RESUMO
Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.
Assuntos
DNA Tumoral Circulante/sangue , DNA Tumoral Circulante/genética , Fragmentação do DNA , Genoma Humano/genética , Neoplasias/diagnóstico , Neoplasias/genética , Estudos de Casos e Controles , Estudos de Coortes , Análise Mutacional de DNA , Humanos , Aprendizado de Máquina , Mutação , Neoplasias/sangue , Neoplasias/patologiaRESUMO
Colorectal cancer is the third most common cancer worldwide, with 1.2 million patients diagnosed annually. In late-stage colorectal cancer, the most commonly used targeted therapies are the monoclonal antibodies cetuximab and panitumumab, which prevent epidermal growth factor receptor (EGFR) activation. Recent studies have identified alterations in KRAS and other genes as likely mechanisms of primary and secondary resistance to anti-EGFR antibody therapy. Despite these efforts, additional mechanisms of resistance to EGFR blockade are thought to be present in colorectal cancer and little is known about determinants of sensitivity to this therapy. To examine the effect of somatic genetic changes in colorectal cancer on response to anti-EGFR antibody therapy, here we perform complete exome sequence and copy number analyses of 129 patient-derived tumour grafts and targeted genomic analyses of 55 patient tumours, all of which were KRAS wild-type. We analysed the response of tumours to anti-EGFR antibody blockade in tumour graft models and in clinical settings and functionally linked therapeutic responses to mutational data. In addition to previously identified genes, we detected mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 as potential mechanisms of primary resistance to this therapy. Novel alterations in the ectodomain of EGFR were identified in patients with acquired resistance to EGFR blockade. Amplifications and sequence changes in the tyrosine kinase receptor adaptor gene IRS2 were identified in tumours with increased sensitivity to anti-EGFR therapy. Therapeutic resistance to EGFR blockade could be overcome in tumour graft models through combinatorial therapies targeting actionable genes. These analyses provide a systematic approach to evaluating response to targeted therapies in human cancer, highlight new mechanisms of responsiveness to anti-EGFR therapies, and delineate new avenues for intervention in managing colorectal cancer.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Genoma Humano/genética , Genômica , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA/genética , Receptores ErbB/química , Receptores ErbB/genética , Exoma/genética , Feminino , Humanos , Proteínas Substratos do Receptor de Insulina/genética , MAP Quinase Quinase 1/genética , Camundongos , Terapia de Alvo Molecular , Mutação/genética , Panitumumabe , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptor ErbB-2/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Genetic changes in repetitive sequences are a hallmark of cancer and other diseases, but characterizing these has been challenging using standard sequencing approaches. We developed a de novo kmer finding approach, called ARTEMIS (Analysis of RepeaT EleMents in dISease), to identify repeat elements from whole-genome sequencing. Using this method, we analyzed 1.2 billion kmers in 2837 tissue and plasma samples from 1975 patients, including those with lung, breast, colorectal, ovarian, liver, gastric, head and neck, bladder, cervical, thyroid, or prostate cancer. We identified tumor-specific changes in these patients in 1280 repeat element types from the LINE, SINE, LTR, transposable element, and human satellite families. These included changes to known repeats and 820 elements that were not previously known to be altered in human cancer. Repeat elements were enriched in regions of driver genes, and their representation was altered by structural changes and epigenetic states. Machine learning analyses of genome-wide repeat landscapes and fragmentation profiles in cfDNA detected patients with early-stage lung or liver cancer in cross-validated and externally validated cohorts. In addition, these repeat landscapes could be used to noninvasively identify the tissue of origin of tumors. These analyses reveal widespread changes in repeat landscapes of human cancers and provide an approach for their detection and characterization that could benefit early detection and disease monitoring of patients with cancer.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Hepáticas , Masculino , Humanos , Neoplasias Hepáticas/genética , Elementos de DNA TransponíveisRESUMO
Circulating cell-free DNA (cfDNA) is emerging as an avenue for cancer detection, but the characteristics of cfDNA fragmentation in the blood are poorly understood. We evaluate the effect of DNA methylation and gene expression on genome-wide cfDNA fragmentation through analysis of 969 individuals. cfDNA fragment ends more frequently contained CCs or CGs, and fragments ending with CGs or CCGs are enriched or depleted, respectively, at methylated CpG positions. Higher levels and larger sizes of cfDNA fragments are associated with CpG methylation and reduced gene expression. These effects are validated in mice with isogenic tumors with or without the mutant IDH1, and are associated with genome-wide changes in cfDNA fragmentation in patients with cancer. Tumor-related hypomethylation and increased gene expression are associated with decrease in cfDNA fragment size that may explain smaller cfDNA fragments in human cancers. These results provide a connection between epigenetic changes and cfDNA fragmentation with implications for disease detection.
Assuntos
Ácidos Nucleicos Livres , Ilhas de CpG , Fragmentação do DNA , Metilação de DNA , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Ácidos Nucleicos Livres/sangue , Animais , Camundongos , Ilhas de CpG/genética , Neoplasias/genética , Epigênese Genética , Feminino , Isocitrato Desidrogenase/genética , Masculino , Regulação Neoplásica da Expressão GênicaRESUMO
PURPOSE: Circulating tumor DNA (ctDNA) has the potential to guide therapy selection and monitor treatment response in patients with metastatic cancer. However, germline and clonal hematopoiesis-associated alterations can confound identification of tumor-specific mutations in cell-free DNA (cfDNA), often requiring additional sequencing of tumor tissue. The current study assessed whether ctDNA-based treatment response monitoring could be performed in a tumor tissue-independent manner by combining ultra-deep targeted sequencing analyses of cfDNA with patient-matched white blood cell (WBC)-derived DNA. EXPERIMENTAL DESIGN: In total, 183 cfDNA and 49 WBC samples, along with 28 tissue samples, from 52 patients with metastatic colorectal cancer participating in the prospective phase III CAIRO5 clinical trial were analyzed using an ultra-deep targeted sequencing liquid biopsy assay. RESULTS: The combined cfDNA and WBC analysis prevented false-positives due to germline or hematopoietic variants in 40% of patients. Patient-matched tumor tissue sequencing did not provide additional information. Longitudinal analyses of ctDNA were more predictive of overall survival than standard-of-care radiological response evaluation. ctDNA mutations related to primary or acquired resistance to panitumumab were identified in 42% of patients. CONCLUSIONS: Accurate calling of ctDNA mutations for treatment response monitoring is feasible in a tumor tissue-independent manner by combined cfDNA and patient-matched WBC genomic DNA analysis. This tissue biopsy-independent approach simplifies sample logistics and facilitates the application of liquid biopsy ctDNA testing for evaluation of emerging therapy resistance, opening new avenues for early adaptation of treatment regimens.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias do Colo , Neoplasias Retais , Humanos , Biomarcadores Tumorais/genética , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Estudos ProspectivosRESUMO
Liver cancer is a major cause of cancer mortality worldwide. Screening individuals at high risk, including those with cirrhosis and viral hepatitis, provides an avenue for improved survival, but current screening methods are inadequate. In this study, we used whole-genome cell-free DNA (cfDNA) fragmentome analyses to evaluate 724 individuals from the United States, the European Union, or Hong Kong with hepatocellular carcinoma (HCC) or who were at average or high-risk for HCC. Using a machine learning model that incorporated multifeature fragmentome data, the sensitivity for detecting cancer was 88% in an average-risk population at 98% specificity and 85% among high-risk individuals at 80% specificity. We validated these results in an independent population. cfDNA fragmentation changes reflected genomic and chromatin changes in liver cancer, including from transcription factor binding sites. These findings provide a biological basis for changes in cfDNA fragmentation in patients with liver cancer and provide an accessible approach for noninvasive cancer detection. SIGNIFICANCE: There is a great need for accessible and sensitive screening approaches for HCC worldwide. We have developed an approach for examining genome-wide cfDNA fragmentation features to provide a high-performing and cost-effective approach for liver cancer detection. See related commentary Rolfo and Russo, p. 532. This article is highlighted in the In This Issue feature, p. 517.
Assuntos
Carcinoma Hepatocelular , Ácidos Nucleicos Livres , Neoplasias Hepáticas , Humanos , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Ácidos Nucleicos Livres/genética , Cirrose Hepática/genética , Cirrose Hepática/patologiaRESUMO
Somatic mutations are a hallmark of tumorigenesis and may be useful for non-invasive diagnosis of cancer. We analyzed whole-genome sequencing data from 2,511 individuals in the Pan-Cancer Analysis of Whole Genomes (PCAWG) study as well as 489 individuals from four prospective cohorts and found distinct regional mutation type-specific frequencies in tissue and cell-free DNA from patients with cancer that were associated with replication timing and other chromatin features. A machine-learning model using genome-wide mutational profiles combined with other features and followed by CT imaging detected >90% of patients with lung cancer, including those with stage I and II disease. The fixed model was validated in an independent cohort, detected patients with cancer earlier than standard approaches and could be used to monitor response to therapy. This approach lays the groundwork for non-invasive cancer detection using genome-wide mutation features that may facilitate cancer screening and monitoring.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Neoplasias , Humanos , Estudos Prospectivos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Taxa de Mutação , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genéticaRESUMO
The impact of thymidylate synthase (TYMS), methylenetetrahydrofolate reductase (MTHFR), and serine hydroxymethyltransferase 1 (SHMT1) gene polymorphisms and that of dihydropyrimidine dehydrogenase (DPD) enzyme activity, serum total homocysteine level, and estimated serum creatinine clearance on first-line 5-fluorouracil, leucovorin, irinotecan, and bevacizumab (FOLFIRI+bevacizumab) regimen efficacy in metastatic colorectal cancer patients was investigated. DNA was extracted from peripheral blood mononuclear cells. Genotyping was performed for TYMS 5'UTR variable number tandem repeat, TYMS 3'UTR ins/del, MTHFR C677T, and SHMT1 C1420T polymorphisms. The DPD activity of peripheral blood mononuclear cells was also determined. The univariate and multivariate analyses demonstrated that the SHMT1 1420T allele was associated with better response (P=0.025) and longer progression-free survival (PFS) (P=0.00004) and overall survival (OS) (P=0.034). Grade ≥2 hypertension was also an independent prognostic factor of longer progression-free survival and OS. Bevacizumab-related hypertension might be a predictive marker of treatment efficacy (P=0.0002 for OS) in the case of wild (CC) SHMT1 1420 genotypes only.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Glicina Hidroximetiltransferase/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Alelos , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab , Camptotecina/administração & dosagem , Camptotecina/efeitos adversos , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Neoplasias Colorretais/genética , Neoplasias Colorretais/secundário , Creatinina/sangue , Di-Hidrouracila Desidrogenase (NADP)/sangue , Intervalo Livre de Doença , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Estudos de Associação Genética , Homocisteína/sangue , Humanos , Hipertensão/induzido quimicamente , Mutação INDEL , Leucovorina/administração & dosagem , Leucovorina/efeitos adversos , Leucovorina/uso terapêutico , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Polimorfismo Genético , Prognóstico , Timidilato Sintase/genética , Resultado do TratamentoRESUMO
Non-invasive approaches for cell-free DNA (cfDNA) assessment provide an opportunity for cancer detection and intervention. Here, we use a machine learning model for detecting tumor-derived cfDNA through genome-wide analyses of cfDNA fragmentation in a prospective study of 365 individuals at risk for lung cancer. We validate the cancer detection model using an independent cohort of 385 non-cancer individuals and 46 lung cancer patients. Combining fragmentation features, clinical risk factors, and CEA levels, followed by CT imaging, detected 94% of patients with cancer across stages and subtypes, including 91% of stage I/II and 96% of stage III/IV, at 80% specificity. Genome-wide fragmentation profiles across ~13,000 ASCL1 transcription factor binding sites distinguished individuals with small cell lung cancer from those with non-small cell lung cancer with high accuracy (AUC = 0.98). A higher fragmentation score represented an independent prognostic indicator of survival. This approach provides a facile avenue for non-invasive detection of lung cancer.
Assuntos
DNA Tumoral Circulante/metabolismo , Fragmentação do DNA , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Diagnóstico Diferencial , Detecção Precoce de Câncer , Feminino , Genoma Humano , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Metástase Neoplásica , Estadiamento de Neoplasias , Carcinoma de Pequenas Células do Pulmão/diagnóstico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Adulto JovemRESUMO
BACKGROUND: Association between rectal or colon cancer risk and serine hydroxymethyltransferase 1 (SHMT1) C1420T or methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms was assessed. The serum total homocysteine (HCY), marker of folate metabolism was also investigated. METHODS: The SHMT1 and MTHFR genotypes were determined by real-time PCR and PCR-RFLP, respectively in 476 patients with rectal, 479 patients with colon cancer and in 461 and 478, respective controls matched for age and sex. Homocysteine levels were determined by HPLC kit. The association between polymorphisms and cancer risk was evaluated by logistic regression analysis adjusted for age, sex and body mass index. The population stratification bias was also estimated. RESULTS: There was no association of genotypes or diplotypes with colon cancer. The rectal cancer risk was significantly lower for SHMT1 TT (OR = 0.57, 95% confidence interval (CI) 0.36-0.89) and higher for MTHFR CT genotypes (OR = 1.4, 95%CI 1.06-1.84). A gene-dosage effect was observed for SHMT1 with progressively decreasing risk with increasing number of T allele (p = 0.014). The stratified analysis according to age and sex revealed that the association is mainly present in the younger (< 60 years) or male subgroup. As expected from genotype analysis, the SHMT1 T allele/MTHFR CC diplotype was associated with reduced rectal cancer risk (OR 0.56, 95%CI 0.42-0.77 vs all other diplotypes together). The above results are unlikely to suffer from population stratification bias. In controls HCY was influenced by SHMT1 polymorphism, while in patients it was affected only by Dukes' stage. In patients with Dukes' stage C or D HCY can be considered as a tumor marker only in case of SHMT1 1420CC genotypes. CONCLUSIONS: A protective effect of SHMT1 1420T allele or SHMT1 1420 T allele/MTHFR 677 CC diplotype against rectal but not colon cancer risk was demonstrated. The presence of SHMT1 1420 T allele significantly increases the HCY levels in controls but not in patients. Homocysteine could be considered as a tumor marker in SHMT1 1420 wild-type (CC) CRC patients in Dukes' stage C and D. Further studies need to clarify why SHMT1 and MTHFR polymorphisms are associated only with rectal and not colon cancer risk.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Glicina Hidroximetiltransferase/genética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Neoplasias Retais/genética , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Despite progress in immunotherapy, identifying patients that respond has remained a challenge. Through analysis of whole-exome and targeted sequence data from 5,449 tumors, we found a significant correlation between tumor mutation burden (TMB) and tumor purity, suggesting that low tumor purity tumors are likely to have inaccurate TMB estimates. We developed a new method to estimate a corrected TMB (cTMB) that was adjusted for tumor purity and more accurately predicted outcome to immune checkpoint blockade (ICB). To identify improved predictive markers together with cTMB, we performed whole-exome sequencing for 104 lung tumors treated with ICB. Through comprehensive analyses of sequence and structural alterations, we discovered a significant enrichment in activating mutations in receptor tyrosine kinase (RTK) genes in nonresponding tumors in three immunotherapy treated cohorts. An integrated multivariable model incorporating cTMB, RTK mutations, smoking-related mutational signature and human leukocyte antigen status provided an improved predictor of response to immunotherapy that was independently validated.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Imunoterapia/métodos , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.
Assuntos
Progressão da Doença , Genômica , Cisto Pancreático/genética , Neoplasias Pancreáticas/genética , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Exoma/genética , Dosagem de Genes , Humanos , Mutação , Cisto Pancreático/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Proteína Smad4/genéticaRESUMO
Liquid biopsies are providing new opportunities for detection of residual disease in cell-free DNA (cfDNA) after surgery but may be confounded through identification of alterations arising from clonal hematopoiesis. Here, we identify circulating tumor-derived DNA (ctDNA) alterations through ultrasensitive targeted sequencing analyses of matched cfDNA and white blood cells from the same patient. We apply this approach to analyze samples from patients in the CRITICS trial, a phase III randomized controlled study of perioperative treatment in patients with operable gastric cancer. After filtering alterations from matched white blood cells, the presence of ctDNA predicts recurrence when analyzed within nine weeks after preoperative treatment and after surgery in patients eligible for multimodal treatment. These analyses provide a facile method for distinguishing ctDNA from other cfDNA alterations and highlight the utility of ctDNA as a predictive biomarker of patient outcome to perioperative cancer therapy and surgical resection in patients with gastric cancer.
Assuntos
Ácidos Nucleicos Livres/química , DNA de Neoplasias/análise , Leucócitos/química , Recidiva Local de Neoplasia/diagnóstico , Análise de Sequência de DNA , Neoplasias Gástricas/diagnóstico , DNA de Neoplasias/química , Hematopoese , Humanos , Prognóstico , Estudo de Prova de Conceito , Ensaios Clínicos Controlados Aleatórios como Assunto , Neoplasias Gástricas/genética , Análise de SobrevidaRESUMO
With the advent of precision oncology, there is an urgent need to develop improved methods for rapidly detecting responses to targeted therapies. Here, we have developed an ultrasensitive measure of cell-free tumor load using targeted and whole-genome sequencing approaches to assess responses to tyrosine kinase inhibitors in patients with advanced lung cancer. Analyses of 28 patients treated with anti-EGFR or HER2 therapies revealed a bimodal distribution of cell-free circulating tumor DNA (ctDNA) after therapy initiation, with molecular responders having nearly complete elimination of ctDNA (>98%). Molecular nonresponders displayed limited changes in ctDNA levels posttreatment and experienced significantly shorter progression-free survival (median 1.6 vs. 13.7 months, P < 0.0001; HR = 66.6; 95% confidence interval, 13.0-341.7), which was detected on average 4 weeks earlier than CT imaging. ctDNA analyses of patients with radiographic stable or nonmeasurable disease improved prediction of clinical outcome compared with CT imaging. These analyses provide a rapid approach for evaluating therapeutic response to targeted therapies and have important implications for the management of patients with cancer and the development of new therapeutics.Significance: Cell-free tumor load provides a novel approach for evaluating longitudinal changes in ctDNA during systemic treatment with tyrosine kinase inhibitors and serves an unmet clinical need for real-time, noninvasive detection of tumor response to targeted therapies before radiographic assessment.See related commentary by Zou and Meyerson, p. 1038.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Tumoral Circulante/análise , DNA de Neoplasias/análise , Terapia de Alvo Molecular , Mutação , Inibidores de Proteínas Quinases/uso terapêutico , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Carga TumoralRESUMO
Despite the initial successes of immunotherapy, there is an urgent clinical need for molecular assays that identify patients more likely to respond. Here, we report that ultrasensitive measures of circulating tumor DNA (ctDNA) and T-cell expansion can be used to assess responses to immune checkpoint blockade in metastatic lung cancer patients (N = 24). Patients with clinical response to therapy had a complete reduction in ctDNA levels after initiation of therapy, whereas nonresponders had no significant changes or an increase in ctDNA levels. Patients with initial response followed by acquired resistance to therapy had an initial drop followed by recrudescence in ctDNA levels. Patients without a molecular response had shorter progression-free and overall survival compared with molecular responders [5.2 vs. 14.5 and 8.4 vs. 18.7 months; HR 5.36; 95% confidence interval (CI), 1.57-18.35; P = 0.007 and HR 6.91; 95% CI, 1.37-34.97; P = 0.02, respectively], which was detected on average 8.7 weeks earlier and was more predictive of clinical benefit than CT imaging. Expansion of T cells, measured through increases of T-cell receptor productive frequencies, mirrored ctDNA reduction in response to therapy. We validated this approach in an independent cohort of patients with early-stage non-small cell lung cancer (N = 14), where the therapeutic effect was measured by pathologic assessment of residual tumor after anti-PD1 therapy. Consistent with our initial findings, early ctDNA dynamics predicted pathologic response to immune checkpoint blockade. These analyses provide an approach for rapid determination of therapeutic outcomes for patients treated with immune checkpoint inhibitors and have important implications for the development of personalized immune targeted strategies.Significance: Rapid and sensitive detection of circulating tumor DNA dynamic changes and T-cell expansion can be used to guide immune targeted therapy for patients with lung cancer.See related commentary by Zou and Meyerson, p. 1038.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , DNA Tumoral Circulante/análise , DNA de Neoplasias/análise , Neoplasias Pulmonares/imunologia , Neoplasia Residual/imunologia , Nivolumabe/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , DNA Tumoral Circulante/genética , Estudos de Coortes , DNA de Neoplasias/genética , Seguimentos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/genética , Neoplasia Residual/patologia , Prognóstico , Taxa de SobrevidaRESUMO
BACKGROUND: High-dose methotrexate (HD-MTX) is one of the most important agents in the therapy of osteosarcoma (OSC). Acute and delayed toxicities still constitute clinical problems. Methylenetetrahydrofolate reductase (MTHFR) has a key role in the folate cycle. In case of homozygosity of the 677C-->T polymorphism, treatment with antimetabolites, such as MTX, can cause additional toxicity. CASE REPORT: In the present work, we describe the case of a 10-year-old boy with OSC. After the first HD-MTX infusion (12 g/m2/6 h) acute neurological disturbances were detected followed by severe hepatotoxicity. Plasma concentrations of MTX and 7-OH-MTX showed delayed clearance. Calcium folinate was administered to the patient until +186 hours. Tha patient was homozygous for the 677 polymorphism and wild-type for the 1298 polymorphism of the MTHFR gene. CONCLUSION: We hypothesize that MTX toxicity can be explained by the association between homozygosity of the MTHFR C677T polymorphism causing disturbances in the folate status and thus an enhanced vulnerability of the central nervous system to antimetabolites and to the prolonged MTX exposure due to delayed MTX clearance.
Assuntos
Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacocinética , Neoplasias Ósseas/tratamento farmacológico , Metotrexato/efeitos adversos , Metotrexato/farmacocinética , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Síndromes Neurotóxicas/etiologia , Osteossarcoma/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Criança , Relação Dose-Resposta a Droga , Humanos , Masculino , Metotrexato/administração & dosagem , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Síndromes Neurotóxicas/metabolismo , Osteossarcoma/enzimologia , Osteossarcoma/genética , Polimorfismo GenéticoRESUMO
To improve our understanding of ovarian cancer, we performed genome-wide analyses of 45 ovarian cancer cell lines. Given the challenges of genomic analyses of tumors without matched normal samples, we developed approaches for detection of somatic sequence and structural changes and integrated these with epigenetic and expression alterations. Alterations not previously implicated in ovarian cancer included amplification or overexpression of ASXL1 and H3F3B, deletion or underexpression of CDC73 and TGF-beta receptor pathway members, and rearrangements of YAP1-MAML2 and IKZF2-ERBB4. Dose-response analyses to targeted therapies revealed unique molecular dependencies, including increased sensitivity of tumors with PIK3CA and PPP2R1A alterations to PI3K inhibitor GNE-493, MYC amplifications to PARP inhibitor BMN673, and SMAD3/4 alterations to MEK inhibitor MEK162. Genome-wide rearrangements provided an improved measure of sensitivity to PARP inhibition. This study provides a comprehensive and broadly accessible resource of molecular information for the development of therapeutic avenues in ovarian cancer.
Assuntos
Epigenômica , Regulação Neoplásica da Expressão Gênica , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Mutação/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The cytotoxic effect of 5-fluorouracil (5-FU) is mediated by the inhibition of thymidylate synthase (TS), however, at the same time 5-FU is catabolized by dihydropyrimidine dehydrogenase (DPD). Efficacy of 5-FU may therefore depend on the TS and DPD activity and on pharmacogenetic factors influencing these enzymes. Our aims were (1) to determine the distribution of DPD activity, the frequency of DPD deficiency and the DPD (IVS14+1G>A) mutation in the peripheral blood mononuclear cells of colorectal cancer (CRC) patients, and study the relationship between DPD deficiency and toxicity of 5-FU; (2) to investigate the influence of TS polymorphisms and DPD activity on the survival of CRC patients receiving 5-FU-based adjuvant therapy. The frequency of DPD deficiency was determined by radiochemical methods in the peripheral blood mononuclear cells (PBMCs) of 764 CRC patients treated with 5-FU. The relationship between the TS polymorphisms, DPD activity and the disease-free and overall survival was studied in 166 CRC patients receiving 5-FU-based adjuvant therapy. TS polymorphisms were determined in the DNA samples separated from the PBMCs, by PCR-PAGE and PCR-RFLP-PAGE (restriction fragment length polymorphism) methods. Low DPD values (<10 pmol/min/106 PBMCs) were demonstrated in 160/764 patients (20.9%), and of those DPD deficiency (<5 pmol/min/106 PBMCs) was verified in 38 patients (4.9%). In the latter group severe (>Gr 3) toxicity was found in 87%. The prevalence of the DPD IVS14+1G>A mutation among the 38 DPD-deficient patients was 7.8% (3/38) and was accompanied by severe Gr 4 toxic symptoms (neutropenia, mucositis, diarrhea). TS polymorphisms showed a relationship with the survival of CRC patients. It is important to mention that by combining the 3-3 genotypes of 5'-TSER and 3'-TSUTR polymorphisms the obtained 8 genotype combinations showed significantly different Kaplan-Meier survival curves. The evaluation of these curves with Cox regression analysis resulted in two prognostically different groups: "A" good prognosis (RR<1) and "B" bad prognosis (RR>1). The disease-free- and overall survival of these two groups were significantly different. DPD activity also showed correlation with the survival; patients with DPD activity <10 pmol/min/106 PBMCs showed significantly longer disease-free and overall survival. The determination of DPD activity proved to be a more valuable parameter in the evaluation of serious 5-FU-related toxicity compared to the IVS14+1G>A mutation analysis. According to the Cox multivariate analysis the combination of germline TS polymorphisms and DPD activity is/an independent prognostic marker of survival in CRC patients treated with adjuvant 5-FU therapy.
Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Deficiência da Di-Hidropirimidina Desidrogenase , Fluoruracila/uso terapêutico , Polimorfismo Genético , Timidilato Sintase/genética , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia Adjuvante , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/mortalidade , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Intervalo Livre de Doença , Feminino , Fluoruracila/efeitos adversos , Fluoruracila/metabolismo , Humanos , Estimativa de Kaplan-Meier , Leucócitos Mononucleares/enzimologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Modelos de Riscos Proporcionais , Timidilato Sintase/antagonistas & inibidores , Resultado do TratamentoRESUMO
Purpose: Analysis of genomic alterations in cell-free DNA (cfDNA) is evolving as an approach to detect, monitor, and genotype malignancies. Methods to separate the liquid from the cellular fraction of whole blood for circulating tumor DNA (ctDNA) analyses have been largely unstudied, although these may be a critical consideration for assay performance.Experimental Design: To evaluate the influence of blood processing on cfDNA and ctDNA quality and yield, we compared the cfDNA levels in serum with those in plasma. Given the limitations of serum for ctDNA analyses, we evaluated the effects of two plasma processing approaches, K2EDTA and Cell-Free DNA BCT (BCT) tubes, on cfDNA and ctDNA recovery. A total of 45 samples from nine patients with cancer were collected in both tube types. Once collected, blood was processed into plasma immediately or kept at room temperature and processed into plasma at 1, 3, 5, or 7 days.Results: As early as 24 hours after collection, plasma isolated from blood collected in K2EDTA tubes contained an elevated level of cfDNA that increased over time compared with BCT tubes where no significant increase in cfDNA levels was observed. When samples from an additional six patients with cancer, collected in the same manner, were stored at 4°C in K2EDTA tubes over the course of 3 days, total cfDNA and ctDNA levels were comparable between samples collected in BCT tubes. At day 3, there was a trend toward a decrease in ctDNA levels in both tubes that was more pronounced when measuring the mutant allele fraction for cases stored at 4°C in K2EDTA tubes.Conclusions: In summary, methods of blood processing have a strong influence on cfDNA and ctDNA levels and should be a consideration when evaluating ctDNA in peripheral circulation. Clin Cancer Res; 23(10); 2471-7. ©2016 AACR.
Assuntos
DNA Tumoral Circulante/isolamento & purificação , DNA de Neoplasias/isolamento & purificação , Neoplasias/sangue , Manejo de Espécimes/métodos , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/isolamento & purificação , DNA Tumoral Circulante/sangue , DNA de Neoplasias/sangue , Humanos , Mutação , Neoplasias/genética , Neoplasias/patologia , TemperaturaRESUMO
Purpose: Ovarian cancer is the leading cause of death from gynecologic malignancy in the United States, with high rates of recurrence and eventual resistance to cytotoxic chemotherapy. Model systems that allow for accurate and reproducible target discovery and validation are needed to support further drug development in this disease.Experimental Design: Clinically annotated patient-derived xenograft (PDX) models were generated from tumor cells isolated from the ascites or pleural fluid of patients undergoing clinical procedures. Models were characterized by IHC and by molecular analyses. Each PDX was luciferized to allow for reproducible in vivo assessment of intraperitoneal tumor burden by bioluminescence imaging (BLI). Plasma assays for CA125 and human LINE-1 were developed as secondary tests of in vivo disease burden.Results: Fourteen clinically annotated and molecularly characterized luciferized ovarian PDX models were generated. Luciferized PDX models retain fidelity to both the nonluciferized PDX and the original patient tumor, as demonstrated by IHC, array CGH, and targeted and whole-exome sequencing analyses. Models demonstrated diversity in specific genetic alterations and activation of PI3K signaling pathway members. Response of luciferized PDX models to standard-of-care therapy could be reproducibly monitored by BLI or plasma markers.Conclusions: We describe the establishment of a collection of 14 clinically annotated and molecularly characterized luciferized ovarian PDX models in which orthotopic tumor burden in the intraperitoneal space can be followed by standard and reproducible methods. This collection is well suited as a platform for proof-of-concept efficacy and biomarker studies and for validation of novel therapeutic strategies in ovarian cancer. Clin Cancer Res; 23(5); 1263-73. ©2016 AACR.