Formalin-fixed Paraffin-embedded Biospecimens Research Articles

Utility of formalin-fixed, paraffin-embedded prostate biospecimens from low-resource settings for use in next-generation sequencing studies in African-descent populations

Background Men of African ancestry experience higher burden from prostate cancer compared to men of other ancestral backgrounds. Limitations in the availability of high-quality biospecimens hinder the inclusion of this population in genetic studies of prostate cancer. The use of formalin-fixed paraffin-embedded (FFPE) tissues represent a potential rich source of genetic material particularly in some international settings, where fresh frozen tissue is difficult to obtain. In this study, we investigate the feasibility of using FFPE biospecimens acquired from various international sites for utility in next-generation sequencing. Methods A total of 976 FFPE blocks were collected between 2002 and 2017 from six international sites in Africa and the Caribbean representing three consortia: Prostate Cancer Transatlantic Consortium; African-Caribbean Cancer Consortium; and Men of African Descent and Carcinoma of the Prostate. Genomic DNA was checked for quality and quantity. Differences in mean quality control (QC) for pre-and-post pathology training were assessed using t-test. Pearson chi-square with trend analysis examined association between time-category and QC success status. Association of continuous DNA quality (Q129/Q41 ratio) and time of specimen collection was estimated with linear regression. Samples with a DNA quantity >0.2µg and a Q129/Q41 ratio >0.00225 were submitted for whole exome sequencing (WES). Results There was a positive relative percentage change in DNA quantity from 2002 to 2017 for Jamaica, Kenya and Senegal. There was a decline in DNA quantity over the same time period for Nigeria. There was a statistically significant improvement in quality of samples from Kenya (P=0.032), Nigeria (P<.001) and Senegal (P=0.043). There was a significant improvement in the collected DNA sample quality over time with an R2 of 0.12. Conclusions FFPE samples from low-resource settings could potentially provide sufficient DNA for WES. Improvements in biospecimen collection processing and storage for research are needed in some of these settings.

Abstract 3313: A novel RNA aptamer-based assay for quantifying fractional occupancy of PD1 by PDL1 in formalin fixed biospecimens

Abstract Background: There is a need for better prognostic biomarkers for PD1-based therapy. Quantifying the fractional occupancy (FO) of PD1 by PDL1, and PDL1 by PD1, in tumor can be a potential biomarker. We previously reported on an RNA aptamer-based assay platform called the Ligand-Receptor Complex Aptamer (LIRECAP) assay that quantifies FO of a receptor by its ligand. Here, we describe a 3-color LIRECAP assay designed to quantify PD1 occupancy by PDL1 and PDL1 occupancy by PD1 in formalin fixed paraffin embedded (FFPE) tissue. Methods: (A) Selection of aptamers: RNA aptamers with preferential binding to PD1 (“P” aptamers), PDL1 (“L” aptamers) or PD1-PDL1 complex (“C” aptamers) were selected from single starting library using a variation of the SELEX, we reported previously (PMID: 31383650). Selected aptamers were tested for binding to formalin fixed proteins that stabilized the complex and assured that selected aptamers bound to the targets in FFPE biospecimens. Lead aptamers, namely P17, L42 and C113, were selected for further study based on their preferential binding to formalin fixed PD1, PDL1 and complex respectively. Three TaqMan-based calorimetric probes were designed targeting the unique central regions of P17, L42 and C113. (B) 3-color LIRECAP assay on FFPE tissue: Cell culture samples containing PD1, PDL1 or PD1-PDL1 complex were created by mixing equal numbers of (1) PD1+ Jurkat with PDL1- Raji cells, (2) PD1- Jurkat with PDL1+ Raji cells or (3) PD1+ Jurkat with PDL1+ Raji cells, respectively. Cell blocks were produced using the collodion bag technique, fixed in formalin and embedded in paraffin using standard clinical techniques. Scrolls of 10-um were cut, deparaffinized, rehydrated, washed and non-specific binding blocked. An equimolar mix of P17, L42 and C113 aptamers was added, bound aptamers were extracted and their relative concentrations were quantified simultaneously by 3-color TaqMan-based RT-qPCR using a single set of primers. The proportion of each aptamer was calculated as the percentage of each aptamer (P or L or C) relative to the amount of total bound aptamers (P+L+C). Results: Each aptamer bound preferentially (underlined) to its designated target as determined by 3-color assay (mean of 2 independent sets). Unoccupied PD1 only: P17=44%; L42=34%; C18=22% Unoccupied PDL1 only: P17=24%; L42=48%; C18=28% PD1-PDL1 complex: P17=19%; L42=17%; C18=64% Although preferential binding of aptamers was not absolute, simultaneous evaluation of three aptamers in a single assay provided an internal control that can allow for correction for this cross-reactivity. Conclusions and significance: The 3-color LIRECAP assay is capable of quantifying FO of PD1 by PDL1 and PDL1 by PD1 in FFPE samples. Ongoing studies are exploring use of this assay to quantify FO in clinical biospecimens. It will then be used to determine whether PD1 FO might serve as a novel predictive biomarker for anti-PD1 therapy. Citation Format: Suresh Veeramani, Nanmeng Yu, Xian Jin Xie, Kristen Coleman, George J. Weiner. A novel RNA aptamer-based assay for quantifying fractional occupancy of PD1 by PDL1 in formalin fixed biospecimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3313.

New protocol for kinetic assay seeding ability recovery "KASAR" from formalin-fixed paraffin-embedded tissues.

The real-time quaking-induced conversion (RT-QuIC) alpha-synuclein (aSyn) protein kinetic seeding assay has been very useful for detecting pathological aggregates in various synucleinopathies including Parkinson's disease (PD). This biomarker assay relies on fresh frozen tissue to effectively seed and amplify aSyn aggregating protein. With vast repositories of formalin-fixed paraffin-embedded (FFPE) tissues, it is paramount to harness the power of kinetic assays to unlock the diagnostic potential of archived FFPE biospecimens. However, the major challenge posed by significantly reduced amplification of formalin-fixed tissues in the assay suggests that formalin fixation deterred monomer interaction with the sample seed and depressed subsequent protein aggregation. To overcome this challenge, we developed a kinetic assay seeding ability recovery (KASAR) protocol to maintain the integrity of the tissue and seeding protein. For this, we implemented a series of heating steps with the brain tissue suspended in a buffer composed of 500mM tris-HCl (pH 7.5) and 0.02% SDS after the standard deparaffinization of the tissue sections. Initially, samples from seven human brain samples, including four samples from patients diagnosed with dementia with Lewy bodies (DLB) and three samples from healthy controls without DLB, were compared to fresh frozen samples under three different, but clinically common sample storage conditions: formalin-fixed, FFPE, and FFPE slices cut 5µm thick. The KASAR protocol was able to recover seeding activity for all positive samples in all storage conditions. Next, 28 FFPE samples from the submandibular gland (SMG) of patients diagnosed with PD, incidental Lewy body disease (ILBD), or healthy controls were tested with 93% of results replicating when blinded. With samples of only a few milligrams, this protocol recovered the same quality of seeding in formalin-fixed tissue as fresh frozen tissue. Moving forward, protein aggregate kinetic assays, in conjunction with the KASAR protocol, can be used to understand and diagnose neurodegenerative diseases more comprehensively. Overall, our KASAR protocol unlocks and restores the seeding ability of formalin-fixed paraffin-embedded tissues for the amplification of biomarker protein aggregates in kinetic assays.

Why Formalin-fixed, Paraffin-embedded Biospecimens Must Be Used in Genomic Medicine: An Evidence-based Review and Conclusion.

Fresh-frozen tissue is the “gold standard” biospecimen type for next-generation sequencing (NGS). However, collecting frozen tissue is usually not feasible because clinical workflows deliver formalin-fixed, paraffin-embedded (FFPE) tissue blocks. Some clinicians and researchers are reticent to embrace the use of FFPE tissue for NGS because FFPE tissue can yield low quantities of degraded DNA, containing formalin-induced mutations. We describe the process by which formalin-induced deamination can lead to artifactual cytosine (C) to thymine (T) and guanine (G) to adenine (A) (C:G > T:A) mutation calls and perform a literature review of 17 publications that compare NGS data from patient-matched fresh-frozen and FFPE tissue blocks. We conclude that although it is indeed true that sequencing data from FFPE tissue can be poorer than those from frozen tissue, any differences occur at an inconsequential magnitude, and FFPE biospecimens can be used in genomic medicine with confidence:

Quantitative analysis of noncoding RNA from paired fresh and formalin-fixed paraffin-embedded brain tissues.

Formalin-fixed paraffin-embedded (FFPE) tissues are commonly used both clinically and in forensic pathology. Recently, noncoding RNA (ncRNA) has attracted interest among molecular medical researchers. However, it remains unclear whether newly identified ncRNAs, such as long noncoding RNA (lncRNA) and circular RNA (circRNA), remain stable for downstream molecular analysis in FFPE tissues. Here, we assessed the feasibility of using autoptic FFPE brain tissues from eight individuals to perform quantitative molecular analyses. Selected RNA targets (9 mRNAs and 15 ncRNAs) with different amplicon lengths were studied by RT-qPCR in paired fresh and FFPE specimens. For RNA quality assessment, RNA purity and yield were comparable between the two sample cohorts; however, the RNA integrity number decreased significantly during FFPE sampling. Amplification efficiency also displayed certain variability related with amplicon length and RNA species. We found molecular evidence that short amplicons of mRNA, lncRNA, and circRNA were amplified more efficiently than long amplicons. With the assistance of RefFinder, 5S, SNORD48, miR-103a, and miR-125b were selected as reference genes given their high stability. After normalization, we found that short amplicon markers (e.g., ACTB mRNA and MALAT1 lncRNA) exhibited high consistency of quantification in paired fresh/FFPE samples. In particular, circRNAs (XPO1, HIPK3, and TMEM56) presented relatively consistent and stable expression profiles in FFPE tissues compared with their corresponding linear transcripts. Additionally, we evaluated the influence of prolonged storage time on the amplification of gene transcripts and found that short amplicons still work effectively in archived FFPE biospecimens. In conclusion, our findings demonstrate the possibility of performing accurate quantitative analysis of ncRNAs using short amplicons and standardized RT-qPCR assays in autopsy-derived FFPE samples.

Using FFPE Tissue in Genomic Analyses: Advantages, Disadvantages and the Role of Biospecimen Science

Formalin-fixed, paraffin-embedded (FFPE) tissue blocks are considered by some to be suboptimal for Next Generation Sequencing (NGS) because, compared with fresh-frozen (FF) biospecimens, the nucleic acid obtained from them is fragmented and low in quantity, containing formalin-induced modifications and mutations. However, clinical workflows do not typically have the capability to collect FF tissue, so there is an inherent necessity to perform NGS using FFPE tissue. We review relevant literature that addresses the issue of extracting DNA from FFPE tissue biospecimens then applying it to NGS. Practical and straightforward optimisation experiments and quality control assays can be performed by laboratories who extract DNA from FFPE biospecimens to maximise its amenability to NGS. FFPE biospecimens can be used in genomic medicine with confidence because the disadvantages associated with DNA quality are outweighed by the ease of sample collection and can be addressed by protocol optimisation.

The Biospecimen Preanalytical Variables Program: A Multiassay Comparison of Effects of Delay to Fixation and Fixation Duration on Nucleic Acid Quality

Despite widespread use of formalin-fixed, paraffin-embedded (FFPE) tissue in clinical and research settings, potential effects of variable tissue processing remain largely unknown. To elucidate molecular effects associated with clinically relevant preanalytical variability, the National Cancer Institute initiated the Biospecimen Preanalytical Variables (BPV) program. The BPV program, a well-controlled series of systematic, blind and randomized studies, investigated whether a delay to fixation (DTF) or time in fixative (TIF) affects the quantity and quality of DNA and RNA isolated from FFPE colon, kidney, and ovarian tumors in comparison to case-matched snap-frozen controls. DNA and RNA yields were comparable among FFPE biospecimens subjected to different DTF and TIF time points. DNA and RNA quality metrics revealed assay- and time point-specific effects of DTF and TIF. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was superior when assessing RNA quality, consistently detecting differences between FFPE and snap-frozen biospecimens and among DTF and TIF time points. RNA Integrity Number and DV200 (representing the percentage of RNA fragments longer than 200 nucleotides) displayed more limited sensitivity. Differences in DNA quality (Q-ratio) between FFPE and snap-frozen biospecimens and among DTF and TIF time points were detected with a qPCR-based assay. DNA and RNA quality may be adversely affected in some tumor types by a 12-hour DTF or a TIF of 72 hours. Results presented here as well as those of additional BPV molecular analyses underway will aid in the identification of acceptable delays and optimal fixation times, and quality assays that are suitable predictors of an FFPE biospecimen's fit-for-purpose.

Abstract A32: Prognostic stratification of stage I lung adenocarcinoma patients by HOXA9 promoter methylation ddPCR and blood vessel invasion analysis in FFPE tissues

Abstract Surgery with curative intent is the standard of care for stage I lung adenocarcinoma. However, disease recurrence eventually occurs in about a third of the patients. Consequently, prognostic biomarkers are urgently needed to predict recurrence following surgery and potentially guide the decision to administer adjuvant chemotherapy for high-risk patients. The type of biospecimens and the choice of assay platform are key issues in the process of cancer biomarker clinical development. Here, we demonstrate the application of a droplet digital PCR (ddPCR)-based assay to analyze DNA methylation in formalin-fixed, paraffin-embedded (FFPE) biospecimens. In recent years, ddPCR has become increasingly used clinically due to its ability to reliably detect and quantitate rare alleles, as well as its technical simplicity, rapidity, and cost-effectiveness. In addition, FFPE tissues are generated during routine pathologic assessment of resected patients, and are generally available for testing molecular markers. We developed a ddPCR assay to quantify HOXA9 promoter methylation in FFPE samples, with high sensitivity, specificity, and reproducibility. High HOXA9 promoter methylation has been associated with worse outcome of resected stage I lung adenocarcinoma patients in multiple studies. Here, the prognostic value of HOXA9 promoter methylation alone or in combination with blood vessel invasion (BVI), another well-described prognostic biomarker for patients with early stage lung adenocarcinoma, was evaluated by Kaplan-Meier survival analyses and Cox regression modeling, using 188 FFPE biospecimens, in two independent sets. We replicated previous observations that HOXA9 promoter is methylated de novo in stage I tumors (P <0.0001), and that high methylation is associated with worse prognosis (Hazard Ratio [HR], 3.37; P=0.0002). HOXA9 methylation was associated with a transcriptome signature enriched in genes marked by Polycomb in Embryonic Stem Cells, a signature previously associated with poor differentiation and worse overall patient survival. Addition of this molecular marker improved a risk model comprising clinical and pathologic parameters (age, sex, smoking status, and tumor stage and size; P=0.003, nested likelihood ratio test). As expected, BVI was independently associated with poor outcome (HR, 2.62; P=0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (Trend P=0.0001 comparing 0, 1 or 2 positive markers). Collectively, our results support the use of ddPCR to quantify HOXA9 promoter methylation and the analysis of BVI from routine pathology FFPE specimens to identify patients at high risk of recurrence. Our findings pave the way for future clinical evaluation of these promising prognostic markers. If validated in a larger independent study, these biomarkers should be considered in prospective trials to evaluate the benefit of adjuvant chemotherapy in high-risk early-stage lung adenocarcinoma patients. Citation Format: Delphine Lissa, Teruhide Ishigame, Rintaro Noro, Marguerite J. Tucker, Elise D. Bowman, Curtis C. Harris, Ana I. Robles. Prognostic stratification of stage I lung adenocarcinoma patients by HOXA9 promoter methylation ddPCR and blood vessel invasion analysis in FFPE tissues [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr A32.

A Critical Evaluation of the PAXgene Tissue Fixation System

To evaluate the PAXgene tissue fixation system. Clinical biospecimens (n = 46) were divided into PAXgene-fixed paraffin-embedded (PFPE), formalin-fixed paraffin-embedded (FFPE), and fresh-frozen (FF) blocks. PFPE and FFPE sections were compared for histology (H&E staining) and immunohistochemistry (14 antibodies) using tissue microarrays. PFPE, FFPE, and FF samples were compared in terms of RNA quality (RNA integrity number, polymerase chain reaction [PCR] amplicon length, and quantitative reverse transcription PCR), DNA quality (gel electrophoresis and methylation profiling) and protein quality (liquid chromatography-mass spectrometry [LC-MS/MS]). PFPE protocol optimization was required in most cases and is described. RNA extracted from PFPE sections was considerably less degraded than that from FFPE sections but more degraded than that from FF blocks. Genomic-length DNA was extracted from PFPE and FF biospecimens, and methylation profiling showed PFPE and FF biospecimens to be almost indistinguishable. Only degraded DNA was extracted from FFPE biospecimens. PFPE sections yielded peptides that were slightly less amenable to LC-MS/MS analysis than FFPE sections, but FF gave slightly better results. While it cannot be envisaged that PAXgene will replace formalin in a routine clinical setting, for specific projects or immunodiagnostics involving biospecimens destined for immunohistochemical or histologic staining and DNA or RNA analyses, PAXgene is a viable option.

Loss of antigenicity with tissue age in breast cancer

Archived tumor specimens, particularly those collected by large cooperative groups and trials, provide a wealth of material for post hoc clinical investigation. As these tissues are rigorously collected and preserved for many decades, subsequent use of the specimens to answer clinical questions must rely on the assumption that expression and detection of target biomarkers are not degraded with time. To test this assumption, we measured the expression of estrogen receptor (ER), human epidermal growth receptor 2 (HER2), and Ki67 in human breast carcinoma using quantitative immunofluorescence (QIF) in a series of formalin-fixed paraffin-embedded (FFPE) tissues from 1295 individual patients preserved for 7 to 53 years in four cohorts on tissue microarrays. Protein expression was measured using the automated quantitative analysis method for QIF. Change in quantitative protein expression over time was estimated in positive cases using both Pearson's correlation and a polynomial regression analysis with a random effects model. The average signal decreased with preservation time for all biomarkers measured. For ER and HER2, there was an average of 10% signal loss after 9.9 years and 8.5 years, respectively, compared with the most recent tissue. Detection of Ki67 expression was lost more rapidly, with 10% signal loss in just 4.5 years. Overall, these results demonstrate the need for adjustment of tissue age when studying FFPE biospecimens. The rate of antigenicity loss is biomarker specific and should be considered as an important variable for studies using archived tissues.

Abstract 304: Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues

Abstract Next Generation Sequencing (NGS) technologies are being used for detection of somatic mutations in tumors and studies of germline variation. However, most NGS studies used DNA isolated either from whole blood or fresh frozen tissue specimens. Meanwhile, the tissue specimens available from most National Cancer Institute (NCI) funded cohorts and the Surveillance, Epidemiology and End Results (SEER) registries (http://seer.cancer.gov/biospecimen/) are primarily formalin fixed paraffin embedded (FFPE). There are limited data, on a small number of FFPE tissue samples, which suggest NGS is feasible. Much less is known about the feasibility of these technologies for large scale studies or using older FFPE specimens (e.g. 5-30 years old). The main objective of this project was to conduct a pilot study to determine whether the DNA obtained from archival FFPE tissue is of sufficient quality and quantity to conduct NGS. Sixty high-grade serous ovarian adenocarcinomas from FFPE tissues which were between 7 and 31 years old were obtained from three SEER registries. DNA was extracted, quantified, quality assessed, and subjected to whole exome sequencing. DNA extraction (yields and quality) and whole exome sequencing (depths of coverage and exome coverage obtained) results from this study will be presented. Ultimately, data derived from this analysis could serve as the basis for determining the utility of archival FFPE biospecimens for characterization and discovery projects utilizing NGS technologies instead of relying on frozen biospecimens. Citation Format: Danielle Mercatante Carrick, Sean Altekruse, Corrine Camalier, Wendy Cozen, Brenda Hernandez, Charles Lynch, Paul McGregor, Michele G. Mehaffey, Lisa McShane, JoyAnn Phillips Rohan, Mickey Williams, Elizabeth M. Gillanders, Leah E. Mechanic, Sheri Schully. Feasibility study of next-generation sequencing on residual formalin-fixed paraffin-embedded tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 304. doi:10.1158/1538-7445.AM2014-304

Biospecimen use correlates with emerging techniques in cancer research: impact on planning future biobanks.

The average cohort size for tissue biospecimens used in cancer research studies has increased significantly over the last 20 years. To understand some of the factors behind changes in biospecimen use, we examined cancer research publications to characterize the relationship between specific assay techniques and biospecimen formats and products. We assessed a representative cross section of 378 publications in the journal Cancer Research that used tissue biospecimens, selected from 6 intervals between 1988 and 2010. Publications were categorized by biospecimen utilization, format type (Frozen, Formalin-Fixed Paraffin-Embedded, and Fresh), product type (RNA, DNA, Protein, Cells, and Metabolites), and types of research techniques performed. There was an increase in average biospecimen cohort size (p=0.001); relative use of Formalin-Fixed Paraffin-Embedded biospecimens (24%-68%, p<0.0001); and the proportion of techniques assaying RNA products from biospecimens (Frozen and Fresh formats, p<0.05), from 1988 to 2008. However, these trends have not continued and there has been no further increase from 2008 to 2010. While specific techniques such as 'tissue microarray' analysis appear to have driven some changes in format requirements, there is an overall trend towards techniques requiring RNA products across all formats of biospecimens in basic cancer research. Since pre-analytical variables influence gene expression (RNA levels) more than gene structure (DNA sequence), recognition of these research trends is important for biobanks when deciding priorities for the optimal preservation format and annotation of biospecimens.

Biospecimen use in cancer research over two decades.

Demand for biospecimens in cancer research has increased but there are relatively few data on the trends in biospecimen usage. These data are needed to enable projection of future demand. We analyzed biospecimen usage in publications published at five-year intervals (2008, 2003, 1998, 1993, and 1988) in four cancer research journals (Cancer Research, Clinical Cancer Research, British Journal of Cancer and International Journal of Cancer). We categorized publications in three ways: 1) biospecimen utilization yes/no; 2) biospecimen cohort size; and 3) format of biospecimens used including frozen tissue, Formalin-Fixed Paraffin-Embedded (FFPE) tissue, fresh tissue, fluids, and hematological biospecimens. Biospecimens were used in 1292/3307 (39%) of publications analyzed and sufficient information was available to further classify biospecimen usage in 1228 publications. The proportion of publications in each journal using biospecimens ranged from 23% to 61% between journals, with no significant change within each journal over time. A more detailed review of tissue biospecimen use showed a significant increase in cohort sizes from 1988 to 2008 (mean 52 to 198, respectively; P < 0.0001). This reflected increased cohort sizes for both frozen and FFPE tissues from 1993 to 2008 (frozen, 59 to 119; FFPE, 66 to 194) but not fresh tissues. The relative proportion of studies using frozen or fresh tissues alone has decreased (71% to 24%) while those using FFPE alone or combined FFPE/frozen tissue cohorts has increased (24% to 72%) over this period. We conclude that the overall demand for biospecimens in cancer research has increased significantly (almost fourfold) over the past 20 years. We predict that average cohort sizes will increase by at least twofold for frozen and FFPE biospecimens over the next ten years, and that the majority of studies will be based on FFPE tissues or combined FFPE/Frozen tissue cohorts.