UTILIZING INFRARED IMAGING TO EVALUATE THE AGGRESSIVENESS OF PROSTATE CANCER.

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Formalin-fixed paraffin-embedded (FFPE) tissue samples are widely used for genomic analyses in clinical and research settings. However, prolonged storage at room temperature may compromise nucleic acid quality, potentially affecting the reliability of molecular testing. In this study, we investigated the impact of various storage temperatures and repeated temperature changes on nucleic acid quality in FFPE samples. Four surgically resected tumor cases were analyzed. Following cold ischemia times of 29-60min, tumor tissues were sampled upon arrival at the pathology department, divided into five equal parts, and fixed in 10% neutral buffered formalin. The resulting FFPE blocks were stored at 18 °C, 4 °C, -20 °C, -80 °C, or-150 °C. After storage, the blocks were returned to room temperature for sectioning and analysis. DNA and RNA quality were assessed at 3, 6, and 12months using DNA integrity number (DIN), short-to-long cycle threshold (S/L Ct) ratio, RNA integrity number (RIN), and DV200. Samples stored at 18 °C and 4 °C exhibited time-dependent deterioration in certain indicators, whereas those stored at -20 °C or below maintained stable nucleic acid quality. Despite multiple freeze-thaw cycles, no significant degradation was observed under colder conditions. Our findings suggest that -20 °C storage is a feasible and effective approach for preserving nucleic acid integrity in FFPE tissues. Although limited by a small sample size (n=4), this study provides practical insights into tissue archiving strategies and supports selective implementation of refrigerated storage for long-term molecular analysis.

Translational prostate cancer research is hampered by long intervals from diagnosis to patient progression and difficulty in obtaining cancer tissue for investigation. As such, it is imperative to utilise aging formalin-fixed paraffin-embedded (FFPE) tissue samples from the pathology archive with linked patient outcome data to allow current day research. This study aimed to assess the adequacy and quantity of mRNA extracted from formalin fixed paraffin embedded (FFPE) prostate tissue, including prostate biopsies, up to 15 years old. The decay of mRNA over time and under differing storage conditions was also assessed. Archived FFPE benign prostatic tissue up to 15 years old from transurethral resection of the prostate (TURP) and transrectal ultrasound guided (TRUS) biopsies as well as fresh tissue obtained from patients undergoing TURP for benign bladder outlet obstruction were used. Following mRNA extraction beta-actin real-time PCR was carried out using a set of 4 different primer/probes to assess mRNA quality and quantity. There was no difference in mRNA quantity/quality extracted from "fresh" FFPE tissue from the same patient over a 4-month period following surgery. The temperature of block storage did not alter quality/quantity of the mRNA (P > 0.05, unpaired t test). Fresh tissue had a higher quality/quantity, indicated by a lower C ( T ) value, than FFPE samples from the same patient (P ≤ 0.03, one-way ANOVA). Despite being up to 15 years old, all archived FFPE TURP and TRUS biopsy samples had "high" or "very high" levels of expression making them suitable for further analysis. However, the quality of the mRNA in archived FFPE samples did significantly decline with increasing sample age. It is possible to extract mRNA of sufficient standard for further transcriptomic analysis from minute FFPE samples up to 15 years old. This work adds to the literature suggesting that exploitation of retrospective prostate tissue collections with robust associated clinical data is possible.

BackgroundArchival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation.MethodsHigh-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering.ResultsWe created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50 % reduction over 20 years) but not in CRC. Formalin fixation for 2–6 days decreased miRNA expression 30–65 %. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results.ConclusionsWe identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-2030-2) contains supplementary material, which is available to authorized users.

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

Reportedly, the pyrosequencing methylation assay can produce inconsistent results between paired snap-frozen and formalin-fixed paraffin-embedded archival tissue samples. In this study, we assayed the methylation levels at four individual CpG sites of L1 using pyrosequencing and found that the methylation levels at individual CpG sites were different but were closely correlated between paired snap-frozen and formalin-fixed paraffin-embedded tissue samples. We aimed to determine whether low methylation status of L1 is associated with gastric cancer patient prognosis. We analyzed 434 formalin-fixed paraffin-embedded tissue samples of advanced gastric cancer for their methylation status at four CpG sites of L1 [nucleotide positions 328, 321, 318, and 306 of X58075 (Genbank)] using pyrosequencing, and correlated the L1 methylation level with clinicopathological features. Older age at onset, males, tumor location at antrum or lower body, intestinal type, and lymphatic or venous invasion were associated with a low average methylation level of L1 at the two CpG sites 1 and 4 combined. The average methylation level of L1 at CpG sites 1 and 4 combined was significantly lower in microsatellite-stable and EBV-negative gastric cancers than in EBV-positive or microsatellite-unstable gastric cancers. Low methylation status of L1 was independently correlated with shorter overall survival and disease-free survival time. Our findings indicate that the discrepancy in the methylation level of L1 between fresh tissue and formalin-fixed paraffin-embedded tissue samples depends on the CpG sites considered, and that the methylation status of L1 at CpG sites 1 and 4 combined could be utilized as a prognostic parameter for advanced gastric cancers.

Background/Objectives: Fresh frozen (FF) samples are routinely used to isolate high-molecular-weight intact genomic DNA. However, when FF samples are not available, archived formalin-fixed paraffin-embedded (FFPE) tissue samples often represent the only available material in clinical research. Due to formaldehyde-induced degradation of nucleic acids they pose special challenges for genetic investigations. In this study we compare whole-genome sequencing results on intact DNA versus fragmented DNA derived from FFPE samples of three dogs. Methods: We prepared matched libraries from FF and FFPE samples of three dogs affected by an inherited disease, EFNB3-related congenital mirror movement disorder 1 (CMM1). Paired-end short-read sequencing data were obtained on an Illumina sequencer and analyzed with adapted workflows for FF or FFPE data, respectively. Results: The data between FF and FFPE samples were largely consistent. FF data showed a superior variant call accuracy, as expected. However, the data quality from the FFPE samples was sufficient to correctly identify the causal variant in EFNB3. Conclusions: This pilot study demonstrates the feasibility of using FFPE samples from dogs for whole-genome sequencing and the detection of germline variants. Using FFPE samples in the analysis of suspected inherited diseases in domestic animals may represent a valuable approach in veterinary genetics if no other samples are available.

Formalin-fixed paraffin-embedded (FFPE) tissue samples often suffer from RNA degradation due to fixation, embedding, and storage processes, affecting the integrity of transcripts for RNA sequencing (RNA-seq). Herein, we developed a novel deep learning model to reconstruct gene expression profiles from FFPE-derived RNA-seq data. We first compared the transcript integrity numbers (TINs) of two in-house datasets derived from paired fresh frozen (FF) and FFPE tissues, with different fixation durations and library preparation strategies. We found that tissue samples fixed for 24 hours and subjected to the rRNA depletion-based library preparation strategy exhibited higher TINs. Using RNA-seq data for 832 FFPE samples across seven cancer types, we identified genes with consistently low TINs on the genome-wide scale. Interestingly, the signature genes of immune cells are more prone to degradation, compared to other cell types such as tumor epithelial cells. To reconstruct gene expression profiles, we developed a deep learning framework, based on 9568 FF tumor samples across 28 cancer types in the TCGA database. The framework demonstrated remarkable performance on both simulated data (672 transcriptomes) and in-house cancer cohorts with RNA-seq data, including one FFPE and three FF-FFPE-matched cohorts. On the simulated data with noise levels ranging from 10% to 60%, the reconstructed data achieved consistently high correlation coefficients (> 0.85) with the ‘clean’ data without noise. For the FF-FFPE-matched datasets, compared to the original FFPE-derived data, the reconstructed gene expression profiles significantly improved the correlation with matched FF profiles, even for immune cell signature genes (all P < 0.05). In our application to cancer subtyping, the gene expression profiles reconstructed improved the classification accuracy from 67% to 92%. Furthermore, the reconstructed profiles recapitulated the subtype-specific biological properties lost in the FFPE-derived data and showed more significant association with patient survival. As the first deep learning model to rectify FFPE-derived RNA-seq data, our framework enables more efficient use of archived FFPE tissue samples that are widely available in the clinic. Citation Format: Lingli He, Kai Song, Ying Li, Celia Yujuan Dong, Cherry Y. Wong, Lin Qi, Xianrui Zhang, Yufeng Chen, Louis Vermeulen, Shentao Zhou, Simon Siu Ng, Xin Wang. A deep learning framework for the reconstruction of gene expression profiles derived from formalin-fixed paraffin-embedded samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2444.

Relationship Among Initial Serum Prostate Specific Antigen, Prostate Specific Antigen Progression and Prostate Cancer Detection at Repeat Screening Visits

Mitochondrial DNA analysis of formalin-fixed paraffin-embedded tissue samples: Effect of formalin on DNA stability and its implications in genetic studies

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Identification of methods for use of formalin-fixed, paraffin-embedded tissue samples in RNA expression profiling

US Trial shows no early mortality benefit from annual prostate cancer screening

BackgroundFormalin-fixed paraffin-embedded (FFPE) tissue samples are routinely archived in the course of patient care and can be linked to clinical outcomes with long-term follow-up. However, FFPE tissues have degraded RNA which poses challenges for analyzing gene expression. Next-generation sequencing (NGS) is rapidly becoming accepted as an effective tool for measuring gene expressions for research and clinical use. However, the feasibility of NGS has not been firmly established when using FFPE tissue.ResultsWe optimized strategies for whole transcriptome sequencing (RNA-seq) using FFPE tissue. Ribosomal RNA (rRNA) was successfully depleted by competitive hybridization using the Ribo-zero™ Kit (Epicentre Biotechnologies), and rRNA sequence content was less than one percent for each library. Gene expression measured by FFPE RNA-seq was compared to two different standards: RNA-seq from fresh frozen (FF) tissue and quantitative PCR (qPCR). Both FF and FFPE tumors were sequenced on an Illumina Genome Analyzer IIX with an average of 10 million reads. The distribution of FPKMs (fragments per kilobase of exon per million fragments mapped) and number of detected genes were similar between FFPE and FF. RNA-seq expressions from FF and FFPE samples from the same renal cell carcinoma (RCC) correlated highly (r = 0.919 for tumor 1 and r = 0.954 for tumor 2). On hierarchical cluster analysis, samples clustered by patient identity rather than method of preservation. TaqMan qPCR of 424 RCC-related genes correlated highly with FFPE RNA-seq expressions (r = 0.775 for FFPE tumor 1, r = 0.803 for FFPE tumor 2). Expression fold changes were considered, to assess biologic relevance of gene expressions. Expression fold changes between FFPE tumors (tumor 1/tumor 2) correlated well when comparing qPCR and RNA-seq (r = 0.890). Expression fold changes between tumors from different risk groups (our high risk RCC/The Cancer Genome Atlas, TCGA, low risk RCC) also correlated well when comparing RNA-seq from FF and FFPE tumors (r = 0.887).ConclusionsFFPE RNA-seq provides reliable genes expression data, comparable to that obtained from fresh frozen tissue. It represents a useful tool for discovery and validation of biomarkers.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1087) contains supplementary material, which is available to authorized users.

Background: The current project aims to explore the possibility to use formalin fixed paraffin embedded (FFPE) samples for exome sequencing, which would open up a large collection of tissue samples for molecular studies. Usage of formalin is known to degrade and modify the DNA both during treatment and storage resulting in problems in downstream molecular analyses. The aim of the present study is to evaluate exome sequencing of FFPE samples by comparing data from FFPE normal tissue to corresponding snap frozen (SF) blood controls with focus on data output such as amount of data removed due to low sequencing quality, amount of data mapped to the genome, GC-content, and duplicate levels. Methods: In the current study we used the western Swedish biobank of neural tumors archived at the Sahlgrenska Hospital, Gothenburg. We performed exome sequencing of FFPE normal adrenal tissue samples and corresponding SF blood samples from five patients diagnosed with pheochromocytoma/paraganglioma. Two of the patients had known heterozygous germline mutations and were included to determine if these mutations could be found in both sample types. Libraries for SF samples were prepared according to protocol using Agilent Technologies SureSelect Human All Exon 50 Mb library prep kit. FFPE samples were prepared with slight modifications in the protocol, with additional PCR cycles used to increase amplification. 75 bp paired end reads were generated on Illumina HiScan SQ according to manufacturer's protocol (v.3). Results: Between 92-99% of the raw data were kept after removal of data due to low sequencing quality independent of sample type. For the SF samples 99-100% of the trimmed data mapped to the genome, the number for FFPE samples were slightly lower ranging from 87-98%. The GC content in the exome enriched DNA from FFPE and SF tissue were similar, ranging between 45-51%. Looking at the mapped data we found higher levels of duplicates in the FFPE samples compared to the SF samples, 19-78% vs. 5-15%. For one of the two patients with a known germline mutation the variant was detected at approximately the same frequency in both FFPE and SF sample (39% in FFPE vs. 44% in SF). For the other patient the variant were found at higher frequency in the FFPE sample compared to SF sample (80% vs. 50%). Conclusions: We saw no difference in amount data removed due to low sequencing quality between the five FFPE and SF samples evaluated in this study. Also, we saw only slightly lower mapping frequencies for the FFPE samples. The GC-content for both FFPE and SF samples were within the range of what's expected for exome sequencing data for libraries prepared with this kit and sequenced on Illumina HiScan SQ platform. Due to the additional PCR cycles used for FFPE samples we saw higher duplicate levels in these samples, suggesting that deeper sequencing is necessary for FFPE samples to get the same amount of unique reads as for the SF samples. A probable explanation to the higher allele frequency of the variant observed in the FFPE sample from one of the patients with a known germline mutation is the presence of contaminant tumor cells in the normal tissue sample, resulting in a shifted allele frequency. To conclude, SF samples are to prefer for exome sequencing. However, if SF material is not available FFPE tissue is a good alternative to increase the sample cohort when studying rare and complex diseases. Citation Format: Annica Wilzen, Heidi Ottesen, Anna Larsson, Bo Wangberg, Andreas Muth, Ola Nilsson, Frida Abel. Exome sequencing of FFPE material: An evaluation. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A11.