Sarcoma Subgrouping by Detection of Fusion Transcripts Using NanoString nCounter Technology

2018 ◽  
Vol 22 (3) ◽  
pp. 205-213 ◽  
Author(s):  
Javal Sheth ◽  
Anthony Arnoldo ◽  
Yunan Zhong ◽  
Paula Marrano ◽  
Carlos Pereira ◽  
...  
Keyword(s):  
Rt Pcr ◽  
Fusion Transcripts ◽  
Pediatric Sarcoma ◽  
Formalin Fixed Paraffin ◽  
Pediatric Sarcomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Nanostring Technology ◽  
Future Work ◽  
Formalin Fixed

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.

scholarly journals Detection of Newcastle Disease Virus RNA by Reverse Transcription-Polymerase Chain Reaction Using Formalin-Fixed, Paraffin-Embedded Tissue and Comparison with Immunohistochemistry and in situ Hybridization

2007 ◽  
Vol 19 (4) ◽  
pp. 396-400 ◽  
Author(s):  
Nobuko Wakamatsu ◽  
Daniel J. King ◽  
Bruce S. Seal ◽  
Corrie C. Brown
Keyword(s):  
Virus Isolation ◽  
Rt Pcr ◽  
Matrix Gene ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Virulent Virus ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Formalin Fixed

The usefulness of reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of 2 NDV isolates: a low virulent virus (LaSota) and a virulent virus (from the 2002–2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just before euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By seminested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-μm sections and to those of virus isolation from swabs. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens that were no longer shedding virus detectable by virus isolation. The RT-PCR was an effective and sensitive method to detect NDV in FFPE tissues. To the authorsn' knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.

scholarly journals Characterization of epididymal and testicular histologic lesions and use of immunohistochemistry and PCR on formalin-fixed tissues to detect Brucella canis in male dogs

2021 ◽  
pp. 104063872098688
Author(s):  
Andrea M. Camargo-Castañeda ◽  
Lauren W. Stranahan ◽  
John F. Edwards ◽  
Daniel G. Garcia-Gonzalez ◽  
Leonardo Roa ◽  
...  
Keyword(s):  
Accurate Diagnosis ◽  
Testicular Atrophy ◽  
Detection Techniques ◽  
Formalin Fixed Paraffin ◽  
Brucella Canis ◽  
Formalin Fixed Paraffin Embedded ◽  
Variable Sensitivity ◽  
Ffpe Tissues ◽  
Formalin Fixed

In male dogs, Brucella canis frequently causes epididymitis, ultimately resulting in testicular atrophy and infertility. Although B. canis predominantly affects the epididymis, the misleading term “orchitis” is still commonly used by clinicians. Of additional concern, diagnosis in dogs remains challenging because of variable sensitivity and specificity of serologic assays and fluctuations in bacteremia levels in infected dogs, reducing the sensitivity of blood culture. We describe here the histologic lesions in the scrotal contents of 8 dogs suspected of being infected with B. canis and clinically diagnosed with orchitis. We explored the possibility of using immunohistochemistry (IHC) and real-time PCR (rtPCR) in formalin-fixed, paraffin-embedded (FFPE) tissues to detect the presence of B. canis. Epididymitis of variable chronicity was identified in all 8 dogs, with only 3 also exhibiting orchitis. Using rtPCR, the presence of B. canis was identified in 4 of 8 dogs, with 3 of these 4 dogs also positive by IHC. These results suggest that rtPCR and IHC are promising techniques that can be used in FFPE tissues to detect B. canis when other detection techniques are unavailable. Additionally, accurate recognition of epididymitis rather than orchitis in suspect cases could aid in accurate diagnosis.

scholarly journals Apolipoprotein D Expression in Primary Brain Tumors: Analysis by Quantitative RT-PCR in Formalin-fixed, Paraffin-embedded Tissue

2005 ◽  
Vol 53 (8) ◽  
pp. 963-969 ◽  
Author(s):  
Stephen B. Hunter ◽  
Vijay Varma ◽  
Bahig Shehata ◽  
J.D.L. Nolen ◽  
Cynthia Cohen ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Tumor Type ◽  
Rt Pcr ◽  
Who Grade ◽  
Primary Brain Tumors ◽  
Formalin Fixed Paraffin ◽  
Pilocytic Astrocytomas ◽  
Formalin Fixed Paraffin Embedded ◽  
Apolipoprotein D ◽  
Formalin Fixed

Apolipoprotein D (apoD) expression has been shown to correlate both with cell cycle arrest and with prognosis in several types of malignancy, including central nervous system astrocytomas and medulloblastomas. ApoD expression was investigated by real-time quantitative RT-PCR using RNA extracted from 68 formalin-fixed, paraffin-embedded brain specimens. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Quantitation was achieved on all specimens. Sixteen poorly infiltrating WHO grade I glial neoplasms (i.e., pilocytic astrocytomas and gangliogliomas) showed an average 20-fold higher apoD expression level compared with the 20 diffusely infiltrating glial neoplasms (i.e., glioblastoma, anaplastic astrocytoma, oligodendrogliomas; p=0.00004). A small number of exceptions (i.e., two high-expressing glioblastomas and three low-expressing gangliogliomas) were identified. Analyzed as individual tumor groups, poorly infiltrating grade I pilocytic astrocytomas and gangliogliomas differed significantly from each tumor type within the diffusely infiltrating higher-grade category ( p<0.05 for each comparison) but not from each other ( p>0.05). Conversely, each individual tumor type within the diffusely infiltrating category differed significantly from both pilocytic astrocytomas and gangliogliomas ( p<0.05) but did not vary from other infiltrating tumors ( p>0.05). Ependymomas, non-infiltrating grade II neoplasms, expressed levels of apoD similar to or lower than levels expressed by the diffusely infiltrating gliomas. Ten medulloblastomas with survival longer than 3 years averaged slightly higher apoD expression than four fatal medulloblastomas; however, this result was not statistically significant and individual exceptions were notable. In 17 of the medulloblastomas, MIB-1 proliferation rates quantitated by image cytometry did not correlate with apoD expression. In addition, apoD expression was 5-fold higher in the slowly proliferating grade I glial neoplasms compared with non-proliferating normal brain tissue ( p=0.01), suggesting that apoD expression is not simply an inverse measure of proliferation. ApoD expression measured by quantitative RT-PCR may be useful in the differential diagnosis of primary brain tumors, particularly pilocytic astrocytomas and gangliogliomas.

Detection of West Nile virus in formalin-fixed, paraffin-embedded human tissues by RT-PCR: A useful adjunct to conventional tissue-based diagnostic methods

2007 ◽  
Vol 38 (2) ◽  
pp. 106-111 ◽  
Author(s):  
Julu Bhatnagar ◽  
Jeannette Guarner ◽  
Christopher D. Paddock ◽  
Wun-Ju Shieh ◽  
Robert S. Lanciotti ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Diagnostic Methods ◽  
Human Tissues ◽  
Rt Pcr ◽  
West Nile ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

scholarly journals Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Tamara Sequeiros ◽  
Marta García ◽  
Melania Montes ◽  
Mireia Oliván ◽  
Marina Rigau ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Molecular Markers ◽  
Tumor Grade ◽  
Developed Countries ◽  
Response To Therapy ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

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