A SpliceAI-guided workflow for transcript validation using archived formalin-fixed paraffin-embedded skin tissue: Application to ATP2C1 splice-region variation in Hailey-Hailey disease.

Proteomic analysis of formalin-fixed paraffin-embedded (FFPE) tissue would enable retrospective biomarker investigations of this vast archive of pathologically characterized clinical samples that exist worldwide. These FFPE tissues are, however, refractory to proteomic investigations utilizing many state of the art methodologies largely due to the high level of covalently cross-linked proteins arising from formalin fixation. A novel tissue microdissection technique has been developed and combined with a method to extract soluble peptides directly from FFPE tissue for mass spectral analysis of prostate cancer (PCa) and benign prostate hyperplasia (BPH). Hundreds of proteins from PCa and BPH tissue were identified, including several known PCa markers such as prostate-specific antigen, prostatic acid phosphatase, and macrophage inhibitory cytokine-1. Quantitative proteomic profiling utilizing stable isotope labeling confirmed similar expression levels of prostate-specific antigen and prostatic acid phosphatase in BPH and PCa cells, whereas the expression of macrophage inhibitory cytokine-1 was found to be greater in PCa as compared with BPH cells.

Many liquid chromatography-mass spectrometry (LC-MS) studies have compared formalin-fixed paraffin-embedded (FFPE) tissues with matched fresh-frozen (FF) tissues to examine the effect of preservation techniques on the proteome; however, few studies have included the phosphoproteome. A high degree of overlap and correlation between the two preservation techniques would demonstrate the importance of FFPE tissues as a valuable biomedical resource. Our aim was to quantitatively compare the proteome and phosphoproteome of matched FFPE and FF tissues using data-independent acquisition LC-MS. Four organs from three rats were cut in half to produce matched FFPE and FF tissue pairs. Excellent overlaps of 85-97% for the proteome and 82-98% for the phosphoproteome were observed, depending on the organ type, between the two preservation techniques. Most of the unique identifications were found in FF with less than 0.3% being unique to FFPE tissues. Strong agreement between FFPE and FF matched tissue pairs was observed with Pearson correlation coefficients of 0.93-0.97 and 0.79-0.87 for the proteome and phosphoproteome, respectively. Digestion efficiency was slightly higher in FFPE (92-94%) than in FF tissues (86-89%), and a search of a data subset for formaldehyde induced chemical modifications revealed that only 0.05% of precursors were unique to FFPE tissues. This suggests that with quality sample preparation methods it is not necessary to include formaldehyde induced chemical modifications when analyzing FFPE tissues. We attribute the lower number of identifications in FFPE tissues to inaccurate peptide quantitation, which resulted in a lower MS peptide load and tryptic peptide enrichment load. Our results demonstrate that both proteomic and phosphoproteomic analyses of FFPE and FF tissues are highly comparable and highlight the suitability of FFPE tissues for both proteomic and phosphoproteomic analysis.

Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.

Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues

The metabolic landscape of the prostate changes with malignant transformation, and metabolomics is a means of identifying diagnostic and prognostic biomarkers for prostate cancer. Epidemiology and registry studies containing archival formalin-fixed paraffin-embedded (FFPE) specimens are a rich resource for this work but this method of tissue preservation presents a challenge due to loss of metabolites. We optimized global metabolic profiling in FFPE tissue and identified metabolites distinguishing tumor from normal prostate tissue, and aggressive from non-aggressive prostate cancer. For optimization of metabolomics in FFPE tissues, we obtained matched frozen and FFPE tumor tissue from 62 prostate cancer cases who underwent radical prostatectomy (RP) at the University of North Carolina (UNC) hospital, of which 24 also had adjacent normal prostate tissue. To identify metabolites associated with tumor aggressiveness, we obtained matched tumor and normal FFPE tissue from 60 prostate cancer patients (Gleason ≥4+3, n=30; Gleason ≤3+4, n=30) who underwent RP participating in the UNC Health Registry/Cancer Survivorship Cohort. Following log transformation and imputation of missing values with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between groups. Random forest analysis evaluated predictive accuracy of metabolites for distinguishing tumor from normal prostate tissue. We identified 768 compounds of known identity in frozen and 119 in matched FFPE tissues. Therefore, only 15% of the metabolites detected in frozen samples were recovered from corresponding FFPE tissue, with 80% of these common to both FFPE and matched frozen tissues. We found a predictive accuracy of 78% and 67% for frozen and FFPE tissue, respectively, for distinguishing tumor from normal. Of 39 metabolites significantly altered between matched tumor and normal FFPE tissue, 47% belonged to the lipid class, and despite a greater number of significantly altered metabolites in frozen tissue (246), the proportion belonging to the lipid class was similar (50%). Citrate, integral to normal prostate metabolism, and citrulline, involved in nitric oxide synthesis, had significantly lower levels in aggressive versus non-aggressive tumors. Cell membrane phospholipids were found at significantly higher levels in aggressive tumors. Common patterns of altered metabolites in tumor vs. normal prostate were observed irrespective of tissue preservation method, despite loss of a large proportion of metabolites during the process of FFPE. As such, FFPE studies are feasible particularly when lipid metabolism is of interest as this class appears one of the best preserved in FFPE. This work will pave the way for incorporating metabolomics profiling of FFPE specimens into epidemiology and registry-based studies of prostate cancer. Citation Format: Sophia Rachael Halliday, Linnea T. Olsson, Alina Hamilton, Sivapriya Ramamoorthy, Jason Kitchen, Erin Kirk, Laura Farnan, Adrian Gerstel, Melissa A. Troester, Jannette T. Bensen, Sara E. Wobker, Emma Allott. Metabolomics profiling of formalin-fixed paraffin-embedded prostate tissues [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 2347.

Use of Formalin-fixed Paraffin-embedded Tumor Tissue as a DNA Source in Molecular Epidemiological Studies of Pediatric CNS Tumors

The ability to accurately quantify proteins in formalin-fixed paraffin-embedded tissues using targeted mass spectrometry opens exciting perspectives for biomarker discovery. We have developed and evaluated a selectedreaction monitoring assay for the human receptor tyrosine-protein kinase erbB-2 (HER2) in formalin-fixed paraffin-embedded breast tumors. Peptide candidates were identified using an untargeted mass spectrometry approach in relevant cell lines. A multiplexed assay was developed for the six best candidate peptides and evaluated for linearity, precision and lower limit of quantification. Results showed a linear response over a calibration range of 0.012 to 100 fmol on column (R(2): 0.99-1.00).The lower limit of quantification was 0.155 fmol on column for all peptides evaluated. The six HER2 peptides were quantified by selected reaction monitoring in a cohort of 40 archival formalin-fixed paraffin-embedded tumor tissues from women with invasive breast carcinomas, which showed different levels of HER2 gene amplification as assessed by standard methods used in clinical pathology. The amounts of the six HER2 peptides were highly and significantly correlated with each other, indicating that peptide levels can be used as surrogates of protein amounts in formalin-fixed paraffin-embedded tissues. After normalization for sample size, selected reaction monitoring peptide measurements were able to correctly predict 90% of cases based on HER2 amplification as defined by the American Society of Clinical Oncology and College of American Pathologists. In conclusion, the developed assay showed good analytical performance and a high agreement with immunohistochemistry and fluorescence in situ hybridization data. This study demonstrated that selected reaction monitoring allows to accurately quantify protein expression in formalin-fixed paraffin-embedded tissues and represents therefore a powerful approach for biomarker discovery studies. The untargeted mass spectrometry data is available via ProteomeXchange whereas the quantification data by selected reaction monitoring is available on the Panorama Public website.

Background: Next generation sequencing (NGS)-based multi-gene panel tests have been performed to predict the treatment response and prognosis in patients with colorectal cancer (CRC). Whether the multi-gene mutation results of formalin-fixed paraffin-embedded (FFPE) tissues are identical to those of fresh frozen tissues remains unknown.Methods: A 22-gene panel with 103 hotspots was used to detect mutations in paired fresh frozen tissue and FFPE tissue from 118 patients with CRC.Results: In our study, 117 patients (99.2%) had one or more variants, with 226 variants in FFPE tissue and 221 in fresh frozen tissue. Of the 129 variants identified in this study, 96 variants were present in both FFPE and fresh frozen tissues; 27 variants were found in FFPE tissues only; 6 variants were found only in fresh frozen tissues. The mutation results demonstrated >94.0% concordance in all variants, with Kappa coefficient >0.500 in 64.3% (83/129) of variants. At the gene level, concordance ranged from 73.8 to 100.0%, with Kappa coefficient >0.500 in 81.3% (13/16) of genes.Conclusions: The results of mutation analysis performed with a multi-gene panel and FFPE and fresh frozen tissue were highly concordant in patients with CRC, at both the variant and gene levels. There were, however, some important differences in mutation results between the two tissue types. Therefore, fresh frozen tissue should not routinely be replaced with FFPE tissue for mutation analysis with a multi-gene panel. Rather, FFPE tissue is a reasonable alternative for fresh frozen tissue when the latter is unavailable.

Formalin-fixed paraffin-embedded (FFPE) tissues are important resources for molecular medical research. However, long-chain RNA analysis is restricted in FFPE tissues due to high levels of degradation. To explore the possibility of long RNA quantification in FFPE tissues, we selected 14 target RNAs (8 mRNAs and 6 long noncoding RNAs) from literatures, and designed short (~60 bp) and long (~200 bp) amplicons for each of them. Colorectal carcinomas with adjacent normal tissues were subjected to quantitative reverse-transcription PCR (quantitative RT-PCR) in 3 cohorts, including 18 snap-frozen and 83 FFPE tissues. We found that short amplicons were amplified more efficiently than long amplicons both in snap-frozen (P = 0.0006) and FFPE (P = 0.0152) tissues. Nonetheless, comparison of colorectal carcinomas with their adjacent normal tissues demonstrated that the consistency of fold-change trends in a single short amplicon between snap-frozen and FFPE tissues was only 36%. Therefore, we innovatively performed quantitative RT-PCR with 3 non-overlapping short amplicons for 14 target RNAs in FFPE tissues. All target RNAs showed a concordance of 100% of fold-change trends in at least two short amplicons, which offers sufficient information for accurate quantification of target RNAs. Our findings demonstrated the possibility of long-chain RNA analysis with 3 non-overlapping short amplicons in standardized-preserved FFPE tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue specimens comprise a potentially valuable resource for retrospective biomarker discovery studies, and recent work indicates the feasibility of using shotgun proteomics to characterize FFPE tissue proteins. A critical question in the field is whether proteomes characterized in FFPE specimens are equivalent to proteomes in corresponding fresh or frozen tissue specimens. Here we compared shotgun proteomic analyses of frozen and FFPE specimens prepared from the same colon adenoma tissues. Following deparaffinization, rehydration, and tryptic digestion under mild conditions, FFPE specimens corresponding to 200 μg of protein yielded ∼400 confident protein identifications in a one-dimensional reverse phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The major difference between frozen and FFPE proteomes was a decrease in the proportions of lysine C-terminal to arginine C-terminal peptides observed, but these differences had little effect on the proteins identified. No covalent peptide modifications attributable to formaldehyde chemistry were detected by analyses of the MS/MS datasets, which suggests that undetected, cross-linked peptides comprise the major class of modifications in FFPE tissues. Fixation of tissue for up to 2 days in neutral buffered formalin did not adversely impact protein identifications. Analysis of archival colon adenoma FFPE specimens indicated equivalent numbers of MS/MS spectral counts and protein group identifications from specimens stored for 1, 3, 5, and 10 years. Combination of peptide isoelectric focusing-based separation with reverse phase LC-MS/MS identified 2554 protein groups in 600 ng of protein from frozen tissue and 2302 protein groups from FFPE tissue with at least two distinct peptide identifications per protein. Analysis of the combined frozen and FFPE data showed a 92% overlap in the protein groups identified. Comparison of gene ontology categories of identified proteins revealed no bias in protein identification based on subcellular localization. Although the status of posttranslational modifications was not examined in this study, archival samples displayed a modest increase in methionine oxidation, from ∼17% after one year of storage to ∼25% after 10 years. These data demonstrate the equivalence of proteome inventories obtained from FFPE and frozen tissue specimens and provide support for retrospective proteomic analysis of FFPE tissues for biomarker discovery.

BackgroundAn absence of reliable molecular markers has hampered individualised breast cancer treatments, and a major limitation for translational research is the lack of fresh tissue. There are, however, abundant banks of formalin-fixed paraffin-embedded (FFPE) tissue. This study evaluated two platforms available for the analysis of DNA copy number and gene expression using FFPE samples.MethodsThe cDNA-mediated annealing, selection, extension, and ligation assay (DASL™) has been developed for gene expression analysis and the Molecular Inversion Probes assay (Oncoscan™), were used for copy number analysis using FFPE tissues. Gene expression and copy number were evaluated in core-biopsy samples from patients with breast cancer undergoing neoadjuvant chemotherapy (NAC).ResultsForty-three core-biopsies were evaluated and characteristic copy number changes in breast cancers, gains in 1q, 8q, 11q, 17q and 20q and losses in 6q, 8p, 13q and 16q, were confirmed. Regions that frequently exhibited gains in tumours showing a pathological complete response (pCR) to NAC were 1q (55%), 8q (40%) and 17q (40%), whereas 11q11 (37%) gain was the most frequent change in non-pCR tumours. Gains associated with poor survival were 11q13 (62%), 8q24 (54%) and 20q (47%). Gene expression assessed by DASL correlated with immunohistochemistry (IHC) analysis for oestrogen receptor (ER) [area under the curve (AUC) = 0.95], progesterone receptor (PR)(AUC = 0.90) and human epidermal growth factor type-2 receptor (HER-2) (AUC = 0.96). Differential expression analysis between ER+ and ER– cancers identified over-expression of TTF1, LAF-4 and C-MYB (p ≤ 0.05), and between pCR vs non-pCRs, over-expression of CXCL9, AREG, B-MYB and under-expression of ABCG2.ConclusionThis study was an integrative analysis of copy number and gene expression using FFPE core biopsies and showed that molecular marker data from FFPE tissues were consistent with those in previous studies using fresh-frozen samples. FFPE tissue can provide reliable information and will be a useful tool in molecular marker studies.Trial RegistrationTrial registration number ISRCTN09184069 and registered retrospectively on 02/06/2010.

MicroRNAs (miRNAs) are small (19-23nt), highly conserved noncoding RNAs that posttranscriptionally regulate target gene expression. Altered expression of miRNAs has been demonstrated in many different human diseases, including cancer. The large archives of formalin-fixed, paraffin-embedded (FFPE) tissue specimens with clinical follow-up information that exist are a highly valuable source of tissue to study human diseases. However, RNA in the FFPE tissue is fragmented and may be chemically modified. In this study, we prepared miRNA preserving total RNA from matched pairs of FFPE and respective fresh-frozen clinical samples, and used that in microarray experiments to compare miRNA expression profiles between FFPE and fresh-frozen tissue from the same tissue samples. We demonstrate that miRNA expression profile from FFPE tissues closely resembles that from fresh tissues. These results underscore the suitability of FFPE tissues as appropriate resources for miRNA expression analyses.

Background and Objective: The availability of formalin-fixed paraffin-embedded (FFPE) tissue provides an enormous resource for the discovery of molecular markers. Several reports indicate that microRNA (also referred to as miRNA or miR, a class of small noncoding RNAs 19-24 nucleotides in length that negatively regulate translation of protein-coding mRNAs) is preserved and readily extracted from FFPE tissue. Our goal in this study was to identify a method for optimal recovery of miRNA from FFPE prostate tissue. Experimental Approach: We compared five commercially available kits that are designed to isolate miRNA from FFPE tissue: SABiosciences RT2 FFPE RNA Extraction Kit (kit 1), Applied Biosystems RecoverAll Total Nucleic Acid Isolation Kit (kit 2), Roche High Pure miRNA Isolation Kit (kit 3), Qiagen miRNeasy FFPE Kit (kit 4), and MO BIO UltraClean FFPE RNA Isolation Kit (kit 5). All kits were used to process serial sections (10 μm thickness) from the same FFPE tissue block. We assessed purity and yield of total RNA based on NanoDrop analysis of absorbance at 230, 260, and 280 nm. Equal amounts of total RNA were converted to cDNA using reverse transcriptase (RT) and miR-specific primers, and real-time PCR using TaqMan miR probes was used to quantitate 378 miRs and 3 small nucleolar RNA species (TaqMan Low Density Array). Results: The total RNA yield/section was 5.3, 1.6, 3.0, 2.5, and 3.2 μg from kits 1-5, respectively (average of two independent extractions of serial sections from the same FFPE tissue block). Nevertheless, as expected, analysis of the same amount of RNA from each kit showed that all samples had the same level (based on CT) of the small nucleolar RNA RNU48; thus, the recovery of RNU48 per unit amount of total RNA was the same for all kits. Unexpectedly, however, only a limited number of miRs were present at the same level (same CT) in all kit preparations; thus, per unit amount of total RNA, the recovery of only a limited number of miRs was independent of the method used to isolate RNA. For a substantial proportion of miRs, recovery was dependent on the kit used, and recovery differed among miRs. To quantitate differences in recovery, we normalized transcript levels to RNU48 and calculated the ratio of the level of each miR in different kits. Ratios of miRx(kit a)/miRx(kit b) = 1.0 vs. miRy(kit a)/miRy(kit b) = 2.0 would mean that the recovery of miRs x and y is not uniform. Comparing kit 4 vs. kit 1, 10% of miRs had a kit 4/kit 1 ratio <0.75, 17% had a ratio ∼1, 28% had a ratio between 1 and 2, 33% had a ratio between 2 and 4, and 12% had a ratio >4; these data indicate non-uniform recovery of different miRs. This non-uniform recovery of miRs was independent of relative abundance, affecting both high abundance and low abundance transcripts. Notably, the observation of non-uniform recovery of different miRs required the analysis of different kit preparations. Conclusions: Different miRs are not uniformly recovered from total RNA of FFPE tissue. This suggests that a miR profile in an RNA extract may not accurately represent the tissue in vivo, and that the profile observed may depend on the RNA extraction method used. We suggest that analysis of miRs in tumor relative to nontumor tissue of the same patient will provide a way to normalize for differences in miR recovery, and thus provide a reliable approach to identify miRs as markers of disease. The basis for non-uniform recovery of miRs from FFPE tissue is not known, nor whether this effect is unique to FFPE tissue. [Supported by a grant from the DOD Prostate Cancer Research Program.] Citation Format: Zizheng Hou, Evelyn R. Barrack. Non-uniform recovery of microRNAs from formalin-fixed paraffin-embedded tissue [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C32.

Objective To estimate the value of immunohistochemical staining for C3d and C4d in paraffin-embedded tissue sections for the auxiliary diagnosis of bullous pemphigoid (BP). Methods Paraffin-embedded tissue sections from 20 patients with BP served as the experiment group, and those from lesions of 9 patients with familial benign pemphigus and 4 patients with epidermolysis bullosa as well as from normal skin served as the controls. Immunohistochemical SP method was used to measure the expressions of C3d and C4d in these paraffin sections. Results The deposition of C3d and C4d was observed in the basement membrane at the dermo-epidermal junction in 95% (19/20) of BP tissue sections, but undetected in any of the familial benign pemphigus or epidermolysis bullosa tissue sections. Conclusion Immunohistochemical staining for C3d and C4d in paraffin-embedded tissue sections may serve as an auxiliary method for the diagnosis of BP. Key words: Pemphigoid, bullous; Paraffin embedding; Microtomy; Immunohistochemistry; Complement C3d; Complement C4d

Genotyping of DNA Samples Isolated from Formalin-Fixed Paraffin-Embedded Tissues Using Preamplification