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Clinical Chemistry 50: 500-508, 2004. First published January 15, 2004; 10.1373/clinchem.2003.025221
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(Clinical Chemistry. 2004;50:500-508.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Evaluation of Oligonucleotide Arrays for Sequencing of the p53 Gene in DNA from Formalin-Fixed, Paraffin-Embedded Breast Cancer Specimens

Melissa Cooper1, Shu-Qiu Li1, Tajinder Bhardwaj1, Thomas Rohan2 and Rita A. Kandel1,a

1 Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada.2 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY.

aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave. Suite 600, Toronto, Ontario M5G 1X5, Canada. Fax 416-586-8628; e-mail rkandel{at}mtsinai.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues.

Methods: DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip®; Affymetrix), and the results were compared with the results obtained from direct sequencing.

Results: DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016).

Conclusions: A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Carcinogenesis models suggest that tumor development is caused by the accumulation of mutations in genes that are critical for regulating cell growth (1)(2)(3)(4). The genetic alterations that lead to the development of breast cancer have yet to be clearly defined (5), but changes in the p53 gene are potentially relevant given that it codes for a protein that is involved in regulating the cell cycle, DNA repair, and apoptosis (6). The p53 gene is one of the commonly altered genes in breast cancer (7), and p53 mutation rates in breast cancer vary from 15% to 71% depending on the geographic population (8).

Benign breast disease (BBD) 1 is associated with an increased risk of developing breast cancer (9)(10), and it has been hypothesized that the genetic events that predispose to breast cancer development may also be present in some benign breast lesions. It has been shown that p53 protein can accumulate in BBD such as intraductal hyperplasia with or without atypia, fibroadenomas, fibrocystic disease, and fibrosis (11)(12)(13)(14)(15) as well as in healthy tissue (16) and phylloides tumors(17). Furthermore, p53 protein accumulation appears to be associated with a 2.5-fold increased risk of developing breast cancer in women with BBD (18). Mutations in p53 have also been discovered in healthy and benign breast tissue. One study showed that 2% of cytologically benign fine-needle aspirates had p53 mutations in the breast epithelium (15). Millikan et al.(19) showed that 8% of sporadic forms of BBD have p53 mutations. Another study using single-strand conformation polymorphism (SSCP) and sequencing analysis examined exons 4–10 of p53 and observed that 59% (16 of 27) of p53 immunopositive and 27% (4 of 15) of p53 immunonegative healthy breast tissue or tissue with BBD showed genetic alterations in p53 (16). These results demonstrate that genetic abnormalities can occur in preneoplastic breast lesions. Furthermore, it has been suggested that p53 alterations may be conserved during the development of breast carcinoma (20).

Retrospective studies using molecular analysis of paired tissue samples from the same patient, such as BBD and breast carcinoma, may help clarify the type and timing of molecular events that occur during breast cancer progression. Routine tissue processing has generated banks of formalin-fixed, paraffin-embedded (FFPE) tissue from patients that can be used in these types of studies. Although the yield of DNA from FFPE tissue is less [four times less than that from fresh tissue (21) and 30% of the amount that can be extracted from frozen tissue (22)] or can be fragmented (21)(23), many of these problems can be circumvented by PCR, which can amplify a small segment of DNA (23), suggesting that this tissue would be suitable for these types of studies.

Direct sequencing is commonly used for detecting p53 alterations (24), but this method is time-consuming and costly; therefore, other methods of screening for p53 changes have been developed, such as immunohistochemistry and SSCP analysis. Immunohistochemical detection of p53 protein can underestimate the frequency of p53 gene changes because not all sequence alterations lead to stabilization of the protein (25). It can also overestimate p53 gene changes if p53 accumulates for a reason other than mutation of the gene (26). The sensitivity of SSCP is altered by formalin fixation and paraffin embedding and has been shown to have a sensitivity of only 62% in detecting p53 mutations in DNA extracted from FFPE tissue (27) compared with a sensitivity of 90% in DNA extracted from frozen tissue (28).

An alternative method to evaluate the p53 gene is the p53 GeneChip® (Affymetrix), which uses oligonucleotide microarray technology to detect mutations (29). The chip contains >50 000 oligonucleotide probes, each of which is 25 nucleotides in length and synthesized by light-directed combinatorial chemistry (30). The probes were created to screen the sense and antisense strands of exons 2–11 for missense mutations, single-base deletions, and the splice sites of the human p53 coding sequence. The p53 GeneChip has been compared with direct sequencing for identifying p53 gene alterations in DNA extracted from frozen tissue of 108 ovarian cancers. In that study, the p53 GeneChip had a 94% accuracy rate, 92% sensitivity, and 100% specificity compared with 87% accuracy, 82% sensitivity, and 100% specificity for direct sequencing (31). In another study, the p53 GeneChip was also shown to be comparable to direct sequencing when DNA was extracted from frozen tumor tissue or blood (32). However, its ability to detect p53 gene alterations in DNA extracted from FFPE tissues is not known. A recent study using arrayed primer extension microarray suggested that it might be possible to assess DNA extracted from FFPE by microarray (33). The purpose of the present study was to determine whether the p53 GeneChip could be used to sequence the p53 gene in DNA extracted from FFPE breast cancer tissues. The results were compared with those obtained from direct sequencing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
tissue analyzed
The sequencing and screening methods were performed on paired DNA samples for each case in the study. Seventy breast cancer cases were studied, for which one paraffin block containing breast cancer tissue was selected from the Mt. Sinai Hospital archives for the years 1983–1995. The breast tissue had been fixed in 10% formalin and embedded in paraffin under standard conditions. The diagnosis of breast cancer was confirmed by reviewing representative sections stained with hematoxylin and eosin from each block. All evaluations were done in the absence of any identifying information.

preparation of dna
We cut 5-µm sections from the paraffin blocks, which were then dewaxed and stained briefly in hematoxylin. The cancer tissue was microdissected out, collected in a microcentrifuge tube, and digested with proteinase K [Gibco BRL; 0.5 g/L in 50 mmol/L Tris-HCl (pH 8.5), 10 mmol/L EDTA, 5 mL/L Tween 20] for at least 48 h at 55 °C. The proteinase K was inactivated by heating to 95 °C for 15 min. Eight of the 70 cases were discarded because of poor DNA yield. The DNA was then divided in two parts for analysis by direct manual sequencing and oligonucleotide microarray (p53 GeneChip).

p53 microarray
Aliquots of DNA were purified with a MiniElute Agarose Gel Purification Kit (Qiagen) according to the manufacturer’s protocol. The sample was eluted in 10–15 µL depending on the amount of tissue that had been microdissected. The DNA was amplified in a multiplex PCR as recommended by the manufacturer, and the primers are listed in Table 1 . Each 100-µL PCR included 1x PCR buffer (PE Biosystems); 2.5 mM MgCl2 (PE Biosystems); 0.2 mM each of dATP, dCTP, dGTP, and dTTP (Eppendorf); 1x p53 GeneChip primer set (Affymetrix); and 0.8 U of AmpliTaq Gold (PE Biosystems). The PCR was performed in a PE 9600 thermal cycler. A 5-µL aliquot of the multiplex PCR reaction mixture was visualized on a 10% polyacrylamide gel to confirm amplification of 10 PCR products of the correct size. These were then fragmented with DNase I and labeled with fluorescein-dideoxy-CMP. Each 50-µL fragmentation reaction included 45 µL of the multiplex PCR mixture, 0.005 U of fragmentation reagent [DNase I in 10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L CaCl2, 10 mmol/L MgCl2, 500 mL/L glycerol; Affymetrix], 0.03 mmol/L EDTA, 0.05 U of calf intestinal alkaline phosphatase (Roche), and 0.5 mmol/L Tris acetate, pH 8.2. The reaction was incubated for 15 min at 25 °C, followed by heat-inactivation of the enzyme at 95 °C for 10 min. To confirm the fragmentation, a 5-µL aliquot of the sample was visualized in a 2% agarose gel, which showed collapse of the 10 PCR products to fragments of ~50 bp.


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  		Table 1. PCR primers for p53 GeneChip analysis.

Each terminal labeling reaction contained 50 µL of the amplified and fragmented target, 1x reaction buffer, 1x CoCl2, 1x fluorescein-dideoxy-CTP, and 1x terminal deoxynucleotide transferase (all from Enzo Diagnostics). The reaction was incubated at 37 °C for 45 min, and 5 µL of 0.2 mol/L EDTA was added to stop each reaction. To confirm the labeling of the multiplex PCR product, a 3-µL aliquot of the sample was visualized on 2% agarose gel (UVP Gel DocSystem). DNA was hybridized to the p53 GeneChip, washed, and scanned (GeneChip Microarray Facility, Albert Einstein College of Medicine). Data analysis was performed with the Affymetrix Microarray Suite to generate a score for each sample. A score >=12 was considered indicative of a gene alteration. When an alteration detected by the p53 microarray was not confirmed by direct sequencing, the DNA underwent repeat PCR and processing to repeat the evaluation by p53 GeneChip.

direct manual sequencing
DNA was amplified by use of PCR and primers designed to contain each coding exon of p53 and the splice sites flanking each exon. The PCR primers and conditions are summarized in Table 2 . The 15-µL PCR included 1.5x PCR buffer; 1.5 mM MgCl2 (Qiagen); 1x Q solution (Qiagen); 0.1 mM each of dATP, dCTP, dGTP or dITP, and dTTP (Eppendorf); 0.3 µM the appropriate forward and reverse primers (Gibco BRL); 0.09 µCi of [{alpha}-33P]-dATP (Dupont NEN); and 0.07 U of Hotstart Taq (Qiagen) (16). The PCR products were visualized after agarose gel electrophoresis (1% gel; 30 min at 150 V). The amount of DNA was determined by comparing the PCR product with a low-mass DNA ladder (Gibco BRL). Products that were >50 ng were excised and purified by use of the QiaQuick Agarose Gel Extraction Kit (Qiagen). The purified PCR product was sequenced by use of the ThermoSequenase Radiolabelled terminator cycle sequencing reagent set according to the manufacturer’s protocol (Amersham Life Sciences). The sequencing reaction PCR was performed using the conditions described in Table 2 . After amplification, 4 µL of stop/loading buffer (950 mL/L formamide, 20 mmol/L EDTA, 0.5 g/L bromphenol blue, 0.5 g/L xylene cyanol blue) was added to each reaction, the sample was denatured for 3 min at 95 °C, and 2.5 µL was loaded on a 6% denaturing polyacrylamide gel (8.3 mol/L urea). The gel was processed for autoradiography (Biomax MR film; Kodak). If an alteration was detected, the DNA underwent repeat PCR and sequencing. The product of each sequencing reaction was compared with the p53 sequence provided by the International Agency for Cancer Research (IARC) database.


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  		Table 2. PCR primers and conditions for direct sequencing of exons 2–11.

The cases in which clear bands were not obtained for exons 5 and 10 were repeated with dITP instead of dGTP. For 22 samples, the exon 4 portion of the exon 3–4 partial PCR product was unreadable. For these samples, PCR and sequencing were performed with primers that were specific for exon 4 only to read the 3' portion of exon 4 (Table 2Up ). If separation was poor for any of the exons, that exon was sequenced in the reverse direction.

statistical analysis
The McNemar test was used to determine whether there were differences between methods in their ability to detect p53 changes. The resulting test statistics were referred to a {chi}2 distribution (df = 1). All statistical analyses were performed with the SigmaStat program. P <=0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
direct sequencing
In the 62 samples analyzed, there were a total of 43 genetic alterations detected by direct sequencing in 35 individuals. These consisted of 26 polymorphisms in 26 individuals and 33 mutations in 17 individuals. An additional 24 alterations were detected in introns, sequences that are not on the microarray and therefore were eliminated from further analysis. Twenty-four of the samples were identified as wild type for all exons. No changes were detected in exons 2, 3, 9, 10, and 11.

Of the 26 polymorphisms, 25 were a G->C change in exon 4 at codon 72 (Fig. 1 ), and 1 was an A->G change in exon 6 at codon 213 (see Table 1Up in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol50/issue3/). The frequencies of polymorphisms in exons 4 and 6 were 40% (25 of 62) and 2% (1 of 62), respectively.



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  		Figure 1. Sequencing gels showing samples that are wild type (A) or have the G->C nucleotide change (B and C) indicative of the polymorphism at codon 72 (exon 4).

The sample in C is homozygous for this polymorphism.

Mutations were identified in 17 cases, giving a mutation frequency of ~27% (17 of 62). This may be an underestimation because not all exons in all samples could be sequenced for technical reasons (see below). The details of the 17 mutations are summarized in Table 3 , which shows that the changes consisted of single nucleotide base pair changes in 16 of 17 cases. Three individuals had a single nucleotide base pair change at splice sites (intron 5 at nucleotide position 13239, intron 6 at nucleotide position 13432, and intron 7 at nucleotide position 14451). An insertion was detected in one case. Eight cases had a polymorphism in exon 4 as well as a mutation elsewhere.


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  		Table 3. p53 mutations.

No PCR product could be obtained for six samples for exon 2, eight samples for exon 3, and one sample for exon 11. Eleven changes in exon 2 and 8 changes in exon 3/portion of exon 4 could not be confirmed because after multiple attempts no repeat PCR product could be generated. These samples were not included in the analysis for these exons. There were 12 changes that were identified by the first round of PCR and direct sequencing that were not confirmed in the repeat analysis, and these cases were considered negative.

p53 microarray
A total of 26 genetic alterations were identified in 24 cases by the p53 GeneChip, giving a mutation frequency of 40% (24 of 62; Table 3Up ). There were 22 cases with one change only and 2 cases with two changes. A 2-bp deletion was detected for one case (1.11) as indicated by a single deletion at two contiguous nucleotides, each with a score of 13. Fifteen of the 26 changes were also detected by direct sequencing. Thirty-seven samples were identified as wild type for all exons. No polymorphisms were detected. There were no changes detected in exons 2, 3, 4, 9, 10, or 11.

A total of 21 samples had selected exons that could not be analyzed for technical reasons. These included exon 4 (20 cases), exon 5 (4 cases), exon 9 (2 cases), intron 10 (4 cases), exon 10 (2 cases), and exon 11 (3 cases). Twenty-four changes could not be confirmed on repeat analysis, and these were considered wild type.

Microarray analysis failed to detect two mutations identified by direct sequencing; an insertion in exon 6 and a single base pair change in exon 4. In the latter case, exon 4 did not amplify sufficiently and therefore could not be analyzed by the p53 GeneChip; it thus is not a true negative.

comparison of direct sequencing and p53 GENECHIP results
There were nine missense mutations and two deletions identified and confirmed by the p53 GeneChip that were not identified by direct sequencing (Fig. 2 ). The sequencing gels were re-reviewed for these 11 cases. In one case the change had been missed initially because of high background. Although this change could be seen in retrospect, it was not included as a positive case in the data analysis because background is a factor in interpreting direct sequencing gels. The remaining 10 mutations still could not be detected. For these cases, the PCR and direct sequencing were repeated again, and all but two cases were still negative. The two cases that became positive on repeat analysis showed an abnormal sequence pattern at the same position as identified by the microarray (codon 249, exon 7). In the other case (single base pair change at codon 144 in exon 5), the change would have been considered background without the p53 GeneChip information and therefore was still considered a negative for the statistical analysis.



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  		Figure 2. Sequencing gel showing the wild-type sequence of sample 1.36 for codon 144 (exon 5).

The p53 GeneChip had showed a mutation in the region, indicated by * (CAG->CTG).

The ability to detect different types of mutations varied between the two sequencing methods. The p53 GeneChip showed a higher mutation detection rate for missense mutations (100%; 19 of 19) than direct sequencing (52%; 10 of 19). Although the numbers are small, the two sequencing methods were able to identify other types of mutations equally well. This included nonsense and splice junction mutations with mutation detection rates of 100% (2 of 2 and 3 of 3, respectively) for direct sequencing and the p53 GeneChip.

The ability of each method to identify changes in each exon/intron was calculated by determining the number of alterations detected by the individual method relative to the combined number of confirmed alterations detected by direct sequencing and the p53 GeneChip. This approach was taken because each method appeared to identify some different alterations. Direct sequencing detected 80% of total changes (43 of a total of 54 possible alterations), and the p53 GeneChip detected 48% (26 of a total of 54 possible alterations). To determine whether oligonucleotide microarray analysis or direct sequencing is better at identifying p53 alterations, the proportion of cases that showed alterations by each method was calculated together with the corresponding 95% confidence intervals. Direct sequencing was significantly better than the chip in detecting all alterations (P = 0.018). However, if exon 4 results were eliminated from the analysis because the PCR product for this exon could not always be amplified, then the p53 GeneChip detected more mutations (26 of 27 mutations) than direct sequencing (16 of 27 mutations; P = 0.016).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we showed that DNA extracted from FFPE tissue can be used for p53 GeneChip analysis. Under our conditions, direct sequencing detected significantly more gene alterations than the p53 GeneChip. This difference could be attributed in part to the difficulty in reliably amplifying exon 4 with the primers provided with the GeneChip. The product generated for this exon is the largest (366 bp) of the 10 PCR products, and because of its size was frequently not successfully amplified (20 of 62 cases) and limited the changes that could be detected by the p53 GeneChip for this exon. This was not a problem for direct sequencing because we designed multiple primer sets to allow for amplification of smaller fragments of exon 4, which appeared to eliminate the problem of amplifying a single large product from DNA extracted from FFPE. This can not be done for the microarray because the system is optimized for a multiplex reaction. When the results for exon 4 were excluded from the analysis, the p53 GeneChip detected 37% more alterations, most of which were missense mutations, than direct sequencing which is an important finding when choosing the optimum method for p53 gene analysis. This observation is in keeping with the results of other studies, which showed that oligonucleotide microarray evaluation of the p53 gene in tissues that were not formalin-fixed and paraffin-embedded had a sensitivity that was equal to or better than that of direct sequencing (31)(32).

There are several reasons for the equivalent or superior performance of microarray evaluation (after exclusion of exon 4). One reason is that the array analysis may be less affected than direct sequencing by the fragmentation of DNA caused by the processing of the tissue because the PCR products undergo a step in which the product is reduced to 50 bp in preparation for the p53 GeneChip analysis. Another reason is that it is possible that we did not design the optimum primers for direct sequencing that would identify all abnormalities because this likely would require multiple primer sets and an unlimited supply of DNA. A third reason is that it has been shown that direct sequencing can only detect a mutation when at least 30% of the total DNA is mutant (34), whereas the p53 GeneChip can detect mutations if <2% of cells are altered (35). Finally, structural reasons might also explain why some alterations were not detected by direct sequencing. DNA sequences containing GC-rich regions may not denature completely during electrophoresis because of the strong hydrogen bonds that form between these two nucleotides. Incomplete denaturing of the strands can disturb the migration pattern of the DNA fragments and cause bands to compress. Although dGTP was replaced with dITP, a nucleotide analog that forms weaker bonds with dCTP than dGTP, this substitution can lead to other problems. For example, one consequence of using dITP is that it may impair the ability of direct sequencing to identify heterozygous changes because dITP gives less uniform band intensities (36). Because one-half of the changes missed by direct sequencing were in exon 5, the exon for which dITP was used most frequently, it is possible that it compromised our ability to identify changes in small populations of cells.

Other factors should be considered when comparing these two methods. The GeneChip cannot detect intronic changes outside of the splice sites, and this could be a limitation in a study in which it is necessary to evaluate intronic DNA sequences. The GeneChip also cannot detect insertions and large deletions. In addition, use of the arrays is costly. However, the p53 GeneChip analysis takes approximately one-fifth of the time that it takes to perform direct sequencing, giving cost savings through a reduced need for personnel.

The p53 GeneChip analysis and direct sequencing had similar results in terms of the number of nonreproducible changes. No similarities were seen in the nonreproducible changes identified by either sequencing method. False positives are expected because both sequencing methods are PCR based and changes that can not be confirmed are most likely attributable to PCR artifacts. Taq polymerase has been shown to misincorporate 1 bp in every 10 000 bp (37) or as many as 1 in 500 bp in DNA extracted from small amounts of FFPE tissues (38). Our results would suggest that, similar to direct sequencing, all samples considered to have a gene alteration by microarray analysis should undergo repeat PCR and GeneChip analysis.

An unexpected limitation identified in this study was the inability of the oligonucleotide microarray to detect polymorphisms. There were 26 polymorphisms identified by sequencing. Of these, 13 were present in samples for which exon 4 could not be analyzed. In the case with a polymorphism in exon 6, the p53 GeneChip had a score of 6, which according to our cutoff criteria indicated a negative result. However, if there was alternative tiling for this change present on the chip, our cutoff score may have been too high (Affymetrix; personal communication). It has been suggested by Wikman et al. (35) that fixed cutoff scores may not be appropriate and that the amount of background present for each probe should be factored into the determination of the cutoff value rather than having a single score for all exons. However, it is not clear why, in the 12 cases in which exon 4 could be examined, the polymorphisms were not detected by the p53 GeneChip. This is a particularly unexpected finding because the polymorphism would be present in at least 50% of the cells if it were a heterozygous change. It is possible that the secondary structure of the gene in this region affects the ability of the DNA to hybridize to the chip (39). Because exon 4 has a 78% G:C content and the region in and around codon 72 is GC rich, the formation of stable secondary structures may explain why the p53 GeneChip did not detect this polymorphism under our conditions. Another study has reported detection of polymorphisms by the oligonucleotide microarray (31), but in that study the DNA was extracted from frozen tissue and a lower score was used to indicate the presence of an alteration. The authors did not state which polymorphisms were detected, and it is possible that the polymorphisms were different because the authors evaluated ovarian cancers and not breast cancers.

The chip identified four samples with a mutation in codon 144 that produced a glutamine-to-leucine change. This change is present in the p53 database but has only been detected in lymphoma and cancers of the prostate, brain, and pancreas. This mutation was not identified by direct sequencing in any of the cases, perhaps because it was present in too few of the cells. Alternatively, it is possible that it represents an artifact of the chip detection method.

It could be argued that direct sequencing of DNA extracted from frozen (or fresh) tissue should be the standard against which DNA extracted from FFPE tissue and analyzed by p53 GeneChip should be evaluated. It may be that the use of FFPE tissue in this analysis generated false positives and negatives, a particular problem when the amount of tissue to be analyzed is limited (38)(40). However, the purpose of this study was to evaluate these methodologies in this type of processed tissue. Furthermore, there have been several studies suggesting that in practice, artifacts introduced by FFPE may not be a major problem because similar genetic alterations were detected in DNA extracted from FFPE tissue compared with DNA extracted from frozen tissue from the same tumor (41)(42)(43)(44)(45). Furthermore, all of the exonic mutations identified in this study were in the database of p53 mutations maintained by the IARC (www.iarc.fr/p53). Direct sequencing detected mutations in ~27% of cases, a finding in keeping with the reported frequencies of p53 mutations in breast cancer, which vary between 15% and 71% (8). The IARC database shows that there is a wide range of frequencies for polymorphisms at codon 72, and the frequency of this polymorphism in the current study was within this range (46). Similarly, the frequency of the codon 213 polymorphism is up to 11% in this database, and our results were in keeping with this. The absence in these cases of insertions/large deletions, changes that are known to occur in breast cancer, may reflect the detection method, but it is more likely that the absence of this change is related to the size of the study. The proportion of p53 mutations in breast cancer that are deletions and insertions is relatively low, 8% and 3%, respectively (47).

Furthermore, several technical precautionary steps were taken in the current study to minimize the possibility of over- and/or undercalling of sequence abnormalities. Every case with a possible alteration was confirmed by a repeat PCR, and it is very unlikely that the same artifact would occur in two separate reactions. In addition, it has been shown that if the tissue is digested sufficiently and the products are small, this will minimize PCR artifacts (48). In this study, all PCR products were <300 bp in length except for those for exon 4.

In conclusion, direct sequencing detects more alterations in DNA extracted from FFPE breast cancer tissue than the p53 GeneChip. However, if exon 4 is eliminated from the evaluation, the p53 GeneChip detects significantly more mutations than direct sequencing. At present it would seem most appropriate to screen the p53 gene with the p53 GeneChip. In those cases in which exon 4 cannot be evaluated by the GeneChip or in which there is a need to sequence introns, these can be done by direct sequencing. The microarray cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.


   Acknowledgments
 
We thank N. Tran for help in performing the microarray hybridization and data collection in the GeneChip Microarray Facility at the Albert Einstein College of Medicine. We also thank Isabelle Schell for technical assistance. This work was supported by US Army Materiel Research Grant DAMD 17-99-1-9310 and the NIH.


   Footnotes
 
1 Nonstandard abbreviations: BBD, benign breast disease; SSCP, single-strand conformation polymorphism; FFPE, formalin-fixed, paraffin-embedded; and IARC, International Agency for Cancer Research.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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