RESUMO
BACKGROUND: Despite a growing number of publications highlighting the potential impact on the therapy outcome, rare genetic variants (minor allele frequency < 1%) in genes associated to drug adsorption, distribution, metabolism, and elimination are poorly studied. Previously, rare germline DPYD missense variants were shown to identify a subset of fluoropyrimidine-treated patients at high risk for severe toxicity. Here, we investigate the impact of rare genetic variants in a panel of 54 other fluoropyrimidine-related genes on the risk of severe toxicity. METHODS: The coding sequence and untranslated regions of 54 genes related to fluoropyrimidine pharmacokinetics/pharmacodynamics were analyzed by next-generation sequencing in 120 patients developing grade 3-5 toxicity (NCI-CTC vs3.0) and 104 matched controls. Sequence Kernel Association Test (SKAT) analysis was used to select genes with a burden of genetic variants significantly associated with risk of severe toxicity. The statistical association of common and rare genetic variants in selected genes was further investigated. The functional impact of genetic variants was assessed using two different in silico prediction tools (Predict2SNP; ADME Prediction Framework). RESULTS: SKAT analysis highlighted DPYS and PPARD as genes with a genetic mutational burden significantly associated with risk of severe fluoropyrimidine-related toxicity (Bonferroni adjusted P = 0.024 and P = 0.039, respectively). Looking more closely at allele frequency, the burden of rare DPYS variants was significantly higher in patients with toxicity compared with controls (P = 0.047, Mann-Whitney test). Carrying at least one rare DPYS variant was associated with an approximately fourfold higher risk of severe cumulative (OR = 4.08, P = 0.030) and acute (OR = 4.21, P = 0.082) toxicity. The burden of PPARD rare genetic variants was not significantly related to toxicity. Some common variants with predictive value in DPYS and PPARD were also identified: DPYS rs143004875-T and PPARD rs2016520-T variants predicted an increased risk of severe cumulative (P = 0.002 and P = 0.001, respectively) and acute (P = 0.005 and P = 0.0001, respectively) toxicity. CONCLUSION: This work demonstrated that the rare mutational burden of DPYS, a gene strictly cooperating with DPYD in the catabolic pathway of fluoropyrimidines, is a promising pharmacogenetic marker for precision dosing of fluoropyrimidines. Additionally, some common genetic polymorphisms in DPYS and PPARD were identified as promising predictive markers that warrant further investigation.
Assuntos
Fluoruracila , Neoplasias , Humanos , Fluoruracila/efeitos adversos , Antimetabólitos Antineoplásicos/efeitos adversos , Neoplasias/genética , Di-Hidrouracila Desidrogenase (NADP)/genética , Di-Hidrouracila Desidrogenase (NADP)/metabolismo , Frequência do GeneRESUMO
BACKGROUND: The isolation of circulating tumor cells (CTCs) requires rapid processing of the collected blood due to their inherent fragility. The ability to recover CTCs from peripheral blood mononuclear cells (PBMCs) preserved from cancer patients could allow for retrospective analyses or multicenter CTC studies. METHODS: We compared the efficacy of CTC recovery and characterization using cryopreserved PMBCs vs fresh whole blood from patients with non-small cell lung cancer (NSCLC; n = 8) and sarcoma (n = 6). Two epithelial cellular adhesion molecule (EpCAM)-independent strategies for CTC enrichment, based on Parsortix® technology or immunomagnetic depletion of blood cells (AutoMACS®) were tested, followed by DEPArray™ single-cell isolation. Phenotype and genotype, assessed by copy number alterations analysis, were evaluated at a single-cell level. Detection of target mutations in CTC-enriched samples from frozen NSCLC PBMCs was also evaluated by digital PCR (dPCR). RESULTS: The use of cryopreserved PBMCs from cancer patients allowed for the retrospective enumeration of CTCs and their molecular characterization, using both EpCAM-independent strategies that performed equally in capturing CTC. Cells isolated from frozen PBMCs were representative of whole blood-derived CTCs in terms of number, phenotype, and copy number aberration profile/target mutations. Long-term storage (≥3 years) did not affect the efficacy of CTC recovery. Detection of target mutations was also feasible by dPCR in CTC-enriched samples derived from stored PBMCs. CONCLUSIONS: Isolating CTCs from longitudinally collected PBMCs using an unbiased selection strategy can offer a wider range of retrospective genomic/phenotypic analyses to guide patients' personalized therapy, paving the way for sample sharing in multicenter studies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Sarcoma , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Molécula de Adesão da Célula Epitelial/genética , Humanos , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Estudos RetrospectivosRESUMO
The presence in the blood of patients with solid tumors of circulating cells expressing both epithelial and leukocyte markers (dual-positive cells, DPcells), has often been reported, though it has never been investigated in detail. A recent study suggested that DPcells are hybrid cells derived from the fusion of tumor cells with macrophages. Such fusion hybrids acquire macrophage-associated features endowing them with accelerated growth, increased motility, enhanced invasion activity and thus, a higher efficiency in metastasis formation. However, no direct evidence proving the tumor origin of circulating DPcells was provided in patients. Here we contribute a review of literature data on DPcells and on the hybrid theory with the aim of putting the current evidence both in a biological and clinical perspective and to generate new hypotheses on the mechanisms underlying tumor progression. To add further biological and clinical context to our literature review, we also report some preliminary data from our laboratory on the identification of DPcells in several solid tumors and confirmation of their malignant genotype, thus classifying them as DP-CTCs.
Assuntos
Biomarcadores Tumorais , Neoplasias/diagnóstico , Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Suscetibilidade a Doenças , Testes Genéticos/métodos , Humanos , Biópsia Líquida/métodos , Neoplasias/etiologiaRESUMO
In biliary tract cancer (BTC), tissue biopsies to guide treatment are rarely feasible, thus implementing liquid biopsy approaches to improve patient management represents a priority. So far, studies on circulating tumor cells (CTCs) in BTC are insufficient to promote their use in patient clinical management and are limited to EpCAM-enriched CTCs evaluated with the CellSearch. We applied a single-cell protocol allowing identification not only of epithelial CTCs (eCTCs), but also of nonconventional CTCs (ncCTCs) lacking epithelial and leukocyte markers, but presenting aberrant genomes as confirmed by copy number alterations and therefore representing a distinct subpopulation of bona fide CTCs. In 41 blood samples longitudinally collected from 21 patients with advanced-stage BTC, addition of ncCTC to classic eCTC led to a CTC-positivity increase from 19% to 83%. Patients presenting with at least 1 eCTC/10 ml of blood at baseline prior to treatment start had a significantly shorter median disease-specific survival (DSS) compared to those lacking eCTCs (9 months vs. 19 months, p = 0.03 by log-rank test). No differences in DSS were observed according to ncCTC-positivity, conversely, variations in ncCTC counts during, and at the end of treatment, were associated with the RECIST response supporting their role in treatment monitoring. Moreover, in 88 ncCTCs collected at different times during treatment, unsupervised clustering evidenced segregation of cells by patient's best response, allowing identification of genomic regions possibly involved in resistance mechanisms. The presence of ncCTCs beside eCTCs opens the way to exploiting liquid biopsy for optimizing clinical management in BTC.
Assuntos
Neoplasias do Sistema Biliar/diagnóstico , Colangiocarcinoma/diagnóstico , Células Neoplásicas Circulantes/patologia , Análise de Célula Única , Idoso , Neoplasias do Sistema Biliar/sangue , Neoplasias do Sistema Biliar/mortalidade , Neoplasias do Sistema Biliar/terapia , Colangiocarcinoma/sangue , Colangiocarcinoma/mortalidade , Colangiocarcinoma/terapia , Feminino , Seguimentos , Humanos , Estimativa de Kaplan-Meier , Biópsia Líquida/métodos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Critérios de Avaliação de Resposta em Tumores SólidosRESUMO
Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood samples serially collected from 10 patients with metastatic RCC entering the TARIBO trial, we overcame this limitation using the marker-independent Parsortix™ approach for CTC-enrichment coupled with positive and negative selection with the DEPArray™ with single cell recovery and analysis for copy number alterations (CNA) by next generation sequencing NGS. Two CTC subpopulations were identified: epithelial CTC (eCTC) and non-conventional CTC (ncCTC) lacking epithelial and leukocyte markers. With a threshold ≥1CTC/10 mL of blood, the positivity rates were 28% for eCTC, 62% for ncCTCs, and 71% considering both CTC types. In two patients with detectable eCTCs at baseline, progression free survival was less than 5 months. In an index case, hierarchical structure by translational oncology (TRONCO) identified three clones among 14 CTCs collected at progression and at baseline, each containing cells with a 9p21.3loss, a well-known metastasis driving subclonal alteration. CTCs detection in RCC can be increased by marker-independent approaches, and CTC molecular characterization can allow detection of subclonal events possibly related to tumor progression.
Assuntos
Biomarcadores Tumorais , Carcinoma de Células Renais , Deleção Cromossômica , Cromossomos Humanos Par 9/genética , Neoplasias Renais , Células Neoplásicas Circulantes , Análise de Célula Única , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologiaRESUMO
Circulating microRNA (ct-miRNAs) are able to identify patients with differential response to HER2-targeted therapy. However, their dynamics are largely unknown. We assessed 752 miRNAs from 52 NeoALTTO patients with plasma pairs prior and two weeks after trastuzumab. Increased levels of ct-miR-148a-3p and ct-miR-374a-5p were significantly associated with pathological complete response (pCR) (p = 0.008 and 0.048, respectively). At a threshold ≥ the upper limit of the 95%CI of the mean difference, pCR resulted 45% (95%CI 24%-68%), and 44% (95%CI 22%-69%) for ct-miR-148a-3p and ct-miR-374a-5p, respectively. Notably, ct-miR-148a-3p retained its predictive value (OR 3.42, 95%CI 1.23-9.46, p = 0.018) in bivariate analysis along with estrogen receptor status. Combined information from ct-miR-148a-3p and ct-miR140-5p, which we previously reported to identify trastuzumab-responsive patients, resulted in greater predictive capability over each other, with pCR of 54% (95%CI 25%-81%) and 0% (95%CI 0%-31%) in ct-miR-148a/ct-miR-140-5p high/present and low/absent, respectively. GO and KEGG analyses showed common enriched terms between the targets of these ct-miRNAs, including cell metabolism regulation, AMPK and MAPK signaling, and HCC progression. In conclusion, early modulated ct-miR-148-3p may inform on the functional processes underlying treatment response, integrate the information from already available predictive biomarkers, and identify patients likely to respond to single agent trastuzumab-based neoadjuvant therapy.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , MicroRNA Circulante/sangue , Terapia Neoadjuvante , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Adulto , Idoso , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Análise MultivariadaRESUMO
BACKGROUND: Among wheat gluten proteins, the α-type gliadins are the major responsible for celiac disease, an autoimmune disorder that affects about 1% of the world population. In fact, these proteins contain several toxic and immunogenic epitopes that trigger the onset of the disease. The α-type gliadins are a multigene family, encoded by genes located at the complex Gli-2 loci. RESULTS: Here, three bread wheat deletion lines (Gli-A2, Gli-D2 and Gli-A2/Gli-D2) at the Gli-2 loci were generated by the introgression in the bread wheat cultivar Pegaso of natural mutations, detected in different bread wheat cultivars. The molecular characterization of these lines allowed the isolation of 49 unique expressed genes coding α-type gliadins, that were assigned to each of the three Gli-2 loci. The number and the amount of α-type gliadin transcripts were drastically reduced in the deletion lines. In particular, the line Gli-A2/Gli-D2 contained only 12 active α-type gliadin genes (-75.6% respect to the cv. Pegaso) and a minor level of transcripts (-80% compared to cv. Pegaso). Compensatory pleiotropic effects were observed in the two other classes of gliadins (ω- and γ-gliadins) either at gene expression or protein levels. Although the comparative analysis of the deduced amino acid sequences highlighted the typical structural features of α-type gliadin proteins, substantial differences were displayed among the 49 proteins for the presence of toxic and immunogenic epitopes. CONCLUSION: The deletion line Gli-A2/Gli-D2 did not contain the 33-mer peptide, one of the major epitopes triggering the celiac disease, representing an interesting material to develop less "toxic" wheat varieties.
Assuntos
Epitopos/imunologia , Gliadina/genética , Gliadina/imunologia , Triticum/genética , Doença Celíaca/induzido quimicamente , Gliadina/química , Humanos , Filogenia , Análise de Sequência de Proteína , Triticum/crescimento & desenvolvimento , Triticum/metabolismoRESUMO
The role of circulating tumor cell (CTC) clusters in the metastatic dissemination process is gaining increased attention. Besides homotypic clusters, heterotypic clusters that contain tumor cells admixed with normal cells are frequently observed in patients with solid tumors. Current methods used for cluster detection and enumeration do not allow an accurate estimation of the relative fractions of tumor cells. Here we describe a method for estimating tumor fraction of clusters including isolation and collection of single clusters, assessment of copy number alterations of single clusters by low-pass whole genome sequencing, and bioinformatic analysis of sequencing data.
Assuntos
Células Neoplásicas Circulantes , Humanos , Genômica , Biologia Computacional , Sequenciamento Completo do GenomaRESUMO
Capitalizing on the widespread adoption of smartphones among farmers and the application of artificial intelligence in computer vision, a variety of mobile applications have recently emerged in the agricultural domain. This paper introduces GranoScan, a freely available mobile app accessible on major online platforms, specifically designed for the real-time detection and identification of over 80 threats affecting wheat in the Mediterranean region. Developed through a co-design methodology involving direct collaboration with Italian farmers, this participatory approach resulted in an app featuring: (i) a graphical interface optimized for diverse in-field lighting conditions, (ii) a user-friendly interface allowing swift selection from a predefined menu, (iii) operability even in low or no connectivity, (iv) a straightforward operational guide, and (v) the ability to specify an area of interest in the photo for targeted threat identification. Underpinning GranoScan is a deep learning architecture named efficient minimal adaptive ensembling that was used to obtain accurate and robust artificial intelligence models. The method is based on an ensembling strategy that uses as core models two instances of the EfficientNet-b0 architecture, selected through the weighted F1-score. In this phase a very good precision is reached with peaks of 100% for pests, as well as in leaf damage and root disease tasks, and in some classes of spike and stem disease tasks. For weeds in the post-germination phase, the precision values range between 80% and 100%, while 100% is reached in all the classes for pre-flowering weeds, except one. Regarding recognition accuracy towards end-users in-field photos, GranoScan achieved good performances, with a mean accuracy of 77% and 95% for leaf diseases and for spike, stem and root diseases, respectively. Pests gained an accuracy of up to 94%, while for weeds the app shows a great ability (100% accuracy) in recognizing whether the target weed is a dicot or monocot and 60% accuracy for distinguishing species in both the post-germination and pre-flowering stage. Our precision and accuracy results conform to or outperform those of other studies deploying artificial intelligence models on mobile devices, confirming that GranoScan is a valuable tool also in challenging outdoor conditions.
RESUMO
In clinical trials evaluating antibody-conjugated drugs (ADCs), HER2-low breast cancer is defined through protein immunohistochemistry scoring (IHC) 1+ or 2+ without gene amplification. However, in daily practice, the accuracy of IHC is compromised by inter-observer variability. Herein, we aimed to identify HER2-low breast cancer primary tumors by leveraging gene expression profiling. A discovery approach was applied to gene expression profile of institutional INT1 (n = 125) and INT2 (n = 84) datasets. We identified differentially expressed genes (DEGs) in each specific HER2 IHC category 0, 1+, 2+ and 3+. Principal Component Analysis was used to generate a HER2-low signature whose performance was evaluated in the independent INT3 (n = 95), and in the publicly available TCGA and GSE81538 datasets. The association between the HER2-low signature and HER2 IHC categories was evaluated by Kruskal-Wallis test with post hoc pair-wise comparisons. The HER2-low signature discriminatory capability was assessed by estimating the area under the receiver operating characteristic curve (AUC). Gene Ontology and KEGG analyses were performed to evaluate the HER2-low signature genes functional enrichment. A HER2-low signature was computed based on HER2 IHC category-specific DEGs. The twenty genes included in the signature were significantly enriched with lipid and steroid metabolism pathways, peptidase regulation, and humoral immune response. The HER2-low signature values showed a bell-shaped distribution across IHC categories (low values in 0 and 3+; high values in 1+ and 2+), effectively distinguishing HER2-low from 0 (p < 0.001) to 3+ (p < 0.001). Notably, the signature values were higher in tumors scored with 1+ as compared to 0. The HER2-low signature association with IHC categories and its bell-shaped distribution was confirmed in the independent INT3, TCGA and GSE81538 datasets. In the combined INT1 and INT3 datasets, the HER2-low signature achieved an AUC value of 0.74 (95% confidence interval, CI 0.67-0.81) in distinguishing HER2-low vs. the other categories, outperforming the individual ERBB2 mRNA AUC value of 0.52 (95% CI 0.43-0.60). These results represent a proof-of-concept for an observer-independent gene-expression-based classifier of HER2-low status. The herein identified 20-gene signature shows promise in distinguishing between HER2 0 and HER2-low expressing tumors, including those scored as 1+ at IHC, and in developing a selection approach for ADCs candidates.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/genética , Receptor ErbB-2/metabolismo , Genes erbB-2 , Imuno-Histoquímica , Expressão Gênica , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismoRESUMO
Human Epidermal growth factor Receptor 2 (HER2) assessment is crucial for breast cancer treatment. Therapeutic decisions for recurrent cases often rely on primary tumor status. However, mounting evidence suggests that tumors show dynamic changes and up to 10% of breast cancer modify their initial status during progression. It is still debated whether these changes reflect a biological evolution of the disease or are secondary to primary tumor heterogeneity. Certainly, repeating HER2 assessment during breast cancer trajectory is important for the increasing availability of effective anti-HER2 drugs. In response to this need, circulating biomarkers such as circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) offer the potential to safely and repeatedly assess HER2 status over time. This chapter outlines current methods for testing HER2 in CTCs and ctDNA, and reviews clinical trials evaluating its prognostic and predictive value in patients with breast cancer, as well as recent advances in the field.
Assuntos
Neoplasias da Mama , Feminino , Humanos , Evolução Biológica , Neoplasias da Mama/tratamento farmacológico , Biópsia LíquidaRESUMO
BACKGROUND: Cholangiocarcinoma (CC) is a rare and aggressive disease with limited therapeutic options and a poor prognosis. All available public records of cohorts reporting transcriptomic data on intrahepatic cholangiocarcinoma (ICC) and extrahepatic cholangiocarcinoma (ECC) were collected with the aim to provide a comprehensive gene expression-based classification with clinical relevance. METHODS: A total of 543 patients with primary tumor tissues profiled by RNAseq and microarray platforms from seven public datasets were used as a discovery set to identify distinct biological subgroups. Group predictors developed on the discovery sets were applied to a single cohort of 131 patients profiled with RNAseq for validation and assessment of clinical relevance leveraging machine learning techniques. RESULTS: By unsupervised clustering analysis of gene expression data we identified both in the ICC and ECC discovery datasets four subgroups characterized by a distinct type of immune infiltrate and signaling pathways. We next developed class predictors using short gene list signatures and identified in an independent dataset subgroups of ICC tumors at different prognosis. CONCLUSIONS: The developed class-predictor allows identification of CC subgroups with specific biological features and clinical behavior at single-sample level. Such results represent the starting point for a complete molecular characterization of CC, including integration of genomics data to develop in clinical practice.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Transcriptoma , Colangiocarcinoma/patologia , Prognóstico , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/patologiaRESUMO
Triple negative breast cancer (TNBC) is characterized by clinical aggressiveness, lack of recognized target therapy, and a dismal patient prognosis. Several studies addressed genomic changes occurring during neoadjuvant chemotherapy (NAC) focusing on somatic variants, but without including copy number alterations (CNAs). We analyzed CNA profiles of 31 TNBC primary tumor samples before and after NAC and of 35 single circulating tumor cells (CTCs) collected prior, during and after treatment by using next-generation sequencing targeted profile and low-pass whole genome sequencing, respectively. In pre-treatment tissue samples, the most common gains occurred on chromosomes 1, 2 and 8, and SOX11 and MYC resulted the most altered genes. Notably, amplification of MSH2 (4/4 versus 0/12, p < 0.01) and PRDM1 and deletion of PAX3 (4/4 versus 1/12, p < 0.01) significantly characterized primary tumors of patients with pathological complete response. All patients with paired pre- and post-NAC samples reported a change in post-treatment CNAs compared to baseline, despite they showed at least one common alteration. CNAs detected after treatment involved genes within druggable pathways such as EGFR, cell cycle process and Ras signaling. In two patients, CTCs shared more alterations with residual rather than primary tumor involving genes such as MYC, BCL6, SOX2, FGFR4. The phylogenetic analysis of CTCs within a single patient revealed NAC impact on tumor evolution, suggesting a selection of driver events under treatment pressure. In conclusion, our data showed how chemoresistance might arise early from treatment-induced selection of clones already present in the primary tumor, and that the characterization of CNAs on single CTCs informs on cancer evolution and potential druggable targets.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Variações do Número de Cópias de DNA/efeitos dos fármacos , Terapia Neoadjuvante , Células Neoplásicas Circulantes/patologia , Neoplasias de Mama Triplo Negativas/terapia , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia , Mama/patologia , Mama/cirurgia , Estudos de Coortes , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Mastectomia , Pessoa de Meia-Idade , Mutação , Estadiamento de Neoplasias , Filogenia , Prognóstico , Neoplasias de Mama Triplo Negativas/diagnóstico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Sequenciamento Completo do GenomaRESUMO
Preemptive targeted pharmacogenetic testing of candidate variations in DPYD is currently being used to limit toxicity associated with fluoropyrimidines. The use of innovative next generation sequencing (NGS) approaches could unveil additional rare (minor allele frequency <1%) genetic risk variants. However, their predictive value and management in clinical practice are still controversial, at least partly due to the challenges associated with functional analyses of rare variants. The aim of this study was to define the predictive power of rare DPYD variants burden on the risk of severe fluoropyrimidine-related toxicity. The DPYD coding sequence and untranslated regions were analyzed by NGS in 120 patients developing grade 3-5 (NCI-CTC vs3.0) fluoropyrimidine-related toxicity and 104 matched controls (no-toxicity). The functional impact of rare variants was assessed using two different in silico predictive tools (i.e., Predict2SNP and ADME Prediction Framework) and structural modeling. Plasma concentrations of uracil (U) and dihydrouracil (UH2) were quantified in carriers of the novel variants. Here, we demonstrate that the burden of rare variants was significantly higher in patients with toxicity compared to controls (p = 0.007, Mann-Whitney test). Carriers of at least one rare missense DPYD variant had a 16-fold increased risk in the first cycle and an 11-fold increased risk during the entire course of chemotherapy of developing a severe adverse event compared to controls (p = 0.013 and p = 0.0250, respectively by multinomial regression model). Quantification of plasmatic U/UH2 metabolites and in silico visualization of the encoded protein were consistent with the predicted functional effect for the novel variations. Analysis and consideration of rare variants by DPYD-sequencing could improve prevention of severe toxicity of fluoropyrimidines and improve patients' quality of life.
Assuntos
Di-Hidrouracila Desidrogenase (NADP) , Qualidade de Vida , Antimetabólitos , Antimetabólitos Antineoplásicos/efeitos adversos , Di-Hidrouracila Desidrogenase (NADP)/genética , Fluoruracila/efeitos adversos , Frequência do Gene , Genótipo , HumanosRESUMO
Background: The absence of breast cancer cells in surgical specimens, i.e., pathological complete response (pCR), is widely recognized as a favorable prognostic factor after neoadjuvant therapy. In contrast, the presence of disease at surgery characterizes a prognostically heterogeneous group of patients. Here, we challenged circulating microRNAs (miRNAs) at the end of neoadjuvant therapy as potential prognostic biomarkers in the NeoALTTO study. Methods: Patients treated within the trastuzumab arm (i.e., pre-operative weekly trastuzumab for 6 weeks followed by the addition of weekly paclitaxel for 12 weeks; post-operative FEC for 3 cycles followed by trastuzumab up to complete 1 year of treatment) were randomized into a training (n= 54) and testing (n= 72) set. RT-PCR-based high-throughput miRNA profile was performed on plasma samples collected at the end of neoadjuvant treatment of both sets. After normalization, circulating miRNAs associated with event free survival (EFS) were identified by univariate and multivariate Cox regression model. Results: Starting from 23 circulating miRNAs associated with EFS in the training set, we generated a 3-circulating miRNA prognostic signature consisting of miR-185-5p, miR-146a-5p, miR-22-3p, which was confirmed in the testing set. The 3-circulating miRNA signature showed a C-statistic of 0.62 (95% confidence interval [95%CI] 0.53-0.71) in the entire study cohort. By resorting to a multivariate Cox regression model we found a statistical significant interaction between the expression values of miR-194-5p and pCR status (p.interaction =0.005) with an estimate Hazard Ratio (HR) of 1.83 (95%CI 1.14- 2.95) in patients with pCR, and 0.87 (95%CI 0.69-1.10) in those without pCR. Notably, the model including this interaction along with the abovementioned 3-circulating miRNA signature provided the highest discriminatory capability with a C-statistic of 0.67 (95%CI 0.58-0.76). Conclusions: Circulating miRNAs are informative to identify patients with different prognosis among those with heterogeneous response after trastuzumab-based neoadjuvant treatment, and may be an exploitable tool to select candidates for salvage adjuvant therapy.
RESUMO
Circulating tumor microemboli (CTMs) are clusters of cancer cells detached from solid tumors, whose study can reveal mechanisms underlying metastatization. As they frequently comprise unknown fractions of leukocytes, the analysis of copy number alterations (CNAs) is challenging. To address this, we titrated known numbers of leukocytes into cancer cells (MDA-MB-453 and MDA-MB-36, displaying high and low DNA content, respectively) generating tumor fractions from 0-100%. After low-pass sequencing, ichorCNA was identified as the best algorithm to build a linear mixed regression model for tumor fraction (TF) prediction. We then isolated 53 CTMs from blood samples of six early-stage breast cancer patients and predicted the TF of all clusters. We found that all clusters harbor cancer cells between 8 and 48%. Furthermore, by comparing the identified CNAs of CTMs with their matched primary tumors, we noted that only 31-71% of aberrations were shared. Surprisingly, CTM-private alterations were abundant (30-63%), whereas primary tumor-private alterations were rare (4-12%). This either indicates that CTMs are disseminated from further progressed regions of the primary tumor or stem from cancer cells already colonizing distant sites. In both cases, CTM-private mutations may inform us about specific metastasis-associated functions of involved genes that should be explored in follow-up and mechanistic studies.
RESUMO
Claudin-low cancer (CL) represents a rare and biologically aggressive variant of epithelial tumor. Here, we identified a claudin-low molecular profile of ovarian high-grade serous carcinoma (HGSOC), which exhibits the main characteristics of the homonym breast cancer subtype, including low epithelial differentiation and high mesenchymal signature. Hierarchical clustering and a centroid based algorithm applied to cell line collection expression dataset labeled 6 HGSOC cell lines as CL. These have a high energy metabolism and are enriched in CD44+/CD24- mesenchymal stem-like cells expressing low levels of cell-cell adhesion molecules (claudins and E-Cadherin) and high levels of epithelial-to-mesenchymal transition (EMT) induction transcription factors (Zeb1, Snai2, Twist1 and Twist2). Accordingly, the centroid base algorithm applied to large retrospective collections of primary HGSOC samples reveals a tumor subgroup with transcriptional features consistent with the CL profile, and reaffirms EMT as the dominant biological pathway functioning in CL-HGSOC. HGSOC patients carrying CL profiles have a worse overall survival when compared to others, likely to be attributed to its undifferentiated/stem component. These observations highlight the lack of a molecular diagnostic in the management of HGSOC and suggest a potential prognostic utility of this molecular subtyping.
RESUMO
High-grade serous ovarian carcinoma (HGSOC) usually spreads directly into the peritoneal cavity following a transcoelomic dissemination route, although distant hematogenous metastasis exist and have been reported. However, no tumor markers can currently predict the risk of distant metastases in HGSOC. Claudins, belonging to tight-junction proteins, are dysregulated in HGSOC and functionally related to cancer progression. Here we analyzed claudin-3, -4, and -7 expression as potential markers of distant metastases. Using quantitative RT-PCR and immunohistochemistry we assessed the expression of claudins in primary HGSOC tissues, normal ovarian, and normal fallopian tube epithelia and correlated it with clinicopathological features, including the site of metastasis and the route of dissemination. Gene set enrichment analysis was performed on microarray-generated gene expression data to investigate key pathways in patients with distant metastases. We found the overall expression level of claudin-3, -4, and -7 mRNA decreased in HGSOC compared to normal tubal epithelium, currently considered the potential site of origin of many HGSOC. The reduced expression of claudin-7 is significantly associated with the development of distant metastases (p = 0.016), mainly by hematogenous route (p = 0.025). In patients with diminished expression of claudin-7, immunohistochemical staining revealed a heterogeneous pattern of membranous staining with discontinuous expression of claudin-7 along the cell border, indicative of a dischoesive architecture. The estimated reduction in the probability of distant disease is of 39% per unit increase in the level of claudin-7 (p = 0.03). Genes involved in epithelial to mesenchymal transition, hypoxia, and angiogenesis processes resulted strongly associated to hematogenous recurrence. Our data suggest a potential role of claudin-7 in discriminating distant metastatic events in HGSOC patients. The quantification of its expression levels could be a useful tool to identify patient deserving a personalized follow-up in terms of clinical and radiological assessment.
RESUMO
Adenosine-to-Inosine (A-to-I) RNA editing is the most prevalent post-transcriptional modification of RNA molecules. Researchers have attempted to find reliable RNA editing using next generation sequencing (NGS) data. However, most of these attempts suffered from a high rate of false positives, and they did not consider the clinical relevance of the identified RNA editing, for example, in disease progression. We devised an effective RNA-editing discovery pipeline called CREDO, which includes novel statistical filtering modules based on integration of DNA- and RNA-seq data from matched tumor-normal tissues. CREDO was compared with three other RNA-editing discovery pipelines and found to give significantly fewer false positives. Application of CREDO to breast cancer data from the Cancer Genome Atlas (TCGA) project discovered highly confident RNA editing with clinical relevance to cancer progression in terms of patient survival. RNA-editing detection using DNA- and RNA-seq data from matched tumor-normal tissues should be more routinely performed as multiple omics data are becoming commonly available from each patient sample. We believe CREDO is an effective and reliable tool for this problem.
Assuntos
Adenosina , Neoplasias da Mama/genética , Biologia Computacional/métodos , Inosina , Edição de RNA , Adenosina/genética , Neoplasias da Mama/mortalidade , Bases de Dados Genéticas , Feminino , Humanos , Inosina/genética , Estimativa de Kaplan-Meier , Navegador , Sequenciamento do ExomaRESUMO
3D biomaterial manufacturing strategies show an extraordinary driving force for the development of innovative therapies in the tissue engineering field. Here, the behaviour of 3D printed chitosan (CH)-based scaffolds was explored as a function of the post-printing gelation process. To this purpose, gel forming properties of different media were tested on their capability to retain 3D structure, water content, mechanical resistance and surface/internal porosity. Three different gelation media (i.e. KOH 1.5 M, Na2CO3 1.5 M, ammonia vapours) were selected and the 3D CH scaffolds were tested in terms of biocompatibility toward fibroblast as skin associated human cell line.