Littérature scientifique sur le sujet « CKIT D816V »

A patient with a diagnosis of myelodysplastic syndrome (MDS) with isolated 5q deletion underwent repeat bone marrow biopsy to assess haematological response after 6 months of initial lenalidomide therapy. Subsequent bone marrow biopsies revealed persistent MDS with del(5q) in addition to a small atypical mast cell population with >25% of mast cells with spindle-shaped morphology and immunohistochemistry characteristics consistent with mastocytosis. Molecular testing on the bone marrow was positive for cKIT D816V and the patient was diagnosed with systemic mastocytosis (SM) with an associated haematological neoplasm. MDS with SM is well known to be associated; however, to the best of our knowledge, only one prior case report identifies MDS with del(5q) and associated cKIT D816V positive mastocytosis. While the exact clonal origin of both chromosomal aberrations is unclear, this case illustrates the therapeutic efficacy of lenalidomide in a patient with MDS with del(5q) and rarely associated cKIT positive SM.

Abstract Background The proto-oncogene c-KIT encodes a receptor tyrosine kinase characterized by five extracellular immunoglobin-like domains, such as a single transmembrane helix, a cytoplasmic juxtamembrane domain (JMD), and a kinase domain. Mutations of c-KIT have been shown to occur in the extracellular portion of the receptor within exon 8, in the JMD domain within exon 11, and in the activation loop of the kinase domain within exon 17. All mutations led to the constitutive activation of the protein and promote development of human cancer, including hematological malignancies. Moreover, they have been frequently described to be associated with poor prognosis in adults with acute myeloid leukemia (AML) harboring aberrancies of the core binding factors (CBF). c-KIT mutations are also found in pediatric CBF-rearranged AML, but their incidence and prognostic impact are still debated. The AIEOP AML2001/02 protocol assigned patients with CBF rearrangements who reach complete remission (CR) at the end of the first course of induction therapy to the standard-risk (SR) group. These patients showed an incidence of relapse higher than expected (24%). New independent prognostic factors are thus desirable for improving the prognosis of this group of AML. In this study, we investigated the prognostic impact of c-KIT mutations. Materials and Methods We retrospectively analyzed the bone marrow of 49 and 30 patients carrying either the t(8;21) or inv(16)(p13;q22). Screening for mutations of c-KIT was assessed by PCR amplification followed by Sanger sequencing of the exons 8, 11 and 17. The prognostic impact was assessed through the calculation of the probability of event-free survival (EFS). Results c-KIT mutations analyzed in the study are: the point mutation at D816 residue known to affect the activation loop of the kinase domain (exon 17); the internal tandem duplication located at exon 11, and small deletions or insertions, or combinations of deletions and insertions, of variable size at exon 8. The screening showed that 2/49 (4%) t(8;21) patients were positive for the point mutation affecting the codon D816V/Y; 2/49 (4%) for internal tandem duplication of exon 11, and 7/49 (14.3%) for small deletions and/or insertions of variable size in the extracellular portion of the receptor (exon 8). We found that the t(8;21) positive patients mutated for cKIT (18.3%) had a significant lower EFS (45.7%) than wild type patients (78.4%; p = 0.025). On the contrary, c-KIT mutations were rarely found in the cohort of inv(16) positive patients. In particular, 1/30 harbored the D816V, and 2/30 (6.6 %) had insertions at exon 8. No mutations at exon 11 were found. Conclusions c-KIT mutations are frequently found in t(8;21) postitive patients and they confer a worse prognosis. Targeted therapy with tyrosine kinase inhibitors may be considered as a new and promising therapeutic strategy for cKIT mutated patients in order to improve their outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by proliferation and hyperactivation of clonal mast cells. Clinical manifestations are heterogeneous and encompass cutaneous lesions, gastrointestinal alterations, osteoporosis, anaphylaxis and involvement of bone marrow and other organs due to neoplastic mast cells (MC) infiltration. As consequence, diagnosis may be difficult and patients (pts) are often evaluated by different specialists before the disease is recognized. To date, only few studies (Lim 2009, Escribano 2009, Cohen 2014) described relatively large series of pts with SM. We performed a multicentre retrospective study to evaluate clinical and biological features and therapeutic management in a large series of pts from 10 Italian centres experienced in management of SM and organized in multidisciplinary groups of specialists. We collected 455 pts diagnosed with SM according to WHO criteria. Additionally 26 pts with mastocytosis in the skin (MIS) evaluated with BM examination did not fulfil criteria for SM, leading to diagnosis of Cutaneous Mastocytosis (CM); however 2/26 pts with CM had both cKITD816V mutation and CD2/CD25 expression on MC in BM, additional 3 showed either cKITD816V or CD2/CD25. Moreover, we found 22 pts without MIS but with features of monoclonal mast cell activation syndrome. Of the 455 pts with WHO-SM (male 56%), 252 (55%) had MIS: median age at MIS diagnosis (dg) was 37 years (y) (range 0-79), while at SM dg it was 46.5 (range 18-82). Time from onset of MIS to dg of SM was 9 y (range 0-43). In 18/252 pts (7%) MIS occurred before age of 18 y (median 9, range 0-17) and persisted over childhood. Median age at dg of SM without MIS (203/455 pts, 45%) was older: 54 y, range 19-79 (p<0.0001). First manifestations of SM were MIS in 46.5% of pts, anaphylaxis in 47.8%, mediator related symptoms in 6.4%, osteoporosis/bone lesions in 5.2%, organomegaly in 3.4%, hematologic alterations in 0.4%. Anaphylaxis was observed in 76/252 (30.1%) pts with MIS, of which 23 had tryptase <20 mcg/L. BM multifocal infiltrates of MC were present in 61.6% of pts, while in 38.4% dg was performed only by using minor criteria. cKIT D816V mutation was detected in BM of 394/432 analysed pts (91.2%). Three D816V-negative pts had different cKIT mutations: M541L, D816H and K546K. In peripheral blood cKIT D816V was evaluated in 165 pts and found in 49.7%. CD2 and/or CD25 expressing MC were found in 99% of pts of the 426 evaluated. Mean tryptase value at dg was 75.9±263 mcg/L. SM subtypes were indolent SM (ISM) 402/455 (88.4%), of which 140 isolated bone marrow mastocytosis (BMM) (34.5% of ISM) and 34 smoldering SM (SSM) (8.4% of ISM), aggressive SM (ASM) 32/455 (7%), SM associated with hematological non mast cell disorders (SM-AHNMD) 20/455 (4.4%), mast cell leukemia (MCL) 1/455 (0.2%). In SM-AHNMD the associated hematologic disease was chronic myelomonocytic leukemia (6/20, 30%), non-Hodgkin lymphoma and refractory anemia with ring sideroblasts and thrombocytosis (3/20 each, 15%), essential thrombocythemia and not otherwise characterized myeloproliferative neoplasm (2/20 each, 10%), myelodysplastic syndrome, myelofibrosis, multiple myeloma and acute myeloid leukemia (AML) (1/20 each, 5%). Median follow up was 23 months (mo), range 2-289. At last follow up, 27/455 pts died (5.9%). 52% of pts had ASM, 18.5% SM-AHNMD, 14.7% SSM, 7.4% ISM and 3.7% each MCL and BMM. Causes of death were disease progression in 21/27 pts (77.8%), other solid neoplasms in 3/27 (11.1%), arterial thrombosis in 2/27 (9.5%), cerebral haemorrhage in 1/27 (3.7%). Disease progression consisted in evolution to AML in 6 pts with ASM, 1 pts with SSM and 1 pts with SM-AHNMD; median time to progression to AML was 30 mo (range 13-149); 2 pts developed other AHNMD: chronic myeloid leukemia and myelofibrosis. 178/455 pts (39%) were treated with anti MC mediators therapies. Of the 60/455 (13%) treated with cytostatic therapy 47% had ASM, 12% SM-AHNMD, 2% MCL, 35% ISM of which 38% were SSM. The ISM cohort was treated mainly due to severe osteoporosis with vertebral fractures not C-findings or disease evolution. First line therapy was interferon (28.3%), hydroxyurea (20%), midostaurine (18.3%), imatinib (13.3%), cladribin (8.3%), dasatinib (6.7%) and masitinib (5%). This is one of the largest series reporting pts with SM that may provide useful information for clinical management of pts with this probably underestimated “rare” disease. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Abstract Background Advanced Systemic Mastocytosis (adSM) still have very poor prognosis. No standard of care is yet available. Midostaurin (PKC412) is a powerful TKI inhibit of VEGFR, FLT3 and most importantly the mutated CKITD816V and D816Y. Preliminarily results from an international phase II trial (Gotlib et al., Blood 2012) reported interesting results in this subgroup of patients. We report herein on the French experience of the compassionate use of PKC412 for the treatment of adSM and compare their outcome to matched adSM historical control patients (pts) who did not receive PKC412 (control group). Methods Over a period of 12 months (from August 2012, date of PKC412 Temporary Approval for Use in France), 22 pts received PKC412 and could be analysed. PKC412 100 mg twice daily was administered orally as continuous 28-day cycles until progression or unacceptable toxicity. Pts were evaluated according to the response criteria used in the recent phase 2 trial on midostaurin (Gotlib et al.). The overall survival (OS) of these 22 patients was compared to the OS of 42 age and WHO subcategories matched control adSM patients who were treated before PKC412 era. Results 2 female and 20 male pts with a median age of 65 years [35-84] received midostaurin. Overall, 21 pts (95%) with adSM (5 ASM; 2 MCL; 1 MCS; 12 ASM-AHNMD; 1 SSM-AHNMD,1) and 1 pt progressive SSM. Median number of C-Findings was 3 (0-4). Hepato- and/or splenomegaly were present in 20 (91%) and 21 (95%) pts. Median tryptase level was 204 ng/l (85-2000). Median haemoglobin level, platelet and PMN counts were respectively 10.1g/dl (6.8-13.9), 117G/L (22-289) and 2.8G/L (0.3-17). Twenty (91%) pts had cKIT D816V mutation, 2 pts had cKITWT. ASXL1 and TET2 were mutated in 3/7 and 2/7 tested, respectively. Steroids, 2-chlorodeoxyadenosine and Interferon were administered in 9, 4 and 2 pts prior to midostaurin. After a median exposure time of 6.9 [1.6-27.5] months, the overall response rate (ORR) was 77.2% including, major responses (n=13; 59%) (incomplete major in 8 pts, pure clinical in 5); partial response (n=5, 23%) (good partial in 3, minor partial in 2). Three patients were rapidly progressive (<2 months) and considered refractory to PKC412 (1 MCL, 1 MCS, 1 ASM-AHNMD). Death occurred in 4 pts (refractoriness 2 pts, AHNMD progression 2 pts). After a median follow-up of 7.4 months from PKC412 start, median OS was 24.4 months [12.6-34.2]. Patients with ASM seemed to better benefit from PKC412, however, no significant difference was found between the ASM and SM-AHNMD, probably due to the low patient’s number. The control group consisted in 42 pts with a median age of 65.5 years [23-84]. There were 17 ASM-AHNMD, 21 ISM-AHNMD, 2 SSM-AHNMD, 1 CM-AHNMD and 1 MCL patients. Median tryptase level was 107ng/l [19-501]. Median haemoglobin level, platelet and PMN counts were respectively 12.1g/dl [8-15], 200G/L [10-1036] and 4.6G/L [0.5-24.3]. Thirty-three (79%) pts had cKIT D816V mutation, 7 pts were cKITWT. ASXL1 and TET2 were mutated in 6/29 (21%) and 7/29 (24%) of pts tested, respectively. Steroids, 2-CdA, interferon, imatinib, thalidomide have been used in 19, 21, 6, 4, and 6 pts respectively. Survival distributions were estimated using the adjusted Kaplan-Meier method from the date of diagnosis to the last follow-up for the PKC412 treated and the control groups. Median survival time for PKC412 treated patients was significantly longer than control patients (p=0.04). Conclusion PKC412 is active in advanced SM. Despite the absence of complete remission, PKC412 gives survival advantage for patients with adSM over pts who did not receive the drug. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 5025 Background: Data on outcomes of patients (pts) with myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), especially MDS/MPN-unclassified (MDS/MPN-U), are scarce. Patients/methods: We retrospectively studied pts followed in our center, with MDS/MPN according to WHO 2008 criteria. Because of overlap characteristics of MPN and MDS, pts with systemic mastocytosis associated with MDS (SM/MDS) were also included. Pts with previous MDS or MPN were excluded. Response and disease progression were defined according to IWG 2006 criteria. Results: Twenty-five pts with MDS/MPN were included. Median age was 70 y (range 19–79). Male/female ratio was 1.77/1. Diagnosis was CMML-1 N=7, CMML-2 N=7, JMML N=1, MDS/MPN-U N=8, systemic mastocytosis (SM)/MDS N=2, with one additional pt with CMML subsequently developing SM. At diagnosis, median WBC count was 18.8 G/L (range 3–120), ANC 15.5 G/L (0.6–70), monocytes 1.9 G/L (0.1–16), left shift 16% (0–28), Hb 11.2 g/dL (6–17), platelets 99 G/L (10–680), peripheral and bone marrow (BM) blasts 5% (0–17) and 7% (2–19), respectively (resp.). 25% of pts had platelets count ≥400 G/L. Splenomegaly, B-symptoms and BM fibrosis were present in 23%, 57% and 27% of pts, resp. Karyotype was fav, int and unfav in 55%, 36% and 9% of pts, with −7, +8, del(12)(p11), del(12)(q14;q21), +10, +21, and previously unreported t(9;12)(q13;q13) in 3, 6, and 1 pt each, resp., while +21 and i(17)(q10) appeared during disease progression other than AML transformation. IPSS was low/int-1 and int-2/high in 50% and 50% of pts, resp. JAK2 V617F and CKIT D816V mutations were detected in 2/6 pts and 2/2 SM/MDS pts, resp. 70% and 29% of pts were transfused at diagnosis with PRBC and platelets, resp. Treatment included erythropoiesis stimulating agents (ESAs), low dose chemotherapy, intensive chemotherapy (IC) and azacitidine (AZA) in 40%, 36%, 16% and 48% of pts resp. Response rate to ESAs, IC and AZA was 60%, 14% and 14% resp. Response rate to AZA in CMML-1 pts was 33%. Dasatinib yielded no response in 1 SM/MDS pt with CKIT D816V. 3-year cumulative incidence of AML and median overall survival (OS) in pts with CMML-1, CMML-2 and MDS/MPN-U were 20%, 40% and 0 (P=0.059) and 39, 8, and 20 mo (P=0.50), resp. The pt with JMML died from AML transformation 3 months after diagnosis. 2/3 pts with SM/MDS died from disease progression w/o AML at a median of 10 mo after diagnosis. Median survival after disease progression other than AML transformation was 35, 15 and 14 mo in pts with CMML-1, CMML-2 and MDS/MPN-U, resp. (P=0.88). Cause of death was disease progression other than AML, AML transformation and unrelated to disease in 50%, 50%, and 0 and 80%, 0 and 20% of cases in CMML and MDS/MPN-U, resp. (P=0.10). Percentage of circulating blasts ≥5% was the only independent factor affecting risk of AML transformation in the overall population (P=0.0004). Diagnosis other than CMML-1, WBC ≥30 G/L, % of circulating blasts ≥5% and IPSS high/int-2 were associated with worse survival in univariate analysis (P=0.06, 0.03, 0.04 and 0.08, resp.). No predictive factor of OS was found in multivariate analysis. Conclusion: MDS/MPN are heterogeneous disorders with respect to disease progression and AML transformation. MDS/MPN-U tended to differ from CMML-1 by shorter survival after disease progression other than AML, and from CMML-2 by lower risk of AML transformation. Mortality of pts with MDS/MPN-U was mainly attributed to disease progression without AML transformation. Alternatively to hypomethylating agents, therapeutic options in pts with MDS/MPN-U could include JAK2 inhibitors. Disclosures: No relevant conflicts of interest to declare.

Abstract Introduction Recent studies utilizing NGS demonstrated that residual allelic burden at complete remission (CR) is associated with worse overall survival (OS) and relapse incidence in AML. Quantitative PCR (qPCR) based disease monitoring is current practice in CBF-AML. However, qPCR requires standardization and the result for the same sample may vary depending on several factors. Also, other known prognostic factors such as cKIT mutation require an additional test. As RNA-seq can detect gene rearrangement as well as somatic mutations, we hypothesized that RNA-seq on samples taken at diagnosis and at remission can be used to monitor these genetic alterations simultaneously and can be utilized for minimal residual disease (MRD) monitoring in CBF-AML. Patients and Methods This study included 42 CBF-AML patients (23 RUNX1-RUNX1T1 and 19 CBFB-MYH11 AML). Overall, 84 bone marrow samples (42 diagnosis-CR pairs) were subjected to targeted RNA-seq using Illumina TruSight Pan-Cancer panel. After read mapping, gene count was measured using HTSeq followed by DEseq2 for gene expression quantification. Average number of sequenced reads was 3.5M reads with 87% overall mapping rate. Gene fusions in diagnostic samples were detected using EricScript. All 84 samples as well as 42 samples from T-cell fraction (CD3+, as a control) were also subjected to DNA sequencing, targeting a panel of 84 genes (Agilent SureSelect custom gene panel). Average on-target coverage was 1,606x. All other computational analyses were done using R and python. Results In diagnostic samples, class-defining gene fusion events were detected in all 42 patients. In CR samples, we tracked identical junctions identified in corresponding diagnostic samples. As expected, both CBFB-MYH11 and RUNX1-RUNX1T1 showed significant reduction in all CR samples compared to their corresponding diagnostic samples (p < 2.2e-13 and p < 6.3e-05, Fig A and B). CBFB-MYH11 was detectable in 6/19 CR samples (32%) and RUNX1-RUNX1T1 was detectable in 15/23 CR samples (65%). Reduction level of RUNX1-RUNX1T1 measured by RNA-seq showed positive correlation with the reduction level measured by qPCR (Pearson's Rho = 0.74, p < 5.4e-05, Fig C). As per mutational profile at diagnosis, we detected 74 mutations in 38 samples (n=38/42, 90%). NRAS (36%), KIT (36%), KRAS (17%) and, ASXL2 (17%) were commonly mutated. Survival analyses on each gene and each protein locus identified cKIT-D816 mutation as an adverse prognostic factor (HR = 3.57, [1.15 - 11.11], p = 0.028). We were able to detect all cKIT-D816 mutations in RNA-seq. Using information from NGS, we built a prognostic model for RUNX1-RUNX1T1 AML (n = 23). Decision tree analysis identified three distinct subgroups of RUNX1-RUNX1T1 AML on the basis of reduction level of RUNX1-RUNX1T1 and mutation profile (Fig D). Consistent with previous studies, 3-log or deeper reduction of RUNX1-RUNX1T1 transcript level was the most significant prognostic factor (low risk group). The algorithm further divided the patients who failed to achieve 3-log reduction according to the presence of cKIT-D816 mutation at diagnosis (intermediate and high risk group). For three defined groups, 2-year OS rates were 87%, 74%, and 33% (p = 0.08, Fig E) and 2-year relapse incidence rates were 13%, 42%, and 67% (p = 0.048, Fig F). Conclusion RNA-seq can be utilized to quantify RUNX1-RUNX1T1 and CBFB-MYH11 transcripts on diagnostic and CR samples in CBF-AML. We also showed that RNA-seq can stratify RUNX1-RUNX1T1 AML patients into three risk groups according to their long-term prognosis. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Abstract INTRODUCTION Mast cell disease is a clonal haematological disorder resulting in organ infiltration by mast cells and uncontrolled degranulation (mast cell activation syndrome, MCAS). Although not characteristic and poorly explained, both primary (platelet’s adhesion and aggregation) and secondary (coagulation cascade leading to thrombin formation) clotting abnormalities are observed among some patients. METHODS We retrospectively identified patients with established diagnosis of mastocytosis and related clotting abnormalities (clinical and/or biological) using the French National Reference Centre for Mastocytosis database. RESULTS AND DISCUSSION Our cohort included 14 adult patients (median age of 42 [range 26-78] years, sex ratio 3.7, CKIT D816V in 13/14 cases, median tryptase of 200 [4-1240] ng/mL). Four patients had typical symptoms and/or clotting tests (PFA, von Willebrand’s disease [vWD] screening) indicating abnormalities of primary haemostasis [Table, patient 1], with n=1 confirmed and n=2 highly suspicious of vWD. They presented with either nil (n=1) or mild (n=3) mucocutaneous bleeding. This could be due to heparin binding to von Willebrand’s factor, therefore preventing platelet aggregation. Ten patients had abnormalities of coagulation cascade (prolonged PT, aPTT, reduced clotting factors II, V, VII, X), usually transient but with bleeding in 7 cases (severe/life-threatening in 5 of them ) [Table, patient 3]. Clotting abnormalities were typically accompanied by anaphylactoid symptoms of MCAS and increased by the presence of hematopoietic organ infiltration [Table, patients 2-4]. Haemostatic management (red blood cells and platelets transfusion, fresh frozen plasma, vitamin K, antifibrinolytic) was unsuccessful, while control of mast cell activity (e.g. with and steroids) would achieve haemostasis, indicating the crucial role of mast cell mediators. These mainly include tryptase and heparin, which inhibit the intrinsic pathway, inducing an anticoagulant state. The exact prevalence of clotting abnormalities in mastocytosis remains unclear and probably underestimated. However, we found a predominance of aggressive types of mastocytosis in the population with coagulation cascade abnormalities: 50% had aggressive systemic mastocytosis associated or not with a clonal haematological non mast cell lineage disease (ASM/AHNMD), 30% had mast cell leukaemia (MCL). All deceased patients (n=5, 36% of the overall population) belonged to this group. In contrast, mastocytosis was indolent in 75% of cases in the group with primary haemostasis abnormalities. Abstract 2856. Table: Description of 4 selected patients Patient number / gender; Diagnosis Acute phase of MCAS H/SMG Bleeding Biology Clotting after episode PLT 150-300 PTr ≥70% aPTTr ≤1.20 #1 / M; CM no no Cutaneous N 89% 1.23 Spontaneously resolved #2 / M; SSM-AHNMD† yes no GI bleed N 37% 1.75 Resolved after steroids yes no GI bleed 20 62% 1.41 Resolved after symptomatic control of MCAS. #3 / M; aMCL yes yes Acute SDH 87 61% 1.46 Resolved after steroids #4 / M; ASM-AHNMD† no yes None 138 *14 62% *43% 1.39 *1.97 Worsening clotting alongside ASM progression and unrelated to the AHNMD. Response to steroids not assessable AHNMD: associated clonal haematological non mast cell lineage disease; aMCL: acute mast cell leukemia; aPTTr: activated partial thromboplastin time ratio; CM: cutaneous mastocytosis; H/SMG: hepato or splenomegaly; GI: gastrointestinal; ISM: indolent systemic mastocytosis; N: normal; SSM: smouldering systemic mastocytosis; PLT: platelet, in x103/µL; PTr: prothrombin time ratio; SDH: subdural haematoma. Reference values are indicated below PLT, PTr, aPTTr. †: patient deceased from the disease or complications of the latter. *6 months later. CONCLUSIONS AND PERSPECTIVES Blood coagulation abnormalities in mastocytosis appear secondary to a hyperactivity of the clonal mast cell, resulting in an anticoagulant state and typically presenting in the context of MCAS and/or significant mast cell proliferation. They are particularly prevalent in aggressive types of mastocytosis, and are only successfully controlled by mast cell inhibitors, notably steroids. More data are required to better correlate clinical and biological pictures, and to assess whether standard or specific clotting tests could be used as a marker of mast cell activity and potentially of poor prognosis in patients with systemic mastocytosis. Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 4913 Background: Among tyrosine kinase inhibitors, midostaurin (PKC412) takes place in the treatment of patients presenting with advanced systemic mastocytosis and, specifically aggressive systemic mastocytosis (ASM) or mast cell leukemia (MCL) with or without associated clonal hematological non-mast cell lineage disease (AHNMD). Our knowledge of the pharmacokinetic (PK) behavior of this major drug remains unfortunately sporadic. We developed a powerful liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, which is able to sensitively detect PKC412 as its two major metabolites CGP52421 and CGP62221 in human plasma. The method was validated in clinical situation and, especially in the aim to monitor plasma concentrations of PKC412 in 11 patients, already enrolled in a Phase II clinical trial (Novartis Lab.). In addition, we also determined the PK profile of PKC412 at steady-state for one specific patient. Methods: A sensitive LC-MS/MS assay was developed for the simultaneous quantification of the product PKC412 and its two major metabolites in human plasma. Midostaurin-D5 was used as internal standard. The method was then validated in accordance with international guidelines and within the bioanalytical ±15% acceptance limits. In any case, PKC412 regimen was 100 mg bid. A total of 61 blood samples were collected from 11 patients and, between 15 min to 12 hrs following oral administration. Sampling took place over a period of 1 to 924 days following the beginning of treatment. PKC412 residual concentration at steady-state, range and mean concentration and, intra- and inter-individual variability coefficients were also calculated. On the other hand, a model independent method was used to determine the PK profile of PKC412 for a 12th patient (using 22 samples collected within 24 hrs): maximal concentration post-dosing (Cmax – ng/mL), time to Cmax (Tmax - hrs) were experimental values; terminal half-life (t1/2lz - hrs) and AUC0–12 were calculated during each 12 hrs interval post-dosing. Data are expressed as means ± SD. Results: This LC-MS/MS method is satisfactory for the key validation criteria i. e. trueness, bias, precision (repeatability and intermediate precision), accuracy and, linearity, over a range of concentrations of 75 to 2500 ng/mL for both PKC412, CGP52421 and, CGP62221. Patients monitored (8/11 males) were 57±14 yrs-old. All except one were treated for ASM±AHNMD (the last one had MCL), 10/11 patients carrying the ckit D816V mutation. The main characteristics at inclusion were as followed: body weight = 65±11 kg, BMI = 22±3 kg/m2, albumin = 31±6 g/L, GFR = 64±12 mL/min, bilirubin = 14±6 mg/L, GGT = 103±78 UI/L, ALP = 264±158 UI/L, tryptase = 412±411 μg/L. At the last follow-up visit i. e. between 54 to 924 day after initiation therapy, 8/11 patients were either in major or partial response with the following clinical picture: body weight 74±16 kg, BMI 24±4 kg/m2, albumin 40±2 g/L, DFG 59±11 mL/min, bilirubin 12±8 mg/L, GGT 56±57 UI/L, ALP 128±59 UI/L, tryptase 256±264 μg/L. It is important to point out that the tolerability profile was very good. The PKC412 residual concentrations value determined at steady-state were between 487 to 3254 ng/mL (1333±867 ng/mL). The intra- and inter-individual coefficients of variations were 18 and 65% respectively. About our 12th subject, major PK parameters were: Cmax = 3692±447 ng/mL, Tmax = 2, 5±2 hrs, t1/2lz = 95±88 hrs, AUC0–12= 34326±2197 ng/mL. hrs. Conclusions: The LC-MS/MS method is efficient and enables a sensitive and discriminative analysis of PKC412 and its major metabolites in human plasma. These first results highlight the high intra- and inter-individual variability of the PK behavior of the drug and, suggest the interest of its routine therapeutic monitoring vs. both adherence to the treatment and, its tolerability profile. Further, it has been shown that the 2 major metabolites of PK412 are active; under these conditions, we are working to the simultaneous therapeutic drug monitoring of the 3 compounds. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemias with t(8;21) or inv(16)/t(16;16) are commonly referred to as Core-Binding Factor AML and represent 5-8% of all AMLs . These genomic rearrangements are characterized by disruption of the CBF and CBF genes respectively. Both genes encode a subunit of core-binding factor complex, an important transcriptional regulator of hematopoiesis involved in myeloid differentiation. CBF AML are considered a favorable AML risk group based on high remission rate and survival probabilities. However, the presence of the cKIT D816V mutation has been reported to harbor an unfavorable prognostic implication in CBF-AML. The proto-oncogene cKIT encodes a receptor for SCF. It belongs to the type-III receptor of the tyrosine kinase subfamily, that is characterized by five extracellular immunoglobulin-like domains, a single transmembrane helix, a cytoplasmic juxtamembrane domain, and a kinase domain. The cKIT D816V mutation is found in 20–25% of patients with t(8;21) and in about 30% with inv(16)/t(16;16). cKIT D816V causes the indipendent-ligand activation of the tyrosine kinase receptor cKIT, and the activation of downstream pathways supporting cell proliferation and survival. CBF AML patients exhibiting D816V mutation in cKIT gene, are classified in the intermediate risk category, since they present an high incidence of relapse and a low overall survival. Moreover the replacement of the aspartic acid residue with valine at codon 816 causes resistence to Imatinib, the tyrosine kinase inhibitor usually used in the treatment of CBF AML to suppress the overexpressed cKIT. In this case, patients are treated with TKIs of second generation, such as Dasatinib or Nilotinib. So, the single point mutation D816V in cKIT gene, represents a critical prognostic factor for CBF AML patients and an important indicator to define the appropriate treatment. Several PCR-based methods are developed for the detection of D816V mutation, such as PCR followed by direct DNA sequencing, enzymatic digestion with HinfI enzyme (RFLP), or dHPLC. However, most of the PCR-based methods result expensive, time-consuming and labor-intensive. At the moment Sanger sequencing is still the gold-standard for D816V detection, even if it presents a low sensitivity (10–15%). The aim of this work is to improve the detection of cKIT D816V mutation in CBF AML patients by the development of rapid, user-friendly and cost-effective assay based on Allele-Specifc Loop mediated isothermal AMPlification (AS-LAMP) technology. The AS-LAMP assay is based on LAMP technology that amplifies target DNA at costant temperature thanks to the employment of the DNA polymerase with strand-displacement activity. AS-LAMP assay is able to detect the single point mutation thanks to the cooperation of two elements: a loop primer specific for the mutated cKIT sequence, and a blocker that binds only the wild type gene. While the blocker suppresses the aspecific amplification of wild type alleles, the LB primer recognizes the mutated codon and allows its amplification. The AS-LAMP assay includes also the amplification of the housekeeping ABL gene as internal control, that is informative to exclude false-negative results due to the failure of DNA extraction procedure. The target and the IC are detected in real-time in 500 nm and 530 nm channels respectively, thanks the use of two labeled probes specifically binding cKIT or ABL genes. The AS-LAMP assay was optimized on synthetic plasmid containing the cKIT sequence and was successfully validated on negative and positive clinical samples, confirming in all cases the results obtained with PCR-based methods. In conclusion the AS-LAMP assay is able to detect the D816V mutation with high sensitivity and specificity in only 40 minutes and it is easy-to-use. For these reasons the AS-LAMP assay represents a powerful tool for molecular diagnostics applications in clinical routine.