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Carcinogenesis Advance Access originally published online on August 19, 2005
Carcinogenesis 2006 27(2):307-310; doi:10.1093/carcin/bgi215
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Carcinogenesis vol.27 no.2 © Oxford University Press 2005; all rights reserved.

Accurate genotyping from paraffin-embedded normal tissue adjacent to breast cancer

Bin Xie 1, Jo L. Freudenheim 2, Simone S. Cummings 1, Baljit Singh 1, 3, Hong He 1, Susan E. McCann 2, Kirsten B. Moysich 2 and Peter G. Shields 1, *

1 Cancer Genetics and Epidemiology Program, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC 20007, USA and 2 Department of Social and Preventive Medicine, University at Buffalo, State University of New York, Buffalo, NY 14260, USA
3 Present address: Department of Pathology, New York University School of Medicine, New York, NY 10016, USA

* To whom correspondence should be addressed. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3800 Reservoir Road. NW, LL (S) Level, Room 150, Box 571465, Washington, DC 20057-1465, USA. Tel: +1 202 687 0003; Fax: +1 202 687 0004; Email: pgs2{at}georgetown.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic polymorphism analysis for disease risk is widely used in epidemiology studies; blood or oral cavity cells are the most widely used source of DNA. However, these types of samples are not always available, particularly for studies that were conducted years ago. An alternative potential source of patient DNA exists in the form of paraffin-embedded normal tissue adjacent to tumor samples, which are collected and stored routinely for clinical use. The use of such samples can be conceptually problematic, however, due to the presence of field cancerization in the surrounding normal tissue, with the possible presence of chromosomal loss. Specifically, loss of heterozygosity (LOH) might bias the genotyping results and cause genotype misclassification. However, field cancerization and LOH might not be an issue because LOH is not easily found unless there is careful microdissection of only tumor cells (leaving stromal, inflammatory and fat cells), for example, laser-capture microdissection. In this study, we set out to determine the degree of genotype misclassification from normal tissues adjacent to tumors, if any, by comparing these results with blood genotyping. We examined samples from 106 subjects with breast cancer, analyzing five different genotypes selected from regions commonly known to have LOH in breast cancer. These genotypes were methylenetetrahydrofolate reductase (MTHFR), oxoguanosine glycosylase 1 (hOGG1), dopamine ß-hydroxylase (DBH), dopamine receptor D2 (DRD2) and NAD(P)H dehydrogenase quinone 1 (NQO1), conducted by using real-time PCR and TaqMan genotyping analyses. We found that among these five genotypes and 106 comparisons, there was a 100% concordance for genotyping from normal tissue adjacent to tumor and from blood. Our findings indicate that the use of adjacent normal tissues provides accurate genotyping results with high specificity. Although this study only used breast tumor samples, and may be applicable only to breast cancer studies, we expect the results to be applicable to other types of cancers also.

Abbreviations: DBH, dopamine ß-hydroxylase; DRD2, dopamine receptor D2; LOH, loss of heterozygosity; MTHFR, methylenetetrahydrofolate reductase; NQO1, NAD(P)H dehydrogenase quinone 1; hOGG1, oxoguanosine glycosylase 1


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Data regarding genetic polymorphisms is an important tool in molecular epidemiology. DNA is generally obtained from either blood or sloughed cells from the oral cavity. However, these sources of DNA are not always available, particularly for studies that were conducted years ago. An alternative source of study subject DNA is the paraffin-embedded normal tissue adjacent to tumor samples. There are many clinical trials where tissue is available and blood may not have been stored. However, because it is known that adjacent normal-appearing cells may harbor genetic lesions, a process known as field cancerization is used (1–3), there are conceptual reasons for the finding that the use of adjacent normal tissue may not be appropriate. For example, if loss of heterozygosity (LOH) occurs in the adjacent normal-appearing tissue on the same chromosome as a polymorphism of interest, then a person who is heterozygous might be incorrectly classified as a homozygote. Field cancerization by LOH has been reported to occur in as many as 30–60% of breast cancer cases. The genetic changes are (i) at specific loci and not global across the entire genome; (ii) tend to be only single abnormalities rather than the multiple LOH lesions typically found in tumors; (iii) occur at a substantially lower frequency for any single defect than in the tumor; and (iv) vary in frequency depending on the locus.

In this study, we examined adjacent, apparently normal, tissue from archived paraffin-embedded tumor blocks to determine whether it can be used to identify genotype or whether the presence of LOH would prevent such use. To test this hypothesis, we performed genetic polymorphism analysis of blood and paraffin blocks for 106 individuals. The genes selected were methylenetetrahydrofolate reductase (MTHFR), oxoguanosine glycosylase 1 (hOGG1) (4–6), dopamine ß-hydroxylase (DBH) (7), dopamine receptor D2 (DRD2) (8,9) and NAD(P)H dehydrogenase quinone 1 (NQO1) (10). These polymorphic loci are among the most commonly lost in breast cancer (Table I). In addition, all of these genes have specifically demonstrated LOH in breast cancer or other cancer types.


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  		Table I. List of SNP studied and primers/probes used to detect genetic polymorphism

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects and samples
Paraffin-embedded tissue and matching blood samples are from a subset of women (cases only) who participated in a case–control study of breast cancer and alcohol drinking in Erie and Niagara counties in western New York (11). Slides were cut from the blocks with adequate precaution to prevent contamination between cases, including replacement of blades between each block. Microtome holders were cleaned using xylene between cases to prevent contamination of tissue from one block to the next. Glass slides were treated to prevent contamination with DNases and RNases.

Materials
Primer and probe sequences for MTHFR, hOGG1, DBH and NQO1 came from SNP500 (http://snp500cancer.nci.nih.gov/) (29); rs numbers are listed in Table I. Primer and probe sequences for DRD2 were designed and synthesized by BioServe Biotechnologies, Laurel, MD. TaqMan Universal Master Mix was purchased from Applied Biosystems (Foster City, CA). For regular PCR, oligonucleotide primers were purchased from Qiagen (Valencia, CA). dNTP was purchased from Invitrogen (Carlsbad, CA). Taq polymerase was purchased from Applied Biosystems.

DNA extraction from blood
DNA was extracted from stored frozen blood clots (–80°C) using a modified protocol provided with the GeneQuick DNA extraction kit (BioServe Biotechnologies, Laurel, MD). Briefly, blood clots were thawed at 37°C and all red blood cells were lysed by incubating the samples for 10 min with a blood lysis solution. Samples were then centrifuged for 10 min and the supernatant was discarded. Sample lysis solution and proteinase K were then added and samples incubated at 55°C for 1 h to overnight to lyse white cells and all the remaining cells. Protein was precipitated out using protein-out solution. DNA was precipitated using 100% isopropanol.

DNA extraction from adjacent normal tissue
Breast carcinoma (both invasive and in situ) was identified and circled by a breast cancer pathologist (B.S.) on a hematoxylin/eosin stained slide. One unstained adjacent section of the slide (20 µm) was deparaffinizated and used for microdissecting adjacent normal tissue (Figure 1). Briefly, tumor tissue was dissected first, according to the circled area as specified on the hematoxylin/eosin stained slide. All of the rest of the tissue on the slide was counted as adjacent normal tissue. To prevent cross contamination, tumor and adjacent normal tissue was dissected separately and different needles were used for dissection. After dissection of the tissue from the unstained slide, adjacent normal tissue was subsequently digested by proteinase K overnight at 50°C. DNA was extracted from the clear lysate using the MagAttract DNA Mini M48 Kit, using a Qiagen BioRobot M48 workstation, according to the manufacturer's protocols.


Figure 1
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  		Fig. 1. Illustration of adjacent normal tissue used for DNA extraction. (A), the hematoxylin/eosin stained slide, with tumor and adjacent normal tissue clearly marked. (B), unstained adjacent section of the slide (20 µm), used for microdissecting adjacent normal tissue. (C), after tumor removal, the rest of the tissue was counted as adjacent normal tissue, which includes stromal, fibroblast, fat and lymphatic cells. (D), the slide after removal of adjacent normal tissue.

 
TaqMan real-time PCR condition
MTHFR, hOGG1, DBH, DRD2 and NQO1 genotyping were performed using TaqMan real-time PCR and carried out on an ABI PRISM 7900HT Sequence Detection System. DNA (15 ng) was used in a 5 µl reaction. Real-time PCR reaction conditions were [50°C (2 min), 95°C (10 min), 40 cycles of 92°C (30 s) and 60°C (1 min)] as specified by SNP500 (http://snp500cancer.nci.nih.gov/). After completion of thermal-cycles, the reaction plate was post-read by the ABI9700 sequence detection system. Genotyping result was generated automatically by autocall (software attached with ABI 7900) with 95% confidence. Quality control 15% random repeats were performed.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Five SNP [MTHFR, hOGG1, DBH, DRD2 and NQO1] assays, as listed in Table I, were performed on 106 pairs of DNA from adjacent normal tissue and matching blood. Out of these 212 DNA samples, the percentage of samples that were amplified by TaqMan method ranged from 95 to 99% (Table II). In a particular SNP assay, those samples that were not amplified came from either blood or tissue, or both. For those few samples for which neither blood nor tissue DNA were amplified, the amplification was carried out in another SNP assay. Overall, those samples that were not amplified were scattered across genotypes and were assay-specific. Genotyping results from adjacent normal tissue and matching blood were compared for those samples with both results. A 100% concordance was found in all the five assays (Table II).


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  		Table II. Genotype matching from blood and adjacent tissue

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
One of the main concerns for using adjacent normal tissue for genotyping is the fear of LOH confounding the genotyping result. The concept of field cancerization or field effect has been well documented, including for breast cancer (1–3). It is thought that morphologically normal cells harbor early genetic lesions that allow for a selective clonal expansion leading to cancer. This is most often demonstrated through LOH studies in adjacent normal tissue. When LOH is found, it is at the same loci as found in the tumor, and the presence of genetic abnormalities depends on the type and chromosomal location seen in that tumor (2,3,12). In breast tumors, LOH is not random, occurring mostly at chromosomal arms 1p, 1q, 3p, 6q, 11p, 11q, 13q, 16q, 17p, 17q and 22q (3,12–14). In this study, we chose five SNPs, four of which are located on chromosomes that are frequently lost in breast cancer (Table I), and did not find genotyping misclassification due to field cancerization.

There are several reasons for this finding. Assaying for LOH is not a qualitative determination that is yes or no, but is actually carried out quantitatively by determining whether one allele is quantitatively decreased in an amount by a percentage (15,16). In most cases, both alleles are still visible and the person can be identified as a heterozygote, even using the tumor analysis. This is because of the presence of genetically normal DNA from stromal, fibroblast, fat and lymphatic cells, which have germline DNA. Also, as discussed in the introduction, only a fraction of ducts in normal tissue is affected by field cancerization, so that there is also unmodified DNA from epithelial cells. Thus, genotyping from adjacent normal tissue uses mostly DNA from stromal cells that would not harbor LOH.

There is some data in the literature to support our findings (17,18). In an analysis of colon cancer among 180 subjects and 11 markers, where adjacent tissue was examined for allelic imbalances on chromosomes 8p and 18q, not one adjacent normal tissue had an abnormality (19). Separately, our study is also supported by a recent publication of a smaller study of pharmacogenetic SNPs in tumors, where there was 100% concordance for four different SNPs (two on CYP2D6 on chromosome 22, one each on CYP2C8 on chromosome 10 and MDR1 on chromosome 7) between tumor and blood in only 10 subjects (20).

We did not examine genotype in the tumor samples in the study, as the purpose of this study was to determine the feasibility of using adjacent normal tissue for genotyping; evidence demonstrating concordance with blood is more convincing. In order to detect precursor lesions in the histologically adjacent normal tissue that may harbor LOH, it is necessary to examine only carefully microdissected ducts, carried out most recently through laser-capture detection (3,18). Even though 30–60% of the cases are positive, most ducts within a tumor are negative. The LOH for the loci inspected herein are so common that we would expect 100% of the samples to have LOH at some locus for heterozygotes of at least one polymorphism.

There are limitations in this study. Not all chromosomal arms that have been reported lost in the tumor-adjacent normal breast tissue are covered in the study. In addition, only breast tissue was tested in this study and caution should be exercised to other types of tissues.

In conclusion, the data provides sufficient evidence to justify the use of normal tissue adjacent to breast cancer tissue from paraffin-embedded tumor blocks for genotyping. It may be that these results are not applicable to other tumor types, and this might depend on the amount of surrounding stromal cells that are not cells of the same origin as the tumor.


   Acknowledgments
 
The authors thank Dr Zumei Feng and Leonidas D.Leondaridis for providing technical expertise. P.G.S was supported by the Department of Defense Breast Cancer Center of Excellence (DAMD17-03-1-0446); S.S.C. was supported by the Department of Defense Predoctoral student awards (DAMD17-03-1-0344); and B.X was supported by a fellowship from the Susan G. Komen Breast Cancer Foundation (PDF0402573).

Conflict of Interest Statement: None declared.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 2, 2005; revised August 7, 2005; accepted August 12, 2005.


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