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OBJECTIVES: Although fluorescence in situ hybridization (FISH) technology is adequate, demand exists for additional recycling and long-term storage of FISH slides. METHODS: Formalin-fixed paraffin-embedded slides derived from breast cancer cases were used for this study. Each slide was probed, and then procedures for removing probes were performed, such as removing the fluorescent probe and diamidino-2-phenylindole signals. Formamide was used for removing probes, and then slides were stored dry at room temperature (22°C), 4°C, -20°C, or -80°C for 101 days. Following storage, each slide was probed in a similar manner to the initial probing. Evaluation was performed using automatic signal count software. Tiles and spots were counted immediately after the initial probing. Reprobed spots for each slide were then compared with the initial probing. RESULTS: Slides stored at -20°C and -80°C for 101 days showed the best recovery of probing. CONCLUSIONS: Our approach for probe removal and recycling allows repeated examination of even a limited number of slides.
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Hibridación Fluorescente in Situ/métodos , Manejo de Especímenes , Humanos , Adhesión en Parafina , ReciclajeRESUMEN
Low-grade lung adenocarcinoma of fetal lung type, which is well characterized by its unique clinicopathologic and molecular features, is recognized as a distinct variant of lung cancer. In contrast, high-grade lung adenocarcinoma with fetal lung-like morphology (HG-LAFM) has not been studied widely. To characterize this subset better, we analyzed 17 high-grade adenocarcinomas with at least focal component resembling a developing epithelium in the pseudoglandular phase of the fetal lung. These rare (ca. 0.4%) carcinomas occurred predominantly in elderly men with a heavy smoking history, who showed elevated serum α-fetoprotein in 4 of 5 cases tested. Histologic examination revealed a fetal lung-like component as a focal finding accounting for 5% to 60% of the total tumor volume. It was invariably admixed with tissues having a morphology not resembling that of a fetal lung. A coexisting non-fetal lung-like element was quite heterogenous in appearance, showing various growth patterns. However, clear-cell (88%), hepatoid (29%), and large cell neuroendocrine carcinoma (24%) histology seemed overrepresented. HG-LAFM was characterized immunohistochemically by frequent expression of α-fetoprotein (41%), glypican-3 (88%), SALL-4 (59%), neuroendocrine markers (82%), CDX-2 (35%), and p53 (65%). HG-LAFM was molecularly heterogenous in that EGFR or KRAS mutation was observed in 22% of cases tested for both. Our data indicate that HG-LAFMs might form a coherent subgroup of lung adenocarcinomas. However, the uniformly focal nature of the fetal lung-like element, widely diverse coexisting non-fetal lung-like histology, and inhomogenous molecular profiles lead us to believe that HG-LAFM is best regarded as a morphologic pattern showing characteristic association with several clinicopathologic parameters rather than a specific tumor entity.
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Adenocarcinoma/genética , Adenocarcinoma/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Adenocarcinoma/mortalidad , Adenocarcinoma del Pulmón , Adulto , Anciano , Biomarcadores de Tumor/análisis , Análisis Mutacional de ADN , Femenino , Genes erbB-1 , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Masculino , Persona de Mediana Edad , Mutación , Estadificación de Neoplasias , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas p21(ras) , alfa-Fetoproteínas/análisis , Proteínas ras/genéticaRESUMEN
We established our bio-repository in October 2002. One of the unique aspects of our bio-repository is that it is based on post-clinical test samples. Although the post-clinical test sample-based storage is beneficial because ample clinical information is available for each sample and samples are to be refreshed regularly, many bio-bankers are hesitant to store them because of the possible problems of de-identification procedures and sample quality. Currently, we have two different types of sample, not de-identified status and de-identified status. Most of the samples for storage are not de-identified status. A portion of the samples are transferred to new tubes before and after being frozen, without clinical patient identifications and the status is de-identified. This tube transfer is the only de-identification procedure in our bio-repository so far. After a procedure of de-identification, these samples are stored. The de-identified samples are collected under various project-based schemes. The quality standards of our samples are established by two factors: pre-analytical quality control efforts, and record keeping of sample history by sample tracking system. All the samples have been phlebotomized under strict control of pre-analytical requirements. By record keeping of sample history with the sample tracking system, we can tell the exact temperature and elapsed time in various situations since the phlebotomy procedure or on arrival at the clinical laboratory. In this article, we will disclose how we have dealt with the de-identification procedure and sample quality.
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Bancos de Muestras Biológicas/normas , Confidencialidad , Neoplasias , Manejo de Especímenes , Instituciones Oncológicas , Humanos , Control de Calidad , TokioRESUMEN
We established the Bio-repository at the National Cancer Center Hospital in October 2002. The main purpose of this article is to show the importance and usefulness of a bio-repository of post-clinical test samples not only for translational cancer research but also for routine clinical oncology by introducing the experience of setting up such a facility. Our basic concept of a post-clinical test sample is not as left-over waste, but rather as frozen evidence of a patient's pathological condition at a particular point. We can decode, if not all, most of the laboratory data from a post-clinical test sample. As a result, the bio-repository is able to provide not only the samples, but potentially all related laboratory data upon request. The areas of sample coverage are the following: sera after routine blood tests; sera after cross-match tests for transfusion; serum or plasma submitted at a patient's clinically important time period by the physician; and samples collected by the individual investigator. The formats of stored samples are plasma or serum, dried blood spot (DBS) and buffy coat. So far, 150 218 plasmas or sera, 35 253 DBS and 536 buffy coats have been registered for our bio-repository system. We arranged to provide samples to various concerned parties under strict legal and ethical agreements. Although the number of the utilized samples was initially limited, the inquiries for sample utilization are now increasing steadily from both research and clinical sources. Further efforts to increase the benefits of the repository are intended.
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Bancos de Muestras Biológicas/normas , Instituciones Oncológicas , Neoplasias/epidemiología , Manejo de Especímenes , Bancos de Muestras Biológicas/economía , Humanos , Neoplasias/diagnóstico , Neoplasias/etiología , Selección de Paciente , Proyectos de Investigación , TokioRESUMEN
Pleomorphic liposarcoma (PLS) is an aggressive subtype of liposarcoma composed of high-grade sarcoma with pleomorphic lipoblasts. PLS usually exhibits a heterogeneous histology and sometimes has a myxoid or round cell area similar to myxoid/round cell liposarcomas (MLS/RCs). Using fluorescence in situ hybridization (FISH) analysis, we investigated the existence of CHOP split signals in various histological areas of PLS including the MLS/RC-like feature and also estimated the distribution of various signals with polyploidy and amplification. Moreover, to detect CHOP fusion transcripts we performed nested reverse transcription-polymerase chain reaction (RT-PCR). Seven PLSs and three MLS/RCs were selected for FISH analysis using the locus-specific indicator CHOP (12q13) dual color, break apart probe (Vysis, USA). The FISH analysis was applied to formalin-fixed, paraffin-embedded tissue sections of representative areas in all cases. Six of seven PLS cases showed the CHOP split signal ranging from 0.5% to 3% of counted nuclei, while all cases of MLS/RC exhibited CHOP rearrangement in more than 50% of counted nuclei. All cases of PLS showed a varied distribution of extra signals with polyploidy and amplification in each histological area. No CHOP fusion transcript was found in any case of PLS by nested RT-PCR. A CHOP rearrangement in PLS should be recognized only as a representative part of complex karyotypes, because the number of cells with split signals was minute compared with that of MLS/RC, and the signals were found in any area despite their histological differences. The cytogenetic background of PLS and that of MLS/RC are obviously different despite histological similarity.
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Reordenamiento Génico , Hibridación Fluorescente in Situ/métodos , Liposarcoma/genética , Factor de Transcripción CHOP/genética , Anciano , Femenino , Humanos , Liposarcoma/patología , Masculino , Persona de Mediana Edad , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Mutations of the epidermal growth factor receptor (EGFR), particularly deletional mutations (DEL) in exon 19 and L858R in exon 21, are reportedly correlated with clinical outcome in patients with non-small cell lung cancer (NSCLC) receiving the EGFR tyrosine kinase inhibitors gefitinib and erlotinib, suggesting that detection of EGFR mutations would have an important role in clinical decision making. We established and validated an easy, inexpensive, and rapid method for detecting DEL and L858R from cytologic material by high-resolution melting analysis (HRMA). Dilution for sensitivity studies revealed that DEL and L858R were detectable in the presence of at least 10% and 0.1% EGFR-mutant cells, respectively. We analyzed 37 archived cytological slides of specimens from 29 patients with advanced NSCLC and compared the results with direct sequencing data obtained previously. Of 37 samples, 34 (92%) yielded consistent results with direct sequencing, 2 were false negative, and 1 was indeterminate. The sensitivity of this analysis was 90% (19/21) and specificity, 100% (15/15). These results suggest that HRMA of archived cytologic specimens of advanced NSCLC is useful for detecting EGFR mutations in clinical practice.