Wholistic approach: Transcriptomic analysis and beyond using archival material for molecular diagnosis

Abstract

Many neoplasms remain unclassified after histopathological examination, which requires further molecular analysis. To this regard, mesenchymal neoplasms are particularly challenging due to the combination of their rarity and the large number of subtypes, and many entities still lack robust diagnostic hallmarks. RNA transcriptomic profiles have proven to be a reliable basis for the classification of previously unclassified tumors and notably for mesenchymal neoplasms. Using exome-based RNA capture sequencing on more than 5000 samples of archival material (formalin-fixed, paraffin-embedded), the combination of expression profiles analyzes (including several clustering methods), fusion genes, and small nucleotide variations has been developed at the Centre Léon Bérard (CLB) in Lyon for the molecular diagnosis of challenging neoplasms and the discovery of new entities. The molecular basis of the technique, the protocol, and the bioinformatics algorithms used are described herein, as well as its advantages and limitations.

A subset of lung adenofibromas are morphological variants of solitary fibrous tumour

“…Other mutations included non?synonymous single?nucleotide variants of ACVR1 , PBRM1 , and APC , or affected the splicing of TACC1 , ACTG1 , or FGFR1 (Table?1). Unsupervised clustering of expression profiles revealed that both components matched perfectly together and within the group of SFTs 4 (Figure?1). Among cancer genes of interest, STAT6 , FGFR1 and AKT2 were highly expressed in both components.…”

“…Whole?exome RNA sequencing (TrueSeq RNA Exome; Illumina, Evry, France) on separately macrodissected components confirmed the NAB2 :: STAT6 fusion in the AF and SFT areas 4 . AF showed a frameshift mutation of FGFR2 (p.E768fs), which was lacking in SFT.…”

“…Whole-exome RNA sequencing (TrueSeq RNA Exome; Illumina, Evry, France) on separately macrodissected components confirmed the NAB2:: STAT6 fusion in the AF and SFT areas. 4 AF showed a frameshift mutation of FGFR2 (p.E768fs), which was lacking in SFT. Other mutations included non-synonymous single-nucleotide variants of ACVR1, PBRM1, and APC, or affected the splicing of TACC1, ACTG1, or FGFR1 (Table 1).…”

Cutaneous Melanocytic Tumor With CRTC1::TRIM11 Translocation

“…The number of duplicate reads was assessed using PICARD tools. The fusion transcripts were called by 5 different algorithms as previously described 12…”

“…The fusion transcripts were called by 5 different algorithms as previously described. 12 Fluorescence In Situ Hybridization Fluorescence in situ hybridization (FISH) was performed on 4-μm formalin-fixed paraffin-embedded sections using the ZytoLight FISH Tissue Implementation Kit (Zytovision #Z-2028-20, Bremerhaven, Germany). The TRIM11 break-apart probe was obtained from Empire Genomics (# TRIM11BA-20-GROR; Amplitech, Compiègne, France).…”