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The Androgen-Excess Theory of Breast Cancer, 2012: 79-109 ISBN: 978-81-308-0507-8 Editors: Giorgio Secreto, Barnett Zumoff

6. Endocrine Classification of

Postmenopausal Breast Cancers

Giorgio Secreto

Hormone Research Laboratory, Department of Preventive and Predictive Medicine

Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1

20133 Milan, Italy

Abstract. To further investigate the role of androgens in breast

cancer, we examined the relationship of serum levels of

testosterone to patient and tumor characteristics in a cohort of

about 600 postmenopausal patients. We found a significant

association of elevated testosterone levels with estrogen-receptor

(ER)-positive status and with high androgen-receptor (AR)-

positivity of tumors. These findings provide further evidence that

testosterone levels are a marker for hormone-dependent breast

cancer, as suggested in our previous studies (Chapter 4, section 2).

Evaluating both the serum testosterone levels (high/low) of the

patient and the ER status (positive/negative) of the tumor, we

identified four different groups of postmenopausal breast cancer

patients, who could be further divided in two subgroups by AR

status (positive/negative). This endocrine classification (based on

testosterone levels, ER status, and AR status), identifies different

subsets of ER-positive and ER-negative breast cancers, possibly

with different prognoses and requiring different treatments.

Preliminary data on the outcomes in the patients of our cohort

validate the clinical implications of our classification. We also discuss the possible mechanisms by which androgens stimulate cancer

growth in the different subsets of patients. Correspondence/Reprint request: Prof. Giorgio Secreto, Hormone Research Laboratory, Department of

Preventive and Predictive Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Venezian 1, 20133

Milan, Italy. E-mail: Giorgio.Secreto@istitutotumori.mi.it

Giorgio Secreto 80

Breast cancer originates in the epithelial cells of the mammary gland. In

the past decades, basic research has focused on the study of the cancerous

cell, and has obtained important knowledge of molecular mechanisms leading

to cancer growth, invasiveness, and metastasis [1]. Concurrently with studies

on the cell, it has become evident that malignant transformation of the

epithelium does not result only from a sequel of genetic abnormalities of the

epithelial cells but also from a profound disruption of the epithelial-

mesenchymal interaction. The role of the tumor microenvironment is now

well documented, although the complex interrelationship among the different

types of stromal cells (fibroblasts, myo-epithelial cells, macrophages,

endothelial cells, adipocytes etc), the extra-cellular matrix, and the epithelial

cells has not been fully clarified [2-10]. The reciprocal communication

between epithelium and stroma in the control of mammary epithelial growth

is regulated by sex steroids, whose action on the epithelium is mediated by

stromal growth factors and components of the extra-cellular matrix [3,4]. In

turn, the cancerous cell stimulates the production of large amount of

estrogens by stromal fibroblasts and macrocrophages via paracrine

mechanisms [2,4,11,12]. Sex steroids also directly support cancer growth

through binding to intracellular receptors.

Therefore, breast cancer growth largely depends on the endocrine

environment in which the tumor develops, and the anomaly of the

microenvironment that favors cancer growth reflects a more general

endocrine abnormality of the woman with cancer, which we have identified

as androgen excess (Table 1).

Table 1. Abnormal endocrine status of women that favors cancer growth both directly

and indirectly through a disruption of the normal mesenchymal-epithelial

communication.

Comprehensive patient-and-tumor characterization

Generalized Endocrine Abnormality of the Woman

Abnormal Endocrine Milieu of the Breast

Breast Cancer

Endocrine classification of breast cancer 81

Studies of the cancerous cell and its microenvironment are useful for

improving our knowledge of the development and behavior of breast cancer;

however, application of the important findings of basic research to clinical

practice has been fairly limited so far.

What we need now is to evaluate the hormonal status of the patient

together with the characteristics of the tumor. At present, measurement of

circulating testosterone levels is the best way to identify women with

hormone-dependent breast cancer. We believe that a comprehensive

characterization of the patient and her tumor might be helpful in selecting

different subsets of breast cancers to submit to different treatments. Better

categorization of the patient/tumor disorder would be a further step toward

personalized therapy, which is the objective of modern oncology.

1. The TPM study (testosterone, prognosis, mammary cancer)

To further investigate the role of androgens in breast cancer, we initiated a

prospective study in women with operable breast cancer specifically designed

with statistical power sufficient for conclusively assessing the role of

endogenous testosterone as a predictor of breast cancer progression (TPM

study). A secondary aim of the study was to examine the relationship of serum

levels of testosterone and other sex steroids to patient and tumor characteristics.

The study design is shown in figure 1. All patients admitted to the Breast

Surgery Unit of the National Cancer Institute (INT) of Milan, Italy, were

evaluated for eligibility for the study. From January 2004 to the end of March

2011, we recruited 3599 patients, of whom 2475 satisfied the criteria of

eligibility for the study: the inclusion criterion was histologically confirmed

non-metastatic breast carcinoma (any T, any N, M0); the exclusion criteria

were non-epithelial cancer, previous cancer diagnosis (except in situ cervical

cancer or non-melanoma skin cancer), and history of neo-adjuvant

chemotherapy or hormone therapy. Written informed consent was obtained

from all included patients. The study was approved by the Scientific and

Ethical Committee of the INT.

Each patient provided a sample of blood to constitute a biological bank

for hormonal and genetic analyses. Results of the analyses were added into

the data bank already containing the relevant data of patients extracted from

the clinical records: personal data (age and BMI), menopausal status,

pharmacological treatment affecting circulating hormone levels (hormone

therapy, cortisone, etc), date and type of surgery, histology, pathological

TNM stage, tumor grade, hormone receptor status (estrogen receptor

(ER), progesterone receptor (PR), epidermal-growth-factor-receptor-type2

(HER2) overexpression), adjuvant therapy (chemotherapy, hormone therapy),

Giorgio Secreto 82

Figure 1

radiotherapy, date of latest check-up, date of recurrence, site of recurrence,

contralateral breast cancer, primary non-breast cancer and date and cause of

death. A follow-up of 5 years has been programmed.

Among the 2475 eligible patients, 782 were premenopausal, 186 peri-

menopausal and 1057 postmenopausal. Surgery and adjuvant therapies were

done according to the standard protocols used in INT.

2. The postmenopausal TPM cohort

The cohort of 592 postmenopausal patients enrolled in the first 3 years of

recruitment (January 2004-December 2006) has been extensively studied, and

the findings of these studies are the basis for the discussion in this chapter.

Postmenopausal status was defined as: last menstruation ≥12 months before

enrollment, bilateral oophorectomy, hysterectomy without oophorectomy, or

unilateral oophorectomy in women 50 or more years old.

Endocrine classification of breast cancer 83

2.1. Relationships of both serum estradiol and testosterone levels

to patient and tumor characteristics

In a previous report [13], we examined the relationship of circulating

testosterone levels to age, BMI, and selected tumor characteristics. An

interesting result of the study was that testosterone levels did not decline with

age in postmenopausal women, in contrast with the constant decline of

adrenal androgens with increasing age reported in the literature [14-16]. This

finding confirms that the postmenopausal ovary is an important source of

androgens in postmenopausal breast cancer patients, consistent with previous

reports in healthy women [14-18], and explains the efficacy of ovariectomy

in patients with elevated androgen levels, as shown in our previous studies

(Chapter 4, sections 1 and 2).

As regards BMI, we found a positive significant relationship between

circulating testosterone levels and BMI. Obesity is a known risk factor for

postmenopausal breast cancer [19,20] and the association of high BMI with

high testosterone levels in women may provide an explanation for that risk

relationship. Testosterone levels by category of age and BMI are reported in

Table 2.

Other interesting observations were obtained by comparing the tumor

characteristics with circulating testosterone levels. We found that elevated

testosterone levels were associated with larger tumor size, but the association

was limited to older women (65 years), a result suggesting that testosterone

may favor tumor growth in late postmenopausal patients.

Table 2. Testosterone levels (mean±SD) by age and BMI categories in the TPM

postmenopausal cohort (592 patients) (modified from Secreto et al. CEBP 2009: 18,

2942).

Patients Testosterone

(ng/mL)

Patients Testosterone

(ng/mL)

N* % Mean±SD P† N* % Mean±SD P

†

Age, y BMI

40-49 14 2.4 0.401 ± 0.202 0.288 <25 250 47.0 0.383 ± 0.164 0.001

50-59 163 27.5 0.396 ± 0.167 25-30 182 34.2 0.402 ± 0.201

60-69 223 37.7 0.403 ± 0.192 >30 100 18.8 0.469 ± 0.219

≥ 70 192 32.4 0.428 ± 0.214

*=Number of patients, †=Nonparametric Cuzick test for trend.

Giorgio Secreto 84

We also found significantly higher testosterone levels in ER-positive

than in ER-negative patients. This finding suggests that testosterone levels

are a marker for hormone-dependent disease in general, and provides further

evidence for the androgen excess theory (Chapter 4 section 2). No significant

relationship was found between testosterone levels and tumor grade, axillary

nodal involvement, or HER2 status. Relationships of testosterone levels to

tumor characteristics in the whole cohort and in the cohort divided by age

categories (<65 and 65 y) are reported in Tables 3 and 4.

In a subsequent publication [21], we focus our attention on the relationship

of serum estradiol and serum testosterone levels to tumor size and ER status in

492 patients of the same cohort in whom we had already measured testosterone

Table 3. Testosterone levels (mean±SD) by categories of tumor characteristics in the

TPM postmenopausal cohort (592 patients) (modified from Secreto et al. CEBP 2009:

18, 2942).

Patients Testosterone (ng/mL)

N* (%) Mean±SD P

Tumor size, cm

<2 385 (66.2) 0.393 ± 0.327 0.001†

>2 196 (33.7) 0.445 ± 0.191

Grade

1 39 (6.7) 0.443 ± 0.282 0.672§

2 325 (55.6) 0.404 ± 0.181

>2 220 (37.7) 0.417 ± 0.194

Nodal status

negative 358 (62.2) 0.410 ± 0.200 0.696†

positive 218 (37.8) 0.412 ± 0.184

ER/PR status ≠

positive 508 (86.0) 0.420± 0.197 0.001†

negative 83 (14.0) 0.346± 0.158

HER2 status

positive 206 (48.0) 0.399± 0.174 0.423†

negative 223 (52.0) 0.418± 0.188

*=Number of patients, †=Fisher's test, §=Nonparametric Cuzick test for trend, ≠ Negative=ER-negative and

PR-negative; positive=ER-positive and PR-positive or ER-negative and PR-positive or ER-positive and

PR-negative.

Endocrine classification of breast cancer 85

Table 4. Testosterone levels (mean±SD) by categories of tumor characteristics in the

TPM postmenopausal patients according to age (<65 and ≥65 y) (modified from

Secreto et al. CEBP 2009: 18, 2942).

Age <65 Age ≥65

Patients Testosterone

(ng/mL)

Patients Testosterone

(ng/mL)

N* (%) Mean±SD P N* (%) Mean±SD P

Tumor size, cm

<2 200 (71.9) 0.398 ± 0.166 0.768† 185 (61.1) 0.389 ± 0.221 0.001

§

>2 78 (28.1) 0.407 ± 0.172 118 (38.9) 0.470 ± 0.199

Grade

1 20 (7.1) 0.413 ± 0.211 0.925§ 19 (6.2) 0.474 ± 0.346 0.706

§

2 146 (52.1) 0.400 ± 0.170 179 (58.9) 0.407 ± 0.190

>2 114 (40.8) 0.397 ± 0.158 106 (34.9) 0.438 ± 0.226

Nodal status

negative 171 (60.8) 0.403 ± 0.173 0.467† 187 (63.4) 0.415 ± 0.233 0.306

§

positive 110 (39.2) 0.389 ± 0.160 108 (36.6) 0.436 ± 0.203

ER/PR status ≠

positive 236 (83.4) 0.409 ± 0.166 0.014† 272 (88.3) 0.430 ± 0.220 0.024

§

negative 47 (16.6) 0.348± 0.163 36 (11.7) 0.343± 0.153

HER2 status

positive 142 (51.1) 0.402 ± 0.169 0.725† 64 (42.4) 0.392 ± 0.187 0.134

†

negative 136 (48.9) 0.397 ± 0.167 87 (57.6) 0.451 ± 0.213

*=Number of patients, †=Fisher's test, §=Nonparametric Kruskal-Wallis test, ≠ Negative=ER-negative and

PR-negative; positive=ER-positive and PR-positive or ER-negative and PR-positive or ER-positive and

PR-negative.

[13], and who had sufficient serum remaining for estrogen assay. We found a

significant relationship of estradiol levels to BMI, but not to age, grade, axillary

nodal involvement, PR status or HER2 status (Table 5).

We found that the relationship to ER status was highly significant for

testosterone levels, as expected [13], but it was surprisingly weak for estradiol

levels. High levels of both testosterone and estradiol were significantly

related to larger tumor size (≥ 2 cm), with a somewhat stronger relationship

for estradiol (Table 6).

Giorgio Secreto 86

Multivariate analysis, adjusting for age and BMI, supported the

relationships of circulating sex steroid levels to ER status and tumor size.

However the relationship of testosterone levels to tumor size weakened and

lost statistical significance after adjustment for estradiol levels (Tables 7, 8).

Dividing patients by age categories (<65 and 65 y), the tumor size was

significantly related to estradiol levels in both age groups (p=0.044 in the

<65 y and p=0.001 in the 65 y groups), while the relationship of tumor size

to testosterone levels was limited to the older patients (p=0.098 in the <65 y

and p=<0.001 in the 65 y groups) and weakened substantially after

adjustment for estradiol levels (p=0.059). ER status was significantly related

to testosterone levels only in the oldest patients (p=0.077 in the <65 y and

p=0.030 in the 65 y); the significance persisted after adjustment for estradiol

levels (p=0.048). No significant relationship was found between ER status

and estradiol levels in either age group (p=0.057 in the <65 y and p=0.343 in

the 65 y groups).

The relationship of estradiol and testosterone levels to tumor size

suggests that both hormones are involved in promoting tumor growth, but our

findings suggest that testosterone exerts its action mainly through conversion

into estradiol. The finding of a relationship of testosterone levels to tumor

Table 5. Estradiol levels (pg/mL) by categories of age, BMI, grade, axillary nodes

and HER2 status in the TPM postmenopausal cohort (492 patients) (modified from

Secreto et al. IJBM 2011: 26, 241).

Patients Estradiol

(pg/mL)

Patients Estradiol

(pg/mL)

N* (%) Mean ± SD P N* (%) Mean ± SD P

Age, y Grade

50-59 133 27.0 8.07 ± 4.29 0.202† 1 33 (6.8) 8.52 ± 4.30 0.883

†

60-69 188 38.2 7.78 ± 4.31 2 269 (55.1) 8.15 ± 4.49

≥70 171 34.8 8.58 ± 4.14 ≥2 186 (38.1) 8.41 ± 4.39

BMI, kg/m2 Nodes

<25 212 47.5 6.92 ± 3.57 <0.001† Negative 301 (62.2) 8.00 ± 4.32 0.113

§

25-30 160 35.9 8.05 ± 4.06 Positive 183 (37.8) 8.67 ± 4.62

≥30 74 16.6 11.52±4.37

PR HER2

Positive 331 67.6 8.43±4.22 0.768 Positive 172 (47.9) 7.61 ± 3.83 0.337§

Negative 159 32.4 7.54±4.29 Negative 187 (52.1) 8.15 ± 4.62

*=Number of patients, †=Nonparametric Cuzick test for trend, §Nonparametric Kruskal-Wallis test.

Endocrine classification of breast cancer 87

Table 6. Estradiol and testosterone levels (mean±SD) by categories of tumor size and

ER status in the TPM postmenopausal cohort (492 patients) (modified from Secreto

et al. IJBM 2011: 26, 241).

*=Number of patients, †=Nonparametric Cuzick test for trend, §= Fisher’s test.

Table 7. Odds ratios of tumor size (2/<2 cm) by estradiol and testosterone quartiles

in the TPM postmenopausal cohort (492 patients) (modified from Secreto et al. IJBM

2011: 26, 241).

Estradiol quartiles (pg/mL)

≤5.07 5.08-7.73 7.74-10.40 >10.40 P trend

Tumor size ≥2/<2 cm 18/86 36/75 39/76 49/58

Age and BMI-adjusted

OR (95% CI)

1 2.36

(1.23-4.56)

2.30

(1.20-4.42)

3.84

(1.99-7.40)

<0.001

Testosterone quartiles (ng/mL)

<0.270 0.270-0.390 0.390-0.527 >0.527 P trend

Tumor size ≥2/<2 cm 22/89 35/73 42/69 43/64

Age and BMI-adjusted

OR (95% CI)

1 2.01

(1.07-3.78)

2.53

(1.36-4.69)

2.45

(1.32-4.56)

0.005

Age, BMI and estradiol-

adjusted OR (95% CI)

1 1.66

(0.87-3.18)

2.05

(1.08-3.88)

1.68

(0.87-3.28)

0.151

Giorgio Secreto 88

Table 8. Odds ratios of ER status (positive/negative) by estradiol and testosterone

quartiles in the TPM postmenopausal cohort (492 patients) (modified from Secreto

et al. IJBM 2011: 26, 241).

Estradiol quartiles (pg/mL)

≤5.07 5.07-7.73 7.74-10.40 >10.40 P trend

ER status positive/negative 96/27 105/17 104/19 108/15

Age-adjusted OR (95% CI) 1 1.80

(0.92-3.53)

1.50

(0.78-2.89)

2.02

(1.01-4.04)

0.070

Testosterone quartiles (ng/mL)

<0.270 0.270-0.390 0.390-0.527 >0.527 P trend

ER status positive/negative 95/29 98/23 109/14 111/12

Age-adjusted OR (95% CI) 1 1.29

(0.69-2.39)

2.42

(1.20-4.87)

2.69

(1.30-5.59)

0.002

Age and estradiol-adjusted

OR (95% CI)

1 1.23

(0.66-2.32)

2.28

(1.11-4.67)

2.42

(1.10-5.34)

0.008

size only in the old postmenopausal group supports the view that the

biological characteristics of breast cancer may differ in the elderly compared

with the early postmenopausal patients [22]. It is well known that the risk of

developing breast cancer increases markedly with advancing age [23,24], and

signs of virilization – markers of enhanced androgenic activity – are often

present in older women [25], suggesting a possible link between androgens

and increased risk of breast cancer in old age.

The most relevant finding of the study was the strong relationship of

circulating testosterone levels to the ER content of the tumor, in contrast to

the weak relationship of circulating estradiol levels to ER content, suggesting

that testosterone level, but not estradiol level, is a marker for hormone-

dependent breast cancer.

Two observations can explain the weak relationship between estradiol

levels and ER content. First, circulating estradiol levels do not reflect the

intratumoral levels of the hormone; higher levels of estradiol in tumor tissue

than in blood have been repeatedly reported in the literature, with a

tissue/plasma ratio for estradiol of about 10 [26-33]. Secondly, it is largely

Endocrine classification of breast cancer 89

accepted that hormones stimulate the synthesis of their own receptors

[34-36]. Assuming that intratumoral estradiol up-regulates the synthesis of its

receptor, the ER content of the tumor has to be only weakly related to the

estradiol level in the blood.

In an attempt to explain the significant relationship between circulating

testosterone and ER, let us consider the biosynthetic pathway of sex steroids

(Tables 9 and 10).

Testosterone can be synthesized through different pathways (the 5

pathway, the 4 pathway, and all the intermediate steps of conversion from

5 to 4 steroids), while estradiol can only be formed either by direct

aromatization of testosterone or by conversion from estrone. Estrone, in turn,

can derive only from androstenedione, by the action of the enzyme

aromatase. Estrone can be converted to estrone sulfate by the enzyme

sulfotransferase in peripheral tissues. Estrone sulfate is the most abundant

estrogen circulating in the blood of pre- and postmenopausal women [37]; it

is biologically inactive and can be converted to the active estrone by the

enzyme sulfatase, either in peripheral organs or in breast cancer tissue [38].

Androstenedione is a steroid with weak androgenic activity; it is, however,

easily converted into the stronger androgen testosterone by the enzyme 17-

Table 9. Biosynthetic pathway of sex steroids.

*=dihydrotestosterone

Giorgio Secreto 90

Table 10. Enzymes involved in sex steroid production in breast-cancer tissue.

*=17-hydroxysteroid dehydrogenase; †a comprehensive review of 17 -HSD family

is reported by Nagasaki et al [40].

HSD5 [39], a reaction that is reversible. In contrast, the reaction catalyzed by

aromatase is irreversible -- estrogens cannot be reverted to androgens once

they are formed. Estrogens can derive only from androgen precursors, and the

immediate precursors of estrone and estradiol are androstenedione and

testosterone, respectively. Estradiol is the most important stimulator of breast

epithelial growth, and its concentrations are 10 or more times as high in

breast cancer tissue as in plasma. A small proportion of intratumoral estradiol

comes directly from the blood stream, while a larger proportion derives from

local (autocrine) production via three possible pathways [40]:

androstenedione estrone estradiol; androstenedione testosterone

estradiol; estrone sulfate estrone estradiol. There is clear evidence that

aromatase as well as the other estrogen-producing enzymes (17β-HSD1,

sulfatase) are abundantly expressed in breast tumors [39,41,42 ].

In conclusion, we suggest that the circulating testosterone level is a good

indicator of the level of androgen precursors available for the intratumoral

Endocrine classification of breast cancer 91

synthesis of estradiol. Estradiol in the cancerous tissue up-regulates the

synthesis of ER, which are, therefore, related to circulating testosterone

levels.

2.2. Androgen receptors (AR) in the TPM cohort: Relationship to

tumor characteristics and circulating sex steroid levels

We examined the intratumoral AR content in 534 patients of our cohort

and divided the tumors into four groups according to the level of AR

expression: absent (0% of stained cells), negative (1-10%), moderately

positive (from >10% to 60%) and highly positive (>60%). More than 70% of

our patients were AR-positive, a finding in agreement with the data from the

literature [43-49]. AR were highly expressed (>60%) in about half of AR-

positive tumors and the half of non-AR-positive tumors was AR absent

(Table 11).

The relationship of AR expression to the clinical-pathological parameters

of our patients is reported in Table 12. AR-absent and AR-negative tumors

were considered as a single group.

No significant relationship was found between AR expression and age,

BMI, tumor size, or axillary nodal involvement; the relationship was of

borderline statistical significance with HER-negative status, and was highly

statistically significant with low histological grade, ER-positivity, and PR-

positivity. All these findings are in substantial agreement with reports in the

literature [45-47,49,50].

The relationship between AR and ER expression is made still more

evident by dividing both receptors into three categories: negative, moderately

positive, and highly positive. The frequency of ER-negative tumors decreases

with increasing AR categories, while the frequency of ER-positive tumors

increases with increasing AR categories (Table 13).

Table 11. Androgen receptor (AR) expression in the TPM postmenopausal cohort

(534 patients).

N* (%) AR expression Group

74 (13.9) 0% Absent

73 (13.9) 1% - 10% Negative

197 (36.9) > 10% - ≤60% Moderately positive

190 (35.4) > 60% Highly positive

*=Number of patients

Giorgio Secreto 92

Table 12. Androgen receptor (AR) categories in relation to patient and tumor

characteristics in the TPM postmenopausal patients.

AR expression

≤ 10%

Positive

> 10% - ≤ 60% > 60%

N* % N* % N* % P†

Age, years

< 70 96 27.2 140 39.7 116 32.9 0.114

≥ 70 51 28.0 57 31.3 74 40.7

BMI, kg/m2

< 25 64 28.6 85 37.9 75 33.5 0.584

≥ 25 66 25.8 93 36.3 97 37.9

Tumor size

cm

< 2 91 26.3 123 35.6 132 38.1 0.179

≥ 2 55 30.1 73 39.8 55 30.1

Grade

≤ 2 73 22.2 120 36.5 136 41.3 <0.001

> 2 73 36.5 74 37.0 53 26.5

Axillary

nodal status

Negative 85 26.5 121 37.7 115 35.8 0.827

Positive 58 28.9 72 35.8 71 35.3

HER2 status

Negative 58 29.7 62 31.8 75 38.5 0.050

Positive 60 32.4 75 40.6 50 27.0

ER status

Positive 93 21.0 171 38.6 179 40.2 <0.001

Negative 54 59.3 26 28.6 11 12.1

PR status

Positive 72 20.3 137 38.6 146 41.1

<0.001 Negative 75 41.9 60 33.5 44 24.6

*=Number of patients, †Pearson’s chi-square test.

Endocrine classification of breast cancer 93

Table 13. Relation of Estrogen receptors (ER) and Androgen receptors (AR) by

categories: negative, moderately positive and highly positive.

AR expression

Negative

≤ 10%

Moderately positive

> 10% - ≤ 60%

Highly positive

> 60%

N* % N* % N* % P†

ER expression

Negative

≤ 10%

51 58.0 25 28.4 12 13.6

<0.001

Moderately positive

> 10% - ≤ 66%

14 29.2 21 43.7 13 27.1

Highly positive

> 66%

74 19.8 143 38.3 156 41.8

*=Number of patients, †Pearson’s chi-square test.

We evaluated the relationship of circulating testosterone and estradiol levels to AR categories. Mean testosterone levels were significantly higher in patients with high AR expression (>60%) than in the other 2 groups (<60%). No significant differences among the 3 AR categories was found for estradiol mean concentrations (Table 14). We further evaluated the odds ratio of AR expression (AR >60% vs AR

60%) by testosterone and estradiol tertiles. AR expression >60% was

significantly associated with high levels of both sex steroids, but the association

with estradiol lost statistical significance after adjustment for testosterone

(Table 15). This finding confirms that circulating testosterone levels, but not

estradiol levels, are a marker of hormone-dependent breast cancer.

2.3. Key role of androgen excess in the growth of ER-positive and

ER-negative breast cancers

In this book, it has been repeatedly stated that the role of androgens in

breast cancer, either inhibiting or stimulating tumor growth, is still debated

and largely unknown. In light of the evidence, it seems reasonable to think

that both actions can be carried out by androgens, with the predominance of

inhibitory or stimulatory effect depending on the microenvironment in which

a tumor develops and on the hormonal status of the patient.

Giorgio Secreto 94

Table 14. Association of circulating testosterone and estradiol with Androgen

receptor (AR) categories in the TPM cohort.

Testosterone

ng/mL

Estradiol

pg/mL

N* mean±SD P N* mean±SD P

AR expression

AR ≤ 10%

10% < AR ≤ 60%

AR > 60%

147

197

190

0.404 ± 0.190

0.375 ± 0.170

0.440 ± 0.215

0.028†

137

170

164

8.35 ± 4.36

7.87 ± 4.32

8.57 ± 4.23

0.153†

AR expression

AR ≤ 60%

AR > 60%

344

190

0.388 ± 0.179

0.440 ±0.215

0.022†

0.008§

307

164

8.08 ± 4.34

8.57 ± 4.23

0.092†

*=Number of patients, †Pearson’s chi-square test, § ANOVA Fisher’s test.

Table 15. Odds ratios of Androgen receptor (AR) expression (AR > 60% vs AR

≤ 60%) by testosterone and estradiol tertiles in the TPM postmenopausal patients.

Testosterone tertiles (ng/mL)

< 0.312 0.312 – 0.466 > 0.466 P for trend

AR expression

>60% vs ≤60% *54/125 61/118 75/101

Age adjusted OR (95% CI) 1 1.24 (0.79-1.94) 1.69 (1.09-2.62) 0.017

Estradiol tertiles (pg/mL)

≤ 6.03 6.04 – 9.55 > 9.55 P for trend

AR expression

>60% vs ≤60% *44/113 58/99 62/95

Age-adjusted OR (95% CI) 1 1.43 (0.88-2.31) 1.66 (1.03-2.66) 0.039

Age- and testosterone-

adjusted OR (95% CI)

1

1.32 (0.81-2.16)

1.38 (0.83-2.30)

0.236

*=number of patients

Endocrine classification of breast cancer 95

Studies from our laboratory suggest that androgen excess is the endocrine

abnormality that characterizes women with breast cancer. The mechanisms

by which androgen excess stimulates cancer growth are those reported in

Chapter 3, namely: 1. Conversion to estrogens. 2. Direct stimulation through

binding to AR. 3. Stimulation of the epidermal-growth-factor-receptor (EGR-

R) signaling pathway. A fourth possible mechanism that emerged in the last

years is the stimulation of the HER2 pathway. In the present section we

suggest how these mechanisms might be implicated in the growth of ER-

positive and ER-negative cancers, commonly called hormone-dependent and

non-hormone-dependent tumors, respectively.

The process suggested for the ER-positive tumors is based on the well

documented evidence that: 1) large amounts of inactive androgen precursors

are present in breast-cancer tissue [26,29,51,52], 2) sex steroid-producing

enzymes are abundantly expressed in breast cancer tissue: aromatase

(androstenedione estrone) [12,39,42,53,54]; 5-reductase (testosterone

DHT) [39,53]; steroid sulfatase (estrone-sulfate estrone and DHEA-S

DHEA) [38,41]; 17βHSD-type 1 (estrone estradiol) [40] and 17βHSD-

type 5 (androstenedione testosterone) [39], 3) intratumoral estradiol

concentrations are higher than those of plasma [26, 27, 29-33, 54], 4)

intratumoral DHT concentrations are higher than those of plasma [30,31], 5)

ER are expressed in higher amount in breast-cancer tissue than in normal

breast tissue [55,56], 6) AR are expressed in the large majority of ER-

positive tumors [43, 45-47,49], 7) hormones up-regulate the synthesis of their

own receptors [34-36].

The role of androgen excess in the development of ER-positive breast

cancer is summarized in Figure 2: within the cancer cell, high androgen

levels in the presence of high aromatase activity induce increased estrogen

synthesis; estrogen binds to ER and stimulates cell proliferation, on the one

hand, and up-regulates ER synthesis on the other hand, thus creating an auto-

regulatory loop that operates at a level higher than in normal cells; the

presence of androgen excess plus increased aromatase activity allows

continuous estrogen formation; the auto-regulatory loop permits continuous

binding of estrogen to its receptor, thus stimulating continuous cell

proliferation, still under hormonal control (hormone-dependent tumors)

(Figure 2A); at the same time, the high androgen levels favor increased

conversion of testosterone into the stronger (and non-aromatizable) DHT,

thus explaining reports from the literature of DHT concentrations three times

as high in tumor tissue as in blood [30,31]; testosterone and DHT probably

up-regulate intratumoral AR synthesis, and this would account for the

frequent presence of ER and AR in the same tumor (Figure 2B).

Giorgio Secreto 96

Figure 2. A. Simplified scheme to summarize the possible role of androgen excess in

breast cancer development. The up-regulation of estrogen receptors is shown in bold.

“Testosterone” (also in bold) represents androgen excess and its transformation to

estradiol; the other precursors of estradiol [androstenedione (4), estrone (E1), and

estrone sulfate (E1-S)] are shown in non-bold letters. B. Simplified scheme to

summarize the possible role of androgen excess in breast cancer development. The

up-regulation of androgen receptors is in bold.

The mechanism described applies to most ER-positive tumors that are

also AR-positive. It suggests that androgen excess operates through increased

formation of estrogens, which are the final stimulators of cancer growth.

Under these conditions, high levels of AR and increased synthesis of DHT

Endocrine classification of breast cancer 97

might be interpreted as an attempt to counter the stimulatory effect of

excessive estrogen production, consistent with the apparently paradoxical

effect of androgen excess, both stimulating and inhibiting cancer growth, that

we have discussed in Chapter 4, section 2. Suzuki et al. [42,53] reported a

negative correlation between aromatase expression and DHT concentration in

breast-cancer tissue, and suggested that aromatase acts as a down-regulator of

DHT synthesis by reducing the availability of the DHT precursors

testosterone and androstenedione. Peters et al. [57] showed that AR can

inhibit ER expression and thereby inhibit estrogen-stimulated cell

proliferation. Studies by Suzuki [42,53] and Peters [57] support a possible

role for androgens in countering estrogenic stimulatory effects. Conversely,

however, a synergistic action of androgens and estrogens in stimulating

breast-cancer growth has been observed in animal studies, as summarized in

the review of Liao and Dickson [58].

ER-negative tumors are a very heterogeneous class of tumors. AR are

expressed in about half of ER-negative tumors [43,45,48,50,59], and

emerging evidence suggests a critical role for androgens in breast

carcinogenesis, independent of ER. Several possible mechanisms can be

inferred from data in the literature: 1) Direct stimulation of cell proliferation

by binding to AR is suggested by the findings of Farmer et al. [59], who

identified a subset of AR-positive tumors with increased androgen signaling,

the “molecular apocrine” class, and by the findings of Doane et al. [60], who

identified a subset of ER-negative/AR-positive tumors whose growth was

directly stimulated by androgens in an AR-dependent way. Such direct

stimulation is represented in bold characters in Figure 2B. 2) A significant

association of AR-positive status with HER2 overexpression has been

repeatedly reported in ER-negative cancers, and cross-talk between AR and

HER2 signaling pathways favoring synergistic stimulation of cell

proliferation in these tumors has been suggested [43,47,50,59-62]. 3) HER2

is one of the four known members of the EGF-R family [63], and synthesis of

EGF is stimulated by androgens, as reported in Chapter 3. HER1, commonly

referred to as EGF-R, is the prototype of the EGF-R family. It has been

reported to be expressed in about 30% of breast cancers in several previous

studies (Bolla and Toi, as quoted by Biscardi et al. [63], and Harris et al, as

quoted by Bhargava et al. [64]), and in 6% of cases in a more recent study by

Bhargava et al. [64], who used more stringent criteria in defining EGF-R

overexpression. All these studies reported no correlation between HER1 and

ER, but in none of them was the correlation with AR examined. We

hypothesize that in a least a subset of HER1-positive and ER-negative

tumors, for example in the 6% identified by Bhargava et al, cell proliferation

Giorgio Secreto 98

may be stimulated by the HER1 pathway, independent of the expression of

ER, PR, or AR.

Summing up the reported mechanisms, androgen excess may favor

breast-cancer-cell proliferation in the majority of ER-positive tumors and in a

substantial subset of ER-negative tumors, both groups that are characterized

by overexpression of AR. AR-positivity is associated with a more favorable

prognosis than AR-negativity, and can be regarded as a marker of hormone

(androgen)-dependent disease. We also suggest that androgens might be

involved in the growth of a subset of ER-negative/AR-negative cancers by

stimulating the EGF/HER1 signaling pathway.

2.4. Endocrine classification of postmenopausal breast cancers

We propose an endocrine classification of breast cancer that takes into

account the characteristics of the patient and of her tumor. Our classification

is based on the evidence that testosterone is a suitable marker of hormone-

dependent disease (Chapter 4, section 2 and Chapter 6, section 2.1) and that

the assessment of ER status is the most widely used approach to select

patients for endocrine therapy. Evaluating both the serum testosterone levels

of the patient and the ER status of the tumor, we have identified four different

groups of postmenopausal breast-cancer patients. The cut-off value used to

select high versus low testosterone values was 0.39 ng/ml, the median value

for patients in our cohort (Table 16).

Table 16. Distribution of 534 patients of the postmenopausal TPM cohort in the 4

groups obtained by combining circulating testosterone levels (high/low) and ER status

(positive/negative).

Group Testosterone* and ER status 534 patients

N† %

Group 1. High testosterone and ER-positive 229 42.9

Group 2. High testosterone and ER-negative 30 5.6

Group 3. Low testosterone and ER-positive 214 40.1

Group 4. Low testosterone and ER-negative 61 11.4

*=Cut-off value between high and low testosterone levels: 0.39 ng/mL (median of all values). †=Number of patients.

Endocrine classification of breast cancer 99

Most of the ER-positive patients were PR-positive (77.0%) and most of

the ER-negative patients were PR-negative (84.6%). In the two ER-positive

groups, PR-positivity was observed in 75.6% and in 78.5% of tumors of

groups 1 and 3, respectively. In the 2 ER-negative groups, a PR-negative

status was found in 86.7% and in 83.6% of tumors of groups 2 and 4,

respectively. Within these four large groups, other subgroups can be further

selected according to other tumor characteristics. We examined AR

expression, which became the third parameter of our classification (Table 17).

We consider group 1 as the “complete” hormone-dependent group (high

testosterone, ER-positive, and 77% AR-positive). In this group, cell

proliferation depends on androgen excess and the intratumoral sequence

shown in Figure 2A (androgen excess estrogen excess). Our previous

studies (Chapter 4, sections 1 and 2) have shown that the ovary is the main

source of the elevated testosterone levels, with a minor contribution of

adrenal androgen precursors. Oophorectomy might be considered as a

possible additional therapeutic option in these patients.

In group 3 too, cell proliferation is stimulated by androgen excess through

increased conversion to estrogens (Figure 2A), but the low levels of circulating

Table 17. Androgen receptor (AR) status (positive >10% of stained cells; negative

≤ 10%) in the four groups of our classification.

Groups Testosterone and

ER status

(%) AR-positive AR-negative

N* % N* %

Group 1. High T and

ER-positive

(42.9) 177 77.3 52 22.7

Group 2. High T and

ER-negative

(5.6) 9 30.0 21 70.0

Group 3. Low T and

ER-positive

(40.1) 173 80.8 41 19.2

Group 4. Low T and

ER-negative

(11.4) 28 45.9 33 54.1

*=Number of patients

Giorgio Secreto 100

testosterone suggest a major role of adrenal androgen precursors in favoring

increased autocrine production of estrogens in the cancer tissue. In this group,

the high intratumoral concentration of estradiol derives in part from increased

sulfatase activity (estrone sulfate estrone) and in part from increased

aromatase activity (androstenedione estrone); the conversion estrone

estradiol is catalyzed by the enzyme 17β-HSD1. Suzuki et al. found higher

concentrations of the enzyme sulfatase than of the enzymes aromatase [41] and

sulfotransferase (estrone estrone sulfate) [38] in breast-cancer tissues,

suggesting that the sulfatase pathway might be more important than the

aromatase pathway. Inhibitors of steroid sulfatase inhibit the conversion of

estrone sulfate to estrone and of DHEAS to DHEA, and since DHEA is the

precursor of androstenedione, which is the main substrate of aromatase [65],

steroid sulfatase inhibitors might be effective in the treatment of patients of

Group 3.

Group 2 is the smallest group in our classification, and is further divided

in two subsets by AR status: 1/3 of these tumors are AR-positive and 2/3 are

AR-negative. All of the 9 AR-positive and 81% of the AR-negative tumors

were PR-negative. The AR-positive subset probably corresponds with the

pure apocrine carcinomas, which represent 0.3-4% of all infiltrating breast

cancers [66] and are generally ER-negative, PR-negative, and AR-positive.

We suggest that androgens stimulate the growth of these tumors by binding

to AR, an effect that may be counteracted by antiandrogens. The 21 tumors in

the AR-negative subset show the highest levels of testosterone (mean 0.577±

0.097 ng/ml: median 0.560 ng/ml). These findings prompt us to suggest that

elevated androgen levels induce increased EGF production and that tumor

growth may be stimulated by the EGF-R (HER1) pathway. EGF-R inhibitors

might therefore be useful in the treatment of these tumors.

In Group 4, about half the tumors are AR-positive. We suggest that in

the AR-positive subset increased intratumoral androgen production occurs

from adrenal androgen precursors, which stimulate tumor growth by binding

to AR. This subset too might benefit from anti-androgen therapy. We regard

the AR-negative tumors in Group 4 as the non-hormone-dependent group (low

testosterone levels, ER-negative, AR-negative, and 78.8% PR-negative).

Summing up our reasoning, we suggest that all ER-positive and/or AR-

positive tumors depend on androgens for their growth. Androgen excess is

obvious when the circulating testosterone levels are elevated, but even when

testosterone levels are normal, increased intratumoral androgen production

from adrenal precursors is likely in AR-positive tumors. Lastly, we propose

that cancer growth can be stimulated through the EGF-R pathway when serum

testosterone levels are elevated but tumors are ER-negative and AR-negative.

Endocrine classification of breast cancer 101

This proposed endocrine classification identifies different subsets of

ER-positive and ER-negative breast cancers, possibly with different

prognoses and requiring different treatments. The outcomes in the patients of

our cohort, reported in the next section, validate the clinical implications

from our classification.

2.5. Outcomes of patients according to our endocrine classification:

Preliminary data

All the evaluable patients in our cohort had three or more years of

follow-up. Follow-up information was extracted from the clinical records or

was obtained by telephone. The follow-up is still ongoing, and statistical

analysis of overall survival and disease-free survival will be done when we

have information for all 534 patients in the cohort. Here we report only

preliminary data on the rate of recurrence in the different groups we have

described above.

We have information on 471 (88.2%) of the 534 patients of our cohort

recruited in the first 3 years of the study (January 2004- December 2006),

with a median follow-up of 67.7 months (interquartile range 59.7 to 77.3).

Thirty-seven of the 471 patients were excluded from the evaluation of the

recurrence rate: 13 had died from unrelated disease, without breast-cancer

progression, and 24 had developed second primary tumors at other sites. Of

the remaining 434 evaluable patients, 67 (15.4%) had progression of breast

cancer (Table 18).

The distribution of the 434 evaluable patients into the 4 groups was

similar to the distribution of patients in the whole cohort (Table 19).

Table 18. Follow-up information in the 534 patients of our cohort.

Giorgio Secreto 102

Table 19. Distribution of the 434 patients evaluable for disease progression in the 4

groups of our endocrine classification.

Group Testosterone and ER status 434 pts

with follow-up

534 pts

(whole cohort)

N* % %

Group 1. High testosterone and ER-positive 184 (42.4) (42.9)

Group 2. High testosterone and ER-negative 25 (5.8) ( 5.6)

Group 3. Low testosterone and ER-positive 177 (40.8) (40.1)

Group 4. Low testosterone and ER-negative 48 (11.1) (11.4)

*=Number of patients

In the two ER-positive groups reported in Table 20, the rate of recurrence

was almost twice as high in the high-testosterone group 1 (14.7%) as in the

low-testosterone group 3 (7.9%) (p=0.037). We also evaluated the prognostic

value of AR status in both ER-positive groups (Table 21). Disease

progression was slightly, but not significantly, more frequent in AR-positive

than in AR-negative patients, both in Group 1 and in Group 3. Whether AR-

positivity is indeed a factor for increased risk of breast-cancer relapse needs

to be examined in a larger number of cases.

The standard therapy of ER-positive tumors with antiestrogens and

antiaromatase drugs is effective only in countering increased estrogen

production and activity, which are consequences of androgen excess, but

these drugs are powerless against the androgen excess itself, which originates

from the ovarian interstitial-cell hyperplasia that such patients frequently

manifest (Chapter 4). We suggest that oophorectomy should be done in

ER-positive patients with high levels of testosterone, in addition to standard

therapy. We would expect that this intervention might reduce the recurrence

rate from the 15% level seen in our group 1 patients to the 8% level seen in

our group 3 patients.

The outcomes of patients in the ER-negative groups are reported in Table 22.

Disease progression was as frequent in the high-testosterone group 2 (36%)

as in the low-testosterone group 4 (35.4%), suggesting that circulating

testosterone levels are not prognostic in ER-negative patients.

However, interesting data concerning the importance of serum

testosterone levels emerged when we examined the prognostic value of AR

status in the two ER-negative groups (Table 23).

Endocrine classification of breast cancer 103

Table 20. Outcomes in the two ER-positive groups of our classification.

Group testosterone and ER status (N*) Disease Progression

NO

N*

YES

N* (%)

P†

Group 1. High testosterone and ER positive (184) 157 27 (14.7)

0.037

Group 3. Low testosterone and ER positive (177) 163 14 ( 7.9)

*=Number of patients, †=Pearson’s chi-square test.

*=Number of patients, †=Pearson’s chi-square test.

Table 21. Disease progression in the two ER-positive groups of our classification

according to AR status.

Progression

Groups

N* (%)

NO

N* (%)

YES

N* (%)

P†

Group 1

T high ER+

AR+ 141/184 (76.6%) 118/141 (83.7 ) 23/141 (16.3)

0.284 AR- 43/184 (23.4%) 39/43 (90.7) 4/43 (9.3)

Group 3

T low ER+

AR+ 139/177 (78.4%) 127/139 (91.4) 12/139 (8.6)

n.e.§ AR- 38/177 (21.6%) 36/38 (94.7) 2/38 (5.3)

*=Number of patients, †= Pearson’s chi-square test, §=not evaluable because of the small number of cases.

Table 22. Outcomes in the two ER-negative groups of our classification.

Group testosterone and ER status (N*) Disease Progression

NO

N*

YES

N* (%)

P†

Group 2 High testosterone and

ER-negative (25)

16 9 (36.0)

0.945

Group 4 Low testosterone and

ER-negative (48)

31 17 (35.4)

*=Number of patients, †=Pearson’s chi-square test.

Giorgio Secreto 104

Table 23. Disease progression in the two ER-negative groups of our classification

according to the AR status.

Progression

Groups N* (%) NO

N* (%)

YES

N* (%)

P†

Group 2

T high ER-

AR+ 7/25 (28.0%) 6/7 (85.7) 1/7 (14.3)

n.e.§ AR- 18/25 (72.0%) 10/18 (55.6) 8/18 (44.4)

Group 4

T low ER-

A+ 22/48 (45.8%) 13/22 (59.1) 9/22 (40.9) 0.464

A- 26/48 (54.2%) 18/26 (69.2) 8/26 (30.8)

*=Number of patients, †=Pearson’s chi-square test, §=not evaluable because of the small

number of cases.

In the high-testosterone group 2, only 1 of the 7 AR-positive patients

(14.3%) relapsed, whereas disease progression occurred in 8 of the 18 AR-

negative patients (44.4%). AR-positive status seems, therefore, to be a factor

for better outcome in ER-negative patients with high testosterone levels. Such

patients can still be regarded as hormone-dependent, and therefore have

intrinsically less aggressive tumors.

In contrast, in the low-testosterone group 4, disease progression occurred

in 40.9% of AR-positive and 30.8% of AR-negative patients, suggesting that

AR-positivity is not significantly related to risk of relapse in ER-negative

patients with low testosterone levels. Taken together, these findings suggest

that two different groups of ER-negative patients can be identified by their

circulating levels of testosterone (high vs low) and their AR status: in patients

with high testosterone levels, AR-positive status is protective, while in low-

testosterone patients, it is not. This tentative conclusion needs to be tested in

a larger number of cases.

In conclusion, the endocrine classification obtained by the simultaneous

evaluation of testosterone level, ER status, and AR status, identifies 2

different groups of ER-positive and 2 different groups of ER-negative tumors

and implies that improved selection of patients can enhance the efficacy of

therapy and the quality of life (Table 24). We suggest that serum testosterone

levels should be routinely determined in postmenopausal breast cancer

patients, in order to program therapies with the dual objective of treating the

cancer and correcting the basic hormonal abnormality, thus reducing the

probability of recurrences.

Endocrine classification of breast cancer 105

As a final consideration, we believe that the findings from basic

research could be better understood if interpreted in the light of the

hormonal status of the patient and not only in that of the characteristics of

the tumor. We think that the molecular mechanisms leading to cancer

growth may be different in the four groups of our classification, and we

think it may be necessary to take that into account in order to achieve

targeted therapy (Table 25). A network of biological banks of serum,

plasma, buffy coat, and tissue, joined with data banks containing all the

relevant information on patients and on their tumors, would be highly

desirable for evaluating this conclusion in a short time.

Table 24. Clinical implications of our Endocrine Classification obtained by the

simultaneous evaluation of circulating testosterone levels, ER status, and AR status.

Table 25. Successive steps towards optimization of basic research for arriving at

targeted therapy.

Giorgio Secreto 106

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