RESUMEN
OBJECTIVE: To determine factors that impact erythropoietin (EPO) production in leiomyomas. We have previously implicated EPO production in promoting the growth of some leiomyomas. DESIGN: The relationship between EPO messenger RNA (mRNA) expression and MED12 gene mutations or mRNA expression levels of high-mobility group AT-hook (HMGA) 1 and HMGA2 were analyzed. Effects of 10-8 M 17ß-E2 on EPO mRNA expression were evaluated using leiomyoma cells grown in primary cultures. SETTING: Graduate school of medicine. PATIENT(S): Patients with leiomyoma. INTERVENTION(S): We used tissue samples and clinical data of 108 patients with leiomyomas to analyze the relation between EPO mRNA expression and MED12 mutation. Tissue samples from another 10 patients with leiomyomas were collected for in vitro experimentation using primary cultures of leiomyoma and myometrial cells. MAIN OUTCOME MEASURE(S): Relations between EPO mRNA expression, MED12 exon 2 mutation, and HMGA1/HMGA2 mRNA expression levels in leiomyoma samplings, in addition to effects of estrogen (E) on EPO mRNA expression in cultures of leiomyoma cells. RESULT(S): The EPO mRNA level was threefold higher in leiomyomas with wild-type (vs. mutated) MED12 genes. There was no correlation between EPO and HMGA1 or HMGA2 mRNA expression levels. In wild-type MED12 leiomyomas only, E2 treatment produced a twofold increase in EPO mRNA expression, whereas mutated MED12 leiomyomas were unaffected. CONCLUSION(S): The EPO mRNA expression increased significantly after E2 treatment only in leiomyomas lacking MED12 mutations. In conjunction with prior evidence linking EPO mRNA expression levels and tumor size, E2-stimulated EPO mRNA expression may explain the marked growth disparities seen in these tumors.
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Biomarcadores de Tumor/biosíntesis , Eritropoyetina/biosíntesis , Leiomioma/metabolismo , Complejo Mediador/biosíntesis , ARN Mensajero/biosíntesis , Neoplasias Uterinas/metabolismo , Adulto , Biomarcadores de Tumor/genética , Eritropoyetina/genética , Estradiol/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Leiomioma/genética , Leiomioma/patología , Complejo Mediador/genética , Persona de Mediana Edad , Mutación/efectos de los fármacos , Mutación/genética , ARN Mensajero/genética , Carga Tumoral/efectos de los fármacos , Carga Tumoral/fisiología , Células Tumorales Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologíaRESUMEN
AIM: With the advent of the molecular-targeted therapy, rapid progress has been made in the treatment of advanced or recurrent non-small-cell lung cancer (NSCLC). Although surgical complete resection remains the standard and most promising treatment, the clinical significance of epidermal growth factor receptor (EGFR) gene mutations in early-stage NSCLC remains uncertain. METHODS: We investigated the prognostic value of EGFR mutations in surgically resected pathological stage I NSCLC. A total of 388 consecutive patients with NSCLC who underwent complete tumor resection in our hospital from 2006 through 2008 were studied retrospectively. Formalin-fixed, paraffin-embedded tissue sections were used to isolate DNA from carcinoma lesions. Mutational analyses of EGFR gene were performed by loop-hybrid mobility shift assay, a highly sensitive polymerase chain reaction-based method. RESULTS: Mutations of EGFR were detected in 185 of the 388 patients (47.7%). EGFR mutations were more frequently found in women (110 of 185, 59.5%), adenocarcinoma (183 of 185, 98.9%), patients with no vascular invasion (139 of 185, 75.1%) and nonsmokers (106 of 185, 57.3%). In patients with pathological stage I adenocarcinoma, both overall survival (OS) and disease-free survival (DFS) were significantly higher in patients with EGFR mutation than in those with wild-type EGFR. Furthermore, patients with exon 21 mutation have better DFS than those with exon 19 mutation in stage IB adenocarcinoma. Cox's proportional hazard model indicated that EGFR status was an independent variable for predicting the OS and DFS in patients with pathological stage IB adenocarcinoma. CONCLUSION: Our results suggest that EGFR mutations might be a prognostic factor in patients with pathological stage I lung adenocarcinoma.
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Adenocarcinoma/genética , Adenocarcinoma/cirugía , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirugía , Mutación , Adenocarcinoma/enzimología , Adenocarcinoma del Pulmón , Adulto , Anciano , Anciano de 80 o más Años , Supervivencia sin Enfermedad , Femenino , Humanos , Neoplasias Pulmonares/enzimología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Estudios Retrospectivos , Análisis de SupervivenciaRESUMEN
We report a very rare case of combined thymic carcinomas: undifferentiated thymic carcinoma coexisting with type AB thymoma. The precise mechanism underlying the coexistence of these tumors remains unknown. Therefore, we used clonality analysis to ascertain whether the two tumors were clonally related. A 63-year-old woman with thyroid cancer visited our hospital. Chest computed tomography also revealed an anterior mediastinal tumor. The patient was treated with total thyroidectomy and surgery for mediastinal tumors together with left upper lobe partial resection. The mediastinal tumor was pathologically diagnosed as undifferentiated thymic carcinoma coexisting with type AB thymoma. Multiple pulmonary metastases were detected in the patient and stage IV disease was diagnosed. The tumor was treatment-resistant, and the patient received fourth-line chemotherapy. We conducted clonality analysis using an improved human androgen receptor gene-amplification assay that involves random X-chromosome inactivation through methylation, followed by methylated gene-specific PCR amplification after sample DNA digestion with HpaII, a methylation-sensitive restriction enzyme. Clonality analysis demonstrated identical X-chromosome inactivation in cells present in both thymoma and thymic carcinoma areas, and thus revealed clonal proliferation. The two lesions in the patient might have arisen through the transformation of a preexisting thymoma into a more malignant lesion.
RESUMEN
BACKGROUND: The rapid aging of the population in Japan has been accompanied by an increased rate of surgery for lung cancer among elderly patients. It is thus an urgent priority to map out a treatment strategy for elderly patients with primary lung cancer. Although surgical resection remains standard treatment for early stage non-small-cell lung cancer (NSCLC), it is now essential to confirm the status of epidermal growth factor receptor (EGFR) gene mutations when planning treatment strategies. Furthermore, several studies have reported that EGFR mutations are an independent prognostic marker in NSCLC. However, the relations between age group and the molecular and pathological characteristics of NSCLC remain unclear. We studied the status of EGFR mutations in elderly patients with NSCLC and examined the relations of EGFR mutations to clinicopathological factors and outcomes according to age group. METHODS: A total of 388 consecutive patients with NSCLC who underwent complete tumor resection in our hospital from 2006 through 2008 were studied retrospectively. Formalin-fixed, paraffin-embedded tissue sections were used to isolate DNA from carcinoma lesions. Mutational analyses of EGFR gene exons 19, 20, and 21 and KRAS gene exons 12 and 13 were performed by loop-hybrid mobility shift assay, a highly sensitive polymerase chain reaction-based method. RESULTS: EGFR mutations were detected in 185 (47.7%) and KRAS mutations were detected in 33 (8.5%) of the 388 patients. EGFR mutations were found in a significantly higher proportion of patients younger than 80 years (younger group; 178/359, 49.6%) than in patients 80 years or older (older group; 7/29, 24.1%) (P = 0.008). In contrast, KRAS mutations were more common in the older group (6/29, 20.7%) than in the younger group (27/359, 7.5%) (P = 0.014). The older group showed a trend toward a higher rate of 5-year overall survival among elderly patients with EGFR mutations (100%) than among those with wild-type EGFR (66.2%), but the difference was not significant. CONCLUSIONS: Our results suggest that the EGFR status of patients with NSCLC differs between patients 80 years or older and those younger than 80 years. EGFR mutation status might be a prognostic marker in elderly patients with completely resected NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Exones , Humanos , Neoplasias Pulmonares/cirugía , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Análisis de Supervivencia , Proteínas ras/genéticaRESUMEN
Lung cancers harboring epidermal growth factor receptor (EGFR) mutations depend on constitutive activation of the kinase for survival. Although most EGFR-mutant lung cancers are sensitive to EGFR tyrosine kinase inhibitors (TKIs) and shrink in response to treatment, acquired resistance to TKI therapy is common. We demonstrate here that two EGFR-mutated lung adenocarcinoma cell lines, HCC827 and HCC4006, contain a subpopulation of cells that have undergone epithelial-to-mesenchymal transition and survive independent of activated EGFR. These EGFR-independent cancer cells, herein termed gefitinib-resistant (GR) cells, demonstrate higher levels of basal autophagy than their parental cells and thrive under hypoxic, reduced-serum conditions in vitro; this somewhat simulates the hypoxic environment common to cancerous tissues. We show that depletion of the essential autophagy gene, ATG5, by small interfering RNA (siRNA) or chloroquine, an autophagy inhibitor, markedly reduces GR cell viability under hypoxic conditions. Moreover, we show a significant elevation in caspase activity in GR cells following knockdown of ATG5. These results suggest that GR cells can evade apoptosis and survive in hostile, hypoxic environments with constant autophagic flux. We also show the presence of autophagosomes in some cancer cells from patient samples, even in untreated EGFR-mutant lung cancer tissue samples. Together, our results indicate that autophagy inhibitors alone or in combination with EGFR TKIs may be an effective approach for the treatment of EGFR-mutant lung cancers, where basal autophagy of some cancer cells is upregulated.
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Adenocarcinoma/metabolismo , Autofagia , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmón/metabolismo , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/metabolismo , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/ultraestructura , Adenocarcinoma del Pulmón , Adulto , Anciano , Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Proteína 5 Relacionada con la Autofagia , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular , Resistencia a Antineoplásicos , Transición Epitelial-Mesenquimal , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/ultraestructura , Masculino , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mutantes/antagonistas & inhibidores , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/ultraestructura , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARNRESUMEN
Epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cells in suspension undergo apoptosis to a greater extent than adherent cells in a monolayer when EGFR autophosphorylation is inhibited by EGFR tyrosine kinase inhibitors (TKIs). This suggests that cell adhesion to a culture dish may activate an anti-apoptotic signaling pathway other than the EGFR pathway. Since the microenvironment of cells cultured in a monolayer are substantially different to that of cells existing in three-dimension (3D) in vivo, we assessed whether two EGFR-mutant lung adenocarcinoma cell lines, HCC827 and H1975, were more resistant to EGFR TKI-induced apoptosis when cultured in a 3D extracellular matrix (ECM) as compared with in suspension. The ECM-adherent EGFR-mutant cells in 3D were significantly less sensitive to treatment with WZ4002, an EGFR TKI, than the suspended cells. Further, a marked degradation of IκBα, the inhibitor of nuclear factor (NF)-κB, was observed only in the 3D-cultured cells, leading to an increase in the activation of NF-κB. Moreover, the inhibition of NF-κB with pharmacological inhibitors enhanced EGFR TKI-induced apoptosis in 3D-cultured EGFR-mutant cells. These results suggest that inhibition of NF-κB signaling would render ECM-adherent EGFR-mutant lung adenocarcinoma cells in vivo more susceptible to EGFR TKI-induced cell death.
Asunto(s)
Adenocarcinoma/metabolismo , Apoptosis/genética , Resistencia a Antineoplásicos/genética , Receptores ErbB/genética , Neoplasias Pulmonares/metabolismo , FN-kappa B/metabolismo , Acrilamidas/farmacología , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Amidas/farmacología , Apoptosis/efectos de los fármacos , Adhesión Celular , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Receptores ErbB/antagonistas & inhibidores , Matriz Extracelular , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Neoplasias Pulmonares/genética , Mutación , FN-kappa B/agonistas , FN-kappa B/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Tiofenos/farmacologíaRESUMEN
BACKGROUND: Many studies on thyroid follicular tumors have reported the presence of somatic mutations to three forms of RAS: HRAS, KRAS, and NRAS. However, the frequency and clinical significance of these RAS mutations remain unclear, in large part due to the different methodologies being used for mutation analysis and the limited number of cases featured in studies. To clarify the significance of RAS mutations, we examined a large number of follicular adenomas and carcinomas obtained from a single institute using established methods for the analysis of RAS. METHODS: Tumor samples from 40 follicular adenoma and 58 follicular carcinoma patients treated at the Kanagawa Cancer Center Hospital were analyzed. The three RAS mutations at codons 12 and 61 were assessed with a polymerase chain reaction-based loop-hybrid mobility shift assay followed by confirmation with direct sequencing. The relationships between mutation status and clinicopathological features at the time of the initial operation and the prognosis of the patients were also analyzed. RESULTS: Twelve out of 40 (30%) adenomas harbored RAS mutations. In contrast, 33 out of 58 (57%) follicular carcinomas harbored RAS mutations, and the mutation was predominantly found in the NRAS codon 61 (22/33, 67%, p<0.01). The rate of gene mutations was significantly higher in the carcinomas than in the adenomas (p<0.01). The NRAS codon 61 mutation in follicular carcinomas was positively associated with distant metastases through the entire clinical course of the patients (p<0.05), and RAS mutations were associated with poor overall patient survival (p<0.05). CONCLUSIONS: We investigated the frequency of RAS mutations in follicular thyroid tumors from a large number of cases obtained from a single institute. The predominance of NRAS codon 61 mutations as a feature of carcinomas indicates that the diagnosis of adenoma alongside the presence of this mutation should be made cautiously. Our study raises the possibility that follicular adenomas with the RAS mutations have an inherent malignant potential; however, the clinical significance of this finding should be further investigated in more patients and over a longer follow-up period.
Asunto(s)
Adenocarcinoma Folicular/genética , Adenoma/genética , Genes ras , Mutación , Neoplasias de la Tiroides/genética , Adenocarcinoma Folicular/patología , Adenoma/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Neoplasias de la Tiroides/patologíaRESUMEN
Src has a role in the anoikis resistance in lung adenocarcinomas. We focused on two epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma cell lines, HCC827 (E746-A750 deletion) and H1975 (L858R+T790M), in suspension to elucidate whether suspended lung adenocarcinoma cells are eradicated by long-term treatment with Src tyrosine kinase inhibitors (TKIs). We also examined metastasis-positive lymph nodes from 16 EGFR-mutant lung adenocarcinoma patients for immunohistochemical expression of mutant-specific EGFR. Almost all suspended HCC827 cells underwent apoptosis after 144 h of combination treatment with AZD0530, trichostatin A (TSA), and ABT-263, whereas many suspended H1975 cells survived the treatment. AZD0530 is a Src TKI, TSA is a histone deacetylase inhibitor, and ABT-263 is a Bcl-2 inhibitor. During the therapy, the phosphorylation of EGFR decreased in HCC827 cells and remained stable in H1975 cells. The phosphorylated EGFR of Src TKI-resistant H1975 cells, as well as HCC827 cells, was completely suppressed by the third generation EGFR TKI, WZ4002. Consequently, both the suspended cell lines were almost completely eradicated within 144 h, with the combined therapy of WZ4002, ABT-263, and TSA. Interestingly, treated suspended cells underwent apoptosis to a greater extent than did adherent cells. Intrasinus floating lung adenocarcinoma cells in the lymph nodes expressed a mutant-specific EGFR. These findings suggest that suspended EGFR-mutant lung adenocarcinoma cells depend significantly more on EGFR activation for survival than attached cells do. The tumor cells circulating in vessels, which express mutant-specific EGFR, would be highly susceptible to the combination therapy of WZ4002, ABT-263, and TSA.
Asunto(s)
Acrilamidas/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Anoicis/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Pirimidinas/uso terapéutico , Acrilamidas/farmacología , Adenocarcinoma/genética , Anciano , Anciano de 80 o más Años , Compuestos de Anilina/farmacología , Compuestos de Anilina/uso terapéutico , Benzodioxoles/farmacología , Benzodioxoles/uso terapéutico , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Quimioterapia Combinada , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Fosforilación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Pirimidinas/farmacología , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Sulfonamidas/farmacología , Sulfonamidas/uso terapéutico , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
BACKGROUND: The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. METHODS: We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. RESULTS: Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. CONCLUSIONS: Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications.
Asunto(s)
Codón , Genes ras , Mutación , Secuencia de Bases , Neoplasias Colorrectales/genética , Cartilla de ADN , HumanosRESUMEN
BACKGROUND: Irinotecan, an inhibitor of topoisomerase I, has been widely used as an important anti-cancer therapeutic drug. Deleterious effects of the drug in hypersensitive patients are known to be associated with genetic polymorphisms of the UGT1A1 gene, namely the polymorphic variants, *28 and *6. METHODS: A modified form of loop-hybrid mobility shift assay using a Cy5-tagged loop-hybrid probe was proposed as a precise and easy method of determining TA repeat polymorphisms at the *28 locus. RESULTS: In this modified method, only loop-hybrid bands were detected by a Cy5-fluorescent signal, despite several irregular electrophoretic bands due to TA repeats in the PCR product. CONCLUSIONS: When a loop-hybrid using a Cy5-tagged probe for the *28 locus and *6 locus were combined and used for mobility shift assay, simultaneous typing of the *28 and *6 variants was achieved in a single lane.
Asunto(s)
Glucuronosiltransferasa/genética , Sondas Moleculares , Polimorfismo de Nucleótido Simple , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
MDM4, a homolog of MDM2, is considered a key negative regulator of p53. Gene amplification of MDM4 has been identified in a variety of tumors. MDM2 or MDM4 gene amplification is only associated with the wild-type TP53 gene in retinoblastomas, thus the amplification of the two genes is mutually exclusive. Previously, we demonstrated that MDM2 amplification and TP53 alteration were not mutually exclusive in colorectal cancer, and we identified a subset of colorectal cancer patients without alterations in either the TP53 or the MDM2 gene. In this study, we investigated the gene amplification status of MDM4 in the same set of colorectal cancer cases. Unexpectedly, MDM4 amplification was rare, detected in only 1.4% (3 out of 211) of colorectal cancer cases. All the three gene-amplified tumors also harbored TP53-inactivating mutations. This contradicts the simple mutually exclusive relationship observed in retinoblastomas. Surprisingly, two of the three MDM4-amplified tumors also demonstrated MDM2 amplification. Paradoxically, the MDM4 protein levels were decreased in the tumor tissue of the gene-amplified cases compared with levels in the matched normal mucosa. We speculate that MDM4 might play a role in colorectal carcinogenesis that is not limited to negative regulation of p53 in combination with MDM2. The functional significance of MDM4 is still unclear and further studies are needed.
Asunto(s)
Neoplasias Colorrectales/genética , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Genes p53 , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína p53 Supresora de Tumor/genética , Anciano , Anciano de 80 o más Años , Proteínas de Ciclo Celular , Neoplasias Colorrectales/metabolismo , Femenino , Humanos , Masculino , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Mutación , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The tyrosine kinase Src plays an important role in the development of anoikis resistance in lung adenocarcinomas. Several suspension lung adenocarcinoma cell lines, which express phosphorylated Src, undergo apoptosis, or anoikis, in the presence of Src kinase inhibitors. However, lung adenocarcinoma cell lines vary in their sensitivity to Src inhibitors. We hypothesized that the addition of ABT-263, a potent Bcl-2 inhibitor, should significantly enhance the degree of anoikis in lung adenocarcinoma cells treated with Src inhibitors. In this study, we treated four suspension lung adenocarcinoma cell lines with ABT-263, an Src inhibitor (bosutinib or PP1), or a combination of both. In LC-KJ and HCC827 cells, combined treatment with ABT-263 and an Src inhibitor effectively induced anoikis, and the extent of anoikis was significantly greater than that induced by each agent alone; the synergy between the two drugs was apparent. Although we did not observe a marked increase in anoikis in LC-KJ and HCC827 cells treated with ABT-263 alone, H1650 and H1975 cells treated with ABT-263 (1 µM) upon detachment significantly underwent apoptosis, the levels of which were much greater than those observed upon attachment. However, the levels of anoikis induced by combination treatment were still greater than those by the individual agents in H1650 and H1975 cells. These findings provide a biological rationale to test combination therapy with ABT-263 and Src inhibitors in patients with lung adenocarcinoma.
Asunto(s)
Compuestos de Anilina/farmacología , Anoicis/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas pp60(c-src)/antagonistas & inhibidores , Sulfonamidas/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Compuestos de Anilina/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/administración & dosificaciónRESUMEN
The fusion gene EML4-ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) was recently identified as a novel genetic alteration in non-small-cell lung cancer. The clinicopathological features of EML4-ALK-positive adenocarcinoma are reported to include its high incidence in young, non-smoking patients, tumors that show distinct solid or acinar growth patterns with or without signet-ring cell histology, and its mutually exclusive occurrence with mutations in EGFR and KRAS. However, the clinical findings have not been well described. Here, we report a case of EML4-ALK-positive lung adenocarcinoma that showed multiple metachronous lesions on the pleura and pulmonary field, suspected to be a recurrence of lung adenocarcinoma after a 20-year disease-free interval. The slow clinical course may be characteristic of EML4-ALK-positive lung adenocarcinoma. Therefore, long-term observation of patients with EML4-ALK-positive lung adenocarcinomas is required after surgery.
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Adenocarcinoma/diagnóstico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pleurales/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Adulto , Biomarcadores de Tumor/genética , Diagnóstico Diferencial , Receptores ErbB/genética , Femenino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/fisiopatología , Mutación/genética , Recurrencia Local de Neoplasia , Proteínas de Fusión Oncogénica/genética , Neoplasias Pleurales/genética , Neoplasias Pleurales/patología , Neoplasias Pleurales/fisiopatología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , Factores de Tiempo , Tomografía Computarizada por Rayos X , Proteínas ras/genéticaRESUMEN
MDM2 is a crucial negative regulator of the TP53 tumor suppressor and almost 10% of human tumors exhibit MDM2 amplification. Although TP53 pathway perturbation has been extensively examined in colorectal cancer (CRC), only one previous report has evaluated MDM2 amplification in relation to clinicopathological factors. In that report, MDM2 amplification was shown to be associated with disease progression from Dukes' Stages A to D. In this study, we investigated MDM2 amplification by quantitative PCR and fluorescence in situ hybridization (FISH) together with the SNP309 genotypes, and analyzed the correlations with TP53 and KRAS mutations and clinicopathological features in 211 Japanese CRC patients. MDM2 amplification was detected in 8% of the specimens and its incidence was significantly higher in Dukes' stage C than in the combined earlier Stages A and B (P = 0.025). Unexpectedly, the incidence was significantly decreased in Stage D metastatic disease (P = 0.043). The copy number gain ranged from four to eight copies and was generally concordant with gain of centromere 12 using FISH analysis. Together with the results of centromere 1 FISH and TP53 copy number assessment, the MDM2 increment most likely resulted from chromosome 12 gain. The mechanism of the copy number gain and incidence in Dukes' Stage D differed considerably from the previous report. Ethnic or geographic factors could be responsible for these differences. Several promising therapeutic strategies targeting the TP53-MDM2 system are being developed. Further understanding of the significance of MDM2 and MDM2 amplification in CRC is required to facilitate personalized treatment for CRC patients.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Amplificación de Genes , Genes ras , Genotipo , Humanos , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas c-mdm2RESUMEN
The ability to resist anoikis is critical for carcinoma cells to metastasize. Although several lung adenocarcinoma cell lines were shown to repress anoikis through the activation of Src, it remains unknown whether Src actually plays a crucial role in anoikis resistance in lung adenocarcinoma tissues. We examined 20 human lung adenocarcinoma tissues with lymphatic permeation and nine cell lines to investigate whether intralymphatic floating carcinoma cells in the tissues, used as an in vivo model of anoikis resistance, actually suppressed anoikis and whether cell lines in suspension culture, an in vitro model of anoikis resistance, survived through Src activation. We observed that the intralymphatic carcinoma cells aggregated tightly to form nests expressing E-cadherin and phosphorylated Src (p-Src). The apoptotic indices of these cells were comparable to those of extracellular matrix adhesive cells in all tissues, indicating that the intralymphatic cells actually evaded anoikis. Next, we found that the nine cell lines in suspension aggregated loosely (five cell lines) or tightly (four cell lines), and all cells resisted anoikis. Upon detachment, four cell lines (LC-KJ, HCC827, H1650, and H1975) formed compact spheroids that expressed E-cadherin and p-Src. The spheroids were similar to intralymphatic tumour nests and were thus considered to be a suitable model of the nests. The spheroids of the four cell lines underwent apoptosis after treatment with the Src/Abl/Kit inhibitor PP1 or Src/Abl inhibitor bosutinib. On the other hand, the Abl/Kit inhibitor imatinib did not affect cell growth or apoptosis in the four types of spheroids. These results indicate that Src, but not Abl or Kit, plays an essential role in the development of anoikis resistance in lung adenocarcinomas.
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Adenocarcinoma/patología , Anoicis/fisiología , Neoplasias Pulmonares/patología , Vasos Linfáticos/patología , Células Neoplásicas Circulantes/patología , Familia-src Quinasas/metabolismo , Adenocarcinoma/metabolismo , Adulto , Anciano , Compuestos de Anilina/farmacología , Anoicis/efectos de los fármacos , Antineoplásicos/farmacología , Benzamidas , Cadherinas/metabolismo , Femenino , Humanos , Mesilato de Imatinib , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Células Neoplásicas Circulantes/metabolismo , Nitrilos/farmacología , Proteínas Oncogénicas v-abl/antagonistas & inhibidores , Fosforilación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Quinolinas/farmacología , Esferoides Celulares , Células Tumorales Cultivadas , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
BACKGROUND: Although postoperative chemotherapy is widely accepted as the standard modality for Dukes' stage C or earlier stage colorectal cancer (CRC) patients, biomarkers to predict those who may benefit from the therapy have not been identified. Previous in vitro and clinical investigations reported that CRC patients with wild-type p53 gene (TP53)-tumors benefit from 5-fluorouracil (5-FU) based chemotherapy, while those with mutated TP53-tumors do not. However, these studies evaluated the mutation-status of TP53 by immunohistochemistry with or without single-strand conformation polymorphism, and the mutation frequency was different from study to study. In addition, the polymorphic status at p53 codon 72, which results in arginine or proline residues (R72P) and is thought to influence the function of the protein significantly, was not examined. METHODS: To evaluate the significance of the TP53 mutation as a molecular marker to predict the prognosis of CRC patients, especially those who received postoperative chemotherapy, we examined the mutation by direct sequencing from fresh CRC tumors and evaluated the R72P polymorphism of the mutated TP53 by a combined mutant allele- and polymorphic allele-specific polymerase chain reaction (PCR). RESULTS: The TP53 mutation occurred in 147 (70%) of 211 Japanese CRC tumors. The mutation was observed in 93 (63%) tumors on the R72 allele and in 54 (37%) tumors on the P72 allele. Although the alterations to TP53 have no prognostic significance for CRC patients overall, we found that Dukes' stage C CRC patients who did not receive postoperative chemotherapy and carried the mutated TP53-R72 showed significantly longer survival times than those with the mutated TP53-P72 when evaluated by overall survival (p = 0.012). CONCLUSION: Using a combined mutant allele- and polymorphic allele-specific PCR, we defined the codon 72 polymorphic status of the TP53 mutated allele in Japanese CRC patients. We raised a possibility that Dukes' stage C colorectal cancer patients with tumors carrying TP53 mutation, especially the P72 allele, benefited from 5-FU based postoperative chemotherapy.
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Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Fluorouracilo/uso terapéutico , Genes p53 , Anciano , Alelos , Biomarcadores de Tumor/genética , Quimioterapia Adyuvante , Codón/genética , Neoplasias Colorrectales/patología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Prolina/genéticaRESUMEN
Diagnostic criteria to identify small lung adenocarcinomas that relapse after resection have yet to be established. For this purpose, we developed a mathematical logistic model in the present study. We collected data for patients with lung adenocarcinoma of 2 cm or less in size: the original cohort comprised 28 men and 25 women and the validation cohort comprised 11 men. By entering five clinicopathological factors (vascular invasion, lymphatic permeation, histological subtype, papillary carcinoma component, and smoking status) into the logistic model, we calculated a predictive function for relapse after surgery. The obtained predictive function accurately classified the patients into a recurrence or non-recurrence group: the overall accuracy of the predictive model for recurrence established from the male patients in the original cohort was 86%. Our predictive model is, however, currently limited to male patients only, because the original cohort included only one female patient with relapse. By applying the logistic model to the validation cohort, six patients were classified into a recurrence group and the other five into a non-recurrence group: four of the six patients in a recurrence group had relapsed, while all five patients in the non-recurrence group were well during their follow-up periods. Although the predictive ability of the logistic model did not reach a statistical significance (P=0.0606), nine of the 11 (82%) patients in the validation cohort were correctly classified. Consequently, using a logistic predictive model consisting of the five clinicopathological factors might enable us to predict the recurrence of resected small-sized lung adenocarcinomas in male patients.
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Adenocarcinoma/cirugía , Neoplasias Pulmonares/cirugía , Modelos Estadísticos , Adenocarcinoma/patología , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Cohortes , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Modelos Logísticos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Reproducibilidad de los Resultados , Factores Sexuales , Resultado del Tratamiento , Adulto JovenRESUMEN
About 10-40% of testicular germ cell tumours (TGCTs) have been reported to have activating c-kit gene mutations. A European study group has reported that most bilateral TGCTs from European patients have c-kit mutations at codon 816, although few unilateral cases harbour the mutations. This implies that the presence of a c-kit mutation in a unilateral TGCT predicts the development of TGCT in the contralateral testis (bilateral disease). However, since little is known about on c-kit gene mutational frequencies in bilateral TGCTs from patients of Asian origin, we examined 12 bilateral TGCTs from seven Japanese patients, along with 39 unilateral TGCTs from Japanese patients, for the presence of c-kit mutations. We analyzed c-kit exons 11 and 17 by PCR followed by direct sequencing, and also analyzed the hotspot mutations at codon 816 by loop-hybrid mobility shift assay, a sensitive PCR-based method. We found c-kit mutations in seven of 39 (18%) unilateral TGCTs: two of the seven mutations were in exon 11 and the others, including four point mutations at codon 816, were in exon 17. No mutations, however, were observed in bilateral TGCTs. Thus, the presence of c-kit gene mutations in TGCTs may not be associated with bilateral diseases, at least in Japan.
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Pueblo Asiatico/genética , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias de Células Germinales y Embrionarias/genética , Proteínas Proto-Oncogénicas c-kit/genética , Seminoma/genética , Neoplasias Testiculares/genética , Adulto , Análisis Mutacional de ADN , Exones , Humanos , Japón , Masculino , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Activating epidermal growth factor receptor (EGFR) gene mutations are frequently detected in lung adenocarcinomas, especially adenocarcinomas with a nonmucinous bronchioloalveolar carcinoma component. EGFR-mutated lung adenocarcinomas respond well to EGFR tyrosine kinase inhibitors. We previously found that most (88%) pure nonmucinous bronchioloalveolar carcinomas (adenocarcinoma in situ) already harbor EGFR mutations, indicating that the mutations are an early genetic event in the pathogenesis. We examined 54 atypical adenomatous hyperplasias, precursor lesions of lung adenocarcinomas, obtained from 28 Japanese patients for the hotspot mutations of EGFR exons 19 and 21 and K-ras codon 12. EGFR mutations were observed in 17 of the 54 (32%) atypical adenomatous hyperplasias examined: Ten and seven atypical adenomatous hyperplasias had deletion mutations at exon 19 or point mutations (L858R) at exon 21, respectively. We did not observe apparent histological differences between atypical adenomatous hyperplasias with and without EGFR mutations. K-ras mutation (G12S) was detected in only one atypical adenomatous hyperplasia. As EGFR mutational frequency of atypical adenomatous hyperplasias was much lower than that of nonmucinous bronchioloalveolar carcinomas, we surmise that EGFR-mutated atypical adenomatous hyperplasias, but not atypical adenomatous hyperplasias with wild-type EGFR, are likely to progress to nonmucinous bronchioloalveolar carcinomas.
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Adenocarcinoma Bronquioloalveolar/genética , Adenomatosis Pulmonar/genética , Receptores ErbB/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/genética , Mutación , Adenocarcinoma Bronquioloalveolar/patología , Adenomatosis Pulmonar/patología , Adulto , Anciano , Pueblo Asiatico/genética , Codón , Análisis Mutacional de ADN , Progresión de la Enfermedad , Exones , Femenino , Genes ras , Humanos , Hiperplasia , Japón , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: The presence of somatic mutations in epidermal growth factor receptor (EGFR) predicts the effectiveness of EGFR tyrosine kinase inhibitors (TKIs). It would be ideal if an EGFR mutation could be detected in biopsy samples, since the majority of non-small cell lung cancer patients are inoperable at the time of presentation. We have reported the usefulness of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the lymph node staging of lung cancer. EBUS-TBNA enables the sampling of histologic cores, which can be used for genetic analysis. METHODS: The purpose of this study was to develop and analyze the feasibility of detecting EGFR mutations in samples obtained by EBUS-TBNA. Forty-six patients with primary lung cancer in whom metastatic adenocarcinoma in the hilar and/or mediastinal lymph node was diagnosed by EBUS-TBNA were enrolled into the study. DNA was extracted from paraffin-embedded samples, and the EGFR mutation was analyzed in exons 19 and 21 using a newly developed loop-hybrid mobility shift assay. The results were confirmed by direct sequencing. RESULTS: Forty-three cases were eligible for analysis and in 11 cases, EGFR mutation (25.6%) was detected; one case was an in-frame deletion (E746-A750del) of exon 19, nine cases were point mutations (L858R) of exon 21, and one case was a double point mutation (L858R+L861V). All cases with EGFR mutations were confirmed by direct sequencing. CONCLUSIONS: EGFR mutation can easily be detected in metastatic lymph nodes sampled by EBUS-TBNA. EBUS-TBNA allows genetic evaluations of tumor cells within the lymph node and may provide us with indications for EGFR-TKI therapy in the near future.