WHOLE GENOME AMPLIFICATION AND MOLECULAR GENETIC ANALYSIS OF DNA FROM PARAFFIN-EMBEDDED PROSTATE ADENOCARCINOMA TUMOR TISSUE

0022-5347/99/1624-1512/0

Vol. 162, 1512-1518, October 1999 Printed in U.S.A.

'hiE JOURNAL OF UROLOOY

Copyright 0 1999 by AMERICAN UROLOGICAL ASS~CIATION, hc.

WHOLE GENOME AMPLIFICATION AND MOLECULAR GENETIC ANALYSIS OF DNA FROM PARAFFIN-EMBEDDED PROSTATE ADENOCARCINOMA TUMOR TISSUE SUN-HUN KIM, TONY GODFREY AND RONALD H. J E N S E " From the Cancer Center, Department of Laboratory Medicine, University of California, Sari Francisco, California

ABSTRACT

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 suff1cient 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. KEY WORDS:prostate cancer, PCR, loss of heterozygosity, allelic imbalance Genetic alterations in somatic tissue are important phenomena in the development of most neoplasias. Thus, a large number of molecular analyses are aimed toward determining alterations in the cellular genome as tumors progress. To determine which genomic lesions occur early in tumor development, it is necessary to analyze DNA of malignant cells microdissected from small tumors. Comparative Genomic Hybridization (CGH),' a genome wide screening technique, and allelic imbalance measurements (AI)of multiple loci,2 another genome wide scanning method, each require on the order of one microgram of tumor DNA for analysis. Typically, microdissected samples contain only hundreds or thousands of cells, yielding only nanograms of DNA. In these cases one can potentially overcome the limiting amount of DNA by using PCR-based whole genome amplification techniques. One such technique, termed DOP-PCR,3 uses a single degenerate PCR primer with low stringency PCR cycle parameters and has been shown to achieve uniform amplification at a

large number of microsatellite loci when starting with fresh human lymphocyte DNA.* This technique has been used by a number of laboratories to perform molecular genetic analysis on tumor tissues that are microdissected to provide histologically uniform specimen^.^-^ However, these studies did not systematically test whether DOP-PCR amplified product gives reliable results with A1 or with CGH. In addition, 2 other labs performed small studies that indicate artifactual allelic imbalance can occur when small quantities of DNA are DOP-PCR amplified and analyzed for allelic i m b a l a n ~ e . ~ . We sought to further investigate conditions under which DOP-PCR gives uniform amplification of DNA extracted from formalin-fixed and paraffin-embedded human prostate tumors by performing molecular genetic characterization of the tumor genome using fluorescence allelic imbalance and comparative genomic hybridization. Comparison of the results of these molecular genetic analyses on DOP-PCR amplified DNA with those for non-amplified DNA from the same specimens was used to determine whether DOP-PCR gwes Accepted for publication May 11, 1999. * Requests for reprints: Cancer Center, Department of Laboratory sufficient material of good genome representation. Based on Medicine, Box 0808, University of California, San Francisco, San our results, conditions were established in which these anaFrancisco, CA 94143-0808. Supported by the Korean Research Foundation and NCI Grant lytical techniques can be routinely applied to specimens miR01 CAIES68637. crodissected from paraffin-embedded prostate tumor tissue. 1512

MOLECULAR GENETICS OF PCR AMPLIFIED PROSTATE TUMOR DNA

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MATERIALS AND M E T H O D S 9600 thermocycler in 2 phases. The composition of each PCR Samples and DNA extraction. Samples were obtained from reaction was 0.33 ELM each Primer, 250 P M each dNTP, 0.6 conventional formalin-fixed and parafin-embedded pathol- units AmPliTaq Gold Polymerase (Applied BiosystemflerFoster CA), 2.5 mM M&129 50 mM KCl, lo ogy specimens prepared from prostatectomies performed at kin UCSF from 1990 to 1994. These specimens had been stored mM Tris PH 8.3 in total volume 15 pL. Phase 1 started with minutes at by lo Of l5 in the Department of Pathology tissue storage facility for 2 to 5 years prior t o being retrieved. As a histopatholo@c refer- seconds at 94c, 15 seconds at 55C, and 30 seconds at 72C. Of l5 seconds at 89c, l5 ence for defining tumor regions, a 5 micron section was cut Phase was 23 onto a microscope slide and stained with conventional H & E seconds at 55c7 and 30 seconds at 72c7 followed by 10 minat lot. stain. Samples were selected t o be representative of those utes at 72cand On an AB1 377 autoroutinely obtained during prostatectomies, based on typical Amp1ified loci were Biosystemflerkin-E1mer, tumor stages (T2 and T3) and Gleason grades (range 3 + 3 to mated DNA sequencer On 5% Long Ranger acvlamide gel (JT 4 +5). For DNA analysis without prior DOP-PCR, only large Foster City, under denaturing conditions. lanes Haward, Baker, tumors were selected to be sectioned, Although more that 50 with an size standard and were specimens were analyzed, close comparison of data obtained were with Genescan 2.1 Biosystemd from analysis of DOP-PCR with those from non-DOP DNA Perkin-Elmer, Foster City, CA). Two parameters, Allelic Rawere obtained only on a subset 8 samples. Approximately 20 to 30 sections (5 microns thick) were cut tio (AR)and Allelic Imbalance (AI), were calculated for each and placed in a polypropylene tube for subsequent DNA Ratio was calculated by: extraction. Paraffin was removed by incubating with Americlear (Baxter, Inc. Deerfield, IL) at 37C for 20 minutes. After ~ e ~ c ( f ~ ~ ~ ~ ~ e centrifugation and supernatant removal, the pellet was deAI = AR(Tumor)/AR(Benign) hydrated by resuspension in 100% ethanol twice followed by or AI = AR(Benign)/AR(Tumor) air drying. Precipitate was then resuspended in 2 ml. digesAI can be calculated as the ratio in either direction, detion buffer SDS in lomM NaC1725 mM EDTA, lomM pending on which allele is lost, peak 1 or peak 2. We prefer to Tris pH 8.0) with 0.3 to 0.5 mg./ml. proteinase K (Sigma, St. report the ratio as less than or equal to l.o, Louis, MO) and incubated at 55C overnight. On subsequent c~~~~~~~~~~ genomic hybridization. comparative genomic days an equal amount of fresh proteinase K was added for hybridization (cGH) was performed using directly overnight incubation each day until tissue particles were no fluorochrome-conjuga~d DNA as described previously.io longer visible. This usually required 3 to 5 days of proteinase Briefly, DNA samples were labeled with FITC-dmP (DuK treatment. DNA was then extracted using a conventional Pant, Boston, and male DNA was labeled with phenol: chloroform: isoamyl alcohol procedure, precipitated Texas red ( T R ) - d m (Dupont, B ~ MA) ~using ~nick ~ in and in TE buffer (lomM Tris, mM translation. From each differentially labeled genome, 500 ng. EDTA, pH 7.5) for subsequent analysis. was precipitated together with an excess (30 pg.) of unlaFor microdissection 2 sequential 5 micron sections were beled Cot-l fraction ofhumanDNA (Gibco BRL, Grand lScut onto separate microscope slides. The first was stained land, ~ y )mis . probe DNA was resuspended in 10 p~ of with conventional H & E and the second was stained with hybridization solution (50% fomamide, 10% dextran sulfate, 0.1% methyl green. Using the H 8~E stained slide as a well- 2 x SSC), denatured at 70C for 5 minutes and pre-annealed defined tissue descriptor, the methyl green stained section for 1hour at 37c. A normal metaphase spread mounted on a was microdissected with a sterile #11 scalpel blade under a microscopeslide was denatured separatelyin a formamide dissecting microscope. Microdissected cells were collected solution (70% formamide, 2 s s c , p~ 7.0) for 8 minutes at into PCR buffer (Boehringer-Mannheim Corp., Indianapolis, 73C and dehydrated through a series of graded solutions of IN) Plus 0.4 mg./ml. Proteinase K and incubated overnight at 70%, 85% and 100% ethanol. Proteinase K (0.1 mg./ml. in 20 55C. On each of 3 subsequent days an equal amount of fresh mM Ws-HCl, 2 nM CaCl,, pH 7.5) was added at room temProteinase K was added. Finally, proteinase K was inacti- perature and the spread was dehydrated again as described vated by incubating for 5 minutes at 95c9 and the sample above. Hybridization of probe DNA to the metaphase spread was stored for subsequent DOP-PCR amplification. was conducted under a cover slip for 2 to 4 days at 37C. After D o p - p c R . DOP-PCR was Performed on an ABI 2400 ther- hybridization, the slides were washed 3 times in 50% formmocycler (Applied Biosystemd'erkin Elmer, Foster City, amide, 2 x SSC pH 7; twice in 2 x SSC; once in 0.1 x SSC at CA) in 2 phases. The composition of each PCR reaction was 45c; once in 2 x SSC plus 0.1% NP 40,O.l M NaH,PO, pH 200 PM each dNTP, 1.5 pM degenerate primer, 2.5 units Taq 8.0, and finally in distilled water at room temperature. The polymerase (Boehringer Mannheim Corp., Indianapolis, IN), slides were then counter stained with 4', 6-diamidino-21to 20 ng. DNA template, 1.5 mM MgC12,50 mM KCl, 10 mM phenylindole (DAPI) and mounted in phenylenediamine anTris pH 8.3 in a total of 50 pL. The degenerate primer was 5' tifade solution." CCGACTCGAGNNNNNNATGTGG3'. Phase 1 started with Microscopy and image analysis. Fluorescence images were 4 minutes at 94C, followed by 5 cycles composed of30 seconds acquired and analyzed using a quantitative image processing at 94C, 30 seconds at 32C, ramp to 72C over 3 minutes, and system (QUIPS) as described by Piper et a1.12 In general, 1.5 minutes at 72C. Phase 2 was 30 cycles composed of 30 images of 4 to 7 different metaphase spreads were captured seconds at 94C, 30 seconds at 52C, 1 minute at 72C with 5 and analyzed to provide profiles of normalized ratios of the seconds added to the 72C extension time for each cycle. After intensity of green fluorescence t o red fluorescence at each the final cycle, phase 2 was completed with 7 minutes at 72C, data channel along the length of the entire genome. In our then held at 1OC. Product was eluted through a n S-300 HR experience the greedred ratios measured 1.0 & 0.15 for DNA column (Pharmacia Biotech, Piscataway, NJ) to remove re- with no copy number alterations. Thus, a threshold of 0.85 m i n i n g primer and dNTPs. was set to determine significant DNA copy number loss and Allelic imbalance. PCR primer pairs flanking polymorphic 1.15 was set to define significant DNA copy number gain. microsatellite loci were obtained from ABI (Applied Biosystems/Perkin-Elmer, Foster City, CA). All pairs used RESULTS were from the ABI PRISM Linkage Mapping Set, Version 1 Although no micromanipulators or specifically designed in which one of the 2 primers was labeled with a fluorophor; FAM, TET, or HEX. Amplification was performed in an ABI microdissection equipment was available, speeific glands of 95c7

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MOLECULAR GENETICS O F PCR AMPLIFIED PROSTATE TUMOR DNA

tissue specimens could be extracted by manual manipulation of specimens stained with methyl green to allow recognition of tissue structure under a dissecting microscope (for details see Materials and Methods). Figure 1illustrates the results obtained when isolating a specific region of a section by scraping the tissue surrounding that region. The isolated region was then lifted from the slide and processed for DOPPCR amplification. 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. The results of these measurements a t 4 different microsatellite loci (table 1)indicate that, in general, the allelic ratios are comparable. Only 2 of the 9 measurements showed significantly different values for DOP-PCR DNA than for non-DOP DNA. The 2 comparisons that showed differences were from 2 different tissue specimens ( B and C ) , and at 2 different microsatellite loci (D7S515 and D7S519). In addition one of these samples was tested at a different locus (sample C a t D8S279)

FIG. 1. Microdissection of prostate tumor tissue. A, H & E stained section immediately adjacent to microdissected section. B , methyl green stained section prior to microdissection. C, same section as in B after microdissection. Isolated gland in center is then removed and DNA is extracted from it. Magnification is X60.

which no~difference in allelic ratio for DOP-PCR . _ showed ~ . ~ versus non-DOP. To further investigate measurements on DOP-PCR DNA, w.e.measured sDecific microsatellite allelic imbalance (AI)on tumor and n o A a l DNA from 3 different prostate cancer ~~~~

TABLE1. Comparison of allelic ratios measured on DOP-PCR amplifwd DNA and non-DOP D N A Allelic RatioDOPb Code ND'

LOCUS

Sample

Mean

Std Dev

Mean

Std Dev

Sign# Diff,

1.18 0.205 No 0.296 A 1.43 D7S515 2.24 0.52 Yes 0.141 C 4.13 D7S515 No 4.12 1.350 2.616 E 4.25 D7S515 0.926 No 1.30 0.275 A 1.40 D7S486 1.95 0.537 No 0.898 D 2.04 D7S486 0.88 0.028 Yes 0.098 B 1.35 D7S519 1.68 0.523 No 0.190 C 1.64 D8S279 1.24 0.127 No 0.098 D 1.39 D8S279 1.87 0.777 No 0.098 E 2.01 D8S279 a ND = non-DOP DNA from conventionally sectioned prostate tissue. DOP = DOP-PCR amplified DNA from the same tissue sections as was used for ND. Signif Diff. = Significant Difference. To define any differences in means, the 95% confidence interval = mean C 1.96 (StdDev/n). To be called significantly different (=Yes) the means being compared must fall outside the 95% confidence interval of the mean being compared.

Allelic Imbalance

FIG. 2. Histograms of allelic imbalance frequencies for prostate tumor specimens. A, for tissues sectioned by conventional sectioning and no DOP-DNA amplification. B, for microdissected tissue and DOP-PCR amplified DNA.

MOLECULAR GENETICS OF PCR AMPLIFIED PROSTATE TUMOR DNA

cases using DOP-PCR amplified DNA from microdissected tissue specimens in parallel with non-DOP DNA from conventionally sectioned specimens. Results of the measurements showed that AI was equally likely to be above or below 1 for both DOP-PCR amplified DNA and non-DOP DNA (12 of 20 values were greater than 1.0 for each DNA type). These results indicate that the specific locus PCR used for allelic imbalance measurements developed no unexpected bias based on allelic size. To define a threshold to differentiate between retained and lost alleles, a histogram of A1 or its inverse was generated (fig. 2). It is apparent that AI tended to cluster in 2 groups for both DOP and non-DOP DNA, with a separation between these groups at AI = 0.7. Thus, we defined retained as 0.67 5 AI 5 1.5 and lost as AI <0.67 or >1.5. By inspection of the data depicted in fig. 2, it can be seen that this threshold applies equally well for DNA isolated from both microdissected and conventionally sectioned tissues. Allelic imbalance measurements were performed by PCR of 20 different specific microsatellite loci on both DOP-PCR amplified and non-DOP DNA from 3 different tumors. The results of these measurements are shown in table 2. Two of the tumors (F and G ) were small, so samples with no DOPPCR contained only enough DNA to measure AI a t 2 microsatellite loci. However, a lymph node metastasis (sample H) was large and histologically homogeneous, thus providing sufficient DNA to extensively test comparative allelic imbalance measurements. By comparing retained and lost loci on

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the 2 DNA types (columns 4 and 6). we note qualitative agreement for every locus. In addition, because the tumor in sample H was very large, microdissection could be performed to isolate samples of the tumor from 3 clearly separate areas, each encompassing roughly 10% of the tumor. For 8 of the microsatellite loci, AI measurements were performed on DOP-PCR DNA from 2 of these 3 areas. For 6 of those loci the AIs of the 2 regions agree in their interpretation, with 3 loci (D18S452, D18S68 and D7S6636) showing allelic balance (retained) and 3 loci (D8S282, D8S258 and D8S277) showing imbalance (lost). For 2 loci (D18S61 and D18S59) AI was sufficiently different between the 2 regions to result in different interpretation of allelic imbalance. Thus, microdissection revealed tumor genomic heterogeneity. Another important observation is that allelic imbalance values for regions that are lost is generally more extreme for DOP-PCR amplified DNA from microdissected tissue than for DNA from conventionally sectioned tissue. To illustrate this effect, profiles of electrophoretic analysis of one sample a t one locus are shown in fig. 3. From these results, it can be seen that for tumor DNA the larger allele (151 bp) was lost and that DOP-PCR amplified DNA displayed a more definitive loss from all 3 subregions of microdissection than shown for non-DOP DNA obtained from conventional sectioning. This illustrates that even for a large, relatively homogenous tumor, conventional sectioning includes a significant heterogeneity of cells with differing genomic alterations (probably from benign epithelial and/or stromal cells), so as to make allelic imbalance measurements less sensitive for detecting tumorigenic alterations.

TABLE2. Comparison of allelic imbalance measured for DOP-PCR

amolitied . . DNA and non-DOP DNA Sample 'Ode

Nan DOP

AI Indexb

DOP-PCR

AI'

AI Index

AI

Aread

D7S507 D7S510 D7S669 D7S515 D7S636

H H H F H

0.89 0.89 1.0 1.18 1.16

R R R R R

1.26 1.01 0.84 1.32 0.96 0.71

R R R R R R

T3 T3 T1

D8S504 D8S277

H H

0.3 0.35

L L

D8S258

H

2.41

L

F H

1.17 3.27

R L

D8S270

F

1.13

R

L L L L L L L R L L L R

T1 T2 T2 T1 T3 T2 T1

D8S283 D8S283

0.46 0.32 0.18 0.09 5.98 >10 >10 1.32 3.03 4.9 3.15 0.81

H

0.85

R 0.98 L 0.47 R D18S452 H 1.1 R 1.01 1.03 R 0.98 R D18S464 H 0.91 R 1.15 R D18S53 H 1.03 R 1.46 R D18S57 H 0.81 R 1.04 R Dl85474 H 0.76 R R 0.71 D18S68 H 1.11 0.8 R R 1.09 R R D18S61 H 0.95 R 1.33 2.33 L G 1.73 L >10 L D18S61 D18S462 G 5.28 L >10 L D18S70 H 1.01 R 1.0 R a Microsatellite loci are listed in order from 5' to 3' along genome. AI Index = Ratio of peaks (tumorYRatio of peaks (normal). A1 = allelic imbalance; R = retained, 0.67 5 AI Index 5 1 . 5 and L AI Index ~ 0 . 6 or 7 >1.5. Area = Tumor H was microdissected at 3 different regions. T1 repion; T2 = second region; T3 = third region.

D18S59

R

-

T3 T1

-

T3 T2 T1 -

T2 T3 T3 T2 T1 T1 T2 T3 T2 T2 T1 T2 T1 T3

=

lost,

=

first

A

n

FIG.3. Fluorescent LOH analysis of D8S258 on DNA from microdissected tissue in comparison with conventionally dissected tissue. A, DNA from conventionally dissected benign tissue; B, DNA from conventionally dissected tumor tissue; C , DNA from microdissected benign tissue followed by DOP-PCR amplification; D , DOP-PCR amplified DNA from microdissected region T3 of tumor sample H (see table 2 for details);E , DOP-PCR amplified DNA from m i d i s sected region T2 of tumor sample H; F, DOP-PCR amplified DNA from microdissected region T1 of tumor sample H.

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MOLECULAR GENETICS OF PCR AMPLIFIED PROSTATE TUMOR DNA

6 1

I

C

12

FIG. 4. Comparative genomic analysis of microdissected tissue in comparison with conventionally dissected tissue. Profiles of ratio of green (test) to red (reference) fluorescence intensity for each chromosome Large numbers that appear on left are chromosome identifylng numbers. Small numbers on l e e (for example, n = 6) indicate number of metaphase chromosomes analyzed to produce each profile Heavy line in each profile is mean fluorescence ratio at each channel and thin lines are 5 1standard deviation. Horizontal dotted lines slgnify fluorescence ratios of 1.2 and 0.8, which are thresholds for defining region of copy number gain or region of copy number loss, respectively. Small vertical bar on X axis for each rofile signifies position of chromosome centromere Samples analyzed were. A, DNA from conventionally dissected tumor; B, DOP-PCR ampEfied DNA from microdissected repon T2 of tumor sample H , C, DOP-PCR amplified DNA from microdissected benign lymph node tissue from sample H.

MOLECULAR GENETICS OF PCR AMPLIFIED PROSTATE TUMOR DNA

To further illustrate the comparison of DOP-PCR amplified DNA with non-DOP DNA, we also performed comparative genomic hybridization (CGH) on both DNA types from the large lymph node metastasis (sampleH). A comparison of the 2 sets of chromosomal profiles (fig. 4) indicates that DOPPCR amplified DNA shows more variation in measurements (note the larger separation o f ? standard deviation) but also shows much larger ratio alterations a t regions of altered copy number. For example the gain in chromosome 8q shows a maximum ratio of 1.7 in non-DOP DNA as compared with 2.7 for DOP-PCR amplified DNA from a microdissected section. Also the allelic loss in chromosome 8p is obvious in the profile for DOP-PCR amplified DNA (minimum ratio of 0.7) while it is very subtle (minimum of 0.9) in the profile for non-DOP DNA. One other region which shows a copy number alteration of interest is chromosome Xq, which is on the border line indicating a true regional alteration but shows no altered profile for non-DOP DNA. Other small amplitude changes which appear in the DOP DNA profiles (for example, chromosome l q , 3 , 9 centromere, 13p telomere, 14p telomere, 15p telomere, 16 centromere, 19, 21p telomere, 22 centromere and Y), occur primarily in regions which tend to show artifactual CGH alterations due to large amounts of repetitive DNA. For comparison, CGH profiles obtained from a microdissection of histological normal lymph tissue from sample H also are shown in fig. 4. These show no DNA copy number alterations beyond the threshold values for losses (ratio = 0.8) or gains (ratio = 1.2). DISCUSSION

Since tissue microdissection allows more homogeneous cellular samples to be obtained from prostate tissue sections, genetic aberrations in cells from small tumors can be detected with high sensitivity. The use of microdissected tissue for molecular analysis usually requires PCR amplification of whole genome DNA so as t o obtain sufficient material. Several other laboratories previously have used this method to provide sufficient material to perform allelic imbalance studies or comparative genomic hybridizati~n~-~-', l3 with variable results. Sanchez-Cesedes et a1 as well as Faulkner and Leigh were dissatisfied with their results for DOP-PCR DNA and suggested other means for amplifying the DNA target, while the other groups appeared satisfied with their results. Our systematic comparison using AI and CGH helps to demonstrate that the product of DOP-PCR amplification is generally very similar to the original DNA. Comparison of allelic ratios and allelic imbalance by fluorescence allelotyping showed that DOP-PCR amplified DNA gave very similar results to non-DOP DNA from a large, homogeneous lymph node tumor (sample H . ) In addition, the difference between allelic imbalance (lost) and balance (retained) is more distinctive when performing microdissection followed by DOPPCR amplification. Since this procedure directly compares tumor DNA with normal DNA from the same sample, alterations in AI Index are often quite dramatic. For example, AI Indices in table 2 for DOP DNA were greater than 10 for a number of loci. Another advantage in performing molecular genetic analysis on DOP-PCR amplified DNA from microdissected tissue is the ability to detect heterogeneous genomic alterations in multiple regions of a tumor. When we performed allelic imbalance studies on 3 different areas from the same lymph node tumor, the results showed that D18S59 was retained for region T2 and lost for region T3 and also that D18S61 was retained for T2 and lost for T1. Presumably these differences in genomic alterations in different parts of a tumor are occurring due to genetic instability of neoplastic tissue leading to multiple clones among the progeny of invasive cells. Monitoring of biopsies from primary tumors by these microdis-

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section methods should provide a means to detect clones that have acquired DNA alterations indicative of invasion and/or metastases. Since a large fraction of elderly men carry small prostate foci which appear to be precursors of ne~plasia,'~. lS a method for discerning whether such foci are genetically altered toward progression would be very beneficial for determining possible therapies. Ultrasound or magnetic resonance mediated biopsy acq~isition'~ might well be followed by molecular genetic analysis of DOP-PCR amplified DNA obtained from such samples. It should be noted that we did find differences between allelic ratios for DOP DNA compared with non-DOP DNA for 2 specimens (samples C and B; see table 1).For sample B it is likely that the conventionally sectioned specimen contained a significant amount of normal tissue mixed with the tumor tissue. Thus, DOP DNA provided a more accurate representation of the original DNA isolated from the microdissected specimen. However, this explanation does not apply to sample C, since it was tested at 2 different loci (D7S515 & D8S279) with one showing agreement and the other showing a difference. No obvious reason for this difference can be given. It may be that the amount of DNA available in this sample for DOP-PCR amplification was small enough to give preferential allelic amplification as has been reported previo ~ s l y . ~ In , ' our experience, such a phenomenon occurs less frequently when DOP amplification is performed on larger amounts of target DNA. Thus we recommend using a minimum of 20 ng. DNA for DOP-PCR in 50 pL solution. As can be seen from the profiles shown in fig. 4, DOP-PCR amplified DNA can be used for CGH analyses to show more distinctive alterations than can DNA from conventional sections. The abnormal profiles from DOP DNA are easily distinguished from CGH profiles for DNA taken from microdissections of normal tissue, indicating that this method should be effective at revealing molecular genetic alterations. Several examples of such applications were published previously for oral squamous cell carcinomas,6 for breast ~ a n c e rand ,~ for renal neop1asms.l6 Acknowledgment. The authors wish to acknowledge the assistance of Dr. Vivek Bhargava, San Francisco VA hospital for providing histopathologic examination of prostate tissues. REFERENCES

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