RESUMO
BACKGROUND: In Lung adenocarcinoma (LUAD), targeted therapies and immunotherapies have moved from metastatic to early stage and stratification of the relapse risk becomes mandatory. Here we identified a miR-200 based RNA signature that delineates Epithelial-to-mesenchymal transition (EMT) heterogeneity and predicts survival beyond current classification systems. METHODS: A miR-200 signature was identified using RNA sequencing. We scored the miR-200 signature by WISP (Weighted In Silico Pathology), used GSEA to identify pathway enrichments and MCP-counter to characterize immune cell infiltrates. We evaluate the clinical value of this signature in our series of LUAD and using TCGA and 7 published datasets. RESULTS: We identified 3 clusters based on supervised classification: I is miR-200-sign-down and enriched in TP53 mutations IIA and IIB are miR-200-sign-up: IIA is enriched in EGFR (p < 0.001), IIB is enriched in KRAS mutation (p < 0.001). WISP stratified patients into miR-200-sign-down (n = 65) and miR-200-sign-up (n = 42). Several biological processes were enriched in MiR-200-sign-down tumors, focal adhesion, actin cytoskeleton, cytokine/receptor interaction, TP53 signaling and cell cycle pathways. Fibroblast, immune cell infiltration and PDL1 expression were also significantly higher suggesting immune exhaustion. This signature stratified patients into high-vs low-risk groups, miR-200-sign-up had higher DFS, median not reached at 60 vs 41 months and within subpopulations with stage I, IA, IB, or II. Results were validated on TCGA data on 7 public datasets. CONCLUSION: This EMT and miR-200-related prognostic signature refines prognosis evaluation independently of tumor stage and paves the way towards assessing the predictive value of this LUAD clustering to optimize perioperative treatment.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , MicroRNAs , Humanos , Transcriptoma/genética , Neoplasias Pulmonares/patologia , Adenocarcinoma de Pulmão/patologia , Prognóstico , Microambiente Tumoral/genética , MicroRNAs/genética , RecidivaRESUMO
BACKGROUND: No circulating biomarker is available for endometrial carcinoma (EC). We aimed to identify DNA positions universally hypermethylated in EC, and to develop a digital droplet PCR (ddPCR) assay for detection of hypermethylated circulating tumor DNA (meth-ctDNA) in plasma from patients with EC. METHODS: DNA positions hypermethylated in EC, and without unspecific hypermethylation in tissue/cell types releasing circulating cell-free DNA in plasma, were identified in silico from TCGA/Gene Expression Omnibus (GEO) data. A methylation-specific ddPCR (meth-ddPCR) assay following bisulfite conversion of DNA extracted from plasma was optimized for detection of meth-ctDNA according to dMIQE guidelines. Performances were validated on a retrospective cohort (n = 78 tumors, n = 30 tumor-adjacent tissues), a prospective pilot cohort (n = 33 stage I-IV patients), and 55 patients/donors without cancer. RESULTS: Hypermethylation of zinc finger and SCAN domain containing 12 (ZSCAN12) and/or oxytocin (OXT) classified EC samples from multiple noncancer samples with high diagnostic specificity/sensitivity [>97%; area under the curve (AUC) = 0.99; TCGA/GEO tissues/blood samples]. These results were confirmed in the independent retrospective cohort (AUC = 0.99). Meth-ddPCR showed a high analytical specificity (limit of blank = 2) and sensitivity (absolute lower threshold of detection = 50 pgmethDNA/mLplasma). In the pilot cohort, meth-ctDNA was detected in pretreatment plasma samples from 9/11 and 5/20 patients with advanced and non-advanced EC, respectively. 2 of 9 patients had ctDNA detected after macroscopic complete surgery and experienced progression within 6 months. No healthy donors had any copy of hypermethylated DNA detected in plasma. CONCLUSIONS: Meth-ddPCR of ZSCAN12/OXT allows a highly specific and sensitive detection of ctDNA in plasma from patients with EC and appears promising for personalized approaches for these patients.
Assuntos
DNA Tumoral Circulante , Neoplasias do Endométrio , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/genética , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Estudos RetrospectivosRESUMO
BACKGROUND: Patients with non-small cell lung cancer (NSCLC) receiving curative surgery have a risk of relapse, and adjuvant treatments only translate into a 5% increase in 5-year survival. We assessed the clinical significance of epithelial-mesenchymal transition (EMT) and explored its association with the [SNAIL/miR-34]:[ZEB/miR-200] regulation hub to refine prognostic information. METHODS: We validated a 7-gene EMT score using a consecutive series of 176 resected NSCLC. We quantified EMT transcription factors, microRNAs (miRs) of the miR-200, miR-34 families and miR-200 promoter hypermethylation to identify outcome predictors. RESULTS: Most tumours presented with an EMT-hybrid state and the EMT score was not predictive of outcome. Individually, all miR-200 were inversely associated with the EMT score, but only chromosome-1 miRs, miR-200a, b, 429, were associated with disease-free survival (p = 0.08, 0.05 and 0.025) and overall survival (p = 0.013, 0.003 and 0.006). We validated these associations on The Cancer Genome Atlas data. Tumour unsupervised clustering based on miR expression identified two good prognostic groups, unrelated to the EMT score, suggesting that miR profiling may have an important clinical value. CONCLUSION: miR-200 family members do not have similar predictive value. Core EMT-miR, regulators and not EMT itself, identify NSCLC patients with a low risk of relapse after surgery.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , MicroRNAs/genética , Fatores de Transcrição da Família Snail/genéticaRESUMO
In metastatic colorectal cancer (mCRC), circulating tumor DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. Patients with a RAS mutated tumor included in the prospective PLACOL study were monitored for ctDNA. Analyses were based on optimized targeted next-generation sequencing and/or droplet-based digital polymerase chain reaction (ddPCR). For plasma samples without detectable mutations at progression disease, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. Among the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only two out of the eight patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only 2 samples among 36 were confirmed for clearance of RAS mutation in our series. In conclusion, this study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored.
Assuntos
Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/análise , Neoplasias Colorretais/sangue , Neoplasias Colorretais/tratamento farmacológico , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Reação em Cadeia da Polimerase , Estudos ProspectivosRESUMO
We describe a technology to perform sizing and concentration analysis of double stranded DNA with a sensitivity of 10 fg/µL in an operating time of 20 min. The technology is operated automatically on a commercial capillary electrophoresis instrument using electro-hydrodynamic actuation. It relies on a new capillary device that achieves online concentration of DNA at the junction between two capillaries of different diameters, thanks to viscoelastic lift forces. Using a set of DNA ladders in the range of 100-1500 bp, we report a sizing accuracy and precision better than 3% and a concentration quantification precision of â¼20%. When the technology is applied to the analysis of clinical samples of circulating cell-free DNA (cfDNA), the measured cfDNA concentrations are in good correlation with those measured by digital PCR. Furthermore, the cfDNA size profiles indicate that the fraction of low molecular weight cfDNA in the range of 75-240 bp is a candidate biomarker to discriminate between healthy subjects and cancer patients. We conclude that our technology is efficient in analyzing highly diluted DNA samples and suggest that it will be helpful in translational and clinical research involving cfDNA.
Assuntos
Ácidos Nucleicos Livres/sangue , Eletroforese Capilar/instrumentação , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/sangue , Ácidos Nucleicos Livres/análise , Desenho de Equipamento , Humanos , Hidrodinâmica , Limite de Detecção , Neoplasias/sangue , Neoplasias/diagnóstico , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) is an approved noninvasive biomarker to test for the presence of EGFR mutations at diagnosis or recurrence of lung cancer. However, studies evaluating ctDNA as a noninvasive "real-time" biomarker to provide prognostic and predictive information in treatment monitoring have given inconsistent results, mainly due to methodological differences. We have recently validated a next-generation sequencing (NGS) approach to detect ctDNA. Using this new approach, we evaluated the clinical usefulness of ctDNA monitoring in a prospective observational series of patients with non-small cell lung cancer (NSCLC). METHODS AND FINDINGS: We recruited 124 patients with newly diagnosed advanced NSCLC for ctDNA monitoring. The primary objective was to analyze the prognostic value of baseline ctDNA on overall survival. ctDNA was assessed by ultra-deep targeted NGS using our dedicated variant caller algorithm. Common mutations were validated by digital PCR. Out of the 109 patients with at least one follow-up marker mutation, plasma samples were contributive at baseline (n = 105), at first evaluation (n = 85), and at tumor progression (n = 66). We found that the presence of ctDNA at baseline was an independent marker of poor prognosis, with a median overall survival of 13.6 versus 21.5 mo (adjusted hazard ratio [HR] 1.82, 95% CI 1.01-3.55, p = 0.045) and a median progression-free survival of 4.9 versus 10.4 mo (adjusted HR 2.14, 95% CI 1.30-3.67, p = 0.002). It was also related to the presence of bone and liver metastasis. At first evaluation (E1) after treatment initiation, residual ctDNA was an early predictor of treatment benefit as judged by best radiological response and progression-free survival. Finally, negative ctDNA at E1 was associated with overall survival independently of Response Evaluation Criteria in Solid Tumors (RECIST) (HR 3.27, 95% CI 1.66-6.40, p < 0.001). Study population heterogeneity, over-representation of EGFR-mutated patients, and heterogeneous treatment types might limit the conclusions of this study, which require future validation in independent populations. CONCLUSIONS: In this study of patients with newly diagnosed NSCLC, we found that ctDNA detection using targeted NGS was associated with poor prognosis. The heterogeneity of lung cancer molecular alterations, particularly at time of progression, impairs the ability of individual gene testing to accurately detect ctDNA in unselected patients. Further investigations are needed to evaluate the clinical impact of earlier evaluation times at 1 or 2 wk. Supporting clinical decisions, such as early treatment switching based on ctDNA positivity at first evaluation, will require dedicated interventional studies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , DNA de Neoplasias/genética , Neoplasias Pulmonares/genética , Mutação , Células Neoplásicas Circulantes/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/genética , DNA de Neoplasias/metabolismo , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos ProspectivosRESUMO
BACKGROUND: Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing. METHODS: We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer. RESULTS: Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90). CONCLUSIONS: Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.
Assuntos
DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Pulmonares/genética , Mutação , Neoplasias Pancreáticas/genética , Análise de Sequência de DNA , Alelos , Humanos , Neoplasias Pulmonares/diagnóstico , Mutagênese Insercional , Neoplasias Pancreáticas/diagnóstico , Polimorfismo de Nucleotídeo Único/genética , Deleção de SequênciaRESUMO
BACKGROUND: Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS: We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS: Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS: These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Biomarcadores Tumorais , Neoplasias Colorretais/genética , Metilação de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/química , Neuropeptídeo Y/genética , Proteínas Repressoras/genética , Idoso , Biomarcadores Tumorais/genética , Metilação de DNA/genética , DNA de Neoplasias/genética , Feminino , Humanos , Masculino , Reação em Cadeia da PolimeraseRESUMO
Patients with EGFR-mutated non-small cell lung cancer (NSCLC) benefit from treatment with tyrosine kinase inhibitors (TKI) targeting EGFR. Despite improvements in patient care, especially with the 3rd generation TKI osimertinib, disease relapse is observed in all patients. Among the various processes involved in TKI resistance, epithelial-to-mesenchymal transition (EMT) is far from being fully characterized. We hypothesized that the cellular prion protein PrPC could be involved in EMT and EGFR-TKI resistance in NSCLC. Using 5 independent lung adenocarcinoma datasets, including our own cohort, we document that the expression of the PRNP gene encoding PrPC is associated with EMT. By manipulating the levels of PrPC in different EGFR-mutated NSCLC cell lines, we firmly establish that the expression of PrPC is mandatory for cells to maintain or acquire a mesenchymal phenotype. Mechanistically, we show that PrPC operates through an ILK-RBPJ cascade, which also controls the expression of EGFR. Our data further demonstrate that PrPC levels are elevated in EGFR-mutated versus wild-type tumours or upon EGFR activation in vitro. In addition, we provide evidence that PRNP levels increase with TKI resistance and that reducing PRNP expression sensitizes cells to osimertinib. Finally, we found that plasma PrPC levels are increased in EGFR-mutated NSCLC patients from 2 independent cohorts and that their longitudinal evolution mirrors that of disease. Altogether, these findings define PrPC as a candidate driver of EMT-dependent resistance to EGFR-TKI in NSCLC. They further suggest that monitoring plasma PrPC levels may represent a valuable non-invasive strategy for patient follow-up and warrant considering PrPC-targeted therapies for EGFR-mutated NSCLC patients with TKI failure.
RESUMO
In eukaryotic cells, DNA is packaged into chromatin and this compact storage in the nucleus promotes transcriptional repression of genes. Chromatin remodeling complexes such as the SWI/SNF complex are involved in making DNA accessible to transcription factors and thereby are implicated in the regulation of gene expression. Mutations and altered expression of chromatin remodeling complex genes have been described in cancer cells. Indeed, non-small cell lung cancer cell lines have been shown to harbor mutations in SMARCA4 (BRG1), a member of the SWI/SNF complex, but evidence has been less clear in primary tumors. Recently, inactivating mutations in AT-rich interaction domain 2 (ARID2) were found in liver cancer related to HCV infection and in melanoma. Here, we explored, using a genome-wide strategy and subsequent sequencing of targeted genes, whether chromatin remodeling is implicated in primary lung adenocarcinoma. Two genes were individualized from the genome screening as homozygously deleted in a set of samples: JARID2 and ARID2. Subsequent analysis of the entire coding sequences showed that ARID2 loss-of-function mutations were found in 5% of nonsmall cell lung cancers, thereby constituting one of the most frequently mutated genes in this cancer type after TP53, KRAS, EGFR, CDKN2A and STK11.
Assuntos
Adenocarcinoma Bronquioloalveolar/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Grandes/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Mutação/genética , Fatores de Transcrição/genética , Adenocarcinoma Bronquioloalveolar/patologia , Adulto , Idoso , Sequência de Bases , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Montagem e Desmontagem da Cromatina , DNA de Neoplasias/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Complexo Repressor Polycomb 2/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA. METHODS: Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues. RESULTS: One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants. CONCLUSIONS: The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.
Assuntos
Fragmentação do DNA , DNA de Neoplasias/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Análise de Sequência de DNA/métodos , Genoma Humano , Humanos , Neoplasias Pulmonares/química , Reprodutibilidade dos TestesRESUMO
BACKGROUND: The development of targeted therapies has created a need for robust molecular characterization of cancer and it has become a challenge to validate methods to ensure accuracy in tumor mutation testing. METHODS: The current study was designed to evaluate KRAS, BRAF and EGFR genotyping by Competitive Allele Specific hydrolysis probes (TaqMan) PCR technology (CAST), on suboptimal formalin fixed paraffin embedded (FFPE) tumor samples. Assays were calibrated on FFPE samples and a minimal quantification cycle (Cq) cut-off was determined to standardize analyses and avoid over-interpretation of degraded material. Sensibility, specificity and blinded clinical sample screenings (n=63) were evaluated. RESULTS: CAST PCR allowed efficient amplification of FFPE samples, probes were highly specific and all assays had a sensibility inferior to 1% except for the EGFR p.T790M assay. 60/63 samples were correctly typed. The three missed mutations were EGFR exon 19 deletions that were not recognized by the DEL19 assays that were used. CONCLUSIONS: This technology is less laborious and prevent crossover of PCR products as compared to multistep methods. TaqMan® Mutation Detection assay is an important technology to consider in the field of mutation detection for KRAS, BRAF and EGFR point mutation screening. Assay calibration on FFPE samples may prevent erroneous interpretations that will ultimately harm clinical oncology practice.
Assuntos
Receptores ErbB/genética , Mutação , Neoplasias/genética , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas ras/genética , Linhagem Celular Tumoral , Sondas de DNA/genética , Formaldeído , Genótipo , Técnicas de Genotipagem/métodos , Células HT29 , Humanos , Neoplasias/patologia , Inclusão em Parafina , Reprodutibilidade dos Testes , Fixação de TecidosRESUMO
Gastrointestinal (GI) cancers are major health burdens worldwide and biomarkers are needed to improve the management of these diseases along their evolution. Circulating tumor DNA (ctDNA) is a promising non-invasive blood and other bodily-fluid-based biomarker in cancer management that can help clinicians in various cases for the detection, diagnosis, prognosis, monitoring and personalization of treatment in digestive oncology. In addition to the well-studied prognostic role of ctDNA, the main real-world applications appear to be the assessment of minimal residual disease to further guide adjuvant therapy and predict relapse, but also the monitoring of clonal evolution to tailor treatments in metastatic setting. Other challenges such as predicting response to treatment including immune checkpoint inhibitors could also be among the potential applications of ctDNA. Although the level of advancement of ctDNA development in the different tumor localizations is still inhomogeneous, it might be now reliable enough to be soon used in clinical routine for colorectal cancers and shows promising results in other GI cancers.
RESUMO
The CMS4 mesenchymal subtype of colorectal cancer (CRC) is associated with poor prognosis and resistance to treatment. The cellular prion protein PrPC is overexpressed in CMS4 tumors and controls the expression of a panel of CMS4-specific genes in CRC cell lines. Here, we sought to investigate PrPC downstream pathways that may underlie its role in CMS4 CRC. By combining gene set enrichment analyses and gain and loss of function approaches in CRC cell lines, we identify the integrin-linked kinase ILK as a proximal effector of PrPC that mediates its control on the CMS4 phenotype. We further leveraged three independent large CRC cohorts to assess correlations in gene expression pattern with patient outcomes and found that ILK is overexpressed in CMS4 mesenchymal tumors and confers a poor prognosis, especially when combined with high expression of the PrPC encoding gene PRNP. Of note, we discovered that the PrPC-ILK signaling axis controls the expression and activity of the tryptophan metabolizing enzyme indoleamine 2,3 dioxygenase IDO1, a key player in immune tolerance. In addition, we monitored alterations in the levels of tryptophan and its metabolites of the kynurenine pathway in the plasma of metastatic CRC patients (n = 325) and we highlight their prognostic value in combination with plasma PrPC levels. Thus, the PrPC-ILK-IDO1 axis plays a key role in the mesenchymal subtype of CRC. PrPC and IDO1-targeted strategies may represent new avenues for patient stratification and treatment in CRC.
Assuntos
Neoplasias Colorretais , Neoplasias Colorretais/diagnóstico , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase , Proteínas Priônicas , Prognóstico , Proteínas Serina-Treonina QuinasesRESUMO
BACKGROUND: In non-metastatic colorectal cancer (CRC), we evaluated prospectively the pertinence of longitudinal detection and quantification of circulating tumor DNA (ctDNA) as a prognostic marker of recurrence. METHOD: The presence of ctDNA was assessed from plasma collected before and after surgery for 184 patients classified as stage II or III and at each visit during 3-4 years of follow-up. The ctDNA analysis was performed by droplet-based digital polymerase chain reaction, targeting mutation and methylation markers, blindly from the clinical outcomes. Multivariate analyses were adjusted on age, gender, stage, and adjuvant chemotherapy. RESULTS: Before surgery, 27.5% of patients were positive for ctDNA detection. The rate of recurrence was 32.7% and 11.6% in patients with or without detectable ctDNA respectively (P = 0.001). Time to recurrence (TTR) was significantly shorter in patients with detectable ctDNA before (adjusted hazard ratio [HR] = 3.58, 95% confidence interval [CI] 1.71-7.47) or immediately after surgery (adjusted HR = 3.22, 95% CI 1.32-7.89). The TTR was significantly shorter in patients with detectable ctDNA during the early postoperative follow-up (1-6 months) (adjusted HR = 5, 95% CI 1.9-12.9). Beyond this period, ctDNA remained a prognostic marker with a median anticipated diagnosis of recurrence of 13.1 weeks (interquartile range 28 weeks) when compared to imaging follow-up. The rate of ctDNA+ might be underestimated knowing that consensus pre-analytical conditions were not described at initiation of the study. CONCLUSION: This prospective study confirms the relevance of ctDNA as a recurrence risk factor in stage II and III CRC before surgery and as a marker of minimal residual disease after surgery that may predict recurrence several months before imaging techniques.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Recidiva Local de Neoplasia/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Prognóstico , Estudos ProspectivosRESUMO
Background: Cellular-cell free-DNA (ccfDNA) is being explored as a diagnostic and prognostic tool for various diseases including cancer. Beyond the evaluation of the ccfDNA mutational status, its fragmentation has been investigated as a potential cancer biomarker in several studies. However, probably due to a lack of standardized procedures dedicated to preanalytical and analytical processing of plasma samples, contradictory results have been published. Methods: ddPCR assays allowing the detection of KRAS wild-type and mutated sequences (KRAS p.G12V, pG12D, and pG13D) were designed to target different fragments sizes. Once validated on fragmented and non-fragmented DNA extracted from cancer cell lines, these assays were used to investigate the influence of the extraction methods on the non-mutated and mutated ccfDNA integrity reflected by the DNA integrity index (DII). The DII was then analyzed in two prospective cohorts of metastatic colorectal cancer patients (RASANC study n = 34; PLACOL study n = 12) and healthy subjects (n = 49). Results and Discussion: Our results demonstrate that ccfDNA is highly fragmented in mCRC patients compared with healthy individuals. These results strongly suggest that the characterization of ccfDNA integrity hold great promise toward the development of a universal biomarker for the follow-up of mCRC patients. Furthermore, they support the importance of standardization of sample handling and processing in such analysis.
RESUMO
Immune checkpoint inhibitors (ICIs) have demonstrated to be highly efficient in treating solid tumors; however, many patients have limited benefits in terms of response and survival. This rapidly led to the investigation of combination therapies to enhance response rates. Moreover, predictive biomarkers were assessed to better select patients. Although PD-L1 expression remains the only validated marker in clinics, molecular profiling has brought valuable information, showing that the tumor mutation load and microsatellite instability (MSI) status were associated to higher response rates in nearly all cancer types. Moreover, in lung cancer, EGFR and MET mutations, oncogene fusions or STK11 inactivating mutations were associated with low response rates. Cancer progression towards invasive phenotypes that impede immune surveillance relies on complex regulatory networks and cell interactions within the tumor microenvironment. Epigenetic modifications, such as the alteration of histone patterns, chromatin structure, DNA methylation status at specific promoters and changes in microRNA levels, may alter the cell phenotype and reshape the tumor microenvironment, allowing cells to grow and escape from immune surveillance. The objective of this review is to make an update on the identified epigenetic changes that target immune surveillance and, ultimately, ICI responses, such as histone marks, DNA methylation and miR signatures. Translational studies or clinical trials, when available, and potential epigenetic biomarkers will be discussed as perspectives in the context of combination treatment strategies to enhance ICI responses in patients with solid tumors.
Assuntos
Epigênese Genética/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias/terapia , Animais , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Imunoterapia/métodos , MicroRNAs/genética , Neoplasias/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
Proteostasis imbalance is emerging as a major hallmark of cancer, driving tumor growth and aggressiveness. Endoplasmic Reticulum (ER) stress has been documented in most major cancers, and the ability to tolerate persistent ER stress through an effective unfolded protein response enhances cancer cell survival, angiogenesis, metastasis, drug resistance and immunosuppression. The ER stress sensor IRE1α contributes to tumor progression through XBP1 mRNA splicing and regulated IRE1α-dependent decay of mRNA and miRNA. The aim of this study was to perform a molecular characterization of series of tumor samples to explore the impact of intratumoral IRE1 signaling in non-small cell lung cancer characteristics. To monitor IRE1 splicing activity, we adopted a fragment length analysis to detect changes in the length of the XBP1 mRNA before and after splicing as a method for measuring sXBP1 mRNA levels in tumors because sXBP1 mRNA is not probed by standard transcriptomic analyses. We demonstrate for the first time that XBP1 splicing is a valuable marker of lung cancer aggressiveness, and our results support a model in which IRE1 downstream signaling could act as a regulator of Epithelial to Mesenchymal Transition (EMT). Our findings study highlights the role of IRE1α downstream signaling in non-small cell lung cancer and opens a conceptual framework to determine how IRE1α endoribonuclease activity shapes the EMT program.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Neoplasias Pulmonares/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Sobrevivência Celular/fisiologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Endorribonucleases/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , Splicing de RNA/fisiologia , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Resposta a Proteínas não Dobradas/fisiologiaRESUMO
Colorectal cancer is a major health burden with a prognosis that has been improved with the progresses in diagnosis and the advance of chemotherapy and personalized medicine. However, because of intra-tumor heterogeneity, clonal evolution and selection, tumors often develop resistance to treatments. "Liquid biopsy" is a minimally invasive method, based on analysis of tumor-specific material in peripheral blood samples of patients. Analysis of tumor specific genetic or epigenetic alterations in cell-free circulating nucleic acids may reflect the molecular heterogeneity of the underlying disease process and serial testing could allow to monitor its temporal genomic changing without using re-biopsy. In this review, we focused on the role of circulating tumor DNA (ctDNA) as a biomarker in the management of patients with colorectal cancer at early and advanced stages. Through recent studies, we described its promising clinical applications for diagnosis, detection of recurrence after surgery and monitoring for tumor response or therapeutic resistance in metastatic setting. Such recent developments offer new perspectives for personalized medicine in colorectal cancer but still needs some standardized detection methods and further studies to validate its use in clinical routine.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Neoplasias Colorretais/sangue , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Humanos , Biópsia LíquidaRESUMO
BACKGROUND: Unicentric mediastinal Castleman disease (CD) is a rare condition, poorly characterized due to the small number of cases and the absence of genomic study. We analyzed clinical, radiological, histological and genomic patterns associated with mediastinal CD in a substantial case series.Methods: We retrospectively reviewed cases of unicentric mediastinal CD managed in 2 French thoracic surgery departments between 1988 and 2012. Clinical, radiological, surgical and pathological data were recorded. On available FFPE blocks we performed mutation screening by next-generation-sequencing, using AmpliSeq™ Cancer Hotspot v2 (Life Technologies) and immunohistochemistry (IHC) (AKT-mTOR pathway). RESULTS: Eleven patients were identified (mean age 41±15 years, sex-ratio 0.8, median follow-up 78 months). Surgical approach was thoracotomy (n=6), sternotomy (n=4), and VATS (n=1). Additional procedures included thymectomy in three cases, mediastinal lymphadenectomy in two cases, and bilobectomy in one case. One patient presented local relapse as a follicular dendritic cell sarcoma, leading to death 48 months after the first resection. Within 9 patients whose FFPE blocks were available, 2 mutations were found: VHL (p.F119L, 35%, n=1) and JAK3 (p.V718L, 53%, n=1). Phospho-AKT and phospho-mTOR stainings were negative in all cases, whereas phospho-S6RP staining was positive in eight cases, mainly in interfollicular cell cytoplasm. CONCLUSIONS: From this series of patients with unicentric mediastinal CD, we observed 2 cases of potential driver mutations and 8 cases of phospho-S6RP activation not related to AKT-mTOR. Larger studies are required to decipher more precisely the molecular abnormalities and potential therapeutic targets underlying this uncommon condition.