RESUMO
OBJECTIVES: Tumor programmed death ligand 1 (PD-L1) expression is associated with improved clinical benefit from immunotherapies targeting the PD-1 pathway. We conducted a global, multicenter, retrospective observational study to determine real-world prevalence of tumor PD-L1 expression in patients with NSCLC. MATERIALS AND METHODS: Patients ≥18 years with histologically confirmed stage IIIB/IV NSCLC and a tumor tissue block (≤5 years old) obtained before treatment were identified in 45 centers across 18 countries. Tumor samples from eligible patients were selected consecutively, when possible. PD-L1 expression was evaluated at each center using the PD-L1 IHC 22C3 pharmDx kit (Agilent, Santa Clara, CA, USA). RESULTS: Of 2617 patients who met inclusion criteria, 2368 (90%) had PD-L1 data; 530 (22%) patients had PD-L1 TPSâ¯≥â¯50%, 1232 (52%) had PD-L1 TPSâ¯≥â¯1%, and 1136 (48%) had PD-L1 TPSâ¯<â¯1%. The most common reason for not having PD-L1 data (nâ¯=â¯249) was insufficient tumor cells (<100) on the slide (nâ¯=â¯170 [6%]). Percentages of patients with PD-L1 TPSâ¯≥â¯50% and TPSâ¯≥â¯1%, respectively were: 22%/52% in Europe; 22%/53% in Asia Pacific; 21%/47% in the Americas, and 24%/55% in other countries. Prevalence of EGFR mutations (19%) and ALK alterations (3%) was consistent with prior reports from metastatic NSCLC studies. Among 1064 patients negative for both EGFR mutation and ALK alteration, the percentage with PD-L1 TPSâ¯≥â¯50% and TPSâ¯≥â¯1%, respectively, were 27% and 53%. CONCLUSIONS: This is the largest real-world study in advanced NSCLC to date evaluating PD-L1 tumor expression using the 22C3 pharmDx kit. Testing failure rate was low with local evaluation of PD-L1 TPS across a large number of centers. Prevalence of PD-L1 TPSâ¯≥â¯50% and TPSâ¯≥â¯1% among patients with stage IIIB/IV NSCLC was similar across geographic regions and broadly consistent with central testing results from clinical trial screening populations.
Assuntos
Antígeno B7-H1/genética , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/genética , Expressão Gênica , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/diagnóstico , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Prevalência , Estudos RetrospectivosRESUMO
Numerous single nucleotide polymorphisms (SNPs) have been identified in the human genome, yet the functional significance of most is unknown. CYP3A4 is a key enzyme in the metabolism of numerous compounds. An A-->G substitution 290 bp upstream of the CYP3A4 transcription start site (CYP3A4*1B) has been associated with cancer phenotypes, but its phenotypic effects are unclear. To investigate the functional significance of CYP3A4*1B, we generated two luciferase reporter constructs: 1-kb (denoted L, long) and 0.5-kb (denoted S, short) promoter fragments containing either the variant (V(L),V(S)) or the wild-type (W(L), W(S)) sequences. We evaluated the effect of the variant sequence in the HepG2 and MCF-7 cell lines, and in primary human hepatocytes from three donors. Reporter constructs with the variant sequence had 1.2- to 1.9-fold higher luciferase activity than constructs with wild-type sequence in the cell lines (P < 0.0001) and hepatocytes (P = 0.021, P = 0.027, P = 0.061). The ratio of transcriptional activity for V(S):W(S) was similar to the V(L):W(L) ratio in HepG2 cells, but the V(S):W(S) ratio was consistently less than the V(L):W(L) ratio in MCF-7 cells. This suggests that CYP3A4 expression is higher from the variant promoter and that a repressor sequence may exist in the longer constructs. Electrophoretic mobility shift assays demonstrated specific binding of a component of HepG2 nuclear extract to both wild-type and variant promoters with consistently higher binding affinities to the wild-type sequence. This suggests the existence of a transcriptional repressor responsible for the lower CYP3A4*1A activity. Therefore, the phenotypic effects of the variant CYP3A4*1B may be associated with enhanced CYP3A4 expression due to reduced binding of a transcriptional repressor.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Transcrição Gênica , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citocromo P-450 CYP3A , Genes Reporter , Vetores Genéticos , Genoma Humano , Hepatócitos/metabolismo , Humanos , Luciferases/metabolismo , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Transfecção , beta-Galactosidase/metabolismoRESUMO
Heterocyclic amines found in well-done meat require host-mediated metabolic activation before initiating DNA mutations and tumors in target organs. Polymorphic N-acetyltransferase-2 (NAT2) catalyzes the activation of heterocyclic amines via O-acetylation, suggesting that NAT2 genotypes with high O-acetyltransferase activity (rapid/intermediate acetylator phenotype) increase the risk of breast cancer in women who consume well-done meat. To test this hypothesis, DNA samples and information on diet and other breast cancer risk factors were obtained from a nested case-control study of postmenopausal women. Twenty-seven NAT2 genotypes were determined and assigned to rapid, intermediate, or slow acetylator groups based on published characterizations of recombinant NAT2 allozymes. NAT2 genotype alone was not associated with breast cancer risk. A significant dose-response relationship was observed between breast cancer risk and consumption of well-done meat among women with the rapid/intermediate NAT2 genotype (trend test, P = 0.003) that was not evident among women with the slow acetylator genotype (trend test, P = 0.22). These results suggest an interaction between NAT2 genotype and meat doneness, although a test for multiplicative interaction was not statistically significant (P = 0.06). Among women with the rapid/intermediate NAT2 genotype, consumption of well-done meat was associated with a nearly 8-fold (odds ratio, 7.6; 95% confidence interval, 1.1-50.4) elevated breast cancer risk compared with those consuming rare or medium-done meats. These results are consistent with a role for O-acetylation in the activation of heterocyclic amine carcinogens and support the hypothesis that the NAT2 acetylation polymorphism is a breast cancer risk factor among postmenopausal women with high levels of heterocyclic amine exposure.
Assuntos
Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Carne/efeitos adversos , Acetilação , Idoso , Aminas/efeitos adversos , Biotransformação , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/etiologia , Carcinógenos , Estudos de Casos e Controles , Culinária , Feminino , Compostos Heterocíclicos/efeitos adversos , Humanos , Funções Verossimilhança , Modelos Logísticos , Pessoa de Meia-Idade , Razão de Chances , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Pós-Menopausa/fisiologia , Fatores de RiscoRESUMO
N-Acetyltransferase 1 (NAT1), encoded by the polymorphic NAT1 gene, has been shown to be one of the major enzymes in human breast tissue that activates aromatic and heterocyclic amines. Humans are mainly exposed to these carcinogens through cigarette smoking and consumption of well-done meat. To test the hypothesis that variations in the NAT1 gene are related to breast cancer risk, particularly among women who smoke or consume high levels of well-done meat, a nested case-control study was conducted in a prospective cohort study of 41,837 postmenopausal Iowa women. Information on cigarette smoking and other breast cancer risk factors was obtained at the baseline survey conducted in 1986. DNA samples and information on the consumption of well-done meat were obtained, in the case-control study, from breast cancer cases diagnosed from 1992 to 1994 and a random sample of cancer-free cohort members. Genomic DNA samples obtained from 154 cases and 330 controls were assayed for 11 NAT1 alleles (NAT1*3, *4, *5, *10, *11, *14, *15, *16, *17, *19, and *22). The NAT1*4 allele was the predominant allele observed in this study population, accounting for 73.2% (72.4% in cases versus 73.8% in controls) of the total alleles analyzed. Compared to controls, breast cancer cases had a slightly higher frequency of the NAT1*10 allele (18.8% in cases versus 17.3% in controls) and a substantially higher frequency of the NAT1*11 allele (3.6% versus 1.2%). In multivariate analyses, we found a 30% [95% confidence interval (CI) = 0.8-1.9] elevated risk of breast cancer associated with the NAT1*10 allele and a nearly 4-fold (95% CI = 1.5-10.5) elevated risk associated with the NAT1*11 allele. The positive association of breast cancer with the NAT1*11 allele was more evident among smokers [odds ratio (OR) = 13.2, 95% CI = 1.5-116.0] and those who consumed a high level of red meat (OR = 6.1, 95% CI = 1.1-33.2) or consistently consumed their red meat well done (OR = 5.6, 95% CI = 0.5-62.7). The association of the NAT1*10 allele with breast cancer was mainly confined to former smokers (OR = 3.3, 95% CI = 1.2-9.5). These findings are consistent with a role for the NAT1 gene in the etiology of human breast cancer.
Assuntos
Arilamina N-Acetiltransferase/genética , Neoplasias da Mama/epidemiologia , Comportamento Alimentar , Isoenzimas/genética , Carne , Polimorfismo Genético/genética , Fumar/epidemiologia , Idoso , Alelos , Aminas/metabolismo , Mama/enzimologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Carcinógenos/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Intervalos de Confiança , Culinária , DNA/genética , Feminino , Frequência do Gene , Humanos , Iowa/epidemiologia , Pessoa de Meia-Idade , Análise Multivariada , Razão de Chances , Pós-Menopausa , Estudos Prospectivos , Fatores de RiscoRESUMO
Currently there is much interest in the N-acetyltransferase-1 (NAT1) genetic polymorphism and its relationship to cancer. Previous studies have described methods to distinguish NAT1 alleles through polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) and/or allele-specific amplification. However, these methods detect at most only four of the NAT1 alleles identified in human populations. In this paper we describe a PCR-RFLP-based assay that differentiates among eight human NAT1 alleles (NAT1*3, *4, *5, *10, *11, *14, *15, *16). This method should prove useful in molecular epidemiological studies investigating associations between NAT1 genotype and cancer.
Assuntos
Alelos , Arilamina N-Acetiltransferase/química , Isoenzimas/química , Polimorfismo de Fragmento de Restrição , Colo/química , Primers do DNA , Enzimas de Restrição do DNA/química , Eletroforese em Gel de Ágar , Humanos , Masculino , Reação em Cadeia da Polimerase/métodos , Próstata/químicaRESUMO
The acetylation polymorphism is associated with differential susceptibility to drug toxicity and cancers related to aromatic and heterocyclic amine exposures. N-Acetylation is catalyzed by two cytosolic N-acetyltransferases (NAT1 and NAT2) which detoxify many carcinogenic aromatic amines. NAT1 and NAT2 also activate (via O-acetylation) the N-hydroxy metabolites of aromatic and heterocyclic amine carcinogens to electrophilic intermediates which form DNA adducts and initiate cancer. The classical N-acetylation polymorphism is regulated at the NAT2 locus, which segregates individuals into rapid, intermediate, and slow acetylator phenotypes. Some human epidemiological studies associate slow acetylator and rapid acetylator phenotypes with increased susceptibility to urinary bladder and colorectal cancers, respectively. The acetylation polymorphism has been characterized in three rodent species (mouse, Syrian hamster, and rat) to test associations between NAT2 acetylator phenotype and susceptibility to aromatic and heterocyclic amine-induced cancers in various tumor target organs. NAT1 and NAT2 from rapid and slow acetylator mouse, Syrian hamster, and rat each have been cloned and sequenced. Recombinant NAT1 and NAT2 enzymes enzymes encoded by these genes have been characterized with respect to their catalytic activities for both activation (O-acetylation) and deactivation (N-acetylation) of aromatic and heterocyclic amine carcinogens. The acetylation polymorphisms in mouse, Syrian hamster, and rat are herein reviewed and compared as models of the human acetylation polymorphism.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Mutagênicos/metabolismo , Acetilação , Animais , Cricetinae , Temperatura Alta , Humanos , Isomerismo , Cinética , Camundongos , Desnaturação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Humans possess two N-acetyltransferase isozymes (NAT1 and NAT2). We cloned and sequenced a novel NAT1 allele (Genbank HSU 80835) that contained nucleotide substitutions at -344 (C-->T), -40 (A-->T), 445 [G-->A(Val-->Ile)], 459 [G-->A(silent)], 640 [T-->G(Ser-->Ala)], a 9 base pair deletion between nucleotides 1065 and 1090, and 1095 (C-->A). The novel NAT1 allele which we have designated NAT1*17 is similar to NAT1*11 except for a G445A substitution (Val149-->Ile) in the NAT1 coding region. The G445A (Val149-->Ile) substitution yielded no significant changes in levels of immunoreactivity, as detected by Western blot, nor in intrinsic stability of the recombinant N-acetyltransferase protein. However, the G445A (Val149-->Ile) substitution yielded expression of recombinant NAT1 protein that catalyzed the N-acetylation of aromatic amines and the O- and N,O-acetylation of their N-hydroxylated metabolites at rates up to 2-fold higher than wild-type recombinant human NAT1.
Assuntos
Alelos , Arilamina N-Acetiltransferase/genética , Isoenzimas/genética , Acetilação , Sequência de Aminoácidos , Arilamina N-Acetiltransferase/imunologia , Arilamina N-Acetiltransferase/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Quimera/genética , Clonagem Molecular , Primers do DNA/genética , Estabilidade Enzimática , Engenharia Genética , Humanos , Isoenzimas/imunologia , Isoenzimas/metabolismo , Cinética , Masculino , Reação em Cadeia da Polimerase , Polimorfismo Genético , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismoRESUMO
Polymorphic aromatic amine N-acetyltransferase (NAT2) catalyzes the N-acetylation of aromatic amines and the metabolic activation of N-hydroxyarylamines (via O-acetylation) and N-hydroxy-N-acetylarylamines (via N,O-acetylation) to electrophilic intermediates that mutate DNA. Acetylation capacity in humans and other mammalian species such as Syrian hamsters is subject to a genetic polymorphism. NAT2 is regulated by a single gene (NAT2) containing a single coding exon of 870 bp. Syrian hamster slow acetylator differs from the rapid acetylator NAT2 coding region by three nucleotide substitutions at T36C, A633G, and C727T. We measured expression of immunoreactive NAT2 protein and aromatic amine N-acetylation. N-hydroxyarylamine O-acetylation and N-hydroxy-N-acetylarylamine N,O-acetylation by recombinant NAT2 proteins expressed from alleles containing all combinations of the T36C, A633G, and C727T substitutions. The C727T substitution, which creates an opal stop codon in slow acetylator NAT2, was the sole mutation responsible for substantial reduction in expression of a truncated NAT2 protein with reduced capacity for the deactivation of aromatic amines (N-acetylation) and the metabolic activation of N-hydroxyarylamines (O-acetylation) and N-hydroxy-N-acetylarylamines (N,O-acetylation). The reductions in aromatic amine N-acetylation correlated very highly with the reductions in metabolic activation of the corresponding N-hydroxyarylamines and N-hydroxy-N-acetylarylamines.
Assuntos
Arilamina N-Acetiltransferase/genética , Regulação da Expressão Gênica , Recombinação Genética , Acetilação , Alelos , Animais , Arilamina N-Acetiltransferase/metabolismo , Western Blotting , Quimera , Clonagem Molecular , Cricetinae , Técnicas In Vitro , Mesocricetus , Reação em Cadeia da Polimerase , Polimorfismo GenéticoRESUMO
DNA adduct formation is an important step in initiation of the carcinogenic process. 3,2'-Dimethyl-4-aminobiphenyl (DMABP) is a well-documented multiorgan carcinogenic aromatic amine in rodents. In the present study, DMABP-DNA adduct levels were measured in rapid (Bio. 82.73/H-Pat(r)) and slow (Bio. 82.73/H-Pat(s)) acetylator Syrian hamsters congenic at the NAT2 locus following a single injection of 33 or 100 mg/kg body wt DMABP. Two DNA adducts, N-(deoxyguanosin-8-yl)-DMABP and 5-(deoxyguanosin-N2-yl)-DMABP, were identified and quantitated by 32P-postlabeling assay. After injection of 33 mg/kg, DMABP-DNA adducts were detected in urinary bladder at 6, 18, 24, and 48 hr with adduct levels increasing up to 48 hr postinjection. DMABP-DNA adducts were not detected in liver, colon, and heart. After injection of 100 mg/kg, DMABP-DNA adducts were detected in urinary bladder, liver, prostate, colon, and heart at 48 hr postinjection. DMABP-DNA adduct levels were significantly higher in urinary bladder (primary tumor target organ) than in the other organs of both rapid and slow acetylator congenic hamsters. N-(deoxyguanosin-8-yl)-DMABP levels were significantly higher in liver and prostate than in colon and heart of rapid and slow acetylator congenic hamsters, whereas 5-(deoxyguanosin-N2-yl)-DMABP levels were significantly higher in prostate than in colon and heart of rapid and slow acetylator congenic hamsters. DMABP-DNA adduct levels in each tissue examined did not differ significantly between rapid and slow acetylator hamsters following either 33 or 100 mg/kg injection. The tissue-dependent differences in DMABP-DNA adduct levels observed in the Syrian hamster differ from those reported in the rat and are consistent with previous studies that show DMABP induces primarily urinary bladder tumors in the Syrian hamster.
Assuntos
Compostos de Aminobifenil/toxicidade , Arilamina N-Acetiltransferase/genética , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/genética , Adutos de DNA/efeitos dos fármacos , Neoplasias da Bexiga Urinária/induzido quimicamente , Neoplasias da Bexiga Urinária/genética , Acetilação/efeitos dos fármacos , Animais , Arilamina N-Acetiltransferase/metabolismo , Neoplasias do Colo/metabolismo , Cricetinae , Adutos de DNA/química , Mesocricetus , Especificidade de Órgãos/efeitos dos fármacos , Neoplasias da Bexiga Urinária/metabolismoRESUMO
These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzidine/ benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.
Assuntos
Arilamina N-Acetiltransferase/metabolismo , Benzidinas/metabolismo , Isoenzimas/metabolismo , Acetilação , Sequência de Bases , Benzidinas/farmacocinética , Feminino , Genótipo , Humanos , Cinética , Fígado/anatomia & histologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fenótipo , Proteínas Recombinantes/metabolismoRESUMO
The human N-acetylation polymorphism, encoded by the NAT2 gene locus, has been associated with higher incidence and/or severity to the adverse effects of therapeutic drugs, and to the carcinogenic actions of environmental and occupational chemicals. In this paper, we describe an efficient method of restriction fragment-length polymorphism and allele-specific amplification analysis which distinguishes between each of 15 (NAT2*4, *5A, *5B, *5C, *6A, *6B, *7A, *7B, *12A, *12B, *13, *14A, *14B, *17, *18) NAT2 alleles that have been identified in human populations. The method should have broad applicability to improvement of drug therapy and to molecular epidemiology investigations of genetic predisposition to cancer and other diseases.