RESUMO
Homozygous deletion of the chromosomal region 9p21.3 is common in urothelial carcinoma (UC) and leads to loss of several genes, including CDKN2A and MTAP, resulting in loss of MTAP protein expression. Here, we aimed to explore the diagnostic potential of MTAP immunohistochemistry (IHC) as a surrogate marker for homozygous 9p21.3 deletion (9p21 homozygous deletion [HD]) in UC. MTAP status was determined by IHC on 27 UC tissue specimens with known 9p21.3 status as defined by fluorescence in situ hybridization in matched cytological specimens, by IHC and fluorescence in situ hybridization on a tissue microarray (TMA) containing 359 UC at different stages, and by IHC on 729 consecutive UC from routine practice. Moreover, we analyzed a longitudinal series of matched specimens from 38 patients with MTAP-negative recurrent UC. MTAP loss by IHC was found in all 17 patients with 9p21 HD and in 2/8 cases without 9p21 HD. In the TMA, MTAP loss was more common in metastases (53%) than in muscle-invasive (33%) and non-muscle-invasive UC (29%) (P = .03). In the consecutive series, 164/729 (22%) cases showed loss of MTAP expression. In 41 of these 164 cases (25%), loss of MTAP expression was heterogenous. We also discovered loss of MTAP expression in flat urothelium adjacent to MTAP-negative low-grade UC, suggesting true flat low-grade neoplasia that could not be diagnosed by morphology alone. Longitudinal analysis of recurrences showed persistent negative MTAP status over time in 37/38 (97%) patients. MTAP IHC can serve as a surrogate marker for 9p21 HD in UC and as a diagnostic tool to differentiate reactive urothelium from urothelial neoplasia. It also provides a unique opportunity to study clinicopathological associations and the heterogeneity of 9p21 HD across the whole spectrum of UC manifestations.
Assuntos
Biomarcadores Tumorais , Cromossomos Humanos Par 9 , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Purina-Núcleosídeo Fosforilase , Neoplasias da Bexiga Urinária , Humanos , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Cromossomos Humanos Par 9/genética , Feminino , Masculino , Pessoa de Meia-Idade , Idoso , Purina-Núcleosídeo Fosforilase/análise , Purina-Núcleosídeo Fosforilase/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologia , Neoplasias da Bexiga Urinária/metabolismo , Deleção Cromossômica , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Carcinoma de Células de Transição/metabolismo , Adulto , Análise Serial de Tecidos , Idoso de 80 Anos ou mais , HomozigotoRESUMO
CDKN2A homozygous deletion has occasionally been reported in atypical and anaplastic meningiomas and is considered as one of the genetic alterations commonly involved in their recurrence and malignant progression. Methylthioadenosine phosphorylase (MTAP) immunohistochemistry is a promising surrogate marker for CDKN2A homozygous deletion in different cancers but has not been examined in meningiomas. We performed CDKN2A FISH and MTAP immunohistochemistry on specimens from 30 patients with CNS WHO grade 2 (n = 27) and 3 (n = 3) meningiomas, including specimens from primary and recurrent tumors and then determined whether MTAP immunohistochemistry correlated with CDKN2A homozygous deletion and clinicopathological features. CDKN2A homozygous deletion was detected in 12% (3/26) of CNS WHO grade 2 and 67% (2/3) of CNS WHO grade 3 meningiomas; 3 cases exhibited temporal and/or spatial heterogeneity. MTAP loss was in excellent concordance with CDKN2A homozygous deletion (sensitivity; 100%, specificity; 100%). MTAP loss/CDKN2A homozygous deletion correlated with cellular proliferation (mitotic rate; p = 0.001, Ki-67 labeling index; p = 0.03) and poor prognosis (overall survival; p = 0.01, progression free survival; p < 0.001). Thus, MTAP immunostaining can be a surrogate marker for CDKN2A homozygous deletion in meningiomas, and MTAP loss/CDKN2A homozygous deletion may be an important prognostic factor for meningiomas.
Assuntos
Biomarcadores Tumorais/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Neoplasias Meníngeas/patologia , Meningioma/patologia , Purina-Núcleosídeo Fosforilase/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Neoplasias Meníngeas/genética , Meningioma/genética , Pessoa de Meia-Idade , Purina-Núcleosídeo Fosforilase/análise , Sensibilidade e EspecificidadeRESUMO
Homozygous deletion (HD) of CDKN2A is one of the most promising biomarkers for predicting poor prognosis of IDH-mutant diffuse gliomas. The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) recommendations propose that IDH-mutant lower-grade astrocytomas with CDKN2A/B HD be classified as grade IV tumors. Loss of methylthioadenosine phosphorylase (MTAP) immunohistochemistry staining has been proposed as a surrogate of CDKN2A HD in various tumors but its performance has not been fully investigated in diffuse glioma. This study determined whether MTAP immunoreactivity could serve as a proxy for CDKN2A HD in adult-type diffuse glioma, thereby contributing to stratifying patient outcome. MTAP immunohistochemistry staining using clone EPR6893 was scored in 178 diffuse glioma specimens consisting of 77 IDH-mutant astrocytomas, 13 IDH-mutant oligodendrogliomas, and 88 IDH-wildtype glioblastomas. The use of MTAP immunohistochemical deficiency to predict CDKN2A HD was good for IDH-mutant astrocytomas (sensitivity, 88%; specificity, 98%) and IDH-wildtype glioblastomas (sensitivity, 89%; specificity, 100%), but poor for IDH-mutant oligodendrogliomas (sensitivity, 67%; specificity, 57%). Both CDKN2A HD and MTAP immunohistochemical deficiency were significant adverse prognostic factors of overall survival for IDH-mutant astrocytoma (P < 0.001 each), but neither were prognostically significant for oligodendroglioma or IDH-wildtype glioblastoma. IDH-mutant lower-grade astrocytoma with CDKN2A HD and deficient MTAP immunoreactivity exhibited overlapping unfavorable outcome with IDH-mutant glioblastoma. MTAP immunostaining was easily interpreted in 61% of the cases tested, but scoring required greater care in the remaining cases. An alternative MTAP antibody clone (2G4) produced identical scoring results in all but 1 case, and a slightly larger proportion (66%) of cases were considered easy to interpret compared to using EPR6893. In summary, loss of MTAP immunoreactivity could serve as a reasonable predictive surrogate for CDKN2A HD in IDH-mutant astrocytomas and IDH-wildtype glioblastomas and could provide significant prognostic value for IDH-mutant astrocytoma, comparable to CDKN2A HD.
Assuntos
Astrocitoma/enzimologia , Astrocitoma/genética , Biomarcadores Tumorais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Deleção de Genes , Imuno-Histoquímica , Purina-Núcleosídeo Fosforilase/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astrocitoma/patologia , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Feminino , Homozigoto , Humanos , Hibridização in Situ Fluorescente , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Mutação , Valor Preditivo dos Testes , Adulto JovemRESUMO
The separation of benign from malignant mesothelial proliferations is a morphologically difficult problem. Mutations/deletions of components of the Hippo pathway are frequent in malignant mesotheliomas, and one downstream effect of aberrant Hippo signaling is increased production of cyclin D1. We examined expression of cyclin D1 nuclear staining in two tissue microarrays containing 52 reactive epithelial mesothelial proliferations, 51 reactive spindle cell mesothelial proliferations, 54 epithelial mesotheliomas, and 22 sarcomatous/desmoplastic mesotheliomas. When present, cyclin D1 staining was always strong, hence the arrays were scored as 0, 1-25%, 26-50%, 51-75%, and 76-100% staining. Both arrays showed a similar pattern. Reactive epithelial proliferations generally showed no staining (42/52 cases) or 1-25% staining (10/52 cases) with no cases showing >25% staining. Overall for reactive epithelial proliferations the maximum staining was 14.8% and mean 1.1 ± 2.9%. For epithelial mesotheliomas 39/54 (72%) cases demonstrated >25% staining, with 8/54 in the 26-50% staining range, 9/54 in the 51-75% range, and 22/54 in the >75% range. Combinations of staining using cyclin D1 >50% plus BAP1 or MTAP loss in epithelial mesotheliomas produced about a 10% increase in sensitivity. Reactive spindle cell proliferations showed a broader range of staining with 27/51 in the 1-25% range, 5/51 in the 26-50% range, and 1/51 >50%. Eleven of 22 sarcomatous/desmoplastic mesotheliomas scored 50% or greater. We conclude that for epithelial mesothelial proliferations, the finding of >50% of tumor cells staining supports a diagnosis of epithelial mesothelioma with 100% specificity but only modest (57%) sensitivity.
Assuntos
Biomarcadores Tumorais/análise , Proliferação de Células , Ciclina D1/análise , Células Epiteliais/química , Imuno-Histoquímica , Mesotelioma Maligno/química , Neoplasias Pleurais/química , Diagnóstico Diferencial , Células Epiteliais/patologia , Humanos , Mesotelioma Maligno/patologia , Neoplasias Pleurais/patologia , Valor Preditivo dos Testes , Purina-Núcleosídeo Fosforilase/análise , Reprodutibilidade dos Testes , Estudos Retrospectivos , Análise Serial de Tecidos , Proteínas Supressoras de Tumor/análise , Ubiquitina Tiolesterase/análiseRESUMO
BACKGROUND: The separation of benign from malignant mesothelial proliferations on effusion cytology can be difficult. Loss of methylthioadenosine phosphorylase (MTAP) by immunohistochemistry is an established marker of malignancy in mesothelial proliferations, but to the authors' knowledge largely has been applied only to biopsies. The current study was conducted to determine the usefulness of MTAP immunohistochemistry in the diagnosis of malignant mesothelioma in effusion cytology specimens. METHODS: A total of 21 effusion cytology cases of malignant mesothelioma were stained for MTAP and BRCA-associated protein 1 (BAP1), with 15 reactive mesothelial cytology cases used as a control. Fourteen cases had a paired surgical specimen for comparison, and 7 cases were run for CDKN2A deletion by fluorescence in situ hybridization. RESULTS: Complete loss of MTAP cytoplasmic staining was noted in 7 of 21 effusion samples (33%), and no loss was observed in 11 effusion samples (52%); 11 of these cases had a matching surgical specimen and all 11 specimens demonstrated the same MTAP pattern. Partial loss was observed in 3 effusion specimens (80%, 40%, and 40% intact staining, respectively), but in all 3 the surgical specimen demonstrated 100% staining. None of the 15 reactive mesothelial cytology specimens demonstrated MTAP cytoplasmic loss. CDKN2A FISH demonstrated concordance in 5 of 7 cases (71%). MTAP immunohistochemistry had a sensitivity of 33% and a specificity of 100% for this differential diagnosis. CONCLUSIONS: MTAP staining demonstrated generally good concordance between the cytologic and surgical specimens and appears to be useful in the diagnosis of mesothelioma on effusion specimens. Complete loss of MTAP is a reliable marker of malignancy, but the significance of partial loss of MTAP staining is unclear.
Assuntos
Biomarcadores Tumorais/análise , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Derrame Pleural Maligno/diagnóstico , Purina-Núcleosídeo Fosforilase/análise , Proteínas Supressoras de Tumor/análise , Ubiquitina Tiolesterase/análise , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Biópsia , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Deleção de Genes , Humanos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/cirurgia , Mesotelioma/genética , Mesotelioma/patologia , Mesotelioma/cirurgia , Mesotelioma Maligno , Cavidade Pleural/patologia , Derrame Pleural Maligno/genética , Derrame Pleural Maligno/patologia , Derrame Pleural Maligno/cirurgia , Purina-Núcleosídeo Fosforilase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismoRESUMO
Ancillary studies facilitate accurate diagnosis of morphologically challenging mesothelial proliferations. The current diagnostic algorithm proceeds from BAP1 immunohistochemistry to CDKN2A fluorescence in situ hybridization. While MTAP immunohistochemistry has recently shown promise as a surrogate for CDKN2A fluorescence in situ hybridization, it has been examined in only a few single-institution studies. Furthermore, there are no published reports on interobserver agreement or interlaboratory reproducibility for MTAP immunohistochemistry. We performed MTAP immunohistochemistry on 20 benign mesothelial lesions and 99 malignant mesotheliomas from five mesothelioma centers in four countries, and each MTAP stain was independently interpreted by four pathologists. CDKN2A fluorescence in situ hybridization data were available for a subset of cases, and a subset of cases was subjected in MTAP immunohistochemistry in multiple laboratories to assess interlaboratory reproducibility. Interobserver agreement in MTAP immunostain interpretation was excellent for all mesothelial lesions (kappa: 0.85) and for malignant mesothelioma cases only (kappa: 0.82). Interlaboratory reproducibility was also excellent (kappa values for paired protocols: 0.77-0.89). MTAP loss by immunohistochemistry was 78% sensitive and 96% specific for CDKN2A homozygous deletion. MTAP immunohistochemistry is a reliable surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant mesothelioma. Interobserver agreement is excellent for interpretation of MTAP staining, and protocols performed in different laboratories yield concordant MTAP staining results. Rare cases with immunohistochemical MTAP loss may retain normal CDKN2A copy number, and the MTAP staining results should be correlated with clinicopathologic findings and other ancillary studies.
Assuntos
Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Mesotelioma Maligno/enzimologia , Mesotelioma Maligno/genética , Neoplasias Pleurais/enzimologia , Neoplasias Pleurais/genética , Purina-Núcleosídeo Fosforilase/análise , França , Humanos , Mesotelioma Maligno/patologia , América do Norte , Variações Dependentes do Observador , Neoplasias Pleurais/patologia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , TóquioRESUMO
Neurofibromatosis type 2 (NF2) gene, a tumor suppressor gene located on chromosome 22q12.2, is frequently abnormal in mesothelioma. Recent studies have revealed the effectiveness of diagnostic assays for differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. These include detection of homozygous deletion of the 9p21 locus by fluorescence in situ hybridization (FISH) (9p21 FISH), loss of expression of BAP1 as detected by immunohistochemistry, and loss of expression of methylthioadenosine phosphorylase (MTAP) as detected by immunohistochemistry. However, the application of FISH detection of NF2 gene deletion (NF2 FISH) in differentiation of malignant pleural mesothelioma from reactive mesothelial hyperplasia has not been fully evaluated. In this study, we investigated whether NF2 FISH, either alone or in a combination with other diagnostic assays (9p21 FISH, MTAP immunohistochemistry, and BAP1 immunohistochemistry), is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia. This study cohort included malignant pleural mesothelioma (n = 47) and reactive mesothelial hyperplasia cases (n = 27) from a period between 2001 and 2017. We used FISH to examine deletion status of NF2 and 9p21 and immunohistochemistry to examine expression of MTAP and BAP1 in malignant pleural mesothelioma and in reactive mesothelial hyperplasia. Hemizygous NF2 loss (chromosome 22 monosomy or hemizygous deletion) was detected in 25 of 47 (53.2%) mesothelioma cases. None of the mesothelioma cases showed homozygous NF2 deletion. Hemizygous NF2 loss showed 53.2% sensitivity and 100% specificity in differentiating malignant pleural mesothelioma from reactive mesothelial hyperplasia. A combination of NF2 FISH, 9p21 FISH, and BAP1 immunohistochemistry yielded greater sensitivity (100%) than that detected for either diagnostic assay alone (53.2% for NF2 FISH, 78.7% for 9p21 FISH, 70.2% for MTAP immunohistochemistry, or 57.4% for BAP1 immunohistochemistry). Thus, NF2 FISH in combination with other diagnostic assays is effective for distinguishing malignant pleural mesothelioma from reactive mesothelial hyperplasia.
Assuntos
Biomarcadores Tumorais/genética , Deleção de Genes , Hibridização in Situ Fluorescente , Mesotelioma Maligno/genética , Neurofibromina 2/genética , Neoplasias Pleurais/genética , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos Par 9 , Feminino , Predisposição Genética para Doença , Hemizigoto , Humanos , Hiperplasia , Imuno-Histoquímica , Masculino , Mesotelioma Maligno/química , Mesotelioma Maligno/mortalidade , Mesotelioma Maligno/patologia , Pessoa de Meia-Idade , Fenótipo , Neoplasias Pleurais/química , Neoplasias Pleurais/mortalidade , Neoplasias Pleurais/patologia , Valor Preditivo dos Testes , Prognóstico , Purina-Núcleosídeo Fosforilase/análise , Reprodutibilidade dos Testes , Estudos Retrospectivos , Proteínas Supressoras de Tumor/análise , Ubiquitina Tiolesterase/análiseRESUMO
The existence of an in situ phase of malignant mesothelioma has long been postulated but until recently has been impossible to prove. Here we describe ten patients with mesothelioma in situ, defined by a single layer of surface mesothelial cells showing loss of BAP1 nuclear immunostaining, no evidence of tumor by imaging and/or by direct examination of the pleura/peritoneum, and no invasive mesothelioma developing for at least 1 year. Nine cases were pleural and one peritoneal. Most patients were biopsied for repeated effusions of unknown etiology; in two patients mesothelioma in situ was found incidentally in lung cancer resections. In addition to surface mesothelium with BAP1 loss, one case had a surface papillary proliferation with BAP1 loss, and two cases had a small (few millimeter) nodule with BAP1 loss. CDKN2A was deleted by FISH in one of eight cases. Methylthioadenosine phosphorylase showed partial loss in the surface mesothelium by immunohistochemistry in three cases. Invasive malignant mesothelioma developed in seven patients with time between biopsy and invasive disease from 12 to 92 (median 60) months. Invasive mesothelioma has not developed in the other three patients at 12, 57, and 120 months, but the latter patient, who has pleural plaques, still has repeated pleural effusions, probably representing a so-called "benign asbestos effusion." We conclude that mesothelioma in situ, as diagnosed using the criteria outlined above, is associated with a high risk of developing invasive mesothelioma, but typically over a relatively protracted time, so that curable interventions maybe possible.
Assuntos
Mesotelioma Maligno/patologia , Neoplasias Peritoneais/patologia , Neoplasias Pleurais/patologia , Idoso , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Biópsia , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Progressão da Doença , Feminino , Humanos , Masculino , Mesotelioma Maligno/enzimologia , Mesotelioma Maligno/genética , Mesotelioma Maligno/terapia , Pessoa de Meia-Idade , Invasividade Neoplásica , Neoplasias Peritoneais/enzimologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/terapia , Neoplasias Pleurais/enzimologia , Neoplasias Pleurais/genética , Neoplasias Pleurais/terapia , Purina-Núcleosídeo Fosforilase/análise , Fatores de Tempo , Resultado do Tratamento , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genéticaRESUMO
Malignant pleural mesothelioma (MPM), associated with unfavorable outcomes, is closely associated with asbestos exposure. Early detection and treatment are critical to prolong survival of patients with MPM because of the rapid progression and resistance to treatment. The recently defined malignant mesothelioma in situ (MIS) has been gaining increasing attention with advances in genome-based methods including fluorescence in situ hybridization (FISH) as well as immunohistochemistry. We herein report the case of a MIS in a 73-year-old male with a history of asbestos exposure presenting with massive pleural effusion in the right thoracic cavity. Video-assisted thoracoscopic surgery with pleural biopsy of the right side revealed a single layer of atypical mesothelial cells without invasive lesions by hematoxylin and eosin staining. However, these mesothelial cells exhibited a loss of methylthioadenosine phosphorylase (MTAP) by immunohistochemistry and homozygous deletion of CDKN2A (p16) by FISH, leading to the diagnosis of MIS.
Assuntos
Biomarcadores Tumorais/análise , Diagnóstico Precoce , Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Neoplasias Pleurais/diagnóstico , Idoso , Inibidor p16 de Quinase Dependente de Ciclina/análise , Inibidor p16 de Quinase Dependente de Ciclina/genética , Genes p16 , Humanos , Masculino , Mesotelioma Maligno , Purina-Núcleosídeo Fosforilase/análise , Purina-Núcleosídeo Fosforilase/biossíntese , Deleção de SequênciaAssuntos
Biomarcadores Tumorais/análise , Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Neoplasias Pleurais/diagnóstico , Purina-Núcleosídeo Fosforilase/análise , Idoso , Idoso de 80 Anos ou mais , Cromossomos Humanos Par 9 , Citoplasma/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/genética , Masculino , Mesotelioma/genética , Mesotelioma Maligno , Pessoa de Meia-Idade , Derrame Pleural Maligno/diagnóstico , Derrame Pleural Maligno/genética , Neoplasias Pleurais/genética , Deleção de SequênciaRESUMO
BACKGROUND: Homozygous deletion of 9p21 detected by fluorescence in situ hybridization (FISH) and loss of BRCA1-associated protein 1 (BAP1) expression detected by immunohistochemistry (IHC) are useful for the differentiation between malignant pleural mesothelioma (MPM) and reactive mesothelial hyperplasia. The authors previously described that IHC expression of the protein product of the methylthioadenosine phosphorylase (MTAP) gene, which is localized in the 9p21 chromosomal region, was correlated with the deletion status of 9p21 FISH in MPM tissues. In the current study, the authors investigated whether a combination of MTAP and BAP1 IHC could distinguish MPM from reactive mesothelial cells (RMC) in cell blocks obtained from pleural effusions. METHODS: The authors examined IHC expression of MTAP and BAP1 in cell blocks obtained from pleural effusions of 45 cases of MPM and 21 cases of reactive mesothelial hyperplasia. Furthermore, IHC expression of MTAP was compared with the deletion status of 9p21 FISH. RESULTS: MTAP and BAP1 IHC differentiated MPM from RMC with 100% specificity for both and sensitivities of 42.2% and 60.0%, respectively. The combination of MTAP and BAP1 IHC yielded a sensitivity of 77.8%, which was higher than that of BAP1 IHC alone or 9p21 FISH alone (62.2%). Moreover, a high degree of concordance was observed between the results of MTAP IHC and 9p21 FISH in cell blocks. CONCLUSIONS: A combination of MTAP and BAP1 IHC in cell blocks from pleural effusions appears to be a reliable and useful method for differentiating MPM cells from RMC and can be used in the routine diagnosis of MPM. Cancer Cytopathol 2018;126:54-63. © 2017 American Cancer Society.
Assuntos
Neoplasias Pulmonares/diagnóstico , Mesotelioma/diagnóstico , Derrame Pleural/patologia , Neoplasias Pleurais/diagnóstico , Purina-Núcleosídeo Fosforilase/análise , Proteínas Supressoras de Tumor/análise , Ubiquitina Tiolesterase/análise , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Neoplasias Pulmonares/patologia , Masculino , Mesotelioma/patologia , Mesotelioma Maligno , Pessoa de Meia-Idade , Neoplasias Pleurais/patologiaRESUMO
A novel capillary electrophoresis (CE) method was developed for simultaneous analysis of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) in red blood cells (RBCs). The developed method considered and took advantage of the natural conversion from the ADA product, inosine to hypoxanthine. The transformation ratio was introduced for ADA and PNP analysis to obtain more reliable results. After optimizing the enzymatic incubation and electrophoresis separation conditions, the determined activities of ADA and PNP in 12 human RBCs were 0.237-0.833 U/ml and 9.013-10.453 U/ml packed cells, respectively. The analysis of ADA in mice RBCs indicated that there was an apparent activity difference between healthy and hepatoma mice. In addition, the proposed method was also successfully applied in the inhibitor screening from nine traditional Chinese medicines, and data showed that ADA activities were strongly inhibited by Rhizoma Chuanxiong and Angelica sinensis. The inhibition effect of Angelica sinensis on ADA is first reported here and could also inhibit PNP activity.
Assuntos
Adenosina Desaminase/análise , Eletroforese Capilar/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/análise , Adenosina Desaminase/metabolismo , Relação Dose-Resposta a Droga , Humanos , Medicina Tradicional Chinesa , Purina-Núcleosídeo Fosforilase/metabolismo , Relação Estrutura-AtividadeRESUMO
We describe a continuous, spectrophotometric, enzyme-coupled assay useful to monitor reactions catalyzed by nucleoside triphosphohydrolases. In particular, using Escherichia coli deoxynucleoside triphosphohydrolase (Dgt), which hydrolyzes dGTP to deoxyguanosine and tripolyphosphate (PPPi) as the enzyme to be tested, we devised a procedure relying on purine nucleoside phosphorylase (PNPase) and xanthine oxidase (XOD) as the auxiliary enzymes. The deoxyguanosine released by Dgt can indeed be conveniently subjected to phosphorolysis by PNPase, yielding deoxyribose-1-phosphate and guanine, which in turn can be oxidized to 8-oxoguanine by XOD. By this means, it was possible to continuously detect Dgt activity at 297 nm, at which wavelength the difference between the molar extinction coefficients of 8-oxoguanine (8000 M(-1) cm(-1)) and guanine (1090 M(-1) cm(-1)) is maximal. The initial velocities of Dgt-catalyzed reactions were then determined in parallel with the enzyme-coupled assay and with a discontinuous high-performance liquid chromatography (HPLC) method able to selectively detect deoxyguanosine. Under appropriate conditions of excess auxiliary enzymes, the activities determined with our continuous enzyme-coupled assay were quantitatively comparable to those observed with the HPLC method. Moreover, the enzyme-coupled assay proved to be more sensitive than the chromatographic procedure, permitting reliable detection of Dgt activity at low dGTP substrate concentrations.
Assuntos
Nucleosídeo-Trifosfatase/análise , Espectrofotometria Ultravioleta/métodos , Fosfatase Alcalina/análise , Cromatografia Líquida de Alta Pressão , Escherichia coli/enzimologia , Purina-Núcleosídeo Fosforilase/análise , Xantina Oxidase/análiseRESUMO
Purine nucleoside phosphorylase (PNP) is an ubiquitous enzyme which plays an important role in arsenic (As) detoxification. As is a toxic metalloid present in air, soil and water; is abundant in the environment and is readily transferred along the trophic chain, being found even in human breast milk. Milk is the main nutrient source for the growth and development of neonates. Information on breast milk synthesis and its potential defense mechanism against As toxicity is scarce. In this study, PNP and antioxidant enzymes activities, as well as glutathione (GSH) and total arsenic (TAs) concentrations, were quantified in breast milk samples. PNP, superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR) activities and GSH concentration were determined spectrophotometrically; TAs concentration ([TAs]) was measured by atomic absorption spectrometry. Data suggest an increase in PNP activity (median = 0.034 U mg protein-1) in the presence of TAs (median = 1.16 g L(-1)). To explain the possible association of PNP activity in breast milk with the activity of the antioxidant enzymes as well as with GSH and TAs concentrations, generalized linear models were built. In the adjusted model, GPx and GR activities showed a statistically significant (p<0.01) association with PNP activity. These results may suggest that PNP activity increases in the presence of TAs as part of the detoxification mechanism in breast milk.
Purina nucleósido fosforilasa (PNP) es una enzima ubicua que desempeña un papel importante en la desintoxicación del arsénico (As). As es un metaloide tóxico presente en el aire, el suelo y el agua; es abundante en el medio ambiente y se transfiere fácilmente a lo largo de la cadena trófica, encontrándose incluso en la leche materna humana. Información sobre la síntesis de la leche materna y su potencial mecanismo de defensa contra tóxicos es escasa. En este estudio, se cuantificó la actividad de PNP y de las enzimas antioxidantes así como la concentración de glutatión (GSH) y de arsénico total ([TAs]) en muestras de leche materna. La actividad de PNP, superóxido dismutasa (SOD), catalasa (CAT), glutatión S-transferasa (GST), glutatión peroxidasa (GPx), glutatión reductasa (GR) y la concentración de GSH se determinaron por espectrofotometría; la [TAs] se midió por espectrometría de absorción atómica. Los datos sugieren un incremento en la actividad de PNP (mediana= 0.034 U mg proteína-1) con la presencia de TAs (mediana= 1.16 g L-1). Para explicar la posible asociación de la actividad de las enzimas antioxidantes y la concentración de GSH, así como [TAs], con la actividad de PNP en la leche materna, se construyeron modelos lineales generalizados. En el modelo ajustado, la actividad de GPx y GR presentó una asociación estadística (p.
Assuntos
Antioxidantes/análise , Arsênio/efeitos adversos , Leite Humano/química , Purina-Núcleosídeo Fosforilase/análise , Adulto , Feminino , Humanos , Inativação Metabólica , México , Leite Humano/enzimologiaRESUMO
The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.
Assuntos
Bioensaio/instrumentação , Avaliação Pré-Clínica de Medicamentos/instrumentação , Purina-Núcleosídeo Fosforilase/antagonistas & inibidores , Purina-Núcleosídeo Fosforilase/química , Schistosoma mansoni/enzimologia , Esquistossomicidas/química , Espectrofotometria Ultravioleta/instrumentação , Adsorção , Animais , Desenho de Fármacos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/química , Enzimas Imobilizadas/química , Desenho de Equipamento , Análise de Falha de Equipamento , Purina-Núcleosídeo Fosforilase/análise , Reprodutibilidade dos Testes , Esquistossomicidas/administração & dosagem , Esquistossomicidas/análise , Sensibilidade e EspecificidadeAssuntos
Biomarcadores Tumorais/análise , Melanoma/enzimologia , Nevo/enzimologia , Purina-Núcleosídeo Fosforilase/análise , Neoplasias Cutâneas/enzimologia , Progressão da Doença , Regulação para Baixo , Humanos , Imuno-Histoquímica , Melanoma/patologia , Estadiamento de Neoplasias , Nevo/patologia , Neoplasias Cutâneas/patologiaRESUMO
OBJECTIVE: The pathophysiology underlying the chromosome (Chr) 9p21 locus of atherosclerosis susceptibility is presently unknown. Here, we sought to determine whether protein coding genes in the Chr9p21 region, i.e. cyclin-dependent kinase inhibitors CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)) and methylthioadenosine phosphorylase (MTAP) were expressed in human atherosclerotic lesions and whether expression was correlated with lesion composition. METHODS AND RESULTS: Protein expression of p15(INK4b), p16(INK4a), p14(ARF) and MTAP was demonstrated by immunostaining in normal and atherosclerotic coronary arteries and co-localized with CD68 and smooth muscle alpha-actin positive cells. Quantitative RT-PCR in human endarteryectomy specimens (n = 57) revealed increased p16(INK4a) and decreased MTAP expression in macrophage-rich lesions (P<0.001 and P = 0.007, respectively). Functional studies suggest that decreased MTAP expression in macrophage-rich lesions might be mediated through down-regulation by TNF-alpha. No clear association of p15(INK4b), p16(INK4a), p14(ARF), and MTAP expression in plaque tissue with Chr9p21 haplotypes was found. The latter finding was corroborated by the lack of correlation of RNA expression of 9p21-regulated transcripts EU741058 and NR_003529 of antisense non-coding RNA in the INK4 locus (ANRIL) with mRNA expression of these genes. In contrast, ANRIL DQ485454 which is not genetically determined by the 9p21 genotype was significantly correlated with MTAP expression (P = 0.01). CONCLUSION: CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)), and MTAP are abundantly expressed in atherosclerotic lesions. While expression levels showed no clear association with Chr9p21 genotype, association of high p16(INK4a) and low MTAP expression with a less stable plaque phenotype suggests a more general role of these proteins in atherogenesis.
Assuntos
Cromossomos Humanos Par 9 , Doença da Artéria Coronariana/genética , Inibidor de Quinase Dependente de Ciclina p15/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Purina-Núcleosídeo Fosforilase/genética , Proteína Supressora de Tumor p14ARF/genética , Actinas/análise , Antígenos CD/análise , Antígenos de Diferenciação Mielomonocítica/análise , Autopsia , Doença da Artéria Coronariana/enzimologia , Doença da Artéria Coronariana/patologia , Doença da Artéria Coronariana/cirurgia , Inibidor de Quinase Dependente de Ciclina p15/análise , Inibidor p16 de Quinase Dependente de Ciclina/análise , Endarterectomia , Predisposição Genética para Doença , Células HEK293 , Haplótipos , Humanos , Imuno-Histoquímica , Macrófagos/química , Fenótipo , Purina-Núcleosídeo Fosforilase/análise , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Necrose Tumoral alfa/análise , Proteína Supressora de Tumor p14ARF/análiseRESUMO
In this paper, an electrochemical biosensor to assay purine nucleoside phosphorylase (PNP) activity is reported. Due to the facilitation of sliver nanoparticles, which are modified on an electrode surface, to the electron transfer reactivity of guanosine and guanine, the electrochemical response of these species can clearly reveal the activity of the enzyme PNP. This electrochemical biosensor for the assay of PNP activity may have a broad linear range of 4-20 unit/mL with a detection limit of 0.1 unit/mL, which is good enough for clinical applications. Meanwhile, on the basis of the finding that guanosine and guanine can induce silver nanoparticles to different agglomeration degrees, we have also developed a rapid UV-vis spectroscopy assay method for PNP activity. This work may show acceptable reliability and specificity for the assay of PNP activity, and may avoid the utilization of coupled enzymes or radiochemical reagents, which are required to the previous reports.
Assuntos
Eletroquímica/instrumentação , Microquímica/instrumentação , Nanopartículas/química , Nanotecnologia/instrumentação , Purina-Núcleosídeo Fosforilase/análise , Purina-Núcleosídeo Fosforilase/química , Prata/química , Técnicas Biossensoriais/instrumentação , Ativação Enzimática , Desenho de Equipamento , Análise de Falha de Equipamento , Nanopartículas/ultraestrutura , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A simple and in principle universal method is proposed for measuring enzyme/ligand dissociation constants. The method is based on measuring enzyme activity remaining after heat treatment in the absence and in the presence of ligands. The method is especially suitable for enzymes interacting with nucleosides, nucleosides and oligonucleotides since for such enzymes convenient spectrophotometric assays are available.
Assuntos
Purina-Núcleosídeo Fosforilase/análise , Adenina/análogos & derivados , Adenina/química , Bioquímica/métodos , Estabilidade Enzimática , Guanina/química , Temperatura Alta , Cinética , Ligantes , Compostos Organofosforados/química , Fosfatos/química , Purina-Núcleosídeo Fosforilase/química , EspectrofotometriaRESUMO
Methylthioadenosine phosphorylase (MTAP) is an important enzyme in the salvage pathway of adenosine and methionine synthesis. MTAP is ubiquitously present in all normal cells and tissues, but deficient in a variety of malignant tumors. The enzyme deficiency is caused by either MTAP gene deletion or promoter hypermethylation. We investigated MTAP expression, MTAP gene deletion and promoter abnormality in 40 primary tumor samples from Japanese osteosarcoma patients and determined the frequency of the enzyme deficiency. We also tested whether or not the enzyme deficiency can be exploited for tumor-specific chemotherapy using osteosarcoma cell lines. For MTAP expression, immunohistochemistry (IHC) and Western blotting were used. Real-time quantitative PCR assay was used for the analysis of MTAP gene deletion in fifteen osteosarcoma samples. MTAP promoter abnormality was analyzed by methylation-specific PCR. Then, the relationship between MTAP expression and sensitivity to the inhibitors of de novo AMP synthesis was confirmed in an MTAP-negative and -positive osteosarcoma cell line. The MTAP protein was negative in 11 of 40 samples (27.5%) by IHC and in 4 of 6 osteosarcoma cell lines (66.7%) by Western blot analysis. Among 40 samples, 15 were subjected to quantitative real-time PCR and promoter methylation analysis. Of 6 samples that were negative by IHC, the MTAP gene was deleted in 3 and the MTAP promoter was methylated in 2. These results indicated that MTAP deficiency was caused by MTAP gene deletion or promoter methylation in all MTAP-negative samples except one that was negative with IHC although no deletion or promoter methylation was detected. In in vitro experiments using transfectoma along with the MTAP-negative parental cell line, the MTAP-negative parental cell line was more chemosensitive to the inhibitors of de novo AMP synthesis than MTAP-positive transfectoma. MTAP deficiency frequently found in osteosarcoma can be exploited for selective chemotherapy in MTAP-negative osteosarcoma patients with the inhibitors of de novo purine synthesis.