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JMD 2001, Vol. 3, No. 2
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology

Tissue Microdissection and Degenerate Oligonucleotide Primed-Polymerase Chain Reaction (DOP-PCR) Is an Effective Method to Analyze Genetic Aberrations in Invasive Tumors

Yuichi Hirose*§, Kenneth Aldape{ddagger}§, Michelle Takahashi{ddagger}§, Mitchel S. Berger*§ and Burt G. Feuerstein{dagger}§

From the Departments of Neurological Surgery * and Lab Medicine, {dagger} the Division of Neuropathology, {ddagger} the Brain Tumor Research Center, § and the Cancer Genetics Program, University of California-San Francisco, San Francisco, California


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We amplified various amounts of DNA derived from frozen SF210 and U251NCI human glioblastoma cells, carried out comparative genomic hybridization (CGH) using degenerate oligonucleotide primed-PCR (DOP-PCR) products as test probes, and compared results to analyses performed with probes prepared by standard nick translation. Next we extracted DNA from hematoxylin-eosin (HE)- and methyl green (MG)-stained, microdissected sections of formalin-fixed and paraffin-embedded U251NCI cells, amplified and labeled it by DOP-PCR, and subjected it to CGH. Finally, we used the same methods in multiple samples from a single human mixed glioma tissue. DOP-PCR products from 50 pg to 250 ng of DNA were equally effective in generating the same CGH profiles as the standard method. DOP-PCR products from microdissected pieces of MG-stained cells were effective probes for CGH, but HE-stained samples were not desirable. As the proportion of HE-stained sample increased, CGH profiles deteriorated. DOP-PCR products from microdissected pieces of MG-stained paraffin sections of glioma tissue produced CGH profiles compatible with their histological features. CGH performed with DOP-PCR products from microdissected paraffin blocks allows for the accurate investigation of the cytogenetic characteristics from invasive tumors and of cytogenetic heterogeneity within neoplastic tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To evaluate the genetic aberrations in an invasive tumor, the normal tissue adjacent to the tumor must be carefully removed. Tissue microdissection based on microscopic observation is one way to accomplish this. Recently, degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) has been reported to be a good method to produce a representation of small amounts of DNA.1, 2, 3, 4, 5, 6 The method allows analysis of less than 1 ng of DNA. These studies suggest that DOP-PCR is useful for amplifying DNA from microdissected tissue to use for comparative genomic hybridization (CGH). CGH produces a map of relative DNA copy number as a function of chromosomal location by comparing the hybridization of test and reference DNA to metaphase chromosomes.7, 8, 9 The results are consistent with fluorescence in situ hybridization (FISH) and flow cytometry when DNA taken directly from the test and the reference is nick translated.10 We have also found that CGH data are compatible with spectral karyotyping, and that conventional karyotyping of complex chromosome aberrations can be unreliable (Pellarin M, Shapiro J, Feuerstein BG, unpublished data). However, it is not clear whether DOP-PCR products of DNA from microdissected tissue faithfully represent each chromosomal abnormality in a sample. To develop a method of cytogenetic investigation in microdissected tissues of known histology, we first studied limits on the amount of DNA which could be reproducibly amplified by DOP-PCR for CGH analysis. Ten to 250 pg of DNA from human glioblastoma cell lines SF210 and U251NCI were used to generate DOP-PCR products. CGH profiles from samples >=50 pg were nearly identical to profiles generated from nick translated probes without DNA amplification. Ten-picogram samples produced profiles of poorer quality. We also found that tissue microdissection using formalin-fixed, paraffin-embedded cell pellets of U251NCI and DOP-PCR produced faithful CGH profiles. This report confirms that DOP-PCR produces a faithful enough representation of small amounts of DNA (>=50 pg) to allow the use of microdissected tissue for CGH. We also discuss sample conditions which can affect CGH results.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA Extraction
SF210 and U251NCI human glioblastoma cells were grown in Dulbecco’s minimal essential medium supplemented with 10% fetal bovine serum and non-essential amino acids. Genomic DNAs from these cells and leukocytes from a normal male donor were extracted using standard protocols.11 DNA concentration was measured with a fluorometer (Hoefer Scientific, San Francisco, CA) using Hoechst 33258 (Sigma, St. Louis, MO).

U251NCI was trypsinized and centrifuged in a polyethylene tube. The cell pellet was fixed in 10% neutral buffered formalin and processed in paraffin. The paraffin block was cut into sections 5 µm thick, deparaffinized, and stained with hematoxylin and eosin (HE; Fisher Scientific, Pittsburgh, PA) or methyl green (MG, Sigma). MG was used at 1% concentration in distilled water, sections were immersed for 15 to 60 seconds, washed in water for 1 to 5 minutes (depending on how blue/green the tissue was), and air-dried. Small (2 mm x 2 mm) and large (6 mm x 6 mm) amounts of tissue were microdissected and scraped with a 22-gauge needle. The tissue was incubated in 60 µl 1x PCR buffer (Roche, Indianapolis, IN) containing 0.5% Tween 20 and 0.5 mg/ml proteinase K (Life Technologies, Inc., Rockville, MD) for 3 days at 55°C. Proteinase K was added twice a day (1 µg per 2.5 µl sample). After 3 days’ incubation, proteinase K was inactivated by heating for 10 to 15 minutes at 95°C.

We also extracted DNAs from microdissected MG-stained pieces of formalin-fixed, paraffin-embedded sections of mixed oligo-astrocytoma in the same manner. The regions for microdissection were marked according to consecutive HE-stained sections. Aliquots of these crude DNA extracts were subjected to DOP-PCR.

DOP-PCR
DOP-PCR amplification was performed in two phases using SF210 and U251NCI genomic DNA as templates. We used amounts ranging from 1 pg to 250 ng in 1 µl volume or 1 µl DNA extract from microdissected specimens. In the first phase (low stringency reaction), 1 µl of sample was added to 4 µl of buffer A (2.5 µl of 600 µmol/L dNTPs (dATP, dCTP, dGTP, and dTTP; Roche), 0.5 µl of 10 µmol/L DOP primer (5'-CCGACTCGAGNNNNNNATGTGG-3', where N = A, C, G, or T)1 and 1 µl of 5x Sequenase Reaction Buffer (Amersham, Cleveland, OH). The reaction was carried out using 5 cycles of 30°C for 5 minutes, 37°C for 2 minutes, and 96°C for 2 minutes, adding 0.65 units Sequenase at every 30°C step. The first phase product was subjected to the second phase reaction in 1x PCR buffer (Roche) with dNTP (200 µmol/L final concentration), DOP primer (1.4 µmol/L final concentration) and 2.5 units of Taq polymerase (Roche). PCR conditions were as follows: 95°C for 5 minutes, 35 cycles at 94°C for 1 minute, 56°C for 1 minute, and 72°C for 2 minutes, followed by a final extension at 72°C for 5 minutes.

Test (cell line) DNA was labeled with a PCR reaction using 5 µl of DOP-PCR product in 1x PCR buffer with dNTP (100 µmol/L final concentration), DOP primer (1.4 µmol/L final concentration), Mg (4.5 mmol/L final concentration), digoxygenin (DIG)-11-dUTP (50 µmol/L final concentration; Roche) and 2.5 units of Taq polymerase. PCR conditions were as follows: 95°C for 10 minutes, 25 cycles at 94°C for 70 seconds, 56°C for 70 seconds, and 72°C for 3 minutes, followed by a final extension at 72°C for 10 minutes. Reference (normal) DNA was amplified from 50 ng normal male DNA and labeled as described above except that fluorescein isothiocyanate-dUTP (DuPont Inc., Wilmington, DE) was used instead of DIG-dUTP.

CGH
CGH was performed according to Mohapatra et al9 with some modifications. For standard CGH experiments, sample and normal DNAs were labeled with fluorescein isothiocyanate-dUTP and Texas Red-dUTP (DuPont), respectively, by nick translation using DNA polymerase I (Life Technologies). Two hundred nanograms each of labeled tumor and reference DNAs and 20 µg of human Cot-1 DNA (Life Technologies) were ethanol-precipitated and dissolved in 50% formamide, 10% dextran sulfate. The probe mixture was denatured and hybridized to normal metaphase spreads (Vysis Inc., Downers Grove, IL). For CGH experiments using DOP-PCR products, 40 µl DIG-labeled test probe, 20 µl biotin-labeled reference probe, and 20 µg human Cot-1 DNA were precipitated in ethanol, dissolved, denatured, and hybridized as above. After unhybridized probes were washed, the metaphase spread was incubated in blocking buffer (1% bovine serum albumin in 4x SSC) at room temperature for 5 minutes, followed by 1 µg/ml rhodamine-conjugated anti-DIG-antibody (Roche) in blocking buffer at room temperature for 45 minutes. The preparations were washed and counterstained with 4,6-diamino-2-phenylinodole (DAPI) in antifade solution. Red, green, and blue images were acquired with a Quantitative Image Processing System (QUIPS), and the ratios of fluorescence intensity along the chromosomes were quantitated as described by Piper et al.12 A relative gain was scored when the mean green:red ratio was above 1.2 and relative loss was scored when the mean green:red ratio was below 0.8. Chromosomal copy number aberrations (CNAs) were not scored at or near the centromeres. Amplifications were scored only when visual inspection revealed a bright and discrete signal confined to a subchromosomal region. Hybridizations produced from DOP-PCR products were more granular than those produced from nick translated probes. If signals were too dim, the hybridization was repeated. If ratios were simultaneously altered at chromosomes 1p, 19, and 22, the experiment was repeated as described in Mohapatra et al.10


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CGH profiles of SF210 human glioblastoma cell line by nick translation, showed gains of 3p16-pter, 5p, 5q31.3-qter, 6p23-pter, 11q13–22 and 16q22.2-qter; amplification of 14q31-qter; and losses on 2, 4p, 4q11.2–28, 4q32-qter, 7p, 9p21, 12q12–21, 15q25-qter (Figure 1A)Go . DOP-PCR products derived from 50 pg to 250 ng DNA produced equivalent CGH profiles (compare Figure 1AGo 1BGo 1CGo 1DGo 1EGo 1FGo igure 1B). We found that DOP-PCR product from as little as 10 pg DNA could produce a CGH profile; however, the latter profiles were not faithful representations of the results recognized in the nick translation experiment. For example, Figure 1CGo shows CGH profiles produced by products from 10 pg DNA; gains on chromosomal arms 3p and 6p were not detected clearly and the quality of the profiles were generally poor (note the large standard deviations).



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  		Figure 1. Ratio profiles from DNA prepared from unfixed SF210 human glioblastoma cells. The x-axis represents the position along the chromosome (p arm to the left and q arm to the right). The centromeres are marked by a crosshatch on the x-axis. The y-axis represents normalized test/reference fluorescence intensity ratios (copy number aberrations). The mean and SD of the fluorescence intensity ratios for the indicated chromosomes are shown. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation (A) were seen in those with DOP-PCR product derived from 50 pg (B) to 250 ng DNA (data not shown). However, experiments performed with DOP-PCR product from 10 pg DNA (C) did not show gain on chromosomal arm 6p (arrowheads), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on chromosomes not shown in the figure.

 
To confirm the sensitivity of DOP-PCR CGH, we performed CGH experiments by nick translation and DOP-PCR using DNA extracted from another human glioblastoma cell line, U251NCI. CGH profiles using nick-translated probes showed gains of 1p, 3q23-qter, 7p21-pter, 9p, 15q, 17p, and 17q25; amplification of 10q26; and losses of 2q32.3–35, 4q32-qter, 10p, 13q31–34, and 18q (Figure 2A)Go . The results of experiments using DOP-PCR products from 50 pg to 250 ng DNA were similar (Figure 2B)Go to profiles derived from nick-translated products. DOP-PCR product derived from 10 pg DNA were not (Figure 2C)Go . In both series of experiments, fluorescence intensity on metaphase spreads produced by the amplified test probes was dim when DOP-PCR products were derived from less than 1 ng DNA (data not shown). When products were derived from 10 pg or less, signal intensity was too dim to give reproducible results.



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  		Figure 2. Ratio profiles from DNA prepared from unfixed U251NCI human glioblastoma cells. All chromosomal aberrations in experiments using DNA extracted without amplification and labeled by nick translation (A) were seen in those with DOP-PCR product from 50 pg (B) to 250 ng DNA (data not shown). Experiments performed with DOP-PCR product from 10 pg DNA (C) did not show gains on chromosomal arm 9p (arrowheads), and ratio profiles were of poor quality (note the large standard deviations). There were no aberrations on the chromosomes not shown in the figure.

 
We also performed CGH using DOP-PCR product from normal DNA as a test probe and normal DNA labeled by nick translation as a reference and found there was no distinguishable gain or loss in the profile (data not shown). These results were obtained with experiments using DOP-PCR products derived from DNA ranging from 50 pg to 250 ng, and they are in agreement with the study by Kuukasjärvi et al.4

Because products derived from very small amounts of DNA (50 pg, corresponding to 5–10 normal human cells) reproducibly yield sensitive and specific CGH profiles, we tested the efficacy of DOP-PCR CGH using microdissected formalin-fixed, paraffin-embedded cell pellets of U251NCI cells stained with HE and MG. Crude DNA extract of MG-stained tissue was a homogeneous blue solution (color intensity depended on the tissue content in the buffer), and those of HE-stained tissue were in two phases; one was a pink solution and the second was a purple precipitate. The former was separated from the latter by brief centrifugation and used as crude DNA extract. Agarose gel electrophoresis of DOP-PCR products from these tissues suggested DNAs could be amplified although their size was smaller than products amplified from fresh cellular DNA (Figure 3)Go . These DOP-PCR products were used as probes for CGH experiments. DOP-PCR products from small amounts (2 mm x 2 mm) of both MG- and HE-stained samples produced CGH profiles equivalent to those produced by nick translation (Figures 4and 5). On the other hand, larger (6 mm x 6 mm) HE-stained samples (ie, a more concentrated sample) produced unfaithful CGH profiles (Figure 5AGo and 5B)Go , even though the DOP-PCR product sizes were adequate (Figure 3Go , lane 11). Increasing the Mg2+ concentration for the second phase of DOP-PCR improved the CGH profile (Figure 3Go , lane 10; profile data not shown).



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  		Figure 3. Agarose gel electrophoresis of DOP-PCR products. Five microliters of each DOP-PCR product were applied to 1.2% agarose gel. M, {phi}X 174/HaeIII. The sizes of the molecular weight markers are indicated at the left. Lanes 1-6 : DOP-PCR product from 10 pg (1), 100 pg (2), 1 ng (3), 10 ng (4), 50 ng (5), and 250 ng (6) of DNA from unfixed U251NCI cells. Size of DOP-PCR products ranged from approximately 400 to 1500 bp, and amplification was better when template DNA was 1 ng or more. Lanes 7-11 show DOP-PCR product from DNA extracted from fixed, embedded, and microdissected piece of MG-stained (7 and 8) and HE-stained (9–11) U251NCI cell pellet. DNA s were extracted from smaller (2 mm x 2 mm, lanes 7 and 9) and larger (6 mm x 6 mm; lanes 8, 10, and 11) pieces in the same amount of buffer (60 µl) and amplified by DOP-PCR. DNA from the larger HE piece was amplified with (lane 10) or without (lane 11) increased magnesium. Size of DOP-PCR products from fixed and microdissected samples (apparently <1000 bp) was smaller than that amplified from freshly prepared DNA.

 


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  		Figure 5. Ratio profiles from HE-stained and microdissected U251NCI human glioblastoma cell pellets. DOP-PCR product from the smaller piece (A, 2 mm x 2 mm in 60 µl extraction buffer) produced a CGH profile similar to that by nick translation (see Figure 2AGo ). On the other hand, the product from the larger piece (6 mm x 6 mm in 60 µl extraction buffer) did not produce faithful CGH profiles (B). However, DOP-PCR using an increased magnesium concentration produced a faithful profile (data not shown).

 
We microdissected histologically normal and neoplastic regions from MG-stained paraffin sections of human oligo-astrocytoma. CGH profiles from the normal regions showed no aberration, but profiles from neoplastic regions had losses on chromosome arms 1p and 19q (Figure 6)Go .



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  		Figure 6. Ratio profiles from fixed and microdissected human mixed oligo-astrocytoma. DNAs were extracted from histologically neoplastic (*) and normal (**) regions and amplified by DOP-PCR. A: MG-stained section where microdissected areas are indicated by the dotted squares. B: HE-stained section where areas to be microdissected are indicated by the dotted squares. C: CGH ratio profile from histologically normal and neoplastic regions. The former showed no aberrations, whereas the latter showed losses on chromosome arms 1p and 19q.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Both SF210 and U251NCI cells have a variety of chromosomal aberrations, and CGH was useful in detecting each of them if >=50 pg DNA was used for DOP-PCR amplification. Thus, our method is effective for investigating the whole genome by CGH. Our studies do not bear on the controversy concerning the effectiveness of DOP-PCR for microsatellite analysis.3, 13, 14 In the SF210 and U251NCI series of experiments, fluorescence on the metaphase spreads produced by the test probes was dim, grainier, and more patchy when <1 ng DNA was used for DOP-PCR. This suggests that the whole genome may not be amplified by DOP-PCR in a universal manner when the amount of template DNA was small (<1 ng). Another possibility is that some pieces of the genome are not included in a sample of less than 1 ng. Certainly, if the amount of template DNA is very small (<10 pg), genomic regions not included in the sample might be quite large, and aberrations (especially gains) in those regions may not be faithfully detected. However, when the sample contains DNA extracted from 5 to 10 cells (>=50 pg), DOP-PCR can provide appropriate probes for CGH, although it may not be suitable for experiments such as fine mapping of chromosomes or microsatellite analysis. As shown in Figure 3Go , the proportion of small (<100 bp) fragments in DOP-PCR products increased as the amount of template DNA decreased. These small fragments may produce unfaithful CGH profiles when unaccompanied by larger fragments. Approximately 200 cells were included in the microdissected region of mixed oligo-astrocytoma tissue that we used as sample in Figure 6Go . Tissue microdissection by needle can isolate cells within a region larger than 1 mm, and the number of cells in the tissue piece depends on the cellularity of the tissue. Recently, a study on CGH from a single cell was published,5, 15 and laser-assisted tissue microdissection16 might enable us to use 10 to 20 cells in a tissue section for DOP-PCR CGH.

Recently, several studies of cancers by CGH using tissue microdissection and DOP-PCR have been published.16, 17, 18 These studies found that DOP-PCR was effective in obtaining CGH profiles, but they have not shown that DOP-PCR product from microdissected tissue gives the same CGH profile as standard nick translation. We made formalin-fixed, paraffin-embedded thin sections of cell pellet from U251NCI cells, microdissected small areas from them, and amplified DNA for CGH. We have previously verified CGH aberrations in nick-translated DNA from nine glioblastoma cell lines including U251NCI, using FISH and flow cytometry.10 We find that DOP-PCR CGH from this tissue can produce faithful CGH profiles if >=50 pg of DNA is used.

The method of tissue staining had an influence on the CGH results. MG-stained tissue was an excellent material for producing faithful CGH profiles, irrespective of the amount of microdissected tissue. On the other hand, HE-stained tissue can produce false CGH results. Thus, for an experiment using DOP-PCR, hematoxylin stains should be avoided when preparing tissues as starting material, because hematoxylin interferes unpredictably with the reaction.19 Our result suggests that the efficacy of DOP-PCR from HE-stained tissue depends on the proportion of sample tissue to volume of the lysate: more tissue gave poorer results, even though the size of the DOP-PCR product from a larger HE-stained piece was bigger than those from a MG-stained piece and from a smaller HE-stained piece (Figure 3Go , lanes 7–11). This suggests that higher concentrations of hematoxylin dye in the sample degrades the quality of DOP-PCR results. Diluting the crude DNA extract (tissue lysate) might help produce successful DOP-PCR experiments, but at the same time lowering DNA concentrations might produce invalid CGH profiles. In our controls for DOP-PCR CGH using SF210 and U251NCI, 50 pg of DNA produces accurate CGH profiles. Even though increasing magnesium during the second phase of DOP-PCR helped improve CGH results, practical difficulties in measuring hematoxylin concentration for each sample makes this solution unworkable.

As noted above, CGH from DNA extracted from fresh cells has been verified,10 and CGH of microdissected pieces of paraffin-embedded cell pellet provides faithful cytogenetic data under appropriate conditions (Figures 4Go and 5)Go . These data encouraged us to study cytogenetic characteristics from multiple regions within a single tissue sample using tissue microdissection and DOP-PCR CGH. When a single microdissected MG-stained section of oligo-astrocytoma was used, there were no chromosomal aberrations in a histologically normal region, but there were losses on 1p and 19q in a neoplastic region. These losses are commonly found in tumors of this type,20, 21 and we have previously confirmed that CGH profiles at these sites from paraffin-embedded tumor sections where histologically normal tissue was removed are compatible with FISH and loss of heterozygosity studies.21 In cases of infiltrative tumors such as oligo-astrocytoma, normal tissue is often intermixed in a surgically excised tumor sample. This might mask chromosomal aberrations in tumor cells. However, because microdissection allows one to pick regions of relatively pure tumor, DOP-PCR CGH on microdissected samples may reveal the true cytogenetic characteristics of tumor cells. Furthermore, since retrospective investigations often require paraffin-embedded tissue, this technique might provide important information when accurate analysis of clinical data is needed. We have investigated grade II diffuse astrocytomas using microdissection and DOP-PCR in paraffin-embedded tissue and found CNAs in more than 80% of cases, although CGH using frozen tumor samples detected CNAs in only 40% of the cases (Hirose Y, Aldape K, Chang S, Larson D, Lamborn K, Berger MS, and Feuerstein BG, submitted data; and Mohupatru G, and Feuerstein BG, unpublished data). This discrepancy could be due to intermixed normal tissue within surgically resected samples. We compared CGH profiles from the central region of tumor with those from infiltrated region. Tissue microdissection together with DOP-PCR enabled us to find greater numbers of CNAs, and the clarity of existing CNAs was improved in regions of pure tumor (submitted). It is possible that cytogenetic analysis will provide important criteria for classifying astrocytomas, and that excluding intermixed normal tissue to obtain accurate information will increase the sensitivity of analysis.



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  		Figure 4. Ratio profiles from MG-stained and microdissected U251NCI human glioblastoma cell pellets. DNA extracted in 60 µl extraction buffer from both smaller (A, 2 mm x 2 mm) and larger (B, 6 mm x 6 mm) pieces produced CGH profiles similar to those by nick translation (see Figure 2AGo ).

 
Furthermore, microdissection might be useful to investigate genetic heterogeneity. To date, most previous CGH studies on gliomas used nick-translated DNA extracted from relatively large samples of frozen or paraffin-embedded tissues.22, 23, 24, 25, 26 Data from these studies could detect a CNA only if it were present in a high proportion of cells. If cytogenetic subpopulations of cells existed within these large samples, these methods might have missed them. This would be unfortunate if such minor subpopulations represent higher-grade or drug-resistant populations. Thus, sampling small regions of tumor might reveal these subpopulations and lead to a more certain diagnosis. Studies of heterogeneity in gliomas are underway in our laboratories to investigate these possibilities using DOP-PCR CGH of microdissected samples.

An important limitation of CGH is its inability to assay chromosome aberrations of a single cell. However, its strength is its ability to screen the whole genome for CNAs. This study provides evidence that very small regions of tissue can be assayed by CGH. These methods may find important applications in evaluating small samples and in investigating the role of heterogeneity as a prognostic factor.


   Footnotes
 
Address reprint requests to Burt G. Feuerstein, Cancer Genetics Program, Box 0808, UCSF, San Francisco, CA 94143-0808. E-mail: feuer{at}cc.ucsf.edu

Supported in part by the National Institutes of Health grants CA13525 and CA 64898, and by funds from the National Brain Tumor Foundation and the Farber Foundation.

Accepted for publication February 21, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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