Tumor mutational burden (TMB) is a measure of the number of somatic mutations per megabase of the genome in a tumor, and it has emerged as a useful biomarker for predicting response to immunotherapy. TMB can be measured using various sequencing technologies, including whole-exome sequencing (WES) and targeted gene panel sequencing. However, the use of formalin-fixed paraffin-embedded (FFPE) samples for TMB measurement in multiple myeloma (MM) poses certain limitations that must be taken into account.
FFPE samples are a common source of clinical specimens and are widely used for diagnostic and research purposes. However, the fixation process can introduce artefacts that can affect the quality and quantity of nucleic acids, including DNA, in the sample. FFPE samples are also known to have a high degree of fragmentation and DNA damage, which can affect the accuracy of TMB measurement.
One of the main limitations of using FFPE samples for TMB measurement in MM is the low tumor purity. MM is a bone marrow-based malignancy, and the tumor cells are often mixed with non-tumor cells, such as stromal cells, immune cells, and hematopoietic cells. In FFPE samples, the tumor cells are often poorly preserved, and the non-tumor cells can dominate the DNA content, leading to a lower tumor purity. This can result in an underestimation of the TMB, as the non-tumor cells will dilute the number of somatic mutations detected.
Another limitation of using FFPE samples for TMB measurement in MM is the low DNA yield and quality. FFPE samples are often associated with low DNA yield and degraded DNA due to the fixation process. This can result in a low coverage depth and uneven coverage across the genome, which can affect the accuracy of TMB measurement. Additionally, FFPE samples are prone to DNA fragmentation, which can lead to the loss of larger genomic regions and the introduction of sequencing errors.
The sequencing technology used for TMB measurement can also affect the accuracy of the results obtained from FFPE samples. WES is a powerful tool for TMB measurement, as it surveys the entire exome and can detect a large number of somatic mutations. However, WES requires a high DNA input and can be challenging to perform on FFPE samples due to the low DNA yield and quality. Targeted gene panel sequencing is an alternative approach that can be used for TMB measurement in FFPE samples, as it focuses on a smaller set of genes and requires a lower DNA input. However, the coverage depth and uniformity of the panel can affect the accuracy of TMB measurement, particularly in regions of low coverage.
Finally, the choice of bioinformatics pipeline used for TMB measurement can also affect the accuracy of the results obtained from FFPE samples. Different pipelines use different algorithms and thresholds for defining somatic mutations and calculating TMB, and the choice of pipeline can affect the sensitivity and specificity of TMB measurement. Additionally, FFPE samples may require specific preprocessing steps, such as removal of artefacts and correction for DNA damage, to improve the accuracy of TMB measurement.
In conclusion, the use of FFPE samples for TMB measurement in MM poses certain limitations that must be taken into account. Low tumor purity, low DNA yield and quality, the sequencing technology used, and the choice of bioinformatics pipeline can all affect the accuracy of TMB measurement. Despite these limitations, TMB measurement in FFPE samples remains a valuable tool for predicting response to immunotherapy in MM and other cancers, and ongoing efforts are focused on improving the accuracy and reliability of TMB measurement in FFPE samples.