An improved and standardized KIT p.D816V PCR‐based assay performed at multiple time points might help establish the diagnosis of systemic mastocytosis (SM), as opposed to basal serum tryptase (BST) values and low-sensitivity sequencing methods, according to an article published in Blood Advances.
To evaluate the available SM testing methodologies and possible diagnostic difficulties, the researchers performed a retrospective mast cell registry review of patients who underwent evaluation for SM using 2017 World Health Organization (WHO) criteria and tryptase genotyping for hereditary‐alpha tryptasemia.
Of 183 cases, they identified 15 patients who met the criteria: 12 had discordant KIT p.D816V results, 4 had discordant BST results comparing different time points, 5 had discordant CD25 mast cell expression comparing immunohistochemistry to flow cytometry or immunohistochemistry between labs, 2 had discordance when assessing mast cell spindling, and 3 had discordance when assessing mast cell aggregates. Nine of the 15 patients had repeat testing and a second hematopathology evaluation to confirm their SM diagnosis.
The review identified 14 patients whose BST values did not provide value within the diagnostic framework. They were diagnosed with SM based on other minor WHO diagnostic criteria regardless of having BST values over 20 ng/mL. Furthermore, BST values were consistently below 20 ng/mL in 8 patients and above 20 ng/mL in 2 patients. In 5 other patients, they shifted below and above 20 ng/mL over time, highlighting the need for sampling at multiple time points.
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Sequencing methods, including Sanger in 7 patients and next-generation sequencing in 10 patients, were unable to detect KIT p.D816V. PCR methods such as qualitative allele‐specific oligonucleotide PCR from 1 lab and quantitative PCR from 3 additional labs successfully detected the pathogenic mutation, however, the PCR results obtained by different methods varied in 7 patients. There were 6 patients with SM with discordant PCR results that had variant allele frequency near the lower limit of detection, highlighting the need for high‐sensitivity PCR to improve KIT p.D816V detection.
“Interestingly, one patient with no history of anaphylaxis who tested positive for hereditary‐alpha tryptasemia with BST values less than 20 ng/mL, and no histopathological evidence of SM, tested positive for KIT p.D816V by one quantitative PCR test and negative by another. This may well be the first false positive result reported for any KIT p.D816V PCR‐based assay and shows that KIT p.D816V positive cases should always be re‐evaluated for the presence of other WHO minor criteria such as CD25 mast cells expression and spindling” Boggs and colleagues stated.
People with SM might have a low fraction of neoplastic mast cells and other hematopoietic cells carrying the acquired activating pathogenic variant KIT p.D816V. Therefore, sensitive assays are required for the detection of both KIT p.D816V and aberrant expression of CD25 on neoplastic mast cells by flow cytometry and immunohistochemistry.
Reference
Boggs NA, Sun X, Lyons JJ, et al. Challenges in applying diagnostic criteria for systemic mastocytosis. Blood Adv. Published online February 27, 2023. doi:10.1182/bloodadvances.2023009826
