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1.
J Mol Diagn ; 26(5): 413-422, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38490303

RESUMO

Blood-based liquid biopsy is increasingly used in clinical care of patients with cancer, and fraction of tumor-derived DNA in circulation (tumor fraction; TFx) has demonstrated clinical validity across multiple cancer types. To determine TFx, shallow whole-genome sequencing of cell-free DNA (cfDNA) can be performed from a single blood sample, using an established computational pipeline (ichorCNA), without prior knowledge of tumor mutations, in a highly cost-effective manner. We describe assay validation of this approach to facilitate broad clinical application, including evaluation of assay sensitivity, precision, repeatability, reproducibility, pre-analytic factors, and DNA quality/quantity. Sensitivity to detect TFx of 3% (lower limit of detection) was 97.2% to 100% at 1× and 0.1× mean sequencing depth, respectively. Precision was demonstrated on distinct sequencing instruments (HiSeqX and NovaSeq) with no observable differences. The assay achieved prespecified 95% agreement of TFx across replicates of the same specimen (repeatability) and duplicate samples in different batches (reproducibility). Comparison of samples collected in EDTA and Streck tubes from single venipuncture in 23 patients demonstrated that EDTA or Streck tubes were comparable if processed within 8 hours. On the basis of a range of DNA inputs (1 to 50 ng), 20 ng cfDNA is the preferred input, with 5 ng minimum acceptable. Overall, this shallow whole-genome sequencing of cfDNA and ichorCNA approach offers sensitive, precise, and reproducible quantitation of TFx, facilitating assay application in clinical cancer care.


Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Reprodutibilidade dos Testes , Ácido Edético , Neoplasias/diagnóstico , Neoplasias/genética , DNA , Biomarcadores Tumorais/genética
2.
JCO Precis Oncol ; 8: e2300368, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38237100

RESUMO

PURPOSE: Somatic chromosomal alterations, particularly monosomy 3 and 8q gains, have been associated with metastatic risk in uveal melanoma (UM). Whole genome-scale evaluation of detectable alterations in cell-free DNA (cfDNA) in UM could provide valuable prognostic information. Our pilot study evaluates the correlation between genomic information using ultra-low-pass whole-genome sequencing (ULP-WGS) of cfDNA in UM and associated clinical outcomes. MATERIALS AND METHODS: ULP-WGS of cfDNA was performed on 29 plasma samples from 16 patients, 14 metastatic UM (mUM) and two non-metastatic, including pre- and post-treatment mUM samples from 10 patients treated with immunotherapy and one with liver-directed therapy. We estimated tumor fraction (TFx) and detected copy-number alterations (CNAs) using ichorCNA. Presence of 8q amplification was further analyzed using the likelihood ratio test (LRT). RESULTS: Eleven patients with mUM (17 samples) of 14 had detectable circulating tumor DNA (ctDNA). 8q gain was detected in all 17, whereas monosomy 3 was detectable in 10 of 17 samples. TFx generally correlated with disease status, showing an increase at the time of disease progression (PD). 8q gain detection sensitivity appeared greater with the LRT than with ichorCNA at lower TFxs. The only patient with mUM with partial response on treatment had a high pretreatment TFx and undetectable on-treatment ctDNA, correlating with her profound response and durable survival. CONCLUSION: ctDNA can be detected in mUM using ULP-WGS, and the TFx correlates with DS. 8q gain was consistently detectable in mUM, in line with previous studies indicating 8q gains early in primary UM and higher amplification with PD. Our work suggests that detection of CNAs by ULP-WGS, particularly focusing on 8q gain, could be a valuable blood biomarker to monitor PD in UM.


Assuntos
DNA Tumoral Circulante , Melanoma , Neoplasias Uveais , Feminino , Humanos , Projetos Piloto , Melanoma/genética , Melanoma/diagnóstico , Monossomia , DNA Tumoral Circulante/genética
3.
J Clin Oncol ; 41(13): 2382-2393, 2023 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-36724417

RESUMO

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.


Assuntos
DNA Tumoral Circulante , Rabdomiossarcoma Alveolar , Rabdomiossarcoma , Humanos , Criança , Prognóstico , Rabdomiossarcoma/patologia , Nucleotídeos , Rabdomiossarcoma Alveolar/genética , Biomarcadores Tumorais/genética
4.
Blood ; 141(15): 1817-1830, 2023 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-36706355

RESUMO

The challenge of eradicating leukemia in patients with acute myelogenous leukemia (AML) after initial cytoreduction has motivated modern efforts to combine synergistic active modalities including immunotherapy. Recently, the ETCTN/CTEP 10026 study tested the combination of the DNA methyltransferase inhibitor decitabine together with the immune checkpoint inhibitor ipilimumab for AML/myelodysplastic syndrome (MDS) either after allogeneic hematopoietic stem cell transplantation (HSCT) or in the HSCT-naïve setting. Integrative transcriptome-based analysis of 304 961 individual marrow-infiltrating cells for 18 of 48 subjects treated on study revealed the strong association of response with a high baseline ratio of T to AML cells. Clinical responses were predominantly driven by decitabine-induced cytoreduction. Evidence of immune activation was only apparent after ipilimumab exposure, which altered CD4+ T-cell gene expression, in line with ongoing T-cell differentiation and increased frequency of marrow-infiltrating regulatory T cells. For post-HSCT samples, relapse could be attributed to insufficient clearing of malignant clones in progenitor cell populations. In contrast to AML/MDS bone marrow, the transcriptomes of leukemia cutis samples from patients with durable remission after ipilimumab monotherapy showed evidence of increased infiltration with antigen-experienced resident memory T cells and higher expression of CTLA-4 and FOXP3. Altogether, activity of combined decitabine and ipilimumab is impacted by cellular expression states within the microenvironmental niche of leukemic cells. The inadequate elimination of leukemic progenitors mandates urgent development of novel approaches for targeting these cell populations to generate long-lasting responses. This trial was registered at www.clinicaltrials.gov as #NCT02890329.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Humanos , Ipilimumab/uso terapêutico , Decitabina/uso terapêutico , Síndromes Mielodisplásicas/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Recidiva
5.
Clin Cancer Res ; 28(23): 5066-5078, 2022 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-36215125

RESUMO

PURPOSE: Sensitivity to endocrine therapy (ET) is critical for the clinical benefit from the combination of palbociclib plus ET in hormone receptor-positive/HER2-negative (HR+/HER2-) advanced breast cancer. Bazedoxifene is a third-generation selective estrogen receptor (ER) modulator and selective ER degrader with activity in preclinical models of endocrine-resistant breast cancer, including models harboring ESR1 mutations. Clinical trials in healthy women showed that bazedoxifene is well tolerated. PATIENTS AND METHODS: We conducted a phase Ib/II study of bazedoxifene plus palbociclib in patients with HR+/HER2- advanced breast cancer who progressed on prior ET (N = 36; NCT02448771). RESULTS: The study met its primary endpoint, with a clinical benefit rate of 33.3%, and the safety profile was consistent with what has previously been seen with palbociclib monotherapy. The median progression-free survival (PFS) was 3.6 months [95% confidence interval (CI), 2.0-7.2]. An activating PIK3CA mutation at baseline was associated with a shorter PFS (HR = 4.4; 95% CI, 1.5-13; P = 0.0026), but activating ESR1 mutations did not impact the PFS. Longitudinal plasma circulating tumor DNA whole-exome sequencing (WES; N = 68 plasma samples) provided an overview of the tumor heterogeneity and the subclonal genetic evolution, and identified actionable mutations acquired during treatment. CONCLUSIONS: The combination of palbociclib and bazedoxifene has clinical efficacy and an acceptable safety profile in a heavily pretreated patient population with advanced HR+/HER2- breast cancer. These results merit continued investigation of bazedoxifene in breast cancer.


Assuntos
Neoplasias da Mama , Feminino , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sequenciamento do Exoma , Biópsia Líquida , Receptor ErbB-2/análise , Receptores de Estrogênio/genética , Resultado do Tratamento
6.
J Clin Oncol ; 40(2): 189-201, 2022 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-34793200

RESUMO

PURPOSE: Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact on recipient outcomes and graft alloimmune function is uncertain. PATIENTS AND METHODS: We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant. RESULTS: CH was present in 22.5% of donors, with DNMT3A (14.6%) and TET2 (5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01. DNMT3A-CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio [HR], 0.79; P = .042) and progression-free survival (HR, 0.72; P = .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor DNMT3A-CH was associated with reduced relapse (subdistribution HR, 0.59; P = .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36; P = .042), and higher interleukin-12p70 levels in recipients. No recipient of sole DNMT3A or TET2-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare TP53 or splicing factor mutations or from donors carrying germline DDX41 mutations. CONCLUSION: Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor DNMT3A-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.


Assuntos
Hematopoiese Clonal/genética , DNA Metiltransferase 3A/genética , Proteínas de Ligação a DNA/genética , Dioxigenases/genética , Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Adolescente , Adulto , Idoso , Alelos , Inibidores de Calcineurina/uso terapêutico , Criança , Pré-Escolar , Doença Crônica , Citocinas/sangue , Feminino , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Leucemia/etiologia , Masculino , Pessoa de Meia-Idade , Mutação , Intervalo Livre de Progressão , Recidiva , Taxa de Sobrevida , Fatores de Tempo , Transplante Homólogo , Doadores não Relacionados , Adulto Jovem
7.
Genome Med ; 13(1): 89, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-34016182

RESUMO

BACKGROUND: Circulating tumor DNA (ctDNA) offers minimally invasive means to repeatedly interrogate tumor genomes, providing opportunities to monitor clonal dynamics induced by metastasis and therapeutic selective pressures. In metastatic cancers, ctDNA profiling allows for simultaneous analysis of both local and distant sites of recurrence. Despite the promise of ctDNA sampling, its utility in real-time genetic monitoring remains largely unexplored. METHODS: In this exploratory analysis, we characterize high-frequency ctDNA sample series collected over narrow time frames from seven patients with metastatic triple-negative breast cancer, each undergoing treatment with Cabozantinib, a multi-tyrosine kinase inhibitor (NCT01738438, https://clinicaltrials.gov/ct2/show/NCT01738438 ). Applying orthogonal whole exome sequencing, ultra-low pass whole genome sequencing, and 396-gene targeted panel sequencing, we analyzed 42 plasma-derived ctDNA libraries, representing 4-8 samples per patient with 6-42 days between samples. Integrating tumor fraction, copy number, and somatic variant information, we model tumor clonal dynamics, predict neoantigens, and evaluate consistency of genomic information from orthogonal assays. RESULTS: We measured considerable variation in ctDNA tumor faction in each patient, often conflicting with RECIST imaging response metrics. In orthogonal sequencing, we found high concordance between targeted panel and whole exome sequencing in both variant detection and variant allele frequency estimation (specificity = 95.5%, VAF correlation, r = 0.949), Copy number remained generally stable, despite resolution limitations posed by low tumor fraction. Through modeling, we inferred and tracked distinct clonal populations specific to each patient and built phylogenetic trees revealing alterations in hallmark breast cancer drivers, including TP53, PIK3CA, CDK4, and PTEN. Our modeling revealed varied responses to therapy, with some individuals displaying stable clonal profiles, while others showed signs of substantial expansion or reduction in prevalence, with characteristic alterations of varied literature annotation in relation to the study drug. Finally, we predicted and tracked neoantigen-producing alterations across time, exposing translationally relevant detection patterns. CONCLUSIONS: Despite technical challenges arising from low tumor content, metastatic ctDNA monitoring can aid our understanding of response and progression, while minimizing patient risk and discomfort. In this study, we demonstrate the potential for high-frequency monitoring of evolving genomic features, providing an important step toward scalable, translational genomics for clinical decision making.


Assuntos
Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/genética , DNA Tumoral Circulante , Evolução Clonal/genética , Adulto , Idoso , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida/métodos , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Sequenciamento do Exoma
8.
Blood ; 137(23): 3212-3217, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-33720354

RESUMO

Relapsed myeloid disease after allogeneic stem cell transplantation (HSCT) remains largely incurable. We previously demonstrated the potent activity of immune checkpoint blockade in this clinical setting with ipilimumab or nivolumab. To define the molecular and cellular pathways by which CTLA-4 blockade with ipilimumab can reinvigorate an effective graft-versus-leukemia (GVL) response, we integrated transcriptomic analysis of leukemic biopsies with immunophenotypic profiling of matched peripheral blood samples collected from patients treated with ipilimumab following HSCT on the Experimental Therapeutics Clinical Trials Network 9204 trial. Response to ipilimumab was associated with transcriptomic evidence of increased local CD8+ T-cell infiltration and activation. Systemically, ipilimumab decreased naïve and increased memory T-cell populations and increased expression of markers of T-cell activation and costimulation such as PD-1, HLA-DR, and ICOS, irrespective of response. However, responding patients were characterized by higher turnover of T-cell receptor sequences in peripheral blood and showed increased expression of proinflammatory chemokines in plasma that was further amplified by ipilimumab. Altogether, these data highlight the compositional T-cell shifts and inflammatory pathways induced by ipilimumab both locally and systemically that associate with successful GVL outcomes. This trial was registered at www.clinicaltrials.gov as #NCT01822509.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4 , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Ipilimumab/administração & dosagem , Proteínas de Neoplasias , Células Alógenas , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Feminino , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Mieloide/terapia , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo
9.
Clin Cancer Res ; 27(18): 5049-5061, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323402

RESUMO

PURPOSE: Whole-exome (WES) and RNA sequencing (RNA-seq) are key components of cancer immunogenomic analyses. To evaluate the consistency of tumor WES and RNA-seq profiling platforms across different centers, the Cancer Immune Monitoring and Analysis Centers (CIMAC) and the Cancer Immunologic Data Commons (CIDC) conducted a systematic harmonization study. EXPERIMENTAL DESIGN: DNA and RNA were centrally extracted from fresh frozen and formalin-fixed paraffin-embedded non-small cell lung carcinoma tumors and distributed to three centers for WES and RNA-seq profiling. In addition, two 10-plex HapMap cell line pools with known mutations were used to evaluate the accuracy of the WES platforms. RESULTS: The WES platforms achieved high precision (> 0.98) and recall (> 0.87) on the HapMap pools when evaluated on loci using > 50× common coverage. Nonsynonymous mutations clustered by tumor sample, achieving an index of specific agreement above 0.67 among replicates, centers, and sample processing. A DV200 > 24% for RNA, as a putative presequencing RNA quality control (QC) metric, was found to be a reliable threshold for generating consistent expression readouts in RNA-seq and NanoString data. MedTIN > 30 was likewise assessed as a reliable RNA-seq QC metric, above which samples from the same tumor across replicates, centers, and sample processing runs could be robustly clustered and HLA typing, immune infiltration, and immune repertoire inference could be performed. CONCLUSIONS: The CIMAC collaborating laboratory platforms effectively generated consistent WES and RNA-seq data and enable robust cross-trial comparisons and meta-analyses of highly complex immuno-oncology biomarker data across the NCI CIMAC-CIDC Network.


Assuntos
Sequência de Bases , DNA de Neoplasias/análise , Sequenciamento do Exoma , Neoplasias/genética , RNA Neoplásico/análise , Humanos , Monitorização Imunológica , Neoplasias/imunologia
10.
Clin Cancer Res ; 26(11): 2556-2564, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32170028

RESUMO

PURPOSE: Existing cell-free DNA (cfDNA) methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. We developed a test for tracking hundreds of patient-specific mutations to detect MRD with a 1,000-fold lower error rate than conventional sequencing. EXPERIMENTAL DESIGN: We compared the sensitivity of our approach to digital droplet PCR (ddPCR) in a dilution series, then retrospectively identified two cohorts of patients who had undergone prospective plasma sampling and clinical data collection: 16 patients with ER+/HER2- metastatic breast cancer (MBC) sampled within 6 months following metastatic diagnosis and 142 patients with stage 0 to III breast cancer who received curative-intent treatment with most sampled at surgery and 1 year postoperative. We performed whole-exome sequencing of tumors and designed individualized MRD tests, which we applied to serial cfDNA samples. RESULTS: Our approach was 100-fold more sensitive than ddPCR when tracking 488 mutations, but most patients had fewer identifiable tumor mutations to track in cfDNA (median = 57; range = 2-346). Clinical sensitivity was 81% (n = 13/16) in newly diagnosed MBC, 23% (n = 7/30) at postoperative and 19% (n = 6/32) at 1 year in early-stage disease, and highest in patients with the most tumor mutations available to track. MRD detection at 1 year was strongly associated with distant recurrence [HR = 20.8; 95% confidence interval, 7.3-58.9]. Median lead time from first positive sample to recurrence was 18.9 months (range = 3.4-39.2 months). CONCLUSIONS: Tracking large numbers of individualized tumor mutations in cfDNA can improve MRD detection, but its sensitivity is driven by the number of tumor mutations available to track.


Assuntos
Neoplasias da Mama/patologia , DNA Tumoral Circulante/genética , Receptor alfa de Estrogênio/metabolismo , Recidiva Local de Neoplasia/patologia , Neoplasia Residual/patologia , Adulto , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , DNA Tumoral Circulante/sangue , Terapia Combinada , Feminino , Seguimentos , Humanos , Recidiva Local de Neoplasia/sangue , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/terapia , Neoplasia Residual/sangue , Neoplasia Residual/genética , Neoplasia Residual/terapia , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Taxa de Sobrevida
11.
Nature ; 570(7762): 474-479, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31142838

RESUMO

How the genomic features of a patient's cancer relate to individual disease kinetics remains poorly understood. Here we used the indolent growth dynamics of chronic lymphocytic leukaemia (CLL) to analyse the growth rates and corresponding genomic patterns of leukaemia cells from 107 patients with CLL, spanning decades-long disease courses. We found that CLL commonly demonstrates not only exponential expansion but also logistic growth, which is sigmoidal and reaches a certain steady-state level. Each growth pattern was associated with marked differences in genetic composition, the pace of disease progression and the extent of clonal evolution. In a subset of patients, whose serial samples underwent next-generation sequencing, we found that dynamic changes in the disease course of CLL were shaped by the genetic events that were already present in the early slow-growing stages. Finally, by analysing the growth rates of subclones compared with their parental clones, we quantified the growth advantage conferred by putative CLL drivers in vivo.


Assuntos
Progressão da Doença , Evolução Molecular , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Proliferação de Células/efeitos dos fármacos , Células Clonais/efeitos dos fármacos , Células Clonais/patologia , Estudos de Coortes , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Masculino , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Recidiva , Reprodutibilidade dos Testes
12.
Cancer Cell ; 35(2): 283-296.e5, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30712845

RESUMO

SF3B1 is recurrently mutated in chronic lymphocytic leukemia (CLL), but its role in the pathogenesis of CLL remains elusive. Here, we show that conditional expression of Sf3b1-K700E mutation in mouse B cells disrupts pre-mRNA splicing, alters cell development, and induces a state of cellular senescence. Combination with Atm deletion leads to the overcoming of cellular senescence and the development of CLL-like disease in elderly mice. These CLL-like cells show genome instability and dysregulation of multiple CLL-associated cellular processes, including deregulated B cell receptor signaling, which we also identified in human CLL cases. Notably, human CLLs harboring SF3B1 mutations exhibit altered response to BTK inhibition. Our murine model of CLL thus provides insights into human CLL disease mechanisms and treatment.


Assuntos
Linfócitos B/imunologia , Senescência Celular , Deleção de Genes , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Neoplasias Experimentais/genética , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Receptores de Antígenos de Linfócitos B/imunologia , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Processamento Alternativo , Animais , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Senescência Celular/efeitos dos fármacos , Dano ao DNA , Predisposição Genética para Doença , Instabilidade Genômica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Piperidinas , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Fatores de Processamento de RNA/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas
13.
Genet Med ; 20(8): 855-866, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29144510

RESUMO

PURPOSE: As massively parallel sequencing is increasingly being used for clinical decision making, it has become critical to understand parameters that affect sequencing quality and to establish methods for measuring and reporting clinical sequencing standards. In this report, we propose a definition for reduced coverage regions and describe a set of standards for variant calling in clinical sequencing applications. METHODS: To enable sequencing centers to assess the regions of poor sequencing quality in their own data, we optimized and used a tool (ExCID) to identify reduced coverage loci within genes or regions of particular interest. We used this framework to examine sequencing data from 500 patients generated in 10 projects at sequencing centers in the National Human Genome Research Institute/National Cancer Institute Clinical Sequencing Exploratory Research Consortium. RESULTS: This approach identified reduced coverage regions in clinically relevant genes, including known clinically relevant loci that were uniquely missed at individual centers, in multiple centers, and in all centers. CONCLUSION: This report provides a process road map for clinical sequencing centers looking to perform similar analyses on their data.


Assuntos
Sequenciamento do Exoma/métodos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Genoma/métodos , Sequência de Bases , Mapeamento Cromossômico , Exoma , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/normas , Software
14.
Nat Commun ; 8(1): 2185, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29259203

RESUMO

Treatment of chronic lymphocytic leukemia (CLL) has shifted from chemo-immunotherapy to targeted agents. To define the evolutionary dynamics induced by targeted therapy in CLL, we perform serial exome and transcriptome sequencing for 61 ibrutinib-treated CLLs. Here, we report clonal shifts (change >0.1 in clonal cancer cell fraction, Q < 0.1) in 31% of patients during the first year of therapy, associated with adverse outcome. We also observe transcriptional downregulation of pathways mediating energy metabolism, cell cycle, and B cell receptor signaling. Known and previously undescribed mutations in BTK and PLCG2, or uncommonly, other candidate alterations are present in seventeen subjects at the time of progression. Thus, the frequently observed clonal shifts during the early treatment period and its potential association with adverse outcome may reflect greater evolutionary capacity, heralding the emergence of drug-resistant clones.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Evolução Clonal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Adenina/análogos & derivados , Adulto , Tirosina Quinase da Agamaglobulinemia , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Evolução Clonal/genética , Progressão da Doença , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/mortalidade , Leucemia Linfocítica Crônica de Células B/patologia , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular/métodos , Mutação , Fosfolipase C gama/genética , Piperidinas , Prognóstico , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Rituximab/farmacologia , Rituximab/uso terapêutico , Transdução de Sinais , Resultado do Tratamento , Sequenciamento do Exoma
15.
Nature ; 547(7661): 55-60, 2017 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-28658208

RESUMO

Genomic analysis of tumours has led to the identification of hundreds of cancer genes on the basis of the presence of mutations in protein-coding regions. By contrast, much less is known about cancer-causing mutations in non-coding regions. Here we perform deep sequencing in 360 primary breast cancers and develop computational methods to identify significantly mutated promoters. Clear signals are found in the promoters of three genes. FOXA1, a known driver of hormone-receptor positive breast cancer, harbours a mutational hotspot in its promoter leading to overexpression through increased E2F binding. RMRP and NEAT1, two non-coding RNA genes, carry mutations that affect protein binding to their promoters and alter expression levels. Our study shows that promoter regions harbour recurrent mutations in cancer with functional consequences and that the mutations occur at similar frequencies as in coding regions. Power analyses indicate that more such regions remain to be discovered through deep sequencing of adequately sized cohorts of patients.


Assuntos
Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos de Coortes , Fatores de Transcrição E2F/metabolismo , Exoma/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Ligação Proteica/genética , RNA Longo não Codificante/genética , Receptores de Estrogênio/antagonistas & inibidores
16.
Genet Med ; 19(7): 787-795, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28125075

RESUMO

PURPOSE: Implementing cancer precision medicine in the clinic requires assessing the therapeutic relevance of genomic alterations. A main challenge is the systematic interpretation of whole-exome sequencing (WES) data for clinical care. METHODS: One hundred sixty-five adults with metastatic colorectal and lung adenocarcinomas were prospectively enrolled in the CanSeq study. WES was performed on DNA extracted from formalin-fixed paraffin-embedded tumor biopsy samples and matched blood samples. Somatic and germ-line alterations were ranked according to therapeutic or clinical relevance. Results were interpreted using an integrated somatic and germ-line framework and returned in accordance with patient preferences. RESULTS: At the time of this analysis, WES had been performed and results returned to the clinical team for 165 participants. Of 768 curated somatic alterations, only 31% were associated with clinical evidence and 69% with preclinical or inferential evidence. Of 806 curated germ-line variants, 5% were clinically relevant and 56% were classified as variants of unknown significance. The variant review and decision-making processes were effective when the process was changed from that of a Molecular Tumor Board to a protocol-based approach. CONCLUSION: The development of novel interpretive and decision-support tools that draw from scientific and clinical evidence will be crucial for the success of cancer precision medicine in WES studies.Genet Med advance online publication 26 January 2017.


Assuntos
Sequenciamento do Exoma/métodos , Exoma/genética , Medicina de Precisão/métodos , Adenocarcinoma/genética , Adenocarcinoma de Pulmão , Adulto , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Genômica/métodos , Mutação em Linhagem Germinativa/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Neoplasias Pulmonares/genética , Mutação/genética , Estudos Prospectivos , Análise de Sequência de DNA/métodos
17.
Artigo em Inglês | MEDLINE | ID: mdl-32913968

RESUMO

PURPOSE: Patients with castration-resistant prostate cancer (CRPC) receive second-generation androgen-deprivation therapy, but frequently experience relapse or do not respond. Understanding the genetic mechanisms of resistance will help to identify strategies and biomarkers that are essential for the next line of therapy. PATIENTS AND METHODS: We analyzed whole exomes of patient-matched pre- and post-treatment tumors from patients with CRPC. These patients had received the secondary androgen-deprivation therapy agent, abiraterone, which suppresses androgens to below castration levels, or enzalutamide, which competitively inhibits the key androgen signaling effector, androgen receptor. RESULTS: We observed that abiraterone-resistant tumors harbored alterations in AR and MYC, whereas enzalutamide-resistant tumors gained alterations in cell-cycle pathway genes, such as mutation in cyclin-dependent kinase N2A (CDKN2A) or amplification of CDK6. Experimentally, overexpressing cell-cycle kinases promoted enzalutamide resistance in androgen-sensitive LnCAP cells that was mitigated via CDK4/6 blockade-palbociclib and ribociclib. CONCLUSION: CDK4/6-mediated resistance observed in preclinical experiments suggests that CDK4/6 amplifications may sufficiently promote enzalutamide resistance in CRPC, and that these patients may respond to palbociclib or ribociclib. The overall observations suggest that, in genomically selected advanced CRPC, clinical strategies against abiraterone- or enzalutamide-resistant tumors may require treatment strategies that are tailored to the resistance mechanisms that are specific to those patient subpopulations.

18.
Blood Adv ; 1(10): 619-623, 2017 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29296704

RESUMO

Although common risk alleles for multiple myeloma (MM) were recently identified, their contribution to familial MM is unknown. Analyzing 38 familial cases identified primarily by linking Swedish nationwide registries, we demonstrate an enrichment of common MM risk alleles in familial compared with 1530 sporadic cases (P = 4.8 × 10-2 and 6.0 × 10-2, respectively, for 2 different polygenic risk scores) and 10 171 population-based controls (P = 1.5 × 10-4 and 1.3 × 10-4, respectively). Using mixture modeling, we estimate that about one-third of familial cases result from such enrichments. Our results provide the first direct evidence for a polygenic etiology in a familial hematologic malignancy.

19.
Cancer Cell ; 30(5): 750-763, 2016 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-27818134

RESUMO

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.


Assuntos
Processamento Alternativo , Perfilação da Expressão Gênica/métodos , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosfoproteínas/genética , Fatores de Processamento de RNA/genética , Linhagem Celular Tumoral , Proteínas Desgrenhadas/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores Notch/genética , Transdução de Sinais
20.
Nat Genet ; 48(6): 607-16, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27158780

RESUMO

To compare lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SqCC) and to identify new drivers of lung carcinogenesis, we examined the exome sequences and copy number profiles of 660 lung ADC and 484 lung SqCC tumor-normal pairs. Recurrent alterations in lung SqCCs were more similar to those of other squamous carcinomas than to alterations in lung ADCs. New significantly mutated genes included PPP3CA, DOT1L, and FTSJD1 in lung ADC, RASA1 in lung SqCC, and KLF5, EP300, and CREBBP in both tumor types. New amplification peaks encompassed MIR21 in lung ADC, MIR205 in lung SqCC, and MAPK1 in both. Lung ADCs lacking receptor tyrosine kinase-Ras-Raf pathway alterations had mutations in SOS1, VAV1, RASA1, and ARHGAP35. Regarding neoantigens, 47% of the lung ADC and 53% of the lung SqCC tumors had at least five predicted neoepitopes. Although targeted therapies for lung ADC and SqCC are largely distinct, immunotherapies may aid in treatment for both subtypes.


Assuntos
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Genoma Humano , Neoplasias Pulmonares/genética , Antígenos de Neoplasias , Variações do Número de Cópias de DNA , Humanos , Recidiva
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