Immunohistochemical expression of the mutant p53 protein and nuclear DNA content during the transition from benign to malignant breast disease

lmmunohistochemical Expression of the Mutant ~53 Protein and Nuclear DNA Content During the Transition From Benign to Malignant Breast Disease ELINA T. ERIKSSON, MD, HENDRIK SCHIMMELPENNING, MD, ULLA ASPENBLAD, BS, ANDERS ZEllERBERG, MD, PHD, AND GERT U. AUER, MD, PHD Immunohistochemical

expression

of the cellular phosphoprotein

was investigated in archival, formalin-fixed,

p53

and paraffin-embedded

surgical breast tissue specimens from 543 patients using the polyclonal antibody CM-l.

Cytometric DNA assessments were performed

on histopathologically or cytopathologically identified cell nuclei using image aualysis. The series included five samples of normal resting breast parenchyma, 35 benign lesions including benign tumors, 54 hyperplastic lesions with and without atypia, 109 carcinomas in situ, and 340 invasive adenocarcinomas. In 56 of the latter cases specimens from corresponding

lymph node metastases also were investigated.

Mutant p53 protein expression was absent in normal resting parenchyma and in benign lesions, including benign tumors and epithelial hyperplasias. However,

14 of the 54 hyperplasias (26%) were found

to be of DNA aneuploid type. Thirteen of 109 (12%) carcinomas in situ and 79 of 340 (23%) invasive neoplasms expressed the mutant p53 protein. Eight of nine (89%) p53 immunoreactive carcinomas in situ and 62 of 78 (80%) invasive carcinomas with p53 expression were DNA

aneuploid. In invasive carcinomas p53 expression was absent

in well differentiated neoplasms. Jn contrast, 58 of 158 (37%) poorly differentiated invasive carcinomas immunoreacted. Intraductal carcinomas of comedo type and poorly differentiated invasive carcinomas of comedo type expressed the mutaut p53 protein in seven of 18 cases (39%)

and in 14 of 22 cases (64%), respectively. The stain-

ing behavior of lymph node metastases was the same as that of the corresponding

primary tumors. The present lindings suggest that

chromosomal

alterations as indicated by DNA

precancerous

lesions. However,

aneuploidy occur in

immunohistochemically

detectable

accumulation of mutant p53 protein cannot be observed before the carcinoma in situ phase. The highest levels of p53 protein overexpression are found in invasive carcinomas and are closely associated with aneuploidy pression

and poor

differentiation.

However,

apparently does not increase further

p53 overex-

during

spread to lymph nodes. HUM PATHOL 25:1228-1233.

the tumor

Copyright 0

1994 by W.B. Saunders Company

The development of human cancer is a complex process in which genetic alterations play an important role. More knowledge about chromosomal aberrations and specific gene functions might improve our understanding of tumor growth and progression. From the Department of Tumor Pathology, Karolinska Hospital and Institute, Stockholm, Sweden: and the Department of General Surgery. Johann-Wolfgang Goethe University, Frankfurt, Germany. Accepted for publication April 8, 1994. Supported by the Swedish Medical Research Council, The Swedish Cancer Society, the Cancer Society of Stockholm, the King Gustav V Jubilee Fund, and the Research Funds of the Faculty of Medicine at the Karolinska Institute, Stockholm, Sweden. ZL+ words: mammary neoplasms, DNA cytometry, ~53. Address correspondence and reprint requests to Elina T. Eriksson, MD, Department of Tumor Pathology, Karolinska Hospital and Institute, S-104 01 Stockholm, Sweden. Copyright 0 1994 by W.B. Saunders Company 0046-8177/94/2511-0013$5.00/0

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Mutations of the p53 suppressor gene seem to be among the most common genetic alterations in human malignant tumors.‘.’ The p53 gene was first identified by Lane3 and is localized on the short arm of chromosome 17.” The gene encodes a 53-kd nuclear phosphoprotein that is expressed in all normal cells at low levels. Wild type p53 protein does not accumulate because of a short half-life of 20 to 30 minutes. Thus, it is virtually undetectable by means of immunohistochemistry. Mutant p53 protein, in contrast, has a significantly longer half-life, does accumulate in the nucleus, and thus can be stained with immunohistochemical methods. The function of the normal p53 protein is still not entirely clear, but it appears to inhibit the progression of cells from the G, to the Sphase.s One of the mechanisms causing alterations of the cellular genotype is allele loss on the short arm of chromosome 17, often accompanied by a mutation of the remaining allele.’ The tumor suppressor gene p53 was first associated with mammary carcinomas in 1982 when Crawford et al’ reported that 9% of a series of breast cancer patients had circulating p53 protein antibodies. Since then both monoclonal and polyclonal antibodies a ainst the hu&I By use of man p53 protein have been generated. these antibodies evidence has been obtained that overexpression of the p53 protein is significantly associated with tumor cell dedifferentiation”-IS and even with cell proliferation in human mammary carcinomas.16.17 In several studies an association between p53 overex ression and prognosis in breast cancer was foundJ.‘.?~‘7 Crude alterations of nuclear DNA distribution patterns (DNA aneuploidy) represent one of the earliest detectable cellular changes during malignant transformation in experimental models. When squamous cell carcinomas were induced in the bronchial epithelium of beagle dogs an increasing degree of cellular atypia paralleled an increasing degree of aneuploidy.‘” Convincing evidence also has been accrued that the nuclear DNA content has significant prognostic information in breast cancer patients. Patients with carcinomas of DNA diploid type generally have a more favorable clinical course than patients with tumors of DNA aneuploid type.‘“gg Against this background we performed the present study to characterize the presence of mutant p53 protein in various mammary conditions, ranging from normal resting parenchyma to invasive carcinomas with regional lymph node involvement. This broad spectrum

BREAST

CANCER

GENESIS-P53

AND DNA ALTERATIONS

lmmunohistochemistry

of different breast lesions was chosen to reflect the transition from benign to malignant breast disease.

MATERIALS AND METHODS Patient Material In the present study 543 archival, formalin-fixed, and paraffinembedded surgical mammary specimens were analyzed. The series included 40 specimens of normal parenchyma and benign conditions, 54 isolated epithelial hyperplastic lesions without coexisting carcinomas, 109 carcinomas in situ, and 340 invasive mammary carcinomas (Table 1). In 56 of the latter cases specimens from regional lymph node metastases at the time of the initial operation also were investigated. The histopathological classification was based on the WHO (World Health Organization) histological typing of breast tumors.” Epithelial hyperplasias were classified according to the criteria described by Page and Rogers.?4

TABLE Parenchyma

(Eriksson et al)

Three consecutive 4pm thick sections were cut from each par&n block. The first was used for routine hematoxylin-eosin (HE) staining, the second for immunohistochemistry, and the third for DNA staining according to the Feulgen procedure. Immunohistochemical p53 staining was performed by means of the avidin-biotin-peroxidase complex technique. The sections were dewaxed and dehydrated and then incubated with 0.05% pronase (Sigma Chemical Co., St. Louis, MO) for 15 minutes at room temperature. Endogenous peroxidase activity was blocked in 0.5% hydrogen peroxide in distilled water for 30 minutes and nonspecific staining was blocked with 1% bovine serum albumin (BSA) in TRIS-buffered saline for 45 minutes. The sections then were incubated overnight with the primary polyclonal antibody CM-1 (Novocastra Laboratories, Newcastle-upon-Tyne, UK) at a dilution of 1:700 in BSA at 4°C in a humidified chamber. After rinsing the slides were incubated with the second, biotinylated antibody (Rabbit IgC, Vector Laboratories, Burlingham, CA) at

1.

lmmunohistochemical Expression of ~53 and Histopathological Subtypes of Normal Breast (n = 5), Benign Samples (n = 35). Epithelial Hyperplasias (n = 541, Carcinomas In Situ (n = 1091, and Primary Invasive Adenocarcinomas of the Breast (n = 340) p53 Immunoreactivity* Present Total No. of Samples

Normal parenchyma Normal breast parenchyma Benign epithelial tumors Fibroadenoma Papilloma, intraductal Adenoma of nipple Tubular adenoma Other nonmalignant lesions Fibrocystic disease Sclerosing adenosis Mastitis Radial scar Epithelial hyperplasias Intraductal hyperplasia without atypia Mild Moderate Florid Intraductal hyperplasia with atypia Lobular hyperplasia with atypia Carcinomas in situ Ductal of comedo type Ductal, micropapillaly Ductal, cribriform Ductal, solid Ductal, mixed Ductal, papillary Lobular Paget’s disease Invasive carcinomas Ductal, well differentiated Ductal, moderately differentiated Ductal, poorly differentiated Ductal of comedo type+ I.obular Papillary Mutinous Medullary

26 109 136 22 33 2 8 4

Total

543

Total No. of Immunoreactive Samples

5

+

ii

++

-t

Absent

0

(1

5

0

0

(I (I

0 0

(I (1

9 4 2 2

0 0 0 0

(I (I 0 0

7 4 3 4

3 25 2 21 3

0 0 0 0 0

0 0 0 0 0

3 2.5 2 21 3

35 13 17 16 12 6 8 2

1 0 1 0 0 0 0 0

7 0 0 0 2 0 0 0

26 13 16 16 9 6 8 2

0 4 9 0 1 0 0 0

0 4 29 12 1 0

26 93 92 8 30 2

I I

7 3

0

16 44 14 3 0 1 1

* p53 immunoreactivity was recorded as described in the Materials and Methods section. t Invasive mammary carcinomas in which the extent of a comedo in situ component qualifies the designation of the tumor.

1229

HUMAN

PATHOLOGY

Volume 25, No. 11 (November

a dilution of 1:200. Diaminobenzidine was used as a chromogen and the sections were slightly counterstained with Mayer’s hematoxylin. Samples from the MDA-231 breast carcinoma cell line served as positive controls. Specimens were recorded as immunoreactive when a distinct staining of the nucleus was observed. A semiquantitative assessment of the staining behavior was performed by estimating the distribution pattern of stained cell nuclei and the percentage of immunoreactive cell nuclei on each slide.“’ The staining results were classified on this basis from absent (-) to 3+ immunoreactive (- = 0%; l+ = sporadic, >O% to 10%; 2+ = sparse/uniform, 10% to 50%; and 3+ = dense/ uniform, >50%) (Fig 1). In a series of 50 invasive carcinomas the staining results of the polyclonal antibody (Pab) CM-l were compared with those of the monoclonal antibody (Mab) DO-7 on parallel sections of the same cases. The immunohistochemical staining procedure for Mab DO-7 was identical to that described above. Twelve cases were found to be immunoreactive with both antibodies and 38 cases did not immunoreact with either antibody. However, the staining results with the Pab CM-l were superior to those with the Mab DO-7. Thus, all further stainings were performed with the Pab CM-l. To test the reliability of the polyclonal antibody CM-l on formalin-fixed material, a comparative study was performed comprised of 19 cases in which both paraffinembedded and frozen sections from the same tumor were immunostained. Frozen sections were fixed in chloroform and acetone (1:l) for 10 minutes. Otherwise they were treated in the same way as described above, except that the application of pronase was omitted. Identical staining results were observed in three p53 immunoreactive and 16 nonimmunoreactive cases.

Nuclear DNA Assessments The measurements of the nuclear DNA distribution patterns were performed on Feulgen-stained preparations in a tissue-adapted manner. To be able to select individual cell nuclei by means of morphological criteria in normal parenchyma, benign (n = 40) and hyperplastic lesions (n = 54), and mammary carcinomas in situ (n = 62), image cytometry was performed on 4pm thick tissue sections by guidance of an HE-stained section. The nuclear DNA content of invasive carcinomas was assessed using fine-needle aspirates from the tumors (n = 337). Details of the cytometric DNA analysis

1994)

1001

“Diploid” Type

w

Proliferating

m f

1

“diploid”/aneuploid 60

Type

III

z -J J W

0

20

Aneuploid

60

Type 20-

IV

3 I II 2c

I I 4c

6c

8c

DNA

FIGURE 2. Examples of distinctly diploid (type I), proliferating diploid/aneuploid (lype Ill), and clearly aneuploid (type IV) nuclear DNA histograms. The 2c-value denotes the DNA content in normal Go/G, cells.

equipment, staining techniques, internal standardization, and cell selection have been described previously.‘”

Interpretation

of DNA Histograms

The nuclear DNA histograms were subdivided into four different types (Fig 2). Type I (diploid) histograms showed a single peak in or close to the diploid region of normal cells. Type II histograms were characterized by a single peak in the tetraploid region of a normal cell population or by two distinct peaks within the diploid and the tetraploid region, where the latter had to contain 20% of all cell counts. Type 111 histograms showed a distribution pattern comparable with that found in proliferating normal populations, ie, a main peak in the diploid region and a considerable number of cells in the Sphase region of that peak. Histograms of type IV had a highly aneuploid distribution pattern exceeding that of the tetraploid region.

RESULTS p53 lmmunostaining

FIGURE 1. Low grade invasive mammary carcinoma of ductal tVpe with strong (3+) Pab CM-l immunostaining pattern in the neoplastic cell nuclei. (Avidin-biotin-peroxidase complex techique-immunoperoxidase stain; original magnification x200.)

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The immunohistochemical p53 staining results in relationship to the various histopathological subgroups of mammary conditions are summarized in Table 1. Normal resting parenchyma, benign epithelial tumors, and other nonmalignant lesions, such as epithelial hyperplasias, did not show any p53 immunostaining. In

BREAST

CANCER

GENESIS-P53

AND DNA ALTERATIONS

contrast, carcinomas in situ and invasive carcinomas immunostained in 13 of 109 (12%) and 79 of 340 (23%) cases, respectively. In invasive carcinomas poorly differentiated variants had a significantly higher frequency of p.53 protein overexpression than well differentiated tumors (37% ZJ
~53 lmmunostaining and DNA Cytometrical Results

TABLE 2. Interrelationships Between the Different Subtypes of Epithelial Hyperplasias and the Nuclear DNA Histogram Type

Intraductal hvperplasias without atypia Mild Moderate Florid Intraductal hyperplasias with atypia Lobular hyperplasias with atypia DNA histogram

typing according

There was no difference in ~53 overexpression and ploidy between lymph node-negative and lymph node-positive carcinomas. As mentioned above, the exact lymph node status was known in 179 of the 340 invasive neoplasms. Node-negative carcinomas of DNA histogram type I overexpressed p53 in two of 33 (6%) cases and node-positive ones overexpressed ~53 in one of 17 (6%) cases. In contrast, 13 of 35 (37%) nodenegative neoplasms and 14 of 44 (32%) node-positive carcinomas of DNA type IV were p53 immunoreactive. All of the results are illustrated in Fig 3, which shows the occurrence of DNA aneuploidy on the one hand and the incidence of ~53 protein overexpression on the other hand in the whole spectrum of mammary lesions from benign to malignant breast disease. It can be noted that in situ and invasive carcinomas of DNA aneuploid type overexpress p53 much more frequently than DNA diploid carcinomas. Finally, Fig 4 summarizes the frequency distribution of DNA aneuploidy and ~53 immunoreactivity. The diagram shows that distinct aneuploidy generally precedes the onset of p53 immunoreactivity. DISCUSSION

Normal resting parenchyma, benign epithelial tumors, and other nonmalignant lesions all had diploid DNA histograms. Among the epithelial hyperplasias, mild intraductal hyperplasias without atypia and lobular hyperplasias also were exclusively of DNA diploid type (Table 2). However, four of 25 (16%) moderate, both florid intraductal hyperplasias without atypia, and eight of 21 (38) intraductal hyperplasias with atypia were characterized by neardiploid histograms of type III or distinctly aneuploid histograms of type IV (Table 2). The detailed interrelationships between the immunohistochemical ~53 overexpression and the nuclear DNA histograms of in situ and infiltrating carcinomas are summarized in Table 3. There was a close association between p53 overexpression and ploidy in both types of neoplasms. Only one of 19 (5%) carcinomas in situ and six of 82 (7%) invasive carcinomas of DNA histogram type I (diploid) were immunoreactive. In contrast, seven of 33 (21.2%) carcinomas in situ and 55 of 187 (29.4%) invasive carcinomas of histogram type IV (aneuploid) overexpressed the ~53 protein (Table 3).

DNA Histogram

(Eriksson et al)

Type

I

II

III

Iv

3 21 0 13 3

0 0 0 0 0

0 3 1 5 0

0 1 1 ?J 0

to Auer et al.”

1231

Evidence from a number of studies indicates that mutations of the ~53 gene represent one of the most common alterations in the genotype of human breast cancer cells.“,“* However, it is not yet entirely clear at what stage of the transition from benign to malignant breast disease p53 gene mutations occur and how the levels of p53 protein overexpression change during tumor progression. Using the Pab CM-l on routinely formalin-fixed sections we investigated the role of the mutant ~53 gene in a series of benign, hyperplastic, dysplastic, and malignant mammary lesions. The choice of the material was based on the concept that mammary carcinomas arise through different stages of cell proliferation, ranging from completely benign resting parenchyma to infiltrating cancer with spread to distant sites. In this investigation none of the benign or dysplastic breast lesions overexpressed the ~53 protein. This indicates that p53 gene mutations are strictly associated with true breast malignancies. Bartek et al’” found four of 14 atypical ductal hyperplasias adjacent to p53 positive mammary carcinomas to be similarly immunoreactive using the Mab 1801 on frozen sections. The number of immunoreactive hyperplasias was reduced to only one case when paraffin sections of the same cases were studied. In the present study the frequency of epithelial hyperplasias was higher than in other studies. This is caused by the fact that we selected breast hyperplasias to emphasize this otherwise relatively rare but interesting histopathological subgroup. The mammary hyperplasias included were never accompanied by invasive carcinomas but very often occurred together with proliferative breast diseases, eg, fibrocystic disease, intraductal papillomas, fibrosing adenosis, and radial scars. Point mutations of the ~53 gene were found in dysplastic lesions of the colon, for example. Some adenomas of the colon with severe dysplasias and even morphologically normal squamous epithelium of the esophagus

HUMAN

TABLE

3.

Interrelationships

PATHOLOGY

Volume 25, No.

11 (November

1994)

Between ~53 Protein Overexpression in Carcinomas In Situ and in Invasive Mammary Carcinomas and the Nuclear DNA Histogram Types Immunohistochemical

p53 Protein

Overexpression

Present

Carcinoma in situ Invasive carcinomas DNA histogram

typing

Absent

Total No. of Specimens

I

II

III

lv

I

II

III

Iv

62 337

1 6

0 10

I 7

7 55

18 76

0 24

9 27

26 132

according

to Auer et al.‘!’

adjacent to malignant tumors have been shown to be mutated.‘Z’gB30However, absence of faulty p53 protein in these lesions does not exclude deletions or p53 binding through proteins (eg, HPV E6 and MDM2).“lX3” Despite the lack of p53 gene mutations in epithelial hyperplasias of the breast there is considerable evidence that these lesions, especially atypical ductal hyperplasias, are associated with an increased risk of cancer development.3”36 These observations are supported by our DNA cytometrical findings in this type of mammary lesion and similar results have been reported earlier by two other groups.3’.38 In this study 7% of the hyperplasias without atypias were composed of cells with distinctly aneuploid nuclei (type IV). This figure increased to 13% in atypical hyperplasias. Interestingly, near-diploid lesions of DNA type III that are probably composed of either proliferating diploid or moderately aneuploid cells were found in 13% and 21% of lesions, respectively. This confirms the concept that chromosomal alterations occur in breast tissue at an early stage. p53 overexpression was first detected in mammary carcinomas in situ, a finding that is in close agreement with those of two previous studies on limited numbers of cases. Thor et al’” investigated 31 mammary carcino-

FIGURE 3. Overview of the immunohistochemicalp53 protein overexpression and frequency of DNA histograms type I (diploid) and type IV (aneuploid) according to Auer et aLI In tumors of DNA diploid type I the levels of ~53 overexpression stay at a rather low level of approximately 6%. In contrast, tumors with aneuploid cell nuclei of type IV are significantly more often p53 immunoreactive. However, there is no significant difference in ~53 protein overexpression between lymph node-negative and lymph node-positive invasive carcinomas and between primary carcinomas and corresponding lymph node metastases.

1232

mas in situ using the Mab 1801 on paraffin-embedded material and found the same percentage of p53 immunoreactive cases as we did (16%). Davidoff et al” assessed 15 carcinomas in situ with the Mab 1801 and found p53 protein overexpression in 13% of the frozen sections. It is notable that about half of the carcinomas in situ exhibit crude chromosomal alterations as indicated by DNA aneuploidy. However, only a minor fraction of mutations in mammary carcinomas in situ seem to involve the p53 gene. The levels of immunohistochemical p53 protein overexpression found in invasive carcinomas in this study are in close a reement with what has been reported previously. “J 8.‘4.‘5.‘7 In a number of studies even higher levels of p53 immunoreactivity have been reported, ranging from 40% to 60% of the cases.‘3.3y One of the presumed explanations for this discordance might be the different properties of the various antibodies in current use (Mab 1801, Pab 421, Pab 240, Mab DO-7, and Pab CM-l). Varying fixation methods, length of storage time, and even the subjective interpretation of the staining patterns could contribute to variations in the results. In light of these considerations we performed parallel pilot studies to find the most reliable staining procedure.

FIGURE 4. Frequency distribution of distinct DNA aneuploidy (type IV) and ~53 immunoreactivity in the whole spectrum of mammary lesions from benign to malignant breast disease. Note that DNA aneuploidy occurs in precancerous lesions. Overexpression of the ~53 protein is first detectable in mammary carcinomas in situ and increases in invasive carcinomas.

BREAST

CANCER

GENESIS-P53

AND DNA ALTERATIONS

p53 protein overexpression in invasive mammary carcinomas was associated closely with a high tumor grade and with DNA aneuploidy, which is consistent with earlier reports.“,‘” These interrelationships imply that mutations of the p53 gene might play an important role as a mechanism for the dedifferentiation and aggressive biological behavior of some mammary neoplasms. Our results indicate that p53 protein overexoression renresents a later event durinp0 breast cancer ;levelopme;lt. In conclusion, the present findings suggest that chromosomal aberrations as indicated by DNA aneuploidy occur in precancerous mammary lesions. Alterations of the p53 gene are probably not involved in the earliest steps of breast cancer tumorigenesis. However, they might play an important role in the later events of malignant transformation, ie, immortalization, clonal expansion, invasive growth, and onset of distant spread.

REFERENCES 1. Bgrtek J, Iggo R, Cannon J, et al: Genetic and immunohistczchemical analysis of mutant p53 in human breast cancer cell lines. Oncogene 5:89%399, 1990 2. Purdie CA. O’Grady J, Piris J, et al: p53 Expression in color-ectal tumors. Am J Patho1 138:807-813, 1991 3. Lane DP, Benchimol S: ~53: Oncogene or anti-oncogene? (ienes Dev 4:1-X, 1990 4. Miller C. Mohandas T, Wolf D, et al: Human p53 gene localized to short arm of chromosome 17. Nature 319:783-784, 1986 5. Levine AJ, Momand J. Finlay CA The ~5.1 tumour suppressolgene. Nature 351:453456, 1991 6. Cannon JV, Greaves R, Iggo R, et al: Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J 9:1595-1602, 1990 7. Crawford LV. Pim DC:. Bulhrook RD: Detection of antibodies against the cellular protein p53 in sera from patients with breast cancer. Int J Cancer 30:403-408, 1982 8. Banks I,, Matlashewski G, Crawford L: Isolation of humanp5.3-specific monoclonal antibodies and their use in the studies of human p53 expression. Eur J Biochem 159:529-534, 1986 9. Midgley CA, Fisher CJ, Bartek J, et al. Analysis of p53 expression in human turnours: An antibody raised against human p53 expressed in Escherichia coli. J Cell Sci 101:185189. 1992 10. Vojtesek B, Birtek J. Midgley CA, et al: An immunohistochemical analysis of the human nuclear phosphoprotein ~53. New monoclonal antibodies and epitope mapping using recombinant ~53. .I Immunol Methods 151:237:244, 1992 11. Iwaya K, Tsuda H. Hiraide H, et al: Nuclear p53 immunoreaction associated with poor prognosis of breast cancer. Jpn J Cancer Res 82:835-840, 1991 12. Martinazzi M. Crivelli F, Zampetti C, et al: Low, intermediate and high grade hrrast carcinomas as determined by histotyping, immunohistochemical prognosticators and histological grading. Pathologica 84:3347, 1992 13. Ostrowski JL, Sawan A, Henry L, et al: p53 Expression in human breast cancer related to survival and prognostic factors: An imtnur~otlistorhemical study. J Pathol 164:75-81, 1991 l-1. Thor .AD, Moore DH, Edgerton SM, et al: Accumulation of ~53 tumor suppressor gene protein: An independent marker of prognosis in breast cancers. J Nat1 Cancer Inst 84:X45-855, 1992 15. Isola J, Visokorpi T, Holli K, et al: Association of overexprcssion of tumor supr-essor protein ~53 with rapid cell proliferation and poor prognosis in node-negative breast cancer patients. J Nat1 (:ancer Inst 84: 1109-l114, 1992 16. Chen I.-C. Ncubauer A, Kurisu W. et al: Loss of hrtcrozygos-

(Eriksson et al)

ity on the short arm of chromosome 17 is associated with high proliferative capacity and DNA aneuploidy in primary human breast cancer. Proc Nat1 Acad Sci U S A 88:3847-3851, 1991 17. DavidoffAM, Herndon JE, Glover NS, et al: Relation between ~5.1 overexpression and established prognostic factors in hreast cancer. Surgery 110:259-264, 1991 18. Konaka C, Auer G, Nasiell M. et al: Sequential cytomorphcp logical and cytochemical changes during developmenr of bronchial carcinoma in beagle dogs exposed to 2O-methylcholanthrene. Acta Histochem Cytochem 15:779-797. 1982 19. Auer GU, Caspersson TO, Wallgren AS: DNA content and survival in mammary c&cinoma. Analyt &ant Cytol 2:161-165, 1980 20. Clark GM, Dressier LG, Owens MA, et al: Prediction of relapse or survival in patients with node-negative breasf. cancer hy DNA flowcytometry. N Engl J Med 320:627-633, 1989 21. Fallenius AG, Auer GU. Carstensen JM: Prognostic significance of DNA measurements in 409 consecutive breast cancer patients. Cancer 62:331-341, 1988 22. Ewers SB, LangstrCim E, Baldetorp B, et al: Flow cytometric DNA analysis in primary breast carcinomas and clinicopathological correlations. Cytometry 5:408-419, 1984 23. World Health Organization: Histological Typing of Breast Tumors (ed 2). Geneva, Switzerland. World Health Organization, 1981 24. Page DL, Rogers LW Epithelial hyperplaslas, in Page DL, Anderson TJ (eds): Diagnostic Histopathology of the Breast. EdinburEh, UK Churchill Livinestone, 1987, DD 120-156 “25. Eriksson ET, SchiGmelpenning H, Rutqvist I.-E, et al: Immunohistochemicai expression of the mucin-type glycoprotein A-80 and prognosis in human breast cancer. Br J Cancer 67:1418-3422, 1993 26. Fallenius A. Zetterberg A, Auer G: Effect of storage time, destaining, and fixation on Frulgen DNA stdinabilitv of archival MGG slide preparations, in Fallenius A (ed): DNA Content and Prognosis in Breast Cancer. Thesis. Stockholm. Sweden, Karolinska Institute, 1986, pp 2:1-2:13 27. Cattoretti G, Rilke F, Andreoh S. et al: ~53 Expression in breast cancer. Int J Cancer 41:178-183, 1988 ?8. Birtek J, BirtkovA J, Vojtesek B, et al: Patterns of expression of the p53 tumom suppressor in human breast tissues and tumours in situ and in vitro. Int J Cancer 46:839-844, 1990 29. Pignatelli M. Stamp GWH, Kafiri G, ct al: Over-expression of p53 nuclear oncoprotein in colorectal adenomas. Int .J Cancer 50:683688, 1992 30. Sasano H, Miyazaki S, Gooukon ‘T. et al: Expression of p53 in human esophageal carcinoma: An immunohistochemical study with correlation to proliferating cell nuclear antigen expression. HUM PAL‘HOL. 23:1238-1243, 1992 31. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with ~53. Sriencr 248:7& 79, 1990 32. Momand J, Zamhetti GP, Olson DC, et al: The MDM-2 onctr gene product forms a complex with the p53 protein and inhibits p5.3mediated transactivation. Cell 69:1237-1245. 1992 33. Page DL, Vander Zwaag R, Rogers LWw,t’t al: Relation IXtween component parts of fibrocystic disease complex and hreast cancer. J Nat1 Cancer Inst 61:1055-1063, 1978 34. Page DL, DuPont WD, Rogers LW, et al: Atypical hyperplastic lesions of the female breast. A long term follow-up study. Cancer 55:269&2708, 1985 35. Page DL, DuPont WD: Anatomic markers of human premalignancy and risk of breast cancer. Cancer 66: I32&1335. 1990 36. Palli D, Rosselli de1 Turco M, Simoncini R. et al: Benign breast disease and breast cancer: A case-control srudv in a cohort in Italy. Int J Cancer 47:705706, 1991 37. Carpenter R, Gibbs N, Matthews J, et al: Importance of celhlar DNA content in pre-malignant hreast disease and pre-invasive carcinoma of the female hreast. Br J Surg 74:905-906, 1987 38. Crissman JD, Visscher DW, Kubus J: Image cytophotometric DNA analysis of atypical hyperplasias and intrdducl.al czarcinomas of the breast. Arch Pathol Lab Med 114:1249-1253, 1990 39. Horak E, Smith K, Bromley I., et al: Mutant ~5.7. E(;F recey tar and c-erbB-2 expression in human hreasr cancer. Oncogenr 6:“277-““84 _ ..* . 1991

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