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BACKGROUND: The EGFR pathway is involved in intrinsic and acquired resistance to a wide variety of targeted therapies in cancer. Vaccination represents an alternative to the administration of anti-EGFR monoclonal antibodies, such as cetuximab or panitumumab. Here, we tested if anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) could potentiate the activity of drugs targeting the ERK/MAPK and PI3K/Akt pathways. METHODS: Non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and melanoma cell lines harboring KRAS, NRAS, BRAF and PIK3CA mutations were used. Anti-EGF VacAbs were obtained by immunizing rabbits with a fusion protein containing a synthetic, highly mutated variant of human EGF. Cell viability was determined by MTT, total and phosphorylated proteins by Western blotting, cell cycle distribution and cell death by flow cytometry and emergence of resistance by microscopic examination in low density cultures. RESULTS: Anti-EGF VacAbs potentiated the antiproliferative effects of MEK, KRAS G12C, BRAF, PI3K and Akt inhibitors in KRAS, NRAS, BRAF and PIK3CA mutant cells and delayed the appearance of resistant clones in vitro. The effects of anti-EGF VacAbs were comparable or superior to those of panitumumab and cetuximab. The combination of anti-EGF VacAbs with the targeted inhibitors effectively suppressed EGFR downstream pathways and sera from patients immunized with an anti-EGF vaccine also blocked activation of EGFR effectors. CONCLUSIONS: Anti-EGF VacAbs enhance the antiproliferative effects of drugs targeting the ERK/MAPK and PIK3CA/Akt pathways. Our data provide a rationale for clinical trials testing anti-EGF vaccination combined with inhibitors selected according to the patient's genetic profile.
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BACKGROUND: Drugs targeting the spindle assembly checkpoint (SAC), such as inhibitors of Aurora kinase B (AURKB) and dual specific protein kinase TTK, are in different stages of clinical development. However, cell response to SAC abrogation is poorly understood and there are no markers for patient selection. METHODS: A panel of 53 tumor cell lines of different origins was used. The effects of drugs were analyzed by MTT and flow cytometry. Copy number status was determined by FISH and Q-PCR; mRNA expression by nCounter and RT-Q-PCR and protein expression by Western blotting. CRISPR-Cas9 technology was used for gene knock-out (KO) and a doxycycline-inducible pTRIPZ vector for ectopic expression. Finally, in vivo experiments were performed by implanting cultured cells or fragments of tumors into immunodeficient mice. RESULTS: Tumor cells and patient-derived xenografts (PDXs) sensitive to AURKB and TTK inhibitors consistently showed high expression levels of BH3-interacting domain death agonist (BID), while cell lines and PDXs with low BID were uniformly resistant. Gene silencing rendered BID-overexpressing cells insensitive to SAC abrogation while ectopic BID expression in BID-low cells significantly increased sensitivity. SAC abrogation induced activation of CASP-2, leading to cleavage of CASP-3 and extensive cell death only in presence of high levels of BID. Finally, a prevalence study revealed high BID mRNA in 6% of human solid tumors. CONCLUSIONS: The fate of tumor cells after SAC abrogation is driven by an AURKB/ CASP-2 signaling mechanism, regulated by BID levels. Our results pave the way to clinically explore SAC-targeting drugs in tumors with high BID expression.
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Neoplasias , Proteínas Serina-Treonina Quinasas , Humanos , Animales , Ratones , Proteínas Serina-Treonina Quinasas/genética , Aurora Quinasa B/genética , Aurora Quinasa B/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Línea Celular Tumoral , ARN Mensajero , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas de Ciclo Celular/genéticaRESUMEN
BACKGROUND: The analysis of liquid biopsies brings new opportunities in the precision oncology field. Under this context, extracellular vesicle circular RNAs (EV-circRNAs) have gained interest as biomarkers for lung cancer (LC) detection. However, standardized and robust protocols need to be developed to boost their potential in the clinical setting. Although nCounter has been used for the analysis of other liquid biopsy substrates and biomarkers, it has never been employed for EV-circRNA analysis of LC patients. METHODS: EVs were isolated from early-stage LC patients (n = 36) and controls (n = 30). Different volumes of plasma, together with different number of pre-amplification cycles, were tested to reach the best nCounter outcome. Differential expression analysis of circRNAs was performed, along with the testing of different machine learning (ML) methods for the development of a prognostic signature for LC. RESULTS: A combination of 500 µL of plasma input with 10 cycles of pre-amplification was selected for the rest of the study. Eight circRNAs were found upregulated in LC. Further ML analysis selected a 10-circRNA signature able to discriminate LC from controls with AUC ROC of 0.86. CONCLUSIONS: This study validates the use of the nCounter platform for multiplexed EV-circRNA expression studies in LC patient samples, allowing the development of prognostic signatures.
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EGFR tyrosine kinase inhibitor (TKI) resistance in non-small cell lung cancer (NSCLC) patients is inevitable. Identification of resistance mechanisms and corresponding targeting strategies can lead to more successful later-line treatment in many patients. Using spectrometry-based proteomics, we identified increased fibroblast growth factor receptor 1 (FGFR1) expression and Akt activation across erlotinib, gefitinib, and osimertinib EGFR-TKI-resistant cell line models. We show that while combined EGFR-TKI and FGFR inhibition showed some efficacy, simultaneous inhibition of FGFR and Akt or PI3K induced superior synergistic growth inhibition of FGFR1-overexpressing EGFR-TKI-resistant NSCLC cells. This effect was confirmed in vivo. Only dual FGFR and Akt inhibition completely blocked the resistance-mediating signaling pathways downstream of Akt. Further, increased FGFR1 expression was associated with significantly lower PFS in EGFR-TKI-treated NSCLC patients, and increased FGFR1 were demonstrated in a few post- vs. pre-EGFR-TKI treatment clinical biopsies. The superior therapeutic benefit of combining FGFR and Akt inhibitors provide the rationale for clinical trials of this strategy.
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Advanced NSCLC patients harboring EML4-ALK and CCDC6-RET rearrangements derive benefit from treatment with ALK and RET TKIs but not immune checkpoint inhibitors. New immunotherapeutic approaches, such as immunization against growth factors, can be of particular interest for combination treatment in these patients. Here, we investigated the effects of anti-EGF antibodies generated by vaccination (anti-EGF VacAbs), TKIs and combinations in EML4-ALK and CCDC6-RET NSCLC cell lines. We found that EGF and tumor growth factor alpha (TGFα) significantly decreased the antiproliferative activity of the RET inhibitor BLU-667 in CCDC6-RET cells and brigatinib, alectinib and crizotinib in EML4-ALK translocated cells. The addition of anti-EGF VacAbs reversed the effects of EGF and TGFα, potentiated the antitumor effects of the kinase inhibitors and delayed the appearance in vitro of resistant clones. Western blotting demonstrated that the combination of anti-EGF VacAbs with ALK or RET TKIs effectively suppressed EGFR downstream pathways in EML4-ALK translocated and CCDC6-RET cells, respectively. In conclusion, anti-EGF VacAbs significantly increased the antitumor activity of TKIs in ALK and RET-positive cell lines. Clinical trials of an EGF vaccine in combination with ALK and RET TKIs are warranted.
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Endometrial cancer (EC) is the most common gynecologic malignancy in developed countries. Although most patients are diagnosed at early stages, 15-20% will relapse despite local treatment. Presently, there are no reliable markers to identify patients with worse outcomes who may benefit from adjuvant treatments, such as chemotherapy, and liquid biopsies may be of use in this setting. Peritoneal lavages are systematically performed during endometrial surgery but little data are available about their potential as liquid biopsies. We analyzed KRAS and PIK3CA mutations in paired surgical biopsies, blood and cytology-negative peritoneal lavages in a cohort of 50 EC patients. Surgical biopsies were submitted to next-generation sequencing (NGS) while circulating-free DNA (cfDNA) purified from plasma and peritoneal lavages was analyzed for KRAS and PIK3CA hotspot mutations using a sensitive quantitative polymerase chain reaction (PCR) assay. NGS of biopsies revealed KRAS, PIK3CA or concomitant KRAS + PIK3CA mutations in 33/50 (66%) EC patients. Of those, 19 cases carried hotspot mutations. Quantitative PCR revealed KRAS and/or PIK3CA mutations in the lavages of 9/19 (47.4%) hotspot EC patients. In contrast, only 2/19 (10.5%) blood samples from hotspot EC patients were positive. Mutations found in cfDNA consistently matched those in paired biopsies. One of the two patients positive in plasma and lavage died in less than 6 months. In conclusion, mutational analysis in peritoneal lavages and blood from early stage EC is feasible. Further studies are warranted to determine if it might help to identify patients with worse prognosis. Human genes discussed: KRAS, KRAS proto-oncogene, GTPase; PIK3CA, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.
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Neoplasias Endometriales/genética , Mutación , Anciano , Anciano de 80 o más Años , Alelos , Fosfatidilinositol 3-Quinasa Clase I/genética , Análisis Mutacional de ADN , ADN Circular/sangre , ADN Circular/genética , Neoplasias Endometriales/sangre , Neoplasias Endometriales/patología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biopsia Líquida , Persona de Mediana Edad , Lavado Peritoneal/métodos , Prueba de Estudio Conceptual , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas p21(ras)/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Introduction: The therapy of patients with lung adenocarcinoma has significantly changed after the discovery of epidermal growth factor receptor (EGFR) mutations. EGFR mutations occur in 10-15% of Caucasian lung cancer patients and are associated with favorable outcome to orally administered EGFR tyrosine kinase inhibitors (TKIs), like erlotinib. However, as soon as the tumor cells are under the pressure of the specific inhibitor, compensatory signaling pathways are activated and resistance emerges. Areas covered: In this review we will focus on the mechanisms of resistance to the first-generation EGFR TKI, erlotinib, and will mainly summarize the findings throughout the last 10 years in the field of EGFR-mutant lung cancer. Expert opinion: Widespread research has been performed and several mechanisms of resistance to EGFR TKIs, especially first- and second-generation, have been identified. Still, no adequate combinatory therapies have received regulatory approval for the treatment of EGFR-mutant patients at the time of resistance. The third-generation EGFR TKI, osimertinib has been approved for patients whose tumor has become resistant through the secondary T790M resistant EGFR mutation. The identification of the mechanisms of resistance and the application of the adequate therapy to each patient is still an unmet need.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Resistencia a Antineoplásicos , Clorhidrato de Erlotinib/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/genética , Reparación del ADN , Receptores ErbB/genética , Humanos , Neoplasias Pulmonares/genética , MutaciónRESUMEN
Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations.
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Antineoplásicos/farmacología , Aurora Quinasa B/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/uso terapéutico , Aurora Quinasa B/antagonistas & inhibidores , Aurora Quinasa B/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/genética , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Histonas/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Ratones , Persona de Mediana Edad , Organofosfatos/farmacología , Organofosfatos/uso terapéutico , Fosforilación/efectos de los fármacos , Poliploidía , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Análisis de Supervivencia , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: In a significant percentage of advanced non-small cell lung cancer (NSCLC) patients, tumor tissue is unavailable or insufficient for genetic analyses at time to progression. We prospectively analyzed the appearance of genetic alterations associated with resistance in liquid biopsies of advanced NSCLC patients progressing to targeted therapies using the NGS platform. METHODS: A total of 24 NSCLC patients were included in the study, 22 progressing to tyrosine kinase inhibitors and two to other treatments. Liquid biopsies samples were obtained and analyzed using the GeneReadTM QIAact Lung DNA UMI Panel, designed to enrich specific target regions and containing 550 variant positions in 19 selected genes frequently altered in lung cancer tumors. Previously, a retrospective validation of the panel was performed in clinical samples. RESULTS: Of the 21 patients progressing to tyrosine kinase inhibitors with valid results in liquid biopsy, NGS analysis identified a potential mechanism of resistance in 12 (57%). The most common were acquired mutations in ALK and EGFR, which appeared in 8/21 patients (38%), followed by amplifications in 5/21 patients (24%), and KRAS mutations in one patient (5%). Loss of the p.T790M was also identified in two patients progressing to osimertinib. Three of the 21 (14%) patients presented two or more concomitant alterations associated with resistance. Finally, an EGFR amplification was found in the only patient progressing to immunotherapy included in the study. CONCLUSIONS: NGS analysis in liquid biopsies of patients progressing to targeted therapies using the GeneReader platform is feasible and can help the oncologist to make treatment decisions.
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INTRODUCTION: Mutations in EGFR correlate with impaired response to immune checkpoint inhibitors and the development of novel immunotherapeutic approaches for EGFR mutant NSCLC is of particular interest. Immunization against epidermal growth factor (EGF) has shown efficacy in a phase III trial including unselected NSCLC patients, but little was known about the mechanisms involved in the effects of the anti-EGF antibodies generated by vaccination (anti-EGF VacAbs) or their activity in tumor cells with EGFR mutations. METHODS: The EGFR-mutant, NSCLC cell lines H1975, and PC9, together with several gefitinib and osimertinib-resistant cells derived from PC9, were treated with anti-EGF VacAbs and/or EGFR tyrosine kinase inhibitors (TKIs). Cell viability was analyzed by proliferation assays, cell cycle by fluorescence-activated cell sorting analysis, and levels of RNA and proteins by quantitative retro-transcription polymerase chain reaction and Western blotting. RESULTS: Anti-EGF VacAbs generated in rabbits suppressed EGF-induced cell proliferation and cycle progression and inhibited downstream EGFR signaling in EGFR-mutant cells. Sera from patients immunized with an EGF vaccine were also able to block activation of EGFR effectors. In combination, the anti-EGF VacAbs significantly enhanced the antitumor activity of all TKIs tested, suppressed Erk1/2 phosphorylation, blocked the activation of signal transducer and activator of transcription 3 (STAT3) and downregulated the expression of AXL receptor tyrosine kinase (AXL). Finally, anti-EGF VacAbs significantly delayed the emergence in vitro of EGFR TKI resistant clones. CONCLUSIONS: EGFR-mutant patients can derive benefit from immunization against EGF, particularly if combined with EGFR TKIs. A phase I trial of an EGF vaccine in combination with afatinib has been initiated.
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Neoplasias Pulmonares/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/patología , Mutación , Inhibidores de Proteínas Quinasas/farmacología , ConejosRESUMEN
Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Análisis Mutacional de ADN , Resistencia a Antineoplásicos , Activación Enzimática , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Ratones , Ratones SCID , Mutación , Trasplante de Neoplasias , Proteómica , Transducción de SeñalRESUMEN
Liquid biopsy analyses are already incorporated in the routine clinical practice in many hospitals and oncology departments worldwide, improving the selection of treatments and monitoring of lung cancer patients. Although they have not yet reached its full potential, liquid biopsy-based tests will soon be as widespread as "standard" biopsies and imaging techniques, offering invaluable diagnostic, prognostic, and predictive information. This review summarizes the techniques available for the isolation and analysis of circulating free DNA and RNA, exosomes, tumor-educated platelets, and circulating tumor cells from the blood of cancer patients, presents the methodological challenges associated with each of these materials, and discusses the clinical applications of liquid biopsy testing in lung cancer.
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BIM is a proapoptotic protein that initiates apoptosis triggered by EGFR tyrosine kinase inhibitors (TKI). mTOR negatively regulates apoptosis and may influence response to EGFR TKI. We examined mRNA expression of BIM and MTOR in 57 patients with EGFR-mutant NSCLC from the EURTAC trial. Risk of mortality and disease progression was lower in patients with high BIM compared with low/intermediate BIM mRNA levels. Analysis of MTOR further divided patients with high BIM expression into two groups, with those having both high BIM and MTOR experiencing shorter overall and progression-free survival to erlotinib. Validation of our results was performed in an independent cohort of 19 patients with EGFR-mutant NSCLC treated with EGFR TKIs. In EGFR-mutant lung adenocarcinoma cell lines with high BIM expression, concomitant high mTOR expression increased IC50 of gefitinib for cell proliferation. We next sought to analyse the signalling pattern in cell lines with strong activation of mTOR and its substrate P-S6. We showed that mTOR and phosphodiesterase 4D (PDE4D) strongly correlate in resistant EGFR-mutant cancer cell lines. These data suggest that the combination of EGFR TKI with mTOR or PDE4 inhibitors could be adequate therapy for EGFR-mutant NSCLC patients with high pretreatment levels of BIM and mTOR.
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Proteínas Reguladoras de la Apoptosis/genética , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Clorhidrato de Erlotinib/uso terapéutico , Neoplasias Pulmonares/genética , Proteínas de la Membrana/genética , Mutación/genética , Proteínas Proto-Oncogénicas/genética , Serina-Treonina Quinasas TOR/genética , Anciano , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Estudios de Cohortes , Supervivencia sin Enfermedad , Clorhidrato de Erlotinib/farmacología , Femenino , Gefitinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Proteínas Proto-Oncogénicas/metabolismo , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/metabolismo , Resultado del TratamientoRESUMEN
Testing of tumor tissue remains the recommended method for detecting the presence of somatic mutations in human malignancies. V600E is the most frequent somatic point mutation in metastatic melanoma, providing a unique molecular marker for this malignancy. In addition, tumors carrying this mutation are primary candidates for BRAF-targeted therapy. Although metastatic melanoma patients usually have sufficient tumor tissue available for genetic analyses, the detection of V600E in blood can have prognostic and predictive value. In addition, patients are rarely re-biopsied and genetic testing in blood can be useful for monitoring response to therapy. Cell-free DNA (cfDNA) and cell-free RNA (cfRNA), RNA associated to platelets and circulating tumor cells (CTCs) are some of the materials that can be derived from the blood of cancer patients. cfDNA can be easily purified from serum and plasma and contains DNA fragments of tumor origin. For this reason, it is the most widely used material for the detection of somatic mutations in blood. Several methodologies have been used to determine V600E status in the cfDNA of metastatic melanoma and some studies have demonstrated that the identification and follow-up of V600E in cfDNA can have prognostic and predictive value.
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BRAFV600E is a unique molecular marker for metastatic melanoma, being the most frequent somatic point mutation in this malignancy. Detection of BRAFV600E in blood could have prognostic and predictive value and could be useful for monitoring response to BRAF-targeted therapy. We developed a rapid, sensitive method for the detection and quantification of BRAFV600E in circulating free DNA (cfDNA) isolated from plasma and serum on the basis of a quantitative 5'-nuclease PCR (Taqman) in the presence of a peptide-nucleic acid. We validated the assay in 92 lung, colon, and melanoma archival serum and plasma samples with paired tumor tissue (40 wild-type and 52 BRAFV600E). The correlation of cfDNA BRAFV600E with clinical parameters was further explored in 22 metastatic melanoma patients treated with BRAF inhibitors. Our assay could detect and quantify BRAFV600E in mixed samples with as little as 0.005% mutant DNA (copy number ratio 1 : 20 000), with a specificity of 100% and a sensitivity of 57.7% in archival serum and plasma samples. In 22 melanoma patients treated with BRAF inhibitors, the median progression-free survival was 3.6 months for those showing BRAFV600E in pretreatment cfDNA compared with 13.4 months for those in whom the mutation was not detected (P=0.021). Moreover, the median overall survival for positive versus negative BRAFV600E tests in pretreatment cfDNA differed significantly (7 vs. 21.8 months, P=0.017). This finding indicates that the sensitive detection and accurate quantification of low-abundance BRAFV600E alleles in cfDNA using our assay can be useful for predicting treatment outcome.
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Análisis Mutacional de ADN/métodos , ADN de Neoplasias/sangre , Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Adulto , Anciano , Anciano de 80 o más Años , Antineoplásicos/uso terapéutico , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Melanoma/tratamiento farmacológico , Melanoma/mortalidad , Persona de Mediana Edad , Mutación , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Resultado del TratamientoRESUMEN
PURPOSE: TP53 mutations in early-stage non-small cell lung cancer (NSCLC) may be associated with worse survival but their prognostic role in advanced NSCLC is controversial. In addition, it remains unclear whether mutated patients represent a clinically homogeneous group. EXPERIMENTAL DESIGN: We retrospectively examined TP53 mutations and outcome in a training cohort of 318 patients with stage IIIB-IV NSCLC: 125 epidermal growth factor receptor (EGFR) wild-type (wt) and 193 EGFR mutated (mut). An independent validation cohort of 64 EGFR-mut patients was subsequently analyzed. Mutations were classified as "disruptive" and "nondisruptive" according to their predicted degree of disturbance of the p53 protein structure and function. RESULTS: In the training cohort, TP53 mutations were found in 43 of the 125 EGFR-wt patients (34.4%). Of these, 28 had nondisruptive TP53 mutations and a median overall survival (OS) of 8.5 months, compared with 15.6 months for the remaining 97 patients (P=0.003). In the EGFR-mut group, TP53 mutations were found in 50 of the 193 patients (25.9%). The OS for the 26 patients with TP53 nondisruptive mutations was 17.8 months versus 28.4 months for the remaining 167 patients (P=0.04). In the validation cohort, the 11 patients with nondisruptive TP53 mutations had a median OS of 18.1 months compared with 37.8 months for the 53 remaining patients (P=0.006). In multivariate analyses, nondisruptive TP53 mutations had an independent, significant association with a shorter OS. CONCLUSIONS: Nondisruptive mutations in the TP53 gene are an independent prognostic factor of shorter survival in advanced NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas/genética , Mutación/genética , Pronóstico , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia sin Enfermedad , Receptores ErbB , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de NeoplasiasRESUMEN
PURPOSE: Concomitant genetic alterations could account for transient clinical responses to tyrosine kinase inhibitors of the EGF receptor (EGFR) in patients harboring activating EGFR mutations. EXPERIMENTAL DESIGN: We have evaluated the impact of pretreatment somatic EGFR T790M mutations, TP53 mutations, and Bcl-2 interacting mediator of cell death (BCL2L11, also known as BIM) mRNA expression in 95 patients with EGFR-mutant non-small-cell lung cancer (NSCLC) included in the EURTAC trial (trial registration: NCT00446225). RESULTS: T790M mutations were detected in 65.26% of patients using our highly sensitive method based on laser microdissection and peptide-nucleic acid-clamping PCR, which can detect the mutation at an allelic dilution of 1 in 5,000. Progression-free survival (PFS) to erlotinib was 9.7 months for those with T790M mutations and 15.8 months for those without, whereas among patients receiving chemotherapy, it was 6 and 5.1 months, respectively (P < 0.0001). PFS to erlotinib was 12.9 months for those with high and 7.2 months for those with low/intermediate BCL2L11 expression levels, whereas among chemotherapy-treated patients, it was 5.8 and 5.5 months, respectively (P = 0.0003). Overall survival was 28.6 months for patients with high BCL2L11 expression and 22.1 months for those with low/intermediate BCL2L11 expression (P = 0.0364). Multivariate analyses showed that erlotinib was a marker of longer PFS (HR = 0.35; P = 0.0003), whereas high BCL2L11 expression was a marker of longer PFS (HR = 0.49; P = 0.0122) and overall survival (HR = 0.53; P = 0.0323). CONCLUSIONS: Low-level pretreatment T790M mutations can frequently be detected and can be used for customizing treatment with T790M-specific inhibitors. BCL2L11 mRNA expression is a biomarker of survival in EGFR-mutant NSCLC and can potentially be used for synthetic lethality therapies.
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Proteínas Reguladoras de la Apoptosis/biosíntesis , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Proteínas de la Membrana/biosíntesis , Proteínas Proto-Oncogénicas/biosíntesis , Anciano , Anciano de 80 o más Años , Proteína 11 Similar a Bcl2 , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia sin Enfermedad , Clorhidrato de Erlotinib , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Quinazolinas/administración & dosificación , ARN Mensajero/biosíntesis , Resultado del TratamientoRESUMEN
OBJECTIVES: Epidermal growth factor receptor (EGFR) mutations have been identified in lung adenocarcinomas and are associated with high response chance to EGFR tyrosine kinase inhibitors. EGFR mutations can be detected in tumour tissue, cytology specimens and blood from lung cancer patients. Thus far, EGFR mutation analysis has not been systematically demonstrated for sputum samples. The aim of the present study was to determine whether EGFR mutation analysis is attainable on sputum samples, employing different assays in a multicenter study. MATERIALS AND METHODS: Sputum DNA from 10 lung cancer patients with confirmed EGFR mutation in their tumour tissue, 10 lung cancer patients without evidence of an EGFR mutation, and 10 patients with chronic obstructive pulmonary disease (COPD) was used for mutation analysis by Cycleave PCR, COLD-PCR, PangaeaBiotech SL Technology (PST), and High Resolution Melting, respectively. Targeted resequencing (TruSeq Amplicon Cancer Panel) and droplet digital PCR were additionally performed on the 10 samples with EGFR mutation. RESULTS: Dependent on the assay, EGFR mutations could be detected in 30-50% of the sputum samples of patients with EGFR mutations. The different techniques revealed consistent results, with slightly higher sensitivity for PST. Neither the lung cancer patients without EGFR mutation nor the COPD controls tested positive for EGFR mutations in their sputum samples, indicating high clinical specificity of all assays. CONCLUSION: EGFR mutations can be detected in sputum samples from patients with EGFR-mutated non-small cell lung cancer, which may replace biopsy procedure for some patients.
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Adenocarcinoma/diagnóstico , Receptores ErbB/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias de Células Escamosas/diagnóstico , Esputo/metabolismo , Adenocarcinoma/genética , Anciano , Estudios de Casos y Controles , ADN/genética , ADN/aislamiento & purificación , Análisis Mutacional de ADN , Humanos , Neoplasias Pulmonares/genética , Persona de Mediana Edad , Técnicas de Diagnóstico Molecular , Neoplasias de Células Escamosas/genética , Reacción en Cadena de la Polimerasa , Enfermedad Pulmonar Obstructiva Crónica/genética , Sensibilidad y Especificidad , Método Simple Ciego , Temperatura de TransiciónRESUMEN
INTRODUCTION: Lung adenocarcinoma patients harboring EGFR activating mutations attain improved progression-free survival (PFS) with treatment with epidermal growth factor receptor tyrosine kinase inhibitors. However, patients ultimately relapse, indicating that other genetic factors could influence outcome in such patients. We hypothesized that PFS could be influenced by the expression of genes in DNA repair pathways. METHODS: We examined the mRNA expression of C terminus-binding protein-interacting protein and Lin11, Isl-1, and Mec-3 domain only 4 (LMO4) in pretreatment tumor samples from 91 erlotinib-treated advanced non-small-cell lung cancer patients with EGFR mutations in whom breast cancer gene 1 (BRCA1) expression and the concomitant presence of the EGFR T790M mutation had previously been assessed. Gene expression was analyzed by polymerase chain reaction, using ß-actin as endogenous gene. Results were correlated with PFS and overall survival. RESULTS: In patients with low LMO4 levels, PFS was 13 months, whereas it was not reached for those with high LMO4 levels (p = 0.03). In patients with low levels of both BRCA1 and LMO4, PFS was 19 months whereas it was not reached in those with low BRCA1 and high LMO4 mRNA levels (p = 0.04). In patients with high BRCA1 and low LMO4 levels, PFS was 8 months, whereas it was 18 months in those with high levels of both genes (p = 0.03). CONCLUSIONS: Low BRCA1 and high LMO4 levels were associated with longer PFS to erlotinib. Baseline assessment of BRCA1 and LMO4 mRNA expression can help predict outcome to erlotinib.