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OBJECTIVE: A mobile handheld snapshot hyperspectral imaging device was developed and tested for in vivo skin evaluation using a new spectral imaging technology. METHODS: The device is equipped with four different LED light sources (VIS, 810 nm, 850 nm, and 940 nm) for illumination. Based on a diffractive optical element (DOE) combined with a CMOS sensor chip, a snapshot hyperspectral imager is achieved for the application on human skin. The diffractive optical element (DOE) consists of a two-dimensional array of identically repeated diffractive microstructures. One hyperspectral image for all wavelength regions is taken within a few seconds. Complex recalculation of the VIS spectral distribution and image information from the received DOE image requires several minutes, depending on computing performance. A risk assessment on the irradiation sources shows no risk of harm due to the LED radiation. RESULTS: Skin tone color patches experiments reproducibly deliver images and spectra of different skin tones. First in vivo use of the device identified pigmentation changes within the field of view. CONCLUSION: We present a working mobile snapshot hyperspectral imaging tool based on diffractive optical elements. This device or future developments thereof can be used for broad skin evaluation in vivo.
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Imágenes Hiperespectrales , Pigmentación de la Piel , Diagnóstico por Imagen , Humanos , Iluminación , Piel/diagnóstico por imagenRESUMEN
OBJECTIVE: A handheld device was developed and qualified for in vivo human skin evaluation using laser speckle imaging technology. METHODS: Each laser speckle device prototype allows the choice of up to three different laser wavelengths in the range of 400 nm to 800 nm in total. Speckle pattern analysis gives various speckle parameters, for example, speckle contrast, speckle size, speckle modulation or fractal dimension. The developed laser speckle device prototypes were evaluated investigating three skin issues. RESULTS: We receive reproducible results from the speckle imaging device. For skin ageing, we found significant changes within three age groups. The effect of a methyl nicotinate treatment was clearly visible and quantifiable using a moorFLPI device as well as our speckle imaging device. In terms of basal cell carcinoma diagnosis, we found significant differences between normal and diseased skin, even though the number of samples was limited. CONCLUSION: As shown with first application examples, it was possible to demonstrate the potential of the method for skin evaluation in vivo.
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Envejecimiento de la Piel , Piel , Humanos , Imágenes de Contraste de Punto Láser , Rayos Láser , Piel/diagnóstico por imagenRESUMEN
We present a variational approach to reconstruct a multispectral image from an array of diffraction patterns captured in one monochromatic image. The mathematical model relies on the superposition of wavelength dependent calibration diffraction patterns and a spectral regularization with second order differences. The necessary preprocessing steps which influence the mathematical setting are discussed in detail. For computing a minimizer of our model we propose an active set method with model specific modifications. We validate our approach by experimental results for spectra within the range of 400 nm to 700 nm.
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Digital inline holographic microscopy is applied for lens-free imaging with high lateral resolution. Microfluidic chambers for the imaging of cells in water-like or native solutions, e.g., thick layers of glass and other materials with different refractive index, cause aberrations that limit the spatial resolution and change the magnification scale. In this Letter, a fast reconstruction technique considering parallel layer systems of different refractive indices is presented. In the experiments, properly scaled images of microbeads and red human blood cells with an optical resolution corresponding to a numerical aperture of about 0.62 were reconstructed.
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PURPOSE: Epithelial-mesenchymal transition enhances tumor cell motility and has a critical role in invasion and metastasis in a number of carcinomas. A set of transcription factors acts as a master regulator of the epithelial-mesenchymal transition process. To our knowledge it is unknown whether epithelial-mesenchymal transition is important for clear cell renal cell carcinoma progression. Therefore, we comprehensively assessed mRNA levels of epithelial-mesenchymal transition associated genes in renal cell carcinoma as well as their prognostic relevance. MATERIALS AND METHODS: We determined the expression of a set of 46 epithelial-mesenchymal transition related genes by oligonucleotide microarray and gene set enrichment analyses using RNA from 14 samples each of normal kidneys, and G1 and G3 primary renal cell carcinomas. Expression of select epithelial-mesenchymal transition genes was validated by real-time polymerase chain reaction in normal kidneys, primary renal cell carcinomas and metastases in an independent cohort of 112 patients. Results were combined with followup data for survival analysis. RESULTS: The epithelial-mesenchymal transition gene set was preferentially expressed in primary renal cell carcinoma compared to normal tissue (false discovery rate 0.01). No difference was found between G1 and G3 tumors. Quantitative reverse transcriptase-polymerase chain reaction revealed down-regulation of critical epithelial-mesenchymal transition genes such as CDH2 and ZEB1 in metastases, suggesting epithelial-mesenchymal transition reversal during metastasis. Kaplan-Meier analysis demonstrated a better outcome in patients with low CXCR4, vimentin, fibronectin and TWIST1 mRNA levels. Multivariate analyses revealed that CXCR4 and VIM up-regulation represents an independent prognostic marker for poor cancer specific survival in patients with renal cell carcinoma. CONCLUSIONS: Taken together, our data provide strong evidence that epithelial-mesenchymal transition occurs in renal cell carcinoma. Thus, interference with epithelial-mesenchymal transition in renal cell carcinoma might represent a future therapeutic option.
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Carcinoma de Células Renales/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Renales/genética , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/patología , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Regulación de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Neoplasias Renales/mortalidad , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Mensajero/biosíntesis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
A self-calibration technique for lensless compact chip-microscopes based on inline holography with pinhole illumination is presented. The pinhole illumination wave acts as reference and is needed for the reconstruction process. This reference wave is assumed to be spherical, so that its phase is already determined by the position of the pinhole in relation to the image sensor. It is shown that the reconstructed spatial resolution is strongly dependent on the estimation for the pinhole to sensor distance. A precision in the range of tens of microns was reached for microscopic imaging with a spatial resolution in the range of one micron. Therefore additional reference crosses are prepared lithographically on the sample holder. The hologram, which contains the optical information about the sample as well as the reference crosses, is used for calibration and image reconstruction at the same time. The presented technique was tested to allow the reconstruction of a spatial resolution corresponding to the limit of detection apertures of about 0.66. The technique was applied to holograms of test beads and blood smear samples.
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The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function, we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16(-/-) mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal days 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16(-/-) mice tectorial membranes were significantly more often stretched out as compared with wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxyl-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 can probably form higher order structures with other tectorial membrane proteins such as α-tectorin and ß-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea, allowing hearing over an extended frequency range.
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Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Animales , Cóclea/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Proteínas Ligadas a GPI/metabolismo , Células Ciliadas Auditivas/metabolismo , Audición , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Mutación , Cadenas Pesadas de Miosina/genética , Miosina Tipo II/genética , Recombinación Genética , Membrana Tectoria/metabolismoRESUMEN
Digital inline holographic microscopy using a pinhole for sample illumination allows lensless imaging. To overcome restrictions of the sample size and density in the setup additional reference waves are generated by extending the single pinhole to a regular 2D pinhole array illumination. A technique is presented that uses phase shifting between the pinhole waves. Multiple foci with stable phase differences and a phase error (rms) of 0.027 rad generate pinhole waves which illuminate an undiluted, dense blood smear sample. Amplitude and phase images of the blood sample were successfully reconstructed.
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Holografía/instrumentación , Aumento de la Imagen/instrumentación , Lentes , Iluminación/instrumentación , Microscopía de Contraste de Fase/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Sistemas en LíneaRESUMEN
The light of a light-emitting diode or a common thermal source, such as a tungsten filament lamp, is known to be quasi-incoherent. We generated partially coherent light of these sources with a volume of coherence in the micrometer range of 5-100 µm3 by spatial and spectral filtering. The corresponding degree of partial coherence was adapted for microscopic interference setups, such as a digital in-line holographic microscope. The practicability of the sources was determined by the spectral emittance and the resulting signal-to-noise ratio (SNR) of the detector. The microscale coherence in correlation with the SNR and its resolution for microscopy were analyzed. We demonstrate how low-light-level, non-laser sources enable holographic imaging with a video frame rate (25 frames/s), an intermediate SNR of 8 dB, and a volume of coherence of 3.4×10(4) µm3. Holograms of objects with a lateral resolution of 1 µm were achieved using a microscope lens (50×/NA=0.7) and a CCD camera featuring a 4-12 bit dynamic range.
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For lensless digital in-line holographic microscopy a new state-of-the-art spatial resolution corresponding to an NA of 0.8 is shown based on the tile superposition propagation. The result is proved using a common glass sample carrier with a refraction index of 1.52. Single-shot high-resolution imaging is possible by suppression of coherent reflections in an optimized arrangement using partially coherent laser light illumination.
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BACKGROUND & AIMS: The ubiquitously expressed basic helix-loop-helix transcription factor ITF-2B has an important role in differentiation processes, and its transcription is regulated by beta-catenin. The ITF-2 gene is located in the chromosomal region 18q21; allelic loss of this locus occurs in 70% of colorectal cancers. We analyzed the expression, regulation, and function of ITF-2B in colorectal carcinogenesis. METHODS: The loss-of-heterozygosity (LOH) status of 18q21 and expression of ITF-2B were studied in colorectal carcinomas using polymerase chain reaction-based methods and immunohistochemistry. The biologic effects of ITF-2B were studied in colorectal cancer cells. Reporter gene assays and chromatin immunoprecipitation were utilized to analyze effects of ITF-2B on gene transcription. RESULTS: ITF-2B is strongly expressed in colon adenomas but frequently down-regulated in carcinomas because of LOH at 18q21. ITF-2B induces cell cycle arrest and regulates the expression of p21(Cip1) via newly identified E-boxes in the CDKN1A gene, independently of p53. Loss of ITF-2B expression correlates with loss of p21(Cip1) expression in primary colon carcinomas. CONCLUSIONS: Accumulation of mutations and allelic losses are driving forces of colorectal carcinogenesis. ITF-2B, which is up-regulated during early colorectal carcinogenesis because of loss of adenomatous polyposis coli, is a target for LOH on chromosome 18q, along with deleted in colorectal carcinoma and Smad4. This finding, along with the fact that ITF-2B is a regulator of the key cell cycle inhibitor p21(Cip1), indicates that ITF-2B is a tumor suppressor that has an important function at the adenoma to carcinoma transition.
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Adenocarcinoma/genética , Poliposis Adenomatosa del Colon/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Transformación Celular Neoplásica/genética , Cromosomas Humanos Par 18/genética , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Adenocarcinoma/patología , Poliposis Adenomatosa del Colon/patología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Neoplasias Colorrectales/patología , Proteínas de Unión al ADN/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Humanos , Inmunohistoquímica , Pérdida de Heterocigocidad , Mutación , Proteínas de Neoplasias/genética , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Factor de Transcripción 4RESUMEN
For applications in the domain of digital holographic microscopy, we present a fast algorithm to propagate scalar wave fields from a small source area to an extended, parallel target area of coarser sampling pitch, using the first Rayleigh-Sommerfeld diffraction formula. Our algorithm can take full advantage of the fast Fourier transform by decomposing the convolution kernel of the propagation into several convolution kernel patches. Using partial overlapping of the patches together with a soft blending function, the Fourier spectrum of these patches can be reduced to a low number of significant components, which can be stored in a compact sparse array structure. This allows for rapid evaluation of the partial convolution results by skipping over negligible components through the Fourier domain pointwise multiplication and direct mapping of the remaining multiplication results into a Fourier domain representation of the coarsly sampled target patch. The algorithm has been verified experimentally at a numerical aperture of 0.62, not showing any significant resolution limitations.
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Algoritmos , Holografía/métodos , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía/métodos , Luz , Dispersión de RadiaciónRESUMEN
PURPOSE: The inflammatory enzyme indoleamine 2,3-dioxygenase (IDO) participates in immune tolerance and tumor immune escape processes by degradation of the essential amino acid tryptophan and formation of toxic catabolites. Here, we analyzed the role of IDO in tumor growth and disease progression in patients with clear cell renal cell carcinoma (RCC). EXPERIMENTAL DESIGN: Expression of IDO mRNA was analyzed by quantitative reverse transcription-PCR in 55 primary and 52 metastatic RCC, along with 32 normal kidneys. Western blot and immunohistochemistry analyses were used to semiquantitatively determine IDO proteins in a subset of tumor samples, in RCC cell lines, and microvessel endothelial cells. IDO expression was correlated with expression of the proliferation marker Ki67 in tumor cells and survival of patients with tumor. RESULTS: More than 75% of the clear cell RCC in comparison to normal kidney contained elevated levels of IDO mRNA, which correlated with their IDO protein content. Low IDO mRNA levels in primary tumors represented an unfavorable independent prognostic factor (hazard ratio, 3.8; P = 0.016). Unexpectedly, immunohistochemical analyses revealed that IDO is nearly exclusively expressed in endothelial cells of newly formed blood vessels and is virtually absent from tumor cells, although RCC cells could principally synthesize IDO as shown by in vitro stimulation with IFN-gamma. A highly significant inverse correlation between the density of IDO-positive microvessels and the content of proliferating Ki67-positive tumor cells in primary and metastatic clear cell RCC was found (P = 0.004). CONCLUSIONS: IDO in endothelial cells might limit the influx of tryptophan from the blood to the tumor or generate tumor-toxic metabolites, thus restricting tumor growth and contributing to survival.
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Carcinoma de Células Renales/irrigación sanguínea , Carcinoma de Células Renales/enzimología , Células Endoteliales/enzimología , Indolamina-Pirrol 2,3,-Dioxigenasa/biosíntesis , Neoplasias Renales/irrigación sanguínea , Neoplasias Renales/enzimología , Anciano , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Células Endoteliales/patología , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/farmacología , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Neovascularización Patológica/enzimología , Neovascularización Patológica/patología , Pronóstico , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Estudios Retrospectivos , Triptófano/metabolismoRESUMEN
OBJECTIVE: This prospective study is the first immunocytochemical investigation of the frequency and prognostic value of CK+ tumour cells in the bone marrow of patients with transitional cell carcinoma (TCC). METHODS: Bone marrow aspirates from 228 TCC patients were taken preoperatively. Cytospins were made and stained by immunocytochemistry using the monoclonal antibodies CK2 and A45-B/B3. 27 patients with no evidence of any malignant disease served as control group. RESULTS: CK+ tumour cells were detected in 28% (63/228) of the TCC patients. No CK+ cells (0/27) were detected in the control group. In multivariate analysis the detection of > or =3 CK+ cells in bone marrow was an independent prognostic factor (hazard ratio=2.7, p<0.05) in patients with T2-4 tumour classification. CONCLUSION: Disseminated CK+ cells play a role in the biology of tumour spread of TCC, and their immunocytochemical detection can be useful in assessing the prognosis of TCC patients with an invasive tumour.
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Neoplasias de la Médula Ósea/secundario , Carcinoma de Células Transicionales/secundario , Neoplasias Renales , Neoplasias de la Vejiga Urinaria , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos/análisis , Examen de la Médula Ósea , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Queratinas/inmunología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios ProspectivosRESUMEN
The measurement of tumor-associated proteins is of high diagnostic value in the follow-up of cancer patients. Most tests ignore that various forms of the protein can exist; especially in epithelial cancers and the soluble receptors they produce. We choose EpCAM as model-antigen to analyze whether tests recognizing different domains of the protein give different results in patients' sera. EpCAM-reactive autoantibodies are present in the sera of patients with colorectal carcinoma, however little is known about the existence and possible relevance of circulating soluble EpCAM protein. Most monoclonal EpCAM-antibodies recognize the first EGF-like repeat and fail to detect N-terminal trimmed protein. We developed a novel ELISA to determine the concentration of serum EpCAM with mAbs recognizing the second EGF-like repeat. In 59 healthy controls, EpCAM concentrations ranged from 232 to 8893ng/ml (mean 1525ng/ml). Levels of EpCAM in 412 patients with adenocarcinoma were somewhat higher with concentrations ranging from 176 to 36,259ng/ml (mean 1971ng/ml). In direct comparison, the untrimmed protein specific ELISA detected lower levels and frequencies as compared to the EGFII-specific ELISA. Only sera with less than 1µg/ml circulating EGFII-EpCAM (66% of the sera) contained EpCAM-specific IgG antibodies. The absence of IgG antibodies in the sera with more than 1µg/ml circulating EpCAM was not due to immune complex formation. Anti-EpCAM IgA and IgM antibodies did not show such a correlation. It will be important to assess whether the presence of high levels of circulating EGFII-EpCAM is associated with side effects in patients given immunotherapy.
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Anticuerpos Monoclonales/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Moléculas de Adhesión Celular/química , Moléculas de Adhesión Celular/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Animales , Antígenos de Neoplasias/sangre , Moléculas de Adhesión Celular/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Molécula de Adhesión Celular Epitelial , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Neoplasias/diagnóstico , Neoplasias/inmunología , Reproducibilidad de los ResultadosRESUMEN
Clear cell renal cell carcinoma (ccRCC) is an aggressive and difficult to manage cancer. Immunotherapy has the potential to induce long-lasting regression in a small group of patients. However, severe side effects limit broad application which highlights the need for a marker to distinguish responder from nonresponder. TNMG staging, referring to tumor size, lymph node involvement, presence of metastasis, and grade of tumor differentiation, represents an important prognostic system but is not useful for predicting responders to immunotherapy. NK cells are potent antitumor effector cells, and a role as prognostic marker in some solid tumors has been suggested. As NK cells are responsive to various immune modifiers, they may be important mediators of patient response to immunotherapies, in particular those including IL-2. We report that the NK cell percentage within RCC-infiltrating lymphocytes, as determined by flow cytometry, allows ccRCC subgrouping in NK(high)/NK(low) tissues independent of TNMG classification. Quantitative reverse transcriptase polymerase chain reaction using whole-tissue RNA identified four markers (NKp46, perforin, CX(3)CL1, and CX(3)CR1) whose transcript levels reproduced the NK(high)/NK(low) tissue distinction identified by flow cytometry with high selectivity and specificity. Combined in a multiplex profile and analyzed using neural network, the accuracy of predicting the NK(high)/NK(low) groups was 87.8%, surpassing that of each single marker. The tissue transcript signature, based on a robust high-throughput methodology, is easily amenable to archive material and clinical translation. This now allows the analysis of large patient cohorts to substantiate a role of NK cells in cancer progression or response to immunotherapy.
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Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Perfilación de la Expresión Génica , Neoplasias Renales/genética , Neoplasias Renales/patología , Células Asesinas Naturales/patología , Adulto , Anciano , Anciano de 80 o más Años , Receptor 1 de Quimiocinas CX3C , Carcinoma de Células Renales/clasificación , Carcinoma de Células Renales/mortalidad , Quimiocina CX3CL1/genética , Femenino , Humanos , Inmunofenotipificación , Neoplasias Renales/clasificación , Neoplasias Renales/mortalidad , Linfocitos Infiltrantes de Tumor/patología , Masculino , Persona de Mediana Edad , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Estadificación de Neoplasias , Perforina/genética , Pronóstico , Receptores de Quimiocina/genéticaRESUMEN
The interaction of human mesenchymal stem cells (hMSCs) and tumor cells has been investigated in various contexts. HMSCs are considered as cellular treatment vectors based on their capacity to migrate towards a malignant lesion. However, concerns about unpredictable behavior of transplanted hMSCs are accumulating. In malignant gliomas, the recruitment mechanism is driven by glioma-secreted factors which lead to accumulation of both, tissue specific stem cells as well as bone marrow derived hMSCs within the tumor. The aim of the present work was to study specific cellular interactions between hMSCs and glioma cells in vitro. We show, that glioma cells as well as hMSCs differentially express connexins, and that they interact via gap-junctional coupling. Besides this so-called functional syncytium formation, we also provide evidence of cell fusion events (structural syncytium). These complex cellular interactions led to an enhanced migration and altered proliferation of both, tumor and mesenchymal stem cell types in vitro. The presented work shows that glioma cells display signs of functional as well as structural syncytium formation with hMSCs in vitro. The described cellular phenomena provide new insight into the complexity of interaction patterns between tumor cells and host cells. Based on these findings, further studies are warranted to define the impact of a functional or structural syncytium formation on malignant tumors and cell based therapies in vivo.
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Conexina 43/metabolismo , Expresión Génica/fisiología , Células Gigantes/fisiología , Glioma/fisiopatología , Células Madre Mesenquimatosas/fisiología , Aminoácidos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Conexina 43/genética , Medios de Cultivo Condicionados/farmacología , Dextranos , Expresión Génica/efectos de los fármacos , Células Gigantes/efectos de los fármacos , Células Gigantes/patología , Glioma/patología , Humanos , ARN Mensajero/metabolismo , Rodaminas , Fracciones SubcelularesRESUMEN
BACKGROUND: The prognostic relevance of disseminated cytokeratin-positive (CK+) tumor cells in the bone marrow of patients with different types of carcinoma has been demonstrated in several studies. In this prospective study, the frequency and prognostic value of CK+ tumor cells was investigated in the bone marrow of 55 consecutive patients with metastatic renal cell carcinoma (M1 RCC) in comparison with 256 M0 RCC patients from a previous study. METHODS: Aspiration of bone marrow from the anterior iliac crest was performed immediately before tumor resection in RCC patients. Cytospins were made and stained by immunocytochemistry using the APAAP (alkaline phosphatase-antialkaline phosphatase) protocol and monoclonal antibodies CK2 and A45-B/B3. Twenty-seven patients with no evidence of any malignant disease served as a control group. RESULTS: CK+ tumor cells were detected in 42% (23 of 55 patients) of the M1 patients and 25% (63 of 256 patients) of the M0 patients (P <.01). No CK+ cells (0 of 27 patients) were detected in the control group. In the M1 group, CK- patients demonstrated a trend toward a better outcome compared with CK+ patients (log-rank test, P = .19). This difference was significant when applying a higher threshold (0-2 CK+ cells vs. > or = 3 CK+ cells; P <.05). On multivariate analysis, the detection of > or = 3 CK+ cells in the bone marrow was found to be an independent prognostic factor (P <.001). CONCLUSIONS: The results of the current study indicate that disseminated CK+ cells play a role in the biology of tumor spread of RCC, and that their immunocytochemical detection can be useful in assessing the prognosis of patients with M1 disease.
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Neoplasias de la Médula Ósea/secundario , Carcinoma de Células Renales/patología , Neoplasias Renales/patología , Células Neoplásicas Circulantes , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Queratinas/análisis , Masculino , Persona de Mediana Edad , Pronóstico , Análisis de SupervivenciaRESUMEN
The human epithelial cell adhesion molecule (EpCAM) is expressed on normal epithelial cells and is overexpressed in most carcinomas. EpCAM-targeted immunotherapy has been tried in several clinical studies. High titers of autoantibodies against EpCAM have been reported by different authors. We have generated large amounts of purified protein in S2 Drosophila cells (S2-EpCAM) with a purity of >96%. In contrast, the protein produced in baculovirus-dependent systems (baculo-EpCAM) that has been used in previous studies shows a purity of 79%. (1)H nuclear magnetic resonance spectrum of S2-EpCAM is typical of folded protein, whereas the baculo-EpCAM sample shows a spectrum corresponding to a partially unfolded protein. Using S2-EpCAM, denatured S2-EpCAM, and baculo-EpCAM, we measured EpCAM Abs of different isotypes in the serum of healthy controls and cancer patients. We found Ab titers against EpCAM in a much lower percentage of sera as published previously, and support the hypothesis that Ab reactivity in some published studies might be due to reactivity against denatured protein, to contaminating proteins in the baculovirus preparations, and to reactivity with BSA. Tetanus toxoid-reactive IgG Abs are present in 1000-fold higher titers compared with EpCAM-reactive Abs. Only IgA Abs were found in higher proportions and in higher concentrations than tetanus toxoid-specific Abs. Our study shows that EpCAM only rarely induces autoantibodies against native protein and emphasizes the importance of using extremely purified Ag preparations when evaluating Abs against tumor-associated Ags.
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Antígenos de Neoplasias/inmunología , Autoanticuerpos/sangre , Autoantígenos/inmunología , Biomarcadores de Tumor/inmunología , Moléculas de Adhesión Celular/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/normas , Animales , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/sangre , Antígenos de Neoplasias/aislamiento & purificación , Autoanticuerpos/biosíntesis , Autoantígenos/aislamiento & purificación , Baculoviridae/genética , Baculoviridae/inmunología , Biomarcadores de Tumor/aislamiento & purificación , Biomarcadores de Tumor/normas , Neoplasias de la Mama/inmunología , Moléculas de Adhesión Celular/aislamiento & purificación , Moléculas de Adhesión Celular/normas , Línea Celular , Neoplasias Colorrectales/inmunología , Relación Dosis-Respuesta Inmunológica , Drosophila melanogaster/genética , Drosophila melanogaster/inmunología , Contaminación de Medicamentos , Molécula de Adhesión Celular Epitelial , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Desnaturalización Proteica , Pliegue de Proteína , Control de Calidad , Proteínas Recombinantes/aislamiento & purificación , Neoplasias Gástricas/inmunologíaRESUMEN
PURPOSE: Several investigations indicate the prognostic value of disseminated cytokeratin positive tumor cells in bone marrow of patients with carcinoma of different origin. In this study we evaluated the prognostic significance of epithelial cells in bone marrow of patients with renal cell carcinoma (RCC). MATERIALS AND METHODS: Aspiration of bone marrow was performed preoperatively in 335 patients with RCC between 1990 and 1998. A total of 287 patients fulfilled all study inclusion (eg M0 R0 tumor stage) and exclusion (eg second malignancy during followup) criteria for the final analysis. Cytospin preparations were made after density gradient centrifugation of bone marrow samples and incubated with monoclonal antibodies directed against cytokeratin 18 (CK2) and pan-cytokeratin. Staining was performed using the alkaline phosphatase-anti-alkaline phosphatase method and 256 samples were evaluated. RESULTS In 25% (63 of 256) of the patients cytokeratin positive (CK+) cells were detected in bone marrow. Tumor progression (defined as tumor associated death, local recurrence or new metastases) was present in 12% (31 of 256) during the followup period (median 40 months), and 14% (9 of 63) with CK+ cells and 11% (22 of 193) with negative bone marrow status exhibited tumor progression. Survival analysis (log-rank test) showed no significant difference between the CK+ and cytokeratin negative group. The detection of CK+ cells was not an independent prognostic parameter in multivariate analysis (Cox regression model). CONCLUSIONS: These results indicate that the immunocytochemical detection of disseminated cytokeratin positive tumor cells in the bone marrow of patients with RCC has no prognostic significance.