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1.
Jpn J Clin Oncol ; 51(1): 60-69, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32844218

RESUMO

BACKGROUND: The prevalence and molecular characteristics of defective DNA mismatch repair endometrial cancers in the Japanese population have been underexplored. Data supporting clinical management of patients with Lynch-like syndrome and germline variant of uncertain significance of mismatch repair genes are still lacking. METHODS: Immunohistochemistry of mismatch repair proteins (MLH1, MSH2, MSH6 and PMS2) was performed on formalin-fixed paraffin-embedded sections prepared from resected primary endometrial cancers in 395 women with a median age of 59 years. Genetic and/or epigenetic alterations of the mismatch repair genes were also investigated. RESULTS: Loss of expression of one or more mismatch repair proteins was observed in 68 patients (17.2%). A total of 17 out of 68 patients (25%, 4.3% of all cases) were identified as candidates for genetic testing for Lynch syndrome after excluding 51 patients with MLH1 hypermethylated cancer. Fourteen of these 17 patients subjected to genetic testing were found to have Lynch syndrome (n = 5), germline variant of uncertain significance (n = 2) or Lynch-like syndrome (n = 7). Compared with patients with Lynch syndrome, those with germline variant of uncertain significance and Lynch-like syndrome tended to demonstrate an older age at the time of endometrial cancer diagnosis (P = 0.07), less fulfillment of the revised Bethesda guidelines (P = 0.09) and lower prevalence of Lynch syndrome-associated tumors in their first-degree relatives (P = 0.01). CONCLUSIONS: This study provides useful information for management in patients with DNA mismatch repair endometrial cancer. Specifically, cancer surveillance as recommended in patients with Lynch syndrome might not be necessary in patients with germline variant of uncertain significance and Lynch-like syndrome and their relatives.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/genética , Hospitais , Adulto , Distribuição por Idade , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais Hereditárias sem Polipose/genética , Metilação de DNA/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Imuno-Histoquímica , Japão/epidemiologia , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL/genética , Prevalência
2.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(11): 1645-1650, 2020 Nov 30.
Artigo em Chinês | MEDLINE | ID: mdl-33243738

RESUMO

OBJECTIVE: To explore the clinicopathological features and types of genic mutations in DNA mismatch repair (MMR) in colorectal cancer (CRC). METHODS: Immunohistochemistry was used to determine the expression of MMR proteins in 1394 patients with CRC, and PCR-capillary electrophoresis (PCR-CE) was used to detect microsatellite instability (MSI) in 106 cases of defective MMR (dMMR), 46 cases of proficient MMR (pMMR) with heterogeneous expression and 147 randomly selected cases of pMMR. The relationship between the expressions of MMR proteins and the clinicopathological features of the patients was evaluated. The consistency between the results of immunohistochemistry and PCR-CE was assessed. RESULTS: Immunohistochemical staining showed an incidence of dMMR of 7.6% in the patients. The main type of dMMR was co-deletion of MLH1 and PMS2, accounting for 55.7% of the total dMMR cases. The deletion of MMR proteins was significantly correlated with the patients' age, tumor location, tumor size, gross type, histological type, degree of differentiation, lymph node status and TNM stage (P < 0.05), but not with gender (P > 0.05). The total accordance rate of immunohistochemistry and PCR-CE was 98.7% in these patients. CONCLUSIONS: The main type of dMMR is co-deletion of MLH1 and PMS2 in patients with colorectal cancer. dMMR colorectal cancer has typical clinicopathological features and a lower incidence in China than in Western countries. The results of immunohistochemistry and PCR-CE are highly consistent for detecting dMMR in colorectal cancer patients.


Assuntos
Neoplasias Colorretais , Instabilidade de Microssatélites , China , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Humanos
3.
PLoS Genet ; 16(10): e1009041, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33119594

RESUMO

Through transfection of short single-stranded oligodeoxyribonucleotides (ssODNs) small genomic alterations can be introduced into mammalian cells with high precision. ssODNs integrate into the genome during DNA replication, but the resulting heteroduplex is prone to detection by DNA mismatch repair (MMR), which prevents effective gene modification. We have previously demonstrated that the suppressive action of MMR can be avoided when the mismatching nucleotide in the ssODN is a locked nucleic acid (LNA). Here, we reveal that LNA-modified ssODNs (LMOs) are not integrated as intact entities in mammalian cells, but are severely truncated before and after target hybridization. We found that single additional (non-LNA-modified) mutations in the 5'-arm of LMOs influenced targeting efficiencies negatively and activated the MMR pathway. In contrast, additional mutations in the 3'-arm did not affect targeting efficiencies and were not subject to MMR. Even more strikingly, homology in the 3'-arm was largely dispensable for effective targeting, suggestive for extensive 3'-end trimming. We propose a refined model for LMO-directed gene modification in mammalian cells that includes LMO degradation.


Assuntos
Replicação do DNA/genética , DNA de Cadeia Simples/genética , Oligonucleotídeos/genética , Animais , Linhagem Celular , Reparo de Erro de Pareamento de DNA/genética , Edição de Genes , Marcação de Genes , Humanos , Mamíferos/genética , Mutação/genética , Transfecção
4.
Sci Rep ; 10(1): 16386, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009475

RESUMO

Microsatellite instability (MSI) status is routinely assessed in patients with colorectal and endometrial cancers as it contributes to Lynch syndrome initial screening, tumour prognosis and selecting patients for immunotherapy. Currently, standard reference methods recommended for MSI/dMMR (deficient MisMatch Repair) testing consist of immunohistochemistry and pentaplex PCR-based assays, however, novel molecular-based techniques are emerging. Here, we aimed to evaluate the performance of a custom capture-based NGS method and the Bio-Rad ddPCR and Idylla approaches for the determination of MSI status for theranostic purposes in 30 formalin-fixed paraffin embedded (FFPE) tissue samples from patients with endometrial (n = 15) and colorectal (n = 15) cancers. All samples were previously characterised using IHC and Promega MSI Analysis System and these assays set as golden standard. Overall agreement, sensitivity and specificity of our custom-built NGS panel were 93.30%, 93.75% and 92.86% respectively. Overall agreement, sensitivity and specificity were 100% with the Idylla MSI system. The Bio-Rad ddPCR MSI assay showed a 100% concordance, sensitivity and specificity. The custom capture-based NGS, Bio-Rad ddPCR and Idylla approaches represent viable and complementary options to IHC and Promega MSI Analysis System for the detection of MSI. Bio-Rad ddPCR and Idylla MSI assays accounts for easy and fast screening assays while the NGS approach offers the advantages to simultaneously detect MSI and clinically relevant genomic alterations.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Biomarcadores Tumorais/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Formaldeído/química , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imuno-Histoquímica/métodos , Instabilidade de Microssatélites , Pacientes , Reação em Cadeia da Polimerase/métodos , Prognóstico , Sensibilidade e Especificidade , Fixação de Tecidos/métodos
5.
PLoS Med ; 17(9): e1003263, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32941469

RESUMO

BACKGROUND: Lynch syndrome (LS) predisposes to endometrial cancer (EC), colorectal cancer, and other cancers through inherited pathogenic variants affecting mismatch-repair (MMR) genes. Diagnosing LS in women with EC can reduce subsequent cancer mortality through colonoscopic surveillance and aspirin chemoprevention; it also enables cascade testing of relatives. A growing consensus supports LS screening in EC; however, the expected proportion of test positives, and optimal testing strategy is uncertain. Previous studies from insurance-based healthcare systems were limited by narrow selection criteria, failure to apply reference standard tests consistently, and poor conversion to definitive testing. The aim of this study was to establish the prevalence of LS and the diagnostic accuracy of LS testing strategies in an unselected EC population. METHODS AND FINDINGS: This was a prospective cross-sectional study carried out at a large United Kingdom gynaecological cancer centre between October 2015 and January 2017. Women diagnosed with EC or atypical hyperplasia (AH) were offered LS testing. Tumours underwent MMR immunohistochemistry (IHC), microsatellite instability (MSI), and targeted MLH1-methylation testing. Women <50 years, with strong family histories and/or indicative tumour molecular features, underwent MMR germline sequencing. Somatic MMR sequencing was performed when indicative molecular features were unexplained by LS or MLH1-hypermethylation. The main outcome measures were the prevalence of LS in an unselected EC population and the diagnostic accuracy of clinical and tumour testing strategies for risk stratifying women with EC for MMR germline sequencing. In total, 500 women participated in the study; only 2 (<1%) declined. Germline sequencing was indicated and conducted for 136 and 135 women, respectively. A total of 16/500 women (3.2%, 95% CI 1.8% to 5.1%) had LS, and 11 more (2.2%) had MMR variants of uncertain significance. Restricting testing to age <50 years, indicative family history (revised Bethesda guidelines or Amsterdam II criteria) or endometrioid histology alone would have missed 9/16 (56%), 8/13 (62%) or 9/13 (69%), and 5/16 (31%) cases of LS, respectively. In total 132/500 tumours were MMR deficient by IHC of which 83/132 (63%) had MLH1-hypermethylation, and 16/49 (33%) of the remaining patients had LS (16/132 with MMR deficiency, 12%). MMR-IHC with targeted MLH1-methylation testing was more discriminatory for LS than MSI with targeted methylation testing, with 100% versus 56.3% (16/16 versus 9/16) sensitivity (p = 0.016) and equal 97.5% (468/484) specificity; 64% MSI-H and 73% MMR deficient tumours unexplained by LS or MLH1-hypermethylation had somatic MMR mutations. The main limitation of the study was failure to conduct MMR germline sequencing for the whole study population, which means that the sensitivity and specificity of tumour triage strategies for LS detection may be overestimated, although the risk of LS in women with no clinical or tumour predictors is expected to be extremely low. CONCLUSIONS: In this study, we observed that age, family history, and histology are imprecise clinical correlates of LS-EC. IHC outperformed MSI for tumour triage and reliably identified both germline and somatic MMR mutations. The 3.2% proportion of LS-EC is similar to colorectal cancer, supporting unselected screening of EC for LS.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Detecção Precoce de Câncer/métodos , Neoplasias do Endométrio/genética , Adulto , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Estudos Transversais , Metilação de DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Neoplasias do Endométrio/diagnóstico , Feminino , Testes Genéticos/métodos , Humanos , Imuno-Histoquímica , Programas de Rastreamento/métodos , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Reino Unido
6.
Proc Natl Acad Sci U S A ; 117(38): 23597-23605, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900932

RESUMO

Trinucleotide repeat (TNR) expansions cause nearly 20 severe human neurological diseases which are currently untreatable. For some of these diseases, ongoing somatic expansions accelerate disease progression and may influence age of onset. This new knowledge emphasizes the importance of understanding the protein factors that drive expansions. Recent genetic evidence indicates that the mismatch repair factor MutSß (Msh2-Msh3 complex) and the histone deacetylase HDAC3 function in the same pathway to drive triplet repeat expansions. Here we tested the hypothesis that HDAC3 deacetylates MutSß and thereby activates it to drive expansions. The HDAC3-selective inhibitor RGFP966 was used to examine its biological and biochemical consequences in human tissue culture cells. HDAC3 inhibition efficiently suppresses repeat expansion without impeding canonical mismatch repair activity. Five key lysine residues in Msh3 are direct targets of HDAC3 deacetylation. In cells expressing Msh3 in which these lysine residues are mutated to arginine, the inhibitory effect of RGFP966 on expansions is largely bypassed, consistent with the direct deacetylation hypothesis. RGFP966 treatment does not alter MutSß subunit abundance or complex formation but does partially control its subcellular localization. Deacetylation sites in Msh3 overlap a nuclear localization signal, and we show that localization of MutSß is partially dependent on HDAC3 activity. Together, these results indicate that MutSß is a key target of HDAC3 deacetylation and provide insights into an innovative regulatory mechanism for triplet repeat expansions. The results suggest expansion activity may be druggable and support HDAC3-selective inhibition as an attractive therapy in some triplet repeat expansion diseases.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Histona Desacetilases , Expansão das Repetições de Trinucleotídeos/genética , Acetilação/efeitos dos fármacos , Acrilamidas/farmacologia , Linhagem Celular , Células Cultivadas , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Fenilenodiaminas/farmacologia
7.
Exp Mol Pathol ; 116: 104519, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32822722

RESUMO

INTRODUCTION: Current methods to detect mismatch repair (MMR) status of colorectal cancer include immunohistochemistry (IHC) and molecular analysis of microsatellite instability (MSI) markers. In this study, we evaluated the performance of a novel Biocartis Idylla™ MSI cartridge-based assay. METHODS: The Biocartis Idylla™ MSI Assay determines MSI status using seven homopolymers frequently mutated in MSI-H cancers. We tested 47 formalin-fixed paraffin-embedded (FFPE) colon cancer tissues previously characterized for MMR deficiency by IHC at our institution from 2013 to 2018. These included 23 MMR-proficient carcinomas with positive/retained nuclear staining of MLH1, PMS2, MSH2, and MSH6 proteins and 24 MMR-deficient carcinomas with loss of nuclear staining for at least one of these proteins. Five µm thick tissue sections were collected from FFPE tissue blocks for each tumor, and the tumor regions were macrodissected, run on the Idylla™ instrument and compared with the Promega panel result. In addition, we evaluated the precision and LOD by using a MSI-H commercially available control and MSS patient samples. A positive result was considered MSI-H when two or more mutations were detected. RESULTS: The overall percent agreement among MMR IHC, the Idylla™ and Promega MSI assays was 100% (47/47). The agreement was 100% in both IHC-determined MMR-proficient tumors (23/23) and IHC-determined MMR-deficient tumors (24/24), compared with molecular MSI assays. CONCLUSION: The novel Biocartis Idylla™ cartridge-based MSI assay showed complete concordance with MMR IHC status and Promega MSI assay. These findings support the use of the Biocartis Idylla™ cartridge-based MSI assay as a rapid and cost-effective alternative method to determine MMR status.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Instabilidade de Microssatélites , Repetições de Microssatélites/genética , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/isolamento & purificação , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA/genética , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade
8.
Nucleic Acids Res ; 48(16): 9124-9134, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32756902

RESUMO

Substitutions in the exonuclease domain of DNA polymerase ϵ cause ultramutated human tumors. Yeast and mouse mimics of the most common variant, P286R, produce mutator effects far exceeding the effect of Polϵ exonuclease deficiency. Yeast Polϵ-P301R has increased DNA polymerase activity, which could underlie its high mutagenicity. We aimed to understand the impact of this increased activity on the strand-specific role of Polϵ in DNA replication and the action of extrinsic correction systems that remove Polϵ errors. Using mutagenesis reporters spanning a well-defined replicon, we show that both exonuclease-deficient Polϵ (Polϵ-exo-) and Polϵ-P301R generate mutations in a strictly strand-specific manner, yet Polϵ-P301R is at least ten times more mutagenic than Polϵ-exo- at each location analyzed. Thus, the cancer variant remains a dedicated leading-strand polymerase with markedly low accuracy. We further show that P301R substitution is lethal in strains lacking Polδ proofreading or mismatch repair (MMR). Heterozygosity for pol2-P301R is compatible with either defect but causes strong synergistic increases in the mutation rate, indicating that Polϵ-P301R errors are corrected by Polδ proofreading and MMR. These data reveal the unexpected ease with which polymerase exchange occurs in vivo, allowing Polδ exonuclease to prevent catastrophic accumulation of Polϵ-P301R-generated errors on the leading strand.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase III/genética , DNA Polimerase II/genética , Neoplasias/enzimologia , Sequência de Aminoácidos/genética , Reparo do DNA/genética , Replicação do DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Mutagênese/genética , Mutação/genética , Neoplasias/genética , Plasmídeos/genética , Saccharomyces cerevisiae/genética
9.
Nat Genet ; 52(9): 958-968, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32747826

RESUMO

Certain mutagens, including the APOBEC3 (A3) cytosine deaminase enzymes, can create multiple genetic changes in a single event. Activity of A3s results in striking 'mutation showers' occurring near DNA breakpoints; however, less is known about the mechanisms underlying the majority of A3 mutations. We classified the diverse patterns of clustered mutagenesis in tumor genomes, which identified a new A3 pattern: nonrecurrent, diffuse hypermutation (omikli). This mechanism occurs independently of the known focal hypermutation (kataegis), and is associated with activity of the DNA mismatch-repair pathway, which can provide the single-stranded DNA substrate needed by A3, and contributes to a substantial proportion of A3 mutations genome wide. Because mismatch repair is directed towards early-replicating, gene-rich chromosomal domains, A3 mutagenesis has a high propensity to generate impactful mutations, which exceeds that of other common carcinogens such as tobacco smoke and ultraviolet exposure. Cells direct their DNA repair capacity towards more important genomic regions; thus, carcinogens that subvert DNA repair can be remarkably potent.


Assuntos
Citidina Desaminase/genética , Reparo de Erro de Pareamento de DNA/genética , Mutação/genética , Neoplasias/genética , Citosina Desaminase/genética , DNA de Cadeia Simples/genética , Genoma/genética , Humanos , Mutagênese/genética
10.
BMC Med Genet ; 21(1): 141, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611331

RESUMO

BACKGROUND: Lynch syndrome (LS), which is known as a hereditary cancer syndrome, is distinguished by microsatellite instability, represented by the altered number of repetitive sequences in the coding and/or non-coding region. Immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins (e.g., MLH1, MSH2, MSH6, and PMS2) has been recognized as an useful technique for screening of LS. Previous study has shown that the assessment of IHC, however, requires specific caution due to variable staining patterns even without germline mutations in MMR genes. CASE PRESENTATION: A 48-year-old man, who had been treated for anaplastic astrocytoma, was referred to our department for the precise examination of progressing anemia. Whole-body examination revealed two advanced carcinomas in descending colon and stomach. A hypo-vascular mass lesion was detected in liver as well. Pathological diagnosis (on surgical specimens) was poorly differentiated adenocarcinoma in descending colon, moderately differentiated tubular adenocarcinoma in stomach, and liver metastasis, which is possibly from colon. It was suspected that this case would be Turcot's syndrome-type-1 due to its specific family history having two cases of colon cancer within the second relatives. Pathogenic frameshift mutations in codon 618 of MLH1 gene was identified. Immunohistochemical analyses (IHC) demonstrated complete loss of MLH1 immuno-expression as well as of PMS2 except for those in brain tumor. Although frameshift mutation was not found in MSH6 gene, histological expression of MSH6 was patchy in primary colon carcinoma and was completely lost in the metastatic site in liver. MSH6 expression in gastric carcinoma, a coincidental cancer in this case, was intact. An abnormal (C)8 region was identified by the cloned PCR of colon and liver tumors but not from gastric cancer. Frameshift mutation in a (C)8 tract in exon 5 of the MSH6 gene was also detected in liver metastasis. CONCLUSION: This case supports a plausible mechanism, proposed by a previous literature, for the reduced expression of MSH6 in a somatic mutation manner, which might preferentially happen in colon cancer rather than in stomach carcinoma in MLH1/PMS2-deficient type of Turcot's syndrome type 1.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/imunologia , Predisposição Genética para Doença , Neoplasias Hepáticas/secundário , Mutação/genética , Síndromes Neoplásicas Hereditárias/genética , Adulto , Sequência de Bases , Reparo de Erro de Pareamento de DNA/genética , Feminino , Humanos , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Linhagem
11.
Nat Commun ; 11(1): 3304, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620809

RESUMO

A main assumption of molecular population genetics is that genomic mutation rate does not depend on sequence function. Challenging this assumption, a recent study has found a reduction in the mutation rate in exons compared to introns in somatic cells, ascribed to an enhanced exonic mismatch repair system activity. If this reduction happens also in the germline, it can compromise studies of population genomics, including the detection of selection when using introns as proxies for neutrality. Here we compile and analyze published germline de novo mutation data to test if the exonic mutation rate is also reduced in germ cells. After controlling for sampling bias in datasets with diseased probands and extended nucleotide context dependency, we find no reduction in the mutation rate in exons compared to introns in the germline. Therefore, there is no evidence that enhanced exonic mismatch repair activity determines the mutation rate in germline cells.


Assuntos
Éxons/genética , Mutação em Linhagem Germinativa , Íntrons/genética , Taxa de Mutação , Algoritmos , Reparo de Erro de Pareamento de DNA/genética , Evolução Molecular , Células Germinativas/metabolismo , Humanos , Modelos Genéticos , Mutação , Sequenciamento Completo do Exoma/métodos
12.
Sci Rep ; 10(1): 9690, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32546739

RESUMO

In the era of intensity-modulated radiotherapy (IMRT), it is important to analyse the prognostic value of deficient mismatch repair (dMMR) in nasopharyngeal carcinoma (NPC). In this study, in pretreatment biopsies of 69 patients with stage II-IVa NPC, the expression levels of MMR proteins, including MLH1, MSH2, MSH6 and PMS2, were assessed by immunohistochemistry (IHC). The median follow-up time was 37.5 months (3.1-87.4 months). 50.7% of cases (35/69) showed preserved expression of all 4 MMR proteins, which was interpreted as proficient mismatch repair (pMMR). Only 1.5% of cases (1/69) lost expression of all 4 MMR proteins, 26.1% of cases (18/69) have PMS2 loss alone and 21.7% of cases (15/69) lost expression of both PMS2 and MLH1. Thus, 49.3% of cases (34/69) lost expression of one or more MMR proteins, which was interpreted as dMMR. There was no significant difference (P > 0.05) in terms of sex, age, clinical stage, T category, N category or therapy regimens between the dMMR and pMMR groups. The multivariate Cox regression analysis revealed that dMMR was an independent significant prognostic factor for distant metastasis-free survival (DMFS) (dMMR vs pMMR: P = 0.01, HR = 0.25, 95% CI: 0.09~0.75). Therefore, NPC patients with dMMR had significantly superior DMFS compared with patients with pMMR. It can be expected that dMMR will become a new independent prognostic factor for NPC.


Assuntos
Reparo de Erro de Pareamento de DNA , Carcinoma Nasofaríngeo/diagnóstico , Neoplasias Nasofaríngeas/diagnóstico , Radioterapia de Intensidade Modulada , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Criança , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/radioterapia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Neoplasias Nasofaríngeas/radioterapia , Estadiamento de Neoplasias , Prognóstico , Adulto Jovem
13.
Mol Cell ; 78(6): 1166-1177.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32497495

RESUMO

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Alelos , Linhagem Celular Tumoral , Reparo de Erro de Pareamento de DNA/fisiologia , Humanos , Mutagênese/genética , Mutação/genética , Neoplasias/genética , Neoplasias/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo
14.
Cutis ; 105(3): E34-E37, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32352449

RESUMO

Mutations in the genes encoding the DNA mismatch repair (MMR) are identified in most sebaceous neoplasms. Sebaceous tumors are rare in the general population; however, they are common in patients with inherited or acquired mutations in the MMR system. This article describes the DNA MMR system and its implication in sebaceous tumors as well as discusses the recent recommendations for screening for Muir-Torre syndrome (MTS) in patients presenting with sebaceous tumors.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Síndrome de Muir-Torre/diagnóstico , Síndrome de Muir-Torre/genética , Detecção Precoce de Câncer , Humanos , Imuno-Histoquímica , Instabilidade de Microssatélites , Mutação
15.
BMC Genomics ; 21(1): 319, 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32326906

RESUMO

BACKGROUND: Ashbya gossypii naturally overproduces riboflavin and has been utilized for industrial riboflavin production. To improve riboflavin production, various approaches have been developed. In this study, to investigate the change in metabolism of a riboflavin-overproducing mutant, namely, the W122032 strain (MT strain) that was isolated by disparity mutagenesis, genomic analysis was carried out. RESULTS: In the genomic analysis, 33 homozygous and 1377 heterozygous mutations in the coding sequences of the genome of MT strain were detected. Among these heterozygous mutations, the proportion of mutated reads in each gene was different, ranging from 21 to 75%. These results suggest that the MT strain may contain multiple nuclei containing different mutations. We tried to isolate haploid spores from the MT strain to prove its ploidy, but this strain did not sporulate under the conditions tested. Heterozygous mutations detected in genes which are important for sporulation likely contribute to the sporulation deficiency of the MT strain. Homozygous and heterozygous mutations were found in genes encoding enzymes involved in amino acid metabolism, the TCA cycle, purine and pyrimidine nucleotide metabolism and the DNA mismatch repair system. One homozygous mutation in AgILV2 gene encoding acetohydroxyacid synthase, which is also a flavoprotein in mitochondria, was found. Gene ontology (GO) enrichment analysis showed heterozygous mutations in all 22 DNA helicase genes and genes involved in oxidation-reduction process. CONCLUSION: This study suggests that oxidative stress and the aging of cells were involved in the riboflavin over-production in A. gossypii riboflavin over-producing mutant and provides new insights into riboflavin production in A. gossypii and the usefulness of disparity mutagenesis for the creation of new types of mutants for metabolic engineering.


Assuntos
Eremothecium/genética , Genoma Fúngico/genética , Genômica/métodos , Mutação , Riboflavina/metabolismo , Acetolactato Sintase/genética , Ciclo do Ácido Cítrico/genética , Reparo de Erro de Pareamento de DNA/genética , Eremothecium/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genótipo , Engenharia Metabólica/métodos , Mutagênese
16.
Nat Med ; 26(4): 566-576, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32251400

RESUMO

PD-1 plus CTLA-4 blockade is highly effective in advanced-stage, mismatch repair (MMR)-deficient (dMMR) colorectal cancers, yet not in MMR-proficient (pMMR) tumors. We postulated a higher efficacy of neoadjuvant immunotherapy in early-stage colon cancers. In the exploratory NICHE study (ClinicalTrials.gov: NCT03026140), patients with dMMR or pMMR tumors received a single dose of ipilimumab and two doses of nivolumab before surgery, the pMMR group with or without celecoxib. The primary objective was safety and feasibility; 40 patients with 21 dMMR and 20 pMMR tumors were treated, and 3 patients received nivolumab monotherapy in the safety run-in. Treatment was well tolerated and all patients underwent radical resections without delays, meeting the primary endpoint. Of the patients who received ipilimumab + nivolumab (20 dMMR and 15 pMMR tumors), 35 were evaluable for efficacy and translational endpoints. Pathological response was observed in 20/20 (100%; 95% exact confidence interval (CI): 86-100%) dMMR tumors, with 19 major pathological responses (MPRs, ≤10% residual viable tumor) and 12 pathological complete responses. In pMMR tumors, 4/15 (27%; 95% exact CI: 8-55%) showed pathological responses, with 3 MPRs and 1 partial response. CD8+PD-1+ T cell infiltration was predictive of response in pMMR tumors. These data indicate that neoadjuvant immunotherapy may have the potential to become the standard of care for a defined group of colon cancer patients when validated in larger studies with at least 3 years of disease-free survival data.


Assuntos
Adenocarcinoma/terapia , Antineoplásicos Imunológicos/efeitos adversos , Neoplasias do Colo/terapia , Reparo de Erro de Pareamento de DNA/genética , Imunoterapia/efeitos adversos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/administração & dosagem , Células Cultivadas , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Terapia Combinada , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Procedimentos Cirúrgicos do Sistema Digestório , Esquema de Medicação , Estudos de Viabilidade , Feminino , Humanos , Imunoterapia/métodos , Ipilimumab/administração & dosagem , Ipilimumab/efeitos adversos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/efeitos adversos , Terapia Neoadjuvante/métodos , Estadiamento de Neoplasias , Nivolumabe/administração & dosagem , Nivolumabe/efeitos adversos , Falha de Tratamento
17.
Nature ; 580(7804): 517-523, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32322066

RESUMO

A high tumour mutational burden (hypermutation) is observed in some gliomas1-5; however, the mechanisms by which hypermutation develops and whether it predicts the response to immunotherapy are poorly understood. Here we comprehensively analyse the molecular determinants of mutational burden and signatures in 10,294 gliomas. We delineate two main pathways to hypermutation: a de novo pathway associated with constitutional defects in DNA polymerase and mismatch repair (MMR) genes, and a more common post-treatment pathway, associated with acquired resistance driven by MMR defects in chemotherapy-sensitive gliomas that recur after treatment with the chemotherapy drug temozolomide. Experimentally, the mutational signature of post-treatment hypermutated gliomas was recapitulated by temozolomide-induced damage in cells with MMR deficiency. MMR-deficient gliomas were characterized by a lack of prominent T cell infiltrates, extensive intratumoral heterogeneity, poor patient survival and a low rate of response to PD-1 blockade. Moreover, although bulk analyses did not detect microsatellite instability in MMR-deficient gliomas, single-cell whole-genome sequencing analysis of post-treatment hypermutated glioma cells identified microsatellite mutations. These results show that chemotherapy can drive the acquisition of hypermutated populations without promoting a response to PD-1 blockade and supports the diagnostic use of mutational burden and signatures in cancer.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , Mutação , Animais , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/imunologia , Reparo de Erro de Pareamento de DNA/genética , Frequência do Gene , Genoma Humano/efeitos dos fármacos , Genoma Humano/genética , Glioma/imunologia , Humanos , Masculino , Camundongos , Repetições de Microssatélites/efeitos dos fármacos , Repetições de Microssatélites/genética , Mutagênese/efeitos dos fármacos , Mutação/efeitos dos fármacos , Fenótipo , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Análise de Sequência de DNA , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Pathol ; 250(5): 518-531, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32141610

RESUMO

Lynch syndrome (LS) is characterised by predisposition to colorectal, endometrial, and other cancers and is caused by inherited pathogenic variants affecting the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2. It is probably the most common predisposition to cancer, having an estimated prevalence of between 1/100 and 1/180. Resources such as the International Society for Gastrointestinal Hereditary Cancer's MMR gene variant database, the Prospective Lynch Syndrome Database (PLSD), and the Colon Cancer Family Register (CCFR), as well as pathological and immunological studies, are enabling advances in the understanding of LS. These include defined criteria by which to interpret gene variants, the function of MMR in the normal control of apoptosis, definition of the risks of the various cancers, and the mechanisms and pathways by which the colorectal and endometrial tumours develop, including the critical role of the immune system. Colorectal cancers in LS can develop along three pathways, including flat intramucosal lesions, which depend on the underlying affected MMR gene. This gives insights into the limitations of colonoscopic surveillance and highlights the need for other forms of anti-cancer prophylaxis in LS. Finally, it shows that the processes of autoimmunisation and immunoediting fundamentally constrain the development of tumours in LS and explain the efficacy of immune checkpoint blockade therapy in MMR-deficient tumours. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias do Endométrio/patologia , Predisposição Genética para Doença/genética , Patologia Molecular , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Feminino , Humanos , Patologia Molecular/métodos
19.
Oncogene ; 39(15): 3041-3055, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32066879

RESUMO

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.


Assuntos
Neoplasias Encefálicas/terapia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/terapia , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Estudos de Coortes , Metilação de DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/mortalidade , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína 2 Homóloga a MutS/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
20.
J Am Coll Surg ; 230(4): 669-675, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32007537

RESUMO

BACKGROUND: Because of increased risk of metachronous colorectal cancer (CRC), all patients with Lynch syndrome (LS) are offered a total colectomy. However, because metachronous CRC rate by mismatch repair (MMR) gene is uncertain, and total colectomy negatively impacts quality of life, it remains unclear whether segmental resection is indicated for lower penetrance MMR genes. We evaluated metachronous CRC incidence according to MMR gene in LS patients who underwent a segmental colectomy. STUDY DESIGN: Single-center, retrospective cohort study in patients with an earlier colectomy for CRC and an MMR germline mutation in MLH1, MSH2, MSH6, or PMS2 followed prospectively in a hereditary CRC family registry. All patients underwent surveillance colonoscopy. Metachronous CRC was defined as one detected more than 1 year after index resection. Primary end point was cumulative incidence of metachronous CRC overall and by MMR gene. RESULTS: One hundred and ten patients were included: 35 with MLH1 likely pathogenic/pathogenic (LP/P) variants (32%), 42 MSH2 (38%), 20 MSH6 (18%), and 13 PMS2 (12%). Median follow-up 4.26 years (range 0.53 to 19.92 years). Overall, metachronous CRC developed in 22 patients (20%). At 10-year follow-up, incidence was 12% (95% CI 6% to 23%), with no metachronous CRC detected in patients with a PMS2 or MSH6 LP/P variant. CONCLUSIONS: After index segmental resection, metachronous CRC is less likely to develop in LS patients with MSH6 or PMS2 LP/P variant than in MLH1 or MSH2 carriers. Our data support segmental resection and long-term colonoscopic surveillance rather than total colectomy in carefully selected, well-informed LS patients with MSH6 or PMS2 LP/P variant.


Assuntos
Colectomia/métodos , Neoplasias Colorretais Hereditárias sem Polipose/cirurgia , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Mutação em Linhagem Germinativa , Segunda Neoplasia Primária/genética , Adolescente , Adulto , Idoso , Criança , Estudos de Coortes , Neoplasias Colorretais/epidemiologia , Feminino , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Segunda Neoplasia Primária/epidemiologia , Estudos Retrospectivos , Medição de Risco , Adulto Jovem
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