The KIT D816V Allele Burden in Systemic Mastocytosis Is Strongly Associated with Disease Subtype.
Authors: Schwaab Juliana , Erben Philipp , Horny Hans-Peter , Teichmann Martina , Metzgeroth Georgia , Ernst Thomas , Sotlar Karl , Mueller Martin C , Marx Alexander , Hartmann Karin , Hochhaus Andreas , Cross Nicholas C.P. , Hofmann Wolf Karsten , Reiter Andreas 
Published: October 2019

Abstract
Abstract 2859
Point mutations in the kinase domain of the receptor tyrosine kinase KIT at position 816 (D816V >95%; D816H, D816Y, others <5%) play a central role in the pathogenesis, diagnosis and targeted treatment of systemic mastocytosis (SM). However, accurate numbers on the overall frequency of KIT D816V in the diverse SM subtypes remain largely unknown because of i) the rarity of disease, ii) highly variable mast cell burden in bone marrow (BM) and peripheral blood (PB) and iii) inadequate sensitivity of disparate assays. We therefore developed an allele-specific quantitative RT-PCR (RQ-PCR) with an enhanced sensitivity of 0.01–0.1% which was superior to D-HPLC (mutation detection rate: 0.5%-1%) or conventional sequencing (mutation detection rate: 10–20%). Classification of the patients into disease subtypes was performed according to the WHO 2008 classification. Irrespective of subtype, KIT D816 mutations were identified in 143/144 (99.3%) of patients (D816V, n=141, 98.6%; D816H, n=1; D816Y, n=1) with SM (indolent SM [ISM], n=62, 43.1%; smoldering SM [SSM], n=7, 4.9%; SM with associated hematological non-mast cell disease [SM-AHNMD], n=15, 10.4%, and aggressive SM/mast cell leukemia [ASM/MCL], n=59, 41%). As control groups, seven patients with cutaneous mastocytosis, 20 healthy individuals and 80 patients with chronic myeloid leukemia in molecular remission were negative for the KIT D816V mutation in BM and PB. All patients with SSM (n=7), SM-AHNMD (n=14) and ASM/MCL (n=56) were positive in BM and also in PB. In contrast, 25/51 (49%) ISM patients were positive in BM but negative in PB. The KIT D816V RQ-PCR was subsequently used for quantitative assessment of the KIT D816V allele burden, expressed as the ratio KIT D816V/total KIT, in a total of 207 samples from PB (n=126) and BM (n=81) of 141 KIT D816V positive patients. In BM, the median allele burden was 8.9% (range 1.1–48.3%) in ISM, 29.7% (range 13.6–39.4%, p=0.007) in SSM, 23.3% (range 1.8–45.8%, p=0.09) in SM-AHNMD and 30.7% (range 1–99%, p<0.0001) in ASM/MCL. In PB, the allele burden clearly allowed differentiation of ISM (median 0, range 0–15.2%) vs. SSM (median 8.8%, range 0.3–42.5%, p=0.001), ISM vs. SM-AHNMD (median 11.5%, range 0.2–64.9%, p<0.001) and ISM vs. ASM/MCL (median 33%, range 0.53–74.0%; p<0.001). The vast majority of ISM patients (47/51; 92%) had an allele burden in PB <10%. ISM patients with an allele burden ≥10% collectively showed overlapping symptoms of diarrhea with (n=2) or without (n=1) histologically confirmed bowel infiltration, mild cytopenia (n=1), splenomegaly (n=1) and/or abdominal lymphadenopathy (n=1) potentially indicating more advanced disease. The wide heterogeneity of the allele burden in SSM and SM-AHNMD patients reflects the clinical heterogeneity in these subtypes. Of interest, the correlation of clinical findings and allele burden also revealed that patients with ASM-CMML had a significantly higher allele burden than ASM patients without monocytosis (45.5% vs. 30.9%, p=0.0029). In order to evaluate the use of RQ-PCR for monitoring of residual disease, serial measurements of the allele burden in PB were performed in patients after chemotherapy with cladribine (n=1) or allogeneic stem cell transplantation (SCT, n=2). The patient on cladribine revealed a decrease from 42% to 5% within 9 months which was associated with a decrease of serum tryptase levels from 610 μg/l to 129 μg/l and a decrease of mast cells in BM from 70–80% to 10%. When the patient relapsed 18 months after completion of chemotherapy with progressive organomegaly and thrombocytopenia, the allele burden had also risen to 35%. In the two allografted patients, allele burden was 35% and 45%, respectively, prior to SCT. Both patients are currently in complete remission without detectable KIT D816V mutation 17 and 12 months after allogeneic SCT. We therefore conclude that the measurement of the KIT D816V allele burden in SM patients from cDNA is a useful complementary tool for diagnosis and subclassification of SM. In the new treatment era of successful use of tyrosine kinase inhibitors and allogeneic SCT, it may also become very useful for monitoring of response to treatment in addition to established parameters like C-findings, serum tryptase and BM histology.

Disclosures:
No relevant conflicts of interest to declare.