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We tested the hypothesis that polymorphisms in cytochrome P450c17a (CYP17), aromatase (CYP19), 17ß-hydroxysteroid dehydrogenase type I (17ß-HSD1) and sex hormone-binding globulin (SHBG) genes may modify the association between isoflavone intake and breast cancer risk. We conducted hospital-based, casecontrol studies in Nagano, Japan and São Paulo, Brazil. A total of 846 pairs (388 Japanese, 79 Japanese Brazilians, and 379 non- Japanese Brazilians) completed validated food frequency questionnaires. Four single nucleotide polymorphisms (SNPs) in CYP17 (rs743572), CYP19 (rs10046), 17ß-HSD1 (rs605059), and SHBG (rs6259) genes were genotyped. We found no association between the 4 SNPs and breast cancer risk. In combination analyses of isoflavone intake and SNPs, an inverse association between intake and risk was limited to women with at least one A allele of the rs605059 polymorphism for all 3 populations, albeit without statistical significance. For the rs6259 polymorphism, the inverse association was limited to postmenopausal Japanese with the GG genotype (odds ratio [OR] for highest vs. lowest tertile = 0.50, 95% confidence interval [CI] = 0.29-0.87; P for trend < 0.01), and to non-Japanese Brazilians with at least one A allele (OR for consumers vs. nonconsumer = 0.21, 95% CI = 0.06-0.77). We found no remarkable difference for the rs743572 and rs10046 polymorphisms. Our findings suggest that polymorphisms in the 17ß-HSD1 and SHBG genes may modify the association between isoflavone intake and breast cancer risk. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the exogenous progesterone (P4) effect on the luteal function from Day 16 to Day 21 of the oestrous cycle in inseminated goats with unknown pregnancy status. A total of 54 does passed through a short progestin-based synchronization protocol and, on Day 16 of the following oestrous cycle, 27 does received a new P4 device which was retained until Day 21. Blood samples were collected daily from all does during this period, as well as on Day 24. Pregnancy diagnoses were performed on Day 30. Serum P4 values from 26 animals (G NPSP : Group of non-pregnant does with second sponge: n = 8; G NPNSP : Group of non-pregnant does without second sponge: n = 6; G PSP : Group of pregnant does with second sponge: n = 5; G PNSP : Group of pregnant does without second sponge: n = 7) were determined by radioimmunoassay commercial kits. No P4 differences were found between groups (G NPSP : 3.1 ± 2.8; 1.7 ± 1.8; 0.4 ± 1.0; and 0.0 ± 0.0 vs. G NPNSP : 4.4 ± 1.8; 3.0 ± 2.2; 0.8 ± 0.8; and 0.0 ± 0.0 or G PSP : 4.2 ± 1.0; 3.4 ± 0.6; 3.3 ± 1.6; 3.2 ± 0.9; 3.6 ± 1.2; 3.5 ± 1.3; 2.7 ± 1.3 vs. G PNSP : 4.4 ± 1.6; 3.6 ± 1.5; 3.7 ± 1.5; 3.8 ± 1.4; 3.2 ± 1.2; 3.1 ± 1.2; 3.6 ± 1.1; D16, D17, D18, D19, D20, D21, D24, respectively) or for the interaction of group and time. In conclusion, a second progestogen device had no effect on luteolysis or early pregnancy in the following oestrous cycle., (© 2020 Wiley-VCH GmbH.)

This study assessed the efficiency of synchronous oestrous induction by light programme followed by two doses of cloprostenol in acyclic Saanen goats of different parity orders. Primiparous (n = 22) and multiparous (n = 33) goats were subjected to 16 hr of light and 8 hr of darkness for 60 days (D0-D60), starting 10 days after the winter solstice. All goats received 120 µg cloprostenol doses on D130 (morning) and D141.5 (afternoon) (11.5 days apart). Oestrus behaviour, ovarian follicular dynamics and serum progesterone (P4) analyses were recorded from D0 to D174 at different intervals. Animals in oestrus after D141.5 were randomly assigned into two groups: assisted natural mating (NM) or artificial insemination (AI; 10-24 hr after oestrus onset with frozen-thawed semen). From D57 to D120, 89.0% of goats presented large follicles (5-8 mm) and P4 concentrations were subluteal from D0 to D120. More multiparous compared to primiparous goats (54.5%, 18/33 vs. 18.2%, 4/22) exhibited oestrus after both injections. More primiparous compared to multiparous goats (54.5%, 12/22 vs. 12.1%, 4/33) did not exhibit oestrus after any injection. A total of 35 goats (64%) were in oestrus after the second prostaglandin injection and were subjected to NM or AI. The conception rate was similar among primiparous (70.0%, 7/10) and multiparous (68.0%, 17/25) goats but the pregnancy rate differed, being 31.8% (7/22) and 51.5% (17/33), respectively. No interaction was found between parity order and P4 concentrations in does that became pregnant or not. Thus, the association between light programme (60 days, starting at the beginning of winter) and two cloprostenol administrations 11.5 days apart (starting 70 days after the end of the light treatment) resulted in sufficient synchronous oestrous response in multiparous acyclic Saanen goats to reach satisfactory fertility levels after both NM and AI., (© 2020 Blackwell Verlag GmbH.)

Immediate breast reconstruction with a latissimus dorsi musculocutaneous (LDM) flap is still not much reported, particularly in studies of patients' views. The aim of this study was to assess the level of patients' satisfaction with the technique. All patients (n = 257) who had had a mastectomy and immediate breast reconstruction with a LDM flap at a single hospital between January 1999 and December 2005 were identified, and 196 patients free of recurrence were included. The operations were done by the same surgical team in a standard manner. Clinical data, technical details, and outcome were collected prospectively. To assess the patients' degree of satisfaction with the aesthetic results in relation to clinical characteristics and treatment, a study-specific questionnaire and a visual analogue scale were sent to 196 patients; 178 forms were completed and returned. To analyse general satisfaction and aesthetic results we used the Mann-Whitney, Kruskal-Wallis, chi square, or Fisher's exact test, as appropriate. The median reconstruction follow-up period was 34 months. Patients who were 51 years or older at the time of reconstruction were less likely to opt for immediate breast reconstruction with LDM flap than younger patients. Patients who were divorced and those who had postoperative complications were less likely to be satisfied. One hundred and sixty-four patients (92%) were satisfied, and 161 (90%) said they would recommend the reconstruction. The median satisfaction score was 9 (range 1-10, mean 8.5). The technique provided satisfactory immediate breast reconstruction according to the patients' evaluation.

An increasing number of studies have shown altered expression of secreted protein acidic and rich in cysteine (SPARC) and N-myc down-regulated gene (NDRG1) in several malignancies, including breast carcinoma; however, the role of these potential biomarkers in tumor development and progression is controversial. In this study, NDRG1 and SPARC protein expression was evaluated by immunohistochemistry on tissue microarrays containing breast tumor specimens from patients with 10 years of follow-up. NDRG1 and SPARC protein expression was determined in 596 patients along with other prognostic markers, such as ER, PR, and HER2. The status of NDRG1 and SPARC protein expression was correlated with prognostic variables and patient clinical outcome. Immunostaining revealed that 272 of the 596 cases (45.6%) were positive for NDRG1 and 431 (72.3%) were positive for SPARC. Statistically significant differences were found between the presence of SPARC and NDRG1 protein expression and standard clinicopathological variables. Kaplan-Meier analysis showed that NDRG1 positivity was directly associated with shorter disease-free survival (DFS, P < 0.001) and overall survival (OS, P < 0.001). In contrast, patients expressing low levels of SPARC protein had worse DFS (P = 0.001) and OS (P = 0.001) compared to those expressing high levels. Combined analysis of the two markers indicated that DFS (P < 0.001) and OS rates (P < 0.001) were lowest for patients with NDRG1-positive and SPARC-negative tumors. Furthermore, NDRG1 over-expression and SPARC down-regulation correlated with poor prognosis in patients with luminal A or triple-negative subtype breast cancer. On multivariate analysis using a Cox proportional hazards model, NDRG1 and SPARC protein expression were independent prognostic factors for both DFS and OS of breast cancer patients. These data indicate that NDRG1 over-expression and SPARC down-regulation could play important roles in breast cancer progression and serve as useful biomarkers to better define breast cancer prognosis.

Substantial experimental evidence indicates that PAWR gene (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) is a central player in cancer cell survival and a potential target for cancer-selective targeted therapeutics. However, little is known about the role of PAR-4 in breast cancer. We investigated the possible role of PAR-4 expression in breast cancer. IHC results on tissue microarrays containing 1,161 primary breast tumor samples showed that 57% (571/995) of analyzable cases were negative for PAR-4 nuclear staining. Down-regulation of nuclear PAR-4 protein expression predicted a poor prognosis for breast cancer patients (OS; P=0.041, log-rank test). PAR-4 down-regulation also correlates with poor survival in the group of patients with luminal A subtype breast cancer (P=0.028). Additionally, in this large series of breast cancer patients, we show that ERBB2/HER2, EGFR and pAKT protein expression are significantly associated with shorter disease-free survival and overall survival, but the prognosis was even worse for HER2-positive, EGFR-positive or pAKT-positive breast cancer patients with tumors negative for nuclear PAR-4 expression. Furthermore, using three-dimensional (3D) cell culture we provide preliminary results showing that PAR-4 is highly expressed in the MCF10A cells inside the acini structure, suggesting that PAR-4 might have a role in the lumen acini formation. Taken together, our results provide, for the first time, evidence that PAR-4 may have a role in the process of the mammary gland morphogenesis and its functional inactivation is associated with tumor aggressive phenotype and might represent an additional prognostic and predictive marker for breast cancer.

Context and Objective: Lobular carcinoma is the second most common type of breast neoplasia and has unique clinical and pathological features. Our aim was to evaluate prognostic factors for this type of breast cancer., Design and Setting: Retrospective study at a tertiary oncological institution., Methods: 162 patients diagnosed and treated between January 1985 and January 2002 were included. The inclusion criteria were: absence of previous treatment, histological diagnosis of lobular carcinoma, no previous history of breast cancer and minimum follow-up of 36 months., Results: In univariate analysis, the following factors were statistically significant: clinical stage T (P = 0.0005), clinical stage N (P = 0.0014), neoadjuvant chemotherapy (P = 0.0008), primary tumor size (P < 0.0001), vascular invasion (P < 0.0001), lymphatic invasion (P = 0.0004), neural invasion (P = 0.0004), skin invasion (P < 0.0001), capsular transposition (P = 0.0008), lymph node ratio (P < 0.0001), estrogen receptor expression (P = 0.0186), progesterone receptor expression (P = 0.0286), pathological stage T (P < 0.0001), pathological stage N (P < 0.0001), adjuvant chemotherapy (P < 0.0001) and postoperative hormone therapy (P = 0.0367). After grouping the variables, multivariate analysis was performed. Presence of lymph node metastases, capsular transposition, lymph node ratio and postoperative hormone therapy remained significant., Conclusion: In this series, the most important prognostic factors for lobular carcinoma of the breast seemed to relate to lymph node status and presence of capsular transposition. Factors relating to axillary involvement, capsular transposition and hormone therapy were significant for survival.

In breast cancer patients, primary chemotherapy is associated with the same survival benefits as adjuvant chemotherapy. Residual tumors represent a clinical challenge, as they may be resistant to additional cycles of the same drugs. Our aim was to identify differential transcripts expressed in residual tumors, after neoadjuvant chemotherapy, that might be related with tumor resistance. Hence, 16 patients with paired tumor samples, collected before and after treatment (4 cycles doxorubicin/cyclophosphamide, AC) had their gene expression evaluated on cDNA microarray slides containing 4,608 genes. Three hundred and eighty-nine genes were differentially expressed (paired Student's t-test, pFDR<0.01) between pre- and post-chemotherapy samples and among the regulated functions were the JNK cascade and cell death. Unsupervised hierarchical clustering identified one branch comprising exclusively, eight pre-chemotherapy samples and another branch, including the former correspondent eight post-chemotherapy samples and other 16 paired pre/post-chemotherapy samples. No differences in clinical and tumor parameters could explain this clustering. Another group of 11 patients with paired samples had expression of selected genes determined by real-time RT-PCR and CTGF and DUSP1 were confirmed more expressed in post- as compared to pre-chemotherapy samples. After neoadjuvant chemotherapy some residual samples may retain their molecular signature while others present significant changes in their gene expression, probably induced by the treatment. CTGF and DUSP1 overexpression in residual samples may be a reflection of resistance to further administration of AC regimen.

Although epidemiologic studies have shown an inverse association between isoflavones and breast cancer risk, little evidence for a dose-response relation is available. We conducted hospital-based case-control studies of patients aged 20-74 years with primary, incident, histologically confirmed invasive breast cancer, and matched controls from medical checkup examinees in Nagano, Japan and from cancer-free patients in São Paulo, Brazil. A total of 850 pairs (390 Japanese, 81 Japanese Brazilians and 379 non-Japanese Brazilians) completed validated food frequency questionnaires. The odds ratio of breast cancer according to isoflavone intake was estimated using a conditional logistic regression model. We found a statistically significant inverse association between isoflavone intake and the risk of breast cancer for Japanese Brazilians and non-Japanese Brazilians. For Japanese, a non-significant inverse association was limited to postmenopausal women. In the three populations combined, breast cancer risk linearly decreased from 'no' to 'moderate' isoflavone intake and thereafter leveled off. Compared to non-consumers, adjusted odds ratios (95% confidence interval) for consumers in increasing quintile intake categories (median intake in each category: 8.7, 23.1, 33.8, 45.7, and 71.3 mg/day) were 0.69 (0.44-1.09), 0.54 (0.31-0.94), 0.45 (0.26-0.77), 0.34 (0.19-0.62), and 0.43 (0.24-0.76), respectively. Overall, we found an inverse association between dietary isoflavone intake and risk of breast cancer. Our finding suggests a risk-reducing rather than risk-enhancing effect of isoflavones on breast cancer within the range achievable from dietary intake alone. In addition, women may benefit from risk reduction if they consume at least moderate amounts of isoflavones.

Epidemiologic studies have shown an inverse association between isoflavones and breast cancer risk. Because isoflavones bind estrogen receptors, we hypothesized that polymorphisms in the estrogen receptor genes might modify the association between isoflavone intake and breast cancer risk. We conducted hospital-based case-control studies of patients aged 20-74 years with primary, incident, histologically confirmed invasive breast cancer, and matched controls from among medical checkup examinees in Nagano, Japan, and from cancer-free patients in São Paulo, Brazil. A total of 846 pairs (388 Japanese, 79 Japanese Brazilians and 379 non-Japanese Brazilians) completed validated food frequency questionnaires, and provided blood samples. Five single nucleotide polymorphisms in the estrogen receptor alpha (rs9340799, rs1913474, and rs2234693) and beta (rs4986938 and rs1256049) genes were genotyped. We found no consistent association between the five single nucleotide polymorphisms and breast cancer risk among the three populations. In analyses of combinations of isoflavone intake and single nucleotide polymorphisms, an inverse association between intake and risk was limited to women with the GG genotype of the rs4986938 polymorphism for postmenopausal Japanese (odds ratio for highest versus lowest tertile = 0.47; P for trend = 0.01), Japanese Brazilians (odds ratio for highest versus lowest median = 0.31) and non-Japanese Brazilians (odds ratio for consumers versus non-consumers = 0.37) (P for interaction = 0.11, 0.08, and 0.21, respectively). We found no remarkable difference for the other four polymorphisms. Our findings suggest that polymorphisms in the estrogen receptor beta gene may modify the association between isoflavone intake and breast cancer risk.

Previously we found that levels of LRRC49 (leucine rich repeat containing 49; FLJ20156) transcripts were elevated in ER-positive breast tumors compared with ER-negative breast tumors. The LRRC49 gene is located on chromosome 15q23 in close proximity to the THAP10 (THAP domain containing 10) gene. These two genes have a bidirectional organization being arranged head-to-head on opposite strands, possibly sharing the same promoter region. Analysis of the promoter region of this gene pair revealed the presence of potential estrogen response elements (EREs), suggesting the potential of this promoter to be under the control of estrogen. We used quantitative real-time PCR (qPCR) to evaluate the expression of LRRC49 and THAP10 in a series of 72 primary breast tumors, and found reduced LRRC49 and THAP10 expression in 61 and 46% of the primary breast tumors analyzed, respectively. In addition, the occurrence of LRRC49/THAP10 promoter hypermethylation was examined by methylation specific PCR (MSP) in a sub-group of the breast tumors. Hypermethylation was observed in 57.5% of the breast tumors analyzed, and the levels of mRNA expression of both genes were inversely correlated with promoter hypermethylation. We investigated the effects of 17beta-estradiol on LRRC49 and THAP10 expression in MCF-7 breast cancer cells and found both transcripts to be up-regulated 2- to 3-fold upon 17beta-estradiol treatment. Our results show that the transcripts of LRRC49/THAP10 bidirectional gene pair are co-regulated by estrogen and that hypermethylation of the bidirectional promoter region simultaneously silences both genes. Further studies will be necessary to elucidate the role of LRRC49/THAP10 down-regulation in breast cancer.

In a previous study, using differential display reverse transcriptase-PCR (DDRT-PCR) we showed that down-regulation of the PHLDA1 (pleckstrin homology-like domain, family A, member 1; also named TDAG51) mRNA was down-regulated in breast tumors compared with normal breast tissue. The present study was conducted to determine the expression pattern and predictive prognostic value of PHLDA1 in breast cancer. A series of 720 primary invasive breast tumors were examined for PHLDA1 expression. PHLDA1 mRNA expression was determined in 74 breast tumors using quantitative Real Time PCR analysis (qPCR). PHLDA1 protein expression was evaluated by immunohistochemistry (IHC) using Tissue Microarrays (TMA) containing 699 primary invasive breast tumors. Reduced PHLDA1 mRNA expression was identified in 72% (53/74) of the primary breast tumors analyzed. Seventy-three percent (512/699) of cases analyzed showed negative PHLDA1 protein expression. Down-regulation of PHLDA1 protein was a strong predictor of poor prognosis for breast cancer patients. Breast cancer patients with tumors that were negative for PHLDA1 protein expression had shorter disease free survival (P < 0.001) and overall survival (P < 0.001) than patients with tumors that were positive for PHLDA1 protein expression. In addition patients with tumors exhibiting reduced PHLDA1 expression and paucity for ER had the worse outcome (P < 0.001). Multivariate analysis indicated that PHLDA1 protein expression is an independent prognostic factor of patient survival. To our knowledge, the expression pattern of PHLDA1 in breast cancer has not previously been investigated. Our results provide strong evidence that reduced PHLDA1 expression is important in breast cancer progression and could serve as useful prognostic marker of disease outcome.

The definition of high risk patients with early stage breast cancer is still controversial. We evaluated the ability of galectin-3, c-erbB-2 and p53 immunohistochemical expression to predict recurrence and survival in a homogeneous set of 92 patients with T1N0M0 ductal carcinoma with a long-term follow-up. In normal breast tissue, the epithelial and fibroblast components were positive for galectin-3 mostly showing nuclear and cytoplasmic reactivity. At the tumor epithelial component, galectin-3 expression was found in 46.7% of the samples with a predominant cytoplasmic staining. Similar results were presented by concurrent in situ lesions. Tumor stromal fibroblasts maintained positivity in 70 out of 92 cases (76%). We found expression of p53 in only 16 cases (17.4%), and c-erbB-2 in 17 (18.48%). A marginal association was found between co-expression of p53 and galectin-3 (p=0.055) and a significant correlation between p53 accumulation and c-erbB-2 expression (p=0.009). There was no significant association between galectin-3 protein expression with disease-free survival or overall survival. C-erbB2 and p53 expression correlated with recurrence (p=0.002, p=0.02; respectively). Diminished overall survival at 10 years was associated with c-erbB-2 (p=0.010), but marginally with p53 expression (p=0.076). Epithelial galectin-3 expression cannot be considered a prognostic factor for patients with T1N0M0 breast cancer, p53 seems to be of minor relevance and c-erbB-2 expression was the best discriminator and may be a marker for aggressive clinical behavior in patients with early stage breast cancer.

We employ Fourier-transform Raman spectroscopy to study normal and tumoral human breast tissues, including several subtypes of cancers. We analyzed 194 Raman spectra from breast tissues that were separated into 9 groups according to their corresponding histopathological diagnosis. The assignment of the relevant Raman bands enabled us to connect the several kinds of breast tissues (normal and pathological) to their corresponding biochemical moieties alterations and distinguish among 7 groups: normal breast, fibrocystic condition, duct carcinoma in situ, duct carcinoma in situ with necrosis, infiltrating duct carcinoma not otherwise specified, colloid infiltrating duct carcinoma, and invasive lobular carcinomas. We were able to establish the biochemical basis for each spectrum, relating the observed peaks to specific biomolecules that play a special role in the carcinogenesis process. This work is very useful for the premature optical diagnosis of a broad range of breast pathologies. We noticed that we were not able to differentiate inflammatory and medullary duct carcinomas from infiltrating duct carcinoma not otherwise specified.

Purpose: This study was designed to identify genes that could predict response to doxorubicin-based primary chemotherapy in breast cancer patients., Experimental Design: Biopsy samples were obtained before primary treatment with doxorubicin and cyclophosphamide. RNA was extracted and amplified and gene expression was analyzed using cDNA microarrays., Results: Response to chemotherapy was evaluated in 51 patients, and based on Response Evaluation Criteria in Solid Tumors guidelines, 42 patients, who presented at least a partial response (> or =30% reduction in tumor dimension), were classified as responsive. Gene profile of samples, divided into training set (n = 38) and independent validation set (n = 13), were at first analyzed against a cDNA microarray platform containing 692 genes. Unsupervised clustering could not separate responders from nonresponders. A classifier was identified comprising EMILIN1, FAM14B, and PBEF, which however could not correctly classify samples included in the validation set. Our next step was to analyze gene profile in a more comprehensive cDNA microarray platform, containing 4,608 open reading frame expressed sequence tags. Seven samples of the initial training set (all responder patients) could not be analyzed. Unsupervised clustering could correctly group all the resistant samples as well as at least 85% of the sensitive samples. Additionally, a classifier, including PRSS11, MTSS1, and CLPTM1, could correctly distinguish 95.4% of the 44 samples analyzed, with only two misclassifications, one sensitive sample and one resistant tumor. The robustness of this classifier is 2.5 greater than the first one., Conclusion: A trio of genes might potentially distinguish doxorubicin-responsive from nonresponsive tumors, but further validation by a larger number of samples is still needed.

This article reports results of the in vitro study for potential evaluation of the laser-induced fluorescence spectroscopy in the differentiation between normal and neoplastic human breast tissue. A coumarine dye laser pumped by nitrogen laser generated an excitation light centered at 458 nm. In order to collect the fluorescence signal was used an optical fiber catheter coupled to a spectrometer and CCD detector. Fluorescence spectra were recorded from normal and neoplastic (benign and malignant) human breast tissue, adding up 94 different areas. The discrimination between normal and neoplasm groups reach a sensitivity and specificity of 100%.

Ultraviolet resonance Raman spectra of bacteriorhodopsin have been obtained using 229 nm excitation from a hydrogen-shifted neodymium yttrium aluminum garnet (Nd: YAG) laser. High signal-to-noise spectra are observed exhibiting vibrational bands at 762, 877, 1011, 1175, 1356, 1552 and 1617 cm-1 which are assigned to scattering from tryptophan and tyrosine side chains. This demonstrates the feasibility of using UV resonance Raman spectroscopy to monitor aromatic amino acid structural changes during the bacteriorhodopsin photocycle.