.png){kind=icon}
12.1. Numerous sarcomas are associated with chromosomal translocation. Which diagnostic tools could you use to aid the precise diagnosis in these cases?
Chromosomal translocations, generating fusion transcripts, can be targeted using FISH – break apart or RT-PCT.
The use of FISH is provide fast and targeted information that can be read under a florescence microscope. However, the use of break-apart proves only provide an indirect evidence of a translocation. With the use of break-apart probes a gene rearrangement is detected and the results must be correlated with additional clinical, histopathological and immunohistochemical information before a definitive diagnosis is provided.
RT-PCR involves RNA extraction and therefore the sample needs optimal fixation and non-acid based decalcification if bone samples are used. The use of RNA and not genomic DNA is necessary as the majority of break-points occur in variable places within intronic sequenced making it very difficult to target on genomic DNA. The reverse transcription with generate a cDNA (complimentary DNA) composed of exomic sequences. The break-points can be reasonably easily detected when targeting the closest exonst involved. The use of RT-PCR will provide information on the specific fusion transcript present in a given disease/
12.2 Which techniques could you use to diagnose the main molecular alterations in sarcomas?
Chromosomal transclocations can be detected by RT-PCR and or FISH. NGC assays with specific pull-down design may also be used, The use of FISH is provide fast and targeted information that can be read under a florescence microscope. However, the use of break-apart proves only provide an indirect evidence of a translocation. With the use of break-apart probes a gene rearrangement is detected and the results must be correlated with additional clinical, histopathological and immunohistochemical information before a definitive diagnosis is provided. RT-PCR involves RNA extraction and therefore the sample needs optimal fixation and non-acid based decalcification if bone samples are used. The use of RNA and not genomic DNA is necessary as the majority of break-points occur in variable places within intronic sequenced making it very difficult to target on genomic DNA. The reverse transcription with generate a cDNA (complimentary DNA) composed of exomic sequences. The break-points can be reasonably easily detected when targeting the closest exonst involved. The use of RT-PCR will provide information on the specific fusion transcript present in a given disease.
Point mutations may be detected by PCR-based assays including direct sequencing, use of specific restriction enzyme digestion and digital PCR. Multiple NGS panels also include detection of common point mutations.
Gene amplification is usually detected by use of DNA labelled probes: FISH, CISH or DISH. In sarcomas, FISH is the preferred method. Atypical lipomatous tumours / well-differentiated liposarcomas may be dissificult to target using CISH.