Ein unterschätzter Schatz in Paraffin – Etablierung einer globalen Transkriptomanalyse an kaninen Tumoren aus FFPE-Material basierend auf der QuantSeq 3‘-Technologie

2021 ◽  
Author(s):  
AFH Haake ◽  
AK Langenhagen ◽  
AD Gruber
Keyword(s):  
2014 ◽  
Author(s):  
Annica Wilzen ◽  
Heidi Ottesen ◽  
Anna Larsson ◽  
Bo Wangberg ◽  
Andreas Muth ◽  
...  

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 403-403
Author(s):  
G. Hutchins ◽  
K. Handley ◽  
F. L. Baehner ◽  
M. Lopatin ◽  
H. Yaziji ◽  
...  

403 Background: Colorectal cancer patients with deficient mismatch repair (dMMR) have significantly fewer recurrences and may respond less well to chemotherapy. Immunohistochemical (IHC) determination of MMR status is therefore recommended to identify patients in whom adjuvant therapy is not indicated, but little is known regarding the variability of assay results. We aimed to define MMR IHC assay variability in formalin-fixed, paraffin-embedded (FFPE) material from the QUASAR-1 colorectal cancer trial contained within heterogeneity-prone tissue microarrays (TMAs) Methods: TMA sections of FFPE material from the QUASAR-1 trial were distributed to 2 independent laboratories (Leeds, UK and Vitro Molecular Laboratories [VML], FL, USA) for MMR IHC assays. Serial TMA sections were stained with MLH1/MSH2 using techniques blinded to the other laboratory. Each stained section was double-scored independently and results compared to determine inter-assay variability. Results: Matched MMR data were available for 1224 cases of MLH1 and 1223 cases of MSH2. Of these, loss of expression of MMR (dMMR) was reported in 160 cases (13.1%) by VML and 179 cases (14.6%) by Leeds. 140 (11.4%) were dMMR in both labs, 20 cases (1.6%) by VML alone and 39 (3.2%) by Leeds alone. Discordant dMMR status was observed in 56/166 (34%) dMMR cases identified with MLH1 and 10/34 (29%) cases identified with MSH2. Kappa coefficients for inter-assay agreement were 0.798 (95% CI = 0.748 - 0.848) for MMR status overall, 0.778 (95% CI = 0.722 - 0.835) for MLH1 and 0.823 (95% CI = 0.714 - 0.932) for MSH2. 85% (33/39) of discordant Leeds dMMR colon cancers were in the right colon where dMMR is more common, compared to 47% of VML discordant dMMR cases. Conclusions: Independent determination of MMR status by IHC on TMAs is associated with excellent inter-assay agreement. The reasons for MMR case discordance are under further investigation. These results further support routine MMR testing by IHC. No significant financial relationships to disclose.


2012 ◽  
Vol 48 ◽  
pp. S142
Author(s):  
B. Wojtas ◽  
A. Pfeifer ◽  
T. Stokowy ◽  
M. Eszlinger ◽  
M. Jarzab ◽  
...  

2012 ◽  
Vol 50 (1) ◽  
pp. 191-199 ◽  
Author(s):  
R. Klopfleisch ◽  
M. von Deetzen ◽  
A. Th. Weiss ◽  
J. Weigner ◽  
F. Weigner ◽  
...  

Formalin fixation and paraffin embedding (FFPE) is the standard method for tissue storage in histopathology. However, FFPE has disadvantages in terms of user health, environment, and nucleic acid integrity. Weigners fixative has been suggested as an alternative for embalming cadavers in human and veterinary anatomy. The present study tested the applicability of Weigners for histology and immunohistochemistry and the preservation of nucleic acids. To this end, a set of organs was fixed for 2 days and up to 6 months in Weigners (WFPE) or formalin. WFPE tissues from the skin, brain, lymphatic tissues, liver, and muscle had good morphologic preservation, comparable to formalin fixation. The quality of kidney and lung samples was inferior to FFPE material due to less accentuated nuclear staining and retention of proteinaceous interstitial fluids. Azan, Turnbull blue, toluidin, and immunohistochemical stainings for CD79a, cytokeratin, vimentin, and von Willebrand factor led to comparable results with both fixates. Of note, immunohistochemical detection of CD3 was possible after 6 months in WFPE but not in FFPE tissues. mRNA, miRNA, and DNA from WFPE tissues had superior quality and allowed for amplification of miRNA, 400-bp-long mRNA, and 1000-bp-long DNA fragments after 6 months of fixation in WFPE. In summary, Weigners fixative is a nonhazardous alternative to formalin, which provides a good morphologic preservation of most organs, a similar sensitivity for protein detection, and a superior preservation of nucleic acids. Weigners may therefore be a promising alternative to cryopreservation and may be embraced by people affected by formalin allergies.


Der Pathologe ◽  
2021 ◽  
Vol 42 (1) ◽  
pp. 83-85
Author(s):  
Torsten Goldmann ◽  
Doris Hillemann ◽  
Florian Maurer ◽  
Barbara Kalsdorf ◽  
Rosemarie Krupar ◽  
...  

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii167-ii167
Author(s):  
Radwa Sharaf ◽  
Dean C Pavlick ◽  
Garrett M Frampton ◽  
Maureen Cooper ◽  
Jacqueline Jenkins ◽  
...  

Abstract BACKGROUND Genomic profiling of gliomas is vital to ensure diagnostic accuracy, inform prognosis, and identify therapeutic options for primary and recurrent tumors. The integration of genomic biomarkers into brain tumor classification has improved the diagnostic accuracy and led to the development of molecularly stratified clinical trials. DESIGN Comprehensive genomic profiling (CGP) was performed on FFPE material from 310 (162 FoundationOne® and 148 FoundationOne® CDx) samples of brain tumors with available 1p19q FISH results, initially diagnosed by submitting institutions based on histology. Via CGP, we analyzed tumors in up to 395 cancer-associated genes (including IDH1/2) and predicted 1p19q codeletion using a custom research-use only algorithm. Progression-free (PFS) and overall survival (OS) were determined for 519 patients based on computationally predicted 1p19q codeletion status. RESULTS For all samples, regardless of their IDH1/2 mutation status, concordance between 1p19q status based on FISH and our algorithm was 97.1%, (301/310), with a positive predictive value (PPV) of 100% (133/133) and sensitivity of 93.7% (133/142). All discordant samples were called as positive for codeletion by FISH and negative by our CGP-based algorithm. Discordant samples were either IDH1/2 wild-type (2) or IDH1/2, ATRX, and TP53 altered (7), consistent with the genomic profile of diffuse astrocytomas. For IDH1/2-mutated samples, concordance was 96.7% (238/246). In the clinical outcomes dataset, median PFS was 35 months for the codeletion group compared to 13 months for the non-codeletion group (hazard ratio (HR): 0.60; 95% CI: 0.40-0.88; p=0.009). Similarly, median OS was 160 and 34 months respectively for codeleted versus intact (HR: 0.46; 95% CI: 0.28-0.76; p=0.002). CONCLUSIONS 1p19q codeletion status derived from CGP is highly concordant with FISH results suggesting that CGP-derived 1p19q codeletion status may be a reliable substitute for traditional FISH testing. Patients with CGP-derived 1p19q codeletion showed increased PFS and OS compared to non-codeleted counterparts.


Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4637-4637
Author(s):  
Konstantinos Lilakos ◽  
Maria K. Angelopoulou ◽  
George Georgiou ◽  
Vassilios Salpeas ◽  
Penelope Korkolopoulou ◽  
...  

Abstract BACKGROUND: FFPE tissues are widely used as a source of genomic DNA (gDNA), as well as for immunohistochemistry. RNA amplification and its usage for gene expression studies from archived FFPE material has been hampered due to RNA degradation. AIM: The description of a protocol for RNA extraction from archived FFPE tissues followed by reverse transcription (RT) and real time PCR and its implementation in the study of reference genes. METHODS: RNA was extracted from single 15μm sections from 18 samples 30 months old and 6 samples 10 years old from patients with diffuse large B-cell lymphoma (DLBCL). The FFPE RNA Extraction kit (Roche) was used, followed by DNA digestion and run on an Agilent2100 Bioanalyzer. RT was performed using the Transcriptor System (Roche) with modified temperatures followed by real time PCR for the c-abl and Survivin genes using the Universal Library probes and primers (Roche). In addition for the 10-year old samples, beta2 microglobulin (b2m) and G6PDH were also amplified. RESULTS: RNA that was yielded, was around 250 bp. c-abl was successfully amplified in all 18 samples up to 30 months old with a median cycle (Ct) number of 33.74. All 3 control genes were successfully amplified from the 10-year old samples with a median Ct number of 30.3 for G6PDH, 23.4 for b2m and 28.6 for c-abl. To further evaluate the amplification potency we successfully amplified Survivin transcripts with a median Ct number of 35.2. Survivin/abl ratio was calculated at a median of 0.5 for all DLBCL samples. gDNA interferenence was excluded, since no amplification signal was observed when gDNA was used as a template. The special RNA extraction methods, elevated temperatures for RT, as well as the use of the Universal Library probes, which are specifically designed to be short (8–9bp) and hybridize to a short amplicon (72bp for c-abl and 97bp for Survivin) were the major novelties of this method. Conclusions: Specialized RNA extraction methods and the use of Universal Library Probes can succesfully lead to several target gene amplification from archived FFPE material and potentially provide templates for gene expression studies.


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