摘要:

In situ hybridization (ISH) allows for the histologic and cytologic localization of DNA and RNA targets. However, the application of ISH techniques can be limited by their inability to detect targets with low copies of DNA and RNA. During the last few years, several strategies have been developed to improve the sensitivity of ISH by amplification of either target nucleic acid sequences prior to ISH or signal detection after the hybridization is completed. Current approaches involving target amplification (in situ PCR, primed labeling, self-sustained sequence replication), signal amplification (tyramide signal amplification, branched DNA amplification), and probe amplification (padlock probes and rolling circle amplification) are reviewed with emphasis on their applications to bright field microscopy. More recent developments such as molecular beacons and in situ strand displacement amplification continue to increase the sensitivity of in situ hybridization methods. Application of some of these techniques has extended the utility of ISH in diagnostic pathology and in research because of the ability to detect targets with low copy numbers of DNA and RNA.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Immunohistochemistry (IHC) is frequently used to detect plasma cell (PC) or B cell monoclonality in histologic sections, but its interpretation is often confounded by background staining. We evaluated a new automated method for colorimetric in situ hybridization (CISH) detection of clonality in PC dyscrasias and small B cell lymphomas. Cases of PC dyscrasia included multiple myeloma (MM; 31 cases), plasmacytoma (seven cases), or amyloidosis (one case), while cases of lymphoma included small lymphocytic (three cases), marginal zone (four cases), lymphoplasmacytic (three cases), and mantle cell lymphomas (three cases). Tissue sections were stained for kappa and lambda light chains by IHC and for light chain mRNA by automated CISH using haptenated probes. Twenty-eight of 31 MM cases had detectable light chain restriction by IHC. Thirty of 31 MM cases demonstrated light chain restriction by CISH, including 2 cases with uninterpretable IHC and one case of nonsecretory myeloma, which was negative for light chains by IHC. Seven of 7 plasmacytoma cases had detectable light chain restriction by CISH, including one case of nonsecretory plasmacytoma in which IHC was noninformative. Automated CISH demonstrated monoclonality in 9 of 13 cases of B cell non-Hodgkin lymphoma and had a slightly higher sensitivity than IHC (6 of 13 cases), especially in cases of lymphoplasmacytic and marginal zone lymphoma. Overall, there were no discrepancies in light chain restriction results between IHC, CISH, or serum paraprotein analysis. Automated CISH is useful in detecting light chain expression in paraffin sections and appeared superior to IHC for light chain detection in PC dyscrasias and B cell non-Hodgkin lymphomas, predominantly due to lack of background staining.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Identification of fungi in tissue sections can be difficult. In particular, species ofAspergillus, Fusarium,andPseudallescheriaall appear as septate, branched hyphae. However, their differentiation can have significant clinical implications, as the latter two groups are often resistant to commonly used antifungal agents. In situ hybridization may assist in rapidly distinguishing these organisms in the absence of available culture. Oligonucleotide DNA probes were directed against the 5S, 18S, or 28S rRNA sequences of three groups of fungi with a high degree of specificity for each. Probes were tested on 26 formalin-fixed, paraffin-embedded tissue specimens, each with culture-proven involvement by one of these organisms:Fusariumspecies, n = 12;Pseudallescheria boydii,n = 5;Aspergillusspecies, n = 9 (Aspergillusprobe set validated in an earlier study). Accuracy of both ISH and morphology was compared with culture.Morphologic examination (GMS and PAS) showed a greater sensitivity in detecting fungi (100%) as compared with in situ hybridization (84.6%). When detected, however, DNA probes allowed definitive identification of organisms. While there was no ability to distinguish between the three groups of organisms by morphologic features, ISH probes showed 100% positive predictive value (PPV, 19/19 organisms identified correctly). No cross-reactivity was observed when the probes were tested against other genera (100% specificity). Furthermore, the use of ISH allowed the detection of mixed fungal infections involving multiple organism types in two cases, demonstrating another advantage over morphology. In situ hybridization, directed against rRNA sequences, provides a rapid and accurate technique for distinguishing commonly encountered, nonpigmented filamentous fungi in histologic sections. While less sensitive than morphology, ISH is highly accurate and may help to distinguish between organisms that have similar or identical morphologic features by light microscopy.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

The histopathologic classification of breast cancer stratifies tumors based on tumor grade, stage, and type. Despite an overall correlation with survival, this classification is poorly predictive and tumors with identical grade and stage can have markedly contrasting outcomes. Recently, breast carcinomas have been classified by their gene expression profiles on frozen material. The validation of such a classification on formalin-fixed paraffin-embedded tumor archives linked to clinical information in a high-throughput fashion would have a major impact on clinical practice. The authors tested the ability of tumor tissue microarrays (TMAs) to sub-classify breast cancers using a TMA containing 107 breast cancers. The pattern of expression of 13 different protein biomarkers was assessed by immunohistochemistry and the multidimensional data was analyzed using an unsupervised two-dimensional clustering algorithm. This revealed distinct tumor clusters which divided into two main groups correlating with tumor grade (P<0.001) and nodal status (P= 0.04). None of the protein biomarkers tested could individually identify these groups. The biological significance of this classification is supported by its similarity with one derived from gene expression microarray analysis. Thus, molecular profiling of breast cancer using a limited number of protein biomarkers in TMAs can sub-classify tumors into clinically and biologically relevant subgroups.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Overexpression of cyclin D1 is necessary but by itself insufficient for the development of mantle cell lymphoma (MCL). To identify pathways in the pathogenesis of MCL and the blastoid variant (MLC-BV), we compared the gene-expression profiles of microdissected normal mantle cells, MCL, and MCL-BV by oligonucleotide microarrays and quantitative reverse transcriptase PCR (QRT-PCR). We identified and confirmed the overexpression of several genes in MCL-BV that are involved in the cell cycle control at the G1/S and G2/M checkpoints or inhibit apoptotic cell death. The highly expressed cyclin dependent kinase 4 (CDK4) is a cell cycle kinase that associates with cyclin D1 for the progression through the G1/S checkpoint, whereas overexpression of cdc28 protein kinase 1 (CKS1) blocks the inhibition of the cyclin D1/CDK4 complex by the CDK inhibitor p27/Kip1. Other highly expressed genes in MCL-BV that promote the cells through the G1/S-checkpoint include the oncogenes B-Myb, PIM1, and PIM2, and passage through the G2/M-checkpoint is enhanced by high levels of cdc25B. Furthermore, two highly expressed genes that inhibit apoptosis are defender against cell death (DAD1) and RSK1. In summary, our microarray and QRT-PCR analyses identified several candidate genes whose expression increased when comparing normal follicular mantles with MCL and MCLBV, suggesting a potential pathogenic role in the evolution of MCL-BV.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Our group has previously demonstrated an association between ret/PTC-1 activation and decreased E-cadherin mRNA levels in papillary thyroid carcinoma. We also observed similarities in the E-cadherin expression profiles of Hashimoto thyroiditis and ret/PTC-1–positive papillary thyroid carcinomas and have hypothesized that ret/PTC-1 activation might cause not only the structural and nuclear peculiarities of PTC but also an immune reaction to thyroid epithelium. The objective of this study was to examine the expression of E-cadherin's ligands, &bgr;- and &ggr;-catenin, in various thyroid tissue types in the context of ret/PTC-1 positivity using laser capture microdissection and TaqMan (Applied Biosystems, Foster City, CA). One-Step RT-PCR. &bgr;-catenin mRNA levels were found to be consistently decreased in both papillary and anaplastic carcinomas when compared with a normal/follicular adenoma group. A significant difference in expression levels was observed between papillary and follicular thyroid carcinomas with the latter having elevated mRNA levels of &bgr;-catenin. &ggr;-Catenin mRNA was decreased in anaplastic carcinomas compared with normal/follicular adenoma groups. A similar expression profile of &ggr;-catenin as &bgr;-catenin was observed in papillary and follicular carcinomas with the latter once again having higher mRNA levels. These results therefore suggest that although &bgr;- and &ggr;-catenin may play a role in the progression of thyroid cancer in general, they do not appear to be associated with ret/PTC-1–modulated pathways.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

Head and neck squamous cell carcinoma (HNSCC) is a multistage process during which adverse genetic alterations accumulate resulting in loss of cell cycle control, selective cell overgrowth, and ultimately formation of malignancy. Among various genetic alterations in HNSCC is increased microsatellite instability (MSI).hMLH1is one of the major mismatch DNA repair genes, the inactivation of which caused increased MSI in a variety of human cancers including HNSCC. While somatic mutation is a major mechanism of thehMLH1gene inactivation in hereditary form of human cancer, promoter hypermethylation appears to be primarily involved in the inactivation of thehMLH1gene in sporadic form of human cancers. In the current study, we analyzed 78 cases of HNSCC forhMLH1protein expression and promoter hypermethylation by IHC and methylation-specific PCR (MSP). Twenty-four of 78 cases (31%) of HNSCC contained markedly reduced levels of thehMLH1protein. Based on the IHC results, 8 cases without and 8 withhMLH1protein expression (total of 16) were further analyzed by MSP. Seven of 8 cases (88%) that were negative for thehMLH1protein displayed promoter hypermethylation, whereas 7 of 7 cases (100%) strongly positive for the protein were free of promoter methylation. This study confirms our previous conclusion that promoter hypermethylation represents a major mechanism of thehMLH1gene inactivation in HNSCC.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID

摘要:

IGFIIandH19genes are expressed only from one allele due to genomic imprinting, biallelic expression (loss of imprinting) being associated with the tumorigenic process of different types of tumors. The mechanism responsible for genomic imprinting is not yet determined, although DNA methylation has been considered the main genetic event for an imprinted mark. In the current study, the authors analyzed the imprinting status and expression levels of theIGFIIandH19genes in 27 cases of Juvenile Nasopharyngeal Angiofibroma (JNA) using RFLPs, RT-PCR, and Southern and Northern Blots. The authors found that four out of eight informative cases (50%) forApaI/IGFIIpolymorphism showed biallelic expression ofIGFII,whereas none of the nine informative cases for theRsaI/H19polymorphism showed biallelic expression of theH19gene. Overexpression ofIGFIIwas observed in 8 out of 22 cases (36.4%), and 7 out of 19 cases (36.8%) showedH19overexpression. Hypomethylation was found only in theH19gene in six out of eight cases analyzed. Therefore, our results demonstrate that alterations in theIGFII/H19imprinted region occur in JNA.

ISSN:1052-9551    

出版商:OVID    

年代:2003

数据来源: OVID