Laboratory Assessment of Patients with Systemic Mastocytosis

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Abstract

Systemic mastocytosis (SM) is a rare disease that can be challenging to diagnose. Difficulties arise because the fraction of neoplastic cells carrying the acquired activating pathogenic variant KIT p.D816V can be low, necessitating the use of sensitive assays for detection of both KIT mutation and aberrant expression of CD25 on MCs using flow cytometry (FC) and immunohistochemistry (IHC). Testing methodology has been improved over time, but challenges remain. In FY 2023, in collaboration with Dr. Boggs, we investigated 15 cases in which resolution of discrepant laboratory results was needed to establish a diagnosis. We retrospectively identified patients from a MC registry who underwent evaluation for SM using 2017 WHO criteria and tryptase genotyping for hereditary- tryptasemia (HT) and met at least 1 of the following 4 criteria: (1) 2 methods of KIT p.D816V testing had discordant results, (2) basal serum tryptase (BST) values were variably >20 ng/mL, (3) there were discordant results of MC CD25 expression assessed using FC and IHC, or (4) there were <25% spindled MCs or <2 MC aggregates in patients meeting other diagnostic criteria. 15/183 patients from the MC registry had discordant results in 1WHO criteria. 5 patients were women and 10 were men. Ages ranged from 26-68 years. Twelve patients had indolent SM, 1 had SM with an associated hematologic neoplasm, 1 had HT, 1 had well-differentiated SM (WDSM). 12/15 patients had discordant KIT p.D816V results, 4 discordant BST results, 5 discordant CD25 MC expression comparing IHC to FC or IHC between laboratories, 2 had discordance when assessing MC spindling, and 3 had discordance when assessing MC aggregates. Nine of the 15 patients required repeat testing and a second hematopathology evaluation to be diagnosed with SM. 13/15 patients were positive for the KIT p.D816V mutation; 12/13 patients had discordance among different tests. Sequencing methods including Sanger sequencing in 7 patients and next generation sequencing (NGS) in 10 patients did not detect KIT p.D816V. In contrast, polymerase chain reaction (PCR) methods including qualitative allelespecific oligonucleotide PCR (asoPCR) from one laboratory and quantitative PCR (qPCR) from 3 additional laboratories (qPCR1, qPCR2, and qPCR3) detected KIT p.D816V but 7 patients had discordance among PCR results performed using different methods. 6/7 patients with discordant PCR results had SM with a variant allele frequency near the lower limit of detection. There was no evidence of SM disease progression or remission in any patient which might account for the discordant test results. These results demonstrate challenges in detecting KIT p.D816V when the fraction of cells with the mutation approaches the assay sensitivity limit. One patient with a positive result failed to meet the 2017 WHO criteria for SM when reviewed by two hematopathologists. This patient also had no history of anaphylaxis and had a diagnosis of HT. This patient had KIT p.D816V originally detected using qPCR2 but not via repeat qPCR1 sample 58 days later, suggesting a false positive original qPCR2 result. 8 patients had BST values consistently below 20 ng/mL. 5 patients had BST values that over time were neither consistently above nor below 20 ng/mL. A tryptase level >20 ng/mL did not contribute to the diagnosis of SM in any of the 14 patients because all met other minor WHO diagnostic criteria regardless of their BST values. These data support prior reports that sampling multiple timepoints may be needed to assess the dynamic BST range. However, in all 14 SM cases, BST did not provide value within the diagnostic framework. 5 patients had discordant results for MC CD25 IHC when compared among different laboratories. 4 patients had detectable KIT p.D816V, but negative CD25 MC expression, raising concern for errors based on reported concordance of KIT p.D816V with positive MC CD25 expression. In 3 patients, FC showed CD25 expression, but IHC did not. In two of these patients, CD25 IHC on the core was positive on re-staining in a different laboratory. In third patient, CD25 IHC on the marrow clot but not on biopsy core was positive on re-staining, suggesting that the decalcification procedure contributed to a false-negative result. Finally, in patient diagnosed with KIT p.D816V-negative WDSM, FC did not show surface MC CD25 expression but CD25 was positive in cytoplasm using IHC. Overall, KIT p.D816V positive SM with negative CD25 results were shown to be erroneous. Pathology reports for three patients noted absence of MC spindling. One patient had WDSM with round, mature appearing MCs. After second hematopathology re-examination, the other two patients showed >25% spindled MCs in both original and recut marrow sections. In addition, 3/10 patients without originally reported MC aggregates were found to have them on re-examination. These data highlight several pitfalls in SM WHO criteria interpretation. First, assay sensitivity and specificity affect detection of KIT p.D816V. NGS and Sanger sequencing lacked sensitivity compared to PCR methods. Relative to this issue, the recently published International Consensus Classification of myeloid neoplasms and acute leukemias revised SM criteria recommends high-sensitivity PCR testing to avoid false-negative results. Considering PCR, we further found 6 patients with SM with discordant PCR results with variant allele frequencies near the lower limit of detection. Therefore, these findings support this need for high-sensitivity PCR for improvement in KIT p.D816V detection to both aid in diagnosis and predict treatment responses to KIT p.D816V selective tyrosine kinase inhibitors. Until new methods are developed and available, multiple timepoints and qPCR methods should be assessed in blood and marrow samples if the initial measurement is negative and there is a high pretest probability of disease. In summary, SM evaluations would benefit from improved and standardized KIT p.D816V assays, potential elimination of BST from SM diagnostic criteria, and consultations at a reference center where there is hematopathology expertise in this rare disease. A separate study was conducted in collaboration with Dr. Lyons to investigate whether genetically defined individual reference ranges for tryptase can limit unnecessary procedures and unmask myeloid neoplasms. Serum tryptase is a biomarker used to aid in the identification of certain myeloid neoplasms, most notably systemic mastocytosis, where basal serum tryptase (BST) levels >20 ng/mL are a minor criterion for diagnosis. Although clonal myeloid neoplasms are rare, the common cause for elevated BST levels is the genetic trait hereditary -tryptasemia (HT) caused by increased germline TPSAB1 copy number. To date, the precise structural variation and mechanism(s) underlying elevated BST in HT and the general clinical utility of tryptase genotyping, remain undefined. Through cloning, long-read sequencing, and assembling of the human tryptase locus from an individual with HT, and validating our findings in vitro and in silico, the study demonstrated that BST elevations arise from overexpression of replicated TPSAB1 loci encoding canonical -tryptase protein owing to coinheritance of a linked overactive promoter element. Modeling BST levels based on TPSAB1 replication number, study generated new individualized clinical reference values for the upper limit of normal. Using this personalized approach, the results demonstrated the clinical utility of tryptase genotyping, finding that in the absence of HT, BST levels >11.4 ng/mL frequently identify indolent clonal mast cell disease. Moreover, substantial BST elevations (eg, >100 ng/mL), which would ordinarily prompt bone marrow biopsy, can result from TPSAB1 replications alone and thus be within normal limits for certain individuals with HT.