Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

Search BRD Papers

Detection of fusion events by RNA sequencing in FFPE versus freshly frozen colorectal cancer tissue samples.

Author(s): Sorokin M, Lyadov V, Suntsova M, Garipov M, Semenova A, Popova N, Guguchkin E, Heydarov R, Zolotovskaia M, Zhao X, Yan Q, Wang Y, Karpulevich E, Buzdin A

Publication: Front Mol Biosci, 2024, Vol. 11, Page 1448792

PubMed ID: 39906487 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the detection of fusions between case-matched formalin-fixed, paraffin-embedded (FFPE) and frozen RNAlater-preserved colorectal cancer specimens by next-generation RNA sequencing (NGS). The presence of a single fusion was verified by PCR followed by Sanger sequencing. Tumor specimens were collected from 29 patients (age 18-75; 17 men and 12 women) with colorectal carcinoma. Case-matched specimens were immediately placed in RNAlater and stored at -70°C or fixed in formalin for 16 h and embedded in paraffin (no further details provided). RNA was extracted using the QIAGEN RNeasy Kit, and libraries were constructed using the KAPA RNA Hyper with rRNA Erase for ribosomal depletion. Libraries were pair-end sequenced with the Genolab M Engine. The files were processed using the STAR aligner in GeneCounts mode. For gene expression clustering and PCA, data were quantile normalized. Fusions were detected using STAR-Fusion software and included if they had a JunctionReadCount or a SpanningFragCount > 1. Statistical analysis and PCA were conducted using R.

   Summary of Findings:

   FFPE specimens had a longer median insert size (186 bp versus 206 bp, P=3.9 x 10^-7) and a higher number of uniquely mapped reads (5.4x 10^-8) than to the matched frozen RNAlater preserved specimen, but a comparable number of fusions (P=0.2). A total of 113 fusions were detected, with 69 of the fusions located in protein-coding genes and 44 in non-coding RNAs. Of the identified fusions, KANSL1-ARL17A/B was the most common (present in 20 patients, 69%) followed by MACC1-AC005062.1 (7 patients, 24%), LEPROT-LEPR (5 patients, 17%), SMG1-NPIPB13 (3 patients, 10%), and AL353138.1-PTCHD4 (3 patients, 10%), with the remaining fusions detected in specimens from only 1 or 2 patients. In 17 of the 29 patients evaluated, one or more fusions were detected in both the FFPE and the frozen RNAlater specimen, but detection of all fusions in both specimen types only occurred in one patient.  More fusions were detected in the frozen RNAlater specimen than the FFPE specimen in 13 patients, while more fusions were detected in the FFPE specimen than the frozen RNAlater specimen in 10 patients. The number of fusions detected was not correlated with the number of uniquely mapped reads in either frozen RNAlater or FFPE specimens. In principal component analysis based on the gene expression profile, the frozen RNAlater and FFPE specimens clustered separately; but in bootstrap dendograms of pairwise distances, clustering was primarily by patient source and not preservation method. A novel fusion of the ALK gene and LRRFIP2 was detected in the frozen RNAlater-preserved specimen but not the matched FFPE specimen from one patient using NGS; PCR followed by Sanger sequencing validated the presence of this fusion in both specimen types. Further, ALK gene exon coverage was also increased from exon 20 onward in both FFPE and frozen RNAlater specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin
   • RNAlater

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Next generation sequencing
   RNA	DNA sequencing

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Formalin (buffered) Frozen
   Next generation sequencing Specific	Technology platform	PCR followed by Sanger sequencing NGS

   View more