Abstract 1664: Comparison of RNA sequencing data generated from formalin-fixed, paraffin-embedded (FFPE) papillary thyroid carcinoma samples using different library preparation methods

Identification of BRAFV600E in thyroid neoplasia may be useful because it is specific for malignancy, connotes a worse prognosis, and is the target of novel therapies currently under investigation. Sanger sequencing is the ‘gold standard’ for mutation detection but is subject to sampling error and requires resources beyond many diagnostic pathology laboratories. In this study, we compared immunohistochemistry (IHC) using a BRAFV600E mutation-specific MAB to Sanger sequencing on DNA from formalin-fixed paraffin-embedded tissue, in a well-characterized cohort of 101 papillary thyroid carcinoma (PTC) patients. For all cases, an IHC result was available; however, five cases failed Sanger sequencing. Of the 96 cases with molecular data, 68 (71%) were BRAFV600E positive by IHC and 59 (61%) were BRAFV600E positive by sequencing. Eleven cases were discordant. One case was negative by IHC and initially positive by sequencing. Repeat sequencing of that sample and sequencing of a macrodissected sample were negative for BRAFV600E. Of ten cases positive by IHC but negative by sequencing on whole sections, repeat sequencing on macrodissected tissue confirmed the IHC result in seven cases (suggesting that these were false negatives of sequencing on whole sections). In three cases, repeat sequencing on recut tissue remained negative (including using massive parallel sequencing), but these cases demonstrated relatively low neoplastic cellularity. We conclude that IHC for BRAFV600E is more sensitive and specific than Sanger sequencing in the routine diagnostic setting and may represent the new gold standard for detection of BRAFV600E mutation in PTC.

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Different stability of miRNAs and endogenous control genes in archival specimens of papillary thyroid carcinoma

Molecular Medicine ◽

10.1186/s10020-020-00218-7 ◽

2020 ◽

Vol 26 (1) ◽

Author(s):

Daina Pamedytyte ◽

Enrika Leipute ◽

Birute Zilaitiene ◽

Valdas Sarauskas ◽

Dalia Dauksiene ◽

...

Keyword(s):

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Mirna Expression ◽

Endogenous Control ◽

Papillary Thyroid ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

The Stability ◽

The Impact

Abstract Background The most popular miRNA quantitation technique is RQ-PCR with relative gene expression method that requires an endogenous control (EC) gene for data normalization. However, there are insufficient data and selection criteria on the most suitable ECs for miRNA expression studies in many cancer types including papillary thyroid carcinoma (PTC). Therefore, in this study we evaluated the impact of chosen EC and archival formalin-fixed, paraffin-embedded (FFPE) PTC tissue age on estimated target miRNA expression. Methods RQ-PCR was used to determine expression levels of five miRNAs (miR-146b, miR-222, miR-21, miR-221 and miR-181b) and three different endogenous controls (RNU48, let-7a, miR-16), which were used to normalize the data. In total, 400 FFPE PTC tissues were analyzed that have been stored from 1 to 15 years. Results The stability of commonly used ECs RNU48 and let-7a significantly differs from the stability of target miRNA in archival FFPE PTC tissues. Moreover, these differences have a great impact on miRNA expression results when FFPE tissue samples have been stored for a different period of time. Conclusions It is important to select an ECs not only stable in the tissue of interest but also with similar stability to target miRNA, especially when working with samples of different age.

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Papillary Thyroid Carcinoma Tissue miR-146b, -21, -221, -222, -181b Expression in Relation with Clinicopathological Features

Diagnostics ◽

10.3390/diagnostics11030418 ◽

2021 ◽

Vol 11 (3) ◽

pp. 418

Author(s):

Aistė Kondrotienė ◽

Albertas Daukša ◽

Daina Pamedytytė ◽

Mintautė Kazokaitė ◽

Aurelija Žvirblienė ◽

...

Keyword(s):

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Univariate Analysis ◽

High Expression ◽

Clinicopathological Parameters ◽

Papillary Thyroid ◽

Tissue Samples ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Node Metastases

We analyzed miR-146b, miR-21, miR-221, miR-21, and miR-181b in formalin fixed paraffin-embedded papillary thyroid carcinoma (PTC) tissue samples of 312 individuals and evaluated their expression relationship with clinicopathological parameters. A higher expression of miR-21 was related to unifocal lesions (p < 0.011) and autoimmune thyroiditis (0.007). miR-221, miR-222 expression was higher in the PTC tissue samples with extrathyroidal extension (p = 0.049, 0.003, respectively). In a group of PTC patients with pT1a and pT1b sized tumors, the expression of miR-146b, miR-21, miR-221, and miR-222 in PTC tissue samples was lower than in patients with pT2, pT3, and pT4 (p = 0.032; 0.0044; 0.003; 0.001; 0.001, respectively). Patients with lymph node metastases had higher expression of miR-21, -221, -222, and -181b (p < 0.05). A high expression of miR-146b, miR-21, miR-221 panel was associated with decreased overall survival (OS) (Log rank p = 0.019). Univariate analysis revealed that presence of metastatic lymph nodes and high expression of miR-146b, miR-21, and miR-221 panels were associated with increased hazard of shorter OS. After multivariate analysis, only sex (male) and age (≥55 years) emerged as independent prognostic factors associated with shorter OS (HR 0.28 (95% CI 0.09–0.86) and HR 0.05 (95% CI 0.01–0.22), respectively). In conclusion, 5 analyzed miRs expression have significant relations to clinicopathologic parameters so further investigations of these molecules are expedient while searching for prognostic PTC biomarkers.

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Single-cell RNA sequencing demonstrates the intratumoral heterogeneityof papillary thyroid carcinoma

10.1101/2021.02.24.21251881 ◽

2021 ◽

Author(s):

Zhengshi Wang ◽

Youlutuziayi Rixiati ◽

Wenli Jiang ◽

Caiguo Huang ◽

Binghua Jiao ◽

...

Keyword(s):

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Single Cell ◽

Rna Sequencing ◽

Immune Therapy ◽

Thyroid Tissue ◽

Papillary Thyroid ◽

Sequencing Data ◽

Tissue Samples ◽

Single Cell Rna Sequencing

AbstractPapillary thyroid carcinoma(PTC) is the most common thyroid malignancy, to investigate the intratumoral heterogeneity of PTC, we analyzed single-cell RNA-sequencing data and identified 10 major cell types from primary papillary thyroid carcinoma, lymph metastatic, or paired normal thyroid tissue samples. In this study, we verified that the increase in the proportion of CD4+Tregs may be key factor responsible for the immunosuppressive property of PTC. Inhibitory checkpoints, such as TIGIT and CD96 may be better targets for immune therapy in lymph metastatic papillary thyroid carcinoma. Our results will further the understanding of the heterogeneity among papillary thyroid carcinoma and provide an essential resource for drug discovery in the future.

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PATH-26. RNA SEQUENCING OF FORMALIN-FIXED PARAFFIN-EMBEDDED SPECIMENS IN DIAGNOSTIC ROUTINE IDENTIFIES CLINICALLY RELEVANT GENE FUSIONS

Neuro-Oncology ◽

10.1093/neuonc/noaa222.661 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii429-iii430

Author(s):

Damian Stichel ◽

Daniel Schrimpf ◽

Jochen Meyer ◽

Annika Wefers ◽

Philipp Sievers ◽

...

Keyword(s):

Brain Tumor ◽

Rna Sequencing ◽

Fusion Gene ◽

Detection Accuracy ◽

Gene Fusions ◽

Sequencing Data ◽

Methylation Array ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Abstract Pediatric brain tumor entities harbor a variety of gene fusions. Whilst other molecular parameters like somatic mutations and copy number alterations have become pivotal for brain tumor diagnostics, gene fusions are only less well covered by routinely applied methylation arrays or targeted next-generation sequencing of DNA. In a routine diagnostic setting we established and optimized a workflow for investigation of gene fusions in formalin-fixed paraffin-embedded (FFPE) tumor tissues by using RNA sequencing. Assessing different tools for calling fusions from raw data, we found relevant fusions in 66 out of 101 (65%) analyzed cases in a prospective cohort collected over 26 months. In 43 (43%) cases the fusions were of decisive diagnostic relevance and in 40 (40%) cases the fusion genes rendered a druggable target. Besides the relevance of pathognomonic fusions for diagnostics, especially the detection of druggable gene fusions yields direct benefit to the patients. This approach allows for an unbiased search for fusion events in the tested samples. Besides rare variants of established fusions which were not detected by prior targeted analyses, we identified previously unreported fusion events. Exemplified on KIAA1549:BRAF fusion, we in addition provide an overview of the detection accuracy of different methods, including breakpoint detection in DNA methylation array data and fusion gene detection in DNA panel sequencing data. Our data show that RNA sequencing has great diagnostic as well as therapeutic value by clinically detecting relevant alterations.

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Fusion transcript discovery using RNA sequencing in formalin-fixed paraffin-embedded specimen

Critical Reviews in Oncology/Hematology ◽

10.1016/j.critrevonc.2021.103303 ◽

2021 ◽

Vol 160 ◽

pp. 103303

Author(s):

Amin Talebi ◽

Jean Paul Thiery ◽

Mohammad Amin Kerachian

Keyword(s):

Rna Sequencing ◽

Fusion Transcript ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Transcript Discovery ◽

Formalin Fixed

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The impact of RNA extraction method on accurate RNA sequencing from formalin-fixed paraffin-embedded tissues

BMC Cancer ◽

10.1186/s12885-019-6363-0 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 4

Author(s):

Michal Marczyk ◽

Chunxiao Fu ◽

Rosanna Lau ◽

Lili Du ◽

Alexander J. Trevarton ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Rna Sequencing ◽

Rna Extraction ◽

Formalin Fixed Paraffin ◽

Ffpe Samples ◽

Formalin Fixed Paraffin Embedded ◽

The Impact ◽

Formalin Fixed

Abstract Background Utilization of RNA sequencing methods to measure gene expression from archival formalin-fixed paraffin-embedded (FFPE) tumor samples in translational research and clinical trials requires reliable interpretation of the impact of pre-analytical variables on the data obtained, particularly the methods used to preserve samples and to purify RNA. Methods Matched tissue samples from 12 breast cancers were fresh frozen (FF) and preserved in RNAlater or fixed in formalin and processed as FFPE tissue. Total RNA was extracted and purified from FF samples using the Qiagen RNeasy kit, and in duplicate from FFPE tissue sections using three different kits (Norgen, Qiagen and Roche). All RNA samples underwent whole transcriptome RNA sequencing (wtRNAseq) and targeted RNA sequencing for 31 transcripts included in a signature of sensitivity to endocrine therapy. We assessed the effect of RNA extraction kit on the reliability of gene expression levels using linear mixed-effects model analysis, concordance correlation coefficient (CCC) and differential analysis. All protein-coding genes in the wtRNAseq and three gene expression signatures for breast cancer were assessed for concordance. Results Despite variable quality of the RNA extracted from FFPE samples by different kits, all had similar concordance of overall gene expression from wtRNAseq between matched FF and FFPE samples (median CCC 0.63–0.66) and between technical replicates (median expression difference 0.13–0.22). More than half of genes were differentially expressed between FF and FFPE, but with low fold change (median |LFC| 0.31–0.34). Two out of three breast cancer signatures studied were highly robust in all samples using any kit, whereas the third signature was similarly discordant irrespective of the kit used. The targeted RNAseq assay was concordant between FFPE and FF samples using any of the kits (CCC 0.91–0.96). Conclusions The selection of kit to purify RNA from FFPE did not influence the overall quality of results from wtRNAseq, thus variable reproducibility of gene signatures probably relates to the reliability of individual gene selected and possibly to the algorithm. Targeted RNAseq showed promising performance for clinical deployment of quantitative assays in breast cancer from FFPE samples, although numerical scores were not identical to those from wtRNAseq and would require calibration.

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