Value of ER-D5 immunocytochemical determination in routine tissue sections of human breast cancer

ER-D5 is a recently identified protein related to estrogen receptors (ER). Generally ER measurement requires fresh frozen tissue and for ER-D5 assay ethanol (E) fixation of the specimen is recommended. We evaluated the possibility of immunocytochemical detection of ER-D5 in routine formalin-fixed (F) sections in 51 breast cancers comparing the results with those obtained in the same specimens using E as fixative. The results of ER-D5 assay were expressed by the staining index (SI) taking values greater than or equal to 5 as positive. In all tumors ER was also assayed by a biochemical method (DCCA). The sensitivity of ER-D5 detection in F was only 33.3%, while the specificity was 94.4%. A lower cut-off value of SI for F sections (greater than or equal to 2) increased the sensitivity to 66.6%, leaving the specificity unchanged. A strong correlation was found between the SI of ER-D5 in E and F (p less than 0.001). The SI of ER-D5 in F sections was also well correlated with ER concentrations (p less than 0.001). These results suggest that immunocytochemical determination of ER-D5 in routine sections may be useful in retrospective studies of hormone dependence in breast cancer.     

Genome-wide 5-Hydroxymethylcytosine Profiling Analysis Identifies MAP7D1 as A Novel Regulator of Lymph Node Metastasis in Breast Cancer

Although DNA 5-hydroxymethylcytosine(5 hmC) is recognized as an important epigenetic mark in cancer, its precise role in lymph node metastasis remains elusive. In this study, we investigated how 5 hmC associates with lymph node metastasis in breast cancer. Accompanying with high expression of TET1 and TET2 proteins, large numbers of genes in the metastasis-positive primary tumors exhibit higher 5 hmC levels than those in the metastasis-negative primary tumors. In contrast, the TET protein expression and DNA 5 hmC decrease significantly within the metastatic lesions in the lymph nodes compared to those in their matched primary tumors. Through genomewide analysis of 8 sets of primary tumors, we identified 100 high-confidence metastasis-associated5 hmC signatures, and it is found that increased levels of DNA 5 hmC and gene expression of MAP7 D1 associate with high risk of lymph node metastasis. Furthermore, we demonstrate that MAP7 D1, regulated by TET1, promotes tumor growth and metastasis. In conclusion, the dynamic5 hmC profiles during lymph node metastasis suggest a link between DNA 5 hmC and lymph node metastasis. Meanwhile, the role of MAP7 D1 in breast cancer progression suggests that the metastasis-associated 5 hmC signatures are potential biomarkers to predict the risk for lymph node metastasis, which may serve as diagnostic and therapeutic targets for metastatic breast cancer.     

Cell kinetics of human tumors by in vitro bromodeoxyuridine labeling

We labeled active S-phase cells in primary breast carcinomas with a modified 5-bromo-2'-deoxyuridine (BrdU) procedure using a silver-enhanced colloidal gold visualization step. Separate samples of 29 tumors were labeled with BrdU or tritiated thymidine ([3H]-dThd), and the labeling indices (LI) from the two methods were equivalent (Spearman's correlation coefficient = 0.96). Three breast carcinomas were incubated in various mixes of both BrdU and [3H]-dThd and developed sequentially for each. Paired photomicrographs showed that the same nuclei were labeled by either precursor. The in vitro method yielded LIs similar to those reported after in vivo pulse BrdU labeling for tumors of the central nervous system. The BrdU LI correlated significantly (r = 0.76, p less than 0.001) with % S-phase by DNA flow cytometry in 33 breast carcinomas. The BrdU labeling method is simpler and more rapid than the [3H]-dThd procedure (1-2 days for completion for the former, 7-10 days for the latter), and it provides an equivalent measurement of proliferative index.     

Parallel assessment of circulatory cell-free DNA by PCR and nucleosomes by ELISA in breast tumors

OBJECTIVES: In order to assess the potential biomolecules for breast cancer, we analyzed in parallel the levels of cell-free glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cell-free nucleosomes in serum samples from patients with benign and malignant breast tumors. The levels of cell-free DNA obtained by quantitative PCR were compared with those obtained by enzyme-linked immunosorbent assay (ELISA). METHODS: Twenty-three patients with benign breast tumors, 27 patients with breast cancer, and 32 age-matched healthy women were recruited. The amounts of serum nucleosomes were analyzed by ELISA and the levels of cell-free GAPDH were measured by real-time quantitative PCR. The correlation between nucleosome and cell-free GAPDH levels was examined using the Spearman rank test. RESULTS: The levels of cell-free GAPDH were significantly higher in the serum samples of patients with benign and malignant breast tumors than in those of the control group (median 37,966 GE/mL, range 3,802-130,104 versus 11,770 GE/mL, range 2,198-73,522, p=0.035 and median 40,698 GE/mL, range 3,644-192,482 versus 11,770 GE/mL range 2,198-73,522, p=0.001). The concentration of cell-free GAPDH correlated significantly with the quantities of nucleosomes in serum samples (r=0.451, p=0.000). There was, however, no significant difference between healthy individuals and women with benign breast tumors or breast cancer in terms of nucleosomes determined by ELISA. CONCLUSION: Our data suggest that the cell-free serum GAPDH DNA assayed by quantitative PCR is a better biomarker than nucleosomes assayed by ELISA in patients with breast tumors.     

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.     

Inhibition of the genesis of spontaneous mammary tumors in C3H mice: effects of selenium and of selenium-antagonistic elements and their possible role in human breast cancer.

The inhibitory effect of selenium on the genesis of spontaneous mammary tumors in C3H mice is statistically significant even at toxic levels of selenium (5 and 15 ppm of Se in form of selenite added to the supply water), no evidence for stimulation of tumor growth by selenium has been obtained. Arsenite lowers the tumor incidence at higher dosage (80 ppm of As in supply water) as well, but animals developing tumors under these conditions demonstrate significantly enhanced tumor growth rates. The addition of subtoxic concentrations of zinc (200 ppm in form of ZnCl2) to supply water containing 5 ppm of Se abolishes the cancer-protecting effect of selenium. The latter result is of possible importance with respect to the human breast cancer mortality experience: The calculated dietary zinc intakes of average adults in 28 countries correlate with the female age-corrected mortalities from breast cancer directly, with P less than 0.005. The zinc concentrations in whole blood from donors in different parts of the U.S.A. are also directly correlated with the female breast cancer mortalities.     

Determination of estrogen receptors in cytologic imprints of breast cancers using immunocytochemical methods.

Immunostaining of estrogen receptors (ERs) was carried out on imprints of 62 breast carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique (the Abbott ER-ICA kit). The results were compared to those obtained by the conventional biochemical analysis of cytosol proteins and to the degree of tumor differentiation. The cytologic specimens were insufficient for analysis in 6 cases; of the remaining 56 cases, 37 (66%) showed a positive ER reaction. In 51 cases with both types of ER analysis, the immunocytochemical staining of the imprints correlated strongly with the biochemical analysis in 44 cases and weakly in 3. Four cases were negative immunocytochemically and positive biochemically. Among the ductal carcinomas, well-differentiated tumors had higher percentages of ER-positive cells than did poorly differentiated tumors. These results show that the immunoperoxidase method is a highly specific and sensitive technique for the evaluation of ER content; it may be applicable to small samples of tumor tissue and may provide additional information for identifying hormonally responsive breast tumors.     

Allelic imbalance in primary breast carcinomas and metastatic tumors of the axillary lymph nodes

Axillary lymph node status is the most important prognostic factor in predicting disease outcome in women with breast cancer. A number of chromosomal aberrations in primary breast tumors have been correlated with lymph node status and clinical outcome, but chromosomal changes particular to metastatic lymph node tumors have not been well studied. DNA samples isolated from laser-microdissected primary breast and metastatic axillary lymph node tumors from 25 women with invasive breast cancer were amplified using 52 microsatellite markers defining 26 chromosomal regions commonly deleted in breast cancer. Levels and patterns of allelic imbalance (AI) within and between breast and lymph node tumors were assessed to identify chromosomal alterations unique to primary or metastatic tumors and to examine the timing of metastatic potential. The overall frequency of AI in primary breast tumors (0.24) was significantly greater (P < 0.001) than that in lymph node tumors (0.10), and congruent AI events were observed for < 20% of informative markers. AI at chromosomes 11q23.3 and 17p13.3 occurred significantly more frequently (P < 0.05) in primary breast tumors alone; no chromosomal regions showed a significantly higher AI frequency in lymph nodes. Higher rates of AI in primary versus metastatic lymph node tumors suggest that acquisition of metastatic potential may be an early event in carcinogenesis, occurring before significant levels of AI accumulate in the primary tumor. In addition, patterns of AI were highly discordant between tumor types, suggesting that additional genetic alterations accumulated independently in the two cell populations.     

Differential expression of Bax and Bcl-2 in the assessment of cellular dynamics in fine-needle samples of primary breast carcinomas

The rates of cell proliferation and programmed cell death (apoptosis) reflect tumor cell dynamics and are considered to directly influence biological progression and tumor response to therapy. Bax and Bcl-2 are members of a gene family that influence apoptosis and have been used as surrogate markers in the evaluation of this process. Sixty-three fine-needle tumor samples from an equal number of patients with breast carcinomas were analyzed for Bax, Bcl-2, and DNA content by flow cytometry. The results were correlated with classical clinicopathological parameters. Bax values varied widely among tumors and showed no significant correlation with any of the clinicopathological parameters analyzed. Bcl-2 levels ranged from 4% to 91%, correlated positively with estrogen (P = 0.0004) and progesterone (P = 0.0045) receptor positivity, and were more associated with low S-phase tumor values. In contrast, high S-phase values correlated with estrogen receptor negativity, high grade, and DNA aneuploidy. The study results indicate that Bcl-2 and S-phase analysis of fine-needle samples of breast carcinomas provide a convenient tool for the assessment of these tumors.     

Relationship between RUNX1 and AXIN1 in ER-negative versus ER-positive Breast Cancer

RUNX1 plays opposing roles in breast cancer: a tumor suppressor in estrogen receptor-positive (ER⁺) disease and an oncogenic role in ER-negative (ER^?) tumors. Potentially mediating the former, we have recently reported that RUNX1 prevents estrogen-driven suppression of the mRNA encoding the tumor suppressor AXIN1. Accordingly, AXIN1 protein expression was diminished upon RUNX1 silencing in ER⁺ breast cancer cells and was positively correlated with AXIN1 protein expression across tumors with high levels of ER. Here we report the surprising observation that RUNX1 and AXIN1 proteins are strongly correlated in ER^? tumors as well. However, this correlation is not attributable to regulation of AXIN1 by RUNX1 or vice versa. The unexpected correlation between RUNX1, playing an oncogenic role in ER^? breast cancer, and AXIN1, a well-established tumor suppressor hub, may be related to a high ratio between the expression of variant 2 and variant 1 (v2/v1) of AXIN1 in ER^? compared with ER⁺ breast cancer. Although both isoforms are similarly regulated by RUNX1 in estrogen-stimulated ER⁺ breast cancer cells, the higher v2/v1 ratio in ER^? disease is expected to weaken the tumor suppressor activity of AXIN1 in these tumors.