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PubMed2005

Laser-assisted microdissection of membrane-mounted sections following immunohistochemistry and in situ hybridization.

Laser microbeam microdissection (LMM) is an increasingly important histological technique for obtaining homogeneous cell populations and tissue components in order to analyze target-specific changes in genes, gene expression, and proteins. The quality of data obtained with LMM is heavily dependent on the precision with which the target for microdissection can be identified. Since no cover slip is used during LMM, tissue morphology is poor compared with traditional light microscopy. This hampers morphological recognition of targets for microdissection in routinely stained sections and can be a limiting factor in the use of this technique. Immunohistochemistry (IHC) and in situ hybridization (ISH) can improve the identification of specific cell populations in situ in tissue sections, but there are a number of problems in applying these methods to slides prepared for LMM. In this chapter, we present optimized protocols that allow IHC to be performed for detecting a wide range of antigens in conjunction with LMM, both on formalin-fixed paraffin-embedded and on frozen sections. In addition, we present a quick, versatile protocol for performing ISH on archival material suitable for LMM.

Animals↗

PubMed2005

Laser-assisted cell microdissection using the PALM system.

Laser-assisted microdissection has enabled the collection of morphologically defined cell populations from a tissue section. The PALM Robot MicroBeam laser microdissection system provides a robust system for the retrieval of specified cells (including single cells). Due to the fragile nature of DNA, and in particular RNA, robust protocols are required to obtain reliable data from a limited number of cells (1-10.000 cells). This chapter describes the application of the PALM MicroBeam system to isolate RNA and DNA from cells in a complex tissue for subsequent molecular analysis. Protocols for successful analysis of RNA from 500 to 1000 cells, including steps to produce cDNA for subsequent polymerase chain reaction analysis, are given. The cDNA could also be used as a template for linear amplification in order to perform gene array analysis. Furthermore, a protocol for genomic analysis of p53 mutations from single cells is given. The described procedures emphasize preparation of tissue, laser microdissection including catapulting of cells, and extraction of RNA and DNA. Downstream experiments for validation are also shown.

Animals↗

PubMed2005

Laser microdissection and RNA analysis.

Microdissection techniques have become an important tool to link histomorphology and pathophysiological events using modern methods of molecular biology. They allow isolation of cell clusters or even single cells precisely under optical control from complex tissue structures for further analysis of DNA, RNA, and proteins. In particular, the fragile RNA molecules can be preserved during microdissection so that gene expression and regulation measurement become feasible in a cell type-specific manner within complex tissues. This report focuses on and outlines the procedures for RNA investigation, from tissue fixation, sectioning, and staining to downstream applications (RT-PCR, mRNA quantification, and mRNA preamplification). Standards for the preparation of RNA from frozen and formalin-fixed tissues are presented. Specific protocols are given for both the isolation of RNA from small numbers of cells (50 cells) as well as for larger cell numbers. While most of the procedures are identical for the microdissection systems, special features of each technique are mentioned.

Animals↗

PubMed2005

Noncontact laser microdissection and pressure catapulting: sample preparation for genomic, transcriptomic, and proteomic analysis.

The understanding of the molecular mechanisms of cellular metabolism and proliferation necessitates accurate identification, isolation, and finally characterization of a specific cell or a population of cells and subsequently their subsets of biomolecules. For the simultaneous analysis of thousands of molecular parameters within a single experiment, as realized by DNA, RNA, and protein microarray technologies, a defined number of homogeneous cells derived from a distinct morphological origin is required. Sample preparation is therefore a very crucial step for high-resolution downstream applications. Laser microdissection and laser pressure catapulting (LMPC) enables such pure and homogeneous sample preparation, resulting in an eminent increase in the specificity of molecular analyses. For microdissection, the force of focused laser light is used to excise selected cells or large tissue areas from object slides or from living cell culture down to a resolution of individual single cells and subcellular components like organelles or chromosomes, respectively. After microdissection this sample is directly catapulted into an appropriate collection device. As the entire process works without any mechanical contact, it enables pure sample retrieval from morphologically defined origin without cross contamination. Wherever homogenous samples are required for subsequent analysis of, e.g., cell areas, single cells, or chromosomes, the PALM MicroBeam system is an indispensable tool. The integration of image analysis platforms fully automates screening, identification, and finally subsequent high-throughput sample handling. These samples can be directly linked into versatile downstream applications, such as single-cell mRNA-extraction, different PCR methods, microarray techniques, and many others. Acceleration in sample generation vastly increases the throughput in molecular laboratories and leads to an increasing knowledge about differentially regulated mRNAs and expressed proteins, providing new insights into cellular mechanisms and therefore enabling the development of systems for tumor biomarker identification, early detection of disease-causing alterations, therapeutic targeting and/or patient-tailored therapy.

Animals↗

PubMed2006

Laser-assisted microdissection and isolation of DNA and RNA.

One of the major challenges in molecular analysis of breast cancer specimens is tissue heterogeneity. The admixture of contaminating bystander cells might distort the results of quantitative molecular analyses. Therefore, pure tumor cell populations have to be isolated in order to obtain reliable molecular data. In this chapter, we present protocols for the laser-assisted microdissection of breast cancer tissue sections (using a laser microdissection microscope from P.A.L.M., Bernried, Germany) and the subsequent isolation of genomic DNA or total RNA. The protocols presented in here have been used in our laboratory for the exact quantification of gene copy numbers in intraductal and invasive tumor cells and for the quantitative assessment of promoter hypermethylation during breast cancer progression. We have added some guidelines for the organization of the laser-microdissection and polymerase chain reaction laboratory, which prevent crosscontamination of samples and carry-over contamination because of polymerase chain reaction products.

Breast Neoplasms↗

PubMed2006

Differential gene expression profiling in aggressive bladder transitional cell carcinoma compared to the adjacent microscopically normal urothelium by microdissection-SMART cDNA PCR-SSH.

Identifying novel and known genes that are differentially expressed in aggressive bladder transitional cell carcinoma (BTCC) has important implications in understanding the biology of bladder tumorigenesis and developing new diagnostic and therapeutic agents. In this study we identified the differential gene expression profiles comparing tumor to the adjacent microscopically normal mucosa by manual microdissection on frozen sections. The RNAs extracted from microdissected tissues were amplified by SMART cDNA PCR technology to generate forward subtractive cDNA library by suppressive subtractive hybridization (SSH). We obtained 376 positive clones, one hundred clones of aggressive BTCC subtracted cDNA library were selected at random and inserts were reamplified by PCR. After differential screening by reverse dot blotting, 73 positive clones, that contend inserts putatively upregulated in aggressive BTCC, were further analysed by DNA sequencing, GenBank and EST database searching. Sequencing results showed that 66 clones stand for 23 known genes and 7 clones for three new EST (Genbank number: DN236875, DN236874 and DN236873). In conclusion, microdissection-SMART cDNA PCR-SSH allowed for an efficient way to identify aggressive BTCC-specific differential expressed genes that may potentially be involved in the carcinogenesis and/or progression of aggressive BTCC. These differentially expressed genes may be of potential utility as therapeutic and diagnostic targets for aggressive BTCC.

Carcinoma, Transitional Cell↗

PubMed2004

Microdissection genotyping of mixed glial and primitive neuroectodermal central nervous system neoplasm.

A 22-year-old man with previous radiation treatment for childhood astrocytoma underwent resection of a right parietooccipital lesion. Histopathology revealed a malignant neoplasm with areas of astrocytic and primitive neuroectodermal components. To resolve the relationship and cellular origin, representative tissue was microdissected from several targets, obtaining a balanced mixture of each element. Nonneoplastic brain parenchyma was separately microdissected to determine polymorphic marker informativeness and to serve as an internal negative control. Despite the relatively small quantity of tissue removed for each microdissection target, sufficient material was available for reliable, balanced, polymerase chain reaction-format genotyping encompassing a panel of tumor suppressor genes and genetic loci associated with these forms of neoplasia. The findings revealed distinct discordant genotypic profiles for each of the neoplastic components. The efficacy of the approach used for molecular analysis of this complex neoplasm and the implication of the genotypic findings are discussed.

Adult↗

PubMed2004

[Detection of IgH gene rearrangement in Reed-Sternberg cells microdissected from classical Hodgkin's lymphoma].

OBJECTIVE: To study the origins and clonality of Hodgkin and Reed-Sternberg (H/RS) cells and their relations with the background lymphocytes in classical Hodgkin's lymphoma. METHOD: IgH gene rearrangement was detected in paffin-embedded tissues from 33 patients with Hodgkin's lymphoma and a further analysis of the gene rearrangement was conducted in 6 of the positive cases identified after immunostaining of the sections with B-cell-specific activator protein (BSAP) followed by microdissection of the positivity labeled H/RS cells and background lymphocytes. RESULTS: IgH gene rearrangement was identified in 16 of the 33 cases. Microdissection of the lymphoma tissues was successfully performed in the 6 positive cases, and of the 19 tubes of H/RS cells obtained, 14 presented clonal bands of the rearrangement, and difference in cell numbers did not significantly influence the positive rate (P=0.290); in the 12 tubes of microdissected background lymphocytes obtained, 2 were positive for the rearrangement, and the positive rates for the rearrangement significantly differed between H/RS cells and the background lymphocytes (P=0.002). CONCLUSION: The results appear to support the hypothesis that H/RS cells originates from B cells, and a part of the background lymphocytes may possess neoplastic proliferation potentials to function as the precursors of H/RS cells.

Adolescent↗

PubMed2005

Laser-assisted microdissection, techniques and applications in pathology (review).

Innovations in molecular medicine provided sophisticated tools for analysis of the pathogenesis of diseases. Much emphasis is put on examination of alterations in affected organ systems. However, since in vivo tissues are inherently complex mixtures of different cell types, specific molecular data on individual cell populations are difficult to obtain. The advent of laser assisted microdissection (LAM) now allows efficient isolation of pure cell populations and even of single cells from mixed tissues. The most promising aspect of LAM is its combination with different molecular downstream analyses of microdissected cells at the levels of the genome, the transcriptome and the proteome. This review focuses on the two basic techniques of laser-assisted microdissection and on its applications in molecular pathology.

Genomics↗

PubMed2005

Forensic applications of laser capture microdissection: use in DNA-based parentage testing and platform validation.

AIM: To report on the successful use of Laser Capture Microdissection (LCM) as a tool for isolation of human chorionic villi from admixed maternal tissue. Subsequent DNA isolation for forensic short tandem repeat (STR) analysis for parentage testing was performed in two cases of alleged sexual assault of female victims. We also performed validation of the LCM instrument platform, using archival formalin-fixed human fetal products of conception (POC), for which microdissection was utilized to separate maternal (decidua) and fetal (chorionic villus) components. METHODS: To isolate DNA from placental chorionic villi admixed with maternal decidua recovered after spontaneous or therapeutic abortion, LCM was used to separate fetal from maternal cells. In contrast to the relatively crude conventional microdissection performed using a narrow pipette, needle, or scalpel blade, LCM allows cell- or tissue-specific isolation of placental chorionic villi from archival paraffin-embedded tissue sections, leaving the maternal tissue intact. RESULTS: After polymerase chain reaction (PCR) amplification of villi after LCM of 9-15 STR loci, the quantity and quality of DNA yielded from fetal cells isolated by LCM was sufficient for PCR analysis and successful forensic parentage testing. The validation data obtained on two sets of formalin-fixed archival POC tissues from anonymous donors demonstrated the encouraging reproducibility of these protocols and procedures. CONCLUSION: We demonstrated the reliability and utility of LCM for forensic applications when high specificity of a particular analyzed cell population or tissue is required. Care must be taken during routine pathology procedures to avoid contamination of tissues with admixture of extraneous DNA.

Adolescent↗

PubMed2006

Microdissection-based allelotyping: a novel technique to determine the temporal sequence and biological aggressiveness of colorectal cancer.

Pathologic staging in colorectal adenocarcinoma (CA) is based on the concept that the timing of metastatic tumor spread is directly related to the depth of the primary tumor invasion. To evaluate the temporal sequence of CA metastasis, we performed microdissection mutational profiling at multiple microscopic sites of primary and metastatic CA specimens. Twenty-one cases of CA were selected from fixed-tissue archives. Primary tumors were microdissected at the deepest point of invasion. Comparative mutational profiling for different genomic loci [1p36(CCM = cutaneous malignant melanoma], 3p26(OGGI = 8 oxoguanine DNA glycosylase), 5q23 (APC, MCC = mutated in colorectal cancer), 9p21(p16/CDKN2A = cyclin-dependent kinase 2A), 10q23(PTEN = phosphatase and tensin homolog [mutated in multiple advanced cancers 11), 12p12(K-ras-2 point mutation), 17p13(TP53), 18q25(DCC= deleted in colorectal cancer) was carried out on each microdissected tissue target using microsatellite loss of heterozygosity determination or DNA sequencing. All primary and metastatic sites of CA manifested acquired mutational change in 18 to 91 per cent of the genomic markers. In 15/21 (71%) cases, metastatic sites lacked a specific allelic loss seen in the corresponding primary tumor, indicating that the metastasis occurred before maximal depth of primary invasion. This was further supported by discordant mutational profiles between primary and secondary tumors, requiring divergent clonal evolution. This is the first report describing the temporal sequence and significance of sequential mutational acquisition in clinical tissue specimens with potential implications for a new molecular pathology approach to classify human cancer.

Adenocarcinoma↗

PubMed2000

Molecular identification of metastatic cancer to the skin using laser capture microdissection: a case report.

BACKGROUND: In the current study the authors report a 57-year-old woman with a scalp tumor and cervical lymphadenopathy who had a previously resected duodenal carcinoid. Histologic and immunophenotypic characteristics of the duodenal carcinoid differed from those of the scalp and cervical lymph node tumors, prompting the use of molecular methodologies to make the diagnosis. METHODS: Paraffin embedded tissues from the duodenal carcinoid, scalp, and lymph node tumors were dissected using microscopic visualization and laser capture microdissection. DNA was extracted and polymerase chain reaction (PCR) was performed to evaluate loss of heterozygosity and microsatellite alterations using primers flanking 22 polymorphic microsatellite markers from 9 chromosomal regions, including genes associated with MEN-1 (11q), CDKN2 (9p), p53 (17p), and bronchial carcinoid (3p). Microdissected lymphocytes from the three tissues were used as source of constitutional DNA (controls). RESULTS: Fourteen of the 22 markers were informative (heterozygous in control lymphocytes). A marker on 3p12 showed loss of the same parental allele in the three tumors. A different marker on 3p14.2 showed an identical shifted band in the three tumors indicative of a common microsatellite alteration. CONCLUSIONS: The shared molecular abnormalities among the three tumors indicated a common clonal origin, leading to a diagnosis of primary duodenal carcinoid with clear cell metastases to the scalp and cervical lymph nodes. These findings led to radiation therapy and immunotherapy rather than chemotherapy. This case illustrates the novel application of laser capture microdissection combined with PCR-based analyses of genomic markers for the identification of the origin of metastatic disease.

Alleles↗

PubMed2000

Proteomic analysis of laser capture microdissected human prostate cancer and in vitro prostate cell lines.

Specific populations of normal and malignant epithelium from three radical prostatectomy tissue specimens were procured by laser capture microdissection (LCM) and analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Six proteins that were only seen in malignant cells and two proteins that were only seen in benign epithelium were reproducibly observed in two of two cases examined. Furthermore, these proteins were not observed in the 2-D PAGE profiles from the patient-matched microdissected stromal cell populations, but were seen in the protein profiles from the undissected whole cryostat sections. One of these proteins was determined to be prostate-specific antigen (PSA) by Western blot analysis, and intriguingly the remaining protein candidates were found to be at least as abundant as the PSA protein. Comparison of 2-D PAGE profiles of microdissected cell with matched in vitro cell lines from the same patient, and metastatic prostate cancer cell lines (LnCaP and PC3) showed striking differences between prostate cells in vivo and in vitro with less than 20% shared proteins. The data demonstrate that 2-D PAGE analysis of LCM-derived cells can reliably detect alterations in protein expression associated with prostate cancer, and that these differentially expressed proteins are produced in high enough levels which could allow for their clinical utility as new targets for therapeutic intervention, serum markers, and/or imaging markers.

Blotting, Western↗

PubMed1996

Demonstration of local clonality of mucosal T cells in human colon using DNA obtained by microdissection of immunohistochemically stained tissue sections.

Intraepithelial lymphocytes have been shown to be oligoclonal and to be disseminated widely along the human intestine. However, studies using monoclonal antibodies have suggested that superimposed on the widespread clones, there is local variability in the mucosal T cell population. We have investigated the possibility that local dominant clones of T cells are present in the colonic mucosa by polymerase chain reaction (PCR) amplification of T cell receptor beta chain junctional regions using DNA extracted from microdissected fragments of tissue sections. Colon from two right hemicolectomy specimens was sampled at 7-cm intervals. Adjacent areas of mucosa were microdissected from sections from each colon sample. When the PCR products were separated according to size on polyacrylamide gels, bands of identical size were often observed when DNA extracted from adjacent fragments of mucosa had been used. Different bands were present when the different samples of colon had been studied. Sequencing of the PCR products confirmed that clonally related T cells were present in adjacent areas of mucosa, whereas different clones dominated at distant sites. DNA extracted from cells microdissected from the T cell zone of Peyer's patch was treated identically. The sequences obtained from the Peyer's patch, as expected, were diverse. However, one of the sequences identified was identical to that of one of the clones in the colon, implying that this clone was either trafficking through the Peyer's patch or possibly originated from the Peyer's patch. In this study, we also identified the Peyer's patches as a site of proliferation of CD4+ T cells. No T cell division was observed in the lamina propria. The molecular and immunohistochemical observations together support the hypothesis that the Peyer's patches are a source of mucosal T cells.

Adenocarcinoma↗

PubMed2003

HLA-G positive trophoblastic cells in transcervical samples and their isolation and analysis by laser microdissection and QF-PCR.

OBJECTIVE: To assess the frequency of cytotrophoblastic cells in endocervical samples collected by lavage at early stages of gestation using a specific anti-HLA-G McAb (G233). From a set of four selected samples, cells identified by immunostaining were collected by laser microdissection and then tested by quantitative fluorescent polymerase chain reaction (QF-PCR) for the presence of paternally derived DNA markers, in order to establish their fetal origin. METHODS: Syncytial fragments and cytotrophoblastic cells from 23 transcervical samples were identified by immunostaining with McAb G233 reacting against HLA-G antigen and with antibodies against cytokeratin. Slides from the same samples were also tested by fluorescent in situ hybridization (FISH), while selected samples were analysed by QF-PCR. Slides from four samples retrieved from mothers with male fetuses were immunolabelled and then cytotrophoblastic cells, syncytial fragments and maternal epithelial cells were collected by laser microdissection and tested by QF-PCR. RESULTS: All endocervical samples retrieved from mothers with male fetuses were found to contain some cells with chromosome Y-specific signals when tested by FISH. Using McAb anti- HLA-G, cytotrophoblastic cellular elements were detected in about 50% of the samples. From four samples, cellular elements identified by immunostaining as cytotrophoblast or syncytial fragments were collected by laser microdissection and shown to be of fetal origin when tested by QF-PCR for the presence of fetal DNA markers. CONCLUSIONS: These results confirm that, during an early phase of gestation, fetal cells are released in the lower uterine cavity and that they can be isolated and analysed for prenatal diagnosis of single gene defects and aneuploidies.

Adult↗

PubMed2005

Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometry.

Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing approximately 200 000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues.

Breast Neoplasms↗

PubMed1996

Clonal analysis by chromosome 11 microsatellite-PCR of microdissected parathyroid tumors from MEN 1 patients.

Loss of heterozygosity (LOH) at chromosome 11q13 loci has been described in the majority of larger parathyroid tumors from patients affected by Multiple Endocrine Neoplasia type 1 (MEN 1) syndrome. Since classical histology of the whole parathyroid gland does not permit a clear morpho-genetic correlation, the clonal composition of abnormal parathyroid tissue was analyzed in DNA obtained from single nodules and non-nodular areas within MEN 1 parathyroid lesions. Microdissected sections were analyzed by chromosome 11q13 microsatellite-PCR for LOH and by patterns of X-inactivation. We detected LOH for chromosome 11q13 loci in at least one microdissected area for each familial MEN 1 patient, but not in the single sporadic case. X-inactivation pattern of two "clonal" tumors exhibited a polyclonal cell composition of these microdissected samples, indicating the existence of a genetic heterogeneity in MEN 1 parathyroid microareas exhibiting a "clonal" pattern for allelic losses.

Adolescent↗

PubMed1993

Isolation of region-specific cosmids by hybridization with microdissection clones from human chromosome 10q11.1-q21.1.

A region-specific plasmid library composed of 20,000 recombinants was constructed by microdissection of human chromosome 10 (10q11.2-q21.1) and subsequent amplification with the primer-linker method of polymerase chain reaction (PCR). Hybridization with total human DNA showed that 32 of 217 microclones studied contained highly repetitive sequences. Further analysis of the remaining 185 microclones proved that 43 microclones, each having an insert longer than 200 bp, contained unique sequences of human chromosome 10 origin. Twenty-five microclones randomly selected from the 43 were used directly as probes to isolate corresponding cosmid clones, resulting in 32 cosmids corresponding to 14 microclones. Of the 25 cosmids that could be mapped by fluorescence in situ hybridization, 24 proved to originate from the microdissected or adjacent region (10p11.2-q22.3) and 1 from a rather distal region (10q24.3-q25.1). In addition, 15 of the 32 cosmids revealed restriction fragment length polymorphisms, including 1 with a variable number of tandem repeats marker. The microdissection library and the obtained cosmids are valuable resources for constructing high-resolution physical and linkage maps of the pericentromeric region of chromosome 10, where the gene predisposing to multiple endocrine neoplasia type 2A (MEN2A) has been mapped.

Base Sequence↗