Pathological and Biological Differences Between Screen-Detected and Interval Ductal Carcinoma in situ of the Breast

The Dutch screening program for breast cancer has been gradually implemented in the North Netherlands since 1991. It offered biennial mammography to women 50–69 years old, and since 1999 women 70–74 years old have also been included. Women received mammography in the cranio-caudal and medio-latero-oblique direction for each breast. Two radiologists evaluated the mammograms by a double independent reading.

From January 1992 to December 2001, 128 consecutive patients were treated for pure DCIS at our institution. To identify patients for inclusion in the study, all women who had actually attended the screening program at least two subsequent rounds with a two-year interval at the time of diagnosis were considered as attenders. Patients who had skipped one or more screening rounds previous to the diagnosis and patients who had not been attending the program at all were considered nonattenders. Patients’ records were checked to obtain this information, and if there was no information regarding the participation of the screening program at the time of diagnosis the general practitioner was consulted. Out of the 128 consecutive patients, 102 attenders and 26 nonattenders could be identified. For immunohistochemistry, patients were selected on the availability of sufficient paraffin-embedded tissue. Thirteen out of the 26 nonattenders and 74 out of the 102 attenders remained, respectively, for evaluation of Her2/neu overexpression, estrogen receptor (ER) expression, progesterone receptor (PR) expression, p53 expression, and cyclin D1 expression using tissue microarray analysis as part of a project protocol that had been approved by the medical ethics committee. The patients in the study-group (n = 74) were divided into two groups. Patients with DCIS that had been detected by screening mammography were classified as screen-detected patients, when the examination two years earlier failed to reveal an abnormality (n = 54). Patients with DCIS that had been detected within the two-year interval between two subsequent screening rounds were classified as interval patients, when the earlier examination failed to reveal an abnormality (n = 20).

Mammographic and pathological characteristics were derived from mammography and pathological reports, respectively. Data were delivered and evaluated anonymously. If data were missing, mammography and pathological slides were reevaluated. Mammographic appearances were scored as microcalcifications, a mass, a combination of the two, or as occult. Microcalcifications were scored as fine granular, coarse granular, or as linear branching. Pathological size had been estimated, and the grade had been scored according to the European Pathologists Working Group (EPWG15) and according to the Van Nuys classification.16

Slides from all blocks were evaluated for representative areas with DCIS, and tissue microarrays were prepared as described earlier.17 In brief, the most representative area of DCIS was marked on the original hematoxylin and eosin (H&E) stained section. With this marked section as an orientation, three 0.6-mm punches were taken from the selected area in the donor blocks and mounted in a recipient block containing approximately 110 biopsies, using a manual tissue microarray device (Beecher Instruments, Silver Springs, MD). The presence of DCIS in the arrayed samples was verified on hematoxylin eosin stained sections.

For immunohistochemistry, 3 μm sections of the paraffin-embedded tissue arrays were deparaffinized in 2 changes of xylene for 5 minutes each and gradually rehydrated through changes of graded ethanol from 100% to distilled water. Antigen retrieval methods and antibodies are summarized in Table 1. The endogenous peroxidase reaction was blocked by incubating the sections in 3% perhydrol for 30 minutes. Primary antibodies were diluted in phosphate-buffered saline (PBS) containing 1% bovine serum albumin and incubated at room temperature for 1 hour. Samples were then washed in PBS and incubated with secondary and tertiary antibodies. For visualization of the antibody-antigen complex, the diaminobenzidine tetrahydrochloride/peroxidase reaction was used. After a final wash with distilled water, sections were counterstained with hematoxylin. Sections were dehydrated through rising concentrations of ethanol and mounted. Immunohistochemistry was successful in 81/87 cases for Her2/neu staining, 73/87 cases for ER staining, 71/87 for PR and cyclin D1 staining, and 70/87 for p53 staining.

All slides stained for molecular markers were read by two authors (MdR and BvdV). The slides were randomly reviewed by a third author (JW) and in case of disagreement between the other two authors. ER, PR, and p53 were graded based on the percentage of cells showing positive nuclear staining in the ducts with DCIS. ER and PR were considered positive if nuclear staining was present in ≥10% of the cases, and p53 was considered positive in case of a substantial percentage of positively stained nuclei (>30%). Her2/neu expression was graded as recommended by the HercepTest^TM scoring guidelines: 0: no staining at all or membrane staining in <10% of tumor cells; 1+: a faint/barely perceptible partial membrane staining in >10% of the tumor cells; 2+: weak to moderate complete membrane staining in >10% of tumor cells; 3+: strong complete membrane staining in >10%. Her-2/neu was considered to be overexpressed if the score was 3+. Cyclin D1 expression was scored using a semiquantitative system as described by Vos et al.18 This system was based on the staining intensity scored as 0 (none), 1 (weak), 2 (moderate), and 3 (strong), and the percentage of positive tumor cell nuclei scored as 0 (0%), 1 (1–25%), 2 (25–50%), 3 (50–75%), and 4 (>75%). The cyclin D1 staining score was calculated as the sum of the intensity and the percentage of positive tumor cells.

Differences in clinicopathological characteristics between screen-detected and interval patients with DCIS in the study group, and between the study group and the excluded group of patients were analyzed by chi-square analysis. Differences in clinicopathological and biological characteristics between the study group and the nonattenders were also analyzed by chi-square analysis. Differences in age were tested by using the Mann-Whitney U test. Univariate analyses, investigating differences in pathological and biological features, was performed by logistic regression, using screen detection as a dependent variable. Multivariate analyses were performed with a logistic-regression model. The elimination of variables in a stepwise manner identified the statistically significant pathological and biological parameters. A p value of ≤.050 was considered as significant. All calculations were performed with SPSS 12.01 (SPSS inc., Chicago, IL).