scholarly journals Comparison of RNA-Based Versus DNA-Based Quantitative KIT D816V Mutation Analysis Reveals Significant Disparity in Patients with Advanced Systemic Mastocytosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2955-2955
Author(s):  
Mohamad Jawhar ◽  
Nicole Naumann ◽  
Johannes Lübke ◽  
Juliana Schwaab ◽  
Oliver Hofmann ◽  
...  
Keyword(s):  
Real Time ◽  
Research Funding ◽  
Limit Of Detection ◽  
Systemic Mastocytosis ◽  
Digital Pcr ◽  
Coefficient Of Determination ◽  
Lower Percentage ◽  
Molecular Profile ◽  
Number Of Patients ◽  
Kit D816v

Systemic mastocytosis (SM) is characterized by activating mutations in KIT, usually KIT D816V in >90% of patients. According to the WHO classification, detection of KIT D816V is a minor diagnostic criterion. Moreover, a reduction of the RNA-based KIT D816V expressed allele burden (EAB) of ≥25% at month 6 is a favorable marker for improved survival in midostaurin-treated advanced SM (AdvSM) patients. However, only limited data exist upon the comparison between RNA-based and DNA-based quantitative KIT D816V mutation analyses. We therefore collected peripheral blood samples from 161 SM patients (indolent SM [ISM], n=40; AdvSM, n=121) at time of referral. We applied a real time reverse-transcriptase polymerase chain reaction (qRT-PCR) assay for assessment of the KIT D816V EAB and a chip-based digital PCR (dPCR) for the quantification of the DNA-based KIT D816V variant allele frequency (VAF, QuantStudio 3D dPCR System, ThermoFisher Scientific, Massachusetts, USA). The limit of detection (LOD) was assessed through serial dilution experiments with DNA from healthy individuals and DNA from a SM patient with a KIT D816V VAF of approximately 50%. All samples were analyzed in two independent dPCR runs. In an inital round-robin test for an inter-laboratory (n=4) comparison on 30 DNA samples (ISM, n=7; AdvSM, n=19), we found a very good correlation between 3 different chip-based digital PCR systems (dPCR; droplet-based dPCR, ddPCR, BioRad Laboratories, California, USA; quantitative real-time PCR, qPCR, California, USA; dPCR vs. ddPCR r=0.99, R2=0.99; dPCR vs. qPCR r=0.99, R2=0.98). In ISM patients, the comparison between EAB and VAF revealed a strong linear relationship with a correlation (r) of 0.91 and a coefficient of determination (R2) of 0.84, which was significantly inferior (r=0.71; R2=0.5) in AdvSM patients. In 45/121 (37%) and 76/121 (63%) of AdvSM patients, the EAB/VAF ratio was ≤2 (cohort A) or >2 (cohort B). To confirm the significant disparity between EAB and VAF in individual patients, serial analyses of at least 3 samples in 12 patients revealed a stable EAB/VAF ratio during follow-up. The comparison between cohort A and cohort B revealed significant differences in terms of a higher median hemoglobin level (p=0.006), a lower percentage of patients with hemoglobin <10g/dL (p=0.02), a lower median monocyte level (p=0.01), a lower median alkaline phosphatase level (p=0.03), and a lower number of patients with a high risk molecular profile (at least one gene mutation in SRSF2, ASXL1, and/or RUNX1, S/A/R, p=0.02) in cohort A. Moreover, patients of cohort A had a significantly better overall survival (OS) (median OS 12.9 versus 3.3 years; hazard ratio 2.1; 95% confidence interval 1.2-3.5; p=0.005; Figure). We conclude that a) KIT D816V EAB and VAF are significantly different in AdvSM patients but not in ISM and b) AdvSM patients with an EAB/VAF ratio >2 present with an aggressive phenotype and adverse prognosis as compared to patients with EAB/VAF ratio ≤2. We therefore recommend to routinely determine KIT D816V EAB and VAF in AdvSM patients. Disclosures Fabarius: Novartis: Research Funding. Reiter:Blueprint: Consultancy, Honoraria, Other: Travel reimbursement; Deciphera: Consultancy, Honoraria, Other: Travel reimbursement; Novartis: Consultancy, Honoraria, Other: Travel reimbursement, Research Funding.

scholarly journals Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis

2018 ◽  
Vol 64 (3) ◽  
pp. 547-555 ◽  
Author(s):  
Georg Greiner ◽  
Michael Gurbisz ◽  
Franz Ratzinger ◽  
Nadine Witzeneder ◽  
Ingrid Simonitsch-Klupp ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Limit Of Detection ◽  
Systemic Mastocytosis ◽  
Prognostic Significance ◽  
Melting Curve ◽  
Digital Pcr ◽  
New Method ◽  
Sensitive Detection ◽  
Melting Curve Analysis ◽  
Kit D816v

Abstract BACKGROUND The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P &lt; 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P &lt; 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P &lt; 0.001). CONCLUSIONS dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.

Performance evaluation of cobas HBV real-time PCR assay on Roche cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test

2018 ◽  
Vol 56 (7) ◽  
pp. 1133-1139 ◽  
Author(s):  
Hanah Kim ◽  
Mina Hur ◽  
Eunsin Bae ◽  
Kyung-A Lee ◽  
Woo-In Lee
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Nucleic Acid Amplification ◽  
Coefficient Of Determination ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Cobas Taqman ◽  
Limit Of Quantitation ◽  
Two Systems

Abstract Background: Hepatitis B virus (HBV) nucleic acid amplification testing (NAAT) is important for the diagnosis and management of HBV infection. We evaluated the analytical performance of the cobas HBV NAAT (Roche Diagnostics GmbH, Mannheim, Germany) on the cobas 4800 System in comparison with COBAS AmpliPrep/COBAS TaqMan HBV Test (CAP/CTM HBV). Methods: Precision was evaluated using three levels of cobas HBV/HCV/HIV-1 Control Kit, and linearity was evaluated across the anticipated measuring range (10.0–1.0×109 IU/mL) at seven levels using clinical samples. Detection capability, including limit of blank (LOB), limit of detection (LOD) and limit of quantitation (LOQ), was verified using the 4th WHO International Standard for HBV DNA for NAT (NIBSC code: 10/266). Correlation between the two systems was compared using 205 clinical samples (102 sera and 103 EDTA plasma). Results: Repeatability and total imprecision (coefficient of variation) ranged from 0.5% to 3.8% and from 0.5% to 3.5%, respectively. Linearity (coefficient of determination, R2) was 0.999. LOB, LOD and LOQ were all acceptable within the observed proportion rate (85%). Correlation was very high between the two systems in both serum and plasma samples (correlation coefficient [r]=0.995). Conclusions: The new cobas HBV real-time PCR assay on the cobas 4800 System showed reliable analytical performances.

CCL-2 Is a KIT D816V-Dependent Modulator of Bone Marrow Remodeling and Microenvironmental Alterations in Systemic Mastocytosis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1635-1635
Author(s):  
Georg Greiner ◽  
Nadine Witzeneder ◽  
Klaus Schmetterer ◽  
Theresia Popow-Kraupp ◽  
Wolfgang R Sperr ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conditioned Medium ◽  
Research Funding ◽  
Systemic Mastocytosis ◽  
Tissue Remodeling ◽  
Neutralizing Antibody ◽  
Boyden Chamber ◽  
Boyden Chamber Assay ◽  
Baseline Levels ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloproliferative neoplasm (MPN) characterized by mast cell (MC) infiltration in the bone marrow (BM) and/or other organs. Most patients present with the activating KIT mutation D816V. Increased production of cytokines is typically found in patients with classical MPN and has been linked to alterations of the BM microenvironment (e.g. enhanced angiogenesis and fibrosis) that are also commonly found in SM. However, little is known about mechanisms contributing to microenvironment alterations in the BM in SM and the role of MC-derived cytokines. The aim of our study was to identify cytokines that are produced in neoplastic MC in a KIT-dependent manner and contribute to increased BM angiogenesis and fibrosis in SM. In a first step, we screened for KIT D816V-dependent production of cytokines relevant to inflammation and microenvironment alterations using growth factor-dependent human cell lines (TF-1 and Mo7e). In these experiments, expression of CCL-2, IL-8, OSM, and VEGF mRNA was induced by KIT D816V but not by wild type KIT. Based on its pleiotropic effects, we focused on CCL-2, also referred to as monocyte chemotactic protein 1 (MCP-1), a CC chemokine that recruits inflammatory cells to sites of inflammation and enhances angiogenesis. KIT D816V+ HMC-1.2 cell were found to express and secrete substantial amounts of CCL-2. Midostaurin, a multikinase inhibitor suppressing the kinase activity of KIT D816V, was found to reduce expression of CCL-2 similar to RNAi-mediated knockdown of KIT. Furthermore, knockdown of STAT5, a key transcription factor downstream of KIT D816V, reduced expression of CCL-2 in HMC-1.2 cells. These results confirmed that CCL-2 expression in neoplastic MC is dependent on KIT D816V. Since CCL-2 has been reported to promote angiogenesis, we analyzed effects of conditioned medium obtained from HMC-1.2 cells on human umbilical vein endothelial cells (HUVEC) known to express the CCL-2 receptor CCR-2. Indeed, MC-derived conditioned medium induced migration of HUVEC in a wound healing assay (214% ± 38% of control, mean ± SD, p<0.01) as well as in a boyden chamber assay (126% ± 8% of control, p<0.05). Moreover, pre-incubation with a neutralizing antibody against CCL-2 significantly reduced migratory responses (p<0.01), and RNAi-mediated knockdown of CCL-2 in neoplastic MC reduced the effect of conditioned medium to baseline levels (p<0.01). These results confirmed that the migration was CCL-2 dependent. Furthermore, patients with advanced SM often present with marked eosinophilia; and eosinophils are often located in the vicinity of, or even within, BM MC infiltrates. Therefore, we also studied the effect of CCL-2 on eosinophils. Normal human eosinophils as well as the eosinophilic cell line EOL-1 were found to express CCR-2, and to migrate against recombinant CCL-2 in a modified boyden chamber assay. Conditioned medium from KIT D816V+ MC also induced a migratory response in eosinophils (200% ± 30% of control, p<0.05), and a neutralizing antibody against CCL-2 reduced this effect to baseline levels (p<0.05). Finally, SM-patients were found to have significantly elevated serum CCL-2 levels (n=35, p=0.0048, 407.4 ± 42.3 pg/ml, mean ± SEM) compared to controls (274.3 ± 15.9 pg/ml). The highest serum levels of CCL-2 were detected in patients with advanced SM where tissue remodeling in the BM is often a prominent feature. Moreover, CCL-2 levels were found to correlate with the grade of MC infiltration in the BM (r=0.656, p=0.0002). In summary, KIT D816V induces the expression of various cytokines potentially involved in tissue remodeling and microenvironment alterations in SM. Moreover, we have identified CCL-2 as a critical, KIT D816V-dependent, cytokine-mediator that interacts with structural BM cells and thereby may be involved in disease evolution and progression in SM. Whether CCL-2 may also serve as a therapeutic target in SM is currently being examined. This study was supported by Austrian Science Fund (FWF) grant P26079-B13. Disclosures Sperr: Novartis: Honoraria. Valent:Pfizer: Honoraria; Celgene: Honoraria; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Novartis: Consultancy, Honoraria, Research Funding.

scholarly journals A 3-Part, Phase 2 Study of Bezuclastinib (CGT9486), an Oral, Selective, and Potent KIT D816V Inhibitor, in Adult Patients with Nonadvanced Systemic Mastocytosis (NonAdvSM)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3642-3642
Author(s):  
Frank Siebenhaar ◽  
Jason Gotlib ◽  
Michael W. Deininger ◽  
Daniel J. DeAngelo ◽  
Francis Payumo ◽  
...  
Keyword(s):  
Mast Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Systemic Mastocytosis ◽  
Symptom Burden ◽  
Phase 2 ◽  
Advisory Committees ◽  
Management Committee ◽  
Study Management ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is characterized by mast cell infiltration of ≥ 1 extracutaneous organs and encompasses a spectrum of diagnoses that can range from a non-advanced to advanced disease (Shomali et al, 2018). There are two nonadvanced variants of SM: indolent systemic mastocytosis (ISM), which accounts for approximately 85% and smoldering systemic mastocytosis (SSM), which includes about 5% of the general SM population (Cohen et al, 2014, Jennings et al, 2014). ISM is characterized by 0 or 1 B-findings and SSM by 2 or more B findings and absence of organ damage or an associated hematologic neoplasm (Gotlib et al, 2018). There are currently no approved therapies to treat the underlying disease of ISM or SSM. Although anti-mediator therapies (e.g. anti-H1 and H2 antihistamines, leukotriene receptor antagonists, cromolyn sodium, corticosteroids) are used to control symptoms such as anaphylaxis, GI intolerance, and flushing to improve quality of life, their effectiveness and tolerability are variable. Many patients experience a persistently high symptom burden despite maximized anti-mediator therapies. Because the molecular pathogenesis of SM is driven by KIT D816V mutations in 95% of patients (Garcia-Montero et al, 2006, Jara-Acevedo et al, 2015, Vaes et al, 2017), other agents targeting this mutated kinase have been used to treat the spectrum of SM variants; however, toxicities such as cognitive impairment, GI effects, intracranial hemorrhage, and edema may limit dosing and thus efficacy. In addition to targeting KIT D816V, bezuclastinib was designed to avoid other closely related kinases with known liabilities, such as PDGFRα, PDGFRβ, wild-type KIT, VEGFR2 (KDR), and CSF1R (FMS). Furthermore, bezuclastinib has demonstrated minimal brain penetration and no CNS toxicities have been identified in preclinical studies. Preliminary clinical activity with bezuclastinib has been observed in patients with advanced solid tumors or locally advanced, unresectable, or metastatic gastrointestinal stromal tumor (GIST). A reduction in KIT exon 17 mutational burden was observed in patients treated with bezuclastinib. This reduction was temporally associated with a reduction in tumor burden supporting bezuclastinib as an active therapy in KIT-driven diseases (Wagner et al, 2018). This is a multi-center, Phase 2, double blind, placebo-controlled, 3-part clinical study to evaluate the safety, efficacy, and biomarker correlates (e.g. bone marrow mast cell percentage, serum tryptase level, and KIT D816V mutation burden) of the KIT inhibitor bezuclastinib in patients with ISM and SSM. This study will enroll patients with SSM and moderate-to-severe ISM who have inadequate control of their symptoms despite at least 2 anti-mediator treatments. Part 1 of the study is intended to determine the recommended dose of bezuclastinib in Part 2. Subjects will be randomized to placebo or 1 of 3 doses of bezuclastinib which will be administered in combination with a baseline regimen of best supportive care (BSC). Part 2 will evaluate the efficacy of bezuclastinib at the selected dose as compared with placebo. Efficacy will be measured by the reduction of symptom burden as assessed by the mastocytosis activity score (MAS), a disease specific patient reported outcome tool (Siebenhaar et al, 2018). In Part 3, patients who have completed treatment in Part 1 or Part 2 of the study, including those initially randomized to placebo, may participate in a long-term extension and receive open-label bezuclastinib in combination with BSC. The study will enroll approximately 138 subjects. Data from this study will support further development of bezuclastinib in SM. Disclosures Gotlib: Cogent Biosciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Chair for the Eligibility and Central Response Review Committee, Research Funding; PharmaEssentia: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kartos: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Deciphera: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Allakos: Consultancy; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Blueprint Medicines: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Deininger: Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees. DeAngelo: Incyte: Consultancy; Jazz: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Servier: Consultancy; Takeda: Consultancy; Abbvie: Research Funding; Forty-Seven: Consultancy; Autolus: Consultancy; Amgen: Consultancy; Agios: Consultancy; Blueprint: Research Funding; Glycomimetrics: Research Funding. Payumo: Cogent Biosciences, Inc: Current Employment. Mensing: Cogent Biosciences, Inc.: Current Employment. Jolin: Cogent Biosciences: Current Employment. Sachs: Cogent Biosciences: Current Employment. George: Bristol Meyers Squibb: Consultancy; Blueprint Medicines: Consultancy; Celgene: Consultancy; Incyte Corporation: Consultancy. OffLabel Disclosure: study of investigational agent

scholarly journals Midostaurin (PKC412) Demonstrates a High Rate of Durable Responses in Patients with Advanced Systemic Mastocytosis: Results from the Fully Accrued Global Phase 2 CPKC412D2201 Trial

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 636-636 ◽  
Author(s):  
Jason Gotlib ◽  
Hanneke C. Kluin-Nelemans ◽  
Tracy I. George ◽  
Cem Akin ◽  
Karl Sotlar ◽  
...  
Keyword(s):  
Mast Cell ◽  
Research Funding ◽  
Systemic Mastocytosis ◽  
Organ Damage ◽  
High Rate ◽  
Advisory Committees ◽  
Laboratory Services ◽  
Grade 3 ◽  
Stage 1 ◽  
Kit D816v

Abstract Background: Patients (pts) with advanced systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell leukemia (MCL) ± associated clonal hematologic non–mast cell lineage disease (AHNMD), have a poor prognosis with few effective treatment options. More than 80% of pts express the activating KIT D816V mutation. The oral multikinase inhibitor midostaurin inhibits wild-type and D816-mutated KIT. In this global phase 2 trial (NCT00782067), the largest prospective trial in advanced SM, a planned interim analysis of stage 1 pts demonstrated a high overall response rate (ORR) with reductions in marrow mast cell (MC) burden and a favorable safety profile (Gotlib et al, Blood 2012). Updated stage 1 results showed improvements in symptoms and quality of life (QOL) (Gotlib et al, Blood 2013). Here, we report primary results for the fully accrued trial. Methods: This single-arm trial consisted of an adapted Fleming 2-stage design. Midostaurin 100 mg twice daily was administered in continuous 28-day cycles until progression or unacceptable toxicity. Eligibility and responses (modified from Valent et al,Leuk Res 2003) were adjudicated by study steering committee (SSC). Histopathology was centrally assessed. Eligible pts had ≥ 1 measurable C-finding (CF) defined as SM-related organ damage. The primary endpoint was ORR (major response [MR] + partial response [PR]) occurring in the first 6 cycles and maintained for ≥ 8 weeks (wk). Results: Of 116 enrolled pts, 89 (77%) were eligible for efficacy evaluation; pts were considered ineligible by the SSC due to absence of measurable CF (n = 14) or organ damage considered unrelated to SM (n = 13). Median age was 64 years (range 25-82). Overall, 73 pts (82%) had ASM, including 57 (78%) with an AHNMD (ASM-AHNMD); 16 (18%) had MCL, including 6 (38%) with an AHNMD (MCL-AHNMD). AHNMDs included chronic myelomonocytic leukemia (n = 25), myelodysplastic/myeloproliferative neoplasm unclassifiable (n = 22), hypereosinophilic syndrome/chronic eosinophilic leukemia (HES/CEL; n = 5), and 11 other subtypes. A total of 37 pts (42%) had received ≥ 1 prior therapy (median 1; range 1-4). Seventy-seven pts (87%) were positive and 10 pts (11%) were negative for KIT D816V/Y/L mutation; 2 pts were not evaluable. The ORR was 60% (53/89); most responses were MRs (40/53, 75%), subtyped as incomplete remission (IR) in 36%, pure clinical response in 28%, and unspecified in 11%. PRs included good PR (GPR) in 21% and minor PR in 4% of pts. Overall, 12%, 11%, and 17% of pts had stable disease, progressive disease, or were not evaluable for response, respectively. After a median follow-up of 26 months (mo; range 12-54), the median duration of response (DOR) and median overall survival (OS) were 24.1 and 28.7 mo, respectively (Figure). Median OS in responders was 44.4 mo. Of 16 MCL pts, 8 responded, including 7 MRs (44%); median DOR was not reached (NR), with 3 IRs ongoing (49+, 33+, and 19+ mo). Median OS was 9.4 mo among all pts with MCL and NR among responders. Median change in MC burden in 72 evaluable pts was -59% (range -96% to 160%); 41 pts (57%) had ≥ 50% reduction. Median best change in serum tryptase level in 89 evaluable pts was -58% (range -99% to 185%); 32 pts (36%) had ≥ 50% reduction lasting ≥ 8 wk. Four of 5 responding ASM/MCL-HES/CEL pts (1 IR, 3 GPR) also had complete resolution of eosinophilia (mean % and absolute eosinophils at baseline: 57% and 12.4 × 109/L). Improvements in symptoms and QOL were consistent with those reported previously (Gotlib et al, Blood 2013). All 116 pts received ≥ 1 midostaurin dose and were evaluable for safety. Grade 3/4 drug-related hematologic adverse events (AEs) were neutropenia (5%), leukopenia (4%), febrile neutropenia (3%), anemia (3%), and thrombocytopenia (3%). Low-grade nausea was common but usually manageable with antiemetics. The most frequent grade 3/4 drug-related non-hematologic AEs included nausea (6%), vomiting (6%), fatigue (4%), and increased lipase/amylase (4%/3%). As of July 9, 2013, 65 pts had discontinued therapy, most commonly due to progression (n = 31) and AEs (n = 18; 11 were considered drug related [3 hematologic and 8 non-hematologic AEs]). Seven patients developed acute myeloid leukemia related to a prior AHNMD. Conclusions: Midostaurin exhibits a high rate of durable responses in pts with advanced SM, particularly in MCL, which has a dismal prognosis. The reductions in MC burden suggest that midostaurin may impact the natural history of the disease. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Gotlib: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees, Research Funding, Travel Support Other. George:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Akin:Novartis: Consultancy. Sotlar:Novartis Pharma: Laboratory Services, Laboratory Services Other; Nanostring Technologies: Consultancy. Hermine:Novartis: Research Funding. Awan:Boehringer Ingelheim: Consultancy; Lymphoma Research Foundation: Research Funding. Mauro:Ariad: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding; Novartis Oncology: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Speakers Bureau. Sternberg:Novartis Pharmaceuticals Corporation: Employment, Equity Ownership. Villeneuve:Novartis Pharma AG: Employment. Emery-Salbert:Novartis: Employment. Stanek:Novartis Pharmaceuticals Corporation: Employment. Hartmann:Novartis: Consultancy. Horny:Novartis: Consultancy. Valent:Novartis: Consultancy, Honoraria, Research Funding. Reiter:Novartis: Consultancy, Honoraria.

Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4290-4290
Author(s):  
Daniela Drandi ◽  
Lenka Kubiczkovà ◽  
Nadia Dani ◽  
Simone Ferrero ◽  
Luigia Monitillo ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Research Funding ◽  
Residual Disease ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Chain Gene ◽  
Attractive Alternative ◽  
Standard Curve ◽  
Real Time Quantitative Pcr

Abstract Background In mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. Methods Bone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. Results Overall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed major qualitative discordance. 28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR. Conclusions Here we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. Disclosures: Kubiczkovà: GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.

Digital PCR (dPCR) Overcomes the Limitations in Detection and in Quantification of Quantitative PCR (qPCR) and Reveals Different Levels of BCR-ABL1 Copies/µl Among the Chronic Myeloid Leukemia (CML) Patients Achieving Major (MR3.0) or DEEP (MR4.0, MR4.5 and MR5.0) Molecular Response with Tyrosin Kynase Inhibitors (TKIs)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4028-4028
Author(s):  
Simona Bernardi ◽  
Andrea Di Palma ◽  
Mario Tiribelli ◽  
Erika Codarin ◽  
Giuseppina Ruggeri ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Peripheral Blood ◽  
Research Funding ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Molecular Response ◽  
Healthy Controls ◽  
Deep Molecular Response ◽  
Different Levels ◽  
Detection And Quantification

Abstract Monitoring BCR-ABL1 transcript by quantitative PCR (qPCR) is essential for the management of CML patients treated with TKIs. Currently, up to 40-50% of CML patients treated with TKIs can achieve a deep molecular response (DMR = BCR-ABL1 minor or equal to0,01% IS), but only 50% of them are reported to maintain a stable Treatment Free Remission (TFR). Since qPCR has some intrinsic limitations in detection and quantification of BCR-ABL1 transcript, this method does not appear optimal in the selection of patients eligible for TKIs cessation. A precise monitoring of BCR-ABL1 transcript levels can help even better the clinicians in managing CML patients treated with TKIs and in selecting the best candidates for discontinuation of TKIs without relapse. Digital PCR (dPCR) can be more advantageous than qPCR. It gives the absolute quantification of target nucleic acidsby partitioning the PCR reaction mix over a large number of wells, each containing a single copy or no copies of the target region. Based on the assumption of Poisson's distribution, the number of template copies originally presenting in the sample can be calculated from the number of partitions in which amplification has successfully occurred. In that way, standard curves cannot be necessary and data are more accurate. In this study we set a dPCR assay to quantify the BCR-ABL1 transcript in a preliminary cohort of CML patients with major (MMR or MR3.0) or deep molecular response (MR4.0, MR4.5 and MR5.0). The analysis by dPCR were based on a TaqMan-MGB probes targeting the BCR-ABL1 transcript. Custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. The experiments were performed on a QuantStudio 3D Digital PCR System (Life Technologies). A commercial BCR-ABL1 Mbcr standard dilutions (Qiagen) and 10 replicates of blank samples (DNA-free, RNA-free water) were used to set dPCR assay and to determine the Limit of Detection and the Limit of Quantification of our method. The values of absolute quantities of BCR-ABL1 transcript assessed by dPCR were expressed as number of copies/ul. Samples for dPCR testing were obtained from peripheral blood (10 ml in EDTA tubes) of CML patients treated with TKIs, namely imatinib, nilotinib, or dasatinib, on time-checks planned for monitoring MMR or DMR through the conventional qPCR. To the purpose of the study, we evaluated 10 cases with stable MR3.0, 7 cases with stable MR4.0, 6 cases with stable MR4.5, 7 cases with stable MR5.0. It has been considered as stable any MR3.0, MR4.0, MR4.5, or MR5.0 molecular response measured by conventional qPCR and detected in the last three consecutive checks performed during the last 12 months. Blank samples served as negative controls, while 10 peripheral blood samples of healthy donors served as normal controls. Digital PCR (dPCR) revealed different levels of BCR-ABL1 copies/µl among the CML patients achieving the major (MR3.0) or deep (MR4.0, MR4.5 AND MR5.0) molecular response with TKIs. Moving from MR3.0 to MR5.0 molecular response the median of BCR-ABL1 copies/µl assessed by dPCR were progressively decreasing (Figure 1), with blanks and healthy controls approximately to zero. Medians with ranges were 0.957 (0.472-1.692) for MR3.0; 0.319 (0.072-0.906) for MR4.0; 0.231 (0.063-0.651) for MR4.5; 0.219 (0.074-0.399) for MR5.0; 0.076 (0.000-0.118) for healthy controls, and 0.000 (0.000-0.129) for blanks. Moreover, dPCR revealed different BCR-ABL1 copies/µl among the patients of each class of molecular response. Importantly, in patients with MR4.5, MR5.0 and with undetectable levels of BCR-ABL1 % IS as measured with qPCR, discrete variable levels of BCR-ABL1 copies/µl have been detected by dPCR. These data, revealing different levels of BCR-ABL1 copies/µl beyond the limit of detection and quantification of conventional qPCR, may explain why no correlation was observed between BCR-ABL1 % IS levels measured by qPCR and numbers of BCR-ABL1 copies/µl measured by dPCR. We are screening CML patients with MMR and DMR, but these preliminary results show that dPCR appears to be more accurate than qPCR for detection and quantification of BCR-ABL1 transcript and it should be seen as a useful step forward in order to better manage the TKI therapy and to better select the candidates for TFR. Acknowledgments: Department of Clinical and Experimental Sciences, University of Brescia; PRIN2009; European Leukaemia Net; BCC "Pompiano e Franciacorta". Figure 1. Figure 1. Disclosures Tiribelli: Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Martinelli:Roche: Consultancy; BMS: Speakers Bureau; MSD: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

The Multi-Kinase Inhibitor DCC-2618 Inhibits Proliferation and Survival of Neoplastic Mast Cells and Other Cell Types Involved in Systemic Mastocytosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1965-1965 ◽  
Author(s):  
Mathias A Schneeweiss ◽  
Barbara Peter ◽  
Katharina Blatt ◽  
Daniela Berger ◽  
Gabriele Stefanzl ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Mast Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Kinase Inhibitor ◽  
Vascular Endothelial Cells ◽  
Systemic Mastocytosis ◽  
Leukemia Cell ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) is a myeloid neoplasm defined by abnormal growth and pathologic accumulation of neoplastic mast cells (MC) in various internal organs. The indolent variant of SM (ISM) is associated with an almost normal life expectancy. By contrast, the prognosis in advanced SM, including SM with an associated hematologic neoplasm (AHN), aggressive SM (ASM), and MC leukemia (MCL) is poor with short survival times. Most patients with SM express the D816V-mutated variant of KIT, which confers resistance against several tyrosine kinase inhibitors (TKI), including imatinib. Midostaurin is a TKI that is effective against KIT D816V. However, despite encouraging clinical efficacy, this drug cannot produce continuous complete remission in all patients. One problem in advanced SM is that the AHN component of the disease, especially when progressing into acute myeloid leukemia (AML) is often drug-resistant. The aims of this study were to evaluate the effects of the multi-kinase inhibitor DCC-2618 on proliferation and survival of primary neoplastic mast cells, various mast cell lines and other malignant and non-malignant cell types that may play a role in advanced SM. As assessed by 3H-thymidine-uptake, DCC-2618 was found to inhibit the proliferation of all human MC lines tested, with lower IC50 values measured in HMC-1.1 cells lacking KIT D816V (11.2±4.3 nM) and ROSAKIT WT cells (61±11 nM) than in KIT D816V+ HMC-1.2 cells (147±68 nM) and ROSAKIT D816V cells (133±43 nM). DCC-2618 also produced growth inhibition in the multi-resistant MCL lines MCPV-1.1 (164±72 nM), MCPV-1.2 (256±167 nM), MCPV-1.3 (124±46 nM), and MCPV-1.4 (235±114 nM). In addition, DCC-2618 was found to inhibit the proliferation of primary neoplastic bone marrow MC obtained from patients with SM including MCL (Figure). We also found that DCC-2618 induces apoptosis in HMC-1 cells and ROSA cells, and to a lesser degree in MCPV-1 cells as determined by light microscopy and AnnexinV/PI staining. Moreover, DCC-2618 was found to block phosphorylation of KIT in all MC lines tested. In a next step, we explored the effects of DCC-2618 on growth of other leukemia cell lines as well on vascular endothelial cells. In these experiments, we were able to show that DCC-2618 inhibits the proliferation of the FIP1L1-PDGFRA+ eosinophilic leukemia cell line EOL-1 (IC50 2±0.6 nM) and the FLT3 ITD-mutated AML cell lines MV4-11 (IC50 130±18 nM) and MOLM-13 (IC50 110±26 nM). DCC-2618 also induced apoptosis in EOL-1, MV-411, and MOLM-13 cells. Moreover, DCC-2618 was found to inhibit the growth of cultured human vascular endothelial cells, suggesting that the drug may also counteract SM-related neo-angiogenesis in SM. DCC-2618 did not inhibit the proliferation of the immature AML cell line KG1 and the monoblastic cell line U937, but was found to block proliferation in primary leukemic monocytes in patients with monoblastic AML or chronic myelomonocytic leukemia (CMML) which may have clinical implications as CMML and AML are the most prevalent types of AHN in advanced SM. Finally, we were able to show that the major DCC-2618-metabolite, DP-5439, is equally effective in producing growth inhibition in all cell lines tested as well as in primary neoplastic MC compared to DCC-2618 (Figure). In summary, our data show that DCC-2618 is a new potent multi-targeted TKI that counteracts growth of neoplastic MC as well as growth and survival of leukemic monocytes, AML blasts, eosinophils, and endothelial cells in vitro. Whether DCC-2618 is also able to inhibit the growth of neoplastic MC and other leukemic (AHN) cells in vivo in patients with advanced SM remains to be determined in clinical trials. Indeed, a first Phase I clinical trial examining the effects of DCC-2618 in SM has recently been initiated. Figure Figure. Disclosures Valent: Novartis: Honoraria, Research Funding; Amgen: Honoraria; Celgene: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Deciphera Pharmaceuticals: Research Funding.

Impact of Molecular Markers on Response and Resistance in Midostaurin-Treated Patients with Advanced Systemic Mastocytosis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 945-945 ◽  
Author(s):  
Mohamad Jawhar ◽  
Juliana Schwaab ◽  
Nicole Naumann ◽  
Sebastian Kluger ◽  
Hans-Peter Horny ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Median Time ◽  
Research Funding ◽  
Sequential Analysis ◽  
Target Genes ◽  
Systemic Mastocytosis ◽  
Molecular Response ◽  
Secondary Resistance ◽  
The Impact ◽  
Kit D816v

Abstract Systemic mastocytosis (SM) arises as a consequence of aberrant activation of KIT signaling, most commonly by acquisition of the KIT D816V mutation (>80-90% of patients). A recently reported phase-II-study exploring the efficacy and safety of midostaurin, a multi-targeted KIT inhibitor, in patients with advanced SM (advSM) has demonstrated major and partial responses in 60% of patients (Gotlib et al., NEJM 374, 2016). However, recent data have highlighted that the molecular pathogenesis of SM is complex. In particular, additional mutations in SRSF2, ASXL1 and/or RUNX1 (S/A/Rpos), seen in 60-70% of patients with advSM, have a significant adverse impact on disease phenotype and prognosis (Jawhar et al., Leukemia 30, 2016). In the current study, we evaluated the impact of molecular markers on response, resistance, progression and overall survival (OS) in midostaurin-treated advSM patients: 1) the S/A/R mutation profile at diagnosis, 2) the reduction of the KIT D816V allele burden (EAB) and 3) the dynamics of the clonal architecture. Between 2009 and 2015, 38 patients (68% male; median age 67 years; range, 48-76) with advSM [SM with an associated hematological neoplasm (SM-AHN), n=22; aggressive SM (ASM), n=3; mast cell leukemia (MCL), n=4; MCL-AHN, n=9] received midostaurin at our institution and were monitored for the KIT D816V EAB by quantitative RT-PCR (RT-qPCR). In addition, serial Next Generation Sequencing (NGS) analyses were performed in 16/25 multi-mutated patients. Median time from diagnosis of advSM to start of midostaurin was 12 months (range, 1-60). Twenty-one of 38 (55%) patients died; median OS was 29 months (range, 0-88) from start of midostaurin and 40 months (range, 5-115) from diagnosis. Three patients (8%) stopped midostaurin early (median 2 months; range, 1-4) because of intolerance. In the remaining 35 (92%) patients, median treatment duration was 13 months (range, 1-88). Progression/death occurred in 6/35 (17%) patients within the first 6 months and 5 (83%) patients were S/A/Rpos. Overall response rate (ORR) according to IWG-MRI-ECNM consensus criteria and OS were significantly different between S/A/Rpos (n=23) vs. S/A/Rneg (n=12) patients [ORR: 35% vs. 75%, P=0.01; OS: P=0.01, HR 4.5 (1.3-16.2), Figure 1A]. The maximal median reduction of KIT D816V EAB in 28 patients treated for >6 months was -29% (range, -100 to 71). Depending on the KIT D816V EAB at month 6, patients were classified as responders (≥25%, n=17) or non-responders (<25%, n=11). Responders were significantly associated with a longer median time on midostaurin (25 vs. 9 months, P=0.01) and achievement of any clinical response according to IWG-MRI-ECNM criteria (13/17 vs. 2/11 patients, P=0.006). All 4 patients who lost their KIT D816V EAB response were S/A/Rpos. In univariate analyses of multiple clinical and molecular response parameters at month 6, three parameters were significantly associated with inferior OS: reduction of KIT D816V EAB <25% (P=0.0004), serum tryptase <50% (P=0.03) and alkaline phosphatase <50% (P=0.04). In multivariate analysis, only KIT D816V EAB reduction <25% remained an independent poor prognostic marker for reduced OS (P=0.004, HR 6.8 [1.8-25.3]) and was superior to the IWG-MRI-ECNM consensus criteria (P=0.005, HR 4.7 [1.5-15.5]) (Figure 1B). Serial NGS analysis of 7 deceased patients revealed acquisition of additional mutations in RUNX1 (n=2), K/NRAS (n=3), IDH2 (n=1) or NPM1 (n=1) and/or increasing mutation allele burdens while KIT D816V EAB was either low (n=2), stable (n=1) or increasing (n=4). In contrast, two patients acquired a JAK2 V617F mutation but remained in remission on midostaurin. In summary, we have found that S/A/Rneg at diagnosis and reduction of the KIT D816V EAB ≥25% at month 6 were the most favorable predictors for survival in midostaurin-treated advSM patients. S/A/Rpos at diagnosis was associated with a more aggressive phenotype, (early) resistance/progression and poor survival. Serial sequencing during treatment also revealed that secondary resistance/transformation may be caused by expansion of sub-clones exhibiting new mutations in critical target genes independent of KIT D816V. In addition to established response parameters, sequential analysis of KIT D816V EAB and other molecular aberrations add relevant information for optimal management and response assessment in midostaurin-treated patients with advSM. Disclosures Valent: Celegene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Amgen: Honoraria. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

scholarly journals Probe-Based Multiplex Real-Time PCR as a Diagnostic Tool to Distinguish Distinct Fungal Symbionts Associated With Euwallacea kuroshio and Euwallacea whitfordiodendrus in California

Plant Disease ◽  
2020 ◽  
Vol 104 (1) ◽  
pp. 227-238 ◽  
Author(s):  
Joseph D. Carrillo ◽  
Joey S. Mayorquin ◽  
Jason E. Stajich ◽  
Akif Eskalen
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Absolute Quantification ◽  
Coefficient Of Determination ◽  
Fungal Isolation ◽  
Real Time Quantitative Pcr ◽  
Field Samples ◽  
Symbiotic Fungi ◽  
Fusarium Dieback ◽  
Platanus Racemosa

California has been invaded by two distinct Euwallacea spp. that vector unique plant pathogenic symbiotic fungi on multiple hosts and cause Fusarium dieback. The objective of this study was to develop multiplex real-time quantitative PCR assays using hydrolysis probes targeting the β-tubulin gene to detect, distinguish, and quantify fungi associated with the polyphagous shot hole borer (PSHB; Euwallacea whitfordiodendrus, Fusarium euwallaceae, Graphium euwallaceae, and Paracremonium pembeum) as well as the Kuroshio shot hole borer (KSHB; Euwallacea kuroshio, Fusarium kuroshium, and Graphium kuroshium) from various sample types. Absolute quantification reaction efficiencies ranged from 88.2 to 104.3%, with a coefficient of determination >0.992 and a limit of detection of 100 copies µl−1 for all targets across both assays. Qualitative detection using the real-time assays on artificially inoculated avocado shoot extracts showed more sensitivity compared with conventional fungal isolation from wood. All symbiotic fungi, except P. pembeum, from PSHB and KSHB female heads were detectable and quantified. Field samples from symptomatic Platanus racemosa, Populus spp., and Salix spp. across 17 of 26 city parks were positively identified as PSHB and KSHB through detection of their symbiotic fungi, and both were found occurring together on five trees from three different park locations. The molecular assays presented here can be utilized to accurately identify fungi associated with these invasive pests in California.

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