Prognostic value of NDRG1 and SPARC protein expression in breast cancer patients.

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.

Dietary isoflavone intake, polymorphisms in the CYP17, CYP19, 17beta-HSD1, and SHBG genes, and risk of breast cancer in case-control studies in Japanese, Japanese Brazilians, and non-Japanese Brazilians.

We tested the hypothesis that polymorphisms in cytochrome P450c17alpha (CYP17), aromatase (CYP19), 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1) and sex hormone-binding globulin (SHBG) genes may modify the association between isoflavone intake and breast cancer risk. We conducted hospital-based, case-control studies in Nagano, Japan and Sao 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), 17beta-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 17beta-HSD1 and SHBG genes may modify the association between isoflavone intake and breast cancer risk.

Isoflavone, polymorphisms in estrogen receptor genes and breast cancer risk in case-control studies in Japanese, Japanese Brazilians and non-Japanese Brazilians.

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.

Dietary isoflavone intake and breast cancer risk in case-control studies in Japanese, Japanese Brazilians, and non-Japanese Brazilians.

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.

Gene expression profile of residual breast cancer after doxorubicin and cyclophosphamide neoadjuvant chemotherapy.

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.

C-erbB-2 expression is a better predictor for survival than galectin-3 or p53 in early-stage breast cancer.

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.

Biochemical analysis of human breast tissues using Fourier-transform Raman spectroscopy.

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.

Gene expression profile associated with response to doxorubicin-based therapy in breast cancer.

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.

A multivariate analysis on prognostic factors for lobular carcinoma of the breast.

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.

Down-regulation of the candidate tumor suppressor gene PAR-4 is associated with poor prognosis in breast cancer.

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.

Down-regulation of PHLDA1 gene expression is associated with breast cancer progression.

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.

A multivariate analysis on prognostic factors for lobular carcinoma of the breast / Uma análise multivariada de fatores prognósticos para o carcinoma lobular de mama

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.

CONTEXTO E OBJETIVO: O carcinoma lobular é o segundo tipo de neoplasia mais frequente na mama e tem características clínicas e patológicas próprias. Nosso objetivo foi avaliar fatores prognósticos para esse tipo de câncer de mama. TIPO DE ESTUDO E LOCAL: Estudo retrospectivo em instituição terciária oncológica. MÉTODOS: 162 pacientes diagnosticadas e tratadas entre janeiro de 1985 e janeiro de 2002 foram incluídas. Os critérios de inclusão foram: ausência de tratamento prévio, diagnóstico histológico de carcinoma lobular, ausência de história prévia de câncer de mama e acompanhamento mínimo de 36 meses. RESULTADOS: Em análise univariada, os seguintes fatores foram estatisticamente significativos: estágio T clínico (P = 0,0005), estágio N clínico (P = 0,0014), quimioterapia neoadjuvante (P = 0,0008), tamanho do tumor primário (P < 0,0001), invasão vascular (P < 0,0001), invasão linfática (P = 0.0004), invasão neural (P = 0,0004), invasão de pele (P < 0,0001), transposição capsular (P = 0,0008), relação linfonodal (P < 0,0001), expressão de receptor estrogênico (P = 0,0168), expressão de receptor de progesterona (P = 0,0286), estágio T patológico (P < 0,0001), estágio N patológico (P < 0,0001), quimioterapia adjuvante (P < 0,0001) e hormonioterapia pós-operatória (P = 0.0367). Agrupando-se as variáveis, realizou-se análise multivariada. Presença de metástases linfonodais, transposição capsular, razão linfonodal e hormonioterapia pós-operatória permaneceram significantes. CONCLUSÃO: Nesta série, os fatores prognósticos mais importantes para carcinoma lobular de mama parecem ser aqueles relacionados com status linfonodal e presença de transposição capsular. Fatores relacionados ao comprometimento axilar, transposição capsular e terapia hormonal foram significativos para sobrevida.

Intracystic papillary carcinoma of the breast: risks in diagnostic, staging and treatment

Intracystic papillary carcinoma (IPC) of the breast is a rare form of breast in situ carcinoma that comprises 0.5-2% of all breast tumors. IPC of the breast occurs in women around the sixth decade of life and presents an injury of great dimensions, more frequently retroareolar. Clinical and radiological findings can be confused with advanced-staged invading carcinomas (T2 or T3), leading to unnecessarily aggressive treatment. The correlation of clinical, radiological and anatomopathological findings is necessary for a correct diagnosis. We present two cases of IPC and discuss diagnostic characteristics.