The neuromuscular transform in a single segment of a segmented heart tube.

Leech hearts are hybrids; they are myogenic but need entrainment by a heartbeat central pattern generator (CPG) to execute functional constriction patterns. Leech hearts are modular: two lateral segmented heart tubes running the length of the animal. Moving blood through the segmented heart tubes of leeches requires sequential constrictions, timed by a heartbeat CPG and relayed to each heart segment by likewise segmental motor neurons. The heartbeat CPG produces bilaterally asymmetric coordinations: rear-to-front peristaltic on one side and nearly synchronous on the other, periodically switching sides. We examined the neuromuscular transform of isolated heart segments in response to electrical nerve stimulation to identify the range of parameters (burst duration, intraburst pulse frequency, period) allowing the heart to constrict continuously and reliably. Constriction amplitudes increased with increasing intraburst frequencies and decreased with decreasing burst durations. Similar amplitudes were achieved with longer burst durations combined with lower frequencies or with shorter burst durations combined with higher frequencies. Long burst durations delayed relaxation, leading to summation and tetanus. The time, and its variability, between stimulus onset and time to constriction onset or to peak decreased with increasing frequency. Data previously obtained in vivo showed that the heart excitatory motor neurons fired longer bursts at lower frequencies at long periods moving to shorter bursts with higher intraburst frequencies as the period shortened. In this scenario, active constriction started earlier and the time to reach full systole shortened, allowing more time for relaxation. Relaxation time before the next motor neuron burst appears critical for maintaining constriction amplitude.NEW & NOTEWORTHY Moving blood through the segmented heart tubes of leeches requires sequential constrictions driven by motor neurons controlled by a central pattern generator. In a single heart segment, we varied stimuli to explore the neuromuscular transform. Decreasing the cycle period, e.g., to increase volume pumped over time, without altering motor burst duration and intraburst spike frequency shortens relaxation time and decreases amplitude. The likely strategy to preserve constriction amplitude is to shorten burst duration while increasing spike frequency.

Mononuclear cell dynamics in M. tuberculosis infection provide opportunities for therapeutic intervention.

Mycobacterium tuberculosis causes chronic infection of mononuclear phagocytes, especially resident (alveolar) macrophages, recruited macrophages, and dendritic cells. Despite the importance of these cells in tuberculosis (TB) pathogenesis and immunity, little is known about the population dynamics of these cells at the sites of infection. We used a combination of congenic monocyte adoptive transfer, and pulse-chase labeling of DNA, to determine the kinetics and characteristics of trafficking, differentiation, and infection of mononuclear phagocytes during the chronic, adaptive immune phase of M. tuberculosis infection in mice. We found that Ly6Chi monocytes traffic rapidly to the lungs, where a subpopulation become Ly6Clo and remain in the lung vascular space, while the remainder migrate into the lung parenchyma and differentiate into Ly6Chi dendritic cells, CD11b+ dendritic cells, and recruited macrophages. As in humans with TB, M. tuberculosis-infected mice have increased numbers of blood monocytes; this is due to increased egress from the bone marrow, and not delayed egress from the blood. Pulse-chase labeling of dividing cells and flow cytometry analysis revealed a T1/2 of ~15 hrs for Ly6Chi monocytes, indicating that they differentiate rapidly upon entry to the parenchyma of infected lungs; in contrast, cells that differentiate from Ly6Chi monocytes turn over more slowly, but diminish in frequency in less than one week. New cells (identified by pulse-chase labeling) acquire bacteria within 1-3 days of appearance in the lungs, indicating that bacteria regularly encounter new cellular niches, even during the chronic stage of infection. Our findings that mononuclear phagocyte populations at the site of M. tuberculosis infection are highly dynamic provide support for specific approaches for host-directed therapies directed at monocytes, including trained immunity, as potential interventions in TB, by replacing cells with limited antimycobacterial capabilities with newly-recruited cells better able to restrict and kill M. tuberculosis.

Limited Antimycobacterial Efficacy of Epitope Peptide Administration Despite Enhanced Antigen-Specific CD4 T-Cell Activation.

Background: Infection with Mycobacterium tuberculosis is associated with inconsistent and incomplete elimination of the bacteria, despite development of antigen-specific T-cell responses. One mechanism used by M tuberculosis is to limit availability of antigen for activation of CD4 T cells. Methods: We examined the utility of systemic administration of epitope peptides to activate pre-existing T cells in mice infected with M tuberculosis. Results: We found that systemic peptide administration (1) selectively activates T cells specific for the epitope peptide, (2) loads major histocompatibility complex class II on lung macrophages and dendritic cells, (3) activates CD4 T cells in the lung parenchyma, (4) and has little antimycobacterial activity. Conclusions: Further studies revealed that CD4 T cells in lung lesions are distant from the infected cells, suggesting that, even if they can be activated, the positioning of CD4 T cells and their direct interactions with infected cells may be limiting determinants of immunity in tuberculosis.

Output variability across animals and levels in a motor system.

Rhythmic behaviors vary across individuals. We investigated the sources of this output variability across a motor system, from the central pattern generator (CPG) to the motor plant. In the bilaterally symmetric leech heartbeat system, the CPG orchestrates two coordinations in the bilateral hearts with different intersegmental phase relations (Δϕ) and periodic side-to-side switches. Population variability is large. We show that the system is precise within a coordination, that differences in repetitions of a coordination contribute little to population output variability, but that differences between bilaterally homologous cells may contribute to some of this variability. Nevertheless, much output variability is likely associated with genetic and life history differences among individuals. Variability of Δϕ were coordination-specific: similar at all levels in one, but significantly lower for the motor pattern than the CPG pattern in the other. Mechanisms that transform CPG output to motor neurons may limit output variability in the motor pattern.

The neural control of heartbeat in invertebrates.

The neurogenic heartbeat of certain invertebrates has long been studied both as a way of understanding how automatic functions are regulated and for how neuronal networks generate the inherent rhythmic activity that controls and coordinates this vital function. This review focuses on the heartbeat of decapod crustaceans and hirudinid leeches, which remain important experimental systems for the exploration of central pattern generator networks, their properties, network and cellular mechanisms, modulation, and how animal-to-animal variation in neuronal and network properties are managed to produce functional output.

Tweet Now, See You In the ED Later? Examining the Association Between Alcohol-related Tweets and Emergency Care Visits.

BACKGROUND: Alcohol use is a major and unpredictable driver of emergency department (ED) visits. Regional Twitter activity correlates ecologically with behavioral outcomes. No such correlation has been established in real time. OBJECTIVES: The objective was to examine the correlation between real-time, alcohol-related tweets and alcohol-related ED visits. METHODS: We developed and piloted a set of 11 keywords that identified tweets related to alcohol use. In-state tweets were identified using self-declared profile information or geographic coordinates. Using Datasift, a third-party vendor, a random sample of 1% of eligible tweets containing the keywords and originating in state were downloaded (including tweet date/time) over 3 discrete weeks in 3 different months. In the same time frame, we examined visits to an urban, high-volume, Level I trauma center that receives > 25% of the emergency care volume in the state. Alcohol-related ED visits were defined as visits with a chief complaint of alcohol use, positive blood alcohol, or alcohol-related ICD-9 code. Spearman's correlation coefficient was used to examine the hourly correlation between alcohol-related tweets, alcohol-related ED visits, and all ED visits. RESULTS: A total of 7,820 tweets (representing 782,000 in-state alcohol-related tweets during the 3 weeks) were identified. Concurrently, 404 ED visits met criteria for being alcohol-related versus 2939 non-alcohol-related ED visits. There was a statistically significant relationship between hourly alcohol-related tweet volume and number of alcohol-related ED visits (rs = 0.31, p < 0.00001), but not between hourly alcohol-related tweet volume and number of non-alcohol-related ED visits (rs = -0.07, p = 0.11). CONCLUSION: In a single state, a statistically significant relationship was observed between the hourly number of alcohol-related tweets and the hourly number of alcohol-related ED visits. Real-time Twitter monitoring may help predict alcohol-related surges in ED visits. Future studies should include larger numbers of EDs and natural language processing.

Low doses of imatinib induce myelopoiesis and enhance host anti-microbial immunity.

Imatinib mesylate (Gleevec) inhibits Abl1, c-Kit, and related protein tyrosine kinases (PTKs) and serves as a therapeutic for chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib also has efficacy against various pathogens, including pathogenic mycobacteria, where it decreases bacterial load in mice, albeit at doses below those used for treating cancer. We report that imatinib at such low doses unexpectedly induces differentiation of hematopoietic stem cells and progenitors in the bone marrow, augments myelopoiesis but not lymphopoiesis, and increases numbers of myeloid cells in blood and spleen. Whereas progenitor differentiation relies on partial inhibition of c-Kit by imatinib, lineage commitment depends upon inhibition of other PTKs. Thus, imatinib mimics "emergency hematopoiesis," a physiological innate immune response to infection. Increasing neutrophil numbers by adoptive transfer sufficed to reduce mycobacterial load, and imatinib reduced bacterial load of Franciscella spp., which do not utilize imatinib-sensitive PTKs for pathogenesis. Thus, potentiation of the immune response by imatinib at low doses may facilitate clearance of diverse microbial pathogens.

Variation in motor output and motor performance in a centrally generated motor pattern.

Central pattern generators (CPGs) produce motor patterns that ultimately drive motor outputs. We studied how functional motor performance is achieved, specifically, whether the variation seen in motor patterns is reflected in motor performance and whether fictive motor patterns differ from those in vivo. We used the leech heartbeat system in which a bilaterally symmetrical CPG coordinates segmental heart motor neurons and two segmented heart tubes into two mutually exclusive coordination modes: rear-to-front peristaltic on one side and nearly synchronous on the other, with regular side-to-side switches. We assessed individual variability of the motor pattern and the beat pattern in vivo. To quantify the beat pattern we imaged intact adults. To quantify the phase relations between motor neurons and heart constrictions we recorded extracellularly from two heart motor neurons and movement from the corresponding heart segments in minimally dissected leeches. Variation in the motor pattern was reflected in motor performance only in the peristaltic mode, where larger intersegmental phase differences in the motor neurons resulted in larger phase differences between heart constrictions. Fictive motor patterns differed from those in vivo only in the synchronous mode, where intersegmental phase differences in vivo had a larger front-to-rear bias and were more constrained. Additionally, load-influenced constriction timing might explain the amplification of the phase differences between heart segments in the peristaltic mode and the higher variability in motor output due to body shape assumed in this soft-bodied animal. The motor pattern determines the beat pattern, peristaltic or synchronous, but heart mechanics influence the phase relations achieved.

Chronic but not acute virus infection induces sustained expansion of myeloid suppressor cell numbers that inhibit viral-specific T cell immunity.

Resolution of acute and chronic viral infections requires activation of innate cells to initiate and maintain adaptive immune responses. Here we report that infection with acute Armstrong (ARM) or chronic Clone 13 (C13) strains of lymphocytic choriomeningitis virus (LCMV) led to two distinct phases of innate immune response. During the first 72 hr of infection, dendritic cells upregulated activation markers and stimulated antiviral CD8(+) T cells, independent of viral strain. Seven days after infection, there was an increase in Ly6C(hi) monocytic and Gr-1(hi) neutrophilic cells in lymphoid organs and blood. This expansion in cell numbers was enhanced and sustained in C13 infection, whereas it occurred only transiently with ARM infection. These cells resembled myeloid-derived suppressor cells and potently suppressed T cell proliferation. The reduction of monocytic cells in Ccr2(-/-) mice or after Gr-1 antibody depletion enhanced antiviral T cell function. Thus, innate cells have an important immunomodulatory role throughout chronic infection.

Animal-to-animal variability of connection strength in the leech heartbeat central pattern generator.

The heartbeat central pattern generator (CPG) in medicinal leeches controls blood flow within a closed circulatory by programming the constrictions of two parallel heart tubes. This circuit reliably produces a stereotyped fictive pattern of activity and has been extensively characterized. Here we determined, as quantitatively as possible, the strength of each inhibitory synapse and electrical junction within the core circuit of the heartbeat CPG. We also examined the animal-to-animal variability in strengths of these connections and, for some, determined the correlations between connections to the same postsynaptic target. The core CPG is composed of seven bilateral pairs of heart interneurons connected via both inhibitory chemical synapses and electrical junctions. Fifteen different connections within the core CPG were measured for strength using extracellular presynaptic recordings and postsynaptic voltage-clamp recordings across a minimum of seven individuals each, and the animal-to-animal variability was characterized. Connection strengths within the core network varied three to more than sevenfold among individuals (depending on the specific connection). The balance between two inputs onto various postsynaptic targets was explored by within-individual comparisons and correlation across individuals. Of the seven comparisons made within the core CPG, three showed a clear correlation of connection strengths, while the other four did not. We conclude that the leech heartbeat CPG can withstand wide variability in connection strengths and still produce stereotyped output. The network appears to preserve the relative strengths of some pairs of inputs, despite the animal-to-animal variability.

Randomized trial of tamoxifen versus combined tamoxifen and octreotide LAR Therapy in the adjuvant treatment of early-stage breast cancer in postmenopausal women: NCIC CTG MA.14.

PURPOSE: Somatostatin analogs act directly on breast cancer cells and indirectly on insulin and insulin-like growth factor 1 (IGF-1) levels. This trial was undertaken to assess whether octreotide would lower insulin and IGF-1 levels and reduce risk of breast cancer recurrence. PATIENTS AND METHODS: The NCIC CTG MA.14 (NCIC Clinical Trials Group MA.14) trial randomly assigned postmenopausal women to 5 years of tamoxifen 20 mg daily (TAM) or TAM plus 2 years of octreotide 90 mg depot intramuscular injections monthly (TAM-OCT) as adjuvant therapy. The primary end point was event-free survival (EFS). Secondary end points were relapse-free survival (RFS), overall survival (OS), toxicity, and effects of treatment on IGF physiology. RESULTS: Among 667 women with a median follow-up of 7.9 years, 220 events occurred-108 with TAM-OCT and 112 with TAM. Adjusted hazard ratios (HRs; TAM-OCT to TAM) were 0.93 for EFS (95% CI, 0.71 to 1.22; P = .62), 0.84 for RFS (95% CI, 0.59 to 1.18; P = .31), and 0.97 for OS (95% CI, 0.69 to 1.37; P = .86). Among patients with normal baseline gallbladder imaging, cholecystectomy was required in 23.0% of those receiving TAM-OCT but in only 1.4% of those receiving TAM (P < .001). At 4 months, TAM-OCT had significantly (P < .001) lowered IGF-1, IGF binding protein 3, and C-peptide levels. Older age (P = .02), tumor size (P = .001), nodal status (P = .01), high C-peptide levels (P < .001), and higher body mass index (BMI) in models excluding C-peptide (P < .001) were associated with poorer EFS in multivariate analysis. CONCLUSION: Octreotide-related changes in circulating IGF-1 and C-peptide levels were statistically significant. Octreotide did not add significant clinical benefit. High C-peptide levels (surrogate for insulin secretion rate) and high BMI were associated with poor outcome.

Coping with variability in small neuronal networks.

Experimental and corresponding modeling studies indicate that there is a 2- to 5-fold variation of intrinsic and synaptic parameters across animals while functional output is maintained. Here, we review experiments, using the heartbeat central pattern generator (CPG) in medicinal leeches, which explore the consequences of animal-to-animal variation in synaptic strength for coordinated motor output. We focus on a set of segmental heart motor neurons that all receive inhibitory synaptic input from the same four premotor interneurons. These four premotor inputs fire in a phase progression and the motor neurons also fire in a phase progression because of differences in synaptic strength profiles of the four inputs among segments. Our work tested the hypothesis that functional output is maintained in the face of animal-to-animal variation in the absolute strength of connections because relative strengths of the four inputs onto particular motor neurons is maintained across animals. Our experiments showed that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in strength of particular inputs do not correlate strongly with output phase. Further experiments measured the precise temporal pattern of the premotor inputs, the segmental synaptic strength profiles of their connections onto motor neurons, and the temporal pattern (phase progression) of those motor neurons all in the same animal for a series of 12 animals. The analysis of input and output in this sample of 12 individuals suggests that the number (four) of inputs to each motor neuron and the variability of the temporal pattern of input from the CPG across individuals weaken the influence of the strength of individual inputs. Moreover, the temporal pattern of the output varies as much across individuals as that of the input. Essentially, each animal arrives at a unique solution for how the network produces functional output.

Bringing up the rear: new premotor interneurons add regional complexity to a segmentally distributed motor pattern.

Central pattern generators (CPGs) pace and pattern many rhythmic activities. We have uncovered a new module in the heartbeat CPG of leeches that creates a regional difference in this segmentally distributed motor pattern. The core CPG consists of seven identified pairs and one unidentified pair of heart interneurons of which 5 pairs are premotor and inhibit 16 pairs of heart motor neurons. The heartbeat CPG produces a side-to-side asymmetric pattern of activity of the premotor heart interneurons corresponding to an asymmetric fictive motor pattern and an asymmetric constriction pattern of the hearts with regular switches between the two sides. The premotor pattern progresses from rear to front on one side and nearly synchronously on the other; the motor pattern shows corresponding intersegmental coordination, but only from segment 15 forward. In the rearmost segments the fictive motor pattern and the constriction pattern progress from front to rear on both sides and converge in phase. Modeling studies suggested that the known inhibitory inputs to the rearmost heart motor neurons were insufficient to account for this activity. We therefore reexamined the constriction pattern of intact leeches. We also identified electrophysiologically two additional pairs of heart interneurons in the rear. These new heart interneurons make inhibitory connections with the rear heart motor neurons, are coordinated with the core heartbeat CPG, and are dye-coupled to their contralateral homologs. Their strong inhibitory connections with the rearmost heart motor neurons and the small side-to-side phase difference of their bursting contribute to the different motor and beating pattern observed in the animal's rear.

Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization.

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.

Constancy and variability in the output of a central pattern generator.

Experimental and corresponding modeling studies have demonstrated a twofold to fivefold variation of intrinsic and synaptic parameters across animals, whereas functional output is maintained. These studies have led to the hypothesis that correlated, compensatory changes in particular parameters can at least partially explain the biological variability in parameters. Using the leech heartbeat central pattern generator (CPG), we selected three different segmental motor neurons that fire in a functional phase progression but receive input from the same four premotor interneurons. Previous work suggested that the phase progression arises because the pattern of relative strength of the four inputs varies systematically across the segmental motor neurons. Nevertheless, there was considerable animal-to-animal variation in the absolute strengths of these connections. We tested the hypothesis that functional output is maintained in the face of variation in the absolute strength of connections because relative strengths onto particular motor neurons are maintained. We found that relative strength is not strictly maintained across animals even as functional output is maintained, and animal-to-animal variations in relative strength of particular inputs do not correlate strongly with output phase. In parallel with this variation in synaptic strength, the firing phase of the premotor inputs to these motor neurons varies considerably across individuals. We conclude that the number (four) of inputs to each motor neuron, which each vary in strength, and the phase diversity of the temporal pattern of input from the CPG diminish the influence of individual inputs. We hypothesize that each animal arrives at a unique solution for how the network produces functional output.

A role for compromise: synaptic inhibition and electrical coupling interact to control phasing in the leech heartbeat CpG.

How can flexible phasing be generated by a central pattern generator (CPG)? To address this question, we have extended an existing model of the leech heartbeat CPG's timing network to construct a model of the CPG core and explore how appropriate phasing is set up by parameter variation. Within the CPG, the phasing among premotor interneurons switches regularly between two well defined states - synchronous and peristaltic. To reproduce experimentally observed phasing, we varied the strength of inhibitory synaptic and excitatory electrical input from the timing network to follower premotor interneurons. Neither inhibitory nor electrical input alone was sufficient to produce proper phasing on both sides, but instead a balance was required. Our model suggests that the different phasing of the two sides arises because the inhibitory synapses and electrical coupling oppose one another on one side (peristaltic) and reinforce one another on the other (synchronous). Our search of parameter space defined by the strength of inhibitory synaptic and excitatory electrical input strength led to a CPG model that well approximates the experimentally observed phase relations. The strength values derived from this analysis constitute model predictions that we tested by measurements made in the living system. Further, variation of the intrinsic properties of follower interneurons showed that they too systematically influence phasing. We conclude that a combination of inhibitory synaptic and excitatory electrical input interacting with neuronal intrinsic properties can flexibly generate a variety of phase relations so that almost any phasing is possible.

A phase II trial of a neoadjuvant platinum regimen for locally advanced breast cancer: pathologic response, long-term follow-up, and correlation with biomarkers.

PURPOSE: The purpose of this study is to determine the response, tolerability, and long-term outcome of a neoadjuvant platinum-containing regimen for locally advanced breast cancer (LABC) and to search for a correlation between pathologic complete response (pCR) and predefined biomarkers in this cohort. PATIENTS AND METHODS: Patients with LABC received 8 cycles of either sequence A or B. Sequence A was doxorubicin 60 mg/m(2) and paclitaxel 175 mg/m(2) (AT) every 3 weeks x 4 followed by cisplatin (C) 60 mg/m(2) and paclitaxel 90 mg/m(2) (CT) every 2 weeks x 4. Sequence B was CT x 4 (with paclitaxel dose escalation) followed by AT x 4. In addition to estrogen receptor (ER) and HER2, immunohistochemistry for MDR-1, MRP-1, topoisomerase IIalpha (topo IIalpha), and p53 was performed. RESULTS: A total of 88 patients were evaluable for response and toxicity. Median follow-up was 97 months. The overall pCR rate was 21.5%. For subgroups ER+/HER2-, HER2+ and double negative (ER-/HER2-) disease, the pCR rates were 5.9%, 23.3%, and 35%, respectively (P = .006). Five-year overall survival for the entire cohort was 71.1%. Five-year overall survival was 88.1% (95% CI, 77.1%-99.1%) for the ER+/HER2- group compared with 68.5% (95% CI, 51.3%-85.7%) and 49.5% (95% CI, 27.4%-71.6%) in the HER2+ and "double-negative" group, respectively (P = .0077). Overexpression of topo IIalpha was correlated with pCR (P < .001). There were no toxic deaths. CONCLUSION: A platinum-containing neoadjuvant regimen was well tolerated and achieved a pCR comparable to other recent studies of multiagent chemotherapy. Further studies tailored for specific breast cancer subtypes are required.

Randomized phase II study comparing two schedules of everolimus in patients with recurrent/metastatic breast cancer: NCIC Clinical Trials Group IND.163.

PURPOSE: To evaluate the safety and efficacy of oral everolimus, a mammalian target of rapamycin (mTOR) inhibitor, in two different schedules in minimally pretreated patients with metastatic breast cancer and to explore for possible biologic correlates of response. PATIENTS AND METHODS: Patients who received no or one prior chemotherapy regimen for metastatic breast cancer were entered onto this multicenter, noncomparative, randomized phase II study of everolimus 10 mg daily versus 70 mg weekly; the multinomial end points of response and progression were evaluated at 8 weeks. A two-stage accrual design was used, with 15 evaluable patients in each schedule in stage 1. Only daily therapy met criteria for continuing, and another 15 patients were added. pAKT, PTEN, carbonic anhydrase 9, estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) were evaluated for possible correlation with response. RESULTS: The most common drug-related toxicities were fatigue, rash, anorexia, diarrhea, stomatitis, cough, and pneumonitis. Pneumonitis occurred at higher than expected rates and seemed to be schedule dependent, with the highest incidence on the daily schedule. Response rate with daily therapy was 12% (95% CI, 3.4% to 28.2%) compared with 0% (95% CI, 0.0% to 20.6%) for weekly therapy. Twenty-seven percent of patients on daily therapy discontinued treatment compared with 13% on weekly therapy (16% v 6% with pneumonitis, respectively). No biologic correlates of response could be identified, although there were trends favoring benefit in ER-positive and HER2-negative metastatic breast cancer. CONCLUSION: Oral everolimus has activity in metastatic breast cancer that is schedule dependent. Daily therapy with 10 mg is worthy of further study in this patient population.

Human epidermal growth factor receptor 2 overexpression as a prognostic factor in a large tissue microarray series of node-negative breast cancers.

PURPOSE: Human epidermal growth factor receptor 2 gene (HER2) is associated with a poorer outcome in node-positive breast cancer, but the results are conflicting in node-negative disease. This study assessed the prognostic impact of HER2 overexpression/amplification in a large series of node-negative breast cancers. PATIENTS AND METHODS: A tissue microarray (TMA) series was constructed consisting of 4,444 invasive breast cancers diagnosed in British Columbia from 1986 to 1992. Within this series, 2,026 patients were node negative, of whom 70% did not receive adjuvant systemic therapy. The TMA series was assessed for estrogen receptor (ER) and HER2. Logistic regression modeling was used to estimate odds ratios at the 10-year follow-up. RESULTS: HER2 was positive in 10.2% of the node-negative cohort. In this cohort, an inferior outcome was seen in patients with HER2-positive tumors compared with HER2-negative tumors for 10-year relapse-free survival (RFS; 65.9% v 75.5%, respectively; P = .01), distant RFS (71.2% v 81.8%, respectively; P = .004), and breast cancer-specific survival (BCSS; 75.5% v 86.3%, respectively; P = .001). A trend for a worse overall survival was also seen (P = .06). HER2 was an independent poor prognostic factor for RFS and BCSS at 10 years, with odds ratios of 1.71 (P = .01) and 2.03 (P = .003), respectively. The number of HER2-positive tumors that were

Risk of early recurrence among postmenopausal women with estrogen receptor-positive early breast cancer treated with adjuvant tamoxifen.

BACKGROUND: Adjuvant aromatase inhibitors (AIs), instead of or after tamoxifen, are effective in decreasing recurrence in postmenopausal women with estrogen receptor (ER)-positive breast cancer. An understanding of which patients are at risk of early recurrence while they are receiving tamoxifen may improve clinical decision making. METHODS: The patients who were included in this study were women aged >or= 50 years with early-stage, ER-positive breast cancer diagnosed between 1986 and 1999 and had been treated with tamoxifen. Characteristics of the patients with early recurrences (within 2.5 years of diagnosis), late recurrences (between 2.5 years and 5 years) and no recurrence within 5 years were compared. Logistic regression analyses were conducted to identify which groups were at risk of early recurrence. RESULTS: Among 3844 women, 304 women (7.9%) developed disease recurrence within 2.5 years. Higher than average rates of recurrence within 2.5 years were observed in cohorts with lymph node (N)-positive tumors (11.5%), grade 3 histology (14.3%), or low-positive ER levels, ie, 10-49 fmol/mg or 10%-20% staining (14.9%). In multivariate analyses, only pathologically N-positive tumors (1-3 vs 0 positive lymph nodes: odds ratio [OR], 1.6; 4-9 vs 0 positive lymph nodes: OR, 2.23 [P= .03]) and low-positive ER status (OR, 2.04; P= .01) were associated with recurrence within 2.5 years compared with recurrence between 2.5 years and 5 years. Other clinical and pathologic variables were not predictive of early recurrence. CONCLUSIONS: Subgroups of women with early ER-positive breast cancer may be identified who are at increased risk of recurrence within 2.5 years of diagnosis despite tamoxifen. It remains to be proven whether upfront AI therapy results in an advantage to these women.