scholarly journals An Optimized Protocol for total RNA isolation from archived formalin-fixed paraffin-embedded tissues to identify the long non-coding RNA in Oral Squamous cell carcinomas

MethodsX ◽  
2021 ◽  
pp. 101602
Author(s):  
Dr. Kiran Kumar ◽  
Dr. Ajay Oli ◽  
Dr. Kaveri Hallikeri ◽  
Dr. Shilpasree AS ◽  
Mr. Mallikarjun Goni
Keyword(s):  
Squamous Cell ◽  
Rna Isolation ◽  
Squamous Cell Carcinomas ◽  
Non Coding Rna ◽  
Formalin Fixed Paraffin ◽  
Optimized Protocol ◽  
Formalin Fixed Paraffin Embedded ◽  
Long Non Coding Rna ◽  
Formalin Fixed ◽  
Total Rna Isolation

Combined expression of bcl-2, bcl-6, HIF, HGF, c-kit, neu/cerb2 and VEGF-C and their clinical significance in laryngeal squamous cell carcinoma

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 20079-20079
Author(s):  
I. O. Kara ◽  
A. Uguz ◽  
B. Sahin ◽  
E. Kilic-Bagir ◽  
M. Erkisi
Keyword(s):  
Clinical Significance ◽  
Squamous Cell ◽  
Tumor Grade ◽  
Squamous Cell Carcinomas ◽  
Regional Lymph Nodes ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Postoperative Irradiation ◽  
Predicting Outcome ◽  
Formalin Fixed

20079 Background: Proteins regulating the cell cycle and cell death are frequently abnormally expressed in cancer. Several of these, particularly Bcl-2, Bcl-6, neu/cerb2 have been widely suggested as possible prognostic markers in diverse human malignancies. Their role in predicting outcome in squamous cell carcinomas of the head and neck is unclear. In addition c-kit protein has been shown in tumorogenesis. Because HNSCC preferentially metastasizes to regional lymph nodes, we investigated the expression of bcl-2, bcl-6, HIF, HGF, c-kit, neu/cerb2 and VEGF-C and their clinical significance in laryngeal squamous cell carcinomas by semiquantitative immunohistochemistry. Methods: We studied 115 patients with stage I o III tumors, all were treated with surgery ± postoperative irradiation/chemotherapy by a single institute. We studied the patients retrospectively to test the association between expression of Bcl-2, bcl-6, HIF, HGF, c-kit, neu/cerb2 and VEGF-C, as assessed by immunohistochemistry in formalin-fixed paraffin-embedded tissue and evaluated by two pathologist. We scored the expression of the proteins from negativ to severe expression. In addition we evaluated the degree of tumor grade, necrosis and also inflamation, respectively. Results: Within 115 patients we found severe expression of bcl-2 in 2 (1%), bcl-6 in 16 (13%), HIF in 57 (49%), HGF in 38 (33%), c-kit in 4 (3%), neu/cerb2 in 17 (14%) and VEGF-C in 11 (9%) patients, respectively. We found significant correlation between bcl-2 and necrosis (p = 0.003), HGF and inflamation (p = 0.05), c-kit and necrosis (p = 0.04), c-kit and tumor grade (p = 0.03) respectively. We found that the significant relation between bcl-6 and HIF (p = 0.000), bcl-6 and HGF (p = 0.02), bcl-6 and c-kit (p = 0.005), bcl-6 and VEGF-C (p = 0.000), HGF and VEGF-C (p = 0.005) respectively. Also we found that the significant correlation between tumor grade and HGF (p = 0.05). Conclusion: These data indicate that assessing expression of bcl-2 or bcl-6, c-kit and HGF is unlikely to be prognostically useful for surgically treated laryngeal carcinoma. No significant financial relationships to disclose.

scholarly journals Multiclass cancer classification in fresh frozen and formalin-fixed paraffin-embedded tissue by DigiWest multiplex protein analysis

2020 ◽  
Vol 100 (10) ◽  
pp. 1288-1299
Author(s):  
Teresa Bockmayr ◽  
Gerrit Erdmann ◽  
Denise Treue ◽  
Philipp Jurmeister ◽  
Julia Schneider ◽  
...  
Keyword(s):  
Squamous Cell ◽  
Squamous Cell Carcinomas ◽  
Cancer Classification ◽  
Protein Profiling ◽  
Formalin Fixed Paraffin ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Fresh Frozen ◽  
Formalin Fixed

Abstract Histomorphology and immunohistochemistry are the most common ways of cancer classification in routine cancer diagnostics, but often reach their limits in determining the organ origin in metastasis. These cancers of unknown primary, which are mostly adenocarcinomas or squamous cell carcinomas, therefore require more sophisticated methodologies of classification. Here, we report a multiplex protein profiling-based approach for the classification of fresh frozen and formalin-fixed paraffin-embedded (FFPE) cancer tissue samples using the digital western blot technique DigiWest. A DigiWest-compatible FFPE extraction protocol was developed, and a total of 634 antibodies were tested in an initial set of 16 FFPE samples covering tumors from different origins. Of the 303 detected antibodies, 102 yielded significant correlation of signals in 25 pairs of fresh frozen and FFPE primary tumor samples, including head and neck squamous cell carcinomas (HNSC), lung squamous cell carcinomas (LUSC), lung adenocarcinomas (LUAD), colorectal adenocarcinomas (COAD), and pancreatic adenocarcinomas (PAAD). For this signature of 102 analytes (covering 88 total proteins and 14 phosphoproteins), a support vector machine (SVM) algorithm was developed. This allowed for the classification of the tissue of origin for all five tumor types studied here with high overall accuracies in both fresh frozen (90.4%) and FFPE (77.6%) samples. In addition, the SVM classifier reached an overall accuracy of 88% in an independent validation cohort of 25 FFPE tumor samples. Our results indicate that DigiWest-based protein profiling represents a valuable method for cancer classification, yielding conclusive and decisive data not only from fresh frozen specimens but also FFPE samples, thus making this approach attractive for routine clinical applications.

scholarly journals Maximizing RNA Yield from Archival Renal Tumors and Optimizing Gene Expression Analysis

2009 ◽  
Vol 15 (1) ◽  
pp. 80-85 ◽  
Author(s):  
Sean T. Glenn ◽  
Karen L. Head ◽  
Bin T. Teh ◽  
Kenneth W. Gross ◽  
Hyung L. Kim
Keyword(s):  
Gene Expression ◽  
Expression Analysis ◽  
Reverse Transcription ◽  
Gene Expression Analysis ◽  
Rna Isolation ◽  
Proteinase K ◽  
Specific Primers ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan ® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen’s TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers’ protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan ® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription.

scholarly journals Label-Free Semiquantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics Analysis of Laryngeal/Hypopharyngeal Squamous Cell Carcinoma on Formalin-Fixed, Paraffin-Embedded Tissue Samples - a Pilot Study

2020 ◽  
Vol 26 (4) ◽  
pp. 2801-2807
Author(s):  
Andras Burian ◽  
Laszlo Lujber ◽  
Imre Gerlinger ◽  
Tamas Jarai ◽  
Eva Orosz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Squamous Cell Carcinoma ◽  
Liquid Chromatography ◽  
Cell Carcinoma ◽  
Tumor Markers ◽  
Squamous Cell ◽  
Label Free ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Formalin Fixed

Abstract Squamous cell carcinoma (SCC) of the head and neck region is the sixth most frequent malignancy with high mortality rate. Due to its poor prognosis it is considered a growing public health problem worldwide inspite of existing treatment modalities. Thus, early diagnosis of new diseases and recurrences is emerging on one hand, but on the other hand troublesome in the lack of reliable tumor markers in this field. The rapid development of proteomics has opened new perspectives in tumor marker discovery. Liquid chromatography/mass spectrometry (LC/MS) as the gold standard in proteomics enables the semi-quantitative analysis of proteins within various tissues. Abundance differences between tumor and normal tissue also can be interpreted as tumor specific changes. The aim of this study was to identify potential tumor markers of laryngeal/hypopharyngeal SCC by revealing abundance changes between cancerous and the surrounding phenotypically healthy tissue. After separating the phenotypically cancerous and healthy parts of formalin-fixed paraffin-embedded tissues, each sample underwent protein recovery process and tryptic digestion for label-free semi-quantitative LC/MS analysis. Eight proteins showed significantly higher abundance in tumor including tenascin, transmembrane emp24 domain-containing protein 2, cytoplasmic dynein light chain 1, coactosin-like protein, small proline-rich protein 2D, nucleolin, U5 small nuclear RNP 200-kDa helicase and fatty aldehyde dehydrogenase. Desmoglein-1 and keratin type I cytoskeletal 9 were down-regulated in tumor. Using Ingenuity Pathway Analysis we mapped the signaling pathways these proteins play role in regarding other tumors. Based on these findings these proteins may serve as promising biomarkers in the fight against laryngeal/hypopharyngeal SCCs.

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