Role of Nek2 on centrosome duplication and aneuploidy in breast cancer cells.

Breast cancer is the most common solid tumor and the second most common cause of death in women. Despite a large body of literature and progress in breast cancer research, many molecular aspects of this complex disease are still poorly understood, hindering the design of specific and effective therapeutic strategies. To identify the molecules important in breast cancer progression and metastasis, we tested the in vivo effects of inhibiting the functions of various kinases and genes involved in the regulation/modulation of the cytoskeleton by downregulating them in mouse PyMT mammary tumor cells and human breast cancer cell lines. These kinases and cytoskeletal regulators were selected based on their prognostic values for breast cancer patient survival. PyMT tumor cells, in which a selected gene was stably knocked down were injected into the tail veins of mice, and the formation of tumors in the lungs was monitored. One of the several genes found to be important for tumor growth in the lungs was NIMA-related kinases 2 (Nek2), a cell cycle-related protein kinase. Furthermore, Nek2 was also important for tumor growth in the mammary fat pad. In various human breast cancer cell lines, Nek2 knockdown induced aneuploidy and cell cycle arrest that led to cell death. Significantly, the breast cancer cell line most sensitive to Nek2 depletion was of the triple negative breast cancer subtype. Our data indicate that Nek2 has a pivotal role in breast cancer growth at primary and secondary sites, and thus may be an attractive and novel therapeutic target for this disease.

Factors associated with negative margins of lumpectomy specimen: potential use in selecting patients for intraoperative radiotherapy.

BACKGROUND: Lumpectomy followed by radiation is standard treatment for early breast cancer. Recently, the use of partial breast intraoperative radiation (IORT) has been developed, and patients selected for IORT should not have positive margins. This study's purpose was to identify factors predicting negative margins after lumpectomy. METHODS: Patient age, preoperative investigations, surgery, final pathology, and margin status were examined using a prospective database between 1999 and 2005. Univariate and multivariate logistic regression analysis were performed to identify patient and tumor factors predicting an increased rate of negative margins. The results were used to generate a patient selection algorithm. RESULTS: The rate of positive margins at first resection was 17% in 730 lumpectomies (708 patients). Multivariate analysis revealed that older age (P = .0006), smaller tumor size (P < .0025), type of surgery (OR = 3.4 for ultrasound vs mammogram-guided wire localization, P = .003), and having a core needle biopsy (CNB) with preoperative cancer diagnosis (P < .0001) were predictive for having a negative margin. Patients older than age 50 with a preoperative CNB showing invasive cancer less that 3 cm that can be localized under ultrasound had a negative margin rate of 98% (n = 178). These patients would be ideal for consideration of IORT. CONCLUSIONS: Negative margin rates after lumpectomy are predicted by age, tumor size, preoperative investigations, and localization technique. These variables can be used to select patients for IORT with a 2.2% chance of positive margins.

Examination of POU homeobox gene expression in human breast cancer cells.

Abnormal expression of homeobox genes may lead to the development of leukemias, lymphomas, and solid tumors. Expression of homeobox genes in mammary glands, however, has not been studied actively until recently. We have examined the expression of POU homeobox genes in human breast cancer cell lines and human breast tissue samples. Using a pair of degenerate primers for reverse transcription-polymerase chain reaction (RT-PCR) followed by DNA sequencing, we found that the human breast cancer cell line, MCF7, expresses at least 4 POU gene products: OCT1, OCT2, OCT3 and OCT11 (Skn-1a/i, Epoc-1). The expression of OCT1 and OCT2 in other human breast epithelial cell lines was further determined by Western blot analyses and electrophoretic mobility shift assay. We were unable to detect OCT11 in human breast cancer cell lines using the anti rat Skn-1a/i antibody, although the expression of this gene in both human breast cancer cell lines and human primary breast tumors was detected by RT-PCR. OCT3 is an embryonic transcription factor. We found that this gene is also expressed in human breast cancer cell lines and all human primary breast carcinomas examined, but not in normal human breast tissue. Taken together, we have shown that several POU genes are expressed in human breast epithelial cells. As OCT3 expression was detected only in the breast cancerous cells, this embryonic transcription factor could play an important role in mammary gland carcinogenesis.

Microdissection and molecular genetic analysis of HER2/neu in breast carcinoma.

Precise correlation of histomorphology with molecular genetic analysis is difficult in tissues composed of heterogeneous cell populations. We describe here a novel microdissection technique employed to correlate HER2/neu (HER2) immunohistochemical staining with HER2 genetic analysis in formalin-fixed, paraffin-embedded breast tissue. Fourteen invasive ductal carcinomas were selected from the pathology files of Memorial Sloan-Kettering Cancer Center that had been immunostained for HER2. Seven tumors showed typical membrane immunoreactivity and seven were negative. A dissecting microscope was then used to isolate minute (< or = 1 mm x 1 mm) areas of invasive carcinoma and normal breast tissue for molecular study. To document the type of cell sample submitted for polymerase chain reaction (PCR) analysis, each microdissected piece of tissue was photographed prior to removal from the glass slide. A preliminary study of four cases compared the results of PCR and genetic analysis using microdissected hematoxylin and eosin (H & E)-stained tissue, unstained dewaxed tissue, and destained dewaxed tissue in four specimens. Similar results were obtained with all three tissue preparations. Thereafter, H & E stained sections were selected as the tissue preparation of choice because tissue details were seen more clearly. There was complete correlation of immunohistochemical staining and HER2 analysis by PCR in all 14 cases. In the final 10 cases, the PCR product was resolved by gel electrophoresis and quantified by optical densitometry. Fourfold to eightfold amplification of HER2 was found in the five tumor specimens that immunohistochemically stained for HER2. A single copy of HER2 was found in all HER2-negative tumors and in normal breast tissue. We conclude that it is possible to quantify gene amplification of HER2 in minute samples of H & E-stained normal and malignant breast tissue. This microdissection technique can be applied to correlative histologic--molecular genetic analysis in a wide variety of tumor types.

Mutations of the p53 gene in male breast cancer.

BACKGROUND: Cancer of the male breast (MBC) is rare, accounting for less than 1% of cancer in males and representing less than 1% of all breast cancers. Reports of abnormalities in the expression of the tumor suppressor gene p53 in MBC have been few. METHODS: To assess the expression and mutations of the p53 gene, 35 patients with 36 MBC (one patient with bilateral breast carcinoma) were examined using immunohistochemical methods, polymerase chain reaction (PCR)-single strand conformation polymorphism and DNA sequencing. RESULTS: Thirty-one of the 36 carcinomas were studied by immunohistochemistry and by the PCR-based approach. Five patients were studied by immunohistochemistry only. Twelve patients (41.4%) of the 29 studied by molecular analysis presented an altered pattern in the single strand conformation polymorphism gel and point mutations were confirmed in all by direct DNA sequencing. Thirty-six tumors were studied by immunohistochemistry and 2 (5.5%) patients showed overexpression of the p53 protein. There were no statistically significant differences in p53 status with respect to: age, stage, estrogen receptors, progesterone receptors, tumor type. Patients with normal p53 showed a predisposition, although not statistically significant, for a longer disease free survival (5.6 years versus 4.2 years) and overall survival (5.9 years versus 4.8 years) than did patients with genetically altered p53. CONCLUSIONS: The incidence of male patients detected with p53 mutations (41.4%) in this series is concordant with the incidence of p53 mutations in female breast cancer, supporting the idea that cancer of the male breast is similar to the female counterpart.

Displacement of carcinomatous epithelium in surgical breast specimens following stereotaxic core biopsy.

A variety of needling procedures in the breast may lead to dislodgment and displacement of fragments of breast carcinoma into tissue outside the target lesion of the needling procedure. To assess how frequently displaced fragments of carcinoma are seen in surgical breast specimens following stereotaxic core needle biopsies, slides were reviewed from 43 consecutive cases of breast carcinoma in which surgical excision and/or mastectomy had been performed following an initial diagnostic stereotaxic 14-gauge core biopsy procedure. In 12 of 43 (28%) cases, displaced carcinomatous fragments were identified outside of the main tumor mass. These patients were subjected to other needling procedures that included local anesthetic injection at the time of core biopsy (43 cases), needle localization (22 of 43 cases), suture placement (18 of 43 cases), and fine-needle aspiration (1 of 43 cases). Attributing carcinomatous displacement solely to the core needle biopsy is complicated by these additional needling procedures. In 18 instances, local anesthetic injection by 25-gauge needle was the only needling procedure other than the core biopsy. In 7 of these 18 (39%) cases, fragments of displaced carcinoma were observed outside the main tumor mass. The authors have previously observed only one case in which a 25-gauge needle was associated with epithelial displacement, suggesting that the core biopsy was more likely to have been the cause of displaced epithelium in these cases. Long-term clinical follow-up will be necessary to determine the biologic and clinical significance of these findings.

Epithelial displacement in surgical breast specimens following needling procedures.

Lesional breast tissue is often subjected to various needling procedures prior to resection and formal histologic examination. In this article, we describe a series of 29 surgical breast specimens in which histologic examination revealed fragments of benign or malignant epithelium displaced in breast stroma or in lymphovascular channels, associated with the traumatic effects of a needling procedure. Follow-up revealed that a variety of different interventions, including needle localization, fine-needle aspiration, infiltration with local anesthetic, core biopsy, and suture placement, preceded the surgical resection in these instances. The displaced epithelial fragments mimic stromal invasion to a variable degree and may represent a potential source of misdiagnosis. The incidence and biological significance of epithelial displacement in this context are as yet unknown.

Calcification in bronchopulmonary sequestration.

We report an unusual complication in a patient with intralobar bronchopulmonary sequestration. Chest tomography, computed tomography, and specimen radiography showed a mass containing numerous round calcifications in the posterior basal segment of the left lower lobe. Pathologic examination disclosed an Exophiala fungus infection and broncholiths within bronchiectatic cavities in the sequestered segment.

Modulation of positive inotropy and metabolic coronary dilatation by myocardial alpha-adrenoceptors.

Cardiac hyperactivity and its consequent metabolically induced coronary vasodilation (MCD) were studied in isolated, perfused, electrically paced rat hearts. The alpha-adrenoceptor agonists, phenylephrine and methoxamine, produced a concentration-dependent inhibition of the inotropic responses to noradrenaline, dobutamine, isoprenaline, tyramine, and glucagon, while relatively potentiating their MCD reactions. This inhibition was unrelated to the alpha-agonists' known inotropic action and was not affected by catecholamine depletion of the heart. Withdrawal of the alpha-agonists or administration of the alpha-adrenoceptor antagonists phentolamine, phenoxybenzamine, or prazosin returned the inotropic and MCD reactions to normal. Neither the MCD response to electrically induced tachycardia nor the inotropic reactions produced by calcium chloride were affected by alpha-adrenoceptor agonists or antagonists. Alone, alpha-adrenoceptor antagonists were shown to potentiate the inotropic responses to noradrenaline and isoprenaline while the MCD was relatively diminished. The responses to glucagon were unaltered by alpha-antagonists. We postulate that myocardial reactivity to sympathetic stimulation can be modulated through alpha-adrenoceptors by the inhibition of processes that mediate cardiostimulation at post-beta-adrenoceptor sites, together with facilitation of those leading up to MCD. Accordingly, this modulation would act to prevent ischaemic damage to the heart by acting to limit the inotropic responses to increasing sympathetic stimulation while maximizing the blood supply to the myocardium.