Recolección de datos de campo

LOGAA 4 3 2 1 0 1 0 1 FAMILY HISTORY

Legend: 0 no family history.

1 positive temily history.

The mean aromatase activity is indicated by the bar. There was a significant difference between

the two groups. Those patients with no family history had greater levels of aromatase activity

4J2J3. Gestation and Parity.

The majority of patients were nulliparous and there were no statistical differences between the aromatase activities in the lesion in relation to the number of pregnancies and live births (one way ANOVA). This was also true of the aromatase activities in the adipose tissue (one way ANOVA).

4.2.4. Association with malignancy.

The lesions were categorised into four classes based upon relative risk of development of malignant disease. Class 1 were no risk lesions (64 cases), including cysts, mild hyperplasia and fibroadenomas. Slight risk lesions fell into the class 2 category (12 cases) and included epithelial hyperplasia, papillomatosis and epithetiosis. Moderate risk/class 3, lesions were the atypical

hyperplasias (1 case) and high risk/class 4 lesions were carcinomas in situ {see chapter 1,

section 4-2). The histological report from each patient was examined and the lesion awarded a classification according to these categories. No class 4 lesions were registered in this chapter since they were treated as malignant and are therefore discussed in the next chapter. The remaining two classes (the 1 class 3 lesion was excluded) were analysed against aromatase activity and in a one way analysis of variance test, no significant relationship was found between aromatase activity and histological class of the lesion.

4.2.5. Endocrine Therapies.

The presence or absence of detectable aromatase activities, either in the lesion or in the surrounding adipose tissue, did not relate to the use of oral contraceptives or hormone replacement therapy.

4-3. mRNA Encoding Aromatase.

13 of the patients had the mRNA levels in the lesions measured using the comparative PCR technique. Figures 4.4 a & b show the results of these assays. The tables depict the patients in rank order (highest to lowest) for mRNA content of the lesion. The corresponding autoradiographs show the human and rat cDNA probed samples. The rank order of the mRNA levels in these lesions on intra-assay comparison did not appear to reflect the histology of the lesion. Insufficient tissue was available to study the aromatase activity in parallel with the mRNA levels.

4-4. Benign Breast Disease in Male Patients.

The 4 cases of male benign disease were all classified as gynaecomastia. All had normal hormonal profiles (serum testosterone and oestradiol) and their aromatase activity levels were within the female range (undetectable to 43.39 fmol oestrogen/mg protein/3hr). There was insufficient data to allow the statistical analysis of these results.

Figure 4.4a. Relative aromatase mRNA levels in 6 patients with benign breast disease. BG3454 330054 073726 ! 008162 c R N A — 530729 117373

Patient number. % density (nANA) Histology.

330054 3.04 fibroadenoma

008162 1.65 mild epithelial hyperplasia

117373 0.79 fibrocystic disease

BG3454 0.66 fibroadenoma

530729 0.62 fibroadenoma

073726 0.11 macro and micro cysts ||

4

R at Human

Figure 4.4b. Relative aromatase mRNA levels in 7 patients (8 samples) with benign breast disease. 326703 BG4765 BG3152 064414 434300 434300 2 532512

....

Q * . '

Pafiem number. % deneily (mRNA). Histotogy.

BG4765 0.722 Dbroadenmatoid hyperplasia 434300 1. 0.650 fibroadenomas 2. 0.331 064414 0.147 fibrocystic disease 245824 0.109 benign adenosis 326703 0.099 fibroadenoma 532512 0.059 fibroadenoma BG3152 0.052 fibroadenomatoid hyperplasia M J # , Rat Human

Table 4.1. Gynaecomastia Patients.

Patient No. Age. Type of Gynea. AA-Lesion AA-Adipose

104654 19 NA 1.89 ND

435755 29 drug-related ND 43.39

905328 63 hormonal UD ND

524858 70 ND UD ND

Legend: AA-Lesion aromatase activity in the lesion sample

AA-Adipose aromatase activity in the adipose.

UD undetectable.

DISCUSSION.

70% of the patients with benign breast disease had detectable aromatase activity in the lesion or adipose tissue. These activities were similar to others reported in the literature

1987).

The distribution of these lesions is of interest since benign disease, is mainly located in the upper outer quadrant of the breast, similar to its malignant counterpart (Donegan, 1988). There was a bias towards this quadrant being the most likely to contain the lesion (42.9%) and this appears to influence the aromatase activity levels in the adipose tissue and the lesion.

Aromatase activity both in the lesion and the adipose tissue would appear not to correlate with age and menopausal status of the patient. The majority of patients with benign disease did not report a positive family history of malignant breast disease, and the activity levels in the lesion for aromatase in the two groups did not vary significantly. However, the aromatase activity levels between the two groups did vary significantly in terms of adipose tissue aromatase activity, the negative family history group displaying a higher activity than the positive family history group. This result presents a paradox, since on the basis of this observation, one would expect patients with a positive family history to synthesise less oestrogens within the breast and hence to experience less of the paracrine/autocrine oestrogenic influence on breast cancer induction and/or maintenance. However, clinical data clearly indicate that patients with a positive family history of breast cancer are at greater risk of developing malignant breast disease than women with no family history. Since this genetic risk may play a greater role in those with a positive family history, local biochemical changes may be more important in the development of breast lesions in those without a family history.

The majority of the lesions are in the class 1 category, no risk lesions and there was no statistical differences between the histological classes and aromatase activity of the lesion.

There was no relationship between gestation and parity and aromatase activity except that most of the cases where aromatase activity was positive in both the lesion and adipose tissue occurred in nulliparous women. This is most likely a reflection of the fact that benign disease is most common in young, premenopausal women.

No relationship was discovered to exist between the administration of oral contraceptives and/or hormone replacement therapy and aromatase activity in either the lesion or the adipose tissue. The link between benign disease and hormonal therapies is controversial (see Chapter 1, section 7-2.5). However, if there is an association between benign disease and these therapies, this data would suggest that it does not involve the aromatase enzyme.

Aromatase mRNA levels were investigated in lesion samples from 13 patients and 100% were identified positive, indicating that all of these lesions had the potential to produce their own oestrogens from circulating androgens. It can be speculated that the oestrogens may be used as an autocrine growth factor for the lesion or as a paracrine stimulus for the surrounding adipose tissue to produce growth factors which may play a role in growth and maintenance of the disease.

To summarise, there was no significant correlation between aromatase activity and any clinical indices, except that aromatase activity in the adipose tissue was higher in patients with no family history of breast cancer than in those patients with a positive family history. There was a significant bias for the lesion to occur in the upper outer quadrant of the breast and for aromatase activity to be higher in this quadrant, suggesting an association between aromatase activity and the location of the lesion.

MAUGNANT BREAST DISEASE AROMATASE ASSAY RESULTS. CHAPTER 5.

INTRODUCTION.

The tumours from malignant lumpectomies, segmental and total mastectomies were assayed for aromatase activity and mRNA encoding the aromatase enzyme. For the malignant lumpectomies the adipose tissue immediately surrounding the tumour (peri-tumour adipose) was assayed for aromatase activity and the adipose from the quadrant containing the tumour was examined for enzyme activity in the segmental mastectomy samples. In addition to this, axillary adipose tissue from the segmental and total mastectomies, where available, was measured for aromatase activity, along with any involved axillary lymph nodes.

The results of the aromatase activity have been analysed with respect to various indices associated with malignant breast disease. Appendix 2 contain the details of the malignant lumpectomies, segmental mastectomies and total mastectomies, in table format.

RESULTS.

5-1. Aromatase Activity Analysis.

There was a total of 134 patients with malignant breast disease, of which 4 were male and 130 female. Of the female patients, 24 were pre-menopausal, 13 peri-menopausal and 91 post­

menopausal, 6 cases did not have their menopausal status noted.

The age range of the patients was 20-89 years, with a mean (±SEM) of 61.43±1.33 years.

Aromatase activity was detectable in the adipose tissue derived from 67% of the upper outer quadrant samples; 70.5% of the upper inner quadrant samples; 76.5% of the lower outer quadrant samples and 74.1% of the lower inner quadrant samples. 81.9% of the tumours contained detectable aromatase activity.

The ranges and means of aromatase activities in the adipose and tumour samples were calculated for all 134 patients. The results are depicted in Table 5.1.

Table 5.1. Aromatase activity range and means in adipose and tumour samples from malignant specimens.

AA Maximum Mean ±SEM

UOQ L(aC) 251.8 5.01 1.20 UIQ 220.2 5.49 1.23 LOO 196.61 5.25 1.23 LIQ 179.14 3.38 1.23 TUMOUR 201.15 8.71 1.20 NODE Cl-'S') 28.48 4.26 1.26 AXILLA 87.2 4.67 1.28 PERITUM L\s) 52.07 7.76 1.23 Legend:

AA aromatase activity (fmol oestrogen/mg protein/3hr).

UOQ upper outer quadrant

UIQ upper inner quadrant

LOO lower outer quadrant

LIQ lower inner quadrant

AXILLA axillary adipose

Aromatase activity levels were examined by sector to compare the activity levels in the medial and lateral breast sections. There was no statistically significant difference in aromatase activities between the medial and lateral breast adipose.

The tumours were distributed as in Fig 5.1. 60 were in the right breast, 57 in the left, and 17 were either non palpable, diffuse or not their location was not noted.

As described for the benign lesions, there was a bias in tumour location towards the upper outer quadrant. 57 of the carcinomas (42.5%, which was statistically significant, Chi-Squared) were located in the upper outer quadrant. 10 were in the upper inner quadrant, 14 were in the lower outer quadrant and 5 were in the lower inner quadrant. 3 were located centrally, 11 were spanning the upper outer and inner quadrants; 13 spanning the upper and lower outer

quadrants, 2 spanning the upper and lower inner quadrants, 1 spanning the lower outer and

inner quadrants and 7 were diffuse or had no associated mass. For 11 of the samples, the location of the tumour was not noted. There was no relationship between the position of the tumour and its level of aromatase activity. There was also no relationship between the location of the tumour and the aromatase activity in the adipose tissue from the tumour bearing quadrant, examined in 65 mastectomy patients. Table 5.2.

Figure 5.1.