RESUMEN
In colorectal cancer, KRAS (exons 2, 3, and 4) and NRAS (exons 2, 3, and 4) mutations are associated with resistance to antiepidermal growth factor receptor monoclonal antibodies, and BRAF mutation is a molecular marker of poor prognosis. KRAS exon 2 and BRAF-mutated colorectal cancers have well-known distinct clinicopathological characteristics. Comparison of tumors with different RAS status (exons 2, 3, and 4 of KRAS and NRAS) based on their clinicopathological characteristics has never been established. All colorectal cancer patients with RAS and BRAF testing from 2011 to 2015 were included in this observational retrospective study. Patient and tumor characteristics were collected and correlation with RAS and BRAF status was evaluated. A total of 1735 patients with colorectal cancer were included. RAS-mutated colorectal cancers (n=1002), compared with RAS wild-type colorectal cancers (n=733), were significantly associated with male gender, classical adenocarcinoma subtype, well/moderately differentiated tumors, and microsatellite stable phenotype. KRAS codon 13-mutated colorectal cancers (n=171), compared with RAS wild-type colorectal cancers, more frequently presented classical adenocarcinoma subtype and microsatellite stable phenotype. In comparison with other RAS mutations, KRAS exon 3-mutated colorectal cancers (n=23) were associated with mucinous/rare histological subtypes and, most likely to located in the rectum. KRAS exon 4-mutated colorectal cancers (n=33) were more frequently associated with mucinous/rare histological subtypes. There was no significant association between NRAS mutation (n=37) and clinicopathological features. Colorectal cancers are associated with different clinicopathological features according to the type of RAS mutation. Consequently, these particular characteristics must be considered when assessing the prognostic value of RAS status in colorectal cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , GTP Fosfohidrolasas/genética , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Pronóstico , Estudios RetrospectivosRESUMEN
In advanced colorectal carcinoma (CRC) patients, extended RAS mutations testing (KRAS exons 2 to 4 and NRAS exons 2 to 4) is a prerequisite for patient stratification to anti-EGFr therapy. Accurately distinguishing mutant patients from potential responders has a clinically critical impact, and thus effective and low cost methods are needed for identification of the mutation status. We have developed quantitative pyrosequencing assays for sensitive and rapid detection of mutant RAS alleles in formalin-fixed, paraffin-embedded tissues. Exons 2 to 4 of KRAS and NRAS genes were PCR amplified and analyzed by pyrosequencing. For validation, PCR products were sequenced by conventional Sanger sequencing. Analytical sensitivity of these assays was determined by calculating the limit of detection. The results showed that low levels of mutant RAS alleles (2-13%) can be detected with pyrosequencing assays.
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Neoplasias Colorrectales/genética , Análisis Mutacional de ADN/métodos , Receptores ErbB/genética , Pruebas Genéticas/métodos , Mutación/genética , Reacción en Cadena de la Polimerasa/métodos , Proteínas ras/genética , Neoplasias Colorrectales/diagnóstico , Neoplasias Colorrectales/tratamiento farmacológico , Humanos , Evaluación de Resultado en la Atención de Salud , Adhesión en ParafinaRESUMEN
Glioblastoma (GBM) is the most malignant type of primary brain tumor with a very poor prognosis. The actual standard protocol of treatment for GBM patients consists of radiotherapy and concomitant temozolomide (TMZ). However, the therapeutic efficacy of this treatment is limited due to tumor recurrence and TMZ resistance. Recently isolated, glioma stem-like cells (GSCs) are thought to represent the population of tumorigenic cells responsible for GBM resistance and recurrence following surgery and chemotherapy. In addition, MGMT (O6-methylguanine-methyltransferase) methylation is considered as one of the principal mechanisms contributing to TMZ sensitivity of GBM. In this study we have isolated GSCs from 10 adult GBM patients and investigated the relationship between MGMT methylation status and Temozolomide (TMZ) sensitivity of these lines grown either in stem-like or differentiation promoting conditions. Sensitivity to TMZ was significantly associated with MGMT methylation status in cells committed to differentiation but not in stem-like cells. In addition, patients harboring highly methylated MGMT promoters had a longer overall survival. These results reveal the importance of the differentiation process when considering the predictive value of MGMT status in GSCs for clinical response to TMZ.
Asunto(s)
Neoplasias Encefálicas/genética , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Glioma/genética , Células Madre Neoplásicas/citología , Regiones Promotoras Genéticas , Proteínas Supresoras de Tumor/genética , Anciano , Algoritmos , Diferenciación Celular , Línea Celular Tumoral , Dacarbazina/análogos & derivados , Dacarbazina/química , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Microscopía Confocal , Persona de Mediana Edad , Análisis de Secuencia de ADN , TemozolomidaRESUMEN
Glioblastoma (GBM), the highest-grade form of gliomas, is the most frequent and the most aggressive. Recently, a subpopulation of cells with stem cells characteristics, commonly named "tumor-initiating stem cells" (TISCs) or "cancer stem cells" (CSCs) were identified in GBM. These cells were shown to be highly resistant to chemotherapeutic drugs and to ionizing radiations. Consequently, the knowledge of the signals that regulate the functions and survival of TISCs is crucial. In our work, we describe a neurosphere-initiating cell (NS-IC) assay to quantify TISC/CSCs from patients with GBM and show that these cells are tumorigenic in vivo. We demonstrate that the intracellular signal transducer and activator of transcription STAT3 is constitutively activated by phosphorylation preferentially on serine 727 in these cells. Moreover, we demonstrate that the selective inhibition of STAT3 by the chemical compound Stattic or by siRNA STAT3 abrogates TISC/CSC proliferation and NS-IC suggesting that self-renewal of GBM "stem-like" cells depends on the presence of STAT3 for their maintenance. Finally, we show that inhibition of STAT3 by Stattic sensitizes TISC/CSCs to the inhibitory action of Temozolomide with a strong synergistic effect of both drugs. Overall, these results suggest that strategies focused on STAT3 inhibition are efficient at the level of "stem-like" cells and could be of interest for therapeutic purposes in patients with malignant GBM.
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Glioblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Antineoplásicos Alquilantes/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Óxidos S-Cíclicos/farmacología , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Sinergismo Farmacológico , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glioblastoma/genética , Glioblastoma/patología , Humanos , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/patología , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/antagonistas & inhibidores , Temozolomida , Células Tumorales CultivadasRESUMEN
BACKGROUND: In most countries, participation in colorectal cancer (CRC) screening programs with the immunological fecal occult blood test (iFOBT) is low. Mutations of RAS and BRAF occur early in colorectal carcinogenesis and "liquid biopsy" allows detection of mutated circulating tumor DNA (ctDNA). This prospective study aims to evaluate the performance of RAS and BRAF-mutated ctDNA in detecting CRC and advanced adenomas (AA). METHODS: One hundred and thirty patients who underwent colonoscopy for suspicion of colorectal lesion were included and divided into four groups: 20 CRC, 39 AA, 31 non-advanced adenoma and/or hyperplastic polyp(s) (NAA) and 40 with no lesion. Mutated ctDNA was analyzed by droplet digital PCR. RESULTS: ctDNA was detected in 45.0% of CRC, in 2.6% of AA and none of the NAA and "no-lesion" groups. All patients with stage II to IV mutated CRC had detectable ctDNA (n = 8/8). Among the mutated AA, only one patient had detectable ctDNA (4.3%), maybe due to limited technical sensitivity or to a low rate of ctDNA or even the absence ctDNA in plasma. Specificity and sensitivity of KRAS- and BRAF-mutated ctDNA for the detection of all CRC and AA were 100% and 16.9%, respectively. CONCLUSIONS: ctDNA had high sensitivity in detection of advanced mutated CRC but was unable to sensitively detect AA. ctDNA analysis was easy to perform and readily accepted by the population but requires combination with other circulating biomarkers before replacing iFOBT.
RESUMEN
Background: Colorectal cancers (CRC) with brain metastases (BM) are scarcely described. The main objective of this study was to determine the molecular profile of CRC with BM. Methods: We included 82 CRC patients with BM. KRAS, NRAS, BRAF and mismatch repair (MMR) status were investigated on primary tumors (n = 82) and BM (n = 38). ALK, ROS1, cMET, HER-2, PD-1, PD-L1, CD3 and CD8 status were evaluated by immunohistochemistry, and when recommended, by fluorescence in situ hybridization. Results: In primary tumors, KRAS, NRAS and BRAF mutations were observed in 56%, 6%, and 6% of cases, respectively. No ROS1, ALK and cMET rearrangement was detected. Only one tumor presented HER-2 amplification. Molecular profiles were mostly concordant between BM and paired primary tumors, except for 9% of discordances for RAS mutation. CD3, CD8, PD-1 and PD-L1 expressions presented some discordance between primary tumors and BM. In multivariate analysis, multiple BM, lung metastases and PD-L1+ tumor were predictive of poor overall survival. Conclusions: CRCs with BM are associated with high frequency of RAS mutations and significant discordance for RAS mutational status between BM and paired primary tumors. Multiple BM, lung metastases and PD-L1+ have been identified as prognostic factors and can guide therapeutic decisions for CRC patients with BM.
RESUMEN
Glioblastoma stem cells (GSCs) are believed to be involved in the mechanisms of tumor resistance, therapeutic failures, and recurrences after conventional glioblastoma therapy. Therefore, elimination of GSCs might be a prerequisite for the development of successful therapeutic strategies. ALK, ROS1, and MET are targeted by Crizotinib, a tyrosine kinase inhibitor which has been approved for treatment of ALK-rearranged non-small-cell lung cancer. In this study we investigated ALK, ROS1, and MET status in nine glioblastoma stem cell lines and tumors from which they arise. Fluorescent in situ hybridization (FISH), Sanger's direct sequencing, and immunohistochemistry were used to screen genomic rearrangements (or amplifications), genomic mutations, and protein expression, respectively. The immunohistochemical and FISH studies revealed no significant dysregulation of ROS1 in GSCs and associated tumors. Neither amplification nor polysomy of ALK was observed in GSC, but weak overexpression was detected by IHC in three of nine GSCs. Similarly, no MET amplification was found by FISH but three GSCs presented significant immunohistochemical staining. No ALK or MET mutation was found by Sanger's direct sequencing. In this study, we show no molecular rearrangement of ALK, ROS1, and MET that would lead us not to propose, as a valid strategy, the use of crizotinib to eradicate GSCs. However, MET was overexpressed in all GSCs with mesenchymal subtype and three GSCs presented an overexpression of ALK. Therefore, our study corroborates the idea that MET and ALK may assume a role in the tumorigenicity of GSC.
Asunto(s)
Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas , Proteínas Proto-Oncogénicas c-met , Proteínas Proto-Oncogénicas , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Proteínas Tirosina Quinasas Receptoras , Anciano , Quinasa de Linfoma Anaplásico , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/genética , Neoplasias del Sistema Nervioso Central/metabolismo , Crizotinib , Análisis Mutacional de ADN , Femenino , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Células Madre Neoplásicas/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Pirazoles/metabolismo , Piridinas/metabolismo , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismoRESUMEN
Two cases of atypical chronic myeloid leukaemia (CML) carrying the t(4;22)(q12;q11) translocation involving the breakpoint cluster region (BCR) and platelet-derived growth factor alpha receptor (PDGFRA) genes have been recently characterized. We report a third case of atypical CML with the same translocation but with a distinct breakpoint fusing BCR exon 1 with PDGFRA exon 13. The patient had a clinical presentation of CML with progressive transformation in B-cell acute lymphoblastic leukaemia. The involvement of PDGFRA led us to treat the patient with the small organic compound imatinib mesylate/STI571 (Glivec) that blocks the ATP binding site of tyrosine kinases such as Abelson, KIT and platelet-derived growth factor receptors. The patient subsequently achieved a rapid clinical and molecular response clearly demonstrating, for the first time, that Glivec is active against PDGFRA in vivo. Therefore, our study expands the list of Glivec targets and has direct biological and also clinical implications.
Asunto(s)
Reordenamiento Génico , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Piperazinas/farmacología , Pirimidinas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Benzamidas , Cromosomas Humanos Par 22 , Cromosomas Humanos Par 4 , Proteínas de Fusión bcr-abl/genética , Humanos , Mesilato de Imatinib , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Masculino , Persona de Mediana Edad , Translocación GenéticaRESUMEN
FISH and multicolor FISH (M-FISH) techniques have greatly enhanced the resolution of conventional cytogenetic analysis, thus enabling the identification of novel regions of rearrangement in hematological malignancies. We report on the analysis of cells from 24 chronic myelogenous leukemia (CML) patients, in either accelerated phase (14 cases) or blast crisis (10 cases) aimed at searching for previously unidentified additional abnormalities related to disease evolution. Indeed, in 6 of 24 cases (25%) M-FISH allowed a more precise description of chromosomal aberrations, the finding of cryptic rearrangements, characterization of markers, identification of additional material and a better interpretation of complex aberrations. However, new recurrent aberration did not emerge from M-FISH analysis.
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Aberraciones Cromosómicas , Bandeo Cromosómico , Hibridación Fluorescente in Situ , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Recent efforts have been made to isolate molecular targets that could explain different outcome between histological subtypes of lymphomas and to understand the molecular mechanisms underlying oncogenic events. Using the SSH technique, we compared the transcriptome of 2 cases of ALK+ and ALK- anaplastic large cell lymphoma (ALCL) and of 2 cases of classical Hodgkin's lymphoma (cHL) with opposite behavior. Regarding ALCL, we showed that ALK-positive tumors overexpressed genes involved in different signaling pathways such as activation or signaling of T-cells, regulation of apoptosis, phospholipase Cgamma and phosphatidyl inositol-3 Kinase. In addition, the characterization of a specific molecular signature may be of clinical relevance since ALK+ tumors generally have a better prognosis than ALK- ones. Similar problems of differential prognosis is observed in cases of cHL, which in addition, may be morphologically and immunologically indistinguishable. Therefore, we applied the same SSH technique to 2 cHL samples from patients with favorable and poor outcome, respectively. Forty-four cDNAs were significantly overexpressed in the poor outcome case. In addition to the defender against death cell 1 (DAD1) gene, overexpressed clones corresponded mostly to expressed sequence tags (ESTs). Interestingly, the present study identifies new genes which may be involved in the pathogenesis and/or clinical outcome of cHL and deserve further investigations.
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Perfilación de la Expresión Génica , Enfermedad de Hodgkin/genética , Linfoma Anaplásico de Células Grandes/genética , Enfermedad de Hodgkin/diagnóstico , Humanos , Linfoma Anaplásico de Células Grandes/diagnóstico , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , PronósticoRESUMEN
It is now well established that metastatic colorectal cancer patients without KRAS mutation (codon 12) benefit from treatment with an epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb). Recently, EFGR and KRAS mutations have been shown to exist in patients who developed resistance to anti-EGFR mAb. We analyzed KRAS, BRAF V600E and EGFR S492R mutations in 37 post-anti-EGFR mAb tumor samples from 23 patients treated with chemotherapy plus anti-EGFR mAb. No EGFR S492R mutation was detected. A KRAS mutation was found after anti-EGFR mAb in only one tumor. Our results suggest that acquired EGFR S492R and KRAS mutations do not constitute the main mechanism of resistance to anti-EGFR mAb in combination with chemotherapy.
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Antineoplásicos/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Receptores ErbB/genética , Receptores ErbB/inmunología , Mutación/genética , Mutación/fisiología , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Anciano , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/patología , Terapia Combinada , Progresión de la Enfermedad , Femenino , Humanos , Neoplasias Hepáticas/secundario , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas p21(ras) , Resultado del TratamientoRESUMEN
The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER-2 mutations in brain metastases from non-small cell lung carcinomas (BM-NSCLC). A total of 77 samples of BM-NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER-2 mutations in BM-NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM-NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM-NSCLC whereas KRAS mutations are as frequent in BM-NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundario , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas/genética , Receptor ErbB-2/genética , Proteínas ras/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/patología , Femenino , Genes ras , Humanos , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Proteínas Proto-Oncogénicas p21(ras)RESUMEN
The murine equivalent of neutrophil gelatinase-associated lipocalin (NGAL) was previously found to be increased by BCR-ABL expression in murine models of chronic myeloid leukemia (CML). Our study evaluates, in CML patients at various clinical stages, the levels of NGAL mRNA in blood samples and protein in sera. A highly significant increase of mRNA expression and protein secretion was shown in patients at diagnosis. The parallel expression of NGAL and BCR-ABL at the early stage of CML process allows us to suggest that NGAL could play an important role in the physiopathology of CML.
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Proteínas de Fusión bcr-abl/análisis , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Lipocalinas/sangre , Proteínas Proto-Oncogénicas/sangre , Proteínas de Fase Aguda/genética , Adulto , Células Cultivadas , Expresión Génica , Granulocitos , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Lipocalina 2 , Lipocalinas/genética , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/sangreRESUMEN
In this study, we used subtractive suppression hybridization to compare gene expression between an ALK-positive anaplastic large cell lymphoma (ALCL)-derived cell line and a clinical case of ALK-negative ALCL. Construction and screening of a subtracted library resulted in the cloning of 29 cDNAs which were differentially expressed. Most of these clones corresponded to novel genes with unknown function (EST) or to genes implicated in the differentiation, activation or signalling of T cells such as Ran/TC4, interleukin 1-receptor, thymosin beta4, thymosin beta10, moesin and cytohesin-1. Other genes involved in the regulation of apoptosis, such as human inhibitor of apoptosis-1 (HIAP-1), Bax inhibitor-1 and MCL-1, or DNA repair, such as poly (ADP-ribose) polymerase 1 (PARP-1), X-associated protein-1 (XAP-1), SUMO-1 (sentrin-1) and RanGTPase-activating protein 1 (RanGAP-1), were isolated. Interestingly, we found that both RNA and protein levels of human sterol isomerase (hSI), also referred to as emopamil binding protein (EBP), were overexpressed in ALK+ tumours. This protein is involved in the biosynthesis of cholesterol and may be activated by NPM-ALK. Overall, our results suggest that all the genes described above are upregulated in the NPM-ALK-driven transformation process, and that moesin and cytohesin-1 may be more specifically implicated in a signalling pathway involving PLCgamma and PI3K.