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1.
Isr Med Assoc J ; 22(1): 32-36, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31927803

RESUMO

BACKGROUND: Evaluation of mismatch repair (MMR) deficiency is conducted via immunohistochemistry or by microsatellite instability (MSI) analysis. Heterogeneous immunohistochemistry staining for MMR proteins may show different patterns; however, according to current guidelines, all of those patterns should be interpreted as MMR proficient. This conclusion might lead to false negative results because although most cases of heterogeneity stem from technical factors and biological variability, other types of heterogeneity represent true MMR deficiency. OBJECTIVES: To identify a unique heterogeneity pattern that is associated with true MMR loss. METHODS: We analyzed 145 cases of colorectal carcinoma. Immunohistochemistry staining for MLH1, PMS2, MSH2, and MSH6 were performed. We defined geographic heterogeneity as areas of tumor nuclear staining adjacent to areas of loss of tumor nuclear staining with intact staining in the surrounding stroma. All cases were evaluated for the presence of geographic heterogeneity. In addition, 24 cases were also evaluated by MSI testing. RESULTS: Of the 145 cases, 24 (16.5%) were MMR deficient. Of the 24 cases for which MSI analysis was also available, 10 cases (41.7%) demonstrated biological heterogeneity, 5 (20.8%) demonstrated technical heterogeneity, and 2 (8.3%) demonstrated geographic heterogeneity. Only the two cases with geographic heterogeneity were MSI-high via MSI analysis. In addition, a germline mutation in MSH-6 was identified in one of these cases. CONCLUSIONS: Geographic heterogeneity may raise a suspicion for a MMR-deficient case, which should be further analyzed using additional methodologies such as MSI analysis.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Proteínas MutS/genética , Adenoma/genética , Adenoma/patologia , Adulto , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Corantes , Heterogeneidade Genética , Humanos , Masculino
2.
Medicine (Baltimore) ; 98(51): e18279, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31860975

RESUMO

RATIONALE: Lynch syndrome (LS) is an autosomal dominant cancer predisposition condition caused by germline heterozygous mutations in mismatch repair (MMR) genes. However, as one of the MMR genes, PMS2 mutation-induced LS-associated endometrial cancer (LSAEC) was rarely reported. PATIENT CONCERNS: A 26-year-old female patient suffered from prolonged menstrual period and increased menstrual flow for 2 months. DIAGNOSES: The patient was diagnosed with cervix CIN III, endometrial cancer (EC), anemia, and LS. INTERVENTIONS: Total hysterectomy, bilateral salpingectomy, pelvic lymphadenectomy were performed for treating EC, while ovariectomy was refused by the patient. The patient underwent postoperative chemotherapy with paclitaxel combined with carboplatin for 6 courses of treatment. Laparoscopic partial enterectomy was applied for treating colon cancer 5 years later after the surgery treatment for EC. Besides, Sanger sequencing and high-throughput genome sequencing were employed to detect the genetic status of the family that included two generations with four members. Immunohistochemistry (IHC) staining was used to identify the function of PMS2 mutation. OUTCOMES: The 26-year-old Chinese patient suffered from LSAEC and recovered well after surgery. A PMS2 germline heterozygous mutation (c.1577delA) was confirmed by gene sequencing 5 years later. In addition, PMS2 mutation was verified by IHC. The patient was followed up for 7 years. LESSONS: Carrying PMS2 germline mutation (c.1577delA) confers an extremely high susceptibility of suffering from LS-associated cancers. Thus, close clinical monitoring and prophylactic surgery are highly recommended to reduce the morbidity and mortality of LS-associated cancers.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias do Endométrio/genética , Mutação em Linhagem Germinativa/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Adulto , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/patologia , Endométrio/patologia , Feminino , Humanos
3.
J Cancer Res Clin Oncol ; 145(12): 2891-2899, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31617076

RESUMO

PURPOSE: Microsatellites are widely distributed repetitive DNA motifs, accounting for approximately 3% of the genome. Due to mismatch repair system deficiency, insertion or deletion of repetitive units often occurs, leading to microsatellite instability. In this review, we aimed to explore the relationship between MSI and biological behaviour of colorectal carcinoma, gastric carcinoma, lymphoma/leukaemia and endometrial carcinoma, as well as the application of frameshift peptide vaccines in cancer therapy. METHODS: The relevant literature from PubMed and Baidu Xueshu were reviewed in this article. The ClinicalTrials.gov database was searched for clinical trials related to the specific topic. RESULTS: Microsatellite instability is divided into three subtypes: high-level, low-level microsatellite instability, and stable microsatellites. The majority of tumour patients with high-level microsatellite instability often show a better efficacy and prognosis than those with low-level microsatellite instability or stable microsatellites. In coding regions, especially for genes involved in tumourigenesis, microsatellite instability often results in inactivation of proteins and contributes to tumourigenesis. Moreover, the occurrence of microsatellite instability in coding regions can also cause the generation of frameshift peptides that are thought to be unknown and novel to the individual immune system. Thus, these frameshift peptides have the potential to be biomarkers to raise tumour-specific immune responses. CONCLUSION: MSI has the potential to become a key predictor for evaluating the degree of malignancy, efficacy and prognosis of tumours. Clinically, MSI patterns will provide more valuable information for clinicians to create optimal individualized treatment strategies based on frameshift peptides vaccines.


Assuntos
Mutação da Fase de Leitura/genética , Repetições de Microssatélites/genética , Neoplasias/genética , Animais , Carcinogênese/genética , Ensaios Clínicos como Assunto , Reparo de Erro de Pareamento de DNA/genética , Humanos , Instabilidade de Microssatélites , Prognóstico
4.
Presse Med ; 48(9): 904-914, 2019 Sep.
Artigo em Francês | MEDLINE | ID: mdl-31561847

RESUMO

About 5% of colorectal cancer (CRC) cases occurred in the context of an underlying hereditary predisposition syndrome. Lynch syndrome is the main causes of hereditary CRC but is also associated with a higher risk of other cancers (such as endometrial cancer and ovarian cancer). It is the consequence of constitutional mutation in a MisMatch Repair (MMR) gene, involved in DNA repair: MLH1, MSH2, MSH6 or PMS2; or of the EPCAM gene (MSH2 promotor). If a mutation predisposing to Lynch Syndrome is identified in an individual, special monitoring should be initiated, adapted to estimated cancer risk. Clinical criteria (Amsterdam II and Bethesda) have been validated to identify the patients who should be referred for genetic counseling in order to initiate constitutional DNA testing. Furthermore, the French National Cancer Institute (INCa) systematically recommend tumoral testing looking for MMR system failure in case of CRC diagnosed under 60, endometrial cancer diagnosed under 50 or whatever the age in patients diagnosed with CRC or endometrial cancer harbouring personal or familal history of Lunch Syndrome cancers. In this review, we will discuss how to detect Lynch syndrome (identification of the index case and family screening) and how to monitor it in 2019.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Neoplasias Colorretais Hereditárias sem Polipose/terapia , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Fatores Etários , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/genética , Detecção Precoce de Câncer , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Intestino Grosso , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/genética , Linhagem , Vigilância da População/métodos , Guias de Prática Clínica como Assunto , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Urológicas/diagnóstico , Neoplasias Urológicas/genética
5.
Turk Neurosurg ; 29(5): 778-784, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31529454

RESUMO

AIM: To evaluate the frequency and prognostic significance of microsatellite instability (MSI) in patients with glioblastoma (GBM), an immunohistochemical analysis of mismatch repair (MMR) proteins was performed. MATERIAL AND METHODS: A total of 71 patients with GBM who underwent surgery between 2011 and 2019, were included in the study. MMR protein expression was examined using immunohistochemistical analysis of tumor tissue samples; the association between the MMR status and clinicopathological findings was evaluated. RESULTS: Immunohistochemical analysis revealed expressions of MLH1, MSH2, MSH6, and PMS2 proteins in 67 (94.4%), 65 (91.5%), 67 (94.4%), and 64 (90.1%) patients, respectively. Among the 71 patients, 64 (90.1%) expressing all MMR proteins were considered microsatellite stable (MSS), and 7 (9.9%) patients showing loss of at least one of the MMR proteins were considered to show MSI. Tumor recurrence was noted in 25 (39.1%) patients in the MSS GBM group, and 4 (57.1%) patients in the MSI GBM group (p=0.433). The overall median survival was 30.65 ± 5.1 and 10.71 ± 5.2 months in the MSS GBM and MSI GBM groups, respectively (p=0.059). CONCLUSION: The results of this study showed no significant relationships between MMR protein expression and recurrence rates or overall survival in patients with GBM.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Instabilidade de Microssatélites , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Criança , Pré-Escolar , Reparo de Erro de Pareamento de DNA/genética , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Adulto Jovem
6.
Nat Commun ; 10(1): 3925, 2019 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-31477700

RESUMO

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.


Assuntos
Proliferação de Células/genética , Replicação do DNA/genética , Fibroblastos/metabolismo , Instabilidade de Microssatélites , Mutação , Animais , Células Cultivadas , Instabilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Reparo de Erro de Pareamento de DNA/genética , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Células HCT116 , Células HeLa , Humanos , Camundongos Knockout
7.
Korean J Gastroenterol ; 74(1): 4-10, 2019 Jul 25.
Artigo em Coreano | MEDLINE | ID: mdl-31344768

RESUMO

The incidence of colorectal cancer (CRC) is increasing among young adults in several countries including the United States. Early-onset colorectal cancer (EO-CRC) is heterogenous group and appears to have a distinct clinical, pathologic, and molecular presentation compared to CRC diagnosed in older patients. EO-CRC patients tend to be diagnosed at later stage and with symptomatic disease. Tumors arise predominantly in the distal colon and rectum and have poorer histological features. Microsatellite and chromosome stable molecular pathway is frequently observed in EO-CRC. Survival data of EO-CRC is conflicting and younger patients tend to be treated more aggressively. It is important to identify high risk and have them screened appropriately to address increasing EO-CRC. Screening and treatment strategies for EO-CRC should be developed through further investigation of molecular mechanism and various environmental risk factors. In Korea, EO-CRC and late-onset CRC have both declined in recent several years, probably due to many screening colonoscopies in young population. Close observation and analysis of EO-CRC in Korea is necessary.


Assuntos
Neoplasias Colorretais/diagnóstico , Fatores Etários , Instabilidade Cromossômica , Colonoscopia , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Humanos , Incidência , Prognóstico , República da Coreia/epidemiologia , Fatores de Risco
8.
Nat Commun ; 10(1): 3190, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320627

RESUMO

Brain metastases (BM) of colorectal cancer (CRC) are rare but lethal, and an understanding of their genomic landscape is lacking. We conduct an analysis of whole-exome sequencing (WES) and whole-genome sequencing (WGS) data on 19 trios of patient-matched BMs, primary CRC tumors, and adjacent normal tissue. Compared with primary CRC, BM exhibits elevated mutational signatures of homologous recombination deficiency (HRD) and mismatch repair deficiency (MMRD). Further analysis reveals two DNA damage response (DDR) signatures could emerge early and are enhanced in BM tissues but are eliminated eventually in matched primary CRC tissues. BM-specific mutations in DDR genes and elevated microsatellite instability (MSI) levels support the importance of DDR in the brain metastasis of CRC. We also identify BM-related genes (e.g., SCN7A, SCN5A, SCN2A, IKZF1, and PDZRN4) that carry frequent BM-specific mutations. These results provide a better understanding of the BM mutational landscape and insights into treatment.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Colorretais/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/secundário , Neoplasias Colorretais/patologia , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/genética , Recombinação Homóloga/genética , Humanos , Instabilidade de Microssatélites , Mutação/genética , Sequenciamento Completo do Exoma
9.
Int J Clin Oncol ; 24(9): 999-1011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31273487

RESUMO

Lynch syndrome is a cancer-predisposing syndrome inherited in an autosomal-dominant manner, wherein colon cancer and endometrial cancer develop frequently in the family, it results from a loss-of-function mutation in one of four different genes (MLH1, MSH2, MSH6, and PMS2) encoding mismatch repair proteins. Being located immediately upstream of the MSH2 gene, EPCAM abnormalities can affect MSH2 and cause Lynch syndrome. Mismatch repair proteins are involved in repairing of incorrect pairing (point mutations and deletion/insertion of simple repetitive sequences, so-called microsatellites) that can arise during DNA replication. MSH2 forms heterodimers with MSH6 or MSH3 (MutSα, MutSß, respectively) and is involved in mismatch-pair recognition and initiation of repair. MLH1 forms a complex with PMS2, and functions as an endonuclease. If the mismatch repair system is thoroughly working, genome integrity is maintained completely. Lynch syndrome is a state of mismatch repair deficiency due to a monoallelic abnormality of any mismatch repair genes. The phenotype indicating the mismatch repair deficiency can be frequently shown as a microsatellite instability in tumors. Children with germline biallelic mismatch repair gene abnormalities were reported to develop conditions such as gastrointestinal polyposis, colorectal cancer, brain cancer, leukemia, etc., and so on, demonstrating the need to respond with new concepts in genetic counseling. In promoting cancer genome medicine in a new era, such as by utilizing immune checkpoints, it is important to understand the genetic and genomic molecular background, including the status of mismatch repair deficiency.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA/fisiologia , Neoplasias Encefálicas/genética , Criança , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/genética , Molécula de Adesão da Célula Epitelial/genética , Feminino , Aconselhamento Genético , Testes Genéticos , Humanos , Instabilidade de Microssatélites , Endonuclease PMS2 de Reparo de Erro de Pareamento/genética , Endonuclease PMS2 de Reparo de Erro de Pareamento/metabolismo , Proteína 1 Homóloga a MutL/genética , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Mutação
10.
Med Sci (Paris) ; 35(6-7): 535-543, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31274083

RESUMO

The human tumor phenotype referred to as MSI (Microsatellite Instability) is associated with inactivating alterations in MMR genes (Mismatch Repair). MSI was first observed in inherited malignancies associated with Lynch syndrome and later in sporadic colon, gastric and endometrial cancers. MSI tumors develop through a distinctive molecular pathway characterized by genetic instability in numerous microsatellite DNA repeat sequences throughout the genome. In this article, french researchers and physicians who have been recently awarded by the Fondation de France (Jean and Madeleine Schaeverbeke prize) make a sum of their activity in the MSI cancer field for more than 20 years. Their findings have greatly contributed to increase our knowledge of this original cancer model, laying the foundation for a personalized medicine of MSI tumors.


Assuntos
Instabilidade Genômica/fisiologia , Instabilidade de Microssatélites , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisão/métodos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Choque Térmico HSP110/genética , Humanos , Neoplasias/diagnóstico , Neoplasias/imunologia , Medicina de Precisão/tendências
11.
Urology ; 134: 24-31, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31302137

RESUMO

Lynch Syndrome (LS) entails a defective DNA mismatch repair system, which is the postreplicative proofreading and editing system, ensuring our genome's integrity. LS predisposes to several cancers, most commonly colorectal and endometrial cancers. LS occurs in approximately 1 in 250-1000 people. LS is associated with urological malignancies with upper tract urothelial carcinoma the most common, although still clinically underestimated. Other urologic malignancies possibly associated with LS include bladder, prostate, testis, and renal cell carcinoma. Ascertaining their true prevalence in LS is mandatory for their and their relatives' diagnosis and treatment. Awareness regarding identifying patients at risk for LS through assessment of personal and familial oncologic history is critical among urologists.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose , Neoplasias Urogenitais , Neoplasias Colorretais Hereditárias sem Polipose/genética , Neoplasias Colorretais Hereditárias sem Polipose/terapia , Reparo de Erro de Pareamento de DNA/genética , Humanos , Administração dos Cuidados ao Paciente , Medição de Risco , Neoplasias Urogenitais/genética , Neoplasias Urogenitais/patologia , Neoplasias Urogenitais/terapia
12.
Indian J Pathol Microbiol ; 62(3): 405-412, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361228

RESUMO

Background: There are several DNA repair pathways that protect cellular DNA from injury, such as nucleotide excision repair (NER) and mismatch repair (MMR). The protein product of the excision repair cross-complementation group 1 (ERCC1) gene plays a pivotal role in NER. The exact relationship between MMR proteins and ERCC1 is not well known in colorectal carcinoma (CRC). Aim of the Study: To investigate expression of ERCC1 and MMR proteins in colorectal mucinous carcinoma (MA) and non-mucinous carcinoma (NMA) using tissue microarray technique. Material and Methods: We studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and non-mucinous adenocarcinoma (NMA). Tissue microarrays were constructed using modified mechanical pencil tips technique and immunohistochemistry for ERCC1, MLH1, MSH2, MSH6, and PMS2. Results: NMA showed a significantly more frequent aberrant cytoplasmic expression than MA while MA showed a more frequent intact nuclear expression than NMA. There were no significant differences between the NMA and MA groups in the expression of MMR proteins. In NMA cases, ERCC1 expression was significantly related to MMR status while was not significantly related in MA cases. ERCC1 expression was not significantly related to overall and disease-free survival in both NMA and MA groups. Conclusion: this study is the first to investigate the relation between MMR status and ERCC1 expression in colorectal MA and NMA. ERCC1 expression was significantly related to MMR status only in NMA cases. Hence, the current study emphasizes that further research about the relation between various DNA repair pathways is needed.


Assuntos
Adenocarcinoma Mucinoso/genética , Adenocarcinoma/genética , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Adenocarcinoma/patologia , Adenocarcinoma Mucinoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Prognóstico , Análise Serial de Tecidos , Adulto Jovem
13.
Cancer Sci ; 110(8): 2620-2628, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31152682

RESUMO

Tumor mutational burden (TMB) and mutational signatures reflect the process of mutation accumulation in cancer. However, the significance of these emerging characteristics remains unclear. In the present study, we used whole-exome sequencing to analyze the TMB and mutational signature in solid tumors of 4046 Japanese patients. Eight predominant signatures-microsatellite instability, smoking, POLE, APOBEC, UV, mismatch repair, double-strand break repair, and Signature 16-were observed in tumors with TMB higher than 1.0 mutation/Mb, whereas POLE and UV signatures only showed moderate correlation with TMB, suggesting the extensive accumulation of mutations due to defective POLE and UV exposure. The contribution ratio of Signature 16, which is associated with hepatocellular carcinoma in drinkers, was increased in hypopharynx cancer. Tumors with predominant microsatellite instability signature were potential candidates for treatment with immune checkpoint inhibitors such as pembrolizumab and were found in 2.8% of cases. Furthermore, based on microarray analysis, tumors with predominant signatures were classified into 2 subgroups depending on the expression of immune-related genes reflecting differences in the immune context of the tumor microenvironment. Tumor subpopulations differing in the content of infiltrating immune cells might respond differently to immunotherapeutics. An understanding of cancer characteristics based on TMB and mutational signatures could provide new insights into mutation-driven tumorigenesis.


Assuntos
Carcinogênese/genética , Mutação/genética , Neoplasias/genética , Carcinogênese/patologia , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/genética , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Japão , Instabilidade de Microssatélites , Neoplasias/patologia , Carga Tumoral/genética , Microambiente Tumoral/genética , Sequenciamento Completo do Exoma/métodos
14.
Indian J Med Res ; 149(2): 146-150, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-31219078

RESUMO

The mismatch amplification assay is a modified version of polymerase chain reaction (PCR) that permits specific amplification of gene sequences with single base pair change. The basis of the technique relies on primer designing. The single nucleotide mismatch at the 3' proximity of the reverse oligonucleotide primer makes Taq DNA polymerase unable to carry out extension process. Thus, the primers produce a PCR fragment in the wild type, whereas it is not possible to yield a product with a mutation at the site covered by the mismatch positions on the mismatch amplification mutation assay (MAMA) primer from any gene. The technique offers several advantages over other molecular methods, such as PCR-restriction fragment length polymorphism (RFLP) and oligonucleotide hybridization, which is routinely used in the detection of known point mutations. Since multiple point mutations in the quinolone resistance determining region play a major role in high-level fluoroquinolone resistance in Gram-negative bacteria, the MAMA-PCR technique is preferred for detecting these mutations over PCR-RFLP and sequencing technology.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Farmacorresistência Bacteriana/genética , Fluoroquinolonas/efeitos adversos , Bactérias Gram-Negativas/genética , Sequência de Bases , Fluoroquinolonas/uso terapêutico , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/patogenicidade , Humanos , Hibridização de Ácido Nucleico/métodos , Oligonucleotídeos/genética , Mutação Puntual/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição/genética
15.
Diagn Pathol ; 14(1): 62, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31221175

RESUMO

The use of immunohistochemistry (IHC) as a companion diagnostic is an increasingly important part of the case workup by pathologists and is often central to clinical decision making. New predictive molecular markers are constantly sought for to improve treatment stratification parallel to drug development. Unfortunately, official biomarker guidelines lag behind, and pathologists are often left hesitating when medical oncologists request off-labelled biomarker testing. We performed a literature review of five commonly requested off-label IHC predictive biomarkers in gastrointestinal tract (GIT) malignancies: HER2, mismatch repair (MMR), PD-L1, BRAF V600E and ROS1. We found that HER2 amplification is rare and poorly associated to IHC overexpression in extracolonic and extragastric GIT cancers; however in KRAS wild type colorectal cancers, which fail conventional treatment, HER2 IHC may be useful and should be considered. For MMR testing, more evidence is needed to recommend reflex testing in GIT cancers for treatment purposes. MMR testing should not be discouraged in patients considered for second line checkpoint inhibitor therapy. With the exception of gastric tumors, PD-L1 IHC is a weak predictor of checkpoint inhibitor response in the GIT and should be replaced by MMR in this context. BRAF inhibitors showed activity in BRAF V600E mutated cholangiocarcinomas and pancreatic carcinomas in non-first line settings. ROS1 translocation is extremely rare and poorly correlated to ROS1 IHC expression in the GIT; currently there is no role for ROS1 IHC testing in GIT cancers. Overall, the predictive biomarker literature has grown exponentially, and official guidelines need to be updated more regularly to support pathologists' testing decisions.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias Colorretais/terapia , Reparo de Erro de Pareamento de DNA/genética , Uso Off-Label , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Humanos , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas/genética
16.
Science ; 364(6439): 485-491, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31048490

RESUMO

Tumors with mismatch repair deficiency (MMR-d) are characterized by sequence alterations in microsatellites and can accumulate thousands of mutations. This high mutational burden renders tumors immunogenic and sensitive to programmed cell death-1 (PD-1) immune checkpoint inhibitors. Yet, despite their tumor immunogenicity, patients with MMR-deficient tumors experience highly variable responses, and roughly half are refractory to treatment. We present experimental and clinical evidence showing that the degree of microsatellite instability (MSI) and resultant mutational load, in part, underlies the variable response to PD-1 blockade immunotherapy in MMR-d human and mouse tumors. The extent of response is particularly associated with the accumulation of insertion-deletion (indel) mutational load. This study provides a rationale for the genome-wide characterization of MSI intensity and mutational load to better profile responses to anti-PD-1 immunotherapy across MMR-deficient human cancers.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Imunoterapia/métodos , Instabilidade de Microssatélites , Neoplasias/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Anticorpos/uso terapêutico , Variação Genética , Melanoma Experimental/genética , Melanoma Experimental/terapia , Camundongos , Proteína 2 Homóloga a MutS/genética , Mutação , Resultado do Tratamento
17.
Histopathology ; 75(2): 236-246, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31062389

RESUMO

AIMS: Beta2-microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair-deficient colorectal cancer (dMMR CRC), with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. METHODS AND RESULTS: Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Deleterious B2M mutations were detected in 39 of 121 (32%) dMMR tumours. Five contained missense B2M-variants of unknown significance, so were excluded from further analyses. With median follow-up of 7.4 years, none of the 39 B2M-mutant tumours recurred, compared with 14 of 77 (18%) B2M-wild-type tumours (P = 0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87 and 71%, respectively. Significantly (P < 0.0001) fewer (three of 104, 2.9%) of the 108 pMMR CRCs demonstrated deleterious B2M mutations. One pMMR tumour, containing a frameshift mutation, later recurred. CONCLUSION: B2M mutations were detected in nearly one-third of dMMR cancers, none of which recurred. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC. The mechanism of protection against recurrence and whether this protection extends to stage III disease remains unclear.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Microglobulina beta-2/genética , Adulto , Idoso , Reparo de Erro de Pareamento de DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/genética
18.
Gastroenterology ; 157(2): 421-431, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30998989

RESUMO

BACKGROUND & AIMS: Approximately 75% of patients with suspected Lynch syndrome carry variants in MLH1 or MSH2, proteins encoded by these genes are required for DNA mismatch repair (MMR). However, 30% of these are variants of unknown significance (VUS). A assay that measures cell response to the cytotoxic effects of a methylating agent can determine the effects of VUS in MMR genes and identify patients with constitutional MMR-deficiency syndrome. We adapted this method to test the effects of VUS in MLH1 and MSH2 genes found in patients with suspected Lynch syndrome. METHODS: We transiently expressed MLH1 or MSH2 variants in MLH1- or MSH2-null human colorectal cancer cell lines (HCT116 or LoVo), respectively. The MMR process causes death of cells with methylation-damaged DNA bases, so we measured proportions of cells that undergo death following exposure to the methylating agent; cells that escaped its toxicity were considered to have variants that affect function of the gene product. Using this assay, we analyzed 88 variants (mainly missense variants), comprising a validation set of 40 previously classified variants (19 in MLH1 and 21 in MSH2) and a prospective set of 48 VUS (25 in MLH1 and 23 in MSH2). Prediction scores were calculated for all VUS according to the recommendations of the American College of Medical Genetics and Genomics, based on clinical, somatic, in silico, population, and functional data. RESULTS: The assay correctly classified 39 of 40 variants in the validation set. The assay identified 12 VUS that did alter function of the gene product and 28 VUS that did not; the remaining 8 VUS had intermediate effects on MMR capacity and could not be classified. Comparison of assay results with prediction scores confirmed the ability of the assay to discriminate VUS that affected the function of the gene products from those that did not. CONCLUSIONS: Using an assay that measures the ability of the cells to undergo death following DNA damage induction by a methylating agent, we were able to assess whether variants in MLH1 and MSH2 cause defects in DNA MMR. This assay might be used to help assessing the pathogenicity of VUS in MLH1 and MSH2 found in patients with suspected Lynch syndrome.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/diagnóstico , Metilação de DNA/genética , Testes Genéticos/métodos , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Bioensaio/métodos , Linhagem Celular Tumoral , Neoplasias Colorretais Hereditárias sem Polipose/genética , Simulação por Computador , Metilação de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Estudos de Viabilidade , Mutação em Linhagem Germinativa , Guanina/análogos & derivados , Guanina/farmacologia , Humanos , Metilnitronitrosoguanidina/toxicidade
19.
DNA Repair (Amst) ; 78: 60-69, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30959407

RESUMO

An important role for the DNA mismatch repair (MMR) pathway in maintaining genomic stability is embodied in its conservation through evolution and the link between loss of MMR function and tumorigenesis. The latter is evident as inheritance of mutations within the major MMR genes give rise to the cancer predisposition condition, Lynch syndrome. Nonetheless, how MMR loss contributes to tumorigenesis is not completely understood. In addition to preventing the accumulation of mutations, MMR also directs cellular responses, such as cell cycle checkpoint or apoptosis activation, to different forms of DNA damage. Understanding this MMR-dependent DNA damage response may provide insight into the full tumor suppressing capabilities of the MMR pathway. Here, we delve into the proposed mechanisms for the MMR-dependent response to DNA damaging agents. We discuss how these pre-clinical findings extend to the clinical treatment of cancers, emphasizing MMR status as a crucial variable in selection of chemotherapeutic regimens. Also, we discuss how loss of the MMR-dependent damage response could promote tumorigenesis via the establishment of a survival advantage to endogenous levels of stress in MMR-deficient cells.


Assuntos
Dano ao DNA , Reparo de Erro de Pareamento de DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética
20.
Gene ; 704: 103-112, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30974197

RESUMO

Lynch syndrome (LS) is a common cancer syndrome that is inherited in an autosomal dominant manner. Its pathogenesis is thought to be closely related to germline mutations of mismatch repair (MMR) genes such as the MLH1, MSH2, PMS2 and MSH6 genes. This study identifies a Chinese family with LS clinically diagnosed according to the Amsterdam II criteria. In these patients, immuno-histochemical staining showed negative MSH6 expressions but positive MLH1, MSH2, and PMS2 expressions. In order to further explore the molecular biology of this LS family, we used targeted next-generation sequencing (NGS) and Multiplex ligation dependent probe amplification (MLPA) to identify the mutation and verify the authenticity of the mutation in 15 family members. For NGS, two panels have been used, one is of MLH1, MSH2, PMS2 and MSH6 genes, the other one is of 139 cancer genetic susceptibility genes. And for the large deletions/duplications can also be identified by NGS panel, an adjusted data analysis strategy of NGS has been used. As a result, we identified a novel heterozygous large deletion in MSH6 gene that was found to be co-segregated among affected family members. This deletion results in the loss of a 3246 bp-sized fragment in MSH6 gene exons 5-9 which represents the coding regions of the MSH6 ATPase domain. This novel mutation has yet to be documented in the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) database. This mutation resulted in MSH6 protein losing gene mismatch repair function, and further caused the microsatellite instable. We speculate that this mutation may significantly impact MMR function through impaired ATP domain function. Theoretically, this proband would likely benefit from PD-1 immune check-point blockade therapy, but conversely, we observed that tumors appeared to rapidly progress after 4 sessions of anti-PD-1 treatment. Further studies to validate the effectiveness of anti-PD-1 treatments in carriers of this mutation are necessary.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa , Deleção de Sequência , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Grupo com Ancestrais do Continente Asiático/genética , China , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Reparo de Erro de Pareamento de DNA/genética , Família , Feminino , Testes Genéticos , Heterozigoto , Humanos , Masculino , Instabilidade de Microssatélites , Linhagem
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