RESUMO
Malignant melanoma (MM) is known for its abundance of genetic alterations and a tendency for rapid metastasizing. Identification of novel plasma biomarkers may enhance non-invasive diagnostics and disease monitoring. Initially, we examined copy number variations (CNV) in CDK genes (CDKN2A, CDKN2B, CDK4) using MLPA (gDNA) and ddPCR (ctDNA) analysis. Subsequently, low-coverage whole genome sequencing (lcWGS) was used to identify the most common CNV in plasma samples, followed by ddPCR verification of chosen biomarkers. CNV alterations in CDK genes were identified in 33.3% of FFPE samples (Clark IV, V only). Detection of the same genes in MM plasma showed no significance, neither compared to healthy plasmas nor between pre- versus post-surgery plasma. Sequencing data showed the most common CNV occurring in 6q27, 4p16.1, 10p15.3, 10q22.3, 13q34, 18q23, 20q11.21-q13.12 and 22q13.33. CNV in four chosen genes (KIF25, E2F1, DIP2C and TFG) were verified by ddPCR using 2 models of interpretation. Model 1 was concordant with lcWGS results in 54% of samples, for model 2 it was 46%. Although CDK genes have not been proven to be suitable CNV liquid biopsy biomarkers, lcWGS defined the most frequently affected chromosomal regions by CNV. Among chosen genes, DIP2C demonstrated a potential for further analysis.
Assuntos
Biomarcadores Tumorais , Variações do Número de Cópias de DNA , Melanoma , Humanos , Melanoma/genética , Melanoma/patologia , Melanoma/diagnóstico , Biópsia Líquida/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/sangue , Inibidor p16 de Quinase Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/genética , Idoso de 80 Anos ou mais , Sequenciamento Completo do Genoma/métodos , Inibidor de Quinase Dependente de Ciclina p15/genéticaRESUMO
BACKGROUND: Cytokines are involved in cancer invasion and metastasis. Circulating tumor cells (CTCs) play key role in tumor dissemination and are an independent survival predictor in breast cancer patients. The aim of this study was to assess correlation between CTCs and plasma cytokines in primary breast cancer (PBC) patients. METHODS: This study included 147 chemotherapy naïve PBC patients. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoetic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (Twist1, Snail1, Slug, Zeb1) and epithelial (Ck19) gene transcripts by qRT-PCR. The concentrations of 51 plasma cytokines were measured using multiplex bead arrays. RESULTS: CTCs were detected in 25.2% patients. CTCs exhibiting only epithelial markers (CTC_EP) and only EMT markers (CTC_EMT) were present evenly in 11.6% patients, while CTCs co-expressing both markers were detected in 2.0% patients. Patients with presence of CTC_EP in peripheral blood had significantly elevated levels of plasma IFN-α2, IL-3, MCP-3, ß-NGF, SCF, SCGF-ß, TNF-ß and SDF-1 compared to patients without CTC_EP. CTC_EP exhibited overexpression of SDF-1 receptor and CXCR4, but not other corresponding cytokine receptor, and in multivariate analysis SDF-1 was independently associated with CTC_EP. There was an inverse correlation between CTC_EMT and plasma cytokines CTACK, ß-NGF and TRAIL, while presence of either subtype of CTCs was associated with increased level of TGF-ß2. CONCLUSION: Using cytokine profiling, we identified cytokines associated with CTCs subpopulations in peripheral blood of PBC. Our data suggest that CXCR4-SDF-1 axis is involved in mobilization and trafficking of epithelial CTCs.
Assuntos
Neoplasias da Mama/patologia , Quimiocina CXCL12/genética , Células Neoplásicas Circulantes/patologia , Receptores CXCR4/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quimiocina CXCL12/sangue , Feminino , Células HCT116 , Células HeLa , Humanos , Células MCF-7 , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/metabolismo , Receptores CXCR4/sangueRESUMO
BACKGROUND: Circulating tumor cells (CTCs) play a crucial role in tumor dissemination and are an independent survival predictor in breast cancer (BC) patients. Epithelial to mesenchymal transition (EMT) is involved in cancer invasion and metastasis. The aim of this study was to assess correlation between CTCs and expression of EMT transcription factors TWIST1 and SLUG in breast tumor tissue. METHODS: This study included 102 early BC patients treated by primary surgery. Peripheral blood mononuclear cells (PBMC) were depleted of hematopoietic cells using RossetteSep™ negative selection kit. RNA extracted from CD45-depleted PBMC was interrogated for expression of EMT (TWIST1, SNAIL1, SLUG, FOXC2 and ZEB1) and epithelial (KRT19) gene transcripts by qRT-PCR. Expression of TWIST1 and SLUG in surgical specimens was evaluated by immunohistochemistry and quantified by multiplicative score. RESULTS: CTCs were detected in 24.5 % patients. CTCs exhibiting only epithelial markers were present in 8.8 % patients, whereas CTCs with only EMT markers were observed in 12.8 % of pts and CTCs co-expressing both markers were detected in 2.9 % pts. We observed lack of correlation between CTCs and expression of TWIST1 and SLUG in breast cancer cells or cancer associated stroma. Lack of correlation was observed for epithelial CTCs as well as for CTCs with EMT. CONCLUSIONS: In this translational study, we showed a lack of association between CTCs and expression of EMT-inducing transcription factors, TWIST1 and SLUG, in breast tumor tissue. Despite the fact that EMT is involved in cancer invasion and metastasis our results suggest, that expression of EMT proteins in unselected tumor tissue is not surrogate marker of CTCs with either mesenchymal or epithelial features.