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After two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no single chromosome has been finished end to end, and hundreds of unresolved gaps persist1,2. Here we present a human genome assembly that surpasses the continuity of GRCh382, along with a gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome3, we reconstructed the centromeric satellite DNA array (approximately 3.1 Mb) and closed the 29 remaining gaps in the current reference, including new sequences from the human pseudoautosomal regions and from cancer-testis ampliconic gene families (CT-X and GAGE). These sequences will be integrated into future human reference genome releases. In addition, the complete chromosome X, combined with the ultra-long nanopore data, allowed us to map methylation patterns across complex tandem repeats and satellite arrays. Our results demonstrate that finishing the entire human genome is now within reach, and the data presented here will facilitate ongoing efforts to complete the other human chromosomes.
Assuntos
Cromossomos Humanos X/genética , Genoma Humano/genética , Telômero/genética , Centrômero/genética , Ilhas de CpG/genética , Metilação de DNA , DNA Satélite/genética , Feminino , Humanos , Mola Hidatiforme/genética , Masculino , Gravidez , Reprodutibilidade dos Testes , Testículo/metabolismoRESUMO
BTK inhibitors, Bcl-2 inhibitors, and other targeted therapies have significantly improved the outcomes of patients with chronic lymphocytic leukemia (CLL). With increased survivorship, monitoring disease and deciphering potential mechanisms of resistance to these agents are critical for devising effective treatment strategies. We used duplex sequencing, a technology that enables detection of mutations at ultra-low allelic frequencies, to identify mutations in five genes associated with drug resistance in CLL and followed their evolution in two patients who received multiple targeted therapies and ultimately developed disease progression on pirtobrutinib. In both patients we detected variants that expanded and reached significant cancer cell fractions (CCF). In patient R001, multiple known resistance mutations in both BTK and PLCG2 appeared following progression on zanubrutinib (BTK p.L528W, p.C481S; PLCG2 S707F, L845F, R665W, and D993H). In contrast, patient R002 developed multiple BTK mutations following acalabrutinib treatment, including known resistance mutations p.C481R, p.T474I and p.C481S. We found that pirtobrutinib was able to suppress, but not completely eradicate, BTK p.C481S mutations in both patients, but other resistance mutations such as mutations in PLCG2 and new BTK mutations increased while the patients were receiving pirtobrutinib. For example, BTK p.L528W in patient R001 increased in frequency more than 1,000-fold (from a CCF of 0.02% to 35%), and the CCF in p.T474I in patient R002 increased from 0.03% to 4.2% (more than 100-fold). Our data illuminate the evolutionary dynamics of resistant clones over the patients' disease course and under selective pressure from different targeted treatments.
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Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Células Clonais , Frequência do GeneRESUMO
BACKGROUND: Individuals with germline BRCA1 and BRCA2 pathogenic variants (BRCA carriers) are at high risk of developing high grade serous ovarian carcinoma (HGSC). HGSC is predominantly driven by TP53 mutations, but mutations in this gene are also commonly found in non-cancerous tissue as a feature of normal human aging. We hypothesized that HGSC predisposition in BRCA carriers may be related to increased TP53 somatic evolution, which could be detectable by ultra-deep sequencing of TP53 mutations in gynecological liquid biopsies. METHODS: Duplex sequencing was used to identify TP53 mutations with high sensitivity in peritoneal washes and cervical liquid-based cytology (LBC) collected at surgery from 60 individuals including BRCA1 and BRCA2 carriers, and non-carriers. TP53 mutation pathogenicity was compared across groups and with TP53 cancer mutations. RESULTS: TP53 mutations were more abundant in cervical LBC than in peritoneal washes but increased with age in both sample types. In peritoneal washes, but not in cervical LBC, pathogenic TP53 mutation burden was increased in BRCA1 carriers compared to non-carriers, independently of age. Five individuals shared identical pathogenic TP53 mutations in peritoneal washes and cervical LBC, but not in blood. CONCLUSIONS: Ultra-deep sequencing of TP53 mutations in peritoneal washes collected at surgery reveals increased burden of pathogenic TP53 mutations in BRCA1 carriers. This excess of pathogenic TP53 mutations might be linked to the elevated risk of HGSC in these individuals. In some patients, concordant TP53 mutations were found in peritoneal washes and cervical LBCs, but the cell of origin remains unknown and deserves further investigation.
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Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions. To overcome this limitation, we developed PolyG-DS, a next-generation sequencing (NGS) method that combines the error-correction capabilities of duplex sequencing (DS) with enrichment of PolyG loci using CRISPR-Cas9-targeted genomic fragmentation. PolyG-DS markedly reduces technical artifacts by comparing the sequences derived from the complementary strands of each original DNA molecule. We demonstrate that PolyG-DS genotyping is accurate, reproducible, and highly sensitive, enabling the detection of low-frequency alleles (<0.01) in spike-in samples using a panel of only 19 PolyG markers. PolyG-DS replicated prior results based on PolyG fragment length analysis by capillary electrophoresis, and exhibited higher sensitivity for identifying clonal expansions in the nondysplastic colon of patients with ulcerative colitis. We illustrate the utility of this method for resolving the phylogenetic relationship among precancerous lesions in ulcerative colitis and for tracing the metastatic dissemination of ovarian cancer. PolyG-DS enables the study of tumor evolution without prior knowledge of tumor driver mutations and provides a tool to perform cost-effective and easily scalable ultra-accurate NGS-based PolyG genotyping for multiple applications in biology, genetics, and cancer research.
Assuntos
Linhagem da Célula , DNA/genética , Guanina/química , Neoplasias/genética , Poli G/genética , Diferenciação Celular , Evolução Clonal , DNA/química , Genótipo , HumanosRESUMO
Next-generation sequencing methods suffer from low recovery, uneven coverage, and false mutations. DNA fragmentation by sonication is a major contributor to these problems because it produces randomly sized fragments, PCR amplification bias, and end artifacts. In addition, oligonucleotide-based hybridization capture, a common target enrichment method, has limited efficiency for small genomic regions, contributing to low recovery. This becomes a critical problem in clinical applications, which value cost-effective approaches focused on the sequencing of small gene panels. To address these issues, we developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion that produces DNA fragments of similar length. These fragments can be enriched by a simple size selection, resulting in targeted enrichment of up to approximately 49,000-fold. Additionally, homogenous length fragments significantly reduce PCR amplification bias and maximize read usability. We combined this novel target enrichment approach with Duplex Sequencing, which uses double-strand molecular tagging to correct for sequencing errors. The approach, termed CRISPR-DS, enables efficient target enrichment of small genomic regions, even coverage, ultra-accurate sequencing, and reduced DNA input. As proof of principle, we applied CRISPR-DS to the sequencing of the exonic regions of TP53 and performed side-by-side comparisons with standard Duplex Sequencing. CRISPR-DS detected previously reported pathogenic TP53 mutations present as low as 0.1% in peritoneal fluid of women with ovarian cancer, while using 10- to 100-fold less DNA than standard Duplex Sequencing. Whether used as standalone enrichment or coupled with high-accuracy sequencing methods, CRISPR-based fragmentation offers a simple solution for fast and efficient small target enrichment.
Assuntos
Sistemas CRISPR-Cas , Neoplasias Ovarianas/genética , Análise de Sequência de DNA/métodos , Proteína Supressora de Tumor p53/genética , DNA/genética , Fragmentação do DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
OBJECTIVES: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes. METHODS: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance. RESULTS: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome. CONCLUSIONS: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type.
Assuntos
Genes BRCA1/fisiologia , Genes BRCA2/fisiologia , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Proteína Supressora de Tumor p53/metabolismoRESUMO
DNA mutations are inevitable. Despite proficient DNA repair mechanisms, somatic cells accumulate mutations during development and aging, generating cells with different genotypes within the same individual, a phenomenon known as somatic mosaicism. While the existence of somatic mosaicism has long been recognized, in the last five years, advances in sequencing have provided unprecedented resolution to characterize the extent and nature of somatic genetic variation. Collectively, these new studies are revealing a previously uncharacterized aging phenotype: the accumulation of clones with cancer driver mutations. Here, we summarize the most recent findings, which converge in the novel notion that cancer-associated mutations are prevalent in normal tissue and accumulate with aging.
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Envelhecimento/genética , Neoplasias/genética , Células Clonais , Reparo do DNA/genética , Bases de Dados Genéticas , Humanos , Mosaicismo , Mutação/genética , FenótipoRESUMO
OBJECTIVE: Pap tests hold promise as a molecular diagnostic for serous ovarian cancer, but previous studies reported limited sensitivity. Furthermore, the presence of somatic mutations in normal tissue is increasingly recognized as a challenge to the specificity of mutation-based cancer diagnostics. We applied an ultra-deep sequencing method with the goal of improving sensitivity and characterizing the landscape of low-frequency somatic TP53 mutations in Pap tests. METHODS: We used CRISPR-DS to deeply sequence (mean Duplex depth ~3000×) the TP53 gene in 30 Pap tests from 21 women without cancer and 9 women with serous ovarian carcinoma with known TP53 driver mutations. Mutations were annotated and compared to those in the TP53 cancer database. RESULTS: The tumor-derived mutation was identified in 3 of 8 Pap tests from women with ovarian cancer and intact tubes. In addition, 221 low-frequency (â²0.001) exonic TP53 mutations were identified in Pap tests from women with ovarian cancer (94 mutations) and without ovarian cancer (127 mutations). Many of these mutations resembled TP53 mutations found in cancer: they impaired protein activity, were predicted to be pathogenic, and clustered in exons 5 to 8 and hotspot codons. Cancer-like mutations were identified in all women but at higher frequency in women with ovarian cancer. CONCLUSIONS: Pap tests have low sensitivity for ovarian cancer detection and carry abundant low-frequency TP53 mutations. These mutations are more frequently pathogenic in women with ovarian cancer. Determining whether low-frequency TP53 mutations in normal gynecologic tissues are associated with an increased cancer risk warrants further study.
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Cistadenocarcinoma Seroso/genética , DNA/genética , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Coortes , Cistadenocarcinoma Seroso/patologia , DNA/isolamento & purificação , Análise Mutacional de DNA , Feminino , Humanos , Pessoa de Meia-Idade , Mutação , Neoplasias Ovarianas/patologia , Teste de Papanicolaou , Adulto JovemRESUMO
Current sequencing methods are error-prone, which precludes the identification of low frequency mutations for early cancer detection. Duplex sequencing is a sequencing technology that decreases errors by scoring mutations present only in both strands of DNA. Our aim was to determine whether duplex sequencing could detect extremely rare cancer cells present in peritoneal fluid from women with high-grade serous ovarian carcinomas (HGSOCs). These aggressive cancers are typically diagnosed at a late stage and are characterized by TP53 mutations and peritoneal dissemination. We used duplex sequencing to analyze TP53 mutations in 17 peritoneal fluid samples from women with HGSOC and 20 from women without cancer. The tumor TP53 mutation was detected in 94% (16/17) of peritoneal fluid samples from women with HGSOC (frequency as low as 1 mutant per 24,736 normal genomes). Additionally, we detected extremely low frequency TP53 mutations (median mutant fraction 1/13,139) in peritoneal fluid from nearly all patients with and without cancer (35/37). These mutations were mostly deleterious, clustered in hotspots, increased with age, and were more abundant in women with cancer than in controls. The total burden of TP53 mutations in peritoneal fluid distinguished cancers from controls with 82% sensitivity (14/17) and 90% specificity (18/20). Age-associated, low frequency TP53 mutations were also found in 100% of peripheral blood samples from 15 women with and without ovarian cancer (none with hematologic disorder). Our results demonstrate the ability of duplex sequencing to detect rare cancer cells and provide evidence of widespread, low frequency, age-associated somatic TP53 mutation in noncancerous tissue.
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Líquido Ascítico , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Neoplasias Ovarianas/genética , Proteína Supressora de Tumor p53/genética , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Accurate annotation of genomic variants in human diseases is essential to allow personalized medicine. Assessment of somatic and germline TP53 alterations has now reached the clinic and is required in several circumstances such as the identification of the most effective cancer therapy for patients with chronic lymphocytic leukemia (CLL). Here, we present Seshat, a Web service for annotating TP53 information derived from sequencing data. A flexible framework allows the use of standard file formats such as Mutation Annotation Format (MAF) or Variant Call Format (VCF), as well as common TXT files. Seshat performs accurate variant annotations using the Human Genome Variation Society (HGVS) nomenclature and the stable TP53 genomic reference provided by the Locus Reference Genomic (LRG). In addition, using the 2017 release of the UMD_TP53 database, Seshat provides multiple statistical information for each TP53 variant including database frequency, functional activity, or pathogenicity. The information is delivered in standardized output tables that minimize errors and facilitate comparison of mutational data across studies. Seshat is a beneficial tool to interpret the ever-growing TP53 sequencing data generated by multiple sequencing platforms and it is freely available via the TP53 Website, http://p53.fr or directly at http://vps338341.ovh.net/.
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Bases de Dados Genéticas , Variação Genética/genética , Software , Proteína Supressora de Tumor p53/genética , Biologia Computacional/tendências , Genômica/tendências , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Internet , Anotação de Sequência Molecular , MutaçãoRESUMO
Cumulative evidence indicates that a significant proportion of cancer evolution may occur before the development of histological abnormalities. While recent improvements in DNA sequencing technology have begun to reveal the presence of these early preneoplastic clones, the concept of 'premalignant field' was already introduced by Slaughter more than half a century ago. Also referred to as 'field effect', 'field defect' or 'field cancerization', these terms describe the phenomenon by which molecular alterations develop in normal-appearing tissue and expand to form premalignant patches with the potential to progress to dysplasia and cancer. Field effects have been well-characterized in ulcerative colitis, an inflammatory bowel disease that increases the risk of colorectal cancer. The study of the molecular alterations that define these fields is informative of mechanisms of tumor initiation and progression and has provided potential targets for early cancer detection. Herein, we summarize the current knowledge about the molecular alterations that comprise the field effect in ulcerative colitis and the clinical utility of these fields for cancer screening and prevention.
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Colite Ulcerativa/complicações , Neoplasias Colorretais , Lesões Pré-Cancerosas , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Humanos , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/genética , Lesões Pré-Cancerosas/patologiaRESUMO
Neoplasms are thought to progress to cancer through genetic instability generating cellular diversity and clonal expansions driven by selection for mutations in cancer genes. Despite advances in the study of molecular biology of cancer genes, relatively little is known about evolutionary mechanisms that drive neoplastic progression. It is unknown, for example, which may be more predictive of future progression of a neoplasm: genetic homogenization of the neoplasm, possibly caused by a clonal expansion, or the accumulation of clonal diversity. Here, in a prospective study, we show that clonal diversity measures adapted from ecology and evolution can predict progression to adenocarcinoma in the premalignant condition known as Barrett's esophagus, even when controlling for established genetic risk factors, including lesions in TP53 (p53; ref. 6) and ploidy abnormalities. Progression to cancer through accumulation of clonal diversity, on which natural selection acts, may be a fundamental principle of neoplasia with important clinical implications.
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Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Adenocarcinoma/patologia , Progressão da Doença , Neoplasias Esofágicas/patologia , Genes p16 , Genes p53 , Humanos , Hibridização in Situ Fluorescente , Perda de HeterozigosidadeRESUMO
Somatic TP53 mutations are prevalent in normal tissue but little is known about their association with cancer risk. Cervical liquid-based cytology (LBC), commonly known as Pap test, provides an accessible gynecological sample to test the value of TP53 somatic mutations as a biomarker for high-grade serous ovarian cancer (HGSC), a cancer type mostly driven by TP53 mutations. We used ultra-deep duplex sequencing to analyze TP53 mutations in LBC and blood samples from 70 individuals (30 with and 40 without HGSC) undergoing gynecologic surgery, 30 carrying BRCA1 or BRCA2 germline pathogenic variants (BRCApv). Only 30% of the tumor mutations were found in LBC samples. However, TP53 pathogenic mutations were identified in nearly all LBC and blood samples, with only 5.4% of mutations in LBC (20/368) also found in the corresponding blood sample. TP53 mutations were more abundant in LBC than in blood and increased with age in both sample types. BRCApv carriers with HGSC had more TP53 clonal expansions in LBC than BRCApv carriers without cancer. Our results show that, while not useful for direct cancer detection, LBC samples capture TP53 mutation burden in the gynecological tract, presenting potential value for cancer risk assessment in individuals at higher hereditary risk for ovarian cancer.
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Proteína BRCA1 , Proteína BRCA2 , Mutação em Linhagem Germinativa , Neoplasias Ovarianas , Proteína Supressora de Tumor p53 , Humanos , Feminino , Proteína Supressora de Tumor p53/genética , Mutação em Linhagem Germinativa/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/sangue , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/diagnóstico , Proteína BRCA2/genética , Pessoa de Meia-Idade , Proteína BRCA1/genética , Adulto , Idoso , Colo do Útero/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , CitologiaRESUMO
Chronic inflammation predisposes to a variety of human cancers. Affected tissues slowly accumulate mutations, some of which affect growth regulation and drive successive waves of clonal evolution, whereas a far greater number are functionally neutral and serve only to passively mark expanding clones. Ulcerative colitis (UC) is an inflammatory bowel disease, in which up to 10% of patients eventually develop colon cancer. Here we have mapped mutations in hypermutable intergenic and intronic polyguanine tracts in patients with UC to delineate the extent of clonal expansions associated with carcinogenesis. We genotyped colon biopsies for length altering mutations at 28 different polyguanine markers. In eight patients without neoplasia, we detected only two mutations in a single individual from among 37 total biopsies. In contrast, for 11 UC patients with neoplasia elsewhere in the colon, we identified 63 mutations in 51 nondysplastic biopsies, and every patient possessed at least one mutant clone. A subset of clones were large and extended over many square centimeters of colon. Of these, some occurred as isolated populations in nondysplastic tissue, considerably distant from neoplastic lesions. Other large clones included regions of cancer, suggesting that the tumor arose within a preexisting clonal field. Our results demonstrate that neutral mutations in polyguanine tracts serve as a unique tool for identifying fields of clonal expansions, which may prove clinically useful for distinguishing a subset of UC patients who are at risk for developing cancer.
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Colite Ulcerativa/patologia , Neoplasias do Colo/diagnóstico , Proliferação de Células , Células Clonais , Neoplasias do Colo/patologia , Eletroforese em Gel de Ágar , Genótipo , Guanina/metabolismo , Humanos , Modelos Biológicos , Mutação/genéticaRESUMO
Although somatic mutations in colorectal cancer are well characterized, little is known about the accumulation of cancer mutations in the normal colon before cancer. Here, we have developed and applied an ultrasensitive, single-molecule mutational test based on CRISPR-DS technology, which enables mutation detection at extremely low frequency (<0.001) in normal colon from patients with and without colorectal cancer. This testing platform revealed that normal colon from patients with and without colorectal cancer carries mutations in common colorectal cancer genes, but these mutations are more abundant in patients with cancer. Oncogenic KRAS mutations were observed in the normal colon of about one third of patients with colorectal cancer but in none of the patients without colorectal cancer. Patients with colorectal cancer also carried more TP53 mutations than patients without cancer and these mutations were more pathogenic and formed larger clones, especially in patients with early-onset colorectal cancer. Most mutations in the normal colon were different from the driver mutations in tumors, suggesting that the occurrence of independent clones with pathogenic KRAS and TP53 mutations is a common event in the colon of individuals who develop colorectal cancer. These results indicate that somatic evolution contributes to clonal expansions in the normal colon and that this process is enhanced in individuals with cancer, particularly in those with early-onset colorectal cancer. SIGNIFICANCE: This work suggests prevalent somatic evolution in the normal colon of patients with colorectal cancer, highlighting the potential of using ultrasensitive gene sequencing to predict disease risk.
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Neoplasias Colorretais , Proteínas Proto-Oncogênicas p21(ras) , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genes ras , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Current screening methods for ovarian cancer (OC) have failed to demonstrate a significant reduction in mortality. Uterine lavage combined with TP53 ultra-deep sequencing for the detection of disseminated OC cells has emerged as a promising tool, but this approach has not been tested for early-stage disease or non-serous histologies. In addition, lavages carry multiple background mutations, the significance of which is poorly understood. Uterine lavage was collected preoperatively in 34 patients undergoing surgery for suspected ovarian malignancy including 14 patients with benign disease and 20 patients with OC (6 non-serous and 14 high grade serous-like (serous)). Ultra-deep duplex sequencing (~3000x) with a panel of common OC genes identified the tumor mutation in 33% of non-serous (all early stage) and in 79% of serous cancers (including four early stage). In addition, all lavages carried multiple somatic mutations (average of 25 mutations per lavage), more than half of which corresponded to common cancer driver mutations. Driver mutations in KRAS, PIK3CA, PTEN, PPP2R1A and ARID1A presented as larger clones than non-driver mutations and with similar frequency in lavages from patients with and without OC, indicating prevalent somatic evolution in all patients. Driver TP53 mutations, however, presented as significantly larger clones and with higher frequency in lavages from individuals with OC, suggesting that TP53-specific clonal expansions are linked to ovarian cancer development. Our results demonstrate that lavages capture cancer cells, even from early-stage cancers, as well as other clonal expansions and support further exploration of TP53 mutation burden as a potential OC risk factor.
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Neoplasias Ovarianas , Irrigação Terapêutica , Humanos , Feminino , Neoplasias Ovarianas/genética , Mutação/genética , Evolução Clonal , Proteína Supressora de Tumor p53/genéticaRESUMO
BACKGROUND: Breast cancer is a leading cause of death in premenopausal women. Progesterone drives expansion of luminal progenitor cells, leading to the development of poor-prognostic breast cancers. However, it is not known if antagonising progesterone can prevent breast cancers in humans. We suggest that targeting progesterone signalling could be a means of reducing features which are known to promote breast cancer formation. METHODS: In healthy premenopausal women with and without a BRCA mutation we studied (i) estrogen and progesterone levels in saliva over an entire menstrual cycle (n = 20); (ii) cancer-free normal breast-tissue from a control population who had no family or personal history of breast cancer and equivalently from BRCA1/2 mutation carriers (n = 28); triple negative breast cancer (TNBC) biopsies and healthy breast tissue taken from sites surrounding the TNBC in the same individuals (n = 14); and biopsies of ER+ve/PR+ve stage T1-T2 cancers and healthy breast tissue taken from sites surrounding the cancer in the same individuals (n = 31); and (iii) DNA methylation and DNA mutations in normal breast tissue (before and after treatment) from clinical trials that assessed the potential preventative effects of vitamins and antiprogestins (mifepristone and ulipristal acetate; n = 44). RESULTS: Daily levels of progesterone were higher throughout the menstrual cycle of BRCA1/2 mutation carriers, raising the prospect of targeting progesterone signalling as a means of cancer risk reduction in this population. Furthermore, breast field cancerization DNA methylation signatures reflective of (i) the mitotic age of normal breast epithelium and (ii) the proportion of luminal progenitor cells were increased in breast cancers, indicating that luminal progenitor cells with elevated replicative age are more prone to malignant transformation. The progesterone receptor antagonist mifepristone reduced both the mitotic age and the proportion of luminal progenitor cells in normal breast tissue of all control women and in 64% of BRCA1/2 mutation carriers. These findings were validated by an alternate progesterone receptor antagonist, ulipristal acetate, which yielded similar results. Importantly, mifepristone reduced both the TP53 mutation frequency as well as the number of TP53 mutations in mitotic-age-responders. CONCLUSIONS: These data support the potential usage of antiprogestins for primary prevention of poor-prognostic breast cancers. TRIAL REGISTRATION: Clinical trial 1 Mifepristone treatment prior to insertion of a levonorgestrel releasing intrauterine system for improved bleeding control - a randomized controlled trial, clinicaltrialsregister.eu, 2009-009014-40 ; registered on 20 July 2009. Clinical trial 2 The effect of a progesterone receptor modulator on breast tissue in women with BRCA1 and 2 mutations, clinicaltrials.gov, NCT01898312 ; registered on 07 May 2013. Clinical trial 3 A pilot prevention study of the effects of the anti- progestin Ulipristal Acetate (UA) on surrogate markers of breast cancer risk, clinicaltrialsregister.eu, 2015-001587-19 ; registered on 15 July 2015.
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Neoplasias da Mama , Neoplasias de Mama Triplo Negativas , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Epigênese Genética , Feminino , Humanos , Mifepristona , Mutação , Progesterona , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Microarrays and high-throughput sequencing methods can be used to measure the expression of thousands of genes in a biological sample in a few days, whereas PCR-based methods can be used to measure the expression of a few genes in thousands of samples in about the same amount of time. These methods become more costly as the number of biological samples increases or as the number of genes of interest increases, respectively, and these factors constrain experimental design. To address these issues, we introduced 'vertical arrays' in which RNA from each biological sample is converted into multiple, overlapping cDNA subsets and spotted on glass slides. These vertical arrays can be queried with single gene probes to assess the expression behavior in thousands of biological samples in a single hybridization reaction. The spotted subsets are less complex than the original RNA from which they derive, which improves signal-to-noise ratios. Here, we demonstrate the quantitative capabilities of vertical arrays, including the sensitivity and accuracy of the method and the number of subsets needed to achieve this accuracy for most expressed genes.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Linhagem Celular , Regulação da Expressão Gênica , Humanos , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND & AIMS: Telomere shortening is implicated in cancer and aging and might link these 2 biologic events. We explored this hypothesis in ulcerative colitis (UC), a chronic inflammatory disease that predisposes to colorectal cancer and in which shorter telomeres have been associated with chromosomal instability and tumor progression. METHODS: Telomere length was measured by quantitative polymerase chain reaction in colonocytes and leukocytes of 2 different sets of UC patients and compared with normal controls across a wide range of ages. For a subset of patients, telomere length was measured in epithelium and stroma of right and left colon biopsy specimens. A third set of biopsy specimens was analyzed for phosphorylation of histone H2AX (gammaH2AX), a DNA damage signal, by immunofluorescence and for telomere length by quantitative fluorescence in situ hybridization. Relationships between telomere length, gammaH2AX intensity, age, disease duration, and age of disease onset were explored. RESULTS: Colonocyte telomeres shorten with age almost twice as rapidly in UC patients as in normal controls. This extensive shortening occurs within approximately 8 years of disease duration. Leukocyte telomeres are slightly shorter in UC patients than in controls, but telomeres of colon stromal cells are unaffected. gammaH2AX intensity is higher in colonocytes of UC patients than in controls and is not dependent on age or telomere length. CONCLUSIONS: Colonocytes of UC patients show premature shortening of telomeres, which might explain the increased and earlier risk of cancer in this disease. Shorter leukocyte telomeres and increased gammaH2AX in colonocytes might reflect oxidative damage secondary to inflammation.