Elsevier

The Breast

Volume 15, Issue 1, February 2006, Pages 81-89
The Breast

ORIGINAL ARTICLE
Ageing, hormonal behaviour and cyclin D1 in ductal breast carcinomas

https://doi.org/10.1016/j.breast.2004.12.008Get rights and content

Summary

Owing to the gradual modification of breast tissue in postmenopausal women, there can be differential effects on local oestrogen receptor (ER) expression, with potential impingement on the biological behaviour of cancer cells in the ageing. A series of 45 ductal carcinoma (DC) cases were selected in postmenopausal women who were not being treated with HRT. Immunohistochemical analyses were performed for hormone receptors and Ki67 expression. Fluorescence in situ hybridisation (FISH) analysis was carried out to study CCND1 amplification. The selected population was subdivided into three groups by age and was subjected to statistical studies: linear model analysis, estimation of relative incidence (RI), multivariate analysis, and nonparametric tests were performed to investigate whether there were any links between age and molecular variables in DCs. The results show a low rate of proliferation and high ER expression in the oldest age group. In the same group a close correlation was found between high ER expression and CCN in the older age group D1 amplification (P=0.000), as was a more advanced phenotype in terms of tumour size and presence of positive lymph nodes than in the other age groups considered. The results suggest that ductal breast cancer has a favourable molecular prognosis, especially in extreme old age. In particular, there is an inverse correlation between ageing and proliferation rate despite the presence of an accentuated proliferation stimulus (high ER with CCD1 amplifications) in the oldest group relative to the other groups considered.

Introduction

Owing to the gradual modification of breast tissue in postmenopausal women, in whom there is a small component of fibrous breast stroma and epithelial cells and a larger component of adipose tissue, there can be differential effects on local oestrogen receptor (ER) expression, with potential impingement on the biological behaviour of cancer cells.

Stromal cells can modulate the growth of normal and neoplastic breast epithelial cells and can secrete growth factors in response to various levels of endogenous hormones.1 Adipose cells contain aromatase enzymes that produce oestradiol from circulating precursors.2 Owing to a greater presence of adipose cells, the levels of oestradiol in the breast tissue of postmenopausal women are much greater than plasma levels.3, 4

Previous studies on the molecular profile of breast carcinomas in pre- and postmenopausal women have observed that the prognosis for younger women is less favourable owing to a lower expression of ER5, 6, 7, 8 in neoplastic tissue and to a higher proliferation rate9, 10, 11 and a higher grade of histological differentiation despite the real hormonal levels both in circulation and locally.12, 13

The few studies carried out on women over 80–85 years of age affected by breast cancer show an adverse prognosis for women in extremes of age,14, 15 i.e., for premenopausal women under 33 years of age12 and postmenopausal women aged 80–85. Our study concentrated on a group of postmenopausal Calabrian women not being treated with HRT who were affected by ductal carcinoma (DC) with a lower nuclear grading; our aim was to evaluate the biological and molecular profile, taking into account age and the consequences of ageing.

This choice arose from the need to evaluate specific variables, which will be outlined later, to reduce interference from other variables, such as the histological type, to a minimum.

We evaluated the biological variables of DC, specifically, hormonal receptors, CCND1 amplification and rate of cellular proliferation, with reference to age, the principal variable. To this end, the selected group of women was divided into three subgroups aged <65, 65–85, >85 for evaluation and comparison of the biological and molecular variables considered in the different groups.

Section snippets

Patients and methods

A series of 45 patients with DC and not being treated with HRT were selected after each had given informed consent following diagnosis in the Department of Histopathology at Lamezia Terme (Italy) Hospital from 1995 to 1998. These cases were classified according to nuclear grading (NG) and necrosis at the time of diagnosis. The tumour blocks were placed in 10% buffered formalin for more than 24 h and then embedded in paraffin. Areas rich in viable tumour cells were located and marked in 4-μm

Collection of clinical data

Clinical information was obtained by review of medical records in specific electronic registers obtained from the Department of Histopathology at Lamezia Terme Hospital. Patient information included: date of birth, age at diagnosis, age at last menstrual period, and number of lymph nodes involved and stage at diagnosis.

The median age of these patients was 68 years (range 41–94); 36 patients (80%) underwent total mastectomy and 9 (20%), breast-conserving surgery. No adjuvant chemotherapy was

Immunohistochemical staining

Our method of performing the immunohistochemical analysis has been reported elsewhere.16, 17

All sections (4 μm thick) were cut 1 day before immunostaining to avoid antigen decay, and they were attached to the slide by heating in an oven at 60 °C oven for 1 h. The sections were deparaffinized in xylene, dehydrated in graded alcohols and placed in Tris-buffered saline. Sections were subjected to enzymatic pretreatment.16, 17 The slides were cooled and rinsed with deionised water. All sections were

Evaluation

Two independent observers performed a blind analysis. All requisite techniques involved were previously validated, and results of scoring were found to be reproducible between the two independent operators.

The intensity of nuclear immunostaining of individual cells was scored on a scale of 0 (no staining) to +4 (strongest intensity), and the percentage of cells with nuclei staining at each intensity was estimated. The proportion of cells at each intensity was multiplied by the corresponding

Fluorescence in situ hybridisation

To determine gene and chromosome copy numbers, levels of gene amplification or deletion in situ, a locus-specific DNA probe and a chromosome enumeration probe for 11 (site of CCND1 locus) in a dual hybridisation reaction were used. The probes were applied to 4-μm tissue sections of tumour specimens. The FISH methodology was followed as outlined here: tissue sections were incubated at 65 °C for 4 h, deparaffinised in three xylene washes for 10 min, and dehydrated in 100% ethanol. After incubation

Evaluation

The evaluation was carried out as stated above. Cases with a normal disomic signal pattern for CCND1 and centromere 11 showed ratios of gene copy number to centromere copy number of 2/2 and 1/1, respectively. Increased copy number was assessed when at least 30% of nuclei showed increased centromere and/or gene FISH signals.

Amplification was assessed when at least half of these nuclei (15% of total nuclei) showed an unbalanced increase of gene copy numbers (ratio of gene copy number to

Statistical analysis

Hormonal receptor expression was assessed as high or low according to the median immunostaining score (based on both extent and intensity of staining) of all 45 cases for each antigen as a cut-off value. Scores above the median were assessed as high and those below the median as low (the median percentage of cells with hormonal receptor immunostaining was 30%).

Proliferation, as reflected by immunohistochemical staining for Ki67, was similarly assessed as high or low, using the median percentage

Characteristics of the study population

The average age of the first group (<65 years) was 53.07 (range 41–62), that of the second group (65–85), 73.83 (range 66–81) and that of the third group (>85), 90.40 (range 86–94). Tumour size, nuclear grading and lymph node positivity are outlined in Table 1.

Fluorescence in situ hybridisation

Amplification of CCND1 (Fig. 1) was detected in 25 (56%) of the 45 DC cases. In these 25 cases the ratio of copy number to cells was between 4.70 and 12.18.

Only 4 of the 25 cases (16%) presented polysomy 11, and their copy number/cell

Discussion

In this study we checked whether the most important molecular factors studied in breast cancer undergo modifications with the ageing process. We considered 65 years the cut-off age, as this is today's fastest growing population sector, defined also as the young elderly as against the old in the age group >85 years. To reduce negative environmental and genetic factors, we selected the region of Italy with the lowest general cancer mortality rate.20

Talley's study5 is one of the few that shows an

Acknowledgements

The authors are grateful to the Regional Neurogenetic Centre in Lamezia Terme Hospital, for concession of their laboratory instruments for FISH execution.

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