Cancer Letters 197 (2003) 53–61 www.elsevier.com/locate/canlet
Application of laser capture microdissection in genetic analysis of neuroblastoma and neuroblastoma precursor cells Katleen De Pretera, Jo Vandesompelea, Pierre Heimannb, Mark M. Kockxc, Mireille Van Gelea, Jasmien Hoebeecka, Els De Smeta, Martine Demarched, Genevie`ve Laureyse, Nadine Van Roya, Anne De Paepea, Frank Spelemana,* a
Department of Medical Genetics, Ghent University Hospital, 1K5, De Pintelaan 185, B-9000 Ghent, Belgium Department of Medical Genetics, University Hospital Erasme, Route de Lennik 808, B-1070 Brussels, Belgium c Department of Pathology, Middelheim Hospital, Lindendreef 1, B-2020 Antwerp, Belgium d Department of Pediatric Surgery, Centre Hospitalier Re´gional de la Citadelle, Boulevard du 12e`me Ligne 1, B-4000 Liege, Belgium e Department of Pediatric Oncology, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium b
Received 7 November 2002; accepted 21 November 2002
Abstract Recently developed quantitative and high-throughput technologies that allow automated and rapid screening of the whole genome, transcriptome and proteome have revolutionized the field of cancer genetics. At the same time, new challenges are met, e.g. the need for improved data analysis and standardization of tumor sample handling. Even if these issues are resolved, an ‘old’ problem in genetic tumor analysis remains, i.e. contamination of tumor samples by stromal and surrounding normal cells. To overcome this obstacle, laser capture microdissection (LCM) has been developed in order to procure the cells of interest from stained tissue sections with retention of morphology. In this review we describe the possible down-stream applications of LCM in the genetic analysis of neuroblastoma (NB). Special focus is given to MYCN copy number determination using realtime quantitative polymerase chain reaction (Q-PCR), analysis of 1p-, 3p- and 11q-deletions using loss of heterozygosity analysis and Q-PCR expression analysis of microdissected normal neuroblast cells and NB cells. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neuroblastoma; Neuroblast; Amplification; Real-time quantitative polymerase chain reaction; Loss of heterozygosity; Microarray; Comparative genomic hybridization; Microdissection; Intratumoral heterogeneity; MYCN; 1p; 3p; 11q; Deletion
1. Introduction In the past decade, new methods for quantitative high-throughput analysis of genes, transcripts and * Corresponding author. Tel.: þ 32-9-2402451; fax: þ 32-92404970. E-mail address:
[email protected] (F. Speleman).
proteins have been introduced and applied in the field of cancer genetics. It has been demonstrated that genome wide detection of DNA low copy number changes is feasible using comparative genomic hybridization (CGH) arrays [1,2]. At the mRNA level, gene expression profiling has become feasible through the introduction of cDNA [3] and oligonucleotide microarrays [4], which allow simultaneous
0304-3835/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3835(03)00084-3
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analysis of thousands of genes. Real-time quantitative polymerase chain reaction (Q-PCR) has evolved as the new standard for accurate quantification of selected subsets of gene specific DNA or RNA sequences [5]. In parallel with the genomics and transcriptomics research areas, proteomics is also coming to the forefront of cancer research as a result of new and powerful analytical methods. Obviously, these new methods offer great promise for further studies, but at the same time create new challenges. These include the need for additional and more sophisticated bioinformatic algorithms for data mining and the standardization of collection and storage of tumor samples. Another important factor that may influence the reliability of genetic analysis of tumor biopsies is the purity and homogeneity of the investigated cells. Indeed, even the most sophisticated genetic testing methods will be of limited value if the input material (nucleic acids or proteins) is not derived from sufficiently pure populations of the cells of interest. To overcome this problem, several methods have been developed to obtain specific cells from a heterogeneous tissue section. This review will deal with the application of laser capture microdissection (LCM) in the genetic analysis of neuroblastoma (NB) with particular focus on QPCR as the down-stream application of LCM par excellence.
available by Arcturus Engineering (http://www. arctur.com) as the PixCell system. Soon after the first commercial LCM microscope became available, other companies developed microdissection systems varying in the cell-capture method, system configuration and intended applications (Table 1). Nowadays, the LCM system (Arcturus Engineering) and the LPC (laser pressure catapulting) system from PALM Microlaser Technologies (http://www. palm-microlaser.com) are the most widely used laserbased microdissection systems. The PALM Microlaser system is based on LPC, which allows non-contact cell isolation based on a laser beam which is used for microdissection (laser microbeam microdissection: LMM) and catapulting of selected cell clusters or single cells directly into a microcentrifuge tube [7]. In the LCM procedure of the PixCell II system a cap that is coated with a special thermoplastic film, is placed on the tissue section. The LCM system is then used to direct an infrared laser through the cap to melt the film onto the cells of interest. When the cap is lifted, the selected cells remain attached and are ready for nucleic acid or protein extraction. Recently, a new
2. Laser capture microdissection 2.1. Principle Initially, isolation of specific cells out of a complex environment was performed through mechanical microscopic recovery of cells from tissue sections. As an alternative, negative selection was used by ablation of unwanted areas of the tissue on the slide employing ultraviolet light. However, none of these methods provide the ease, precision and efficiency needed in routine diagnostic and research applications. In 1996, Emmert-Buck and colleagues of the National Institute of Health (NIH) [6] introduced the LCM system. This simple, reliable and rapid technique allowed microdissection of cells with retention of cell morphology and was made commercially
Fig. 1. LOH profiles of three different polymorphic markers on tumor DNA of three different NB samples extracted from microdissected cells or bulk tumor material. Allelic imbalance factors (AIF) [22] are measured: N, normal (AIF , 2); AI, allelic imbalance (2 , AIF , 5); LOH, loss of heterozygosity (AIF . 5)). Samples originally found heterozygous for the marker are scored as AI or LOH after LCM; markers originally having AI can be scored as LOH after LCM (not shown).
http://www.mmi-micro.com
2.2. Advantages and limitations of LCM LCM is user friendly as it is easy to learn and integrate in routine and research applications. Furthermore, LCM is characterized by the preservation of morphology of transferred and not-transferred cells and a short hands-on time for microdissection of conventional tissue sections on glass slides. The limitations of LCM reflect the difficulties of microdissection in general. (1) Complete dehydration of the tissue section and the absence of a cover-slip lead to a poor visualization of cell morphology. However, an optical light diffuser is available on the commercial LCM system that improves resolution. (2) Only small amounts of nucleic acids or proteins can be isolated using microdissection. Fortunately, DNA and RNA amplification protocols have been developed and validated, and sensitive down-stream applications (such as PCR) allow to obtain accurate results from LCM extracted material. (3) The traditionally long staining protocols for tissue sections (especially in immunohistochemical and immunofluorescent assays) are not beneficial for RNA and protein quality and must be adjusted thoroughly by significantly reducing the washing and staining time.
Adhesive layer on collection cap
http://www.palm-microlaser.com
http://www.arctur.com http://www.cellrobotics.com http://www.leica-microsystems.com
CapSure cap (EVA polymer film) Pick-up sticks Excised tissue falls down by gravity Laser pressure catapulting UV PALM Microbeam
mCUT
P.A.L.M. Microlaser Technologies
MMI AG
UV
IR UV UV PixCell II Laser-Scissors Pro300 Leica AS LMD Arcturus Engineering Cell Robotics Leica Microsystems
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type of caps was developed to allow non-contact LCM, thus further reducing possible contamination of not-targeted cells. The use of these caps is especially recommended for isolation of smaller number of cells, including single and rare cells.
Laser hitting of desired cells Laser hitting of desired cells Laser cutting around tissue of interest Laser cutting around tissue of interest Laser cutting around tissue of interest
3. LCM in cancer research and diagnostics
IR, infrared; UV, ultraviolet.
Collection Excision Laser Instrument Company
Table 1 List of commercially available laser-based tissue microdissection systems with their respective laser configuration, and cell-capture method
Website
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The success of laser-based microdissection is illustrated by the large number of studies using this technique for a broad range of down-stream applications, such as loss of heterozygosity analysis (LOH), CGH and CGH array analysis, methylation-specific PCR, real-time Q-PCR, expression microarrays, cDNA library construction and 2D-PAGE (twodimensional polyacrylamide gel electrophoresis) (for references see http://allserv.rug.ac.be/ , fspelema/ neubla/cancerletters/index.htm). Application of LCM allows to exclude contaminating stromal cells (e.g. fibroblasts, myofibroblasts and endothelial cells) and normal surrounding cells
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from the analysis. In addition, intratumoral heterogeneity can be studied. 3.1. Gene copy number changes in neuroblastoma It is well known that the clinical course of NB is highly variable ranging from disseminated disease to spontaneous regression. Consequently, many studies aimed at identification of the clinical and biological parameters, which accurately predict outcome in NB patients. Currently, age at diagnosis, tumor stage, DNA-index, amplification of the proto-oncogene MYCN and 17q-gain are the most important risk indicators [8]. Depending on the status of the genetic parameters, different treatment modalities are chosen. In view of the importance of accurate assessment of these biological parameters, a recent quality control study of the SIOP Europe Neuroblastoma Biology Group recommended to investigate MYCN amplification and 1p-deletion using a second independent technique in parallel with fluorescence in situ hybridization (FISH) (Ambros et al., submitted). 3.1.1. PCR-based down-stream applications 3.1.1.1. Quantification of MYCN copy number. In parallel with FISH analysis, our group developed a QPCR assay as an alternative for Southern blot analysis (SB) [9]. Q-PCR offers major advantages compared to conventional methods such as SB and former PCRbased quantification strategies, such as a large dynamic range of quantification, the exclusion of post-PCR manipulations, the possibility to perform the assay on only minimal amounts of tumor material (such as needle biopsies), the speed and the highthroughput capacity (e.g. for retrospective studies on many samples). We have developed a Q-PCR assay based on two different detection chemistries (i.e. SYBR Green I and TaqMan hydrolysis probe). The high accuracy of our assay for the detection of MYCN copy numbers was illustrated by the possibility to detect MYCN single gene copy changes as demonstrated in DNA samples from patients with a constitutional 2p-deletion or duplication. For this purpose, choice of primers and control genes (BCMA and SDC4, on chromosomal regions 16p and 20q rarely showing genetic abnormalities in NB [10]) was shown to be critical. Subsequent analysis of 175 NB
samples with known MYCN status yielded highly concordant results with previous FISH and SB data. The sensitivity of the Q-PCR assay for detection of MYCN amplification was illustrated by the finding of a positive result in a tumor, which was assessed as a case with MYCN single copy by initial FISH analysis. Upon re-evaluation of the FISH slide, a few cells with MYCN amplification restricted to one particular area of the slide were detected. However, the Q-PCR results for this sample were near the threshold value for amplification. Therefore, DNA extracted from multiple samples of microdissected material is preferably used in the Q-PCR assay for detection of MYCN copy number changes in tumors containing a mixture of cells or revealing intratumoral heterogeneity [11,12]. An ordinary haematoxylin and eosin (H&E) staining of archival paraffin sections or cryo-sections of NB tumors with subsequent dehydration steps is sufficient to recognize the NB cells to be microdissected. The need for only minimal amounts of input DNA (no more than 100 pg) makes the Q-PCR assay particularly compatible with LCM. A detailed protocol can be found on our website (allserv.rug.ac.be/, fspelema/ neubla/cancerletters/index.htm). Starting from archival paraffin or freshly prepared cryo-sections of NB samples, we were able to perform accurate and sensitive MYCN copy number determination on the microdissected material (unpublished data). 3.1.1.2. Detection of 1p-, 3p- and 11q-deletion. Loss of 1p, 3p and 11q are important recurrent changes in NB and the status of these chromosome regions may be relevant for clinical study protocols or for the genetic classification of tumor samples, e.g. in the context of gene expression profiling studies. Although detection of deletions is straightforward by FISH, combination with LOH analysis may be required in order to determine if one of both parental alleles is effectively deleted. This may be the case when for a given chromosome three copies are present of which one is partly deleted. In contrast to FISH, however, LOH studies may be hampered by the presence of contaminating cells or clonal variation. Previous studies [13] and results presented here show that LCM improves the LOH sensitivity. Measuring the intensity of the allelic decrease, it was shown that the mean decrease of the lost allele is 34% using whole
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tumor samples and 67% for microdissected samples. LOH analysis for 1p, 3p and 11q markers in NB samples showed in most cases an improved result when using DNA from microdissected material. Fig. 1 illustrates that LCM facilitates and improves LOH interpretation.
reverse transcriptase real-time Q-PCR provides expression data in a large panel of samples for smaller series of genes [5]. The use of microdissection in expression profiling studies is recommended for the same reasons as for the analysis of DNA alterations.
3.1.2. Whole genome profiling of microdissected tumor cells CGH allows genome wide screening of genomic imbalances, i.e. identification of chromosome regions that are preferentially lost, overrepresented or amplified. This approach was proven to be valuable in the classification of NB. CGH, however, is cumbersome and labor-intensive and suffers from a rather limited resolution (5 –10 Mb). Recently, CGH-arrays were developed that offer at least a 10-fold increase in resolution [1,2]. However, both conventional and array-based CGH detection of single copy changes are sometimes hampered by lack of sufficiently high tumor percentage of the biopsy under investigation. Again, LCM can circumvent this, but is in itself limited by the amount of ‘pure’ DNA that can be extracted. Three different whole genome amplification protocols are currently in use to generate sufficient amounts of DNA for further investigation while retaining the original complexity and representation of the microdissected DNA sample, i.e. degenerate oligonucleotide-primed PCR [14], adaptor ligation mediated PCR [15] and multiple displacement amplification based on the rolling circle amplification [16]. Given these technical advances, we expect that CGH-array analysis on microdissected NB cells will replace conventional CGH for sensitive whole genome aberration profiling thus offering the possibility to study genetic heterogeneity of NB and search for hitherto unknown genomic alterations.
3.2.1. Q-PCR and microarray analysis Q-PCR is particularly convenient for gene expression analysis of microdissected cells as this method requires only minute amounts of RNA. We previously showed that intercalating SYBR Green I is the detection format of choice for accurate and reproducible Q-PCR quantification of multiple genes [17]. An optimized two-step Q-PCR based on DNase treated RNA was shown to be a prerequisite for accurate results. Using the described protocol, we further showed that reproducible DNase treatment and cDNA synthesis could be performed using as little as 100 pg total RNA (sufficient for 10 Q-PCR reactions of moderately abundant genes) (Fig. 2). In order to control for differences in amount of starting material, enzymatic efficiencies and overall cellular transcriptional activity, an appropriate normalization strategy is required. We therefore recommend to normalize gene expression levels with the geometric mean of at
3.2. Gene expression profiling in neuroblastoma Gene expression analysis plays an increasingly important role in many areas of biological research. Two recently developed methods for measurement of transcript abundance have gained much popularity and are frequently applied. Expression microarrays allow the parallel analysis of thousands of genes in two differentially labeled cDNA samples [3,4], while
Fig. 2. Efficient and reproducible DNase treatment and cDNA synthesis for reverse transcriptase Q-PCR from picogram amounts of total RNA. RNA quantities used for DNase treatment and cDNA synthesis were 100 pg (A), 1 ng (B), 10 ng (C), 100 ng (D) and 1 mg (E). For the Q-PCR reaction (housekeeping gene RPL13A expression) 1/10 of the cDNA (A, B and C: 10 pg, 100 pg and 1 ng) or 1/40 of the cDNA (D and E: 2.5 ng and 25 ng) was used. This graph clearly shows that the starting amount of RNA in cDNA synthesis does not affect the efficiency of the Q-PCR reaction, and therefore reliable expression analysis is possible in microdissected material.
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least three carefully selected housekeeping genes as described [18]. In contrast to Q-PCR, microarray experiments require large amounts of good quality RNA (20 – 200 mg). Therefore, it is necessary to include an RNA amplification step following the RNA extraction of microdissected cells. Two RNA amplification protocols can be distinguished, i.e. linear RNA amplification using in vitro transcription of cDNA [19] and PCR based low cycle exponential cDNA amplification after incorporation of an anchor sequence to the 50 end (SMART cDNA technology, BD Biosciences, http://www.clontech.com/smart/). Validation of the RNA amplification procedures can easily be performed using Q-PCR. Tooled up with these amplification methods, the future challenge is to perform expression profiling of NB tumor cells from distinct regions within the tumor using expression microarrays. Especially for microarray experiments, high-qual-
ity full-length RNA is required. The starting material for LCM-based expression analysis is usually fixed and embedded in paraffin, or frozen. Several groups have reported RNA extraction from paraffin sections. However, paraffin embedding requires previous tissue fixation which has been shown to affect the RNA integrity. Therefore, frozen tissue sections are highly recommended for RNA recovery. Staining protocols for frozen sections have to be modified with minimal staining and washing times in order to reduce RNase activity. In our laboratory, an adapted H&E staining protocol was developed guaranteeing good RNA quality. Sections from snap-frozen tissues were obtained with an RNase free knife (treated with NaOH) followed by a fast H&E staining in RNase free recipients and solutions. Immediately after staining and dehydration of the sections, LCM was performed. RNA isolated from not-targeted tissue that was scraped off the slide was demonstrated to be of sufficiently good quality, as shown by ratio analysis of
Fig. 3. Microdissection and expression analysis on adrenal neuroblasts from an H&E stained adrenal cryo-section (19 weeks gestational age). (A) Section before microdissection; (B) after; (C) microdissected cells on the cap; (D) mounted and cover-slipped H&E section provides highquality visualization of the neuroblastic cell clusters; (E,F) sympathetic nervous system marker gene expression analysis in four NB cell lines, neuroblast cells (18 or 19 weeks gestational age) and surrounding cortical cells. CGHA (chromogranin A) (E) and HAND2 (dHAND) (F) are absent in cortical cells and clearly expressed in both NB cell lines and normal adrenal neuroblast, confirming published data [20, 21].
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the ribosomal 18S/28S RNA bands using the Agilent 2100 Bioanalyzer (www.agilent.com). This apparatus requires only minimal amounts of total RNA (5 – 500 ng) to evaluate integrity (and quantity) and is therefore ideally suited for LCM applications. A detailed protocol for staining and further processing can be found on our website (http://allserv.rug.ac. be/ , fspelema/neubla/cancerletters/index.htm). 3.2.2. Normal neuroblast expression analysis An intrinsic aspect of many cancer gene expression profiling studies is the inclusion of the normal cellular counterparts to identify significant tumor associated changes in mRNA expression. For NB, the normal counterparts are pluripotent neural crest derived cells that amongst others give rise to the sympathetic nervous system. These precursor cells are not readily accessible, as they are predominantly found in prenatal life. In fetal adrenals these cells appear as small clusters of darkly stained cells (in H&E stained sections). In order to have an appropriate normal control for gene expression analysis of (adrenal) NB tumors, we have therefore performed LCM on frozen sections from snap-frozen human fetal adrenals which were H&E stained with the above-mentioned modified protocol (Fig. 3). Preliminary experiments indicated the feasibility of high quality pure neuroblastic RNA extraction. RNA from two large microdissected clusters of approximately 300 neuroblasts allowed expression analysis of more than 20 genes. In addition, we dissected pure populations of neuroblasts from different gestational time-points (15 –20 weeks). Q-PCR analysis of known neuronal marker genes and recently identified MYCN transcriptional target genes were performed (De Preter et al., in preparation). Where available, Q-PCR data correlated well with previous expression studies using in situ hybridization on sections from human embryos [20,21] (Fig. 3). These studies will shed more light onto normal fetal neuroblast development. Moreover, we expect that by comparing the expression patterns of normal neuroblasts with those obtained from a large series of tumors and cell lines, we will be able to determine the point of developmental arrest in the different subtypes of adrenal NB.
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4. Conclusions Due to technical advances and the availability of commercial microdissection systems, genetic studies on microdissected cells can now be implemented on a broader scale. In this review we have summarized the possible application of LCM with particular reference to studies in NB. Fig. 4 gives an overview as to how LCM can be integrated in the study of genetic aberrations or gene expression level changes. We conclude that LCM makes a more tumor cell-directed
Fig. 4. Integration of laser-based microdissection in current genetic analysis of NB. Comprehensive genome and transcriptome profiling of pure tumor cells and normal precursors (A: NB tumor paraffin section, B: NB tumor cryo-section, C: fetal adrenal cryo-section) will lead to improved diagnosis and prediction of patient outcome, and help the identification of relevant tumor subgroups and candidate genes.
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approach possible in cancer research, in addition to the frequent use of relatively homogeneous tumor cell lines.
Acknowledgements We would like to thank the members of the BENG group (Belgian Neuroblastoma Group) for providing us with NB tumor material, Vera Schelfhout and Bart Lelie (Pathology Department, Ghent University Hospital, Belgium) for helping us with tumor cell recognition on H&E stained sections, Ann Neesen and Indra Deborle (Pneumology Department, Ghent University Hospital, Belgium) for help with the preparation of the paraffin, and cryo-sections and Steven Verberckmoes for help with the LOH analysis. Katleen De Preter is an aspirant with the Fund for Scientific Research, Flanders (FWO-Vlaanderen). Nadine Van Roy is a postdoctoral researcher with the FWO. The work was also supported by VEO-grant 011V1302, BOF-grant 011F1200 and 011B4300, GOA-grant 12051203 and FWO-grant G.0028.00.
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