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PubMed1997

Laser beam microdissection in the diagnosis of cutaneous B-cell lymphoma.

Interpretation of molecular analyses of cutaneous lymphoid infiltrates may be difficult because a heterogeneous group of cells in usually present within the neoplasms. Extraction of DNA from tissue sections does not provide exact information about which cell population has been analysed. We present a laser microscope system that allows selective molecular analysis of single cells or small groups of cells in cases of cutaneous lymphoma. An ultraviolet (UV)-laser microscope system (PALM, Wolfratshausen, Germany) was used to isolate particular populations of cells from a routinely processed specimen of a cutaneous follicular lymphoid proliferation. Using the UV-laser beam, a circle was cut around a target germinal centre in order to separate it from neighbouring tissues and to isolate a pure population of germinal centre cells. Isolated cells were scraped off with a micromanipulator and placed in a proteinase-K solution. DNA was extracted and amplified by the polymerase chain reaction (PCR) technique. Analysis of immunoglobulin JH gene rearrangement showed a distinct monoclonal band. In a second phase, using the same procedure in the same specimen, mantle zone cells around a germinal centre and single interfollicular B lymphocytes were isolated for PCR analysis of immunoglobulin JH gene rearrangement. In this population of cells, no clonality could be detected. This new technique allows the selective elimination of undesired cells and tissue from cutaneous neoplasms. By destruction of unwanted tissues with laser-beam energy a contamination-free sample is obtained. Analysis of isolated cells in our case demonstrated a clonal rearrangement derived from germinal centre cells and not from other B lymphocytes in the specimen, confirming the diagnosis of cutaneous follicle centre lymphoma. The method described has exciting implications for dermatology and dermatopathology, allowing precise correlation of morphological features with findings by molecular genetics.

Cell Separation↗
PubMed1995

Homozygous deletions at chromosome 9p21 and mutation analysis of p16 and p15 in microdissected primary non-small cell lung cancers.

Loss of heterozygosity on chromosome 9p has been detected in many primary human tumors and cell lines, suggesting that this chromosomal arm harbors one or more tumor suppressor genes. The recently cloned p16 and p15 genes, mapped to 9p21, are likely candidates for such tumor suppressors. To map the deletion at chromosome 9p21 in non-small cell lung tumors, we analyzed DNA from 25 tumors and matching normal DNAs at six microsatellite markers that flank the region occupied by the p16 and p15 genes. Loss of heterozygosity of at least one microsatellite marker on chromosome 9p21 was detected in 13 (52%) of 25 tumors, including one tumor that exhibited homozygous deletion of both human IFNalpha and D9S171. Six tumors analyzed by a comparative multiplex PCR technique showed homozygous deletions of the sequence tag site marker c5.1 (within p16). Screening for mutations in p16 and p15 revealed one tumor with a non-sense mutation in exon 2 of p16, but no mutations were detected in p15 in any of the tumors. Thus, in these analyses approximately one-half of the non-small cell lung tumors had loss of heterozygosity at chromosome 9p21, and of these tumors, one-half had homozygous deletions of the region that includes p16. This appears to confirm the importance of a locus in this region critical to growth control in lung. The apparent lack of other mutations in p16 and p15 in the tumors with loss of heterozygosity leaves open the possibility of an unidentified gene in this region that may function as a tumor suppressor.

Adenocarcinoma↗
PubMed2005

Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

Carcinoma↗
PubMed2001

Rapid, efficient genotyping of clinical tumor samples by laser-capture microdissection/PCR/SSCP.

BACKGROUND: Mutation analysis is becoming increasingly important in clinical practice, since sporadic mutations in tumors often correlate with prognosis and/or therapeutic response. However, the labor-intensive nature of the molecular analyses has limited the routine clinical use of tumor genotyping. Laser-capture microdissection (LCM) allows procurement of relatively pure tumor cell populations. We have investigated the possibility that the use of laser-capture microdissection would allow elimination of time-consuming intermediate steps in tumor genotyping. Design. Archival formalin-fixed, paraffin-embedded tissues from seven cases of colorectal adenocarcinoma were laser- and hand-microdissected and subsequently evaluated by PCR/SSCP/sequencing for Ki-ras exon 1 and p53 exons 5, 7, and 8. Results. Mutations in Ki-ras exon 1 and/or p53 exons 5 and 7 were detected in five of the seven samples. In the hand-microdissected samples, confident identification of mutations was possible in several cases only after band excision, DNA elution, reamplification, and verification of mutant enrichment by a second SSCP analysis prior to sequencing. In the laser-microdissected samples, confident mutation identification was possible in all cases with direct sequencing of the original PCR product, reducing the time required for molecular analysis to 3 days. Conclusion. Using laser-capture microdissection, mutant signals are strong enough to sequence directly from original PCR products. With rapid, efficient genotyping by LCM/PCR/SSCP, results can be incorporated directly into the surgical pathology report.

Base Sequence↗
PubMed2001

Allelic imbalance of the mutant and wild-type RET allele in MEN 2A-associated medullary thyroid carcinoma.

Germline mutations of the RET proto-oncogene are responsible for the familial tumor syndrome called multiple endocrine neoplasia type 2 (MEN 2) that includes medullary thyroid carcinoma (MTC). Although inherited mutations of RET lead to tumor formation in patients with MEN 2, it is not understood why only selected cells develop into tumors. We have recently shown that duplication of the mutated RET allele or loss of the wild-type allele might represent mechanisms of tumorigenesis in patients with MEN 2A-related pheochromocytoma. We now analysed 19 DNA samples of MTC (15 of which were non-microdissected, four of which were microdissected) from patients with MEN 2A. Using polymorphic marker and phosphorimage densitometry analyses, we found allelic imbalance of the mutated and wild-type RET allele in six of 19 DNA MTC samples. Of note, two of the four microdissected tumor DNA samples showed allelic imbalance of RET, whereas only four of the 15 non-microdissected MTC samples did. These results underscore the significance of microdissection in the analysis of tumor DNA. In our study, some of the non-microdissected tumor DNA samples may have failed to display allelic imbalance of RET, because of contamination of tumor DNA with nonneoplastic DNA or noninformative microsatellite marker analysis. Taken together, our results suggest allelic imbalance between mutated and wild-type RET as a possible mechanism for tumor formation in some patients with MEN 2A-related MTC.

Alleles↗
PubMed2002

[Testicular sperm extraction for non-obstructive azoospermia: results with conventional and microsurgical techniques].

Testicular sperm extraction (TESE) was performed on patients with non-obstructive azoospermia using the conventional or microdissection technique. First, conventional TESE was attempted on all patients. If the sperm was retrieved successfully, the TESE procedure was terminated. Microdissection TESE was indicated only for patients for whom the conventional sperm retrieval was unsuccessful. Sperm was successfully retrieved with conventional TESE from four (24%) of 17 patients with non-obstructive azoospermia. The remaining 13 patients underwent microdissection TESE and sperm was successfully retrieved from four of them. Application of the microdissection technique resulted in an improvement in sperm retrieval rates from 24% with conventional TESE to 48% when combined with the microdissection technique. None of the patients showed any acute or chronic complications after TESE. Intracytoplasmic sperm injection (ICSI) was performed on five of the eight patients with successful sperm retrieval and the partner of four of these patients became pregnant and progressed to delivery. These results indicate the usefulness of microdissection for improving the sperm retrieval rate in men with non-obstructive azoospermia.

Adult↗
PubMed2000

Quantitation of DNA extracted after micropreparation of cells from frozen and formalin-fixed tissue sections.

Quantitation of DNA from microdissected fresh-frozen or paraffin-embedded tissue sections would be not only a valuable tool for ensuring optimum reaction conditions for many types of qualitative polymerase chain reaction (PCR) analyses, but also a prerequisite for any kind of subsequently performed genetic analyses aimed at the absolute quantitation of target sequences. The present study describes the quantitation of DNA after microdissection and extraction of cells with the PicoGreen fluorescence method. The limits of detection and of quantitative determination, respectively, have been determined by measuring dilutional series of three different DNA extractions, using either a medium-scale preparation from a solid tissue specimen or a known number of leukocytes or microdissected cells from frozen tumor sections. As corresponding limits of detection, 26, 24, and about 40 diploid genomes, and as limits of quantitative determination, 80, 73, and about 120 diploid genomes were obtained. Furthermore, it was shown that formalin fixation as well as hematoxylin staining of frozen sections with Delafield's and Mayer's alum or Weigert's iron hematoxylin before microdissection significantly diminishes the amount of extractable DNA and may lead to less reliable results, even of qualitative PCR analysis. In conclusion, the PicoGreen method allows precise quantitation of DNA corresponding to a minimum of about 120 diploid cells. It provides the basis for reliable qualitative analyses as well as the precondition for further quantitative genetic measurements from microdissected frozen or formalin-fixed and paraffin-embedded tissue sections.

DNA, Neoplasm↗
PubMed1997

Identification of a novel transcript up-regulated in a clinically aggressive prostate carcinoma.

OBJECTIVES: To identify differentially expressed genes in tumor cells of patients with prostate cancer by means of tissue microdissection and targeted differential display. METHODS: RNA was recovered from pure populations of microdissected normal epithelium and invasive tumor from frozen tissue sections of a radical prostatectomy specimen. Reverse transcription-polymerase chain reaction (PCR) using arbitrary and zinc finger PCR primers was performed. RESULTS: A 130-base pair product was identified that appeared selectively in the tumor sample. DNA sequence analysis revealed it to be a clone from the expressed sequence tag database (GenBank accession R00504). Microdissection of normal epithelium and the corresponding invasive tumor was subsequently performed on a test panel of 10 prostate carcinoma specimens. Comparison of R00504 levels in normal epithelium and invasive carcinoma, using beta-actin as an internal control, showed the transcript to be substantially overexpressed in 5 of 10 carcinomas. Northern blotting revealed R00504 to be a 2.6-kilobase gene. CONCLUSIONS: A novel transcript up-regulated in an aggressive prostate carcinoma was identified using degenerate zinc finger primers in microdissected tissue samples. The approach used in this study may be helpful in quantitative comparison of known genes and identification of novel genes in microdissected human tissue samples.

Humans↗
PubMed2005

Positive association of heparanase expression with tumor invasion and lymphatic metastasis in gastric carcinoma.

Tumor invasion and metastasis are the most common causes of death in gastric carcinoma. Human heparanase influences tumor invasiveness and angiogenesis. Analysis of its expression in gastric carcinoma has been hindered by our inability to procure pure cancer cells from heterogeneous tissue. In the present study, we analyzed heparanase expression in human primary and metastatic gastric carcinoma cells as well as in paired normal gastric epithelial cells by laser capture microdissection coupled with reverse transcription-polymerase chain reaction (RT-PCR). Tumor tissues, metastatic lymph nodes, and apparently uninvolved normal gastric tissues were collected from 30 patients who had undergone gastrectomy with radical lymph node dissection for gastric carcinoma without preoperative treatment. Bulk tissues and laser capture microdissected cell groups were separately subjected to RT-PCR analysis with heparanase-specific primers. For bulk tissues, heparanase-specific transcripts were detectable in all primary tumor tissues, metastatic lymph nodes, and almost all matching normal tissues. RT-PCR analysis after laser capture microdissection showed no detectable heparanase expression in matching normal epithelial cell groups. Of the laser capture microdissected primary gastric carcinoma cells, 47% (14/30) were heparanase positive. Expression was closely associated with greater tumor invasiveness, including Borrmann gross type and depth of wall infiltration. For metastatic cell groups dissected from lymph nodes, 95% showed clear heparanase expression. Furthermore, the extent of lymphatic spread was directly correlated to heparanase expression at the primary site. In conclusion, laser capture microdissection coupled with RT-PCR is a reliable approach for molecular analysis of heparanase expression in gastric carcinoma. Heparanase may facilitate invasion and metastasis of gastric carcinoma cells.

Adult↗
PubMed2004

Identification of genes whose expression is upregulated in lung adenocarcinoma cells in comparison with type II alveolar cells and bronchiolar epithelial cells in vivo.

To identify genes whose expression is upregulated in lung adenocarcinoma (AdC) cells in comparison with noncancerous peripheral lung epithelial cells, type II alveolar cells and bronchiolar epithelial cells, as well as AdC cells, were isolated by laser capture microdissection, and subjected to cDNA microarray analysis of 637 human cancer-related genes. Each of the component cells was obtained from several different individuals and analysed independently. As a comparison, two lung AdC cell lines and two primarily cultured normal lung epithelial cell lines were also subjected to cDNA microarray analysis. Four genes, TOP2A, MMP15, MX2 and KOC1, were commonly upregulated in microdissected AdC cells in comparison with microdissected epithelial cells. Hierarchical clustering analysis revealed that differences in gene-expression profiles were more evident between cultured and uncultured cells than between cancerous and noncancerous cells. To further identify the common molecular targets of AdC cells in vivo, quantitative real-time RT-PCR was performed against the four genes upregulated by cDNA microarray analysis. The TOP2A, MMP15, MX2 and KOC1 genes were overexpressed in 10/10 (100%), 8/10 (80%), 5/10 (50%) and 3/10 (30%) microdissected AdC cell samples, respectively, in comparison with any of nine independently microdissected noncancerous epithelial cell samples. The TOP2A gene was commonly overexpressed in lung AdC cells, as previously reported. In addition, the MMP15 and MX2 genes were identified, for the first time, as being commonly overexpressed in lung AdC cells. These results strongly indicate that the MMP15 and MX2 genes could be novel markers for molecular diagnosis and therapy of lung AdC.

Adenocarcinoma↗
PubMed2004

Multilineage progression of genetically unstable tumor subclones in cutaneous T-cell lymphoma.

Molecular analysis of solid malignant tumors has suggested multilineage progression of genetically unstable subclones during early stages of tumorigenesis as a common mechanism of tumor cell evolution. We have investigated whether multilineage progression is a feature of cutaneous T-cell lymphoma (CTCL). To identify individual tumor cell subclones, we determined the pattern of mutations within microsatellite DNA obtained from multiple histomorphologically confined tumor cell nests of mycosis fungoides (MF) and lymphomatoid papulosis (LyP) lesions. Tumor cells were isolated by laser microdissection, and allelotypes were determined at microsatellite markers D6S260, D9S162, D9S171, D10S215, TP53.PCR15, and D18S65. Nine cases of MF and one patient with anaplastic large cell lymphoma (ALCL) originating from LyP were analyzed at 277 different microdissected areas obtained from 31 individual lesions. Three specimens of cutaneous lichen planus microdissected at 26 areas served as the control tissue. Microsatellite instability in microdissected tissue [MSI(md-tissue)] was detected in tumor tissues of all CTCL patients. One hundred and fifty-seven of 469 analyzed polymerase chain reaction (PCR) amplifications contained mutated microsatellite alleles (34%). In lichen planus, MSI(md-tissue) was seen in only four of 76 PCR products (5%) (P < 0.0001). The distribution of allelotypes in tumor cells from different disease stages was consistent with multilineage progression in five MF cases, as well as in the LyP/ALCL patient. Our results suggest that CTCL may evolve by multilineage progression and that tumor subclones in MF can be detected in early disease stages by mutation analysis of microsatellite DNA obtained from multiple microdissected areas.

Alleles↗
PubMed1999

Whole genome amplification and molecular genetic analysis of DNA from paraffin-embedded prostate adenocarcinoma tumor tissue.

PURPOSE: Often tissues obtained from prostate adenocarcinoma tumors embedded in paraffin are heterogeneous in cell type and must be carefully microdissected to acquire tissue fragments that provide homogeneous aliquots of tumor clones. Such tissue fragments rarely contain sufficient DNA to perform genomic characterization needed as an early step in localizing relevant oncogenes or tumor suppressor genes. We report that PCR using a degenerate oligonucleotide primer (DOP-PCR) can be applied to DNA samples from microdissected paraffin-embedded prostate adenocarcinomas, and this provides sufficient product for fluorescent allelic imbalance measurements or comparative genomic hybridization (CGH). MATERIALS AND METHODS: Samples were selected to be representative of those routinely obtained during prostatectomies, based on typical tumor stages (T2 and T3) and Gleason grades (range 3 +3 to 4 +5). For DNA analysis without prior DOP-PCR, only large tumors were selected to be sectioned. More than 50 specimens were analyzed. Close comparison of data obtained from analysis of DOP-PCR with those from non-DOP DNA was obtained on a subset 8 samples. To compare the allelic balance of DOP-PCR amplified DNA with that measured for non-DOP DNA, we analyzed allelic ratios on DNA from 5 different tissue samples processed by both microdissection and conventional sectioning. RESULTS: Systematic comparison of allelic imbalance results shows close similarity between DOP-PCR amplified product and non-DOP DNA, indicating that PCR product is a valid representation of the tumor genome. In addition, the difference between allelic balance and imbalance is more distinctive when microdissection followed by DOP-PCR is performed. Performing CGH on products of DOP-PCR also shows distinctive regional copy number alterations in DNA from microdissected tumor tissue. CONCLUSION: Either of these procedures allows distinction between benign and malignant genomes, and also allows independent analysis of genomic alterations in different portions of tumors. They also may be applied clinically for genomic characterization of small foci that frequently appear in prostates of elderly men who are showing no obvious pathological symptoms of adenocarcinoma.

Adenocarcinoma↗
PubMed1996

Allelic deletions on chromosome 11q13 in multiple tumors from individual MEN1 patients.

Familial multiple endocrine neoplasia type 1 is an autosomal dominant hereditary disorder characterized by multiple parathyroid, pancreatic, duodenal, and pituitary tumors. The parathyroid tumors may arise as diffuse areas of hyperplasia, whereas the pancreatic and duodenal tumors usually form as discrete nodules. Except for a single report, tumor loss of heterozygosity (LOH) mapping of the putative MEN1 suppressor gene on chromosome 11q13 in the past has been restricted by analysis of a single tumor from individual patients and somatic cellular contamination. For this reason, it has not been possible to analyze the clonality of the emerging MEN1 neoplasms. Furthermore, it has been previously unknown whether the LOH pattern varies between individual MEN1 tumors in a given patient or among tumors of different histological origins within unrelated patients. To address these previous limitations, the present study introduces a refinement in microdissection in which endothelial cells are stained and selectively excluded. Tissue microdissection was applied to study LOH patterns on chromosome 11q13 using 8 polymorphic DNA markers in 44 different MEN1 tumors from parathyroid, pancreas, and duodenum in nine unrelated patients. In addition, X-chromosome inactivation clonal analysis was applied to 16 individual microdissected regions from seven parathyroid glands in three female patients. The LOH rates of parathyroid lesions (100%) and endocrine tumors of the pancreas (83%) were strikingly different from the LOH rate of gastrinomas (21%), suggesting that the mechanism that drives LOH may be influenced by the tissue context. Moreover, combined LOH and X-chromosome inactivation scoring of the same microdissected region revealed that parathyroid MEN1 neoplasms can consist of more than one clone. In this study, the centromeric boundary of the putative MEN1 gene was PYGM. Analysis of differential LOH patterns in multiple microdissected tumors in the same patient constitutes a novel approach to suppressor gene mapping.

Adult↗
PubMed2004

Microgenomics: Identification of new expression profiles via small and single-cell sample analyses.

BACKGROUND: Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high-throughput platforms. But these mainly chip-based methods can only generate biologically relevant data if the samples investigated consist of homogeneous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results. METHODS: Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser-assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available. RESULTS: These laser-assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high-throughput RNA expression profiling by microdissecting and processing even single-cell samples. CONCLUSIONS: Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient-specific drugs tailored to the requirements assessed by assaying only a few biopsy cells.

Cell Separation↗
PubMed2006

Detection and identification of heat shock protein 10 as a biomarker in colorectal cancer by protein profiling.

Although colorectal cancer is one of the best-characterized tumors with regard to the multistep progression, it remains one of the most frequent and deadly neoplasms. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, changes in protein expression between microdissected normal and tumorous colonic epithelium were analyzed. Cryostat sections from colorectal tumors, adenoma tissue, and adjacent normal mucosa were laser-microdissected and analyzed using ProteinChip Arrays. The derived MS profiles exhibited numerous statistical differences. One peak showing significantly high expression in the tumor was purified by reverse-phase chromatography and SDS-PAGE. The protein band of interest was passively eluted from the gel and identified as heat shock protein 10 (HSP 10) by tryptic digestion, peptide mapping, and MS/MS analysis. This tumor marker was further characterized by immunohistochemistry. Analysis of HSP 10-positive tissue by ProteinChip technology confirmed the identity of this protein. This work demonstrates that biomarker in colorectal cancer can be detected, identified, and assessed by a proteomic approach comprising tissue microdissection, protein profiling, and immunological techniques. In our experience, histological defined microdissected tissue areas should be used to identify proteins that might be responsible for tumorigenesis.

Biomarkers, Tumor↗