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MOTIVATION: Detection of genomic alterations in circulating tumor DNA (ctDNA) is currently used for active clinical monitoring of cancer progression and treatment response. While methods for analysis of small mutations are more developed, strategies for detecting structural variants (SVs) in ctDNA are limited. Additionally, reproducibly calling small-scale mutations, copy number alterations, and SVs in ctDNA is challenging due to the lack to unified tools for these different classes of variants. RESULTS: We developed a unified pipeline for the analysis of ctDNA [Pipeline for the Analysis of ctDNA (PACT)] that accurately detects SVs and consistently outperformed similar tools when applied to simulated, cell line, and clinical data. We provide PACT in the form of a Common Workflow Language pipeline which can be run by popular workflow management systems in high-performance computing environments. AVAILABILITY AND IMPLEMENTATION: PACT is freely available at https://github.com/ChrisMaherLab/PACT.
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DNA Tumoral Circulante , Neoplasias , Humanos , DNA Tumoral Circulante/genética , Mutação , Neoplasias/genética , Genômica , Linhagem Celular , Biomarcadores Tumorais/genéticaRESUMO
BACKGROUND: The leading cause of mortality for patients with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome is the development of malignant peripheral nerve sheath tumor (MPNST), an aggressive soft tissue sarcoma. In the setting of NF1, this cancer type frequently arises from within its common and benign precursor, plexiform neurofibroma (PN). Transformation from PN to MPNST is challenging to diagnose due to difficulties in distinguishing cross-sectional imaging results and intralesional heterogeneity resulting in biopsy sampling errors. METHODS AND FINDINGS: This multi-institutional study from the National Cancer Institute and Washington University in St. Louis used fragment size analysis and ultra-low-pass whole genome sequencing (ULP-WGS) of plasma cell-free DNA (cfDNA) to distinguish between MPNST and PN in patients with NF1. Following in silico enrichment for short cfDNA fragments and copy number analysis to estimate the fraction of plasma cfDNA originating from tumor (tumor fraction), we developed a noninvasive classifier that differentiates MPNST from PN with 86% pretreatment accuracy (91% specificity, 75% sensitivity) and 89% accuracy on serial analysis (91% specificity, 83% sensitivity). Healthy controls without NF1 (participants = 16, plasma samples = 16), PN (participants = 23, plasma samples = 23), and MPNST (participants = 14, plasma samples = 46) cohorts showed significant differences in tumor fraction in plasma (P = 0.001) as well as cfDNA fragment length (P < 0.001) with MPNST samples harboring shorter fragments and being enriched for tumor-derived cfDNA relative to PN and healthy controls. No other covariates were significant on multivariate logistic regression. Mutational analysis demonstrated focal NF1 copy number loss in PN and MPNST patient plasma but not in healthy controls. Greater genomic instability including alterations associated with malignant transformation (focal copy number gains in chromosome arms 1q, 7p, 8q, 9q, and 17q; focal copy number losses in SUZ12, SMARCA2, CDKN2A/B, and chromosome arms 6p and 9p) was more prominently observed in MPNST plasma. Furthermore, the sum of longest tumor diameters (SLD) visualized by cross-sectional imaging correlated significantly with paired tumor fractions in plasma from MPNST patients (r = 0.39, P = 0.024). On serial analysis, tumor fraction levels in plasma dynamically correlated with treatment response to therapy and minimal residual disease (MRD) detection before relapse. Study limitations include a modest MPNST sample size despite accrual from 2 major referral centers for this rare malignancy, and lack of uniform treatment and imaging protocols representing a real-world cohort. CONCLUSIONS: Tumor fraction levels derived from cfDNA fragment size and copy number alteration analysis of plasma cfDNA using ULP-WGS significantly correlated with MPNST tumor burden, accurately distinguished MPNST from its benign PN precursor, and dynamically correlated with treatment response. In the future, our findings could form the basis for improved early cancer detection and monitoring in high-risk cancer-predisposed populations.
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Ácidos Nucleicos Livres/análise , Neurofibroma Plexiforme/diagnóstico , Neurofibrossarcoma/diagnóstico , Sequenciamento Completo do Genoma , Adulto , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: The standard of care treatment for muscle-invasive bladder cancer (MIBC) is radical cystectomy, which is typically preceded by neoadjuvant chemotherapy. However, the inability to assess minimal residual disease (MRD) noninvasively limits our ability to offer bladder-sparing treatment. Here, we sought to develop a liquid biopsy solution via urine tumor DNA (utDNA) analysis. METHODS AND FINDINGS: We applied urine Cancer Personalized Profiling by Deep Sequencing (uCAPP-Seq), a targeted next-generation sequencing (NGS) method for detecting utDNA, to urine cell-free DNA (cfDNA) samples acquired between April 2019 and November 2020 on the day of curative-intent radical cystectomy from 42 patients with localized bladder cancer. The average age of patients was 69 years (range: 50 to 86), of whom 76% (32/42) were male, 64% (27/42) were smokers, and 76% (32/42) had a confirmed diagnosis of MIBC. Among MIBC patients, 59% (19/32) received neoadjuvant chemotherapy. utDNA variant calling was performed noninvasively without prior sequencing of tumor tissue. The overall utDNA level for each patient was represented by the non-silent mutation with the highest variant allele fraction after removing germline variants. Urine was similarly analyzed from 15 healthy adults. utDNA analysis revealed a median utDNA level of 0% in healthy adults and 2.4% in bladder cancer patients. When patients were classified as those who had residual disease detected in their surgical sample (n = 16) compared to those who achieved a pathologic complete response (pCR; n = 26), median utDNA levels were 4.3% vs. 0%, respectively (p = 0.002). Using an optimal utDNA threshold to define MRD detection, positive utDNA MRD detection was highly correlated with the absence of pCR (p < 0.001) with a sensitivity of 81% and specificity of 81%. Leave-one-out cross-validation applied to the prediction of pathologic response based on utDNA MRD detection in our cohort yielded a highly significant accuracy of 81% (p = 0.007). Moreover, utDNA MRD-positive patients exhibited significantly worse progression-free survival (PFS; HR = 7.4; 95% CI: 1.4-38.9; p = 0.02) compared to utDNA MRD-negative patients. Concordance between urine- and tumor-derived mutations, determined in 5 MIBC patients, was 85%. Tumor mutational burden (TMB) in utDNA MRD-positive patients was inferred from the number of non-silent mutations detected in urine cfDNA by applying a linear relationship derived from The Cancer Genome Atlas (TCGA) whole exome sequencing of 409 MIBC tumors. We suggest that about 58% of these patients with high inferred TMB might have been candidates for treatment with early immune checkpoint blockade. Study limitations included an analysis restricted only to single-nucleotide variants (SNVs), survival differences diminished by surgery, and a low number of DNA damage response (DRR) mutations detected after neoadjuvant chemotherapy at the MRD time point. CONCLUSIONS: utDNA MRD detection prior to curative-intent radical cystectomy for bladder cancer correlated significantly with pathologic response, which may help select patients for bladder-sparing treatment. utDNA MRD detection also correlated significantly with PFS. Furthermore, utDNA can be used to noninvasively infer TMB, which could facilitate personalized immunotherapy for bladder cancer in the future.
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Biomarcadores Tumorais/análise , Cistectomia/estatística & dados numéricos , DNA de Neoplasias/análise , Neoplasia Residual/diagnóstico , Neoplasias da Bexiga Urinária/diagnóstico , Urina/química , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Missouri , Invasividade Neoplásica/patologia , Neoplasia Residual/etiologia , Intervalo Livre de Progressão , Neoplasias da Bexiga Urinária/etiologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pmed.1003732.].
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Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.
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Antineoplásicos/síntese química , Benzamidinas/síntese química , Proteínas de Neoplasias/antagonistas & inibidores , Inibidores de Proteases/síntese química , Serina Endopeptidases/química , Amidinas/síntese química , Antineoplásicos/farmacologia , Arginina/química , Benzamidinas/farmacologia , Domínio Catalítico , Desenho de Fármacos , Ensaios Enzimáticos , Guanidinas/química , Ensaios de Triagem em Larga Escala , Humanos , Cinética , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/química , Peptidomiméticos/química , Fenilalanina/análogos & derivados , Fenilalanina/síntese química , Piperazinas/síntese química , Inibidores de Proteases/farmacologia , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Ureia/análogos & derivados , Ureia/químicaRESUMO
PURPOSE: Our goal was to demonstrate that lymphatic drainage fluid (lymph) has improved sensitivity in quantifying postoperative minimal residual disease (MRD) in locally advanced human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) compared with plasma, and leverage this novel biofluid for patient risk stratification. EXPERIMENTAL DESIGN: We prospectively collected lymph samples from neck drains of 106 patients with HPV (+) OPSCC, along with 67 matched plasma samples, 24 hours after surgery. PCR and next-generation sequencing were used to quantify cancer-associated cell-free HPV (cf-HPV) and tumor-informed variants in lymph and plasma. Next, lymph cf-HPV and variants were compared with TNM stage, extranodal extension (ENE), and composite definitions of high-risk pathology. We then created a machine learning model, informed by lymph MRD and clinicopathologic features, to compare with progression-free survival (PFS). RESULTS: Postoperative lymph was enriched with cf-HPV compared with plasma (P < 0.0001) and correlated with pN2 stage (P = 0.003), ENE (P < 0.0001), and trial-defined pathologic risk criteria (mean AUC = 0.78). In addition, the lymph mutation number and variant allele frequency were higher in pN2 ENE (+) necks than in pN1 ENE (+) (P = 0.03, P = 0.02) or pN0-N1 ENE (-) (P = 0.04, P = 0.03, respectively). The lymph MRD-informed risk model demonstrated inferior PFS in high-risk patients (AUC = 0.96, P < 0.0001). CONCLUSIONS: Variant and cf-HPV quantification, performed in 24-hour postoperative lymph samples, reflects single- and multifeature high-risk pathologic criteria. Incorporating lymphatic MRD and clinicopathologic feature analysis can stratify PFS early after surgery in patients with HPV (+) head and neck cancer. See related commentary by Shannon and Iyer, p. 1223.
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Neoplasias de Cabeça e Pescoço , Neoplasias Orofaríngeas , Infecções por Papillomavirus , Humanos , Papillomavirus Humano , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/cirurgia , Neoplasia Residual/patologia , Prognóstico , Estadiamento de Neoplasias , Neoplasias Orofaríngeas/diagnóstico , Neoplasias Orofaríngeas/cirurgia , Neoplasias Orofaríngeas/patologia , Neoplasias de Cabeça e Pescoço/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/diagnóstico , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/cirurgia , Estudos RetrospectivosRESUMO
Traditional cold chain systems of collection, transportation, and storage of biofluid specimens for eventual analysis pose a huge financial and environmental burden. These systems are impractical in pre-hospital and resource-limited settings, where refrigeration and electricity are not reliable or even available. Here, we develop an innovative technology using metal-organic frameworks (MOFs), a novel class of organic-inorganic hybrids with high thermal stability, as encapsulates for preserving the integrity of protein biomarkers in biofluids under ambient or non-refrigerated storage conditions. We encapsulate prostate-specific antigen (PSA) in whole patient plasma using hydrophilic zeolitic imidazolate framework-90 (ZIF-90) for preservation at 40 °C for 4 weeks and eventual on-demand reconstitution for antibody-based assays with recovery above 95% compared to storage at -20 °C. Without ZIF-90 encapsulation, only 10-30% of the PSA immunoactivity remained. Furthermore, we demonstrate encapsulation of multiple cancer biomarker proteins in whole patient plasma using ZIF-8 or ZIF-90 encapsulants for eventual on-demand reconstitution and analysis after 1 week at 40 °C. Overall, MOF encapsulation of patient biofluids is important as climate change may be affecting the stability and increase costs of maintaining biospecimen cold chain custody for the collection, transportation, and storage of biospecimens prior to analysis or for biobanking regardless of any countries' affluence.
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Estruturas Metalorgânicas , Humanos , Masculino , Antígeno Prostático Específico , Bancos de Espécimes BiológicosRESUMO
Circulating tumor DNA (ctDNA) sensitivity remains subpar for molecular residual disease (MRD) detection in bladder cancer patients. To remedy this problem, we focused on the biofluid most proximal to the disease, urine, and analyzed urine tumor DNA in 74 localized bladder cancer patients. We integrated ultra-low-pass whole genome sequencing (ULP-WGS) with urine cancer personalized profiling by deep sequencing (uCAPP-Seq) to achieve sensitive MRD detection and predict overall survival. Variant allele frequency, inferred tumor mutational burden, and copy number-derived tumor fraction levels in urine cell-free DNA (cfDNA) significantly predicted pathologic complete response status, far better than plasma ctDNA was able to. A random forest model incorporating these urine cfDNA-derived factors with leave-one-out cross-validation was 87% sensitive for predicting residual disease in reference to gold-standard surgical pathology. Both progression-free survival (HR = 3.00, p = 0.01) and overall survival (HR = 4.81, p = 0.009) were dramatically worse by Kaplan-Meier analysis for patients predicted by the model to have MRD, which was corroborated by Cox regression analysis. Additional survival analyses performed on muscle-invasive, neoadjuvant chemotherapy, and held-out validation subgroups corroborated these findings. In summary, we profiled urine samples from 74 patients with localized bladder cancer and used urine cfDNA multi-omics to detect MRD sensitively and predict survival accurately.
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Metastatic castration-resistant prostate cancer (mCRPC) resistant to androgen receptor (AR)-targeted agents is often lethal. Unfortunately, biomarkers for this deadly disease remain under investigation, and underpinning mechanisms are ill-understood. Here, we applied deep sequencing to â¼100 mCRPC patients prior to the initiation of first-line AR-targeted therapy, which detected AR /enhancer alterations in over a third of patients, which correlated with lethality. To delve into the mechanism underlying why these patients with cell-free AR /enhancer alterations developed more lethal prostate cancer, we next performed genome-wide cell-free DNA epigenomics. Strikingly, we found that binding sites for transcription factors associated with developmental stemness were nucleosomally more accessible. These results were corroborated using cell-free DNA methylation data, as well as tumor RNA sequencing from a held-out cohort of mCRPC patients. Thus, we validated the importance of AR /enhancer alterations as a prognostic biomarker in lethal mCRPC, and showed that the underlying mechanism for lethality involves reprogramming developmental states toward increased stemness.
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Numerous cell states are known to comprise the pancreatic ductal adenocarcinoma (PDAC) tumor microenvironment (TME). However, the developmental stemness and co-occurrence of these cell states remain poorly defined. Here, we performed single-cell RNA sequencing (scRNA-seq) on a cohort of treatment-naive PDAC time-of-diagnosis endoscopic ultrasound-guided fine needle biopsy (EUS-FNB) samples (n = 25). We then combined these samples with surgical resection (n = 6) and publicly available samples to increase statistical power (n = 80). Following annotation into 25 distinct cell states, cells were scored for developmental stemness, and a customized version of the Ecotyper tool was used to identify communities of co-occurring cell states in bulk RNA-seq samples (n = 268). We discovered a tumor microenvironmental community comprised of aggressive basal-like malignant cells, tumor-promoting SPP1+ macrophages, and myofibroblastic cancer-associated fibroblasts associated with especially poor prognosis. We also found a developmental stemness continuum with implications for survival that is present in both malignant cells and cancer-associated fibroblasts (CAFs). We further demonstrated that high-dimensional analyses predictive of survival are feasible using standard-of-care, time-of-diagnosis EUS-FNB specimens. In summary, we identified tumor microenvironmental and developmental stemness characteristics from a high-dimensional gene expression analysis of PDAC using human tissue specimens, including time-of-diagnosis EUS-FNB samples. These reveal new connections between tumor microenvironmental composition, CAF and malignant cell stemness, and patient survival that could lead to better upfront risk stratification and more personalized upfront clinical decision-making.
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PURPOSE: Low-dose computed tomography (LDCT) screening of high-risk patients decreases lung cancer-related mortality. However, high false-positive rates associated with LDCT result in unnecessary interventions. To distinguish non-small-cell lung cancer (NSCLC) from benign nodules, in the present study, we integrated cellular liquid biomarkers in patients with suspicious lung nodules (lung cancer screening reporting and data system [Lung-RADS] 4). METHODS: Prospectively, 7.5 mL of blood was collected from 221 individuals (training set: 90 nonscreened NSCLC patients, 74 high-risk screening patients with no/benign nodules [Lung-RADS 1-3], and 20 never smokers; validation set: 37 patients with suspicious nodules [Lung-RADS 4]). Circulating tumor cells (CTCs), CTC clusters, and tumor-macrophage fusion (TMF) cells were identified by blinded analyses. Screening patients underwent a median of two LDCTs (range, 1-4) with a median surveillance time of 30 (range, 11-50) months. RESULTS: In the validation set of 37 Lung-RADS 4 patients, all circulating cellular biomarker counts (P < .005; Wilcoxon test) and positivity rates were significantly higher in 23 biopsy-proven NSCLC patients (CTCs: 23 of 23 [100%], CTC clusters: 6 of 23 [26.1%], and TMF cells: 15 of 23 [65.2%]) than in 14 patients with biopsy-proven benign nodules (6 of 14 [42.9%], 0 of 14 [0%], and 2 of 14 [14.3%]). On the basis of cutoff values from the training set, logistic regression with receiver operating characteristic and area under the curve analyses demonstrated that CTCs (sensitivity: 0.870, specificity: 1.0, and area under the curve: 0.989) and TMF cells (0.652; 0.880; 0.790) complement LDCT in diagnosing NSCLC in Lung-RADS 4 patients. CONCLUSION: Cellular liquid biomarkers have a potential to complement LDCT interpretation of suspicious Lung-RADS 4 nodules to distinguish NSCLC from benign lung nodules. A future prospective, large-scale, multicenter clinical trial should validate the role of cellular liquid biomarkers in improving diagnostic accuracy in high-risk patients with Lung-RADS 4 nodules.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Lesões Pré-Cancerosas , Biomarcadores , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Detecção Precoce de Câncer/métodos , Humanos , Pulmão/patologia , Neoplasias Pulmonares/diagnóstico , Macrófagos/patologia , Células Neoplásicas Circulantes/patologia , Tomografia Computadorizada por Raios X/métodosRESUMO
Severe immune-related adverse events (irAEs) occur in up to 60% of patients with melanoma treated with immune checkpoint inhibitors (ICIs). However, it is unknown whether a common baseline immunological state precedes irAE development. Here we applied mass cytometry by time of flight, single-cell RNA sequencing, single-cell V(D)J sequencing, bulk RNA sequencing and bulk T cell receptor (TCR) sequencing to study peripheral blood samples from patients with melanoma treated with anti-PD-1 monotherapy or anti-PD-1 and anti-CTLA-4 combination ICIs. By analyzing 93 pre- and early on-ICI blood samples and 3 patient cohorts (n = 27, 26 and 18), we found that 2 pretreatment factors in circulation-activated CD4 memory T cell abundance and TCR diversity-are associated with severe irAE development regardless of organ system involvement. We also explored on-treatment changes in TCR clonality among patients receiving combination therapy and linked our findings to the severity and timing of irAE onset. These results demonstrate circulating T cell characteristics associated with ICI-induced toxicity, with implications for improved diagnostics and clinical management.
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Inibidores de Checkpoint Imunológico , Melanoma , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Melanoma/tratamento farmacológico , Estudos Retrospectivos , Linfócitos TRESUMO
The detection of circulating tumor DNA via liquid biopsy has become an important diagnostic test for patients with cancer. While certain commercial liquid biopsy platforms designed to detect circulating tumor DNA have been approved to guide clinical decisions in advanced solid tumors, the clinical utility of these assays for detecting minimal residual disease after curative-intent treatment of nonmetastatic disease is currently limited. Predicting disease response and relapse has considerable potential for increasing the effective implementation of neoadjuvant and adjuvant therapies. As a result, many companies are rapidly investing in the development of liquid biopsy platforms to detect circulating tumor DNA in the minimal residual disease setting. In this review, we discuss the development and clinical implementation of commercial liquid biopsy platforms for circulating tumor DNA minimal residual disease detection of solid tumors. Here, we aim to highlight the technological features that enable highly sensitive detection of tumor-derived genomic alterations, the factors that differentiate these commercial platforms, and the ongoing trials that seek to increase clinical implementation of liquid biopsies using circulating tumor DNA-based minimal residual disease detection.
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DNA Tumoral Circulante , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Genômica , Humanos , Biópsia Líquida , Recidiva Local de Neoplasia , Neoplasia Residual/diagnóstico , Neoplasia Residual/genéticaRESUMO
We hypothesized that circulating tumor DNA (ctDNA) molecular residual disease (MRD) analysis without prior mutational knowledge could be performed after neoadjuvant chemotherapy to assess oligometastatic colorectal cancer (CRC) treated surgically with curative intent. We also investigated urine as an alternative analyte for ctDNA MRD detection in this nongenitourinary setting. PATIENTS AND METHODS: We applied AVENIO targeted next-generation sequencing to plasma, tumor, and urine samples acquired on the day of curative-intent surgery from 24 prospectively enrolled patients with oligometastatic CRC. Age-related clonal hematopoiesis was accounted for by removing variants also present in white blood cells. Plasma and urine ctDNA MRD were correlated with tumor cells detected in the surgical specimen, and adjuvant treatment strategies were proposed based on ctDNA-inferred tumor mutational burden (iTMB) and targetable alterations. RESULTS: Seventy-one percent of patients were treated with neoadjuvant chemotherapy. Tumor-naive plasma ctDNA analysis detected MRD at a median level of 0.62% with 95% sensitivity and 100% specificity, and 94% and 77% sensitivity when only considering patients treated with neoadjuvant chemotherapy and putative driver mutations, respectively. In urine, ctDNA MRD detection specificity remained high at 100%, but sensitivity decreased to 64% with median levels being 11-fold lower than in plasma (P < .0001). Personalized ctDNA MRD oncogenomic analysis revealed 81% of patients might have been candidates for adjuvant immunotherapy based on high iTMB or targeted therapy based on actionable PIK3CA mutations. CONCLUSION: Tumor-naive plasma ctDNA analysis can sensitively and specifically detect MRD in patients with oligometastatic CRC after neoadjuvant chemotherapy. Urine-based ctDNA MRD detection is also feasible; however, it is less sensitive than plasma because of significantly lower levels. Oligometastatic patients with detectable MRD may benefit from additional personalized treatment based on ctDNA-derived oncogenomic profiling.
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DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/urina , Neoplasia Residual/sangue , Neoplasia Residual/genética , Neoplasias Colorretais/tratamento farmacológico , Correlação de Dados , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante , Metástase NeoplásicaRESUMO
Efforts to refine the SAR of the piperazinyl-glutamate-pyridines for more potent analogs with improved pharmacokinetic profiles are described. Exploring substituted piperidines and other ring systems at the 4-pyridyl position led to compounds with improved potency and pharmacokinetic properties over candidate I. In particular, compounds 4t and 5t were discovered with a 10-fold improvement over potency and improved pharmacokinetic profiles in both the rat and dog.
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Fibrinolíticos/farmacologia , Ácido Glutâmico/síntese química , Piperidinas/síntese química , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Piridinas/síntese química , Piridinas/farmacologia , Administração Oral , Animais , Células CHO , Cricetinae , Cricetulus , Cães , Fibrinolíticos/síntese química , Fibrinolíticos/química , Ácido Glutâmico/química , Ácido Glutâmico/farmacologia , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Piperidinas/química , Piperidinas/farmacologia , Piridinas/química , Ratos , Receptores Purinérgicos P2Y12 , Relação Estrutura-AtividadeRESUMO
PURPOSE: Cell-free DNA (cfDNA) and circulating tumor cell (CTC) based liquid biopsies have emerged as potential tools to predict responses to androgen receptor (AR)-directed therapy in metastatic prostate cancer. However, due to complex mechanisms and incomplete understanding of genomic events involved in metastatic prostate cancer resistance, current assays (e.g. CTC AR-V7) demonstrate low sensitivity and remain underutilized. The recent discovery of AR enhancer amplification in >80% of metastatic patients and its association with disease resistance presents an opportunity to improve upon current assays. We hypothesized that tracking AR/enhancer genomic alterations in plasma cfDNA would detect resistance with high sensitivity and specificity. METHODS: We developed a targeted sequencing and analysis method as part of a new assay called Enhancer and neighboring loci of Androgen Receptor Sequencing (EnhanceAR-Seq). We applied EnhanceAR-Seq to plasma collected from 40 patients with metastatic prostate cancer treated with AR-directed therapy to monitor AR/enhancer genomic alterations and correlate these events with therapy resistance, progression-free survival (PFS) and overall survival (OS). RESULTS: EnhanceAR-Seq identified genomic alterations in the AR/enhancer locus in 45% of cases, including a 40% rate of AR enhancer amplification. Patients with AR/enhancer alterations had significantly worse PFS and OS than those without (6-month PFS: 30% vs. 71%, P=0.0002; 6-month OS: 59% vs. 100%, P=0.0015). AR/enhancer alterations in plasma cfDNA detected 18 of 23 resistant cases (78%) and outperformed the CTC AR-V7 assay which was also run on a subset of patients. CONCLUSION: cfDNA-based AR locus alterations, including of the enhancer, are strongly associated with resistance to AR-directed therapy and significantly worse survival. cfDNA analysis using EnhanceAR-Seq may enable more precise risk stratification and personalized therapeutic approaches for metastatic prostate cancer.
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Circulating tumor DNA (ctDNA) is a component of cell-free DNA that is shed by malignant tumors into the bloodstream and other bodily fluids. Levels of ctDNA are typically low, particularly in patients with localized disease, requiring highly sophisticated methods for detection and quantification. Multiple liquid biopsy methods have been developed for ctDNA analysis in solid tumor malignancies and are now enabling detection and assessment of earlier stages of disease, post-treatment molecular residual disease (MRD), resistance to targeted systemic therapy, and tumor mutational burden. Understanding ctDNA biology, mechanisms of release, and clearance and size characteristics, in conjunction with the application of molecular barcoding and targeted error correction, have increased the sensitivity and specificity of ctDNA detection techniques. Combinatorial approaches including integration of ctDNA data with circulating protein biomarkers may further improve assay sensitivity and broaden the scope of ctDNA applications. Circulating viral DNA may be utilized to monitor disease in some virally induced malignancies. In spite of increasingly accurate methods of ctDNA detection, results need to be interpreted with caution given that somatic mosaicisms such as clonal hematopoiesis of indeterminate potential (CHIP) may give rise to genetic variants in the bloodstream unrelated to solid tumors, and the limited concordance observed between different commercial platforms. Overall, highly precise ctDNA detection and quantification methods have the potential to transform clinical practice via non-invasive monitoring of solid tumor malignancies, residual disease detection at earlier timepoints than standard clinical and/or imaging surveillance, and treatment personalization based on real-time assessment of the tumor genomic landscape.
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Biomarcadores Tumorais , DNA Tumoral Circulante , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , Neoplasias/diagnóstico , Neoplasias/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Biópsia Líquida , Técnicas de Diagnóstico Molecular , Neoplasia Residual/sangue , Neoplasias/sangueRESUMO
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Assuntos
Desenho de Fármacos , Agonismo Inverso de Drogas , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/agonistas , Piridinas/administração & dosagem , Piridinas/farmacologia , Administração Oral , Animais , Disponibilidade Biológica , Avaliação Pré-Clínica de Medicamentos , Humanos , Camundongos , Piridinas/farmacocinética , Células Th17/efeitos dos fármacos , Células Th17/metabolismoRESUMO
Upregulation of the HGF and MSP growth-factor processing serine endopeptidases HGFA, matriptase and hepsin is correlated with increased metastasis in multiple tumor types driven by c-MET or RON kinase signaling. We rationally designed P1' α-ketobenzothiazole mechanism-based inhibitors of these proteases. Structure-activity studies are presented, which resulted in the identification of potent inhibitors with differential selectivity. The tetrapeptide inhibitors span the P1-P1' substrate cleavage site via a P1' amide linker off the benzothiazole, occupying the S3' pocket. Optimized inhibitors display sub-nanomolar enzyme inhibition against one, two, or all three of HGFA, matriptase, and hepsin. Several compounds also have good selectivity against the related trypsin-like proteases, thrombin and Factor Xa. Finally, we show that inhibitors block the fibroblast (HGF)-mediated migration of invasive DU145 prostate cancer cells. In addition to prostate cancer, breast, colon, lung, pancreas, gliomas, and multiple myeloma tumors all depend on HGF and MSP for tumor survival and progression. Therefore, these unique inhibitors have potential as new therapeutics for a diverse set of tumor types.
Assuntos
Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Oligopeptídeos/farmacologia , Inibidores de Serina Proteinase/farmacologia , Antineoplásicos/síntese química , Benzotiazóis/síntese química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Fator Xa/metabolismo , Humanos , Modelos Moleculares , Oligopeptídeos/síntese química , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/síntese química , Transdução de Sinais , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
The p68 DEAD box helicases comprise a widely conserved protein family involved in a large range of biological processes including transcription, splicing and translation. The genome of the ciliate Tetrahymena thermophile encodes two p68-like helicases, Drh1p and Lia2p. We show that DRH1 is essential for growth and completion of development. In growing cells, Drh1p is excluded from the nucleus and accumulates near cortical basal bodies. In contrast, during sexual reproduction, this protein localizes to meiotic micronuclei, initially in punctate foci in regions where centromeres and telomeres are known to reside and later in post-zygotic differentiating somatic macronuclei. Differentiation of the macronuclear genome involves extensive DNA rearrangements including fragmentation of the five pairs of germline-derived chromosomes into 180 chromosomal sub-fragments that are stabilized by de novo telomere deletion. In addition, thousands of internal eliminated sequences (IESs) are excised from loci dispersed throughout the genome. Strains with DRH1 deleted from the germline nuclei, which do not express the protein during post-zygotic development, fail to fragment the developing macronuclear chromosomes. IES excision still occurs in the absence of DRH1 zygotic expression; thus, Drh1p is the first protein found to be specifically required for chromosome breakage but not DNA elimination.