Expression of nm23-H1 gene product in thyroid,ovary, and breast cancers

The nm23 gene product is one of several possible mediators of cancer invasion and metastasis. As the amounts of nm23-H1 mRNA and gene product are reduced in metastatic lymph nodes from patients with papillary carcinoma of the thyroid, we examined the expression of nm23 gene product in 115 thyroid cancers, 78 ovarian cancers, 63 breast cancers, and metastatic lymph node tissues by immunohistochemistry. It was found that nm23-H1, but not nm23-H2 gene product, was expressed in primary sites of thyroid, ovarian, and breast cancers, except for medullary and anaplastic carcinomas of the thyroid, but expressed only weakly or poorly in metastatic lymph nodes. Although nm23-H1 gene product expression was lower in anaplastic and medullary carcinomas of the thyroid, there was no significant difference in nm23-H1 gene product expression among histological types of ovarian and breast cancers. Our data indicate that the nm23-H1 gene product may play a role in metastasis in these hormone-producing organs and that other factors may be involved in metastasis of anaplastic and medullary carcinomas of the thyroid.     

Demonstration of mucinous-like carcinoma-associated antigen in bone marrow and lymph node biopsies from patients with breast carcinoma.

Two hundred and fifty bone marrow and 140 lymph nodal biopsies were analyzed immunocytochemically, using a mouse monoclonal antibody b-12 (M Ab b-12), which reacts with MCA (mucinous-like carcinoma-associated antigen). The presence of MCA in bone marrow specimens was demonstrated in 102 out of 105 (97.1%) breast cancer metastases, 5 out of 8 (62.5%) gastric cancers, 5 out of 6 (83.3%) colon cancers, 3 out of 5 (60%) prostate cancers, 11 out of 26 (42.3%) lung cancers and 25 out of 30 (83.3%) unknown primary cancers, while no positivity to anti-MCA antibody was found in 30 cases of normal bone marrow biopsies, 5 cases of non epithelial malignancies and 30 cases of hemolymphoproliferative disease. Normal lymph nodes and non-epithelial lymph node metastases did not show any reaction to M Ab b-12; on the contrary MCA positive staining was observed in 75 out of 75 (100%) lymph nodal metastases in breast cancer. These results suggest that application of M Ab b-12 in immunohistochemistry is valid for the detection of bone marrow and lymph nodal micrometastases of epithelial origin.     

High Prevalence of Human Cytomegalovirus Proteins and Nucleic Acids in Primary Breast Cancer and Metastatic Sentinel Lymph Nodes

Background

Breast cancer is a leading cause of death among women worldwide. Increasing evidence implies that human cytomegalovirus (HCMV) infection is associated with several malignancies. We aimed to examine whether HCMV is present in breast cancer and sentinel lymph node (SLN) metastases.

Materials and Methods

Formalin-fixed paraffin-embedded tissue specimens from breast cancer and paired sentinel lymph node (SLN) samples were obtained from patients with (n = 35) and without SLN metastasis (n = 38). HCMV immediate early (IE) and late (LA) proteins were detected using a sensitive immunohistochemistry (IHC) technique and HCMV DNA by real-time PCR.

Results

HCMV IE and LA proteins were abundantly expressed in 100% of breast cancer specimens. In SLN specimens, 94% of samples with metastases (n = 34) were positive for HCMV IE and LA proteins, mostly confined to neoplastic cells while some inflammatory cells were HCMV positive in 60% of lymph nodes without metastases (n = 35). The presence of HCMV DNA was confirmed in 12/12 (100%) of breast cancer and 10/11 (91%) SLN specimens from the metastatic group, but was not detected in 5/5 HCMV-negative, SLN-negative specimens. There was no statistically significant association between HCMV infection grades and progesterone receptor, estrogen receptor alpha and Elston grade status.

Conclusions

The role of HCMV in the pathogenesis of breast cancer is unclear. As HCMV proteins were mainly confined to neoplastic cells in primary breast cancer and SLN samples, our observations raise the question whether HCMV contributes to the tumorigenesis of breast cancer and its metastases.     

PRC2/EED-EZH2 Complex Is Up-Regulated in Breast Cancer Lymph Node Metastasis Compared to Primary Tumor and Correlates with Tumor Proliferation In Situ

Background

Lymph node metastasis is a key event in the progression of breast cancer. Therefore it is important to understand the underlying mechanisms which facilitate regional lymph node metastatic progression.

Methodology/Principal Findings

We performed gene expression profiling of purified tumor cells from human breast tumor and lymph node metastasis. By microarray network analysis, we found an increased expression of polycomb repression complex 2 (PRC2) core subunits EED and EZH2 in lymph node metastatic tumor cells over primary tumor cells which were validated through real-time PCR. Additionally, immunohistochemical (IHC) staining and quantitative image analysis of whole tissue sections showed a significant increase of EZH2 expressing tumor cells in lymph nodes over paired primary breast tumors, which strongly correlated with tumor cell proliferation in situ. We further explored the mechanisms of PRC2 gene up-regulation in metastatic tumor cells and found up-regulation of E2F genes, MYC targets and down-regulation of tumor suppressor gene E-cadherin targets in lymph node metastasis through GSEA analyses. Using IHC, the expression of potential EZH2 target, E-cadherin was examined in paired primary/lymph node samples and was found to be significantly decreased in lymph node metastases over paired primary tumors.

Conclusions/Significance

This study identified an over expression of the epigenetic silencing complex PRC2/EED-EZH2 in breast cancer lymph node metastasis as compared to primary tumor and its positive association with tumor cell proliferation in situ. Concurrently, PRC2 target protein E-cadherin was significant decreased in lymph node metastases, suggesting PRC2 promotes epithelial mesenchymal transition (EMT) in lymph node metastatic process through repression of E-cadherin. These results indicate that epigenetic regulation mediated by PRC2 proteins may provide additional advantage for the outgrowth of metastatic tumor cells in lymph nodes. This opens up epigenetic drug development possibilities for the treatment and prevention of lymph node metastasis in breast cancer.     

Distinct Ezrin Truncations Differentiate Metastases in Sentinel Lymph Nodes from Unaffected Lymph Node Tissues,from Primary Breast Tumors,and from Healthy Glandular Breast Tissues

BACKGROUND: Lymph node metastasis status is a prognostic factor for further lymph node involvement and for patient survival in breast cancer patients. Frozen section analysis of lymph nodes is a reliable method for detection of macro-metastases. However, this method is far less effective in detecting micro-metastases, requesting improved diagnostic procedures. METHODS: We investigated expression and truncation of ezrin in (i) sentinel lymph node metastases, (ii) unaffected axillary lymph nodes, (iii) primary breast tumors, and (iv) healthy glandular breast tissues using 2D gel electrophoresis, SDS-PAGE, and mass spectrometry in addition to Western blotting. RESULTS: Full-length ezrin (E1; amino acids 1–586) is present in all four investigated tissues. Two truncated ezrin forms, one missing about the first hundred amino acids (E2a) and the other lacking about 150 C-terminal amino acids (E2b) were detectable in primary tumor tissues and in sentinel lymph node metastases but not in glandular tissues. Strikingly, an ezrin truncation (E3) which consists approximately of amino acids 238–586 was found strongly expressed in all sentinel lymph node metastases. Moreover, an N-terminal ezrin fragment (E4) that consists approximately of amino acids 1–273 was identified in sentinel lymph node metastases as well. CONCLUSIONS: We show for the first time the existence of tissue-dependent specific ezrin truncations. The distinguished strong Western blot staining of ezrin E3 in sentinel lymph node metastases underlines its capability to substantiate the occurrence of lymph node (micro)metastases in breast cancer patients.     

Common protein biomarkers assessed by reverse phase protein arrays show considerable intratumoral heterogeneity in breast cancer tissues

Proteins are used as prognostic and predictive biomarkers in breast cancer. However, the variability of protein expression within the same tumor is not well studied. The aim of this study was to assess intratumoral heterogeneity in protein expression levels by reverse-phase-protein-arrays (RPPA) (i) within primary breast cancers and (ii) between axillary lymph node metastases from the same patient. Protein was extracted from 106 paraffin-embedded samples from 15 large (≥3 cm) primary invasive breast cancers, including different zones within the primary tumor (peripheral, intermediate, central) as well as 2-5 axillary lymph node metastases in 8 cases. Expression of 35 proteins including 15 phosphorylated proteins representing the HER2, EGFR, and uPA/PAI-1 signaling pathways was assessed using reverse-phase-protein-arrays. All 35 proteins showed considerable intratumoral heterogeneity within primary breast cancers with a mean coefficient of variation (CV) of 31% (range 22-43%). There were no significant differences between phosphorylated (CV 32%) and non-phosphorylated proteins (CV 31%) and in the extent of intratumoral heterogeneity within a defined tumor zone (CV 28%, range 18-38%) or between different tumor zones (CV 24%, range 17-38%). Lymph node metastases from the same patient showed a similar heterogeneity in protein expression (CV 27%, range 18-34%). In comparison, the variation amongst different patients was higher in primary tumors (CV 51%, range 29-98%) and lymph node metastases (CV 65%, range 40-146%). Several proteins showed significant differential expression between different tumor stages, grades, histological subtypes and hormone receptor status. Commonly used protein biomarkers of breast cancer, including proteins from HER2, uPA/PAI-1 and EGFR signaling pathways showed higher than previously reported intratumoral heterogeneity of expression levels both within primary breast cancers and between lymph node metastases from the same patient. Assessment of proteins as diagnostic or prognostic markers may require tumor sampling in several distinct locations to avoid sampling bias.     

Metastatic breast cancer from an occult primary provisionally identified by steroid receptor content and two-dimensional protein electrophoresis: some problems associated with the use of the latter technique

Occult primary breast cancer in a patient presenting with undifferentiated metastatic adenocarcinoma in an inguinal lymph node was provisionally diagnosed by the steroid receptor content of its metastases and by the sclerotic pattern of osseous metastases. This diagnosis was supported by the presence of several proteins electrophoresed in 2 dimensions that may be common to other identified adenocarcinomas of the breast. These proteins appear to be absent from a representative adenocarcinoma of the ovary and of the uterus, at least at the level of sensitivity provided by the dye used to detect them. Some clinical implications for the use of two-dimensional protein electrophoresis in identifying undifferentiated cancers are considered.     

Expression of the insulin-like growth factor-I receptor in primary breast cancer and lymph node metastases: correlations with estrogen receptors alpha and beta.

Numerous laboratory studies and some epidemiological data have suggested the involvement of the insulin-like growth factor-I receptor (IGF-IR) in breast cancer development and progression. However, data on IGF-IR expression in human tissues, including breast cancer sections, are limited and often inconsistent. We therefore examined by immunohistochemistry the expression of IGF-IR in primary tumors and breast cancer metastases to lymph nodes, and correlated IGF-IR positivity with estrogen receptor (ER) status and selected clinicopathological features. We found that 1) IGF-IR was expressed in primary tumors as well as in lymph node metastases, but the expression in primary tumors was more frequent (56 % vs. 44.4 %); 2) IGF-IR expression in primary tumors was associated with negative node status (p < 0.033); 3) in node-negative primary tumors, IGF-IR positively correlated with ERbeta (p < 0.008; r = 0.538), but not with ERalpha, tumor size or grade; 4) both IGF-IR-positive and IGF-IR-negative primary tumors were found to produce IGF-IR-positive as well as IGF-IR-negative metastases; 5) in metastases, IGF-IR expression did not associate with ERalpha, ERbeta or any of the studied pathobiological markers. The results suggest that IGF-IR could become a viable pharmaceutical target in breast cancer therapy, but such therapy should be based on IGF-IR assessment in primary tumor and metastasis in each potential patient.     

Adjacent thoracic lymph node metastases originating from two separate primary cancers: case report

Reported is an unusual case of adjacent thoracic lymph nodes demonstrating metastases from two different primary malignancies. A 51 year-old woman with a previous history of bilateral breast cancer underwent a radical gastro-oesophagectomy for adenocarcinoma of the lower third of the oesophagus. The resection specimen demonstrated breast and oesophageal metastases in adjacent thoracic lymph nodes. Mechanisms for this phenomenon, including the known local immune suppression on lymphoid cells by oesophageal carcinoma cells, are discussed.     

Quantitative Molecular Analysis of Sentinel Lymph Node May Be Predictive of Axillary Node Status in Breast Cancer Classified by Molecular Subtypes

To determine the performance of intraoperative one-step nucleic acid amplification (OSNA) assay in detecting sentinel lymph node metastases compared to postoperative histology taking into account breast cancer molecular classification and to evaluate whether the level of cytokeratin 19 mRNA copy number may be useful in predicting the likelihood of a positive axillary lymph node dissection. OSNA assay was performed in a prospective series of 903 consecutive sentinel lymph nodes from 709 breast cancer patients using 2 alternate slices of each sentinel lymph node. The remaining 2 slices were investigated by histology. Cytokeratin 19 mRNA copy number, which distinguishes negative cases (<250 copies), micrometastases (+, ≥250≤5000 copies) and macrometastases (++, >5000 copies), was compared to axillary lymph node dissection status and to the biological tumor profile. Concordance between OSNA and histopathology was 95%, specificity 95% and sensitivity 93%. Multiple Corresponce Analysis and logistic regression evidenced that positive axillary lymph node dissection was significantly associated with a higher cytokeratin 19 mRNA copy number (>5000; p<0.0001), HER2 subtype (p = 0.007) and lymphovascular invasion (p<0.0001). Conversely, breast cancer patients with cytokeratin 19 mRNA copy number <2000 mostly presented a luminal subtype and a negative axillary lymph node dissection. We confirmed that OSNA assay can provide standardized and reproducible results and that it represents a fast and quantitative tool for intraoperative evaluation of sentinel lymph node. Omission of axillary lymph node dissection could be proposed in patients presenting a sentinel lymph node with a cytokeratin 19 mRNA copy number <2000 and a Luminal tumor phenotype.     

Evaluation of Tumor Cell Proliferation by Ki-67 Expression and Mitotic Count in Lymph Node Metastases from Breast Cancer

Few studies have addressed the risk of recurrence by assessing proliferation markers in lymph node metastasis from breast cancer. Here, we aimed to examine Ki-67 expression and mitotic count in lymph nodes in comparison with primary tumors. A cohort of node positive breast cancer (n = 168) was studied as a part of the prospective Norwegian Breast Cancer Screening Program (1996–2009). The percentage of Ki-67 positivity was counted per 500 tumor cells in hot-spot areas (x630). Mitotic count was conducted in the most cellular and mitotic active areas in 10 high power fields (x400). Our results showed that Ki-67 and mitotic count were significantly correlated between primary tumor and lymph nodes (Spearman`s correlation 0. 56 and 0.46, respectively) and were associated with most of the histologic features of the primary tumor. Univariate survival analysis (log-rank test) showed that high Ki-67 and mitotic count in the primary tumor and lymph node metastasis significantly predicted risk of recurrence. In multivariate analysis, mitotic count in the lymph node metastasis was an independent predictor of tumor recurrence. In conclusion, proliferation markers in lymph node metastases significantly predicted disease free survival in node positive breast cancer.     

NY-BR-1 protein expression in breast carcinoma: a mammary gland differentiation antigen as target for cancer immunotherapy

NY-BR-1 is a recently identified differentiation antigen of the mammary gland. To use NY-BR-1 for T-cell-based immunotherapy, analysis of its co-expression with HLA class I antigens is required. In the present tissue microarray study, primary breast cancers (n = 1,444), recurrences (n = 88), lymph node (n = 525) and distant metastases (n = 91) were studied for NY-BR-1 expression using a novel monoclonal antibody. NY-BR-1 expression was compared with prognosis, estrogen receptor, HER2-status, EGFR and HLA class I antigen expression. NY-BR-1 was more frequently expressed in grade 1 (82%) than in grade 2 (69%) and grade 3 (46%) carcinomas (P < 0.0001). Moreover, NY-BR-1 expression correlated directly with estrogen receptor expression (P < 0.0001) and inversely correlated with HER2-status and EGFR expression (P < 0.0001 for both). Considering high expression level of co-expression, 198/1,321 (15%) primary breast carcinomas and 4/65 (6%) distant metastases expressed NY-BR-1 and HLA class I, suggesting that active immunotherapy can be applied to about 10% of breast cancer patients. Survival analysis showed an association of NY-BR-1 expression with better patient outcome (P = 0.015). No difference between NY-BR-1 expression of primary tumors and metastases could be found, indicating that the presence of NY-BR-1 in metastases can be deduced from their corresponding primary. Forty-three paired biopsies taken from patients before and after chemotherapy suggest that NY-BR-1 expression is not influenced by preceding chemotherapy (kappa = 0.89, P < 0.0001). In summary, the co-expression of NY-BR-1 with HLA class I antigens and its expression in metastases without modification by chemotherapy suggest that NY-BR-1 targeted immunotherapy represents a viable strategy in addition to other targeted cancer drug therapies of breast cancer.     

Epigenetic Silencing of ITGA2 by MiR-373 Promotes Cell Migration in Breast Cancer

The loss of ITGA2 plays an important role in cancer metastasis in several solid cancers. However, the molecular mechanism of ITGA2 loss in primary cancers remains unclear. In this study, we found that a lower ITGA2 protein level was observed in breast cancers compared to adjacent non-cancerous breast tissues. Interestingly, the reduction degree of ITGA2 at the protein level was far more than that at the mRNA level. We further showed that the translation of ITGA2 mRNA was directly inhibited by miR-373 through binding to ITGA2-3’UTR. Silencing of ITGA2 detached cell-cell interactions, induced the deploymerization of stress fiber F-actin and stimulated cancer cell migration, similar to the effect of miR-373 over-expression. The co-expression of ITGA2, not ITGA2-3’UTR, could abrogate miR-373-induced cancer cell migration because that the expression of ITGA2-3’UTR was inhibited by co-transfected miR-373. ITGA2 protein level was inversely associated with miR-373 level in breast cancers (r = -0.663, P<0.001). 73.33% of breast cancer patients with high miR-373 and low ITGA2 expression exhibited the lymph node-positive metastases. Together, our results show that epigenetic silencing of ITGA2 by miR-373 stimulates breast cancer migration, and miR-373^high/ITGA2^low may be as a prognosis biomarker for breast cancer patients.     

Lower expression of CXCR4 in lymph node metastases than in primary breast cancers: potential regulation by ligand-dependent degradation and HIF-1alpha

Stromal-derived factor-1 (SDF-1) is a unique ligand of the CXC chemokine receptor 4 (CXCR4), which is critically involved in the metastasis of breast cancer. High levels of SDF-1 in the common destination organs of metastasis, such as the lymph nodes, lungs, liver, and bones, attract CXCR4-positive tumor cells. The interaction between SDF-1 and CXCR4 leads to the activation of specific signaling pathways, allowing for homing and metastatic progression. However, regulation of CXCR4 expression at the metastatic organ site is not well-documented. We detected the expression of CXCR4 and hypoxia inducible factor (HIF)-1alpha in breast tumor tissues by immunohistochemical staining and analyzed SDF-1 in primary tumors and lymph nodes using real-time RT-PCR. Compared to the corresponding metastasized tumors in the lymph nodes, primary invasive carcinomas showed more intense staining for CXCR4, particularly on the cellular membrane. Both primary tumors and lymph node metastases exhibited higher levels of CXCR4 expression compared to non-neoplastic breast tissues. Therefore, we hypothesized that the tumor environment in the lymph nodes may cause the reduction of CXCR4 levels in the metastatic tumor cells because of: (1) high SDF-1 levels and (2) lower levels of HIF-1alpha. Our in vitro data demonstrated that high levels of SDF-1 can induce the internalization and degradation of CXCR4 through the lysosome pathway. In addition, lower levels of HIF-1alpha in the lymph node metastases, probably induced by the less hypoxic environment, further lowered CXCR4 levels. These results indicate that ligand-dependent degradation and lower HIF-1alpha levels may be potential causes of lowered levels of CXCR4 in the lymph nodes compared to the primary tumors. Our study suggests that CXCR4 levels in tumor cells are regulated by its microenvironment. These findings may enhance our ability to understand the biological behavior of breast cancers.     

Antigen levels of urokinase type plasminogen activator and its inhibitors in primary breast cancer

Primary Breast Cancer, Urokinase-Type Plasminogen Activator, Inhibitors The aim of the study was to monitor urokinase plasminogen activator antigen concentrations and its type 1 (PAI-1) and type 2 (PAI-2) inhibitors in histologically defined forms of primary breast cancer and a comparison with these antigens levels in normal tissue. Another goal was a search for a relationship/or its lack/between the occurrence of the new generation markers of neoplastic disease and a presence/or absence/of lymph node metastases. U-PA, PAI-1 and PAI-2 antigen levels were determined by ELISA tests in protein extracts of breast cancer tissues. Among the studied breast tumors 32 specimens were ductal carcinomas, 15 specimens were lobular carcinomas and the remaining 13 were other rare histological forms. In comparison to the obtained values of u-PA antigen levels in normal tissue, the values in neoplastic tissues were elevated several times: 11-fold, 6-fold and 15-fold in ductal c., lobular c. and other rare neoplasms. The values of PAI-1 antigen levels were about 20-fold higher for all studied, histologically defined primary breast cancers. The greatest differences of PAI-2 antigen levels growth was observed in histologically defined primary breast cancer forms. It was augmented 10-fold, 40-fold and 20-fold, respectively, for ductal carcinoma, lobular carcinoma and rare forms of neoplasms. In various forms of invasive breast cancer and those without lymph node metastases the content of u-PA, PAI-1 and PAI-2 were also significantly elevated. Among the new generation of independent markers of the neoplastic process, PAI-2 seems to be the most reliable marker for the identification of primary breast cancer. The goal of the present study was to evaluate a possible combined prognostic value of the three major components of the u-PA system (u-PA, PAI-1 and PAI-2) in patients with defined histopathological forms of primary breast cancer.     

P-gp、MMP-2、C-erbB-2在乳腺癌原发灶和转移淋巴结中的表达对比研究

目的：探讨乳腺癌侵袭转移和多药耐药之间的关系,为治疗方案的个体化提供依据。方法：采用免疫组化方法检测46例乳腺浸润性导管癌患者乳腺原发灶及相应腋淋巴结转移灶中P-gp、MMP-2、c-erbB-2的表达,结合临床表现、病理学指标,分析其相关性。结果：46例原发灶P-gp阳性表达35例（76.1%）,MMP-2阳性表达25例（54.3%）,c-erbB-2高表达18例（39.1%）;相应腋淋巴结转移灶P-gp阳性表达28例（60.9%）,MMP-2阳性表达16例（34.8%）,c-erbB-2高表达16例（34.8%）;P-gp、MMP-2蛋白表达水平与肿块大小、淋巴结转移数目均呈正相关（P〈0.05）,c-erbB-2蛋白表达水平与腋窝淋巴结转移数量呈正相关,与ER、PR表达呈负相关,P-gp阳性表达与MMP-2和c-erbB-2的表达呈正相关（P〈0.05）。结论：肿瘤原发灶与转移灶存在异质性,P-gp、MMP-2、c-erbB-2的表达与乳腺癌的多药耐药和侵袭转移有关,检测上述基因在原发灶与转移灶的表达,为乳腺癌选择个体化的化疗、内分泌治疗及分子靶向治疗提供了分子生物学依据。     

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.