REPORT

p53 Missense Mutations in Microdissected High-Grade Ductal Carcinoma In Situ of the Breast

Susan J. Done, Sasha Eskandarian, Shelley Bull, Mark Redston, Irene L. Andrulis

Affiliations of authors: S. J. Done, M. Redston (Department of Laboratory Medicine and Pathobiology and Samuel Lunenfeld Research Institute), S. Eskandarian (Samuel Lunenfeld Research Institute), S. Bull (Department of Public Health Sciences and Samuel Lunenfeld Research Institute), I. L. Andrulis (Departments of Molecular and Medical Genetics and Laboratory Medicine and Pathobiology and Samuel Lunenfeld Research Institute), Mount Sinai Hospital, Toronto, ON, Canada.

Correspondence to: Irene L. Andrulis, Ph.D., Samuel Lunenfeld Research Institute, Rm. 984, Mount Sinai Hospital, 600 University Ave., Toronto, ON, Canada, M5G 1X5 (e-mail: andrulis{at}mshri.on.ca).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: To understand the role of sporadic mutations in the tumor suppressor gene p53 (also known as TP53) in the pathogenesis of breast cancer, it is important to identify at which histologic stage such mutations first occur. We previously showed that a p53 mutation present in invasive breast cancer was found in all surrounding areas of ductal carcinoma in situ (DCIS) but not in areas of hyperplasia or normal breast epithelium. In the present investigation, we studied patients with DCIS, but without invasive breast cancer, to determine the spectrum of DCIS types that can harbor a p53 mutation. Methods: Formalin-fixed, paraffin-embedded tissues from 94 patients with DCIS were evaluated histologically for the predominant cellular architectural pattern, degree of necrosis, and nuclear grade. Each specimen was also assigned an overall histologic grade (with the use of the Van Nuys Prognostic Index pathologic classification). Tissue specimens were stained immunohistochemically with an anti-p53 antibody. Positively stained tissue areas were analyzed for the presence of p53 mutations by single-strand conformation polymorphism and direct sequencing. All statistical tests were two-sided. Results: DCIS from 10 of 94 patients were found to contain p53 missense mutations. All 10 were of a solid or a comedo histologic pattern and contained cells of nuclear grade 2 or 3 (i.e., more abnormal nuclei). The frequency of p53 missense mutations was statistically significantly different among the three overall histologic grade categories (zero [0%] of 49 with low-grade DCIS, one [4.35%] of 23 with intermediate-grade DCIS, and nine [40.9%] of 22 with high-grade DCIS; df = 2 and P<.0001). Conclusion: The DCIS types in patients in this series are representative of clinically detected DCIS. Our finding that p53 mutations can occur before the development of invasive breast cancer, particularly in DCIS of high histologic grade, has potentially important implications for prevention and treatment.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mutation of the tumor suppressor gene p53 (also known as TP53) is one of the most frequent somatic alterations in sporadic breast cancer. Our earlier studies (1,2) indicate that p53 mutation occurs at least as early as ductal carcinoma in situ (DCIS). However, the following questions remain: If a p53 mutation is present in DCIS, did it occur before the advent of invasion or does it merely represent invasive carcinoma that has breached the basement membrane, gained access to the ductal system, and spread along it? Several small studies (310) have identified mutations in DCIS, but these studies are mainly confined to frozen tissues. It seems likely that studies on frozen tissue consist of predominantly large, grossly visible cases of DCIS. In most patients, the DCIS is currently detected mammographically as small areas of suspicious microcalcifications giving rise to microscopic areas of DCIS that cannot be detected grossly. To understand the role of p53 mutation in the spectrum of DCIS observed clinically, it is necessary to look at p53 mutations in formalin-fixed, paraffin-embedded DCIS tissues. Only four mutations leading to amino acid alterations have been described in formalin-fixed, paraffin-embedded DCIS tissues (9,10); three of these four were observed in comedo-type DCIS.

To assess the mutational status of a large group of formalin-fixed, paraffin-embedded DCIS tissues not selected on the basis of size and thus without an invasive component, it was necessary to devise a screening strategy. It is technically challenging to perform mutational analysis of exons 4 through 10 because of the poor quality of the tissue material and the small size of many of the lesions seen. Because loss of a tumor suppressor gene may give in situ carcinoma a growth advantage, allowing it to progress to invasion, the frequency of p53 mutation in solitary DCIS is likely to be lower than the frequency of 15%–50% detected in invasive cancers (11). We have found p53 missense mutations to stain strongly with a widely used anti-p53 antibody (DO7 clone; Novocastra Laboratories Ltd., Newcastle, U.K.) (2). For these reasons, tissues of patients with DCIS were screened first with immunohistochemistry to identify those cases that were likely to harbor missense mutations. In this way, we were able to perform mutational analysis only in those cases with a high likelihood of containing a detectable mutation.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Selection of Patients

Formalin-fixed, paraffin-embedded tissue sections from all patients with DCIS without invasive cancer occurring during the period from January 1993 through August 1998 were retrieved from the surgical pathology files of Mount Sinai Hospital, Toronto, ON, Canada. Patient consent was not required because patient identifiers were removed and no follow-up information was obtained. One pathologist (S. J. Done) reviewed all surgical pathology slides on each case, and the block containing the greatest extent of DCIS, by surface area, was selected for immunohistochemical staining. Tissues from patients for which only a core-needle biopsy was available were excluded on the basis of insufficient material for mutational analysis and the fact that all patients having DCIS on a core biopsy are then referred for excision of the lesion. Patients with histologic evidence of recent surgery were excluded because there may have been an associated invasive carcinoma resected in a previous specimen. Patient identifiers were removed from the samples before analysis. For each tissue sample, the predominant architectural pattern of DCIS (i.e., comedo, solid, cribriform, micropapillary, or papillary intracystic) was noted. The degree of necrosis and nuclear pleomorphism was assessed for each tissue specimen semiquantitatively on a 3-point scale (1–3 points). Each specimen from the patient was also assigned an overall histologic grade that incorporated the scores for nuclear grade and degree of necrosis. The Van Nuys Prognostic Index (VNPI) pathologic classification was used as described by Silverstein et al. (12).

p53 Immunohistochemistry

Immunohistochemistry was performed with an anti-p53 antibody (DO7 clone). Sections were stained in the Department of Pathology and Laboratory Medicine at Mount Sinai Hospital. Formalin-fixed, paraffin-embedded tissue sections (5 µm thick) were placed on slides coated with aminopropyltriethoxysilane (Sigma Chemical Co., St. Louis, MO), which prevents sections from falling off the slides during processing. Sections were immersed in a citrate buffer (10 mM, pH 6.0) and heated to boiling in a microwaveable pressure cooker. Endogenous peroxidase activity was inactivated with hydrogen peroxide (3% solution in water), and the sections were treated with a protein blocker (Signet Laboratories Inc., Dedham, MA) to minimize background staining. The selected sections, together with appropriate controls, were incubated for 1 hour with a mouse monoclonal antibody to p53 (DO7 clone). After being washed, the sections were incubated with biotinylated horse anti-mouse immunoglobulin G, followed by the avidin–biotin peroxidase complex (Elite kit; Vector Laboratories, Burlingame, CA), and developed with diaminobenzidine (Sigma Chemical Co.). Sections were counterstained with hematoxylin. One observer (S. J. Done) reviewed the histopathology of all of the patients' tissues. For each tissue specimen, the proportion of positively staining cells was estimated, and the intensity of staining was rated semiquantitatively on a 4-point scale (0 = no staining, 1 = light staining, 2 = staining of moderate intensity, and 3 = intense or maximum staining). The maximum intensity of staining was noted for each tissue specimen.

Microdissection and DNA Extraction

Areas of intense p53 staining (scoring 3/3, i.e., score of 3 out of maximum possible score of 3 or maximum intensity staining ) were selected for microdissection. The microdissections and the tissue processing were done as described previously (1). For each selected tissue area, approximately 20–40 000 cells were manually microdissected, transferred to a microtube, and digested with Proteinase K (Qiagen, Hilden, Germany) for 72 hours.

Mutational Analysis

Mutations in exons 4 through 10 of p53 gene were detected by single-strand conformation polymorphism (SSCP), and any shifted bands were excised from the gel and sequenced. Reagents in each SSCP reaction included the following: 0.6 mM deoxynucleoside triphosphates, 0.3 µM of each polymerase chain reaction (PCR) primer, 2.0 µCi [{alpha}-33P]deoxyadenosine triphosphate (2000 Ci/mmol), 1 U Advantage 2 Taq DNA polymerase (Clontech Laboratories, Inc., Palo Alto, CA), and Advantage 2 PCR Buffer (Clontech Laboratories, Inc.). Annealing temperatures were optimized for each primer pair (Table 1Go). DNA in 2–5 µL of digested tissue sample was amplified in a reaction volume of 30 µL with the use of a 9600 Thermal Cycler (The Perkin-Elmer Corp., Foster City, CA). Reaction conditions were 94 °C for 15 seconds, annealing temperature for 15 seconds, and 72 °C for 20 seconds. The number of reaction cycles varied between 35 and 45, depending on the quality of the tissue sample and the guanosine–cytosine content of the primer pair. Sequencing was performed with the ThermoSequenase cycle sequencing kit (Amersham Pharmacia, Baie d'Urfé, Québec, Canada).


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Table 1. Primers used for p53 mutational analysis of specimens with ductal carcinoma in situ
 
All mutations detected were confirmed by microdissecting additional cells from DCIS lesions from consecutive sections on a separate occasion and by sequencing the exon containing the mutation.

Statistical Analysis

The frequency of p53 mutations was compared among the three histologic grades of DCIS with the use of two-sided exact tests for independence in two-by-three and two-by-two tables. The Cochran–Armitage test for trend with grade was also performed with exact calculation of a two-sided P value, odds ratio, and 95% confidence interval (CI).


    RESULTS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Identification of DCIS

Specimens from 103 patients were confirmed to contain DCIS by histologic review of the original surgical pathology slides. DCIS was also detected in specimens from 94 of these patients after immunohistochemical staining with an anti-p53 antibody. In tissues of nine patients, the original area of DCIS had been cut through and was not present in the sections submitted for immunohistochemistry. The patients' specimens displayed predominantly a solid architectural pattern, with a cribriform pattern being the second most commonly seen pattern (Table 2Go). These specimens included a range of grades of DCIS and are representative of the spectrum of DCIS seen in routine clinical practice.


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Table 2. Description of ductal carcinoma in situ in breast specimens from 94 patients and their immunohistochemical staining patterns with the anti-p53 antibody DO7
 
p53 Immunohistochemical Staining

The analysis of DCIS in immunohistochemically stained sections of tissue specimens from 94 patients is shown in Table 2Go. Specimens from 16 patients showed maximum intensity staining (grade 3/3). This level of staining intensity was previously demonstrated in our laboratory to be associated with missense mutations (2). The proportion of DCIS cells staining with grade 3/3 intensity in these 16 patients ranged from 1% to 100%.

Specimens from 11 patients with greater than one third of cells having grade 3/3 staining intensity were selected for mutational analysis. In one of these patients (#2), staining with grade 3/3 intensity was observed in 100% of cells in only a single duct, and the other ducts in this case had staining of moderate intensity (grade 2/3) (Fig. 1, A, B, and CGo). Panels B and C of Fig. 1Go are photographs taken from areas close to those marked in panel A but are not the exact same areas marked in panel A. All photographs are taken from sections of the same tissue block, and panels B and C are from the areas of the ducts indicated. Panel A is a photograph of an immunohistochemically stained section, and panels B and C are photographs of a section stained with hematoxylin–eosin. The single duct with maximal intensity staining was microdissected and analyzed separately from the other ducts. The cells in the positively staining duct were notable for having a higher nuclear-to-cytoplasmic ratio than the cells in the surrounding ducts as well as slightly higher grade nuclei.



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Fig. 1. Area of ductal carcinoma in situ in specimen from patient #2 with p53 mutation found in a single duct with higher grade cellular features. Part of the immunohistochemically stained (with anti-p53 DO7 clone) section showing a duct containing cells stained at grade 3/3 intensity is shown in panel A. Panel B is a higher power view of the same duct displaying a staining intensity of grade 3/3 in 100% of cells. Panel C is a higher power view of an area of the surrounding ducts displaying an immunohistochemical staining intensity of grade 2/3 in 100% of cells of lower grade features. Panels B and C are photographs of a section adjacent to that shown in panel A and are stained with hematoxylin–eosin.

 
Mutational Analysis

p53 mutations were found in specimens from 10 of the 11 patients that showed intense staining in more than one third of the cells (Table 3Go). The percentage of positive cells did not influence the likelihood of finding a mutation, since we were able to identify mutations in patients' tissues in which 35%, 70%, 80%, and 90% of cells stained strongly. Eight of the 10 samples found to contain missense mutations showed high histologic grade DCIS; the other two showed intermediate grade. The DCIS showed either a comedo or a solid pattern. In eight specimens with a mutation, all of the original surgical pathology slides were available for review. The DCIS in these patients' specimens ranged in greatest extent from 2.8 to 10.5 mm (extent refers to the distance between the outer edges of the furthest apart ducts containing DCIS) on a single slide and involved from three to six blocks. The longest continuous length of duct involved by DCIS ranged from 1.0 to 2.6 mm. The greatest number of ducts involved by DCIS in one patient found to contain a mutation ranged from four to more than 100.


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Table 3. Mutations found in ductal carcinoma in situ (DCIS)
 
In one patient, we were not able to investigate the presence or absence of a p53 mutation because of poor amplification of the archival formalin-fixed, paraffin-embedded material. No mutation was identified in the ducts in the specimen from patient #2 in which staining of intermediate intensity (score 2/3) was seen, although a mutation was found in a single duct with cellular features of higher grade. As an additional control, a specimen from a patient in which 20% of DCIS cells stained with a maximal intensity of 2/3 was analyzed; no p53 mutation was found.

The frequency of p53 missense mutations differed statistically significantly among the three histologic grade categories (0 [0%] in 49 in low-grade DCIS, one [4.35%] in 23 intermediate-grade DCIS, and nine [40.9%] in 22 in high-grade DCIS) by an overall 2-df test for independence with a P value of <.0001. The mutation frequency is statistically significantly higher for high-grade DCIS than for intermediate-grade DCIS and for high-grade DCIS than for low-grade DCIS (exact P values of .004 and .0001, respectively). The Cochran–Armitage 1-df test for trend with grade was also statistically significant (P<.0001), with an odds ratio estimate of 12.2 (95% CI = 2.48 to 414.2).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To our knowledge, this is the largest series of DCIS without invasive breast cancer that has been screened for mutations in the p53 gene to date. We chose to study a series of archival clinical samples that had been routinely processed because we wanted to look at a series that contained the spectrum of types and grades of DCIS seen in routine clinical practice. After immunohistochemical staining, p53 missense mutations were found in predominantly high histologic grade DCIS from 10 of 94 patients.

The tumor suppressor gene, p53, has an important role in protecting the integrity of the cellular genome. Loss of functional p53 can result in apoptosis or dysregulated growth. Cells with aberrant karyotypes may be allowed to continue to grow and to divide. It is perhaps not surprising that, in this series, mutations were found only in cases of intermediate and high histologic grades. However, in our previous study (1) of "early" lesions (hyperplasia and DCIS) surrounding invasive cancers known to harbor p53 mutations, we were able to find mutations in a broader spectrum of grades of DCIS (low histologic grade as well as high histologic grade). Despite similar p53 missense mutation frequencies in DCIS without invasion, as are found in invasive breast cancer, there appears to be a relative reduction in p53 mutation frequency in low histologic grade DCIS without invasion. This suggests that, if a p53 mutation occurs in low-grade DCIS, the growth advantage afforded may allow more rapid progression to invasion. The relative growth advantage to high-grade DCIS may be less. Larger studies will be needed to investigate this hypothesis further and to determine whether these breast cancer patients have a poorer prognosis.

The link between p53 mutation and higher histologic grade was further emphasized by the presence in patient #2 of a p53 mutation in a single duct that contains higher grade cells (higher nucleusto-cytoplasm ratio) than the surrounding ducts with wild-type p53. Another group of investigators (9) also described the presence of p53 mutations in some, but not all, ducts of DCIS within a given patient's specimen. In our previous series of "early" lesions surrounding invasive cancers known to harbor p53 mutations, focal, intense immunohistochemical positivity was seen in a patient with micropapillary DCIS with a small focus of invasive cancer (2). This mutational heterogeneity suggests that p53 mutation may occur during the stage of DCIS. Mutations have also been reported to occur prior to DCIS in normal breast epithelium (13); however, in an earlier study of p53 mutations (1), we were unable to detect p53 mutations in any areas of hyperplasia or normal breast epithelium.

In this group of patients with DCIS but without invasive breast cancer, we found a frequency of p53 missense mutations of 10 (10.6%) of 94, similar to the frequency of p53 missense mutations found by our group in invasive breast cancers (Özçelik H, Andrulis IL: unpublished data). The data indicate that p53 mutations can occur before invasion.

This study of a large group of clinically representative DCIS lesions, not associated with invasive breast cancer and comprising a spectrum of types and grades, found an appreciable frequency of missense mutations, suggesting that mutations occur prior to invasion. Furthermore, most of the mutations were found in DCIS of high histologic grade, which has been shown to behave more aggressively than low-grade DCIS. The presence of a p53 mutation in DCIS may have prognostic value; in addition, it may be useful for further refining the classification of DCIS.


    NOTES
 
Present address: S. J. Done, OCI/PMH-University Health Network, Toronto, ON, Canada.

Supported in part by grants from the Canadian Breast Cancer Research Initiative and the National Cancer Institute of Canada (to M. Redston and I. L. Andrulis). S. J. Done was a Research Fellow of the National Cancer Institute of Canada supported with funds provided by the Terry Fox Run.

We thank the surgeons and pathologists of Mount Sinai Hospital (Toronto, ON, Canada) for the specimens and Kelvin So and Suzanna Tjan in the Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, for their excellent technical assistance.


    REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received October 19, 2000; revised January 19, 2001; accepted February 28, 2001.


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