Detection of SS18-SSX fusion transcripts in formalin-fixed paraffin-embedded neoplasms: analysis of conventional RTPCR, qRT-PCR and dual color FISH as diagnostic tools for synovial sarcoma

Background NanoString technology is an innovative barcode-based system that requires less tissue than traditional techniques and can test for multiple fusion transcripts in a single reaction. The objective of this study was to determine the utility of NanoString technology in the detection of sarcoma-specific fusion transcripts in pediatric sarcomas. Design Probe pairs for the most common pediatric sarcoma fusion transcripts were designed for the assay. The NanoString assay was used to test 22 specific fusion transcripts in 45 sarcoma samples that had exhibited one of these fusion genes previously by reverse transcription polymerase chain reaction (RT-PCR). A mixture of frozen (n = 18), formalin-fixed, paraffin-embedded (FFPE) tissue (n = 23), and rapid extract template (n = 4) were used for testing. Results Each of the 22 transcripts tested was detected in at least one of the 45 tumor samples. The results of the NanoString assay were 100% concordant with the previous RT-PCR results for the tumor samples, and the technique was successful using both FFPE and rapid extract method. Conclusion Multiplexed interrogation for sarcoma-specific fusion transcripts using NanoString technology is a reliable approach for molecular diagnosis of pediatric sarcomas and works well with FFPE tissues. Future work will involve validating additional sarcoma fusion transcripts as well as determining the optimal workflow for diagnostic purposes.

Download Full-text

Detection of COL1 A1-PDGFB Fusion Transcripts in Dermatofibrosarcoma Protuberans by Reverse Transcription-Polymerase Chain Reaction Using Archival Formalin-Fixed, Paraffin-Embedded Tissues

Diagnostic Molecular Pathology ◽

10.1097/00019606-199909000-00002 ◽

1999 ◽

Vol 8 (3) ◽

pp. 113-119 ◽

Cited By ~ 90

Author(s):

Jian Wang ◽

Masanori Hisaoka ◽

Shohei Shimajiri ◽

Yosuke Morimitsu ◽

Hiroshi Hashimoto

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Dermatofibrosarcoma Protuberans ◽

Chain Reaction ◽

Fusion Transcripts ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Polymerase Chain ◽

Formalin Fixed

Download Full-text

Agreement in Breast Cancer Classification between Microarray and Quantitative Reverse Transcription PCR from Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Tissues

Clinical Chemistry ◽

10.1373/clinchem.2006.083725 ◽

2007 ◽

Vol 53 (7) ◽

pp. 1273-1279 ◽

Cited By ~ 48

Author(s):

Michael Mullins ◽

Laurent Perreard ◽

John F Quackenbush ◽

Nicholas Gauthier ◽

Steven Bayer ◽

...

Keyword(s):

Breast Cancer ◽

Training Set ◽

Pcr Assay ◽

Gene Set ◽

Qrt Pcr ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Ffpe Tissues ◽

Formalin Fixed

Abstract Background: Microarray studies have identified different molecular subtypes of breast cancer with prognostic significance. To transition these classifications into the clinical laboratory, we have developed a real-time quantitative reverse transcription (qRT)-PCR assay to diagnose the biological subtypes of breast cancer from fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: We used microarray data from 124 breast samples as a training set for classifying tumors into 4 previously defined molecular subtypes: Luminal, HER2+/ER−, basal-like, and normal-like. We used the training set data in 2 different centroid-based algorithms to predict sample class on 35 breast tumors (test set) procured as FF and FFPE tissues (70 samples). We classified samples on the basis of large and minimized gene sets. We used the minimized gene set in a real-time qRT-PCR assay to predict sample subtype from the FF and FFPE tissues. We evaluated primer set performance between procurement methods by use of several measures of agreement. Results: The centroid-based algorithms were in complete agreement in classification from FFPE tissues by use of qRT-PCR and the minimized “intrinsic” gene set (40 classifiers). There was 94% (33 of 35) concordance between the diagnostic algorithms when comparing subtype classification from FF tissue by use of microarray (large and minimized gene set) and qRT-PCR data. We found that the ratio of the diagonal SD to the dynamic range was the best method for assessing agreement on a gene-by-gene basis. Conclusions: Centroid-based algorithms are robust classifiers for breast cancer subtype assignment across platforms and procurement conditions.

Download Full-text

Detection of KIAA1549-BRAF Fusion Transcripts in Formalin-Fixed Paraffin-Embedded Pediatric Low-Grade Gliomas

Journal of Molecular Diagnostics ◽

10.1016/j.jmoldx.2011.07.002 ◽

2011 ◽

Vol 13 (6) ◽

pp. 669-677 ◽

Cited By ~ 56

Author(s):

Yongji Tian ◽

Benjamin E. Rich ◽

Natalie Vena ◽

Justin M. Craig ◽

Laura E. MacConaill ◽

...

Keyword(s):

Low Grade ◽

Low Grade Gliomas ◽

Fusion Transcripts ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Braf Fusion ◽

Formalin Fixed

Download Full-text

Five-Gene Model to Predict Survival in Mantle-Cell Lymphoma Using Frozen or Formalin-Fixed, Paraffin-Embedded Tissue

Journal of Clinical Oncology ◽

10.1200/jco.2007.12.0410 ◽

2008 ◽

Vol 26 (30) ◽

pp. 4966-4972 ◽

Cited By ~ 67

Author(s):

Elena Hartmann ◽

Verònica Fernàndez ◽

Victor Moreno ◽

Joan Valls ◽

Luis Hernández ◽

...

Keyword(s):

Cell Lymphoma ◽

Survival Prediction ◽

Gene Model ◽

Qrt Pcr ◽

Formalin Fixed Paraffin ◽

Mantle Cell ◽

Ffpe Tumor ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Formalin Fixed

Purpose Despite the common underlying translocation t(11;14) involving cyclin D1 that is present in nearly all cases of mantle-cell lymphoma (MCL), the clinical course of the disease is highly variable. The aim of the present study was to develop a quantitative gene expression–based model to predict survival in newly diagnosed patients with MCL that involves a minimum number of genes and is applicable to fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tumor samples. Patients and Methods The expression of 33 genes with potential prognostic and pathogenetic impact in MCL was analyzed using quantitative reverse-transcription polymerase chain reactions (qRT-PCR) in a low-density array format in frozen tumor samples from 73 patients with MCL. Multivariate Cox methods and stepwise algorithms were applied to build gene expression-based survival predictors. An optimized five-gene model was subsequently applied to FFPE tumor samples from 13 patients with MCL from the initial series and to 42 independent MCL samples. Results The optimized survival predictor was composed of the five genes RAN, MYC, TNFRSF10B, POLE2, and SLC29A2 and was validated for application in FFPE tissue samples. It allowed the survival prediction of patients with MCL with widely disparate clinical outcome and was superior to the immunohistochemical marker Ki-67, an established prognostic factor in MCL. Conclusion We here present a validated qRT-PCR–based test for survival prediction in patients with MCL that is applicable to fresh frozen as well as to FFPE tissue specimens. This test may prove useful to guide individualized treatment approaches for patients with MCL.

Download Full-text