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DNA-methylation alterations are common in cancer and display unique characteristics that make them ideal markers for tumor quantification and classification. Here we present MIMESIS, a computational framework exploiting minimal DNA-methylation signatures composed by a few dozen informative DNA-methylation sites to quantify and classify tumor signals in tissue and cell-free DNA samples. Extensive analyses of multiple independent and heterogenous datasets including >7200 samples demonstrate the capability of MIMESIS to provide precise estimations of tumor content and to enable accurate classification of tumor type and molecular subtype. To assess our framework for clinical applications, we designed a MIMESIS-informed assay incorporating the minimal signatures for breast cancer. Using both artificial samples and clinical serial cell-free DNA samples from patients with metastatic breast cancer, we show that our approach provides accurate estimations of tumor content, sensitive detection of tumor signal and the ability to capture clinically relevant molecular subtype in patients' circulation. This study provides evidence that our extremely parsimonious approach can be used to develop cost-effective and highly scalable DNA-methylation assays that could support and facilitate the implementation of precision oncology in clinical practice.
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Neoplasias de la Mama , Ácidos Nucleicos Libres de Células , Humanos , Femenino , Ácidos Nucleicos Libres de Células/genética , Medicina de Precisión , Metilación de ADN , Neoplasias de la Mama/genética , Biomarcadores de Tumor/genética , ADN de Neoplasias/genéticaRESUMEN
BACKGROUND: Circulating tumor cells (CTCs) are prognostic in patients with advanced breast cancer (ABC). However, no data exist about their use in patients treated with palbociclib. We analyzed the prognostic role of CTC counts in patients enrolled in the cTREnd study, a pre-planned translational sub-study of TREnd (NCT02549430), that randomized patients with ABC to palbociclib alone or palbociclib plus the endocrine therapy received in the prior line of treatment. Moreover, we evaluated RB1 gene expression on CTCs and explored its prognostic role within the cTREnd subpopulation. METHODS: Forty-six patients with ER-positive, HER2-negative ABC were analyzed. Blood samples were collected before starting palbociclib treatment (timepoint T0), after the first cycle of treatment (timepoint T1), and at disease progression (timepoint T2). CTCs were isolated and counted by CellSearch® System using the CellSearch™Epithelial Cell kit. Progression-free survival (PFS), clinical benefit (CB) during study treatment, and time to treatment failure (TTF) after study treatment were correlated with CTC counts. Samples with ≥ 5 CTCs were sorted by DEPArray system® (DA). RB1 and GAPDH gene expression levels were measured by ddPCR. RESULTS: All 46 patients were suitable for CTCs analysis. CTC count at T0 did not show significant prognostic value in terms of PFS and CB. Patients with at least one detectable CTC at T1 (n = 26) had a worse PFS than those with 0 CTCs (n = 16) (p = 0.02). At T1, patients with an increase of at least three CTCs showed reduced PFS compared to those with no increase (mPFS = 3 versus 9 months, (p = 0.004). Finally, patients with ≥ 5 CTCs at T2 (n = 6/23) who received chemotherapy as post-study treatment had a shorter TTF (p = 0.02). Gene expression data for RB1 were obtained from 19 patients. CTCs showed heterogeneous RB1 expression. Patients with detectable expression of RB1 at any timepoint showed better, but not statistically significant, outcomes than those with undetectable levels. CONCLUSIONS: CTC count seems to be a promising modality in monitoring palbociclib response. Moreover, CTC count at the time of progression could predict clinical outcome post-palbociclib. RB1 expression analysis on CTCs is feasible and may provide additional prognostic information. Results should be interpreted with caution given the small studied sample size.
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Neoplasias de la Mama/tratamiento farmacológico , Células Neoplásicas Circulantes/patología , Piperazinas/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Recuento de Células , Progresión de la Enfermedad , Femenino , Humanos , Células Neoplásicas Circulantes/efectos de los fármacos , Células Neoplásicas Circulantes/metabolismo , Supervivencia sin Progresión , Receptor ErbB-2/deficiencia , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Proteínas de Unión a Retinoblastoma/metabolismo , Resultado del Tratamiento , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Motivation: Controlling for tumor purity in molecular analyses is essential to allow for reliable genomic aberration calls, for inter-sample comparison and to monitor heterogeneity of cancer cell populations. In genome wide screening studies, the assessment of tumor purity is typically performed by means of computational methods that exploit somatic copy number aberrations. Results: We present a strategy, called Purity Assessment from clonal MEthylation Sites (PAMES), which uses the methylation level of a few dozen, highly clonal, tumor type specific CpG sites to estimate the purity of tumor samples, without the need of a matched benign control. We trained and validated our method in more than 6000 samples from different datasets. Purity estimates by PAMES were highly concordant with other state-of-the-art tools and its evaluation in a cancer cell line dataset highlights its reliability to accurately estimate tumor admixtures. We extended the capability of PAMES to the analysis of CpG islands instead of the more platform-specific CpG sites and demonstrated its accuracy in a set of advanced tumors profiled by high throughput DNA methylation sequencing. These analyses show that PAMES is a valuable tool to assess the purity of tumor samples in the settings of clinical research and diagnostics. Availability and implementation: https://github.com/cgplab/PAMES. Contact: matteo.benelli@uslcentro.toscana.it or f.demichelis@unitn.it. Supplementary information: Supplementary data are available at Bioinformatics online.
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Islas de CpG , Metilación de ADN , Epigenómica/métodos , Neoplasias/genética , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Motivos de NucleótidosRESUMEN
BACKGROUND: New single-cell isolation technologies are facilitating studies on the transcriptomics of individual cells. Bio-Rad ddSEQ is a droplet-based microfluidic system that, when coupled with downstream Illumina library preparation and sequencing, enables the monitoring of thousands of genes per cell. Sequenced reads show unique features that do not permit the use of freely available tools to perform single cell demultiplexing. RESULTS: We present ddSeeker, a tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina experiments. Its application to the Illumina test dataset demonstrates that ddSeeker performs better than Illumina BaseSpace software, enabling a higher recovery of valid reads. We also show its utility in the analysis of an in-house dataset including two read sets characterized by low and high sequencing quality. ddSeeker and its source code are available at https://github.com/cgplab/ddSeeker . CONCLUSIONS: ddSeeker is a freely available tool to perform initial processing and quality metrics of reads generated through Bio-Rad ddSEQ/Illumina single cell transcriptomic experiments.
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ARN/genética , Análisis de Secuencia de ARN , Análisis de la Célula Individual/métodos , Programas Informáticos , Transcriptoma/genética , Algoritmos , Biología Computacional , Perfilación de la Expresión GénicaRESUMEN
PURPOSE: To identify prognostic circulating biomarkers to cyclin-dependent kinase 4 and 6 inhibitors (CDK4/6i), we performed a mutational analysis on circulating tumor DNA (ctDNA) samples from patients included in the TREnd trial, which randomly assigned patients to receive the CDK4/6i palbociclib alone or with the endocrine treatment (ET) to which they had progressed. METHODS: Forty-six patients were enrolled in this substudy. Plasma was collected before treatment (T0), after the first cycle of therapy (T1), and at the time of progression (T2). ctDNA hybridization and capture were performed using the Illumina TruSight Tumor 170 Kit. Acquired mutations were confirmed by digital polymerase chain reaction. Progression-free survival analysis was estimated using the Kaplan-Meier method and compared with the log-rank test. RESULTS: The most frequently mutated genes at T0 were ESR1 (23%), PIK3CA (17%), AR, FGFR2, and TP53 (10%). Mutations in ESR1 at T0 conferred higher risk of progression in the entire population (P = .02) and in patients treated with palbociclib + ET (P = .04). ESR1 mutation effect remained significant after correction for clinical variables (P = .03). PIK3CA mutations at T0 were not prognostic, but higher risk of progression was observed when a broader analysis of PI3K pathway was performed (P = .04). At T2, we observed the emergence of nine new mutations in seven genes. CONCLUSION: Mutations in ESR1 and in PI3K pathway genes at T0 were associated with worse prognosis in palbociclib-treated patients. We describe the emergence of newly acquired mutations in palbociclib-treated patients, which might potentially affect subsequent treatment.
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Neoplasias de la Mama , ADN Tumoral Circulante , Piperazinas , Piridinas , Receptor ErbB-2 , Humanos , Femenino , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , ADN Tumoral Circulante/genética , Fosfatidilinositol 3-Quinasas , Receptores de Estrógenos/genéticaRESUMEN
Despite negative results of clinical trials conducted on the overall population of patients with gastric cancer, PARP inhibitor (PARPi) therapeutic strategy still might represent a window of opportunity for a subpopulation of patients with gastric cancer. An estimated 7% to 12% of gastric cancers exhibit a mutational signature associated with homologous recombination (HR) failure, suggesting that these patients could potentially benefit from PARPis. To analyze responsiveness of gastric cancer to PARPi, we exploited a gastroesophageal adenocarcinoma (GEA) platform of patient-derived xenografts (PDX) and PDX-derived primary cells and selected 10 PDXs with loss-of-function mutations in HR pathway genes. Cell viability assays and preclinical trials showed that olaparib treatment was effective in PDXs harboring BRCA2 germline mutations and somatic inactivation of the second allele. Olaparib responsive tumors were sensitive to oxaliplatin as well. Evaluation of HR deficiency (HRD) and mutational signatures efficiently stratified responder and nonresponder PDXs. A retrospective analysis on 57 patients with GEA showed that BRCA2 inactivating variants were associated with longer progression-free survival upon platinum-based regimens. Five of 7 patients with BRCA2 germline mutations carried the p.K3326* variant, classified as "benign." However, familial history of cancer, the absence of RAD51 foci in tumor cells, and a high HRD score suggest a deleterious effect of this mutation in gastric cancer. In conclusion, PARPis could represent an effective therapeutic option for BRCA2-mutated and/or high HRD score patients with GEA, including patients with familial intestinal gastric cancer. SIGNIFICANCE: PARP inhibition is a potential strategy for treating patients with gastric cancer with mutated BRCA2 or homologous repair deficiency, including patients with familial intestinal gastric cancer, for whom BRCA2 germline testing should be recommended.
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Antineoplásicos , Neoplasias Ováricas , Neoplasias Gástricas , Humanos , Femenino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Mutación de Línea Germinal , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Estudios Retrospectivos , Proteína BRCA1/genética , Proteína BRCA2/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológicoRESUMEN
Differentially DNA methylated regions (DMRs) inform on the role of epigenetic changes in cancer. We present Rocker-meth, a new computational method exploiting a heterogeneous hidden Markov model to detect DMRs across multiple experimental platforms. Through an extensive comparative study, we first demonstrate Rocker-meth excellent performance on synthetic data. Its application to more than 6,000 methylation profiles across 14 tumor types provides a comprehensive catalog of tumor type-specific and shared DMRs, and agnostically identifies cancer-related partially methylated domains (PMD). In depth integrative analysis including orthogonal omics shows the enhanced ability of Rocker-meth in recapitulating known associations, further uncovering the pan-cancer relationship between DNA hypermethylation and transcription factor deregulation depending on the baseline chromatin state. Finally, we demonstrate the utility of the catalog for the study of colorectal cancer single-cell DNA-methylation data.
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Biología Computacional/métodos , Metilación de ADN , Epigénesis Genética , Neoplasias/genética , Humanos , Cadenas de MarkovRESUMEN
AURORA aims to study the processes of relapse in metastatic breast cancer (MBC) by performing multi-omics profiling on paired primary tumors and early-course metastases. Among 381 patients (primary tumor and metastasis pairs: 252 targeted gene sequencing, 152 RNA sequencing, 67 single nucleotide polymorphism arrays), we found a driver role for GATA1 and MEN1 somatic mutations. Metastases were enriched in ESR1, PTEN, CDH1, PIK3CA, and RB1 mutations; MDM4 and MYC amplifications; and ARID1A deletions. An increase in clonality was observed in driver genes such as ERBB2 and RB1. Intrinsic subtype switching occurred in 36% of cases. Luminal A/B to HER2-enriched switching was associated with TP53 and/or PIK3CA mutations. Metastases had lower immune score and increased immune-permissive cells. High tumor mutational burden correlated to shorter time to relapse in HR+/HER2- cancers. ESCAT tier I/II alterations were detected in 51% of patients and matched therapy was used in 7%. Integration of multi-omics analyses in clinical practice could affect treatment strategies in MBC. SIGNIFICANCE: The AURORA program, through the genomic and transcriptomic analyses of matched primary and metastatic samples from 381 patients with breast cancer, coupled with prospectively collected clinical data, identified genomic alterations enriched in metastases and prognostic biomarkers. ESCAT tier I/II alterations were detected in more than half of the patients.This article is highlighted in the In This Issue feature, p. 2659.
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Neoplasias de la Mama , Biomarcadores de Tumor/genética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Detección Precoz del Cáncer , Femenino , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Recurrencia Local de Neoplasia/patología , Proteínas Proto-Oncogénicas/genética , TranscriptomaRESUMEN
Temozolomide is an oral alkylating agent used for treating several cancers including glioblastoma and melanoma. Promising, albeit limited, activity and efficacy of temozolomide have been reported in pretreated patients with metastatic colorectal cancer bearing MGMT promoter methylation. MGMT silencing and proficiency of the mismatch repair system were considered the major predictive biomarkers of sensitivity to temozolomide. Refinement of established biomarkers and integration with those related to alteration in specific DNA-damage response pathways such as base excision repair are promising strategies for selecting metastatic colorectal patients to this old drug with several potential novel applications. Then, mounting preclinical and clinical observations have linked acquired resistance to temozolomide to emergence of alterations in the mismatch repair system. Whilst accounting for tumor cells capability of escaping apoptosis when exposed to temozolomide, inactivation of key mismatch-repair proteins will ultimately lead to increasing tumor mutational burden. This drug-induced mismatch deficient-like phenotype is being exploited in proof-of-concept trials combining temozolomide and immune checkpoint inhibitors in metastatic colorectal cancer.
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Neoplasias Colorrectales , Temozolomida/farmacología , Antineoplásicos/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Reparación de la Incompatibilidad de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Humanos , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteínas Supresoras de Tumor/genéticaRESUMEN
Liquid biopsy based on cell-free DNA (cfDNA) enables non-invasive dynamic assessment of disease status in patients with cancer, both in the early and advanced settings. The analysis of DNA-methylation (DNAm) from cfDNA samples holds great promise due to the intrinsic characteristics of DNAm being more prevalent, pervasive, and cell- and tumor-type specific than genomics, for which established cfDNA assays already exist. Herein, we report on recent advances on experimental strategies for the analysis of DNAm in cfDNA samples. We describe the main steps of DNAm-based analysis workflows, including pre-analytics of cfDNA samples, DNA treatment, assays for DNAm evaluation, and methods for data analysis. We report on protocols, biomolecular techniques, and computational strategies enabling DNAm evaluation in the context of cfDNA analysis, along with practical considerations on input sample requirements and costs. We provide an overview on existing studies exploiting cell-free DNAm biomarkers for the detection and monitoring of cancer in early and advanced settings, for the evaluation of drug resistance, and for the identification of the cell-of-origin of tumors. Finally, we report on DNAm-based tests approved for clinical use and summarize their performance in the context of liquid biopsy.
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Ácidos Nucleicos Libres de Células/genética , Metilación de ADN/genética , Oncología Médica/métodos , Animales , Biología Computacional , Humanos , Biopsia LíquidaRESUMEN
PURPOSE: Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC. EXPERIMENTAL DESIGN: Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor-positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial (n = 46). RESULTS: Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase-correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05). CONCLUSIONS: TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.
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Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Piperazinas/uso terapéutico , Piridinas/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Línea Celular Tumoral , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/sangre , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/sangre , Resistencia a Antineoplásicos , Femenino , Humanos , Persona de Mediana Edad , Metástasis de la Neoplasia , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptores de Estrógenos/metabolismo , Tasa de Supervivencia , Timidina Quinasa/sangre , Cambio de TratamientoRESUMEN
The recent arrival of CDK4/6 inhibitor agents, with an approximate doubling of progression-free survival (PFS) associated with their use in hormone receptor-positive, HER2-negative advanced breast cancer (BC), has radically changed the approach to managing this disease. However, resistance to CDK4/6 inhibitors is considered a near-inevitability in most patients. Mechanisms of resistance to these agents are multifactorial, and research in this field is still evolving. Biomarkers with the ability to identify early resistance, or to predict the likelihood of successful treatment using CDK4/6 inhibitors are yet to be identified, and represent an area of unmet clinical need. Here we present selected mechanisms of resistance to CDK4/6 inhibitors, largely focussing on roles of Rb, cyclin E1, and the PIK3CA pathway, with discussion of associated biomarkers which have been investigated and applied in recent pre-clinical and clinical studies. These biological drivers may furthermore influence clinical treatment strategies adopted beyond CDK4/6 resistance.
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Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).
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MicroARNs/genética , Próstata/citología , ARN Largo no Codificante/genética , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Células Epiteliales/metabolismo , Edición Génica , Humanos , Masculino , Regiones Promotoras Genéticas , Próstata/metabolismo , Ribonucleasa III/metabolismo , TranscriptomaRESUMEN
BACKGROUND: Thymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome. PATIENTS AND METHODS: The Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression. RESULTS: The median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91-5.89) versus 2.57 months (95% CI: 2.04-3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14-4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01-6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker. CONCLUSION: sTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.
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Androstadienos/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Fulvestrant/uso terapéutico , Timidina Quinasa/uso terapéutico , Anciano , Androstadienos/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/complicaciones , Femenino , Fulvestrant/farmacología , Humanos , Metástasis de la Neoplasia , Pronóstico , Timidina Quinasa/farmacologíaRESUMEN
BACKGROUND: Chemotherapy added to anti-HER2 agents (H) is the treatment of choice in patients with HER2+ early breast cancer. However, HER2+ tumours are clinically and biologically heterogeneous, and treatment response varies significantly by hormone receptor (HR) status and molecular subtype. Predictive biomarkers are needed in this context. This study assessed whether an RB-1 loss of function gene signature (RBsig) is predictive of response to neoadjuvant chemotherapy in combination with trastuzumab, lapatinib or both, within the NeoALTTO trial. METHODS: We collected RNA-sequencing data from pretreatment biopsies derived from the NeoALTTO trial. RBsig expression was computed retrospectively and correlated with pathological complete response (pCR) using receiver-operating characteristic (ROC) curves. The RBsig was dichotomised as High/Low in correspondence to the 25th percentile. Reported p values resulted from Fisher's exact test. RESULTS: Of 455 NeoALTTO patients, 244 were eligible for this substudy (HR+ n = 129; HR- n = 115). Overall, pCR rate was significantly higher in patients with RBsig High tumours than those with RBsig Low (35% versus 18% respectively; p = 0.01). The area under the ROC curve (AUC) was 0.60 (95% CI 0.52-0.67). A remarkably low pCR rate of 11% was seen in HR+/RBsig Low patients versus 28% in HR+/RBsig High. CONCLUSIONS: These results indicate RBsig may add valuable information to HER2 and HR expression, which may in turn inform treatment choices. HR+/HER2+/RBsig Low breast cancers exhibited the poorest pathological response following chemotherapy plus H. Accordingly, in such patients, endocrine therapy in combination with H and, possibly, a CDK4/6 inhibitor, may potentially prove to be a more effective treatment.
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CDK4/6 inhibitors represent a new treatment standard for hormone receptor-positive (HR+), HER2-negative advanced breast cancer (BC) patients. Although efficacious, resistance to these agents is universal. Here, we profiled a large panel of HR+ BC cell lines with conditioned resistance to the CDK4/6 inhibitor palbociclib, and analyzed cell cycle-related markers by gene expression profiles (GEP) and western blot (WB). GEP showed high molecular heterogeneity among the models, with E2F targets being significantly enriched both during treatment and at the time of resistance. By both WB and GEP, a common molecular feature at the time of palbociclib resistance was the concomitant overexpression of cyclin E1 and down-regulation of Rb. CCNE1 was the only significantly up-regulated gene among E2F targets at resistance with CCNE1 genomic amplification being observed in two resistant models. Rb was downregulated in all resistant models; a reduction of RB1 copy number was observed in three resistant cell lines. In silico analyses showed that CCNE1/RB1 ratio correlated with palbociclib IC50 in different datasets of both breast and non-breast cancer cell lines, performing better than CCNE1 or RB1 taken separately. Finally, the CCNE1/RB1 ratio was shown to be an adverse prognostic factor in patients with ER+ BC and to be able to discriminate palbociclib-sensitive versus resistant among patients enrolled in the NeoPalAna trial, a neoadjuvant trial testing palbociclib, performing better than CCNE1 or RB1 alone. Our data suggest that the CCNE1/RB1 ratio may be a viable biomarker of palbociclib resistance, warranting further clinical validation.