A Study On Molecular Classification And Its Association With Prognosis In Breast Cancer

Objective:To summarize clinicopathological and morphological features of basal-like breast carcinoma (BLBC); To perfect the standards of immunohistochemistry (IHC) classification in breast cancer. To evaluate the prognostic differences among subtypes of breast cancer using four IHC classifications (CK/TN, TN, CK, ER/HER2), and guide clinical treatment and prognosis evaluaton; To study GEP features of breast cancer in Chinese people and screen differentially expressed genes among subtypes of breast cancer; To validate several differentially expressed genes of triple negative breast cancer (TNBC) and analyze their correlation with clinicopathological parameters and prognosis, and provide new clue of looking for specific molecular markers and targeted therapy of TNBC.Materials and methods:Partâ… :(1) The formalin-fixed paraffin-embedded samples of 543 cases of breast cancer with complete clinicopathologic and follow-up data were collected in the Department of Pathology, Cancer Hospital, Fudan University. The cases were initially diagnosed between 1997 and 2002. Immunohistochemical staining of ER, PR, HER2, CK5/6, CK14, CK17, EGFR, SMA, P63, vimentin was performed. Four classifications of CK/TN, TN, CK, ER /HER2 were used to IHC phenotyping analysis. (2)The clinicopathological and morphological characteristics of BLBC were summarized; the expression of basal-like and myoepithelial markers were analyzed in BLBC. (3) The relationship between clinicopathological, morphological, immunohistochemical parameters and prognosis in BLBC was investigated by survival analysis. The prognostic differences of breast cancer subtypes were compared using CK/TN, TN, CK, ER/HER2 classifications.Partâ…¡:(1) The frozen tissues and corresponding formalin-fixed paraffin-embedded samples of 50 cases of breast cancer from 2002-2009 were obtained from Cancer Hospital, Fudan University. Immunohistochemistry study of ER, PR, HER2, CK5/6, CK14, CK17 and EGFR was performed. According to CK/TN classification, the cases were divided into five subtypes:luminal-A, luminal-B, HER2-overexpression, basal-like and null subtype. (2)The total RNA of 50 frozen samples was extracted and purified, and RNA quality was controlled by agarose gel electrophoresis and Alignent 2100 bioanalyzer. (3)Gene expression profiling of 50 cases of breast cancer was studied using genomewide microarray technology. The GEP classification was completed by hierarchical clustering analysis, to study GEP characteristics of Chinese breast cancer. Differentially expressed genes between different subtypes were screened and their biological functions were analyzed by Pathway and Gene Ontology (GO) analysis.Partâ…¢:(1) The frozen tissue samples of 92 cases of breast cancer acquired from 2002-2009 were collected in Cancer Hospital, Fudan University. The expression of SLC6A14, PTX3, FOXC1 mRNA in 92 cases were detected by Real-time RT-PCR, and the expression of these genes was compared between TNBC and luminal subtype to validate the results of microarray. (2) The formalin-fixed paraffin-embedded samples of 94 cases of breast cancer with complete clinicopathologic and follow-up data acquired from 1997 to 2003 were collected, including BLBC (57 cases) and luminal subtype (37 cases). The expressin of FOXC1 protein was detected by IHC staining, to compare its expression difference between TNBC and luminal subtype and study its association with clinicopathologic parameters and prognosis in breast cancer.Results:Partâ… :(1) According to CK/TN classification, BLBC, luminal-A, luminal-B HER2-overexpression and null subtype constituted 19.9%,42.2%,10.5%,16.6%,10.9% of breast cancer, respectively. (2) The average tumor size was 2.9cm, significantly bigger than luminal-A (P>0.05). The metastasis rate of lymph node was 47.6%, lower than luminal-A, HER2-over expression and null type.75.1% of BLBCs were histologically grade 3, higher than other non-basal-like subtypes. (3) The histopathologic features of BLBC: 70.4% of BLBCs showed expansive growth,72.2% showed diffusely solid pattern; Syncytial cells, vesicular nuclei and prominent nucleoli, nuclei of grade 3 and metaplasia was seen in 67.3%,61.1%,58.3%,74.1%,21.3% of BLBC, respectively. The necrosis rate of BLBC was high and predominant of geographic and central necrosis. The incidence of geographic necrosis was significantly higher than luminal and HER2-overexpression type. (4)The expression rate of CK5/6 was highest among basal-like markers, up to 63.0%. The expression rate of CK17, CK14, EGFR was 49.1%,47.2%,37.0%, respectively. The expression rate of myoepithelial markers was 23.1%. (5) Survival analysis:(a) The disease-free survival and overall survival of 543 cases were 61.7% and 80.8% respectively. Local recurrence, distant metastasis and fatality rate were 9.8%,34.8%,19.2%, respectively. (b)The recurrence rate was highest in null type (18.6%), the second was BLBC. Pulmonary and brain metastasis rate of BLBC were 22.2% and 10.2% respectively, higher than other subtypes. (c) Univariate analysis demonstrated the following as poor prognostic factors for overall and disease-free survival:high clinical staging, positive lymph node, vascular invasion, positivity with CK5/6; the following as good prognostic factors for overall and disease-free survival:expansive growth and lymphocytic infiltrates. Multivariate analysis indicated that lymphocytic infiltrates and expression of CK5/6 were independent prognostic factors of BLBC. (3) The evaluation results of prognosis were essentially consistent using CK/TN, TN, ER/HER2 classifications, but CK classification was different from other three classifications.Partâ…¡:(1) The purity and integrity of RNA in all samples was good. (2) 50 cases of breast cancer can be divided into three groups predominant of luminal-A, HER2-overexpression breast cancer (including HER2-overexpression subtype and luminal-B) and TNBC. There was similar expression pattern between HER2-overexpression and luminal-B, basal-like and null subtype. (3) TNBC, non-TNBC, luminal-A subtype defined by IHC was consistent with GEP classification, there was certain overlapping in distinguishing luminal-A and luminal-B, luminal-B and HER2-overexpression type, HER2-overexpression and TNBC, basal-like and null subtype.(4)Screening of differentially expressed genes showed that the number of differentially expressed genes of luminal-A vs. luminal-B subtype, luminal-A vs. HER2-overexpression subtype, luminal-B vs. HER2-overexpression subtype, TNBC vs. non-TNBC, TNBC and luminal subtype was 190,137,8,611,411, respectively.Partâ…¢:(1) The expression of SLC6A14, PTX3 and FOXC1 mRNA in TNBC and luminal subtypes of breast cancer is consistent in Real-time RT-PCR and microarray detection. (2) The expression of SLC6A14 and FOXC1 mRNA in TNBC was significantly higher than luminal subtype of breast cancer. There was no significan difference of PTX3 mRNA expression between TNBC and luminal subtype of breast cancer. (3) No relationship was found between SLC6A14, PTX3 and FOXC1 mRNA expression in TNBC and other clinicopathologic factors of such as tumor size, histological grade, clinical stage, lymph node metastasis and age of patients. (4) FOXC1 protein was located in the cytoplasm and was occasionally seen in the cell membrane. The normal mammary epithelium and myoepithelium were strongly positive for FOXC1 protein. The epresssion rate of FOXC1 protein in TNBC and BLBC was significantly higher than luminal subtype of breast cancer (66.7% vs.32.4%; 76.6% vs.32.4%). (5) There was significant difference of FOXC1 protein expression between the high age group and low age group. No relationship was found FOXC1 protein expression and other clinicopathologic parameters such as tumor size, histological grade, clinical stage, lymph node metastasis in TNBC. (6) The disease-free and overall survival of FOXC1-positive group was lower than FOXC1-negative group, but there was no statistical significance.Conclusions:(1) BLBC shows distinctive clinicopathological and morphological features; CK5/6 is most sensitive of all basal-like markers and the specificity of CK14 is highest. (2)Clinical stage, metastasis of lymph node, expansive growth, lymphocytotic infiltrates, high mitotic index and expression of CK5/6 are associated with prognosis of BLBC. (3)There are some disparities in recurrence and metastasis rate, and distribution of metastasis sites between different subtypes of breast cancer. (4)CK/TN, TN, CK and ER/HER2 classifications have certain differences on evaluation of the prognosis of each subtype of breast carcinoma. (5) SLC6A14 and FOXC1 mRNA expression in TNBC is significantly higher than luminal subtype of breast cancer. There is possibly an association between these genes and developing of TNBC. (6)FOXC1 protein expression in TNBC and BLBC is significantly higher than luminal subtype of breast cancer. It is promising to become a specific molecular marker of TNBC and BLBC.