Survival among women with triple receptor-negative breast cancer and brain metastases.

BACKGROUND: The purpose of this study was to determine the incidence of and survival following brain metastases among women with triple receptor-negative breast cancer. PATIENTS AND METHODS: In all, 679 patients with nonmetastatic triple receptor-negative breast cancer diagnosed from 1980 to 2006 were identified. Cumulative incidence of brain metastases was computed. Cox proportional hazards models were fitted to explore factors that predict for development of brain metastases. Survival was computed using the Kaplan-Meier product limit method. RESULTS: Median follow-up was 26.9 months. In all, 42 (6.2%) patients developed brain metastases with a cumulative incidence at 2 and 5 years of 5.6% [95% confidence interval (CI) 3.8% to 7.9%] and 9.6% (95% CI 6.8% to 13%), respectively. A total of 24 (3.5%) patients developed brain metastases as the first site of recurrence with cumulative incidence at 2 and 5 years of 2.0% (95% CI 2.6% to 6.0%) and 4.9% (95% CI 3.2% to 7.0%), respectively. In the multivariable model, no specific factor was observed to be significantly associated with time to brain metastases. Median survival for all patients who developed brain metastases and those who developed brain metastases as the first site of recurrence was 2.9 months (95% CI 2.0-7.6 months) and 5.8 months (95% CI 1.7-11.0 months), respectively. CONCLUSION: In this single-institutional study, patients with nonmetastatic triple receptor-negative breast tumors have a high early incidence of brain metastases associated with poor survival and maybe an ideal cohort to target brain metastases preventive strategies.

Defining prognosis for women with breast cancer and CNS metastases by HER2 status.

BACKGROUND: The purpose of this retrospective study was to determine, in a cohort of patients with breast cancer and central nervous system (CNS) metastases, the effect of trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-positive disease and to compare this with that of patients with HER2-negative disease. METHODS: Five hundred and ninety-eight patients with invasive breast cancer, CNS metastases and known HER2 status were identified. Time to CNS metastases and survival after CNS metastases were estimated by the Kaplan-Meier method, and Cox models were fitted to determine the association between HER2 status, trastuzumab treatment and outcomes after adjustment for other patient characteristics. RESULTS: In the multivariable model, patients with HER2-negative disease [Hazard ratio (HR) 1.50, 95% confidence interval (CI) 1.15-1.95, P = 0.003] and patients with HER2-positive disease who did not receive trastuzumab (HR 2.13, 95% CI 1.51-3.00, P < 0.0001) had shorter times to CNS metastases compared with patients with HER2-positive disease who had received trastuzumab as first-line therapy for metastases. Furthermore, patients with HER2-negative disease (HR 1.66, 95% CI 1.31-2.12, P < 0.0001) and patients with HER2-positive disease who had never received trastuzumab (HR 1.34, 95% CI 0.78-2.30, P = 0.28) had an increased hazard of death compared with patients with HER2-positive disease who had received trastuzumab before or at the time of CNS metastases diagnosis. CONCLUSION: In our cohort of patients with breast cancer and CNS metastases, patients with HER2-positive disease treated with trastuzumab had longer times to development of and better survival from CNS metastases compared with patients with HER2-positive disease who had never received trastuzumab and patients with HER2-negative breast cancer.

Cytokine-induced cytotoxic function expressed by lymphocytes of the innate immune system: distinguishing characteristics of NK and LAK based on functional and molecular markers.

Several molecular events are now identifiable during the activation, recognition, and killing by natural killer (NK) cells that are distinct from those differentiated in response to cytokines during the generation of lymphokine-activated killer (LAK) cells or during lymphocyte proliferation. Because LAK and MHC-unrestricted killing activities also include the prototypic NK targets as part of their broad recognition spectra, accurate identification of the complete function being studied is critical. In many publications, past and present, only NK-sensitive target cells were used (K562, Molt-4, others), and, therefore, the results do not necessarily indicate whether the effectors are NK or have differentiated into LAK cells. Such a consideration becomes critical when the effectors are grown in interleukin-2 (IL-2), and an attempt is made to define receptor recognition, signal transduction pathways, and specificity at the molecular level. In some instances, effector cells are likely to have stopped, therefore merely expressing NK activity, and have also acquired LAK function. The identified receptors may not have been unique to NK cells or NK function. Not until the targets employed are also confirmed to be NK sensitive, and the effectors do not kill NK-resistant targets can the results of molecular studies be proposed to represent aspects unique to NK. Reports of the use of IL-2-expanded NK clones are most likely providing data concerning the biology of LAK and not of classic NK. The classic NK activity surveying our blood apparently performs an important function, including the ability to respond rapidly to certain cytokines and to acquire additional functions and receptors for use in destroying a vast array of target cells. It is critical for scientists to appreciate the functional distinctions and to identify the molecules and pathways unique to each of these curious cytolytic systems.

Wide-field imaging of fluorescent deoxy-glucose in ex vivo malignant and normal breast tissue.

Rapid in situ determination of surgical resection margins during breast cancer surgery would reduce patient time under anesthesia. We present preliminary data supporting the use of a fluorescent glucose analog (2-NBDG) as an optical contrast agent to differentiate freshly excised breast tissue containing cancerous cells from normal breast tissue. Multi-spectral images of 14 breast cancer specimens acquired before and after incubation with 2-NBDG demonstrated increased fluorescent signal in all of the malignant tissue due to increased 2-NBDG consumption. We demonstrate that 2-NBDG has potential as an optical contrast agent to differentiate cancerous from non-cancerous tissue.

Effects of hypercholesterolaemia on physiological recruitment of coronary vascular reserve in swine.

1. The endothelium participates in the regulation of coronary vascular tone. As evidence exists from studies performed on epicardial vessels that hypercholesterolaemia impairs endothelial function, we tested the hypothesis that hypercholesterolaemia impairs coronary vascular reserve in an intact animal. 2. Domestic swine, maintained on a regular (n = 9) or a 2% high-cholesterol (n = 9) diet for 3 months were instrumented with a catheter in the left atrium for microsphere injection, a catheter in the anterior interventricular vein for venous sampling and an 82% stenosis in the left anterior descending artery. Papaverine was used to determine coronary vascular reserve. Regional coronary flow as reflected by perfusion (microsphere measurement), lactate consumption, oxygen consumption and haemodynamics were obtained at baseline, after 10 mg of papaverine and after atrial pacing at a rate of 120 beats/min and 150 beats/min. 3. Cholesterol was elevated in animals on the high cholesterol diet (350 +/- 50 mg/dl versus 99 +/- 10 mg/dl, P < 0.001). Baseline haemodynamics were similar between groups. Baseline transmural flow and its augmentation with papaverine were comparable in the two groups in the control (circumflex) and stenosed (left anterior descending artery) zones. In both groups, perfusion increased in the control zone in response to increased oxygen demand, whereas in the stenosis zone no increase was observed in either group (P not significant for normal versus high cholesterol diet). Endocardial flow reserve in the stenosis zone was exhausted in both groups. Epicardial flow in the stenosis zone increased significantly in the normal (P < 0.02) but not in the hypercholesterolaemic animals (P not significant). 4. The endocardial/epicardial ratio in the control zone at baseline revealed greater endocardial dominance in the normal compared with the hypercholesterolaemic animals (1.35 versus 1.10, P < 0.01). With papaverine, similar ratios indicated a similar reserve potential in both groups. During increased oxygen demand, normal animals continued to demonstrate endocardial dominance whereas it diminished in the hypercholesterolaemic group. In the stenosis zone, endocardial blood flow dominated at baseline in the normal animals and to a lesser extent in the hypercholesterolaemic animals (1.30 versus 1.10, P = 0.10). During increased oxygen demand, endocardial dominance decreased significantly in both groups of animals; however, it remained greater than 1.0 only in the normal animals. 5. Exposure to elevated cholesterol levels did not impair an animal's ability to augment coronary blood flow in response to an increase in oxygen demand. In contrast to this lack of effect on recruitment of coronary reserve, regional coronary blood flow was altered in the hypercholesterolaemic animals.

Augmentation of coronary blood flow by ACE inhibition: role of angiotensin and bradykinin.

Inhibition of the angiotensin converting enzyme (ACE) is known to enhance coronary blood flow via partial suppression of angiotensin II and potentiation of bradykinin. The purpose of these experiments was to evaluate the contribution of each of these mechanisms to the ACE inhibition induced changes in blood flow in myocardial regions perfused by intact or stenotic coronary arteries. Seven domestic swine were submitted to an 82% stenosis of the left anterior descending artery with the circumflex artery left intact to serve as control area. Regional coronary blood flows were measured by the radioactive microsphere technique in the total area perfused by each coronary artery and in the subepicardial and subendocardial regions of each area separately, at rest and after treatments with captopril, losartan and a bradykinin antagonist given consecutively. We found a significant increase of total flow in both the stenotic and intact areas after captopril. Losartan caused a significant fall in systemic blood pressure with no further changes in overall coronary blood flow and the bradykinin antagonist produced a small but nonsignificant decline in total coronary flow. However, further separate analysis of subregions showed that subendocardial regions had a sharper increase in flow after captopril, and a significantly greater decline after bradykinin inhibition than subepicardial regions, whereas losartan tended to shunt blood from the subendocardial to the subepicardial regions. The results indicate that augmentation of coronary blood flow after ACE inhibition is not further enhanced by angiotensin II blockade and is in part mediated via potentiation of endogenous bradykinin, which exerts a preferential vasodilatory effect on the subendocardial regions of the myocardium.

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway.

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.

Characterization of human copine III as a phosphoprotein with associated kinase activity.

The copines, first described by Creutz et al. [(1998) J. Biol. Chem. 273, 1393-1402], comprise a two C2 domain-containing protein family and are known to aggregate phosphatidylserine membranes in a calcium-dependent manner. No enzymatic function has been attributed to copines yet. Due to a cross-reacting activity of Mikbeta1, an antibody to the IL-2Rbeta chain, we were able to serendipitously purify, partially microsequence, and clone human copine III. The 5 kb copine III transcript is expressed ubiquitously as determined by a multitissue Northern blot analysis. Phosphoamino acid analysis revealed phosphorylation of copine III on serine and threonine residues. In vitro kinase assays were performed with immunoprecipitated endogenous copine III, chromatography-purified endogenous copine III, and recombinant copine III expressed in Saccharomyces cerevisiae. The exogenous substrate myelin basic protein was phosphorylated in all in vitro kinase assays containing copine III immunoprecipitate or purified copine III. A 60-kDa band was observed in corresponding in gel kinase assays with staurosporine-activated cells. Cell lines expressing high levels of copine III protein had correspondingly high kinase activity in copine III antiserum immunoprecipitate. However, the copine amino acid sequences lack the traditional kinase catalytic domain. Therefore, the data suggest copine III may possess an intrinsic kinase activity and represent a novel unconventional kinase family.

Protein tyrosine kinase inhibition blocks granule exocytosis and cytolytic function of lymphokine-activated killer cells.

Short-term pretreatment of human lymphokine-activated killer cells (LAK) with the protein tyrosine kinase-specific inhibitor Herbimycin A (Herb A) blocked cytotoxic function against the NK-resistant (LAK-sensitive) tumor targets, SK-Mel-1 (human melanoma) and Daudi (human lymphoma). Greater than 50% inhibition of LAK activity was observed after a 2.5-h pretreatment with 0.125 microgram/ml (ca. 0.2 microM) Herb A. Inhibition of LAK occurred over a time interval in which LAK were not dependent upon IL-2 for maintenance of killing function, supporting the conclusion that the drug interfered with mobilization of cytotoxic function. Conjugate formation between LAK and tumor targets was unaffected by Herb A, indicating that inhibition was occurring at a post-binding step. Granule exocytosis as measured by BLT-esterase release was detected from LAK after coincubation with tumor targets, and was inhibited by Herb A pretreatment. The majority of LAK killing was dependent upon extracellular calcium, supporting the hypothesis that granule exocytosis rather than Fas ligand was the principal pathway leading to target cell death. The data suggest that protein tyrosine kinases play a pivotal role in LAK cytolytic function by regulating granule exocytosis.