RESUMO
PURPOSE: RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. MATERIALS: We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. RESULTS: 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. CONCLUSION: Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias do Sistema Nervoso Central/secundário , Rearranjo Gênico , Receptores Proteína Tirosina Quinases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Quinase do Linfoma Anaplásico , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias do Sistema Nervoso Central/genética , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
BACKGROUND: The mitogen-activated protein kinases 1 and 2 (MEK1, MEK2) are fundamental partners in the RAS-RAF-MEK-ERK pathway that is involved in regulation of cell proliferation, differentiation and survival. Downregulation of the MEK cascades has been implicated in acquiring of the malignant phenotype in various cancers. Somatic mutations in MEK1 gene (substitutions K57N, Q56P, D67N) were described in <1 % of non-small cell lung cancer (NSCLC) and they were more commonly reported in adenocarcinoma patients with current or former smoking status. MATERIALS AND METHODS: In the following study, we assessed the MEK1 gene mutations in 145 FFPE tissue samples from central nervous system (CNS) metastases of NSCLC using HRM-PCR and ASP-qPCR techniques. The studied group was heterogeneous in terms of histopathology and smoking status. The prevalence of the MEK1 gene mutation was correlated with the occurrence of mutations in KRAS, EGFR, DDR2, PIK3CA, NRAS, HER2, AKT1 and PTEN genes. RESULTS: Using HRM and ASP-qPCR methods we identified one (0.7 %; 1/145) MEK1 substitution (Q56P) in CNS metastases of NSCLC. The mutation was identified in a single, 50-year-old, current smoking men with adenocarcinoma (1.25 %; 1/80 of all adenocarcinomas). CONCLUSIONS: According to the current knowledge, the incidence of MEK1 gene mutation in CNS metastatic lesion of NSCLC is the first such report worldwide. The analysis of gene profile in cancer patients may extend the scope of molecularly targeted therapies used both in patients with primary and metastatic tumors of NSCLC.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias do Sistema Nervoso Central/genética , Análise Mutacional de DNA/métodos , Neoplasias Pulmonares/genética , MAP Quinase Quinase 1/genética , Mutação/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Neoplasias do Sistema Nervoso Central/secundário , Detecção Precoce de Câncer/métodos , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Reação em Cadeia da Polimerase em Tempo RealRESUMO
INTRODUCTION: The possibility of detection of suppressor genes methylation in circulating free DNA (cf-DNA) of cancer patients and the lack of methylation in healthy individuals makes this epigenetic alternation an ideal diagnostic marker of neoplastic processes. Moreover, hypermethylation in several genes promoter was described as a biomarker of lung cancer. Methylation in the gene encoding doublecortin-like kinase 1 (DCLK1) is observed in patients with colorectal cancer and cholangiocarcinoma. However, there are no studies concerning DCLK1 methylation in lung cancer patients. The aims of the study was to evaluate the frequency of DCLK1 promoter methylation in cf-DNA of lung cancer patients and of healthy persons as well as the usefulness of this test for predicting the lung cancer course. MATERIALS AND METHODS: DCLK1 methylation status was evaluated in DNA isolated from peripheral blood plasma from 65 lung cancer patients and 95 healthy individuals. After DNA bisulfitation, DCLK1 methylation was determined using the qMSP-PCR technique. Moreover, the presence of DCLK1 methylation was correlated with the overall survival (OS) probability of lung cancer patients. RESULTS: DCLK1 promoter methylation was detected in 32 lung cancer patients (49.2 %) and 8 healthy individuals (8.4 %). The methylation of the region before transcription start site (TSS) and the region after TSS of DCLK1 gene was detected in 28 and 11 patients, respectively. In seven cases (10.8 %), the DCLK1 promoter methylation in both regions was reported simultaneously. The methylation was observed slightly frequently in patients with small cell lung cancer (75 % of SCLC patients). The median overall survival of patients with DCLK1 promoter methylation was lower than that of patients without DCLK1 gene modification (p = <0.001, HR = 4.235). CONCLUSIONS: The evaluation of DCLK1 promoter region methylation may be useful in both early diagnosis and prediction of the course of lung cancer.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Metilação de DNA , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Células Neoplásicas Circulantes/patologia , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Adenocarcinoma/sangue , Adenocarcinoma/genética , Adenocarcinoma/patologia , Biomarcadores Tumorais/sangue , Carcinoma de Células Grandes/sangue , Carcinoma de Células Grandes/genética , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Estudos de Casos e Controles , Quinases Semelhantes a Duplacortina , Feminino , Seguimentos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Serina-Treonina Quinases/sangue , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de SobrevidaRESUMO
PURPOSE: Chemotherapy with platinum compounds and gemcitabine is frequently used in first-line treatment of advanced non-small cell lung cancer (NSCLC) patients in which tyrosine kinase inhibitors (EGFR or ALK) cannot be administered. Unfortunately, less than half of the patients achieve the benefit from chemotherapy. Gemcitabine is an analog of deoxycytidine (pyrimidine antimetabolite) with antitumor activity. The excess of deoxycytidine synthesized by RRM1 enzyme activity may be a cause of competitive displacement of gemcitabine, which reduces the efficacy of this cytostatic. The aim of this study was to determine the association between single nucleotide polymorphisms (SNPs) of the RRM1 promoter (-37C>A, -524C>T) and the effectiveness of first-line chemotherapy based on platinum compounds and gemcitabine in NSCLC patients. PATIENTS AND METHODS: SNPs were determined by SNaPshot PCR(®) in DNA isolated from peripheral blood of 91 NSCLC patients. RESULTS: The median progression-free survival (PFS) was significantly longer in carriers of AA (-37C>A) as well as CC (-524C>T) genotype of RRM1 compared to patients with other genotypes (10.5 vs 3.5 months, p = 0.0437; HR = 2.17, 95 % CI 1.02-4.62 and 10.5 vs 3.5 months, p = 0.0343; HR = 2.12, 95 % CI 1.06-4.27). In addition, the CC genotype carriers (-37C>A) showed a significant increase in the risk of shortening overall survival (OS) in comparison to patients with AA or AC genotypes (9.5 vs 18 months, p = 0.0193; HR = 2.13, 95 % CI 1.13-4.03). CONCLUSIONS: Presence of rare AA (-37C>A) and CC (-524C>T) genotypes of the RRM1 may be favorable predictive factors for chemotherapy with platinum compounds and gemcitabine in NSCLC patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Desoxicitidina/uso terapêutico , Intervalo Livre de Doença , Feminino , Genótipo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Compostos de Platina/administração & dosagem , Regiões Promotoras Genéticas , Modelos de Riscos Proporcionais , Ribonucleosídeo Difosfato Redutase , GencitabinaRESUMO
PURPOSE: Second-line chemotherapy of advanced non-small cell lung cancer (NSCLC) with docetaxel or pemetrexed allows to achieve objective response rate only in 5-10 % of patients. Recent studies have shown that single nucleotide polymorphisms (SNPs) in genes encoding proteins which regulate dynamics of microtubules may be considered as predictive factors of response to taxane-based chemotherapy. STMN1 gene encodes stathmin 1, which plays role in cell division by regulation of microtubules depolarisation, and this process may be associated with taxanes' effectiveness. MATERIALS AND METHODS: Using HRM-PCR technique, we evaluated the -2166C>T SNP of STMN1 gene in DNA from peripheral blood leucocytes of 54 advanced NSCLC patients treated in second-line monotherapy with docetaxel or paclitaxel. RESULTS: Patients with TT genotype of STMN1 gene demonstrated significantly longer progression-free survival (PFS) and the lower risk of early disease progression after second-line treatment compared to patients with other STMN1 genotypes (median PFS: 7 and 2 months; p = 0.0154; HR = 0.371; 95 % CI 0.184-0.743). Early disease progression during second-line chemotherapy was significantly more frequently observed in patients with CC genotype of STMN1 in contrast to patients with presence of T allele (median PFS: 2 and 4 months; p = 0.0385; HR = 1.776; 95 % CI 0.905-3.445). CONCLUSION: Only selected NSCLC patients could benefit from second-line chemotherapy. Therefore, investigations of novel predictive molecular factors for proper qualification of patients to second-line taxane-based chemotherapy are justified. Studied SNP of STMN1 gene may have potential predictive role in such therapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Paclitaxel/uso terapêutico , Estatmina/genética , Taxoides/uso terapêutico , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Quimioterapia Adjuvante , Progressão da Doença , Docetaxel , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Estudos Retrospectivos , Taxoides/administração & dosagem , Resultado do TratamentoRESUMO
INTRODUCTION: Platinum-based chemotherapy and 3rd generation drugs is still the main treatment option for advanced non-small cell lung cancer (NSCLC) patients without activating EGFR mutations or ALK rearrangements. However, the side effects associated with cytostatics are well known. Changes in the genes (e.g. single nucleotide polymorphisms, SNPs) encoding proteins regulating DNA repair or cell division could potentially influence on both the susceptibility of cancer cells to chemotherapy, and the occurrence of toxicities. MATERIALS AND METHODS: In presented study, the relationship between the fourteen SNPs in nine DNA repair and cell division regulating genes: ERCC1, XPD, XPA, XPC, XRCC1, XPG, RRM1, BRCA1, STMN1 and the toxicity of first-line chemotherapy in NSCLC patients were investigated. SNPs were determined by SNaPshot PCR® in DNA isolated from peripheral blood of 55 NSCLC patients treated with platinum compound and vinorelbine. The toxicity of therapy was evaluated according to the Common Toxicity Criteria (CTC) Version 4.03. RESULTS: The odds ratio (OR) of severe haematological toxicity was significantly lower in carriers of the T allele of XRCC1 gene (1196A > G, OR = 0.22, 95 % CI: 0.06-0.82, p = 0.018) and higher in the carriers of the T allele (2704C > A) of XPC gene (OR: 7.50, 95 % CI: 0.89-63.17, p = 0.036) compared to the remaining patients. Risk of severe hepatotoxicity was significantly lower in carriers of the C allele of STMN1 (-2166T > C, OR = 0.09, 95 % CI: 0.01-1.12, p = 0.025) than in patients with T allele of this gene. In carriers of G allele (2251A > C, OR: 0.24, 95 % CI: 0.07-0.81, p = 0.017) and T (934G > A, OR: 0.26, 95 % CI: 0.07-0.90, p = 0.029) of XPD gene, risk of severe nephrotoxicity was significantly lower than in other patients. CONCLUSIONS: Selected SNPs of genes encoding DNA repair enzymes and cell division regulation proteins could be useful biomarkers for prediction of platinum and vinorelbine-based chemotherapy toxicity in patients with advanced NSCLC.
Assuntos
Antineoplásicos/efeitos adversos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Divisão Celular/genética , Reparo do DNA/genética , Neoplasias Pulmonares/tratamento farmacológico , Polimorfismo de Nucleotídeo Único , Idoso , Carcinoma Pulmonar de Células não Pequenas/genética , Feminino , Genótipo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Razão de Chances , Compostos de Platina/efeitos adversos , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Vimblastina/efeitos adversos , Vimblastina/análogos & derivados , VinorelbinaRESUMO
In this study, the one- and two-photon absorption spectra of seven azoaromatic compounds (five pseudostilbenes-type and two aminoazobenzenes) were theoretically investigated using the density functional theory combined with the response functions formalism. The equilibrium molecular structure of each compound was obtained at three different levels of theory: Hartree-Fock, density functional theory (DFT), and Møller-Plesset 2. The effect of solvent on the equilibrium structure and the electronic transitions of the compounds were investigated using the polarizable continuum model. For the one-photon absorption, the allowed pi-->pi(*) transition energy showed to be dependent on the molecular structures and the effect of solvent, while the n-->pi(*) and pi-->pi(*)(n) transition energies exhibited only a slight dependence. An inversion between the bands corresponding to the pi-->pi(*) and n-->pi(*) states due to the effect of solvent was observed for the pseudostilbene-type compounds. To characterize the allowed two-photon absorption transitions for azoaromatic compounds, the response functions formalism combined with DFT using the hybrid B3LYP and PBE0 functionals and the long-range corrected CAM-B3LYP functional was employed. The theoretical results support the previous findings based on the three-state model. The model takes into account the ground and two electronic excited states and has already been used to describe and interpret the two-photon absorption spectrum of azoaromatic compounds. The highest energy two-photon allowed transition for the pseudostilbene-type compounds shows to be more effectively affected (approximately 20%) by the torsion of the molecular structure than the lowest allowed transition (approximately 10%). In order to elucidate the effect of the solvent on the two-photon absorption spectra, the lowest allowed two-photon transition (dipolar transition) for each compound was analyzed using a two-state approximation and the polarizable continuum model. The results obtained reveal that the effect of solvent increases drastically the two-photon cross-section of the dipolar transition of the pseudostilbene-type compounds. In general, the features of both one- and two-photon absorption spectra of the azoaromatic compounds are well reproduced by the theoretical calculations.