Formalin-fixed And Paraffin-embedded Samples Research Articles

Evaluation of DNAmAge in paired fresh, frozen, and formalin-fixed paraffin-embedded heart tissues.

The continued development in methylome analysis has enabled a more precise assessment of DNA methylation, but treatment of target tissue prior to analysis may affect DNA analysis. Prediction of age based on methylation levels in the genome (DNAmAge) has gained much interest in disease predisposition (biological age estimation), but also in chronological donor age estimation in crime case samples. Various epigenetic clocks were designed to predict the age. However, it remains unknown how the storage of the tissues affects the DNAmAge estimation. In this study, we investigated the storage method impact of DNAmAge by the comparing the DNAmAge of the two commonly used storage methods, freezing and formalin-fixation and paraffin-embedding (FFPE) to DNAmAge of fresh tissue. This was carried out by comparing paired heart tissue samples of fresh tissue, samples stored by freezing and FFPE to chronological age and whole blood samples from the same individuals. Illumina EPIC beadchip array was used for methylation analysis and the DNAmAge was evaluated with the following epigenetic clocks: Horvath, Hannum, Levine, Horvath skin+blood clock (Horvath2), PedBE, Wu, BLUP, EN, and TL. We observed differences in DNAmAge among the storage conditions. FFPE samples showed a lower DNAmAge compared to that of frozen and fresh samples. Additionally, the DNAmAge of the heart tissue was lower than that of the whole blood and the chronological age. This highlights caution when evaluating DNAmAge for FFPE samples as the results were underestimated compared with fresh and frozen tissue samples. Furthermore, the study also emphasizes the need for a DNAmAge model based on heart tissue samples for an accurate age estimation.

Quantifying mRNA in Highly Degraded Fixed Tissues by Nanostring Technology: A Comparative Study.

Archive tissues are the most available source of human tissues useful for molecular analysis in translational research. The main issues for those specimens are the modification and degradation of biomolecules, namely proteins, DNA, and RNA. In the last decade, several high-throughput analytical methods have been applied to archive tissues. Although histological tissues are fixed in neutral-buffered formalin nowadays, in the recent past, Bouin's solution was also used in tissue processing. The present study aims to investigate the feasibility of nCounter Nanostring hybridization in quantifying mRNA in highly degraded samples, such as Bouin's fixed and paraffin-embedded (BFPE) tissues, in comparison to the standard formalin-fixed and paraffin-embedded (FFPE) tissues as a source of RNA. A total of 16 paraffin-embedded tissue blocks from eight patients were analyzed (8 were FFPE and 8 were BEPE). Nanostring technology was applied to 300 ng of each RNA sample, whereas 360 ng of the same templates were retrotranscribed and submitted to qPCR and ddPCR. Our results show that the Nanostring technology outperforms the reference methods (ddPCR and qPCR) in detecting target mRNA in FFPE and BFPE samples. However, even Nanostring technology does not escape the limitation imposed by the degradation of the RNA templates, which could lead to misleading conclusions on the gene expression level.

Characteristics of innate and adaptive immune disorder in IgA nephropathy based on integrated bioinformatics.

Our study aims to investigate the immunological pathogenesis underlying immunoglobulin A nephropathy (IgAN) and explore potential biomarkers for IgAN diagnosis. Differentially expressed genes (DEGs) of formalin-fixed and paraffin-embedded (FFPE) samples were screened between IgAN patients and healthy people based on GSE115857. Gene oncology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment was performed to identify related biological processes and pathways. CIBERSORT was utilized to seek the relationship of immune cell infiltration with IgAN. Finally, the expression of paraoxonase 2 (PON2) related to innate immune response was verified in FFPE samples of minimal change disease and IgAN patients by immunohistochemistry and PAS staining. 25 down-regulated genes and 12 up-regulated genes were identified in IgAN patients, which mainly responded to endothelial cell proliferation, inflammatory response, and angiogenesis. Toll-like receptor signaling pathway and Epstein-Barr virus (EBV) infection might be involved in IgAN pathogenesis. In addition, the infiltration of macrophages M0, naïve B cells, and follicular helper T (Tfh) cells was positively correlated in IgAN patients. Macrophages M1 and M2 infiltration were up-regulated in IgAN patients, which indicated that innate immune response was closely associated with IgAN. Besides, the results of immunohistochemistry showed that PON2 was obviously positively expressed in acute and chronic lesions of IgAN patients. In addition to abnormalities in the adaptive immune response, macrophages M1/M2 and innate immune disorder may participate in IgAN pathogenesis. PON2 may become the feasible targets for further investigation of IgAN.

A comprehensive assessment of exome capture methods for RNA sequencing of formalin-fixed and paraffin-embedded samples

RNA-Seq analysis of Formalin-Fixed and Paraffin-Embedded (FFPE) samples has emerged as a highly effective approach and is increasingly being used in clinical research and drug development. However, the processing and storage of FFPE samples are known to cause extensive degradation of RNAs, which limits the discovery of gene expression or gene fusion-based biomarkers using RNA sequencing, particularly methods reliant on Poly(A) enrichment. Recently, researchers have developed an exome targeted RNA-Seq methodology that utilizes biotinylated oligonucleotide probes to enrich RNA transcripts of interest, which could overcome these limitations. Nevertheless, the standardization of this experimental framework, including probe designs, sample multiplexing, sequencing read length, and bioinformatic pipelines, remains an essential requirement. In this study, we conducted a comprehensive comparison of three main commercially available exome capture kits and evaluated key experimental parameters, to provide the overview of the advantages and limitations associated with the selection of library preparation protocols and sequencing platforms. The results provide valuable insights into the best practices for obtaining high-quality data from FFPE samples.

Proteomic analyses identify HK1 and ATP5A to be overexpressed in distant metastases of lung adenocarcinomas compared to matched primary tumors

Lung cancer is the leading cause of cancer-related deaths worldwide with lung adenocarcinoma (LUAD) being the most common type. Genomic studies of LUAD have advanced our understanding of its tumor biology and accelerated targeted therapy. However, the proteomic characteristics of LUAD are still insufficiently explored. The prognosis for lung cancer patients is still mostly determined by the stage of disease at the time of diagnosis. Focusing on late-stage metastatic LUAD with poor prognosis, we compared the proteomic profiles of primary tumors and matched distant metastases to identify relevant and potentially druggable differences. We performed high-performance liquid chromatography (HPLC) and electrospray ionization tandem mass spectrometry (ESI–MS/MS) on a total of 38 FFPE (formalin‐fixed and paraffin‐embedded) samples. Using differential expression analysis and unsupervised clustering we identified several proteins that were differentially regulated in metastases compared to matched primary tumors. Selected proteins (HK1, ATP5A, SRI and ARHGDIB) were subjected to validation by immunoblotting. Thereby, significant differential expression could be confirmed for HK1 and ATP5A, both upregulated in metastases compared to matched primary tumors. Our findings give a better understanding of tumor progression and metastatic spreads in LUAD but also demonstrate considerable inter-individual heterogeneity on the proteomic level.

Transcriptomic profiling of nuclei from paraformaldehyde-fixed and formalin-fixed paraffin-embedded brain tissues

BackgroundParaformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal. ResultsWe evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition. SignificanceThis represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1–3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.

P-325 Evidence for dysregulation of oncogene pathways at the gene expression level in the epithelium of deep infiltrating endometriosis compared to adenomyosis

Abstract Study question How does the gene expression profile of epithelium cells differ between deep infiltrating endometriosis (DIE) and adenomyosis (FA)? Summary answer The gene expression of epithelial cells from DIE reveals a significant upregulation of the PI3K-pathway and downregulation of the RAS-pathway compared to epithelium from FA. What is known already Both DIE and FA, are histologically considered benign but have features of malignant diseases, such as a tendency for local tissue invasion. Recent next generation sequencing studies have detected driver mutations in cancer-associated genes such as PIK3CA, ARID1A, PPP2R1A and KRAS in the epithelium of DIE, as well as in KRAS in epithelium of FA. Furthermore, identical mutations in the KRAS gene were detected in coexisting adenomyotic and endometriotic lesions, supporting the theory of a common molecular mechanism. The gene expression differences between these two entities were therefore investigated in the present study. Study design, size, duration This is an experimental analysis of 7 adenomyosis and 19 DIE samples (histologically confirmed), that were formalin-fixed and paraffin-embedded (FFPE) collected between 2003 and 2018. These were provided by the tissue bank of the National Centre for Tumour Diseases (Heidelberg, Germany), in accordance with the Ethics Committee of the University of Heidelberg (approval number S-362/2017). In addition, the medical and epidemiological data of the corresponding patients were retrospectively analyzed based on the clinical data collected. Participants/materials, setting, methods For this study, the epithelia of FFPE samples were microdissected in a laser-guided fashion. After RNA extraction, the expression of 770 genes was analyzed using the nCounter Technology with the Human PanCancer Pathways Panel (Nanostring). All genes with an adjusted false discovery rate of p < 0.05 and a fold change of < 0.66 or > 1.5 were considered differentially expressed and subjected to functional annotation and clustering using DAVID bioinformatics resources. Main results and the role of chance Our analysis revealed a total of 162 differentially expressed genes, that were either significantly increased (n = 116) or decreased (n = 46) in epithelium of DIE compared to that of FA (with log2 fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05). Gene ontology and KEGG pathway analysis for genes with increased expression in DIE compared with FA revealed significant upregulation of genes belonging to the PI3K-pathway and the focal adhesion pathway, as well as pathways related to viral infection, endocrine resistance, and malignancy. The upregulated pathways in adenomyosis mainly included the RAS-pathway. Limitations, reasons for caution The average age of DIE patients was lower, but many of them were hormonally inactive due to GnRH analogues, which mitigates any age-related differences. Furthermore, only relative expression was studied between the groups without comparison to normal endometrium. Overall, the sample size is relatively small and further studies are needed. Wider implications of the findings The expression of different signaling pathways in the two entities could, for example, explain the different sensitivity of the two diseases to certain therapeutic approaches. While DIE responds well to therapy with the progestogen dienogest, progesterone resistance is often described in adenomyosis patients. Trial registration number not applicable

Abstract 6021: Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas

Abstract Background: Uterine leiomyosarcoma (LMS) comprises 60% to 70% of the uterine sarcoma (US). These tumors present high rates of recurrence and metastasis, and the patients’ survival rates are very low (20% in five years). It is known that somatic mutations and modifications in the DNA methylation levels are associated with several types of neoplasms. Previously, our group identified missense and loss of function mutations in KMT2D, CREBBP, ATM, TSC2, and GNAS, using a genetic screening method in LMS samples. Based on these results, we decided to investigate whether a genetic and epigenetic crosstalk could help to understand the biological processes involved in the LMS origin, development, and pathogenesis. The present study aimed to evaluate the KMT2D, CREBBP, ATM, TSC2 and GNAS gene expression profile in LMS cell line, and their methylation levels in formalin-fixed and paraffin-embedded (FFPE) patients´ samples. Methods: We selected 15 LMS - FFPE samples obtained via surgical procedures performed between 2012 and 2019 at the Instituto do Cancer do Estado de São Paulo (ICESP). Genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit and we evaluated methylation levels using the Illumina Infinium Methylation EPIC BeadChip system 850k, including samples treated with bisulfite-treated DNA. Leiomyoma (LM) (THESCs CRL-4003) and LMS (SK-UT-1) cell lines were cultivated for gene expression analysis. Cells were growth for 24, 48, 72, and 96 hours, and the total RNA was extracted by TRIzol. High-Capacity cDNA Reverse Transcription Kit was used for cDNA synthesis and for gene expression evaluation, real-time PCR reactions were performed using TaqMan Universal PCR Master Mix and inventoried TaqMan probes. Results: KMT2D and TSC2 showed increased expression in LMS compared to LM (cut-off ≤ -2 and ≥ 2 for down and upregulation, respectively), with higher expression in 24 hours of KMT2D [Fold Regulation (FR): 2.77] and TSC2 (FR: 2.58). In contrast, CREBBP was downregulated, with lowest expression in 72 hours (FR: -6.57). ATM was upregulated at 24, 48 with higher expression in 72 hours (FR: 2.32), and GNAS was downregulated, with lowest expression in 96 hours (FR: -4.31). Moreover, the methylation analyzes showed an expressive hypomethylation in LMS samples, compared to myometrium, for KMT2D (β value: 0.45; p= 0.004); CREBBP (β value: 0.51 p < 0.0001); ATM (β value: 0.47 p < 0.0001); TSC2 (β value: 0.53 p < 0.0001) and GNAS (β value: 0.41 p < 0.0001). In conclusion, our study showed that potentially pathogenic mutations may be associated with an aberrant hypomethylation profile, as well as increased expression of KMT2D, TSC2, ATM and loss expression of CREBBP and GNAS in LMS. The interconnection of these genetic and epigenetic events is essential for understanding the complex biology of these tumors, in addition to enabling identification of biomarkers. Citation Format: Laura G. dos Anjos, Daniela Bizinelli, Katia C. Carvalho. Genetic and epigenetic features OF KMT2D, CREBBP, ATM, TSC2 and GNAS in uterine leyomiosarcomas [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 6021.

808 Analysis of Medulloblastomas Subgroups in Pediatric Patients in Latin-America

INTRODUCTION: The 4 groups molecular-based classification of medulloblastomas is widely accepted. However, technology inaccessibility and low-quality formalin-fixed and paraffin-embedded (FFPE) samples, unable its application for most Latin-American cancer centers. METHODS: The goal of this study was to perform a feasibility analysis based on 18 mRNA expression by qPCR integrated with immunohistochemistry (IH) and fluorescent hybridization in situ (FISH) (c-MYC, n-MYC and monosomy 6) for molecular stratification to create an International Treatment Protocol at a low cost for low-income centers. RESULTS: Fifty-four FFPE samples were classified according to histologic and molecular aspects. Classic medulloblastoma was the commonest histology (53.7%), followed by desmoplastic/extensive nodularity (24.1%), NOS (16,7%) and anaplastic (5,6%). IH markers classified patients in three groups (WNT, SHH and non-WNT/non-SHH) in 98% of cases. PCR-based method confirmed results from IH in 81.5%. Additionally, it detected WNT activation in 2 more patients, previously classified as SHH. For both cases, the presence of monosomy 6 confirmed WNT subgroup. The integration of these three techniques resulted in the following frequencies: WNT (13.0%), SHH (38.9%), group 3 (9.3%), group 4 (20.3%) and non-WNT/non-SHH (18.5%). From 40 patients with clinical follow-up available, 3-year overall survival (n = 40) for low, intermediate and high-risk groups were 100%, 60% and 20%, respectively (p < 0.05), based only in molecular criteria, which confirmed the prognostic importance of this method at a low cost. CONCLUSIONS: The prognosis of the treatment of medulloblastomas has changed after novel studies on the molecular subgroups and the introduction of target-based therapy. The current protocol will enable different centers to implement treatment based on the knowledge of molecular subgroups.

Selection and Evaluation of mRNA and miRNA Reference Genes for Expression Studies (qPCR) in Archived Formalin-Fixed and Paraffin-Embedded (FFPE) Colon Samples of DSS-Induced Colitis Mouse Model

The choice of appropriate reference genes is essential for correctly interpreting qPCR data and results. However, the majority of animal studies use a single reference gene without any prior evaluation. Therefore, many qPCR results from rodent studies can be misleading, affecting not only reproducibility but also translatability. In this study, the expression stability of reference genes for mRNA and miRNA in archived FFPE samples of 117 C57BL/6JOlaHsd mice (males and females) from 9 colitis experiments (dextran sulfate sodium; DSS) were evaluated and their expression analysis was performed. In addition, we investigated whether normalization reduced/neutralized the influence of inter/intra-experimental factors which we systematically included in the study. Two statistical algorithms (NormFinder and Bestkeeper) were used to determine the stability of reference genes. Multivariate analysis was made to evaluate the influence of normalization with different reference genes on target gene expression in regard to inter/intra-experimental factors. Results show that archived FFPE samples are a reliable source of RNA and imply that the FFPE procedure does not change the ranking of stability of reference genes obtained in fresh tissues. Multivariate analysis showed that the histological picture is an important factor affecting the expression levels of target genes.

Establishment and evaluation of digital PCR methods for HER2 copy number variation in breast cancer

Accurate measurement of human epidermal growth factor receptor 2 (HER2) copy number variation (CNV) is very important for guiding the tumor target therapy in breast cancer. Digital PCR (dPCR) is a sensitive and an absolute quantitative method, which can be used to detect HER2 CNV. Three HER2 exon-specific digital PCR assays along with three new reference genes assays (homo sapiens ribonuclease P RNA component H1 (RPPH1), glucose-6-phosphate isomerase (GPI), and chromosome 1 open reading frame 43 (C1ORF43), on different chromosomes) were established and validated by using standard reference material, 8 different cell lines and 110 clinical Formalin-fixed and paraffin-embedded (FFPE) samples. DPCR can achieve precise quantification of HER2 CNV by calculating the ratio of HER2/reference gene. The positive and negative coincidence rates were 98% (53/54) and 95% (53/56), respectively, compared with fluorescence in situ hybridization (FISH) diagnostic result 110 of FFPE samples. The common reference gene CEP17 used for FISH diagnostic was not suitable as single reference gene for HER2 CNV measurements by dPCR. The best practice of HER2 CNV determination by dPCR is to conduct the three duplex assays of H1 (HER2 exon 4) with the proposed three new reference genes, with a positive cut-off value of H1/RPPH1 ≥ 2.0 or H1/averaged reference gene ≥ 2.0. The proposed dPCR method in our study can accurately provide absolute copy number of HER2 and reference gene on an alternative chromosome, thus avoiding false negative caused by polysomy of chromosome 17. The improved molecular typing and diagnosis of breast cancer will better guide clinical medication.

Developing an effective quality evaluation strategy of next-generation sequencing for accurate detecting non-small cell lung cancer samples with variable characteristics: a real-world clinical practice.

Next-generation sequencing (NGS) has been widely used in determining molecular profiling of non-small cell lung cancer (NSCLC). However, low-quality sequencing data may be generated with formalin-fixed and paraffin-embedded (FFPE) samples that have passed pre-sequencing quality control (QC). Therefore, quality evaluation of sequencing data is also critical for accurate tissue genotyping. Herein, we aimed to developed a grading QC algorithm, and provide a recommendation to refine and optimize NGS-based molecular diagnostic strategies. We interrogated 1260 NSCLC samples using hybrid capture-based targeted DNA NGS, and quantified the sequencing data as high, medium and low quality, according to a grading QC algorithm. Then, we explored the relationship between sequencing quality and sample characteristics, and compared the concordance rates of results between NGS and conventional molecular tests for FFPE samples with variable characteristics. We found that high-quality data were associated with samples with shorter storage time and lower DNA degradation in resection samples, and were associated with intra-hospital samples, adequate DNA quantity, and lower DNA degradation in biopsy samples. Moreover, accurate NGS results can be achieved in samples with high-quality data, but not samples with medium-quality data, especially for rearrangements detection. Our study demonstrates that the real-world clinical adoption of an effective QC strategy for NGS is necessary to ensure accurate results from FFPE samples of NSCLC with variable characteristics. Validation of actionable alterations by additional methods is highly recommended in cases with low QC score, particularly for the detection of rearrangements.

P454 Massive parallel fungal sequencing on formalin-fixed tissues: development and contribution in integrated histomolecular diagnosis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesHistopathology is the gold standard for distinguishing between colonization and fungal infection, but it does not provide a precise diagnosis of genera/species. However, if the culture is negative or if no specimen is sent to the Mycology laboratory, only the specimen sent to the Pathology department is available. Formalin fixation and paraffin embedding (FFPE) cause DNA damage, making it difficult to perform molecular techniques.The objective was to develop and evaluate the contribution of massive parallel sequencing (MPS) to FFPE tissues.MethodsHistopathological review of all cases was performed. Then, DNA extraction from FFPE tissues was optimized by: (1) macrodissecting the fungal-rich area on the paraffin block; (2) comparing the efficiency of two DNA extraction kits (QIAamp DNAmicro-kit, QIAGEN; Maxwell 16 LEVRNA FFPE Purification kit, Promega, optimized for RNA and DNA extraction), by comparison of Aspergillus fumigatus and Mucorale specific PCR results for 30 cases. For 124 other cases, the sensitivity of two primer pairs (ITS3/4 and MITS2A/2B) was tested for identification by Sanger sequencing and then MPS. Finally, a histomolecular comparison was performed. This work was funded by the Société de Pathologie Infectieuse de Langue Française (SPILF).ResultsTo optimize extraction, DNA was extracted by both kits from samples of 16 mucormycoses and 14 A. fumigatus infections. PCR sensitivity was better with the QIAGEN extraction kit [100% (30/30)] compared to the Promega kit [86.7% (26/30)].PCR amplification of fungal DNA from an additional 124 FFPE samples was performed. The primer pairs ITS3/4 and MITS2A/2B, allowed: (1) identification by Sanger sequencing-histopathological analysis in 38.7% (48/124) of the cases in total, and more specifically 33% (41/124) of cases with the ITS3/4 primers and 32.3% (40/124) of cases with the MITS2A/B primers; and (2) identification by integrated SMP-histopathological analysis in 75% (93/124) of all cases (primers ITS3/4 and MITS2A/2B), and more specifically 66.1% (82/124) for ITS3/4 and 62.1% (77/124) for MITS2A/B (both primer pairs did not detect/amplify the same fungal genera/species). The combination of all results from Sanger sequencing and MPS led to fungal identification in 75.8% (94/124) of cases. In total, the addition of NGS to Sanger sequencing increased the diagnostic proportion by 36.3% (45/124; P <.0001). Example of integrated histomolecular diagnosis (Fig. 1): patient with a pseudotumor presentation of pulmonary invasive aspergillosis (A: thoracic CT scan; B: macroscopic examination of lesion after formalin fixation; C, D, E: Hematoxylin-eosin-safran, x20 and x400: observation of a necrotic mass caused by hyalohyphomycetes; F: Grocott, x400) with no culture or molecular identification available on fresh tissue. In contrast, identification by MPS on FFPE tissue was compatible with morphological analysis: Aspergillus section Fumigati, leading to the integrated histomolecular diagnosis of pulmonary invasive aspergillosis.ConclusionThe development of the fungal MPS on FFPE tissues is innovative and unprecedented for the achievement of an integrated histomolecular diagnosis in fungal pathology. It increases significantly the diagnostic proportion by 36.3%. This strategy can be used in hospitals and could improve patient management, especially when no sample is sent to the Mycology laboratory or when the culture is negative.

The mutational signatures of formalin fixation on the human genome

Clinical archives of patient material near-exclusively consist of formalin-fixed and paraffin-embedded (FFPE) blocks. The ability to precisely characterise mutational signatures from FFPE-derived DNA has tremendous translational potential. However, sequencing of DNA derived from FFPE material is known to be riddled with artefacts. Here we derive genome-wide mutational signatures caused by formalin fixation. We show that the FFPE-signature is highly similar to signature 30 (the signature of Base Excision Repair deficiency due to NTHL1 mutations), and chemical repair of DNA lesions leads to a signature highly similar to signature 1 (clock-like signature due to spontaneous deamination of methylcytosine). We demonstrate that using uncorrected mutational catalogues of FFPE samples leads to major mis-assignment of signature activities. To correct for this, we introduce FFPEsig, a computational algorithm to rectify the formalin-induced artefacts in the mutational catalogue. We demonstrate that FFPEsig enables accurate mutational signature analysis both in simulated and whole-genome sequenced FFPE cancer samples. FFPEsig thus provides an opportunity to unlock additional clinical potential of archival patient tissues.

The Presence of EGFR T790M in TKI-Naïve Lung Cancer Samples of Patients Who Developed a T790M-Positive Relapse on First or Second Generation TKI Is Rare.

Simple SummaryTreatment outcomes for non-small cell lung cancer (NSCLC) patients have significantly improved since the introduction of targeted therapy using tyrosine kinase inhibitors (TKI). Despite the initial promising results, most patients develop resistance due to on- or off-target mechanisms. On-target mutations prevent the effective binding of TKIs. In this study, we determined whether the most common on-target resistance mutation for treatment with EGFR-TKI mutation, i.e., the EGFR T790M mutation, can be detected in pre-treatment tissue samples and can predict treatment outcome. We included 33 patients who progressed with a T790M positive relapse upon treatment with EGFR-TKI between 2013 to 2019. Progression-free survival (PFS) time of the single T790M positive patient was 9 months, while the T790M negative patients had a median PFS of 10 months (range 2–27). Our results show that the presence of EGFR T790M mutations in pre-TKI samples is rare, even in patients who subsequently progressed with an EGFR T790M mutation.EGFR-mutated non-small cell lung cancer (NSCLC) patients can be effectively treated with tyrosine kinase inhibitors (TKI) but frequently present with an EGFR T790M resistance mutation at relapse. We aimed to screen for T790M in pre-treatment formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients with a confirmed T790M mutation at progression. We analyzed 33 pre-treatment DNA samples of NSCLC patients who progressed upon TKI between 2013 to 2019. To establish storage-time dependent formalin fixation-induced background levels for C>T mutations, we analyzed DNA isolated from archival (stored >1 year, n = 22) and recently generated (stored <1 month, n = 11) FFPE samples and included DNA isolated from white blood cells (WBC) (n = 24) as controls. DNA samples were analyzed by droplet digital (dd)PCR, and positivity was defined by outlier detection according to Grubb’s criterion. The T790M background allele frequency levels were 0.160% in DNA isolated from archival-FFPE, 0.100% in fresh FFPE, and 0.035% in WBC. Progression-free survival (PFS) time of the single T790M positive patient was 9 months, while T790M negative patients had a median PFS of 10 months (range 2–27). Proper storage time matched FFPE control samples are essential for reliable detection of T790M mutation at low VAF. The presence of EGFR T790M mutations in pre-TKI samples is rare, even in patients who progressed with EGFR T790M mutations.

The significance of micro-EGFR T790M mutation on EGFR-TKI efficacy in patients with NSCLC: The WJOG13119L study.

e21177 Background: The significance of micro-pretreatment EGFR T790M mutation on EGFR-tyrosine kinase inhibitor (TKI) efficacy in patients with non-small cell lung cancer (NSCLC) remains uncertain. In addition, there has been no method to examine the micro-T790M mutation after excluding formalin-fixed and paraffin-embedded (FFPE)-derived artificial mutations. Therefore, we used a novel method to examine the variant allele frequency (VAF) of T790M mutation in the FFPE samples after excluding the artificial mutations. Subsequently, we retrospectively investigated the association between micro-T790M mutations and time to treatment failure (TTF) in patients treated with 1st, 2nd, or 3rd generation (1G, 2G, 3G) EGFR-TKIs. Methods: We enrolled patients with NSCLC who harbored EGFR exon 19 deletion (Del19) or L858R without T790M mutation using conventional methods. Patients who received 1G or 2G EGFR-TKIs until June 2018 or 3G EGFR-TKI until June 2019 from 31 institutions were included. Droplet digital PCR was performed using pretreatment FFPE tumor samples. We defined cases as micro-T790M positive when the VAF of T790M was higher than that of F795F, owing to the FFPE-derived artificial mutations. The ratio of micro-T790M mutation was calculated as follows: {(VAF of T790M) – (VAF of F795F)} /VAF of (Del19 or L858R). The lowest value was set as 0. Results: A total of 110, 102, and 103 patients received 1G, 2G, and 3G EGFR-TKIs, respectively. Of these patients, 48.1%, 47.0%, and 47.6% were classified as micro-T790M positive, respectively. The ratios of micro-T790M mutation, represented as median (%) (interquartile range), in 1G, 2G, and 3G EGFR-TKI groups were 0.27 (0.12-0.87), 0.28 (0.13-0.47), and 0.42 (0.20-0.68), respectively. The VAF of T790M or F795F in all patients, represented as median (%) (interquartile range), was 0.26 (0.19-0.35) or 0.25 (0.19-0.33), respectively. The TTF was not significantly different between the T790M-positive and negative groups in analysis based on each generation TKIs. In the Del19 group, the TTF of the 1G EGFR-TKI was significantly shorter, while that of the 3G EGFR-TKI was significantly longer in the T790M-positive group than in the negative group ( p= 0.02, 0.04, respectively). In the T790M-positive group, the TTF of the 2G or 3G EGFR-TKI group was significantly longer than that of the 1G EGFR-TKI group ( p= 0.04, 0.01, respectively). This result was observed in the Del19 group but not in the L858 group. Furthermore, TTF did not significantly differ between the 1G, 2G, and 3G EGFR-TKIs in the T790M-negative group. Conclusions: This study showed that micro- EGFR T790M mutations influence the EGFR-TKI efficacy. Detection of micro-T790M mutations will facilitate selection of the optimal EGFR-TKI for patients with EGFR mutated NSCLC.

Development of a Novel Reference Material for Tumor Mutational Burden Measurement Based on CRISPR/Cas9 Technology.

As a biomarker that affects treatment decisions of immune checkpoint inhibitors, the accuracy, reliability, and comparability of tumor mutational burden (TMB) estimation is of paramount importance. To improve the consistency and reliability of these tests, qualified reference materials providing ground-truth data are crucial. In this study, we developed a set of formalin-fixed and paraffin-embedded (FFPE) samples with different TMB values as the novel reference materials for TMB estimation. By introducing several clinically relevant variants in MutS Homolog 2 (MSH2) gene and DNA polymerase epsilon (POLE) gene into human cell lines using CRISPR/Cas9 technology, we first constructed four typical cell lines which verified with hypermutator or ultramutator phenotype. Followed by cell mixing and paraffin embedding, the novel FFPE samples were prepared. It was confirmed that our novel FFPE samples have sufficient quantity of cells, high reproducibility, and they can provide matched wild type sample as the genetic background. The double-platform whole exome sequencing validation showed that our FFPE samples were also highly flexible as they containing different TMB values spanning a clinically relevant range (2.0–106.1 mut/Mb). Without limitations on production and TMB values, our novel FFPE samples based on CRISPR/Cas9 editing are suitable as candidate reference materials. From a practical point of view, these samples can be used for the validation, verification, internal quality control, and proficiency testing of TMB assessment.

A Non-Hazardous Deparaffinization Protocol Enables Quantitative Proteomics of Core Needle Biopsy-Sized Formalin-Fixed and Paraffin-Embedded (FFPE) Tissue Specimens.

Most human tumor tissues that are obtained for pathology and diagnostic purposes are formalin-fixed and paraffin-embedded (FFPE). To perform quantitative proteomics of FFPE samples, paraffin has to be removed and formalin-induced crosslinks have to be reversed prior to proteolytic digestion. A central component of almost all deparaffinization protocols is xylene, a toxic and highly flammable solvent that has been reported to negatively affect protein extraction and quantitative proteome analysis. Here, we present a ‘green’ xylene-free protocol for accelerated sample preparation of FFPE tissues based on paraffin-removal with hot water. Combined with tissue homogenization using disposable micropestles and a modified protein aggregation capture (PAC) digestion protocol, our workflow enables streamlined and reproducible quantitative proteomic profiling of FFPE tissue. Label-free quantitation of FFPE cores from human ductal breast carcinoma in situ (DCIS) xenografts with a volume of only 0.79 mm3 showed a high correlation between replicates (r2 = 0.992) with a median %CV of 16.9%. Importantly, this small volume is already compatible with tissue micro array (TMA) cores and core needle biopsies, while our results and its ease-of-use indicate that further downsizing is feasible. Finally, our FFPE workflow does not require costly equipment and can be established in every standard clinical laboratory.

The Current State of Chromatin Immunoprecipitation (ChIP) from FFPE Tissues.

Cancer cells accumulate epigenomic aberrations that contribute to cancer initiation and progression by altering both the genomic stability and the expression of genes. The awareness of such alterations could improve our understanding of cancer dynamics and the identification of new therapeutic strategies and biomarkers to refine tumor classification and treatment. Formalin fixation and paraffin embedding (FFPE) is the gold standard to preserve both tissue integrity and organization, and, in the last decades, a huge number of biological samples have been archived all over the world following this procedure. Recently, new chromatin immunoprecipitation (ChIP) techniques have been developed to allow the analysis of histone post-translational modifications (PTMs) and transcription factor (TF) distribution in FFPE tissues. The application of ChIP to genome-wide chromatin studies using real archival samples represents an unprecedented opportunity to conduct retrospective clinical studies thanks to the possibility of accessing large cohorts of samples and their associated diagnostic records. However, although recent attempts to standardize have been made, fixation and storage conditions of clinical specimens are still extremely variable and can affect the success of chromatin studies. The procedures introduced in the last few years dealt with this problem proponing successful strategies to obtain high-resolution ChIP profiles from FFPE archival samples. In this review, we compare the different FFPE-ChIP techniques, highlighting their strengths, limitations, common features, and peculiarities, as well as pitfalls and caveats related to ChIP studies in FFPE samples, in order to facilitate their application.

Development of a method for isolation of melanin from archival FFPE tissues of human melanoma for structural studies by pyrolysis-gas chromatography-tandem mass spectrometry

Abstract Introduction There is some evidence that pheomelanin produced in skin melanocytes may be involved in the development of cutaneous melanoma, particularly in individuals with a light skin/red hair phenotype. However, nothing is known about possible correlation between the type and/or clinical stage of melanoma and the content of pheomelanin in the tumor tissue. We believe that archival formalin-fixed and paraffin-embedded (FFPE) melanoma tissues could be a good source of melanin pigment for future large-scale research on that issue. Aim The aim of this work was to develop a method for isolation and purification of melanin from FFPE samples of human melanoma. To test the suitability of the isolation protocol for planned structural studies, the obtained melanin was analyzed for pheomelanin content by the method based on pyrolysis (Py) coupled with gas chromatography and tandem mass spectrometry (GC/MS/MS). Material and methods For melanin isolation, microtome sections of FFPE tissue of primary lesion and lymph node metastases were subjected to a multistep procedure of paraffin removal, tissue rehydration, homogenization, and digestion with the set of proteolytic enzymes. The pigment samples were then pyrolyzed at 500 °C, and the GC-separated thermal degradation products were identified using a triple quadrupole mass spectrometer operating in the multiple reaction monitoring mode. Results GC/MS/MS analysis of the pyrolysis products revealed the presence of pheomelanin markers, which allowed quantitation of a pheomelanin component of each of the isolated pigments. Melanin from the FFPE primary melanoma was found to contain 6.6%, and the pigment from FFPE metastatic lymph node 7.5% of pheomelanin. Conclusions The developed protocol allows for the isolation of melanin from FFPE melanoma specimens. The pigment can be successfully studied for pheomelanin content by Py-GC/MS/MS method. The results of our study indicate that archival FFPE tumor tissues can be used as a good source of melanin for future structural studies aimed at shedding more light on the role of pheomelanin in the pathomechanism of cutaneous melanoma.