Contract no.: IDEI345/2011
Project Manager: Marius RAICA

Short description

Validation of human breast cancer molecular classification. Classification of malignant breast tumors included in the present study based on the accepted markers used for malecular stratification and establishment of subgroups based on ER, PR, Ki67 and HER2/neu expression. This objective is crucial for the project because the distribution of positive and negative cases is less studied until now and proliferation marker Ki67 is not included in the present classification. It si necessary to compare the molecular profile of the malignant breast cancer with molecular profile of normal mammary tissue and benign tumors and to predict the intermediate steps of carcinogenesis linked to these profiles. Study of the not well known characterized markers of molecular classification: androgen receptors, CD117, p53, Bcl-2, E- /P-cadherin, PSA, mammaglobin A, GCDFP-15, BAX, ApoBrdu and anti-thymine dimers expression.

Project Abstract

Breast cancer is a heterogeneous disease and five major distinct molecular types have been characterized by gene analysis and immunohistochemistry. The molecular types of breast cancer have different behavior, a particular profile of response to therapy, reflected in the differential survival of patients. Breast tumors have been classified based on their gene expression profile and immunohistochemical expression of hormone receptors, HER2, cytokeratin 5/6, epidermal growth factor receptor(EGFR), p53 and BCL-2. This classification recognized basal-like, luminal, HER2 and unclassified types. Although these data bring new information on the biology of breast cancer, the molecular profile of different types is far to be complete. This concept emerged many years ago, but only in last five years it was demonstrated by gene analysis and IHC. In these conditions, there are some problems that wait to be solved: if the profile of breast cancer differs from the normal mammary tissue, if it has predictive value for progression/metastases and for the response to adjuvant therapy. There were identified 5 distinct types by genomic analysis: ER positive that include luminal A and B types, and ER negative that include basal-like, HER2 and unclassified types. It is not clear if the last type represents a distinct entity or includes itself subtypes not yet characterized. Gene analysis results were overlapped on IHC, and therefore, IHC may be used as a surrogate method in the current diagnosis of these types, mainly because it can be performed in a large number of cases. Basal-like carcinoma is more frequent in premenopausal women, is characterized by the absence of ER/PR/HER2 expression and positive reaction for CK5/6 and/or EGFR. Based on the IHC profile these tumors are also called triple negative, they show a high rate of p53 mutation, and are more frequentrly found in BRCA1 carriers. It is thought that basal-like carcinoma represents 8 to 20% from all breast cancers, is associated with high grade, high prognostic score, rarely disseminates in axillary lymph nodes, liver and bone, but develops brain and lung metastases. The majority of medullary carcinoma also belong to this type, based on the expression of CK5/6 and EGFR. Some tumors of this type also express CK8/18, which led to a particular subtype, called baso-luminal, but which is not characterized and it is not known if it really exists. The triple negative profile is not enough to characterize the basal-like carcinoma, additional methods are need (as vimentin, CK14, CK17, EGFR), but significance of present results is uncertain. The most available specific markers are thought to be vimentin and CK5/6, and both correlate with bad prognosis. Although the basal-like carcinoma shows some distinctive features, at present it is not clear if it represents an individual entity or includes many subtypes, and moreover, specific precursors are largely unknown. A controversial aspect refers to as the unfavorable prognosis, with high rate of recurrence and metastases, apparently independent from the lymph node status in the moment of diagnosis and size of the tumor. Luminal breast carcinoma is characterized by the expression of ER, PR, Bcl-2 and CK8/18, and additional, GATA-3. Luminal tumors are usually well or moderate differentiated, and does not express HER2. Luminal subtype A (56-61% of all breast carcinoma) is associated with relatively good prognosis. This subtype includes the majority of lobular carcinomas (in situ and invasive). Luminal B subtype (9-16%) can show a more aggressive phemotype with high grade, and besides ER/PR, express EGFR, HER2 and cyclin E1. HER2 type (8-16%) includes 2 distinct subtypes based on the expression of ER: ER negative close to the basal-like carcinoma (HER2+ ER-) and ER positive, which clusters with luminal B type (HER2+ ER+). Unclassified carcinoma (6-10%) is a triple negative tumor, close to the basal-like carcinoma and HER2 type, but which does not respond to neoadjuvant chemotherapy. In this type were included all tumors without expression of specific markers. Triple negative tumors do not express ER, PR and HER2 and represent 10-17% of all breast carcinomas, and 96% from them are G3. The aggresive character of triple negative tumors is demonstrated by the increased rate of recurrence in first 1 to 3 years, and many specific deaths in first 5 years after therapy. It becomes evident that triple negative tumors constitute a heterogeneous group, 56-84% express CK5/6 and EGFR, do not respond to hormone therapy, and the only available method, with partial efficiency, is chemotherapy (although the rate of proliferation and mutation of TP53 gene are frequent). The most recently identified tumor group is the claudin-low subtype. The subtype name is based on the defining feature of this group, specifically that these tumors exhibit low expression of many of the claudin genes, including 3, 4, and 7. The claudin-low tumors are also triple negative, and so can be considered another subtype of triple-negative disease, in addition to the basal-like group. Other important features of claudin-low tumors is that they almost always have an intense immune cell infiltrate, and they also have stem cell features and features of epithelial–mesenchymal transition (EMT). The claudin-low group represents the most primitive tumors that are the most similar to the mammary stem cell, and the next step on the pathway is what is sometimes referred to as the luminal progenitor, which is the basal-like phenotypeExtensive evaluation of the molecular types of breast cancer has shown significant differences not only in their molecular profile, but also in their metastasis susceptibility. Previous findings showed a particular preference for lymph node and distant metastases of different molecular types, but the specific lymphangiogenic profile of these types is less characterized.

Lymphangiogenesis in breast cancer was extensively investigated in recent years, and lymphatic microvascular density (LMVD) has been shown to be a good predictor of lymph node metastasis. Although the role of vascular endothelial growth factor-C and D (VEGF-C and VEGF-D), and their binding to the cognate receptor VEGFR-3 in the induction of lymphangiogenesis is largely accepted, their predictive value for lymph node metastasis in breast cancer is still controversial. Recently, it has been shown that lymphangiogenesis assessed by LMVD predicts lymph node metastasis in ductal carcinoma in situ with microinvasion. Few data are available concerning the specific profile of lymphangiogenesis in different molecular subtypes of breast cancer, despite it has been shown that their metastatic behaviour is different. More recently, it has been shown that triple-negative breast cancer correlates with higher LMVD and expression of VEGF-C and D. The HER2 subtype is one of the most aggressive molecular variants of breast cancer, frequently associated with lymph node metastasis and poor prognosis. The aggressive behaviour of these tumors may be explained in part by VEGF-C expression in tumor cells. More recently, Schoppmann et al. have shown that HER2 overexpression is associated with high VEGF-C expression and LMVD. These data support the clinical relevance of the association between HER2 and VEGF-C expression, and blocking HER2 may reduce not only tumor progression, but also lymphangiogenic metastasis. Recently, indirect evidences of claudin involvement in lymphangiogenesis from breast cancers were done by Szasz and co. who reported that lymph node metastases developed a notable increase of claudin-5. Also, they observed that the expression of claudins, is different in primary breast carcinomas compared to their lymph node metastases as demonstrated by immunohistochemistry. The study was done on conventional invasive ductal and lobular carcinomas, not on molecular subtypes of breast cancer. In other tumor types, recent evidences have shown that tumor cells gain entry to lymphatics by using VE-cadherin and tight junction proteins including claudin-5 expressed at the initial openings of lymphatics. The problem of the basic mechanisms involved and the strategy for an anti-lymphangiogenic therapy is even more complicated, as it was shown that the neutralizing anti-VEGF-A antibody suppresses tumor lymphangiogenesis and reduces VEGFR-3 expression in an orthotopic breast tumor model. Although a lot of evidences has been accumulated in recent years to support the contribution of the VEGF C /VEGFR-3 axis to lymphangiogenesis in breast cancer, the clinical significance remains controversial and the results of experimental studies have not yet been translated into practice.

In the light of the above mentioned known data, several questions remain to be solved about lymphangiogenesis in molecular types of breast cancer as:

1. onset time of lymphangiogenesis in breast cancer;
2. if lymphangiogenesis mechanism are the same for all molecular types of breast cancer or different;
3. molecular markers of lymphangiogenesis differentially described in molecular types of breast cancer;
4. role of angiogenic growth factors (VEGF A; PDGF and their receptors) in lymphangiogenesis from molecular types of breast cancer;
5. dynamic features of lymphangiogenesis appearance in cases with or without metastasis (lymph node first or tumor first, or both);
6. lymphatic endothelial cells proliferative index and lymphatic progenitor cells involvement in lymphangiogenesis from molecular types of breast cancer;
7. claudin involvement in the inititation and sustaining of lymphangiogenesis in primary breast tumors and their nodal metastasis.

Objectives

1. Validation of human breast cancer molecular classification. Classification of malignant breast tumors included in the present study based on the accepted markers used for malecular stratification and establishment of subgroups based on ER, PR, Ki67 and HER2/neu expression. This objective is crucial for the project because the distribution of positive and negative cases is less studied until now and proliferation marker Ki67 is not included in the present classification. It si necessary to compare the molecular profile of the malignant breast cancer with molecular profile of normal mammary tissue and benign tumors and to predict the intermediate steps of carcinogenesis linked to these profiles. Study of the not well known characterized markers of molecular classification: androgen receptors, CD117, p53, Bcl-2, E- /P-cadherin, PSA, mammaglobin A, GCDFP-15, BAX, ApoBrdu and anti-thymine dimers expression.
2. Claudins expression in molecular types of breast cancer and their involvement in tumor progression, lymphangiogenesis and nodal metastasis. We will use the retrospective and prospective cases to find correlations between conventional histopathology, grade and claudins expressions followed by study of claudins in molecular types of breast cancer and its correlation with lymph node metastasis and lymphangiogenesis. Also, it will be evaluated the presence and distribution of stem cells among molecular types of breast cancer correlated with claudins expression.
3. haracterization of lymphangiogenesis in accepted molecular types of breast cancer and their nodal metastasis and defining the new potential therapeutic targets. This objective will include the assessement of lymphatic microvascular density by immunohistochemistry (with D2-40), specific for each molecular type of breast cancer. This will be followed by evaluation of angiogenic growth factors (VEGF A, bFGF, PDGF, EG VEGF and their receptors-VEGFR2, FGFR2, PDGFRalpha, beta) and lymphangiogenic growth factors (VEGF C, VEGF D, and correspondent receptor VEGFR3), proliferative index of lymphatic endothelial cells and PROX1 expression in molecular types of breast cancer.
4. To establish an experimental model for each molecular type necessary to test the chemotherapy and anti-lymphangiogenic drugs efficiency alone or in combination with Rapamycin. Several breast cancer cell lines will be used to design experimental models of breast tumors on chick embryo chorioallantoic membrane. These models will represent an experimental support for testing antitumor and anti-lymphangiogenic agents.
5. To disseminate the research results by publication of research articles and reviews in ISI journals and also by participation at national and international conferences.

Publication List

1. Raica M, Cimpean AM, Ceausu R, Ribatti D, Gaje P. Interplay between mast cells and lymphatic vessels in different molecular types of breast cancer. Anticancer Res. 2013 Mar;33(3):957-63. PMID:23482767 [PubMed - indexed for MEDLINE] Read more
2. Comşa S, Ciuculescu F, Raica M. Mesenchymal stem cell-tumor cell cooperation in breast cancer vasculogenesis. Mol Med Rep. 2012 May;5(5):1175-80. doi: 10.3892/mmr.2012.796. Epub 2012 Feb 17. PMID: 22367291 [PubMed - indexed for MEDLINE] Read more
3. Avram SF, Cimpean AM, Raica M. Behavior of the P1.HTR mastocytoma cell line implanted in the chorioallantoic membrane of chick embryos. Braz J Med Biol Res. 2013 Jan;46(1):52-7. PMID: 23314344 [PubMed - in process] Read more
4. ŞERBAN COMŞA, ANCA MARIA CIMPEAN, RALUCA CEAUŞU,CRISTIAN SUCIU and MARIUS RAICA. CORRELATIONS BETWEEN VASCULAR ENDOTHELIAL GROWTH FACTOR EXPRESSION, MICROVASCULAR DENSITY IN TUMOR TISSUES AND TNM STAGING IN BREAST CANCER. Arch. Biol. Sci., Belgrade, 64 (2), 409-417, 2012 Read more

CONTACT

Project Manager: Marius RAICA

Office address:
Post Address: Piata E. Murgu 2, 300041 Timisoara, Romania
Tel/Fax: +40 256 220482 int. 484, 490

E-mail: raica@umft.ro
Office Hours: 12:30 to 14:30, Monday to Friday