Riccardo Ponzone

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.

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.

Prostate specific antigen in breast cancer, benign breast disease and normal breast tissue

Prostate specific antigen (PSA) is a tumor marker used widely for the diagnosis and monitoring of prostatic adenocarcinoma. Recently, we provided evidence that PSA may also be produced by breast tumors. In this report we examined quantitatively the PSA levels in 199 breast tumors, 48 tissues with benign breast disease (BBD, 34 fibroadenomas), and 36 normal breast tissues. Significant amounts of PSA (≥ 0.030 ng of PSA per mg of total protein) were found in 28% of breast tumors, 65% of BBD tissues, and 33% of normal breast tissues. PSA positivity in breast tumors was highest in stage I disease (34%) and decreased with disease stage (24% in stage II and 18% in stage III–IV). Using polymerase chain reaction amplification we have shown PSA mRNA presence in patients with PSA protein-positive tissues (benign and malignant) but not in patients with PSA protein-negative tissues. Our data suggest that PSA is expressed frequently by normal breast tissue, by tissue of benign breast diseases, and by breast cancer tissue. Highest expression is seen in benign breast disease and lowest expression in advanced stage cancerous tissue. As PSA production is mediated by steroid hormones and their receptors, we propose that PSA may be a new marker of steroid hormone action in the normal or diseased female breast. The role of this enzyme in the development of breast diseases including breast cancer is currently unknown.

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

Prostate-specific antigen in serum of women with breast cancer

Prostate-specific antigen (PSA) was recently found in 30% of female breast tumours. In this study we have examined if PSA circulates in the blood of breast cancer patients and if serum PSA has any clinical application. We have compared serum PSA levels between women with and without breast cancer, between women with PSA-positive and PSA-negative breast cancer and between women with breast cancer before and after surgical removal of the tumour. We found that for women > or = 50 years, there is no difference in serum PSA between normal or breast cancer patients. We also could not find any difference in presurgical or post-surgical serum PSA between women who have PSA-positive or PSA-negative breast cancer. We found no correlation between PSA concentrations in matched presurgical and post-surgical sera, between presurgical sera and tumour cytosols and between post-surgical sera and tumour cytosols. High-performance liquid chromatography has shown that PSA in normal male serum consists mostly of PSA bound to alpha 1-antichymotrypsin (molecular weight approximately 100,000), and PSA in breast tumours and presurgical and post-surgical serum consists mostly of free PSA (molecular weight approximately 33,000). These data suggest that female serum PSA is not associated with tumour PSA levels. We speculate that most of the circulating PSA in women originates from the normal breast. It appears that serum PSA in women does not have potential for breast cancer diagnosis or monitoring, but our previous data are consistent with the view that tumour PSA concentration is a favourable prognostic indicator in women with breast cancer.

Medium-Chain Acyl-CoA Dehydrogenase (MCAD) Deficiency: The Prevalent Mutation G985 (K304E) Is Subject to a Strong Founder Effect from Northwestern Europe

Medium-chain acyl CoA dehydrogenase (MCAD) deficiency is a potentially fatal inherited defect of fatty acid beta-oxidation. Approximately 90% of the disease-causing alleles in diagnosed patients are due to a single base mutation, an A (adenine) to G (guanine) transition at position 985 of MCAD cDNA (G985). In a limited number of cases it was found that this mutation was always associated with a particular haplotype, defined by three intragenic restriction fragment length polymorphisms, indicating a founder effect [Kølvraa et al.; Hum Genet 1991; 87: 425-429]. In addition, recent studies of American patients and their ancestors suggested the existence of a founder from northern Europe [Yokota et al.; Am J Hum Genet 1991; 49: 1280-1291]. In the present study we document (1) that the G985 heterozygous frequency in the Caucasian population of North Carolina in the USA is 1/84, which is 5- to 10-fold higher than in non-Caucasian Americans; (2) that there exists a 100% association of the G985 mutation in 17 families with MCAD-deficient patients to a certain haplotype, defined by the restriction endonucleases BanII, PstI and TaqI; (3) that MCAD deficiency due to the G985 mutation is more frequent in the Netherlands, Ireland, England, Belgium and Denmark than in other western European countries, and (4) that the frequency distribution of G985 mutation carriers is 1/68-1/101 in newborns in the United Kingdom and Denmark, and 1/333 in Italy. These results support the notion of a founder effect in northwestern Europe.

Human kallikrein gene 13 (KLK13) expression by quantitative RT - PCR: an independent indicator of favourable prognosis in breast cancer

Kallikreins are a group of serine proteases with diverse physiological functions. KLK13 (previously known as KLK-L4) is a novel kallikrein gene located on chromosome 19q13.4 and shares a high degree of homology with other kallikrein family members. Many kallikrein genes were found to be differentially expressed in various malignancies, and their regulation is controlled by steroid hormones in prostate and breast cancer cell lines. We studied the expression of KLK13 by quantitative reverse transcriptase–polymerase chain reaction in 173 patients with epithelial breast carcinoma. An optimal cutoff point equal to the 40th percentile was defined, based on the ability of KLK13 to predict disease-free survival. KLK13 values were then associated with other established prognostic factors and with disease-free survival and overall survival. Higher positivity for KLK13 expression was found in older, oestrogen receptor positive patients. In univariate analysis, KLK13 expression is a significant predictor of improved disease-free survival and overall survival (P<0.001 and P=0.009, respectively). Cox multivariate analysis indicated that KLK13 was an independent prognostic variable in the subgroups of patients with Grade I–II tumours and in patients who were oestrogen receptor and progesterone receptor positive, and node positive. Hazard ratios derived from Cox analysis, related to disease-free survival and overall survival were 0.22 (P=0.001) and 0.24 (P=0.008), respectively, for the Grade I–II group; 0.36 (P=0.008) and 0.44 (P=0.038), respectively, for the node positive group and 0.36 (P=0.008) and 0.18 (P=0.008), respectively, for the oestrogen receptor positive group. The adjusted hazard ratio for progesterone receptor positive patients for disease-free survival was 0.25 (P=0.012). For patients in the node positive and oestrogen receptor positive subgroup (n=51) the adjusted hazard ratio was 0.25 (P=0.006) and for the node positive and progesterone receptor positive subgroup (n=46) the hazard ratio was 0.24 (P=0.008). Taken together, these data suggest that higher KLK13 expression in these subgroups of breast cancer patients is associated with an approximately 55 to 80% reduction in the risk of relapse or death. We conclude that KLK13 expression, as assessed by quantitative reverse transcriptase–polymerase chain reaction, is an independent favourable prognostic marker for breast carcinoma.

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.