AP-IR-LA-PPI), with the first proof-of-concept examples of MSI for FFPE and fresh-frozen tissues, with no post-sectioning sample preparation. We present ion images from FFPE and fresh tissues in positive and negative ion modes. Molecular annotations (via the Metaspace annotation engine) and on-tissue MS/MS provide additional confidence that the detected ions arise from a broad range of metabolite and lipid classes from both FFPE and fresh-frozen tissues.

Abstract 3864: Cytometric analysis of immune cell populations in clinical tumor biopsy tissue microarrays for immuno-oncology

Abstract Over the past decade, a new focus on the interactions between the immune system and the development and progression of cancer has led to a wide array of research into medical treatments which leverage activation or modulation of the immune system to aid in treatment. Research in the field of immuno-oncology and elucidation of the biological mechanisms by which cancer cells suppress immunological function has led to new biological treatments for cancer, such as pembrolizumab, which have shown great clinical success since their advent. Population-level analysis of immune cell populations is critical for understanding the immune-component of disease progression, and flow cytometry is a commonly utilized technique for this purpose. However, flow cytometry requires dissolution of tissue specimens, and is incompatible with formalin-fixed paraffin-embedded (FFPE) samples, the gold standard in tissue archives. In order to allow for cytometric analysis of FFPE tissues, we have developed a digital pathology technique for the quantitative assessment of immune-cell populations within tissue microarrays prepared from archived FFPE clinical tissue specimens from patient biopsies. The technique involves a combination of HALO® image analysis using the Highplex FL, Spatial Analysis, and TMA modules, and custom scripting to develop imaging cytometry scatterplots. Bioinformatics techniques were utilized to assess features correlated with tumor progression from the quantitative data extracted from the tissue microarrays. Citation Format: Tom Villani, Jigar Patel, Kim Collins, Kate Lillard Tunstall, Bradley Krisanits. Cytometric analysis of immune cell populations in clinical tumor biopsy tissue microarrays for immuno-oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3864.

Spatial Whole Transcriptome Profiling of the Tumor Microenvironment in Prostate Carcinomas

BackgroundDespite years of studies and effort, the best strategies for treating prostate cancer and minimizing the complications of treatment remain unanswered questions. This gap in knowledge is partially due to the inability to dissect the complex heterogeneous tumor microenvironment (TME) and immune compartment. Spatially resolved molecular profiling of tumor sections will enhance our understanding of these complexities; However, it has been particularly challenging to do spatial molecular profiling in formalin‐fixed paraffin‐embedded (FFPE) tissues due to RNA degradation associated with this tissue‐embedding method, which is routinely used in oncology workflows.The 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue overcomes these limitations, enabling spatial gene expression analysis of FFPE tissues combined with classical histology staining techniques such as Hematoxylin & Eosin (H&E) staining and immunofluorescence.MethodsWe used the 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue to analyze and resolve tumorigenic profiles in sections of normal and adenocarcinoma prostate samples. This assay incorporates ~5,000 molecularly barcoded, spatially encoded capture probes in spots over which the tissue is placed, imaged, and permeabilized. Imaging and sequencing data are processed together, resulting in a spatially resolved transcriptional readout.ResultsWe profiled the whole transcriptome in normal, invasive adenocarcinoma, and acinar cell carcinoma FFPE human prostate tissues.Unsupervised clustering of the whole transcriptome data from normal, invasive adenocarcinoma, and acinar cell prostate carcinoma FFPE sections enabled the identification of 2 different regions, which had a well defined spatial distribution within the tissues. Well known prostate gland and prostate‐cancer markers were over‐expressed in the corresponding healthy and cancerous portions of the tissue, validating the performance of this method. We found that, while in healthy tissues basal cells and luminal cells are spatially organized, this pattern is lost in tumor samples, where luminal cells are greatly expanded in the invasive carcinoma region and do not colocalize with basal cells. Moreover, T lymphocytes are dispersed throughout the whole tissue section in the adenocarcinoma, while plasma B cells are located in the peritumoral region which could impact prognosis.ConclusionsSpatial whole transcriptome analysis opens new opportunities for better understanding the TME which can not only help discover novel predictive tumor biomarkers, but also enable identifying cell type and tumor region specific drug targets.

Evaluation of Disposable Trap Column nanoLC-FAIMS-MS/MS for the Proteomic Analysis of FFPE Tissue.

Proteomic biomarker discovery using formalin-fixed paraffin-embedded (FFPE) tissue requires robust workflows to support the analysis of large cohorts of patient samples. It also requires finding a reasonable balance between achieving a high proteomic depth and limiting the overall analysis time. To this end, we evaluated the merits of online coupling of single-use disposable trap column nanoflow liquid chromatography, high-field asymmetric-waveform ion-mobility spectrometry (FAIMS), and tandem mass spectrometry (nLC-FAIMS-MS/MS). The data show that ≤600 ng of peptide digest should be loaded onto the chromatographic part of the system. Careful characterization of the FAIMS settings enabled the choice of optimal combinations of compensation voltages (CVs) as a function of the employed LC gradient time. We found nLC-FAIMS-MS/MS to be on par with StageTip-based off-line basic pH reversed-phase fractionation in terms of proteomic depth and reproducibility of protein quantification (coefficient of variation ≤15% for 90% of all proteins) but requiring 50% less sample and substantially reducing sample handling. Using FFPE materials from the lymph node, lung, and prostate tissue as examples, we show that nLC-FAIMS-MS/MS can identify 5000-6000 proteins from the respective tissue within a total of 3 h of analysis time.

72 Spatial whole transcriptome profiling of the tumor microenvironment in prostate carcinomas

BackgroundDespite years of studies and effort, the best strategies for treating prostate cancer and minimizing the complications of treatment remain unanswered questions. This gap in knowledge is partially due to the inability to dissect the complex heterogeneous tumor microenvironment (TME) and immune compartment. Spatially resolved molecular profiling of tumor sections will enhance our understanding of these complexities; However, it has been particularly challenging to do spatial molecular profiling in formalin-fixed paraffin-embedded (FFPE) tissues due to RNA degradation associated with this tissue-embedding method, which is routinely used in oncology workflows. The 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue overcomes these limitations, enabling spatial gene expression analysis of FFPE tissues combined with classical histology staining techniques such as Hematoxylin & Eosin (H&E) staining and immunofluorescence.MethodsWe used the 10x Genomics Visium Spatial Gene Expression Solution for FFPE tissue to analyze and resolve tumorigenic profiles in sections of normal and adenocarcinoma prostate samples. This assay incorporates ~5,000 molecularly barcoded, spatially encoded capture probes in spots over which the tissue is placed, imaged, and permeabilized. Imaging and sequencing data are processed together, resulting in a spatially resolved transcriptional readout.ResultsWe profiled the whole transcriptome in normal, invasive adenocarcinoma, and acinar cell carcinoma FFPE human prostate tissues. Unsupervised clustering of the whole transcriptome data from normal, invasive adenocarcinoma, and acinar cell prostate carcinoma FFPE sections enabled the identification of 2 different regions, which had a well defined spatial distribution within the tissues. Well known prostate gland and prostate-cancer markers were over-expressed in the corresponding healthy and cancerous portions of the tissue, validating the performance of this method. We found that, while in healthy tissues basal cells and luminal cells are spatially organized, this pattern is lost in tumor samples, where luminal cells are greatly expanded in the invasive carcinoma region and do not colocalize with basal cells. Moreover, T lymphocytes are dispersed throughout the whole tissue section in the adenocarcinoma, while plasma B cells are located in the peritumoral region which could impact prognosis.ConclusionsSpatial whole transcriptome analysis opens new opportunities for better understanding the TME which can not only help discover novel predictive tumor biomarkers, but also enable identifying cell type and tumor region specific drug targets.

Technical challenges regarding the use of formalin-fixed paraffin embedded (FFPE) tissue specimens for the detection of bacterial alterations in colorectal cancer

BackgroundFormalin-fixed paraffin embedded (FFPE) tissues may provide an exciting resource to study microbial associations in human disease, but the use of these low biomass specimens remains challenging. We aimed to reduce unintentional bacterial interference in molecular analysis of FFPE tissues and investigated the feasibility of conducting quantitative polymerase chain reaction (qPCR) and 16S rRNA amplicon sequencing using 14 colorectal cancer, 14 normal adjacent and 13 healthy control tissues.ResultsBacterial contaminants from the laboratory environment and the co-extraction of human DNA can affect bacterial analysis. The application of undiluted template improves bacterial DNA amplification, allowing the detection of specific bacterial markers (Escherichia coli and Faecalibacterium prausnitzii) by qPCR. Nested and non-nested PCR-based 16S rRNA amplicon sequencing approaches were employed, showing that bacterial communities of tissues and paired paraffin controls cluster separately at genus level on weighted Unifrac in both non-nested (R2 = 0.045; Pr(> F) = 0.053) and nested (R2 = 0.299; Pr(> F) = 0.001) PCR datasets. Nevertheless, considerable overlap of bacterial genera within tissues was seen with paraffin, DNA extraction negatives (non-nested PCR) or PCR negatives (nested PCR). Following mathematical decontamination, no differences in α- and β diversity were found between tumor, normal adjacent and control tissues.ConclusionsBacterial marker analysis by qPCR seems feasible using non-normalized template, but 16S rRNA amplicon sequencing remains challenging. Critical evaluation of laboratory procedures and incorporation of positive and negative controls for bacterial analysis of FFPE tissues are essential for quality control and to account for bacterial contaminants.

Comparison of different digestion methods for proteomic analysis of isolated cells and FFPE tissue samples

Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients’ treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.

The Immune Checkpoint Inhibitor LAG-3 and Its Ligand GAL-3 in Vulvar Squamous Neoplasia.

Vulvar squamous cell carcinoma (vSCC), although rare, carries significant morbidity and a high rate of recurrence. Treatment options beyond surgical excision remain limited. Lymphocyte activation gene-3 (LAG-3) and its binding partner galectin-3 (GAL-3) are an immuno-inhibitory checkpoint pair that represent potential immunotherapy targets for the treatment of vSCC. This study examined the expression of LAG-3 and GAL-3 alongside programmed cell death ligand-1 expression in invasive SCC and vulvar intraepithelial neoplasia (VIN) by immunohistochemical analysis of formalin-fixed paraffin-embedded tissue. A total of 35 cases were selected for evaluation: 13 VIN3 [human papillomavirus (HPV)-associated VIN/usual-type VIN], 2 differentiated VIN (dVIN), 16 HPV-associated vSCC, and 4 dVIN-associated vSCC. LAG-3+ tumor-infiltrating lymphocytes were identified in 91% (32/35) of cases of vulvar squamous neoplasia. Tumor cells were positive for GAL-3 in 71% of the vulvar neoplasia cases. HPV-associated vSCC was more likely to demonstrate GAL-3 tumoral positivity when compared with dVIN-associated vSCC (24/29 vs. 1/6, P=0.004). We observed co-expression of all 3 immunomarkers in 40% (14/35) of cases evaluated. In light of these findings, use of immunomodulatory drugs that target the LAG-3/GAL-3 pathway may be potentially beneficial in vSCC and efficacy may be increased when combined with anti-programmed cell death ligand-1 therapy.

Molecular alterations in oral cancer using high-throughput proteomic analysis of formalin-fixed paraffin-embedded tissue.

Loss of cell differentiation is a hallmark for the progression of oral squamous cell carcinoma (OSCC). Archival Formalin-Fixed Paraffin-Embedded (FFPE) tissues constitute a valuable resource for studying the differentiation of OSCC and can offer valuable insights into the process of tumor progression. In the current study, we performed LC-MS/MS-based quantitative proteomics of FFPE specimens from pathologically-confirmed well-differentiated, moderately-differentiated, and poorly-differentiated OSCC cases. The data were analyzed in four technical replicates, resulting in the identification of 2376 proteins. Of these, 141 and 109 were differentially expressed in moderately-differentiated and poorly differentiated OSCC cases, respectively, compared to well-differentiated OSCC. The data revealed significant metabolic reprogramming with respect to lipid metabolism and glycolysis with proteins belonging to both these processes downregulated in moderately-differentiated OSCC when compared to well-differentiated OSCC. Signaling pathway analysis indicated the alteration of extracellular matrix organization, muscle contraction, and glucose metabolism pathways across tumor grades. The extracellular matrix organization pathway was upregulated in moderately-differentiated OSCC and downregulated in poorly differentiated OSCC, compared to well-differentiated OSCC. PADI4, an epigenetic enzyme transcriptional regulator, and its transcriptional target HIST1H1B were both found to be upregulated in moderately differentiated and poorly differentiated OSCC, indicating epigenetic events underlying tumor differentiation. In conclusion, the findings support the advantage of using high-resolution mass spectrometry-based FFPE archival blocks for clinical and translational research. The candidate signaling pathways identified in the study could be used to develop potential therapeutic targets for OSCC.

Comparative evaluation of two methods for LC-MS/MS proteomic analysis of formalin fixed and paraffin embedded tissues

The proteomics of formalin-fixed, paraffin-embedded (FFPE) samples has advanced significantly during the last two decades, but there are many protocols and few studies comparing them directly. There is no consensus on the most effective protocol for shotgun proteomic analysis. We compared the in-solution digestion with RapiGest and Filter Aided Sample Preparation (FASP) of FFPE prostate tissues stored 7 years and mirroring fresh frozen samples, using two label-free data-independent LC-MS/MS acquisitions. RapiGest identified more proteins than FASP, with almost identical numbers of proteins from fresh and FFPE tissues and 69% overlap, good preservation of high-MW proteins, no bias regarding isoelectric point, and greater technical reproducibility. On the other hand, FASP yielded 20% fewer protein identifications in FFPE than in fresh tissue, with 64–69% overlap, depletion of proteins >70 kDa, lower efficiency in acidic and neutral range, and lower technical reproducibility. Both protocols showed highly similar subcellular compartments distribution, highly similar percentages of extracted unique peptides from FFPE and fresh tissues and high positive correlation between the absolute quantitation values of fresh and FFPE proteins. In conclusion, RapiGest extraction of FFPE tissues delivers a proteome that closely resembles the fresh frozen proteome and should be preferred over FASP in biomarker and quantification studies. SignificanceHere we analyzed the performance of two sample preparation methods for shotgun proteomic analysis of FFPE tissues to give a comprehensive overview of the obtained proteomes and the resemblance to its matching fresh frozen counterparts. These findings give us better understanding towards competent proteomics analysis of FFPE tissues. It is hoped that it will encourage further assessments of available protocols before establishing the most effective protocol for shotgun proteomic FFPE tissue analysis.

Quantitative Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded (FFPE) Human Cardiac Tissue.

Clinical tissue archives represent an invaluable source of biological information. Formalin-fixed, paraffin-embedded (FFPE) tissue can be used for retrospective investigation of biomarkers of diseases and prognosis.Recently, the number of studies using proteome profiling of samples from clinical archives has markedly increased. However, the application of conventional quantitative proteomics technologies remains a challenge mainly due to the harsh fixation process resulting in protein cross-linking and protein degradation. In the present chapter, we demonstrate a protocol for label-free proteomic analysis of FFPE tissue prepared from human cardiac autopsies. The data presented here highlight the applicability and suitability of FFPE heart tissue for understanding the molecular mechanism of cardiac injury using a proteomics approach.

FiTAc-seq: fixed-tissue ChIP-seq for H3K27ac profiling and super-enhancer analysis of FFPE tissues

Fixed-tissue ChIP-seq for H3K27 acetylation (H3K27ac) profiling (FiTAc-seq) is an epigenetic method for profiling active enhancers and promoters in formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a modified ChIP-seq protocol (FiT-seq) for chromatin profiling in FFPE. FiT-seq produces high-quality chromatin profiles particularly for methylated histone marks but is not optimized for H3K27ac profiling. FiTAc-seq is a modified protocol that replaces the proteinase K digestion applied in FiT-seq with extended heating at 65 °C in a higher concentration of detergent and a minimized sonication step, to produce robust genome-wide H3K27ac maps from clinical samples. FiTAc-seq generates high-quality enhancer landscapes and super-enhancer (SE) annotation in numerous archived FFPE samples from distinct tumor types. This approach will be of great interest for both basic and clinical researchers. The entire protocol from FFPE blocks to sequence-ready library can be accomplished within 4 d.

SWATH-MS Analysis of FFPE Tissues Identifies Stathmin as a Potential Marker of Endometrial Cancer in Patients Exposed to Tamoxifen

A specific form of endometrial cancer (EC) can develop in breast cancer patients previously treated with tamoxifen (ET), an antagonist of estrogen receptor (ER) that inhibits proliferation of ER positive breast cancer. ET tumors have a different phenotype than endometrial tumors, which typically develop de novo without previous exposure to tamoxifen (EN). Here we aimed to identify specific protein markers that could serve as specific molecular targets in either phenotype. A set of total 45 formalin-fixed paraffin-embedded (FFPE) endometrial tumor tissues and adjacent myometrium tissue samples were analyzed using LC-MS/MS in SWATH-MS mode. We found that calcyphosin (CAPS) levels were elevated in EN tumors compared to ET tumors. The higher CAPS level in EC tissue invading to myometrium supports its relationship to EC aggressiveness. Further, stathmin (STMN1) levels were found significantly elevated in ET versus EN tumors and significantly associated with patient survival. This finding connects elevated levels of this cell cycle regulating, proliferation-associated protein with tamoxifen exposure. In summary, using SWATH-MS we show that CAPS and STMN1 should be recognized as clinicopathologically different EC markers of which STMN1 is specifically connected with a previous tamoxifen exposition.

Proteomics Analysis of Formalin Fixed Paraffin Embedded Tissues in the Investigation of Prostate Cancer.

Prostate cancer (PCa) is one of the leading causes of death in men worldwide. The molecular features, associated with the onset and progression of the disease, are under vigorous investigation. Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources for large-scale studies; however, their application in proteomics is limited due to protein cross-linking. In this study, the adjustment of a protocol for the proteomic analysis of FFPE tissues was performed which was followed by a pilot application on FFPE PCa clinical samples to investigate whether the optimized protocol can provide biologically relevant data for the investigation of PCa. For the optimization, FFPE mouse tissues were processed using seven protein extraction protocols including combinations of homogenization methods (beads, sonication, boiling) and buffers (SDS based and urea-thiourea based). The proteome extraction efficacy was then evaluated based on protein identifications and reproducibility using SDS electrophoresis and high resolution LC-MS/MS analysis. Comparison between the FFPE and matched fresh frozen (FF) tissues, using an optimized protocol involving protein extraction with an SDS-based buffer following beads homogenization and boiling, showed a substantial overlap in protein identifications with a strong correlation in relative abundances (rs = 0.819, p < 0.001). Next, FFPE tissues (3 sections, 15 μm each per sample) from 10 patients with PCa corresponding to tumor (GS = 6 or GS ≥ 8) and adjacent benign regions were processed with the optimized protocol. Extracted proteins were analyzed by GeLC-MS/MS followed by statistical and bioinformatics analysis. Proteins significantly deregulated between PCa GS ≥ 8 and PCa GS = 6 represented extracellular matrix organization, gluconeogenesis, and phosphorylation pathways. Proteins deregulated between cancerous and adjacent benign tissues, reflected increased translation, peptide synthesis, and protein metabolism in the former, which is consistent with the literature. In conclusion, the results support the relevance of the proteomic findings in the context of PCa and the reliability of the optimized protocol for proteomics analysis of FFPE material.

Identification of Coiled-Coil Domain–Containing Protein 180 and Leucine-Rich Repeat–Containing Protein 4 as Potential Immunohistochemical Markers for Liposarcoma Based on Proteomic Analysis Using Formalin-Fixed, Paraffin-Embedded Tissue

Recent technical improvements in both mass spectrometry and protein extraction have made it possible to use formalin-fixed, paraffin-embedded (FFPE) tissues for proteome analysis. In this study, comparable proteome analysis of FFPE tissues revealed multiple candidate marker molecules for differentiating atypical lipomatous tumor/well-differentiated liposarcoma (ALT/WDL) from lipoma. A total of 181 unique proteins were identified for ALT/WDL. Of the identified proteins, coiled-coil domain-containing protein 180 (CCDC180) and leucine-rich repeat-containing protein 4 (LRRC4) were studied as candidate markers of ALT/WDL. CCDC180 and LRRC4 immunohistochemistry clearly stained tumor cells of ALT/WDL and dedifferentiated liposarcoma and could differentiate them from lipoma with high accuracy. Cell biological methods were used to further examine the expression of the candidate marker molecules in liposarcoma cells. In liposarcoma cells, knockdown of CCDC180 and LRRC4 inhibited cell proliferation. CCDC180 inhibited cell migration, invasion, and apoptosis resistance in WDL cells. Adipogenic differentiation suppressed the expression of CCDC180 and LRRC4 in WDL cells. These results indicated that LRRC4 and CCDC180 are novel immunohistochemical markers for differentiating ALT/WDLs. Their expression was associated with adipocyte differentiation and contributed to malignant potentials of WDL cells. Proteome analysis using a standard stock of FFPE tissues can reveal novel biomarkers for various diseases, which contributes to the progress of molecular pathology.

Abstract A074: Spatially resolved deep antigen profiling of single cells in FFPE tissue samples through CODEXTM

Abstract Spatially resolved, deep antigen profiling of tissue specimens is crucial for investigating the architecture and cell diversity in complex matrices, such as the tumor microenvironment (TME). The correlation of these two parameters with the progression of a variety of pathologies, ranging from cancer to autoimmune diseases, is still vastly unknown due to the lack of technologies that enable the detection of both high content and spatial resolution. Formalin-fixed, paraffin-embedded (FFPE) tissues constitute the ideal sample for these investigations as they retain tissue morphology at room temperature for long periods of time, which is convenient and cost-effective, and are available in vast specimen archives. Akoya Biosciences, Inc., is commercializing CODEXTM (CO-Detection by indEXing), a multiparameteric imaging platform that performs the concurrent detection and quantification of dozens of antigens with single cell resolution within a tissue specimen. The CODEX platform uses a DNA-based barcode library to label antibodies and iterative cycles of adding and removing cognate dye-labeled oligonucleotides to reveal the staining pattern for a subset of the target markers per cycle. The entire data acquisition process is fully automated using the CODEX instrument, which integrates with existing microscopes. To support high-multiplexing capabilities, more than 45 different antibody clones have been screened and validated for the detection of target epitopes in human FFPE secondary lymphoid tissues for both healthy and cancerous specimens using the CODEX platform. Proof-of-concept data have also been obtained for different cancer types, including melanoma and head and neck cancer in both normal and TMA formats. Analysis software has been developed to extract meaning from CODEX datasets and is included as part of the CODEX platform. The analysis pipeline includes image drift compensation, deconvolution and segmentation to measure integrated fluorescence intensity for each cell across tens of parameters. In this manner, infiltrating lymphocytes and other immune cells have been identified within the TME. With the high multiplexing capabilities, a variety of T-cells can be identified to detect the ratios of both immunosuppressive and immune-activating subtypes. Analysis of FFPE tissues using the CODEX platform demonstrates the unparalleled opportunities for simultaneously detecting tens of antigens with single-cell resolution within a tissue specimen. Citation Format: Maria Elena Gallina, Atri Choksi, Nadya Nikulina, Jaskirat Singh, Gajalakshmi Dakshinamoorthy, Joseph Kim, Sejal Mistry, Julia Kennedy-Darling. Spatially resolved deep antigen profiling of single cells in FFPE tissue samples through CODEXTM [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A074.