KIT D816V Allele Research Articles

KIT D816V Mutation Burden Predicts Prognosis and Survival In Patients With Mastocytosis and Correlates With The WHO Type Of The Disease

Mastocytosis is characterized by abnormal expansion and accumulation of neoplastic mast cells in one or more organ systems. Traditionally, mastocytosis is divided into cutaneous mastocytosis (CM) and systemic mastocytosis (SM). In most patients with SM, the transforming somatic mutation KIT D816V is detected. However, only few studies have quantified the KIT D816V allele burden in CM and SM. The aim of the present study was to quantify KIT D816V in various forms of mastocytosis and to correlate the allele burden of KIT D816V with the disease category, serum tryptase levels and clinical outcomes. KIT D816V was quantified in bone marrow (BM) and peripheral blood (PB) cells by a real-time PCR (qPCR) assay based on allele-specific primers. In addition, BM and PB cells were also examined for the presence of KIT D816V by melting curve analysis after PCR clamping in all patients. Overall, 225 DNA samples (BM, n=112; PB, n=113) from 107 patients with mastocytosis (females: n=57; males, n=50; median age 49 years; range 18-73 years) were analyzed. Based on WHO criteria, 14 patients had CM, 3 the provisional diagnosis of mastocytosis in the skin (MIS), 66 indolent SM (ISM), 6 smouldering SM (SSM), 7 aggressive SM (ASM), one mast cell leukemia (MCL) and 10 patients SM with an associated hematologic non-mast cell lineage disorder (SM-AHNMD). KIT D816V was found in in 76/107 patients (71%) by melting curve analysis after PCR clamping, and in 92/107 patients (86%) by qPCR (p<0.005). In paired BM and PB samples of 43 patients an excellent correlation of the KIT D816V burden with almost identical results was found (r=0.98, p<0.001). When examining the KIT D816V allele burden in KIT D816V+ patients (n=92) in various categories of the disease, significant differences were found between CM (median KIT D816V allele fraction: 0.042%), MIS (median: 0.084%), ISM (median: 0.286%), SSM (median: 3.012%), ASM (median: 9.346%) and SM-AHNMD (median: 3.761%) (p<0.001). Moreover, we found that the KIT D816V allele burden correlates significantly with the serum tryptase level in our patients (r=0.50, p<0.005). Consecutive studies revealed that the KIT D816V allele fraction is of prognostic significance concerning survival as determined by Cox regression (p=0.015). As assessed by Kaplan Meier estimates and log rank testing, patients with a KIT D816V allele burden of ≥2% were found to have a significantly shorter survival than those with an allele burden of less than 2% (p=0.001) (Figure 1). Thirty patients were evaluated at diagnosis and during the follow up. In untreated patients with stable disease, the KIT D816V allele burden remained within a constant range. By contrast, in patients with disease progression, an increase in the KIT D816V burden over time was detectable. In patients responding to cytoreductive agents (cladribine n=4; hydroxyurea n=1) a significant decrease in the median KIT D816V allele burden (by 91.6%) after therapy compared to pre-therapeutic samples was observed (p=0.027). In summary, our data show that qPCR is a highly sensitive approach for the detection and quantification of KIT D816V in patients with mastocytosis and that the KIT D816V mutation burden differs significantly among patients in different WHO subtypes. Moreover, the KIT D816V allele burden correlates with serum tryptase levels and is of prognostic significance concerning survival in patients with mastocytosis. Finally, quantification of KIT D816V may serve as follow up parameter useful for determining the natural course and treatment responses in patients with mastocytosis. We recommend that the KIT D816V mutation burden is included as a novel parameter in daily practice and clinical trials in advanced SM. [Display omitted] Disclosures:Valent:Novartis: Consultancy, Honoraria, Research Funding.

The KIT D816V Allele Burden in Systemic Mastocytosis Is Strongly Associated with Disease Subtype.

AbstractAbstract 2859Point 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.

Improved Detection of the KIT D816V Mutation in Patients with Systemic Mastocytosis Using a Quantitative and Highly Sensitive Real-Time qPCR Assay

The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM.

Aggressive Systemic Mastocytosis Is Associated with a High Kit D816V Allele Burden in Peripheral Blood.

Abstract 2899Poster Board II-875According to 2008 WHO definitions, diagnosis of systemic mastocytosis (SM) is based on the presence of one major criteria (multifocal dense infiltrates of mast cells in bone marrow or organ biopsies) and at least one of the following minor criteria (i) >25% are atypical cells on bone marrow smears or are spindle-shaped in mast cell infiltrates of visceral organs, ii) KIT point mutation at codon 816 in the bone marrow or other extracutaneous organs, iii) mast cells express CD2 and/or CD25, iv) baseline serum tryptase concentration >20 ng/mL) or the presence of at least three minor criteria. Patients with aggressive SM (ASM) have to present with one or more C-findings (i) neutrophils <1,000/μL, Hb <10g/dL, or platelets <100,000/μL, ii) hepatomegaly with impaired liver function – elevated transaminases and/or bilirubin levels and/or hypoalbuminemia, iii) palpable splenomegaly with signs of hypersplenism, iv) malabsorption with significant hypoalbuminemia and/or significant loss. ASM and MCL are clearly associated with an inferior survival. The activating KIT D816V point mutation is thought to be pivotal for pathogenesis and potential targeted therapy with novel tyrosine kinase inhibitors. However, conventional sequencing (CS) of RNA/DNA extracted from bone marrow (BM) samples of SM patients reveals the mutation in less than 50-60% of patients while peripheral blood (PB) samples are frequently negative as consequence of low numbers of malignant cells and poor assay sensitivity. For better qualitative and additional quantitative assessment of the KIT D816V allele burden (expressed as ratio KIT D816V vs. KIT wildtype), we sought to establish (i) a D-HPLC (denaturing-high performance liquid chromatography) assay combined with direct sequencing in case of a positive D-HPLC signal and (ii) a LightCyclerTM based quantitative PCR (RQ-PCR). The assay sensitivities were calculated through serial cell (KIT D816V positive HMC-1 cells in NB4 cells) and RNA dilutions. The detection limit was estimated between 0.1 and 0.5% for both assays which was thus significantly improved in comparison to the detection limit of CS which was at 10–15%. Patient material was available from BM (n=134) and PB (n=93) from 173 patients (88 m, 85 f, median age 54 years) with diagnosis of SM including 10 patients with confirmed ASM (n=9) or aleukemic variant of MCL (n=1) according to WHO definitions. At diagnosis, D-HPLC, RQ-PCR and CS were positive in 78% (104/134), 84% (112/134) and 60% (80/134) of BM samples, respectively. Two patients had a KIT D816H and one patient had a KIT D816L mutation. In 54 cases with contemporaneously collected BM and PB samples, the KIT D816V mutation was found in PB of BM positive patients by D-HPLC, RQ-PCR and CS in 47% (22/46), 55% (27/49) and 38% (14/37) of cases, respectively. This information allowed the detection of the KIT D816V mutation in an additional 27 of 39 (69%) patients without available bone marrow biopsies. All ASM patients were KIT D816V positive in PB with a median KIT D816V allele burden of 36% (range 26–98%) vs. 7.8% (range 0.1–61%) in other stages of SM (p=0.0021). Eleven of 124 (8.9%) SM patients with a KIT D816V allele burden >20% are currently explored for the potential diagnosis of ASM or MCL. In conclusion, D-HPLC is a reliable and sensitive method for the screening of variable KIT mutations in BM and PB of SM patients and is clearly superior to CS. Application of RQ-PCR assays for the most common D816V mutation may overlook rare mutations but allows quantification of the KIT D816V allele burden which may be useful for diagnosis of ASM and monitoring of residual disease during treatment with novel tyrosine kinase inhibitors, e.g. midostaurin or dasatinib. Disclosures:No relevant conflicts of interest to declare.

Prognostically Relevant Breakdown of 138 Patients with Systemic Mastocytosis Associated with Another Hematological Malignancy

Background: Systemic mastocytosis (SM) sometimes coexists with an associated clonal hematological non mast cell lineage disease and such an occurrence is recognized by the 2001 World Health organization (WHO) classification system as SM-AHNMD (Leuk Res. 2001 25:603). However, SM-AHNMD has not been further stratified into prognostically relevant subgroups. This was the major objective of the current study, which also provides information on prevalence and relevance of detecting KITD816V and JAK2V617F mutations.Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those cases where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. SM- and AHNMD-components were classified according to WHO criteria.Results:AHNMD sub-classification: Of 342 consecutive cases of SM that were screened, 138 (40%) were identified as having SM-AHNMD (median age 65 years, range 20–87). The most frequent AHNMD was chronic myelomonocytic leukemia (SM-CMML) seen in 38 (28%) patients. 23 (17%) patients had SM associated with myelodysplastic syndrome (SM-MDS), 22 (16%) with hypereosinophilic syndrome/chronic eosinophilic leukemia (SM-HES/CEL) and 33 (24%) with another myeloproliferative neoplasm (SM-MPN). The number of cases with other SM-AHNMD was much lower and included lymphoma 8 (6%), acute leukemia 3 (2%), MDS/MPN 3 (2%), and 8 (6%) cases of multiple myeloma, primary amyloidosis or chronic lymphocytic leukemia. Of the 33 SM-MPN patients, 2 had polycythemia vera, 6 essential thrombocythemia, and 2 myelofibrosis; the remaining 23 patients had an unclassified MPN in association with SM.Clinical characteristics at presentation: The combined incidence of urticaria pigmentosa (UP) was 18%, mast cell mediator release symptoms 28% and constitutional symptoms 62%. 76 patients (57%) had palpable splenomegaly. 99 patients (72%) had anemia, 18 (13%) leukopenia (43% with SM-MDS; p<0.0001) and 76 (55%) thrombocytopenia. 70 (51%) patients had leukocytosis and 63 (55%) displayed circulating immature myeloid cells (78% with CMML; p=0.001). 59 (54%) patients had elevated serum alkaline phosphatase level: SM-CMML 70%, SM-MPN 60%, SM-HES/CEL 40%, and SM-MDS 30% (p=0.01). Where evaluated, serum tryptase was elevated in 92% of the patients (median=75 ng/mL; range=4–1360). BM blasts were ≥5% in 25 patients (22%), including 48% with SM-MDS (p=0.004). 12 of 35 patients tested carried the FIP1L1-PDGFRA mutation (all had SM-HES/CEL; p<0.0001).Disease course and prognostic factors At a median follow up of 15 months (range 0–182), 99 deaths were recorded and the combined median survival was 24 months: SM-CMML 15 months; SM-MDS 15 months, SM-MPN 30 months; and SM-HES/CEL 87 months (p=0.003). Fifteen patients (11%) transformed to acute leukemia or mast cell leukemia. Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and SM-AHNMD sub-type (p=0.007), presence of constitutional symptoms (p=0.04), thrombocytopenia (p=0.01), presence of circulating immature myeloid cells (p=0.006), and hypoalbuminemia (p<0.0001). In SM-CMML, hypoalbuminemia was associated with inferior survival (p=0.03).KITD816V andJAK2V617F analysis: Archived BM was available in 88 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 33%: SM-CMML 48%, SM-MDS 21%, SM-HES 8%, and SM-MPN 45% (p=0.03). In patients with SM-HES/CEL (p=0.0009) and SM-MPN (p=0.04), detection of KITD816V was associated with inferior survival. Greater than 1% (range 5–57) JAK2V617F allele burden was detected in 6 patients (9%): 4 in SM-MPN, 1 in SM-CMML and 1 in SM-MDS.Conclusions: The WHO-defined SM-AHNMD comprises prognostically diverse subcategories among which SM-CMML is the most frequent. Survival is favorable in SM-HES/CEL and significantly worse in SM-CMML and SM-MDS. Additional independent predictors of inferior survival include constitutional symptoms, thrombocytopenia, circulating immature myeloid cells, and hypoalbuminemia. In SM-HES/CEL and SM-MPN, BM KITD816V allele burden may influence survival. JAK2V617F occurs infrequently in SM-AHNMD and when it does, it clusters with SM-MPN.

Revisiting Mastocytosis in Adults: A Mayo Clinic Case Series of 342 Patients Including Information on KITD816V and JAK2V617F.

Background: Systemic mastocytosis (SM) has a varied presentation ranging from indolent forms with limited morbidity over many years to the rapidly fatal mast cell leukemia (MCL). This heterogeneity complicates clinical decision making regarding choice and timing of therapy. The aim of this study was to evaluate the prognostic value of the 2001 World Health Organization (WHO) classification of SM, and to refine risk stratification within SM sub-groups to facilitate clinical decision making. Information on KITD816V and JAK2V617F mutation analysis and clinical correlates is also provided.Methods: Patient records in the institutional electronic database from January 1976 to October 2007 were reviewed and SM patients 18 years of age or older identified; the date of diagnosis ranged from July 1964 to October 2007. Only those patients where diagnosis was confirmed by bone marrow (BM) histology were included in the analysis. KITD816V mutation analysis was performed by DNA sequencing. JAK2V617F was screened by allele-specific quantitative PCR analysis.Results: i. Clinical characteristics at presentation: A total of 342 SM patients were identified (154 female; median age at diagnosis=57 years; range 19 to 87 years). Per the 2001 WHO classification, 159 had indolent SM (ISM; median age=49 years), 41 aggressive SM (ASM; median age=65 years), 138 SM with associated clonal hematological non-mast cell lineage disease (SM-AHNMD; median age=65 years), and 4 MCL (median age=55 years) (p-value for age <0.0001). 140 (41%) patients had urticaria pigmentosa (UP) (63% with ISM; p<0.0001), 160 (47%) mast cell (MC) mediator release symptoms (69% with ISM; p<0.0001), 107 (31%) skeletal symptoms, and 142 (42%) constitutional symptoms (61% with ASM/SM-AHNMD versus 19% with ISM; p<0.0001). 123 patients (36%) had palpable splenomegaly (57% with SM-AHNMD; p<0.0001). 56 patients (16%) exhibited prominent eosinophilia (absolute eosinophil count >1500/mcl) – 31% and 22% with SM-AHNMD and ASM, respectively, versus 3% with ISM (p<0.0001); 12 of 23 patients (52%) with eosinophilia who were tested carried the FIP1L1-PDGFRA mutation. Serum tryptase (normal <11.5 ng/mL) was measured in 160 patients (47%) and almost all (96%) had an elevated level (median=64 ng/mL; range=4 to 2000 ng/mL; 33 patients had >200 ng/mL). ii. Disease course and prognostic factors After a median follow-up of 20.7 months (range=0–417), 153 deaths were recorded. 17 patients (5%) transformed to acute myeloid leukemia (AML; n=14) or MCL (n=3), most frequently with SM-AHNMD (11% versus 0% for ISM; p<0.0001). The median overall survival was 63 months (ISM=198 months, ASM=41 months; SM-AHNMD=24 months, and MCL=2 months; p<0.0001). Multivariate analysis of parameters at the time of diagnosis showed a significant and independent association between inferior survival and WHO sub-type (p<0.0001), age at diagnosis (p<0.0001), history of weight loss (p=0.01), anemia (p=0.007), thrombocytopenia (p=0.0008), hypoalbuminemia (p=0.0008), and excess BM blasts (>5%; p=0.004). A similar analysis in ISM identified anemia (p=0.04), hypoalbuminemia (P=0.002), and BM MC ≥ 30% (p=0.03) as independent predictors of inferior survival; the corresponding parameters in ASM were age at diagnosis (p=0.002) and history of weight loss (p=0.003). iii)KITD816V andJAK2V617F analysis Archived bone marrow was available in 165 patients for KITD816V and JAK2V617F analysis. By DNA sequencing, overall KITD816V detection rate was 28%: ISM 20%, ASM 29%, SM-AHNMD 33% (p=0.4). In patients with ASM (p=0.003) and SM-AHNMD (p=0.001), detection of KITD816V was associated with inferior survival. Greater than 1% (range 1–57) JAK2V617F allele burden was detected in 6 patients (4%); all belonged to SM-AHNMD including 4 with non-MC lineage myeloproliferative neoplasm.Conclusions: The current study confirms the prognostic value of the WHO subclassification of SM and identifies advanced age, weight loss, anemia, thrombocytopenia, hypoalbuminemia, and excess BM blasts as additional adverse prognostic factors. In ISM, presence of anemia, hypoalbuminemia, or BM MC ≥ 30% is associated with inferior survival. In ASM and SM-AHNMD, BM KITD816V allele burden might influence survival. JAK2V617F is rare in SM and when found, it is almost always in the context of SM-AHNMD.