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1.
Proc Natl Acad Sci U S A ; 115(8): 1871-1876, 2018 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-29432176

RESUMO

Aneuploidy is a feature of most cancer cells, and a myriad of approaches have been developed to detect it in clinical samples. We previously described primers that could be used to amplify ∼38,000 unique long interspersed nucleotide elements (LINEs) from throughout the genome. Here we have developed an approach to evaluate the sequencing data obtained from these amplicons. This approach, called Within-Sample AneupLoidy DetectiOn (WALDO), employs supervised machine learning to detect the small changes in multiple chromosome arms that are often present in cancers. We used WALDO to search for chromosome arm gains and losses in 1,677 tumors and in 1,522 liquid biopsies of blood from cancer patients or normal individuals. Aneuploidy was detected in 95% of cancer biopsies and in 22% of liquid biopsies. Using single-nucleotide polymorphisms within the amplified LINEs, WALDO concomitantly assesses allelic imbalances, microsatellite instability, and sample identification. WALDO can be used on samples containing only a few nanograms of DNA and as little as 1% neoplastic content and has a variety of applications in cancer diagnostics and forensic science.


Assuntos
Aneuploidia , Elementos Nucleotídeos Longos e Dispersos/genética , Neoplasias/genética , Aberrações Cromossômicas , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos
2.
Gut ; 68(4): 663-671, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29420226

RESUMO

OBJECTIVE: For patients with locally advanced rectal cancer (LARC), adjuvant chemotherapy selection following surgery remains a major clinical dilemma. Here, we investigated the ability of circulating tumour DNA (ctDNA) to improve risk stratification in patients with LARC. DESIGN: We enrolled patients with LARC (T3/T4 and/or N+) planned for neoadjuvant chemoradiotherapy. Plasma samples were collected pretreatment, postchemoradiotherapy and 4-10 weeks after surgery. Somatic mutations in individual patient's tumour were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. We then designed personalised assays to quantify ctDNA in plasma samples. Patients received adjuvant therapy at clinician discretion, blinded to the ctDNA results. RESULTS: We analysed 462 serial plasma samples from 159 patients. ctDNA was detectable in 77%, 8.3% and 12% of pretreatment, postchemoradiotherapy and postsurgery plasma samples. Significantly worse recurrence-free survival was seen if ctDNA was detectable after chemoradiotherapy (HR 6.6; P<0.001) or after surgery (HR 13.0; P<0.001). The estimated 3-year recurrence-free survival was 33% for the postoperative ctDNA-positive patients and 87% for the postoperative ctDNA-negative patients. Postoperative ctDNA detection was predictive of recurrence irrespective of adjuvant chemotherapy use (chemotherapy: HR 10.0; P<0.001; without chemotherapy: HR 22.0; P<0.001). Postoperative ctDNA status remained an independent predictor of recurrence-free survival after adjusting for known clinicopathological risk factors (HR 6.0; P<0.001). CONCLUSION: Postoperative ctDNA analysis stratifies patients with LARC into subsets that are either at very high or at low risk of recurrence, independent of conventional clinicopathological risk factors. ctDNA analysis could potentially be used to guide patient selection for adjuvant chemotherapy.


Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Neoplasias Retais/genética , Neoplasias Retais/terapia , Austrália , Terapia Combinada , Diagnóstico por Imagem , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Estudos Prospectivos , Neoplasias Retais/sangue , Neoplasias Retais/patologia , Sistema de Registros , Fatores de Risco , Análise de Sobrevida
3.
Proc Natl Acad Sci U S A ; 113(35): 9846-51, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27528664

RESUMO

We present the bottleneck sequencing system (BotSeqS), a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.


Assuntos
Genoma Humano/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Núcleo Celular/genética , Criança , Pré-Escolar , DNA Mitocondrial/química , DNA Mitocondrial/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
4.
Nature ; 486(7404): 537-40, 2012 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-22722843

RESUMO

Colorectal tumours that are wild type for KRAS are often sensitive to EGFR blockade, but almost always develop resistance within several months of initiating therapy. The mechanisms underlying this acquired resistance to anti-EGFR antibodies are largely unknown. This situation is in marked contrast to that of small-molecule targeted agents, such as inhibitors of ABL, EGFR, BRAF and MEK, in which mutations in the genes encoding the protein targets render the tumours resistant to the effects of the drugs. The simplest hypothesis to account for the development of resistance to EGFR blockade is that rare cells with KRAS mutations pre-exist at low levels in tumours with ostensibly wild-type KRAS genes. Although this hypothesis would seem readily testable, there is no evidence in pre-clinical models to support it, nor is there data from patients. To test this hypothesis, we determined whether mutant KRAS DNA could be detected in the circulation of 28 patients receiving monotherapy with panitumumab, a therapeutic anti-EGFR antibody. We found that 9 out of 24 (38%) patients whose tumours were initially KRAS wild type developed detectable mutations in KRAS in their sera, three of which developed multiple different KRAS mutations. The appearance of these mutations was very consistent, generally occurring between 5 and 6 months following treatment. Mathematical modelling indicated that the mutations were present in expanded subclones before the initiation of panitumumab treatment. These results suggest that the emergence of KRAS mutations is a mediator of acquired resistance to EGFR blockade and that these mutations can be detected in a non-invasive manner. They explain why solid tumours develop resistance to targeted therapies in a highly reproducible fashion.


Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Evolução Molecular , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , DNA de Neoplasias/sangue , Resistencia a Medicamentos Antineoplásicos/genética , Genes ras/genética , Humanos , Mutação/genética , Panitumumabe , Proteínas Proto-Oncogênicas p21(ras) , Seleção Genética/efeitos dos fármacos , Fatores de Tempo
5.
Proc Natl Acad Sci U S A ; 112(31): 9704-9, 2015 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-26195750

RESUMO

Cell-free DNA shed by cancer cells has been shown to be a rich source of putative tumor-specific biomarkers. Because cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we studied whether the cerebrospinal fluid (CSF) that bathes the CNS is enriched for tumor DNA, here termed CSF-tDNA. We analyzed 35 primary CNS malignancies and found at least one mutation in each tumor using targeted or genome-wide sequencing. Using these patient-specific mutations as biomarkers, we identified detectable levels of CSF-tDNA in 74% [95% confidence interval (95% CI) = 57-88%] of cases. All medulloblastomas, ependymomas, and high-grade gliomas that abutted a CSF space were detectable (100% of 21 cases; 95% CI = 88-100%), whereas no CSF-tDNA was detected in patients whose tumors were not directly adjacent to a CSF reservoir (P < 0.0001, Fisher's exact test). These results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.


Assuntos
Neoplasias Encefálicas/líquido cefalorraquidiano , DNA de Neoplasias/líquido cefalorraquidiano , Neoplasias da Medula Espinal/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , DNA de Neoplasias/genética , Demografia , Éxons/genética , Feminino , Genoma Humano , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação/genética , Neoplasias da Medula Espinal/genética
6.
Gastroenterology ; 149(6): 1501-10, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26253305

RESUMO

BACKGROUND & AIMS: The management of pancreatic cysts poses challenges to both patients and their physicians. We investigated whether a combination of molecular markers and clinical information could improve the classification of pancreatic cysts and management of patients. METHODS: We performed a multi-center, retrospective study of 130 patients with resected pancreatic cystic neoplasms (12 serous cystadenomas, 10 solid pseudopapillary neoplasms, 12 mucinous cystic neoplasms, and 96 intraductal papillary mucinous neoplasms). Cyst fluid was analyzed to identify subtle mutations in genes known to be mutated in pancreatic cysts (BRAF, CDKN2A, CTNNB1, GNAS, KRAS, NRAS, PIK3CA, RNF43, SMAD4, TP53, and VHL); to identify loss of heterozygozity at CDKN2A, RNF43, SMAD4, TP53, and VHL tumor suppressor loci; and to identify aneuploidy. The analyses were performed using specialized technologies for implementing and interpreting massively parallel sequencing data acquisition. An algorithm was used to select markers that could classify cyst type and grade. The accuracy of the molecular markers was compared with that of clinical markers and a combination of molecular and clinical markers. RESULTS: We identified molecular markers and clinical features that classified cyst type with 90%-100% sensitivity and 92%-98% specificity. The molecular marker panel correctly identified 67 of the 74 patients who did not require surgery and could, therefore, reduce the number of unnecessary operations by 91%. CONCLUSIONS: We identified a panel of molecular markers and clinical features that show promise for the accurate classification of cystic neoplasms of the pancreas and identification of cysts that require surgery.


Assuntos
Algoritmos , Biomarcadores Tumorais/genética , Pâncreas/patologia , Cisto Pancreático/classificação , Cisto Pancreático/patologia , Adulto , Feminino , Predisposição Genética para Doença , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Cisto Pancreático/genética , Cisto Pancreático/cirurgia , Fenótipo , Valor Preditivo dos Testes , Prognóstico , Estudos Retrospectivos
7.
Mod Pathol ; 29(5): 511-5, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26965579

RESUMO

TERT promoter mutations (TERT-mut) are detectable in the majority of urothelial carcinomas. The detection of TERT-mut in urine is under investigation as a potential urine-based molecular-screening assay for bladder cancer. A small but significant number of bladder carcinomas are pure squamous cell carcinoma. We sought to assess the incidence of TERT-mut in squamous cell carcinoma of the urinary bladder. A retrospective search of the institutional pathology archives yielded 15 cystectomy specimens performed for squamous cell carcinoma (2000-2014). Histologic slides were reviewed by a senior urologic pathologist to confirm the diagnosis and select a representative formalin-fixed paraffin-embedded tissue block for mutational analysis. All cases yielded adequate material for DNA analysis. Sequencing for TERT-mut was performed using previously described SafeSeq technique. We detected TERT-mut in 12/15 (80%) of bladder squamous cell carcinomas. TERT promoter mutations, commonly found in conventional urothelial carcinoma, are also highly prevalent in urinary bladder squamous cell carcinoma suggesting a common tumorigenesis and potential utility as a molecular urine-based-screening assay.


Assuntos
Carcinoma de Células Escamosas/genética , Telomerase/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos
8.
Genes Chromosomes Cancer ; 53(1): 15-24, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24190505

RESUMO

Bone and soft tissue sarcomas are a group of histologically heterogeneous and relatively uncommon tumors. To explore their genetic origins, we sequenced the exomes of 13 osteosarcomas, eight myxoid liposarcomas (MLPS), and seven synovial sarcomas (SYN). These tumors had few genetic alterations (median of 10.8). Nevertheless, clear examples of driver gene mutations were observed, including canonical mutations in TP53, PIK3CA, SETD2, AKT1, and subclonal mutation in FBXW7. Of particular interest were mutations in H3F3A, encoding the variant histone H3.3. Mutations in this gene have only been previously observed in gliomas. Loss of heterozygosity of exomic regions was extensive in osteosarcomas but rare in SYN and MLPS. These results provide intriguing nucleotide-level information on these relatively uncommon neoplasms and highlight pathways that help explain their pathogenesis.


Assuntos
Neoplasias Ósseas/genética , Lipossarcoma Mixoide/genética , Osteossarcoma/genética , Sarcoma Sinovial/genética , Sarcoma/genética , Adolescente , Adulto , Idoso , Criança , Exoma , Humanos , Perda de Heterozigosidade , Pessoa de Meia-Idade , Mutação
9.
Proc Natl Acad Sci U S A ; 108(23): 9530-5, 2011 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-21586637

RESUMO

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Although massively parallel sequencing instruments are in principle well suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. The keys to this approach, called the Safe-Sequencing System ("Safe-SeqS"), are (i) assignment of a unique identifier (UID) to each template molecule, (ii) amplification of each uniquely tagged template molecule to create UID families, and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are considered mutant ("supermutants") only if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.


Assuntos
Análise Mutacional de DNA/métodos , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Análise Mutacional de DNA/instrumentação , Humanos , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase/instrumentação , Reprodutibilidade dos Testes , beta Catenina/genética
10.
Science ; 369(6499)2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32345712

RESUMO

Cancer treatments are often more successful when the disease is detected early. We evaluated the feasibility and safety of multicancer blood testing coupled with positron emission tomography-computed tomography (PET-CT) imaging to detect cancer in a prospective, interventional study of 10,006 women not previously known to have cancer. Positive blood tests were independently confirmed by a diagnostic PET-CT, which also localized the cancer. Twenty-six cancers were detected by blood testing. Of these, 15 underwent PET-CT imaging and nine (60%) were surgically excised. Twenty-four additional cancers were detected by standard-of-care screening and 46 by neither approach. One percent of participants underwent PET-CT imaging based on false-positive blood tests, and 0.22% underwent a futile invasive diagnostic procedure. These data demonstrate that multicancer blood testing combined with PET-CT can be safely incorporated into routine clinical care, in some cases leading to surgery with intent to cure.


Assuntos
Detecção Precoce de Câncer/métodos , Testes Hematológicos , Programas de Rastreamento/métodos , Neoplasias/sangue , Neoplasias/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Idoso , Estudos de Coortes , Feminino , Humanos
11.
JAMA Oncol ; 5(8): 1118-1123, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31070668

RESUMO

IMPORTANCE: For patients with resected, nonmetastatic colorectal cancer (CRC), the optimal surveillance protocol remains unclear. OBJECTIVE: To evaluate whether serial circulating tumor DNA (ctDNA) levels detected disease recurrence earlier, compared with conventional postoperative surveillance, in patients with resected CRC. DESIGN, SETTING, AND PARTICIPANTS: This study included patients (n = 58) with stage I, II, or III CRC who underwent radical surgical resection at 4 Swedish hospitals from February 2, 2007, to May 8, 2013. Eighteen patients received adjuvant chemotherapy at the discretion of their clinicians, who were blinded to the ctDNA results. Blood samples were collected at 1 month after the surgical procedure and every 3 to 6 months thereafter for ctDNA analysis. Patients were followed up until metachronous metastases were detected, or for a median of 49 months. Data analysis was performed from March 1, 2009, to June 23, 2018. MAIN OUTCOMES AND MEASURES: Sensitivity and timing of ctDNA positivity were compared with those of conventional surveillance modalities (computed tomographic scans and serum carcinoembryonic antigen tests) for the detection of disease recurrence. RESULTS: This study included 319 blood samples from 58 patients, with a median (range) age of 69 (47-83) years and 34 males (59%). The recurrence rate among patients with positive ctDNA levels was 77% (10 of 13 patients). Positive ctDNA preceded radiologic and clinical evidence of recurrence by a median of 3 months. Of the 45 patients with negative ctDNA throughout follow-up, none (0%; 95% CI, 0%-7.9%) experienced a relapse, with a median follow-up of 49 months. However, 3 (6%; 95% CI, 1.3%-17%) of the 48 patients without relapse had a positive ctDNA result, which subsequently fell to undetectable levels during follow-up. CONCLUSION AND RELEVANCE: Although these findings need to be validated in a larger, prospective trial, they suggest that ctDNA analysis could complement conventional surveillance strategies as a triage test to stratify patients with resected CRC on the basis of risk of disease recurrence.

12.
Hum Pathol ; 85: 1-9, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30447301

RESUMO

Activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Although the cadherin 1 (CDH1) gene is commonly mutated in the clinically aggressive plasmacytoid variant of urothelial carcinoma (PUC), little is known about their TERT promoter mutation status. A retrospective search of our archives for PUC and UC with plasmacytoid and/or signet ring cell features (2007-2014) was performed. Ten specimens from 10 patients had archived material available for DNA analysis and were included in the study. Intratumoral areas of nonplasmacytoid histology were also evaluated when present. Samples were analyzed for TERT promoter mutations with Safe-SeqS, a sequencing error-reduction technology, and sequenced using a targeted panel of the 10 most commonly mutated genes in bladder cancer on the Illumina MiSeq platform. TERT promoter mutations were detected in specimens with pure and focal plasmacytoid features (6/10). Similar to conventional UC, the predominant mutation identified was g.1295228C>T. In heterogeneous tumors with focal variant histology, concordant mutations were found in plasmacytoid and corresponding conventional, glandular, or sarcomatoid areas. Co-occurring mutations in tumor protein p53 (TP53, 2 cases) and kirsten rat sarcoma (KRAS) viral proto-oncogene (1 case) were also detected. TERT promoter mutations are frequently present in PUC, which provides further evidence that TERT promoter mutations are common events in bladder cancer, regardless of histologic subtype, and supports their inclusion in any liquid biopsy assay for bladder cancer.


Assuntos
Carcinoma de Células de Transição/genética , Mutação , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias Urológicas/genética , Urotélio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células de Transição/metabolismo , Carcinoma de Células de Transição/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Proto-Oncogene Mas , Estudos Retrospectivos , Neoplasias Urológicas/metabolismo , Neoplasias Urológicas/patologia , Urotélio/patologia
13.
J Mol Biol ; 373(2): 355-66, 2007 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-17826792

RESUMO

The CA domain of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein plays critical roles in both the early and late phases of viral replication and is therefore an attractive antiviral target. Compounds with antiviral activity were recently identified that bind to the N-terminal domain of CA (CA N) and inhibit capsid assembly during viral maturation. We have determined the structure of the complex between CA N and the antiviral assembly inhibitor N-(3-chloro-4-methylphenyl)-N'-{2-[({5-[(dimethylamino)-methyl]-2-furyl}-methyl)-sulfanyl]ethyl}-urea) (CAP-1) using a combination of NMR spectroscopy and X-ray crystallography. The protein undergoes a remarkable conformational change upon CAP-1 binding, in which Phe32 is displaced from its buried position in the protein core to open a deep hydrophobic cavity that serves as the ligand binding site. The aromatic ring of CAP-1 inserts into the cavity, with the urea NH groups forming hydrogen bonds with the backbone oxygen of Val59 and the dimethylamonium group interacting with the side-chains of Glu28 and Glu29. Elements that could be exploited to improve binding affinity are apparent in the structure. The displacement of Phe32 by CAP-1 appears to be facilitated by a strained main-chain conformation, which suggests a potential role for a Phe32 conformational switch during normal capsid assembly.


Assuntos
Fármacos Anti-HIV/química , Furanos/química , HIV-1/química , Compostos de Fenilureia/química , Compostos de Enxofre/química , Montagem de Vírus/efeitos dos fármacos , Produtos do Gene gag do Vírus da Imunodeficiência Humana/química , Alanina/química , Alanina/metabolismo , Fármacos Anti-HIV/farmacologia , Proteínas do Capsídeo/antagonistas & inibidores , Proteínas do Capsídeo/química , Cristalografia por Raios X , Furanos/metabolismo , HIV-1/efeitos dos fármacos , HIV-1/metabolismo , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Fenilalanina/química , Fenilalanina/metabolismo , Compostos de Fenilureia/metabolismo , Relação Estrutura-Atividade , Compostos de Enxofre/metabolismo
14.
Elife ; 72018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29557778

RESUMO

Current non-invasive approaches for detection of urothelial cancers are suboptimal. We developed a test to detect urothelial neoplasms using DNA recovered from cells shed into urine. UroSEEK incorporates massive parallel sequencing assays for mutations in 11 genes and copy number changes on 39 chromosome arms. In 570 patients at risk for bladder cancer (BC), UroSEEK was positive in 83% of those who developed BC. Combined with cytology, UroSEEK detected 95% of patients who developed BC. Of 56 patients with upper tract urothelial cancer, 75% tested positive by UroSEEK, including 79% of those with non-invasive tumors. UroSEEK detected genetic abnormalities in 68% of urines obtained from BC patients under surveillance who demonstrated clinical evidence of recurrence. The advantages of UroSEEK over cytology were evident in low-grade BCs; UroSEEK detected 67% of cases whereas cytology detected none. These results establish the foundation for a new non-invasive approach for detection of urothelial cancer.


Assuntos
Aneuploidia , Carcinoma de Células de Transição/diagnóstico , Detecção Precoce de Câncer/métodos , Mutação , Neoplasias da Bexiga Urinária/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/urina , Criança , Pré-Escolar , Feminino , Testes Genéticos/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Telomerase/genética , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/urina , Adulto Jovem
15.
Sci Transl Med ; 10(433)2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563323

RESUMO

We report the detection of endometrial and ovarian cancers based on genetic analyses of DNA recovered from the fluids obtained during a routine Papanicolaou (Pap) test. The new test, called PapSEEK, incorporates assays for mutations in 18 genes as well as an assay for aneuploidy. In Pap brush samples from 382 endometrial cancer patients, 81% [95% confidence interval (CI), 77 to 85%] were positive, including 78% of patients with early-stage disease. The sensitivity in 245 ovarian cancer patients was 33% (95% CI, 27 to 39%), including 34% of patients with early-stage disease. In contrast, only 1.4% of 714 women without cancer had positive Pap brush samples (specificity, ~99%). Next, we showed that intrauterine sampling with a Tao brush increased the detection of malignancy over endocervical sampling with a Pap brush: 93% of 123 (95% CI, 87 to 97%) patients with endometrial cancer and 45% of 51 (95% CI, 31 to 60%) patients with ovarian cancer were positive, whereas none of the samples from 125 women without cancer were positive (specificity, 100%). Finally, in 83 ovarian cancer patients in whom plasma was available, circulating tumor DNA was found in 43% of patients (95% CI, 33 to 55%). When plasma and Pap brush samples were both tested, the sensitivity for ovarian cancer increased to 63% (95% CI, 51 to 73%). These results demonstrate the potential of mutation-based diagnostics to detect gynecologic cancers at a stage when they are more likely to be curable.


Assuntos
Neoplasias do Endométrio/diagnóstico , Biópsia Líquida/métodos , Neoplasias Ovarianas/diagnóstico , Teste de Papanicolaou/métodos , Adolescente , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Esfregaço Vaginal/métodos , Adulto Jovem
16.
Elife ; 52016 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-27421040

RESUMO

We determined whether the mutations found in ovarian cancers could be identified in the patients' ovarian cyst fluids. Tumor-specific mutations were detectable in the cyst fluids of 19 of 23 (83%) borderline tumors, 10 of 13 (77%) type I cancers, and 18 of 18 (100%) type II cancers. In contrast, no mutations were found in the cyst fluids of 18 patients with benign tumors or non-neoplastic cysts. Though large, prospective studies are needed to demonstrate the safety and clinical utility of this approach, our results suggest that the genetic evaluation of cyst fluids might be able to inform the management of the large number of women with these lesions.


Assuntos
Líquido Cístico/química , DNA/análise , Mutação , Cistos Ovarianos/diagnóstico , Cistos Ovarianos/patologia , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/patologia , DNA/genética , Diagnóstico Diferencial , Feminino , Humanos , Estudos Prospectivos
17.
Virchows Arch ; 469(4): 427-34, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27520411

RESUMO

Somatic activating mutations in the promoter of the telomerase reverse transcriptase (TERT) gene are the most common genetic alterations in urothelial carcinoma (UC) of the bladder and upper urinary tract. Little is known, however, about TERT-mutation status in the relatively uncommon but clinically aggressive micropapillary (MPC) variant. We evaluated the presence of TERT promoter mutations in MPC of the bladder and upper urinary tract. A retrospective search of our archives for MPC and UC with micropapillary features (2005-2014) was performed. All slides were reviewed to confirm the histologic diagnosis. Thirty-three specimens from 31 patients had FFPE blocks available for DNA analysis and were included in the study. Intratumoral areas of non-micropapillary histology were also evaluated when present. Samples were analyzed with Safe-SeqS, a sequencing error reduction technology, and sequenced using the Illumina MiSeq platform. TERT promoter mutations were detected in all specimens with pure MPC (18 of 18) and UC with focal micropapillary features (15 of 15). Similar to conventional UC, the predominant mutations identified occurred at positions -124 (C228T) (85 %) and -146 (C250T) (12 %) bp upstream of the TERT ATG start site. In heterogeneous tumors with focal variant histology, intratumoral concordant mutations were found in variant (MPC and non-MPC) and corresponding conventional UC. We found TERT promoter mutations, commonly found in conventional UC, to be frequently present in MPC. Our finding of concordant intratumoral mutational alterations in cases with focal variant histology lends support to the common oncogenesis origin of UC and its variant histology.


Assuntos
Carcinoma Papilar/genética , Carcinoma de Células de Transição/epidemiologia , Mutação/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Neoplasias da Bexiga Urinária/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Papilar/patologia , Carcinoma de Células de Transição/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Neoplasias da Bexiga Urinária/patologia
18.
Hum Pathol ; 53: 8-13, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26980028

RESUMO

TERT promoter mutations (TERT-mut) have been detected in 60% to 80% of urothelial carcinomas. A molecular urine-based screening assay for the detection of TERT-mut is currently being pursued by our group and others. A small but significant number of bladder carcinomas are adenocarcinoma. The current study assesses the incidence of TERT-mut in primary adenocarcinomas of urinary bladder. A retrospective search of our institutional pathology records identified 23 cystectomy specimens with a diagnosis of adenocarcinoma (2000-2014). All slides were reviewed by a senior urologic pathologist to confirm tumor type and select a representative formalin-fixed, paraffin-embedded block for mutational analysis. Adequate material for DNA testing was available in 14 cases (7 enteric type and 7 not otherwise specified). TERT-mut sequencing analysis was performed using previously described SafeSeq technique. Overall, 28.5% of primary adenocarcinoma harbored TERT-mut. Interestingly, 57% of nonenteric adenocarcinomas were mutation positive, whereas none of the enteric-type tumors harbored mutations. Similar to urothelial carcinoma, we found a relatively higher rate of TERT-mut among nonenteric-type adenocarcinomas further supporting the potential utility of TERT-mut urine-based screening assay for bladder cancer.


Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Mutação , Regiões Promotoras Genéticas , Telomerase/genética , Neoplasias da Bexiga Urinária/genética , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Adulto , Idoso , Baltimore , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Neoplasias da Bexiga Urinária/enzimologia , Neoplasias da Bexiga Urinária/patologia
19.
Sci Transl Med ; 8(346): 346ra92, 2016 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-27384348

RESUMO

Detection of circulating tumor DNA (ctDNA) after resection of stage II colon cancer may identify patients at the highest risk of recurrence and help inform adjuvant treatment decisions. We used massively parallel sequencing-based assays to evaluate the ability of ctDNA to detect minimal residual disease in 1046 plasma samples from a prospective cohort of 230 patients with resected stage II colon cancer. In patients not treated with adjuvant chemotherapy, ctDNA was detected postoperatively in 14 of 178 (7.9%) patients, 11 (79%) of whom had recurred at a median follow-up of 27 months; recurrence occurred in only 16 (9.8 %) of 164 patients with negative ctDNA [hazard ratio (HR), 18; 95% confidence interval (CI), 7.9 to 40; P < 0.001]. In patients treated with chemotherapy, the presence of ctDNA after completion of chemotherapy was also associated with an inferior recurrence-free survival (HR, 11; 95% CI, 1.8 to 68; P = 0.001). ctDNA detection after stage II colon cancer resection provides direct evidence of residual disease and identifies patients at very high risk of recurrence.


Assuntos
DNA Tumoral Circulante/genética , Neoplasias do Colo/genética , Neoplasia Residual/genética , DNA Tumoral Circulante/análise , Neoplasias do Colo/sangue , Neoplasias do Colo/cirurgia , Intervalo Livre de Doença , Humanos , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Neoplasia Residual/sangue , Neoplasia Residual/cirurgia , Modelos de Riscos Proporcionais , Estudos Prospectivos
20.
J Mol Biol ; 327(5): 1013-20, 2003 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-12662926

RESUMO

During the assembly stage of the human immunodeficiency virus (HIV) replication cycle, several thousand copies of the viral Gag polyprotein associate at the cell membrane and bud to form an immature, non-infectious virion. Gag is subsequently cleaved by the protease, which liberates the capsid proteins for assembly into the polyprotein shell of the central core particle (or capsid) of the mature virus. Viral infectivity is critically dependent on capsid formation and stability, making the capsid protein a potentially attractive antiviral target. We have identified compounds that bind to an apical site on the N-terminal domain of the HIV-1 capsid protein and inhibit capsid assembly in vitro. One compound, N-(3-chloro-4-methylphenyl)-N'-[2-[([5-[(dimethylamino)-methyl]-2-furyl]-methyl)-sulfanyl]ethyl]urea) (CAP-1), is well tolerated in cell cultures, enabling in vivo antiviral and mechanistic studies. CAP-1 inhibits HIV-1 infectivity in a dose-dependent manner, but does not interfere with viral entry, reverse transcription, integration, proteolytic processing, or virus production, indicating a novel antiviral mechanism. Significantly, virus particles generated in the presence of CAP-1 exhibit heterogeneous sizes and abnormal core morphologies, consistent with inhibited CA-CA interactions during virus assembly and maturation. These findings lay the groundwork for the development of assembly inhibitors as a new class of therapeutic agents for the treatment of AIDS.


Assuntos
Fármacos Anti-HIV/farmacologia , Capsídeo/efeitos dos fármacos , Furanos/farmacologia , Compostos de Fenilureia/farmacologia , Fármacos Anti-HIV/uso terapêutico , Sítios de Ligação , Western Blotting , Capsídeo/química , Capsídeo/metabolismo , Furanos/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , Humanos , Microscopia Eletrônica , Modelos Moleculares , Compostos de Fenilureia/uso terapêutico , Conformação Proteica , Compostos de Enxofre , Virulência
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