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1.
Am J Surg Pathol ; 44(5): 626-632, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32141886

RESUMO

Papillary fibroelastoma (PFE) is an increasingly recognized cardiac tumor. Despite its prevalence, controversy exists as to whether it represents a reactive or neoplastic process due to histopathologic similarities with Lambl excrescences (LEs), an accepted reactive phenomenon. Recently, KRAS mutations were reported in a small collection of PFEs, but the incidence of mutations and conditions in which they arise in are unknown. Furthermore, the relationship between PFE and LE has yet to be investigated. Institutional archives were queried for cases of PFE (2001-2017). Paraffin-embedded tissue was microdissected for tumor isolation. Prospectively identified LEs (2018) were collected and wholly isolated. Extracted DNA underwent droplet digital polymerase chain reaction analysis of the most common KRAS mutations (codons 12/13 and 61). Relevant clinical information was abstracted from the medical record. Fifty-two PFEs were tested from 50 patients (32 women). The median patient age was 67 years. Seventeen (33%) PFEs harbored pathogenic variants in tested KRAS codons (12 in codons 12/13; 5 in codon 61). Mutations were mutually exclusive. No clinical or pathologic correlates differed significantly from cases without detectable pathogenic variants. No pathogenic mutation were detected in LEs (n=20; P=0.002). Herein, we report on the largest series of PFE tested for KRAS mutations and present the largest cohort of KRAS-mutant PFEs to date, providing evidence in support of the notion that at least a subset of PFEs represents neoplasia. Moreover, the lack of KRAS mutations in LEs provides evidence as to the separate etiology of this accepted reactive lesion.

2.
Eur J Endocrinol ; 182(2): K7-K13, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31804968

RESUMO

Introduction: Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. Case description: A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5'-CYP21A1P/CYP21A2-3' hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. Conclusions: The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

3.
J Proteome Res ; 19(1): 186-193, 2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31736316

RESUMO

Insulin-like growth factor-1 (IGF-1) measurement by high-resolution accurate mass-mass spectrometry (HRAM-MS) is replacing IGF-1 immunoassays and allows for identification of single amino acid variants; by contrast, both normal and deleterious sequence variants might be missed by immunoassays or non-HRAM-MS methods. We have developed an intact molecule HRAM-MS method to identify IGF-1 variants, distinguishing them by a center of mass (COM) calculation, followed by various tandem-MS activation techniques (HCD, ETD, ETciD, EThcD, UVPD). We found single amino acid variants in 841 of 146 620 patient samples (0.57%). Most were benign (A67T, A70T). We also observed a pathogenic variant (V44M), likely pathogenic variants (A38V, V17M), and a likely benign variant (A67V). For 207 samples from unique patients with residual serum, the MS variant results were confirmed by cell-free DNA sequencing. Our approach allows accurate quantitative reporting of functional IGF-1 in the presence of single amino acid variants. The COM approach potentially enables omission of tandem-MS for known, common variants, while the combination of COM and tandem-MS allows accurate identification in all cases we encountered. This approach should be applicable to qualitative and quantitative analyses of other peptides/proteins in clinical and research settings and might lend itself to the characterization of other protein variations.

4.
J Neuropathol Exp Neurol ; 78(11): 1011-1021, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31562743

RESUMO

MAPK pathway activation has been recurrently observed in desmoplastic infantile ganglioglioma/astrocytoma (DIG/DIA) with reported disproportionally low mutation allele frequencies relative to the apparent high tumor content, suggesting that MAPK pathway alterations may be subclonal. We sought to expand the number of molecularly profiled cases and investigate if tumor cell composition could account for the observed low mutation allele frequencies. Molecular (targeted neuro-oncology next-generation sequencing/RNA sequencing and OncoScan microarray) and immunohistochemical (CD68-PGM1/CD163/CD14/CD11c/lysozyme/CD3/CD20/CD34/PD-L1) studies were performed in 7 DIG. Activating MAPK pathway alterations were identified in 4 (57%) cases: 3 had a BRAF mutation (V600E/V600D/V600_W604delinsDQTDG, at 8%-27% variant allele frequency) and 1 showed a TPM3-NRTK1 fusion. Copy number changes were infrequent and nonrecurrent. All tumors had at least 30% of cells morphologically and immunophenotypically consistent with microglial/macrophage lineage. Two subtotally resected tumors regrew; 1 was re-excised and received adjuvant treatment (chemotherapy/targeted therapy), with clinical response to targeted therapy only. Even with residual tumor, all patients are alive (median follow-up, 83 months; 19-139). This study further supports DIG as another MAPK pathway-driven neuroepithelial tumor, thus expanding potential treatment options for tumors not amenable to surgical cure, and suggests that DIG is a microglia/macrophage-rich neuroepithelial tumor with frequent low driver mutation allele frequencies.

5.
Mod Pathol ; 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31481664

RESUMO

Lipomatosis of nerve is a rare malformation characterized by a fibrolipomatous proliferation within peripheral nerve. Lipomatosis of nerve most frequently involves the median nerve, and manifests clinically as a compressive neuropathy. However, 30-60% of cases are associated with tissue overgrowth within the affected nerve's territory (e.g., macrodactyly for lipomatosis of nerve in the distal median nerve). Somatic activating PIK3CA mutations have been identified in peripheral nerve from patients with lipomatosis of nerve with type I macrodactyly, which is now classified as a PIK3CA-related overgrowth spectrum disorder. However, the PIK3CA mutation status of histologically confirmed lipomatosis of nerve, including cases involving proximal nerves, and cases without territory overgrowth, has not been determined. Fourteen histologically confirmed cases of lipomatosis of nerve involving the median (N = 6), brachial plexus (N = 1), ulnar (N = 3), plantar (N = 2), sciatic and superficial peroneal nerves (N = 1 each) were included. Ten cases had nerve territory overgrowth, ranging from macrodactyly to hemihypertrophy; and four cases had no territory overgrowth. Exome sequencing revealed "hotspot" activating PIK3CA missense mutations in 6/7 cases. Droplet digital polymerase chain reaction for the five most common PIK3CA mutations (p.H1047R, p.H1047L, p.E545K, p.E542K, and p.C420R) confirmed the exome results and identified an additional six cases with mutations (12/14 total). PIK3CA mutations were found in 8/10 cases with territory overgrowth (N = 7 p.H1047R and N = 1 p.E545K), including two proximal nerve cases with extremity overgrowth, and 4/4 cases without territory overgrowth (p.H1047R and p.H1047L, N = 2 each). The variant allele frequency of PIK3CA mutations (6-32%) did not correlate with the overgrowth phenotype. Three intraneural lipomas had no detected PIK3CA mutations. As PIK3CA mutations are frequent events in lipomatosis of nerve, irrespective of anatomic site or territory overgrowth, we propose that all phenotypic variants of this entity be classified within the PIK3CA-related overgrowth spectrum and termed "PIK3CA-related lipomatosis of nerve".

6.
Hum Pathol ; 91: 114-122, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31299266

RESUMO

Mutations of the succinate dehydrogenase (SDHX) enzyme subunits commonly lead to a loss of function of the holoenzyme complex, and germline SDHX mutations lead to a genetic predisposition to SDH-deficient neoplasms, including renal cell carcinomas (RCC). Similarly, loss-of-function alterations of fumarate hydratase (FH) leads to a genetic predisposition to hereditary leiomyomatosis and renal cell cancer (HLRCC)-associated RCC. Loss of FH leads to an accumulation of fumarate and aberrantly high levels of S-(2-succino)-cysteine (2SC). Subtype-specific consecutively diagnosed renal cell neoplasms were selected for the study and cases were not otherwise selected based on clinicopathologic features. Tissue microarrays were constructed from 1009 renal cell neoplasms (papillary: 400, clear cell: 203, chromophobe: 87, oncocytomas [original diagnosis]: 273, unclassified: 46) and these cases were immunostained for SDHA/SDHB to screen for SDH loss. A smaller subset (n = 730; oncocytomas, papillary and unclassified RCCs) were screened for FH-deficiency using immunohistochemistry for FH/2SC. Loss of SDHA/SDHB was seen in three of 273 tumors originally diagnosed as oncocytomas (1.1%). Diffuse nuclear and cytoplasmic 2SC staining, with retained FH expression was seen in one case (suggestive of dysfunctional FH protein), while absent FH was seen in 3 cases (2/400 papillary RCCs, 0.5% and 2/46 unclassified RCCs, 4.35%). No aberrant FH/2SC expression was noted in 273 cases originally diagnosed as oncocytomas. SDH-deficient RCCs were identified only in the cases originally diagnosed as oncocytomas (1.1%), while FH-deficient RCCs were identified in the papillary (0.5%) and unclassified RCC cohorts (4.35%). These results can help guide immunohistochemistry-based screening strategies for these tumors.

7.
J Med Biochem ; 38(2): 164-171, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30867644

RESUMO

Background: Diabetes mellitus (DM) with its micro- and macrocomplications is the leading global epidemic of the 21st century. The aim of the research is to determine possible changes in the complete blood count (CBC) parameters depending on glycemic controlin patients with Type 2 diabetes mellitus (T2DM). Methods: The study included a total of 178 patients with T2DM, both gender over the age of 40 years, from the Health Care Center ¼Dr Milorad Mika Pavlovic« Indjija, Serbia. To notice the possible correlation between the CBC parameters and glucose control in T2DM, the subjects were divided in two groups with HbA1c ≤ 7% and with HbA1c>7%. We analysed CBC parameters, parameters of glycoregulation, lipid status using standard biochemical methods, performed anthropometric measurements and collected patients data by questionnaire and electronic patient card. Results: There was statistical difference between HbA1c groups for PMDW (p=0.045), HDL (p=0.0067). Using univariate linear regression it is shown that PCT was correlated with WBC (p=0.0005), neutrophils (p=0.046), monocytes (p=0.003); MPM was associated with MPV (p=0.0005); MPC (p=0.0005), PDW (P=0.0005), GLU0 (p=0.034), HDL-C (p=0.005); PMDW was correlated with HbA1c% (p=0.049), GLU0 (p=0.013), HDL-C (p=0.001), BW (p=0.043) in all patients. Conclusions: Based on our study results it may be concluded that some of the parameters of CBC could be useful tool in following glycemic control of diabetics.

8.
Clin Chem ; 64(12): 1732-1742, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30237149

RESUMO

BACKGROUND: Droplet digital PCR (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, assay performance criteria must be established in a standardized manner to harness this potential. We reasoned that standard protocols used in clinical chemistry assay validation should be able to fill this need. METHODS: We validated KRAS, EGFR, and BRAF quantitative ddPCR assays based on the Clinical Laboratory Improvement Act regulations for laboratory-developed tests in clinical chemistry and the matching Clinical and Laboratory Standards Institute guidelines. This included evaluation of limit of the blank (LOB), limit of detection (LOD), limit of quantification (LOQ), intraassay and interassay imprecision, analytical range, dilution linearity, accuracy (including comparison with orthogonal platforms), reference range study, interference, and stability studies. RESULTS: For the ddPCR assays, the LOB was 4 mutant copies, LODs were 12 to 22 copies, and LOQs were 35 to 64 copies. The upper limit of the dynamic range was 30000 copies, and dilutions were linear down to the LOQs with good accuracy of spike recovery of Horizon reference material. Method comparisons with next-generation sequencing and an alternative ddPCR platform showed complete qualitative agreement and quantitative concordance, with slopes of 0.73 to 0.97 and R 2s of 0.83 to 0.99. No substantial interferences were discovered. Wild-type copy numbers in plasma ranged from 462 to 6169/mL in healthy individuals. CONCLUSIONS: Standard clinical chemistry assay validation protocols can be applied to quantitative ddPCR assays. This should facilitate comparison of the performance of different assays and allow establishment of minimal significant change thresholds in monitoring applications.


Assuntos
Química Clínica/normas , Análise Mutacional de DNA/normas , Biópsia Líquida/normas , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Adulto , Idoso , Ácidos Nucleicos Livres , Análise Mutacional de DNA/métodos , Receptores ErbB/genética , Feminino , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Valores de Referência
9.
J Clin Endocrinol Metab ; 103(9): 3169-3182, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29846633

RESUMO

Objective: To investigate the molecular underpinnings of thyroid cancer, preclinical cell line models are crucial; however, ∼40% of these have been proven to be either duplicates of existing thyroid lines or even nonthyroid-derived lines or are not derived from humans at all. Therefore, we set out to establish procedures and guidelines that should proactively avoid these problems, which facilitated the creation of criteria to make valid preclinical models for thyroid cancer research. Design: Based on our recommendations, we systematically characterized all new cell lines that we generated by a standardized approach that included (1) determination of human origin, (2) exclusion of lymphoma, (3) DNA fingerprinting and histological comparisons to establish linkage to presumed tissue of origin, (4) examining thyroid differentiation by screening two to three thyroid markers, (5) examination of biological behavior (growth rate, tumorigenicity), and (6) presence of common thyroid cancer genetic changes (TP53, BRAF, PTEN, PIK3CA, RAS, TERT promoter, RET/PTC, PAX8/PPARγ, NF1, and EIF1AX). Results: We established seven new thyroid cell lines (LAM136, EAM306, SDAR1, SDAR2, JEM493, THJ529, and THJ560) out of 294 primary culture attempts, and 10 patient-derived tumor xenografts (PDTXs; MC-Th-95, MC-Th-374, MC-Th-467, MC-Th-491, MC-Th-493, MC-Th-504, MC-Th-524, MC-Th-529, MC-Th-560, and MC-Th-562) out of 67 attempts. All were successfully validated by our protocols. Conclusions: This standardized approach for cell line and PDTX characterization should prevent (or detect) future cross-contamination and ensure that only valid preclinical models are used for thyroid cancer research.


Assuntos
Neoplasias da Glândula Tireoide/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular , Divisão Celular , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Impressões Digitais de DNA/métodos , DNA de Neoplasias/genética , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos Nus , Pessoa de Meia-Idade , Mutação , Transplante de Neoplasias , Neoplasias da Glândula Tireoide/genética , Células Tumorais Cultivadas
10.
Clin Gastroenterol Hepatol ; 16(10): 1632-1640.e1, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29526691

RESUMO

BACKGROUND & AIMS: Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) associates with markers of tumoremia. METHODS: We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS-FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell-free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2-fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. RESULTS: Peripheral blood concentrations of cfDNA were 1200 ng/ml (500-3300 ng/ml) before FNA vs 1400 ng/ml (900-4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2-fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8-14.9 months vs 11.1 months for patients without tumoremia; range, 5.5-14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS-FNA (range, 19.9-25 months after EUS-FNA) vs none of the patients with tumoremia. CONCLUSION: In patients with PDAC, EUS-FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.


Assuntos
Adenocarcinoma/diagnóstico , DNA Tumoral Circulante/sangue , Testes Diagnósticos de Rotina/efeitos adversos , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico/efeitos adversos , Metástase Neoplásica/fisiopatologia , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plasma/química , Estudos Prospectivos , Medição de Risco , Adulto Jovem
11.
J Mol Diagn ; 19(5): 755-765, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28826609

RESUMO

Severe combined immunodeficiency (SCID) benefits from early intervention via hematopoietic cell transplantation to reverse T-cell lymphopenia (TCL). Newborn screening (NBS) programs use T-cell receptor excision circle (TREC) levels to detect SCID. Real-time quantitative PCR is often performed to quantify TRECs in dried blood spots (DBSs) for NBS. Yet, real-time quantitative PCR has inefficiencies necessitating normalization, repeat analyses, or standard curves. To address these issues, we developed a multiplex, droplet digital PCR (ddPCR) method for measuring absolute TREC amounts in one DBS punch. TREC and RPP30 levels were simultaneously measured with a Bio-Rad AutoDG and QX200 ddPCR system. DBSs from 610 presumed-normal, 29 lymphocyte-profiled, and 10 clinically diagnosed infants (1 X-linked SCID, 1 RAG1 Omenn syndrome, and other conditions) were tested. Control infants showed 14 to 474 TREC copies/µL blood. SCID infants, and other TCL conditions, had ≤15 TREC copies/µL. The ddPCR lower limit of quantitation was 14 TREC copies/µL, and the limit of detection was 4 TREC copies/µL. Intra-assay and interassay imprecision was <20% CV for DBSs at 54 to 60 TREC copies/µL. Testing 29 infants with known lymphocyte profiles resulted in a sensitivity of 88.9% and a specificity of 100% at TRECs <20 copies/µL. We developed a multiplex ddPCR method for the absolute quantitation of DBS TRECs that can detect SCID and other TCL conditions associated with absent or low TRECs and validated this method for NBS.


Assuntos
Triagem Neonatal/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Imunodeficiência Combinada Severa/diagnóstico , Imunodeficiência Combinada Severa/genética , Estudos de Casos e Controles , Feminino , Marcadores Genéticos , Humanos , Lactente , Recém-Nascido , Linfócitos/metabolismo , Masculino , Reação em Cadeia da Polimerase Multiplex , Receptores de Antígenos de Linfócitos T/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Fluxo de Trabalho
12.
Endocr Pathol ; 28(3): 253-268, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28646318

RESUMO

Alterations of von Hippel-Lindau (VHL), succinate dehydrogenase (SDHX), and TMEM127 have been associated with the development of pheochromocytomas (PCs) and paragangliomas (PGLs) and are also associated with the development of renal neoplasms. This study involved 2 primary renal PGL and 12 cases of PC/PGL with associated renal neoplasia with a mean follow up of 74 months. Germline VHL and SDHX mutation status was obtained from the medical record. Immunohistochemistry for SDHB and mutation analysis for TMEM127 was performed, in addition to analysis of The Cancer Genome Atlas datasets for SDHX and TMEM127 mutated renal cell carcinomas (RCCs). The spectrum of renal neoplasia included clear cell and tubulocystic and papillary RCC, as well as a case of multiple papillary adenomas. Three patients had metastatic PC/PGL and three patients had VHL syndrome. Previously unreported TMEM127 alterations were identified in two patients, both without evidence of VHL syndrome or SDH-deficiency, and were classified as variants of uncertain significance. Primary renal PGL and neoplasia was associated with about 2% of 710 cases of PC/PGL. These were diagnosed concurrently or on average 27 months prior to the PC/PGL, and most were low-grade, low-stage clear cell RCCs. Up to half of patients with PC/PGL and renal neoplasia had VHL syndrome, SDH deficiency, or alterations in TMEM127. One (of three) case of metastatic PC/PGL had SDHB mutation and loss of SDHB by immunohistochemistry. The other two cases had retained SDHB expression.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Neoplasias Renais/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Análise Mutacional de DNA , Feminino , Mutação em Linhagem Germinativa , Humanos , Neoplasias Renais/patologia , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Paraganglioma/patologia , Feocromocitoma/patologia , Succinato Desidrogenase , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Adulto Jovem
13.
Mar Pollut Bull ; 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26806663

RESUMO

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

15.
J Clin Endocrinol Metab ; 99(6): E936-43, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24628546

RESUMO

BACKGROUND: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. APPROACH AND METHODS: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2-7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 µM pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. RESULTS: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. CONCLUSIONS: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem.


Assuntos
Carcinoma/tratamento farmacológico , Carcinoma/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/patologia , Animais , Carcinoma/genética , Carcinoma Papilar , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Análise Mutacional de DNA , Humanos , Camundongos , Terapia de Alvo Molecular , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas p21(ras) , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/genética , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras/genética
16.
Food Chem ; 145: 1072-5, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24128586

RESUMO

In order to detect presence and quantity of Roundup Ready (RR) soybean in crude oil extracted from soybean seed with a different percentage of GMO seed two extraction methods were used, CTAB and DNeasy Plant Mini Kit. The amplifications of lectin gene, used to check the presence of soybean DNA, were not achieved in all CTAB extracts of DNA, while commercial kit gave satisfactory results. Comparing actual and estimated GMO content between two extraction methods, root mean square deviation for kit is 0.208 and for CTAB is 2.127, clearly demonstrated superiority of kit over CTAB extraction. The results of quantification evidently showed that if the oil samples originate from soybean seed with varying percentage of RR, it is possible to monitor the GMO content at the first stage of processing crude oil.


Assuntos
Produtos Agrícolas/química , Contaminação de Alimentos , Plantas Geneticamente Modificadas/química , Sementes/química , Óleo de Soja/química , Soja/química , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , DNA de Plantas/isolamento & purificação , DNA de Plantas/metabolismo , Resistência a Medicamentos , Manipulação de Alimentos , Inspeção de Alimentos/métodos , Rotulagem de Alimentos/legislação & jurisprudência , Glicina/análogos & derivados , Glicina/farmacologia , Herbicidas/farmacologia , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/metabolismo , Sérvia , Proteínas de Soja/genética , Proteínas de Soja/metabolismo , Soja/efeitos dos fármacos , Soja/genética , Soja/metabolismo
17.
Blood ; 120(11): 2280-9, 2012 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-22855598

RESUMO

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.


Assuntos
Rearranjo Gênico , Linfoma de Células T Periférico/genética , Mutação , Fatores de Transcrição/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Inibidor p16 de Quinase Dependente de Ciclina/química , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Análise Mutacional de DNA , Feminino , Estudo de Associação Genômica Ampla , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/mortalidade , Linfoma Difuso de Grandes Células B/patologia , Linfoma de Células T Periférico/metabolismo , Linfoma de Células T Periférico/mortalidade , Linfoma de Células T Periférico/patologia , Masculino , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Oxirredutases/química , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Estados Unidos , Oxidorredutase com Domínios WW
18.
Eur J Cancer ; 48(11): 1739-49, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22036874

RESUMO

BACKGROUND: There are currently no reliable biomarkers for malignant pheochromocytomas and paragangliomas (PPGLs). This study examined whether measurements of catecholamines and their metabolites might offer utility for this purpose. METHODS: Subjects included 365 patients with PPGLs, including 105 with metastases, and a reference population of 846 without the tumour. Eighteen catecholamine-related analytes were examined in relation to tumour location, size and mutations of succinate dehydrogenase subunit B (SDHB). RESULTS: Receiver-operating characteristic curves indicated that plasma methoxytyramine, the O-methylated metabolite of dopamine, provided the most accurate biomarker for discriminating patients with and without metastases. Plasma methoxytyramine was 4.7-fold higher in patients with than without metastases, a difference independent of tumour burden and the associated 1.6- to 1.8-fold higher concentrations of norepinephrine and normetanephrine. Increased plasma methoxytyramine was associated with SDHB mutations and extra-adrenal disease, but was also present in patients with metastases without SDHB mutations or those with metastases secondary to adrenal tumours. High risk of malignancy associated with SDHB mutations reflected large size and extra-adrenal locations of tumours, both independent predictors of metastatic disease. A plasma methoxytyramine above 0.2nmol/L or a tumour diameter above 5cm indicated increased likelihood of metastatic spread, particularly when associated with an extra-adrenal location. CONCLUSION: Plasma methoxytyramine is a novel biomarker for metastatic PPGLs that together with SDHB mutation status, tumour size and location provide useful information to assess the likelihood of malignancy and manage affected patients.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Biomarcadores Tumorais/sangue , Dopamina/análogos & derivados , Paraganglioma/secundário , Feocromocitoma/secundário , Fatores de Risco , Succinato Desidrogenase/genética , Neoplasias das Glândulas Suprarrenais/genética , Adulto , Dopamina/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Normetanefrina/sangue , Paraganglioma/genética , Paraganglioma/patologia , Feocromocitoma/genética , Feocromocitoma/patologia
19.
J Clin Oncol ; 29(31): 4137-42, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21969497

RESUMO

PURPOSE: To present data on the high rate of SDHB mutations in patients with metastatic pheochromocytoma/paraganglioma whose initial tumor presentation began in childhood or adolescence. PATIENTS AND METHODS: From 2000 to 2010, 263 patients with pheochromocytoma/paraganglioma were evaluated through the National Institutes of Health (NIH), Bethesda, MD. Of the 263 patients, 125 patients were found to have metastatic disease; of these 125 patients, 32 patients presented with a tumor before 20 years of age. An additional 17 patients presented with a tumor before 20 years of age but demonstrated no development of metastatic disease. Genetic testing for mutations in the VHL, MEN, and SDHB/C/D genes was performed on patients without previously identified genetic mutations. RESULTS: Of the 32 patients who presented with metastatic disease and had their primary tumor in childhood or adolescence, sequence analysis of germline DNA showed SDHB mutations in 23 patients (71.9%), SDHD mutations in three patients (9.4%), VHL mutations in two patients (6.3%), and an absence of a known mutation in four patients (12.5%). The majority of these 32 patients (78.1%) presented with primary tumors in an extra-adrenal location. CONCLUSION: The majority of patients with metastatic pheochromocytoma/paraganglioma who presented with a primary tumor in childhood/adolescence had primary extra-adrenal tumors and harbored SDHB mutations. Except for primary tumors located in the head and neck where SDHD genetic testing is advised, we recommend that patients who present with metastatic pheochromocytoma/paraganglioma with primary tumor development in childhood or adolescence undergo SDHB genetic testing before they undergo testing for other gene mutations, unless clinical presentation or family history suggests a different mutation.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Testes Genéticos , Mutação em Linhagem Germinativa , Paraganglioma/genética , Feocromocitoma/genética , Succinato Desidrogenase/genética , Adolescente , Adulto , Idade de Início , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Paraganglioma/secundário , Feocromocitoma/secundário , Valor Preditivo dos Testes , Adulto Jovem
20.
Genes Cancer ; 2(1): 46-55, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21779480

RESUMO

In vitro studies have demonstrated that the PAX8/PPARγ fusion protein (PPFP), which occurs frequently in follicular thyroid carcinomas (FTC), exhibits oncogenic activity. However, paradoxically, a meta-analysis of extant tumor outcome studies indicates that 68% of FTC-expressing PPFP are minimally invasive compared to only 32% of those lacking PPFP (χ(2) = 6.86, P = 0.008), suggesting that PPFP favorably impacts FTC outcomes. In studies designed to distinguish benign thyroid neoplasms from thyroid carcinomas, the previously identified tumor suppressor miR-122, a major liver micro-RNA (miR) that is decreased in hepatocellular carcinoma, was increased 8.9-fold (P < 0.05) in all FTC versus normal, 9.2-fold in FTC versus FA (P < 0.05), and 16.8-fold (P < 0.001) in FTC + PPFP versus FTC - PPFP. Constitutive expression of PPFP in the FTC-derived cell line WRO (WRO-PPFP) caused a 5-fold increase of miR-122 expression (P < 0.05) and a striking 5.1-fold reduction (P < 0.0001) in tumor progression compared to WRO-vector cells in a mouse xenograft model. Constitutive expression of either miR-122 or a dominant-negative PPARγ mutant in WRO cells was less effective than PPFP at inhibiting xenograft tumor progression (1.8-fold [P < 0.001] and 1.7-fold [P < 0.03], respectively). PPFP-induced up-regulation of miR-122 expression was independent of its known dominant-negative PPARγ activity. Up-regulation of miR-122 negatively regulates ADAM-17, a known downstream target, in thyroid cells, suggesting an antiangiogenic mechanism in thyroid carcinoma. This latter inference is directly supported by reduced CD-31 expression in WRO xenografts expressing PPFP, miR-122, and DN-PPARγ. We conclude that, in addition to its apparent oncogenic potential in vitro, PPFP exhibits paradoxical tumor suppressor activity in vivo, mediated by multiple mechanisms including up-regulation of miR-122 and dominant-negative inhibition of PPARγ activity.

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