Irmgard Costa

Histopathologic characterization of mammary neoplastic lesions induced with 7,12 dimethylbenz(alpha)anthracene in the rat: a comparative analysis with human breast tumors.

CONTEXT The dimethylbenz(alpha)anthracene (DMBA) breast cancer model induced in the rat is used for the study of mammary carcinogenesis because it closely mimics human breast disease. OBJECTIVE To analyze the histopathologic features of mammary carcinomas induced in the DMBA experimental model, in a manner similar to that used in human pathology, to allow a comparative analysis between both systems. DESIGN Three experimental series of 20 animals were used. At 53 days of age, a single dose of 5 mg of DMBA per rat was given. Mammary tumors were collected when the rats were killed. Several histopathologic parameters were studied. For grading, the parameters described in the modified Scarff-Bloom-Richardson scheme were used, adapted to rat mammary tumors. RESULTS More than 50% of the carcinomas presented a pattern grade I, a nuclear grade I or II, and fewer than 10 mitoses/10 high-power fields (P <.05). Although the tumors were generally well differentiated, they showed a range of differentiation. More than 85% of carcinomas did not display tumoral necrosis (P <.05). This feature was observed mostly in high-grade carcinomas. There was no or scanty lymphoplasmacytic infiltration in more than 70% of carcinomas (P <.05). The degree of infiltration increased with the histologic grade. Microcalcifications were found rarely (P <.05). The carcinomas exhibited a mixed structural pattern, most with a predominant cribriform pattern (P <.05). No or light (+) stromal response was seen in most cases (P <.05). Some carcinomas, especially when poorly differentiated, presented a desmoplastic reaction. Most carcinomas presented scanty mast cell infiltration (P <.05), no features of secretion (P <.05), and absence of microcribriform pattern (P <.05). These features were seen more often in low-grade carcinomas. CONCLUSIONS Despite the presence of some structural differences, rat mammary adenocarcinomas and the most common human breast carcinomas share several morphologic similarities. Moreover, some features could be related to the aggressive behavior of the tumor. The analysis carried out in this study, similar to that done in human pathology, allows a more extensive understanding of mammary tumors in rats, as well as a more accurate use of this animal model, and has made it possible to develop an innovative classification of rat mammary lesions.

K-ras mutations in nonmucinous ovarian epithelial tumors†

To assess the putative prognostic value of K‐ras mutations in nonmucinous ovarian tumors, the authors looked for K‐ras point mutations at codons 12 and 13 in 144 nonmucinous ovarian tumors.

Identification of novel differentially expressed genes by the effect of a high-fat n-6 diet in experimental breast cancer

In previous studies, we demonstrated that high corn oil diets promote the development of 7,12‐dimethylbenz(α)anthracene (DMBA)‐induced mammary tumors. In this study, we have investigated whether modulation of gene expression is one of the mechanisms by which this high‐fat diet exerts such effects. Female Sprague‐Dawley rats were induced with DMBA and fed normolipidic (3% corn oil) or high‐fat (20% corn oil) diet. Screening of genes differentially expressed in adenocarcinomas from the high corn oil diet group compared to the control diet group was performed with cDNA microarrays. The resulting six upregulated and nine downregulated genes were validated by Northern blot and/or reverse transcription (RT)‐polymerase chain reaction (PCR). Further investigation in a higher number of adenocarcinomas showed that in the high‐fat n‐6 diet group, where the tumor phenotype was verified to be more aggressive, the expression of submaxillary gland α‐2u globulin, vitamin D3‐upregulated protein 1 (VDUP1), H19, and the unknown function gene that codifies the expressed sequence tag (EST)‐Rn.32385 was significantly decreased in comparison with the control group (C). These results, together with the fact that VDUP1, H19, and this globulin have been associated with cell proliferation and differentiation, open a new line of research about how the underexpression of these genes contributes to the stimulating effect of a high corn oil diet on experimental mammary carcinogenesis.

High-Fat Corn Oil Diet Promotes the Development of High Histologic Grade Rat DMBA-Induced Mammary Adenocarcinomas, While High Olive Oil Diet Does Not

Effects of a high corn oil and a high olive oil diet on the histopathologic characteristics of rat dimethylbenz(α)anthracene-induced mammary adenocarcinomas were investigated in comparison with those of a control low-fat diet. Two experimental series (A and B) studied the influence of a high corn oil diet on the initiation and the promotion of mammary carcinogenesis, while another one (C) assessed the effects of the two dietary lipids on the promotion. Nine parameters have been analyzed and a new histologic grading method, adapted to rat tumors, has been applied in each carcinoma. High corn oil diets, particularly when acting as promoters, associated with higher-grade carcinomas than control (p < 0.05) and high olive oil groups. Stromal invasion and tumoral necrosis were more prominent and a prevailing cribriform pattern was observed (p < 0.05). High olive oil diet adenocarcinomas exhibited a predominantly low histologic grade and few necrotic and invasive areas, similar to the control, and they presented the highest percentage of papillary areas. Lymphoplasmacytic and mast cell infiltration were also influenced by the dietary lipids. Thus, high corn oil diet adenocarcinomas presented a higher degree of morphological malignancy than control and high olive oil tumors, which is in line with the greater clinical malignancy described in rats from the former group and the non-promoting effect of the high olive oil diet. As far as we are concerned, a similar histopathologic approach of the effects of the dietary lipids on experimental breast cancer has not been carried out up to now.

Epithelial to mesenchymal transition markers are associated with an increased metastatic risk in primary cutaneous squamous cell carcinomas but are attenuated in lymph node metastases

BACKGROUND Cutaneous squamous cell carcinoma (cSCC) is the second most common malignancy in humans and approximately 5% metastasize, usually to regional lymph nodes. Epithelial to mesenchymal transition (EMT) is a process involving loss of intercellular adhesion, acquisition of a mesenchymal phenotype and enhanced migratory potential; epithelial markers, such as E-cadherin, are down-regulated and mesenchymal proteins (Vimentin), increased. OBJECTIVE To investigate the expression of EMT markers in metastatic SCC (MSCC) and their corresponding metastases, and to correlate them with clinico-pathological factors associated with an increased risk of metastasis. METHODS We performed a retrospective study that included 146 cSCC samples (51 primary non-metastatic, 56 primary metastatic, 39 lymphatic metastases). Immunohistochemistry for E-cadherin, Vimentin, Snail, beta-catenin, Twist, Zeb1 and Podoplanin was performed. RESULTS Loss of membranous E-cadherin was observed in 77% cSCCs, with no differences between MSCC and non-MSCC. Among the transcriptional factors controlling EMT, no significant Snail1 expression was detected. Twist, Zeb1, Vimentin, beta-catenin and Podoplanin were significantly overexpressed in MSCCs. Twist ectopic expression in SCC13 cells induced Zeb1, Vimentin and Podoplanin expression and E-cadherin delocalization. These changes resulted in a scattered migration pattern in vitro. Expression of EMT markers was decreased in the metastases when compared with the corresponding primary tumors. CONCLUSION These results suggest that a partial EMT, characterized by the expression of Twist but without a total E-cadherin depletion, is involved in the acquisition of invasive traits by cSCC, but the process is downregulated in lymph node metastases.

Diets High in Corn Oil or Extra-Virgin Olive Oil Provided From Weaning Advance Sexual Maturation and Differentially Modify Susceptibility to Mammary Carcinogenesis in Female Rats

Based on the importance of early-life events in breast cancer risk, we have investigated the effects of high-fat diets on maturation, mammary gland development, and its susceptibility to transformation. Female Sprague-Dawley rats were fed a lowfat (LF), high corn oil (HCO), or high extra-virgin olive oil (HOO) diet from weaning and gavaged with 7,12-dimethylbenz[a]anthracene. Body weight and mass increased in the HCO group compared to the LF group. The vaginal opening was advanced in both high-fat groups, especially in the HCO group. This HCO group also had increased body weight around puberty, more corpora lutea at post-puberty, and tended to have higher kisspeptin levels in the hypothalamus. Both high-fat diets induced subtle modifications in the morphology of the mammary gland, with no changes on β-casein or hormone receptors expression in the gland. The HCO diet had a clearly stimulating effect of carcinogenesis, inducing the earliest appearance of tumors and the highest tumor incidence and yield, whereas the HOO diet seemed to have a weak enhancing effect, increasing tumor yield. Our data suggest a strong influence of the HCO diet in sexual maturation and mammary cancer risk, while rats fed the HOO diet were more similar to the controls.

D2-40 immunohistochemical overexpression in cutaneous squamous cell carcinomas: A marker of metastatic risk

BACKGROUND Approximately 4% of cutaneous squamous cell carcinomas (cSCCs) develop lymphatic metastases. The value of lymphatic endothelial markers to enhance the detection of lymphatic tumor invasion in cSCC has not been assessed previously. OBJECTIVE We sought to evaluate the use of the antibody D2-40, a podoplanin immunohistochemical marker, to identify tumor lymph vessel invasion in cSCC and to assess its expression in tumor cells. METHODS This was a retrospective case-control study. A series of 101 cSCC, including 51 cases that developed lymphatic metastatic spread (metastasizing cSCC [MSCC]) and 50 cases that resolved definitely after surgical excision (non-MSCC) were included in the study. Lymph vessel invasion using D2-40 was evaluated on all primary biopsy specimens. The percentage of tumor cells showing D2-40 positivity and intensity scoring were recorded. All the immunohistochemical findings were correlated with the clinicopathological features. RESULTS Lymph vessel invasion was observed in 8% of non-MSCCs and in 25.5% of MSCCs (P = .031). D2-40 expression was significantly increased, both in intensity (odds ratio 4.42 for intensity ++/+++) and in area (odds ratio 2.29 for area >10%), in MSCC when compared with non-MSCC. Interestingly, almost half (49%) of the MSCC had moderate to intense D2-40 positivity compared with 16% of non-MSCC. D2-40 immunohistochemical expression was increased in tumors with an infiltrative pattern of extension. In the multivariate analysis, histologically poorly differentiated tumors, recurrent lesions, and cSCC showing D2-40 overexpression (in intensity) were significantly associated with lymphatic metastases development (odds ratios 15.67, 14.72, and 6.07, respectively). LIMITATIONS This was a retrospective study. CONCLUSION The expression of podoplanin associates with high metastatic risk in cSCC.

Deregulated expression of the PCPH proto-oncogene in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene

The PCPH proto‐oncogene was identified by its frequent activation in Syrian hamster fetal cells exposed to 3‐methylcholanthrene. We previously isolated human PCPH cDNA and studied its expression in normal human tissues. We report herein the pattern of PCPH expression in normal rat tissues. Each tissue expressed one major PCPH polypeptide that varied in molecular mass in different tissues. Normal mammary gland expressed a single PCPH polypeptide of 27 kDa. This PCPH form also was expressed in lactating mammary glands but at significantly greater levels. These results suggest the existence of tissue‐specific regulatory mechanisms for PCPH expression that may be influenced by the differentiation stage. Our previous studies on the involvement of PCPH in human cancer showed that human breast tumor cell lines have frequent alterations in PCPH, including multiple PCPH polypeptide forms that are not expressed in normal cells. These cell lines also have frequent loss of a 27‐kDa form identified as the only PCPH polypeptide expressed by normal human breast epithelial cells. In this study, we found that these same alterations occurred in vivo during mammary carcinogenesis in Sprague‐Dawley rats treated with 7,12‐dimethylbenz[a]anthracene, in both benign and malignant tumors, indicating that stable changes in PCPH expression took place early in the neoplastic process. Results showed that this experimental system is relevant to human breast carcinogenesis and provides an excellent model to study the molecular basis of the regulation of PCPH expression during normal differentiation and pathologic stages of neoplasia of the mammary gland and to analyze the role of PCPH in the carcinogenic process. Furthermore, the detection of atypical PCPH polypeptides in tumors suggests that PCPH immunodetection may be applied as a diagnostic tool for the early identification of neoplastic breast epithelial cells.

Dietary extra-virgin olive oil and corn oil differentially modulate the mRNA expression of xenobiotic-metabolizing enzymes in the liver and in the mammary gland in a rat chemically induced breast cancer model.

High extra-virgin olive oil (EVOO) and corn oil diets differentially modulate experimental mammary carcinogenesis. We have investigated their influence on the initiation stage through the modulation of the expression of xenobiotic-metabolizing enzymes (XMEs) in the liver and the mammary gland. Female Sprague–Dawley rats were fed a low-fat (LF), high corn oil (HCO), or high EVOO (HOO) diet from weaning and gavaged with 7,12-dimethylbenz(a)anthracene (DMBA). The HCO diet increased the mRNA levels of the phase I enzymes CYP1A1, CYP1A2 and, to a lesser extent, CYP1B1, in the liver. The Aryl hydrocarbon receptor (AhR) seemed to be involved in this upregulated CYP1 expression. However, a slight trend toward an increase in the mRNA levels of the phase II enzymes GSTP1 and NQO1 was observed with the HOO diet. At least in the case of GSTP1, this effect was linked to an increased Nrf2 transactivation activity. This different regulation of the XMEs expression led, in the case of the HCO diet, to a balance between the production of active carcinogenic compounds and their inactivation tilted toward phase I, which would stimulate DMBA-induced cancer initiation, whereas the HOO diet was associated with a slower phase I metabolism accompanied by a faster phase II detoxification, thus reducing the output of the active compounds to the target tissues. In the mammary gland, the differential effects of diets may be conditioned by the state of cell differentiation, sexual maturity, and hormone metabolism.

High corn oil and extra virgin olive oil diets and experimental mammary carcinogenesis: clinicopathological and immunohistochemical p21Ha-Ras expression study.

Dietary lipids have a role in the aetiology of breast cancer, acting at several cellular levels. We investigated the effects of a high corn oil and a high extra virgin olive oil diet on the clinical and histopathological characteristics of rat dimethylbenz(α)anthracene-induced mammary carcinogenesis and on the expression of p21Ha-Ras, detected by immunohistochemistry, in one experimental series including a low-fat corn oil diet (LFCO) and two high-fat diet groups: HFCOP, rich in corn oil, and HFOOP, rich in extra virgin olive oil. Whereas the high corn oil diet tended to reduce latency time, to raise tumour incidence and to increase total tumour yield, the high extra virgin olive oil diet led to a latency time similar to that of LFCO and to a lower tumour incidence than HFCOP and lower total tumour yield, even than LFCO. HFCOP tumours displayed a higher histological grade and profile than LFCO tumours, while adenocarcinomas in HFOOP were similar to LFCO ones. Although no significant differences in p21Ha-Ras expression among dietary groups was found, we detected a significant p21Ha-Ras decreasing expression as grade increased, in groups LFCO and HFCOP. HFOOP tumours exhibited a higher staining in high-grade carcinomas compared to the similar malignant tumours of the two other dietary groups. These data suggest that dietary lipids influence the clinical behaviour and the morphological malignancy of the experimental mammary carcinogenesis, according to the type of fat, without altering p21Ha-Ras expression. Nevertheless, this expression could be affected by the malignancy of tumours, probably through a post-translational event.