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1.  The RpoB H481Y Rifampicin Resistance Mutation and an Active Stringent Response Reduce Virulence and Increase Resistance to Innate Immune Responses in Staphylococcus aureus 
The Journal of Infectious Diseases  2012;207(6):929-939.
The occurrence of mutations in methicillin-resistant Staphylococcus aureus (MRSA) during persistent infection leads to antimicrobial resistance but may also impact host-pathogen interactions. Here, we investigate the host-pathogen consequences of 2 mutations arising in clinical MRSA during persistent infection: RpoB H481Y, which is linked to rifampicin resistance, and RelA F128Y, which is associated with an active stringent response. Allelic exchange experiments showed that both mutations cause global transcriptional changes, leading to upregulation of capsule production, with attenuated virulence in a murine bacteremia model and reduced susceptibility to both antimicrobial peptides and whole-blood killing. Disruption of capsule biosynthesis reversed these impacts on innate immune function. These data clearly link MRSA persistence and reduced virulence to the same mechanisms that alter antimicrobial susceptibility. Our study highlights the wider consequences of suboptimal antimicrobial use, where drug resistance and immune escape mechanisms coevolve, thus increasing the likelihood of treatment failure.
doi:10.1093/infdis/jis772
PMCID: PMC3633451  PMID: 23255563
Staphylococcus aureus; antibiotic resistance; virulence; persistence
2.  A Catalyst for Change: The European Cancer Patient’s Bill of Rights 
The Oncologist  2014;19(3):217-224.
The European Cancer Concord is a unique patient-centered partnership that will act as a catalyst to achieve improved access to an optimal standard of cancer care and research for European citizens. In order to provide tangible benefits for European cancer patients, the partnership proposes the creation of a “European Cancer Patient’s Bill of Rights,” a patient charter that will underpin equitable access to an optimal standard of care for Europe’s citizens.
doi:10.1634/theoncologist.2013-0452
PMCID: PMC3958470  PMID: 24493667
3.  Pretreatment anti-Müllerian hormone predicts for loss of ovarian function after chemotherapy for early breast cancer☆ 
European Journal of Cancer  2013;49(16):3404-3411.
Aim
Improving survival for women with early breast cancer (eBC) requires greater attention to the consequences of treatment, including risk to ovarian function. We have assessed whether biochemical markers of the ovarian reserve might improve prediction of chemotherapy related amenorrhoea.
Methods
Women (n = 59, mean age 42.6 years [(range 23.3–52.5]) with eBC were recruited before any treatment. Pretreatment ovarian reserve markers (anti-Müllerian hormone [AMH], follicle-stimulating hormone [FSH], inhibin B) were analysed in relation to ovarian status at 2 years.
Results
Pretreatment AMH was significantly lower in women with amenorrhoea at 2 years (4.0 ± 0.9 pmol/L versus 17.2 ± 2.5, P < 0.0001), but FSH and inhibin B did not differ between groups. By logistic regression, pretreatment AMH, but not age, FSH or inhibin B, was an independent predictor of ovarian status at 2 years (P = 0.005; odds ratio 0.013). We combined these data with a similar cohort (combined n = 75); receiver–operator characteristic analysis for AMH gave area under curve (AUC) of 0.90 (95% confidence interval (CI) 0.82–0.97)). A cross-validated classification tree analysis resulted in a binary classification schema with sensitivity 98.2% and specificity 80.0% for correct classification of amenorrhoea.
Conclusion
Pretreatment AMH is a useful predictor of long term post chemotherapy loss of ovarian function in women with eBC, adding significantly to the only previously established individualising predictor, i.e. age. AMH measurement may assist decision-making regarding treatment options and fertility preservation procedures.
doi:10.1016/j.ejca.2013.07.014
PMCID: PMC3807650  PMID: 23968732
AMH; Ovarian reserve; Chemotherapy; Amenorrhoea; Fertility; Breast cancer
4.  Serine/Threonine Phosphatase Stp1 Contributes to Reduced Susceptibility to Vancomycin and Virulence in Staphylococcus aureus 
The Journal of Infectious Diseases  2012;205(11):1677-1687.
The genetic mechanisms that contribute to reduced susceptibility to vancomycin in Staphylococcus aureus are complex and heterogeneous. In addition, debate is emerging as to the true effect of reduced susceptibility to vancomycin on staphylococcal virulence. To investigate this, comparative genomics was performed on a collection of vancomycin-exposed isogenic S. aureus pairs (14 strains in total). Previously described mutations were observed in genes such as vraG, agrA, yvqF, and rpoB; however, a new mechanism was identified involving a serine/threonine phosphatase, Stp1. After constructing an stp1 deletion mutant, we showed that stp1 is important in vancomycin susceptibility and cell wall biosynthesis. Gene expression studies showed that stp1 also regulates virulence genes, including a hemolysin, superantigen-like protein, and phenol-soluble modulin, and that the deletion mutant is attenuated in virulence in vivo. Stp1 provides a new link between vancomycin susceptibility and virulence in S. aureus.
doi:10.1093/infdis/jis252
PMCID: PMC3415852  PMID: 22492855
5.  Is TIMP-1 immunoreactivity alone or in combination with other markers a predictor of benefit from anthracyclines in the BR9601 adjuvant breast cancer chemotherapy trial? 
Introduction
Predictive cancer biomarkers to guide the right treatment to the right patient at the right time are strongly needed. The purpose of the present study was to validate prior results that tissue inhibitor of metalloproteinase 1 (TIMP-1) alone or in combination with either HER2 or TOP2A copy number can be used to predict benefit from epirubicin (E) containing chemotherapy compared with cyclophosphamide, methotrexate and fluorouracil (CMF) treatment.
Methods
For the purpose of this study, formalin fixed paraffin embedded tumor tissue from women recruited into the BR9601 clinical trial, which randomized patients to E-CMF versus CMF, were analyzed for TIMP-1 immunoreactivity. Using previously collected data for HER2 amplification and TOP2A gene aberrations, we defined patients as "anthracycline non-responsive", that is, 2T (TIMP-1 immunoreactive and TOP2A normal) and HT (TIMP-1 immunoreactive and HER2 negative) and anthracycline responsive (all other cases).
Results
In total, 288 tumors were available for TIMP-1 analysis with (183/274) 66.8%, and (181/274) 66.0% being classed as 2T and HT responsive, respectively. TIMP-1 was neither associated with patient prognosis (relapse free survival or overall survival) nor with a differential effect of E-CMF and CMF. Also, TIMP-1 did not add to the predictive value of HER2, TOP2A gene aberrations, or to Ki67 immunoreactivity.
Conclusion
This study could not confirm the predictive value of TIMP-1 immunoreactivity in patients randomized to receive E-CMF versus CMF as adjuvant treatment for primary breast cancer.
doi:10.1186/bcr3411
PMCID: PMC3672718  PMID: 23570501
6.  Phase III Multicenter Clinical Trial of the Sialyl-TN (STn)-Keyhole Limpet Hemocyanin (KLH) Vaccine for Metastatic Breast Cancer 
The Oncologist  2011;16(8):1092-1100.
Results of a double-blind, randomized, phase III clinical trial evaluating time to progression and overall survival in women with metastatic breast cancer who received sialyl-TN keyhole limpet hemocyanin vaccine are reported.
Purpose.
This double-blind, randomized, phase III clinical trial evaluated time to progression (TTP) and overall survival in women with metastatic breast cancer (MBC) who received sialyl-TN (STn) keyhole limpet hemocyanin (KLH) vaccine. Secondary endpoints included vaccine safety and immune response.
Experimental design.
The study population consisted of 1,028 women with MBC across 126 centers who had previously received chemotherapy and had had either a complete or a partial response or no disease progression. All women received one-time i.v. cyclophosphamide (300 mg/m2) 3 days before s.c. injection of 100 μg STn-KLH plus adjuvant (treatment group) or 100 μg KLH plus adjuvant (control group) at weeks 0, 2, 5, and 9. Subsequently, STn-KLH without adjuvant or KLH without adjuvant was then administered monthly for 4 months, and then quarterly until disease progression, without cyclophosphamide.
Results.
STn-KLH vaccine was well tolerated; patients had mild to moderate injection-site reactions and reversible flu-like symptoms. Week-12 antibody testing revealed high specific IgG titers and a high rate of IgM-to-IgG seroconversion; the median IgG titers in STn-KLH recipients were 320 (anti-ovine submaxillary mucin) and 20,480 (anti-STn), with no detectable antimucin antibodies in the control group. The TTP was 3.4 months in the treatment group and 3.0 months in the control group. The median survival times were 23.1 months and 22.3 months, respectively.
Conclusions.
Although STn-KLH was well tolerated in this largest to date metastatic breast cancer vaccine trial, no overall benefit in TTP or survival was observed. Lessons were learned for future vaccine study designs.
doi:10.1634/theoncologist.2010-0307
PMCID: PMC3228158  PMID: 21572124
Metastatic breast cancer; MUC-1 antibody; Antiestrogen; Vaccine; Chemotherapy
7.  Hierarchical clustering of gene expression patterns in the Eomes + lineage of excitatory neurons during early neocortical development 
BMC Neuroscience  2012;13:90.
Background
Cortical neurons display dynamic patterns of gene expression during the coincident processes of differentiation and migration through the developing cerebrum. To identify genes selectively expressed by the Eomes + (Tbr2) lineage of excitatory cortical neurons, GFP-expressing cells from Tg(Eomes::eGFP) Gsat embryos were isolated to > 99% purity and profiled.
Results
We report the identification, validation and spatial grouping of genes selectively expressed within the Eomes + cortical excitatory neuron lineage during early cortical development. In these neurons 475 genes were expressed ≥ 3-fold, and 534 genes ≤ 3-fold, compared to the reference population of neuronal precursors. Of the up-regulated genes, 328 were represented at the Genepaint in situ hybridization database and 317 (97%) were validated as having spatial expression patterns consistent with the lineage of differentiating excitatory neurons. A novel approach for quantifying in situ hybridization patterns (QISP) across the cerebral wall was developed that allowed the hierarchical clustering of genes into putative co-regulated groups. Forty four candidate genes were identified that show spatial expression with Intermediate Precursor Cells, 49 candidate genes show spatial expression with Multipolar Neurons, while the remaining 224 genes achieved peak expression in the developing cortical plate.
Conclusions
This analysis of differentiating excitatory neurons revealed the expression patterns of 37 transcription factors, many chemotropic signaling molecules (including the Semaphorin, Netrin and Slit signaling pathways), and unexpected evidence for non-canonical neurotransmitter signaling and changes in mechanisms of glucose metabolism. Over half of the 317 identified genes are associated with neuronal disease making these findings a valuable resource for studies of neurological development and disease.
doi:10.1186/1471-2202-13-90
PMCID: PMC3583225  PMID: 22852769
Transcriptome; Migration; Cortex; Profiling; Excitatory neuron
8.  Layer 6 cortical neurons require Reelin-Dab1 signaling for cellular orientation, Golgi deployment, and directed neurite growth into the marginal zone 
Neural Development  2012;7:25.
Background
The secreted ligand Reelin is believed to regulate the translocation of prospective layer 6 (L6) neocortical neurons into the preplate, a loose layer of pioneer neurons that overlies the ventricular zone. Recent studies have also suggested that Reelin controls neuronal orientation and polarized dendritic growth during this period of early cortical development. To explicitly characterize and quantify how Reelin controls this critical aspect of neurite initiation and growth we used a new ex utero explant model of early cortical development to selectively label a subset of L6 cortical neurons for complete 3-D reconstruction.
Results
The total neurite arbor sizes of neurons in Reelin-deficient (reeler mutant) and Dab1-deficient (Reelin-non-responsive scrambler mutant) cortices were quantified and unexpectedly were not different than control arbor lengths (p = 0.51). For each mutant, however, arbor organization was markedly different: mutant neurons manifested more primary processes (neurites emitted directly from the soma) than wild type, and these neurites were longer and displayed less branching. Reeler and scrambler mutant neurites extended tangentially rather than radially, and the Golgi apparatus that normally invests the apical neurite was compact in both reeler and scrambler mutants. Mutant cortices also exhibited a neurite “exclusion zone” which was relatively devoid of L6 neuron neurites and extended at least 15 μm beneath the pial surface, an area corresponding to the marginal zone (MZ) in the wild type explants. The presence of an exclusion zone was also indicated in the orientation of mutant primary neurite and neuronal somata, which failed to adopt angles within ~20˚ of the radial line to the pial surface. Injection of recombinant Reelin to reeler, but not scrambler, mutant cortices fully rescued soma orientation, Golgi organization, and dendritic projection defects within four hrs.
Conclusions
These findings indicate Reelin promotes directional dendritic growth into the MZ, an otherwise exclusionary zone for L6 neurites.
doi:10.1186/1749-8104-7-25
PMCID: PMC3466444  PMID: 22770513
Preplate; Neurite; Orientation; Epilepsy; Migration; Cortex; Dendrite
9.  Whole Genome Characterization of the Mechanisms of Daptomycin Resistance in Clinical and Laboratory Derived Isolates of Staphylococcus aureus 
PLoS ONE  2012;7(1):e28316.
Background
Daptomycin remains one of our last-line anti-staphylococcal agents. This study aims to characterize the genetic evolution to daptomycin resistance in S. aureus.
Methods
Whole genome sequencing was performed on a unique collection of isogenic, clinical (21 strains) and laboratory (12 strains) derived strains that had been exposed to daptomycin and developed daptomycin-nonsusceptibility. Electron microscopy (EM) and lipid membrane studies were performed on selected isolates.
Results
On average, six coding region mutations were observed across the genome in the clinical daptomycin exposed strains, whereas only two mutations on average were seen in the laboratory exposed pairs. All daptomycin-nonsusceptible strains had a mutation in a phospholipid biosynthesis gene. This included mutations in the previously described mprF gene, but also in other phospholipid biosynthesis genes, including cardiolipin synthase (cls2) and CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (pgsA). EM and lipid membrane composition analyses on two clinical pairs showed that the daptomycin-nonsusceptible strains had a thicker cell wall and an increase in membrane lysyl-phosphatidylglycerol.
Conclusion
Point mutations in genes coding for membrane phospholipids are associated with the development of reduced susceptibility to daptomycin in S. aureus. Mutations in cls2 and pgsA appear to be new genetic mechanisms affecting daptomycin susceptibility in S. aureus.
doi:10.1371/journal.pone.0028316
PMCID: PMC3253072  PMID: 22238576
10.  An epifluorescent attachment improves whole-plant digital photography of Arabidopsis thaliana expressing red-shifted green fluorescent protein 
AoB Plants  2012;2012:pls003.
Filter cubes made with machine-vision dichroic filters and illuminated with a royal blue light emitting diode can be used to produce an epifluorescent digital camera attachment that improves whole organism green fluorescent protein (GFP) photography. Mean pixel intensity responds linearly to purified GFP titration.
Background and aims
Studies have shown that levels of green fluorescent protein (GFP) leaf surface fluorescence are directly proportional to GFP soluble protein concentration in transgenic plants. However, instruments that measure GFP surface fluorescence are expensive. The goal of this investigation was to develop techniques with consumer digital cameras to analyse GFP surface fluorescence in transgenic plants.
Methodology
Inexpensive filter cubes containing machine vision dichroic filters and illuminated with blue light-emitting diodes (LED) were designed to attach to digital single-lens reflex (SLR) camera macro lenses. The apparatus was tested on purified enhanced GFP, and on wild-type and GFP-expressing arabidopsis grown autotrophically and heterotrophically.
Principal findings
Spectrum analysis showed that the apparatus illuminates specimens with wavelengths between ∼450 and ∼500 nm, and detects fluorescence between ∼510 and ∼595 nm. Epifluorescent photographs taken with SLR digital cameras were able to detect red-shifted GFP fluorescence in Arabidopsis thaliana leaves and cotyledons of pot-grown plants, as well as roots, hypocotyls and cotyledons of etiolated and light-grown plants grown heterotrophically. Green fluorescent protein fluorescence was detected primarily in the green channel of the raw image files. Studies with purified GFP produced linear responses to both protein surface density and exposure time (H0: β (slope) = 0 mean counts per pixel (ng s mm−2)−1, r2 > 0.994, n = 31, P < 1.75 × 10−29).
Conclusions
Epifluorescent digital photographs taken with complementary metal-oxide-semiconductor and charge-coupled device SLR cameras can be used to analyse red-shifted GFP surface fluorescence using visible blue light. This detection device can be constructed with inexpensive commercially available materials, thus increasing the accessibility of whole-organism GFP expression analysis to research laboratories and teaching institutions with small budgets.
doi:10.1093/aobpla/pls003
PMCID: PMC3296078  PMID: 22479674
11.  Clinical benefit of lapatinib-based therapy in patients with HER2-positive breast tumors co-expressing the truncated p95HER2 receptor 
Purpose
A subgroup of HER2 overexpressing breast tumors co-expresses p95HER2, a truncated HER2 receptor that retains a highly functional HER2 kinase domain but lacks the extracellular domain and results in intrinsic trastuzumab resistance. We hypothesized that lapatinib, a HER2 tyrosine kinase inhibitor, would be active in these tumors. We have studied the correlation between p95HER2 expression and response to lapatinib, both in preclinical models and in the clinical setting
Experimental design
Two different p95HER2 animal models were used for preclinical studies. Expression of p95HER2 was analyzed in HER2 overexpressing breast primary tumors from a first line lapatinib monotherapy study (EGF20009) and a second line lapatinib in combination with capecitabine study (EGF100151). p95HER2 expression was correlated with overall response rate (complete + partial response), clinical benefit rate (complete response + partial response + stable disease ≥ 24 weeks) and progression-free survival using logistic regression and Cox-proportional hazard models.
Results
Lapatinib inhibited tumor growth and HER2 downstream signaling of p95HER2 expressing tumors. A total of 68 and 156 tumors from studies EGF20009 and EGF100151 were evaluable, respectively, for p95HER2 detection. The percentage of p95HER2 positive patients was 20.5% in the EGF20009 study and 28.5% in the EGF100151 study. In both studies there was no statistically significant difference in progression-free survival, clinical benefit rate and overall response rate between p95HER2-positive and p95HER2-negative tumors.
Conclusions
Lapatinib as a monotherapy or in combination with capecitabine appears to be equally effective in patients with p95HER2-positive and p95HER2-negative HER2-positive breast tumors.
doi:10.1158/1078-0432.CCR-09-3407
PMCID: PMC3243489  PMID: 20406840
lapatinib; p95HER2; breast cancer; trastuzumab; HER2
12.  Lapatinib Plus Capecitabine in Women with HER-2–Positive Advanced Breast Cancer: Final Survival Analysis of a Phase III Randomized Trial 
The Oncologist  2010;15(9):924-934.
The article presents final analyses of overall survival from a phase III trial of lapatinib and capecitabine in patients with human epidermal growth factor receptor 2–positive locally advanced or metastatic breast cancer that progressed following prior therapy including trastuzumab.
Objectives.
A planned interim analysis of study EGF100151 prompted early termination of enrollment based on a longer time to progression with lapatinib and capecitabine than with capecitabine alone in patients with human epidermal growth factor receptor (HER)-2+ previously treated advanced breast cancer or metastatic breast cancer (MBC). Here, we report final analyses of overall survival.
Patients and Methods.
Women with HER-2+ MBC who progressed after regimens that included, but were not limited to, anthracyclines, taxanes, and trastuzumab, were randomized to lapatinib (1,250 mg/day) plus capecitabine (2,000 mg/m2) or capecitabine monotherapy (2,500 mg/m2) on days 1–14 of a 21-day cycle.
Results.
At enrollment termination, 399 patients were randomized, and nine were being screened and were offered combination treatment. In total, 207 and 201 patients were enrolled to combination therapy and monotherapy, respectively. Thirty-six patients receiving monotherapy crossed over to combination therapy following enrollment termination. The median overall survival times were 75.0 weeks for the combination arm and 64.7 weeks for the monotherapy arm (hazard ratio [HR], 0.87; 95% confidence interval [CI], 0.71–1.08; p = .210). A Cox regression analysis considering crossover as a time-dependent covariate suggested a 20% lower risk for death for patients treated with combination therapy (HR, 0.80; 95% CI, 0.64–0.99; p = .043). The low incidence of serious adverse events was consistent with previously reported rates.
Conclusions.
Although premature enrollment termination and subsequent crossover resulted in insufficient power to detect differences in overall survival, exploratory analyses demonstrate a trend toward a survival advantage with lapatinib plus capecitabine. These data continue to support the efficacy of lapatinib in patients with HER-2+ MBC.
doi:10.1634/theoncologist.2009-0181
PMCID: PMC3228041  PMID: 20736298
Breast cancer; Lapatinib; Metastatic; Capecitabine; Survival; HER-2
13.  Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure 
Background
Retinoic acid (RA) is important for vertebrate eye morphogenesis and is a regulator of photoreceptor development in the retina. In the zebrafish, RA treatment of postmitotic photoreceptor precursors has been shown to promote the differentiation of rods and red-sensitive cones while inhibiting the differentiation of blue- and UV-sensitive cones. The roles played by RA and its receptors in modifying photoreceptor fate remain to be determined.
Results
Treatment of zebrafish embryos with RA, beginning at the time of retinal progenitor cell proliferation and prior to photoreceptor terminal mitosis, resulted in a significant alteration of rod and cone mosaic patterns, suggesting an increase in the production of rods at the expense of red cones. Quantitative pattern analyses documented increased density of rod photoreceptors and reduced local spacing between rod cells, suggesting rods were appearing in locations normally occupied by cone photoreceptors. Cone densities were correspondingly reduced and cone photoreceptor mosaics displayed expanded and less regular spacing. These results were consistent with replacement of approximately 25% of positions normally occupied by red-sensitive cones, with additional rods. Analysis of embryos from a RA-signaling reporter line determined that multiple retinal cell types, including mitotic cells and differentiating rods and cones, are capable of directly responding to RA. The RA receptors RXRγ and RARαb are expressed in patterns consistent with mediating the effects of RA on photoreceptors. Selective knockdown of RARαb expression resulted in a reduction in endogenous RA signaling in the retina. Knockdown of RARαb also caused a reduced production of rods that was not restored by simultaneous treatments with RA.
Conclusions
These data suggest that developing retinal cells have a dynamic sensitivity to RA during retinal neurogenesis. In zebrafish RA may influence the rod vs. cone cell fate decision. The RARαb receptor mediates the effects of endogenous, as well as exogenous RA, on rod development.
doi:10.1186/1471-213X-11-51
PMCID: PMC3189157  PMID: 21878117
14.  Light Responses in Rods of Vitamin A–Deprived Xenopus 
Purpose
Accumulation of free opsin by mutations in rhodopsin or insufficiencies in the visual cycle can lead to retinal degeneration. Free opsin activates phototransduction; however, the link between constitutive activation and retinal degeneration is unclear. In this study, the photoresponses of Xenopus rods rendered constitutively active by vitamin A deprivation were examined. Unlike their mammalian counterparts, Xenopus rods do not degenerate. Contrasting phototransduction in vitamin A–deprived Xenopus rods with phototransduction in constitutively active mammalian rods may provide new understanding of the mechanisms that lead to retinal degeneration.
Methods
The photocurrents of Xenopus tadpole rods were measured with suction electrode recordings, and guanylate cyclase activity was measured with the IBMX (3-isobutyl-1-methylxanthine) jump technique. The amount of rhodopsin in rods was determined by microspectrophotometry.
Results
The vitamin A–deprived rod outer segments were 60% to 70% the length and diameter of the rods in age-matched animals. Approximately 90% of its opsin content was in the free or unbound form. Analogous to bleaching adaptation, the photoresponses were desensitized (10- to 20-fold) and faster. Unlike bleaching adaptation, the vitamin A–deprived rods maintained near normal saturating (dark) current densities by developing abnormally high rates of cGMP synthesis. Their rate of cGMP synthesis in the dark (15 seconds−1) was twofold greater than the maximum levels attainable by control rods (~7 seconds−1).
Conclusions
Preserving circulating current density and response range appears to be an important goal for rod homeostasis. However, the compensatory changes associated with vitamin A deprivation in Xenopus rods come at the high metabolic cost of a 15-fold increase in basal ATP consumption.
doi:10.1167/iovs.08-3186
PMCID: PMC2792892  PMID: 19407019
15.  Mammostrat® as a tool to stratify breast cancer patients at risk of recurrence during endocrine therapy 
Introduction
Patients with early-stage breast cancer, treated with endocrine therapy, have approximately 90% 5-year disease-free survival. However, for patients at higher risk of relapse despite endocrine therapy, additional adjuvant therapy, such as chemotherapy, may be indicated. The challenge is to prospectively identify such patients. The Mammostrat® test uses five immunohistochemical markers to stratify patients on tamoxifen therapy into risk groups to inform treatment decisions. We tested the efficacy of this panel in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up.
Methods
Tissue microarrays from a consecutive series (1981 to 1998) of 1,812 women managed by wide local excision and postoperative radiotherapy were collected following appropriate ethical review. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57, 71% were postmenopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat® risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score.
Results
Increased Mammostrat® scores were significantly associated with reduced DRFS, RFS and OS in estrogen receptor (ER)-positive breast cancer (P < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (P < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 ± 1.5% in the low-risk group versus 20.0 ± 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and untreated patients.
Conclusions
This is the fifth independent study providing evidence that Mammostrat® can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer. In addition, this study revealed for the first time a possible association with outcome regardless of node status and ER-negative tumors. When viewed in the context of previous results, these data provide further support for this antibody panel as an aid to patient management in early-stage breast cancer.
doi:10.1186/bcr2604
PMCID: PMC2949634  PMID: 20615243
16.  Dynamic changes in gene expression in vivo predict prognosis of tamoxifen-treated patients with breast cancer 
Introduction
Tamoxifen is the most widely prescribed anti-estrogen treatment for patients with estrogen receptor (ER)-positive breast cancer. However, there is still a need for biomarkers that reliably predict endocrine sensitivity in breast cancers and these may well be expressed in a dynamic manner.
Methods
In this study we assessed gene expression changes at multiple time points (days 1, 2, 4, 7, 14) after tamoxifen treatment in the ER-positive ZR-75-1 xenograft model that displays significant changes in apoptosis, proliferation and angiogenesis within 2 days of therapy.
Results
Hierarchical clustering identified six time-related gene expression patterns, which separated into three groups: two with early/transient responses, two with continuous/late responses and two with variable response patterns. The early/transient response represented reductions in many genes that are involved in cell cycle and proliferation (e.g. BUB1B, CCNA2, CDKN3, MKI67, UBE2C), whereas the continuous/late changed genes represented the more classical estrogen response genes (e.g. TFF1, TFF3, IGFBP5). Genes and the proteins they encode were confirmed to have similar temporal patterns of expression in vitro and in vivo and correlated with reduction in tumour volume in primary breast cancer. The profiles of genes that were most differentially expressed on days 2, 4 and 7 following treatment were able to predict prognosis, whereas those most changed on days 1 and 14 were not, in four tamoxifen treated datasets representing a total of 404 patients.
Conclusions
Both early/transient/proliferation response genes and continuous/late/estrogen-response genes are able to predict prognosis of primary breast tumours in a dynamic manner. Temporal expression of therapy-response genes is clearly an important factor in characterising the response to endocrine therapy in breast tumours which has significant implications for the timing of biopsies in neoadjuvant biomarker studies.
doi:10.1186/bcr2593
PMCID: PMC2917034  PMID: 20569502
17.  Bridging the gaps between research, policy and practice in low- and middle-income countries: a survey of researchers 
Background
Many international statements have urged researchers, policy-makers and health care providers to collaborate in efforts to bridge the gaps between research, policy and practice in low- and middle-income countries. We surveyed researchers in 10 countries about their involvement in such efforts.
Methods
We surveyed 308 researchers who conducted research on one of four clinical areas relevant to the Millennium Development Goals (prevention of malaria, care of women seeking contraception, care of children with diarrhea and care of patients with tuberculosis) in each of 10 low- and middle-income countries (China, Ghana, India, Iran, Kazakhstan, Laos, Mexico, Pakistan, Senegal and Tanzania). We focused on their engagement in three promising bridging activities and examined system-level, organizational and individual correlates of these activities.
Results
Less than half of the researchers surveyed reported that they engaged in one or more of the three promising bridging activities: 27% provided systematic reviews of the research literature to their target audiences, 40% provided access to a searchable database of research products on their topic, and 43% established or maintained long-term partnerships related to their topic with representatives of the target audience. Three factors emerged as statistically significant predictors of respondents’ engagement in these activities: the existence of structures and processes to link researchers and their target audiences predicted both the provision of access to a database (odds ratio [OR] 2.62, 95% CI 1.30–5.27) and the establishment or maintenance of partnerships (OR 2.65, 95% CI 1.25–5.64); stability in their contacts predicted the provision of systematic reviews (OR 2.88, 95% CI 1.35–6.13); and having managers and public (government) policy-makers among their target audiences predicted the provision of both systematic reviews (OR 4.57, 95% CI 1.78–11.72) and access to a database (OR 2.55, 95% CI 1.20–5.43).
Interpretation
Our findings suggest potential areas for improvement in light of the bridging strategies targeted at health care providers that have been found to be effective in some contexts and the factors that appear to increase the prospects for using research in policy-making.
doi:10.1503/cmaj.081164
PMCID: PMC2882466  PMID: 20439449
18.  Bridging the gaps among research, policy and practice in ten low- and middle-income countries: Development and testing of a questionnaire for researchers 
Background
A questionnaire could assist researchers, policymakers, and healthcare providers to describe and monitor changes in efforts to bridge the gaps among research, policy and practice. No questionnaire focused on researchers' engagement in bridging activities related to high-priority topics (or the potential correlates of their engagement) has been developed and tested in a range of low- and middle-income countries (LMICs).
Methods
Country teams from ten LMICs (China, Ghana, India, Iran, Kazakhstan, Laos, Mexico, Pakistan, Senegal, and Tanzania) participated in the development and testing of a questionnaire. To assess reliability we calculated the internal consistency of items within each of the ten conceptual domains related to bridging activities (specifically Cronbach's alpha). To assess face and content validity we convened several teleconferences and a workshop. To assess construct validity we calculated the correlation between scales and counts (i.e., criterion measures) for the three countries that employed both and we calculated the correlation between different but theoretically related (i.e., convergent) measures for all countries.
Results
Internal consistency (Cronbach's alpha) for sets of related items was very high, ranging from 0.89 (0.86-0.91) to 0.96 (0.95-0.97), suggesting some item redundancy. Both face and content validity were determined to be high. Assessments of construct validity using criterion-related measures showed statistically significant associations for related measures (with gammas ranging from 0.36 to 0.73). Assessments using convergent measures also showed significant associations (with gammas ranging from 0.30 to 0.50).
Conclusions
While no direct comparison can be made to a comparable questionnaire, our findings do suggest a number of strengths of the questionnaire but also the need to reduce item redundancy and to test its capacity to monitor changes over time.
doi:10.1186/1478-4505-8-4
PMCID: PMC2825187  PMID: 20205837
19.  Targeted effects of retinoic acid signaling upon photoreceptor development in zebrafish 
Developmental biology  2005;287(1):157-167.
Retinoic acid (RA) is a signaling molecule important for photoreceptor development in vertebrates. The purpose of this study was to examine the mechanisms of the effects of RA upon developing rod and cone photoreceptors in the embryonic zebrafish. Exposure to exogenous RA increased the number of photoreceptors expressing rod opsin and red cone opsin, and decreased the number of photoreceptors expressing the blue and UV cone opsins, suggesting targeted effects of RA on photoreceptor development. RA exposure also increased opsin expression in individual rods and red cones, but decreased opsin expression in individual blue and UV cones, as indicated by differences in the strength of opsin hybridization in identified photoreceptors. RA exposure did not, however, significantly alter quantitative measures of photoreceptor pattern in a manner expected for changes in photoreceptor fate. These observations collectively indicate that RA treatment does not affect photoreceptor fate, but rather differentially influences opsin transcription in determined photoreceptors. An enzyme involved in RA synthesis, RALDH2, was immunocytochemically localized to retinal progenitor cells and the retinal pigmented epithelium (RPE), suggesting the presence of RA in the vicinity of developing photoreceptors. However, expression of an RA response element-driven transgene was restricted to the RPE, retinal progenitors, and a small population of neurons in ventral retina, suggesting that the endogenous RA signaling system is spatially limited within the eye.
doi:10.1016/j.ydbio.2005.08.045
PMCID: PMC2804901  PMID: 16197938
Photoreceptor; Opsin; Cell fate; Retinoic acid; Danio rerio; Retina; Cone; Nuclear receptor signaling; Differentiation
20.  Retinal ectopias and mechanically weakened basement membrane in a mouse model of muscle-eye-brain (MEB) disease congenital muscular dystrophy 
Molecular Vision  2010;16:1415-1428.
Purpose
Some forms of congenital muscular dystrophy are associated with cortical and retinal dysplasias. Protein O-mannose N-acetylglucosaminyltransferase 1 (POMGnT1) knockout mice, one of the mouse models of muscular dystrophy, exhibit a thinner retina with reduced density of retinal ganglion cells. This study is aimed to further characterize the knockout retina, with special emphasis on the inner limiting membrane, the basement membrane of the retina.
Methods
Immunofluorescence staining and transmission electron microscopy were used to analyze the retinas. Atomic force microscopy was performed on the inner limiting membrane preparations to examine their mechanical properties.
Results
The inner limiting membrane of the knockout mice exhibited frequent breaks with protrusions of the Müller glial processes and ectopic placement of retinal ganglion cells into the vitreous humor. Disruptions in inner limiting membrane integrity developmentally precede the cellular abnormalities. Regions of disrupted inner limiting membrane were also associated with molecular abnormalities of Müller glia that included diminished presence of the integral membrane proteins Kir4.1 (an inwardly rectifying potassium channel) and aquaporin-4. When measured with atomic force microscopy, the POMGnT1 knockout mouse inner limiting membrane (ILM) exhibited significantly reduced Young’s modulus and is therefore mechanically weaker than the ILM from controls.
Conclusions
Deficiency of POMGnT1-mediated glycosylation of dystroglycan is implicated in reduced stiffness of the ILM. The weakened ILM results in the disruption of the membrane and subsequent reduction in retinal integrity.
PMCID: PMC2913139  PMID: 20680099
21.  Foundation clinical trials 
Breast Cancer Research : BCR  2009;11(Suppl 3):S23.
doi:10.1186/bcr2442
PMCID: PMC2797703  PMID: 20030875
22.  Phase III Study Comparing Exemestane With Tamoxifen As First-Line Hormonal Treatment of Metastatic Breast Cancer in Postmenopausal Women: The European Organisation for Research and Treatment of Cancer Breast Cancer Cooperative Group 
Journal of Clinical Oncology  2008;26(30):4883-4890.
Purpose
This phase III randomized open-label clinical trial was designed to evaluate the efficacy and safety of the steroidal aromatase inactivator exemestane versus the antiestrogen tamoxifen as first-line treatment for metastatic breast cancer (MBC) in postmenopausal women.
Patients and Methods
The study was conducted at 81 centers and enrolled postmenopausal patients with measurable hormone-sensitive metastatic or locally advanced breast cancer. Prior adjuvant chemotherapy and/or tamoxifen were allowed. One previous chemotherapy regimen and no prior hormone therapy for advanced disease were permitted. Patients were randomly assigned to receive exemestane 25 mg or tamoxifen 20 mg orally once daily until disease progression or unacceptable toxicity occurred.
Results
A total of 371 patients enrolled at 79 sites (182 exemestane, 189 tamoxifen) were included in the analysis. Both treatments were generally well tolerated without major toxicity. Overall response rate was greater for exemestane than for tamoxifen treatment (46% v 31%; odds ratio = 1.85; 95% CI, 1.21 to 2.82; P = .005). Median progression-free survival (PFS) was longer with exemestane (9.9 months; 95% CI, 8.7 to 11.8 months) than with tamoxifen (5.8 months; 95% CI, 5.3 to 8.1 months). However, these early differences (Wilcoxon P = .028) did not translate to a longer-term benefit in PFS, the primary study end point (log-rank P = .121). There was also no difference in survival between both study arms.
Conclusion
Exemestane is an effective and well-tolerated first-line hormonal treatment for postmenopausal women with MBC and offers significant early improvement in time to tumor progression when compared with tamoxifen.
doi:10.1200/JCO.2007.14.4659
PMCID: PMC2652082  PMID: 18794551
23.  Sequential docetaxel as adjuvant chemotherapy for early breast cancer (TACT): an open-label, phase III, randomised controlled trial 
Lancet  2009;373(9676):1681-1692.
Summary
Background
Incorporation of a taxane as adjuvant treatment for early breast cancer offers potential for further improvement of anthracycline-based treatment. The UK TACT study (CRUK01/001) investigated whether sequential docetaxel after anthracycline chemotherapy would improve patient outcome compared with standard chemotherapy of similar duration.
Methods
In this multicentre, open-label, phase III, randomised controlled trial, 4162 women (aged >18 years) with node-positive or high-risk node-negative operable early breast cancer were randomly assigned by computer-generated permuted block randomisation to receive FEC (fluorouracil 600 mg/m2, epirubicin 60 mg/m2, cyclophosphamide 600 mg/m2 at 3-weekly intervals) for four cycles followed by docetaxel (100 mg/m2 at 3-weekly intervals) for four cycles (n=2073) or control (n=2089). For the control regimen, centres chose either FEC for eight cycles (n=1265) or epirubicin (100 mg/m2 at 3-weekly intervals) for four cycles followed by CMF (cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, and fluorouracil 600 mg/m2 at 4-weekly intervals) for four cycles (n=824). The primary endpoint was disease-free survival. Analysis was by intention to treat (ITT). This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN79718493.
Findings
All randomised patients were included in the ITT population. With a median follow-up of 62 months, disease-free survival events were seen in 517 of 2073 patients in the experimental group compared with 539 of 2089 controls (hazard ratio [HR] 0·95, 95% CI 0·85–1·08; p=0·44). 75·6% (95% CI 73·7–77·5) of patients in the experimental group and 74·3% (72·3–76·2) of controls were alive and disease-free at 5 years. The proportion of patients who reported any acute grade 3 or 4 adverse event was significantly greater in the experimental group than in the control group (p<0·0001); the most frequent events were neutropenia (937 events vs 797 events), leucopenia (507 vs 362), and lethargy (456 vs 272).
Interpretation
This study did not show any overall gain from the addition of docetaxel to standard anthracycline chemotherapy. Exploration of predictive biomarker-defined subgroups might have the potential to better target the use of taxane-based therapy.
Funding
Cancer Research UK (CRUK 01/001), Sanofi-Aventis, Pfizer, and Roche.
doi:10.1016/S0140-6736(09)60740-6
PMCID: PMC2687939  PMID: 19447249
24.  Age-Related Cone Abnormalities in Zebrafish with Genetic Lesions in Sonic Hedgehog 
Purpose
Sonic hedgehog (Shh) signaling is essential for photoreceptor differentiation and retinal cell survival in embryonic zebrafish. The study was conducted to determine whether adult heterozygous carriers of mutant alleles for the shh gene display retinal abnormalities.
Methods
Retinal cryosections from young, middle-aged, and senescent wild-type and sonic-you+/− (syu+/−) zebrafish were probed with retinal cell type–specific markers. Contralateral retinal flatmounts from these fish, and from adult albino zebrafish subjected to light-induced photoreceptor damage followed by regeneration, were hybridized with blue cone opsin cRNA for quantitative analysis of the blue cone pattern. Retinal expression of shh mRNA was measured by quantitative RT-PCR.
Results
Regions of cone loss and abnormal cone morphology were observed in the oldest syu+/− zebrafish, although no other retinal cell type was affected. This phenotype was age-related and genotype-specific. Cone distribution in the oldest syu+/− zebrafish was predominantly random, as assessed by measuring the short-range pattern, whereas that of wild-type fish and the younger syu+/− zebrafish was statistically regular. A measure of long-range pattern revealed atypical cone aggregation in the oldest syu+/− zebrafish. The light-treated albino zebrafish displayed random cone patterns immediately after light toxicity, but showed cone aggregation on regeneration. Retinas from the syu+/− fish showed reduced expression of shh mRNA compared with those of wild-type siblings.
Conclusions
The syu+/− zebrafish presents a model for the study of hereditary age-related cone abnormalities. The syu+/− retinas most likely experience progressive cone photoreceptor loss, accompanied by cone regeneration. Shh signaling may be required to maintain cone viability throughout life.
doi:10.1167/iovs.07-1224
PMCID: PMC2584603  PMID: 18502998
25.  Hindpaw incision in the rat produces long-lasting colon hypersensitivity 
Visceral injury has been shown to alter somatic sensitivity, but little is known about the effect of somatic insult on the viscera. In the present study we examined: 1) the effect of colon inflammation on somatic sensitivity and 2) the affect of hindpaw incision on colon sensitivity. After intracolonic administration of trinitrobenzene sulfonic acid (TNBS) or zymosan, visceromotor responses (VMR) to colorectal distension (CRD) were increased to post-treatment day 8. Mechanical withdrawal thresholds in the hindpaw were decreased in TNBS- and in zymosan-treated rats until post intracolonic treatment day 2. There was no change in hindpaw heat withdrawal latency in either group. Plantar incision of the hindpaw resulted in a decrease in both hindpaw mechanical withdrawal threshold and heat withdrawal latency and significantly increased the VMR to CRD from post-incision day 1 to 8. The colon hypersensitivity was of longer duration than hyperalgesia at the site of hindpaw incision. These results support the hypothesis that somatic injury and visceral inflammation can alter central processing of visceral and somatic inputs, respectively.
Perspective
Surgical procedures are common and typically associated with hyperalgesia at and around the site of incision. This report establishes in a model of post-surgical pain and hyperalgesia that a long-lasting visceral hypersensitivity may also accompany post-surgical hyperalgesia.
doi:10.1016/j.jpain.2007.10.017
PMCID: PMC2396331  PMID: 18088562
Colorectal distension; central sensitization; somatic pain; somatovisceral convergence; visceral pain; zymosan; incision

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