RESUMEN
Because of the increasing application of ionizing radiation in medicine, quantitative data on effects of low-dose radiation are needed to optimize radiation protection, particularly with respect to cataract development. Using mice as mammalian animal model, we applied a single dose of 0, 0.063, 0.125 and 0.5 Gy at 10 weeks of age, determined lens opacities for up to 2 years and compared it with overall survival, cytogenetic alterations and cancer development. The highest dose was significantly associated with increased body weight and reduced survival rate. Chromosomal aberrations in bone marrow cells showed a dose-dependent increase 12 months after irradiation. Pathological screening indicated a dose-dependent risk for several types of tumors. Scheimpflug imaging of the lens revealed a significant dose-dependent effect of 1% of lens opacity. Comparison of different biological end points demonstrated long-term effects of low-dose irradiation for several biological end points.
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Catarata/genética , Traumatismos Experimentales por Radiación/genética , Animales , Catarata/etiología , Aberraciones Cromosómicas/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Femenino , Estimación de Kaplan-Meier , Masculino , Ratones , Traumatismos Experimentales por Radiación/etiología , Protección Radiológica , Medición de Riesgo , Telómero/efectos de la radiación , Factores de TiempoRESUMEN
Triple-negative breast cancer (TNBC), lacking the steroid hormone receptors ER and PR and the oncoprotein HER2, is characterized by its aggressive pattern and insensitivity to endocrine and HER2-directed therapy. Human kallikrein-related peptidases KLK1-15 provide a rich source of serine protease-type biomarkers associated with tumor growth and cancer progression for a variety of malignant diseases. In this study, recombinant KLK4 protein was generated and affinity-purified KLK4-directed polyclonal antibody pAb587 established to allow localization of KLK4 protein expression in tumor cell lines and archived formalin-fixed, paraffin-embedded TNBC tumor tissue specimens. For this, KLK4 protein expression was assessed by immunohistochemistry in primary tumor tissue sections (tissue microarrays) of 188 TNBC patients, mainly treated with anthracycline- or CMF-based polychemotherapy. KLK4 protein is localized in the cytoplasm of tumor and stroma cells. In this patient cohort, elevated stroma cell KLK4 expression, but not tumor cell KLK4 expression, is predictive for poor disease-free survival by univariate analysis (hazard ratio: 2.26, p=0.001) and multivariable analysis (hazard ratio: 2.12, p<0.01). Likewise, univariate analysis revealed a trend for statistical significance of elevated KLK4 stroma cell expression for overall survival of TNBC patients as well.
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Biomarcadores de Tumor/metabolismo , Calicreínas/metabolismo , Neoplasias de la Mama Triple Negativas/enzimología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/biosíntesis , Humanos , Calicreínas/biosíntesis , Análisis Multivariante , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Due to lack of a targeted therapy for the triple-negative breast cancer (TNBC) patients, it is important to explore this aggressive breast cancer type in more detail and to establish novel therapeutic approaches. TNBC is defined negative for the protein expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). One prominent feature of this cancer type is the frequent overexpression of major components of the urokinase-type plasminogen activator system (uPAS) including uPA, its receptor uPAR and the inhibitor PAI-1, which may be valuable as therapeutic targets. METHODS: Direct interactions of uPAR with interactors were demonstrated by immunoprecipitations and proximity ligation assays. For stable knockdowns of target proteins, lentiviral vectors were used and the effects were analysed by immunoblottings and using in vitro cell viability, migration and invasion assays. Immunohistochemical and statistical analyses of biomarkers and clinical parameters were conducted in a TNBC cohort (n = 174). RESULTS: Direct tumour-promoting interactions of uPAR with uPA and the insulin-like growth factor receptor 1 (IGF1R) were shown in TNBC cells and these interactions were significantly reduced (p = 0.001) when uPAR was downregulated. The combined knockdown of uPAR and uPA or IGF1R additively and significantly reduced cell viability, migration and invasion of the model cell lines. In TNBC tissue, the complexes formed by uPAR with uPA or with IGF1R significantly correlated with the histological grade (p = 0.0019) as well as with cathepsin B and D (p ≤ 0.0001) that are implicated in cell invasion and metastasis. CONCLUSIONS: Our outcomes show that not only overexpressed biomarkers promote tumourigenesis, but rather their interactions further potentiate tumour progression. This study emphasises the potential of combined approaches targeting uPAR and its interactors with regard to an improved therapy of TNBC.
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Receptores de Somatomedina/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Clasificación del Tumor , Receptor IGF Tipo 1 , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
BACKGROUND: Triple-negative breast cancer (TNBC) with a BRCA1-like molecular signature has been demonstrated to remarkably respond to platinum-based chemotherapy and might be suited for a future treatment with poly(ADP-ribose)polymerase (PARP) inhibitors. In order to rapidly assess this signature we have previously developed a multiplex-ligation-dependent probe amplification (MLPA)-based assay. Here we present an independent validation of this assay to confirm its important clinical impact. METHODS: One-hundred-forty-four TNBC tumor specimens were analysed by the MLPA-based "BRCA1-like" test. Classification into BRCA1-like vs. non-BRCA1-like samples was performed by our formerly established nearest shrunken centroids classifier. Data were subsequently compared with the BRCA1-mutation/methylation status of the samples. T-lymphocyte infiltration and expression of the main target of PARP inhibitors, PARP1, were assessed on a subset of samples by immunohistochemistry. Data acquisition and interpretation was performed in a blinded manner. RESULTS: In the studied TNBC cohort, 63 out of 144 (44 %) tumors were classified into the BRCA1-like category. Among these, the MLPA test correctly predicted 15 out of 18 (83 %) samples with a pathogenic BRCA1-mutation and 20 of 22 (91 %) samples exhibiting BRCA1-promoter methylation. Five false-negative samples were observed. We identified high lymphocyte infiltration as one possible basis for misclassification. However, two falsely classified BRCA1-mutated tumors were also characterized by rather non-BRCA1-associated histopathological features such as borderline ER expression. The BRCA1-like vs. non-BRCA1-like signature was specifically enriched in high-grade (G3) cancers (90 % vs. 58 %, p = 0.0004) and was also frequent in tumors with strong (3+) nuclear PARP1 expression (37 % vs. 16 %; p = 0.087). CONCLUSIONS: This validation study confirmed the good performance of the initial MLPA assay which might thus serve as a valuable tool to select patients for platinum-based chemotherapy regimens. Moreover, frequent PARP1 upregulation in BRCA1-like tumors may also point to susceptibility to treatment with PARP inhibitors. Limitations are the requirement of high tumor content and high-quality DNA.
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Proteína BRCA1/genética , Biomarcadores de Tumor , Mapeo Cromosómico , Neoplasias de la Mama Triple Negativas/genética , Adulto , Anciano , Proteína BRCA1/metabolismo , Terapia Combinada , Metilación de ADN , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Clasificación del Tumor , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Regiones Promotoras Genéticas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Neoplasias de la Mama Triple Negativas/diagnóstico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/terapia , Carga TumoralRESUMEN
An essential and so far unresolved factor influencing the evolution of cancer and the clinical management of patients is intratumour clonal and phenotypic heterogeneity. However, the de novo identification of tumour subpopulations is so far both a challenging and an unresolved task. Here we present the first systematic approach for the de novo discovery of clinically detrimental molecular tumour subpopulations. In this proof-of-principle study, spatially resolved, tumour-specific mass spectra were acquired, using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry, from tissues of 63 gastric carcinoma and 32 breast carcinoma patients. The mass spectra, representing the proteomic heterogeneity within tumour areas, were grouped by a corroborated statistical clustering algorithm in order to obtain segmentation maps of molecularly distinct regions. These regions were presumed to represent different phenotypic tumour subpopulations. This was confirmed by linking the presence of these tumour subpopulations to the patients' clinical data. This revealed several of the detected tumour subpopulations to be associated with a different overall survival of the gastric cancer patients (p = 0.025) and the presence of locoregional metastases in patients with breast cancer (p = 0.036). The procedure presented is generic and opens novel options in cancer research, as it reveals microscopically indistinct tumour subpopulations that have an adverse impact on clinical outcome. This enables their further molecular characterization for deeper insights into the biological processes of cancer, which may finally lead to new targeted therapies.
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Neoplasias de la Mama/patología , Tumores del Estroma Gastrointestinal/patología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Algoritmos , Neoplasias de la Mama/mortalidad , Análisis por Conglomerados , Femenino , Tumores del Estroma Gastrointestinal/mortalidad , Humanos , Masculino , Fenotipo , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
We present the first analytical approach to demonstrate the in situ imaging of metabolites from formalin-fixed, paraffin-embedded (FFPE) human tissue samples. Using high-resolution matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR MSI), we conducted a proof-of-principle experiment comparing metabolite measurements from FFPE and fresh frozen tissue sections, and found an overlap of 72% amongst 1700 m/z species. In particular, we observed conservation of biomedically relevant information at the metabolite level in FFPE tissues. In biomedical applications, we analysed tissues from 350 different cancer patients and were able to discriminate between normal and tumour tissues, and different tumours from the same organ, and found an independent prognostic factor for patient survival. This study demonstrates the ability to measure metabolites in FFPE tissues using MALDI-FT-ICR MSI, which can then be assigned to histology and clinical parameters. Our approach is a major technical, histochemical, and clinicopathological advance that highlights the potential for investigating diseases in archived FFPE tissues.
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Biomarcadores de Tumor/metabolismo , Fijadores/química , Formaldehído/química , Metabolómica/métodos , Neoplasias/metabolismo , Adhesión en Parafina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Fijación del Tejido/métodos , Análisis por Conglomerados , Biología Computacional , Ciclotrones , Diagnóstico Diferencial , Supervivencia sin Enfermedad , Femenino , Análisis de Fourier , Alemania , Humanos , Masculino , Neoplasias/mortalidad , Neoplasias/patología , Neoplasias/terapia , Países Bajos , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Reproducibilidad de los Resultados , Factores de Tiempo , Resultado del TratamientoRESUMEN
MALDI mass spectrometry imaging (MSI) has rapidly established itself as a powerful biomarker discovery tool. To date, no formal investigation has assessed the center-to-center comparability of MALDI MSI experiments, an essential step for it to develop into a new diagnostic method. To test such capabilities, we have performed a multicenter study focused on biomarkers of stromal activation in breast cancer. MALDI MSI experiments were performed in two centers using independent tissue banks, infrastructure, methods, and practitioners. One of the data sets was used for discovery and the other for validation. Areas of intra- and extratumoral stroma were selected, and their protein signals were compared. Four protein signals were found to be significantly associated with tumor-associated stroma in the discovery data set measured in Munich. Three of these peaks were also detected in the independent validation data set measured in Leiden, all of which were also significantly associated with intratumoral stroma. Hierarchical clustering displayed 100% accuracy in the Munich MSI data set and 80.9% accuracy in the Leiden MSI data set. The association of one of the identified mass signals (PA28) with stromal activation was confirmed with immunohistochemistry performed on 20 breast tumors. Independent and international MALDI MSI investigations could identify validated biomarkers of stromal activation.
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Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células del Estroma/metabolismo , Neoplasias de la Mama/clasificación , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Alemania , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Países BajosRESUMEN
Chemotherapeutic drugs kill cancer cells, but it is unclear why this happens in responding patients but not in non-responders. Proteomic profiles of patients with oesophageal adenocarcinoma may be helpful in predicting response and selecting more effective treatment strategies. In this study, pretherapeutic oesophageal adenocarcinoma biopsies were analysed for proteomic changes associated with response to chemotherapy by MALDI imaging mass spectrometry. Resulting candidate proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and investigated for functional relevance in vitro. Clinical impact was validated in pretherapeutic biopsies from an independent patient cohort. Studies on the incidence of these defects in other solid tumours were included. We discovered that clinical response to cisplatin correlated with pre-existing defects in the mitochondrial respiratory chain complexes of cancer cells, caused by loss of specific cytochrome c oxidase (COX) subunits. Knockdown of a COX protein altered chemosensitivity in vitro, increasing the propensity of cancer cells to undergo cell death following cisplatin treatment. In an independent validation, patients with reduced COX protein expression prior to treatment exhibited favourable clinical outcomes to chemotherapy, whereas tumours with unchanged COX expression were chemoresistant. In conclusion, previously undiscovered pre-existing defects in mitochondrial respiratory complexes cause cancer cells to become chemosensitive: mitochondrial defects lower the cells' threshold for undergoing cell death in response to cisplatin. By contrast, cancer cells with intact mitochondrial respiratory complexes are chemoresistant and have a high threshold for cisplatin-induced cell death. This connection between mitochondrial respiration and chemosensitivity is relevant to anticancer therapeutics that target the mitochondrial electron transport chain.
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Adenocarcinoma/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Neoplasias Esofágicas/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Adenocarcinoma/enzimología , Adenocarcinoma/genética , Adenocarcinoma/patología , Anciano , Biomarcadores de Tumor/genética , Biopsia , Línea Celular Tumoral , Quimioterapia Adyuvante , Cromatografía Liquida , Cisplatino/administración & dosificación , Regulación hacia Abajo , Resistencia a Antineoplásicos , Complejo IV de Transporte de Electrones/genética , Neoplasias Esofágicas/enzimología , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Fluorouracilo/administración & dosificación , Humanos , Persona de Mediana Edad , Mitocondrias/enzimología , Mitocondrias/patología , Terapia Neoadyuvante , Medicina de Precisión , Proteómica/métodos , Interferencia de ARN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en Tándem , Transfección , Resultado del TratamientoRESUMEN
BACKGROUND: CRIP1 (cysteine-rich intestinal protein 1) has been found in several tumor types, its prognostic impact and its role in cellular processes, particularly in breast cancer, are still unclear. METHODS: To elucidate the prognostic impact of CRIP1, we analyzed tissues from 113 primary invasive ductal breast carcinomas using immunohistochemistry. For the functional characterization of CRIP1, its endogenous expression was transiently downregulated in T47D and BT474 breast cancer cells and the effects analyzed by immunoblotting, WST-1 proliferation assay and invasion assay. RESULTS: We found a significant correlation between CRIP1 and HER2 (human epidermal growth factor receptor 2) expression levels (p = 0.016) in tumor tissues. In Kaplan Meier analyses, CRIP1 expression was significantly associated with the distant metastases-free survival of patients, revealing a better prognosis for high CRIP1 expression (p = 0.039). Moreover, in multivariate survival analyses, the expression of CRIP1 was an independent negative prognostic factor, along with the positive prognosticators nodal status and tumor size (p = 0.029). CRIP1 knockdown in the T47D and BT474 breast cancer cell lines led to the increased phosphorylation of MAPK and Akt, to the reduced phosphorylation of cdc2, and to a significantly elevated cell proliferation in vitro (p < 0.001). These results indicate that reduced CRIP1 levels may increase cell proliferation and activate cell growth. In addition, CRIP1 knockdown increased cell invasion in vitro. CONCLUSIONS: Because the lack of CRIP1 expression in breast cancer tissue is significantly associated with a worse prognosis for patients and low endogenous CRIP1 levels in vitro increased the malignant potential of breast cancer cells, we hypothesize that CRIP1 may act as a tumor suppressor in proliferation and invasion processes. Therefore, CRIP1 may be an independent prognostic marker with significant predictive power for use in breast cancer therapy.
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Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Proteínas con Dominio LIM/metabolismo , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/mortalidad , Proteínas Portadoras/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Silenciador del Gen , Humanos , Proteínas con Dominio LIM/genética , Invasividad Neoplásica , Pronóstico , Receptor ErbB-2/metabolismoRESUMEN
Accidental nuclear scenarios lead to environmental contamination of unknown level. Immediate radiation-induced biological responses that trigger processes leading to adverse health effects decades later are not well understood. A comprehensive proteomic analysis provides a promising means to identify and quantify the initial damage after radiation exposure. Early changes in the cardiac tissue of C57BL/6 mice exposed to total body irradiation were studied, using a dose relevant to both intentional and accidental exposure (3 Gy gamma ray). Heart tissue protein lysates were analyzed 5 and 24 h after the exposure using isotope-coded protein labeling (ICPL) and 2-dimensional difference-in-gel-electrophoresis (2-D DIGE) proteomics approaches. The differentially expressed proteins were identified by LC-ESI-MS-MS. Both techniques showed similar functional groups of proteins to be involved in the initial injury. Pathway analyses indicated that total body irradiation immediately induced biological responses such as inflammation, antioxidative defense, and reorganization of structural proteins. Mitochondrial proteins represented the protein class most sensitive to ionizing radiation. The proteins involved in the initial damage processes map to several functional categories involving cardiotoxicity. This prompts us to propose that these early changes are indicative of the processes that lead to an increased risk of cardiovascular disease after radiation exposure.
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Rayos gamma/efectos adversos , Corazón/efectos de la radiación , Proteínas/análisis , Proteoma/efectos de la radiación , Animales , Western Blotting , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Cromatografía Liquida , Electroforesis en Gel Bidimensional , Peroxidación de Lípido/efectos de la radiación , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Carbonilación Proteica/efectos de la radiación , Mapeo de Interacción de Proteínas , Proteínas/metabolismo , Proteoma/análisis , Proteómica , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/patología , Reproducibilidad de los Resultados , Remodelación Ventricular/efectos de la radiación , Irradiación Corporal TotalRESUMEN
BACKGROUND: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. MATERIAL AND METHODS: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. RESULTS: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). CONCLUSION: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation.
RESUMEN
Formalin-fixed paraffin-embedded (FFPE) tissue has recently gained interest as an alternative to fresh/frozen tissue for retrospective protein biomarker discovery. However, during the fixation process, proteins undergo degradation and cross-linking, making conventional protein analysis technologies problematic. In this study, we have compared several extraction and separation methods for the analysis of proteins in FFPE tissues. Incubation of tissue sections at high temperature with a novel extraction buffer (20 mM Tris-HCl, pH 8.8, 2% SDS, 1% beta-octylglucoside, 200 mM DTT, 200 mM glycine, and a mixture of protease inhibitors) resulted in improved protein recovery. Protein separation by 1-DE followed by LC-ESI MS/MS analysis was the most effective approach to identify proteins, based on the number of peptides reliably identified. Interestingly, a number of peptides were identified in regions of the 1DE not corresponding to their native molecular weights. This is an indication of the formation of protein-protein complexes by cross-linking, and of protein fragmentation due to prolonged sample storage. This study will facilitate the development of future proteomic analysis of FFPE tissue and provide a tool for the validation in archival samples of biomarkers of exposure, prognosis and disease.
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Biomarcadores/química , Cromatografía Liquida/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Adhesión en Parafina , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Formaldehído , Immunoblotting , Focalización Isoeléctrica , Masculino , Ratones , Datos de Secuencia Molecular , Miocardio/química , Proteínas/química , Proteínas/aislamiento & purificación , Fijación del TejidoRESUMEN
Clinical laboratory testing for HER2 status in breast cancer tissues is critically important for therapeutic decision making. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating proteins through the direct and morphology-driven analysis of tissue sections. We hypothesized that MALDI-IMS may determine HER2 status directly from breast cancer tissues. Breast cancer tissues (n = 48) predefined for HER2 status were subjected to MALDI-IMS, and protein profiles were obtained through direct analysis of tissue sections. Protein identification was performed by tissue microextraction and fractionation followed by top-down tandem mass spectrometry. A discovery and an independent validation set were used to predict HER2 status by applying proteomic classification algorithms. We found that specific protein/peptide expression changes strongly correlated with the HER2 overexpression. Among these, we identified m/z 8404 as cysteine-rich intestinal protein 1. The proteomic signature was able to accurately define HER2-positive from HER2-negative tissues, achieving high values for sensitivity of 83%, for specificity of 92%, and an overall accuracy of 89%. Our results underscore the potential of MALDI-IMS proteomic algorithms for morphology-driven tissue diagnostics such as HER2 testing and show that MALDI-IMS can reveal biologically significant molecular details from tissues which are not limited to traditional high-abundance proteins.
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Biomarcadores de Tumor/química , Neoplasias de la Mama/enzimología , Fragmentos de Péptidos/química , Proteómica/métodos , Receptor ErbB-2/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Algoritmos , Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/química , Proteínas Portadoras , Análisis por Conglomerados , Femenino , Histocitoquímica , Humanos , Proteínas con Dominio LIM , Fragmentos de Péptidos/metabolismo , Receptor ErbB-2/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
In normal physiology, kallikrein-related peptidase 7 (KLK7), together with other members of the kallikrein-related peptidase family, is mainly involved in skin desquamation and keratinization processes. Moreover, expression of KLK7 was shown in various tumor types to be dysregulated and to correlate to patients' survival time. However, there are contradictory reports in breast cancer whether KLK7 represents an unfavorable or favorable prognostic biomarker. In the present study, we examined the prognostic value of KLK7 protein expression in triple-negative breast cancer (TNBC), determined by immunohistochemistry (IHC). A cohort encompassing 133 TNBC specimens, present on tissue microarrays, was analyzed. For quantification of the staining intensity, an automated digital IHC image analysis algorithm was applied. In both Kaplan-Meier and univariate Cox analyses, elevated KLK7 protein levels were significantly linked with prolonged overall survival (OS). In multivariable Cox analysis, addition of KLK7 immunoreactivity scores to the base model (including the clinical parameters age, tumor size, and nodal status) demonstrated that KLK7 protein expression remained as a statistically significant, independent parameter for prolonged OS. These results strongly indicate that KLK7 is a favorable prognostic biomarker in triple-negative breast cancer.
RESUMEN
Triple-negative breast cancer (TNBC) constitutes a heterogeneous breast cancer subgroup with poor prognosis; survival rates are likely to be lower with TNBC compared to other breast cancer subgroups. For this disease, systemic adjuvant chemotherapy regimens often yield suboptimal clinical results. To improve treatment regimens in TNBC, identification of molecular biomarkers may help to select patients for individualized adjuvant therapy. Evidence has accumulated that determination of the methylation status of the PITX2 gene provides a predictive value in various breast cancer subgroups, either treated with endocrine-based therapy or anthracycline-containing chemotherapy. To further explore the validity of this novel predictive candidate biomarker, in the present exploratory retrospective study, determination of the PITX2 DNA-methylation status was assessed for non-metastatic TNBC patients treated with adjuvant anthracycline-based chemotherapy by molecular analysis of breast cancer tissues. The PITX2 DNA-methylation status was determined in fresh-frozen tumor tissue specimens (n=56) by methylation-specific qRT-PCR (qMSP) and the data related to disease-free and overall survival, applying an optimized DNA-methylation score of 6.35%. For non-metastatic TNBC patients treated with adjuvant systemic anthracycline-based chemotherapy, a low PITX2 DNA-methylation status (<6.35) defines TNBC patients with poor disease-free and overall survival. Univariate and multivariate analyses demonstrate the statistically independent predictive value of PITX2 DNA-methylation. For non-metastatic TNBC patients, selective determination of the PITX2 DNA-methylation status may serve as a cancer biomarker for predicting response to anthracycline-based adjuvant chemotherapy. The assay based on methylation of the PIXT2 gene can be applied to frozen and routinely available formalin-fixed, paraffin-embedded (FFPE) breast cancer tumor tissues that will not only define those TNBC patients who may benefit from anthracycline-based chemotherapy but also those who should be spared the necessity of such potentially toxic treatment. Such patients should be allocated to alternative treatment options.
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Antraciclinas/uso terapéutico , Antibióticos Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/genética , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/terapia , Adulto , Anciano , Mama/patología , Mama/cirugía , Quimioterapia Adyuvante/métodos , Metilación de ADN , Supervivencia sin Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Selección de Paciente , Proyectos Piloto , Estudios Retrospectivos , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Proteína del Homeodomínio PITX2RESUMEN
Background: Breast cancer patients at high risk for recurrence are treated with anthracycline-based chemotherapy, but not all patients do equally benefit from such a regimen. To further improve therapy decision-making, biomarkers predicting outcome are of high unmet medical need. Methods: The percent DNA methylation ratio (PMR) of the promoter gene coding for the Paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time polymerase chain reaction (PCR) test. The multicenter study was conducted in routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue from 205 lymph node-positive breast cancer patients treated with adjuvant anthracycline-based chemotherapy. Results: The cut-off for the PITX2 methylation status (PMR = 12) was confirmed in a randomly selected cohort (n = 60) and validated (n = 145) prospectively with disease-free survival (DFS) at the 10-year follow-up. DFS was significantly different between the PMR ≤ 12 versus the PMR > 12 group with a hazard ratio (HR) of 2.74 (p < 0.001) in the validation cohort and also for the patient subgroup treated additionally with endocrine therapy (HR 2.47; p = 0.001). Conclusions: Early-stage lymph node-positive breast cancer patients with low PITX2 methylation do benefit from adjuvant anthracycline-based chemotherapy. Patients with a high PITX2 DNA methylation ratio, approximately 30%, show poor outcome and should thus be considered for alternative chemotherapy regimens.
RESUMEN
High-risk breast cancer comprises distinct tumor entities such as triple-negative breast cancer (TNBC) which is characterized by lack of estrogen (ER) and progesterone (PR) and the HER2 receptor and breast malignancies which have spread to more than three lymph nodes. For such patients, current (inter)national guidelines recommend anthracycline-based chemotherapy as the standard of care, but not all patients do equally benefit from such a chemotherapy. To further improve therapy decision-making, predictive biomarkers are of high, so far unmet, medical need. In this respect, predictive biomarkers would permit patient selection for a particular kind of chemotherapy and, by this, guide physicians to optimize the treatment plan for each patient individually. Besides DNA mutations, DNA methylation as a patient selection marker has received increasing clinical attention. For instance, significant evidence has accumulated that methylation of the PITX2 (paired-like homeodomain transcription factor 2) gene might serve as a novel predictive and prognostic biomarker, for a variety of cancer diseases. This review highlights the current understanding of treatment modalities of high-risk breast cancer patients with a focus on recommended treatment options, with special attention on the future clinical application of PITX2 as a predictive biomarker to personalize breast cancer management.
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Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Metilación de ADN , Proteínas de Homeodominio/genética , Factores de Transcripción/genética , Neoplasias de la Mama Triple Negativas/genética , Antineoplásicos/normas , Femenino , Humanos , Guías de Práctica Clínica como Asunto , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Proteína del Homeodomínio PITX2RESUMEN
A 3D microtissues using T47D and JIMT-1 cells were generated to analyze tissue-like response of breast cancer cells after combined human epidermal growth factor receptor 2 (HER2)-targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2-depleted T47D cells and 3D microtissues using trastuzumab (anti-HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab-resistant JIMT-1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs--as single agents or in combination--here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug-combined radiotherapy.
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Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Evaluación Preclínica de Medicamentos , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/radioterapia , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Radiación , Receptor ErbB-2/deficiencia , Técnicas de Cultivo de Tejidos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Western Blotting , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/genética , Femenino , Humanos , Inmunohistoquímica , Células MCF-7 , Unión Proteica , Interferencia de ARN , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
Formalin-fixed and paraffin-embedded (FFPE) tissue specimens are the gold standard for histological examination, and they provide valuable molecular information in tissue-based research. Metabolite assessment from archived tissue samples has not been extensively conducted because of a lack of appropriate protocols and concerns about changes in metabolite content or chemical state due to tissue processing. We present a protocol for the in situ analysis of metabolite content from FFPE samples using a high-mass-resolution matrix-assisted laser desorption/ionization fourier-transform ion cyclotron resonance mass spectrometry imaging (MALDI-FT-ICR-MSI) platform. The method involves FFPE tissue sections that undergo deparaffinization and matrix coating by 9-aminoacridine before MALDI-MSI. Using this platform, we previously detected â¼1,500 m/z species in the mass range m/z 50-1,000 in FFPE samples; the overlap compared with fresh frozen samples is 72% of m/z species, indicating that metabolites are largely conserved in FFPE tissue samples. This protocol can be reproducibly performed on FFPE tissues, including small samples such as tissue microarrays and biopsies. The procedure can be completed in a day, depending on the size of the sample measured and raster size used. Advantages of this approach include easy sample handling, reproducibility, high throughput and the ability to demonstrate molecular spatial distributions in situ. The data acquired with this protocol can be used in research and clinical practice.