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Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.
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Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Evolução Molecular , Variação Genética/genética , Instabilidade Genômica/genética , Transcrição Gênica/genética , Neoplasias da Mama/patologia , Proliferação de Células , Forma Celular , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Variação Genética/efeitos dos fármacos , Instabilidade Genômica/efeitos dos fármacos , Humanos , Células MCF-7 , Reprodutibilidade dos TestesRESUMO
Utilization of tumor-only sequencing has expanded in pediatric cancer patients, which can lead to identification of pathogenic variants in genes that may be germline and/or have uncertain relevance to the tumor in question, such as the homologous recombination (HR) pathway genes BRCA1/2. We identified patients with pathogenic BRCA1/2 mutations from somatic tumor sequencing, and performed additional germline sequencing to assess for the presence of loss of heterozygosity (LOH). Of seven patients identified, four (57.1%) mutations were found in the germline and none had associated LOH. Our data suggest that BRCA1/2 mutations identified in this context are likely incidental findings.
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Neoplasias do Sistema Nervoso Central , Neoplasias Ovarianas , Feminino , Humanos , Criança , Proteína BRCA1/genética , Neoplasias Ovarianas/patologia , Mutação em Linhagem Germinativa , Proteína BRCA2/genética , Perda de HeterozigosidadeRESUMO
BACKGROUND: Ewing sarcoma (EWS) is an aggressive sarcoma with no validated molecular biomarkers. We aimed to determine the frequency of STAG2 protein loss by immunohistochemistry (IHC) and whether loss of expression is associated with outcome. METHODS: We performed a retrospective cohort study of patients with EWS enrolled to Children's Oncology Group studies. We obtained unstained slides from 235 patients and DNA for sequencing from 75 patients. STAG2 expression was tested for association with clinical features and survival was estimated using Kaplan-Meier methods with log-rank tests. RESULTS: In total, 155 cases passed quality control for STAG2 IHC. STAG2 expression in 20/155 cases could not be categorised with the limited available tissue, leaving 135 patients with definitive STAG2 IHC. In localised and metastatic disease, STAG2 was lost in 29/108 and 6/27 cases, respectively. Among patients with IHC and sequencing, 0/17 STAG2 expressing cases had STAG2 mutations, and 2/7 cases with STAG2 loss had STAG2 mutations. Among patients with localised disease, 5-year event-free survival was 54% (95% CI 34-70%) and 75% (95% CI 63-84%) for patients with STAG2 loss vs. expression (P = 0.0034). CONCLUSION: STAG2 loss of expression is identified in a population of patients without identifiable STAG2 mutations and carries a poor prognosis.
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Sarcoma de Ewing , Criança , Humanos , Prognóstico , Sarcoma de Ewing/genética , Estudos Retrospectivos , Proteínas de Ciclo CelularRESUMO
The authors have noticed that the final paragraph of the Results section contains errors in the number of patients involved. The correct number of patients is included in the text below. These errors do not affect the Figure referenced.In osteosarcoma, we focused on 8q gain as a specific biological feature of interest. Among the 41 patients with detectable ctDNA in the osteosarcoma cohort, 8q gain was detected in 73.2% (30/41). The 3-year EFS for patients with 8q gain (n = 30) in ctDNA was 60.0% (95% CI 40.5-75.0) compared to 80.8 (95% CI 42.4-94.9) in patients without 8q gain (n = 11) in ctDNA (p = 0.18; Fig. 3).
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BACKGROUND & AIMS: Barrett's esophagus (BE) is the greatest risk factor for esophageal adenocarcinoma (EAC), but only a small proportion of patients with BE develop cancer. Biomarkers might be able to identify patients at highest risk of progression. We investigated genomic differences in surveillance biopsies collected from patients whose BE subsequently progressed compared to patients whose disease did not progress. METHODS: We performed a retrospective case-control study of 24 patients with BE that progressed to high-grade dysplasia (HGD, n = 14) or EAC (n = 10). The control group (n = 73, called non-progressors) comprised patients with BE and at least 5 years of total endoscopic biopsy surveillance without progression to HGD or EAC. From each patient, we selected a single tissue sample obtained more than 1 year before progression (cases) or more than 2 years before the end of follow-up (controls). Pathogenic mutations, gene copy numbers, and ploidy were compared between samples from progressors and non-progressors. RESULTS: TP53 mutations were detected in 46% of samples from progressors and 5% of non-progressors. In this case-control sample set, TP53 mutations in BE tissues increased the adjusted risk of progression 13.8-fold (95% confidence interval, 3.2-61.0) (P < .001). We did not observe significant differences in ploidy or copy-number profile between groups. We identified 147 pathogenic mutations in 57 distinct genes-the average number of pathogenic mutations was higher in samples from progressors (n = 2.5) than non-progressors (n = 1.2) (P < .001). TP53 and other somatic mutations were recurrently detected in samples with limited copy-number changes (aneuploidy). CONCLUSIONS: In genomic analyses of BE tissues from patients with or without later progression to HGD or EAC, we found significantly higher numbers of TP53 mutations in BE from patients with subsequent progression. These mutations were frequently detected before the onset of dysplasia or substantial changes in copy number.
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Adenocarcinoma/genética , Esôfago de Barrett/genética , Neoplasias Esofágicas/genética , Lesões Pré-Cancerosas/genética , Proteína Supressora de Tumor p53/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Esôfago de Barrett/patologia , Biópsia , Estudos de Casos e Controles , Progressão da Doença , Neoplasias Esofágicas/patologia , Esofagoscopia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Lesões Pré-Cancerosas/patologia , Prognóstico , Estudos RetrospectivosRESUMO
We characterize the genetic diversity of Xenopus laevis strains using RNA-seq data and allele-specific analysis. This data provides a catalogue of coding variation, which can be used for improving the genomic sequence, as well as for better sequence alignment, probe design, and proteomic analysis. In addition, we paint a broad picture of the genetic landscape of the species by functionally annotating different classes of mutations with a well-established prediction tool (PolyPhen-2). Further, we specifically compare the variation in the progeny of four crosses: inbred genomic (J)-strain, outbred albino (B)-strain, and two hybrid crosses of J and B strains. We identify a subset of mutations specific to the B strain, which allows us to investigate the selection pressures affecting duplicated genes in this allotetraploid. From these crosses we find the ratio of non-synonymous to synonymous mutations is lower in duplicated genes, which suggests that they are under greater purifying selection. Surprisingly, we also find that function-altering ("damaging") mutations constitute a greater fraction of the non-synonymous variants in this group, which suggests a role for subfunctionalization in coding variation affecting duplicated genes.
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Variação Genética , Fases de Leitura Aberta/genética , Transcriptoma/genética , Xenopus laevis/genética , Animais , Sequência de Bases , Cruzamentos Genéticos , Duplicação Gênica , Genoma , Hibridização Genética , Endogamia , Espectrometria de Massas , Mutação de Sentido Incorreto/genética , Polimorfismo de Nucleotídeo Único/genética , Reprodutibilidade dos Testes , Análise de Sequência de RNA , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
BACKGROUND: Sample index cross-talk can result in false positive calls when massively parallel sequencing (MPS) is used for sensitive applications such as low-frequency somatic variant discovery, ancient DNA investigations, microbial detection in human samples, or circulating cell-free tumor DNA (ctDNA) variant detection. Therefore, the limit-of-detection of an MPS assay is directly related to the degree of index cross-talk. RESULTS: Cross-talk rates up to 0.29% were observed when using standard, combinatorial adapters, resulting in 110,180 (0.1% cross-talk rate) or 1,121,074 (0.29% cross-talk rate) misassigned reads per lane in non-patterned and patterned Illumina flow cells, respectively. Here, we demonstrate that using unique, dual-matched indexed adapters dramatically reduces index cross-talk to ≤1 misassigned reads per flow cell lane. While the current study was performed using dual-matched indices, using unique, dual-unrelated indices would also be an effective alternative. CONCLUSIONS: For sensitive downstream analyses, the use of combinatorial indices for multiplexed hybrid capture and sequencing is inappropriate, as it results in an unacceptable number of misassigned reads. Cross-talk can be virtually eliminated using dual-matched indexed adapters. These results suggest that use of such adapters is critical to reduce false positive rates in assays that aim to identify low allele frequency events, and strongly indicate that dual-matched adapters be implemented for all sensitive MPS applications.
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Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normasRESUMO
BACKGROUND: New prognostic markers are needed to identify patients with Ewing sarcoma (EWS) and osteosarcoma unlikely to benefit from standard therapy. We describe the incidence and association with outcome of circulating tumour DNA (ctDNA) using next-generation sequencing (NGS) assays. METHODS: A NGS hybrid capture assay and an ultra-low-pass whole-genome sequencing assay were used to detect ctDNA in banked plasma from patients with EWS and osteosarcoma, respectively. Patients were coded as positive or negative for ctDNA and tested for association with clinical features and outcome. RESULTS: The analytic cohort included 94 patients with EWS (82% from initial diagnosis) and 72 patients with primary localised osteosarcoma (100% from initial diagnosis). ctDNA was detectable in 53% and 57% of newly diagnosed patients with EWS and osteosarcoma, respectively. Among patients with newly diagnosed localised EWS, detectable ctDNA was associated with inferior 3-year event-free survival (48.6% vs. 82.1%; p = 0.006) and overall survival (79.8% vs. 92.6%; p = 0.01). In both EWS and osteosarcoma, risk of event and death increased with ctDNA levels. CONCLUSIONS: NGS assays agnostic of primary tumour sequencing results detect ctDNA in half of the plasma samples from patients with newly diagnosed EWS and osteosarcoma. Detectable ctDNA is associated with inferior outcomes.
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Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , DNA Tumoral Circulante/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Osteossarcoma/genética , Adolescente , Biomarcadores Tumorais/sangue , Neoplasias Ósseas/sangue , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Masculino , Osteossarcoma/sangue , Prognóstico , Estudos Retrospectivos , Sarcoma de Ewing/sangue , Sarcoma de Ewing/genética , Análise de Sequência de DNA/métodos , Análise de SobrevidaAssuntos
Angiomiolipoma/genética , Neoplasias Renais/genética , Neoplasias Pulmonares/genética , Linfangioleiomiomatose/genética , Mosaicismo , Neoplasias Primárias Múltiplas/genética , Neoplasias Retroperitoneais/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Adulto , Idoso , Ácidos Nucleicos Livres/genética , Feminino , Humanos , Linfangioleiomiomatose/sangue , Pessoa de Meia-Idade , Pneumotórax , Fator D de Crescimento do Endotélio Vascular/sangue , Adulto JovemRESUMO
Over 200,000 individuals are diagnosed with lung cancer in the U.S. every year, with a growing proportion of cases, especially lung adenocarcinoma, occurring in individuals who have never smoked. Women over the age of 50 comprise the largest affected demographic. To understand the genomic drivers of lung adenocarcinoma and therapeutic response in this population, we performed whole genome and/or whole exome sequencing on 73 matched lung tumor/normal pairs from post-menopausal women who participated in the Women's Health Initiative. Somatic copy number alterations showed little variation by smoking status, suggesting that aneuploidy may be a general characteristic of lung cancer regardless of smoke exposure. Similarly, clock-like and APOBEC mutation signatures were prevalent but did not differ in tumors from smokers and never-smokers. However, mutations in both EGFR and KRAS showed unique allelic differences determined by smoking status that are known to alter tumor response to targeted therapy. Mutations in the MYC-network member MGA were more prevalent in tumors from smokers. Fusion events in ALK, RET, and ROS1 were absent, likely due to age-related differences in fusion prevalence. Our work underscores the profound impact of smoking status, age, and sex on the tumor mutational landscape and identifies areas of unmet medical need.
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Lung cancer in never-smokers disproportionately affects older women. To understand the mutational landscape of this cohort, we performed detailed genome characterization of 73 lung adenocarcinomas from participants of the Womenâ™s Health Initiative (WHI). We find enrichment of EGFR mutations in never-/light-smokers and KRAS mutations in heavy smokers as expected, but we also show that the specific variants of these genes differ by smoking status, with important therapeutic implications. Mutational signature analysis revealed signatures of clock, APOBEC, and DNA repair deficiency in never-/light-smokers; however, the mutational load of these signatures did not differ significantly from those found in smokers. Last, tumors from both smokers and never-/light-smokers shared copy number subtypes, with no significant differences in aneuploidy. Thus, the genomic landscape of lung cancer in never-/light-smokers and smokers is predominantly differentiated by somatic mutations and not copy number alterations.
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PURPOSE: Novel biomarkers are needed to differentiate outcomes in intermediate-risk rhabdomyosarcoma (IR RMS). We sought to evaluate strategies for identifying circulating tumor DNA (ctDNA) in IR RMS and to determine whether ctDNA detection before therapy is associated with outcome. PATIENTS AND METHODS: Pretreatment serum and tumor samples were available from 124 patients with newly diagnosed IR RMS from the Children's Oncology Group biorepository, including 75 patients with fusion-negative rhabdomyosarcoma (FN-RMS) and 49 with fusion-positive rhabdomyosarcoma (FP-RMS) disease. We used ultralow passage whole-genome sequencing to detect copy number alterations and a new custom sequencing assay, Rhabdo-Seq, to detect rearrangements and single-nucleotide variants. RESULTS: We found that ultralow passage whole-genome sequencing was a method applicable to ctDNA detection in all patients with FN-RMS and that ctDNA was detectable in 13 of 75 serum samples (17%). However, the use of Rhabdo-Seq in FN-RMS samples also identified single-nucleotide variants, such as MYOD1L122R, previously associated with prognosis. Identification of pathognomonic translocations between PAX3 or PAX7 and FOXO1 by Rhabdo-Seq was the best method for measuring ctDNA in FP-RMS and detected ctDNA in 27 of 49 cases (55%). Patients with FN-RMS with detectable ctDNA at diagnosis had significantly worse outcomes than patients without detectable ctDNA (event-free survival, 33.3% v 68.9%; P = .0028; overall survival, 33.3% v 83.2%; P < .0001) as did patients with FP-RMS (event-free survival, 37% v 70%; P = .045; overall survival, 39.2% v 75%; P = .023). In multivariable analysis, ctDNA was independently associated with worse prognosis in FN-RMS but not in the smaller FP-RMS cohort. CONCLUSION: Our study demonstrates that baseline ctDNA detection is feasible and is prognostic in IR RMS.
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DNA Tumoral Circulante , Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Humanos , Criança , Prognóstico , Rabdomiossarcoma/patologia , Nucleotídeos , Rabdomiossarcoma Alveolar/genética , Biomarcadores Tumorais/genéticaRESUMO
In a genetic screen in a drnl-2 background, we isolated a loss-of-function allele in miR319a (miR319a(129)). Previously, miR319a has been postulated to play a role in leaf development based on the dramatic curled-leaf phenotype of plants that ectopically express miR319a (jaw-D). miR319a(129) mutants exhibit defects in petal and stamen development; petals are narrow and short, and stamens exhibit defects in anther development. The miR319a(129) loss-of-function allele contains a single-base change in the middle of the encoded miRNA, which reduces the ability of miR319a to recognize targets. Analysis of the expression patterns of the three members of the miR319 gene family (miR319a, miR319b, and miR319c) indicates that these genes have largely non-overlapping expression patterns suggesting that these genes have distinct developmental functions. miR319a functions by regulating the TCP transcription factors TCP2, TCP3, TCP4, TCP10, and TCP24; the level of RNA expression of these TCP genes is down-regulated in jaw-D and elevated in miR319a(129). Several lines of evidence demonstrate that TCP4 is a key target of miR319a. First, the tcp4(soj6) mutant, which contains a mutation in the TCP4 miRNA-binding site complementary to the miR319a(129) mutation, suppresses the flower phenotype of miR319a(129). Second, expression of wild-type TCP4 in petals and stamens (i.e., AP3:TCP4) has no effect on flower development; by contrast, a miRNA-resistant version of TCP4, when expressed in petals and stamens (i.e., pAP3:mTCP4) causes these organs not to develop. Surprisingly, when AP3:TCP4 is present in a miR319a(129) background, petal and stamen development is severely disrupted, suggesting that proper regulation by miR319a of TCP4 is critical in these floral organs.
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Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , MicroRNAs/genética , RNA de Plantas/genética , Fatores de Transcrição/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Sequência de Bases , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Marcação de Genes , Genes de Plantas , Mutação , Fenótipo , Plantas Geneticamente Modificadas , Fatores de Transcrição/metabolismoRESUMO
PURPOSE: The utility of circulating tumor DNA (ctDNA) analyses has not been established in the risk stratification of Wilms tumor (WT). We evaluated the detection of ctDNA and selected risk markers in the serum and urine of patients with WT and compared findings with those of matched diagnostic tumor samples. PATIENTS AND METHODS: Fifty of 395 children with stage III or IV WT enrolled on Children's Oncology Group trial AREN0533 had banked pretreatment serum, urine, and tumor available. Next-generation sequencing was used to detect ctDNA. Copy-number changes in 1q, 16q, and 1p, and single-nucleotide variants in serum and urine were compared with tumor biopsy data. Event-free survival (EFS) was compared between patients with and without ctDNA detection. RESULTS: ctDNA was detected in the serum of 41/50 (82%) and in the urine in 13/50 (26%) patients. Agreement between serum ctDNA detection and tumor sequencing results was as follows: 77% for 1q gain, 88% for 16q deletions, and 70% for 1p deletions, with ĸ-coefficients of 0.56, 0.74, and 0.29, respectively. Sequencing also demonstrated that single-nucleotide variants detected in tumors could be identified in the ctDNA. There was a trend toward worse EFS in patients with ctDNA detected in the serum (4-year EFS 80% v 100%, P = .14). CONCLUSION: ctDNA demonstrates promise as an easily accessible prognostic biomarker with potential to detect tumor heterogeneity. The observed trend toward more favorable outcome in patients with undetectable ctDNA requires validation. ctDNA profiling should be further explored as a noninvasive diagnostic and prognostic tool in the risk-adapted treatment of patients with WT.
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DNA Tumoral Circulante , Neoplasias Renais , Tumor de Wilms , Biomarcadores Tumorais/genética , Criança , Aberrações Cromossômicas , DNA Tumoral Circulante/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Estadiamento de Neoplasias , Nucleotídeos , Tumor de Wilms/genética , Tumor de Wilms/patologiaRESUMO
PURPOSE: We sought to determine whether the detection of circulating tumor DNA (ctDNA) in samples of patients undergoing chemotherapy for advanced leiomyosarcoma (LMS) is associated with objective response or survival. EXPERIMENTAL DESIGN: Using ultra-low-passage whole-genome sequencing (ULP-WGS) of plasma cell-free DNA from patients treated on a prospective clinical trial, we tested whether detection of ctDNA evaluated prior to the start of therapy and after two cycles of chemotherapy was associated with treatment response and outcome. Associations between detection of ctDNA and pathologic measures of disease burden were evaluated. RESULTS: We found that ctDNA was detectable by ULP-WGS in 49% patients prior to treatment and in 24.6% patients after two cycles of chemotherapy. Detection of pretreatment ctDNA was significantly associated with a lower overall survival [HR, 1.55; 95% confidence interval (CI), 1.03-2.31; P = 0.03] and a significantly lower likelihood of objective response [odds ratio (OR), 0.21; 95% CI, 0.06-0.59; P = 0.005]. After two cycles of chemotherapy, patients who continued to have detectable levels of ctDNA experienced a significantly worse overall survival (HR, 1.77; 95% CI, 1-3.14; P = 0.05) and were unlikely to experience an objective response (OR, 0.05; 95% CI, 0-0.39; P = 0.001). CONCLUSIONS: Our results demonstrate that detection of ctDNA is associated with outcome and objective response to chemotherapy in patients with advanced LMS. These results suggest that liquid biopsy assays could be used to inform treatment decisions by recognizing patients who are likely and unlikely to benefit from chemotherapy. See related commentary by Kasper and Wilky, p. 2480.
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DNA Tumoral Circulante , Leiomiossarcoma , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Humanos , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/genética , Mutação , Estudos ProspectivosRESUMO
In mammalian cells, maternal and paternal alleles usually have similar transcriptional activity. Epigenetic mechanisms such as X-chromosome inactivation (XCI) and imprinting were historically viewed as rare exceptions to this rule. Discovery of autosomal monoallelic autosomal expression (MAE) a decade ago revealed an additional allele-specific mode regulating thousands of mammalian genes. Despite MAE prevalence, its mechanistic basis remains unknown. Using an RNA sequencing-based screen for reactivation of silenced alleles, we identified DNA methylation as key mechanism of MAE mitotic maintenance. In contrast with the all-or-nothing allelic choice in XCI, allele-specific expression in MAE loci is tunable, with exact allelic imbalance dependent on the extent of DNA methylation. In a subset of MAE genes, allelic imbalance was insensitive to DNA demethylation, implicating additional mechanisms in MAE maintenance in these loci. Our findings identify a key mechanism of MAE maintenance and provide basis for understanding the biological role of MAE.
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Impressão Genômica , Inativação do Cromossomo X , Alelos , Animais , Cromossomos , Metilação de DNA/genética , Análise de Sequência de RNA , Inativação do Cromossomo X/genéticaRESUMO
Although somatic mutations influence the pathogenesis, phenotype, and outcome of myeloproliferative neoplasms (MPNs), little is known about their impact on molecular response to cytoreductive treatment. We performed targeted next-generation sequencing (NGS) on 202 pretreatment samples obtained from patients with MPN enrolled in the DALIAH trial (A Study of Low Dose Interferon Alpha Versus Hydroxyurea in Treatment of Chronic Myeloid Neoplasms; #NCT01387763), a randomized controlled phase 3 clinical trial, and 135 samples obtained after 24 months of therapy with recombinant interferon-alpha (IFNα) or hydroxyurea. The primary aim was to evaluate the association between complete clinicohematologic response (CHR) at 24 months and molecular response through sequential assessment of 120 genes using NGS. Among JAK2-mutated patients treated with IFNα, those with CHR had a greater reduction in the JAK2 variant allele frequency (median, 0.29 to 0.07; P < .0001) compared with those not achieving CHR (median, 0.27 to 0.14; P < .0001). In contrast, the CALR variant allele frequency did not significantly decline in those achieving CHR or in those not achieving CHR. Treatment-emergent mutations in DNMT3A were observed more commonly in patients treated with IFNα compared with hydroxyurea (P = .04). Furthermore, treatment-emergent DNMT3A mutations were significantly enriched in IFNα-treated patients not attaining CHR (P = .02). A mutation in TET2, DNMT3A, or ASXL1 was significantly associated with prior stroke (age-adjusted odds ratio, 5.29; 95% confidence interval, 1.59-17.54; P = .007), as was a mutation in TET2 alone (age-adjusted odds ratio, 3.03; 95% confidence interval, 1.03-9.01; P = .044). At 24 months, we found mutation-specific response patterns to IFNα: (1) JAK2- and CALR-mutated MPN exhibited distinct molecular responses; and (2) DNMT3A-mutated clones/subclones emerged on treatment.
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Hidroxiureia , Transtornos Mieloproliferativos , Genômica , Humanos , Hidroxiureia/uso terapêutico , Interferon-alfa/uso terapêutico , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/genéticaRESUMO
INTRODUCTION: A subset of lung adenocarcinomas (ADs) has been found to have somatic activating mutations in the tyrosine kinase domain of the EGFR gene, associated with response to EGFR tyrosine kinase inhibitor therapy. Rare germline mutations within this domain, including EGFR T790M, have been associated with genetic susceptibility to lung ADs. Using high-throughput sequencing, we elucidate the genomic evolution in tissues from a patient with lung AD carrying a germline EGFR T790M mutation. METHODS: We performed microdissection, targeted panel, and whole-exome sequencing to molecularly characterize multiple foci of atypical adenomatous hyperplasia (AAH), in situ and invasive components of AD, normal lung tissue, and whole blood from the patient. Normal lung tissue was analyzed for potential acquired somatic genome alterations ("field effect"). RESULTS: All lesions harbored a secondary somatic EGFR mutation, either L858R or L861Q, in addition to the germline T790M mutation. Clear overlap was observed between the somatic profiles of in situ and invasive AD components, confirming clonal relatedness. AAH lesions shared few to no somatic alterations with the AD, suggesting clonal independence. No robust evidence of field effect was identified in the normal lung tissue. CONCLUSIONS: Somatic EGFR mutations are early events in the pathogenesis of lung ADs arising in the context of germline EGFR T790M. Synchronous AAH lesions seem to be independent. Stepwise genomic evolution is observed in association with invasiveness of the neoplastic cell population.
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Inherited lung cancer risk, particularly in nonsmokers, is poorly understood. Genomic and ancestry analysis of 1,153 lung cancers from Latin America revealed striking associations between Native American ancestry and their somatic landscape, including tumor mutational burden, and specific driver mutations in EGFR, KRAS, and STK11. A local Native American ancestry risk score was more strongly correlated with EGFR mutation frequency compared with global ancestry correlation, suggesting that germline genetics (rather than environmental exposure) underlie these disparities. SIGNIFICANCE: The frequency of somatic EGFR and KRAS mutations in lung cancer varies by ethnicity, but we do not understand why. Our study suggests that the variation in EGFR and KRAS mutation frequency is associated with genetic ancestry and suggests further studies to identify germline alleles that underpin this association.See related commentary by Gomez et al., p. 534.This article is highlighted in the In This Issue feature, p. 521.
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Biomarcadores Tumorais/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Hispânico ou Latino/genética , Neoplasias Pulmonares/epidemiologia , Neoplasias Pulmonares/genética , Mutação , Alelos , Estudos de Associação Genética/métodos , Genômica/métodos , Humanos , América Latina/epidemiologia , Taxa de Mutação , Vigilância da PopulaçãoRESUMO
Bladder cancer is a genetically heterogeneous disease, and novel therapeutic strategies are needed to expand treatment options and improve clinical outcomes. Here, we identified a unique subset of urothelial tumors with focal amplification of the RAF1 (CRAF) kinase gene. RAF1-amplified tumors had activation of the RAF/MEK/ERK signaling pathway and exhibited a luminal gene expression pattern. Genetic studies demonstrated that RAF1-amplified tumors were dependent upon RAF1 activity for survival, and RAF1-activated cell lines and patient-derived models were sensitive to available and emerging RAF inhibitors as well as combined RAF plus MEK inhibition. Furthermore, we found that bladder tumors with HRAS- or NRAS-activating mutations were dependent on RAF1-mediated signaling and were sensitive to RAF1-targeted therapy. Together, these data identified RAF1 activation as a dependency in a subset making up nearly 20% of urothelial tumors and suggested that targeting RAF1-mediated signaling represents a rational therapeutic strategy.