Formalin-fixed And Paraffin-embedded Research Articles

Epigenetic imprinting alterations as effective diagnostic biomarkers for early-stage lung cancer and small pulmonary nodules

BackgroundEarly lung cancer detection remains a clinical challenge for standard diagnostic biopsies due to insufficient tumor morphological evidence. As epigenetic alterations precede morphological changes, expression alterations of certain imprinted genes could serve as actionable diagnostic biomarkers for malignant lung lesions.ResultsUsing the previously established quantitative chromogenic imprinted gene in situ hybridization (QCIGISH) method, elevated aberrant allelic expression of imprinted genes GNAS, GRB10, SNRPN and HM13 was observed in lung cancers over benign lesions and normal controls, which were pathologically confirmed among histologically stained normal, paracancerous and malignant tissue sections. Based on the differential imprinting signatures, a diagnostic grading model was built on 246 formalin-fixed and paraffin-embedded (FFPE) surgically resected lung tissue specimens, tested against 30 lung cytology and small biopsy specimens, and blindly validated in an independent cohort of 155 patients. The QCIGISH diagnostic model demonstrated 99.1% sensitivity (95% CI 97.5–100.0%) and 92.1% specificity (95% CI 83.5–100.0%) in the blinded validation set. Of particular importance, QCIGISH achieved 97.1% sensitivity (95% CI 91.6–100.0%) for carcinoma in situ to stage IB cancers with 100% sensitivity and 91.7% specificity (95% CI 76.0–100.0%) noted for pulmonary nodules with diameters ≤ 2 cm.ConclusionsOur findings demonstrated the diagnostic value of epigenetic imprinting alterations as highly accurate translational biomarkers for a more definitive diagnosis of suspicious lung lesions.

DNA methylation-based classification of malformations of cortical development in the human brain

Malformations of cortical development (MCD) comprise a broad spectrum of structural brain lesions frequently associated with epilepsy. Disease definition and diagnosis remain challenging and are often prone to arbitrary judgment. Molecular classification of histopathological entities may help rationalize the diagnostic process. We present a retrospective, multi-center analysis of genome-wide DNA methylation from human brain specimens obtained from epilepsy surgery using EPIC 850 K BeadChip arrays. A total of 308 samples were included in the study. In the reference cohort, 239 formalin-fixed and paraffin-embedded (FFPE) tissue samples were histopathologically classified as MCD, including 12 major subtype pathologies. They were compared to 15 FFPE samples from surgical non-MCD cortices and 11 FFPE samples from post-mortem non-epilepsy controls. We applied three different statistical approaches to decipher the DNA methylation pattern of histopathological MCD entities, i.e., pairwise comparison, machine learning, and deep learning algorithms. Our deep learning model, which represented a shallow neuronal network, achieved the highest level of accuracy. A test cohort of 43 independent surgical samples from different epilepsy centers was used to test the precision of our DNA methylation-based MCD classifier. All samples from the test cohort were accurately assigned to their disease classes by the algorithm. These data demonstrate DNA methylation-based MCD classification suitability across major histopathological entities amenable to epilepsy surgery and age groups and will help establish an integrated diagnostic classification scheme for epilepsy-associated MCD.

The effect of sample age on the metabolic information extracted from formalin-fixed and paraffin embedded tissue samples using desorption electrospray ionization mass spectrometry imaging

Background: Metabolites, especially lipids, have been shown to be promising therapeutic targets. In conjugation with genes and proteins they can be used to identify phenotypes of disease and support the development of targeted treatments. The majority of clinically collected tissue samples are stored in formalin-fixed and paraffin embedded (FFPE) blocks due to their tissue conservation ability and indefinite storage capacity. For metabolic analysis, however, fresh frozen (FF) samples are currently preferred over FFPE samples due to concerns of metabolic information being lost when preparing the samples. With little or no sample preparation, desorption electrospray ionisation mass spectrometry imaging (DESI-MSI) allows for the study of spatial as well as spectral information.Methods: DESI-MSI analysis was performed on FFPE breast cancer tissue microarray samples from 213 patients collected between the years 1935-2013. Logistic regression (LR) models were built to classify samples based on age and FF samples were used for feature validation.Results: LR models developed on the FFPE samples achieved an average classification accuracy of 96% when predicting their age with a 10-year grouping. Closer examination of the metabolic change over time revealed that the mean signal intensities for the lower mass range (100 - 500 m/z) linearly decrease over time, while the mean intensities for the higher mass range (500 - 900 m/z), remained relatively constant.Conclusions: In our samples, which span over 70 years, sample age has a weak yet quantifiable impact on metabolite content in FFPE samples, while the higher mass range is seemingly unaffected. FFPE samples thus provide an alternative avenue for metabolic analysis of lipids.

Expression analysis of p16 and TOP2A protein biomarkers in cervical cancer lesions and their correlation with clinico-histopathological characteristics in a referral hospital, Tanzania.

Biomarkers yield important information for early diagnosis of cervical cancer. However, they are rarely applied for prognosis of cervical cancer in Tanzania, where visual inspection assay with acetic acid or Lugol's iodine and Pap test are being used as the standard screening/ diagnostic methods. This was a retrospective hospital-based cross-sectional study that was conducted to assess cyclin-dependent kinase inhibitor (p16) and topoisomerase II-alpha (TOP2A) proteins expression among women seeking cervical cancer care at Kilimanjaro Christian Medical Centre, Tanzania between May 1, 2017 and May 10, 2018. Immunohistochemistry technique was used to detect the expressions of p16 and TOP2A proteins from the retrieved formalin-fixed and paraffin-embedded (FFPE) cervical biopsies. A total of 145 patients, with a mean age of 52.1 ± 12.9 years, were included in this study. Upon immunohistochemistry staining, 103 (71.0%) and 90 (62.1%) were p16 and TOP2A positive respectively. There was a strong association between histopathological class and p16/TOP2A expression levels (Fisher's exact test, p<0.001). Moreover, there was a strong positive correlation between p16/TOP2A and cancerous cervical lesions (Spearman's rank correlation coefficients = 0.833 and 0.687, p = 0.006 and 0.005, respectively). The age-adjusted odds ratio for predicting cervical cancer lesions were independently significant for p16/TOP2A biomarkers in FFPE cervical tissues [p16: OR = 1.142 (95% CI: 1.059-1.232, p<0.001) and TOP2A: OR = 1.046 (95% CI: 1.008-1.085, p = 0.015)]. Importantly, the diagnostic performance of p16 was higher than that of TOP2A in the diagnosis of cancerous lesions from non-cancerous cervical lesions (sensitivity: 97.2% versus 77.6%, accuracy: 92.8% versus 87.8%, respectively). Our study has highlighted that over-expression of TOP2A is related to the grade of cervical intraepithelial neoplasia but does not predict prognosis in cervical cancer. Similarly, expression of p16 is related to degree of histological dysplasia and malignancy, suggesting its prognostic and predictive value in the management of cervical cancers. Further bigger studies are needed to validate their applications in the early diagnosis of cervical cancer.

P66.01 Characterize the Heterogeneity of the Immunophenotype in Different Neuroendocrine (NE) Subtypes of Small-Cell Lung Cancer (SCLC)

SCLC is a typical neuroendocrine carcinoma, however, a variant subtype of SCLC with low expression of NE markers and a variant morphology has been reported mainly based on cell lines and xenograft models. The difference of the tumor immune microenviroment (TIME) and other clinicopathological characteristics between the two subtypes in human specimens remain to be investigated. Forty-eight resected Formalin-Fixed and Paraffin-Embedded (FFPE) SCLC tissues were collected for mRNA detection using Nanostring technology. Patients were assigned into NE-high and NE-low group according to NE gene signature score, and the expression level of immune-related genes and abundance of immunocytes estimated by CIBERSORT was compared between the two groups. Nanostring digital spatial profiling (DSP) was performed in 6 NE score paired tumors to dissect the heterogeneity of spatial distribution of immune cells and immunotherapeutic drug targets between the two subtypes. Kaplan-meier method and COX regression model was used to compare the survival of the two subtypes and identify the independent prognostic factors and potential immunotherapeutic targets respectively. Forty-eight patients were assigned into the NE-high and NE-low group respectively based on NE score. NE-low group had earlier clinical stages (P=0.030), higher positive rates of lymph nodes (P=0.017) and higher frequency of variant morphology (P=0.047) than that of NE-high group. Compared with NE-high tumors, most of the detected immune-related genes were up-regulated in NE-low tumors. CIBERSORT results indicated NE-high subtypes had a higher relative proportion of Treg cells and M2 macrophages. However, higher absolute abundance of activated mast cells, CD8+ T cells and M1 macrophages enriched in NE-low TME. DSP results showed NE-high tumors were lack of dendritic cells and macrophages in peritumor area and normal tissue compartments and also lack of effector T cells in tumor center and surrounding area. However, a set of targetable negative immune checkpoints were up-regulated in NE-low TME. The prognosis was better for NE-low patients with 5-year DFS% 75% vs.38.6% (hazard ratio [HR] 0.35, 95% confidence interval [CI] 0.08-1.46; P=0.130) and 5-year OS% 87.5% vs.53.9% (HR 0.19, 95% CI 0.03-1.43; P=0.073). SCLC patients exhibited heterogeneous NE gene expression profile, implicating two entities with distinct TIME and prognosis. Compared with NE-high subtypes, patients with NE-low tumors had higher expression of immune-related genes, showing T cell-inflamed immunophenotypes, which might be one of causes for longer survival and suggested a higher propensity to benefit from immunotherapy.

Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study.

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available.Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells.Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550–8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease.Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

PD42-03 HIGH LEVELS OF BASELINE IGG1, IGG3, AND IFN-G MAY BE ASSOCIATED WITH RESISTANCE TO BCG THERAPY FOR TREATMENT OF NMIBC

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (PD42)1 Sep 2021PD42-03 HIGH LEVELS OF BASELINE IGG1, IGG3, AND IFN-G MAY BE ASSOCIATED WITH RESISTANCE TO BCG THERAPY FOR TREATMENT OF NMIBC Jorge Daza, Yuanshuo Wang, Andrew Charap, Berengere Salome, Rachel Brody, Emilie Grasset, Giuliana Magri, Dominic LaRoche, Yong Lee, Rafael Cabal, Adam Farkas, Li Wang, Jun Zhu, Kristin Beaumont, Ketan Badani, Reza Mehrazin, Peter Wiklund, Robert Sebra, Matthew Galsky, Nina Bharwadj, Amir Horowitz, and John Sfakianos Jorge DazaJorge Daza More articles by this author , Yuanshuo WangYuanshuo Wang More articles by this author , Andrew CharapAndrew Charap More articles by this author , Berengere SalomeBerengere Salome More articles by this author , Rachel BrodyRachel Brody More articles by this author , Emilie GrassetEmilie Grasset More articles by this author , Giuliana MagriGiuliana Magri More articles by this author , Dominic LaRocheDominic LaRoche More articles by this author , Yong LeeYong Lee More articles by this author , Rafael CabalRafael Cabal More articles by this author , Adam FarkasAdam Farkas More articles by this author , Li WangLi Wang More articles by this author , Jun ZhuJun Zhu More articles by this author , Kristin BeaumontKristin Beaumont More articles by this author , Ketan BadaniKetan Badani More articles by this author , Reza MehrazinReza Mehrazin More articles by this author , Peter WiklundPeter Wiklund More articles by this author , Robert SebraRobert Sebra More articles by this author , Matthew GalskyMatthew Galsky More articles by this author , Nina BharwadjNina Bharwadj More articles by this author , Amir HorowitzAmir Horowitz More articles by this author , and John SfakianosJohn Sfakianos More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002056.03AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: The mechanisms that contribute to protection from tumor recurrence after M.Bovis BCG are not fully elucidated. We aim to evaluate the role of Antibody Dependent Cell Cytotoxicity (ADCC) in BCG therapy for NMIBC. METHODS: Formalin-fixed and paraffin-embedded (FFPE) tissue sections before and after BCG therapy were used for bulk targeted gene expression analysis. Differential gene expression analysis was performed on targeted RNAseq data. Gene Set Enrichment Analysis (GSEA) was performed using the “Hallmark Gene Sets” from the Broad Intitute’s Molecular Signatures Database (MSigDB) and customized assembled gene sets. Enzyme linked immunosorbent assay (ELISA) and OLink Proteomics® inflammation panel were used to profile IgG1/3, and Interferon Gamma (IFN-g), respectively on prospectively collected plasma from BCG-treated patients (N=14) at specific timepoints throughout the treatment. RESULTS: B cell markers and IFN-g signatures were the two most enriched pathways after BCG therapy in the GSEA (Fig. 1A). We also observed significantly increased gene expression of IgG1 and IgG3 after BCG therapy (Fig. 1B), which may induce ADCC reactivity via FcgRIIIA signaling. In non-recurrent cases, we observed an increase in plasma IgG1/3 concentration that preceded IFN-g hyperactivity after 1st and 3rd BCG doses (Fig. 2). In contrast, patients that recur after BCG therapy had higher levels of plasma IFN-g at baseline, which remained elevated throughout the induction phase unrelated to IgG1/3 concentrations (Fig. 2). While the median recurrence time was 3.9 months, the median follow-up for non-recurrent patients was 36.7 months. CONCLUSIONS: We observed increased expression of B cell-related genes and an IFN-g signature after BCG therapy. Moreover, we found that response to BCG may include activation of Immune cells through IgG1/IgG3-mediated ADCC reactivity. Future studies in larger patient cohorts should measure IFN-g during BCG therapy and consider a role for ADCC in mediating protection from tumor recurrence BCG therapy. Source of Funding: Self-funded © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e725-e725 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jorge Daza More articles by this author Yuanshuo Wang More articles by this author Andrew Charap More articles by this author Berengere Salome More articles by this author Rachel Brody More articles by this author Emilie Grasset More articles by this author Giuliana Magri More articles by this author Dominic LaRoche More articles by this author Yong Lee More articles by this author Rafael Cabal More articles by this author Adam Farkas More articles by this author Li Wang More articles by this author Jun Zhu More articles by this author Kristin Beaumont More articles by this author Ketan Badani More articles by this author Reza Mehrazin More articles by this author Peter Wiklund More articles by this author Robert Sebra More articles by this author Matthew Galsky More articles by this author Nina Bharwadj More articles by this author Amir Horowitz More articles by this author John Sfakianos More articles by this author Expand All Advertisement Loading ...

Reproducible Lipid Alterations in Patient-Derived Breast Cancer Xenograft FFPE Tissue Identified with MALDI MSI for Pre-Clinical and Clinical Application.

The association between lipid metabolism and long-term outcomes is relevant for tumor diagnosis and therapy. Archival material such as formalin-fixed and paraffin embedded (FFPE) tissues is a highly valuable resource for this aim as it is linked to long-term clinical follow-up. Therefore, there is a need to develop robust methodologies able to detect lipids in FFPE material and correlate them with clinical outcomes. In this work, lipidic alterations were investigated in patient-derived xenograft of breast cancer by using a matrix-assisted laser desorption ionization mass spectrometry (MALDI MSI) based workflow that included antigen retrieval as a sample preparation step. We evaluated technical reproducibility, spatial metabolic differentiation within tissue compartments, and treatment response induced by a glutaminase inhibitor (CB-839). This protocol shows a good inter-day robustness (CV = 26 ± 12%). Several lipids could reliably distinguish necrotic and tumor regions across the technical replicates. Moreover, this protocol identified distinct alterations in the tissue lipidome of xenograft treated with glutaminase inhibitors. In conclusion, lipidic alterations in FFPE tissue of breast cancer xenograft observed in this study are a step-forward to a robust and reproducible MALDI-MSI based workflow for pre-clinical and clinical applications.

Immunohistochemical features of gastrointestinal stromal tumors and the role of expression of p16ink4A, Ki-67, VEGF and MMP-9 in their behavior

Aim: to clarify the prognostic value of cytoplasmic p16ink4A, VEGF, MMP-9 and Ki-67 expressions in gastrointestinal stromal tumors (GISTs) and connection of different levels of these markers expression with aggressive transformation of GISTs. Materials and methods. Our study included 36 samples of primary tumors and 10 relapses of GIST and metastases in liver after primary combined treatment (surgery and chemotherapy with imatinib). The immunohistochemical study was performed with 4 primary antibodies: Ki-67, p16ink4A, VEGF and MMP-9. We used formalin fixed and paraffin embedded (FFPE) tissue samples for immunohistochemical study. Results. In our study we showed significant connection between levels of cytoplasmic expression of p16ink4A in primary GISTs and such markers of tumor aggressive behaviour as Ki-67, MMP-9 and VEGF (Fisher’s exact P-value = 0.000753; 0.000101 and 0.000048 respectively). Between cytoplasmic expression of p16ink4A and VEGF and also between p16ink4A and MMP-9 strong direct correlation was found (γ = 0.829, P &lt; 0.05 and rs = 0.961, P &lt; 0.05 respectively). The correlation between expression of Ki-67 and p16ink4A was also direct and strong (rs = 0.754, P &lt; 0.05), but with some exclusions, that’s why this correlation needs further investigation in larger groups with preciser molecular analysis. Analysis of metastatic GISTs samples showed prominent levels of MMP-9 and VEGF expression. Conclusions. Our study has shown very important role of cytoplasmic expression of p16ink4A in GIST as one of the markers of aggressive behavior, which can be used in complex with other markers for more accurate prognosis of GISTs progression. Prominent levels of MMP-9 and VEGF expression in metastatic GISTs can be a marker of resistance to imatinib. So probably evaluation of MMP-9 and VEGF expression can be used as a tool for correct choice of chemotherapy for patients with GISTs.

Targeted transcriptomic analyses of RNA isolated from formalin-fixed and paraffin-embedded human menisci.

Formalin-fixed and paraffin-embedded (FFPE) biospecimens are a valuable and widely-available resource for diagnostic and research applications. With biobanks of tissue samples available in many institutions, FFPE tissues could prove to be a valuable resource for translational orthopaedic research. The purpose of this study was to characterize the molecular profiles and degree of histologic degeneration on archival fragments of FFPE human menisci obtained during arthroscopic partial meniscectomy. We used FFPE menisci for multiplexed gene expression analysis using the NanoString nCounter® platform, and for histological assessment using a quantitative scoring system. In total, 17 archival specimens were utilized for integrated histologic and molecular analyses. The median patient age was 22 years (range: 14-62). We found that the genes with the highest normalized counts were those typically expressed in meniscal fibrocartilage. Gene expression differences were identified in patient cohorts based on age (≤40 years), including genes associated with the extracellular matrix and tissue repair. The majority of samples showed mild to moderate histologic degeneration. Based on these data, we conclude that FFPE human menisci can be effectively utilized for molecular evaluation following a storage time as long as 11 years.Statement of Clinical Significance: The integration of histological and transcriptomic analyses described in this study will be useful for future studies investigating the basis for biological classification of meniscus specimens in patients. Further exploration into the genes and pathways uncovered by this study may suggest targets for biomarker discovery and identify patients at greater risk for osteoarthritis once the meniscus is torn.

Drivers and suppressors of triple-negative breast cancer

To identify regulators of triple-negative breast cancer (TNBC), gene expression profiles of malignant parts of TNBC (mTNBC) and normal adjacent (nadj) parts of the same breasts have been compared. We are interested in the roles of estrogen receptor β (ERβ) and the cytochrome P450 family (CYPs) as drivers of TNBC. We examined by RNA sequencing the mTNBC and nadj parts of five women. We found more than a fivefold elevation in mTNBC of genes already known to be expressed in TNBC: BIRC5/survivin, Wnt-10A and -7B, matrix metalloproteinases (MMPs), chemokines, anterior gradient proteins, and lysophosphatidic acid receptor and the known basal characteristics of TNBC, sox10, ROPN1B, and Col9a3. There were two unexpected findings: 1) a strong induction of CYPs involved in activation of fatty acids (CYP4), and in inactivation of calcitriol (CYP24A1) and retinoic acid (CYP26A1); and 2) a marked down-regulation of FOS, FRA1, and JUN, known tethering partners of ERβ. ERβ is expressed in 20 to 30% of TNBCs and is being evaluated as a target for treating TNBC. We used ERβ+ TNBC patient-derived xenografts in mice and found that the ERβ agonist LY500703 had no effect on growth or proliferation. Expression of CYPs was confirmed by immunohistochemistry in formalin-fixed and paraffin-embedded (FFPE) TNBC. In TNBC cell lines, the CYP4Z1-catalyzed fatty acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) increased proliferation, while calcitriol decreased proliferation but only after inhibition of CYP24A1. We conclude that CYP-mediated pathways can be drivers of TNBC but that ERβ is unlikely to be a tumor suppressor because the absence of its main tethering partners renders ERβ functionless on genes involved in proliferation and inflammation.

Preoperative detection of the TERT promoter mutations in papillary thyroid carcinomas.

Telomerase reverse transcriptase promoter (TERT-p) mutations are strongly associated with tumour aggressiveness and worse prognosis in papillary thyroid carcinomas (PTCs). Since the TERT-p mutations have been reported to be subclonal, it is unclear how accurately they can be detected by preoperative fine-needle aspiration (FNA). The objective of this study was to analyse the concordance rate of the TERT-p mutations between preoperative FNA and corresponding postoperative surgical specimens. Ninety-six cases of PTC aged 55 years or older were studied. The mutational status of TERT-p was detected by droplet digital polymerase chain reaction assay. The mutational status of the TERT-p in FNA samples was highly concordant with that in postoperative formalin-fixed and paraffin-embedded (FFPE) specimens. The TERT-p mutation was significantly associated with age, tumour size, extrathyroidal extensionand the Ki-67 labelling index in multivariate analysis in both FNA and FFPE samples. The detection of the TERT-p mutations using FNA samples has a good ability to predict disease aggressiveness and, therefore, could be clinically useful in the determination of PTC management.

Effects of processing, storage time, and tissue volume in the retrieval and quality of RNA and the expression level of RNU6

Background: Molecular analyses of tumor RNA expression have become widely used both for research and clinical purposes. Tumoral tissue preservation is a critical step to ensure accuracy of molecular-based diagnostics, for which, formalin-fixed and paraffin-embedded (FFPE) tissues represent a valuable source of clinical samples. MicroRNAs are ideal biomarkers in FFPE-tissues, in whose expression evaluation RNU6 is one of the genes used as a normalizer. Our aim was to determine, in FFPE tissue samples, the effects of length of storage and corresponding volume of each studied sample, on the RNA retrieval, quality and concentration, as well as their correlation to the expression level of RNU6. Methods: Fifty tissue blocks with a mean length of tissue storage of 30 months (SD=±12.07, 95% CI= 27.4-34.3). were included. Total RNA was isolated, absorbance and concentrations were determined and correlated with length of storage and volume of tissue. RT-qPCR for RNU6 was performed and their Ct results were correlated to the same parameters. Results: There was a direct correlation between the concentration and quality of the obtained RNA, and an inverse correlation between the tissue storage time and the RNA quality. The volume of tissue studied was not correlated with the RNA quality or concentration. The RNA quality and the length of tissue storage directly correlated to the RNU6 expression level, while RNA concentration and the volume of tissue studied did not affect it. Conclusions: There is an association between longer FFPE tissue storage time with lower RNA quality and lower RNU6 expression level.

Abstract 2283: Validation &amp; application of next-generation sequencing panel YuanSu S for gene rearrangements/fusions detection

Abstract Introduction: Gene rearrangement/fusion play an important role in cancer diagnosis and treatment. Comparing with routine diagnostic methods, Sanger sequencing, fluorescence in situ hybridization (FISH) &amp; immunohistochemistry (IHC), next-generation sequencing (NGS)-based panel can provide genetic information with robust sensitivity and precision. Moreover, panel-based RNAseq can detect novel fusions and only requires a small amount of RNA. We have developed a 450-gene DNA &amp; RNA NGS panel, YuanSu S, for clinical sample detection. In this study, YuanSu S was applied to detect gene rearrangements/fusions and determine its limit of detection (LOD) and concordance with FISH/IHC, and difference between DNAseq &amp; RNAseq. Methods: A total of 184 formalin fixed and paraffin embedded (FFPE) sarcoma samples were detected by YuanSu S at OrigiMed, a laboratory certified by College of American Pathologists (CAP) &amp; Clinical Laboratory Improvement Amendments (CLIA). Gene fusions were analyzed by OriFusion, an OrigiMed's proprietary algorithm. For LOD determination, gradient amounts of RNA or mixed cell line samples were processed for sequencing. To confirm its concordance with FISH/IHC, 36 sarcomas FFPE samples were examined by FISH (33/36) or IHC (3/36). A cohort of 148 tumor samples was used to analyze rearrangement/fusion detection capability. Results: For RNAseq in YuanSu S, LOD was determined as 5 copies/1ng RNA input and was verified by ddPCR. Samples with rearrangement/fusion ≥ 5% detected by DNAseq were confirmed by ddPCR. For the interfering substance validation, results of samples treated with ethanol or necrotic tissue were consistent with untreated samples. Furthermore, two different Illumina platforms, Nextseq 500 &amp; Novaseq 6000, showed no difference in sequencing abilities. 36 sarcoma FFPE samples were assessed by NGS and verified by FISH or IHC. All the 19 samples with FISH or IHC positive were detected the same rearrangements/fusions by YuanSu S, whereas the negative samples had no fusion detected. In the cohort of 148 samples, 426 rearrangements/fusions were detected by YuanSu S. 101 (23.7%) were detected both by DNAseq &amp; RNAseq; 204 (47.9%) were determined by DNAseq only; while 121 (28.4%) were determined by RNAseq only. Rearrangements were detected by DNAseq in 97 (65.5%) samples, and 40 (41.2%) may benefit from FDA-approved drugs. Importantly, 51 (34.5%) samples had fusions detected only by RNAseq, and 33 (64.7%) may benefit from targeted therapies. Conclusion: Our results confirmed the reliability and advantages of YuanSu S, a DNA plus RNA NGS panel. Experience from YuanSu S indicated that integrative DNA &amp; RNA sequencing could increase sensitivity of fusion detection. Therefore, the combination of DNAseq &amp; RNAseq is able to screen more patients carrying fusion targets who could benefit from corresponding inhibitors and play an important role in guiding treatment decisions. Citation Format: Jiacheng Cai, Feng Li, Haiyan Wu, Weifeng Wang, Qiuyue Pan, Lin Zhang, Kai Wang. Validation &amp; application of next-generation sequencing panel YuanSu S for gene rearrangements/fusions detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2283.

Abstract 2288: Identification of gene fusions in soft tissue sarcoma improved by integrative DNA and RNA sequencing

Abstract Background: More than 50 different histologic subtypes of soft tissue sarcoma (STS) with mesenchymal origin have been recognized, and their overlapping morphology and histology make clinical diagnosis challenging. Gene fusion has been described as an important molecular biological feature of STS, which can assist in its diagnosis and treatment. Conventional DNAseq methods have limitations for gene fusion detection, while the RNAseq method might circumvent them. In this study, we evaluated the clinical value of integrated DNA and RNA sequencing in the detection of STS gene fusions. Methods: Formalin-fixed and paraffin embedded (FFPE) tumor samples and matched blood of 142 Chinese STS patients were collected and tested by the next-generation sequencing (NGS)-based 825-gene DNA panel and 395-gene RNA panel at Genetronhealth, a laboratory accredited by College of American Pathologists and Clinical Laboratory Improvement Amendments. Results: The genetic landscape of 75 male and 67 female Chinese patients with STS was analyzed. The median age of enrolled patients was 39 years (range, 1-82 years). Multiple histological subtypes were collected, including rhabdomyosarcoma (n = 15), liposarcoma (n = 15), myofibroblastic sarcoma (n = 11) and other types which were all less than 10 cases. Overall, 83 different fusions were detected in 60 patients. Of these, 25 fusions could be detected both by DNA and RNA panels in 18 patients, five fusions were detected only by the DNA panel in four patients, and 53 fusions only by the RNA panel in 39 patients. Compared to DNA-NGS individual testing, integrative panels increased the detection rate of fusion in patients with STS by 177% (53/30). The five gene fusions detected only by the DNA panel, including FOSL1-RELA, intergenic-RUNX2, ADAMTS16-TERT, NCR3LG1-KMT2A and YAP1-MAMLD1, remain clinical significance uncertain in STS. Fusion genes, such as PAX3-FOXO1, ASPSCR1-TFE3, COL1A1-PDGFB, EWSR1-ERG, were detected separately by the RNA panel, which could assist the diagnosis or targeted therapy. Additionally, unreported DIP2B-EWSR1 and AMLD1-SSX1 gene fusions were detected in a patient with clear cell sarcoma and a patient with synovial sarcoma respectively, which might be helpful in clinical diagnosis. Conclusions: Our results showed that the integrated DNA and RNA sequencing can effectively improve the detection rate of STS gene fusions, which might benefit more STS patients. Citation Format: Jiayong Liu, Shu Li, Tian Gao, Chunyang Wang, Qifan He, Xiaojuan Wang, Tonghui Ma. Identification of gene fusions in soft tissue sarcoma improved by integrative DNA and RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2288.

A new method to accurately identify single nucleotide variants using small FFPE breast samples.

Most tissue collections of neoplasms are composed of formalin-fixed and paraffin-embedded (FFPE) excised tumor samples used for routine diagnostics. DNA sequencing is becoming increasingly important in cancer research and clinical management; however it is difficult to accurately sequence DNA from FFPE samples. We developed and validated a new bioinformatic pipeline to use existing variant-calling strategies to robustly identify somatic single nucleotide variants (SNVs) from whole exome sequencing using small amounts of DNA extracted from archival FFPE samples of breast cancers. We optimized this strategy using 28 pairs of technical replicates. After optimization, the mean similarity between replicates increased 5-fold, reaching 88% (range 0–100%), with a mean of 21.4 SNVs (range 1–68) per sample, representing a markedly superior performance to existing tools. We found that the SNV-identification accuracy declined when there was less than 40 ng of DNA available and that insertion–deletion variant calls are less reliable than single base substitutions. As the first application of the new algorithm, we compared samples of ductal carcinoma in situ of the breast to their adjacent invasive ductal carcinoma samples. We observed an increased number of mutations (paired-samples sign test, P < 0.05), and a higher genetic divergence in the invasive samples (paired-samples sign test, P < 0.01). Our method provides a significant improvement in detecting SNVs in FFPE samples over previous approaches.

KRAS gene status in gastric signet-ring cell carcinoma patients and acts as biomarker of MEK inhibitor.

Signet-ring cell carcinoma (SRCC) is a specific subtype of stomach cancer with unique epidemiology. Here, we sought to explore the role of KRAS in SRCC. KRAS status was studied both in The Cancer Genome Atlas (TCGA) and internal cohorts. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) were performed in formalin-fixed and paraffin-embedded (FFPE) samples. We explored patients' survival and clinicopathological characteristics in terms of KRAS mutation and expression. We also explored KRAS status and drug response curve of MEK/mTOR inhibitors in SRCC cell lines. Patients with KRAS mutations and copy number variation (CNV) showed higher mRNA level compared to non-mutant cases (P=0.003 and P<0.001). In internal cohort, 15 samples harbored KRAS mutations. Survival analysis showed that these patients had significantly lower overall survival (OS) (P=0.048). We further analyzed 75 patients with sufficient FFPE samples. Eight patients showed KRAS mutations and one patient showed KRAS amplification. The median OS was 12.5 months for patients with KRAS mutation, and 19.5 months for patients without KRAS mutation (P=0.005). Positive expression of KRAS as shown by IHC was detected in majority of SRCC samples, which was higher than our intestinal cohort (28% vs. 12.6%, P=0.033). We further explored the correlation between KRAS status and drug sensitivity in 4 SRCC cell lines. SNU601 and SNU668, which harbored KRAS mutation, were hypersensitive to MEK and mTOR inhibitors than KRAS wide type cell lines KATO-III and NUGC-4. Our findings demonstrate that KRAS gene plays an important role in SRCC and reveals therapeutic potential of targeting tumors by inhibiting MEK and mTOR pathways.

FC 017DEEP-LEARNING ENABLED QUANTIFICATION OF SINGLE-CELL SINGLE-MRNA TRANSCRIPTS AND CORRELATIVE SUPER-RESOLVED PODOCYTE FOOT PROCESS MORPHOMETRY IN ROUTINE KIDNEY BIOPSY SPECIMEN

Abstract Background and Aims Although high-throughput single-cell transcriptomic analysis, super-resolution light microscopy and deep-learning methods are broadly used, the gold-standard to evaluate kidney biopsies is still the histologic assessment of formalin-fixed and paraffin embedded (FFPE) samples with parallel ultrastructural evaluation. Recently, we and others have shown that super-resolution fluorescence microscopy can be used to study glomerular ultrastructure in human biopsy samples. Additionally, in the last years mRNA in situ hybridization techniques have been improved to increase specificity and sensitivity to enable transcriptomic analysis with single-mRNA resolution (smFISH). Method For smFISH, we used the fluorescent multiplex RNAscope kit with probes targeting ACE2, WT1, PPIB, UBC and POLR2A. To find an on-slide reference gene, the normfinder algorithm was used. The smFISH protocol was combined with a single-step anti-podocin immunofluorescence enabled by VHH nanobodies. Podocytes were labeled by tyramide-signal amplified immunofluorescence using recombinant anti-WT1 antibodies. Slides were imaged using confocal laser scanning, as well as 3D structured illumination microscopy. Deep-learning networks to segment glomeruli and cell nuclei (UNet and StarDist) were trained using the ZeroCostDL4Mic approach. Scripts to automate analysis were developed in the ImageJ1 macro language. Results First, we show robust functionality of threeplex smFISH in archived routine FFPE kidney biopsy samples with single-mRNA resolution. As variations in sample preparation can negatively influence mRNA-abundance, we established PPIB as an ideal on-slide reference gene to account for different RNA-integrities present in biopsy samples. PPIB was chosen for its most stable expression in microarray dataset of various glomerular diseases determined by the Normfinder algorithm as well as its smFISH performance. To segment glomeruli and to label glomerular and tubulointerstitial cell subsets, we established a combination of smFISH and immunofluorescence. As smFISH requires intense tissue digestion to liberate cross-linked RNAs, immunofluorescence protocols had to be adapted: For podocin, a small-sized single-step label approach enabled by small nanobodies and for WT1, tyramide signal amplification was used. For enhanced segmentation performance, we used deep learning: First, a network was customized to recognize DAPI+ cell nuclei and WT1/DAPI+ podocyte nuclei. Second, a UNet was trained to segment glomeruli in podocin-stained tissue sections. Using these segmentation masks, we could annotate PPIB-normalized single mRNA transcripts to individual cells. We established an ImageJ script to automatize transcript quantification. As a proof-of-principle, we demonstrate inverse expression of WT1 and ACE2 in glomerular vs. tubulointerstitial single cells. Furthermore, in the podocyte subset, WT1 highly clustered whereas no significant ACE2 expression was found under baseline conditions. Additionally, when imaged with super-resolution microscopy, podocyte filtration slit morphology could be visualized The optical resolution was around 125 nm and therefore small enough to resolve individual foot processes. The filtration slit density as a podocyte-integrity marker did not differ significantly from undigested tissue sections proving the suitability for correlative podocyte foot process morphometry with single-podocyte transcript analysis. Conclusion Here we present a modular toolbox which combines algorithms for multiplexed, normalized single-cell gene expression with single mRNA resolution in cellular subsets (glomerular, tubulointerstitial and podocytes). Additionally, this approach enables correlation with podocyte filtration slit ultrastructure and gross glomerular morphometry.

Digital PCR-based cell-free DNA K-Ras mutations analysis and clinical implication in pancreatic cancer.

e16215 Background: Pancreatic cancer is one of the most fatal cancers worldwide. In addition to patients presenting later in the disease, limitations in current testing modalities pose a challenge to early diagnosis and treatment. Somatic mutations of the K-Ras gene have been suggested as a key driver of pancreatic carcinogenesis and thus proposed as a biomarker for diagnosis and therapy. Majority of these studies utilize tissue-based methods for analyzing K-Ras mutations. In recent years, liquid biopsy assay, in particular, analysis of cell-free DNA (cfDNA), has emerged as a promising noninvasive diagnostic approach in oncology, with the respect to the identification of minimal residual diseases, monitoring treatment response, detection recurrence and metastasis, and identification of chemo-resistance mechanisms. The application of digital PCR based plasma cfDNA K-Ras mutation assay in pancreatic cancer has not been reported. Methods: We included patients who underwent evaluation of a pancreatic lesion detected by conventional radiography (CT/MRI) and confirmed by endoscopic ultrasound (EUS) and biopsy. The biopsies from pancreatic tissues were formalin-fixed and paraffin-embedded (FFPE) for pathological diagnosis, and the total DNA was extracted from the FFPE slides. In addition, matched blood from the same patient was collected at the time of biopsy, and plasma cfDNA was extracted. Both DNA quality and concentration were evaluated. 4.0 ng of DNA was used for droplet digital PCR (ddPCR) analysis. K-Ras G12D, G12V, G12R, G12C, G12A, G12S, and G13D mutations were analyzed by multiplexed assay reagents. The sensitivity cut-off of the multiplexed K-Ras G12/G13 assay was 1.0%. Results: cfDNA could be detected in the initiation of pathological diagnosis. Among the twelve patients with pancreatic lesions, eight (67%) were pancreatic ductal adenocarcinoma (PDAC) (head (4), body (2), tail (2) of the pancreas), one was acute pancreatitis, one was a tail cyst and two were unspecified non-malignant lesions. All the eight PDAC (100%) FFPE tissues exhibited K-Ras G12/13 mutations (Scores: 4.2-41.5%), while none of the non-malignant lesions (0%) demonstrated K-Ras mutations. Interestingly, K-Ras mutations were detected from seven of the eight PDAC patient’s plasma cfDNA (87.5%) (Scores: 1.2-20.7%) while the plasma cfDNA from patients with non-malignant lesions did not show any positive K-Ras mutations. Conclusions: Digital PCR-based plasma cfDNA assay for K-Ras mutation is a promising tool for diagnosis of PDAC and is comparable to tissue-based assays. Larger prospective studies can substantiate this and explore their roles in the prediction and early detection of recurrence of PDAC.

An ultrasensitive method for noninvasive pan-cancer early detection based on targeted methylation sequencing of cell-free DNA.

10544 Background: Screening the biomarkers from the cell-free DNA (cfDNA) of peripheral blood is a non-invasive and promising method for cancer diagnosis. Among diverse types of biomarkers, epigenetic biomarkers have been reported to be one of the most promising ones. Epigenetic modifications are widespread on the human genome and generally have strong signals due to the similar methylation patterns shared by adjacent CpG sites. Although some epigenetic diagnostic methods have been developed based on cfDNAs, few of them could be applied to pan-cancer and their sensitivities are barely satisfactory for early cancer detection. Methods: Targeted methylation sequencing was performed using our in-house-designed panel targeting regions with abundant cancer-specific methylation CpGs. The cfDNA samples from 80 healthy individuals and 549 cancer patients of 14 cancer types were separately sequenced. The dataset was randomly split into one discovery dataset and one validation dataset. Moreover, cfDNA samples from four cancer patients were diluted with the healthy cfDNAs to generate 12 in vitro simulated samples with low circulating tumor DNA (ctDNA) fraction. Additionally, DNAs extracted from 130 unmatched tumor formalin fixation and paraffin embedding (FFPE) samples of 10 cancer types were sequenced to screen the diagnostic biomarkers. Adjacent CpG sites were first merged into methylation-correlated blocks (MCB) according to their correlations of methylation levels in tumor DNAs. The MCBs with higher methylation levels in tumor DNAs than that of healthy cfDNAs (from the discovery dataset) were defined as our hypermethylation biomarkers. For each cfDNA sample, a hypermethylation score (HM-score) was computed to measure the overall methylation level difference of selected biomarkers. The performance of our method was evaluated with the real-world dataset, while the limit of detection was estimated using the simulated low-ctDNA samples. Results: Our model based on 37 hypermethylation MCB biomarkers achieved an area under the curve (AUC) of 0.89 and 0.86 in the real-world pan-cancer discovery and validation cfDNA datasets, respectively. Furthermore, the overall specificity and sensitivity are 100% and 76.19% in the discovery dataset, and 96.67% and 72.86% in the validation dataset. In the validation dataset, 28/40 (70%) of early-stage colorectal cancer patients and 10/20 (50%) of non-small-cell lung cancer patients were successfully diagnosed. Additionally, all the simulated samples with theoretical ctDNA factions over 0.5% were predicted as diseased, demonstrating the ability of our method to detect tumor signals at early stages. Conclusions: Our cfDNA-based epigenetic method outperforms currently available methods in various cancer types, and is promising to be applied to early-stage cancer detection and samples with low ctDNA fractions.