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1.
Br J Haematol ; 194(1): 53-60, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34114218

RESUMO

Measurement of BCR activator of RhoGEF and GTPase -ABL proto-oncogene 1, non-receptor tyrosine kinase (BCR-ABL1) mRNA levels by reverse transcription quantitative polymerase chain reaction (RTqPCR) has been critical to treatment protocols and clinical trials in chronic myeloid leukaemia; however, interlaboratory variation remains a significant issue. Reverse transcriptase droplet digital PCR (RTddPCR) has shown potential to improve testing but a large-scale interlaboratory study is required to definitively establish this. In the present study, 10 BCR-ABL1-positive samples with levels ranging from molecular response (MR)1·0 -MR5·0 were tested by 23 laboratories using RTddPCR with the QXDX BCR-ABL %IS kit. A subset of participants tested the samples using RTqPCR. All 23 participants using RTddPCR detected BCR-ABL1 in all samples to MR4·0 . Detection rates for deep-response samples were 95·7% at MR4·5 , 78·3% at MR4·7 and 87·0% at MR5·0 . Interlaboratory coefficient of variation was indirectly proportional to BCR-ABL1 level ranging from 29·3% to 69·0%. Linearity ranged from 0·9330 to 1·000 (average 0·9936). When results were compared for the 11 participants who performed both RTddPCR and RTqPCR, RTddPCR showed a similar limit of detection to RTqPCR with reduced interlaboratory variation and better assay linearity. The ability to detect deep responses with RTddPCR, matched with an improved linearity and reduced interlaboratory variation will allow improved patient management, and is of particular importance for future clinical trials focussed on achieving and maintaining treatment-free remission.


Assuntos
Proteínas de Fusão bcr-abl/sangue , Ensaio de Proficiência Laboratorial , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Ásia , Biomarcadores Tumorais/sangue , Europa (Continente) , Células HL-60/química , Humanos , Células K562/química , Laboratórios Clínicos , Modelos Lineares , América do Norte , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes
3.
J Mol Diagn ; 22(1): 81-89, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669230

RESUMO

Real-time quantitative PCR (qPCR) is routinely used to detect minimal residual disease in chronic myeloid leukemia patients. The absolute quantification with droplet digital PCR (ddPCR) could reduce the inherent variability of qPCR. We established a duplex ddPCR assay using the Europe against Cancer (EAC) primer/probe system for breakpoint cluster region protein-tyrosine-protein kinase ABL1 (BCR-ABL1) and ABL1 and compared the results with qPCR. cDNA samples (n = 230) from patients with chronic myeloid leukemia were analyzed using both procedures. A second, commercially developed ddPCR assay for BCR-ABL1 was also evaluated. ABL1 and BCR-ABL1 transcript levels were similar with all assays, but the proportion of deep molecular responses was lower with ddPCR than with qPCR. The EAC ddPCR assay had a false-positive rate of 4% using a cutoff of three BCR-ABL1 copies per duplicate, compared with 2% without cutoff for the commercial ddPCR. The detection rate for molecular response 4.5 was 100, and a shift toward more minimal residual disease was seen in patient samples. In conclusion, using the EAC protocol for BCR-ABL1 quantification with ddPCR is feasible and shows low intra-assay and interassay variation but requires a cutoff that reduces sensitivity. The commercial ddPCR assay is highly sensitive and specific for BCR-ABL1. The use of either ddPCR assay resulted in a shift to lower molecular response classes compared with qPCR aligned to international scale.


Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas Proto-Oncogênicas c-abl/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estudos de Coortes , Estudos de Viabilidade , Células HeLa , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Monitorização Fisiológica/métodos , Neoplasia Residual/genética , Inibidores de Proteínas Quinases/uso terapêutico , Sensibilidade e Especificidade
4.
J Cancer Res Clin Oncol ; 145(6): 1645-1650, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30941573

RESUMO

PURPOSE: The advent of tyrosine kinase inhibitor (TKI) therapies has revolutionized the treatment of chronic myeloid leukemia (CML). The European LeukemiaNet (ELN) recommends quantification of BCR-ABL1 transcripts by real-time quantitative PCR every 3 months during TKI treatment. Since a proportion of patients in deep molecular response (DMR: MR4, MR4.5, MR5) maintain remission after treatment stop, assessment of DMR is crucial. However, systematically collected molecular data, monitored with sensitive standardized assays, are not available outside clinical trials. METHODS: Data were collected on the standardized assessment of molecular response in the context of real-life practice. BCR-ABL1 transcript levels after > 2 years of TKI therapy were evaluated for DMR by local laboratories as well as standardized EUTOS laboratories. Since standardized molecular monitoring is a prerequisite for treatment discontinuation, central surveillance of the performance of the participating laboratories was carried out. RESULTS: Between 2014 and 2017, 3377 peripheral blood samples from 1117 CML patients were shipped to 11 standardized reference laboratories in six European countries. BCR-ABL1 transcript types were b3a2 (41.63%), b2a2 (29.99%), b2a2/b3a2 (3.58%) and atypical (0.54%). For 23.72% of the patients, the initial transcript type had not been reported. Response levels (EUTOS laboratory) were: no MMR, n = 197 (6.51%); MMR, n = 496 (16.40%); MR4, n = 685 (22.64%); MR4.5, n = 937 (30.98%); MR5, n = 710 (23.47%). With a Cohen's kappa coefficient of 0.708, a substantial agreement between EUTOS-certified and local laboratories was shown. CONCLUSIONS: Multicenter DMR assessment is feasible in the context of real-life clinical practice in Europe. Information on the BCR-ABL1 transcript type at diagnosis is crucial to accurately monitor patients' molecular response during or after TKI therapy.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide de Fase Crônica/tratamento farmacológico , Leucemia Mieloide de Fase Crônica/genética , Inibidores de Proteínas Quinases/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Europa (Continente) , Humanos , Laboratórios/normas , Laboratórios/estatística & dados numéricos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mieloide de Fase Crônica/sangue , Pessoa de Meia-Idade , Sistema de Registros , Adulto Jovem
5.
Int J Cancer ; 145(8): 2292-2303, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30882891

RESUMO

This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.


Assuntos
DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA de Neoplasias/sangue , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde/métodos , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêutico
6.
J Mol Diagn ; 21(1): 27-37, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30347270

RESUMO

Quantitative real-time PCR methods are commonly used to monitor BCR-ABL transcript levels in patients with chronic myelogenous leukemia. However, standard techniques involve separate measurements of target and reference DNAs, require standard curves, and are susceptible to PCR inhibition. An optimized duplex droplet digital PCR (ddPCR) should provide absolute quantification without the need for standard curves. The combination of high sensitivity and low background is particularly important for reliable monitoring of minimal residual disease. In this report, we describe primer probe set testing and step by step optimization of a duplex ddPCR for BCR-ABL/ABL. The optimization of ddPCR parameters increased ABL and BCR-ABL fluorescence signals by two- and fivefold, respectively, and enhanced the resolution between positive and negative drops. The optimized procedure generates a background false-positive rate of 5% of samples and reliably detects BCR-ABL/ABL down to 1/100,000 (CV <10%), with a single BCR-ABL copy being detected in 54% of reactions performed in duplicate from a molecular remission (MR) 5 sample (limit of detection = 1). Assay of duplicates resulted in a detection rate of 100% and 92% for MR4 and MR4.5, respectively. Detection of MR4.5 was increased to 100% by analyzing quadruplicates. Selection of an optimal primer and probe combination and stepwise optimization of the ddPCR conditions has yielded a robust low-background duplex ddPCR procedure for BCR-ABL/ABL.


Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Reação em Cadeia da Polimerase/métodos , Linhagem Celular Tumoral , Primers do DNA/genética , Sondas de DNA/genética , Dosagem de Genes , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética
7.
Oncotarget ; 9(17): 13870-13883, 2018 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-29568401

RESUMO

BACKGROUND: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic KIT mutations that cluster in two exon 11 hotspots. The aim of this study was to develop a single, sensitive, quantitative digital droplet PCR (ddPCR) assay for the detection of common exon 11 mutations in both GIST tumor tissue and in circulating tumor DNA (ctDNA) isolated from GIST patients' plasma. METHODS: A ddPCR assay was designed using two probes that cover both hotspots. Available archival FFPE tumor tissue from 27 consecutive patients with known KIT exon 11 mutations and 9 randomly selected patients without exon 11 mutations were tested. Plasma samples were prospectively collected in a multicenter bio-databank from December 2014. ctDNA was analyzed of 22 patients with an exon 11 mutation and a baseline plasma sample. RESULTS: The ddPCR assay detected the exon 11 mutation in 21 of 22 tumors with exon 11 mutations covered by the assay. Mutations in ctDNA were detected at baseline in 13 of 14 metastasized patients, but in only 1 of 8 patients with localized disease. In serial plasma samples from 11 patients with metastasized GIST, a decrease in mutant droplets was detected during treatment. According to RECIST 1.1, 10 patients had radiological treatment response and one patient stable disease. CONCLUSION: A single ddPCR assay for the detection of multiple exon 11 mutations in ctDNA is a feasible, promising tool for monitoring treatment response in patients with metastasized GIST and should be further evaluated in a larger cohort.

8.
Clin Cancer Res ; 19(17): 4854-67, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23833305

RESUMO

PURPOSE: In gastrointestinal stromal tumor (GIST), there is no biomarker available that indicates success or failure of therapy. We hypothesized that tumor-specific v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (CKIT)- or platelet-derived growth factor receptor-α (PDGFRA)-mutant DNA fragments can be detected and quantified in plasma samples of patients with GIST. EXPERIMENTAL DESIGN: We prospectively collected 291 plasma samples from 38 subjects with GIST harboring activating mutations of CKIT or PDGFRA detected in tumor tissue, irrespective of current disease status or treatment. We used allele-specific ligation PCR to detect mutant free circulating DNA (fcDNA). RESULTS: We were able to detect fcDNA harboring the tumor mutation in 15 of 38 patients. Patients with active disease displayed significantly higher amounts of mutant fcDNA compared with patients in complete remission (CR). The amount of mutant fcDNA correlated with disease course. We observed repeated positive test results or an increase of mutant fcDNA in five patients with progressive disease or relapse. A decline of tumor fcDNA or conversion from positive to negative was seen in five patients responding to treatment. A negative to positive conversion was seen in two patients with relapse and one patient with progression. In two cases, we aimed to identify additional mutations and found four additional exchanges, including mutations not known from sequentially conducted tumor biopsies. CONCLUSIONS: Our results indicate that fcDNA harboring tumor-specific mutations in the plasma of patients with GIST can be used as tumor-specific biomarker. The detection of resistance mutations in plasma samples might allow earlier treatment changes and obviates the need for repeated tumor biopsies.


Assuntos
Biomarcadores Tumorais/sangue , DNA/sangue , Tumores do Estroma Gastrointestinal/sangue , Tumores do Estroma Gastrointestinal/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adulto , Animais , DNA/genética , DNA/isolamento & purificação , Feminino , Tumores do Estroma Gastrointestinal/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Células Neoplásicas Circulantes , Prognóstico
9.
Blood ; 121(20): 4175-83, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23543457

RESUMO

Genomic instability is a hallmark of chronic myeloid leukemia in chronic phase (CML-CP) resulting in BCR-ABL1 mutations encoding resistance to tyrosine kinase inhibitors (TKIs) and/or additional chromosomal aberrations leading to disease relapse and/or malignant progression. TKI-naive and TKI-treated leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) accumulate high levels of reactive oxygen species (ROS) and oxidative DNA damage. To determine the role of TKI-refractory LSCs in genomic instability, we used a murine model of CML-CP where ROS-induced oxidative DNA damage was elevated in LSCs, including quiescent LSCs, but not in LPCs. ROS-induced oxidative DNA damage in LSCs caused clinically relevant genomic instability in CML-CP-like mice, such as TKI-resistant BCR-ABL1 mutations (E255K, T315I, H396P), deletions in Ikzf1 and Trp53, and additions in Zfp423 and Idh1. Despite inhibition of BCR-ABL1 kinase, imatinib did not downregulate ROS and oxidative DNA damage in TKI-refractory LSCs to the levels detected in normal cells, and CML-CP-like mice treated with imatinib continued to accumulate clinically relevant genetic aberrations. Inhibition of class I p21-activated protein kinases by IPA3 downregulated ROS in TKI-naive and TKI-treated LSCs. Altogether, we postulate that genomic instability may originate in the most primitive TKI-refractory LSCs in TKI-naive and TKI-treated patients.


Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Instabilidade Genômica , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Células-Tronco Neoplásicas/fisiologia , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Animais , Células Cultivadas , Dano ao DNA/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Instabilidade Genômica/fisiologia , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos , Camundongos Transgênicos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo
10.
Haematologica ; 98(5): 722-8, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23300178

RESUMO

The risk profile and prognosis of patients with myelofibrosis is well described by the Dynamic International Prognostic Scoring System risk categorization. Allogeneic stem cell transplantation is considered for intermediate-2/high risk disease. However, indicators of prognosis after transplantation are still lacking. Seventy simultaneously collected pairs of trephine and blood samples were quantified for JAK2 p.V617F allele burden to compare test sensitivity. The course of 30 patients with JAK2 p.V617F-positive myeloproliferative neoplasia was correlated with allele burden after transplantation. Monitoring can be performed on full blood samples as well as trephine biopsies, provided that techniques with ample sensitivity (0.01% to 0.001%) are available. Measurement of allele burden on day 28 after transplantation discriminates two prognostic groups: patients with a JAK2 p.V617F allele burden >1% have a significantly higher risk of relapse of JAK2 p.V617F positive neoplasia (P=0.04) and a poorer overall survival (P<0.01). In conclusion, measurement of JAK2 p.V617F allele burden early after transplantation is an important predictive parameter in monitoring patients following this treatment. As this might provide an important tool in early management of imminent early relapse it will be important to define consensus guidelines for optimal monitoring.


Assuntos
Alelos , Janus Quinase 2/genética , Mutação , Transtornos Mieloproliferativos/genética , Idoso , Feminino , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/mortalidade , Transtornos Mieloproliferativos/terapia , Prognóstico , Recidiva , Transplante Homólogo , Resultado do Tratamento
11.
Haematologica ; 98(5): 714-7, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23065514

RESUMO

The BCR-ABL T315I mutation causes resistance to imatinib, nilotinib and dasatinib in chronic myeloid leukemia. Forty BCR-ABL positive patients with imatinib resistance were analyzed for T315I mutated clones after six months on nilotinib or dasatinib treatment by quantitative allele-specific ligation polymerase chain reaction with a sensitivity of 0.05%. Ligation polymerase chain reaction revealed 10 patients with more than 10(-5) BCR-ABL(T315I%)/GUS (high levels), none of whom achieved major molecular response after 12 months, and a further 8 patients with 10(-5) or below BCR-ABL(T315I%)/GUS (low levels) who all achieved major molecular response (P<0.001). A second independent group showed molecular response in one of 12 patients with high levels and 5 of 8 patients with low levels (P=0.018). Combining the groups resulted in a sensitivity and specificity of 92.9% and 87.5%, respectively. We conclude that the quantitative level of mutant T315I allele is predictive of major molecular response at 12 months on second-line nilotinib or dasatinib treatment. www.clinicaltrials.gov: CT00109707, NCT00384228, CA180013, CA180005 CA180006.


Assuntos
Antineoplásicos/uso terapêutico , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Pirimidinas/uso terapêutico , Tiazóis/uso terapêutico , Adulto , Idoso , Alelos , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Inibidores de Proteínas Quinases/uso terapêutico , Resultado do Tratamento
12.
Haematologica ; 94(9): 1227-35, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19608684

RESUMO

BACKGROUND: Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations. DESIGN AND METHODS: We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed. RESULTS: One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography. CONCLUSIONS: Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/genética , Pirimidinas/administração & dosagem , Tiazóis/administração & dosagem , Adulto , Idoso , Benzamidas , Análise Mutacional de DNA , Dasatinibe , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Masculino , Pessoa de Meia-Idade , Piperazinas/administração & dosagem , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/antagonistas & inibidores
13.
Nat Clin Pract Endocrinol Metab ; 3(10): 713-20, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17893690

RESUMO

Somatic mutations can be identified in two-thirds of papillary and follicular thyroid carcinomas and 'hot' thyroid nodules, whereas equivalent mutations relevant for benign 'cold' thyroid nodules are unknown. This Review summarizes current knowledge about early molecular conditions for nodular and tumor transformation in the thyroid gland. We reconstruct a line of events that could explain the predominant neoplastic character (i.e. originating from a single mutated cell) of thyroid nodular lesions. This process might be triggered by the oxidative nature of thyroid hormone synthesis or additional oxidative stress caused by iodine deficiency or smoking. If the antioxidant defense is not effective, this oxidative stress can cause DNA damage followed by an increase in the spontaneous mutation rate, which is a platform for tumor genesis. The hallmark of thyroid physiology--H2O2 production during hormone synthesis--is therefore very likely to be the ultimate cause of frequent mutagenesis in the thyroid gland. DNA damage and mutagenesis could provide the basis for the frequent nodular transformation of endemic goiters.


Assuntos
Dano ao DNA , Peróxido de Hidrogênio/metabolismo , Mutagênese , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo , Animais , Predisposição Genética para Doença , Humanos
14.
Thyroid ; 15(9): 1089-94, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16187920

RESUMO

OBJECTIVE: Hereditary nonautoimmune hyperthyroidism is caused by activating thyrotropin receptor (TSHR) germline mutations. We describe a family from Saxony, Germany, with this condition. Recurrent thyrotoxicosis and goiter were prevalent in three generations, affecting in addition to the 16-year-old index patient, her father and late paternal grandmother. Hyperthyroidism in the girl was remarkable for its poor response to methimazole (40-60 mg/d) treatment. METHODS AND RESULTS: Molecular analysis of genomic DNA extracted from peripheral blood leucocytes showed a TSHR germline mutation in the girl and her father. This mutation results in a new amino acid exchange of valine for isoleucine in TSHR codon 568 (Ile568Val). Only the wild-type TSHR sequence was found in the girl's mother. On functional characterization in COS-7 cells, the novel Ile568Val TSHR mutation conferred constitutive activation of the cAMP pathway (2- to 3-fold increase of basal cyclic adenosine monophosphate [cAMP]), but not of the inositol phosphate cascade. As a consequence of the molecular findings, total thyroidectomy has been performed in the girl. She is now euthyroid on levothyroxine replacement therapy. CONCLUSION: This is the second Saxonian family with autosomal-dominant nonautoimmune hyperthyroidism, adding to a total of 13 families and 11 individuals with activating TSHR germline mutations worldwide. We suggest that the condition may indeed be more frequent than previously thought and that consequent assessment of a family history in children as well as adults with thyroid autonomy will contribute to ensure correct diagnosis and adequate treatment of patients with activating TSHR germline mutations.


Assuntos
Hipertireoidismo/genética , Mutação/fisiologia , Receptores da Tireotropina/genética , Tireoidite Autoimune/genética , Adolescente , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , DNA/genética , Feminino , Citometria de Fluxo , Alemanha , Humanos , Fosfatos de Inositol , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tireotropina/metabolismo
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