Nicoletta Biglia

Monitoring Response to Primary Chemotherapy in Breast Cancer using Dynamic Contrast-enhanced Magnetic Resonance Imaging

AbstractPurpose. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) allows analysis of both tumor volume and contrast enhancement pattern using a single tool. We sought to investigate whether DCE-MRI could be used to predict histological response in patients undergoing primary chemotherapy (PCT) for breast cancer. Patients and methods. Thirty patients with breast cancer, clinical diameter >3 cm or stage III A/B, received anthracycline and taxane based PCT. DCE-MRI was performed at the baseline, after two cycles and after four cycles of PCT, before surgery. Histological response was assessed using a five-point scheme. Grade 4 (small cluster of dispersed residual cancer cells) and grade 5 (no residual viable cancer cell) were defined as a major histopathological response (MHR). Results. Univariate analysis showed that a >65% reduction in the tumor volume and a reduction in the early enhancement ratio (ECU) after two cycles of PCT were associated with a MHR. Multivariate analysis revealed that tumor volume reduction after two cycles of PCT was independently associated with a MHR (odds ratio [OR] 39.968, 95% confidence interval [CI] 3.438–464.962, p < 0.01). ECU reduction was still associated with a MHR (OR 2.50, 95% CI 0.263–23.775), but it did not retain statistical significance (p = 0.42). Combining tumor volume and ECU reduction after two cycles of PCT yielded a 93% diagnostic accuracy in identifying tumors achieving a pathological complete response (pCR) (histopathological grade 5). Conclusions. DCE-MRI allows prediction of the effect of neoadjuvant chemotherapy in breast cancer. Although in our study tumor volume reduction after two cycles had the strongest predictive value, DCE-MRI has the potential to provide functional parameters that could be integrated to optimize neoadjuvant chemotherapy strategies.

Angiopoietin-2 expression in breast cancer correlates with lymph node invasion and short survival

Angiogenic factors produced by tumor cells are essential for tumor growth and metastasis. In our study, the expression of Angiopoietin‐1 (ANG1) and Angiopoietin‐2 (ANG2) mRNA in archival human breast cancer tumor samples and in 6 breast cancer cell lines was investigated. Total RNA from biopsies of 38 breast cancer patients was extracted and ANG1 and ANG2 mRNA expression was measured by means of quantitative real‐time RT‐PCR (Taqman®). Matching data with available clinicopathologic and biochemical data revealed a significant association between ANG2 expression and axillary lymph node invasion. Univariate and multivariate survival analysis, by means of Kaplan‐Meier method and Coxs proportional hazards model, showed significant and independent association between ANG2 mRNA level and both disease‐free (p < 0.0001) and overall survival (p < 0.0003). An important fact is that, notwithstanding the small number of cases examined, this association was confirmed also in the group of lymph node‐negative patients (DFS, p < 0.003; OS, p < 0.020). Immunohistochemical analysis demonstrated that Ang2 is expressed by both tumor cells and endothelial elements. Expression in tumor cells was confirmed by studying a panel of human breast carcinoma cell lines in culture by RT‐PCR. In ZR75.1 and T47D cells, expression of ANG2 mRNA was increased up to 10‐fold by treatment with estrogen within 24 hr. Although preliminary, these data suggest a possible role of ANG2 as a prognostic factor for primary breast cancer.

Differential effects of oral conjugated estrogens and transdermal estradiol on insulin-like growth factor 1, growth hormone and sex hormone binding globulin serum levels

In postmenopausal women oral ethinylestradiol causes a reduction in circulating insulin-like growth factor 1 (IGF-1) and an increase in serum growth hormone levels. There are no data on the effect of conjugated estrogens, the preparation most often used in estrogen replacement treatment (ERT), on these parameters. We evaluated serum IGF-1 and growth hormone levels, together with the levels of sex hormone binding globulin (SHBG), an indicator of estrogen hepatocellular action, before and after 6 months of ERT in two comparable groups of postmenopausal women. Sixteen women were treated with oral conjugated estrogens, 0.625 mg/day, and 14 with transdermal estradiol, 0.05 mg/day. In the women treated with oral conjugated estrogens, an increase in SHBG (p < 0.001), a decrease in IGF-1 (p < 0.001) and an increase in growth hormone (p < 0.05) serum levels were observed. No such effects were seen with the use of transdermal estradiol, devoid of hepatocellular effects. Undoubtedly, oral conjugated estrogens, 0.625 mg/day, through a hepatocellular effect, cause marked modifications in the IGF-1/growth hormone axis, which may have clinical relevance. For instance, the decreased IGF-1 level, together with the increased level of SHBG, might provide some explanation of the favorable epidemiological data on breast cancer risk in women receiving oral conjugated estrogens.

Menopause after breast cancer: a survey on breast cancer survivors

UNLABELLED Due to the younger age and the ever wider use of adjuvant chemotherapy and antiestrogens, menopausal symptoms are a frequent cause of concern for breast cancer patients. OBJECTIVES To determine the prevalence of menopausal symptoms, and to explore the attitudes toward Hormone Replacement Therapy (HRT) or other treatments and the willingness to take oestrogen in breast cancer patients. METHODS A questionnaire-based survey on 250 breast cancer patients treated and followed-up at our department. Of them 144 (Group A) were in postmenopause and 106 (Group B) were in premenopause at time of diagnosis. RESULTS Adjuvant therapy with tamoxifen or tamoxifen plus chemotherapy is associated with a significant worsening of menopause-related symptoms of women belonging to Group A. These women are more concerned about risk of breast cancer recurrence than about risk of osteoporosis (P=0.05) and heart disease (P=0.006). Seventy-eight percent are against the use of HRT; only 22% would consider taking HRT mainly for vasomotor symptoms relief and osteoporosis prevention. The incidence of vasomotor and dystrophic symptoms is significantly higher in women belonging to Group B treated with chemotherapy and/or hormonotherapy as compared with postmenopausal women (P<0.000 and P=0.02, respectively). Premenopausal women are more concerned about risk of breast cancer recurrence than older women (P=0.09) and at the same time are significantly more worried about the impairment of the quality of life due to adjuvant therapy (P=0.005). Younger women are more prone to consider HRT than postmenopausal women (P=0.05). Sixty-six percent are against HRT use, and 34% would consider taking HRT to alleviate vasomotor and dystrophic symptoms and to prevent osteoporosis. CONCLUSIONS Breast cancer survivors are interested to treatments that may improve their quality of life, but fear of HRT persists among these women and their doctors, despite new evidence suggesting the low probability of detrimental effects.

Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion.

Background Estrogen is an established enhancer of breast cancer development, but less is known on its effect on local progression or metastasis. We studied the effect of estrogen receptor recruitment on actin cytoskeleton remodeling and breast cancer cell movement and invasion. Moreover, we characterized the signaling steps through which these actions are enacted. Methodology/Principal Findings In estrogen receptor (ER) positive T47-D breast cancer cells ER activation with 17β-estradiol induces rapid and dynamic actin cytoskeleton remodeling with the formation of specialized cell membrane structures like ruffles and pseudopodia. These effects depend on the rapid recruitment of the actin-binding protein moesin. Moesin activation by estradiol depends on the interaction of ERα with the G protein Gα13, which results in the recruitment of the small GTPase RhoA and in the subsequent activation of its downstream effector Rho-associated kinase-2 (ROCK-2). ROCK-2 is responsible for moesin phosphorylation. The Gα13/RhoA/ROCK/moesin cascade is necessary for the cytoskeletal remodeling and for the enhancement of breast cancer cell horizontal migration and invasion of three-dimensional matrices induced by estrogen. In addition, human samples of normal breast tissue, fibroadenomas and invasive ductal carcinomas show that the expression of wild-type moesin as well as of its active form is deranged in cancers, with increased protein amounts and a loss of association with the cell membrane. Conclusions/Significance These results provide an original mechanism through which estrogen can facilitate breast cancer local and distant progression, identifying the extra-nuclear Gα13/RhoA/ROCK/moesin signaling cascade as a target of ERα in breast cancer cells. This information helps to understand the effects of estrogen on breast cancer metastasis and may provide new targets for therapeutic interventions.

Low-dose vaginal estrogens or vaginal moisturizer in breast cancer survivors with urogenital atrophy: a preliminary study

The study aim is to evaluate the efficacy and safety of two low-dose vaginal estrogen treatments (ETs) and of a non-hormonal vaginal moisturizer in postmenopausal breast cancer survivors with urogenital atrophy. Eighteen patients receiving estriol cream 0.25 mg (n = 10) or estradiol tablets 12.5 μg (n = 8) twice/week for 12 weeks were evaluated and compared with eight patients treated with polycarbophil-based moisturizer 2.5 g twice/week. Severity of vaginal atrophy was assessed using subjective [Vaginal Symptoms Score (VSS), Profile of Female Sexual Function (PFSF)] and objective [Vaginal Health Index (VHI), Karyopycnotic Index (KI)] evaluations, while safety by measuring endometrial thickness and serum sex hormones levels. After 4 weeks, VSS and VHI were significantly improved by both vaginal ETs, with further improvement after 12 weeks. PFSF improved significantly only in estriol group (p = 0.02). Safety measurements did not significantly change. Vaginal moisturizer improved VSS at week 4 (p = 0.01), but score returned to pre-treatment values at week 12; no significant modification of VHI, KI, PFSF was recorded. Both low-dose vaginal ET are effective for relieving urogenital atrophy, while non-hormonal moisturizer only provides transient benefit. The increase of serum estrogens levels during treatment with vaginal estrogen at these dosages is minimal.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse‐associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR‐148b, down‐regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR‐148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR‐148b in the control of breast cancer progression.—Cimino, D., De Pittà, C., Orso, F., Zampini, M., Casara, C., Penna, E., Quaglino, E., Forni, M., Damasco, C., Pinatel, E., Ponzone, R., Romualdi, C., Brisken, C., De Bortoli, M., Biglia, N., Provero, P., Lanfranchi, G., Taverna, D. miR148b is a major coordinator of breast cancer progression in a relapse‐associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1. FASEB J. 27, 1223–1235 (2013). www.fasebj.org

Increased incidence of lobular breast cancer in women treated with hormone replacement therapy: implications for diagnosis, surgical and medical treatment

A growing body of evidence support the association between the use of hormone replacement therapy (HRT) and a higher risk of both invasive lobular carcinoma (ILC) and invasive ductal-lobular mixed carcinoma (IDLC). Overall biological and clinical features of ILC entail a more cautious diagnostic and therapeutic approach as compared with invasive ductal carcinoma (IDC). ILCs are more frequently multifocal, multicentric and/or bilateral. Mammography and ultrasound show, therefore, significant limitations, while the higher sensitivity of magnetic resonance imaging in the detection of multifocal and/or multicentric lesions seems to improve the accuracy of preoperative staging of ILCs. Early diagnosis is even more challenging because the difficult in the localization and the sparse cellularity of lobular tumours may determine a false negative core biopsy. ILC is characterized by low proliferative activity, C-ErbB-2 negativity, bcl-2 positivity, p53 and VEGF negativity, oestrogen and progesterone positive receptors, low grade and low likelihood of lymphatic-vascular invasion. However, this more favourable biological behaviour does not reflect into a better disease-free and overall survival as compared with IDC. Since lobular histology is associated with a higher risk of positive margins, mastectomy is often preferred to breast conservative surgery. Moreover, only few patients with ILC achieve a pathologic response to preoperative chemotherapy and, therefore, in most patients mastectomy can be regarded as the safer surgical treatment. The preoperative staging and the follow-up of patients with ILC are also complicated by the particular metastatic pattern of such histotype. In fact, metastases are more frequently distributed to the gastrointestinal tract, peritoneum/retroperitoneum and gynaecological organs than in IDC.

Identification of new genes associated with breast cancer progression by gene expression analysis of predefined sets of neoplastic tissues

Gene expression profiles were studied by microarray analysis in 2 sets of archival breast cancer tissues from patients with distinct clinical outcome. Seventy‐seven differentially expressed genes were identified when comparing 30 cases with relapse and 30 cases without relapse within 72 months from surgery. These genes had a specific ontological distribution and some of them have been linked to breast cancer in previous studies: AIB1, the two keratin genes KRT5 and KRT15, RAF1, WIF1 and MSH6. Seven out of 77 differentially expressed genes were selected and analyzed by qRT‐PCR in 127 cases of breast cancer. The expression levels of 6 upregulated genes (CKMT1B, DDX21, PRKDC, PTPN1, SLPI, YWHAE) showed a significant association to both disease‐free and overall survival. Multivariate analysis using the significant factors (i.e., estrogen receptor and lymph node status) as covariates confirmed the association with survival. There was no correlation between the expression level of these genes and other clinical parameters. In contrast, SERPINA3, the only downregulated gene examined, was not associated with survival, but correlated with steroid receptor status. An indirect validation of our genes was provided by calculating their association with survival in 3 publicly available microarray datasets. CKMT1B expression was an independent prognostic marker in all 3 datasets, whereas other genes confirmed their association with disease‐free survival in at least 1 dataset. This work provides a novel set of genes that could be used as independent prognostic markers and potential drug targets for breast cancer.

Body mass index (BMI) and breast cancer: impact on tumor histopathologic features, cancer subtypes and recurrence rate in pre and postmenopausal women.

The study aims to analyze the association between body mass index (BMI) at time of diagnosis, breast cancer histopathologic features (tumor size, nuclear grade, estrogen and progesterone receptor (ER and PgR) and HER-2/neu expression, histological subtypes, Ki-67 index, lymphatic/vascular invasion, axillary nodes involvement) and incidence of different subtypes defined using hormone receptors and HER2/neu expression, according to menopausal status; to evaluate the impact of BMI on disease free survival (DFS) at multivariate analysis. A total of 2148 patients (592 premenopausal, 1556 postmenopausal) were classified into subgroups according to BMI distribution. High BMI was significantly associated with larger size tumor both in pre (p = 0.01) and postmenopausal women (p = 0.00). Obese premenopausal women showed worse histopathologic features (more metastatic axillary lymphnodes, p = 0.017 and presence of vascular invasion, p = 0.006) compared to under/normal weight group. Postmenopausal patients with BMI > 25 developed more frequently ER/PgR positive cancers (87% versus 75%, p 0.017), while no association was found in premenopausal women. We could not found any statistically significant correlation between breast cancer subtypes (luminal A, B, HER-2 and basal-like) and BMI both in pre and postmenopause. Higher BMI was significantly associated with a shorter DR-FS in postmenopausal women but the independent prognostic role of obesity was not confirmed in our analysis.