Formalin-fixed And Paraffin-embedded Tissue Research Articles

Quantitative Profiling of the Human Substantia Nigra Proteome from Laser-capture Microdissected FFPE Tissue

Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

A streamlined mass spectrometry-based proteomics workflow for large-scale FFPE tissue analysis.

Formalin fixation and paraffin-embedding (FFPE) is the most common method to preserve human tissue for clinical diagnosis, and FFPE archives represent an invaluable resource for biomedical research. Proteins in FFPE material are stable over decades but their efficient extraction and streamlined analysis by mass spectrometry (MS)-based proteomics has so far proven challenging. Herein we describe a MS-based proteomic workflow for quantitative profiling of large FFPE tissue cohorts directly from histopathology glass slides. We demonstrate broad applicability of the workflow to clinical pathology specimens and variable sample amounts, including low-input cancer tissue isolated by laser microdissection. Using state-of-the-art data dependent acquisition (DDA) and data independent acquisition (DIA) MS workflows, we consistently quantify a large part of the proteome in 100 min single-run analyses. In an adenoma cohort comprising more than 100 samples, total workup took less than a day. We observed a moderate trend towards lower protein identification in long-term stored samples (>15 years), but clustering into distinct proteomic subtypes was independent of archival time. Our results underscore the great promise of FFPE tissues for patient phenotyping using unbiased proteomics and they prove the feasibility of analyzing large tissue cohorts in a robust, timely, and streamlined manner. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Fast Cancer Molecular Diagnosis from FFPE Tissues Based on Metabolic Profiles Using SpiderMass Technology

Formalin-fixation and paraffin-embedding (FFPE) is the worldwide gold standard for tissue preservation and routine material for general diagnostics and cancer in particular. Despite the fact that histology is complemented by other modalities such as in situ hybridization (ISH) or immunohistochemistry (IHC), the pathology can fail to provide the right diagnosis in a certain number of cases. Overall, the standard process is time-consuming, labor intensive and doesn’t reach the right sensitivity and specificity. Lipids are central players in cancer. Unfortunately, to this date lipids aren’t used for diagnostics and lipid analysis from FFPE has never been reported due to tissue processing which limits their detection. Here, we report a new method for cancer diagnostics based on their unique tissue-lipidomic signatures through rapid molecular screening of FFPE sections without dewaxing using Water-Assisted Laser Desorption Ionization Mass Spectrometry (WALDI-MS) - SpiderMass. Advantageously, the method does not require human intervention. The annotated tissues are first analyzed to train the system and then blind samples can be diagnosed in less than a few minutes. The platform was demonstrated for the diagnostics of canine and human sarcoma, including cases which are known to be problematic in the gold standard workflow. Interrogation of blind samples against the built models revealed over 95% probability values to discriminate specific subtypes

Diagnostic 2-Gene Classifier in Early-Stage Mycosis Fungoides: A Retrospective Multicenter Study

Introduction Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma and represents more than 50% of all primary cutaneous lymphomas. It is typically an indolent disorder with limited patches and plaques. One third, however, experience progression with ulcerating tumors and possible further systemic dissemination. Currently, the diagnosis of MF is based on clinical and histological examinations, but this has proven, especially in early-stage disease, to be challenging due to similarities with several benign skin conditions such as psoriasis, pityriasis lichenoides chronica, and dermatitis. Despite intensive research, reliable diagnostic biomarkers for early-stage MF are still needed. This study aims to identify a diagnostic classifier that could support the diagnostic workup leading to an exact diagnosis in the early-stage of this complex and potentially lethal disease. Methods We analyzed 43 formalin-fixed and paraffin-embedded (FFPE) skin biopsies from 36 patients with early-stage MF. Seven patients had 2 longitudinal biopsies available for analysis. These were compared with FFPE skin biopsies from patients with unspecified dermatitis (n=29) and healthy skin (n=12). All samples were collected from the archives of the Department of Pathology, Region Zealand, Denmark in the period 1990-2016. The histological samples were revised and clinical records were reviewed to establish the diagnosis and stage for each patient. Total RNA was extracted from ten 10-μm sections of FFPE tissue, and 50-100 ng of RNA was analyzed on the NanoString nCounter platform by applying the Myeloid Innate Immunity Panel, which quantifies the expression of 800 immune related genes. Differentially expressed genes (DEG, 2-fold change, p<0.05), were assessed by ANOVA, and a Support Vector Machine (SVM) diagnostic classifier was built based on 2-10 DEG and evaluated by 10-fold cross-validation. The classifier was tested on an independent, early-stage MF patient cohort (n=27). Protein expression was validated with immunohistochemistry and digitally analyzed by applying a specifically designed algorithm with the Leica Tissue IA 2.0 software. Double immunofluorescence staining protocols were developed to identify subtypes of TRAF1 positive cells in combination with various macrophage/dendritic cell markers (CD168, CD63, CD11c, CD1a, CD14, and S100) as well as T-cell (CD3) and B-cell (CD20) markers. Results A diagnostic classifier consisting of TOX and TRAF1 was able to distinguish early-stage MF from dermatitis with an overall accuracy of 85% in the discovery cohort and 80% in an independent validation cohort. TOX and TRAF1 protein levels were significantly elevated in early-stage MF compared to the dermatitis group (p < 0.0001). TOX and TRAF1 were also significantly increased in the progression from early-stage MF to tumor stage MF (p=0.003 and p=0.004, respectively). Subtypes of TRAF1-positive dendritic cells in early-stage MF consisted primarily of S100+ cells in both the epidermal and dermal compartment. A few TRAF1+ cells in the Pautrier microabscesses stained double positive with CD11c. In tumor stage MF the majority of TRAF1+ dendritic cells counterstained with CD1a and CD11c. Both neoplastic and reactive T-cells (CD3+) expressed TRAF1 in a minor degree in early-stage MF, while T-cells in tumor stage MF expressed TRAF1 in a much higher degree and the majority of the neoplastic T-cells were TRAF1 positive. No macrophages (CD68+ or CD163+) or B-cells double stained with TRAF1. Conclusion In the present study, we developed a two gene mRNA diagnostic classifier discriminating early-stage MF from dermatitis. The protein expression level of TOX and TRAF1 confirmed our gene expression levels and identified a highly significant difference between early-stage MF and dermatitis, which can prove useful in diagnostics of early-stage MF. Disclosures Andersen: Novo Nordisk: Other: Holds stock in Novo Nordisk; Hologic Deutchland GmbH: Research Funding. Odum:Micreos human Health B.V: Consultancy. Litman:Leo Pharma A/S: Research Funding. Gjerdrum:Nanostring Technologies: Other: Recieves founding from NanoString regarding a B-cell lymphoma research project., Research Funding; Celgene: Other: Celgene has funded the participation in ASH 2019.

P2.14-48 Clinical Sequencing Using a NGS-Based Multiple Gene Assay in Patients with Non-Small Cell Lung Cancer

Patients with non-small cell lung cancer (NSCLC) often harbor driver mutations in multiple oncogenes, including EGFR, ALK, ROS1, BRAF, HER2, MET, RET, etc. The presence of gene alterations can impact the selection of and the response to targeted therapies. Testing of lung cancer for multi-gene alterations is important for identification of potentially efficacious targeted therapies. Therefore, identifying mutations in oncogenes and tailoring therapy become a standard in clinical cancer management. A genetic testing assay based on next-generation sequencing (NGS), named AmoyDx Essential NGS Panel, has been developed for multiplexed and targeted deep sequencing of variants in 10 driver genes. Clinical validation using FFPE tissue was conducted in the present study. A total of 294 formalin-fixed and paraffin-embedded (FFPE) tissue samples collected from NSCLC patients were tested by AmoyDx Essential NGS Panel, which enables the simultaneous detection of single-nucleotide variants (SNVs), insertions/deletions and fusions in 10 driver genes (EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, ROS1, HER2, RET, and MET) in DNA samples. DNA sequencing was performed on the Illumina sequencer. Golden standard Sanger sequencing was used as a reference method to test the same cohort. The concordance of alterations determined with the AmoyDx Essential NGS Panel was assessed compared to the reference method. In total of 294 samples, 98.30% (289/294) of patients were successfully detected by both AmoyDx Essential NGS Panel and Sanger sequencing. 68.86% (199/289) were identified with positive mutation/fusion by NGS assay (Table 1). The overall concordance rate of alterations determined with AmoyDx Essential NGS Panel compared with reference was 90.66%.Table 1Alterations detected by NGSVariant typeNumber of patientsPositive rateEGFR mutation12643.60%KRAS mutation4013.84%NRAS mutation10.35%BRAF mutation10.35%PIK3CA mutation31.04%ALK fusion227.61%ROS1 fusion20.69%HER2 mutation62.08%RET fusion10.35%MET mutation00.00%Total19968.86% Open table in a new tab The NGS analysis with AmoyDx Essential NGS Panel represents an accurate and efficient approach to detect genetic alterations in 10 driver genes with high concordance rate of 90.66% compared with Sanger sequencing.

Histopathological study on the prevalence of trichosporonosis in formalin-fixed and paraffin-embedded tissue autopsy sections by in situ hybridization with peptide nucleic acid probe.

Trichosporon species are some of the most common pathogenic yeasts in Asia, and many are resistant to echinocandin antifungal drugs. Effective treatment of fungal infections requires the selection of appropriate antifungals and the accurate identification of the causal organism. However, in histopathological specimens Trichosporon spp. are often misidentified as Candida species due to morphological similarities. In situ hybridization (ISH) is a useful technique for identifying fungal species in formalin-fixed and paraffin-embedded (FFPE) tissue sections. Although many novel probes for ISH are available, the practical use of ISH for identification of fungi remains limited, in part due to the lack of adequate verifications. We conducted a two-center retrospective observational study in which the ISH technique was used to differentiate Trichosporon spp. and C. albicans in FFPE tissue from autopsy specimens. The study included 88 cases with blood stream yeast infection without Cryptococci extracted from 459 autopsy files of cases with proven invasive fungal infection (IFI). Positive signals for the Trichosporon spp. protein nucleic acid (PNA) probe and C. albicans PNA probe were seen for 7 and 35 cases, respectively, whereas the remaining 46 were negative for both. For the Trichosporon spp.- positive specimens, 5/7 were reported as candidiasis in autopsy records. Our results suggested that accurate histological identification of fungal infections remains challenging, but ISH may be a suitable approach to support histological findings. In addition, this retrospective study suggested that trichosporonosis may have high prevalence among cases of bloodstream yeast infections in Japan.

MiR-221-5p acts as an oncogene and predicts worse survival in patients of renal cell cancer

BackgroundRenal cell carcinoma(RCC) is one of the most common malignancies in kidney, and usually leads to poor prognosis. Therefore, identifying novel biomarkers for predicting the progression and prognosis of RCC is essential. The purpose of this study is aimed to evaluate the function of miR-221-5p in RCC and the clinical value of miR-221-5p in RCC prognosis after surgery. Materials and methodsIn our study, RT-qPCR, wound scratch assay, cell proliferation assay, transwell assay, and flow cytometry assay were performed to explore miR-221-5p expression level and its proliferation, migration and apoptosis in clear cell RCC(ccRCC). Besides, we collected 196 formalin-fixed and paraffin-embedded (FFPE) tissue samples of patients who received partial or radical nephrectomy from May 2006 to October 2016 at Shenzhen Traditional Chinese Medicine Hospital and People's Liberation Army 303 Hospital. The relative levels of miR-221-5p from the FFPE tissue samples was detected by RT-qPCR. The Kaplan-Meier method, Cox regression analyses, and ROC curve analysis were performed to approve the effect of the miR-221-5p expression on patient survival. ResultsIn our study, we found that miR-221-5p is significantly upregulated in ccRCC tissues and ccRCC cell lines. Moreover, miR-221-5p promotes cell proliferation, mobility, and inhibits cell apoptosis in 786-O and ACHN cell lines. The Kaplan-Meier analysis indicated that patients with high expression of miR-221-5p had a significantly poor prognosis (P = 0.013). The Cox regression analyses showed that patients with high expression of miR-221-5p remained to have a shorter overall survival (P = 0.025). The ROC curve of miR-221-5p expression combined with tumor stage showed an area under the curve of 0.658 (P < 0.001). ConclusionOur results indicated that miR-221-5p might not only be an oncogene in ccRCC cells but also might be an independent prognosis factor of ccRCC.

Histopathological factors affecting the extraction of high quality genomic DNA from tissue sections for next-generation sequencing.

To enable the widespread application of genomic medicine, the extraction of genomic DNA from thin sections of archived formalin-fixed and paraffin-embedded (FFPE) tissue blocks for next-generation sequencing (NGS) is often necessary. However, there are currently no guidelines available on which specific regions of the microtome sections to use for macrodissection with respect to the histopathological factors observed under microscopic examination. The aim of this study was to clarify the relationship between histopathological factors and DNA quality, and to standardize the macrodissection method for more efficient implementation of NGS. FFPE tissue specimens of 218 patients from the Biomarker Research for Anti-EGFR Monoclonal Antibodies by Comprehensive Cancer Genomics study were used to investigate the relationship between 15 histopathological factors and the quantitative ratio of double-stranded DNA (dsDNA) to total nucleic acids, as well as the ∆ crossing point value of each tissue specimen. Multivariate logistic regression analysis revealed that specimen storage of ≥3 years was negatively associated with dsDNA quality (P=0.0007, OR: 4.30, 95% CI: 1.85-10.04). In contrast, the presence of a mucus pool was positively associated with dsDNA quality (P=0.0308, OR: 0.23, 95% CI: 0.06-0.87). Metastatic tumors and longer specimen storage periods were significantly associated with lower ∆Cp values (P=0.0007, OR: 4.43, 95% CI: 1.87-10.49; and P=0.0003, OR: 5.51, 95% CI: 2.18-13.95, respectively). Therefore, macrodissection should not be performed on specimens exhibiting histopathological factors associated with poor DNA quality. In particular, the use of tissue blocks with a storage period of <3 years allows the extraction of genomic DNA suitable for NGS.

Whole genome and transcriptome sequencing of post-mortem cardiac tissues from sudden cardiac death victims identifies a gene regulatory variant in NEXN.

Sudden cardiac death (SCD) is a major public health problem and constitutes a diagnostic and preventive challenge in forensic pathology, especially for cases with structural normal hearts at autopsy, so-called sudden arrhythmic death syndrome (SADS). The identification of new genetic risk factors that predispose to SADS is important, because they may contribute to establish the diagnosis and increase the understanding of disease pathways underlying SADS. Pathogenic mutations in the protein coding regions of cardiac genes were found in relation to SADS. However, much remains unknown about variants in non-coding regions of the genome. In this study, we explored the potential of whole genome sequencing (WGS) and whole transcriptome sequencing (WTS) to find DNA variants in SCD victims with structural normal hearts. With focus on the non-coding regulatory regions, we re-examined a cohort of 13 SADS and sudden unexplained death in infancy (SUDI) victims without disease causing DNA variants in recognized cardiac genes. The genetic re-examination of DNA was carried out using frozen tissue samples and WTS was carried out using five distinct formalin fixed and paraffin embedded (FFPE) cardiac tissue samples from each individual, including anterior and posterior walls of the left ventricle, ventricular papillary muscle, septum, and the right ventricle. We identified 23 candidate variants in regulatory sequences of cardiac genes, including a variant in the promotor region of NEXN, c.-194A>G, that was found to be statistically significantly (p < 0.05) associated with decreased expression of NEXN and cardiac hypertrophy. With the use of post-mortem FFPE tissues, we highlight the potential of using WTS investigations and compare gene expression levels with DNA variation in regulatory non-coding regions of the genome for a better understanding of the genetics of cardiac diseases leading to SCD.

Hypermethylation of APC2 Is a Predictive Epigenetic Biomarker for Chinese Colorectal Cancer.

Objective To investigate methylation of the adenomatosis polyposis coli homologue (APC2) promoter and its correlation with prognostic implications in Chinese colorectal cancer (CRC). Methods The mRNA expression of APC2 in colorectal tissues was evaluated using the database of The Cancer Genome Atlas (TCGA). Methylation analysis of APC2 in tumor (n = 66) and corresponding adjacent formalin-fixed and paraffin-embedded (FFPE) tissues (n = 44) was performed by Sequenom EpiTYPER® and verified by cloning-based bisulfite sequencing analysis. Demethylation and retrieval of APC2 expression in cell lines HT29, HCT116, and SW480 were treated with 5-aza-2′-deoxycytidine (5-AZC). Results Analysis of TCGA showed that APC2 mRNA was significantly downregulated in primary tumors when compared to normal tissues (p < 0.05). APC2 methylation was upregulated (43.93% vs 7.31%, p < 0.05) in tumors compared to adjacent FFPE tissues. In vitro experiments demonstrated that 5-AZC downregulated the methylation of APC2 and retrieved its expression of mRNA and protein levels (p < 0.05). Multivariate Cox regression indicated that APC2_CPG_14 was an independent risk factor for overall survival (HR = 6.38, 95% CI: 1.59–25.64, p < 0.05). Conclusion This study indicates that APC2 is hypermethylated and may be a tumorigenesis biomarker for Chinese CRC patients.

Progression-specific genes identified in microdissected formalin-fixed and paraffin-embedded tissue containing matched ductal carcinoma in situ and invasive ductal breast cancers

BackgroundThe transition from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) is an important step during breast carcinogenesis. Understanding its molecular changes may help to identify high-risk DCIS that progress to IBC. Here, we describe a transcriptomic profiling analysis of matched formalin-fixed and paraffin-embedded (FFPE) DCIS and IBC components of individual breast tumours, containing both tumour compartments. The study was performed to validate progression-associated transcripts detected in an earlier gene profiling project using fresh frozen breast cancer tissue. In addition, FFPE tissues from patients with pure DCIS (pDCIS) were analysed to identify candidate transcripts characterizing DCIS with a high or low risk of progressing to IBC.MethodsFifteen laser microdissected pairs of DCIS and IBC were profiled by Illumina DASL technology and used for expression validation by qPCR. Differential expression was independently validated using further 25 laser microdissected DCIS/IBC sample pairs. Additionally, laser microdissected epithelial cells from 31 pDCIS were investigated for expression of candidate transcripts using qPCR.ResultsMultiple statistical calculation methods revealed 1784 mRNAs which are differentially expressed between DCIS and IBC (P < 0.05), of which 124 have also been identified in the gene profiling project using fresh frozen breast cancer tissue. Nine mRNAs that had been selected from the gene list obtained using fresh frozen tissues by applying pathway and network analysis (MMP11, GREM1, PLEKHC1, SULF1, THBS2, CSPG2, COL10A1, COL11A1, KRT14) were investigated in tissues from the same 15 microdissected specimens and the 25 independent tissue samples by qPCR. All selected transcripts were also detected in tumour cells from pDCIS. Expression of MMP11 and COL10A1 increased significantly from pDCIS to DCIS of DCIS/IBC mixed tumours.ConclusionWe confirm differential expression of progression-associated transcripts in FFPE breast cancer samples which might mediate the transition from DCIS to IBC. MMP11 and COL10A1 may characterize pure DCIS with a high risk developing IDC.

Abstract 1427: Comprehensive investigation of false mutation discoveries in FFPE samples

Abstract Next generation sequencing (NGS) has emerged as a primary tool for “precision” medicine, especially in the rapidly evolving field of cancer care. However NGS-based somatic variant calling in a clinical oncology setting is susceptible to numerous sources of technical error impeding optimal patient management. One important source of error in variant reporting is formalin fixation and paraffin embedding (FFPE). Of note, FFPE-derived errors are commonly present at clinically actionable loci and can occur at allelic frequencies (5-65%) that meet treatment criteria. Compounding this problem, degree of FFPE error is highly susceptible to inter-specimen and inter-laboratory variations. In this context, static variant calling thresholds to control for “general FFPE-effect” are likely inadequate; resulting in both False-Negatives (ignoring true somatic variation) and False-Positives (reporting technical error as truth). Despite the importance of fresh tissue for molecular studies, routine tissue histology (derived from FFPE) continues to be the gold standard for cancer staging/grading. And, as emphasis on smaller sample sizes becomes the norm, routine histology will be prioritized in many scenarios. Thus, the need to understand how molecular testing performs in FFPE tissue is a driving imperative for optimal cancer care. In this study, our aim was to develop and evaluate “best” quality control practices that would minimize False-Positive or -Negative somatic variant calling in FFPE specimens. Reference samples (paired breast carcinoma and “normal” lymphoblast cell-lines) from the FDA-led Sequencing Quality Control Phase II (SEQC2) consortium were used. These specimens were subjected to varying FFPE conditions (n=8) mimicking the pathology laboratory setting. For each condition, multiple replicates of whole genome and exome sequencing (WGS/WES) were performed with ~100X coverage across the entire human genome. Observed somatic variation frequencies for each genomic position for each FFPE condition were compared to ground truth and quality control measures derived from matched fresh materials using numerous statistical models. Applying rigorous analytical strategies, we aim to identify genomic regions that serve as markers (internal control regions) for the full range of FFPE-effect, develop QC measures and statistics to define lower limit of detection for variant calling for each genomic position, and provide general guidance for the community on how to accurately report mutations in FFPE processed clinical cancer specimens. Citation Format: Thomas M. Blomquist, Wenming Xiao, Somatic Mutation Working Group of the SEQC2 Consortium. Comprehensive investigation of false mutation discoveries in FFPE samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1427.

Abstract 5592: HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method

Abstract In around 10% of gastric cancer, amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) is observed and trastuzumab is adopted as a target therapy agent for HER2-positive gastric cancer. Because of efficiency and accuracy, droplet digital PCR (ddPCR) is one of the best methods for measuring HER2 amplification, especially in cell-free plasma samples. In this study, we aimed to investigate the correlation between tissue and plasma HER2 status by using ddPCR method in gastric cancer patients. We collected formalin-fixed and paraffin-embedded tissue (FFPE) and cell-free plasma samples of 81 gastric cancer patients, including 30 HER2 immunohistochemistry (IHC)-negative (0 or 1+) cases and 51 HER2 IHC-positive cases (IHC 2+, n=20; IHC 3+, n=31), with informed consents. We performed ddPCR of a target gene HER2 and a reference gene EIF2C1 in both tissue and cell-free plasma samples, and gene copy number (GCN) of HER2 was calculated. The median HER2 GCN of tissue DNA ddPCR was 2.61 (1.47-49.00) and the median GCN of plasma ddPCR was 1.83 (0.97-7.67). HER2 IHC results showed moderate correlation with HER2 GCN of tissue ddPCR (r=0.455, p&amp;lt;0.001) and weak correlation with plasma ddPCR (r=0.321, p=0.003). A ratio &amp;gt;2.405, which showed the best discriminating level from a receiver operating characteristic curve, was defined as a cut-off for tissue HER2 amplification using ddPCR method. Comparing the HER2 amplification results of tissue DNA ddPCR to HER2 IHC results, the concordance rate was 86.4% with Cohen's kappa of 0.715. Sensitivity and specificity of tissue ddPCR were 86.3% and 86.7%, respectively. HER2 amplification by tissue ddPCR was significantly associated with advanced gastric cancer and lymphatic invasion (p&amp;lt;0.05). HER2 GCN of tissue ddPCR was weakly correlated with plasma ddPCR (r=0.250, p=0.025). Sensitivity and specificity of plasma ddPCR for HER2 IHC positivity were 35.3% and 93.3%, respectively. However, sensitivity and specificity of plasma ddPCR were 66.7% and 60.0% with another cut-off of 1.715. In 51 HER2 IHC-positive cases, intratumoral heterogeneity was observed in 100% (5/5) of both tissue and plasma ddPCR-negative cases, 100% (2/2) of tissue-negative and plasma positive cases, 64.3% (18/28) of tissue-positive and plasma-negative cases, and 50.0% (8/16) of both positive cases (p=0.026). In conclusion, HER2 GCN of plasma ddPCR showed a weak correlation with tissue ddPCR, and the sensitivity of plasma ddPCR was not high. Intratumoral heterogeneity is suggested to be one of the possible causes, and these findings may contribute to developing plasma HER2 testing useful in daily practice. Citation Format: Boram Kim, Soo Kyung Nam, Soo Hyun Seo, Sang-Hoon Ahn, Do Joong Park, Hyung-Ho Kim, Kyoung Un Park, Hye Seoung Lee. HER-2 copy number measurement in tissue and cell-free plasma DNA of gastric cancer patients using droplet digital PCR method [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5592.

Profiling of lymphoma from formalin-fixed paraffin-embedded tissue

Molecular profiling of lymphoma samples has contributed enormously to our understanding of disease biology leading to detailed descriptions of diagnostic categories. These studies have also helped the field to recognize different subtypes of disease, different diseases that share similar cellular pathway perturbations, different immune responses, and different prognostic groups. While nearly all of these discoveries were made using unfixed, snap-frozen materials, with few exceptions, clinical biopsy materials are comprised of formalin-fixed and paraffin-embedded (FFPE) tissues. Here, we describe the impact of molecular profiling on the field of lymphoma, the challenges associated with using FFPE tissues for downstream molecular diagnostic testing, the various molecular profiling techniques, and also provide an example of the clinical application of a molecular profiling test of lymphoma using FFPE tissues.

Reproducible proteomics sample preparation for single FFPE tissue slices using acid-labile surfactant and direct trypsinization

BackgroundProteomic analyses of clinical specimens often rely on human tissues preserved through formalin-fixation and paraffin embedding (FFPE). Minimal sample consumption is the key to preserve the integrity of pathological archives but also to deal with minimal invasive core biopsies. This has been achieved by using the acid-labile surfactant RapiGest in combination with a direct trypsinization (DTR) strategy. A critical comparison of the DTR protocol with the most commonly used filter aided sample preparation (FASP) protocol is lacking. Furthermore, it is unknown how common histological stainings influence the outcome of the DTR protocol.MethodsFour single consecutive murine kidney tissue specimens were prepared with the DTR approach or with the FASP protocol using both 10 and 30 k filter devices and analyzed by label-free, quantitative liquid chromatography–tandem mass spectrometry (LC–MS/MS). We compared the different protocols in terms of proteome coverage, relative label-free quantitation, missed cleavages, physicochemical properties and gene ontology term annotations of the proteins. Additionally, we probed compatibility of the DTR protocol for the analysis of common used histological stainings, namely hematoxylin & eosin (H&E), hematoxylin and hemalaun. These were proteomically compared to an unstained control by analyzing four human tonsil FFPE tissue specimens per condition.ResultsOn average, the DTR protocol identified 1841 ± 22 proteins in a single, non-fractionated LC–MS/MS analysis, whereas these numbers were 1857 ± 120 and 1970 ± 28 proteins for the FASP 10 and 30 k protocol. The DTR protocol showed 15% more missed cleavages, which did not adversely affect quantitation and intersample comparability. Hematoxylin or hemalaun staining did not adversely impact the performance of the DTR protocol. A minor perturbation was observed for H&E staining, decreasing overall protein identification by 13%.ConclusionsIn essence, the DTR protocol can keep up with the FASP protocol in terms of qualitative and quantitative reproducibility and performed almost as well in terms of proteome coverage and missed cleavages. We highlight the suitability of the DTR protocol as a viable and straightforward alternative to the FASP protocol for proteomics-based clinical research.

USP8 Mutations in Pituitary Cushing Adenomas-Targeted Analysis by Next-Generation Sequencing.

Gain-of-function somatic mutations in the ubiquitin specific protease 8 (USP8) gene have recently been reported as a cause of pituitary adenomas in Cushing disease. Molecular diagnostic testing of tumor tissue may aid in the diagnosis of specimens obtained through therapeutic transsphenoidal surgery; however, for small tumors, availability of fresh tissue is limited, and contamination with normal tissue is frequent. We performed molecular testing of DNA isolated from single formalin-fixed and paraffin-embedded (FFPE) tissue sections of 42 pituitary adenomas from patients with Cushing disease (27 female patients and 15 male patients; mean age at surgery, 42.5 years; mean tumor size, 12.2 mm). By Sanger sequencing, we identified previously reported USP8 missense mutations in six tumors. Targeted next-generation sequencing (NGS) revealed known or previously undescribed missense mutations in three additional tumors (two with two different mutations each), with mutant allele frequencies as low as 3%. Of the nine tumors with USP8 mutations (mutation frequency, 21.4%), seven were from female patients (mutation frequency, 25.9%), and two were from male patients (mutation frequency, 13.3%). Mutant tumors were on average 11.4 mm in size, and patients with mutations were on average 43.9 years of age. The overall USP8 mutation frequency in our cohort was lower than in previously described cohorts, and we did not observe USP8 deletions that were frequent in other cohorts. We demonstrate that testing for USP8 variants can be performed from small amounts of FFPE tissue. NGS showed higher sensitivity for USP8 mutation detection than did Sanger sequencing. Assessment for USP8 mutations may complement histopathological diagnosis.

Standardization and validation of real time PCR assays for the diagnosis of histoplasmosis using three molecular targets in an animal model.

Histoplasmosis is considered one of the most important endemic and systemic mycoses worldwide. Until now few molecular techniques have been developed for its diagnosis. The aim of this study was to develop and evaluate three real time PCR (qPCR) protocols for different protein-coding genes (100-kDa, H and M antigens) using an animal model. Fresh and formalin-fixed and paraffin-embedded (FFPE) lung tissues from BALB/c mice inoculated i.n. with 2.5x106 Histoplasma capsulatum yeast or PBS were obtained at 1, 2, 3, 4, 8, 12 and 16 weeks post-infection. A collection of DNA from cultures representing different clades of H. capsulatum (30 strains) and other medically relevant pathogens (36 strains of related fungi and Mycobacterium tuberculosis) were used to analyze sensitivity and specificity. Analytical sensitivity and specificity were 100% when DNAs from the different strains were tested. The highest fungal burden occurred at first week post-infection and complete fungal clearance was observed after the third week; similar results were obtained when the presence of H. capsulatum yeast cells was demonstrated in histopathological analysis. In the first week post-infection, all fresh and FFPE lung tissues from H. capsulatum-infected animals were positive for the qPCR protocols tested except for the M antigen protocol, which gave variable results when fresh lung tissue samples were analyzed. In the second week, all qPCR protocols showed variable results for both fresh and FFPE tissues. Samples from the infected mice at the remaining times post-infection and uninfected mice (controls) were negative for all protocols. Good agreement was observed between CFUs, histopathological analysis and qPCR results for the 100-kDa and H antigen protocols. We successfully standardized and validated three qPCR assays for detecting H. capsulatum DNA in fresh and FFPE tissues, and conclude that the 100-kDa and H antigen molecular assays are promising tests for diagnosing this mycosis.

P3.02-055 Detecting ALK, ROS1 and RET Gene Translocations in Non-Small Cell Lung Cancer (NSCLC) with the NanoString Platform

Identification of ALK, ROS1 and RET fusions is critical for guiding target therapy of lung cancers. In this study, we evaluated the transcript-based nanoString assay for detection of ALK fusions compared to immunohistochemistry (IHC) and FISH methods, and the simultaneous detection of ROS1 and RET transcripts in formalin-fixed and paraffin embedded tissues (FFPE).

Multiplexed analysis of fixed tissue RNA using Ligation in situ Hybridization.

Clinical tissues are prepared for histological analysis and long-term storage via formalin fixation and paraffin embedding (FFPE). The FFPE process results in fragmentation and chemical modification of RNA, rendering it less suitable for analysis by techniques that rely on reverse transcription (RT) such as RT-qPCR and RNA-Seq. Here we describe a broadly applicable technique called ‘Ligation in situ Hybridization’ (‘LISH’), which is an alternative methodology for the analysis of FFPE RNA. LISH utilizes the T4 RNA Ligase 2 to efficiently join adjacent chimeric RNA–DNA probe pairs hybridized in situ on fixed RNA target sequences. Subsequent treatment with RNase H releases RNA-templated ligation products into solution for downstream analysis. We demonstrate several unique advantages of LISH-based assays using patient-derived FFPE tissue. These include >100-plex capability, compatibility with common histochemical stains and suitability for analysis of decade-old materials and exceedingly small microdissected tissue fragments. High-throughput DNA sequencing modalities, including single molecule sequencing, can be used to analyze ligation products from complex panels of LISH probes (‘LISH-seq’), which can be amplified efficiently and with negligible bias. LISH analysis of FFPE RNA is a novel methodology with broad applications that range from multiplexed gene expression analysis to the sensitive detection of infectious organisms.

Immunohistochemistry on old archival paraffin blocks: is there an expiry date?

Few studies have focused on antigen preservation in formalin-fixed and paraffin-embedded (FFPE) tissue in old archival material and additional studies are required, especially considering that these samples are an irreplaceable...