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Comparison of the miRNA expression profiles in fresh frozen and formalin-fixed paraffin-embedded tonsillar tumors.

Author(s): Vojtechova Z, Zavadil J, Klozar J, Grega M, Tachezy R

Publication: PLoS One, 2017, Vol. 12, Page e0179645

PubMed ID: 28644855 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the miRNA expression profile in matched frozen and FFPE specimens from 10 tonsilar tumors and 5 non-malignant tonsil specimens using TLDA arrays and two different normalization methods. miRNA expression differences between tumor and non-tumor specimens and between tumor specimens based on known HPV status were further investigated in a second cohort by real-time PCR. Five of the tumor specimens were previously shown to be HPV positive while the other five were known to be HPV negative. Details of specimen processing were not included. Frozen sections were cut on a cryostat and the percentage of tumor cells were determined (>50% for all specimens). RNA was extracted from frozen specimens using the miRVana kit and from four macrodissected 10 µm sections of FFPE specimens using Ambion RecoverAll Total Nucleic Acid Isolation kit for FFPE. RNA yield was determined by spectrophotometer and chip electrophoresis. miRNA expression was investigated using the TaqMan Array Human MicroRNA A+B Cards Set v3.0. Differences in expression of 11 selected miRNAs were confirmed by individual TaqMan real-time PCR in the initial cohort and a larger cohort of 64 FFPE tonsil specimens for which no details of handling were provided.

   Summary of Findings:

   As expected, the RIN for frozen specimens were >7 but only 1.9-2.6 for the matched FFPE specimens. Principal component analysis showed that specimens clustered by preservation method and tumor status. The correlation in expression between matched FFPE and frozen specimens was modest to strong (R=0.4642-0.7361), but consideration of only the miRNAs on card A slightly increased the strength of the correlations (R=0.5194-0.8302). There was no apparent effect of tumor percentage on the level of correlation.

   Using a threshold of >1.33-fold change and P<0.05 with the 50th percentile shift normalization method, 83 miRNAs were differentially expressed between frozen tumor and non-tumor specimens and 58 were differentially expressed between tumor and non-tumor FFPE specimens. However, only 27-38% overlap was found between the lists.  When global normalization was also applied, 72 miRNAs were differentially expressed between frozen tumor and non-tumor specimens and 57 were differentially expressed between tumor and non-tumor FFPE specimens with 28-37% overlap. The overlap between lists generated by the two different normalization methods was 58-67% for frozen specimens and 62-63% for FFPE specimens.  

   In the same 15 specimens, real-time PCR confirmed the TLDA findings of higher expression of three miRNAs in tonsillar tumors compared to non-tumor specimens and higher expression of two miRNAs and lower expression of six miRNAs in HPV-positive than HPV-negative tumors. Real-time PCR analysis of 64 additional FFPE specimens confirmed increased expression of miR-155 and miR-126 in tonsillar tumor specimens compared to non-tumor tissue, but contrary to the results in the initial cohort, expression of miR-205 was found to be non-significantly higher in non-tumor specimens in the larger cohort. Similarly, only three of six miRNAs found to be expressed at higher levels in HPV-negative tumors in the initial study were also found to have higher expression in HPV-negative tumors in the larger cohort by real-time PCR (miR-16 and miR-34a significantly and miR-193b not significantly) with miR-31, miR-221, and miR-21 having non-significantly lower expression in HPV-negative tumors in the larger cohort. The initial study and the confirmatory study both found higher expression of miR-106b and miR-9 in HPV-positive tumors.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Frozen
   • Formalin

   Diagnoses:

   • Neoplastic - Not specified
   • Other diagnoses
   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   RNA	Low density array

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Frozen
   Preaquisition	Diagnosis/ patient condition	HPV-positive tonsillar tumor HPV-negative tonsillar tumor Non-tumor
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-106b miR-9 miR-16 miR-34a miR-193b miR-31 miR-221 miR-21 miR-155 miR-126 miR-205
   Low density array Specific	Technology platform	Real-time PCR
   Low density array Specific	Data handling	50'th percentile shift normalization only 50'th percentile shift normalization with global normalization

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