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Following the publication of the above article, an interested reader drew an anomaly associated with the presentation of Fig. 7 to our attention. Essentially, the panel showing the Cis 0.001 data at T0 for the BRC-230 cell line was the same as that showing the Cis 0.01 data. After having re-examined our data, we realized that an error must have occurred when preparing Fig. 7. A duplication of the photo corresponding to BRC-230 Cis 0.001 T0 was incorrectly placed in the position corresponding to BRC-230 Cis 0.01 T0, while the correct BRC-230 Cis 0.01 T0 figure was erroneously positioned in the BRC-230 Cis 0.01 T1 slot. Fortunately, we had retained all of the data on our computer and were able to find the correct photos representing the conditions of BRC-230 Cis 0.01 T0 and T1. The findings and conclusions of this paper are still supported by our experimental data and are not affected by this error. We sincerely apologize for this oversight and thank the reader for drawing this matter to our attention. We regret any inconvenience caused by our mistake. [the original article was published in the International Journal of Oncology 42: 1263-1270, 2013; DOI: 10.3892/ijo.2013.1809].
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BACKGROUND: We evaluated the role of single nucleotide polymorphisms in the CYP17A1 gene for predicting clinical outcome in castration-resistant prostate cancer (CRPC) patients treated with abiraterone. METHODS: Sixty-four patients were genotyped for the selected polymorphisms (rs743572, rs10883783, rs17115100 and rs284849) in CYP17A1. We hypothesized that different genotypes could be associated with progression-free survival (PFS) and overall survival (OS). RESULTS: Statistical analyses highlighted no significant associations between these polymorphisms and clinical outcome. However, individuals with the most common TT genotype for rs10883783 had a 3 months' longer PFS than individuals with the TA + AA genotype. CONCLUSIONS: With the limitation of the retrospective study design and the small sample size, the analyzed polymorphisms do not seem to be correlated with clinical outcome of CRPC patients treated with abiraterone.
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Androstenos/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Esteroide 17-alfa-Hidroxilase/genética , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Inibidores Enzimáticos/uso terapêutico , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/genética , Estudos Retrospectivos , Fatores de Risco , Esteroide 17-alfa-Hidroxilase/antagonistas & inibidores , Esteroide 17-alfa-Hidroxilase/metabolismo , Resultado do TratamentoRESUMO
INTRODUCTION: The detection of tumor-specific markers in urine has paved the way for new early noninvasive diagnostic approaches for prostate cancer. We evaluated the DNA integrity in urine supernatant to verify its capacity to discriminate between prostate cancer and benign diseases of the urogenital tract. PATIENTS AND METHODS: A total of 131 individuals were enrolled: 67 prostate cancer patients and 64 patients with benign diseases of the urogenital tract (control group). Prostate-specific antigen (PSA) levels were determined. Urine cell-free (UCF) DNA was isolated and sequences longer than 250 bp corresponding to 3 genes (c-MYC, HER2, and AR) were quantified by Real-Time PCR to assess UCF-DNA integrity. RESULTS: UCF-DNA was quantifiable in all samples, while UCF-DNA integrity was evaluable in all but 16 samples. Receiver operating characteristic analysis showed an area under the curve of 0.5048 for UCF-DNA integrity and 0.8423 for PSA. Sensitivity was 0.58 and 0.95 for UCF-DNA integrity and PSA, respectively. Specificity was 0.44 and 0.69, respectively. CONCLUSIONS: UCF-DNA integrity showed lower accuracy than PSA and would not seem to be a reliable marker for early prostate cancer diagnosis. Despite this, we believe that UCF-DNA could represent a source of other biomarkers and could detect gene alterations.
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Biomarcadores Tumorais/urina , DNA/urina , Neoplasias da Próstata/urina , Idoso , Biomarcadores Tumorais/química , Estudos de Casos e Controles , DNA/química , Detecção Precoce de Câncer , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Receptor ErbB-2/genética , Receptores Androgênicos/genéticaRESUMO
BACKGROUND: Bevacizumab plus chemotherapy is a widely used therapeutic option for first-line treatment of metastatic colorectal cancer (mCRC). However, molecular predictors of bevacizumab efficacy have not yet been identified. We analyzed vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) polymorphisms in relation to response to bevacizumab. METHODS: Two hundred and thirty-seven patients with mCRC enrolled onto the phase III prospective multicentre randomized "Italian Trial in Advanced Colorectal Cancer (ITACa)" trial were evaluated. One hundred fourteen patients received chemotherapy plus bevacizumab (CT + B) and 123 received chemotherapy (CT) alone. Five single nucleotide polymorphisms (SNPs) (-2578, -1498, -1154, -634 and +936) for VEGF and 2 SNPs (-786, +894) and one variable number tandem repeat in intron 4 for eNOS were analyzed for each patient. The polymorphisms were assessed in relation to progression-free survival (PFS), objective response rate (ORR) and overall survival (OS). RESULTS: VEGF 936C/T, eNOS +894 G/T and VNTR were significantly correlated with outcome in CT + B patients, but not in CT-only patients. In particular, patients with a specific haplotype combination of the 2 eNOS polymorphisms (defined eNOS Haplo1/Haplo1 and eNOS Haplo 2/Haplo2) showed significantly longer PFS (15.0 vs 9.1 months, P = 0.001) and OS (34.5 vs 20.5 months P = 0.002), and a higher ORR (71 vs 45.9%, P = 0.013) than those with the other genotypes, respectively. CONCLUSIONS: Specific eNOS polymorphisms may be capable of identifying a subset of mCRC patients who are more responsive to bevacizumab-based chemotherapy. If confirmed, these results would permit individually tailored treatment with bevacizumab.
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Bevacizumab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Óxido Nítrico Sintase Tipo III/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/enzimologia , Intervalo Livre de Doença , Feminino , Estudos de Associação Genética , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Resultado do Tratamento , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
BACKGROUND: Acute myeloid leukemia (AML) is an incurable disease with fatal infections or relapse being the main causes of death in most cases. In particular, the severe infections occurring in these patients before or during any treatment suggest an intrinsic alteration of the immune system. In this respect, IL-17-producing T helper (Th17) besides playing a key role in regulating inflammatory response, tumor growth and autoimmune diseases, have been shown to protect against bacterial and fungal pathogens. However, the role of Th17 cells in AML has not yet been clarified. METHODS: T cell frequencies were assessed by flow cytometry in the peripheral blood of 30 newly diagnosed AML patients and 30 age-matched healthy volunteers. Cytokine production was determined before and after culture of T cells with either Candida Albicans or AML blasts. Statistical analyses were carried out using the paired and unpaired two-tailed Student's t tests and confirmed with the non parametric Wilcoxon signed-rank test. RESULTS: A strong increase of Th17 cells producing immunosuppressive IL-10 was observed in AML patients compared with healthy donors. In addition, stimulation of AML-derived T cells with a Candida albicans antigen induced significantly lower IFN-γ production than that observed in healthy donors; intriguingly, depletion of patient Th17 cells restored IFN-γ production after stimulation. To address the role of AML blasts in inducing Th17 alterations, CD4+ cells from healthy donors were co-cultured with CD33+ blasts: data obtained showed that AML blasts induce in healthy donors levels of IL-10-producing Th17 cells similar to those observed in patients. CONCLUSIONS: In AML patients altered Th17 cells actively cause an immunosuppressive state that may promote infections and probably tumor escape. Th17 cells could thus represent a new target to improve AML immunotherapy.
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Candidíase/imunologia , Terapia de Imunossupressão , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/microbiologia , Linfócitos T/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Crise Blástica/imunologia , Candida albicans/imunologia , Candidíase/complicações , Candidíase/microbiologia , Técnicas de Cocultura , Citocinas/metabolismo , Feminino , Humanos , Interferon gama/biossíntese , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/complicações , Contagem de Linfócitos , Masculino , Pessoa de Meia-Idade , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Células Th17/imunologiaRESUMO
Targeted therapy of metastatic colorectal cancer (mCRC) with monoclonal antibody anti-epidermal growth factor receptor (EGFR) agents, such as cetuximab (CTX) or panitumumab, is the treatment strategy of choice in patients characterised by a wild-type (wt) RAS gene status. However, despite selection based on RAS status, a high proportion of patients do not respond to therapy. EGFR methylation has been reported to have a role in predicting the response to anti-EGFR agents. The present study aimed to evaluate the role of EGFR methylation in association with the clinical outcome of patients with mCRC treated with CTX. In total, 64 patients with mCRC were assessed in the present study. Genomic DNA was extracted from tumoral tissue and EGFR methylation and mutation of the KRAS, BRAF and PIK3CA genes were analysed by pyrosequencing. EGFR expression was assessed by immunohistochemistry. The various alterations were analysed by assessing the objective response rate (ORR), progression free survival (PFS) and overall survival (OS) rates. In total, 42 cases (66%) exhibited >10% EGFR methylation and there was no correlation with EGFR expression. Mean EGFR methylation of 41 and 9% was observed in KRAS-mutated and -wt patients, respectively (P=0.05). Conversely, a high EGFR methylation was observed in BRAF-wt patients with compared with patients possessing the mutated gene (18 vs. 3%, respectively; P=0.07). EGFR methylation was significantly correlated with the OS rate [hazard ratio, 0.98; 95% confidence interval (CI), 0.96-1.00; P=0.019], but not PFS rate. In patients with a methylation rate <10 and >10%, the median OS rate was 7.5 months (95% CI, 4.4-9.4 months) and 12.0 months (95% CI, 8.7-13.9 months), respectively (P=0.034). In conclusion, the present study revealed a correlation between EGFR methylation and improved OS rate in patients treated with CTX-based chemotherapy. The presence of EGFR methylation is inversely correlated with BRAF and PIK3CA mutations, indicating that the prognostic value of gene methylation may be worth verifying in further studies.
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The present study deals with the preparation of albumin nanocapsules containing fenretinide and their evaluation in experimental models of human non-small cell lung cancer. These nanocapsules showed enhanced antitumor activity with respect to free fenretinide due to the solubilization effect of albumin on the hydrophobic drug, known to improve bioavailability. The high expression of caveolin-1 on the A549 cell surface further enhanced the antitumor activity of the nanoencapsulated fenretinide. Caveolin-1 favored albumin uptake and improved the efficacy of the fenretinide-loaded albumin nanocapsules, especially in 3-D cultures where the densely packed 3-D structures impaired drug diffusibility and severely reduced the activity of the free drug. The efficacy of the fenretinide albumin nanocapsules was further confirmed in tumor xenograft models of A549 by the significant delay in tumor progression observed with respect to control after intravenous administration of the novel formulation. FROM THE CLINICAL EDITOR: This study describes the preparation of fenretinide containing albumin nanocapsules and their evaluation in experimental models of non-small cell lung cancer, showing enhanced antitumor activity compared to free fenretinide.
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Antineoplásicos/química , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Fenretinida/administração & dosagem , Nanocápsulas/administração & dosagem , Albuminas/administração & dosagem , Albuminas/química , Animais , Antineoplásicos/administração & dosagem , Disponibilidade Biológica , Linhagem Celular Tumoral , Humanos , Camundongos , Nanocápsulas/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied.
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Cell counting is one of the basic needs of most biological experiments. Numerous methods and systems have been studied to improve the reliability of counting. However, at present, manual cell counting performed with a hemocytometer still represents the gold standard, despite several problems limiting reproducibility and repeatability of the counts and, at the end, jeopardizing their reliability in general. We present our own approach based on image processing techniques to improve counting reliability. It works in two stages: first building a high-resolution image of the hemocytometer's grid, then counting the live and dead cells by tagging the image with flags of different colours. In particular, we introduce GridMos (http://sourceforge.net/p/gridmos), a fully-automated mosaicing method to obtain a mosaic representing the whole hemocytometer's grid. In addition to offering more significant statistics, the mosaic "freezes" the culture status, thus permitting analysis by more than one operator. Finally, the mosaic achieved can thus be tagged by using an image editor, thus markedly improving counting reliability. The experiments performed confirm the improvements brought about by the proposed counting approach in terms of both reproducibility and repeatability, also suggesting the use of a mosaic of an entire hemocytometer's grid, then labelled trough an image editor, as the best likely candidate for the new gold standard method in cell counting.
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Contagem de Células/métodos , Processamento de Imagem Assistida por Computador/métodos , Neoplasias Pulmonares/diagnóstico , Neoplasias Gástricas/diagnóstico , Algoritmos , Automação , Contagem de Células/instrumentação , Linhagem Celular Tumoral , Processamento Eletrônico de Dados , Humanos , Processamento de Imagem Assistida por Computador/instrumentação , Neoplasias Pulmonares/patologia , Microscopia/métodos , Reprodutibilidade dos Testes , Software , Neoplasias Gástricas/patologia , Azul Tripano/química , Interface Usuário-ComputadorRESUMO
BACKGROUND: Epigenetic alterations of specific genes have been reported to be related to colorectal cancer (CRC) transformation and would also appear to be involved in the early stages of colorectal carcinogenesis. Little data are available on the role of these alterations in determining a different risk of colorectal lesion recurrence. The aim of the present study was to verify whether epigenetic alterations present in pre-neoplastic colorectal lesions detected by colonoscopy can predict disease recurrence. METHODS: A retrospective series of 78 adenomas were collected and classified as low (35) or high-risk (43) for recurrence according to National Comprehensive Cancer Network guidelines. Methylation alterations were analyzed by the methylation-specific multiplex ligation probe assay (MS-MLPA) which is capable of quantifying methylation levels simultaneously in 24 different gene promoters. MS-MLPA results were confirmed by pyrosequencing and immunohistochemistry. RESULTS: Higher levels of methylation were associated with disease recurrence. In particular, MLH1, ATM and FHIT gene promoters were found to be significantly hypermethylated in recurring adenomas. Unconditional logistic regression analysis used to evaluate the relative risk (RR) of recurrence showed that FHIT and MLH1 were independent variables with an RR of 35.30 (95% CI 4.15-300.06, P = 0.001) and 17.68 (95% CI 1.91-163.54, P = 0.011), respectively. CONCLUSIONS: Histopathological classification does not permit an accurate evaluation of the risk of recurrence of colorectal lesions. Conversely, results from our methylation analysis suggest that a classification based on molecular parameters could help to define the mechanisms involved in carcinogenesis and prove an effective method for identifying patients at high risk of recurrence.
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Neoplasias Colorretais/genética , Metilação de DNA , Genes Supressores de Tumor , Recidiva Local de Neoplasia/genética , Lesões Pré-Cancerosas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese/genética , Carcinogênese/patologia , Neoplasias Colorretais/patologia , Epigenômica , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Lesões Pré-Cancerosas/patologia , Estudos RetrospectivosRESUMO
Single nucleotide polymorphisms (SNPs) may be associated with the response or toxicity to different types of treatment. Although SNP analysis is usually performed on DNA from peripheral blood, formalin fixed paraffin-embedded (FFPE) tissue is often used for retrospective studies. We analyzed VEGF (-2578C>A, -1498C>T, -1154G>A, -634C>G, +936C>T) and eNOS (+894G>T, -786T>C, VNTR (variable number of tandem repeats) 27bp intron 4) polymorphisms by direct sequencing or Real Time PCR in 237 patients with advanced colorectal cancer. Peripheral blood was used for 153 patients, whereas only FFPE tumor tissue was available for 84 patients. All SNP frequencies were in Hardy-Weinberg Equilibrium (HWE), with the exception of VEGF -1154, which was only in HWE in peripheral blood specimens. We therefore analyzed this SNP in DNA extracted from FFPE tumor tissue compared to FFPE healthy tissue and peripheral blood from 20 patients. Numerous heterozygous patients in peripheral blood DNA were homozygous for the A-allele in both tumor and healthy FFPE tissues. Our findings indicate that, although FFPE tissue might be a suitable specimen for genotyping, VEGF -1154 does not give reliable results on this type of material. As other SNPs may also have this limitation, genotype concordance should first be confirmed by comparing results obtained from FFPE and fresh sample analyses.
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Neoplasias Colorretais/genética , DNA/sangue , Polimorfismo de Nucleotídeo Único , Fator A de Crescimento do Endotélio Vascular/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Sequência de Bases , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Óxido Nítrico Sintase Tipo III/genética , Análise de Sequência com Séries de OligonucleotídeosRESUMO
BACKGROUND: DNA integrity analysis could represent an alternative approach to the early detection of colorectal cancer. Previously, fluorescence long DNA (FL-DNA) in stools was extracted using a manual approach and analyzed by capillary electrophoresis assay (CE FL-DNA). We aimed to improve diagnostic accuracy using a simpler and more standardized method [Real Time PCR FL-DNA (RT FL-DNA)] for the detection of early malignant lesions in a population undergoing colorectal cancer screening. METHODS: From 241 stool samples, DNA was extracted using manual and semiautomatic extraction systems and analyzed using FL-DNA tests by CE and RT assays. The RT FL-DNA approach showed slightly higher sensitivity and specificity compared with the CE FL-DNA method. Furthermore, we compared the RT FL-DNA approach with the iFOBT report. RESULTS: Nonparametric ranking statistics were used to analyze the relationship between the median values of RT FL-DNA and the clinicohistopathologic characteristics. The median values of both variables were significantly higher in patients with cancer than in patients with noncancerous lesions. According to the Fagan nomogram results, the iFOBT and FL-DNA methods provided more accurate diagnostic information and were able to identify subgroups at varying risks of cancer. CONCLUSIONS: The combination of the semiautomatic extraction system and RT FL-DNA analysis improved the quality of DNA extracted from stool samples. IMPACT: RT FL-DNA shows great potential for colorectal cancer diagnosis as it is a reliable and relatively easy analysis to perform on routinely processed stool samples in combination with iFOBT.
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Neoplasias Colorretais/genética , DNA/genética , Fezes/química , Detecção Precoce de Câncer , Feminino , Humanos , Masculino , Programas de RastreamentoRESUMO
Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher's test p=0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p=0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.
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Biomarcadores Tumorais/genética , Variações do Número de Cópias de DNA , Recidiva Local de Neoplasia/genética , Proteínas Nucleares/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias da Bexiga Urinária/genética , Idoso , Proteínas de Ciclo Celular , Feminino , Humanos , Masculino , Recidiva Local de Neoplasia/diagnóstico , Resultado do Tratamento , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/cirurgiaRESUMO
BACKGROUND: Preclinical studies have shown that several chemotherapeutic agents at low doses may affect the vascular system. Here we report the case of a patient with long-term cancer control by metronomic chemotherapy. CASE PRESENTATION: A 62-year-old woman with breast cancer underwent a left mastectomy in July 2007. For a liver metastasis she was given first-line chemotherapy with doxorubicin plus paclitaxel every 21 days. A CT scan after the sixth cycle showed a partial response. It was decided to stop the treatment with doxorubicin and paclitaxel, and start metronomic therapy with cyclophosphamide 50 mg daily orally and methotrexate 2.5 mg twice daily, 2 days a week. After 6 months of this maintenance treatment, CT scan showed a complete response. We examined the expression of vascular endothelial growth factor receptor 2 (VEGFR2) in histological sections of the primary tumor of our patient, finding evidence of overexpression of the receptor. The metronomic treatment is still ongoing, and after 60 months the patient maintains a complete response. CONCLUSION: This clinical case highlights how suitable metronomic chemotherapy can be used as maintenance therapy, allowing long-term treatment with no significant toxicity. This case suggests that the level of VEGFR2 is predictive of best response to antiangiogenic therapy.
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Administração Metronômica , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/análise , Carcinoma Ductal de Mama/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/análise , Administração Oral , Carcinoma Ductal de Mama/secundário , Ciclofosfamida/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/diagnóstico , Quimioterapia de Manutenção , Mastectomia Radical Modificada , Metotrexato/administração & dosagem , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/administração & dosagem , Valor Preditivo dos Testes , Tomografia Computadorizada por Raios X , Resultado do Tratamento , Regulação para CimaRESUMO
BACKGROUND: CHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer. CASE PRESENTATION: Here, we describe an Italian family with a high number of cases of breast cancer and other types of tumour subjected to the MLPA test to verify the presence of BRCA1, BRCA2 and CHEK2 deletions and duplications. We identified a new 23-kb duplication in the CHEK2 gene extending from intron 5 to 13 that was associated with breast cancer in the family. The presence and localisation of the alteration was confirmed by a second analysis by Next-Generation Sequencing. CONCLUSIONS: This finding suggests that CHEK2 mutations are heterogeneous and that techniques other than sequencing, such as MLPA, are needed to identify CHEK2 mutations. It also indicates that CHEK2 rare variants, such as duplications, can confer a high susceptibility to cancer development and should thus be studied in depth as most of our knowledge of CHEK2 concerns common mutations.
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Quinase do Ponto de Checagem 2/genética , Duplicação Gênica , Predisposição Genética para Doença , Adulto , Idoso , Neoplasias da Mama/genética , Feminino , Estudos de Associação Genética , Variação Genética , Mutação em Linhagem Germinativa , Humanos , Itália , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNARESUMO
Lung cancer is a leading cause of cancer death and late diagnosis is one of the most important reasons for the high mortality rate. Circulating microRNAs (miRNAs) represent stable and reproducible markers for numerous solid tumors, including lung cancer, and have been hypothesized as non-invasive diagnostic markers. Serum, plasma or whole peripheral blood can be used as starting material, and several methodological approaches have been proposed to evaluate miRNA expression. The present review provides an in depth summary of current knowledge on circulating miRNAs in different types of biological samples used as diagnostic markers of lung cancer. We also evaluate the diagnostic accuracy of each miRNA or group of miRNAs in relation to the different housekeeping miRNAs used. Finally, the limitations and potential of miRNA analysis are discussed.
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Biomarcadores Tumorais/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , MicroRNAs/genética , HumanosRESUMO
Metastatic bone disease has a major impact on the morbidity and mortality of breast cancer patients, and studies on bone metastasis biology have led to the development of the most widely used drugs for bone metastases treatment: zoledronate (Zol) and denosumab (Den). The aim of the present study was to assess the effect of soluble mediators produced by breast cancer cells on human osteoclast maturation in a co-culture model. We also tested the ability of zoledronate, denosumab and 5H4, an antibody directed against CSF-1, to interfere with the osteoclastogenic potential of breast cancer. The study was performed on the triple negative cell line MDA-MB-231 and on human osteoclasts obtained from the differentiation of peripheral blood monocytes of a healthy volunteer. Osteoclastogenesis was evaluated by TRAP assay after 14days of differentiation with 10% MDA-MB-231-conditioned media or with CSF-1 and RANKL. Den, Zol and 5H4 were administered after 7days of differentiation. MDA-MB-231-conditioned media doubled the differentiation of monocytes into osteoclasts. MDA-MB-231 secreted CSF-1, especially when cells were cultured to confluence. Induced osteoclasts were sensitive to bone-targeted drugs: Den and 5H4 blocked osteoclast differentiation and survival, while Zol induced osteoclast apoptosis. Osteoclasts differentiated by breast cancer cells were less sensitive to Zol than those induced by differentiation factors, whereas sensitivity to Den was similar. Conversely, breast cancer-induced osteoclast activation resulted in a higher sensitivity to 5H4. A significant increase in CSF-1 secretion was observed in osteoclast precursors after treatment with the highest concentration of Den. Further research is ongoing to evaluate the efficacy of 5H4 combination with Den.
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Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Osteoclastos/metabolismo , Osteoclastos/patologia , Fosfatase Ácida/metabolismo , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular Tumoral , Técnicas de Cocultura , Denosumab , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Fator Estimulador de Colônias de Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Osteoclastos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Fosfatase Ácida Resistente a TartaratoRESUMO
The aim of the present study was to identify sensitive and noninvasive biomarkers of early carcinogenic effect at target organ to use in biomonitoring studies of workers at risk for previous occupational exposure to potential carcinogens. Standard urine cytology (Papanicolaou staining test), comet assay, and quantitative telomerase repeat amplification protocol (TRAP) assay were performed in 159 ex-rubber workers employed in tyres production and 97 unexposed subjects. In TRAP positive cases, a second level analysis using FISH (Urovysion) was done. Cystoscopy results were available for 11 individuals whose 6 FISH/TRAP/comet positive showed in 3 cases a dysplastic condition confirmed by biopsy, 1 comet positive resulted in infiltrating UBC to the biopsy and with hyperplasia and slight dysplasia to the urinary cytology, 1 comet positive resulted in papillary superficial UBC to the biopsy, 1 FISH/TRAP positive showed a normal condition, and 2 TRAP positive showed in one case a phlogosis condition. The results evidenced good concordance of TRAP, comet, and FISH assays as early biomarkers of procarcinogenic effect confirmed by the dysplastic condition and UBC found by cystoscopy-biopsy analysis. The analysis of these markers in urine cells could be potentially more accurate than conventional cytology in monitoring workers exposed to mixture of bladder potential carcinogens.
Assuntos
Biomarcadores Tumorais/metabolismo , Dano ao DNA , Proteínas de Neoplasias/metabolismo , Exposição Ocupacional/efeitos adversos , Telomerase/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Indústria Química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , BorrachaRESUMO
Although combination chemotherapy and radiotherapy have become the standard of care in numerous tumors, the mechanisms of interaction are often still unclear. The purpose of this study was to analyze the efficacy of radiation treatment and cisplatin sequences and to investigate their mechanisms of interaction. Three melanoma cell lines were used to evaluate in vitro radiation-induced cytotoxicity before and after cisplatin treatment. Expression levels of a panel of genes were determined by real-time RT-PCR. Cytotoxic effect was evaluated by flow cytometry analysis and Comet assay. We also used normal human dermal fibroblasts (HUDE) to evaluate the cytotoxicity of the two treatments by clonogenic assay. Radiation and cisplatin used singly were not particularly effective in reducing proliferation in melanoma cells. Conversely, radiation treatment followed by cisplatin showed a strong synergistic interaction in all cell lines, with a ratio index ranging from 16 to >100. The synergistic effect was accompanied by apoptosis induction (up to 40%) and an increase in the percentage of comet-shaped nucleoids from 85% to 99%. In parallel, our results also showed that radiation treatment of HUDE fibroblasts followed by cisplatin only induced weak cytotoxicity. Our findings highlight the efficacy of the sequence radiation â cisplatin in reducing cell proliferation and in inducing apoptosis in melanoma cell lines. This sequence also modulated a network of proteins involved in DNA damage repair.
Assuntos
Antineoplásicos/farmacologia , Quimiorradioterapia/métodos , Cisplatino/farmacologia , Melanoma/patologia , Neoplasias Cutâneas/patologia , Antineoplásicos/toxicidade , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Quimiorradioterapia/efeitos adversos , Cisplatino/toxicidade , Ensaio Cometa , Dano ao DNA , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Esquema de Medicação , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Concentração Inibidora 50 , Melanoma/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Fatores de TempoRESUMO
Anti-EGFR therapy appears to be a potential treatment option for squamous cell anal carcinoma (SCAC). KRAS mutation is a rare event in SCAC, indicating the absence of the principal mechanism of resistance to this type of therapy. However, no information is available from the literature regarding the status of BRAF or PIK3CA in this cancer type. We analysed KRAS, BRAF and PIK3CA status in SCAC patients in relation to the clinical-pathological characteristics of patients and to the presence of the human papilloma virus (HPV). One hundred and three patients were treated with the Nigro scheme for anal cancer from March 2001 to August 2012. Fifty patients were considered for the study as there was insufficient paraffin-embedded tumour tissue to perform molecular analysis the remaining 53. DNA was extracted from paraffin-embedded sections. KRAS, BRAF and PIK3CA gene status and HPV genotype were evaluated by pyrosequencing. KRAS and BRAF genes were wild-type in all cases. Conversely, PIK3CA gene was found to be mutated in 11 (22%) cases. In particular, 8 mutations occurred in exon 9 and 3 in exon 20 of the PIK3CA gene. These findings suggest that SCAC could potentially respond to an anti-EGFR drug. PIK3CA mutation may be involved in the process of carcinogenesis in some cases of SCAC.